CN113061524A - Double-layer micro-fluidic chip, breast cancer miRNA detection kit containing same, preparation method and detection method - Google Patents
Double-layer micro-fluidic chip, breast cancer miRNA detection kit containing same, preparation method and detection method Download PDFInfo
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Abstract
The invention belongs to the technical field of nucleic acid detection, and relates to a double-layer micro-fluidic chip for nucleic acid detection, a detection kit containing the chip, a preparation method of the detection kit and a detection method of the detection kit. A double-layer micro-fluidic chip comprises a valve control layer on the upper layer, a flow channel layer in the middle and a functional substrate, wherein the flow channel layer is provided with a micro-cavity, a micro-channel connected with the micro-cavity, a sample inlet and a sample outlet; the valve control layer comprises a control valve and a valve sample inlet connected with the control valve; the functional base is divided into a detection area and a reaction area, the detection area corresponds to the micro-cavity, and the reaction area corresponds to the micro-channel. The invention also provides a breast cancer miRNA detection kit. The double-layer micro-fluidic chip has a simple structure, is easy to prepare, and the chip and the kit are used for detecting the miRNA of the breast cancer, so that the time consumption is short, the steps are simple, the sensitivity is high, the required amount of a detected sample is small, and the quick and efficient detection of the miRNA of the breast cancer can be realized.
Description
Technical Field
The invention belongs to the technical field of nucleic acid detection, and relates to a double-layer micro-fluidic chip for nucleic acid detection, a detection kit containing the chip, a preparation method of the detection kit and a detection method of the detection kit.
Background
Breast cancer, one of the most common malignancies in women, severely affects women's life health safety, with rising incidence and a trend toward youthfulness. Recent investigation in 2011 shows that breast cancer is the first to affect the mortality rate of female malignant tumors all over the world. As the early-stage manifestation symptoms of the breast cancer are not obvious, most patients have already developed to middle and late stages when seeing a doctor, and the early diagnosis of the breast cancer has important value in the aspects of relieving the pain of the patients in the follow-up treatment, prolonging the life time of the patients and the like. Therefore, the method has important significance for improving the early detection level of the breast cancer and exploring a good early diagnosis index. Conventionally, Qrt-PCR technology, Western blotting, etc. have been mainly used for early diagnosis of breast cancer, and these methods have disadvantages such as low detection sensitivity, long time consumption, and high sample consumption, and thus are rarely used for early diagnosis of clinical cancer.
MiRNA is single-stranded RNA of 19-23 bp non-coding protein, participates in expression regulation of genes after transcription in animals and plants, and plays an important regulating role in the animals and plants. Many studies indicate that mirnas can participate in different biological processes in organisms, such as development, angiogenesis, differentiation, immune cell function, proliferation, apoptosis, etc., and the expression level of mirnas is different in different individuals, even in the same body, the expression level is different in health and disease. Because of the difference in expression levels, mirnas can be used as markers to detect abnormalities in organisms. Mostafa Azimzadeh et al detected breast cancer miR-155 gene by using an electrochemical biosensor, and the gene can be used as a marker gene of breast cancer; kseniia Boriachek et al detected miR-21 gene by electrochemical method, and showed the difference of expression between normal and patient. The miRNA has the specific advantages of being stable in serum/plasma, free of wound during sampling and the like, and has potential application value in early detection of cancers.
The discovery of miRNA provides a new method for early detection of cancer, the existing detection method based on miRNA is generally based on the principles of nucleotide hybridization and amplification, and mainly comprises PCR, Northern blotting, microarray chip and the like, and the methods convert miRNA hybridization signals into measurable signals so as to achieve the purpose of quantification or qualification. However, the methods have many problems in the using process, such as long time consumption of Northern blotting technology, complex operation, large sample demand and the like; the PCR technology needs to amplify a target object and then carries out result judgment through gel electrophoresis, and the detection process consumes longer time and has complicated steps. Therefore, in order to fully utilize the advantages of miRNA in cancer detection, it is necessary to develop a rapid, efficient and highly accurate miRNA-based early cancer detection method to provide technical support for early detection of breast cancer.
