A method for determining total antioxidant capacity based on nano-gold cone

Abstract

The application provides a total antioxidant capacity determination method based on nano gold cones, which comprises the following steps: synthesizing nano gold cones through a seed-mediated method to obtain a nano gold cone solution; mixing diethanolamine buffer, the nano gold cone solution and silver nitrate under room temperature conditions, and adding the mixture into an enzyme-labeled hole; adding a sample containing an antioxidant to be measured into the enzyme-labeled hole to mix and react; inducing silver to deposit on the surface of the nano gold cone through the antioxidant; irradiating the enzyme-labeled hole with a near-infrared laser to convert the molecular concentration information of the antioxidant into a temperature signal; taking a temperature measurement of the enzyme-labeled hole by using a digital thermal imager, and recording the temperature value in the laser irradiation process; and determining the total antioxidant capacity according to the temperature value. In the application, the silver is induced to deposit on the surface of the nano gold cone, the appearance of the nano gold cone is changed, and a unique temperature response to the antioxidant is induced. The sensitivity of the temperature change depends on the thickness of the silver deposited on the surface of the nano gold cone, so that the total antioxidant capacity can be directly determined by the temperature change of the enzyme-labeled hole during laser irradiation.

Description

Technical Field

[0001] This invention relates to the field of nanobiosensors, and in particular to a method for determining the total antioxidant capacity based on nanogold cones. Background Technology

[0002] Excessive free radicals in the body can lead to oxidative stress and redox imbalance, increasing the risk of cardiovascular disease, inflammation, and various degenerative diseases.

[0003] Antioxidants with the ability to scavenge free radicals can prevent undesirable oxidation reactions, counteract oxidative damage, and protect redox homeostasis.

[0004] To date, various techniques have been used to measure total antioxidant capacity. This information can be converted into electrochemical, fluorescence, or chromatographic signals.

[0005] Subsequently, these signals are read using specialized instruments such as electrochemical analyzers, fluorescence spectrophotometers, and high-performance liquid chromatographs. However, current methods require large, expensive, and inconvenient equipment; moreover, these instruments require operation by specialized technicians, demanding high levels of skill from the operators, making the entire process extremely complex. Summary of the Invention

[0006] In view of the above, the main objective of this invention is to propose a method for determining the total antioxidant capacity based on nano-gold cones, so as to solve the above-mentioned technical problems.

[0007] This invention provides a method for determining the total antioxidant capacity based on nano-gold cones, characterized in that the method includes the following steps:

[0008] Step 1: Synthesize gold nanoparticles using a seed-mediated method to obtain a gold nanoparticle solution;

[0009] Step 2: At room temperature, diethanolamine buffer, gold nanoparticle solution and silver nitrate are mixed and added to the enzyme label wells. Then, the test sample containing antioxidant is dropped into the label wells and mixed. The antioxidant induces silver deposition on the surface of the gold nanoparticles, so as to regulate the conical structure at both ends of the gold nanoparticles into an elliptical structure, which in turn leads to the blue shift of the local surface plasmon resonance peak.

[0010] Step 3: Irradiate the enzyme-labeled wells with a near-infrared laser to convert the molecular concentration information of the antioxidant into a temperature signal. Use a digital thermal imager to measure and photograph the enzyme-labeled wells and record the temperature value during the laser irradiation process. Measure the total antioxidant capacity based on the temperature value.

[0011] In this invention, an antioxidant is used to induce silver deposition on the surface of gold nanoparticles, which changes the morphology of the gold nanoparticles and induces a unique temperature response to the antioxidant. The sensitivity of temperature change depends on the thickness of silver deposited on the surface of the gold nanoparticles, so the total antioxidant capacity can be determined directly by the temperature change of the enzyme label well during laser irradiation.

[0012] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0013] 1. The synthesis method of uniform gold nanoparticles is very simple and reliable. Uniform size and morphological uniformity endow gold nanocrystals with narrower peak widths, which is beneficial for absorbing light of specific wavelengths and achieving higher photothermal conversion.

