A dual-mode detection test strip for clenbuterol hydrochloride constructed with natural nanoparticles of cuttlefish ink and its application

Abstract

For the first time, this invention uses natural nanoparticles from cuttlefish ink (CINPs) as a multifunctional carrier to design a colorimetric and photothermal dual-mode immunochromatographic strip assay (CPLFIA) for the sensitive detection of the lean meat powder clenbuterol (CL). Research shows that the biocompatibility of CINPs is significantly better than that of Au NPs, thus greatly improving the sensitivity of the immunosensor. At the same time, CINPs exhibit a remarkable photothermal conversion efficiency (η is about 30.24%), enabling dual-signal detection. In addition, CINPs are essentially food scraps and can be obtained by simple washing, avoiding problems such as the complex manufacture and high cost of artificial nanomaterials. This invention integrates a smartphone and a thermal imager to construct a portable sensing system. After optimization, the detection limits of this invention are 0.179 ng mL-1 (colorimetric mode) and 0.076 ng mL-1 (photothermal mode) respectively, significantly lower than the detection limit of traditional gold-based LFIA (0.786 ng mL-1), and the sensitivity is increased by 10 times. This invention explores the hidden value of natural resources and provides new insights for the construction of multimode portable detection.

Description

Technical Field

[0001] The present invention belongs to the field of biological detection, and particularly relates to a colorimetric photothermal integrated and portable immunoassay method constructed by natural nanoparticles from cuttlefish ink; and relates to the application of specific monoclonal antibody labeling, immunochromatographic test strip preparation and actual detection of clenbuterol hydrochloride in meat products. Background Art

[0002] Clenbuterol hydrochloride (CL), commonly known as lean meat powder, is a synthetic β 2 -adrenergic agonist, which is widely used in the treatment of bronchus, asthma and lung diseases. At the same time, lean meat powder has the effect of promoting the decomposition and transformation of fat, and adding it to feed can promote animal growth and increase the lean meat rate. However, lean meat powder has excellent stability in animal tissues and is difficult to decompose, thus accumulating along the food chain. The residual lean meat powder in meat products poses a great threat to human health. If humans overdose on such drugs, they will experience symptoms such as muscle tremors and palpitations, leading to acute poisoning, and in severe cases, even death. Many countries have clearly prohibited the use of clenbuterol hydrochloride to promote growth in food production or have set strict limit standards for it. Therefore, there is an urgent need for a rapid, simple and sensitive method for the qualitative and quantitative detection of lean meat powder to ensure food safety in China.

[0003] Immunochromatographic test strips (LFIA) have been widely used due to their advantages such as rapidity, portability, user-friendliness and specific detection. Traditional test strips are mostly based on visual colorimetric qualitative and semi-quantitative detection, which is simple and rapid, but is easily affected by environmental factors (especially light). At the same time, compared with dual-signal qualitative and quantitative detection, the single-signal mode has poor fault tolerance and sensitivity. Therefore, it is of certain significance and value to develop a dual-mode immunochromatographic test strip detection platform with the aid of portable instruments. In addition, immunochromatographic test strips have been widely promoted due to their low cost, while the synthesis of artificial nanomaterials is often time-consuming and laborious, requiring strict control of experimental conditions (high temperature and high pressure), and the raw material cost is relatively high. Therefore, exploring and using natural nanomaterials with excellent biological and physicochemical properties can not only reduce the complexity and cost of detecting label synthesis, but also promote resource recycling, reduce the environmental burden, extend the industrial chain and develop hidden value. Summary of the Invention

