A microfluidic chip, microfluidic device and detection method for detecting Escherichia coli

Abstract

The present invention provides a microfluidic chip, a microfluidic device and a detection method for detecting Escherichia coli, which belong to the field of microbial detection technology. In the present invention, the microfluidic chip is a three-layer tightly combined stacked structure, including an acrylic plastic bottom plate, an acrylic plastic cover plate, a reaction chamber sandwiched between the acrylic plastic bottom plate and the acrylic plastic cover plate, and two metal electrode sheets; the two metal electrode sheets are fixed on the acrylic plastic bottom plate at intervals to form a micro-reaction channel; the reaction chamber includes an injection chamber and an outlet chamber; the injection chamber, the micro-reaction channel and the outlet chamber are connected in sequence. After the sample to be tested and the glucose aqueous solution are injected into the injection chamber, when the phase difference of the AC signal applied to the metal electrode sheet is 180°, the mixing efficiency of the sample to be tested and the glucose solution is improved, and the efficiency and sensitivity of Escherichia coli detection are also improved.

Description

Technical Field

[0001] The present invention relates to the technical field of microorganism detection, and in particular to a microfluidic chip, a microfluidic device and a detection method for detecting Escherichia coli. Background Art

[0002] Escherichia coli is a bacterial group widely distributed in nature (environment and water bodies) and feces of warm-blooded animals. It is a conditional pathogen that can cause gastrointestinal infections, urinary tract infections, arthritis, meningitis, septicemia and other local tissue and organ infections in humans and various animals. It is also the main pathogenic microorganism that causes food contamination. Therefore, the detection of Escherichia coli content in food and the environment (tableware and air) has become an important part of current food safety and one of the key indicators of the Food and Drug Administration.

[0003] The method for detecting Escherichia coli in the prior art includes the following steps: mixing the sample to be tested with a glucose solution, allowing the Escherichia coli in the sample to be tested to decompose glucose to generate lactic acid, adding a phenol red solution to the mixture, and detecting the lactic acid content in the mixture at a wavelength of 610nm, thereby calculating the content of Escherichia coli in the sample to be tested. The higher the OD value, the more glucose is decomposed by Escherichia coli, and the more Escherichia coli is detected. However, the rate at which Escherichia coli decomposes glucose is low, so the detection efficiency needs to be improved. Summary of the invention

[0004] The purpose of the present invention is to provide a microfluidic chip, a microfluidic device and a detection method for detecting Escherichia coli. The microfluidic chip, the microfluidic device and the detection method of the present invention are used to detect Escherichia coli with high detection efficiency.

[0005] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0006] The present invention provides a microfluidic chip for detecting Escherichia coli, which is a three-layer tightly combined laminated structure, including an acrylic plastic bottom plate 3, an acrylic plastic cover plate 1, a reaction chamber 2 sandwiched between the acrylic plastic bottom plate 3 and the acrylic plastic cover plate 1, and two metal electrode sheets;

[0007] The two metal electrode sheets are fixed on the acrylic plastic bottom plate 3 at intervals to form a micro-reaction channel 22;

[0008] The reaction chamber 2 includes an injection chamber 21 and an outlet chamber 23; the injection chamber 21, the micro-reaction channel 22 and the outlet chamber 23 are connected in sequence.

[0009] Preferably, a sample inlet 11 is provided on the acrylic plastic cover plate 1 at a position corresponding to the sample inlet chamber 21 , and a sample outlet 12 is provided at a position corresponding to the outlet chamber 23 .

[0010] Preferably, a dielectric is filled in the area between the acrylic plastic bottom plate 3 and the acrylic plastic cover plate 1 except the reaction chamber 2 and the metal electrode sheet; the thickness of the dielectric is the same as the thickness of the metal electrode sheet.

[0011] Preferably, the micro-reaction channel 22 is in the shape of a cuboid; the length of the micro-reaction channel 22 is 3 to 8 mm, the width is 100 to 300 μm, and the height is 100 to 300 μm.

