CN111781259A - Preparation of electrochemical luminescence sensor capable of simultaneously detecting two sialylated glycans - Google Patents

Preparation of electrochemical luminescence sensor capable of simultaneously detecting two sialylated glycans Download PDF

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CN111781259A
CN111781259A CN202010630194.9A CN202010630194A CN111781259A CN 111781259 A CN111781259 A CN 111781259A CN 202010630194 A CN202010630194 A CN 202010630194A CN 111781259 A CN111781259 A CN 111781259A
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于超
朱启豪
毛巍然
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Chongqing Medical University
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Abstract

And (3) preparing an electrochemiluminescence sensor capable of detecting two sialylated glycans simultaneously. The invention successfully prepares novel luminescent composite materials Au @ BSA MSs-Luminol and ZIF-8-TCPP, and develops a potential resolution type electrochemical luminescence biosensor for simultaneously detecting alpha 2, 3-sialylated glycan and alpha 2, 6-sialylated glycan. Luminol (Luminol) and meso-tetrakis (4-carboxyphenyl) porphine (TCPP) are capable of emitting optical signals at positive and negative potentials, respectively. The bovine serum albumin-doped gold microspheres (Au @ BSA MSs) have a large number of active sites and excellent conductivity, can immobilize more biological materials and obviously improve the signal of luminol. The zeolite imidazolate framework-8 (ZIF-8) has a stable framework structure and a rough surface, and can immobilize and provide more active sites for immobilizing biological materials and stabilize an optical signal of meso-tetra (4-carboxyphenyl) porphine. In addition, based on the design of a magnetic capture probe 3-aminophenylboronic acid-magnetic microspheres (APBA-MMs), the sensor construction process is carried out in solution, and compared with the construction on electrodes, the sensor has a wider linear range.

Description

Preparation of electrochemical luminescence sensor capable of simultaneously detecting two sialylated glycans
Technical Field
The invention relates to a preparation method and application of an electrochemical luminescence sensor for clinically and quantitatively detecting alpha 2, 3-sialylated glycan and alpha 2, 6-sialylated glycan, and belongs to the field of electrochemical detection.
Background
Sialic acids are reported to be commonly present on glycoconjugates on cell surfaces. Variations in α 2, 3-sialylation and α 2, 6-sialylation may be associated with the development and progression of a variety of cancers, such as gastric, pancreatic, liver and colon cancers, among others. When cancer cells undergo apoptosis, sialylated glycans on their cell surfaces are released into the blood, which leads to elevated levels of α 2, 3-sialylated glycans and α 2, 6-sialylated glycans in the blood. Sensitive detection of α 2, 3-sialylated glycans and α 2, 6-sialylated glycans in blood would therefore be of great significance for early diagnosis of relevant cancers. Both α 2, 3-sialylated glycans and α 2, 6-sialylated glycans have now been considered as key biological targets for certain cancers in early clinical diagnosis. Therefore, it is important to explore a new method for sensitive detection of cancer in early diagnosis and treatment.
To date, only a few conventional assays have been available for the detection of α 2, 3-sialylated glycans and α 2, 6-sialylated glycans. The main reason is that sialylated glycans are chemically diverse by various enzymatic reactions and therefore difficult to isolate and quantify. Moreover, these methods require expensive equipment, take a lot of testing time, and require specialized staff, which greatly limits their clinical applications. In recent years, some researchers have developed electrochemical sensor methods to detect these two saccharides, but most of these methods can only detect one glycan. If only one sialylated glycan is used as a target, false positive results may result, thereby affecting the accuracy of the clinical diagnosis. Therefore, it is highly desirable to develop a new method to improve the efficiency and reliability of detection of sialylated glycans.
In recent years, since the electrochemical luminescence sensor has many excellent characteristics such as high sensitivity, low cost, good specificity and easy operability, it has gained much attention in the field of trace analysis. We have found that in electrochemiluminescence experiments different luminophores will emit light signals at different potentials, which will give the possibility to detect two targets simultaneously. Inspired by the fact, we intend to design a potential-resolved electrochemical biosensor to detect both α 2, 3-sialylated glycans and α 2, 6-sialylated glycans. The key element is to find two kinds of luminophors capable of generating different electric potentials to generate light signals.
