Micro-fluidic chip, detection method and device for detecting 5-HMF (5-hydroxymethylfurfural)

Abstract

The invention provides a micro-fluidic chip, a detection method and a device for rapidly detecting 5-HMF (5-hydroxymethylfurfural), and relates to the technical field of biological detection. The method comprises the following steps: firstly, combining 5-HMF in a sample with a 5-HMF antibody site on an immunomagnetic bead chain; enabling the biotin to be combined with a 5-HMF antibody site which is not occupied by the 5-HMF on the immunomagnetic bead chain to form a complex of the immunomagnetic bead chain-5-HMF-biotin; then peroxidase is added into a channel of a micro-fluidic chip, and the peroxidase is specifically combined with biotin on the immunomagnetic bead chain-5-HMF-biotin complex, so that an immunomagnetic bead chain-5-HMF-biotin-peroxidase complex is formed; the peroxidase and the color developing agent are subjected to a color developing reaction; and judging the concentration of 5-HMF in the sample to be detected according to the chromogenic reaction. The technical effects of rapid, high-sensitivity, high-selectivity, low-cost and full-automatic detection of 5-HMF can be achieved, and the corresponding detection device has the characteristics of portability, wide popularization and low professional requirement on operators.

Description

Technical Field

[0001] The present invention relates to the field of biological detection technology, and in particular to a microfluidic chip, a detection method and a device for rapidly detecting 5-HMF. Background Art

[0002] Honey is widely used in food and medicine. However, during the honey refining process, the 5-hydroxymethylfurfural (5-HMF) content is very likely to increase significantly, triggering food and drug safety risks caused by excessive 5-HMF content.

[0003] For 5-HMF in refined honey, the 2020 version of the Chinese Pharmacopoeia stipulates that 5-HMF detection must use high-performance liquid chromatography. Although this method has the advantages of high sensitivity and good accuracy, it has high requirements for instruments and operators and is not suitable for widespread promotion. At the same time, due to the large size of the instrument, it does not have the advantage of being portable and is not suitable for instant rapid testing. In addition, the 2019 edition of the International Food Codex "Honey" standard stipulates the use of ultraviolet spectrophotometry for 5-HMF detection. Although this method has the advantages of simple method, low cost and fast speed, it has disadvantages. For complex samples, it is necessary to improve the accuracy and reliability of the method through pre-enrichment or chemical derivatization. Due to the complex pre-treatment steps, it is impossible to quickly detect the 5-HMF content.

[0004] Therefore, there is an urgent need for a method for detecting 5-HMF content with fast detection speed and low detection cost. Summary of the invention

[0005] In view of the above problems, the present invention provides a microfluidic chip, a detection method and a device for rapid detection of 5-HMF to solve at least one problem existing in the prior art.

[0006] To achieve the above object, the present invention provides a 5-HMF detection method based on a microfluidic chip, the method comprising:

[0007] Adding 5-HMF antibody-modified immunomagnetic beads into the channel of the microfluidic chip, wherein the immunomagnetic beads form an immunomagnetic bead chain at the main channel of the microfluidic chip under the action of the magnetic sensing structure;

[0008] Continuously injecting the sample to be tested into the channel of the microfluidic chip, so that 5-HMF in the sample binds to the 5-HMF antibody site on the immunomagnetic bead chain;

[0009] Passing biotin into the channel of the microfluidic chip, so that the biotin binds to the 5-HMF antibody sites on the immunomagnetic bead chain that are not occupied by 5-HMF, to form a complex of the immunomagnetic bead chain-5-HMF-biotin;

[0010] Adding peroxidase into the channel of the microfluidic chip, the peroxidase specifically binds to the biotin on the immunomagnetic bead chain-5-HMF-biotin complex to form an immunomagnetic bead chain-5-HMF-biotin-peroxidase complex;

[0011] The peroxidase and the color developer produce a color reaction; the concentration of 5-HMF in the sample to be tested is determined according to the color reaction.

