CN111474218B

CN111474218B - Integrated micro-fluidic electrochemical sensor chip for BOD rapid detection, preparation method thereof and BOD detection method

Info

Publication number: CN111474218B
Application number: CN202010327183.3A
Authority: CN
Inventors: 胡敬芳; 周兴辉; 胡世炜; 宋钰; 高国伟
Original assignee: Beijing Information Science and Technology University
Current assignee: Beijing Information Science and Technology University
Priority date: 2020-04-23
Filing date: 2020-04-23
Publication date: 2022-07-01
Anticipated expiration: 2040-04-23
Also published as: AU2020103437A4; CN111474218A

Abstract

The invention provides an integrated micro-fluidic electrochemical sensor chip for BOD rapid detection, a preparation method thereof and a BOD detection method, and relates to the technical field of BOD detection. The chip provided by the invention comprises: a single crystal silicon substrate; a PDMS layer which is overlapped on the surface of the monocrystalline silicon substrate and is internally provided with a microflow channel system, wherein the microflow channel system comprises a liquid inlet to be detected, a microflow channel, a biomembrane reactor, an electrochemical detection pool and a waste liquid outlet; a three-electrode system arranged between the monocrystalline silicon substrate and the PDMS layer and distributed in an interdigital ultramicro electrode array; and a platinum film temperature sensor arranged on the bottom surface of the monocrystalline silicon substrate, wherein a micro heating copper electrode is adhered to the surface of the platinum film temperature sensor. The invention adopts the micro-fluidic technology to realize the effective integration of two functional units of the biofilm reactor and the electrochemical electrode on the same chip, and realizes the miniaturization of the sensor and the rapid, stable, accurate and continuous automatic monitoring of BOD of a water sample.

Description

Integrated micro-fluidic electrochemical sensor chip for BOD rapid detection, preparation method thereof and BOD detection method

Technical Field

The invention relates to the technical field of biochemical oxygen demand detection, in particular to an integrated micro-fluidic electrochemical sensor chip for BOD rapid detection and a preparation method and a BOD detection method thereof.

Background

Biochemical oxygen demand (also called BOD) is a comprehensive index representing the content of aerobic pollutants such as organic matters in water, and it indicates the total amount of dissolved oxygen in water consumed in the process of oxidative decomposition of organic matters in water due to the biochemical action of microorganisms, and the higher the value, the more organic pollutants in water, the more serious the pollution. The measurement of BOD is a biological degradation and aerobic biochemical treatment process simulating a natural water body, is a main means for evaluating the biochemical treatment of sewage, and is also a necessary measurement project for evaluating water quality. The rapid and accurate determination of BOD in water has always been a major problem in environmental monitoring.

In recent decades, BOD rapid measurement methods have been developed rapidly, with the microbial membrane electrode method being the most studied. According to this method, the microbial membrane is fixed on the surface of the oxygen electrode, the reading of the dissolved oxygen electrode is kept stable in a sample containing no organic matter, and when the sample contains organic matter, the dissolved oxygen is consumed by the microbes fixed in the membrane to decompose the organic matter, so that the dissolved oxygen concentration decreases with the increase in the concentration of the organic matter, and the BOD in water can be calculated from the quantitative relationship between the difference in the organic matter concentration and the decrease in the dissolved oxygen. In 2002, the method is listed as an industrial standard for BOD rapid determination by China, and a standard is established for market admittance of the instruments. Although such instruments and methods are simple to operate and rapid to detect (typically within 30 minutes), they have their own problems: firstly, dissolved oxygen is taken as a detection object, and the efficiency of degrading organic matters by microorganisms is limited due to the low solubility of the dissolved oxygen in water, so that an oxygen electrode signal is weak, and a challenge is brought to a detection system of a sensor rear end circuit; secondly, the compacted microbial film covers the surface of the oxygen electrode, the microbial system is influenced while the electrode is replaced, the long-term stability of the sensor is difficult to ensure, and the continuous online monitoring of BOD cannot be realized; thirdly, because the thickness of the microbial film on the surface of the fixed oxygen electrode is thin, the types and the quantity of microbes on the film are limited, and organic matter components which exceed the capability of decomposing the fixed microbes are easily encountered in practical application, thereby causing the problem of low test result.

In the early 90 s of the 20 th century, researchers have studied and utilized a bioreactor method to achieve rapid BOD determination in a water sample, the principle of which is to place microorganisms in a closed container containing a test water sample, after a period of incubation reaction, detect the consumption of dissolved oxygen with an oxygen electrode to obtain the BOD concentration. However, as the microorganisms are dispersed and suspended in the water sample, the time and labor are consumed for cleaning the microorganisms in the detection process of different water samples, so that the method cannot be applied to field detection. In the beginning of 2012, dongjun research group of Changchun applied chemistry research institute of Chinese academy of sciences proposed a BOD electrochemical detection method based on a biofilm reactor, which has the principle that microbial flora is fixed into a film and placed in a sealed glass tube to form the biofilm reactor, so that the microbes can be conveniently and rapidly cleaned, and the detection life of the sensor reaches 17 months. However, both the conventional bioreactor and the currently proposed bioreactor have complicated structure and large volume, and it is difficult to miniaturize the detection instrument.

Disclosure of Invention

In view of this, the present invention aims to provide an integrated micro-fluidic electrochemical sensor chip for BOD rapid detection, a preparation method thereof, and a BOD detection method. The invention adopts the micro-fluidic technology to realize the effective integration of two functional units of the biofilm reactor and the electrochemical electrode on the same chip, and realizes the miniaturization of the sensor and the rapid, stable and continuous automatic monitoring of BOD of a water sample.

In order to achieve the above object, the present invention provides the following technical solutions:

the invention provides an integrated micro-fluidic electrochemical sensor chip for rapidly detecting biochemical oxygen demand, which comprises:

a single-crystal silicon substrate 1 which is,

a PDMS layer 3 superposed on the surface of the monocrystalline silicon substrate 1; a microflow channel system is arranged in the PDMS layer 3, the microflow channel system comprises a liquid inlet 4 to be detected, a biomembrane reactor 6 with an inlet communicated with the liquid inlet 4 to be detected through a first microflow channel 5-1, an electrochemical detection cell 7 with an inlet communicated with an outlet of the biomembrane reactor through a second microflow channel 5-2, and a waste liquid outlet 8 communicated with an outlet of the electrochemical detection cell 7 through a third microflow channel 5-3, and the waste liquid outlet 8 and the liquid inlet 4 to be detected both penetrate out of the upper surface of the PDMS layer; the biomembrane reactor 6 comprises an open biomembrane reaction tank 6-1, a microbial membrane 6-3 which is arranged in the biomembrane reaction tank during application and a cover plate 6-2 which is used for packaging an opening at the top end of the biomembrane reaction tank;

the three-electrode system is arranged between the monocrystalline silicon substrate 1 and the PDMS layer 3; the three-electrode system comprises a working gold electrode 2-1, a counter gold electrode 2-2 and a reference gold electrode 2-3, wherein the surface of the reference gold electrode is modified with an Ag/AgCl material; the three electrode bodies are arranged in an interdigital ultramicro electrode array and are contacted with an electrochemical detection cell 7;

and a platinum film temperature sensor 9 arranged on the bottom surface of the monocrystalline silicon substrate 1, wherein the platinum film temperature sensor 9 is right opposite to the position of the biomembrane reaction tank 6-1; a micro heating copper electrode is adhered to the surface of the platinum film temperature sensor 9;

when the integrated microfluidic electrochemical sensor chip is used, the integrated microfluidic electrochemical sensor chip is packaged on a PCB, and three electrodes on the integrated microfluidic electrochemical sensor chip are connected with circuit pins of the PCB.

