Colorimetric-fluorescence dual-mode detection method for salmonella and escherichia coli

Abstract

The invention discloses a colorimetric-fluorescence dual-mode detection method for salmonella and escherichia coli, and belongs to the technical field of biological detection. According to the invention, concanavalin A (ConA) is coupled with magnetic nanoparticles (MNPs) to form MNPs (at) ConA as a capture probe by recognizing the characteristic of capturing bacteria by sugar residues, and in addition, an antibody (Ab) can specifically recognize bacteria and is coupled with quantum dots (QDs) to form QDs (at) Ab as a fluorescence signal probe. The invention finds that MNPs can catalyze 3, 3 ', 5, 5'-tetramethyl benzidine (TMB) to generate blue oxTMB which can be observed by naked eyes; and the QDs have strong fluorescence and can also be observed by naked eyes, so that obvious distinguishing is realized in Sal and Esc detection. And the signal is linearly related to the target bacteria, so that the visibility is good. The practicability of the method is verified by detecting milk tea, milk and fruit juice samples, and the recovery rate is 93.5-106%. The feasibility of detecting Sal and Esc by the method is verified, and the method has important application potential in food safety monitoring.

Description

Technical Field

[0001] The invention relates to a colorimetric-fluorescence dual-mode detection method for Salmonella and Escherichia coli, and belongs to the technical field of biological detection. Background Art

[0002] Foodborne pathogens, including Salmonella ( Sal ) and Escherichia coli ( Esc ), are the leading causes of foodborne illness worldwide, resulting in significant public health and economic burdens. Traditional detection methods, such as culture-based testing and polymerase chain reaction (PCR), are reliable but often time-consuming, cumbersome, and require specialized equipment and expertise. Therefore, there is an urgent need to develop a rapid, sensitive, and cost-effective strategy to effectively monitor these pathogens in food samples.

[0003] Recent advances in nanotechnology have opened up new avenues for bacterial detection, with the development of innovative biosensing platforms utilizing fluorescent quantum dots (QDs), nanozymes, and magnetic nanoparticles (MNPs). As semiconductor nanocrystals, quantum dots possess excellent optical properties, such as high fluorescence intensity, tunable emission wavelength, and excellent photostability, making them ideal candidates for fluorescence-based pathogen detection. Nanozymes are a class of artificial enzymes based on nanomaterials that have attracted widespread attention due to their superior catalytic performance, stability, and cost-effectiveness compared to natural enzymes. They possess excellent catalytic activity (e.g., peroxidase- and oxidase-like) and can amplify colorimetric signals, thereby improving the sensitivity of pathogen detection.

[0004] MNPs are widely used in biosensors due to their low synthesis cost, stable performance, good biocompatibility, and environmental friendliness. Their surfaces have a large number of modifiable groups, making them easy to couple with various inorganic substances, enzymes, proteins, antibodies, and other substances. Furthermore, MNPs play a vital role in the separation and enrichment of bacteria, separating target bacteria from food matrices. By combining MNPs with quantum dots, multifunctional biosensing platforms can be developed, utilizing the POD activity of MNPs and the fluorescent properties of QDs for highly sensitive colorimetric and fluorescent detection, enabling simple, equipment-free visual output.

[0005] Sandwich immunoassays have been a commonly used analyte detection technique for decades. In this technique, signal amplification of the analyte is crucial to further enhance sensitivity. Summary of the Invention

[0006] In order to solve or partially solve the problems existing in the related art, one of the purposes of the present invention is to provide a method for preparing a composite sandwich nanomaterial. The present invention prepares MNPs@ConA / E. coli / RQDs@Ab- Esc and MNPs@ConA / Salmonella / OQDs@Ab- Sal The sandwich complex combines multiple signal probes with bacteria, so that the signal output to the target bacteria is effectively amplified. In addition, due to the inherent catalytic properties of the assembled MNPs, the oxidation of TMB to oxTMB is achieved. Therefore, dual-mode detection is achieved by relying on the excellent performance of MNPs and QDs.

[0007] In order to achieve the purpose of the present invention, it is necessary to perform the following steps: (1) Activating the carboxyl groups on RQDs, OQDs, and MNPs to obtain activated RQDs solution, OQDs solution, and MNPs solution.

[0008] (2) The activated MNPs solution was coupled with the concanavalin A solution to obtain the MNPs@ConA solution.

[0009] (3) The activated RQDs solution was coupled with the Ab-Esc solution to obtain the RQDs@Ab-Escs solution, and the OQDs solution was coupled with the Ab-Sal solution to obtain the OQDs@Ab-Sal solution.

[0010] (4) The MNPs@ConA solution was mixed with Escherichia coli solution of known concentration for the first incubation. After incubation, magnetic separation was performed to remove excess liquid. RQDs@Ab-Esc solution was added for the second incubation to obtain the MNPs@ConA / Escherichia coli / RQDs@Ab-Esc sandwich complex.

[0011] (5) The MNPs@ConA solution was mixed with Salmonella of known concentration for the first incubation. After incubation, magnetic separation was performed to remove excess liquid. The OQDs@Ab-Sal solution was then added for the second incubation to obtain the MNPs@ConA / Salmonella / OQDs@Ab-Sal sandwich complex.

[0012] (6) Detect the fluorescence intensity or UV absorbance of the MNPs@ConA / Escherichia coli / QDs@Ab-Esc sandwich complex and the MNPs@ConA / Salmonella / OQDs@Ab-Sal sandwich complex, respectively, and establish a standard curve between the bacterial solution concentration and the fluorescence intensity or the bacterial solution concentration and the UV absorbance.

[0013] (7) The MNPs@ConA solution was mixed with the sample to be tested for the first incubation. After incubation, magnetic separation was performed to remove excess liquid. RQDs@Ab-Esc and OQDs@Ab-Sal were added respectively for the second incubation. Finally, MNPs@ConA / to-be-tested Escherichia coli / RQDs@Ab-Esc sandwich complex and MNPs@ConA / to-be-tested Salmonella / OQDs@Ab-Sal sandwich complex were obtained.

