r. almog biosensors ביו-חיישנים – עקרונות ויישומים מרפואה...
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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