Method and kit for detecting content of D-amino acid based on Pt / Ti3C2Tx nano material and application of method and kit

Abstract

The invention belongs to the technical field of nano material application and chemical detection, and particularly discloses a method and a kit for detecting the content of D-amino acid based on a Pt / Ti3C2Tx nano material and application of the method and the kit. The method comprises the following steps: incubating a sample to be detected and D-amino acid oxidase together; after the incubation is completed, adding a Pt / Ti < 3 > C < 2 > T < x > nano material and a chromogenic substrate TMB (Tetramethylbenzidine), and reacting under laser irradiation; and after the reaction is finished, determining the content of D-amino acid by a colorimetric detection method. The kit comprises D-amino acid oxidase, a Pt / Ti < 3 > C < 2 > T < x > nanometer material and TMB (Tetramethylbenzidine). The Pt / Ti < 3 > C < 2 > T < x > nanometer material has local surface plasma resonance effect and peroxidase-like catalytic activity, a colorimetric detection platform constructed based on the Pt / Ti < 3 > C < 2 > T < x > nanometer material is applied to clinical saliva sample detection, has the characteristics of high sensitivity, high selectivity and the like, and can provide a rapid, simple and painless new path for early screening of gastric cancer.

Description

Technical Field

[0001] The present invention relates to the field of nanomaterial application and chemical detection technology, in particular to a nanomaterial based on Pt / Ti3C2T x A method and kit for detecting D-amino acid content using nanomaterials and their application. Background Art

[0002] Gastric cancer is one of the common malignant tumors, and its morbidity and mortality rate rank fifth and third among malignant tumors, respectively. Therefore, early detection and timely treatment are of great significance for gastric cancer patients. At present, the clinical diagnosis method for gastric cancer is tissue sectioning, which not only has a long detection cycle, but also brings strong discomfort and even great pain to patients, and also creates the risk of infection. Therefore, it is particularly important and urgent to establish a simple, fast, and non-invasive method to achieve early screening of gastric cancer.

[0003] Studies have shown that saliva can be used as a "mirror of the body" like blood to reflect the physiological and pathological state of an individual. Analysis and identification of the content of relevant biomarkers in human saliva is an important step in discovering and monitoring human health and disease. At the same time, studies have also shown that the content of D-amino acids (DAA) in the saliva of patients with early gastric cancer is significantly higher than that of normal people. The content of D-amino acids in the saliva of healthy people is 0-25.3μM D-alanine (D-Ala) / 0-11.3μM D-proline (D-Pro), and the content of D-amino acids in the saliva of gastric cancer patients is 126.3-285.3μM D-Ala / 46.1-114.5μM D-Pro. D-amino acids in saliva can be oxidized to α-keto acids and ammonia under the action of D-amino acid oxidase (DAAO), and a large amount of hydrogen peroxide (H2O2) is produced at the same time. Therefore, by detecting the content of D-amino acids in saliva, a simple, rapid and non-invasive method can be established to achieve early screening of gastric cancer.

[0004] At present, the main methods for detecting D-amino acids in saliva include surface enhanced Raman scattering sensors, liquid chromatography, biosensors and colorimetry. Among these methods, colorimetry is favored in clinical laboratory testing because of its advantages such as simple operation, rapidity, non-invasiveness, low cost and intuitive results. Its detection principle is: based on the fact that D-amino acid oxidase can oxidize D-proline and D-alanine in saliva to decompose into H2O2, combined with 3,3',5,5'-tetramethylbenzidine (TMB) colorimetric detection of D-amino acids in saliva. However, how to improve the detection sensitivity and ensure high selectivity is still the main bottleneck faced by colorimetry in clinical testing. Summary of the invention

[0005] In view of the above-mentioned shortcomings of the prior art, the object of the present invention is to provide a Pt / Ti3C2Tx A method, a kit and its application for detecting the content of D-amino acids using nanomaterials, using a Pt / Ti3C2T nanoparticle with localized surface plasmon resonance effect and peroxidase-like catalytic activity. x Nanomaterials are used as nanoprobes to construct a new colorimetric detection method for gastric cancer marker D-amino acids in saliva with high sensitivity and selectivity, breaking through the above bottleneck.

