A kit for rapid detection of etomidate, a detection method and application

Abstract

The application discloses a rapid detection kit, a detection method and application of etomidate, and the kit comprises a fluorescent indicator preloaded tube and an immunochromatography test strip, wherein the fluorescent indicator preloaded tube is quantum dot fluorescent microspheres and etomidate monoclonal antibody chemical coupling preparation quantum dot fluorescent microsphere labeled antibody probe as a fluorescent indicator, the immunochromatography test strip comprises a sample pad, a nitrocellulose membrane and a water absorption pad, the nitrocellulose membrane is provided with a T line coated with etomidate antigen and a C line coated with a sheep anti-mouse IgG antibody; the application can be applied to rapid and efficient screening of etomidate in hair samples, is accurate in quantification, high in sensitivity, wide in detection time window, simple and convenient in operation, suitable for various on-site detection scenes, and has a very good application prospect.

Description

Technical Field

[0001] This invention relates to a rapid detection kit for etomidate, as well as a detection method using the above-described detection kit and its application in hair detection. Background Technology

[0002] Etomidate is an imidazole compound that activates γ-aminobutyric acid type A (GABAA) receptors and inhibits excitation transmission, thereby providing induction and short-term anesthesia for general anesthesia in clinical practice. It belongs to the non-barbiturate class of intravenous short-acting hypnotics. Due to its rapid onset, stable recovery, and good cardiovascular stability, it is widely used in clinical anesthesia induction and maintenance.

[0003] Currently, commonly used biological samples for drug testing mainly include urine, blood, saliva, and hair. Of the first three, drugs are metabolized relatively quickly, typically reflecting drug use over only a few hours to a few days. In contrast, hair can stably preserve drug use records for up to 3 to 6 months at room temperature, and the collection process is non-invasive and convenient, making it an important medium for long-term tracing, on-site screening, and judicial evidence collection by public security organs. However, traditional laboratory testing methods, such as high-performance liquid chromatography (HPLC), gas chromatography-mass spectrometry (GC-MS), and liquid chromatography-tandem mass spectrometry (LC-MS), generally suffer from problems such as long testing times, large equipment investment, cumbersome sample pretreatment, and stringent environmental requirements, making it difficult to meet the needs of on-site, real-time testing. While colloidal gold immunochromatography (CIGM) has advantages such as low cost, ease of operation, and rapid response, its detection sensitivity is limited, and it cannot perform quantitative analysis.

[0004] Quantum dots, as a novel type of semiconductor fluorescent nanomaterial, possess excellent optical properties such as broad excitation spectrum, narrow emission spectrum, high fluorescence quantum yield, good photostability, and large Stokes shift. Immunochromatography using quantum dots as fluorescent markers combines the high specificity of immunoreactions with the high sensitivity of quantum dot labeling, enabling rapid and accurate quantitative detection of target analytes. However, the sensitivity of current quantum dot fluorescence immunochromatography still needs improvement and is not suitable for detecting trace amounts of drugs in hair. Therefore, there is an urgent need for a method for rapid quantitative screening of individuals suspected of etomidate abuse, in order to provide frontline law enforcement personnel with a scientific and timely basis for judgment. Summary of the Invention

[0005] Purpose of the invention: The purpose of this invention is to provide a rapid detection kit for etomidate, as well as a detection method and application using the above detection kit, so as to achieve the purpose of simple operation, high sensitivity, accurate quantification, and rapid detection of etomidate in hair.

[0006] Technical Solution: The present invention provides a quantum dot fluorescent immunochromatographic kit for detecting etomidate, comprising a fluorescent indicator pre-filled tube and an immunochromatographic test strip. The fluorescent indicator pre-filled tube is an etomidate-quantum dot fluorescent microsphere conjugated antibody probe. The immunochromatographic test strip includes a sample pad, a nitrocellulose membrane, and an absorbent pad. The nitrocellulose membrane has a T line coated with etomidate antigen and a C line coated with goat anti-mouse IgG antibody. The fluorescent indicator pre-filled tube and the immunochromatographic test strip together constitute the quantum dot fluorescent immunochromatographic test strip QDFMs-LFIA.

[0007] The etomidate-quantum dot fluorescent microsphere conjugated antibody probe (QDFMs-mAb) is prepared as follows: Quantum dot fluorescent microspheres are washed with borate (BB) buffer, centrifuged, and the precipitate is resuspended. 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS) are added for activation. After activation, the precipitate is centrifuged, resuspended, and then etomidate monoclonal antibody is added for conjugation. Bovine serum albumin (BSA) is added for blocking. After centrifugation, the precipitate is resuspended in preservation solution to obtain the etomidate-quantum dot fluorescent microsphere conjugated antibody probe. The mass ratio of quantum dot fluorescent microspheres to etomidate monoclonal antibody is 10:1-30:1, preferably 12.5:1.

