Application of CTAB-Au NCs in antibacterial and / or enzymatic chemiluminescence analysis, substrate solution

Abstract

The present invention relates to the field of chemiluminescent immunoassay technology, and particularly to the use of CTAB-Au NCs in antibacterial and / or enzymatic chemiluminescent assays and a substrate solution. The present invention provides the use of CTAB-Au NCs in antibacterial and / or enzymatic chemiluminescent assays, a substrate solution, and the use of the substrate solution in preparing an enzymatic chemiluminescent assay kit. The chemiluminescent substrate solution of the present invention has the advantages of high luminescence intensity, a long luminescence plateau phase, high detection sensitivity, good stability, and long shelf life.

Description

Technical Field

[0001] The present invention relates to the technical field of chemiluminescent immunoassay, and in particular to application of CTAB-AuNCs in antibacterial and / or enzymatic chemiluminescent analysis and a substrate solution. Background Art

[0002] Chemiluminescent immunoassays have become the fastest-growing and most widely used immunoassay method, and are currently the most advanced labeled immunoassay technology. They offer advantages such as high sensitivity, strong specificity, stable reagents with a long shelf life, a stable and rapid method, a wide detection range, simple operation, and a high degree of automation. Chemiluminescent immunoassays have been used for the detection and analysis of a variety of antigens, haptens, antibodies, hormones, enzymes, fatty acids, vitamins, and drugs, and are currently the most ideal method for quantitative immunoassay analysis.

[0003] Commonly used luminescent substances in chemiluminescent immunoassays include luminol, isoluminol, acridinium esters, and 1,2-dioxetane compounds. Isoluminol and acridinium esters are directly labeled as tracer molecules, representing a flash-type chemiluminescent reaction. Luminol and 1,2-dioxetane compounds rely on enzymes as tracer molecules, representing an enzymatic glow-type chemiluminescent reaction. 1,2-dioxetane compounds are the latest ultrasensitive alkaline phosphatase substrates. They can be hydrolyzed by alkaline phosphatase in a suitable buffer, emitting a light signal that can last for over 20 hours, making them ideal chemiluminescent substances.

[0004] Currently, the most commonly used 1,2-dioxetane derivatives are AMPPD, CSPD, and CDP-Star. AMPPD, a 1,2-dioxetane derivative, is a new, ultrasensitive alkaline phosphatase substrate in biochemistry. It is also extremely stable. When stored in a solid state at low temperatures, AMPPD exhibits virtually no degradation and produces virtually no luminescent background. However, under the catalytic action of alkaline phosphatase, AMPPD undergoes hydrolysis of its phosphate group, forming an unstable intermediate, AMP-D. This intermediate undergoes intramolecular electron transfer, cleaving the -OO- four-membered ring into an adamantanone molecule and an excited methyl m-oxybenzoate anion. The excited methyl m-oxybenzoate anion generates light radiation as it transitions from the excited state to the ground state, stimulating a chemiluminescent reaction and emitting photons. The light quantum yield, which is proportional to the amount of the analyte in the sample, can be measured using a luminescence signal measurement instrument. This quantum yield can be used to establish a standard curve and calculate the analyte content in the sample.

[0005] However, in an aqueous system, the energy of AMPPD will be damaged into water molecules, resulting in low luminescence intensity. Currently, there are mainly methods such as adding surfactant-dye adducts, indirect luminescence, self-degradation and substrate modification to enhance the chemiluminescence signal of AMPPD. However, the chemiluminescence enhancement factors of these methods are relatively low, resulting in low precision of the measured luminescence values ​​and uneven luminescence rates. Summary of the Invention

[0006] In view of this, the present invention provides the use of CTAB-AuNCs in antibacterial and / or enzymatic chemiluminescent assays and a substrate solution. The present invention also provides the use of CTAB-AuNCs in antibacterial and / or enzymatic chemiluminescent assays, a substrate solution, and the use of the substrate solution in preparing an enzymatic chemiluminescent assay kit. Beneficial Effects of the Invention: The chemiluminescent substrate solution of the present invention has the advantages of high luminescence intensity, a long luminescence plateau phase, high detection sensitivity, good stability, and long shelf life.

[0007] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0008] The present invention provides the use of CTAB-AuNCs in antibacterial and / or enzymatic chemiluminescence analysis.

[0009] In some specific embodiments of the present invention, the preparation method of the CTAB-AuNCs comprises: taking MPA-AuNCs, CTAB and Tris-HCl buffer solution, shaking the reaction, and then centrifuging and washing in a 10KDa ultrafiltration centrifuge tube to obtain the CTAB-AuNCs;

[0010] The molar ratio of [Au] to the CTAB comprises 60:1;

[0011] The concentration of the Tris-HCl buffer solution includes 0.1M;

[0012] The shaking reaction includes reacting at 1000 rpm for 30 min;

[0013] The centrifugal conditions include centrifugation at 1000 rpm for 30 min.

[0014] In some specific embodiments of the present invention, the method for preparing MPA-AuNCs comprises the following steps:

[0015] Step 1: Mix 1 M NaOH and DMF, add 0.03 M HAuCl4·3H2O, and obtain AuNPs;

[0016] Step 2: Take the AuNPs described in step 1, mix them with 100 mM phosphate buffer solution (pH = 7.0) and 4 M MPA, keep them in the dark, centrifuge to remove undissolved thiol ligands, filter through a 0.22 μm filter membrane, and then centrifuge in a 10 KDa ultrafiltration centrifuge tube to obtain the MPA-AuNCs;

[0017] The time of standing still in the dark includes 4 hours;

[0018] The conditions for removing undissolved thiol ligands by centrifugation include centrifugation at 7000 rpm for 30 min;

[0019] The centrifugation conditions of the ultrafiltration centrifuge tube include centrifugation at 1000 rpm for 30 minutes.

