CN108424844A - A kind of single-molecule sequencing chip and preparation method thereof - Google Patents
A kind of single-molecule sequencing chip and preparation method thereof Download PDFInfo
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Abstract
The present invention provides a kind of single-molecule sequencing chip and preparation method thereof, the chip includes runner plate, substrate and cover board, and the runner plate is arranged between substrate and cover board；Flow cell is set on the runner plate, and the flow cell includes multiple runners disposed in parallel；Substrate is arranged in the top setting cover board of the flow cell, the lower section of flow cell；The bottom of the runner is connected to substrate surface；Sequencing primer and positioning fluorescent marker are fixed in the surface of the substrate contacted with runner plate.Chip surface load has fluorescence lifetime length, fluorescence intensity high, and the quantum dot or fluorescent microsphere that fluorescence property is stablized can position target dna unimolecule in sequencing procedure.Since quantum dot or fluorescent microsphere are maintained to higher fluorescence intensity after being shone by exciting light many times, solve the problems, such as that positioning fluorescence signal can not be kept for a long time, it significantly improves single-molecule sequencing and reads length, reduce the error rate of sequencing, and effectively reduce sequencing cost.
Description
Technical field
The present invention relates to the design preparing technical fields of sequence testing chip, and in particular to a kind of single-molecule sequencing chip and its system
Preparation Method.
Background technology
Although two generation sequencing technologies still occupy leading position in the market in current sequencing, it is known as third generation survey
The single-molecule sequencing technology development of sequence technology is very rapid.Three generations's DNA sequencing includes mainly unimolecule synthesis order-checking (Single-
Molecule Sequencing by Synthesis, SMSBS), (Single-Molecule Real- are sequenced in unimolecule in real time
Time Sequencing, SMRTS) and three kinds of technologies of nano-pore sequencing (Nanopore Sequencing).Wherein unimolecule closes
At sequencing technologies (Helicos is known as SMS technologies)3-5Basic principle it is similar to two generation synthesis order-checking (SBS), be all using glimmering
Four kinds of nucleotide that signal is formed carry out Single base extension on DNA primer successively, using highly sensitive Imaging-PAM
Detection is extended the fluorescence signal of primer to identify the base of corresponding position in template to be measured, then removes fluorophor, and
It is repeated in above-mentioned sequencing cyclic process, to realize the measurement of DNA sequence dna.In this single-molecule sequencing engineering philosophy without pair
DNA sample to be measured is expanded, can be at single point so as to avoid the GC preference equal errors for amplifying due to DNA cloning and bringing
Original DNA sequence dna information to be measured is directly read in sub- level, detection sensitivity is high, and sample preparation is simple.It is low to realize
Cost, high-throughput direct Sequencing.
DNA sequencing chip is one of core technology of DNA sequencing and the important component of sequenator.Unimolecule is surveyed
Sequence chip not yet forms large-scale production at present, since when carrying out single-molecule sequencing, the object of detection is single DNA molecules,
Fine degree is high, and the slight vibration of sample stage or chip is likely to cause target dna that can not lock, and sequencing can not continue
Serious problems.Existing single-molecule sequencing technology realizes positioning by marking fluorescent molecular in template to be measured, each
Sequencing cycle is required for the positioning fluorescent molecular to the label to be imaged, and generation fluorescence is easy to after repeatedly exciting and is quenched
It goes out, can not be kept for a long time in the case where adding anti-fluorescence quenching and imaging agents.Unimolecule synthesizes at present
Sequencing is average to read long only 25-30, and it is a major reason that positioning fluorescent molecular, which is quenched,.If in single-molecule sequencing chip surface
The fluorescence lifetime that loads particular arrangement is long, other fluorescent markers that fluorescence intensity is high are to be substituted in mark fluorescent in template to be measured
For positioning, will be expected to solve the above problems.
Invention content
The technical problem to be solved by the present invention is to, in view of the shortcomings of the prior art, provide a kind of single-molecule sequencing chip and its
Preparation method, this method is simple for process, and cost is relatively low, and the single-molecule sequencing chip prepared using this method disclosure satisfy that unimolecule
The requirement to positioning fluorescence signal detection is sequenced, the good fluorescent marker of area load fluorescence property especially can be to mesh
Mark DNA unimolecules are positioned, and to improve the reading length and accuracy of single-molecule sequencing, are had a very important significance and very high
Application value.
