WO2024000622A1

WO2024000622A1 - Device for performing enzyme electrochemical fluid analysis by using short-life enzyme

Info

Publication number: WO2024000622A1
Application number: PCT/CN2022/104630
Authority: WO
Inventors: 库科斯路迪米特里; 塞茨彼得
Original assignee: 上海大学
Priority date: 2022-06-29
Filing date: 2022-07-08
Publication date: 2024-01-04
Also published as: CN115058337A

Abstract

An enzyme electrochemical measurement device for determining the concentration of an analyte in a fluid by using a short-life enzyme. The device is formed by generating a film on a substrate (1), the film being immersed in a fluid under test (3). The film contains a microbial culture capable of producing in situ a required short-life enzyme (10), the film accommodating a micro-bioreactor. The produced enzyme (10) diffuses towards a nearby electrode (12) after leaving the micro-bioreactor. The molecules of a receptor (16) are immobilized on the surface of the electrode (12) and have a specificity for a protease. Thus, the enzyme (10) is bonded to the surface of the electrode (12). When a target analyte arrives at the bonded enzyme, electrons are transferred between the electrode (12) and the enzyme (10). Charges on the electrode (12) are sensed by using a known electronic circuit (14) so as to measure the current or the charges. The total charges have linear correlation with the number of analyte molecules (5) arriving at the enzyme (10), and therefore have linear correlation with the measured concentration of the analyte in the fluid under test (3).

Description

Device for enzymatic electrochemical fluid analysis using short-lived enzymes

Technical field

The present invention relates to a device for the continuous electrochemical determination of target analyte concentration in a fluid, wherein short-lived enzymes are used.

In particular, the present invention relates to a consumable having a microfluidic chamber housing a microbial culture that produces the enzymes necessary for the continuous electrochemical analysis of fluids.

Background technique

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 a liquid sample and subsequently analyzing it in the laboratory using advanced methods such as chromatography, mass spectrometry or immunoassays. Electrochemical methods can be used for continuous analysis without having to analyze liquids with extremely high specificity and sensitivity. This allows for continuous reading of analyte concentration values, rather than repeated measurements of analyte concentration from a sample by immersing a suitable sensor head in a liquid.

A typical electrochemical method for continuous determination of analyte molecular concentration is enzymatic electrochemistry. It involves functionalizing the electrode by coupling oxidoreductase enzymes to the electrode surface. In this way, the presence of the analyte causes an electron transfer between the electrode and the enzyme's cofactors, which is required for the activity of the enzyme as a catalyst. Therefore, the determination of transferred charge is a direct measurement of the amount of analyte that is reduced or oxidized by the catalytic properties of the enzyme, and therefore the concentration of this analyte can be measured directly and continuously.

Most enzymes are composed primarily of proteins, often with some non-protein components called cofactors incorporated into them. Some of these proteases are not very stable at room temperature, and they may degrade rapidly over months, weeks, days, or even hours. Therefore, enzyme electrochemical sensor systems utilizing such short-lived enzymes can only be used for a short period of time given by the lifetime of the corresponding enzyme protein, which is shorter than the expected lifetime of the electrochemical fluid analysis system.

Contents of the invention

In order to overcome the limitations of current enzymatic electrochemical methods using short-lived enzymes, the present invention proposes a device for continuous enzymatic electrochemical measurement of analyte concentration in fluids, utilizing short-lived enzymes, which is characterized by:

The device includes a base, a membrane is included on the base, the membrane encloses a certain volume on the base, and the membrane is immersed in the fluid to be measured; the membrane accommodates a micro bioreactor and an electrochemical sensor, and the micro The bioreactor contains a microbial culture and nutrients necessary for the longevity 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 the receptor molecules on the electrode surface of the electrochemical sensor, causing the short-lived enzyme to be tightly bound to the electrode surface; the specific catalytic reaction of the short-lived enzyme leads to the binding of the short-lived enzyme The oxidation or reduction of the target analyte results in electron transfer between the short-lived enzyme and the electrode; the current of the electrode is measured, and the current is linearly related to the concentration of the analyte in the measured fluid.

Preferably, the electrochemical sensor is placed within the microbioreactor;

Preferably, said microbioreactor is separated from said electrochemical sensor by a semi-permeable membrane or conduit allowing fluid flow from said microbioreactor to said electrochemical sensor;

Preferably, said measuring the current of the electrode includes measuring using a charge sensitive amplifier or a current sensor;

Preferably, the upper part of the base is provided with a cover, so that the device becomes a microfluidic system in the flow direction of the measured fluid.

Preferably, the microbioreactor and the electrochemical sensor in the microfluidic system are each contained in their own fluidic compartments, separated by a semipermeable membrane or fluid flow controller.

