CN106124476A - Based on surface enhanced raman spectroscopy and the glucose sensing approach of bi-molecular probe - Google Patents

Abstract

The invention provides a glucose detection method based on surface-enhanced Raman scattering (SERS) and bimolecular probes, which provides a method with strong specificity and high sensitivity for glucose detection, which can be used not only for direct blood glucose detection, but also for For the detection of human samples with low glucose content such as urine, saliva and sweat. SERS technology realizes high-sensitivity and non-invasive glucose detection. Gold-silver core-shell nanorods with highly controllable properties such as shape and size are used to prepare SERS active substrates, and the plasmon wavelength of the substrate can be precisely adjusted to match the incident light. Resonance, so as to achieve the highest Raman enhancement factor, and maximize the detection sensitivity. The dual-molecular probe technology of 4-mercaptophenylboronic acid and 4-cyanophenylboronic acid is used to realize the specific detection of glucose. The characteristic Raman peak of 4-cyanophenylboronic acid is located in the biological quiet zone, thus effectively avoiding the The interference of other endogenous biomolecules ensures the specificity and accuracy of the detection.

Description

Glucose detection method based on surface-enhanced Raman scattering and bimolecular probes

technical field

The invention relates to the field of blood glucose detection, more specifically, to a glucose quantitative detection method based on surface-enhanced Raman scattering and bimolecular probes.

Background technique

Direct blood testing is commonly used clinically and in household blood glucose detectors, and frequent blood collection will bring discomfort to patients. Urine glucose test is the simplest method of non-invasive diabetes screening. There is almost no glucose in the urine of healthy people. Only when the blood glucose concentration is higher than the renal glucose threshold (8.88~9.99 mmol/l), glucose will appear in the urine. And the concentration has a good correlation with the blood glucose concentration. However, due to the influence of various factors, the screening test strip cannot accurately reflect the blood glucose concentration, so its reference value is limited. Studies have confirmed that for individuals, the glucose concentration in body fluids such as saliva and sweat before meals also has a good correlation with blood glucose concentration, which can be used to indirectly reflect blood glucose concentration. If the glucose content can be accurately measured, it will be used in Diabetes early screening, blood glucose detection and other fields have great application value, but because of its low glucose concentration (generally 2*10 ^-2 mmol/l ~ 2.4*10 ^-1 mmol/l), it is not yet possible to use commercially available blood glucose The detection instrument detects it accurately. Therefore, a glucose quantitative detection method with high detection sensitivity and fast speed will bring new opportunities for clinical application.

Surface-enhanced Raman scattering (SERS) detection technology is a non-invasive, high-sensitivity fingerprint-type spectral detection technology, which can give information about the structure of substances at the molecular level, and can enhance the Raman signal by up to 10 ¹⁴ times. Therefore, SERS detection technology has wide application value in the field of biomedical sensing and detection. Chinese patent CN103411949A discloses a method for indirect detection of glucose content in serum using SERS technology. The method is to produce hydrogen peroxide molecules through glucose under the action of glucose oxidase, and in the presence of the produced hydrogen peroxide molecules, through The surface-enhanced Raman signal intensity of the oxidized chromogenic substrate molecule is used to indirectly obtain the glucose content in the serum. The feasibility of the surface-enhanced Raman technique in the quantitative detection of glucose was proved.

Malini Olivo et al. (Journal Papers, Biosensors and Bioelectronics, 56 (2014)186– 191; J. Am. Chem. Soc. 2013, 135, 18028−18031) proposed the use of sandwich bimolecular probe technology to achieve the specificity of glucose Sexual detection, using 4-mercaptobenzoic acid-triosmium carbonyl cluster conjugates or alkynylphenylboronic acid in combination with 4-mercaptophenylboronic acid to selectively capture glucose molecules for SERS detection to achieve highly selective quantification of glucose detection. However, since this method prepares a relatively uniform nanostructured SERS active substrate on a glass slide by spin-coating polymer microspheres and sputtering silver and gold films, the stability time of the substrate is short, and due to the plasmonic excitation of its nanostructure The meta-wavelength cannot be adjusted, resulting in limited detection sensitivity, which is not conducive to clinical application.

Contents of the invention

In order to overcome the defect of limited detection sensitivity in the above prior art, the present invention provides a glucose detection method based on surface-enhanced Raman scattering and bimolecular probes with strong specificity, high sensitivity and non-invasiveness.