Preferably, the thickness of the monocrystalline silicon substrate 1 is 200-300 μm; the thickness of the PDMS layer 3 is 500-1000 μm; the thickness of the first micro-flow channel 5-1, the thickness of the second micro-flow channel 5-2 and the thickness of the third micro-flow channel 5-3 are 100-300 mu m independently, and the width of the first micro-flow channel 5-1, the width of the second micro-flow channel 5-2 and the width of the third micro-flow channel 5-3 are 300-600 mu m independently.

The invention provides a preparation method of the integrated microfluidic electrochemical sensor chip, which comprises the following steps:

(1) insulating two sides of a monocrystalline silicon substrate, throwing positive photoresist on the surface of the insulated monocrystalline silicon substrate, and constructing a three-electrode gold interdigital ultramicroelectrode array on the surface of the insulated monocrystalline silicon substrate;

throwing positive photoresist on the bottom surface of the monocrystalline silicon substrate on which the three-electrode gold interdigital ultramicroelectrode array is formed, constructing a platinum film temperature sensor on the bottom surface of the insulated monocrystalline silicon substrate, and adhering a micro heating copper electrode on the surface of the platinum film temperature sensor;

(2) throwing negative photoresist on the surface of a quartz glass sheet, and engraving a microflow channel system pattern on the surface of the quartz glass sheet as a male die by adopting a photoetching method; then, the PDMS is cast on the surface of the male mold, and the PDMS casting layer is taken down from the male mold after curing molding to obtain a PDMS layer with a microfluidic channel system inside;

(3) bonding the monocrystalline silicon substrate with the three-electrode gold interdigital ultramicroelectrode array and the platinum film temperature sensor formed in the step (1) with the PDMS layer with the microfluidic channel system inside, which is obtained in the step (2), so that the three-electrode gold interdigital ultramicroelectrode array is contacted with an electrochemical detection pool in the microfluidic channel system, and the platinum film temperature sensor is right opposite to the position of a biomembrane reaction pool in the microfluidic channel system, thus obtaining the integrated microfluidic electrochemical sensor chip;

there is no time sequence restriction between the step (1) and the step (2).

Preferably, the method for insulating the double sides of the monocrystalline silicon substrate in the step (1) comprises the following steps: cleaning the monocrystalline silicon substrate, and then sequentially depositing SiO on the two sides of the monocrystalline silicon substrate₂Layer and Si₃N₄A layer; the SiO₂The thickness of the layer is 200nm, the Si₃N₄The thickness of the layer was 300 nm.

Preferably, the method for constructing the three-electrode gold interdigital ultramicroelectrode array on the surface of the insulated monocrystalline silicon substrate in the step (1) comprises the following steps:

(a) after positive photoresist is thrown on the surface of an insulated monocrystalline silicon substrate, a three-electrode gold interdigital ultramicro electrode array graph is engraved on the surface of the insulated monocrystalline silicon substrate through photoetching processes including pre-baking, exposure, development and hardening;

(b) cleaning the monocrystalline silicon substrate surface without the protection of the positive photoresist by adopting an oxygen plasma etching method, and then sequentially depositing tantalum and gold on the patterned monocrystalline silicon substrate surface obtained in the step (a) by utilizing a radio frequency magnetron sputtering method; the thickness of the tantalum is 20nm, and the thickness of the gold is 200 nm;

(c) soaking the monocrystalline silicon substrate deposited with tantalum and gold obtained in the step (b) in acetone for stripping, removing tantalum and gold films on the positive photoresist and the positive photoresist, and forming a three-electrode gold interdigital ultramicroelectrode array on the surface of the insulated monocrystalline silicon substrate;

(d) and coating Ag/AgCl slurry on the surface of a reference gold electrode in the three-electrode gold interdigital ultramicroelectrode array, and then annealing the monocrystalline silicon substrate.

Preferably, the method for constructing the platinum film temperature sensor on the bottom surface of the monocrystalline silicon substrate in the step (1) comprises the following steps:

(A) after positive photoresist is thrown on the bottom surface of a monocrystalline silicon substrate on which a three-electrode gold interdigital ultramicroelectrode array is formed, a platinum film temperature sensor graph is coated and etched on the bottom surface of the monocrystalline silicon substrate through photoetching processes including pre-baking, exposure, development and hardening;

(B) cleaning the bottom surface of the monocrystalline silicon substrate without the protection of the positive photoresist by adopting an oxygen plasma etching method, and then sequentially depositing chromium and platinum on the bottom surface of the patterned monocrystalline silicon substrate obtained in the step (A) by utilizing a radio frequency magnetron sputtering method; the thickness of the chromium is 30nm, and the thickness of the platinum is 200 nm;

(C) and (C) soaking the monocrystalline silicon substrate deposited with the chromium and the platinum obtained in the step (B) in acetone for stripping, removing chromium and platinum films on the positive photoresist and the positive photoresist, and forming a platinum film temperature sensor on the bottom surface of the insulated monocrystalline silicon substrate.

Preferably, the step (2) of engraving the microchannel system pattern on the surface of the quartz glass sheet comprises the following steps: after throwing negative photoresist on the surface of the quartz glass sheet, photoetching processes including pre-baking, exposure, development and hardening are carried out, and a microchannel channel system graph is engraved on the surface of the quartz glass sheet; and then cleaning the surface of the quartz glass sheet without the protection of the negative photoresist by adopting an oxygen plasma etching method to obtain the male die.

Preferably, the bonding method in step (3) is: and cleaning the PDMS layer by using a plasma processor, aligning and combining the cleaned PDMS layer with the monocrystalline silicon substrate, and heating at 80 ℃ for 30 min.

The invention provides a method for rapidly detecting the biochemical oxygen demand by adopting the integrated microfluidic electrochemical sensor chip or the integrated microfluidic electrochemical sensor chip prepared by the preparation method, which comprises the following steps:

preparing a water sample to be detected containing potassium ferricyanide with the pH value of 7.0, injecting the water sample to be detected from a liquid inlet 4 to be detected until a biofilm reactor is filled with a solution, and decomposing the water sample to be detected by the biofilm reactor under the conditions of heating by a micro heating copper electrode and temperature control by a platinum film temperature sensor;

the obtained decomposition treatment solution enters an electrochemical detection pool 7, and a three-electrode system is used for detecting an oxidation current signal of ferrocyanide ions in the decomposition treatment solution to obtain an oxidation current value of a water sample to be detected;

and calculating the BOD value of the water sample to be detected according to a calibration curve and the oxidation current value of the water sample to be detected, wherein the calibration curve is a linear curve of the oxidation current value and the BOD value of the BOD standard solution.

Preferably, the molar concentration of the potassium ferricyanide in the water sample to be detected is 10-40 mmol/L; the temperature of the decomposition treatment is 30-50 ℃, and the time is 10-30 min.

The invention provides an integrated micro-fluidic electrochemical sensor chip for rapidly detecting biochemical oxygen demand, which comprises: a monocrystalline silicon substrate; the micro-flow channel system comprises a liquid inlet to be detected, a micro-flow channel, a biomembrane reactor, an electrochemical detection pool and a waste liquid outlet; the three-electrode system is arranged between the monocrystalline silicon substrate and the PDMS layer and comprises a working gold electrode, a counter gold electrode and a reference gold electrode, and the three-electrode system is arranged in an interdigital ultramicro electrode array and is contacted with an electrochemical detection cell; the surface of the platinum film temperature sensor is adhered with a micro heating copper electrode; when the integrated microfluidic electrochemical sensor chip is used, the integrated microfluidic electrochemical sensor chip is packaged on a PCB, and three electrodes on the integrated microfluidic electrochemical sensor chip are connected with circuit pins of the PCB. The invention adopts the micro-fluidic technology to realize the effective integration of two functional units of the biofilm reactor and the electrochemical electrode on the same chip, can realize the rapid and continuous BOD monitoring, and simultaneously, the biofilm reactor is separated from the electrochemical three-electrode, and the cleaning process is not influenced by the two electrodes, thereby effectively solving the problem of instability of the sensor caused by the traditional cleaning and electrode or microbial film replacement and effectively improving the long-term stability of the sensor; the heating micro copper electrode and the temperature sensor are integrated on the chip, so that the heating and temperature control functions of the bio-membrane reactor are realized, and additional constant-temperature heating equipment is not needed; the electrochemical detection electrode adopts the structural design of an interdigital ultramicro electrode array, so that the detection sensitivity of the sensor is effectively improved. The integrated micro-fluidic electrochemical sensor chip provided by the invention realizes the miniaturization of the sensor and the rapid, stable, accurate and continuous automatic monitoring of BOD of a water sample.

