integration of micro fluidics and fluorescence in situ
TRANSCRIPT
INTEGRATION OF MICROFLUIDICS AND FLUORESCENCE IN SITU HYBRIDIZATION (FISH) FOR THE RAPID IDENTIFICATION OF MICROORGANISMS
CÁTIA DANIELA CRUZ MOREIRA DISSERTAÇÃO DE MESTRADO APRESENTADA À FACULDADE DE ENGENHARIA DA UNIVERSIDADE DO PORTO EM ENGENHARIA BIOMÉDICA
M 2014
i
This thesis was supervised by:
Professor Nuno Filipe Azevedo
Faculdade de Engenharia, Universidade do Porto
Professor João Mário Miranda
Faculdade de Engenharia, Universidade do Porto
The host institution of this thesis was:
FEUP – Faculdade de Engenharia da Universidade do Porto
LEPABE – Laboratório de Engenharia de Processos, Ambiente, Biotecnologia e Energia
The research described in this thesis was financially supported by:
FEDER funds through the Operational Programme for
Competitiveness Factors – COMPETE, ON.2 - O Novo Norte - North
Portugal Regional Operational Programme and National Funds
through FCT - Foundation for Science and Technology under the
projects: PEst-C/EQB/UI0511, NORTE-07-0124-FEDER-000025 -
RL2_ Environment&Health and DNA mimics Research Project
PIC/IC/82815/2007.
ii
“The two most powerful warriors are patience and time”
Lev Tolstoy, War and peace
iii
Acknowledgements
iv
Acknowledgments
This work was made possible by the valuable contributions of an important number of
people. Firstly, I would like to express my gratitude to my supervisor, Prof. Nuno
Azevedo, and co-supervisor Prof. João Mário Miranda, for their help and support,
excellent advice and encouragement during the development of this work. I also wish
to express my sincere gratitude to Laura Cerqueira, for all her help and support.
I would like to acknowledge the collaboration of several persons from the following
institutions:
Laboratório de Engenharia de Processos, Ambiente, Biotecnologia e Energia
(LEPABE) – Andreia Azevedo, who provided me with the best support I could have
had, for being a friend more than a co-worker, and for always being there for me –
thank you for your patience and understanding. To my colleagues at LEPABE that
have in anyway contributed to this work and provided a good working environment:
Bruno Pereira, Rui Rocha, André Ferreira, Sílvia Fontenete, Nuno Guimarães, Luciana
Gomes, Rita Santos, and Carolina Lima.
Centro de Estudos de Fenómenos de Transporte (CEFT) – Maha Ponmozhi, Lúcia
Sousa, Romeu Matos and João Carneiro for their assistance and technical support.
Mariline Pinto, from who I learnt my first steps on microfluidics, for the suggestions and
assistance, for sharing his scientific knowledge, for all the helpful discussions and for
providing me the friendship I needed.
Finally, I wish to thank my close friends and family, namely to:
Mom: I can’t thank you enough.
My father: thanks for being always there!
BabyBro: I’m sorry for the lack of patience and attention over these last months!
And you can’t imagine how important was your joy every time we jumped on the bed
singing «I’m on top of the world»
A last and very special thanks to my friends José Pedro Gonçalves, Marlene
Morgado, Tatiana Teixeira, and Claudia Tonelo who offer me great moments, and for
having the amazing ability to laugh on my busiest and annoying days!
Abstract
v
Abstract The frequency of invasive fungal infections caused by yeasts has increased in
intensive care units, being Candida spp. reported among the ten leading causes of
bloodstream infections in Europe and in the USA. Candida sepsis has proven to be a
life-threatening infection with high prevalence and crude morbidity and mortality rates.
A rapid identification of the microorganism leads to an efficient intervention and a
significant reduction of the intake of broad spectrum antibiotics, with results in overall
cost savings. Peptide nucleic acid fluorescence in situ hybridization (PNA-FISH) is
already used as a highly precise and sensitive molecular method for microbial
identification. However, the entire procedure can take 1-2 days, since an enrichment
step of blood culture is necessary before applying the technique. Microfluidic devices
appear as a solution to reduce the time of the diagnosis by just consuming the time of
the PNA-FISH methodology, without wasting it on sample enrichment.
Therefore, the aim of this work is to develop a microfluidic system capable of retain
yeasts from a sample in a specific place within, increasing this way the sample
concentration, so the PNA-FISH procedure can be performed and the detection of the
pathogen can be made directly. Saccharomyces cerevisiae, a generally recognized as
safe (GRAS) microorganism, was chosen as a surrogate microorganism, in order to
avoid the contact with pathogens during this study. The construction of the microfluidics
system comprised micro-channels produced in polydimethylsiloxane (PDMS) using soft
lithography and a fluid injection system (including tubing, needles and a syringe pump).
To monitor the yeast retention, an optical microscope was used for the experiments of
flow visualization and to assess the hybridization an epifluorescence microscope
equipped with a red filter, sensitive to the fluorochrome attached to the probe, was
used.
PNA-FISH protocol was optimized in terms of fixation, temperature of hybridization
and the composition of hybridization solution. Paraformaldehyde/ethanol fixation, 54ºC
and a simplified hybridization solution without formamide was set as the optimal
procedure. Several microchannel geometries were conceived and a CFD simulation for
each one was performed in order to evaluate the best option. In some cases, the
exclusion of possibilities was made by geometrical characterization of the channels or
experimental microfluidics. Finally, the PNA-FISH was performed within the
microchannel with limited success.
In the future, deeper optimizations in the hybridization process within the microfluidic
device and the flow rate and yeast concentration need to be also optimized in order to
Abstract
vi
increase, as much as possible, the efficiency of the retention process. Additionally, the
time of hybridization and washing must be reduced without compromising the output.
Nonetheless, a microchannel geometry that makes feasible the rapid identification of S.
cerevisiae inside a microchip was achieved.
Resumo
vii
Resumo
A frequência de infeções fúngicas invasivas causadas por leveduras tem
aumentado nas unidades de cuidados intensivos, estando as espécies de Candida
reportadas entre as dez principais causas de infeções sistémicas na Europa e nos
EUA. A sepses tem proado ser uma infeção que ameaça a vida com elevada
prevalência e com índices de morbidade e mortalidade brutos. Uma identificação
rápida dos microrganismos leva a uma intervenção eficiente e a uma redução
significativa da toma de antibióticos de largo espectro, com resultados numa poupança
global. A técnica de hibridação in situ fluorescente com utilização de ácidos péptido-
nucleicos (PNA-FISH) é já usada como um método molecular para identificação de
microrganismos. No entanto, o processo completo pode levar 1-2 dias, uma vez que é
necessário um passo de enriquecimento da cultura sanguínea, antes de aplicar a
técnica. Os dispositivos de microfluídica aparecem como a solução para reduzir o
tempo do diagnóstico para unicamente o consumo do tempo da metodologia do PNA-
FISH, sem o gastar no enriquecimento da amostra.
Portanto, o objetivo deste trabalho é desenvolver um sistema de microfluídica
capaz de reter leveduras de uma amostra num local específico no seu interior,
aumentando, desta forma, a concentração da amostra, para que o procedimento de
PNA-FISH possa ser realizado e a deteção dos patogénios possa ser feita
diretamente. Saccharomyces cerevisiae, um microrganismo geralmente reconhecido
como seguro (GRAS), foi escolhida como o microrganismo substituto, para evitar o
contacto com patogénios em estudos preliminares. A construção destes sistemas de
microfluídica compreendem a produção de microcanais em polidimetilsiloxano
(PDMS), usando a litografia suave e um sistema de injeção de fluidos (incluindo
tubagem, agulhas e uma bomba de seringa). Para monitorizar a retenção das
leveduras, um microscópio ótico foi usado para os ensaios de visualização de
escoamento e para avaliar a hibridação foi utilizado um microscópio epifluorescente
equipado com um filtro vermelho, sensível à sonda usada.
O protocolo de PNA-FISH foi otimizado em termos de fixação, da temperatura
de hibridação e da composição da solução de hibridação. A fixação de
paraformaldeído/etanol, 54ºC e uma solução de hibridação simplificada sem
formamida foi estabelecida como ótima. Várias geometrias de microcanais foram
concebidas e foi realizada uma simulação CFD para cada, com o objetivo de avaliar a
melhor opção. Em alguns casos, foi feita a exclusão de possibilidades pela
caracterização geométrica do canal ou pela microfluídica experimental. Finalmente,
Resumo
viii
procedeu-se ao PNA-FISH dentro do microcanal.
Os resultados são promissores para a obtenção de bons resultados, utilizando
o método proposto, mas precisam de otimizações profundas no processo de
hibridação no interior do dispositivo de microfluídica assim como a concentração de
leveduras precisam, também, de ser otimizados, com o objetivo de aumentar, tanto
quanto possível, a eficiência do processo de retenção. Adicionalmente, o tempo de
hibridação e lavagem deve ser reduzido, sem comprometer o resultado. Conclui-se
que foi encontrada uma geometria de microcanal que torna fazível a identificação
rápida de S. cerevisiae dentro do microchip.
Contents
ix
Contents
Acknowledgments .......................................................................................................... iv
Abstract ............................................................................................................................ v
Resumo ........................................................................................................................... vii
Contents .......................................................................................................................... ix
List of figures .................................................................................................................. xi
List of tables ................................................................................................................... xii
Acronyms ...................................................................................................................... xiii
Chapter I – Introduction .................................................................................................. 1 1. Framework ...................................................................................................................... 2 2. Aim ................................................................................................................................... 3 3. Thesis Outline ................................................................................................................ 3
Chapter II - Literature review .......................................................................................... 5 1. Candida spp. .................................................................................................................. 6
1.1 Microbiology and pathogenesis ............................................................................... 6 1.2 Epidemiology and treatment ..................................................................................... 9
2. Diagnosis of Candida spp. ......................................................................................... 12 2.1 Phenotypic methods ................................................................................................ 12 2.2 Molecular Techniques ............................................................................................. 13
3. Fluorescence in situ hybridization ............................................................................. 14 3.1 Brief historical overview .......................................................................................... 17 3.2 Nucleic acid mimics ................................................................................................. 18 3.2.1 Peptide nucleic acid (PNA).................................................................................. 19
4. Microfluidics for miniaturizing biological assays ..................................................... 20 4.1 Entrapment strategies ............................................................................................. 20 4.2 Microfabrication ........................................................................................................ 22 4.2.1 Photolithography (SU-8) ...................................................................................... 22 4.2.2 Xurography ............................................................................................................ 23 4.2.3 Soft Lithography .................................................................................................... 24 4.2.3.1 PDMS .................................................................................................................. 25 4.2.4 Other techniques ................................................................................................... 26 4.3 Concepts in fluid mechanics ................................................................................... 27 4.4 Liquid handling systems .......................................................................................... 29
5. Computational fluid dynamics .................................................................................... 31 5.1 Finite volume method .............................................................................................. 33
Chapter III – Materials and methods ........................................................................... 35 1. Yeast characterization ................................................................................................ 36
1.1. Surrogate microorganism selection, yeast strain and culture maintenance . 36 1.2 S. cerevisiae growth ................................................................................................. 36
2. PNA-FISH protocol optimization ............................................................................... 37 2.1 Probe sequence ....................................................................................................... 37 2.2 Hybridization conditions optimization .................................................................... 37 2.3 Fixation conditions optimization ............................................................................. 38 2.4 Assessment of dextran sulfate influence on hybridization .................................. 39
Contents
x
3. Material preparation .................................................................................................... 40 3.1 Microchip design ...................................................................................................... 40 3.1.1 Production of molds – channels with a dam-like structure .............................. 40 3.1.2 Production of molds – channels with pillar-based structure ............................ 41 3.2 Microfabrication of microchannels .......................................................................... 41
4. CFD simulation ............................................................................................................ 42 5. Characterization of the microchannels ..................................................................... 43
5.1 Geometrical characterization of the microchannels ............................................ 43 5.2 Volumetric measurement of channels capacity ................................................... 44 5.3 Material testing ......................................................................................................... 44
6. Microfluidic channels retention test........................................................................... 44 7. PNA-FISH methodology within the microfluidic channel ....................................... 44 8. Microscopic visualization ............................................................................................ 46
Chapter IV – Results and Discussion ......................................................................... 48 1. S. cerevisiae growth .................................................................................................... 49 2. PNA-FISH protocol optimization ............................................................................... 51 3. Microchannels geometry design ............................................................................... 58
3.1 Channels with a dam-like structure ....................................................................... 59 3.2 Channels with a pillar-based filter .......................................................................... 59 3.2.1 Single channel devices ......................................................................................... 60 3.2.2 Two inlet devices ................................................................................................... 62
4. Computational fluid dynamics analysis .................................................................... 64 5. Retention tests in channels with a dam-like structure ........................................... 67 6. Geometrical characterization of channels with pillar-based filter ......................... 69 7. Retention tests in single channels with pillar-based filter ...................................... 72 8. PNA-FISH methodology within the microfluidic channel ....................................... 74
Chapter V – Conclusions and Future Work ................................................................ 77 Conclusions ........................................................................................................................... 78 Future work ........................................................................................................................... 79
References ..................................................................................................................... 80
List of Figures
xi
List of figures Figure 1| Morphologies assumed by Candida albicans ____________________________________________________ 6 Figure 2| Candida albicans tissue invasion ________________________________________________________________ 7 Figure 3| Schematic of cell biology of yeast, pseudohyphal and hyphal development ___________________ 8 Figure 4| Fluconazole drug resistance by Candida species and country ________________________________ 11 Figure 5| CHROMagar Candida ___________________________________________________________________________ 13 Figure 6| API Candida System strips ______________________________________________________________________ 13 Figure 7| Basics steps of FISH ______________________________________________________________________________ 15 Figure 8| Chemical structure of DNA, RNA LNA and PNA ________________________________________________ 18 Figure 9| Examples of microfilters _________________________________________________________________________ 21 Figure 10| Schematic of photolithography technique ____________________________________________________ 23 Figure 11| Xurography technique__________________________________________________________________________ 24 Figure 12| Schematic of soft lithography technique ______________________________________________________ 24 Figure 13| Interconnected channels _______________________________________________________________________ 25 Figure 14| Structure of PDMS polymer chain _____________________________________________________________ 26 Figure 15| Motion of cylindrical fluid element within a pipe _____________________________________________ 28 Figure 16| Schematic of equipment network for pressure driven laminar flow _________________________ 30 Figure 17| Flow focusing structure ________________________________________________________________________ 30 Figure 18| Steps in CFD simulation ________________________________________________________________________ 32 Figure 19| Bi-dimensional finite volume P and its neighbors. ____________________________________________ 33 Figure 20| Construction of the microfluidic device with a dam-like structure with 8-µm gap __________ 41 Figure 21| Cross section of the microchannel _____________________________________________________________ 43 Figure 22| Schematic illustration of the concept to perform FISH assay in the microfluidic device ____ 46 Figure 23| Saccharomyces cerevisiae growth curve ______________________________________________________ 50 Figure 24| Saccharomyces cerevisiae calibration curve __________________________________________________ 51 FIGURE 25| Fluorescence microscope results for in suspension PNA-FISH with different hybridization solutions at different temperatures _________________________________________________ 53 Figure 26| Fluorescence microscope results for in suspension PNA-FISH with simplified hybridization
solution (0% formamide) at 54˚C __________________________________________________________________________ 54 Figure 27| Fluorescence microscope results for in suspension PNA-FISH simplified hybridization
solution (0% formamide) at 54˚C after different fixation protocols _____________________________________ 56 Figure 28| Fluorescence microscope results for in suspension PNA-FISH simplified hybridization solution (0% formamide) at 54˚C with presence or absence of dextran sulfate. _________________________ 58 Figure 29| Mask structure __________________________________________________________________________________ 59 Figure 30| Geometries of cell-filtering single microfluidic devices _______________________________________ 60 Figure 31| Geometries of enlarged cell-filtering microfluidic devices ____________________________________ 61 Figure 32| Geometries of two streams cell-filtering microfluidic devices ________________________________ 63 Figure 33| Geometries of three streams cell-filtering microfluidic devices ______________________________ 63 FIGURE 34| Velocity contours and streamlines in the conceived geometries for microfluidic devices along the horizontal plane of symmetry as predicted by the CFD simulation
performed __________________________________________________________________________________________________ 66 FIGURE 35| Contour plot in a vertical plane in the middle of the horseshoe structure from Simulation X _______________________________________________________________________________________________ 67 Figure 36| Retention test in channels with a dam-like structure _________________________________________ 68 Figure 37| Usage of PS particles as filter for retention of S. cerevisiae in channels with a dam-like structure ____________________________________________________________________________________________________ 69 Figure 38| Geometrical characterization of a channel with a nominal height of 50µm ________________ 70 Figure 39| Geometrical characterization of a channel with a nominal height of 30µm ________________ 70 Figure 40| Channel with diamond-like pillars exhibiting barrier imperfections ________________________ 71 Figure 41| Channel with 15x5 µm rectangular pillars exhibiting barrier imperfections _______________ 71 Figure 42| Channel with pyramidal structures ____________________________________________________________ 72 Figure 43| Microspheres retention test for channels with pyramidal structures ________________________ 73 Figure 44| S. cerevisiae retention test for channels with pyramidal structures _________________________ 74 Figure 45| Channel with pyramidal structures ____________________________________________________________ 75 Figure 46| Positive for PNA-FISH methodology within the microfluidic channel. _______________________ 75
List of Tables
xii
List of tables
Table 1| Risk factors for development of invasive Candidiasis ____________________________________________ 9 Table 2| Probe thermodynamic parameters177 ____________________________________________________________ 37 Table 3| Comparison of different fixation protocols ______________________________________________________ 39 Table 4| Characteristics of each 2D mesh ____________________________________________________________ 43
Acronyms
xiii
Acronyms
BSI – Bloodstream infection
CFD – Computational fluid dynamics
DAPI – 4',6-diamidino-2-phenylindole
FISH – Fluorescence in situ hybridization
ICU – Intensive care unit
LNA – Locked nucleic acid
MALDI-TOF – matrix-assisted laser desorption ionization – time of flight
MEMS – Microelectromechanical system
MRI – Magnetic resonance imaging
MS – Mass spectrometry
NAC – Non-albicans Candida
OD - Optical Density
PC – Polycarbonate
PCR – Polymerase chain reaction
PDMS – Polydimethylsiloxane
PE – Polyethylene
PMMA – Polymethylmethacrylate
PNA – Peptide Nucleic Acid
PVC – Polyvinylchloride
Re –Reynolds number
2’-OMe – 2’-O-methyl nucleic acid
3D – Three dimensional
Chapter I – Introduction
1. Framework
2. Aim
3. Thesis Outline
Integration of microfluidics and FISH for the rapid identification of microorganisms
2 Daniela Cruz Moreira
1. Framework
The World Health Organization (WHO) has recently warned against what can be the
entry into the ominous “post-antibiotic” era, in its first global report on antibiotic
resistance in microoganisms1. This first-of-its-kind report has reached prominence in
the press and rang the alarm bell regarding the need to start a global effort to fight
against emerging resistance. According to the information included in the report, this
public health threat can be tackled essentially by avoiding the overuse of antibiotics,
preventing the quick proliferation of drug-resistant pathogens. In spite of not being a
new problem, modern medicine is now facing an apocalyptic scenario in which
common infections can kill, and healthcare professionals should act in order to use
antibiotics effectively. The consciousness of antimicrobial-resistant emergence in every
part of the world provides the kick-start needed to the urgent and consolidated
development of rapid and effective diagnostic tools1.
