A microfluidic paper chip for detecting freshness of fresh meat and a preparation method thereof

Abstract

The present application relates to the technical field of food detection, and particularly relates to a microfluidic paper chip for detecting freshness of fresh meat and a preparation method thereof. The microfluidic paper chip comprises a hydrophobic upper paper base material with a scale, a lower paper base material with a hydrophilic flow channel, and DNA hydrogel in a storage tank of the lower paper base material. The paper base material is a carrier and a reactor, and the pH-sensitive DNA hydrogel is a signal sensing and output unit. When the meat is spoiled, the volatile nitrogen base salt (TVB-N) which is a basic characteristic metabolite can induce the DNA hydrogel to transform from a gel state to a solution state, and move in the hydrophilic channel of the lower paper base material, so as to generate a readable moving distance signal, thereby realizing accurate indication of different freshness grades of fresh meat.

Description

Technical Field

[0001] This invention relates to the field of food testing technology, specifically to a microfluidic paper chip for detecting the freshness of fresh meat products and its preparation method. Background Technology

[0002] my country is a major consumer of fresh meat globally. In recent years, influenced by the rapid development of e-commerce, more and more consumers prefer to purchase packaged fresh meat products online. Packaged fresh meat typically undergoes a complex storage and transportation process, including packaging, distribution, transshipment, and delivery, which severely tests its freshness. It is well known that fresh meat is rich in nutrients and has a high free water content, making it prone to bacterial growth and spoilage, potentially leading to safety issues. However, current consumer judgments of the freshness of packaged fresh meat rely primarily on personal sensory evaluation and the expiration date or best-before date on the packaging. The former is easily influenced by individual subjective factors, while the latter ignores the impact of improper storage and transportation on food quality, failing to reflect its true freshness and posing food safety risks. Furthermore, inaccurate assessments of food freshness can lead to food waste. In my country, food waste during post-production processing and storage reaches as high as 175 million tons. Therefore, developing reliable, non-destructive, and end-consumer-oriented technologies for assessing the freshness of packaged fresh meat is of great significance for ensuring food quality and safety in my country and building a resource-conserving society.

[0003] Currently, freshness sensors for packaged fresh meat products mainly fall into two categories: those based on synthetic dyes and those based on natural dyes. Their sensing principle involves using volatile basic nitrogen (TVB-N), produced by the microbial degradation of proteins and amino acids during the spoilage process of fresh meat, as signal molecules. This TVB-N alters the environmental pH, causing a color change in the sensor, thus indicating changes in food freshness. While synthetic dye-based sensors offer advantages such as high sensitivity and good stability, they also pose safety risks and have low consumer acceptance. The latter, using plant extracts, is inherently safe and non-toxic. However, most natural dyes have limited color-changing sensitivity and are not readily apparent in response to subtle pH fluctuations caused by different stages of spoilage, often making it difficult to accurately distinguish different freshness levels. Therefore, improving the sensitivity and accuracy of fresh meat sensors while ensuring the safety and non-toxicity of the materials remains a critical scientific problem and urgent need in the field of food safety. Summary of the Invention

[0004] To overcome the shortcomings and deficiencies of existing technologies, this invention aims to provide a microfluidic paper chip for detecting the freshness of fresh meat and its preparation method. The aforementioned microfluidic paper chip uses DNA hydrogel as the sensing and signal output element, and is safe and non-toxic. Its sensing principle is significantly different from existing colorimetric freshness sensors. It converts the meat spoilage signal into the movement distance of the DNA hydrogel in the hydrophilic channels of the microfluidic paper chip. Consumers can intuitively and accurately determine the freshness level of the meat based on this movement distance, thus exhibiting significant advantages in signal readability and accuracy. Accurate determination of meat freshness not only effectively avoids food safety incidents caused by consumers mistakenly consuming spoiled meat, but also helps guide sellers to promptly dispose of meat that is about to spoil, avoiding food waste.