[0014] (8) Detect the fluorescence intensity or UV absorbance of the MNPs@ConA / to-be-detected Escherichia coli / RQDs@Ab-Esc sandwich complex and the MNPs@ConA / to-be-detected Salmonella / OQDs@Ab-Sal sandwich complex respectively, and substitute them into the standard curve obtained in step (6) to obtain the concentrations of Escherichia coli and Salmonella in the sample to be detected.

[0015] Preferably, the preparation method of RQDs and OQDs in step (1) is as follows: cadmium chloride, sodium citrate and 3-mercaptopropionic acid are mixed evenly, NaOH is added to adjust the pH, and then sodium borohydride and sodium tellurite are added and mixed evenly to obtain a mixture, the mixture is stirred in an oil bath for reaction, ultrafiltration is performed after cooling, and the substance in the tube is freeze-dried to obtain RQDs or OQDs.

[0016] Preferably, the mass ratio of cadmium chloride, sodium citrate, sodium borohydride and sodium tellurite is 1:(1.9-2.2):(0.3-0.7):(0.1-0.4); the concentration of cadmium chloride in pure water is 0.2-0.4 mg / mL, and the concentration of 3-mercaptopropionic acid in pure water is 0.95-1.05 g / L.

[0017] Preferably, when the pH is adjusted to 9-10, RQDs are prepared; and when the pH is adjusted to 11-12, OQDs are prepared.

[0018] Preferably, the temperature of the oil bath is 90° C. to 97° C., the stirring reaction time is 5 to 7 hours, and the freeze-drying temperature is -60° C.

[0019] Preferably, the preparation method of MNPs in step (1) is as follows: dissolving ferric chloride hexahydrate in ethylene glycol, then adding polyethylene glycol-6000, sodium acetate and citric acid and stirring until completely dissolved, then heating to react, after the reaction is completed, cooling, purifying and vacuum drying to collect the solid to obtain MNPs material.

[0020] Preferably, the mass ratio of ferric chloride hexahydrate, polyethylene glycol-6000, sodium citrate and sodium acetate is 1:(0.15-0.35):(0.2-0.3):(1-1.5); the concentration of ferric chloride hexahydrate in ethylene glycol is 30-60 mg / mL.

[0021] Preferably, the heating reaction conditions are: heating reaction at 195-210° C. for 15-20 h, and the vacuum drying temperature is 60° C.

[0022] Preferably, in step (1), the EDC / NHS method is used to activate the carboxyl groups on the surfaces of RQDs, OQDs and MNPs.

[0023] Preferably, the specific steps of the EDC / NHS activation method are: RQDs, OQDs and MNPs are dispersed in a buffer solution, and then EDC (1-ethyl-3-[3-dimethylaminopropyl]carbodiimide) and NHS (N-hydroxysuccinimide) are added and reacted under shaking conditions for 58-62 minutes.

[0024] Preferably, the buffer is HEPES or PBS with a concentration of 10 mmol / L.

[0025] Preferably, the volume ratio of the activated MNPs solution to the ConA solution in step (2) is 1:(0.1-0.3).

[0026] Preferably, the mass concentration of ConA in the ConA solution in step (2) is 4.8-5.1 mg / mL.

[0027] Preferably, in step (3), the volume ratio of the activated RQDs solution to Ab-Esc is 1:(0.005-0.015); the volume ratio of the activated OQDs solution to Ab-Sal is 1:(0.005-0.015).

[0028] Preferably, in step (3), the concentration of Ab-Esc in the Ab-Esc solution is 0.9-1.1 mg / mL; the concentration of Ab-Sal in the Ab-Sal solution is 0.9-1.1 mg / mL.

[0029] Preferably, in step (3), the concentration of RQDs@Ab-Escs in the RQDs@Ab-Escs solution is greater than or equal to 0.6 mg / mL; the concentration of OQDs@Ab-Sal solution in the OQDs@Ab-Sal solution is greater than or equal to 0.6 mg / mL.

[0030] Preferably, the amount of MNPs@ConA added to the bacterial solution in step (4), step (5) and step (7) is 2.5~3.5 mg / mL; the solid-liquid ratio of MNPs@ConA to RQDs@Ab-Esc solution is: (2.5~3.5):1. The unit is mg:mL; the solid-liquid ratio of MNPs@ConA to OQDs@Ab-Sal solution is: 2.5~3.5:1. The unit is mg:mL.

[0031] Preferably, the first incubation time in step (4), step (5) and step (7) is greater than or equal to 60 minutes; the second incubation time is greater than or equal to 45 minutes.

[0032] Preferably, the pH of the solution is maintained between 7.2 and 9.0 during the second incubation in step (4), step (5) and step (7).

[0033] The technical solution provided by the present invention can have the following beneficial effects: (1) The present invention is based on Esc and Sal The magnetic nanoparticles are used as targets and a sandwich structure is formed by combining MNPs and QDs. Firstly, the magnetic separation performance of the magnetic composite nanomaterial is utilized to quickly separate bacteria from food. Secondly, the colorimetric and fluorescence sensing signals of the magnetic composite nanomaterial can be used to perform short-time, high-precision and high-stability dual-mode quantitative detection of bacteria.

[0034] (2) Existing Esc and Sal Most detection technologies are single-mode detection, and there is little development of dual-mode quantitative detection methods. The present invention chooses to use colorimetric and fluorescence dual-mode signals and utilizes the prepared magnetic composite nanomaterials to perform quantitative detection of bacteria.

