Preparation method and application of gold nanocluster / antigen aggregate

Abstract

The invention discloses a preparation method and application of a gold nanocluster / antigen aggregate, and the preparation method comprises the following steps: dropwise adding a Prot solution into ATT-AuNCs, incubating to synthesize Proton-AuNCs, carrying out centrifugal treatment, and storing in a dark place to prepare the Proton-AuNCs; the preparation method comprises the following steps: mixing Proton-AuNCs with CAR-BSA (Chimeric Antigen Receptor-Bovine Serum Albumin) to prepare a gold nano cluster / antigen aggregate; the pairing of FeNPs and the aggregate enables the FONLFA detection system to achieve high sensitivity, the sensitivity of the FONLFA detection system is respectively improved by 5 times and 100 times compared with the sensitivity of an off mode, the sensitivity of the FONLFA detection system is respectively improved by 5 times and 200 times compared with the sensitivity of a traditional colorimetric mode, and the FONLFA detection system can be used for carrying out naked eye detection on a large batch of samples.

Description

Technical Field

[0001] The invention belongs to the field of food safety detection and nano-biosensor technology, and specifically relates to a preparation method and application of a gold nanocluster / antigen aggregate. Background Art

[0002] Lateral flow analysis (LFA) is a paper-based rapid detection tool with the advantages of simplicity, speed and economy. At present, it has been widely used in food safety, biomedicine, toxic chemical detection and environmental monitoring. Lateral flow analysis (LFA) has a detection speed far beyond that of laboratory detection and has great applications in the field of rapid detection, but LFA also has obvious limitations. The sensitivity of LFA detection is far from the level of laboratory detection. The LFA detection principle of small molecule targets is usually competitive, outputting an "off" signal, in which the concentration of the analyte is inversely proportional to the signal intensity. Due to the high background signal, this detection mode is not conducive to the "naked eye" qualitative detection of LFA. The low detection sensitivity and specificity of traditional LFA greatly limit its application in large-scale field detection.

[0003] LFA has its irreplaceable advantages in the field of rapid detection. In order to improve the sensitivity and accuracy of LFA, researchers have invested a lot of energy to improve the technology. Some studies apply fluorescent nanomaterials and metal nanoclusters to LFA to form a signal "on" lateral flow immunoassay mode based on the fluorescence quenching mechanism, which shows higher naked eye detection sensitivity than traditional LFA. The fluorescent donors currently used mainly include fluorescein, fluorescent groups, quantum dots and time-resolved fluorescent nanospheres, but the preparation process of these fluorescent nanomaterials is complicated and requires tedious modification before they can be further applied to LFA. The modification process may also affect the stability of the fluorescent material, which is not conducive to the development of the signal "on" lateral flow immunoassay method based on the fluorescence quenching mechanism.

[0004] Metal nanoclusters are an emerging class of fluorescent materials that are less toxic and easier to synthesize than previous fluorescent materials. The structure and fluorescence properties of metal nanoclusters are closely related to ligands, metal-metal interactions, and assembly methods. Stable ligands usually play an important role in guiding their structure and properties. In addition to the commonly used thiol molecules as protective ligands for metal nanoclusters, more ligands, such as proteins, can be easily further coupled, thereby improving their applications in food, biology, and medicine. In the synthesis of self-assembled nanoparticles, the collective interactions of the assembled components can be controlled to effectively give new materials special properties, making them more widely used.

[0005] Therefore, it is of great research significance to prepare a simple, stable and highly sensitive gold nanocluster antigen aggregate and apply it to a rapid detection platform. Summary of the invention

[0006] The purpose of this section is to summarize some aspects of embodiments of the present invention and briefly introduce some preferred embodiments. Some simplifications or omissions may be made in this section and the specification abstract and the invention title of this application to avoid blurring the purpose of this section, the specification abstract and the invention title, and such simplifications or omissions cannot be used to limit the scope of the present invention.

[0007] In view of the above problems and / or the problems existing in the prior art, the present invention is proposed.

[0008] Therefore, the purpose of the present invention is to overcome the deficiencies in the prior art and provide a method for preparing gold nanocluster / antigen aggregates.

[0009] In order to solve the above technical problems, the present invention provides the following technical solutions: a method for preparing gold nanocluster / antigen aggregates, comprising:

[0010] The Prot solution was added dropwise to the ATT-AuNCs, incubated to synthesize the Prot-AuNCs, centrifuged, and stored in the dark to obtain the Prot-AuNCs;

[0011] Prot-AuNCs were mixed with CAR-BSA to prepare gold nanocluster / antigen aggregates.