[0004] Aiming at the defects and deficiencies in the prior art, the purpose of the present invention is to provide a colorimetric photothermal integrated and portable immunoassay method using natural nanoparticles (CINPs) derived from cuttlefish ink; and to realize the actual detection of clenbuterol hydrochloride in meat products by using a probe labeled with a specific monoclonal antibody and an immunochromatographic test strip. The invention verifies the possibility of black-brown cuttlefish ink natural nanoparticles providing colorimetric and photothermal dual-mode detection, and constructs a portable detection platform based on a smartphone and an infrared thermal imager. The nanomaterial can be obtained only by simple washing, and can be mixed with a monoclonal antibody simply to prepare an immunoprobe. The simple treatment without chemical reagents reduces the damage to the antibody, and the realization of dual signals improves the sensitivity of the analysis system, which has important practical significance and application value for the sensitive and rapid detection of residual clenbuterol hydrochloride in meat products. The calculated detection limit of the invention is 0.179 ng mL -1 (colorimetric mode) and 0.076 ng mL -1 (photothermal mode), and its sensitivity is 10 times higher than that of the traditional gold standard method (0.786 ng mL -1 ).

[0005] To achieve the above object, the technical solutions adopted by the present invention include:

[0006] A probe based on natural nanoparticles, comprising melanin nanoparticles obtained by washing from cuttlefish ink and a specific monoclonal antibody, and the antibody is an anti-clenbuterol hydrochloride monoclonal antibody.

[0007] Specifically, cuttlefish ink natural nanoparticles (CINPs) are used as signal tags and mixed with a monoclonal antibody at a mixing ratio of 0.5 mg:5 μg in 1 mL of deionized water to prepare an immunoprobe by simple mixing.

[0008] Specifically, the CINPs derived from biological resources are obtained from cuttlefish ink in food scraps by a simple water washing and centrifugation method, and the average particle size is 125 nm.

[0009] A test strip for detecting clenbuterol hydrochloride, and qualitative and quantitative detection of clenbuterol hydrochloride is realized on the test strip by using the immunoprobe prepared above.

[0010] Specifically, the test strip is composed of a backing board, a nitrocellulose membrane, a water absorption pad, and a sample pad. The water absorption pad and the sample pad are respectively placed on both sides of the nitrocellulose membrane, and a detection line and a quality control line are arranged horizontally along the non-covered surface of the nitrocellulose membrane.

[0011] Further, the preparation methods of the detection line coated with antigen CL-BSA and the quality control line coated with goat anti-mouse immunoglobulin are as follows: The 1.5 mg / mL CL-BSA antigen is coated on the detection line at a scribing rate of 0.5 μL / cm; the 5 mg / mL goat anti-mouse immunoglobulin is coated on the quality control line at a scribing rate of 0.5 μL / cm.

[0012] The described test strip is used for the application of actually detecting clenbuterol hydrochloride residues in pork and beef.

[0013] Compared with the prior art, its advantages and positive effects are as follows:

[0014] (1) Portable colorimetric and photothermal dual-signal mode. The present invention uses squid ink nanoparticles as signal tags, deeply explores their potential physical and chemical properties, and constructs a colorimetric and photothermal dual-signal immunoassay mode mainly based on qualitative colorimetric signals and quantitative photothermal signals under near-infrared light. The invention combines a smartphone and a thermal imager portable device to construct an intelligent sensing system.

[0015] (2) Green, clean, and economically recyclable. Squid ink is the waste material discarded during squid processing. The present invention does not add additional chemical reagents and only obtains natural nanoparticles with antibody affinity and photothermal conversion performance through simple washing, and applies them to immunoassay, avoiding the disadvantages of time-consuming, laborious, and high-cost synthesis of artificial nanomaterials, realizing the recycling of renewable resources, creating new economic value, being environmentally friendly, green and clean, and turning waste into treasure.