[0035] (3) Concanavalin A (ConA) can recognize and capture glycoproteins. The main component of bacterial cell walls is peptidoglycan; the main components of cell membranes are lipids, proteins, and carbohydrates, so ConA can recognize but not specifically capture bacteria. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 The characterization data of various materials in Example 1 are as follows: FT-IR spectra of MNPs and MNPs@ConA (a); VSM spectra of MNPs and MNPs@ConA (b); MNPs, MNPs@ConA, RQDs, RQDs@Ab, OQDS, OQDS@Ab, MNPs@ConA / Esc / RQDs@Ab、MNPS@ConA / Sal Zeta potential diagram of / OQDs@Ab (c).

[0037] Figure 2 Michaelis-Menten kinetics curves of MNPs (a, b) and MNPs@ConA (c, d) in Example 1.

[0038] Figure 3 For detection in Example 2 Esc Colorimetric-fluorescence dual-mode results; including EscColorimetric linear results (a, b) and fluorescence linear results (c, d); Esc Colorimetric-specific results (e, f) and fluorescence-specific results (g, h) of .

[0039] Figure 4 For detection in Example 3 Sal Colorimetric-fluorescence dual-mode results; including Sal Colorimetric linear results (a, b) and fluorescence linear results (c, d); Sal Colorimetric-specific results (e, f) and fluorescence-specific results (g, h) of .

[0040] Figure 5 Comparison diagram of colorimetric and fluorescence optimization in the composite material in Example 4; including: different MNPs (a), QDs (b) concentrations, different pH conditions (c, d), first incubation stage (e) and second incubation stage (f) time optimization diagram. DETAILED DESCRIPTION

[0041] The present invention is further described in detail below with reference to the accompanying drawings and specific embodiments, but the protection scope of the present invention is not limited to the contents described above.

[0042] Unless otherwise specified, all reagents used were of commercially available analytical grade.

[0043] Antibody against Salmonella (Ab-Sal) and antibody against Escherichia coli (Ab-Esc) were purchased from Huamei Biotechnology (Wuhan, China) (Cat. No.: CSB-PA16479A0Rb, CSB-PA359577HA01ENV).

[0044] Example 1 The specific steps for the preparation of different materials are as follows: (1) Preparation of MNPs: Ferric chloride hexahydrate was dissolved in ethylene glycol and stirred for 10 min. Then polyethylene glycol-6000, sodium acetate and sodium citrate were added and stirred until completely dissolved. The mixture was then placed in a high-pressure reactor and heated at 200°C for 18 h. After the reaction was completed, the mixture was cooled, the solid was separated and collected, and washed alternately with water and ethanol until there were no unreacted impurities. The MNPs material was then vacuum-dried at 60°C. The mass ratio of ferric chloride hexahydrate, polyethylene glycol-6000, sodium citrate and sodium acetate was 1:0.2:0.2:1.2. The concentration of ferric chloride hexahydrate in ethylene glycol was 54 mg / mL.

[0045] (2) Preparation of RQDs: Cadmium chloride and sodium citrate were dispersed in ultrapure water, and 3-mercaptopropionic acid was added and stirred at room temperature for 15 min. NaOH was added to adjust the pH of the solution to 10, and then sodium borohydride and sodium tellurite were added to obtain a mixture. The mixture was magnetically stirred in an oil bath at 95°C for 6 h. After cooling, the excess unreacted material was filtered and purified using an ultrafiltration tube (3 kDa). The remaining material in the tube was freeze-dried at -60°C and RQDs were collected; the mass ratio of cadmium chloride, sodium citrate, sodium borohydride and sodium tellurite was 1:2:0.5:0.4, the concentration of cadmium chloride in pure water was 0.52 mg / mL, and the concentration of 3-mercaptopropionic acid in pure water was 1 g / L.

[0046] (3) Preparation of OQDs: Cadmium chloride and sodium citrate were dispersed in ultrapure water, and 3-mercaptopropionic acid was added and stirred at room temperature for 15 min. NaOH with a concentration of 1 mol / L was added to adjust the pH of the solution to 11, and then sodium borohydride and sodium tellurite were added to obtain a mixture. The mixture was reacted in an oil bath at 95°C with magnetic stirring for 6 h. After cooling, the excess unreacted material was filtered and purified with an ultrafiltration tube (3 kDa). The remaining material in the tube was freeze-dried at -60°C and OQDs were collected; the mass ratio of cadmium chloride, sodium citrate, sodium borohydride and sodium tellurite was 1:2:0.5:0.4, the concentration of cadmium chloride in pure water was 0.52 mg / mL, and the concentration of 3-mercaptopropionic acid in pure water was 1 g / L.

[0047] (4) Activation of MNPs, RQDs, and OQDs: MNPs, RQDs, and OQDs were dispersed in 10 mmol / L HEPES solution, respectively, and reacted on a shaker at 180 rpm and 38°C for 60 min. The solution was then purified using magnetic separation to remove excess unreacted EDC / NHS, thereby obtaining activated MNPs solution, RQDs solution, and OQDs solution, wherein the mass ratio of MNPs, EDC, and NHS was 1:0.7:0.7; the mass ratio of RQDs, EDC, and NHS was 1:0.7:0.7; and the mass ratio of OQDs, EDC, and NHS was 1:0.7:0.7.

[0048] (5) Preparation of MNPs@ConA: The activated MNPs solution was added to the ConA solution (the mass concentration of ConA in the ConA solution was 5 mg / mL), mixed and incubated for 60 min, and then magnetic separation and purification were performed to obtain MNPs@ConA, where the volume ratio of the activated MNPs solution to the ConA solution was 1:0.1.

[0049] (5) Preparation of RQDs@Ab-Esc and OQDs@Ab-Sal: The activated RQDs solution (the concentration of RQDs in the activated RQDs solution was 0.3 mg / mL) was mixed with the Ab-Esc solution (the concentration of Ab-Esc in the Ab-Esc solution was 1 mg / mL) and the activated OQDs solution (the concentration of OQDs in the activated OQDs solution was 0.6 mg / mL) was mixed with the Ab-Sal solution (the concentration of Ab-Sal in the Ab-Sal solution was 1 mg / mL) and incubated for 60 min. After the mixture was separated and purified, the RQDs@Ab-Esc solution and the OQDs@Ab-Sal solution were obtained. The volume ratio of the activated RQDs solution to the Ab-Esc solution was 1:0.005; the volume ratio of the activated OQDs solution to the Ab-Sal solution was 1:0.005.