[0012] As a preferred embodiment of the preparation method of the present invention, the preparation method of the ATT-AuNCs comprises:

[0013] Dissolve 6-aza-2-thiothymine ATT in NaOH solution;

[0014] The ATT solution was mixed with the HAuCl4 solution, and the mixed solution was continuously stirred at 25°C in the dark to prepare ATT-AuNCs.

[0015] As a preferred embodiment of the preparation method of the present invention, the concentration of the Prot solution is 15 μmol / L, and the volume ratio of ATT-AuNCs to Prot solution is 2:3.

[0016] Another object of the present invention is to overcome the deficiencies in the prior art and provide the use of the gold nanocluster / antigen aggregates prepared by the preparation method in the detection of carbendazim.

[0017] As a preferred solution of the application of the present invention, it includes:

[0018] Mix the carbendazim sample to be tested with the running buffer, take 50-100 μL of the mixed solution and drop it onto the Ab-FONLFA test paper sample pad, and read the test result after 5-10 minutes;

[0019] The running buffer included 4×SSC, 0.5% Tween-20, pH 7.4;

[0020] Wherein, the Ab-FONLFA test paper contains gold nanoclusters / antigen aggregates.

[0021] As a preferred embodiment of the application of the present invention, the method for preparing the Ab-FONLFA test paper comprises:

[0022] Preparation of signal probe Ab-AuNPs: AuNPs were synthesized by citrate reduction method, and Ab-AuNPs were prepared by physical adsorption and condensation reaction;

[0023] Preparation of signal probe Ab-FePNs: Preparation of Ab-FePNs by physical adsorption and condensation reaction;

[0024] Assembly of the test strip: stick the sample pad, gold label pad, NC membrane and absorption pad on the PVC adhesive backing in sequence, overlapping each by 2 mm;

[0025] Gold nanoclusters / antigen aggregates were sprayed on the T line of the NC membrane using a three-dimensional spraying platform;

[0026] Prot-AuNCs and BSA were sprayed on line C and dried, and the distance between the test line and the control line was 4-8 mm.

[0027] As a preferred embodiment of the application of the present invention, the Ab-FONLFA test paper comprises a PVC base plate, a sample pad, a gold label pad, an NC membrane and an absorption pad;

[0028] Wherein, the sample pad, the gold label pad, the NC film and the absorption pad are sequentially adhered on the PVC bottom plate.

[0029] As a preferred solution of the application of the present invention, wherein: the overlap length between the sample pad and the PVC adhesive back is 1 to 2 mm, and the sample pad is placed above the PVC adhesive back;

[0030] The overlapping length between the sample pad, gold label pad and NC membrane is 1 to 2 mm, the sample pad membrane is placed above the gold label pad, and the gold label pad is placed above the NC membrane; the overlapping length between the NC membrane and the absorption pad is 1 to 3 mm, and the absorption pad is placed above the NC membrane.

[0031] As a preferred embodiment of the application of the present invention, the method for preparing the signal probe Ab-AuNPs comprises:

[0032] Take 1 mL of colloidal gold solution, adjust the pH of the system with 0.1 M K2CO3 solution, shake well, add 5 μg of carbendazim antibody, mix gently, place on a shaker and oscillate slowly for 45 min, and add 1 mL of colloidal gold sealing solution to each tube of solution;

[0033] After mixing evenly, seal at room temperature for 2 h;

[0034] After blocking, centrifuge at 8000r / min for 15min, discard the supernatant, add gold-labeled antibody washing solution, shake well, resuspend, centrifuge again to remove the supernatant, repeat 2 times, wherein the formula of gold-labeled antibody washing solution is: 0.01M PBS buffer, 5% PEG-2000, pH=7.4;

[0035] The obtained precipitate was redispersed in 1 ml of gold-labeled antibody resuspension solution and stored at 4° C., wherein the formula of the gold-labeled antibody resuspension solution was: 0.01 M boric acid buffer, 0.1% Tween 20, 5% sucrose, 1% BSA, and 0.05% sodium azide.

