Biosensors and Their Applications (BIEN 515) • Introductions – Name – Background • Review of syllabus and class objectives – Tailor this class to your individual needs! What is a Biosensor? • Definitions – A device used to measure biologically relevant information • Oxygen electrodes, neural interfaces, – A device using a biological component as part of the transduction mechanism • Antibodies • Enzymes • DNA, RNA • Whole cells • Whole organs/systems What is a Biosensor? • Configuration – Can be developed from any basic sensor by adding a biological component – Usually incorporates a biomembrane • Transduction – Electrical – Optical – Mechancial • Mass • Acoustic – Thermal – Chemical – Magnetic
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Biosensors and Their Applications (BIEN 515)
• Introductions – Name – Background
• Review of syllabus and class objectives – Tailor this class to your individual needs!
What is a Biosensor? • Definitions
– A device used to measure biologically relevant information • Oxygen electrodes, neural interfaces,
– A device using a biological component as part of the transduction mechanism
Conclusion• Theory predicts benefits for miniaturization of
EFFF systems
• Design, fabrication and characterization of aminiaturized EFFF system was performed
• Experiment confirmed basic theory
• Basic separations demonstrated
• Separation of particles with attached proteins
• Separation of some blood components
Acknowledgments
• National Science Foundation GraduateResearch Fellowship
• Whitaker Foundation Biobased Internship
• University of Utah Technology InnovationGrant
DNA
b Contains genetic material for all livingorganisms
b Double stranded helixb Made up of 4 different nucleotides- A,T,C,Gb Each nucleotide a 5 carbon sugarb Sequences of nucleotides define proteinsb Each sequence is a “gene”
Problem
b Typical molecular analysis problemsrequire statistically significant quantitiesand must pass detection limits on the orderof millions and billions of molecules
PCR
b Technique used to produce a large numberof copies from a target DNA sequence
b Template DNAb Complementary Primers (~20 nucleotides)b Thermostable Polymerase Enzyme (TAQ)b Single nucleotides (A,C,G,T)b Buffers (pH and ionic concentrations)
PCR Applications
b Creates 2n copies (typically 30 cycles)
b Genetic analysisb Viral diagnosisb Start with just one sample molecule
Why Apply Micromachining?
b Small reagent costsb Fast cycling time
• Low thermal mass• High surface to volume ratio
b System integration• Electrophoresis• Point of care system
b Low cost
Design Considerations
b Biocompatibilityb Chamber volumeb Control systemb Bulk or surface micromachiningb Bonding method (if necessary)b Move the fluid or cycle in positionb External equipment
Mechanical Pressure Sensors
• Designs– Absolute
– Gauge
– Sealed Gauge
– Differential
Piezoresistive Pressure Sensors
• Piezoresistivity is a material property where bulkresistivity is influenced by mechanical stress appliedto material
• Common piezoresistors: Si, poly Si, SiO2, ZnO
• Typical design: 4 piezoresistors in a Wheatstonebridge on a diaphragm
• Pressure sensitivity (mV/V-bar): S = (∆R/∆P)(1/R)
Capacitive Pressure Sensors
• Capacitive sensors convert charge into change incapacitance
• Advantages:– more sensitive than piezoresistive
– less temperature dependent
• Disadvantages:– gap fabrication
– diaphragm mechanical properties
Capacitive Pressure Sensors (cont)
• Basic concept: C = ε S/d
• Sensitivity: ∆C/∆d = -ε S/d2
• Small Gaps:– larger capacitance
– easier capacitance detection
– plates may stick together
• Large Gaps:– small capacitance
– may require wafer bonding
Microphones
• Convert acoustic energy into electrical energy
• High sensitivity pressure sensors
