Biosensors

Definition of a biosensor• A device that uses specific biochemical reactions mediated by

isolated enzymes, immunosystems, tissues, organelles or whole cells to detect chemical compounds usually by electrical, thermal or optical signals.

Source: PAC, 1992, 64, 148 (Glossary for chemists of terms used in biotechnology.) (http://goldbook.iupac.org/B00663.html)

History of Biosensors

• 1916 First report on immobilization of proteins : adsorption of invertase on activated charcoal

• 1922 First glass pH electrode • 1956 Clark published his definitive paper on the oxygen electrode.• 1962 First description of a biosensor: an amperometric enzyme

electrodre for glucose (Clark)• 1969 Guilbault and Montalvo – First potentiometric

biosensor:urease immobilized on an ammonia electrode to detect urea

• 1970 Bergveld – ion selective Field Effect Transistor (ISFET)• 1975 Lubbers and Opitz described a fibre-optic sensor with

immobilised indicator to measure carbon dioxide or oxygen

History of Biosensors (contd.)

• 1975 First commercial biosensor ( Yellow springs Instruments glucose biosensor)

• 1975 First microbe based biosensor, First immunosensor• 1976 First bedside artificial pancreas (Miles)• 1980 First fibre optic pH sensor for in vivo blood gases (Peterson)• 1982 First fibre optic-based biosensor for glucose• 1983 First surface plasmon resonance (SPR) immunosensor• 1984 First mediated amperometric biosensor: ferrocene used with

glucose oxidase for glucose detection

History of Biosensors (contd.)

• 1987 Blood-glucose biosensor launched by MediSense ExacTech• 1990 SPR based biosensor by Pharmacia BIACore• 1992 Hand held blood biosensor by i-STAT• 1996 Launching of Glucocard• 1998 Blood glucose biosensor launch by LifeScan FastTake• 1998 Roche Diagnostics by Merger of Roche and Boehringer

mannheim• Current: Quantom dots, nanoparicles, nanowire, nanotube, etc

Application of Biosensor• Food Analysis• Study of biomolecules and their interaction• Drug Development• Crime detection• Medical diagnosis (both clinical and laboratory use)• Environmental field monitoring• Quality control• Industrial Process Control• Detection systems for biological warfare agents• Manufacturing of pharmaceuticals and replacement organs

Components of Biosensor

Detector

Substrate

Product

Required Characteristics• Sensitivity• Low detection limits• Cost• Simplicity• Reliability• Speed • Accuracy • Precision

• Utility• Field portability• Ruggedness• Reproducibility• Ease of calibration• Stability• Room for

improvement

Biosensor breakdown

Analyte

Sample handling/ preparation

Detection

SignalAnalysis

ResponseThe Analyte

What do you want to detect?

MoleculeProtein, toxin, peptide, vitamin, sugar, metal ion

Cholera toxin Glucose

Sample handling

How to do deliver the analyte to the sensitive region?

•(Micro) fluidics•Concentration (increase/decrease)•Filtration/selection

How to do deliver the analyte to the sensitive region? Detection/Recognition

How do you specifically recognize the analyte?

Antibody Enzyme

Active site

Fab

Fc

Cell

Membrane receptors

Polymer/ Hydrogel

Competitive binding

Signal

How do you know there was a detection?Specific recognition?

Often the detector is immobilized on a solid support/sensor

Common signaling principlesOptical (SPR, ELM, IR)Electrical (Voltammetry, Potentiometry, Conductivity)Electromechanical (QCM)ThermalMagneticPressure

Avoiding false signals

Specific recognition

False specific recognition?

Non- specific signal

Secondary signal amplifier

Magnectic bead, fluorecent dye, enzyme etc

Inert background

Highly specific detection

Improving performanceRegeneration or single use?

Low and high pH bufferspH~1 & pH~13

Break binding

Types of Biosensors1. Calorimetric Biosensor: If the enzyme catalyzed reaction is exothermic, two

thermistors may be used to measure the difference in resistance between reactant and product and, hence,the analyte concentration.