Luminol (Luminol) is widely used in electrochemiluminescence analysis due to its high quantum yield. Based on relevant literature and preliminary experimental results, we found that it can be combined with H2O2In response, an optical signal is generated at a positive potential. Meso-tetrakis (4-carboxyphenyl) porphine (TCPP) is a porphyrin derivative with strong photoluminescence and is rarely used for sensor construction due to instability of the emitted light signal. In experiments we found that it can be used with K2S2O8Reacts and then emits an optical signal at a negative potential. Through extensive testing, we observed that when dual coreactant H was present in the reaction system2O2And K2S2O8The lumineol and TCPP may generate optical signals at positive and negative potentials, respectively. This lays the foundation for the potential resolution sensor that we designed.
Gold nanomaterials have gained extensive attention and application in the field of biosensors due to their excellent conductivity, catalytic activity and biocompatibility. In this study, we successfully synthesized bovine serum albumin-doped gold microspheres-luminol (Au @ BSA MSs), which not only maintained good conductivity and stability of gold nanomaterials, but also provided many useful functional groups to link biomolecules. In addition, we can see from the scanning electron microscope and transmission electron microscope images, the surface of Au @ BSA MSs is very rough. Protrusions, tips and corner points on these rough surfaces in catalysis H2O2May have good catalytic activity in reactions that generate Reactive Oxygen Species (ROSs). It can also be seen by experiments that after the ligation of Luminol to Au @ BSA MSs, Luminol was obtainedHigher ECL signal than pure luminel. Therefore, the signal probe is made of the Au @ BSA MSs-Luminol composite luminescent material, so that the sensitivity of the biosensor is greatly improved. Zeolitic imidazolium salt frameworks (ZIFs) are a subclass of Metal Organic Frameworks (MOFs). In this study, we prepared zeolitic imidazolate framework-8 (ZIF-8) crystals to attach TCPP to form a composite luminescent material ZIF-8-TCPP. In the following experiments, it was found that the ECL signal of ZIF-8-TCPP was more stable than that of pure TCPP (FIG. 2D), probably because ZIF-8 could improve the stability and solubility of TCPP in water.
The signal probe constructed by the materials greatly enhances the sensitivity and stability of the sensor, the specific detection capability of the sensor on a target object is improved by the design of a sandwich method, and two target objects can be simultaneously detected by the design based on potential resolution, so that the clinical efficiency and the reliability of the sensor are greatly improved.
Disclosure of Invention
1. The invention aims to provide a preparation method and application of an electrochemiluminescence sensor for detecting alpha 2, 3-sialylated glycan and alpha 2, 6-sialylated glycan, which provide a basis for early detection and treatment of various cancers clinically and are characterized by comprising the following steps of:
(1) preparing a bovine serum albumin-doped gold microsphere-Luminol (Au @ BSA MSs-Luminol) composite material and preparing a signal probe 1;
(2) preparing a zeolite imidazole acid ester framework-8-meso-tetra (4-carboxyphenyl) porphin (ZIF-8-TCPP) composite material and preparing a signal probe 2;
(3) preparing 3-aminophenylboronic acid-magnetic microspheres (APBA-MMs) used as capture probes;
(4) establishing a potential-discrimination type electrochemical luminescence sensor, simultaneously detecting alpha 2, 3-sialylated glycan and alpha 2, 6-sialylated glycan, and drawing a standard curve.
2. The preparation process of the two composite materials Au @ BSA MSs-Luminol and ZIF-8-TCPP, the two corresponding signal probes and the magnetic capture probe comprises the following steps:
(1) preparation of bovine serum albumin-doped gold microsphere-luminol composite material and signal probe 1:
a25 mL beaker was charged with 10mL of ultrapure water, and then 50mg of Bovine Serum Albumin (BSA) powder was added to the beaker. The mixture was stirred with a magnetic stirrer at 600r for 10 minutes. 10mL of a 10mM solution of chloroauric acid (HAuCl4) was then added to the solution and stirring was continued. After mixing well, 50mg of Ascorbic Acid (AA) was quickly poured into the above mixture and we can see that the solution turned gray rapidly. After stirring for 15 minutes, the mixture was washed three times by centrifugation with ultrapure water to obtain Au @ BSA MSs.