[0006] To achieve the above object and other related objects, the present invention provides a method for detecting the content of D-amino acids, comprising:

[0007] Incubating the sample to be tested with D-amino acid oxidase;

[0008] After incubation, Pt / Ti3C2T x Nanomaterials and a color developing substrate 3,3',5,5'-tetramethylbenzidine are reacted under laser irradiation; during the reaction, the Pt / Ti3C2T x The nanomaterial catalyzes the reaction of D-amino acid and D-amino acid oxidase to produce hydrogen peroxide, which is converted into reactive oxygen free radicals (ROS), and catalyzes the oxidation of the color-developing substrate 3,3',5,5'-tetramethylbenzidine into oxidized TMB (oxTMB);

[0009] After the reaction is completed, the content of D-amino acid in the sample to be tested is determined by colorimetric detection.

[0010] Furthermore, the D-amino acid includes D-proline and / or D-alanine.

[0011] Furthermore, during and after the incubation process, a buffer is added, and the buffer is selected from a phosphate buffer and / or an acetate buffer; preferably, the buffer is selected from a phosphate buffer with a pH of 5 to 6.8 and an acetate buffer with a pH of 3.5 to 4.5; more preferably, the buffer is selected from a phosphate buffer with a pH of 6.0 and an acetate buffer with a pH of 4.0.

[0012] Furthermore, during incubation, the pH is controlled to be 4 to 7.5, preferably 5 to 6.8, and more preferably 5 to 6.5.

[0013] Furthermore, the concentration of the color developing substrate 3,3',5,5'-tetramethylbenzidine is controlled at 0.1 to 0.35 mM, preferably 0.15 to 0.35 mM, and more preferably 0.15 to 0.30 mM.

[0014] Furthermore, the Pt / Ti3C2T xThe concentration of the nanomaterial is controlled at 10 to 60 μg / mL, preferably 10 to 40 μg / mL, and more preferably 10 to 35 μg / mL.

[0015] Furthermore, the laser irradiation reaction time is 1 to 15 minutes, preferably 3 to 12 minutes, and more preferably 4 to 8 minutes.

[0016] Furthermore, the laser is selected from near-infrared light, preferably 780 to 850 nm.

[0017] Furthermore, the Pt / Ti3C2T x Nanomaterial is Pt / Ti3C2T x Nanosheets, including two-dimensional nanosheets Ti3C2T x As well as the Ti3C2T2 nanosheets grown on the x Pt nanoparticles (Pt NPs) on .

[0018] Furthermore, the Pt / Ti3C2T x The preparation method of the nano material comprises: x The solution is mixed with a Pt source precursor for a reduction reaction; after the reduction reaction is completed, the mixture is centrifuged and the precipitate is washed to obtain Pt / Ti3C2T x Nanomaterials.

[0019] Furthermore, the Pt / Ti3C2T x The preparation method of the nano material comprises: x The solution is mixed with a Pt source precursor for a pre-reaction, and then a reducing agent is added to perform a reduction reaction. After the reduction reaction is completed, the mixture is centrifuged, and the precipitate is washed to obtain Pt / Ti3C2T x Nanomaterials.

[0020] Furthermore, the reducing agent is selected from any one of sodium borohydride, sodium citrate and ascorbic acid.

[0021] Furthermore, the Pt source precursor is selected from any one of chloroplatinic acid, chloroplatinic acid hexahydrate, sodium chloroplatinate, potassium chloroplatinate, ammonium chloroplatinate, platinum chloride and other soluble salts of platinum.

[0022] The present invention also provides a kit for detecting the content of D-amino acids, comprising: D-amino acid oxidase, Pt / Ti3C2T x A nano material and a color developing substrate, wherein the color developing substrate is 3,3',5,5'-tetramethylbenzidine.

[0023] Furthermore, the kit further comprises a buffer, and the buffer is selected from phosphate buffer and / or acetate buffer.

[0024] Furthermore, the kit further comprises a phosphate buffer with a pH of 5 to 6.8 and an acetate buffer with a pH of 3.5 to 4.5; preferably, the kit further comprises a phosphate buffer with a pH of 6.0 and an acetate buffer with a pH of 4.0.

[0025] The present invention also provides Pt / Ti3C2T x Application of nanomaterials in the preparation of a kit for detecting the content of D-amino acids, a gastric cancer marker, in saliva.

[0026] Furthermore, the kit for detecting the gastric cancer marker D-amino acid content in saliva also includes at least one of the following components: D-amino acid oxidase, a chromogenic substrate and a buffer; the chromogenic substrate is 3,3',5,5'-tetramethylbenzidine, the buffer is selected from phosphate buffer and / or acetate buffer, the phosphate buffer has a pH of 5 to 6.8, preferably 6.0, and the acetate buffer has a pH of 3.5 to 4.5, preferably 4.0.

