CN115266894B - Ratio type electrochemical detection method of glycoconjugate - Google Patents
Ratio type electrochemical detection method of glycoconjugate Download PDFInfo
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Classifications
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
- G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
- G01N27/416—Systems
- G01N27/48—Systems using polarography, i.e. measuring changes in current under a slowly-varying voltage
Abstract
The invention relates to the technical field of electrochemical detection, and discloses a ratio type electrochemical detection method of a glycoconjugate, which comprises the following steps: s1: immobilizing the aptamer with the end modified with the electroactive probe for specific recognition and capture of the glycoconjugate, and adding a boric acid derivative of a second electroactive probe after the aptamer is combined with the target glycoconjugate; s2: the target glycoconjugates were quantitatively analyzed by measuring the ratio of the magnitude of the response currents of the two electroactive probes. The ratio electrochemical detection method of the saccharide conjugates is characterized in that a target saccharide conjugate is identified and captured by taking a nucleic acid aptamer with an end modified with an electroactive probe as an immobilized molecular recognition element, then a second electroactive probe is targeted and marked on the saccharide conjugate captured by the nucleic acid aptamer through borate affinity recognition interaction, and the content of the saccharide conjugate is detected by means of the ratio of the response current magnitudes of the two electroactive probes.
Description
Technical Field
The invention relates to the technical field of electrochemical detection, in particular to a ratio type electrochemical detection method of a glycoconjugate.
Background
The basic principle of the existing commercial methods for detecting glycoconjugates is antigen-antibody immune recognition reaction. However, antibodies suffer from the disadvantages of complex preparation process, large batch-to-batch variation, difficult modification, high price, poor stability, and the like. In contrast, the aptamer has the excellent characteristics of chemical synthesis, easy modification, low cost, good stability and the like, and has wider application prospect in the aspect of high-selectivity detection of the glycoconjugate. The electrochemical method has the advantages of simple equipment, quick response, low cost, high sensitivity, good selectivity, easy miniaturization and the like, and is attracting attention in the detection of the glycoconjugates. To achieve high sensitivity electrochemical aptamer detection of glycoconjugates.
The existing methods are generally by means of signal amplification by enzymes or nanomaterials. However, the enzymatic signal amplification has the defects of high cost, poor stability, complex labeling process and the like; the signal amplification assisted by the nano material has the defects of complex operation, high cost and the like due to the synthesis and surface biological functionalization of the nano material. Thus, achieving simple, rapid, low cost, high sensitivity, high selectivity electrochemical detection of glycoconjugates remains a challenge to be addressed.
Disclosure of Invention
The invention aims to provide a ratio-type electrochemical detection method of a glycoconjugate, in particular to a ratio-type electrochemical detection method of a glycoconjugate. The method is characterized in that a aptamer with an end modified with an electroactive probe is used as an immobilized molecular recognition element to specifically recognize and capture a glycoconjugate, after the glycoconjugate is combined with a target glycoconjugate, a second electroactive probe is further marked on the glycoconjugate captured by the aptamer in a targeted manner by means of borate affinity recognition interaction, and the simple, rapid, low-cost, high-sensitivity and high-selectivity ratio electrochemical detection of the glycoconjugate can be realized by measuring the ratio of the response currents of the two electroactive probes.
In order to achieve the above purpose, the present invention provides the following technical solutions:
a method for the ratiometric electrochemical detection of a glycoconjugate comprising the steps of:
s1: the aptamer with the end modified electroactive probe is immobilized for specific recognition and capture of the glycoconjugate, and after it has bound to the target glycoconjugate, a boronic acid derivative of the second electroactive probe is added.
S2: the target glycoconjugates were quantitatively analyzed by measuring the ratio of the magnitude of the response currents of the two electroactive probes.
Preferably, the aptamer in S1 is an oligonucleotide fragment capable of specifically recognizing and capturing the target glycoconjugate.
Preferably, the electroactive probe in S1 includes one of substances having redox activity, such as ferrocene, methylene blue, thionine, and the like.