Disclosure of Invention
The invention aims to solve the problems in the prior art and provide a double-layer micro-fluidic chip, a kit and a preparation method thereof, wherein the double-layer micro-fluidic chip has the advantages of short time consumption, high sensitivity and less sample detection requirement and is used for early diagnosis of breast cancer.
The invention solves the technical problem and adopts a technical scheme that: a double-layer micro-fluidic chip comprises a valve control layer on the upper layer, a flow channel layer in the middle and a functional substrate, wherein the flow channel layer is provided with a micro-cavity, a micro-channel connected with the micro-cavity, a sample inlet and a sample outlet; the valve control layer comprises a control valve and a valve sample inlet connected with the control valve; the functionalized substrate is divided into a detection area and a reaction area, the detection area corresponds to the microcavity, and the reaction area corresponds to the micro-channel.
As a preferred mode of the present invention, the reaction region is modified with nano graphene oxide or nano graphene material, and the detection region is modified with polylysine.
Further preferably, the reaction region is immobilized with a detection probe.
In a preferred embodiment of the present invention, the micro-cavity control valve is provided at an inlet of the micro-cavity, and the micro-channel control valve is provided between the micro-channel and the sample outlet.
The invention solves the technical problem and also provides a breast cancer miRNA detection kit, which comprises the double-layer microfluidic chip, a breast cancer miRNA standard product and a fluorescence-labeled miRNA probe.
As a preferable mode of the invention, the breast cancer miRNA standard is a product synthesized by taking breast cancer markers miR-125, miR-126, miR-155, miR-191 and miR-21 as templates.
Further preferably, the kit further comprises a dilution reagent, wherein the dilution reagent comprises 1M TE Buffer and an RNA protection solution and is used for diluting the breast cancer miRNA standard or probe.
The invention further provides a preparation method of the breast cancer miRNA detection kit, which comprises the following steps:
preparing a double-layer micro-fluidic chip;
synthesizing a breast cancer miRNA standard product;
and (3) synthesizing a fluorescent probe.
Further preferably, the preparation steps of the double-layer microfluidic chip are as follows:
(1) preparing a valve control layer and a flow channel layer by adopting two reagents RTV 615A and RTV 615B respectively;
(2) preparing a functionalized substrate;
(3) laminating the valve control layer and the flow channel layer, and then bonding with the functionalized substrate;
(4) and laying fluorescent probes in the reaction area of the functionalized substrate.
The invention also provides a method for detecting the breast cancer miRNA by adopting the kit, which comprises the following steps:
(1) diluting the breast cancer miRNA standard substance into gradient concentration, respectively injecting miRNA standard substance solutions with various concentrations into a sample inlet of a double-layer micro-fluidic chip with a laid fluorescent probe, and blowing nitrogen into a micro-channel from the sample inlet;
(2) incubating in the micro flow channel for 20-30 min; blowing liquid in the micro-channel into the microcavity from the sample inlet by using nitrogen, flushing the micro-channel by using a flushing reagent, and flushing the micro-channel into the microcavity;
(3) incubating the liquid in the micro-cavity for 10-15 min;
(4) then immersing the whole chip in 3% BSA, removing the double-layer PDMS on the substrate, and immersing the substrate for 10-15 min for sealing;
(5) soaking the sealed substrate in 100% PBS, 50% PBS and ultrapure water in sequence, and drying for fluorescence detection;
(6) carrying out linear fitting on the logarithms of different concentrations and fluorescence values to prepare a standard curve;
(7) and carrying out fluorescence detection on the miRNA in the sample to be detected, and substituting the measured fluorescence value according to the standard curve to obtain the content of the miRNA in the sample to be detected.
The invention has the beneficial effects that: the double-layer micro-fluidic chip has the advantages of simple structure, easiness in preparation and convenience in use, and the chip and the kit are adopted for detecting the miRNA of the breast cancer, so that the time consumption is short, the steps are simple, the sensitivity is high, the demand of a detection sample is low, the rapid and efficient detection can be realized, and a technical platform is provided for early discovery and early diagnosis of the breast cancer.