[0005] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0006] A microfluidic paper chip for detecting the freshness of fresh meat products comprises an upper hydrophobic paper base material and a lower paper base material. Both the upper and lower paper base materials have circular holes and elongated patterns connected to the circular holes, and the patterns overlap. The circular holes and elongated patterns of the upper paper base material are hollowed out. The area of ​​the lower paper base material, except for the circular holes and elongated patterns, is coated with wax to act as a hydrophobic barrier. The circular hole portion of the lower paper base material serves as a storage tank containing a pH-sensitive DNA hydrogel. The elongated portion of the lower paper base material serves as a hydrophilic reaction flow channel.

[0007] As a preferred embodiment, distance markings are printed next to the elongated strip of the upper paper-based material.

[0008] The pH-sensitive hydrogel is formed by acidifying i-motif-functionalized DNA nanoscaffolds or other natural polymers, such as polyethylene glycol and hyaluronic acid.

[0009] pH-sensitive hydrogels are in a colloidal state under acidic conditions and in a solution state under neutral or alkaline conditions.

[0010] The DNA nanoscaffold is a DNA nanostructure containing at least two sticky ends, such as a DNA nanowire with two sticky ends, a Y-shaped DNA nanostructure with three sticky ends, or a cross-shaped DNA nanostructure with four sticky ends.

[0011] The preferred DNA nanoscaffold of this invention is a Y-shaped DNA nanostructure.

[0012] The DNA nanoscaffold has a deconstruction temperature of not less than 37°C.

[0013] The sticky ends of the DNA nanoscaffold are short nucleotide sequences rich in cytosine, i.e., i-motif sequences. These short nucleotide sequences rich in cytosine cannot form intramolecular i-motif structures under acidic pH conditions, but they can form intermolecular i-motif structures, and the deconstruction temperature of the formed intermolecular i-motif structures is not lower than 37°C.

[0014] The length of the cytosine-rich short nucleotide sequence must not exceed the length of any arm or double-stranded fragment of the DNA nanoscaffold. The preferred cytosine-rich short nucleotide sequence of this invention is 5'-CCCCCCAATCCCCCC-3'. However, this invention is not limited to this; the number of consecutive, repeating cytosines in the cytosine-rich short nucleotide sequence can be adjusted according to the actual situation of the meat being tested, but it must not be less than 3 bp, as it determines the pH response range and stability of the entire DNA hydrogel. The linker sequence located in the middle of the cytosine cluster in the cytosine-rich short nucleotide sequence can be composed of any bases, but its length must not be less than 1 bp.

[0015] The pH-sensitive DNA hydrogel cannot produce liquid movement traces on filter paper before reacting with alkaline substances; after reacting with alkaline substances, it will transform from a gel state to a solution state, thereby producing liquid movement distance on the filter paper.

[0016] The pH-sensitive DNA hydrogel used must have a volume of not less than 4 μL.

[0017] The pH-sensitive DNA hydrogel can be supplemented with safe, non-toxic dyes for easy observation.

[0018] Preferably, the added dye is a natural dye extracted from animals, plants, or microorganisms, such as lycopene.

[0019] The flow channel is formed by two layers of paper-based material stacked and fixed. The upper layer is made of paper-based material with a certain hardness and thickness, such as 0.1 to 0.5 mm, and the lower layer is made of absorbent dry filter paper.

[0020] The length of the flow channel must not be shorter than the distance required for the DNA hydrogel to be completely converted into a solution.

[0021] Preferably, the upper paper base material is coated paper.

[0022] The lower paper base material is left untreated at the location corresponding to the cutout of the upper paper base material to ensure that it is a hydrophilic area, while the parts outside the hydrophilic area are coated with wax to make them a hydrophobic barrier.

[0023] Preferably, the lower paper-based material is Whatman Grade 1 chromatography filter paper.

[0024] This invention also provides a method for preparing a microfluidic paper chip for detecting the freshness of fresh meat products. The method includes the following steps.

