CN113025477B - Micro-fluidic chip and method for double-color fluorescence double detection - Google Patents
Micro-fluidic chip and method for double-color fluorescence double detection Download PDFInfo
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- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
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- B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
- B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
- B01L3/502707—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip characterised by the manufacture of the container or its components
Abstract
The invention belongs to the technical field of nucleic acid detection, and particularly relates to a microfluidic chip and a method for detecting nucleic acid. The micro-fluidic chip for double-color fluorescence detection comprises a cover plate and a substrate, wherein the cover plate is of a double-layer structure and comprises a valve control layer and a micro-channel layer; the valve control layer is used for controlling the trend of the liquid in the flow channel of the micro-channel layer; the micro-channel layer comprises a plurality of detection units, and each detection unit comprises two micro-channels; one end of the micro-channel is provided with a sample inlet, and the other end of the micro-channel is provided with a micro-cavity and a sample outlet; in the substrate, nano-scale graphene oxide or graphene materials are modified at positions corresponding to the micro-channels, and glutaraldehyde is modified at positions corresponding to the micro-cavities. The chip of the invention is matched with different fluorescent marking technologies, and measures the fluorescent signal change in a micro-channel area and the fluorescent signal change in a micro-cavity area, thereby achieving the effects of double detection and mutual verification, and further achieving the rapid, sensitive and accurate double-color double detection of miRNAs.
Description
Technical Field
The invention belongs to the technical field of nucleic acid detection, and particularly relates to a microfluidic chip and a method for detecting nucleic acid.
Background
Cancer is one of the leading causes of death worldwide and gives rise to great pain for patients. The traditional detection means mostly adopt Computer Tomography (CT), nuclear magnetic resonance, radioactive detection, pathological detection, blood examination and the like, and most of the methods use huge instruments, are complex in operation, are time-consuming and labor-consuming, have low sensitivity and poor specificity. In addition, in most cases, the traditional detection technology can only detect larger tumors, often when diagnosis is confirmed, patients are already in the middle or late stage of cancer, early diagnosis cannot be carried out, and treatment time is missed.
In order to achieve early diagnosis and early treatment of tumors, more researchers have focused on detecting Tumor markers, which are chemical substances reflecting the presence of tumors. They are either not present in normal adult tissue but only in embryonic tissue, or are present in tumor tissue in amounts substantially exceeding those present in normal tissue, and their presence or amount may be indicative of tumor properties, thereby understanding tumor tissue development, cell differentiation, cell function, to aid in tumor diagnosis, classification, prognosis and therapeutic guidance. Tumor marker detection has the advantages that: the sensitivity is high, and a tumor patient can be detected early; the specificity is good, and the tumor/non-tumor patients can be accurately identified; the specificity of organs is realized, so that the tumor can be conveniently positioned; serum levels correlated with tumor volume size and clinical staging for prognosis; the half-life is short, the dynamic change of the tumor can be reflected, the detection method for monitoring the treatment effect, recurrence and metastasis has high precision and accuracy, and the operation is convenient.
mirnas are a class of endogenous single-stranded small molecule RNAs that are themselves not functional for encoding proteins, about 18-25 nucleotides in length. Studies have shown that abnormal expression of certain mirnas is closely related to tumorigenesis, tumor staging and tumor treatment, and that the presence of mirnas in serum can be clearly detected. All these findings indicate that mirnas can be used as tumor markers, with positive clinical significance for early diagnosis of malignant diseases.
Currently, widely used methods for miRNA analysis, including real-time reverse transcription Polymerase Chain Reaction (PCR), northern blotting (Northern blotting) and miRNA microarray techniques, can meet the detection requirements to some extent. However, these methods require transcription and amplification, which is time consuming and laborious, some require expensive kits and complex handling, and the detection throughput is low. Thus, there is a need to establish a miRNA detection system that is sensitive, fast, low cost and easy to operate.
Disclosure of Invention
The invention aims to provide a micro-fluidic chip and a method for detecting miRNA, which can simultaneously perform double-color fluorescence dual detection of different tumor markers on the same detection sample, greatly improve the sensitivity and precision of detection, realize rapid and high-flux detection of the tumor markers and provide an effective detection means for early diagnosis of tumors.
