Application of light-emitting retarder in aspect of improving development quality of solid-phase membrane immunodetection

Abstract

The invention belongs to the technical field of chemiluminescence immunoassay, and particularly relates to application of a luminescence retarder in improving development quality of solid-phase membrane immunodetection. According to the invention, when the concentration of the delay agent is fixed, the time of luminescence starting delay and the concentration of the enzyme catalyst are in nonlinear negative correlation, that is, the delay time is obviously increased along with the reduction of the enzyme concentration. The nonlinear negative correlation is suitable for luminescent systems of different reinforcing agents. Benefited from the enzyme concentration dependent luminescence delay effect, the chemiluminescence liquid with the delay effect is used for improving the development quality of solid-phase membrane immunodetection for the first time, noise and background signals are suppressed, and the chemiluminescence liquid has wide application value in the aspect of solid-phase membrane immunodetection.

Description

Technical Field

[0001] This invention belongs to the field of chemiluminescence immunoassay technology, specifically relating to the application of luminescence delay agents in improving the imaging quality of solid-phase membrane immunoassay. Background Technology

[0002] Luminol and its derivatives, catalyzed by peroxidases (such as horseradish peroxidase and soybean peroxidase), can react with oxidants such as hydrogen peroxide to produce chemiluminescence, with a spectral peak at 425 nm. This reaction is widely used in chemiluminescent immunodiagnostics, especially in solid-phase membrane immunoassay techniques, such as Western blotting and dot blotting, where it serves as a core signal output mechanism. To improve detection sensitivity, enhanced chemiluminescent solutions (ECL solutions) are commonly used. The enhancers in these solutions (such as 4-iodophenol, or the dual-enhancer system SPTZ (sodium phenothiazine-10-propylsulfonate) and MORP (4-morpholinopyridine)) can increase the luminescence intensity of luminol by thousands of times and significantly extend the luminescence time, thereby significantly enhancing the practicality of peroxidase-based immunodiagnostic techniques.

[0003] Solid-phase membrane immunoassay, an important tool in biomedical research and clinical diagnosis, achieves signal detection through the specific binding of antigens and antibodies and the catalytic activity of labeled enzymes (such as horseradish peroxidase (HRP)). It boasts high sensitivity and specificity. Immunoblotting can identify and analyze target antigens based on molecular weight, while dot blotting is simple to operate and suitable for rapid screening. However, in practice, this technique often suffers from significant noise or high background signals on the membrane due to factors such as antibody cross-reaction, antigen degradation, and inadequate blocking. These interfering signals usually originate from the local catalytic effect of low-concentration HRP on the membrane. Although their intensity is weaker than the target band, they severely interfere with the accurate interpretation of results, becoming a key bottleneck restricting the signal-to-noise ratio and reliability of the detection.

[0004] In existing technologies, some approaches have attempted to optimize the enzymatic chemiluminescence process to improve detection stability. For example, a Chinese patent application discloses a method to adjust the onset time of chemiluminescence by introducing a delay agent, thereby achieving a stable long plateau period and effectively reducing the coefficient of variation (CV) in repeated detections, thus improving reproducibility. However, this strategy is primarily designed for plate-based chemiluminescence immunoassay systems, whose reaction environment, substrate diffusion kinetics, and signal readout mechanisms differ significantly from solid-phase membrane systems. On solid-phase membrane supports, noise and background signals typically originate from the catalysis of non-specifically contaminated HRP; extending the luminescence plateau period does not eliminate noise and background. Therefore, existing delay agent approaches are difficult to directly transfer to the membrane plateau, and they do not reveal how to effectively suppress local noise and background caused by low-concentration HRP catalysis on the membrane surface. Currently, there is a lack of dedicated chemiluminescence systems or substrate optimization strategies that can effectively suppress non-specific background while maintaining clear signal intensity, tailored to the characteristics of solid-phase membrane systems. This has become a key obstacle restricting the further development of this technology. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to overcome the defects and shortcomings of the existing technology in lacking a dedicated chemiluminescence system or substrate optimization strategy that can effectively suppress non-specific background while maintaining clear signal intensity, based on the characteristics of solid membrane system. The primary objective is to provide the application of luminescence delay agents in improving the imaging quality of solid membrane immunoassay.

[0006] A second objective of this invention is to provide the application of delayed chemiluminescent solutions in improving the imaging quality of solid-phase membrane immunoassays.

[0007] A third objective of this invention is to provide a method for improving the imaging quality of solid-phase membrane immunoassay.

[0008] The above-mentioned objective of this invention is achieved through the following technical solution: This invention protects the application of luminescence delay agents in improving the imaging quality of solid-phase membrane immunoassay. The luminescence delay agent is an alkyl radical scavenger, which is selected from one or more of the following substances: compounds containing a catechol structure or their polymers, and vitamin C.

