CN113025477A - 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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Abstract
The invention belongs to the technical field of nucleic acid detection, and particularly relates to a micro-fluidic chip and a method for nucleic acid detection. The micro-fluidic chip for double-color fluorescence double 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 direction of liquid in the 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 flow channel is provided with a sample inlet hole, and the other end of the micro flow channel is provided with a micro cavity and a sample outlet hole; in the substrate, the positions corresponding to the micro-channels are modified with nano-scale graphene oxide or graphene materials, and the positions corresponding to the micro-cavities are modified with glutaraldehyde. The chip of the invention is matched with different fluorescence labeling technologies to measure the change of fluorescence signals in a micro-channel area and the change of fluorescence signals in a micro-cavity area, thereby achieving the effects of double detection and mutual evidence, and further achieving the quick, 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 micro-fluidic chip and a method for nucleic acid detection.
Background
Cancer is one of the leading causes of death worldwide and causes significant pain to patients. The traditional detection means mostly adopts Computed Tomography (CT), nuclear magnetic resonance, radioactive detection, pathological detection, blood examination and the like, and most of the methods use large instruments, are complex to operate, waste time and labor, have low sensitivity and poor specificity. In addition, in most cases, the traditional detection technology can only detect larger tumors, and often the patients are in the middle or late stage of cancer at the time of diagnosis, so that early diagnosis cannot be carried out, and the treatment time is delayed.
In order to realize early diagnosis and treatment of tumors, more researchers are focusing on detecting Tumor markers, which are chemical substances reflecting the existence of tumors. They are not existed in normal adult tissue but only in embryonic tissue, or their content in tumor tissue is greatly greater than that in normal tissue, and their existence or quantity can indicate the nature of tumor, so that it can know the tissue generation, cell differentiation and cell function of tumor, and can help diagnosis, classification, prognosis and treatment guidance of tumor. Tumor marker detection has the advantages that: the sensitivity is high, and the tumor patient can be detected early; the specificity is good, and tumor/non-tumor patients can be accurately identified; the tumor locating kit has organ specificity and is convenient for locating tumors; serum levels are correlated with tumor volume, clinical stage, and used to determine prognosis; the half-life period is short, the dynamic change of the tumor can be reflected, the precision and the accuracy of the measuring method for monitoring the treatment effect, the recurrence and the metastasis are high, and the operation is convenient.
miRNAs are endogenous RNAs which do not have the function of coding proteins per se, and are endogenous single-stranded small RNA molecules with the length of about 18 to 25 nucleotides. Research shows that the abnormal expression of some miRNA is closely related to tumorigenesis, tumor stage and tumor treatment, and the existence of miRNA in serum can be definitely detected. All these findings indicate that miRNA can be used as tumor marker, and has positive clinical significance for early diagnosis of malignant diseases.
At present, widely applied miRNA analysis methods, including real-time reverse transcription Polymerase Chain Reaction (PCR), Northern blotting technology (Northern blotting), miRNA microarray technology and the like, can meet detection requirements to a certain extent. However, these methods require transcription and amplification, which are time-consuming and laborious, some require expensive kits and complex processing, and the detection throughput is low. Therefore, there is a need to establish a sensitive, rapid, low-cost and easy-to-operate miRNA detection system.
Disclosure of Invention
The invention aims to provide a micro-fluidic chip and a method for detecting miRNA, which can simultaneously carry out double-color fluorescence double detection on 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 purpose, the invention adopts a first technical scheme that: the micro-fluidic chip for double-color fluorescence double 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 flow channel is provided with a sample inlet hole, and the other end of the micro flow channel is provided with a micro cavity and a sample outlet hole; the substrate is modified with nano-scale graphene oxide or graphene materials at the position corresponding to the micro-channel, and is modified with glutaraldehyde at the position corresponding to the microcavity.
In a preferred embodiment of the present invention, the sample inlet includes a probe sample inlet and a sample inlet, and the two microchannels are respectively connected to two different probe sample inlets and to the same sample inlet.
In a preferred embodiment of the present invention, the two microchannels are connected to two different microcavities, respectively, and the two microcavities are not in communication with each other.
In a preferred embodiment of the present invention, two microchannels are connected to the same microcavity.
As a preferred mode of the present invention, the valve control layer includes a plurality of valves for controlling liquid to flow into and out of the microchannel, water inlets/outlets, and a channel connecting the water inlets/outlets and the valves.
