CN115561222A

CN115561222A - Method for detecting fructose in urine based on SERS combined with functionalized substrate

Info

Publication number: CN115561222A
Application number: CN202211118705.4A
Authority: CN
Inventors: 陈国庆; 沈嘉璐; 朱纯; 吴亚敏; 辜姣; 马超群; 李磊; 高辉; 吴慧; 杨太群
Original assignee: Jiangnan University
Current assignee: Jiangnan University
Priority date: 2022-09-13
Filing date: 2022-09-13
Publication date: 2023-01-03

Abstract

The invention discloses a method for detecting fructose in urine based on SERS combined with a functional substrate, and belongs to the field of analysis and detection. The method comprises the following steps: (1) Adding silver nitrate crystals into water, incubating by heating, and adding a sodium citrate solution for reduction after incubation is finished to obtain an AgNPs substrate; (2) Adding a 4-MPBA aqueous solution into the AgNPs substrate obtained in the step (1) and uniformly mixing to obtain a functionalized AgNPs-4MPBA composite substrate; (3) Uniformly mixing urine to be detected and the SERS substrate obtained in the step (2) to obtain a sample solution to be detected; (4) Performing Raman detection on the sample solution to be detected to obtain a Raman spectrum; the characteristic peak in the Raman spectrum is 1570cm ^‑1 ±5cm ^‑1 Substituting the peak intensity into a standard curve model to obtain the concentration of fructose in the urine to be detected. The AgNPs-4MPBA can be used as an SERS substrate to detect fructose in urine within 10 minutes, and the detection limit is as low as 0.535 mu mol/L。

Description

Method for detecting fructose in urine based on SERS combined with functionalized substrate

Technical Field

The invention relates to a method for detecting fructose in urine based on SERS combined with a functional substrate, belonging to the field of analysis and detection.

Background

Fructose is monosaccharide with the highest sweetness, fruits and honey contain a large amount of fructose, and the fructose has important influence on daily life and body health of human beings. The fructose is mainly widely used in various foods as a sweetening agent, and is used for increasing the sweetness of the foods and improving the mouthfeel of the foods. Numerous medical studies have shown that excessive fructose intake causes a heavy burden on the internal organs, further leading to the development of many modern common diseases including cancer, heart disease, and the like. The intake and metabolic amount of fructose can be monitored clinically by measuring the fructose content in urine. Therefore, a sensitive and reliable fructose detection method is needed in the medical clinical and biological detection fields.

Currently, the main methods for detecting fructose in urine are: high performance liquid chromatography, fluorescence spectroscopy, and electrochemical method. These methods have a wide detection range and high reliability, but most of them are complicated and time-consuming in detection steps, and require sample pretreatment and professional instrumentation and manual work. Therefore, a simpler, faster and more sensitive method for detecting fructose is urgently needed.

The Surface Enhanced Raman Spectroscopy (SERS) technology combines the fingerprint identification capability of Raman spectroscopy and the characteristic of high sensitivity of plasma enhancement, so that the method has the effectiveness of ultra-sensitive detection. At present, the SERS technology is applied to the detection of fructose in urine, and the sensitivity and the detection limit are not superior to those of the traditional method.

Disclosure of Invention

The technical problem is as follows: the traditional method for detecting the fructose in the urine is complex and time-consuming, needs sample pretreatment, and professional instruments and manpower; at present, the SERS can be used for detecting fructose in water environment, and no document specifically discloses how to sensitively and rapidly detect fructose in urine by SERS.

The technical scheme is as follows: in order to solve at least one problem, the SERS substrate with the AgNPs-4MPBA functional composite structure is applied to detecting fructose in urine, so that the effects of short detection time, high detection precision, low detection limit and low substrate material cost are realized.