[0016] (3) Sensitive detection of clenbuterol hydrochloride. The detection limits of the immunochromatographic test strip constructed by the present invention for clenbuterol hydrochloride are 0.179 ng / mL in the colorimetric mode -1 and 0.076 ng / mL in the photothermal mode -1 , and its sensitivity is 10 times higher than that of the traditional gold standard method (0.786 ng / mL -1 ), realizing the rapid and simple detection of lean meat powder in food. Description of the Drawings

[0017] Figure 1 is a schematic diagram of the signal tag acquisition and dual-mode detection principle of the present invention;

[0018] Figure 2 is the evaluation of the photothermal properties of the signal tag of the present invention

[0019] Figure 3 is the performance evaluation of the immunochromatographic test strip of the present invention;

[0020] Figure 4 is the specificity evaluation of the immunochromatographic test strip of the present invention;

[0021] Figure 5 It is the application evaluation of the immunochromatographic test strip of the present invention;

[0022] The following further elaborates on the specific embodiments of the present invention with reference to the accompanying drawings. Specific Embodiments

[0023] The present invention has obtained natural melanin nanoparticles from food waste cuttlefish ink through simple water washing and used them as signal tags. Monoclonal antibodies are conjugated without chemical reagents through simple mixing and then used as detection probes, turning production waste into treasures and integrating the development concepts of circular economy and green chemistry; a colorimetric and photothermal dual-mode portable immunoassay platform is constructed using the photothermal properties of dark brown cuttlefish ink nanoparticles, avoiding the interference of environmental light sources, increasing the operation error tolerance, and improving the actual detection sensitivity, showing high potential in monitoring the residue of clenbuterol in meat products; in the detection and specificity evaluation of actual samples of pork and beef, the prepared competitive test strips all have excellent performance, indicating that the present invention has certain practical value and development prospects.

[0024] As a new type of signal tag, the natural nanoparticles obtained from cuttlefish ink can be obtained by simply washing with water, and have good protein enrichment ability and photothermal conversion performance. On the one hand, the complex synthesis process is omitted, the use of chemical reagents is avoided, the preparation difficulty is reduced, and it is helpful for the protection of antibody activity and large-scale industrial production; on the other hand, the cuttlefish ink natural nanoparticles are obtained from food waste, almost achieving "zero cost", alleviating the pressure of natural degradation and waste treatment, and excavating potential economic value. In addition, it also provides the possibility of dual-signal detection, providing a new development idea for immunoassay.

[0025] Preparation of signal tag cuttlefish ink natural nanoparticles (CINPs): See Figure 1 In order to remove unwanted impurities (pectin, NaCl, sodium carboxymethylcellulose, etc.), the cuttlefish ink is appropriately diluted with deionized water first. After sufficient stirring, it is centrifuged and washed.

[0026] Preparation of a new immunoassay probe: Mix the optimal content of monoclonal antibody against clenbuterol hydrochloride (Ab CL ) with the prepared CINPs solution, incubate at room temperature for 5 h, and then store in an environment at 4 °C.

[0027] The anti-clenbuterol hydrochloride monoclonal antibody used in the present invention was screened by the applicant's research group using cell fusion technology and purified from ascites. Briefly described as: using commercially available clenbuterol CL-bovine serum albumin (CL-BSA) complete antigen to immunize female Balb / c mice to produce antiserum, fusing positive mouse spleen cells with SP2 / 0 myeloma cells, screening and culturing hybridoma cells and preparing corresponding ascites, and finally purifying the collected ascites by the octanoic acid-ammonium sulfate method.

[0028] Preparation of immunochromatographic test strip: See Figure 1 , the test strip is composed of a backing plate, a nitrocellulose membrane, a blotting pad, and a sample pad. The blotting pad and the sample pad are respectively placed on both sides of the nitrocellulose membrane, and a test line (T) and a quality control line (C) are arranged horizontally along the non-covered surface of the nitrocellulose membrane.

