CN212845415U - Micro-fluidic chip for detecting tumor marker - Google Patents
Micro-fluidic chip for detecting tumor marker Download PDFInfo
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- CN212845415U CN212845415U CN202021122337.7U CN202021122337U CN212845415U CN 212845415 U CN212845415 U CN 212845415U CN 202021122337 U CN202021122337 U CN 202021122337U CN 212845415 U CN212845415 U CN 212845415U
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Abstract
The utility model discloses a micro-fluidic chip for tumor marker detection, this chip includes reaction layer substrate and the miniflow channel application of sample layer that is located above that, the reaction layer substrate includes slide glass base plate and the oxidation graphite alkene quantum dot nano-material basement of assembling on the slide glass base plate, it has bar detection area and quality control area to print on the oxidation graphite alkene quantum dot nano-material basement; the bottom on microchannel application of sample layer is equipped with a plurality of microchannels recess, and is a plurality of the microchannels recess is with a plurality of the bar is detected area and quality control area and is arranged perpendicularly and form a plurality of reaction dot matrixes, still be equipped with the inflow hole and the outlet port that link up from top to bottom on the microchannel application of sample layer, the both ends of microchannel recess respectively with inflow hole and outlet port intercommunication. The utility model discloses a chip can detect the tumour marker of multiple difference simultaneously, has fast, stable, but on-the-spot high flux detection's advantage.
Description
Technical Field
The utility model relates to a micro-fluidic chip field, in particular to micro-fluidic chip for tumour marker detects.
Background
Over the years, various cancers have severely affected human health, and early treatment and intervention of tumors, if discovered, can greatly improve patient survival and survival. In the early stages of tumor development, the concentrations of certain biomolecules in human serum begin to change. For example, CEA, CA125, CA199, and CA153 in the serum of a breast cancer patient are important markers for breast cancer, and have an important relationship with the diagnosis and prognosis of breast cancer. They are antigens associated with breast cancer, are present in breast cancer cells and can be released into the blood, and the detection of CEA, CA125, CA199, CA153 levels in the blood can be used to detect breast cancer. The detection of tumor markers in blood or tissue provides important information for early diagnosis, clinical monitoring, disease treatment and rehabilitation monitoring of cancer patients.
Due to the increasing need for various disease control, sensitive, low cost, point-of-care detection methods have attracted considerable interest to related researchers. The traditional tumor marker detection method mainly comprises enzyme-linked immunoassay, radioimmunoassay, chemiluminescence analysis, mass spectrometry and the like. However, these methods do not meet the requirement of instantaneous detection. The traditional detection method, namely the ELISA kit, consumes a large amount of samples to complete the detection of various biomarkers by one-by-one detection. Electrochemical methods have high sensitivity but have limited high-throughput detection capabilities.
In recent years, nanomaterial-integrated biosensors have played an important role in biomolecule detection due to their specific interactions with bioactive molecules, cells and organisms. Recently, carbon-based nanomaterials have attracted the attention of researchers. Some carbon-based luminescent nano materials have excellent photoelectric characteristics and good biocompatibility. Due to the single-atom layered structure, the material has the advantages of unique physical adsorption, luminescent property, low toxicity, stability, easy preparation and the like. Some carbon-based nanomaterials have a large number of oxygen-containing groups present to make them more compatible in polar solvents (i.e., water). Therefore, the nano material is combined with technologies such as a microfluid chip and the like, and has wide application prospects in the aspects of improving the protein detection sensitivity, improving the protein detection reliability and improving high flux.
SUMMERY OF THE UTILITY MODEL
In order to solve the technical problem, the utility model provides a micro-fluidic chip for tumor marker detects to reach and to detect multiple different tumor markers simultaneously, but have the purpose quick, stable, on-the-spot high flux detection.
In order to achieve the above purpose, the technical scheme of the utility model is as follows:
a microfluidic chip for detecting tumor markers comprises a reaction layer substrate and a microfluidic channel sample adding layer positioned on the reaction layer substrate, wherein the reaction layer substrate comprises a glass slide substrate and a graphene oxide quantum dot nano material substrate assembled on the glass slide substrate, and a strip-shaped detection band and a quality control band are printed on the graphene oxide quantum dot nano material substrate; the bottom on microchannel application of sample layer is equipped with a plurality of microchannels recess, and is a plurality of the microchannels recess is with a plurality of the bar is detected area and quality control area and is arranged perpendicularly and form a plurality of reaction dot matrixes, still be equipped with the inflow hole and the outlet port that link up from top to bottom on the microchannel application of sample layer, the both ends of microchannel recess respectively with inflow hole and outlet port intercommunication.
