Norfloxacin visual ratio fluorescence detection method based on integrated detection device and application of norfloxacin visual ratio fluorescence detection method

Abstract

The invention belongs to the field of food safety detection, and particularly relates to a norfloxacin visual ratio fluorescence detection method based on an integrated detection device and application of the norfloxacin visual ratio fluorescence detection method. The integrated detection device provided by the invention comprises a filtering module and a detection module, the filtering module comprises a first container as well as modified sodium alginate hydrogel and glass wool which are filled in the first container; the modified sodium alginate hydrogel comprises a sodium alginate matrix and polyvinyl alcohol doped in the sodium alginate matrix; the detection module comprises a second container and a sodium alginate hydrogel bead which is filled in the second container and comprises a probe; the probe comprises a copper nano-cluster which is coordinated with terbium ions and modified by dithioerythrine. The integrated detection device provided by the invention is simple to operate and reliable in result, realizes rapid detection and visual response of norfloxacin, and has very high practicability and wide applicability.

Description

Technical Field

[0001] The present invention belongs to the technical field of food safety detection, and in particular relates to a norfloxacin visual ratio fluorescence detection method based on an integrated detection device and an application thereof. Background Art

[0002] The reliance on antibiotics in intensive chicken farming significantly increases the risk of drug residues in eggs. Norfloxacin (NOR), a fluoroquinolone antibiotic (FQ), is a simple, broad-spectrum, and inexpensive fluoroquinolone antibiotic widely used in the poultry industry to prevent and treat bacterial infections and other diseases. However, due to improper use and poor biodegradability, NOR is easily retained and enters the human body through the food chain. This can lead to the development of antibiotic resistance and cause diseases such as tendon damage, hepatotoxicity, adverse central nervous system reactions, and gastrointestinal disorders. Currently, many countries have banned the use of some antibiotics in poultry farming and have strictly established antibiotic residue limits. Therefore, establishing accurate and sensitive methods for detecting NOR residues is crucial for ensuring egg quality and safety and promoting the healthy development of the poultry and egg industry.

[0003] In recent years, fluorescent probe-based sensing technologies have attracted widespread attention due to their rapid response and ease of use. However, existing methods still face challenges such as insufficient stability of solution probes in the presence of complex egg matrices (such as proteins and lipids), easy aggregation of probe materials, and reliance on laboratory fluorescence spectrophotometers, making them difficult to meet the needs of rapid on-site screening. Summary of the Invention

[0004] The present invention aims to provide a method and application for the visual ratiometric fluorescence detection of norfloxacin based on an integrated detection device. The integrated detection device provided by the present invention is simple to operate, provides reliable results, exhibits good stability, and is not susceptible to interference. It enables rapid detection and visual response of norfloxacin, and has strong practicality and wide applicability.

[0005] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0006] An integrated detection device comprises a filtration module and a detection module; the filtration module comprises a first container and modified sodium alginate hydrogel and glass wool filled in the first container; the modified sodium alginate hydrogel comprises a sodium alginate matrix and polyvinyl alcohol doped in the sodium alginate matrix; the detection module comprises a second container and sodium alginate hydrogel beads containing probes filled in the second container, wherein the probes comprise copper nanoclusters modified with dithioerythrocyanide coordinated with terbium ions.

[0007] Preferably, the first container in the filtration module is a syringe or a chromatography column, and the modified sodium alginate hydrogel and glass wool are filled in the first container from bottom to top, the filling height of the modified sodium alginate hydrogel is 1 to 3 cm, and the filling height of the glass wool is 1 to 3 cm; the filling height of the sodium alginate hydrogel beads containing probes in the detection module is 1 to 3 cm; the filtration module is arranged above the detection module.

[0008] Preferably, the method for preparing the sodium alginate hydrogel beads containing the probe comprises the following steps:

[0009] performing a first mixing of erythrodithiocarbamate-modified copper nanoclusters, terbium nitrate, and water to obtain a first dispersion;

[0010] performing a second mixing of the first dispersion liquid and the sodium alginate solution to obtain a second dispersion liquid;

[0011] The second dispersion is cross-linked with a calcium chloride solution to obtain the sodium alginate hydrogel beads containing the probe.