[0014] 2. Antioxidant-induced silver deposition on gold nanoparticles can modulate surface plasmon resonance and photothermal effects without background interference. The well-defined optical properties and high exponential sensitivity of gold nanoparticles make them suitable for highly sensitive photothermal analysis, as low concentrations of analytes can lead to significant changes in photothermal conversion efficiency and the resulting temperature.

[0015] 3. Low cost and low skill requirements. Total antioxidant capacity can be measured without expensive instruments and professional personnel. Furthermore, the laser and thermal imager of this invention can be directly used as portable devices, achieving high portability.

[0016] Additional aspects and advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by means of embodiments of the invention. Attached Figure Description

[0017] Figure 1 This is a schematic diagram of the principle of a method for determining the total antioxidant capacity based on nano-gold cones proposed in this invention;

[0018] Figure 2 This is a TEM characterization image of the gold nanoparticles of this invention;

[0019] Figure 3 This is the ultraviolet-visible absorption spectrum of the nano-gold cone of the present invention;

[0020] Figure 4 This is a comparison diagram of the photothermal feasibility test of the method proposed in this invention and the nano-gold cone under different conditions;

[0021] Figure 5 This is a linear graph showing the process of determining the total antioxidant capacity in this invention. Detailed Implementation

[0022] Embodiments of the present invention are described in detail below. Examples of these embodiments are shown in the accompanying drawings, wherein the same or similar reference numerals denote the same or similar elements or elements having the same or similar functions throughout. The embodiments described below with reference to the accompanying drawings are exemplary and are only used to explain the present invention, and should not be construed as limiting the present invention.

[0023] These and other aspects of the embodiments of the present invention will become clear from the following description and accompanying drawings. In these descriptions and drawings, some specific embodiments of the present invention are specifically disclosed to illustrate some ways of implementing the principles of the embodiments of the present invention; however, it should be understood that the scope of the embodiments of the present invention is not limited thereto.

[0024] Example 1

[0025] Please see Figure 1 and Figure 2 A method for determining the total antioxidant capacity based on gold nanoparticles, the method comprising the following steps:

[0026] Step 1: Mix 1 mL of 0.01 M citric acid solution and 40 mL of 0.8 M chloroauric acid solution, and dissolve them completely in a 30°C water bath to obtain a mixed solution containing gold ions and chloride ions.

[0027] 0.01M sodium borohydride (0.6 mL) was added to a mixed solution containing gold and chloride ions and stirred rapidly for 2 min. After stirring, the solution was allowed to stand and age in the dark for 5 h to obtain the seed solution.

[0028] The growth solution was prepared by mixing 200 mL of 0.1 M hexadecyltrimethylammonium bromide solution, 10 mL of 0.01 M tetrachloroauric acid solution, 2 mL of 0.01 M silver nitrate solution, 4 mL of 1 M hydrochloric acid solution, and 1.6 mL of 0.1 M ascorbic acid solution.

[0029] Add 0.9 mL of seed culture to the growth medium and let it stand overnight in a 30°C water bath to obtain a nano-gold cone (nano-gold cone) solution.

[0030] Step 2: At room temperature, mix 50 μL of 0.1 M diethanolamine buffer (pH 8.4), 50 μL of 808 nm gold nanoparticle solution, and 50 μL of 0.4 mM silver nitrate solution and add them to the enzyme label wells. Then, add the test sample containing antioxidant to the wells and mix for 2 min. The antioxidant induces silver deposition on the surface of the gold nanoparticles, thereby regulating the conical structure at both ends of the gold nanoparticles into an elliptical structure, which in turn leads to a blue shift of the local surface plasmon resonance peak.

[0031] Step 3: Place the enzyme-labeled wells directly below the near-infrared laser, with a distance of 10 cm between the wells and the laser source. Irradiate the wells with an 808 nm near-infrared laser to convert the molecular concentration information of the antioxidant into a temperature signal. Place a portable digital thermal imager directly in front of the wells to measure and photograph the temperature. Record the temperature value after 8 minutes of laser irradiation. Measure the total antioxidant capacity based on the temperature value.