In a preferred embodiment of the present invention, a probe with a fluorescent group or a probe stained by fluorescence is laid on the nano-scaled graphene oxide or graphene material modified on the substrate.
The invention also provides a double-color fluorescence double detection method, which comprises the following steps:
closing a valve between the micro-channel and the microcavity;
injecting different DNA probes into the two micro-channels through two different probe sample injection holes respectively to fill the channels with the DNA probes, and incubating for 0.5-2 h;
discharging the redundant liquid through the sample outlet, and flushing the micro-flow channel with PBS to remove the single-stranded DNA which is not adsorbed on the substrate;
detecting the fluorescent signal of the DNA probe in the flow channel;
opening a valve between the micro-channel and the microcavity, closing the valve at the sample outlet and the DNA sample inlet, injecting a sample to be detected from the sample inlet, and pushing the sample to be detected into the two channels by using nitrogen gas;
the sample to be detected reacts with the DNA probe in the micro-channel and then flows into the micro-cavity through the micro-channel;
carrying out fluorescence detection on the liquid in the flow channel and entering the micro cavity;
and judging whether the sample to be detected contains the target miRNA or not according to different fluorescence colors and fluorescence intensities.
Preferably, the detection result of the fluorescence of the liquid 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.
In a preferred embodiment of the present invention, the 3' end of the DNA probe is modified with NH2C6A group.
Further preferably, the NDA probe is dyed 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 following beneficial effects: the invention combines the development of nano graphene oxide and glutaraldehyde, assembles the nano graphene oxide and glutaraldehyde onto a glass slide through an autonomous loading technology, integrates with a high-flux double-layer microfluidic chip technology, and can detect different tumor markers simultaneously. The chip can control the flow through a control valve, has strong controllability, and is matched with different fluorescence labeling technologies, thereby achieving the quick, sensitive and accurate double-color double detection of miRNAs.
Drawings
FIG. 1 is a schematic diagram of a microfluidic chip experiment for double-color dual fluorescence detection of miRNA according to the present invention;
FIG. 2 is a perspective view of a red-green double-color dual fluorescence detection microfluidic chip provided by the invention;
FIG. 3 is a structural front view of a red-green double-color dual fluorescence detection microfluidic chip provided by the invention;
FIG. 4 is a schematic view of a structure of a micro-channel layer of a red-green double-color dual fluorescence detection micro-fluidic chip provided by the invention
FIG. 5 is a schematic view of a valve control layer structure of a red-green double-color dual fluorescence detection microfluidic chip provided by the invention;
FIG. 6 is a perspective view of a blue-green two-color dual fluorescence detection microfluidic chip provided by the present invention;
FIG. 7 is a structural front view of a blue-green two-color dual fluorescence detection microfluidic chip provided by the present invention;
fig. 8 is a schematic view of a micro-channel layer structure of a blue-green double-color dual fluorescence detection micro-fluidic chip provided by the invention;
fig. 9 is a schematic view of a valve control layer structure of a blue-green two-color dual fluorescence detection microfluidic chip provided by the invention.
Detailed Description
In order to facilitate an understanding of the invention, the invention is described in more detail below with reference to the accompanying drawings and specific examples. 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 as follows: the structure of the micro-fluidic chip for red-green double-color fluorescence double detection is shown in figure 2, and the micro-fluidic chip sequentially comprises the following components from bottom to top: the glass slide comprises a glass slide substrate 1 modified with graphene and glutaraldehyde, a micro-flow channel layer 2 and a valve control layer 3. Wherein the micro-flow channel layer 2 and the valve control layer 3 adopt Polydimethylsiloxane (PDMS) and a curing agent matched with the PDMS, and the mass ratio of the PDMS to the curing agent is 10: 1 proportion, and the material has good biocompatibility and chemical inertness. The micro-channel layer 2 is mainly used for fixing DNA probes and detecting samples, and as shown in FIGS. 3 and 4, the micro-channel layer 2 comprises a plurality of detection units, so that high-throughput detection can be realized on one chip. Each of the detecting units includes 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 and are both in a "Y" shape, and the two are mirror images of each other. A DNA probe inlet hole 13 and a sample inlet hole 15 are provided at one end of the first flow channel 11 and the second flow channel 16, respectively. The sample inlet hole 15 is arranged 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 inlet hole 15 is a shared sample inlet 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 respectively through the sample inlet hole 15.
