Biosensor for measuring nitroreductase, preparation method for biosensor, and quantitative measurement method for nitroreductase

Abstract

A biosensor for measuring nitroreductase, a preparation method for a biosensor, and a quantitative measurement method for nitroreductase. The preparation method for the biosensor comprises the following steps: uniformly mixing an ethanol solution of Cy-No2 and a hydrochloric acid solution of Fmoc-H, then adding a Zn2+ aqueous solution, uniformly stirring, adjusting the pH value of a system to 7, continuing stirring to react for 55-65 min, and then centrifuging, washing, and drying to prepare the biosensor for measuring nitroreductase. The preparation method is simple, and the biosensor can be prepared by simple one-step self-assembly, can be used for quantitative measurement of nitroreductase, and can be used for monitoring the nitroreductase of hypoxic cancer cells by means of an in-vitro fluorescence biological imaging technology.

Description

A biosensor for detecting nitroreductase, a preparation method thereof, and a quantitative detection method of nitroreductase Technical Field

[0001] The present invention belongs to the technical field of biosensors, and particularly relates to a biosensor for detecting nitroreductase, a preparation method thereof, and a quantitative detection method of nitroreductase. Background Art

[0002] Nitroreductases (NTRs) are a family of flavin-containing enzymes that reduce the levels of nitro compounds in the presence of reduced nicotinamide adenosine nucleotide (NADH). Furthermore, nitroreductases have been reported to play a role in the activation of nitro compound-based prodrugs for cancer therapy. Therefore, sensitive and selective detection and imaging of nitroreductases in cancer cells is of great significance for a better understanding of their biological functions or the development of cancer therapeutic probes.

[0003] Numerous methods are currently available for nitroreductase detection. Fluorescent probes, among them, can provide sensitive and highly sensitive imaging of abnormally expressed molecules in tissues, making them key tools in molecular biology and cancer therapy. However, most of these fluorescent probes are free small organic molecule dyes, which suffer from limitations such as rapid metabolism and weak signal contrast between normal tissue and tumor sites.

[0004] Therefore, sensitive and selective detection and imaging of nitroreductase in cancer cells is of great significance for a deeper understanding of its biological functions or the development of better cancer therapeutic probes. The ability to achieve simple, sensitive, and selective quantitative detection of nitroreductase in cancer cells is an urgent problem that needs to be solved.

[0005] Summary of the Invention

[0006] To solve the above technical problems, the present invention provides a biosensor for detecting nitroreductase, a preparation method thereof, and an application thereof. The biosensor is a multi-stimulus-responsive near-infrared fluorescence biosensor based on metal ion-amino acid coordination-driven multi-stimulus-responsive near-infrared fluorescence, which can be synthesized through a simple one-step self-assembly; it can be used to achieve quantitative detection of nitroreductase.

[0007] The present invention also provides a method for quantitative detection of nitroreductase. Using the biosensor provided by the present invention as a sensor, quantitative detection of nitroreductase can be achieved. During detection, the biosensor releases a fluorescent dye that can catalyze the cleavage of nitroreductase in response to pH stimulation, and its fluorescence signal switches from off to on. As the concentration of nitroreductase increases, the fluorescence signal of the detection system gradually increases. A linear curve can be constructed with the concentration of the nitroreductase solution as the horizontal coordinate and the fluorescence intensity of the detection system at a specific emission wavelength as the vertical coordinate to achieve quantitative detection of nitroreductase. The detection method is simple to operate and has high sensitivity.

[0008] To achieve the above object, the technical solution adopted by the present invention is as follows:

[0009] A method for preparing a biosensor for detecting nitroreductase, the method comprising the following steps:

[0010] Mix the ethanol solution of Cy-NO2 and the hydrochloric acid solution of Fmoc-H evenly, then add Zn 2+ The aqueous solution is stirred evenly and the pH of the system is adjusted to 7. The stirring reaction is continued for 55 to 65 minutes. Then, the solution is centrifuged, washed, and dried to prepare the biosensor ZFC NPs for detecting nitroreductase.

[0011] The molecular structures of the Fmoc-H and Cy-NO2 are as follows:

[0012] Fmoc-H:C 21 H 19 N3O4

[0013] Cy-NO2:C 48 H 58 N5O4

[0014] In the reaction system, Cy-NO2, Fmoc-H and Zn 2+ The molar ratio is 1:2:1.

[0015] Cy-NO2 ethanol solution, Fmoc-H hydrochloric acid solution, Zn 2+ The molar concentration of the aqueous solution is the same.

[0016] The drying is freeze-drying.

