A SERS rapid analysis method for energy intake

Abstract

The present invention discloses a SERS rapid analysis method for energy intake, comprising the following steps: (1) filling a pre-filled liquid; (2) collecting SERS signals; (3) establishing a urine SERS database; and (4) principal component analysis based on the urine SERS database. The present invention collects urine surface enhanced Raman spectroscopy (SERS) signals of people with different energy intakes and combines them with a principal component analysis algorithm to establish a relationship between the SERS signals of urine and energy intake, that is, by collecting the SERS signals of the urine of the person to be tested, the energy intake information of the person to be tested can be obtained; at the same time, the present invention designs an integrated sol test tube, which solves the problem of cumbersome operation caused by carrying reagents such as nano-enhanced particle sol and nano-particle aggregating agent during use, and the test can be completed without professional technicians and any large-scale instruments and equipment; the operation is simple, the cost is low, the speed is fast, the instrument is portable, and it is easy to promote. It is a SERS method that is very suitable for on-site rapid evaluation of energy intake.

Description

Technical Field

[0001] The invention relates to the field of analysis and testing, and in particular to a SERS rapid analysis method for energy intake. Background Art

[0002] The mechanism of human aging has always been a hot topic of research. In the past few decades, research on the mechanism of aging has focused on the study of energy intake. Excessive calorie intake will produce reactive oxygen species in the body, causing damage to DNA, phospholipids or proteins, and accelerating the aging of the organism. Continuous low calorie intake while maintaining necessary nutrient intake will have a beneficial effect on the body's health and anti-aging.

[0003] At present, the calculation of energy intake is mainly assessed through the dietary recall method. The substances in food that can provide energy are carbohydrates, fats and proteins. The physiological energy values ​​of each gram of carbohydrates, fats and proteins are 3.99 kcal, 9.01 kcal and 3.99 kcal respectively. By calculating the mass of carbohydrates, fats and proteins in the diet, the energy intake of an individual can be roughly assessed. Although the dietary recall method can make a rough assessment of an individual's energy intake, it is cumbersome and time-consuming. There is an urgent need to develop a method that can quickly and easily analyze energy intake.

[0004] SERS (surface enhanced Raman spectroscopy) is a molecular vibration and rotation spectrum based on the Raman scattering effect. It can provide structural information of sample molecules without loss, and analyze the composition and molecular structure of the sample, so as to achieve qualitative analysis of the molecules. The intensity of the Raman spectrum is positively correlated with the number of scattered molecules, which can achieve semi-quantitative-quantitative analysis of the molecules. The sol method is one of the test methods of surface enhanced Raman spectroscopy. The sol method test process generally mixes three components of nanoparticle aggregators, nano-enhanced particles and the substance to be tested in a certain proportion. Therefore, the sol method test requires the preparation of nanoparticle aggregators and nano-enhanced particles for on-site use. The process is cumbersome and there is a problem of spontaneous agglomeration of the sol during storage and transportation. Summary of the invention

[0005] The purpose of the present invention is to overcome the defects of the prior art and provide a SERS rapid analysis method for energy intake. By developing a SERS sol test kit integrating storage, transportation and testing functions, the kit is used to collect SERS signals of urine of people with different energy intakes and perform principal component analysis. It is found that the analysis results are significantly different for different energy intakes. Therefore, the energy intake can be evaluated by analyzing the SERS signals of the urine of the tested person.

[0006] In order to achieve the above objectives, the technical solution of the present invention is: a SERS rapid analysis method for energy intake, specifically comprising the following steps:

[0007] (1) Pre-filling solution: silver nanoparticle sol and nanoparticle aggregating agent are respectively filled into the sol test tube;

[0008] (2) Collecting SERS signals: Collect urine from people with different energy intakes, mix the urine with the silver nanoparticle sol in the sol test tube, and then mix them with the nanoparticle aggregator in the sol test tube. Then place the sol tube on the Raman instrument and collect the SERS signal of the lower layer of the tube.

