Intermolecular electrochemiluminescence co-reactant drying fixation method and application thereof in ECL chip

Abstract

The application discloses a kind of intermolecular electrochemiluminescence co-reactant drying fixation method and its application in ECL chip, which comprises the following steps: co-reactant solution is sprayed on the sample pad of ECL chip, and is dried, namely obtained;The co-reactant is one or more of di(2-hydroxyethyl) imino tri(hydroxymethyl) methane, 1,3-bis(trihydroxymethyl) methyl amino) propane or bis(2-hydroxyethyl) amino-tri(hydroxymethyl) methane.The application first dries and fixes intermolecular ECL co-reactant on the sample pad of ECL chip, and is successfully applied to immunodetection.The method of the application overcomes the problems of traditional co-reactant, such as strong volatility, toxicity and inability to dry fixation.

Description

Technical Field

[0001] This invention belongs to the field of microfluidic chips, specifically relating to a method for drying and fixing intermolecular electrochemiluminescence (ECL) co-reactants and its application in ECL chips. Background Technology

[0002] Ruthenium complexes, as ECL (electroluminescence ionization) markers, have been widely studied and applied in clinical diagnostics and scientific research due to their high sensitivity, wide dynamic range, good stability, simple operation, and wide range of applications. The electrochemical reaction between ruthenium complexes and their co-reactants produces the ECL phenomenon. The ECL efficiency is mainly related to the charge transfer between the ruthenium complex and the co-reactant. The co-reactant generates an active intermediate through electrochemical reaction, which further reacts with the oxidized luminescent group to form an excited state, thus producing the ECL phenomenon. Under conditions of better free radical ion supply and sufficient co-reactant supply, ECL with good stability and high luminescence intensity can be generated. Therefore, finding suitable co-reactants for effective ECL detection has significant research significance and application value in the field of bioanalysis.

[0003] ECL co-reactants can be divided into intramolecular ECL co-reactants and intermolecular ECL co-reactants. Intramolecular ECL co-reactants act as a "bridge," forming the desired ruthenium-intramolecular ECL co-reactant-biomarker complex with ruthenium and biomarkers (such as antibodies, antigens, and nucleic acid fragments) through amino / carboxyl condensation. This complex formation has strict requirements on the types of groups on the co-reactants, requiring at least one carboxyl group and one amino group; therefore, amino acids are often used as intramolecular ECL co-reactants. However, because ECL efficiency is higher under alkaline conditions, the range of suitable amino acid-based intramolecular ECL co-reactants is greatly narrowed. Furthermore, the complex synthesis process is relatively cumbersome (requiring two amide bond connections), resulting in waste of co-reactants.

[0004] In contrast, intermolecular ECL co-reactants do not participate in the synthesis of ruthenium-biomarker complexes based on amide bonds, offering a wider range of choices and more flexible application methods, allowing for the substitution of co-reactant types according to actual needs. Furthermore, the synthesis process of ruthenium-biomarker complexes is simpler and faster than that of ruthenium-intramolecular ECL co-reactant-biomarker complexes. Intermolecular ECL co-reactants include tripropylamine (TPA), diethylaminoethanol (DBAE), and n-butyldiethanolamine (NBEA). TPA is the most common, but it suffers from low water solubility, volatility, and slow ECL oxidation. DBAE and NBEA have high water solubility and low volatility, but commercially available DBAE and NBEA are in liquid form, making them unsuitable for dry ECL immunoassay. Therefore, finding a dry-fixable intermolecular ECL co-reactant is essential.

[0005] In immunoassays based on intermolecular ECL co-reactants, the co-reactant is typically added using a wet method. The wet method usually involves adding the test sample solution first, allowing the target and ruthenium-biomarker complex to undergo an immunoreaction for a period of time, and then adding the intermolecular ECL co-reactant to initiate the ECL reaction. One drawback of this method is that adding the test sample solution first can wash away the complex, leading to insufficient reaction between the subsequently added co-reactant and ruthenium, thus reducing ECL efficiency. Another drawback is that, with the wet method, the co-reactant is easily affected by the adverse microenvironment of the buffer solution used for rinsing, ultimately resulting in inaccurate ECL detection results. Furthermore, this method involves multiple steps of solution addition and rinsing, making the detection process cumbersome and unable to achieve a truly "sample-in-result" detection.

[0006] In conclusion, exploring safe, efficient, and convenient methods for the dry fixation of intermolecular ECL co-reactants and their application in dry immunoassay is an urgent problem for researchers to solve. Summary of the Invention

[0007] To address the problems faced by existing technologies, the present invention aims to provide a method for drying and fixing intermolecular electrochemiluminescence (ECL) co-reactants and its application in ECL chips. A class of solid powder-like, environmentally friendly intermolecular ECL co-reactants was selected to investigate a series of problems encountered in drying and fixing them on test strips. This method solves the problems of conventional intermolecular ECL co-reactants being unable to be dried and the impact of their toxicity and volatility on the health of operators.

