CN111617814A - Microfluidic chip and detection method for survivable biological detection in water body - Google Patents

Abstract

The invention discloses a microfluidic chip used for survivable biological detection in a water body, relates to the technical field of environmental monitoring, and comprises a channel layer, a dielectric layer and a base layer which are sequentially bonded from top to bottom; the channel layer An inlet unit, a bacterial electrophoresis control unit, and an outlet unit are arranged in sequence; the inlet unit and the outlet unit pass through the channel layer and the dielectric layer in sequence. The invention also provides a detection method of a microfluidic chip used for the detection of viable organisms in a water body. The invention has the advantages of single bacteria analysis capability, high integration and simple operation. According to the advantages of the microfluidic chip device of the present invention, it is simple to manufacture, easy to operate, can control the flow state of the fluid in the channel, can realize the qualitative and quantitative detection and analysis of the type and quantity of bacteria in water, especially in ballast water, and is suitable for environmental Viable bioanalysis and drug monitoring in water, especially ballast water.

Description

Microfluidic chip and detection method for survivable biological detection in water body

technical field

The invention relates to the technical field of environmental monitoring, in particular to a microfluidic chip and a detection method for detecting viable organisms in a water body.

Background technique

Bacterial pollution indicators are a series of indicators used to measure whether water sources are contaminated by bacteria and to describe the degree of pollution. Humans often come into contact with pathogenic bacteria in water through drinking, touching, etc., causing harm to human health. The most common diseases include cholera, typhoid, dysentery, etc. In order to ensure the hygienic quality of water bodies, different types of water quality should be tested for microorganisms. Due to the low content of pathogenic bacteria in the water, the detection steps are complex and time-consuming, so it is difficult. Therefore, all countries in the world generally test the unified health indicator bacteria first, and then test each pathogenic bacteria one by one when necessary. This health indicator bacteria can be used as a bacterial contamination indicator. At the same time, ships’ ballast water often carries a large number of exotic marine species, including phytoplankton, bacteria and viruses. According to statistics, the ballast water carried by ships around the world contains an average of 110 million phytoplankton individuals per cubic meter of ballast water. Ship's ballast water can spread various bacteria around the world during the ship's voyage, which has threatened the safety of marine ecosystems, and even led to catastrophic consequences. Therefore, it is often necessary to detect viable organisms, especially bacteria (germs) in water bodies. At present, the detection of microorganisms, especially bacteria, mainly involves methods such as culture, chemiluminescence, bioluminescence, mass spectrometry and polymerase chain reaction (PCR). The 3M microbiological test sheet was applied in the sanitary monitoring of ballast water, and it was found that it was suitable for the determination of the total number of bacteria in water, but not suitable for the detection of coliform bacteria in ballast water. The detection of microorganisms is limited by the uncertainty of the growth rate of microorganisms and the time of water discharge, and its effectiveness has not been achieved. Therefore, there is an urgent need to establish a rapid detection method for microorganisms in water, so as to effectively carry out the assessment of water.

The microfluidic chip has the characteristics of high throughput, integration and low cost. Since the size of its channel structure is the same as that of bacteria (micron scale), the use of microfluidic chip technology can realize the capture, identification and identification of bacteria. One-button closed operation such as detection has the advantages of high efficiency and small error. Therefore, microfluidic chip technology can be used as a powerful tool for rapid detection of bacteria in ballast water in situ. At present, bacterial chip analysis systems mainly include optical analysis and detection systems and electrochemical analysis and detection systems. The optical analysis and detection system hinders the miniaturization development of the system due to its complex system and huge volume; while the electrochemical analysis and detection system has the advantages of high sensitivity, relatively simple peripheral equipment such as signal processing system, and easy matching with the micro-electrodes on the chip. It has good prospects in terms of miniaturization and portability, as well as efficient and fast detection. Wang ShuQi achieved 71.8% ± 5% capture of E. coli in blood and milk by immobilizing antibodies that specifically bind to E. coli surface proteins on a microfluidic chip. Renjie Wang et al. built a simple microfluidic chip platform, and realized the in situ rapid detection of Salmonella typhimurium by immunocapture technology combined with the corresponding light-emitting diode-induced fluorescence detection system. On a portable in situ real-time PCR analysis chip, S Furutani et al. can detect herbaceous Escherichia coli within 7 minutes through the Taq Man fluorescent probe. Fronczek et al. realized the detection of Salmonella typhimurium in 5 minutes on a microfluidic paper chip using LED lights and a smartphone as a fluorescence imaging system, which is simple and convenient to operate.

