CN114675027A - Bladder cancer protein marker activity detection kit and detection method thereof - Google Patents

Abstract

The invention discloses a bladder cancer protein marker activity detection kit and a detection method thereof, wherein the kit comprises: the detection kit and the detection method can simultaneously detect at least two bladder cancer protein markers based on potential resolution, and improve the controllability and the combination efficiency of the immunological combination of an electrode interface through a DNA tetrahedral structure so as to improve the detection sensitivity.

Description

Bladder cancer protein marker activity detection kit and detection method thereof

Technical Field

The invention relates to the technical field of biological detection and chemical analysis, in particular to a kit for detecting activity of bladder cancer protein markers and a method for simultaneously detecting multiple bladder cancer protein markers for non-diagnosis purposes.

Background

Bladder cancer is a malignant tumor disease with high morbidity and mortality, is one of the most common malignant tumors of the male urinary system, and currently, cystoscopy is the gold standard for bladder cancer diagnosis, but the method has certain invasiveness, relates to traumatic examination, is easy to cause urinary tract infection of patients, and in early diagnosis of bladder cancer, the traditional detection method has low sensitivity and specificity and is easy to have high false positive rate.

The occurrence of bladder cancer can directly influence the components of blood and urine, the specificity of the non-invasive liquid biopsy is obviously superior to that of cystoscopy, the combined analysis and detection of various biomarkers can be realized, the liquid biopsy has higher efficiency, the detection time is generally within several minutes, and the rapid detection of tumor markers can be realized; in addition, the liquid biopsy is easy to carry, and has good application prospect.

At present, a plurality of related reagents based on liquid biopsy are developed for detecting bladder cancer markers, including enzyme-linked immunosorbent assay, fluorescence and the like, but the related reagent detection depends on expensive instruments, the detection process is time-consuming and labor-consuming, a large amount of samples are required, and the detection sensitivity and specificity still have great promotion space.

The ECL immunosensor is a biosensor developed by combining an electrochemiluminescence technology with an immunoassay method and has the advantages of high sensitivity, high selectivity, low background and the like, immune antigen antibodies are used as recognition elements and fixed on an electrode through a self-assembly method, and quantitative detection to be detected is realized through conversion of signal molecules and the concentration of an analyte to be detected. In addition, the ECL immunosensor also has the advantages of relatively simple equipment, suitability for integration, easiness in miniaturization and the like, and is successfully applied to the fields of medicine, environment, food safety detection and the like; in the prior art, a technical scheme for applying the electrochemiluminescence ECL immunosensor to liquid biopsy has been developed, for example, a patent with publication number CN 104483481B provides a bladder cancer marker-NMP 22 immunosensor constructed based on a carbon nanotube/PdPt nanocage, and the carbon nanotube/PdPt nanocage composite material is used as a detection antibody marker, so that the sensitivity and stability of the immunosensor are improved to a certain extent, and the detection limit of the bladder cancer marker NMP22 can reach 0.21pg/mL, but the detection immunosensor and the detection method can not realize simultaneous detection of multiple bladder cancer markers and have limited sensitivity, and are not suitable for screening early bladder cancer markers.

Disclosure of Invention

Aiming at the technical problems of expensive instruments, large sample amount, complex operation and low detection sensitivity of the existing bladder cancer marker detection reagent and method, the invention provides a bladder cancer protein marker activity detection kit and a detection method thereof.

The technical scheme adopted by the invention is as follows:

accordingly, in a first aspect, the present invention provides a kit for detecting activity of a bladder oncoprotein-based marker, comprising: the kit comprises electrodes, a functional luminescent probe for specifically recognizing bladder cancer protein markers, and a capture probe for specifically recognizing bladder cancer protein markers; the capture probe, the bladder cancer protein marker and the functionalized luminescent probe can form a sandwich structure through immunological binding;

wherein the electrode is formed by gold electrodeposition on a glassy carbon electrode;

wherein, as shown in fig. 1a, the functionalized luminescent probe at least comprises: a first functionalized luminescent probe and a second functionalized luminescent probe; the first functionalized luminescent probe is made of a first luminophor modified by a class I secondary antibody of a first protein marker; the second functionalized luminescent probe is prepared by modifying a second luminophore by a II-class secondary antibody of a second protein marker; the specific modification mode is that gold is grown in situ on the first luminophor or the second luminophor, and the Au-N bond effect between the nano gold particles and protein is utilized to modify the secondary antibody on the first luminophor or the second luminophor;

