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SENSORS
by
Luciana V. Ilao
Associate Professor in ChemistryDepartment of Physical Sciences and Mathematics
University of the Philippines Manila
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What is a sensor?
It is a device that detects or measures a physical property and records, indicates or otherwise responds to it.
Types of sensorsA. physical sensors: for measuring distance, mass,
temperature, pressure, etc.; B. chemical sensors: measure chemical
substances by chemical or physical responses; and
C. biosensors which measure chemical substances by using a biological sensing element.
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BiosensorsA biosensor is an analytical device which converts a biological response into an electrical signal (Figure 1).
The term 'biosensor' is often used to cover sensor devices used in order to determine the concentration of substances and other parameters of biological interest even where they do not utilize a biological system directly.
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Figure 1. Schematic of a Biosensor
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Analogy with the nose as asensor (actually a biosensor),
Olfactory membrane - biological recognition element
Nerve cell - transducer, Nerve fibre - actuator, i.e., the device that produces
the displayBrain - measuring element.
*Eggins, B. R., Biosensors: An Introduction, Copyright
1996. John Wiley & Sons Limited.
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Figure 2. Schematic diagram showing the main components of a biosensor. The biocatalyst (a) converts the substrate to product. This reaction is determined by the transducer (b) which converts it to an electrical signal. The output from the transducer is amplified (c), processed (d) and displayed (e).
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Aspects of Sensors
Recognition Element• Key component of any sensor device. • Impart the selectivity that enables the sensor to respond selectively to a particular analyte or group of analytes, thus avoiding interferences from other substances. • In biosensors, the most common recognition element is an enzyme. Others include antibodies, nucleic acids and receptors.
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Figure 3. Biosensor/Biochip Classification Scheme
Biosensors/Biochips
Bioreceptor Transducer
Antibody
Enzyme
DNA
Cell/Tissue
Optical
CellularSystems
Other
Mass–based
Electrochemical
Biomimetic
Non–enzymaticproteins
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(Recognition Element)
A. Biocatalytic Recognition Element The biosensor is based on a reaction catalysed
by macromolecules, which can be: present in their original biological
environment; have been isolated previously; or have been manufactured.
A continuous consumption of substrate(s) is achieved by the immobilized biocatalyst incorporated into the sensor.
Transient or steady-state responses are monitored by the integrated detector.
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Recognition Element Types of biocatalyst commonly used:
A. Enzyme (mono-or multi-enzyme): the most common and well-developed recognition system;
B. Whole cells (micro-organisms, such as bacteria, fungi, eukaryotic cells or yeast) or cell organelles or particles (mitochondria, cell walls);
C. Tissue (plant or animal tissue slice).
One or more analytes react in the presence of enzyme(s), whole cells or tissue culture and yield one or several products according to the general reaction scheme:
biocatalystS + S0 P + P0
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(Recognition Element)
Strategies that use adjacent transducers for monitoring the analyte consumption by biocatalysed reaction:
1. detection of the co-substrate (S0) consumption, e.g. oxygen depleted by oxidase, bacteria or yeast reacting layers, and the corresponding signal decrease from its initial value;
2. recycling of one of the reaction products (P), e.g. hydrogen peroxide, H, CO2, NH3, etc., production by oxidoreductase, hydrolase, lyase, etc., and corresponding signal increase;
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(Recognition Element)Strategies that use adjacent transducers
3. detection of the state of the biocatalyst redox active centre, cofactor, prosthetic group evolution in the presence of substrate S, using an immobilized mediator which reacts sufficiently rapidly with the biocatalyst and is easily detected by the transducer; e.g. various ferrocene derivatives, as well as tetrathiafulvalene-tetracyanoquinodimethane (TTF+; TCNQ-) organic salt, quinones, quinoid dyes, Ru or Os complexes in a polymer matrix
4. direct electron transfer between the active site of a redox enzyme and the electrochemical transducer.
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(Recognition Element) Strategies for improving biosensor performancewhen several enzymes are immobilized within the same reaction layer:1. Several enzymes facilitate the biological
recognition sequentially converting the product of a series of
enzymatic reactions into a final electroactive form set-up allows a much wider range of possible
biosensor analytes;2. Multiple enzymes, applied in series may regenerate the first enzyme co-substrate a real amplification of the biosensor output signal
may be achieved by efficient regeneration of another co-substrate of the first enzyme.
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(Recognition Element) Strategies for improving biosensor performance (cont’d)
3. multiple enzymes, applied in parallel may improve the biosensor selectivity by
decreasing the local concentration of electrochemical interfering substance
an alternative to the use of either a permselective membrane or a differential set-up, i.e. subtraction of the output signal generated by the biosensor and by a reference sensor having no biological recognition element
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Biocomplexing or bioaffinity recognition element
biosensor operation is based on the interaction of the analyte with macromolecules or organized molecular assemblies that have either been isolated from their original biological environment or engineered;
Equilibrium is usually reached and there is no further net consumption of the analyte by the immobilized biocomplexing agent.
