Antioxidant system and application thereof

Abstract

The invention discloses an anti-oxidation system. The anti-oxidation system is prepared from 0.05 v / v% to 1 v / v% of squalene and 0.2 mM to 5 mM of Trolox. According to the anti-oxidation system provided by the invention, the squalene and the Trolox are combined for use, so that the anti-oxidation function of the Trolox is exerted to the greatest extent; and by using squalene, the problem of pH value fluctuation caused by using a Gloxy and glucose mixed solution can be solved, and the antioxidant system provided by the invention can realize an excellent oxygen removal effect under the condition of not influencing the pH value of the system.

Description

Technical Field

[0001] The present invention relates to the field of biological detection technology, and in particular to an antioxidant system and application thereof. Background Art

[0002] The emergence and development of single-molecule science in the 1990s enabled the observation and manipulation of the microscopic world of matter at the atomic and molecular scales. Optical measurements at the single-molecule level can reveal the properties of individual molecules' activities that are often obscured by the average of a larger molecular ensemble. High-resolution fluorescence imaging based on single molecules, used for molecular probes and fluorescent dye labeling, is a crucial technique for studying molecular diffusion, genetic recombination, and protein dynamics.

[0003] To achieve single-molecule detection, the background fluorescence of the solution needs to be significantly reduced, but the laser cannot be directly irradiated into the sample. When light is reflected on the surfaces of two materials with different refractive indices, the evanescent wave only excites the sample tens of nanometers below the interface, which can ensure that fewer molecules are activated and the noise is reduced. Completely removing oxygen free radicals can increase the lifespan of fluorescent molecules and reduce free radical-induced quenching. The current technical solutions use a mixed solution of Gloxy and glucose to remove oxygen free radicals. In addition, the blinking of fluorescent molecules is also a major factor affecting the data results. The application of Trolox can greatly reduce or even eliminate the blinking of fluorescent molecules. Therefore, in single-molecule fluorescence imaging systems, it is very important to solve the problem of excessive oxygen free radicals and blinking of fluorescent molecules. However, in the process of using a mixed solution of Gloxy and glucose for oxygen scavenging, the hydrogen peroxide and acid produced will lower the pH value of the system, thereby affecting subsequent reactions. Summary of the Invention

[0004] The present invention aims to address at least one of the technical problems existing in the prior art. To this end, the present invention proposes an antioxidant system that can enhance deoxygenation reactions in single-molecule fluorescence imaging systems while avoiding impacts on the pH of the entire reaction system and eliminating Cy5 fluorescence flickering. This antioxidant system also enables multi-channel detection, enabling rapid, accurate, low-cost, and multi-sample fluorescence single-molecule detection.

[0005] The present invention also provides an oxygen scavenging method for a single-molecule fluorescence imaging system.

[0006] The present invention also provides a multi-channel single-molecule fluorescence detection device.

[0007] The present invention also provides an application.

[0008] According to a first aspect of the present invention, an antioxidant system is provided. The antioxidant system comprises 0.05 v / v% to 1 v / v% and 0.2 to 5 mM Trolox.

[0009] In some embodiments of the present invention, the antioxidant system includes 0.3 v / v% to 0.9 v / v% and 1 to 3 mM Trolox.

[0010] Existing solutions use a mixed solution of Gloxy and glucose to scavenge oxygen free radicals. Gloxy is an antioxidant system composed of glucose oxidase and catalase. While Gloxy affects the pH of the reaction system, the antioxidant system provided by the present invention maintains a stable pH without affecting subsequent reactions.

[0011] According to a second aspect of the present invention, an oxygen scavenging method for a single-molecule fluorescence imaging system is proposed. The oxygen scavenging method for a single-molecule fluorescence imaging system adopts the antioxidant system described in the first aspect of the present invention.

[0012] In some embodiments of the present invention, the oxygen scavenging method comprises the following steps:

[0013] S1: Immobilize the biotin-carrying polymer on the surface of the slide, and then add streptavidin for incubation;

[0014] S2: Add the sample to be tested carrying biotin and fluorescent groups to the surface of the slide for incubation;

[0015] S3: Prepare the antioxidant system as described in claim 1 and add it to the sample chamber where the slide is located, and then form an image and observe.