In order to solve the problems of the technologies described above, the technical solution of the present invention is as follows:

A glucose detection method based on surface-enhanced Raman scattering (SERS) and bimolecular probes, the method comprising the following steps:

S1: uniformly deposit the prepared metal nanorods on the surface of the carrier to prepare a surface-enhanced Raman scattering (SERS) active substrate, and adjust the size and aspect ratio of the metal nanorods to make the plasmon of the SERS active substrate The excimer wavelength resonates with the incident light;

S2: modifying the SERS active substrate with primary glucose receptor molecules to immobilize it on the SERS active substrate;

Soak the SERS active substrate in different concentrations of glucose standard aqueous solution, and capture the glucose in the solution onto the SERS active substrate;

Adding secondary glucose receptor molecules to the SERS active substrate, the secondary glucose receptor molecules selectively covalently bind to the glucose molecules on the substrate;

Carry out SERS spectrum test, use the Raman characteristic peak intensity of the secondary glucose receptor molecule to calibrate the standard glucose aqueous solution of known concentration, and establish a quantitative analysis standard curve;

S3: Collect the sample to be tested with unknown glucose concentration, after appropriate dilution, process and SERS spectrum test with the method of step S2, and substitute the Raman characteristic peak intensity of the obtained secondary grape receptor molecule into the quantitative analysis established in step S2 standard curve to obtain the glucose concentration value of the sample to be tested.

In a preferred solution, the coin group metal nanorods are gold-silver core-shell nanorods.

In a preferred solution, the carrier is a cover glass.

In a preferred solution, the carrier is filter paper or filter membrane.

In a preferred solution, the instrument used in the SERS spectrum test is a Raman spectrometer, and the wavelength range of the excitation light is 400-1000 nm.

In a preferred solution, the samples to be tested include blood, urine, saliva, and sweat.

In a preferred solution, the primary glucose acceptor molecule is 4-mercaptophenylboronic acid molecule, which is immobilized on the SERS active substrate.

In a preferred solution, the secondary glucose acceptor molecule is 4-cyanophenylboronic acid, which binds to the glucose molecule on the SERS active substrate.

In a preferred scheme, the glucose concentration is quantitatively detected by using the cyano-Raman characteristic peak of 4-cyanophenylboronic acid.

Compared with the prior art, the beneficial effect of the technical solution of the present invention is: the present invention provides a glucose detection method based on surface-enhanced Raman scattering and bimolecular probes, which provides a specificity and sensitivity for glucose detection. High means can be used not only for blood sugar detection, but also for detection of human samples with low glucose content such as urine, saliva and sweat. SERS technology realizes high-sensitivity and non-invasive glucose detection. Gold-silver core-shell nanorods with highly controllable properties such as shape and size are used to prepare SERS active substrates. The plasmon wavelength of the substrate can be precisely adjusted to match the incident light. Resonance, so as to achieve the highest Raman enhancement factor, and maximize the detection sensitivity. The dual molecular probe technology of 4-mercaptophenylboronic acid and 4-cyanophenylboronic acid is used to realize the specific detection of glucose. The characteristic Raman peak of the cyano group of 4-cyanophenylboronic acid is located in the biological quiet area, thus effectively The interference of other endogenous biomolecules is avoided, and the specificity and accuracy of detection are guaranteed.

Description of drawings

Fig. 1 is a schematic diagram of the glucose detection method of the present invention.

Figure 2 is a schematic diagram of a gold-silver core-shell nanorod SERS substrate.

Figure 3 is a schematic diagram of glucose detection.

detailed description

The accompanying drawings are for illustrative purposes only and cannot be construed as limiting the patent;

In order to better illustrate this embodiment, some parts in the drawings will be omitted, enlarged or reduced, and do not represent the size of the actual product;

For those skilled in the art, it is understandable that some well-known structures and descriptions thereof may be omitted in the drawings.

The technical solutions of the present invention will be further described below in conjunction with the accompanying drawings and embodiments.

Example 1

As shown in Figures 1-3, the glucose detection method based on surface-enhanced Raman scattering and bimolecular probes, the method comprises the following steps:

S1: The prepared gold-silver core-shell nanorods (silver-coated gold nanorods) were uniformly deposited on the surface of the cover glass to prepare a SERS active substrate, and the size and aspect ratio of the gold-silver core-shell nanorods were adjusted to make The plasmon wavelength of the SERS active substrate resonates with the incident light;

S2: modifying the SERS active substrate with 4-mercaptophenylboronic acid, so that 4-mercaptophenylboronic acid is immobilized on the SERS active substrate as the primary glucose receptor;

Soak the SERS active substrate in different concentrations of glucose standard aqueous solution, and capture the glucose in the solution onto the SERS active substrate;

Adding the secondary glucose acceptor 4-cyanophenylboronic acid to the SERS active substrate, the 4-cyanophenylboronic acid molecule selectively covalently binds to the glucose molecule on the substrate;

Carry out SERS spectrum test, use the Raman characteristic peak intensity of 4-cyanophenylboronic acid to calibrate the standard glucose aqueous solution of known concentration, and establish a quantitative analysis standard curve;

S3: Use a Raman spectrometer with an excitation light wavelength range of 400-1000 nm to collect blood, urine, saliva or sweat samples with unknown glucose concentration, and after appropriate dilution, use the method of step S2 for processing and SERS spectrum testing. The obtained Raman characteristic peak intensity of 4-cyanophenylboronic acid is substituted into the quantitative analysis standard curve established in step S2 to obtain the glucose concentration value of the sample to be tested.

