Notes Biosensor

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What are biosensors?

A biosensor is an analytical device which converts a biological response into an electrical signal (Figure 6.1). 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 utilise a biological system directly. This very broad definition is used by some scientific

ournals (e.g. !iosensors" #lsevier Applied $cience) but will not be applied to the coverage here. The emphasis of this %hapter

concerns en&ymes as the biologically responsive material" but it should be recognised that other biological systems may be

utilised by biosensors" for eample" whole cell metabolism" ligand binding and the antibodyantigen reaction. !iosensors represent

a rapidly epanding field" at the present time" with an estimated 6* annual growth rate+ the maor impetus coming from the

healthcare industry (e.g. 6* of the western world are diabetic and would benefit from the availability of a rapid" accurate and

simple biosensor for glucose) but with some pressure from other areas" such as food ,uality appraisal and environmental

monitoring. The estimated world analytical mar-et is about 1/""" year1of which 0* is in the health care area. There

is clearly a vast mar-et epansion potential as less than .1* of this mar-et is currently using biosensors. esearch and

development in this field is wide and multidisciplinary" spanning biochemistry" bioreactor science" physical chemistry"

electrochemistry" electronics and software engineering. 2ost of this current endeavour concerns potentiometric and amperometric

biosensors and colorimetric paper en&yme strips. 3owever" all the main transducer types are li-ely to be thoroughly eamined" for

use in biosensors" over the net few years.

A successful biosensor must possess at least some of the following beneficial features4

1. The biocatalyst must be highly specific for the purpose of the analyses" be stable under normal storage conditions and"

ecept in the case of colorimetric en&yme strips and dipstic-s (see later)" show good stability over a large number of

assays (i.e. much greater than 1).

2. The reaction should be as independent of such physical parameters as stir ring" p3 and temperature as is manageable.

This would allow the analysis of samples with minimal pretreatment. 5f the reaction involves cofactors or coen&ymes

these should" preferably" also be coimmobilised with the en&yme (see %hapter ).

0. The response should be accurate" precise" reproducible and linear over the useful analytical range" without dilution or

concentration. 5t should also be free f rom electrical noise.

7. 5f the biosensor is to be used for invasive monitoring in clinical situations" the probe must be tiny and biocompatible"

having no toic or antigenic effects. 5f it is to be used in fermenters it should be sterilisable. This is preferably performed

by autoclaving but no biosensor en&ymes can presently withstand such drastic wetheat t reatment. 5n either case" the

biosensor should not be prone to fouling or proteolysis.

8. The complete biosensor should be cheap" small" portable and capable of being used by semis-illed operators.

6. There should be a mar-et for the biosensor. There is clearly little purpose developing a biosensor if other factors (e.g.

government subsidies" the continued employment of s-illed analysts" or poor customer perception) encourage the use

of traditional methods and discourage the decentralisation of laboratory testing.

The biological response of the biosensor is determined by the biocatalytic membrane which accomplishes the conversion of

reactant to product. 5mmobilised en&ymes possess a number of advantageous features which ma-es them particularly applicable

for use in such systems. They may be reused" 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

en&yme otherwise necessary in analytical protocols. 2any en&ymes are intrinsically stabilised by the immobilisation process (see

%hapter 0)" but even where this does not occur there is usually considerable apparent stabilisation. 5t is normal to use an ecess

of the en&yme within the immobilised sensor system. This gives a catalytic redundancy (i.e. 99 1) which is sufficient to ensure
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an increase in the apparent stabilisation of the immobilised en&yme (see" for eample" Figures 0.11"0.1:and8.). #ven where

there is some inactivation of the immobilised en&yme over a period of time" this inactivation is usually steady and predictable. Any

activity decay is easily incorporated into an analytical scheme by regularly interpolating standards between the analyses of

un-nown samples. For these reasons" many such immobilised en&yme systems are reusable up to 1" times over a period of

several months. %learly" this results in a considerable saving in terms of the en&ymes' cost relative to the analytical usage of f ree

soluble en&ymes.