The invention provides a method for detecting fructose in urine based on SERS combined with a functionalized substrate, which comprises the following steps:

(1) Adding silver nitrate crystals into water, heating and incubating to obtain a silver nitrate aqueous solution; adding a sodium citrate solution into a silver nitrate aqueous solution for reduction to obtain an AgNPs substrate solution;

(2) Adding a 4-MPBA (4-mercaptophenylboronic acid) aqueous solution into the AgNPs substrate solution obtained in the step (1), uniformly mixing and reacting to obtain a functionalized AgNPs-4MPBA composite substrate solution;

(3) Uniformly mixing the urine to be detected and the AgNPs-4MPBA composite substrate solution obtained in the step (2) to obtain a sample solution to be detected;

(4) Performing Raman detection on the sample solution to be detected to obtain a Raman spectrum; the characteristic peak in the Raman spectrum is 1570 +/-5 cm ^-1 And substituting the peak intensity into a standard curve model to obtain the concentration of the fructose in the urine to be detected.

In one embodiment of the present invention, the concentration of the aqueous silver nitrate solution in the step (1) is 100 to 300mg/L. More preferably 170mg/L.

In one embodiment of the present invention, the incubation conditions in step (1) are: incubating at 60-100 deg.C for 15-30min. Further preferably, the incubation is carried out at 70 ℃ for 20min.

In one embodiment of the invention, the concentration of the sodium citrate solution in the step (1) is 0.5-1.5 wt%, and the addition amount is 3-5% of the volume of the silver nitrate solution; further preferably, the concentration of the sodium citrate solution is 1wt%.

In one embodiment of the invention, in step (1), the volume fraction of the sodium citrate solution added relative to the silver nitrate solution is 4%.

In one embodiment of the present invention, the conditions of the reduction reaction in step (1) are: reacting at 85-105 deg.C for 35-45min; further preferably, the reaction is carried out at 100 ℃ for 40min.

In one embodiment of the present invention, the volume ratio of the AgNPs substrate solution and the 4-MPBA solution in step (2) is 8-12:1. more preferably 10:1.

in one embodiment of the invention, the method for preparing the functionalized AgNPs-4MPBA composite substrate in the step (2) comprises the following specific steps:

under the condition of room temperature, the AgNPs substrate solution and the 4-MPBA solution are added into a container in sequence without additional heating treatment, the AgNPs substrate and the 4-MPBA solution are fully mixed through magnetic stirring, and the magnetic stirring time is 1 hour; the 4-MPBA is modified on the AgNPs substrate in a self-assembly mode, so that the functionalization of the AgNPs substrate is completed, and the AgNPs-4MPBA composite substrate is obtained.

In one embodiment of the present invention, the preparation method of the 4-MPBA aqueous solution in the step (2) specifically comprises:

adding the frozen 4-MPBA powder into water, and performing ultrasonic treatment to completely dissolve the 4-MPBA powder for 10min. Magnetically stirring for 1-3min to dissolve 4-MPBA in the water solution.

In one embodiment of the present invention, the 4-MPBA solution in the step (2) is an aqueous 4-MPBA solution having a concentration of 80 to 200. Mu. Mol/L, and more preferably 100. Mu. Mol/L.

In one embodiment of the invention, the volume ratio of the urine to be tested to the AgNPs-4MPBA composite substrate solution in the step (3) is 1:1-5. More preferably 1:1.

in one embodiment of the present invention, the blending in step (3) is performed by shaking for 1-3min.

In one embodiment of the present invention, the conditions for performing raman detection in step (4) are: raman detection is carried out by using a Raman spectrometer, the wavelength of an excitation light source of the Raman spectrometer is 532nm, the integration time is 20s, and the laser power is 100%.

In one embodiment of the present invention, the preparation method of the standard curve model in the step (4) comprises the following steps:

preparing fructose standard solutions with different concentration gradients, and mixing the standard solutions with the urine sample according to a volume ratio of 1:1, uniformly mixing to obtain a solution to be detected, and mixing the solution to be detected with AgNPs-4MPBA substrate solution according to the volume ratio of 1:1, uniformly mixing to obtain a sample solution; directly carrying out Raman detection on the sample liquid to obtain a Raman spectrum; finally, the characteristic peak 1570cm in the Raman spectrum is utilized ^-1 ±5cm ^-1 And establishing a linear model, namely a standard curve model, by using the peak intensity and the corresponding fructose concentration in the sample liquid.

