CN101806740B - Detection method of human plasma surface enhanced raman spectroscopy by integrating main component analysis - Google Patents

Abstract

The present invention provides a detection method of human plasma surface-enhanced Raman spectroscopy combined with principal component analysis, the method steps are as follows: obtain a human plasma sample in a physiological state, and prepare silver sol by reduction with hydroxylamine hydrochloride; silver sol and human plasma Samples were mixed uniformly in equal volumes and incubated at 4 ⁰ C for two hours before plasma surface-enhanced Raman spectroscopy measurement; plasma surface-enhanced Raman spectroscopy measurement can also use lasers with different polarization states to excite human plasma samples; establish plasma The surface-enhanced Raman spectrum database uses principal component analysis to obtain the distribution of scatter diagrams corresponding to the surface-enhanced Raman spectra of different human plasma. The invention ensures the activity of biomolecules in blood plasma, and has the advantages of simplicity, rapidity and strong reliability. Moreover, the SERS technology allows the sample to obtain a strong Raman spectral signal at a very low laser power, and the spectral signal has good repeatability, which avoids carbonization and damage to biological samples caused by high-power lasers.

Description

A detection method of human plasma surface-enhanced Raman spectroscopy combined with principal component analysis

technical field

The invention relates to a human plasma surface-enhanced Raman spectrum combined with multivariate analysis and detection method, more specifically, human blood is subjected to plasma centrifugation under aseptic conditions to obtain a human plasma solution in a physiological state, and then the human plasma solution is obtained by surface enhancement. Raman spectroscopy is used to detect the SERS signal of plasma, and a method for statistical analysis using multivariate analysis techniques. Belongs to the field of biomedicine.

Background technique

Raman spectroscopy technology can provide molecular vibration spectra, which contain molecular fine structure information and characteristic fingerprints. Raman spectroscopy has become one of the important technologies for item identification and molecular detection. The application of Raman spectroscopy technology to the research of life sciences can analyze the structure or components of human tissues or cells by detecting the Raman spectra of biological macromolecules in human tissues or cells, such as proteins, nucleic acids, lipids, etc. Changes have become a hot topic in related research fields. However, conventional Raman spectroscopy has the disadvantages of weak signal and being easily interfered by autofluorescence. Therefore, it is necessary to enhance and amplify the Raman signal. Surface-enhanced Raman spectroscopy (SERS for short) is one of the commonly used means to enhance Raman signals. This technology utilizes the adsorption effect of molecules on the surface of some rough metals (such as Au, Ag, Cu and Pt, etc.), increases the Raman scattering intensity of these molecules by 10 ⁴ ~10 ¹⁴ times, and effectively suppresses autofluorescence. Signal. SERS technology has the characteristics of high spatial resolution, high sensitivity, and rich information content, and has been widely used in the fields of substance identification and molecular structure detection.

Chirality is a basic attribute of nature. In nature, many molecules often have two structural forms that are mirror images of each other but cannot be completely overlapped. These two forms of molecules are like the left and right hands of humans. This compound with chiral factors Molecules are called enantiomers or optical isomers. Life activities are closely related to the chirality of biomolecules. For example, among the more than 20 amino acids needed in the human body, only glycine is not chiral, and the others are all chiral molecules. Another example: the specificity of enzyme-catalyzed reactions reflects The requirements of molecular three-dimensionality, and many other processes are also permeated with the influence of molecular three-dimensionality factors. When biomolecules are diseased, the three-dimensional structure of biomolecules may undergo slight changes, which may further cause changes in the chirality of biomolecules. However, the small changes in the three-dimensional structure of biomolecules are difficult to detect with ordinary non-polarized laser light. The superhelical electromagnetic field generated by the circularly polarized laser can sensitively detect small changes in the chirality of biomolecules. Therefore, the use of circularly polarized lasers to detect the changes in the chirality of biomolecules is of great significance for revealing the mysteries of life and information about pathological changes.

  At present, scholars at home and abroad use Raman spectroscopy technology for blood testing mainly to conduct direct Raman testing on the obtained blood and plasma samples, but they have not achieved ideal results. The shortcomings of these studies are that conventional Raman spectroscopy signals are weak, autofluorescence interference is strong, and conventional laser Raman detection requires a large excitation light power and takes a long time, which is easy to cause damage to the sample. However, the use of non-polarized laser, linearly polarized laser or circularly polarized laser as excitation light and silver colloid as a reinforcing matrix surface-enhanced Raman spectroscopy combined with principal component analysis to detect and analyze human plasma has not yet been reported.

