CN107505302B

CN107505302B - Application of rice-shaped leaf nitrogen-doped carbon nanoribbon in biological thiol detection

Info

Publication number: CN107505302B
Application number: CN201710857274.6A
Authority: CN
Inventors: 孟祥英; 伊正君; 乔晋娟; 楚海荣; 王颖; 陈祥雨
Original assignee: Weifang Medical University
Current assignee: Weifang Medical University
Priority date: 2017-09-21
Filing date: 2017-09-21
Publication date: 2020-07-07
Anticipated expiration: 2037-09-21
Also published as: CN107505302A

Abstract

The invention discloses a fluorescence detection method of unmarked biological mercaptan based on rice leafy nitrogen-doped carbon nanoribbon and application thereof, wherein the detection steps comprise that ① phosphate buffer solution, rice leafy nitrogen-doped carbon nanoribbon fluorescence probe solution and silver ion solution are sequentially added into a container;② adding standard solutions containing biological thiol with different concentrations and a sample to be tested containing biological thiol with unknown concentration, ③ diluting and mixing uniformly, incubating, ④ determining the change of fluorescence intensity of each group of solutions, ⑤ drawing a standard curve with the concentration of biological thiol added in the standard solution group as the horizontal axis and the fluorescence recovery degree of the standard solution as the vertical axis, ⑥ comparing the fluorescence recovery degree of the sample to be tested with the standard curve to determine the biological thiol concentration of the sample, wherein NRPCNRs-Ag is used for determining the biological thiol concentration of the sample⁺The reversible complexation of (2) can realize fluorescence recovery in the biological thiol without fluorescence labeling and can realize real-time quantitative analysis.

Description

Application of rice-shaped leaf nitrogen-doped carbon nanoribbon in biological thiol detection

Technical Field

The invention relates to the field of trace detection, in particular to a rice leaf-shaped nitrogen-doped carbon nanoribbon-based unmarked biological mercaptan fluorescence detection method and preparation and application thereof.

Background

Biological thiol mercapto compounds, typically include cysteine (Cys), homocysteine (Hcy), Glutathione (GSH). As these molecules play important physiological roles in organisms, they have attracted increasing attention from researchers. Numerous studies have demonstrated that imbalances in the organism of sulfhydryl compounds are closely related to many diseases. For example, excessive concentrations of cysteine in humans can cause neurotoxicity, while cysteine deficiency can lead to edema, slow growth, leukopenia, hair loss, and other disorders of the body. Elevated homocysteine concentrations are also closely associated with many diseases such as cardiovascular disease, osteoporosis, senile dementia, and the like. In addition, if the glutathione level in the human body is abnormal, the functions of the immune system of the body can be inhibited, the aging is accelerated, and the glutathione level is related to some diseases such as diabetes, tuberculosis, AIDS and the like. Since, by detecting the levels of these compounds in biological samples such as plasma, living cells or urine, diagnostic basis can be provided for the diagnosis of clinically relevant diseases. Therefore, the development of an effective detection method for biological thiol has very important practical significance.

Currently, many methods have been developed for the detection of biological thiols, including high performance liquid chromatography-mass spectrometry, zonal capillary electrophoresis, electrochemical methods, and inductively coupled plasma-mass spectrometry (ICP-MS for short), among others. However, these detection techniques often have some unavoidable disadvantages, such as expensive and heavy instruments, complicated operation procedures, long measurement time, high skill requirements for operators, the need for a skilled person to operate, and the like. To overcome the above-mentioned main problems, the technology for detecting bio-thiol based on fluorescent probe has received more and more attention and has been widely developed. However, despite their high selectivity and sensitivity to biological thiols, these methods often involve complex and expensive modification of molecular beacon probes and labeling of fluorophores and fluorescence quenchers, so such sensors tend to be time consuming and require cumbersome pre-processing procedures. Therefore, it is needed to develop a simple and rapid fluorescence sensing detection technology with high sensitivity and good selectivity for the analysis and detection of biological thiol.