[0008] Furthermore, the quantum dot fluorescent microspheres have a particle size of 200-300 nm, and the BB buffer used for washing the quantum dot fluorescent microspheres has a concentration of 30-80 mM and a pH of 5.0-8.0; the concentrations of EDC and NHS are both 0.05-0.15 mg / mL; the preservation solution is a borate buffer containing NaCl and BSA, with a concentration of 80-120 mM and a pH of 7.0-8.0; the EDC and NHS are dissolved in a BB buffer containing 0.01% Tween 20 (w / v) at a pH of 5.0-8.0; when adding etomidate monoclonal antibody for conjugation, a 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid (HEPES) buffer with a pH of 7.0-8.0 is used. Preferably, the amount of etomidate monoclonal antibody added is 8 μg, and the precipitate is resuspended using HEPES buffer, 40-60 mM, pH 7.0-8.0. The blocking step involving BSA involves adding 1% BSA (w / v) HEPES buffer at a concentration of 100 mM and a pH of 7.5. Preferably, the concentrations of EDC and NHS are both 0.1 mg / mL, and the added volumes are 50 μL and 100 μL, respectively. The EDC and NHS are dissolved in 30-80 mM BB buffer containing 0.01% Tween 20 (w / v) at a pH of 5.0-8.0. Preferably, the blocking step involving BSA involves adding 1% BSA (w / v) HEPES buffer at 50 mM and a pH of 8.0. Preferably, the activation step involves a shaking reaction at room temperature for 20-50 min.

[0009] The immunochromatographic test strip includes a base plate, a sample pad, a nitrocellulose membrane, and an absorbent pad. The nitrocellulose membrane, sample pad, and absorbent pad are sequentially pasted onto the base plate along the length of the test strip, with the sample pad and absorbent pad each pressing down on the nitrocellulose membrane by 1-5 mm. Etomidate antigen and goat anti-mouse IgG antibody are sequentially sprayed onto the detection line (T line) and control line (C line) of the nitrocellulose membrane using a membrane scratching device.

[0010] Furthermore, the immunochromatographic test strip is 2-5 mm wide, and the concentrations of etomidate antigen and goat anti-mouse IgG antibody are 0.1-0.3 mg / mL and 0.2-0.5 mg / mL, respectively. Preferably, the streaking concentrations of etomidate antigen and goat anti-mouse IgG antibody are 0.2 mg / mL and 0.3 mg / mL, respectively. The spray volume is 2-10 μL / cm. The sample pad is thoroughly soaked in Tris-HCl buffer containing NaCl, surfactant S9, trehalose, and BSA, and then dried for later use. The concentration of Tris-HCl buffer is 80-120 mM, and the pH is 8.0-8.5. Preferably, the sample pad treatment solution contains 0.9% NaCl, 1% S9, 1% trehalose, and 1% BSA (w / v) in Tris-HCl buffer (80-120 mM, pH 8.0-8.5). The nitrocellulose membrane is of type SS12, SN12, or SS40.

[0011] The aforementioned quantum dot fluorescence immunochromatography kit also includes an EP tube, a shell coated with immunochromatographic test strips, a UV lamp, and a dry fluorescence immunoassay analyzer. The UV lamp is a 365nm UV flashlight. The immunochromatographic test strips and the shell together form a test card.

[0012] The preparation method of a quantum dot fluorescence immunochromatographic kit for detecting etomidate includes the following steps:

[0013] (a) Preparation of fluorescent indicator: Quantum dot fluorescent microspheres were washed with BB buffer, centrifuged, and the precipitate was resuspended. EDC and NHS were added for activation, followed by centrifugation, resuspending of the precipitate, conjugation with monoclonal antibody, and then blocking with BSA. After centrifugation, the precipitate was resuspended in storage solution to obtain the quantum dot fluorescent microsphere-conjugated antibody probe. The monoclonal antibody was etomidate monoclonal antibody. The quantum dot fluorescent microsphere-conjugated antibody probe was stored in a pre-filled fluorescent indicator tube.

[0014] (b) Assembly of the test strip: Etomidate antigen and goat anti-mouse IgG antibody are sequentially sprayed onto the test line T and control line C of the nitrocellulose membrane of the test strip using a membrane coating instrument. The coated nitrocellulose membrane is dried and then attached to the base plate along with the sample pad and absorbent pad. The membrane is then cut into test strips with a width of 4 mm. The test strips are installed in a plastic housing.

[0015] (c) The fluorescent indicator described in step (a), the detection card described in step (b), the EP tube, the 365 nm ultraviolet flashlight, and the dry fluorescence immunoassay analyzer together constitute the quantum dot fluorescence immunochromatographic kit of etomidate.

[0016] The BB buffer used for cleaning the quantum dot fluorescent microspheres has a concentration of 30-80 mM and a pH of 5.0-8.0. The concentrations of EDC and NHS are both 0.05-0.15 mg / mL; the EDC and NHS are dissolved in a BB buffer containing 0.01% Tween 20 (w / v) at a pH of 5.0-8.0; preferably, the EDC and NHS are dissolved in a 30-80 mM BB buffer containing 0.01% Tween 20 (w / v) at a pH of 5.0-8.0.