wherein, as shown in FIG. 1c, the capture probe at least comprises: a first capture probe and a second capture probe; the first capture probe is made of a class I primary anti-modification DNA tetrahedron; the second capture probe is made of a type II primary-antibody modified DNA tetrahedron; FIG. 1b shows that the DNA tetrahedron is formed by self-assembly of four DNA fragments, and the formed DNA tetrahedron structure has a sulfhydryl functional group and an amino functional group, and the sulfhydryl functional group can be modified on the electrode for electrodepositing gold through Au-S covalent bond; the amino functional group can be used for carrying out tetrahedral structure modification on the primary antibody modified DNA by glutaraldehyde;

further, the first and second luminophors can be selected from Ru (bpy)₃ ²⁺Luminol, AuAgNCs, Ru (bpy)₃ ²⁺-any of MOF or CdS quantum dots; further preferably, when the first luminophore is AuAgNCs, the second luminophore is luminol; or further preferably, the first light emitter is Ru (bpy)₃ ²⁺-MOF, the second luminophore is AuAgNCs; ru (bpy)₃ ²⁺The MOF ECL signal has a relative standard deviation of 2.5% and the AuAgNCs ECL signal has a relative standard deviation of 2.7%, both luminophores have excellent ECL performance and can be used as the most preferred luminophor combination of the present invention;

further, when the detection kit is used for liquid biopsy and is used for detecting the mitotic protein NuMA1 and the complement-associated factor CFHR1, the first functionalized luminescent probe is made of a second antibody modified first luminescent body of NuMA 1; the second functional probe is made of a second luminophore modified by a secondary antibody of CFHR 1.

As a preferred embodiment of the present invention, the kit for detecting the activity of a bladder cancer protein marker of the present invention comprises:

a first reagent container comprising the electrodeposited gold modified electrode;

a second reagent container comprising the capture probe;

a third reagent container comprising a functionalized luminescent probe.

A fourth reagent container comprising phosphate buffered saline, PBS.

A fifth reagent container comprising phosphate buffered saline PBS, TPrA, and K₂S₂O₈。

To say thatObviously, the use method of the detection kit comprises the following steps: dropwise adding a second reagent container to the electrode in the first reagent container, reacting at room temperature, cleaning the electrode by using PBS in a fourth reagent container after reaction, dropwise adding the electrode into a sample extracting solution to be detected, reacting at room temperature, cleaning the electrode by using PBS in the fourth reagent container after reaction, dropwise adding the electrode into a reagent in a third reagent container, mixing, taking out after reaction, cleaning by using PBS, and placing the electrode into a fifth reagent container containing TPrA and K₂S₂O₈In PBS solution (b), for ECL detection.

As the same technical concept of the above tumor marker detection kit, in a second aspect, the present invention provides a method for simultaneously detecting multiple bladder cancer protein markers for non-diagnostic purposes, as shown in FIG. 1d, comprising the following steps:

step S1, obtaining a sample extracting solution to be detected;

step S2, modifying the two capture probes on an electrode by utilizing Au-S covalent bond to construct an ECL sensing platform;

step S3, dropwise adding the sample extracting solution to be detected on the ECL sensing platform constructed in the step S2 for incubation;

step S4, washing the ECL sensing platform incubated in the step S3 by PBS, dripping the two constructed functional luminescent probes, and continuing incubation;

and step S5, washing the ECL sensing platform incubated in the step S4 by PBS, and then carrying out ECL detection.

Has the advantages that:

according to the activity detection reagent and the detection method for the bladder cancer protein markers, the antibodies are respectively marked by using different functionalized luminescent probes, and then the two antibodies are respectively combined by using a DNA tetrahedron and then are modified on the electrodes, so that the combination efficiency and the controllability of the capture probe on the interface of the electrodes are effectively improved, the detection sensitivity is improved, the high-sensitivity activity simultaneous detection of the two tumor markers is realized based on electrochemical detection, the reagent and the method are suitable for the simultaneous detection of multiple markers, the diagnosis accuracy is improved, and compared with the existing sensing platform, the sensitivity is higher, and the detection limit is low; compared with the traditional bladder cancer marker detection method, the method is simple, the sensor is easy to prepare, and the detection sample amount is small.