1.5mg of prepared Au @ BSA MS was added to 1mL of ultrapure water and dispersed with sonication. Then 250 μ L of 15% Glutaraldehyde (GA) was added to the solution and shaken at room temperature for 3 hours. The product was centrifuged and washed 3 times and then dissolved in 1mL of ultrapure water. Next, 1mL of 0.01M luminol solution was added to the above solution and shaken in the dark for 4 hours. And centrifugally washing the solution for three times by using ultrapure water to obtain Au @ BSA MSs-Luminol.
Au @ BSA MSs-Luminol was dissolved in 2mL of water and 50. mu.L of 50mg mL was added-1NHS and 50. mu.L 50mgmL-1And EDC. The BSA was activated by shaking at 4 ℃ for 30 minutes. Then 40. mu.L of 2.1mg mL-1Huai lectin (MAL) was added to the above solution and shaken for 8 hours. After washing and centrifugation, the mixture was dispersed in 2mL of a PBS solution having a pH of 7.4. Finally, to block non-specific binding sites, 100. mu.L of 0.25 wt% BSA was added to the above solution and shaken for 1 hour. After washing and centrifugation, the final product of signal probe 1 was redispersed in 1mL PBS (pH 7.4).
(2) Preparation of zeolite imidazolate framework-8-meso-tetrakis (4-carboxyphenyl) porphine composite and signal probe 2:
0.074g of zinc nitrate hexahydrate (Zn (NO)3)2·6H2O) and 1.23g of 2-methylimidazole were dissolved in 1mL and 9mL of ultrapure water, respectively. The former solution was then added to the latter solution and after stirring for a few minutes we can see that the solution gradually turned milky white. After stirring for a further 24 hours, the precipitate was washed with ultrapure water and centrifuged, and then placed under vacuum at 60 ℃In the cabinet for 12h, zeolite imidazate framework-8 crystal (ZIF-8) is obtained.
2mg of the prepared ZIF-8 crystals were dissolved in 1mL of ultrapure water by sonication. Then 1mL of 0.01M TCPP was added to the above solution and stirred in the dark for 6 hours. After 3 times of centrifugal washing, the luminophore ZIF-8-TCPP obtained was redispersed in 2mL of ultrapure water and 50. mu.L of 50mg mL was added-1NHS and 50. mu.L 50mg mL-1And EDC. The carboxyl groups of TCPP were activated by shaking at 4 ℃ for 30 minutes. Next, 40. mu.L of 2.1mg mL-1SNA was added to the above solution and after gentle shaking at 4 ℃ for 8 hours, washed and centrifuged, dispersed in 2mL of PBS solution at pH 7.4. Finally, to block non-specific binding sites, 100. mu.L of 0.25 wt% BSA was added to the above solution and shaken for 1 hour. After washing and centrifugation, the final product of signal probe 2 was redispersed in 1mL PBS (pH 7.4).
(3) Preparation of a capture probe 3-aminophenylboronic acid-magnetic microsphere (APBA-MMs):
first, 1mL of 5mg mL-1MMs，50μL 50mg mL-1NHS and 50. mu.L 50mg mL-1EDC was gently mixed at 4 ℃ for 30 minutes to activate the carboxyl groups on the surface of the MMs. Next, 50. mu.L of 50mgmL-1APBA was added to the above mixture and stirred for 4 h. Then, 100. mu.L of 0.25 wt% BSA solution was added to the above mixture and shaken gently at 4 ℃ for 1 h. Finally, the black precipitate was washed several times with ultrapure water and collected by magnetic separation. Finally, the capture probe 3-aminophenylboronic acid-magnetic microspheres (APBA-MMs) were redispersed in 1mL of ultrapure water for future use.