[0017] The present invention also provides a biosensor for detecting nitroreductase prepared by the preparation method. This fluorescent sensor is basically non-fluorescent in a reaction system without nitroreductase. After adding nitroreductase and in a reaction system with a pH of 6.5, it can emit strong fluorescence at an excitation wavelength of 680nm.

[0018] The present invention also provides application of the biosensor for detecting nitroreductase in detecting nitroreductase.

[0019] The present invention also provides a method for detecting nitroreductase, which comprises the following steps:

[0020] 1) Multiple groups of ZFC NPs were dispersed in a pH 6.5 PBS buffer solution, and then a series of nitroreductase solutions were added and reacted in the dark for 1 h.

[0021] 2) Testing the fluorescence spectrum of each reaction system at an excitation wavelength of 680 nm;

[0022] 3) A linear curve is constructed with the concentration of the nitroreductase solution as the abscissa and the fluorescence intensity at 785 nm as the ordinate, and a linear equation is derived. According to the linear equation, the concentration of the nitroreductase to be tested corresponding to any fluorescence intensity at 785 nm can be obtained.

[0023] In step 1), the concentrations of the nitroreductase solution were 10 μg / mL, 5 μg / mL, 1.5 μg / mL, 1.2 μg / mL, 0.8 μg / mL, 0.6 μg / mL, 0.4 μg / mL, 0.25 μg / mL, and 0 μg / mL, respectively.

[0024] In step 3), the linear equation is y=1050.16x-10.5763, wherein y is the fluorescence intensity at 785 nm, and x is the nitroreductase concentration, and the linear correlation coefficient is 0.994.

[0025] In the preparation method of the biosensor for detecting nitroreductase provided by the present invention, uniform ZFC NPs nanoreagents are obtained on the basis of coordination and multiple non-covalent interactions by using metal-binding amino acids, metal ions and Cy-NO2 as building blocks.

[0026] In the present invention, 9-fluorenylmethyloxycarbonyl modified histidine (Fmoc-H) with good biocompatibility is selected as the bridging ligand, and zinc ion (Zn 2+ ) is a trace element required for normal physiological processes and is easily metabolized and cleared without obvious side effects. Cy-NO2 is sensitive and selective to NTR overexpressed in hypoxic cells. The nanoreagent has a high loading capacity and shows responsive release characteristics in the tumor microenvironment. The application of ZFC NPs nanoreagents can be used to monitor the NTR of hypoxic cells through in vitro fluorescence bioimaging technology.

[0027] The biosensor provided by the present invention can be used for the quantitative detection of NTR concentration. The reaction solution of the detection needs to be carried out in an acidic environment of pH 6.5 to ensure the effective release of the fluorescent probe. The imidazole group on Fmoc-H in the biosensor will be protonated in an acidic solution, thereby releasing Cy-NO2 with high fluorescence intensity. Cy-NO2 consists of a fluorescent reporter unit (aminoamide dye) and an NTR recognition unit (p-nitrobenzyl carbamate). The electron transfer process induced by the electron-withdrawing group in the biosensor quenches its fluorescence emission. As long as the nitro group in the biosensor is successfully reduced to an amino group by NTR catalysis, the fluorescent signal will turn from off to on. If it is not carried out in acid, the fluorescence intensity does not change much after adding NTR. As a result, this results in that the fluorescence intensity cannot establish a corresponding relationship with the NTR concentration, and thus cannot achieve quantitative detection of the NTR concentration.

[0028] When quantitatively detecting NTR concentration, as the concentration of nitroreductase increases, the fluorescence signal of the detection system gradually increases. A linear curve can be constructed with the concentration of the nitroreductase solution as the horizontal axis and the fluorescence intensity of the detection system at a specific emission wavelength as the vertical axis to achieve quantitative detection of nitroreductase. The detection method is simple to operate and has high sensitivity.

[0029] Compared with the prior art, the present invention has the following beneficial effects:

[0030] The preparation method of the biosensor for detecting nitroreductase provided by the present invention is simple and can be prepared by a simple one-step self-assembly. It can be used for the quantitative detection of nitroreductase and can be used to monitor the NTR of hypoxic cancer cells through in vitro fluorescence bioimaging technology, and has a very good application prospect. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] FIG1 is a schematic diagram of a method for quantitatively detecting nitroreductase concentration using a biosensor provided by the present invention;

[0032] FIG2 is a TEM image of the biosensor prepared in Example 1;

[0033] FIG3 is a fluorescence spectrum of the biosensor prepared in Example 1 in PBS buffer solutions of different pH values ​​before and after the addition of nitroreductase;