Equilibrium responses are monitored by the integrated detector.
In some cases, this biocomplexing reaction is monitored using a complementary biocatalytic reaction.
Steady-state or transient signals are then monitored by the integrated detector.
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Antibody–antigen interaction
The most developed examples of biosensors using biocomplexing receptors are based on immunochemical reactions, i.e. binding of the antigen (Ag) to a specific antibody (Ab).
Formation of such Ab±Ag complexes has to be detected under conditions where non-specific interactions are minimized.
Each Ag determination requires the production of a particular Ab, its isolation and, usually, its purification.
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Aspects of Sensors (cont’d)
Transducer – the detector device (shown as the 'black box' in Figure 2) which makes use of a physical change accompanying the reaction.
A. Electrochemical Transducers 1. Potentiometric. • Based on changes in the distribution of charges
causing an electrical potential to be produced (potentiometric biosensors)
• Involve the measurement of the emf (potential) of a cell at zero current.
• The emf is proportional to the logarithm of the concentration of the substance being determined.
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Aspects of Sensors (cont’d)
2. Voltammetric • Based on the movement of electrons produced
in a redox reaction • An increasing (decreasing) potential is applied to
the cell until oxidation (reduction) of the substance to be analyzed occurs and there is a sharp rise (fall) in the current to give a peak current.
• The height of the peak current is directly proportional to the concentration of the electroactive material.
• If the appropriate oxidation (reduction) potential is known the current may be observed at the potential. This mode is known as amperometric.
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Aspects of Sensors (cont’d)
3. Conductometric. Involves measurement of change in electrical conductivity that results from the composition of the solution.
4. FET-based sensors. Electrochemical transducers on a silicon chip-based field-effect transistor.
• Mainly been used with potentiometric sensors, but could also be used with voltammetric or conductometric sensors.
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Aspects of Sensors (cont’d)
B. Optical Transducers • Based on the light output during the reaction
or a light absorbance difference between the reactants and products
• include absorption spectroscopy, fluorescence spectroscopy, luminescence spectroscopy, internal reflection spectroscopy, surface plasmon spectroscopy and light scattering.
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Aspects of Sensors (cont’d)
C. Piezo-electric Devices • Based on effects due to the mass of the
reactants or products (piezo-electric biosensors).
• Devices that involve the generation of electric currents from a vibrating crystal.
• The frequency of vibration is affected by the mass of material adsorbed on its surface, which could be related to changes in a reaction.
• Includes surface acoustic wave devices
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Aspects of Sensors (cont’d)
D. Thermal Sensors • Based on the production or absorption of heat
during chemical and biochemical processes. • The heat can be measured by sensitive
thermistors and hence be related to the amount of substance to be analyzed.
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Fig. 3. Configuration of a biosensor showing biorecognition, interface, and transduction elements.
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Table 1. Biosensor Components
Transducer Measurement Typical System Mode Applications
Ion-Selective Potentiometric Ions in biological Electrode media, enzyme
electrodes
Gas-Sensing Potentiometric Gases, enzyme, Electrodes organelle, cell or tissue electrodes
Field-Effect Potentiometric Ions, gases,Transistors enzyme substrates immunological
analytes
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Optoelectronic Optical pH; enzymes; and Fiber–Optic immunologicalDevices analytes
Thermistors Calorimetric Enzyme, organelle, gases, pollutants,
antibiotics, vitaminsEnzyme Electrodes Amperometric Enzymes,
immunological systems
ConductimetricConductance Enzyme substrates
Piezoelectric Acoustic (mass) Volatile gases and Crystals vapors, antibodies
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Beneficial features of a Biosensor:
1. Biocatalyst highly specific for the purpose of the
analyses, stable under normal storage conditions, and show good stability over a large number of
assays, i.e., much greater than 100 (except in the case of colorimetric enzyme strips and
dipsticks)
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Beneficial features of a Biosensor(cont’d):
2. Reaction should be as independent of physical parameters
such as stirring, pH and temperature as is manageable for minimal samples pre-treatment
3. Response determined by the biocatalytic membrane which
accomplishes the conversion of reactant to product
should be accurate, precise, reproducible and linear over the useful analytical range, without dilution or concentration
should be free from electrical noise.
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Beneficial features of a Biosensor (cont’d):
4. The probe must be: tiny and biocompatible, having no toxic or
antigenic effects should be sterilisable (preferably by autoclaving
but no biosensor enzymes can presently withstand such drastic wet-heat treatment)
should not be prone to fouling or proteolysis. be cheap, small, portable and capable of being
used by semi-skilled operators.
