Integrated micro-fluidic chip as well as preparation method and application thereof

Abstract

The invention provides an integrated micro-fluidic chip as well as a preparation method and application thereof, and belongs to the technical field of electrochemical and fluorescent dual-mode detection. The integrated micro-fluidic chip disclosed by the invention consists of a micro-fluidic chip and a peristaltic pump, the micro-fluidic chip comprises a chip upper layer, a chip lower layer and a middle sealing layer, the chip lower layer comprises a conductive indium tin oxide / polyethylene glycol terephthalate flexible substrate film and an electrode layer constructed on the conductive indium tin oxide / polyethylene glycol terephthalate flexible substrate film; the middle sealing layer comprises a liquid flowing layer and an acrylic plate provided with three circular detection cells; the upper layer of the chip is a visible cover plate, and the visible cover plate is formed by polydimethylsiloxane reverse mold synthesis. The method can realize simultaneous detection of electrochemical signals and fluorescence signals of various target objects, and has the advantages of simplicity and convenience in operation, high detection speed and low cost.

Description

Technical Field

[0001] The present invention relates to the technical field of electrochemical and fluorescence dual-mode detection, and in particular to an integrated microfluidic chip and a preparation method and application thereof. Background Art

[0002] Electrochemistry is a simple and fast signal recognition and conversion technology. Through the interaction between the electrode surface modification material and the analyte in the sample, changes in chemical / physical properties are generated, and the sensory information such as the composition, content, concentration, etc. of the inorganic or organic substance is converted into a measurable electrical signal in the form of impedance, resistance, potential or current by using a transducer. By analyzing the collected electrical signal, the composition and concentration of the analyte can be determined, that is, the sensing of the analyte is achieved. Among them, the electrical signal is generated by the redox reaction of the electrically active analyte on the electrode surface under a fixed or variable voltage. In short, the working principle of the electrochemical sensor is based on the redox reaction to convert the chemical signal into an electrical signal. The basic structure of the electrochemical sensor consists of two core parts connected in series: one is the electrode sensing system; the other is the signal conversion unit. Among them, the electrode sensing system is used for analyte recognition, and the three-electrode system is a widely used electrochemical sensing system, which mainly includes a working electrode, a counter electrode and a reference electrode. The working electrode is the site of the electrocatalytic reaction. Different biomaterials and nanomaterials can be modified on the electrode surface according to the detection requirements to enhance the recognition ability of the analyte; the counter electrode is used to complete the circuit and enable the continuous transmission of electrons in the circuit; the reference electrode is used to accurately calibrate the potential of the working electrode. The signal conversion unit is responsible for transferring the signal from the output domain of the recognition system to the electronic domain to complete the signal processing and analysis; in layman's terms, it is responsible for converting the signal generated by the interaction between the electrode modification material and the analyte into a readable value.

[0003] From the perspective of electron transition, fluorescence refers to the transition of some electrons from the lowest vibrational energy level in the ground state to certain higher vibrational energy levels after certain substances absorb light with the same characteristic frequency as themselves. Fluorescence is the process of dropping from the lowest vibrational energy level to a different energy level in the ground state, while emitting light with a lower frequency and longer wavelength than the original absorption. Its wavelength is always longer than the wavelength of ultraviolet light absorbed by the molecule, and is usually within the visible light range. The properties of fluorescence are closely related to the molecular structure. Molecules of different structures do not all emit fluorescence after being excited.

[0004] Fluorescence detection is a device that converts specific molecular recognition events into detectable fluorescent signals, and analyzes the visualized signals to achieve sensitive detection of the target. Fluorescence sensors generally include biomolecular recognition elements and fluorescent signal generators. Among them, the biorecognition element directly binds specifically to the target. Through the above-mentioned binding method, the change in the sensor fluorescence signal value is caused. Then, the quantitative analysis of the target is achieved based on a certain relationship between the change in the obtained fluorescence signal and the concentration of the target. Generally speaking, the constructed fluorescent sensor is carried out in a homogeneous solution, so it is easy to achieve biological recognition, and the operation steps are relatively simple. At the same time, fluorescent sensors also have the advantages of low cost, high sensitivity, simplicity, and fast response speed, so they have received widespread attention from researchers.

[0005] Based on this, there is an urgent need for an integrated microfluidic chip that can realize simultaneous fluorescence detection and electrochemical detection. Summary of the invention

[0006] The purpose of the present invention is to provide an integrated microfluidic chip and a preparation method and application thereof, so as to solve the technical problem that the prior art cannot simultaneously realize visualized fluorescence detection and accurate electrochemical detection.