[0017] Preferably, the precipitate is resuspended using HEPES buffer, 40-60 mM, pH 7.0-8.0. When adding etomidate monoclonal antibody for conjugation, HEPES buffer with pH 7.0-8.0 is used. Preferably, when adding etomidate monoclonal antibody for conjugation, HEPES buffer, 40-60 mM, pH 7.0-8.0 is used. The blocking with BSA is performed by adding 1% BSA (w / v) HEPES buffer. Preferably, the blocking with BSA is performed by adding 1% BSA (w / v) HEPES buffer, 50 mM, pH 7.0. The preservation solution is a borate buffer containing NaCl and BSA, with a concentration of 80-120 mM and a pH of 7.0-8.0; preferably, the preservation solution is a 100 mM borate buffer containing 0.5% BSA and 0.9% NaCl, pH 7.5.

[0018] The detection method using the above-mentioned quantum dot fluorescence immunochromatographic kit for detecting etomidate includes the following steps:

[0019] (1) Mix etomidate reference solutions of different concentrations with quantum dot fluorescent microsphere-labeled antibody probes and add them to the sample pad of the test strip. Measure the fluorescence signal values ​​of the T and C lines and calculate their F values. T / F C Value, 0 ng / mL F T / F C F values ​​and other concentration points T / F C The values ​​are represented as B0 and B respectively. X Plot the logarithm of the concentration of the reference standard on the x-axis, and substitute the measured values ​​of different reference standard concentrations into Logit(Y) = Ln[(B X / B0) / (1-B X Using the result of / B0)] as the ordinate, a standard curve is established;

[0020] (2) After mixing the sample to be tested with the quantum dot fluorescent microsphere-labeled antibody probe, add it to the sample pad of the test strip, measure the fluorescence signal values ​​of the T and C lines, and calculate the F. T / FC The value is then substituted into the standard curve to calculate the concentration of the sample to be tested.

[0021] The quantum dot fluorescence immunochromatographic kit or detection method for detecting etomidate described above can be applied to the detection of etomidate in hair samples.

[0022] The processing of hair samples includes, but is not limited to, the following methods: put the hair sample into a grinding tube, add 1 mL of sample pretreatment reagent (10 mM PBS containing 0.05% Tween20), freeze-grind, centrifuge, and take the supernatant for later use.

[0023] Invention Principle: The present invention relates to a quantum dot fluorescence immunochromatographic kit and detection method for etomidate. Due to the abundance of carboxyl functional groups on the surface of the quantum dot fluorescent microspheres, an EDC / NHS-mediated carbodiimide chemical method is used to achieve covalent coupling of antibodies. Given that etomidate, as a hapten, possesses only a single antigenic epitope, the QDFMs-LFIA detection mode employs a competitive immunochromatographic principle. In this detection system, the target analyte competes with the coating antigen immobilized on the T-line for binding to the specific antibody labeled on the quantum dot fluorescent microspheres. This results in a decrease in the fluorescence signal intensity of the T-line as the concentration of the target analyte in the sample increases, while the C-line provides a reference signal by capturing excess labeled probes. The ratio of the fluorescence intensity of the T-line to the C-line (F...) is used as the reference signal. T / F C As a quantitative response parameter, this ratio is negatively correlated with the concentration of etomidate in the sample; that is, the higher the concentration of etomidate in the sample, the higher the F ratio. T / F C The smaller the value, the better. By constructing F... T / F C By using a standard curve of the target analyte concentration, accurate quantitative analysis of etomidate content in unknown samples can be achieved.

[0024] On-site operation procedure: Take 100 µL of hair treatment solution into an EP tube, add 5 µL of fluorescent indicator and vortex mix well, then add 80 µL to the sample addition area of ​​the detection card. After chromatography at room temperature for 15 min, you can choose to visually identify or semi-quantitatively interpret the results using a UV flashlight, or insert the instrument into a dry fluorescence immunoassay analyzer to automatically read the T and C line signal values ​​for quantitative detection.

[0025] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages: The quantum dot fluorescence immunochromatographic kit and detection method for detecting etomidate of the present invention achieve rapid detection of etomidate, improve screening efficiency, and based on quantum dot fluorescence immunochromatographic technology, the sensitivity of etomidate is increased to 0.051 ng / mL, which is about 20 times higher than the prior art; it has good stability, stable quantum dot fluorescence signal, and excellent photobleaching resistance; the detection kit is easy to operate, portable, and does not require professional technicians; the detection time window is wide, which can trace drug use history over a longer period of time and meet the needs of long-term retrospective investigation. Attached Figure Description

[0026] Figure 1 This is a schematic diagram of the quantum dot fluorescent immunochromatographic test strip of the present invention;

[0027] Figure 2 TEM images of QDFMs;

[0028] Figure 3 TEM images of QDFMs-mAb;

[0029] Figure 4 Characterization results of quantum dot fluorescent microspheres; (A) Zeta potential of QDFMs and QDFMs-mAb; (B) Particle size analysis of TQDFMs and QDFMs-mAb;

[0030] Figure 5 The results show the optimization of the conjugation conditions between quantum dot fluorescent microspheres and etomidate monoclonal antibody; (A) optimization of activation buffer; (B) optimization of activation buffer pH; (C) optimization of labeling buffer pH.

[0031] Figure 6 The results show the optimization of the conjugation conditions between quantum dot fluorescent microspheres and etomidate monoclonal antibody; (A) optimization of antibody type; (B) optimization of antibody dosage; (C) optimization of EDC / NHS concentration.