[0012] Preferably, the dosage ratio of the dithioerythrocyanide-modified copper nanoclusters and terbium nitrate is 9 mg:0.0005 mmol; the mass concentration of the sodium alginate solution is 1-3%; the dosage ratio of the dithioerythrocyanide-modified copper nanoclusters and the sodium alginate solution is 9 mg:10 mL; the mass concentration of the calcium chloride solution is 1-3%, and the cross-linking temperature is 15-30°C.

[0013] Preferably, the method for preparing the modified sodium alginate hydrogel comprises the following steps:

[0014] mixing polyvinyl alcohol, sodium alginate and water to obtain a mixture;

[0015] The mixture is dried and then immersed in a calcium chloride solution for cross-linking to obtain the modified sodium alginate hydrogel.

[0016] Preferably, the mass ratio of the polyvinyl alcohol to sodium alginate is 3:1, the mixing temperature is 95-105° C., the drying temperature is 50-60° C., the drying time is 7-9 hours, and the cross-linking time is 25-35 minutes.

[0017] The present invention provides application of the integrated detection device described in the above technical solution in visual ratio fluorescence detection of norfloxacin.

[0018] The present invention provides a method for detecting norfloxacin by visual ratio fluorescence, comprising the following steps:

[0019] The test solution is mixed with trichloroacetic acid solution, and the pH value is adjusted to neutral to obtain a treatment solution;

[0020] Adding the treated liquid to the filtration module of the integrated detection device described in the above technical solution for filtration, and adding the obtained filtrate to the detection module for coordination reaction;

[0021] irradiating the detection module after the coordination reaction with ultraviolet light to obtain an ultraviolet fluorescence photograph;

[0022] The fluorescence information in the ultraviolet irradiation fluorescence photograph is converted into RGB data, and the content of norfloxacin in the test solution is obtained according to the RGB data and a standard curve of norfloxacin; the ordinate of the standard curve of norfloxacin is the intensity ratio of green fluorescence to red fluorescence of sodium alginate hydrogel beads containing the probe, and the abscissa of the standard curve of norfloxacin is the concentration of norfloxacin.

[0023] Preferably, the test liquid is homogenized egg liquid containing norfloxacin; the volume concentration of the trichloroacetic acid solution is 1-3%, and the volume ratio of the test liquid to the trichloroacetic acid solution is 1:10.

[0024] Preferably, the coordination reaction time is 2 to 4 minutes, and the wavelength of the ultraviolet light is 365 nm.

[0025] The present invention provides an integrated detection device, including a filtration module and a detection module; the filtration module includes a first container and modified sodium alginate hydrogel and glass wool filled in the first container; the modified sodium alginate hydrogel includes a sodium alginate matrix and polyvinyl alcohol doped in the sodium alginate matrix; the detection module includes a second container and sodium alginate hydrogel beads containing probes filled in the second container, and the probes include copper nanoclusters modified with dithioerythrocyanide coordinated with terbium ions. The modified sodium alginate hydrogel (PVA-SA hydrogel) and glass wool in the filtration module of the present invention form a double-layer filtration structure, which is beneficial to the separation and purification of impurities such as proteins in the test liquid, and solves the current problem of insufficient probe stability caused by interference from proteins and lipids; at the same time, it can reduce the loss of NOR. The detection module is filled with sodium alginate hydrogel beads (TD-SA hydrogel beads) containing probes, and the probes include copper nanoclusters modified with dithioerythrocyanide coordinated with terbium ions (Tb 3+ / DTE-CuNCs). Tb 3+ The / DTE-CuNCs probe showed red fluorescence of dithioerythrocyanide-modified copper nanoclusters (DTE-CuNCs) under 365nm ultraviolet light, and the TD-SA hydrogel beads containing the probe also showed strong red fluorescence. When NOR was present, the carboxyl and carbonyl groups of NOR reacted with the terbium ions (Tb 3+ ) coordination, sensitized Tb 3+This caused it to emit intrinsic green fluorescence, while the red fluorescence of DTE-CuNCs was not disturbed.

[0026] Compared with the existing fluorescence technology for detecting NOR, the present invention uses an integrated detection device for ratiometric fluorescence visual detection of NOR, which has at least the following advantages:

[0027] 1. The present invention is simple to operate, has reliable results, and is low in cost. It only takes 3 minutes to complete the NOR response and has good application prospects.

