CN108822005B - Reversible colorimetric probe based on malachite green and bisulfite addition product, and preparation and application thereof - Google Patents
Reversible colorimetric probe based on malachite green and bisulfite addition product, and preparation and application thereof Download PDFInfo
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- 229940107698 malachite green Drugs 0.000 title claims abstract description 47
- LSNNMFCWUKXFEE-UHFFFAOYSA-M Bisulfite Chemical compound OS([O-])=O LSNNMFCWUKXFEE-UHFFFAOYSA-M 0.000 title claims abstract description 34
- 230000002441 reversible effect Effects 0.000 title claims abstract description 25
- 238000002360 preparation method Methods 0.000 title claims abstract description 8
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- 239000000047 product Substances 0.000 claims description 25
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- 238000011534 incubation Methods 0.000 claims description 3
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- OWEGMIWEEQEYGQ-UHFFFAOYSA-N 100676-05-9 Natural products OC1C(O)C(O)C(CO)OC1OCC1C(O)C(O)C(O)C(OC2C(OC(O)C(O)C2O)CO)O1 OWEGMIWEEQEYGQ-UHFFFAOYSA-N 0.000 description 4
- GUBGYTABKSRVRQ-PICCSMPSSA-N Maltose Natural products O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@@H](CO)OC(O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-PICCSMPSSA-N 0.000 description 4
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- GUBGYTABKSRVRQ-QUYVBRFLSA-N beta-maltose Chemical compound OC[C@H]1O[C@H](O[C@H]2[C@H](O)[C@@H](O)[C@H](O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@@H]1O GUBGYTABKSRVRQ-QUYVBRFLSA-N 0.000 description 4
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- 238000005160 1H NMR spectroscopy Methods 0.000 description 1
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 1
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- GZCGUPFRVQAUEE-SLPGGIOYSA-N aldehydo-D-glucose Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@@H](O)C=O GZCGUPFRVQAUEE-SLPGGIOYSA-N 0.000 description 1
- 239000012491 analyte Substances 0.000 description 1
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 description 1
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- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C309/00—Sulfonic acids; Halides, esters, or anhydrides thereof
- C07C309/01—Sulfonic acids
- C07C309/02—Sulfonic acids having sulfo groups bound to acyclic carbon atoms
- C07C309/24—Sulfonic acids having sulfo groups bound to acyclic carbon atoms of a carbon skeleton containing six-membered aromatic rings
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Abstract
The invention belongs to the technical field of colorimetric probes, and particularly relates to a reversible colorimetric probe based on a malachite green and bisulfite addition product, and preparation and application thereof. The structural formula of the reversible colorimetric probe is as follows:
. The colorimetric probe has high sensitivity, good selectivity, simplicity, convenience and reusability, and is expected to be popularized and used in the field of biomedicine.
Description
Technical Field
The invention belongs to the technical field of colorimetric probes, and particularly relates to a reversible colorimetric probe based on a malachite green and bisulfite addition product, and preparation and application thereof.
Background
Glucose is a main carbon source and energy source for cell metabolism, plays a crucial role in the natural growth process of cells, and the glucose level in blood is closely related to diseases such as diabetes or hypoglycemia. While over the past decades, diabetes has become one of the largest public health threats. Therefore, monitoring blood glucose levels is of great importance for the prevention and treatment of diabetes or other related diseases.
At present, many glucose detection methods such as a fluorescence sensing method, a colorimetric sensing method, a chemiluminescence sensing method, and high performance liquid chromatography have been reported. Among these analysis methods, the colorimetric method has attracted much attention, and in addition to the characteristics of accuracy, easy operation, low cost, and the like, the colorimetric method can analyze an analyte by observing a color change with the naked eye, which requires less labor and even does not require a detection device. However, most of the existing colorimetric probes are disposable consumables, and no glucose reversible colorimetric probe with the recycling capability exists, so that the glucose reversible colorimetric probe is not beneficial to sustainable development.
