CN115452817B - Method for detecting homocysteine based on functionalized nanoparticle-paper chip system - Google Patents
Method for detecting homocysteine based on functionalized nanoparticle-paper chip system Download PDFInfo
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- CN115452817B CN115452817B CN202211174950.7A CN202211174950A CN115452817B CN 115452817 B CN115452817 B CN 115452817B CN 202211174950 A CN202211174950 A CN 202211174950A CN 115452817 B CN115452817 B CN 115452817B
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Abstract
The invention discloses a method for detecting cysteine based on a functionalized nanoparticle-paper chip system, belonging to the field of biological detection. The functionalized nanoparticle-paper chip system comprises aspartic acid modified nano gold particles and a paper chip loaded with the aspartic acid modified nano gold particles. The invention establishes a novel method for rapidly detecting homocysteine by taking a paper chip as an analysis platform. Hcy causes the nano gold system modified by aspartic acid to agglomerate, the solution is gradually changed from red to blue, the color ratio of red/blue channels and the Hcy concentration show good linear relation within the range of 0.74 mu mol/L to 74 mu mol/L, and the lowest detection limit is 0.37 mu mol/L. The method has the characteristics of small sample consumption, rapid detection and good accuracy, and can rapidly detect Hcy in an actual sample.
Description
Technical Field
The invention relates to the field of biological detection, in particular to a method for detecting cysteine based on a functionalized nanoparticle-paper chip system.
Background
Small molecule biological thiols play an important role in physiological and pathological processes. Homocysteine (Hcy) is an important intermediate in methionine and cysteine metabolism, and abnormal levels of Hcy in plasma or serum are associated with a range of human diseases such as dementia, parkinson's disease, cardiovascular disease, and the like. Therefore, quantitative determination of Hcy in physiological systems is of great importance for the clinical early diagnosis and assessment of relevant disease progression. However, since Hcy is structurally similar to cysteine (Cys) and Glutathione (GSH), selective detection thereof presents a major challenge. Of the general assay methods for biological thiols, only a few methods are able to selectively identify Hcy.
As a novel instant detection system, the microfluidic paper-based analysis device (mu PADS) has the advantages of low cost, portability, disposability, rapidness, simpleness, high flux, low sample consumption and the like, and provides a platform with wide prospect for biochemical detection. Compared with the traditional analysis method, the mu PADS does not need a large-scale precise analysis instrument, and is suitable for being used in areas with poor resources. Currently, various paper-based colorimetric assays have been developed for the determination of different disease factors, such as glucose, uric acid, creatinine, and the like. To date, quantitative detection of Hcy using μpad colorimetry has not been reported.
Disclosure of Invention
The invention aims to provide a method for detecting homocysteine based on a functionalized nanoparticle-paper chip system, which solves the problems in the prior art, and establishes a low-cost, rapid and accurate method for detecting homocysteine content based on the color reaction of an aspartic acid modified nanoparticle solution and homocysteine by adopting a paper chip colorimetric detection method.
In order to achieve the above object, the present invention provides the following solutions:
the invention provides a functionalized nanoparticle-paper chip system, which comprises aspartic acid modified nano gold particles and a paper chip loaded with the aspartic acid modified nano gold particles.
Preferably, the paper chip is circular chromatographic paper with the diameter of 6mm, and the volume of the paper chip loaded with the aspartic acid modified nano gold particles is 3-10 mu L.
The invention also provides a preparation method of the functionalized nanoparticle-paper chip system, which comprises the following steps:
(1) Heating and stirring chloroauric acid solution and deionized water to boil, and then adding trisodium citrate solution to react until the color of the solution becomes wine red to obtain a nano gold solution;
(2) Adding an aspartic acid solution into the nano gold solution, and stirring at room temperature to obtain aspartic acid modified nano gold particles;
(3) And (3) dropwise adding the aspartic acid modified nano gold particles onto a paper chip to obtain a functionalized nano particle-paper chip system.
Preferably, in the step (1), the volume ratio of the chloroauric acid solution to the deionized water is 3.5:96.5, wherein the concentration of the chloroauric acid solution is 25.6mmol/L;
in the step (2), the volume ratio of the nano gold solution to the aspartic acid solution is 5:0.02, and the concentration of the aspartic acid solution is 0.8nmol/L-0.2 mu mol/L.