At the other ends of the first flow path 11 and the second flow path 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. Sample outlet holes 6 are respectively arranged near one ends of the two micro channels close to the micro-cavity. The valve control layer 3 mainly functions to control the fluid flow direction in the lower micro flow channel layer 2 by using the fluid pressure. As shown in fig. 5, the valve control layer 3 also has a plurality of control units corresponding to the micro flow channel layer 2 therebelow. Each control unit comprises two microcavity control valves 8, two sample outlet control valves 9 and 2 sample inlet control valves 14. Two microcavity control valves 8 are arranged at the corresponding positions between the two microcavities and the two microchannels, two sample outlet control valves 9 are arranged at the corresponding positions between the two sample outlet holes 6 and the microchannels, and two sample inlet control valves 14 are arranged at the corresponding positions between the two DNA probe sample inlet holes 13 and the microchannels. The two microcavity control valves 8 are connected to the first water inlet/outlet 7 through a microchannel; the two sample outlet control valves 9 are connected to a second water inlet/outlet 10 through a micro-channel; two sample injection control valves 14 are connected to the third water inlet/outlet port 12 through a microchannel. Ultrapure water is injected into and pumped out of the micro-channel through the water inlet/outlet, and the valves can be controlled to be closed and opened by matching with nitrogen, so that the trend of fluid in the lower micro-channel is controlled.
As shown in fig. 2, the slide glass 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 DNA probe for fluorescent staining is laid on the nano graphene material. The nano graphene has good biocompatibility, low biotoxicity and strong chemiluminescence sensitization effect. The size of the nano-graphene is smaller than 14nm, and the quenching capability is reduced along with the reduction of the size, so that the nano-graphene is very weak in quenching capability and does not quench the fluorescence of a fluorescent group carried by a DNA single chain with the length of 20 bp. Due to the action of pi-pi bond, it still retains the characteristics of single-strand adsorption and double-strand desorption. The position of the glass slide substrate 1 corresponding to the microcavity is modified with glutaraldehyde, and NH is modified for fixing the 3' end2C6The DNA probe of the group is combined with the target miRNA to form a double-stranded structure. Amino groups in the DNA probes and aldehyde groups on the glass slide substrate 1 form stable chemical bonds through covalent crosslinking, and the stable chemical bonds are fixed on the substrate of the microcavity part so as to facilitate subsequent fluorescence detection.
The detection method and the principle of the micro-fluidic chip for red-green double-color fluorescence double detection provided in the embodiment are shown in fig. 1, and are specifically described as follows:
1. adopts Acridine Orange (AO) dye and 3' end to modify NH2C6Uniformly mixing the DNA probes of the groups at equal concentration, and carrying out fluorescent dyeing;
2. injecting a proper amount of ultrapure water into the first water inlet/outlet 7 through a micro-injection pump, then continuously injecting nitrogen, pressing down the micro-cavity control valve 8, and blocking the liquid in the micro-channel from flowing into the micro-cavity;
3. 1uL of DNA probes (NH at the 3' end) having different base sequences were injected from the two DNA probe inlet holes 132C6Group, AO dye), filling the first flow channel 11 and the second flow channel 16, and incubating for 1h to sufficiently fix the two on the graphene substrate; 4. after the incubation is completed, the excess liquid is drained from the outlet 6,and washed with PBS to wash out the single-stranded DNA probes that are not adsorbed on the substrate. At the moment, the DNA probe is adsorbed and fixed on the substrate by virtue of a large pi bond of graphene, and emits green fluorescence; 5. detecting a fluorescence signal of a DNA probe combined on a base in a flow channel 6, then closing nitrogen, pumping ultrapure water out from a first water inlet/outlet 7, opening a micro-cavity control valve 8, simultaneously injecting a proper amount of ultrapure water into a second water inlet/outlet 10 and a third water inlet/outlet 12, then continuously injecting nitrogen, pressing a sample outlet control valve 9 and a sample injection control valve 14 down, and blocking the corresponding flow channel;
7. injecting 1uL of sample to be detected from the sample inlet hole 15, pushing the sample to be detected into the first flow channel 11 and the second flow channel 16 by using nitrogen gas, and detecting the same sample by using two different DNA probes;
8. the sample to be detected reacts with the DNA probe in the micro-channel and then flows into the micro-cavity through the micro-channel;
9. carrying out fluorescence detection on the liquid in the flow channel and entering the micro cavity; judging whether the sample to be detected contains the target miRNA according to different fluorescence colors and fluorescence intensities;
10. and comparing the detection result of the 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 miRNA complementary to the DNA probe exists in the sample, the miRNA and the DNA probe form double chains, the double chains are desorbed on the graphene substrate and respectively flushed into the two micro-cavities, and the double-chain DNA-miRNA heterozygote is fixed on the substrate modified with glutaraldehyde through the chemical reaction of amino and aldehyde groups.
