Molecular colorimetric ratio detection and analysis method based on metal ions

Abstract

The invention discloses a molecular colorimetric ratio detection and analysis method based on metal ions, which utilizes color change generated by reaction of metal ions (strontium ions Sr < 2 + >) and specific chemical substances and gas pressure effect generated by catalysis of decomposition of hydrogen peroxide by platinum nano-enzyme to detect the molecular colorimetric ratio when pathological cells exist. According to the technical scheme, obvious color precipitates appear in the solution through a series of chemical reactions, so that signal output is realized, the detection range is widened, and the technical scheme has higher accuracy and practicability in pathological cell detection.

Description

Technical Field

[0001] The invention relates to biosensing technology, in particular to a molecular absorption ratio sensing method and application based on metal ions, specifically a molecular colorimetric ratio detection and analysis method based on metal ions. Background Art

[0002] MCF-7 cells have been widely used in scientific research, spanning multiple fields such as genetics and molecular biology. These studies have not only deepened our understanding of how human genes work and their mutual regulatory mechanisms. Using MCF-7 cells as a research platform, scientists can gain insight into subtle changes at the genetic level, thereby providing innovative insights and solutions to the medical community. To achieve this goal, the development of detection technologies with high sensitivity and wide range response has become key. Although a variety of analytical technologies such as electrochemistry, nanopores, and optics have been applied to cancer cell detection, these methods often have problems with insufficient sensitivity or limited detection range.

[0003] In terms of detection, high sensitivity is the key to achieve early low-level, accurate quantitative detection of MCF-7 cells. The amount of MCF-7 cells remains variable, and accordingly, a wide range of detection capabilities is critical for monitoring changes in diseased cells. Considering these criteria, it is very urgent to highly pursue analytical techniques with high sensitivity and wide range response.

[0004] So far, a large number of analytical techniques based on electrochemistry, nanopores, optics, magnetism and pressure have been developed for cell analysis, among which visual biosensors based on molecular colorimetry are promising due to their easy recognition by the naked eye, low cost and point-of-care testing (POCT). Despite these significant advantages, most of them still have many disadvantages. For example, the sensitivity of traditional single-color systems is always unsatisfactory because the human eye is insensitive to intensity changes of similar colors; the introduction of metal nanoparticles allows the generation of multiple colors to obtain higher sensitivity, while the localized surface plasmon resonance peak (LSPR) responds sensitively to the slight changes in the morphology or shape of the metal nanoparticles, which results in the detection range being limited to a certain extent.

[0005] Therefore, the quantitative detection of MCF-7 cells has become a research hotspot for diseased cell detection. The ability to achieve simple and highly sensitive quantitative detection of MCF-7 cells is a problem that needs to be solved at present. Summary of the invention

[0006] The purpose of the present invention is to provide a molecular colorimetric ratio detection and analysis method based on metal ions in view of the shortcomings of the prior art. This method has the advantages of simple operation, low cost, and instant POCT detection, and can rapidly and highly sensitively selectively visualize the detection of MCF-7 cells in living cells, providing an important tool for related biomedical research.