From the analysis performance of microfluidic chip, its future application fields will be very wide, but the current key application field is obviously in the field of life sciences. Its advantages in miniaturization, integration and portability of analytical instruments provide broad application in many fields such as environmental monitoring and protection, biomedicine, high-throughput drug synthesis screening, crop optimization and breeding, health and immunity, etc. prospect.

SUMMARY OF THE INVENTION

In order to solve the above technical problems, the present invention provides a microfluidic chip for the detection of viable organisms in a water body. Microbial detection and quantitative analysis of the bacterial content in water.

For achieving the above object, the present invention provides the following scheme:

The invention provides a microfluidic chip for detecting viable organisms in a water body, comprising a channel layer, a dielectric layer and a base layer which are sequentially bonded from top to bottom; the channel layer is provided with sequentially connected an inlet unit, a bacterial electrophoresis manipulation unit and an outlet unit; the inlet unit and the outlet unit all run through the channel layer and the dielectric layer in sequence.

Optionally, the inlet unit includes a bacterial mixed liquid tank, a buffer tank and a side channel; the outlet unit includes two waste liquid tanks; the bacterial electrophoresis control unit includes a main channel, a metal microelectrode group and a flat plate drive electrode ; The bacterial mixed liquid tank is arranged at one end of the main channel, and the two waste liquid tanks are arranged on the other end of the main channel; the buffer tank and the side channel are located on both sides of the bacterial mixed liquid tank and communicated with the main channel, the main channel is communicated with the bacterial mixed liquid tank, the buffer tank and the waste liquid tank through a side channel; the metal microelectrode group is arranged on the base. On the bottom layer and extending into the dielectric layer, the input pins of the plate driving electrodes are arranged on the base layer for connecting with external driving circuits.

Optionally, the metal micro-electrode group includes 9 metal micro-electrodes.

Optionally, the metal micro-electrodes are rectangular or strip-shaped, with a width of 30 μm, and the distance between adjacent metal micro-electrodes 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 bacterial mixed solution tank is 70 μm.

Optionally, the bacterial mixture tank, the buffer tank and the waste liquid tank are all cylindrical, with a diameter of 60 μm and a depth of 130 μm.

Optionally, the side channel is 40 μm wide and 150 μm long, and the depth is the same as that of the main channel.

Optionally, both the metal micro-electrode group and the flat plate driving electrode are made of 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 base layer is a glass or silicon or ceramic or polymer layer with a thickness of 0.5mm-3mm.

The present invention also discloses a detection method comprising the above-mentioned microfluidic chip for detecting survivable organisms in a water body, comprising the following steps:

First, the bacterial mixture enters from the bacterial mixture tank and flows through the bacterial electrophoresis control unit, under the action of the electric drive plate driving electrode with the frequency F1 applied, the cells are moved forward electrophoretically between the microelectrodes;

Secondly, the buffer enters from the buffer tank, and the convection through the side channel forms a radial extrusion force, which makes the bacteria flow in a straight line. Bacteria near the electrodes undergo electrofocusing and individual arrays advance axially along the main channel;

Finally, the buffer tank is refilled with buffer to continuously dilute the bacteria, pass through the detection zone and then be removed from the outlet unit.

Optionally, the waveforms of the electric drive of the frequency F1 and the electric drive of the frequency F2 are triangular waves, square waves or sine waves, the frequency is 1 Hz-0.6 MHz, and the peak value is 5V-20V.

The present invention has achieved the following technical effects with respect to the prior art:

The microfluidic chip used for the detection of viable organisms in the water body in the present invention utilizes the electrophoresis characteristics of bacteria to drive and separate different types of bacteria, and then uses hydrodynamics to control the flow state of the bacteria, and then uses the microelectrodes to electro-focus the bacteria, so that the Bacteria flow through the detection zone individually. Compared with the common electrophoresis chip, the detection accuracy of the chip is improved; compared with the three-dimensional electrode, the complicated preparation process of the three-dimensional electrode is avoided.

The microfluidic chip used for the detection of viable organisms in the water body in the present invention, and the three-dimensional microfluidic chip device used for the detection of bacteria in the ballast water, exhibits some significant advantages, including: simple fabrication, easy operation, and biological materials used. The combination of good compatibility, low conductivity, high optical transparency, air permeability, biological inertness, easy processing, and low cost combined with the traditional advantages of microfluidic chips makes detection efficient and reliable.