In one embodiment of the present invention, in the process of constructing the standard curve, the standard solution is a solution prepared by using 500 μmol/L fructose solution as a mother solution, diluting the mother solution to 4, 20, 40, 60, 80, 100, 120, 140, 160, 200, and 400 μmol/L, and mixing the standard solution with the urine sample to obtain 2, 10, 20, 30, 40, 50, 60, 70, 80, 100, and 200 μmol/L as the solution to be measured.

In one embodiment of the invention, in the construction process of the standard curve, the fructose standard solution and the urine sample are mixed for 1-3min in a shaking way, and the urine solution of the fructose and the AgNPs-4MPBA base solution are mixed for 1-3min in a shaking way.

In one embodiment of the present invention, the standard curve model in step (4) is: i =79.90C +5251.46, correlation coefficient R ² Is 0.990, and C is the fructose concentration in the sample liquid and has a unit of mu mol/L; i is Raman 1570cm ^-1 Characteristic peak intensity.

A second object of the invention is the use of the method according to the invention in the field of biological detection.

Has the advantages that:

(1) The functionalized Raman enhanced substrate with the AgNPs-4MPBA composite structure is applied to detection, and the functionalized groups of the functionalized Raman enhanced substrate are specifically combined with a target detection object, so that the grabbing of the substrate to a target molecule to be detected is enhanced, and the detection capability is improved.

(2) The nano silver reduced by the sodium citrate has surface plasma resonance performance and plays a role in enhancing Raman signals.

(3) The AgNPs-4MPBA can be used as an SERS substrate to detect fructose in urine, the detection time is within 10 minutes, and the detection limit is as low as 0.535mg/L, which has important significance for monitoring the fructose content in urine.

(4) The invention provides a new detection method for fructose in urine, which improves the detection precision of fructose in urine.

Drawings

FIG. 1 is a flow chart of fructose detection based on surface enhanced Raman spectroscopy.

FIG. 2 shows the fructose concentration in the water environment of 1570cm in example 2 ^-1 The relationship curve of Raman intensity.

FIG. 3 is the concentration of fructose in urine environment and 1570cm in example 3 ^-1 The relationship curve of Raman intensity.

FIG. 4 is a graph showing the effect of different concentrations of 4-MPBA modified AgNPs in example 4 on the Raman characteristic peak intensity and linearity of fructose.

Fig. 5 shows the results of the raman detection using nano silver as a substrate in comparative example 1.

Fig. 6 is a result of raman detection of the composite substrate obtained by modifying AgNPs with phenylboronic acid in comparative example 2.

FIG. 7 shows the results of the test in comparative example 3.

Detailed Description

The following description of the preferred embodiments of the present invention is provided for the purpose of better illustrating the invention and is not intended to limit the invention thereto.

EXAMPLE 1 preparation of AgNPs-4MPBA substrates

The preparation method of the AgNPs-4MPBA substrate comprises the following steps:

adding 36mg of silver nitrate crystal into 100mL of water, and incubating for 20min at the temperature of 70 ℃ to obtain a completely incubated silver nitrate solution with the concentration of 170 mg/L; then, 4mL of sodium citrate aqueous solution with the mass fraction of 1% is added, and reduction reaction is carried out for 40min at 100 ℃ to obtain an AgNPs substrate (solution). Mixing AgNPs substrate solution and 4-MPBA solution with the concentration of 100 mu mol/L in a volume ratio of 10:1, mixing, and magnetically stirring for 1 hour to finally obtain the AgNPs-4MPBA substrate.

