A rapid detection method of nitrite concentration gradient colorimetry using microfluidic technology

Abstract

The present invention discloses a nitrite microfluidic concentration gradient colorimetric rapid detection method, which is based on a microfluidic chip, a colorimetric signal acquisition box and a mobile terminal carrying a color recognition APP. The microfluidic chip is used to automatically obtain multiple concentration gradients of nitrite, and the nitrites are mixed and incubated with AuNPs amplification liquid in a detection chamber, and AuNPs are added to generate colorimetric amplified signals of different red intensities. The colorimetric signals are collected and analyzed by a mobile terminal carrying a color recognition APP. The microfluidic concentration gradient colorimetric detection system based on nitrite inhibiting AuNPs signal amplification of the present invention can detect nitrite as low as 0.425 mg / L within 40 minutes, and has good specificity; rapid, on-site, equipment-free, and visual quantitative detection of nitrite is realized.

Description

Technical Field

[0001] The invention belongs to the technical field of colorimetric detection, and in particular relates to a nitrite microfluidic concentration gradient colorimetric rapid detection method. Background Art

[0002] Nitrite is an important intermediate in the natural nitrogen cycle and is commonly found in human environments such as surface water, soil and food. Nitrite is widely used as an additive, preservative, dye, bleaching agent, fertilizer or vasodilator in food, industry, agriculture and physiological systems. However, nitrite can bind to hemoglobin to form methemoglobin, leading to methemoglobinemia (blue baby syndrome), and can also react with secondary amines under acidic conditions to form strong carcinogens N-nitrosamines, leading to gastric cancer. In addition, excessive or long-term intake of nitrite is more harmful to human health. Cumulative intake of 0.3-0.5 mg of nitrite can cause food poisoning, and more than 3 g can cause death. The World Health Organization (WHO) and the United States Environmental Protection Agency (USEPA) limit the maximum content of nitrite in drinking water to 3 mg / L and 1 mg / L, respectively. Therefore, high sensitivity and rapid detection of nitrite play a vital role in protecting the natural environment and consumer health.

[0003] Currently, spectrophotometry, high-performance ion exchange chromatography, high-throughput headspace gas chromatography and capillary electrophoresis have played an important role in the detection of nitrite. However, these methods require large, expensive and bulky instruments, long analysis time and professional technicians, which limits their practical application. In recent years, methods based on colorimetry, fluorescence, electrochemistry or surface enhanced Raman spectroscopy (SERS) have also been used in the detection of nitrite. Although fluorescence, electrochemistry, SERS and other methods have high detection sensitivity, they all require cumbersome signal acquisition and conversion, and the stability and reproducibility of the methods are relatively poor, which also limits their application in on-site, real-time and rapid detection. The colorimetric method based on the diazo reaction of nitrite with other chemical reagents is the simplest and most accurate method for detecting nitrite. However, due to the low extinction coefficient of organic dyes, the sensitivity of this method is relatively poor.

[0004] Gold nanoparticles (AuNPs) have a high extinction coefficient due to their localized surface plasmon resonance properties. Currently, AuNPs have become the best candidate for colorimetry. AuNPs can act as their own catalysts, transferring and receiving electrons to AuNPs to amplify the Au in the solution. + , and then the reduced Au 0 Wrapped on the surface of AuNPs, signal amplification is then achieved. Nitrite can inhibit the signal amplification of AuNPs. Based on this principle, rapid and highly sensitive colorimetric detection of nitrite can be achieved in two steps without labeling.

[0005] In addition, the establishment of all detection methods requires the establishment of a linear standard curve, which in turn requires the quantitative detection of a series of samples with different concentration gradients. However, manually preparing a series of samples with equal concentration gradients in different proportions is time-consuming and laborious. Microfluidic chip technology has become the most popular technology for automatically generating chemical and physical concentration gradients due to its precise control of flow rate and stable spatiotemporal distribution. In the past few decades, microfluidic concentration gradient chips have developed from T-type channels to Christmas tree-shaped channels, and then evolved into channels with various improved configurations. These microfluidic concentration gradient chips have been used as important research tools for drug screening or evaluation, antimicrobial susceptibility testing, crystal nucleation and growth, cell microenvironment, etc. However, these microfluidic concentration gradient chips generally take a long time to form concentration gradients, the stability of the concentration gradient is easily affected by small fluctuations in flow or pressure, fluid drive requires precision devices, and the channel structure is cumbersome, so they have not yet been widely used in the quantitative detection of targets.