[0020] In some specific embodiments of the present invention, the preparation method of the CTAB-AuNCs comprises: taking 150 μL of MPA-AuNCs, 200 μL of CTAB (50 μM) and 200 μL of Tris-HCl buffer solution, diluting the volume to 1 mL with ultrapure water, reacting at 1000 rpm for 30 minutes, and then centrifuging and washing at 10000 rpm for 30 minutes in a 10 KDa ultrafiltration centrifuge tube to obtain the CTAB-AuNCs;

[0021] The molar ratio of [Au] to CTAB is 60:1

[0022] The concentration of the Tris-HCl buffer solution is 0.1 M, and the pH value is 9.0.

[0023] In some specific embodiments of the present invention, the method for preparing MPA-AuNCs comprises the following steps:

[0024] Step 1: 1 M NaOH and DMF were mixed and stirred for 10 min, and 0.03 M HAuCl4·3H2O was added to obtain AuNPs;

[0025] Step 2: Take the AuNPs, 100 mM phosphate buffer solution (pH = 7.0) and 4 M MPA and mix them. Keep them in the dark for 4 hours, centrifuge them at 7000 rpm for 30 minutes to remove undissolved thiol ligands, filter them through a 0.22 μm filter membrane to remove Au NPs aggregates, and centrifuge them at 10000 rpm for 30 minutes in a 10 KDa ultrafiltration centrifuge tube to remove unreacted products to obtain the MPA-AuNCs.

[0026] In some specific embodiments of the present invention, the antibacterial activity includes inhibiting and / or lysing E. coli.

[0027] The present invention also provides a substrate solution comprising AMPPD, CTAB-AuNCs, an enzyme stabilizer and a buffer solution.

[0028] In some specific embodiments of the present invention, the preparation method of the CTAB-AuNCs comprises: taking MPA-AuNCs, CTAB and Tris-HCl buffer solution, shaking the reaction, and then centrifuging and washing in a 10KDa ultrafiltration centrifuge tube to obtain the CTAB-AuNCs;

[0029] The molar ratio of [Au] to the CTAB comprises 60:1;

[0030] The concentration of the Tris-HCl buffer solution includes 0.1M;

[0031] The shaking reaction includes reacting at 1000 rpm for 30 min;

[0032] The centrifugal conditions include centrifugation at 1000 rpm for 30 min.

[0033] In some specific embodiments of the present invention, the method for preparing MPA-AuNCs comprises the following steps:

[0034] Step 1: Mix 1 M NaOH and DMF, add 0.03 M HAuCl4·3H2O, and obtain AuNPs;

[0035] Step 2: Take the AuNPs described in step 1, mix them with 100 mM phosphate buffer solution (pH = 7.0) and 4 M MPA, keep them in the dark, centrifuge to remove undissolved thiol ligands, filter through a 0.22 μm filter membrane, and then centrifuge in a 10 KDa ultrafiltration centrifuge tube to obtain the MPA-AuNCs;

[0036] The time of standing still in the dark includes 4 hours;

[0037] The conditions for removing undissolved thiol ligands by centrifugation include centrifugation at 7000 rpm for 30 min;

[0038] The centrifugation conditions of the ultrafiltration centrifuge tube include centrifugation at 1000 rpm for 30 minutes.

[0039] In some specific embodiments of the present invention, the preparation method of the CTAB-AuNCs comprises: taking 150 μL of MPA-AuNCs, 200 μL of CTAB (50 μM) and 200 μL of Tris-HCl buffer solution, diluting the volume to 1 mL with ultrapure water, reacting at 1000 rpm for 30 minutes, and then centrifuging and washing at 10000 rpm for 30 minutes in a 10 KDa ultrafiltration centrifuge tube to obtain the CTAB-AuNCs;

[0040] The concentration of the Tris-HCl buffer solution is 0.1 M; and its pH value is 9.0.

[0041] In some specific embodiments of the present invention, the method for preparing MPA-AuNCs comprises the following steps:

[0042] Step 1: 1 M NaOH and DMF were mixed and stirred for 10 min, and 0.03 M HAuCl4·3H2O was added to obtain AuNPs;

[0043] Step 2: Take the AuNPs, 100 mM phosphate buffer solution (pH = 7.0) and 4 M MPA and mix them. Keep them in the dark for 4 hours, centrifuge them at 7000 rpm for 30 minutes to remove undissolved thiol ligands, filter them through a 0.22 μm filter membrane to remove Au NPs aggregates, and centrifuge them at 10000 rpm for 30 minutes in a 10 KDa ultrafiltration centrifuge tube to remove unreacted products to obtain the MPA-AuNCs.

[0044] In some specific embodiments of the present invention, the substrate solution comprises:

[0045]

[0046] In some specific embodiments of the present invention, the substrate solution comprises:

[0047]

[0048] In some specific embodiments of the present invention, the substrate solution comprises:

[0049]

[0050] In some embodiments of the present invention, the enzyme stabilizer comprises a magnesium salt and / or a zinc salt.

