A method for detecting oxidation free radical absorption capacity

Abstract

The present invention discloses a method for detecting oxidative free radical absorption capacity, which is based on FLIPR-TETRA real-time fluorescence detection technology, and utilizes the fluorescence decay produced by AAPH oxidant contained in sodium fluorescein, and with the aid of water-soluble vitamin E antioxidant, establishes an oxidative free radical absorption capacity (ORAC) detection model; performs ORAC detection on the test agent and antioxidant, and directly derives the area under the fluorescence curve of each well using ScreenWorks™ system software in the FLIPR-TETRA detection system; establishes antioxidant concentration, test agent concentration-net area standard curve, and judges the antioxidant capacity of the test agent by the ratio of the net area of ​​the antioxidant at the same concentration. The present invention is based on FLIPR-TETRA technology, can realize whole plate loading and whole plate detection, avoids errors in the operation process, is simple to extract whole data, reduces the time of data processing, and has reliable detection results.

Description

Technical Field

[0001] The invention belongs to the technical field of antioxidant methods, and specifically relates to a method for detecting oxidation free radical absorption capacity. Background Art

[0002] Oxidative Radical Absorption Capacity (ORAC) is also known as antioxidant capacity. The ORAC analysis method is based on the principle that free radicals damage fluorescent probes, causing changes in fluorescence intensity. Trolox, a water-soluble analog of vitamin E, is used as a quantitative standard and analyzed using a fluorescent microplate analyzer. The change in fluorescence intensity reflects the degree of free radical damage. In the presence of antioxidants, it can inhibit the fluorescence changes caused by free radicals, and the degree of inhibition reflects its antioxidant capacity against free radicals.

[0003] Currently, ORAC detection is mainly performed by detecting fluorescence decay using an enzyme-labeled instrument. However, this method has certain limitations, such as the need for manual sample addition, which leads to reaction time errors during operation, single-well detection, which leads to inconsistent detection times between the initial well and the final well, and complicated data processing. Summary of the invention

[0004] In view of this, the present invention hopes to provide a method for detecting the oxidative free radical absorption capacity. Compared with the existing method of detecting fluorescence through an ELISA instrument, it can achieve whole-plate sampling and whole-plate detection, avoid errors in the operation process, and easily extract the whole data, greatly reducing the time for data processing, and the detection results are reliable and the operation and analysis are simple.

[0005] To achieve the above object, the present invention adopts a method for detecting the oxidation free radical absorption capacity, using FLIPR-TETRA real-time fluorescence detection technology, which specifically includes the following steps:

[0006] a) dissolving the antioxidant in a phosphate buffer of pH 7.4 and diluting it to a concentration of 0.1 μM to 50 μM, adding it to a 96-well black light-transmitting bottom plate, and placing it at the Read plate in the FLIPR-TETRA detection system, then adding 10 nM to 100 nM fluorescein sodium dissolved in phosphate buffer to the first 96-well V-bottom drug plate, adding 100 mM AAPH dissolved in phosphate buffer to the second 96-well V-bottom drug plate, and finally transferring the liquid in the first 96-well V-bottom drug plate to the 96-well black light-transmitting bottom plate, and transferring the liquid in the second 96-well V-bottom drug plate to the 96-well black light-transmitting bottom plate after 5 minutes, detecting the fluorescence signal every 1 second to 60 seconds, and detecting 60 to 500 times, as a control group sample;

[0007] b) dissolving the test agent in a phosphate buffer of pH 7.4 and diluting it to a concentration of 0.5 μM to 10 μM, adding it to a 96-well black light-transmitting bottom plate, and placing it at the Read plate in the FLIPR-TETRA detection system, then adding 10 nM to 100 nM fluorescein sodium dissolved in phosphate buffer to the first 96-well V-bottom drug plate, adding 100 mM AAPH dissolved in phosphate buffer to the second 96-well V-bottom drug plate, and finally transferring the liquid in the first 96-well V-bottom drug plate to the 96-well black light-transmitting bottom plate, and transferring the liquid in the second 96-well V-bottom drug plate to the 96-well black light-transmitting bottom plate after 5 minutes, and the time interval and number of detections for detecting a fluorescence signal are consistent with the time interval and number of detections for detecting a fluorescence signal in step a), as the detection group sample;

