A melamine detection method based on DNA hydrogel and microfluidic chip

Abstract

The present invention belongs to the field of small molecule detection, and provides a melamine detection method and microfluidic chip based on DNA hydrogel. The present invention combines stimulus-responsive DNA hydrogel with microfluidic chip, wherein the DNA hydrogel uses melamine aptamer as a cross-linking agent, and encapsulates nanogold as a basis for colorimetric detection and quantitative analysis. When melamine is present, it competitively binds to its aptamer, resulting in the disintegration of the DNA hydrogel and the release of the encapsulated nanogold, thereby achieving visual detection and quantitative measurement of melamine. Furthermore, a microfluidic chip is introduced in combination with the properties of DNA hydrogel, and quantitative detection of melamine can be achieved by taking pictures and analyzing the grayscale value of the copolymer solution after the reaction according to the different color depths. The method does not require skilled operators or large instruments, and can achieve portable and sensitive on-site detection of the target, and has great potential in food safety, clinical testing, environmental monitoring, etc.

Description

Technical Field

[0001] The invention belongs to the field of small molecule detection, and in particular relates to a melamine detection method based on DNA hydrogel, and a microfluidic chip used for the detection method. Background Art

[0002] Melamine (MEL) is often used as a chemical raw material and is one of the raw materials for making plastics, coatings, adhesives, etc. The Kjeldahl method is often used to detect the protein content in food or feed. Because melamine has a high nitrogen content, it is added to dairy products or animal feed to increase the apparent protein content in the measurement. Studies have shown that the intake of melamine is the cause of stone formation and disease in animals and infants. The United Nations Food Standards Commission has set the safety limit of melamine in food at 2.5 mg / kg (19.8 μM) and infant formula at 1 mg / kg (7.9 μM), and the World Health Organization's International Agency for Research on Cancer classifies it as a Class 2B carcinogen. Therefore, the detection of melamine has attracted the attention of more and more researchers.

[0003] Traditional detection methods for melamine include liquid chromatography, gas chromatography, gas chromatography-mass spectrometry, atomic absorption spectroscopy, etc. These methods rely on expensive large instruments, require strict sample pretreatment, and require high operator proficiency. In order to overcome the shortcomings of traditional technologies in melamine detection, detection methods based on nanotechnology have greatly promoted the development of rapid detection technology due to their high sensitivity, low detection limit, and simple process. Chanchal Hazra and his collaborators achieved sensitive detection of melamine by selective quenching of the luminescence signal of upconversion nanocrystals with melamine. Liao et al. replaced Fe with melamine. 3+The fluorescent signal of β-cyclodextrin modified carbon nanoparticles was turned on and off, and excellent results were achieved in the detection of melamine in milk. Xie et al. prepared microfluidic paper-based analytical devices (μPADs) based on gold nanoparticles (AuNPs) to achieve low-cost, portable and sensitive detection of melamine, which is very friendly to resource-limited areas. Among various detection methods, point-of-care testing (POCT) is very attractive. Common POCT technologies include lateral flow immunoassay (e.g., virus detection strips), sensor technology (e.g., blood glucose meter) and microfluidic technology that has developed rapidly in recent years. Microfluidic chips have been widely used in POCT detection platforms due to their low cost and the ability to realize the conversion of different signals. Compared with quantitative analysis, colorimetric detection can perform real-time qualitative or semi-quantitative analysis without relying on any advanced or complex instruments. It is worth noting that in colorimetric detection applications, AuNPs have long been favored by researchers due to their mature synthesis process and stability.

[0004] Stimuli-responsive DNA hydrogels have the programmable ability of DNA and the good carrying capacity of hydrogels, and have received increasing attention in the preparation of biosensors. Through the rational design and precise application of DNA chains, hydrogels can have good responsiveness, including the ability to respond to non-biological (light, heat, magnetism, pH, etc.) and biological (nucleic acids, proteins, small molecules, etc.) stimuli. Usually, nucleic acid aptamers are introduced into hydrogels to achieve specific responses to targets. Nucleic acid aptamers are oligonucleotide chains isolated by the systematic evolution of ligands by exponential enrichment (SELEX) technology, which have the advantages of a wide range of targets, easy in vitro large-scale synthesis, good repeatability, high stability, and low cost. Summary of the invention

[0005] In order to achieve the purpose of colorimetric detection and quantitative detection at the same time, the present invention provides a melamine detection method based on DNA hydrogel, which combines DNA hydrogel with microfluidic chip to develop a portable, inexpensive, easy-to-operate detection platform that does not rely on large-scale instrument quantitative analysis. The depth of the color of the copolymer solution after the reaction provides the basis for colorimetric detection, which does not rely on large-scale instruments, and can achieve the purpose of quantitative detection by only analyzing the gray value through software.