Contents of the invention

The purpose of the present invention is to aim at the deficiencies and problems existing in the current blood Raman spectrum detection, and to provide a method using plasma surface-enhanced Raman spectrum technology combined with principal component analysis, which firstly performs plasma centrifugation on human blood under sterile conditions , obtain the human plasma solution under the physiological state, and use the SERS detection method to realize the detection of the surface-enhanced Raman spectrum of the human plasma. The time required for the plasma sample pretreatment process of the present invention is 2 hours, and the detection time is only 10 seconds, so the activity of biomolecules in the plasma is guaranteed during the measurement process, and has the advantages of simplicity, speed, and strong reliability. Moreover, the SERS technology allows the sample to obtain a strong Raman spectral signal at a very low laser power, and the spectral signal has good repeatability, which avoids carbonization and damage to biological samples caused by high-power lasers. Based on the plasma surface-enhanced Raman spectra of different people, the plasma surface-enhanced Raman spectrum database was established, and a new method was provided for the analysis of human plasma surface-enhanced Raman spectra by using the multivariate statistical analysis method.

For realizing the purpose of the present invention, adopt technical scheme as follows:

(1) Extract human blood and add anticoagulant for centrifugation under sterile conditions to obtain human plasma samples under physiological conditions, and prepare silver sol by reduction with hydroxylamine hydrochloride;

(2) Silver sol and human plasma samples were mixed uniformly in equal volumes and incubated at ^40C for two hours before plasma surface-enhanced Raman spectroscopy was measured;

(3) Lasers with different polarization states can be used to excite human plasma samples for plasma surface-enhanced Raman spectroscopy;

(4) Establish a plasma surface-enhanced Raman spectrum database, and use principal component analysis and T-test to obtain the scatter diagram distribution corresponding to the surface-enhanced Raman spectrum of different human plasma.

The plasma surface-enhanced Raman spectrum measurement described in step (2) is to drop the mixed solution of human plasma and silver colloid on an aluminum sheet with a purity of 99.99% for surface-enhanced Raman measurement, focusing on the detection of 450-4000cm ^-1 wave number scope.

The lasers with different polarization states in step (3) can be used for conventional Raman spectroscopy detection and analysis of human plasma samples, purified protein, DNA, and RNA samples.

The lasers with different polarization states in step (3) may be non-polarized lasers, linearly polarized lasers, left-handed circularly polarized lasers or right-handed circularly polarized lasers. Information about chiral changes in biomolecules can be obtained using circularly polarized laser excitation.

The plasma surface-enhanced Raman spectrum database described in step (4) is composed of plasma surface-enhanced Raman spectrum detection data of different human bodies; before the plasma surface-enhanced Raman spectrum database is established, the plasma SERS spectra of different human Combined to eliminate the fluorescent background and perform area normalization processing to remove the influence of inconsistent experimental conditions such as excitation light power fluctuations and aggregation differences.

The substitute of the blood plasma described in the technical solution adopted by the present invention can be urine, serum, lymph fluid, cerebrospinal fluid, urine, saliva, tears, sweat, cell extract, tissue homogenate, vaginal secretion fluid or semen, or Can be purified protein, DNA or RNA samples.

The advantage of the present invention is that the method of using non-polarized laser, linearly polarized laser and circularly polarized laser as excitation light for surface-enhanced Raman spectroscopy measurement can obtain high-quality plasma surface-enhanced Raman signals, and can obtain relevant Information on chiral changes in biomolecules. Combining with principal component analysis, the distribution of scatter diagrams corresponding to the surface-enhanced Raman spectra of different human plasmas was obtained. It provides an important reference for the rapid and non-destructive detection of different human plasma surface-enhanced Raman spectra.