[0009] (3) Establishing a urine SERS database: Using the principal component analysis algorithm, the scatter plot corresponding to the surface enhanced Raman spectra of urine of people with different energy intakes is obtained, and cluster analysis of energy intake is performed to establish a SERS database;

[0010] (4) Principal component analysis based on urine SERS database: including the following steps:

[0011] a. Collect urine from the person to be tested and acquire SERS spectral signals;

[0012] b. performing power-time normalization operation on the collected SERS spectral signal and calculating the first-order derivative difference spectrum; merging the spectrum with the previously established SERS database to obtain a new spectral data set;

[0013] c. Calculate the principal component score matrix of the spectral data set using the principal component analysis algorithm;

[0014] d. Draw the scatter plots of principal component 1 (PC1) and principal component 2 (PC2), mark the scatter point classification in the database and the scatter points corresponding to the tested spectrum, and obtain the scatter point distribution map of the principal component analysis of human urine SERS.

[0015] In a preferred embodiment of the present invention, the sol test tube in step (1) comprises an upper and lower layer of high borosilicate glass tubes, which are respectively used to contain silver nanoparticle sol and nanoparticle aggregating agent, and the upper layer and the lower layer are separated by an elastic membrane, and the elastic membrane is used to seal the lower layer of nanoparticle aggregating agent and carry the upper layer of silver nano-enhanced particle sol; the top of the sol test tube is also provided with a soft rubber cap containing an elastic membrane, which is used to seal the sol test tube for convenient storage and transportation; the sol test tube is made of glass material, which is conducive to light transmission and convenient for collecting Raman signals of samples in the tube.

[0016] In a preferred embodiment of the present invention, the silver nanoparticle sol in step (1) is prepared by sodium citrate reduction method. The specific preparation method is as follows: 100 mL of 1-5 mM silver nitrate solution is heated to boil, and 5 mL of 1 wt% sodium citrate solution is added at once, and the boiling is continued for 3 hours to obtain a silver nanoparticle sol with a particle size of 80 to 100 nm.

[0017] In a preferred embodiment of the present invention, the nanoparticle aggregating agent in step (1) is one or more of HCl, NaCl, KCl, and NaOH, and the concentration of the nanoparticle aggregating agent is 1 mol / L, wherein the optimal aggregating agent is 1 mol / L HCl.

[0018] In a preferred embodiment of the present invention, the urine collected in step (2) can be directly used for testing without any pretreatment.

[0019] In a preferred embodiment of the present invention, the ratio of the silver nanoparticle sol, the nanoparticle aggregating agent and the urine in step (2) is 150-450uL:40-120uL:100-300uL.

[0020] In a preferred embodiment of the present invention, the Raman spectrometer in step (2) is a portable Raman spectrometer, and the laser excitation wavelength is 785 nm.

[0021] In a preferred embodiment of the present invention, the SERS database in step (3) is composed of surface enhanced Raman spectroscopy detection data of urine of people with different energy intakes.

[0022] In a preferred embodiment of the present invention, before the SERS database of urine is established in step (3), the surface enhanced Raman spectrum of urine is first normalized to eliminate the deviation caused by laser power and acquisition time; and then the first-order derivative difference spectrum of the spectrum is calculated to avoid interference caused by fluorescence background.

[0023] In a preferred embodiment of the present invention, the method of collecting SERS spectral signals in step (4)a is the same as that in step (3).

[0024] Compared with the prior art, the present invention has the following beneficial effects:

[0025] 1. The sol test tube designed by the present invention has the advantages of small size, light weight, easy storage, transportation and use; it can avoid the inconvenience caused by carrying nano-enhanced particle sol and nano-particle aggregating agent during on-site testing, and also saves operation steps, which is conducive to rapid and convenient testing; at the same time, it can be prepared in batches for use at any time.

[0026] 2. The urine test in the present invention does not require pre-treatment, the spectral test time is short, the spectral data analysis can get the result within 1 minute, the overall test analysis time does not exceed 2 minutes, the operation is simple and efficient.

[0027] 3. The test data and numerical calculation data obtained in the present invention are all obtained based on objective test analysis, avoiding errors caused by human subjective factors.