Compared with the conventional analysis process, the invention is used for bacterial detection, and only needs one device and one chip to complete the detection, reduces the number of devices, and reduces the hardware purchase cost; improves the automation of the analysis process and improves work efficiency , avoids the transfer of samples between different equipment, simplifies manual operations, and reduces the risk of sample contamination; the microfluidic chip reduces the consumption of samples and reagents, and reduces operating costs; the device of the invention is small in size, Portable equipment can be formed to meet the needs of real-time and on-site detection.

Description of drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the accompanying drawings required in the embodiments will be briefly introduced below. Obviously, the drawings in the following description are only some of the present invention. In the embodiments, for those of ordinary skill in the art, other drawings can also be obtained according to these drawings without any creative effort.

Fig. 1 is the chip A-A' cross-sectional schematic diagram involved in the present invention;

2 is a schematic structural diagram of a three-dimensional microfluidic chip device involved in the present invention;

3 is a chip design diagram and an electrode system diagram involved in the present invention;

FIG. 4 is the movement trajectory of bacterial particles generated by the simulation of the microfluidic channel according to the present invention by the Comsol software.

5 is a comparison diagram of bacterial particle separation by applying an electric field frequency to the microfluidic channel involved in the present invention;

6 is a comparison diagram of bacterial particle separation by applying another electric field frequency to the microfluidic channel involved in the present invention;

Fig. 7 is a diagram showing the spatial variation of the electric potential in the microfluidic channel generated by the simulation of the microfluidic channel involved in the present invention by Comsol software.

Description of reference numerals: 1. Base layer; 2. Dielectric layer; 3. Main channel; 4. Channel layer; 5. Bacterial mixed liquid tank; 6. Side channel; 7. Buffer tank;

Detailed ways

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, but not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

Example 1: Preparation of chips

As shown in FIG. 1 , the microfluidic chip used for the detection of viable organisms in a water body in this embodiment is fabricated by using the “one-shot exposure” technique and by using the ultraviolet lithography method. After the silicon wafer was treated with sulfuric acid and hydrogen peroxide solution, washed and dried, a layer of SU-82035 negative light glue was thrown on the surface with a glue spinner at a speed of 3000 rpm, and baked at 65 °C for 10 minutes and 95 °C for 4 minutes. Then, after UV exposure, secondary curing, development, and nitrogen drying, patterns of each cell chamber and microchannel network are obtained. Then silanized to make the surface of the mold hydrophobic, polydimethylsiloxane (PDMS) prepolymer and initiator were mixed with a mass ratio of 10:1 to cover the mold, and after removing air bubbles, the mold was cured at 75°C. The polymerized PDMS sheet was carefully peeled off, cut into shape, punched with a flat-headed syringe needle, and then treated with oxygen plasma to bond with the treated substrate, and the chip was successfully fabricated.

Example 2: Structure of the chip

As shown in FIG. 1 , the microfluidic chip used for the detection of viable organisms in a water body in this embodiment includes a channel layer 4 , a dielectric Layer 2 and base layer 1; the channel layer 4 is provided with an inlet unit, a bacterial electrophoresis control unit and an outlet unit that are connected in sequence; the inlet unit and the outlet unit all run through the channel layer 4 and the medium in sequence. Electrical layer 2.

The inlet unit includes a bacterial mixed liquid tank 5, a buffer tank 7 and a side channel 6; the outlet unit includes two waste liquid tanks; the bacterial electrophoresis control unit includes a main channel 3, 8 groups of metal microelectrodes and a plate drive electrode; the bacterial 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 tank 7 and the side channel 6 are located in the Both sides of the bacterial mixed liquid tank 5 are communicated with the main channel 3, and the main channel 3 is communicated with the bacterial mixed liquid tank 5, the buffer tank 7 and the waste liquid tank through the side channel 6. 8 groups of the metal micro-electrodes are arranged on the base layer 1 and extend into the dielectric layer 2, and the input pins of the flat plate drive electrodes are arranged on the base layer 1 for connecting with an external drive circuit connect.

The group of metal microelectrodes 8 includes 9 metal microelectrodes 8 .

The metal micro-electrodes 8 are rectangular or strip-shaped, with a width of 30 μm, and the distance between adjacent metal micro-electrodes 8 is 10 μm. The width of the flat plate driving electrodes is 30 μm, the distance between adjacent flat plate driving electrodes is 30 μm, and the thickness is the same as that of the metal micro-electrodes 8 .

The depth of the main channel 3 is 20-50 μm, preferably 30 μm, the width is 30-70 μm, preferably 50 μm, and the length is 380-450 μm, preferably 420 μm.