2. Potentiometric Biosensor: For voltage, change in distribution of charge is detected using ion-selective electrodes, such as pH-meters.

3. Optical Biosensor: Colorimetric for color (Measure change in light adsorption) or Photometric for light intensity (Photon output for a luminescent or fluorescent process can be detected with photomultiplier tubes or photodiode systems).

4. Piezo-electric Biosensor: Piezo-electric devices use gold to detect the specific angle at which electron waves are emitted when the substance is exposed to laser light or crystals, such as quartz, which vibrate under the influence of an electric field. The change in frequency is proportional to the mass of absorbed material.

5. Electro- chemical Biosensor: For applied current, movement of e- in redox reactions detected when a potential is applied between two electrodes.

Electrochemical DNA Biosensor

• Steps involved in electrochemical DNA hybridization biosensors:• Formation of the DNA recognition layer• Actual hybridization event• Transformation of the hybridization event into an electrical signal

• DNA Biosensor: Motivated by the application to clinical diagnosis and genome mutation detection

• Types DNA Biosensors• Electrodes• Chips• Crystals

1. LINEARITY Linearity of the sensor should be high for the detection of high substrate concentration.

2. SENSITIVITY Value of the electrode response per substrate concentration.

3. SELECTIVITY Chemicals Interference must be minimised for obtaining the correct result.

4.RESPONSE TIME Time necessary for having 95% of the response.

Basic Characteristics of a Biosensor

Data AnalysisResponse variable (R) vs time(t):Example of response variables:Refractive indexPotentialCurrentFrequencyMassPressureTemperature

t

RStable baseline

t

Quantifying NoiseRoot mean square (RMS) of a sample of data points for a given time

Should be stable when there is no binding

Drift baseline

t

Quantifying DriftShift in the baseline (RMS) shown as response units per time

Baseline

Sensitivity

• Inhomogenous sample• Bubbles/flow artifacts• Temperature• Electromagnetic interferance• Electronic unstability• Unstable chip/detection layer

Signal-to-noise ratio Per time unit

t

t

t

SpikesRapid (1 datapoint!) shift in signal

Baseline shiftRapid (1 datapoint!) shift in baseline (offset)

Common signal error sources

Active sensordetects the analyte

Reference sensorCoated with inert material does not detect the analyte

R1 R2

Improved sensitivity

Output signalR=R1-R2 or R=R1/R2The reference is exposed to the same kind of disturbances as the active sensor. These effects are cancelled out by taking the difference between the two sensors

t t

R1 R2Sample

t

R

Signal interpretation• Visual (example pregnancy test)• Automatic (Software)• Manual (Research Biosensor)

Biacore Biosensor platform (example of Research Biosensor)General and flexible, good tool for development of specific biosensors

For a comprehensive list of research biosensor suppliers see:www.realtimebiosensor.com

Pregnancy test

Detects the hCG protein in urine. Interpretation and data analysis performed by the user

Typical Sensing Techniques for Biosensors

• Fluorescence• DNA Microarray• SPR Surface plasmon resonance• Impedance spectroscopy• SPM (Scanning probe microscopy, AFM, STM)• QCM (Quartz crystal microbalance)• SERS (Surface Enhanced Raman Spectroscopy)• Electrochemical

Nanosensors

If I want to measure something small, I need

something small…

Sensibility range

Antigen Concentration Organ Confinement

PSA 4-10 ng/ml 75%

PSA >10ng/ml >50%

PSA (prostate specific antigen) is a biomarker related to the existence of prostate cancer…

Need of Nano-Biosensor

• Quick and specific detection methods BoNT/A in nanogram (ng) quantity are of great importance to control the outbreaks of botulinum

• Detection prevents the spreading of botulinum disease even before the customers table.

• Among the other detection methods as array biosensor, PCR reaction, sandwich immunoassay, SYBR Green real time PCR method, the Fluorescent nanoparticles (GNPs)/ quantum dots (QD) based nano-ELISA detection techniques provides the best nanobiosensor with respect to specificity, sensitivity, user-friendly, cheap and time saving

Nanosensor • An extremely small device capable of detecting and

responding to physical stimuli (biological and chemical substances, displacement, motion, force, mass, acoustic, thermal, and electromagnetic) with dimensions on the order of one billionth of a meter and used to convey information to the macroscopic world.