[0034] FIG4 is a fluorescence spectrum of the detection system after adding different concentrations of nitroreductase in Example 2;

[0035] FIG5 is a linear relationship diagram constructed with the concentration of the NTR solution as the abscissa and the fluorescence intensity as the ordinate in Example 2;

[0036] FIG6 is a TEM image of the product obtained at different reaction times in Comparative Example 1;

[0037] FIG7 is a TEM image of the product obtained in Comparative Example 2 with different reaction raw materials;

[0038] FIG8 is a TEM image of the product prepared in Comparative Example 3;

[0039] FIG9 is a TEM image of the product obtained in Comparative Example 4 at different pH values ​​of the reaction system;

[0040] FIG10 shows the results of in vitro cell experiments, wherein (a) shows the experimental results of the control group, (b) shows the experimental results of the experimental group, and (c) shows the experimental results of the inhibition group. DETAILED DESCRIPTION

[0041] The present invention is described in detail below with reference to the embodiments.

[0042] In the embodiment, ethanol solution of Cy-NO2, hydrochloric acid solution of Fmoc-H, Zn 2+ The aqueous solution was prepared as follows:

[0043] Ethanol solution of Cy-NO2: Dissolve Cy-NO2 in anhydrous ethanol;

[0044] Fmoc-H hydrochloric acid solution: Dissolve Fmoc-H in 50 mM hydrochloric acid solution;

[0045] Zn 2+ Aqueous solution: Dissolve ZnCl2 in deionized water.

[0046] Nitroreductase solution: Nitroreductase was dissolved in a PBS buffer solution at pH 7.4.

[0047] Fmoc-H was purchased from Bidex Pharmaceuticals, batch number CMV973.

[0048] For the preparation method of Cy-NO2, see Near-infrared off-on fluorescence probe activated by NTR for in vivo hypoxia imaging, Biosensors and Bioelectronics 119(2018)141-148.

[0049] Example 1

[0050] A method for preparing a biosensor for detecting nitroreductase, the method comprising the following steps:

[0051] Mix the ethanol solution of Cy-NO2 and the hydrochloric acid solution of Fmoc-H with equal molar concentrations, and then add the Zn 2+The aqueous solution was stirred evenly and then the pH of the system was adjusted to 7 with 10mM sodium hydroxide solution. In the reaction system, Cy-NO2, Fmoc-H and Zn 2+ The molar ratio of 1:2:1 was 1:2:1, and the reaction was continued with stirring for 1 hour. Then, the mixture was centrifuged, washed, and freeze-dried to prepare the biosensor ZFC NPs for detecting nitroreductase.

[0052] The TEM image is shown in FIG2 , from which it can be seen that the particles are spherical with an average particle size of 100 nm.

[0053] 100 μg of the biosensor ZFC NPs prepared in this embodiment were dispersed in 1 mL of 10 mM PBS buffer solution at pH 6.5 and PBS buffer solution at pH 7.4, respectively. The fluorescence spectra at different pH values ​​were then tested at an excitation wavelength of 680 nm. Nitroreductase solution was then added to each of the solutions. The final concentration of nitroreductase in the reaction system was 1.0 μg / mL. After the reaction was protected from light for 1 hour, the results were tested at different pH values ​​at an excitation wavelength of 680 nm. As shown in FIG3 , before the addition of the nitroreductase solution, the biosensor ZFC NPs had no fluorescence at both pH values. However, after the addition of the nitroreductase solution, the reaction system had a strong fluorescence intensity at 785 nm in an environment of pH 6.5, and still had no fluorescence in an environment of pH 7.4. This indicates that the biosensor constructed in this embodiment can be cleaved and release the fluorescent probe in an acidic environment. After the addition of nitroreductase (NTR), NTR reacts with Cy-NO2, resulting in a significant fluorescence enhancement at 785 nm.

[0054] Example 2

[0055] A method for quantitatively detecting nitroreductase, comprising the following steps:

[0056] 1) Multiple groups of 80 μg ZFC NPs were dispersed in 1 mL of pH 6.5 PBS buffer solution, and then a series of nitroreductase solutions were added. The reaction was carried out in the dark for 1 h. The final concentrations of nitroreductase in each system were 10 μg / mL, 5 μg / mL, 1.5 μg / mL, 1.2 μg / mL, 0.8 μg / mL, 0.6 μg / mL, 0.4 μg / mL, 0.25 μg / mL, and 0 μg / mL, respectively.

