CN109628557A - Dual signal enhances application of the paper base biosensor in miRNA detection - Google Patents
Dual signal enhances application of the paper base biosensor in miRNA detection Download PDFInfo
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Abstract
The invention discloses a kind of at low cost, high sensitivity, fluorescence/colorimetric double mode sensor preparation method of Visual retrieval miRNA and it is used successfully to miRNA detection.Paper chip is prepared using wax printing technique, nano platinum particle is grown in fluorescence coating working region, the amplification of nucleic acid cycle signal is realized to the quenching effect of palladium nanocluster, and duplexing specific nucleic acid enzyme using graphite phase carbon nitride nanometer sheet, to realize the precise measurement of small molecule miRNA.The paper chip prepared is folded, chromophoric solution is added dropwise, it can be achieved that the pre- measurement of visualization in conversion zone；Then paper chip is put into fluorescence ware, realizes that fluorescence accurately measures miRNA.This is easy to operate quickly, time saving and energy saving, provides a kind of new reliable method for the detection of miRNA.
Description
Technical field
The present invention relates at low cost, high sensitivities, visual biological micromolecule technical field of analysis and detection, more specifically
Say it is a kind of fluorescence for being able to detect miRNA/colorimetric double mode sensor preparation, the invention further relates to use nucleic acid digestion to believe
Number amplifying technique.
Background technique
MiRNA belongs to endogenous small molecule non-coding RNA, and length is about 19 ~ 22 mature nucleotide.It is so far
Only, miRNA is found in all animals and plants, accounts for about the 4% of all genomes.Meanwhile miRNA has been demonstrated to cell point
Split, growth and apoptosis have a major impact, and some common diseases include cancer, cardiovascular and cerebrovascular disease, virus infection etc. with it is certain
The improper expression of specific miRNA is closely related.Definite effect of each miRNA in specific organ, tissue or cell and
Function still requires study.However, the series of characteristics of miRNA, such as short sequence, low content, it is degradable and with family member it
Between similar sequence become the limiting factor that accurately detects.Therefore, either on tissue or cellular level, sensitive miRNA
The development of detection method will all become an important milestone of miRNA biology.
So far, miRNA detection means is increasingly valued by people, and various detection techniques have been employed
To among the accurate expression of miRNA.Wherein Northern trace is considered as exemplary standard method, other many crucial detection skills
Art, such as hybridization array technology, quantitative reverse transcription polymerase chain reaction (RT-PCR), isothermal exponential amplification techniques etc. all exist
Huge practical value has been played in the detection of miRNA.Even so, highly sensitive, high-throughput, the good detection technique of specificity
It is still a challenge.Therefore, it there is an urgent need to develop an effective platform, can not only solve the above problems, and may be implemented
Reliable and portable miRNA detection.
In order to improve the sensitivity of miRNA detection, we imitate the quenching of noble metal nano cluster in graphite phase carbon nitride
A kind of novel and multifunctional fluorescence/colorimetric dual-mode biological sensor is established on the basis of should amplifying with nucleic acid cycle signal.Simultaneously
Noble metal nanometer material is grown using micro-fluidic paper base platform, this operates the specific surface area and table for not only considerably increasing paper chip
Surface roughness, and effectively reduce the background fluorescence of paper chip.Noble metal nano cluster is unique as fluorescent bio-probes
Physics and chemical property, such as good biocompatibility, charging performance and luminescent properties are excellent, it is easily prepared the advantages that so that its
There is better application prospect than organic fluorescence molecule；In addition, in distinctive class horseradish peroxidase in noble metal nano cluster
Catalytic performance has equally caused huge concern in science and technology field.Most of all, being based on dual signal output mode, simultaneously
The Preliminary Determination of colorimetric method and the further quantitative detection of fluorescence method are realized, in addition nucleic acid cycle signal amplifying technique, realized
The hypersensitive of miRNA is expressed.Our invention is that design sensor opens unique visual angle, and provides a kind of economy
Efficiently, convenient and efficient and disposable paper chip, can detect to quicklook miRNA, and this method has very strong practicability, can
With the detection for various miRNA and DNA, to having in the bioanalysis detection and clinical medicine for starting hypersensitive, high stable
Good prospect.