[0036] As a preferred embodiment of the application of the present invention, the method for preparing Ab-FePNs comprises:

[0037] Evenly mix 50 μL of FeCl3·H2O solution 6.7 mM and 50 μL of DA·HCl 100 mM, and then quickly add 5 mL of Tris solution 0.1 M into the mixed solution;

[0038] Subsequently, FePNs were collected by centrifugation after 100 min of reaction and then resuspended in ultrapure water;

[0039] Dilute 60 μg of FePNs with 1 mL of 0.02 mol / LBB solution, then add 200 μL of 50 μg / mL CAR antibody and stir for 1.5 h;

[0040] Then 200 μL of 5% casein was added to the mixture for blocking for 2 h, and the Ab-FePNs were purified by centrifugation and resuspended in 100 μL of ultrapure water and stored at 4°C.

[0041] Beneficial effects of the present invention:

[0042] (1) The present invention uses gold nanoclusters as fluorescence donors to form an aggregate with protamine, which can significantly enhance the fluorescence of the gold nanoclusters. In addition, the preparation of this aggregate is relatively simple and has low toxicity.

[0043] (2) The aggregates formed by the gold nanoclusters and protamine of the present invention can be fixed on the NC membrane by simply mixing and spraying with the antigen, which greatly simplifies the process flow.

[0044] (3) The pairing of FeNPs and aggregates in the present invention enables FONLFA to achieve ultra-high naked eye detection sensitivity, which is 5 times and 100 times higher than the sensitivity of colorimetric detection in the "off" mode of the corresponding nanoprobe, and 5 times and 200 times higher than the sensitivity of the traditional colloidal gold-colorimetric mode, respectively, and can be used for naked eye detection of large quantities of samples. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following briefly introduces the drawings required for describing the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative labor. Among them:

[0046] Figure 1 TEM images, UV-visible absorption spectra and Prot concentration optimization results of the synthetic raw materials in the embodiments of the present invention; wherein A is the TEM image of Prot-AuNCs, B is the UV-visible absorption spectra of ATT-AuNCs and Prot-AuNCs, and C is the Prot concentration optimization diagram.

[0047] Figure 2 These are TEM images of AuNPs and FeNPs, where A is the TEM image of AuNPs and B is the TEM image of FeNPs.

[0048] Figure 3 Characterization of the self-assembly results of the present invention; wherein, A is the TEM image of Prot-AuNCs and CAR-BSA after self-assembly, B is the particle size spectrum of Prot-AuNCs, CAR-BSA, BSA, AuNPs / CAR-BSA and AuNPS / BSA, and C is the fluorescence intensity diagram of Prot-AuNCs, AuNPs / CAR-BSA and AuNPS / BSA.

[0049] Figure 4 Optimization diagram of the dilution ratio of Prot-AuNCs.

[0050] Figure 5 It is the optimization diagram of the FONLFA detection system of the present invention; wherein, A and B are the optimization result diagrams of the Ab-AuNPS volume and CAR-BSA concentration of the AuNPs-FONLFA detection system, and C and D are the optimization result diagrams of the Ab-FeNPs volume and CAR-BSA concentration of the FeNPs-FONLFA detection system.

[0051] Figure 6It is a linear range diagram detected by the present invention and a standard curve diagram for detecting carbendazim; wherein, (A) is the visual detection range of carbendazim of AuNPs-LFA, AuNPs-FONLFA and FeNPs-FONLFA, and (B) is the standard curve diagram for detecting carbendazim of AuNPs-LFA, AuNPs-FONLFA and FeNPs-FONLFA.

[0052] Figure 7 is the extinction coefficient and quenching efficiency diagram, where A represents FeNPs with a higher extinction coefficient, and B represents FeNPs with a higher quenching efficiency.

[0053] Figure 8 It is a graph showing the specificity results of the FeNPs-FONLFA test paper of the present invention. DETAILED DESCRIPTION

[0054] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, the specific implementation methods of the present invention are described in detail below in conjunction with the embodiments of the specification.

[0055] In the following description, many specific details are set forth to facilitate a full understanding of the present invention, but the present invention may also be implemented in other ways different from those described herein, and those skilled in the art may make similar generalizations without violating the connotation of the present invention. Therefore, the present invention is not limited to the specific embodiments disclosed below.

[0056] Secondly, the term "one embodiment" or "embodiment" as used herein refers to a specific feature, structure, or characteristic that may be included in at least one implementation of the present invention. The term "in one embodiment" that appears in different places in this specification does not necessarily refer to the same embodiment, nor does it refer to a separate or selective embodiment that is mutually exclusive with other embodiments.