[0028] 2. The present invention uses ratio fluorescence detection to improve sensitivity and accuracy through self-calibration, which is more accurate and stable than single signal detection and is convenient for visual detection.

[0029] 3. The present invention integrates sample pre-treatment and signal readout, breaking through the traditional method's dependence on laboratory instruments, and has strong practicality and wide applicability. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 The comparison chart of protein removal rate and FQs loss rate of glass wool with different filling heights;

[0031] Figure 2 The fluorescence photos of TD-SA hydrogel beads immersed in NOR solution at different times. 3+ Fluorescence spectra of / DTE-Cu NCs probe solution and the changes in G / R values ​​of TD-SA hydrogel beads in the presence of different concentrations of NOR;

[0032] Figure 3 A physical picture of the integrated detection device and the response of the integrated detection device to NOR solution and egg liquid containing NOR;

[0033] Figure 4 Fluorescence photos of TD-SA hydrogel beads in the presence of different concentrations of NOR and the corresponding G / R value curves. DETAILED DESCRIPTION

[0034] The present invention provides an integrated detection device, comprising a filtration module and a detection module; the filtration module comprises a first container and modified sodium alginate hydrogel and glass wool filled in the first container; the modified sodium alginate hydrogel comprises a sodium alginate matrix and polyvinyl alcohol doped in the sodium alginate matrix; the detection module comprises a second container and sodium alginate hydrogel beads containing probes filled in the second container, wherein the probes comprise copper nanoclusters modified with dithioerythrocyanide coordinated by terbium ions.

[0035] In the present invention, unless otherwise specified, all preparation raw materials / components are commercially available products well known to those skilled in the art.

[0036] The integrated detection device provided by the present invention includes a filtration module, which includes a first container and a modified sodium alginate hydrogel and glass wool filled in the first container; the modified sodium alginate hydrogel includes a sodium alginate matrix and polyvinyl alcohol doped in the sodium alginate matrix. As one embodiment of the present invention, the first container in the filtration module is a syringe or a chromatography column, specifically a 10mL syringe; the modified sodium alginate hydrogel and glass wool are filled in the first container from bottom to top, and the modified sodium alginate hydrogel can be filled to a height of 1 to 3 cm, specifically 2 cm; the glass wool can be filled to a height of 1 to 3 cm, specifically 1 cm, 1.5 cm, 2 cm, 2.5 cm, or 3 cm.

[0037] As an embodiment of the present invention, the preparation method of the modified sodium alginate hydrogel comprises the following steps:

[0038] mixing polyvinyl alcohol, sodium alginate and water to obtain a mixture;

[0039] The mixture is dried and then immersed in a calcium chloride solution for cross-linking to obtain the modified sodium alginate hydrogel.

[0040] The present invention mixes polyvinyl alcohol, sodium alginate, and water to obtain a mixture. As one embodiment of the present invention, the mass ratio of the polyvinyl alcohol to the sodium alginate can be 3:1, and the amount ratio of the polyvinyl alcohol to the water can be 9g:108mL. The mixing is performed under stirring conditions at a temperature of 95 to 105°C, specifically 100°C, and the resulting mixture is a uniform viscous liquid.

[0041] After obtaining the mixture, the present invention dries the mixture and then soaks it in a calcium chloride solution for cross-linking to obtain the modified sodium alginate hydrogel. As one embodiment of the present invention, before drying the mixture, the mixture is further cooled to 20-30°C; the drying can be oven drying, the drying temperature can be 50-60°C, specifically 55°C, and the drying time can be 7-9 hours, specifically 8 hours; the mass concentration of the calcium chloride solution can be 1-3%, specifically 2%, and the cross-linking time can be 25-35 minutes, specifically 30 minutes.

[0042] The integrated detection device provided herein includes a detection module, comprising a second container and sodium alginate hydrogel beads containing a probe within the second container. The probe comprises copper nanoclusters modified with terbium ion-coordinated dithioerythrocyanide. In one embodiment, the second container is a glass cuvette. The sodium alginate hydrogel beads containing the probe within the detection module can be filled to a height of 1 to 3 cm, specifically 2 cm. In one embodiment, the filtration module in the integrated detection device is positioned above the detection module.