Summary of the invention
It paper chip the object of the present invention is to provide one kind with nano platinum particle (Pt NPs) and is nitrogenized based on graphite-phase
Carbon nanosheet (g-C3N4NSs) the nucleic acid cycle signal amplifying technique of the quenching effect of palladium nanocluster (Pd NCs) is realized glimmering
Light/colorimetric double mode biosensor preparation method, quick, Sensitive Detection for small molecule miRNA.The biosensor
Preparation step it is as follows:
(1) paper chip fluorescence coating and hydrophobic wax print area and hydrophilic working region (attached drawing 1) than chromatograph are designed；
(2) Pt NPs is grown in fluorescence area and hydrophilic working region；
(3) by g-C3N4NSs is fixed on the working region of step (2) resulting paper chip, then by the palladium nanometer of DNA modification
Chain (DNA-Pd NCs) is fixed, and completes fluorescent quenching；
(4) miRNA chain to be measured is fixed on to the working region of step (3) resulting paper chip, introduces duplexing specific nucleic acid enzyme
(DSN) amplification of nucleic acid cycle signal is carried out；
(5) colorimetric measures in advance: the resulting paper chip of step (4) being folded (attached drawing 2), colour developing is added dropwise in the working region than chromatograph
Substrate, the buffer solution and hydrogen peroxide that pH is 4 ~ 6, carry out colorimetric analysis；
(6) accurate fluoremetry: step (4) resulting paper chip is put into fluorescence equipment, in 380 nm of excitation wavelength and hair
Fluoremetry is carried out under long 460 nm of ejected wave.
Paper material of the present invention is chromatographic paper, fluorescence obtained/colorimetric double mode paper chip pattern Adobe
Illustrator CS4 software design, the micro-fluidic paper chip preparation process are as follows: designing micro-fluidic paper chip on computers
Hydrophobic wax bulk print pattern, style is as shown in Fig. 2, which includes left side chrominance response region, intermediate
Working region, the right fluorescence reaction region, wherein hydrophilic region diameter dimension is 6 mm, and channel width is 2 mm.
The step of fluorescence coating of the present invention and working region grow Pt NPs are as follows: by freshly prepared 10-100 μ L chlorine
Platinic acid (H2PtCl6, concentration is 20 mM) and 10-100 μ L sodium borohydride (NaBH4, concentration is 20 mM) quickly it is uniformly mixed, so
Mixed liquor is added dropwise to paper chip working region afterwards and dries 20 min at room temperature.Then, it is thoroughly washed using ultrapure water
It states paper chip 3 times, is then dried at room temperature for 30 min.
It is of the present invention by g-C3N4NSs is fixed on the working region of step (2) resulting paper chip, then by DNA-Pd
The step of NCs fixes, and completes fluorescent quenching are as follows:
(1) g-C is synthesized3N4NSs: it is placed in the porcelain crucible with lid, is divulging information firstly, weighing 1-5 g melamine powder
2 h are heated under state at 600 DEG C, wherein the rate of heating and cooling procedure is 3 DEG C/min, synthesizing yellow bulk graphite
Phase carbon nitride (g-C3N4).Then, by g-C3N4It is ground to superfines, weighing 10-200 mg, to be dispersed in 10-200 mL ultrapure
In water, 18 h of ultrasound.The suspension obtained after ultrasound is centrifuged with the speed of 8000 r/min, to remove remaining unstripped g-
C3N4.Finally, collecting simultaneously concentrated supernatant under 60 DEG C of vacuum conditions, obtained cream is g-C3N4NSs；
(2) synthetic DNA-Pd NCs: by 10-200 μ L PdCl2Solution (concentration is 0.1 M) and 10-20 mL dimethyl formyl
Amine (DMF) mixes, and after 5 min, mixed solution is heated to reflux 6 h at 150 DEG C, obtains yellow solution.Then,
Solution is centrifuged with 8000 r/min to obtain Pd NCs.Then, by 50-300 μ L DNA(concentration be 10 μM) introduce it is made
In standby Pd NCs solution (1-5 mL), 45 min are reacted, at room temperature to obtain DNA-Pd NCs；
(3) quenching reaction: in the paper chip of growth Pt NPs, 10-200 μ L g-C is introduced3N4NSs is stood at room temperature
20 min are added dropwise 10-200 μ L DNA-Pd NCs, are then incubated at room temperature 30 min after its drying.Finally, using super
Pure water thoroughly washs above-mentioned paper chip 3 times, dries 30 min at room temperature.
The working region of the present invention that miRNA chain to be measured is fixed on to step (3) resulting paper chip, introduce DSN into
The step of row nucleic acid cycle signal amplifies are as follows: 10-200 μ L standard miRNA solution is added drop-wise to step (3) resulting paper chip
Working region, and be incubated at room temperature 30 min to form DNA-RNA heteroduplex.10-200 μ L DSN is then added
(the DSN property of can choose DNA) in cutting double-stranded DNA or DNA-RNA heteroduplex destroys above-mentioned heteroduplex, release
MiRNA can be reused in subsequent reaction cycle, carry out cycle signal amplification.