[0016] In some embodiments of the present invention, the biotin-carrying polymer in step S1 comprises biotin-polyethylene glycol-succinimidyl carbonate (biotin-PEG-SC).

[0017] In some embodiments of the present invention, step S1 further uses polyethylene glycol-succinimidyl carbonate (PEG-SC).

[0018] In some embodiments of the present invention, the mixing mass ratio of the biotin-PEG-SC and PEG-SC is 1:(25-30).

[0019] In some embodiments of the present invention, the glass slide in step S1 is modified by aminosilanization.

[0020] In some embodiments of the present invention, step S1 further includes performing a quality inspection on the fluorescent preparation before adding streptavidin, wherein the quality inspection includes: adding a fluorescently labeled protein sample, incubating for 5 to 15 minutes, then washing, and observing whether there is fluorescence; if all fluorescence is detected, the preparation is unqualified, and if there is no fluorescent signal, subsequent operations are continued.

[0021] In some embodiments of the present invention, the incubation time in step S1 is 5 to 15 minutes.

[0022] In some embodiments of the present invention, the fluorescent group in step S2 includes at least one of Cy3, Cy5, Alexa Fluor 488, Alexa Fluor 594 and FITC.

[0023] In some embodiments of the present invention, the incubation time in step S2 is 1 to 8 minutes.

[0024] In some embodiments of the present invention, the sample chamber in step S3 is a sandwich structure, which includes a cover glass, a double-sided tape and a slide from top to bottom.

[0025] The sandwich-structured sample chamber can greatly reduce the contact area between the reaction system and the air, thereby reducing oxidation.

[0026] In some embodiments of the present invention, at least two independent reaction channels are provided on the glass slide of the sample chamber.

[0027] In some embodiments of the present invention, the slide has at least two holes for introducing and exporting reagents.

[0028] According to a third aspect of the present invention, a multi-channel single-molecule fluorescence detection device is provided, the detection device comprising: a sample chamber, an excitation light source, an inverted fluorescence microscopy optical path module and an imaging device;

[0029] The sample chamber is composed of a cover glass, double-sided tape and a glass slide, and the sample chamber is loaded with the antioxidant system according to claim 1.

[0030] In some embodiments of the present invention, the glass slide of the sample chamber is provided with at least two independent reaction channels; the glass slide has at least two holes for introducing and exporting reagents.

[0031] In some embodiments of the present invention, the sample chamber is loaded with the antioxidant system described in the first aspect of the present invention.

[0032] According to a fourth aspect of the present invention, the use of squalene in preparing an antioxidant system for a single-molecule fluorescence imaging system is proposed.

[0033] In some embodiments of the present invention, squalene is used in combination with Trolox in an antioxidant system of a single molecule fluorescence imaging system.

[0034] In some embodiments of the present invention, the antioxidant system includes 0.05 v / v% to 1 v / v% squalene and 0.2 to 5 mM Trolox.

[0035] In some embodiments of the present invention, the antioxidant system includes 0.3 v / v% to 0.9 v / v% and 1 to 3 mM Trolox.

[0036] The present invention has at least the following beneficial effects:

[0037] 1) The antioxidant system provided by the present invention combines squalene with Trolox to maximize the antioxidant function of Trolox. The use of squalene can overcome the pH fluctuation problem caused by the use of a mixture of Gloxy and glucose. The antioxidant system provided by the present invention can achieve excellent oxygen scavenging effects without affecting the pH value of the system.

[0038] 2) The oxygen scavenging method for a single-molecule fluorescence imaging system provided by the present invention improves the oxygen scavenging efficiency in the single-molecule fluorescence imaging system by using the above-mentioned antioxidant system and the sandwich-structured sample chamber, thereby extending the lifespan of the fluorescent molecules, reducing the fluorescence quenching induced by free radicals, and ultimately achieving accurate and rapid single-molecule fluorescence detection.