[0050] (6) Preparation of MNPs@ConA / E. coli / RQDs@Ab-Esc composite sandwich material: 3 mg of MNPs@ConA was mixed with 1 mL of E. coli and incubated for 75 min. After the excess liquid was separated by magnetic separation, 1 mL of RQDs@Ab-Esc solution (the concentration of RQDs@Ab-Esc in the RQDs@Ab-Esc solution was 0.6 mg / mL) was added and incubated for 60 min to form MNPs@ConA / E. coli / RQDs@Ab-Esc composite nanomaterial (MNPs@ConA / Esc / RQDs@Ab).

[0051] (7) Preparation of MNPs@ConA / Salmonella / OQDs@Ab composite sandwich nanomaterials: 3 mg of MNPs@ConA was mixed with 1 mL of Salmonella solution and incubated for 75 min. After the excess liquid was separated by magnetic separation, 1 mL of OQDs@Ab-Sal solution (the concentration of OQDs@Ab-Sal in the OQDs@Ab-Sal solution was 0.6 mg / mL) was added and incubated for 60 min to form MNPs@ConA / Salmonella / OQDs@Ab-Sal composite nanomaterials (MNPS@ConA / Sal / OQDs@Ab).

[0052] The MNPs, MNPs@ConA, RQDs, RQDs@Ab, OQDS, OQDS@Ab, MNPs@ConA / Esc / RQDs@Ab、MNPS@ConA / Sal / OQDs@Ab were subjected to relevant characterization analysis. Figure 1 As shown, Figure 1a is the FT-IR spectrum of MNPs and MNPs@ConA. It can be seen from the figure that at 3415 cm −1 and 2000cm −1 There are absorption peaks at 2932 cm, which correspond to the stretching vibration of -OH groups on the surface of MNPs; −1 The peak at 1394 cm is attributed to the bending vibration of CH; in addition, the absorption of C-N bond is located at 1394 cm −1 ; Figure 1 b is the VSM spectrum of MNPs and MNPs@ConA, which shows that the magnetic saturation values ​​of MNPs and MNPs@ConA are 25.82 and 24.73 emu / g, respectively. It is worth noting that the saturation magnetization intensity of MNPs is higher than that of MNPs@ConA, indicating that the magnetic saturation value does not decrease significantly after the coupling of MNPs and ConA. In addition, when the MNPs@ConA suspension comes into contact with a permanent magnet, it responds quickly to the magnetic field within 6 seconds. Therefore, the prepared MNPs@ConA has good magnetic response properties and can be used as an excellent separation tool for detecting and analyzing target bacteria. Figure 1 c is MNPs, MNPs@ConA, RQDs, RQDs@Ab, OQDS, OQDS@Ab, MNPs@ConA / Esc / RQDs@Ab、MNPS@ConA / Sal The zeta potential plot of the OQDs@Ab / OQDs@Ab shows a small difference in potential between OQDs and RQDs, at -35.1±2.12 mV and -33.2±0.93 mV, respectively. After conjugation with the antibody, their respective potentials increased to -10.8±1.1 mV and -7.93±0.6 mV, respectively. This increase in potential indicates successful conjugation of OQDs and RQDs to the antibody, and the bacteria-containing sandwich structures exhibit varying degrees of zeta potential reduction.

[0053] The Michaelis-Menten kinetics curves of MNPs and MNPs@ConA were measured. Figure 2 The results showed that the catalytic rate of peroxidase-like activity of MNPs@ConA was slightly higher than that of MNPs in both H2O2 and TMB. However, the K m The values ​​were higher than those of MNPs, which indicated that the affinity of MNPs to TMB and H2O2 was higher than that of MNPs@ConA.

[0054] Example 2 A colorimetric-fluorescence dual-mode analysis and detection method for Escherichia coli was constructed based on the colorimetric and fluorescence properties of magnetic composite nanomaterials. The specific steps are as follows: (1) Preparation of MNPs: Ferric chloride hexahydrate was dissolved in ethylene glycol and stirred for 10 min. Then polyethylene glycol-6000, sodium acetate and sodium citrate were added and stirred until completely dissolved. The mixture was then placed in a high-pressure reactor and heated at 200°C for 18 h. After the reaction was completed, the mixture was cooled, the solid was separated and collected, and washed alternately with water and ethanol until there were no unreacted impurities. The MNPs material was then vacuum-dried at 60°C. The mass ratio of ferric chloride hexahydrate, polyethylene glycol-6000, sodium citrate and sodium acetate was 1:0.2:0.2:1.2. The concentration of ferric chloride hexahydrate in ethylene glycol was 54 mg / mL.

[0055] (2) Preparation of RQDs: Cadmium chloride and sodium citrate were dispersed in ultrapure water, and 3-mercaptopropionic acid was added and stirred at room temperature for 15 minutes. NaOH was added to adjust the pH of the solution to 10, and then sodium borohydride and sodium tellurite were added to obtain a mixture. The mixture was stirred in an oil bath at 95°C for 6 hours. After cooling, the excess unreacted material was purified by filtration using an ultrafiltration tube (3kDa). The remaining material in the tube was then freeze-dried at -60°C to collect RQDs; the mass ratio of cadmium chloride, sodium citrate, sodium borohydride and sodium tellurite was 1:2:0.5:0.4. The concentration of cadmium chloride in pure water was 0.52 mg / mL, and the concentration of 3-mercaptopropionic acid in pure water was 1 g / L.