[0029] Working principle: See Figure 1 , the present invention uses the competitive immunoassay method to detect clenbuterol hydrochloride, that is, the target molecule in the sample competitively binds to the monoclonal antibody loaded with the signal tag with the antigen immobilized on the test line. The sample solution containing the target molecule is pre-mixed with the immunoprobe and then dropped onto the sample pad of the test strip. Due to the capillary force, the mixed solution will move along the test strip towards the blotting pad. When it moves to the test line, the target molecule in the mixed solution will compete with the immobilized antigen for the limited antigen binding sites on the antibody in the probe. Therefore, the color depth of the test line is inversely proportional to the content of the target molecule. When there are more target molecules in the sample, less immobilized antigen binds to the probe, and the test line is weaker; conversely, when the content of the target molecule decreases, the color of the test line is darker; and when the number of target molecules is too large, the immobilized antigen can hardly bind to the immunoprobe, and at this time the test line is almost invisible. In addition, a quality control line is set up to verify whether the test strip works properly. Whether there is a target molecule or not, the goat anti-mouse antibody on the quality control line can tightly bind to the monoclonal antibody, thereby reflecting the antibody activity and whether the test strip works properly. Therefore, in the present invention, when there is no clenbuterol hydrochloride in the sample (negative), the test strip shows two dark brown bands; when there is clenbuterol hydrochloride (positive), the depth of the test line of the test strip changes trendily with the content. In addition, since the depth of the test line is determined by the content of the enriched signal tag, the natural nanoparticles of cuttlefish ink have the characteristic of photothermal conversion, that is, under the stimulation of near-infrared light, they can convert the invisible quantity signal into a photothermal signal that can be captured by a thermal imager, which is reflected in the form of temperature, realizing the quantitative detection of the test strip and avoiding the environmental interference and semi-quantitative defects of relying only on colorimetric single-signal detection.

[0030] Example 1:

[0031] Evaluation of the photothermal performance of natural nanoparticles of cuttlefish ink

[0032] Study the photothermal properties of CINPs at different concentrations. Under 808 nm laser irradiation with an irradiation area of 2 W cm -2 , study the maximum temperature that can be achieved by nanoparticles at different concentrations and calculate the temperature difference (concentrations are 0, 0.1, 0.3, 0.5 mg mL -1 ); study the cycling effect and photothermal conversion ability of CINPs at a concentration of 0.3 mg mL -1 . Specifically, when the CINPs reach the peak temperature, turn off the laser and let it cool naturally for 10 min, repeat 3 times, record the temperature at different time points with an infrared thermal imaging camera and draw the relevant curves, and calculate the photothermal conversion efficiency according to the cooling curve theory (Formula 1). (1)

[0034] In the formula, h is the heat transfer coefficient, S is the specific surface area of the container, T max and T surr are the maximum temperature of CINP dispersion and the ambient temperature respectively, Q dis is the heat change amount (J*S -1 ) when the reagent is blank, I is the laser power (W), A λ is the absorbance of the CINP aqueous solution at a wavelength of 808 nm. According to the cooling stage, h and S can be obtained. (2)

[0036] Among them, τ s is the time constant of the system (dimensionless), obtained from the linear relationship between t and ln(θ), θ is the thermal drive constant (dimensionless), m i and C p,i represent the specific heat capacity and mass of the solvent (water).

[0037] Evaluation results: As Figure 2 , after optimizing the excitation conditions, the photothermal transduction behavior of CINPs at different concentrations was evaluated using a near-infrared laser of 808 nm, 2 W cm -2 . The CINPs solution showed a concentration-dependent temperature change and gradually tended to be stable with the increase of time ( Figure 2 A and B). Therefore, the photothermal properties can be adjusted by changing the concentration and irradiation time of the biosensor. The maximum temperatures of CINPs at different concentrations can reach 69.6 °C (0.5 mg mL -1 ) and 62.5 °C (0.3 mg mL -1 ). The temperature differences between PBS buffer and CINPs (0.3 mg mL -1 ) are 1.4 and 35.0 respectively ( Figure 2 C). Select CINPs (0.3 mg mL -1)Cyclic tests were carried out on the samples, and natural cooling for about 10 minutes when the temperature reached the peak was regarded as one cycle. The results showed that CINPs had high photostability during 3 on / off irradiation cycles ( Figure 2 D). Subsequently, to quantify the photothermal performance of CINPs, the photothermal conversion efficiency (η) was calculated according to the recorded temperature curves and formulas. The results showed that the photothermal performance of CINPs (η = 30.24%) was comparable to that of the representative photothermal material Bi 2 S 3 nanorods (η = 33.58%), and was superior to the signal tags Cu 2-x Se nanocrystals (η = 13.2%) used in immunoassays ( Figure 2 E).