In order to achieve the above object, the present invention adopts a first technical scheme that: the micro-fluidic chip for double-color fluorescence detection comprises a cover plate and a substrate, wherein the cover plate is of a double-layer structure and comprises a valve control layer and a micro-channel layer; the valve control layer is used for controlling the opening and closing of the liquid flow channel of the micro-flow channel layer; the micro-channel layer comprises a plurality of detection units, and each detection unit comprises two micro-channels; one end of the micro-channel is provided with a sample inlet, and the other end of the micro-channel is provided with a micro-cavity and a sample outlet; the substrate is modified with nanoscale graphene oxide or graphene materials at positions corresponding to the micro-channels, and glutaraldehyde is modified at positions corresponding to the micro-cavities.
As a preferred mode of the invention, the sample injection hole comprises a probe sample injection hole and a sample injection hole, and the two micro-channels are respectively connected with two different probe sample injection holes and connected with the same sample injection hole.
As a preferred mode of the present invention, the two micro flow channels are respectively connected with two different micro cavities, and the two micro cavities are not communicated.
As a preferred mode of the present invention, two micro flow channels are connected to the same micro cavity.
As a preferred mode of the present invention, the valve control layer includes a plurality of valves for controlling the flow of liquid into and out of the micro flow channel, a water inlet/outlet, and a flow channel connecting the water inlet/outlet and the valve.
As a preferred mode of the invention, probes with fluorescent groups or fluorescent staining are paved on the nanoscale graphene oxide or graphene material modified on the substrate.
The invention also provides a double-color fluorescence dual detection method, which comprises the following steps:
closing a valve between the micro-channel and the micro-cavity;
injecting different DNA probes into the two micro-channels through two different probe injection holes respectively to enable the micro-channels to be full of the channels, and incubating for 0.5-2h;
discharging excessive liquid through a sample outlet, and flushing the micro-channel by using PBS to remove single-stranded DNA which is not adsorbed on the substrate; detecting a fluorescent signal of the DNA probe in the flow channel;
opening a valve between the micro-channel and the micro-cavity, closing a valve at a sample outlet and a DNA sample inlet, then injecting a sample to be detected from the sample inlet, and pushing the sample into the two channels simultaneously by using nitrogen;
after reacting with the DNA probe in the micro-channel, the sample to be tested flows into the micro-cavity through the micro-channel;
carrying out fluorescence detection on the liquid in the flow channel and the liquid entering the microcavity;
and judging whether the sample to be detected contains the target miRNA according to different fluorescent colors and fluorescent intensities.
Further preferably, the detection result of the liquid fluorescence in the flow channel is compared with the fluorescence signal of the DNA probe, or the content of the target miRNA in the sample to be detected is judged according to the fluorescence intensity in the microcavity.
As a preferred embodiment of the present invention, the 3' -end of the DNA probe is modified with NH 2 C 6 A group.
Further preferably, the NDA probe is stained with a fluorescent dye or a fluorescent group is modified at the 5' end of the DNA probe.
Compared with the prior art, the invention has the beneficial effects that: the invention combines the development of nano graphene oxide and glutaraldehyde, assembles the nano graphene oxide and glutaraldehyde on a glass slide through an autonomous assembling technology, integrates the nano graphene oxide and glutaraldehyde with a high-flux double-layer microfluidic chip technology, and can detect different tumor markers at the same time. The chip can control flow through the control valve, has strong controllability and is matched with different fluorescent marking technologies, thereby achieving the rapid, sensitive and accurate double-color double detection of the miRNAs.
Drawings
Fig. 1 is a schematic diagram of a microfluidic chip experiment for dual-color dual-fluorescence detection of miRNA provided by the invention;
fig. 2 is a perspective view of a red-green dual fluorescence detection microfluidic chip provided by the invention;
fig. 3 is a front view of the structure of the red-green dual fluorescence detection micro-fluidic chip provided by the invention;
fig. 4 is a schematic diagram of a micro-fluidic layer structure of a red-green dual-fluorescence detection micro-fluidic chip provided by the invention
FIG. 5 is a schematic diagram of a valve control layer structure of the red-green dual fluorescence detection micro-fluidic chip provided by the invention;
fig. 6 is a perspective view of a blue-green dual fluorescence detection microfluidic chip provided by the invention;
fig. 7 is a front view of a structure of a blue-green dual-color fluorescence detection micro-fluidic chip provided by the invention;
FIG. 8 is a schematic diagram of a micro-channel layer structure of the blue-green dual fluorescence detection micro-fluidic chip provided by the invention;
fig. 9 is a schematic diagram of a valve control layer structure of the blue-green dual fluorescence detection microfluidic chip provided by the invention.
