Colorimetric immunochromatography test strip based on gold-bismuth nanoparticles as well as preparation method and application of colorimetric immunochromatography test strip

Abstract

The invention discloses a colorimetric immunochromatography test strip based on gold-bismuth nanoparticles as well as a preparation method and application of the colorimetric immunochromatography test strip, and belongs to the technical field of rapid detection of heavy metals. The test strip comprises a PVC base plate and a gold-bismuth immune probe, a sample pad, an NC film and a water absorption pad are sequentially arranged on the PVC base plate from left to right, the upper end face of one end of the NC film is attached to one end of the sample pad, the upper end face of the other end of the NC film is attached to one end of the water absorption pad, and a C line and a T line are fixed to the NC film; the gold-bismuth immune probe comprises a cadmium monoclonal antibody and a gold-bismuth nano material. The preparation method comprises the following steps: preparing the gold-bismuth nano material, developing the gold-bismuth immune probe, and assembling the test strip. The colorimetric immunochromatography test strip disclosed by the invention has the advantages of high sensitivity and high specificity, is simple in preparation process, is an economical and high-sensitivity real-time rapid detection technology, can be used for rapidly detecting heavy metal cadmium in vegetable agricultural products, and has important practical application value in detection of the vegetable agricultural products.

Description

Technical Field

[0001] The invention belongs to the technical field of rapid heavy metal detection, and specifically relates to a colorimetric immunochromatographic test strip based on gold-bismuth nanoparticles, and a preparation method and application thereof. Background Art

[0002] Due to human activities such as sewage sludge, cement industry, fossil fuel combustion and agricultural fertilization, heavy metal pollution is becoming increasingly serious in the atmosphere, soil, water and food, which has a serious impact on food safety, human health and economic benefits. Cadmium (Cd) is a harmful heavy metal that is mutagenic and weakly genotoxic, relatively non-biodegradable, and persists in the environment for a long time. Therefore, it is necessary to develop a highly sensitive real-time rapid detection technology to effectively monitor the distribution of heavy metal cadmium in the environment and the pollution control.

[0003] Existing cadmium detection methods include atomic absorption spectrometry, atomic emission spectrometry (AES), inductively coupled plasma mass spectrometry (ICP-MS), atomic fluorescence spectrometry (AFS), ultraviolet visible spectrophotometry (UV-VIS), laser induced breakdown spectroscopy (LIBS), etc. However, these methods have high professional requirements, long analysis time, and expensive analysis prices, which are not conducive to promotion and on-site detection. At present, known rapid detection methods for heavy metal cadmium include the following: (1) electrochemical sensor method, such as patent document CN115901733A discloses a preparation and application method of cadmium ion detection test paper, based on the principle that glutathione aggregation-induced gold is released after adding cadmium ions, and the strength of the generated chemiluminescent signal is used to detect cadmium. (2) Immunochromatographic test strips. For example, patent document CN113030466A discloses a colloidal gold immunochromatographic test strip for detecting heavy metal cadmium in soil, with a detection limit of 200 ng / mL, an antibody spraying concentration of 0.5 mg / mL, and a T-line antigen spraying concentration of 0.5 mg / mL. The antibody and antigen dosage per unit sample is calculated to be 0.2 μg; patent document CN105242047A discloses an aflatoxin B1 graphene oxide immunochromatographic test strip with a detection limit of 0. 6ng / mL, the probe-coupled antibody concentration is 1mg / mL, the T-line antigen spraying concentration is 0.7mg / mL, and the antibody dosage per unit sample is calculated to be 0.2μg, and the antigen dosage is 0.126μg; Patent document CN114518449A discloses a latex microsphere immunochromatographic test strip for detecting heavy metal mercury ions, with a detection limit of 0.78ng / mL, a needle-coupled antibody concentration of 1.25mg / mL, and the antibody dosage per unit sample is calculated to be 0.75μg. However, the antigen and antibody dosage of these test strips is relatively large, and there is currently no report on the use of economical gold-bismuth nanomaterials for rapid detection of heavy metal cadmium. Summary of the invention

[0004] The main purpose of the present invention is to provide a colorimetric immunochromatographic test strip based on gold-bismuth nanoparticles, which can effectively monitor the distribution and pollution of heavy metal cadmium in the environment, and overcome the problems of existing heavy metal cadmium detection technology, such as the need for instrument assistance, low sensitivity, and poor specificity.

