jortec workshop : biossensores fct-unl 21/02/2013 · jortec - workshop em biossensores 21/02/2013...

Abel González Oliva

Laboratório Diagnóstico Biomolecular

ITQB-UNL / IBET

JORTEC – WORKSHOP : BIOSSENSORES

FCT-UNL 21/02/2013

• Imunoensaios e imunosensores

• Testes rápidos

• Sensores enzimáticos

• Sensores baseados em células

• Sensores de DNA (DNA-chips)

• Instrumentos para a saúde humana

• Instrumentos para aplicação em

biotecnologia

The affinity and specificity of an antigen - antibody interaction to form a

stable complex, are the basis properties for an immunoassay

antibody

antigens

support

label

Immunoassay: A laboratory technique that makes use of

the binding between an antigen and its homologous

antibody in order to identify and quantify the specific

antigen or antibody in a sample.

Imunoensaio

JORTEC - WORKSHOP EM BIOSSENSORES 21/02/2013 Abel G. Oliva Lab. Diagnostico Biomolecular/ITQB-UNL

An assay that quantifies antigen or antibody by immunochemical means.

An antigen can be a relatively simple substance such as a drug, or a complex one

such as a protein or a virus.

• Immunofluorescence

• Immunonephelometry

• Radioimmunoassay

Immunofluorescence

human skin prepared for direct immunofluorescence

Imunoensaio


Antigens from the sample are attached to a surface of a plate (e.g. 96 wells). Then, a

specific antibody is applied over the surface so it can bind to the antigen. This antibody is

linked to an enzyme, and, in the final step, a substance (enzyme’s substrate) is added.

The subsequent reaction produces a detectable signal, most commonly a color change,

quantified by the ELISA reader.

ELISA (Enzyme-linked immunosorbent assay)

ELISA, Enzyme Linked Immuno Assay is a biochemical technique used

mainly to detect the presence of an antibody or an antigen in a sample.

The ELISA has been used as a diagnostic tool in clinical medicine, plant

pathology, environmental monitoring, as well as a quality control check

in various industries.

ELISA reader. 96 wells polystyrene microtiter plate

Plate well

E E E E

S S S S P

P P P


Immunosensors transduce antigen-antibody interactions directly into physical

signals. The design and preparation of an optimum interface between the

biocomponents and the detector material is the key part of sensor development.

Immunosensors can be applied in diagnostic of diseases in clinical health of

humans and animals; in the detection of drugs and environmental pollutants

and in the food quality control, allowing applications that need a rapid

detection with high sensitivity and specificity.

Imunosensor

antibody

antigens

support

label blocking agent


• Antibodies are proteins

• Synthesized by plasma cells, i.e. mature B-lymphocytes

• Response to immunogen

• Immunoglobulins (G, A, M, D, and E)

• Immunoglobulin G (IgG), also known as -globulin, 150 kd

• Principal antibody in serum.

Antibodies


Immunosensors


DNA-chip

A DNA microarray is used in molecular biology and consists of an arrayed series of thousands

of microscopic spots of DNA oligonucleotides, each containing picomoles (10−12 moles) of a

specific DNA sequence, known as probes (or reporters).

Example of an approximately 37,500 probe spotted oligo

DNA-chip


DNA-chip


High density, oligonucleotide-based DNA arrays.

DNA-chip


Sensores baseados

em células

Sensores baseados em células

Cell-based biosensors (CBBs) have emerged as powerful functional tools for

the rapid detection of hazards and threats associated with food, agriculture,

environment and biosecurity.


Relative advantages of cell-based sensors


Relative disadvantages of cell-based sensors

• cells to respond to complex mixtures

• Receptors, channels, and enzymes active

• Alternatives to animal testing

• Better tolerance for solute and pH variations

• Longer life than enzyme biosensors

• Need optimized environment for long periods to be effective

• Need physical and chemical controlled conditions

• Slower reaction / measurement time than biosensors

• Longer Recovery time


hazard-induced

physiological responses

receptor–ligand interactions, signal transduction,

gene expression, membrane damage, apoptosis

and oncosis of living sensing organisms

toxicity

array-formatted impedance biosensor

(a) The biosensing chip with a microelectrode array and cell-culture

wells. (b) Cells on the electrode, (left) before and (right) after attachment.

Whole cell based electrical impedance sensing approach for a rapid nanotoxicity assay. Evangelia

Hondroulis et al 2010 Nanotechnology 21 315103 doi: 10.1088/0957-4484/21/31/315103



Field Effect Transistor (FET)

Peter Fromherz. Solid-State Electronics. Volume 52, Issue 9, September 2008, Pages 1364-1373

Papers Selected from the 37th European Solid-State Device Research Conference - ESSDERC’07



Real-time measurements of the shift in resonance frequency and energy dissipation due to

changes in mass and viscoelastic properties give quantitative information about the state

of the cell adhesion process

(D. Johannsmann, Viscoelastic analysis of organic thin films on quartz resonators - 2000)

Quarz Crystal Microbalance (QCM)

J.Elsom, M.I.Lethem, G.D. Rees and A. Christy Hunter. Novel quartz crystal microbalance

based biosensor for detection of oral epithelial cell–microparticle interaction in real-time

Biosensors and Bioelectronics 23(8)1259-1265, 2007 DOI:10.1016/j.bios.2007.11.020



Light Addressable Potentiometric Sensor (LAPS)

Myocytes

or neurons

pH-sensitive LAPS that uses LEDs in combination with (semiconducting) silicon and pH-sensitive Ta2O5.



cell- and tissue-based sensors


Sensores enzimáticos



substrate product

Enzyme

Specificity

Selectivity

Change in proton conc.

