CN107764803B - Biomarker detection method using electrochemiluminescence imaging recognition technology - Google Patents
Biomarker detection method using electrochemiluminescence imaging recognition technology Download PDFInfo
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Abstract
The invention discloses a biomarker detection method applying an electrochemiluminescence imaging recognition technology, which comprises the steps of firstly constructing an electrochemiluminescence amplification probe, adding the electrochemiluminescence amplification probe into an extracted sample, and connecting a biomarker with the electrochemiluminescence signal amplification probe to obtain a system 1; adding a capture probe into the system 1 to connect the biomarker with the capture probe to obtain a system 2; adding streptavidin magnetic beads into the system 2, fully swirling and uniformly mixing to connect the biomarker with the streptavidin magnetic beads, separating by using a magnetic separator, and washing by using PBS1X buffer solution; and (3) uniformly mixing the product obtained in the step one with ultrapure water, and then placing the product in an electrochemical luminescence imaging system to detect a luminescence signal. The invention has the advantages that: 1) the signal acquisition sensitivity is high, the visualization is realized, and quantitative statistical analysis can be realized; (2) the sample compatibility is good; (3) the operation is simple and quick, and the sample amplification is not needed; the detection process is simple, the biomarker monitoring can be carried out through a simple sample extraction process, the time consumption is short, the amplification step is omitted, and the detection is rapid.
Description
Technical Field
The invention belongs to the technical field of biomarker detection, and particularly relates to a biomarker detection method based on a single-molecule electrochemiluminescence recognition system and application thereof.
Background
With the change of life science and technology, more and more biomarkers play an increasingly important role in related technical fields. Wherein the major markers such as microRNA, telomerase, CEA protein and related coding RNA become the star molecules in the disciplines of life science, medicine and the like. However, the existing detection technologies for these molecular markers have the disadvantages of non-uniform technical routes, poor compatibility of instruments and equipment, and incapability of realizing cooperation among technical systems. Therefore, the development of a universal, high-sensitivity, good-compatibility and instrument-reagent-sharable multifunctional detection platform has important significance for the detection of the existing biomarkers.
The invention develops a novel single-molecule electrochemical luminescence imaging identification method by taking major biomarkers such as microRNA, telomerase, CEA protein and related coding RNA as research objects and taking a high-efficiency electrochemical luminescence amplification method as technical support, and realizes the biomarker detection method with high sensitivity, simple operation, rapidness and low cost. The method for amplifying the signal based on the electrochemical luminescence of the polymer can provide powerful technical support for a single molecule recognition technology, and the system can provide accurate, objective and systematic monitoring indexes for researchers by constructing systems such as a specific biomarker recognition probe, a specific biomarker capture probe and a specific signal amplification probe.
Disclosure of Invention
In order to overcome the disadvantages and shortcomings of the prior art, the invention provides a biomarker detection method using an electrochemiluminescence imaging identification technology. The invention takes the terpyridyl ruthenium-lysine polymer as an entry point to synthesize linear and tree-shaped terpyridyl ruthenium-lysine polymer molecules, thereby constructing a polymer amplification system, providing an amplified electrochemical luminescence signal for a single-molecule electrochemical luminescence imaging system and finally realizing the aim of single-molecule identification. In order to further improve the synthesis convenience of linear terpyridyl ruthenium-lysine polymer and dendritic terpyridyl ruthenium-lysine polymer, the inventor designs automatic terpyridyl ruthenium-lysine polymer synthesis equipment in the earlier stage, and realizes the synthesis of linear and dendritic polymer molecules by circulating a monomer synthesis step. The electrochemical luminescence imaging recognition system developed by the invention consists of an electrochemical module, a microscopic imaging and image acquisition system: the electrochemical light-emitting module adopts a chip mode and is assembled with a detachable printed circuit to overcome the problems of easy oxidation and poor reproducibility of the traditional circuit; the microscopic imaging system adopts a high-resolution microscope and is matched with a high-precision stepping displacement table and a chip fixing support to finally obtain stable and complete microscopic image information; the image acquisition system adopts a high-sensitivity Charge Coupled Device (CCD) as a core component and is matched with a temperature control and gain adjustment module to realize an efficient image acquisition process. On the basis of a single-molecule electrochemical luminescence imaging recognition system, the invention simultaneously designs systems such as specific marker recognition, capture and signal amplification probes and the like aiming at microRNA, telomerase and CEA protein respectively, and finally achieves the purpose of high-sensitivity detection of the biomarker.
