CN111617814A - Microfluidic chip for detecting living organisms in water body and detection method - Google Patents
Microfluidic chip for detecting living organisms in water body and detection method Download PDFInfo
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Abstract
The invention discloses a micro-fluidic chip for detecting living organisms in a water body, which relates to the technical field of environmental monitoring and comprises a channel layer, a dielectric layer and a substrate layer which are sequentially bonded from top to bottom; an inlet unit, a bacterial electrophoresis control unit and an outlet unit which are sequentially communicated are arranged in the channel layer; the inlet unit and the outlet unit sequentially penetrate through the channel layer and the dielectric layer. The invention also provides a detection method of the micro-fluidic chip for detecting the survivable organisms in the water body. The invention has the advantages of single bacterium analysis capability, high integration and simple operation. The microfluidic chip device has the advantages of simple manufacture and easy operation, can control the flowing state of fluid in a channel, can realize qualitative and quantitative detection and analysis on the types and the quantity of bacteria in water, particularly ballast water, and is suitable for the analysis of survivable organisms and the drug monitoring of environmental water, particularly ballast water.
Description
Technical Field
The invention relates to the technical field of environmental monitoring, in particular to a micro-fluidic chip for detecting living organisms in a water body and a detection method.
Background
Bacterial contamination indicators are a series of indicators used to measure whether a water source is contaminated with bacteria and to describe the degree of contamination. Human beings usually contact pathogenic bacteria in water by drinking, touching and the like, so that the human bodies are harmed, and the most common diseases comprise cholera, typhoid fever, dysentery and the like. In order to ensure the sanitary quality of water, the detection of microorganisms is carried out on different types of water quality. The pathogenic bacteria content in water is less, the detection steps are complex, and the time consumption is long, so the difficulty is higher. Therefore, countries in the world generally detect the uniform health indicator bacteria first and detect various pathogenic bacteria one by one when necessary. The health indicator bacterium can be used as a bacterial pollution index. Meanwhile, ship ballast water often carries a large number of foreign marine organism species, including phytoplankton, bacteria, viruses, and the like. According to statistics, the ballast water carried by ships around the world contains 1.1 hundred million floating swimming plants per cubic meter of ballast water on average. Ship ballast water is capable of disseminating various bacteria to various parts of the world during the course of a ship's voyage, and has threatened the safety of marine ecosystems, even leading to catastrophic consequences. Therefore, it is often desirable to detect viable organisms, particularly bacteria (pathogens), in a body of water. At present, methods for detecting microorganisms, especially bacteria, mainly comprise culture, chemiluminescence, bioluminescence, mass spectrometry, Polymerase Chain Reaction (PCR) and the like, and the methods have the defects of long detection period, complex procedure, high price and the like. The 3M microorganism test piece is applied to ballast water sanitation monitoring, and the result shows that the test piece is suitable for measuring the total number of bacteria in water, but is not suitable for detecting coliform groups in a ballast water body. The detection of microorganisms is limited by the growth rate of microorganisms and the uncertainty of water discharge time, and the effectiveness of the detection cannot be achieved. Therefore, it is urgently needed to establish a rapid detection method for microorganisms in water bodies so as to effectively evaluate the water bodies.
The microfluidic chip has the characteristics of high flux, integration and low cost, and the size of the channel structure of the microfluidic chip and bacteria are in the same scale (micron scale), so that the microfluidic chip technology can be used for realizing one-key closed operation such as capture, identification, detection and the like of the bacteria, and the microfluidic chip has the advantages of high efficiency, small error and the like. Therefore, the microfluidic chip technology can be used as a powerful tool for rapidly detecting bacteria in ballast water on site. At present, the bacterial chip analysis system mainly comprises an optical analysis detection system and an electrochemical analysis detection system. The optical analysis and detection system hinders the miniaturization development of the system due to the complexity and the large volume of the system; the electrochemical analysis and detection system has the advantages of high sensitivity, simple peripheral equipment such as a signal processing system and the like, easiness in matching with a microelectrode on a chip and the like, and has good prospects in the aspects of miniaturization, portability, high detection efficiency, rapidness and the like. By fixing the antibody specifically combined with the surface protein of the escherichia coli on the microfluidic chip, the Wang ShuQi realizes 71.8% +/-5% capture of the escherichia coli in blood and milk. Renjie Wang and the like build a simple micro-fluidic chip platform, and realize the in-situ rapid detection of the salmonella typhimurium by combining an immunocapture technology and a corresponding light-emitting diode induced fluorescence detection system. S Furutani et al detected grassy Escherichia coli in 7min by Taq Man fluorescent probe on a portable in situ real-time PCR analysis chip. Fronczek and the like are arranged on the microfluidic paper chip, an LED lamp and a smart phone are used as a fluorescence imaging system, the detection of the salmonella typhimurium is realized within 5min, and the operation is simple and convenient.