[0005] Another object of the present invention is to provide a method for preparing the colorimetric immunochromatographic test strip, which has simple materials and probe synthesis and significantly reduces costs.

[0006] Another object of the present invention is to provide an application of the colorimetric immunochromatographic test strip for rapid detection of heavy metal cadmium, which has high sensitivity, simple and rapid detection, can obtain the test result in 10 minutes, is less affected by the matrix, has high specificity, and is comparable to other metal ions such as Cr. 3+ , Pb 2+ , Hg 2+ 、Ni + , Cu 2+ , Fe 3+ 、As 3+ The crossover rate is below 5%.

[0007] The above-mentioned object of the present invention can be achieved by the following technical solutions:

[0008] The first aspect of the present invention is a colorimetric immunochromatographic test strip based on gold-bismuth nanoparticles, comprising a PVC base plate and a gold-bismuth immune probe, wherein: a sample pad, an NC membrane, and a water-absorbing pad are arranged on the PVC base plate from left to right, and the upper end surface of one end of the NC membrane is in contact with one end of the sample pad, and the upper end surface of the other end is in contact with one end of the water-absorbing pad, and a C line and a T line are fixed on the NC membrane; the gold-bismuth immune probe comprises a cadmium monoclonal antibody and a gold-bismuth nanomaterial.

[0009] Preferably, the width of the colorimetric immunochromatographic test strip is 3.8 mm, and the lengths of the sample pad, NC membrane, and absorbent pad are 14 mm, 25 mm, and 45 mm, respectively.

[0010] Preferably, the C line and T line are parallel to each other and have a spacing of 6 mm; the T line is the coated original OVA-Cd, close to the sample pad; the C line is the goat anti-mouse antibody, close to the absorbent pad.

[0011] Preferably, the sample pad is made of polyester fiber, and the NC membrane is a nitrocellulose membrane.

[0012] Preferably, the particle size of the gold-bismuth nanomaterial is 4.8 nm, and the bismuth ion concentration is 4 mmol / L; the antibody coupling concentration of the gold-bismuth immune probe is 4 μg / mL.

[0013] In a second aspect of the present invention, the method for preparing the colorimetric immunochromatographic test strip comprises the following steps:

[0014] Step A: adding PVP to HAuCl4 solution, placing in an ice water bath to obtain a mixed solution; rapidly injecting a cold NaBH4 solution into the mixed solution under vigorous stirring, adding ascorbic acid, reacting fully to obtain a gold nano solution; adding a bismuth ion solution and mixing well, allowing to stand to obtain a gold-bismuth nano material;

[0015] Step B: Take the gold-bismuth nanomaterial obtained in step A, adjust the pH to 6 with K2CO3, add EDC, shake and mix at room temperature, add cadmium monoclonal antibody, shake at room temperature, add trehalose, mix, and obtain a gold-bismuth immune probe;

[0016] Step C: Assemble according to the structure of the colorimetric immunochromatographic test strip.

[0017] Preferably, in step A, the T line is sprayed with competing antigen (OVA-Cd) and antibodies on the antigen (Cd) competing probe complex Au-Bi-mAb, and the spraying concentration of the competing antigen is 0.2 mg / mL, that is, the required antigen coating amount per centimeter of the detection line is 0.2 μg;

[0018] and / or the goat anti-mouse antibody sprayed on the C line is combined with Au-Bi-mAb and Au-Bi-mAb-Cd conjugates, and the spraying concentration of the goat anti-mouse antibody is 1 mg / mL, that is, the amount of goat anti-mouse antibody required per centimeter of the detection line is 1 μg;

[0019] And / or the added amount of the gold-bismuth immune probe is 30 μL.

[0020] Preferably, in step A, the content of PVP is 555 mg and the molecular weight is 10000; the concentration of HAuCl4 solution is 1.0×10 -3 mol / L, content is 50mL; the content of the NaBH4 solution is 5mL, the concentration is 100mM; the content of the ascorbic acid is 0.016g.