The purpose of the invention is realized by the following technical scheme: a biomarker detection method applying an electrochemiluminescence imaging recognition technology mainly comprises the following steps:
1. general technical route and flow
The invention aims to construct a single-molecule electrochemiluminescence imaging recognition system and finally realize a high-sensitivity detection method based on microRNA, telomerase and CEA protein. The technical route of the invention is shown in figure 1:
1.1 the invention takes terpyridyl ruthenium-lysine polymer as an entry point to synthesize linear and tree-shaped terpyridyl ruthenium-lysine polymer molecules, thereby constructing a polymer amplification system, providing amplified electrochemical luminescence signals for a single-molecule electrochemical luminescence imaging system and finally realizing the aim of single-molecule identification. In order to further improve the synthesis convenience of linear terpyridyl ruthenium-lysine polymer and dendritic terpyridyl ruthenium-lysine polymer, automatic terpyridyl ruthenium-lysine polymer synthesis equipment is designed, and the synthesis of linear and dendritic polymer molecules is realized through a cyclic monomer synthesis step.
1.2 the electrochemical luminescence imaging recognition system developed by the invention comprises an electrochemical module, a microscopic imaging and image acquisition system: the electrochemical light-emitting module adopts a chip mode and is assembled with a detachable printing electrode so as to overcome the problems of easy oxidation and poor reproducibility of the traditional electrode; the microscopic imaging system adopts a high-resolution microscope and is matched with a high-precision stepping displacement table and a chip fixing support to finally obtain stable and complete microscopic image information.
1.3 the image acquisition system adopts a high-sensitivity Charge Coupled Device (CCD) as a core component and is matched with a temperature control and gain adjustment module to realize an efficient image acquisition process.
1.4 on the basis of a single-molecule electrochemiluminescence imaging recognition system, the invention simultaneously designs systems such as specific biomarker recognition, capture and signal amplification probes and the like aiming at microRNA, telomerase and CEA protein respectively, and finally achieves the purpose of high-sensitivity detection of the biomarker.
1.5 in order to further improve the convenience and widen the application range, the invention develops a series of technologies such as sample collection, extraction and signal amplification matched with the single-molecule electrochemiluminescence imaging recognition system, and finally verifies the feasibility, improves the stability and the operability of the scheme by detecting the biomarkers in samples such as serum, body fluid and tissues, so as to realize the development and the application of the standard biomarker detection technology. The technical flow chart of the invention is shown in fig. 2.
2. Construction of Polymer electrochemiluminescence probes
The construction of the polymer electrochemiluminescence probe adopts an automatic and controllable synthesis method, and reaction raw materials are pumped into the reaction tank in a circulating reciprocating manner through a program control flow control device, so that the continuous growth of the terpyridyl ruthenium-lysine polymer monomer is realized, the polymerization degree of the terpyridyl ruthenium-lysine polymer can be controlled by controlling the circulating number, namely the number of the terpyridyl ruthenium is combined on a single polymer molecule, and the consistency of the luminous intensity of the single terpyridyl ruthenium-lysine polymer molecule is finally realized. In order to obtain a better result of single molecule recognition, the polymerization degree of the polymer in the present invention is preferably 10, taking high sensitivity CCD performance into consideration.
3. The invention relates to a single-molecule electrochemiluminescence imaging recognition system, which is the core content of the invention and mainly comprises the following components: the device comprises an electrochemical module and a microscopic imaging and image acquisition system.
3.1 electrochemical luminescence Module
The electrochemical light-emitting module mainly comprises an electrochemical workstation, a flow control system and an electrochemical light-emitting chip. The electrochemical workstation adopts a commercial mature product, and can provide stable voltage for the electrochemical luminescence chip (figure 3). The electrochemiluminescence chip adopts etchable polystyrene as a chip mode (figure 5), and is assembled with a detachable printed electrode to overcome the problems that the traditional electrode is very easy to oxidize and has poor reproducibility.