[0009] In solid-phase membrane immunoassay, taking immunoblotting as an example, noise and background signals often appear due to problems such as low antibody specificity, antigen degradation, incomplete blocking, and other improper operations, resulting in poor development quality. Compared to the main band signal, these noise and background signals are usually caused by relatively low concentrations of HRP catalysis. Using this delayed-type ECL luminescent solution for immunoblotting development can complete the acquisition of luminescence signals in the relatively high concentration HRP region (target main band) before the luminescence initiation in the low concentration HRP region (noise or background), thereby suppressing noise and background signals. This method of suppressing noise and background signals differs from previous methods that suppress background signals by adding metal ion chelating agents and antioxidants (usually reactive oxygen species quenchers). The latter mainly reduces background signals by inhibiting free radicals catalyzed by non-peroxidases (such as iron ions, copper ions, etc.). This method is suitable for liquid-phase luminescence detection, such as multi-well plate immunochemiluminescence detection, but it is not suitable for eliminating noise and background signals in solid-phase membrane immunoassay development. The reason is that these noise and background signals come from the reaction between non-specifically contaminated HRP and the luminescent liquid, and are essentially consistent with the target signal. Suppressing the signals generated by non-HRP catalysis cannot eliminate the noise and background generated by HRP catalysis.

[0010] Regarding the issue of solid-phase membrane immunoassay development, this invention is the first to discover that the concentration of the retardant agent and the reciprocal of the luminescence onset delay time (dT) are positively correlated with the concentration of the enzyme catalyst; that is, dT is non-linearly related to the concentration of the enzyme catalyst. Utilizing this characteristic, a delayed-type chemiluminescent solution containing the aforementioned luminescence retardant agent can suppress or completely eliminate noise and background signals in solid-phase membrane immunoassay development.

[0011] Preferably, the compound containing the catechol structure is selected from at least one of protocatechuic aldehyde, protocatechuic acid, methyl protocatechuate, ethyl protocatechuate, gallic acid, methyl gallate, ethyl gallate, 3-O-methylgallic acid, pyrogallic acid, and tannin.

[0012] Furthermore, the solid-phase membrane immunoassay includes immunoblotting and / or dot blotting.

[0013] This invention protects the application of delayed chemiluminescence liquid in improving the imaging quality of solid-phase membrane immunoassay. The components of the delayed chemiluminescence liquid include a luminescent substrate, an enzyme catalyst, an oxidant, a luminescence enhancer, and a luminescence delay agent. The luminescence delay agent is an alkyl radical scavenger, which is selected from one or more of the following substances: compounds containing a catechol structure or their polymers, and vitamin C.

[0014] Preferably, the compound containing the catechol structure is selected from at least one of protocatechuic aldehyde, protocatechuic acid, methyl protocatechuate, ethyl protocatechuate, gallic acid, methyl gallate, ethyl gallate, 3-O-methylgallic acid, pyrogallic acid, and tannin.

[0015] Furthermore, the components in the chemiluminescent liquid are mixed or stored separately according to actual conditions, and then mixed and blended when needed.

[0016] In experiments such as immunoblotting, chemiluminescent solutions are typically divided into two parts: solution A and solution B. Solution A mainly includes the luminescent substrate and luminescence enhancer, and may also contain a separately stored luminescence retarder; solution B mainly consists of an oxidant. The enzyme-labeled antibody (such as primary or secondary antibody) used in the experiment provides the enzyme catalyst required for the luminescence reaction. During development, solution A and solution B are mixed in a specific ratio to prepare a working solution, which is then incubated onto the blot membrane for chemiluminescent development and imaging analysis.

[0017] Furthermore, both solution A and solution B use buffer solution as solvent.

[0018] Preferably, the buffer solution includes at least one of Tris buffer, phosphate buffer, carbonate buffer, and acetate buffer.

[0019] Furthermore, the pH of the buffer solution is 6-9, and the salt concentration in the buffer solution is 10-100 mM.

[0020] Furthermore, the luminescence retarder is dissolved in an organic solvent during use, the organic solvent being selected from at least one of dimethyl sulfoxide (DMSO), N,N-dimethylformamide (DMF), ethanol, and acetonitrile.

[0021] Preferably, the concentration of the luminescence delay agent in the chemiluminescent solution is 1~100μM.

[0022] Preferably, the luminescent substrate is a phthalohydrazide luminescent molecule or a derivative thereof.

[0023] More preferably, the luminescent substrate is luminol.

[0024] Preferably, the concentration of the luminescent substrate in the chemiluminescent solution is 1 μM to 5 mM, more preferably 1 to 100 μM. The concentration of the luminescent substrate can be selected according to the actual situation.

[0025] Furthermore, the solid-phase membrane immunoassay includes immunoblotting and / or dot blotting.

[0026] Further, the enzyme catalyst is a peroxidase; preferably, the enzyme catalyst includes at least one of horseradish peroxidase, potato peroxidase, and soybean peroxidase.

[0027] Preferably, the concentration of the enzyme catalyst in the chemiluminescent solution is (0, 100] ng / mL, more preferably 0.0001~10 ng / mL. Parentheses “()” indicate that the endpoint is not included, and square brackets [] indicate that the endpoint is included. Therefore, (0, 100] clearly means greater than 0 ng / mL and less than or equal to 100 ng / mL. The enzyme catalyst has a delaying effect at various concentrations, but when the enzyme catalyst concentration is greater than 100 ng / mL, the reaction is very fast, and the delay is not significant. Theoretically, the lower the concentration, the more obvious the delay effect. The concentration of the enzyme catalyst can be adjusted according to the actual situation.

