Paper-based microfluidic ratiometric fluorescent chip as well as preparation method and visual detection application thereof

Abstract

The invention discloses a paper-based microfluidic ratiometric fluorescent chip for visually detecting the residual quantity of tetracycline (TC) in animal-derived food as well as preparation and application of the paper-based microfluidic ratiometric fluorescent chip. The chip is developed on the basis of a ratio fluorescent probe (Eu / Tb) composed of europium and terbium rare earth complexes, and shows a'double response-off 'fluorescence quenching effect on TC concentration. The preparation method comprises the following steps: synthesizing Eu and Tb complexes; mixing the two methanol solutions to prepare a probe; drawing a channel pattern containing a sample introduction area and a detection area on filter paper by using a hydrophobic solution to form a paper-based microfluidic substrate; and soaking the substrate in a probe solution or dropwise adding the probe solution into a sample introduction area to prepare a single-concentration type chip or a multi-concentration simultaneous detection type chip. A sample to be detected is dropped to a chip detection area, and after airing, fluorescence color change is observed in a 365 nm ultraviolet camera obscura analyzer. The RGB value of the fluorescent region is extracted by a mobile phone, and the TC residual concentration can be quantitatively analyzed according to a pre-established linear working curve. The chip has the advantages of good specificity, strong interference resistance, simplicity and convenience in operation, low cost, accurate result and the like.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of food safety rapid detection, and particularly relates to a paper-based microfluidic ratio fluorescent chip for visually detecting tetracycline concentration residues in animal-derived food. BACKGROUND

[0002] Tetracycline (TC) is a kind of organic compound with broad-spectrum antibacterial activity and containing polycyclic tetracene skeleton. The unique antibacterial mechanism and potential drug resistance risk of tetracycline make it continue to attract attention in clinical antibacterial therapy. Tetracycline can bind to the 30S subunit of ribosomes, interfere with protein synthesis, affect normal cell function, induce reactive oxygen species (ROS) burst at high concentrations, and cause DNA oxidative damage and cell apoptosis.

[0003] The main purpose of using veterinary drugs is to prevent and treat animal diseases to promote the growth of animals. However, if veterinary drugs are used improperly, a series of adverse effects will occur: first, harm to human health: the chemical components in veterinary drug residues can have toxic reactions on the human body, and long-term consumption or consumption of too much such food can damage human organs and increase the risk of cancer. In addition, most veterinary drugs are antibiotics, which may cause allergic reactions in some people, and long-term consumption of food with excessive antibiotic residues can also cause antibiotic resistance, leading to immune system disorders. Second, it triggers a public safety crisis: food safety is a hot issue of global concern, and veterinary drug residues account for a large proportion of food safety and are closely related to human dietary health. Excessive veterinary drug residues can easily cause human panic in food selection and reduce consumer confidence. Third, it affects the ecological environment: veterinary drug residues enter the environment with livestock excreta, accumulate and react in the soil or water, causing soil and river pollution. Therefore, it is very important to develop a rapid, accurate, and convenient visual detection technology for veterinary drug residues.

[0004] Traditional fluorescence detection methods have the problems of no specificity for veterinary drug residue detection, high detection cost, low sensitivity, strong professional operation, poor selectivity, etc. Paper-based microfluidic chips have the advantages of low cost, environmental protection, and control of liquid flow direction; probes prepared from rare earth complexes have long fluorescence lifetime, high stability, low toxicity, and excellent anti-photobleaching characteristics, and can identify veterinary drugs according to the internal filter effect (IFE) mechanism; the ratio fluorescent sensor is a detection method that measures the change in the intensity ratio of two or more different fluorescence signal emission peaks, which can effectively avoid background interference and increase the detection range, and has excellent selectivity and accuracy. The paper-based microfluidic ratio fluorescent chip is a new type of analysis tool that combines paper-based microfluidic technology with ratio fluorescent detection methods, and has wide application prospects in the fields of environmental monitoring and biological analysis.