[0006] Therefore, how to provide a microfluidic concentration gradient colorimetric rapid detection method for nitrite is an urgent problem to be solved by those skilled in the art. Summary of the invention

[0007] In view of this, the present invention provides a nitrite microfluidic concentration gradient colorimetric rapid detection method, characterized in that it comprises the following steps:

[0008] Step 1: Hardware preparation: prepare a microfluidic chip, a colorimetric signal acquisition box, and a mobile terminal with a color recognition APP. The microfluidic chip is placed on a mobile platform inside the colorimetric signal acquisition box. A collection hole is set on the top of the colorimetric signal acquisition box. The camera of the mobile terminal collects the colorimetric information of the microfluidic chip on the mobile platform through the collection hole.

[0009] Step 2: A plurality of microchannels are arranged in parallel and spaced apart on the microfluidic chip, and two adjacent microchannels have a volume difference. Samples are injected into the plurality of microchannels to form different concentration gradient values. Detection holes are respectively provided on the microfluidic chip corresponding to different microchannels, and the samples in the plurality of microchannels are pushed into the corresponding detection holes by air pressure;

[0010] Step 3: Push diluent PB into the detection well, then add 200 μL AuNPs amplification solution. After 20 minutes of reaction, add 35 μL AuNPs, and the detection chamber will produce colorimetric amplification signals of different red intensities.

[0011] Step 4: Collect the colorimetric signal of the amplified AuNPs through the color recognition APP of the mobile terminal.

[0012] The beneficial effects of the present invention are as follows: the colorimetric information on the microfluidic chip is collected based on the color recognition APP of the mobile terminal, and the nitrite concentration in the sample can be quickly obtained by comparing with the analysis results of the multifunctional microplate reader, so as to realize fast and reliable gradient quantitative detection of nitrite. The present invention is based on the principle that nitrite inhibits the signal amplification of AuNPs. In the test, the nitrite sample is automatically diluted into a plurality of different concentration gradients by the microfluidic chip, and then the nitrites of the plurality of concentration gradients obtained after dilution are mixed and incubated with the AuNPs amplification solution in the detection chamber; then, AuNPs are added to generate colorimetric amplified signals of different red intensities. Finally, the colorimetric signal is captured and analyzed by the colorimetric signal acquisition box and the color recognition APP on the mobile terminal, so as to realize the gradient quantitative detection of nitrite.

[0013] Preferably, the microfluidic chip is stacked with a surface layer, a channel layer, a detection layer and a bottom layer in sequence from top to bottom, injection ports are opened at both end edges of the surface layer, a plurality of detection holes are staggeredly arranged on the surface layer and the plurality of detection holes are located on the same oblique line, the detection holes penetrate downward through the channel layer and the detection layer, microchannel lumens are horizontally arranged on the channel layer and on both sides corresponding to each detection hole, there is a length difference between two adjacent microchannel lumens on one side of the detection hole, sampling pipes are arranged at both ends of the channel layer, one end of the sampling pipe is connected to the injection port, and the other end of the sampling pipe is connected to the pipelines between the plurality of microchannel lumens on the corresponding side.

[0014] Preferably, the surface layer, channel layer, detection layer and bottom layer are all made of polymethyl methacrylate, the thickness of the surface layer, channel layer and bottom layer is 1 mm, the thickness of the detection layer is 3 mm, the length difference between two adjacent microchannel lumens on the same side of the detection hole is 10 mm, the total length of the microchannel lumens on both sides of each detection hole is 70 mm, the length of the two microchannel feed lumens connected to the detection chamber is 4.6 mm, and the detection hole cooperates with the bottom layer to form a detection chamber, which has a diameter of 10 mm and a depth of 5 mm.