Description of drawings

Fig. 1 is the average surface-enhanced Raman spectrum of the A group blood plasma that the present invention records;

Fig. 2 is the average surface-enhanced Raman spectrum of B group blood plasma measured by the present invention;

Fig. 3 is the first, fourth and eighth principal component spectra required by the present invention for calculating parameters a ₁ , a ₄ , a ₈

Fig. 4 is the scatter diagram distribution of the surface-enhanced Raman spectrum PCA of the A group plasma and the B group plasma surface-enhanced Raman spectrum drawn by PC1 and PC4 in the present invention, and the circular black point in the figure represents the surface-enhanced Raman spectrum of the human plasma of the A group, red The triangle tip represents the surface-enhanced Raman spectrum of human plasma in group B;

Fig. 5 is the scatter diagram distribution of the surface-enhanced Raman spectrum PCA of the A group plasma and the B group plasma surface-enhanced Raman spectrum drawn by PC1 and PC8 in the present invention, and the circular black point in the figure represents the surface-enhanced Raman spectrum of the human plasma of the A group, red The triangle tip represents the surface-enhanced Raman spectrum of human plasma in group B;

Figure 6 is the comparison of the average SERS spectra of the first human plasma under the excitation of unpolarized laser, left-handed circularly polarized laser and right-handed circularly polarized laser;

Figure 7 is a comparison of the average SERS spectrum of the second human plasma under the excitation of unpolarized laser, left-handed circularly polarized laser and right-handed circularly polarized laser;

Detailed ways

The present invention is set forth as follows according to specific implementation details:

(1) Silver sol pre-preparation and plasma sample pre-treatment

Add 4.5 ml of sodium hydroxide solution (0.1 mol) to 5 ml of hydroxylamine hydrochloride solution (0.06 mol), then quickly add the mixture to 90 ml of silver nitrate solution (0.0011 mol), and stir evenly until a uniform milky gray solution is obtained. Use a centrifuge at 10,000 rpm for 10 minutes to separate the silver colloids, discard the supernatant, and remove the concentrated silver colloid from the lower layer and store it in the dark at room temperature for later use.

The overnight fasting blood of different people between 7 am and 8 am was drawn under sterile conditions, EDTA was added to prevent blood coagulation and centrifuged (2000 rpm) for 15 minutes. The upper layer of serum was discarded, and the lower layer of plasma was taken as a sample. Two groups of different human plasma samples (group A and group B) were obtained by using this method. Use a pipette gun to take 200 μl of plasma from each sample in group A and add it to a sterile test tube. And use a pipette gun to add 200 μl of the previously prepared centrifuged silver sol into the test tube, and mix according to the volume ratio of plasma and silver sol at 1:1. The mixed solution was fully stirred, so that the plasma and the silver sol were mixed as evenly as possible, and a group A plasma-silver sol mixed solution was prepared. Use a pipette to take out 200 μl of each sample plasma from group B and add it into a sterile test tube. And use a pipette to add 200 μl of the previously prepared centrifuged silver sol into the test tube, and mix according to the volume ratio of plasma and silver sol at 1:1. The mixed solution was fully stirred, so that the blood plasma and the silver sol were mixed as evenly as possible, and the plasma-silver sol mixed solution of group B was prepared. All the prepared mixed solutions were incubated in a refrigerator set at 4°C for two hours.

(2) Surface-enhanced Raman spectroscopy detection sample process

Use a pipette gun to transfer the mixed plasma-silver sol mixture to an aluminum sample stand with a purity of 99.99%, let it dry naturally, and use a Raman spectrometer to detect the sample. The excitation light used is non-polarized laser or linearly polarized laser. Left-handed circularly polarized laser or right-handed circularly polarized laser, focusing on detecting the wavenumber range of 450-4000cm ^-1 to obtain the surface-enhanced Raman spectrum of plasma. Set measurement parameters: integration time 10s, excitation wavelength 785nm, excitation light power 5mw.

(3) Principal component analysis of surface-enhanced Raman spectra of different human plasma

To perform principal component analysis on different plasmas, it is necessary to establish a database of plasma surface-enhanced Raman spectra. Before establishing the spectral database model, the surface-enhanced Raman spectrum of plasma should be normalized by area to remove the influence of inconsistent experimental conditions such as excitation light power fluctuations and aggregation differences. When establishing the database, considering some individual differences among different people, in order to ensure the statistics, it is necessary to collect the surface-enhanced Raman spectrum data of a sufficient number of different human plasma. The plasma surface-enhanced Raman spectroscopy database consists of plasma surface-enhanced Raman spectroscopy detection data from different human bodies. On the basis of establishing the database, use PCA analysis to obtain the scores corresponding to each principal component (PC score), and then use the Independent-Sample T test in SPSS to select the three PCA scores with the most significant differences to further draw Scatter plot distribution of surface-enhanced Raman spectra of different human plasma in groups A and B.

