UNIT 1 ------------------------Principles of Enzyme Biosensors

Explanation of the Introduction to Enzyme Biosensors

An enzyme biosensor is a special type of analytical device used to detect and measure specific substances (called analytes). An enzyme
biosensor uses an enzyme to specifically recognize a substance and a transducer to convert that biochemical reaction into a measurable
signal. It works by combining two key components:

1. Enzyme (biological recognition element):

o Enzymes are proteins that speed up chemical reactions with high specificity.

o In a biosensor, the enzyme reacts only with the target analyte (for example, glucose oxidase reacts specifically with
glucose).

o During this enzymatic reaction, physical or chemical changes occur such as change in proton concentration (pH),
release/uptake of gases (oxygen, ammonia, carbon dioxide), production of light, heat, or change in optical
properties.

2. Transducer (signal converter):

o The transducer takes these biochemical changes and converts them into a measurable signal (like current, voltage,
light intensity, or temperature change).

o The signal is then amplified, processed, and displayed so we can analyze it

Why Enzymes are Used in Biosensors?

 Specificity: Enzymes recognize only their specific substrate (e.g., glucose oxidase works only with glucose). This ensures highly
accurate sensing.

 Catalytic ability: A single enzyme molecule can convert many substrate molecules, producing an amplified signal.

These biosensors are now widely used in:

 Healthcare: Glucose monitoring, cholesterol detection, disease diagnostics.

 Veterinary medicine: Detecting infections or metabolic disorders in animals.

 Food industry: Checking freshness, detecting contaminants, monitoring fermentation.

 Environmental monitoring: Measuring pollutants, toxins, pesticides.

 Defense: Detecting hazardous biological or chemical agents.

2. Transducers in Enzyme Biosensors

Transducers are the core components of enzyme biosensors.

They convert biochemical signals (from enzyme reactions) into measurable electrical or physical outputs like current, voltage,
light intensity, or temperature change.

According to the text, the main transducer technologies are:

 Electrochemical (most common)

o Potentiometric

o Amperometric

o Conductimetric

 Optical (color, luminescence, fluorescence, etc.)

 Calorimetric (heat-based, e.g., thermistors)

2.1 Electrochemical Transducers

2.1.1 Potentiometric Biosensors

  How it works:

o Uses ion-selective electrodes (ISEs) such as pH, ammonium, fluoride, or gas electrodes.

o The enzyme is immobilized on the electrode surface.

o When the enzyme reacts with the analyte, ions are either produced or consumed → this changes the electrical
potential across the electrode.

o This change in potential is measured (almost no current flows).

 Applications:

o Detection of urea, glucose, creatinine, and amino acids.

 Limitations:

 Requires a stable reference electrode, which can be difficult to maintain.

 Advanced versions:

 ENFET (Enzyme-based Field Effect Transistor):

o An enzyme-coated ion-sensitive FET (ISFET) is used.

o Smaller, compact, and uses less enzyme.

o Example: pH-sensitive ISFET coated with urease or glucose oxidase.

 pF-FET biosensors:

o Use special reactions that produce fluoride ions, which the FET detects.

 EIS (Electrolyte Insulator Semiconductor) chips:

o Detect ions like H⁺ and F⁻ using multilayer structures.

2.1.2 Amperometric Biosensors

 How it works:

o Operates at a fixed potential with respect to a reference electrode.

o Measures the current generated by oxidation or reduction reactions at the electrode surface.

o Most suitable with redox enzymes (oxidoreductases).

 Enzymes used:

o Glucose oxidase, lactate oxidase, urate oxidase, cholesterol oxidase, etc.

o These enzymes typically use oxygen as an electron acceptor → producing hydrogen peroxide (H₂O₂) as a byproduct.

 Measurement options:

1. Oxygen consumption

2. Hydrogen peroxide production

 Problems:

o Oxygen dependence: fluctuations in O₂, pH, temperature, or ionic strength affect results.

o Hydrogen peroxide detection requires a high potential (0.6–0.7 V vs Ag/AgCl) → causes interference from ascorbic
acid, uric acid, cysteine, glutathione (present in biological samples).

 Solutions:

o Chemically modified electrodes with mediators (like ferrocene, methylene blue, quinones).
  Lower the operating potential → reduce interference.

o Mediator incorporation methods:

1. Adsorption on electrode surface.

2. Entrapment in polymer films.

3. Covalent bonding to polymers.

4. Mixing with graphite paste electrodes.

o Peroxidase-modified electrodes:

 Detect H₂O₂ at much lower potentials using metals like ruthenium, iridium, or rhodium, minimizing
interference.

 Applications:

o Widely used in commercial glucose biosensors (diabetes testing strips/meters).