[0021] Preferably, in step A, the ice water bath time is 30 minutes; after the NaBH4 is injected into the mixed solution, the ascorbic acid is added for 5 minutes; and the reaction time after the ascorbic acid is added is 24 hours.

[0022] Preferably, in step A, the gold nanoparticle solution is 1 mL, the concentration of the bismuth ion solution is 4 mmol / L, and the added amount is 300 μL.

[0023] Preferably, in step B, the amount of the gold bismuth solution added is 0.5 mL, the concentration of K2CO3 is 0.1 mol / L, the room temperature oscillation time is 2 h, the concentration of trehalose is 10 mg / mL, the volume is 100 μL, the optimal amount of EDC is 24 μg / mL, and the optimal amount of the cadmium monoclonal antibody is 4 μg / mL.

[0024] In a third aspect of the present invention, the colorimetric immunochromatographic test strip is used for rapid detection of heavy metal cadmium in vegetable agricultural products.

[0025] Preferably, the quantitative range of the colorimetric immunochromatographic test strip is 1 to 20 ng / mL, IC 50 It is 5.21ng / mL and LOD is 0.86ng / mL.

[0026] Compared with the prior art, the present invention has the following beneficial effects:

[0027] (1) The gold-bismuth immune probe of the present invention comprises a cadmium monoclonal antibody and a gold-bismuth nanomaterial, the T line comprises OVA-Cd, and the C line comprises sheep anti-mouse antibody. The materials and probes are simple to synthesize, and the cost can be significantly reduced.

[0028] (2) The colorimetric immunochromatographic test strip of the present invention uses an indirect competitive method to detect heavy metal cadmium in vegetable agricultural products. The minimum detection sensitivity for asparagus samples is 73.93 ng / g, which is comparable to other metal ions Cr 3+ , Pb 2+ , Hg 2+ 、Ni + , Cu 2 + , Fe 3+ 、As 3+ The crossover rate was below 5%, which had good specificity.

[0029] (3) The colorimetric immunochromatographic test strip of the present invention is an economical and highly sensitive real-time rapid detection technology with high sensitivity, high specificity, and a simple preparation process. It can quickly detect heavy metal cadmium in vegetable agricultural products and has important practical application value in the detection of vegetable agricultural products. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 This is a schematic diagram of the structure of the colorimetric immunochromatographic test strip prepared in Example 1.

[0031] Figure 2 This is the 96-well plate shown in Example 1.

[0032] Figure 3 Schematic diagram of the process of adding the gold-bismuth immunoprobe shown in Example 1 into the microwells of a 96-well plate.

[0033] Figure 4 This is the TEM image of the gold-bismuth nanomaterial shown in Example 1.

[0034] Figure 5 Sensitivity and specificity detection of the colorimetric immunochromatographic test strip prepared in Example 1; (a) photo of sensitivity determination results; (b) sensitivity fitting curve; (c) photo of specificity detection results. DETAILED DESCRIPTION

[0035] In order to more fully understand and demonstrate the technical solutions, purposes and advantages of the present invention, the technical effects produced by the present invention are further described in detail and completely in conjunction with the accompanying drawings and specific embodiments. Obviously, the described embodiments are only part of the embodiments of the present invention, not all. It should be pointed out that for ordinary technicians in this field, other embodiments obtained without departing from the concept of the present invention all belong to the protection scope of the present invention.

[0036] Example 1

[0037] In this embodiment, a colorimetric immunochromatographic test strip based on gold-bismuth nanoparticles is prepared, which is used in conjunction with a gold-bismuth immune probe, including a PVC base plate, a sample pad, an NC membrane, and a water-absorbing pad are sequentially and interlacedly attached to the PVC base plate, a C line and a T line are fixed on the NC membrane, the C line is a sheep anti-mouse antibody and the T line is OVA-Cd, the test strip width is 3.8 mm, the particle size of the gold-bismuth nanomaterial is 4.8 nm, the bismuth ion concentration is 4 mmol / L, the addition amount is 300 μL, and the antibody coupling concentration of the gold-bismuth immune probe is 4 μg / mL. Figure 1 shown.