• Types:
– Capacitive• variable gap capacitor; most common
• require DC bias
• sensitivity: 0.2 to 25 mV/Pa
• response: 10 Hz to 15 kHz
Microphones (cont)
– Piezoresistive• diaphragm with 4 pezoresistors in a Wheatsone
bridge
• sensitivity: ~25 µV/Pa
• response: 100 Hz to 5 kHz
– Piezoelectric• use piezoelectric material mechanically coupled to
diaphragm
• sensitivity: 50 to 250 µV/Pa
• response: 10 Hz to 10 kHz
DNA • Contains code for operation of all living organisms • Made up of 4 (5) bases in long chains • Each nucleotide a 5 carbon sugar • Double helix of complementary strands • Sequences of nucleotides define “genes” • Genes define proteins
RNA • Converts DNA codes into proteins • Similar to RNA with additional oxygen • Matches up with DNA strands (complementary) • Three types
– Messenger (mRNA) • Carries genetic code out of nucleus
– Ribosomal (rRNA) • Interacts with mRNA to generate protein
– Transfer • Transports amino acids to ribosome • Translates mRNA to generate protein • Series of 3 RNA bases correspond to amino acid
Proteins • Main functional units of all cells • Provide structure and function • Catalytic proteins are called enzymes • Structure of protein is critical to function
The Cell • Basic building block of
biology • Made up of smaller
organelles – Nucleus
• Site of DNA storage • RNA synthesis takes place here
– Endoplasmic Reticulum • Smooth
– Processing of phospholipids and fats
• Rough – Processing of proteins – Studded with ribosomes –
– Golgi Body • Process and sort secretory and
membrane proteins – Lysosomes
• Degrade particles and dysfunctional units
– Mitochondria • Site of ATP production (power
generation) • ATP powers most functions in
cell – Plasma membrane
• Lipid bilayer • Gatekeeper • Communication
– Cytosol • Contains structural proteins
1) Immobilization a) Adsorption
i) SAM’s b) Entrapment c) Cross-linking d) Covalent bonding
2) Transduction efficiency a) Highly dependent on immobilization method b) Loading factor
i) Usually an optimal density c) Thin layers
i) Mass transport effects d) Specificity of attachment
i) Random attachment leads to “blocked” areas ii) Orientation important
3) Surface modification techniques a) Self assembled monolayers (SAM’s) b) Light directed synthesis c) Photolithography d) Micromachining e) Ink jet deposition f) Microscopic patterning g) Printing
4) Selectivity and Recognition a) Structural complements
i) Hydrogen ii) Ionic iii) Van der Waals
b) Phase partitioning c) Size or charge exclusion
5) Arrays a) Multianalyte detection and analysis b) Amplification c) Spatial resolution d) Averaging
6) Dimensional considerations a) Where is measurement being made? b) Nano-, micro-, and ultra electrodes c) Scaling considerations and limits
i) Macro analysis may not apply at microscale ii) Concentrations
d) Which dimension is important e) Controllability and repeatability
7) Calibration and Figures of Merit a) Quantifiable Performance Measures b) Calibration uses known concentrations or samples
i) Model formation c) Zero-order
i) One data point ii) Comparison to theory or empirical data
d) First order i) Array of zero order ii) Multiple measurements in time
e) Second order, etc
f) Increasing order allows better multicomponent analysis g) In situ calibration h) Figures of Merit
i) Sensitivity (1) Slope
ii) Limit of determination (1) Based on standard deviations and sensitivity
iii) Linear range iv) Selectivity
(1) Signal overlap (2) Separation schemes (3) Interferents (4) Methods to enhance
(a) Transduction (b) Molecular recognition
v) Response time (1) May depend on “normal” (2) Depends on both instrument and signal processing (3) Measured by 95% or time constant (4) How is sample introduced? (5) Equilibration (6) Refreshing time?