Drawings

FIG. 1a is a schematic structural diagram of a functionalized luminescent probe according to the present invention and a modification process thereof;

FIG. 1b shows the structure and fabrication process of the DNA tetrahedron according to the present invention;

FIG. 1c shows a capture probe according to the present invention and a process for making the same;

FIG. 1d is a schematic diagram of the construction of the sensing platform and the detection process of the protein tumor marker according to the present invention;

FIG. 2 shows Ru (bpy)₃ ²⁺-MOF TEM representation;

FIG. 3 is a representation and an element mapping of AuAgNCsTEM according to an embodiment, wherein A is a representation of high resolution TEM of AuAgNCs; b is element mapping diagram of AuAgNCs;

FIG. 4 shows Ru (bpy)₃ ²⁺-ECL profile of MOF modified electrode;

FIG. 5 shows Ru (bpy)₃ ²⁺ECL curve stability of MOF modified electrodes;

FIG. 6 is an ECL graph of an AuAgNCs modified electrode in accordance with an embodiment;

FIG. 7 illustrates the stability of the ECL curve of an AuAgNCs modified electrode in accordance with an embodiment;

FIG. 8 is a sensor build process CV characterization in accordance with an embodiment;

FIG. 9 is an EIS characterization of a sensor construction process in accordance with an embodiment;

FIG. 10 is an ECL profile for different modified electrodes in accordance with an embodiment;

FIG. 11 is a graph showing the simultaneous detection of ECL for tumor markers at different concentrations in accordance with an embodiment;

figure 12 is a graph of ECL signal intensity versus NuMA1 antigen concentration in a linear fashion in accordance with embodiments;

FIG. 13 is a graph of the linear relationship between ECL signal intensity and CFHR1 antigen concentration in accordance with certain embodiments;

figure 14 is a graph of the results of the stability test in a specific embodiment to simultaneously detect NuMA1 and CFHR 1.

Detailed Description

The invention is further described below with reference to the accompanying drawings.

In the present invention, the test materials and test equipment are described as follows:

oligonucleotide sequences were synthesized in Shanghai; tris (2, 2' -dipyridyl) dichlorRuthenium (II) hexahydrate (Ru (bpy)₃ ²⁺)、HAuCl₄·3H₂O、AgNO₃Purchased from Sigma-Aldrich; 6-aze-2-thionine (ATT) available from AlfaAesar Co., Ltd. (China); l-arginin (Arg) available from national chemical group, Inc.; CFHR1 (complete factor H-related 1) antigen and antibody, NuMA1(Nuclear viral associated protein 1) antigen and antibody were purchased from Abcam; and diluting the antigen and the antibody by using a phosphate buffer solution to prepare solutions with different gradient concentrations.

The ultra-weak light-emitting measuring instrument BPCL is purchased from micro-optical technology limited, and the electrochemical workstation is purchased from Shanghai Chenghua. The scanning voltage range applied in the electrochemical luminescence detection experiment is-2.0-1.3V, the scanning speed is 0.5V/s, and the PMT high voltage is 800V.

The detection process of the invention is specifically illustrated by using bladder cancer markers NuMA1 and CFHR1 as detection samples and adopting a better ECL sensor.

Construction of capture probes

The first capture probe is made of a type I primary anti-modification DNA tetrahedron; in the embodiment, the I-type primary antibody is a mitotic nuclear protein NuMA1 antibody;

the second capture probe is made of a II-type primary anti-modification DNA tetrahedron; in this example, class II primary antibodies are the complement associated factor CFHR1 antibody;

the DNA tetrahedron is formed by self-assembling four DNA fragments, and the formed DNA tetrahedron structure has a mercapto functional group and an amino functional group, wherein the mercapto functional group can be modified on the electrode of the electrodeposited gold through Au-S covalent bond; the amino functional group can be used for carrying out tetrahedral structure modification on the primary antibody modified DNA by glutaraldehyde; the four DNA fragments are shown in the present example as SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3 and SEQ ID NO. 4; these four DNA fragments can form a tetrahedral structure by base pairing.