[0051] In some specific embodiments of the present invention, the magnesium salt comprises magnesium chloride; and the zinc salt comprises zinc chloride.

[0052] In some specific embodiments of the present invention, the buffer comprises one or more of a carbonate buffer, a diethanolamine buffer or 1-amino-2-methyl-1-propanol (AMP); or

[0053] The pH value of the substrate solution is 9.0-10.0.

[0054] The present invention also provides the use of the substrate solution in preparing an enzymatic chemiluminescence analysis kit.

[0055] The present invention also provides an enzymatic chemiluminescence analysis kit, which comprises the substrate solution.

[0056] In some embodiments of the invention, the enzyme comprises alkaline phosphatase.

[0057] The present invention includes but is not limited to the following beneficial effects:

[0058] The present invention creatively selects CTAB-coated AuNCs, which can not only enhance the chemiluminescence intensity, chemiluminescence stability and chemiluminescence reaction rate, but also has excellent antibacterial properties that can extend the shelf life of the reagent. In addition, it can also avoid the generation of a large number of bubbles, thereby avoiding the impact of bubbles on the luminescence value. The fully automatic immunoassay system containing CTAB-coated AuNCs uses a substrate solution. The CTAB on the surface of the AuNCs generates a large number of micelles to shape the internal hydrophobic microenvironment, thereby shortening the distance between the gold nanoclusters and the AMPPD substrate. This leads to light energy transfer. The quantum yield of AuNCs is much higher than that of chemiluminescent molecules, thereby greatly enhancing the chemiluminescence intensity; and through the large specific surface area, high surface and catalytic activity of AuNCs, the chemiluminescence stability is greatly improved and the chemiluminescence reaction rate is accelerated. The positively charged quaternary ammonium salt and long hydrophobic carbon chain on the CTAB adsorbed on the surface of the AuNCs are efficiently adsorbed on the negatively charged bacterial surface and destroy their cell membranes to achieve the sterilization effect, thereby extending the shelf life of the reagent. Therefore, the chemiluminescent substrate solution of the present invention has the advantages of high luminescence intensity, long duration of luminescence plateau, high detection sensitivity, good stability, long storage time, etc. BRIEF DESCRIPTION OF THE DRAWINGS

[0059] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for describing the embodiments or the prior art.

[0060] Figure 1 The standard curve shows that the volume of the required SDS standard solution changes with the concentration of CTAB;

[0061] Figure 2 coli suspension was plated on an agar plate and the bacterial colonies on the plate were imaged;

[0062] Figure 3 Shown are the changing curves of the killing process of E. coli in the presence of 5 μM CTAB, AuNCs and CTAB-AuNCs respectively;

[0063] Figure 4 The luminescence kinetic curves of the substrate solution of the present invention, the comparison substrate solution and the Beckmann chemiluminescence substrate solution under the catalysis of alkaline phosphatase (3 ng / mL) are shown;

[0064] Figure 5 The linear relationship curves between AP enzyme concentration and luminescence intensity of the substrate solution of the present invention, the comparison substrate solution and the Beckman chemiluminescence substrate solution under different AP enzyme concentration conditions are shown. DETAILED DESCRIPTION

[0065] The present invention discloses the use of CTAB-AuNCs in antibacterial and / or enzymatic chemiluminescent assays and substrate solutions. Those skilled in the art can refer to the contents herein and appropriately modify the process parameters to achieve the desired results. It is particularly important to note that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in the present invention. The methods and applications of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit, and scope of the present invention to implement and apply the technology of the present invention.

[0066] Gold nanoclusters (AuNCs) are clusters of gold atoms, typically less than 2 nm in size and composed of several to hundreds of gold atoms. They exhibit quantum confinement and surface effects similar to those of quantum dots, with high fluorescence intensity, strong photostability, and high surface activity. The chemiluminescence of AuNCs has been demonstrated and is widely used in the analysis and detection of plasma, urine, and tissue fluids. Furthermore, AuNCs are simple to synthesize and have good biocompatibility, making them suitable for practical in vitro applications. Therefore, modifying AuNCs with surfactants for in vitro chemiluminescence detection can both ensure detection accuracy and improve detection efficiency.

[0067] In order to overcome the deficiencies in the prior art, the present invention achieves enhanced chemiluminescence and antibacterial efficacy through CTAB-AuNCs. By utilizing the quantum confinement effect and surface effect of AuNCs, and combining its high fluorescence intensity, strong photostability, high surface activity, and easy binding to ligands, by using MPA-AuNCs as a template and utilizing the superposition effect of CTAB's hydrophobic interaction and electrostatic force, a double-layer CTAB-protected AuNCs is formed. The surface of AuNCs is coated with a large amount of positively charged CTAB. The internal hydrophobic microenvironment of the micelles formed by CTAB shortens the distance between AuNCs and the AMPPD substrate, allowing AuNCs to receive the energy transmitted by AMPPD, generating light energy transfer, and combining the quantum confinement characteristics of AuNCs to obtain a higher quantum yield, greatly enhancing the chemiluminescence intensity, thereby obtaining a substrate solution for a fully automatic immunoassay system with significantly enhanced precision, sensitivity, and stability and a stable luminescence rate.

[0068] The invention provides a substrate liquid for a fully automatic immunoassay system. The substrate liquid for the fully automatic immunoassay system uses water as a solvent and contains the following components: AMPPD, a buffer solution, CTAB-AuNCs, and an enzyme stabilizer.