[0006] The present invention provides a melamine detection method based on DNA hydrogel, comprising the following steps:

[0007] S1. Preparation of linear polyacrylamide DNA polymer monomers:

[0008] SA or SB is mixed with acrylamide monomer and then vacuum deoxygenated, and then an initiator and a catalyst are added and mixed to perform polymerization reaction to obtain a polymer P-SA or P-SB;

[0009] The SA and SB are both single-stranded oligonucleotides, the 3' end of the SA and the 3' end of the SB are fully complementary to the two ends of the melamine aptamer (MA), and the 5' end of the SA and the 5' end of the SB are functionalized with a methyl propylene group;

[0010] S2. Preparation of melamine-responsive DNA hydrogel:

[0011] AuNPs, P-SA and P-SB are fully mixed in a buffer solution, and then MA is added and mixed evenly, so that MA hybridizes with SA in P-SA and SB in P-SB to obtain melamine-responsive DNA hydrogel; hybridization is generally achieved by annealing at a temperature higher than the annealing temperature of the complementary pairing part;

[0012] S3. Sample testing:

[0013] The melamine-responsive DNA hydrogel is mixed with a sample to be tested, and whether the sample contains melamine is qualitatively detected by observing the color of the supernatant with naked eyes; and / or, the melamine in the sample is quantitatively detected by measuring the ultraviolet absorption peak of the released AuNPs using an ultraviolet spectrophotometer (UV-Vis);

[0014] and / or,

[0015] A microfluidic chip is used for detection, wherein the microfluidic chip comprises a liquid storage area, a reaction area, a liquid channel and a detection area which are connected in sequence, wherein the liquid channel is serpentine and smaller in size than the unreacted DNA hydrogel (i.e., the DNA hydrogel cracked after the reaction with melamine is allowed to pass through but the unreacted DNA hydrogel is blocked from passing through); the detection method comprises: injecting the melamine-responsive DNA hydrogel and the sample to be tested into the reaction area of ​​the microfluidic chip, tilting the chip after a period of time, the unreacted DNA hydrogel will block the liquid channel, and the reacted mixed solution will flow through the liquid channel to the detection area below by gravity; qualitatively detecting whether the sample contains melamine by visually observing the position and color of the DNA hydrogel in the microfluidic chip; and / or quantitatively detecting the melamine in the sample by measuring the change in the color of the sample solution.

[0016] The synthesis of the target-responsive DNA hydrogel of the present invention and the detection principle of melamine in combination with the microfluidic chip are as follows: Figure 1 As shown. First, two single-stranded DNAs (SA and SB) complementary to partial regions of MA were designed and grafted to acrylamide to form polymer chains (P-SA and P-SB). Subsequently, MA was used as a cross-linker to hybridize with SA and SB to form a melamine-responsive DNA hydrogel, and AuNPs were embedded in the DNA hydrogel. When melamine is present, due to the high affinity between the aptamer and the target, the specific aptamer competitively binds to melamine to form a target-aptamer complex, resulting in the disintegration of the hydrogel and the release of the preloaded AuNPs. Visual detection and quantitative detection can be achieved by changing the color of the supernatant and UV detection. Similarly, the DNA hydrogel is placed in a designed microfluidic chip. The unreacted DNA hydrogel will block the liquid channel. In the presence of melamine, the DNA hydrogel gradually breaks down and shrinks in volume. By tilting the chip, the sample solution after the reaction flows into the detection area through the liquid channel under the action of gravity. The grayscale value is analyzed according to the color change caused by the released AuNPs content to achieve the effect of indicating the concentration of the target.

[0017] According to the above principles of the present invention, a sequence that meets the requirements can be designed. Preferably, the lengths of SA and SB are independently 15-20 nt; the length of MA is 30-40 nt; the lengths of the two ends of MA that are fully complementary to one end of SA and SB can be the same or different, and are independently 10-18 nt.

[0018] According to a preferred embodiment of the present invention, the nucleotide sequence of SA is shown in SEQ ID NO: 1, the nucleotide sequence of SB is shown in SEQ ID NO: 2, and the nucleotide sequence of MA is shown in SEQ ID NO: 3. The melamine aptamer (MA) used in the present invention is improved on the basis of the existing aptamer, maintaining a considerable affinity and better selectivity. By changing the aptamer in the system, the detection platform of the present invention can be further extended to the portable and quantitative detection of other targets.

[0019] 5'-Acrydite-AAAACCATCGGTGTGC-3'(SEQ ID NO: 1)

[0020] 5'-Acrydite-AAAAGCACACCCCCTAA-3' (SEQ ID NO: 2)

[0021] 5'-GCACACCGATGGCGGTCCTGTTTAGGGGGTGTGC-3' (SEQ ID NO: 3)

[0022] According to the present invention, the polymerization reaction of acrylamide monomer can select conventional components in the art, for example, the initiator is APS, and the catalyst is TEMED. The reaction conditions can also be conventional conditions in the art, and the present invention has no particular limitation on this.