[0025] (1) Preparation of pH-sensitive DNA hydrogel: Three single-stranded oligonucleotide chains (Y1, Y2, Y3) with cytosine-rich sequences at their ends were added to PBS in an equimolar ratio. After mixing, the mixture was incubated at 95°C for 5 min, then at 65°C for 2 min, at 25°C for 3 min, and at 4°C for 2 h to obtain a solution containing Y-type DNA nanostructures. Based on this, high-concentration HCl and lycopene were added to the above solution, mixed, and incubated at 4°C for 2 h to obtain a pH-sensitive DNA hydrogel.

[0026] (2) Preparation method of flow channel: Prepare upper and lower paper base materials, design round holes and long strips using AutoCAD, print them, cut out round holes and long strips in the upper paper base material, and print distance scales on one side of the long strips. The lower paper base material except for the round holes and long strips is sealed with wax. Then, place the lower paper base material in a 60℃ oven for 2 to 10 minutes to allow the wax to melt and penetrate into the pores of the paper base material.

[0027] (3) Fix the upper and lower paper base materials, and add more than 4 μL of the pH-sensitive DNA hydrogel to the circular hole to obtain the microfluidic paper chip.

[0028] Compared with the prior art, the advantages of the present invention are:

[0029] (1) Using a cytosine-rich sequence that is highly sensitive to pH and has a flexible and adjustable response range as a signal recognition element is expected to improve the response sensitivity to putrefaction signals (volatile basic nitrogen).

[0030] (2) The sensing raw material used is nucleic acid, which is a macromolecule in living organisms. It is safe and there is no need to worry about safety issues caused by leakage of sensing materials or contact with meat products.

[0031] (3) Compared with colorimetric signals, using the movement distance after DNA hydrogel liquefaction as the signal output mode is convenient for establishing a quantitative relationship between the movement distance and the concentration of volatile basic nitrogen, which is beneficial for accurately determining the freshness grade. On the other hand, it is convenient for users to read and understand, eliminating the influence of individual subjective factors. Attached Figure Description

[0032] Figure 1 Design of microfluidic paper chips.

[0033] Figure 2 The schematic diagram of the microfluidic paper chip of this invention for detecting the freshness of chilled meat. (a) Preparation and response principle of DNA hydrogel; (b) Relationship between the degree of meat spoilage and the distance the DNA hydrogel moves in the hydrophilic flow channel.

[0034] Figure 3 (a) Circular dichroism spectrum of the cytosine-rich sequence (Stand I) at different pH values; (b) Melting curves obtained by plotting the changes in the circular dichroism absorption peak of Strand I (pH 5.0) at 261 nm at different temperatures.

[0035] Figure 4 (a) DNA nanowires characterized by 15% natural polyacrylamide gel electrophoresis; (b) Images of DNA nanowire hydrogels (1: before gel formation; 2: after gel formation); (c) Y-DNA nanoscaffold formation characterized by 15% natural polyacrylamide gel electrophoresis; (d) Images of Y-DNA hydrogels (1: before gel formation; 2: after gel formation).

[0036] Figure 5 Microstructure of a microfluidic paper chip. (a) Scanning electron microscope image of the hydrophilic channel; (b) Scanning electron microscope image of the hydrophobic barrier.

[0037] Figure 6 The graph shows the change in the distance the DNA hydrogel moves on the microfluidic paper chip in response to different ammonia concentrations at 4℃.

[0038] Figure 7 The graph shows the change in the distance the DNA hydrogel moves on the microfluidic paper chip in response to different ammonia concentrations at 25°C.

[0039] Figure 8 The graph shows the change in the storage time of fresh pork and the distance the DNA hydrogel moves on the microfluidic paper chip at 25°C.