• Benefits• Particles in nanoregime display new properties,• Sensibility increase due to better conduction property• Detection level become lower,• Direct detection possible with out labels

Nanosensor technology

LABEL-FREE LABEL

In labeled technology, some sort of label has to be attached to the biomarker, which otherwise would pass by undetected…

Labeled technology examples

• Quantum dots

• Gold nanoparticles

• Radioactive inks

labeled Non-labeled

Why would I prefer a label-free approach?

1. One fewer step to worry about (labeling)

2. I do not need a device to excite and image the sample

3. I might create a lab-on-a-chip device

4. I would end-up with point of care (POC) testing

Point of care testing

test results treatment

SCENARIO A

SCENARIO B

Wait results treatment

Target Molecule

1. Chen, G.Y., Thundat, T. Wachter, E. A., Warmack, R. A., “Adsorption-induced surface stress and its effects on resonance frequency of microcantilevers,” J. Appl. Phys 77, pp. 3618-3622 (1995).

2. Ratierri, R. et al., “Sensing of biological substances based on the bending of microfabricated cantilevers,” Sensors and Actuators B 61, 213-217 (1999).

3. Fritz, J. et al. “Translating Biomolecular Recognition into Nanomechanics,” Science 288, 316-318 (2000).

4. Wu, G. et al. “Origin of nanomechanical cantilever motion generated from biomolecular interactions,” PNAS 98(4), 1560-1564 (2001).

Courtesy: Prof. A. Majumdar, U.C. Berkeley

FET Nanosensor

Based on a conventional MOSFET…

Source:wikipedia

2001 Major Breakthroughs

Proof-of conceptPerformed by Lieber et al (Science 293, 1289, 2001)…

nanowire

Science 293, 1289, 2001

2001 Major Breakthroughs (contd.)

pH SensingIt is a good start to demonstrate the sensibility to

‘superficial’ charge changes…

Science 293, 1289, 2001

2001 Major Breakthroughs (contd.)

Antibody sensingStudy of the biotin-streptavidin system…

biotin

streptavidin

Science 293, 1289, 2001

250nM Unmodified SiNW

d-biotin 25 pM

2001 Major Breakthroughs (contd.)

Antibody sensingOnce again, the biotin-streptavidin system is studied… the nanoribbon is functionalized with biotin (biotinalized) and the solution contains streptavidin at different concentrations…

biotin

streptavidin

Si Nanoribbon

Nanoletters, 8, 3, 945-949 (2008)

Modified from Science 293, 1289, 2001

2001 Major Breakthroughs (contd.)

Thiolated ssDNA

5’-HS ATCCGCATTACGTCAATC

TAGGCGTAATGCAGTTAG-5’(Complementary Strand)

AuSelf-Assembly of ssDNA

PB = Sodium Phosphate Buffer Solution

-----

---+

++ +

+ ++

Wu, G. et al. “Origin of nanomechanical cantilever motion generated from biomolecular interactions,” PNAS 98(4), 1560-1564 (2001). Courtesy: Prof. A. Majumdar, U.C. Berkeley

Probe ssDNA Target ssDNA

Wu, G. et al. “Origin of nanomechanical cantilever motion generated from biomolecular interactions,” PNAS 98(4), 1560-1564 (2001).

Courtesy: Prof. A. Majumdar, U.C. Berkeley

Time [min]

0 60 120 180 240

Def

lect

ion

, h

[n

m]

-40

-20

0

20

40

60

80

Injections

[HSA] = 1 mg/ml[fPSA]

6 ng/ml

60 ng/ml

No PSA Ab ([fPSA] = 60 g/ml)

HP only ([HP] = 1 mg/ml)

No PSA

Time [min]

0 60 120 180 240 300

Def

lect

ion

, h

[n

m]

-50

0

50

100

150

200

[BSA] = 1 mg/ml

Injections

60 g/ml

6 g/ml

60 ng/ml

6 ng/ml

No fPSANo PSA Ab

([fPSA] = 60 g/ml)