[0032] Example 2

[0033] Please see Figure 1 and Figure 2 A method for determining the total antioxidant capacity based on gold nanoparticles, the method comprising the following steps:

[0034] Step 1: Mix 0.01M, 0.8mL citric acid solution and 0.8M, 30mL chloroauric acid solution, and dissolve them completely in a 30℃ water bath to obtain a mixed solution containing gold ions and chloride ions.

[0035] 0.01M sodium borohydride (0.4 mL) was added to a mixed solution containing gold and chloride ions and stirred rapidly for 1 min. After stirring, the solution was aged in the dark for 7 h to obtain the seed solution.

[0036] The growth solution was prepared by mixing 180 mL of 0.1 M hexadecyltrimethylammonium bromide solution, 8 mL of 0.01 M tetrachloroauric acid solution, 1 mL of 0.01 M silver nitrate solution, 3 mL of 1 M hydrochloric acid solution, and 1 mL of 0.1 M ascorbic acid solution.

[0037] Add 0.5 mL of seed culture to the growth medium and let it stand overnight in a 30°C water bath to obtain a nano gold cone solution.

[0038] Step 2: At room temperature, mix 40 μL of 0.1 M diethanolamine buffer (pH 8.4), 40 μL of 808 nm gold nanoparticle solution, and 40 μL of 0.4 mM silver nitrate solution and add them to the enzyme label wells. Then, add the test sample containing antioxidant to the wells and mix for 1 min. The antioxidant induces silver deposition on the surface of the gold nanoparticles, thereby regulating the conical structure at both ends of the gold nanoparticles into an elliptical structure, which in turn leads to a blue shift of the local surface plasmon resonance peak.

[0039] Step 3: Place the enzyme-labeled wells directly below the near-infrared laser, with a distance of 8 cm between the wells and the laser source. Irradiate the wells with an 808 nm near-infrared laser to convert the molecular concentration information of the antioxidant into a temperature signal. Place a portable digital thermal imager directly in front of the wells to measure and photograph the temperature. Record the temperature value after 8 minutes of laser irradiation. Measure the total antioxidant capacity based on the temperature value.

[0040] Example 3

[0041] Please see Figure 1 and Figure 2 A method for determining the total antioxidant capacity based on gold nanoparticles, the method comprising the following steps:

[0042] Step 1: Mix 1.2 mL of 0.01 M citric acid solution and 50 mL of 0.8 M chloroauric acid solution, and dissolve them completely in a 30°C water bath to obtain a mixed solution containing gold ions and chloride ions.

[0043] 0.01M sodium borohydride (0.8 mL) was added to a mixed solution containing gold and chloride ions and stirred rapidly for 4 min. After stirring, the solution was allowed to stand and age in the dark for 10 h to obtain the seed solution.

[0044] The growth solution was prepared by mixing 200 mL of 0.1 M hexadecyltrimethylammonium bromide solution, 12 mL of 0.01 M tetrachloroauric acid solution, 3 mL of 0.01 M silver nitrate solution, 6 mL of 1 M hydrochloric acid solution, and 2 mL of 0.1 M ascorbic acid solution.

[0045] Add 1.5 mL of seed culture to the growth medium and let it stand overnight in a 30°C water bath to obtain a nano gold cone solution.

[0046] Step 2: At room temperature, mix 60 μL of 0.1 M diethanolamine buffer (pH 8.4), 60 μL of 808 nm gold nanoparticle solution, and 60 μL of 0.4 mM silver nitrate solution and add them to the enzyme label wells. Then, add the test sample containing antioxidants to the wells and mix for 3 min. The antioxidants induce silver deposition on the surface of the gold nanoparticles, thereby regulating the conical structure at both ends of the gold nanoparticles into an elliptical structure, which in turn leads to a blue shift of the local surface plasmon resonance peak.