[0028] 4. The present invention collects SERS signals of urine of people with different energy intakes and performs principal component analysis, thereby establishing a relationship between the SERS signals of urine and energy intake, and achieving direct analysis of the SERS signals of urine to obtain information on energy intake, avoiding complex and tedious calculations, and providing a reference for the balance of energy intake of the human body; this method does not require professional technicians and any large-scale instruments and equipment to complete the test; it is simple to operate, low cost, fast, and the instrument is portable and easy to promote. It is a SERS method that is very suitable for on-site rapid evaluation of energy intake and has very broad application prospects in the biomedical field. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 Schematic diagram of the sol test tube designed by the present invention, Figure A is a stereogram, and Figure B is a cross-sectional diagram;

[0030] Among them: 1-soft rubber cap 1, 2-upper high borosilicate glass tube, 3-lower high borosilicate glass tube, 4-elastic diaphragm in the middle of the soft rubber cap, 5-elastic diaphragm between the two layers of high borosilicate glass tubes

[0031] Figure 2 is a scanning electron microscope image of the silver nanoparticles used in the present invention;

[0032] Figure 3 It is the ultraviolet visible absorption spectrum of the silver nanoparticle sol prepared by the present invention;

[0033] Figure 4 is the average surface enhanced Raman spectrum of urine of people with different energy intakes measured by the present invention, the energy intake of group A is 2300 kcal / day, and the energy intake of group B is 1500 kcal / day;

[0034] Figure 5 It is a scatter plot of principal component analysis of urine with different energy intakes measured by the present invention, wherein the energy intake of group A is 2300 kcal / day; and the energy intake of group B is 1500 kcal / day. DETAILED DESCRIPTION

[0035] The present invention is further explained below in conjunction with the accompanying drawings and specific embodiments.

[0036] 1. Design of Sol Test Tube

[0037] The sol test tube includes a soft rubber cap 1 (for sealing the reagent), an upper high borosilicate glass tube 2 (for containing nano-enhanced particles), and a lower high borosilicate glass tube 3 (for containing nano-particle aggregating agent). Figure 1 (A) is shown. The sol test tube also includes an elastic diaphragm 4 in the middle of the soft rubber cap and an elastic diaphragm 5 between two layers of high borosilicate glass tubes, which can resist a certain pressure and carry and seal reagents, and facilitate the pipette tip to pierce the elastic membrane to achieve reagent mixing, as shown in Figure 1 (B). Pre-filled liquid can be loaded into the sol test tube for use, and the specific steps are as follows: use a pipette to transfer the prepared nanoparticle aggregator to the lower layer of the sol test tube, seal it with an elastic diaphragm and connect the upper and lower layers of high borosilicate glass tubes with a heat shrink tube; then transfer the silver nano sol to the upper layer of the sol test tube, seal the upper tube with a soft rubber cap with an elastic diaphragm, and complete the entire pre-filled liquid loading process; wherein the ratio of silver nanoparticle sol, nanoparticle aggregator and urine is 150-450uL:40-120uL:100-300uL.

[0038] 2. Preparation of Silver Nanoparticle Sol

[0039] The synthesis of silver nanoparticle sol adopts sodium citrate reduction method. 100mL 1-5mM silver nitrate solution is heated to boil, and 5mL 1wt% sodium citrate solution is added at one time. The boiling is continued for 3 hours to obtain silver sol. The particle size of silver nanoparticles is 80-100nm.

[0040] 3. Urine Collection

[0041] There were 11 participants in Group A, with an energy intake of 2,300 kcal / day; there were 11 participants in Group B, with an energy intake of 1,500 kcal / day. Each experiment lasted for 3 days, and the subjects' morning urine was collected, 3 mL each. In order to achieve metabolic balance, the first two days were used as a metabolic buffer period, and no samples were collected. Instead, urine samples from the stable period on the third day were selected for signal collection. Urine does not require any pre-treatment and can be directly used for subsequent testing.