[0057] Example 1

[0058] Preparation and identification of AuNPs, FePNs, and Prot-AuNCs:

[0059] 1. Synthesis of Gold Nanoparticles

[0060] Gold nanoparticles were synthesized by the seed growth method. The specific steps are as follows:

[0061] Sodium citrate solution (2.2 mM, 75 mL) was injected into a three-necked round-bottom flask and heated for 10 min;

[0062] Then, 0.8 mL HAuCl4 (25 mM) was added and reacted for 8 min;

[0063] Observe the color change from yellow to dark pink, and immediately cool the prepared gold seeds to 90°C;

[0064] Then, at a constant temperature of 90 °C, 0.8 mL of sodium citrate solution (60 mM) was added, and after 2 minutes, 0.8 mL of HAuCl4 (25 mM) was added, and after reacting for 30 min, 25 nm AuNPs were obtained;

[0065] TEM images such as Figure 2 As shown in A.

[0066] 2. Preparation of signal probe Ab-AuNPs

[0067] Take 1 mL of colloidal gold solution and adjust the pH of the system with 0.1 M K2CO3 solution;

[0068] After shaking, add 5 μg of carbendazim antibody (purchased from Di Tengmin Biotechnology Co., Ltd.), mix gently, place on a shaker and oscillate slowly for 45 min, and add 1 mL of colloidal gold sealing solution to each tube of solution;

[0069] After mixing evenly, seal at room temperature for 2 h;

[0070] After blocking, centrifuge at 8000r / min for 15min, discard the supernatant, add gold-labeled antibody washing solution (0.01M PBS buffer, 5% PEG-2000, pH=7.4), shake well, resuspend, centrifuge again and remove the supernatant, repeat twice;

[0071] The obtained precipitate was redispersed in 1 ml of gold-labeled antibody resuspension solution (0.01 M boric acid buffer, 0.1% Tween 20, 5% sucrose, 1% BSA, 0.05% sodium azide) and stored at 4°C.

[0072] 3. Preparation of FePNs

[0073] First, 50 μL of FeCl3·H2O solution (6.7 mM) and 50 μL of DA·HCl (100 mM) were evenly mixed, and then 5 mL of Tris solution (0.1 M) was quickly added to the mixed solution;

[0074] Subsequently, after 100 min of reaction, FePNs were collected by centrifugation (12000 r / min, 15 min) and then resuspended in ultrapure water;

[0075] TEM images such as Figure 2 As shown in B.

[0076] The extinction coefficient and quenching efficiency of FeNPs are shown in Figure 7 , the FeNPs shown in A have a higher extinction coefficient and a wider spectral overlap range, and the FeNPs in B have a higher quenching efficiency.

[0077] 4. Preparation of Signal Probe Ab-FeNPs

[0078] First, FePNs (60 μg) were diluted with 1 mL of BB (0.02 mol / L) solution, and then 200 μL of CAR antibody (50 μg / mL) was added and stirred (300 r / min, 20 °C) for 1.5 h;

[0079] Then, 200 μL of 5% casein was added to the mixture for blocking for 2 h. Ab-FePNs were purified by centrifugation (7000 r / min, 15 min, 4° C.) and resuspended in 100 μL of ultrapure water and stored at 4° C.

[0080] 5. Synthesis and identification of Prot-AuNCs

[0081] (1) Preparation of ATT-AuNCs

[0082] Preparation of ATT-AuNCs, the specific steps are as follows:

[0083] Dissolve 6-aza-2-thiothymidine (ATT) in NaOH (0.2 M) solution;

[0084] ATT (3 mL, 80 mM) solution was mixed with HAuCl4 (3 mL, 10 mg / mL) solution, and the mixed solution was stirred continuously for 1 h at 25 °C in the dark;

[0085] Then ATT-AuNCs were purified by ultrafiltration (Millipore, 50 kDa);

[0086] The UV-visible absorption spectrum of ATT-AuNCs is shown in Figure 1 B.

[0087] (2) Preparation of Prot-AuNCs

[0088] 1 mL of Prot (protamine, purchased from Sigma, 15 μmol / L) was added dropwise to 1.5 mL of ATT-AuNCs (diluted 2 times, 2x);

[0089] After incubation at 37 °C for 2 min, the synthesized Prot-AuNCs were centrifuged and stored at 4 °C in the dark;

[0090] TEM images of Prot-AuNCs are shown in Figure 1 A.