Example 2 construction of an in-Water quantitative determination model

(1) Preparing a sample solution:

taking 500 mu mol/L fructose solution as mother solution, and taking the mother solution to dilute to 0.2, 1, 2, 10, 20, 30, 40, 50, 60, 100, 150, 200 and 400 mu mol/L solution as standard solution;

the AgNPs-4MPBA substrate of example 1 and a standard solution are mixed according to the volume ratio of 1:1, mixing, and performing vortex oscillation for 1min to obtain solutions with concentrations of 0.1, 0.5, 1, 5, 10, 15, 20, 25, 30, 50, 75, 100 and 200 mu mol/L respectively as sample solutions;

(2) Carrying out Raman detection:

the obtained sample liquid does not need any pretreatment procedure, and Raman detection is directly carried out by utilizing a Raman spectrometer under the conditions that the wavelength of an excitation light source is 532nm, the integration time is 20s and the laser power is 100 percent, so that a Raman spectrum is obtained;

(3) Drawing a relation curve:

drawing the characteristic peak intensity and the fructose concentration into a relation curve, which is concretely as follows:

characteristic peak 1570cm ^-1 Treating: the Raman intensity of the fructose concentration of 1-30 mu mol/L is in linear relation with the fructose concentration, the linear regression equation is I =95.28C +5723.08, and the correlation coefficient R ² Is 0.994, and C is fructose concentration with unit of mu mol/L; i is Raman 1570cm ^-1 Characteristic peak intensity. The detection limit was calculated to be 0.084. Mu. Mol/L, as shown in FIG. 2.

Example 3 construction of a model for quantitative detection in urine

(1) Preparing a sample solution:

taking 500 mu mol/L fructose solution as mother solution, and taking the mother solution to dilute to 4, 20, 40, 60, 80, 100, 120, 140, 160, 200 and 400 mu mol/L solution as standard solution;

and (3) mixing the standard solution with the urine sample according to the volume ratio of 1:1 to obtain 2, 10, 20, 30, 40, 50, 60, 70, 80, 100 and 200 mu mol/L solution as solution to be detected;

the AgNPs-4MPBA substrate of example 1 and the solution to be tested are mixed according to the volume ratio of 1:1, mixing, and performing vortex oscillation for 1min to obtain solutions with the concentrations of 1, 5, 10, 15, 20, 25, 30, 35, 40, 50 and 100 mu mol/L respectively as sample solutions;

(2) Carrying out Raman detection:

(3) Constructing a standard curve:

and (3) constructing a standard curve by using the characteristic peak intensity and the fructose concentration, wherein the standard curve comprises the following specific steps:

characteristic peak 1570cm ^-1 Treating: the Raman intensity of the fructose concentration of 5-35 mu mol/L is in linear relation with the fructose concentration, the linear regression equation is I =79.90C +5251.46, and the correlation coefficient R ² Is 0.990, and C is fructose concentration with unit of mu mol/L; i is Raman 1570cm ^-1 Characteristic peak intensity. The detection limit was calculated to be 0.535. Mu. Mol/L, as shown in FIG. 3.

Example 4 Effect of modifying AgNPs with 4-MPBA at different concentrations on detection of Raman characteristic peak intensity and linearity of fructose

Adjusting the concentration of the 4-MPBA solution for modifying AgNPs in the step (1) of the example 3 to be 1, 10, 50, 100, 500 and 1000 mu mol/L according to the vortex shaking time of the step (1) of the example 3 to be 1 min; raman detection is carried out according to the step (2) of the embodiment 3, and the detection results are as follows:

FIG. 4 is the Raman spectrum of fructose detected after modification of AgNPs with 4-MPBA at different concentrations in example 4, and it can be seen that: when the concentration of the 4-MPBA solution is 1, 10, 50 and 1000 mu mol/L, the AgNPs-4MPBA substrate obtained after modification does not have the capability of detecting fructose, or the AgNPs-4MPBA substrate which can be used for detection cannot be formed; when the concentration of the 4-MPBA solution is 500 mu mol/L, the characteristic peak intensity is obviously increased after fructose is added, but the linearity of the characteristic peak intensity is poor at the moment, and the capability of distinguishing the fructose concentration in a low concentration interval is not provided, so that the 4-MPBA solution with the best linearity and the high Raman characteristic peak intensity of 100 mu mol/L is finally selected to modify AgNPs.

