Lecture 13: Introduction to Biosensors

What is a Biosensor?

• Biosensors are analytical devices that combine a biological detecting elements with a transducer to produce a signal

• Sensor is very specific for analyte

Elements of a Biosensor

• Bioelement: Enzyme, Ab, Nucleic acid, tissue, microbial, polysaccharide

• sensor element: electric potential/ current/ conductance/ impedance, intesity and phase of EM radiation, mass, temperature, viscosity

Coupling the Biorecognition Element to the Transducer

• Membrane Entrapment

  • A semipermeable membrane separates the analyte and the bioelement

  • only allows analyte of interest to diffuse through: eg gas

  • inconsistent signals

• Physical Adsorption

  • Dependent on van der Waals forces, hydrophobic forces, hydrogen bonds, and ionic forces to attach the biomaterial to the surface of the sensor

  • weak bonds = easy to dislodge biomaterial, flow through system not ideal

• Porous Entrapment

  • Based on forming a porous encapsulation matrix around the biological material which helps bind it to the sensor

  • more stable, but when carbon dries it can distort the analyte, less sensitive sensor

• Covalent Bonding

  • The sensor surface is treated as a reactive group to which the biological materials can bind

  • stronger, best

Essential Performance Characteristics of a Biosensor

• The biocatalyst must be highly specific for analyte of interest

• The sensor should be independent of physical parameters

  • eg stirring, pH and temperature

• The sensor response should be accurate, precise, reproducible and linear over an appropriate analytical range

• The biosensor should be small and biocompatible if it is to be used in vivo

• The biosensor should be cheap, portable and capable of being used by semi-skilled operators (eg glucose sensors)

• There should be a commercial market for the biosensor

Cholesterol Monitoring (CardioChek)

• blood sample is applied to test strip and chemical reaction occurs producing a colour change

• reflectance photometrynused to measure the colour reaction and compares the info to the calibration curve stored in the unit.

• Cholesterol need to be measured at 6 monthly intervals – So no market

Market for biosensors

• Clinical biosensors: Medical applications for patient diagnostics and monitoring

• In Vivo (inside the body)

  • Long-term implantable: Artificial organs

  • Short-term invasive: Bedside glucose monitoring

• In Vitro (outside the body/lab)

  • Singleshot: Home blood glucose monitoring

  • Multi-analysis: Pathology laboratory glucose monitoring

• Non-clinical biosensors: Applications outside medical settings

  • Single analysis: Fruit ripening sensors

  • Reactive monitoring: Pollution monitoring, fermentation processes

  • Environmental bioagent detection: Detection of pathogens like anthrax, plague, and cholera

The Physical Characterisation of Biosensor Sensing Properties

• Optical: direct optical dectection

• Physical: thermometric

• Electrochemical: conductometric, potentiometric, amperometric

Electrochemical Biosensors

• Many chemical reactions produce or consume ions or electrons, causing some change in the electrical properties of the solution that can be used as a measuring parameter

• no electrochemical reactions

  • Conductometric - measures conductance (inverse of resistance)

• Electrochemical biosensors with electrochemical reactions.

  • Potentiometric - measures changes in potential difference (consumption of ions)

  • Amperometric - measures current (consumption of electrons)

Ohm’s Law

• Ohm's Law deals with the relationship between voltage and current.

• ‘The potential difference (voltage) across an ideal conductor (i.e. no resistance) is proportional to the current through it.’

• Ohm's Law: V = I R

• V = potential difference between two points

• I = current flowing through the resistance.

• R = resistance to current flow.

Conductometric Biosensors

• Conductometry is a method with no electrochemical reactions for the electrodes to detect.

• The most important property of the electrolytic solution is its conductivity

  • varies with a wide range of biological reactions.

• Based on measuring changes in resistance of a selective material.

• Inverse of resistivity = conductivity

• called conductometric sensors or chemiresistors

• There are two types of these sensors:

  • measuring gas: A selective material, which can change its conductivity upon interaction with chemical species is clamped between two contact electrodes and the resistance of the entire device is measured

  • no gas = no current = maximum resistance

  • measuring solution: the chemically interactive layer is at the top of an electrode, which is immersed in the solution of electrolyte

Potentiometric (pH) Biosensors

• Potentiometric biosensors use ion-selective electrodes

• transduce the biological reaction into an electrical signal.

• This consists of an immobilised enzyme membrane surrounding the probe from a pH-meter where the catalysed reaction generates or absorbs hydrogen ions.

• The reaction occurring next to the thin sensing glass membrane causes a change in pH

Amperometric Biosensors

• Amperometric is a high-sensitivity biosensor that can detect electroactive species present in biological test samples.

• Since the biological test samples may not be intrinsically electroactive, enzymes are needed to catalyze the production of reactive species.

• In this case, the measured parameter is current.