[0027] Furthermore, the Pt / Ti3C2T x The nanomaterial has localized surface plasmon resonance effect and peroxidase-like catalytic activity. The Pt / Ti3C2T x The nanomaterial is used for catalyzing the conversion of hydrogen peroxide generated by the reaction of D-amino acid and D-amino acid oxidase into active oxygen free radicals, and catalyzing the oxidation of the color-developing substrate 3,3',5,5'-tetramethylbenzidine into an oxidized state TMB.

[0028] As mentioned above, the present invention is based on Pt / Ti3C2T x The method, kit and application of nanomaterials for detecting D-amino acid content have the following beneficial effects:

[0029] The present invention develops a two-dimensional nanosheet Ti3C2T x Composite nanomaterials Pt / Ti3C2T with small-sized Pt NPs grown on x , the material has a localized surface plasmon resonance effect and excellent POD-like activity, which can catalyze the conversion of H2O2 produced by the reaction of D-amino acids and D-amino acid oxidase into a large amount of active ROS, significantly accelerating the catalytic oxidation of the chromogenic substrate molecule TMB to oxTMB, and the above catalytic process can be further accelerated under laser (such as near-infrared light) irradiation. Based on this principle, the present invention has built a colorimetric platform with high sensitivity and selectivity for detecting the content of D-amino acids in human saliva, and applied the colorimetric detection platform to the detection of clinical saliva samples, achieving the expected effect. Therefore, the present invention provides a new path for rapid, simple, non-invasive and painless screening and diagnosis of gastric cancer in humans. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 It is shown as Pt / Ti3C2T in Example 1 of the present invention x Morphology and structural characteristics of Pt / Ti3C2T x Particle size distribution diagram of Pt / Ti3C2T x AFM image of nanosheets; (g) AFM image of Ti3C2T x 、Pt / Ti3C2T x Thickness distribution of nanosheets.

[0031] Figure 2 It is shown as Pt / Ti3C2T in Example 1 of the present invention x The photothermal performance and conversion efficiency characterization results are shown in Figure 1: (a) Pt / Ti3C2T with different concentrations x UV absorption spectra of different concentrations of Pt / Ti3C2T x tT curve after 10min irradiation (808nm, 2W / cm 2 ); (c) Pt / Ti3C2T laser irradiation with different power densities at 808 nm x (d) NIR laser on / off cycle six times, Pt / Ti3C2T x Photothermal stability test; (e) Pt / Ti3C2T x UV absorption spectra before and after 6 consecutive irradiations; (f) Pt / Ti3C2T x Linear fit plot of ln(q)-t during cooling.

[0032] Figure 3 It is shown as 808nm, 0.80W / cm in Example 1 of the present invention. 2 Under laser irradiation, the absorbance change value (ΔA 652 ) changes with different conditions: (a) pH value; (b) concentration of chromogenic substrate TMB; (c) material Pt / Ti3C2T x concentration; (d) reaction time.

[0033] Figure 4 It is shown that in Example 1 of the present invention, the photothermal enhancement of Pt / Ti3C2T xPOD-like activity was used to detect D-Ala: (a) UV-visible absorption spectrum of the system with increasing D-Ala concentration; (b) The absorbance change value ΔA of the corresponding solution in (a) at 652 nm 652 The curve of the change with the concentration c of D-Ala; (c) is the ΔA in (b) 652 Linear fitting plot between and c.

[0034] Figure 5 It is shown that in Example 1 of the present invention, the photothermal enhancement of Pt / Ti3C2T x POD-like activity of D-Pro was used to detect D-Pro: (a) UV-visible absorption spectrum of the system with the concentration of D-Pro; (b) The absorbance change value ΔA of the corresponding solution in (a) at 652 nm 652 The curve of the change with the concentration c of D-Pro; (c) is the ΔA in (b) 652 Linear fitting plot between and c.

[0035] Figure 6 It is shown in Example 1 of the present invention, based on Pt / Ti3C2T x Selectivity and anti-interference test results of the constructed colorimetric method for detecting DAA: (a) selectivity; (b) anti-interference. DETAILED DESCRIPTION

[0036] The following describes the embodiments of the present invention through specific examples, and those skilled in the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and the details in this specification can also be modified or changed in various ways based on different viewpoints and applications without departing from the spirit of the present invention.

[0037] One embodiment of the present invention provides a method for detecting the content of D-amino acids, comprising:

[0038] Incubating the sample to be tested with D-amino acid oxidase;

[0039] After incubation, Pt / Ti3C2T x Nanomaterials and a color developing substrate 3,3',5,5'-tetramethylbenzidine are reacted under laser irradiation; during the reaction, the Pt / Ti3C2T x The nanomaterial catalyzes the reaction of D-amino acid and D-amino acid oxidase to produce hydrogen peroxide, which is converted into active oxygen free radicals, and catalyzes the oxidation of the color-developing substrate 3,3',5,5'-tetramethylbenzidine into an oxidized state TMB;

[0040] After the reaction is completed, the content of D-amino acid in the sample to be tested is determined by colorimetric detection.

