Method for jointly detecting creatinine and human serum albumin based on fluorescence colorimetric double signals

Abstract

The invention discloses a method for jointly detecting creatinine and human serum albumin based on fluorescence colorimetric double signals, in the method, creatinine and molybdenum diselenide are used as cocatalysts and Cu < 2 + > are used for co-catalyzing hydrogen peroxide to decompose and etch Au (at) Ag NRs, and solutions present different colors. Free-state silver ions generated by etching the silver shell layer are reduced by taking the human serum albumin as a template in the presence of a reducing agent to generate Ag NCs with fluorescence, so that creatinine and human serum albumin are sequentially detected through colorimetric fluorescence double signals, cross interference is avoided, and the advantages of multi-target detection, high sensitivity, simple operation, low cost and the like are achieved.

Description

Technical Field

[0001] The present invention belongs to the technical fields of nanomaterials science and optical detection, and relates to a method for jointly detecting creatinine and human serum albumin based on fluorescence and colorimetric dual signals. Background Art

[0002] Colorimetric visual sensors are simple and convenient and can be used for on-site instant detection, which is a common detection method. However, traditional colorimetric visual methods detect based on the color depth of the chromogenic substrate, and the color is relatively single, making it difficult for naked-eye quantitative research. Due to their unique local surface plasmon resonance optical properties, noble metal nanomaterials cause rich and bright color changes in the macroscopic solution and are widely used. Among them, colorimetric visualization based on noble metal etching has rich color changes compared with aggregation and growth methods and is widely used in constructing multicolor visual sensors without being affected by the substrate nanoparticles. However, in the detection of multiple targets by current multicolor visual colorimetric sensors, the signals (such as color or spectral changes) triggered by different targets have small signal discrimination and can only be semi-quantitatively studied, unable to meet the application scenarios with high requirements for detection accuracy.

[0003] In addition, some multi-signal detection methods (such as colorimetric and fluorescence dual-mode detection) have cross-interference between different signals, affecting the accuracy and reliability of the detection results. For example, traditional methods may not be able to effectively separate the contributions of different targets to the signals, resulting in detection errors.

[0004] There are also some detection methods that involve complex nanomaterial synthesis, multi-step reactions, or expensive instrument equipment, resulting in cumbersome operations and high costs, and it is difficult to meet the requirements for simplicity and economy in practical applications.

[0005] Therefore, how to provide a simple and accurate method to improve the discrimination of multi-target detection signals has great research significance. Summary of the Invention

[0006] The purpose of the present invention is to provide a method for jointly detecting creatinine and human serum albumin based on fluorescence and colorimetric dual signals in view of the deficiencies of the prior art. Among them, Cu 2+ combines with creatinine to have certain peroxidase-like properties, and combines with molybdenum diselenide as a co-catalyst to improve the catalytic reaction efficiency, constructs a reaction for etching Au@Ag NRs (gold-core silver-shell nanorods), generates rich color changes, and performs multicolor visual detection on creatinine. The silver ions generated after further etching synthesize fluorescent silver nanoclusters (Ag NCs) using human serum albumin as a template, and perform fluorescence quantitative detection on human serum albumin.

[0007] To achieve the above purpose, the present invention adopts the following technical solutions:

[0008] Step (1): Mix creatinine, molybdenum diselenide, and copper chloride with different concentrations evenly in a buffer solution, add hydrogen peroxide and TMB (3,3',5,5'-tetramethylbenzidine), mix evenly, and incubate at room temperature for 5 - 30 minutes to obtain mixture A1;

[0009] Step (2): Add CTAB (cetyltrimethylammonium bromide) and Au@AgNRs to mixture A1, mix evenly, and incubate at room temperature for 5 - 30 minutes to obtain mixture A2;

[0010] Step (3): Measure the absorption spectrum of Au@AgNRs using an ultraviolet spectrophotometer, and detect creatinine with different concentrations through the ultraviolet spectrum and color;

[0011] Step (4): Add an excessive amount of creatinine to mixture A2 to completely etch Au@Ag NRs, add human serum albumin, stir vigorously for 1 - 10 minutes, then add NaOH, mix evenly, and let it stand at room temperature for 10 - 40 minutes to obtain mixture A3;

[0012] Step (5): Add NaBH4 to mixture A3, mix evenly, and react at 85°C for 20 - 80 minutes to obtain a mixture A4 containing AgNCs;

[0013] Step (6): Measure the fluorescence spectrum of Ag NCs using a fluorescence spectrophotometer, and detect human serum albumin with different concentrations through the fluorescence intensity.

[0014] Preferably, in step (1), the concentration of molybdenum diselenide is 0.1 - 1 mg / mL, the concentration of copper chloride is 2 - 20 mM, the concentration of hydrogen peroxide is 10 - 50 mM, and the concentration of TMB is 1 - 10 mM.

