CN109001177A - The detection method of tyrosine in a kind of urine - Google Patents

Abstract

The invention relates to a method for detecting tyrosine in urine, in particular to a method for detecting surface-enhanced Raman spectroscopy. Azotyrosine is mixed with cubic nano-silver and dropped on the superhydrophobic silver film, and the Raman signal of azotyrosine is detected under a Raman spectrometer after it dries. The advantage of this method is that the concentration of tyrosine can be indirectly detected by using the enhancement effect of cubic nano-silver and the contraction effect of superhydrophobic silver film combined with the diazo coupling reaction of tyrosine. The method has a detection limit concentration of 10 ^{‑ 12} mol/L for tyrosine solution and 10 ^‑8 mol/L for tyrosine in urine, realizing ultra-trace detection with high accuracy. sex and sensitivity.

Description

A kind of detection method of tyrosine in urine

technical field

The invention relates to the technical field of amino acid detection in urine, in particular to a method for detecting tyrosine in urine.

Background technique

Tyrosine is an important semi-essential amino acid for the human body. It is an important raw material for the synthesis of various protein hormones in the human body and a precursor substance for the biosynthesis of catecholamines in the body. Diseases caused by abnormal tyrosine metabolism include albinism, Alzheimer's disease, phenylketonuria, vitiligo, and Parkinson's, among others. Its content is relatively stable in normal individuals, but in the body fluids of tumor patients, the content of tyrosine will be quite different from that of healthy people. Body fluid metabolites can also be used as markers for early detection and monitoring of various tumor processes, and the excretion of phenolic metabolites in the urine of tumor patients increases. In the study of tyrosine, there are relatively many studies on tyrosine in serum and its metabolites, but there are relatively few studies on tyrosine in human urine. Serum tumor markers have disadvantages such as cumbersome operation, high cost, invasiveness, and low early detection rate. Therefore, it is of great clinical significance to explore the new analysis technology of tyrosine content and apply it to the determination of urine samples for the auxiliary diagnosis, treatment and monitoring of cancer.

Surface-enhanced Raman scattering (SERS) technology is widely used because of its high sensitivity, high selectivity, non-destructive, simple operation and so on. Cubic nano-silver has 8 sharp corners and 12 edges. Due to the hot spot effect, the light field is localized at the corner, which greatly enhances the electric field intensity there. Its regular planar structure makes cubic nano-silver dispersive. Better characteristics, these characteristics make the cubic nano silver substrate have a good reinforcing effect. The shrinking process of the superhydrophobic silver film can enrich analytes for trace detection. The dynamic process of the adsorption of the analyte on the superhydrophobic silver film after mixing with the nano-silver colloid can be monitored in real time.

Chinese Patent No. (CN 107014806A) discloses a detection test paper for tyrosine in urine, which utilizes biological complex enzymes to catalyze tyrosine to generate specific color reaction; Chinese Patent (CN 201510962013.1) discloses a test paper for detecting human A reagent for tyrosine tumor markers in urine is composed of mercury nitrate solution, mercury sulfate solution, nickel nitrate solution, and cobalt sulfate solution; Chinese patent (CN 201510537446.2) discloses a p-hydroxyphenylalanine tyrosine Urine detection reagent, the reagent contains H ⁺ , Ni ²⁺ , Hg ⁺ , NO ^3- ions and phosphomolybdic acid and/or soluble salt of phosphomolybdic acid. The existing patented reaction principle for the detection of tyrosine in urine is generally through color reaction. These methods still have many shortcomings, such as low sensitivity, inaccurate detection results, and easy to be interfered by urine color.

Contents of the invention

In order to solve the above problems, the present invention provides a method of mixing azotized tyrosine with cubic nano-silver and dropping it on the super-hydrophobic silver film through diazo coupling reaction, and using surface-enhanced Raman spectroscopy to detect urine A new method of tyrosine. The invention is fast and simple, has high sensitivity, uniform shape of cubic nano-silver Raman base material, and high Raman activity.