[0023] According to a preferred embodiment of the present invention, step S1 further comprises a purification step: the polymerization product is subjected to ultrafiltration centrifugal tube centrifugation purification to remove unpolymerized monomers and polymers with a small molecular weight. Step S2 further comprises a purification step: washing the hybridized DNA hydrogel to remove AuNPs on the polymer surface. Purification can improve the sensitivity and accuracy of subsequent reactions.

[0024] According to a preferred embodiment of the present invention, the average particle size of the AuNPs particles is 10-30 nm, preferably 15 nm. The AuNPs particles can be prepared by the method described in the prior art.

[0025] The cross-linking of DNA has a direct impact on the network structure of the hydrogel. After fixing the concentrations of chain SA and SB in the complex, in order to achieve the highest performance of DNA hydrogel for melamine detection, the formation conditions of DNA hydrogel, reaction time and volume of encapsulated AuNPs were optimized. The following optimization features were determined: the concentration of MA was selected as 40μM, and the reaction reached maximum efficiency at 60min. AuNPs were encapsulated in the DNA hydrogel at a volume ratio of 8:21. The reaction efficiency was highest when the pH value of the supernatant was 7.5.

[0026] Based on this, a preferred embodiment of step S2 includes: mixing 4-10 μL, 0.4-0.6 mg / mL AuNPs, 3-6 μL P-SA with 100 μM SA, and 3-6 μL P-SB with 100 μM SB in a pH 7.5 Tris-HCl buffer, shaking vigorously at 55-65°C for 4-8 min, then adding 3-6 μL, 38-42 μM MA, shaking vigorously at 55-65°C for 8-12 min to ensure uniform mixing; after slowly cooling to room temperature, washing the prepared DNA hydrogel with Tris-HCl buffer to remove AuNPs on the polymer surface, and obtaining a melamine-responsive DNA hydrogel. The prepared DNA hydrogel can be stored at room temperature overnight to ensure sufficient cross-linking between DNAs.

[0027] According to the present invention, the step of measuring the color change of the sample solution to quantitatively detect melamine in the sample may include: under certain external lighting conditions, using a handheld camera (such as a mobile phone) to take a picture of the detection area of ​​the chip and uploading it to ImageJ software for average gray value analysis to achieve quantitative detection.

[0028] The present invention also provides a microfluidic chip for the above detection method, such as Figure 2 As shown, the microfluidic chip includes a liquid storage area, a reaction area, a liquid channel and a detection area which are connected in sequence.

[0029] The liquid storage area is used for storing the reaction solution, for example, pre-loaded with the melamine-responsive DNA hydrogel.

[0030] The sample reaction area is used for the reaction of DNA hydrogel and melamine.

[0031] The liquid channel is serpentine and smaller than the unreacted DNA hydrogel, so that the DNA hydrogel cannot pass through. After the DNA hydrogel reacts with melamine, the gel breaks, and the copolymer flows through the liquid channel to the bottom.

[0032] The detection area is used to photograph the solution flowing into the detection area after the reaction, and quantitatively analyze the concentration of melamine.

[0033] According to a preferred embodiment of the present invention, the material of the microfluidic chip is polydimethylsiloxane.

[0034] The present invention designs a portable and sensitive POCT detection platform and detection method, combines the stimulus-responsive DNA hydrogel based on the aptamer and the microfluidic chip, develops a POCT method and equipment, and realizes the sensitive detection of melamine. The polyacrylamide DNA hydrogel is bridged by the melamine aptamer and wraps the AuNPs inside. In the presence of melamine, the competitive binding with its aptamer causes the rupture of the gel and the release of AuNPs. The AuNPs provide the basis for colorimetric detection and quantitative detection. Under optimal conditions, the detection range of the DNA hydrogel for melamine is 0.1μM-100μM, and the detection limit is 42nM. On this basis, in order to achieve the purpose of immediate analysis at the sampling site, the present invention introduces a microfluidic chip to construct a POTC detection platform. The DNA hydrogel cannot flow directly from the reaction zone into the detection zone below. After the reaction with melamine is completed, the copolymer liquid flows into the detection zone below under the action of gravity. At this time, the mobile phone is used to capture the photo and use the Image J software to analyze the gray value to achieve the purpose of quantitative analysis of the detection concentration. It can be seen from the data of the embodiment that the concentration of the target is positively correlated with the amount of AuNPs released by the DNA hydrogel and negatively correlated with the gray value. The detection range of melamine by the POCT detection device and method is 0.2μM-50μM, and the detection limit is 37nM. The method of the present invention does not require skilled operators or large instruments, and can achieve portable and sensitive on-site detection of the target, which has great potential in food safety, clinical testing, environmental monitoring and other aspects.

[0035] Other features and advantages of the present invention will be described in detail in the following detailed description. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] The above and other objects, features and advantages of the present invention will become more apparent through a more detailed description of exemplary embodiments of the present invention with reference to the accompanying drawings.