[0040] Figure 9 (a) The change in the distance the DNA hydrogel moves on the microfluidic paper chip during the storage time of fresh pork at 4°C; (b) The correlation between the distance moved and the volatile basic nitrogen in the meat in Figure a. Detailed Implementation

[0041] To better understand the present invention, a more complete description will be given below with reference to the accompanying drawings. Several embodiments of the invention are shown in the drawings. However, the invention can be implemented in many forms and is not limited to the embodiments described herein. Rather, these embodiments are provided so that the invention can be understood more thoroughly.

[0042] This invention provides a DNA hydrogel-assisted microfluidic paper chip for detecting the freshness of fresh meat, the design and working principle of which are as follows: Figure 1 As shown, this microfluidic paper chip consists of an upper hydrophobic paper-based material, a lower paper-based material with hydrophilic flow channels, and a DNA hydrogel located in the lower paper-based reservoir. Figure 1 The DNA hydrogel is composed of DNA nanoscaffolds cross-linked with terminal cytosine-rich nucleic acid fragments. In the absence of putrefactive characteristic gases—volatile basic nitrogen—the DNA nanoscaffolds can stably cross-link by forming i-motif structures, maintaining a gel state and preventing movement within the hydrophilic channels. Figure 2 (b) When fresh meat spoils, the alkaline volatile basic nitrogen gas produced induces the disintegration of the i-motif structure, causing the DNA nanostructures to move away from each other. The DNA hydrogel transitions from a gel state to a solution state, and then moves within the hydrophilic channels. Since the amount of spoilage gas produced is directly proportional to the degree of liquefaction of the DNA hydrogel, the freshness of the fresh meat can be quantitatively analyzed based on the distance the liquid travels within the hydrophilic channels.

[0043] Table 1. DNA sequences of the hydrogels prepared according to this invention

[0044]

[0045]

[0046] In some embodiments of the present invention, the experimental water was all ultrapure water prepared from Milli-Q (18.25 MΩ·cm) ultrapure water (Millipore, Bedford, MA). Unless otherwise specified, all purchased reagents were used directly without further treatment. The main reagents used included: DNA sequences synthesized by Sangon Biotech (Shanghai) Co., Ltd.; hydrochloric acid (Beijing Dingguo Changsheng Biotechnology Co., Ltd.); magnesium chloride, tris(hydroxymethyl)aminomethane, and boric acid (Sinopharm Chemical Reagent Co., Ltd.).

[0047] In some embodiments of the present invention, the main instruments used include: an electrophoresis apparatus (Tanon EPS 600, Shanghai Tianneng Technology Co., Ltd.); a gel imaging system (ChemiDoc XRS+Bio-RAD, Lenovo Biotechnology Co., Ltd.); an ultraviolet spectrophotometer (UV-2700, Shimadzu, Japan); and a circular dichroism spectrometer (J-1500, Japan).

[0048] During the spoilage of fresh meat, volatile basic nitrogen (TVB-N, the only physicochemical indicator for evaluating meat freshness in national standards) produced by the degradation of proteins and amino acids by the body's enzyme system and surface microorganisms serves as a signal molecule. It can change the pH of the environment, thereby causing the paper chip to generate a distance signal, which indicates the freshness of the fresh meat.

[0049] Example 1

[0050] In an embodiment of the present invention, a design concept and characterization method for cytosine-rich nucleic acid sequences are provided.

[0051] Following the design concept of microfluidic paper chips, the DNA hydrogel is composed of DNA nanoscaffolds cross-linked with C-rich nucleic acid fragments at the ends. Therefore, we first investigated the pH response of a simple cytosine-rich nucleic acid sequence (Stand I, 5'-CCCCCCAATCCCCCC-3'). Stand I (2 μM) was reacted with PBS at different pH values ​​at 4°C for 120 min, followed by analysis using circular dichroism spectroscopy (CD). The results are as follows... Figure 3 As shown in (a), when pH ≥ 7, Stand I exhibits a typical positive peak at ~280 nm, while when pH ≤ 6, the positive peak at ~280 nm disappears, and new positive and negative peaks appear at ~290 nm and ~261 nm, respectively, indicating that Stand I can form an i-motif structure under acidic conditions, consistent with expectations. Based on this, we investigated the deconstruction temperature of the i-motif structure formed in pH 5.0 PBS. According to the circular dichroism melting curve, (… Figure 3 (b) The i-motif structure maintains its stability within the temperature range of 0–40℃. These results indicate that the DNA hydrogel prepared using this i-motif structure will possess good thermal stability, thus avoiding false positive signals caused by temperature increases in practical applications.