Release or uptake of gas

Light emission, absortion or

reflectance

Heat emission…

cofactor

Reaction catalized

by the enzyme

Transducer


two possibilities to measure the glucose concentration: oxygen consumption or the

hydrogen-peroxide production. As a third option it would be also possible to measure the

temperature increase due to heat released by the two reactions.

The presence of the second enzyme (catalase), although not really essential for the assay,

enhances the first reaction. This happens due to the decomposition of the hydrogen-

peroxide.



Enzymes used in different biosensors based in enzymatic inhibition



Testes rápidos


Testes rápidos

In vitro diagnostic test strips based on the principles of immunochromatography exist

for a wide array of target analytes. The first tests were made for the detection of

human chorionic gonadotropin (hCG).

Today, there are commercially available tests for monitoring ovulation, detecting

infectious disease organisms, analyzing drugs of abuse, and measuring other analytes

important to human physiology. Products have also been introduced for veterinary

testing, agricultural applications, environmental testing, and product quality evaluation.


Most immunochromatographic test strips are modeled after existing immunoassay

formats. Thus, test strips for hCG are typically sandwich assays; and drugs of

abuse are analyzed by competitive or inhibition assays. In serum assays,

antibodies are detected as an indicator of various disease states.

Many variations are possible, but they all have in common the formation of a

complex between a detector particle that is free in the sample stream and a capture

reagent that is bound to the membrane at the test line.

Testes rápidos


Testes rápidos


mLabsTM Cardiac Panel

based on immunoassay

technique, is a point-of-care

(POC) diagnostic chip to

diagnose acute myocardial

infarction. It can simultaneously

detect three myocardial markers

including creatine kinase-MB,

myoglobin, and Troponin

General Description:

Visitect Syphilis test, kit of 25 tests.

Technical Specifications:

Technology: Immunochromatographic

Antigen type: TpN17, TpN47

Solid phase: Membrane strip in cassette

Specimen type: Whole blood/plasma/serum

Volume of sample: 50 µl whole blood or 25 µl serum/plasma

Results available for reading: 15 minutes

Shelf life: 24 months at +4° to +30°C.

Temperature requirements for storage: +4° to +30°C –

DO NOT FREEZE.

Testes rápidos


Instrumentos para

a saúde humana


•optical spectroscopy

•mass spectrometry

•electrophoresis and other

separation techniques

•microscopy

•lasers and optics

•X-ray tools and techniques

•nuclear magnetic resonance

spectroscopy

Biomedical Applications

•bioreactors and other forms of cell

culture

•proteomics

•genomic sequencing

•functional genomics

•comparative genomics

•microarrays

•human sequence variation (e.g.

genotyping).

•biosensors (POC)

Ceramic Foams

Instrumentos para a saúde humana


epidermal electronics

•Lysine

•Acetylcholine

•Uric acid

•Urea

•Glutamate

•Ethanol

•DNA diagnostics

Disease diagnostic – Clinical analysis - Intensive care monitorization

•Viral diseases

•Bacterial diseases

•Genetic diseases

•Cancer

•Diabetes glucose

•Lactate

•Cholesterol

Clinical Diagnosis



Eestimated 554 million adults in 2007



Oxidase enzymes such as

Gox act by oxidising their

substrates, accepting

electrons in the process

and thereby changing to

an inactivated reduced

state. These enzymes are

normally returned to their

active oxidised state by

transferring these

electrons to molecular

oxygen, resulting in the

production of hydrogen

peroxide (H2O2)

Many inorganic redox

couples (mediators) and

organic dyes have been

successfully utilised as

electron sinks for the

glucose oxidase catalysed

oxidation of glucose.

These redox couples, or

mediators, are able to

shuttle electrons between

the redox centre of the

enzyme and the electrode.

J.D. Newman, A.P.F. Turner / Biosensors and Bioelectronics 20 (2005) 2435–2453



Subcutaneously implantable glucose sensor

Miniature needle-type

amperometric glucose sensors

The Guardian RT system, recently approved by the U.S. Food and Drug Administration for

patients over the age of 18 with Type 1 and Type 2 diabetes, is a doctor-prescribed, patient-

owned continuous glucose monitoring system that displays an updated REAL-Time glucose

value every five minutes and alerts patients when glucose levels become too high or too low,

this information helps patients take action before they experience a problem.