[0037] Figure 1 The synthesis of the target-responsive DNA hydrogel of the present invention and the detection principle of melamine in combination with a microfluidic chip are shown.

[0038] Figure 2 The figure is a schematic diagram of the structure of a microfluidic chip according to a specific embodiment of the present invention.

[0039] Figure 3 The results of characteristic detection of the prepared AuNPs are shown. (A) and (B) TEM images of AuNPs. (C) DLS analysis result of AuNPs.

[0040] Figure 4The feasibility verification results of DNA hydrogel are shown. (A) 20% Native-PAGE electrophoresis, a: SA, b: SB, c: MA, d: SA+SB+MA, e: SA+SB+MA+MEL (10μM), f: SA+SB+MA+MEL (100μM). The concentrations of SA, SB and MA are 2.0μM, 2.0μM and 1.0μM, respectively. (B) CD spectrum of DNA copolymer, where the concentrations of SA, SB, MA and MEL are 10μM, 10μM, 10μM and 20μM, respectively.

[0041] Figure 5 The results of optimization of the process conditions for DNA hydrogel preparation are shown. (AD) Effect of MA concentration on the detection efficiency of DNA hydrogel, dissociation kinetics of DNA hydrogel at 524 nm when MA concentrations were 30 μM, 35 μM, 40 μM and 50 μM, respectively. (E) Effect of the volume of encapsulated AuNPs on absorbance. (F) Effect of pH value on melamine detection. Error bars represent the standard deviation of three repeated experiments.

[0042] Figure 6 The characterization results of DNA hydrogels are shown. (A) and (B) SEM images of DNA hydrogels. (C) TEM image of DNA hydrogel diluted with water. (D) Rheological study of DNA hydrogels.

[0043] Figure 7 Figure 2 shows the sensitivity of DNA hydrogel to melamine. (A) UV-visible absorption spectra of the supernatant after reaction with different concentrations of melamine. (B) Standard curve of DNA hydrogel reaction with melamine at different concentrations. Error bars represent the standard deviation of three repeated experiments.

[0044] Figure 8 Shows the selectivity results of DNA hydrogel detection. a. Glucose, b. Sucrose, c. Pb 2+ , d.Al 3+ , e. lysine, f. L(+)-cysteine, g. histidine, h. adenine, i. thymine, j. melamine. Error bars represent the standard deviation of three replicate experiments.

[0045] Fig. 9 The results of microfluidic chip detection of melamine are shown. (A) Color change images of the microfluidic chip detection area when the melamine concentration is 0μM, 0.2μM, 0.5μM, 1.5μM, 5.0μM, 15μM, and 50μM. (B) Standard curve of microfluidic chip detection of different concentrations of melamine. The error bars represent the standard deviation of three repeated experiments. DETAILED DESCRIPTION

[0046] The preferred embodiments of the present invention will be described in more detail below. Although the preferred embodiments of the present invention are described below, it should be understood that the present invention can be implemented in various forms and should not be limited to the embodiments set forth herein.

[0047] If the specific conditions are not specified in the examples, all the experiments were carried out under conventional conditions or the conditions recommended by the manufacturer. If the manufacturers of the reagents or instruments are not specified, all of them are conventional products that can be purchased from the market.

[0048] Example

[0049] Materials and Equipment

[0050] Ammonium persulfate (APS), N,N,N',N'-tetramethylethylenediamine (TEMED) and Tris-HCl buffer were purchased from Shanghai Aike Biochemical Co., Ltd. (China). Melamine, glucose, sucrose, lysine, L(+)-cysteine, histidine, VB4, and thymine were purchased from Alta Scientific Co., Ltd. They were all standard products with a purity greater than 99%. Other reagents were purchased from Sigma-Aldrich. All oligonucleotide sequences were purified by high-performance liquid chromatography (HPLC) and synthesized by Shanghai Sangon Biotechnology Co., Ltd. (Shanghai, China). The oligonucleotide sequences are shown in Table 1.

[0051] Table 1 DNA sequences used in the present invention

[0052]

[0053] Instruments and features

[0054] The absorbance was measured by UV-Vis spectrophotometer (TU-1901, Percy Co., Ltd., China). The DNA hydrogel was freeze-dried and then observed using scanning electron microscopy (SEM) to observe its morphological characteristics. Transmission electron microscopy (TEM) and dynamic and static laser light scattering were used to analyze whether the synthesized AuNPs were qualified. Circular dichroism spectrometer (CD, CHIRASCAN, Applied Photophysics Co., Ltd., England) and electrophoresis instrument (DYY-11, Liuyi Instrument Factory, China) were also used in this experiment.