[0015] Preferably, in step three, the colorimetric quantitative detection of nitrite is achieved based on a colorimetric method in which nitrite inhibits AuNPs signal amplification.

[0016] Preferably, a mobile platform is provided inside the colorimetric signal acquisition box, the concentration gradient dilution chip is placed on the mobile platform, LED lights with adjustable brightness are arranged on the two inner side walls of the colorimetric signal acquisition box, the mobile terminal is a smart phone, and the camera of the smart phone corresponds to the collection hole and collects the colorimetric detection information on the concentration gradient dilution chip.

[0017] The beneficial effects of the present invention are: rapid detection of test samples is achieved based on the microfluidic chip, the colorimetric signal acquisition box and the mobile terminal. The detection principle is a colorimetric method based on nitrite inhibiting AuNPs signal amplification, which can specifically realize the quantitative detection of nitrite in river and lake water samples. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 A hardware structure diagram of a nitrite microfluidic concentration gradient colorimetric rapid detection method used in the present invention;

[0019] Figure 2 The overall structure diagram of the microfluidic chip used in the present invention;

[0020] Figure 3 A hierarchical structure diagram of the microfluidic chip used in the present invention;

[0021] Figure 4 The UV-visible spectrum of the nitrite-inhibited AuNPs signal amplification of the present invention;

[0022] Figure 5 This is the mechanism by which nitrite inhibits AuNPs signal amplification in the present invention;

[0023] Figure 6 This is a comparison diagram of the theoretical concentration and the actual concentration of the sample in the eight parallel channels of the microfluidic chip verified by the protein quantification kit of the present invention;

[0024] Figure 7 The sensitivity and selectivity of the colorimetric method designed for the present invention for detecting nitrite based on inhibiting AuNPs signal amplification;

[0025] Figure 8 This is a result diagram of detecting nitrite using the UV-Vis based microfluidic concentration gradient colorimetry method of the present invention;

[0026] Fig. 9 This is a diagram of the microfluidic concentration gradient colorimetric detection results of nitrite under different light source conditions of the present invention.

[0027] 1 surface layer, 2 channel layer, 3 detection layer, 4 bottom layer, 5 injection port, 6 detection hole, 7 microchannel lumen, 8 sampling pipeline, 9 microchannel material passing lumen, 10 microvalve, 11 outlet, 12 drainage cavity, 13 hose microvalve, 14 colorimetric signal acquisition box, 15 mobile detector, 16 LED lamp, 17 mobile platform. DETAILED DESCRIPTION

[0028] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. 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.

[0029] See the attached Figures 1 to 9 According to an embodiment of the present invention, a nitrite microfluidic concentration gradient colorimetric rapid detection method comprises the following steps:

[0030] Step 1: Hardware preparation: prepare a microfluidic chip, a colorimetric signal acquisition box, and a mobile terminal with a color recognition APP. The microfluidic chip is placed on a mobile platform inside the colorimetric signal acquisition box. A collection hole is set on the top of the colorimetric signal acquisition box. The camera of the mobile terminal collects the colorimetric information of the microfluidic chip on the mobile platform through the collection hole.

[0031] Step 2: A plurality of microchannels are arranged in parallel and spaced apart on the microfluidic chip, and two adjacent microchannels have a volume difference. Samples are injected into the plurality of microchannels to form different concentration gradient values. Detection holes are respectively provided corresponding to different microchannels on the microfluidic chip, and the samples in the plurality of microchannels are pushed into the corresponding detection holes by air pressure;

[0032] Step 3: Push diluent PB into the detection well, then add 200 μL AuNPs amplification solution. After reacting for 20 minutes, add 35 μL AuNPs.

[0033] Step 4: Collect the colorimetric signal of the amplified AuNPs through the color recognition APP of the mobile terminal.

[0034] Reference Figure 3 , 3a is the surface layer; 3b is the channel layer, 3c is the detection layer; 3d is the bottom layer.

