Time-resolved colorimetric immunosensor for detecting antipyrine concentration and application

Abstract

This invention discloses a time-resolved colorimetric immunosensor for detecting antipyrine concentration. The time-resolved colorimetric immunosensor includes a colorimetric recognition module, a signal processing module, a timing module, and an immune competitive reaction module. Based on the photothermal properties of chiral gold nanoparticles and combined with thermochromic pigments, this invention achieves photothermal visualization. To avoid visual errors, the color change process is converted into a time-based process, successfully constructing a timing-colorimetric sensor. A portable device was also developed to achieve sensitive and rapid detection of antipyrine.

Description

Technical Field

[0001] This invention belongs to the field of immunoassay, specifically relating to a time-resolved colorimetric immunosensor for detecting antipyrine concentration and its application. Background Technology

[0002] With the continuous improvement of living standards and rapid economic development, people are paying increasing attention to the nutritional and health benefits of food. Various herbal teas and health products claiming to lower uric acid and treat arthritis have appeared on the market. However, pure herbal treatments often require a long treatment period to achieve good results. Therefore, some unscrupulous merchants may illegally add antipyretic, analgesic, and anti-inflammatory drugs such as antipyrine (ATAP) in pursuit of efficacy. However, long-term use of such drugs has significant side effects and can cause harm to the human body. Common methods for detecting ATAP content include high-performance liquid chromatography (HPLC) and Raman spectroscopy. While these methods can accurately detect ATAP content, they often require expensive laboratory equipment and skilled operators, making them unsuitable for point-of-care testing (POCT) in practical applications.

[0003] In POCT applications, photothermal sensing technology is widely favored due to its convenience and ease of operation. Research has found that thermochromic materials change color by altering their structure in response to temperature. Color change, as a visual perception, can be directly identified with the naked eye without external equipment, reducing detection costs and further improving convenience. While simple color changes are prone to errors due to variations in the inspector, combining visual visualization with timing sensing technology can effectively avoid such errors. This inspires the development of timing-based colorimetric sensing based on the photothermal properties of materials and combined with thermochromic pigments. Therefore, finding a material with stable photothermal properties is crucial.

[0004] Nanomaterials of noble metals play a significant role in photothermal detection due to their excellent biocompatibility and synergistic optical properties. Among them, gold nanomaterials of various morphologies are widely used in photothermal immunosensing. However, research indicates that current gold nanomaterials still face the following challenges:

[0005] 1) The photothermal conversion efficiency of gold nanoparticles is relatively low. Gold nanoparticles with symmetrical and uniform morphology have low light absorption and utilization. Changing their morphology can enhance their light absorption and utilization, thereby improving the photothermal conversion efficiency. For example, Huang and his team discussed the morphology of gold nanoparticles. [1] The photothermal conversion efficiency of simple spherical and rod-shaped gold nanoparticles under 808nm irradiation is only 21.6% and 20.4%, respectively, while that of gold nanoparticles is 46.2%.

[0006] 2) Gold nanoparticles exhibit poor photothermal stability. After prolonged irradiation, gold nanoparticles tend to aggregate and form clusters as they condense, altering their size and causing changes in the distribution of surface plasmon resonance. This results in a loss of photothermal conversion capacity, thereby reducing the ability to absorb and utilize light. [2] .

[0007] 3) The small surface area of ​​gold nanoparticles limits their absorption and utilization of light. This is because there is a proportional relationship between the photothermal response and absorption cross-section of nanostructures. At the same particle size, gold nanoparticles with smooth, undistorted surfaces have smaller absorption cross-sections, thus limiting their light response. [3][4] .

[0008] References:

[0009] [1]Yang W,Xia B,Wang L,et al.Shape Effects of Gold Nanoparticles inPhotothermal CancerTherapy[J].Materials Today Sustainability,2021,13:100078.

[0010] [2]Esporrín-Ubieto D,Huck-Iriart C,Picco AS,et al.Hybrid Nanogel-Wrapped Anisotropic GoldNanoparticles Feature Enhanced Photothermal Stability[J].Small,2024,20(48):2404097.

[0011] [3]Zhang H, Zhu T, Li M. Quantitative analysis of the shape effect ofthermoplasmonics in goldnanostructures[J].The Journal of Physical ChemistryLetters,2023,14(16):3853-3860.