3.2 microscopic imaging System
The microscopic imaging system adopts a high-resolution optical microscope as a main body part of the system, and in order to achieve a better imaging effect, the microscope is provided with a high-transmittance high-power objective lens. The microscopic imaging system also needs to have an acquisition window that is matched with the image acquisition system so as to be coupled with the image acquisition. On the basis of a high-resolution optical microscope, a high-precision stepping displacement table (micron-sized) and a chip fixing support are matched, so that imaging information of an XY plane is realized, the moving speed of the stepping displacement table needs to be matched with the acquisition frequency of an image acquisition system, and an image is acquired by moving a distance of one field of view. And the image acquisition system can form image information on the Z axis according to the light intensity, and finally stable and complete microscopic image information is obtained.
3.3 image acquisition System
The image acquisition system adopts a scientific research grade high-sensitivity Charge Coupled Device (CCD) as a core component, and the component can identify single photons, so that the sensitivity of the whole system is ensured. The temperature control module is mainly used for controlling the temperature of the CCD imaging component and maintaining the working temperature of the photoelectric system so as to prevent the CCD from overheating. The gain adjusting module is used for adjusting the capacity of the CCD for responding to weak light, and can adjust the gain module according to the luminous intensity of a sample so as to adapt to different luminous intensities. In addition, the image acquisition system is provided with a time sequence photographing program, and can acquire images at specific time points.
4. Biomarker detection
1) MicroRNA expression level detection
The invention is based on the electrochemiluminescence imaging technology of polymer amplification, and makes up the problem of insufficient sensitivity of the non-amplification microRNA detection method. The technical process mainly comprises the following steps (figure 6):
taking 10 mu M of linear or tree-shaped terpyridyl ruthenium-lysine polymer, adding an equivalent amount of DNA recognition domain (the concentration is 100 mu M), and incubating overnight at 37 ℃ for 12 hours to complete the connection of a signal amplification group, namely the linear or tree-shaped terpyridyl ruthenium-lysine polymer and the DNA recognition domain.
And secondly, taking an ultrafiltration tube of 50K daltons (Da), transferring the initial product obtained in the step I to the ultrafiltration tube, and carrying out refrigerated centrifugation at 5000 rpm for 5 minutes until the liquid in the ultrafiltration tube is centrifuged to the lower layer.
And thirdly, adding 500 mu LPBS buffer solution (1X) into the ultrafiltration tube in the second step, freezing and centrifuging for 5 minutes at 5000 rpm until the liquid in the ultrafiltration tube is centrifuged to the lower layer. The procedure was repeated 3 times and the free DNA recognition domain was centrifuged to the lower layer.
Dissolving the electrochemiluminescence signal amplification probe in the upper layer of the product with 200. mu.L of ultrapure water, and repeatedly washing the bottom of the upper layer tube to fully dissolve the electrochemiluminescence signal amplification probe.
Fifthly, placing the product obtained in the step (iv) in a refrigerator at the temperature of 20 ℃ below zero, freezing and drying the product for 6 hours to obtain a solid product, and storing the solid product in the refrigerator at the temperature of 20 ℃ below zero for later use.
Sixthly, taking 1 mu L of microRNA extraction sample, adding into 44.5 mu L of ultrapure water, adding into 2.5 mu L of phosphate buffer solution (PBS, 20X), and vortexing and shaking.
Seventhly, adding 1 mu L of electrochemiluminescence signal amplification probe (labeled electrochemiluminescence polymer with the concentration of 0.25 mu M) into the mixture obtained in the step II, performing vortex oscillation, and incubating for 15 minutes at 37 ℃ to ensure that the electrochemiluminescence signal amplification probe is fully hybridized with the target microRNA.
Adding 1 mu L of capture probe (labeled biotin with the concentration of 0.25 mu M) into the system, vortexing, and incubating at 37 ℃ for 15 minutes to ensure that the capture probe is fully hybridized with the target microRNA.
Ninthly, adding excessive streptavidin magnetic beads (with the concentration of 1mg/mL), which can be 10 mu L of streptavidin magnetic beads (with the concentration of 1mg/mL), fully and uniformly swirling, and incubating for 10 minutes at 37 ℃.
