Use of a carboxyl-modified trispyridine ruthenium complex in electrochemiluminescence immunoassays for non-diagnostic or non-therapeutic purposes

Abstract

The application discloses application of a carboxyl-modified terpyridine ruthenium complex in electrochemiluminescence immunoassay for non-diagnostic or non-therapeutic purposes, and belongs to the field of high polymer synthesis chemistry.The carboxyl-modified terpyridine ruthenium complex is shown as follows: The application further includes a preparation method of the carboxyl-modified terpyridine ruthenium complex, which has low preparation cost, simple purification and high efficiency.The terpyridine ruthenium complex can be used for specific modification of proteins due to the introduction of the carboxyl group, and can also be used as an electrochemiluminescence reagent.The terpyridine ruthenium complex is efficiently labeled with proteins by using DMT-MM as a condensing agent, and has high labeling efficiency, simple operation and low cost, and has a wide application prospect in the field of labeled immunoassay.

Description

Technical Field

[0001] This invention relates to the field of polymer synthetic chemistry, and in particular to the application of a carboxyl-modified terpyridine ruthenium complex in electrochemiluminescence immunoassay for non-diagnostic or non-therapeutic purposes. Background Technology

[0002] Electro-Chemiluminescence immunoassay (ECLIA) is a labeled immunoassay method that combines highly sensitive electrochemiluminescence with highly specific immunoreaction. ECLIA features high sensitivity, a wide linear range, long label validity, no radioactivity, and ease of automation, and is widely used in clinical diagnostics, food, environmental protection, and military fields for the detection of proteins, hormones, fatty acids, vitamins, drugs, viruses, and other components.

[0003] Ruthenium terpyridine, a commonly used electrochemiluminescent agent, possesses high luminescence intensity, high quantum yield, and good stability, making it one of the most widely applied electrochemiluminescence systems. Currently, commercially available ruthenium terpyridine is modified with N-hydroxysuccinimide ester, enabling direct coupling with proteins. However, N-hydroxysuccinimide-modified ruthenium terpyridine is prone to hydrolysis during synthesis, resulting in low yields. Summary of the Invention

[0004] The purpose of this invention is to provide a carboxyl-modified terpyridine ruthenium complex. By introducing a carboxyl group, it can be used for specific protein modification and as an electrochemiluminescence reagent. The terpyridine ruthenium complex of this invention has the advantages of low preparation cost, simple purification, and high efficiency.

[0005] One objective of this invention is to provide a carboxyl-modified terpyridine ruthenium complex, the structural formula of which is shown below;

[0006] ;

[0007] The second objective of this invention is to provide a method for preparing the above-mentioned carboxyl-modified terpyridine ruthenium complex, comprising the following steps:

[0008] Step 1: Preparation of ruthenium intermediate: (2,2'-bipyridine)-4,4'-disulfonate and ruthenium trichloride (RuCl3) were mixed in dimethylformamide (DMF) and lithium chloride (LiCl), and the mixture was heated and stirred under nitrogen protection. After cooling, the mixture was added to tertiary methyl ether at room temperature, and centrifuged to obtain a dark purple solid, which is the ruthenium intermediate.

[0009] Step 2: The ruthenium intermediate from Step 1 is mixed with 4-methyl-(2,2'-bipyridine)-4-hexanoic acid in ethanol (EtOH). The mixture is heated and stirred under nitrogen protection and refluxed. After cooling, excess acid is added at room temperature, and excess acetone is added while stirring. The resulting orange-red flocculent precipitate is collected, dissolved in a small amount of water, evaporated to dryness, and purified by column chromatography to obtain the carboxyl-modified terpyridine ruthenium complex.

[0010] In step one, the molar ratio of (2,2'-bipyridine)-4,4'-disulfonate to RuCl3 is 2:1.

[0011] In step one, each millimole of RuCl3 corresponds to 5-6 mL of DMF.

[0012] In step one, the molar amount of LiCl is 6–7 mmol.

[0013] The heating temperature in step one is 140-145℃.

[0014] The reflux time in step one is 20 to 24 hours.

[0015] In step two, the molar ratio of the ruthenium intermediate to 4-methyl-(2,2'-bipyridine)-4-hexanoic acid is 1:1.2 mmol.