[0035] The specific structure of the microfluidic chip is as follows: a surface layer 1, a channel layer 2, a detection layer 3 and a bottom layer 4 are stacked in sequence from top to bottom, a total of four layers, injection ports 5 are opened at the edges of both ends of the surface layer 1, a plurality of detection holes 6 are staggeredly arranged on the surface layer 1 and the plurality of detection holes 6 are located on the same oblique line, the detection holes 6 penetrate the channel layer 2 and the detection layer 3 downward, microchannel lumens 7 are horizontally arranged on the channel layer 2 and on both sides corresponding to each detection hole 6, there is a length difference between two adjacent microchannel lumens 7 on one side of the detection hole 6, and sampling pipes 8 are arranged at both ends of the channel layer 2, one end of the sampling pipe 8 is connected to the injection port 5, and the other end of the sampling pipe 8 is connected to the pipelines between the plurality of microchannel lumens 7 on the corresponding side.

[0036] The specific process of quantitative detection of nitrite by microfluidic chip is as follows: close outlet c and microvalve b, and inject the sample into 8 parallel microchannel lumens from the injection port. Close microvalve a, and discharge the excess sample between microvalve a and the injection port from outlet c. Close outlets c and d, open microvalves a and b, and push the sample in the channel into the detection hole by air pressure. In the same way, push the diluent PB into the detection hole. Then add 200μL AuNPs amplification solution. After 20 minutes of reaction, add 35μL AuNPs. Finally, the UV-Vis and solution color change images of the amplified AuNPs were collected by a multifunctional microplate reader and a smart phone, respectively, and the results of the multifunctional microplate reader detection were used for comparison.

[0037] More specifically, the surface layer 1, the channel layer 2, the detection layer 3 and the bottom layer 4 are all made of polymethyl methacrylate, the thickness of the surface layer 1, the channel layer 2 and the bottom layer 4 is 1 mm, the thickness of the detection layer 3 is 3 mm, the length difference between two adjacent microchannel lumens on the same side of the detection hole 6 is 10 mm, the total length of the microchannel lumens 7 on both sides of each detection hole 6 is 70 mm, the length of the two microchannel feed lumens connected to the detection hole is 4.6 mm, the diameter of the detection hole 6 is 10 mm, and the depth is 5 mm.

[0038] The present invention consists of a microfluidic chip, a colorimetric signal acquisition box, and a smart phone with a color recognition APP. It uses a microfluidic chip to perform automatic gradient dilution and colorimetric detection of nitrite samples; specifically, nitrite with an initial concentration of C0 can be automatically diluted to 0, 0.20C0, 0.33C0, 0.46C0, 0.59C0, 0.72C0, 0.86C0 and C08 concentration gradients on the microfluidic chip. Subsequently, the nitrites of the eight concentration gradients obtained after dilution are mixed and incubated with AuNPs amplification liquid in a detection chamber respectively; then, AuNPs are added to generate colorimetric amplified signals of different red intensities. Finally, the colorimetric signal is captured and analyzed by a colorimetric signal acquisition box and a smart phone, thereby realizing gradient quantitative detection of nitrite. The colorimetric signal is inversely proportional to the content of nitrite in the sample.

[0039] The specific process of colorimetric quantitative detection of nitrite is as follows: manually prepare different concentrations of nitrite standard samples in the range of 0 to 12.94 mg / L; then add different concentrations of nitrite standards to 200 μL AuNPs amplification solution. After 20 minutes of reaction, add 35 μL AuNPs. Finally, the UV-Vis spectrum of the amplified AuNPs and the color change of the solution are collected by a multifunctional microplate reader and a smartphone, respectively.

[0040] In other embodiments, in step three, colorimetric quantitative detection of nitrite is achieved based on a colorimetric method in which nitrite inhibits AuNPs signal amplification.