Chromogenic substrate of the present invention is 3,3,5,5-tetramethyl benzidines, o-phenylenediamine, 2,2- azine groups-bis--(3-
Ethyl benzo thiazoline quinoline -6- sulfonic acid) di-ammonium salts.
The buffer solution that pH of the present invention is 4 ~ 6 is NaAc_HAc buffer solution, phosphate buffer solution,
Tris hydrochloric acid buffer solution.
Chromogenic substrate, the buffer solution that pH is 4 ~ 6 and mistake is added dropwise in the pre- determination step of miRNA colorimetric of the present invention respectively
Hydrogen oxide carries out the measurement of sample, draws the standard curve of gray scale and miRNA concentration, realizes the visualization preliminary examination of miRNA.
MiRNA fluoremetry step of the present invention, paper chip is put into fluorescence ware, in 380 nm of excitation wavelength and hair
The measurement that sample is carried out under long 460 nm of ejected wave, draws the standard curve of fluorescence intensity and miRNA concentration, realizes the essence of miRNA
Really detection；
Beneficial effects of the present invention
(1) Pd NCs replaces fluorescent dye, has better fluorescent characteristic than organic fluorescence molecule, and it is with excellent peroxide
Compound enzymatic activity has started the bioanalysis detection technique of hypersensitive, high stable and long luminescence lifetime；
(2) use of Pt NPs increases paper chip specific surface area and effectively reduces the background fluorescence of paper, improves detection
Sensitivity；
(3) nucleic acid circulation is carried out using DSN, effectively realizes the dual amplification of fluorescence and colorimetric signal；
(4) by the combination of colorimetric/fluorescent dual module formula biosensor, it is based on dual signal output mode, realizes the super of miRNA
Sensitive Detection；
(5) by changing aptamers sequence, the visualization colorimetric initial survey of different miRNA and DNA and accurate glimmering may be implemented
Light measurement.
Detailed description of the invention
Fig. 1: paper chip hydrophobic wax print pattern.
Fig. 2: the size of paper chip and each regional function；The folding mode of paper chip.
Specific embodiment
For a better understanding of the present invention, below with reference to the embodiment and attached drawing content that the present invention is further explained, but this
The content of invention is not limited solely to following implementation.
Embodiment 1
Application of the fluorescence/colorimetric double mode paper chip in detection let-7a, it is characterized in that the following steps are included:
(1) paper chip fluorescence coating and hydrophobic wax print area and hydrophilic working region (attached drawing 1) than chromatograph are designed.Institute of the present invention
The paper material stated is chromatographic paper, and fluorescence obtained/colorimetric double mode paper chip pattern is soft with Adobe Illustrator CS4
Part design, the micro-fluidic paper chip preparation process are as follows: designing the hydrophobic wax bulk print of micro-fluidic paper chip on computers
Pattern, style is as shown in Fig. 1, which includes left side chrominance response region, intermediate working region, the right fluorescence
Conversion zone, wherein hydrophilic region diameter dimension is 6 mm, and channel width is 2 mm；
(2) Pt NPs is grown in fluorescence area and hydrophilic working region.By freshly prepared 50 μ L H2PtCl6(concentration is 20
) and 50 μ L NaBH mM4Then mixed liquor is added drop-wise to paper chip working region and in room by (concentration be 20 mM) quickly mixing
Lower dry 20 min of temperature.Then, above-mentioned paper chip is thoroughly washed using ultrapure water 3 times, be dried at room temperature for 30 min；
(3) by g-C3N4NSs is fixed on the working region of step (2) resulting paper chip, then will be in DNA-Pd NCs fixation
It goes, completes fluorescent quenching.The first step synthesizes g-C3N4NSs: weighing 3 g melamines and be placed in the porcelain crucible with lid,
2 h are heated under ventilation state at 600 DEG C, wherein the rate of heating and cooling procedure is 3 DEG C/min, synthesizing yellow bulk
g-C3N4.Then, by g-C3N4It is ground to superfines, 100 mg is weighed and is dispersed in 100 mL ultrapure waters, and 18 h of ultrasound.