The purpose of the present invention is achieved through the following technical solutions:

A method for detecting tyrosine in urine, comprising the following steps:

(1) cubic nano-silver is prepared by a one-step hydrothermal method to obtain cubic nano-silver with uniform particle size distribution;

(2) React p-aminothiophenol with sodium nitrite under ice bath conditions to generate a diazo compound, then add sodium carbonate to adjust the pH so that the entire solution is in an alkaline environment, and react with 5-7 phenols with different concentration gradients. Amino acid mixed to form azo dyes;

(3) prepare superhydrophobic silver film;

(4) Mix the azo dyes with different concentration gradients prepared in step (2) with the cubic nano-silver prepared in step (1), drop them on the superhydrophobic silver film in step (3), and let stand for 1000s-2000s Use the Renishaw Raman spectrometer to detect the Raman signal, collect the SERS spectrum of the azo dye, and establish the relationship between the intensity of the Raman spectrum and the concentration of tyrosine to draw a standard curve;

(5) Detection of unknown sample concentration: Mix the unknown concentration of urine to be detected with the cubic nano-silver prepared in step (1), drop it on the superhydrophobic silver film, and use the Renishaw Raman spectrometer to test after it dries , to obtain the Raman spectral line intensity, and calculate the concentration of tyrosine by the formula.

Preferably, the standing time of the step (4) is 1500s.

Further, the specific steps of step (1) are: first configure the silver ammonia solution, then mix 5mL silver ammonia solution with 5mL concentration of cetyltrimethylammonium bromide of 20-50mmol/L, 10mL concentration of 1- After the 10mmol/L glucose solution was fully mixed, the above mixed solution was poured into a hydrothermal reaction kettle and placed in an oven for heating reaction. The reaction time was 8 hours and the reaction temperature was 120°C to obtain nano-silver colloid.

Further, the specific steps of step (2) are: react 1mL p-aminothiophenol (10 ^-3 mol/L) with 1mL sodium nitrite 5%-8% (mass fraction) in an ice bath to generate a diazo compound , and then add 1 mL of sodium carbonate 8%-10% (mass fraction) to adjust the pH to make the whole solution under alkaline conditions, and mix with 2 mL of tyrosine of different concentrations to form azo dyes.

Further, the specific steps of step (3) are: the superhydrophobic silver film is to coat a layer of silver on the glass sheet through the silver mirror reaction, first configure the silver ammonia solution, and add a certain amount of ammonia water to 50mL of 2% (mass fraction) AgNO ₃ solution, then add 2mL of 6% (mass fraction) glucose solution to the silver ammonia solution, then put the cleaned glass plate into the above solution and heat it in a water bath at 75°C for 10 minutes, then soak the prepared silver film in After 12 hours in ethanol solution (10 ^-3 mol/L) of dodecylmercaptan, it was taken out from the solution and air-dried.

Further, in step (4), the concentration change value of the 5-7 concentration gradient tyrosine solution samples is ^10-6 mol/L ^{- 10-12} mol/L.

Further, the concentration of tyrosine in step (4) is 10 ^-6 mol/L, 10 ^-7 mol/L, 10 ^-8 mol/L, 10 ^-9 mol/L, 10 ^-10 mol/L, 10 ^-11 mol/L, 10 ^-12 mol/L.

Further, the particle size of the cubic nano silver is 40-80nm.

Further, the excitation wavelength of the Raman test is 785nm.

Compared with existing methods, the present invention has the beneficial effects of:

1. The present invention uses the diazo coupling reaction of tyrosine to indirectly detect the concentration of tyrosine, and the obtained azo dye is used as a probe molecule, and the presence of tyrosine is indirectly judged through the characteristic peak of the azo dye. The method is simple, efficient, and highly sensitive. It can not only qualitatively analyze the presence of tyrosine in urine, but also quantitatively detect the content of tyrosine in human urine. It has potential value for early screening of tumor patients.