Identification of microorganisms causing sepsis is a determinant outcome because it
can lead to an appropriate antimicrobial therapy, instead of an empiric broad-spectrum
antibiotic treatment. Prior antibiotic use in patients with fungal infection is a risk factor
since it will not lead to fungus eradication but to its opportunistic spread. Rapid
diagnostic methods can revolutionize modern medicine by avoiding those current
misdirected practices that can worsen the patient’s condition and also by softening the
selective pressure of intense antibiotic use that promotes antibiotic resistance.
Molecular techniques require prior enrichment the biosample to reach detectable levels
of target cells. These assays are generally traditional cultural techniques, that can take
several hours. Miniaturization technology provides facilities for creating tools with
feature sizes matching the dimensions of cells. A particular cell is fixed and labeled by
its position, enabling high spatial resolution. Bypassing the enrichment step, the time
consumed during the entire procedure can be reduced to the time of the technique
alone.
Diagnosis delay also implies a heavy and unnecessary economic burden for the
hospital, due to treatments inefficiency, increased patient’s hospitalization time and
resources2. Despite the progress and improvement of hygiene and sanitary conditions,
hospital-acquired infections, also called nosocomial infections, are a major cause of
morbidity and mortality3. This fact is due to the hospital environment, that facilitates the
transmission of microorganisms among patients. Bloodstream infections (BSI) are
frequent in intensive care units (ICU), essentially because of immunocompromised
patient’s vulnerability and their prolonged hospital stay4. Although Gram-positive
CHAPTER I - INTRODUCTION
Daniela Cruz Moreira 3
bacterial pathogens persist as the most common cause of sepsis, fungal organisms
infections are increasing rapidly5,6.
Development of rapid diagnostic technology can be the golden key to positively impact
the economic and social issues described above, considering that can provide faster,
cheaper and more precise tools than time-consuming procedures available nowadays7.
Raising new advances in the diagnosis of infectious diseases is the first step to
generate significant progresses in healthcare.
2. Aim
This work is part of a project, whose ultimate goal is the conception of a fully integrated
and automated lab-on-a-chip sensor platform capable of being clinically implemented
for point-of-care pathogen detection where the sensing relies on nucleic acid
hybridization.
The aim of this work is the development of a microfluidic filter-based device to achieve
rapid detection of yeast cells in which peptide nucleic acid fluorescence in situ
hybridization (PNA-FISH) assays can be performed. The low number of cells present in
clinical samples implies an enrichment step before the PNA-FISH procedure can be
carried out. This system should allow the effective entrapment and concentration of
non-adherent yeast cells in defined locations, in order to bypass the prior enrichment
step required by standard FISH. All geometries developed should be conceived to trap
Candida spp., the most common cause of fungal infection worldwide5. Although
Candida spp. is the targeted microorganism, a morphologically similar non-pathogenic
surrogate microorganism must be carefully chosen to facilitate the testing phase of the
product.
This represents one of the first attempts to integrate PNA-FISH procedures applied to
microorganisms into miniaturized fluidic devices.
3. Thesis Outline
This thesis is composed by 6 chapters. On this chapter (Chapter I), a brief introduction
to the work in question is presented, and the framework and description of the thesis
structure is mentioned, to allow a better understanding of each chapter’s content.
Chapter II is dedicated to the literature review, where concepts of microbiology and
epidemiology of Candida spp., fluorescence in situ hybridization, microfluidics,
Integration of microfluidics and FISH for the rapid identification of microorganisms
4 Daniela Cruz Moreira
computational fluids dynamics (CFD) and current available methods in the market for
microorganism infections are discussed
Chapter III describes the experimental methodology, providing the necessary
information to replicate the procedures. . An introduction about the microorganism that
was selected as a Candida surrogate, S. cerevisiae, is also presented.
Chapter IV presents the experimental results obtained.
Chapter V discusses the results in detail.
Finally, chapter VI summarizes the main conclusions of this thesis and presents an
outlook for future work.
Chapter II - Literature review
1. Candida spp.
2. Diagnosis of Candida spp.
3. Fluorescence in situ hybridization
4. Microfluidics for miniaturizing
biological assays
5. Computational fluid dynamics
Integration of microfluidics and FISH for the rapid identification of microorganisms
6 Daniela Cruz Moreira
1. Candida spp.
1.1 Microbiology and pathogenesis
Mammalian mucosal surfaces and skin harbor millions of microorganisms that live as
benign commensals. Most of these microorganisms are beneficial to their host but a
few of them are considered opportunistic pathogens since they are capable of causing
illness when they cross the host’s protective barriers and colonize internal organs8.
Candida spp. are naturally present in the skin and the mucosal surfaces of the oral
cavity, gastrointestinal and urogenital tracts and vagina of the human host8. The genus
Candida belongs to the Fungi kingdom, class of Deuteromycetes, and includes several
species such as yeasts with ascomycetous and basidiomycetous affinities9. Some of
them can assume a variety of cell morphologies, as Figure 1 shows, ranging from
yeast-like cells (blastospore) to a variety of elongated growth forms, including germ
tubes (transition between yeast and hyphae), hyphae and pseudohyphae10. Only
Candida albicans and Candida dubliniensis are able to form true hyphae and these two
species are considered to be polymorphic11. This phenotypic plasticity is crucial to C.
albicans pathogenesis12,13, since hyphal cells may promote tissue invasion (Fig. 2),
whereas yeast cells facilitate dissemination of the pathogen (see below)14,15.
The yeast form is the one most commonly found in the laboratory because the
transition to elongated growth has to be stimulated by a wide range of environmental
conditions that mimics the microenvironment that it encounters in the host10. Hyphal
growth can be experimentally induced, at 37˚C (physiological temperature), by the
presence of serum16; a neutral pH17; 5% CO2 partial pressure, experienced in the
bloodstream18 and N-acetyl-D-glucosamine19. There are two synthetic growth media
that often induce hyphal growth due to the presence of amino acids20 or mannitol as a
carbon source21.
FIGURE 1| Morphologies assumed by Candida albicans
Candida albicans can assume a variety of morphologies: Yeast (Y), pseudohyphal (P) or hyphal (H) forms10 .
CHAPTER II – LITERATURE REVIEW
Daniela Cruz Moreira 7
FIGURE 2| Candida albicans tissue invasion
The several steps in tissue invasion by Candida
albicans, for a stylized epithelial cell surface:
adhesion to the epithelium; epithelial penetration
and invasion by hyphae; vascular dissemination,
which involves hyphal penetration of blood vessels
and seeding of yeast cells into the bloodstream; and,
finally, endothelial colonization and penetration
during disseminated disease22
.
The growth behavior of yeast is also similar to bacteria. Yeast cells display a lag
phase prior to an exponential period of division. As some nutrients become depleted,
the increase in cell number slows and stops. If refrigerated in this stationary phase,
cells can remain alive for months23.
Figure 3 shows fundamental differences in the cell cycle progression of a yeast,
pseudohyphal and hyphal cells. It is observed that what is common in all types is the
evagination of a single germ tube from the mother cell, that exhibit highly polarized
growth. First, the fixation site of the septin ring24,25 and the nucleus migration26,27 of
yeast (Figure 3a) and pseudohyphal (Fig. 3b) cells are very similar to each other but
different from hyphal cells28 (Fig. 3c). The main difference among these cells types are
related to cytokinesis. In budded yeast cells results on a constriction between adjacent
cells until they separate and both reenter the cycle29. In pseudohyphae, daughter cell
remained associated with the mother cell in both types but the attachment is easily
disrupted by mechanical agitation30. In hyphae, cytokinesis does not result in any
constriction, but in the formation of a primary and secondary septum, and the two
daughter compartments remain firmly attached to each other, being hyphae shape a
Integration of microfluidics and FISH for the rapid identification of microorganisms
8 Daniela Cruz Moreira
sparsely branched tube-like structure29. After hyphal cytokinesis, the subapical
compartment of hypha becomes highly vacuolated and remains in G1 and the apical
compartment is continuing dividing.
FIGURE 3| Schematic of cell biology of yeast, pseudohyphal and hyphal development
(a) Yeast cell. As the mother cell germinates, septin bars (orange) are visible at the germ tube base, as well as
a septin cap at the tip (red). The nucleus (blue) moves to the neck, assisted by astral microtubules (green), followed
by division across the neck. (b) Pseudohyphal cell. Similar features to yeast cells are observed in the beginning of
the cycle. The main exception is that cells do not separate following by cytokinesis, and the polarisome persists for
longer. (c) Hyphal cell. As the germ tube extends, the septin bars disappear and a septin ring appears in the hypha.
The nucleus migrates out of the mother cell to undergo mitosis in the germ tube. Septum (yellow) is formed
between the two rings of septin. The apical compartment remains in the cell cycle and the subapical remains in G1
phase29
.
The alteration of cell wall proteins composition during the yeast-to-hypha transition
that triggers a potent defense mechanism. The human host mucosa reveals
immunological tolerance to colonizing microorganisms but when C. albicans infects the
host, hypha specific cell wall proteins are major adhesins and invasins that function as
antigens capable of modulate immune response31.
The disease can be caused by the toxins that pathogen releases in the host
organism, or due to the direct destruction of living tissues23. Disseminated candidiasis
is fatal in immunocompromised individuals. Kidney and brain are the primary target
organs of this yeast during infection. Within the kidney, massive fungal invasion and
growth can occur, resulting in inflammatory reactions that lead to tissue necrosis32.
CHAPTER II – LITERATURE REVIEW
Daniela Cruz Moreira 9
1.2 Epidemiology and treatment
Candida spp. are considered the most frequent fungi in the hospital. The frequency of
invasive fungal infections caused by yeasts has increased in intensive care units (ICU),
being Candida reported among the ten leading causes of bloodstream infections (BSI)
in Europe and USA33,34. Fungal infections in immunocompromised patients are
increasing and are associated with immunosuppression, illness, prematurity, exposure
to broad-spectrum antibiotics and patients of older age35 (Table 1). Fungal infections
may be superficial, such as in skin, hair and nails; subcutaneous and systemic,
affecting several internal organs, blood, and internal epithelia. Candida sepsis has
proven to be a life-threatening infection with high prevalence and high morbidity and
crude mortality rates36,37.
Table 1| Risk factors for development of invasive Candidiasis38
Risk factors for development of invasive Candidiasis
1. Length of stay in the ICU
2. Use of central venous catheters
3. Use of broad-spectrum antimicrobial agents
4. Parenteral nutrition
5. Development of acute renal insufficiency,
hemodialysis
6. Severe pancreatitis
7. Diabetes mellitus
8. Neutropenia
9. Gastrointestinal tract surgery
10. Solid organ or stem cell transplantation
11. Candida colonization
There are about 150 species of Candida, but only a small number are human
pathogens. C. albicans colonizes the mucus membranes of 30-60% on humans39 and
is the cause of vaginal infections, diaper rash in infants, and thrush in the mouth and
throat. The infections caused by C. albicans affect mostly immunocompromised
patients that suffer from acquired immunodeficiency syndrome, for instance8. C.
albicans also invades the brain during acute infections and causes
Integration of microfluidics and FISH for the rapid identification of microorganisms
10 Daniela Cruz Moreira
meningoencephalitis40. Navarathna et al.41 recently confirmed in vivo, using magnetic
resonance imaging (MRI), that the integrity of the blood-brain barrier is lost during C.
albicans invasion. Although C. albicans is the most prevalent species involved in
invasive mycoses, the incidence of infections due to non-albicans Candida (NAC)
species is increasing42. The most common NAC species are C. tropicalis, C. glabrata,
C. krusei and C. parapsilosis, which as a group, represents about one-half of all
Candida spp. isolates from blood cultures43.
C. tropicalis infections are usually preceded by a band of progressive necrotic tissue,
which is not observed with C. albicans44. Moreover, infections due to C. tropicalis have
increased dramatically on a global scale because of its resistance to fluconazole. C.
tropicalis candidaemia has been associated with cancer, especially in patients with
leukemia or neutropenia45.
Candidaemia due to C. glabrata has been reported as related to the use of
fluconazole46. C. rugosa has been described in the oral cavity of diabetic patients47.
C. parapsilosis has emerged as a significant nosocomial pathogen with manifestations
in endophthalmitis, endocarditis, septic arthritis, peritonitis and fungaemia, usually
associated with invasive procedures48.
In Europe, in 2006, more than half of the cases of candidaemia were caused by C.
albicans, and the incidence rates for NAC infections were 14% each for C. glabrata and
C. parapsilosis, 7% for C. tropicalis and 2% for C. krusei37. In North America a
predominance of NAC species was observed, although C. albicans was the most
frequently isolated species, followed by C. glabrata38. Latin American Countries
showed changes in prevalence of C. albicans, and a progressive increase of NAC
infections. C. parapsilosis was the most frequent species, followed by C. tropicalis and
C. glabrata49. According to the Brazilian Network Candidaemia Study, C. albicans
accounted for 40.9% of cases in Brazil, followed by C. tropicalis (20.9%), C.
parapsilosis (20.5%) and C. glabrata (4.9%)47,49. In a four years retrospective study in a
Portuguese hospital it was observed that C. albicans was the most frequently yeast,
with an overall incidence of 69.9% followed by C. glabarata, C. tropicalis, C.
parapsilosis and C. krusei50.
Response to antifungal therapy differs for each Candida spp.. Currently, there are
only three classes of antifungal agents available to treat Candida infections: azoles,
echinocandins and the polyenes51. The main reason to discriminate between different
Candida species is to proceed with an effective treatment, especially because there are
some species resistant to some antifungals like azoles, such as fluconazole, the
standard antifungal drug of choice in many countries52,48. Fig. 4 shows resistance rates
CHAPTER II – LITERATURE REVIEW
Daniela Cruz Moreira 11
against fluconazole for Candida albicans, non-C. albicans, and all Candida isolates
combined in selected countries from which data are available1. Significant cost, toxicity
and absence of an oral formulation can present barriers to the use of drugs to treat
azole resistant Candida infections.
FIGURE 4| Fluconazole drug resistance by Candida species and country
A rapid microorganism identification leads to an efficient intervention, a significant
reduction of the intake of broad spectrum antibiotics with an overall cost savings. The
early diagnosis is determinant in critically ill patients because the indiscriminate intake
of antibiotics usually kills bacteria that are essential for normal digestion and favors the
opportunistic spread of Candida spp. on the digestive tract walls, which can be
worsened when associated with a diet rich in sugars and carbohydrates53,54. Once
colonizing the intestinal walls, the fungus is very difficult to eradicate and infection
treatment is usually followed by recurrence23. According to a review from intensive care
units in USA and Canadian cities, the risk of death from sepsis increases by 6 to 10%
with every hour that passes from the onset of septic shock until the start of effective
antimicrobial therapy55.
Integration of microfluidics and FISH for the rapid identification of microorganisms
12 Daniela Cruz Moreira
2. Diagnosis of Candida spp.
Invasive candidiasis is difficult to diagnose. Tissue sampling has a low diagnostic
yield in patients who have received empirical therapy. Cultures other than blood (e.g.
bronchoalveolar lavage samples) sometimes reflect colonization instead of invasion
and blood cultures have a great incidence of false negatives56.
Several methods have been developed for the identification of Candida spp.. These
methods can be based on phenotypic aspects using culturing methods or can be
molecular-based methods. General insights regarding each of these methods will be
hereafter discussed in detail.