[0056] (3) Activation of MNPs and RQDs: MNPs and RQDs were dispersed in 10 mmol / L HEPES solution, and EDC and NHS were added. The mixture was shaken at 180 rpm and 38°C for 60 min. The solution was then purified by magnetic separation to remove excess unreacted EDC / NHS. The activated MNPs solution and activated RQDs solution were obtained, wherein the mass ratio of MNPs, EDC, and NHS was 1:0.7:0.7; the mass ratio of RQDs, EDC, and NHS was 1:0.7:0.7.

[0057] (4) Preparation of MNPs@ConA: The activated MNPs solution was added to the ConA solution (the mass concentration of ConA in the ConA solution was 5 mg / mL), mixed and incubated for 60 min, and then separated and purified to obtain MNPs@ConA, where the volume ratio of activated MNPs to ConA solution was 1:0.1.

[0058] (5) Preparation of RQDs@Ab: The activated RQDs solution (the concentration of RQDs in the activated RQDs solution was 0.3 mg / mL) and the Ab-Esc solution (the concentration of Ab-Esc in the Ab-Esc solution was 1 mg / mL) were mixed and incubated for 60 min to obtain the RQDs@Ab-Esc solution. The volume ratio of the activated RQDs solution to the Ab-Esc solution was 1:0.005.

[0059] (6) Prepare a concentration gradient of 10 1 , 10 2 , 10 3 , 10 4 and 10 5 CFU / mL of Escherichia coli liquid, 3 mg of MNPs@ConA was mixed with 1 mL of Escherichia coli liquid of different concentrations and incubated for 70 minutes. After magnetic separation to remove the excess liquid, 1 mL of RQDs@Ab-Esc (the concentration of RQDs@Ab-Esc in the RQDs@Ab-Esc solution was 0.6 mg / mL) solution was added and incubated for 65 minutes to obtain MNPs@ConA / Escherichia coli / RQDs@Ab-Esc sandwich complexes incubated with different concentrations of Escherichia coli.

[0060] (7) Detect the absorbance of the MNPs@ConA / E. coli / RQDs@Ab-Esc sandwich complex obtained in step (6): 50 μL of the MNPs@ConA / E. coli / RQDs@Ab-Esc sandwich complex, 50 μL of H2O2 (50 mmol / L), 50 μL of TMB (5 mmol / L), and 100 μL of HAc-NaOAc solution (10 mmol / L, pH 4.0) were mixed and incubated for 5 min. The absorbance of the solution was measured at 654 nm. The results were as follows: Figure 4 a, b show: in 10 1 -10 5 CFU / mL( Esc ) has a good linear relationship within the range. The fitted regression equation is y=-0.09x+1.06, and the correlation coefficient R 2 =0.98; the absorbance decreased with the increase of the number of target bacteria in the sample, indicating that the more the number of target bacteria, the more the active sites of MNPs were masked, resulting in a weaker ability to catalyze TMB to form oxTMB. To verify the specificity of this method, Str, Sta and Lis were selected as interfering strains, and the concentration was set to 10 2 CFU / mL. Figure 4 e,f( Esc ) It can be seen that the signals of the interfering strains are significantly different from those of the target strains and can be clearly distinguished, indicating that the method has good specificity.

[0061] (8) Detection of the fluorescence intensity of the MNPs@ConA / E. coli / RQDs@Ab-Esc sandwich complex obtained in step (6): In the fluorescence mode, the more sandwich structures are formed, the higher the fluorescence intensity is, which is exactly the opposite of the colorimetric mode. The results show that when λ Ex / λ Em When the fluorescence intensity is 365 / 640nm, Esc Concentration (10 1 ~10 5 CFU / mL) increased with the increase of ( Figure 4 c, d), the established linear scale is y = 26.29x + 296.84, and the correlation coefficient R 2 is 0.99.

[0062] (9) Esc Inoculate into water, milk (Mengniu), milk tea and juice respectively, with the spiked amounts shown in Table 1, to obtain test samples containing Escherichia coli.

[0063] (10) 3 mg of MNPs@ConA was incubated with 1 mL of the test sample containing E. coli obtained in step (9), and then magnetic separation was performed to remove excess liquid. The mixture was then incubated with 1 mL of RQDs@Ab-Esc solution (the concentration of RQDs@Ab-Esc in the RQDs@Ab-Esc solution was 0.6 mg / mL) to finally obtain a MNPs@ConA / test E. coli / RQDs@Ab-Esc sandwich complex.

[0064] (11) The fluorescence intensity of the MNPs@ConA / tested E. coli / RQDs@Ab-Esc sandwich complex obtained in step (10) was detected. The detected fluorescence intensity was substituted into the equation obtained in step (8): y = 26.29x + 296.84 to obtain the concentration of E. coli in the test sample. The results are shown in Table 1.

[0065] Table 1: Real samples tested using this method Esc The recovery rate ( n = 3) Example 3 A colorimetric-fluorescence dual-mode analysis and detection method for Salmonella was constructed based on the colorimetric and fluorescence properties of magnetic composite nanomaterials. The specific steps are as follows: (1) Preparation of MNPs: Ferric chloride hexahydrate was dissolved in ethylene glycol and stirred for 10 min. Then polyethylene glycol-6000, sodium acetate and sodium citrate were added and stirred until completely dissolved. The mixture was then placed in a high-pressure reactor and heated at 200°C for 18 h. After the reaction was completed, the mixture was cooled, the solid was separated and collected, and washed alternately with water and ethanol until there were no unreacted impurities. The MNPs material was then vacuum-dried at 60°C. The mass ratio of ferric chloride hexahydrate, polyethylene glycol-6000, sodium citrate and sodium acetate was 1:0.2:0.2:1.2. The concentration of ferric chloride hexahydrate in ethylene glycol was 54 mg / mL.

