4.2. Design Variables

4.2.1. Immobilization Methods

Four methods are used for immobilizing enzyme for use in a biosensor: (1) adsorption; (2) entrapment; (3) covalent coupling; and (4) cross-linking. These four methods are compared in Table 4.1. Among various methods, the cross-linking method is most frequently used because it has the advantage of the covalent bonding yet the cost is inexpensive.

Effects of Immobilization. With immobilized enzymes the measured reaction rate depends not only on the substrate concentration and the kinetic constants KM and Vmax but also on so-called immobilization effects. These effects are due to the following alterations of the enzyme by the immobilization process.

1. Change in Conformation. Conformational changes of the enzyme caused by immobilization usually decrease the affinity to the substrate (increase of KM). Furthermore, a partial inactivation of all, or the complete inactivation of a part of the enzyme molecules may occur (decrease of Vmax). These two cases of a conformation-induced drop of Vmax may be distinguished by measuring the activity of the resolubilized enzyme or by titration of the active center with an irreversible inhibitor.

2. Change in Microenvironment. Ionic, hydrophobic, or other interactions between the enzyme and the matrix (microenvironmental effects) may also result in changed KM and Vmax values These essentially reversible effects are caused by variations in the dissociation equilibria of charged groups of the active center.

3. Non-Uniform Distribution. A non-uniform distribution of substrate and/or product between the enzyme matrix and the surrounding solution affects the measured (apparent) kinetic constants.

4. Reaction and Diffusion. In biosensors the biocatalyst and the signal transducer are spatially combined, i.e., the enzyme reaction proceeds in a layer separated from the measuring solution. The substrates reach the membrane system of the biosensor by convective diffusion from the solution. The rate of this external transport process depends essentially on the degree of mixing. In the multilayer system in front of the sensor the substrates and products are transferred by diffusion. Slow mass transfer to and within the enzyme matrix leads to different concentrations of the reaction partners in the measuring solution and in the matrix. Diffusion and the enzyme reaction do not proceed independently of one another; they are coupled in a complex manner.

4.2.2. Outer Membrane

Requirements

1. The outer membrane has to be compatible with the medium into which it will be placed. Therefore, the requirement will be different depending on the nature of the measurement medium. For example, the outer membrane for biosensor used in liquid samples should be different from that intended for implantation application. For the latter application, bio-compatibility becomes an issue (the rejection of the sensor by body may occur).

2. The outer membrane should offer low diffusional resistance to analytes while the resistance should be high for macromolecules.

3. For long-term continuous use applications, the fouling of the membrane must be minimal. The fouling causes an increase in the diffusional resistance of the analyte and thus the signal of the sensor changes as the fouling progresses. If microorganism grows on the surface of the outer membrane, the passage of oxygen to the enzyme layer is hindered which makes the sensor to behave erroneously.

Table 4.1. Comparison of four enzyme immobilization methods.


1. Adsorption

2. Entrapment

3. Covalent coupling

4.Crosslinking

Matrix material

ion exchange resins,
active charcoal,
silica gel,
clay,
aluminum oxide,
porous glass
alginate,
carageenan,
collagen,
polyacrylamide,
gelatine,
silicon rubber,
polyurethens
agarose,
cellulose,
PVC,
ion
exchange
resins,
porous glass
Crosslinking agents:
glutaraldehyde,
bisisocyanate,
bisdiazobenzidine

Nature of bonding

reversible;
changes in pH, ionic strength may detach the enzyme
physical
entrapment

chemical bonding

entrapment;
functionally inert proteins are often used together (BSA, gelatin)

Enzyme loading

low

low

high

high

Enzyme leakage

some

some

very low

low

Loss of activity

negligible

negligible

significant

small

Cost

inexpensive

inexpensive

expensive

inexpensive

Optimization of Biosensor. Variations of the diffusion resistance of the semipermeable membrane are being used to optimize the sensor performance. A semipermeable membrane with a molecular cutoff of 10, 000 and a thickness of 10 m only slightly influences the response time and sensitivity. In contrast, thicker membranes such as polyurethane or charged material, significantly increase the measuring time, but may also lead to an extension of the linear measuring range. Table 4.2. lists some of the commercially available membranes that can be used as the outer membrane.

Table 4.2. Available pre-cast membranes

Collagen

Polycarbonate (Nucleopore)

Cellulose acetate

Characteristics

a hydroxylic natural protein

uniform pore size

slightly negative due to -COO-

Derivatizability

easy

easy

Temperature stability

O.K. at room T

unstable at 37oC

Stable

stable

Permeselectivity

exclude protein

exclude protein

exclude protein, retard transport of anionic species

Source

Sigma

Nucleopore

Amicon

4.2.3. Inner Membrane

Requirements. The inner membrane should be permeselective to target species only (for example H2O2 only for the current glucose sensor). Also, it should be as thin as possible and stable for long-term use. Some of the solution castable membranes and their characteristics are compared in Table 4.3.