2.1 Phenotypic methods
Phenotypic methods allow the identification of the microorganism based on
observable characteristics such as morphology, development, and biochemical or
physiological properties resulting from gene expression57. Phenotypic methods use
culturing methods such as dextrose agar medium, germ-tube test, chlamydospore-
inducing media-based test, fermentation test, and CHROMagar test.
A dextrose agar medium can be used and a direct observation of yeast colonies on
slants or under the microscope is performed by an observer which leads to a subjective
reliance on fungal morphological aspects58. The germ-tube test is considered a simple,
economical and efficient procedure to differentiate C. albicans from other Candida spp.
and it is based on the observation of germ tubes formed by C. albicans59. This
technique, nevertheless, can lead to a misidentification since some NAC species such
as C. dubliniensis, C. tropicalis, and C. parapsilosis can also generate germ tubes or
pseudohyphae60. Cornmeal and rice extract agar are the best-known chlamydospore-
inducing media. Chlamydospore formation is a peculiarity of C. albicans, C.
dubliniensis and more rarely of C. tropicalis. The chlamydospore is a highly refractile,
thick walled, asexual fungal spore that is derived from of hyphal cell and can function
as a resting spore.61 The difference between C. dubliniensis chlamydospore and those
formed by C. albicans is that in C. dublinienses they are attached in pairs or larger
clusters. However, the method may not be consistent and may require additional
tests.62 Another method to differentiate C. albicans from C. dublienensis is the
fermentation test, based on the demonstration of acid or carbon dioxide production that
occurs in liquid media63. CHROMagar Candida (Fig. 5) is a plate-based test for the
simultaneous isolation and identification of various Candida species. This medium is
CHAPTER II – LITERATURE REVIEW
Daniela Cruz Moreira 13
useful to differentiate the species C. dubliniensis, C. albicans, C. krusei, C. tropicalis
and C. glabrata based on colour development64. API Candida system (bioMérieux,
Marcy l’Etoile, France) (Fig. 6) is based on the detection of enzyme reactions and
acidification of sugars. The reactions are visually interpreted by spontaneous color
changes.65
FIGURE 5| CHROMagar Candida
Different isolations of Candida spp. appear in different colors. C. albicans, C.
tropicalis and C. krusei appea as green, blue and pink, respectivelya.
FIGURE 6| API Candida System strips
The system consists on a strip of tubes, which allows
identification after 18-24h of incubation at 35ºCb
2.2 Molecular Techniques
Conventional culture-based methods are mostly employed because of their user-
friendliness and low cost. However these methods are time-consuming and do not give
promptly useful results59. In addition, in critically-ill patients, to whom pre-emptive
therapy is administered, the presence of fastidious microorganisms, that sometimes
are in low densities, can lead to false negative cultures66. Molecular identification
methods have been used as suitable alternatives due to their high accuracy, sensitivity
and specificity for the identification of microorganisms 67. From these, the most
frequently used are the polymerase chain reaction (PCR)-based methods.
PCR-based methods are based on the amplification and detection of microbial nucleic
acid directly from clinical samples. This method comprises repeated DNA denaturation
a Source:http://www.frilabo.pt/fcms//index.php?option=content&task=view&id=172 b Source: http://www.biomerieux- diagnostics.com/servlet/srt/bio/clinical-diagnostics/dynPage?open=CNL_CLN_PRD&doc=CNL_PRD_CPL_G_PRD_CLN_11&pubparams.sform=5&lang=en
Integration of microfluidics and FISH for the rapid identification of microorganisms
14 Daniela Cruz Moreira
cycles, annealing of two oligonucleotide primers to the target DNA, flanking a region of
interest, and extension of a new strand by the DNA polymerase enzyme. The
synthesized sequence in one cycle can serve as template in the next and the number
of target DNA copies doubles approximately at every cycle68.
Multiplex-PCR combines different species-specific primers in a single PCR tube.
Carvalho et al. 69, described a method based on the amplification of two fragments from
ITS1 and ITS2 regions that made possible the identification of eight Candida spp. with
high specificity. Nested PCR consists of two primers sets for specific amplification of
Candida spp. DNA that are used in two successive PCR runs to improve specificity of
Candida identification70. The TaqMan assay (Perkin-Elmer Corp. Applied Biosystems,
Foster City, CA, USA) is a real-time procedure that can also be applied to identify
Candida spp.. Guiver et al. 71, used this method for the rapid identification of clinically
relevant Candida spp. within 4-5h.
Non PCR-based methods identify specific genotypes, without needing DNA
amplification. For instance, matrix-assisted laser desorption ionization – time of flight
mass spectrometry (MALDI-TOF MS) is based on protein fingerprints. Using Burker
Daltonic’s MALDI BioTyper system (Burker Daltonics, Bremen, Germany), fungi are
submitted to a thermal degradation, and the resulting small molecules are cleaved at
their frailest points, producing volatile fragments. Using mass spectrometry, a pyrolysis
mass spectrum is produced, which can be analyzed as a chemical fingerprint of the
yeast72. It is considered a rapid, reliable and cost-effective alternative for the
identification of Candida spp.73.
Another non-PCR-based method is fluorescence in situ hybridization (FISH) that will
be discussed in detail in the next chapter.
3. Fluorescence in situ hybridization
Fluorescence in situ hybridization (FISH) is a molecular method used to identify and
quantify microbial populations, attaching a fluorescent label to nucleic acids74,75. Three
essential steps illustrated in Fig. 7, fixation and permeabilization, hybridization and
washing, define FISH procedure76.
CHAPTER II – LITERATURE REVIEW
Daniela Cruz Moreira 15
FIGURE 7| Basics steps of FISH
FISH follows three basic steps: 1| Cells are fixed and the cell membrane permeabilized. 2| The labelled probe
hybridize to the target rRNA sequence. 3| The unbound probe is washed away. Then the sample is ready for
visualization or quantification76
.
The fixation and permeabilization step is one of the most crucial steps assuring the
accuracy of detection protocols and is therefore decisive in determining the subsequent
success of a given experiment. An effective sample fixation preserves cellular
structures by rapidly ending all enzymatic and other metabolic activities maintaining the
cellular morphology77. An ideal fixative should reflect the in vivo situation by preventing
autolysis and stabilizing cellular structures. However, a compromise must be attained
since over fixation leads to fixation artifacts, loss of signal and increased nonspecific
background78.
Glutaraldehyde, a five-carbon dialdehyde, and formaldehyde, a one-carbon
monoaldehyde, are the most commonly used fixative agents, and act by cross-linking
proteins78. Glutaraldehyde is a harsh fixative due to its ability to polymerize and form
extended cross-linkings. However, formaldehyde is a small molecule that penetrates
quickly in cellular structures, stabilizing them and has proven to be the most effective
fixative for nucleic acids78. Paraformaldehyde is a polymerized form of formaldehyde,
and is preferred for most immunohistochemical procedures because it provides a
fixative free of extraneous additives79.
Integration of microfluidics and FISH for the rapid identification of microorganisms
16 Daniela Cruz Moreira
In addition to fixation, it is also necessary to permeabilize the cell wall, so probes can
diffuse and attach to intracellular target molecules. Some of the more commonly used
permeabilization agents are used after fixation, but some protocols suggest fixing and
permeabilizing cells at the same time80. Cell membrane permeabilizing agents can be
divided in two major groups: organic solvents and detergents. Alcohols, such as
methanol and ethanol, and acetone are harsh organic solvents that dissolve lipids from
the cell wall and also precipitate proteins and carbohydrates79. These reagents have
the advantage of performing rapid one-step fixation at low temperatures and they are
also considered permeabilizing agents. However, shrinkage of the sample can occur
due to simultaneous dehydration and fixation81. At low temperatures (0 to -20ºC)
ethanol precipitate proteins without denaturing them81.
Detergents are used especially after fixation with cross-linking agents. Triton-X is a
non-ionic detergent with uncharged, hydrophilic head groups of polyoxyethylene
moiety. It solubilizes phospholipids from the plasma membrane but, as it is non-
selective, may extract proteins along with lipids82. The importance of fixation step relies
on its osmotic and ionic effects on cellular structures, particularly organelles83.
Hybridization, the following step, is when the annealing between a fluorescently
labeled probe with the complementary target sequence occurs. In order to identify
microbial species, the preferred targets used are sequences in 16 or 23S rRNA that
comprises highly conserved regions, specific for each microorganism, being accepted
as discrimination criteria of microorganisms84. There are nearly 200 000 ribosomes in a
single Saccharomyces cerevisiae cell 85, and due to its abundance in fixed cells,
targeting rRNA specific sequences facilitate the visualization and quantification of
individual cells86. Nevertheless, one possible limitation of the method is associated with
probe inaccessibility to the target region within the secondary structure of ribosome,
which may hinder probe hybridization87. It is necessary to adequately design a probe
that targets sites located in the rRNA regions already known to be accessible. Other
limitations of this technique can be explained by decreased probe permeabilization and
low cellular ribosome content88. To guarantee that the probe accesses and hybridizes
with the target sequence, parameters like temperature, pH, ionic strength and
formamide concentration should be optimized74. When added to the hybridization
buffer, formamide act as a denaturant solvent that weakens the secondary structures of
rRNA89.
To avoid false positives, the washing step, as the name suggests, washes away
unbound or loosely bound probes. The sample is then ready for identification and
CHAPTER II – LITERATURE REVIEW
Daniela Cruz Moreira 17
quantification by fluorescence microscopy or flow cytometry depending on the aim of
the analysis.
3.1 Brief historical overview
Watson and Crick’s discovery of the three-dimensional double-helice model for the
structure of DNA was a landmark on the history of genetic research 90. This discovery
marked a milestone in science and gave rise to modern molecular biology, yielding the
understanding of genes control, chemical processes within cells and the development
of powerful molecular techniques.
In 1961, Schildkraut et al.91 introduces the technique of DNA-DNA hybridization, used
to study genetic relationships among several species and boosted the first genome
studies92,93. Later in the same decade, Pardue and Gall94 show that hybridization can
be used to identify the position of a DNA sequence in situ, hybridizing a radioactive
labelled DNA probe to DNA within a cytological preparation. In 1977, Rudkin and
Stollar95 use fluorescent labeled probes instead of radioactive labeled probes
demonstrating the significant advantages over autoradiographic procedures, like ease
of use, safety, specificity, stability, diminished hybridization and microscopic analysis
times, sensitivity and resolution. The first application of fluorescence in situ
hybridization came in 1980, when a specific DNA sequence was hybridized with RNA
labeled with a fluorophore96. The following year, to overcome the difficulty in detecting
short segments of DNA, Harper and Saunders97 demonstrated the suggestion of Wahl
et al.98 that the use of dextran sulfate could accelerate the rate of hybridization. Langer
et al.99 incorporated enzymatically, amino-allyl modified bases throughout the length of
the RNA, which in turn conjugates to haptens or fluorophores allowing the production of
low-noise probes. Biotin covalently attaches to the nitrogenous base ring and then
avidin is conjugated. The interaction between biotin and avidin has a very high binding
constant and when avidin is coupled to indicator molecules, a small quantity of biotin
can be detected. It is considered an indirect detection since there are secondary
reporters that bind to the hybridization probe. Secondary detection by fluorescent
streptavidin conjugate assays were also performed for the detection of DNA and
mRNA100,101. In order to achieve a rapid phylogenetic identification of single microbial
cells, DeLong102 and co-workers described fluorescence in situ hybridization (FISH)
technique with rRNA-target probes.
Integration of microfluidics and FISH for the rapid identification of microorganisms
18 Daniela Cruz Moreira
Although traditionally used in FISH procedures, DNA probes suffers from limitations
related to cell permeability due to its negatively charged sugar-phosphate backbone
and the fact that the hydrophobic core of the lipid bilayer of the membrane is
impermeable to charged species. Other disadvantages of DNA-FISH are associated
with hybridization affinity and target site accessibility, leading to poor signal-to-noise
ratio and lack of target site specificity and sensitivity75. In the early 1990s, synthetic
single-stranded PNA probes were designed and synthesized, allowing direct detection
and circumventing DNA-FISH shortcomings103. In the next section, synthetic single-
stranded DNA mimic probes will be discussed in detail, with particular focus on peptide
nucleic acid (PNA) probes.
3.2 Nucleic acid mimics
FISH probes target specific sequences of nucleic acids by complementarity. Nucleic
acid analogues appear as a breakthrough to improve sensitivity of FISH. Different
types of nucleic acid mimics, which were reviewed by Cerqueira et al.74, have been
reported to potentially have application on FISH procedures, being the most common
peptide nucleic acid (PNA), locked nucleic acid (LNA) and 2’-O-methyl (2’-OMe) RNA
(Fig. 8).
FIGURE 8| Chemical structure of DNA, RNA LNA and PNA
A, B| DNA and RNA have a negatively charged sugar-phosphate backbone C| The ribose ring of LNA is locked to a
C3’ endo-conformation. D| PNA have a neutral polyamide backbone composed of N-(2-aminoethyl) glycine units 74
.
CHAPTER II – LITERATURE REVIEW
Daniela Cruz Moreira 19
LNA resembles RNA, being the ribose ring locked to a C3’ endo-conformation. The
ribose is linked to a methylene bridge between 2’-Oxigen and 4’-carbon (Fig.8C)104.
LNA forms duplexes with complementary DNA and RNA. The most important
characteristics of this mimic for FISH implementation are the increased thermal stability
and improved selectivity74,105. 2’O-Me RNA is another modified oligonucleotide that
shows a high affinity towards an RNA106.
3.2.1 Peptide nucleic acid (PNA)
PNA probes contain the same nucleotide bases as DNA. However, the negatively
charged sugar-phosphate backbone of DNA is replaced by a non-charged polyamide
backbone composed of N-(2-aminoethyl) glycine units, as shown in Figure 8D.
An important aspect is the physical configuration of nucleobases in PNA, that are
positioned at the same distance from each other, similarly to what occurs in the DNA
molecular structure, enabling the hybridization by complementarity, still obeying the
Watson-Crick hydrogen bonding rules107.
PNA/DNA complementary bounds are stronger than DNA/DNA. Due to the lack of
charge of PNA molecules, electrostatic repulsions are not verified and the hybridization
is rapid, more stable and more specific108. However, it is important to bear in mind the
self-complementarity of the probe design since complementary PNA sequences
hybridize strongly109. Moreover, the neutrally charged nature of the molecule implies
relatively low aqueous solubility which means that PNA tends to aggregate.
Fortunately, solubility-enhancing modifications to the molecule are possible to make110.
The relatively smaller molecular size, about 15 base pairs, and the hydrophobic
nature of the peptide backbone facilitates the diffusion through the lipidic cell wall86.
Also, shorter sequences mean a higher destabilization by a single-base mismatch, and
an effective discrimination of single-base differences109.
PNA probes thermodynamic properties were studied by Ylmaz and Noguera111,
providing an essential tool to study the mismatches effect. Due to its synthetic
backbone, PNA revealed an extended lifespan because of its increased resistance to
nucleases and proteases, which is a great advantage in contrast to DNA probes112.
As described below, PNA probes own such characteristics that make it so favorable to
many molecular biology applications. However, the required enrichment steps that
some clinical samples need, to assure considerable number of cells in the sample, can
hinder PNA-FISH use for microorganism clinical diagnosis since it can be more time-
Integration of microfluidics and FISH for the rapid identification of microorganisms
20 Daniela Cruz Moreira
consuming. To overcome this difficulty, a promising solution appears as a lab-on-a-chip
device form, a miniaturized and automated system, where a significant time and cost
reduction is achieved. PNA-FISH adapted to fit microfluidic channels is believed to
become a new rapid and extremely effective tool in clinical diagnosis. For better
understanding the advantages of these two techniques (PNA-FISH and microfluidics)
integration, microfluidic systems are introduced in the next section.
4. Microfluidics for miniaturizing biological assays
Science has been driven to develop new technology and miniaturization has been a
central component. When applied to the analytical and diagnostic assays field, the
main goals of miniaturized devices are to lower the cost per test (because the amount
of materials and reagent consumption decreases by scaling down the assay volume),
to shorten the time to obtain a result, provide higher sensitivity, portability and to
decrease the amount of laboratory space needed113. In vitro diagnostics (e.g. point-of-
care and central laboratory-based testing) offer huge potential for microfluidics since is
possible to fabricate a hand-held device with low energy consumption and high
precision.
Microfluidics is the handling and analyzing of fluids in structures of micrometer scale,
namely microfluidic devices, which are a tool with an integrated channel where a fluid
circulates 114. This concept can be applied to obtain ideal conditions for well-defined
microenvironments and offers spatial control and manipulation of cells in microfluidic
systems115. The microdomain provides conditions for performing biological experiments
on cells and offers precise definition of compound concentrations in a confined space
with high resolution115. By implementing these systems to concentrate cell
suspensions, the time consuming enrichment step of standard procedures can be
bypassed.
4.1 Entrapment strategies
Moving cell-based studies into a microfluidic network means achieving new strategies
to retain cells at defined locations over time. Culturing cells in microfluidic environments
requires fundamental knowledge of multiple topics, including cell biology, cell culture
techniques, fluid dynamics and the physics of the microscale.