The purpose of the present invention is what is be achieved through the following technical solutions：
The present invention provides a kind of single-molecule sequencing chip, including runner plate, substrate and cover board, the runner plate setting exists
Between substrate and cover board；Flow cell is set on the runner plate, and the flow cell includes multiple runners disposed in parallel；The stream
Substrate is arranged in the top setting cover board in logical pond, the lower section of flow cell；The runner is connected to substrate surface.
Preferably, sequencing primer and positioning fluorescent marker are fixed in the surface of the substrate contacted with runner plate.
Preferably, it is the alkynyl-modified primers of-N3 or 5 '-that the primer, which is 5 ',；The positioning fluorescent marker is glimmering
One kind in light quanta point, nanometer carbon dots and fluorescent microsphere, emission wavelength and the label of the positioning fluorescent marker are being surveyed
Fluorescein wavelength difference on sequence reagent.
Preferably, the fluorescence quantum surface with can react to each other with the group or substance of substrate surface or with
The functional group that non-covalent bond combines, the preferred cadmiumsulfide quantum dot of the fluorescence quantum, CdSe quantum dots, zinc selenide quantum
Point；The fluorescent microsphere is the polystyrene fluorescent microsphere of diameter 20-100nm, and polystyrene fluorescent microsphere surface is with can be with
The functional group that the group or substance of substrate surface are reacted to each other or combined with non-covalent bond.
Preferably, the method for fixing sequencing primer and positioning fluorescent marker in the substrate surface includes following step
Suddenly：
A1, substrate surface is activated and is modified, substrate surface is made to carry the group or substance of reactivity；
A2, sequencing primer is connect with the substrate surface after modification using water-soluble difunctional connection unit；
A3, positioning fluorescent marker is connect with the substrate surface after modification with water-soluble difunctional connection unit.
It is highly preferred that in step A1, the group or substance with reactivity include amino, carboxyl, alkynyl, fold
At least one of nitrogen, acid anhydrides, active ester, acid imide, biotin, protein, but not limited to this.
Preferably, the middle part of the runner is cuboid-type, and both ends are tapered, and both ends are respectively set first fluid and enter
Mouth and first fluid outlet；
Position corresponding with first fluid entrance and first fluid outlet is respectively arranged with second fluid and enters on the cover board
Mouth and second fluid outlet, the first fluid entrance are connected to second fluid entrance, and second fluid outlet goes out with second fluid
Mouth connection；
The runner quantity is 2-16, and each width of flow path is 2-8mm, length 5-10cm, adjacent channels
Spacing is 2-5mm.
Preferably, the thickness of the substrate is 500-1000 μm, and the thickness of the cover board is 100-500 μm；
The material of the runner plate is one kind in silicon chip, glass (i.e. slide) or ceramics；The material of the substrate and cover board
Matter is quartz slide or high borosilicate slide.
Preferably, the runner is prepared using photoetching process, specifically includes following steps：
S1, photoresist is evenly applied to by flow field plate surfaces according to runner pattern, forms mask；
S2, the runner plate that mask is covered using ultraviolet light, are then removed mask, are heat-treated；
S3, it after being cooled to room temperature the runner plate after heat treatment, performs etching, then wash residue, that is, forms runner.
It is highly preferred that in step S1, the thickness of the runner plate is 80-500 μm；The mask thicknesses are 200-600 μm；
In step S2, the ultraviolet light wave a length of 248nm or 365nm used in the ultraviolet light processing, illumination power
For 15-30J/cm2, light application time is 60-180 seconds；500-600 DEG C of the temperature of the heat treatment, time 5-10min；
In step S3, the etching liquid used that etches is hydrofluoric acid solution.
The present invention also provides a kind of preparation methods of single-molecule sequencing chip, include the following steps：
After runner plate, substrate and cover board are cleaned, it is pressed to get the list by assembling using adhesive in order
Molecule sequence testing chip.
Preferably, the adhesive is selected from dimethyl silicone polymer, polyurethane, epoxy resin, but not limited to this.