Disclosure of Invention
In view of this, the present invention aims to provide a reversible colorimetric probe based on an addition product of malachite green and bisulfite, and a preparation method and an application thereof, wherein the reversible colorimetric probe can be reused.
In order to achieve the purpose, the technical scheme adopted by the invention is as follows:
a reversible colorimetric probe based on a malachite green and bisulfite addition product has a structural formula as follows:
。
the preparation method of the reversible colorimetric probe based on the addition product of malachite green and bisulfite comprises the following steps: 1) taking raw materials of malachite green and hydrosulfite;
2) adding malachite green and bisulfite into a phosphate buffer solution (with the concentration of 10 mmol/LPBS), reacting for 3min at room temperature after mixing with the malachite green of 20 mu M and the bisulfite of 25 mu M, and obtaining a reversible colorimetric probe solution based on the addition product of the malachite green and the bisulfite.
The mass ratio of malachite green to bisulfite is 1: (1-1.25).
The bisulfite is sodium bisulfite, potassium bisulfite or ammonium bisulfite.
Use of a reversible colorimetric probe for detecting glucose in a sample.
In specific application, a sample to be tested is mixed with glucose oxidase for incubation (incubation is carried out for 30min at 37 ℃) to obtain a reaction solution, then the reaction solution is added into a reversible colorimetric probe solution, and then the color and the ultraviolet-visible spectrum change after reaction are respectively tested.
The experiment shows that: with the increase of the glucose content in the sample to be detected, the reaction solution is added into the reversible colorimetric probe solution and gradually changes from colorless to blue, and the change of the ultraviolet visible spectrum is as follows: the absorbance at 618 nm increases with increasing glucose content.
According to the invention, an addition product obtained by 1, 4-addition reaction of malachite green and bisulfite is used as a colorimetric probe, and researches show that glucose can generate hydrogen peroxide H under the catalytic action of glucose oxidase2O2And H is2O2The method comprises the steps of oxidizing an addition reaction product of malachite green and bisulfite to enable the product to turn back to the blue malachite green again, if a reaction solution obtained by mixing and incubating a sample to be tested and glucose oxidase is added into a probe solution, the probe solution turns from colorless to blue, which indicates that the addition reaction product turns back to the malachite green, and at the moment, indicates that the reaction solution contains H catalyzed by the glucose oxidase2O2Further, it is stated that the sample to be tested contains glucose, so that it can be measured according to H2O2The resulting color change from colorless to blue or the corresponding change in the ultraviolet-visible absorption spectrum indirectly detects glucose. After the colorimetric probe detects glucose, sufficient bisulfite can be continuously added into the probe solution, and the product of the probe reaction, namely malachite green, is changed back to the addition product of the malachite green and the bisulfite, so that the probe is realizedCan be repeatedly used.
The invention has the following beneficial effects: 1) the adopted raw materials of malachite green and hydrosulfite are cheap and easy to obtain, and the cost is not high; 2) the reversible colorimetric probe is simple and convenient in preparation, can be used for conveniently detecting glucose in solution and human serum, and has the advantages of good selectivity, high sensitivity and 70 nM minimum detection limit; 3) after the probe detects glucose, the probe can also continuously detect the glucose again after the bisulfite is added for reaction, only trace and very cheap bisulfite is consumed in the process, and the cost is greatly reduced; 4) the colorimetric probe has high sensitivity, good selectivity, simplicity, convenience and reusability, and is expected to be popularized and used in the field of biomedicine.
Drawings
Figure 1 is a high resolution mass spectrum of malachite green.
FIG. 2 is a high resolution mass spectrum of malachite green after reaction with bisulfite.
FIG. 3 is a high resolution mass spectrum of the reaction of malachite green with bisulfite and continued addition of hydrogen peroxide.
FIG. 4 is a UV-VIS spectrum and a color change photograph (1.5 mL of the final mixed solution containing glucose at a concentration of 0 to 1000. mu.M) after adding the reaction solution of glucose (0 to 1000. mu.M) and glucose oxidase at different concentrations to the probe solution.
