BIOSENSING

- BIOSENSORS AND NANOMATERIALS -

Prof.dr.eng. Gabriel-Lucian RADU

28, February 2013 Bucharest

Analytical system

ideal

selectivespecificreproducible

reliable

compactcompact

rapidrapid

cheepcheep

friendlyfriendly useuse

Classification of chemical sensors

Massloading Mechanical

Conductingpathways

Electrochemicalreactions

Electrical

Refractiveindex

Fluorescence

Optical

Heattransfer Thermal

Non-exhaustive list…

Commerical (nano)sensors

NoseChipTM

Sensor Technology:nanocompositesensor arrayweight: ~0.5 ozPower: nanowatts

ArtinosSensor Technology:nanocrystalline tin oxide gradientmicroarraySize: 3x4 mm2

power consumption@ 300oC: 1 W

Cyranose 320

Cantilever based sensors

A surface acoustic wavepropagates over a coatedsurface.

Absorption of gas moleculeschange in the mass of thesensor coating -> change inthe resonant frequency

HAZMATCAD™ by Microsensor Systems

Surface Acoustic Wave Sensor

Cantilever is coated with achemically selective layer.Cantilever bends due to surfacestressDeflection of cantilever can bemeasured precisely by deflectinga light beam from the surface.

Cantilever sensor array by Concentris

Nanomechanical Sensor

µChemLabTM

Preconcentrator accumulatesspecies of interest

Gas chromatographseparates species in time SAW sensor detects gas

Conductivity sensors

Common sensing materials: conducting polymercomposites and metal oxides and CNTs

n-type metal oxide sensor operation: ambient O2 moelcules chemisorb onto the sensing film

surface Reducing target gas (e.g. CO) reacts with O- and release e-

Oxidizing agents (e.g. NO2) remove more e-

Conductive polymer composite

Conductive polymercomposite

1. Vapors pass over the polymer andswelling produces a change in resistance

2. Resistance change is measuredfor each sensor.

3. Using pattern recognition algorithms, the datais converted into a unique response pattern.

Carbon nanotube sensor

Hollow nanostructure and high specific surface areaprovides excellent sensitivities and fast response.

Can be functionalized to reversibly adsorb molecules ofpollutants undergoing a modulation of electrical,geometrical and optical properties.

Nanomix: SensationTechnology

NASA SWNT conductive gas andorganic vapor detector

CNT field ionization sensors

Ionization gas sensor

Different gases have aspecific ionization potential.Sharp tips of nanotubesgenerate very high electricfield at low voltages.No adsorption/desorptioninvolved -> fast response

• CNT tips are at the scale close tomolecules

• Dramatically reduced backgroundnoise

Traditional Macro- orMicro- Electrode

NanoelectrodeArray

Nanoscale electrodes create a dramatic improvement in signaldetection over traditional electrodes

Electrode

• Scale difference betweenmacro-/micro- electrodes andmolecules is tremendous

• Background noise onelectrode surface is thereforesignificant

• Significant amount of targetmolecules required

• Multiple electrodes results in magnifiedsignal and desired redundance forstatistical reliability

• Can be combined with other electrocatalyticmechanism for magnified signals

Nano-Electrode

Insulator

Nanoelectrode for sensors

Miniaturization

Micro technology Nano technology Nanostructures have high catalytic surface area: High

sensitivity, selectivity and response time Reduction in size, weight and power consumption Multiplexing capability to distinguish multiple chemical

species.

Micro Gas ChromatographHydrogen sensorusing palladiumnanoparticles

Rack sizedmeasuringinstrument

Shrinking technology

Microfabrication using MEMs-based technologyallows minimal size, weight and powerconsumption.

Construction of three dimensional structures arehighly desirable for chemical and electrochemicalsensors and microsystems.

Enable ease of integration with electronic circuitry

Micromachining

MEMS for rapid localized temperature control inMicro-hotplate (NIST)

What is a biosensor?

Biosensor = Any device that uses specific biochemicalreactions to detect (bio)chemical compounds in biological

samples.

Biosensor is not a bioanalytical system.