Table 4.3. Solution castable membranes

Cellulose acetate

Nafion

Polyurethane*

Characteristics

perfluorosulphonic

acid ionomer,

negatively charged

widely varying MW dep. on source

Solvent

acetone,
cyclohexanone

low MW alcohols

dissolves in 98% tetrahydrofuran and 2% dimethylform-amide

Coating method

dip coating,
in-situ formation

dip coating as thin as 1000A (with 5% solution)

dip coating

Other characteristics

tend to adsorb proteins & cations,
not useful as an outer membrane
biocompatible,
retard glucose access to enzyme layer, passes O2 well

* More useful as an outer membrane.

4.2.4. Effect of Enzyme Loading

Internal Diffusion Is Important. Usually in the operation of biosensors the flow conditions are adjusted to provide a mass transfer rate from the solution to the membrane system faster than that of in the enzyme layer (the internal mass transfer). In the immobilized enzyme layer, reaction and diffusion occur simultaneously. Therefore, rigorous modeling is required to fully characterize the behavior of a biosensor. The key question in designing a biosensor is: (1) how thick should the enzyme layer be? and (2) how much enzyme has to be placed in the layer? Although rigorous modeling is required to fully characterize the behavior of biosensors, the design can be carried out by considering limiting cases.

Enzyme Loading Factor, fE. The key variable for determining limiting cases is the enzyme loading factor defined by fE:

where Ds and d are diffusivity of the substrate and the enzyme immobilization layer thickness. The enzyme loading factor is essentially the ratio of the reaction rate (for the case when KM >> S; or first order reaction) to the diffusion rate (when k = Ds/d and the mass flux J is expressed as k(dS/dx)).

Diffusion Control: fE > 25 When the rate of reaction is much faster than the rate of diffusion, the bottle neck of the transport process is the diffusion. In such a case, we say, the biosensor is operated in a diffusion-controlled regime. The condition is:

When this condition is met, the sensor signal depends on the diffusion process. This means the sensor output signal is linear with the analyte concentration, and is independent of the reaction rate of the enzyme layer.

Reaction Control: When the rate of reaction is much slower than the rate of diffusion, the bottle neck of the transport process is the reaction. In such a case, we say, the biosensor is operated in a reaction-controlled regime. The condition is:

When this condition is met, the sensor signal depends on the reaction rate and independent of the diffusion rate. This means that the sensor output signal depends on the reaction rate expression - i.e. the Michaelis-Menten equation. Therefore, the sensor output signal will be non-linear with the analyte concentration.

Advantages of Diffusion Control. There are many advantages of diffusion-controlled biosensor. These include:

1. Sensitivity is independent of enzyme content and activity
2. Sensitivity is independent of inhibitors and pH variations.
3. There is small temperature effect because the activation energy of diffusion is much less than that of reaction.
4. Extended linear range. Extendible beyond [S] = KM. For kinetics controlled case, linear range is achieved only for [S] < KM
5. Slower response time. The response time is determined by the diffusion time of the reaction product (sensed): d2/Dp This is the major disadvantage of the diffusion controlled biosensor.
6. Greater functional stability. Due to the excess of enzyme in the membrane, the sensor will have higher functional stability. With diffusion controlled sensor, 2000-10,000 measurement is possible, while kinetically controlled sensors permit only 200-500 measurement.

Apparent KM When an enzyme is immobilized, the KM value changes because of the changes in enzyme conformation and the microenvironment. Table 4.4. lists the KM values for some of the immobilized enzyme systems.

Table 4.4. Apparent enzyme activity and KM values of adsorbed layers and enzyme membranes.

Enzyme

Immobilization

Apparent enzyme activity, mU/cm2

KM value

GOD*

gelatine entrapment

110

7.5

GOD

collagen, covalent

60-80

3.0

GOD

cellulose acetate

340

GOD

cellulose acetate

>1000

GOD

PVA entrapment

160-700

GOD

spectral carbon, adsorbed

150-200

GOD

carbon, covalent

50-170

3.1-19.1

-galactosidase

gelatin entrapment

1000

Urease

cellulose triacetate, entrapment

3-30

2.4

Cholesterol oxidase

collagen, crosslinked

3

Creatinine amidohydrolase

cellulose acetate, covalent

1140

278

Creatine amidohydrolase

cellulose acetate, covalent

110

64.9

Sarcosine oxidase

cellulose acetate, covalent

13

2.4

* GOD stands for glucose oxidase.