The present invention also provides a kind of methods carrying out single-molecule sequencing using chip above-mentioned, include the following steps：
B1, template solution to be measured is added in runner by second fluid entrance, is existed with the primer for being fixed on substrate surface
It incubates 5 minutes and is hybridized at 65 DEG C, exported by second fluid and extract substrate surface residual liquid out；
B2, by second fluid entrance by the reversible terminator nucleotide, polymerase, buffering of fluorescein-labeled different bases
The mixture of liquid is added in runner, and extension is carried out under the action of polymerase, and the time is 15 minutes, and temperature is 37 DEG C；
After B3, extension, buffer solution for cleaning runner is injected from second fluid entrance, exports and extracts out from second fluid
Residual liquid after cleaning；
B4, using suitable wavelength exciting light exposure label(l)ing substrate surface positioning fluorescent marker, to participation prolong
The primer stretched/template composite carries out fluorescence localization, then irradiates drawing after participating in extending with the exciting light of other wavelength again
Object/template composite, the recognizable corresponding base to be measured of fluorescence signal by participating in extension products；
Cleavage reagent solution is added dropwise in B5, the substrate surface in runner bottom, is reacted 5 minutes at 37 DEG C, then protection is added dropwise
The reaction was continued 3 minutes for agent solution, injects buffer solution for cleaning runner from second fluid entrance, after second fluid exports extraction cleaning
Residual liquid；
B6, above step is repeated, the sequencing to template to be measured can be completed.
Compared with prior art, the present invention has following advantageous effect：
1. single-molecule sequencing chip of the present invention is prepared using the technique of three-decker pressing assembling, can be respectively to three
Layer material is processed, and is then combined using simple and practicable method, and not high to equipment requirement, cost is relatively low.
2. single-molecule sequencing chip surface load of the present invention has, fluorescence lifetime is long, fluorescence intensity is high, and fluorescence property is steady
Fixed quantum dot or fluorescent microsphere can position target dna unimolecule in sequencing procedure.Due to quantum dot or fluorescence
Microballoon is maintained to higher fluorescence intensity after being shone by exciting light many times, and solving positioning fluorescence signal can not
The problem of keeping for a long time can significantly improve single-molecule sequencing and read length, so as to avoid anti-fluorescence quenching and imaging agents
Use, the reading for being conducive to greatly improve single-molecule sequencing grows, and reduces the error rate of sequencing, and effectively reduces sequencing cost.
Description of the drawings
Upon reading the detailed description of non-limiting embodiments with reference to the following drawings, other feature of the invention,
Objects and advantages will become more apparent upon：
Fig. 1 is the horizontal cross-section structure diagram of single-molecule sequencing chip of the present invention；
Fig. 2 is the decomposition texture schematic diagram of single-molecule sequencing chip of the present invention；
Fig. 3 is the schematic diagram of a runner of single-molecule sequencing Microchip flow cell of the present invention；
Fig. 4 is in the case of single sequence to be measured, the primer on single-molecule sequencing chip extends for the first time in sequencing recycles
Fluorescence photo after reaction；
Fig. 5 is primer second of extension in sequencing recycles on single-molecule sequencing chip in the case of single sequence to be measured
Fluorescence photo after reaction；
Fig. 6 is the fluorescence photograph of the primer first time extension on single-molecule sequencing chip in the case of four templates to be measured
Piece, 1 is sequence to be measured (1), and 2 be sequence to be measured (2), and 3 be sequence to be measured (3), and 4 be sequence to be measured (4)；
Fig. 7 is to fix 5 ' the terminal modified primers for having nitrine and surface with azido group in the substrate surface with alkynyl
The schematic diagram of fluorescent marker；
Fig. 8 is the fluorescence photograph of the 50th extension of primer on single-molecule sequencing chip in the case of four templates to be measured
Piece；
Wherein, in Fig. 1-Fig. 3：1- cover boards；2- runner plates；3- substrates；4- single-molecule sequencing chips；11- second fluids enter
Mouthful；12- second fluids export；21- runners；22- first fluid entrances；23- first fluids export；24- DNA unimolecules moulds to be measured
Plate；31- positioning fluorescent markers.
Specific implementation mode
With reference to specific embodiment, the present invention is described in detail.Following embodiment will be helpful to the technology of this field
Personnel further understand the present invention, but the invention is not limited in any way.It should be pointed out that the ordinary skill of this field
For personnel, without departing from the inventive concept of the premise, several changes and improvements can also be made.These belong to the present invention
Protection domain.
Following embodiment provides a kind of single-molecule sequencing chip, as shown in Figure 1-Figure 3, including runner plate 2,3 and of substrate
Cover board 1, the runner plate 2 are arranged between substrate 3 and cover board 1；Flow cell, the flow cell packet are set on the runner plate 2
Include multiple runners disposed in parallel 21；Substrate 3 is arranged in the top setting cover board 1 of the flow cell, the lower section of flow cell；The stream
21 bottom of road is connected to 3 surface of substrate.