[0008] c) adding a phosphate buffer of pH 7.4 to a 96-well black light-transmitting bottom plate, and placing it at the Read plate in the FLIPR-TETRA detection system, then adding 10nM to 100nM fluorescein sodium dissolved in phosphate buffer to the first 96-well V-bottom drug plate, adding 100mM AAPH dissolved in phosphate buffer to the second 96-well V-bottom drug plate, and finally transferring the liquid in the first 96-well V-bottom drug plate to the 96-well black light-transmitting bottom plate, and transferring the liquid in the second 96-well V-bottom drug plate to the 96-well black light-transmitting bottom plate after 5 minutes, and the time interval and number of detections for detecting a fluorescence signal are consistent with the time interval and number of detections for detecting a fluorescence signal in step a), as a blank group;

[0009] d) The fluorescence signal intensities detected in the control group, the test group, and the blank group were respectively analyzed by ScreenWorks in the FLIPR-TETRA detection system. TM The system software can directly export the area under the fluorescence curve (AUC) of each well, where the net area = AUC 样品 -AUC 磷酸盐缓冲液 , and draw a standard curve of antioxidant concentration, test agent concentration-net area. The basis for judging the antioxidant capacity of the test agent is the ratio of the net area of ​​the antioxidant at the same concentration.

[0010] Furthermore, the antioxidant is water-soluble vitamin E.

[0011] Furthermore, the test agent is EGCG (epigallocatechin gallate), EGC (epigallocatechin), and Ferulic acid.

[0012] Furthermore, in the steps a), b) and c), 100 nM fluorescein sodium dissolved in phosphate buffer is added to the first 96-well V-bottom drug plate, and the fluorescence signal is detected every 60 seconds for 60 times.

[0013] Furthermore, in the steps a), b) and c), 10 nM sodium fluorescein dissolved in phosphate buffer is added to the first 96-well V-bottom drug plate, and the fluorescence signal is detected every 10 seconds for 360 times.

[0014] The invention has the following beneficial effects: compared with the existing fluorescence detection method using an enzyme marker, the invention uses FLIPR-TETRA real-time fluorescence detection technology to achieve whole-plate loading and whole-plate detection, thus avoiding errors in the operation process. The data results are displayed in the FLIPR-TETRA detection system through ScreenWorks. TM The system software can directly obtain the area under the fluorescence curve, greatly reducing the time for data processing, and the detection results are reliable and the operation and analysis are simple. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 The graphs are respectively the fluorescence signal intensity curves detected by the control group, the test group and the blank group in Example 1 of the present invention;

[0016] Figure 2 This is a standard curve diagram of water-soluble vitamin E, EGC, EGCG, and Ferulic acid concentration-net area in Example 1 of the present invention. DETAILED DESCRIPTION

[0017] In order to more thoroughly understand the features and technical contents of the present invention, the implementation of the present invention is described in detail below.

[0018] The present invention provides a method for detecting the oxidative free radical absorption capacity, using FLIPR-TETRA real-time fluorescence detection technology, and specifically comprises the following steps:

[0019] a) dissolving the antioxidant in a phosphate buffer of pH 7.4 and diluting it to a concentration of 0.1 μM to 50 μM, adding it to a 96-well black light-transmitting bottom plate, and placing it at the Read plate in the FLIPR-TETRA detection system, then adding 10 nM to 100 nM fluorescein sodium dissolved in phosphate buffer to the first 96-well V-bottom drug plate, adding 100 mM AAPH dissolved in phosphate buffer to the second 96-well V-bottom drug plate, and finally transferring the liquid in the first 96-well V-bottom drug plate to the 96-well black light-transmitting bottom plate, and transferring the liquid in the second 96-well V-bottom drug plate to the 96-well black light-transmitting bottom plate after 5 minutes, detecting the fluorescence signal every 1 second to 60 seconds, and detecting 60 to 500 times, as a control group sample;