vi) Accuracy vii) Precision
(1) S/N viii) Confidence level
(1) Errors ix) Robustness x) Ease of use xi) Economics xii) Availability
Application Overview
• Each application has its own challenges – Applications similar to analytical chemistry
• Design and characterization (performance) heavily dependent on application – Example of oxygen – Environmental variations
• Concentrations • Media • Temperature • Pressure • Interferents • Matrix effects
• In situ measurements limited by – Sterility – Calibration – Lifetime
• Contamination a big issue
Clinical Applications • Medical diagnostics and monitoring • Trend toward decentralized and immediate results • Generally high priority • 10% GNP goes to healthcare • Difficulties
– Measurements outside of controlled environment – Miniaturization – Stability – Biocompatibility – Glucose sensors good example
Ion Selective Membranes • Allow movement of one ion while restricting another • Allow ion selective sensing • Membranes derived from polymers • Complex chemistry to derive selectivity
Acoustic Wave Devices • Mass sensitive technique • Quartz Crystal Microbalance (QCM) • Usually use antibodies • Other factors affect response
– Enzyme load – Membrane thickness – Alternative enzymes
• Two catalytic reactions – One substrate, one product – Ping Pong (Two phase)
Current and Future • Many biosensors still not commercialized • Mostly single analyte devices • Some arrays in production • Unlikely to develop perfect biosensor • Future directions
– Improved immobilization methods – Fine tuning of molecular selectivity
• Gene and protein engineering • Replacement of biocomponents which are expensive, unstable, and difficult to derive
– New materials • Designed membranes to improve characteristics • Improve connections for molecular recognition agents
– Specific binding sites on membrane • Understanding and improving interfaces between analyte and transducer • Transducer improvement (carbon composites, etc)
– Multianalyte sensors – High density arrays (especially for genomics) – Miniaturization – Implantation – Integration of components – Small volumes
• Cell interiors • Between nerves
– High spatial resolution – Non aqueous media – Non invasive measurements – Improved material characterization and equipment – Data interpretation (neural networks) – Microseparation systems
1) Enzymes a) Catalyze reactions b) Kinetics
i) Study of reaction rates c) Groups
i) Oxidoreductases (1) Transfer electrons: H-
ii) Transferases (1) Transfer functional groups
iii) Hydrolases (1) Transfer functional group to water
iv) Lyases (1) Transfer groups to or from double bonds
v) Isomerases (1) Transfer groups within molecules
vi) Ligases (1) Transfer by joining groups (2) ATP cleavage
d) Optional conversion routes i) What is final product ii) Where is group transferred to
2) Terminology a) Cofactor or coenzyme
i) Bond with enzyme to allow function ii) When bound called prosthetic group
b) Holoenzyme i) Enzyme with bound coenzyme
c) Apoenzyme i) Enzyme without cofactor or prosthetic group
d) Activity i) Measure of purity and ability of enzyme ii) Given as units of activity per milligram iii) Unit (U) defined as:
(1) Amount to convert 1 µmole of substrate/minute (2) Given a specific pH and temperature
3) Rational design a) Range of enzymes
i) Some enzymes catalyze same reaction but with additional advantages
b) End products c) Reagents d) Consumables e) What is being measured
i) Product ii) Consumption
f) Interferents i) Can they be removed?