In the scheme, a plurality of strip-shaped detection bands and quality control bands are periodically paved on a graphene oxide quantum dot nano material substrate to form a probe area.
In the above scheme, the plurality of strip-shaped detection zones and the plurality of quality control zones are uniformly arranged and arranged in parallel at intervals, and the plurality of micro-channel grooves are uniformly perpendicular to the plurality of strip-shaped detection zones and the plurality of quality control zones.
In the scheme, the glass slide substrate is made of calcium silicate, sodium silicate or silicon dioxide.
In the scheme, the graphene oxide quantum dot nano-material substrate is obtained by reacting the glass slide substrate with the graphene oxide quantum dot nano-material, washing and drying.
In the scheme, the strip-shaped detection band and the quality control band are formed by physically adsorbing biomolecules and a graphene oxide quantum dot nano material substrate on a glass slide substrate through functional groups or pi-pi bonds.
A method for detecting a tumor marker by using the microfluidic chip for detecting the tumor marker comprises the following steps:
(1) injecting different samples to be detected from the plurality of inflow holes respectively, incubating for 15-30min to ensure that the antigen to be detected in the samples to be detected is fully combined with the tumor marker detection antibodies of the strip-shaped detection band and the quality control band on the reaction layer substrate, taking out the redundant samples to be detected from the outflow holes after the incubation is finished, and at the moment, specifically combining the antigen to be detected and the tumor marker detection antibodies and fixing the antigen to be detected and the tumor marker detection antibodies on the reaction layer substrate;
(2) uncovering the sheet in DPBS containing BSA with the mass concentration of 0.5-5%, and washing the antigen to be detected which is not adsorbed on the substrate of the reaction layer clean by using BSA with the mass concentration of 0.5-5%;
(3) spreading the mixed detection antibody solution on the reaction layer substrate, and incubating for 10-30 min;
(4) and (2) washing the substrate by using BSA (bovine serum albumin) with the mass concentration of 0.5-5%, then fully spreading the APC-streptavidin conjugate on the reaction layer substrate, specifically binding and fixing the APC-streptavidin conjugate and a tumor marker detection antibody with a biotin label on the reaction layer substrate, and then carrying out fluorescence detection on the tumor marker.
Through the technical scheme, the utility model provides a pair of a micro-fluidic chip for tumour marker detects has following beneficial effect:
the utility model discloses a self-assembling graphene oxide quantum dot nanomaterial basement is integrated with the detection chip of micro-fluidic technique, and the microchannel application of sample layer contains a plurality of microchannels recess and is evenly perpendicular to a plurality of bar detection areas and quality control area align to grid, can carry out the detection of a plurality of biochemical or chemical indexes to the sample simultaneously; when the chip is used, only the outflow hole and the inflow hole are communicated with the outside, most of reaction processes are completed in a closed micro-channel system, and the interference of the external environment on the reaction and detection processes is effectively reduced; and the chip has simple structure, and is easy to integrate and realize automatic detection by combining with matched automatic equipment.
The chip can detect various different tumor markers simultaneously, has the functions of rapidness, stability and on-site high-flux detection, and enables the detection of on-site samples to be more convenient, simpler and quicker.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below.
Fig. 1 is a schematic view of the whole structure of a microfluidic chip for detecting tumor markers according to an embodiment of the present invention;
FIG. 2 is a schematic structural diagram of a reaction layer substrate disclosed in an embodiment of the present invention;
FIG. 3 is a schematic view of a microchannel sample application layer disclosed in the embodiment of the present invention;
fig. 4 is a standard curve diagram of an embodiment of the present invention.
In the figure, 1, a reaction layer substrate; 2. a micro-channel sample addition layer; 3. a glass slide substrate; 4. a graphene oxide quantum dot nanomaterial substrate; 5. detecting a belt; 6. a quality control band; 7. an inflow hole; 8. an outflow hole; 9. micro flow channel groove.
Detailed Description
The technical solution in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention.