5. There should be a market for the biosensor.
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Immobilization of Enzymes
The biorecognition element has to be properly attached to the transducer to make a functional biosensor
Qualities of a Good Immobilization Technique:1. It should not reduce the activity or the
specificity of the biorecognition element.2. The stability of the element should be
maintained or increased in order to make the biosensor reusable.
3. The method should be easily repeatable so that large scale production is possible.
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Principal methods for enzyme immobilization
a. Adsorption to the surface can either be physical or chemical generally used only for short term
applications because it is a relatively simple process that requires no reagents making it easy to setup
Disadvantage: Not easily reproduced and the resulting biosensor does not keep its properties over time(Diamond; Eggins).
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• Physical adsorption
* can take place on solid surfaces such as alumina, charcoal, clay, cellulose, silica gel, glass, and collagen.
* Uses hydrogen bonds, van der Waals forces, salt linkages, and hydrophobic interactions.
* The resulting bonds are not stable and are susceptible to changes in pH, temperature, and ionic strength
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Chemical adsorption * Utilizes a nucleophilic group to couple the
biorecognition material to the transducer or membrane.
* The resulting coupling bonds are typically covalent bonds.
* The nucleophilic group is specifically chosen so that it does not contribute to the overall catalytic activity of the biorecognition element.
* Common nucleophilic groups used: NH2, CO2H, OH, C6H4OH, SH, and the heterocyclic imidazole..
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b. Covalent binding: Covalent chemical bonds are formed between the selective component and the transducer.
* Cross-linking, a multifunctional crosslinking reagent is used to bind the biomaterial to solid supports on the transducer surface.
* Often used in the stabilization of adsorbed proteins, where a reagent is used to link inert proteins together.
* Adds greater stability to the system. * Generally damage to the enzyme and has poor
mechanical strength. * Should only be used in short term application.
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covalent binding(cont’d)
* Should not be used when antibodies are the biorecognition element because it can orient the antibody binding site so that antibody-antigen binding will not be favored and will result in an inaccurate sensor (Diamond; Eggins).
*Common molecules used for cross-linking (Eggins).
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Enzyme Immobilization (cont’d)
c. Entrapment * Where the selective element is physically
entrapped in a matrix of a gel, paste or polymer * Typical materials used for this outer gel matrix
are polyacrylamide, polyvinyl alcohol, polyvinyl chloride, epoxy, sol-gel processed glass, or a Langmuir-Blodgett film (Prasad).
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d. Membrane confinement (Microencapsulation) * Trapping between membranes – one of the
earliest methods to be employed.* Membranes with varying porosities are used to
entrap the biorecognition material close to the transducer surface.
* Keeps the biorecognition material close to the transducer while not actually immobilizing the material.
* Tends to be very reliable and adaptable. * Disadvantage: Diffusional resistance through the
membrane which can be used as an advantage by reducing the pore size so only molecules of the target analyte can pass through the membrane.
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Fig.5. The four principal methods for enzyme immobilization
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Advantageous features of immobilized enzymes:
1. They may be re-used, which ensures that the same catalytic activity is present for a series of analyses. This is an important factor in securing reproducible results and avoids the pitfalls associated with the replicate pipetting of free enzyme otherwise necessary in analytical protocols.
2. Many are intrinsically stabilized by the immobilization process
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Advantageous features of immobilized enzymes(cont’d):
3. An excess of enzyme within the immobilised sensor system gives a catalytic redundancy (i.e. h << 1) that is sufficient to ensure an increase in the apparent stabilization of the immobilized enzyme.
4. Lower cost relative to the analytical usage of free
soluble enzymes.
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Performance Factors of a Biosensor
1. Selectivity. • The most important characteristic of sensors • The ability to discriminate between different substances. • Principally a function of the selective component, although
sometimes the operation of the transducer contributes to the selectivity.
2. Sensitivity range. • The change in response per unit change in concentration of
analyte • Usually needs to be sub-millimolar, but in special cases can
go down to the femtomolar (10-15 M) range.3. Accuracy. This needs to be better than ±5%.
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Performance Factors (cont’d)
4. Nature of solution. Conditions such as pH, temperature and ionic strength.
5. Response time. Usually much longer (30 s or more) with biosensors than with chemical sensors.
6. Recovery time. The time that elapses before the sensor is ready to analyze the next sample – it must not be more than a few minutes.
7. Working lifetime is usually determined by the stability of the selective material. For biological materials this can be a short as a few days, although it is often several months or more.
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Generations of biosensors (Fig.4):
First generation biosensors: the normal product of the reaction diffuses to the transducer and causes the electrical response;
Second generation biosensors: involve specific 'mediators' between the reaction and the transducer in order to generate improved response; and
Third generation biosensors: the reaction itself causes the response and no product or mediator diffusion is directly involved.
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Fig.4. Amperometric biosensors for flavo-oxidase enzymes illustrating the three generations in the development of a biosensor. The biocatalyst is shown schematically by the cross-hatching.