g) Multiple sequential reactions 4) Kinetics
a) Factors related to reaction rate b) Plots of v vs. [S]
i) Michaelis-Menten plots ii) Assembled using multiple experiments iii) Identical temp, pH, and enzyme concentration iv) Assumptions (Requirements)
(1) Soluble enzymes (2) Optimal pH for enzyme (3) Initial substrate still in high concentration (4) Increase in [E] increases plateau height
5) Reactions a) E+SàESàP+E
i) k1 is reaction rate 1 ii) k2 is reaction rate 2
iii) [ ]ESkdt
Pdv 2
][==
iv) totEkV ][2max = v) Km is [S] when ½ Vmax occurs
vi) After derivation [ ]][
][][][ 220 SK
SEkESk
dt
PdV
m
tot
+===
b) Lineweaver Burke Plot
i) Derived from above equation ii) Constants can be read right off of plot
iii) ][
111
maxmax SV
K
VVm+=
c) Eadie-Hofstree i) Specific to amperometric biosensors
ii) ][max S
VKVV m−=
d) More complex reactions also occur and modeling has been done for them
6) Immobilization Effects a) Enzymatic behavior altered b) No general trends c) Causes
i) Random orientation ii) Shielding of active site iii) Denaturation iv) Environmental effects v) Microenvironment effects
(1) Local pH (a) Possibly caused by polymer for entrapment
(2) Accessibility/tortuosity (a) Cross linking of polymer
(3) Ionic strength (4) Polarity of membrane/medium (5) Product accumulation
7) Enzyme Inhibition a) Determine concentration of inhibitor b) Competitive and noncompetitive inhibition c) Some loss of selectivity
8) Sequenced Reactions a) Improve detection
i) Easier to detect b) Amplification
i) Heat generation
c) Elimination of interferents 9) Bioaffinity sensors
a) Immunoassays b) Kinetics of “adsorption” or binding c) On and off rates d) Determine “Association” constants
e) Similar kinetics to enzymes f) F is fraction of available sites bound g) Equilibrium based
i) Take minutes to hours
Design Considerations • Use type:
– Disposable, single use, no reagents, no training – Portable, hand held, multiple use with a disposable component,
minimal training – Batch testing or sample injection measurements (Large labs) – In situ devices
• Sterilizable • Compatible with process
– Leaching • Minimal calibration • No additiional reagents
– Research devices
Transduction Modes • Electrochemical
– Potentiometric • ADV: Easily miniaturized, easy translation • DA: Reference required, limited linear range, pH sensitive
– Amperometric • ADV: Variety of analytes, easily mini, dynamic range, selectivity, • DA: Reference required, multiple membranes can be required
– Conductimetric • ADV: Simple, easy to fab, no ref, low frequency source • DA: Non-selective
• Optical – Advantages
• No reference required • Multiple modes: intensity, phase, frequency, polarization • Real time using evanescent waves • Multianalyte arrays simple • Wide range of EM spectra
– Disadvantages • Ambient light and scattering • Limited dynamic range • Miniatuization affects magnitude of signal • Limited selection of chromophores and fluorophores
• Thermal – Advantages
• Works with all reactions • Works with all solutions • Great for offline measurments • Multiple analytes easily
• Requires sorption • SAWs have been used • Neural networks for determination • Enzyme inhibition
Biocatalysis
• See book Pg. 99 for typical reactions • Optical, electrical, and thermal sensing available • Multienzyme systems
– Amplification – Recycling – Sensitivity – Elimination of interferents and products – Step beyond interferents – Suitability of sensor
Bioligand Binding
• Antibodies • DNA/RNA • Lectin and carbohydrates • May have problems with dissociation and regeneration
– Increase steps for washing, etc – May be damaging
Other Considerations
• Trace concentrations – Ultralow concentrations
• Less than 10-9 M
– Fluorescence – Recycling amperometry – ELISA
• Enzyme linked immunoassays
• Temporal Resolution – Dependent on needs – Typically 1 minute is OK
• Spatial Resolution – Confined spaces – “Near” locations – Often require micropositioning and microscopic observation
– Interactions between “sites” • Cross talk • Diffusion
Mass Transport
• Analyte must reach sensor – Internal and external components – Time required
• Equilibrium • Flow rate/reaction rate
• Diffusion – Concentration gradients
• Convection – Stirring
• Migration – External field
• Mass transport changes in vivo • Partitioning and permeability • Modeling and limits