[0038] The preparation process of the above colorimetric immunochromatographic test strip is as follows:

[0039] (1) Preparation of gold-bismuth nanomaterials

[0040] Add 555 mg PVP to 50 mL 1.0 × 10 -3 mol / L HAuCl4 solution, ice water bath for 30 minutes. Then, under vigorous stirring, 5mL 100mM cold NaBH4 solution was quickly injected into the mixed solution, and 0.016g ascorbic acid was added after 5 minutes, and fully reacted for 24 hours. Then, 1mL of gold nano solution was taken, 300μL of 4mmol / L bismuth ion solution was added, mixed, and placed at 4℃ for 24 hours to obtain gold-bismuth nanomaterials.

[0041] (2) Development of gold-bismuth immunoprobe

[0042] The gold-bismuth immunoprobe adopts the electrostatic adsorption-chemical coupling method. Take 0.5 mL of gold-bismuth solution and adjust the pH to 6 with 0.1 mol / L K2CO3. Then add 24 μg / mL EDC and shake at room temperature. Then add 4 μg cadmium monoclonal antibody and shake at room temperature for 2 hours. Then add 100 μL trehalose (10 mg / mL) and mix for 0.5 hours. Finally, store at 4°C for use.

[0043] (3) Preparation of colorimetric immunochromatographic test strips

[0044] OVA-Cd with a concentration of 0.2 mg / mL was fixed on the NC membrane as the T line for detection, and goat anti-mouse antibody with a concentration of 1 mg / mL was fixed as the C line for quality control and control. The NC membrane with fixed T and C lines was placed in a 37°C oven for drying for 12 hours. The polyester fiber pad was cut into a size of 1.6 cm × 30 cm using a manual strip cutter, completely immersed in the sample pad treatment solution for 3 minutes, and then dried in a 60°C oven for 2 hours. After drying, it was placed in a moisture-proof box for storage and standby use. The absorbent paper was processed into a size of 4.5 cm × 30 cm by a manual strip cutter and placed in a moisture-proof box for storage and standby use.

[0045] The sample pad, NC membrane and absorbent paper were pasted on the back plate in order with an overlap of 2mm, and then OVA-Cd and goat anti-mouse antibody were sprayed on the NC membrane at a spraying amount of 1μL / cm by the X, Y, Z three-dimensional fully automatic gold spray film scribing instrument, respectively, as the T line and C line of the test strip. Among them, the C line and the T line are parallel to each other and have a spacing of 6mm. The C line is goat anti-mouse antibody with a spraying concentration of 1mg / mL, and the T line is OVA-Cd with a spraying concentration of 0.2mg / mL. The probe dosage is 30μL. Then the assembled test strip was placed in a 37℃ oven to dry for 18h. After drying, the test strip was cut into 3.8mm width using a fully automatic strip cutter, collected and placed in a plastic bag with desiccant, and stored in a 4℃ refrigerator.

[0046] (4) Detection of colorimetric immunochromatographic test strips

[0047] like Figure 2 and 3 As shown, in a 96-well microplate, 30 μL of coupled probe and 90 μL of test solution (PBS-EDTA buffer at pH = 7) were added to each well successively. After mixing for 3 minutes, the test strip was taken out of the plastic bag and inserted into the microwell. The chromatography reaction was carried out for 8 minutes, and the test strip was removed to read the result.

[0048] like Figure 4As shown, the synthesized gold-bismuth nanoparticles have a relatively round shape and a relatively uniform particle size of only 4.8±0.6 nm, with good dispersibility. A small amount of bismuth particles are conjugated on the surface of the gold nanoparticles, which increases the biological activity of the gold-bismuth material, makes its local surface plasmon resonance peak sharper, and has a higher absorption intensity.

[0049] like Figure 5 (a) shows the actual picture of gold-bismuth test strips with different spiked concentrations. 0-50 represents the Cd content in the solution during the test. 2+ The concentration is in ng / mL. When the spike concentration is 5ng / mL, the color of the T line is obviously lighter than the C line. At this time, the sensitivity of naked eye detection is 5ng / mL. Figure 5 (b) shows the sensitivity fitting curve of the gold-bismuth test strip, with a quantitative range of 1 to 20 ng / mL, and the standard curve of the test strip is y = -0.222ln(x) + 0.0262 (R 2 is 0.982), IC 50 is 5.21ng / mL, and LOD is 0.86ng / mL; Figure 5 (c) shows that the heavy metal cadmium, gold and bismuth immunochromatographic test strips and other heavy metal Cr 3+ , Pb 2+ , Hg 2+ 、Ni + , Cu 2+ , Fe 3+ 、As 3+ The specificity of Cd 2+ There is no cross-reaction with other heavy metals and the test strips have good specificity.