[0066] (2) Preparation of OQDs: Cadmium chloride and sodium citrate were dispersed in ultrapure water, and 3-mercaptopropionic acid was added and stirred at room temperature for 15 minutes. NaOH was added to adjust the pH of the solution to 12, and then sodium borohydride and sodium tellurite were added to obtain a mixture. The mixture was stirred in an oil bath at 95°C for 6 hours. After cooling, the excess unreacted material was purified by filtration using an ultrafiltration tube (3 kDa). The remaining material in the tube was then freeze-dried at -60°C to collect OQDs; the mass ratio of cadmium chloride, sodium citrate, sodium borohydride, and sodium tellurite was 1:2:0.5:0.4. The concentration of cadmium chloride in pure water was 0.52 mg / mL, and the concentration of 3-mercaptopropionic acid in pure water was 1 g / L.

[0067] (3) Activation of MNPs and OQDs: MNPs and OQDs were dispersed in HEPES solution, and EDC / NHS was added. The mixture was shaken at 180 rpm and 38°C for 60 min. The solution was then purified by magnetic separation to remove excess unreacted EDC / NHS. Activated MNPs solution and activated OQDs solution were obtained. The mass ratio of MNPs, EDC, and NHS was 1:0.7:0.7; the mass ratio of OQDs, EDC, and NHS was 1:0.7:0.7.

[0068] (4) Preparation of MNPs@ConA: The activated MNPs solution (the concentration of MNPs in the activated MNPs solution is 3 mg / mL) was added to the ConA solution (the mass concentration of ConA in the ConA solution is 5 mg / mL), mixed and incubated for 60 min, and then magnetic separation and purification were performed to obtain MNPs@ConA, where the volume ratio of the activated MNPs solution to the ConA solution was 1:0.1.

[0069] (5) Preparation of OQDs@Ab: The activated OQDs solution was mixed with Ab-Sal solution (the mass concentration of Ab-Sal in the Ab-Sal solution was 1 mg / mL) and incubated for 60 min. After that, OQDs@Ab was separated and purified. The volume ratio of the activated OQDs to the Ab-Sal solution was 1:0.005.

[0070] (6) Prepare a concentration gradient of 10 1 , 10 2 , 10 3 , 10 4 and 10 5 CFU / mL of Salmonella liquid, 3 mg of MNPs@ConA was mixed with 1 mL of Salmonella liquid of different concentrations and incubated for 80 minutes. After the incubation, magnetic separation was performed, and the excess liquid was removed and then incubated with 1 mL of OQDs@Ab-Sal (the concentration of OQDs@Ab-Sal in the OQDs@Ab-Sal solution was 0.6 mg / mL) for 55 minutes to obtain MNPs@ConA / Salmonella / OQDs@Ab-Sal sandwich complexes incubated with different concentrations of Escherichia coli.

[0071] (7) Detect the absorbance of the MNPs@ConA / Salmonella / OQDs@Ab-Sal sandwich complex obtained in step (6): 50 μL of the MNPs@ConA / Salmonella / OQDs@Ab-Sal sandwich complex, 50 μL of H2O2 (50 mmol / L), 50 μL of TMB (5 mmol / L), and 100 μL of HAc-NaOAc solution (10 mmol / L, pH 4.0) were mixed and incubated for 5 min. The absorbance of the solution was measured at 654 nm. The results were as follows: Figure 5 a, b show: Sal The linear range is 10 1 ~10 6 CFU / mL. Similarly, the results have good linear detection, the regression equation is y=-0.15x+0.96, and the correlation coefficient R 2 =0.95; the absorbance decreased with the increase of the number of target bacteria in the sample, indicating that the more the number of target bacteria, the more the active sites of MNPs were masked, resulting in a weaker ability to catalyze TMB to form oxTMB. To verify the specificity of this method, Str, Sta and Lis were selected as interfering strains, and the concentration was set to 10 2 CFU / mL, from Figure 5 As can be seen from e and f, the signals of the interfering strains are significantly different from those of the target strains and can be clearly distinguished, indicating that the method has good specificity.

[0072] (8) Detection of the fluorescence intensity of the MNPs@ConA / Salmonella / OQDs@Ab-Sal sandwich complex obtained in step (6): In the fluorescence mode, the more sandwich structures are formed, the higher the fluorescence intensity is, which is exactly the opposite of the colorimetric mode. The results show that when λ Ex / λ Em When the test target is 365 / 640nm, Sal When, such as Figure 5 c, d, λ Ex / λ Em is 365 / 585nm. It can be seen that at 10 1 ~10 6 There is a good linear relationship within the concentration range of CFU / mL. The established linear calibration is y=19.722x+271.12, and the correlation coefficient R 2 is 0.98.

[0073] (9) Sal The samples were inoculated into water, milk (Mengniu), milk tea and juice respectively, with the spiked amounts shown in Table 2, to obtain the test samples containing Salmonella.

[0074] (10) 3 mg of MNPs@ConA was incubated with 1 mL of the sample containing Salmonella obtained in step (9), and then magnetic separation was performed to remove excess liquid, and then incubated with 1 mL of OQDs@Ab-Sal to finally obtain an MNPs@ConA / Salmonella to be tested / OQDs@Ab-Sal sandwich complex.

[0075] (11) The fluorescence intensity of the MNPs@ConA / Salmonella to be tested / OQDs@Ab-Sal sandwich complex obtained in step (10) was detected. The detected fluorescence intensity was substituted into the equation obtained in step (8): y = 19.722x + 271.12 to obtain the concentration of Salmonella in the sample to be tested. The results are shown in Table 2.

[0076] Table 2: Real samples tested using this method Sal The recovery rate ( n = 3) Example 4 The sandwich structure dual-mode method was optimized and verified (1) Optimizing the dosage of MNPs@ConA MNPs@ConA and OQDs@Ab-Sal solutions were prepared according to the method described in Example 1.