[0039] 3) The multi-channel single-molecule fluorescence detection device provided by the present invention improves the oxygen scavenging efficiency in the single-molecule fluorescence imaging system by using the above-mentioned antioxidant system and the sandwich-structured sample chamber, thereby extending the lifespan of the fluorescent molecules and reducing the fluorescence quenching induced by free radicals, and ultimately achieving accurate and rapid single-molecule fluorescence detection; at the same time, the present invention designs at least two channels of independent reaction units in the above-mentioned sample chamber, which can perform quality inspection on the fluorescent preparation before formal sample loading, thereby avoiding the problem of sample waste.

[0040] Additional aspects and advantages of the present invention will be set forth in part in, and in part will be apparent from, the description which follows. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] The present invention will be further described below with reference to the accompanying drawings and embodiments, in which:

[0042] Figure 1 Schematic diagram of the sample chamber of the multi-channel single-molecule fluorescence detection device in Example 1 of the present invention;

[0043] Figure 2 DPPH antioxidant test results in the test examples of the present invention;

[0044] Figure 3 This is a diagram showing the structure of a four-stranded DNA intermediate obtained by fluorescence detection in a test example of the present invention;

[0045] Figure 4 The graph shows the acidity test results in the test examples of the present invention, wherein a is the probe detection standard solution, b is the traditional enzymatic oxygen scavenging system, c is the antioxidant system of the present invention, and d is a statistical graph of the change in pH value 30 minutes before and after treatment. The first group (GOT) is the traditional enzymatic oxygen scavenging system, the second group is the system containing only squalene, and the third group is the antioxidant system of the present invention;

[0046] Figure 5 The results of single-molecule fluorescence imaging in the experimental example of the present invention are shown in Figure 1, where (a) is the experimental schematic diagram and (b) is the result;

[0047] Figure 6 Graph showing the change in average fluorescence signal intensity in the experimental example of the present invention. DETAILED DESCRIPTION

[0048] The following will clearly and completely describe the concept and technical effects of the present invention in conjunction with the embodiments to fully understand the purpose, features and effects of the present invention. Obviously, the embodiments described are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, other embodiments obtained by those skilled in the art without creative work are all within the scope of protection of the present invention.

[0049] Example 1

[0050] This embodiment provides a multi-channel single-molecule fluorescence detection device, which includes a sample chamber, an excitation light source, an inverted fluorescence microscopy optical path module, and an imaging device; the sample chamber is a sandwich structure, which includes a cover glass, double-sided tape, and a slide from top to bottom. The slide of the sample chamber is provided with four independent reaction channels, and the slide has two holes for introducing and exporting reagents; the surface of the slide is modified with aminosilanization; the structural schematic diagram and mechanism schematic diagram of the above-mentioned sample chamber are shown as follows Figure 1 shown.

[0051] The sample chamber is loaded with an antioxidant system comprising 0.7 v / v% squalene and 2 mM Trolox. The specific preparation method is as follows:

[0052] 6 mL of 1 mol / L DMSO was diluted with 94 mL of Tris-HCl buffer to obtain 6% DMSO, and squalene was then diluted with 6% DMSO to obtain a 4 v / v% squalene stock solution (squalene is an oily liquid at room temperature, so its concentration is expressed as volume fraction). The squalene stock solution was then mixed with Tris-HCl buffer containing Trolox to obtain an antioxidant system; wherein the final squalene concentration in the antioxidant system was 0.7 v / v%, and the final Trolox concentration was 2 mM.

[0053] The above-mentioned method for pre-treating and surface modifying the slide comprises the following steps:

[0054] 1) Place the coverslip and slide in a glass container with a slot and prepare a 250 mL conical flask.

[0055] 2) Add chromatography-grade ethanol to glassware and Erlenmeyer flasks, heat to 40°C, and sonicate for 10 minutes.

[0056] 3) Wash with ultrapure water three times and add 3 mol / L KOH and sonicate for 40 min;

[0057] 4) Wash the slides with ultrapure water 6 times and blow dry the slides with nitrogen;

[0058] 5) Add 100 mL of methanol, 5 mL of acetic acid, and 1 mL of aminosilane to a 250 mL conical flask and mix thoroughly to obtain a mixture;

[0059] 6) Pour the mixed solution prepared in step 5) into the above-mentioned glassware, immerse the glass slide in it, seal it, and incubate overnight;

[0060] 7) Wash with methanol 3 times and then with ultrapure water 6 times, and finally blow dry with nitrogen for later use.