[0057] 2) Fluorescence spectra of each reaction system at an excitation wavelength of 680 nm were measured, as shown in FIG4 . It can be seen from the figure that as the concentration of nitroreductase increases, the fluorescence signal of the detection system gradually increases;

[0058] 3) A linear curve was constructed with the concentration of the nitroreductase solution as the abscissa and the fluorescence intensity at 785 nm as the ordinate, as shown in FIG5 . A linear equation was then derived: y = 1050.16x - 10.5763, where y is the fluorescence intensity at 785 nm and x is the nitroreductase concentration. The linear correlation coefficient was 0.994. Based on the linear equation, the concentration of the nitroreductase to be measured corresponding to any fluorescence intensity at 785 nm could be obtained.

[0059] Example 3

[0060] To further verify the specific response of the biosensor provided by the present invention to nitroreductase, in vitro cell experiments were conducted using human cervical cancer cells (HeLa) and human breast cancer cells (MCF-7), as well as human embryonic kidney cells (HEK293T).

[0061] The experimental process is as follows:

[0062] According to literature reports, cobalt chloride (CoCl2) is often used to induce hypoxia models in tumor cells due to its advantages such as low price and fast induction rate. Therefore, the present invention simulates 1% O2 conditions by incubating cells with 150μM CoCl2 for 8h.

[0063] Experimental group:

[0064] (1) Cobalt chloride induced hypoxia in Hela, MCF-7, and 293T cells: After Hela, MCF-7, and 293T cells were growing well and their cell population reached 80%, the cell waste was aspirated and different concentrations of cobalt chloride 1640 culture medium dispersion (0 μM, 150 μM) were added to simulate different oxygen conditions (20% O2, 1% O2) for incubation of cells. The cells were incubated in a carbon dioxide incubator for 8 h. The morphology of the three cells was observed using an inverted microscope for subsequent studies.

[0065] (2) Hypoxia fluorescence imaging of Hela cells: The residual cobalt chloride in the Hela cell glass-bottomed culture dish was aspirated, and 1 mL of culture medium solution containing 6.125 μg of ZFC NPs 1640 was added and incubated with the Hela cells in a hypoxic state for 8 h. After the incubation, the culture medium containing the fluorescent probe in the Hela cells was aspirated and washed twice with PBS phosphate buffer solution. Then, the cell nucleus staining reagent Hoechst was added, incubated for 15 min, and washed three times with PBS phosphate buffer solution. Finally, 1 mL of PBS phosphate buffer solution was added to each glass-bottomed culture dish, and fluorescence imaging of the Hela cells was recorded using a Leica laser confocal microscope. The excitation filter was 405 nm to collect the emission signal of the blue channel (420-480 nm); 638 nm to collect the emission signal of the red channel (750-800 nm), as shown in (b) of Figure 10.

[0066] Hypoxic fluorescence imaging of MCF-7 and 293T cells: Residual cobalt chloride was removed from the glass-bottomed culture dishes containing MCF-7 and 293T cells. 1 mL of DMEM culture medium containing 6.125 μg of ZFC NPs was added and incubated with the hypoxic MCF-7 and 293T cells for 8 hours. After incubation, the medium containing the fluorescent probe was removed from the MCF-7 and 293T cells, and the cells were washed twice with PBS. Hoechst, a nuclear stain, was then added, incubated for 15 minutes, and washed three times with PBS. Finally, 1 mL of PBS was added to each glass-bottomed dish, and fluorescence imaging of the MCF-7 and 293T cells was performed using a Leica laser confocal microscope. The excitation filter wavelength is 405 nm, and the emission signal of the blue channel (420-480 nm) is collected; the excitation filter wavelength is 638 nm, and the emission signal of the red channel (750-800 nm) is collected, as shown in (b) of FIG10 .

[0067] Control group: Hela cells, MCF-7 cells, and HEK293T cells were incubated under normoxic conditions with 20% O₂ for 8 h, then added to a 6.125 μg / mL ZFC NPs solution and incubated for 8 h. Cell imaging was then performed using a Leica confocal microscope. The ZFC NPs solution was prepared by dispersing the ZFC NPs prepared in Example 1 in a 1640 culture medium solution and a DMEM culture medium solution, respectively. This is shown in Figure 10(a).

[0068] Inhibitor group: Hela cells, MCF-7 cells, and HEK293T cells were cultured with dicoumarol for 2 h. Other conditions were maintained the same as those of the other two groups. Cell imaging was then performed using a Leica confocal microscope. The results are shown in Figure 10 (c). The fluorescence intensity was significantly different from that of the group without inhibitor addition, and all three cell types showed no fluorescence.