[0007] The technical solution for achieving the purpose of the present invention is: A molecular colorimetric ratio detection and analysis method based on metal ions comprises the following steps: 1) Preparation of PtNPs: including: 1-1) Add 2 mL of 18.9 mM HO 2 PtCl 6 6H 2 O, 30 μL, 5 M NaOH, 0.2 mL, 1 M L-ascorbic acid, and 2 mL ultrapure water were added to the beaker and mixed well; 1-2) After heating the mixed solution in step 1-1) in a 60 °C water bath for 10 min, the color of the solution changed from light yellow to brown-black, thereby obtaining a PtNPs solution. The PtNPs solution was then cooled to room temperature and stored at 4 °C. 2) Preparation of AS1411 aptamer-functionalized PtNPs: including: 2-1) PtNPs were functionalized with aptamers using the salt aging method, i.e., 125 μL, 5 μM thiolated aptamer AS1411 and 2.5 mL PtNPs were mixed and incubated overnight at room temperature, wherein the concentration of PtNPs was 5 mg / mL and the diameter of PtNPs was 54±14 nm; 2-2) 1 M sodium chloride and 0.1 M PBS with a pH of 7.4 were slowly added to the mixed solution in five times within 12 h, stirred for 24 h, and then centrifuged at 14,000 rpm for 30 min at 4 °C to remove the unreacted aptamer AS1411, thereby obtaining functionalized AS1411-PtNPs; 2-3) Finally, the functionalized AS1411-PtNPs were resuspended in 10 mM PBS, pH 7.4; 3) Preparation of cell suspension: including: 3-1) The cells in the logarithmic phase were digested with trypsin, centrifuged at 1000 rpm for 5 min, and then the cells at different concentrations were placed in a 96-well plate; 3-2) After 24 h of culture, discard the culture medium and wash with 10 mM PBS, pH 7.4; 3-3) Add 100 μL of AS1411-PtNPs cell culture medium with a final concentration of 0.49 mg / mL to each well, incubate for 4 h, wash the cells three times with PBS, digest and collect the cells to obtain a cell suspension; 4) co-incubating the AS1411 aptamer-functionalized PtNPs prepared in step 2) with the cell suspension prepared in step 3) to obtain a connection complex of PtNPs and cells; 5) The PtNPs and cell complex obtained in step 4) was transferred to a flask containing 110 μL of 30 wt % H 2 O 2 The gas generating tube is provided with a closed drainage device consisting of two spaced-apart but interconnected tubes, one of which is used for targeted mediated gas generation and the other for overflow of concentrated Sr 2+ The gas generating tube contained 1 ml of 50 mM Sr 2+ solution, PtNPs induced hydrogen peroxide H 2 O 2 decomposes to produce oxygen O 2 , resulting in an increase in the pressure of the closed system of the gas generating tube, collecting the spilled Sr 2+ The solutions were subsequently analyzed using the molecular colorimetric detection mode; 6) The overflowed 50 mM Sr 2+ The solution was treated with H 2 O volume was set to 860 μL; 7) Take another 10 μL of the diluted solution and react with 1 mL, 3 mM NaRho for precipitation reaction; 8) Photo identification: Take a photo of the precipitate obtained in step 7) to read the color, analyze the RGB value, and compare it with the detection mode of the molecular colorimetry to obtain the accurate value of the MCF-7 cell number.

[0008] This technical solution provides an innovative molecular absorption ratio sensing method, which aims to use the advantages of molecular colorimetry with a wide detection range to achieve high sensitivity and wide range visual detection of diseased cells. 2+ ) produces color changes when it reacts with specific chemicals, and the gas pressure effect produced by the decomposition of hydrogen peroxide catalyzed by platinum nanozymes. When diseased cells are present, a series of chemical reactions can lead to obvious color precipitation in the solution, thereby achieving signal output. This technical solution broadens the detection range and makes the technical solution more accurate and practical in the detection of diseased cells.

[0009] This method has the advantages of simple operation, low cost, and instant detection POCT. It can quickly and highly sensitively selectively visualize the number of MCF-7 cells in living cells, providing an important tool for related biomedical research. BRIEF DESCRIPTION OF THE DRAWINGS