[0055] Synthesis of AuNPs

[0056] AuNPs were synthesized according to the literature (Ohta, S.; Kikuchi, E.; Ishijima, A.; Azuma, T.; Sakuma, I.; Ito, T., Investigating the optimum size of nanoparticles for their delivery into the brain assisted by focused ultrasound-induced blood-brain barrier opening. Scientific reports 2020, 10(1), 18220), ensuring that the glassware used to prepare AuNPs was soaked in aqua regia (HCl:HNO3=3:1, v / v) for 120 min, then washed with ultrapure water and dried. First, 100 mL of 0.01% (w / v) HAuCl4 was heated to a boil in a 250 mL round-bottom flask, and then 2.6 mL of 1% freshly prepared sodium citrate solution (Na3C6H5O7·2H2O) was quickly added. The mixed solution was boiled and stirred for 30 minutes to obtain an AuNPs solution with a concentration of 2.5 nM and a particle size of 15 nm. Finally, the AuNPs were modified with polyethylene glycol thiol (PEG-SH, Mw 5000) and coated with 0.01 wt% Tween-20 to avoid the aggregation of the AuNPs. The obtained AuNPs were centrifuged and redispersed to the required concentration. The synthesized AuNPs were characterized as follows. Figure 3 The TEM images show that the synthesized AuNPs have a particle size of 15 nm and are well dispersed. Figure 3 The hydrated particle size of AuNPs was further analyzed by dynamic light scattering (DLS). Figure 3 The C results are slightly larger than the TEM results.

[0057] Preparation of DNA hydrogel

[0058] First, a linear polyacrylamide DNA polymer is prepared. SA or SB is mixed with 2.4% acrylamide in an Eppendorf tube and then vacuum deoxygenated at 37°C for 6 minutes. Then, 1.4% APS (freshly prepared, 10 wt%) and 2.8% TEMED (freshly prepared, 5% by volume) are added and mixed. The polymer P-SA or P-SB is vacuum deoxygenated at 37°C for 8 minutes. The obtained P-SA or P-SB (the final concentration of SA or SB is 100 μM) is purified by centrifugation in an ultrafiltration centrifuge tube with a molecular weight of 100 KD to remove unpolymerized monomers and polymers with a smaller molecular weight.

[0059] Next, melamine-responsive DNA hydrogels were prepared. First, 8 μL of AuNPs, 5 μL of P-SA, and 5 μL of P-SB were mixed in Tris-HCl buffer and shaken vigorously at 60°C for 5 min. Then, 5 μL of a certain concentration of MA (denatured at 95°C and slowly cooled to room temperature) was added and shaken vigorously at 60°C for 10 min to ensure that the sample was evenly mixed. After the sample was slowly cooled to room temperature, the prepared DNA hydrogel was rinsed three times with Tris-HCl buffer to remove AuNPs on the polymer surface. The prepared DNA hydrogel was stored at room temperature overnight to ensure sufficient cross-linking between DNA.

[0060] Feasibility verification

[0061] The degree of cross-linking of the hydrogel network is determined by the degree of binding between MA and its complementary sequence. 20% native polyacrylamide gel electrophoresis (PAGE) was used to verify the binding of MA to the target, as well as the hybridization and dehybridization behavior of MA with the complementary chain. Figure 4 As shown in A, channels a and b (chains SA and SB) differ in molecular weight from channel c (MA), while chains SA and SB also clearly bind to MA (channel d). After the addition of melamine, the MA band can be seen (channel e), which proves the breakage of the three complementary chains. By further increasing the concentration of melamine, it can be seen from channel f that the dehybridization of the three-chain complex is more obvious.

[0062] To further verify the feasibility of the experiment, circular dichroism (CD) spectroscopy was used to verify the hybridization of complementary DNA and the conformational changes caused by the binding of melamine to its aptamer. Figure 4 As shown in Figure B, melamine does not show the peak of its secondary structure. It is observed that MA has the largest positive peak at 284nm and the largest negative peak at 241nm. Chain SA+SB has the largest positive peak at 277nm and the largest negative peak at 251nm. The peak of the hybrid chain of SA+SB+MA has red-shifted compared to chain MA and blue-shifted compared to chain SA+SB, and the peak sizes are also different, proving that MA hybridizes with its complementary chain and changes the secondary structure. After melamine was subsequently introduced into the hybrid chain, the positive peak (277nm) was significantly red-shifted and the peak increased, indicating that melamine competed with its aptamer from the hybrid chain, further changing the configuration of the system.