[0041] Reference to the mechanism of nitrite inhibiting AuNPs signal amplification Figure 4-5 HAuCl4 itself has no obvious absorption peak. 3+ Can be directly reduced to Au by AA 0 , so there is an absorption peak at 550nm. 4 After adding NaNO2 to the reaction solution with AA, the absorbance of the absorption peak at 550nm decreased significantly, indicating that NO2 - It has strong oxidizing property under acidic conditions, which can inhibit AA from converting Au to 3+ Reduction to Au 0 When HAuCl4 combines with the stabilizer CTAB to form CTA + [AuBr4]-, due to ligand exchange, there are obvious absorption peaks at 390nm and 460nm. When AA is added, CTA + Au in [AuBr4]- 3+ Can be quickly restored to Au + , and there is no obvious absorption peak in the solution.

[0042] AuNPs act as a self-catalyst that can transfer and receive electrons to Au + When there is no nitrite in the sample, Au + Reduced to Au 0 . Due to Au 0 When nitrite is present in the sample, it may hinder the electron transfer to Au due to its strong oxidizing effect in acidic solution. + Since there is no obvious absorption peak, there may be Au in the solution. + or Au 2+ Therefore, the particle size of AuNPs will not increase. In other words, nitrite can inhibit the signal amplification of AuNPs.

[0043] In the present invention, a mobile platform 17 is provided inside the colorimetric signal acquisition box 14, and a concentration gradient dilution chip is placed on the mobile platform 17. LED lights 16 with adjustable brightness are arranged on the two inner side walls of the colorimetric signal acquisition box 14. The mobile terminal 15 is a smart phone, and the camera of the smart phone corresponds to the collection hole and collects the colorimetric detection information on the concentration gradient dilution chip.

[0044] The purpose of setting up the colorimetric signal acquisition box is to avoid the influence of external environment on the colorimetric analysis results. Fig. 9 , attached Fig. 9Figure 9a shows the result of detecting 10 mg / L nitrite by microfluidic concentration gradient colorimetry based on smartphone APP under natural light; Figure 9b shows the result of detecting 5 mg / L nitrite. Figure 9c shows the result of detecting 10 mg / L nitrite using a signal acquisition box; Figure 9d shows the result of detecting 5 mg / L nitrite.

[0045] The present invention verifies the set concentration:

[0046] Since the volume of a 10 mm long microchannel is about 8 μL, the internal volume of the two stainless steel tubes (5 mm long, 1 mm outer diameter, 0.5 mm inner diameter) at the interfaces at both ends of the microchannel is about 1.96 μL. Therefore, the theoretical volume gradient values ​​of the sample and PB in the 8 parallel microchannels are 0, 10, 18, 26, 34, 42, 50, 58 μL and 58, 50, 42, 34, 26, 18, 10, 0 μL, respectively. When the concentration of the sample is set to C0, the theoretical concentration gradient values ​​of the sample in the 8 parallel microchannels are 0, 0.17C0, 0.30C0, 0.43C0, 0.57C0, 0.70C0, 0.83C0 (Table 1), respectively, and the concentration gradient difference (C n+1 / C0-C n / C0) is approximately 0.13C0.

[0047] In order to obtain the actual concentration gradient values ​​of the 8 parallel microchannels, we used the designed microfluidic concentration gradient chip and protein quantification kit to quantitatively measure 250μg / mL BSA. A linear standard curve was established with the theoretical concentration gradient of the 250μg / mL BSA standard. The actual concentration gradient detection results of 250μg / mL BSA detected by the designed microfluidic concentration gradient chip are shown in Table 2. The actual concentration gradient values ​​of the 8 parallel microchannels were -7.600, 49.956, 81.956, 115.511, 147.289, 178.844, 213.956 and 242.622μg / mL, respectively. Therefore, the actual concentration gradients of the samples in the 8 parallel microchannels were -0.03C0, 0.20C0, 0.33C0, 0.46C0, 0.59C0, 0.72C0, 0.86C0 and 0.97C0, respectively. C n+1 / C0-C n / C0 is also approximately 0.13C0.

[0048] Comparing the theoretical value with the actual value, we found that the actual concentration of the 8 parallel microchannels was close to the theoretical value, C n+1 / C0-C n The / C0 values ​​are all around 0.13. These results show that the samples form a good concentration gradient in the 8 parallel microchannels.