[0015] Preferably, the buffer solution used is Britton-Robinson buffer solution (B-R buffer).

[0016] Preferably, in step (1), the volume ratio of creatinine, molybdenum diselenide, copper chloride, hydrogen peroxide, and TMB is 2:2:1:(2:-2.5), more preferably 2:2:1:2:2.

[0017] Preferably, in step (2), the concentration of CTAB is 0.1 - 1 M.

[0018] Preferably, in step (2), the volume ratio of mixture A1, CTAB, and Au@Ag NRs is 17:(3 - 5):5, more preferably 17:3:5.

[0019] Preferably, in step (4), the concentration of NaOH is 0.1 - 5 M.

[0020] Preferably, in step (4), the volume ratio of the mixed solution A2, human serum albumin, and NaOH is 25:(10 - 12):(1 - 3), and more preferably 25:10:1.

[0021] Preferably, in step (5), the concentration of NaBH4 is 0.1 - 20 mM.

[0022] Preferably, in step (5), the volume ratio of the mixed solution A3 and NaBH4 is 23:(4 - 6), and more preferably 23:5.

[0023] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0024] (1) The present invention designs a fluorescence - colorimetric dual - signal combined detection system. Using Au@Ag NRs as the etching material, creatinine and molybdenum diselenide are used to synergistically etch Au@Ag NRs to reduce and generate fluorescent Ag NCs. Silver nanomaterials have a low reduction potential, exhibit stronger surface plasmon resonance, and the reaction is more sensitive; while gold nanomaterials as the core ensure the high stability of the material.

[0025] (2) The present invention uses a fluorescence - colorimetric dual - mode to achieve sequential detection of multiple targets through silver ions. Compared with a single colorimetric signal, it improves the signal discrimination, enhances the detectability, and effectively solves the problems of low signal discrimination, difficult quantification, cross - interference, and complex operation in the above - mentioned prior art. Brief Description of the Drawings

[0026] Figure 1 is the TEM image of Au NRs, and the scale bar is 50 nm.

[0027] Figure 2 is the TEM image of Au@Ag NR, and the scale bar is 50 nm.

[0028] Figure 3 is the UV - Vis absorption change of Au@Ag NR after etching for different times.

[0029] Figure 4 is the UV - Vis absorption change of Au@Ag NRs etched by hydrogen peroxide with different concentrations.

[0030] Figure 5 is the UV - Vis absorption change of Au@Ag NRs etched after reacting with creatinine at different concentrations.

[0031] Figure 6 is the color change of Au@Ag NRs etched after reacting with creatinine at different concentrations.

[0032] Figure 7 is the fluorescence spectrum change caused by reacting with human serum albumin at different concentrations. Detailed implementation manners

[0033] As described above, in view of the deficiencies of the prior art, the inventors of this case, through long-term research and a large number of practices, have proposed the technical solution of the present invention, and its main bases include at least:

[0034] The present invention uses the color change cascade generated by etching Au@Ag NRs to generate a reaction of Ag NCs with fluorescence, and sequentially detects creatinine and human serum albumin. The present invention detects creatinine according to the different colors and ultraviolet peaks shown in the results, and detects human serum albumin according to the different fluorescence spectrum intensities, which is simple, convenient, efficient and sensitive.

[0035] In order to make the purpose, technical solution and advantages of the present invention clearer, the following further details the present invention in conjunction with the drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention. The reagents used therein, including serum, can be obtained by conventional commercial purchases without special instructions.

[0036] The specific implementation scheme of the present invention is as follows:

[0037] Step (1): Mix 10 μL of creatinine with different concentrations, 10 μL of molybdenum diselenide and 5 μL of copper chloride with a concentration of 10 mM evenly in 125 μL of buffer solution, add 10 μL of hydrogen peroxide with a concentration of 40 mM and 10 μL of TMB with a concentration of 5 mM and mix evenly, and incubate at room temperature for 5 to 30 minutes to obtain a mixed solution A1;

[0038] Step (2): Add 30 μL of CTAB with a concentration of 0.1 M and 50 μL of Au@Ag NRs to the mixed solution A1, mix evenly and incubate at room temperature for 5 to 30 minutes to obtain a mixed solution A2;

[0039] Step (3): Use a UV spectrophotometer to measure the absorption spectrum of Au@AgNRs, and detect creatinine with different concentrations through the UV spectrum and color.