2. The present invention adopts a one-step hydrothermal method to prepare a cubic nano-silver substrate with uniform particle size. The cubic nano-silver has 8 sharp corners and 12 edges. Due to the hot spot effect, the light field is localized at the corners, which is extremely large. The electric field strength at this place is enhanced to produce a hot spot effect, which makes it have higher activity and enhancement effect.

3. The present invention selects the superhydrophobic silver film as the substrate for Raman detection. The detection limit of tyrosine was improved by utilizing the contraction effect of the superhydrophobic silver film. Because of the shrinkage process of the superhydrophobic silver film, diffuse analytes can be enriched for detection of trace molecules. At the same time, the shrinking process of probe molecules on the superhydrophobic silver film is a dynamic process from wet state to dry state, and the dry state is the best detection state.

Description of drawings

Fig. 1 is the transmission electron microscope (TEM) figure of the cubic nano-silver of embodiment 1;

Fig. 2 is the dynamic Raman spectrogram of the tyrosine azo dye (10 ^-6 mol/L) of different time periods in Example 1;

Fig. 3 is three characteristic peak intensity changes with time of the tyrosine azo dye of embodiment 2;

Figure 4 is the different concentrations of Example 2 (10 ^-6 mol/L, 10 ^-7 mol/L, 10 ^-8 mol/L, 10 ^-9 mol/L, 10 ^-10 mol/L, 10 ^-11 mol/L L, 10 ^-12 mol/L) the surface enhanced Raman spectrogram of tyrosine;

Fig. 5 is a schematic diagram of the linear relationship between the characteristic peak intensity (1142±2cm ^-1 , 1393±2cm ^-1 1432±2cm ^-1 ) of the azo compound of Example 1 and the concentration of tyrosine;

Fig. 6 is the concentration (10 ^-4 mol/L, 10 ^-5 mol/L, 10 ^-6 mol/L, 10 ^-7 mol/L, 10 ^{- 8} mol/L) in urine of application example 1 Surface-enhanced Raman spectroscopy of azo salts of tyrosine;

Fig. 7 is a schematic diagram of the linear relationship between the characteristic peak intensity of azo compounds in urine at 1393±2cm ^-1 and the concentration of tyrosine in urine according to Example 1.

Detailed ways

In order to better understand the present invention, the present invention will be further described through the following examples, which are only used to explain the present invention and do not constitute any limitation to the present invention.

The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, and the raw materials used are all commercially available products.

Example 1

(1) Preparation of cubic nano-silver by hydrothermal method

First configure silver ammonia solution, take 0.01mol/L silver nitrate aqueous solution, add dropwise dilute ammonia water 2% (mass fraction, the same below) solution from clarification to precipitation until the precipitation just disappears. 5mL silver ammonia solution and 5mL cetyltrimethylammonium bromide (20mmol/L) plus 10mL glucose solution (10mmol/L), fully stirred and mixed evenly, poured into a 25mL hydrothermal reaction kettle , put the reactor in an oven at 120°C for 8h. After the reaction was completed, it was naturally cooled to room temperature, centrifuged at 10,000 rpm for 20 min, and washed three times to remove excess cetyltrimethylammonium bromide. A cubic nanosilver with a diameter of 75nm and uniform particle size was obtained, as shown in FIG. 1 .

(2) Diazo coupling reaction of tyrosine

In an ice bath, first take 1 mL of p-aminothiophenol (10 ^-3 mol/L) in a test tube, add 1 mL of sodium nitrite aqueous solution (5%), and stir for 1 min. Then mix 1 mL of sodium carbonate (8%) with 2 mL Tyrosine with a concentration of 10 ^-6 mol/L was quickly added into the test tube at the same time and stirred for 1 min to generate an azo dye. The structure looks like this:

(3) Determination of tyrosine concentration

Take 1.5 μL of azotized tyrosine mixed with cubic nano-silver and drop it on the superhydrophobic silver film, use Renishaw Raman spectrometer to detect Raman signal, the excitation wavelength is 785nm, collect the SERS of azo salt spectrum. Using 10 ^-6 mol/L azotyrosine as the probe molecule, the Raman signal is the most stable when the probe molecule is in the dry state, as shown in Figure 2. Before 1000s, there is almost no Raman signal. This is because in the initial stage, the nanoparticles in the sol are doing random Brownian motion, and they are in a wet state at this time; the Raman intensity reaches the maximum at 1500s, but then the Raman intensity The time of this process is too short to accurately measure the signal of the probe molecule; when standing for 1500s, the Raman signal is the strongest, and after 2000s, the Raman signal of the probe molecule is in a stable state, and the peak intensity is at a plateau. Then fluctuate, at this time in a dry state.

Example 2

(1) Preparation of cubic nano-silver by hydrothermal method

First configure silver ammonia solution, take 0.01mol/L silver nitrate aqueous solution, add dropwise dilute ammonia water 2% (mass fraction, the same below) solution from clarification to precipitation until the precipitation just disappears. 5mL silver ammonia solution and 5mL cetyltrimethylammonium bromide (20mmol/L) plus 10mL glucose solution (10mmol/L), fully stirred and mixed evenly, poured into a 25mL hydrothermal reaction kettle , put the reaction kettle in an oven at 120°C for 8 hours, cool down to room temperature naturally after the reaction, centrifuge at 10,000 rpm for 20 minutes, wash three times, remove excess cetyltrimethylammonium bromide, and obtain a particle with a diameter of 50nm and a particle size of Uniform cubic nanosilver, as shown in Figure 1.

(2) Diazo coupling reaction of tyrosine

In an ice bath, first take 1 mL of p-aminothiophenol (10 ^-3 mol/L) in a test tube, add 1 mL of sodium nitrite aqueous solution (5%), stir for 1 min, then mix 1 mL of sodium carbonate (8%) with 2 mL Different concentrations of tyrosine (10 ^-6 mol/L, 10 ^-7 mol/L, 10 ^-8 mol/L, 10 ^-9 mol/L, 10 ^-10 mol/L, 10 ^-11 mol/L, 10 ^-12 mol/L) was quickly added into the test tube and stirred for 1 min to generate azo dyes.

(3) Determination of tyrosine concentration

Take 1.5 μL of azotized tyrosine mixed with cubic nano-silver and drop it on the super-hydrophobic silver film. After standing for 1500s, use the Renishaw Raman spectrometer to detect the Raman signal. The excitation wavelength is 785nm. The SERS spectrum of the azo salt is shown in Figure 3, and the surface-enhanced Raman spectrum of different concentrations of tyrosine is measured. As shown in Figure 4, as the concentration of tyrosine gradually increases, the corresponding Raman spectrum signal also gradually increases, according to the characteristic peaks of azo compounds 1142±2cm ^-1 , 1393±2cm ^-1 , 1432±2cm ^-1 Draw a standard curve with respect to the concentration of tyrosine. As shown in Figure 5, the linear fitting degree between the concentration and signal intensity of each characteristic peak is good, and the standard curve drawn with the characteristic peak at 1432±2cm ^-1 has the best linear fitting degree.

Example 3

(1) Preparation of cubic nano-silver by hydrothermal method

First configure silver ammonia solution, take 0.01mol/L silver nitrate aqueous solution, add dropwise dilute ammonia water 2% (mass fraction, the same below) solution from clarification to precipitation until the precipitation just disappears. 5mL silver ammonia solution and 5mL cetyltrimethylammonium bromide (30mmol/L) plus 10mL glucose solution (5mmol/L), fully stirred and mixed evenly, poured into a 25mL hydrothermal reaction kettle , put the reactor in an oven at 120°C for 8h. After the reaction was completed, it was naturally cooled to room temperature, centrifuged at 10,000 rpm for 20 min, and washed three times to remove excess cetyltrimethylammonium bromide. A cubic nanosilver with a diameter of 75nm and uniform particle size was obtained, as shown in FIG. 1 .