All electrode potentials (E0) are relative to the Cl-/AgCl,Ag0 electrode.
The following reaction occurs at the enzyme in all three biosensors:
Substrate(2H) + FAD-oxidase Product + FADH2-oxidase
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Fig.4a. First generation electrode utilising the H2O2 produced by the reaction. (E0=+0.68 V).
biocatalyst:
FADH2-oxidase + O2 FAD-oxidase + H2O2
electrode: H2O2 O2 + 2H+ + 2e-
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Fig 4b. Second generation electrode utilising a mediator (ferrocene) to transfer the electrons, produced by the reaction, to the electrode. (E0= +0.19 V).
Biocatalyst:
FADH2-oxidase + 2 Ferricinium+ FAD-oxidase + 2
Ferrocene + 2H+
electrode : 2 Ferrocene 2 Ferricinium+ + 2e-
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Fig 4c.Third generation electrode directly utilising the electrons produced by the reaction. (E0= +0.10 V).
biocatalyst/electrode
FADH2-oxidase FAD-oxidase + 2H+ + 2e-
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Calorimetric Biosensors
The most generally applicable type of biosensor
based on heat evolved by exothermic reactions (Table 2).
may be used as a basis for measuring the rate of reaction and the analyte concentration.
Temperature changes are usually determined by means of thermistors at the entrance and exit of small packed bed columns containing
immobilised enzymes within a constant temperature environment (Figure 5).
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Calorimetric Biosensors
Registers up to 80% of the heat generated in the reaction under closely controlled conditions as a temperature change in the sample stream.
Can cause a change in temperature of the solution amounting to approximately 0.02°C (the temperature change commonly encountered)
Necessitates a temperature resolution of 0.0001°C for the biosensor to be generally useful.
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Table 2. Molar enthalpies of enzyme catalyzed reactions.Reactant Enzyme H (kJ mole-1)
Cholesterol Cholesterol oxidase – 53
Esters Chymotrypsin – 4 – 16
Glucose Glucose oxidase – 80
Hydrogen peroxide Catalase – 100
Penicillin G Penicillinase – 67
Peptides Trypsin – 10 - 30
Starch Amylase – 8
Sucrose Invertase – 20
Urea Urease – 61
Uric acid Uricase – 49
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Figure 5. Schematic diagram of a calorimetric biosensor. The sample stream (a) passes through the outer insulated box (b) to the heat exchanger (c) within an aluminium block (d). From there, it flows past the reference thermistor (e) and into the packed bed bioreactor (f, 1mL volume) containing the biocatalyst, where the reaction occurs. The change in temperature is determined by the thermistor (g) and the solution passed to waste (h). External electronics (l) determines the difference in the resistance, and hence temperature, between the thermistors.
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The thermistors used to detect the temperature change, function by changing their electrical resistance with the temperature, obeying the relationship
ln(R1/R2)=B(1/T1 - 1/T2)
therefore:(R1/R2)=e(B(1/T1 – 1/T2))
where:R1 and R2 are the resistances of the thermistors at
absolute temperatures T1 and T2 respectively; and
B is a characteristic temperature constant for the thermistor.
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When the temperature change is very small
B(1/T1) – (1/T2) << 1
the relationship may be simplified by the approximation
when x<<1 that ex 1 + x (where x is B(1/T1) – (1/T2),
R1=R2 {1 + B [(T2 – T1) / T1T2]}
As T1 T2, they both may be replaced in the
denominator by T1.
R/R = – (B/T12)T
The relative decrease in the electrical resistance (R/R) of the thermistor is proportional to the increase in temperature (T).
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The sensitivity (10-4 M) and range (10-4 - 10-2 M) of thermistor biosensors are both quite low for the majority of applications although greater sensitivity is possible using the more exothermic reactions (e.g. catalase).
The low sensitivity of the system can be increased substantially by increasing the heat output by the reaction. This is achieved by linking together several reactions in a reaction pathway, all of which contribute to the heat output.
Ex. The sensitivity of glucose analysis using glucose oxidase can be more than doubled by co-immobilisation of catalase within the column reactor in order to disproportionate the hydrogen peroxide produced. An extreme case of this amplification is shown in the following recycle scheme for the detection of ADP.
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Potentiometric Biosensors
These make use of ion-selective electrodes in order to transduce the biological reaction into an electrical signal.
Consists of an immobilised enzyme membrane surrounding the probe from a pH-meter (Figure 4), where the catalysed reaction generates or absorbs hydrogen ions (Table 3).
The reaction occurring next to the thin sensing glass membrane causes a change in pH which may be read directly from the pH-meter's display.
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Figure 6. A simple potentiometric biosensor. A semi-permeable membrane (a) surrounds the biocatalyst (b) entrapped next to the active glass membrane (c) of a pH probe (d). The electrical potential (e) is generated between the internal Ag/AgCl electrode (f) bathed in dilute HCl (g) and an external reference electrode (h).