[0016] In step two, each millimole of ruthenium intermediate corresponds to 1.6 to 1.8 mL of EtOH.

[0017] The heating temperature in step two is 80-85℃.

[0018] The reflux time in step two is 20 to 24 hours.

[0019] The acid added in step two is preferably one of ammonium hexafluorophosphate (NH4PF6) or trifluoroacetic acid.

[0020] The third objective of this invention is to provide a method for labeling proteins with carboxyl-modified terpyridine ruthenium complexes, comprising the following steps:

[0021] (1) Dissolve the carboxyl-modified terpyridine ruthenium complex in morpholine ethanesulfonic acid (MES) buffer, add 4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholineonium chloride hydrate (DMT-MM) and the protein to be labeled, mix thoroughly and incubate with shaking;

[0022] (2) Add the reaction solution to an ultrafiltration centrifuge tube and centrifuge to purify it;

[0023] (3) Collect ruthenium tripyridine and protein markers, i.e. ruthenium-labeled proteins, from the filter membrane of ultrafiltration centrifuge tubes.

[0024] The MES buffer solution is 100 mM and pH 6.0.

[0025] The concentration of DMT-MM is 200 mg / mL, and it is added at a molar ratio of 25:1 between the terpyridine ruthenium complex and DMT-MM.

[0026] The protein to be labeled can be an antigen, antibody, streptavidin, or other detection proteins used in the field of biotechnology, and the initial concentration of the protein to be labeled is 2~20 mg / mL.

[0027] The protein to be labeled was added at a molar ratio of 1:150 to the carboxyl-modified terpyridine ruthenium complex, and the final concentration after adding the protein to be labeled was 1~2 mg / mL.

[0028] The shaking incubation conditions were 25℃, 700~800rpm, and 2~3h.

[0029] The molecular rejection rate of the ultrafiltration centrifuge tube membrane is 30 kDa.

[0030] The centrifugation speed was 1000 g, and the time was 8 to 10 minutes.

[0031] This invention also relates to the application of the carboxyl-modified terpyridine ruthenium complex in electrochemiluminescence immunoassay.

[0032] In this application, 4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholinium chloride hydrate (DMT-MM) was used as a condensing agent, and the carboxyl-modified terpyridine ruthenium complex was used to efficiently label the protein to obtain ruthenium-labeled protein.

[0033] The application in electrochemiluminescence immunoassay involves the following steps:

[0034] S1, the capture protein was added to the graphene electrode plate and incubated overnight at 4°C;

[0035] S2, remove uncoated capture proteins through a washing step, add blocking solution, and block at room temperature;

[0036] S3, remove excess blocking solution through the washing step, add the protein to be tested, and incubate with shaking at room temperature;

[0037] S4, remove unbound test protein through a washing step, add ruthenium-labeled protein, and incubate with shaking at room temperature;

[0038] S5, the free ruthenium-labeled protein is removed by a washing step, tri-n-propylamine is added as a photogenerating co-reactant, and the signal is detected in an electrochemiluminescence analyzer.

[0039] The coating diluent was 10 mM PBS buffer, pH 7.2.

[0040] The coating concentration of the capture protein is 1–20 μg / mL.

[0041] The incubation time for the coating is 16–20 hours.

[0042] The washing solution was PBST buffer (10 mM PBS, 0.05% Tween-20, pH 7.2).

[0043] The blocking solution consisted of 10 mM PBS, 3% BSA, and pH 7.2.

[0044] The closure period is 1 to 2 hours.

[0045] The shaking incubation conditions are 500-600 rpm for 1-2 hours.

[0046] The mass percentage of tri-n-propylamine added is 5% to 6%.

[0047] The present invention has the following beneficial effects:

[0048] The carboxyl-modified terpyridine ruthenium complex of this invention is formed through coordination of a polypyridine ligand with a ruthenium(II) center. This complex exhibits a strong absorption band in aqueous solution and emits orange-red light in the visible region, demonstrating a large Stokes shift and good luminescence quantum yield. Due to the introduction of the carboxyl group, the terpyridine ruthenium complex of this invention can be specifically modified into proteins. This complex possesses unique photophysical properties and the advantage of easy group modification, providing new possibilities for applications in chemical sensing, biological detection, and materials science.