The strategy adopted to concentrate cell suspensions depends on the cell adherence
to the material116. Most of the experiments with bacteria inside microchannels do not
CHAPTER II – LITERATURE REVIEW
Daniela Cruz Moreira 21
need specific structures for entrapment. Bacteria adhere to the substratum surface due
to hydrophobic interactions, stronger than repulsive forces, and include irreversible
interaction as hydrogen, ionic and covalent bonding117. In the case of non-adherent
cells a trapping mechanism is mandatory. The most commonly used technologies for
cell trapping in microfluidic systems are based on surface bound cell assays118,
localized surface modifications119 or chemical immobilization120. Current trends and
technologies make use of hydrodynamic121, electrical122, optical123, magnetic124 or
acoustic125 fields. However, incorporation of these principles in complex biomedical
microfluidic platforms requires a set of components that need to be combined together.
These components are expensive and are not widely available; therefore the general
trend in commercial devices has been to fabricate simple, disposable devices that are
designed to interface with external equipment that houses the required control
electronics, reagent supply, detectors and programming126.
Various techniques have been adopted to incorporate within the microdevice a
simple-to-obtain 3D architecture to confine micrometer-sized particles to specific
regions in a microfluidic network. These microfluidic filters (Fig. 9) can be microporous
membranes incorporated as one of the walls of a microfluidic channel127, arrays of
pillars arranged in a regular geometric pattern128 and porous beds distributed by
sizes129.
FIGURE 9| Examples of microfilters
a| Porous membranes Schematic of microfluidic blood filtration device, in which top and bottom pieces are made
up of PDMS and the membrane is a commercially-available filter129
. b| Arrays of pillars SEM image of pillar-type
microfilter on silicon. These pillars serve as filters with a well-defined size130
. c| Packed beds Schematic of
microfluidic filter of 20 and 10 µm beads, which inhibit the passage of cells out of the culture chamber preventing
blockage of the flow131
.
Micro and nanoporous membranes (Fig. 9a) may either be manufactured using
microfabrication techniques130 or commercial routinely used membranes may be
bought and incorporated into microfluidic systems. The integration is made by having
two separated open microchannels that are glued together sandwiching the
membrane127. Although the design has a high degree of flexibility relatively to pore size
Integration of microfluidics and FISH for the rapid identification of microorganisms
22 Daniela Cruz Moreira
distribution, the main drawbacks of the method are expensiveness and limitation to few
materials.
Arrays of pillars (Fig. 9b) are designed and fabricated along with the channels. Pillars
have well-defined gaps size that can be easily manipulated, considering the size
resolution of the fabrication process128. The ratio between channel depth and the size
of the space among pillars is a critical concern, because pillars are liable to buckle
when this ratio is increased too much. The main disadvantage of the method is that the
failure of even a few pillars may compromise the filtration process131.
Porous beads (Fig. 9c) consist of a large number of solid particles packed by sizes.
They are typically held in a dam-like structure, and beads are packed by size at high
pressure, maintaining a network of interconnected pores through which fluid is
driven129. The main advantage of this type of filters is that porous characteristics can be
rapidly modified with minimum changes in fabrication procedure.
4.2 Microfabrication
Microfabrication is how three dimensional (3D) microstructures are produced132. The
fabrication technique and material chosen are dependent on the application and are
fundamental to achieve the goal of the microfluidic device at a low cost and high
throughput. Microelectromechanical systems (MEMS) technologies, like molding and
embossing techniques, are the most cost-effective for mass production, since the fixed
cost associated with the expensive tools can be spread over a large number of
devices.
Replica molding process is used to replicate with high reproducibility topographical
features from a rigid or elastomeric mold into another material by solidifying a liquid in
contact with the original patterned master133. Several procedures are used to obtain a
master and their characteristics are determinant to choose which is the most
appropriate. The most popular techniques to produce microfluidic devices are
photolithography and soft lithography. Other techniques are xurography, hot imprinting
or embossing, injection molding or laser ablation134.
4.2.1 Photolithography (SU-8)
Photolithography is the most common technique to produce patterned masters with
high accuracy. The development of complex 3D geometries in microfluidic devices are
CHAPTER II – LITERATURE REVIEW
Daniela Cruz Moreira 23
made possible through the implementation of photoactivated polymers, such as SU-
8135.
As illustrated by Figure 10, the SU-8 photolithographic process consists on spin-
coating a SU-8 photoresist on a silicon wafer and then soft-bake on a hot plate to
evaporate the solvent and harden the photoresist. The UV exposure onto a
photopatternable polymer, initiate a cross-linking reaction across exposed regions and
the pattern on the photomask is replicated into the photoresist. Excess polymer and
reactants need to be washed away leaving the microstructure136.
SU-8 was made a popular photoresist due to its harsh environmental durability and
lithographic contrast, that enables the production of high resolution structures for direct
molding137. An important feature of SU-8 prototyping is the ability to control layers
thickness and quality varying the solvent, which will be reflected on channel depth.
Also, tight control of structures width and height has elevated SU-8 to the photoresist of
choice for creating microchannels molds138. However, several baking sessions to
condition the photoresist and clean room facilities to reduce dust and particles that may
compromise film quality are required.
FIGURE 10| Schematic of photolithography technique
A silicon wafer is spin-coated with SU-8 photoresist and soft-baked on a hot plate to evaporate the solvent and
harden the photoresist; a photomask is aligned above the photoresist on silicon wafer and exposed to UV to
replicate the pattern into the photoresist; following agitation in SU-8 developer the unexposed photoresist is
removed; finally, the mold is rinsed and dried with clean air136
.
4.2.2 Xurography
Xurography is a photolithography replacement technique for rapid prototyping of
microstructures using a cutting plotter (Fig. 11). The mold is designed using a software
Integration of microfluidics and FISH for the rapid identification of microorganisms
24 Daniela Cruz Moreira
tool and the limits of the mold are cut on film with a cutting plotter. The surrounding film
is removed and the pattern is adherently attached to a glass substrate.
The control settings of the cutting plotter need to be considered, since cutting speed
and force influence the features precision. Cutting force has to be enough to
completely cut the film, but it only must be slightly scored. High cutting speed and a dull
blade are closely related to material tearing, resulting in rougher edges139,140.
Xurography is cheaper because it uses low-cost equipment and materials and can
rapidly prototype microfluidic devices for higher resolution devices141. In spite of being a
fast, effective and an easy method, dimensions obtainable with this technique can be
limiting because patterns with small dimensions not only are difficult to handle but also
depends on the resolution of the equipment142. Xurography proved to have poor
accuracy. Due to small dimensions of the structures, irregularities are critical and a
more precise technique has to be adopted to create the master.
FIGURE 11| Xurography technique
First the film is cut with a plotter blade and then unnecessary film around pattern is weed
145.
4.2.3 Soft Lithography
After mold fabrication, soft lithography (Fig.12) represents a non-photolithographic
strategy based on replica molding. An elastomeric stamp with patterned relief
structures is used to generate microstructures. In order to avoid damage of molded
structures, the molded part should have elastic properties so that demolding can be
performed under reversible deformation143. Usually a two-part polymer in a liquid form
is poured over the prefabricated mold at room temperature and cured at a slightly
elevated temperature. Once cured, the polymer is separated from the mold and access
points are made with a needle (Fig. 13) and finally sealed to a flat surface143.
FIGURE 12| Schematic of soft lithography
technique
Elastomer is poured on master, polymerized and removed
from the mold. Ports are pouched and the block is bond to a
glass slide previously spin-coated with a thin layer of
elastomer136
.
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Daniela Cruz Moreira 25
Polymer molding is a powerful tool for microfabrication, allowing the manufacture of
many transparent polymer chips from a single mold. The practicability of polymers for
fabrication with both rapid prototyping and mass production as well as lower cost
relative to silicon and glass make them particularly attractive144. Elastic properties of
elastomers have proven to reduce breakage of structures during demolding, due to
reversible deformation. Taking advantage of soft elastomers properties, whose Young’s
modulus makes it moderately stiff (~1MPa), micropatterns formed on the walls of
microfluidic channels have proven to enhance cell capturing and cell cultivation. A
variety of polymers are candidates for low cost, mass production of microfluidic
devices, such as polycarbonate (PC), polymethylmethacrylate (PMMA),
polyvinylchloride (PVC), polyethylene (PE), and polydimethylsiloxane (PDMS)145.
FIGURE 13| Interconnected channels
1| Using a twist motion, the needle punch the block from the outside of the PDMS to the buried channel 2| A hole is cut with a diameter identical to the inner diameter of the needle. 3| When the needle is removed, the hole diameter readjusts 4| When a second needle is inserted into the hole, a compression seals is formed around the needle, preventing leakage
146.
4.2.3.1 PDMS
Poly(dimethylsiloxane) or PDMS is an optically transparent, non-toxic, soft silicone
elastomer, widely used to fabricate highly reproducible microfluidic devices by replica
molding147. PDMS base monomers are in the liquid phase at room temperature and
cure at 80ºC, after being combined with a curing agent. The PDMS base monomer is
vinyl terminated, while the crosslinking monomers are methyl terminated and contain
silicone hydride units. PDMS cure process is due to the crosslinking of PDMS
monomer vinyl group and the crosslinker silicon hydride groups148. Structure of PDMS
polymer chain is illustrated on Figure 14, and is composed by repeating –O-Si(CH3)2-
units.
Hydrophobicity of this material is due to the CH3, which may become a disadvantage
since it makes cell adhesion harder and also microchannels are susceptible to air
bubbles trapping149. The surface may become hydrophilic if it is exposed to oxygen
plasma147.
Integration of microfluidics and FISH for the rapid identification of microorganisms
26 Daniela Cruz Moreira
FIGURE 14| Structure of PDMS polymer chain150
A large number of PDMS devices can be made from a single master, reducing the time
and cost of fabrication149. PDMS can be molded easily to form simple, complex, or
multilayered channel systems. It is a soft elastomer with a low Young’s modulus, with
great demolding behavior and reduced breakage of structures. However, depending on
the ratio of the designed microstructures, they can collapse because of the low
modulus of elasticity of PDMS and it can be a problem when dealing with structures of
a few micrometers147. Thin PDMS structures become crumpled easily, making it very
difficult to handle.
Experimental feasibility depends on the intrinsic properties of the device, like optical
transparency and imaging compatibility, low water permeability to prevent leakage,
thermal stability and is chemically inert to provide conditions to a controlled
environment151. An important characteristic as a diagnostic tool is that PDMS-based
microfluidic devices are easily sterilized through conventional means and are
biocompatible151.
PDMS offers versatility of the substrate, since it can be reversibly or irreversibly sealed
to glass, polystyrene or silicone. Substrate properties can be chosen taking in account
hydrophobicity, stiffness, topography, and the ability to be functionalized with bound
biomolecules or substrate patterning.
4.2.4 Other techniques
Hot embossing, injection molding and laser ablation are other low cost methods for
manufacturing of MEMS.
Hot embossing uses the replication of a micromachined embossing master to
generate microstructures on a polymer substrate. The plastic substrate is heated, then
a mold is insert to above plastic and forces are applied to emboss plastic substrate.
The substrate is cooled to solidify and de-molding152. It is a very quickly and simple
CHAPTER II – LITERATURE REVIEW
Daniela Cruz Moreira 27
fabrication for small structures with high aspect ratios. However, because of the high
residual stress, created by the use of temperature in conjunction with pressure, can
cause cracks and broken edges152.
Injection molding, as the name suggests, utilizes a plunger to force the molten
polymer into a mold cavity, which forms the shape of the desired component. Polymer
solidifies, acquiring the contour of the mold153. This method offers, above all, the
opportunity of a fully automated production. Limitations for injection molding are high
tooling costs, skrinkage and partial mold filling. For microscale components, tighter
tolerances are required and mold inserts are often fabricated by costly high-precision
micromachining techniques, such as laser ablation. Laser ablation uses CO2 lasers to
cut through a polymer sheet and quickly generate patterns on it154.
4.3 Concepts in fluid mechanics
Flow properties and laws that govern the microscale are different of those
experienced in everyday life. Some applications can only be performed due to the
physics of the scale. Learning to think on an entirely different scale is a new and
exciting challenge in microfluidics. Forces that work on the microscale result in other
dominant effects such as laminar flow, diffusion, fluidic resistance, surface area to
volume ratio, and surface tension155. Projecting complex microfluidic systems requires
a study of flow inside microchannels.
In a microchannel, the flow rate, Q (m3/s), is given by:
� = � × � × � (1)
being �(m/s) the mean fluid velocity, � (m) the thickness of the channel and �(m) the
width of channel155.
The shear stress, � (Pa), represents the hydrodynamic force associated to a surface
which can cause detachment of particles. On a channel with rectangular cross section
it is given by:
� = 3�2�(�/2) × �
(2)
where � (kg/(m.s)) is the viscosity of the fluid156.
Due to shear stress at a stationary surface, e.g. the wall of a pipe, the fluid touching
the surface is brought to rest (no-slip condition). The region where there is a velocity
profile in the flow, due to the shear stress at the wall, is called the boundary layer. The
Integration of microfluidics and FISH for the rapid identification of microorganisms
28 Daniela Cruz Moreira
velocity increases from the wall to a maximum in the mainstream of the flow and,
between two fixed walls, the flow profile is parabolic (Fig. 15).
FIGURE 15| Motion of cylindrical fluid element within a pipe157
The velocity profile in fully developed laminar flow in pipe is parabolic with a maximum at the centerline and
minimum near to zero at the pipe wall. These flow leads to the deformation of any fluid element in the pipe.
Another important aspect is the Reynolds number, which give us information about
fluid behavior and is calculated by:
�� = ����
(3)
where � (kg/m3) is the density and � (kg/(m.s)) the dynamic viscosity of the fluid, and l
(m) a characteristic dimension of the flow. It is known that for cylindrical tubes the flow
is laminar for �� < 2300, else is transient or turbulent, and this threshold is used as
approximate reference for other geometries158. Inside microchannels a laminar flow is
observed, without turbulence, due to drastic decrease of Reynolds on microliter volume
scale159. One consequence of laminar flow is that fluid particles move in distinct and
separated layers and the streams flowing in contact without mixing except by diffusion.
Diffusion is a transport phenomenon by which a concentrated solute spreads out in a
volume, over time, , through a random movements, until becomes uniformly distributed.
In the case of particles or cells the random motion is due to Brownian motion.
Therefore, laminar flow conditions and controlled diffusion enable temporally and
spatially highly resolved reactions with little reagent consumption.
The distance d a particle moves in a time t, can be modeled in one dimension by:
� = √2�� (4)
where D is the diffusion coefficient of the particle. The hydrodynamic properties of
spherical particles can be express in the translational self-diffusion coefficient form, Dt
(cm2/s), by Einstein’s equation :
�� = � × ���
(5)
CHAPTER II – LITERATURE REVIEW
Daniela Cruz Moreira 29
where � (K) is the absolute temperature, � (1,38 x 10-23 m2.kg.s-2.K-1) is the
Boltzmann’s constant and �� (m2/s)) the translational frictional coefficient.
The frictional coefficient for rod-like molecules was developed by Tirado and Garcia
de la Torre160, employing a bead-model procedure that accounted rigorously for the
hydrodynamic interactions and expressing the end-effect term as an interpolating
equation.
�� = 3����ln ! + 0,312 + 0,565!'( − 0,100!' (6)
Another factor that becomes important at the microscale is surface area to volume
(SAV) ratio. Surface phenomena become increasingly dominant over volume
phenomena. A very large SAV ratio and low thermal mass makes heat transfer
between the fluid and the environment more efficient161. That fact enables precise
temperature control due to quick temperature changes. However, large SAV can also
be a disadvantage in processes involving transporting fluids, because it allows
macromolecules to quickly diffuse and adsorb to channel surfaces162.
4.4 Liquid handling systems
Microfluidic platforms can be defined by its liquid handling system. Different
microfluidic platforms were summarized in a critical review by Mark et al.163, focusing
on the main requirements and characteristics of lateral flow tests, linear actuated
devices, pressure driven laminar flow, microfluidic large scale integration, segmented
flow microfluidics, centrifugal microfluidics, electrokinetics, electrowetting, surface
acoustic waves and dedicated systems for massively parallel analysis. The principal
parameter that differentiates these devices is the fluid propulsion system within
microchannels with sub-millimeter cross section. For instance, in pregnancy tests,
liquids are driven by capillary forces, and controlled by wettability and feature size of
the microstructured substrate, and it is known as a lateral flow test (e.g. test strips).
A special attention here will be given to pressure driven laminar flow, whose fluid
moves due to pressure gradients. The samples and reagents are injected into the chip
inlets in a continuous mode using syringe pumps and tubes (Fig. 16)164. Pressure
driven laminar flow offers predictable velocity profiles and it is a key requirement for
several cell-based assays.
Integration of microfluidics and FISH for the rapid identification of microorganisms
30 Daniela Cruz Moreira
FIGURE 16| Schematic of equipment network for pressure driven laminar flow
Reagents are stored in a syringe and are connected to the microfluidic device by a tube. Syringe pump injects the
fluid at a constant flow rate. All the process can be observed, using a microscope165
.
The strictly laminar flow effect can be utilized to implement hydrodynamic focusing
technology, illustrated by Fig. 17, whereby particles or cells suspended in the liquid
flowing through the central channel are focused and aligned to a well-defined
streamline position. The network consists of a junction of more than one inlet channels,
connected at a junction to form a common outlet channel. By varying the ratio of flow
rates, the width of streamlines within the common outlet channel can be adjusted very
accurately.
