Device for enzymatic electrochemical fluid analysis using short-lived enzymes

Abstract

An enzyme electrochemical measurement device for determining the concentration of an analyte in a fluid using a short-lived enzyme. The device consists of a substrate on which a membrane is produced, which is immersed in the fluid to be measured. The membrane contains a culture of microorganisms capable of producing the desired short-lived enzyme in situ. Thus, the membrane houses a micro-bioreactor. The produced enzyme leaves the micro-bioreactor and diffuses towards a nearby electrode. Receptor molecules are immobilized on the surface of the electrode and are specific to the protease. In this way, the enzyme binds to the electrode surface. When the target analyte reaches the bound enzyme, electrons are transferred between the electrode and the enzyme. The charge on the electrode is sensed with known electronic circuitry to measure the current or charge. The total charge is linearly related to the number of analyte molecules that reach the enzyme and thus also to the concentration of the sought analyte in the fluid to be measured.

Description

Technical Field

[0001] This invention relates to an apparatus for continuous electrochemical determination of the concentration of a target analyte in a fluid, wherein a short-lived enzyme is used.

[0002] In particular, the present invention relates to a consumable having a microfluidic cavity for containing microbial cultures, which produces enzymes necessary for continuous electrochemical analysis of fluids. Background Technology

[0003] To ensure the safety and quality of liquids, chemical analysis is often required to determine the concentration of relevant analytes. This can be done by collecting liquid samples and then analyzing them in the laboratory using advanced methods such as chromatography, mass spectrometry, or immunoassay. In cases where it is not necessary to analyze liquids with extremely high specificity and sensitivity, continuous analysis can be performed using electrochemical methods. This allows for the continuous reading of analyte concentration values, rather than repeatedly measuring the analyte concentration from the sample by immersing a suitable sensor head in the liquid.

[0004] A typical electrochemical method for the continuous determination of analyte molecular concentration is enzyme electrochemistry. This involves functionalizing the electrode by coupling a redox enzyme to the electrode surface. The presence of the analyte induces electron transfer between the electrode and the enzyme's cofactor, which is necessary for the enzyme's catalytic activity. Therefore, the determination of the transferred charge is a direct measurement of the amount of analyte reduced or oxidized by the enzyme's catalytic properties, thus allowing for direct and continuous measurement of the analyte concentration.

[0005] Most enzymes are primarily composed of proteins, often incorporating non-protein components called cofactors. Some of these proteases are not very stable at room temperature and can degrade rapidly over months, weeks, days, or even hours. Therefore, enzyme electrochemical sensor systems utilizing such short-lived enzymes can only be used for short time periods determined by the lifetime of the corresponding enzyme protein, which is shorter than the expected lifetime of an electrochemical fluid analysis system. Summary of the Invention

[0006] To overcome the limitations of current enzyme electrochemical methods using short-lived enzymes, this invention proposes a device for continuous enzyme electrochemical measurement of analyte concentrations in fluids, utilizing short-lived enzymes, characterized by:

[0007] The device includes a substrate on which a membrane is enclosed to a certain volume on the substrate and is immersed in a fluid to be measured; the membrane houses a microbioreactor and an electrochemical sensor, the microbioreactor containing a microbial culture and nutrients necessary for the lifespan of the electrochemical sensor; the microbial culture consists of microorganisms prepared by appropriate DNA insertion, and the microbioreactor continuously produces short-lived enzymes in situ;

[0008] The short-lived enzyme diffuses to acceptor molecules on the electrode surface of the electrochemical sensor, causing the short-lived enzyme to bind tightly to the electrode surface; the specific catalytic reaction of the short-lived enzyme leads to the oxidation or reduction of the target analyte that has bound the short-lived enzyme, thereby generating electron transfer between the short-lived enzyme and the electrode; the current of the electrode is measured, and the current is linearly correlated with the concentration of the analyte in the analyte fluid.

[0009] Preferably, the electrochemical sensor is placed inside the microbioreactor;

[0010] Preferably, the current of the measuring electrode is measured using a charge-sensitive amplifier or a current sensor;

[0011] Preferably, the upper part of the substrate has a cover, making the device a microfluidic system in the direction of the flow of the fluid being measured.

[0012] Preferably, the microbioreactor and the electrochemical sensor in the microfluidic system are each contained in their own fluid compartments, separated by a fluid flow controller.

[0013] This invention comprises a substrate on which a membrane is generated, which is then immersed in the analyte fluid. The membrane contains a microbial culture capable of producing the desired protease in situ. Therefore, the membrane houses a microbioreactor. The generated protease exits the microbioreactor and diffuses towards a nearby electrode. Acceptor molecules specific to the protease are immobilized on the electrode surface, causing the enzyme to bind tightly to the electrode surface. When the target analyte reaches the binding enzyme, electrons are transferred between the electrode and the enzyme's cofactor. The charge on the electrode is sensed using known electronic circuitry to measure the current or charge. The total charge is linearly correlated with the number of analyte molecules reaching the enzyme, and therefore also linearly correlated with the analyte concentration in the analyte fluid. This invention effectively solves the technical problem of enzyme protein lifetimes being shorter than the expected lifetime of electrochemical fluid analysis systems. Attached Figure Description

[0014] The invention will be better understood when the following detailed description is considered, and objectives other than those described above will become apparent. This description refers to the accompanying drawings, in which:

[0015] Figure 1 A schematic cross-section of the enzyme electrochemical fluid analysis system according to the present invention is shown. Detailed Implementation

[0016] The main objective of this invention is to provide an enzyme electrochemical fluid analysis system for continuously measuring the concentration of analytes contained in the test fluid.

