CN113061524B - Double-layer microfluidic chip and breast cancer miRNA detection kit - Google Patents
Double-layer microfluidic chip and breast cancer miRNA detection kit Download PDFInfo
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Abstract
The invention belongs to the technical field of nucleic acid detection, and relates to a double-layer microfluidic 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. The double-layer microfluidic chip comprises an upper valve control layer, a middle runner layer and a functional substrate, wherein the runner layer is provided with a microcavity, a microchannel connected with the microcavity, 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 microcavity, and the reaction area corresponds to the micro-channel. The invention also provides a kit for detecting the miRNA of the breast cancer. The double-layer microfluidic chip provided by the invention has the advantages of simple structure and easiness in preparation, 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 requirement on detection samples is small, and the rapid 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 microfluidic 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 malignant tumors in women, severely affects the life health safety of women, and its incidence appears to rise and has a tendency to younger. Recent surveys in 2011 have found that breast cancer is the leading one in affecting global female malignant tumor mortality. Since the early symptoms of breast cancer are not obvious, most patients already develop to middle and late stages when taking a doctor, and the early diagnosis of breast cancer has important value in relieving the pain of the subsequent treatment of the patients, prolonging the life time of the patients and the like. Therefore, the method improves the early detection level of the breast cancer and has important significance in exploring good early diagnosis indexes. Conventionally, qrt-PCR technology, western blotting, and the like are mainly used for early diagnosis of breast cancer, and these methods have the disadvantages of low detection sensitivity, long time consumption, high sample consumption, and the like, and therefore are rarely applied to early diagnosis of clinical cancer.
MiRNA is single-stranded RNA with the length of 19-23 bp of non-coding protein, and participates in the expression regulation of genes transcribed in animals and plants, thereby playing an important role in the regulation of the animals and plants. Many studies have shown that mirnas can be involved in different biological processes in organisms, such as development, angiogenesis, differentiation, immune cell function, proliferation, apoptosis, etc., while their expression levels are different in different individuals, even in the same body, and the amounts of expression in healthy and diseased conditions are different. Just as the expression levels differ, mirnas can be used as markers to detect abnormalities in organisms. Mostafa Azimzadeh et al detected the breast cancer miR-155 gene by means of an electrochemical biosensor, which could be used as a marker gene for breast cancer; kseniia Boriachek et al detected the miR-21 gene electrochemically, and showed a difference in expression levels between normal and patient. miRNA can stably exist in serum/plasma with the special advantages, has no wound during sampling, and has potential application value in early detection of cancers.
The discovery of miRNA provides a new method for early detection of cancer, and the current detection method based on miRNA is generally based on the principles of nucleotide hybridization and amplification, mainly comprises PCR, northern blotting, a microarray chip and the like, and converts miRNA hybridization signals into measurable signals so as to achieve the purpose of quantification or characterization. However, the methods have a plurality of problems in the use process, such as long time consumption, complex operation, large sample demand and the like of Northern blotting technology; the PCR technology needs to amplify the target object, then judges the result 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 accurate early detection method based on miRNA, which provides 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 is short in time consumption, high in sensitivity and small in detection sample demand, and is used for early diagnosis of breast cancer.
The invention solves the technical problems by adopting a technical scheme that: the double-layer microfluidic chip comprises an upper valve control layer, a middle runner layer and a functional substrate, wherein a microcavity, a microchannel connected with the microcavity, a sample inlet and a sample outlet are arranged on the runner layer; the valve control layer comprises a control valve and a valve sample inlet connected with the control valve; the functional 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 preferable mode of the invention, the reaction area is modified with nano graphene oxide or graphene material, and the detection area is modified with polylysine.
Further preferably, the reaction zone is immobilized with a detection probe.
As a preferable mode of the invention, the microcavity control valve is arranged at the inlet of the microcavity, and the microchannel control valve is arranged between the microchannel 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 substance and a fluorescence-marked miRNA probe.
As a preferable mode of the invention, the breast cancer miRNA standard products are respectively products 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 diluting reagent, wherein the diluting reagent comprises 1M TE Buffer and RNA protection liquid 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;
synthesis of fluorescent probes.