In the Y-shaped micro-channel at the bottom of the graphene substrate, due to double-strand desorption, the fluorescence value of green light is reduced after double strands are formed, and the phenomenon from existence to nonexistence is generated; the red fluorescence value generates a phenomenon from non-existence to existence, the double chains are adsorbed by the glutaraldehyde substrate after the red fluorescence value is increased and enters the microcavity, and the phenomenon from non-existence to existence of the red fluorescence also occurs in the microcavity. The fluorescence values of green and red in the micro-channel are changed oppositely, and whether the sample to be detected contains the target miRNA or not is judged by observing the change of the fluorescence, so that the aim of performing red-green double-color double-fluorescence detection on the miRNA is fulfilled.
In addition, the content of the target miRNA in the sample to be detected can be judged and calculated by detecting the fluorescence of the liquid in the flow channel and comparing the change of the fluorescence signal intensity before and after the double strand is formed by combining the fluorescence intensity of the DNA probe measured in the step 5. 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 miRNA in the sample to be detected can be accurately quantified.
The second embodiment provided by the invention is as follows: the structure of the micro-fluidic chip for blue-green double-color fluorescence double detection is shown in figure 6, and the micro-fluidic chip sequentially comprises from bottom to top: a glass slide substrate 19 modified with graphene and glutaraldehyde, a micro-channel layer 20 and a valve control layer 21. Wherein the micro-flow channel layer 20 and the valve control layer 21 are made of Polydimethylsiloxane (PDMS) and a curing agent matched with the PDMS, and the ratio of the PDMS to the curing agent is 10: 1 proportion, and the material has good biocompatibility and chemical inertness. The micro channel layer 20 is mainly used for fixing DNA probes and detecting samples, and as shown in FIGS. 7 and 8, the micro channel layer 20 includes a plurality of detecting units, so that high-throughput detection can be realized on one chip. Each of which includes a first flow channel 28, a second flow channel 33. The first flow channel 28 and the second flow channel 33 have the same structure and are both in a "Y" shape, and the two are mirror images of each other. At one ends of the first flow path 28 and the second flow path 33, a DNA probe inlet 30 and a sample inlet 32 are provided, respectively. The sample inlet 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 inlet hole 32 is a common sample inlet 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 respectively through the sample inlet hole.
The other ends of the first flow path 28 and the second flow path 33 are connected to a microcavity 22. Sample outlet holes 23 are arranged near one end of the two micro channels close to the micro-cavity. The valve control layer 21 is primarily used to control the fluid flow direction in the underlying microchannel layer 20 by fluid pressure. As shown in fig. 9, the valve control layer 21 also has a plurality of control units corresponding to the micro flow channel layer 20 therebelow. Each of the control units includes two microcavity control valves 25, two sample outlet control valves 26, and 2 sample inlet control valves 31. Two microcavity control valves 25 are disposed at corresponding positions between the microcavity 22 and the two microchannels, two sample outlet control valves 26 are disposed at corresponding positions between the two sample outlet holes 23 and the channels, and two sample inlet control valves 31 are disposed at corresponding positions between the two DNA probe sample inlet holes 30 and the two channels. Two microcavity control valves 25 are connected to the first water inlet/outlet port 24 through a microchannel; the two sample outlet control valves 26 are connected to the second water inlet/outlet 27 through a micro flow channel; two sample injection control valves 31 are connected to the third water inlet/outlet port 30 through microchannels. Ultrapure water is injected into and pumped out of the micro-channel through the water inlet/outlet, and the valves can be controlled to be opened and closed by matching with nitrogen, so that the trend of fluid in the lower micro-channel is controlled.
As shown in fig. 6, the slide glass substrate 19 is located below the micro flow channel layer 20, and a nano graphene oxide material is decorated (laid) 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 good biocompatibility, low biotoxicity and strong chemiluminescence sensitization effect. The size of the nano-graphene is smaller than 14nm, and the quenching capability is reduced along with the reduction of the size, so that the nano-graphene is very weak in quenching capability and does not quench the fluorescence of a fluorescent group carried by a DNA single chain with the length of 20 bp. Due to the action of pi-pi bond, it still retains the characteristics of single-strand adsorption and double-strand desorption.