Drawings
FIG. 1 is a schematic structural diagram of a double-layered microfluidic chip according to an embodiment of the present invention;
FIG. 2 is a top view of a dual-layer microfluidic chip according to an embodiment of the present invention;
FIG. 3 is a view of the valve control layer;
FIG. 4 is a schematic view of a flow channel layer structure;
FIG. 5 is a schematic view of a functionalized substrate structure;
FIG. 6 is a fluorescence value diagram of the breast cancer serum miRNA detection kit for detecting miR-125 with different concentrations in the embodiment of the invention;
FIG. 7 is a standard curve graph plotting miRNA concentration in breast cancer serum versus fluorescence value in an example of the present invention;
FIG. 8 is a comparison graph of the actual concentration detection result and the theoretical concentration of the breast cancer serum miRNA detection kit and the detection method according to the embodiments of the present 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: a double-layer micro-fluidic chip is shown in a structural diagram 1 and a structural diagram 2, and comprises a valve control layer 1, a flow channel layer 2 and a functionalized substrate 3 from top to bottom in sequence.
As shown in fig. 2 and 3, a valve injection port 4, a microcavity control valve 5, and a microchannel control valve 6 are provided on the valve control layer 1, and the valve injection port 4 is connected to the microcavity control valve 5 and the microchannel control valve 6 via a microchannel, respectively.
As shown in fig. 2 and 4, the micro-cavity 8, the sample micro-channel 10, and the sample micro-channel 9 are provided on the channel layer 2. One end of the sample micro-channel is connected with the micro-cavity 8, and the other end is connected with the sample inlet 11. One end of the sampling micro-channel 9 is connected with the end of the sampling micro-channel close to the microcavity, and the other end is connected with the sampling outlet 7.
As shown in fig. 2, a microcavity control valve 5 is located at the outlet channel of the microcavity 8 for controlling the liquid ingress and egress from the microcavity. The micro-channel control valve is positioned above the sample outlet micro-channel 9 and is used for controlling the on-off of the sample outlet micro-channel.
As shown in fig. 5, a functionalized substrate 3 is disposed below the flow channel layer 2, and the functionalized substrate 3 is divided into a reaction region and a detection region, wherein the reaction region corresponds to a region of a sample injection micro flow channel 10 on the flow channel layer 2, nano graphene oxide is modified in the reaction region, a fluorescent probe for detecting a breast cancer miRNA marker is fixed in the reaction region through the adsorption of the nano graphene oxide, and as shown in the figure, FAM fluorophore molecules 13 are labeled on a breast cancer marker probe molecule 12. The detection area corresponds to the micro-cavity 8 area on the flow channel layer 2, is modified with polylysine, and is used for combining a double chain formed by combining a fluorescent probe for fixing a breast cancer miRNA marker and miRNA, and is used for subsequent fluorescence detection.
The second embodiment of the invention provides a breast cancer miRNA detection kit, which comprises the double-layer microfluidic chip, a detection reagent, a detection probe, a dilution reagent and a flushing reagent provided in the embodiment. The detection reagent is used for calibrating the microfluidic chip, and the dilution reagent is used for diluting a breast cancer miRNA standard product and comprises 1M TE Buffer (10 mM Tris-HCl, 1 mM EDTA, pH = 8.0) and an RNA protection solution. Washing reagents were used to wash the flow channels, including 1M TE Buffer (10 mM Tris-HCl, 1 mM EDTA, pH = 8.0).
In the kit of this embodiment, the detection reagent includes 5 breast cancer miRNA standards, a blocking solution (3% BSA), and a cleaning solution (100% PBS, 50% PBS, and ultrapure water).
Breast cancer miRNA standard: the breast cancer miRNA standard in the kit is a synthesized breast cancer marker which is designed and synthesized by Takara company according to nucleotide sequences miR-125, miR-126, miR-155, miR-191 and miR-21 reported by a microRNA database as templates, and the sequence of the marker is shown in Table 1.
