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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

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?

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?

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

Electrical biosensor design

Sandwich Immunoassay

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

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

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

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

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?

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)

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

Antibody detection: ELISA


Immobilized antigen Target antibody

Gold-labeled secondary antibody

Signal amplification

Liquid handling

Does it work?

70 specimens tested 15 minutes, 1L blood One false positive (96% specificity)

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

Biosensing with nanopipettes

Nanopipettes as sensors

Fabrication Ionic current Current rectification

Ion current through nanopipettes

An electrical DNA nanosensor


Detected 15-base oligomers Signal detected by current rectification

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

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

Label-free immunosensing with nanopipettes (STING)

Art by David Liao

Selective sensing with STING

Signal Transduction by Ion Nano-Gating


Binding to receptors modulates current through the conical nanopore

Electrical antibody recognition

1.1 1.0

+VEGF

I, normalized

0.9 0.8 -0.8 -0.9 -1.0 -1.1 -20 -10 0 10 20

Anti-VEGF Anti-ferritin

Time [min]

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 [Ca2+] 1,2

1. Xu, et al. Anal. Biochem. 2010

2. Li et al. Integr. Biol. 2009

Li, 2009

A receptor for calcium: Calmodulin

Quartz surface functionalization

Change bath

Ca2+ (0.1 mM)

buffer (pH 7)

buffer

Calcium sensor is reversible

Ca2+

Ca2+

buffer

buffer

buffer

And selective

Summary

Nanopipettes modified with Calmodulin as a recepter Reversible calcium signal Selective over magnesium Detection limit 2x10-5 M Ca2+. 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