Detailed Description
In order that the invention may be readily understood, a more particular description thereof will be rendered by reference to specific embodiments that are illustrated in the appended drawings. Preferred embodiments of the present invention are shown in the drawings. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.
The first embodiment provided by the invention is: the structure of the micro-fluidic chip for dual detection of red and green fluorescence is shown in fig. 2, and the micro-fluidic chip sequentially comprises: the glass slide comprises a glass slide substrate 1 modified with graphene and glutaraldehyde, a micro-channel layer 2 and a valve control layer 3. Wherein the micro-channel layer 2 and the valve control layer 3 adopt Polydimethylsiloxane (PDMS) and a curing agent matched with the PDMS according to the following weight ratio of 10: the material is prepared by mixing in proportion, and has good biocompatibility and chemical inertness. The micro flow channel layer 2 is mainly used for fixing DNA probes and detecting samples, and as shown in fig. 3 and 4, the micro flow channel layer 2 comprises a plurality of detection units, and high-throughput detection can be realized on one chip. Each of the detection units comprises a first flow channel 11 and a second flow channel 16. The first flow channel 11 and the second flow channel 16 have the same structure, are Y-shaped and are mirror images of each other. A DNA probe introduction hole 13 and a sample introduction hole 15 are provided at one ends of the first flow channel 11 and the second flow channel 16, respectively. The sample injection hole 15 is disposed between the first flow channel 11 and the second flow channel 16 and is communicated with the first flow channel 11 and the second flow channel 16, the sample injection hole 15 is a common injection hole of the first flow channel 11 and the second flow channel 16, and the same sample to be detected can be simultaneously added into the two flow channels for detection.
At the other ends of the first flow channel 11 and the second flow channel 16, a first microcavity 4 and a second microcavity 5 are connected, respectively. The first microcavity 4 and the second microcavity 5 are separated from each other and do not communicate with each other. And a sample outlet hole 6 is respectively arranged beside one end of each micro-channel close to the micro cavity. The valve control layer 3 mainly uses the fluid pressure to control the direction of the fluid in the lower micro-channel layer 2. As shown in fig. 5, the valve control layer 3 also has a plurality of control units corresponding to the fluidic channel layer 2 thereunder. Each control unit comprises two microcavity control valves 8, two sample outlet control valves 9 and 2 sample inlet control valves 14. The two microcavity control valves 8 are arranged at the corresponding positions between the two microcavities and the two micro-channels, the two sample outlet control valves 9 are arranged at the corresponding positions between the two sample outlet holes 6 and the micro-channels, and the two sample inlet control valves 14 are arranged at the corresponding positions between the two DNA probe sample inlet holes 13 and the micro-channels. The two microcavity control valves 8 are connected with the first water inlet/outlet 7 through a microchannel; the two sample outlet control valves 9 are connected with the second water inlet/outlet 10 through micro-channels; two sample injection control valves 14 are connected to the third water inlet/outlet 12 through a micro-channel. Ultrapure water is injected and pumped into the micro-flow channel through the water inlet/outlet and is matched with nitrogen, so that the closing and opening of each valve can be controlled, and the trend of fluid in the micro-flow channel at the lower layer is controlled.
As shown in fig. 2, the slide substrate 1 is located below the micro flow channel layer 2, and a nano graphene oxide material is decorated (laid) at a position corresponding to the micro flow channel, and a fluorescent dyed DNA probe is laid on the nano graphene material. The nano graphene has better biocompatibility, low biotoxicity and strong chemiluminescent sensitization. The size of the nano graphene is smaller than 14nm, and the quenching capacity is reduced along with the reduction of the size, so that the nano graphene has weak quenching capacity and does not quench the fluorescence of a fluorescent group carried by a DNA single strand with the length of 20 bp. Due to pi-pi bond, it still maintains the single-chain adsorption and double-chain desorption characteristics. Glutaraldehyde is modified at the position of the glass slide substrate 1 corresponding to the microcavity for fixing NH modified at the 3' end 2 C 6 The DNA probe of the group is combined with the target miRNA to form a double-chain structure. The amino groups in the DNA probe are covalently cross-linked to aldehyde groups on the slide substrate 1 to form stable chemical bonds, which are immobilized on the substrate of the microcavity moiety for subsequent fluorescence detection.