[fPSA]

SiNx

AuDTSSP

Rabbit Anti-Human PSA

Glass

Analyte

SiNx

AuDTSSP

Rabbit Anti-Human PSA

Glass

Analyte

PSA

Wu, G. et al., “Bioassay of Prostate Specific Antigen (PSA) Using Microcantilevers,”

Nature Biotechnology (Sept., 2001)

HSA: Human Serum Albumin

HP: Human Plasminogen

fPSA: free PSA

cPSA: complex PSA

Courtesy: Prof. A. Majumdar, U.C. Berkeley

Why it worksAntigens appear during disease and can be used as biomarkers

Nature has made the binding between antibodies and antigens very specific

30 dies on a 4” Si wafer

200 mm300 mm

Potential applications:(1) Lab-on-a-chip applications(2) Early cancer detection(3) Infectious disease detection(4) Environmental monitoring(5) Pathogen detection

2002 Major Breakthrough

Functionalization of DNA

CO2H

NC

N

CH3

N

CH3

H

Cl-

O

CH3

HN

H2N ATGCCTTCCy3

ATGCCTTCCy3

CH3

H

Cl-

TACGGAAGGGGGGGGGGCy5

N

O

O

HO

SO3Na

CH3

C

O

NH

C

N

CH3

N

O

O

O N

O

O

SO3Na

O

HN ATGCC TTCCy3TACGGAAGGGGGGGGGGCy5

+

EDC

+

Sulfo-NHS

DNA probe

Target DNA

Cy3 image

Cy5 imageC. Nguyen et al, NanoLett., 2002, Vol. 2, p. 1079.

2002 Major Breakthrough

A simple model

Analytical Chemistry (2006), 2093-2099

2006 Major Breakthrough

Some thoughts•Nanowires are sensitive to the antigen-antibody binding, because the local charge transfer is a strong enough effect for the nanowire dimensions

• Building nanosensors is complicated, involving either top-down approaches using sophisticated litographic techniques, or bottom up techniques

• Residual effects during fabrication causes spurious effects during functioning

• Find a more process friendly substitute, but that is expected to be as sensitive

Si Nanoribbons as nanosensorsIntroduced by Linnros et al: Nanoletters, 8, 3, 945-949 (2008)

Nanoletters, 8, 3, 945-949 (2008)

It behaves like a Schottky barrier

2008 Major Breakthrough

The sensorSensor geometry and behavior similar to the one reported by Linnros et al (2008)

Schottsky barrier

2008 Major Breakthrough

The performanceThe performance described in previous studies is retained

2008 Major Breakthrough

Conclusions• The advances in label-free nanosensing have been plausible

during the last decade

• Nanoribbon sensors appears to have the necessary sensitivity and are less troublesome than nanowires

• The current sensitivity of nanosensors is in the appropriate range for early cancer detection

2008 Major Breakthrough

Good newsOnly there was response to

StreptavidinThere is a concentration dependant

response

Sensitivity can be manipulated

Nanoletters, 8, 3, 945-949 (2008)

2008 Major Breakthrough

Are we done yet?

The short answer is NO!!!...

For the nanosensor to be effective, the sensing has to be performed in the presence of a pure buffer solution. On the other hand, the human blood is nothing like it.

The ‘long’ answer is ...

2009 Major Breakthrough

The deviceTwo separate chambers. The big one has a chip functionalized with antibody-photocleavable groups. The small one has the nanosensors.

chip

Nature Nanotechnology, 5, 153 (2010)

2010 Major Breakthrough

First bloodSpiked blood containing the antigens PSA (prostate cancer) and CA15.3 (breast cancer) flow into the big chamber…

Nature Nanotechnology, 5, 153 (2010)

2010 Major Breakthrough (contd.)

Wash and sunbatheThe buffer solution is added to leave the chamber blood-free. UV light breaks the photocleavable-antigen pair.

Now I have a buffer solution of antigen!!!