[0012] [4]Hao C, Xu L, Sun M, et al. Chirality on Hierarchical Self-Assembly ofAu@AuAg Yolk-Shell Nanorods into Core-Satellite Superstructures for Biosensingin Human Cells[J]. Advanced Functional Materials, 2018, 28(33):1802372. Summary of the Invention

[0013] The purpose of this invention is to overcome at least one deficiency of the prior art and to provide a time-resolved colorimetric immunosensor for detecting antipyrine concentration and its application.

[0014] The technical solution adopted in this invention is:

[0015] In a first aspect, the present invention provides a time-resolved colorimetric immunoassay sensor for detecting antipyrine concentration. The time-resolved colorimetric immunoassay sensor includes a colorimetric recognition module, a signal processing module, a timing module, and an immune competitive reaction module. The time-resolved colorimetric immunoassay sensor uses the following method to detect the concentration of antipyrine:

[0016] 1) The antigen was immobilized on a 96-well plate and then incubated with known concentrations of antipyrine solution, primary antibody, and Ab2-modified chiral gold nanocomposite.

[0017] 2) When PBS solution and thermochromic pigment are added to the well plate, the concentration is maintained at 805–810 nm and 1.0–5.0 W / cm². 2 The solution was irradiated with infrared light and the color change time was recorded to construct a standard curve of antipyrine concentration and color change time.

[0018] 3) Replace the known different concentrations of antipyrine in step 1) with the antipyrine solution to be tested, and use the same method as in steps 1) and 2) to calculate the concentration of the antipyrine solution to be tested based on the standard curve.

[0019] In some instances, the preparation method of the Ab2-modified chiral gold nanocomposite includes the following steps:

[0020] 1) Take a certain amount of gold salt solution, add surfactant, stir and then add reducing agent to prepare octahedral gold nanoparticle solution;

[0021] 2) Add a chiral organic compound, a reducing agent, and a quaternary ammonium salt to the octahedral gold nanoparticle solution, and modify the solution by reaction to obtain a chiral gold nanoparticle solution;

[0022] 3) The chiral gold nanoparticle solution is subjected to amination treatment, and the carboxyl groups on the surface of the secondary antibody are activated. Finally, the treated chiral gold nanoparticle solution is reacted with the secondary antibody to obtain the Ab2-modified chiral gold nanocomposite.

[0023] In some instances, the reaction temperature in step 2) is 30–40°C.

[0024] The temperature, reactant purity, and reactant concentration during the reaction process all affect the degree of distortion of chiral gold nanoparticles, further influencing their photothermal properties. Reaction temperatures below 25℃ easily lead to the precipitation of quaternary ammonium salts in the solution, forming crystals that coat the surface of the gold nanoparticles and blocking the reaction between the gold nanoparticles and the chiral organic compounds. Impurities in the reactants result in non-uniform morphology of the generated chiral gold nanoparticles. Similarly, if the concentration of intermediate reactants is too low, the generated gold nanoparticles will exhibit a "split" shape; if the concentration of intermediate reactants is too high, the added chiral organic compounds will only induce the formation of surface-distorted gold nanoparticles.

[0025] In some instances, the gold salt includes one or more of gold chloride, gold chlorohydrate, chloroauric acid, chloroauric acid trihydrate, chloroauric acid tetrahydrate, potassium chloroaurate dihydrate, and sodium chloroaurate dihydrate.

[0026] In some instances, the quaternary ammonium salt is selected from one or more of hexadecyltrimethylammonium bromide, dodecyltrimethylammonium chloride, and dodecyldimethylbenzylammonium chloride.

[0027] In some instances, the reducing agent is selected from at least one of sodium borohydride, potassium borohydride, ascorbic acid, or potassium iodide.

[0028] In some instances, the chiral organic compound includes one or more of cysteine, glutathione, glucose, cysteylglycine, 1,1-binaphthyl-2,2-diamine, and adenine polymers.

[0029] In some instances, the amination process involves adding a chiral gold nanoparticle solution to anhydrous ethanol and 3-aminopropyltriethoxysilane and reacting at 28–35°C for 20–30 h to obtain amination-treated chiral gold nanoparticles.

[0030] In some instances, the activation treatment is as follows: N-ethyl-N′-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide are weighed, dissolved in PBS solution, mixed with secondary antibody, and reacted with shaking at 35–40°C for 2 hours.

[0031] Secondly, in addition to the first aspect, this invention proposes an innovative application of a time-resolved colorimetric immunoassay sensor in the field of antipyrine point detection.