[0028] Preferably, the oxidant is selected from at least one of hydrogen peroxide, urea peroxide, sodium perborate, or other compounds that can generate peroxides on-site.

[0029] Furthermore, the concentration of the oxidant in the chemiluminescent liquid is 0.1~50 mM, preferably 0.2~20 mM, and more preferably 0.5~5 mM.

[0030] Furthermore, the luminescence enhancer includes phenothiazine compounds, pyridine compounds, phenylboronic acid compounds, or phenolic compounds; Preferably, the phenothiazine compound includes sodium phenothiazine-10-propylsulfonate or a derivative thereof; Preferably, the pyridine compound includes 4-morpholinopyridine or its derivatives.

[0031] Preferably, the phenylboronic acid compound includes p-iodophenylboronic acid; Preferably, the phenolic compound includes 4-iodophenol.

[0032] Preferably, the concentration of the luminescence enhancer in the chemiluminescent liquid is 100 μM to 10 mM, and more preferably 1 to 3 mM.

[0033] Optionally, the chemiluminescent liquid may also include surfactants and / or viscosity modifiers. These components are typically added to the A-component of the chemiluminescent liquid.

[0034] Preferably, the surfactant is selected from one or more of sodium dodecyl sulfate (SDS), Triton X-100, and Tween 80.

[0035] Preferably, the surfactant has a mass concentration of (0~2wt%) in the chemiluminescent liquid.

[0036] Preferably, the viscosity modifier is selected from one or more of ethylene glycol, oligoethylene glycol, glycerol, and cyclodextrin.

[0037] Preferably, the viscosity modifier has a mass concentration of (0~10wt%) in the chemiluminescent liquid.

[0038] This invention also protects a method for improving the development quality of solid-phase membrane immunoassay, which uses a delayed-type chemiluminescent solution for solid-phase membrane immunoassay development; The components of the delayed chemiluminescent liquid include a luminescent substrate, an enzyme catalyst, an oxidant, a luminescence enhancer, and a luminescence delay agent. The luminescence delay agent is an alkyl radical scavenger, which is selected from one or more of the following substances: compounds containing a catechol structure or their polymers, vitamin C; Preferably, the compound containing the catechol structure is selected from at least one of protocatechuic aldehyde, protocatechuic acid, methyl protocatechuate, ethyl protocatechuate, gallic acid, methyl gallate, ethyl gallate, 3-O-methylgallic acid, pyrogallic acid, and tannin.

[0039] Furthermore, the delayed chemiluminescent liquid is consistent with the aforementioned limitations.

[0040] Compared with the prior art, the present invention has the following beneficial effects: This invention is the first to discover a nonlinear correlation between the luminescence onset delay time and the enzyme catalyst concentration when the concentration of the retardant is fixed. This nonlinear correlation applies to luminescence systems with different enhancers. Based on this nonlinear correlation between the luminescence onset time of the luminescent liquid and the enzyme catalyst concentration, this invention is the first to utilize this chemiluminescent liquid with a delay effect to improve the development quality of solid-phase membrane immunoassay, achieving noise and background signal suppression, and has broad application value in solid-phase membrane immunoassay. Attached Figure Description

[0041] Figure 1 (a) shows the kinetic curves of HRP / luminol enzyme-catalyzed luminescence intensity-reaction time for compounds containing catechol structures and vitamin C; (b) shows the kinetic curves of HRP / luminol enzyme-catalyzed luminescence intensity-reaction time for phenolic compounds containing a single phenolic hydroxyl group and compounds containing resorcinol structures: the luminescence on-off has a limited delay effect or no delay effect; the final concentrations of H2O2 and HRP in the reaction solution are 2 mM and 5 ng / mL, respectively, the concentrations of vitamin C and phenolic compounds are 100 μM, and the concentrations of other additives are as described in Example 1.

[0042] Figure 2(a) is a graph showing the change in luminescence onset delay time with HRP concentration, and the concentration of protocatechuic acid (PCA) in the luminescent solution is 30 μM; (b) is a graph showing the linear correlation between the reciprocal of the delay time (in seconds) and the HRP concentration, and the PCA concentration is 30 μM; (c) is a graph showing the change in luminescence onset delay time with HRP concentration, and the PCA concentration is 50 μM; (d) is a graph showing the linear correlation between the reciprocal of the delay time (in seconds) and the HRP concentration, and the PCA concentration is 50 μM; the concentrations of other substances in the luminescent solution used in the above tests are luminol 100 μM, MORP 2 mM, SPTZ 1 mM, and H2O2 2 mM, respectively.