FIG. 5 is the relative absorbance at 618 nm (A/A)0) Linear dependence on glucose concentration, where A and A0Representing the absorbance at 618 nm of the probe-containing PBS buffered system in the presence of different concentrations of glucose and in the absence of glucose, respectively.
FIG. 6 is a color photograph of the system in which the potential interfering substance in serum coexists with glucose, showing the relative absorbance of the probe-containing PBS buffer system, with the abscissa from left to right corresponding to the color of the system in which the interfering substance coexists with glucose.
FIG. 7 is a color photograph of the PBS buffer system containing the probe solution and the system when other carbohydrate interfering substances (galactose, sucrose, maltose) and glucose are added, wherein the color photographs correspond to the system without sugar and with galactose, sucrose, maltose and glucose from left to right in the figure (the concentration of galactose, sucrose and maltose in the final mixed solution of 1.5 mL is 1200 μ M, and the concentration of glucose is 120 μ M).
FIG. 8 is a graph showing the results of examination of reversible cycling ability of the probe, and the inset corresponds to the color change of the reversible cycling test sample.
FIG. 9 shows the addition product of malachite green with bisulfite of example 11H NMR spectrum.
Detailed Description
The following examples are given to illustrate specific embodiments of the present invention, but are not intended to limit the scope of the present invention in any way.
Example 1:
to a 1.5 mL centrifuge tube was added 300. mu.L of 50 mM phosphate buffer solution (PBS, pH 6.8), 150. mu.L of 200. mu.M malachite green and 30. mu.L of 1.25mM sodium bisulfite were added to make up a mixture, the total volume of which was 1.5 mL supplemented with ultrapure water, containing 10mM phosphate buffer solution, 20. mu.M malachite green and 25. mu.M NaHSO3Reacting the mixed solution for 3 minutes to obtain the product containing
(hereinafter referred to as addition product) in the presence of a probe solution (hereinafter referred to as solution B).
HRMS（ESI）：[M+H]+(Malachite Green C23H25N2 +）calcd 329.2012, found 329.2018; [M-H]+(Probe C obtained by reacting Malachite Green with bisulfite)23H26N2 +O3S）calcd 409.1664, found 409.1595；[M+H]+(Malachite Green C whose Probe was regenerated by oxidation with Hydrogen peroxide23H25N2 +）calcd 329.2012, found 329.2017。
1. Investigation of reversible reaction mechanism of probe
Addition of HSO via malachite Green3 −And then adding H2O2The reversible reaction mechanism of the probe is verified by the subsequent high-resolution mass spectrum change. As shown in fig. 1, the mass spectrum data for malachite green was found to be 329.2018, corresponding to a molecular weight of 329.2012 for malachite green. Adding malachite green into HSO3 −After completion of the reaction, negative ion peak mass spectra data 409.1595 (fig. 2) appeared on the mass spectra, which coincided with the molecular weight 410.1664 (409.1664) of the addition product expected to form by the addition reaction; addition product addition H2O2And after fully reacting, mass spectral data 329.2017 (FIG. 3) was obtained, consistent with the molecular weight of malachite green, further illustrating that the addition product was substituted by H2O2Reoxidizes to malachite green. Therefore, through the experimental results and analysis, and the combination of the ultraviolet and visible absorption spectrum data, the malachite green and the HSO can be determined3 −And H2O2A redox reversible reaction occurs, i.e. HSO3 −Nucleophilic addition reaction with the C = C double bond of malachite green to form an addition product, and the addition product can be substituted with H2O2Oxidized to the original malachite green. These results indicate that the probes, malachite green and HSO3 −In the presence of an addition product of (2) with H2O2After the reaction has taken place, HSO can also be added3 −Is continued with H2O2And (4) reacting.
2. Color and UV-Vis spectra of the probe solution as a function of the concentration of glucose added
First, 40. mu.L of PBS buffer (50 mM, pH 6.8), 20. mu.L of glucose oxidase (10 mg/mL) and 140. mu.L of glucose at different concentrations (0, 1.07, 5.36, 10.71, 53.57, 107.14, 214.29, 428.57, 642.86, 857.14, 1071.43, 1500. mu.M) were mixed well, and then the mixed solution was incubated at 37 ℃ for 30 minutes to generate H at different concentrations2O2At this point, solution a was obtained.