From the analysis performance of the microfluidic chip, the future application field of the microfluidic chip is quite wide, but the current important application field is obviously in the life science field. The advantages of the method in the aspects of miniaturization, integration, portability and the like of analytical instruments provide wide prospects for the application of the method in a plurality of fields such as environmental monitoring and protection, biomedicine, high-throughput drug synthesis screening, optimal breeding of crops, health immunity and the like.
Disclosure of Invention
In order to solve the technical problems, the invention provides the micro-fluidic chip for detecting the living organisms in the water body, which has the advantages of simple structure, miniaturization, low cost, easy preparation and operation, capability of realizing the detection of the microorganisms in the water body, particularly in the ballast water, and capability of quantitatively analyzing the content of each bacterium in the water.
In order to achieve the purpose, the invention provides the following scheme:
the invention provides a micro-fluidic chip for detecting living organisms in a water body, which comprises a channel layer, a dielectric layer and a substrate layer which are sequentially bonded from top to bottom; an inlet unit, a bacterial electrophoresis control unit and an outlet unit which are sequentially communicated are arranged in the channel layer; the inlet unit and the outlet unit sequentially penetrate through the channel layer and the dielectric layer.
Optionally, the inlet unit comprises a bacteria mixture liquid tank, a buffer liquid tank and a side channel; the outlet unit comprises two waste liquid tanks; the bacterial electrophoresis control unit comprises a main channel, a metal micro-electrode group and a flat plate driving electrode; the bacteria mixed liquid tank is arranged at one end of the main channel, and the two waste liquid tanks are arranged at the other end of the main channel; the buffer liquid tank and the side channels are positioned at two sides of the bacteria mixed liquid tank and are communicated with the main channel, and the main channel is communicated with the bacteria mixed liquid tank, the buffer liquid tank and the waste liquid tank through the side channels; the metal micro-electrode group is arranged on the substrate layer and extends into the dielectric layer, and an input pin of the flat plate driving electrode is arranged on the substrate layer and is used for being connected with an external driving circuit.
Optionally, the metal microelectrode group comprises 9 metal microelectrodes.
Optionally, the metal microelectrodes are rectangular or strip-shaped, the width of each metal microelectrode is 30 μm, and the distance between every two adjacent metal microelectrodes is 10 μm.
Optionally, the depth of the main channel is 20-50 μm, the width is 30-70 μm, and the length is 380-450 μm.
Optionally, the height difference between the main channel and the bacteria mixed liquid bath is 70 μm.
Optionally, the bacteria mixed liquid tank, the buffer liquid tank and the waste liquid tank are all cylindrical, and have a diameter of 60 μm and a depth of 130 μm.
Optionally, the side channels are 40 μm wide and 150 μm long, and have a depth corresponding to the main channel.
Optionally, the metal microelectrode group and the flat plate driving electrode both adopt conductive metal copper or silver.
Optionally, the channel layer is a polydimethylsiloxane layer.
Optionally, the dielectric layer is a photoresist layer, preferably SU-8 photoresist.
Optionally, the substrate layer is a glass or silicon or ceramic or polymer layer with a thickness of 0.5mm-3 mm.
The invention also discloses a detection method of the microfluidic chip for detecting the survivable organisms in the water, which comprises the following steps:
firstly, bacteria mixed liquid enters from a bacteria mixed liquid groove, and when the bacteria mixed liquid flows through a bacteria electrophoresis control unit, cells are subjected to forward electrophoresis movement to be between microelectrodes under the action of an electrically driven flat plate driving electrode with the frequency F1;
secondly, buffer solution enters from a buffer solution tank, forms radial extrusion force through side channel convection, enables bacteria to flow along a straight line, and enables the bacteria near a metal microelectrode to be electrically focused and singly arranged to move along the axial direction of a main channel under the action of the metal microelectrode electrically driven by applying frequency F2 for capturing target cells;
finally, the buffer solution is injected into the buffer solution tank again to continuously dilute the bacteria, and the bacteria are moved out of the outlet unit through the detection area.
Optionally, the waveforms of the electric drive with the frequency F1 and the electric drive with the frequency F2 are triangular waves, square waves or sine waves, the frequency is 1Hz-0.6MHz, and the peak value is 5V-20V.