[0032] Figure 7 The results show the optimization of the conjugation conditions between quantum dot fluorescent microspheres and etomidate monoclonal antibody; (A) optimization of blocking solution concentration; (B) optimization of NC membrane type; (C) optimization of streak concentration.

[0033] Figure 8 To optimize the detection time of quantum dot immunochromatographic test strips;

[0034] Figure 9 The results of sensitivity analysis of quantum dot immunochromatographic test strips; (A) Test strip detection results; (B) Standard curve;

[0035] Figure 10 The results are for the specific analysis of the quantum dot immunochromatographic test strip;

[0036] Figure 11 A commercially available rapid on-site detection kit for etomidate;

[0037] Figure 12 The results are from 28 real hair samples. Detailed Implementation

[0038] The technical solution of the present invention will be further described below with reference to the accompanying drawings.

[0039] Example 1

[0040] This invention relates to a quantum dot fluorescent immunochromatographic kit for detecting etomidate, comprising a pre-filled fluorescent indicator tube and an immunochromatographic test strip. The preparation of the etomidate-quantum dot fluorescent microsphere conjugated antibody probe (QDFMs-mAb) is as follows:

[0041] Materials selected: Potassium chloride and disodium hydrogen phosphate dodecahydrate were purchased from Nanjing Chemical Reagent Co., Ltd. Sodium chloride was purchased from Xilong Scientific & Technical Co., Ltd. Potassium dihydrogen phosphate was purchased from Shanghai Lingfeng Chemical Reagent Co., Ltd. Surfactant S9 was purchased from Shanghai Yuanye Biotechnology Co., Ltd. Boric acid (BB) and Tween 20 were purchased from Sinopharm Chemical Reagent Co., Ltd. N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES), sodium N-hydroxythiosuccinimide (Sulfo-NHS), 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC), bovine serum albumin, and D-trehalose were purchased from Shanghai Aladdin Biochemical Technology Co., Ltd. Etomidate reference standard was provided by the Narcotics Control Intelligence Technology Center of the Ministry of Public Security. Quantum dot fluorescent microspheres (QDFMs) were purchased from Shanghai Huizhi Biotechnology Co., Ltd. Etomidate antibody, etomidate antigen, and goat anti-mouse immunoglobulin (IgG) were purchased from Changsha Boyou Biotechnology Co., Ltd. The NC membrane was purchased from Advanced Microdevices Pvt. Ltd. (MDI), and the sample pad, PVC base plate, and absorbent paper were purchased from Shanghai Jieyi Biotechnology Co., Ltd.

[0042] Preparation method: (1) Cleaning microspheres: 100 μg of fluorescent microspheres were suspended in 500 μL of activation buffer (BB 50 mM, pH 8.0) and centrifuged at 15000 rpm for 10 min at 4℃. After discarding the supernatant, the precipitate was resuspended in 500 μL of activation buffer and dispersed by sonication;

[0043] (2) Activation: Dissolve EDC in 50 mM pH 8.0 BB (containing 0.01% Tween 20) to a concentration of 0.1 mg / ml and add 50 μL of EDC to the microsphere solution. Dissolve Sulfo-NHS in 50 mM pH 8.0 BB (containing 0.01% Tween 20) to a concentration of 0.1 mg / ml and add 100 μL of Sulfo-NHS to the microsphere solution. Shake and react for 30 min at room temperature. Centrifuge at 15000 rpm for 10 min at 4℃ and discard the supernatant.

[0044] (3) Wash the microspheres again: Resuspend the quantum dot fluorescent microspheres in 500 μL HEPES buffer (50 mM, pH 8.0) and disperse by sonication;

[0045] (4) Coupling: Add 8 μg of antibody and react on a shaker for 2 h;

[0046] (5) Blocking: Add 50 µL of 1% BSA in HEPES (50 mM, pH 8.0) solution and react on a shaker for 1 h. After the reaction is complete, centrifuge at 13000 rpm for 10 min at 4 °C;

[0047] (6) Resuscitation: The precipitate was resuspended in 500 µL of BB buffer (100 mM, pH 7.5) containing 0.5% BSA and 0.9% NaCl. The resuspension was stored at 4°C in the dark for later use.

[0048] Characterization of the above QDFMs-mAb: The prepared quantum dot fluorescent microsphere-conjugated antibody probe was characterized, and the TEM electron microscopy results are as follows. Figure 2 and Figure 3 As shown, the unconjugated quantum dot fluorescent microspheres exhibit a regular spherical morphology, uniform particle size distribution, and smooth surface; while after antibody conjugation, the microsphere edges show irregular features, with obvious protrusions and increased surface roughness, indicating that the antibody molecules have successfully bound to the microsphere surface; the zeta potential is as follows. Figure 4 As shown in Figure A, the surface potential of the original quantum dot fluorescent microspheres was -24.59 mV, while the surface potential of the microsphere probe coupled with etomidate antibody became -8.61 mV. This significant decrease in absolute potential value confirms the alteration of the microsphere surface charge properties by the antibody protein. DLS results are shown in Figure A. Figure 4 As shown in Figure B, the hydrodynamic diameter of the antibody-conjugated microsphere probe is significantly larger than that of the unconjugated microspheres, confirming the effective labeling of etomidate monoclonal antibody on the surface of quantum dot fluorescent microspheres. The comprehensive characterization results demonstrate the successful acquisition of antibody probes labeled with quantum dot fluorescent microspheres.