[0043] Figure 3 The following are statistical graphs showing the luminescence delay effect of the retardant on ECL luminescent liquid containing 4-iodophenol as an enhancer: (a) Statistical graph showing the effect of different concentrations of protocatechuic aldehyde (PCAL) on luminescence kinetics, with the blank being the conventional luminescent liquid without PCAL and HRP concentration of 20 ng / mL; (b) Statistical graph showing the effect of different concentrations of protocatechuic acid (PCA) on luminescence kinetics, with the blank being the conventional luminescent liquid without PCA and HRP concentration of 20 ng / mL; (c) Curve showing the delay time versus HRP concentration with PCA fixed at 100 μM; (d) Linear correlation between the reciprocal of the delay time 1 / dT and HRP concentration. In the above tests, the concentrations of luminol, 4-iodophenol, and H2O2 were 100 μM, 1 mM, and 2 mM, respectively.

[0044] Figure 4 A schematic diagram illustrating the possible mechanism of luminescence enhancement in chemiluminescent liquids and the delay effect produced by retarders.

[0045] Figure 5 The general formula for the reaction of the quenching agent (QH) with DPPH· is shown in the statistical graph, along with the data on the scavenging rate of the measured compounds against DPPH·.

[0046] Figure 6 (a) shows the curve of luminescence delay time versus HRP concentration with LAA fixed at 50 μM; (b) shows the linear correlation between the reciprocal of delay time 1 / dT and HRP concentration; the concentrations of other components in the luminescent solution are luminol 100 μM, MORP 2 mM, SPTZ 1 mM, and H2O2 2 mM, respectively.

[0047] Figure 7(a) is a statistical chart of the immunoblotting results of ECL chemiluminescent solutions containing different concentrations of PCA (0, 5, 10, 15 μM), imaged using the automatic exposure mode of a chemiluminescence imager; (b) is a statistical chart of the immunoblotting results of enhanced chemiluminescent solutions containing different concentrations of PCA (0, 5 μM), imaged using the cumulative exposure mode of a chemiluminescence imager; PCA5 / PCA0 is the ratio of the signal-to-noise ratio of the imaging results with PCA (5 μM) containing the retardant to the signal-to-noise ratio of the imaging results without the retardant under the corresponding exposure conditions; Figure 7 (b) The red selection box is used to select the corresponding area for signal-to-noise ratio calculation. The mean of the signal (target signal) of the main strip in the selection box and the signal (noise signal) of the adjacent blank area are quantified using Image J software, and then the signal-to-noise ratio is calculated. The concentrations of other components in the luminescent liquid are: luminol 1mM, MORP 2mM, SPTZ 1mM, and H2O2 2mM.

[0048] Figure 8 The images show the WB development results of the chemiluminescent liquid containing a retarder and a conventional chemiluminescent liquid. (a) is a statistical chart of the development results of ECL without a retarder; (b) is a statistical chart of the development results of ECL containing 5 μM protocatechuic aldehyde (images a and b are taken from the same WB film); (c) is a statistical chart of the development results of ECL without a retarder; (d) is a statistical chart of the development results of ECL containing 5 μM gallic acid (images c and d are taken from the same WB film); (e) is the development result of ECL without a retarder; and (f) is a statistical chart of the development results of ECL containing 2 μM methyl gallate (images e and f are taken from the same WB film). All development was performed using automatic exposure mode for chemiluminescence development. Other components of the chemiluminescent liquid were described in the experimental section. Figure 7 The conditions are the same.

[0049] Figure 9 The image shows the statistical results of immunoblotting of enhanced luminescent solutions containing different concentrations of LAA (0, 5, 10, 20 μM), and the imaging was performed using the cumulative exposure mode of a chemiluminescence imager. The concentrations of other components in the luminescent solution were: luminol 1 mM, MORP 2 mM, SPTZ 1 mM, and H2O2 2 mM.

[0050] Figure 10 (a) is a WB chemiluminescence imaging image without any delay agent; (b) is a WB chemiluminescence imaging image containing 10 μM resveratrol; (c) is a WB chemiluminescence imaging image containing 10 μM LAA; chemiluminescence development was performed using automatic exposure mode; the concentrations of other components in the luminescent solution were: luminol 1 mM, MORP 2 mM, SPTZ 1 mM, and H2O2 2 mM. Detailed Implementation

[0051] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but the embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in this technical field.

[0052] Unless otherwise specified, all reagents and materials used in the following examples are commercially available.

[0053] Figure 1 a represents Figure 1 Figure (a) in the middle, Figure 2 a represents Figure 2 Figure (a) in the middle, Figure 2 b represents Figure 2 The naming meanings of the other figures are similar to those of Figure (b).

[0054] Example 1: Discovery of a luminescence retarder The delayed effects of each component (protocatechuic aldehyde, protocatechuic acid, methyl protocatechuate, ethyl protocatechuate, gallic acid, methyl gallate, ethyl gallate, 3-O-methylgallic acid, pyrogallol, tannin, vitamin C, vanillin, vanillic acid, 4-hydroxybenzoic acid, 4-iodophenol, 3,5-dihydroxybenzoic acid, 2,4-dihydroxybenzoic acid, phloroglucinol, resveratrol, 100 μM) were determined according to the method disclosed in CN119044155A.

[0055] The final concentrations of each substance used in the test were: luminol (100 μM), MORP (2 mM), SPTZ (1 mM), delay agent (100 μM), H2O2 (2 mM), and HRP (5 ng / mL). The specific test methods are as follows.