The invention provides a preparation method of the integrated microfluidic electrochemical sensor chip, and the integrated microfluidic electrochemical sensor chip is processed by adopting an MEMS technology to form the microfluidic chip with the microbial membrane reactor, the microfluidic channel and the electrochemical detection cell, and has the advantages of simple process and easy operation.

The invention also provides a method for rapidly detecting the biochemical oxygen demand by using the integrated microfluidic electrochemical sensor chip, which takes potassium ferricyanide as an electronic medium for BOD detection, the potassium ferricyanide medium can substitute dissolved oxygen to promote electrons to be transferred from a microbial biochemical reaction center, the microbial metabolic process is accelerated, and Fe is detected by electrodes³⁺Consumption of or Fe²⁺The yield of BOD can be rapidly measured.

Drawings

Fig. 1 is a schematic front structural diagram of an integrated microfluidic electrochemical sensor chip provided by the present invention;

fig. 2 is a schematic diagram of a back structure of an integrated microfluidic electrochemical sensor chip provided in the present invention;

FIG. 3 is a schematic diagram of a PDMS layer with a microfluidic channel system disposed therein according to an embodiment of the present invention;

in FIGS. 1 to 3, 1 denotes a single-crystal silicon substrate, 2-1 denotes a working gold electrode, 2-2 denotes a counter gold electrode, 2-3 denotes a reference gold electrode, 3 denotes a PDMS layer, 4-1 and 4-2 denote inlets for a solution to be measured, 5-1 denotes a first microfluidic channel, 5-2 denotes a second microfluidic channel, 5-3 denotes a third microfluidic channel, 6 denotes a biofilm reactor, 6-1 denotes a biofilm reaction tank, 6-2 denotes a cover plate, 6-3 denotes a microbial membrane, 7-an electrochemical detection tank, 8-a waste liquid outlet, 9-a platinum film temperature sensor, and 10-an air injection port;

FIG. 4 is a flow chart of the present invention for preparing an integrated microfluidic electrochemical sensor chip;

FIG. 5 is a schematic diagram of a monocrystalline silicon substrate with a three-electrode gold-interdigitated microelectrode array formed on the surface thereof according to the present invention;

FIG. 6 is a schematic view of a single crystal silicon substrate having a platinum thin film temperature sensor formed on the bottom surface thereof (back surface of the single crystal silicon substrate) according to the present invention;

FIG. 7 is a diagram of an integrated microfluidic electrochemical sensor chip formed after bonding according to the present invention;

FIG. 8 is a diagram of a packaged integrated microfluidic electrochemical sensor chip of the present invention, wherein, a-a monocrystalline silicon substrate with a three-electrode gold interdigital ultramicroelectrode array formed on the surface, b-a PDMS layer with a microfluidic channel system disposed therein, c-a microbial film, d-a cover plate, e-the back of a PCB, and f-a micro-heating copper electrode;

FIG. 9 is a graph of the calibration curve obtained in example 2;

FIG. 10 is a graph of electrochemical current signals over time for BOD standard concentration water samples tested in example 3.

Detailed Description

a single-crystal silicon substrate 1 is formed,

a PDMS layer 3 superposed on the surface of the monocrystalline silicon substrate 1; a microfluidic channel system is arranged in the PDMS layer 3, the microfluidic channel system comprises a to-be-detected liquid inlet 4, a biofilm reactor 6 with an inlet communicated with the to-be-detected liquid inlet 4 through a first microfluidic channel 5-1, an electrochemical detection cell 7 with an inlet communicated with an outlet of the biofilm reactor through a second microfluidic channel 5-2, and a waste liquid outlet 8 communicated with an outlet of the electrochemical detection cell 7 through a third microfluidic channel 5-3, wherein the waste liquid outlet 8 and the to-be-detected liquid inlet 4 penetrate through the upper surface of the PDMS layer; the biomembrane reactor 6 comprises an open biomembrane reaction tank 6-1, a microbial membrane 6-3 which is arranged in the biomembrane reaction tank during application and a cover plate 6-2 which is used for packaging an opening at the top end of the biomembrane reaction tank;

the three-electrode system is arranged between the monocrystalline silicon substrate 1 and the PDMS layer 3; the three-electrode system comprises a working gold electrode 2-1, a counter gold electrode 2-2 and a reference gold electrode 2-3, wherein the surface of the reference gold electrode is modified with an Ag/AgCl material; the three electrode systems are arranged in an interdigital ultramicroelectrode array and are in contact with an electrochemical detection cell 7;

and a platinum film temperature sensor 9 arranged on the bottom surface of the monocrystalline silicon substrate 1, wherein the platinum film temperature sensor 9 is right opposite to the position of the biomembrane reaction tank 6-1; the surface of the platinum film temperature sensor 9 is adhered with a micro heating copper electrode;

The structure schematic diagrams of the integrated microfluidic electrochemical sensor chip for rapid biochemical oxygen demand detection provided by the invention are shown in fig. 1 and fig. 2, wherein fig. 1 is a front structure schematic diagram of the integrated microfluidic electrochemical sensor chip, and fig. 2 is a back structure schematic diagram of the integrated microfluidic electrochemical sensor chip.

The integrated microfluidic electrochemical sensor chip provided by the invention comprises a monocrystalline silicon substrate 1. In the invention, the thickness of the monocrystalline silicon substrate 1 is preferably 200-300 μm, more preferably 270 μm, and the monocrystalline silicon substrate has no special requirement and can be prepared by a monocrystalline silicon substrate known to those skilled in the art.

The integrated microfluidic electrochemical sensor chip provided by the invention comprises a PDMS (polydimethylsiloxane) layer 3 superposed on the surface of the monocrystalline silicon substrate 1. In the invention, the thickness of the PDMS layer is preferably 500-1000 μm. In the invention, a microflow channel system is arranged in the PDMS layer 3 and provides channels and places for the inlet and outlet and flow of samples, the incubation of microbial membrane reaction and BOD detection. In the invention, the microflow channel system comprises a liquid inlet 4 to be detected, a biofilm reactor 6, an electrochemical detection pool 7 and a waste liquid outlet 8:

in the present invention, the microfluidic channel system includes a to-be-detected liquid inlet 4, in order to fill the biofilm reactor with the to-be-detected liquid quickly during detection in the specific embodiment of the present invention, the number of the to-be-detected liquid inlets is preferably set to 2, as shown in 4-1 and 4-2 in fig. 3, and fig. 3 is a diagram of a PDMS layer in which the microfluidic channel system is disposed in the embodiment of the present invention; the liquid inlets 4-1 and 4-2 to be detected are converged by a microflow channel and then communicated to the biomembrane reactor. In the embodiment of the present invention, in order to facilitate the solution in the biofilm reactor to smoothly enter the electrochemical detection cell 7, an air injection port is preferably further provided, as shown at 10 in fig. 3; the air injection port 10 is arranged between the liquid inlet 4 to be detected and the biofilm reactor 6 and communicated to the biofilm reactor 6 through a microflow channel. In the specific embodiment of the invention, the port parts of the liquid inlets 4-1 and 4-2, the air injection port 10 and the waste liquid outlet 8 are respectively provided with a cover, and the opening and the closing of the inlet and the outlet are realized through the opening and closing of the covers. When the device is applied, after the biomembrane reactor finishes the decomposition treatment of a water sample, the to-be-detected liquid inlets 4-1 and 4-2 are kept closed, the air injection port 10 and the waste liquid outlet 8 are opened, then air is injected from the air injection port 10, and the liquid in the biomembrane reactor enters the electrochemical detection pool 7.