[0049] Table 1 Theoretical concentration values ​​in 8 parallel microchannels

[0050]

[0051] Table 2 Actual concentration gradient values ​​of 8 parallel microchannels

[0052]

[0053] Reference Figure 7 , 7a is the UV-Vis spectra of the enlarged AuNPs under different nitrite concentrations; 7b is the absorbance value of the enlarged AuNPs under different nitrite concentrations; 7c is the UV-Vis spectra of the enlarged AuNPs under different interfering substances; 7d is the absorbance value of the enlarged AuNPs under different interfering substances.

[0054] In order to investigate the sensitivity of the established colorimetric detection method based on inhibiting AuNPs signal amplification, standard nitrite samples with different concentrations were manually prepared in different proportions within the range of 0-12.94 mg / L for detection. - With the increase of concentration, the absorbance decreased significantly, and the red intensity of the amplified AuNPs solution decreased rapidly. - The concentration ranges from 0 to 6.90 mg / L and shows a good linear relationship. The linear equation is Ab = -0.077C + 0.726, and the correlation coefficient is R 2 It is 0.993, and the detection limit is 0.164 mg / L.

[0055] In addition to sensitivity, the selectivity of the detection method also plays a crucial role in practical applications. Therefore, in order to examine its selectivity, we further applied the colorimetric detection method to detect a variety of possible interfering substances in the water environment, including PO4 2- 、NO3 - , Cl - 、C5H7O5COO - 、H2BO3 - 、SO4 2- Mg 2+ 、CO3 2- , K + , Fe 2+ and NH4 + The concentration of these interfering substances is NO2 - The concentration is 10 times higher. But from the test results Figure 7 c and 7d, the absorbance values ​​of these interfering substances were similar to those of the control group. Therefore, these results clearly showed that the established colorimetric method was effective for NO2 - The detection has good selectivity.

[0056] Attached Figure 8 In the figure, A1, B1, C1, D1, E1, F, G and H are the UV-Vis spectra of AuNPs in 8 parallel microchannel detection chambers when the nitrite concentration is 10, 5, 2.5, 1.25, 0.63, 0.31, 0.156 and 0.08 mg / L, respectively. A2, B2, C2, D2 and E2 are the absorbance of AuNPs solution in 8 parallel microchannel detection chambers when the nitrite concentration is 10, 5, 2.5, 1.25 and 0.63 mg / L, respectively. I1 and I2 are the UV-Vis spectra and absorbance values ​​of AuNPs solution in 8# microchannel when the nitrite concentration is 0.078~10 mg / L, respectively.

[0057] Under natural light, the detection results of the microfluidic concentration gradient chip with initial nitrite concentrations of 10 mg / L and 5 mg / L were collected and processed by a smartphone app. Fig. 9 a and 9b. In the 8 parallel channels of the microfluidic chip, as the gradient dilution of nitrite from 0 to C0, the red intensity and HSB (%) of the amplified AuNPs solution gradually decreased. A good linear relationship was also observed in the concentration ranges of 2-8.6 mg / L and 1.65-5 mg / L. The linear equations were HSB (%) = -14.194lgC + 20.292 and HSB (%) = -1.720C + 18.219, respectively, with a correlation coefficient R 2 They are 0.997 and 0.994, respectively. However, there is a large and bright light spot in the acquired image, which will affect the acquisition of the colorimetric signal;

[0058] After using the signal acquisition box, the microfluidic concentration gradient colorimetric detection method further detected 10 mg / L and 5 mg / L nitrite. Fig. 9 As shown in Fig. 9c and 9d, the light spot on the captured image is very small, so it hardly affects the acquisition of the colorimetric signal. A good linear relationship was also observed in the range of 2-8.6 mg / L and 1.65-5 mg / L. The linear equations are HSB(%) = -28.496lgC + 254.08 and HSB(%) = -4.27C + 252.41, and the correlation coefficient R 2 They are 0.998 and 0.992 respectively. The detection limit is 0.425 mg / L.

