Biological chip for detecting bacteria in food

Abstract

The invention discloses a biochip for detecting bacteria in food, which comprises a chip substrate and a chip cover plate, and a plurality of detection units are arranged on the chip substrate around the central point; the detection unit comprises a lysis solution pool, a sample pool, an enzyme solution pool and a reaction pool which are formed on the chip substrate, the reaction pool is farthest away from the central point, and the distances between the lysis solution pool, the sample pool and the reaction pool and the central point are sequentially increased; the lysate pool is communicated with the sample pool through a first micro-channel, the sample pool is communicated with the reaction pool through a second micro-channel, the enzyme solution pool is communicated with the reaction pool through a third micro-channel, and breakthrough valves are arranged on the second micro-channel and the third micro-channel; a plurality of sample adding holes are formed in the chip cover plate. The invention further discloses a detection system comprising the micro-fluidic chip, a fluorescence detection method and application of the micro-fluidic chip. The biochip for detecting nitrite in food has the advantages of fast detection speed, high precision and high automation degree, and can realize on-site real-time detection.

Description

Technical Field

[0001] The present invention relates to the technical field of biochips, and particularly relates to a biochip for detecting bacteria in food and its application. Background Art

[0002] Bacterial foodborne diseases (bacterial food poisoning) often cause group diarrhea, vomiting and other harms, which pose a threat to people's physical health. The total number of colonies is an important indicator for food safety and environmental monitoring ("GB 4789.2-2016 National Food Safety Standard Microbiological Examination of Foods - Determination of Total Number of Colonies", colony culture, time 1 - 3 days). There are many methods for detecting the total number of bacteria in food. Common methods include: 1) Plate counting method: Divide food samples into small pieces and place them on agar plates rich in nutrients for culture, and estimate the total number of bacteria in the food by counting the number of colonies. 2) Membrane filtration method: Pass food samples through a special filter membrane to filter out particulate matter and large bacteria, then place the filter membrane on an agar plate rich in nutrients for culture, and count the number of colonies to estimate the total number of bacteria in the food. 3) Spectral method: By detecting the absorption of light with a specific wavelength in food samples by bacteria, and estimating the total number of bacteria in the food by measuring the change in light absorption. These methods have their own advantages and disadvantages in practical applications, and the selection of a suitable detection method needs to be based on specific situations and requirements. Different food types and bacterial species may require different methods for detection. The national standard method is time-consuming, requires large equipment, and has complicated operations, which is not conducive to application fields such as inspection and quarantine that require on-site real-time detection.

[0003] In recent years, as a new type of analysis platform, biochips have the advantages of miniaturization, automation, integration, etc., and have received extensive attention in related fields such as environmental detection. There is no report on building a biochip platform for the detection of nitrite. These chip detections do not fully utilize the advantages of disk-type chips in design, have complex actual operations, are not conducive to the simultaneous detection of multiple samples, and have a narrow chip application range and poor flexibility. There has been no substantial breakthrough in achieving high-resolution and high-sensitivity bacterial detection applications on chips. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to provide a biochip for detecting bacteria in food to overcome the deficiencies of the prior art.

[0005] To solve the above technical problem, the present invention provides a biochip for detecting bacteria in food, including a chip substrate that can rotate around a center point and a chip cover plate attached to the chip substrate. A plurality of detection units are arranged around the center point on the chip substrate;

[0006] The detection unit includes a lysis solution pool, a sample pool, an enzyme solution pool, and a reaction pool formed on the chip substrate. The reaction pool is the farthest from the center point, and the distances between the lysis solution pool, the sample pool, and the reaction pool and the center point increase in sequence. The lysis solution pool is communicated with the sample pool through a first microchannel, the sample pool is communicated with the reaction pool through a second microchannel, and the enzyme solution pool is communicated with the reaction pool through a third microchannel. Break valves are provided on both the second microchannel and the third microchannel.

[0007] A plurality of sample addition holes are provided on the chip cover plate. Each of the lysis solution pool, the sample pool, and the enzyme solution pool is respectively communicated with a corresponding sample addition hole, and samples can be added to each pool through the sample addition hole.