Adding the solution A with different concentrations into the solution B obtained in the example 1 to react for 20 minutes, then supplementing the total volume to 1.5 mL by ultrapure water, and measuring and recording the ultraviolet visible absorption spectrum and the color change graph of the corresponding solution after uniformly mixing. The results are shown in FIGS. 4-5, in which the numbers 1-12 represent glucose concentrations of 0, 0.1, 0.5, 1, 5, 10, 20, 40, 60, 80, 100, 140. mu.M, respectively, and the solution gradually changes from colorless to blue with increasing glucose concentration; the change of the ultraviolet visible spectrum is as follows: the absorbance at 618 nm increases with increasing glucose content. The linear fitting is carried out on the relative absorbance and the concentration of the glucose, and the result shows that when the concentration range of the glucose in the probe solution is 0.1-140 mu M, the relative absorbance and the concentration have a good linear relation, the detection limit is 70 nM, the sensitivity is very high, the blood glucose content is far lower than that of fasting blood glucose (3.89-6.11 mM), and the linear fitting method can be applied to the detection of the glucose in a life sample.
3. Effect of different interfering substances on the Probe
First, 40. mu.L of PBS buffer solution (50 mM, pH 6.8), 20. mu.L of glucose oxidase (10 mg/mL) and 140. mu.L of 1.29 mM glucose were mixed well, and then the mixed solution was incubated at 37 ℃ for 30 minutes to obtain solution C. Then, solution C and various potential interferents in serum (the concentration of the potential interferent in serum was 1% of the concentration of the corresponding interferent in human serum) were added to solution B prepared in example 1 to react for 20 minutes, and the UV-visible absorption spectrum and the color change thereof were measured and recorded after the total volume was made up to 1.5 mL and mixed well. The results are shown in FIG. 6, where a-u in FIG. 6 represent samples from left to right of the abscissa of the graph, respectively, and the experimental results show that other interferents cause only a negligible effect. Next, examining the ability of the present invention to distinguish between glucose and its analogs, v-z in fig. 7 represent samples from left to right in the abscissa of the graph, respectively, and fig. 7 shows that other common sugars (galactose, sucrose and maltose) with a content of 10 times the glucose concentration produce a signal response much lower than that of glucose, even nearly as low as the background signal. In conclusion, the method has high specificity to glucose and strong anti-interference capability.
4. Reversible cycling capability review of probes
To a 10 mL centrifuge tube were added 300. mu.L of 50 mM phosphate buffer solution (PBS, pH 6.8) and 778. mu.L of ultrapure water, and 150. mu.L of the solution was addedL of 200. mu.M malachite green and 72. mu.L of 1.25mM sodium hydrogen sulfite were reacted for 3 minutes to form a mixture D, and its ultraviolet-visible absorption spectrum was measured and its color change pattern was recorded. Then, 40. mu.L of PBS buffer solution (50 mM, pH 6.8), 20. mu.L of glucose oxidase (10 mg/mL) and 140. mu.L of 2.14 mM glucose were mixed well, and the mixed solution was incubated at 37 ℃ for 30 minutes to generate H2O2At this point, solution E was obtained. Solution E was added to solution D and reacted for 20 minutes to give 1.5 mL of solution F, whose UV-VIS absorption spectrum and color change pattern of the corresponding solution were then measured and recorded. To the solution F were further added 300. mu.L of 50 mM phosphate buffer solution (PBS, pH 6.8), 72. mu.L of 1.25mM sodium hydrogen sulfite and 928. mu.L of ultrapure water, reacted for 3 minutes to form a mixture G, and the ultraviolet-visible absorption spectrum thereof was measured and the color change thereof was recorded. The solution E described above was then prepared. Solution E was added to solution G and reacted for 20 minutes to give 3 mL of solution H, whose UV-vis absorption spectrum and color change pattern of the corresponding solution were then measured and recorded. And so on to prepare solution I, J, K, L, M, N. The absorbance of their absorption peak at 618 nm was recorded, and a photograph of the color change at each cycle was additionally recorded with a camera. As a result, as shown in FIG. 8, H generated when the probe was oxidized with glucose2O2And HSO3 −After 5 times of repeated oxidation reduction, the ultraviolet visible absorption of the probe is only slightly weakened, and the color of the solution is only slightly faded as shown in the inset, which shows that the probe has good reversibility and still retains H generated by oxidation of glucose after multiple times of oxidation reduction2O2Reactivity and good spectral properties. Therefore, the probe of the present invention can be reused to detect H2O2And glucose, thus greatly reducing the cost, being beneficial to the further popularization of the method of the invention and having important application value in the biological and medical fields.