SEQ ID NO.1 is as follows:

5’-CTCAACTGCCTGGTGATACGAGGATGGGCATGCTCTTCCCGACGGTATTGGACCCTCGCATG-3’；

SEQ ID NO.2 is as follows:

5’-CGATTACAGCTTGCTACACGATTCAGACTTAGGAATGTTCGACATGCGAGGGTCCAATACCG-3’；

SEQ ID NO.3 is as follows:

5’-CTACTATGGCGGGTGATAAAACGTGTAGCAAGCTGTAATCGACGGGAAGAGCATGCCCATCC-3’；

SEQ ID NO.4 is as follows:

5’-TTTATCACCCGCCATAGTAGACGTATCACCAGGCAGTTGAGACGAACATTCCTAAGTCTGAA-3’；

the four precisely designed oligonucleotide chains with complementary first sites are formed by annealing and self-assembling, the concentration of each DNA sequence is quantified by an ultraviolet spectrophotometer, and 4 sequences are prepared according to the molar ratio of 1: 1: 1: 1, can ensure that most of DNA can be self-assembled into a DNA tetrahedron, and after the DNA is quantified, the DNA tetrahedron is expressed by the following formula of SEQ ID NO. 1: SEQ ID NO. 2: SEQ ID NO. 3: SEQ ID No.4 ═ 1: 1: 1: 1, uniformly mixing the four sequences in a TM buffer solution, annealing at 95 ℃ for 5min, and then reducing the temperature to 4 ℃ to form a DNA tetrahedral structure.

And as shown in figure 1c, adding a glutaraldehyde solution into the synthesized tetrahedron, reacting at room temperature for 30min, then respectively adding a class I primary antibody and a class II primary antibody, continuing to react for 30min, and ultrafiltering the obtained solution through an ultrafiltration tube to obtain the capture probe.

Preparation of the first functionalized luminescent Probe

The first functionalized luminescent probe is made of a first luminophor modified by a class I secondary antibody; in the embodiment, the class I secondary antibody is a mitotic nuclear fission protein NuMA1 antibody, the first luminophore is Ru (bpy)₃ ²⁺-a MOF; the specific modification is that the first illuminator Ru (bpy)₃ ²⁺In situ gold growth on MOF, using gold nanoparticles and proteinsInterval Au-N bond action the secondary antibody was modified in Ru (bpy)₃ ²⁺-on the MOF;

in this embodiment, Ru (bpy)₃ ²⁺The specific MOF preparation process is as follows:

75mg of ZrOCl₄·8H₂O, 25mg of meso-Tetra (4-carboxyphenyl) porphine, 550mg of BDC and 75mg of Ru (bpy)₃ ²⁺The solution is stirred and reacted for 5h at 95 ℃ in 25mLDMF, and after centrifugation and washing, Ru (bpy) is obtained₃ ²⁺-a MOF product;

in this embodiment, the first light emitter Ru (bpy)₃ ²⁺The specific process of native gold length on MOF is as follows:

40mg of Ru (bpy) prepared above₃ ²⁺-MOF dispersed in 1mL of ultrapure water, 60. mu.L of this was taken and added to 25mL of ultrapure water, and 100. mu.L of HAuCl was added, respectively, with constant stirring₄(1%) and 80. mu.L of fresh NaBH₄(3.8mg mL^-1) Stopping stirring until the solution is purple red; finally, the resulting Ru (bpy) is centrifuged₃ ²⁺Washing MOF/AuNPs twice with ultrapure water, dispersing in ultrapure water, and storing at 4 ℃;

in this example, the NuMA1 secondary antibody was modified in Ru (bpy)₃ ²⁺The specific process on MOFs is as follows:

250 μ L of 0.6mg mL^-1Ru(bpy)₃ ²⁺Buffer solution of MOF/AuNPs (pH 7.4) 10. mu.L 5. mu.g mL^-1After adding 100. mu.L of 1% BSA to block non-specific sites on the MOFs surface, after incubation at 37 ℃ for 30min, the resulting precipitate was collected by centrifugation and rinsed once with a buffer solution (pH 7.4). Finally, the first functionalized luminescent probe obtained was dispersed in 250. mu.L of a buffer solution (pH 7.4) and stored at 4 ℃.

As shown in FIG. 2, is Ru (bpy)₃ ²⁺TEM representation of MOFs, from which it can be seen that the morphology is ellipsoidal.