[0050] Example 2

[0051] The process is basically the same as that of Example 1, except that the amount of bismuth ions added in step (1) is adjusted to 100 μL.

[0052] Example 3

[0053] The process is basically the same as that of Example 1, except that the amount of bismuth ions added in step (1) is adjusted to 200 μL.

[0054] Example 4

[0055] The process is basically the same as that of Example 1, except that the amount of bismuth ions added in step (1) is adjusted to 400 μL.

[0056] Example 5

[0057] The process is basically the same as that of Example 1, except that the amount of bismuth ions added in step (1) is adjusted to 500 μL.

[0058] Example 6

[0059] The process is basically the same as that of Example 1, except that the amount of bismuth ions added in step (1) is adjusted to 600 μL.

[0060] Example 7

[0061] The process is basically the same as that of Example 1, except that the amount of bismuth ions added in step (1) is adjusted to 700 μL.

[0062] Example 8

[0063] The process is basically the same as that of Example 1, except that the amount of bismuth ions added in step (1) is adjusted to 800 μL.

[0064] Example 9

[0065] The process is basically the same as Example 1, except that the concentration of EDC added in step (2) is adjusted to 16 μg / mL.

[0066] Example 10

[0067] The process is basically the same as Example 1, except that the concentration of EDC added in step (2) is adjusted to 20 μg / mL.

[0068] Embodiment 11

[0069] The process is basically the same as Example 1, except that the concentration of EDC added in step (2) is adjusted to 28 μg / mL.

[0070] Example 12

[0071] The process is basically the same as Example 1, except that the concentration of EDC added in step (2) is adjusted to 32 μg / mL.

[0072] Example 13

[0073] The process is basically the same as Example 1, except that the concentration of EDC added in step (2) is adjusted to 36 μg / mL.

[0074] Embodiment 14

[0075] The process is basically the same as Example 1, except that the amount of cadmium monoclonal antibody added in step (2) is adjusted to 2 μg.

[0076] Embodiment 15

[0077] The process is basically the same as Example 1, except that the amount of cadmium monoclonal antibody added in step (2) is adjusted to 3 μg.

[0078] Example 16

[0079] The process is basically the same as Example 1, except that the concentration of OVA-Cd in step (3) is adjusted to 0.2 mg / mL.

[0080] Embodiment 17

[0081] The process is basically the same as Example 1, except that the concentration of OVA-Cd in step (3) is adjusted to 0.3 mg / mL.

[0082] Example 16

[0083] The process is basically the same as Example 1, except that the amount of probe in step (3) is adjusted to 20 μL.

[0084] Embodiment 17

[0085] The process is basically the same as Example 1, except that the amount of probe in step (3) is adjusted to 40 μL.

[0086] Embodiment 18

[0087] The process is basically the same as Example 1, except that the pH of the PBS-EDTA buffer is adjusted to 5 in step (4).

[0088] Embodiment 19

[0089] The process is basically the same as Example 1, except that the pH of the PBS-EDTA buffer is adjusted to 6 in step (4).

[0090] Embodiment 20

[0091] The process is basically the same as Example 1, except that the pH of the PBS-EDTA buffer is adjusted to 8 in step (4).

[0092] Embodiment 21

[0093] The process is basically the same as Example 1, except that the pH of the PBS-EDTA buffer is adjusted to 9 in step (4).

[0094] The above are only preferred embodiments of the present invention and are not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A colorimetric immunochromatographic test strip based on gold-bismuth nanoparticles, characterized in that: It comprises a PVC base plate and a gold-bismuth immune probe, wherein: a sample pad, a NC membrane, and a water-absorbing pad are arranged on the PVC base plate from left to right in sequence, and the upper end surface of one end of the NC membrane is in contact with one end of the sample pad, and the upper end surface of the other end is in contact with one end of the water-absorbing pad, and a C line and a T line are fixed on the NC membrane; the gold-bismuth immune probe comprises a cadmium monoclonal antibody and a gold-bismuth nanomaterial.