，其中

为平均谱。为方便阐述，我们用下标m标记不同的光谱，用下标i和 j表示波数。 The first step in building a plasma SERS database is to normalize the spectra. Because the information in the spectrum mainly comes from the relative intensity of each spectral peak, and the absolute intensity of the spectral peak is related to the fluctuation and aggregation of laser power, and normalization can eliminate this effect. The present invention adopts the method of normalizing the spectral lines according to the integral area to carry out normalization processing. The second step in building the model is to plot the mean spectrum of group A versus the mean spectrum of group B that make up the database. The formula for calculating the average spectrum is:

,in

is the average spectrum. For the convenience of illustration, we use the subscript m to mark the different spectra, and the subscripts i and j to denote the wavenumbers.

Next, the standard algorithm of principal component analysis is used to establish the principal element spectrum that characterizes the spectral variation. First, calculate the covariance matrix of each wavenumber after normalization, namely:

，

…

}和其相对应的本征矢量{ 

,

…. 

}组成主元素光谱。由于本征矢量的正交归一性，可对定标光谱进行按主元素光谱的线性分解，即： Calculation matrix The eigenvalues and eigenvectors of , since only a few eigenvalues in the front rank in order of magnitude have values higher than the noise, and the latter ones can be ignored because they are weaker than the noise, so we only take the first 20 eigenvalues Levy {

,

…

} and its corresponding eigenvector {

,

....

} form the main element spectrum. Due to the orthonormality of the eigenvectors, the calibration spectrum can be linearly decomposed according to the principal element spectrum, namely:

，

,

 .

.

是归一化后的光谱，

是展开系数（score）。{

}组成了正交归一的基失集,张成一个新的空间，称为主元素空间，这个空间可由之前的矩阵

做空间变化得到。经过上面的变化，每条光谱映射为主元素空间的一个点。 in

is the normalized spectrum,

is the expansion coefficient (score). {

} constitutes an orthonormalized basis loss set, and forms a new space called the principal element space, which can be obtained by the previous matrix

Do space changes to get. After the above changes, each spectrum is mapped to a point in the principal element space.

The following is the specific calculation process of applying PCA analysis:

(1) Firstly, polynomial fitting was used to eliminate the fluorescent background of different human plasma SERS spectra;

(2) Normalize the area of the SERS spectra of different human plasma whose fluorescent background has been eliminated;

(3) Use SPSS software to conduct PCA analysis on the SERS spectra of different human plasmas processed by (1) and (2);

(4) Use the T test to obtain the three PCA scores with the most significant differences and draw a scatter diagram distribution;

After trial and error, under the excitation of non-polarized laser, the present invention confirms that the three main components of main component 1, main component 4 and main component 8 have significant differences after PCA analysis through T-test. We further draw the scatter diagram of principal component 1 and principal component 4, principal component 1 and principal component 8.

The above non-polarized laser can be replaced by linearly polarized laser, left-handed circularly polarized laser or right-handed circularly polarized laser.

(4) The specific steps for PCA analysis of the plasma SERS database are as follows:

1. Prepare silver sol-plasma mixed solution according to step (1) for different human plasma;

2. Measure plasma surface-enhanced Raman spectra according to step (2); measure the plasma spectra of no less than 30 cases in group A and group B respectively.

3. Perform area normalization for each plasma SERS spectrum.

4. For the plasma SERS spectrum in the spectral database, use the PCA standard algorithm to calculate the scores of each principal component. .

5. The principal components with the most significant differences calculated by T test are PC1, PC4, and PC8.

6. The surface-enhanced Raman spectrum PCA distribution of different human plasma is the measurement result of the present invention.

All the data and numerical values in the present invention are obtained by actual measurement, and the identification conclusion of plasma is given objectively by the surface-enhanced Raman spectrum data, and does not depend on the subjective judgment of the observer. The plasma sample preparation can be completed within 2 hours, and the spectral measurement time can be controlled within two minutes. Principal component analysis calculations on spectra can get results within 10 minutes. Therefore, surface-enhanced Raman spectroscopy of human plasma combined with principal component analysis has a very broad application prospect in the field of biomedicine.