An enzyme electrode is a biosensor.

chemical and bioanalytical techniques

chemical sensorschemical sensors biosensorsbiosensors bioanalysisbioanalysis

Molecular bio-recognition principle

signalsignal

++

Biosensor

general concept

analytical information

analyteanalyte

transducertransducer

bioreceptorbioreceptor

output: physical signal

input: molecular recognition

Components of a biosensorComponents of a biosensor

Detector

Basic structure of biosensor

BioreceptorBioreceptor InterfaceEnergy

transducer

Signalprocessing

& dataoutput

Bioreceptor = selectivity

Transducer = sensitivity

Current definition

A sensor that integrates a biological element with aphysiochemical transducer to produce an electronicsignal proportional to a single analyte which is then

conveyed to a detector.

Analyte

Samplehandling/preparation

Detection

Signal

Analysis

Response

“Father” and “Mother”of the Biosensor

Professor Leland C Clark Jnr Professor George G. Guilbault

1956 Clark published his definitive paper on the oxygenelectrode

1965 First description of a biosensor: an amperometricenzyme electrodre for glucose - Guilbault

1970 Bergveld – ion selective Field Effect Transistor(ISFET)

1975 First commercial biosensor (Yellow springsInstruments glucose biosensor)

1987 Blood-glucose biosensor launched by MediSenseExacTech

1998 Launching of Glucocard by Roche Diagnostics

2004 Biochip by Affimetrix

Present Quantom dots, nanoparicles, nanowire, nanotube, etc.

History of biosensors

1. LINEARITY Linearity of the sensor should be highfor the detection of high substrateconcentration

2. SENSITIVITY Value of the sensor response persubstrate concentration

3. SELECTIVITY Chemicals interference must beminimised for obtaining the correctresult

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

LOD, QL, etc…..

Basic characteristics of a biosensor

Food Analysis Study of biomolecules and their interaction Drug Development Crime detection Medical diagnosis (both clinical and laboratory use)(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

Potential application of biosensors

2003 = 7.3 Bilion $

Biosensor Market 2007= 10.2 Bilion $

2012 = a growth rate of about 11%

Example of biosensors

Pregnancy testDetects the hCG protein in urine

Glucose monitoring device(for diabetes patients)

Monitors the glucose level in the blood

Example of biosensors

Infectous diseasebiosensor from RBS

Old time coal miners’ biosensor

Industrialbiosensors

Biacore Biosensor platform

Electrochemical

Fluorescence

DNA Microarray

SPR Surface plasmon resonance

QCM (Quartz crystal microbalance)

Impedance spectroscopy

SPM (Scanning probe microscopy, AFM, STM)

SERS (Surface Enhanced Raman Spectroscopy)

Typical sensing techniquesfor biosensors

Types of biosensors

1. Amperometric Biosensor

2. Potentiometric Biosensor

3. Optical Biosensor

4. Calorimetric Biosensor

5. Piezo-electric Biosensor

6. etc.

Data AcquisitionSample Acquisition

Analysis system architecture

Analytical systemAnalyte

separationBio-Chemical

detection

Pumps & valves Fluidic interfaces

ChromatographyElectrophoresis BiosensorsBiosensors

Sampleconcentration

Environmentalsampling

Extraction,Pyrolysis

etc.

Calibration,Self-check

RefreshRegeneration

Commu-nication

Display

Biosensors on the nanoscale

Molecular sheaths around the nanotube are developed thatrespond to a particular chemical and modulate thenanotube's optical properties.

A layer of olfactory proteins on a nanoelectrode react withlow-concentration odorants (SPOT-NOSED).Doctors can use to diagnose diseases at earlier stages.

Nanosphere lithography (NSL) derived triangular Agnanoparticles are used to detect streptavidin down toone picomolar concentrations.

Developed an anti- body based piezoelectric nanobiosensorto be used for anthrax, HIV, hepatitis detection.

DS

BOX

DS

BOX

++ +

Operation principle of SiNW-FETbiosensors

Silicon nanowire field effect transistor

Detection of Non-chargeAnalytes on SiNW-FET

Protein design Reporting molecule (probe)Fabrication andmodification of

SiNWs

Gene construction&

Over expressionProtein modified by probe

proteinimmobilization

Improve and Optimizeprotein function

Detection system

SiNWs

Design of a nano-device for steroids detection

steroids

Real-time biosensing on steroid

Linear correlation 1 fM~0.1Linear correlation 1 fM~0.1 pMpM

What wants to measure in medicine?