[0012] Further, preferably, the biotin is 5-HMF-BSA-Sulfo-NHS-Biotin;

[0013] The peroxidase is horseradish peroxidase-streptavidin (HRP-Streptavidin, HRP-SA);

[0014] The color developer is 3,3',5,5'-tetramethylbenzidine (TMB) color developer.

[0015] The present invention also provides an application of the above-mentioned 5-HMF detection method based on a microfluidic chip in detecting the 5-HMF content in traditional Chinese medicine or food.

[0016] The present invention also provides a microfluidic chip for 5-HMF detection, comprising a chip channel for carrying immunomagnetic beads; the chip channel comprises a channel inlet, an inlet channel, a channel outlet and an outlet channel; the channel inlet and the channel outlet are respectively arranged at two ends of the microfluidic chip; the channel inlet is connected to the inlet channel, and the channel outlet is connected to the outlet channel; a detection hole and a main channel are arranged between the inlet channel and the outlet channel;

[0017] A magnetic sensing structure corresponding to the immunomagnetic beads is arranged below the microfluidic chip.

[0018] Further, preferably, the inlet channel and the outlet channel respectively form an angle of 20°-40° with the main channel.

[0019] Further, preferably, the material of the microfluidic chip is one of PDMS, PMMA, PP, COC, COP and PC.

[0020] Furthermore, preferably, the width of the main channel is 0.8-1.2 cm, and the depth is 150-250 μm; the width of the inlet channel and the outlet channel is 0.5-1 cm, and the depth is 150-250 μm.

[0021] The present invention also protects a small molecule compound detection device based on a microfluidic chip, which is used for automatic detection of small molecule compounds; including the microfluidic chip for 5-HMF detection as described above;

[0022] The magnetic induction structure is a magnetic grid separator; the magnetic grid separator includes a magnetic grid shell, a NdFeB magnet and a serrated iron sheet; a serrated iron sheet and at least two NdFeB magnets are arranged in a chamber formed by the magnetic grid shell; adjacent NdFeB magnets are arranged to repel each other and are separated by the serrated iron sheet.

[0023] Furthermore, preferably, it also includes lofting holes for the cylinder; the number of the lofting holes is 3 to 9.

[0024] Further, preferably, the Nd-boron magnet is a square sheet with a side length of 8-12 mm and a thickness of 0.5-1.5 mm; the serrated iron sheet is a square sheet with a side length the same as that of the Nd-boron magnet and a thickness of 0.4-0.6 mm.

[0025] The beneficial effects of the present invention are as follows:

[0026] The microfluidic chip, detection method and device for rapid detection of 5-HMF provided by the present invention utilize the principles of microfluidic immunomagnetic separation technology, antibody-antigen specific binding, and peroxidase-developer specific reaction; the technical effect of detecting the 5-HMF content in the target sample within 2 hours is achieved; compared with liquid chromatography, the 5-HMF detection method based on the microfluidic chip of the present invention has a more miniaturized detection device and is more portable; compared with ultraviolet spectrophotometry, because various specific binding principles are utilized, the influence of other substances with the same absorption wavelength in the complex material system on the detection result is avoided; moreover, the 5-HMF detection method based on the microfluidic chip of the present invention utilizes the fact that the microfluidic chip has less contact and transfer with the external environment during the detection process, thereby reducing the probability of cross contamination and sample loss; the small molecule compound detection device based on the microfluidic chip of the present invention can complete the full-automatic detection of the 5-HMF content, has low professional requirements for operators, and the detection environment is not limited to the laboratory, and can achieve clinical instant rapid detection requirements; it has high detection accuracy and high selectivity and is suitable for various samples such as Chinese patent medicine raw material powder and refined honey. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 A schematic structural diagram of a microfluidic chip for 5-HMF detection according to Example 1 of the present invention.

[0028] Figure 2 A schematic diagram of the circuit structure of a small molecule compound detection device based on a microfluidic chip according to an embodiment of the present invention.

[0029] Figure 3 A schematic structural diagram of a small molecule compound detection device based on a microfluidic chip according to an embodiment of the present invention.

[0030] Figure 4 A standard curve diagram for detecting 5-HMF according to one embodiment of the present invention.