[0008] The break valve, also called a siphon valve, has a fixed critical rotation speed. In the present invention, when the rotation speed of the microfluidic chip is lower than the critical rotation speed of the break valve, the sample pool and the reaction pool, and the enzyme solution pool and the reaction pool will not be conducted; when the rotation speed reaches or exceeds the critical rotation speed, the passages between the sample pool, the enzyme solution pool and the reaction pool are conducted, and the solution begins to enter the reaction pool. Therefore, when the rotation speed of the biochip is controlled to be lower than the critical rotation speed of the break valve, under the drive of centrifugal force, the lysis solution in the lysis solution pool will flow into the sample pool along the first microchannel and mix with the sample, while the passages between the sample pool, the enzyme solution pool and the reaction pool will not be conducted at this time. Then, the rotation speed of the microfluidic chip is controlled to reach or exceed the critical rotation speed of the break valve. At this time, the passages between the sample pool, the enzyme solution pool and the reaction pool are conducted, and the mixed solution in the sample pool and the enzyme solution in the enzyme solution pool respectively enter the reaction pool along the second microchannel and the third microchannel, so that a fluorescence reaction occurs in the sample pool.

[0009] Preferably, the plurality of detection units are uniformly arranged around the center point on the chip substrate.

[0010] Preferably, the chip substrate is in a disc shape, and a mounting hole for mounting on a rotating shaft is provided at its central part.

[0011] Preferably, a plurality of exhaust holes are further provided on the chip cover plate. Each of the lysis solution pool, the sample pool, the enzyme solution pool, and the reaction pool is respectively communicated with a corresponding exhaust hole. The exhaust holes are used to discharge the bubbles in the added sample solution.

[0012] Preferably, the first microchannel is a channel with a plurality of S-shaped bends. The bent channel can not only play a role in fully mixing the lysis solution, but also increase the dissolved oxygen in the solution.

[0013] Preferably, the chip substrate sequentially includes a first chip layer and a second chip layer that are attached to each other from bottom to top. The lysis solution pool, the sample pool, the enzyme solution pool, the reaction pool, and the break valve all penetrate through the second chip layer.

[0014] Preferably, the first microchannel, the second microchannel, and the third microchannel are flow channel grooves recessed in the upper surface of the second chip layer. In another embodiment, the first microchannel, the second microchannel, and the third microchannel are recessed in the lower surface of the chip cover plate.

[0015] The present invention also provides a fluorescence detection system for bacterial detection, including the aforementioned bacterial detection biochip and a fluorescence detection device. The fluorescence detection device can be any device capable of detecting fluorescence intensity, including but not limited to a fluorescence spectrophotometer, a handheld fluorescence detector, and a desktop fluorescence detector. When the fluorescence detection device is a desktop fluorescence detector, preferably, it has both centrifugation and detection functions at the same time.

[0016] The present invention also provides a method for fluorescence detection using the aforementioned biochip for bacterial detection in food, including the following steps:

[0017] (1) Add lysate, sample, and enzyme solution into the lysate pool, sample pool, and enzyme solution pool respectively through the sample addition hole;

[0018] (2) Centrifuge the bacterial detection biochip at a first centrifugal speed so that the lysate enters the sample pool and mixes with the sample;

[0019] (3) Centrifuge the bacterial detection biochip at a second centrifugal speed so that the enzyme solution and the mixed solution in the sample pool enter the reaction pool and a fluorescence reaction occurs;

[0020] (4) Use a fluorescence detection device to detect the intensity of fluorescence.

[0021] In the present invention, the second centrifugal speed is greater than the first centrifugal speed, and its specific value needs to be determined according to the structure of the chip.

[0022] Preferably, in step (2), after the lysate enters the sample pool, control the biochip to rotate forward and backward alternately to fully mix the lysate and the sample, so as to fully extract bacteria in the sample. More preferably, the mixing time is 1 - 3 min.

[0023] Since the intensity of fluorescence decays very quickly, it is necessary to detect the fluorescence intensity in time after the fluorescence reaction. Preferably, it is detected within 5 min after the fluorescence reaction, and more preferably within 1 min.

[0024] In addition, the present invention also provides the application of the aforementioned biochip for bacterial detection in food in food detection.

[0025] Advantages of the present invention:

[0026] The bacterial detection biochip of the present invention has less reagent consumption and high efficiency. One chip can meet the online separation and parallel detection of multiple groups of samples.

[0027] The fluorescence detection method of the present invention has a fast detection speed, high accuracy, and high automation degree, and can realize on-site real-time detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 It is a schematic structural view of the first chip layer of the biochip for detecting bacteria in food according to an embodiment of the present invention;

[0029] Figure 2 It is a schematic structural view of the second chip layer;

[0030] Figure 3 is Figure 2 a schematic structural view of the detection unit in

[0031] Figure 4 a schematic structural view of the chip cover plate;

[0032] Figure 5 It is a schematic structural view of the fluorescence detector used in conjunction with the biochip for detecting bacteria in food of this embodiment;

[0033] Description of the reference numerals in the drawings:

[0034] 100, the first chip layer; 110, mounting holes;

[0035] 200, the second chip layer; 210, detection units; 211, lysis solution pool; 212, sample pool; 213, enzyme solution pool; 214, reaction pool; 215, breakthrough valve; 216, first microchannel; 217, second microchannel; 218, third microchannel;

[0036] 300, chip cover plate; 310, sample addition hole; 320, exhaust hole. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0037] The present invention will be further described below in conjunction with the drawings and specific embodiments, so that those skilled in the art can better understand the present invention and implement it, but the embodiments cited are not intended to limit the present invention.

