Method for quantitatively detecting creatinine in serum by pH-assisted ratio type SERS (Surface Enhanced Raman Scattering)

Abstract

The invention discloses a method for quantitatively detecting creatinine in serum by pH-assisted ratio type SERS (Surface Enhanced Raman Scattering), which comprises the following steps: preparing silver-coated gold core-shell nanoparticles Au (at) MPA (at) Ag NPs with internal standard thiohydracrylic acid as an SERS substrate, and normalizing an SERS signal of the creatinine by using an internal standard signal. In addition, the pH of the creatinine is adjusted to enhance the adsorption effect of the creatinine and the nanoparticles, so that a Raman signal is enhanced. The detection method disclosed by the invention has the advantages of effectively eliminating interference of environmental factors, non-uniformity of samples, high dependence on a substrate and the like. Moreover, 2.5 mol / L of creatinine can be detected by the creatinine detection method based on the pH-assisted ratio type SERS technology, the adding standard recovery rate in a serum sample is 98.1%-107.4%, the relative standard deviation is smaller than 5.8%, and the quantitative accuracy of creatinine detection is improved.

Description

TECHNICAL FIELD

[0001] The application relates to a method for pH-assisted ratio type SERS quantitative detection of creatinine in serum and belongs to the technical field of analysis and detection. BACKGROUND

[0002] Creatinine (CR) as an important biomarker for evaluating renal function has been widely concerned. If the concentration of blood creatinine is abnormal, it can be used to indicate various pathological states. When the blood creatinine level is significantly higher than the normal level, it means that the ability of glomerular filtration of blood is decreased, which may be acute kidney injury or chronic kidney disease (CKD) becoming more serious. When the blood creatinine level is lower than the normal range, it may be muscle atrophy or metabolic disease. The combination of blood creatinine concentration and thyroid function index can be used as an auxiliary diagnosis of related diseases such as endocrine disorders. Creatinine is an important indicator reflecting renal function, which is closely related to the nutritional status of the human body, muscle content and the occurrence of various chronic diseases. Therefore, the detection of creatinine is widely used in clinical practice, which provides important information for doctors about the renal function and overall health status of patients.

[0003] At present, the detection methods of creatinine include picric acid method, enzyme method, electrochemical method and spectroscopic method. However, these methods have poor specificity and low sensitivity, the enzyme method has poor stability and high detection cost, is interfered by other substances and is limited by complex near-infrared spectroscopy modeling.

[0004] The patent with the publication number CN119409226A "Method for synthesizing porous carbon / vanadium sulfide nanometer enzyme assisted by yeast and application thereof in detection of serum creatinine" discloses a method for synthesizing porous carbon / vanadium sulfide nanometer enzyme assisted by yeast and the application thereof in detection of serum creatinine. However, this method has extremely high requirements for the stability and temperature control precision of equipment, and it is difficult to ensure the accurate reproduction of experimental conditions each time. The patent with the publication number CN119972099A "Fe3O4-V2O5 ultra-micro composite foam nanometer enzyme for detection of serum creatinine and preparation method and application thereof" discloses a preparation method of Fe3O4-V2O5 ultra-micro composite foam nanometer enzyme for detection of serum creatinine. The method achieves creatinine detection by preparing Fe3O4-V2O5 ultra-micro composite foam nanometer enzyme, but has poor anti-interference property.

[0005] The ratio type surface enhanced Raman scattering (SERS) technology is applied to creatinine detection, and the fluctuation of SERS signal intensity caused by different measurement conditions and different aggregation states of the substrate can be solved. After adding a known concentration of an internal standard substance in the detection, the intensity ratio of the SERS signal of the internal standard substance to the signal of the analyte is used for quantitative analysis, so that the influence of external factors on the measurement result can be effectively reduced. By normalizing the signal intensity of the analyte and the signal intensity of the internal standard substance, the corrected intensity can be obtained, so that accurate quantitative analysis can be realized. Therefore, a creatinine detection method based on the ratio type SERS technology is indispensable. SUMMARY

[0006] In order to solve the above problems, the application provides a pH-assisted ratio type SERS quantitative detection method for creatinine in serum, an internal standard molecule is introduced, silver-coated gold core-shell nanoparticle colloid is prepared and optimized, the colloid is used as a SERS substrate, and a ratio type SERS detection method is constructed. In the detection process, the pH value of the creatinine solution is adjusted to ensure that the creatinine molecules are effectively adsorbed on the surface of the SERS substrate, so that the signal is enhanced. The stability and accuracy of the detection signal are improved, and finally the high sensitivity and high accuracy detection of creatinine is realized.