2.1.3 Conductimetric Biosensors

 How it works:

o Measures change in electrical conductivity of the solution.

o Many enzyme-catalyzed reactions produce or consume ions, altering conductivity.

 Advantages:

o Extremely sensitive.

 Limitations:

o Non-specific, because conductivity can be affected by many factors (not just the target analyte).

o Therefore, less common compared to potentiometric and amperometric.

2.2 Thermal Enzyme Biosensors

 Principle:

o These biosensors work by measuring the heat released (enthalpy change) during enzyme-catalyzed reactions.

o Every enzymatic reaction either releases or absorbs some amount of heat (ΔH = 4–100 kJ/mol).

o The more substrate present, the more heat is produced → this heat is measured to determine substrate
concentration.

 How it works:

1. The enzyme is immobilized either:

 Directly on a thermistor (a temperature-sensitive resistor), or

 Inside a column where sample flows through.

2. When the substrate reacts with the enzyme, the temperature increases.

3. The thermistor measures this temperature change, which is proportional to the analyte concentration.

 Challenges/Disadvantages:

o Nonspecific thermal effects: Heat may come from sample flow or environment, not just the enzymatic reaction.

o Baseline drift: Continuous heating may shift the baseline temperature over time.
2.3 Optical Enzyme Biosensors

 Principle:

o Enzyme reactions often cause changes in optical properties like:

 UV/Visible absorption

 Luminescence (light emission from chemical or biological reactions)

 Fluorescence (light re-emitted after excitation)

 Reflectance

o These optical changes are measured to determine the analyte concentration.

 Examples of Optical Detection Approaches:

 NAD(P)H Fluorescence

 Some enzyme reactions use NAD(P)H.

 NAD(P)H gives fluorescence (excites at 360 nm, emits at 450 nm).

 Measuring fluorescence tells us substrate concentration.

 Fluorescent Dyes (pH-sensitive)

 Example: FITC dye changes fluorescence with pH.

 If the enzyme reaction changes pH (by releasing/using protons), the dye detects it.

 Luminescence-based Sensors

 Certain bacteria naturally emit light when NADH is present.

 Enzymes produce NADH → luminescence increases → analyte detected.

 Chemiluminescence

 Example: Luminol + H₂O₂ + HRP enzyme → produces light.

 Used with oxidase enzymes that generate hydrogen peroxide.

 Absorbance / Color Change

 Some enzyme reactions make colored products.

 Light absorbance is measured (like a color test).

 Advantages of Optical Biosensors:

o High sensitivity (nanomolar levels detectable).

o Fiber-optic biosensors are small, flexible, resistant to electrical noise, and compact.

o Calibration is stable, especially when using ratiometric (two-wavelength) detection.

 Disadvantages:

o Instability of dyes (fluorescent or chromophoric) over time reduces long-term reliability.

Enzyme Immobilization in Biosensors

Principle:
Immobilization means fixing enzymes onto a solid support or transducer so they can be reused, remain stable, and keep working for long
periods. The method of immobilization directly affects the stability, sensitivity, and lifetime of the biosensor.
1. Physical Methods

 Entrapment:

o Enzymes are trapped inside gels or polymers (e.g., polyacrylamide, alginate, agar, agarose, chitosan).

o Advantages: Gentle on the enzyme, structure and activity mostly retained.

o Disadvantages:

 Pores in gels may restrict movement of substrates/products → diffusion barrier.

 Weak mechanical strength, enzymes may leak out.

 Leads to low sensitivity.

 Adsorption:

o Enzymes stick to surfaces by weak forces (electrostatic, van der Waals, hydrogen bonds).

o Advantages: Simple, cheap, no enzyme modification.

o Disadvantages: Weak attachment → enzyme can wash away in water, poor long-term stability.

o To avoid leakage, an extra protective membrane/film is often added.

2. Chemical Methods

 Covalent Binding:

o Enzyme is chemically bonded to electrode or membranes using reagents like glutaraldehyde or cyanuric chloride.

o Advantages: Strong, stable bonds, resistant to pH and temperature changes.

o Disadvantages: Harsh chemicals may damage enzyme, membranes not regenerable.

 Crosslinking:

o Enzyme molecules are linked to each other (enzyme–enzyme bonding) instead of to a support.

o Often used along with entrapment to reduce enzyme loss.

o Disadvantage: Can reduce enzyme activity, harsh conditions may harm enzymes.

 Electrochemical Polymer Films:

o Thin polymer layers (nanometer thickness) made by electro-polymerization of compounds like pyrrole, thiophene,
phenol, etc.

o Enzyme is either trapped inside or attached to the film.

o Advantages:

 Film is very thin → high sensitivity.

 Provides a permselective barrier that blocks interfering substances (like ascorbate, uric acid, cysteine).