[0017] Another object of the present invention is to provide an enzyme electrochemical fluid analysis system for short-lived enzymes with a shorter service life than typical system usage time.

[0018] In view of the above objectives, the present invention provides... Figure 1 The enzyme electrochemical fluid analysis system is schematically illustrated. This system is implemented on a substrate 1. Optionally, the substrate 1 has a cap 2, which implements a microfluidic system in which the test fluid 3 flows in direction 4. Optionally, the substrate 1 is simply immersed in the test fluid 3 without microfluidic flow guidance. The test fluid 3 contains analyte molecules 5 plus several additional molecules 6. The measurement task is to determine the concentration of only the analyte molecules 5. Specificity is obtained by confining a certain volume on the substrate 1 with a semi-permeable membrane 7. The test fluid 3 and the analyte 5 can move freely through the membrane 7, while the membrane 7 blocks the passage of larger entities such as microorganisms 8 and their nutrients 9. Microorganism 8 is a cell capable of producing short-lived enzymes 10 and simultaneously generating waste 11 by supplying nutrients 9. A preferred embodiment of microorganism 8 is the bacterium *Escherichia coli*, which can provide modified DNA to produce the desired proteins, particularly the short-lived enzymes required for electrochemical sensing of analyte 5.

[0019] The complexity of enzyme molecules that can be expressed by bacteria is limited because bacteria are prokaryotic cells, meaning their organelles and nuclei are not contained within a membrane. Therefore, another preferred embodiment of microorganism 8 is a eukaryotic cell whose DNA has been modified to express the desired short-lived enzymes. Fungi, particularly yeasts such as *Saccharomyces cerevisiae*, are well-suited for this purpose, and the DNA modification processes used for them are well-known.

[0020] To better contain the microbial culture, the microorganisms 8 and their nutrients 9 can be enclosed by another semi-permeable membrane or in a separate section of the microfluidic system. The aim is to retain the microbial culture and its raw materials while allowing waste products 11 and enzyme proteins 10 to freely enter the volume contained in the membrane 7.

[0021] A short-lived enzyme 10 diffuses from a microbial culture into the volume contained in membrane 7, which is placed near conductive electrode 12. Electrode 12 is functionalized with a surface immobilizer 16 specific to enzyme 10. Once enzyme 10 approaches the binding receptor 16, the enzyme protein also binds to electrode 12, near its surface. The bound enzyme protein 17 is now ready for electrochemical transduction: once analyte molecule 5 reaches the immobilized enzyme 17, the catalytic properties of enzyme 17 cause the reactant analyte 19 to be oxidized or reduced to product 20. This catalytic reaction is accompanied by electron transfer between the electrode and the enzyme's cofactor. Electron transfer 21 alters the charge state of electrode 12. This can be sensed by an electronic circuit 14 electrically connected to electrode 12 via conductive leads 13. Electronic circuit 14 is implemented as a known charge-sensitive amplifier or current sensor, generating a corresponding readout signal at its output 15.

[0022] A first preferred embodiment of the enzyme electrochemical measurement system according to the invention is a system that can continuously determine potentially toxic analytes in drinking water, industrial water, wastewater, or purified water. For this purpose, it may be advantageous to generate a small side branch of the water being measured, in which the electrochemical measurement system is placed.

[0023] Another preferred embodiment of the enzyme electrochemical measurement system according to the invention is a miniaturized, wearable sensor system for the continuous analysis of biomarkers in biological, particularly human, sweat. For this purpose, the wearable system comprises a microfluidic system that performs the following tasks: collecting sweat on the user's skin, delivering the sweat to a measurement channel of the microfluidic system, and then transferring the sweat to a waste reservoir or evaporating it into the environment. The measurement channel contains the enzyme electrochemical sensor system according to the invention for the continuous determination of the concentration of analytes in the sweat.

[0024] The above are merely preferred embodiments of the present invention and do not constitute any limitation on the present invention. Any equivalent substitutions or modifications made by those skilled in the art to the technical solutions and content disclosed in the present invention without departing from the scope of the present invention shall be deemed to have remained within the protection scope of the present invention.

Claims

1. An apparatus for continuous enzymatic electrochemical measurement of analyte concentration in fluids, utilizing short-lived enzymes, characterized in that: The device includes a substrate on which a membrane is enclosed to a certain volume on the substrate and is immersed in a fluid to be measured; the membrane houses a microbioreactor and an electrochemical sensor, the microbioreactor containing a microbial culture and nutrients necessary for the lifespan of the electrochemical sensor; the microbial culture consists of microorganisms prepared by appropriate DNA insertion, and the microbioreactor continuously produces short-lived enzymes in situ; The short-lived enzyme diffuses to acceptor molecules on the electrode surface of the electrochemical sensor, causing the short-lived enzyme to bind tightly to the electrode surface; the specific catalytic reaction of the short-lived enzyme leads to the oxidation or reduction of the target analyte that has bound the short-lived enzyme, thereby generating electron transfer between the short-lived enzyme and the electrode; the current of the electrode is measured, and the current is linearly correlated with the concentration of the analyte in the analyte fluid.

2. The apparatus for continuous enzyme electrochemical measurement of analyte concentration in a fluid according to claim 1, wherein the current of the measuring electrode is measured using a charge-sensitive amplifier or a current sensor.

3. The apparatus for continuous enzyme electrochemical measurement of analyte concentration in a fluid according to claim 1, wherein the substrate has a cap on the upper part, making the apparatus a microfluidic system in the direction of flow of the fluid being measured.

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