The invention also provides a method for detecting cysteine based on the functionalized nanoparticle-paper chip system, which comprises the following steps:
and adding a sample to be detected containing homocysteine into the functionalized nanoparticle-paper chip system prepared by the preparation method, fixing and carrying out a color development reaction for 1-30min, and quantitatively detecting the concentration of homocysteine in the sample to be detected according to the relation between the color change and the concentration of homocysteine.
Preferably, the volume ratio of the sample to be tested and the aspartic acid modified nanoparticle in the functionalized nanoparticle-paper chip system is (5-10): 3-10.
Preferably, the volume ratio of the sample to be tested and the aspartic acid modified nanoparticles in the functionalized nanoparticle-paper chip system is 8:7.
Preferably, the fixed color reaction time is 5 minutes.
Preferably, the homocysteine-containing test sample comprises a plasma sample.
The invention also provides application of the functionalized nanoparticle-paper chip system in preparing a product for detecting homocysteine.
The invention discloses the following technical effects:
experiments show that Hcy causes an Aspartic acid modified nano gold (Asp-AuNPs) system to agglomerate, the solution is gradually changed from Red to Blue, the Red/Blue channel color ratio (delta Red/Blue) and the Hcy concentration show good linear relation within the range of 0.74 mu mol/L-74 mu mol/L, the lowest detection limit is 0.37 mu mol/L, and the sample adding recovery rate is 98.7% -100.3%. The interference error of coexisting reductive substances is within 2%, and the repeatability and the stability are good. The invention establishes a novel method for detecting the Hcy content with low cost, high speed and accuracy, and has a profound significance for clinical detection of Hcy and prevention and diagnosis of related diseases. The method combines the characteristics of low cost, portability, simple operation and the like of the paper chip method, and combines the good stability, optical effect and unique biological affinity of the nano gold.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are needed in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 is a schematic diagram of paper chip analysis detection Hcy;
FIG. 2 is a graph showing the effect of reaction time on Hcy ΔRed/Blue values at different concentrations;
FIG. 3 is a graph of Hcy standard.
Detailed Description
Various exemplary embodiments of the invention will now be described in detail, which should not be considered as limiting the invention, but rather as more detailed descriptions of certain aspects, features and embodiments of the invention.
It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. In addition, for numerical ranges in this disclosure, it is understood that each intermediate value between the upper and lower limits of the ranges is also specifically disclosed. Every smaller range between any stated value or stated range, and any other stated value or intermediate value within the stated range, is also encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference for the purpose of disclosing and describing the methods and/or materials associated with the documents. In case of conflict with any incorporated document, the present specification will control.
It will be apparent to those skilled in the art that various modifications and variations can be made in the specific embodiments of the invention described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from consideration of the specification of the present invention. The specification and examples are exemplary only.
As used herein, the terms "comprising," "including," "having," "containing," and the like are intended to be inclusive and mean an inclusion, but not limited to.
Example 1 Selective detection of homocysteine by Asp-modified nanogold-paper chip System
1. Preparation of aspartic acid (Asp) -nanogold (AuNPs) system
3.5mL of chloroauric acid solution (25.6 mmol/L) and 96.5mL of deionized water are sequentially poured into a three-necked round bottom flask, heated and stirred until boiling, then 10mL of trisodium citrate solution is rapidly added, the color of the solution is observed to be changed from pale yellow to purple, and finally, the solution is changed into the special wine red of nano gold; heating and stirring are continued, heating is stopped after 10min, and stirring is continued for about 15min at room temperature to obtain the nano gold solution. 5mL of the prepared nano gold solution is taken, 20 mu L of Asp solution (0.2 mu mol/L) is added under slow stirring, and stirring is carried out for 2 hours at room temperature, so as to prepare Asp-modified nano gold particles (Asp-AuNPs), and the Asp-modified nano gold particles are stored in a refrigerator at 4 ℃ in a dark place.
2. Colorimetric detection of Hcy
Round paper chips with a diameter of 6mm were punched out on Whatman grade 1 chromatographic paper with a punch. Paper chip analysis the principle of homocysteine detection is shown in figure 1.
An Asp-AuNP solution with a volume of 7 mu L is dripped on the round paper-based platform, 8 mu L of Hcy solution is loaded, and after 5min, an OPPO R9 mobile phone is used for scanning, photographing and recording the result in a self-made light-shielding box in a laboratory by taking an LED lamp (3W) as an auxiliary light source. The color intensity (Mean value) of the Red and Blue channels was analyzed using Photoshop CS2 software to obtain a Red/Blue channel color ratio (Red/Blue value) to subtract the blank control, Δred/Blue value, as an evaluation index.