[0008] The objective of this invention is achieved through the following technical solution:

[0009] A method for drying and fixing intermolecular ECL co-reactants includes the following steps:

[0010] The co-reactant solution is sprayed onto the sample pad of the ECL chip and dried to obtain the final product.

[0011] A method for drying and fixing intermolecular ECL co-reactants includes the following steps:

[0012] The co-reactant solution was reacted with Ru(II)([Ru(bpy)3) 2+ A ruthenium complex-labeled antibody solution is mixed and sprayed onto the binding pad of an ECL chip, then dried to obtain the final product.

[0013] Preferably, the co-reactant solution and the labeled antibody solution are mixed in equal volumes.

[0014] A method for drying and fixing intermolecular ECL co-reactants includes the following steps:

[0015] First, the Ru(II) labeled antibody solution is dried and fixed onto the binding pad of the ECL chip. Then, the co-reactant solution is sprayed onto the binding pad and dried to obtain the final product.

[0016] The co-reactant is one or more of bis(2-hydroxyethyl)aminotris(hydroxymethyl)methane (BIS-TRIS methane), 1,3-bis[tris(hydroxymethyl)methylamino]propane (BIS-TRIS propane), or bis(2-hydroxyethyl)amino-tris(hydroxymethyl)methane (2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)-,hydrochloride(1:1) (BIS-TRIS hydrochloride), preferably BIS-TRIS methane;

[0017] The concentration of the co-reactant solution is 10–110 mM;

[0018] The pH value of the co-reactant solution is 5.0–8.5;

[0019] The solvent for the co-reactant solution is deionized water (ddH2O), phosphate buffer (PB, 0.1M), and PBS (0.1M), preferably PBS (0.1M).

[0020] The Ru(II)-labeled antibody is prepared by the following steps:

[0021] First, 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC), N-hydroxysuccinimide (NHS), and Ru(II) were dissolved in buffer solution and mixed to activate the carboxyl group, resulting in carboxyl-activated Ru(II). Next, the carboxyl-activated Ru(II) was mixed evenly with an antibody solution (prepared in buffer solution) and reacted thoroughly for several hours to obtain a Ru(II)-antibody complex. The complex was purified using a dextran gel to remove impurities, and the eluent was collected to obtain a pure Ru(II)-antibody complex. The pure Ru(II)-antibody complex was lyophilized at -80°C for several hours. The lyophilized complex was in the form of a dry powder. Different volumes of buffer solution were used to dissolve the dry powder to obtain Ru(II)-labeled antibodies with different concentrations.

[0022] The buffer solution has a pH of 7.2–7.4, preferably PBS;

[0023] The carboxyl activation temperature is 25℃, and the reaction time is 30 min.

[0024] The reaction time between the carboxyl-activated Ru(II) and the antibody is 1 to 10 hours, preferably 4 hours.

[0025] The aforementioned method for drying and immobilizing intermolecular ECL co-reactants can be applied to the preparation of ECL chips;

[0026] The ECL chip refers to a device that utilizes the ECL phenomenon generated by the electrochemical reaction between ruthenium complexes and their co-reactants to perform qualitative and / or quantitative analysis of the biomarkers to be tested by collecting and analyzing the generated ECL signals. Its components mainly include electrodes, microfluidic reaction cells, and microchannels.

[0027] The microfluidic reaction cell includes a binding pad and a detection pad;

[0028] The microchannel includes a sample application pad, a binding pad, a detection pad, and an absorbent pad.

[0029] The ECL chips mentioned above are generally divided into three-electrode ECL chips and bipolar ECL chips (bipolar electrode ECL chips, bipolar electrode ECL chips);

[0030] The three-electrode ECL chip described above has electrodes including a working electrode, a counter electrode, and a reference electrode.

[0031] The bipolar ECL chip described above has electrodes including a driving electrode and a bipolar electrode.

[0032] The bipolar ECL chip includes ECL test strips, ECL cloth chips, etc.

[0033] The ECL test strips include ECL lateral flow immunoassay strips.

[0034] The ECL lateral flow immunoassay strip includes a sample application pad, a conjugate pad, a detection pad, an absorbent pad, and a base plate. The sample application pad, conjugate pad, detection pad, and absorbent pad are microchannels that trigger capillary flow. In addition, the conjugate pad and the detection pad are also microfluidic reaction cells, with the former undergoing an immune reaction and the latter undergoing both an immune reaction and an electrochemical reaction.