[0063] Optimization of experimental conditions

[0064] The cross-linking of DNA has a direct impact on the network structure of the hydrogel. After fixing the concentrations of chain SA and SB in the complex, in order to achieve the highest performance of DNA hydrogel in detecting melamine, the formation conditions of DNA hydrogel, reaction time and volume of encapsulated AuNPs were optimized. MA, as a bridge of nucleic acid hydrogel, specifically responds to melamine, leading to the dissociation of nucleic acid hydrogel and the release of AuNPs. Therefore, it is unquestionable to optimize the concentration of MA first. Figure 5 As shown in the AD, the dissociation kinetics at different MA concentrations were observed by monitoring the absorbance at 524 nm (maximum absorption of AuNPs with a particle size of 15 nm). When the MA concentration was 30 μM, the system gradually disintegrated over time after the addition of the target, but even in the absence of the target, the hydrogel gradually dissociated as it swelled with water. Obviously, the low concentration of the bridging chain made the overall cross-linking degree of the system too low ( Figure 5 As the concentration of MA increased to 35 μM, the cross-linking degree of the DNA hydrogel was strengthened, but the gel system gradually dissociated in the absence of melamine ( Figure 5 B). When the MA concentration was 40 μM, melamine was added, and the absorbance of the system basically stopped increasing after 60 minutes of reaction, and the reaction reached the maximum efficiency. In addition, the dissociation degree of the blank control was very low, which means that the system is stable and has a high sensitivity to the target ( Figure 5 However, as the concentration of MA increased to 50 μM, the degree of cross-linking in the system was further enhanced, resulting in insensitivity to the presence of melamine ( Figure 5 D). In summary, the concentration of MA was selected to be 40 μM, and the reaction reached the maximum efficiency at 60 min.

[0065] Obviously, the concentration determines the depth of the colloidal gold color. In order to smoothly carry out the colorimetric analysis of the subsequent experimental results, the concentration of AuNPs was determined to be 0.5 mg / mL. Because the amount of encapsulated AuNPs has a significant effect on the experimental sensitivity, the volume of AuNPs loaded by DNA hydrogel was further optimized. Figure 5 As shown in Figure E), as the amount of AuNPs loaded in the DNA hydrogel increases, the absorbance monitoring value after the experiment also increases, reaching the maximum value when the encapsulation volume is 8 μL. However, as the loading amount increases further, the background signal increases, resulting in a decrease in the absorbance result. Therefore, the volume of AuNPs encapsulated in the DNA hydrogel was finally selected to be 8 μL.

[0066] Finally, the pH of the reaction system was optimized to explore the optimal reaction conditions. By adjusting the pH value of the supernatant, the reaction efficiency was tested at pH 3.5, 5.5, 7.5, 9.5 and 11.5. Figure 5It can be seen from the F that the reaction efficiency is highest when the pH value of the supernatant is 7.5.

[0067] Characterization of DNA hydrogels

[0068] In order to verify the successful synthesis of DNA hydrogel, the optimized DNA hydrogel was observed by scanning electron microscopy. The DNA hydrogel was freeze-dried, such as Figure 6 As shown in AB, it can be observed that the SEM image is a porous cross-linked network, which is consistent with the morphology of the hydrogel matrix. Next, the synthesized DNA hydrogel was dissolved and diluted with water and characterized by TEM. The results showed that AuNPs were regularly dispersed in the system ( Figure 6 C).

[0069] To gain insight into the physical state of the DNA hydrogels, rheological studies were performed ( Figure 6 D). The ability of the composite to recover its original geometry and the material's flow tendency were evaluated by comparing the optimized DNA hydrogel storage modulus (G') and loss modulus (G") . The moduli (G' and G") showed a slight frequency dependence over the entire tested frequency range. And over the entire tested range, the two values ​​did not cross each other, which means that the elastic response dominated and the material had a permanent network with a high degree of cross-linking. G" was always lower than G', indicating that the system had a gel-like behavior.

[0070] Ultrasensitive detection of melamine

[0071] A series of 75 μL melamine standard solutions with gradient concentrations were added to the DNA hydrogel and reacted at 37°C and 150 rpm for 1 hour. The color of the supernatant changed. Obviously, measuring the absorbance of the supernatant can obtain more accurate results.

[0072] DNA hydrogel colorimetric and quantitative detection

[0073] The ability of the DNA hydrogel to detect melamine can be determined by the color depth of the supernatant and quantitatively expressed by detecting the absorbance of the supernatant, that is, colorimetric analysis and quantitative detection can be performed under different concentrations of melamine. Figure 7 As expected, increasing the concentration of melamine resulted in a decrease in the crosslinker density of the DNA hydrogel, thereby promoting the dissociation of the hydrogel and the subsequent release of AuNPs into the supernatant. Figure 7 A) shows that the absorbance is positively correlated with the melamine concentration, and the melamine concentration in the range of 0.1 μM-100 μM is logarithmically linear with the absorbance of the supernatant ( Figure 7B). The linear equation is Y=0.1977LgX+1.1487. Obviously, as the concentration of melamine increases further, MA is consumed in the reaction and the reaction reaches a plateau. The detection limit of the sensor is 42nM, and the result is obtained by the formula 3σ / slope, where σ is the standard deviation of the blank sample (n=11), and slope represents the slope of the linear equation, which is 0.1977. Compared with the previously reported detection method of melamine, the present invention has a more excellent detection performance, as shown in Table 2.

