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BIOSENSORS AND BIOCONJUGATION

Boaz Vilozny October 4, 2011

BME 140 Bioengineering

What is a sensor?

A sensor is any device that converts a measurement into a signal

What is a biosensor?

A sensor in which the receptor is a biomolecule

A sensor in which the analyte is a biomolecule

A sensor in which the signal is produced by

biological interactions

Optical Biosensors

Optical Biosensors

Indicators and immobilized sensors

Indicators and immobilized sensors homepages.wmich.edu/~rossbach/bios312
Indicators and immobilized sensors homepages.wmich.edu/~rossbach/bios312

Optical biosensor: DNA microarray

What is the receptor?

What is the analyte?

How is the signal transduced?

Is this a “true sensor”?

 How is the signal transduced?  Is this a “true sensor”? http://www.wormbook.org/chapters/www_germlinegenomics

http://www.wormbook.org/chapters/www_germlinegenomics

Fluorescent biosensor: Cameleon

What is the receptor?

What is the reporter?

How is the signal transduced?

How is sensor delivered to cells?

How is the signal transduced?  How is sensor delivered to cells? http://probes.invitrogen.com/media/pis/mp36207.pdf

http://probes.invitrogen.com/media/pis/mp36207.pdf

How is the signal transduced?  How is sensor delivered to cells? http://probes.invitrogen.com/media/pis/mp36207.pdf

Electrical Biosensors

What is the electrical signal?

Potentiometry

Amperometry

Voltammetry

Conductance, etc.

No optical components

Miniaturizable

Sensitivity increases with nanofabrication

Receptors for electrical biosensors

Enzymes

Antibodies

DNA/RNA

Receptors for electrical biosensors  Enzymes  Antibodies  DNA/RNA

Electrical biosensor design

Sandwich Immunoassay

Electrical biosensor design Sandwich Immunoassay Urban, 2009 Measurement Science and Technology

Urban, 2009 Measurement Science and Technology

Bioconjugation and biosensor fabrication

How is bioconjugation used?

Requirements of bioconjugation

Selective

Stable

Physiological conditions

Non-chemical affinity methods

Nucleic acid hybridization

Protein/substrate interactions

Bioconjugation: chemical crosslinking

Why a chemical bond? Chemistry for special surfaces

Metal oxides

Gold

Organic polymers

Charged surfaces

Optical immunoassay on a chip

Surface treatment

Amino-Silanization

Crosslinker Capture antibody

Protein-blocking

Analyte (IgG) Labeled antibody

Optical measurement

• Analyte (IgG) • Labeled antibody • Optical measurement Raj, J. et al , Biosens. Bioelectron.

Raj, J. et al, Biosens. Bioelectron. 2009

Voltammetric aptamer-biosensor

Gold surface on glassy

carbon electrode

Doxorubicin as indicator:

redox active, dsRNA

intercalator

Why not a sandwich assay?

How to achieve selectivity?

How is signal generated?

assay? • How to achieve selectivity? • How is signal generated? Zhao, G-C. et al ,

Zhao, G-C. et al, Electrochem. Commun. 2010

Putting it all together: the biosensor as a self-contained diagnostics lab

together: the biosensor as a self-contained diagnostics lab http://www.technologyreview.com/biomedicine/38201/ Chin,

http://www.technologyreview.com/biomedicine/38201/

diagnostics lab http://www.technologyreview.com/biomedicine/38201/ Chin, C.D.; Sia, S.K. et al . Nature Medicine, 2011
diagnostics lab http://www.technologyreview.com/biomedicine/38201/ Chin, C.D.; Sia, S.K. et al . Nature Medicine, 2011

Chin, C.D.; Sia, S.K. et al. Nature Medicine, 2011

The problem: low-cost diagnostics

Resource-limited settings

Lack of medical facilities

Lack of doctors

Lack of money

Health epidemic

Diagnostics can make a difference

HIV, siphylis treatable; avoids transmission to newborns

Why bioengineering?