[0069] The CAS number of the AMPPD substrate solution for the fully automatic immunoassay system is 122341-56-4, and it was purchased from Wuhan Hanhai New Enzyme Biotechnology Co., Ltd. in a purchase specification of 1 g.

[0070] The structural formula of AMPPD used in the substrate solution of the fully automatic immunoassay system is shown in Formula I below:

[0071]

[0072] The AMPPD structure consists of four parts: a phenolic group, a methoxy group, an adamantyl group, and a dioxetane ring. The phenolic group, after inductive triggering, becomes an electron donor and forms the primary portion of the emitter. The methoxy group likely contributes to the formation of an aromatic ester by combining with the phenolic hydroxyl group of the phenol and the CO bond of the dioxetane, thereby converting the singlet state to the lowest excited state. The adamantyl group does not directly participate in chemical excitation, but acts as a stabilizer to enhance the thermal stability of the luminescent substrate.

[0073] The pH of the substrate solution used in the fully automatic immunoassay system is 9.0-10.0.

[0074] Preferably, the buffer is one of carbonate buffer, diethanolamine buffer, and 2-amino-2-methyl-1-propanol (AMP). The buffer has a pH that maintains the detection environment.

[0075] Preferably, the CTAB-AuNCs have the dual functions of a chemiluminescence enhancer and an antibacterial agent. The AuNCs are based on MPA-AuNCs as a template. The superposition effect of the hydrophobic interaction and electrostatic force of CTAB is utilized to remove the MPA ligands on the surface of MPA-AuNCs and adsorb CTAB onto the surface of Au NCs to form AuNCs protected by a double-layer CTAB.

[0076] The reagents required for the preparation of the CTAB-AuNCs are: sodium hydroxide (NaOH), N,N-dimethylformamide (DMF), chloroauric acid trihydrate (HAuCl4·3H2O), phosphate buffer (pH=7.0), mercaptopropionic acid (MPA), and cetyltrimethylammonium bromide (CTAB).

[0077] The specific steps for preparing CTAB-AuNCs are as follows: 1M NaOH and DMF are added to an ultrapure aqueous solution and stirred for 10 minutes, followed by the addition of 0.03M HAuCl4·3H20. After the solution gradually turns reddish-brown, the AuNPs are stored at 4°C until ready for use. A certain amount of the stored AuNPs solution is then added to 100mM phosphate buffer (pH 7.0), followed by the addition of 4M MPA (dissolved in an equal amount of 1M NaOH). The mixed solution is stirred thoroughly and allowed to stand in the dark at 25°C±5°C for 4 hours to obtain a yellow solution. The solution is then centrifuged at 7000 rpm for 30 minutes to remove undissolved thiol ligands. A small amount of AuNP aggregates is removed using a 0.22μm filter membrane. Finally, the solution is passed through a 10KDa ultrafiltration centrifuge tube at 10000 rpm for 30 minutes to remove excess unreacted products, yielding MPA-AuNCs. Take a certain amount of MPA-AuNCs and add 50 μM CTAB and 0.1 M Tris-HCl buffer (pH 9.0) to a volume of 1 mL. Incubate on a shaker at 1000 rpm for 30 min. Remove the solution and place it in a 10 kDa ultrafiltration centrifuge tube. Centrifuge repeatedly at 10,000 rpm for 30 min until no visible bubbles are visible when the solution is vigorously shaken. Dilute the resulting solution to the original volume with ultrapure water to obtain CTAB-AuNCs. Store at 25°C ± 5°C.

[0078] The technical principle of this invention is that CTAB adsorbed on the surface of AuNCs gives them an extremely high positive charge density and extremely high local CTAB concentration. The large amount of CTAB micellarization creates an internal hydrophobic microenvironment, which shortens the distance between the gold nanoclusters and the AMPPD substrate. The energy level structure of the AuNCs accepts the energy transmitted by AMPPD, generating light energy transfer. Due to the quantum confinement characteristics of AuNCs, they have a high quantum yield, which is far higher than that of chemiluminescent molecules, thereby greatly enhancing the chemiluminescence intensity. AuNCs also have a large specific surface area and high surface energy, which can improve the stability of chemiluminescence and possess a certain degree of catalytic activity, greatly accelerating the chemiluminescence reaction rate. Therefore, the luminescence value, measurement precision, sensitivity, and luminescence rate stability of the substrate solution used in fully automated immunoassay systems are significantly enhanced. Furthermore, CTAB, with its positively charged quaternary ammonium salt and long hydrophobic carbon chain, can efficiently adsorb onto the negatively charged bacterial surface, destroying its cell membrane and achieving a sterilizing effect. Furthermore, CTAB adsorbed on the surface of Au NCs exhibits superior antibacterial properties compared to CTAB dispersed in solution, resulting in an antibacterial effect superior to that of conventional antimicrobial agents and extending the shelf life of the reagent. Furthermore, since CTAB is encapsulated within Au NCs, the generation of large amounts of bubbles is avoided, eliminating the need for defoaming agents and minimizing their impact on the properties of the substrate solution.

[0079] Preferably, the enzyme stabilizer is one or both of magnesium chloride and zinc chloride.

[0080] As a preferred embodiment, the substrate solution for the fully automatic immunoassay system of the present invention uses water as a solvent and contains the following components:

[0081]

[0082] The pH was adjusted to 9.5 with hydrochloric acid.