[0041] In some embodiments, the D-amino acid comprises D-proline and / or D-alanine.

[0042] In some embodiments, the sample to be tested includes but is not limited to saliva, a sample known to contain D-amino acids, an unknown sample, and the like.

[0043] The present invention utilizes Pt / Ti3C2T x Nanomaterials catalyze the conversion of H2O2 generated by the reaction of D-amino acids and D-amino acid oxidase into a large amount of active ROS, which can significantly accelerate the catalytic oxidation of the chromogenic substrate molecule TMB to oxTMB. At the same time, the above catalytic process can be further accelerated under laser irradiation. Based on this principle, the present invention establishes a simple, rapid, and non-invasive colorimetric method to detect the content of D-amino acids in saliva, which can not only achieve early screening of gastric cancer, but also improve the detection sensitivity and ensure high selectivity.

[0044] In some embodiments, a buffer is added during and after the incubation process, and the buffer is selected from phosphate buffer and / or acetate buffer; in some embodiments, the buffer is selected from phosphate buffer with a pH of 5 to 6.8 and acetate buffer with a pH of 3.5 to 4.5; in a specific embodiment, the buffer is selected from phosphate buffer with a pH of 6.0 and acetate buffer with a pH of 4.0.

[0045] In some embodiments, during incubation, the pH is controlled at 4 to 7.5, preferably 5 to 6.8, more preferably 5 to 6.5, for example 4, 5, 6, 6.5, 6.8, 7, 7.5, etc.

[0046] In some embodiments, the concentration of the chromogenic substrate 3,3',5,5'-tetramethylbenzidine is controlled at 0.1-0.35 mM, preferably 0.15-0.35 mM, more preferably 0.15-0.30 mM, for example 0.1, 0.15, 0.2, 0.25, 0.3, 0.35 mM, etc.

[0047] In some embodiments, the Pt / Ti3C2T x The concentration of the nanomaterial is controlled at 10-60 μg / mL, preferably 10-40 μg / mL, more preferably 10-35 μg / mL, for example 10, 20, 30, 40, 50, 60 μg / mL, etc.

[0048] In some embodiments, the laser irradiation reaction time is 1 to 15 min, preferably 3 to 12 min, more preferably 4 to 8 min, for example 1, 3, 6, 10, 12, 15 min, etc.

[0049] In some embodiments, the laser is selected from near infrared light, preferably 780-850 nm, such as 808 nm.

[0050] In order to further improve the detection sensitivity and speed up the detection speed, the present invention also optimizes the above detection conditions. At the same time, the determination and mutual coordination of the above detection conditions promote the application of the colorimetric detection method provided by the present invention in clinical detection and realize the rapid detection of the gastric cancer marker D-amino acid content in saliva.

[0051] In one embodiment, the Pt / Ti3C2T x Nanomaterial is Pt / Ti3C2T x Nanosheets, including two-dimensional nanosheets Ti3C2T x As well as the Ti3C2T2 nanosheets grown on the x In one embodiment, the two-dimensional nanosheet Ti3C2T x The thickness is 3-4 nm, and the particle size of the Pt NPs is 2.70 nm.

[0052] In one embodiment, the Pt / Ti3C2T x The preparation method of the nano material comprises: x The solution is mixed with a Pt source precursor for a reduction reaction; after the reduction reaction is completed, the mixture is centrifuged and the precipitate is washed to obtain Pt / Ti3C2T x Nanomaterials.

[0053] In one embodiment, the Pt / Ti3C2T x The preparation method of the nano material comprises: x The solution is mixed with a Pt source precursor for a pre-reaction, and then a reducing agent is added to perform a reduction reaction. After the reduction reaction is completed, the mixture is centrifuged, and the precipitate is washed to obtain Pt / Ti3C2T x Nanomaterials.

[0054] In some embodiments, the reducing agent includes but is not limited to sodium borohydride, sodium citrate, ascorbic acid, and the like.

[0055] In some embodiments, the Pt source precursor includes but is not limited to chloroplatinic acid, chloroplatinic acid hexahydrate, sodium chloroplatinate, potassium chloroplatinate, ammonium chloroplatinate, platinum chloride and other soluble salts of platinum.

[0056] In some embodiments, the pre-reaction and the reduction reaction are both carried out at room temperature, the pre-reaction time is 1 to 2.5 hours, and the reduction reaction time is 10 to 24 hours.