[0091] Example 2

[0092] Preparation of signal "on" lateral flow chromatography test strips based on fluorescence quenching mechanism:

[0093] 1. Preparation of test strips

[0094] Five materials were used to prepare the lateral flow chromatography device, i.e., the sample pad, gold label pad, NC membrane, and absorbent pad were sequentially attached to the PVC adhesive backing, with each overlapping by 2 mm;

[0095] 20xProt-AuNCs and 0.05 mg / mL CAR-BSA were sprayed on the test line (T line) of the NC membrane using a three-dimensional spraying platform;

[0096] 20xProt-AuNCs and 0.05 mg / mL BSA were sprayed on the control line (C line) of the NC membrane and dried at 37 °C for 4 h, and the distance between the T line and the C line was 5 mm;

[0097] Finally, the test strips were cut into 4 mm width and stored at 4 °C.

[0098] 2. Establishment of the detection method based on Ab-FONLFA test paper

[0099] 80 μL of different concentrations of CAR (0, 0.05, 0.1, 0.2, 0.5, 1, 2, 5, 10 ng / mL) was mixed with 20 μL of Running buffer and added to the sample pad. The results were detected with a portable reader after 8 minutes.

[0100] In the absence of CAR, the Ab-AuNPs and Ab-FeNPs immunoprobes were captured by the antigen (CAR-BSA) on the T line, forming red and black stripes, respectively, and quenching the green fluorescence on the T line;

[0101] In the presence of CAR, CAR competes with CAR-BSA on the T line to bind to limited Ab-AuNPs and Ab-FeNPs. At this time, the color signal on the T line is "off", while the fluorescence signal on the T line is "on";

[0102] Regardless of the presence or absence of CAR, the fluorescence on the C line is always in the signal "on" state.

[0103] 3. Characterization of Self-Assembly Results

[0104] TEM images of Prot-AuNCs and CAR-BSA after self-assembly are shown in Figure 3 A, it can be seen that aggregates are easily formed by simply mixing Prot-AuNCs with CAR-BSA;

[0105] Particle size profiles of Prot-AuNCs, CAR-BSA, BSA, AuNPs / CAR-BSA, and AuNPS / BSA are shown in Figure 3B. It can be seen that after self-assembly, DLS measurement results showed that the average hydrated particle sizes of AuNCs / CAR-BSA and AuNCs / BSA were 48.1±6.07nm and 52.8±7.25nm, respectively, which were significantly larger than BSA (5.6±1.06nm), CAR BSA (3.6±0.82nm) and Prot-AuNCs (7.5±1.86nm).

[0106] The fluorescence intensities of Prot-AuNCs, AuNPs / CAR-BSA and AuNPS / BSA are shown in Figure 3 C, it can be seen that the fluorescence intensity of Prot-AuNCs is almost unaffected before and after self-assembly.

[0107] Example 3

[0108] Optimization of Ab-FONLFA detection method:

[0109] 1. Optimization of Prot modified ATT-AuNs

[0110] While keeping other conditions unchanged (CAR-BSA concentration 0.05 mg / mL, Prot-AuNs dilution ratio 20×), the fluorescence intensity of the T line was detected when different concentrations of Prot (5μM, 10μM, 15μM, 20μM) and ATT-AuNs conjugates were mixed with antigens and fixed on the T line, and the fluorescence intensity of the T line was detected in a portable reader.

[0111] The results are as follows Figure 1 As shown in C, when the Prot concentration is 15 μM, the fluorescence signal intensity reaches the highest.

[0112] 2. Synthesis of AuNPs and FePNs:

[0113] Under the condition of keeping other conditions unchanged (CAR-BSA concentration 0.05 mg / mL, Prot-AuNCs dilution ratio 20×), different quenchers AuNPs and FePNs were synthesized for quenching. The TEM images after synthesis are shown in Figure 2. Figure 2 A is AuNPs, Figure 2 B is FePNs.

[0114] 3. Optimization of Ab-FONLFA experimental conditions

[0115] (1) Concentration of CAR-BSA

[0116] While keeping other conditions unchanged (Prot-AuNCs dilution ratio 20×, Ab-AuNPs volume 5 μL), only the concentration of CAR-BSA (0.01 mg / mL-0.05 mg / mL) was changed. CAR-BSA was purchased from Di Tengmin Biotechnology Co., Ltd. (Wuxi, China). The signal strength was determined by detecting the fluorescence intensity of the T line using a portable reader. The results are shown in Figure 2. Figure 5 As shown in B, the optimal CAR-BSA concentration is 0.04 mg / mL.

