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BiosensorsBiosensorsביו-חיישנים – עקרונות ביו-חיישנים – עקרונות

ויישומים מרפואה ביתית ועד ויישומים מרפואה ביתית ועד לניטור סביבתילניטור סביבתי

Dr. Ronen AlmogDr. Ronen Almog

מכון טכנולוגי חולוןמכון טכנולוגי חולון

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•Biosensors •Detection principles:

•Electrochemical•Optical•Mechanical

•Lab on a chip/BioMEMS•Examples:

•Diabetes - glucose monitoring•Water toxicity detection

OutlineOutline

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Elements of a biosensorElements of a biosensor

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An integrated device consisting of •a biological recognition element and •a transducer

capable of detecting specific biological/chemical compound and converting it into an electronic signal.

Biosensor definition

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Biosensors applications and importanceBiosensors applications and importance

•Medical/health monitors

•Homeland security

•Pharmaceutical industry

•Food industry

•Simplicity

•Selectivity

•Sensitivity

•Miniaturization

•Rapid BiosensorsFeatures

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•Enzymes and their substrates•Antibodies and their antigens•Nucleic acids and their complementary sequences•Whole cell

Biological recognition elementsBiological recognition elements

A - TG - C

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On Size and Scale

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EnzymesEnzymes• Large protein molecules. • Catalyze chemical reactions. • Used as tools to perform various biochemical reactions in the cell.

Participates actively in the transformation of chemical A (the substrate) to chemical B (the product) but remains unchanged at the end of the reaction.

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Immunosensors: Antibody – Antigen recognitionImmunosensors: Antibody – Antigen recognition

Antibody - proteins, produced by the immune system of higher animals in response to the entry of “foreign” materials into the body, eg. viruses, bacteria

Bind tightly to the foreign material (the antigen) that provoked the response and mark it for attack by other elements of the immune system.

Antibodies are also very specific- they need to be, in recognizing and binding to the foreign substance only and not to materials native to the organism.

This will change some physicochemical parameter (usually mass or an optical parameter) of the environment at the transducer surface of the sensor and that change is subsequently detected.

If an antigen is present in that medium, it will be bound by the antibody to form a larger, antigen-antibody complex.

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Antibody structure

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1. Electrochemical-Amperometric : Measures currents generated when electrons are exchanged between a biological system (in solution) and an electrode in a constant potential.

4. Conductometric/Impedimetric : detect changes in conductivity/impedance between two electrodes.

The transducerThe transducer

Chemical change Physical change

2 .Optical detection - refractive index change or fluorescence

3 .Mechanical detection

The most common are:

The key part of a biosensor is the transducer which makes use of a physical change accompanying the reaction.

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Transducers - sensing methods in biosensorsTransducers - sensing methods in biosensors

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MEMS - MicroElectroMechanichal sensorsMEMS - MicroElectroMechanichal sensors

Use: Bioassay of prostate-specific antigen (PSA)

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Optical sensors - Biacore exampleOptical sensors - Biacore example

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Reasons for MiniaturizationReasons for Miniaturization

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Biochips Microelectronic-inspired devices that are used for delivery, processing, analysis, or detection of biological molecules and species.These devices are used to detect cells, microorganisms, viruses, proteins, DNA and related nucleic acids, and small molecules of biochemicalimportance and interest.

BioMEMS Biomedical or biological applications of MEMS (micro electro mechanical systems)

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Lab-on-a-chipLab-on-a-chip

Burns et al. Science 282, 484, 1998

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BioChip/BioMEMS Materials

•Silicon

• Glass, Quartz

• Polymers– Poly (dimethylsiloxane) (PDMS)– Poly (methyl methacrylate) (PMMA)– Teflon, etc.

Considerations • Biocompatibility, ideal for biomedical devices• Transparent within the visible spectrum• Rapid fabrication• Photo-definable• Chemically modifiable

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Biochip - microfluidics

Key Attributes of Biochips1. Small length scale2. Small thermal mass3. Laminar flow4. High surface-to-volume ratio

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The Oxygen electrode

Clark’s Glucose electrode

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Biosensors History

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Technology evolution

6 analyses from a drop of blood in about one minute.

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Diabetes - Glucose Biosensor

• The user carries a wallet sized case that contains the testing equipment

• A lancet pierces the skin on the finger

• The user places this blood sample on a test strip and inserts it into the reader

• Electrochemical detection

http://www.diabetesuffolk.com/Managing%20Diabetes/Meters/LifeScan%20Ultrasmart.asp

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Alcohol Test – Drager Alcotest 7110Alcohol Test – Drager Alcotest 7110 הינשוףהינשוף

/www.draeger-safety.com

The Alcotest 7110 Standard is a highly developed measuring instrument for precise determination of breath alcohol concentration.

Two different and independent measuring systems:

• Infrared spectroscopy – λ=9.5µm • Electrochemical measurement

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Example - insect MEMS hybridExample - insect MEMS hybrid

The University of California, Berkeley

IEEE MEMS, January 2009

A radio-controlled beetle

1.3g electronic module

Max weight: 3g

6 electrodes affixed to the brain and muscles

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Example: Toxichip

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Water toxicity detection - motivation

1. Homeland security

THE THREAT:

Intentional poisoning of a drinking water source

2. Enviromental pollution

THE NEED: A rapid early toxicity warning device

3. Pharmaceutical screening applications

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Toxicity detection systems

Standard toxicity bioassays, mostly designed for environmental purposes, are unsuitable for our needs: size, response time.