Compared with the prior art, the invention has the following technical effects:
the micro-fluidic chip for detecting the living organisms in the water body drives and separates different types of bacteria by utilizing the electrophoresis characteristic of the bacteria, controls the flowing state of the bacteria by utilizing the hydrodynamics, and enables the bacteria to generate electric focusing through the microelectrode, thereby enabling the bacteria to flow through the detection area singly. Compared with the common electrophoresis chip, the detection precision of the chip is improved; compared with the three-dimensional electrode, the complex preparation process of the three-dimensional electrode is avoided.
The three-dimensional microfluidic chip device for detecting bacteria in ballast water of the microfluidic chip for detecting living organisms in water body in the invention has some remarkable advantages, including: the detection method has the advantages that the preparation is simple, the operation is easy, the characteristics of good biocompatibility, low conductivity, high optical transparency, air permeability, biological inertia, easy processing, low cost and the like of the used material are combined with the traditional advantages of the micro-fluidic chip, so that the detection is efficient and reliable.
Compared with the conventional analysis process, the method is used for detecting bacteria, and can finish detection only by one device and one chip, so that the number of devices is reduced, and the hardware acquisition cost is reduced; the automation of the analysis process is improved, the working efficiency is improved, the transfer of the sample among different devices is avoided, the manual operation is simplified, and the risk of sample pollution is reduced; the micro-fluidic chip reduces the consumption of samples and reagents and reduces the operation cost; the device of the invention has small volume, can form portable equipment and meets the detection requirements of real-time and on-site detection.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings needed in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings without creative efforts.
FIG. 1 is a schematic cross-sectional view of a chip A-A' according to the present invention;
FIG. 2 is a schematic structural diagram of a three-dimensional microfluidic chip device according to the present invention;
FIG. 3 is a chip layout and electrode system diagram according to the present invention;
FIG. 4 is a movement track of bacterial particles generated by Comsol software simulation of a microfluidic channel according to the present invention.
FIG. 5 is a graph comparing the separation of bacterial particles by applying an electric field frequency to a microfluidic channel according to the present invention;
FIG. 6 is a graph comparing the separation of bacterial particles when another frequency of electric field is applied to the microfluidic channel according to the present invention;
FIG. 7 is a graph of the spatial variation of the electrical potential in a microfluidic channel generated by Comsol software simulation according to the microfluidic channel of the present invention.
Description of reference numerals: 1. a base layer; 2. a dielectric layer; 3. a main channel; 4. a channel layer; 5. a bacteria mixing liquid tank; 6. a side channel; 7. a buffer tank; 8. a metal microelectrode.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example 1: preparation of the chip
As shown in fig. 1, the microfluidic chip for detecting living organisms in a water body in this embodiment is manufactured by an ultraviolet lithography method using a "one-shot exposure" technique. Treating a silicon wafer with a sulfuric acid-hydrogen peroxide solution, cleaning, drying, throwing a layer of SU-82035 negative photoresist on the surface of the silicon wafer by a spin coater at the rotating speed of 3000rpm, baking at 65 ℃ for 10min, baking at 95 ℃ for 4min, performing ultraviolet exposure, secondary curing, developing, and drying by nitrogen to obtain patterns of each cell and the micro-channel network. And performing silanization to make the surface of the mold hydrophobic, wherein the mass ratio of Polydimethylsiloxane (PDMS) prepolymer to initiator is 10: 1 mixing and covering a mould, removing bubbles, and standing at 75 ℃ for curing. And carefully removing the polymerized PDMS sheet, cutting and forming, punching by using a flat-head syringe needle, then carrying out oxygen plasma treatment, and bonding with the treated substrate, thus obtaining the chip.
Example 2: chip structure
As shown in fig. 1, the microfluidic chip for detecting living organisms in a water body in the present embodiment includes a channel layer 4, a dielectric layer 2 and a substrate layer 1 bonded in sequence from top to bottom; an inlet unit, a bacterial electrophoresis control unit and an outlet unit which are sequentially communicated are arranged in the channel layer 4; the inlet unit and the outlet unit both penetrate the channel layer 4 and the dielectric layer 2 in sequence.
The inlet unit comprises a bacteria mixed liquid tank 5, a buffer liquid tank 7 and a side channel 6; the outlet unit comprises two waste liquid tanks; the bacterial electrophoresis control unit comprises a main channel 3, a metal microelectrode 8 group and a panel drive electrode; the bacteria mixed liquid tank 5 is arranged at one end of the main channel 3, and the two waste liquid tanks are arranged at the other end of the main channel 3; the buffer liquid tank 7 and the side channel 6 are positioned at two sides of the bacteria mixed liquid tank 5 and communicated with the main channel 3, and the main channel 3 is communicated with the bacteria mixed liquid tank 5, the buffer liquid tank 7 and the waste liquid tank through the side channel 6; the metal microelectrode 8 group is arranged on the basal layer 1 and extends into the dielectric layer 2, and an input pin of the flat plate driving electrode is arranged on the basal layer 1 and is used for being connected with an external driving circuit.