In the invention, the micro-flow channel system comprises a biofilm reactor 6, the inlet of which is communicated with the liquid inlet 4 to be detected through a first micro-flow channel 5-1; the biomembrane reactor 6 comprises a biomembrane reaction tank 6-1, a microbial membrane 6-3 which is arranged in the biomembrane reaction tank during application and a cover sheet 6-2 for packaging the top end of the biomembrane reaction tank. In the present invention, the inner diameter of the biofilm reaction tank 6-1 is preferably 500 μm, and the height is preferably 600 μm. In the invention, the microbial film 6-3 is preferably a reduced graphene oxide/polypyrrole/bacillus subtilis microbial film, wherein polypyrrole is deposited on the reduced graphene oxide and forms a three-dimensional porous composite material with the reduced graphene oxide, and bacillus subtilis is electrostatically adsorbed on the polypyrrole; the reduced graphene oxide/polypyrrole/bacillus subtilis microbial film (rGO/PPy/b.subtilis microbial film) is prepared according to the method of example 1 in patent CN107037105A, and the preparation process is as follows:

preparing 1mg/L GO solution, adding Py to 1mM into 10mL GO solution, and uniformly mixing by ultrasonic for 1 h; pouring the mixed solution into a hydrothermal reaction kettle to react for 12 hours at a constant temperature of 180 ℃ to obtain a black columnar rGO/Py hydrogel composite material;

(II) taking rGO/Py hydrogel as a working electrode, a platinum electrode as a counter electrode, an Ag/AgCl electrode as a reference electrode and an electrolyte solution of 0.005M PBS (pH 7.0), performing in-situ electropolymerization of Py by adopting an electrochemical timing current method, wherein the timing potential is 0.8V, and the timing time is 30min to obtain an rGO/PPy composite material; freezing and drying the rGO/PPy to obtain a three-dimensional porous rGO/PPy composite material;

(III) putting the three-dimensional porous rGO/PPy composite material into 100mL of B.subtilis culture solution with the concentration of 9.8 x 107CFU/mL, keeping the temperature at 37 ℃, culturing for 12h at the oscillation frequency of 100rpm, taking out the three-dimensional porous rGO/PPy composite material fixed with the B.subtilis, naturally airing to obtain an rGO/PPy/B.subtilis microbial membrane, and storing at 4 ℃ for later use.

In the present invention, the microfluidic channel system comprises an electrochemical detection cell 7, the inlet of which is communicated with the outlet (located at the lower part of the biofilm reaction cell) of the biofilm reactor 6 through a second microfluidic channel 5-2. In the present invention, the electrochemical detection cell 7 preferably has an inner length of 2000 μm, an inner width of 1500 μm, and an inner height of 600 μm.

In the invention, the microfluidic channel system comprises a waste liquid outlet 8 communicated with the outlet of the electrochemical detection cell 7 through a third microfluidic channel 5-3; the solution that completes the electrochemical detection in the electrochemical detection cell 7 flows out from the waste liquid outlet 8. In the embodiment of the present invention, after the electrochemical detection is completed, air is preferably injected through the air injection port 10, so that the solution in the electrochemical detection cell can smoothly flow out through the waste liquid outlet 8.

In the invention, the thickness of the first microfluidic channel 5-1, the thickness of the second microfluidic channel 5-2 and the thickness of the third microfluidic channel 5-3 in the microfluidic channel system are preferably 100-300 μm and more preferably 150 μm independently, and the width of the first microfluidic channel 5-1, the thickness of the second microfluidic channel 5-2 and the width of the third microfluidic channel are preferably 300-600 μm and more preferably 400 μm independently.

The integrated microfluidic electrochemical sensor chip provided by the invention comprises a three-electrode system arranged between the monocrystalline silicon substrate 1 and the PDMS layer 3. In the invention, the three-electrode system comprises a working gold electrode 2-1, a counter gold electrode 2-2 and a reference gold electrode 2-3, wherein the surface of the reference gold electrode is modified with an Ag/AgCl material. In the invention, the three electrode bodies are arranged in an interdigital ultramicro electrode array and are contacted with an electrochemical detection cell 7; the electrochemical detection electrode adopts the structural design of an interdigital ultramicro electrode array, so that the detection sensitivity of the sensor is effectively improved. The invention adopts the design scheme that the biomembrane reactor and the electrochemical three electrodes are separated, effectively solves the problem of instability of the sensor caused by traditional cleaning and electrode or microbial membrane replacement, and can effectively improve the long-term stability of the sensor.

The integrated microfluidic electrochemical sensor chip provided by the invention comprises a platinum film temperature sensor 9 arranged on the bottom surface of the monocrystalline silicon substrate 1, wherein the platinum film temperature sensor 9 is right opposite to the position of a biological film reaction tank 6-1; the invention has no special requirement on the platinum film temperature sensor, and the platinum film temperature sensor well known by the technical personnel in the field can be adopted, and the platinum film temperature sensor is also preferably connected with a high-precision multimeter and used for measuring the platinum film resistance so as to monitor the temperature of the biomembrane reactor and play a role in controlling the temperature. In the invention, a micro heating copper electrode is adhered to the surface of the platinum film temperature sensor 9, the micro heating copper electrode is preferably connected with a stabilized voltage power supply, and the stabilized voltage power supply is used for supplying power to the micro heating copper electrode, thereby realizing the heat supply of the biofilm reactor. The invention integrates the heating micro copper electrode and the temperature sensor on the chip, realizes the heating and temperature control functions of the bio-membrane reactor, and does not need additional constant temperature heating equipment.

The invention adopts the micro-fluidic technology to realize the effective integration of two functional units of the biomembrane reactor and the electrochemical electrode on the same chip, and realizes the miniaturization of the sensor and the rapid, accurate, stable and continuous automatic monitoring of BOD of the water sample.

When the integrated microfluidic electrochemical sensor chip is applied, the integrated microfluidic electrochemical sensor chip is packaged on a PCB, and the three electrodes on the integrated microfluidic electrochemical sensor chip are connected with circuit pins of the PCB. The present invention has no particular requirement for the PCB, and the PCB known to those skilled in the art may be used.

throwing positive photoresist on the bottom surface of the monocrystalline silicon substrate on which the three-electrode gold interdigital ultramicroelectrode array is formed, constructing a platinum film temperature sensor on the bottom surface of the insulated monocrystalline silicon, and adhering a micro heating copper electrode on the surface of the platinum film temperature sensor;

there is no time sequence restriction between the step (1) and the step (2).

The flow chart of the preparation method of the integrated microfluidic electrochemical sensor chip provided by the invention is shown in fig. 4.