[0083] In a specific embodiment, the enzyme stabilizers are MgCl2 and ZnCl2.

[0084] The present invention relates to a substrate solution for a fully automated immunoassay system. This substrate solution is an alkaline phosphatase-based enzymatic chemiluminescent substrate solution. The substrate solution uses water as a solvent and contains AMPPD, a buffer, gold nanoclusters (CTAB-AuNCs), and an enzyme stabilizer. The AuNCs in this invention are coated with a large amount of positively charged cetyltrimethylammonium bromide (CTAB), providing dual functions as a chemiluminescence enhancer and an antibacterial agent. CTAB-AuNCs use MPA (mercaptopropionic acid)-AuNCs as a template. Utilizing the combined effects of CTAB's hydrophobic interaction and electrostatic forces, the MPA ligands on the surface of MPA-AuNCs are removed and CTAB is adsorbed onto the AuNCs surface, thereby forming a bilayer of CTAB-protected AuNCs. Consequently, the AuNCs surface is coated with a large amount of positively charged CTAB, giving the cationic surfactant-stabilized AuNCs an extremely high positive charge density and a very high local CTAB concentration. The large amount of CTAB micellization shapes the internal hydrophobic microenvironment, thereby shortening the distance between the AuNCs and the AMPPD substrate. The AuNCs utilize their energy level structure to accept the energy transferred by AMPPD, generating light energy transfer. Due to the quantum confinement characteristics of AuNCs, they have a high quantum yield, which is much higher than that of chemiluminescent molecules, thereby greatly enhancing the chemiluminescence intensity. AuNCs also have a large specific surface area and high surface energy, which can improve the stability of chemiluminescence and possess a certain degree of catalytic activity, greatly accelerating the chemiluminescent reaction rate. This significantly enhances the luminescence value, measurement precision, sensitivity, and luminescence rate stability of the substrate solution used in fully automated immunoassay systems. Furthermore, CTAB, a positively charged quaternary ammonium salt with a long hydrophobic carbon chain, can efficiently adsorb on the surface of negatively charged bacteria, destroying their cell membranes and achieving a sterilizing effect. Furthermore, CTAB adsorbed on the surface of AuNCs exhibits superior antibacterial properties compared to CTAB dispersed in solution, surpassing those of conventional antibacterial agents and significantly extending the shelf life of the reagent. Furthermore, because CTAB is coated on the surface of AuNCs, the generation of large amounts of bubbles is avoided, eliminating the need for defoaming agents and minimizing their impact on the properties of the substrate solution. The substrate solution for the fully automated immunoassay system of the present invention boasts advantages such as a short time to reach the luminescence plateau, high intensity, a long duration of the luminescence plateau, high sensitivity, good stability, and a long shelf life, fully meeting the needs of clinical testing.

[0085] Unless otherwise specified, the application of CTAB-AuNCs provided by the present invention in antibacterial and / or enzymatic chemiluminescence analysis, and the raw materials and reagents used in the substrate solution can all be purchased from the market.

[0086] The present invention will be further described below in conjunction with the embodiments:

[0087] Example 1 Preparation of CTAB-Au NCs and Calibration of CTAB Content

[0088] (1) Preparation of CTAB-AuNCs

[0089] 1.25 mL of 1 M NaOH and 12.5 mL of DMF were added to 45 mL of ultrapure water solution and stirred for 10 min. Then 12.5 mL of HAuCl4·3H2O (0.03 M) was added. The solution gradually turned red-brown, indicating that Au 3+ Gradually reduced to Au 0 Thus, AuNPs of about 3 nm were formed and stored at 2-8°C until use.

[0090] To 25 mL of the AuNPs solution, 5 mL of 100 mM phosphate buffer (pH 7.0) was added, followed by 1.25 mL of 4 M MPA (dissolved in an equal amount of 1 M NaOH). The mixture was stirred and allowed to stand at 25°C ± 5°C in the dark for 4 h. The resulting yellow solution was centrifuged at 7000 rpm for 30 min to remove undissolved thiol ligands. The solution was then filtered through a 0.22 μm filter to remove small amounts of AuNP aggregates. Finally, the solution was centrifuged at 10,000 rpm for 30 min through a 10 kDa ultrafiltration tube to remove excess unreacted products, yielding MPA-AuNCs. To 150 μL of the MPA-AuNCs solution, 200 μL of 50 μM CTAB and 200 μL of 0.1 M Tris-HCl (pH 9.0) buffer were added, with a molar ratio of [Au] to CTAB of approximately 60:1. Add ultrapure water to maintain the total volume of the solution at 1 mL. Incubate the mixture on a shaker at 1000 rpm for 30 min. Remove the solution and place it in a 10 kDa ultrafiltration centrifuge tube. Repeat the centrifugation at 10,000 rpm for 30 min until no visible bubbles are formed when the solution is shaken vigorously. Dilute the resulting solution to the original volume with ultrapure water to obtain 0.36 μg / mL CTAB-AuNCs. Store at 25°C ± 5°C.