[0007] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0008] The present invention provides an integrated microfluidic chip, which is composed of a microfluidic chip and a peristaltic pump;

[0009] The microfluidic chip comprises an upper chip layer, a lower chip layer and a middle sealing layer;

[0010] The lower layer of the chip comprises a conductive indium tin oxide / polyethylene terephthalate flexible substrate film and an electrode layer constructed on the conductive indium tin oxide / polyethylene terephthalate flexible substrate film;

[0011] The middle sealing layer comprises a liquid circulation layer and an acrylic plate provided with three circular detection pools;

[0012] The upper layer of the chip is a visible cover plate, and the visible cover plate is synthesized by reverse molding of polydimethylsiloxane.

[0013] Furthermore, the electrode layer is composed of a counter electrode, a working electrode and a reference electrode from left to right, wherein there are three working electrodes;

[0014] The three detection cells correspond one to one to the three working electrodes.

[0015] Furthermore, the preparation method of the working electrode and the counter electrode is as follows: attaching a laser-engraved tape mask 1 to a conductive indium tin oxide / polyethylene terephthalate flexible substrate film, etching to remove the conductive layer in the non-electrode area, and after removing the mask, attaching a mask 2 to the substrate, applying ink to the electrode area by screen printing, and then treating with ion sputtering to obtain;

[0016] The preparation method of the reference electrode is as follows: after removing the mask 2, the mask 3 is covered on the conductive indium tin oxide / polyethylene terephthalate flexible substrate film, the Ag / AgCl slurry is uniformly coated on the exposed substrate area as the reference electrode on the right side, the mask 3 is removed, and the final electrode substrate is cleaned and dried, and finally immersed in 6-hydroxy-1-hexanethiol to obtain the reference electrode.

[0017] The present invention provides a method for preparing the integrated microfluidic chip, comprising the following steps:

[0018] The electrode layer in the lower layer of the chip is constructed on a conductive indium tin oxide / polyethylene terephthalate flexible substrate film; then three detection pools are engraved on an acrylic plate, and another acrylic plate is engraved into a liquid circulation layer, and two small holes are engraved at the center positions on both sides. The detection pool layer is located between the liquid circulation layer and the electrode layer; the visible cover plate on the upper layer of the chip is synthesized by polydimethylsiloxane reverse molding; the layers are bonded by thermal curing of polydimethylsiloxane to obtain a microfluidic chip.

[0019] Furthermore, the circular hole at the left center of the liquid circulation layer is a liquid communication port for injecting or sucking out liquid.

[0020] Furthermore, the circular hole in the center of the right side of the liquid circulation layer is an air communication hole.

[0021] The present invention also provides an application of the integrated microfluidic chip in on-site electrochemical and fluorescence dual-mode detection of samples, comprising the following steps:

[0022] (1) During the detection process, a sample mixture is added to the integrated microfluidic chip, and the sample mixture is injected simultaneously and fills the three detection pools in sequence;

[0023] (2) During the testing process, the excess sample mixture is aspirated;

[0024] (3) After the test is completed, the integrated microfluidic chip is turned upside down. The liquid in the test pool will flow to the circulation layer due to gravity. As the peristaltic pump sucks out the liquid, the liquid in the test pool is gradually discharged.

[0025] Beneficial effects of the present invention:

[0026] (1) The present invention uses an integrated microfluidic chip to quickly screen positive samples from a large number of samples, and uses electrochemical quantitative detection of targets, saving detection time and cost, and achieving simultaneous detection of electrochemical signals and fluorescence signals of multiple targets.

[0027] (2) The integrated microfluidic chip of the present invention combines the advantages of visualized fluorescence and electrochemistry through a dual-mode biosensor, and uses homemade screen-printed electrodes (ITO) to assemble and construct an integrated microfluidic chip. The peristaltic pump automatically injects or draws out liquid to achieve integrated enrichment, capture, and detection of the target, and can be used for on-site screening and simultaneous differentiation and precise quantification of multiple targets. It has the advantages of simple operation, fast detection speed, and low cost, and can be widely used in food safety monitoring, disease diagnosis, and drug screening. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 A cross-sectional view of an integrated microfluidic chip prepared according to an embodiment of the present invention;

[0029] Figure 2 The mask model of the electrode layer preparation process prepared by the present invention, wherein A is mask 1, B is mask 2, and C is mask 3;

[0030] Figure 3 The integrated microfluidic chip prepared by the present invention, wherein A is a top view, B is a left side view, and C is a right side view;

[0031] Figure 4 A color simulation demonstration diagram of the workflow of the integrated microfluidic chip prepared by the present invention, wherein A, B, and C are diagrams of the liquid injection process, D, E, and F are diagrams of the liquid discharge process of the flow layer, and G, H, and I are diagrams of the liquid discharge process of the detection pool;