[0056] The basic procedure for testing using a handheld chemiluminescence analyzer is as follows: 100 μL of luminol (600 μM), 100 μL of MORP (12 mM), 100 μL of SPTZ (6 mM), 100 μL of delay agent (protocatechuic aldehyde, protocatechuic acid, methyl protocatechuate, ethyl protocatechuate, gallic acid, methyl gallate, ethyl gallate, 3-O-methylgallic acid, pyrogallol, tannin, vitamin C, vanillin, vanillic acid, 4-hydroxybenzoic acid, 4-iodophenol, 3,5-dihydroxybenzoic acid, 2,4-dihydroxybenzoic acid, phloroglucinol, resveratrol, 600 μM) and 100 μL of HRP (30 ng / mL) are mixed and allowed to stand at room temperature. 100 μL of H2O2 (12 mM) is added to the above solution, and after shaking and mixing for 2 seconds, the solution is placed in the chemiluminescence recorder to read the luminescence intensity. When conducting luminescence dynamics tests, the luminometer is used to read the values ​​every 5 seconds.

[0057] Example 2: Preparation of a conventional enhanced ECL luminescent liquid (without retarder) Prepare Solution A: Dissolve luminol (2mM), MORP (4mM), and SPTZ (2mM) in Tris buffer (20mM, pH 8.5). This is Solution A. Prepare Solution B: Prepare a peroxide solution (H2O2, 4mM) in Tris buffer (20mM, pH 8.5). Both solutions were stored at 4°C, protected from light. Luminescence intensity and luminescence kinetics data were acquired using a System SUREPlus luminescence detector (Hygiena, UK).

[0058] Example 3: Preparation of Noise-Eliminating ECL Luminescent Liquid This novel luminescent liquid can be prepared as a luminescent liquid containing three reagents, A, B, and C. The preparation of solutions A and B is based on the conventional ECL formulation in Example 2. A retarding agent (0.1–50 mM) is dissolved in one or more mixed solvents such as DMSO, DMF, ethanol, and acetonitrile. The retarding agent can be a catechol compound, vitamin C (LAA), or other control components to be tested (such as resveratrol). This is solution C. Alternatively, the retarding agent (1–100 μM) can be directly dissolved in solution A to prepare a dual-reagent luminescent liquid containing reagents A and B.

[0059] Example 4: Chemiluminescent Development Taking Western blotting (WB) development as an example, (1): When developing with ordinary ECL luminescent solution, mix solution A and solution B in a 1:1 ratio, take an appropriate amount of the mixed luminescent solution and apply it to the Western blotting membrane, or immerse the Western blotting membrane in the luminescent solution and then quickly remove it, and develop it using a chemiluminescence imaging system. (2) When developing with noise-reducing ECL luminescent solution, first mix solution A and solution B in a 1:1 ratio, then add a certain volume of solution C to make the retarder concentration between 1-100 μM. Select different retarder concentrations according to the strength of the signal to be eliminated. Take an appropriate amount of the mixed luminescent solution and apply it to the Western blotting membrane, or immerse the Western blotting membrane in the luminescent solution and then quickly remove it, and develop it using a chemiluminescence imaging system. In order to facilitate comparison of development quality, the same Western blotting membrane was developed multiple times using luminescent solutions with different components. Before the next development, the membrane was first immersed in PBS buffer (pH 7.4) to wash away the original luminescent solution.

[0060] Example 5: Quenching efficiency test of 2,2-diphenyl-1-picric hydrazine radical (DPPH·) Prepare 500 μM DPPH·ethanol solution and 100 μM ethanol solution of the test compound. Accurately transfer 100 μL of DPPH solution into each well of a 96-well plate, then add 100 μL of the test compound solution, mix well, wrap with aluminum foil, and incubate in the dark with shaking for 30 min. Measure the optical density at 517 nm using a microplate reader. OD 1). The optical density of a mixture of 100 μL LPPH solution and 100 μL anhydrous ethanol was determined using the same method. OD 2), the optical density value of a mixture of 100 μL of the test compound solution and 100 μL of anhydrous ethanol ( OD 0), calculate the clearance rate using the formula:

[0061] Each sample was measured in triplicate, and the average value was taken.

[0062] Results Analysis (1) The delay time is linearly correlated with the concentration of the delay agent. Table 1. Compounds tested and their effect on the delay time of luminescence initiation.

[0063] Note: Delay time (s) is defined as the time required for the luminescence intensity to increase from 0 to 1000 (RLU), in seconds; the compound test concentration is 100 μM.

[0064] The results are shown in Table 1 and Figure 1 As shown, compounds containing catechol structures (protocatechuic aldehyde (PCAL), protocatechuic acid (PCA), methyl protocatechuate, ethyl protocatechuate, gallic acid, methyl gallate, ethyl gallate, 3-O-methylgallic acid, pyrogallic acid, and tannins) can significantly prolong the luminescence onset time and can be used as retarding agents for chemiluminescent liquids. Furthermore, the addition of these retarding agents significantly prolongs the luminescence plateau period. As indicated in the inventors' aforementioned patent applications, the luminescence delay time of these retarding agents is linearly correlated with the concentration of the retarding agent within a certain concentration range.