[0047] Step 3: Place the enzyme-labeled wells directly below the near-infrared laser, with a distance of 12cm between the wells and the laser source. Irradiate the wells with an 808nm near-infrared laser to convert the molecular concentration information of the antioxidant into a temperature signal. Place a portable digital thermal imager directly in front of the wells to measure and photograph the temperature. Record the temperature value after 8 minutes of laser irradiation. Measure the total antioxidant capacity based on the temperature value.

[0048] pass Figure 3 It can be seen that the nano-gold cone of the present invention has good light absorption for 808nm near-infrared laser.

[0049] To verify that the present invention can convert the molecular concentration information of antioxidants into a temperature signal, the photothermal feasibility of the present invention and the nano gold cone under different states was compared.

[0050] like Figure 4 As shown, 'a' represents the temperature curve recorded after laser irradiation following the addition of buffer solution and gold nanoparticles.

[0051] b represents the temperature curve recorded after laser irradiation following the addition of buffer solution, nano-gold cones, and ascorbic acid;

[0052] c represents the temperature curve recorded after laser irradiation following the addition of buffer solution, gold nanoparticles, and silver nitrate.

[0053] d represents the temperature curve recorded by the method of Embodiment 1 of the present invention;

[0054] As can be seen from the above, the present invention utilizes antioxidants to induce silver deposition on the surface of gold nanoparticles, thereby changing the morphology of the gold nanoparticles and inducing a unique temperature response to the antioxidants. This allows the molecular concentration information of the antioxidants to be converted into a temperature signal, which is suitable for the determination of total antioxidant capacity.

[0055] Please see Figure 5 Total antioxidant capacity is usually determined using standard antioxidants such as ascorbic acid. To demonstrate that this invention is applicable to the determination of total antioxidant capacity, ascorbic acid is used as the test antioxidant in this embodiment. Following the method of Example 1 above, ascorbic acid is used as the test sample. Different concentrations of ascorbic acid are added to the enzyme-labeled wells and irradiated with an 808nm near-infrared laser for 8 minutes. As the concentration of ascorbic acid increases, the temperature change ΔT caused by the photothermal effect of the mixed solution increases rapidly. The change in ΔT shows a strong linear relationship with the ascorbic acid concentration in the range of 0.1-100μM. The limit of detection (LOD) is 0.08μM. The linear equation is:

[0056] ΔT = 2.43 + 0.38c AA ;

[0057] Among them, the determination coefficient R 2 =0.999, sample size n=3, ΔT represents temperature change, c AA This indicates the concentration of ascorbic acid.

[0058] Compared to other existing sensors for detecting total antioxidant capacity, this invention exhibits a wider linear range and higher sensitivity in detecting total antioxidant capacity. These results strongly suggest that this invention possesses ideal sensitivity and detection limit for detecting total antioxidant capacity.

[0059] To test the applicability of this invention in practical analysis, honey and vitamin C effervescent tablets were used as test samples for total antioxidant capacity determination. To evaluate the accuracy and practicality of the results, ascorbic acid at concentrations of 10 μM, 20 μM, and 30 μM was directly added to actual samples that underwent only simple dissolution for testing. As shown in Table 1, the recoveries of honey and vitamin C effervescent tablets determined by this invention were 95.0%–107.0% (Table 1), respectively (Table 1). The acceptable recoveries in the spiked experiments indicate that this invention can accurately detect ascorbic acid and demonstrate its feasibility in practical samples.

[0060] Table 1. Comparison of the recovery rate and corresponding RSD of AA (ascorbic acid) added to vitamin C effervescent tablets and honey using the present invention.

[0061]

[0062]

[0063] To demonstrate the reliability of this invention in practical applications, the concentration of ascorbic acid in honey was determined using a spiked method. Before use, the honey was dissolved in 5 ml of water, diluted 100 times, and then ascorbic acid at concentrations of 10 μM, 20 μM, and 30 μM was added as test samples. 50 μL of each sample with different ascorbic acid concentrations was added to the wells of the enzyme-labeled enzyme. After reacting at room temperature for 2 minutes, the mixture was irradiated with an 808 nm near-infrared laser, and the temperature was recorded at 8 minutes using a portable digital thermal imager. Finally, the concentration of ascorbic acid in the actual sample was calculated based on the ascorbic acid standard curve.