[0032] Figure 5 The results of the integrated integrated microfluidic chip prepared in the embodiment of the present invention detecting three different concentrations of target bacteria, wherein A is three different concentrations (0CFUmL -1 , 10 2 CFUmL -1 , 10 4 CFUmL -1 ) is a curve graph of the DPV response of the target bacteria detection, B is a visualized fluorescence graph of the target bacteria detection of Escherichia coli (E.coli), C is a visualized fluorescence graph of the target bacteria detection of Salmonella typhimurium (ST), D is a visualized fluorescence graph of the target bacteria detection of Staphylococcus aureus (SA), and E is a visualized fluorescence graph of the three target bacteria detections. DETAILED DESCRIPTION

[0033] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0034] Example 1

[0035] Preparation of electrode layer: sticking laser-engraved tape mask 1 on a conductive indium tin oxide / polyethylene terephthalate flexible substrate film, etching and removing the conductive layer in the non-electrode area, after removing the mask, sticking mask 2 on the substrate, applying ink on the electrode area by screen printing, and then treating it with ion sputtering to obtain a working electrode and a counter electrode; after removing mask 2, covering mask 3 on the conductive indium tin oxide / polyethylene terephthalate flexible substrate film, uniformly coating Ag / AgCl slurry as a reference electrode on the right side on the exposed substrate area, removing mask 3, washing and drying, and finally immersing the final electrode substrate in 6-hydroxy-1-hexanethiol to obtain a reference electrode;

[0036] The electrode layer in the lower layer of the chip is constructed on a conductive indium tin oxide / polyethylene terephthalate flexible substrate film; then three detection pools are engraved on an acrylic plate, and another acrylic plate is engraved into a liquid circulation layer, and two small holes are engraved at the center positions on both sides. The detection pool layer is located between the liquid circulation layer and the electrode layer; the visible cover plate on the upper layer of the chip is synthesized by polydimethylsiloxane reverse molding; the layers are bonded by thermal curing of polydimethylsiloxane to obtain a microfluidic chip.

[0037] Use an injection needle to inject 300μL of three magnetic capture probes into the three detection pools in sequence, let it stand for 1 minute, fix the capture probes through the magnetic attraction of the magnetic patch to the magnetic beads, turn the chip upside down, and the excess liquid will flow from the reaction pool into the liquid circulation layer. Turn on the peristaltic pump and the device will automatically suck out the excess liquid. Use a peristaltic pump to automatically inject standard pathogenic bacteria solutions containing different concentrations of Escherichia coli (E.coli), Salmonella typhimurium (ST) and Staphylococcus aureus (SA) or a mixture of the three into the microfluidic chip, fill the three detection pools with the detection solution, suck out the excess pathogenic bacteria solution in the circulation layer, and let it stand for 10 minutes to incubate, so that the magnetic capture probes in each detection pool can fully and specifically bind to the corresponding target bacteria;

[0038] Invert the chip, and the excess liquid flows into the liquid circulation layer and is sucked out by the peristaltic pump. Use the peristaltic pump to inject the signal label mixture into the chip, and the three detection pools are filled with the signal label solution. Let it stand for 1 minute to incubate so that the signal label with a specific aptamer is specifically bound to the corresponding target bacteria to form a sandwich structure. Invert the chip to suck out all the liquid, and repeat the injection, incubation, and suction of the signal label solution three times, so that the signal label can be combined with the target bacteria to the greatest extent at a high concentration;

[0039] Use an injection needle to inject 200 μL of an equal amount of a mixed reaction solution containing 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, MgCl2 (100 μM), CuSO4 (0.1 μM), and AHC (0.1 μM) into the three detection pools respectively. React for 10 minutes and observe the visualized fluorescence signal in the detection pool. Differential pulse voltammetry (DPV) was used for electrochemical measurement with a potential increment of 4 mV, a pulse amplitude of 50 mV, a pulse width of 60 ms, a sampling width of 20 ms, a pulse period of 500 ms, and a standing time of 2 s. The signal tags in the detection pool can trigger the electrochemical signals of different target bacteria. By switching the output site of the working electrode, the electrochemical signals corresponding to different concentrations of bacteria can be obtained. The results are shown in the figure. Figure 5 As shown. Figure 5 A shows that in the absence of target bacteria, the sensor shows no electrochemical signal. 2 CFUmL -1 When 10 4 CFUmL -1 When the three types of bacteria are present at the same time, an electrochemical reaction of Fc will occur when any working electrode is connected, and the signal value is the same whether a single target or a mixed target is detected. The results show that the electrochemical method can detect E.coli, ST and SA in the biosensor at the same time without mutual interference. The visualized fluorescence detection of the chip was discussed using a mobile phone APP and a five-point sampling method. Figure 5 As shown in B to E, B is a visualized fluorescence image of Escherichia coli (E.coli) target bacteria detection, C is a visualized fluorescence image of Salmonella typhimurium (ST) target bacteria detection, D is a visualized fluorescence image of Staphylococcus aureus (SA) target bacteria detection, and E is a visualized fluorescence image of three target bacteria detections. Figure 5 From B to E, it can be seen that when there is no bacteria, none of the three detection pools will emit fluorescent signals. 4 CFUmL -1 of bacterial solution as the verification concentration, when only 10 4 CFUmL -1 When E.coli, ST and SA exist separately, only the corresponding detection pool, that is, the left detection pool, the middle detection pool or the right detection pool, emits a fluorescent signal. When the three bacteria exist at the same time, all three detection pools of the chip will emit fluorescent signals. The fluorescent signals generated by the three target bacteria will not affect each other.