[0020] b) dissolving the test agent in a phosphate buffer of pH 7.4 and diluting it to a concentration of 0.5 μM to 10 μM, adding it to a 96-well black light-transmitting bottom plate, and placing it at the Read plate in the FLIPR-TETRA detection system, then adding 10 nM to 100 nM fluorescein sodium dissolved in phosphate buffer to the first 96-well V-bottom drug plate, adding 100 mM AAPH dissolved in phosphate buffer to the second 96-well V-bottom drug plate, and finally transferring the liquid in the first 96-well V-bottom drug plate to the 96-well black light-transmitting bottom plate, and transferring the liquid in the second 96-well V-bottom drug plate to the 96-well black light-transmitting bottom plate after 5 minutes, and the time interval and number of detections for detecting a fluorescence signal are consistent with the time interval and number of detections for detecting a fluorescence signal in step a), as the detection group sample;

[0021] c) adding a phosphate buffer of pH 7.4 to a 96-well black light-transmitting bottom plate, and placing it at the Read plate in the FLIPR-TETRA detection system, then adding 10nM to 100nM fluorescein sodium dissolved in phosphate buffer to the first 96-well V-bottom drug plate, adding 100mM AAPH dissolved in phosphate buffer to the second 96-well V-bottom drug plate, and finally transferring the liquid in the first 96-well V-bottom drug plate to the 96-well black light-transmitting bottom plate, and transferring the liquid in the second 96-well V-bottom drug plate to the 96-well black light-transmitting bottom plate after 5 minutes, and the time interval and number of detections for detecting a fluorescence signal are consistent with the time interval and number of detections for detecting a fluorescence signal in step a), as a blank group;

[0022] d) The fluorescence signal intensities detected in the control group, the test group, and the blank group were respectively analyzed by ScreenWorks in the FLIPR-TETRA detection system. TMThe system software can directly export the area under the fluorescence curve (AUC) of each well, where the net area = AUC 样品 -AUC 磷酸盐缓冲液 , and draw a standard curve of antioxidant concentration, test agent concentration-net area. The basis for judging the antioxidant capacity of the test agent is the ratio of the net area of ​​the antioxidant at the same concentration.

[0023] Furthermore, the antioxidant is water-soluble vitamin E.

[0024] Furthermore, the test agent is EGCG, EGC, or Ferulic acid.

[0025] Furthermore, in the steps a), b) and c), 100 nM fluorescein sodium dissolved in phosphate buffer is added to the first 96-well V-bottom drug plate, and the fluorescence signal is detected every 60 seconds for 60 times.

[0026] Furthermore, in the steps a), b) and c), 10 nM sodium fluorescein dissolved in phosphate buffer is added to the first 96-well V-bottom drug plate, and the fluorescence signal is detected every 10 seconds for 360 times.

[0027] FLIPR-TETRA real-time fluorescence detection technology is a detection system that combines an automatic liquid adding robot with a CCD camera. It can add liquid to the entire plate at the same time, CCD image the entire plate, and have an imaging interval of 0.2 to 60 seconds. It can detect high-quality fluorescence data. Compared with the existing fluorescence detection method using an enzyme marker, it can achieve whole-plate loading and detection, avoiding errors in the operation process. The data results are displayed through ScreenWorks in the FLIPR-TETRA detection system. TM The system software can directly obtain the area under the fluorescence curve, greatly reducing the time for data processing.

[0028] Embodiment 1:

[0029] Materials: 96-well black transparent bottom plate and 96-well V-bottom drug plate were purchased from Nunc; fluorescein sodium was purchased from Shanghai Yuanye Company; AAPH (2,2'-azobis(2-amidinopropane) dihydrochloride) was purchased from APEBIO; PBS (phosphate buffer pH 7.4) was purchased from Gbico; EGCG, EGC, and Ferulic acid were purchased from MCE; the detection platform was FLIPR-TETRA, and the detected signal was relative fluorescence intensity (RFU), which was detected using ScreenWorks in the detection platform. TM The system software detects the area under the fluorescence curve (AUC).