Further preferably, the preparation steps of the double-layer microfluidic chip are as follows:
(1) Preparing a valve control layer and a flow passage layer respectively by adopting two reagents, namely RTV 615A and RTV 615B;
(2) Preparing a functionalized substrate;
(3) Combining the valve control layer and the runner layer, and then bonding with the functionalized substrate;
(4) And paving 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 a breast cancer miRNA standard substance into gradient concentrations, respectively injecting miRNA standard substance solutions with various concentrations into a sample inlet of a double-layer microfluidic chip on which a fluorescent probe is paved, and blowing the miRNA standard substance solutions into a micro-channel from the sample inlet by adopting nitrogen;
(2) Incubating for 20-30 min in the micro-channel; blowing liquid in the micro-channel into the micro-cavity from the sample inlet by adopting nitrogen, flushing the micro-channel by adopting a flushing reagent, and flushing the micro-channel into the micro-cavity;
(3) Incubating the liquid in the microcavity for 10-15 min;
(4) Then the whole chip is immersed in 3% BSA, and the double-layer PDMS on the substrate is removed, and the substrate is immersed for 10-15 min for sealing;
(5) Soaking the sealed substrate in 100% PBS,50% PBS and ultrapure water in sequence, and blow-drying for fluorescence detection;
(6) Linearly fitting the logarithm of the fluorescence value and the different concentrations to prepare a standard curve;
(7) And carrying out fluorescence detection on the miRNA in the sample to be detected, and carrying in the measured fluorescence value according to a standard curve to obtain the content of the miRNA in the sample to be detected.
The beneficial effects of the invention are as follows: the double-layer microfluidic chip is simple in structure, easy to prepare and convenient to use, and the chip and the kit are used for detecting the miRNA of the breast cancer, are short in time consumption, simple in steps, high in sensitivity and small in detection sample demand, can realize rapid and efficient detection, and provide a technical platform for early discovery and early diagnosis of the breast cancer.
Drawings
Fig. 1 is a schematic structural diagram of a dual-layer 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 diagram of a valve control layer structure;
FIG. 4 is a schematic view of a flow channel layer structure;
FIG. 5 is a schematic diagram of a functionalized substrate structure;
FIG. 6 is a graph of fluorescence values of a breast cancer serum miRNA detection kit for detecting miR-125 with different concentrations in an embodiment of the invention;
FIG. 7 is a standard graph plotting breast cancer serum miRNA concentration versus fluorescence values in an example of the present invention;
fig. 8 is a graph showing the comparison between the actual concentration detection result and the theoretical concentration of the breast cancer serum miRNA detection kit and the detection method according to the embodiment of the present 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: a double-layer micro-fluidic chip is shown in a structural diagram 1 and a structural diagram 2 of the chip, and comprises a valve control layer 1, a runner layer 2 and a functional substrate 3 from top to bottom.
As shown in fig. 2 and 3, a valve sample inlet 4, a microcavity control valve 5 and a microchannel control valve 6 are arranged on the valve control layer 1, and the valve sample inlet 4 is connected with the microcavity control valve 5 and the microchannel control valve 6 through microchannels respectively.
As shown in fig. 2 and 4, a microcavity 8, a sample injection microchannel 10 and a sample discharge microchannel 9 are provided on the flow channel layer 2. One end of the sample injection micro-channel is connected with the micro-cavity 8, and the other end is connected with the sample injection port 11. One end of the sample outlet micro-channel 9 is connected with the position of the sample inlet micro-channel close to the micro-cavity end, and the other end is connected with the sample outlet 7.
As shown in fig. 2, a microcavity control valve 5 is located on the outlet channel of microcavity 8 for controlling the ingress and egress of fluid 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, the functionalized substrate 3 is arranged below the runner layer 2, and is divided into a reaction area and a detection area on the functionalized substrate 3, wherein the reaction area corresponds to a sample injection micro runner 10 area on the runner layer 2, nano graphene oxide is modified in the reaction area, and a fluorescent probe for detecting a breast cancer miRNA marker is fixed in the reaction area through the adsorption of the nano graphene oxide, as shown in the figure, FAM fluorescent group molecules 13 are marked on breast cancer marker probe molecules 12. The detection area corresponds to the microcavity 8 area on the runner layer 2, is modified with polylysine, and is used for combining and fixing a double strand formed by combining a fluorescent probe of a breast cancer miRNA marker and miRNA for subsequent fluorescence detection.
The second and embodiment of the invention provides a breast cancer miRNA detection kit, which comprises the double-layer microfluidic chip, the detection reagent, the detection probe, the dilution reagent and the flushing reagent provided in the embodiment. The detection reagent is used for calibrating the microfluidic chip, and the dilution reagent is used for diluting the breast cancer miRNA standard substance and comprises 1M TE Buffer (10 mM Tris-HCl,1 mM EDTA,pH =8.0) and RNA protection liquid. The wash reagent was 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 comprises 5 breast cancer miRNA standards, blocking solution (3% BSA), washing solution (100% PBS,50% PBS, ultrapure water).
Breast cancer miRNA standard: the breast cancer miRNA standard in the kit is a synthesized breast cancer marker, the marker 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 sequences are shown in table 1.