Toxicity bioassays

The only question need to be asked is “Is the sample toxic?”

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The goal

To develop a portable system that

can detect the presence of unknown acute toxicity chemicals in drinking water within 20 minutes.

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Whole-cell biosensor:The biological material is an intact, living, functioning cell.

Toxichip whole cell biosensors:•Bacterial cells •Genetically modified•Bioluminescent •Tailored to respond to different cell stress factors

Three elements in the solution

3. Analyzer: a small mobile instrument into which the chip will be inserted, and which will provide the reading.

1. Reporter cells: live cells “tailored” to detect toxicity. We use Escherichia coli (E. coli) bacteria as a whole cell sensor.

2. Biochips: disposable, credit card size, containing the cells.

E. Coli bacteria

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Biochip arrayE. Coli bacteria

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Bioluminescence

The emission of light by a living organism as the result of a chemical reaction. Chemical energy is converted to light energy.

Marine organisms Insects

Fungee

Bacteria

Emission spectrum: Visible - blue-green (490-500 nm)

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The one that drives or catalyzes the reaction is called a "luciferase."

Each organism has its own luciferin and luciferase compounds.

Bioluminescence

At least two chemicals are required:

The one which produces the light is generically called a "luciferin“.

Luciferin(substrate)

Luciferase(enzyme)

+Product:Oxyluciferin + light+ O2

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Bacteria engineering for toxins detection

Two types of biochemical response to toxins :

Type I

Type II

Normally doesn’t emit light.

Toxin exposure induces light emission.

Sensitive to low concentration of toxins with a dose-dependent signal.

Constitutive test

Normally emits light.

In the presence of a toxin, the signal intensity decreases.

Respond to high concentration of toxins

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Sensing element: A promoter of a gene involved in the response to the desired target.

Light

gene promoter

Sensing element

luxCDABE

Reporting element

Light

gene promoter

Sensing element

gene promoter

Sensing element

luxCDABE

Reporting element

luxCDABE

Reporting element

Type I - bacteria engineering for toxins detection

The fusion of two genetic elements inside a host E. coli bacteria:

Reporting element: A Bioluminescent gene. – generates the Luciferin and Luciferase when experssed

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Species LD50

1 Sodium arsenite Heavy metal, carcinogen. Use as :Herbicide, Insecticide, Rodenticide, Fungicide.

Rat (oral) 41 mg/kg,

11.2 ppmמשמש

בחקלאות

2 Cadmium chloride Heavy metal, Used as stabilizer and pigment in plastics Rat (oral) 88 mg/kg

3 Parathion Cholinesterase Inhibitor.Very potent insecticide and acaricide used on cotton, rice and fruit trees

Rat (oral) 3.6-13 mg/kg

4 Paraquat Quaternary ammonium herbicide (non selective). superoxide generating compoundRat (oral) 100 mg/kgקוטל

עשבים

5 Botulinum Toxin A It is the most poisonous naturally occurring substance in the world.A muscle-nerve paralyzing bacterial toxin, used in minute doses both to treat painful muscle spasms, and as a cosmetic treatment.

Rat 1-0.03 ng/kg טרור ביולוגי

6Clostridium difficile Toxin A

An enterotoxin. Responsible with toxin B for the Pseudomembranous colitis (diarrhea and inflammation)- severe gastrointestinal disease.

Mice 500 ng/kg טרור ביולוגי

Acute ToxicityChemical Description

Toxins listToxins list

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Bacteria response to mitomycin C - chemotherapeutic agent

Example - cell bioluminescence responseExample - cell bioluminescence response

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Effect of toxin concentration:

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0

5000

10000

15000

20000

25000

30000

0 20 40 60 80 100 120 140 160 180 200 220Time (min.)

Lum

ines

cenc

e (R

LU

)

Bacteria + 10 ppm NA

Bacteria + 5 ppm NA

Bacteria + 0 ppm NA

No Bacteria + 10 ppm NA

Measurement of the bacterial bioluminescence response to different NA concentrations under static condition (no flow). Here the bacteria were suspended in LB.

•We use Nalidixic Acid (NA) as the model toxin

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PDMSBiochip

Elastomer, Simple, fast, modular,cheap, reproducible, disposable

Microfluidics interface system for the PDMS biochip

InletOutlet

Glass cover PMMA

base

10mm

PDMS

PMMA

Glass

Inlet OutletSPADsStainless steel

Bacteria immobilized in Agar

SinglePhotonAvalanchephotoDiode

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Sample

Positive

Negative

Constitutive

Bacteria wells4 channelsSample

Positive

Negative

Constitutive

Bacteria wells4 channels

The biochip layout:

4 main channels: sample, positive, negative and constitutive.

The constitutive channel consists of “Normally On” bacteria.

The sample, positive and negative channels consist of four different bacteria strains immobilized in agar.

4 strains

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Bacteria panel

2 toxins:Nitrogen mustard, Potassium cyanide

2 strains (promoters):nhoA, grpE

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Acknoledgments

• Prof. Yosi Shacham• Ramiz Daniel• Klimentiy Levkov• Matan Peer • Yaniv Chen• Ragini Raj Singh • Sefi Vernick• Amit Ron• Mordechai Aharonson• Tsvi Shmilovich• Arthur Rabner

HUJI - Shimshon Belkin’s group:• Sharon Yagur-kroll

• Tal Elad

• Sahar Melamed

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Biochip arrayE. Coli bacteria

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Optical sensors - Biacore exampleOptical sensors - Biacore example