TABLE 1 miRNA marker sequences
miRNA markers are in a powdered state after synthesis and need to be diluted before use: (1) before dilution, the vial lid was opened and the vial was placed in a 4 ℃ low temperature centrifuge (3000 rpm) for centrifugation for a period of time; (2) adding 1M TE buffer with v (mu.l) = nmol number x10 in a dark environment; (3) centrifuging at 4 deg.C and low temperature (3000 rpm) for a period of time, taking out, and storing at-20 deg.C. The MiRNA marker also requires the addition of a certain amount of RNA protecting solution after dilution.
Preparation of a sealing liquid: and (3) diluting BSA according to a certain proportion by using PBS.
In the kit of the embodiment, the detection probes comprise the probes of the 5 breast cancer markers miR-125, miR-126, miR-155, miR-191 and miR-21. The 3' -end of the probe is modified with a fluorescent group such as FAM, Cy3, etc. The sequences and Tm values of the probes are shown in Table 2.
TABLE 2 sequences of Breast cancer marker probes, Tm values
The invention also provides a preparation method of the kit, which comprises the following specific steps:
(1) preparation of valve control layer of double-layer micro-fluidic chip
Adopting two reagents of RTV 615A and RTV 615B, mixing the two reagents according to a certain proportion, pouring the mixture into a mould with a valve control layer pattern, baking the mixture for 1 to 2 hours at the temperature of 80 ℃, cooling the mixture to room temperature, and taking the mixture off the mould.
(2) Preparation of middle flow channel layer of double-layer micro-fluidic chip
Mixing two reagents RTV 615A and RTV 615B according to a certain proportion, pouring the mixture on a mold with a middle runner layer pattern, uniformly covering the mixed reagent on the mold by using a glue homogenizer (3000 rpm, 1-2 min), baking for 30-40 min at 70 ℃, taking out, standing for 10-20min at room temperature, and storing.
(3) Preparation of double-layer micro-fluidic chip functional substrate
Carrying out scribing treatment on the clean substrate to distinguish a detection functional area and a reaction functional area; pretreating the substrate in a piranha solution, and cleaning the substrate with deionized water; plasma treating the surface of the substrate (10-20 min); growing a coupling agent APTMS on the surface of the substrate and then cleaning the substrate with deionized water; growing graphene oxide in a substrate reaction functional area, taking out after growth, and cleaning with deionized water; and growing and modifying polylysine in the detection functional region, taking out after growth, cleaning with deionized water, and finally drying by blowing, thereby completing the preparation of the double-layer microfluidic chip functionalized substrate.
(4) The valve control layer and the middle flow channel layer of the double-layer micro-fluidic chip are combined: and (3) bonding the prepared valve control layer with the flow channel layer under a microscope, baking for 1-2h at the temperature of 70 ℃, and cooling to room temperature.
(5) And bonding the combined body of the two layers with the functionalized substrate.
(6) Diluting the fluorescent probe reagent with a diluting reagent
The probe reagents were centrifuged for a period of time (4 ℃, 3000 rpm) without uncapping; the cap was opened in a sterile environment protected from light, and a certain amount of the diluent reagent (liquid amount: ul = nmol number x 100) was added to the probe reagent bottle, and centrifugation (4 ℃, 3000 rpm) was performed.
(7) Laying marker probes in the reaction functional area of the double-layer microfluidic chip
Taking 1-2ul of diluted probe reagent, and adding the diluted probe reagent into a sample inlet 11 of the double-layer micro-fluidic chip; closing the microcavity control valve 5, opening the micro-channel control valve 6, and blowing the probe reagent in the sample inlet 11 into the sample outlet 7 through the sample inlet micro-channel 10 and the sample outlet micro-channel 9 by adopting nitrogen; closing the micro-channel control valve 6, keeping the state of the micro-cavity control valve 5 unchanged, and incubating for 30-40 min; opening the micro-channel control valve 6, keeping the state of the micro-cavity control valve 5 unchanged, and sucking out redundant liquid in the sampling micro-channel 10 from the sample outlet 7; and at this moment, the laying of the marker probe is finished, and the preparation of the detection chip is finished.