C, separating products obtained in the step ninthly by using a magnetic separator, washing by using PBS buffer solution (1X), and repeating for three times; the obtained product is mixed uniformly with ultrapure water, and signals are detected by an electrochemical luminescence imaging recognition system finally.
2) Telomerase activity detection
I, telomerase extraction step, namely processing by adopting a commercialized kit to obtain a telomerase extraction sample, and performing telomerase extension treatment to obtain a telomerase extension product.
II, constructing an electrochemiluminescence signal amplification probe by adopting the steps from 1) to fifthly in microRNA expression level detection.
III, taking 1 mu L of the electrochemiluminescence signal amplification probe obtained in the step II, adding 44.5 mu L of ultrapure water, adding 2.5 mu L of phosphate buffer solution (PBS, 20X), and carrying out vortex shaking.
IV, adding 1 mu L of telomerase extraction sample into the product obtained in the step III, vortexing, fully mixing uniformly, and incubating for 15 minutes at 37 ℃ to fully hybridize the electrochemical luminescence signal amplification probe and the telomerase.
V, adding 1 mu L of capture probe (labeled biotin with the concentration of 0.25 mu M) into the system IV, vortexing, and incubating at 37 ℃ for 15 minutes to ensure that the capture probe is fully hybridized with telomerase.
VI to the system of step V, an excessive amount of streptavidin magnetic beads (concentration 1mg/mL), which may be 10. mu.L streptavidin magnetic beads (concentration 1mg/mL), is added, vortexed thoroughly, mixed, and incubated at 37 ℃ for 10 minutes.
VII separating the product obtained in the step VI by using a magnetic separator, washing by using PBS buffer solution (1X) and repeating the steps for three times. And mixing with ultrapure water, and finally detecting signals by using an electrochemical luminescence imaging recognition system. The schematic diagram is shown in fig. 7.
3) CEA protein and detection of coding RNA thereof
The CEA protein adopts the traditional immunoassay principle as the technical basis, the detection of the CEA protein is realized by constructing a polymer-antibody electrochemiluminescence signal amplification probe (figure 8), and the detection of the coding RNA adopts the experimental steps with the same microRNA detection mode. The detection steps of the CEA protein are as follows:
A. uniformly mixing a linear or tree-shaped terpyridyl ruthenium-lysine polymer and an antibody 1 according to the proportion of 1:1, and incubating for 6 hours at 37 ℃ to fully connect the polymer and the antibody 1 so as to construct an electrochemiluminescence signal amplification probe.
B. A sample of CEA protein (oncofetal protein, CEA) to be tested was taken at 1. mu.L, and added to 44.5. mu.L of ultrapure water, and 2.5. mu.L of phosphate buffer solution (PBS, 20X) and vortexed.
C. And (3) adding 1 mu L of the electrochemiluminescence signal amplification probe (the concentration is 0.25 mu M) into the system in the step (B), vortexing, and incubating for 15 minutes at 37 ℃ to combine the electrochemiluminescence signal amplification probe with the CEA protein.
D. Adding 1 mu L of antibody 2 (labeled biotin) into the system obtained in the step C, vortexing, fully mixing, and then incubating for 15 minutes at 37 ℃ to enable the capture probe to be combined with the CEA protein.
E. To the system in step D, an excess amount of streptavidin magnetic beads (concentration 1mg/mL), optionally 10. mu.L of streptavidin magnetic beads (concentration 1mg/mL), was added, and after thorough vortex mixing, the mixture was incubated at 37 ℃ for 10 minutes.
F. The resulting product was separated with a magnetic separator and washed with PBS buffer (1X) and repeated three times. And finally, mixing the mixture with ultrapure water uniformly, and detecting signals by using an electrochemical luminescence imaging recognition system.
Compared with the prior art, the invention has the following advantages and effects:
(1) high signal acquisition sensitivity, visualization and quantitative statistical analysis
The invention adopts the high-sensitivity CCD as the receiving system of the electrochemical luminescence signal, has the advantages of high sensitivity and visualized experimental result, and can carry out quantitative statistical analysis on the graphical experimental result.