[0035] The sample pad for fixing the co-reactant is used to assemble the ECL lateral flow immunoassay strip (ZL202410829788.0). The assembled test strip is placed in the cartridge, the cartridge is placed in the tray, and the tray is placed in the ECL point detection instrument for dry immunoassay.

[0036] The dry immunoassay process is as follows: A sample solution containing the target is added to the sample pad. The solution carries the co-reactants on the sample pad towards the binding pad, where the target binds to the labeled antibody on the binding pad to form a "target-labeled antibody" complex. As the solution flows, this complex and co-reactants continue to flow towards the detection pad, forming a "capture antibody-target-labeled antibody" sandwich immunocomplex on the T line and a "capture antibody-labeled antibody" complex on the C line. Next, PBS is added to the sample pad for further immunoassay. Simultaneously, excess labeled antibody is washed onto the absorption pad to connect the negative driving electrode and the detection anode. PBS solution is also added to the connection pad to connect the positive driving electrode and the shared cathode. The driving electrode of the electrode pad is electrically connected to the ECL point-of-care instrument, triggering the ECL reaction at a given driving voltage. During the reaction, a CMOS camera acquires ECL signals on the T and C lines respectively, and the ratio (T / C) is used for quantitative analysis of the LH target.

[0037] Compared with the prior art, the present invention has the following advantages:

[0038] 1. This invention proposes a method for drying and immobilizing intermolecular ECL co-reactants. For the first time, intermolecular ECL co-reactants (BIS-TRIS methane, BIS-TRIS propane, and BIS-TRIS hydrochloride) are dried and immobilized on the sample application pad of an ECL chip, and successfully applied to immunoassay. The invented method overcomes the problems of traditional co-reactants being highly volatile, toxic, and unable to be dried and immobilized.

[0039] 2. The method of the present invention fixes the intermolecular ECL co-reactant and the labeled antibody onto the sample pad and the binding pad of the test strip, respectively, realizing the integration of multi-step reactions on a single chip.

[0040] 3. The luminescence intensity obtained by the intermolecular ECL co-reactant drying fixation method proposed in this invention is higher than that obtained by the corresponding wet method. Therefore, it has the potential to achieve high-performance detection, broaden application scenarios, and simplify operation steps.

[0041] 4. The method of the present invention achieves direct linear fitting and double logarithmic linear fitting between target concentration and T / C, which has higher detection accuracy compared with the single logarithmic fitting of the prior art.

[0042] 5. Compared with the wet method, the method of the present invention only requires the addition of the sample solution to be tested and the buffer solution, which is convenient and quick.

[0043] 6. The Ru(II)-antibody complex proposed in this invention can effectively shorten the synthesis time and improve the synthesis efficiency when used as a labeling antibody.

[0044] 7. The method of the present invention uses gel column chromatography to purify labeled antibodies. Compared with the existing ultrafiltration method, it has high purification efficiency, can purify large volumes of labeled antibodies, has high reproducibility, wide application range, and mild operating conditions. Attached Figure Description

[0045] Figure 1 This is an optimization diagram of the drying and fixation method for intermolecular ECL co-reactants.

[0046] Figure 2 This is a graph showing the optimized concentration of intermolecular ECL co-reactants.

[0047] Figure 3 Optimization diagram for pH value of ddH2O.

[0048] Figure 4 Optimized pH value for PB (0.1M).

[0049] Figure 5 The pH optimization diagram for PBS (0.1M).

[0050] Figure 6 Comparison of ECL signal intensities for ddH2O, PB (0.1M), and PBS (0.1M) at the optimal pH value.

[0051] Figure 7 Optimization diagrams for different types of intermolecular ECL co-reactants.

[0052] Figure 8 The diagram shows the structures of three intermolecular ECL co-reactants: (a) BIS-TRIS methane, (b) BIS-TRIS hydrochloride, and (c) BIS-TRIS propane.

[0053] Figure 9 This is a comparison chart of ECL signal intensity under dry and wet conditions.

[0054] Figure 10 Optimization plot for amide linkage time of Ru(II) and antibody.

[0055] Figure 11 The diagram shows the optimization of the concentration factor of the Ru(II)-antibody complex.

[0056] Figure 12 This is a structural diagram of the ECL lateral flow immunoassay strip; where 1—lateral flow test strip, 1-1—sample application pad, 1-2—conjugation pad, 1-3—detection pad, 1-3-1—C line, 1-3-2—T line, 1-4—absorbent pad, 1-5—backplate; 2—electrode pad, 2-1—negative driving electrode, 2-2—integrated closed bipolar electrode (2-2-1—quality control anode, 2-2-2—detection anode), 2-3—shared cathode, 2-4—positive driving electrode; 3—connecting pad, 4—hydrophobic backplate.