[0052] Implementation Case 2

[0053] In embodiments of the present invention, a method for preparing a DNA hydrogel is provided. Specifically, the feasibility of preparing a DNA hydrogel using two types of DNA nanomaterials—DNA nanowires and Y-DNA nanoscaffolds—is demonstrated.

[0054] First, DNA nanowires were prepared using two types of single-stranded DNA (L1, L2), and Y-DNA nanoscaffolds were prepared using three types of single-stranded DNA (Y1, Y2, Y3). The specific nucleic acids are shown in Table 1. First, L1 and L2 or Y1, Y2, Y3 were mixed in equimolar ratios in PBS (8mM MgSO4). 2 +In a solution (pH 7.0), the concentration of each single strand was maintained at 500 nM. The mixture was then treated at 95℃ (5 min), 65℃ (2 min), 25℃ (3 min), and 4℃ (2 h). The assembly of the DNA nanowires and γ-DNA nanoscaffolds was then characterized by polyacrylamide gel electrophoresis. The DNA nanowires were characterized using four lanes: L: DNA ladder; lanes 1-2: L1, L2; lane 3: L1+L2. The γ-DNA nanoscaffolds were characterized using seven lanes: L: DNA ladder; lanes 1-3: DNA strands Y1-Y3; lane 4: Y1+Y2; lane 5: Y1+Y3; lane 6: Y2+Y3; lane 7: Y1+Y2+Y3. The voltage was set to 90V, and the electrophoresis time was 100 min. After electrophoresis, the sample was stained with the prepared 1×Gelred post-staining solution and shaken on a shaker in the dark for 30 min. Imaging was then performed using a gel electrophoresis apparatus. Figure 4 (a) The molecular weight of the product in lane 3 is significantly larger than that in other lanes, indicating that L1 and L2 can autonomously assemble into DNA nanowires. Similarly, as... Figure 4 (c) The molecular weight of the product in lane 7 is significantly larger than that in other lanes, so the band moves slower than in other lanes, indicating that Y1, Y2 and Y3 can autonomously assemble into a Y-shaped DNA scaffold with an assembly efficiency of more than 90%.

[0055] Subsequently, containing 20mM H + A 1% hibiscus red solution was mixed with DNA nanowires (1000 μM) and γ-DNA (333 μM) solutions, respectively, to achieve a DNA concentration of 250 μM. + The concentration was 5 mM, and then it was placed in a refrigerator at 4°C for 2 hours to obtain a DNA hydrogel as shown. Figure 4 As shown in (b, d)2. If an untreated 1% hibiscus red solution is added, the solution cannot transform into a gel, and the result is as follows. Figure 4 As shown in (b, d)1.

[0056] Example 3

[0057] This invention provides a method for processing the hydrophilic flow region of a microfluidic paper chip.

[0058] First, the pattern of the paper chip is designed using CAD software, such as... Figure 1As shown. Then, the hydrophobic barrier on the filter paper surface was coated with wax using a wax pen. After heating in a 60℃ constant temperature oven for 2 minutes, the melted paraffin penetrated the filter paper surface, thus forming a paraffin barrier with hydrophobic properties, used to separate the hydrophilic circular reservoirs and flow channels. Then, conductive adhesive was used to attach the hydrophilic and hydrophobic regions of the lower paper base to the sample stage, and the sample was placed in an ion spectrometer for gold sputtering for 30 seconds. Finally, the morphology of the sample was observed using a scanning electron microscope at different magnifications, revealing the hydrophilic channels (…). Figure 5 a) Contains abundant pores, which allow liquid to flow, but are protected by a hydrophobic barrier ( Figure 5 The gap in b) is blocked, and the liquid cannot flow.

