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
Substrate

Product

Detector

Required Characteristics
Sensitivity Low detection limits Cost Simplicity Reliability Speed Accuracy Precision Utility Field portability Ruggedness Reproducibility Ease of calibration Stability Room for improvement

Response

Analyte

The Analyte
What do you want to detect? Molecule Protein, toxin, peptide, vitamin, sugar, metal ion

Biosensor breakdown
Analysis

Detection Signal

Sample handling/ preparation

Sample handling
How to do deliver the analyte to the sensitive region? How to do deliver the analyte to the sensitive region? (Micro) fluidics Concentration (increase/decrease) Filtration/selection Cholera toxin Glucose

Detection/Recognition
How do you specifically recognize the analyte? Fab Active site Fc Antibody Enzyme Cell Polymer/ Hydrogel Membrane receptors Competitive binding

Common signaling principles Optical (SPR, ELM, IR) Electrical (Voltammetry, Potentiometry, Signal Conductivity) Electromechanical (QCM) Often the detector is Thermal Magnetic immobilized on a solid support/sensor Pressure

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

Avoiding false signals

Non- specific Specific recognition signal False specific recognition?

Improving performance
Secondary signal amplifier
Highly specific detection

Regeneration or single use?

Break binding Low and high pH buffers pH~1 & pH~13

Magnectic bead, fluorecent dye, enzyme etc

Inert background

Types of Biosensors
1. 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

Basic Characteristics of a Biosensor

1. LINEARITY Linearity of the sensor should be high for the detection of high substrate concentration. Value of the electrode response per substrate concentration. Chemicals Interference must be minimised for obtaining the correct result. Time necessary for having 95% of the response.

2. SENSITIVITY

3. SELECTIVITY

4.RESPONSE TIME

Data Analysis
Response variable (R) vs time(t): Example of response variables: Refractive index R Potential Current Frequency Mass Pressure t Temperature Drift baseline

Baseline
Should be stable when there is no binding
Stable baseline

t Quantifying Noise Root mean square (RMS) of a sample of data points for a given time

t
Quantifying Drift Shift in the baseline (RMS) shown as response units per time

Sensitivity
Signal-to-noise ratio Per time unit t Spikes Rapid (1 datapoint!) shift in signal Baseline shift Rapid (1 datapoint!) shift in baseline (offset)

Common signal error sources

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

Improved sensitivity
t
Active sensor detects the analyte Reference sensor Coated with inert material does not detect the analyte

Output signal R=R1-R2 or R=R1/R2 The 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

R1

R2

R1
t

R2
t

R t Sample

Signal interpretation
Visual (example pregnancy test) Automatic (Software) Manual (Research Biosensor)
Pregnancy test Detects the hCG protein in urine. Interpretation and data analysis performed by the user

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

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

Antigen PSA
PSA

Concentration 4-10 ng/ml

>10ng/ml

Organ Confinement 75%

>50%

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

labeled Non-labeled

Quantum dots Gold nanoparticles Radioactive inks

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

SCENARIO A

test

results

treatment

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

2001 Major Breakthroughs

FET Nanosensor
Based on a conventional MOSFET

Source:wikipedia

2001 Major Breakthroughs (contd.)

Proof-of concept
Performed by Lieber et al (Science 293, 1289, 2001)

nanowire

Science 293, 1289, 2001

2001 Major Breakthroughs (contd.)

pH Sensing
It is a good start to demonstrate the sensibility to superficial charge changes

Science 293, 1289, 2001

2001 Major Breakthroughs (contd.)

Antibody sensing
Study of the biotin-streptavidin system
biotin streptavidin

Science 293, 1289, 2001

250nM

Unmodified SiNW

d-biotin

25 pM

2001 Major Breakthroughs (contd.)

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

streptavidin

biotin

Si Nanoribbon
Modified from Science 293, 1289, 2001

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

Self-Assembly of ssDNA

Thiolated ssDNA
5-HS ATCCGCATTACGTCAATC TAGGCGTAATGCAGTTAG-5 (Complementary Strand)

Au

-+ + + --+ + -- + -+

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

PSA
Analyte Rabbit AntiHuman PSA DTSSP Au SiNx
Deflection, h [nm]