[0038] Example 2:

[0039] Evaluation of the coupling efficiency and affinity of cuttlefish ink natural nanoparticles with antibodies

[0040] The coupling efficiency between monoclonal antibodies and signal labels, including CINPs and AuNPs, was evaluated. First, a standard curve of optical density versus antibody concentration (ranging from 0.001 to 0.25 μg mL 450 nm ) was established. 5, 10, 15, 20, and 25 μg of anti-CL antibodies were added to 1 mL of CINPs and AuNPs solutions respectively. The supernatants were obtained by centrifugation and the coupling efficiency was evaluated after dilution 100-fold. To analyze the effects of different carriers on monoclonal antibodies and antigens, we used enzyme-linked immunosorbent assay (ELISA) to evaluate the affinity constant (k -1 ) and equilibrium dissociation constant (K a ) of natural monoclonal antibodies, CINPs - monoclonal antibodies, and AuNPs - monoclonal antibodies. The higher the k D value and the lower the K a value indicate better affinity. D

[0041] Evaluation results:

[0042] At equilibrium, the k a value of CINPs - mAbs probe (2.43×10 9 M -1 ) was similar to that of natural mAbs (2.16×10 9 M -1 ), and was about one order of magnitude higher than that of AuNPs - mAbs (1.1×10 8 M -1 ). Meanwhile, the K D value was calculated from the binding isotherm. The K D value of CINPs - mAbs (1.94×10​-9 M -1 ) is also significantly lower than the K of AuNPs - mAbs D value (3.26×10 -8 M -1 ), while there is no increase compared with the K D value (2.01×10 -9 M -1 ) of natural mAbs. At different concentrations, the monoclonal antibody conjugation efficiency of CINPs (82.7% - 96.23%) is basically the same as that of Au NPs (78.98% - 94.68%), and even slightly higher. The results show that the influence of CINPs on antigen - antibody recognition can be ignored, and it provides a good carrier for antibodies.

[0043] Example 3:

[0044] Cuttlefish ink natural nanoparticles (CINPs) were prepared by a simple method of washing and centrifuging.

[0045] The specific method is as follows: In cuttlefish ink, without chemical reagents, CINPs can be separated only by simple washing and centrifuging. Briefly, to remove unwanted impurities (pectin, NaCl, sodium carboxymethylcellulose, etc.), the cuttlefish ink is appropriately diluted with deionized water (water:cuttlefish ink = 15:1). After sufficient stirring, it is centrifugally washed with water 4 times at a speed of 10000 r / min (15 min each time). Finally, the clean CINPs are vacuum freeze - dried for 12 h for storage.

[0046] The preparation method of the immunoprobe is as follows: Filter the CINPs solution with the optimized optimal concentration through a 200 - nm filter to remove ultra - large - sized nanoparticles. Mix the optimal content of anti - CL monoclonal antibody (5 μL, 1 mg mL -1 ) with the prepared CINPs solution and incubate at room temperature for 5 h. The final probe is stored at 4°C.

[0047] Example 4:

[0048] Preparation of a rapid immunochromatographic test strip for clenbuterol hydrochloride

[0049] The described test strip consists of four parts: a backing board, a nitrocellulose membrane, an absorbent pad, and a sample pad. The absorbent pad and the sample pad are respectively placed on both sides of the nitrocellulose membrane. A test line (T) and a control line (C) are horizontally arranged along the non - covered surface of the nitrocellulose membrane. The preparation method includes the following steps:

[0050] 1) Preparation of the absorbent pad:

[0051] The absorbent pad is obtained by cutting absorbent paper, with dimensions of 12 mm * 18 mm (length * width).