[0007] In order to achieve the purpose of the application, the following technical solutions are provided: The application provides a pH-assisted ratio type SERS quantitative detection method for creatinine in serum, Au@MPA@AgNPs colloid is prepared by introducing an internal standard mercaptopropionic acid as a SERS substrate, a ratio type SERS detection method is constructed, the pH of the creatinine solution is adjusted in the detection process, Raman detection is performed, and the content of creatinine in serum is obtained.

[0008] Further, the synthesis of the Au@MPA@AgNPs colloid includes the following steps: taking a gold nanoparticle colloid solution, adding mercaptopropionic acid as a Raman signal molecule, stirring and mixing uniformly, centrifuging, resuspending in ultrapure water, and obtaining Au@MPA colloid; measuring the prepared Au@MPA colloid, adding 0.1 mol / L NaOH and 1% sodium citrate, and then adding a silver nitrate solution and an ascorbic acid solution drop by drop, so that the solution continuously reacts for 1-1.5 hours, and the Au@MPA@AgNPs colloid is prepared.

[0009] Further, the ratio type SERS quantitative detection method for the content of creatinine in serum is as follows: creatinine solutions with different pH values are prepared, mixed with concentrated Au@MPA@AgNPs colloid at a volume ratio of 1:1, and then subjected to Raman testing. The Raman spectrum after testing is subjected to normalization processing, the optimal pH value for creatinine detection is studied, a standard curve for the quantitative detection of creatinine in serum is established, and the content of creatinine in serum is calculated.

[0010] Further, the volume ratio of the gold nano colloid solution and the mercaptopropionic acid is 1000:1-2.

[0011] Further, the concentration of the mercaptopropionic acid is 1 mmol / L.

[0012] Further, the volume ratio of the Au@MPA colloid, the silver nitrate solution and the ascorbic acid solution is 100: (6-14): 10.

[0013] Further, the concentration of the silver nitrate solution is 1 mmol / L, and the concentration of the ascorbic acid solution is 10 mmol / L.

[0014] Further, the pH of the creatinine solution is 3-12.

[0015] Further, the method for the pH-assisted ratio type SERS quantitative detection of creatinine in serum comprises the following steps: (1) The pH of the creatinine solution is adjusted to the range of 3-12 by using acid or base, and then the prepared core-shell nanoparticles are mixed, the Raman spectrum is collected, the characteristic peak of MPA is used as an internal standard signal, the characteristic peak of creatinine is used as a target signal, the intensity of the characteristic peak of creatinine is divided by the intensity of the characteristic peak of MPA, and normalization processing is performed; (2) Different concentrations of creatinine are added to serum, a linear curve of different concentrations and the ratio signal intensity is drawn, a linear equation is obtained after fitting, and a standard curve for the quantitative detection of serum creatinine is established; (3) The serum sample is detected, the obtained Raman signal ratio is brought into the standard curve established in step (2), the content of creatinine in the serum is calculated, and a commercial kit is used for verification.

[0016] Further, the standard curve for the quantitative detection of creatinine in serum is established by using creatinine solutions with different concentrations of 50 μmol / L, 60 μmol / L, 70 μmol / L, 80 μmol / L, 90 μmol / L and 100 μmol / L.