[0040] It can be seen from the above embodiments that the present invention provides an integrated microfluidic chip and its preparation method and application. The integrated microfluidic chip prepared by the present invention is composed of a microfluidic chip and a peristaltic pump; the microfluidic chip comprises an upper chip layer, a lower chip layer and an intermediate sealing layer; the lower chip layer comprises a conductive indium tin oxide / polyethylene terephthalate flexible substrate film and an electrode layer constructed on the conductive indium tin oxide / polyethylene terephthalate flexible substrate film; the intermediate sealing layer comprises a liquid circulation layer and an acrylic plate with three circular detection pools; the upper chip layer is a visible cover plate, and the visible cover plate is synthesized by reverse molding of polydimethylsiloxane. The present invention can realize the simultaneous detection of electrochemical signals and fluorescence signals of multiple targets, and has the advantages of simple operation, fast detection speed and low cost.

[0041] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. An integrated microfluidic chip, characterized in that: The integrated microfluidic chip consists of a microfluidic chip and a peristaltic pump; The microfluidic chip comprises an upper chip layer, a lower chip layer and a middle sealing layer; The lower layer of the chip comprises a conductive indium tin oxide / polyethylene terephthalate flexible substrate film and an electrode layer constructed on the conductive indium tin oxide / polyethylene terephthalate flexible substrate film; The middle sealing layer comprises a liquid circulation layer and an acrylic plate provided with three circular detection pools; The upper layer of the chip is a visible cover plate, and the visible cover plate is synthesized by reverse molding of polydimethylsiloxane.

2. The integrated microfluidic chip according to claim 1, characterized in that: The electrode layers are, from left to right, a counter electrode, a working electrode and a reference electrode, wherein there are three working electrodes; The three detection cells correspond one to one to the three working electrodes.

3. The integrated microfluidic chip according to claim 2, characterized in that: The preparation method of the working electrode and the counter electrode is as follows: a laser-engraved tape mask 1 is attached to a conductive indium tin oxide / polyethylene terephthalate flexible substrate film, the conductive layer of the non-electrode area is removed by etching, and after removing the mask, a mask 2 is attached to the substrate, ink is applied to the electrode area by screen printing, and then ion sputtering is used to obtain; The preparation method of the reference electrode is as follows: after removing the mask 2, the mask 3 is covered on the conductive indium tin oxide / polyethylene terephthalate flexible substrate film, the Ag / AgCl slurry is uniformly coated on the exposed substrate area as the reference electrode on the right side, the mask 3 is removed, and the final electrode substrate is cleaned and dried, and finally immersed in 6-hydroxy-1-hexanethiol to obtain the reference electrode.

4. A method for preparing an integrated microfluidic chip according to any one of claims 1 to 3, characterized in that: The following steps are involved: The electrode layer in the lower layer of the chip is constructed on a conductive indium tin oxide / polyethylene terephthalate flexible substrate film; Then, three detection pools are engraved on one acrylic plate, and another acrylic plate is engraved into a liquid circulation layer, and two small holes are engraved at the center positions on both sides. The detection pool layer is located between the liquid circulation layer and the electrode layer; the visible cover plate on the upper layer of the chip is synthesized by polydimethylsiloxane reverse molding; the layers are bonded by thermal curing of polydimethylsiloxane to obtain a microfluidic chip.

5. The integrated microfluidic chip according to claim 4, characterized in that: The circular hole at the left center of the liquid circulation layer is a liquid communication port for injecting or sucking out liquid.

6. The integrated microfluidic chip according to claim 5, characterized in that: The circular hole in the center of the right side of the liquid circulation layer is an air communication hole.

7. Use of the integrated microfluidic chip according to any one of claims 1 to 3 in on-site electrochemical and fluorescence dual-mode detection of samples, characterized in that: The following steps are involved: (1) During the detection process, a sample mixture is added to the integrated microfluidic chip, and the sample mixture is injected simultaneously and fills the three detection pools in sequence; (2) During the testing process, the excess sample mixture is aspirated; (3) After the test is completed, the integrated microfluidic chip is turned upside down. The liquid in the test pool will flow to the circulation layer due to gravity. As the peristaltic pump sucks out the liquid, the liquid in the test pool is gradually discharged.

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