Example 5 detection of fructose in urine sample

A method for detecting fructose in urine based on SERS technology combined with a functionalized substrate comprises the following steps:

urine samples with fructose concentrations of 36. Mu. Mol/L, 56. Mu. Mol/L and the AgNPs-4MPBA substrate of example 1 were mixed in a volume ratio of 1:1, vortex and shake for 1 minute to obtain a sample solution;

raman detection is directly carried out by using a Raman spectrometer under the conditions that the wavelength of an excitation light source is 532nm, the integration time is 20s and the laser power is 100% without pretreatment, so that a Raman spectrum is obtained; the characteristic peak in the Raman spectrum is 1570cm ^-1 ±5cm ^-1 And substituting the peak intensity into a standard curve model to obtain the concentration of the fructose in the urine to be detected.

The results are shown in Table 1. As can be seen from table 1: the detection result is accurate and feasible.

Table 1 test results for example 5

Standard concentration (μmol/L)	Assay concentration (μmol/L)	Recovery (%)
			18	18.43	102.39
28	29.57	105.59

Comparative example 1

Omitting the 4-MPBA ligand in the example 1 to obtain a nano silver substrate; then, mixing the nano silver substrate and the fructose solution according to the volume ratio of 1:1, vortex and shake for 1 minute to obtain a sample solution; the obtained sample solution was subjected to raman detection, and the detection results are shown in fig. 5.

As can be seen from fig. 5: 1570cm enhanced with AgNPs-4MPBA substrate ^-1 The intensity of the characteristic peak is high and clear, and the spectrum enhanced by the traditional nano silver substrate does not show any Raman characteristic peak, so the introduction of 4-MPBA increases the combination ability of the substrate and fructose molecules, and the Raman spectrum shows that the enhancement times are obviously improved, thereby realizing the obvious change of the Raman signal intensity after the fructose to be detected is added.

1570cm ^-1 The characteristic peak belongs to a thiophenol molecule which is a product generated after the 4MPBA molecule is subjected to boron removal reaction on the AgNPs surface, and is not the characteristic peak of the probe molecule 4 MPBA. The introduction of fructose molecule can induce further boron removal reaction to generate more thiophenol, so that thiophenol at 1570cm can be used ^-1 And establishing a corresponding relation between the enhancement of the characteristic peak intensity and the fructose concentration so as to construct a working curve.

Comparative example 2

Instead of the 4-MPBA solution in example 1 being a phenylboronic acid solution, the AgNPs and phenylboronic acid solution were mixed at a 10:1 and the raman spectrum is measured as shown in fig. 6.

As can be seen from fig. 6: the overall Raman signal intensity of the AgNPs substrate modified by phenylboronic acid after fructose addition does not fluctuate significantly. After the phenylboronic acid is added into the AgNPs substrate, partial Raman characteristic peaks of the phenylboronic acid are shown, so that the background baseline is obviously increased, and the detection is interfered. In addition, the Raman signal intensity does not change obviously after the fructose is added, namely, the AgNPs modified by phenylboronic acid do not have the capability of detecting the fructose.

Comparative example 3

In alternative example 1, agNPs was AgNPs @ COF, an AgNPs substrate was mounted with a COF material, the AgNPs @ COF substrate was modified with 4-MPBA, fructose was detected using the modified AgNPs @ COF-4MPBA substrate, and the Raman spectrum was measured as shown in FIG. 7.

A preparation method of the AgNPs @ COF substrate comprises the following steps:

adding an ethyl acetate solution (30 mL) of trimesoyl chloride (TMC, 4 mmol) and an ethyl acetate solution (15 mL) of p-phenylenediamine (PPD, 3 mmol) into two 50mL centrifuge tubes respectively, and performing ultrasonic treatment for 10min to completely dissolve the solid; dropwise adding an ethyl acetate solution of p-phenylenediamine (PPD) into an ethyl acetate solution of trimesoyl chloride (TMC) at a constant speed under a stirring state at a low temperature of 10 ℃; after all the dropwise adding is finished, continuously stirring for 1h at low temperature, and then standing for 24h in a room temperature environment; centrifuging to collect the obtained yellow precipitate, and washing with deionized water and anhydrous ethanol for 3-4 times; finally drying for 8h under vacuum at 65 ℃ to obtain the COF material.