TABLE 1 miRNA marker sequences
miRNA markers are in powder state after synthesis, and need to be diluted before use: (1) The small tube cover is not opened before dilution, and the small tube is placed in a 4 ℃ low-temperature centrifuge (3000 rpm) for centrifugation for a period of time; (2) In a dark environment, add 1M TE buffer with v (μl) =nmol number x 10; (3) Centrifuging at 4deg.C with a low temperature centrifuge (3000 rpm) for a period of time, taking out, and preserving at-20deg.C. The MiRNA markers also require the addition of a certain amount of RNA protection fluid after dilution.
Preparation of a sealing liquid: diluting BSA by PBS according to a certain proportion.
In the kit of the embodiment, the detection probes comprise probes of the 5 breast cancer markers of miR-125, miR-126, miR-155, miR-191 and miR-21. A fluorescent group such as FAM, cy3 and the like is modified at the 3' -end of the probe. 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
Mixing two reagents RTV 615A and RTV 615B according to a certain proportion, pouring the mixture into a mould with a valve control layer pattern, baking for 1-2h at 80 ℃, cooling to room temperature, and taking off the mould.
(2) Preparation of middle runner layer of double-layer micro-fluidic chip
Mixing two reagents RTV 615A and RTV 615B according to a certain proportion, pouring the two reagents on a mold with a middle runner layer pattern, uniformly covering the mixed reagents on the mold by a spin coater (3000 rpm,1-2 min), baking at 70 ℃ for 30-40 min, taking out, standing at room temperature for 10-20min, and preserving.
(3) Preparation of double-layer microfluidic chip functionalized substrate
Scribing the clean substrate to distinguish a detection functional area and a reaction functional area; the substrate is pretreated in a piranha solution and then is cleaned by deionized water; plasma treatment of the substrate surface (10-20 min); after a coupling agent APTMS grows on the surface of the substrate, the substrate is washed by deionized water; growing graphene oxide in a substrate reaction functionalization area, taking out after growth, and cleaning with deionized water; and (3) growing modified polylysine in the detection functional area, taking out the modified polylysine after growing, cleaning the modified polylysine by deionized water, and finally drying the modified polylysine by blowing, so that the preparation of the double-layer microfluidic chip functionalized substrate is finished.
(4) Combining a valve control layer of the double-layer microfluidic chip with an intermediate flow channel layer: and (3) attaching the prepared valve control layer to the runner layer under a microscope, baking for 1-2h at 70 ℃, and cooling to room temperature.
(5) Bonding the combination of the two layers with a functionalized substrate.
(6) Diluting the fluorescent probe reagent with a diluting reagent
Centrifuging the probe reagent for a period of time (4 ℃ C., 3000 rpm) without uncapping; the vial cap was opened under aseptic conditions in the absence of light, a certain amount of diluted reagent (liquid amount: ul=nmol number x 100) was added to the probe reagent vial, and centrifugation was performed (4 ℃,3000 rpm).
(7) Laying a marker probe in a reaction functional area of a double-layer microfluidic chip
1-2ul of diluted probe reagent is added into a sample inlet 11 of the double-layer microfluidic chip; the microcavity control valve 5 is closed, the micro-channel control valve 6 is opened, and the probe reagent in the sample inlet 11 is blown into the sample outlet 7 through the sample injection 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 a micro-channel control valve 6, keeping the state of the micro-cavity control valve 5 unchanged, and sucking out redundant liquid in the sample injection micro-channel 10 from a sample outlet 7; so far, the marker probe is laid, and the detection chip is prepared.
The invention also provides a method for detecting the miRNA of the breast cancer by adopting the kit in the embodiment, taking miR-125 in the serum of the breast cancer as an example, and specifically comprising the following steps:
1. extracting serum from blood: after a certain amount of blood is extracted by an anticoagulant tube, the blood is placed in a sterile environment at room temperature, and is kept stand for about 5 hours, and the light yellow liquid at the uppermost layer is taken. Centrifuging the liquid at low temperature for a period of time at 4deg.C; the upper liquid is taken and put into an EP tube to be preserved at-80 ℃ for standby.