[0031] in,

[0032] 1. Chip body; 2. Channel entrance; 3. Entrance channel; 4. Main channel; 5. Detection hole; 6. CCD; 7. Exit channel; 8. Channel exit; 9. Serrated iron sheet; 10. NdFeB magnet; 11. Circuit; 12. Power supply; 13. Power switch; 14. Voltage stabilizer; 15. Radiator; 16. TTL-485; 17. Peristaltic pump; 18. Buzzer; 19. Stepper motor linear guide; 20. Driver; 21. Relay; 22. Single motor; 23. Solenoid valve; 24. Control switch; 25. Diode; 26. Converter; 27. Computer; 28. Device housing; 29. ​​First to sixth sample holes; 30. Sample injection tube; 31. Waste liquid tube; 32. Waste liquid barrel. DETAILED DESCRIPTION

[0033] The present invention will be described in more detail with reference to the following examples. However, the protection scope of the present invention is not limited to the following examples.

[0034] Example 1

[0035] Figure 1 Schematic diagram of the structure of the microfluidic chip for 5-HMF detection is shown in FIG. Figure 1 As shown, the microfluidic chip for 5-HMF detection of the present invention comprises a chip channel for carrying immunomagnetic beads and a chip body 1; the chip channel comprises a channel inlet 2, an inlet channel 3, a channel outlet 8 and an outlet channel 7; the channel inlet 2 and the channel outlet 8 are respectively arranged at two ends of the microfluidic chip; the channel inlet 2 is connected to the inlet channel 3, and the channel outlet 8 is connected to the outlet channel 7; a detection hole 5 and a main channel 4 are arranged between the inlet channel 3 and the outlet channel 7; a magnetic sensing structure corresponding to the immunomagnetic beads is arranged under the microfluidic chip. Figure 1 Also shown is a CCD 6 for collecting channel information of the microfluidic chip, and the CCD is arranged just above the detection hole 5. Wherein, the material of the microfluidic chip is one of polydimethylsiloxane (PDMS), polymethyl methacrylate (PMMA), polypropylene (PP), cycloolefin copolymer (COC), cycloolefin polymer (COP) and polycarbonate (PC). That is to say, PDMS is preferably selected, and PDMS has the characteristics of excellent biocompatibility, transparency, flexibility, gas permeability, chemical stability and cost-effectiveness. In the specific implementation process, materials such as PP, COC, COP, PMMA and PC can be used as alternative materials.

[0036] That is to say, the microfluidic chip for 5-HMF detection of the present invention applies microfluidic immunomagnetic separation technology. The immunomagnetic separation technology is a process in which immunomagnetic beads in the chip channel and a magnetic sensing structure under the microfluidic chip are used to quickly separate and enrich the target.

[0037] In the specific implementation process, the magnetic induction structure is a magnetic grid separator; the magnetic grid separator includes a magnetic grid shell, a NdFeB magnet 10 and a serrated iron sheet 9; a serrated iron sheet 9 and at least two NdFeB magnets 10 are arranged in the chamber formed by the magnetic grid shell; adjacent NdFeB magnets 10 are arranged to repel each other and are separated by the serrated iron sheet 9. Two adjacent magnets repel each other and are separated by a serrated iron sheet. This structure can ensure that the magnetic field intensity at the tip of the serrated iron sheet is the strongest, and the immunomagnetic beads gather at the tip of the serrated iron sheet and form a vertical chain, which increases the contact area between the immunomagnetic beads and the sample and improves the capture and enrichment efficiency. Furthermore, the grade of the ultra-thin square NdFeB magnet is N52, and the NdFeB magnet is a square sheet with a side length of 8-12 mm and a thickness of 0.5-1.5 mm; the serrated iron sheet is a square sheet with a side length the same as that of the NdFeB magnet and a thickness of 0.4-0.6 mm. Preferably, the side length of the neodymium boron magnet is 10 mm, and the thickness is 1 mm; the side length of the serrated iron sheet is 10 mm, and the thickness is 0.5 mm, and the serration interval is 1 mm. By limiting the shape of the magnet and the iron sheet according to the size of the specific magnetic grid separator, a more accurate magnetic field can be provided for the immunomagnetic beads.

