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