[0043] As an embodiment of the present invention, the method for preparing the sodium alginate hydrogel beads containing the probe comprises the following steps:

[0044] performing a first mixing of erythrodithiocarbamate-modified copper nanoclusters, terbium nitrate, and water to obtain a first dispersion;

[0045] performing a second mixing of the first dispersion liquid and the sodium alginate solution to obtain a second dispersion liquid;

[0046] The second dispersion is cross-linked with a calcium chloride solution to obtain the sodium alginate hydrogel beads containing the probe.

[0047] In the present invention, erythrodithiocarbamate-modified copper nanoclusters, terbium nitrate, and water are first mixed to obtain a first dispersion. In one embodiment of the present invention, the erythrodithiocarbamate-modified copper nanoclusters and terbium nitrate can be used in a ratio of 9 mg:0.0005 mmol. The first mixing is performed under stirring for 25 to 35 seconds, specifically 30 seconds.

[0048] After obtaining the first dispersion, the present invention performs a second mixing of the first dispersion with a sodium alginate solution to obtain a second dispersion. As one embodiment of the present invention, the mass concentration of the sodium alginate can be 1 to 3%, specifically 2%, and the ratio of dithioerythrocyanol-modified copper nanoclusters to the sodium alginate solution in the first dispersion is 9 mg:10 mL. The second mixing is performed under stirring conditions, the temperature of the second mixing can be 20 to 30° C., and the time can be 15 to 25 minutes, specifically 20 minutes.

[0049] After obtaining the second dispersion, the present invention cross-links the second dispersion with a calcium chloride solution to obtain the sodium alginate hydrogel beads containing the probe. As one embodiment of the present invention, the mass concentration of the calcium chloride solution can be 1-3%, specifically 2%. Cross-linking the second mixed solution with the calcium chloride solution specifically includes dripping the second mixed solution into the calcium chloride solution. The cross-linking temperature is 15-30°C.

[0050] As an embodiment of the present invention, the preparation method of the dithioerythrocyanide-modified copper nanoclusters comprises the following steps:

[0051] Dithioerythrocyanide, copper sulfate pentahydrate and water were mixed and the pH value was adjusted to 8 to obtain dithioerythrocyanide-modified copper nanoclusters.

[0052] As an embodiment of the present invention, the usage ratio of dithioerythrocyanide to copper sulfate pentahydrate is 160 mg:0.08 mmol; the dithioerythrocyanide, copper sulfate pentahydrate and water are mixed under stirring conditions for 25 to 35 minutes, specifically 30 minutes; the solution used to adjust the pH value is a sodium hydroxide solution, and the concentration of the sodium hydroxide solution can be 0.2 to 0.3 M, specifically 0.25 M; after adjusting the pH value, a second stirring is performed, and the second stirring time can be 15 to 25 minutes, specifically 20 minutes, to obtain a dithioerythrocyanide-modified copper nanocluster aqueous solution; the present invention also includes purifying the obtained dithioerythrocyanide-modified copper nanocluster aqueous solution, and the purification is performed by filtering with a 3KD ultrafiltration tube; the purified dithioerythrocyanide-modified copper nanocluster aqueous solution is freeze-dried to obtain dithioerythrocyanide-modified copper nanocluster powder; the obtained dithioerythrocyanide-modified copper nanocluster powder is stored in the present invention, and the storage temperature is -20°C.

[0053] The present invention provides application of the integrated detection device described in the above technical solution in visual ratio fluorescence detection of norfloxacin.

[0054] The present invention provides a method for detecting norfloxacin by visual ratio fluorescence, comprising the following steps:

[0055] The test solution is mixed with trichloroacetic acid solution, and the pH value is adjusted to neutral to obtain a treatment solution;

[0056] Adding the treated liquid to the filtration module of the integrated detection device described in the above technical solution for filtration, and adding the obtained filtrate to the detection module for coordination reaction;

[0057] irradiating the detection module after the coordination reaction with ultraviolet light to obtain an ultraviolet fluorescence photograph;

[0058] The fluorescence information in the ultraviolet irradiation fluorescence photograph is converted into RGB data, and the content of norfloxacin in the test solution is obtained according to the RGB data and a standard curve of norfloxacin; the ordinate of the standard curve of norfloxacin is the intensity ratio of green fluorescence to red fluorescence of sodium alginate hydrogel beads containing the probe, and the abscissa of the standard curve of norfloxacin is the concentration of norfloxacin.