[0074] Table 2 Method comparison

[0075]

[0076]

[0077] Specificity and anti-interference performance testing

[0078] After confirming that the sensor has a sensitive response to melamine, the potential substances that affect the sensor were further explored to evaluate its anti-interference and specificity. The substances selected included some coexistent substances of melamine, glucose, sucrose, lysine, L-cysteine, histidine, VB4, thymine, and metal ions that may affect the sensor, such as Pb 2+ 、Al 3+ According to the above melamine detection experimental procedure, the concentration of these substances is 100μM, and compared with the concentration of 10μM melamine (that is, the added amount is 10 times that of melamine), and the absorbance of the supernatant after the reaction is recorded. The results are as follows Figure 8 As shown, it can be seen that under the same reaction conditions, only melamine has a significant impact on the sensor, and the absorbance changes of other interferences are very small. It can be seen that the established melamine detection method has a high specificity.

[0079] Detection of melamine in real samples

[0080] In order to test the detection ability of DNA hydrogel in actual samples, milk and infant formula purchased from local supermarkets were selected as samples. Different amounts of melamine were added to the samples after pre-treatment. The absorbance of the supernatant after the reaction was recorded according to the above-mentioned melamine detection experimental procedure.

[0081] Two samples were selected, namely milk and infant formula milk powder. After pre-treatment, different concentrations of melamine were added to the actual samples. The results are shown in Table 3 below. The spiked recovery rate of the actual sample detection was 87.3%-112%, and the relative standard deviation (RSD) was 1.4%-5.7%. It is proved that the method of the present invention has good application prospects for the detection of melamine in milk and infant formula milk powder.

[0082] Table 3 Results of using DNA hydrogel to detect melamine in real samples (n=3)

[0083]

[0084] Design and testing of microfluidic chips

[0085] In order to meet the requirements of POCT testing and be as cheap, easy to operate and sensitive as possible, a microfluidic chip was designed in combination with the characteristics of DNA hydrogel. The material for preparing the microfluidic chip is polydimethylsiloxane (PDSM). The pattern was drawn using AutoCAD software and sent to Suzhou Shuotian Automation Technology Co., Ltd. (Suzhou, China) for preparation. The chip is divided into a liquid storage area, a reaction area, a liquid channel and a detection area (such as Figure 2 As shown). DNA hydrogel and melamine solutions of different concentrations are injected into the chip reaction area successively. The unreacted DNA hydrogel will block the liquid channel. After the reaction is completed, the chip is tilted. Relying on gravity, the reacted sample solution flows through the liquid channel to the detection area below. The change in the color of the copolymer can provide colorimetric detection. Under certain external lighting conditions, quantitative detection can be achieved by taking pictures of the detection area of ​​the chip with a mobile phone camera and uploading them to ImageJ software for average grayscale value analysis. First, the average grayscale value of the blank control group was analyzed. As the concentration of melamine increased, the color of the copolymer flowing into the detection area of ​​the microfluidic chip gradually deepened ( Fig. 9 A), further quantitative results such as Fig. 9 As shown in B, the average gray value is linearly related to the logarithm of melamine concentration in the range of 0.2 μM-50 μM.

[0086] In summary, the present invention uses melamine aptamers as bridge chains to construct a DNA hydrogel that specifically responds to melamine, and combines the DNA hydrogel with a microfluidic chip to create a portable, rapid, and sensitive POCT detection platform. In the presence of a target, it will compete with its specific aptamer for binding, resulting in a decrease in the cross-linking density of the DNA hydrogel. The release of encapsulated AuNPs after the DNA hydrogel breaks can achieve the results of colorimetric detection and quantitative detection. For the needs of POCT detection, a mobile phone is used to take a picture of the chip and the numerical value is read out using software. No large instrument is required, and any non-professional can operate well, with a detection limit of 37nM. This is lower than the melamine limit of 0.15mg / kg in liquid infant formula specified by the Codex Alimentarius Commission. The DNA hydrogel has good specificity and anti-interference ability, and has no obvious response to other substances. The spiked recovery rate in milk and infant formula is 87.3%-112.0%. The RSD is 1.4%-5.7%. In addition, based on the good specificity and responsiveness of aptamers to targets, by changing the aptamers in this strategy and designing responsive complementary sequences, this method may be further extended to portable quantitative detection of various targets. Therefore, melamine-responsive DNA hydrogel combined with microfluidic chips provides a user-friendly POCT device that is easy to operate, portable, and does not require other equipment to cooperate. The detection can be completed through a mobile phone, and has great potential in food safety detection and medical diagnosis.