[0049] Example 2

[0050] This invention relies on the establishment of a fluorescence immunochromatographic detection method using imidated ester quantum dots:

[0051] Assembly of quantum dot fluorescent immunochromatographic test strips (QDFMs-LFIA): as follows Figure 1 As shown, etomidate antigen (0.2 mg / mL) and goat anti-mouse IgG (0.3 mg / mL) were sequentially sprayed onto the NC membrane at the positions of the detection line (T line) and control line (C line) using a scrubbing machine at a spray rate of 1 μL / cm. The scrubbing NC membrane was then dried in an oven at 37°C. The sample pad was thoroughly soaked in Tris-HCl buffer (100 mM, pH 8.0-8.5) containing 0.9% NaCl, 1% S9, 1% trehalose, and 1% BSA (w / v) and then dried. The dried NC membrane, sample pad, and absorbent pad were then sequentially glued onto a PVC base plate, with the sample pad and absorbent pad pressing down on the NC membrane by 1-2 mm at each end. The assembled base plate was then cut into test strips with a width of 4 mm using a strip cutter and placed in a dry, sealed aluminum foil bag for later use.

[0052] The quantum dot fluorescent immunochromatographic test strip of the present invention can qualitatively determine or quantitatively detect etomidate in the sample to be tested. After mixing the processed sample to be tested with the quantum dot fluorescent microsphere-labeled antibody probe, add it to the sample pad of the test strip, and proceed as follows:

[0053] Qualitative judgment: Observe the test strip under a UV flashlight. If both the T and C lines are fluorescent, the sample does not contain etomidate. If the T line is not fluorescent but the C line is fluorescent, the sample contains etomidate. If the C line is not fluorescent, the test strip is invalid.

[0054] Quantitative detection: Insert the test strip into the dry fluorescence immunoassay analyzer, read the fluorescence signal values ​​of the T and C lines, calculate the FT / FC value, and perform quantitative detection based on the established standard curve.

[0055] Optimization of quantum dot fluorescence immunochromatography method:

[0056] (1) Optimization of activation buffer: Three common buffers (BB, MES, HEPES, 50 mM, pH 6.0) were selected for the activation of quantum dot fluorescent microspheres, with other conditions remaining the same. The results are as follows: Figure 5 As shown in A, when BB buffer is used as the activation buffer, the FT / FC ratio and inhibition rate of negative samples reach the optimal level. Therefore, BB buffer is selected as the activation buffer.

[0057] (2) Optimization of activation buffer pH: The pH of the activation buffer (50 mM) was further optimized by adjusting the pH of the BB buffer to 7.0, 7.5, and 8.0, while keeping other conditions the same. The results are as follows: Figure 5As shown in B, the inhibition rate was highest when the pH of the BB buffer was 8.0, indicating that the activation of the NHS active ester on the surface of the microspheres was most thorough under this condition.

[0058] (3) Optimization of labeling buffer pH: Etomidate antibody was conjugated using HEPES buffer (50 mM) at pH 7.0, 7.5, and 8.0, respectively, while keeping other conditions the same. Results are as follows: Figure 5 As shown in C, although the negative sample had the highest FT / FC value at pH 7.0, it also had the lowest inhibition rate, indicating that the signal difference between the positive and negative samples was not significant. At pH 7.5 and 8.0, the difference in FT / FC values ​​between the two samples was small, but the inhibition rate was relatively high at pH 8.0, suggesting that the antibody bioactivity was effectively maintained and the conjugation efficiency was optimal under this condition.

[0059] (4) Optimization of antibody type: Given that immunochromatography is based on the specific recognition of antigen and antibody, antibody affinity is the fundamental factor determining the performance of the test strip. For example... As shown in Figure A, after conjugating monoclonal antibodies ABET00901 and ABET00904 to fluorescent microspheres and comparing them, the test strip modified with ABET00901 showed a higher FT / FC ratio and inhibition rate, confirming that it has the best affinity activity with the target antigen.

[0060] (5) Optimization of antibody dosage: The antibody dosage was set to 2 μg, 5 μg, 8 μg, and 10 μg, respectively, while keeping other conditions the same. The results are as follows: Figure 6 In the study, the FT / FC ratio increased with increasing antibody concentration (B), but at a dosage of 2 μg, although the inhibition rate was highest, the difference in FT / FC values ​​between positive and negative samples was too small to be easily interpreted visually. Considering both inhibition rate and signal intensity, 8 μg was determined to be the optimal antibody dosage.

[0061] (6) Optimization of EDC / NHS concentration: The EDC / NHS concentrations were set to 0.1 mg / mL, 0.2 mg / mL, 0.5 mg / mL, and 1.0 mg / mL, respectively, while keeping other conditions the same. The results are as follows: Figure 6 As shown in Figure C, the FT / FC value gradually decreases with increasing conjugate concentration, indicating that excessively high conjugate concentrations lead to decreased conjugation efficiency between the antibody and fluorescent microspheres or deterioration of probe performance. Considering both signal intensity and inhibition rate, 0.1 mg / mL was determined to be the optimal activation concentration for EDC and NHS.