[0032] The beneficial effects of this invention are:

[0033] This invention develops a colorimetric-chronometric sensor for sensitive detection of ATAP concentration. This sensor combines temperature and thermochromic pigments to establish a relationship between the solution's color change time and the analyte concentration, thereby achieving detection. Specifically, the heat generated by chiral gold nanoparticles drives electron transfer in the pigment, altering the pigment's molecular structure and producing different color changes. Since different concentrations of the analyte and antigen simultaneously compete for a certain amount of antibody, the concentration of the secondary antibody (Ab2)-modified chiral gold nanocomposite bound to the antibody varies in the well plate. Under 808nm infrared irradiation, different concentrations of Ab2-modified chiral gold nanocomposite generate different amounts of heat, correspondingly resulting in different times for the solution to reach the temperature change temperature. This allows for visual semi-quantitative detection by the naked eye, with quantitative verification through a time signal. Simultaneously, a portable device was constructed, mainly composed of a color probe and a timing component, capable of displaying the detection results on an OLED display in a short time, providing more possibilities for point-of-care testing (POCT). Attached Figure Description

[0034] Figure 1 The basic structure (A) and components (B) of a portable device.

[0035] Figure 2 Figure 1 shows the SEM images (A, B), TEM image (C), circular dichroism chromatogram (D), and g-factor plot (E) of chiral gold nanoparticles. The inset in Figure 1 shows the particle size measured by laser particle size analysis.

[0036] Figure 3 The response temperature of chiral gold nanoparticles after continuous heating for 60 min was measured every five minutes (A); the average temperature and coefficient of variation of chiral gold nanoparticles after 10 min of illumination (B); the average heating rate of chiral gold nanoparticles every 5 min within 60 min (C); and the cycle curve of chiral gold nanoparticles (D).

[0037] Figure 4 Images showing the color changes of ATAP solutions at different concentrations, taken every 10 seconds (A); a standard curve of color change time versus ATAP concentration (B); recovery tests of ATAP at different concentrations (C); and sensor specificity tests, from af to b, for antipyrine, aminopyrine, phenacetin, rosiglitazone, furosemide, and tofenamic acid, with all samples using 10 μg·L⁻¹. -1 (D) Detailed Implementation

[0038] The following disclosure provides many different implementations or examples for different ways of implementing the present invention.

[0039] Example

[0040] 1) Synthesis of chiral gold nanoparticles

[0041] First, octahedral gold nanoparticles were synthesized. (1) 10 mL of a solution containing 0.1–0.3 mM hexadecyltrimethylammonium chloride (CTAC) and 1.0 × 10⁻⁶ mM hexadecyltrimethylammonium chloride (CTAC) was prepared in a 25 mL bottle. -4 ~4.0×10 -4 (1) Add 0.45-0.6 mL of 0.2 mM NaBH4 solution to the bottle under vigorous stirring and stir for 2-5 min. Place the bottle in a 30℃ water bath and let it stand for 1.5-3 h to obtain gold seeds. (2) Take two small bottles, A and B respectively, add 0.32 g of CTAC and 9-10 mL of deionized water to each bottle, sonicate to dissolve and store in a 30-40℃ water bath. Add 200-300 μL of 0.1 mM HAuCl4 solution, 5-15 μL of 0.01 mM potassium iodide solution, and then add 100-300 μL of 0.1 mM ascorbic acid (AA) solution. Add 55 μL of the above gold seed solution to bottle A, shake until light pink, immediately transfer 55 μL of the solution from bottle A to bottle B, shake for 10–40 s, let stand in a water bath at 25–35 °C for 10–30 min, centrifuge at 3000–4500 r / min for 10 min, and store the product in 1–10 mM hexadecyltrimethylammonium bromide (CTAB) solution to obtain octahedral gold nanoparticle solution.

[0042] Next, chiral gold nanoparticles were prepared. 0.5–2 mL of 100 mM CTAB solution and 3–5 mL of deionized water were added to a beaker. Then, 0.1–0.5 mL of 10 mM HAuCl4 solution was added, followed by a rapid addition of 0.2–1.0 mL of 0.1 mM AA solution. At this point, the solution changed from brownish-yellow to colorless. 5–50 μL of 5 mM L-glutathione and 10–100 μL of the above octahedral gold nanoparticle solution were added, and the mixture was shaken well and incubated in a water bath at 30–40 °C for 2 hours. The solution changed from pink to blue. The mixture was centrifuged at 10000–12000 r / min for 5–30 min to remove unreacted reagents. The product was stored in 1–10 mM CTAB solution at 4 °C to obtain the chiral gold nanoparticle solution.