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Types of ion-selective electrodes which are of use in biosensors:
1. Glass electrodes for cations (e.g. normal pH electrodes) in which the sensing element is a very thin hydrated glass membrane that generates a transverse electrical potential due to the concentration-dependent competition between the cations for specific binding sites.
The selectivity of this membrane is determined by the composition of the glass. The sensitivity to H+ is greater than that achievable for NH4
+
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Types of ion-selective electrodes which are of use in biosensors:2. Glass pH electrodes coated with a gas-permeable
membrane selective for CO2, NH3 or H2S. The diffusion of the gas through this membrane causes a
change in pH of a sensing solution between the membrane and the electrode which is then determined.
3. Solid-state electrodes where the glass membrane is replaced by a thin membrane of a specific ion conductor made from a mixture of silver sulfide and a silver halide.
The iodide electrode is useful for the determination of I- in the peroxidase reaction (Table 3c) and also responds to cyanide ions.
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Table 3. Reactions involving the release or absorption of ions that may be utilised by potentiometric biosensors.(a) H+ cation glucose oxidase H2O
D–glucose + O2 D–glucono–1,5–lactone + H2O2 D–gluconate + H+
PenicillinasePenicillin penicilloic acid + H+
urease (pH 6.0)a
H2NCONH2 + H2O + 2H+ 2NH4+ + CO2
urease (pH 9.5)b
H2NCONH2 + 2H2O 2NH3 + HCO3– + H+
Lipaseneutral lipids + H2O glycerol + fatty acids + H+
aCan also be used in NH4+ and CO2(gas) potentiometric biosensors.
bCan also be used in an NH3(gas) potentiometric biosensor
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Table 3. Reactions involving the release or absorption of ions that may be utilised by potentiometric biosensors(cont’d).
(b) NH4+ cation
L–amino acid oxidaseL–amino acid + O2 + H2O keto acid + NH4
+ + H2O2
AsparaginaseL–asparagine + H2O L–aspartate + NH4
+
urease (pH 7.5)H2NCONH2 + 2H2O + H+ 2NH4
+ + HCO3–
(c) I– anion Peroxidase
H2O2 + 2H+ + 2I– I2 + 2H2O
(d) CN-anion,–glucosidase
amygdalin + 2H2O 2glucose + benzaldehyde + H+ + CN–
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The response of an ion–selective electrode is given by
= + RT (ln[i]/zF) where: = the measured potential (in volts)
= characteristic standard potential for the ion–selective/external electrode system
R = the gas constant T = absolute temperature (K) z = the signed ionic charge F = Faraday’s constant [i] = concentration of free uncomplexed ionic species (strictly, [i] should be the activity of the ion but at the concentrations
normally encountered in biosensors, this is effectively equal to the concentration).
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This means, for example, that there is an increase in the electrical potential of 59 mv for every decade increase in the concentration of H+ at 25°C.
The logarithmic dependence of the potential on the ionic concentration is responsible both for the wide analytical range and the low accuracy and precision of these sensors.
Their normal range of detection is 10– 4 – 10– 2 M, a minority are ten–fold more sensitive.
Typical response time are between one and five minutes allowing up to 30 analyses every hour.
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Biosensors that involve H+ release or utilisation necessitate the use of very weakly buffered solutions (i.e. < 5 mM) if a significant change in potential is to be determined.
The relationship between pH change and substrate concentration is complex, including other such non-linear effects as pH-activity variation and protein buffering.
However, conditions can often be found where there is a linear relationship between the apparent change in pH and the substrate concentration.
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A recent development from ion–selective electrodes is the production of ion–selective field effect transistors (ISFETs) and their biosensor use as enzyme–linked field effect transistors (ENFETs, Fig. 7).
Enzyme membranes are coated on the ion–selective gates of these electronic devices; the biosensor responds to the electrical potential change via the current output.
These are potentiometric devices although they directly produce changes in the electric current.
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Main advantage of ENFETS: their extremely small size (<< 0.1 mm2) which allows cheap mass–produced fabrication using integrated circuit technology.
As an example, a urea-sensitive FET (ENFET containing bound urease with a reference electrode containing bound glycine) has been shown to show only a 15% variation in response to urea (0.05 - 10.0 mg ml-1) during its active lifetime of a month.
Several analytes may be determined by miniaturised biosensors containing arrays of ISFETs and ENFETs.
The sensitivity of FETs, however, may be affected by the composition, ionic strength and concentrations of the solutions analysed.
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(0.1 mm thick) of silica (SiO2) which forms the gate of the FET. Above this gate is an equally thin layer of H+ -sensitive material (e.g. tantalum oxide), a protective ion selective membrane, the biocatalyst and the analyte solution, which is separated from sensitive parts of the FET by an inert encapsulating polyimide photopolymer. When a potential is applied between the electrodes, a current flows through the FET dependent upon the positive potential detected at the ion-selective gate and its consequent attraction of electrons into the depletion layer. This current (I) is compared with that from a similar, but non-catalytic ISFET immersed in the same solution. (Note that the electric current is, by convention, in the opposite direction to the flow of electrons).