The height difference between the main channel 3 and the bacterial mixed liquid tank 5 is 70 μm.

The bacterial mixture tank 5, the buffer tank 7 and the waste liquid tank are all cylindrical, with a diameter of 60 μm and a depth of 130 μm.

The side channel 6 is 40 μm wide and 150 μm long, and the depth is the same as that of the main channel 3 .

The 8 groups of metal micro-electrodes and the flat plate driving electrodes are all 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, in this embodiment, SU-82035 photoresist is used.

The base layer 1 is glass with a thickness of 0.5mm-3mm.

Example 3: Dielectrophoresis technology is used for bacterial detection and separation

An electrode system is installed at the bottom of the device, which has different driving effects on different kinds of bacteria during detection. As shown in Figure 4, the electrodes on the bottom side of the sample tank are used to drive the bacteria, so that there is an axial mutual separation between different bacteria, that is, different bacteria are separated due to differences in physical and chemical properties, and focus on a certain zone to advance forward. After separation, the metal microelectrodes 8 staggered on both sides of the main channel 3 perform electric focusing on the separated bacterial zone, so that various bacteria are concentrated in the center of each zone, which is convenient for passing detection. The sensing electrodes on both sides of the detection area monitor and conduct real-time changes in the conductivity of the solution in the detection area for quantitative analysis. As shown in Figure 4, in order to use dielectrophoresis to separate bacterial particles in the microfluidic channel, a positive and negative voltage of 5v was applied, and the particles with a diameter of 3 μm and a particle with a diameter of 5 μm were shown in Fig. marked out.

Example 4: Optimization of electric field frequency for isolation of bacteria in microfluidic chips

Different electric field frequencies are applied to the bacterial solution in the microfluidic channel, and the electric field force on the particles is different. When the frequency is too high, the separation and sorting of bacterial particles of different particle sizes cannot be achieved. Therefore, based on the microfluidic chip designed in the present invention, the conditions of bacterial separation in the range of 1HZ-1MHZ of the applied electric field frequency were respectively tested. The results are shown in the comparison between Figure 5 and Figure 6. When the frequency is 0.6MHZ (Figure 5), the bacteria can be separated; when the frequency is 0.7MHZ (Figure 6), the bacteria cannot be separated.

Example 5: Application of Impedance Detection Technology

The AC impedance method was selected as the electrochemical measurement method for the bacterial impedance detection technology, and the principle and electrode preparation of the impedance measurement technology were analyzed and optimized. The AC impedance method generally uses the electrochemical workstation portable equipment to measure the impedance of the electrochemical system, and usually uses a three-electrode system to perturb the electrode system with a small-amplitude sine wave voltage signal. Now by using the bimetallic microelectrode 8 system and using the microelectrode as the working electrode to realize the miniaturization of the counter electrode detection device, and injecting the bacterial sample to be measured into the liquid storage tank, the total liquid storage volume is only a few cubic millimeters. Compared with the large amount of electrolyte solution wasted by ordinary electrochemical measurement, the electrolyte solution consumption of this detection method is very small, and the consumption of bacterial samples is also very small. The small area of the electrode also saves the time for bacterial detection and greatly improves the detection efficiency.

For the spatial variation of potential and current density simulations during fluid flow and voltage application in the microfluidic channel, the results are shown in Figure 7. Bacterial impedance detection is to apply a small-amplitude AC disturbance signal (voltage or current) to the bacteria to measure the electrical characteristics of the bacteria and analyze the physiological state and changes of the bacteria. This technology does not require labeling of bacteria, and small disturbances will not cause any damage to bacteria. At the same time, it can continuously measure the impedance of bacteria. It can also be combined with microfluidic technology to realize the miniaturization of measurement equipment.

It should be noted that it is obvious to those skilled in the art that the present invention is not limited to the details of the above-mentioned exemplary embodiments, and the present invention can be implemented in other specific forms without departing from the spirit or essential characteristics of the present invention. . Therefore, the embodiments are to be regarded in all respects as illustrative and not restrictive, and the scope of the invention is to be defined by the appended claims rather than the foregoing description, which are therefore intended to fall within the scope of the claims. All changes that come within the meaning and range of equivalents of , are intended to be embraced within the invention, and any reference signs in the claims shall not be construed as limiting the involved claim.

In this specification, specific examples are used to illustrate the principles and implementations of the present invention, and the descriptions of the above embodiments are only used to help understand the method and the core idea of the present invention; There will be changes in the specific implementation manner and application scope of the idea of the invention. In conclusion, the contents of this specification should not be construed as limiting the present 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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