 Prevents electrode fouling.

4. Potential Applications of Enzyme Biosensors

Enzyme biosensors are widely useful in healthcare, environment, food, agriculture, and defense.

1. Medical & Clinical Applications

o Detect substances like glucose, urea, lactate, paracetamol, creatine kinase, AST enzyme, salicylate.

o Useful in emergencies and ICU monitoring (continuous or frequent testing).

 o Growing demand for point-of-care testing (doctor’s office, outpatient, even home testing).

o Benefits: Fast, low-cost, minimal sample preparation, easy to use.

2. Environmental Monitoring

o Detects toxic chemicals (e.g., PCBs, hydrocarbons, phenols, dioxins, peroxides).

o Measures pesticides and heavy metals in air, soil, and water.

o Key features: portable, real-time, cost-effective, on-site testing.

o Needs regulatory approval + field demonstrations before large-scale adoption.

3. Food & Fermentation Industry

o Food quality control: measure amino acids, sugars, antibiotics.

o Freshness indicators: e.g., ATP degradation products or trimethylamine in fish.

o Fermentation monitoring: track substrates, nutrients, and products during production.

o Challenge: steam sterilization in fermenters damages enzyme electrodes

UNIT 3 _______Enzyme Biosensors Based on Gas Electrodes

1. What Are Gas Electrode-Based Enzyme Biosensors?

 These biosensors use gas-sensitive electrodes (usually for CO₂ or NH₃) combined with enzymes to detect specific metabolites.

 Enzymes are immobilized on a gas-permeable membrane over the electrode. They react with a substrate to produce a gaseous
product (like NH₃ or CO₂).

 The gas diffuses through the membrane, changes ion concentration in a thin internal electrolyte layer, and generates a
measurable potential (voltage) proportional to the analyte concentration.

2. How Gas Electrodes Work

 A pH-sensitive glass electrode is placed behind a gas-permeable membrane.

 Gas from the sample (NH₃ or CO₂) dissolves in the thin internal electrolyte layer, altering H⁺ concentration.

 The pH electrode detects this change and converts it to a voltage, which is proportional to the gas concentration.

 Example (ammonia electrode):

Ecell=K−59log⁡[NH3]E_{cell} = K - 59 \log [NH_3]Ecell=K−59log[NH3]

Here, K is a constant, and [NH₃] is the gas produced by enzymatic reaction.

3. Role of pH in Gas Biosensors

 The fraction of gas in its gaseous form depends on the sample pH.

 NH₃ electrode: works best around pH 8.5 (10% ammonia is gaseous).

 CO₂ electrode: more acidic pH favors CO₂ gas formation.

 Compromise: pH must support both enzyme activity and gas formation for accurate measurement.

4. Example: Urease + Ammonia Electrode

  Urease enzyme converts urea → NH₃ + CO₂ + OH⁻.

 NH₃ gas diffuses through the membrane to the electrode.

 The electrode measures NH₃ and gives a voltage reading proportional to urea concentration:

E=K−Slog⁡[urea]E = K - S \log [urea]E=K−Slog[urea]

o S ≈ 45–55 mV (theoretical slope = 59 mV).

IMMOBILISATION

Immobilization of the Enzyme on the Gas Permeable Membrane

Enzymes are attached to a gas-permeable membrane on the electrode. This is necessary because the enzyme reacts with a specific
metabolite (like urea) to produce a gas that the sensor can detect.

Methods:

1. Physical Entrapment:

o Place a small amount of powdered enzyme on the membrane.

o Cover it with a thin dialysis membrane to hold it in place.

o Tighten the membrane using an O-ring.

o Pros: Simple, fast, avoids chemical damage to the enzyme.

o Cons: Enzyme works only for a short time (2–3 days).

2. Chemical Bonding:

o Mix enzyme with BSA (protein) and glutaraldehyde (cross-linker) on the membrane.

o Let it form a thin film, then wash it in glycine solution.

o Store in buffer with preservatives like Kathon or sodium azide.

o Pros: Very stable, can last weeks or months.

o Cons: Chemicals may affect sensitive enzymes if not careful.

3. Using Preactivated Membranes:

o Use commercially available membranes already prepared to bind proteins.

o Soak the membrane in enzyme solution, wash away unbound enzyme, and fix it onto the electrode.

o Pros: Fast, gives strong and stable attachment.

. Assembly of the Gas Electrode

 The gas electrode has an internal pH electrode and a reference electrode (Ag/AgCl).

 Place the electrode in a tube with the gas-permeable membrane at the bottom.

 Fill the tube with the internal electrolyte.

 Tighten the electrode so the pH electrode touches the membrane.

Purpose: This setup allows the electrode to measure the gas produced by the enzyme reaction.