[0049] This invention utilizes DMT-MM as a condensing agent to achieve efficient labeling of terpyridine ruthenium complexes with proteins and applies it to electrochemiluminescence immunoassay. This method has the advantages of high labeling efficiency, simple operation, and low cost, and has broad application prospects in the field of labeled immunoassay. Attached Figure Description

[0050] Figure 1 The 1H NMR spectrum of the carboxyl-modified terpyridine ruthenium complex;

[0051] Figure 2 The carbon NMR spectrum of the carboxyl-modified terpyridine ruthenium complex;

[0052] Figure 3 This is a dose-response curve for the labeled protein. Detailed Implementation

[0053] To facilitate understanding by those skilled in the art, the present invention will be further described below with reference to embodiments. The content mentioned in the embodiments is not intended to limit the present invention.

[0054] Example 1

[0055] Preparation of carboxyl-modified terpyridine ruthenium complexes.

[0056] It is synthesized through a two-step reaction, and the reaction equation is shown below:

[0057]

[0058]

[0059] Step 1: (2,2'-bipyridine)-4,4'-disulfonate (2.00 mmol) and RuCl3 (1 mmol) were mixed in DMF (6 ml, 99.5%) and LiCl (6.0 mmol). The mixture was stirred vigorously with a magnetic stirrer and gradually heated to 143 °C, then refluxed under nitrogen for 24 h. The reaction mixture was then allowed to cool naturally, and at room temperature, it was added to methyl ether. The mixture was centrifuged and dried to obtain a dark purple solid, which is the ruthenium intermediate Ru(L1)2Cl2, where L1 is the (2,2'-bipyridine)-4,4'-disulfonate ligand. The structural formula of the ruthenium intermediate is shown below:

[0060]

[0061] Step 2: The ruthenium intermediate (1.00 mmol) from Step 1 was mixed with 4-methyl-(2,2'-bipyridine)-4-hexanoic acid (1.2 mmol) in EtOH (1.6 mL, 99.5%). The mixture was stirred vigorously with a magnetic stirrer and heated to 80 °C, then refluxed under nitrogen for 24 h. The reaction mixture was then allowed to cool naturally to room temperature. 3 mmol of NH4PF6 and excess acetone were added with stirring. The resulting orange-red flocculent precipitate was collected, dissolved in a small amount of water, and evaporated to dryness. The final product was obtained by column chromatography at an ACN:H2O ratio of 10:1, yielding the carboxyl-modified terpyridine ruthenium complex. The structural formula is shown below:

[0062] ;

[0063] Appendix Figure 1 and Figure 2 The proton and carbon NMR spectra of the carboxyl-modified terpyridine ruthenium complex are shown.

[0064] Hydrogen spectrum data: 1 H NMR (400 MHz, D2O) δ 8.59 (s, 4H), 8.35 (d, J = 6.4 Hz,2H), 7.93 – 7.78 (m, 4H), 7.67 (dd, J = 11.8, 5.8 Hz, 2H), 7.41 (q, J = 5.8Hz, 4H), 7.28 – 7.18 (m, 2H), 4.37 (s, 8H), 3.59 (s, 1H), 2.81 (t, J = 7.4Hz, 2H), 2.52 (s, 3H), 2.42 – 2.29 (m, 2H), 1.77 – 1.55 (m, 4H), 1.44 – 1.28(m, 2H).

[0065] Carbon spectrum data: 13 C NMR (101 MHz, D2O) δ 177.35, 157.01, 156.94, 156.84,151.48, 151.14, 150.78, 150.65, 150.34, 142.87, 142.63, 128.66, 127.38,125.59, 123.91, 55.78, 52.64, 52.00, 34.53, 34.21, 33.43, 28.77, 27.43,23.89, 20.42.

[0066] The data above confirms that the structure of the prepared compound is correct.

[0067] Example 2

[0068] Labeling of carboxylated terpyridine ruthenium complex with mouse anti-human κ light chain antibody.