[0030] Step a): Dissolve water-soluble vitamin E in phosphate buffer at pH 7.4 and dilute to 25 μM, 12.5 μM, 6.25 μM, 5 μM, 3 μM, 2 μM, 1 μM, 0.75 μM, 0.5 μM, 0.25 μM, and 0.1 μM concentrations, 50 μL each, and add to a 96-well black light-transmitting bottom plate, and place it at the Read plate in the FLIPR-TETRA detection system, then add 150 μL of 100 nM fluorescein sodium dissolved in phosphate buffer to the first 96-well V-bottom plate, and add 100 mM AAPH dissolved in phosphate buffer to the second 96-well V-bottom plate. 80 μL, and finally, 100 μL of the liquid in the first 96-well V-bottom medicine plate is transferred to the 96-well black light-transmitting bottom plate by a mechanical pipette. After 5 minutes, 50 μL of the liquid in the second 96-well V-bottom medicine plate is transferred to the 96-well black light-transmitting bottom plate by a mechanical pipette. The fluorescence signal is detected every 60 seconds, and the detection is performed 60 times as a control group sample;

[0031] Step b): EGCG, EGC, and Ferulic acid were dissolved in a phosphate buffer at pH 7.4 and diluted to 10 μM, 5 μM, 3 μM, 1 μM, 0.75 μM, and 0.5 μM concentrations, 50 μL each, and added to a 96-well black transparent bottom plate, and placed in the FLIPR-TETRA detection system Reader plate, then add 150 μL of 100nM sodium fluorescein dissolved in phosphate buffer to the first 96-well V-bottom drug plate, add 80 μL of 100mM AAPH dissolved in phosphate buffer to the second 96-well V-bottom drug plate, finally transfer 100 μL of the liquid in the first 96-well V-bottom drug plate to the 96-well black transparent bottom plate by a mechanical pipette, and after 5 minutes, transfer 50 μL of the liquid in the second 96-well V-bottom drug plate to the 96-well black transparent bottom plate by a mechanical pipette, detect the fluorescence signal every 60 seconds, and detect 60 times as the detection group sample;

[0032] Step c): add 50 μL of phosphate buffer of pH 7.4 to a 96-well black light-transmitting bottom plate and place it at the Read plate in the FLIPR-TETRA detection system, then add 150 μL of 100 nM fluorescein sodium dissolved in phosphate buffer to the first 96-well V-bottom drug plate, add 80 μL of 100 mM AAPH dissolved in phosphate buffer to the second 96-well V-bottom drug plate, finally transfer 100 μL of the liquid in the first 96-well V-bottom drug plate to the 96-well black light-transmitting bottom plate by a mechanical pipette, and after 5 minutes, transfer 50 μL of the liquid in the second 96-well V-bottom drug plate to the 96-well black light-transmitting bottom plate by a mechanical pipette, detect the fluorescence signal every 60 seconds, and detect 60 times as a blank group;

[0033] The fluorescence signal intensities detected in the control group, the test group, and the blank group are as follows: Figure 1 As shown, the FLIPR-TETRA detection system is used in ScreenWorks TM The system software extracts the area under the fluorescence curve AUC from detection points 1 to 60;

[0034] Calculate the net area under the curve = AUC 样品 -AUC 磷酸盐缓冲液 , draw the concentration-net area standard curve of water-soluble vitamin E, EGC, EGCG, and Ferulicacid, such as Figure 2 As shown, the ORAC capacity is: EGCG>EGC>Ferulic acid, which is consistent with the results reported in the literature. The relative ORAC equivalents (net area ratio to water-soluble vitamin E at the same concentration) are shown in the following table.

[0035]

[0036] Embodiment 2:

[0037] Materials: 96-well black transparent bottom plate and 96-well V-bottom drug plate were purchased from Nunc; fluorescein sodium was purchased from Shanghai Yuanye Company; AAPH (2,2'-azobis(2-amidinopropane) dihydrochloride) was purchased from APEBIO; PBS (phosphate buffer pH 7.4) was purchased from Gbico; EGCG, EGC, and Ferulic acid were purchased from MCE; the detection platform was FLIPR-TETRA, and the detected signal was relative fluorescence intensity (RFU), which was detected using ScreenWorks in the detection platform. TM The system software detects the area under the fluorescence curve (AUC).