Preferably, the saccharide conjugates in S1 include glycoproteins (e.g., alpha Fetoprotein (AFP), carcinoembryonic antigen (CEA), carbohydrate antigen 125 (CA 125), carbohydrate antigen 15-3 (CA 15-3), carbohydrate antigen 199 (CA 199), carbohydrate antigen 549 (CA 549), carbohydrate antigen 27.29 (CA 27.29), carbohydrate antigen 72-4 (CA 72-4), carbohydrate antigen 242 (CA 242), carbohydrate antigen 50 (CA 50), non-small cell lung cancer associated antigen (CYFRA 21-1), vascular endothelial growth factor, squamous cell carcinoma antigen, prostate Specific Antigen (PSA), human chorionic gonadotrophin, mucin-like, glycosylated albumin, glycosylated hemoglobin, lectin, pancreatic ribonuclease, immunoglobulin (i.e., antibody), plasminogen, copper blue protein, transferrin, erythropoietin, thyroid stimulating hormone, fibronectin, laminin, horseradish peroxidase (HRP), etc.), glycosylated proteins (e.g., glycosylated albumin and glycosylated hemoglobin), teichoic acid, polysaccharide, peptidoglycan, polysaccharide, membrane, tumor cell, exosome, bacteria, and the like.
Compared with the prior art, the invention has the beneficial effects that:
1. the ratio-type electrochemical detection method of the glycoconjugate has the advantages of simple operation, low cost, short detection time, high sensitivity, good selectivity, good reproducibility and the like.
2. The ratio-type electrochemical detection method of the glycoconjugate can realize high-sensitivity electrochemical detection of the glycoconjugate without adopting an additional signal amplification strategy.
3. The ratio-type electrochemical detection method of the glycoconjugates can be used for high-sensitivity and high-selectivity ratio-type electrochemical detection of other glycoconjugates only by replacing the aptamer sequences.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings that are needed in the description of the embodiments will be briefly described below, it being obvious that the drawings in the following description are only some embodiments of the present invention, and that other drawings may be obtained according to these drawings without inventive effort to a person skilled in the art.
FIG. 1 is a schematic diagram illustrating the process and principle of a ratio-based electrochemical detection method according to an embodiment of the present invention;
FIG. 2 is a square wave voltammogram of a different modified electrode in accordance with an embodiment of the present invention;
FIG. 3 is a graph showing the distribution of P, B and Fe elements according to the embodiment of the present invention;
FIG. 4 is a graph showing the ratio of the selective peak currents according to the embodiment of the present invention.
Detailed Description
The following description of the embodiments of the present invention will be made clearly and completely with reference to the accompanying drawings, in which it is apparent that the embodiments described are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
In the present invention, unless explicitly specified and limited otherwise, the terms "immobilized," "captured," and the like are to be construed broadly and may be, for example, fixedly attached, detachably attached, or integrally formed; can be directly connected or indirectly connected through an intermediate medium. The specific meaning of the above terms in the present invention can be understood by those of ordinary skill in the art according to the specific circumstances.
Embodiment one:
referring to fig. 1-4, the ratio electrochemical detection method of glycoconjugates provided by the embodiment of the invention comprises the following steps:
s1: immobilizing aptamer with end modified with electroactive probe for specific recognition and capture of saccharide conjugate, blocking the residual active site on the surface of electrode with blocking agent after capture, adding boric acid derivative of second electroactive probe after it is combined with target saccharide conjugate, wherein the aptamer is an oligonucleotide fragment capable of specifically recognizing and capturing target saccharide conjugate, the electroactive probe comprises ferrocene, methylene blue, thionine and other substances with oxidation-reduction activity, glycoconjugates include glycoproteins such as Alpha Fetoprotein (AFP), carcinoembryonic antigen (CEA), carbohydrate antigen 125 (CA 125), carbohydrate antigen 15-3 (CA 15-3), carbohydrate antigen 199 (CA 199), carbohydrate antigen 549 (CA 549), carbohydrate antigen 27.29 (CA 27.29), carbohydrate antigen 72-4 (CA 72-4), carbohydrate antigen 242 (CA 242), carbohydrate antigen 50 (CA 50), non-small cell lung cancer-associated antigen (CYFRA 21-1), vascular endothelial growth factor, squamous cell carcinoma antigen, prostate-specific antigen (PSA), human chorionic gonadotrophin, mucin-like, glycosylated albumin, glycosylated hemoglobin, lectin, pancreatic ribonuclease, immunoglobulins (i.e., antibodies), plasminogen, ceruloplasmin, transferrin, erythropoietin, thyroid stimulating hormone, fibronectin, laminin, horseradish peroxidase (HRP), etc.), glycosylated proteins such as glycosylated albumin and glycosylated hemoglobin, teichoic acid, lipopolysaccharide, peptidoglycan, capsular polysaccharide, circulating tumor cells, exosomes, bacteria, viruses, etc., specifically any one of them may be selected.