[0117] (2) Prot-AuNCs dilution ratio

[0118] The specific method is the same as step (1), only the Prot-AuNCs dilution ratio is changed (24X, 48X, 96X). Figure 4 , the optimal dilution ratio is 48X.

[0119] (3) Volume of Ab-AuNPs and Ab-FePNs

[0120] The specific method is the same as step (1), except that the volume of Ab-AuNPs (2 μL-6 μL) and the volume of Ab-FePNs (1 μL-3 μL) are changed.

[0121] When other conditions were kept constant (CAR-BSA concentration 0.05 mg / mL, Prot-AuNCs dilution ratio 20×), different volumes of Ab-AuNPs probes were added. The results were shown in Table 1 according to the degree of T-line signal quenching. Figure 5 A, the optimal probe addition volume is 5 μL;

[0122] When other conditions were kept constant (CAR-BSA concentration 0.04 mg / mL, Prot-AuNCs dilution ratio 20×), different volumes of Ab-FeNPs probes (1-3 μL) were added. The results are shown in Figure 2. Figure 5 C, according to the degree of T line signal quenching, the optimal probe addition amount is 3 μL;

[0123] While keeping other conditions unchanged (probe volume 3 μL, Prot-AuNCs dilution ratio 20×), the CAR-BSA concentration was changed (0.01-0.03 mg / mL). The results were as follows based on the T-line signal response. Figure 5 D, The optimal CAR-BSA concentration is 0.03 mg / mL.

[0124] Example 4

[0125] Detection of Carbendazim:

[0126] (1) Determination of the sensitivity of carbendazim Ab-FONLFA test paper

[0127] Under the optimal experimental conditions determined in Example 3, 80 μL of different concentrations of CAR (0, 0.05, 0.1, 0.2, 0.5, 1, 2, 5, 10 ng / mL) was mixed with 20 μL of Running buffer and added to the sample pad. The results were detected with a portable reader after 8 minutes to determine the sensitivity of carbendazim Ab-FONLFA.

[0128] Visual LOD (vLOD) is defined as: in the traditional colorimetric mode, the color of the T line is weaker than the minimum carbendazim concentration of the control sample; in the "on" mode, the fluorescence is stronger than the minimum carbendazim concentration of the control sample.

[0129] Under the optimal detection conditions (AuNPs-FONLFA detection system: Ab-AuNPs probe volume is 5μL, CAR-BSA concentration is 0.04mg / mL; FeNPs-FONLFA detection system: Ab-FeNPs probe volume is 3μL, CAR-BSA concentration is 0.03mg / mL), the vLOD of carbendazim detected in the traditional colorimetric mode is 10ng / ml, the vLOD of carbendazim detected by Ab-AuNPs probe in the "off" mode and "on" mode are 10ng / mL and 2ng / mL, respectively, and the vLOD of carbendazim detected by Ab-FeNPs probe is 5ng / mL and 0.05ng / mL, respectively. That is, the fluorescence "on" mode of FONLFA shows excellent visual qualitative ability, which is 5 times and 100 times more sensitive than the "off" mode, and 5 times and 200 times more sensitive than the traditional colorimetric mode.

[0130] like Figure 6 A, the vLOD of carbendazim detected in the traditional colorimetric mode is 10ng / ml. With the increase of carbendazim concentration, the T line color signal gradually "turns off" under natural light, and the green fluorescence gradually "turns on" under ultraviolet light. In the "off" mode and "on" mode, the vLOD of carbendazim detected by Ab-AuNPs probe is 10ng / mL and 2ng / mL, respectively, and the vLOD of carbendazim detected by Ab-FeNPs probe is 5ng / mL and 0.05ng / mL, respectively. That is, the fluorescence "on" mode of FONLFA shows excellent visual qualitative ability, which is 5 times and 100 times more sensitive than the "off" mode, and 5 times and 200 times more sensitive than the traditional colorimetric mode, respectively, and can be used for naked eye detection of large quantities of samples.

[0131] For the quantitative detection of carbendazim, a portable reader was used to record the color and fluorescence signals. A quantitative calibration curve for carbendazim detection was constructed by the relationship between the signal intensity ratio and the logarithmic concentration of carbendazim.