[0017] Further, the concentration of creatinine in serum and the SERS signal intensity (I 670 / I 925 ) present an excellent linear relationship, the linear equation is y=0.0055x+0.121, and the correlation coefficient R²=0.9780 Compared with the prior art, the present application has the following beneficial effects: 1.Using Au@MPA@Ag NPs core-shell nanoparticles colloid as SERS substrate, high sensitivity detection of creatinine can be realized, in Au@MPA@Ag NPs, the silver shell enhances the electromagnetic field strength of the nanoparticle surface and the intensity of the SERS signal, in the detection of some biomolecules, the SERS signal intensity of the silver-coated gold structure is several times higher than that of pure gold nanoparticles, under the enhancement of the silver shell, Au@MPA@Ag NPs can produce more obvious characteristic peaks in SERS detection, and the intensity of the characteristic peaks is higher, which is beneficial to the quantitative analysis of target molecules; and by introducing an internal standard, a ratio type SERS detection is constructed, the differences of signals caused by instrument fluctuation and substrate unevenness are calibrated, and the accuracy of creatinine detection is improved.

[0018] 2.Through pH adjustment of creatinine solution, the SERS signal of creatinine can be affected, and the pH value can affect the interaction between the Au@MPA@Ag NPs substrate and the detection molecules; at pH=9, creatinine and Au@MPA@Ag NPs are beneficial to adsorption, thereby enhancing the SERS signal of creatinine; the present application has the characteristics of rapidness, high sensitivity and the like, and can realize creatinine detection in the complex biological system of blood, and is convenient to operate. BRIEF DESCRIPTION OF DRAWINGS

[0019] Figure 1 is a detection principle diagram; Figure 2 is a SERS spectrum diagram of core-shell nanoparticles prepared in examples 1-6 with different amounts of silver nitrate added; Figure 3 is a line graph of I 675 / I 925 of different silver nitrate added amounts in examples 1-6 and I Figure 4 is a SERS spectrum diagram corresponding to creatinine with different pH values mixed with Au@MPA@Ag NPs in examples 1-6; Figure 5 is a line graph of I 675 / I 925 of creatinine with different pH values in examples 1-6 and I Figure 6 is a SERS spectrum diagram of serum creatinine with different concentrations mixed with Au@MPA@Ag NPs in examples 1-6; Figure 7 is a linear relationship diagram of I 670 / I 925 of different concentrations of creatinine in serum in examples 1-6 and I DETAILED DESCRIPTION

[0020] With reference to the drawings of the embodiments of the present application, the technical solutions in the embodiments of the present application will be described clearly and completely. Obviously, the described embodiments are only a part of the embodiments of the present application, rather than all the embodiments; based on the embodiments in the present application, all other embodiments obtained by a person of ordinary skill in the art without creative labor fall within the protection scope of the present application.

[0021] The raw materials used in the following examples are commercially available, unless otherwise specified.

[0022] The methods used in the following examples are conventional operation methods in the art, unless otherwise specified.

[0023] Example 1 The present embodiment provides a pH-assisted ratio type SERS quantitative detection method for creatinine in serum, comprising the following steps: (1) 100 mL of 0.01% chloroauric acid solution is heated to boiling, 1% sodium citrate solution is quickly added, and after 15 min of continuous reaction, it is cooled to prepare AuNPs sol with uniform particle size; 10 mL of prepared AuNPs sol is mixed with 10 μL of 1 mmol / L MPA solution, and after stirring and centrifugation, the supernatant is removed, and the precipitate is resuspended in 10 mL of ultrapure water; while stirring, 20 μL of 0.1 mol / L NaOH and 300 μL of 1% mass fraction of sodium citrate are added in sequence, followed by dropwise addition of 600 μL of 1 mmol / L silver nitrate and 1 mL of 10 mmol / L ascorbic acid, and the solution is continuously reacted for 1 hour to prepare Au@MPA@AgNPs colloid; (2) The prepared creatinine solution is mixed with the prepared Au@MPA@AgNPs colloid at a volume ratio of 1:1, and the Raman spectrum data is collected; (3) The prepared creatinine solution is adjusted to pH=3, and the concentrated Au@MPA@AgNPs colloid prepared in step (1) is mixed with the creatinine solution at a volume ratio of 1:1, and the Raman spectrum data is collected; (4) The creatinine solution with a concentration of 50 μmol / L is added to the serum, the solution pH is adjusted to 9, and then mixed with the Au@MPA@AgNPs colloid for Raman spectrum test, and a standard curve is established.