Optical Spectroscopy for Biosensing - 12/15/99 A. Definitions
1. spectrum: an array of the components of an emission or wave, separated and arranged in the order of some varying characteristic (wavelength, frequency, mass)
2. c = λν, E=� ν, �=Planck's constant=6.626×10 -34 J· s 3. absorption, absorption coefficient 4. scatter, scattering coefficient, anisotropy
B. Absorption-based spectroscopy 1. Infrared spectroscopy
a. Theory 1) All atoms in constant relative vibration 2) Frequency of incident energy = frequency of bond vibration � absorption 3) IR active: vibrations resulting in net change in dipole moment
4) Ranges a) Far IR: 50-1,000µm, 200-10cm-1
Difference, coupling b) Mid IR: 2.5-50µm, 4,000-200cm-1
Fundamental c) Near IR: 0.78-2.5µm, 13,000-4,000cm-1
Combinations, overtones (integral multiples), coupling *Combination of all factors � Unique IR spectrum for each compound
b. Practice 1) Dispersive methods 2) FT methods - Felgett (speed), Jacquinot (throughput), Connes (internal reference) advantages 3) Transmission vs. reflectance c. Quantitation (Beer's law) I=I0e
-εlc 1) Transmittance (T): Ratio of transmitted to incident intensity (I/I0) (0-100%) 2) Absorbance (A): log10(1/T)= log10(I0/I) 3) Attenuated total reflection - evanescent wave � thick or highly absorbing samples d. Applications
MAJOR ADVANTAGES: simple to perform, high SNR (FT-IR), insensitive to scatter (at longer wavelengths), multi-component analysis*
MAJOR DISADVANTAGES: limited pathlength*, *high water absorption, *temperature dependence, *overlapping bands, *weak absorption at short wavelengths
Photoacoustic spectroscopy a. Theory 1) Energy absorbed converts to heat within sample 2) thermal expansion produces pressure waves b. Practice 1) Pulsed laser source a) Tuned to specific wavelength b) Scanned over range � spectrum 2) Focus modulated FTIR beam 2) Sensitive pressure sensor (e.g. piezoelectric) a) single detector vs. array b) time-based signal c. Quantitation d. Applications Strongly-absorbing samples
MAJOR ADVANTAGES: highly sensitive MAJOR DISADVANTAGES: more complex signal processing, prone to saturation at high absorption
2. Emission spectroscopy a. Theory
1) Blackbody radiation 2) Wien's displacement law
b. Practice Heated sample Emission observed by spectrometer Emission bands occur at same frequencies as absorption bands c. Quantitation Similar to absorbance d. Applications
1) Temperature measurement 2) Thermal imaging
MAJOR ADVANTAGES: good for thick samples MAJOR DISADVANTAGES: need to heat
3. Fluorescence Spectroscopy a. Theory 1) Photon absorption � Excitation of molecule to higher energy level 2) Subsequent re-emission of photon, wavelength shifted b. Practice 1) Only small number of molecules exhibit fluorescence 2) Use known "fluorophores" to "tag" target molecules c. Quantitation 1) F ~ concentration, Φ, intensity 2) Quenching - loss of intensity
3) May use energy transfer to advantage (Friday) 4) Photobleaching 5) Fluorescence Lifetime (Friday)
d. Applications 1) Analyte sensing 2) Imaging
MAJOR ADVANTAGES: highly specific, extremely sensitive, simple instrumentation, relatively cheap MAJOR DISADVANTAGES: requires use of exogenous probes, limited choice of chemistry*, *short wavelengths
C. Scatter-based spectroscopy 1. Scattering (Rayleigh) Spectroscopy
a. Theory 1) Rayleigh Scattering occurs with the interaction of light with an atom, or anything producing a refractive index mismatched boundary 2) Scattering strength depends upon size, shape, concentration, and relative refractive index of scatterers (theory by Mie)
b. Practice c. Quantitation d. Applications
MAJOR ADVANTAGES: cheap and easy MAJOR DISADVANTAGES: nonspecific for chemistry
2. Raman Spectroscopy a. Theory
1) Raman Scattering occurs with the interaction of light with a molecular bond. 2) Raman Scattering causes the wavelength of the light to shift
(higher = "Stokes", lower = "anti-Stokes"). 3) The intensity of the Raman Scattered light is much less than the intensity of the Rayleigh scattered light 4) The amount of the wavelength shift and intensity depends on the size, shape, and strength of the molecule. 5) Similar to infrared absorption bands, each Raman shift is a distinct "fingerprint" of the molecule.
b. Practice 1) In order to see the less powerful Raman shifted light, the Rayleigh light needs to be blocked.