[0012] The present invention also provides a microfluidic device, comprising the microfluidic chip described in the above scheme and an arbitrary function generator electrically connected to two metal electrode sheets of the microfluidic chip respectively.

[0013] The present invention also provides the use of the microfluidic chip or the microfluidic device in the above scheme in the detection of Escherichia coli for non-diagnostic purposes.

[0014] The present invention also provides a method for detecting Escherichia coli for non-diagnostic purposes based on the microfluidic device described in the above scheme, comprising the following steps:

[0015] Set the phase difference of the AC signal of the arbitrary function generator to 180°;

[0016] The sample to be tested and the glucose aqueous solution are injected into the injection chamber 21 through the injection port 11 to react and obtain a reaction solution;

[0017] The reaction solution is collected at the sample outlet 12, and after the reaction solution and phenol red are mixed, the OD value is measured at a wavelength of 610 nm.

[0018] Preferably, the AC voltage of the arbitrary function generator is 2 to 10V. p .

[0019] Preferably, the alternating current frequency of the arbitrary function generator is 10 kHz to 10 MHz.

[0020] Preferably, the flow rates of the sample to be tested and the glucose aqueous solution are independently 6 to 30 μL / min.

[0021] The present invention provides a microfluidic chip for detecting Escherichia coli, which is a three-layer tightly combined laminated structure, including an acrylic plastic bottom plate 3, an acrylic plastic cover plate 1, a reaction chamber 2 sandwiched between the acrylic plastic bottom plate 3 and the acrylic plastic cover plate 1, and two metal electrode sheets; the two metal electrode sheets are fixed on the acrylic plastic bottom plate 3 at intervals to form a micro-reaction channel 22; the reaction chamber 2 includes an injection chamber 21 and an outlet chamber 23; the injection chamber 21, the micro-reaction channel 22 and the outlet chamber 23 are connected in sequence. When the sample to be tested and the glucose aqueous solution are injected into the injection chamber 21, when the phase difference of the alternating current signal applied to the metal electrode sheet is 180°, a strong electric field can be formed between the two electrodes under the condition of applying the same voltage, and the mixing of Escherichia coli and the glucose solution can be made more complete by changing the electric field strength, frequency and flow rate, thereby promoting the decomposition of glucose by Escherichia coli, and the efficiency and sensitivity of Escherichia coli detection are also improved. In addition, the microfluidic chip of the present invention has a simple manufacturing process and low material cost. The price of the acrylic plastic plate in its manufacturing material is only 0.5 yuan per piece, and the metal electrode sheet can be reused. In the present invention, the microfluidic channel is small in size, only at the micron level, which can significantly reduce the consumption of reagents and samples, reduce experimental costs and waste generation. The process of Escherichia coli decomposing glucose is successfully realized on the microfluidic chip of the present invention in a fast, continuous, sample-saving, portable and short time-consuming manner, thereby realizing the rapid detection of Escherichia coli. The present invention provides a new method for the detection of Escherichia coli in trace liquid samples, provides important technical support for the rapid, portable and integrated detection and analysis of Escherichia coli and other bacteria, and has important scientific significance and broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 Schematic diagram of the structure of the microfluidic rapid mixing chip, wherein 1-acrylic plastic cover plate, 2-reaction chamber, 3-acrylic plastic bottom plate, 11-inlet, 12-outlet, 21-inlet chamber, 22-micro reaction channel, 23-outlet chamber;

[0023] Figure 2 Physical picture of microfluidic rapid mixing chip;

[0024] Figure 3 Schematic diagram of the E. coli detection device;

[0025] Figure 4 To apply different AC voltages (2, 4, 6, 8V p ) when detecting the effect of Escherichia coli;

[0026] Figure 5This is a diagram showing the effect of E. coli detection when different electric field frequencies (10kHz, 100kHz, 500kHz, 1MHz and 10MHz) are applied;

[0027] Figure 6 This is the E. coli detection effect diagram at different flow rates (6, 12, 18, 24 μL / min). DETAILED DESCRIPTION