;hen the reaction" occurring at the immobilised en&yme membrane of a biosensor" is l imited by the rate of eternal diffusion" the

reaction process will possess a number of valuable analytical assets. 5n particular" it will obey the relationship shown in e,uation

0./" in e,uation 0./


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7. light output during the reaction or a light absorbance difference between the reactants and products (optical

biosensors)" or

8. effects due to the mass of the reactants or products (pie&oelectric biosensors).

There are three socalled 'generations' of biosensors+ First generation biosensors where the normal product of the reaction

diffuses to the transducer and causes the electrical response" second generation biosensors which involve specific 'mediators'

between the reaction and the transducer in order to generate improved response" and third generation biosensors where the

reaction itself causes the response and no product or mediator diffusion is directly involved.

The electrical signal from the transducer is often low and superimposed upon a relatively high and noisy (i.e. containing a high

fre,uency signal component of an apparently random nature" due to electrical interference or generated within the electronic

components of the transducer) baseline. The signal processing normally involves subtracting a 'reference' baseline signal" derived

from a similar transducer without any biocatalytic membrane" from the sample signal" amplifying the resultant signal difference

and electronically filtering (smoothing) out the unwanted signal noise. The relatively slow nature of the biosensor response

considerably eases the problem of electrical noise filtration. The analogue signal produced at this stage may be output directly but

is usually converted to a digital signal and passed to a microprocessor stage where the data is processed" converted to

concentration units and output to a display device or data store.

Calorimetric biosensors

2any en&yme catalysed reactions are eothermic" generating heat (Table 6.1) which may be used as a basis for measuring the

rate of reaction and" hence" the analyte concentration. This represents the most generally applicable type of biosensor. The

temperature changes are usually determined by means of thermistors at the entrance and eit of small pac-ed bed columns

containing immobilised en&ymes within a constant temperature environment (Figure 6./). ?nder such closely controlled

conditions" up to * of the heat generated in the reaction may be registered as a temperature change in the sample stream.This may be simply calculated from the enthalpy change and the amount reacted. 5f a 1 m2 reactant is completely converted to

product in a reaction generating 1 -@ mole1then each ml of solution generates .1 @ of heat. At * efficiency" this will cause a

change in temperature of the solution amounting to approimately ./%. This is about the temperature change commonly

encountered and necessitates a temperature resolution of .1% for the biosensor to be generally useful.

Table 6.1.3eat output (molar enthalpies) of en&yme catalysed reactions.

Reactant EnzymeHeat output

-H !" mole-1#

%holesterol %holesterol oidase 80

#sters %hymotrypsin 7 16

Blucose Blucose oidase


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3ydrogen peroide %atalase 1

Cenicillin B Cenicillinase 6amino acid oidases" cholesterol oidase" galactose oidase" and urate oidase)
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Figure 6.*.$chematic diagram of a simple amperometric biosensor. A potential is applied between the central platinum cathode

and the annular silver anode. This generates a current (5) which is carried between the electrodes by means of a saturated

solution of =%l. This electrode compartment is separated from the biocatalyst (here shown glucose oidase" BI) by a thin

plastic membrane" permeable only to oygen. 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

./8 mm diameter using a Ct wire cathode within a silver plated steel needle anode and utilising dipcoated membranes.


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Figure 6.6.The response of an amperometric biosensor utilising glucose oidase to the presence of glucose solutions. !etween

analyses the biosensor is placed in oygenated buffer devoid of glucose. The steady rates of oygen depletion may be used to

generate standard response curves and determine un-nown samples. The time re,uired for an assay can be considerably

reduced if only the initial transient (curved) part of the response need be used" via a suitable model and software. The washout

time" which roughly e,uals the time the electrode spends in the sample solution" is also reduced significantly by this process.