[0040] Step (4): Add human serum albumin to the mixed solution A2 after the Au@Ag NRs are completely etched by adding an excessive amount of creatinine in step (1), stir vigorously for 1 to 10 minutes, then add 10 μL of NaOH, mix evenly and let stand at room temperature for 10 to 40 minutes to obtain a mixed solution A3;

[0041] Step (5): Add 100 μL of NaBH4 to the mixed solution A3 and mix evenly, and react at 85 °C for 20 to 80 minutes to obtain a mixed solution A4;

[0042] Step (6): Measure the fluorescence spectrum of Ag NCs using a fluorescence spectrophotometer, and detect human serum albumin at different concentrations through fluorescence intensity.

[0043] In addition, it should be noted that the specific embodiments of the present invention described below do not limit the protection scope of the present invention. Any other corresponding changes and deformations made according to the technical concept of the present invention should be included in the protection scope of the claims of the present invention.

[0044] Example 1

[0045] Preparation of Au@AgNRs

[0046] (1) Synthesize Au NRs according to the seed growth method.

[0047] 1.1 Prepare the seed solution: Add 0.25 mL of 0.01 M chloroauric acid solution and 0.60 mL of 0.01 M sodium borohydride solution prepared with ice water to 9.75 mL of 0.10 M cetyltrimethylammonium bromide solution in sequence, stir vigorously at 1200 rpm for 2 minutes, and let it stand at 30 °C for at least 120 min for standby.

[0048] 1.2 Prepare the growth solution: Add 6 mL of 0.01 M chloroauric acid solution, 1.2 mL of 0.01 M silver nitrate solution, 0.96 mL of 0.1 M ascorbic acid solution and 2.4 mL of 1 M hydrochloric acid solution to 120 mL of 0.10 M cetyltrimethylammonium bromide solution in sequence under the condition of 27 °C water bath.

[0049] 1.3 Add 255 μL of the prepared seed solution to the growth solution under the condition of 27 °C water bath, and let it stand for more than 6 h.

[0050] Figure 1 This is the TEM characterization of the obtained Au NRs.

[0051] (2) Synthesize Au@Ag NRs

[0052] Centrifuge the synthesized Au NRs twice at 8000 rpm for 10 min. After the second centrifugation, disperse them evenly in the same volume of 80 mM CTAC solution. Take 10 mL of the dispersed AuNRs, add 0.4 mL of 10 mM AgNO3 and 0.3 mL of 0.1 M AA in sequence, react at 60 °C for 4 h to coat the Ag shell. After the reaction is completed, centrifuge twice at 7000 rpm for 10 min for concentration and standby.

[0053] Figure 2 This is the TEM characterization of the obtained Au@Ag NRs.

[0054] Example 2

[0055] To increase the reaction sensitivity and accuracy, the experimental conditions of the method for the combined detection of creatinine and human serum albumin based on fluorescence colorimetric dual signals were optimized by the single variable method. Since the etching time is directly related to the etching degree, and the hydrogen peroxide concentration is directly related to the degree of etching of Au@AgNRs, the etching time and hydrogen peroxide concentration are two conditions that must be optimized for the present invention.

[0056] Optimization of etching time:

[0057] Copper chloride, creatinine, molybdenum diselenide, hydrogen peroxide and TMB were successively added to the B-R buffer solution for the catalytic oxidation of TMB reaction. After the reaction was complete, CTAB and Au@AgNRs were added for etching for 1, 3, 5, 7, 10, 15 min. The absorption spectrum was measured by an ultraviolet spectrophotometer and the color difference was observed with the naked eye. The optimal etching time was determined to be 7 min.

[0058] Figure 3 It is the ultraviolet change of the etching degree of Au@Ag NRs at different reaction times. As the time prolongs, the etching degree of Au@AgNRs increases continuously and the ultraviolet peak continuously blue-shifts. In order to ensure the complete etching of Au@Ag NRs, a reaction time of 7 min was selected as the subsequent reaction condition.

[0059] Optimization of hydrogen peroxide concentration:

[0060] Copper chloride, creatinine, molybdenum diselenide, hydrogen peroxide and TMB were successively added to the B-R buffer solution for the catalytic oxidation of TMB reaction. The hydrogen peroxide concentration was (1, 5, 10, 20, 40, 60 mM). The absorption spectrum was measured by an ultraviolet spectrophotometer and the color difference was observed with the naked eye. The optimal hydrogen peroxide concentration was determined to be 40 mM.

[0061] Figure 4 It is the ultraviolet absorbance change of TMB oxidation at different hydrogen peroxide concentrations. As the hydrogen peroxide concentration increases, more reactive oxygen species are generated, promoting the catalytic TMB reaction, resulting in an increase in the amount of oxidized TMB and an increase in the absorbance at 652 nm of ultraviolet light, reaching a plateau at 40 mM. A hydrogen peroxide concentration of 40 mM was selected as the subsequent reaction condition.