[0042] 4. Collection of SERS spectral signals

[0043] 200uL of urine collected was drawn with a pipette and mixed with the nano-enhanced particles in the upper layer of the sol test tube, and then mixed with the nano-particle aggregator in the lower layer of the sol test tube. A portable Raman instrument was used to collect the lower layer of the sol test tube at 300-1200cm -1 The Raman signal within the wavenumber range can be used to obtain the surface enhanced Raman spectrum of urine; the test parameters are: excitation light wavelength of 785nm, laser power of 100mW, integration time of 3 to 8s, and accumulation times of 2 times.

[0044] 5. Establishment of urine SERS database based on SERS spectra

[0045] The principal component analysis of the SERS spectra of the collected urine is performed to establish a database; in order to avoid deviations caused by signal fluctuations during the collection process, all spectral data need to be normalized, that is, the spectral intensity value is divided by the collection time and the collection power; the calculated spectrum set can be used for principal component analysis. The following is the specific calculation process:

[0046] (1) Calculate the first-order derivative difference spectrum for all spectra to eliminate the fluorescence background;

[0047] (2) The statistical and machine learning toolkit in MATLAB R2017a software was used to perform principal component analysis on the first-order derivative difference spectra (Group A and Group B);

[0048] (3) Draw a scatter plot of principal component 1 (PC1) and principal component 2 (PC2), and calculate the total contribution rate of the two principal components.

[0049] 6. Principal component analysis based on urine SERS database

[0050] The specific steps are as follows:

[0051] (1) Collect urine from a new person to be tested, use a pipette to transfer 200uL of urine sample, pierce the elastic diaphragm of the soft rubber cap of the sol test tube to achieve sample addition, mix the urine with the silver nanoparticle sol, and then use a pipette tip to pierce the middle elastic diaphragm to mix the urine sample-silver nanoparticle sol mixture with the nanoparticle aggregator, and carry out the test according to the above-mentioned SERS spectrum signal collection conditions;

[0052] (2) performing power-time normalization on the collected SERS spectrum and calculating a first-order derivative difference spectrum; merging the spectrum with a previously established SERS database to obtain a new spectral data set;

[0053] (3) Calculate the principal component score matrix of the spectral data set using the principal component analysis algorithm;

[0054] (4) Draw a scatter plot of principal component 1 (PC1) and principal component 2 (PC2), indicate the scatter point classification in the database and the scatter points corresponding to the tested spectrum, and the obtained scatter point distribution diagram of the principal component analysis of human urine SERS is the measurement result of the present invention.

[0055] The integrated SERS sol detection kit of the present invention has the advantages of light weight, small volume, and is very convenient for storage, transportation and use of sol. The kit is used to collect the SERS spectrum of human urine, and combines the principal component analysis algorithm to obtain an intuitive scatter distribution diagram, which can directly judge the level of human energy intake; the obtained test data and numerical calculation data are all obtained based on objective test analysis, avoiding errors caused by human subjective factors; urine test does not require pre-treatment, the spectrum test time is short, the spectrum data analysis can obtain results within 1 minute, and the overall test analysis time does not exceed 2 minutes. Therefore, the present invention has a very broad application prospect in the biomedical field.

[0056] Example 1

[0057] (1) Preparation of silver nanosol: The synthesis of silver nanosol adopts sodium citrate reduction method. 100 mL of 1.5 mM silver nitrate solution is heated to boil, and 5 mL of 1 wt % sodium citrate solution is added at once. The mixture is boiled for 3 hours to obtain silver sol.

[0058] (2) Filling the sol test tube with pre-filled liquid: Use a pipette to transfer 80uL of nanoparticle aggregating agent to the lower layer of the sol test tube, seal it with an elastic diaphragm and connect the upper and lower layers of high borosilicate glass tubes with a heat shrink tube. Then use a pipette to transfer 300uL of the prepared silver nanosol to the upper layer of the sol test tube. After completion, cover it with a soft rubber cap to complete the entire process of filling the pre-filled liquid.