[0057] In the above embodiments / examples, the Ti3C2T x The solution is a two-dimensional nanosheet Ti3C2T x Solution, two-dimensional nanosheet Ti3C2T x A specific embodiment of the present invention provides a two-dimensional nanosheet Ti3C2T x The synthesis method comprises: a Ti3AlC2 (Ti3AlC2 can be purchased directly) precursor is etched with a mixed solution of LiF and HCl to form a multilayer Ti3C2T x , the layered Ti3C2T x Uniform dispersion to form a single-layer two-dimensional nanosheet Ti3C2T x .

[0058] The Pt / Ti3C2T used in the present invention x The nanomaterial is a two-dimensional nanosheet Ti3C2T x The composite nanomaterial is constructed by growing small-sized Pt NPs on the surface of the nanoparticle. The design idea is as follows: The present invention hopes to build a good colorimetric platform for detecting the content of D-amino acids in human saliva based on nanomaterials with POD-like catalytic activity. At the same time, since Pt NPs are widely used in various biological applications due to their excellent POD-like activity, the present invention chooses Pt NPs. However, precious metal nanoparticles are easy to aggregate. How to solve this problem is the challenge faced by the present invention. Ti3C2T x The extremely high dispersibility in aqueous solution, rich surface functional groups and different terminal types provide support for the reduction of metal ions and countless direct ion change sites, which makes it easier for metal nanoparticles to be inserted and anchored. This feature makes Ti3C2T x It can become a good substrate and carrier for the growth of small-particle metal ions, solving the problem of easy aggregation of Pt NPs. x It also has good photothermal properties. After irradiation with near-infrared light, hot electrons and hot holes will be generated. x The POD-like combination with Pt NPs can not only effectively promote the decomposition of H2O2 to produce a large amount of ROS, significantly accelerate the catalytic oxidation of TMB to oxTMB, but also further accelerate the above catalytic process under near-infrared light irradiation.

[0059] Another embodiment of the present invention provides a kit for detecting the content of D-amino acids, comprising: D-amino acid oxidase, Pt / Ti3C2T x A nano material and a color developing substrate, wherein the color developing substrate is 3,3',5,5'-tetramethylbenzidine.

[0060] In some embodiments, the kit further comprises a buffer selected from phosphate buffer and / or acetate buffer.

[0061] In some embodiments, the kit further comprises a phosphate buffer having a pH of 5 to 6.8 and an acetate buffer having a pH of 3.5 to 4.5; in a specific embodiment, the kit further comprises a phosphate buffer having a pH of 6.0 and an acetate buffer having a pH of 4.0.

[0062] It should be noted that the kit for detecting the content of D-amino acids provided in the above embodiments / examples and the method for detecting the content of D-amino acids provided in the above embodiments / examples belong to the same concept, and the detection principle, detection method and required conditions have been described in detail in the method implementation / examples and will not be repeated here.

[0063] Another embodiment of the present invention provides Pt / Ti3C2T x Application of nanomaterials in the preparation of a kit for detecting the content of D-amino acids, a gastric cancer marker, in saliva.

[0064] In some embodiments, the kit for detecting the content of gastric cancer marker D-amino acids in saliva further comprises at least one of the following components: D-amino acid oxidase, a chromogenic substrate and a buffer; the chromogenic substrate is 3,3',5,5'-tetramethylbenzidine, the buffer is selected from a phosphate buffer and / or an acetate buffer, the phosphate buffer has a pH of 5 to 6.8, and the acetate buffer has a pH of 3.5 to 4.5. In a specific embodiment, the phosphate buffer has a pH of 6.0, and the acetate buffer has a pH of 4.0.

[0065] In some embodiments, the Pt / Ti3C2T x The nanomaterial has localized surface plasmon resonance effect and peroxidase-like catalytic activity. The Pt / Ti3C2T x The nanomaterial is used for catalyzing the conversion of hydrogen peroxide generated by the reaction of D-amino acid and D-amino acid oxidase into active oxygen free radicals, and catalyzing the oxidation of the color-developing substrate 3,3',5,5'-tetramethylbenzidine into an oxidized state TMB.

[0066] The present invention proposes for the first time to combine Pt / Ti3C2T x The application of nanomaterials in the detection of D-amino acid content, a gastric cancer marker, in saliva has improved sensitivity and ensured high selectivity compared to traditional colorimetric detection methods, providing a new, rapid, simple and painless path for human screening and diagnosis of gastric cancer.