[0057] Figure 13 This is a schematic diagram of the ECL lateral flow immunoassay strip detection principle.

[0058] Figure 14 For quantitative detection, a standard curve is directly linearly fitted.

[0059] Figure 15 To quantitatively detect the double logarithmic fitted standard curve. Detailed Implementation

[0060] The present invention will be further described in detail below with reference to the embodiments and accompanying drawings, but the embodiments of the present invention are not limited thereto.

[0061] Example 1

[0062] Intermolecular ECL co-reactant drying fixation method

[0063] First, prepare NaOH (0.1M) and HCl (0.1M) solutions to adjust the pH of the buffer solution (pH 5.0–8.5) for dissolving the intermolecular ECL co-reactants (BIS-TRIS methane, BIS-TRIS propane, BIS-TRIS hydrochloride). The buffer solution consists of ddH2O, PB (0.1M), and PBS (0.1M). Second, take 30 μL of the prepared intermolecular ECL co-reactant solution and fix it onto the sample pad or conjugate pad of the ECL chip using a gold sputtering spectrometer (model HGS510). Then, dry the solution in a forced-air drying oven at 37°C for 1 hour to dry and fix the co-reactants.

[0064] The specific method for drying and fixing intermolecular ECL co-reactants is as follows:

[0065] A. Spray the single co-reactant solution (BIS-TRIS methane solution, BIS-TRIS propane solution, BIS-TRIS hydrochloride solution) onto the sample pad of the ECL lateral flow immunoassay strip, and then dry it at 37°C for 1 hour.

[0066] B. Mix 30 μL of BIS-TRIS methane solution with 30 μL of labeled antibody in equal volume and spray the mixture onto the conjugate pad of the ECL lateral flow immunoassay strip, then dry at 37°C for 1 hour.

[0067] C. First, spray 30 μL of labeled antibody onto the conjugate pad of the ECL lateral flow immunoassay strip and dry it at 37°C for 1 hour to fix the labeled antibody. Then, spray 30 μL of BIS-TRIS methane solution onto the conjugate pad of the ECL lateral flow immunoassay strip and dry it at 37°C for 1 hour.

[0068] Example 2

[0069] The method for drying and fixing intermolecular ECL co-reactants in Example 1 was optimized.

[0070] 1. The co-reactant was BIS-TRIS methane, with concentrations set at 30 mM and 70 mM, respectively. The buffer was PBS (0.1 M) with a pH of 7.2–7.4, and the luteinizing hormone (LH) target concentration was 1 mIU / mL. -1 The volume of the sample solution to be tested was 30 μL, the driving voltage was 11 V, and the immunoreaction time was 6.5 min.

[0071] 2. The methods for drying and fixing the co-reactant BIS-TRIS methane are the three methods A, B, and C in Example 1.

[0072] 3. Experimental results are as follows Figure 1 As shown.

[0073] The experimental results show that when the BIS-TRIS methane concentration is 30mM and 70mM, method A has the highest ECL signal intensity on the C-line, the highest ECL signal intensity on the T-line, and the highest T / C ratio. Method C...

[0074] Method B is the worst. This may be because drying the co-reactant and labeled antibody in the same location can dilute the labeled antibody, leading to a decrease in ECL signal intensity. Therefore, method A is preferred, and any of the three acceptable methods for drying and fixing the intermolecular ECL co-reactant are acceptable.

[0075] Example 3

[0076] Optimize the influencing factors of method A

[0077] (1) Optimization of intermolecular ECL co-reactant concentration

[0078] 1. The buffer solution is PBS (0.1M), with a pH of 7.2–7.4, and the LH target concentration is 1 mIU / mL. -1 The volume of the sample solution to be tested was 30 μL, the driving voltage was 11 V, and the immune reaction time was 6.5 min.

[0079] 2. Several control groups were set up: the BIS-TRIS methane concentration was set to several different values ​​(10mM, 30mM, 50mM, 70mM, 90mM and 110mM).

[0080] 3. Experimental results are as follows Figure 2 As shown.

[0081] The experimental results show that as the BIS-TRIS methane concentration increases from 10 mM to 90 mM, the ECL signal intensity on both the C and T lines generally increases. At 90 mM, the ECL signal intensity on both the C and T lines reaches its maximum, the T / C ratio stabilizes, and the coefficient of variation is minimal. However, as the BIS-TRIS methane concentration further increases to 110 mM, both the ECL signal intensity on the C and T lines and the T / C ratio decrease. This phenomenon may be due to the production of byproducts at excessively high BIS-TRIS methane concentrations, which reduce the ECL signal intensity. Therefore, a BIS-TRIS methane concentration of 90 mM is preferred, with an acceptable range of 50–110 mM.