(2) Diazo coupling reaction of tyrosine

In an ice bath, first take 1mL of p-aminothiophenol (10 ^-3 mol/L) in a test tube, add 1mL of sodium nitrite aqueous solution (8%), and stir for 3min. Then mix 1mL of sodium carbonate (10%) with 2mL Different concentrations of tyrosine (10 ^-6 mol/L, 10 ^-7 mol/L, 10 ^-8 mol/L, 10 ^-9 mol/L, 10 ^-10 mol/L, 10 ^-11 mol/L, 10 ^-12 mol/L) was quickly added into the test tube and stirred for 1 min to generate azo dyes.

(3) Determination of tyrosine concentration

Take 1.5 μL of azotized tyrosine mixed with cubic nano-silver and drop it on the super-hydrophobic silver film. After standing for 1500s, use the Renishaw Raman spectrometer to detect the Raman signal. The excitation wavelength is 785nm. SERS spectra of azo salts.

Application Example 1 detects the content of tyrosine in urine

In this application, the preceding steps are the same as step (1) in Example 2.

(1) Take urine samples from healthy young people and dilute them with different concentrations of tyrosine solutions to 10 ^-4 mol/L, 10 ^-5 mol/L, 10 ^-6 mol/L, 10 ^-7 mol/L, 10 ^-8 mol/L. In an ice bath, first take 1 mL of p-aminothiophenol (10 ^-3 mol/L) in a test tube, add 1 mL of sodium nitrite aqueous solution (5%), and stir for 1 min. Then 1 mL of sodium carbonate (10%) was mixed with 2 mL of tyrosine urine solution of different concentrations (10 ^-4 mol/L, 10 ^-5 mol/L, 10 ^-6 mol/L, 10 ^-7 mol/L, 10 ^-8 mol/L) was quickly added to the test tube and stirred for 1 min to generate an azo dye, and the urine of a blank healthy youth was directly prepared as a control.

(2) Mix 1.5 μL of azotized tyrosine in urine with 1.5 μL of cubic nano-silver and drop it on the super-hydrophobic silver film. Use Renishaw Raman spectrometer to detect the Raman signal. The excitation wavelength is 785nm, collect the SERS spectrum of tyrosine azo salt in urine. As shown in Figure 6, it can be seen from the figure that when the blank urine is directly detected, the Raman signal of the azo compound is not obvious, but it still exists, indicating that there is also a small amount of tyrosine in the urine of normal people; while adding different concentrations of tyrosine Obvious Raman signals of azo compounds can be observed in the urine after tyrosine, indicating that the method can be applied to the analysis and detection of tyrosine in urine. As shown in Figure 7, the linear fit between the concentration of the characteristic peak at 1393±2cm ^-1 and the intensity of the Raman signal is good, indicating that urea in urine has little effect on tyrosine. In order to verify the accuracy of the standard curve, the recovery test was carried out by preparing samples with 3 different concentrations in the linear range to obtain the relevant SERS signal, and then calculate the ratio of the measured concentration to the relevant concentration, that is, the recovery (%). The recovery rate experiment is as shown in list 1, and the recovery rate of tyrosine in the urine calculated by the standard curve drawn by the characteristic peak at 1393 ± 2cm-1 can be seen from the following table. Tyrosine has high sensitivity and small error, and the high recovery rate shows that it is feasible to detect tyrosine in urine by surface-enhanced Raman spectroscopy.

Table 1 is the test of the recovery rate of tyrosine in urine

The above is only an embodiment of the present invention, and does not limit the patent scope of the present invention. Any equivalent structure or equivalent process conversion made by using the content of the description of the present invention, or directly or indirectly used in other related technical fields, shall be The same reasoning is included in the patent protection scope of the present invention.