An alternative method for determining the rate of this reaction is to measure the production of hydrogen peroide directly by

applying a potential of E.6 M to the platinum electrode" relative to the AgDAg%l electrode" and causing the reactions4

Ct anode 3/I/ I/E /3EE /e %6.1*&

Ag cathode /Ag%l E /e /AgE /%l%6.16&

The maor problem with these biosensors is their dependence on the dissolved oygen concentration. This may be overcome by

the use of 'mediators' which transfer the electrons directly to the electrode bypassing the reduction of the oygen cosubstrate. 5n

order to be generally applicable these mediators must possess a number of useful properties.

1. They must react rapidly with the reduced form of the en&yme.

/. They must be sufficiently soluble" in both the oidised and reduced forms" to be able to rapidly diffuse between the

active site of the en&yme and the electrode surface. This solubility should" however" not be so great as to cause

significant loss of the mediator from the biosensor's microenvironment to the bul- of the solution. 3owever soluble" the

mediator should generally be nontoic.

0. The overpotential for the regeneration of the oidised mediator" at the electrode" should be low and independent of p3.

7. The reduced form of the mediator should not readily react with oygen.

The ferrocenes represent a commonly used family of mediators (Figure 6.


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%6.1+&

#lectrodes have now been developed which can remove the electrons directly from the reduced en&ymes" without the necessity

for such mediators. They utilise a coating of electrically conducting organic salts" such as Gmethylphena&inium cation (G2C E"

Figure6.


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Figure 6.,.Amperometric biosensors for flavooidase en&ymes illustrating the three generations in the development of a

biosensor. The biocatalyst is shown schematically by the crosshatching. (a) First generation electrode utilising the 3 /I/produced

by the reaction. (#O E.6 M). (b) $econd generation electrode utilising a mediator (ferrocene) to transfer the electrons" produced

by the reaction" to the electrode. (#O E.1: M). (c) Third generation electrode directly utilising the electrons produced by the

reaction. (#O E.1 M). All electrode potentials (#) are relative to the %lDAg%l"Agelectrode. The following reaction occurs at the

en&yme in all three biosensors4

$ubstrate(/3) E FAoidase Croduct E FA3/oidasefi %6.1,&

This is followed by the processes4


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(a)

biocatalyst

FA3/oidase E I/ FAoidase E 3/I/ %6.1&

electrode

3/I/ I/E /3EE /e %6.$&

(b)

biocatalyst

FA3/oidase E / FerriciniumE FAoidase E / Ferrocene E /3E %6.$1&

electrode

/ Ferrocene / FerriciniumEE /e %6.$$&

(c)

biocatalystDelectrode

FA3/oidase FAoidase E /3EE /e %6.$(&

The current (i) produced by such amperometric biosensors is related to the rate of reaction (vA) by the epression4

i O nFAvA (6.6)

where n represents the number of electrons transferred" A is the electrode area" and F is the Faraday. ?sually the rate of reaction

is made diffusionally controlled (see e,uation 0./


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The evaluation of the dyed strips is best achieved by the use of portable reflectance meters" although direct visual comparison

with a coloured chart is often used. A wide variety of test strips involving other en&ymes are commercially available at the present

time.A most promising biosensor involving luminescence uses firefly luciferase (Photinusluciferin 7monooygenase (ATC

hydrolysing)" #% 1.10.1/.


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Figure 6.1.Crinciples of immunosensors. (a)(i) A tube is coated with (immobilised) antigen. An ecess of specific antibody

en&yme conugate is placed in the tube and allowed to bind. (a)(ii) Af ter 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 antibodyen&yme conugate dependent

upon the antigen's concentration. The amount of antibodyen&yme conugate released is determined by the response from the

biosensor. (b)(i) A transducer is coated with (immobilised) antibody" specific for the antigen of interest. The transducer is

immersed in a solution containing a miture of a -nown amount of antigenen&yme conugate plus un-nown concentration of

sample antigen. (b)(ii) After a suitable period the antigen and antigenen&yme conugate will be distributed between the bound

and free states dependent upon their relative concentrations. (b)(iii) ?nbound material is washed off and discarded. The amount

of antigenen&yme conugate bound is determined directly from the transduced signal.

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