Cocaine

Meth-amphetamines

Ecstasy

Opiates

THC (cannabis)

TNT explosives

Tetryl

DNT

RDX

Nitroglycerin

PETN

The commercial BIOSENS® detects:

BIOSENS® 300

for analysis of narcotics

and explosives

Immunosensors


High Sensitivity detection using a zero reflectance

condition on a coated silicon wafer. A) Thin film

schematic and modeling variables. B) Simulated

reflectance of a silicon dioxide layer on Silicon in an

air ambient for 633 nm laser light – the zero

condition exists for a 1419 Å thick film at 70.7°

incidence. C) biodetection methodology – molecular

binding causes a strong reflactance increase

Experimental AIR setup and reference images. A) The

low-complexity AIR imaging reader. B) Reference

image of a 264 spot photolithographically patterned

array. C) Imaging reference chips are fabricated using

photolithography and highly accurate etching to form

arrays of spots slightly thicker than the remaining film.

Images of arrays with spots heights of 700 pm and 10

pm are shown. D) Relative sizes of human serum

albumin (HSA), FITC dye, and water, compared to a 10

pm step height. (i.e., smal fractions of a mollecular

monolayer can be imaged).

Adarza BioSystems, Inc Rapid and label-free biological assay platform for

measuring clinical and point-of-care (POC) samples.



microfluidics, magnetic particles and gold nanoparticles encoded with

antibodies and DNA (Virigene )





Processor SP

Reader

Nanosphere

VERIGENE

Encapsulated Carbon Nanotubes for Implantable

Biological Sensors to Monitor Blood Glucose Levels

Near-infrared nanoscale sensor that detects

glucose. The sensor could be inserted into

tissue, excited with a laser pointer, and

provide real-time, continuous monitoring of

blood glucose level. The hydrogen peroxide

changes the optical properties of the

nanotubes so that the more hydrogen

peroxide present, the more the tubes will

fluoresce when exposed to near-infrared laser

light.

University of Illinois at Urbana-Champaign and U. Stanford (CA)

Glucose oxidase is sensitive to glucose between 0-10 mmol but, in combination with multi-walled

carbon nanotubes, its detection sensitivity extends to 20 mmol. This range covers the

physiologically relevant values for diabetes.



New sensors are based on single-walled carbon nanotubes: cylindrical

molecules whose sides are formed from a lattice of carbon atoms This

glass capillary tube, shown here on a fingertip, has been loaded with

glucose-sensitive nanotubes. The capillary tube keeps the nanotubes

confined, but has porous walls so that glucose molecules can get to

them.

The idea is to exploit the nanotubes’ ability to fluoresce, or glow, when illuminated by certain

wavelengths of infrared light—“a region of the spectrum where human tissue and biological fluids

are particularly transparent,” (Michael S. Strano, University of Illinois in Urbana-Champaign )



World market

for glucose

biosensors and

test strips 11,5

billions in 2012



Diabetes

world prevalence of diabetes among adults

(20–79 years of age) would be 6.4%,

affecting 285 million adults in 2010

and estimated 439 million adults by 2030



Instrumentos para aplicação

em biotecnologia

Fermenters

Production and Quality control of final Products

Monitoring and Process control

•Biosensor for Industrial Pharmaceutical Research Applications

•Protein Chip-based Biosensor

Optical Biosensors for Protein Studies

Membrane Biosensor

Lab-on-a-chip based Biosensor

Special Purpose Biochip-based Biosensors

Instrumentos para aplicação em biotecnologia


Food Industry Biosensor Applications

Food Safety Monitoring

Rapid Food Diagnostics and Biosensors

Biosensor Technologies that Apply to Food

Single Function-Type Enzyme Sensor

Alcohol Sensor

Glucose Sensor

Lactic Acid Sensor

Peptide Sensor

Multi-Function-Type Enzyme Sensor

Fish & Shellfish's Freshness Sensor

Smell or Taste Ingredient Sensor

Monitoriztion of food and drinks

Food Safety Threats

Botulism

Salmonellosis

Escherichia coli O157:H7

Shigella dysenteriae Type 1

Typhi (typhoid fever)

Vibrio cholerae (cholera)


Attana AB

Quality control of final Products

Consumption of hormone-treated beef may be causing girls to reach

puberty earlier than they used to and making them more susceptible to

breast cancer, say researchers attending a world conference on breast

cancer.

Pediatricians say the onset of menstruation has steadily decreased in

recent decades. The average age for a first period is now 12½, up from

age 14 in 1900.

Hormones are used by cattle farmers in Canada and the United States

to increase the weight of cattle prior to slaughter. They are currently

the focus of a major trade dispute between North American and the

European Union.

Antibiotics used in chicken: After a European Union ruling on

July 1, only two chemicals - flavomycin and avilamycin - are

used to speed up growth. Until then several antibiotics were

routinely used for more than 20 years on poultry and pigs.

Antibiotics are used in pigs to prevent the spread of disease

in often cramped conditions. Their defenders argue that

chickens grow 5% faster, and the 3-4% saved on feed costs

is vital in keeping businesses viable.


Medical

BioDefense

Food &

Beverage Environment

Bio/Pharma

Research Biosensor

Market

Biosensor market


jortec workshop : biossensores fct-unl 21/02/2013 · jortec - workshop em biossensores 21/02/2013...

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