[0082] (2) pH optimization of ddH2O for dissolving BIS-TRIS methane

[0083] 1. BIS-TRIS methane concentration 90mM, buffer solution ddH2O, LH target concentration 1mIU / mL -1 The volume of the sample solution to be tested was 30 μL, the driving voltage was 11 V, and the immune reaction time was 6.5 min.

[0084] 2. Set up several control groups: set the pH value of ddH2O to several different values ​​(5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0 and 8.5).

[0085] 3. Experimental results are as follows Figure 3 As shown.

[0086] The experimental results show that when the pH value is between 5.0 and 7.0, the ECL signal intensity on both the C-line and T-line gradually increases with increasing pH value; however, as the pH value gradually increases to 8.5, the ECL signal intensity on both the C-line and T-line gradually decreases. Simultaneously, when the pH value changes from 5.0 to 6.5, the T / C ratio gradually increases; as the pH value further increases, the T / C ratio fluctuates between 1.0 and 1.2. Therefore, a pH value of 6.5 is preferred for ddH2O, with an acceptable range of 6.0–8.0.

[0087] (3) Optimization of pH value of PB (0.1M) for dissolving BIS-TRIS methane

[0088] 1. The BIS-TRIS methane concentration is 90 mM, the buffer is PB (0.1 M), and the LH target concentration is 1 mIU / mL. -1 The volume of the sample solution to be tested was 30 μL, the driving voltage was 11 V, and the immune reaction time was 6.5 min.

[0089] 2. Set up several control groups: The pH value of PB was set to several different values ​​(5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0 and 8.5).

[0090] 3. Experimental results are as follows Figure 4 As shown.

[0091] The experimental results show that when the pH value is between 5.0 and 7.0, the ECL signal intensity on both the C-line and T-line gradually increases with increasing pH value; however, as the pH value gradually increases to 8.5, the ECL signal intensity on both the C-line and T-line gradually decreases. Meanwhile, the T / C ratio reaches its peak at pH 7 and a second peak at pH 8.0. Therefore, a pH value of 7.0 is preferred for PB (0.1M), with an acceptable range of 6.5–8.0.

[0092] (4) pH optimization of PBS (0.1M) for dissolving BIS-TRIS methane

[0093] 1. The BIS-TRIS methane concentration was 90 mM, the buffer was PBS (0.1 M), and the LH target concentration was 1 mIU / mL. -1 The volume of the sample solution to be tested was 30 μL, the driving voltage was 11 V, and the immune reaction time was 6.5 min.

[0094] 2. Set up several control groups: The pH value of PBS was set to several different values ​​(5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0 and 8.5).

[0095] 3. Experimental results are as follows Figure 5 As shown.

[0096] The experimental results show that the ECL signal intensity is weak under acidic and near-neutral conditions. As the pH increases from 5.0 to 7.5, the ECL signal intensity on both the C and T lines gradually increases, reaching its peak at pH 7.5, where the T / C ratio is also at its maximum. At pH 8.0, the ECL signal intensity on the C line reaches its maximum, while the ECL signal intensity on the T line begins to decrease. As the pH gradually increases to 8.5, both the ECL signal intensity on the C and T lines, as well as the T / C ratio, gradually decrease. Therefore, a pH of 7.5 is preferred for PBS (0.1M), with an acceptable range of 6.0–8.5.

[0097] like Figure 6 As shown, compared to ddH2O and PB under optimal pH conditions, PBS (0.1M) can achieve a relatively large T / C at the optimal pH value. Therefore, the following examples all use PBS (0.1M, pH 7.5) as the buffer.

[0098] Example 4

[0099] The optimization results from Example 3 were used to optimize different types of intermolecular ECL co-reactants.

[0100] 1. The co-reactant concentration was 90 mM, the buffer was PBS (0.1 M), the pH of the PBS was 7.5, and the LH target concentration was 1 mIU / mL. -1 The volume of the sample solution to be tested was 30 μL, the driving voltage was 11 V, and the immune reaction time was 6.5 min.

[0101] 2. Several control groups were set up: the intermolecular ECL co-reactants were several different types (BIS-TRIS methane, BIS-TRIS propane, BIS-TRIS hydrochloride).

[0102] 3. Experimental results are as follows Figure 7 As shown.