[0059] As for the device and the method of use disclosed in the embodiment, since they correspond to the method disclosed in the embodiment, the description is relatively simple, and the relevant parts can be referred to the description of the method part.

[0060] The above description of the disclosed embodiments enables those skilled in the art to implement or use the present invention. Various modifications to these embodiments will be apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but rather to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A nitrite microfluidic concentration gradient colorimetric rapid detection method, characterized in that: The following steps are involved: Step 1: Hardware preparation: prepare a microfluidic chip, a colorimetric signal acquisition box, and a mobile terminal with a color recognition APP. The microfluidic chip is placed on a mobile platform inside the colorimetric signal acquisition box. A collection hole is set on the top of the colorimetric signal acquisition box. The camera of the mobile terminal collects the colorimetric information of the microfluidic chip on the mobile platform through the collection hole. Step 2: A plurality of microchannels are arranged in parallel and spaced apart on the microfluidic chip, and two adjacent microchannels have a volume difference. Samples are injected into the plurality of microchannels to form different concentration gradient values. Detection holes are respectively provided on the microfluidic chip corresponding to different microchannels, and the samples in the plurality of microchannels are pushed into the corresponding detection holes by air pressure; Step 3: Push diluent PB into the detection well, and then add 200μL AuNPs amplification solution. The AuNPs amplification solution is The formula is: After 20 min of reaction, 35 μL of AuNPs were added, and colorimetric amplified signals of different red intensities were generated in the detection chamber; Step 4: Collect the colorimetric signal of the amplified AuNPs through the color recognition APP of the mobile terminal.

2. A nitrite microfluidic concentration gradient colorimetric rapid detection method according to claim 1, characterized in that: The microfluidic chip is stacked in order from top to bottom with a surface layer (1), a channel layer (2), a detection layer (3) and a bottom layer (4); both ends of the surface layer (1) are provided with injection ports (5); a plurality of detection holes (6) are staggeredly arranged on the surface layer (1) and the plurality of detection holes (6) are located on the same oblique line; the detection holes (6) penetrate downward through the channel layer (2) and the detection layer (3); microchannel lumens (7) are horizontally arranged on both sides of the channel layer (2) and corresponding to each detection hole (6); there is a length difference between two adjacent microchannel lumens (7) on one side of the detection hole (6); and injection pipes (8) are arranged at both ends of the channel layer (2); one end of the injection pipe (8) is connected to the injection port (5); and the other end of the injection pipe (8) is connected to a plurality of microchannel lumens (7) on the corresponding side.

3. A nitrite microfluidic concentration gradient colorimetric rapid detection method according to claim 2, characterized in that: The surface layer (1), channel layer (2), detection layer (3) and bottom layer (4) are all made of polymethyl methacrylate. The thickness of the surface layer (1), channel layer (2) and bottom layer (4) is 1 mm. The thickness of the detection layer (3) is 3 mm. The length difference between two adjacent microchannel lumens on the same side of the detection hole (6) is 10 mm. The total length of the microchannel lumens (7) on both sides of each detection hole (6) is 70 mm. The length of the two microchannel feed lumens connected to the detection chamber is 4.6 mm. The detection hole cooperates with the bottom layer to form a detection chamber, which has a diameter of 10 mm and a depth of 5 mm.

4. A nitrite microfluidic concentration gradient colorimetric rapid detection method according to claim 1, characterized in that: In the step three, the colorimetric quantitative detection of nitrite is achieved based on the colorimetric method in which nitrite inhibits the amplification of AuNPs signals.

5. A nitrite microfluidic concentration gradient colorimetric rapid detection method according to claim 1, characterized in that: A mobile platform (17) is provided inside the colorimetric signal acquisition box (14), the concentration gradient dilution chip is placed on the mobile platform (17), LED lights (16) with adjustable brightness are arranged on the two inner side walls of the colorimetric signal acquisition box (14), and the mobile terminal (15) is a smart phone, the camera of the smart phone corresponds to the acquisition hole and collects the colorimetric detection information on the concentration gradient dilution chip.

Images