Comprehensive Notes on COVID-19 Detection Methods, Biosensors, and Electrochemistry

From DNA to Proteins: The Case Study of COVID

Spike Glycoprotein (S), RNA, and N Protein

  • These are components related to viruses, specifically relevant to the study of COVID.
  • Envelope and Hemagglutinin-esterase dimer (HE) are also viral components.

DNA/RNA Nanopore Sequencing

  • MinION is a device used for DNA/RNA Nanopore Sequencing.

Antibodies

  • Antibodies are Y-shaped proteins used by the immune system to neutralize pathogens.
  • Y=antibodyY = antibody

Detecting Proteins

  • The spike protein and antibodies themselves are proteins.
  • Two methods to detect proteins are:
    • ELISA (enzyme-linked immunosorbent assay)
    • Lateral flow assay

Lateral Flow Assay

  • Provides a qualitative (yes or no) answer in 30 minutes without specialized equipment.
  • Gold nanoparticles decorated with SARS-COV-2 antibodies are dried on the strip.
  • Gold nanoparticles decorated with control antibodies are also present.
  • If present, spike proteins bind to the antibodies.
  • The spike-antibody complex flows to the test line.
  • If the spike protein-antibodies complex is formed, it will be captured at the test line by a second set of antibodies specific to the spike protein, and the test line will turn red due to the presence of gold nanoparticles.
  • The control line is functionalized with another set of antibodies designed to bind the antibodies-coated gold nanoparticles. The presence of a control line ensures that the sample has flowed through and the antibodies are active.

ELISA

  • An assay to detect SARS-CoV-2 seroconversion in humans.
  • Antigen (=VIRUS) binds to the surface of a plastic well.
  • Antibodies (if present) bind to the antigen.
  • The results are then read out.
  • ELISA is performed in laboratories by trained personnel.
  • It is a quantitative method.
  • Complete the ELISA virtual lab on Labster to learn more about the details of this assay.

Proton-ELISA

  • Uses an ISFET (Ion-Sensitive Field-Effect Transistor) to detect antigen-antibody interactions.
  • Requires biochemical reactions that release H+ (protons) so an ISFET readout can be used.
  • The process involves:
    • Antigen and antibody interaction.
    • Binding event leading to substrate conversion, which GOX facilitates.
    • GOX releases H+ (protons) creating a signal.
  • The drain current change is measured over time, indicating different concentrations like +0 pg, +25 pg, and -50 pg.
  • Proton-ELISA demonstrates a dose-response relationship with analytes like CRP (C-Reactive Protein) and IgE (Immunoglobulin E).
  • Comparison with standard ELISA shows comparable results.

Biosensors

  • A biosensor is an analytical device that can detect the presence (or absence) of an analyte (often a biological molecule such as a protein, indicative of a disease biomarker) in a certain environment (e.g., patient blood) using biological molecules as part of the sensing mechanism.
  • The most important application area of biosensors in medical electronics is in vitro diagnostics (IVD).
  • IVD devices are used in hospitals and clinical laboratories.
  • Also important are over-the-counter diagnostics like glucose tests or LFD COVID tests.
  • Ideally, the sensor should be self-contained, so that it is not necessary to add reagents to the sample to obtain the desired response.
  • Biosensors use molecular recognition components to detect specific substances.
  • The detector senses changes such as charge, heat, light, pH change, or mass change.
  • A signal transducer converts these changes into an electrical signal.
  • Selectivity is a key requirement; the sensor should only generate a signal if the target molecule of interest is present.
  • Antibodies are an example of a bioreceptor, but there are many others.
  • The information on whether a biological molecule is present needs to be converted into a processable signal.
  • The binding of a biological molecule onto the receptor part can generate a change in the sensor environment (e.g., heat, light, etc.).

Electrochemical Sensors

  • An electronic transducer is ideal as the interfacing with the rest of the electronics in the patient care pathway would be straightforward.
  • A biosensor usually has to work in a liquid environment (e.g., blood or serum).
  • Electrochemical principles are often employed for sensing rather than pure electronic components.
  • An electrochemical sensor is usually a three-electrode system comprising:
    • A working electrode (the actual sensor).
    • A counter electrode (to supply charge/current to the system).
    • A reference electrode (electrochemical reference).