[0059] The present invention mixes a test liquid with a trichloroacetic acid solution and adjusts the pH to neutral to obtain a treated liquid. As one embodiment of the present invention, the test liquid is a homogenized egg liquid containing norfloxacin; the volume concentration of the trichloroacetic acid solution can be 1 to 3%, specifically 2%, and the volume ratio of the test liquid to the trichloroacetic acid solution is 1:10. The mixing is ultrasonic treatment, and the ultrasonic treatment time can be 15 to 25 minutes, specifically 20 minutes. The solution for adjusting the pH value is a sodium hydroxide solution.

[0060] After obtaining the treated liquid, the present invention adds the treated liquid to the filtration module of the integrated detection device described in the above technical solution for filtration, and the resulting filtrate is added to the detection module to perform a coordination reaction. As one embodiment of the present invention, the coordination reaction time can be 2 to 4 minutes, specifically 3 minutes.

[0061] The present invention converts the fluorescence information in the ultraviolet-irradiated fluorescence photograph into RGB data, and determines the content of norfloxacin in the test solution based on the RGB data and a standard curve for norfloxacin. The ordinate of the standard curve for norfloxacin represents the intensity ratio of green to red fluorescence from the sodium alginate hydrogel beads containing the probe, and the abscissa represents the concentration of norfloxacin. In one embodiment of the present invention, the wavelength of the ultraviolet light is 365 nm. The linear range of norfloxacin is 0.01 to 8 μM, and the detection limit is 0.016 μM.

[0062] In order to further illustrate the present invention, the technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0063] Example 1: Constructing a filtration module

[0064] 9 g of polyvinyl alcohol (PVA) and 3 g of sodium alginate (SA) were added to 108 mL of ultrapure water and stirred at 100°C until a uniform viscous liquid was formed; the resulting mixture was cooled to room temperature and poured into a Petri dish, dried in an oven at 55°C for 8 h, and then soaked in a 2 wt% CaCl2 solution for 30 min to obtain a PVA-SA hydrogel.

[0065] A 10 mL syringe was used as a filter column, which was sequentially filled with a 2 cm high PVA-SA hydrogel (lower layer) and glass wool (upper layer) of varying heights (1.0 cm, 1.5 cm, 2.0 cm, 2.5 cm, and 3.0 cm) to form a double-layer filtration system, forming a filtration module. 0.5 mL of a 0.1 mM NOR solution was added to a 0.5 g homogenized egg sample and mixed thoroughly. 5.0 mL of a 2% trichloroacetic acid (TCA) solution was added and ultrasonicated for 20 minutes. The pH was adjusted to neutral with a 0.25 M sodium hydroxide solution to obtain a treated solution. 2 mL of the treated solution was added to the filtration module for filtration, and the initial filtrate was collected. 2 mL of ultrapure water was then added to the filtration module to wash the module, and the washed filtrate was collected. The initial filtrate and the washed filtrate were combined to form the sample to be tested. The absorbance of the sample to be tested at 595 nm was measured by an enzyme-labeled instrument, and the residual protein content was calculated based on the protein standard curve (the protein standard curve was established by Coomassie Brilliant Blue staining). The fluorescence intensity of FQs in the sample to be tested was measured by a fluorescence spectrophotometer to calculate the loss rate of FQs. The results are shown in Figure 2. Figure 1 shown.

[0066] Figure 1 The figure below is a comparison of the protein removal rate and FQs loss rate of glass wool with different filling heights. Figure 1 As can be seen, glass wool with varying fill heights achieved excellent protein removal rates, reaching up to 94%. Glass wool with a fill height of 1 cm showed the best penetration of FQs, with a loss rate of only 4%. The filter modules all demonstrated excellent filtration capacity for FQs. Therefore, the filter modules have the potential to separate and purify egg liquid, replacing the functions of centrifuges and simplifying the complex sample pretreatment steps during testing.