[0091] (2) CTAB content calibration

[0092] The CTAB content on the surface of AuNCs was quantified by an indirect method. After centrifugation in an ultrafiltration centrifuge tube, the CTAB concentration in 200 μL of the filtrate was quantified using the national standard (GB / T5174-2018) method, thereby inferring the CTAB concentration adsorbed on the surface of AuNCs. First, a standard curve was established, and CTAB standard solutions with concentration gradients of 10, 40, 60, 80, and 100 μM were prepared. According to the national standard method, sodium dodecyl sulfate (SDS) was used to titrate it, and the volume of SDS solution required was recorded to construct a standard curve, as shown in the following example. Figure 1 As shown. The same method was used to measure 200 μL of filtrate, and the CTAB concentration in the filtrate was quantified. The amount of CTAB adsorbed on the AuNCs can be preliminarily inferred by subtracting the CTAB content in the filtrate from the total amount of CTAB input. After repeated centrifugation and washing of the CTAB-AuNCs, the CTAB concentration in the 200 μL filtrate was determined to be approximately 45 μM by titration. Since the CTAB concentration was 50 μM during synthesis and the input amount was 200 μL, only 5 μM of the CTAB input in 200 μL was bound to the AuNCs surface. The amount of CTAB adsorbed on the AuNCs surface was calculated to be 0.36 μg / mL, with a molar ratio of [Au][CTAB] of approximately 615:1.

[0093] Example 2 Experimental study on the antibacterial properties of CTAB-Au NCs

[0094] (1) Culture of E. coli

[0095] All instruments used in the experiment were sterilized before use. First, E. coli was added to 10 mL of nutrient agar liquid medium and incubated at 37°C for 24 hours to increase the bacteria. The resulting solution was centrifuged at 11,800 rpm for 20 minutes, and the precipitate was collected. After washing three times by centrifugation, the precipitate was dispersed in PBS. This precipitate was spread on an agar plate and incubated at 37°C for 24 hours. The E. coli colony concentration in the original solution was then counted and calculated. The E. coli solution was stored at 4°C.

[0096] (2) Experiment on the killing effect of CTAB-AuNCs on E. coli

[0097] 5 μM CTAB-AuNCs, AuNCs and CTAB solutions were added to E. coli solution respectively to ensure that the UV absorption value of E. coli at 600 nm in the mixed solution was 0.6 (OD 600 =0.6). During the sterilization process, samples were taken every 5 minutes, 10 μL of the sample was diluted to 10 mL with ultrapure water, and 100 μL of the diluted reaction solution was spread on three agar plates in parallel. After incubation at 37°C for 24 hours, the bacterial colonies were counted using the plate count method. The results are shown in Figure 2. Figure 2 and Figure 3 As shown, from Figure 2The growth of E. coli colonies can be clearly seen in the photos of the culture dishes. It can be found that after the addition of CTAB (5μM), the number of colonies slowly decreased, and even after 30 minutes of sterilization, there were still a large number of colonies. After the addition of AuNCs, the number of colonies showed almost no significant change with the change of sterilization time. Then, after the addition of CTAB-AuNCs, the growth of the colonies was significantly inhibited in the first 15 minutes, and when the sterilization time reached 30 minutes, there were almost no colonies left. We counted the number of colonies, and the number of colonies at 0 minutes was recorded as N0, and the number of colonies at different reaction times was recorded as N. Figure 3 As shown in the figure, after 30 minutes of sterilization, CTAB (5 μM) achieved a sterilization rate of 44%. Notably, CTAB-AuNCs exhibited a very high sterilization efficiency (85%) in the first 15 minutes, which gradually stabilized at 98% after 30 minutes. This demonstrates that our constructed CTAB-AuNCs exhibit superior sterilization performance compared to CTAB at the same concentration.

[0098] Example 3 Preparation of substrate solution for the fully automatic immunoassay system of the present invention

[0099] Using purified water as solvent, substrate solutions 1-4 of the present invention were prepared according to the substrate solution formulas in Tables 1-4.

[0100] Table 1 Formula of substrate solution 1 for fully automated immunoassay system

[0101]

[0102] Table 2 Formulation of substrate solution 2 for fully automated immunoassay system

[0103]

[0104] Table 3 Formula of substrate solution 3 for fully automatic immunoassay system

[0105]

[0106]

[0107] Table 4 Formula of substrate solution 4 for fully automatic immunoassay system

[0108]

[0109] Example 4 Pre-screening of the fully automated immunoassay system using a comparison substrate solution

[0110] Using purified water as the solvent, comparative substrate solutions 1-3 were prepared according to the substrate solution formulas in Tables 5-7.

[0111] Table 5 Formula of comparison substrate solution 1 for fully automatic immunoassay system

[0112]

[0113] Table 6 Formula of comparison substrate solution 2 for fully automatic immunoassay system

[0114]

[0115] Table 7 Formula of comparison substrate solution 3 for fully automatic immunoassay system

[0116]

[0117]

[0118] Example 5 Comparison of the relative luminescence intensity of substrates 1-4 and 1-3 for the fully automated immunoassay system of the present invention and Beckman Access Substrate

[0119] The relative luminescence intensity of the substrate solution itself was measured in the absence of alkaline phosphatase catalysis. Specifically, the substrate solutions 1-4 for the fully automated immunoassay system of the present invention described in Example 3, the comparison substrate solutions 1-3 described in Example 4, and the Beckman luminescent substrate solution were placed in the substrate bottles of a chemiluminescence analyzer. The chemiluminescence analyzer parameters were set to: "Sample position 0, sample volume 0 μL, number of tests 3, substrate volume 200 μL, substrate bottle 1." The chemiluminescence analyzer captured the 470 nm light signal to measure the relative luminescence intensity of the substrate solution background. The results are shown in Table 8.