The group of metal micro-electrodes 8 includes 9 metal micro-electrodes 8.
The metal microelectrodes 8 are rectangular or strip-shaped, the width of each metal microelectrode 8 is 30 micrometers, and the distance between every two adjacent metal microelectrodes 8 is 10 micrometers. The width of the plate driving electrodes is 30 μm, the distance between the adjacent plate driving electrodes is 30 μm, and the thickness is the same as that of the metal micro-electrode 8.
The main channel 3 has a depth of 20-50 μm, preferably 30 μm, a width of 30-70 μm, preferably 50 μm, and a length of 380-450 μm, preferably 420 μm.
The height difference between the main channel 3 and the bacteria mixed liquid tank 5 is 70 μm.
The bacteria mixed liquid tank 5, the buffer liquid tank 7 and the waste liquid tank are all cylindrical, the diameter is 60 mu m, and the depth is 130 mu m.
The side channels 6 are 40 μm wide and 150 μm long and have a depth corresponding to the main channel 3.
The metal microelectrode 8 group and the flat plate driving electrode are both made of conductive metal silver.
The channel layer 4 is a polydimethylsiloxane layer.
The dielectric layer 2 is a photoresist layer, preferably SU-8 photoresist, and SU-82035 photoresist is used in this embodiment.
The substrate layer 1 is made of glass and has a thickness of 0.5mm-3 mm.
Example 3: dielectrophoresis technology for detecting and separating bacteria
The bottom of the device is provided with an electrode system which can generate different driving effects on different bacteria in detection. As shown in FIG. 4, the bacteria are driven by the electrode on the bottom side of the sample groove, so that the axial mutual separation action is generated among different bacteria, namely, different bacteria are separated due to the difference of physicochemical properties and are concentrated in a certain zone to advance. The metal microelectrodes 8 staggered at two sides of the main channel 3 after separation perform electric focusing action on the separated bacterial zones, so that various bacteria are intensively distributed at the centers of the zones, and the passing detection is facilitated. The sensing electrodes on two sides of the detection area are used for real-time monitoring and conducting the change of the conductivity of the solution in the detection area for quantitative analysis. As shown in FIG. 4, to separate the movement trajectory of the bacterial particles in the microfluidic channel by dielectrophoresis, a voltage of 5v alternating positive and negative was applied, and particles having a diameter of 3 μm and particles having a diameter of 5 μm were respectively identified in the figure.
Example 4: optimization of electric field frequency for separation of bacteria in microfluidic chip
Different electric field frequencies are applied to the bacterial solution in the microfluidic channel, the electric field force borne by the particles is different, and when the frequency is too high, the separation and sorting of the bacterial particles with different particle sizes cannot be realized. Therefore, based on the micro-fluidic chip designed by the invention, the separation condition of bacteria is respectively tested when the frequency of the applied electric field is in the range of 1HZ-1 MHZ. The results are shown in a comparison of FIGS. 5 and 6, where the bacteria were isolated at a frequency of 0.6MHz (FIG. 5); at a frequency of 0.7MHz (FIG. 6), the bacteria were unable to achieve separation.
Example 5: application of impedance detection technology
The bacteria impedance detection technology selects an alternating current impedance method in an electrochemical measurement method, and the principle of the impedance measurement technology and the electrode preparation are analyzed and optimized. The ac impedance method generally uses portable equipment of an electrochemical workstation to perform impedance measurement of an electrochemical system, and a three-electrode system is usually adopted to perform small-amplitude sine wave voltage signal disturbance on the electrode system. The micro-electrode detection device is miniaturized by adopting a bimetallic microelectrode 8 system and using the microelectrode as a working electrode, the bacteria sample to be measured is injected into a liquid storage tank, the total liquid storage amount is only a few cubic millimeters, compared with a large amount of electrolyte solution wasted by common electrochemical measurement, the electrolyte solution consumption of the detection method is very little, the consumption of the bacteria sample is very small, the bacteria detection time is saved due to the small area of the electrode, and the detection efficiency is greatly improved.