[0052] 2) Preparation of the test line and the control line:

[0053] Coat 1.5 mg / mL clenbuterol antigen (CL-BSA) and 5 mg / mL goat anti-mouse immunoglobulin on the nitrocellulose membrane at a scribing rate of 0.5 μL / cm respectively. Remove the first and last 1 - 2 mm, and take the evenly scribed part as the test line (T) and the control line (C), avoiding folding, wrinkling and contamination.

[0054] 3) Preparation of the sample pad:

[0055] The sample pad is obtained by cutting a glass fiber membrane, with dimensions of 12 mm * 24 mm (length * width). Pretreat the sample pad before assembly, that is, soak the sample pad completely in the blocking solution, dry it at 37 °C for about 5 h, wrap it with plastic wrap and store it at 4 °C for later use; the composition of the blocking solution is: 2% BSA, 1% PVP-K30 and 0.05% Tween-20.

[0056] 4) Assembly of the test strip:

[0057] First, attach the scribed nitrocellulose membrane flatly to the middle area of the backing board, paste the sample pad at the lower end, press the nitrocellulose membrane by 1 - 3 mm, paste the absorbent pad at the upper end, press the nitrocellulose membrane by 1 - 3 mm, and then put it into a strip cutter after compacting up and down, and cut it at a width of 3 mm to obtain an immunochromatographic test strip for rapid detection of clenbuterol CL.

[0058] Example 5:

[0059] Performance evaluation of the immunochromatographic test strip for rapid detection of clenbuterol hydrochloride

[0060] Dilute the clenbuterol standard (solution) to an appropriate concentration, use PBS without adding the standard as a blank control, and compare the detection performance of the immunochromatographic test strip by the traditional gold-label method (AuNPs-LFIA) and the detection with cuttlefish ink nanoparticles as the signal label (CINPs-LFIA). Prepare 100 μL of detection solutions with different standard contents, immerse the sample pad of the test strip into them, and observe the detection results. It can be clearly observed that as the concentration of the standard increases, the color of the T line is lighter than that of the negative control. Evaluate the analytical performance of the sensor through three indicators: visual limit of detection (vLOD), that is, the lowest target concentration when the signal of the T line is significantly lower than that of the blank; cut-off value, that is, the lowest CL concentration when the T line is colorless; calculated limit of detection (cLOD), which can be obtained by using the four-parameter logistic regression (4-PL) function. In addition, obtain the photothermal signal value by measuring the temperature difference on the T line between the detection test strip and the blank test strip.

[0061] Detection results:

[0062] As Figure 3 shown in A and C, due to the enhanced competition, as the CL concentration increased from 0 to 9 ng mL -1 , the color of the T line in CINPs-LFIA gradually changed from dark brown to colorless, and at the same time, the corresponding photothermal temperature was also reduced. In the colorimetric and photothermal modes, the vLOD and cut-off values were both 1 ng mL -1 and 7 ng mL -1 , while the cut-off value of AuNPs-LFIA reached 24 ng mL -1 ( Figure 3 E). As Figure 3 shown in B and D, to further evaluate the detection performance, the present invention calculated the cLOD according to the fitting curve and formulas (1) and (2). CINPs-LFIA had a good linear relationship in the dual modes, and the cLODs were 0.179 ng mL -1 (colorimetric mode) and 0.076 ng mL -1 (photothermal mode), respectively. At the same time, as Figure 3 shown in F, according to the calculation, the cLOD of the AuNPs-LFIA mode was 0.786 ng mL -1 . The results showed that taking the cLOD as an example, the sensitivity of CINPs-LFIA in the photothermal mode was 2.35 times higher than that in the colorimetric mode and 10 times higher than that of the traditional AuNPs-LFIA.

