Clinical instrumentation II

Question 1

What is a biosensor?

Answer 1

According to IUPAC:

• A device that uses specific biochemical reactions mediated by isolated enzymes, immuno-systems, tissues, organelles or whole cells to detect chemical compounds usually by electrical, thermal or optical signals.
• A biosensor is an analytical device which converts a biological response into an electrical signal.

Question 2

How do biosensors work?

Answer 2

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).

Question 3

Biosensors – why are they important?

Answer 3

Biosensors are:

• important tools in food safety, diagnostics, medical monitors, and detection systems for biological warfare agents.
• important devices offering analytical simplicity both in and outside the analytical laboratory.
• selective, rapid and sensitive instruments for determination of chemical and biochemical targets

Question 4

Biosensors – 1st generation

Answer 4

• The biocatalyst is entrapped between or bound to the membrane & fixed on the surface of the transducer;
• The reaction of the product disperses to the transducer and causes the electrical reaction.

Question 5

Biosensors – 2nd generation

Answer 5

• The covalent fixation of biologically active component to the transducer’s surface permits the elimination of the semi permeable membrane
• Use of particular mediators between the sensor and the response in order to produce a better response.

Question 6

Biosensors – 3rd generation

Answer 6

The direct binding of biocatalyst to an electronic device that transduces and amplifies the signal

The response itself causes the reaction and no mediator is directly involved.

Question 7

What are the requirements of biosensors?

Answer 7

• Biocatalyst must be specific for the target, stable under normal storage conditions, good stability over a large number of assays.
• Reaction must be independent of stirring, pH, temperature to minimise pre-treatments. Cofactors should be immobilised also.
• Response should be sensitive, accurate, linear and reproducible.
• Biosensor must be suitable for the working environment e.g. small and non-immunogenic for in vivo, sterilisable.
• Cheap, small, usable by unskilled operators.
• Must be a market for the biosensor. Technology is not cheap.

Question 8

types of biosensors: Transducers

What can they measure?

Answer 8

• The heat output (or absorbed) by the reaction (calorimetric biosensors),
• Changes in the distribution of charges causing an electrical potential to be produced (potentiometric biosensors),
• Movement of electrons produced in a redox reaction (amperometric biosensors),
• Light output during the reaction or a light absorbance difference between the reactants and products (optical biosensors), or
• Effects due to the mass of the reactants or products (piezo-electric biosensors).

Question 9

How do calorimertric biosensors work?

Answer 9

1. The sample stream (a) passes through the outer insulated box (b) to the heat exchanger (c) within an aluminium block (d).
2. 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.
3. The change in temperature is determined by the thermistor (g) and the solution passed to waste collection (h).
4. External electronics (l) determines the difference in the resistance, and hence temperature, between the thermistors.

Question 10

Calorimetric biosensors: The reactions are exothermic and generate heat

Answer 10

• If 1mM reactant produces 100 kJ mole-1 then each ml of solution generates 0.1 J of heat.
• This will cause a change in temperature of the solution of approx. 0.02°C.
• The biosensor needs a temperature resolution of 0.0001°C to be useful.

Question 11

What is the function of a thermistor?

Answer 11

• Detects temperature change.
• Measures the electrical resistance with the change in temperature.
• The resistance change is converted to a proportional voltage change.
• Downside: A change in the environmental temperature between the two thermistors alters the resistances such that they lose accuracy. → Using a relatively large, well insulated aluminium block to encase the thermistors reduces this effect.
• Sensitivities are in the order of 10^-4M.
• Ranges are limited 10^-4 – 10^-2M.

Question 12

How can the sensitivity of a thermister be improved?

Answer 12

Sensitivity can be improved by coupling other exothermic reactions, especially the more exothermic ones such as catalase

Question 13

What are potentiometric biosensors?

Answer 13

• Potentiometric biosensors make use of ion-selective electrodes that transduce the biological reaction into an electrical signal.
• Example: pH electrode for the detection of hydrogen ions.

Question 14

What are Amperometric biosensors?

Answer 14

Produce a current when a potential is applied between two electrodes.

They generally have similar response times, dynamic ranges and sensitivities to the potentiometric biosensors.

Example 1: the Clark oxygen electrode (1956; Leland C Clark).

Example 2: glucose oxidase biosensor.

Question 15

How do amperometric biosensors work?

Answer 15

• A potential is applied between the central platinum cathode and the reference silver anode.
• This will generate a current (I) which is carried between the electrodes by means of a saturated solution of KCl.
• The electrode compartment is separated from the biocatalyst (here shown glucose oxidase, GOD) by a thin plastic membrane that is permeable only to oxygen.
• The analyte solution is separated from the biocatalyst by another membrane that is permeable to the substrate and the product.

Question 16

Overcoming accuracy issues of oxygen electrodes

Answer 16

Dependent on dissolved oxygen concentration which is small and slow to change. →May be overcome by using ‘mediators’ which transfer the electrons directly to the electrode, bypassing the reduction of the oxygen co-substrate.

Question 17

Overcoming accuracy issues of oxygen electrodes-Mediators must:

Answer 17

• React rapidly with the reduced form of the enzyme;
• Be sufficiently soluble, in both the oxidised and reduced forms, to be able to rapidly diffuse between the active site of the enzyme and the electrode surface;
• Over-potential for the regeneration of the oxidised mediator, at the electrode should be low and independent of pH;
• The reduced form of the mediator should not readily react with oxygen.

Question 18

History of glucose biosensors

Who is the father of the bisensor?

Answer 18

Professor Leland C Clark Jnr was the father of the biosensor

• In 1956, Clark published his definitive paper on the oxygen electrode
• In 1962, he described “how to make electrochemical sensors more intelligent” by adding “enzyme transducers as membrane enclosed sandwiches”
• In 1975, YSI (Yellow Spring Instruments Co., Ohio, USA) produced the first of many biosensor-based laboratory analysers to be built by companies around the world

Question 19

Laboratory glucose biosensors: Model 23 YSI glucose analyser

1975

Answer 19

Question 20

Laboratory glucose biosensors: YSI 2300 STAT Plus automated. Current model

Answer 20

Question 21

What are Electrochemical biosensors?

Answer 21

• With the advent of artificial mediators to remove the oxygen dependence for measurement a new group of meters emerged;
• Personal blood glucose meters based on disposable (screen printed) enzyme electrode test strips

Question 22

Why are commercial biosensors so immportant?

Answer 22

qOver 170 million diabetics worldwide

qExpected to double by 2025

qDiabetic complications are often fatal, but can be prevented by accurate blood glucose control;

qGlobal market $12 billion by 2012

Question 23

What are the two main types of optical biosensors?

What do the commonly used methods of gauging the colour involve?

Answer 23

1. Changes in light absorption between the reactants and products of a reaction.
2. Measuring the light output by a luminescent process.

• Simplest form are colorimetric test strips which are disposable single-use cellulose pads impregnated with enzyme and reagents;

Most common way to measure blood glucose:

• D-glucose + O₂ → D-glucono-1,5-lactone + H₂O₂

Catlysed by glucose oxidase

• chromogen(2H) + H₂O₂ → dye + 2H₂O

Catalysed by peroxidase

Commonly used methods of gauging the colour involve portable reflectance meters, although direct visual comparison with a coloured chart is often used