The invention provides a glucose detection method based on surface-enhanced Raman scattering and bimolecular probes, which provides a method with strong specificity and high sensitivity for glucose detection, which can be used not only for blood sugar detection, but also for urine, Detection of human samples with low glucose content such as saliva and sweat. SERS technology realizes high-sensitivity and non-invasive glucose detection. Gold-silver core-shell nanorods with highly controllable properties such as shape and size are used to prepare SERS active substrates. The plasmon wavelength of the substrate can be precisely adjusted to match the incident light. Resonance, so as to achieve the highest Raman enhancement factor, and maximize the detection sensitivity. The dual molecular probe technology of 4-mercaptophenylboronic acid and 4-cyanophenylboronic acid is used to realize the specific detection of glucose. The characteristic Raman peak of 4-cyanophenylboronic acid is located in the biological quiet area, thus effectively avoiding the The interference of other endogenous biomolecules ensures the specificity and accuracy of the detection.

The same or similar reference numerals correspond to the same or similar components;

The terms describing the positional relationship in the drawings are only for illustrative purposes and cannot be interpreted as limitations on this patent;

Apparently, the above-mentioned embodiments of the present invention are only examples for clearly illustrating the present invention, rather than limiting the implementation of the present invention. For those of ordinary skill in the art, other changes or changes in different forms can be made on the basis of the above description. It is not necessary and impossible to exhaustively list all the implementation manners here. All modifications, equivalent replacements and improvements made within the spirit and principles of the present invention shall be included within the protection scope of the claims of the present invention.

Claims (9)

1. based on surface enhanced raman spectroscopy and the glucose sensing approach of bi-molecular probe, it is characterised in that described method bag Include following steps:

S1: by the coin race metal nano-rod uniform deposition for preparing at carrier surface, is prepared as SERS active-substrate, regulates coin race The size of metal nano-rod and transverse and longitudinal ratio, make the phasmon wavelength of SERS active-substrate resonate with incident illumination；

S2: by primary glucoreceptor molecular modification SERS active-substrate so that it is be fixed in SERS active-substrate；

SERS active-substrate is soaked in the Glucose standards aqueous solution of variable concentrations, the glucose in solution is captured In SERS active-substrate；

Adding two grades of glucoreceptor molecules in SERS active-substrate, two grades of glucoreceptor molecules are selective and in substrate Glucose molecule covalent bond；

Carry out SERS spectra test, demarcate the standard of concentration known with the Raman signatures peak intensity of two grades of glucoreceptor molecules D/W, sets up quantitative analysis standard curve；

After S3: gather the sample to be tested that concentration of glucose is unknown, suitably dilution, carry out processing and SERS light by the method for step S2 Spectrum test, substitutes into, by the Raman signatures peak intensity of obtain two grades of Fructus Vitis viniferae acceptor molecules, the quantitative analysis standard that step S2 is set up Curve, obtains the glucose concentration value of sample to be tested.

The most according to claim 1 based on surface enhanced raman spectroscopy with the glucose sensing approach of bi-molecular probe, institute Shu Bi race metal nano-rod is Jin-silver core-shell nanometer rod.

The most according to claim 1 based on surface enhanced raman spectroscopy with the glucose sensing approach of bi-molecular probe, its Being characterised by, described carrier is coverslip.

The most according to claim 1 based on surface enhanced raman spectroscopy with the glucose sensing approach of bi-molecular probe, its Being characterised by, described carrier is filter paper or filter membrane.

The most according to claim 1 based on surface enhanced raman spectroscopy with the glucose sensing approach of bi-molecular probe, its Being characterised by, the instrument that SERS spectra test uses is Raman spectrometer, and the wave-length coverage of its exciting light is 400 ~ 1000 nm.

The most according to claim 1 based on surface enhanced raman spectroscopy with the glucose sensing approach of bi-molecular probe, its Being characterised by, described sample to be tested includes blood, urine, saliva, perspiration.

The most according to claim 1 based on surface enhanced raman spectroscopy with the glucose sensing approach of bi-molecular probe, its Being characterised by, described primary glucoreceptor molecule uses 4-mercaptophenyl boronic acid molecule, and it is fixed in SERS active-substrate.

The most according to claim 1 based on surface enhanced raman spectroscopy with the glucose sensing approach of bi-molecular probe, its Being characterised by, described two grades of glucoreceptor molecules use 4-cyanophenylboronic acid, itself and the Fructus Vitis viniferae sugar in SERS active-substrate Son combines.

The most according to claim 8 based on surface enhanced raman spectroscopy with the glucose sensing approach of bi-molecular probe, its It is characterised by, uses the cyano group raman characteristic peak of 4-cyanophenylboronic acid that concentration of glucose is carried out detection by quantitative.