FIGURE 17| Flow focusing structure
Three different liquid streams are
symmetrically contacted at an intersection
point. Cells suspended in the liquid flowing
through the central channel are focused
and aligned to streamline position
Another technique allowed by pressure driven laminar flow is the implementation of
active and passive valves166. A disadvantage of pressure driven platforms is the
necessity of a pressure source, which requires manual steps and decreases the
portability of the device.
The requirements on microfluidic devices differ between market segments. Several
aspects need to be considered while designing a microfluidic device, such as selection
of materials, dimensions of the microfluidics devices and fluidic control devices, as
described above. The optimization of the developed device requires several
experimental tests to achieve the best conditions for an effective procedure.
CHAPTER II – LITERATURE REVIEW
Daniela Cruz Moreira 31
5. Computational fluid dynamics
As shown above, the development of microfluidic devices is a process that requires
time and resources to build and test successive prototypes of a selected chip design.
Since the development of lab-on-a-chip devices becomes a highly competitive field,
computational simulation has become the standard method to reduce the time from
concept to chip167. Virtual prototyping can reduce the number of experimental iterations
allowing a rapid determination of how design changes will affect chip performance. A
new trend is the use of computational fluid mechanics (CFD) tools to simulate the flow
combined with mass and heat transfer in the design of a new equipment and
process171.
There are many commercial general-purpose CFD programs available. Fig. 18
illustrates the steps that must be defined in solving a problem using CFD.
A CFD solution displays the result of the specific chosen model with the given mesh,
as color maps in which is possible to obtain detailed local information (pressure,
velocity) on the simulated system that will help building and understanding of the
process.
CFD software uses an iterative solution method where the equation sets for variables
such as pressure, velocity and temperature are solved sequentially and repeatedly until
a converged solution is obtained169.
It is possible to obtain very accurate flow simulations for single-phase laminar flow
systems, the kind of flow behavior present in microfluidic devices. Simulation of heat
and mass transfer is also often very accurate and a good prediction can be easily
obtained168.
Integration of microfluidics and FISH for the rapid identification of microorganisms
32 Daniela Cruz Moreira
FIGURE 18| Steps in CFD simulation169
A CFD simulation starts with geometry drawing in a CAD program. The computational volume must be properly
discretized, then computational domain is meshed. A refined mesh leads to an accurate numerical solution of the
equation. A selection of the most appropriate model should be done. Simple, single-phase laminar flow, can be
simulated very accurately, because the Navier-Stokes equations can be solved directly, and for the most single-
phase laminar flows the simulations are reliable. All physical properties of the fluids must be defined. All inlet and
outlet conditions must be defined, as well as conditions on the wall and other boundaries. The solver and the
quality of an acceptable solution in terms of the convergence criteria must also be defined. Analysis of the final
simulation gives local information about fluid in a colorful display.
Geometry modelling
•Define geometry
•Define boundaries
Mesh Generation
•Divide the geometry into small computational cells
Defining models
•Add models for turbulence, laminar, etc.
Set properties
•Density, viscosity, etc.
Set boundary conditions
•The initial conditions, inlet and outlet conditions and conditions at the walls are set
Solve
•Choose solver, iteration methods, transient or steady state, convergence requirement
Post-Processing
•Analyse results
CHAPTER II – LITERATURE REVIEW
Daniela Cruz Moreira 33
5.1 Finite volume method
There are three distinct streams of numerical solution techniques: finite difference,
finite element and spectral methods. Solely finite volume method (FVM) will be
described because is central to the most well-established CFD codes, like Fluent ® 170.
FVM is a numerical technique that divides the entire computational domain into a
number of cells known as control volumes (CV), each corresponding to a single grid
point (node)171, as shown in Fig. 19.
FIGURE 19| Bi-dimensional finite volume P and its neighbors172.
The balance of a given property in a volume P should take into account the influence of its neighbors at east (E),
west (W), north (N) and south (S), when bi-dimensional problems are considered. Capital letters refer to the center
of the elementary volume and small letters to the frontier between P and its respective neighbor.
The general transport equation for an arbitrary variable ϕ in conservative form, written
using Einstein notation, is:
� *+*� + � *(,-+)*.- = **.- /Γ
*+*.-1 + 23
(7)
where � is the specific mass, ,- is the velocity vector, Γ the diffusion coefficient, and 2 is
the source term.
Since this equation is nonlinear and often contain both spatial and temporal
derivatives, generally is not possible to solve it analytically, and requires a numerical
method. In the finite volume approach, the governing equations are made discrete and
finite, and then numerically integrated over each CV169:
4 �*+*�56�7 + 4 �*(,-+)*.-56
�7 = 4 **.- /Γ
*+*.-1�756
+4 23�756
(8)
Integration of microfluidics and FISH for the rapid identification of microorganisms
34 Daniela Cruz Moreira
where 9 � :(;<3):=<56 �7 represents the convective term, 9 :
:=< >Γ :3:=<?�756 is the diffusion
term that takes into account the transport of ϕ by diffusion, and 9 23�756 , the source
term, takes into account any generation or dissipation of ϕ.
According to Gauss’ theorem it is possible to rewrite eq. 8 as
4 �*+*� �756+4 �,-@+5A
�B = 4 Γ *+*.- @�B5A+4 23�756
(9)
where CS denotes the control-volume surfaces (the faces that surround the cell), dA is
the area that surrounds the volume dV, and n is a normal vector pointing outwards from
dA.
The original transport equation is now transformed into an equation that can be solved
algebraically in an iterative manner and a numerical error is introduced into the
solution. The smaller cell size is, the lowest is magnitude of the error. However, it is a
question of cell size trade-off, because reducing the cell size too much will create an
unnecessarily large number of cells and the consequences are an increased
computational effort, slowing down the process to achieve the solution169.
The FVM is perhaps the simplest to understand and to program, because it does not
require a structured grid and the values of the neighboring nodes are unknown173.
Although unstructured grids make it possible to mesh complex geometries, CFD
programs using unstructured grids are slower and require more memory than those
using structured grids169.
Chapter III – Materials and methods
1. Yeast characterization
2. PNA-FISH protocol optimization
3. Material preparation
4. CFD simulation
5. Characterization of the microchannels
6. Microfluidic channels retention tests
7. PNA-FISH methodology within the
microfluidic channel
8. Microscopic visualization
Integration of microfluidics and FISH for the rapid identification of microorganisms
36 Daniela Cruz Moreira
1. Yeast characterization
1.1. Surrogate microorganism selection, yeast strain and culture maintenance Experimental research involving pathogens requires precautions to prevent infection
and contamination during microbial procedures. Candida spp. are categorized as
biohazard level 2 and LEPABE laboratory available for this project is classified as
Biosafety level 1. In the scope of the project, a surrogate microorganism for Candida
spp. had to be selected.
Saccharomyces cerevisiae, a non-pathogenic yeast well-known from the alimentary
industry174, met all criteria.
Saccharomyces cerevisiae PYCC4072 was kindly provided by Dr. Manuela Rodrigues
from University of Minho - Biology Department and maintained YEPD medium with
25% glycerol at -80ºC. The strain was then streaked onto a YEPD-agar and incubated
at 30ºC, as described by Larsson et al175.
To prepare liquid cultures, a loopful of biomass was transferred into 100mL of YEPD
medium and incubated overnight (~16h) at 30ºC (VELP Scientifica Incubator, FOC
225E model, Usmate, Italy) and 150 rpm (IKA KS 130 basic shaker, Vidrolab,
Portugal), under aerobic conditions, yielding a stationary phase culture176.
YEPD-agar and YEPD media recipes may be found in Appendix I.
1.2 S. cerevisiae growth
The growth curve for S. cerevisiae PYCC4072 strain was carried out by transferring a
small portion of the overnight culture to 100mL of fresh YEPD medium until it reaches
an OD600 of 0.1, and incubated at 30ºC and 150 rpm. Cellular growth was determined
by measuring optical density at 600nm (OD600) every 30 min using a
spectrophotometer (VWR V-1200), until the culture reach the stationary phase. This
experiment determined the OD600 of the culture that corresponds to the mid-log phase.
In order to assess the number of cells present in a given suspension, a calibration
curve was also built where an overnight culture of S. cerevisiae was prepared being
serially diluted in YEPD medium (1:2, 1:4, 1:8, 1:16, 1:32). OD600 of each sample was
measured in triplicates. Cells suspensions (500 µL) were filtered onto black nucleopore
polycarbonate membrane (Ø 25 mm) with a pore size of 0.2µm (Whatman, Japan) and
3 drops of 4’,6-diamidino-2-phenylindole (DAPI) (Sigma-Aldrich, USA) (0,2 mg/mL)
were added. Samples were placed in the dark, incubated at room temperature for 10
CHAPTER III – MATERIALS AND METHODS
Daniela Cruz Moreira 37
min. After that, the samples were filtered by vacuum and mounted with 1 drop of
immersion oil (Merk, Germany) on a slide. Cells were analyzed using an
epifluorescence microscope (Leica DM LB2, Leica Microsystems, Germany) (see used
features in microscopic visualization description below) . For each sample, 10 fields
with an area of 0,0063 mm2 were counted. For every field, the cells were manually
counted and averaged. A calibration curve was prepared by plotting the mean values of
OD600 against the corresponding concentration. All experiments were repeated at least
three times.
2. PNA-FISH protocol optimization
2.1 Probe sequence
A PNA-probe targeting S. cerevisiae 26S rRNA, already designed by Meireles177 and
ordered to Panagene Inc., was used.
The final probe sequence was Alexa 594-OO-AGGCTATAATACTTACC (5’-3’), being
HPLC purified > 90%. The theoretical specificity is 91.15% and sensitivity of 89.47%.
Probe thermodynamic parameters calculated by Meireles are summarized in Table 2.
Table 2| Probe thermodynamic parameters177
Probe ∆H
(Kcal/mol)
∆S
(kcal/K)
∆G
(Kcal/mol)
Tm
(ºC)
Tm PNA
(ºC)
AGGCTATAATACTTACC -115 -321.7 -15.26 62.4 66.2
2.2 Hybridization conditions optimization
Although hybridization conditions for this particular PNA probe were already studied, it
was decided to test several different parameters on the hybridization assays
(description bellow) to simplify the methodology. Hence, different hybridization
temperatures, formamide concentrations and the use of simplified hybridization
solutions were tested to increase the signal to noise ratio (See Chapter II, section 3).
For the hybridization conditions optimization, assays were based on the PNA-FISH
suspension protocol described by Perry-O’Keefe et al178. One mL of 1x107 cells /ml of
S. cerevisiae was pelleted by centrifugation at 10 000G for 5 min (Centrifuge 5418,
Eppendorf, USA), and the pellet was re-suspended in 400µL of 4% paraformaldehyde
Integration of microfluidics and FISH for the rapid identification of microorganisms
38 Daniela Cruz Moreira
(wt/vol) (Acros Organics, UK) and left for 1 hour at room temperature. Then,
suspension was pelleted by centrifugation again at 10 000G for 5 min and resuspended
in 500 µl of 50%(vol/vol) ethanol and placed for at least 30 min at -20ºC. Subsequently,
100 µl of the fixed cells aliquot was pelleted by centrifugation and resuspended in 100
µl of hybridization solution containing the PNA probe at 200nM and incubated for 60
min (FD 23, Binder, Germany). Besides the standardly used hybridization solution179,180
two simplified hybridization solutions with different formamide concentration (30% and
0%) were tested and compared. The detailed composition of these solutions may be
found in Appendix II. Also, different temperatures, ranged between 50 and 60ºC, for
each hybridization solution were tested.
After hybridization, cells were rinsed by centrifugation at 10.000 G for 5 min, and 500
µl of wash solution was added (composition in Appendix III). Cells were incubated at
the same temperature as in hybridization for 30 min. Washed suspension was pelleted
by centrifugation, and resuspended in 500 µl of sterile water. Finally, 20 µl of the cell
suspension were placed on a microscope slide. Samples were allowed to air dry. The
sample is then ready to be observed in the epifluorescent microscope.
2.3 Fixation conditions optimization
The choice of the agents to be used in the fixation step can compromise the
performance of subsequent FISH procedure (See Chapter II, section 3). Different
fixation protocols were performed and the quality of fluorescent signal was compared.
One mL of S. cerevisiae inoculum was pelleted by centrifugation at 10 000G for 5 min
and re-suspended in 1mL of sterile water. 20µL of the suspension was dispensed in 14
mm well slides (Thermo Scientific) and allowed to dry by flame. Subsequently, smears
were chemically fixed using ten different protocols, described in Table 3, based on the
previously described by Guimarães et al. 181, Reller et al.182, Rigby et al.183, Harris and
Hata184, Shepard et al.185, protocols described by the manufacturer abcam®186
(methods 2, 5 and 7) and protocols suggested by the manufacturer Li-Cor ®187
(methods 9 and 10). In protocols with most volatile reagents, such as alcohols, the
procedure was performed at low temperatures (-20ºC).
The subsequent hybridization step was performed according to Guimarães et al.181,
where samples were covered with 20µL of hybridization solution containing 200nM of
PNA probe, covered with cover slips and incubated for 60 min at 54ºC, which was the
temperature with better signal to noise ratio (see chapter IV for these results).
CHAPTER III – MATERIALS AND METHODS
Daniela Cruz Moreira 39
Subsequently coverslips were removed and the slides were immersed in the wash
solution and incubated for 30min at 54ºC. Then, samples were allowed to air dry and
were ready to be observed by epifluorescence microscopy.
2.4 Assessment of dextran sulfate influence on hybridization
In microfluidic devices, excessive solution viscosity should be avoided mostly because
of the difficulty in flowing high viscosity fluids on the microscale (See equations (2) and
(3) of Chapter II-Section 5.3). Also, when cells contact with the hybridization solution
containing the probe, the assessment of the probe to the target is made by diffusion,
and according to equations (4) and (5) (see Chapter II, Section 5.3) the diffusion is
faster as less viscous the solution is. Dextran sulfate is known for accelerating the rate
of hybridization but also to increase the viscosity of the hybridization solution188. This
experiment was performed to evaluate the influence of dextran sulfate on the quality of
the hybridization. For this purpose the protocol in suspension was used (see
description above in point 2.2).
Two simplified hybridization solutions with 10% (w/v) and 0%(w/v) of dextran sulfate
were compared. A negative control was performed simultaneously, where no probe
was added during the hybridization procedure.
Table 3| Comparison of different fixation protocols
Protocol Fixation step in slide
Reagent Time Temperature Reference
Method 1 4% (w/v) Paraformaldehyde 10min Room temp. Guimarães et
al.181 50% (v/v) Ethanol 10min -20˚C
Method 2
4% (w/v) Paraformaldehyde 10min Room temp. Anon. 2014a186 0,2% (v/v) Triton X-100 in
PBS 10min -20˚C
Method 3 4% (w/v) Paraformaldehyde 10min Room temp.
Reller et al.182 100% (v/v) Methanol 10min -20˚C
Method 4
1% (v/v) Triton X-100 in PBS
10min Room temp. Rigby et al.183
96% (v/v) Ethanol 10min -20˚C
Method 5 100% (v/v) Methanol 10min -20˚C Anon. 2014a186
Method 6 100% (v/v) Methanol 10min -20˚C Harris and
Hata184 80% (v/v) Ethanol 10min -20˚C
Method 7 100% (v/v) Acetone 10min -20˚C Anon. 2014a186
Integration of microfluidics and FISH for the rapid identification of microorganisms
40 Daniela Cruz Moreira
Method 8 50% (v/v) Ethanol 10min -20˚C Shepard et al.
(adapted)185
Method 9 100% (v/v) Acetone 10min -20˚C
Anon. 2014b187
100% (v/v) Methanol 10min -20˚C
Method 10 50% (v/v) Acetone in
Methanol 10min -20˚C Anon. 2014b187
3. Material preparation
3.1 Microchip design
Microfluidic systems were conceived to operate by pressure driven laminar flow.
Therefore, solutions were injected into inlets using syringe pumps and flow to the
microchannel due to pressure gradients, coming out in the outlet. To concentrate the
cell suspension, entrapment strategies focused on dam-like structures and filtering
pillars were employed. It is necessary to take special care in the design of geometries
in order to avoid clogging, because exposure of cells to different solutions during PNA-
FISH procedure is performed by passing each solution from the inlet to the outlet of the
cell-trapping microchannels.
3.1.1 Production of molds – channels with a dam-like structure
A microfluidic device possessing an 8 µm gap was planned for cell entrapment. A
cross-sectional view of the microfluidic device is shown in Fig. 20. It is meant that cells
get trapped in the region of the dam, in front of the 8 µm gap, which is called the
“detection zone”.
As can be seen in Fig. 20, the microfluidic device was constructed by bonding of two
(upper and lower) parts. The mold production of the upper part was performed by
xurography technique. Two geometries composed by two macro-to-micro interfaces
(inlet and outlet) and a microchannel connecting them with 25 000µm long and 300µm
wide were drawn by CorelDRAW ®. Geometries were cut with a cutting plotter on a
100 µm thickness self-adhesive paper using GreatCut® software. The desired mold
was placed on a petri dish and the surrounding paper was removed.
A small rectangle was cut and adherently attached to a glass slide substrate. Substrate
was covered with PDMS mixed in 1:20 (or 1:10) ratio, using a spin coater (ω=3000 rpm
for 50s) to obtain a thin layer and was cured in an oven for 20 minutes. The rectangle
CHAPTER III – MATERIALS AND METHODS
Daniela Cruz Moreira 41
was removed, forming a 8µm well, and PDMS block was bonded to glass slide
substrate and connected to an injection system.