2) A spectrograph is used to spread out the Raman shifted light (can do FT also) 3) A photo detector or CCD camera detects the Raman shifted light from the spectrograph.
c. Quantitation Raman Intensity ~ concentration, laser power d. Applications 1) O2, CO2, anesthesia, and other gases expelled from a surgical patient. 2) Emissions of gases into the air by power, oil, steel, and other companies. 3) ANY application where gases, liquids, or solids need to be identified and measured.
MAJOR ADVANTAGES: no sample prep, can get depth-resolved information, PPM detection MAJOR DISADVANTAGES: need laser&expensive filters, weak signals, high fluorescence background, scatter dependence
D. Calibration 1. Univariate a. Peak height b. Area under the curve c. Ratios 2. Multivariate Calibration
a. Classical b. Factor-based methods
3. Wavelength selection
Optical Glucose Monitors - 12/17/99 General Advantages of Optical Biosensors Potentially Noninvasive No Electrical Connections High Bandwidth - Information Density Raman spectroscopy Need low-scattering site - Eye Polarimetry Relies upon rotation of plane of polarization by "chiral" molecules Polarized laser source, polarizers aligned at 90-degrees
àintensity increases as sample rotates polarization Need low-scattering/low-birefringence site - Eye? Other species are chiral, not just glucose Near-infrared spectroscopy Many companies working on this... “Biocontrol” Many papers published No convincing evidence of specific measurements in vivo More useful in process control…monitoring cell culture growthà can measure multiple species
simultaneously Fluorescence spectroscopy
Highly-specific (Probe Required) Invasive - Probe chemistry at tip of optical fiber Minimally Invasive - Implanted glucose-sensitive particles, interrogated transdermally Glucose assay Excited with 488nm
FITC-dextran (520nm emission) and TRITC-ConA (580nm emission) Dextran competes with glucose for ConA binding sites à dextran displaced by glucose Energy transfer from FITC to TRITC à relative shift in peak intensity 520nm peak increases with glucose concentration Currently, poor reversibility with this assay (others are available) Optics of the system relatively good
*Efficiency may be increased by using two-photon excitation
Optical Biosensors • Optical means “electromagnetic” • Types of measurements
– Reflected light from thin film interferes with light reflected from other surface
– Small changes in thickness can be measured • Adsorbed or bound analyte
• Near IR – Chemometric spectroscopy – Heavily analysis dependent
Fiber Optic Devices • Wide range of optical fibers
– Application depends on light and spectral components – Match to source and detector
• Arrays of fibers can give multicomponent info and spatial resolution • Cladding (IR smaller than fiber) used to reduce signal loss down fiber • Guided waves
– Refractive index changes determineactivity – Large angles: refraction – Critical angle: Total internal reflectance – Small angle: reflection – “Cone of acceptance” or Numerical Aperture (NA) – Modes
Evanescent Wave • Total internal reflection generates interference • Creates a standing wave • Wave extends beyond the boundary of the wave guide
– Evanescent wave • Exponentially decays from surface
– Must be close – Less than 100-200 nm – Ideal for chemically bound layers
• Cladding may be stripped and replaced with biomembrane, etc
Fiber Optic Design • See page 274 of text • Optical fiber carries light to biocomponent
– Absorbance or fluorescence is measured • Light brought in by one fiber, collected by another • Evanescent wave along fiber • Planar wave guide • Fiber bundles
• Generally very versatile
Planar Waveguides • Similar to fiber optic devices • Can add coupling prisms
Near Field Sensing • Eliminates need for high energy beams • Improved resolution • Collection through small appertures • Small magnitudes • Micropositioning
Surface Plasmon Resonance • Works with thin films • Most useful for Biospecific Interaction Analysis (BIA)