The following are several specific implementation examples of the present invention to further describe the present invention, but the present invention is not limited thereto.

Example 1

Take overnight fasting blood from two groups of different people between 7:00 and 8:00 in the morning, add EDTA to prevent blood coagulation and centrifuge (2000 rpm) for 15 minutes. The upper layer of serum was discarded, and the lower layer of plasma was taken as a sample. The first group of human plasma totaled 33 copies, which were compiled as group A. The second group consisted of 43 samples of human plasma, which were grouped as group B; 200 μl of each sample plasma was taken from group A with a pipette gun and added to a sterile test tube. And use a pipette gun to add 200 μl of the previously prepared centrifuged silver sol into the test tube, and mix according to the volume ratio of plasma and silver sol at 1:1. The mixed solution was fully stirred, so that the plasma and the silver sol were mixed as evenly as possible, and a group A plasma-silver sol mixed solution was prepared. Use a pipette to take out 200 μl of each sample plasma from group B and add it into a sterile test tube. And use a pipette to add 200 μl of the previously prepared centrifuged silver sol into the test tube, and mix according to the volume ratio of plasma and silver sol at 1:1. The mixed solution was fully stirred, so that the blood plasma and the silver sol were mixed as evenly as possible, and the plasma-silver sol mixed solution of group B was prepared. All the prepared mixed solutions were incubated in a refrigerator set at 4°C for two hours.

Use a pipette gun to move the mixed plasma-silver sol mixture to an aluminum sheet sample stage with a purity of 99.99%, let it dry naturally, and use a confocal Raman spectrometer to detect the sample, focusing on the wavenumber range of 450-4000cm ^-1 , to Obtain surface-enhanced Raman spectra of plasma. Set measurement parameters: integration time 10s, excitation wavelength 785nm, excitation light power 5mw. The excitation light used for measuring SERS spectra is unpolarized laser light.

Before establishing the plasma surface-enhanced Raman spectrum database, the surface-enhanced Raman spectrum of plasma should be normalized by area to remove the influence of inconsistent experimental conditions such as excitation light power fluctuations and aggregation differences. On the basis of establishing the database, draw the average spectrum of Group A (as shown in Figure 1) and the average spectrum of Group B (as shown in Figure 2) in the database. Use the principal component analysis to get the corresponding score (PC score) of each principal component, and then use the Independent-Sample T test in SPSS to select the three PCA scores with the most significant differences, namely PC1, PC4 and PC8, to further draw The distribution of scatter plots of plasma surface-enhanced Raman spectra of group A and group B is shown. Accompanying drawing 3 is the spectrum of the first, fourth and eighth principal components, accompanying drawing 4 is the scatter diagram distribution of PC1 and PC4, and accompanying drawing 5 is the scatter diagram of PC1 and PC8.

Example 2

Take overnight fasting blood from two different people between 7:00 and 8:00 in the morning, add EDTA to prevent blood coagulation and centrifuge (2000 rpm) for 15 minutes. The upper layer of serum was discarded, and the lower layer of plasma was taken as a sample. Use a pipette gun to take 200 μl of sample plasma from the first plasma sample and add it to a sterile test tube. And use a pipette gun to add 200 μl of the previously prepared centrifuged silver sol into the test tube, and mix according to the volume ratio of plasma and silver sol at 1:1. The mixed solution was fully stirred, so that the plasma and the silver sol were mixed as evenly as possible, and the first plasma-silver sol mixed solution was prepared. Use a pipette to take out 200 μl of sample plasma from the second human plasma and add it to a sterile test tube. And use a pipette to add 200 μl of the previously prepared centrifuged silver sol into the test tube, and mix according to the volume ratio of plasma and silver sol at 1:1. The mixed solution was fully stirred, so that the plasma and the silver sol were mixed as evenly as possible, and a silver sol-plasma mixed solution of the control group was prepared. All the prepared mixed solutions were incubated in a refrigerator set at 4°C for two hours.