3. The invention also provides a method for detecting alpha 2, 3-sialylated glycans and alpha 2, 6-sialylated glycans by using the electrochemiluminescence sensor, and a standard curve is drawn, wherein the method comprises the following steps:
(1) mu.L of capture probe (APBA-MMs) was added to 100. mu.L of a mixture containing different concentrations of α 2, 3-sialylated glycan and α 2, 6-sialylated glycan and they were then incubated for 2 hours at 37 ℃.
(2) After magnetic separation and washing, it was redissolved to 100. mu.L. Then, 10. mu.L of Signaling Probe 1 and Signaling Probe 2 were added to the above solution, and incubation was continued for 2 hours.
(3) The non-attached material was removed by magnetic separation and washing, and was redissolved to 100. mu.L. Then, 10. mu.L of the above solution was dropped onto the surface of the electrode and left to dry at room temperature.
(4) The electrode was placed in 3mL of 0.1M PBS (containing 80mM K)2S2O8And 12.5mM H2O2) The experiment was performed to measure the magnitude of two ECL signals it produces. Scanning potential a two-step scanning method of 0V to-1.9V and 0V to 0.6V was employed, and then the voltage of the photomultiplier tube was set to 800V.
(5) Working curves were drawn according to the linear relationship of the resulting ECL signal values with α 2, 3-sialylated glycan and α 2, 6-sialylated glycan concentrations.
Compared with the prior art, the invention establishes the preparation method and the application of the electrochemical luminescence sensor for detecting the alpha 2, 3-sialylated glycan and the alpha 2, 6-sialylated glycan, and has the outstanding characteristics that:
(1) the bovine serum albumin doped gold microsphere-Luminol (Au @ BSA MSs-Luminol) composite material is used as a signal material, so that the Luminol optical signal is effectively improved, the immobilization amount of biomolecules is improved due to the huge surface area of Au @ BSAMSs, and the sensitivity and the detection range of the sensor are further improved;
(2) the zeolite imidazole acid ester framework-8-meso-tetra (4-carboxyphenyl) porphin (ZIF-8-TCPP) based composite material is used as a signal material, so that an optical signal of the TCPP is effectively stabilized, and the stability of the sensor is improved;
(3) the sensor can simultaneously detect the alpha 2, 3-sialylated glycan and the alpha 2, 6-sialylated glycan, and more reliable detection results are brought to clinical early diagnosis of cancer;
(4) the electrochemiluminescence sensor prepared by the method can provide more basis for early diagnosis of various cancers. In addition, the method is simple, convenient and quick, and is convenient for realizing commercialization, thereby promoting the development of precise medicine.
(5) Different antigen antibodies can be immobilized on the signal probe and the capture probe by using the same nano material and the same modification method, so that the detection of various biomolecules is realized, and a more comprehensive basis is provided for the diagnosis of diseases.
Drawings
Fig. 1 is a schematic diagram of the construction of the potential-resolved electrochemiluminescence sensor of the present invention.
FIG. 2 is a scanning electron micrograph, a transmission electron micrograph and an EDS energy spectrogram of two carrier materials of bovine serum albumin-doped gold microspheres and zeolite imidazolate frameworks-8 synthesized by the invention.
Fig. 3 is a verification diagram of the feasibility of the potential-resolved electrochemiluminescence sensor of the present invention and a verification diagram of the advantages of the synthesized material.
FIG. 4 is a linear relationship between the intensity and concentration of an electrochemiluminescence signal obtained by the potential-resolved electrochemiluminescence sensor of the present invention when detecting α 2, 3-sialylated glycan and α 2, 6-sialylated glycan, and the specificity and stability of the sensor.
Detailed Description
The invention is further illustrated by the following examples, which are not intended to be limiting.