[0005] The paper-based microfluidic ratio fluorescent chip is developed, Eu and Tb two rare earth complexes are selected to form a ratio fluorescent probe, the probe has a double-response-off function to the tetracycline (TC) concentration, therefore, the paper-based microfluidic ratio fluorescent chip developed in combination with the intelligent mobile phone identification RGB can be used for visual detection of the tetracycline (TC) concentration in animal-derived food. SUMMARY

[0006] OBJECTIVE

[0007] The main purpose of the present application is to solve how to quickly obtain the amount of veterinary drug residues in animal-derived food, and provide a method for quickly detecting the amount of tetracycline residues in meat products for market supervision departments, manufacturers and consumers. The characteristics of the method mainly include (1) a double-response-off paper-based microfluidic ratio fluorescent chip based on Eu and Tb rare earth complex probes is constructed, and is used for detecting the tetracycline residue concentration in animal-derived food, the method has the characteristics of reducing background interference, sensitive visual detection effect and easy to distinguish; (2) based on the double-response-off ratio fluorescent principle, a paper-based microfluidic ratio fluorescent chip for quickly detecting different concentrations of tetracycline in different foods is developed, which can be used for simultaneous quantitative detection of different tetracycline concentrations in various actual food samples, and solves the problem that the traditional ratio fluorescent paper-based sensor has low quantitative detection accuracy and can only detect a single concentration.

[0008] In order to achieve the above purpose, the present application provides the following technical scheme:

[0009] A preparation method of a paper-based microfluidic ratio fluorescent chip, comprising the following steps:

[0010] (1) Preparation of ratio fluorescent probe

[0011] Step 1: 0.2 mmol of 2,5-dibromothiophene-3,4-dicarboxylic acid and 0.2 mmol of europium nitrate hexahydrate are respectively dissolved in 6 mL of N,N-dimethylformamide (DMF), 4 mL of distilled water is added until the solution is clear, and then 0.2 mmol of terephthalic acid is added to the above solution to obtain a mixture; the mixture is transferred to a polytetrafluoroethylene high-pressure reaction kettle, heated at 100 DEG C for 72 h, cooled to room temperature, and then a colorless crystal is obtained, the crystal is washed with DMF and distilled water three times respectively, and then the colorless crystal is dried in a vacuum oven at 50 DEG C to obtain a fluorescent material product, which is marked as Eu complex fluorescent material, and a 200 μmol / L Eu complex methanol solution is prepared for standby;

[0012] Step 2: 0.2 mmol of 2,5-dibromothiophene-3,4-dicarboxylic acid and 0.2 mmol of terbium nitrate hexahydrate were weighed into 6 mL of N,N-dimethylformamide (DMF), 4 mL of distilled water was added until the solution was clear, and then 0.2 mmol of terephthalic acid was added to the above solution to obtain a mixture; the mixture was transferred to a polytetrafluoroethylene high-pressure reaction kettle, heated at 100°C for 72 h, and after cooling to room temperature, colorless crystals were obtained; the crystals were washed with DMF and distilled water three times, respectively, and then the colorless crystals were dried in a vacuum oven at 50°C to obtain a fluorescent material product, which was recorded as a Tb complex fluorescent material, and a 200 μmol / L Tb complex methanol solution was prepared and used later;

[0013] Step 3: 200 μmol / L of Eu complex methanol solution and 200 μmol / L of Tb complex methanol solution were mixed in a 1:1 volume ratio to obtain an Eu / Tb complex ratio fluorescent probe;

[0014] (2) Preparation of a paper-based microfluidic ratio fluorescent chip

[0015] Step 1: The high-viscosity butter double-sided adhesive tape release paper was peeled off, leaving only the adhesive layer containing the adhesive, 70 mL of toluene solution was measured in a beaker, 8 g of adhesive was dissolved in the toluene solution, and a glass rod was stirred for 5 min to dissolve completely to obtain a hydrophobic solution; the hydrophobic solution was filled into an empty water-based pen, and a large circle with a diameter of 4.5 mm was drawn on a new star qualitative filter paper as a sample inlet area, and a small circle with a diameter of 3.0 mm was drawn as a detection area, and a pattern of 1.2 mm long and 2.0 mm wide flow channels was connected between each detection area; the patterned filter paper was dried in a fume hood for 30 min to establish a hydrophobic channel, and finally an internal hydrophilic and external hydrophobic paper-based microfluidic chip was formed on the filter paper;