The detection method and principle of the microfluidic chip for dual detection of red and green fluorescence provided in the embodiment are shown in fig. 1, and specifically described as follows:
1. NH was modified with Acridine Orange (AO) dye and 3' end 2 C 6 Uniformly mixing the concentration of the DNA probes of the groups and performing fluorescent staining;
2. injecting proper amount of ultrapure water into the first water inlet/outlet 7 through a microinjection pump, then continuously injecting nitrogen, pressing down a microcavity control valve 8, and blocking the liquid in the microchannel from flowing into the microcavity;
3、1uL of DNA probes (3' -terminal NH) having different base sequences were injected from two DNA probe injection holes 13, respectively 2 C 6 Groups, AO dye staining) to fill the first flow channel 11 and the second flow channel 16, and incubating for 1h to enable the groups to be fully fixed on the graphene substrate; 4. after the incubation is completed, excess liquid is drained from the wells 6 and washed with PBS, and the single-stranded DNA probes not adsorbed on the substrate are washed clean. At this time, the DNA probe is fixed on the substrate by means of the large pi bond adsorption of the graphene, and green fluorescence is generated;
5. detecting fluorescent signals of DNA probes bound on the base in the flow channel;
6. then, closing the nitrogen, pumping out the ultrapure water from the first water inlet/outlet 7, opening the microcavity control valve 8, simultaneously, injecting proper amounts of ultrapure water into the second water inlet/outlet 10 and the third water inlet/outlet 12, continuously injecting the nitrogen, pressing down the sample outlet control valve 9 and the sample injection control valve 14, and blocking the corresponding flow channels;
7. 1uL of sample to be detected is injected from a sample injection hole 15, and is pushed into a first flow channel 11 and a second flow channel 16 simultaneously by using nitrogen, and the same sample is detected by adopting two different DNA probes simultaneously;
8. after reacting with the DNA probe in the micro-channel, the sample to be tested flows into the micro-cavity through the micro-channel;
9. carrying out fluorescence detection on the liquid in the flow channel and the liquid entering the microcavity; judging whether the sample to be detected contains the target miRNA according to different fluorescent colors and fluorescent intensities;
10. and comparing the detection result of liquid fluorescence in the flow channel with the fluorescence signal of the DNA probe, or judging the content of the target miRNA in the sample to be detected according to the fluorescence intensity in the microcavity.
If the sample contains miRNA complementary to the DNA probe, the two miRNAs form double chains, the double chains are desorbed on the graphene substrate and respectively flushed into the two microcavities, and the double-chain DNA-miRNA hybrid is fixed on the substrate modified with glutaraldehyde through the chemical reaction of amino groups and aldehyde groups.
In the Y-shaped micro-channel of the graphene substrate part, the fluorescence value of green light is reduced after double chains are formed due to double-chain desorption, so that the phenomenon of 'existence to non-existence' is generated; the fluorescence value of red light generates a 'from nothing to nothing' phenomenon, after the fluorescence value of red light rises and enters the microcavity, double chains are adsorbed by glutaraldehyde substrates, and the 'from nothing to nothing' phenomenon of red fluorescence also occurs in the microcavity. The fluorescence values of green and red in the micro-channel are changed inversely, and whether the sample to be detected contains the target miRNA is judged by observing the change of fluorescence, so that the purpose of red-green double-color fluorescence detection of the miRNA is realized.
In addition, by carrying out fluorescence detection on the liquid in the flow channel and combining the fluorescence intensity of the DNA probe measured in the step 5, the content of the target miRNA in the sample to be measured can be judged and calculated by comparing the change of the fluorescence signal intensity before and after the formation of double chains. Meanwhile, the content of the target miRNA can be judged and calculated according to the fluorescence intensity value in the microcavity, and the two calculation results are mutually verified, so that the accurate quantification of the miRNA in the sample to be measured is realized.