[0120] Table 8 Comparison of relative luminescence intensity of substrate solution 1-4 and comparison substrate solution 1-3 for the fully automatic immunoassay system of the present invention and the Beckman luminescence substrate solution substrate background

[0121]

[0122] The lower the substrate background of the substrate liquid, the better the performance of the substrate liquid. However, if the substrate background is too low, other key performance requirements of the substrate liquid (short time to reach the luminescence plateau, high intensity, and long duration of the luminescence plateau) cannot be met. Therefore, the substrate background of the substrate liquid within a certain range (Beckmann luminescence substrate liquid substrate background RLU value ±1500) can be considered to meet the required conditions and can only be used as a preliminary judgment for the early performance screening of the substrate liquid.

[0123] According to the results in Table 8, the substrate backgrounds of the comparison substrate solutions 1-3 are all lower than that of the Beckmann chemiluminescent substrate solution, while the relative luminescence intensities of the chemiluminescent substrate solutions 1-4 of the present invention are comparable to those of the Beckmann chemiluminescent substrate solution, among which substrate solutions 2 and 3 of the present invention are closest to the Beckmann chemiluminescent substrate solution.

[0124] Example 5 Reaction kinetics study and comparison of the plateau phase of the luminescent substrate solution

[0125] Take 200 μL of the substrate solution 1-4 for the fully automatic immunoassay system of the present invention in Example 3, the comparison substrate solution 1-3 in Example 4, and the Beckman luminescent substrate solution, and add 5 μL of alkaline phosphatase at a concentration of 3 ng / mL to each of the eight solutions. After rapid mixing, the main component AMPPD in the substrate solution for the fully automatic immunoassay system undergoes hydrolysis of the phosphate group under the action of alkaline phosphatase to form an unstable intermediate AMP-D. This intermediate product causes the -OO- four-membered ring to be cleaved into one molecule of adamantane ketone and one molecule of methyl m-oxybenzoate anion in an excited state through intramolecular electron transfer. When the methyl m-oxybenzoate anion in an excited state changes from an excited state to a ground state, it generates 470 nm of light, stimulates a chemiluminescent reaction, emits photons, and captures the light quantum yield through a chemiluminescence meter, thereby measuring the relative luminescence intensity of each chemiluminescent substrate solution. A luminescence kinetics curve showing the change of relative luminescence intensity over time is drawn with relative luminescence intensity as the Y axis and time as the X axis, as shown in FIG. Figure 4 The substrate solution plateau phase was compared, and the experimental results are shown in Table 9.

[0126] Table 9 Comparison of the plateau phase of substrate solutions 1-4 and comparison substrate solutions 1-3 for the fully automatic immunoassay system of the present invention and imported luminescent substrate solutions

[0127]

[0128]

[0129] From Table 9 and Figure 4It can be seen that the luminescence effects of the comparison substrate solutions 1-3 are all lower than the control Beckmann substrate solution, while the luminescence effects of the substrate solutions 1-4 of the present invention are all higher than the control Beckmann substrate solution. Among them, the luminescence effects of the substrate solutions 1 and 3 of the present invention are closer to those of the control Beckmann substrate solution. Compared with the control Beckmann luminescent substrate solution, the chemiluminescent substrate solutions 2 and 4 of the present invention have higher relative luminescence intensity and also have longer plateau phases and shorter plateau phases. Among them, the luminescence time of the Beckmann chemiluminescent substrate solution to reach the plateau phase is 50 minutes, the luminescence duration is 30-80 minutes, and the relative luminescence intensity of the luminescence plateau phase is about 201878. The luminescence time to reach the plateau phase, the luminescence duration, and the relative luminescence intensity of the luminescence plateau phase of the chemiluminescent substrate solutions 2 and 4 of the present invention are significantly better than those of the control Beckmann luminescent substrate solution. Among them, the chemiluminescent substrate solution 2 of the present invention has achieved a relatively high relative luminescence intensity, its luminescence time to reach the plateau phase is shortened relative to other substrate solutions, and its luminescence duration is the longest.

[0130] Example 6 Comparison of the signal intensity and linear relationship between substrate solutions 1-4, comparison substrate solutions 1-3, Beckman luminescent substrate solution and AP enzymes at various gradient concentrations for the fully automated immunoassay system of the present invention

[0131] Prepare different concentration gradients of alkaline phosphatase (0.05ng / mL to 50ng / mL), take 5μL of each and add it to the sample cup, then add 200μL of the substrate solution 1-4 for the fully automatic immunoassay system of the present invention, the comparison substrate solution 1-3 and the Beckman luminescent substrate solution. The luminescent substrate AMPPD in the substrate solution is catalyzed and hydrolyzed by alkaline phosphatase to produce a 470nm light signal. The relative luminescence intensity (RLU) is measured by a chemiluminescence analyzer. The relative luminescence intensity is proportional to the concentration of alkaline phosphatase. The test results are recorded in Table 10, and for the results of AP enzyme concentration and relative luminescence intensity, a curve of the relationship between relative luminescence intensity and AP enzyme concentration is drawn with relative luminescence intensity as the Y axis and AP enzyme concentration as the X axis, and the concentration-relative luminescence intensity data is linearly fitted, and the fitting linear value R is calculated. 2 ,See Figure 5 .