Sequencing primer and positioning fluorescent marker 31 are fixed in the surface of the substrate 3 contacted with runner plate 2.
It is the alkynyl-modified primers of-N3 or 5 '-that the sequencing primer, which is 5 ',；The positioning fluorescent marker 31 is fluorescence
The emission wavelength of one kind in quantum dot, nanometer carbon dots and fluorescent microsphere, the positioning fluorescent marker 31 is different from label
Fluorescein wavelength on sequencing reagent.
The fluorescence quantum surface carries and can react to each other or with non-covalent with the group or substance of substrate surface
It is bonded the functional group closed；The fluorescent microsphere is the polystyrene fluorescent microsphere of diameter 20-100nm, polystyrene fluorescent microsphere
Surface carries the functional group that can be reacted to each other with the group of substrate surface or substance or be combined with non-covalent bond.
Include the following steps in the method that sequencing primer and positioning fluorescent marker 31 are fixed in 3 surface of the substrate：
A1,3 surface of substrate is activated and is modified, 3 surface of substrate is made to carry the group or substance of reactivity；
A2, sequencing primer is connect with 3 surface of substrate after modification using water-soluble difunctional connection unit；
A3, positioning fluorescent marker 31 and 3 surface of substrate after modification are connected using water-soluble difunctional connection unit
It connects.
In step A1, the activation step is specially：Clean matrix is placed in the mixed liquor of hydrogen peroxide and the concentrated sulfuric acid,
1h is heated at 80-90 DEG C, makes matrix surface hydroxylating；
The modification step is specially：By the matrix after activated step in a solvent with aminopropyl triethoxysilane
Heating reaction 2 hours at 60 DEG C, obtain the matrix that surface carries amino；
The group or substance with reactivity include amino, carboxyl, alkynyl, nitrine, acid anhydrides, active ester, acyl Asia
At least one of amine, biotin, protein, but not limited to this.
In step A2, the reactive functional of one end that the difunctional connection unit of water solubility is connect with matrix is carboxylic
Base active ester is alkynyl or azido with the reactive functional of the one end connecting primer P1.
Specifically, the difunctional connection unit of water solubility includes at least one of following structural formula：
Step A2 specifically includes following steps：
Matrix is placed in the solution containing water-soluble difunctional connection unit, reacts at room temperature and core is connected to amido bond
Piece surface；
The sequencing primer of surface modification appropriate functional group is added through step A1 treated matrix surfaces, at room temperature into
Row click chemistry reacts 9h, you can.
Step A3 specifically includes following steps：
The positioning fluorescent marker for having modified nitrine functional group or alkynyl is added dropwise in substrate surface, is passed through at room temperature
Click reacts 9h, and the positioning can be connected to substrate surface with fluorescent marker by covalent bond.
The middle part of the runner 21 is cuboid-type, and both ends are tapered, and first fluid entrance 22 is respectively set in both ends
With first fluid outlet 23；
23 corresponding positions are exported with first fluid entrance 22 and first fluid be respectively arranged with second on the cover board 1
Body entrance 11 and second fluid outlet 12, the first fluid entrance 22 is connected to second fluid entrance 11, second fluid outlet
23 are connected to second fluid outlet 12；
21 quantity of runner is 2-16, and each 21 width of the runner is 2-8mm, length 5-10cm, adjacent flow
The spacing in road 21 is 2-5mm.
The thickness of the substrate 3 is 500-1000 μm, and the thickness of the cover board 1 is 100-500 μm；
The material of the runner plate 2 is one kind in silicon chip, glass or ceramics；The material of the substrate 3 and cover board 1 is stone
English slide or high borosilicate slide.
The runner 21 is prepared using photoetching process, specifically includes following steps：
S1, photoresist is evenly applied to by 2 surface of runner plate according to runner pattern, forms mask；
S2, the runner plate 2 that mask is covered using ultraviolet light, are then removed mask, are heat-treated；
S3, it after being cooled to room temperature the runner plate 2 after heat treatment, performs etching, then wash residue, that is, forms runner
21。
In step S1, the thickness of the runner plate 2 is 80-500 μm；The mask thicknesses are 200-600 μm；
In step S2, the ultraviolet light wave a length of 248nm or 365nm used in the ultraviolet light processing, illumination power
For 15-30J/cm2, light application time is 60-180 seconds；500-600 DEG C of the temperature of the heat treatment, time 5-10min；
In step S3, the etching liquid used that etches is hydrofluoric acid solution.