Disclosure of Invention

In view of the problems of the prior art, the invention aims to provide a fluorescence detection method of unmarked biological thiol based on rice leaf-shaped nitrogen-doped carbon nanoribbons, which utilizes NRPCNRs-Ag⁺The reversible complexation of (2) can realize fluorescence recovery in the biological thiol without fluorescence labeling and can realize real-time quantitative analysis.

A fluorescence detection method of unmarked biological thiol based on rice leaf-shaped nitrogen-doped carbon nanoribbons is provided, and comprises the steps of ① sequentially adding Phosphate Buffered Saline (PBS), rice leaf-shaped nitrogen-doped carbon nanoribbon (NRPCNRs) fluorescence probe solution and silver ion solution into a container, ② grouping and adding standard solutions containing biological thiol with different concentrations and to-be-detected samples containing biological thiol with unknown concentrations, ③ diluting and uniformly mixing the solutions, incubating the solutions, ④ detecting the change of the fluorescence intensity of each group of solutions, ⑤ detecting the change of the fluorescence intensity of each group of solutions according to the standardConcentration of biological thiol added to the solution group is horizontal axis, and fluorescence recovery degree of standard solution [ (FL-FL)₀)]/FL₀And ⑥, comparing the fluorescence recovery degree of the sample to be detected with the standard curve to judge the concentration of the biological thiol of the sample.

Preferably, in step ①, the mass ratio of the rice leaf-shaped nitrogen-doped carbon nanoribbon fluorescent probe to the silver ions is (2-3): 1.

Preferably, in step ③, the concentration of the rice leaf-shaped nitrogen-doped carbon nanoribbon fluorescent probe is 10-25 μ g/ml, and the concentration of silver ions is 70-90 μ M.

Preferably, in step ④, the leaf-shaped nitrogen-doped carbon nanoribbons have a nitrogen doping content of (35-37) wt%, preferably, a fluorescence excitation wavelength of 355nm and an excitation and emission slit width of 3nm, and the bio-thiol comprises at least one of cysteine and glutathione, and more preferably, the sample to be tested is a biological sample containing at least one of serum and cells.

Preferably, in steps ⑤ and ⑥, the standard curve is plotted and the content of the bio-thiol in the sample to be tested is calculated by a standard addition method.

Application of rice leaf nitrogen-doped carbon nanoribbons in biological thiol detection by combining NRPCNRs-Ag through detection method⁺And carrying out label-free fluorescence detection on the sample to be detected.

Preferably, the fluorescent probe is in a rice leaf shape, the particle size is 100-120nm, the doping amount of N element (35-37) wt%, and the excitation wavelength is 355 nm.

Further, the fluorescent probe of the rice-shaped leaf nitrogen-doped carbon nanoribbon is prepared by a hydrothermal synthesis method, and the preparation steps comprise:

1.1g of Uric Acid (Uric Acid), 25mL of absolute ethanol and 25mL of deionized water are mixed and uniformly mixed under the ultrasonic condition to obtain a suspension; transferring the prepared 25mL of suspension into a polytetrafluoroethylene high-temperature reaction kettle, and keeping the suspension at 180 ℃ for 4.5 hours; naturally cooling to room temperature, extracting the obtained product with dichloromethane, refrigerating the aqueous phase solution obtained by extraction for 5-10 days, and standing to remove large-size NRPCNRs with length of more than 1 μm; then, the collected solution was centrifuged at 8000rpm for 15min, and the upper bright yellow aqueous NRPCNRs solution was taken and stored at 4 ℃ until use.

The comprehensive effects brought by the invention are as follows:

1. the invention adopts a simple and rapid hydrothermal method to synthesize the rice leaf-shaped nitrogen-doped carbon nanobelt which has good water solubility, uniform dispersion, fluorescent characteristic and stable fluorescence, and uses the NRPCNRs as a fluorescent probe to construct a fluorescent nano-sensor for detecting biological thiol with high sensitivity and high selectivity.