[0065] This invention also discovered that vitamin C (LAA), an antioxidant that does not contain catechol, also has a delayed effect; therefore, delayed agents are not limited to catechol compounds. Figure 1 a). However, resveratrol, an antioxidant reported to have a strong quenching effect on reactive oxygen species (such as hydroxyl radicals), did not show a significant delay effect under the same conditions, and it significantly reduced the luminescence intensity ( Figure 1 b).

[0066] (2) The delay time is non-linearly related to the peroxidase concentration. The inventors further investigated the relationship between the delay time of these delay agents and the concentration of peroxidase, based on their previous research. The results are as follows: Figure 2 As shown, when the concentration of the delay agent is fixed, the higher the HRP concentration, the shorter the delay time. This is mainly because increasing the enzyme concentration increases the consumption rate of the delay agent. Interestingly, the delay time and enzyme concentration have a non-linear relationship; the delay time increases rapidly as the enzyme concentration gradually decreases. For example, taking a reaction system with a PCA concentration of 30 μM as an example, the delay time is 56.3 seconds when the HRP concentration is 10 ng / mL, and 32.1 seconds when the HRP concentration is 15 ng / mL. Above these concentrations, further increasing the HRP concentration slows down the change in delay time. It can be inferred that as the HRP concentration increases infinitely, the delay time approaches zero, but will not equal zero. When the HRP concentration decreases, the delay time increases rapidly; for example, the delay time reaches 245 seconds when the HRP concentration is 5 ng / mL, and 656 seconds when the HRP concentration is 2.5 ng / mL. It can be inferred that as the HRP concentration continues to decrease, the delay time approaches infinity. Figure 2 a). When the reciprocal of the delay time (1 / dT) is plotted against the HRP concentration, it is found that 1 / dT is related to the HRP concentration (C HRP ) are linearly correlated (R 2 = 0.996)( Figure 2 b). If the concentration of PCA is set to 50 μM, a similar trend can be observed ( Figure 2 c) and Figure 2 d). Based on this, it can be inferred that a similar trend can be obtained by keeping the PCA concentration constant. Therefore, the relationship between the delay time dT and the HRP concentration can be deduced as: dT = 1 / (a ​​× C) HRP + b), where a and b are the slope and intercept of the linear fit between 1 / dT and HRP concentration, respectively, which are affected by the concentration of the delay agent.

[0067] As can be seen from the above, the reciprocal of the delay time (unit S) is linearly correlated with the HRP concentration, that is, the delay time is non-linearly correlated with the peroxidase concentration.

[0068] (3) The delay effect also applies to luminescent liquids using 4-iodophenol as an enhancer. In further research, this invention replaced the SPTZ / MORP two-component reinforcing agent with a 4-iodophenol one-component reinforcing agent and explored the delaying effect of the retarder. Figure 3 As shown, the representative retardant PCAL ( Figure 3 a) and PCA ( Figure 3(b) Both methods produce significant delay effects, which are concentration-dependent. At the same retarder concentration, the luminescent solution containing 4-iodophenol enhancer has a significantly longer luminescence onset delay time than the SPTZ / MOPR luminescent solution system. For example, when PCA is 100 μM and HRP concentration is 10 ng / mL, the luminescence delay time reaches 2579 seconds (approximately 43 minutes). In the SPTZ / MORP system, when PCA is 50 μM and HRP concentration is 10 ng, the delay time is 96.5 seconds (…). Figure 2 c). According to linear estimation, when PCA is 100 μM, the delay time will not exceed 200 seconds, less than one-tenth of that of the 4-iodophenol system. The results show that when the PCA concentration is fixed (100 μM), the delay time exhibits a non-linear correlation with the HRP concentration. Figure 3 c), and the reciprocal of the delay time 1 / dT is linearly correlated with the HRP concentration. Figure 3 d). This trend is largely consistent with that observed in the SPTZ / MORP luminescent liquid system. Therefore, it can be shown that this delay effect and the observed relationship between delay time and HRP concentration are applicable to ECL luminescent liquid systems containing different enhancers.

[0069] (4) Discussion on the delay mechanism The delay agent in this novel chemiluminescent solution can be considered an HRP substrate with higher chemical reactivity than luminol. Generally, within a certain enzyme concentration range, the substrate consumption rate is linearly correlated with the amount of enzyme; however, this invention reveals that the consumption rate of the delay agent is non-linearly correlated with the enzyme concentration. It is generally believed that the luminescence mechanism of enhanced chemiluminescent solutions can be explained by a "ping-pong" mechanism (3-(10′-Phenothiazinyl)propionic acid is a potential primary enhancer of peroxidase-induced chemiluminescence and its application in sensitive ELISA of methylglyoxal-modified low-density lipoprotein). Talanta 2013, 115: 414-417), namely: HRP reacts with H2O2 to form a high-valent iron complex of HRP (HRP-I). HRP-I catalyzes the formation of the reinforcing agent free radical (E·). HRP-I is transformed into HRP-II, which can continue to react with the reinforcing agent E to form E·, while HRP-II is transformed back into HRP, entering a cycle. The reinforcing agent free radical E· and H2O2 co-oxidize luminol to produce chemiluminescence ( Figure 4Through this "ping-pong" mechanism, a non-linear correlation exists between HRP concentration and the rate of consumption of the luminescent substrate, and this relationship also exists between HRP and the retarder.