[0038] Embodiment 1

[0039] Referring to Figures 1-4 As shown, an embodiment of the biochip for detecting bacteria of the present invention includes a chip substrate that can rotate around a center point and a chip cover plate 300 attached to the chip substrate. Six detection units 210 are evenly distributed around the center point on the chip substrate. Of course, in an alternative embodiment, the number of detection units 210 includes but is not limited to 6.

[0040] Each detection unit 210 includes a lysis solution pool 211, a sample pool 212, an enzyme solution pool 213, and a reaction pool 214 formed on a chip substrate. Among them, the reaction pool 214 is the farthest from the center point, and the distances of the lysis solution pool 211, the sample pool 212, and the reaction pool 214 from the center point increase in sequence. The lysis solution pool 211 is communicated with the sample pool 212 through a first microchannel 216, the sample pool 212 is communicated with the reaction pool 214 through a second microchannel 217, and the enzyme solution pool 213 is communicated with the reaction pool 214 through a third microchannel 218. Breakthrough valves 215 are provided on both the second microchannel 217 and the third microchannel 218.

[0041] In this embodiment, the chip substrate is in a disc shape and includes a first chip layer 100 and a second chip layer 200 which are mutually attached from bottom to top.

[0042] In this embodiment, the lysis solution pool 211, the sample pool 212, the enzyme solution pool 213, the reaction pool 214, and the breakthrough valves 215 all penetrate through the second chip layer 200, and the first microchannel 216, the second microchannel 217, and the third microchannel 218 are all flow channel grooves recessed on the upper surface of the second chip layer 200. In other embodiments, the lysis solution pool 211, the sample pool 212, the enzyme solution pool 213, the reaction pool 214, and the breakthrough valves 215 may be grooves recessed on the surface of the second chip layer 200 without penetrating through the second chip layer 200.

[0043] In this embodiment, the first chip layer 100 is a blank substrate, which is attached below the second chip layer 200 to block the through holes on the second chip layer 200, thereby forming the lysis solution pool 211, the sample pool 212, the enzyme solution pool 213, the reaction pool 214, and the breakthrough valves 215.

[0044] The chip cover plate 300 is provided with a plurality of sample addition holes 310 and exhaust holes 320. Each of the lysis solution pool 211, the sample pool 212, and the enzyme solution pool 213 is respectively communicated with a corresponding sample addition hole 310, and each of the lysis solution pool 211, the sample pool 212, the enzyme solution pool 213, and the reaction pool 214 is respectively communicated with a corresponding exhaust hole 320. Among them, samples are added to each pool through the sample addition holes 310, and the exhaust holes 320 are used to discharge the bubbles in the added sample solution.

[0045] In this embodiment, mounting holes 110 for mounting to a rotating shaft are provided at the centers of the first chip layer 100, the second chip layer 200, and the chip cover plate 300.

[0046] In this embodiment, the first microchannel 216 is a flow channel with a plurality of S-shaped bends. The bent flow channel can not only fully mix the lysis solution but also increase the dissolved oxygen amount in the lysis solution.

[0047] In the present invention, the first chip layer 100, the second chip layer 200 and the chip cover plate 300 may be made of PMMA, quartz or glass, preferably PMMA, and the three layers may be bonded together by thermal compression.

[0048] In the present invention, due to the characteristics of the breakthrough valve 215 itself, when the rotation speed of the biochip is lower than the critical rotation speed of the breakthrough valve 215, the passages between the sample pool 212 and the reaction pool 214, and between the enzyme solution pool 213 and the reaction pool 214 will not be conducted; when the rotation speed reaches or exceeds the critical rotation speed, the passages between the sample pool 212, the enzyme solution pool 213 and the reaction pool 214 are conducted, and the solution begins to enter the reaction pool 214. Therefore, when the rotation speed of the microfluidic chip is controlled to be lower than the critical rotation speed of the breakthrough valve 215, driven by centrifugal force, the lysate in the lysate pool 211 will flow into the sample pool 212 along the first microchannel 216 and mix with the sample, while at this time the passages between the sample pool 212, the enzyme solution pool 213 and the reaction pool 214 will not be conducted. Next, the rotation speed of the microfluidic chip is controlled to reach or exceed the critical rotation speed of the breakthrough valve 215. At this time, the passages among the sample pool 212, the enzyme solution pool 213 and the reaction pool 214 are opened, and the mixed solution in the sample pool 212 and the enzyme solution in the enzyme solution pool 213 enter the reaction pool 214 along the second microchannel 217 and the third microchannel 218 respectively, so that a fluorescent reaction occurs in the sample pool 212.