[0103] The experimental results show that when the intermolecular ECL co-reactant is BIS-TRIS methane, the ECL signal intensity is highest on both the C and T lines, followed by BIS-TRIS hydrochloride, and lowest on BIS-TRIS propane. However, the corresponding T / C ratio shows the opposite trend: BIS-TRIS methane is the lowest, followed by BIS-TRIS hydrochloride, and highest on BIS-TRIS propane. Analysis of this phenomenon reveals that different types of co-reactants have a greater impact on the ECL signal intensity on the C line than on the T line, resulting in a greater attenuation of the ECL signal intensity on the C line than on the T line. This leads to a gradual decrease in ECL signal intensity on both the C and T lines, while the T / C ratio increases. The possible reasons for this phenomenon are analyzed below.

[0104] BIS-TRIS methane and BIS-TRIS hydrochloride are tertiary amines and both contain two hydroxyethyl groups (-CH2CH2OH). Figure 8 As shown, hydroxyethyl promotes amine oxidation and significantly increases ECL signal intensity. BIS-TRIS propane, being a secondary amine, has a lower oxidation rate than tertiary amines, which may explain the lower ECL signal intensity corresponding to BIS-TRIS propane. Furthermore, the difference in ECL signal intensity between BIS-TRIS methane and BIS-TRIS hydrochloride can be attributed to the presence of HCl in BIS-TRIS hydrochloride, which makes the original buffer solution slightly acidic. This difference is addressed in the pH optimization of the PBS buffer. Figure 5 This can be verified in [the study]. Therefore, the preferred intermolecular ECL co-reactant is BIS-TRIS methane, and acceptable types are BIS-TRIS methane and BIS-TRIS hydrochloride.

[0105] Example 5

[0106] The optimization results from Example 4 were used to compare the ECL signal intensity under dry and wet conditions.

[0107] 1. The BIS-TRIS methane concentration was 90 mM, the buffer was PBS (0.1 M), the PBS pH was 7.5, and the LH target concentration was 1 mIU / mL. -1 The volume of the sample solution to be tested was 30 μL, the driving voltage was 11 V, and the immune reaction time was 6.5 min.

[0108] 2. Set up several control groups: Set up two different BIS-TRIS methane treatment methods (dry method is Method A, wet method is to add the test sample solution and BIS-TRIS methane solution to the sample pad in sequence).

[0109] 3. Experimental results are as follows Figure 9 As shown.

[0110] The experimental results show that when using the wet method, the ECL signal intensity on the C line is 7.71 × 10⁻⁶. 6 The ECL signal strength on the T line is 10.49 × 10. 6 The T / C ratio is 1.36; when using the dry method, the ECL signal strength on the C line is 8.17 × 10⁻⁶. 6 The ECL signal strength on the T line is 12.72 × 10⁻⁶. 6The T / C ratio is 1.56. This shows that the ECL signal intensity and T / C on the C and T lines corresponding to the dry method are superior to those of the wet method. The possible reason for this phenomenon is that drying and fixing the intermolecular ECL co-reactants is more conducive to the redox reaction between Ru(II) and the co-reactants. Therefore, the dry method is the preferred methane treatment method for BIS-TRIS.

[0111] Example 6

[0112] The labeled antibody described in Example 1 was synthesized and purified.

[0113] First, 7.78 mg EDC, 1.15 mg NHS, and 3.2 mg Ru(II) were dissolved in 500 μL of 0.1 M PBS with a pH of 7.2-7.4. The solution was mixed using a micro vortex mixer (model XW-80A) and then placed on a constant temperature shaker (model ZWY-103B) at 25 °C for 30 min to activate the carboxyl group, thus obtaining carboxyl-activated Ru(II). Next, the carboxyl-activated Ru(II) was mixed evenly with 500 μL of antibody solution at a concentration of 0.5 mg / mL and placed on a constant temperature shaker at 25 °C. After reacting for several hours, the Ru(II)-antibody complex was obtained.

[0114] The synthesized Ru(II)-antibody complex was purified by dextran gel column chromatography to remove impurities, and the eluent (400 μL per tube) was collected to obtain pure Ru(II)-antibody complex. The pure Ru(II)-antibody complex was placed in a vacuum freeze dryer and freeze-dried at -80℃ for 12 h. The freeze-dried complex was in the form of dry powder, and the dry powder was dissolved in 80 μL, 40 μL, and 27 μL of PBS (0.1M) to obtain Ru(II)-labeled antibodies with concentrations of 5-fold, 10-fold, and 15-fold, respectively.

[0115] Example 7

[0116] Optimization of factors affecting the synthesis and purification processes in Example 6

[0117] (1) Optimize the amide linkage time between Ru(II) and the antibody.

[0118] 1. The masses of EDC, NHS, and Ru(II) were 7.78 mg, 1.15 mg, and 3.2 mg, respectively. The antibody concentration was 0.5 mg / mL. The volumes of PBS (0.1 M, pH 7.2-7.4) used to dissolve Ru(II) and prepare the antibody solution were 500 μL each. The temperature of the constant temperature shaker was set to 25℃.