[0070] Based on the above mechanism, it is easy to see that quenching the oxidized HRP (HRP-I and HRP-II) or E· in this reaction may inhibit the oxidation and luminescence of luminol. Therefore, it is speculated that catechol-based retarder may achieve its retarding effect by quenching the above key intermediates. However, considering that the formation of HRP-I is almost unaffected by the type of enhancer, if the retarder works by quenching HRP-I, then changing the enhancer would have a relatively small impact on the retarding effect. However, the inventors observed in experiments that different enhancer systems have a significant impact on the retarding effect. Therefore, it is reasonable to speculate that the retarder mainly works by quenching the enhancer E·. Different E· have different reactivity with the retarder, which may be the main reason for the different retarding effects.

[0071] This invention further notes that E· is a small molecule alkyl radical, but it is unstable and difficult to capture. To investigate the quenching effect of the retarder on the alkyl radical, the inventors selected a commonly used, stable small molecule alkyl radical, DPPH·, for relevant tests. The results are as follows... Figure 5 As shown, catechol compounds with significant delaying effects all exhibit high DPPH· quenching efficiency, while compounds containing a single phenolic hydroxyl group and those containing a resorcinol structure have limited quenching efficiency or almost no quenching effect. Based on the DPPH· quenching phenomenon, it is possible to predict whether a compound will have a delaying effect on HRP enzymatic luminescence. Resveratrol is an antioxidant containing resorcinol. Previous reports have shown that resveratrol has a stronger ability to scavenge reactive oxygen free radicals (such as hydroxyl radicals) than LAA, but its ability to scavenge alkyl free radicals such as DPPH· is significantly weaker than that of LAA. The DPPH· test results suggest that resveratrol may not have a significant delaying effect, which is consistent with... Figure 1 The results are largely consistent. LAA has been reported to have strong DPPH quenching activity. Figure 5 Test results show that LAA can indeed quench DPPH·. Interestingly, LAA can also produce a significant delayed luminescence effect. For example, when the HRP concentration is 5 ng / mL, 50 μL of LAA produces a delay time of up to 200 seconds. Figure 6 When the LAA concentration is fixed (50 μM), a nonlinear correlation also exists between the delay time and the HRP concentration. Figure 6 a), the reciprocal of the delay time 1 / dT is linearly correlated with the HRP concentration ( Figure 6(b) This is consistent with the phenomenon observed in the catechol retarder test. Therefore, it can be said that catechol is not a necessary structure to produce a retarding effect; whether a retarding effect is present depends on whether the compound can quench E·.

[0072] (5) Delayed ECL luminescent solution eliminates the quality issues of immunoblotting by nonlinearly controlling the delay time. This invention creatively discovers that the novel ECL luminescent liquid has a non-linear delay effect on the luminescence onset time under different concentrations of HRP catalysis, and further applies it to immunoblotting development. To verify the development effect, the inventors randomly obtained immunoblotting membranes of relatively low quality from other research groups and developed them according to the method disclosed in Example 4 above. The results are as follows. Figure 7 As shown in Figure a, when the blot film was imaged using a conventional ECL luminescent solution (with a PCA retarder concentration of 0), noise and non-specific weak bands appeared. Adding PCA to the conventional ECL luminescent solution significantly improved the development quality. For example, using an ECL luminescent solution containing 15 μMPa significantly reduced noise and non-specific weak signals, resulting in a substantial improvement in development quality. Furthermore, as... Figure 7 As shown in b, the inventors developed another immunoblot membrane using a cumulative exposure mode. The results clearly show that with conventional ECL, strong background signals can be observed around the main band. Shortening the exposure time is insufficient to completely eliminate these background signals, and it also reduces the signal of the target band, affecting the development quality. In contrast, when developed using an ECL containing 5 μM PCA, these background signals are almost completely suppressed. Under different exposure conditions, the signal-to-noise ratio (SNR) obtained using the delayed-type ECL is generally higher than that of conventional ECL, by more than three times. These Western blotting results indicate that when the target band signal is relatively strong (relatively high HRP concentration), the development time of the PCA-containing ECL under automatic exposure conditions is comparable to that of the conventional ECL without a retardant. This suggests that the addition of a retardant does not affect the signal strength of the target main band, nor does it significantly delay the onset of the main band's luminescence signal. When the target band itself is weak (HRP concentration is relatively low), the addition of the retarder suppresses background and non-specific signals, while also delaying the appearance of the target band signal. However, high-quality development results can still be obtained by increasing the exposure time.