[0069] Dilute DMT-MM to a working solution of 200 mg / mL with 100 mM MES buffer (pH 6.0). Weigh out sufficient amount of the ruthenium tripyridine complex according to the amount of ruthenium tripyridine complex to be used, and dilute it to a working solution of 10 mg / mL (10 mM) with 100 mM MES buffer (pH 6.0). Add sufficient 200 mg / mL DMT-MM working solution (freshly prepared) at a molar ratio of ruthenium tripyridine complex to DMT-MM of 25:1, and vortex to mix. Add sufficient amount of mouse anti-human κ light chain antibody to be labeled at a molar ratio of 1:150 to ruthenium tripyridine complex, and dilute the added mouse anti-human κ light chain antibody to be labeled to 2 mg / mL with 100 mM MES buffer (pH 6.0), vortex to mix, and incubate at 25°C in the dark with shaking at 800 rpm for 2 h. After the reaction is complete, transfer the above mixture to a 30 kDa ultrafiltration centrifuge tube and centrifuge at 1000 g for 10 min at 4°C. Discard the filtrate at the bottom of the tube (Note: retain the solution in the intermediate filter tube; do not discard it). Add 2 mL of 10 mM PBS buffer (pH 7.2) to the intermediate filter tube and centrifuge at 1000 g for 10 min at 4°C. Discard the filtrate at the bottom of the tube. Repeat this step three times. Carefully collect all the solution in the intermediate filter tube (the labeled terpyridine ruthenium complex and protein conjugate solution is usually pale yellow) into a centrifuge tube. Add 200 μL of 10 mM PBS buffer (pH 7.2) to the intermediate filter tube to reabsorb the remaining conjugate (the carboxylated terpyridine ruthenium complex and the label of mouse anti-human κ light chain antibody, i.e., ruthenium-labeled antibody), and let stand for 2 min. Transfer all the solution in the intermediate filter tube into a centrifuge tube. The coupling agent should be stored at 2-8℃ away from light. For long-term storage, it should be stored at -20℃.

[0070] Example 3

[0071] 1. Application of carboxylated terpyridine ruthenium complex and mouse anti-human κ light chain antibody markers in electrochemiluminescence immunoassay:

[0072] (1) Dilute mouse anti-human IgG (Fc fragment) antibody to 2 μg / mL with 10 mM PBS buffer (pH 7.2), and add 100 μL to each well of the graphene electrode plate and incubate at 4 °C for 16 h.

[0073] (2) After coating is completed, discard the coating solution in each well and wash each well three times with 300 μL PBST (10 mM PBS, 0.05% Tween-20, pH 7.2) buffer.

[0074] (3) Add 200 μL of blocking buffer (10 mM PBS, 3% BSA, pH 7.2) to each well and block at room temperature for 2 h. After blocking, discard the blocking buffer in each well and wash each well 3 times with 300 μL of PBST buffer (10 mM PBS, 0.05% Tween-20, pH 7.2).

[0075] (4) Dilute the anti-PD-1 human antibody to 0, 0.1, 1, 10, and 100 ng / mL using 10 mM PBS, 1% Casein, and pH 7.2 buffer, and add 100 μL of each solution to the reaction wells. Incubate at room temperature for 1 h at 600 rpm. After the reaction is complete, discard the reaction solution in each well and wash each well three times with 300 μL of PBST (10 mM PBS, 0.05% Tween-20, pH 7.2) buffer.

[0076] (5) Dilute the carboxylated terpyridine ruthenium complex and the label of mouse anti-human κ light chain antibody (i.e., ruthenium-labeled antibody) 10,000 times with 10 mM PBS, 1% Casein, pH 7.2 buffer, and add 100 μL of the diluted ruthenium-labeled antibody to each well. Incubate at room temperature for 1 h at 600 rpm.

[0077] (6) After the reaction is complete, discard the reaction solution in each well and wash each well three times with 300 μL of PBST (10 mM PBS, 0.05% Tween-20, pH 7.2) buffer. Finally, add 100 μL of 5% tri-n-propylamine solution to each well of the plate and measure the electrochemiluminescence value of each well using an electrochemiluminescence analyzer.

[0078] 2. Application of N-hydroxysuccinimide esterified terpyridine ruthenium complex and mouse anti-human κ light chain antibody markers in electrochemiluminescence immunoassay.