The concentration of a biomolecules (biomarkers) cantell us the nature of a disease and what stage it is in.

WHITE CELLSWHITE CELLS INFECTION

CREATININECREATININEKIDNEY

MALFUNCTION

Biomolecule detection methods

< 1 pM (streptavidin)

< 50 pM (DNA)

Cantilevers Sensor

> 1 fM (antibody)

Nanowire Field Effect Transistor

< 1 fM (DNA, antibody)

I

Surface Plasmon ResonanceEnzyme Linked Immunosorbent Assay

Device surface

Protein-A(cross-linker)

(Not drawn to scale)

Antibody layer

Non-specifictarget

Specific target(antigen)

Antibody immobilization on sensor

Sandwich electrochemicalimmunosensor proteins

H2O2

Ab1

Ag

HRP HRP HRP HRPHRP

Ab1

Ag

HRP

HPR

Ab2

Apply E measure ISWNT forest Conductive polymer

(SPAN)

protein

S DSiO2

Si back gate

polymer

VgVsd

biotin

streptavidin

Ploymer coated,biotin-immobilizeddevice(approximately 50streptavidins)

Polymer coateddevice withoutbiotin

Ligand-receptor bindingwithout false positives

Response tobiotinilatedstreptavidin

Detection limit:10 proteins

Motivated by the application to clinical diagnosisand genome mutation detection

Types DNA Biosensors:

ElectrodesElectrodes ChipsChips CrystalsCrystals

DNA biosensors

1 nm

10 nm

100 nm

1

DNA

Virus

Bacteria

Proteins

0.1 nm

Nanotube

Nanowire

Next Generation CMOS

Current CMOS Technology

DNA detection - electronic

Infineon

HP

Harvard

UCLA

Critical issues:

sensitivity

multiplexing

Critical issues:Critical issues:

sensitivity

multiplexing

How genetic sequencing works inDNA biosensor

Separate ds-DNA (Probe DNA)DNA is denatured by heat or chemicaldenaturant and placed in solution or ona solid substrate, forming a referencesegment

Introduce unknown ss-DNA(Target DNA)Unknown DNA sample is introduced to thereference segment. The complement of thereference segment will hybridize to it.

How hybridization is identifiedAnalyteAnalyte(Target DNA)(Target DNA)

Recognition layerRecognition layer(Probe DNA)(Probe DNA)

Transducer

Signal

Electrochemical devicesCurrent signal of a redoxindicatorOptical devicesEmission signal offluorescent orchemiluminescent lablesSurface optical propertiesNanoparticle basedcolorimetric detection

Mass-sensitive devicesFrequency signal of oscillatingcrystal with DNA probe

DNA Hybridization biosensor

- Immobilization of ss-DNA probe onto the transducer surface

- Tranducing (association of an appropriate hybridization indicator)

ssDNA immobilization approaches

1. Noncovalent anchoring

Aromatic molecule binding, ssDNA tethering

2. Thiol attachment to gild nanoparticles

Au nanoparticle deposition followed by thiol chemistry

3. Tethering to polymer coatingPEI tethering, following our biotin immobilizationapproach

S DSiO2

Si back gate

VSD

Single-strandDNA

ComplementaryDNA

Sequence

Carbon NanotubeTransducer

DNA detection by carbon nanotube transistors

DNADuplex

Formation

AnalyticalSignal

G

VG

DNA Immobilization Strategies

NHO

S

N

O

O

NHO

NNH x y

OOHHn

S

1) Metal Nanoparticles

2) Sticky Labels

3) Polymer Layer

(~2nm diameter)

- Nanopore in membrane- DNA in buffer- Voltage clamp- Measure current

,

-hemolysin pore

Axial View Side View

(very first, natural pore)

DNA sequencing with nanopores

Open nanopore DNA translocation event

• When there is no DNA translocation, there is a background ioniccurrent

• When DNA goes through the pore, there is a drop in thebackground signal

• The goal is to correlate the extent and duration of the drop in thesignal to the individual nucleotides