[0016] Step 2: The paper-based microfluidic chip obtained in step 1 of the preparation of the paper-based microfluidic ratio fluorescent chip in (2) above was soaked in the Eu / Tb complex ratio fluorescent probe solution obtained in step 3 of the preparation of the ratio fluorescent probe in (1) above, and after 10 min, it was taken out and dried to obtain a paper-based microfluidic ratio fluorescent chip, which can detect one concentration of a substance, and is recorded as a single-concentration detection type chip; or the paper-based microfluidic chip obtained in step 1 of the preparation of the paper-based microfluidic ratio fluorescent chip in (2) above was added with the Eu / Tb complex probe solution at the sample inlet area of the paper-based microfluidic chip, and after 10 min, a paper-based microfluidic chip ratio fluorescent sensor was obtained, which can simultaneously detect multiple concentrations of a substance, and is recorded as a multi-concentration simultaneous detection type chip.

[0017] The paper-based microfluidic ratio fluorescence chip prepared according to the above method can be used for visual detection of tetracycline residues in meat products, and is characterized by comprising the following steps:

[0018] For single-concentration detection chip application: 200 uL of a tetracycline-containing solution sample to be detected is added dropwise to the single-concentration detection chip of claim 1, naturally air-dried, observed under a 365 nm ultraviolet dark box analyzer, the RGB value of the fluorescent color on the single-concentration detection chip is extracted by using a smart phone APP color identifier, and then quantitative analysis is carried out according to the pre-established working curve, so as to determine the tetracycline concentration in the sample; For multi-concentration simultaneous detection chip application: 200 uL of a tetracycline-containing solution sample to be detected with different concentrations is added dropwise to each detection area of the multi-concentration detection chip of claim 1, naturally air-dried, observed under a 365 nm ultraviolet dark box analyzer, the RGB value of the fluorescent color on each detection area is extracted by using a smart phone APP color identifier, and then quantitative analysis is carried out according to the pre-established working curve, so as to determine the tetracycline concentration in each sample.

[0019] Technical advantages and effects of the present application: (1) According to the “double response - off” function of the europium (Eu) and terbium (Tb) rare earth complex fluorescent probe to the tetracycline concentration, the paper-based microfluidic ratio fluorescence chip is designed and developed. The advantage is that it does not need to introduce specific biological materials such as antigens, antibodies and aptamers, which simplifies the preparation process and significantly shortens the time and cost of chip preparation. (2) The paper-based microfluidic ratio fluorescence chip prepared by the present application can be used for visual detection of tetracycline residues in actual food samples, and only needs to be combined with a smart phone APP color identifier to realize rapid and accurate detection of tetracycline residues, and the operation is simple. BRIEF DESCRIPTION OF DRAWINGS

[0020] Figure 1 It is a graph of the influence of the Eu / Tb complex fluorescent probe solution on the fluorescence of tetracycline with different concentrations.

[0021] Figure 2 It is a graph of the anti-interference test results of the Eu / Tb complex fluorescent probe solution.

[0022] Figure 3 It is a graph of the stability (a), anti-bleaching (b) and precision (c) test results of the Eu / Tb complex solution.

[0023] Figure 4 It is a scanning electron microscope graph of the paper-based functionalization before and after the present application: (a) (c) SEM before paper-based functionalization; (b) (d) SEM after paper-based functionalization.

[0024] Figure 5The characterization results of the paper-based functionalized before and after the application: (a) (c) SEM images; (b), (d) (k) EDS element distribution map.

[0025] Figure 6 The Eu complex and Tb complex powder XRD chart of the application: (a) Eu complex simulation and test PXRD; (b) Tb complex simulation and test PXRD.

[0026] Figure 7 The linear working curve chart of the Eu / Tb complex functionalized paper-based sensor of the application. DETAILED DESCRIPTION

[0027] The application will be further described in detail below in combination with the drawings and specific embodiments, but the protection scope of the application is not limited thereto.