[0038] In the specific implementation process, the main channel is designed as a hexagonal structure; the inlet channel and the outlet channel form an acute angle with the main channel respectively; wherein the inlet channel and the outlet channel form an angle of 20°-40° with the main channel respectively; in order to further reduce the sample residue in the channel; preferably, the angle is 30°. The width of the main channel is 0.8-1.2cm, and the depth is 150-250μm; the width of the inlet channel and the outlet channel is 0.5-1cm, and the depth is 150-250μm. Preferably, the main channel is 2cm long, 1cm wide, and 200μm high; the width of the inlet channel and the outlet channel is 0.5-1mm, and the depth is 200μm.

[0039] Example 2

[0040] The present invention provides a small molecule compound detection device based on a microfluidic chip. Figure 2 and Figure 3 The overall description of the small molecule compound detection device based on microfluidic chip is given. Specifically, Figure 2 The present invention is a schematic diagram of the circuit structure of a small molecule compound detection device based on a microfluidic chip according to an embodiment of the present invention. Figure 3The figure is a schematic structural diagram of a small molecule compound detection device based on a microfluidic chip according to an embodiment of the present invention.

[0041] The present invention also protects a small molecule compound detection device based on a microfluidic chip, which is used for automatic detection of small molecule compounds; including the microfluidic chip for 5-HMF detection as described above; the microfluidic chip for 5-HMF detection includes a chip channel and a chip body 1 for carrying immunomagnetic beads; the chip channel includes a channel inlet 2, an inlet channel 3, a channel outlet 8 and an outlet channel 7; the channel inlet 2 and the channel outlet 8 are respectively arranged at two ends of the microfluidic chip; the channel inlet 2 is connected to the inlet channel 3, and the channel outlet 8 is connected to the outlet channel 7; a sample hole 5 and a main channel 4 are arranged between the inlet channel 3 and the outlet channel 7; a magnetic sensing structure corresponding to the immunomagnetic beads is arranged under the microfluidic chip.

[0042] That is to say, the small molecule compound detection device based on the microfluidic chip of the present invention is suitable for small molecule detection that uses microfluidic immunomagnetic separation technology to quickly separate and enrich target objects; that is to say, in addition to being used for 5-HMF content detection, the small molecule compound detection device based on the microfluidic chip can also be used for other similar small molecule compound content detection.

[0043] In order to realize the automated detection of small molecule compound detection devices based on microfluidic chips, such as Figure 2 and Figure 3 As shown, the specific implementation may include but is not limited to the following: the small molecule compound detection device based on the microfluidic chip includes a device housing 28, and a detection component disposed in the device housing 28; the detection component includes but is not limited to the first to sixth sample holes 29 for adding reagents, a sample injection tube 30 for connecting the sample holes and the microfluidic chip, and a peristaltic pump 17 is disposed on the sample injection tube 30; it also includes a waste liquid barrel 32 connected to the channel outlet 8 of the microfluidic chip, and a waste liquid pipe 31 for connecting the channel outlet 8 and the waste liquid barrel 32. A power switch 13 is also externally disposed on the device housing 28.

[0044] The device is divided into three modules, namely, a power module, a control module, and a detection module. The power module includes a power supply 12, a power switch 13, and a voltage-stabilized power supply 14. The power supply 12 is used to supply power to the device, and the voltage-stabilized power supply is used to stabilize the voltage of the device. The control module includes a switch 13, a radiator 15, a TTL-485 16, a peristaltic pump 17, a buzzer 18, a stepper motor linear guide 19, a driver 20, a relay 21, a single motor 22, a solenoid valve 23, a control switch 24, and a photodiode 25. The switch is used to control the start-up state of the control module, the peristaltic pump is used to push the liquid through the microfluidic channel, and the stepper motor linear guide is used to carry the magnetic grid to move under the chip to control the position of the immune magnetic beads in the chip. The detection module includes a CCD 6, a converter 26, and a computer 27. The CCD is used to convert the color in the detection hole into an optical signal, the converter is used to convert the optical signal in the CCD into an electrical signal, and the computer end is used to read the signal.