Fig.7. Schematic diagram of the section across the width of an ENFET. The actual dimensions of the active area is about 500 mm long by 50 mm wide by 300 mm thick. The main body of the biosensor is a p-type silicon chip with two n-type silicon areas; the negative source and the positive drain. The chip is insulated by a thin layer
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Amperometric Biosensors
These biosensors function by the production of a current when a potential is applied between two electrodes.
They generally have response times, dynamic ranges and sensitivities similar to the potentiometric biosensors.
The simplest amperometric biosensors in common usage involve the Clark oxygen electrode
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The Response of an Amperometric Biosensor
The current (i) produced by such amperometric biosensors is related to the rate of reaction (vA) by the expression:
i = nFAvA
where: n = the number of electrons transferredA = electrode area; and F = Faraday’s constant.
Usually the rate of reaction is made diffusionally controlled by use of external membranes. Under these circumstances the electric current produced is proportional to the analyte concentration and independent both of the enzyme and electrochemical kinetics.
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Fig. 8. Schematic diagram of a simple amperometric biosensor. A potential is applied between the central platinum cathode and the annular silver anode. This generates a current (I) which is carried between the electrodes by means of a saturated solution of KCl. This electrode compartment is separated from the biocatalyst (here shown glucose oxidase, GOD) by a thin plastic membrane, permeable only to oxygen. The analyte solution is separated from the biocatalyst by another membrane, permeable to the substrate(s) and product(s). This biosensor is normally about 1 cm in diameter but has been scaled down to 0.25 mm diameter using a Pt wire cathode within a silver plated steel needle anode and utilizing dip-coated membranes.
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Methods of Optical Transduction
Properties of optical signal produced by thebiorecognition of an analyte:1. Phase change: Happens when the real part of the
biorecognition material’s refractive index changes. * Shown as a polarization change of the input light
source, a change in the propagation characteristics, or a change in the optical field distribution.
2. Amplitude changes: Caused by absorption or reflection of the input light.
* Shown as a loss of intensity in the output light as compared to the input source.
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Methods of Optical Transduction(cont’d)
3. Frequency changes of the output light: Can be measured using a fluorescent dye that fluoresces in the presence of an analyte with a frequency that is Stokes-shifted away from the original sensing light.
* Raman scattering can also be used which also uses the same principle of a Stokes-shifted frequency but it is due to vibrational excitation.
4. Frequency shift: Can be caused by a nonlinear optical interaction such as second-harmonic generation in which the output beam is doubled from what the input beams were.
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Types of optical biosensors
1. fiber optic2. planar waveguide3. evanescent wave 4. interferometric 5. Surface plasmon resonance
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Diagram of a fiber based optical biosensor Wolfbeis).
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Main Areas of Development
1. Involves determining changes in light absorption between the reactants and products of a reaction;
Usually involve the widely established, if rather low technology, use of colorimetric test strips which are disposable single–use cellulose pads impregnated with enzyme and reagents. The most common use of this technology is for whole–blood
monitoring in diabetes control where the strips include glucose oxidase, horseradish peroxidase (EC 1.11.1.7) and a chromogen (e.g. o-toluidine or 3,3',5,5'-tetramethylbenzidine). The hydrogen peroxide, produced by the aerobic oxidation of glucose oxidising the weakly coloured chromogen to a highly coloured dye.
peroxidasechromogen(2H) + H2O2 dye + 2H2O
The dyed strips are evaluated by the use of portable reflectance meters or direct visual comparison with a colored chart.
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A biosensor involving luminescence uses firefly luciferase (Photinus–luciferin 4–monooxygenase (ATP–hydrolysing) to detect the presence of bacteria in food or clinical samples.
Bacteria are specifically lysed and the ATP released (roughly proportional to the number of bacteria present) react with D–luciferin and oxygen in a reaction that produces yellow light in high quantum yield.
luciferase
ATP + D–luciferin + O2 oxyluciferin + AMP + pyrophosphate +
CO2 + light (562 nm) The light produced may be detected photometrically by use
of high-voltage and expensive photomultiplier tubes or low–voltage cheap photodiode systems.
2. Involves measuring the light output by a luminescent process.
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Piezo-electric crystals (e.g. quartz) vibrate under the influence of an electric field.
The frequency of this oscillation (f) depends on their thickness and cut, each crystal having a characteristic resonant frequency.
This resonant frequency changes as molecules adsorb or desorb from the surface of the crystal, obeying the relationships
f = Kf 2 m/A wheref = the change in resonant frequency (Hz)m = change in mass of adsorbed material (g)K = constant for the particular crystal dependent on such
factors as its density and cut, and A = the adsorbing surface area (cm2).