[0028] Example 1

[0029] In order to further illustrate the application, taking the preparation of tetracycline paper-based microfluidic ratio fluorescent chip and its use in the detection of tetracycline residues in pork as an example, the specific steps are as follows:

[0030] Step 1, preparation of europium (Eu) rare earth complex material: 0.2 mmol of 2,5-dibromo thiophene-3,4-dicarboxylic acid and 0.2 mmol of europium nitrate hexahydrate were respectively dissolved in 6 mL of N,N-dimethylformamide (DMF), 4 mL of distilled water was added until the solution was clear. Then, 0.2 mmol of terephthalic acid was added to the above solution. The obtained mixture was transferred to a polytetrafluoroethylene high-pressure reaction kettle, and heated at 100℃ for 72 h, cooled to room temperature to obtain colorless crystals, which were washed with DMF and distilled water for three times respectively, and then the obtained colorless crystals were dried in a vacuum oven at 50℃ to obtain Eu complex fluorescent material, and prepared into 200 μmol / L Eu complex methanol solution for further use.

[0031] Step 2, preparation of europium (Tb) rare earth complex material: 0.2 mmol of 2,5-dibromo thiophene-3,4-dicarboxylic acid and 0.2 mmol of terbium nitrate hexahydrate were respectively dissolved in 6 mL of N,N-dimethylformamide (DMF), 4 mL of distilled water was added until the solution was clear. Then, 0.2 mmol of terephthalic acid was added to the above solution. The obtained mixture was transferred to a polytetrafluoroethylene high-pressure reaction kettle, and heated at 100℃ for 72 h, cooled to room temperature to obtain colorless crystals, which were washed with DMF and distilled water for three times respectively, and then the obtained colorless crystals were dried in a vacuum oven at 50℃ to obtain Tb complex fluorescent material, and prepared into 200 μmol / L Tb complex methanol solution for further use.

[0032] Step 3, preparation of Eu / Tb complex ratio fluorescent probe

[0033] Mix 200 μmol / L Eu complex methanol solution and 200 μmol / L Tb complex methanol solution at a volume ratio of 1:1 to obtain the Eu / Tb complex ratio fluorescent probe.

[0034] Figure 1 is a fluorescence intensity curve of Eu / Tb complex ratio fluorescent probe solution of the present application; Figure 1 Figure 2 is a fluorescence intensity curve of Eu / Tb complex ratio fluorescent probe solution of the present application; Figure 2 Figure 3 is a fluorescence intensity curve of Eu / Tb complex ratio fluorescent probe solution of the present application; Figure 3 Figure 4 is a fluorescence intensity curve of Eu / Tb complex ratio fluorescent probe solution of the present application; and Figure 1 Figure 5 is a fluorescence intensity curve of Eu / Tb complex ratio fluorescent probe solution of the present application. Figure 2 Figure 6 is a fluorescence intensity curve of Eu / Tb complex ratio fluorescent probe solution of the present application. Figure 3 It can be seen from the figures that the fluorescence intensity of the Eu complex and the Tb complex gradually decreases with the increase of the TC concentration, indicating that the detection mechanism of the ratio fluorescent probe is "double response-off", and the ratio fluorescent probe has good anti-interference, stability, anti-bleaching and precision.

[0035] Step 4, preparation of paper-based microfluidic ratio fluorescent chip

[0036] Single concentration detection type chip preparation: soak the paper base (Xinxing brand qualitative filter paper) in the Eu / Tb complex ratio fluorescent probe solution, so that the Eu / Tb complex is adsorbed on the surface of the paper base to realize the functionalization of the paper base surface, take out after 10 min, and dry to obtain the Eu / Tb complex functionalized paper-based microfluidic ratio fluorescent chip (single concentration detection type), which is stored in a refrigerator; multi-concentration simultaneous detection type chip preparation: add the Eu / Tb complex probe solution to the sample inlet area of the paper-based microfluidic chip to functionalize it, and then add different concentrations of tetracycline (TC) solution to the detection area, wait for 10 min, and then place the functionalized paper-based microfluidic ratio fluorescent chip under the ultraviolet dark box analyzer to observe the phenomenon, thereby obtaining the Eu / Tb complex functionalized paper-based microfluidic ratio fluorescent chip (multi-concentration simultaneous detection type chip).