[0119] 2. Set up several control groups: The amide linkage time between Ru(II) and the antibody was set to several different values ​​(0h, 1h, 2h, 3h, 4h, 5h, 6h, 7h, 8h, 9h, 10h).

[0120] 3. Experimental results are as follows Figure 10 As shown.

[0121] The experimental results show that: when the amide linkage time is between 0 and 3 hours, the ECL signal intensity corresponding to the Ru(II)-antibody complex decreases with increasing time; when the amide linkage time is 4 hours, the obtained ECL signal intensity shows a slight rebound, with the smallest error bar and the smallest coefficient of variation; when the amide linkage time is between 5 and 7 hours, the ECL signal intensity is lower than that at 4 hours; when the amide linkage time is 8 hours, the ECL signal intensity reaches its peak, but the error is large; as the amide linkage time further increases, the ECL signal intensity gradually decreases. Therefore, the preferred amide linkage time is 4 hours, and the acceptable range is 2–7 hours.

[0122] (2) Optimization of the concentration factor of Ru(II)-antibody complex

[0123] 1. Each tube of Ru(II)-antibody complex collected had a volume of 400 μL. The dry powder was dissolved in 80 μL, 40 μL, and 27 μL of PBS (0.1M, pH 7.2-7.4) to restore it to liquid state.

[0124] 2. Set up several control groups: the concentration factor is different (5 times, 10 times, 15 times).

[0125] 3. Experimental results are as follows Figure 11 As shown.

[0126] The experimental results show that as the concentration factor increases from 5 to 10, the corresponding ECL signal intensity ratio increases. However, as the concentration factor further increases to 15, the corresponding ECL signal intensity ratio decreases. Therefore, a concentration factor of 10 is preferred, and the acceptable range is 5 to 15.

[0127] Example 8

[0128] Preprocessing, assembly, and testing of the ECL chip in Example 1

[0129] The ECL chip used in this embodiment is an ECL lateral flow immunoassay strip, which is prepared by the following steps:

[0130] The C-line and T-line labeled antibodies synthesized and purified using the optimized parameters of Example 7 were mixed at a volume ratio of 1:1 to obtain a labeled antibody mixture. Then, the mixture was sprayed onto the conjugate pad using the gold spraying mode of a gold spraying membrane apparatus and dried in an oven at 37°C for 1 hour. Using the optimized parameters of Method 1 in Examples 1 to 5, the BIS-TRIS methane solution was uniformly dried and fixed onto the sample pad. At the same time, the T-line capture antibody and C-line capture antibody were sprayed onto the detection pad using the gold spraying membrane apparatus and dried in an oven at 37°C for 2 hours.

[0131] The processed sample pad, conjugate pad, and detection pad are used to assemble an ECL lateral flow immunoassay strip. The ECL lateral flow immunoassay strip specifically includes a lateral flow strip 1, an electrode pad 2, a connecting pad 3, and a hydrophobic substrate 4. Figure 12 The lateral flow test strip 1 includes a sample application pad 1-1, a conjugate pad 1-2, a detection pad 1-3, a C-line 1-3-1, a T-line 1-3-2, an absorbent pad 1-4, and a base plate 1-5; the electrode pad 2 includes a negative driving electrode 2-1, an integrated closed bipolar electrode 2-2 (including a control anode 2-2-1 and a detection anode 2-2-2), a shared cathode 2-3, and a positive driving electrode 2-4; the lateral flow test strip 1 is placed upside down on the electrode pad 2, with the connecting pad located on the hydrophobic base plate between the shared cathode 2-3 and the positive driving electrode 2-4, and partially in contact with the anode driving electrode 2-4.

[0132] The assembled ECL lateral flow immunoassay strip is placed into the cartridge, which is then placed into the tray of the point-of-care testing instrument for immunoassay of the LH target. Figure 1330 μL of the test sample solution containing the target is added dropwise to the sample pad. The solution carries the BIS-TRIS methane on the sample pad towards the binding pad. The LH target binds to the T-line labeled antibody on the binding pad to form an "LH target-T-line labeled antibody" complex. As the solution flows, this complex and BIS-TRIS methane continue to flow towards the detection pad, forming a "T-line capture antibody-LH target-T-line labeled antibody" sandwich immune complex on the T-line and a "C-line capture antibody-C-line labeled antibody" complex on the C-line. This process continues for approximately 3.5 minutes. Next, 30 μL of PBS (0.1 M, pH 7.2–7.4) was added to the sample pad, and the immunoreaction was further carried out for about 3 minutes. At the same time, excess labeled antibody was washed into the absorbent pad, which was used to connect the negative driving electrode and the detection anode. Simultaneously, 30 μL of PBS (0.1 M, pH 7.2–7.4) solution was added to the connection pad to connect the positive driving electrode and the shared cathode. The driving electrode of the electrode pad was electrically connected to the ECL point detection instrument, and the ECL reaction was triggered at an 11V driving voltage. During the reaction, the CMOS camera acquired the ECL signal intensity on the T line and C line, respectively, and the ratio (i.e., T / C) was used to quantitatively analyze the LH target.