[0045] To achieve automation, the present device also needs to include a local processing and data module that can include an energy-saving processor or controller, and a digital memory, such as flash memory, both of which can be used to help process, cache and store data captured from a sensor operably coupled to the frame, such as a CCD capture device (e.g., a camera); and / or acquire and / or process using a remote processing module and / or a remote data repository, possibly for transmission to a display after such processing or retrieval. The local processing and data module is operably coupled, such as through a wired or wireless communication link, to the remote processing module and the remote data repository, so that these remote modules are operably coupled to each other and can be used as a resource for the local processing and data module. In one embodiment, the remote processing module may include one or more relatively powerful processors or controllers configured to analyze and process data and / or image information. In one embodiment, the remote data repository may include a relatively large-scale digital data storage facility that can be obtained through the Internet or other network configurations in a "cloud" resource configuration. In one embodiment, all data is stored and all calculations are performed in the local processing and data module, allowing full autonomous use from any remote module.

[0046] Furthermore, in order to improve the accuracy of 5-HMF content detection, the magnetic sensing structure is a magnetic grid separator; the magnetic grid separator includes a magnetic grid shell, a NdFeB magnet and a serrated iron sheet; a serrated iron sheet and at least two NdFeB magnets are arranged in the chamber formed by the magnetic grid shell; adjacent NdFeB magnets are arranged to repel each other and are separated by the serrated iron sheet. The NdBo magnet is a square sheet with a side length of 8-12mm and a thickness of 0.5-1.5mm; the serrated iron sheet is a square sheet with the same side length as the NdBo magnet and a thickness of 0.4-0.6mm. The preferred method is that the ultra-thin square NdFeB magnet in the magnetic grid separator is of grade N52, with a side length of 10mm and a thickness of 1mm, and the serrated iron sheet has a side length of 10mm, a thickness of 0.5mm, and a sawtooth spacing of 1mm. The sample holes of the small molecule compound detection device based on the microfluidic chip are cylindrical; the number of sample holes is 3 to 9. Preferably, there are 6. In the detection scenario of 5-HMF content, different concentrations of 5-HMF standard solutions are pre-placed in the first sample hole in sequence, the second sample hole is pre-placed with the detection honey sample, the third sample hole is pre-placed with phosphate buffer PBS as a cleaning solution, the fourth sample hole is pre-placed with 5-HMF-BSA-Sulfo-NHS-Biotin, the fifth sample hole is pre-placed with HRP-SA, and the sixth sample hole is pre-placed with TMB color developer. The sample hole has a diameter of 2 cm and a height of 5 cm; the sample injection tube and the waste liquid tube have a diameter of 2 mm and a length of 10 cm; the waste liquid bucket has a diameter of 5 cm and a height of 10 cm.

[0047] Example 3

[0048] Taking the detection of 5-HMF content in refined honey as an example, the above-mentioned microfluidic chip is used to detect 5-HMF. The microfluidic chip is composed of a 26mm×43mm glass bottom plate and a PDMS cover plate, and the glass bottom plate and the PDMS cover plate are bonded together by plasma.

[0049] The present invention provides a 5-HMF detection method based on a microfluidic chip, and the method comprises steps s110-s150.

[0050] s110, adding 5-HMF antibody-modified immunomagnetic beads into the channel of the microfluidic chip, and the immunomagnetic beads form immunomagnetic bead chains at the main channel of the microfluidic chip under the action of the magnetic sensing structure.

[0051] Specifically, the process of 5-HMF antibody-modified immunomagnetic beads is to wash the immunomagnetic beads with 2-(N-morpholino)ethanesulfonic acid (MES) buffer, then activate the immunomagnetic beads with 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS), bind the 5-HMF antibody to the carboxyl groups on the immunomagnetic beads, and fill the carboxyl vacancies on the immunomagnetic beads that are not bound to the 5-HMF antibody with 10% bovine serum albumin (BSA). The 5-HMF antibody can be modified on the magnetic beads in advance and can be stored in a 4°C refrigerator for 21 days. During the detection, the immunomagnetic beads modified with the 5-HMF antibody are first injected into the chip channel from the entrance hole, and the immunomagnetic beads in the main channel can form an immunomagnetic bead chain under the high gradient magnetic field formed by the magnetic grid below.