[0067] Example 2: Feasibility verification of NOR detection using TD-SA hydrogel beads

[0068] 160 mg of dithioerythroyl alcohol (DTE) was added to 20 mL of ultrapure water and stirred at room temperature for 10 min. Then, 800 μL of 100 mM CuSO4·5H2O solution was added. The resulting mixture was stirred continuously for 30 min, and then 0.25 M NaOH solution was added dropwise to adjust the pH of the system to 8. After stirring for 20 min, a clear yellow-brown solution was obtained. The solution was purified by 3KD ultrafiltration tube and then freeze-dried to obtain DTE-CuNCs powder (stored at -20°C).

[0069] DTE-CuNCs powder was dispersed in water to obtain a DTE-CuNCs dispersion with a concentration of 9 mg / mL. 1 mL of 9 mg / mL DTE-CuNCs dispersion and 1 mL of 0.5 mM Tb(NO3)3·6H2O solution were stirred and mixed for 30 s to obtain Tb 3+ / DTE-CuNCs probe dispersion; the obtained Tb 3+ The TD-SA / DTE-CuNCs probe dispersion was added to 10 mL of 2% sodium alginate solution and stirred at room temperature for 20 min. The resulting mixed dispersion was added dropwise to 2% CaCl2 solution using a rubber-tipped dropper for ionic crosslinking at room temperature to form TD-SA hydrogel beads of uniform size.

[0070] The TD-SA hydrogel beads were immersed in a NOR solution with a concentration of 5 μM, and the color change of the TD-SA hydrogel beads after different reaction times (0 min, 1 min and 3 min) was observed. 3+ / DTE-CuNCs probe dispersion, measured its fluorescence spectrum in the range of 500-700nm, and recorded the fluorescence intensity at 545nm and 650nm. TD-SA hydrogel beads were immersed in NOR solutions of different concentrations (0, 1μM, 2μM, 3μM and 4μM), and the RGB values ​​of the TD-SA hydrogel beads were analyzed by taking photos with a smartphone. The results are shown in Figure 2 shown.

[0071] Figure 2 The fluorescence photos of TD-SA hydrogel beads immersed in NOR solution at different times. 3+ Fluorescence spectra of / DTE-CuNCs probe dispersion and the changes in G / R values ​​of TD-SA hydrogel beads in the presence of different concentrations of NOR; Figure 2 A in the middle is the fluorescence photos of TD-SA hydrogel beads immersed in NOR solution for different time periods; Figure 2 B in the middle is Tb after the immersion liquid is introduced 3+ Fluorescence spectrum of / DTE-CuNCs probe dispersion, Figure 2 Figure C shows the change of G / R value of TD-SA hydrogel beads in the presence of different concentrations of NOR. Figure 2 As shown in middle A, the TD-SA hydrogel beads before immersion showed strong red fluorescence, which may be due to the red fluorescent Tb 3+ / DTE-CuNCs probes were embedded in the hydrogel beads. After immersion, the red fluorescence of the TD-SA hydrogel beads faded in just 1 minute, revealing a faint green color. At 3 minutes, the fluorescence of the TD-SA hydrogel beads almost completely turned green, which may be because the NOR molecules diffused into the hydrogel beads over time and were embedded in the Tb 3+ / DTE-CuNCs probe adsorption, NOR containing β-diketone structure and Tb 3+ The coordination produces an "antenna effect" that enhances the Tb 3+ Furthermore, the immersion solution of TD-SA hydrogel beads was introduced into Tb 3+ / DTE-CuNCs probe dispersion. Figure 2 As shown in B, with the increase of reaction time, the wavelength at 545nm belongs to Tb 3+ The fluorescence intensity of the characteristic peak gradually decreased, indicating that the content of NOR in the NOR solution gradually decreased with time, further verifying that NOR was adsorbed by the TD-SA hydrogel beads. In addition, the fluorescence intensity of DTE-Cu NCs at 650nm increased slightly after the reaction, but the fluorescence intensity after different reaction times was basically the same. This may be because a small amount of DTE-CuNCs leached out of the TD-SA hydrogel beads during the soaking process, but the leaching concentration was limited. Figure 2 As shown in C, with the increase of NOR concentration, the G / R value gradually increases. This is because NOR and Tb in the hydrogel 3+ / DTE-CuNCs specifically coordinate and activate Tb via the “antenna effect” 3+ Therefore, TD-SA hydrogel beads can achieve ratiometric fluorescence response to NOR, and the detection module of an integrated detection device can be constructed for the real-time detection of NOR.