[0062] (7) Optimization of blocking solution concentration: Keeping other conditions the same, tests were conducted at three blocking concentrations: 1%, 2%, and 5% BSA (w / v) in HEPES (50 mM, pH 8.0). The results are as follows: Figure 7 As shown in Figure A, the FT / FC ratio increases with increasing BSA concentration, but the inhibition rate decreases, indicating a reduction in signal differentiation between positive and negative samples. Therefore, 1% BSA, with its superior inhibition rate, was selected as the optimal blocking solution concentration.

[0063] (8) Optimization of NC membrane type: The effects of different NC membrane types, SS12 (97 s / 4 cm), SN12 (117 s / 4 cm), and SS40 (98 s / 4 cm), on the results were tested. The results are as follows: Figure 7 As shown in B, the SS40 membrane and the quantum dot fluorescent microsphere labeled antibody probe have the best fit, and a high FT / FC ratio and inhibition rate can be obtained.

[0064] (9) Optimization of streak concentration: The T and C lines were streaked with the following six groups of mass-volume concentrations (mg / mL): 0.1 / 0.3, 0.1 / 0.5, 0.2 / 0.3, 0.2 / 0.5, 0.5 / 0.3, and 0.5 / 0.5. The effect of streak concentration on detection performance was systematically investigated. The results are as follows: Figure 7 As shown in C, using FT / FC value as the signal response index and inhibition rate as the sensitivity evaluation parameter, a comprehensive comparison of the data from each group shows that when the T-line concentration is 0.5 mg / mL, the FT / FC value of the negative sample is too high, indicating that the T-line fluorescence signal is significantly stronger than the C-line, resulting in insufficient C-line signal intensity, making it difficult to distinguish with the naked eye and unable to determine the effectiveness of the test strip. However, when the T-line and C-line concentrations are 0.2 mg / mL and 0.3 mg / mL, respectively, the inhibition rate is better, and the distinction between positive and negative samples is significant, which is determined to be the optimal spraying concentration.

[0065] (10) Optimization of detection time: After adding the sample, starting 5 minutes after adding the negative sample, the fluorescence signal intensity of the T and C lines on the test strip is dynamically monitored at specific time intervals. For example... Figure 8 As shown, the absolute fluorescence signal values ​​of both the T and C lines exhibit a monotonically increasing trend with increasing reaction time. In the initial stage of the reaction (5-15 min), the fluorescence signal rapidly increases, corresponding to the rapid formation of immune complexes and the sufficient chromatographic migration of the microsphere probe. After 15 min, although the fluorescence signal continues to rise slowly, the rate of increase significantly decreases, indicating that the immune reaction is approaching equilibrium. Notably, the fluorescence intensity ratio of the T and C lines (FT / FC) remains relatively stable throughout the monitoring period. Considering the practical needs of detection sensitivity, signal stability, and rapid on-site detection, to ensure sufficient visual visibility of the fluorescence signal under UV flashlight illumination, this study determined the optimal detection time for the test strip to be 15 min.

[0066] Method sensitivity: Etomidate reference standard was diluted with blank hair treatment solution to 0 ng / mL, 0.02 ng / mL, 0.05 ng / mL, 0.1 ng / mL, 0.2 ng / mL, 0.5 ng / mL, 1.0 ng / mL, 2.0 ng / mL, 5.0 ng / mL, 10.0 ng / mL, 20.0 ng / mL, and 50.0 ng / mL. 5 μL of etomidate quantum dot fluorescent microsphere probe was added to 100 μL of standard curve solution, mixed thoroughly, and then added to the sample pad of the test strip. The T and C line signal values ​​were then measured. Each concentration was repeated 5 times. A standard curve was established with the logarithm of etomidate reference standard concentration as the x-axis and the result of substituting the measured values ​​of different reference standard concentrations into Logit(Y) = Ln[(BX / B0) / (1-BX / B0)] as the y-axis. Test strips were tested using blank hair samples (hair samples were washed three times with acetone and deionized water, then dried at room temperature before extraction. Hair was cut into 1-2 mm fragments, and 20 mg of hair was weighed and placed in a grinding tube containing 8 3 mm zirconium beads. 1 mL of 10 mM PBS (0.05% Tween 20) was added, and the mixture was cryogenically ground at -20°C according to the following procedure: frequency: 70 Hz, working time: 30 s, interval: 10 s, number of cycles: 20. The ground hair sample was centrifuged at 13000 rpm for 5 min. 100 µL of the supernatant was collected to obtain the hair treatment solution. 5 μL of etomidate quantum dot fluorescent microsphere probe was added to 100 μL of the treated hair sample solution, mixed well, and then added to the test strip sample pad. The T and C line signal values ​​were then measured). This was repeated 20 times, and the obtained FT / FC values ​​were substituted into the standard curve and the average value was calculated. The detection limit is the average concentration of the blank sample minus three times the standard deviation.

[0067] The results are as follows Figure 9 As shown, the linear equation for etomidate established in this invention is Logit(Y) = -0.7053 Ln(X) – 1.0631, R = 0.9960, and the detection limit is 0.051 ng / mL.