[0059] (3) Urine collection: There were 11 participants in Group A, with an energy intake of 2,300 kcal / day; there were 11 participants in Group B, with an energy intake of 1,500 kcal / day. Each experiment lasted for 3 days, and the subjects' morning urine was collected, 3 mL per sample. In order to achieve metabolic balance, the first two days were used as a metabolic buffer period, and no samples were collected. Instead, urine samples from the stable period on the third day were selected for signal collection. Urine does not require any pretreatment and can be directly used for subsequent testing.

[0060] (4) Collecting SERS signals: Use a pipette to absorb the collected 200uL urine and use the pipette tip to pierce the elastic membrane at the top of the upper layer of the sol test tube and mix it with the nano-enhanced particles. Then pierce the elastic membrane 5 in the sol test tube and mix it with the nano-particle aggregator in the lower layer of the sol test tube. Finally, use a portable Raman instrument to collect the lower layer of the sol test tube at 300-1200cm -1 The Raman signal within the wave number range can obtain the surface enhanced Raman spectrum of urine. The test parameters are: excitation light wavelength 785nm, laser power 100mW, integration time 5s, accumulation times 2 times.

[0061] (5) Establishing a urine SERS database: Calculate the average spectrum of the SERS spectra of the two groups of urine collected, such as Figure 4 All SERS spectra were subjected to principal component analysis. Before establishing the database, in order to avoid deviations caused by signal fluctuations during the acquisition process, all spectral data needed to be normalized, that is, the spectral intensity value was divided by the acquisition time and the acquisition power. The obtained scatter plot is shown in Figure 5 shown.

[0062] (6) Principal component analysis based on urine SERS database: The urine SERS spectrum of the test person is collected and then preprocessed by power and time normalization and differential background subtraction. The processed data is synthesized with the SERS database and principal component analysis is performed to obtain a scatter plot, indicating the position of the test scatter points in the plot.

[0063] Example 2

[0064] (1) Preparation of silver nanosol: The synthesis of silver nanosol adopts sodium citrate reduction method. 100 mL of 2 mM silver nitrate solution is heated to boil, and 5 mL of 1 wt% sodium citrate solution is added at once. The mixture is boiled for 3 hours to obtain silver sol. The particle size of silver nanoparticles is 80-100 nm. The ultraviolet visible absorption peak of silver nanoparticles is at 404 nm. Figure 2 , Figure 3 shown.

[0065] (2) Filling the sol test tube with pre-filled liquid: Use a pipette to transfer 50uL of nanoparticle aggregating agent to the lower layer of the sol test tube, seal it with an elastic diaphragm and connect the upper and lower layers of high borosilicate glass tubes with a heat shrink tube. Then use a pipette to transfer 300uL of the prepared silver nanosol to the upper layer of the sol test tube. After completion, cover it with a soft rubber cap to complete the entire process of filling the pre-filled liquid.

[0066] (3) Urine collection: There were 11 participants in Group A, with an energy intake of 2,300 kcal / day; there were 11 participants in Group B, with an energy intake of 1,500 kcal / day. Each experiment lasted for 3 days, and the subjects' morning urine was collected, 3 mL per sample. In order to achieve metabolic balance, the first two days were used as a metabolic buffer period, and no samples were collected. Instead, urine samples from the stable period on the third day were selected for signal collection; urine did not require any pretreatment and could be directly used for subsequent testing.

[0067] (4) Collecting SERS signals: Use a pipette to absorb the collected 200uL urine and use the pipette tip to pierce the elastic membrane at the top of the upper layer of the sol test tube and mix it with the nano-enhanced particles. Then pierce the elastic membrane 5 in the sol test tube and mix it with the nano-particle aggregator in the lower layer of the sol test tube. Finally, use a portable Raman instrument to collect the lower layer of the sol test tube at 300-1200cm -1 The Raman signal within the wavenumber range can obtain the surface enhanced Raman spectrum of urine. The test parameters are: excitation light wavelength of 785nm, laser power of 100mW, integration time of 6s, and accumulation times of 2 times.