The suspension obtained after ultrasound is centrifuged with the speed of 8000 r/min, to remove remaining unstripped g-C3N4.Finally,
Simultaneously concentrated supernatant is collected under 60 DEG C of vacuum conditions, obtained cream is g-C3N4NSs.Second step synthetic DNA-
Pd NCs: by 150 μ L PdCl2(concentration 0.1M) and 15 mL DMF are mixed, and after 5 min, mixed liquor is added at 150 DEG C
Heat 6 h of reflux, obtain yellow solution.Then, solution is centrifuged under 8000 r/min to obtain Pd NCs.Then, by 200 μ
L DNA(concentration is 10 μM) it is introduced into prepared Pd NCs solution (3mL), 45 min are reacted, at room temperature to obtain DNA-
Pd NC.It finally carries out quenching reaction: in the paper chip of growth Pt NPs, introducing 100 μ L g-C3N4NSs is stood at room temperature
20 min are added dropwise 100 μ L DNA-Pd NCs, are incubated for 30 min at room temperature after its drying.Then, thorough using ultrapure water
Above-mentioned paper chip 3 times of bottom washing, is then dried at room temperature for 30 min；
(4) let-7a chain to be measured is fixed on to the working region of step (3) resulting paper chip, DSN is introduced and carries out nucleic acid circulation
Signal amplification.100 μ L standard let-7a solution are added drop-wise to the working region of step (3) resulting paper chip, and in room temperature
30 min are to form DNA-RNA heteroduplex for lower incubation.It cuts the 100 μ L DSN(DSN property of can choose then are added double
DNA in chain DNA or DNA-RNA heteroduplex) destroy above-mentioned heteroduplex, the let-7a of release can be subsequent anti-
It is reused in should recycling, carries out cycle signal amplification；
(5) the resulting paper chip of step (4) is folded, 30 μ L chromogenic substrates 3,3,5,5-is added dropwise in the working region than chromatograph
Tetramethyl benzidine (TMB), the NaAc_HAc buffer solution and hydrogen peroxide that pH is 4, carry out colorimetric and measure in advance, draw ash
The standard curve of degree and let-7a concentration, realizes the visualization preliminary examination of let-7a；
(6) accurate fluoremetry: step (4) resulting paper chip is put into fluorescence equipment, in 380 nm of excitation wavelength and hair
Fluoremetry is carried out under long 460 nm of ejected wave, the standard curve of fluorescence intensity and let-7a concentration is drawn, realizes the essence of let-7a
Really detection.
Embodiment 2
Detecting step is a difference in that with example 1: be will test substance let-7a and is changed the small molecules such as other miRNA or DNA, phase into
DNA sequence dna used in the step of answering (3) also needs to change.
Sequence table
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<120>application of the dual signal enhancing paper base biosensor in miRNA detection
<130> 2019
<141> 2019-01-07
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 22
<212> RNA
<213>artificial sequence (Artificial Sequence)
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uugauauguu ggaugaugga gu 22
<210> 2
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<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 2
aactatacaa cctactacct ca 22
Claims (8)
1. dual signal enhance paper base biosensor miRNA detection in application, feature the following steps are included:
1.1 design paper chip fluorescence coatings and hydrophobic wax print area and hydrophilic working region than chromatograph；
1.2 in fluorescence area and hydrophilic working region growth nano platinum particle (Pt NPs)；
1.3 by class graphitic nitralloy carbon nanosheet (g-C3N4NSs it) is fixed on the working region of step 1.2 gained paper chip, then
The palladium nano chain (DNA-Pd NCs) of DNA modification is fixed, completes fluorescent quenching；
1.4 are fixed on miRNA chain to be measured the working region of step 1.3 gained paper chip, introduce duplexing specific nucleic acid enzyme (DSN)
Carry out the amplification of nucleic acid cycle signal；
1.5 fold step 1.4 gained paper chip, molten in the buffering that chromogenic substrate is added dropwise in the working region than chromatograph, pH is 4 ~ 6
Liquid and hydrogen peroxide carry out colorimetric and measure in advance；
1.6 accurate fluoremetries: the resulting paper chip of step 1.4 is put into fluorescence equipment, in 380 nm of excitation wavelength and hair
Fluoremetry is carried out under long 460 nm of ejected wave.
2. enhancing application of the paper base biosensor in miRNA detection, feature according to dual signal described in claims 1
It is, paper material used is chromatographic paper, with Adobe Illustrator CS4 software design fluorescence/colorimetric double mode paper chip
Pattern, the micro-fluidic paper chip preparation process are as follows: designing the hydrophobic wax bulk print figure of micro-fluidic paper chip on computers
Case, the micro-fluidic paper chip include left side chrominance response region, intermediate working region, the right fluorescence reaction region, wherein hydrophilic
For regional diameter having a size of 6 mm, channel width is 2 mm.