Nanopore ion conductance

GGA A A A

G

C CTT

Present FutureA AG

G

G

GC C

Status of nanoporesbased DNA sequencing

After a decade of using protein pores, efforts are underway in manygroups to develop synthetic pores (such as in Si3N4)

• Interaction with single nuclotides- ~20 nucleotides simultaneously

• Slower translocation- 1-5 ms /nucleotide

• Resistance to extreme conditions: temperature, pH, voltage• - hemolysin is toxic and hard to work with

Nanobiosensor technology

LABELLABEL--FREEFREE LABELLABEL

In labeled technology, some sort of label has tobe attached to the biomarker, which otherwisewould pass by undetected…

Labeled technology examples

• Quantum dots

• Gold nanoparticles• Radioactive inks

Labeled Non-labeled

The device

Two separate chambers. The big one has a chip functionalizedwith antibody-photocleavable groups.The small one has the nanobiosensors.

chip

Nature Nanotechnology, 5, 153 (2010)

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)

What is a microarray ?A slide or chip that contains numerous amounts ofbiomolecules in fixed amount of space.

Why microarrays ????? Small volume > miniaturization High throughput analysis Large information generated Less time required to analyze

A microarray is a spatially ordered, miniaturizedarrangement of multitude of immobilized reagents.

Microarray landscape

subarray

Slide

Gene/Protein

spot

pingroup

Microarrays are fabricated by high-speed robotics, generally onglass but sometimes on nylon substrates, for which probes withknown identity are used to determine complementary binding,thus allowing massively parallel screening studies.

Micro-arrayers

How are they made? Non-contact printing

Piezoelectric Syringe Solenoid

Contact Printing

Microarrays

DNA - genomicsCellProteinAntibody arrays – detects proteinsProtein arrays – detects

interactions of proteins orwith small molecules

3 main types3 main types3 main types

Surface modified by chemistries

Steps involved in microarrays

Microarray printing

Hybridization

Detection

Image processing by Laser scanning

The microscope slidecontaining the microarray isplaced inside a microarrayscanner, where the slide isscanned with two lasers todetect the bound green andred cDNAs.

DNA-microarray printing

100-10000 spots Glass slide used as

substrate DNA is attached

covalently to glass slide 96-384 well microtitre

plates used Spot Volume 0.25-1nL Spot size 100-150µm in

diameter

Reporting resultsMicroarray

Gene

The expression ratios for every gene can be organizedinto a table where each column is a microarray and eachrow is a gene.This representation however is overwhelming inexperiments involving thousands of genes and data.

Applications of microarrays

Drug development, drug response,and therapy development

Expressionanalysis

Tumor classification, riskassessment, and prognosis prediction

cDNA arrayApplicationApplication

Future directions: sensitivity enhancement,multiplexing

Biotech applications:gene chips, protein chips,disease identification,bio-threat agent detection …..

Noise reduction

(a) (b)100 nm

100 nm

1 m

Ultradense arrays

Protein microarrays

Analysis of thousands of proteins at one time

Many different types Antibody arrayed - detect many proteins Proteins arrayed - detect interacting proteins Proteins arrayed - detect interacting small molecules Proteins arrayed - detect enzymatic activity (Enzyme

profiling) Peptides arrayed – substrate for enzymes, interaction Small molecules arrayed – detect enzymatic activity

(enzyme profiling) etc.

Protein detection microarray

Protein-chip array surfaces

Design evolution

2nd generation2nd generation

3rd generation3rd generation -- biochipbiochip

1st generation1st generation

biomimeticbiomimetic

Trends in (nano)biosensors

construction

presentpresent

futurefuture

hardware

software

(arbitrary units)(arbitrary units)

elec

tron

ic in

terf

aces

elec

tron

ic in

terf

aces

MULTIFUNCTIONALMULTIFUNCTIONAL

multilevelmultilevel

modemode

dimensiondimension

multimode

multimode mul

tidi

men

sion

alm

ulti

dim

ensi

onal

com

plex

ity

com

plex

ity

Nanobiosensors

Listeria detectionfrom blood

Space Exploration

A range of chemical sensing technologiesto measure several parameters of interestsimultaneously. MEMs-based micro-sensor arrays

Reliability of sensor technologies Harsh environment (during launching) Calibration issues Signal drifting

Broad inclusion into intelligent “smart” systems: Supporting technologies: signal processing, communication.. “Lick and stick” technology (ease of application)

Take advantage of quantum properties of materials for ultra-sensitive detection. CNTs, nanowires,nanopores…..