Piezo-electric Biosensors
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Principles of a direct competitive ELISA. (i) Antibody, specific for the antigen of interest is immobilised on the surface of a tube. A mixture of a known amount of antigen-enzyme conjugate plus unknown concentration of sample antigen is placed in the tube and allowed to equilibrate. (ii) After a suitable period the antigen and antigen-enzyme conjugate will be distributed between the bound and free states dependent upon their relative concentrations. (iii) Unbound material is washed off and discarded. The amount of antigen-enzyme conjugate that is bound may be determined by the rate of the subsequent enzymatic reaction.
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Principles of immunosensors. (a)(i) A tube is coated with (immobilised) antigen. An excess of specific antibody-enzyme conjugate is placed in the tube and allowed to bind. (a)(ii) After a suitable period any unbound material is washed off. (a)(iii) The analyte antigen solution is passed into the tube, binding and releasing some of the antibody-enzyme conjugate dependent upon the antigen's concentration. The amount of antibody-enzyme conjugate released is determined by the response from the biosensor.
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(b) (i) A transducer is coated with (immobilised) antibody, specific for the antigen of interest. The transducer is immersed in a solution containing a mixture of a known amount of antigen-enzyme conjugate plus unknown concentration of sample antigen. (ii) After a suitable period the antigen and antigen-enzyme conjugate will be distributed between the bound and free states dependent upon their relative concentrations. (b)(iii) Unbound material is washed off and discarded. The amount of antigen-enzyme conjugate bound is determined directly from the transduced signal.
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Schematic diagram of an immunosensor device.
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Apparatus for piezoelectric sensor
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What are Molecularly Imprinted Polymers (MIPs)?
MIPs are polymers that contain highly selective recognition sites as a result of polymerization around a template molecule bound covalently or non-covalently to functional monomers
To date a wide range of templates have been used including organic compounds, sugars, peptides, nucleotides, proteins, crystals and even cells
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MIP synthesis is a three step process:1. Assembly of the template with funtional monomer units via covalent bonding, hydrogen bonding,hydrophobic, or ionic interactions;2. Polymerization around the template/monomer complex, incroporating the monomers into the polymerbackbone to create a recognition pocket;3. Removal of the template by washing with organic solvent or acid/base hydrolysis.
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Uses of MIPs
1. Industrial Use Purification: selectively removing reaction
products or by-products 2. Organic Chemistry Stoichiometric Reagents: scavengers, passive
supports, supported reagents, protecting groups Catalysts: C-C bond formation, elimination
reactions, mimics of natural enzymes, transition metal mediated reactions
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Electron Microscopy
What are Electron Microscopes?
Electron Microscopes are scientific instruments that use a beam of highly energetic electrons to examine objects on a very fine scale. This examination can yield the following information:
Topography The surface features of an object or "how it
looks", its texture; direct relation between these features and materials properties (hardness, reflectivity...etc.)
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Morphology: The shape and size of the particles making up the object; direct relation between these structures and materials properties (ductility, strength, reactivity...etc.)
Composition: The elements and compounds that the object is composed of and the relative amounts of them; direct relationship between composition and materials properties (melting point, reactivity, hardness...etc.)
Crystallographic Information: How the atoms are arranged in the object; direct relation between these arrangements and materials properties (conductivity, electrical properties, strength...etc.)
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How do Electron Microscopes Work?
Electron Microscopes(EMs) function exactly as their optical counterparts except that they use a focused beam of electrons instead of light to "image" the specimen and gain information as to its structure and composition.
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Basic steps involved in all EMs:
1. A stream of electrons is formed (by the Electron Source) and accelerated toward the specimen using a positive electrical potential.
2. This stream is confined and focused using metal apertures and magnetic lenses into a thin, focused, monochromatic beam.
3. This beam is focused onto the sample using a magnetic lens.
4. Interactions occur inside the irradiated sample, affecting the electron beam.
These interactions and effects are detected and transformed into an image
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SEMs are patterned after Reflecting Light Microscopes and yield similar information:
Topography: The surface features of an object or "how it looks", its texture; detectable features limited to a few manometers.
Morphology: The shape, size and arrangement of the particles making up the object that are lying on the surface of the sample or have been exposed by grinding or chemical etching; detectable features limited to a few manometers.
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Composition: The elements and compounds the sample is composed of and their relative ratios, in areas ~ 1 micrometer in diameter.
Crystallographic Information: The arrangement of atoms in the specimen and their degree of order; only useful on single-crystal particles >20 micrometers.
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A detailed explanation of how a typical SEM functions follows (refer to the diagram below):
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1. The "Virtual Source" at the top represents the electron gun, producing a stream of monochromatic electrons.
2. The stream is condensed by the first condenser lens (usually controlled by the "coarse probe current knob"). This lens is used to both form the beam and limit the amount of current in the beam. It works in conjunction with the condenser aperture to eliminate the high-angle electrons from the beam.