Electrochemistry

  • Electrochemistry is the branch of chemistry concerned with the interrelation of electrical and chemical effects.
  • It studies chemical changes caused by the passage of an electric current and the production of electrical energy by chemical reactions.
  • The field encompasses a huge array of different phenomena, devices, and technologies.
  • R<em>ox+ne−⇌R</em>redR<em>{ox} + ne^- \rightleftharpoons R</em>{red}
  • LEO the lion says GER: Loss of Electron Oxidation, Gain of Electron Reduction
  • Example: Fe2+⇌Fe3++e−Fe^{2+} \rightleftharpoons Fe^{3+} + e^-

Nernst Equation

  • For thermodynamically controlled systems, the potential at the electrode is given by the Nernst equation:
  • E=E0+RTnFlnC<em>oxC</em>redE = E^0 + \frac{RT}{nF} ln \frac{C<em>{ox}}{C</em>{red}}
    • Where:
      • E0E^0 is the standard potential for the reaction.
      • RR is the universal gas constant (8.314J⋅K−1⋅mol−18.314 J \cdot K^{-1} \cdot mol^{-1}).
      • TT is the temperature in Kelvin.
      • nn is the number of electrons exchanged in the reaction.
      • FF is Faraday's constant (96,485C⋅mol−196,485 C \cdot mol^{-1}).
      • CoxC_{ox} is the concentration of the oxidized species.
      • CredC_{red} is the concentration of the reduced species.

Electrochemical Setup

  • The rate of the reaction depends on the potential, and the current is proportional to the rate of the reaction.
  • In a 2-electrode configuration, the potential at the WE (working electrode) is not well-defined.

Electrochemical Sensors (Three-Electrode System)

  • An electrochemical sensor uses a three-electrode system:
    • Working electrode (the sensor).
    • Counter electrode (to supply charge/current).
    • Reference electrode (electrochemical reference).

Working Electrode (WE)

  • The sensor, usually made of conductive materials like carbon, Au, or Pt.

Reference Electrode (RE)

  • For biological applications, Ag/AgCl (Silver Chloride) is commonly used.
  • Provides a stable potential for controlled regulation of the working electrode potential.
  • Made up of phases with essentially constant composition, so RE has a constant potential if no current is passed through it (Nernst equation).
  • Chemically inert and stable over a wide range of pH and voltages.
  • ECG electrodes, ISFET reference electrodes, and electronic glucose meter reference electrodes are made of Ag/AgCl.

Counter Electrode

  • Needed to close the circuit and to supply charge/current to the system.
  • Usually made of noble metals like Pt or Au.

Potentiostat

  • Electronic hardware required to control a three-electrode cell and run most electrochemical experiments.
  • Maintains the potential of the working electrode at a given level with respect to the reference electrode by adjusting the current at the counter electrode.
  • For more information, consult provided resources.

Types of Electrochemical Sensors

  • Amperometric sensors: Electrons are produced/consumed at the working electrode due to the analyte-to-capture-molecule binding event (capture molecule is an enzyme). Blood glucose sensors are common examples.
  • Potentiometric sensors: The binding of the analyte causes a shift of the electrochemical potential of the working electrode.
  • Impedimetric sensors: The impedance of the working electrode changes as a result of the binding event.

Glucose Sensors

  • The first glucose sensors used the (electro)chemical reaction:
  • Glucose+Gox−FAD⇌Gluconolactone+Gox−FADH2Glucose + Gox-FAD \rightleftharpoons Gluconolactone + Gox-FADH_2
  • Gox−FADH<em>2+O</em>2⇌Gox−FAD+H<em>2O</em>2Gox-FADH<em>2 + O</em>2 \rightleftharpoons Gox-FAD + H<em>2O</em>2
  • H<em>2O</em>2⇌2H++O2+2e−H<em>2O</em>2 \rightleftharpoons 2H^+ + O_2 + 2e^-
  • The number of electrons measured (current) at the electrode correlates with the concentration of glucose.
  • A problem is that H<em>2O</em>2H<em>2O</em>2 requires an electrochemical potential of about 0.7 V to be oxidized, which is very high. Many blood components oxidize (and decompose) as well!

Improved Glucose Sensors

  • Use mediators such as ferrocene or N-methylphenzinium cation (NMP+).
  • These can be covalently attached to the enzyme or electrode.
  • Reactions:
  • Glucose+Gox−FAD⇌Gluconolactone+Gox−FADH2Glucose + Gox-FAD \rightleftharpoons Gluconolactone + Gox-FADH_2
  • Gox−FADH<em>2+Ferrocene</em>ox⇌Gox−FAD+FerroceneredGox-FADH<em>2 + Ferrocene</em>{ox} \rightleftharpoons Gox-FAD + Ferrocene_{red}
  • Ferrocene<em>red⇌Ferrocene</em>ox+e−Ferrocene<em>{red} \rightleftharpoons Ferrocene</em>{ox} + e^-
  • The mediators oxidize at the electrode.

Typical Glucose Sensor Setup

  • Consists of:
    • Substrate (ceramic or plastic).
    • Conducting tracks.
    • Carbon electrode.
    • Glucose oxidase + mediator.
    • Silver reference electrode.