“Lick and stick” smart leakdetector

AstroBioLab for Mars ExplorationExoMars Rover

Mobile laboratory that uses a suite of in situ instruments: Mars OrganicDetector and Oxidant detector, micro-capillary electrophoresisanalyzer.Target compounds are amino acids and Polycyclic AromaticHydrocarbonsElectronics designed for Martian ambient survivability (-145 to 100oC)Low power consumption with broad chemical extraction ability.

Mars Organic Detector

Specifications:Mass:~ 2 kgPower: 24 WSize: 145 x 193 x 112 mm

Uses sublimation at Marsambient pressure andtemperatures to releaseorganic components ofretrieved samples.

Highly sensitive fluorescent detection,detects presence/ absence of aminoacids and PAHInterfaced with microchip-basedcapillary electrophoresis foridentification of amino acids

Mars Oxidant Detector

Mars Oxidant Instrument (MOI) sensorarrays configured into a soil cup

Test the Martian samples andenvironment for their ability todegrade organic compoundsthrough oxidation

Monitor reaction with well-characterized reactants overdays/weeks exposure.

The chemical state ismonitored by measuringelectrical resistivity via achemiresistor transducer.

The carp-shaped robots, costing 20,000 pounds($29,000) a piece, mimic the movement of realfish and are equipped with chemical sensorsto sniff out potentially hazardous pollutants,such as leaks from vessels or underwaterpipelines.They will transmit the information back toshore using Wi-Fi technology.Unlike earlier robotic fish, which neededremote controls, they will be able to navigateindependently without any humaninteraction.The robot fish will be 1.5 meters (nearly 5 feet)long -- roughly the size of a seal.

Robot fish to detect pollution

Environmental biosensors forPersonal Exposure Assessment

•Multiple Compounds•Real-time continuous data acquisition•GPS location data•Health indicators

Implantable biosensors with telemetrybatteries included – testing in WiStar Rats

8 Piezoresistive cantilevers Integrated electronic

readout Telemetry Low power consumption 3 cm X 1 cm (diameter) No pump

Implanted in rats forImplanted in rats for inin--vivovivo detection ofdetection ofalcoholalcohol Ferrell (UT)Ferrell (UT)

Wireless Biosensing

TARGET MOLECULEAcetaminophen

Nanowire biosensor

•1,000 nanowire detectors can be jammed into a few square micrometres — roughly the areataken up by a single cell. Technique called superlattice nanowire pattern transfer which cancreate individual semiconductor nanowires that are as little as 8 nm in diameter with the samedistance between each wire1.

Early detection of cancer, can detect cancer cells when there are only a fewnanowires coated with probes such as an antibody that binds to a target protein.Thus changing the electrical conductivity

Single virus detection

Nanoscale cantilevers

Cantilevers can be used asdetectors of molecules.Specific molecules areattached to the cantilevers.

The molecules selected aremolecules that will bind toa specific molecule.

When that molecule bindsto the cantilever it changesthe physical properties ofthe cantilever and thatchange can be detected.

Biochip - GeneChip(lab on a chip all on a chip)

Detection of protein biomarkers for cancer:• NIH, NIDCR• prostate, squamous cell, and breast cancers

Printed layer

Electrodecontacts,5 x 10 mmand spaced5 mm apart

10 mm

Screen printed carbon arrays

Future work: pattern SWNT forest arrays onto microchip;collaboration withUniv. of Edinburgh Genomics Inst. (GTI)

Pattern SWNT forest arrays onto microchip

Markets and Technologies

Making Sense of Sensors

Produced by the Institute of Bio-Sensing Technology for theMicroelectronics iNet

November 2011

http://www.biosensingtech.co.uk/

http://www.bioanaliza.ro

“Daca stii cum sunt alcatuite lucrurile in interiorul lor,

asta nu inseamna ca esti genial.”Michelangelo