[0010] Figure 1 Figure A is a graph showing Sr 2+ Schematic diagram of the precipitation reaction with sodium rhodamine; Figure 1 Figure B shows Sr in the embodiment 2+ Schematic diagram of the effect of volume and NaRho concentration on precipitation reaction; Figure 1 Figure C in the middle is a schematic diagram of the volume response range of the precipitation reaction in the embodiment; Figure 2 Figure A is a TEM image of PtNPs in the embodiment (showing the effect of platinum nanoparticles on H 2 O 2 Schematic diagram of the catalytic reaction; Figure 2 Figure B in the middle is a schematic diagram showing the effect of aptamer modification on the catalytic activity of PtNPs in the example; Figure 2 Figure C in the middle is a schematic diagram of the Zeta potential of PtNPs and AS1411-PtNPs in the embodiment; Figure 2 Figure D in the middle is a schematic diagram of the atomic flame color in the embodiment; Figure 2 Figure E shows the catalytic H 2 O 2 Schematic diagram of the R / G value of the color signal after the image is taken; Figure 3 Figure A in the middle is a schematic diagram of the viability of HeLa cells in the presence of different concentrations of AS1411-PtNPs in the embodiment; Figure 3 Figure B shows the cells washed once (red line), twice (blue line), and three times (yellow line) with PBS in the example, and the supernatant and blank control were added to TMB-H 2 O 2 Schematic diagram of the UV absorption spectrum in the system; Figure 4 Figure A in the middle is a schematic diagram of the specific binding of AS1411-PtNPs to HeLa cells and the selectivity of the sensing platform in the embodiment; Figure 4 Figure B is a schematic diagram of the linear relationship analysis between cell concentration and R / G value in the embodiment; Figure 5 Schematic diagram of the principle flow of the method in the embodiment. DETAILED DESCRIPTION

[0011] The content of the present invention is further described below in conjunction with the drawings and embodiments, but the present invention is not limited thereto.

[0012] Example:

[0013] Reference Figure 5 , a molecular colorimetric ratio detection and analysis method based on metal ions, comprising the following steps: 1) Preparation of PtNPs: including: 1-1) Add 2 mL of 18.9 mM HO 2 PtCl 6 6H 2 O, 30 μL, 5 M NaOH, 0.2 mL, 1 M L-ascorbic acid, and 2 mL ultrapure water were added to the beaker and mixed well; 1-2) After heating the mixed solution in step 1-1) in a 60 °C water bath for 10 min, the color of the solution changed from light yellow to brown-black, thereby obtaining a PtNPs solution. The PtNPs solution was then cooled to room temperature and stored at 4 °C. 2) Preparation of AS1411 aptamer-functionalized PtNPs: including: 2-1) PtNPs were functionalized with aptamers using the salt aging method, i.e., 125 μL, 5 μM thiolated aptamer AS1411 and 2.5 mL PtNPs were mixed and incubated overnight at room temperature, wherein the concentration of PtNPs was 5 mg / mL and the diameter of PtNPs was 54±14 nm; 2-2) 1 M sodium chloride and 0.1 M PBS with a pH of 7.4 were slowly added to the mixed solution in five times within 12 h, stirred for 24 h, and then centrifuged at 14,000 rpm for 30 min at 4 °C to remove the unreacted aptamer AS1411, thereby obtaining functionalized AS1411-PtNPs; 2-3) Finally, the functionalized AS1411-PtNPs were resuspended in 10 mM PBS, pH 7.4; 3) Preparation of cell suspension: including: 3-1) The cells in the logarithmic phase were digested with trypsin, centrifuged at 1000 rpm for 5 min, and then the cells at different concentrations were placed in a 96-well plate; 3-2) After 24 h of culture, discard the culture medium and wash with 10 mM PBS, pH 7.4; 3-3) Add 100 μL of AS1411-PtNPs cell culture medium with a final concentration of 0.49 mg / mL to each well, incubate for 4 h, wash the cells three times with PBS, digest and collect the cells to obtain a cell suspension; 4) co-incubating the AS1411 aptamer-functionalized PtNPs prepared in step 2) with the cell suspension prepared in step 3) to obtain a connection complex of PtNPs and cells; 5) The PtNPs and cell complex obtained in step 4) was transferred to a flask containing 110 μL of 30 wt % H 2 O 2 The gas generating tube is provided with a closed drainage device consisting of two spaced-apart but interconnected tubes, one of which is used for targeted mediated gas generation and the other for overflow of concentrated Sr 2+ The gas generating tube contained 1 ml of 50 mM Sr 2+ solution, PtNPs induced hydrogen peroxide H 2 O 2 decomposes to produce oxygen O 2 , resulting in an increase in the pressure of the closed system of the gas generating tube, collecting the spilled Sr 2+ The solutions were subsequently analyzed using the molecular colorimetric detection mode; 6) The overflowed 50 mM Sr 2+ The solution was treated with H 2 O volume was set to 860 μL; 7) Take another 10 μL of the diluted solution and react with 1 mL, 3 mM NaRho for precipitation reaction; 8) Photo identification: Take a photo of the precipitate obtained in step 7) to read the color, analyze the RGB value, and compare it with the detection mode of the molecular colorimetry to obtain the accurate value of the MCF-7 cell number.