The second embodiment provided by the invention is: the structure of the microfluidic chip for double detection of blue-green double fluorescence is shown in fig. 6, and the microfluidic chip sequentially comprises: the glass slide substrate 19 modified by graphene and glutaraldehyde, the micro-channel layer 20 and the valve control layer 21. Wherein the micro-channel layer 20 and the valve control layer 21 adopt Polydimethylsiloxane (PDMS) and a curing agent matched with the PDMS according to the following weight ratio of 10: the material is prepared by mixing in proportion, and has good biocompatibility and chemical inertness. The micro flow channel layer 20 is mainly used for fixing a DNA probe and detecting a sample, and as shown in fig. 7 and 8, the micro flow channel layer 20 includes a plurality of detecting units, and high throughput detection can be realized on one chip. Each of the detection units includes a first flow channel 28 and a second flow channel 33. The first flow channel 28 and the second flow channel 33 have the same structure, are Y-shaped and are mirror images of each other. A DNA probe injection hole 30 and a sample injection hole 32 are provided at one ends of the first flow channel 28 and the second flow channel 33, respectively. The sample injection hole 32 is disposed between the first flow channel 28 and the second flow channel 33 and is communicated with the first flow channel 28 and the second flow channel 33, the sample injection hole 32 is a common injection hole of the first flow channel 28 and the second flow channel 33, and the same sample to be detected can be simultaneously added into the two flow channels for detection.
At the other end of the first flow channel 28 and the second flow channel 33, one microcavity 22 is commonly connected. And a sample outlet hole 23 is arranged beside one end of the two micro-channels close to the micro cavity. The valve control layer 21 mainly uses the fluid pressure to control the direction of the fluid in the lower micro flow channel layer 20. As shown in fig. 9, the valve control layer 21 also has a plurality of control units corresponding to the fluidic channel layer 20 thereunder. Each control unit comprises two microcavity control valves 25, two sample outlet control valves 26 and 2 sample inlet control valves 31. Two microcavity control valves 25 set in the microcavity 22 and two micro flow channel between the corresponding position, two sample control valves 26 set in two sample hole 23 and flow channel between the corresponding position, two sample control valves 31 set in two DNA probe sample hole 30 and two flow channel between the corresponding position. Two microcavity control valves 25 are connected to the first water inlet/outlet 24 through microchannels; the two sample outlet control valves 26 are connected with the second water inlet/outlet 27 through micro-channels; two sample injection control valves 31 are connected to the third water inlet/outlet 29 through a micro flow channel. Ultrapure water is injected and pumped into the micro-flow channel through the water inlet/outlet and is matched with nitrogen, so that the opening and closing of each valve can be controlled, and the trend of fluid in the micro-flow channel at the lower layer is controlled.
As shown in fig. 6, the slide substrate 19 is located below the micro flow channel layer 20, and is decorated (laid) with a nano graphene oxide material at a position corresponding to the micro flow channel, and a fluorescence decorated DNA probe is laid on the nano graphene material. The nano graphene has better biocompatibility, low biotoxicity and strong chemiluminescent sensitization. The size of the nano graphene is smaller than 14nm, and the quenching capacity is reduced along with the reduction of the size, so that the nano graphene has weak quenching capacity and does not quench the fluorescence of a fluorescent group carried by a DNA single strand with the length of 20 bp. Due to pi-pi bond, it still maintains the single-chain adsorption and double-chain desorption characteristics.
The slide substrate 19 is modified with glutaraldehyde at the position corresponding to the microcavity 22 for fixing the 3' -end modified with NH 2 C 6 Double-chain formed by combining DNA probe of group and target miRNAStructure is as follows. The amino groups in the DNA probe are covalently cross-linked to aldehyde groups on the slide substrate 19 to form stable chemical bonds that are immobilized on the substrate of the microcavity 22 portion for subsequent fluorescence detection.