[0077] According to the method described in Example 1, 10 5Salmonella with a concentration of 500 CFU / mL was added with different amounts of MNPs@ConA for the first incubation. The incubation time was 70 min. The added amounts of MNPs@ConA were 0.5 mg / mL, 1.0 mg / mL, 1.5 mg / mL, 2.0 mg / mL, 2.5 mg / mL, 3.0 mg / mL, 35 mg / mL and 4.0 mg / mL, and 8 incubation solutions were obtained. After removing the excess liquid from the 8 incubation solutions by magnetic separation, 1 mL of OQDs@Ab-Sal solution (the concentration of OQDs@Ab-Sal in the OQDs@Ab-Sal solution was 0.6 mg / mL) was added and mixed for a second incubation. The second incubation time was 70 min, and the pH of the solution was controlled at 7.2 during the second incubation to obtain 8 MNPs@ConA / Salmonella / OQDs@Ab-Sal sandwich complexes.

[0078] The fluorescence intensities of the eight MNPs@ConA / Salmonella / OQDs@Ab-Sal sandwich complexes were measured according to the method described in Example 1. Figure 5 As shown in a, it can be seen from the figure that when the MNPs concentration is below 3 mg / mL, the fluorescence intensity of the system increases with the increase of concentration because more sandwich structures are formed. However, when the concentration continues to increase, excessive MNPs may quench the fluorescence of the system, resulting in a decrease in fluorescence intensity.

[0079] (2) Optimization of the concentration of OQDs@Ab-Sal in the OQDs@Ab-Sal solution MNPs@ConA and OQDs@Ab-Sal solutions were prepared according to the method described in Example 1, and the concentrations of OQDs@Ab-Sal in the OQDs@Ab-Sal solutions were 0.3 mg / mL, 0.6 mg / mL, 0.9 mg / mL, 1.2 mg / mL, 1.5 mg / mL, and 3.0 mg / mL, respectively.

[0080] According to the method described in Example 1, 10 5 CFU / mL of Salmonella was added to 3.0 mg of MNPs@ConA for the first incubation, and the incubation time was 70 min. After removing the excess liquid in the incubation solution by magnetic separation, 1 mL of OQDs@Ab-Sal solution was added (the concentration of OQDs@Ab-Sal in the OQDs@Ab-Sal solution was 0.3 mg / mL, 0.6 mg / mL, 0.9 mg / mL, 1.2 mg / mL, 1.5 mg / mL, and 3.0 mg / mL) and mixed for a second incubation, and the incubation time was 70 min. During the second incubation, the pH of the solution was controlled at 7.2 to obtain 6 MNPs@ConA / Salmonella / OQDs@Ab-Sal sandwich complexes.

[0081] The fluorescence intensities of the six MNPs@ConA / Salmonella / OQDs@Ab-Sal sandwich complexes were detected. Figure 5 As shown in (b), it can be observed that when the OQDs-Sal concentration reaches 0.6 mg / mL, the fluorescence intensity of the system does not undergo a qualitative change. The possible reason is that the sandwich structure formed by OQDs-Sal has reached saturation.

[0082] (3) Effect of pH in the second incubation on fluorescence intensity and absorbance MNPs@ConA and OQDs@Ab-Sal solutions were prepared according to the method described in Example 1.

[0083] According to the method described in Example 1, 10 5 CFU / mL of Salmonella was added to 3.0 mg of MNPs@ConA for the first incubation, and the incubation time was 70 min. After the excess liquid in the incubation solution was removed by magnetic separation, 1 mL of OQDs@Ab-Sal solution (the concentration of OQDs@Ab-Sal in the OQDs@Ab-Sal solution was 0.6 mg / mL) was added and mixed for a second incubation, and the incubation time was 70 min. During the second incubation, the pH of the solution was controlled at 2.6, 3.8, 4.8, 5.8, 8, 7.2, 8.2, 9.0 and 10, and 9 MNPs@ConA / Salmonella / OQDs@Ab-Sal sandwich complexes were obtained.

[0084] The fluorescence intensity and absorbance values ​​of the six MNPs@ConA / Salmonella / OQDs@Ab-Sal sandwich complexes were detected. Figure 5 c and Figure 5 As shown in Figure d, the optimal pH for both the colorimetric and fluorescence modes is 8.2.

[0085] (4) Optimization of the first incubation time MNPs@ConA and OQDs@Ab-Sal solutions were prepared according to the method described in Example 1.

[0086] According to the method described in Example 1, 10 5CFU / mL of Salmonella was added with 3.0 mg of MNPs@ConA for the first incubation, and the incubation time of the first stage was controlled at 15 min, 30 min, 45 min, 60 min, 75 min and 90 min. After the excess liquid in the incubation solution was removed by magnetic separation, 1 mL of OQDs@Ab-Sal solution (the concentration of OQDs@Ab-Sal in the OQDs@Ab-Sal solution was 0.6 mg / mL) was added and mixed for a second incubation. The incubation time was 70 min. During the second incubation, the pH of the solution was controlled at 8.2 to obtain 6 MNPs@ConA / Salmonella / OQDs@Ab-Sal sandwich complexes.

[0087] The fluorescence intensity and absorbance values ​​of the six MNPs@ConA / Salmonella / OQDs@Ab-Sal sandwich complexes were detected. Figure 5 As shown in Figure e, it can be seen that the optimal first incubation time is 75 minutes.

[0088] (5) Optimization of the second incubation time MNPs@ConA and OQDs@Ab-Sal solutions were prepared according to the method described in Example 1.

[0089] According to the method described in Example 1, 10 5 CFU / mL of Salmonella was added to 3.0 mg of MNPs@ConA for the first incubation, and the first incubation time was controlled at 75 min. After the excess liquid in the incubation solution was removed by magnetic separation, 1 mL of OQDs@Ab-Sal solution was added (the concentration of OQDs@Ab-Sal in the OQDs@Ab-Sal solution was 0.6 mg / mL) and mixed for a second incubation with incubation times of 15 min, 30 min, 45 min, 60 min and 70 min. During the second incubation, the pH of the solution was controlled at 8.2 to obtain five MNPs@ConA / Salmonella / OQDs@Ab-Sal sandwich complexes.