2. Fluorescence quenching of NRPCNR can be induced by interaction of Ag + ions with N atoms on NRPCNRs to form non-fluorescent coordination complexes once in NRPCNRs-Ag⁺The biological mercaptan is added into the reaction system due to Ag⁺Ions more readily form stable Ag-S bonds with S atoms within the bio-thiol molecule, thereby allowing Ag to be bound to NRPCNRs⁺When released, the fluorescence intensity of NRPCNRs is recovered, and the recovery degree of the fluorescence intensity of NRPCNRs is related to the amount of biological thiol added to the reaction system.

3. The novel method for detecting the unlabelled biological mercaptan based on the NRPCNRs can effectively realize high-sensitivity and high-selectivity detection of the biological mercaptan in human serum, is simple and rapid, has wide linear detection range, provides an effective way for efficiently detecting the biological mercaptan in a biological sample, and has potential application value.

Drawings

FIG. 1 is a schematic diagram of fluorescence spectrum and fluorescence brightness of a solution according to an embodiment of the present invention;

wherein, a, NRPCNRs (20 mu g/mL), b, NRPCNRs (20 mu g/mL) + Ag⁺(80μM),c.NRPCNRs (20μg/mL)+Ag⁺(80. mu.M) + Cys (100. mu.M), d.NRPCNRs (20. mu.g/mL) + Cys (100. mu.M); the inset shows from left to right the fluorescence photographs of different solutions 1.NRPCNRs, 2.NRPCNRs + Ag⁺，3. NRPCNRs+Ag⁺+Cys，4.NRPCNRs+Cys。

FIG. 2 is a UV spectrum and an emission spectrum of NRPCNRs under 355nm excitation in a detection method according to an embodiment of the present invention; the inset is a picture of NRPCNRs under 1. natural light and 2. UV lamp 365nm illumination.

FIG. 3 is a Transmission Electron Microscope (TEM) image of a solution in an examination method according to an embodiment of the present invention; wherein A.NRPCNRs, B.NRPCNRs-Ag⁺And C.NRPCNRs-Ag⁺-Cys。

FIG. 4 is a fluorescence intensity spectrum of NRPCNRs obtained at a maximum emission wavelength of 420nm by the detection method according to the embodiment of the present invention; wherein, A, the variable of the detection condition is Ag⁺Concentration, B.PBS buffer pH.

FIG. 5 is a graph of the detection method of the present invention using NRPCNRs to detect Cys and GSH: A. NRPCNRs-Ag⁺A solution, fluorescence spectrum at Cys concentration from 0 to 200 μ Μ; relative fluorescence intensity of NRPCNRs fluorescent Probe [ (FL-FL)₀)/FL₀]A standard curve graph for quantitatively detecting the concentration of Cys along with the change of the concentration of Cys; C.NRPCNRs-Ag⁺A solution, fluorescence spectrum at a GSH concentration from 0 to 200 μ Μ; [ (FL-FL) of NRPCNRs fluorescent probe₀)/FL₀]And quantitatively detecting a standard curve chart of the GSH concentration along with the change of the GSH concentration.

FIG. 6 shows NRPCNRs-Ag as the detection method according to the embodiment of the present invention⁺Detecting [ (FL-FL) s of different biological samples₀)/FL₀]Intensity contrast plots, examining the selectivity of NRPCNRs to Cys in the detection method of the invention.

Detailed Description

The present invention is further explained below with reference to specific embodiments and figures, but it should be understood that the scope of the present invention is not limited thereto. In this embodiment, the spectrophotometer is a Hitachi fluorescence spectrometer, model F-7000. And (3) quantitatively detecting the content of the biological mercaptan in the solution, and processing data by adopting a standard addition method in the prior art. Specifically, the implementation is performed with reference to the standard addition METHOD described in the national standard GB18582 issued by the State quality inspection administration and the standard addition METHOD described in the United states environmental protection agency 'Metal office addition AND EFFECTS OF DILUTION'.