FIGURE 20| Construction of the microfluidic device with a dam-like structure with 8-µm gap
To construct the channel with a dam-like structure the upper part with a 100 µm high barrier is bonded to a lower
part with a 8 µm depth gap
3.1.2 Production of molds – channels with pillar-based structure
The geometry of the channels was drawn using AutoCAD 2013 ® software (Autodesk
Inc, USA). Eight different geometries composed of microchannels with filtering
structures were evaluated. Five of these eight geometries are composed by one inlet
and one outlet and remaining three have two inlets and one outlet, to apply the
hydrodynamic focusing principle. The main differences between these approaches are
the arrangement and the dimensions of the gaps between the filtering pillars, and also
the influence of an enlarged section in which the velocity of the fluid is amended.
SU-8 molds on a silicon wafer were produced by photolithography using chrome masks
by a specialized manufacturer (microLIQUID, Spain). Molds were ordered in two
different depths, 50µm and 30µm, to ascertain what is the most appropriate.
3.2 Microfabrication of microchannels
Microchannels were manufactured by soft lithography. A two-part PDMS kit (Sylgard,
USA) was used to create the liquid polymer. The PDMS used to create a negative of
the molds was hand mixed, using a glass stirring rod, with curing agent at a ratio of 1:5.
To degas, remove air bubbles formed during the mixing, the liquid polymer was placed
in a desiccator connected to a vaccum pump until no air bubbles were observed. The
liquid PDMS was poured over the prefabricated mold at room temperature and pre-
cured for 20 minutes at 80ºC. Then, a PDMS block with the channels imprinted on it
was removed from the mold and access points were created using a needle. The
PDMS used to create a coverslip in the substrate to seal the channel, was hand mixed
Integration of microfluidics and FISH for the rapid identification of microorganisms
42 Daniela Cruz Moreira
with curing agent at a ratio of 1:20 and the air bubbles were removed used the same
conditions as above. A small portion of the liquid polymer was applied on the glass
cover slip and placed on a spin-coater at a speed of 3000 rpm for 50s to form a thin
layer of elastomer, and then was pre-cured for 5 min at 80ºC. Finally the PDMS block
is bound to the spin coated glass slide and cured at 80ºC overnight.
4. CFD simulation
Computational fluid dynamics simulations were made in Ansys Fluent CFD package
(version 14.5, Ansys, Inc., Canonsburg, PA, USA). Channel geometries designed in
AutoCAD were imported to Design Modeller 14.5 (Ansys, Inc., Canonsburg, PA, USA)
and discretized into a grid of quadrilateral cells by Meshing 14.5 (Ansys, Inc.,
Canonsburg, PA, USA).
Table 4 contains the characteristics of each mesh created. A mesh independence
study was performed to make sure that the solution is also independent of the mesh
density. The mesh was refined and the number of cells across the gap was increased
until a certain point in which the solution do not change because of mesh density. To
reduce the simulation run time, the smallest mesh that gives the mesh independent
solution was used.
For each simulation, the initial velocity of the inflow from the inlets was assumed to be
0.003 m/s, corresponding to an inlet flow rate of 1µL/min in a cross section of 100µm x
50 µm, and the fluid was assumed to be water for the sake of simplicity. A pressure
outlet boundary condition was implemented at the downstream and was assumed to be
zero. The boundary condition for the inner walls of the microchannels was defined as
the no slip condition.
In Ansys Fluent, the convergence was defined by reducing Residual Error to an
acceptable value (10-6) to ensure a valid solution is achieved. The number of iterations
was set to 100, ensuring that the main outputs from the simulation have converged to a
steady solution.
Ansys Fluent was used to carry out the two dimensional (2D) simulation of velocity
magnitude and stream function of each channel geometry conceived.
CHAPTER III – MATERIALS AND METHODS
Daniela Cruz Moreira 43
Table 4| Characteristics of each 2D mesh
Cells Across Gap Nodes Elements
Simulation I 20 5275 5003
Simulation II 10 329844 322479
Simulation III 20 994975 985397
Simulation IV 20 313105 303838
Simulation V 10 40358 37507
Simulation VI 10 519249 506888
Simulation VII 10 83092 79682
Simulation VIII 10 240640 235096
Simulation IX 10 40890 37438
5. Characterization of the microchannels
5.1 Geometrical characterization of the microchannels
Several measurements were performed using Leica Application Suite tool, to confirm
if the results from microfabrication were in agreement to the designed.
To confirm if the channel height correspond to the depth ordered to the manufacturer,
a cross section of the channel was cut as displayed in Fig. 21, the cross section was
placed in a glass slide, observed using a microscope (DMI 5000M, Leica Microsystems
GmbH) and its dimensions were measured using Leica Application Suite tool.
FIGURE 21| Cross section of the microchannel189
Integration of microfluidics and FISH for the rapid identification of microorganisms
44 Daniela Cruz Moreira
5.2 Volumetric measurement of channels capacity
Due to the irregular geometry of the microchannels, determining the capacity of each
microchannel geometry seems to be more accurate by experimental methods instead
calculations. Concentrated violet crystal, was pumped into the device at a constant flow
rate and the filling time was clocked. The volume was achieved using a simple
calculation, with the formula 7 = � × �, being V (µL) the volume, Q (µL/s) the flow rate
and t (s) the time.
5.3 Material testing
In order to evaluate the suitability of the PDMS for fabricating the microchannels and
complete the PNA-FISH methodology within its interior without compromising the
characteristics of the channel, a simple test was performed. A 50 µm wide
microchannel with a narrowed section of 20 µm was filled with each reagent of the
PNA-FISH procedure during the specific time of each one, at room temperature. The
channel was visually analyzed using a microscope (DMI 5000M, Leica Microsystems
GmbH) and its dimensions were measured at two points using Leica Application Suite
tool, to assess the resistance of the PDMS under those conditions.
6. Microfluidic channels retention test
In order to test channels ability to entrap yeasts, retention was first evaluated using 10
µm polystyrene microspheres (Sigma-Aldrich, USA). Theoretically, microparticles will
have a behavior similar to yeasts and are integrated in the initial tests because are
considerably easier to handle and require less limitation laboratorial conditions than
microorganisms. The same test was later made using S. cervisiae.
7. PNA-FISH methodology within the microfluidic channel
The concept of trapping and labeling yeast cells within the microfluidic device is
illustrated in Fig 22. For a FISH assay, a new microfluidic chip was washed with sterile
water for 30 s at a constant flow rate of 100 µL/min using a syringe pump (Cetoni,
CHAPTER III – MATERIALS AND METHODS
Daniela Cruz Moreira 45
neMESYS syringe pump, Germany). The flow rate in this first washing step was so
high to drag any dust and remove air bubbles that sometimes remain in the detection
zone. Then, 5 µL of solution containing approximately 1x107 cells/ml of previously fixed
S. cerevisiae were delivered into the device using a micro-syringe. Yeast size is larger
than the gap between any two given pillars, and for this reason, S. cerevisiae were
trapped in front of the pillars and retained in the detection zone by the flow pressure
toward the pillars. The hybridization solution chosen as optimum was pumped at a
constant flow rate of 1µL/min into the device at a constant flow rate until fill the channel,
allowing the probe to access the target. The device was disconnected from the pump
system and placed in the incubator at 54ºC for 1 h. After hybridization, the chip was
reconnected to the pump system and washing solution was injected at a constant flow
rate of 1µL/min until fill the channel, to remove exceed probe. The device was again
disconnected from the pump system and placed in the incubator at 54ºC for 30 min.
Then, the chip was reconnected to the pump system and sterile water was injected to
wash away artifacts that can influence the result and DAPI (0.2 mg/mL) was injected
until fulfill the channel, to counterstain the cells. After 10 min, sterile water was injected
to wash any residuals. Microfluidic device containing labeled yeasts is ready to be
observed by epifluorescence microscopy.
Integration of FISH methodology with these new microfluidic devices requires some
adjustments, performed as a trial and error procedure. The volume of fixed cells
injected was adjusted, as well as the flow rate of the hybridization and washing
solution.
Integration of microfluidics and FISH for the rapid identification of microorganisms
46 Daniela Cruz Moreira
FIGURE 22| Schematic illustration of the concept to perform FISH assay in the microfluidic
device
I| Yeast cells are trapped and retained. II| PNA probe solution is pumped into the device at a constant flow rate . III| After hybridization, cells are washed to remove loosely-bound probes and examined using an epifluorescence microscopy.
8. Microscopic visualization
Fluorescently labeled samples were observed using Leica DM LB2 (Leica
Microsystems, Germany) epifluorescence microscope connected to a Leica DFC300
FX camera (Leica Microsystems, Germany) and equipped with a Live/Dead Filter
(Excitation 530 to 550 nm; Barrier 570 nm; Emission LP 591 nm) sensitive to the
fluorophore Alexa Fluor 594 attached to PNA probe. To confirm the absence of cells
autofluorescence a N2.1 Filter (Excitation 515 to 560 nm; Barrier 580 nm; Emission LP
590 nm) was used. The microscope software parameters (exposure, gain and
saturation) were carefully maintained constant in all the experiments involving FISH.
CHAPTER III – MATERIALS AND METHODS
Daniela Cruz Moreira 47
For DAPI stained cells a Chroma 61000-V2 Filter, consisted of a 545/30 nm excitation
filter combined with a dichromatic mirror at 565 nm and suppression filter 610/75, was
used.
During experimental microfluidics, a Leica DMI 5000 microscope was used.
Chapter IV – Results and Discussion
1. S. cerevisiae growth
2. PNA-FISH protocol optimization
3. Microchannels geometry design
4. Computational fluid dynamics analysis
5. Retention tests in channels with a dam-
like structure
6. Geometrical characterization of
channels with pillar-based filter
7. Retention tests in single channels with
pillar-based filter
8. PNA-FISH methodology within the
microfluidic channel
CHAPTER IV – RESULTS AND DISCUSSION
Daniela Cruz Moreira 49
1. S. cerevisiae growth
. Being the goal of the project the development of a microfluidic filter-based device in
which yeasts can be identified by FISH, the surrogate microorganism has to mimic
especially the morphology of Candida spp.. To correctly evaluate the practicability of
any conceived trapping structures, surrogate must have similar shape, size and life
cycle form.
Phylogenetically, Saccharomyces spp. is closely related to Candida spp., sharing a
common ancestor190. Due to its long history of safe use and consumption, and lack of
production of toxins, most strains of S. cerevisiae have been considered as generally
recognized as safe (GRAS)191. Also, it is easy to obtain and to produce a large number
of samples at low cost192. The morphology of S. cerevisiae is similar to Candida spp.,
nonetheless, small differences in terms of size are verified. S. cerevisiae cells are ovoid
with 5-8 µm whilst Candida spp. diameter is about 2-3µm193. Since microfluidic
channels fabrication may be easily scaled up or down according to the used technique,
small differences in term of size are insignificant.
Under appropriate nutritional conditions that trigger cellular morphogenic programs, S.
cervisiae can alternate between yeast-like growth form and pseudohyphae194,195. Also,
environmental conditions highly influence the thickness of its cell wall that can vary in a
range of 10 to 25% of its total biomass, depending on the nature of the carbon
source196.
As already referred, probe penetration into the cell wall is crucial to an effective
hybridization during FISH procedure (see Chapter I, Section 3). Understanding the
properties of the cell wall can be helpful to optimize the FISH protocol. S. cerevisiae
cell wall resembles C. albicans cell wall197 and is essentially composed by β1,3- linked
glucans and mannoproteins198. β1,6- linked glucan is a minor component of the wall
but has a central role in cross-linking wall components199. Another important minor
component is chitin, which can be covalently joined to β1,6- glycosylated
mannoproteins to form high order complexes and contributes to the insolubility of the
fibers200. Although chitin levels in the cell wall are normally low, cell wall stress activate
the cell integrity pathway, which result in a strong increase in the deposition of chitin in
the lateral wall, increasing the resistance of the cells201. Another interesting fact is that
proteins incorporated in the cell wall depend not only on environmental conditions but it
is observed differences at different phases of the cell cycle202. Cell wall of
logarithmically growing cells has relatively high porosity, that could reflect a lower
Integration of microfluidics and FISH for the rapid identification of microorganisms
50 Daniela Cruz Moreira
degree of cross-linking, whereas walls of stationary-phase cells are less porous203,204. It
is easy to understand that PNA probes would penetrate easier through the cell wall
pores to access the target rRNA in the cytosol in logarithmically growing cells than in
stationary-phase cells with insoluble and resistant cell walls.
To predict an efficient model for the S. cerevisiae growth, OD600 measures were
performed with time (Fig. 23). Obtained results match typical yeast growth curve,
where three essential phases are observed: a lag phase, when newly pitched yeast
acclimate to the environment; a log phase, when cells grow rapidly due to relative
excess of nutrients and insignificant waste in the environment; and a stationary phase,
when cell growth decelerate due to substrate consumption and high waste
concentration205.
In order to obtain the best fluorescence intensity in fluorescent-labeled yeast cells by
PNA-FISH, reducing the possibility of false negatives, some cell cycle-dependent
characteristics of yeasts need to be considered. Ribosomal content are closely linked
to growth rate and, according to Warner’s review85, rapidly growing yeast cells contain
a higher number of ribosomes per cell. Being the targeted sequence present in the 26S
ribosome, higher number of ribosomes per cell leads to more targets and,
consequently, enhanced fluorescence intensity. According to Smits et al.202 study in
cell cycle-regulated incorporation of cell wall proteins, mid-log phase yeast cell wall are
highly porous, what improve the accessibility of the probe to the target by ease the
penetration of probe into the cell through the cell wall pores. Thereupon, it is thought
that intensity of probe-conferred fluorescence reach a higher value in the mid-log
phase.
FIGURE 23| Saccharomyces cerevisiae growth curve
CHAPTER IV – RESULTS AND DISCUSSION
Daniela Cruz Moreira 51
In addition, to determine S. cerevisae concentration used in the experiments, a
calibration curve was obtained (Fig. 24)
FIGURE 24| Saccharomyces cerevisiae calibration curve
To the FISH experiments, S. cerevisiae culture was grown until the mid-log phase with
an OD600 of approximately 0.90 what, according to the obtained calibration curve,
corresponds to a cell density of 8,7x106 cells/mL.
2. PNA-FISH protocol optimization
PNA-FISH protocol optimization main goal is to achieve the best output, in order to
enhance signal-to-noise ratio, to a better discrimination of the results when performed
inside microfluidic devices. Several assays were performed to define the best fixation
and hybridization conditions, such as fixation protocols, hybridization temperature, and
hybridization buffer composition. Results were analyzed comparing the fluorescent
signal-to-noise resulting of each assay.
According to SantaLucia206, thermal stability, also referred as melting temperature, is
related to the probe affinity to the target. It can be estimated by Gibbs free energy
change during hybridization reaction89. However, this theoretic calculation does not
consider, among other parameters, the presence of formamide, a denaturant agent
known to reduce the melting temperature of double-stranded nucleic acids207. Aware
that hybridization stringency is affected by the interplay of several parameters and to
properly adjust hybridization conditions during experimental PNA-FISH methodologies,
a trial and error procedure was performed. Temperatures ranging from 51-59ºC were
y = 1,50E+07x - 4,79E+06R² = 9,18E-01
-5,E+06
0,E+00
5,E+06
1,E+07
2,E+07
2,E+07
3,E+07
3,E+07
0,0 0,5 1,0 1,5 2,0
Ce
ll c
on
cen
tra
tio
n (
cell
s/m
L)
OD600
Integration of microfluidics and FISH for the rapid identification of microorganisms
52 Daniela Cruz Moreira
tested with different hybridization solutions to assess PNA-FISH performance, and the
results are displayed in Fig. 26. Initial temperature optimizations test lower
temperatures than the theoretically melting temperature, because higher temperatures
are not favorable in biological terms208. The chosen temperature is according to
previous results obtained by Meireles177, were the best temperature for hybridization in
suspension was 53ºC and for hybridization in slide was 55ºC. Lima209 also predicted,
with a response surface methodology (RSM) approach, the maximum hybridization
efficiency at a temperature of 53.9ºC. As such, and according to the results observed in
here it seems appropriate to use the medium temperature of 54ºC in the subsequent
tests. Regarding the comparison between hybridization solutions, the difference from
the standard hybridization solution developed by Azevedo179 and simplified one used
by Santos et al.210 is the lack of Denhardt’s solution components (sodium
pyrophosphate, polyvinylpyrrolidone, and FICOLL), NaCl and disodium EDTA.
According to Azevedo179, sodium pyrophosphate, polyvinylpyrrolidone and FICOLL act
as blocking reagents for preventing unspecific binding to nitrocellulose membranes;
NaCl is used to increase salt concentration, increasing reaction rate and stabilize
secondary structures of ribosomes; and disodium EDTA act as a chelator. Also the
presence of formamide, a denaturing agent, in the simplified solutions is evaluated.
Formamide is known to increase the accessibility to rRNA target by weakening
hydrogen bounds responsible for secondary structures of ribosome.