[0049] In this embodiment, the above-mentioned microfluidic channel, lysate pool 211, sample pool 212, enzyme solution pool 213, reaction pool 214 and breakthrough valve 215, injection hole and exhaust hole 320 can be prepared by computer numerical control grinding machine, laser etching, LIGA technology, molding, hot pressing, chemical corrosion, and can also be prepared by soft etching technology.

[0050] Example 2

[0051] This embodiment provides a food bacteria detection system, comprising the bacteria detection biochip described in Example 1 and Figure 5 The fluorescence detector shown in the figure. The fluorescence detector is provided with a detection position for placing the biochip, and has the functions of centrifugation and detection at the same time. When using the fluorescence detector, just put the chip with the sample on the detection position, set the centrifugation and detection parameters, and the centrifugation and detection can be automatically completed.

[0052] The above-described embodiments are only preferred embodiments for fully illustrating the present invention, and the protection scope of the present invention is not limited thereto. Equivalent substitutions or changes made by those skilled in the art based on the present invention are within the protection scope of the present invention. The protection scope of the present invention shall be subject to the claims.

Claims

1. A biochip for detecting bacteria in food, comprising a chip substrate that can rotate around a central point and a chip cover plate attached to the chip substrate. A plurality of detection units are arranged around the central point on the chip substrate. It is characterized in that the detection unit includes a lysis solution pool, a sample pool, an enzyme solution pool, and a reaction pool formed on the chip substrate. The reaction pool is the farthest from the central point, and the distances of the lysis solution pool, the sample pool, and the reaction pool from the central point increase in sequence. The lysis solution pool is connected to the sample pool through a first microchannel, the sample pool is connected to the reaction pool through a second microchannel, and the enzyme solution pool is connected to the reaction pool through a third microchannel. Breakthrough valves are provided on both the second microchannel and the third microchannel. A plurality of sample addition holes are provided on the chip cover plate, and each of the lysis solution pool, the sample pool, and the enzyme solution pool is connected to a corresponding sample addition hole.

2. The bacterial detection biochip for food according to claim 1, characterized in that, The chip substrate is in a disc shape, and a mounting hole for mounting on a rotating shaft is provided at its central part.

3. The bacterial detection biochip for food according to claim 1, characterized in that, A plurality of exhaust holes are further provided on the chip cover plate, and each of the lysis solution pool, the sample pool, the enzyme solution pool, and the reaction pool is connected to a corresponding exhaust hole.

4. The bacterial detection biochip for food according to claim 1, characterized in that, The first microchannel is a channel with a plurality of S-shaped bends.

5. The bacterial detection biochip for food according to claim 1, characterized in that, The chip substrate sequentially includes a first chip layer and a second chip layer that are attached to each other from bottom to top. The lysis solution pool, the sample pool, the enzyme solution pool, the reaction pool, and the breakthrough valve all penetrate through the second chip layer.

6. The bacterial detection biochip for food according to claim 5, wherein The first microchannel, the second microchannel, and the third microchannel are all channel grooves recessed on the upper surface of the second chip layer.

7. A bacterial detection system in food, characterized in that, It includes the biochip for detecting bacteria in food according to any one of claims 1-6 and a fluorescence detection device.

8. A method for fluorescence detection using the bacterial detection biochip for food according to any one of claims 1-6, characterized in that, It includes the following steps: (1) Add lysis solution, sample, and enzyme solution into the lysis solution pool, the sample pool, and the enzyme solution pool respectively through the sample addition holes. (2) Centrifuge the biochip for detecting bacteria in food at a first centrifugal speed so that the lysis solution enters the sample pool and mixes with the sample. (3) Centrifuge the biochip for detecting bacteria in food at a second centrifugal speed greater than the first centrifugal speed so that the enzyme solution in the enzyme solution pool and the mixed solution in the sample pool enter the reaction pool and a fluorescence reaction occurs. (4) Use a fluorescence detection device to detect the intensity of fluorescence.

9. The method for fluorescence detection according to claim 8, wherein, In step (2), after the lysis solution enters the sample pool, control the biochip to alternately rotate forward and backward to fully mix the lysis solution and the sample.

10. The application of the biochip for detecting bacteria in food according to any one of claims 1-6 in food detection.

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