The invention consists of a substrate on which a film is produced, which substrate is immersed in the fluid to be measured. The membrane contains a culture of microorganisms capable of producing the desired protease in situ. Therefore, the membrane houses a microbioreactor. The produced protease leaves the microbioreactor and diffuses toward nearby electrodes. Receptor molecules specific for proteases are immobilized on the electrode surface, allowing the enzyme to bind tightly to the electrode surface. When the target analyte reaches the conjugated enzyme, electrons are transferred between the electrode and the enzyme's cofactors. The charge on the electrodes is sensed using known electronic circuits to measure current or charge. The total charge is linearly related to the number of analyte molecules reaching the enzyme and therefore to the analyte concentration of the fluid being measured. The invention effectively solves the technical problem that the enzyme protein life time is shorter than the expected life time of the electrochemical fluid analysis system.

Description of drawings

The present invention will be better understood, and objects in addition to the above-described objects will become apparent when the following detailed description is considered. Such description refers to the accompanying drawings, in which:

Figure 1 shows a schematic cross-section of an enzymatic electrochemical fluid analysis system according to the invention.

Figure 2 shows a schematic cross-section of a preferred embodiment of an enzymatic electrochemical fluid analysis system in which a bioreactor containing a microbial culture is separated from an electrochemical sensor by a membrane.

Detailed ways

The main purpose of the present invention is to provide an enzymatic electrochemical fluid analysis system for continuously measuring the concentration of analytes contained in the fluid being measured.

It is another object of the present invention to provide an enzymatic electrochemical fluid analysis system with short-lived enzymes that have a service life shorter than typical system usage.

In view of the above objects, the present invention is achieved by an enzyme electrochemical fluid analysis system schematically shown in FIG. 1 . The system is implemented on substrate 1. Optionally, the substrate 1 has a cover 2 which enables a microfluidic system in which the test fluid 3 flows in the direction 4 . Alternatively, the substrate 1 is just immersed in the test fluid 3 without the need for microfluidic flow guidance. Test fluid 3 contains analyte molecules 5 plus several additional molecules 6 . The measurement task is to determine the concentration of only analyte molecules 5 . Specificity is obtained by enclosing a volume on the substrate 1 with a semi-permeable membrane 7 . The test fluid 3 and the analyte 5 are free to move through the membrane 7 while the membrane 7 blocks the passage of larger entities such as microorganisms 8 and their nutrients 9 . Microorganisms 8 are cells capable of producing short-lived enzymes 10 while producing waste products 11 by supplying nutrients 9 . A preferred embodiment of the microorganism 8 is the bacterium E. coli, which can be provided with modified DNA such that the required proteins, in particular the short-lived enzymes required for electrochemical sensing of the analyte 5, can be produced.

The complexity of enzyme molecules that can be expressed by bacteria is limited because bacteria are prokaryotic cells, i.e. their organelles and their 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 enzyme. Fungi, especially yeasts such as Saccharomyces cerevisiae, are well suited for this purpose, and the DNA modification processes used for them are well known.

To better accommodate the microbial culture, the microorganisms 8 and their nutrients 9 can be enclosed by another semipermeable membrane or in a separate part of the microfluidic system. The purpose is to maintain the microbial culture and its feedstock while allowing free access of waste products 11 and enzyme proteins 10 into the volume contained by membrane 7 .

Short-lived enzymes 10 diffuse from the microbial culture into the volume contained by the membrane 7 , which is placed near the conductive electrode 12 . Electrode 12 is functionalized with surface-immobilized receptors 16 specific for enzyme 10 . Once the enzyme 10 approaches the binding receptor 16, the enzyme protein also binds to the electrode 12, approaching its surface. The conjugated enzyme protein 17 is now ready for the electrochemical transduction task: once the analyte molecule 5 reaches the immobilized enzyme 17 , the catalytic properties of the enzyme 17 cause the oxidation or reduction of the reactant analyte 19 to the product 20 . The catalytic reaction is accompanied by electron transfer between the electrode and the enzyme's cofactors. Electron transfer 21 changes the charge state of electrode 12 . This may be sensed using electronic circuitry 14 electrically connected to electrode 12 via conductive leads 13 . The electronic circuit 14 is implemented as a known charge-sensitive amplifier or current sensor, which generates a corresponding readout signal at its output 15 .

A first preferred embodiment of the enzyme electrochemical measurement system according to the invention is a system with which potentially toxic analytes in drinking water, industrial water, sewage or purified water can be continuously determined. For this purpose, it may be advantageous to generate small side branches of the measured water, in which the electrochemical measurement system is placed.