[0028] The present invention provides a microfluidic chip for detecting Escherichia coli, which is a three-layer tightly combined laminated structure, comprising an acrylic plastic base plate 3, an acrylic plastic cover plate 1, a reaction chamber 2 sandwiched between the acrylic plastic base plate 3 and the acrylic plastic cover plate 1, and two metal electrode sheets; the two metal electrode sheets are fixed on the acrylic plastic base plate 3 at intervals to form a micro reaction channel 22; the reaction chamber 2 comprises a sample injection chamber 21 and an outlet chamber 23; the sample injection chamber 21, the micro reaction channel 22 and the outlet chamber 23 are connected in sequence.

[0029] In the present invention, a sample inlet 11 is preferably provided at a position corresponding to the sample inlet chamber 21 on the acrylic plastic cover plate 1, and a sample outlet 12 is preferably provided at a position corresponding to the sample outlet chamber 23. In the present invention, a medium is filled between the acrylic plastic bottom plate 3 and the acrylic plastic cover plate 1, except for the reaction chamber 2 and the metal electrode sheet; the thickness of the medium is the same as that of the metal electrode sheet. In the present invention, the medium is preferably several layers of 3M double-sided tape.

[0030] In the present invention, the shape of the metal electrode sheet is preferably a rectangle, the length of the metal electrode sheet is preferably 2 to 3 cm, the width is preferably 3 to 8 mm, more preferably 5 mm, and the thickness is preferably 100 to 300 μm, more preferably 200 μm. In the present invention, the metal electrode sheet is preferably a copper sheet or a gold sheet, more preferably a gold sheet. In the present invention, the metal electrode sheet is smooth and flat and has a right angle to generate a uniform electric field.

[0031] In the present invention, the metal electrode sheet is preferably fixed to the acrylic plastic base plate 3 by using 3M double-sided tape.

[0032] In the present invention, the shape of the micro-reaction channel 22 is preferably a cuboid; the length of the micro-reaction channel 22 is preferably 3-8 mm, more preferably 5 mm; the width is preferably 100-300 μm, more preferably 200 μm; the height is preferably 100-300 μm, more preferably 200 μm.

[0033] In the present invention, the shapes of the injection port 11 and the outlet port 12 are preferably circular; the diameter is preferably 1 mm; and the number of the injection ports is preferably 2.

[0034] In the present invention, the acrylic plastic cover plate 1 is used to maintain a sealed environment for the microfluidic chip.

[0035] The present invention also provides a microfluidic device, comprising the microfluidic chip described in the above scheme and an arbitrary function generator electrically connected to two metal electrode sheets of the microfluidic chip respectively.

[0036] In the present invention, the arbitrary function generator is preferably Tektronix model AFG3102; the arbitrary function generator is used to provide an electrical signal.

[0037] In the present invention, the microfluidic device preferably further comprises an inlet sample injection component and an outlet sample sampling component;

[0038] The sample injection assembly is connected to the injection port 11;

[0039] The sample sampling assembly is communicated with the sample outlet 12 .

[0040] In the present invention, the microfluidic device preferably also includes a microscopic observation component; the microscopic observation component is arranged directly above the position of the acrylic plastic cover plate 1 corresponding to the microreaction channel 22; the function of the microscopic observation component is to observe whether the channel is leaking and whether the liquid sample in the channel is mixed.

[0041] In the present invention, the sample injection assembly is preferably a syringe and a syringe pump, and the model of the syringe pump is preferably a syringe pump (Harvard PHD 2000); the syringe pump is used to fix the syringe and adjust the flow rate of the bacterial solution and the glucose solution.

[0042] The present invention also provides the use of the microfluidic chip or the microfluidic device in the above scheme in the detection of Escherichia coli for non-diagnostic purposes.