[0072] Example 3: Method for detecting NOR using an integrated detection device

[0073] The present invention uses a 10mL syringe as a filter column, and fills the inside of the 10mL syringe with a modified sodium alginate hydrogel (PVA-SA hydrogel) with a height of 1 to 3 cm and glass wool with a filling height of 1 to 3 cm from bottom to top to construct a filter module; sodium alginate hydrogel beads (TD-SA hydrogel beads) containing probes are filled in a glass cuvette with a filling height of 1 to 3 cm to construct a detection module. The present invention arranges the filter module above the detection module to construct an integrated detection device. The constructed integrated detection device is as follows Figure 3 shown. Figure 3 It is a physical picture of the integrated detection device and the response of the integrated detection device to NOR solution and egg liquid containing NOR.

[0074] 2 mL of NOR solution of different concentrations (0, 0.01 μM, 1 μM, 2 μM, 3 μM, 4 μM, 5 μM, 6 μM, 8 μM and 10 μM) was injected into the filter module respectively. After it was completely infiltrated into the cuvette, 2 mL of ultrapure water was added to the filter module to wash the filter module. After it was completely infiltrated into the cuvette, the cuvette was removed and the reaction was shaken at room temperature for 3 minutes. Subsequently, it was placed under a 365 nm ultraviolet lamp and a fluorescence photo was taken with a smartphone. The fluorescence information in the photo was converted into RGB data using Adobe Photoshop software, and a standard curve of NOR concentration and G / R value was established to achieve quantitative analysis of NOR. The results are shown in Figure 2. Figure 4 shown.

[0075] Figure 4 A in the figure is the fluorescence photograph of TD-SA hydrogel beads in the presence of different concentrations of NOR. Figure 4 B in the figure is the standard curve of NOR concentration and G / R value. Figure 4 As shown in A, under 365nm UV light, as the concentration of NOR increases, the fluorescence of TD-SA hydrogel spheres gradually changes from red to green. This is mainly because NOR and Tb 3+ coordination, effectively sensitizing Tb 3+ , thereby enhancing Tb 3+ Intrinsic fluorescence. Figure 4 Figure B shows that as the NOR concentration increases, the corresponding G / R value also gradually increases, and in the range of 0.01 to 8.0 μM, it shows a good linear relationship with the NOR concentration. The linear regression equation is y = 0.068x + 0.553 (R 2 =0.991), with a detection limit of 0.016 μM. The transition of fluorescence from red to green can be detected by the naked eye without the aid of precision instruments, indicating that the integrated detection device constructed in this invention can achieve sensitive visual analysis and detection of NOR and has broad potential for on-site detection.

[0076] Example 4: Method for detecting NOR residues in eggs using an integrated detection device

[0077] Eggs purchased from a local supermarket were selected, and the practical application potential of the integrated detection device in NOR detection was explored through a spike recovery test. 0.5 mL of a NOR solution of known concentration was added to 0.5 g of a homogenized egg liquid sample. After mixing evenly, 5.0 mL of a 2% volume fraction TCA solution was added. After the mixture was ultrasonically treated for 20 minutes, the pH value was adjusted to neutral using a 0.25 M sodium hydroxide solution to obtain a treated liquid. 2 mL of the above-mentioned treated liquid was added to the integrated detection device constructed in Example 3, and the detection was performed according to the steps of Example 3. The standard curve in Example 3 was substituted to calculate the NOR concentration in the egg liquid. The experiment was set up in 3 parallels, and the average value was taken.

[0078] The experimental results are shown in Table 1. The recovery rate of NOR in actual egg liquid samples is 92.5% to 112.0%, and the relative standard deviation ranges from 0.06% to 0.17%, showing good recovery rate and relative standard deviation. This shows that the visual ratio fluorescence detection method provided by the integrated detection device provided by the present invention performs well in NOR detection, can be used for sensitive and accurate detection of NOR, and has broad application prospects.