Examples

(1) Method for preparing NRPCNRs fluorescent nanoprobe by hydrothermal synthesis

1.1g of uric acid, 25mL of absolute ethanol and 25mL of deionized water are mixed and uniformly mixed under the ultrasonic condition to obtain a suspension; transferring the prepared 25mL of suspension into a polytetrafluoroethylene high-temperature reaction kettle, and keeping the suspension at 180 ℃ for 4.5 hours; naturally cooling to room temperature, extracting the obtained product with dichloromethane, refrigerating the aqueous phase solution obtained by extraction for 5-10 days, and standing to remove large-size NRPCNRs with length of more than 1 μm; then, the collected solution was centrifuged at 8000rpm for 15min, and the upper bright yellow aqueous NRPCNRs solution was taken and stored at 4 ℃ until use.

(2) In this embodiment, the synthetic rice leaf-shaped nitrogen-doped carbon nanoribbon is used as an unmarked fluorescent probe to construct a high-sensitivity and high-selectivity fluorescent nanosensor for detecting biological thiol, and the detection steps include:

① adding 50 μ L PBS buffer solution with concentration of 50mM and pH of 7.4, 5 μ L NRPCNRs fluorescent probe solution with concentration of 2mg/mL, 40 μ L Ag + ion solution with concentration of 1mM into 1.5mL centrifuge tube, ② grouping and adding standard solution containing different concentrations of biological thiol and sample to be tested containing unknown concentration of biological thiol, ③ diluting reaction solution volume to 500 μ L with deionized water, mixing thoroughly on vortex mixer, incubating reaction mixture at room temperature for 20min, ④ measuring change of fluorescence intensity of each group of solutions, ⑤ taking concentration of biological thiol added into standard solution group as horizontal axis and fluorescence recovery degree of standard solution [ (FL-FL)₀)]/FL₀And ⑥, comparing the fluorescence recovery degree of the sample to be tested with the standard curve to judge the concentration of the biological thiol in the sample.

In step ①, the mass ratio of the rice leaf-shaped nitrogen-doped carbon nanoribbon fluorescent probe to the silver ions is 2.3: 1.

In step ③, the rice leaf-shaped nitrogen-doped carbon nanoribbon fluorescent probe has a concentration of 20 μ g/ml and a silver ion concentration of 80 μ M.

In step ④, the rice leaf-shaped nitrogen-doped carbon nanoribbon has a nitrogen doping content of 35-37 wt%, a fluorescence excitation wavelength of 355nm and excitation and emission slit widths of 3nm, and the biological thiol comprises cysteine and glutathione, and the sample to be tested is a biological sample containing serum.

In steps ⑤ and ⑥, the standard curve is plotted and the content of the biological thiol in the sample to be tested is calculated by using a standard addition method.

The preparation method of the NRPCNRs fluorescent probe adopts a hydrothermal synthesis method, the fluorescent probe is in a rice leaf shape, the particle size is 150-180nm, the doping amount of N element is (35-37) wt%, and the excitation wavelength is 355 nm.

The detection mechanism is as follows: due to Ag⁺Interaction with the N atom of NRPCNRs may induce fluorescence quenching of NRPCNRs to form non-fluorescent coordination complexes once in NRPCNRs-Ag⁺The biological mercaptan is added into the reaction system due to Ag⁺Ions more readily form stable Ag-S bonds with S atoms within the bio-thiol molecule, thereby allowing Ag to be bound to NRPCNRs⁺When released, the fluorescence intensity of NRPCNRs is recovered, and the recovery degree of the fluorescence intensity of NRPCNRs is related to the amount of biological thiol added to the reaction system.

(3) The method of the embodiment is used for detecting biological thiol by applying the rice leaf nitrogen-doped carbon nanoribbon and combining NRPCNRs-Ag⁺The sample to be detected is subjected to label-free fluorescence detection, and the sample detection and probe characterization results are shown in figures 1-6:

the result shows that the design of the invention can effectively realize the high-sensitivity and high-selectivity detection of the biological thiol.