The optimal growth conditions (eg temperature, pH) of a microbial culture may differ from the conditions in the fluid or at the electrochemical sensing site for efficient catalytic operation of the enzyme. For this case, another preferred embodiment of the enzyme electrochemical measurement system according to the invention consists of a microfluidic system, in which the bioreactor and the electrochemical sensor are each contained in its own fluidic compartment, consisting of a semipermeable membrane or Fluid flow controller separate. The microfluidic system is shown in cross-section in Figure 2 . The liquid to be measured enters the system from direction 30. Liquid flows into the inlet chamber 31 , from which the liquid is transported into the microfluidic channel 32 . This transport can occur actively, such as through a pump or pressure differential, or passively, such as through the capillary effect. The microfluidic channel contains at least two compartments separated by

semi-permeable membranes

33, 34 and 35. The first compartment 36 contains the microbioreactor and the second compartment 37 contains the electrochemical sensor. Once the sensing process in the electrochemical sensor compartment 37 is achieved, the fluid flows through the membrane 35 into the output reservoir 38 from which it is conveyed to a fluid waste disposal point 39. Microbioreactor 36 contains a microbial culture consisting of microorganisms 40 that are genetically engineered such that they utilize surrounding nutrients 41 to produce required short-lived enzymes 42 while simultaneously producing waste 43. The produced enzymes 42 leave the microorganisms and diffuse into the microbioreactor 36 where they can move freely in the fluid. Free enzyme molecules 44 can diffuse through the membrane 34 into the sensor compartment 37 . Free enzyme molecules 45 in the sensor compartment diffuse close to the conductive electrode 46, whose surface is functionalized with receptor molecules 47. These receptor molecules provide specific binding sites for enzyme molecules 45. Enzyme molecules close to the receptor 46 are accommodated close to the electrode surface 46 as bound enzyme 48 . As described above, the enzyme 48 mediates a catalytic reaction 49 of the analyte molecule, transferring electrons between the electrode 46 and the enzyme's cofactor. The enzyme-catalyzed reaction 49 changes the charge state of the electrode 46 . This may be sensed using electronic circuitry 51 electrically connected to electrode 46 via conductive leads 50 . The electronic circuit 51 is implemented as a known charge-sensitive amplifier or current sensor, which generates a corresponding readout signal at its output 52 . This signal is a direct measurement of the desired analyte concentration in the fluid being measured. By separating the bioreactor 36 and the electrochemical sensor 37 by membranes in the microfluidic system, the chemical and physical conditions in each compartment can be adjusted for optimal performance of each. The membrane allows transport of all molecules through the microfluidic system that need to flow through the system for the correct operation of the electrochemical detection system.

Another preferred embodiment of the enzymatic electrochemical measurement system according to the invention is a miniaturized, wearable sensor system for the continuous analysis of biomarkers in organisms, in particular human sweat. For this purpose, the wearable system contains a microfluidic system that has the following tasks: collect sweat on the skin of the user, convey the sweat into the measurement channels of the microfluidic system, and then transfer the sweat to waste storage or evaporate into the environment. The measurement channel contains an enzymatic electrochemical sensor system according to the invention for the continuous determination of analyte concentration in sweat.

The above are only preferred embodiments of the present invention and do not limit the present invention in any way. Any person skilled in the technical field who makes any form of equivalent substitution or modification to the technical solutions and technical contents disclosed in the present invention shall not deviate from the technical solutions of the present invention. The contents still fall within the protection scope of the present invention.

Claims

An apparatus for continuous enzymatic electrochemical measurement of analyte concentration in fluids utilizing short-lived enzymes, characterized by:

The device includes a base, a membrane is included on the base, the membrane encloses a certain volume on the base, and the membrane is immersed in the fluid to be measured; the membrane accommodates a micro bioreactor and an electrochemical sensor, and the micro The bioreactor contains a microbial culture and nutrients necessary for the longevity 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 the receptor molecules on the electrode surface of the electrochemical sensor, causing the short-lived enzyme to be tightly bound to the electrode surface; the specific catalytic reaction of the short-lived enzyme leads to the binding of the short-lived enzyme The oxidation or reduction of the target analyte results in electron transfer between the short-lived enzyme and the electrode; the current of the electrode is measured, and the current is linearly related to the concentration of the analyte in the measured fluid.
8. An apparatus for continuous enzymatic electrochemical measurement of analyte concentration in a fluid according to claim 1, the electrochemical sensor being placed within the microbioreactor.
The device for continuous enzymatic electrochemical measurement of analyte concentration in a fluid according to claim 1, by allowing the fluid to flow from the micro bioreactor to the electrochemical sensor through a semi-permeable membrane or conduit. The bioreactor is separate from the electrochemical sensor.
2. An apparatus for continuous enzymatic electrochemical measurement of analyte concentration in a fluid according to claim 1, said measuring the current of the electrode comprising measuring using a charge sensitive amplifier or a current sensor.
According to the device for continuous enzymatic electrochemical measurement of analyte concentration in fluid according to claim 1, the upper part of the substrate is provided with a cover, so that the device becomes a microfluidic system in the flow direction of the measured fluid.
The device for continuous enzymatic electrochemical measurement of analyte concentration in a fluid according to claim 5, the microbioreactor and the electrochemical sensor in the microfluidic system are each contained in their own fluidic compartments. chambers, separated by semipermeable membranes or fluid flow controllers.