[0038] Step a): Dissolve water-soluble vitamin E in phosphate buffer at pH 7.4 and dilute to 25 μM, 12.5 μM, 6.25 μM, 5 μM, 3 μM, 2 μM, 1 μM, 0.75 μM, 0.5 μM, 0.25 μM, and 0.1 μM concentrations, 50 μL each, and add to a 96-well black transparent bottom plate, and place it at the Read plate in the FLIPR-TETRA detection system, then add 150 μL of 10 nM fluorescein sodium dissolved in phosphate buffer to the first 96-well V-bottom plate, and add 100 mM AAPH dissolved in phosphate buffer to the second 96-well V-bottom plate. 80 μL, and finally, 100 μL of the liquid in the first 96-well V-bottom medicine plate is transferred to the 96-well black light-transmitting bottom plate by a mechanical pipette. After 5 minutes, 50 μL of the liquid in the second 96-well V-bottom medicine plate is transferred to the 96-well black light-transmitting bottom plate by a mechanical pipette. The fluorescence signal is detected every 10 seconds, and the detection is performed 360 times as a control group sample;

[0039] Step b): EGCG, EGC, and Ferulic acid were dissolved in a phosphate buffer at pH 7.4 and diluted to 10 μM, 5 μM, 3 μM, 1 μM, 0.75 μM, and 0.5 μM, respectively, and 50 μL of each was added to a 96-well black transparent bottom plate, and placed at the Read plate in the FLIPR-TETRA detection system, and then 150 μL of 10 nM fluorescein sodium dissolved in phosphate buffer was added to the first 96-well V-bottom drug plate, and 100 mM AAPH dissolved in phosphate buffer was added to the second 96-well V-bottom drug plate. 80 μL, and finally, 100 μL of the liquid in the first 96-well V-bottom medicine plate is transferred to the 96-well black light-transmitting bottom plate by a mechanical pipette. After 5 minutes, 50 μL of the liquid in the second 96-well V-bottom medicine plate is transferred to the 96-well black light-transmitting bottom plate by a mechanical pipette. The fluorescence signal is detected every 10 seconds, and the detection is performed 360 times as the detection group sample;

[0040] Step c): add 50 μL of phosphate buffer of pH 7.4 to a 96-well black light-transmitting bottom plate and place it at the Read plate in the FLIPR-TETRA detection system, then add 150 μL of 10 nM fluorescein sodium dissolved in phosphate buffer to the first 96-well V-bottom drug plate, add 80 μL of 100 mM AAPH dissolved in phosphate buffer to the second 96-well V-bottom drug plate, finally transfer 100 μL of the liquid in the first 96-well V-bottom drug plate to the 96-well black light-transmitting bottom plate by a mechanical pipette, and after 5 minutes, transfer 50 μL of the liquid in the second 96-well V-bottom drug plate to the 96-well black light-transmitting bottom plate by a mechanical pipette, detect the fluorescence signal every 10 seconds, and detect 360 times as a blank group;

[0041] The fluorescence signal intensities detected in the control group, test group and blank group were respectively measured using ScreenWorks in the FLIPR-TETRA detection system. TM The system software extracts the area under the fluorescence curve AUC from detection point 1 to 360;

[0042] Calculate the net area under the curve = AUC 样品 -AUC 磷酸盐缓冲液 , the concentration-net area standard curves of water-soluble vitamin E, EGC, EGCG, and Ferulic acid were drawn, and the ORAC capacity was: EGCG>EGC>Ferulic acid, which was consistent with the results reported in the literature.

[0043] The technical solution provided by the present invention is compared with the existing performance data as follows:

[0044]

[0045]

[0046] The present invention utilizes FLIPR-TETRA real-time fluorescence detection technology. Compared with the existing fluorescence detection method using an enzyme marker, the present invention realizes whole-plate sample addition and whole-plate detection, thus avoiding errors in the operation process. The data results are displayed in the FLIPR-TETRA detection system through ScreenWorks. TM The system software can directly obtain the area under the fluorescence curve, greatly reducing the time for data processing, and the detection results are reliable and the operation and analysis are simple.