The positions of the glass slide substrate 19 corresponding to the microcavity 22 are modified with glutaraldehyde, and NH is modified for fixing the 3' end2C6The DNA probe of the group is combined with the target miRNA to form a double-stranded structure. The amino group in the DNA probe and the aldehyde group on the glass slide substrate 19 form a stable chemical bond through covalent crosslinking, and the stable chemical bond is fixed on the substrate of the microcavity 22 part so as to facilitate the subsequent fluorescence detection.
The detection method and the principle of the microfluidic chip for blue-green two-color fluorescence dual detection provided in the embodiment are shown in fig. 1, and are specifically described as follows:
1. modifying NH at the 3' end by using fluorescent groups FAM and Cy3 with different excitation wavelengths2C6Carrying out fluorescence modification on the 5' end of the DNA probe of the group; the FAM has the excitation wavelength of 485nm and emits blue light, and the Cy3 has the excitation wavelength of 532nm and emits green light;
2. injecting a proper amount of ultrapure water into the first water inlet/outlet 24 through a micro-injection pump, then continuously injecting nitrogen, pressing down the micro-cavity control valve 25, and blocking the liquid in the micro-channel from flowing into the micro-cavity;
3. 1uL of DNA probes (NH at the 3' end) having different base sequences were injected from the two DNA probe wells 302C6Group, 5' -end fluorophore), filling the first flow channel 28 and the second flow channel 33, and incubating for 1h to sufficiently immobilize the groups on the graphene substrate;
4. after completion of the incubation, excess liquid was drained from the outlet port 23 and washed with PBS, and the single-stranded DNA probes not adsorbed on the substrate were washed clean. At the moment, the DNA probes are fixed on the substrate by virtue of the adsorption of large pi bonds of graphene, and the DNA probes in different flow channels are provided with fluorescent groups with different excitation wavelengths;
5. detecting a fluorescent signal of a DNA probe bound to the base in the flow channel;
6. then, the nitrogen gas is closed, ultrapure water is pumped out from the first water inlet/outlet port 24, the microcavity control valve 25 is opened, meanwhile, a proper amount of ultrapure water is injected into the second water inlet/outlet port 27 and the third water inlet/outlet port 29, then the nitrogen gas is continuously injected, the sample outlet control valve 26 and the sample inlet control valve 31 are pressed down, and the corresponding flow channels are blocked;
7. injecting 1uL of sample to be detected from the sample inlet hole 32, pushing the sample to be detected into the first flow channel 28 and the second flow channel 33 by using nitrogen gas, and detecting the same sample by using two different DNA probes;
8. the sample to be detected reacts with the DNA probe in the micro-channel and then 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 micro cavity; judging whether the sample to be detected contains the target miRNA according to different fluorescence colors and fluorescence 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 miRNA complementary with the DNA probe exists in the sample, the miRNA and the DNA probe form double chains, the double chains are desorbed on the graphene substrate and are flushed into the microcavity 22, the double-chain DNA-miRNA heterozygote is fixed on the glutaraldehyde substrate through chemical reaction of amino and aldehyde groups, and lasers with different wavelengths are used for detecting the fluorescence intensity of different fluorescent groups in the same microcavity, so that double-color detection is completed.
In the Y-shaped micro-channel on the graphene substrate part, due to double-strand desorption, the double-strand desorption is flushed into the microcavity, so that corresponding fluorescence values are reduced before and after the double-strand desorption; as the glutaraldehyde substrate at the microcavity adsorbs double chains, the fluorescence value generates a phenomenon from absent to present, and the fluorescence value is increased. Whether the sample to be detected contains the target miRNA or not is judged by observing the change of the fluorescence intensity in the micro-channel and the micro-cavity, and two different fluorescence colors of blue and green are used for distinguishing different miRNA.
In addition, the content of the target miRNA in the sample to be detected can be judged and calculated by comparing the change of the fluorescence signal intensity before and after the double strand is formed through the fluorescence detection result of the liquid in the flow channel and the fluorescence intensity of the DNA probe measured in the step 5. 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 miRNA in the sample to be detected can be accurately quantified.