5. Application capability investigation of probe in detection of glucose in human serum
First, 40. mu.L of PBS buffer solution (50 mM, pH 6.8), 20. mu.L of glucose oxidase (10 mg/mL), 15. mu.L of human serum sample and 125. mu.L of glucose (0, 60, 120, 240. mu.M) were mixed well, and then the mixed solution was incubated at 37 ℃ for 30 minutes to obtain solution D. Then, to the solution B obtained in example 1, the solution D was added and reacted for 20 minutes, and after the total volume was made up to 1.5 mL with ultrapure water and mixed well, the ultraviolet-visible absorption spectrum and the color change thereof were measured and recorded. The results of the spiked recovery experiments are shown in table 1, the recovery rate of the recovery experiments is between 92.8% and 108.9%, and the relative standard deviation is lower than 5.36%, which indicates that the method is stable, accurate and reliable. In addition, considering the dilution times of the serum samples, the content of glucose in 3 actual serum samples obtained by colorimetric detection is 4.523 mM, 4.998 mM and 4.384 mM, which is basically consistent with the blood glucose level reported in the literature, and the results fully prove the practical application capability of the invention and provide a new choice for detecting glucose in complex biological samples.
TABLE 1 analysis results of glucose detection in 1% human serum using colorimetric method
6. FIG. 9 shows the addition product of malachite green with bisulfite1The H NMR spectrum of the mixture is shown,1H NMR（400 MHz, DMSO-d 6 ）δ 7.34 (t, J = 4 Hz, 2H), 7.24 (s, 3H), 7.15 (d, J =8.8 Hz, 4H), 6.67 (d, J=9.2 Hz, 4H), 4.26 (s, 12H); the structure of the malachite green and bisulfite addition product is verified to be the structural formula shown.
Finally, the above embodiments are only used for illustrating the technical solutions of the present invention and not for limiting, and other modifications or equivalent substitutions made by the technical solutions of the present invention by those of ordinary skill in the art should be covered within the scope of the claims of the present invention as long as they do not depart from the spirit and scope of the technical solutions of the present invention.
Claims (3)
1. The application of the reversible colorimetric probe based on the addition product of malachite green and bisulfite in the aspect of non-disease diagnosis or treatment on the detection of glucose in solution is characterized in that the solution is a solution after adding glucose oxidase for mixing and incubation, the solution is added into the probe solution, the probe solution is changed from colorless to blue, which indicates that the addition reaction product is changed back to malachite green, which indicates that the solution contains glucose, sufficient bisulfite is continuously added into the probe solution, and the product of the probe reaction is changed back to the addition product of malachite green and bisulfite, so that the probe can be repeatedly used; the preparation method of the reversible colorimetric probe comprises the following steps:
1) taking raw materials of malachite green and hydrosulfite;
2) adding malachite green and bisulfite into phosphate buffer solution, and reacting at room temperature for 3min to obtain reversible colorimetric probe solution based on the addition product of malachite green and bisulfite.
2. Use according to claim 1, wherein the malachite green and bisulfite species are present in a ratio of 1: (1-1.25).
3. The use according to claim 1, wherein the bisulfite salt is sodium bisulfite, potassium bisulfite or ammonium bisulfite; the concentration of the phosphate buffer solution is 10 mmol/L.
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