The preparation method of the single-molecule sequencing chip includes the following steps：
After runner plate 2, substrate 3 and cover board 1 are cleaned, it is pressed to get institute by assembling using adhesive in order
State single-molecule sequencing chip 4.
The single-molecule sequencing chip 4 can be used for carrying out sequencing to DNA unimolecules 24 to be measured.
Embodiment 1
A kind of single-molecule sequencing chip designs and prepares method, includes the following steps：
(1) it is 200 μm of silicon chip to take one piece of thickness, is formed on its surface runner using photoetching process, photolithography steps are as follows：
Photoresist is evenly applied to silicon chip surface by 1.1 according to pre-designed flow cell runner pattern, forms thickness
For 500 μm of mask；
1.2 be 365nm with wavelength the above-mentioned silicon chip for covering mask of ultraviolet light, illumination power be 25J/cm2, light
It it is 90 seconds according to the time；
Above-mentioned silicon chip is heated to 520 DEG C of heat treatments for carrying out 10 minutes by 1.3 removal masks；
1.4 after silicon chip naturally cools to room temperature, is performed etching with hydrofluoric acid solution, finally washes residue, obtains
Flow channel layer.
(2) it takes the high borosilicate slide that one piece of thickness is 800 μm as substrate, substrate surface is modified, surface band is made
There are a large amount of amino, and reacted in turn with difunctional connection unit (one end is carboxyl-reactive ester, and the other end is alkynyl), and further
5 ' the terminal modified primers for having azido group are fixed by " click " reaction, it is by similarly chemically reacting, surface modification is folded
The cadmiumsulfide quantum dot of nitrogen is fixed on substrate surface (being specially to be connect with the difunctional link unit of not connected primer), and formation can
Extend the positioning fluorescence of fluorescence localization for unimolecule；In subsequent experimentation, the positioning for being fixed on chip surface is glimmering
Light is after each extension, it is necessary first to measure the signal of the positioning fluorescence, then detect primer/template after extending again
The fluorescence of compound, and latter fluorescence is fluorescein of the label on sequencing reagent, i.e. fluorescence information on extension products.
(3) it takes the high borosilicate slide that one piece of thickness is 500 μm as cover board, stamps size, position and runner two on it
Hold the identical aperture of aperture；
(4) above-mentioned flow channel layer, substrate, cover board are cleaned with oxygen plasma, then utilizes dimethyl silicone polymer
Bonding effect pressing fit together, formed single-molecule sequencing chip.The single-molecule sequencing chip so formed, it is maximum
Feature is that chip surface secures fluorescent microsphere or quantum dot is used as positioning fluorescence to be needed so as to avoid before this
The 3 ends label positioning fluorescein of template to be measured, and the positioning fluorescein at 3 end of template to be measured is in fact marked, in sequencing procedure
In due to irradiating repeatedly, it is easy to be quenched, cause to measure and read length, it is average to read long there was only 25-30.And it of the present invention secures
Quantum dot either fluorescent microsphere single-molecule sequencing chip due to the fluorescence lifetime of quantum dot or fluorescent microsphere it is very long, so
Even if after repeated multiple times irradiation excitation, fluorescence is still not easy to be quenched, so single-molecule sequencing is read length and can be significantly increased.
The single-molecule sequencing chip is composed by three layers, and the flow cell of formation includes 5 runners (as shown in Figure 2), phase
The spacing of adjacent runner is 4mm, and the width of each runner is 5mm, length 8cm, and both ends are respectively equipped with an aperture, one of them
For first fluid entrance, another is exported for first fluid.The cadmiumsulfide quantum dot loaded on chip can be in sequencing procedure
Target dna unimolecule is positioned.Using this chip pair sequence to be measured carry out single-molecule sequencing, first by the sequence to be measured with
It is fixed on the primer hybridization of chip surface, is then prolonged with the reversible terminator nucleotide of four color fluorescent markers under the action of polymerase
Extend object, and the fluorescence signal by detecting extension products obtains the information of sequence to be measured.Fig. 4 is the primer first on sequence testing chip
Single molecular fluorescence photo after secondary extension, it is A that can read corresponding base in sequence to be measured according to fluorescence signal.It utilizes
Cadmiumsulfide quantum dot is positioned, and can be tracked with the monomolecular fluorescence of a batch DNA during reading base sequence one by one
Signal.And because the fluorescence property of cadmiumsulfide quantum dot is extremely stablized, even across repeated multiple times excitation, also it is not easy to quench
It goes out, so can get reliable and stable location information, so design can be avoided completely in 3 ends of template to be measured label positioning fluorescence
Element.And in document before, in the positioning fluorescein that 3 ends of template to be measured mark, it is easy to be quenched, be read so as to cause sequencing
Length, error rate are high.