Preparation of second functionalized luminescent Probe

The second functionalized luminescent probe is prepared by modifying a second luminophor by a II-type secondary antibody of a second protein marker; in this example, the class II secondary antibody is CFHR1 antibody, and the second luminophore is AuAgNCs; the specific modification mode is that the secondary antibody is modified on AuAgNCs by utilizing the Au-N bond effect between the nano gold particles and the protein;

in this example, the preparation process of AuAgNCs is as follows:

one preferred preparation procedure used in this example was to prepare AuAgNCs according to the literature report (modulation engineering of gold-silver nanocomputer super molecular structure end flow electrochemical analysis for ultra-sensitive biological analysis. biosensors and Bioelectronics 190(2021) 113449). specifically, 0.1M NaOH solution was added to a mixture of ATT solution (15mL, 80mM) and HAuCl4(15mL, 24mM) to adjust the pH to 10, and stirred for 1h away from light. Precipitating and washing the obtained solution twice by using isopropanol, and dissolving the solution in ultrapure water to obtain ATT-AuNCs stock solution; 1mL of 10mM AgNO was added with vigorous stirring₃The solution was added dropwise to 1mL of 0.5 MArg solution, triggering complexation of silver ions and amino groups in Arg by Ag-N covalent bonds. After the pH of the solution was adjusted to 10 by 1M NaOH, the resulting solution was left to react at 37 ℃ for 6 hours. Purifying by using a 50kDa ultrafiltration tube; 2mL of the Arg-AgNCs prepared above was mixed with 6mL of ATT AuNCs, and reacted at 37 ℃ for 24 hours to generate a hydrogen bond between ATT and Arg; washing the precipitate obtained after the reaction with isopropanol, precipitating, centrifuging, re-dispersing in 10mL of ultrapure water, and storing at 4 ℃;

fig. 3, a, is a high resolution TEM image of the AuAgNCs, which shows that the grain size of the AuAgNCs is about 4nm, and the elemental composition of the AuAgNCs is analyzed by an energy spectrometer EDS mapping, as shown in fig. 3, B, which shows that the material mainly consists of Au and Ag elements, further indicating the successful preparation of the AuAgNCs.

In this example, the specific process of modifying the secondary antibody on the AuAgNCs is as follows:

200. mu.L of AuAgNCs buffer (pH 7.4) was added to 10. mu.L of 5. mu.g mL^-1And kept stirring for 2 hours to ensure reaction. Thereafter, 100. mu.L of 1% BSA was added to block non-specific sites on the MOFs surface. At 37After incubation at deg.C for 30min, the resulting precipitate was collected by centrifugation and rinsed once with buffer solution (pH 7.4). Finally, the obtained second functionalized luminescent probe was dispersed in 200. mu.L of a buffer solution (pH 7.4) and stored at 4 ℃.

For Ru (bpy)₃ ²⁺ECL performance of MOFs was studied by dropping it on electrodes and after drying at room temperature, placing the electrodes in 0.1M PBS (pH 7.4) containing 10mM TPrA for ECL detection with the highest peak ECL potential at 1.25V as shown in figure 4. The stability of the material was investigated and as shown in fig. 5, 9 consecutive scans with a relative standard deviation of 2.5% indicated good ECL stability of the material.

The ECL performance of AuAgNCs was studied by dropping it on an electrode, air drying at room temperature, and then placing the electrode in a container containing 0.1M K₂S₂O₈In 0.1M PBS (pH 7.4) for ECL detection. The results are shown in FIG. 6, which shows the highest peak ECL potential at-1.94V. The stability of the material was investigated and as shown in fig. 7, the relative standard deviation was 2.7% for 15 consecutive scans, indicating that the ECL stability of the material was good.

From the peak potentials of the two luminescent probes, Ru (bpy)₃ ²⁺The MOF generates an optical signal at the positive electrode, the AuAgNCs generates an optical signal at the negative electrode, and the luminous potentials of the two electrodes are greatly separated, so that the two markers can be simultaneously detected according to potential resolution.

The performance of the ECL sensing platform was studied and, as shown in FIG. 8, the electrode modified with a mixture of two luminescent probes contained 0.1M K as compared to the bare electrode₂S₂O₈And 10mM TPrA in 0.1M PBS (pH 7.4) with higher ECL signal; in addition, after gold is electrodeposited on the glassy carbon electrode, ECL signals displayed by the electrodes modified by the two luminescent probes are obviously improved, and the electrodeposited gold particles have an enhancement effect on the two probes.

Manufacturing method of ECL sensing platform electrode

Polishing, ultrasonically treating and cleaning a glassy carbon electrode to obtain a clean electrode surface; the glassy carbon electrode was placed in 5mM HAuCl₄Applying a constant voltage of-0.2V to carry out electrochemical deposition so as to uniformly deposit a layer of nano gold particles on the surface of the electrode.