[0014] In this example, the volumetric analysis range is: sodium rhodamine (NaRho) can be combined with Sr 2+ Chelation forms a brown-red precipitate, such as Figure 1 As shown in A, the precipitation reaction shows the wide range visual determination of Sr by molecular colorimetry. 2+ Volume prospects, such as Figure 1 As shown in B, as an organic ligand, the solution color of NaRho changes from yellow to orange and then to red as the concentration increases. 2+ When the solution turns reddish brown, it is attributed to the formation of insoluble NaRho-Sr 2+ The precipitate was observed by naked eye, and the ratiometric color conversion was proportional to the solution volume, which verified the molecular colorimetric method of Sr 2+The possibility of visual volume analysis, by comparing the relative contrast and saturation of colors, to demonstrate Sr 2+ The volume response range of the SrO2 solution significantly increases with the decrease of NaRho concentration. In this example, only 15 μL of SrO2 solution is required for 20 mM NaRho. 2+ The solution can reach saturation reaction, and 3.0 mM NaRho can react with Sr in the volume range of 0 to 30 μL. 2+ response, a wider range can be used depending on the NaRho dose and Sr 2+ Dosage product -NaRho-Sr 2+ As mentioned above, both flame reaction and precipitation reaction can be used for Sr 2+ Visual volumetric analysis of solutions, therefore, is expected to apply the advantages of precipitation reactions to sensing platforms to achieve higher sensitivity and a wide response range, e.g. Figure 1 C, under the optimal condition of 50 mM Sr 2+ and 3 mM NaRho, the flame reaction can only effectively distinguish volumes within the range of 0.5 μL; while the precipitation reaction can effectively distinguish volumes within the range of 0.5-20 μL.

[0015] Experimental Example 1: Preparation of PtNPs and visualization of cancer cells, including: 1) Preparation of PtNPs functionalized with AS1411 aptamer: H 2 PtCl 6 6H 2 O (2 mL, 18.9 mM), NaOH (30 μL, 5 M) L-ascorbic acid (0.2 mL, 1 M) and 2 mL ultrapure water were added to a beaker and mixed evenly. After heating in a 60 °C water bath for 10 min, the color of the solution changed from light yellow to brown-black. The PtNPs solution was then cooled to room temperature and stored at 4 °C for further use. Next, the PtNPs were functionalized with the aptamer using the salt aging method. The thiolated aptamer AS1411 (125 μL 5 μM) and PtNPs (2.5 mL) were mixed and incubated at room temperature overnight. Subsequently, sodium chloride (1 M) and PBS (0.1 M, pH 7.4) were slowly added to the mixed solution in multiple times within 12 h. After stirring for 24 h, the solution was centrifuged at 14,000 rpm at 4 °C for 30 min. min to remove the unreacted aptamer AS1411, and finally the functionalized AS1411-PtNPs were resuspended in PBS (10 mM, pH 7.4); 2) Comparison of catalytic ability between PtNPs and AS1411-PtNPs: The amount of PtNPs should be linear with the ratiometric color conversion of NaRho solution, which is crucial to prove the feasibility of the developed visual sensing platform, e.g. Figure 2 As shown in A, ascorbic acid-stabilized platinum nanoparticles with a size of 38.1 ± 14.9 nm were successfully synthesized. Then, the prepared platinum nanoparticles were introduced into 30% (w / v) H 2 O 2 The solution is used for gas generation, and the appearance of a large number of bubbles proves this. Figure 2 As shown in B, due to the pressure increase, 50 mM Sr 2+ The solution is pushed out of the closed system, which can be seen from the Sr 2+ In contrast, in the absence of PtNPs, the yellow-orange flame persists, confirming the importance of PtNP-mediated pressure enhancement for visual signal readout. Notably, the addition of PtNPs leads to a more pronounced color change of both the flame and the NaRho solution, suggesting a PtNP-dependent ratiometric optical signal, such as Figure 2 As shown in C, the thiolated AS1411 aptamer was successfully modified on the surface of PtNPs by the salt aging method, and Figure 2 D. Figure 2 The functional modification process does not affect the catalytic efficiency of PtNPs, as shown in Figure E. Compared with the atomic flame detection method, which is sensitive to a small amount of PtNPs, the molecular colorimetric detection method is sensitive to a large amount of PtNPs, which further proves the possibility of a wide range of responses.