[0059] Example 4

[0060] In this invention, ammonia gas is used to simulate the volatile basic nitrogen produced during meat spoilage. To assess the microfluidic paper chip's response sensitivity to ammonia, a 650 mL container was used to simulate the environment of fresh meat storage. Different concentrations of ammonia (10 mL) and ultrapure water (5 mL) were placed in the container, and the mixtures were incubated at 4°C and 25°C for 5 minutes, respectively. The movement distance of the hydrogel prepared using a Y-DNA nanoscaffold within the hydrophilic channels of the paper chip after liquefaction was recorded. Ammonia concentrations: 1-0.05%, 2-0.1%, 3-0.15%, 4-0.2%, 5-0.25%, 6-0.3%, 7-0.4%, 8-0.5%. Figure 6 As shown, at 4℃, the pH change and the distance the solution moves on the paper chip exhibit a certain linear relationship, with the linear regression equation y = 13.56x - 3.96 (R²). 2 =0.9754). However, when the ambient temperature rises to 25℃, the evaporation rate of ammonia increases, leading to an increase in the ammonia content in the environment within the same time frame. Therefore, at low concentrations, the DNA hydrogel travels a longer distance in the hydrophilic channels. Figure 7 ).

[0061] Example 5

[0062] At 650cm 3 200g of fresh pork was placed in a food storage container and stored at 25℃ for 0h, 9h, and 30h respectively. The freshness of the pork was then detected using a DNA hydrogel-assisted microfluidic paper chip. Figure 8As shown, at 0h, because the pork is very fresh and there is no volatile basic nitrogen in the environment, the upper distance signal in the flow channel of the paper-based chip is 0mm. After 9h of storage at 25℃, the pork begins to spoil under the action of microorganisms. Since the degree of spoilage is low at this time, the generated distance signal is only 2mm. After 30h of storage at 25℃, the pork is severely spoiled, and the movement distance generated in the flow channel after liquefaction of the hydrogel prepared by the Y-DNA nanoscaffold increases to 13mm. The above results indicate that the microfluidic paper chip constructed in this invention can accurately indicate the degree of spoilage of fresh meat, and is expected to provide consumers and regulators with a portable, simple, and reliable tool for judging the freshness of meat.

[0063] Example 6

[0064] At 650cm 3 200g of fresh pork was placed in a food storage container and stored at 4℃ for 0-8 days. The freshness of the pork was then assessed using a DNA hydrogel-assisted microfluidic paper chip. Figure 9 As shown in Figure a, during the first 0-4 days, due to the freshness of the pork and the absence or trace amounts of volatile basic nitrogen in the environment, the distance signal in the flow channel of the paper-based chip was 0 mm. After 5 days, the pork began to spoil, and as the storage time continued, the putrefaction by microorganisms intensified, resulting in an increasingly larger distance signal. By day 7, the pork was severely spoiled, and the movement distance generated in the flow channel after liquefaction of the hydrogel prepared from the Y-DNA nanoscaffold increased to 18 mm. The volatile basic nitrogen content in meat products stored for different days was determined using GB 5009.228-2016 "National Food Safety Standard - Determination of Volatile Basic Nitrogen in Food," and the relationship between the volatile basic nitrogen content and the movement distance in the flow channel of the paper-based chip was obtained as follows: Figure 9 As shown in b, GB2707—2016, the National Food Safety Standard for Fresh (Frozen) Livestock and Poultry Products, stipulates that meat with a TVB-N content ≤15mg / 100g is considered fresh. On day 7, the TVB-N content of the pork was 24.4mg / 100g, indicating it was in a state of spoilage. These results demonstrate that the distance signal generated by the microfluidic paper chip constructed in this invention is positively correlated with the TVB-N content of pork, providing a direct and accurate reflection of the spoilage progress of meat and potentially offering comprehensive guidance for consumers, sellers, and regulators.