The utility model provides a micro-fluidic chip for tumour marker detects, as shown in figure 1, including reaction layer substrate 1 and the miniflow channel application of sample layer 2 that is located above it, as shown in figure 2, reaction layer substrate 1 includes slide glass base plate 3 and the oxidation graphite alkene quantum dot nano-material basement 4 of assembling on slide glass base plate 3, and the printing has bar detection area 5 and quality control area 6 on oxidation graphite alkene quantum dot nano-material basement 4; as shown in fig. 3, the bottom of the microchannel sample application layer 2 is provided with a plurality of microchannel grooves 9, the plurality of strip-shaped detection bands 5 and the quality control bands 6 are vertically arranged to form a plurality of reaction dot matrices, the microchannel sample application layer 2 is further provided with an inflow hole 7 and an outflow hole 8 which are vertically communicated, and two ends of the microchannel grooves 9 are respectively communicated with the inflow hole 7 and the outflow hole 8.
And a plurality of strip-shaped detection bands 5 and quality control bands 6 are periodically paved on the graphene oxide quantum dot nano material substrate 4 to form a probe area. A plurality of bar detection area 5 and quality control area 6 align to grid, and parallel interval sets up, and a plurality of miniflow channel grooves 9 are the even perpendicular to a plurality of bar detection area 5 and quality control area 6 and are arranged.
The material of the glass slide substrate 3 is calcium silicate, sodium silicate or silicon dioxide.
The graphene oxide quantum dot nano-material substrate 4 is obtained by reacting the glass slide substrate 3 with a graphene oxide quantum dot nano-material, washing and drying.
The plurality of strip-shaped detection bands 5 and the plurality of quality control bands 6 are patterns of the strip-shaped detection bands 5 and the plurality of quality control bands 6 printed on the graphene oxide quantum dot nano material substrate 4 by adopting a linear micro-fluidic chip. The strip-shaped detection zone 5 and the quality control zone 6 are formed by physically adsorbing biological molecules and the graphene oxide quantum dot nano material substrate 4 on the glass slide substrate 3 through functional groups or pi-pi bonds.
The micro-channel sample adding layer 2 is made of polydimethylsiloxane material by casting or injection molding. The detection chip can detect 60 samples from different sources, and each sample can detect 50 antibodies simultaneously, so that the detection efficiency is greatly improved.
In order to verify the uniformity of the strip-shaped detection band 5 and the quality control band 6, one end of the short side of the glass slide substrate 3 fixed with the strip-shaped detection band 5 and the quality control band 6 is soaked in a goat anti-mouse fluorescent secondary antibody solution, the incubation time is 10min, then the end is washed by DPBS containing BSA with the mass concentration of 1%, and then the whole glass slide substrate 3 fixed with the bar-shaped detection band 5 and the quality control band 6 is respectively placed in the following solutions of 100% DPBS, 50% DPBS and DI Water for washing, so that the detection background is reduced, and the detection sensitivity is improved. And finally, placing the glass slide substrate 3 fixed with the strip-shaped detection belt 5 and the quality control belt 6 in a glass slide centrifuge for spin-drying for 20-30s, and observing a fluorescence signal by using a microarray gene scanner for later use. At this time, the micro flow channel sample addition layer 2 and the processed reaction layer substrate 1 on which the strip-shaped detection band 5 and the quality control band 6 are fixed are attached by the self-weight, and thus, a complete and usable chip system is prepared.
A method for detecting a tumor marker by using the microfluidic chip for detecting the tumor marker comprises the following steps:
(1) injecting 1uL of samples to be detected with different concentrations from the plurality of inflow holes 7 respectively, incubating for 20min to ensure that the antigen to be detected in the samples to be detected is fully combined with the tumor marker detection antibodies of the strip detection belt 5 and the quality control belt 6 on the reaction layer substrate 1, after the incubation is finished, pumping out the redundant samples to be detected from the outflow holes 8, and at the moment, the antigen to be detected and the tumor marker detection antibodies are specifically combined and fixed on the reaction layer substrate 1;
(2) uncovering the plate in DPBS containing BSA with the mass concentration of 1%, and washing the antigen to be detected which is not adsorbed on the reaction layer substrate 1 with BSA with the mass concentration of 1%;
(3) taking the mixed detection antibody solution by using a liquid transfer gun, fully spreading the mixed detection antibody solution on the reaction layer substrate 1, and incubating for 20 min;
(4) washing with BSA (bovine serum albumin) with the mass concentration of 1%, then taking 200ul of the APC-streptavidin conjugate by using a pipette gun, fully spreading the APC-streptavidin conjugate on the reaction layer substrate 1, specifically binding and fixing the APC-streptavidin conjugate and a tumor marker detection antibody with a biotin label on the reaction layer substrate 1, and then carrying out fluorescence detection on the tumor marker.