[0133] Example 9

[0134] The LH target was quantitatively detected using the ECL lateral flow immunoassay strip described in Example 8.

[0135] 1. The intermolecular ECL co-reactant was BIS-TRIS methane at a concentration of 90 mM, the buffer was PBS (0.1 M) with a pH of 7.5, the volume of the sample solution was 30 μL, the driving voltage was 11 V, and the immunoreaction time was 6.5 min.

[0136] 2. Several control groups were set up: LH target concentrations were 0 mIU / mL. -1 0.001 mIU mL -1 0.005 mIU mL -1 0.01 mIU mL -1 0.1 mIU mL -1 1 mIU mL -1 1.5 mIU mL -1 and 2mIU mL -1 .

[0137] 3. Experimental results are as follows Figure 14 and Figure 15 As shown.

[0138] The experimental results show that as the LH target concentration gradually increases, the T / C ratio also gradually increases, and there is a direct linear relationship between the two. The linear fitting equation is Y = 1.2365X + 0.1729 (where Y represents T / C, and X represents the LH target concentration ([LH]) / mIU mL). -1 Correlation coefficient R 2 =0.9967. Furthermore, a double logarithmic linear fit exists between T / C and LH target concentration, with the linear fitting equation being Y = 0.3874X + 0.2366 (where Y represents Lg{T / C}, and X represents Lg{[LH] / mIU mL)). -1}), correlation coefficient R 2 =0.9928. Based on the linear fitting equation above, the detection limit is estimated to be 0.6975 μIU / mL. -1 1.3678 μIU mL -1 The detection limit is calculated as follows: Y L =Y b +3S b , where Y b S represents the average T / C value in the blank control group. b The standard deviation (in 5 replicates) of the blank control is expressed using the obtained Y. L The corresponding LH concentration is calculated and is the detection limit.

[0139] The intermolecular ECL co-reactant drying and immobilization method, the Ru(II)-antibody complex synthesis method as a labeled antibody, and the dextran gel column chromatography purification method proposed in this invention enable more convenient and accurate detection of characteristic targets, reducing the detection limit from 0.0496 mIU / mL in existing technologies. -1 Reduced to 0.6975 μIU / mL -1 The detection accuracy has been improved by two orders of magnitude.

[0140] 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. A method for drying and fixing intermolecular ECL co-reactants, characterized in that... Includes the following steps: The co-reactant solution is sprayed onto the sample pad of the ECL chip and dried to obtain the final product. The co-reactant is one or more of bis(2-hydroxyethyl)iminotris(hydroxymethyl)methane, 1,3-bis(tris(hydroxymethyl)methylamino)propane, or bis(2-hydroxyethyl)amino-tris(hydroxymethyl)methane.

2. The drying and fixation method according to claim 1, characterized in that: The concentration of the co-reactant solution is 10–110 mM.

3. The drying and fixation method according to claim 1, characterized in that: The pH value of the co-reactant solution is 5.0 to 8.

5.

4. The application of the drying and fixation method according to any one of claims 1 to 3 in the preparation of ECL chips.

5. The application according to claim 4, characterized in that: The ECL chip refers to a device that utilizes the ECL phenomenon generated by the electrochemical reaction between ruthenium complexes and their co-reactants to perform qualitative and / or quantitative analysis of the biomarkers to be tested by collecting and analyzing the generated ECL signals.

6. The application according to claim 5, characterized in that: The ECL chip described above comprises electrodes, a microfluidic reaction cell, and a microchannel.

7. The application according to claim 6, characterized in that: The microfluidic reaction cell includes a binding pad and a detection pad.

8. The application according to claim 6, characterized in that: The microchannel includes a sample application pad, a binding pad, a detection pad, and an absorbent pad.

9. The application according to claim 5, characterized in that: The ECL chip includes a three-electrode ECL chip and a bipolar ECL chip.

10. The application according to claim 9, characterized in that: The bipolar ECL chip includes an ECL test strip and an ECL cloth chip.

11. The application according to claim 10, characterized in that: The ECL test strips include ECL lateral flow immunoassay strips.

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