[0079] Table 1 Determination of NOR in egg liquid samples

[0080]

[0081] The embodiments described above are merely descriptions of preferred implementations of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should fall within the scope of protection determined by the claims of the present invention.

Claims

1. An integrated detection device, comprising a filtration module and a detection module; the filtration module comprises a first container and a modified sodium alginate hydrogel and glass wool filled in the first container; the modified sodium alginate hydrogel comprises a sodium alginate matrix and polyvinyl alcohol doped in the sodium alginate matrix; the detection module comprises a second container and alginate hydrogel beads containing probes filled in the second container, wherein the probes comprise copper nanoclusters modified with dithioerythrocyanide coordinated with terbium ions.

2. The integrated detection device according to claim 1, characterized in that: The first container in the filtration module is a syringe or a chromatography column, and the modified sodium alginate hydrogel and glass wool are filled in the first container from bottom to top. The filling height of the modified sodium alginate hydrogel is 1 to 3 cm, and the filling height of the glass wool is 1 to 3 cm; the filling height of the sodium alginate hydrogel beads containing probes in the detection module is 1 to 3 cm; the filtration module is arranged above the detection module.

3. The integrated detection device according to claim 1 or 2, characterized in that: The method for preparing the sodium alginate hydrogel beads containing the probe comprises the following steps: performing a first mixing of erythrodithiocarbamate-modified copper nanoclusters, terbium nitrate, and water to obtain a first dispersion; performing a second mixing of the first dispersion liquid and the sodium alginate solution to obtain a second dispersion liquid; The second dispersion is cross-linked with a calcium chloride solution to obtain the sodium alginate hydrogel beads containing the probe.

4. The integrated detection device according to claim 3, characterized in that: The dosage ratio of the dithioerythrocyanide-modified copper nanoclusters and terbium nitrate is 9 mg:0.0005 mmol; the mass concentration of the sodium alginate solution is 1-3%, and the dosage ratio of the dithioerythrocyanide-modified copper nanoclusters and the sodium alginate solution is 9 mg:10 mL; the mass concentration of the calcium chloride solution is 1-3%, and the cross-linking temperature is 15-30°C.

5. The integrated detection device according to claim 1 or 2, characterized in that: The preparation method of the modified sodium alginate hydrogel comprises the following steps: mixing polyvinyl alcohol, sodium alginate and water to obtain a mixture; The mixture is dried and then immersed in a calcium chloride solution for cross-linking to obtain the modified sodium alginate hydrogel.

6. The integrated detection device according to claim 5, characterized in that: The mass ratio of polyvinyl alcohol to sodium alginate is 3:1, the mixing temperature is 95-105° C., the drying temperature is 50-60° C., the drying time is 7-9 hours, and the cross-linking time is 25-35 minutes.

7. Use of the integrated detection device according to any one of claims 1 to 6 in the visual ratiometric fluorescence detection of norfloxacin.

8. A method for detecting norfloxacin by visual ratio fluorescence, characterized in that: The following steps are involved: The test solution is mixed with trichloroacetic acid solution, and the pH value is adjusted to neutral to obtain a treatment solution; Adding the treated liquid to the filtration module of the integrated detection device according to any one of claims 1 to 6 for filtration, and adding the obtained filtrate to the detection module for coordination reaction; irradiating the detection module after the coordination reaction with ultraviolet light to obtain an ultraviolet fluorescence photograph; The fluorescence information in the ultraviolet irradiation fluorescence photograph is converted into RGB data, and the content of norfloxacin in the test solution is obtained according to the RGB data and a standard curve of norfloxacin; the ordinate of the standard curve of norfloxacin is the intensity ratio of green fluorescence to red fluorescence of sodium alginate hydrogel beads containing the probe, and the abscissa of the standard curve of norfloxacin is the concentration of norfloxacin.

9. The method according to claim 8, characterized in that The test liquid is homogenized egg liquid containing norfloxacin; the volume concentration of the trichloroacetic acid solution is 1-3%, and the volume ratio of the test liquid to the trichloroacetic acid solution is 1:

10.

10. The method according to claim 8, characterized in that The coordination reaction time is 2 to 4 minutes, and the wavelength of the ultraviolet light is 365 nm.

Images