[0024] Example 2 The present embodiment provides a pH-assisted ratio type SERS quantitative detection method for creatinine in serum, comprising the following steps: (1) 100 mL of 0.01% chloroauric acid solution was heated to boiling, 1% sodium citrate solution was quickly added, and the reaction was continued for 15 min, then cooled to prepare AuNPs sol with uniform particle size; 10 mL of the prepared AuNPs sol was mixed with 20 μL of 1 mmol / L MPA solution, stirred and centrifuged, and the supernatant was removed, and the precipitate was resuspended in 10 mL of ultrapure water; the resuspended nanoparticles were stirred and 20 μL of 0.1 mol / L NaOH and 300 μL of 1% sodium citrate were added in sequence, followed by dropwise addition of 800 μL of 1 mmol / L silver nitrate and 1 mL of 10 mmol / L ascorbic acid, and the solution was continuously reacted for 1.5 hours to prepare Au@MPA@AgNPs colloid; (2) The prepared creatinine solution was mixed with the prepared Au@MPA@AgNPs colloid at a volume ratio of 1:1, and the Raman spectrum data was collected; (3) The prepared creatinine solution was adjusted to pH=5, and the concentrated Au@MPA@AgNPs colloid prepared in step (1) was mixed with the creatinine solution at a volume ratio of 1:1, and the Raman spectrum data was collected; (4) The prepared Au@MPA@AgNPs colloid was mixed with the serum to which 60 μmol / L of creatinine solution was added, and the pH of the solution was adjusted to 9, and the Raman spectrum was tested to establish a standard curve.

[0025] Example 3 The present embodiment provides a method for pH-assisted ratio-type SERS quantitative detection of creatinine in serum, comprising the following steps: (1) 100 mL of 0.01% chloroauric acid solution was heated to boiling, 1% sodium citrate solution was quickly added, and the reaction was continued for 15 min, then cooled to prepare AuNPs sol with uniform particle size; 10 mL of the prepared AuNPs sol was mixed with 20 μL of 1 mmol / L MPA solution, stirred and centrifuged, and the supernatant was removed, and the precipitate was resuspended in 10 mL of ultrapure water; the resuspended nanoparticles were stirred and 20 μL of 0.1 mol / L NaOH and 300 μL of 1% sodium citrate were added in sequence, followed by dropwise addition of 800 μL of 1 mmol / L silver nitrate and 1 mL of 10 mmol / L ascorbic acid, and the solution was continuously reacted for 1.5 hours to prepare Au@MPA@AgNPs colloid; (2) The prepared creatinine solution was mixed with the prepared Au@MPA@AgNPs colloid at a volume ratio of 1:1, and the Raman spectrum data was collected; (3) The prepared creatinine solution was adjusted to pH = 7, mixed with the Au@MPA@AgNPs colloid prepared in step (1) at a volume ratio of 1:1, and then the Raman spectrum data was collected; (4) The prepared creatinine solution with a concentration of 70 μmol / L was added to the serum, the solution was adjusted to pH = 9, mixed with the Au@MPA@AgNPs colloid, and then the Raman spectrum test was performed to establish a standard curve.

[0026] Example 4 The present example provides a method for pH-assisted ratio-type SERS quantitative detection of creatinine in serum, comprising the following steps: (1) The difference between the present example and step (1) of Example 1 is that 1000 μL of silver nitrate solution is added; (2) The prepared creatinine solution was mixed with the Au@MPA@AgNPs colloid at a volume ratio of 1:1, and then the Raman spectrum data was collected; (3) The prepared creatinine solution was adjusted to pH = 9, mixed with the Au@MPA@AgNPs colloid prepared in step (1) at a volume ratio of 1:1, and then the Raman spectrum data was collected, (4) The prepared creatinine solution with a concentration of 80 μmol / L was added to the serum, the solution was adjusted to pH = 9, mixed with the Au@MPA@AgNPs colloid, and then the Raman spectrum test was performed to establish a standard curve.