Embodiment 2
A kind of single-molecule sequencing chip designs and prepares method, includes the following steps：
(1) it is 300 μm of slide to take one piece of thickness, is formed on its surface runner using photoetching process, photolithography steps are as follows：
Photoresist is evenly applied to surface of glass slide by 1.1 according to pre-designed flow cell runner pattern, forms thickness
For 400 μm of mask；
1.2 be 365nm with wavelength the above-mentioned slide for covering mask of ultraviolet light, illumination power be 20J/cm2, light
It it is 120 seconds according to the time；
Above-mentioned slide is heated to 550 DEG C of heat treatments for carrying out 10 minutes by 1.3 removal masks；
1.4 after slide naturally cools to room temperature, is performed etching with hydrofluoric acid solution, finally washes residue, obtains
Flow channel layer.
(2) it takes the quartz slide that one piece of thickness is 600 μm as substrate, substrate surface is modified, surface is made to carry
A large amount of amino, and further exist with difunctional connection unit (one end is carboxyl-reactive ester, and the other end is alkynyl) reaction in turn
5 ' the terminal modified primers for having nitrine are fixed by " click " reaction thereon, then by similarly chemically reacting click reactions,
Polystyrene fluorescent microsphere of the surface with azido group, the blue-fluorescence of a diameter of 50nm is fixed on chip surface；
(3) it takes the quartz slide that one piece of thickness is 200 μm as cover board, it is small in ends to beat size, position and runner on it
The identical aperture in hole；
(4) above-mentioned flow channel layer, substrate, cover board are cleaned with oxygen plasma, then utilizes the bonding of epoxy resin
Effect pressing fits together, and forms single-molecule sequencing chip.
The single-molecule sequencing chip is composed by three layers, and the flow cell of formation includes 12 runners, between adjacent channels
Away from for 2.5mm, the width of each runner is 4mm, length 10cm, and both ends are respectively equipped with an aperture, one of them is first
Fluid inlet, another is exported for first fluid.The blue-fluorescence polystyrene fluorescent microsphere loaded on chip is in sequencing procedure
In target dna unimolecule can be positioned (as shown in Figure 3).Unimolecule survey is carried out using this chip pair sequence to be measured
Sequence, first by the sequence and the primer hybridization for being fixed on chip surface, then under the action of polymerase fluorescent marker it is reversible
Terminating nucleotide extension primer, the fluorescence signal by detecting extension products obtain the information of sequence to be measured.Fig. 5 is sequence testing chip
On primer second extend the single molecular fluorescence photo after experiment, can be read according to fluorescence signal corresponding in sequence to be measured
Base is T.It is positioned, can be tracked with a batch during reading base sequence one by one using polystyrene fluorescent microsphere
The monomolecular fluorescence signals of DNA.In the present embodiment, it is positioned using polystyrene fluorescent microsphere, is existed as positioning fluorescence
In extension, template/primer complex is positioned, then detects the fluorescence letter for the reversible terminator for participating in extending again
Breath.In this experiment, we have found very happily, and the performance for positioning fluorescence is highly stable, by repeatedly irradiating, fluorescent microsphere
Fluorescence is still highly stable, and intensity does not reduce, and so design can be avoided completely in 3 ends of template to be measured label positioning fluorescence
Element.
Embodiment 3
A kind of single-molecule sequencing chip designs and prepares method, includes the following steps：
(1) it is 200 μm of silicon chip to take one piece of thickness, is formed on its surface runner using photoetching process, photolithography steps are as follows：
Photoresist is evenly applied to silicon chip surface by 1.1 according to pre-designed flow cell runner pattern, forms thickness
For 600 μm of mask；
1.2 be 248nm with wavelength the above-mentioned silicon chip for covering mask of ultraviolet light, illumination power be 20J/cm2, light
It it is 60 seconds according to the time；
Above-mentioned silicon chip is heated to 580 DEG C of heat treatments for carrying out 8 minutes by 1.3 removal masks；
1.4 after silicon chip naturally cools to room temperature, is performed etching with hydrofluoric acid solution, finally washes residue, obtains
Flow channel layer.