Claims (9)

1. a method for detecting tyrosine in urine, comprising the following steps: Prepare cubic nano-silver by one-step hydrothermal method to obtain cubic nano-silver with uniform particle size distribution; React p-aminothiophenol with sodium nitrite aqueous solution under ice bath conditions to form a diazo compound, then add sodium carbonate to adjust the pH to make the whole solution in an alkaline environment, and mix with 5-7 different concentration gradients of tyramine respectively Acid mixed to form azo dyes; Preparation of superhydrophobic silver film; Mix the azo dyes with different concentration gradients prepared in step (2) with the cubic nano-silver prepared in step (1), drop them on the super-hydrophobic silver film, let it stand for 1000s-2000s, and then use Renishaw Raman spectrometer to pull To detect the Man signal, collect the SERS spectrum of the azo dye, establish the relationship between the intensity of the Raman spectrum and the concentration of tyrosine, and draw a standard curve; Detection of unknown sample concentration: Mix the unknown concentration of urine to be tested with the cubic nano-silver prepared in step (1), drop it on the super-hydrophobic silver film, and use the Renishaw Raman spectrometer to test it after it dries to obtain Ra The intensity of the Mann spectrum was used to calculate the concentration of tyrosine through the formula. 2. A method for detecting tyrosine in urine according to claim 1, wherein the method for preparing cubic nano-silver by hydrothermal method in step (1) specifically comprises the following steps: first configure silver ammonia solution, Then 5mL of silver ammonia solution and 5mL of cetyltrimethylammonium bromide with a concentration of 20-50mmol/L and 10mL of glucose solution with a concentration of 1-10mmol/L are fully mixed to obtain a mixed solution. The liquid is poured into a hydrothermal reaction kettle, and then placed in an oven for heating reaction. The reaction time is 8 hours, and the reaction temperature is 120° C., centrifuged and washed to obtain nano-silver colloid. 3. The method for detecting tyrosine in urine according to claim 1, characterized in that: the resting time of the step (4) is 1500s. 4. A method for detecting tyrosine in urine according to claim 1, characterized in that the specific steps of step (2) are: adding 1 mL of p-aminothiophenol with a concentration of 10 ^-3 mol/L React with 1mL of sodium nitrite aqueous solution with a mass fraction of 5%-8% in an ice bath and stir for 1min-3min to form a diazo compound, then add 1mL-2mL of sodium carbonate with a mass fraction of 8%-10% to adjust the pH Make the whole solution under alkaline conditions, mix with 2mL of tyrosine with different concentration gradients to generate azo dyes. 5. A method for detecting tyrosine in urine according to claim 1, characterized in that in step (4), the concentration change values of the 5-7 concentration gradient tyrosine solution samples are 10 ^-6 mol/L-10 ^-12 mol/L. 6. The method for detecting tyrosine in a kind of urine according to claim 5, characterized in that: the concentration of the tyrosine solution sample is ^10-6 mol/L, ^10-7 mol/L, 10 ^-8 mol/L, 10 ^-9 mol/L, 10 ^-10 mol/L, 10 ^-11 mol/L, 10 ^-12 mol/L. 7. The method for detecting tyrosine in urine according to claim 1, characterized in that, the specific steps of step (3) are: the superhydrophobic silver film is coated with a layer of silver on the glass sheet through the silver mirror reaction , first configure the silver ammonia solution, drop the ammonia solution with a mass fraction of 2% into 50mL of the AgNO3 solution with a mass fraction of ₂ %, until the solution just disappears from clarification, add 2mL of glucose with a mass fraction of 6% solution in silver ammonia solution; then put the cleaned glass plate into the above solution and heat it in a water bath at 75°C for 10 minutes, then soak the prepared silver film in dodecyl mercaptan with a concentration of 10 ^-3 mol/L After 12 hours in the ethanol solution, remove from the solution and air dry. 8. A method for detecting tyrosine in urine according to claim 1, characterized in that: the particle size of the cubic nanosilver is 40-80nm. 9. A method for detecting tyrosine in urine according to claim 1, characterized in that: the excitation wavelength of the Raman test is 785nm.