[0090] The fluorescence intensity and absorbance values ​​of the five MNPs@ConA / Salmonella / OQDs@Ab-Sal sandwich complexes were detected. Figure 5 As shown in Figure f, it can be seen that the optimal second incubation time is 60 minutes.

[0091] While various embodiments of the present invention have been described above, the foregoing description is intended to be illustrative, non-exhaustive, and not limited to the disclosed embodiments. Many modifications and variations will be apparent to those skilled in the art without departing from the scope and spirit of the described embodiments. The terminology used herein is selected to best explain the principles of the embodiments, their practical applications, or improvements to existing technologies, or to enable others skilled in the art to understand the embodiments disclosed herein.

Claims

1. A colorimetric-fluorescence dual-mode detection method for Salmonella and Escherichia coli, characterized by: The specific steps include: (1) Activating the carboxyl groups on RQDs, OQDs, and MNPs to obtain activated RQDs solution, OQDs solution, and MNPs solution; (2) The activated MNPs solution was coupled with concanavalin A solution and magnetically separated to obtain MNPs@ConA; (3) The activated RQDs solution was coupled with the Ab-Esc solution to obtain the RQDs@Ab-Escs solution, and the OQDs solution was coupled with the Ab-Sal solution to obtain the OQDs@Ab-Sal solution; (4) MNPs@ConA was mixed with E. coli solution of gradient concentrations for the first incubation. After incubation, magnetic separation was performed to remove excess liquid. RQDs@Ab-Esc solution was added for the second incubation to obtain the MNPs@ConA / E. coli / RQDs@Ab-Esc sandwich complex. (5) MNPs@ConA was mixed with Salmonella solution of gradient concentration for the first incubation. After incubation, magnetic separation was performed to remove excess liquid. OQDs@Ab-Sal solution was added for the second incubation to obtain the MNPs@ConA / Salmonella / OQDs@Ab-Sal sandwich complex. (6) Detect the fluorescence intensity or UV absorbance of the MNPs@ConA / Escherichia coli / QDs@Ab-Esc sandwich complex and the MNPs@ConA / Salmonella / OQDs@Ab-Sal sandwich complex, respectively, and establish a standard curve between bacterial solution concentration and fluorescence intensity or between bacterial solution concentration and UV absorbance; (7) MNPs@ConA is mixed with the sample to be tested for the first incubation. After incubation, magnetic separation is performed to remove excess liquid, and RQDs@Ab-Esc solution or OQDs@Ab-Sal is added for the second incubation to finally obtain MNPs@ConA / to-be-tested Escherichia coli / RQDs@Ab-Esc sandwich complex or MNPs@ConA / to-be-tested Salmonella / OQDs@Ab-Sal sandwich complex; (8) Detect the fluorescence intensity or UV absorbance of the MNPs@ConA / to-be-detected Escherichia coli / RQDs@Ab-Esc sandwich complex or the MNPs@ConA / to-be-detected Salmonella / OQDs@Ab-Sal sandwich complex respectively, and substitute it into the standard curve obtained in step (6) to obtain the concentration of Escherichia coli and Salmonella in the sample to be tested.

2. The colorimetric-fluorescence dual-mode detection method for Salmonella and Escherichia coli according to claim 1, characterized in that: In step (1), the EDC / NHS method is used to activate the carboxyl groups on the surfaces of RQDs, OQDs and MNPs.

3. The colorimetric-fluorescence dual-mode detection method for Salmonella and Escherichia coli according to claim 1, characterized in that: The volume ratio of the activated MNPs solution to the concanavalin A solution in step (2) is 1:(0.1-0.3); The mass concentration of ConA in the ConA solution is 4.8~5.1 mg / mL.

4. The colorimetric-fluorescence dual-mode detection method for Salmonella and Escherichia coli according to claim 1, characterized in that: In step (3), the volume ratio of the activated RQDs solution to the Ab-Esc solution is 1:(0.005-0.015); the volume ratio of the activated OQDs solution to the Ab-Sal solution is 1:(0.005-0.015), wherein the concentration of Ab-Esc in the Ab-Esc solution is 0.9-1.1 mg / mL; and the concentration of Ab-Sal in the Ab-Sal solution is 0.9-1.1 mg / mL.

5. The colorimetric-fluorescence dual-mode detection method for Salmonella and Escherichia coli according to claim 1, characterized in that: In step (3), the concentration of RQDs@Ab-Escs in the RQDs@Ab-Escs solution is greater than or equal to 0.6 mg / mL; the concentration of OQDs@Ab-Sal solution in the OQDs@Ab-Sal solution is greater than or equal to 0.6 mg / mL.

6. The colorimetric-fluorescence dual-mode detection method for Salmonella and Escherichia coli according to claim 1, characterized in that: In steps (4), (5) and (7), the amount of MNPs@ConA added to the bacterial solution is 2.5~3.5 mg / mL; the solid-liquid ratio of MNPs@ConA to RQDs@Ab-Esc solution is: (2.5~3.5):

1. The unit is mg:mL; the solid-liquid ratio of MNPs@ConA to OQDs@Ab-Sal solution is: 2.5~3.5:

1. The unit is mg:mL.

7. The colorimetric-fluorescence dual-mode detection method for Salmonella and Escherichia coli according to claim 1, characterized in that: The first incubation time in step (4), step (5) and step (7) is greater than or equal to 60 minutes; the second incubation time is greater than or equal to 45 minutes.

8. The colorimetric-fluorescence dual-mode detection method for Salmonella and Escherichia coli according to claim 1, characterized in that: During the second incubation in step (4), step (5) and step (7), the pH of the solution is maintained between 7.2 and 9.0.

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