[0047] The above description is only a preferred embodiment of the present invention and is not intended to limit the protection scope of the present invention. Therefore, any equivalent changes or modifications made in accordance with the patent scope of the present invention shall fall within the scope of the patent claims of the present invention.

Claims

1. A method for detecting the oxidation free radical absorption capacity, It is characterized in that Using FLIPR-TETRA real-time fluorescence detection technology, the following steps are specifically included: a) dissolving the antioxidant in a phosphate buffer of pH 7.4 and diluting it to a concentration of 0.1 μM to 50 μM, adding it to a 96-well black light-transmitting bottom plate, and placing it at the Read plate in the FLIPR-TETRA detection system, then adding 10 nM to 100 nM fluorescein sodium dissolved in phosphate buffer to the first 96-well V-bottom drug plate, adding 100 mM AAPH dissolved in phosphate buffer to the second 96-well V-bottom drug plate, and finally transferring the liquid in the first 96-well V-bottom drug plate to the 96-well black light-transmitting bottom plate, and transferring the liquid in the second 96-well V-bottom drug plate to the 96-well black light-transmitting bottom plate after 5 minutes, detecting the fluorescence signal every 1 second to 60 seconds, and detecting 60 to 500 times, as a control group sample; b) dissolving the test agent in a phosphate buffer of pH 7.4 and diluting it to a concentration of 0.5 μM to 10 μM, adding it to a 96-well black light-transmitting bottom plate, and placing it at the Read plate in the FLIPR-TETRA detection system, then adding 10 nM to 100 nM fluorescein sodium dissolved in phosphate buffer to the first 96-well V-bottom drug plate, adding 100 mM AAPH dissolved in phosphate buffer to the second 96-well V-bottom drug plate, and finally transferring the liquid in the first 96-well V-bottom drug plate to the 96-well black light-transmitting bottom plate, and transferring the liquid in the second 96-well V-bottom drug plate to the 96-well black light-transmitting bottom plate after 5 minutes, and the time interval and number of detections for detecting a fluorescence signal are consistent with the time interval and number of detections for detecting a fluorescence signal in step a), as the detection group sample; c) adding a phosphate buffer of pH 7.4 to a 96-well black light-transmitting bottom plate, and placing it at the Read plate in the FLIPR-TETRA detection system, then adding 10nM to 100nM fluorescein sodium dissolved in phosphate buffer to the first 96-well V-bottom drug plate, adding 100mM AAPH dissolved in phosphate buffer to the second 96-well V-bottom drug plate, and finally transferring the liquid in the first 96-well V-bottom drug plate to the 96-well black light-transmitting bottom plate, and transferring the liquid in the second 96-well V-bottom drug plate to the 96-well black light-transmitting bottom plate after 5 minutes, and the time interval and number of detections for detecting a fluorescence signal are consistent with the time interval and number of detections for detecting a fluorescence signal in step a), as a blank group; d) The fluorescence signal intensities detected in the control group, the test group, and the blank group are respectively derived using the ScreenWorks™ system software in the FLIPR-TETRA detection system to directly derive the area under the fluorescence curve (AUC) of each well. Net area = AUC 样品 -AUC 磷酸盐缓冲液 , and draw a standard curve of antioxidant concentration, test agent concentration-net area. The basis for judging the antioxidant capacity of the test agent is the ratio of the net area of ​​the antioxidant at the same concentration.

2. A method for detecting the oxidation free radical absorption capacity according to claim 1, It is characterized in that The antioxidant is water-soluble vitamin E.

3. A method for detecting the oxidation free radical absorption capacity according to claim 1, It is characterized in that The test agents are EGCG, EGC, and Ferulic acid.

4. The method for detecting the oxidation free radical absorption capacity according to claim 1, It is characterized in that In the steps a), b) and c), 100 nM fluorescein sodium dissolved in phosphate buffer is added to the first 96-well V-bottom drug plate, and the fluorescence signal is detected every 60 seconds for 60 times.

5. A method for detecting the oxidation free radical absorption capacity according to claim 1, It is characterized in that In the steps a), b) and c), 10 nM fluorescein sodium dissolved in phosphate buffer is added to the first 96-well V-bottom drug plate, and the fluorescence signal is detected every 10 seconds for 360 times.

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