(2) it takes the quartz slide that one piece of thickness is 900 μm as substrate, substrate surface is modified, surface is made to carry
A large amount of active ester groups, and reacted in turn with difunctional connection unit (one end is amino, and the other end is nitrine), and further lead to
It crosses and 5 ' the terminal modified primers for having alkynyl is fixed by " click " reaction on it, then is anti-by similarly chemically reacting click
It answers, CdSe quantum dots of the surface with alkynyl, which are fixed on chip, to be shown to form the single-molecule sequencing that can be used for fluorescence localization
Chip (is reacted by click and connects quantum dot in substrate surface)；Single-molecule sequencing is carried out on the chip, it will from this
The way in 3 ends of template to be measured label positioning fluorescein is taken leave of, and due to the high stability of quantum dot fluorescence performance, and makes list
The reading length of molecule sequencing significantly improves.
(3) it takes the quartz slide that one piece of thickness is 400 μm as cover board, it is small in ends to beat size, position and runner on it
The identical aperture in hole；
(4) above-mentioned flow channel layer, substrate, cover board are cleaned with oxygen plasma, the bonding of polyurethane is then utilized to make
It is fitted together with pressing, forms single-molecule sequencing chip.
The single-molecule sequencing chip is composed by three layers, and the flow cell of formation includes 8 runners, the spacing of adjacent channels
For 3mm, the width of each runner is 6mm, length 9cm, and both ends are respectively equipped with an aperture, one of them is fluid inlet,
Another is fluid outlet.The CdSe quantum dots loaded on chip can join target dna unimolecule in sequencing procedure
It is positioned with primer/template composite after extension.Unimolecule survey is carried out using four different sequences to be measured of this chip pair
Sequence, first by four sequences and the primer hybridization for being fixed on chip surface, then under the action of polymerase fluorescent marker can
Inverse terminating nucleotide is extended in chip surface, by detecting fluorescence signal of the label on sequencing reagent on extension products
The information of sequence to be measured is obtained, it should be noted that the label in each base primers/template composite is on sequencing reagent
Fluorescence signal before, be required to be fixed on chip surface quantum dot fluorescence carry out exciting irradiation, to obtain primer/mould
The location information of plate.Fig. 6 is that the primer on sequence testing chip extends the single molecular fluorescence photo after experiment for the first time, is believed according to fluorescence
It is respectively A (sequence 1), C (sequence 2), T (sequence 3), G (sequence 4) that number can read corresponding base in four sequences to be measured.
It is positioned, can be tracked during reading base sequence one by one monomolecular with a batch DNA using CdSe quantum dots
Fluorescence signal.And because the fluorescence property of quantum dot is extremely stablized, even across repeated multiple times excitation, also it is not easy to be quenched,
So can get reliable and stable location information, so design can be avoided completely in 3 ends of template to be measured label positioning fluorescein.
Embodiment 4
A kind of single-molecule sequencing chip designs and prepares method, includes the following steps：
(1) it is 400 μm of slide to take one piece of thickness, is formed on its surface runner using photoetching process, photolithography steps are as follows：
Photoresist is evenly applied to surface of glass slide by 1.1 according to pre-designed flow cell runner pattern, forms thickness
For 500 μm of mask；
1.2 be 248nm with wavelength the above-mentioned slide for covering mask of ultraviolet light, illumination power be 15J/cm2, light
It it is 110 seconds according to the time；
Above-mentioned slide is heated to 560 DEG C of heat treatments for carrying out 10 minutes by 1.3 removal masks；
1.4 after above-mentioned slide naturally cools to room temperature, are performed etching with hydrofluoric acid solution, finally wash residue,
Obtain flow channel layer.
(2) it takes the quartz slide that one piece of thickness is 700 μm as substrate, substrate surface is modified, surface is made to carry
A large amount of alkynyls fix 5 ' the terminal modified primers (Fig. 7) for having nitrine by " click " reaction on it, then anti-with same chemistry
Answer green fluorescence polystyrene fluorescent microsphere (Fig. 7) of the Random Load surface with azido group, a diameter of 30nm on it；It should
Fluorescent microsphere is fixed on chip and shows that be formed in single-molecule sequencing can be used for determining primer/template composite in the process
The fluorescent marker of position.