Commercially Successful Biosensors

Blood Glucose: Diabetes Monitoring

Pregnancy Test: ClearBlue

Blood Glucose Biosensor

• 85% of biosensors are glucose biosensors (~£2.5billion)

• mainly due to the prevalence of diabetes in developed nations

• Need for repeated measurement – high demand for consumable electrodes

Diabetes complecations

• 270/380 million develop complications

  • 70% eye diseases

  • 70% heart diseases

  • 40% nerve damage

  • 30% kidney disease

• >60% of all non-traumatic limb amputations are due to diabetes

• Routine monitoring of blood glucose is the key to effective management of diabetes

• Dexcom

Redox Enzyme Electrochemistry

• Central to Amperometric, Biosensor Based Glucose Measurement

• most commonly used enzymes in the design of glucose biosensors contain redox groups that change redox state during the biochemical reaction.

• Enzymes of this type include glucose oxidase (GOx) and glucose dehydrogenase (GDH)

Redox Electrochemistry

• Oxidase enzymes oxidise their substrates > accepts electrons > inactivated reduced state

• enzymes return to active oxidised state on electrode

• electrons can be transferred to molecular oxygen, resulting in the production of hydrogen peroxide (H₂O₂):

  • glucose + O₂ → gluconolactone + H₂O₂

• Glucose may also be oxidised by GDH, it relies on NAD+ acting as a cofactor, rather than oxygen as a cosubstrate.

  • glucose + NAD+ → gluconolactone + NADH

Advantages/Disadvantages of redox electrochemistry

• Glucose Oxidase

  • Advantages: Inexpensive

  • Disadvantages: Requires oxygen as a cosubstrate. depleted O₂ in the sample = decrease performance

• Glucose Dehydrogenase

  • Advantages: Oxygen independent

  • Disadvantages: The cofactor (NAD+) is expensive and unstable.

Biosensor ‘Generations’

1. No direct connection between enzyme and electrode. Measures end product of substrate conversion eg H₂O₂

2. The use of a mediator (eg ferrocyanide) to link active site to electrode surface

3. Direct electron transfer between enzyme and electrode surface

1st Generation Glucose Biosensor: amperometric glucose biosensors

• Professor Clark Jr: direct detection of H₂O₂ oxidation at the electrode surface +ve charge

• The earliest approaches to the construction of amperometric glucose biosensors were based on GOx immobilised close to an electrode

• The depletion of oxygen was monitored, using a Clark oxygen electrode

• disadvantage: high potential of electrode other things can be oxidisese (paracetamol) to form current

2nd Generation Glucose Biosensor: Redox Mediators

• The use of redox mediators facilitated a transfer of electrons in enzyme electrodes which:

  • was independent of the local oxygen concentration

  • allowed operation at much lower potentials, minimising detection of interferents (eg ascorbic acid, urate and paracetamol)

• These redox couples, or mediators, are able to shuttle electrons between the redox centre of the enzyme and the electrode

• most important examples of this class are mediators based on ferrocene and its derivatives

  • They have a wide range of redox potentials

  • Their redox potentials are independent of pH

  • They are easy to manufacture

• Disadvantage: mediators are toxic so can’t be used in vivo biosensors

Third Generation Glucose Sensor

• Direct electron transport between enzyme active site and the electrode surface

  • no reagent or mediators required

• Redox enzymes can be:

  • ectrinsic: active site on surface, easy

  • GOx intrinsic: active site deep within structure

• Instead of (toxic) mediators, the electrode can perform direct electron transfers using organic conducting materials based on charge-transfer complexes

The structure of glucose oxidase

• Dimeric enzyme.

• Co-factor FAD/FADH2 (1 per subunit)

• Glucose Oxidase is an intrinsic enzyme with an active site buried deep within the protein structure

• Difficult to gain access to the active site for electrochemistry

Access to GOx active site

• FAD active site 'buried' in cavity 13Å from surface of protein.

• Electrostatic surface at entrance to active site

  • Positively charged lysine residues used to orientate substrate to AS

  • potentially use to bind to electrode

Covalent immobilisation of GOx at a modified gold electrode

• Positively charged residues of GOx (e.g. lysine), in red.

• Suitable for reaction with cross-linkers such as DTSSP (3,3'-dithiobis(sulfosuccinimidyl propionate)

• DTSSP = dimer linked by diS bonds + carboxyl groups

• DTSSP is a water-soluble crosslinker that contains amine-reactive NHS-ester ends around an 8-atom spacer arm, whose central disulfide bond can bind covalently to gold

Binding of Gox to Gold Electrode using DTTSP

1. disulphide group bonds covalently to the gold electrode surface

2. DTSSP dimer split into two monomers

3. carboxyl groups presented

4. GOx, have several primary amines in the side chain of lysine that are available as targets for sulfo-NHS- ester crosslinking reagents

5. Amine groups of Gox bind to carbonyl groups of DTSSP to covalently link the enzyme to the electrode surface

Direct electrochemistry of GOx

• glucose + GOx /FAD → gluconolactone + FADH₂

• GOx /FADH₂ →(electrode)→ GOx /FAD + 2H⁺ + 2e-

• Catalytic oxidation current from DTSSP immobilized GOx electrode plotted as function of [glucose]