The main chemical reagents used in the examples of the present invention are as follows:
α 2, 3-sialylated glycan and α 2, 6-sialylated glycan are available from Tokyo chemical industry, biotinylated Sambucus nigra lectin (bio-SNA) and Huai lectin (MAL) are available from Vector Laboratories, Inc., USA, meso-tetra (4-carboxyphenyl) porphine (TCPP), luminol, zinc nitrate hexahydrate (Zn (NO)3)2·6H2O), 2-methylimidazole (mim), 3-aminophenylboronic acid (APBA), Ascorbic Acid (AA), Bovine Serum Albumin (BSA), N- (3-dimethylaminopropyl) -N-ethylcarbodiimide hydrochloride (EDC), N-hydroxysuccinimide (NHS) and potassium persulfate (K)2S2O8) Both from alatin; carboxy-functionalized Fe3O4Magnetite microspheres (C-MMs) and chloroauric acid trihydrate (HAuCl)4·3H2O) from michelin biochemistry, inc; hydrogen peroxide (H2O2), methanol and ethanolAlcohol was purchased from Chongqing Chundong chemical group, Inc. Phosphate Buffer Solution (PBS) is KH containing 0.15M NaCl2PO4And Na2HPO4Is prepared by the following steps. Other chemical reagents were analytically pure and used without further purification. A healthy human serum sample was provided by university city hospital of Chongqing medical university (Chongqing, China). All aqueous solutions were made using ultra pure water supplied by Millipore Milli-Q system (> 18.2M. OMEGA. cm, USA, www.millipore.com).
The equipment and technical parameters used are as follows:
the electrochemical experiments were all carried out on an electrochemical workstation (CHI660E) (chenhua instruments ltd, shanghai, china). The electrochemiluminescence experiment was performed on an MPI-E electrochemiluminescence analyzer (Remax electronics high-tech. Co., Ltd., Seisan, China). A Saturated Calomel Electrode (SCE) was used as a reference electrode throughout the electrochemical experiment, and an Ag/AgCl (saturated KCl) electrode was used as a reference electrode throughout the ECL detection. The platinum wire electrode was the counter electrode and the bare glass carbon electrode (GCE, 4 mm diameter) was the working electrode. The scan was performed using Cyclic Voltammetry (CV) ranging from 0V to-1.9V and 0V to 0.6V, with the voltage of the photomultiplier tube (PMT) set at 800V.
Example 1
Step 1, dispersing the prepared signal probe 1(MAL-Au @ BSA MSs-Luminol) in 1mL PBS (pH 7.4) for later use.
And 2, dispersing the prepared signal probe 2(SNA-ZIF-8-TCPP) in 1mL of PBS (pH 7.4) for later use.
Step 3. mu.L of prepared capture probes (APBA-MMs) were added to reaction wells containing 100. mu.L of different concentrations of α 2, 3-sialylated glycans and α 2, 6-sialylated glycans, which were then incubated for 2 hours at 37 ℃.
Step 4. after magnetic separation and washing, it was redissolved to 100. mu.L. Then, 10. mu.L of the signal probe 1 and the signal probe 2 prepared in step 1 and step 2, respectively, was added to the above solution, and incubation was continued for 2 hours.
And 5, removing non-attached substances by magnetic separation and washing, and dissolving the substances again to 100 mu L. Then, 10. mu.L of the above solution was dropped onto the surface of the electrode and left to dry at room temperature.
Step 6. Place the electrode in 3mL of 0.1M PBS (containing 80mM K)2S2O8And 12.5mM H2O2) The experiment is carried out, and two optical signal values generated by positive and negative potentials are measured. The scanning method employs Cyclic Voltammetry (CV) in which potentials are scanned in two steps of 0V to-1.9V and 0V to 0.6V, and then the voltage of a photomultiplier is set to 800V.
And 7, drawing a working curve according to the linear relation between the optical signal value obtained by the positive and negative potentials and the concentrations of the alpha 2, 3-sialylated glycan and the alpha 2, 6-sialylated glycan.