[0061] Example 2

[0062] This embodiment provides an oxygen scavenging method for a single-molecule fluorescence imaging system. The oxygen scavenging method is based on the multi-channel single-molecule fluorescence detection device provided in Example 1 and specifically includes the following steps:

[0063] 1) PEGylation modification:

[0064] ① Dissolve 115 mg of PEG-SC and 4 mg of biotin-PEG-SC reagent in 500 μL of 0.1 mol / L sodium bicarbonate solution to obtain a PEG mixture. Place 64 μL of the PEG mixture in the center of the modified glass slide provided in Example 1, gently cover with a coverslip to avoid bubbles, and incubate in a dark room at room temperature for 4 h (3 to 5 h is acceptable).

[0065] ② After incubation, wash with ultrapure water three times, blow dry with nitrogen, place a pair of slides in a 50 mL test tube, vacuum, and store in a -80°C refrigerator;

[0066] ③ Take out the slide and place it at room temperature to restore it to normal temperature. Figure 1 The sandwich structure shown is built into a sample chamber. Figure 1 The sample chamber is shown to have 6 channel cavities.

[0067] 2) Slide quality inspection:

[0068] Directly add 10 μL of protein sample labeled with Cy3 fluorescence into the first channel of the sample chamber; incubate for 10 minutes and rinse with ultrapure water three times to observe whether there is fluorescence. If there is a fluorescence signal, it means that the preparation is unqualified. If there is no fluorescence, proceed to the next step.

[0069] 3) Coupled with streptavidin:

[0070] In the second channel of the sample chamber, add 10 μL of 0.2 mg / mL streptavidin and incubate for 10 min. Add 50 μL of ultrapure water to wash away excess streptavidin and wash at least three times.

[0071] 4) Add sample:

[0072] Add 10 μL of 150 pmol / L four-stranded intermediate structure DNA and incubate for 3 minutes (2 to 5 minutes is acceptable); add 50 μL of ultrapure water to wash away excess four-stranded intermediate structure DNA, and add 50 μL of antioxidant system, place on the stage for imaging observation; the above antioxidant system is Tris-HCl buffer containing 0.7% squalene and 2 mM Trolox.

[0073] Test example

[0074] This test example tested the antioxidant performance of the oxygen scavenging method for the single-molecule fluorescence imaging system provided in Example 2 and the effect of the antioxidant system used on the pH value of the entire reaction system. The photobleaching condition was also detected. The specific test method and results are as follows:

[0075] 1.DPPH antioxidant test:

[0076] DPPH (1,1-diphenyl-2-trinitrophenylhydrazine) is a stable free radical commonly used to assess the antioxidant capacity of samples. Antioxidants can scavenge DPPH radicals, causing the color of the DPPH radical to change from purple to yellow. This change in absorbance can be used to measure antioxidant activity.

[0077] Add 1 mL of ethanol solution containing 100 μmol DPPH and 800 μL of Tris-HCl buffer (pH 7.4) to a test tube. Then add 200 μL of the squalene solution to be tested (0.05, 0.3, 0.5, 0.7, and 1 mol of squalene for every 1 mol DPPH in the reaction system) and mix quickly. Incubate the solution at room temperature for 30 minutes. Record the absorbance of the reaction system at 517 nm. Use a mixed solution containing 1.2 mL of ethanol and 800 μL of Tris-HCl buffer (pH 7.4) as a blank control. Finally, calculate its oxygen scavenging rate according to the following formula:

[0078] Inhibition ratio (%) = (A1-A2) × 100 / A1, where A1 is the initial absorbance of the blank control and A2 is the absorbance of the sample solution to be tested. The results are as follows: Figure 2 shown.

[0079] Depend on Figure 2 It can be seen that the free radical scavenging ability of squalene was determined using the DPPH assay, and it was found that up to 1% squalene had anti-free radical activity, and the free radical scavenging activity of squalene increased in a dose-dependent manner; the free radical scavenging activity was highest at 1% squalene, reaching 32%.