[0067] In addition, it should be noted that the Pt / Ti3C2T x The application of nanomaterials in the preparation of a kit for detecting the content of D-amino acids, a gastric cancer marker, in saliva belongs to the same concept as the method and kit for detecting the content of D-amino acids provided in the above-mentioned implementation modes / examples. The detection principle, detection method and required conditions have been described in detail in the method implementation modes / examples and will not be repeated here.

[0068] The following specific examples are given to illustrate the present invention in detail. It should also be understood that the following examples are only used to specifically illustrate the present invention and cannot be understood as limiting the scope of protection of the present invention. Some non-essential improvements and adjustments made by those skilled in the art based on the above content of the present invention belong to the scope of protection of the present invention. The specific process parameters and the like in the following examples are also only examples within a suitable range, that is, those skilled in the art can make a selection within a suitable range according to the description herein, and are not limited to the specific values ​​​​exemplified below.

[0069] Example 1

[0070] 1. Pt / Ti3C2T x Preparation of nanosheets

[0071] (1) Weigh 1.2 g of LiF and dissolve it in 15 mL of 12 M HCl and stir for 10 min.

[0072] (3) Add 1.0 g Ti3AlC2 to the above solution and stir at 40°C for 30 h.

[0073] (3) After stopping stirring, the resulting suspension was centrifuged at 6000 rpm for 5 min and washed with water several times until the pH of the supernatant was 6 to 7. The sample was then placed in a vacuum drying oven at 60°C and dried for 24 h.

[0074] (4) The above sample was ground into powder, dissolved in distilled water at a ratio of 10 mg / mL, and ultrasonically exfoliated for 3.0 h using an ultrasonic cell crusher at 300 W under room temperature and argon atmosphere. The sample was then centrifuged at 3500 rpm for 1 h, and the upper liquid (i.e., Ti3C2T x Clear liquid) bottled.

[0075] (5) Add 10 mg of polyvinyl pyrrolidone into a 20 mL glass bottle, then add 4.5 mL of Ti3C2T x The clear solution (2 mg / mL), 5 mL of anhydrous ethanol and 0.5 mL of H2PtCl6·6H2O (0.1 M) were completely mixed by ultrasonication and stirred at room temperature for 2 h.

[0076] (6) 1 mL of freshly prepared 0.1 M frozen NaBH4 solution was quickly added to the above solution and stirred for 15 h. The suspension was centrifuged at 12000 rpm for 10 min and washed three times with a mixture of ethanol and water (1:1) to obtain Pt / Ti3C2T x , adjust the volume to 20mL for later use.

[0077] Figure 1 The Pt / Ti3C2T prepared in this example is shown x The morphology and structural characteristics of Pt / Ti3C2T were successfully prepared in this example. x At the same time, from the Pt / Ti3C2T x From the particle size distribution diagram, we can see that the particle size of Pt NPs is about 2.70nm. x AFM images of the nanosheets revealed the presence of Ti3C2T x With Pt / Ti3C2T x The thickness distribution of the nanosheets, as shown in Figure (g), is 3 to 4 nm.

[0078] 2. Pt / Ti3C2T x Photothermal performance evaluation

[0079] (1) 1.7 mL of Pt / Ti3C2T x (50, 80, 100, 150 μg / mL) were added to a 48-well plate and the power density was 2 W / cm 2 The samples were irradiated with 808 nm laser for 10 min.

[0080] (2) 1.7 mL of Pt / Ti3C2T x (100 μg / mL) was added to a 48-well plate and illuminated with an 808 nm laser at 1, 1.5, 2, and 2.5 W / cm 2 Irradiation was performed for 10 min at four different power densities.

[0081] (3) On / Off cycle irradiation experiment on Pt / Ti3C2T x The photothermal stability of Pt / Ti3C2T was evaluated. The specific steps are as follows: 1.7 mL of Pt / Ti3C2T x (100 μg / mL) was added to a 48-well plate and illuminated with an 808 nm laser at 2 W / cm 2Irradiate at the same power density for 10 minutes, turn off the laser for 15 minutes, turn on the laser again, irradiate again at the same power density for 10 minutes, and turn off the laser. Repeat the same operation 6 times. The temperature is recorded with a thermal imager every 20 seconds.

[0082] (4) 1.7 mL of Pt / Ti3C2T x (100 μg / mL) was added to a 48-well plate and illuminated with an 808 nm laser at 2 W / cm 2 The power density was irradiated for 10 min, the laser was turned off for 10 min, and the corresponding photothermal response curve was drawn; and the Pt / Ti3C2T x The light-to-heat conversion effect was calculated using the ln(q)-t linear fitting curve during the cooling period.