Step 8. assay results showed that α 2, 3-sialylated glycan and α 2, 6-sialylated glycan were each present at a concentration of 10fg mL-1-100μg mL-1Has a linear relation in the range of (1), the linear correlation coefficients are respectively 0.9957 and 0.9929, and the detection limit is respectively 2.1fg mL-1And 3.3fg mL-1。
Step 9, storing the sensor at 4 ℃, intermittently detecting the optical signal response of the sensor, and storing the sensor for 28 days until the response values are 90.86% and 90.04% of the initial detection value, which indicates that the sensor has good stability;
step 10, taking 5 immunosensors prepared in the same batch, and carrying out 1pg mL treatment on the immunosensors under the same condition-1The measurement was carried out 3 times for each of α 2, 3-sialylated glycans and α 2, 6-sialylated glycans, respectively, and the results showed good sensor reproducibility.
Step 11, the sensor of the invention is used for detecting the alpha 2, 3-sialylated glycan and the alpha 2, 6-sialylated glycan under the condition that other biomolecules exist in blood, and as a result, the change of optical signals of the alpha 2, 3-sialylated glycan and the alpha 2, 6-sialylated glycan is hardly influenced by the existence of other biomolecules, which shows that the sensor has good specificity and can well distinguish a target object from an interfering object.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.

Claims (3)

1. The preparation of the electrochemical luminescence sensor capable of simultaneously detecting two sialylated glycans is characterized by comprising the following steps:
(1) preparing a bovine serum albumin-doped gold microsphere-Luminol (Au @ BSA MSs-Luminol) composite material and preparing a signal probe 1;
(2) preparing a zeolite imidazole acid ester framework-8-meso-tetra (4-carboxyphenyl) porphin (ZIF-8-TCPP) composite material and preparing a signal probe 2;
(3) preparing a magnetic capture probe 3-aminobenzene boric acid-magnetic microsphere (APBA-MMs);
(4) establishing a potential-discrimination type electrochemical luminescence sensor, simultaneously detecting alpha 2, 3-sialylated glycan and alpha 2, 6-sialylated glycan, and drawing a standard curve.
2. The process for preparing the composite luminescent material Au @ BSA MSs-Luminol, ZIF-8-TCPP and the corresponding signal probe and magnetic capture probe according to claim 1, which comprises the following steps:
(1) preparation of bovine serum albumin-doped gold microsphere-luminol composite luminophore and signal probe 1:
a25 mL beaker was charged with 10mL of ultrapure water, and then 50mg of Bovine Serum Albumin (BSA) powder was added to the beaker. The mixture was stirred with a magnetic stirrer at 600r for 10 minutes. 10mL of a 10mM solution of chloroauric acid (HAuCl) were then added4) Add to the above solution and continue stirring. After mixing well, 50mg of Ascorbic Acid (AA) was quickly poured into the above mixture and we can see that the solution turned gray rapidly. After stirring for 15 minutes, the mixture was washed three times by centrifugation with ultrapure water to obtain Au @ BSA MSs.
1.5mg of prepared Au @ BSAMS was added to 1mL of ultrapure water and dispersed with sonication. Then 250 μ L of 15% Glutaraldehyde (GA) was added to the solution and shaken at room temperature for 3 hours. The product was centrifuged and washed 3 times and then dissolved in 1mL of ultrapure water. Next, 1mL of 0.01M luminol solution was added to the above solution and shaken in the dark for 4 hours. And centrifugally washing the solution for three times by using ultrapure water to obtain Au @ BSA MSs-Luminol.
Au @ BSA MSs-Luminol was dissolved in 2mL of water and 50. mu.L of 50mg mL was added-1NHS and 50. mu.L 50mg mL- 1And EDC. The BSA was activated by shaking at 4 ℃ for 30 minutes. Then 40. mu.L of 2.1mg mL-1Huai lectin (MAL) was added to the above solution and shaken for 8 hours. After washing and centrifugation, the mixture was dispersed in 2mL of a PBS solution having a pH of 7.4. Finally, to block non-specific binding sites, 100. mu.L of 0.25wt% BSA was added to the above solution and shaken for 1 hour. After washing and centrifugation, the final product of Signaling probe 1 was redispersed in 1mLPBS (pH 7.4).
