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Amperometry: Overview01:10

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Amperometry is a technique commonly used to measure the concentration of specific analytes in a solution by monitoring the electric current generated during an electrochemical reaction. It involves applying a constant potential between a working electrode and a reference electrode to measure the resulting current, which is proportional to the concentration of the analyte. The Clark oxygen electrode operates based on this principle of amperometry. It consists of a cathode and an anode enclosed...
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Membrane electrodes, also known as p-ion electrodes, use membranes that selectively interact with free analyte ions, generating a potential difference across the membrane. The resulting membrane potential, known as the asymmetry potential, is not zero even when analyte concentrations on both sides of the membrane are equal. The membrane's response is typically not selective to a single analyte but proportional to the concentration of all ions in the sample solution capable of interacting at...
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Polarography is a classical voltammetric technique used to analyze electrochemical reactions. This method applies a linear potential sweep to a dropping mercury electrode (DME), and the resulting current is measured. A dropping mercury electrode is commonly used as the working electrode in polarography. It consists of a capillary tube filled with mercury, where the tiny droplet forms at the tip. This droplet continuously drops from the capillary, creating a new electrode surface for each...
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An enzyme-centric approach for constructing an amperometric l-malate biosensor with a long and programmable linear

Christopher J Matthews1, Wayne M Patrick1

  • 1Centre for Biodiscovery, School of Biological Sciences, Victoria University of Wellington, Wellington, New Zealand.

Protein Science : a Publication of the Protein Society
|July 29, 2023
PubMed
Summary

Researchers developed a new enzyme for biosensors to accurately measure l-malate in undiluted juices and wine. This enzyme offers a tunable range, improving upon existing malate detection methods for food and beverage analysis.

Keywords:
Ascaris suum malic enzymebioelectronicselectrochemical biosensorelectron mediatorsenzyme characterizationscreen-printed electrodewinemaking

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Area of Science:

  • Biochemistry and biosensor technology
  • Enzyme engineering and directed evolution
  • Food and beverage analysis

Background:

  • L-malate is crucial for flavor and acidity in food and beverages, especially wine.
  • Current enzyme biosensors for l-malate have limited linear ranges, requiring sample dilution.
  • A need exists for more versatile and sensitive l-malate detection methods.

Purpose of the Study:

  • To discover and engineer novel malate-oxidizing enzymes for improved biosensor performance.
  • To develop an l-malate biosensor with a tunable linear range and high sensitivity.
  • To validate the biosensor's functionality in real-world food and beverage samples.

Main Methods:

  • Database mining, gene synthesis, and recombinant expression to identify and produce enzymes.
  • Spectrophotometric assays to characterize enzyme activity and suitability for biosensors.
  • Development and optimization of an amperometric biosensor prototype using a novel enzyme.

Main Results:

  • A bespoke biocatalyst, Ascaris suum malic enzyme with mutation R181Q [AsME(R181Q)], was engineered.
  • The AsME(R181Q) biosensor demonstrated an ultra-wide linear range (50-200 mM) in its initial configuration.
  • Sensitivity was enhanced five-fold with Mn2+ and six-fold with citrate, yielding a 1-10 mM linear range.
  • Interference from ascorbate was mitigated using ascorbate oxidase, enabling accurate measurements in undiluted wine.

Conclusions:

  • An enzyme-centric approach successfully yielded a novel l-malate biosensor with adaptable sensitivity and linear range.
  • The developed biosensor accurately quantifies l-malate in undiluted wine samples, outperforming previous methods.
  • This work paves the way for advanced electrochemical biosensors with enhanced functionality for food and beverage applications.