[0016] Experimental Example 2: Visualization process of MCF-7 cells: 1) MTT experiment: including: 1-1) First, human cervical cancer HeLa cells, human breast cancer MCF-7 cells, and mouse fibroblast L929 cells were cultured in DMEM medium containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin in an environment of 5% CO. 2 , 37°C, wash the cells with sterile PBS (0.01 M, pH 7.4), and digest with 0.25% trypsin when the cell density reaches 80%-90%; 1-2) The cytotoxicity of AS1411-PtNPs complex to HeLa cells was evaluated by MTT method, that is, the logarithmic phase HeLa cells were first washed with PBS, seeded into 96-well plates at a density of 105 cells / mL per well, and cultured in an incubator for 24 hours. Then, different concentrations of AS1411-PtNPs were added, and the final concentrations were 0.05, 0.25, 0.49, and 0.74 mg / mL, respectively. The wells with PBS were used as blank control groups, and cultured for 4 hours. Then, MTT (10 μL, 5 mg / mL) was added, and cultured for another 4 hours. All the liquid in the wells was removed, and then 150 μL DMSO was added to each well. After shaking on a shaker for 10 min, the wells were placed on a microplate reader to measure the absorbance at 490 nm. Figure 3 As shown in Figure A, in order to evaluate the cytotoxicity of the synthesized AS1411-PtNPs complex, HeLa cells were incubated with different concentrations of AS1411-PtNPs, and then the in vitro cell viability was determined by the MTT method. The cell activity in all samples exceeded 80%, proving that AS1411-PtNPs have good biocompatibility. Figure 3 As shown in B, after combining with AS144-PtNPs, HeLa cells remained in a relatively good state, which was well demonstrated by the fact that the cell viability exceeded 80%, indicating that the synthesized AS144-PtNPs had relatively low toxicity to HeLa cells.

[0017] 1-2) Visualization detection of MCF-7 cells: To detect diseased cells, AS1411 aptamer-functionalized PtNPs (AS1411-PtNPs) were first incubated with HeLa cells at 37 °C for 4 h and then washed with PBS (0.01 M, pH 7.4) to remove unbound AS1411-PtNPs. Figure 3 As shown in Figure B, 3,3',5,5'-tetramethylbenzidine hydrochloride (TMB)-H 2 O 2 The supernatant was still a colorless solution, which confirmed that after three washes with PBS, the unbound AS1411-PtNPs could be completely removed. 2 O 2 decompose to initiate pressure-mediated flame / precipitation reactions, e.g. Figure 3As shown in A, the red column in the figure proves the selective binding of AS1411-PtNPs to HeLa cells, and the corresponding R / G value is nearly 2 times higher than that of cells treated with bare PtNPs (gray column) similar to the blank group (green column), while the R / G value of HeLa cells alone is lower, indicating that AS1411-PtNPs specifically bound to cells are the cause of the light signal; Figure 3 As shown in B, the atomic flame detection mode was used in the case of low concentration, and the molecular colorimetric detection mode was used in the case of high concentration. Although the light signals of different cells were different, the signal intensities of all samples showed a similar trend regardless of the detection mode selected.