The invention provides a method for constructing a three-electrode gold interdigital ultramicroelectrode array on the surface of an insulated monocrystalline silicon substrate by insulating two sides of the monocrystalline silicon substrate, throwing positive photoresist on the surface of the insulated monocrystalline silicon substrate and then constructing the three-electrode gold interdigital ultramicroelectrode array on the surface of the insulated monocrystalline silicon substrate. In the present invention, the single crystal silicon baseThe bottom double-side insulation method is preferably as follows: cleaning the monocrystalline silicon substrate, and sequentially depositing SiO on the two sides of the monocrystalline silicon substrate₂Layer and Si₃N₄A layer; the SiO₂The thickness of the layer is preferably 200nm, said Si₃N₄The thickness of the layer is preferably 300 nm. The invention preferably adopts a wet thermal oxidation method to deposit the SiO₂Layer, preferably by low pressure chemical vapor deposition of said Si₃N₄A layer; the present invention does not require any particular implementation of the wet thermal oxidation process and the low pressure chemical vapor deposition process, and the corresponding implementation is well known to those skilled in the art.

In the invention, the type of the positive photoresist is preferably AZ 1500; the spin coating rotating speed of the positive photoresist is preferably 6000rpm, and the spin coating time is preferably 3 min. In the invention, the method for constructing the three-electrode gold interdigital ultramicroelectrode array on the surface of the insulated monocrystalline silicon substrate is preferably as follows:

(c) soaking the monocrystalline silicon substrate deposited with the tantalum and the gold obtained in the step (b) in acetone for stripping (Lift-off), removing tantalum and gold films on positive photoresist and positive photoresist, and forming a three-electrode gold interdigital ultramicroelectrode array on the surface of the insulated monocrystalline silicon substrate;

The present invention has no special requirement on the specific operation methods of pre-baking, exposing, developing and hardening in the step (a), and corresponding operation methods well known to those skilled in the art can be adopted.

The invention has no special requirements on the oxygen plasma etching method and the radio frequency magnetron sputtering method in the step (b), and corresponding methods well known by the technicians in the field can be adopted; in the step (b), the surface of the monocrystalline silicon substrate without the protection of the positive photoresist is cleaned by adopting an oxygen plasma etching method, so that the residual photoresist can be removed, and the adhesive strength between the metal layer (tantalum and gold) and the monocrystalline silicon substrate in the subsequent metal sputtering process is ensured. The tantalum deposited in step (b) of the present invention serves as an adhesion layer for the single crystal silicon substrate and gold.

The present invention does not require any particular method for the peeling (Lift-off) in the step (c), and any method known to those skilled in the art may be used. FIG. 5 is a schematic diagram of a monocrystalline silicon substrate with a three-electrode gold interdigitated microelectrode array formed on the surface.

In the present invention, the temperature of the annealing treatment in the step (d) is preferably 80 ℃ and the time is preferably 1 h. The invention has no special requirements on the Ag/AgCl slurry, and can adopt the commercial conductive Ag/AgCl slurry well known to the technical personnel in the field; the invention has no special requirement on the coating thickness of the Ag/AgCl slurry, and the reference gold electrode can be uniformly coated. The surface of the reference gold electrode is coated with Ag/AgCl slurry, then the monocrystalline silicon substrate is annealed, and an Ag/AgCl material is modified on the surface of the reference gold electrode, so that the on-chip Ag/AgCl reference electrode, the working electrode and the counter electrode form a three-electrode system electrochemical cell to complete the electrochemical detection of BOD.

After the monocrystalline silicon substrate with the three-electrode gold interdigital ultramicroelectrode array constructed on the surface is obtained, positive photoresist is thrown on the bottom surface of the monocrystalline silicon substrate with the three-electrode gold interdigital ultramicroelectrode array formed, then a platinum film temperature sensor is constructed on the bottom surface of the insulated monocrystalline silicon, and a micro heating copper electrode is adhered to the surface of the platinum film temperature sensor. In the present invention, the method of constructing the platinum thin film temperature sensor on the bottom surface of the insulated monocrystalline silicon is preferably as follows:

(C) and (C) soaking the monocrystalline silicon substrate deposited with the chromium and the platinum obtained in the step (B) in acetone for stripping (Lift-off), removing chromium and platinum films on the positive photoresist and the positive photoresist, and forming a platinum thin film temperature sensor on the bottom surface of the insulated monocrystalline silicon substrate.

The present invention has no special requirement on the specific operation methods of pre-baking, exposing, developing and hardening in the step (a), and the corresponding operation methods well known to those skilled in the art can be adopted.

The invention has no special requirements on the oxygen plasma etching method and the radio frequency magnetron sputtering method in the step (B), and adopts a corresponding method well known by the technicians in the field; in the step (B), the bottom surface of the monocrystalline silicon substrate without the protection of the positive photoresist is cleaned by adopting an oxygen plasma etching method, so that the residual photoresist can be removed, and the adhesive strength between the metal layer (chromium and platinum) and the monocrystalline silicon substrate in the subsequent metal sputtering process is ensured.

The chromium deposited in step (B) of the present invention acts as an adhesion layer for the monocrystalline silicon substrate and the platinum. The present invention does not require any particular method for the peeling (Lift-off) in the step (C), and any method known to those skilled in the art may be used. Fig. 6 is a view showing a single crystal silicon substrate (back surface of the single crystal silicon substrate) on which a platinum thin film temperature sensor is formed on the bottom surface.

Throwing negative photoresist on the surface of a quartz glass sheet, and engraving a pattern of a microfluidic channel system on the surface of the quartz glass sheet as a male die by adopting a photoetching method; and then, casting PDMS on the surface of the male mold, and taking the PDMS casting layer off the male mold after curing and forming to obtain the PDMS layer with the microfluidic channel system inside. In the present invention, the method for etching the microchannel system pattern on the surface of the quartz glass sheet is preferably: after throwing negative photoresist on the surface of the quartz glass sheet, photoetching processes including pre-baking, exposure, development and hardening are carried out, and a microchannel channel system graph is engraved on the surface of the quartz glass sheet; and then cleaning the surface of the quartz glass sheet without the protection of the negative photoresist by adopting an oxygen plasma etching method to obtain the male die.

The quartz glass sheet of the present invention has no particular requirement, and can be a quartz glass sheet well known to those skilled in the art; cleaning the quartz glass sheet preferably, and then throwing a negative photoresist on the surface; the spin coating rotating speed of the negative photoresist is 1000rpm, and the spin coating time is preferably 5 min. In the present invention, the negative photoresist is preferably SU8(GM 1075).

The invention adopts an oxygen plasma etching method to clean the surface of the quartz glass sheet without the protection of the negative photoresist, and remove the residual photoresist.

In the present invention, before the PDMS is cast, it is preferable to perform a PDMS degassing treatment, and the present invention does not require any particular degassing method, and may adopt a degassing method well known to those skilled in the art.

After curing and forming, the PDMS casting layer is taken down from the male mold, the invention cuts the PDMS casting layer according to the marking line (the marking line left on the surface of the male mold after demolding), and then punches (the inlet of the liquid to be detected, the waste liquid outlet and the air injection opening are opened) to communicate the two surfaces of the PDMS. The invention cuts the PDMS casting layer according to the marking line, can ensure that the size of the cut PDMS casting layer is matched with the monocrystalline silicon substrate, thus ensuring that the bonded monocrystalline silicon substrate is accurately aligned in the subsequent bonding, and can also obtain the cover plate 6-2 used for packaging the top end of the biomembrane reaction tank through cutting. Fig. 3 shows a schematic diagram of a PDMS layer with a microfluidic channel system inside, which is formed after demolding.