3. Results
(1) Principle of detecting Hcy by Asp-AuNPs
There is an ion pair interaction between Hcy and Asp, the strength of which is directly related to the molecular structure and charged groups. Earlier studies in this laboratory have found that Asp concentrations of 8. Mu. Mol/L respond to Cys. Hcy has two CH's compared to Cys 2 The group, which gives it better flexibility, may form a chelate structure with Asp on the surface of AuNPs, resulting in an easier aggregation of Asp-AuNPs (FIG. 1). When the Asp concentration is 0.8nmol/L, hcy can be selectively responded, and Cys is not interfered. The higher the Hcy concentration is, the faster Asp-AuNPs agglomerate, the more obvious the color change is, the delta Red/Blue value is in direct proportion to the Hcy concentration, and the method can be used for quantitatively detecting the Hcy concentration in a sample.
(2) Characterization of Asp-AuNPs modification solutions
The AuNPs particle diameters before and after modification were (21.86.+ -. 0.15) nm and (28.27.+ -. 0.21) nm, respectively, and the potential values were (-30.07.+ -. 0.86) mV and-45.16.+ -. 0.52) mV, respectively. The particle size of the Asp-AuNPs modification solution is increased, which indicates that Asp is successfully bonded to the surface of the nano gold particles; the potential is increased, and the Asp-AuNPs stability is better.
Example 2 optimization of the detection method
The invention finds that Asp-AuNPs has too small loading volume to cover round paper, and reagent overflow occurs when the volume is excessive. The present invention thus optimizes the loading of Asp-AuNPs. The results showed that the detection zone was completely covered when the Asp-AuNPs loading was 7. Mu.L. The Asp-AuNPs volume was fixed at 7. Mu.L, and the effect of the sample loading volume on the experimental results was examined, and Hcy solutions (7.4 mmol/L, 3.7mmol/L and 0.74 mmol/L) at different concentrations were examined, respectively, to give optimal loadings of 9. Mu.L, 9. Mu.L and 8. Mu.L, respectively. Indicating that Hcy optimum loading is less affected by concentration. Thus, the fixed Asp-AuNPs and Hcy loadings of the present invention were 7. Mu.L and 8. Mu.L, respectively.
After addition of the Hcy solution, the Asp-AuNPs color starts to change from red to blue, and the higher the Hcy concentration, the faster the color change speed. Studies showed that ΔRed/Blue reached a maximum at concentrations of 7.4mM, 0.74mmol/L, 0.074mmol/L, 7.4. Mu. Mol/L and 0.074. Mu. Mol/L of Hcy solutions, respectively, that interacted with Asp-AuNPs for 4min, 4min and 5min (FIG. 2). Therefore, the present invention fixes the color development time to 5min.
EXAMPLE 3 establishment of Hcy Standard Curve
The concentration of the sample Hcy was determined by making a standard curve. mu.L Asp-AuNPs and 8. Mu.L Hcy (0.74. Mu. Mol/L, 18.5. Mu. Mol/L, 37. Mu. Mol/L, 55.5. Mu. Mol/L, 74. Mu. Mol/L) were sequentially added dropwise to the round paper chip, and the results were recorded after 5min. The Hcy concentration was linearly fitted with Δred/Blue to obtain a Hcy standard curve.
The results show that ΔRed/Blue is proportional to Hcy concentration (FIG. 3), in the range of 0.74 to 74. Mu. Mol/L, the linear fitting equation is y=0.0704x+0.5445, the linear correlation coefficient (R) is 0.9995, the linear relationship is good, and LOD is 0.37. Mu. Mol/L. The recovery rate is between 98.7% and 100.3% (table 1), and the accuracy is good.
Table 1 results of recovery experiments by paper chip analysis
Example 4 Selective and repetitive assays
This example examined the effect of coexisting substances on Hcy detection, especially those with high structural similarity, such as Cys and GSH. Preparing 74 mu mol/LHcy, and respectively adding 1000 times of vitamin C, potassium nitrate, GSH, cys, glucose and sodium chloride to obtain a mixed sample. And (3) sequentially loading 7 mu LAsp-AuNPs and 8 mu L of the mixed sample on a round paper chip, recording the result after 5min, measuring the Hcy content, and checking whether the coexisting materials interfere with the detection of the Hcy.