[0027] Example 5 The present example provides a method for pH-assisted ratio-type SERS quantitative detection of creatinine in serum, comprising the following steps: (1) The difference between the present example and step (1) of Example 1 is that 1000 μL of silver nitrate solution is added; (2) The prepared creatinine solution was mixed with the Au@MPA@AgNPs colloid at a volume ratio of 1:1, and then the Raman spectrum data was collected; (3) The prepared creatinine solution was adjusted to pH = 9, mixed with the Au@MPA@AgNPs colloid prepared in step (1) at a volume ratio of 1:1, and then the Raman spectrum data was collected, (4) The prepared creatinine solution with a concentration of 80 μmol / L was added to the serum, the solution was adjusted to pH = 9, mixed with the Au@MPA@AgNPs colloid, and then the Raman spectrum test was performed to establish a standard curve.

[0028] Example 6 The present example provides a method for pH-assisted ratio-type SERS quantitative detection of creatinine in serum, comprising the following steps: (1) The Au@MPA@AgNPs colloid in this embodiment is the same as that in Example 1 (1); (2) After mixing the prepared creatinine solution with the prepared Au@MPA@AgNPs colloid at a volume ratio of 1:1, Raman spectral data were collected; (3) Adjust the pH of the prepared creatinine solution to 9, and mix it with the concentrated Au@MPA@AgNPs colloid and creatinine solution prepared in step (1) at a volume ratio of 1:1. Then, collect Raman spectral data. (4) Add a creatinine solution with a concentration of 100 μmol / L to the serum, adjust the pH of the solution to 9, mix it with the prepared Au@MPA@AgNPs colloid, and then perform Raman spectroscopy to establish a standard curve.

[0029] The Raman spectral data analysis diagrams for Examples 1-6 are shown below; Figure 1 The diagram illustrates the principle of ratiometric SERS detection. Using Au NPs as the gold core, silver shell growth is guided by MPA modification to prepare core-shell structures of Au@MPA@Ag NPs. After creatinine specifically binds to Au@MPA@Ag NPs to form an Au@MPA@Ag + Creatinine complex, it is detected using a handheld Raman spectrometer. MPA serves as an internal standard (IS), and the intensity ratio of the characteristic Raman peak of creatinine to that of the internal standard is used as the detection criterion.

[0030] Figure 2 The creatinine detection spectra of colloidal core-shell nanoparticles prepared with different amounts of silver nitrate are shown in the figure. The amount of silver nitrate added affects the thickness of the silver layer on the core-shell nanoparticles, as shown in the figure. Creatinine levels are highest at 675 cm⁻¹. -1 It exhibits a significant and stable characteristic peak at 925 cm⁻¹, while the characteristic peak of the internal standard molecule is located at 925 cm⁻¹. -1 The two peaks are clearly separated (ΔV≈250cm). -1 The absence of overlapping interference indicates that it can serve as a SERS substrate for ratiometric detection methods. In ratiometric SERS detection, the signal intensity ratio (Ic) of the creatinine characteristic peak to the internal standard molecule characteristic peak needs to be closely monitored. 675 / I 925 ).

[0031] Figure 3 For different amounts of silver nitrate and I 675 / I 925 The line graph is shown in the figure. Under a volume of 600 µL of silver nitrate, I... 675 / I 925 The ratio was most significant, indicating that when 600 µL of silver nitrate was added, the intensity ratio of the creatinine peak to the internal standard peak (I0.05) was significantly higher. 675 / I 925 ) large.

[0032] Figure 4 The SERS spectra of creatinine solution with different pH mixed with Au@MPA@Ag NPs colloid are shown in the figure. It can be observed that the peak intensity is significantly weakened under the condition of pH = 3 or pH = 12. In contrast, under the condition of pH = 9, the characteristic peak intensity reaches the maximum value, and the peak shape is sharp, which indicates that the pH value greatly affects the interaction between the Au@MPA@Ag NPs substrate and the detection molecule. When the pH of the creatinine solution is 9, the interaction between creatinine and the surface of the nanoparticles is the strongest, and the SERS signal enhancement effect is the best.