(2) Good sample compatibility
On the basis of constructing an electrochemical luminescence imaging recognition platform, the invention can simultaneously design a detection process aiming at various biomarkers such as microRNA, telomerase, CEA protein and coding RNA thereof, and the like, and has the characteristics of good sample compatibility and wide application range.
(3) Simple and rapid operation, no need of sample amplification
The detection process is simple, the biomarker monitoring can be carried out through a simple sample extraction process, the time consumption is short, the amplification step is omitted, and the detection is rapid.
Drawings
FIG. 1 is a technical scheme of an electrochemiluminescence imaging identification method.
FIG. 2 is a technical flow chart of an electrochemiluminescence imaging identification method.
FIG. 3 is a structural diagram of an electrochemiluminescence imaging recognition system.
FIG. 4 is an electrochemiluminescence displacement stage;
FIG. 5 an electrochemiluminescence chip.
FIG. 6 is a schematic diagram of a method for detecting microRNA and CEA protein coding RNA based on an electrochemiluminescence imaging recognition system; A. a linear polymer; B. a dendrimer.
FIG. 7 is a schematic diagram of a telomerase activity detection method based on an electrochemiluminescence imaging recognition system. A. A linear polymer; B. a dendrimer.
FIG. 8 is a schematic diagram of the CEA protein detection method based on the electrochemiluminescence imaging identification method. A. A linear polymer; B. a dendrimer.
FIG. 9.A. magnetic bead surface activated ruthenium polymer imaging identification; b, detecting an experimental result by using microRNA; c, CEA protein detection experiment result; D. detecting the activity of telomerase.
Detailed Description
The present invention will be described in further detail with reference to examples and drawings, but the present invention is not limited thereto.
Example 1 electrochemiluminescence imaging identification method
1. General technical route and flow
The invention aims to construct a single-molecule electrochemiluminescence imaging recognition system and finally realize a biomarker detection method based on microRNA, telomerase and CEA protein. The technical route of the invention is shown in figure 1:
1.1 the invention takes terpyridyl ruthenium-lysine polymer as an entry point to synthesize linear and tree-shaped terpyridyl ruthenium-lysine polymer molecules, thereby constructing a polymer amplification system, providing amplified electrochemical luminescence signals for a single-molecule electrochemical luminescence imaging system and finally realizing the aim of single-molecule identification. In order to further improve the synthesis convenience of linear terpyridyl ruthenium-lysine polymer and dendritic terpyridyl ruthenium-lysine polymer, automatic terpyridyl ruthenium-lysine polymer synthesis equipment is designed, and the synthesis of linear and dendritic polymer molecules is realized through a cyclic monomer synthesis step.
1.2 the electrochemical luminescence imaging recognition system developed by the invention comprises an electrochemical luminescence module and a microscopic imaging and image acquisition system: the electrochemical light-emitting module adopts a chip mode and is assembled with a detachable printing electrode 8 so as to overcome the problems of easy oxidation and poor reproducibility of the traditional electrode; the microscopic imaging system adopts a high-resolution microscope 2, and is matched with a high-precision stepping displacement table 7 and a chip fixing support to finally obtain stable and complete microscopic image information.
1.3 the image acquisition system adopts a high-sensitivity charge coupled device CCD1 as a core component and is matched with a temperature control module 5 and a gain adjustment module 4 to realize an efficient image acquisition process.
1.4 on the basis of a single-molecule electrochemiluminescence imaging recognition system, the invention simultaneously designs systems such as specific biomarker recognition, capture and signal amplification probes and the like aiming at microRNA, telomerase and CEA protein respectively, and finally achieves the purpose of high-sensitivity detection of the biomarker.
1.5 in order to further improve convenience and widen the application range of the system, the invention aims to develop a series of technologies such as sample collection, extraction, signal amplification and the like matched with a single-molecule electrochemiluminescence imaging recognition system, and finally verifies the feasibility, improves the stability and the operability of the scheme by detecting biomarker samples from sources such as serum, body fluid, tissues and the like so as to realize the development and the application of a standard biomarker detection product. The technical flow chart of the invention is shown in fig. 2.