The results indicate that Na + 、K + The analysis and detection deviation of vitamin C, glucose, cys and GSH on Hcy is within 2 percent (table 2), which shows that under the optimized experimental conditions, the coexisting substances have no interference on the measurement of Hcy, and the method has good selectivity. The repeatability experiment result shows that the Relative Standard Deviation (RSD) of the daytime (n=5) and the intra-day (n=5) is smallAt 2% (Table 3), this method is shown to have better reproducibility.
TABLE 2 results of the paper chip analysis selectivity and repeatability experiments
TABLE 3 daytime and intra-daytime reproducibility of paper chip assays
EXAMPLE 5 application of Asp-modified nanogold-paper chip System
To 100. Mu.L of fresh plasma, 300. Mu.L of acetone was added and centrifuged at 8000rpm for 10min. Supernatant with N 2 Blow-dry with air, add 1000 μl deionized water to obtain plasma sample. The sample was analyzed and tested according to the test conditions optimized in example 2.
As shown in Table 4, the concentration of Hcy in human plasma was 5.52.+ -. 0.39. Mu. Mol/L, which was in accordance with the reference range of Hcy concentration in human plasma (5. Mu. Mol/L to 15. Mu. Mol/L). The method has the potential of measuring Hcy in actual samples.
Table 4 detection of plasma samples
The results of the embodiment show that the invention establishes a novel paper chip-based Hcy rapid detection method which has high detection sensitivity, good repeatability and high accuracy and can be used for measuring the Hcy content in a plasma sample. In addition, the method has the advantages of low detection cost, high portability and simple operation, is expected to be used for clinical and household Hcy real-time detection, and has important significance for clinical early diagnosis and evaluation of relevant disease progress
The above embodiments are only illustrative of the preferred embodiments of the present invention and are not intended to limit the scope of the present invention, and various modifications and improvements made by those skilled in the art to the technical solutions of the present invention should fall within the protection scope defined by the claims of the present invention without departing from the design spirit of the present invention.
Claims (5)
1. A method for detecting cysteine based on functionalized nanoparticle-paper chip systems for non-disease diagnosis and treatment purposes, comprising the steps of: adding a sample to be detected containing homocysteine into the functionalized nanoparticle-paper chip system, fixing and carrying out a color development reaction for 1-30min, and quantitatively detecting the concentration of homocysteine in the sample to be detected according to the relation between the color change and the concentration of homocysteine;
the functionalized nanoparticle-paper chip system comprises aspartic acid modified nano gold particles and a paper chip loaded with the aspartic acid modified nano gold particles; the paper chip is circular chromatographic paper with the diameter of 6mm, and the volume of the paper chip loaded with the aspartic acid modified nano gold particles is 3-10 mu L;
the preparation method of the functionalized nanoparticle-paper chip system comprises the following steps:
(1) Heating and stirring chloroauric acid solution and deionized water to boil, and then adding trisodium citrate solution to react until the color of the solution becomes wine red to obtain a nano gold solution;
(2) Adding an aspartic acid solution into the nano gold solution, and stirring at room temperature to obtain aspartic acid modified nano gold particles;
(3) Dripping the aspartic acid modified nano gold particles on a paper chip to obtain a functionalized nano particle-paper chip system;
in the step (1), the volume ratio of the chloroauric acid solution to the deionized water is 3.5:96.5, wherein the concentration of the chloroauric acid solution is 25.6mmol/L;
in the step (2), the volume ratio of the nano gold solution to the aspartic acid solution is 5:0.02, and the concentration of the aspartic acid solution is 0.8nmol/L-0.2 mu mol/L.
2. The method of claim 1, wherein the ratio of the volume of the sample to be tested to the volume of the aspartic acid-modified gold nanoparticles in the functionalized nanoparticle-paper chip system is (5-10): 3-10.
3. The method of claim 2, wherein the volume ratio of the sample to be tested to the aspartic acid modified gold nanoparticles in the functionalized nanoparticle-paper chip system is 8:7.
4. The method of claim 1, wherein the fixed chromogenic reaction time is 5 minutes.
5. The method of claim 1, wherein the homocysteine-containing test sample comprises a plasma sample.
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