Uniformly mixing 100mL of silver nitrate solution with the concentration of 300mg/L and 10mg of COF material, and incubating for 20min at 100 ℃; then, 2mL of a 1% sodium citrate aqueous solution was added, and reduction reaction was performed at 100 ℃ for 40min to obtain an AgNPs @ COF substrate (solution).

As can be seen from fig. 7: by introducing the COF material, the overall Raman signal intensity change of the obtained AgNPs @ COF-4MPBA substrate after fructose is added is very weak, and the effect of the AgNPs @ COF-4MPBA substrate is obviously inferior to that of the AgNPs-4MPBA substrate after the detection effect of the AgNPs @ COF-4MPBA substrate is compared.

Comparative example 4

Alternative example 3 characteristic Peak 1570cm ^-1 Is a characteristic peak 1071cm ^-1 Repeating the steps (1) and (2) in example 3 while keeping the same, changing only the characteristic peak in step (3), and using the characteristic peak 1071cm ^-1 And (4) constructing a working curve of the intensity and the fructose concentration. As a result, it was found that: characteristic peak 1071cm ^-1 There is no linear trend between the intensity and the fructose concentration, because the characteristic peak isThe signal contained a signal common to both probe molecule 4MPBA and the product thiophenol produced, and was not characteristic of responding to changes in fructose concentration.

Claims

1. A method for detecting fructose in urine based on SERS combined with a functionalized substrate is characterized by comprising the following steps:

(2) Adding a 4-MPBA aqueous solution into the AgNPs substrate solution obtained in the step (1), uniformly mixing and reacting to obtain a functionalized AgNPs-4MPBA composite substrate solution;

2. The method according to claim 1, wherein the concentration of the aqueous silver nitrate solution in the step (1) is 100-300mg/L.

3. The method according to claim 1, wherein the incubation conditions in step (1) are: incubating at 60-100 deg.C for 15-30min. Further preferably, the incubation is carried out at 70 ℃ for 20min.

4. The method according to claim 1, wherein the concentration of the sodium citrate solution in step (1) is 0.5wt% to 1.5wt%.

5. The method of claim 1, wherein in step (1), the volume fraction of the sodium citrate solution added relative to the silver nitrate solution is 4%.

6. The method of claim 1, wherein the ratio of the AgNPs substrate solution to the 4-MPBA solution in step (2) is 8-12:1.

7. the method according to claim 1, wherein the 4-MPBA solution in the step (2) is an aqueous 4-MPBA solution having a concentration of 80 to 200. Mu. Mol/L.

8. The method of claim 1, wherein the volume ratio of the urine to be tested to the AgNPs-4MPBA composite substrate solution in step (3) is 1:1-5.

9. The method according to any one of claims 1 to 8, wherein the conditions for performing Raman detection in step (4) are as follows: raman detection is carried out by using a Raman spectrometer, the wavelength of an excitation light source of the Raman spectrometer is 532nm, the integration time is 20s, and the laser power is 100%.

10. The method according to claim 1, wherein the preparation method of the standard curve model in the step (4) comprises the following steps:

preparing fructose standard solutions with different concentration gradients, and mixing the standard solutions with the urine sample according to a volume ratio of 1:1, uniformly mixing to obtain a solution to be detected, and mixing the solution to be detected with an AgNPs-4MPBA substrate solution according to a volume ratio of 1:1, uniformly mixing to obtain a sample solution; directly carrying out Raman detection on the sample liquid to obtain a Raman spectrum; finally, the characteristic peak 1570cm in the Raman spectrum is utilized ^-1 ±5cm ^-1 And establishing a linear model, namely a standard curve model, by using the peak intensity and the corresponding fructose concentration in the sample liquid.