Nature Nanotechnology, 5, 153 (2010)

2010 Major Breakthrough (contd.)

The happy endingThe content of the big chamber flows toward the small chamber, where sensing takes place

Nature Nanotechnology, 5, 153 (2010)

2010 Major Breakthrough (contd.)

Verifying the capture

Nature Nanotechnology, 5, 153 (2010)

A modified ELISA test is performed

2010 Major Breakthrough (contd.)

Specific Assessment• Fahmy et al did not performed a control with an antigen not

specific to the selected antibodies.

• The correlation between the introduced concentration and the captured/release concentration must be improved

• An exploration of the optimal operation parameters (potentials, thickness, etc..) must be done

• The technique can be assembled in a self-contained compact design

General assessment to the topic

• Silicon nanowire/nanoribbons are ideally suited for nanosensing, due to sensitivity and ease of functionalization

• A successful implementation of the technique awaits for significant advances in the detection of suitable biomarkers

• Charge screening effects (Debye length) are still a point to be addressed through more clever design of the nanosensors

General assessments

• The research for new and more sensitive materials must not be discarded

• Complete charting of a disease needs more than one antigen, so improvements in microarray arrangements must be made, as well as independent signal detection

• Microfluidics studies must be made to set the fluid parameters to optimize binding, diffusion effects and response times

References• Science 293,1289 (2001) Lieber, et al. Nanowire Nanosensors

for highly Sensitive and Selective Detection of Biological and Chemical Species

• Nanoletters 8, 3, 945 (2008) Linnros, et al. Silicon Nanoribbons for Electrical Detection of Biomolecules

• Nature Nanotechnology, 5, 138 (2010) Fahmy, et al. Label Free biomarker detection from whole blood

• Clinical Chimica Acta, 381, 93 (2007) Chan & Liang. Enzymes and related proteins as cancer biomarkers (REVIEW)

• Clinical Chimica Acta, 385, 37 (2005) Jain, Nanotechnology in clinical laboratory diagnostics (REVIEW)

Medical Imaging for diagnosis

• quantum dots or synthetic chromophores to selected molecules (e.g proteins) for intracellular imaging.

• Fluorescent tagging• Nanoclustes (<3nm)

Nanosensors Detect Cancer Biomarkers In Exhaled Breath

Gene TherapyNano Biosensors

Bioimaging

Nanoparticle Targeting Data

Conventional antibody labeling

Nanoparticle labeling

Note: Nuclei of cells are counter-stained blue with a DNA dye

Targeting strategies already developed can detect one rare cell in a million other cells (similar to the expected frequency of cancer cells in astronauts exposed to space radiation)

• High specificity• Direct, fast

response• High sensitivity• Single molecule

and cell signal capture and detection

3+

2+

e

3+

2+

Ru bPy 3

2

• Probe molecules for a given target can be attached to CNT tips for biosensor development

• Electrochemical approach: requires nanoelectrode development using PECVD grown vertical nanotubes

• The signal can be amplified with metal ion mediator oxidation catalyzed by Guanine.

Courtesy: Jun Li

• CNT tips are at the scale close to molecules

• Dramatically reduced background noise

Traditional Macro- or Micro- Electrode

NanoelectrodeArray

Nanoscale electrodes create a dramatic improvement in signal detection over traditional electrodes

Electrode

• Scale difference between macro-/micro- electrodes and molecules is tremendous

• Background noise on electrode surface is therefore significant

• Significant amount of target molecules required

• Multiple electrodes results in magnified signal and desired redundance for statistical reliability.

• Can be combined with other electrocatalytic mechanism for magnified signals.

Nano-Electrode

Insulator

Source: Jun Li

Nanosensor Roadmap

2002 2005 2015

Mis

sio

n C

om

ple

xity

Sensor Capacity1999

DSI RAX

2003ISPP

Missions too earlyfor nanotechnology impact

Biosensors

Spacestation

Europa Sub

Mars Robot Colony

Sensor Web2020

Nanotube VibrationSensor for Propulsion

Diagnostics

Optical Sensorsfor Synthetic

Vision

Nanopore for in situbiomark-sensor

Multi-sensorArrays (Chemical,optical and bio)

2010

Sharp CJV