Use a pipette gun to transfer the mixed silver sol-plasma mixture to an aluminum sheet sample stage with a purity of 99.99%, let it dry naturally, and use a confocal Raman spectrometer to detect the sample. The excitation light used to measure the SERS spectrum is respectively Unpolarized laser, left-handed circularly polarized laser and right-handed circularly polarized laser. Focus on detecting the wavenumber range of 450-1730cm ^-1 to obtain the surface-enhanced Raman spectrum of plasma. Set measurement parameters: integration time 10s, excitation wavelength 785nm, excitation light power 5mw. Two sets of data obtained from the test are shown in Figure 6 and Figure 7. From the comparison of the average SERS of the excitation light of the two human plasmas in different polarization states, especially the comparison of the band range in the shaded area, it can be seen that using circularly polarized light excitation can obtain more information about molecular chirality changes than non-polarized light excitation . It shows that using circularly polarized laser to detect the change of chirality of biomolecules will be of great significance to reveal the mystery of life and the information of pathological changes.

Claims (8)

1. a detection method of human plasma surface-enhanced Raman spectroscopy combined with principal component analysis, characterized in that the method is made up of the following three steps: (1) Extract human blood and add anticoagulant for centrifugation under sterile conditions to obtain human plasma samples under physiological conditions, and prepare silver sol by reducing with hydroxylamine hydrochloride; (2) Silver sol and human plasma samples were mixed uniformly in equal volumes and incubated at 4 ⁰ C for two hours before plasma surface-enhanced Raman spectroscopy was measured; (3) Establish a plasma surface-enhanced Raman spectrum database, and use principal component analysis and T-test to obtain the scatter diagram distribution corresponding to the surface-enhanced Raman spectrum of different human plasma. 2. The detection method of human plasma surface-enhanced Raman spectroscopy combined with principal component analysis according to claim 1, characterized in that: the measurement of plasma surface-enhanced Raman spectroscopy in step (2) is to combine human plasma samples with silver The mixed solution of the sol was added dropwise on an aluminum sheet with a purity of 99.99% for surface-enhanced Raman spectroscopy measurement, focusing on the wavenumber range of 450-4000cm ^-1 . 3. A human plasma surface-enhanced Raman spectroscopy detection method combined with principal component analysis according to claim 1, characterized in that: the plasma surface-enhanced Raman spectroscopy database in step (3) consists of different human plasma surface Enhanced Raman spectroscopy detects data composition. 4. The detection method of a kind of human plasma surface-enhanced Raman spectrum combined with principal component analysis according to claim 1 or 3, characterized in that: before the establishment of the plasma surface-enhanced Raman spectrum database, different human plasma SERS spectra were processed by polynomial fitting to eliminate fluorescent background and normalize the area. 5. A detection method combining human plasma surface-enhanced Raman spectroscopy with different polarization states of laser excitation, characterized in that: the specific steps are as follows: (1) Extract human blood and add anticoagulant for centrifugation under sterile conditions to obtain human plasma samples under physiological conditions, and prepare silver sol by reducing with hydroxylamine hydrochloride; (2) Silver sol and human plasma samples were mixed uniformly in equal volumes and incubated at 4 ⁰ C for two hours before plasma surface-enhanced Raman spectroscopy was measured; (3) Plasma surface-enhanced Raman spectroscopy measurement uses lasers with different polarization states to excite human plasma samples. (4) Establish a database of plasma surface-enhanced Raman spectra, and use principal component analysis to obtain the distribution of scatter diagrams corresponding to the surface-enhanced Raman spectra of different human plasma. 6. the human plasma surface-enhanced Raman spectrum according to claim 5 is combined with the detection method of different polarization states laser excitation, it is characterized in that: the laser of described different polarization states is unpolarized laser, linearly polarized laser, left-handed circularly polarized laser Or right circularly polarized laser. 7. The detection method of human plasma surface-enhanced Raman spectroscopy combined with different polarization states of laser excitation according to claim 5, characterized in that: said different polarization states of lasers are used for human plasma samples, purified proteins, DNA or RNA The samples were detected and analyzed by surface-enhanced Raman spectroscopy. 8. the human plasma surface-enhanced Raman spectrum according to claim 5 is combined with the detection method of different polarization states laser excitation, it is characterized in that the substitute of described plasma is urine, serum, lymph fluid, cerebrospinal fluid, saliva, Tears, sweat, cell extracts, tissue homogenate, vaginal fluid, semen, purified protein, DNA or RNA samples.

Images