The detection method and principle of the microfluidic chip for dual detection of blue-green double fluorescence provided in the embodiment are shown in fig. 1, and specifically described as follows:
1. the 3' end is modified with NH by using fluorescent groups FAM and Cy3 with different excitation wavelengths respectively 2 C 6 Performing fluorescence modification on the 5' end of the DNA probe of the group; FAM excitation wavelength is 485nm, blue light is emitted, cy3 excitation wavelength is 532nm, and green light is emitted; 2. injecting a proper amount of ultrapure water into the first water inlet/outlet 24 by a micro-injection pump, then continuously injecting nitrogen, pressing down a microcavity control valve 25, and blocking the liquid in the micro-channel from flowing into the microcavity;
3. 1uL of DNA probes (3' -terminal NH) having different base sequences were injected from two DNA probe injection holes 30, respectively 2 C 6 A group, a 5' end fluorescent group) to fill the first flow channel 28 and the second flow channel 33, and incubating for 1h to be sufficiently immobilized on the graphene substrate;
4. after the incubation is completed, excess liquid is drained from the sample outlet 23 and washed with PBS, and the single-stranded DNA probes that are not adsorbed on the substrate are washed clean. At this time, the DNA probes are fixed on the substrate by means of large pi bond adsorption of graphene, and the DNA probes in different flow channels are provided with fluorescent groups with different excitation wavelengths;
5. detecting fluorescent signals of DNA probes bound on the base in the flow channel;
6. then, the nitrogen gas is closed, ultrapure water is pumped out from the first water inlet/outlet 24, the microcavity control valve 25 is opened, meanwhile, a proper amount of ultrapure water is injected into the second water inlet/outlet 27 and the third water inlet/outlet 29, then the nitrogen gas is continuously injected, the sample outlet control valve 26 and the sample injection control valve 31 are pressed down, and corresponding flow channels are blocked;
7. 1uL of sample to be detected is injected from a sample injection hole 32, and is pushed into a first flow channel 28 and a second flow channel 33 simultaneously by using nitrogen, and the same sample is detected by adopting two different DNA probes simultaneously;
8. after reacting with the DNA probe in the micro-channel, the sample to be tested flows into the micro-cavity through the micro-channel;
9. respectively carrying out fluorescence detection on the liquid in the flow channel and the liquid entering the microcavity; judging whether the sample to be detected contains the target miRNA according to different fluorescent colors and fluorescent intensities;
10. and comparing the fluorescence detection result of the liquid in the flow channel with the fluorescence signal of the DNA probe, and judging the content of the target miRNA in the sample to be detected.
If there is a miRNA complementary to the DNA probe in the sample, the two miRNAs form double chains, the double chains are desorbed on the graphene substrate and are flushed into the microcavity 22, the double-chain DNA-miRNA hybrid is fixed on the glutaraldehyde substrate through the chemical reaction of amino groups and aldehyde groups, and the fluorescence intensities of different fluorescent groups are detected in the same microcavity by using lasers with different wavelengths, so that the double-color detection is completed.
In the Y-shaped micro-channel of the graphene substrate, the double-strand is desorbed and flushed into the microcavity, so that the corresponding fluorescence value is reduced before and after the double-strand is formed; the fluorescence value is increased due to the adsorption of double strands by glutaraldehyde substrates at the microcavities, which produces a "no-to-no" phenomenon. And judging whether the sample to be detected contains the target miRNA or not by observing the change of fluorescence intensity in the micro-channel and the micro-cavity, wherein the blue-green two different fluorescence colors are used for distinguishing different miRNAs.
In addition, the content of the target miRNA in the sample to be detected can be judged and calculated by combining the fluorescence detection result of the liquid in the flow channel with the fluorescence intensity of the DNA probe measured in the step 5 and comparing the change of the fluorescence signal intensity before and after the double-chain formation. Meanwhile, the content of the target miRNA can be judged and calculated according to the fluorescence intensity value in the microcavity, and the two calculation results are mutually verified, so that the accurate quantification of the miRNA in the sample to be measured is realized.
The two fluorescent materials emit fluorescence with different colors, so that blue-green double-color fluorescence detection is carried out on different miRNAs.