S2: the target glycoconjugates were quantitatively analyzed by measuring the ratio of the magnitude of the response currents of the two electroactive probes.
Feasibility study of ratio electrochemical detection method of glycoconjugates:
to verify the feasibility of the glycoconjugate ratio electrochemical detection method, saccharide antigen 15-3 (CA 15-3) was selected as the target glycoconjugate. 0.1 mu M of aptamer (MB-Apt) with a mercapto group-and methylene blue probe-modified end is dripped on the surface of a gold electrode and incubated for 30min; subsequently, the active sites remaining on the electrode surface were blocked with 1.0mM mercaptohexanol; subsequently, the electrode surface was incubated with 50U/mL of CA15-3 for 45min and immersed in 0.2mM 4- (ferrocene acetamido) phenyl) boronic acid (FcPBA) solution for 30min; finally, the resulting electrode was inserted into a 25mM lithium perchlorate solution and tested by Square Wave Voltammetry (SWV). As can be seen from fig. 2, for the fully modified gold electrode, two oxidation peaks were observed at-0.3V and 0.4V, corresponding to electrochemical oxidation of the methylene blue probe and ferrocene probe, respectively; if CA15-3 or FcPBA is not added when the surface of the electrode is modified, only an oxidation peak of the methylene blue probe can be observed; furthermore, if MB-Apt is not added for electrode surface modification, neither oxidation peak can be observed. It follows that the method can be used for the ratiometric electrochemical detection of glycoconjugates. Furthermore, as can be seen from the spectral analysis results shown in fig. 3, the electrode surface has a large amount of P, B and Fe elements, which is consistent with the presence of aptamer and FcPBA on the electrode surface.
Embodiment two:
the ratio-type electrochemical detection method of the glycoconjugate provided by the embodiment of the invention comprises the following steps of:
s1: immobilizing a nucleic acid aptamer with an end modified with an electroactive probe for specific recognition and capture of a glycoconjugate, blocking the residual active site on the surface of an electrode by using a blocking agent after capture, and adding a boric acid derivative of a second electroactive probe after the nucleic acid aptamer is combined with the target glycoconjugate, wherein the nucleic acid aptamer is an oligonucleotide fragment capable of specifically recognizing and capturing the target glycoconjugate, and the electroactive probe comprises substances with redox activity such as ferrocene, methylene blue, thionine and the like, and the glycoconjugate is: glycoprotein (such as Alpha Fetoprotein (AFP), carcinoembryonic antigen (CEA), carbohydrate antigen 125 (CA 125), carbohydrate antigen 15-3 (CA 15-3), carbohydrate antigen 199 (CA 199), carbohydrate antigen 549 (CA 549), carbohydrate antigen 27.29 (CA 27.29), carbohydrate antigen 72-4 (CA 72-4), carbohydrate antigen 242 (CA 242), carbohydrate antigen 50 (CA 50), non-small cell lung cancer-associated antigen (CYFRA 21-1), vascular endothelial growth factor, squamous cell carcinoma antigen, prostate-specific antigen (PSA), human chorionic gonadotrophin, mucin, mucoid, glycosylated albumin, glycosylated hemoglobin, lectin, pancreatic ribonuclease, immunoglobulin (i.e., antibody), plasminogen, ceruloplasmin, transferrin, erythropoietin, thyroid stimulating hormone, fibronectin, laminin, horseradish peroxidase (HRP) and the like), glycosylated protein (such as glycosylated albumin and glycosylated hemoglobin), teichoic acid, lipopolysaccharide, peptidoglycan, capsular polysaccharide, membrane polysaccharide, circulating tumor cells, exosomes, and bacteria.
S2: the target glycoconjugates were quantitatively analyzed by measuring the ratio of the magnitude of the response currents of the two electroactive probes.
Selective study of electrochemical aptamer detection method of glycoconjugates:
to evaluate the selectivity of the saccharide conjugate ratio electrochemical detection method, CA15-3 was selected as the target saccharide conjugate, and the ratio of peak current of ferrocene probe to methylene blue probe (I) corresponding to 50U/mL CA15-3, 10ng/mL Lipopolysaccharide (LPS), 10ng/mL hemoglobin (Hb), 10ng/mL carcinoembryonic antigen (CEA), 10ng/mL Glycated Albumin (GA), 10ng/mL Alpha Fetoprotein (AFP) or 100ng/mL glucose (glucose) was collected Ferrocene /I Methylene blue ). As can be seen from FIG. 4, only the target glycoconjugate (i.e., CA 15-3) produced a strong detection signal, while the other interfering components tested produced detection signals that were comparable to the blank without the target glycoconjugate. Thus, the saccharide conjugate ratio type electrochemical detection method has good selectivity.