[0043] The present invention also provides a method for detecting Escherichia coli for non-diagnostic purposes based on the microfluidic device described in the above scheme, comprising the following steps:

[0044] Set the phase difference of the AC signal of the arbitrary function generator to 180°;

[0045] The sample to be tested and the glucose aqueous solution are injected into the injection chamber 21 through the injection port 11 to react and obtain a reaction solution;

[0046] The reaction solution is collected at the sample outlet 12, and after the reaction solution and phenol red are mixed, the OD value is measured at a wavelength of 610 nm.

[0047] In the present invention, the E. coli detection is preferably a qualitative detection.

[0048] In the present invention, the molar concentration of the aqueous glucose solution is preferably 0.05 to 0.15 mol / L, more preferably 0.1 mol / L.

[0049] In the present invention, the phenol red is used to reflect the content of lactic acid produced after Escherichia coli decomposes glucose, and further reflects the efficiency of Escherichia coli in decomposing glucose, thereby obtaining the sensitivity of Escherichia coli detection.

[0050] In the present invention, the AC voltage of the arbitrary function generator is preferably 2 to 10V. p , preferably 4 to 8 V p , preferably 6V p The frequency of the AC power is preferably 10kHz to 10MHz, more preferably 100kHz to 1MHz, and most preferably 500kHz.

[0051] In the present invention, the flow rates of the sample to be tested and the glucose aqueous solution are independently preferably 6 to 30 μL / min, more preferably 12 to 24 μL / min, and most preferably 18 to 20 μL / min.

[0052] In the present invention, a higher OD value means a higher content of the product lactic acid, and more glucose is decomposed by E. coli, that is, the detection sensitivity of E. coli is higher.

[0053] In the specific implementation process of the present invention, the injection pump is adjusted to automatically inject the bacterial suspension and glucose solution into the micro-reaction channel through the syringe and the hose at a certain flow rate to decompose glucose. This process does not require manual operation, and the parameters can be adjusted at any time or the continuation or suspension of this process can be determined. After the product is collected on the enzyme-linked plate, a phenol red solution is added, and the product can be detected and analyzed using an enzyme marker. The degree of automation is high, and the detection process is continuously controllable. In addition, the present invention can achieve efficient Escherichia coli detection by adjusting three parameters: voltage, frequency, and bacterial liquid flow rate. The detection efficiency is high under the conditions of higher voltage, medium frequency range, and smaller flow rate.

[0054] The technical solutions in the present invention will be described clearly and completely below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0055] Example 1

[0056] Schematic diagram of the microfluidic chip structure Figure 1As shown. The chip is mainly divided into three layers: the upper layer is an acrylic plastic cover plate 1, the lower layer is an acrylic plastic bottom plate 3, which is sprayed with a 75% ethanol aqueous solution and wiped with lens paper before use. The middle layer is a sample injection chamber 21, a micro-reaction channel 22, and an outlet chamber 23 composed of 3M double-sided tape and metal electrodes.

[0057] Cut out a copper or gold sheet of about 2 cm in length, 5 mm in width, and 200 μm in thickness as a metal electrode sheet, and use 3M double-sided tape to tightly bond the metal electrode sheet and the acrylic plastic base plate 3 together. The two metal electrode sheets are placed opposite each other with a distance of 200 μm in between, forming a micro-reaction channel of 5 mm in length, 200 μm in width, and 200 μm in height. Take a section of 3M double-sided tape of appropriate length and stick it on the area other than the electrode on the base plate to fill the gap. After compaction, tear off the protective film and cover it with a layer of 3M double-sided tape. Repeat this process until the thickness of the double-sided tape is equal to the thickness of the metal electrode. Take another acrylic plastic plate and place it vertically at the marked position on the acrylic plastic plate with a 1 mm drill bit to drill out an inlet 11 and an outlet 12 with a diameter of 1 mm. The acrylic plastic cover is used to keep the chip in a sealed environment and connect the syringe and the hose. Press the prepared microfluidic chip repeatedly and place it under a heavy object overnight to make the three-layer structure tightly combined.