[0033] Figure 5 The SERS spectra of creatinine with different pH and I 675 / I 925 are shown in the figure. As shown in the figure, when pH = 9, the ratio of I 675 / I 925 is the most significant, and the intensity ratio of creatinine and the internal standard peak (I 675 / I 925 ) is large.

[0034] Figure 6 The SERS spectra of creatinine with different concentrations are shown in the figure. As shown in the figure, the characteristic peak of creatinine in serum is 670 cm -1 , and the characteristic peak of the internal standard molecule is located at 925 cm -1 .

[0035] Figure 7 The linear graph of different creatinine concentrations in serum and I 670 / I 925 is shown in the figure. The best linear equation is fitted by taking I 670 / I 925 as the dependent variable and the creatinine concentration as the independent variable by the linear regression method. In the range of 50-100 µmol / L, I 670 / I 925 has a good linear relationship with the creatinine concentration, and the linear equation is y = 0.0055x + 0.121, R² = 0.9780. The results show that the method exhibits good linear relationship when detecting creatinine in serum.

[0036] Example 7 This example provides the accuracy verification of the ratio type SERS technology. The same sample is detected by using a commercial kit based on the creatinine oxidase method spectrophotometric creatinine content determination kit, and the results are shown in Table 1. The kit results and the SERS detection results are consistent, indicating the reliability of the detection method.

[0037] Table 1 Creatinine spiking recovery experiment

[0038] The above merely illustrates the embodiments of the present application, and is not intended to limit the patent scope of the present application. Any equivalent structure or equivalent process conversion, or direct or indirect application in other related technical fields, made by using the content of the present application, are also included in the patent protection scope of the present application.

Claims

1. A method for quantitative detection of serum creatinine using pH-assisted ratiometric SERS, characterized in that, A ratiometric SERS detection method was constructed by preparing Au@MPA@Ag NPs colloid using mercaptopropionic acid as an internal standard. During the detection process, the pH of the creatinine solution was adjusted, and Raman spectroscopy was performed to obtain the creatinine content in serum.

2. The method for quantitative detection of serum creatinine using pH-assisted ratiometric SERS as described in claim 1, characterized in that, The synthesis of the Au@MPA@AgNPs colloid is as follows: Take the nano-gold colloid solution, add mercaptopropionic acid as the Raman signal molecule, stir and mix evenly, centrifuge, and resuspend in ultrapure water to obtain Au@MPA colloid; add 0.1 mol / L NaOH and 1% sodium citrate to the prepared Au@MPA colloid, and then add silver nitrate solution and ascorbic acid solution dropwise, allowing the solution to react continuously for 1-1.5 hours to obtain Au@MPA@AgNPs colloid.

3. The method for quantitative detection of serum creatinine using pH-assisted ratiometric SERS as described in claim 1, characterized in that, Creatinine solutions with different pH values ​​were prepared and mixed with concentrated Au@MPA@Ag NPs colloid at a volume ratio of 1:1 before Raman spectroscopy. The Raman spectra were normalized to determine the optimal pH value for creatinine detection. A standard curve for quantitative detection of creatinine in serum was established, and the creatinine content in serum was calculated.

4. The method for quantitative detection of serum creatinine using pH-assisted ratiometric SERS as described in claim 2, characterized in that, The volume ratio of the nano-gold colloid solution to mercaptopropionic acid is 1000:1~2.

5. The method for quantitative detection of serum creatinine using pH-assisted ratiometric SERS as described in claim 2, characterized in that, The concentration of the mercaptopropionic acid is 1 mmol / L.

6. The method for quantitative detection of serum creatinine using pH-assisted ratiometric SERS as described in claim 2, characterized in that, The volume ratio of Au@MPA colloid, silver nitrate solution, and ascorbic acid solution is 100:(6~14):

10.

7. The method for quantitative detection of serum creatinine using pH-assisted ratiometric SERS technology as described in claim 2, characterized in that, The concentration of the silver nitrate solution is 1 mmol / L; the concentration of the ascorbic acid is 10 mmol / L.

8. The method for quantitative detection of serum creatinine using pH-assisted ratiometric SERS as described in claim 3, characterized in that, The pH of creatinine solution is 3-12.

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