After a monocrystalline silicon substrate for forming the three-electrode gold interdigital ultramicro electrode array and the platinum film temperature sensor and a PDMS layer with a microfluidic channel system inside are obtained, the monocrystalline silicon substrate and the PDMS layer are bonded, so that the three-electrode gold interdigital ultramicro electrode array is in contact with an electrochemical detection pool in the microfluidic channel system, and the platinum film temperature sensor is right opposite to a position of a biomembrane reaction pool in the microfluidic channel system, thereby obtaining the integrated microfluidic electrochemical sensor chip. In the present invention, the bonding method is preferably: and cleaning the PDMS layer by using a Plasma Treater (Plasma Treater), aligning and combining the cleaned PDMS layer with the monocrystalline silicon substrate, and heating at 80 ℃ for 30 min. The method comprises the steps of cleaning a PDMS layer by using a plasma processor, and generating a silicon hydroxyl group on the surface of the PDMS layer; according to the invention, plasma modification packaging is adopted, the surface property of PDMS is modified by using plasma, so that the surface of PDMS is oxidized to generate a silicon hydroxyl group, then PDMS and a substrate material are packaged together to form a new and stable O-Si-O covalent bond, and the plasma is adopted to modify PDMS, so that the operation time is short, and the surface state of a chip is favorably maintained. The method is used for heating for 30min at the temperature of 80 ℃, so that the PDMS layer and the monocrystalline silicon substrate are deeply bonded. Fig. 7 is a physical diagram of the integrated microfluidic electrochemical sensor chip formed after bonding.

After the integrated microfluidic electrochemical sensor chip is obtained, when the integrated microfluidic electrochemical sensor chip is applied, the integrated microfluidic electrochemical sensor chip is packaged on a PCB, three electrodes on the integrated microfluidic electrochemical sensor chip are connected with circuit pins of the PCB, then a microbial membrane is filled in a biological membrane reaction tank, and a PDMS cover plate is packaged at the top end of the biological reaction tank. Fig. 8 is a physical diagram of the packaged integrated microfluidic electrochemical sensor chip.

The invention adopts MEMS technology to process and form the micro-fluidic chip with the microbial membrane reactor, the micro-fluidic channel system and the electrochemical detection pool, and has simple process and easy operation.

In the present invention, the method of obtaining the obtained calibration curve is preferably: preparing BOD standard solutions with different concentrations and pH value of 7.0 and containing potassium ferricyanide; injecting the BOD standard solutions with different concentrations from a to-be-detected liquid inlet 4 until the biofilm reactor is filled with the solutions, and decomposing the BOD standard solutions by the biofilm reactor under the conditions of micro heating copper electrode heating and temperature control of a platinum film temperature sensor; the obtained treatment solution enters an electrochemical detection pool 7, a three-electrode system is used for detecting an oxidation current signal of ferrocyanide ions in the treatment solution, the measured oxidation current value and the BOD value of the standard solution are recorded, a linear relation curve of the oxidation current value and the BOD value is drawn, and a calibration curve is obtained.

In the present invention, the specific method for preparing the BOD standard solution containing potassium ferricyanide having a pH of 7.0 is preferably: and mixing the PBS solution of potassium ferricyanide with the pH value of 7.0 with the PBS standard solution with the pH value of 7.0 being BOD to obtain the BOD standard solution containing potassium ferricyanide with the pH value of 7.0. In the invention, the molar concentration of potassium ferricyanide in the PBS solution of potassium ferricyanide is preferably 40mmol/L, and the molar concentration of PBS is preferably 5 mmol/L. In the invention, the molar concentration of the potassium ferricyanide in the BOD standard solution is preferably 10-40 mmol/L, and more preferably 20 mmol/L. In the invention, the temperature of the decomposition treatment is preferably 30-50 ℃, more preferably 35 ℃, and the time is preferably 10-30 min, more preferably 15 min. According to the invention, preferably, after the decomposition treatment is finished, the obtained treatment fluid is kept stand and cooled for 10min, and then the treatment fluid enters the electrochemical detection pool from the biofilm reactor.

In the invention, the specific method for preparing the water sample to be tested containing potassium ferricyanide with the pH value of 7.0 is preferably as follows: firstly, adjusting the pH value of a water sample to be detected to 7.0, and then mixing the obtained water sample with a PBS (phosphate buffer solution) solution of potassium ferricyanide with the pH value of 7.0 to obtain the water sample to be detected containing the potassium ferricyanide with the pH value of 7.0. In the present invention, the PBS solution of potassium ferricyanide is the same as the above scheme, and is not described herein again. In the invention, the molar concentration of the potassium ferricyanide in the water sample to be detected is preferably 20 mmol/L. The integrated micro-fluidic electrochemical sensor chip provided by the invention has the advantages that the standard BOD concentration is in a standard concentration range of 4-40 mg/L, the standard BOD concentration and the oxidation current value of the ferricyanide ions are in a linear relation, the lower detection limit is 1.6mg/L, and the signal-to-noise ratio S/N is more than or equal to 3.

The invention adds potassium ferricyanide into a water sample to be detected (and BOD standard solution), the potassium ferricyanide is used as an electronic medium for BOD detection, and the potassium ferricyanide medium has an excitation function for BOD detection: a water sample to be detected is injected into the biofilm reactor from a liquid inlet 4 to be detected, organic matters in the water sample to be detected are oxidized in the microbial metabolism process in the biofilm reactor to lose electrons, and simultaneously potassium ferricyanide (Fe)³⁺) Reduction reaction is carried out to obtain electrons to generate Fe²⁺The reduction reaction further promotes the respiration of microorganisms to enable the organic matters to generate oxidation reaction, and the organic matters are oxidized to further promote Fe³⁺The potassium ferricyanide can replace dissolved oxygen to promote electrons to be transferred from a microorganism biochemical reaction center and accelerate the microorganism metabolic process; after decomposition treatment of the biofilm reactor, the obtained treatment liquid enters an electrochemical detection pool 7, and Fe is detected by three electrodes³⁺Consumption of or Fe²⁺The generated amount of the BOD can be quickly measured by detecting an oxidation current signal of the ferrous cyanide ions by a chronoamperometry.

After the biochemical oxygen demand of the water sample to be detected is detected, the integrated microfluidic electrochemical sensor chip is cleaned. In the present invention, the method of cleaning is preferably: injecting deionized water from a to-be-detected liquid inlet, cleaning the whole microflow channel system by using the deionized water, and then discharging the cleaned deionized water from a waste liquid outlet. After cleaning, the cleaned integrated microfluidic electrochemical sensor chip is preferably placed in an oven for low-temperature drying. The integrated microfluidic electrochemical sensor chip provided by the invention is convenient to clean, and effectively solves the problem of instability of the sensor caused by traditional cleaning and electrode or microbial film replacement.

The integrated microfluidic electrochemical sensor chip for rapid BOD detection and the preparation thereof and the BOD detection method provided by the present invention are described in detail below with reference to the examples, but they should not be construed as limiting the scope of the present invention.

Example 1

Preparing an integrated microfluidic electrochemical sensor chip:

the structure schematic diagram of the integrated microfluidic electrochemical sensor chip is shown in fig. 1 and fig. 2, and the physical diagram is shown in fig. 7, and the integrated microfluidic electrochemical sensor chip comprises a monocrystalline silicon substrate 1;

a PDMS layer 3 superposed on the surface of the monocrystalline silicon substrate 1 (the thickness is 270 μm); the PDMS layer 3 is internally provided with a microflow channel system which comprises a liquid inlet 4 to be detected (divided into a liquid inlet 4-1 to be detected and a liquid inlet 4-2 to be detected), a biomembrane reactor 6 (an air injection port 10 is arranged between the liquid inlet to be detected and the biomembrane reactor), an electrochemical detection pool 7 and a waste liquid outlet 8, wherein the inlet of the biomembrane reactor is communicated with the liquid inlet 4 to be detected through a first microflow channel 5 (the thickness is 150 mu m, and the width is 400 mu m), the electrochemical detection pool 7 is communicated with the outlet of the biomembrane reactor through a second microflow channel 5-2, and the waste liquid outlet 8 is communicated with the outlet of the electrochemical detection pool 7 through a third microflow channel 5-3; the biofilm reactor 6 comprises a biofilm reaction tank 6-1, a microbial film 6-3 (reduced graphene oxide/polypyrrole/bacillus subtilis microbial film prepared according to the method of example 1 in patent CN 107037105A) which is filled in the biofilm reaction tank during application, and a cover plate 6-2 for packaging the top end of the biofilm reaction tank;

and a platinum film temperature sensor 9 arranged on the bottom surface of the monocrystalline silicon substrate 1, wherein the platinum film temperature sensor 9 is right opposite to the position of the biomembrane reaction tank 6-1; the surface of the platinum film temperature sensor 9 is adhered with a micro-heating copper electrode.