The results of the spatial variation of the potential and the current density simulation during the fluid flow and the voltage application in the microfluidic channel are shown in fig. 7. The bacteria impedance detection is to apply a small amplitude AC perturbation signal (voltage or current) to the bacteria to measure the electrical characteristics of the bacteria and analyze the physiological state and change of the bacteria. The technology does not need to mark bacteria, small-amplitude disturbance does not cause any damage to the bacteria, meanwhile, impedance measurement can be continuously carried out on the bacteria, and the technology can be combined with a microfluidic technology to realize the miniaturization of measuring equipment
It will be evident to those skilled in the art that the invention is not limited to the details of the foregoing illustrative embodiments, and that the present invention may be embodied in other specific forms without departing from the spirit or essential attributes thereof. The present embodiments are therefore to be considered in all respects as illustrative and not restrictive, the scope of the invention being indicated by the appended claims rather than by the foregoing description, and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein, and any reference signs in the claims are not intended to be construed as limiting the claim concerned.
The principle and the implementation mode of the present invention are explained by applying specific examples in the present specification, and the above descriptions of the examples are only used to help understanding the method and the core idea of the present invention; meanwhile, for a person skilled in the art, according to the idea of the present invention, the specific embodiments and the application range may be changed. In view of the above, the present disclosure should not be construed as limiting the invention.
Claims (10)
1. A micro-fluidic chip for detecting living organisms in a water body is characterized by comprising a channel layer, a dielectric layer and a substrate layer which are sequentially bonded from top to bottom; an inlet unit, a bacterial electrophoresis control unit and an outlet unit which are sequentially communicated are arranged in the channel layer; the inlet unit and the outlet unit sequentially penetrate through the channel layer and the dielectric layer.
2. The microfluidic chip for detection of viable organisms in a body of water of claim 1, wherein the inlet unit comprises a bacteria mixing tank, a buffer tank and a side channel; the outlet unit comprises two waste liquid tanks; the bacterial electrophoresis control unit comprises a main channel, a metal micro-electrode group and a flat plate driving electrode; the bacteria mixed liquid tank is arranged at one end of the main channel, and the two waste liquid tanks are arranged at the other end of the main channel; the buffer liquid tank and the side channels are positioned at two sides of the bacteria mixed liquid tank and are communicated with the main channel, and the main channel is communicated with the bacteria mixed liquid tank, the buffer liquid tank and the waste liquid tank through the side channels; the metal micro-electrode group is arranged on the substrate layer and extends into the dielectric layer, and an input pin of the flat plate driving electrode is arranged on the substrate layer and is used for being connected with an external driving circuit.
3. The microfluidic chip for detecting the viable organisms in the water body according to claim 2, wherein the metal microelectrode group comprises 9 metal microelectrodes.
4. The microfluidic chip for detecting the survivable organisms in the water body according to claim 3, wherein the metal microelectrodes are rectangular or strip-shaped, the width of the metal microelectrodes is 30 μm, and the distance between every two adjacent metal microelectrodes is 10 μm.
5. The microfluidic chip for detecting the viable organisms in the water body as claimed in claim 2, wherein the depth of the main channel is 20-50 μm, the width is 30-70 μm, and the length is 380-450 μm.
6. The microfluidic chip for detection of viable organisms in a water body according to claim 2, wherein the height difference between the main channel and the bacteria mixed liquid tank is 70 μm.
7. The microfluidic chip for detecting the viable organisms in the water body according to claim 2, wherein the bacteria mixing liquid tank, the buffer liquid tank and the waste liquid tank are all cylindrical, and have a diameter of 60 μm and a depth of 130 μm.
8. The microfluidic chip for detection of viable organisms in a body of water of claim 2, wherein the side channels are 40 μm wide and 150 μm long and have a depth corresponding to the main channel.
9. The method for detecting the microfluidic chip for detecting the viable organisms in the water body according to any one of claims 1 to 8, wherein the method comprises the following steps:
firstly, bacteria mixed liquid enters from a bacteria mixed liquid groove, and when the bacteria mixed liquid flows through a bacteria electrophoresis control unit, cells are subjected to forward electrophoresis movement to be between microelectrodes under the action of an electrically driven flat plate driving electrode with the frequency F1;
secondly, buffer solution enters from a buffer solution tank, forms radial extrusion force through side channel convection, enables bacteria to flow along a straight line, and enables the bacteria near a metal microelectrode to be electrically focused and singly arranged to move along the axial direction of a main channel under the action of the metal microelectrode electrically driven by applying frequency F2 for capturing target cells;
finally, the buffer solution is injected into the buffer solution tank again to continuously dilute the bacteria, and the bacteria are moved out of the outlet unit through the detection area.
10. The method as claimed in claim 9, wherein the waveforms of the electric drive with the frequency F1 and the electric drive with the frequency F2 are triangular wave, square wave or sine wave, the frequency is 1Hz-0.6MHz, and the peak value is 5V-20V.
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