[0052] s120, continuously injecting the sample to be tested into the channel of the microfluidic chip, so that the 5-HMF in the sample binds to the 5-HMF antibody site on the immunomagnetic bead chain.

[0053] The first well is pre-placed with diluted 5-HMF standard solutions of different concentrations, which flow through the immunomagnetic bead chain under the action of the peristaltic pump. The 5-HMF in the solution is captured and enriched, and other interfering substances flow into the waste liquid bucket with the solution. Then the cleaning solution pre-placed in the third well flows through the microfluidic channel under the action of the peristaltic pump to flush the residual sample matrix to reduce interference.

[0054] s130, introducing biotin into the channel of the microfluidic chip, so that the biotin binds to the 5-HMF antibody sites on the immunomagnetic bead chain that are not occupied by 5-HMF, to form an immunomagnetic bead chain-5-HMF-biotin complex.

[0055] Then, the 5-HMF-BSA-Sulfo-NHS-Biotin pre-placed in the fourth sample hole is introduced into the chip channel to fill the 5-HMF antibody vacancies on the immunomagnetic beads that are not occupied by 5-HMF, forming an immunomagnetic bead-5-HMF-BSA-Sulfo-NHS-Biotin complex, and the excess 5-HMF-BSA-Sulfo-NHS-Biotin leaves the chip channel with the fluid.

[0056] s140, adding peroxidase into the channel of the microfluidic chip, the peroxidase specifically binds to the biotin on the immunomagnetic bead chain-5-HMF-biotin complex to form an immunomagnetic bead chain-5-HMF-biotin-peroxidase complex.

[0057] Then, HRP-SA in the fifth sample hole is introduced into the chip channel to form a complex of immunomagnetic beads-5-HMF-BSA-Sulfo-NHS-Biotin-SA-HRP. Excess HRP-SA leaves the chip channel with the fluid to reduce interference.

[0058] s150, the peroxidase and the color developer produce a color reaction; and the concentration of 5-HMF in the sample to be tested is determined according to the color reaction.

[0059] Then start the stepper motor to drive the magnetic grid to move to the bottom of the chip detection hole. Under the action of the magnetic field, the complex of immune magnetic beads-5-HMF-BSA-Sulfo-NHS-Biotin-SA-HRP is concentrated in the detection hole. Then introduce the TMB color developer in the sixth sample hole that can make HRP color. Finally, use CCD to irradiate the color of the detection hole. The converter converts the color signal into the corresponding value. At this time, the instrument buzzes and alarms, indicating that the detection is over. Then connect the computer software to read. The buzzer sounds again after the reading is over. Then the instrument starts to automatically clean the microfluidic chip channel. After the cleaning is completed, the instrument automatically stops. At this time, the magnetic grid stays on the right side of the slide rail, that is, below the detection hole of the chip. When the next detection starts, the magnetic grid resets and automatically moves to the bottom of the chip channel.