(2) Preparation of zeolitic imidazolate framework-8-meso-tetrakis (4-carboxyphenyl) porphine complex luminophore and signal probe 2:
0.074g of zinc nitrate hexahydrate (Zn (NO)3)2·6H2O) and 1.23g of 2-methylimidazole were dissolved in 1mL and 9mL of ultrapure water, respectively. The former solution was then added to the latter solution and after stirring for a few minutes we can see that the solution gradually turned milky white. After stirring was continued for 24 hours, washing with ultrapure water and centrifugation were carried out, and then the precipitate was placed in a vacuum cabinet at 60 ℃ for 12 hours to obtain zeolitic imidazolate framework-8 crystals (ZIF-8).
2mg of the prepared ZIF-8 crystals were dissolved in 1mL of ultrapure water by sonication. Then 1mL of 0.01M TCPP was added to the above solution and stirred in the dark for 6 hours. After 3 times of centrifugal washing, the luminophores ZIF-g-TCPP obtained were redispersed in 2mL of ultrapure water and 50. mu.L of 50mg mL was added-1NHS and 50. mu.L 50mg mL-1And EDC. The carboxyl groups of TCPP were activated by shaking at 4 ℃ for 30 minutes. Next, 40. mu.L 2.1mgmL of the suspension was added-1SNA was added to the above solution and after gentle shaking at 4 ℃ for 8 hours, washed and centrifuged, dispersed in 2mL of PBS solution at pH 7.4. Finally, to block non-specific binding sites, 100. mu.L of 0.25 wt% BSA was added to the above solution and shaken for 1 hour. After washing and centrifugation, the final product of signal probe 2 was redispersed in 1mL PBS (pH 7).4) In (1).
(3) Preparation of a capture probe 3-aminophenylboronic acid-magnetic microsphere (APBA-MMs):
first, 1mL of 5mg mL-1MMs，50μL50mg mL-1NHS and 50. mu.L 50mg mL-1EDC was gently mixed at 4 ℃ for 30 minutes to activate the carboxyl groups on the surface of the MMs. Next, 50. mu.L of 50mgmL-1APBA was added to the above mixture and stirred for 4 h. Then, 100. mu.L of 0.25 wt% BSA solution was added to the above mixture and shaken gently at 4 ℃ for 1 h. Finally, the black precipitate was washed several times with ultrapure water and collected by magnetic separation. Finally, the capture probe 3-aminophenylboronic acid-magnetic microspheres (APBA-MMs) were redispersed in 1mL of ultrapure water for future use.
3. The method for establishing a potential-resolved electrochemiluminescence sensor according to claim 1, wherein the α 2, 3-sialylated glycan and the α 2, 6-sialylated glycan are simultaneously detected, and a standard curve is drawn, the method comprising the steps of:
(1) mu.L of capture probe (APBA-MMs) was added to 100. mu.L of a mixture containing different concentrations of α 2, 3-sialylated glycan and α 2, 6-sialylated glycan and they were then incubated for 2 hours at 37 ℃.
(2) After magnetic separation and washing, it was redissolved to 100. mu.L. Then, 10. mu.L of Signaling Probe 1 and Signaling Probe 2 were added to the above solution, and incubation was continued for 2 hours.
(3) The non-attached material was removed by magnetic separation and washing, and was redissolved to 100. mu.L. Then, 10. mu.L of the above solution was dropped onto the surface of the electrode and left to dry at room temperature.
(4) The electrode was placed in 3mL of 0.1M PBS (containing 80mM K)2S2O8And 12.5mM H2O2) The experiment was performed to measure the magnitude of two ECL signals it produces. Scanning potential a two-step scanning method of 0V to-1.9V and 0V to 0.6V was employed, and then the voltage of the photomultiplier tube was set to 800V.
(5) Working curves were drawn according to the linear relationship of the resulting ECL signal values with α 2, 3-sialylated glycan and α 2, 6-sialylated glycan concentrations.
CN202010630194.9A 2020-06-24 2020-06-24 Preparation of electrochemical luminescence sensor capable of simultaneously detecting two sialylated glycans Pending CN111781259A (en)

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