– Kinetics of binding • Requires polarized light • Only on metal surface
– Sea of electrons – Waves on “sea” absorb light at specific angles
• Quantum mechanical detection • Only occurs with light at an angle
– Angle depends on film thickness and refractive index – Very sensitive
• Experimentally observed as sharp minimum in intensity measurement
SPR Applications • Gold and silver films most often used
– About 5 nm in thickness • Proteins or antibodies bound to surface • Light injected from opposite surface
– Must be polarized- controlled to thousandths of a degree • Light undergoes total internal reflection • Evanescent wave couples to opposite layer • Reflected light monitored
– Shifts indicate change in film thickness
Optical Sol-Gel Sensors • Transparent gels for entrapment of biochemicals • Allow direct spectrophotometric measurements • Reactants diffuse into gel • Absorbance or luminescence measurements possible • Response time diffusion dependent • Entrapment has little effect on biomolecules • Limited to low molecular weight analytes
Luminescence • Requires light generating molecules • High energy molecules or systems • Chemiluminescence most fundamental • Bioluminescence is enzymatically catalyzed chemiluminescence • Energetics
– Blue (400 nm) 70 kcal/mol – Red (700 nm) 40 kcal/mol
– ATP 7 kcal/mol – ROOR- 100 kcal/mol
Chemiluminescence • Most basic is luminol • Generates a peroxide in presence of oxygen • Works in any assay involving oxygen, peroxide or peroxidase • Can be linked with fluorescence to make dual measurements • All chemiluminescent systems are also fluorescent • Need highly exothermic reaction • Quantum mechanical pathway required • Usually ring structures • All use oxygen
a) Basic materials i) Au, Pt, C, Carbon composites, and conducting salts
(1) Material limits range of applied voltages ii) Solvent can also limit range of applied voltage
b) Operation i) Potential step
(1) Applied potential critical to experiment (a) Overpotential may be required
(i) Varies with material (ii) Highly empirical
(b) Scanning methods may be helpful to find optimum
(2) Current measured for ms to seconds (a) Double layer charging and electron transfer
(3) Chronoamperometry (a) Current with time
(4) Chronocoulometry (a) Current is integrated to get charge transfer
(5) Amperometry (a) Sample current at specific time
ii) Mass transport governs (1) Diffusion through membrane (2) Alleviated by allowing electrode to go to
steady state before introducing analyte
iii) iv) Can measure variety of species (see sensing
options) (1) Note limitations of each sensing option
(2)
(3)
(4) Cyclic voltammetry
(5)
1) Electroanalytical Biosensors a) Involve charge and electron transfer
i) Potential ii) Current iii) Conductance iv) Impedance
b) Most common for practical devices c) Wide variety of methods (see chart page
208) d) Amperometry
i) Current measurement e) Voltammetry
i) Current as a function of variable voltage f) Chronoamperometry
i) Current as a function of time at constant voltage
2) Electrochemistry
a) Electrochemical cell b) Galvanic
i) Spontaneous reaction generates current or voltage
c) Electrolytic i) External energy source ii) Current flows related to
oxidation/reduction levels d) Cyclic voltammetry
i) Scan between voltages e) Nernst potential and equation
i) O+neàR ii) [ ]
[ ]R
O
nF
RTEE ln+= o
iii) Standard hydrogen electrode is basis iv) Practical use, silver/silver chloride or
calomel (Hg) (1) Typically difficult in practice,
especially in vivo (2) Short duration
v) Microsystems avoid some of the problem by using low currents
3) Potentiometric sensors
a) Ideal for ions and dissolved gases that produce ions
b) No current flow (critical assumption) c) Systems, while simple in appearance,
often quite complex d) Polymer membrane
i) Limit access ii) Selective to specific ions
e) Solid state i) Must convert ions to electrons ii) Problems with stability and
reproducibility iii) Metal wires with applied films
iv)
4) Field Effect Transistors
a) Current from source to drain related to gate voltage