[0132] The standard curves of AuNPs-LFA, AuNPs-FONLFA and FeNPs-FONLFA for detecting carbendazim. In the traditional colorimetric mode and fluorescence "on" mode, the LODs of carbendazim using Ab-AuNPs probe were 10 ng / mL and 2 ng / mL, respectively, and the detection ranges were 0.2-5 ng / mL and 0.2-10 ng / mL, respectively. Using Ab-FeNPs probe, the LODs of carbendazim were 5 ng / mL and 0.05 ng / mL, respectively, and the detection ranges were 0.1-2 ng / mL and 0.01-0.2 ng / mL, respectively.

[0133] Use a portable reader for quantitative testing. Figure 6 B In the traditional colorimetric mode and fluorescence "on" mode, the LOD of carbendazim using Ab-AuNPs probe was 10 ng / mL and 2 ng / mL, and the detection range was 0.2-5 ng / mL and 0.2-10 ng / mL, respectively. Using Ab-FeNPs probe, the LOD of carbendazim was 5 ng / mL and 0.05 ng / mL, and the detection range was 0.1-2 ng / mL and 0.01-0.2 ng / mL, respectively.

[0134] These results indicate that the sensitivity of the “on” mode of FONLFA is better than that of the “off” mode.

[0135] (2) Specificity of Carbendazim Ab-FONLFA Test Paper

[0136] Several drugs with similar structures and functions to carbendazim, such as benzimidazole, thiophene imidazole, ethylthiophanate, methylthiophanate and 2-mercaptobenzimidazole, were used as potential interfering substances to test the specificity of Ab-FONLFA. The steps are as follows:

[0137] 80 μL of different concentrations of carbendazim structural analogs (10, 20, 50 ng / mL) were mixed with 20 μL of Running buffer and added to the sample pad. The results were detected using a portable reader after 8 minutes.

[0138] The results are as follows Figure 8 As shown, only the test paper with carbendazim added has a positive test result, while the test results with other interfering substances added are all negative. Therefore, the Ab-FONLFA established in the present invention has a strong specificity for carbendazim.

[0139] (3) Testing the recovery rate using apple simulated samples

[0140] The recovery rate was tested using apple simulated samples. The steps are as follows:

[0141] Homogenized apple samples (20 g) were added to the CAR standard solution and 20 mL of acetonitrile, mixed in a 50 mL centrifuge tube, and the samples were vigorously stirred for 2 min;

[0142] Then, 8 g of anhydrous MgSO4 and 2 g of NaCl were added and immediately mixed on a shaker for 2 min;

[0143] Next, the mixture was shaken for another 1 min and centrifuged at 5000 rpm for 5 min;

[0144] The concentrations of the standard solutions were 0, 0.2, 1, and 5 μg / kg;

[0145] Afterwards, the centrifuged supernatant was filtered through a 0.22 μm membrane syringe filter;

[0146] Finally, the filtrates were combined and diluted 3 times with 0.1 M PBS (pH = 7.0) for testing with Ab-FONLFA prepared in Example 2, and the concentration of CAR after spike addition was obtained according to the standard curve obtained using the Ab-FeNPs probe in Example 4, and the spike recovery rate was calculated.

[0147] As shown in Table 1, in the fluorescence "on" mode, the average recovery of CAR in the spiked apple samples was 95.0%-110.0%, and the CVs were lower than 5.3%. In addition, in the conventional colorimetric mode, the average recovery of CAR in the spiked apple samples was 96.0%-105.0%, and the CV was lower than 4.8%. These results show that FePNs-FONLFA is an excellent method for detecting CAR in real samples. Therefore, the Ab-FONLFA method provided by the present invention can be used for the rapid detection of carbendazim in food.

[0148] Table 1

[0149]

[0150] Among them, ND, not detectable; NC, not calculated.

[0151] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present invention can be modified or replaced by equivalents without departing from the spirit and scope of the technical solutions of the present invention, which should all be included in the scope of the present invention.

Claims

1. A method for preparing gold nanocluster / antigen aggregates, characterized in that: include, The Prot solution was added dropwise to the ATT-AuNCs, incubated to synthesize the Prot-AuNCs, centrifuged, and stored in the dark to obtain the Prot-AuNCs; Prot-AuNCs were mixed with CAR-BSA to prepare gold nanocluster / antigen aggregates.