The invention also provides a method for detecting the miRNA of the breast cancer by adopting the kit in the embodiment, taking the detection of miR-125 in the serum of the breast cancer as an example, the method comprises the following specific steps:
1. extraction of serum from blood: after a certain amount of blood is extracted by an anticoagulation tube, the blood is placed in a sterile environment at room temperature for 5 hours or so, and the uppermost layer is taken as yellowish liquid. Centrifuging the liquid at a low temperature of 4 ℃ for a period of time; taking the upper layer liquid, and placing into an EP tube for storage at-80 ℃ for later use.
2. Making a standard curve
(1) Adopting a diluting reagent to make the miR-125 marker standard substance in a powder stateDiluting to liquid state; diluting the miR-125 marker in a liquid state into gradient concentrations by adopting 1M TE Buffer, wherein the gradient concentrations are as follows: 10-8、10-9、10-10、10-11、10-12、10-13 M；
(2) The micro-cavity control valve 5 is closed, the micro-channel control valve 6 is opened, 2 mul of miRNA standard solution with each concentration is respectively taken and injected into the laid miR-125 fluorescent probe (with the concentration of 10)-4 M) injecting the solution into a sample micro-channel 10 from the injection port 11 by adopting nitrogen gas through the injection port 11; closing the micro-channel control valve 6, keeping the state of the micro-cavity control valve 5 unchanged, and incubating the standard solution in the sampling micro-channel 10 for 20-30 min;
(3) opening the microcavity control valve 5, closing the micro-channel control valve 6, blowing liquid in the sampling micro-channel 10 into the microcavity 8 from the sampling port 11 by adopting nitrogen, flushing the sampling micro-channel 10 by adopting a flushing reagent, and flushing the sampling micro-channel 10 into the microcavity 8;
(4) closing the micro-cavity control valve 5, keeping the state of the micro-channel control valve 6 unchanged, and incubating the liquid in the micro-cavity 8 for 10-15 min; (5) immersing the whole double-layer micro-fluidic chip in a sealing liquid (3% BSA), removing PDMS on the substrate, and soaking for 10-15 min;
(6) soaking the sealed chips in 100% PBS in sequence; soaking in 50% PBS; soaking with ultrapure water for many times; drying and carrying out fluorescence detection;
(7) detecting the fluorescence signal and analyzing the fluorescence value; the fluorescence values were log-fitted to the concentrations linearly to prepare a standard curve, and the fitting formula was lg [ f (x) ] =5.60+0.19lgx, using miR-125 as an example, as shown in fig. 6 and 7.
3. Detection of miRNA in serum
Injecting 2 mul serum into the sample inlet 11 of the chip; blowing the serum into a sample micro-channel 10 from a sample inlet 11 by adopting nitrogen; closing the micro-channel control valve 6, keeping the state of the micro-cavity control valve 5 unchanged, and incubating the serum in the sampling micro-channel 10 for 20-30 min; opening the micro-cavity control valve 5, closing the micro-channel control valve 6, blowing liquid in the sampling micro-channel 10 into the micro-cavity 8 from the sampling port 11 by adopting nitrogen, flushing the sampling micro-channel 10 by adopting a flushing reagent, and flushing the flushing reagent into the micro-cavity 8; closing the micro-cavity control valve 5, keeping the state of the micro-channel control valve 6 unchanged, and incubating the liquid in the micro-cavity 8 for 10-15 min; soaking the whole chip in 3% BSA, removing PDMS on the substrate, and soaking the glass substrate for 10-15 min; then soaking the raw materials in 100% PBS in sequence; soaking in 50% PBS; soaking with ultrapure water for many times; drying and carrying out fluorescence detection.
(4) And (3) calculating: and substituting the measured fluorescence value according to a relation between the fluorescence value and the concentration obtained by the standard curve to obtain the miRNA content in the serum to be detected.