[0087] The embodiments of the present invention have been described above, and the above description is exemplary, not exhaustive, and is not limited to the disclosed embodiments. Many modifications and changes will be apparent to those skilled in the art without departing from the scope and spirit of the described embodiments. Sequence Listing <110> Institute of Environmental and Occupational Medicine, Academy of Military Medical Sciences, Academy of Military Sciences <120> A melamine detection method based on DNA hydrogel and microfluidic chip <130> 2200710 <160> 3 <170> SIPOSequenceListing 1.0 <210> 1 <211> 16 <212> DNA <213> Artificial Sequence <400> 1 aaaaccatcg gtgtgc 16 <210> 2 <211> 17 <212> DNA <213> Artificial Sequence <400> 2 aaaagcacac cccctaa 17 <210> 3 <211> 34 <212> DNA <213> Artificial Sequence <400> 3 gcacaccgat ggcggtcctg tttagggggt gtgc 34

Claims

1. A melamine detection method based on DNA hydrogel, characterized in that: The following steps are involved: S1. Preparation of linear polyacrylamide DNA polymer monomer: SA and SB are mixed with acrylamide monomers respectively and then vacuum deoxidized, and then an initiator and a catalyst are added and mixed to perform polymerization reaction to obtain polymers P-SA and P-SB; the initiator is ammonium persulfate, and the catalyst is N,N,N',N'-tetramethylethylenediamine; The SA and SB are both single-stranded oligonucleotides, the 3' end of the SA and the 3' end of the SB are fully complementary to the two ends of the melamine aptamer MA, respectively, and the 5' end of the SA and the 5' end of the SB are functionalized with a methyl propylene group; The nucleotide sequence of SA is: 5′-Acrydite-AAAACCATCGGTGTGC-3′; The nucleotide sequence of SB is: 5′-Acrydite-AAAAGCACACCCCCTAA-3′; The nucleotide sequence of MA is: 5'-GCACACCGATGGCGGTCCTGTTTAGGGGGTGTGC-3'; S2. Preparation of melamine-responsive DNA hydrogel: 4-10 μL, 0.4-0.6 mg / mL AuNPs, 3-6 μL P-SA (100 μM in terms of SA) and 3-6 μL P-SB (100 μM in terms of SB) were mixed in a pH 7.5 Tris-HCl buffer and vigorously shaken at 55-65°C for 4-8 min, then 3-6 μL 38-42 μM MA was added and vigorously shaken at 55-65°C for 8-12 min to ensure uniform mixing; after slowly cooling to room temperature, the prepared DNA hydrogel was rinsed with Tris-HCl buffer to remove AuNPs on the polymer surface, thereby obtaining a melamine-responsive DNA hydrogel; S3. Sample testing: The melamine-responsive DNA hydrogel is mixed with a sample to be tested, and whether the sample contains melamine is qualitatively detected by observing the color of the supernatant with naked eyes; and / or, the melamine in the sample is quantitatively detected by measuring the ultraviolet absorption peak of the released AuNPs using an ultraviolet spectrophotometer (UV-Vis); and / or, A microfluidic chip is used for detection, wherein the microfluidic chip comprises a liquid storage area, a reaction area, a liquid channel and a detection area which are connected in sequence, wherein the liquid channel is serpentine and its size is smaller than that of the unreacted DNA hydrogel; the detection method comprises: injecting the melamine-responsive DNA hydrogel and the sample to be tested into the reaction area of ​​the microfluidic chip, tilting the chip after a period of time, the unreacted DNA hydrogel will block the liquid channel, and the reacted sample copolymer liquid will flow through the liquid channel to the detection area below by gravity; and qualitatively detecting whether the sample contains melamine by observing the position and color of the DNA hydrogel in the microfluidic chip with the naked eye; And / or, the melamine in the sample is quantitatively detected by measuring the change in the color of the sample solution.

2. The melamine detection method based on DNA hydrogel according to claim 1, characterized in that: Step S1 also includes a purification step: the polymerization product is purified by ultrafiltration centrifuge tube centrifugation to remove unpolymerized monomers and polymers with smaller molecular weight; Step S2 also includes a purification step: washing the hybridized DNA hydrogel to remove AuNPs on the polymer surface.

3. The melamine detection method based on DNA hydrogel according to claim 1, characterized in that: The average particle size of the AuNPs particles is 10-30 nm.

4. The melamine detection method based on DNA hydrogel according to claim 1, characterized in that: The steps of quantitatively detecting melamine in the sample by measuring the color change of the sample solution include: under certain external lighting conditions, taking pictures of the detection area of ​​the chip with a handheld camera and uploading them to ImageJ software for average gray value analysis to achieve quantitative detection.

5. A microfluidic chip for use in the detection method according to any one of claims 1 to 4, characterized in that: The microfluidic chip comprises a liquid storage area, a reaction area, a liquid channel and a detection area which are connected in sequence, the liquid channel is serpentine and its size is smaller than that of the unreacted DNA hydrogel; the material of the microfluidic chip is polydimethylsiloxane; The liquid storage area is pre-loaded with the melamine-responsive DNA hydrogel.

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