[0063] Example 6:

[0064] Specificity evaluation of the rapid detection immunochromatographic test strip for clenbuterol hydrochloride

[0065] For the constructed immunochromatographic test strip, salbutamol (SAL), ractopamine (RAC), kanamycin (KAN), tobramycin (TOB), 17β-estradiol (EST), streptomycin sulfate (STR), levofloxacin hydrochloride (LEV), and sodium nitrite (NaNO 2 ) were used for specificity evaluation to measure the anti-interference ability of the prepared test strip.

[0066] Detection results: As Figure 4 shown, the prepared test strip did not cross-react with other structural analogs and was not affected by other possible interfering substances, showing a highly specific detection reaction to clenbuterol hydrochloride.

[0067] Example 7:

[0068] Application of the rapid detection immunochromatographic test strip for clenbuterol hydrochloride

[0069] The research in Examples 1-2 proves that the immunoprobes and immunochromatographic test strips prepared in Examples 3-4 are used for the detection of clenbuterol hydrochloride in actual samples. The specific pretreatment includes the following steps:

[0070] Select pork and beef as actual samples and pretreat them by the acid method, that is, cut them into pieces, accurately weigh 1 g of the sample, add it to 2 mL of 3% trichloroacetic acid, shake well for 10 minutes, then centrifuge (10000 rpm, 10 minutes), take out the supernatant, filter it through a membrane with a size of 0.22 μm, and then adjust the filtrate to neutral with 1 M NaOH for standby. According to the pre-experiment of the actual samples, the two actual samples are diluted 2 times with PBS for detection.

[0071] Detection results: As Figure 5 , the vLODs of pork and beef in the two modes are 2 ng mL -1 and 3 ng mL -1 respectively, and the cut-off values of CL are all around 9 ng mL -1 . By calculation, the recovery rate of the actual samples of the immunochromatographic test strip with CINPs as the signal label is 88.38% - 115.84%, and the RSD within the confidence range is less than 10.25%, which has good reliability and repeatability in the actual detection application of CL.

Claims

1. A colorimetric and photothermal dual-mode detection test strip for clenbuterol hydrochloride constructed by natural cuttlefish ink nanoparticles, characterized in that, it includes a test strip and an immunoprobe, and the immunoprobe is formed by mixing natural cuttlefish ink nanoparticles and monoclonal antibodies at a ratio of 0.5 mg: 5 μg; the average particle size of the natural cuttlefish ink nanoparticles is 125 nm and is prepared by the following steps: first dilute the cuttlefish ink with deionized water, fully stir, and then centrifuge and wash; the test strip is composed of a backing plate, a nitrocellulose membrane, a blotting pad, and a sample pad. The blotting pad and the sample pad are respectively placed on both sides of the nitrocellulose membrane, and a detection line and a quality control line are arranged horizontally along the non-covered surface of the nitrocellulose membrane.

2. The colorimetric and photothermal dual-mode detection test strip for clenbuterol hydrochloride according to claim 1, characterized in that, using the color and photothermal properties of natural cuttlefish ink nanoparticles without additional modification, combined with a smartphone and a thermal imager to capture signals for immunoassay.

3. The colorimetric and photothermal dual-mode detection test strip for clenbuterol hydrochloride according to claim 1, characterized in that, the preparation methods of the detection line coated with antigen CL-BSA and the quality control line coated with goat anti-mouse immunoglobulin are respectively: 1.5 mg / mL CL-BSA antigen is coated on the detection line at a scribing rate of 0.5 μL / cm; 5 mg / mL goat anti-mouse immunoglobulin is coated on the quality control line at a scribing rate of 0.5 μL / cm.

4. The test strip according to any one of claims 1-3 is used for the detection of clenbuterol hydrochloride in pork and beef.

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