2. Construction of Polymer electrochemiluminescence probes
The construction of the polymer electrochemiluminescence probe adopts an automatic and controllable synthesis method, and reaction raw materials are pumped into the reaction tank in a circulating reciprocating manner through a program control flow control device, so that the continuous growth of a terpyridyl ruthenium polymer monomer is realized, the polymerization degree of the terpyridyl ruthenium-lysine polymer can be controlled by controlling the circulating number, namely the number of the terpyridyl ruthenium combined on a single polymer molecule, and the consistency of the luminous intensity of the single terpyridyl ruthenium-lysine polymer molecule is finally realized. In order to obtain a better result of single molecule recognition, the polymerization degree of the polymer in the present invention is preferably 10, taking high sensitivity CCD performance into consideration.
3. The single-molecule electrochemiluminescence imaging recognition system is the core content of the invention (the device principle is shown in figure 1), and mainly comprises the following components: the device comprises an electrochemical luminescence module and a microscopic imaging and image acquisition system.
3.1 electrochemical luminescence Module
The electrochemical light-emitting module mainly comprises an electrochemical workstation 3, a flow control system and an electrochemical light-emitting chip 8. The electrochemical workstation 3 is a commercially mature product, and can provide a stable voltage for the electrochemiluminescence chip 8, as shown in fig. 3. The electrochemiluminescence chip 8 adopts an etchable polystyrene as a chip mode as shown in fig. 5, and is assembled with a detachable printed electrode to overcome the problems of high oxidation and poor reproducibility of the conventional electrode, wherein the printed electrode comprises a counter electrode 81, a reference electrode 82 and a working electrode 83 as shown in fig. 5.
3.2 microscopic imaging System
The microscopic imaging system adopts a high-resolution optical microscope 2 as a main body part of the system, and in order to achieve a better imaging effect, the microscope is provided with a high-transmittance high-power objective lens. The microscopic imaging system also needs to have an acquisition window that is matched with the image acquisition system so as to be coupled with the image acquisition. On the basis of the high-resolution optical microscope 2, a high-precision stepping displacement table (micron-sized) 7 and a chip fixing support shown in fig. 4 are matched, so that the imaging information of an XY plane is realized, the moving speed of the stepping displacement table 7 needs to be matched with the acquisition frequency of an image acquisition system, and an image is acquired by moving the distance of one field of view. And the image acquisition system can form image information on the Z axis according to the light intensity, and finally stable and complete microscopic image information is obtained.
3.3 image acquisition System
As shown in fig. 3, the image acquisition system adopts a scientific research level high-sensitivity charge coupled device CCD1 as a core component, and the component can identify single photons, thereby ensuring the sensitivity of the whole system. The temperature control module 5 is mainly used for controlling the temperature of the imaging part of the CCD1 and maintaining the working temperature of the optoelectronic system to prevent the CCD1 from overheating. The gain adjusting module 4 is used for adjusting the capability of the CCD1 to respond to weak light, and the gain adjusting module 4 can be adjusted according to the luminous intensity of the sample to adapt to different luminous intensities. In addition, the image acquisition system is provided with a time sequence photographing program, and can acquire images at specific time points.
In order to verify the performance and feasibility of the electrochemiluminescence imaging system, an imaging experiment is performed on the electrochemiluminescence probe on the surface of the magnetic bead, and the experimental result is shown in fig. 9A. In the dark field, the platform obtains stable imaging results of the surfaces of the magnetic beads, thereby proving the feasibility of the invention.
Example 2 application of electrochemiluminescence imaging identification method in microRNA expression level detection
1) MicroRNA expression level detection
The invention is based on the electrochemiluminescence imaging technology of polymer amplification, and makes up the problem of insufficient sensitivity of the non-amplification microRNA detection method. The technical process mainly comprises the following steps as shown in fig. 6:
taking 10 mu M of linear or tree-shaped terpyridyl ruthenium-lysine polymer, adding an equivalent amount of DNA recognition domain (the concentration is 100 mu M), and incubating overnight (12 hours) at 37 ℃ to complete the connection of a signal amplification group, namely the linear or tree-shaped terpyridyl ruthenium-lysine polymer and the DNA recognition domain.