Claims

1. A microfluidic paper chip for detecting the freshness of fresh meat products, characterized in that, The material comprises an upper hydrophobic paper-based material and a lower paper-based material. Both the upper and lower paper-based materials have circular holes and elongated patterns connected to the circular holes, and the patterns overlap. The circular holes and elongated patterns of the upper paper-based material are hollowed out. The area outside the circular holes and elongated patterns of the lower paper-based material is coated with wax to act as a hydrophobic barrier. The circular holes of the lower paper-based material serve as storage tanks for storing pH-sensitive DNA hydrogels. The elongated portions of the lower paper-based material serve as hydrophilic reaction flow channels. The pH-sensitive DNA hydrogel is formed by acidification of i-motif-functionalized DNA nanoscaffolds. The DNA nanoscaffold is a Y-shaped DNA nanostructure. The end of the DNA nanoscaffold is connected to a short nucleotide sequence rich in cytosine. The length of the short nucleotide sequence rich in cytosine must not exceed the length of any arm or double-stranded fragment of the DNA nanoscaffold. The number of continuous and repeating cytosines in the short nucleotide sequence rich in cytosine must not be less than 3 bp, and the length of the linking sequence located in the middle of the cytosine cluster must not be less than 1 bp. When fresh meat spoils, the alkaline volatile basic nitrogen gas produced induces the i-motif structure to disintegrate, causing the DNA nanostructures to move away from each other. The DNA hydrogel changes from a gel state to a solution state, and then moves in the hydrophilic channels. The freshness of the fresh meat can be quantitatively analyzed based on the distance the liquid moves in the hydrophilic channels.

2. The microfluidic paper chip according to claim 1, characterized in that, The upper paper-based material has distance markings printed next to the long strip.

3. The microfluidic paper chip according to claim 2, characterized in that, The short nucleotide sequence rich in cytosine is 5'-CCCCCCAATCCCCCC-3'.

4. The microfluidic paper chip according to claim 1, characterized in that, The pH-sensitive DNA hydrogel contains a safe and non-toxic dye.

5. The microfluidic paper chip according to claim 4, characterized in that, The added dye was lycopene.

6. The microfluidic paper chip according to claim 1, characterized in that, The upper paper base material is coated paper with a thickness of 0.1 to 0.5 mm, and the lower layer is absorbent dry filter paper.

7. The method for preparing the microfluidic paper chip for detecting the freshness of fresh meat products according to claim 1, characterized in that, The process includes the following: (1) Preparation of pH-sensitive DNA hydrogel: Three single-stranded oligonucleotide chains with cytosine-rich sequences at the ends were added to PBS in an equimolar ratio, mixed, and then reacted at 95 °C for 5 min, 65 °C for 2 min, 25 °C for 3 min, and 4 °C for 2 h to obtain a solution containing Y-type DNA nanostructures; based on this, high concentration of HCl and lycopene were added to the above solution, mixed, and then placed at 4 °C for 2 h to obtain pH-sensitive DNA hydrogel; (2) Preparation method of flow channel: Prepare upper paper base material and lower paper base material, design round holes and long strips with AutoCAD, print them, cut out round holes and long strips in the upper paper base material, and print distance scale on one side of the long strips. The lower paper base material except for the round holes and long strips is sealed with wax. Then place the lower paper base material in a 60 ℃ oven for 2 to 10 min to melt the wax and penetrate into the pores of the paper base material. (3) The upper and lower paper base materials are bonded and fixed, and more than 4 μL of the prepared pH-sensitive DNA hydrogel is added to the circular hole to obtain the microfluidic paper chip.

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