Analyzing the results presented in Fig. 25, it is clear that the best results occur for
54ºC with a simplified solution without formamide. Contrarily to what was thought,
hybridization without a denaturant agent is stronger compared to hybridization with
formamide. Actually, it is possible and clearly distinguished compared with a negative
control (data not shown). Earlier accessibility studies in Bacteria and Eukarya211
conclude that the absence of formamide in the hybridization buffer result in kinetic
limitations, in agreement with Yilmaz et al.207. However, recently, Santos et al. 210,
proved that the use of formamide in FISH is beneficial for certain species of bacteria,
but could also have a harmful effect on integrity of cells with a relatively thick wall. In
the same study, S. cerevisiae was used to demystify the influence of formamide in the
hybridization buffer, leading to the conclusion that do not affect the hybridization
specificity. Accordingly, the absence of fomamide in PNA-FISH may not affect the
specificity of the method.
CHAPTER IV – RESULTS AND DISCUSSION
Daniela Cruz Moreira 53
Temp.
(ºC)
Standard hybridization
solution
Simplified hybridization solution
(30% formamide)
Simplified hybridization solution
(0% formamide)
51
53
54
55
57
59
FIGURE 25| Fluorescence microscope results for in suspension PNA-FISH with different hybridization solutions at different temperatures Images were obtained with equal exposure times. Original magnification x 1000. Scale bar 20µm.
Integration of microfluidics and FISH for the rapid identification of microorganisms
54 Daniela Cruz Moreira
What is also observable in Fig. 25 is that the hybridization is not perfect and the
distribution of the signal is not uniform. In Fig. 26b is observable the heterogeneity of
the fluorescence signal in different cells of the same samples.
a. b.
Figure 26| Fluorescence microscope results for in suspension PNA-FISH with simplified hybridization solution (0% formamide) at 54˚C a| Smears without probe as negative control b| Positive. Images were obtained with equal exposure times. Original magnification x 1000. Scale bar 20µm.
As already explained, rRNA content depends on physiologic state of the cell which is
directly correlated with growth rate102. Even though the culture was used when reach a
mid-log phase, it is possible that cells appear in different states of growth, since cell
division is not a synchronized event. Depending on the cell cycle phase of a single cell,
low rRNA content or increased wall resistance can thereby result in low signal intensity
or false negatives as well76.
Despite all significant differences in intensity of probe-conferred fluorescent in this set
of temperature optimization tests, even in the optimum temperature, the signal is less
intense than expected. Another consequence of low signal intensity may be insufficient
probe penetration into cell wall. Optimal fixation should result in good probe penetration
and retention of the maximal level of target rRNA, as well as maintenance of cell
integrity and morphologic detail76.
Various fixation protocols were tested to assess the best fixation conditions to S.
cerevisiae in order to obtain a successful hybridization. All the fixation procedures were
performed in slide for the simplicity of the method. Due to the results obtained in
hybridization conditions optimization, simplified hybridization solution with 0% formamide
and hybridization temperature of 54ºC were used in all the subsequent experiments.
The results were displayed in Fig. 27. The negative control was obtained with N2.1
filter that was not able to detect the probe.
CHAPTER IV – RESULTS AND DISCUSSION
Daniela Cruz Moreira 55
Protocol Positive
(Live/Dead Filter) Negative control
(N2.1 Filter)
Method 1
Method 2
Method 3
Method 4
Method 5
Method 6
Method 7
Integration of microfluidics and FISH for the rapid identification of microorganisms
56 Daniela Cruz Moreira
Protocol Positive
(Live/Dead Filter) Negative control
(N2.1 Filter)
Method 8
Method 9
Method 10
Figure 27| Fluorescence microscope results for in suspension PNA-FISH simplified hybridization solution (0% formamide) at 54˚C after different fixation protocols Images were obtained with equal exposure times. Original magnification x 1000. Scale bar 20µm.
Examining all positives, it might be possible to think that Methods 9 (acetone and
methanol) and 10 (50% (v/v) acetone in methanol) offered the best results. However,
looking closely at their negative controls, it is observable that there is an unexpectedly
high signal when looking at any other negative. Autofluorescence is the natural
emission of light by biological structures that masks specific fluorescent signal. It can
be concluded that fixation with acetone and methanol trigger autofluorescence in S.
cerevisiae. Some moulds and yeasts have been reported by Graham212, to be
themselves autofluorescent, therefore the interpretation of fungal fluorescent signals
has to be done with caution213,214. Graf et al.213 state that autofluorescence in C.
albicans depends on the fixation and excitation wavelength. Nevertheless, S.
cerevisiae background do not appear to increase in paraformaldehyde-fixed cells, as
can be seen by previous results (Method 1 by Guimarães et al181 ). When Methods 6
(Methanol and 80%(v/v) ethanol), 7 (acetone) and 8 (50% (v/v) ethanol) were used, a
weaker signal was observed, probably due to alcohols evaporation, before completing
the 10 minutes of fixation. These alcohols were applied at -20˚C to prevent this
situation, but it seems to be inefficient. In addition hybridization results after Method 2
(4% paraformaldehyde and 0.2% (v/v) Triton X-100 in PBS) and 4 (1% Triton X-100 in
CHAPTER IV – RESULTS AND DISCUSSION
Daniela Cruz Moreira 57
PBS and 96% ethanol) fixation protocols were very similar, presenting a low signal-to-
noise ratio, which may be due to Triton X-100 reagent, common in both protocols.
Triton X-100 is a nonionic surfactant widely use to permeabilize cells215. It solubilizes
phospholipids, forming transmembrane pores that serve as channels to periplasm216.
However, if large amounts are added or cells are subject to prolonged exposure to
Triton X-100, disruption of polar head group on the hydrogen bonding present within
the cell’s bilayer could lead to destruction of compactness and integrity of the lipid
membrane217. Miozzari and colleagues215 report that S. cerevisiae are completely
permeabilized by Triton X-100 concentration of 0.05% (vol/vol), reiterated by Vasileva-
Tonkova et al218. Therefore, it leads to the conclusion that maybe the concentration of
Triton X-100 used was high enough to compromise cell wall integrity, that is intrinsically
linked to cell viability76.
In the end, Method 1 and 5 appears to be the most effective among the tested
protocols. Therefore, the fixation method chosen was Method 1 by Guimarães et al.181
since it is the more standard one219,220. The conditions achieved in slides hybridizations
were also used in suspension in all subsequent experiments.
As any of these fixation optimization protocols result in an increased of fluorescence
intensity, it was supposed that the limitation of the method could be related to the
accessibility of the target region of the ribosome. Conformational structure of
ribosomes can be favorable to hinder the targeted sequence and difficult the probe
access and consequent hybridization76. Inácio et al.87 evaluated the accessibility of D1
and D2 domains in the 26S rRNA of S. cerevisiae. To identify D1-D2 position of the
sequence targeted by the probe used in this work [AGGCTATAATACTTACC (5’-3’)], a
multiple sequence alignment software, ClustalW [European Bioinformatics
Institute(http://www.ebi.ac.uk/Tools/msa/clustalw2/)] was used. The entire sequence of
the 26S rRNA D1 and D2 domain of S. cerevisiae (sequence retrieved from GenBank
under accession number U44806) used by Inácio et al87f was aligned with the
probe221. According to results of the referred paper, the probe targets a lower
accessibility target sequence (brightness class V with 6-20 % of relative probe
fluorescence). However, low salt concentration is used, which destabilizes native
nucleic acid structures and results in an improved access to target sequnces.
The last attempt of PNA-FISH protocol optimization was made to evaluate if a less
viscous hybridization solution, more advantageous to flow into microchannels, could
perform an efficient hybridization222. Dextran sulfate is a volume exclusion polymer
incorporated in the hybridization solution that causes increasing viscosities and surface
tensions. Brigatti223 supports that increased viscosity inhibits both probe diffusing in and
Integration of microfluidics and FISH for the rapid identification of microorganisms
58 Daniela Cruz Moreira
out from the target region and during washing steps to wash away probe excess,
making such hybridization solutions non-ideal for capillary gap technology, which can
be a problem when transporting FISH procedure to microfluidic devices.
As hypothesized, to assess dextran sulfate influence on quality of hybridization, cells
were fixed using Guimarães et al.181 protocol, and hybridization was performed at 54ºC
with simplified hybridization solution (0% formamide) and simplified hybridization
solution (0% formamide and 0% dextran sulfate), as shown in Fig 28.
In a brief analysis of the results, is clearly observable that sulfate dextran is crucial for
an efficient hybridization under these conditions and the chemical effect overlaps the
increased viscosity. Dextran sulfate functions as an accelerant by excluding the nucleic
acid from volume of the solution occupied by the polymer, resulting in local
concentration of the probe. This principle had been shown to be successful previously
by Zaytseva et al224 and Ku et al225.
For these reasons, the dextran sulfate concentration of 10% (vol/vol) was used in all
subsequent experiments.
Positive
Negative control (No probe)
Simplified hybridization solution
(0% formamide)
Simplified hybridization solution
(0% formamide and 0%
dextran sulfate)
Figure 28| Fluorescence microscope results for in suspension PNA-FISH simplified hybridization solution (0%
formamide) at 54˚C with presence or absence of dextran sulfate.
Images were obtained with equal exposure times. Original magnification x 1000. Scale bar 20µm.
3. Microchannels geometry design
Most of the forces relevant for microfluidics are negligible at the macro scale, so the
development of new geometries has to be thought having in mind the scale effect.
Several geometries were conceived to obtain various microfluidic systems and choose
CHAPTER IV – RESULTS AND DISCUSSION
Daniela Cruz Moreira 59
the best approach for rapid and efficient microorganism identification by PNA-FISH.
Two types of structures were evaluated: dam-like structures and pillar-like structures
3.1 Channels with a dam-like structure
Geometries designed with CorelDRAW ® software are represented by Fig. 28. The
results are two microchannels composed by one inlet and one outlet carrying out the
macro-to-micro interface. The microchannel connecting them is 25 000 µm long, 300
µm wide. A 250 µm thick stripe was purposefully removed in the middle of the channel
to be filled with PDMS during soft lithography and form the 250µm wide central barrier
of the dam structure in the upper part. Channel represented by Fig. 28a. is 10 times
enlarged to increase the cross-sectional area at dam-like structure and consequently
decrease Re, comparatively to channel represented by Fig. 28b. It was designed in an
attempt for decrease velocity in this region for the fluid do not exert pressure enough to
push cells beyond the dam-like structure through the gap. The gap will be formed
below the barrier, during the microfabrication of the device, and it will have the shape of
a rectangle.
(a)
(b)
FIGURE 29| Mask structure
a| Geometry to produce a dam-like structure; b| Geometry with the same entrapment strategy but enlarged at the
detection zone.
3.2 Channels with a pillar-based filter
Geometries for the pillar filtering strategy guarantee that the gaps between pillars are
smaller than the cell size, so that, the gaps between each two pillars of every designed
structure have 5µm width. In some cases, it was decided to incorporate three (or more)
lines of pillars so if the cells can escape from one line can be entrapped at the next.
Integration of microfluidics and FISH for the rapid identification of microorganisms
60 Daniela Cruz Moreira
3.2.1 Single channel devices
Fig. 30 and 31 represent microfluidic systems with one inlet and one outlet connected
by a 10 000 µm long and 100 µm wide channel, being the channel represented by Fig.
31 enlarged at the detection zone, exactly for the same reasons of the channel
represented by Fig. 29b, to reduce the velocity at this level. The detection zone is
where cells are retained over time. It located in the middle of the channel to be easy to
find when observing the results with the microscope. Total structure was maintained in
Fig. 8 but the geometry of the pillars was altered. Channel represented by Fig. 30a is
100 µm wide, but when the fluid passes through the gaps, the section is reduced to a
quarter, 5 gaps of 5µm wide. The expected consequence is an increased velocity in the
detection zone.
(a)
(b) Mask structure
FIGURE 30| Geometries of cell-filtering single microfluidic devices
a| 15x45µm rectangular pillars ; b| 5x15µm rectangular pillars
CHAPTER IV – RESULTS AND DISCUSSION
Daniela Cruz Moreira 61
Trying to circumvent the abrupt increase of fluid velocity, geometry represented by
Fig. 30b was designed. As can be seen, the number of gaps is increased from 5 to 10.
However, pillars are thinner, which may have as a consequence more unstable and
less effective structures, compared with the robust pillars of Fig. 30a.
Thinner pillars are more susceptible to bend than robust ones, and this possibility is
higher when they suffer from effects of increased velocity. In an attempt to minimize the
effects of increased velocity and reduce not only damages on the structures but also
the loss of cells that are pushed through the gaps by the flow, channels in Fig. 31 were
designed. Channel represented by Fig. 31 is 100 µm wide, but the enlargement
reaches 500 µm wide. The sum of the gaps width (total width available for the flow
when passing a pillar row) 150 µm, 1.5 times the channel width. This conformation will
reduce the velocity of the fluid at the gaps.
(a)
(b)
(c)
Mask Structure
FIGURE 31| Geometries of enlarged cell-filtering microfluidic devices
a| 5x10µm rectangular pillars; b| diamond-shape pillars; b| pyramidal structures
Integration of microfluidics and FISH for the rapid identification of microorganisms
62 Daniela Cruz Moreira
Yeast cells have a cell wall that confers some rigidity to keep its form, but in the inside
they are full of liquid cytoplasm. When pushed through a gap they have the capability
to deform. Wherefore, it is possible, and probable, that cells, when subject to high
pressure gradients near the gap, deform and may be trapped, clogging it. Fig. 31b
represents a geometry designed keeping that in mind. The rectangular pillars were
substituted by diamond shape pillars, to reduce the length of the structure in which the
cell may get stuck. The main advantage is the reduced probability of clogging with the
chance of cells to get trapped on further structures.
Fig. 31c represents a different alternative to filter the cell suspension. The first
geometries developed had the pillars aligned, and the cells were trapped in a plane
perpendicular to the flow. This new geometry has intermediate gaps where the velocity
rate is supposed to decrease. The smallest cells should deposit between the pillars and
the larger ones in the taper gaps.
3.2.2 Two inlet devices
Two inlets-channels (Fig. 32) were also designed, trying to develop alternatives if the
one inlet geometries were prone to clogging.
The aim of the insertion of another inlet, is to use the properties of laminar flow, and
use one auxiliary stream to confine and direct the cell suspension.
For instance, as represented by Fig. 17 (hydrodynamic focus) from Section 5.4 of
Chapter II, it is possible to direct the cell suspension. The strategy employed is
injecting the cell suspension through the one inlet and sterile water through the other
inlet, focusing the cell flow to side of the microchannel, in both cases represented as
Fig. 32a or 32b. Cells would be entrapped in the lower region and, in case of clogging,
the cell suspension, and later on the remaining reactant streams, can be deviated from
the traps switching off the sterile water stream. The channel would not be totally
useless in case of clogging and the reagent would reach the cells by diffusion.
Figure 33 is a typical structure to perform hydrodynamic focusing. Sterile water is
injected by outer inlet and the cells suspension by the inner inlet. The streams contact
symmetrically at an intersection point and cells are focused in the flow central position,
until bump a horseshoe-like structure and be retained there.
CHAPTER IV – RESULTS AND DISCUSSION
Daniela Cruz Moreira 63
Mask Structure
(a)
(b)
FIGURE 32| Geometries of two streams cell-filtering microfluidic devices a| arc aligned rectangular pillars ; b| rectangular pillars
Mask structure
(a)
FIGURE 33| Geometries of three streams cell-filtering microfluidic devices
a| horseshoe structure
Integration of microfluidics and FISH for the rapid identification of microorganisms
64 Daniela Cruz Moreira
4. Computational fluid dynamics analysis
Numerical simulations are able to determine the fluid behavior inside a microchannel
even before the production of the microchip. This is advantageous because save
resources and time. 2D simulation are assumed as a modelling of the central horizontal
section of the fluid. Figure 34 is based entirely on numerical result which shows the
velocity contours (velocity magnitude) and the stream function contours (streamlines)
of all geometries conceived. It is observable in all simulations that the velocity
increases from the wall to a maximum in the center of the channel, in the zone free of
pillars. In almost all simulations, the fluid flows through the traps, except in Simulation
X. Fig. 35 represents a contour plot in a vertical plane in the middle of the horseshoe
structure. As we can interpret, in the middle, the horseshoe structure, the velocity of the
fluid is near to zero, which means that in this zone the fluid is almost stagnant.
Analyzing the streamlines in the stream function contour is perfectly notorious that the
fluids detour from the structure and flow at the sides, not resulting the openings in the
horseshoe structures in a bottleneck effect, as expected. For that reason, the geometry
of three streams cell filtering device is not the best approach for cell entrapment.
Simulation III shows an interesting pattern of velocity magnitude between the pillars.
This pattern was investigated, to try to understand why this happens. Since a grid
independence analysis was made and the gaps have all the same size.
This set of simulations can be divided into subsets, according to the order of magnitude
and position of their points with maximum velocity. Straight channels have maximums
of velocity magnitude between pillars and the order of magnitude is 10-2. It is due to the
narrowing of cross sectional area between the pillars. Maximums of enlarged channels
(IV-VI) not always are in the detection zone, and its magnitude is ten times smaller.
This is an advantage of the channels with an enlarged region compared to the straight
ones because cells will be less prone to be dragged. For that reason, single straight
channels do not seem as appealing as the enlarged and were excluded from the set of
channels to test experimentally.