The preparation method of the integrated microfluidic electrochemical sensor chip comprises the following steps:

(1) double-sided insulation of a silicon wafer:

growing SiO with thickness of 200nm on the two sides of a cleaned 270 mu m thick silicon wafer by adopting wet thermal oxidation and low-pressure chemical vapor deposition (LPCVD) technology in sequence₂Layer and 300nm of Si₃N₄A layer as an insulating layer;

(2) patterning a gold interdigital ultramicro electrode array:

throwing photoresist AZ1500 on an insulated silicon wafer, etching a gold interdigital ultramicroelectrode array pattern on the surface of the silicon wafer through standard photoetching processes such as prebaking, exposing, developing, hardening and the like, then cleaning the surface of a substrate without photoresist protection by adopting an oxygen plasma etching process, and removing the possibly residual photoresist so as to ensure the adhesion strength between a pre-sputtered metal layer and the silicon substrate; depositing 20nm tantalum (Ta) and 200nm gold (Au) on the patterned silicon wafer in sequence by adopting a radio frequency magnetron sputtering process, wherein the tantalum is used as an adhesion layer, then soaking the silicon wafer sputtered with the gold in acetone, performing Lift-off, removing the photoresist and the metal film thereon, and forming a gold interdigital ultramicroelectrode array; coating Ag/AgCl slurry on the surface of a reference gold electrode in a gold interdigital ultramicroelectrode array, and then annealing a silicon substrate at 80 ℃ for 1 h;

(3) patterning of the platinum film temperature sensor:

throwing a positive photoresist AZ1500, performing standard photoetching processes such as prebaking, exposure, developing, hardening and the like to form a platinum temperature sensor pattern on the back of a silicon wafer in a sleeved mode, cleaning the surface of a substrate without photoresist protection by adopting an oxygen plasma etching process, and removing the possibly residual photoresist to ensure the adhesive strength between a pre-sputtered metal layer and the silicon substrate; depositing 30nm chromium (Cr) and 200nm platinum on the patterned silicon wafer in sequence by adopting a radio frequency magnetron sputtering process, wherein the chromium is used as an adhesion layer; then soaking the silicon wafer sputtered with platinum in acetone, performing Lift-off, removing the photoresist and the metal film thereon to form a platinum film temperature sensor;

(4) preparing a PDMS micro-channel, comprising the preparation of a male mold and PDMS casting:

preparing a male die: throwing negative photoresist SU8(GM 1075) on the surface of a cleaned quartz glass sheet, performing standard photoetching processes such as prebaking, exposure, developing, hardening and the like to overlay a micro-channel system pattern on the surface of the glass sheet, then cleaning the surface of a substrate without photoresist protection by adopting an oxygen plasma etching process to remove the possibly residual photoresist, wherein the micro-channel etching thickness is about 150 mu m;

PDMS casting: placing the male mold in a disposable culture dish, slowly casting the degassed PDMS on the surface of the male mold, taking the PDMS off the male mold after curing and molding, cutting the PDMS layer according to the mark line, and then punching and communicating two surfaces of the PDMS;

(5) bonding:

scribing and cleaning the silicon substrate prepared in the steps (1) to (3), cleaning the PDMS layer micro-channel system in the step (4) for 3min by using a Plasma Tree so as to generate silicon hydroxyl groups on the surface of the PDMS layer micro-channel system, aligning and slightly pressing the treated PDMS layer and the silicon substrate so as to tightly combine the PDMS layer and the silicon substrate, and then placing the silicon substrate in an oven at 80 ℃ for drying for 30min so as to carry out deep bonding; a physical diagram of the integrated microfluidic electrochemical sensor chip formed after bonding is shown in fig. 7.

(6) And (3) lead and packaging:

packaging the bonded silicon wafer on a PCB, welding, connecting an electrode on the silicon wafer with a PCB circuit pin, fixing and packaging a micro-flow pipeline, filling the prepared microbial membrane (reduced graphene oxide/polypyrrole/bacillus subtilis), packaging the prepared PDMS circular cover plate on the top of a biological reaction tank to form a micro-biofilm reactor, wherein a real object diagram of the packaged integrated micro-flow control electrochemical sensor chip is shown in FIG. 8.

Example 2

The integrated microfluidic electrochemical sensor chip packaged in the embodiment 1 is used for measuring the Biochemical Oxygen Demand (BOD) of a water sample:

(1) preparing a solution: diluting 100mg/L BOD standard solution with 5mM PBS (pH 7.0) to prepare BOD standard solution with different concentrations (4mg/L, 20mg/L, 40mg/L, 60mg/L and 80mg/L), then respectively mixing 40mM potassium ferricyanide (prepared by 5mM PBS (pH 7.0)) solution, and finally preparing BOD sample solution with different concentrations (2mg/L, 10mg/L, 20mg/L, 30mg/L and 40mg/L) respectively containing 20mM potassium ferricyanide;

(2) solution injection: opening a to-be-detected liquid inlet 4-1, a to-be-detected liquid inlet 4-2 and an air injection port 10, closing a waste liquid outlet 8, simultaneously injecting a standard solution from the to-be-detected liquid inlet 4-1 and the to-be-detected liquid inlet 4-2 until the biofilm reactor is filled with the solution, and closing the to-be-detected liquid inlet 4-1, the to-be-detected liquid inlet 4-2 and the air injection port 10;

(3) decomposing a water sample by using the biofilm reactor: turning on a power supply, loading voltage to the micro heating copper electrode, controlling the temperature to be 35 ℃, starting to perform reaction decomposition treatment on the organic matter sample to be detected, and turning off the power supply after 15 min;

(4) electrochemical calibration curve: after the reaction and decomposition are finished, standing and cooling for 10min, opening an air injection port 10 and a waste liquid outlet 8, injecting air from the air injection port 10 to enable the solution after the reaction to enter an electrochemical detection pool from a reactor, filling the detection pool, performing electrochemical detection on an oxidation current signal of the ferrous cyanide ions through a gold interdigital ultramicroelectrode array, recording the measured electrochemical signal current value and a water sample BOD value, drawing a linear relation curve of the oxidation current value and the BOD value of the BOD standard solution, and obtaining a calibration curve, wherein the BOD standard concentration and the oxidation current value of the ferrous cyanide ions are in a linear relation in a standard concentration range of BOD 4-40 mg/L of the integrated micro-fluidic electrochemical sensor chip as shown in figure 9.

(5) Collecting surface water samples of different places at 3 positions around Beijing, respectively marking as a 1# water sample, a 2# water sample and a 3# water sample, firstly adjusting the pH value of each water sample to be 7.0, and then mixing the water samples with 40mM potassium ferricyanide (prepared from 5mM PBS (pH 7.0)) according to a volume ratio of 1:1 to prepare a water sample to be detected containing 20mM potassium ferricyanide; and (3) obtaining the oxidation current values of the corresponding ferrous cyanide ions of the water samples to be detected according to the processes of the steps (2) to (4), and then calculating the BOD value of the actual water sample according to the calibration curve (because the actual water sample is mixed with the potassium ferricyanide solution according to the volume ratio of 1:1, the calculated BOD concentration value needs to be multiplied by 2). For comparison, the BOD of 3 water samples was also determined by the national Standard BOD5 method. The BOD test results for the 3 water samples are shown in table 1:

the BOD test results of the water samples in Table 13 are shown in Table 1

As can be seen from Table 1, compared with the national standard BOD5 method, the integrated micro-fluidic electrochemical sensor chip provided by the invention has the advantages that the deviation of the measurement result is less than 15% and the accuracy is good when the BOD of the water sample is measured.