The ratio electrochemical detection method of the saccharide conjugates provided by the embodiment of the invention uses the aptamer with the end modified with the electroactive probe as an immobilized molecule recognition element to recognize and capture the target saccharide conjugates, then targets and marks the second electroactive probe site on the saccharide conjugates captured by the aptamer through borate affinity recognition interaction, and detects the content of the saccharide conjugates by means of the ratio of the response current magnitudes of the two electroactive probes.
It is noted that relational terms such as first and second, and the like are used solely to distinguish one entity or action from another entity or action without necessarily requiring or implying any actual such relationship or order between such entities or actions. Moreover, the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus. Without further limitation, an element defined by the phrase "comprising one does not exclude the presence of other like elements in a process, method, article, or apparatus that comprises an element.
Although embodiments of the present invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made therein without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.
Claims (1)
1. A method for the ratiometric electrochemical detection of a glycoconjugate comprising the steps of:
s1: immobilizing the aptamer with the end modified with the electroactive probe for specific recognition and capture of the glycoconjugate, and adding a boric acid derivative of a second electroactive probe after the aptamer is combined with the target glycoconjugate;
s2: quantitatively analyzing the target glycoconjugate by measuring the ratio of the response current magnitudes of the two electroactive probes;
wherein: the aptamer in the step S1 is an oligonucleotide fragment which can specifically identify and capture target sugar conjugates;
the electroactive probe in the step S1 comprises one of ferrocene, methylene blue and thionine;
the glycoconjugate in the step S1 is one of glycoprotein, teichoic acid, lipopolysaccharide, peptidoglycan, capsular polysaccharide, circulating tumor cells, exosomes, bacteria and viruses;
the glycoprotein is one of alpha fetoprotein, carcinoembryonic antigen, saccharide antigen 125, saccharide antigen 15-3, saccharide antigen 199, saccharide antigen 549, saccharide antigen 27.29, saccharide antigen 72-4, saccharide antigen 242, saccharide antigen 50, non-small cell lung cancer related antigen, vascular endothelial growth factor, squamous cell carcinoma antigen, prostate specific antigen, human chorionic gonadotrophin, mucin-like protein, glycosylated albumin, glycosylated hemoglobin, lectin, pancreatic ribonuclease, immunoglobulin, plasminogen, ceruloplasmin, transferrin, erythropoietin, thyroid stimulating hormone, fibronectin, laminin and horseradish peroxidase (HRP);
the glycosylated protein is one of glycosylated albumin and glycosylated hemoglobin.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2013081363A1 (en) * | 2011-11-28 | 2013-06-06 | 에스디 바이오센서 주식회사 | Biosensor for measuring glycosylated haemoglobin using potentiometry |
| CN104977340A (en) * | 2014-04-09 | 2015-10-14 | 南京理工大学 | Nucleic acid electrochemical-biological analysis method with polysaccharides as signal amplification medium |
| CN111004836A (en) * | 2019-12-24 | 2020-04-14 | 山东大学 | Bidirectional amplification ratio type electrochemical aptamer sensor and application thereof |
| CN113740398A (en) * | 2021-08-14 | 2021-12-03 | 云南师范大学 | Ratio-type biosensor and method for detecting MUC1 |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2013081363A1 (en) * | 2011-11-28 | 2013-06-06 | 에스디 바이오센서 주식회사 | Biosensor for measuring glycosylated haemoglobin using potentiometry |
| CN104977340A (en) * | 2014-04-09 | 2015-10-14 | 南京理工大学 | Nucleic acid electrochemical-biological analysis method with polysaccharides as signal amplification medium |
| CN111004836A (en) * | 2019-12-24 | 2020-04-14 | 山东大学 | Bidirectional amplification ratio type electrochemical aptamer sensor and application thereof |
| CN113740398A (en) * | 2021-08-14 | 2021-12-03 | 云南师范大学 | Ratio-type biosensor and method for detecting MUC1 |
Non-Patent Citations (1)
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| A ratiometric electrochemical sensor for the determination of exosomal glycoproteins;Yu An 等;《Talanta》;第122790（1-8）页 * |
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