[0080] 2. Oxygen scavenging ability test of antioxidant system:

[0081] The oxygen scavenging method for single-molecule fluorescence imaging provided in Example 2 was omitted from the antioxidant system, and imaging observation was performed as a control group. The single-molecule fluorescence detection results of the control group and Example 2 for the four-stranded intermediate structure DNA are shown as follows: Figure 3 shown.

[0082] Depend on Figure 3 As can be seen, the fluorescence change curve of the control group fluctuates more than that of Example 2, thus improving the fluorescence stability of the sample after the addition of the antioxidant system. The oxygen scavenging method provided by the present invention is an all-liquid system with a rapid reaction process. Dynamic measurement of the fluorescence signal of the four-stranded DNA intermediate structure in the sample chamber is performed using a micro-single-molecule fluorescence resonance energy transfer imaging microscope. By calculating the relative ratio of the fluorescence signals, a dynamic FRET trajectory diagram of a single molecule is obtained, allowing for accurate and rapid detection.

[0083] 3. Acidity test:

[0084] First, use SNARF-1 as a pH sensor to test the concentration of the standard solution. For example, use hydrochloric acid and sodium hydroxide to adjust the pH to 8 / 7.8 / 7.6 / 7.4 / 7.2 / 7.0 / 6.8 / 6.6 / 6.4 / 6.2 / 6.0 to draw a standard curve. The spectral curve obtained by the test is the peaks A and B (568nm and 640nm) under different acid and alkaline conditions. Figure 4 As shown in a.

[0085] Then, the antioxidant system provided by the present invention was subjected to an acidity test:

[0086] 1) Dissolve SNARF-1: Dissolve in DMSO to a stock solution concentration of 1 mM and dilute with ddH2O before use.

[0087] 2) Add 10 μL of working solution (10 μM SNARF-1, 5 mM MgCl2, 0.8% (w / v) D-glucose, 2 mM Trolox, 0.7% squalene, 10 mM Tris) to the independent chamber of the sample chamber, and record the fluorescence spectrum data every 5 minutes;

[0088] 3) To compare the effects of the system provided by the present invention with those of a conventional enzymatic oxygen scavenging system, 10 μL of a conventional enzymatic oxygen scavenging system working solution, comprising 10 μM SNARF-1, 5 mM MgCl2, 0.8% (w / v) dextrose, 2 mM Trolox, 1 mg / mL glucose oxidase, and 0.04 mg / mL catalase, was added to another channel of the same slide. Fluorescence spectral data were recorded every 5 minutes. The results are shown in FIG. Figure 4 As shown in b~d.

[0089] Depend on Figure 4 It can be seen from a that in the standard solution, the fluorescence spectrum has a peak value of 590nm when pH < 7, and the fluorescence spectrum has a peak value of 640nm when pH > 7.

[0090] Depend on Figure 4 b~ Figure 4 d It can be seen that as time changes, the antioxidant system provided by the present invention does not show a significant decrease in acidity, but maintains a stable solution pH value, while the traditional enzymatic oxygen scavenging system shows severe acidification after 30 minutes, indicating that the antioxidant system provided by the present invention has significantly better anti-acidification performance than the traditional enzymatic oxygen scavenging system.

[0091] In addition, this experimental example also calculated the fluorescence quenching time of the fluorescent molecules to evaluate their photobleaching; the fluorescence signal was imaged by TIRFM (self-built) with an incident angle of 30 degrees and an exposure time of 500 ms; the laser power was 1 mW, the gamma value was 1, the LUT intensity range was 100 to 5000, the EMCCD had 1024×1024 pixels, and more than 20 images were taken from different areas of a single channel; for the experimental groups, the reaction buffer (40 mM Tris-HCl, 60 mM NaCl, 6 mM MgCl2, pH 7.3) and 0.25 mg / mL glucose oxidase (GOD group), 0.7% squalene (SQ group), 0.25 mg / mL glucose oxidase combined with 200 mM bME (GOD-BME group), and 0.7% squalene combined with 2 mM Trolox (SQ-Trolox group) was flowed into different channels for 5 to 15 minutes, and the changes in fluorescence signals before and after the flow were measured. For the negative control group, reaction buffer (40 mM Tris-HCl, 60 mM NaCl, 6 mM MgCl2, pH 7.3) was used for detection. The average fluorescence signal intensity of all ROIs was measured, and all data were normalized by the negative control. The results are shown in Figure 2. Figure 5 and Figure 6 shown.