3. The beam is then constricted by the condenser aperture (usually not user selectable), eliminating some high-angle electrons.
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4. The second condenser lens forms the electrons into a thin, tight, coherent beam and is usually controlled by the "fine probe current knob".
5. A user selectable objective aperture further eliminates high-angle electrons from the beam.
6. A set of coils then "scan" or "sweep" the beam in a grid fashion (like a television), dwelling on points for a period of time determined by the scan speed (usually in the microsecond range).
7. The final lens, the Objective, focuses the scanning beam onto the part of the specimen desired.
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8. When the beam strikes the sample (and dwells for a few microseconds) interactions occur inside the sample and are detected with various instruments.
9. Before the beam moves to its next dwell point these instruments count the number of interactions and display a pixel on a CRT whose intensity is determined by this number (the more reactions the brighter the pixel).
10. This process is repeated until the grid scan is finished and then repeated, the entire pattern can be scanned 30 times per second.
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Mechanically Alloyed Cu-Nb-Fe (500x)
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Organically Deposited Iron Oxide (10,000x)
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Scanning Electron Microscopy (SEM)
The scanning electron microscope (SEM) uses a focused beam of high-energy electrons to
generate a variety of signals at the surface of solid specimens.
The signals that derive from electron-sample interactions reveal information about the sample such as external morphology (texture), chemical composition; and crystalline structure and orientation of materials
making up the sample.
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Fundamental Principles of Scanning Electron Microscopy (SEM) Accelerated electrons in an SEM carry significant
amounts of kinetic energy This energy is dissipated as a variety of signals
produced by electron-sample interactions when the incident electrons are decelerated in the solid sample. secondary electrons (that produce SEM images), backscattered electrons (BSE), diffracted backscattered electrons photons
(characteristic X-rays that are used for elemental analysis and continuum X-rays),
visible light (cathodoluminescence--CL); and heat.
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Information Derived Secondary electrons
for showing morphology and topography on samples backscattered electrons
for illustrating contrasts in composition in multiphase samples (i.e. for rapid phase discrimination)
X-ray generation produced by inelastic collisions of the incident
electrons with electrons in discrete ortitals (shells) of atoms in the sample.
As the excited electrons return to lower energy states, they yield characteristic X-rays of a fixed wavelength (that is related to the difference in energy levels of electrons in different shells for a given element).
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Transmission Electron Microscope (TEM)
TEMs are patterned after Transmission Light Microscopes and will yield similar information.
Morphology: The size, shape and arrangement of the particles which make up the specimen as well as their relationship to each other on the scale of atomic diameters.
Crystallographic Information: The arrangement of atoms in the specimen and their degree of order, detection of atomic-scale defects in areas a few nanometers in diameter
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Compositional Information (if so equipped): The elements and compounds the sample is composed of and their relative ratios, in areas a few nanometers in diameter
A TEM works much like a slide projector. A projector shines a beam of light through (transmits)
the slide. As the light passes through it is affected by the
structures and objects on the slide. These effects result in only certain parts of the light
beam being transmitted through certain parts of the slide.
This transmitted beam is then projected onto the viewing screen, forming an enlarged image of the slide.
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TEMs work the same way except that they shine a beam of electrons (like the light) through the specimen(like the slide).
Whatever part is transmitted is projected onto a phosphor screen for the user to see.
A more technical explanation of a typical TEMs workings is as follows (refer to the diagram):
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1. The "Virtual Source" at the top represents the electron gun, producing a stream of monochromatic electrons.
2. This stream is focused to a small, thin, coherent beam by the use of condenser lenses 1 and 2.
The first lens(usually controlled by the "spot size knob") largely determines the "spot size"; the general size range of the final spot that strikes the sample.
The second lens(usually controlled by the "intensity or brightness knob" actually changes the size of the spot on the sample; changing it from a wide dispersed spot to a pinpoint beam.
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3. The beam is restricted by the condenser aperture (usually user selectable), knocking out high angle electrons (those far from the optic axis, the dotted line down the center).
4. The beam strikes the specimen and parts of it are transmitted.
5. This transmitted portion is focused by the objective lens into an image.
6. Optional Objective and Selected Area metal apertures can restrict the beam;
Objective aperture enhances contrast by blocking out high-angle diffracted electrons
Selected Area aperture enables the user to examine the periodic diffraction of electrons by ordered arrangements of atoms in the sample.
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7. The image is passed down the column through the intermediate and projector lenses, being enlarged all the way.
8. The image strikes the phosphor image screen and light is generated, allowing the user to see the image.
The darker areas of the image represent those areas of the sample that fewer electrons were transmitted through (they are thicker or denser).
The lighter areas of the image represent those areas of the sample that more electrons were transmitted through (they are thinner or less dense)