And secondly, taking an ultrafiltration tube of 50K daltons (Da), transferring the initial product obtained in the step I to the ultrafiltration tube, and carrying out refrigerated centrifugation at 5000 rpm for 5 minutes until the liquid in the ultrafiltration tube is centrifuged to the lower layer.
And thirdly, adding 500 mu LPBS buffer solution (1X) into the ultrafiltration tube in the second step, freezing and centrifuging for 5 minutes at 5000 rpm until the liquid in the ultrafiltration tube is centrifuged to the lower layer. The procedure was repeated 3 times and the free DNA recognition domain was centrifuged to the lower layer.
Dissolving the electrochemiluminescence signal amplification probe in the upper layer of the product with 200. mu.L of ultrapure water, and repeatedly washing the bottom of the upper layer tube to fully dissolve the electrochemiluminescence signal amplification probe.
Fifthly, placing the product obtained in the step (iv) in a refrigerator at the temperature of 20 ℃ below zero, freezing and drying the product for 6 hours to obtain a solid product, and storing the solid product in the refrigerator at the temperature of 20 ℃ below zero for later use.
Sixthly, taking 1 mu L of microRNA extraction sample, adding into 44.5 mu L of ultrapure water, adding into 2.5 mu L of phosphate buffer solution (PBS, 20X), and vortexing and shaking.
Seventhly, adding 1 mu L of electrochemiluminescence signal amplification probe (labeled electrochemiluminescence polymer with the concentration of 0.25 mu M) into the mixture obtained in the step II, performing vortex oscillation, and incubating for 15 minutes at 37 ℃ to ensure that the electrochemiluminescence signal amplification probe is fully hybridized with the target microRNA.
Adding 1 mu L of capture probe (labeled biotin with the concentration of 0.25 mu M) into the system, vortexing, and incubating at 37 ℃ for 15 minutes to ensure that the capture probe is fully hybridized with the target microRNA.
Ninthly, adding excessive (10 mu L) streptavidin magnetic beads (with the concentration of 1mg/mL) into the system of the step VIII, fully whirling and mixing uniformly, and then incubating for 10 minutes at 37 ℃.
C, separating products obtained in the step ninthly by using a magnetic separator, washing by using PBS buffer solution (1X), and repeating for three times; the obtained product is uniformly mixed with ultrapure water, and signals are finally detected by an electrochemical luminescence imaging recognition system. The experimental result is shown in fig. 9B, when the target microRNA exists, the electrochemiluminescence imaging recognition system can obtain a stable response signal.
Example 3 application of electrochemiluminescence imaging identification method to telomerase activity detection
The application of the electrochemiluminescence imaging identification method in telomerase activity detection is based on an electrochemiluminescence cascade amplification principle, and the electrochemiluminescence imaging identification method is used as a signal giving mode (a schematic diagram is shown in figure 7), and the electrochemiluminescence imaging identification method mainly comprises the following steps:
i, telomerase extraction step, namely processing by adopting a commercialized kit to obtain a telomerase extraction sample, and performing telomerase extension treatment to obtain a telomerase extension product.
II the construction of the electrochemiluminescence signal amplification probe continues with the steps (i) to (v) in example 2.
III, taking 1 mu L of the electrochemiluminescence signal amplification probe obtained in the step II, adding 44.5 mu L of ultrapure water, adding 2.5 mu L of phosphate buffer solution (PBS, 20X), and carrying out vortex shaking.
IV, adding 1 mu L of telomerase extraction sample into the product obtained in the step III, vortexing, fully mixing uniformly, and incubating for 15 minutes at 37 ℃ to fully hybridize the electrochemical luminescence signal amplification probe and the telomerase.
V, adding 1 mu L of capture probe (labeled biotin with the concentration of 0.25 mu M) into the system IV, vortexing, and incubating at 37 ℃ for 15 minutes to ensure that the capture probe is fully hybridized with telomerase.
VI to the system of step V, an excess (10. mu.L) of streptavidin magnetic beads (concentration 1mg/mL) was added, vortexed well, and incubated at 37 ℃ for 10 minutes.