[0037] Figure 7 is a TEM image of the paper-based microfluidic ratio fluorescent chip of the present application; Figure 4 Figure 8 is an EDS image of the paper-based microfluidic ratio fluorescent chip of the present application; Figure 5 Figure 9 is an XRD image of the paper-based microfluidic ratio fluorescent chip of the present application. Figure 6 Figure 10 is a fluorescence intensity curve of the paper-based microfluidic ratio fluorescent chip of the present application. Figure 4 Figure 11 is a fluorescence intensity curve of the paper-based microfluidic ratio fluorescent chip of the present application. Figure 5 Figure 12 is a fluorescence intensity curve of the paper-based microfluidic ratio fluorescent chip of the present application. Figure 6 It can be seen from the figures that the Eu complex and the Tb complex have high purity and have been successfully functionalized on the paper-based microfluidic chip.

[0038] Step 5, preparation of working curve

[0039] Different concentrations (60~350 μmoL / L) of tetracycline hydrochloride solution were added dropwise to the paper-based microfluidic ratio fluorescence chip, and after drying, the color reaction was observed carefully by relying on the ultraviolet dark box analyzer, and the color results were recorded by using the camera function of the smart phone, and the professional color analysis application program was used to quantitatively analyze the RGB parameters of the obtained image. Taking the concentration (60~350 μmoL / L) of tetracycline as the abscissa and the extracted paper color R' value as the ordinate, the working curve graph of the Eu / Tb complex functionalized paper-based microfluidic ratio fluorescence chip was drawn. The results show that the concentration of tetracycline and the color R' value of the paper-based microfluidic chip have a good linear relationship, and the linear equation is R'=-0.26129C TC +270.48765(R 2 =0.99503), and the detection limit (LOD) is calculated as 58.32 μmoL / L.

[0040] The paper-based microfluidic ratio fluorescence chip of the present application has a linear working curve graph. As shown in the attached Figure 7 figure, with the increase of the concentration of tetracycline hydrochloride added dropwise, the R' value decreases continuously, and the two show a good linear relationship, and the color of the paper-based microfluidic chip also appears the phenomenon of red to green. Figure 7

[0041] Step 6 Actual sample analysis

[0042] Prepare the pretreated sample solution: weigh 10.0 g of chopped fresh pork in a beaker, add 40 mL of methanol solution, mix and seal with plastic wrap. Stir the pork sample and methanol solution with a magnetic stirrer for 30 min to ensure that the pork sample is fully dispersed in the methanol solution, so as to effectively extract the target analyte in the pork matrix. Finally, the mixture is placed in a centrifugal tube and centrifuged in a high-speed centrifuge at a speed of 4000 r / min for 15 min. The clear solution containing the extracted analyte is collected, and the pretreated sample solution is obtained.

[0043] ​Spiked recovery experiment: The pretreated pork sample solution was subjected to spiked recovery experiment, and the spiked tetracycline concentrations were 60, 180, 200, 250 and 350 μmoL / L. The total volume of 200 uL of the spiked tetracycline solution was added dropwise to the paper-based microfluidic ratiometric fluorescent chip, naturally dried, and the RGB value of the color was extracted by shooting in the 365 nm ultraviolet dark box analyzer. The tetracycline concentration, the recovery rate of tetracycline and the relative standard deviation were calculated by substituting the linear equation obtained in step 5. The results are shown in Table 1. As can be seen from Table 1, through the spiked recovery experiment, the recovery rate of tetracycline in the pretreated pork sample solution was 93.39%~101.05%, and the RSD was less than 3.00%, indicating that the functionalized paper-based microfluidic ratiometric fluorescent chip has high detection accuracy and precision, and can be used for the determination of actual samples.

[0044] Table 1. Detection of TC concentration in animal-derived food by Eu / Tb complex functionalized paper-based microfluidic ratiometric fluorescent chip spiked recovery experiment (n=3)

[0045]