[0083] like Figure 2 As shown in ab, the temperature change of Pt / Ti3C2T under 808nm laser irradiation x The concentration of Pt / Ti3C2T x As the concentration of increases, the corresponding temperature also increases. Figure 2 As shown in c, when Pt / Ti3C2T x When the concentration is 100 μg / mL, as the irradiation power density increases, the temperature of the corresponding solution also gradually increases, which indicates that the temperature of the solution is dependent on the power density. Figure 2 As shown in d, by measuring the corresponding concentration of Pt / Ti3C2T x At 808nm (2W / cm 2 ) laser irradiation conditions, after 6 cycles of illumination-cooling, the temperature increase and decrease are almost the same, which indicates that Pt / Ti3C2T x It has excellent light and heat stability. Figure 2 As shown in e and 2f, the formula is used to calculate the 2 ) Under laser irradiation conditions, Pt / Ti3C2T x The photothermal conversion efficiency during the heating and cooling process was η = 63.24%, which indicates that the two-dimensional sheet material Pt / Ti3C2T x It has a large specific surface area and high utilization rate of near-infrared light, producing a good photothermal effect.

[0084] 3. Optimization of D-amino acid (D-alanine (D-Ala) / D-proline (D-Pro)) detection conditions

[0085] At 808nm, 0.80W / cm 2 Under the laser irradiation condition, the absorbance change value (ΔA 652) varies with the following conditions, the results are as follows Figure 3 shown.

[0086] (1) Optimize the pH value of incubation with D-amino acids. The pH values ​​used in the optimization process are 4, 5, 6, 6.5, 6.8, 7, and 7.5.

[0087] (2) Optimize the concentration of the chromogenic substrate 3,3',5,5'-tetramethylbenzidine. The concentrations during the optimization process were 0.1, 0.15, 0.2, 0.25, 0.3, and 0.35 mM. Figure 3 As shown in (b), the optimal concentration of the chromogenic substrate 3,3',5,5'-tetramethylbenzidine is 0.30 mM.

[0088] (3) Optimization of Pt / Ti3C2T x The concentrations in the optimization process were 10, 20, 30, 40, 50, and 60 μg / mL. Figure 3 (c) shows Pt / Ti3C2T x The optimal concentration is 30 μg / mL.

[0089] (4) Optimize the laser irradiation reaction time. The time in the optimization process is 1, 3, 6, 10, 12, and 15 minutes respectively. Figure 3 As shown in (d), the optimal reaction time of laser irradiation is 6 minutes.

[0090] 4. D-amino acid (D-alanine (D-Ala) / D-proline (D-Pro)) detection

[0091] (1) When detecting D-Ala, 200 μL of phosphate buffered saline (PBS) buffer (pH 6), 10 μL of DAAO (10 U / mL) and 200 μL of different concentrations of D-Ala were taken to form solution A and incubated at 37°C for 60 min.

[0092] (2) Take 200 μL of solution A after incubation, and then add 900 μL acetate buffer (pH 4.0), 300 μL Pt / Ti3C2T x (150 μg / mL), 300 μL TMB. 2 ) for 6 min, and then record the UV-visible absorption spectrum. The detection of D-Pro is the same as the above method, except that D-Pro is used instead of D-Ala.

[0093] like Figure 4 As shown, the photothermal enhancement of Pt / Ti3C2T xThe POD-like activity of D-Ala was detected by 808nm (0.80W / cm 2 ) After 6 min of laser irradiation, the absorbance at 652 nm gradually increased with the increase of DAA concentration (4a), and the absorbance change value at 652 nm (ΔA 652 ) With the increase of D-Ala concentration, it gradually reached a platform (4b) and established a linear relationship. By fitting its linear equation, ΔA = 4.76 × 10 -4 c+0.0165, detection range is 20~500μM, detection limit is 14.1μM (4c). Figure 5 As shown, the photothermal enhancement of Pt / Ti3C2T x The POD-like activity of D-Pro was used to detect D-Pro. Similarly, it was found that as the concentration of D-Pro increased, the absorbance at 652 nm gradually increased with the increase of D-Pro concentration (5a) and reached a corresponding platform ( Figure 5 b), the corresponding linear equation ΔA=4.26×10 -4 c+0.0141, its detection range is 25~600μM, and its detection limit is 15.8μM (5c).