(6) Cleaning: after the test is finished, opening a to-be-tested liquid inlet 4-1, a to-be-tested liquid inlet 4-2 and a waste liquid outlet 8, injecting deionized water from the to-be-tested liquid inlet 4-1 and the to-be-tested liquid inlet 4-2, cleaning the whole microflow channel system, then opening an air injection port 10 to flush air, and discharging the cleaned deionized water from the waste liquid outlet 8; and after cleaning, placing the chip in an oven for low-temperature drying.

Example 3

The test stability of the integrated microfluidic electrochemical sensor chip packaged in example 1 was tested by the following method:

mixing 20mg/L BOD standard solution with 40mM potassium ferricyanide solution (prepared from 5mM PBS (pH 7.0)) according to the volume ratio of 1:1 to prepare a 10mg/L BOD standard concentration water sample containing 20mM potassium ferricyanide, recording electrochemical current signals obtained by testing according to the testing process of the steps (2) to (4) in the example 2, and continuously measuring for 5 times each time to obtain an average value; the measurement is carried out once every 5 days, the total time lasts for 60 days, the stability of the integrated sensor chip is judged by comparing the change of data, and the change curve of the electrochemical current signal of the tested water sample with the standard BOD concentration along with the time is shown in figure 10.

As can be seen from fig. 10, the test data value after 60 days is 80% of the initial value, indicating that the sensor chip has good long-term stability.

The integrated microfluidic electrochemical sensor chip provided by the invention realizes the miniaturization of the sensor and the rapid, stable, accurate and continuous automatic monitoring of BOD of a water sample.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.

Claims

1. An integrated microfluidic electrochemical sensor chip for rapid biochemical oxygen demand detection, comprising:

a monocrystalline silicon substrate (1),

a PDMS layer (3) superposed on the surface of the monocrystalline silicon substrate (1); a microflow channel system is arranged in the PDMS layer (3), the microflow channel system comprises a liquid inlet (4) to be detected, a biomembrane reactor (6) with an inlet communicated with the liquid inlet (4) to be detected through a first microflow channel (5-1), an electrochemical detection cell (7) with an inlet communicated with an outlet of the biomembrane reactor (6) through a second microflow channel (5-2), and a waste liquid outlet (8) communicated with an outlet of the electrochemical detection cell (7) through a third microflow channel (5-3), and the waste liquid outlet (8) and the liquid inlet (4) to be detected both penetrate through the upper surface of the PDMS layer; the biofilm reactor (6) comprises an open biofilm reaction tank (6-1), a microbial film (6-3) which is arranged in the biofilm reaction tank during application and a cover plate (6-2) for packaging an opening at the top end of the biofilm reaction tank;

the three-electrode system is arranged between the monocrystalline silicon substrate (1) and the PDMS layer (3); the three-electrode system comprises a working gold electrode (2-1), a counter gold electrode (2-2) and a reference gold electrode (2-3), wherein the surface of the reference gold electrode is modified with an Ag/AgCl material; the three-electrode system is arranged in an interdigital ultramicro electrode array and is contacted with an electrochemical detection cell (7), and the three-electrode system is separated from the biomembrane reactor;

and a platinum film temperature sensor (9) arranged on the bottom surface of the monocrystalline silicon substrate (1), wherein the platinum film temperature sensor (9) is right opposite to the position of the biomembrane reaction tank (6-1); a micro heating copper electrode is adhered to the surface of the platinum film temperature sensor (9);

2. The integrated microfluidic electrochemical sensor chip according to claim 1, wherein the thickness of the monocrystalline silicon substrate (1) is 200-300 μm; the thickness of the PDMS layer (3) is 500-1000 μm; the thickness of the first micro-flow channel (5-1), the thickness of the second micro-flow channel (5-2) and the thickness of the third micro-flow channel (5-3) are 100-300 mu m independently, and the width of the first micro-flow channel, the second micro-flow channel and the third micro-flow channel is 300-600 mu m independently.

3. A method of making an integrated microfluidic electrochemical sensor chip according to claim 1 or 2, comprising the steps of:

(1) insulating two sides of a monocrystalline silicon substrate, throwing positive photoresist on the surface of the insulated monocrystalline silicon substrate, and constructing a three-electrode gold interdigital ultramicroelectrode array on the surface of the insulated monocrystalline silicon substrate; throwing positive photoresist on the bottom surface of the monocrystalline silicon substrate on which the three-electrode gold interdigital ultramicroelectrode array is formed, constructing a platinum film temperature sensor on the bottom surface of the insulated monocrystalline silicon substrate, and adhering a micro heating copper electrode on the surface of the platinum film temperature sensor;

there is no time sequence restriction between the step (1) and the step (2).

4. The preparation method according to claim 3, wherein the step (1) of insulating the single crystal silicon substrate on both sides comprises the following steps: cleaning the monocrystalline silicon substrate, and sequentially depositing a SiO2 layer and a Si3N4 layer on the two sides of the monocrystalline silicon substrate; the thickness of the SiO2 layer is 200nm, and the thickness of the Si3N4 layer is 300 nm.

5. The preparation method according to claim 3 or 4, wherein the step (1) of constructing the three-electrode gold interdigital ultramicroelectrode array on the surface of the insulated monocrystalline silicon substrate comprises the following steps:

6. The manufacturing method according to claim 3, wherein the step (1) of constructing the platinum thin film temperature sensor on the bottom surface of the insulated monocrystalline silicon substrate is as follows:

7. The method according to claim 3, wherein the step (2) of etching the microchannel system pattern on the surface of the quartz glass sheet comprises: after throwing negative photoresist on the surface of the quartz glass sheet, photoetching processes including pre-baking, exposure, development and hardening are carried out, and a microchannel channel system graph is engraved on the surface of the quartz glass sheet; and then cleaning the surface of the quartz glass sheet without the protection of the negative photoresist by adopting an oxygen plasma etching method to obtain the male die.

8. The manufacturing method according to claim 3, wherein the bonding method in step (3) is: and cleaning the PDMS layer by using a plasma processor, aligning and combining the cleaned PDMS layer with the monocrystalline silicon substrate, and heating at 80 ℃ for 30 min.

9. The method for rapidly detecting the biochemical oxygen demand by adopting the integrated microfluidic electrochemical sensor chip of claim 1 or 2 or the integrated microfluidic electrochemical sensor chip obtained by the preparation method of any one of claims 3 to 8 comprises the following steps:

preparing a water sample to be detected containing potassium ferricyanide with the pH value of 7.0, injecting the water sample to be detected from a liquid inlet (4) to be detected until a biofilm reactor is filled with a solution, and decomposing the water sample to be detected by the biofilm reactor under the conditions of heating by a micro heating copper electrode and temperature control by a platinum film temperature sensor;

the obtained decomposition treatment solution enters an electrochemical detection pool (7), and a three-electrode system is used for detecting an oxidation current signal of ferrocyanide ions in the decomposition treatment solution to obtain an oxidation current value of a water sample to be detected;

10. The method according to claim 9, wherein the molar concentration of the potassium ferricyanide in the water sample to be tested is 10-40 mmol/L; the temperature of the decomposition treatment is 30-50 ℃, and the time is 10-30 min.