[0058] Step 2: Cultivation and treatment of E. coli

[0059] The strain used was Escherichia coli JM109, and the culture medium was LB medium. Streak the existing bacterial solution on the LB solid medium, culture it in a 37°C incubator overnight, pick a single clone and culture it in 5mL LB liquid medium for 18h with a constant temperature shaker set at 37°C, 180-200rpm, and mix the bacterial solution obtained after culture with the LB liquid medium at a ratio of 1:1.

[0060] Step 3: Connection and use of E. coli detection device

[0061] Schematic diagram of E. coli detection device Figure 3 As shown. The treated bacterial solution was injected into a syringe sterilized with 75% alcohol in advance, and the arbitrary function generator (Tektronix model AFG3102) was connected to the microfluidic chip. The syringe was connected to the inlet of the micro-reaction channel through a hose to pass the E. coli bacterial solution and glucose solution (concentration of 0.1 mol / L). A pipette was used to transfer 100 μl of the product to the enzyme-linked plate at the outlet of the micro-reaction channel, and 50 μl of phenol red solution was added. The enzyme-linked plate was immediately placed in an ELISA instrument to measure the lactic acid content at a wavelength of 610 nm.

[0062] Step 4: E. coli detection test

[0063] Before the experiment, the micro-reaction channel, the syringe and the hose used were sterilized with alcohol and rinsed with ultrapure water. The glucose solution and the diluted bacterial solution were sucked into the syringe. After connecting the experimental device, the phase difference of the AC signal of the arbitrary function generator was adjusted to 180°, and the phase difference was adjusted to 2-10V. p The AC voltage is changed within the range, the AC frequency is changed within the range of 10kHz to 10MHz, and the flow rate of the liquid sample is changed within the range of 6 to 30μL / min. After adding 50μL of phenol red reagent to the collected 100μL reaction solution, the ELISA plate is immediately placed in the ELISA reader to measure the lactic acid content at a wavelength of 610nm to determine the detection effect of E. coli. The higher the OD value, the more glucose is decomposed by E. coli, the higher the detection efficiency of E. coli, and the higher the detection sensitivity.

[0064] Test results:

[0065] 1. Effect of AC voltage on Escherichia coli detection efficiency

[0066] Figure 4 To apply different AC voltages (2, 4, 6, 8V p ), the flow rate of the injection pump was controlled to be 18 μL / min, the electric field frequency was 500 kHz, and the phase difference was 180°.

[0067] As can be seen from the figure, when the voltage is 2V p When the voltage is 4V, the absorbance of the product is slightly higher than that before the power is applied, indicating that the yield of the product has increased, indicating that the efficiency of E. coli decomposing glucose has increased, that is, the detected E. coli content has increased, which proves that the improvement of the mixing efficiency of E. coli and glucose can improve the sensitivity of E. coli content detection. p When the power is on, the absorbance of the product is significantly higher than that without power (P<0.1), which is similar to that of 2V p The increase is greater when compared with the time phase, indicating that the product of glucose decomposition by E. coli has increased, that is, the decomposition efficiency has increased, and the sensitivity of E. coli content detection has also increased. When the voltage is 6V p When the power was on, the absorbance of the product increased significantly compared with that without power (P<0.01), which was similar to that of the 2V p , 4V p The improvement is more significant than that of the control group, indicating that the efficiency of E. coli in decomposing glucose has been greatly improved, and the detection sensitivity of E. coli has also been greatly improved. p When the voltage was 6V, the absorbance of the product after power-on was significantly higher than that without power-on (P<0.05), but the OD value was not pFrom this we can conclude that by increasing the applied voltage to improve the mixing efficiency of E. coli and glucose, the sensitivity of E. coli detection can be significantly improved.

[0068] 2. Effect of AC frequency on E. coli detection efficiency

[0069] Figure 5 This is the effect diagram of E. coli detection when different electric field frequencies (10kHz, 100kHz, 500kHz, 1MHz and 10MHz) are applied. The flow rate of the injection pump is controlled to be 18μL / min and the voltage is 6V p , the phase difference is 180°.