2. The colorimetric immunochromatographic test strip based on gold-bismuth nanoparticles according to claim 1, characterized in that: The width of the colorimetric immunochromatographic test strip is 3.8 mm, and the lengths of the sample pad, NC membrane, and water absorbent pad are 14 mm, 25 mm, and 45 mm, respectively.

3. The colorimetric immunochromatographic test strip based on gold-bismuth nanoparticles according to claim 1, characterized in that: The C line and the T line are parallel to each other and have a spacing of 6 mm; the T line is the coated original OVA-Cd, close to the sample pad; the C line is the goat anti-mouse antibody, close to the absorbent pad.

4. The colorimetric immunochromatographic test strip based on gold-bismuth nanoparticles according to claim 1, characterized in that: The sample pad is made of polyester fiber, and the NC membrane is a nitrocellulose membrane.

5. The colorimetric immunochromatographic test strip based on gold-bismuth nanoparticles according to claim 1, characterized in that: The particle size of the gold-bismuth nanomaterial is 4.8 nm, and the bismuth ion concentration is 4 mmol / L; the antibody coupling concentration of the gold-bismuth immune probe is 4 μg / mL.

6. The method for preparing the colorimetric immunochromatographic test strip based on gold-bismuth nanoparticles according to any one of claims 1 to 5, characterized in that: The following steps are involved: Step A: Add PVP to HAuCl4 solution, place in ice water bath to obtain a mixed solution; Rapidly injecting a cold NaBH4 solution into the mixed solution under vigorous stirring, adding ascorbic acid, and reacting sufficiently to obtain a gold nanoparticle solution; Add bismuth ion solution and mix well, let stand, and obtain gold-bismuth nanomaterial; Step B: Take the gold-bismuth nanomaterial obtained in step A, adjust the pH to 6 with K2CO3, add EDC, shake and mix at room temperature, add cadmium monoclonal antibody, shake at room temperature, add trehalose, mix, and obtain a gold-bismuth immune probe; Step C: Assemble according to the structure of the colorimetric immunochromatographic test strip.

7. The method for preparing the colorimetric immunochromatographic test strip according to claim 6, characterized in that: In step A, the T line is sprayed with competing antigen (OVA-Cd) and antibodies on the antigen (Cd) competing probe complex Au-Bi-mAb, and the spraying concentration of the competing antigen is 0.2 mg / mL, that is, the required antigen coating amount per centimeter of the detection line is 0.2 μg; and / or the goat anti-mouse antibody sprayed on the C line is combined with Au-Bi-mAb and Au-Bi-mAb-Cd conjugates, and the spraying concentration of the goat anti-mouse antibody is 1 mg / mL, that is, the amount of goat anti-mouse antibody required per centimeter of the detection line is 1 μg; And / or the added amount of the gold-bismuth immune probe is 30 μL.

8. The method for preparing the colorimetric immunochromatographic test strip according to claim 6, characterized in that: In step A, the content of PVP is 555 mg and the molecular weight is 10000; the concentration of HAuCl4 solution is 1.0×10 -3 mol / L, content is 50mL; the content of the NaBH4 solution is 5mL, the concentration is 100mM; the content of the ascorbic acid is 0.016g.

9. The method for preparing the colorimetric immunochromatographic test strip according to claim 6, characterized in that: In step A, the ice water bath time is 30 min; after the NaBH4 is injected into the mixed solution, the ascorbic acid is added for 5 min; the reaction time after the ascorbic acid is added is 24 h; the gold nanoparticle solution is 1 mL, the concentration of the bismuth ion solution is 4 mmol / L, and the amount added is 300 μL; And / or in step B, the amount of the gold bismuth solution added is 0.5 mL, the concentration of K2CO3 is 0.1 mol / L, the room temperature oscillation time is 2 h, the concentration of trehalose is 10 mg / mL, the volume is 100 μL, the optimal amount of EDC is 24 μg / mL, and the optimal amount of the cadmium monoclonal antibody is 4 μg / mL.

10. The colorimetric immunochromatographic test strip based on gold-bismuth nanoparticles according to any one of claims 1 to 5 is used for rapid detection of heavy metal cadmium in vegetable agricultural products.

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