[0062] Example 3

[0063] 10 mM of copper chloride, different concentrations of creatinine, molybdenum diselenide, 40 mM of hydrogen peroxide and 5 mM of TMB were successively added to the B-R buffer solution and reacted at room temperature for 5 min. After completion, CTAB and Au@Ag NRs were added for etching reaction to detect creatinine. The absorption spectrum was measured by an ultraviolet spectrophotometer, and the color change could also be distinguished with the naked eye.

[0064] Figure 5 and Figure 6 are the UV and color changes of the etching degree of Au@Ag NRs by creatinine at different concentrations. It can be seen that different concentrations of creatinine lead to different degrees of etching of Au@Ag NRs. The UV peak first blue-shifts to 565 nm and then red-shifts to 615 nm. At the same time, the color of the solution changes from yellow-brown to light green.

[0065] Excessive creatinine is added to completely etch Au@Ag NRs to produce the maximum amount of Ag + , different concentrations of human serum albumin are added to the solution after the etching reaction and stirred vigorously for 2 min. Then, 1 M NaOH is added to adjust the pH. After standing at room temperature for 30 min, 10 mM NaBH4 is added to reduce Ag + , and the reaction is carried out at 85 °C for 1 h, and the fluorescence spectrum is measured by a fluorescence spectrophotometer.

[0066] Figure 7 is the change in the fluorescence intensity of the generated Ag NCs by different concentrations of human serum albumin. Different concentrations of human serum albumin result in different amounts of synthesized Ag NCs, thus affecting the fluorescence intensity.

[0067] Application Example 1

[0068] Detect creatinine in serum according to the method in Example 3 above:

[0069] The obtained serum is directly diluted 100 times for standby. In the buffer solution, serum, 10 mM copper chloride, molybdenum diselenide, 40 mM hydrogen peroxide, and 5 mM TMB are added in sequence and reacted at room temperature for 5 min. After completion, CTAB and Au@Ag NRs are added for the etching reaction to detect creatinine. The absorption spectrum is measured by a UV spectrophotometer, and the color change can also be distinguished by the naked eye.

[0070] The above embodiments are not limitations on the present invention. The present invention is not limited to the above embodiments. As long as it meets the requirements of the present invention, it belongs to the protection scope of the present invention.

Claims

1. A method for the combined detection of creatinine and human serum albumin based on fluorescence colorimetric dual signals, characterized in that, The method includes the following steps: Step (1): Mix creatinine with different concentrations, molybdenum diselenide, and copper chloride evenly in a buffer solution, add hydrogen peroxide and TMB and mix evenly, and incubate at room temperature for 5 - 30 minutes to obtain mixture A1; Step (2): Add CTAB and Au@Ag NRs to mixture A1, mix evenly and incubate at room temperature for 5 - 30 minutes to obtain mixture A2; Step (3): Measure the absorption spectrum of Au@AgNRs using an ultraviolet spectrophotometer, and detect creatinine with different concentrations through the ultraviolet spectrum and color; Step (4): Add an excessive amount of creatinine to mixture A2 to completely etch Au@Ag NRs, add human serum albumin and stir vigorously for 1 - 10 minutes, then add NaOH, mix evenly and let stand at room temperature for 10 - 40 minutes to obtain mixture A3; Step (5): Add NaBH4 to mixture A3 and mix evenly, react at 85 °C for 20 - 80 minutes to obtain a mixture A4 containing AgNCs; Step (6): Measure the fluorescence spectrum of Ag NCs using a fluorescence spectrophotometer, and detect human serum albumin with different concentrations through the fluorescence intensity.

2. The method according to claim 1, wherein In step (1), the concentration of molybdenum diselenide is 0.1 - 1 mg / mL, the concentration of copper chloride is 2 - 20 mM, the concentration of hydrogen peroxide is 10 - 50 mM, and the concentration of TMB is 1 - 10 mM.

3. The method according to claim 1 or 2, characterized in that, In step (1), the volume ratio of creatinine, molybdenum diselenide, copper chloride, hydrogen peroxide, and TMB is 2:2:1:(2:-2.5).

4. The method according to claim 1, wherein In step (2), the concentration of CTAB is 0.1 - 1 M.

5. The method according to claim 1 or 4, characterized in that, In step (2), the volume ratio of mixture A1, CTAB, and Au@AgNRs is 17:(3 - 5):

5.

6. The method according to claim 1, wherein In step (4), the concentration of NaOH is 0.1 - 5 M.

7. The method according to claim 1 or 6, characterized in that, In step (4), the volume ratio of mixture A2, human serum albumin, and NaOH is 25:(10 - 12):(1 - 3).

8. The method according to claim 1, characterized in that In step (5), the concentration of NaBH4 is 0.1 - 20 mM.

9. The method according to claim 1 or 8, characterized in that In step (5), the volume ratio of mixture A3 and NaBH4 is 23:(4 - 6).

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