b) Application of membranes to gate allows selective measurements
c) Problems i) Membrane adhesion ii) pH sensitivity iii) Drift iv) Coatings can help eliminate all v) Nonlinear
1) Amperometric Sensors (cont) a) Oxygen species
i) Several potentials at which charge transfer occurs ii) Some byproducts are highly toxic iii) Addition of appropriate enzymes can limit toxicity
b) NADH i) Involve dehydrogenases ii) Potentially hundreds of related reactions iii) Requires soluble coenzyme
(1) External reagent (2) Expensive (3) Difficult
iv) Mediators improve process (1) Enhance electron transfer (2) Lower required voltage (3) Coating on electrode
c) Detection limits i) Very low detection limits
(1) Especially with amplification ii) Immunosensors with attached enzymes
2) Other Electronic Biosensors a) Conductivity and Impedance
i) Inherently non-selective ii) Require modified surfaces for selectivity iii) No reference electrodes required iv) Easy and inexpensive to fabricate
b) Most basic configuration is Wheatstone bridge c) Both time and frequency domain measurements possible d) Involve both capacitive and resistive elements
i) Magnitude and phase measurements e) Added enzyme sensors paired with reference for differential measurement
i) Several relevant enzymes have been used f) One example included pH sensitive hydrogel on surface of electrodes
i) Change in pH changed resistance through membrane g) Impedance Spectroscopy
i) Impedance sweeps ii) Similar applications to optical spectroscopy
(1) Not as “varied” 3) Bioelectric Interfaces
a) Measurement of electrical activity in the body b) Generally very low signal levels c) Non-selective d) Neural Recording Arrays
i) Attempt to access multiple neurons simultaneously ii) Attempts at cortical prostheses iii) Used for both recording (sensor) and stimulation
Microfluidic Sensors Example University of Washington H Sensor
Methods for Fabrication KOH etching of silicon Other etching methods (High Aspect Ratio) LIGA Molding in PDMS Interesting Ideas Bubbles for added power CD systems Systems in Ceramics, Polymers, Silicon Surface tension valves Problems Valves Pumps Seals Mixing Diffusion
1) Antibodies a) Produce by body in response to antigens b) Have specific binding domains
i) Epitope or determinant c) Y shaped in general
i) Symmetric d) Heavy and light chains e) Binding site at tip of Y
i) Made up of 20-30 amino acid sequence f) Base of Y involved in activation of
complement and other immune components g) Polyclonal
i) Antibodies from a variety of immune cells that bind to an antigen
ii) Usually have different epitope h) Monoclonal antibodies
i) All produced by same cell ii) Generated by cloning iii) These are typically used in biosensors
2) Immunoassay
a) Based on Ag-Ab binding b) Homogenous
i) No washing steps ii) Free and bound antibody does not need
to be separated iii) Usually binding causes change that
allows bound and unbound antibody to be distinguished
c) Heterogenous i) Require washing and separation ii) Displacement
(1) All sites are filled with labeled antigen
(2) Unlabeled antigen displaces labeled antigen
iii) Competitive (1) Labeled and unlabeled antigen added
simultaneously (2) Amount of labeled antigen bound
gives info iv) Sandwich
(1) Capture antibody is immobilized (2) Analyte is added and bound to
antibody (3) A labeled antibody is added
generating signal (4) Requires antigen with two
determinants d) ELISA
i) Enzyme linked immunoassay
1) Random Homogenous Immunosensor Types a) CEDIA
i) Clone enzyme donor immunoassay ii) Engineered antibodies and enzymes iii) Competitive assay iv) Existence of analyte allows active
enzymes to form v) Convert chromogenic substrate to dye vi) Linear
(1) Most competitive assays are non-linear
vii) Signal proportional to analyte present b) ARIS
i) Apoenzyme reactivation immunosystem (1) Competitive assay (2) Allows enzyme activation in the
presence of antigen c) SLFIA
i) Optical system ii) Enzyme release fluorogenic substance in
presence of analyte iii) If not bound to antigen, antibody blocks