[0043] 2) Construction of immune sensors

[0044] First, an Ab2-modified chiral gold nanocomposite was prepared. 5–10 mL of chiral gold nanoparticle solution was subjected to amination treatment. The chiral gold nanoparticle solution was added to 10–50 mL of anhydrous ethanol, followed by 10–100 μL of 3-aminopropyltriethoxysilane (APTES). The mixture was sonicated for 30–60 min to completely dissolve the APTES in the ethanol. Then, the mixture was transferred to a water bath at 28–35 °C and stirred for 20–30 h. After centrifugation at 10,000–12,000 r / min for 10–15 min, the mixture was washed 2–3 times with water to remove ethanol and APTES from the solution. The resulting nanocomposite was dispersed and dissolved in deionized water to obtain the amination-modified chiral gold nanoparticles. Correspondingly, the carboxyl groups on the Ab2 surface also required activation treatment. N-ethyl-N′-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) in a mass ratio of 2:1 were dissolved in 80–120 mM phosphate buffered saline (PBS) at pH 7.4. The prepared solution was then mixed with Ab2 at a volume ratio of 2:1 and reacted with shaking at 35–40 °C for 2 h. Finally, the aminated chiral gold nanoparticles were stirred with activated Ab2 at 4 °C for 20–30 h to ensure complete binding of the amino and carboxyl groups. After centrifugation at 10,000–12,000 r / min for 5–10 min and washing 2–3 times, the product was dispersed in 1–5 mL of PBS and stored at 4 °C to obtain the Ab2-modified chiral gold nanocomposite.

[0045] Next, 50–100 μL of antigen was added to a 96-well plate and incubated overnight at 4°C. Then, 40–100 μL of blocking buffer was added to block excess active sites. For the immunocompetitive assay, 50 μL of a solution containing different concentrations of ATAP and primary antibody (Ab1) (v:v = 1:1) was added to the wells and incubated at 35–45°C for 2–3 hours. Afterward, 30–100 μL of Ab2-modified chiral gold nanocomposite was added and incubated at 35–45°C for 2–3 hours. After each step, the plate was washed twice with 0.1 M PBS. For the visualization-chronometry assay, 300–400 μL of 0.1 M PBS and 80–120 μL of thermochromic pigment were added to the wells and incubated at 808 nm and 2 W / cm². 2 Irradiate the solution with infrared light and record the time it takes for the solution to change color.

[0046] 3) Equipment Development

[0047] A portable detection device has been developed based on the photothermal properties of chiral gold nanoparticles, such as... Figure 1 As shown in Figure A, the device consists of a 3D-printed outer shell and upper and lower circuit boards, with a photograph of the printed circuit boards shown below. Figure 1 B, the specific design is as follows.

[0048] (1) OLED display screen Figure 1B(a)): The data on the display screen, from top to bottom, are time reading, color reading, and concentration calculation reading; (2) Timing section: such as Figure 1 As shown in B(b), the timing stops when the colorimetric probe detects that the RGB value of the solution has completely turned blue-purple, and the signal is sent to the main control chip via "DAT"; (3) Switch: The switch on the circuit board is as follows Figure 1 As shown in B(c), the device is started by a switch; the switch is turned down to turn on and turned up to turn off; (4) Arduino Nano development board: Arduino Nano development board ( Figure 1 B(d)) contains the core chip responsible for signal processing and the power supply section, RX and TX ports ( Figure 1 B(e)) is used to receive and send serial communication signals with the timing section and the colorimetric probe module; (5) Optical colorimetric probe: Figure 1 B(f) is the colorimetric recognition module, where the central part is the colorimetric probe. Figure 1 B(g) is used to detect color signals.