[0068] Method Specificity: To verify the specificity of the established method, several other common drugs were tested, including morphine (MOR), heroin (HER), cocaine (COC), ketamine (KET), methadone (MTD), fentanyl (FEN), diazepam (DZP), amphetamine (AMP), trimethoprim-amycin (TMA), and lysergic acid diethylamide (LSD), all at a concentration of 100 ng / mL. Results are as follows: Figure 10 As shown, the etomidate quantum dot fluorescence immunochromatography method established in this invention does not exhibit cross-reactivity with other common drugs.

[0069] Method precision: To verify the precision of the established method, three batches of prepared test strips were selected for testing. Each batch was tested with three spiked samples at different concentrations (0.5, 1.0, and 10.0 ng / mL), and the tests were repeated five times.

[0070] Table 1 Precision Experiment Results

[0071]

[0072] The results are shown in Table 1. The intra-batch coefficient of variation (CV) for etomidate was 5.79%–9.46%, and the inter-batch coefficient of variation (CV) was 5.61%–10.25%, both within a reasonable range. The results indicate that this method has good precision.

[0073] Method accuracy: To verify the accuracy of the established method, the spiked recovery rate was used as the evaluation index. Etomidate mixed standard was added to the blank hair treatment solution to make the spiked sample concentrations 0.5, 1.0, and 10.0 ng / mL, and the determination was repeated 5 times. Spike recovery rate = (actual measured concentration / theoretical concentration) × 100%.

[0074] Table 2 Accuracy Experiment Results

[0075]

[0076] The results are shown in Table 2. The recoveries of etomidate ranged from 86.32% to 102.02%, and the coefficients of variation ranged from 5.79% to 9.46%, all within a reasonable range. These results indicate that the method has good accuracy.

[0077] Method stability: To verify the stability of the established method, the prepared test strips were placed at 4℃ and 25℃ respectively, and the concentration of spiked samples (0.5, 1.0, 10.0 ng / mL) was measured on days 0, 7, and 14, and the measurements were repeated 5 times.

[0078] Table 3 Stability test results

[0079]

[0080] The results are shown in Table 3. Under the conditions of 4℃ and 25℃, the relative deviation of the FT / FC value of the test strip compared with day 0 was less than 20%, indicating good stability.

[0081] Example 3

[0082] Actual sample testing:

[0083] Twenty-eight real hair samples were collected for testing in this study. The 28 hair samples were washed three times with acetone and deionized water, then dried at room temperature before extraction. The hair was cut into 1-2 mm fragments, and 10 mg of hair was weighed and placed in a grinding tube containing eight 3 mm zirconium beads. 1 mL of 10 mM PBS (0.05% Tween 20) was added, and the mixture was cryogenically ground at -20°C according to the following program: frequency: 70 Hz, working time: 30 s, interval: 10 s, number of cycles: 20. The ground hair samples were centrifuged at 13000 rpm for 5 min. 100 µL of the supernatant was collected as the hair treatment solution.

[0084] Actual sample testing used a commercially available etomidate rapid on-site detection kit. Figure 11 This kit features an integrated, portable design, including a pre-filled fluorescent indicator tube, an EP tube, a QDFMs-LFIA detection card, a 365 nm UV flashlight, and a dry fluorescence immunoassay analyzer. The on-site operating procedure is standardized: 100 µL of hair treatment solution is placed in an EP tube, 5 µL of fluorescent indicator is added and vortexed, then 80 µL is added to the sample loading area of ​​the detection card. After chromatography at room temperature for 15 min, qualitative or semi-quantitative readings can be performed visually using the UV flashlight, or quantitative detection can be achieved by automatically reading the T and C line signal values ​​using the dry fluorescence immunoassay analyzer. Simultaneously, LC-MS / MS is used for methodological comparison to verify the consistency of results between this commercial kit and the laboratory-confirmed method.

[0085] Qualitative interpretation results as follows Figure 12 As shown, compared with the blank test strip, the T lines of all samples showed varying degrees of disappearance, and the degree of disappearance was positively correlated with the concentration of etomidate: the T lines of low-concentration samples (<100 pg / mg) showed obvious fluorescent residue, the T lines of medium-concentration samples (100-1000 pg / mg) partially faded, and the T lines of high-concentration samples (>1000 pg / mg) almost completely disappeared while the C lines remained clear. This indicates that the test card has good concentration gradient response characteristics and can achieve semi-quantitative initial screening through visual interpretation.

[0086] Table 4 Sample Test Results

[0087]

[0088] The quantitative results are shown in Table 4. The known concentration range of the 28 positive hair samples detected by LC-MS / MS was 8-2900 pg / mg, with an average of 325.11 pg / mg. The concentration range detected by QDFMs-LFIA was 7.57-2611.43 pg / mg, with an average of 294.20 ± 0.74 pg / mg, indicating good consistency between this method and LC-MS / MS. Furthermore, QDFMs-LFIA showed excellent repeatability, with the coefficient of variation for the 28 samples ranging from 0.10% to 0.76%, and an average of 0.31%. However, the QDFMs-LFIA results were all lower than the LC-MS / MS reference values, with the relative deviations for the 28 samples ranging from -11.54% to -4.28%, and an average relative deviation of -9.58%, suggesting that the results obtained by this method are generally low.