The specific detection process for simultaneously detecting two bladder cancer protein markers in the embodiment is as follows:

constructing an ECL sensing platform: modifying two pre-prepared capture probes on an electrode by utilizing Au-S covalent bond; after PBS is washed, 10 mu L of Mercaptoethanol (MCH) is dripped, and the mixture is kept stand for 30min to seal the non-specific binding sites of the electrode interface, so that an ECL sensing platform is constructed; the construction process of the ECL sensing platform is characterized by CV and EIS respectively, a CV curve of different modification processes is shown in FIG. 9, an EIS curve of different modification processes is shown in FIG. 10, and successful preparation of the sensor is indicated from CV curve current change and EIS impedance value change.

And (3) dropwise adding an antigen and a functional luminescent probe for detection: respectively taking 10 mu L of a series of antigen NuMA1 and AuAgNCs mixtures with concentration (0.001-0.5 ng/mL) and dropwise adding the mixtures on the constructed ECL sensing platform, and incubating for 1h at 37 ℃; after PBS cleaning, respectively dripping 10 μ L of the two functional luminescent probes constructed above, placing at 37 ℃ for further incubation for 1h, after PBS cleaning, placing the modified electrode in a solution containing 10mM TPrA and 0.1M K₂S₂O₈In 0.1M PBS (pH 7.4) for ECL detection.

The prepared ECL sensing platform is used as a working electrode, an Ag/AgCl electrode is used as a reference electrode, a Pt wire is used as an auxiliary electrode, and cyclic voltammetry scanning voltage is used, wherein the voltage range is-2.0-1.3V, the scanning speed is 0.5V/s, and the high voltage of a photomultiplier is 800V.

Setting antigen NuMA1 sample solution and antigen CFHR1 sample solution with different concentration gradients, and setting five test groups in total, wherein the first group is sample solution formed by mixing NuMA1 sample solution with the concentration gradient of 0.001ng/ml and sample solution with the concentration gradient of 0.001ng/ml, the second group is sample solution formed by mixing NuMA1 sample solution with the concentration gradient of 0.005ng/ml and sample solution with the concentration gradient of 0.005ng/ml, the third group is sample solution formed by mixing NuMA1 sample solution with the concentration gradient of 0.01ng/ml, the fourth group is sample solution formed by mixing NuMA1 sample solution with the concentration gradient of 0.05ng/ml and sample solution with the concentration gradient of 0.5ng/ml, and the fifth group is sample solution formed by mixing NuMA1 sample solution with the concentration gradient of 0.5ng/ml and sample solution with the concentration gradient of 0.005 ng/ml; the ECL detection curves detected by the five groups of mixed sample solutions are shown in fig. 11, the left signal curve is an ECL detection curve of antigen NuMA1 with different concentrations, and the right signal curve is an ECL detection curve of antigen CFHR1 with different concentrations; the corresponding ECL signals increased progressively with increasing antigen concentration, indicating that more functionalized probe was introduced to the electrode with increasing antigen concentration, resulting in a stronger ECL signal.

According to the detection results of fig. 11, the linear relationship between ECL signal intensity and the concentrations of two antigens, NuMA1 and CFHR1, was plotted, wherein fig. 12 is a linear relationship between ECL signal intensity and NuMA1 antigen concentration; FIG. 13 is a linear plot of ECL signal intensity versus CFHR1 antigen concentration; as can be seen from fig. 12 and 13, the ECL intensity has good correlation with the concentrations of both NuMA1 and CFHR1 antigens in the range of 0.001-0.5ng/mL, the correlation coefficients are 0.993 and 0.945 respectively, and the detection limits are 0.14pg/mL and 0.11pg/mL respectively (S/N ═ 3), indicating that the constructed sensing platform has good specificity and higher sensitivity for the simultaneous detection of both antigens.

As shown in fig. 14, the ECL signal intensities generated by the two antigen detections in the fourth mixed sample group are less than 3.0% of the relative standard deviation after 23 consecutive scans, which indicates that the prepared ECL sensor has good stability.