[0132] Table 10 Comparison of signal intensity differences of substrate solutions 1-4, comparison substrate solutions 1-3, and Beckman luminescent substrate solution under different AP enzyme concentrations for the fully automated immunoassay system of the present invention

[0133]

[0134]

[0135] Depend on Figure 5The results in Table 10 show that under the action of the same concentration of AP enzyme, the luminescence intensity of the comparison substrate solutions 1-3 is lower than that of the Beckman chemiluminescent substrate solution, while the luminescence intensity of the substrate solutions 1-4 of the present invention is higher than that of the Beckman chemiluminescent substrate solution. Among them, the luminescence intensity of the substrate solution 1 of the present invention is close to that of the Beckman chemiluminescent substrate solution, and the luminescence intensity of the substrate solutions 2-4 of the present invention are significantly higher than that of the Beckman chemiluminescent substrate solution. In addition, under the condition of the same concentration of AP enzyme, the substrate signal intensity is from large to small in the order of substrate solution 4 of the present invention, substrate solution 2 of the present invention, substrate solution 3 of the present invention, and substrate solution 1 of the present invention. By linearly fitting the AP enzyme concentration-relative luminescence intensity data, it was found that the fitting linear value R of the luminescent substrate solution was 2 The values ​​were all greater than 0.99, indicating a good linear correlation, indicating that within the AP enzyme concentration range of 0.05 ng / mL-50 ng / mL, the relative luminescence intensity of the substrate solution was linearly related to the AP enzyme concentration.

[0136] Example 7 Thermal Stability Experiment of Chemiluminescent Substrate Solution

[0137] Thermal stability is an important performance and indicator for the application of chemiluminescent substrate solutions. The present invention tested and evaluated the thermal stability of the prepared substrate solutions 1-4 for the fully automatic immunoassay system, the comparison substrate solutions 1-3, and the Beckman luminescent substrate solution.

[0138] When conducting the stability test, the substrate luminescent liquid 1-4 of the present invention, the comparison substrate liquid 1-3, and the Beckmann luminescent substrate liquid were respectively placed at 37°C and compared with the luminescent substrate liquid stored at 2-8°C at 1, 3, 7, and 14 days to compare the relative luminescence intensity of the two. In the specific experiment, 5 μL of alkaline phosphatase with a concentration of 3 ng / mL was added to the sample cup, and then 200 μL of the substrate liquid 1-4, the comparison substrate liquid 1-3, and the Beckmann luminescent substrate liquid for the fully automatic immunoassay system of the present invention were added respectively. The luminescent substrate AMPPD in the substrate liquid was catalyzed by alkaline phosphatase to generate a light signal of 470 nm. The relative luminescence intensity (RLU) was measured by a chemiluminescence meter, and the test results were recorded in Table 11.

[0139] Table 11 Comparison of thermal stability of luminescent substrate solution at 37°C and 2-8°C

[0140]

[0141]

[0142] From the above data and test results, it can be seen that the substrate solutions 1-4 for the fully automated immunoassay system of the present invention in Example 3 achieved a good luminescence plateau and relative luminescence intensity while achieving excellent thermal stability compared to commercially available products. The luminescent substrates 1-4 of the present invention all retained more than 90% of their activity after 14 days at 37°C. In contrast, the thermal stability test conducted by Beckman's luminescent substrate solution at 37°C showed that the activity was essentially stable at 81.59% after 7 days, but the activity began to decline to 64.68% after 14 days, and its relative luminescence intensity also decreased to a lower level. In addition, when comparing the thermal stability test conducted by substrate solution 1-3 at 37°C, its activity retention rate was far lower than that of the substrate solution 1-4 for the fully automated immunoassay system of the present invention, and even lower than that of Beckman's luminescent substrate solution. This shows that the shelf life of the substrate solution for the fully automated immunoassay system of the present invention is longer than the one-year shelf life guaranteed by commercially available imported reagents.

[0143] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. Application of CTAB-Au NCs in enzymatic chemiluminescence analysis; The preparation method of the CTAB-Au NCs comprises: MPA-Au NCs, CTAB and Tris-HCl buffer solution were taken, shaken for reaction, and then centrifuged and washed in a 10 KDa ultrafiltration centrifuge tube to obtain the CTAB-Au NCs; The molar ratio of [Au] to the CTAB is 60:1; The concentration of the Tris-HCl buffer solution is 0.1M; The shaking reaction includes a reaction at 1000 rpm for 30 min; The centrifugal conditions include centrifugation at 10,000 rpm for 30 min; The substrate solution used in the enzymatic chemiluminescence analysis includes the CTAB-Au NCs and AMPPD.

2. A substrate solution for enzymatic chemiluminescence analysis, characterized in that: include: The enzyme stabilizer includes magnesium salt and / or zinc salt.

3. The substrate solution according to claim 2, wherein include:

4. The substrate solution according to claim 2 or 3, wherein The magnesium salt includes magnesium chloride; the zinc salt includes zinc chloride.

5. The substrate solution according to any one of claims 2 or 3, wherein The buffer comprises one or more of carbonate buffer, diethanolamine buffer or 1-amino-2-methyl-1-propanol; or The pH value of the substrate solution is 9.0-10.

0.

6. Use of the substrate solution according to any one of claims 2 to 5 in the preparation of an enzymatic chemiluminescence analysis kit.

7. An enzymatic chemiluminescence analysis kit, characterized in that: Comprising the substrate solution according to any one of claims 2 to 5.

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