[0049] 4) Characterization and properties of chiral gold nanoparticles

[0050] like Figure 2 As shown in transmission electron microscopy images A and 2B, L-glutathione-induced chiral gold nanoparticles exhibit a cubic structure with a helical morphology around the center axis on the surface, and the average particle size of the chiral gold nanoparticles is approximately 140-160 nm. Figure 2 (Illustration A). By Figure 2 The C-ray transmission electron microscopy (TEM) image shows a clockwise rotation from the white dashed line, and the structure exhibits asymmetry, indicating the material possesses the expected chirality. Circular dichroism spectroscopy is an optical rotation spectroscopy technique used to infer the configuration and conformation of asymmetric molecules; therefore, circular dichroism spectroscopy was employed to determine the synthesized chiral gold nanoparticles. Figure 2 As shown in D and 2E, the gold nanospirals generated by L-glutathione as an inducer exhibit a strong chiral signal, which is more obvious at 612 nm with a value of -1.3; while the g-factor value at 622 nm is -0.14, indicating that the generated chiral gold nanoparticles have a certain degree of asymmetry.

[0051] To evaluate the photothermal effect of chiral gold nanoparticles, performance tests, including stability and heating rate, were conducted at a concentration of 0.5 mg / mL. Figure 3 As shown in Figure A, temperature changes were recorded every 5 minutes during 60 minutes of continuous illumination. The results indicate that the temperature response of chiral gold nanoparticles reached its peak within 10 minutes of illumination and remained relatively stable. Furthermore, as... Figure 3As shown in Figure B, after reaching a stable state, the average temperature of the chiral gold nanoparticles is approximately 85.2℃, and after 10 minutes of illumination, the coefficient of variation of its response temperature is only 0.014, demonstrating excellent stability. Figure 3 As shown in Figure C, the heating rate of chiral gold nanoparticles was close to 15 °C / min during the first 5 minutes of irradiation, demonstrating a high thermal response capability; and during the first 2 minutes of irradiation, the photothermal conversion efficiency η of chiral gold nanoparticles reached as high as 77.1%.

[0052] The photothermal stability was studied by alternately turning the infrared lamp on and off four times. Figure 3 As shown in D, after four cycles, the highest response temperature of chiral gold nanoparticles remained relatively stable, further demonstrating that even after multiple heating and cooling processes, chiral gold nanoparticles can still maintain their photothermal stability.

[0053] 5) Immune performance test

[0054] The visualization is achieved using reversible thermochromic pigments, specifically organic chromophores composed of electron-transfer type organic compounds. At a specific temperature, electron transfer occurs, causing a change in the molecular structure of the organic compounds in the reversible thermochromic pigment, thus achieving a color change. This type of chromatic substance can achieve a reversible "colored to colorless" color change. Two pigments, one turning yellow to colorless at 28℃ and the other turning blue to colorless at 38℃, were mixed in a 3:1 volume ratio, resulting in a green solution. When the temperature reaches 28℃, the yellow pigment gradually fades, and the solution gradually changes from green to blue.

[0055] ATAP was detected using an immune competitive assay. Different concentrations of ATAP competed with the antigen immobilized on the ELISA plate for a fixed amount of antigen. The higher the ATAP concentration, the fewer Ab1 molecules could be immobilized on the ELISA plate, and consequently, the number of Ab2-modified chiral gold nanocomposites bound to Ab1 decreased. This reduced the photothermal effect and thus affected the time it took for the solution to reach the color change temperature. Figure 4 As shown in Figure A, the relationship between the color of solutions with different concentrations of ATAP and time was investigated, with images taken every 10 seconds. Plotting time on the x-axis and ATAP concentration on the y-axis, it was found that the color-changing effect exhibited a certain gradient with ATAP concentration. As the ATAP concentration increased, the amount of Ab2 bound to Ab1 at the bottom of the well decreased, reducing the photothermal effect and thus prolonging the color-changing time. Therefore, as... Figure 4 B, at 1.0 × 10 -2 -1.0×10 2 μg·L -1 Within the concentration range, a linear relationship was established between the solution color change time and the ATAP concentration: T = 48.2 + 7.6lgC(R) 2=0.9785)(C:ATAP concentration), detection limit is 4.03×10 -3 μg·L -1 (S / N = 3). To further investigate the reliability of the device, a recovery test was conducted. As shown in 4C, the sample recoveries ranged from 96.9% to 104.2%, indicating its ability to detect real samples. Simultaneously, the device's specificity for ATAP was tested. Figure 4 D. When detecting ATAP, the system exhibits immune competition, reducing the amount of Ab2 binding to Ab1 and lengthening the time to reach the color change temperature. However, when detecting interfering substances, under the same concentration conditions, the color change time is significantly shorter than that required for ATAP, indicating that the sensor possesses specific selectivity. In conclusion, the established device demonstrates high accuracy and has potential for practical detection applications.