[0018] Experimental Example 3: Analysis of actual samples: including: 1) Analysis of the relationship between R / G value and cell concentration: For molecular colorimetric detection modes, such as Figure 4 As shown in B, the increase in cell concentration is accompanied by a higher R / G value, which remains consistent and has an excellent linear relationship in the concentration range of 200-10000 cells / mL. The colorimetric method can capture subtle color changes, thereby achieving accurate determination of the number of MCF-7 cells. This visual sensing platform not only facilitates high-sensitivity detection and analysis, but also provides a wide range of responses visible to the naked eye. 2) Use this method to analyze actual human blood samples; In this example, HeLa cells of different concentrations (90, 102, 104, 105 cells / mL) were added to 1000-fold diluted human blood, and then 100 μL of culture medium containing AS1411-PtNPs (final concentration: 0.49 mg / mL) was added. After incubation for 4 h, the culture medium was removed, and the cells were washed three times with PBS, digested and collected. Finally, the cell suspension was transferred to a 110 μL H 2 O 2 (30 wt %) gas generating tube, the reaction pushes out Sr 2+ Then, the method in this example is used for visual inspection and analysis. Figure 4 As can be seen from A, the test results in the actual sample are almost the same as Figure 4 B, with recoveries ranging from 95.0% to 104.0%, which verified the analytical sensitivity of the actual samples.

Claims

1. A molecular colorimetric ratio detection and analysis method based on metal ions, characterized in that: The steps include: 1) Preparation of PtNPs: including: 1-1) Add 2 mL, 18.9 mM H2PtCl6·6H2O, 30 μL, 5 M NaOH, 0.2 mL, 1 M L-ascorbic acid and 2 mL ultrapure water into a beaker and mix well. 1-2) After heating the mixed solution in step 1-1) in a 60°C water bath for 10 min, the color of the solution changed from light yellow to brown-black, thereby obtaining a PtNPs solution. The PtNPs solution was then cooled to room temperature and stored at 4°C. 2) Preparation of PtNPs functionalized with AS1411 aptamer: including: 2-1) PtNPs were functionalized with aptamers using the salt aging method, i.e., 125 μL, 5 μM thiolated aptamer AS1411 and 2.5 mL PtNPs were mixed and incubated overnight at room temperature, wherein the concentration of PtNPs was 5 mg / mL and the diameter of PtNPs was 54±14 nm; 2-2) 1 M sodium chloride and 0.1 M PBS with a pH of 7.4 were slowly added to the mixed solution in five times within 12 h, stirred for 24 h, and then centrifuged at 14,000 rpm for 30 min at 4 °C to remove the unreacted aptamer AS1411, thereby obtaining functionalized AS1411-PtNPs; 2-3) Finally, the functionalized AS1411-PtNPs were resuspended in 10 mM PBS, pH 7.4; 3) Preparation of cell suspension: including: 3-1) The cells in the logarithmic phase were digested with trypsin, centrifuged at 1000 rpm for 5 min, and then the cells at different concentrations were placed in a 96-well plate; 3-2) After 24 h of culture, discard the culture medium and wash with 10 mM PBS, pH 7.4; 3-3) Add 100 μL of AS1411-PtNPs cell culture medium with a final concentration of 0.49 mg / mL to each well, incubate for 4 h, wash the cells three times with PBS, digest and collect the cells to obtain a cell suspension; 4) co-incubating the AS1411 aptamer-functionalized PtNPs prepared in step 2) with the cell suspension prepared in step 3) to obtain a connection complex of PtNPs and cells; 5) The PtNPs and cell complex obtained in step 4) was transferred to a gas generating tube containing 110 μL of 30 wt % H2O2. The gas generating tube was equipped with a closed drainage device consisting of two spatially separated but interconnected tubes, one of which was used for targeted mediated gas production and the other for overflow of concentrated Sr 2+ The gas generating tube contains 1 ml of 50 mM Sr 2+ Solution; 6) The overflowed 50 mM Sr 2+ The solution was made up to 860 μL with H O; 7) Take another 10 μL of the diluted solution and react with 1 mL, 3 mM NaRho for precipitation reaction; 8) Photo identification: Take a photo of the precipitate obtained in step 7) to read the color, analyze the RGB value, and compare it with the detection mode of the molecular colorimetry to obtain the accurate value of the MCF-7 cell number.

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