[0089] Therefore, the detection kit and detection method of the present invention can be applied to the rapid screening of etomidate in hair samples. It is simple to operate, fast and convenient, accurate in quantification, and highly sensitive. It is suitable for various clinical testing scenarios such as community drug rehabilitation, entertainment venues, and police checkpoints, and has great application prospects.

Claims

1. A quantum dot fluorescence immunochromatographic assay kit for detecting etomidate, characterized in that, The kit includes a fluorescent indicator pre-filled tube and an immunochromatographic test strip. The fluorescent indicator pre-filled tube is an etomidate-quantum dot fluorescent microsphere coupled antibody probe. The immunochromatographic test strip includes a sample pad, a nitrocellulose membrane, and an absorbent pad. The nitrocellulose membrane has a T line coated with etomidate antigen and a C line coated with goat anti-mouse IgG antibody.

2. The quantum dot fluorescence immunochromatographic assay kit for detecting etomidate according to claim 1, characterized in that, The etomidate-quantum dot fluorescent microsphere conjugated antibody probe is prepared by washing the quantum dot fluorescent microspheres with BB buffer, centrifuging and resuspending the precipitate, adding EDC and NHS for activation, centrifuging again, resuspending the precipitate, adding etomidate monoclonal antibody for conjugation, adding BSA for blocking, centrifuging again and resuspending the precipitate with preservation solution to obtain the etomidate-quantum dot fluorescent microsphere conjugated antibody probe.

3. The quantum dot fluorescence immunochromatographic assay kit for detecting etomidate according to claim 2, characterized in that, The mass ratio of the quantum dot fluorescent microspheres to etomidate monoclonal antibody is 10:1-30:

1.

4. The quantum dot fluorescence immunochromatographic assay kit for detecting etomidate according to claim 2, characterized in that, The BB buffer used for cleaning quantum dot fluorescent microspheres has a concentration of 30-80 mM and a pH of 5.0-8.0; the concentrations of EDC and NHS are both 0.05-0.15 mg / mL; the preservation solution is a borate buffer containing NaCl and BSA with a concentration of 80-120 mM and a pH of 7.0-8.0; the EDC and NHS are dissolved in a BB buffer containing 0.01% Tween 20 by weight / volume at a pH of 5.0-8.0; and the HEPES buffer with a pH of 7.0-8.0 is used when adding etomidate monoclonal antibody for conjugation.

5. The quantum dot fluorescence immunochromatographic assay kit for detecting etomidate according to claim 1, characterized in that, The immunochromatographic test strip includes a base plate, a sample pad, a nitrocellulose membrane, and an absorbent pad. The nitrocellulose membrane, sample pad, and absorbent pad are sequentially pasted onto the base plate along the length of the test strip, with the sample pad and absorbent pad each pressing down on the nitrocellulose membrane by 1-5 mm. Etomidate antigen and goat anti-mouse IgG antibody are sequentially sprayed onto the detection line (T line) and control line (C line) of the nitrocellulose membrane using a membrane scratching device.

6. The quantum dot fluorescence immunochromatographic assay kit for detecting etomidate according to claim 5, characterized in that, The immunochromatographic test strips are 2-5 mm wide, with concentrations of etomidate antigen and goat anti-mouse IgG antibody of 0.1-0.3 mg / mL and 0.2-0.5 mg / mL, respectively, and a spray volume of 2-10 μL / cm.

7. The quantum dot fluorescence immunochromatographic assay kit for detecting etomidate according to claim 5, characterized in that, The sample pads were thoroughly soaked in Tris-HCl buffer containing NaCl, surfactant S9, trehalose, and BSA, and then dried for later use. The concentration of the Tris-HCl buffer was 80-120 mM, and the pH was 8.0-8.

5.

8. The quantum dot fluorescence immunochromatographic assay kit for detecting etomidate according to claim 1, characterized in that, The quantum dot fluorescence immunochromatography kit also includes EP tubes, a shell for coated immunochromatographic test strips, an ultraviolet lamp, and a dry fluorescence immunoassay analyzer.

9. A detection method using the quantum dot fluorescence immunochromatographic assay kit for detecting etomidate as described in claim 1, characterized in that, Includes the following steps: (1) Mix etomidate reference solutions of different concentrations with quantum dot fluorescent microsphere-labeled antibody probes and add them to the sample pad of the test strip. Measure the fluorescence signal values ​​of the T and C lines and calculate their F values. T / F C Value, 0 ng / mL F T / F C F values ​​and other concentration points T / F C The values ​​are represented as B0 and B respectively. X Plot the logarithm of the concentration of the reference standard on the x-axis, and substitute the measured values ​​of different reference standard concentrations into Logit(Y) = Ln[(B X / B0) / (1-B X Using the result of / B0)] as the ordinate, a standard curve is established; (2) After mixing the sample to be tested with the quantum dot fluorescent microsphere-labeled antibody probe, add it to the sample pad of the test strip, measure the fluorescence signal values ​​of the T and C lines, and calculate the F. T / F C The value is then substituted into the standard curve to calculate the concentration of the sample to be tested.

10. The application of the quantum dot fluorescence immunochromatographic kit for detecting etomidate as described in claim 1 or the detection method as described in claim 9 in the detection of etomidate in hair samples.

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