[0060] An example of repeated steps is as follows: the immunomagnetic beads modified with 5-HMF antibodies are injected into the chip channel from the inlet hole again, and the immunomagnetic beads in the main channel can form an immunomagnetic bead chain under the high gradient magnetic field formed by the magnetic grid below; then the refined honey sample liquid pre-placed in the second sampling hole flows through the immunomagnetic bead chain under the action of the peristaltic pump, and the 5-HMF in the sample is captured and enriched, and other interfering substances enter the waste liquid barrel with the liquid flow; then the cleaning solution pre-placed in the third sampling hole flows through the microfluidic channel under the action of the peristaltic pump to flush the residual sample matrix to reduce interference. Then, the 5-HMF-BSA-Sulfo-NHS-Biotin pre-placed in the fourth lofting hole is introduced into the chip channel to fill the 5-HMF antibody vacancy on the immunomagnetic beads that is not occupied by 5-HMF, forming an immunomagnetic bead-5-HMF-BSA-Sulfo-NHS-Biotin complex, and the excess 5-HMF-BSA-Sulfo-NHS-Biotin leaves the chip channel with the fluid, and then the HRP-SA in the fifth lofting hole is introduced into the chip channel to form an immunomagnetic bead-5-HMF-BSA-Sulfo-NHS-Biotin-SA-HRP complex, and the excess HRP-SA leaves the chip channel with the fluid to reduce interference. Then, the stepper motor is started to drive the magnetic grid to move to the bottom of the chip detection hole, and under the action of the magnetic field, the immunomagnetic bead-5-HMF-BSA-Sulfo-NHS-Biotin-SA-HRP complex is concentrated in the detection hole. Then, TMB colorimetric reagent is introduced into the sixth sample hole that can make HRP color. Finally, CCD is used to irradiate the color of the detection hole. The converter converts the colorimetric signal into a corresponding value. At this time, the instrument buzzes and alarms, indicating that the detection is over. Then the computer software is connected for reading. The buzzer sounds again when the reading is over. Then the instrument starts to automatically clean the microfluidic chip channel. After the cleaning is completed, the instrument automatically stops. At this time, the magnetic grid stays on the right side of the slide rail, that is, below the detection hole of the chip. When the next detection starts, the magnetic grid is reset and automatically moves to the bottom of the chip channel. Wherein, the biotin is 5-HMF-BSA-Sulfo-NHS-Biotin; the peroxidase is HRP-SA; and the colorimetric reagent is TMB colorimetric reagent.

[0061] The above steps were repeated to detect 5-HMF standards of different concentrations to obtain corresponding values ​​and draw a standard curve. Figure 4 The standard curve for detecting the dose-effect relationship between 5-HMF and absorbance. Figure 4As shown, by calculating the limit of detection (LOD), that is, when y is equal to the average value of y of the blank sample plus 3 times the standard deviation (SD) of y of the blank sample, the corresponding x, it is known that the LOD of the 5-HMF detection method based on the microfluidic chip of the present invention is 5.72ng; the linear range of detection is 5.72-160ng; through the above detection steps, it can be known that the present invention determines the content of 5-HMF in the refined honey sample, and the 5-HMF content is 54.9ng. The sample is verified by high performance liquid chromatography and the 5-HMF content in the sample is 52.6ng. It can be seen that the determination method of the present invention is used to determine the 5-HMF in the refined honey, which is of the same order of magnitude as the actual value. Moreover, the time to complete the detection of the 5-HMF content in the refined honey is less than 2h. The device using the microfluidic chip can realize rapid, high-sensitivity, high-selectivity, low-cost, and fully automatic detection of 5-HMF, and the device is portable and widely applicable, and has low professional requirements for operators, which provides a more portable and rapid detection method for detecting 5-HMF.

[0062] The present invention provides a method for rapid detection of 5-HMF based on a microfluidic chip and its application in the process of detecting the content of 5-HMF in refined honey. The above-mentioned 5-HMF detection method based on a microfluidic chip is suitable for 5-HMF detection in various scenarios, especially suitable for on-site rapid detection with high requirements for detection sensitivity and timeliness.

[0063] The main sources of reagents are as follows: 2-(N-morpholino)ethanesulfonic acid buffer (MES, Beijing Bio-Ray Biotechnology Co., Ltd.), 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC, Shanghai MacLean Biochemical Technology Co., Ltd.), N-hydroxysuccinimide (NHS, Shanghai MacLean Biochemical Technology Co., Ltd.), bovine serum albumin (BSA, Hebei Bailingwei Ultrafine Materials Co., Ltd.). Other reagents were imported and domestic analytical grade reagents.

[0064] Among the various experimental supplies mentioned in this article (including but not limited to: chemical reagents, biological products, cells, organisms, instruments, etc.), for those that are special or difficult to obtain, the manufacturers, references or detailed preparation methods have been indicated in the article; those not otherwise specified are conventional experimental supplies and can be easily obtained by various means (such as purchase, self-preparation, etc.) before the date of this application.