Sequence listing

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Claims (10)

1. A bladder cancer protein marker activity detection kit is characterized in that: the kit comprises an electrode, a functional luminescent probe for specifically recognizing the bladder cancer protein marker and a capture probe for specifically recognizing the bladder cancer protein marker, wherein the capture probe, the bladder cancer protein marker and the functional luminescent probe can form a sandwich structure through immune combination, and the electrode is formed by a glassy carbon electrode electrodeposited gold;

wherein the functionalized luminescent probe comprises at least: a first functionalized luminescent probe and a second functionalized luminescent probe; the first functionalized luminescent probe is made of a first luminophor modified by a class I secondary antibody of a first protein marker; the second functionalized luminescent probe is prepared by modifying a second luminophor by a II-type secondary antibody of a second protein marker; the specific modification mode is that the second antibody is modified on the first luminophor or the second luminophor by growing gold on the first luminophor or the second luminophor in situ and utilizing the Au-N bond effect between the nano gold particles and protein;

wherein the capture probe comprises at least: the first capture probe is made of a class I primary-resistant modified DNA tetrahedron; the second capture probe is made of a type II primary anti-modification DNA tetrahedron; the DNA tetrahedron is formed by self-assembling four DNA fragments, and the formed DNA tetrahedron structure has a mercapto functional group and an amino functional group, wherein the mercapto functional group is modified on the electrode of the electrodeposited gold through Au-S covalent bond; the amino functional group modifies the primary antibody on the DNA tetrahedron structure through glutaraldehyde.

2. The kit for detecting the activity of the bladder cancer protein marker according to claim 1, wherein the DNA tetrahedron is formed by self-assembly of four DNA fragments, and the sequences of the four DNA fragments are shown as SEQ ID No.1, SEQ ID No.2, SEQ ID No.3 and SEQ ID No. 4.

3. The kit for detecting the activity of the bladder cancer protein marker according to claim 2, wherein the preparation process of the DNA tetrahedron comprises: the four DNA fragment sequences were separated in a molar ratio of 1: 1: 1: 1 in TM buffer solution, annealing at 95 ℃ for 5min, and then cooling to 4 ℃ to form DNA tetrahedron.

4. The kit for detecting the activity of the bladder cancer protein marker according to claim 3, wherein the preparation process of the capture probe comprises: adding glutaraldehyde solution into the DNA tetrahedron, reacting at room temperature for 30min, then respectively adding class I primary antibody and class II primary antibody, continuing to react for 30min, and ultrafiltering the obtained solution through an ultrafiltration tube to obtain the first capture probe and the second capture probe.

5. The bladder cancer protein marker of claim 1The assay kit for detecting the activity of a target is characterized in that the first luminophor and the second luminophor are both selected from Ru (bpy)₃ ²⁺Luminol, AuAgNCs, Ru (bpy)₃ ²⁺-any of MOF or CdS quantum dots.

6. The kit for detecting the activity of a bladder cancer protein marker according to claim 5, wherein when the first luminophore is AuAgNCs, the second luminophore is luminol.

7. The kit for detecting the activity of a bladder cancer protein marker according to claim 5, wherein the first illuminant is Ru (bpy)₃ ²⁺-MOF, the second luminophore is AuAgNCs.

8. The kit for detecting the activity of the bladder cancer protein marker according to claim 1, wherein the electrode preparation process comprises: polishing, ultrasonically treating and cleaning a glassy carbon electrode to obtain a clean electrode surface; the glassy carbon electrode was placed in 5mM HAuCl₄And applying a constant voltage of-0.2V to perform electrochemical deposition so as to uniformly deposit a layer of nano gold particles on the surface of the electrode to form the electrode.

9. The kit for detecting the activity of the bladder cancer protein marker according to claim 1, wherein the kit for detecting the activity of the bladder cancer protein marker comprises:

a first reagent container comprising the electrodeposited gold modified electrode;

a second reagent container comprising the capture probe;

a third reagent container comprising a functionalized luminescent probe;

a fourth reagent container comprising phosphate buffered saline, PBS;

a fifth reagent container comprising phosphate buffered saline PBS, TPrA, and K₂S₂O₈。

10. A method for simultaneously detecting multiple bladder cancer protein markers for non-diagnostic purposes, which is characterized by comprising the following steps:

step S1, obtaining a sample extracting solution to be detected;

step S2, modifying the capture probe of claim 1 by Au-S covalent bond to construct ECL sensing platform on the electrode of claim 1;

step S3, dropwise adding the sample extracting solution to be detected on the ECL sensing platform constructed in the step S2 for incubation;

step S4, washing the ECL sensing platform incubated in the step S3 with PBS, dripping the functionalized luminescent probe of claim 1, and continuing incubation;

and step S5, washing the ECL sensing platform incubated in the step S4 by PBS, and then carrying out ECL detection.

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