Why bioengineering?  Microfluidics  Biochemistry  Nanotechnology  Medicine  Engineering

Microfluidics

Biochemistry

Nanotechnology

Medicine

Engineering

The challenge of integration

Innovations:

1) Chip manufacturing (injection-molded plastic)

2) Reagent delivery (air spacing)

3) Signal amplification (silver reduction)

delivery (air spacing) 3) Signal amplification (silver reduction) Chin, C.D.; Sia, S.K. et al . Nature
delivery (air spacing) 3) Signal amplification (silver reduction) Chin, C.D.; Sia, S.K. et al . Nature

Chin, C.D.; Sia, S.K. et al. Nature Medicine, 2011

Antibody detection: ELISA

Antibody detection: ELISA Immobilized antigen Target antibody Gold-labeled secondary antibody

Immobilized antigen

Target antibody

Gold-labeled secondary antibody

Antibody detection: ELISA Immobilized antigen Target antibody Gold-labeled secondary antibody

Signal amplification

Signal amplification
Signal amplification
Signal amplification

Liquid handling

Liquid handling

Does it work?

Does it work?  70 specimens tested  15 minutes, 1µL blood  One false positive

70 specimens tested

15 minutes, 1µL blood One false positive (96% specificity)

Chin, C.D.; Sia, S.K. et al. Nature Medicine, 2011

Biosensing with nanopipettes

Biosensing with nanopipettes

Nanopipettes as sensors

Fabrication

Ionic current

Current rectification

Ion current through nanopipettes

Ion current through nanopipettes

An electrical DNA nanosensor

Detected 15-base oligomers

Signal detected by current rectification

DNA nanosensor Detected 15-base oligomers Signal detected by current rectification Fu, Baker, et al Chem. Commun.

Fu, Baker, et al Chem. Commun. 2009

A renewable nanosensor

Reporter dye trapped in pipette tip

Fluorescence increases in presence of sodium

Imaged with confocal

microscope

increases in presence of sodium Imaged with confocal microscope Piper, Klenerman et al , J. Am.
increases in presence of sodium Imaged with confocal microscope Piper, Klenerman et al , J. Am.

Piper, Klenerman et al, J. Am. Chem. Soc. 2006

Label-free immunosensing with nanopipettes (STING)

Art by David Liao
Art by David Liao

Selective sensing with STING

Selective sensing with STING S ignal T ransduction by I on N ano- G ating Binding

Signal T ransduction by Ion N ano- G ating Binding to receptors modulates current through the conical nanopore

Electrical antibody recognition

+VEGF 1.1 1.0 0.9 0.8 -0.8 -0.9 -1.0 -1.1 -20 -10 0 10 20 I,
+VEGF
1.1
1.0
0.9
0.8
-0.8
-0.9
-1.0
-1.1
-20
-10
0
10
20
I, normalized

Time [min]

Anti-VEGF
Anti-VEGF

Anti-ferritin

Umehara, Pourmand et al,

Proc. Nat. Acad. Sci. 2009

Calcium ions: a good target for biosensing

Major affector of cellular events

Indicator of cytotoxycity/apoptosis

Single-cell arrays used to monitor cellular events by monitoring cytosolic [Ca 2+ ] 1,2

1.

Xu, et al. Anal. Biochem. 2010

2.

Li et al. Integr. Biol. 2009

2 + ] 1 , 2 1. Xu, et al. Anal. Biochem. 2010 2. Li et

Li, 2009

A receptor for calcium: Calmodulin

A receptor for calcium: Calmodulin

Quartz surface

functionalization

Quartz surface functionalization
Change bath Ca 2+ (0.1 mM) buffer buffer (pH 7)
Change bath
Ca 2+ (0.1 mM)
buffer
buffer (pH 7)

Calcium sensor is reversible…

Ca 2+ Ca 2+ buffer buffer buffer
Ca 2+
Ca 2+
buffer
buffer
buffer

And selective

And selective

Summary

Nanopipettes modified with Calmodulin

as a recepter

Reversible calcium signal

Selective over magnesium

Detection limit 2x10 -5 M Ca 2+ .

Further work: improve surface chemistry, sensitivity, shelf life.

Test with biological systems

Summary

Biosensors: form and function

Bioconjugation: making biomolecules behave

Biosensor case studies

Optical sandwich immunoassay

Electrical aptamer sensor

mCHIP immunoassay

STING bioelectrical sensor

 Optical sandwich immunoassay  Electrical aptamer sensor  mCHIP immunoassay  STING bioelectrical sensor