[0069] These results demonstrate that the biosensor for detecting nitroreductase provided by the present invention achieves an "off-on" effect by specifically binding to NTR. Furthermore, because the onset of the biosensor's fluorescence signal is controlled by both tumor acidity and nitroreductase, it demonstrates excellent ability to distinguish cancer cells from normal cells.

[0070] Comparative Example 1

[0071] The rest of the process was the same as in Example 1, except that the reaction time was changed to 0, 15, 30, 45, 75, 90, 120, and 600 min, respectively. The TEM images of the final products are shown in FIG6 . It can be seen from the figure that ZFC NPs with uniform size and stable morphology can only be obtained when the reaction time is 1 h.

[0072] Comparative Example 2

[0073] The rest is the same as in Example 1, except that the hydrochloric acid solution of Fmoc-H, the ethanol solution of Cy-NO2, and the Zn 2+ The TEM image of the final product is shown in Figure 7. It can be seen from the figure that without any of the above raw materials, nanoparticles with uniform size and stable morphology cannot be prepared.

[0074] Comparative Example 3

[0075] The other conditions were the same as in Example 1, except that the hydrochloric acid solution of Fmoc-H was replaced by the hydrochloric acid solution of His. The TEM image of the final product is shown in FIG8 . As can be seen from the figure, without the hydrophobic effect of the Fmoc group, nanoparticles with uniform size and stable morphology could not be prepared.

[0076] Comparative Example 4

[0077] The other conditions were the same as in Example 1, except that the pH of the reaction system was adjusted to pH = 9 and pH = 11. The TEM images of the final products are shown in FIG9 . As can be seen from the figure, when the pH of the system is adjusted to alkaline, the formed nanoparticles are cross-linked, while the nanoparticles formed only under neutral conditions are more uniformly dispersed.

[0078] The detailed description of a biosensor for detecting nitroreductase, a method for preparing the same, and a method for quantitatively detecting nitroreductase with reference to the above-mentioned embodiments is illustrative rather than restrictive. Several embodiments may be enumerated within the limited scope. Therefore, changes and modifications that do not depart from the overall concept of the present invention are intended to be within the scope of protection of the present invention.

Claims

1. A method for preparing a biosensor for detecting nitroreductase, characterized in that: The preparation method comprises the following steps: Mix the ethanol solution of Cy-NO2 and the hydrochloric acid solution of Fmoc-H evenly, then add Zn 2+ The aqueous solution is stirred evenly, and the pH of the system is adjusted to 7. The stirring reaction is continued for 55 to 65 minutes, and then centrifuged, washed, and dried to prepare the biosensor ZFC NPs for detecting nitroreductase.

2. The preparation method according to claim 1, characterized in that: In the reaction system, Cy-NO2, Fmoc-H and Zn 2+ The molar ratio is 1:2:

1.

3. The preparation method according to claim 1, characterized in that: Cy-NO2 ethanol solution, Fmoc-H hydrochloric acid solution, Zn 2+ The molar concentration of aqueous solutions is the same.

4. A biosensor for detecting nitroreductase prepared by the preparation method according to any one of claims 1 to 3.

5. Use of the biosensor for detecting nitroreductase as claimed in claim 4 in detecting nitroreductase.

6. A method for quantitative detection of nitroreductase, characterized in that: The detection method comprises the following steps: 1) Disperse multiple groups of ZFC NPs in pH 6.5 PBS buffer solution, then add nitroreductase solution of a series of concentrations and react for 1 hour in the dark; 2) Testing the fluorescence spectrum of each reaction system at an excitation wavelength of 680 nm; 3) A linear curve was constructed with the concentration of nitroreductase solution as the horizontal axis and the fluorescence intensity at 785 nm as the vertical axis, and then a linear equation was obtained. According to the linear equation, any The fluorescence intensity at 785 nm corresponds to the concentration of the nitroreductase to be tested.

7. The quantitative detection method of nitroreductase according to claim 6, characterized in that: In step 1), the concentrations of the nitroreductase solution were 10 μg / mL, 5 μg / mL, 1.5 μg / mL, 1.2 μg / mL, 0.8 μg / mL, 0.6 μg / mL, 0.4 μg / mL, 0.25 μg / mL, and 0 μg / mL, respectively.

8. The method for quantitative detection of nitroreductase according to claim 6, characterized in that: In step 3), the linear equation is y=1050.16x-10.5763, wherein y is the fluorescence intensity at 785 nm, and x is the nitroreductase concentration, and the linear correlation coefficient is 0.994.

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