(5) The intensity of the optical signal (which has a detection wavelength of 635nm) was detected by a GenePix 4400 microarray scanner, and the detection result was given. The total detection time is 30min, each standard substance is respectively measured by using 3 detection holes of the microfluidic chip system for 3 times, the average value of the results is obtained, and a standard curve is drawn.
Method for drawing standard curve: performing gradient dilution on the recombinant protein CEA and AFP, namely 50000pg/ml, 5000pg/ml, 500pg/ml, 50pg/ml and 5 pg/ml; the recombinant proteins CA125, CA199 and CA153 were diluted in a gradient of 1000u/ml, 100u/ml, 10u/ml, 1u/ml, 0.1 u/ml. The following operations were performed for each concentration of recombinant protein: and (3) taking 2 mu L of recombinant protein standard substance by using a pipette, injecting the recombinant protein standard substance into a detection sample chip structure of the chip system through a sample inlet hole of the chip, and executing the operation to obtain a standard curve shown in figure 4.
The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.
Claims (5)
1. The microfluidic chip for detecting the tumor marker is characterized by comprising a reaction layer substrate and a micro-channel sample adding layer positioned on the reaction layer substrate, wherein the reaction layer substrate comprises a glass slide substrate and a graphene oxide quantum dot nano-material substrate assembled on the glass slide substrate, and a strip-shaped detection band and a quality control band are printed on the graphene oxide quantum dot nano-material substrate; the bottom on microchannel application of sample layer is equipped with a plurality of microchannels recess, and is a plurality of the microchannels recess is with a plurality of the bar is detected area and quality control area and is arranged perpendicularly and form a plurality of reaction dot matrixes, still be equipped with the inflow hole and the outlet port that link up from top to bottom on the microchannel application of sample layer, the both ends of microchannel recess respectively with inflow hole and outlet port intercommunication.
2. The microfluidic chip for detecting tumor markers according to claim 1, wherein a plurality of strip-shaped detection bands and quality control bands are periodically tiled on the graphene oxide quantum dot nanomaterial substrate to form a probe region.
3. The microfluidic chip for detecting tumor markers according to claim 1, wherein a plurality of the strip-shaped detection strips and the quality control strips are uniformly arranged and spaced in parallel, and a plurality of the micro-channel grooves are uniformly arranged perpendicular to the plurality of the strip-shaped detection strips and the quality control strips.
4. The microfluidic chip for detecting tumor markers according to claim 1, wherein the glass slide substrate is made of calcium silicate, sodium silicate or silicon dioxide.
5. The microfluidic chip for detecting tumor markers according to claim 1, wherein the strip detection zone and the quality control zone are formed by physical adsorption of biomolecules and graphene oxide quantum dot nanomaterial substrate on a glass slide substrate through functional groups or pi-pi bonds.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113009134A (en) * | 2020-06-17 | 2021-06-22 | 山东大学 | Micro-fluidic chip for detecting tumor marker and detection method |
| CN113311160A (en) * | 2021-06-17 | 2021-08-27 | 山东科讯生物芯片技术有限公司 | Micro-fluidic biochip for rapidly detecting SARS-CoV-2 antigen and IgG/IgM antibody |
| CN113654953A (en) * | 2021-07-29 | 2021-11-16 | 山东大学深圳研究院 | Method for detecting environmental behaviors and biological effects of nano-particle pollutants |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113009134A (en) * | 2020-06-17 | 2021-06-22 | 山东大学 | Micro-fluidic chip for detecting tumor marker and detection method |
| CN113311160A (en) * | 2021-06-17 | 2021-08-27 | 山东科讯生物芯片技术有限公司 | Micro-fluidic biochip for rapidly detecting SARS-CoV-2 antigen and IgG/IgM antibody |
| CN113654953A (en) * | 2021-07-29 | 2021-11-16 | 山东大学深圳研究院 | Method for detecting environmental behaviors and biological effects of nano-particle pollutants |
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