The microfluidic chip of the invention has the advantages that:
1. the design of the double-layer chip can better control the flow direction of liquid, and the liquid is integrated on the same chip to finish detection;
2. the Y-shaped flow channel design can compare the change of the fluorescence values before and after the reaction in the same flow channel, so that the characterization is more visual and accurate;
3. two different materials are integrated on the substrate simultaneously, so that the biocompatibility is good, and the fluorescence detection sensitivity is high;
4. the red-green double-color detection chip is suitable for AO modified DNA probe molecules, different DNA probe molecules are dyed by using the same dye, and finally, the DNA probe molecules are respectively fixed in different microcavities for detection, nano graphene oxide adsorbs single chains and emits green light, and the fluorescence value is reduced after double chains are formed; glutaraldehyde adsorbs double chains and emits red light, and fluorescence value is increased after double chains are formed; the fluorescence characterization can be performed simultaneously, so that the dual detection effect is achieved, and the detection result is more reliable;
5. the blue-green double-color detection chip is suitable for DNA probes marked by fluorescent groups with different excitation wavelengths, the fluorescent groups of different types modify the DNA probe molecules of different types, and finally the DNA probe molecules are fixed in the same microcavity, and laser with different wavelengths is adopted for fluorescence detection, and the fluorescence value is changed before and after double chains are formed, so that double-color double-detection is completed;
6. the DNA probe sample inlet and the sample inlet are separated, and two kinds of detection can be simultaneously carried out by one sample adding, so that the detection efficiency is improved;
7. the experimental scheme has the advantages of simple experimental principle, easy operation, high detection sensitivity, short time consumption, stable performance and low cost;
8. the high-flux microfluidic chip can detect multiple miRNAs simultaneously.
Claims (8)
1. The micro-fluidic chip for double-color fluorescence detection comprises a cover plate and a substrate, and is characterized in that: the cover plate is of a double-layer structure and comprises a valve control layer and a micro-channel layer; the valve control layer is used for controlling the trend of the liquid in the flow channel of the micro-channel layer; the micro-channel layer comprises a plurality of detection units, and each detection unit comprises two micro-channels; the two micro-channels are Y-shaped and are mirror images of each other; one end of the micro-channel is provided with a sample inlet, and the other end of the micro-channel is provided with a micro-cavity and a sample outlet; the sample injection hole comprises a probe sample injection hole and a sample injection hole, and two micro-channels which are mirror images of each other are respectively connected with two different probe sample injection holes and share the same sample injection hole; the substrate is modified with nanoscale graphene oxide or graphene materials at positions corresponding to the micro-channels, and glutaraldehyde is modified at positions corresponding to the micro-cavities.
2. The dual-color fluorescence dual-detection microfluidic chip according to claim 1, wherein: the two micro-channels are respectively connected with two different micro-cavities, and the two micro-cavities are not communicated.
3. The dual-color fluorescence dual-detection microfluidic chip according to claim 1, wherein: the two micro-channels are connected with the same micro-cavity.
4. A micro-fluidic chip for dual detection of two-color fluorescence according to any one of claims 1-3, characterized in that: the valve control layer comprises a plurality of valves for controlling liquid to enter and flow out of the micro-flow channel, a water inlet/outlet and a flow channel connected between the water inlet/outlet and the valves.
5. A dual fluorescent detection method for non-diagnostic purposes, using the dual fluorescent dual detection microfluidic chip according to claim 1, comprising:
closing a valve between the micro-channel and the micro-cavity;
injecting different DNA probes into the two micro-channels through two different probe injection holes respectively to enable the micro-channels to be full of the channels, and incubating for 0.5-2h;
discharging excessive liquid through a sample outlet, and flushing the micro-channel by using PBS to remove single-stranded DNA which is not adsorbed on the substrate;
detecting a fluorescent signal of the DNA probe in the flow channel;
opening a valve between the micro-channel and the micro-cavity, closing a valve at a sample outlet and a DNA sample inlet, then injecting a sample to be detected from the sample inlet, and pushing the sample into the two channels simultaneously by using nitrogen;
after reacting with the DNA probe in the micro-channel, the sample to be tested flows into the micro-cavity through the micro-channel;
carrying out fluorescence detection on the liquid in the flow channel and the liquid entering the microcavity;
and judging whether the sample to be detected contains the target miRNA according to different fluorescent colors and fluorescent intensities.
6. The dual-color fluorescence dual-detection method for non-diagnostic purposes according to claim 5, wherein the detection result of liquid fluorescence in the flow channel is compared with the fluorescence signal of the DNA probe, or the content of the target miRNA in the sample to be detected is determined according to the fluorescence intensity in the microcavity.
7. The dual fluorescent detection method for non-diagnostic purposes according to claim 5 or 6, wherein the 3' -end of the DNA probe is modified with NH 2 C 6 A group.
8. The dual color fluorescence dual detection method for non-diagnostic purposes according to claim 5 or 6, wherein the DNA probe is stained with a fluorescent dye or a fluorescent group is modified at the 5' end of the DNA probe.
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