[0064] In the description of this specification, references to terms such as "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples.

[0065] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention. Therefore, the scope of protection of this patent should be determined by the appended claims.

Claims

1. A method for determining the total antioxidant capacity based on nano-gold cones, characterized in that, The method includes the following steps: Step 1: Synthesize gold nanoparticles using a seed-mediated method to obtain a gold nanoparticle solution. This process includes the following steps: Citric acid solution and chloroauric acid solution were mixed and fully dissolved under water bath conditions to obtain a mixed solution containing gold ions and chloride ions; Cold sodium borohydride was added to a mixed solution containing gold ions and chloride ions and stirred rapidly. After stirring, the solution was allowed to stand and age in the dark to obtain the seed solution. A growth solution was obtained by mixing hexadecyltrimethylammonium bromide solution, tetrachloroauric acid solution, silver nitrate solution, hydrochloric acid solution, and ascorbic acid solution. The seed solution was added to the growth solution and left to stand overnight in a water bath to obtain a nano gold cone solution. Step 2: At room temperature, diethanolamine buffer, gold nanoparticle solution and silver nitrate are mixed and added to the enzyme label wells. Then, the test sample containing antioxidant is dropped into the label wells and mixed. The antioxidant induces silver deposition on the surface of the gold nanoparticles, so as to regulate the conical structure at both ends of the gold nanoparticles into an elliptical structure, which in turn leads to the blue shift of the local surface plasmon resonance peak. Step 3: Irradiate the enzyme-labeled wells with a near-infrared laser to convert the molecular concentration information of the antioxidant into a temperature signal. Use a digital thermal imager to measure and photograph the enzyme-labeled wells and record the temperature value during the laser irradiation process. Measure the total antioxidant capacity based on the temperature value.

2. The method for determining the total antioxidant capacity based on nano-gold cones according to claim 1, characterized in that, The mixing ratio of citric acid solution and chloroauric acid solution is 0.01M, 0.8-1.2mL: 0.8M, 30-50mL; When sodium borohydride is added to a mixed solution containing gold and chloride ions, the proportion of sodium borohydride added is 0.01M, 0.4-0.8mL.

3. The method for determining the total antioxidant capacity based on nano-gold cones according to claim 2, characterized in that, The mixing ratios of hexadecyltrimethylammonium bromide solution, tetrachloroauric acid solution, silver nitrate solution, hydrochloric acid solution, and ascorbic acid solution are 0.1M, 180-200 mL: 0.01M, 8-12 mL: 0.01M, 1-3 mL: 1M, 3-6 mL: 0.1M, 1-2 mL; After preparing the growth solution, the amount of seed solution added to the growth solution is 0.5-1.5 mL.

4. The method for determining the total antioxidant capacity based on nano-gold cones according to claim 2, characterized in that, The stirring time is 1-4 minutes, the water bath temperature is 30℃, and the aging time is 5-10 hours.

5. The method for determining the total antioxidant capacity based on nano-gold cones according to claim 4, characterized in that, In step 2, the mixing ratio of diethanolamine buffer, gold nanoparticle solution and silver nitrate is 0.1M, 40-60µL: 808nm, 40-60µL: 0.4mM, 40-60µL, the pH of diethanolamine buffer is 8.4, the room temperature is 26℃, and the mixing reaction time is 1-3min.

6. The method for determining the total antioxidant capacity based on nano-gold cones according to claim 5, characterized in that, In step 3, the emission wavelength of the near-infrared laser is 808nm, the distance between the enzyme label well and the near-infrared laser light source is 8-12cm, and the laser irradiation time recorded by the digital thermal imager is 8min.

7. The method for determining the total antioxidant capacity based on nano-gold cones according to claim 6, characterized in that, In step 3, when irradiating the enzyme-labeled wells with a near-infrared laser, the wells are positioned directly below the near-infrared laser, and a portable digital thermal imager is positioned directly in front of the wells to measure the temperature and take pictures.

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