Claims

1. A method for preparing a paper-based microfluidic ratiometric fluorescent chip, characterized in that, Comprising the following steps: (1) Preparation of the ratio fluorescent probe Step 1: 0.2 mmol of 2,5-dibromothiophene-3,4-dicarboxylic acid and 0.2 mmol of europium nitrate hexahydrate were weighed and dissolved in 6 mL of N,N-dimethylformamide (DMF), 4 mL of distilled water was added until the solution was clear, then 0.2 mmol of terephthalic acid was added to the above solution to obtain a mixture; the mixture was transferred to a polytetrafluoroethylene high-pressure reaction kettle, heated at 100℃ for 72 h, cooled to room temperature to obtain colorless crystals, which were washed with DMF and distilled water three times respectively, and then dried in a vacuum oven at 50℃ to obtain a fluorescent material product, which was recorded as Eu complex fluorescent material, and prepared into a 200 μmol / L Eu complex methanol solution for later use; Step 2: 0.2 mmol of 2,5-dibromothiophene-3,4-dicarboxylic acid and 0.2 mmol of terbium nitrate hexahydrate were weighed and dissolved in 6 mL of N,N-dimethylformamide (DMF), 4 mL of distilled water was added until the solution was clear, then 0.2 mmol of terephthalic acid was added to the above solution to obtain a mixture; the mixture was transferred to a polytetrafluoroethylene high-pressure reaction kettle, heated at 100℃ for 72 h, cooled to room temperature to obtain colorless crystals, which were washed with DMF and distilled water three times respectively, and then dried in a vacuum oven at 50℃ to obtain a fluorescent material product, which was recorded as Tb complex fluorescent material, and prepared into a 200 μmol / L Tb complex methanol solution for later use; Step 3: 200 μmol / L of Eu complex methanol solution and 200 μmol / L of Tb complex methanol solution were mixed in a volume ratio of 1:1 to obtain an Eu / Tb complex ratio fluorescent probe; (2) Preparation of paper-based microfluidic ratio fluorescent chip Step 1: The high-viscosity butter double-sided adhesive tape release paper was peeled off, leaving only the adhesive layer containing the adhesive, 70 mL of toluene solution was measured in a beaker, 8 g of adhesive was dissolved in the toluene solution, and a glass rod was stirred for 5 min to dissolve completely to obtain a hydrophobic solution; the hydrophobic solution was filled into an empty water-based pen, and a large circle with a diameter of 4.5 mm as a sample inlet and a small circle with a diameter of 3.0 mm as a detection zone were drawn on a new star qualitative filter paper, and a pattern of 1.2 mm long and 2.0 mm wide flow channel was connected between each detection zone, the patterned filter paper was dried in a fume hood for 30 min to establish a hydrophobic channel, and finally an internal hydrophilic and external hydrophobic paper-based microfluidic chip was formed on the filter paper; Step 2: The paper-based microfluidic chip obtained in step 1 in the preparation of the paper-based microfluidic ratiometric fluorescent chip above (2) is added with the Eu / Tb complex probe solution at the sample inlet of the paper-based microfluidic chip, and is allowed to stand for 10 min, to obtain a paper-based microfluidic ratiometric fluorescent sensor, which can simultaneously detect multiple concentrations of a substance, and is recorded as a multi-concentration simultaneous detection type chip; if only one concentration of a substance is to be detected, a new star brand qualitative filter paper is cut into a circle with a diameter of 4.5 mm, is soaked in the Eu / Tb complex ratiometric fluorescent probe solution obtained in step 3 in the preparation of the ratiometric fluorescent probe above (1), is taken out after 10 min, and is dried, to obtain a paper-based microfluidic ratiometric fluorescent chip, which only detects one concentration of a substance, and is recorded as a single-concentration detection type chip.

2. Paper-based microfluidic ratiometric fluorescent chip prepared according to the method of claim 1, for the visual detection of tetracycline residues in meat products, characterized by, The method comprises the following steps: For application of the single-concentration detection type chip, 200 uL of a tetracycline solution sample to be detected is added to the single-concentration detection type chip prepared by the method of claim 1, is naturally dried, is observed under a 365 nm ultraviolet dark box analyzer, the RGB value of the fluorescent color on the single-concentration detection type chip is extracted by using a color identifier of a smart phone APP, quantitative analysis is carried out according to a previously established working curve, and the tetracycline concentration in the sample is determined; for application of the multi-concentration simultaneous detection type chip, 200 uL of a tetracycline sample solution with different concentrations to be detected is added to each detection area of the multi-concentration detection type chip of claim 1, is naturally dried, is observed under a 365 nm ultraviolet dark box analyzer, the RGB value of the fluorescent color on each detection area is extracted by using a color identifier of a smart phone APP, quantitative analysis is carried out according to a previously established working curve, and the tetracycline concentration in each sample is determined.

Images