2. Making a standard curve
(1) Diluting a miR-125 marker standard product in a powder state into a liquid state by adopting a diluting reagent; 1M TE Buffer is adopted to dilute the miR-125 marker in a liquid state into gradient concentrations, which are respectively as follows: 10-8, 10-9, 10-10, 10-11, 10-12, 10-13 and M;
(2) The microcavity control valve 5 is closed, the micro flow channel control valve 6 is opened, 2 mu l of miRNA standard substance solution with each concentration is respectively taken and injected into the sample inlet 11 of the double-layer micro flow control chip on which the miR-125 fluorescent probe (with the concentration of 10-4M) is paved, and nitrogen is adopted to blow the solution into the sample injection micro flow channel 10 from the sample inlet 11; closing the micro-channel control valve 6, and incubating the standard solution in the sample injection micro-channel 10 for 20-30 min with the state of the micro-cavity control valve 5 unchanged;
(3) The micro-cavity control valve 5 is opened, the micro-channel control valve 6 is closed, the liquid in the sample injection micro-channel 10 is blown into the micro-cavity 8 from the sample inlet 11 by adopting nitrogen, and the sample injection micro-channel 10 is flushed by adopting a flushing reagent, so that the sample injection micro-channel 10 is flushed into the micro-cavity 8;
(4) Closing the microcavity control valve 5, keeping the state of the microchannel control valve 6 unchanged, and incubating the liquid in the microcavity 8 for 10-15 min; (5) Immersing the whole double-layer microfluidic chip in a sealing liquid (3% BSA), removing PDMS on the substrate, and immersing for 10-15 min;
(6) Sequentially soaking the closed chip with 100% PBS; soaking in 50% PBS; soaking in ultrapure water for multiple times; blow-drying, and performing fluorescence detection;
detecting the signal and analyzing the fluorescence value; taking log of fluorescence values, performing linear fitting with concentration to prepare a standard curve, taking miR-125 as an example, and adopting a fitting formula of lg [ f (x) ]=5.60+0.19 lgx, as shown in fig. 6 and 7.
3. Detection of miRNA in serum
2. Mu.l of serum is injected into a sample inlet 11 of the chip; blowing serum into the sample injection micro-channel 10 from the sample injection port 11 by adopting nitrogen; closing the micro-channel control valve 6, keeping the state of the micro-cavity control valve 5 unchanged, and incubating serum in the sample injection micro-channel 10 for 20-30 min; the micro-cavity control valve 5 is opened, the micro-channel control valve 6 is closed, nitrogen is adopted to blow the liquid in the sample injection micro-channel 10 into the micro-cavity 8 from the sample inlet 11, and a flushing reagent is adopted to flush the sample injection micro-channel 10 into the micro-cavity 8; closing the microcavity control valve 5, keeping the state of the microchannel control valve 6 unchanged, and incubating the liquid in the microcavity 8 for 10-15 min; immersing the whole chip in 3% BSA, removing PDMS on the substrate, and immersing the glass substrate for 10-15 min; then soaking with 100% PBS sequentially; soaking in 50% PBS; soaking in ultrapure water for multiple times; blow-drying, and performing fluorescence detection.
(4) And (3) calculating: and carrying the measured fluorescence value into the relation between the fluorescence value and the concentration obtained by the standard curve to obtain the miRNA content in the serum to be detected.
As shown in FIG. 8, the accuracy of the detection result of the kit is verified, and compared with the theoretical concentration, the actual detection concentration of the kit is smaller in error, so that the accuracy of the kit and the detection method provided by the invention is high.
Claims (6)
1. The double-layer micro-fluidic chip is characterized in that: the device comprises an upper valve control layer, a middle runner layer and a functional substrate, wherein the runner layer is provided with a microcavity, a microchannel connected with the microcavity, 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 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; the reaction area is modified with nano graphene oxide or graphene material, and the detection area is modified with polylysine; the reaction zone is fixed with a detection probe.
2. The dual-layer microfluidic chip of claim 1, wherein: the control valve comprises a microcavity control valve and a micro-channel control valve, the microcavity control valve is arranged at the inlet of the microcavity, and the micro-channel control valve is arranged between the micro-channel and the sample outlet.
3. A breast cancer miRNA detection kit is characterized in that: the kit comprises the double-layer microfluidic chip, the breast cancer miRNA standard substance and the fluorescence-labeled miRNA probe according to claim 1 or 2.
4. The breast cancer miRNA detection kit of claim 3, 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.
5. The breast cancer miRNA detection kit of claim 3, wherein: the kit also comprises a diluting reagent, wherein the diluting reagent comprises 1M TE Buffer and RNA protection liquid and is used for diluting the breast cancer miRNA standard or probe.
6. The preparation method of the breast cancer miRNA detection kit is characterized by comprising the following steps:
preparing the double-layer microfluidic chip according to claim 1;
synthesizing a breast cancer miRNA standard;
synthesizing a fluorescent probe;
the preparation steps of the double-layer micro-fluidic chip are as follows:
(1) Preparing a valve control layer and a flow passage layer respectively by adopting two reagents, namely RTV 615A and RTV 615B;
(2) Preparing a functionalized substrate;
(3) Combining the valve control layer and the runner layer, and then bonding with the functionalized substrate;
(4) And paving fluorescent probes in the reaction area of the functionalized substrate.
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