200
[BSA] = 1 mg/ml [fPSA] 60 g/ml Injections 6 g/ml

150 100 50 0
No fPSA

60 ng/ml 6 ng/ml No PSA Ab ([fPSA] = 60 g/ml)

Glass

-50 0 60 120 180 240 300 Time [min]

HSA: Human Serum Albumin

Deflection, h [nm]

80 60 40
Injections 6 ng/ml [HSA] = 1 mg/ml [fPSA] 60 ng/ml

HP:

Human Plasminogen

fPSA: free PSA cPSA: complex PSA

Wu, G. et al., Bioassay of Prostate Specific Antigen (PSA) Using Microcantilevers, Nature Biotechnology (Sept., 2001)
Courtesy: Prof. A. Majumdar, U.C. Berkeley

20 0 -20 -40 0 60 120 Time [min] 180 240

No PSA Ab ([fPSA] = 60 g/ml) HP only No PSA ([HP] = 1 mg/ml)

Why it works
Antigens appear during disease and can be used as biomarkers

Nature has made the binding between antibodies and antigens very specific

2002 Major Breakthrough

300 m 200 m

30 dies on a 4 Si wafer

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
CH3 CH3 CO2H O HO N O Sulfo-NHS O O SO3Na O N O TACGGAAGGGGGGGGGGCy5 Target DNA O HN TACGGAAGGGGGGGGGGCy5 ATGCC TTCCy3 N C N EDC C O SO3Na C NH CH3 ClN+ H CH3 O N CH3 ClH N+ CH3

Cy3 image

O HN

H2N

ATGCCTTCCy3

DNA probe ATGCCTTCCy3

Cy5 image

C. Nguyen et al, NanoLett., 2002, Vol. 2, p. 1079.

2006 Major Breakthrough

A simple model

Analytical Chemistry (2006), 2093-2099

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

2008 Major Breakthrough

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

It behaves like a Schottky barrier

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

2008 Major Breakthrough

The sensor
Sensor geometry and behavior similar to the one reported by Linnros et al (2008)
Schottsky barrier

2008 Major Breakthrough

The performance
The 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 news

Only there was response to Streptavidin

There is a concentration dependant response

Sensitivity can be manipulated

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

Are we done yet?

The short answer is NO!!!...
The long answer is ...
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.

2009 Major Breakthrough

2010 Major Breakthrough

The device
Two 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 (contd.)

First blood
Spiked 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 sunbathe

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

Nature Nanotechnology, 5, 153 (2010)

Now I have a buffer solution of antigen!!!

2010 Major Breakthrough (contd.)

The happy ending

The 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

A modified ELISA test is performed

Nature Nanotechnology, 5, 153 (2010)

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)

Bioimaging

Gene Therapy Nanosensors Detect Cancer Biomarkers In Exhaled Breath

Nano Biosensors

Nanoparticle Targeting Data

Conventional antibody labeling

Nanoparticle labeling

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

 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 RubPy oxidation catalyzed by Guanine.
2 3

2+

2+

3+

3+ e

High specificity Direct, fast response High sensitivity Single molecule and cell signal capture and detection

Courtesy: Jun Li

Nanoscale electrodes create a dramatic improvement in signal detection over traditional electrodes
Traditional Macro- or Micro- Electrode Electrode Nanoelectrode Array

Insulator

Scale difference between macro/micro- electrodes and molecules is tremendous Background noise on electrode surface is therefore significant Significant amount of target molecules required

CNT tips are at the scale close to molecules

Dramatically reduced background noise

NanoElectrode

Multiple electrodes results in magnified signal and desired redundance for statistical reliability. Can be combined with other electrocatalytic mechanism for magnified signals. Source: Jun Li

Nanosensor Roadmap

2002

2005

2010

2015
Optical Sensors for Synthetic Vision

Mission Complexity

Sensor Web
Nanotube Vibration Sensor for Propulsion Diagnostics

2020

Mars Robot Colony Europa Sub

Multi-sensor Arrays (Chemical, optical and bio)

Spacestation

Sharp CJV

2003 ISPP 1999 DSI RAX Missions too early for nanotechnology impact

Nanopore for in situ biomark-sensor

Sensor Capacity