VII separating the product obtained in the step VI by using a magnetic separator, washing by using PBS buffer solution (1X) and repeating the steps for three times. Mixing with ultrapure water, and finally identifying detection signals by using electrochemical luminescence imaging, wherein a schematic diagram is shown in FIG. 5. The experimental result is shown in fig. 9D, when telomerase exists, the electrochemiluminescence imaging recognition system can obtain a stable response signal.
Example 4 application of electrochemiluminescence imaging identification method in CEA protein and its coding RNA
The CEA protein adopts the traditional immunoassay principle as the technical basis, and the detection of the CEA protein is realized by constructing a polymer-antibody electrochemiluminescence signal amplification probe, as shown in figure 8, the detection of the coding RNA adopts the same experimental steps of a microRNA detection mode. The detection steps of the CEA protein are as follows:
A. uniformly mixing a linear or tree-shaped terpyridyl ruthenium-lysine polymer and an antibody 1 according to the proportion of 1:1, and incubating for 6 hours at 37 ℃ to fully connect the polymer and the antibody 1 so as to construct an electrochemiluminescence signal amplification probe.
B. A sample of CEA protein (oncofetal protein, CEA) to be tested was taken at 1. mu.L, and added to 44.5. mu.L of ultrapure water, and 2.5. mu.L of phosphate buffer solution (PBS, 20X) and vortexed.
C. And (3) adding 1 mu L of the electrochemiluminescence signal amplification probe (the concentration is 0.25 mu M) into the system in the step (B), vortexing, and incubating for 15 minutes at 37 ℃ to combine the electrochemiluminescence signal amplification probe with the CEA protein.
D. Adding 1 mu L of antibody 2 (labeled biotin) into the system obtained in the step C, vortexing, fully mixing, and then incubating for 15 minutes at 37 ℃ to enable the capture probe to be combined with the CEA protein.
E. An excess (10. mu.L) of streptavidin magnetic beads (concentration 1mg/mL) was added to the step D system, vortexed well, and incubated at 37 ℃ for 10 minutes.
F. The resulting product was separated with a magnetic separator and washed with PBS buffer (1X) and repeated three times. And finally, mixing the mixture with ultrapure water uniformly, and detecting signals by using an electrochemical luminescence imaging identification method. The experimental result is shown in FIG. 9C, when the specific CEA protein or its coding RNA exists, the electrochemiluminescence imaging recognition system can obtain stable response signal.
Claims (1)
1. A biomarker detection method using an electrochemiluminescence imaging recognition technology is characterized in that:
the CEA protein is detected according to the following steps:
the method comprises the following steps:
A. firstly, constructing an electrochemiluminescence amplification probe, uniformly mixing a linear or tree-shaped terpyridyl ruthenium-lysine polymer and an antibody 1 according to the ratio of 1:1, and incubating for 6 hours at 37 ℃ to fully connect the polymer and the antibody 1;
B. taking 1 mu L of CEA protein to be detected, namely carcinoembryonic protein CEA sample, adding into 44.5 mu L of ultrapure water, adding into 2.5 mu L of phosphate buffer PBS, 20X, and performing vortex oscillation;
C. adding 1 mu L of the electrochemiluminescence amplification probe into the system obtained in the step B, wherein the concentration is 0.25 mu M, performing vortex oscillation, and incubating for 15 minutes at 37 ℃ to connect the CEA protein with the electrochemiluminescence signal amplification probe to obtain a system 1;
D. adding 1 mu L of capture probe for marking biotin into the system 1, vortexing, fully and uniformly mixing, and then incubating for 15 minutes at 37 ℃ to connect the CEA protein with the capture probe to obtain a system 2;
E. adding streptavidin magnetic beads into the system 2, wherein the concentration of the streptavidin magnetic beads is 1mg/mL, fully swirling and mixing the mixture, incubating the mixture for 10 minutes at 37 ℃ to connect the capture probe with the streptavidin magnetic beads, separating the mixture by using a magnetic separator, and washing the mixture by using PBS1X buffer solution;
step two: and (3) uniformly mixing the product obtained in the step one with ultrapure water, and then placing the product in an electrochemical luminescence imaging system to detect a luminescence signal.
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