[0068] (5) Establishing a urine SERS database: Calculate the average spectrum of the SERS spectra of the two groups of urine collected. Perform principal component analysis on all SERS spectra. Before establishing the database, in order to avoid deviations caused by signal fluctuations during the acquisition process, all spectral data need to be normalized, that is, the spectral intensity value is divided by the acquisition time and the acquisition power.

[0069] (6) Principal component analysis based on urine SERS database: The urine SERS spectrum of the test person is collected and then preprocessed by power and time normalization and differential background subtraction. The processed data is synthesized with the SERS database and principal component analysis is performed to obtain a scatter plot, indicating the position of the test scatter points in the plot.

[0070] The above embodiments are only used to further illustrate a SERS rapid analysis method for energy intake of the present invention, but the present invention is not limited to the embodiments. Any simple modifications, equivalent changes and modifications made to the above embodiments based on the technical essence of the present invention fall within the protection scope of the technical solution of the present invention.

Claims

1. A SERS rapid analysis method for energy intake, characterized in that: The following steps are involved: (1) Pre-filling liquid: silver nanoparticle sol and nanoparticle aggregating agent are respectively filled into the sol test tube; the sol test tube comprises two layers of high borosilicate glass tubes, and the upper and lower layers of the borosilicate glass tubes are separated by an elastic diaphragm; the top of the sol test tube is also provided with a soft rubber cap containing an elastic diaphragm; the sol test tube is made of glass; the silver nanoparticle sol and the nanoparticle aggregating agent are respectively contained in the upper and lower layers of the high borosilicate glass tubes of the sol test tube; (2) Collecting SERS signals: collecting urine from people with different energy intakes, and mixing the urine with the silver nanoparticle sol in the sol test tube, and then mixing it with the nanoparticle aggregator in the sol test tube, and then placing the sol tube on the Raman instrument to collect SERS signals; (3) Establishing a urine SERS database: Using the principal component analysis algorithm, the scatter plot corresponding to the surface enhanced Raman spectra of urine of people with different energy intakes is obtained, and cluster analysis of energy intake is performed to establish a SERS database; (4) Principal component analysis based on urine SERS database: including the following steps: a. Collect urine from the person to be tested and acquire SERS spectral signals; b. performing power-time normalization operation on the collected SERS spectral signal and calculating the first-order derivative difference spectrum; merging the spectrum with the previously established SERS database to obtain a new spectral data set; c. Calculate the principal component score matrix of the spectral data set using the principal component analysis algorithm; d. Draw the scatter plots of principal component 1 (PC1) and principal component 2 (PC2), mark the scatter point classification in the database and the scatter points corresponding to the tested spectrum, and obtain the scatter point distribution map of the principal component analysis of human urine SERS.

2. The SERS rapid analysis method for energy intake according to claim 1, characterized in that: In the step (1), the silver nanoparticle sol is prepared by sodium citrate reduction method.

3. The SERS rapid analysis method for energy intake according to claim 1, characterized in that: In the step (1), the nanoparticle aggregating agent is one or more of HCl, NaCl, KCl, and NaOH.

4. The SERS rapid analysis method for energy intake according to claim 1, characterized in that: In the step (2), the ratio of the silver nanoparticle sol, the nanoparticle aggregating agent and the urine is 150-450uL:40-120uL:100-300uL.

5. The SERS rapid analysis method for energy intake according to claim 1, characterized in that: The urine collected in step (2) can be directly used for testing without any pre-treatment.

6. The SERS rapid analysis method for energy intake according to claim 1, characterized in that: In step (2), the Raman spectrometer is a portable Raman spectrometer, and the laser excitation wavelength is 785 nm.

7. The SERS rapid analysis method for energy intake according to claim 1, characterized in that: Before the SERS database of urine is established in step (3), the surface enhanced Raman spectrum of urine is first normalized, and then the first-order derivative difference spectrum of the spectrum is calculated.

8. The SERS rapid analysis method for energy intake according to claim 1, characterized in that: The method of collecting SERS spectral signals in step (4)a is the same as that in step (3).

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