The accuracy of the detection result of the kit is verified and tested: by adopting the kit and the method of the invention to carry out actual detection on the standard solutions with different concentrations and comparing the detection result with the actual concentration of the standard solution, as shown in figure 8, the error between the two is small, which indicates that the kit and the detection method provided by the invention have high accuracy.
Claims (10)
1. A double-layer micro-fluidic chip is characterized in that: the micro-channel micro-fluidic chip comprises a valve control layer on the upper layer, a flow channel layer in the middle and a functional substrate, wherein the flow channel layer is provided with a micro-cavity, a micro-channel connected with the micro-cavity, a sample inlet and a sample outlet; the valve control layer comprises a control valve and a valve sample inlet connected with the control valve; the functionalized substrate is divided into a detection area and a reaction area, the detection area corresponds to the microcavity, and the reaction area corresponds to the micro-channel.
2. The dual-layer microfluidic chip of claim 1, wherein: the reaction zone is modified with nano graphene oxide or nano graphene materials, and the detection zone is modified with polylysine.
3. The dual-layer microfluidic chip of claim 2, wherein: the reaction area is fixed with a detection probe.
4. The dual-layer microfluidic chip of claim 3, wherein: the control valve comprises a micro-cavity control valve and a micro-channel control valve, the micro-cavity control valve is arranged at the inlet of the micro-cavity, and the micro-channel control valve is arranged between the micro-channel and the sample outlet.
5. A breast cancer miRNA detection kit is characterized in that: the kit comprises the double-layer micro-fluidic chip described in any one of 1-4, a breast cancer miRNA standard and a fluorescence-labeled miRNA probe.
6. The breast cancer miRNA detection kit of claim 5, wherein: the breast cancer miRNA standard products are products synthesized by taking breast cancer markers miR-125, miR-126, miR-155, miR-191 and miR-21 as templates respectively.
7. The breast cancer miRNA detection kit of claim 6, wherein: the kit also comprises a diluting reagent, wherein the diluting reagent comprises 1M TE Buffer and RNA protective solution and is used for diluting a breast cancer miRNA standard substance or probe.
8. A preparation method of a breast cancer miRNA detection kit is characterized by comprising the following steps:
preparing a double-layer micro-fluidic chip;
synthesizing a breast cancer miRNA standard product;
and (3) synthesizing a fluorescent probe.
9. The preparation method of the breast cancer miRNA detection kit according to claim 8, wherein the double-layer microfluidic chip is prepared by the following steps:
(1) preparing a valve control layer and a flow channel layer by adopting two reagents RTV 615A and RTV 615B respectively;
(2) preparing a functionalized substrate;
(3) laminating the valve control layer and the flow channel layer, and then bonding with the functionalized substrate;
(4) and laying fluorescent probes in the reaction area of the functionalized substrate.
10. A detection method for breast cancer miRNA detection is characterized by comprising the following steps:
(1) diluting the breast cancer miRNA standard substance into gradient concentration, respectively injecting miRNA standard substance solutions with various concentrations into a sample inlet of a double-layer micro-fluidic chip with a laid fluorescent probe, and blowing nitrogen into a micro-channel from the sample inlet;
(2) incubating in the micro flow channel for 20-30 min; blowing liquid in the micro-channel into the microcavity from the sample inlet by using nitrogen, flushing the micro-channel by using a flushing reagent, and flushing the micro-channel into the microcavity;
(3) incubating the liquid in the micro-cavity for 10-15 min;
(4) then immersing the whole chip in 3% BSA, removing the double-layer PDMS on the substrate, and immersing the substrate for 10-15 min for sealing;
(5) soaking the sealed substrate in 100% PBS, 50% PBS and ultrapure water in sequence, and drying for fluorescence detection;
(6) carrying out linear fitting on the logarithms of different concentrations and fluorescence values to prepare a standard curve;
(7) and carrying out fluorescence detection on the miRNA in the sample to be detected, and substituting the measured fluorescence value according to the standard curve to obtain the content of the miRNA in the sample to be detected.
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