CHAPTER IV – RESULTS AND DISCUSSION
Daniela Cruz Moreira 65
Simulation Contours of velocity magnitude (m/s) Contours of stream function
(kg/s)
I
Magnitude Range: [0; 5.60x10-2]
II
Magnitude Range: [0; 2.42x10-2]
III
Magnitude Range: [0; 1.06x10-2]
IV
Magnitude Range: [0; 4.49x10-3]
V
Magnitude Range: [0; 4.47x10-3]
Integration of microfluidics and FISH for the rapid identification of microorganisms
66 Daniela Cruz Moreira
VI
Magnitude Range: [0; 8.96x10-3]
VII
Magnitude Range: [0; 1.23x10-2]
VIII
Magnitude Range: [0; 4.98x10-2]
X
Magnitude Range: [0; 1.10x10-2]
FIGURE 34| Velocity contours and streamlines in the conceived geometries for microfluidic
devices along the horizontal plane of symmetry as predicted by the CFD simulation
performed
CHAPTER IV – RESULTS AND DISCUSSION
Daniela Cruz Moreira 67
FIGURE 35| Contour plot in a vertical plane in the middle of the horseshoe structure from
Simulation X
Two inlet channels were conceived as backup options, in the case clogging was
observed in single channel devices. Although their maximum velocity magnitudes are
high, they were not immediately excluded. Simulation I does not represent an
appealing velocity profile because the velocity increases drastically in the gap. This can
result in the bent of the barrier, or the fluid can drag the cells through the gap.
However, this geometry is still being submitted to experimental tests because of its low
manufacturing costs.
5. Retention tests in channels with a dam-like structure
The first set of retention tests was performed in microfluidic devices that integrates
channels with a dam-like structure. The criteria for this choice was the relative low cost
producing the masks, since masks produced by xurography technique use adhesive
paper that are so much cheaper than SU-8 molds.
The elevated height of the barrier in opposition to the slight depth of the well, most of
the times, caused the collapse of the structure, closing the channel. The percentage of
successful fabricated channels was quite low.
Fig. 36a shows the results for the retention tests in channels with dam-like structures
using 10µm polystyrene microspheres. It is possible to see that spheres were retained
in the detection zone, but not where was expected. Fig. 36b supports the idea of similar
behavior between yeasts and PS particles. The channel was designed so that the cells
get trapped upstream the region of the dam, and what is observed is that they get stuck
in the well, the gap below the barrier. The fact can have two explanations. First, due to
the technique low accuracy, it is impossible to guarantee that the gap is 8µm high all
Integration of microfluidics and FISH for the rapid identification of microorganisms
68 Daniela Cruz Moreira
over the well, essentially because the ratio between the height of the barrier and the
height of the gap is very high, making possible a collapse of the structure. Second,
analyzing the image, it is perfectly visible that the well zone is not clean, it has some
residuals that are thought to be adhesive residue, from the adhesive paper. It is
impossible to remove the mold that forms the well without leaving some residuals of
adhesive, essentially because the adhesive paper glue is molten after the step of cure
of the PDMS in the oven for 20min at 80ºC. These residues are randomly and where
there are no residuals, the fluid flows. This molten adhesive problem also makes the
fabrication reproducibility impossible.
a. b.
FIGURE 36| Retention test in channels with a dam-like structure
a| PS particle retention b| S. cerevisiae retention.
Original magnification x 100.
2 µm microspheres were injected and entrapped into the microfluidic device, after the
injection of 10 µm microparticles to form a filter. The gaps tend to be smaller, and it is
expected that S. cerevisiae, injected after the microparticles, will be entrapped in an
easier and more efficient way.
When PS particles were used as a filter (Fig. 37), although yeast retention was
significantly higher and flow were effectively more difficult, because the gaps between
microparticles were so small, after some time, the microchannel clogged and it was
impossible to continue the experiments.
CHAPTER IV – RESULTS AND DISCUSSION
Daniela Cruz Moreira 69
FIGURE 37| Usage of PS particles as filter for retention of S. cerevisiae in channels with a
dam-like structure
.Original magnification x 100.
It was concluded that microparticles as a filter were not a viable method, at least using
this method of microfabrication. As such, the microchannels with dam-like structures
were discarded.
6. Geometrical characterization of channels with pillar-based
filter
Molds of channels with pillar-based filter were ordered in two different depths, 50µm
and 30µm. To confirm the channel height, a cross section of the channels fabricated by
soft lithography technique was measured using Leica Application Suite Tools.
The analysis was made for 50 µm high channels, represented in Fig.38 . The geometry
chosen was the enlarged channel with 5x10µm pillars. As can be observed, the
channel has the expected height, but, pillars are bent. Square pillars are very high and
thin, and can collapse due to lack of support. For that reason, 50 µm channels are
excluded from the range of channels to test.
Integration of microfluidics and FISH for the rapid identification of microorganisms
70 Daniela Cruz Moreira
FIGURE 38| Geometrical characterization of a channel with a nominal height of 50µm
Original magnification x 400.
Analyzing 30 µm channels with other geometries, they seem more stable. Fig. 39
shows two cross sections of different geometries and the differences to the 50µm
channels, in terms of stability, are notorious.
a.
b.
FIGURE 39| Geometrical characterization of a channel with a nominal height of 30µm
a| pyramidal structures; b| diamond-shape pillars
Original magnification x 400.
The first criterium to choose a channel to test was related to the microfabrication
outcome. Channels with frailer pillars are more prone to flaws than channels with
robust ones. When it comes to fragile pillars, it was very frequent to miss a pillar
because it was plucked during demolding. Additionally, the pillars arrangement were
distorted due to the bending of the pillars. For instance, Fig. 40 shows a channel with
diamond-shaped pillars and where it is notorious that two pillars are missing, the fifth of
the first barrier and the fifteenth of the third barrier. The volume of the missing pillars is
a preferential escape to yeasts and has strong repercussions on their entrapment.
CHAPTER IV – RESULTS AND DISCUSSION
Daniela Cruz Moreira 71
FIGURE 40| Channel with diamond-like pillars exhibiting barrier imperfections
Original magnification x100.
Fig. 41 shows a channel with 5x15µm rectangular pillars and, even being 30µm high,
the channels are bent. 5x15µm rectangular pillars proved to be very frail, maybe due to
the towering height in opposition to the reduced dimensions of the base.
FIGURE 41| Channel with 15x5 µm rectangular pillars exhibiting barrier imperfections
Original magnification x200.
From the above, diamond shape and 5x15µm rectangular pillars were excluded from
the set of channels to test.
Fig. 42 is a microphotography of a microchannel with pyramidal structures. Some
pillars can lean but since the gap of the barrier is maintained, no relevant imperfections
Integration of microfluidics and FISH for the rapid identification of microorganisms
72 Daniela Cruz Moreira
are observed. The gaps obtained between the biggest pillars in the last line are
considerably uniform.
FIGURE 42| Channel with pyramidal structures
Original magnification x200.
With the resources available, the volumetric measure of the channel has poor
precision and accuracy, but it was relevant to understand that with an inflow rate of
1µL/min during almost 1 min is enough to fill the channel.
The material testing did not show any relevant PDMS expansion, when in contact with
any FISH reagents.
7. Retention tests in single channels with pillar-based filter
Retention tests were performed in enlarged channels with pyramidal structures using
10µm PS spheres. Fig. 43 shows better results. The microspheres were perfectly
entrapped in the narrowing space between pyramidal structures.
CHAPTER IV – RESULTS AND DISCUSSION
Daniela Cruz Moreira 73
FIGURE 43| Microspheres retention test for channels with pyramidal structures
Original magnification x400.
In Fig. 43, an air bubble is observable in the right side. During retention tests, it was
very frequent to have bubbles dragging all the cells and fluid inside the channel. It is a
common issue in microfluidics procedures, and has to be minimized226.
Given the results for this geometry, the work advanced to similar tests using S.
cerevisiae cells. Fig 44 shows the entrapment of yeast cells. These results were not so
satisfactory as those for the microspheres retention test. S. cerevisiae diameter is
about 5-8µm (see Chapter III, section 1), smaller than 10 µm microspheres, meaning
that S. cerevisiae are more prone to pass through the gaps than the microspheres.
Also, yeasts are not bulky as microspheres. We can compare them with microcapsules,
since the cytoplasm are encapsulated by the membrane, which means that they are
deformed by pressure gradients in the gaps. In this retention test, S. cerevisiae prove
to be able to contract and pass throw the gaps, behavior that is not similar to the
experienced by microspheres. However, this characteristics also are advantageous for
the yeast to entry in the gaps perpendicular to the flow, being safeguarded, since it is
an objective of this work to depend only on hydrodynamic traps to retain the cells in the
detection zone.
Integration of microfluidics and FISH for the rapid identification of microorganisms
74 Daniela Cruz Moreira
FIGURE 44| S. cerevisiae retention test for channels with pyramidal structures
Original magnification x400.
Two inlets devices were conceived as a backup plan, in case of clogging in all single
channel devices tested. As clogging did not appear as a problem, and after the
entrapment, the fluid continues to flow without visible limitations, two inlet devices were
excluded from the set of channels tested. This does not mean they can be used in
different assays in future work, but, for this work, a reasonable solution was achieved.
8. PNA-FISH methodology within the microfluidic channel
The implementation of the full PNA-FISH methodology offers great difficulties,
particularly due to the formations of air bubbles during all the process. Several attempts
failed as a result of bubble formation that dragged all the cells or completely blocked
the channel. Even by the end of this work, bubble formation was impossible to control
at a full extent, which compromises reproducibility. Fig. 45 illustrates the bubble
formation during the retention step. These frequent events can result in the rupture of
the channel and in the abortion of the assays. These problems may be due to the
insertion of bubbles already present in the tube connected to the microchip or, when
the pressure inside the channel is lower than the atmospheric pressure, the air can
enter by the connection between the needle of the tube and the chip.
CHAPTER IV – RESULTS AND DISCUSSION
Daniela Cruz Moreira 75
FIGURE 45| Channel with pyramidal structures
Original magnification x100.
Some changes were adopted, trying to solve this problem. To minimize the number of
times that the chip was disconnected and reconnected to the pumping system, 3µL of
each reagent were inserted in the tube sequentially, and each time that the sample had
to incubate, the syringe was placed inside the incubator together with the microchip.
This procedure was effective during the retention step, but, when the system
experienced a temperature increase during the hybridization step in the incubator,
bubbles were created inside the channel and the cells were dragged. This may occur
due to fluid evaporation in low pressure points. The solution was to immerge the chip in
water previously heated inside the incubator. This minimizes bubble formation but do
not solve it completely.
After controlling to some extent the problem related to air-bubbles, the entire PNA-
FISH methodology was performed within the microfluidic channel. Figure 46 shows the
results for this assay.
a. b.
FIGURE 46| Positive for PNA-FISH methodology within the microfluidic channel.
a| DAPI staining (N2.1 Filter) b| Positive (Dead/Live Filter). Images were obtained with equal exposure times. Original magnification x 100. Scale bar 20µm.
Integration of microfluidics and FISH for the rapid identification of microorganisms
76 Daniela Cruz Moreira
The DAPI staining (Fig. 46a) confirms the presence of cells after all the FISH
procedure. However, when observing the sample with the filter sensitive to the probe
(Fig. 46b), it is notorious that the hybridization only occurs in a few cells in the central
section of the channel. Due to the height of the upper block of the device, the higher
magnification that can be used is 10x (lenses magnifications). It is possible that the
hybridization solution is not passing through the traps very well or the flow is increased
in the central part and this zone is more exposed to the probe. This would explain the
higher fluorescence intensity of the cells trapped in the central part of the
microchannel.
Successful implementation of FISH procedures are reported by Sieben227,228 and
Vedarethinam229, and the work of Zhang126 that uses pillar-based filters to entrap cells
and successfully hybridizes RNA with quantum-dots shows that this procedure is
possible. Despite some limitations, the fact that a cluster of cells fluorescing red is
identified within the microchip works as a proof of concept. A trustable geometry was
developed but the PNA-FISH protocol within the microchip has however to be strongly
optimized.
Chapter V – Conclusions and Future Work
Integration of microfluidics and FISH for the rapid identification of microorganisms
78 Daniela Cruz Moreira
Conclusions
This work describes the development of a novel microfluidic device for the retention
of S. cerevisiae and subsequent identification by PNA-FISH. Unlike other devices, this
system is much less susceptible to clogging, as observed in Meireles177, and is much
simpler to prepare than the other systems128, while retaining a significant amount of S.
cerevisiae cells. Furthermore, it was one of the first times that PNA has been used
successfully in microchannels using simple hydrodynamic methods.
Strategies for successful retention of yeast cells are reported by Li et al230, using U-
shape barriers, which, according to Meireles177, is a strategy more difficult to handle,
since it is susceptible to fluid recess. Zhang et al231also proposes the use of dam-like
structures to retain yeast cells in a study of yeast aging. Some pillar-based geometries
for cell retention was also described232 but there are no evidences of geometries similar
to the ones described in this work. The chosen geometry allows cells to be entrapped
not only in the narrowing space between pyramidal structures but also in the gaps
perpendicular to the flow, as suggested by numerical simulations.
The PNA-FISH proved to be successful for S. cerevisiae identification, in spite of a
better signal being expected. Some new probe design can be performed in an attempt
to achieve a better signal. Regarding the microfabrication, no significant leakages were
observed.
Forrest et al.233, studied the impact of a diagnosis using PNA-FISH on the expenses
related to antifungal treatment. This study proves that although laboratory costs for
doing PNA-FISH test exceed those for blood culture, savings through a decrease in
antifungal drug costs, particularly caspofungin were obtained and can result in
substantial savings for hospitals. This supports the significance of rapid identification of
the microorganism. An appropriated and effective treatment drastically reduces
mortality. Unfortunately, standard microbiology laboratory methods are too slow to
support rapid interventions, requiring approximately 24 hours to detect the presence of
hematopathogens and at least 3 days to confirm the selection of appropriate
antimicrobial therapy due to low density of microbes and the need of an enrichment
step. Empiric treatment is common in these situations and, if it is not effective, may
contribute to the evolution of drug-resistant microbes and increase treatment costs234.
As proposed initially, this work validates the concept proposed. Reproducible
retention of S. cerevisiae was ensured and its detection within the microchannel by
fluorescence microscopy was successful and unequivocally achieved.
CHAPTER V – CONCLUSIONS AND FUTURE WORK
Future work
The formation of air bubble was the main issue during the development of the device.
The process of cells retention could be optimized, if it was possible to perform PNA-
FISH under continuous flow, avoiding reflux. It would be necessary a heated plate with
high accuracy, since the hybridization process is extremely sensitive. Also, in an ideal
situation of continuous flow, the outlet of the channel could be elevated, increasing the
pressure inside the channels and preventing the formation of air bubbles. The problem
of air bubbles is compounded by the fact that the FISH procedure needs to hybridize at
high temperatures, which leads to the evaporation of organic reagents in low pressure
points226. Also, it is thought that if the time of the procedure could be reduced, and
Zhang126 proved it possible, organic reagents would be less prone to evaporate. This
definitely demands a PNA-FISH optimization to the new conditions. The design of a
bubble entrapment device, suggested by Zheng235, is very appealing to solve the
bubble problem. The surface may become hydrophilic if it is exposed to oxygen
plasma, solving the bubble problem147.
As a final remark, to determinate the device’s limit of detection, a flow rate and yeast
concentration optimization should also be performed.
80 Daniela Cruz Moreira
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Appendix
I – YEPD-agar and YEPD medium recipe
a) YEPD-agar medium recipe
Reagent Concentration Manufacturer
Yeast Extract 1% (w/v) Merck, Germany Peptone 2% (w/v) Liofilchem, Italy Dextrose 2% (w/v) Merck, Germany Agar 2% (w/v) Merck, Germany Water - - Sterilize by autoclaving.
b) YEPD medium recipe
Reagent Concentration Manufacturer
Yeast Extract 1% (w/v) Merck, Germany Peptone 2% (w/v) Liofilchem, Italy Dextrose 2% (w/v) Merck, Germany Water - - Sterilize by autoclaving.
II – Composition of FISH Solutions
a) Standard hybridization solution (pH 7.5)
Reagent Concentration Manufacturer
Sodium Chloride 10 mM Sigma-Aldrich, USA Dextran Sulfate 10% (w/v) Fisher Scientific, UK Formamide 30% (v/v) Acros Organics, UK Sodium pyrophosphate 0.1% (w/v) Acros Organics, UK Polyvinylpyrrolidone 0.2% (w/v) Sigma-Aldrich, USA Ficol 0.2% (w/v) Fisher Bioreagents, UK Disodium EDTA 5 mM Panreac Quimica, Spain Triton X-100 0.1% (v/v) Panreac Quimica, Spain Tris-HCL 50mM Fisher Scientific, UK
94 Daniela Cruz Moreira
b) Simplified hybridization solution (30% formamide) (pH 7.5)
Reagent Concentration Manufacturer
Tris-HCL 50mM Fisher Scientific, UK Dextran Sulfate 10% (w/v) Fisher Scientific, UK Formamide 30% (v/v) Acros Organics, UK Triton X-100 0.1% (v/v) Panreac Quimica, Spain
c) Simplified hybridization solution (0% formamide) (pH 7.5)
Reagent Concentration Manufacturer
Tris-HCL 50mM Fisher Scientific, UK Dextran Sulfate 10% (w/v) Fisher Scientific, UK Triton X-100 0.1% (v/v) Panreac Quimica, Spain
II – Composition of washing solutions
a) Standard hybridization solution (pH 10)
Reagent Concentration Manufacturer
Tris Base 5 mM Fisher Scientific, UK Sodium Chloride 15 mM Sigma-Aldrich, USA Triton X-100 1% (v/v) Panreac Quimica, Spain