[0073] It is speculated that these catechol compounds can all improve the quality of WB development. In addition to PCA, the inventors randomly selected protocatechuic aldehyde, gallic acid and methyl gallate for testing. Figure 8 The results showed that adding a low concentration of protocatechuic acid to ECL significantly eliminated nonspecific weak bands while ensuring the development quality of the target bands. Figure 8 a and Figure 8 b). Similarly, using ECL reagent containing 5 μM gallic acid for Western blotting, in automatic exposure mode, not only completely eliminated noise and weak bands, but also significantly enhanced the main band signal. Figure 8 c and Figure 8 d). Similarly, WB development using an ECL luminescent solution containing 2 μM methyl gallate also significantly eliminated nonspecific bands and background signals. Figure 8 e and Figure 8 f).

[0074] This delaying effect is not limited to luminescent solutions containing catechol-based delaying agents; this invention is the first to discover that vitamin C (LAA) also possesses the same function. The inventors have prepared a delayed-type ECL luminescent solution by adding LAA to the ECL luminescent solution, which can also improve development quality. For example... Figure 9 As shown, conventional ECL chemiluminescence solutions without LAA exhibit certain background signals and nonspecific weak bands. However, these signals can be suppressed using chemiluminescence solutions containing LAA. Simultaneously, this invention also investigated the effect of resveratrol in eliminating WB noise and background, with results as follows... Figure 10 As shown, compared with the control group without added retarder ( Figure 10 a) Compared to the addition of resveratrol ( Figure 10 b) Significantly reduced the emission signal, which is consistent with Figure 1 The test results were consistent; noise and non-specific strip signals were not eliminated, and their signal strength was weakened along with the target strip signal strength. However, when LAA was used as a delay agent ( Figure 10 c) can largely eliminate nonspecific signals and noise. These results indicate that, while both are antioxidants, resveratrol cannot eliminate background and noise in WB imaging, while LAA and catechol compounds can achieve the same goal. This may stem from their different antioxidant mechanisms and their different roles in key free radicals during the ECL reaction.

[0075] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. The application of luminescence retarder in improving the imaging quality of solid-phase membrane immunoassay, characterized in that, The luminescence delay agent is an alkyl radical scavenger, which is selected from one or more of the following substances: compounds containing a catechol structure or their polymers, and vitamin C.

2. The application according to claim 1, characterized in that, The compound containing the catechol structure is selected from at least one of protocatechuic aldehyde, protocatechuic acid, methyl protocatechuate, ethyl protocatechuate, gallic acid, methyl gallate, ethyl gallate, 3-O-methylgallic acid, pyrogallic acid, and tannin.

3. The application according to claim 1 or 2, characterized in that, The solid-phase membrane immunoassay includes immunoblotting and / or dot blot.

4. The application of delayed chemiluminescent solutions in improving the imaging quality of solid-phase membrane immunoassay, characterized in that, The components of the delayed chemiluminescence liquid include a luminescent substrate, an enzyme catalyst, an oxidant, a luminescence enhancer, and a luminescence delay agent; wherein the luminescence delay agent is an alkyl radical scavenger, and the alkyl radical scavenger is selected from one or more of the following substances: compounds containing a catechol structure or their polymers, and vitamin C.

5. The application according to claim 4, characterized in that, The compound containing the catechol structure is selected from at least one of protocatechuic aldehyde, protocatechuic acid, methyl protocatechuate, ethyl protocatechuate, gallic acid, methyl gallate, ethyl gallate, 3-O-methylgallic acid, pyrogallic acid, and tannin.

6. The application according to claim 4, characterized in that, The solid-phase membrane immunoassay includes immunoblotting and / or dot blot.

7. The application according to claim 4, characterized in that, The enzyme catalyst is a peroxidase; preferably, the enzyme catalyst includes at least one of horseradish peroxidase, potato peroxidase, and soybean peroxidase.

8. The application according to claim 4, characterized in that, The oxidant is selected from at least one of hydrogen peroxide, urea peroxide, sodium perborate, or other compounds that can generate peroxides on-site.

9. The application according to claim 4, characterized in that, The luminescence enhancer includes phenothiazine compounds, pyridine compounds, phenylboronic acid compounds, or phenolic compounds; Preferably, the phenothiazine compound includes sodium phenothiazine-10-propylsulfonate or a derivative thereof; Preferably, the pyridine compound includes 4-morpholinopyridine or a derivative thereof; Preferably, the phenylboronic acid compound includes p-iodophenylboronic acid; Preferably, the phenolic compound includes 4-iodophenol.

10. A method for improving the imaging quality of solid-phase membrane immunoassay, characterized in that, Solid-phase membrane immunoassay was performed using a delayed-type chemiluminescent solution. The components of the delayed chemiluminescent liquid include a luminescent substrate, an enzyme catalyst, an oxidant, a luminescence enhancer, and a luminescence delay agent. The luminescence delay agent is an alkyl radical scavenger, which is selected from one or more of the following substances: compounds containing a catechol structure or their polymers, vitamin C; Preferably, the compound containing the catechol structure is selected from at least one of protocatechuic aldehyde, protocatechuic acid, methyl protocatechuate, ethyl protocatechuate, gallic acid, methyl gallate, ethyl gallate, 3-O-methylgallic acid, pyrogallic acid, and tannin.

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