[0092] Depend on Figure 5 It can be seen from the single-molecule fluorescence images that the anti-fading effect of the fluorescent molecules in the experimental group with squalene added is better than that of the glucose oxidase system; Figure 6 As can be seen from the changes in mean fluorescence signal intensity, the average on / off ratio of Cy3 ranged from 0.2% to 6% after buffer injection and remained virtually unchanged when using SQ for 5 minutes. The antioxidant system provided by the present invention exhibits superior photostability in single-molecule fluorescence imaging compared to conventional enzymatic oxygen scavenging systems (glucose oxidase).

[0093] In summary, the antioxidant system provided by the present invention and the corresponding oxygen scavenging method for the single-molecule fluorescence imaging system achieve increased photostability and enhanced antioxidant properties during the detection of single-molecule fluorescence signals, overcoming the problem of pH drop caused by traditional enzyme oxygen scavenging systems; for the detection of four-stranded DNA intermediate structures, the oxygen scavenging method provided by the present invention greatly improves antioxidant performance and does not detect pH changes, which is unattainable by other methods under the same conditions; and squalene is relatively inexpensive compared to traditional enzyme reaction systems, greatly reducing application costs. These favorable conditions enable the present invention to be fully promoted and applied.

[0094] While the embodiments of the present invention have been described in detail above with reference to the accompanying drawings, the present invention is not limited to the embodiments described above. Various modifications may be made within the scope of knowledge possessed by a person skilled in the art without departing from the spirit of the present invention. Furthermore, the embodiments of the present invention and the features thereof may be combined with one another unless there is a conflict.

Claims

1. An antioxidant system, characterized in that: The antioxidant system comprises 0.05v / v% to 1v / v% squalene and 0.2 to 5mM Trolox.

2. An oxygen scavenging method for a single-molecule fluorescence imaging system, characterized in that: The oxygen scavenging method for the single-molecule fluorescence imaging system adopts the antioxidant system as claimed in claim 1.

3. The oxygen scavenging method for a single-molecule fluorescence imaging system according to claim 2, characterized in that: The oxygen scavenging method comprises the following steps: S1: Immobilize the biotin-carrying polymer on the surface of the slide, and then add streptavidin for incubation; S2: Add the sample to be tested carrying biotin and fluorescent groups to the surface of the slide for incubation; S3: Prepare the antioxidant system as described in claim 1 and add it into the sample chamber where the slide is located, and then form an image and observe.

4. The oxygen scavenging method according to claim 3, wherein: The biotin-carrying polymer in step S1 includes biotin-polyethylene glycol-succinimidyl carbonate.

5. The oxygen scavenging method according to claim 3, wherein: The glass slide in step S1 is modified by aminosilanization.

6. The oxygen scavenging method according to claim 3, wherein: The fluorescent group in step S2 includes at least one of Cy3, Cy5, Alexa Fluor 488, Alexa Fluor 594 and FITC.

7. The oxygen scavenging method according to claim 3, wherein: The sample chamber in step S3 is a sandwich structure, which includes a cover glass, double-sided tape and a slide from top to bottom; Preferably, the glass slide of the sample chamber is provided with at least two independent reaction channels; Preferably, the slide has at least two holes for introducing and exporting reagents.

8. A multi-channel single-molecule fluorescence detection device, characterized in that: The detection device includes: a sample chamber, an excitation light source, an inverted fluorescence microscopy optical path module and an imaging device; The sample chamber is composed of a cover glass, double-sided tape and a glass slide, and the sample chamber is loaded with the antioxidant system according to claim 1.

9. The detection device according to claim 8, characterized in that The glass slide of the sample chamber is provided with at least two independent reaction channels; the glass slide has at least two holes for introducing and exporting reagents.

10. Application of squalene in the preparation of antioxidant systems for single-molecule fluorescence imaging systems.

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