[0094] 5. D-amino acid selectivity and anti-interference ability test:

[0095] Selectivity: The selectivity and anti-interference ability of amino acids and ions contained in saliva are tested to evaluate the Pt / Ti3C2T x The feasibility of the constructed colorimetric method for detecting D-amino acids in saliva was investigated. First, the selectivity of the above reaction system was tested by using amino acids such as DL-aspartic acid, L-arginine, L-tryptophan, L-threonine, L-phenylalanine, L-alanine, L-proline, L-histidine, L-glutamic acid, and L-glycine, and Mg 2+ , Ca 2+ 、Zn 2+ , K + 、Na + 、Cl-、SO4 2- 、HCO3 - , sodium carboxymethyl cellulose (SCC), and methyl parahydroxybenzoate (MPHB) were used to replace D-amino acids in the system. It was found that the absorbance change at 652 nm was less than that of D-Pro and D-Ala, indicating that the method for detecting D-amino acids proposed in this study has good selectivity ( Figure 6 a).

[0096] Anti-interference: Under the same conditions as above, the above interfering substances were added to the reaction system containing D-Pro and D-Ala at a concentration 10 times that of D-amino acids. It was found that the ΔA of oxTMB 652 There is no significant fluctuation in the change value, indicating that this method has good anti-interference ability in detecting D-amino acids ( Figure 6 b).

[0097] The above embodiments are merely illustrative of the principles and effects of the present invention, and are not intended to limit the present invention. Anyone familiar with the art may modify or alter the above embodiments without departing from the spirit and scope of the present invention. Therefore, all equivalent modifications or alterations made by a person of ordinary skill in the art without departing from the spirit and technical concept disclosed by the present invention shall still be covered by the claims of the present invention.

Claims

1. A method for detecting D-amino acid content for non-disease diagnosis or treatment purposes, characterized in that: include: Incubating the sample to be tested with D-amino acid oxidase; After incubation, Pt / Ti3C2T x Nanomaterials and a color developing substrate 3,3',5,5'-tetramethylbenzidine are reacted under laser irradiation; during the reaction, the Pt / Ti3C2T x The nanomaterial catalyzes the reaction of D-amino acid and D-amino acid oxidase to produce hydrogen peroxide, which is converted into active oxygen free radicals, and catalyzes the oxidation of the color-developing substrate 3,3',5,5'-tetramethylbenzidine into an oxidized state TMB; After the reaction is completed, the content of D-amino acid in the sample to be tested is determined by colorimetric detection.

2. The method according to claim 1, characterized in that: The D-amino acids include D-proline and / or D-alanine.

3. The method according to claim 1, characterized in that: The detection condition is selected from at least one of the following conditions I to VI: Ⅰ. During and after the incubation, a buffer is added, wherein the buffer is selected from a phosphate buffer and / or an acetate buffer; Ⅱ. During incubation, the pH is controlled at 4-7.5; III. The concentration of the color developing substrate 3,3',5,5'-tetramethylbenzidine is controlled at 0.1-0.35 mM; IV. The Pt / Ti3C2T x The concentration of nanomaterials is controlled at 10-60 μg / mL; V. The laser irradiation reaction time is 1 to 15 minutes; VI. The laser is selected from near infrared light.

4. The method according to any one of claims 1 to 3, characterized in that: The Pt / Ti3C2T x Nanomaterial is Pt / Ti3C2T x Nanosheets, including two-dimensional nanosheets Ti3C2T x As well as the Ti3C2T2 nanosheets grown on the x Pt nanoparticles on .

5. The method according to claim 4, characterized in that: The Pt / Ti3C2T x The preparation method of the nano material comprises: x The solution is mixed with a Pt source precursor for a reduction reaction; after the reduction reaction is completed, the mixture is centrifuged and the precipitate is washed to obtain Pt / Ti3C2T x Nanomaterials.

6. A kit for detecting the content of D-amino acids, characterized in that: include: D-amino acid oxidase, Pt / Ti3C2T x Nanomaterial and color developing substrate, wherein the color developing substrate is 3,3',5,5'-tetramethylbenzidine.

7. The kit according to claim 6, characterized in that: The kit further comprises a buffer, which is selected from phosphate buffer and / or acetate buffer.

8. The kit according to claim 7, characterized in that: The kit also includes a phosphate buffer solution with a pH of 5 to 6.8 and an acetate buffer solution with a pH of 3.5 to 4.

5. 9.Pt / Ti3C2T x Application of nanomaterials in the preparation of a kit for detecting the content of D-amino acids, a gastric cancer marker, in saliva.

10. The use according to claim 9, characterized in that: The kit for detecting the content of gastric cancer marker D-amino acids in saliva also includes at least one of the following components: D-amino acid oxidase, a colorimetric substrate and a buffer; The color developing substrate is 3,3',5,5'-tetramethylbenzidine, and the buffer is selected from phosphate buffer and / or acetate buffer.

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