3. application of the paper base biosensor in miRNA detection is enhanced according to dual signal described in claims 1, it is special
Sign is, grows Pt NPs in fluorescence area and working region, preparation step is as follows: by freshly prepared 10-100 μ L chlorine platinum
Acid (H2PtCl6, concentration is 20 mM) and 10-100 μ L sodium borohydride (NaBH4, concentration is 20 mM) quickly it is uniformly mixed, then
Mixed liquor is added dropwise to paper chip working region and dries 20 min at room temperature and is then thoroughly washed using ultrapure water above-mentioned
Paper chip 3 times, it is dried at room temperature for 30 min.
4. application of the paper base biosensor in miRNA detection is enhanced according to dual signal described in claims 1, it is special
Sign is, by g-C3N4NSs is fixed on the working region of step 1.2 gained paper chip, then fixes by DNA-Pd NCs,
Complete fluorescent quenching, preparation process are as follows: the first step synthesizes g-C3N4NSs: it weighs suitable 1-5 g melamine powder and is placed in lid
In the porcelain crucible of son, 2 h are heated at 600 DEG C under ventilation state, wherein the rate of heating and cooling procedure be 3 DEG C/
Min, synthesizing yellow bulk g-C3N4；Then, by g-C3N4It is ground to superfines, 10-200 mg is weighed and is dispersed in 10-200
In mL ultrapure water, 18 h of ultrasound, obtained suspension is centrifuged with the speed of 8000 r/min after ultrasound, remaining is not shelled with removing
From g-C3N4, finally, collecting simultaneously concentrated supernatant under 60 DEG C of vacuum conditions, obtained cream is g-C3N4
NSs；Second step synthetic DNA-Pd NCs: by 10-200 μ L PdCl2Solution (concentration is 0.1 M) and 10-20 mL dimethyl methyl
Amide mixes, and after 5 min, mixed solution is heated to reflux 6 h at 150 DEG C, obtains yellow solution, then, will be molten
Liquid is centrifuged with 8000 r/min to obtain Pd NCs, is 10 μM by 50-300 μ L DNA(concentration then) introduce it is prepared
In Pd NCs solution (1-5 mL), 45 min are reacted at room temperature, to obtain DNA-Pd NCs；Finally carry out quenching reaction:
In the paper chip for growing Pt NPs, 10-200 μ L g-C is introduced3N4NSs stands 20 min at room temperature, after its drying, drop
Add 10-200 μ L DNA-Pd NCs, 30 min is then incubated at room temperature, finally, thoroughly washing above-mentioned core using ultrapure water
Piece 3 times, 30 min are dried at room temperature.
5. application of the paper base biosensor in miRNA detection is enhanced according to dual signal described in claims 1, it is special
Sign is, miRNA chain to be measured is fixed on to the working region of the resulting paper chip of step 1.3, introduces DSN and carries out nucleic acid circulation letter
Number amplification, the process flow are as follows: 10-200 μ L standard miRNA solution is added drop-wise to the workspace of the resulting paper chip of step 1.3
Domain, and 30 min are incubated at room temperature to form DNA-RNA heteroduplex；10-200 μ L DSN(DSN then is added can be with
The selectively DNA in cutting double-stranded DNA or DNA-RNA heteroduplex) destroy above-mentioned heteroduplex, the miRNA of release
It can be reused in subsequent reaction cycle, carry out cycle signal amplification.
6. application of the paper base biosensor in miRNA detection is enhanced according to dual signal described in claims 1, it is special
Sign is that chromogenic substrate used is 3,3,5,5-tetramethyl benzidines, o-phenylenediamine, 2,2- azine groups-bis--(3- ethyl benzo
Thiazoline quinoline -6- sulfonic acid) di-ammonium salts；The buffer solution of pH 4 ~ 6 is NaAc_HAc buffer solution, and phosphate-buffered is molten
Liquid, Tris hydrochloric acid buffer solution.
7. enhancing application of the paper base biosensor in miRNA detection, feature according to dual signal described in claims 1
It is, the pre- determination step of miRNA colorimetric, chromogenic substrate is added dropwise respectively, the buffer solution and hydrogen peroxide that pH is 4 ~ 6, carries out sample
The measurement of product draws the standard curve of gray scale and miRNA concentration, realizes the visualization preliminary examination of miRNA.
8. application of the paper base biosensor in miRNA detection is enhanced according to dual signal described in claims 1, it is special
Sign is that paper chip is put into fluorescence ware by miRNA fluoremetry step, in 380 nm of excitation wavelength and launch wavelength 460
The measurement that sample is carried out under nm, draws the standard curve of fluorescence intensity and cancer cell concentration, realizes the accurate detection of miRNA.
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