[0065] Although the present invention has been described in detail above by general description, specific implementation methods and experiments, it is obvious to those skilled in the art that some modifications or improvements can be made to the present invention. Therefore, these modifications and improvements made without departing from the spirit of the present invention all belong to the scope of protection claimed by the present invention.

Claims

1. A 5-HMF detection method based on a microfluidic chip, characterized in that: Methods include: Adding 5-HMF antibody-modified immunomagnetic beads into the channel of the microfluidic chip, wherein the immunomagnetic beads form an immunomagnetic bead chain at the main channel of the microfluidic chip under the action of the magnetic sensing structure; Continuously injecting the sample to be tested into the channel of the microfluidic chip, so that 5-HMF in the sample binds to the 5-HMF antibody site on the immunomagnetic bead chain; Passing biotin into the channel of the microfluidic chip, so that the biotin binds to the 5-HMF antibody sites on the immunomagnetic bead chain that are not occupied by 5-HMF, to form a complex of the immunomagnetic bead chain-5-HMF-biotin; Adding peroxidase into the channel of the microfluidic chip, the peroxidase specifically binds to the biotin on the immunomagnetic bead chain-5-HMF-biotin complex to form an immunomagnetic bead chain-5-HMF-biotin-peroxidase complex; The peroxidase and the color developer produce a color reaction; the concentration of 5-HMF in the sample to be tested is determined according to the color reaction.

2. The 5-HMF detection method based on a microfluidic chip according to claim 1, characterized in that: The biotin is 5-HMF-BSA-Sulfo-NHS-Biotin; The peroxidase is horseradish peroxidase-streptavidin; The color developer is TMB color developer.

3. An application of the 5-HMF detection method based on a microfluidic chip as claimed in claim 1 or 2 in detecting 5-HMF content in traditional Chinese medicine or food.

4. A microfluidic chip for 5-HMF detection, characterized in that: The invention comprises a chip channel for carrying immunomagnetic beads; the chip channel comprises a channel inlet, an inlet channel, a channel outlet and an outlet channel; the channel inlet and the channel outlet are respectively arranged at two ends of the microfluidic chip; the channel inlet is connected to the inlet channel, and the channel outlet is connected to the outlet channel; a detection hole and a main channel are arranged between the inlet channel and the outlet channel; A magnetic sensing structure corresponding to the immunomagnetic beads is arranged below the microfluidic chip.

5. The microfluidic chip for 5-HMF detection according to claim 4, characterized in that: The inlet channel, the outlet channel and the main channel respectively form an angle of 20°-40°.

6. The microfluidic chip for 5-HMF detection according to claim 4, characterized in that: The material of the microfluidic chip is one of PDMS, PMMA, PP, COC, COP and PC.

7. The microfluidic chip for 5-HMF detection according to claim 4, characterized in that: The width of the main channel is 0.8-1.2 cm, and the depth is 150-250 μm; the width of the inlet channel and the outlet channel is 0.5-1 cm, and the depth is 150-250 μm.

8. A small molecule compound detection device based on a microfluidic chip, characterized in that: Used for automatic detection of small molecule compounds; including the microfluidic chip for 5-HMF detection as claimed in claim 4; The magnetic induction structure is a magnetic grid separator; the magnetic grid separator includes a magnetic grid shell, a NdFeB magnet and a serrated iron sheet; a serrated iron sheet and at least two NdFeB magnets are arranged in a chamber formed by the magnetic grid shell; adjacent NdFeB magnets are arranged to repel each other and are separated by the serrated iron sheet.

9. The small molecule compound detection device based on a microfluidic chip according to claim 8, characterized in that: It also includes cylindrical lofting holes; the number of the lofting holes is 3 to 9.

10. The small molecule compound detection device based on microfluidic chip according to claim 8, characterized in that: The Nd-boron magnet is a square sheet with a side length of 8-12 mm and a thickness of 0.5-1.5 mm; the serrated iron sheet is a square sheet with a side length the same as that of the Nd-boron magnet and a thickness of 0.4-0.6 mm.

Images