The two can emit fluorescence of different colors, thereby carrying out blue-green double-color double fluorescence detection on different miRNA.
The microfluidic chip of the invention has the advantages that:
1. the double-layer chip is designed to 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 value before and after reaction in the same flow channel, and the representation is more visual and accurate;
3. two different materials are integrated on the substrate at the same time, so that the biocompatibility is good, and the fluorescence detection sensitivity is high;
4. the red-green double-color double-detection chip is suitable for AO modified DNA probe molecules, different DNA probe molecules are dyed by using the same dye, and finally the different DNA probe molecules are respectively fixed in different micro-cavities for detection, and the nano graphene oxide absorbs single chains, emits green light, and reduces the fluorescence value after double chains are formed; glutaraldehyde adsorbs double chains, red light is emitted, and the fluorescence value is increased after the double chains are formed; fluorescence characterization can be carried out simultaneously, so that the effect of double detection is achieved, and the detection result is more credible;
5. the blue-green double-color double detection chip is suitable for DNA probes marked by fluorophores with different excitation wavelengths, different types of fluorophores modify different types of DNA probe molecules, and are finally fixed in the same microcavity, and the laser with different wavelengths is adopted for fluorescence detection, so that the fluorescence value is changed before and after the formation of double chains, and the 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-time sample adding, so that the detection efficiency is improved;
7. the experimental principle of the experimental scheme is simple, the operation is easy, the detection sensitivity is high, the time consumption is short, the performance is stable, and the cost is low;
8. the high-flux microfluidic chip can be used for simultaneously detecting multiple miRNAs.
Claims (10)
1. The micro-fluidic chip for double detection of double-color fluorescence 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-flow channel layer; the valve control layer is used for controlling the direction of liquid in the 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 flow channel is provided with a sample inlet hole, and the other end of the micro flow channel is provided with a micro cavity and a sample outlet hole; the substrate is modified with nano-scale graphene oxide or graphene materials at the position corresponding to the micro-channel, and is modified with glutaraldehyde at the position corresponding to the microcavity.
2. The microfluidic chip for bicolor fluorescence double detection according to claim 1, wherein: the sample inlet holes comprise probe sample inlet holes and sample inlet holes, and the two micro-channels are respectively connected with two different probe sample inlet holes and the same sample inlet hole.
3. The microfluidic chip for bicolor fluorescence double detection according to claim 2, wherein: the two micro channels are respectively connected with two different micro cavities, and the two micro cavities are not communicated.
4. The microfluidic chip for bicolor fluorescence double detection according to claim 2, wherein: two micro channels are connected with the same micro cavity.
5. The microfluidic chip for two-color fluorescence dual detection according to any one of claims 1 to 4, wherein: 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.
6. The microfluidic chip for two-color fluorescence dual detection according to any one of claims 1 to 4, wherein: and probes with fluorescent groups or subjected to fluorescent dyeing are laid on the nano-scale graphene oxide or graphene materials modified on the substrate.
7. A two-color fluorescence double detection method is characterized by comprising the following steps:
closing a valve between the micro-channel and the microcavity;
injecting different DNA probes into the two micro-channels through two different probe sample injection holes respectively to fill the channels with the DNA probes, and incubating for 0.5-2 h;
discharging the redundant liquid through the sample outlet, and flushing the micro-flow channel with PBS to remove the single-stranded DNA which is not adsorbed on the substrate;
detecting the fluorescent signal of the DNA probe in the flow channel;
opening a valve between the micro-channel and the microcavity, closing the valve at the sample outlet and the DNA sample inlet, injecting a sample to be detected from the sample inlet, and pushing the sample to be detected into the two channels by using nitrogen gas;
the sample to be detected reacts with the DNA probe in the micro-channel and then flows into the micro-cavity through the micro-channel;
carrying out fluorescence detection on the liquid in the flow channel and entering the micro cavity;
and judging whether the sample to be detected contains the target miRNA or not according to different fluorescence colors and fluorescence intensities.
8. The dual-color fluorescence dual-detection method according to claim 7, wherein 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 determined according to the fluorescence intensity in the microcavity.
9. The dual-color fluorescence dual-detection method according to claim 7 or 8, wherein NH is modified at the 3' end of the DNA probe2C6A group.
10. The dual-color fluorescence dual-detection method according to claim 7 or 8, wherein the NDA probe is dyed with a fluorescent dye, or a fluorescent group is modified at the 5' end of the DNA probe.
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