[0079] The mouse anti-human κ light chain antibody to be labeled was mixed with the N-hydroxysuccinimide esterified terpyridine ruthenium complex at a molar ratio of 1:25 and incubated at 25°C in the dark with shaking at 800 rpm for 2 h. After the reaction was complete, the mixture was transferred to a 30 kDa ultrafiltration centrifuge tube and centrifuged at 1000 g for 10 min at 4°C, discarding the filtrate at the bottom of the tube. 2 mL of 10 mM PBS buffer (pH 7.2) was added to the intermediate filter tube, and the mixture was centrifuged at 1000 g for 10 min at 4°C, discarding the filtrate at the bottom of the tube. This step was repeated 3 times. All the solution in the intermediate filter tube was carefully collected into a centrifuge tube. 200 μL of 10 mM PBS buffer (pH 7.2) was added to the intermediate filter tube to reabsorb the remaining conjugate (the N-hydroxysuccinimide esterified terpyridine ruthenium complex and the mouse anti-human κ light chain antibody label), and the mixture was allowed to stand for 2 min. Add all the solution from the intermediate filter tube to the centrifuge tube. The conjugate should be stored at 2-8℃ in the dark. For long-term storage, it should be stored at -20℃.

[0080] The steps for the electrochemiluminescence immunoassay of N-hydroxysuccinimide esterified ruthenium tripyridine complex and mouse anti-human κ light chain antibody are the same as those for the electrochemiluminescence immunoassay of carboxylated ruthenium tripyridine complex and mouse anti-human κ light chain antibody, except that in step (5), the marker for the carboxylated ruthenium tripyridine complex and mouse anti-human κ light chain antibody is replaced with the marker for the carboxylated ruthenium tripyridine complex and mouse anti-human κ light chain antibody.

[0081] The electrochemiluminescence values ​​of N-hydroxysuccinimide esterified ruthenium terpyridine and the carboxylated ruthenium terpyridine labeled protein of this application were compared, and the dose-response curves are shown in the figure. Figure 3 As shown, carboxylated terpyridine ruthenium exhibits a better electrochemiluminescence signal than N-hydroxysuccinimide esterified terpyridine ruthenium at high concentrations. The carboxylated terpyridine ruthenium of this invention can label proteins under DMT-MM action, exhibiting high labeling efficiency and high luminescence efficiency, and can be used as an electrochemiluminescence reagent.

Claims

1. Use of a carboxyl-modified trispyridine ruthenium complex in an electrochemiluminescence immunoassay for non-diagnostic or non-therapeutic purposes, characterized in that, The method comprises the following steps: S1, adding capture protein into a graphene electrode plate and incubating the coating at 4℃ overnight; S2, removing uncoated capture protein through a washing step, adding blocking solution, and blocking at room temperature; S3, removing excess blocking solution through a washing step, adding the protein to be tested, and oscillating incubation at room temperature; S4, removing unbound protein to be tested through a washing step, adding ruthenium-labeled protein, and oscillating incubation at room temperature; S5, removing free ruthenium-labeled protein through a washing step, adding tri-n-propylamine as a light-generating co-reactant, and performing signal detection in an electrochemiluminescence analyzer; wherein the coating diluent is 10 mM PBS buffer, pH 7.2, and the coating concentration of the capture protein is 1-20 μg / mL; the coating incubation time is 16-20 h; wherein the washing solution is PBST buffer, and the PBST buffer is 10 mM PBS, 0.05% Tween-20, pH 7.2; wherein the blocking solution is 10 mM PBS, 3% BSA, pH 7.2; and the blocking time is 1-2 h; wherein the oscillating incubation condition is 500-600 rpm, 1-2 h; wherein the mass percentage of tri-n-propylamine added is 5%-6%; The structural formula of the carboxyl-modified terpyridine ruthenium complex is as follows: ； The ruthenium-labeled protein is obtained by efficiently labeling the carboxyl-modified terpyridine ruthenium complex with the protein, using 4-(4,6-dimethoxytriazin-2-yl)-4-methyl morpholinium chloride hydrate as a condensing agent.

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