[0070] As can be seen from the figure, when the frequency is 10kHz, the absorbance of the product after power-on is higher than that when power is not applied, indicating that the output of the product lactic acid has increased, that is, the efficiency of E. coli decomposing glucose has increased, and the detection sensitivity of E. coli content has increased, proving that the improvement of the mixing efficiency of E. coli and glucose can improve the efficiency of E. coli decomposing glucose and the sensitivity of E. coli content detection. When the frequencies are 100kHz, 500kHz, 1MHz, and 10MHz, the absorbance of the product after power-on is also significantly improved compared to before power-on, but in comparison, when the frequency is 1MHz, the absorbance of the product is higher than that at other frequencies, indicating that the output of the product in the decomposition process has increased, indicating that the efficiency of E. coli decomposing glucose has increased, and indicating that the sensitivity of E. coli detection has increased. This proves that within a certain range, by increasing the frequency of the applied electric field to improve the mixing efficiency of E. coli and glucose, the efficiency of E. coli decomposing glucose and the sensitivity of E. coli detection can be significantly improved.

[0071] 3. Effect of flow rate on E. coli detection efficiency

[0072] Figure 6 The results of E. coli detection at different flow rates (6, 12, 18, 24 μl / min) are shown. The electric field frequency between the two metal electrodes is controlled to be 500 kHz and the voltage is 6 V. p , the phase difference is 180°.

[0073] As can be seen from the figure, when the flow rate is 6μL / min, the absorbance of the product after power-on is significantly higher than that before power-on (P<0.01), indicating that the production of lactic acid has increased significantly, indicating that the efficiency of E. coli decomposing glucose has been improved, and at the same time, the sensitivity of E. coli detection has also increased. As the flow rate increases to 12μL / min, 18μL / min, and 24μL / min, the absorbance of the product after power-on is higher than that before power-on, but the higher the flow rate, the lower the degree of increase compared with the flow rate of 6μL / min. It is proved that a lower flow rate can make E. coli and glucose mix faster, more fully, and more efficiently, thereby accelerating the efficiency of E. coli decomposing glucose and improving the sensitivity of E. coli detection.

[0074] 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. A method for detecting Escherichia coli for non-diagnostic purposes based on a microfluidic device, comprising the following steps: Set the phase difference of the AC signal of the arbitrary function generator to 180°; Injecting the sample to be tested and the glucose aqueous solution into the injection chamber (21) through the injection port (11) to react and obtain a reaction solution; The reaction solution is collected at the sample outlet (12), the reaction solution is mixed with phenol red, and the OD value is measured at a wavelength of 610 nm; The microfluidic device comprises a microfluidic chip and an arbitrary function generator electrically connected to two metal electrode sheets of the microfluidic chip respectively; The microfluidic chip is a three-layer tightly combined stacked structure, comprising an acrylic plastic base plate (3), an acrylic plastic cover plate (1), a reaction chamber (2) sandwiched between the acrylic plastic base plate (3) and the acrylic plastic cover plate (1), and two metal electrode sheets; The two metal electrode sheets are fixed on the acrylic plastic base plate (3) at intervals to form a micro-reaction channel (22); The reaction chamber (2) comprises an injection chamber (21) and an outlet chamber (23); the injection chamber (21), the micro-reaction channel (22) and the outlet chamber (23) are connected in sequence; An inlet (11) is provided on the acrylic plastic cover plate (1) at a position corresponding to the inlet chamber (21), and an outlet (12) is provided at a position corresponding to the outlet chamber (23); A dielectric is filled in the area between the acrylic plastic bottom plate (3) and the acrylic plastic cover plate (1) except for the reaction chamber (2) and the metal electrode sheet; the thickness of the dielectric is the same as the thickness of the metal electrode sheet; The shape of the micro-reaction channel (22) is a rectangular parallelepiped; the length of the micro-reaction channel (22) is 5 mm, the width is 200 μm, and the height is 200 μm; The AC voltage of the arbitrary function generator is 6V p ; The alternating current frequency of the arbitrary function generator is 1 MHz; The flow rates of the sample to be tested and the glucose aqueous solution are independently 6 μL / min.

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