2. The preparation method according to claim 1, characterized in that: The preparation method of the ATT-AuNCs comprises: Dissolve 6-aza-2-thiothymine ATT in NaOH solution; The ATT solution was mixed with the HAuCl4 solution, and the mixed solution was continuously stirred at 25°C in the dark to prepare ATT-AuNCs.

3. The preparation method according to claim 1 or 2, characterized in that: The concentration of the Prot solution is 15 μmol / L, and the volume ratio of ATT-AuNCs to the Prot solution is 2:

3.

4. Use of the gold nanoclusters / antigen aggregates prepared by the preparation method according to any one of claims 1 to 3 in the detection of carbendazim.

5. The use according to claim 4, characterized in that: include, Mix the carbendazim sample to be tested with the running buffer, take 50-100 μL of the mixed solution and drop it onto the Ab-FONLFA test paper sample pad, and read the test result after 5-10 minutes; The running buffer included 4×SSC, 0.5% Tween-20, pH 7.4; Wherein, the Ab-FONLFA test paper contains gold nanoclusters / antigen aggregates.

6. The use according to claim 5, characterized in that: The preparation method of the Ab-FONLFA test paper comprises: Preparation of signal probe Ab-AuNPs: AuNPs were synthesized by citrate reduction method, and Ab-AuNPs were prepared by physical adsorption and condensation reaction; Preparation of signal probe Ab-FePNs: Preparation of Ab-FePNs by physical adsorption and condensation reaction; Assembly of the test strip: stick the sample pad, gold label pad, NC membrane and absorption pad on the PVC adhesive backing in sequence, overlapping each by 2 mm; Gold nanoclusters / antigen aggregates were sprayed on the T line of the NC membrane using a three-dimensional spraying platform; Prot-AuNCs and BSA were sprayed on line C and dried, and the distance between the test line and the control line was 4-8 mm.

7. The use according to claim 5, characterized in that: The Ab-FONLFA test paper comprises a PVC base plate, a sample pad, a gold label pad, an NC membrane and an absorption pad; Wherein, the sample pad, the gold label pad, the NC film and the absorption pad are sequentially adhered on the PVC bottom plate.

8. The use according to claim 6, characterized in that: The overlapping portion between the sample pad and the PVC adhesive backing is 1 to 2 mm in length, and the sample pad is placed above the PVC adhesive backing; The overlapping length between the sample pad, gold label pad and NC membrane is 1 to 2 mm, the sample pad membrane is placed above the gold label pad, and the gold label pad is placed above the NC membrane; the overlapping length between the NC membrane and the absorption pad is 1 to 3 mm, and the absorption pad is placed above the NC membrane.

9. The use according to claim 6, characterized in that: The method for preparing the signal probe Ab-AuNPs comprises: Take 1 mL of colloidal gold solution, adjust the pH of the system with 0.1 M K2CO3 solution, shake well, add 5 μg of carbendazim antibody, mix gently, place on a shaker and oscillate slowly for 45 min, and add 1 mL of colloidal gold sealing solution to each tube of solution; After mixing evenly, seal at room temperature for 2 h; After blocking, centrifuge at 8000r / min for 15min, discard the supernatant, add gold-labeled antibody washing solution, shake well, resuspend, centrifuge again to remove the supernatant, repeat 2 times, wherein the formula of gold-labeled antibody washing solution is: 0.01M PBS buffer, 5% PEG-2000, pH = 7.4; The obtained precipitate was redispersed in 1 ml of gold-labeled antibody resuspension solution and stored at 4° C., wherein the formula of the gold-labeled antibody resuspension solution was: 0.01 M boric acid buffer, 0.1% Tween 20, 5% sucrose, 1% BSA, and 0.05% sodium azide.

10. The use according to claim 6, characterized in that: The preparation method of Ab-FePNs comprises: Evenly mix 50 μL of FeCl3·H2O solution 6.7 mM and 50 μL of DA·HCl 100 mM, and then quickly add 5 mL of Tris solution 0.1 M into the mixed solution; Subsequently, FePNs were collected by centrifugation after 100 min of reaction and then resuspended in ultrapure water; Dilute 60 μg of FePNs with 1 mL of 0.02 mol / LBB solution, then add 200 μL of 50 μg / mL CAR antibody and stir for 1.5 h; Then 200 μL of 5% casein was added to the mixture for blocking for 2 h, and the Ab-FePNs were purified by centrifugation and resuspended in 100 μL of ultrapure water and stored at 4°C.

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