1) As shown in FIG. 1, the NRPCNRs prepared by the hydrothermal synthesis method of the present invention have good fluorescence properties (a in FIG. 1), and when an appropriate amount of Ag + ions is added to the NRPCNRs solution, the fluorescence of the NRPCNRs is reflected by Ag⁺The ions were quenched (b in fig. 1), and when the addition of the bio-thiol (Cys) to their solution was resumed, 90% of the fluorescence of NRPCNRs was recovered (c in fig. 1), while also confirming that Cys did not have an effect on the fluorescence of NRPCNRs (d in fig. 1). The new nano fluorescence sensing detection method for establishing the biological thiol by using the NRPCNRs as the fluorescent probe is demonstrated.

Before NRPCNRs are used as fluorescent probes for detection, the NRPCNRs are subjected to ultraviolet characterization and TEM characterization, and the characterization results are shown in FIGS. 2 and 3.

2) As can be seen from fig. 2, the ultraviolet absorption spectrum of NRPCNRs shows two characteristic peaks (curve a) at 275 nm and 355nm, respectively, and with 355nm as the optimum excitation wavelength, NRPCNRs can be excited to emit a fluorescence spectrum (420nm) (curve b), and the interpolation graph in fig. 2 also further shows the fluorescence characteristics of NRPCNRs. The results show that the prepared NRPCNRs can be used as fluorescent nanoprobes for quantitative analysis of biological thiol.

3) FIG. 3 is a TEM image of different solutions. As shown in A in FIG. 3, the NRPCNRs fluorescent probe is shaped like a rice leaf and has a particle size of 150-180 nm; due to Ag⁺An Ag — N bond may be formed with a nitrogen atom in NRPCNs, causing NRPCNRs to aggregate (B in fig. 3), resulting in fluorescence quenching of NRPCNRs. When in NRPCNRs-Ag⁺The result that NRPCNRs did not undergo significant aggregation after addition of Cys to the reaction system (C in FIG. 3) indicates that Ag⁺Ions more easily form stable Ag-S bonds with S atoms in the molecules of the biological thiol, so that Ag + bound on the NRPCNRs is released, and the fluorescence intensity of the NRPCNRs is recovered.

4) As shown in FIG. 4, in order to better realize effective detection of biological thiol, Ag was studied in the present invention⁺The concentration of (3) and the pH of the PBS buffer solution on the assay results. The optimized result shows that the optimal Ag of the detection system⁺The concentration of (b) was 80. mu.M, and the optimum pH of the PBS buffer solution was 7.4.

5) Detection of Cys and GSH. Under optimized conditions (50mM, pH 7.4PBS, 20. mu.g/mL NRPCNRs, 80. mu.MAG)⁺) A series of different concentrations of Cys and GSH were determined using the method of the example of the present invention, and the results are shown in FIG. 5, where A in FIG. 5 shows the change in fluorescence intensity of NRPCNRs in the concentration range of 0-200. mu.M for Cys. At 80. mu.M Ag⁺The fluorescence intensity of NRPCNRs at 420nm increased with increasing Cys concentration (B in FIG. 5) and exhibited a good linear relationship in the 0.05-10 μ M concentration range. C in fig. 5 and D in fig. 5 are graphs of GSH detection results. The results showed that the fluorescence intensity of NRPCNRs at 420nm increased with increasing GSH concentration (D in FIG. 5) and ranged from 0.5 to 30. mu.M concentrationThe inner wall presents a good linear relation, and the results fully illustrate that the fluorescence sensor designed by the invention has good response to biological thiol.

6) And (4) selectively inspecting the method. The invention considers the selectivity of the detection system and considers the possible non-thiol amino acid interference substances in a complex biological system, including Pro, Val, Tyr, Ser, His, Try, Arg, Glu, Thr, Phe, Lys, Ala and Gly. As shown in fig. 6, the detection system has high selectivity for Cys and GSH in the context of complex biological systems containing different non-thiol amino acids. The fluorescence sensor constructed by the invention has better selectivity in detecting the biological thiol in a complex biological system.