[0056] 6) Conclusion

[0057] This experiment successfully constructed a point-of-care testing (POCT) immunosensing device based on the photothermal properties of chiral gold nanoparticles. This device drives an immune reaction by driving antibodies labeled with chiral gold nanoparticles, combined with a thermochromic pigment. This allows for simple visual semi-quantitative detection with the naked eye, and also enables accurate and reliable quantitative detection of the target substance, antipyrine, using time signals. The linear range of this method is 1.0 × 10⁻⁶. -2 -1.0×10 2 μg·L -1 The detection limit is 4.03 × 10⁻⁶. -3 μg·L -1 This device features high sensitivity, simple operation, and portability, and has great application potential in POCT detection and thermochromic materials.

[0058] The above is a further detailed description of the present invention and should not be considered as a limitation on the specific implementation of the present invention. For those skilled in the art, simple deductions or substitutions without departing from the concept of the present invention are all within the protection scope of the present invention.

Claims

1. The application of a time-resolved colorimetric immunosensor for detecting antipyrine concentration in the point-of-care detection of antipyrine, characterized in that, The time-resolved colorimetric immunosensor includes a colorimetric recognition module, a signal processing module, a timing module, and an immune competitive reaction module. The time-resolved colorimetric immunosensor uses the following method to detect the concentration of antipyrine: 1) The antigen was immobilized on a 96-well plate, and then antipyrine solutions of known different concentrations, primary antibody and Ab2-modified chiral gold nanocomposites were added for incubation. 2) Two thermochromic pigments, one turning yellow to colorless at 28℃ and the other turning blue to colorless at 38℃, were mixed at a volume ratio of 3:

1. Then, PBS solution and the mixed thermochromic pigments were added to the well plates. The mixture was incubated at 805–810 nm and 1.0–5.0 W / cm². 2 The solution was irradiated with infrared light and the color change time was recorded to construct a standard curve of antipyrine concentration and color change time. 3) Replace the known different concentrations of antipyrine in step 1) with the antipyrine solution to be tested, and use the same method as in steps 1) and 2) to calculate the concentration of the antipyrine solution to be tested based on the standard curve; The preparation method of the Ab2-modified chiral gold nanocomposite includes the following steps: 1) Take a certain amount of gold salt solution, add surfactant, stir and then add reducing agent to prepare octahedral gold nanoparticle solution; 2) Add a chiral organic compound, a reducing agent, and a quaternary ammonium salt to the octahedral gold nanoparticle solution, and modify the solution by reaction to obtain a chiral gold nanoparticle solution; 3) The chiral gold nanoparticle solution is subjected to amination treatment, and the carboxyl groups on the surface of the secondary antibody are activated. Finally, the treated chiral gold nanoparticle solution is reacted with the secondary antibody to obtain the Ab2-modified chiral gold nanocomposite.

2. The application according to claim 1, characterized in that, The reaction modification in step 2) to obtain the chiral gold nanoparticle solution is carried out at a temperature of 30–40 °C.

3. The application according to claim 1, characterized in that, The gold salt includes one or more of gold chloride, gold chlorohydrate, chloroauric acid, chloroauric acid trihydrate, chloroauric acid tetrahydrate, potassium chloroaurate dihydrate, and sodium chloroaurate dihydrate.

4. The application according to claim 1, characterized in that, The quaternary ammonium salt is selected from one or more of hexadecyltrimethylammonium bromide, dodecyltrimethylammonium chloride, and dodecyldimethylbenzylammonium chloride.

5. The application according to claim 1, characterized in that, The reducing agent is selected from at least one of sodium borohydride, potassium borohydride, ascorbic acid, or potassium iodide.

6. The application according to claim 1, characterized in that, The chiral organic compounds include one or more of cysteine, glutathione, glucose, cysteylglycine, 1,1-binaphthyl-2,2-diamine, and adenine polymers.

7. The application according to claim 1, characterized in that, The amination treatment is as follows: chiral gold nanoparticle solution is added to anhydrous ethanol and 3-aminopropyltriethoxysilane and reacted at 28-35 °C for 20-30 h to obtain amination-treated chiral gold nanoparticles.

8. The application according to claim 1, characterized in that, The activation treatment was performed as follows: N-ethyl-N′-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide were weighed, dissolved in PBS solution, mixed with secondary antibody, and reacted with shaking at 35-40°C for 2 h.

Images