7) And (4) analyzing an actual sample. The invention takes the example of measuring Cys in serum of normal human by a standard addition method to verify the practicability of the fluorescence sensor designed by the invention in the analysis and detection of actual samples. The results are shown in the attached table 1, where the recovery rate of Cys was between 96.2% and 104.8% and the relative standard deviation was less than 5.0%, indicating that the method of the example of the invention has better reliability. Meanwhile, the content of Cys in serum of normal human is measured to be within a normal range. The results show that the novel method for detecting the unlabelled biological thiol based on the NRPCNRs can effectively realize high-sensitivity and high-selectivity detection of the biological thiol in human serum, is simple and rapid, has a wide linear detection range, provides an effective way for efficiently detecting the biological thiol in a biological sample, and has potential application value.

TABLE 1 detection of Cys in samples to be tested as fluorescent probes for NRCNRs

Although the present invention has been described in detail, modifications within the spirit and scope of the invention will be apparent to those skilled in the art. Further, it should be understood that the various aspects recited herein, portions of different embodiments, and various features recited may be combined or interchanged either in whole or in part. In the various embodiments described above, those embodiments that refer to another embodiment may be combined with other embodiments as appropriate, as will be appreciated by those skilled in the art. Furthermore, those skilled in the art will appreciate that the foregoing description is by way of example only, and is not intended to limit the invention.

Claims

1. The application of the rice leaf nitrogen-doped carbon nanobelt in biological thiol detection is characterized in that a sample to be detected is a biological sample and contains at least one of serum and cells;

unmarked biological mercaptan fluorescence detection method based on rice-shaped leaf nitrogen-doped carbon nanoribbon and combined with NRPCNRs-Ag⁺①, adding phosphate buffer solution, rice leaf-shaped nitrogen-doped carbon nanoribbon fluorescent probe solution and silver ion solution into a container in sequence, ② grouping and adding standard solution containing biological thiol with different concentrations and a sample to be detected containing biological thiol with unknown concentration, ③ diluting, uniformly mixing and incubating, ④ determining the change of the fluorescence intensity of each group of solutions, ⑤ drawing a standard curve by taking the concentration of the biological thiol added into the standard solution group as a horizontal axis and the fluorescence recovery degree of the standard solution as a vertical axis, ⑥ comparing the fluorescence recovery degree of the sample to be detected with the standard curve to determine the biological thiol concentration of the sample;

in step ④, the nitrogen doping content of the rice leaf-shaped nitrogen-doped carbon nanoribbon is (35-37) wt%;

the rice-shaped leaf nitrogen-doped carbon nanoribbon fluorescent probe is prepared by a hydrothermal synthesis method, and the preparation steps comprise: 1.1g of uric acid, 25mL of absolute ethanol and 25mL of deionized water are mixed and uniformly mixed under the ultrasonic condition to obtain a suspension; transferring the prepared 25mL of suspension into a polytetrafluoroethylene high-temperature reaction kettle, and keeping the suspension at 180 ℃ for 4.5 hours; naturally cooling to room temperature, extracting the obtained product with dichloromethane, refrigerating the aqueous phase solution obtained by extraction for 5-10 days, and standing to remove large-size NRPCNRs with length of more than 1 μm; then, the collected solution was centrifuged at 8000rpm for 15min, and the upper bright yellow aqueous NRPCNRs solution was taken and the resulting bright yellow solution was stored at 4 ℃.

2. The use of claim 1, wherein the mass ratio of the rice leaf-shaped nitrogen-doped carbon nanoribbon fluorescent probe to silver ions is (2-3):1 in step ①.

3. The use of claim 2, wherein in step ③, the concentration of the fluorescent probe of the rice leaf-shaped nitrogen-doped carbon nanoribbon is 10-25 μ g/ml, and the concentration of the silver ion is 70-90 μ M.

4. Use according to claim 3, wherein the fluorescence excitation wavelength is 355nm and the excitation and emission slit widths are both 3 nm.

5. The use of claim 3, wherein the biological thiol comprises at least one of cysteine and glutathione.

6. The use of claim 3, wherein in steps ⑤ and ⑥, the standard curve is plotted and the amount of biological thiol in the test sample is calculated using standard addition methods.

7. The use according to claim 1, wherein the fluorescent probe has a rice leaf-shaped profile, a particle size of 150-180nm, a doping amount of N element (35-37) wt%, and an excitation wavelength of 355 nm.