(3) it takes the quartz slide that one piece of thickness is 300 μm as cover board, it is small in ends to beat size, position and runner on it
The identical aperture in hole；
(4) above-mentioned flow channel layer, substrate, cover board are cleaned with oxygen plasma, then utilizes dimethyl silicone polymer
Bonding effect pressing fit together, formed single-molecule sequencing chip.
The single-molecule sequencing chip is composed by three layers, and the flow cell of formation includes 10 runners, between adjacent channels
It is 5mm away from the width for 2.5mm, each runner, length 10cm, depth is 200 μm, and both ends are respectively equipped with an aperture,
In one be fluid inlet, another is fluid outlet.The green fluorescence polystyrene fluorescent microsphere loaded on chip is being sequenced
Target dna unimolecule can be positioned in the process.Single-molecule sequencing is carried out using this chip pair sequence to be measured, first should
Sequence and the primer hybridization for being fixed on chip surface, then under the action of polymerase fluorescent marker reversible terminator nucleotide
Primer/template composite of formation is extended, the fluorescence signal by detecting extension products obtains the information of sequence to be measured,
It should be noted that before fluorescence signal of the label in each base primers/template composite on sequencing reagent, it is both needed to
Exciting irradiation is carried out to the quantum dot fluorescence for being fixed on chip surface, to obtain the location information of primer/template.Using green
Color fluorescence polystyrene fluorescent microsphere is positioned, and can be tracked during reading base sequence one by one mono- with a batch DNA
The fluorescence signal of molecule.And because the fluorescence property of green fluorescence polystyrene fluorescent microsphere is extremely stablized, even across anti-
Multiple repeatedly excitation, is also not easy to be quenched, so reliable and stable location information is can get, so as to be avoided completely in template to be measured
3 ends label positioning fluorescein.
Embodiment 5
A kind of single-molecule sequencing chip designs and prepares method, includes the following steps：
(1) it is 500 μm of silicon chip to take one piece of thickness, is formed on its surface runner using photoetching process, photolithography steps are as follows：
Photoresist is evenly applied to silicon chip surface by 1.1 according to pre-designed flow cell runner pattern, forms thickness
For 250 μm of mask；
1.2 be 365nm with wavelength the above-mentioned silicon chip for covering mask of ultraviolet light, illumination power be 25J/cm2, light
It it is 100 seconds according to the time；
Above-mentioned silicon chip is heated to 580 DEG C of heat treatments for carrying out 9 minutes by 1.3 removal masks；
1.4 after above-mentioned silicon chip naturally cools to room temperature, are performed etching with hydrofluoric acid solution, finally wash residue,
Obtain flow channel layer.
(2) it takes the high borosilicate slide that one piece of thickness is 900 μm as substrate, substrate surface is modified, surface band is made
There is the albumen that can be combined with biotin, fixes 5 ' the terminal modified primers for having biotin, then random carrier surface band simultaneously on it
There are the red fluorescence polystyrene fluorescent microsphere of biotin, a diameter of 20nm；
(3) it takes the quartz slide that one piece of thickness is 500 μm as cover board, it is small in ends to beat size, position and runner on it
The identical aperture in hole；
(4) above-mentioned flow channel layer, substrate, cover board are cleaned with oxygen plasma, then utilizes the bonding of epoxy resin
Effect pressing fits together, and forms single-molecule sequencing chip.
The single-molecule sequencing chip is composed by three layers, and the flow cell of formation includes 12 runners, between adjacent channels
It is 4mm away from the width for 3.5mm, each runner, length 10cm, depth is 180 μm, and both ends are respectively equipped with an aperture,
In one be fluid inlet, another is fluid outlet.The red fluorescence polystyrene fluorescent microsphere loaded on chip is being sequenced
Target dna unimolecule can be positioned in the process.And because of the fluorescence property of red fluorescence polystyrene fluorescent microsphere
Extremely stablize, even across repeated multiple times excitation, be also not easy to be quenched, so can get reliable and stable location information.
Embodiment 6
A kind of single-molecule sequencing chip designs and prepares method, includes the following steps：
(1) it is 400 μm of potsherd to take one piece of thickness, is formed on its surface runner using photoetching process, photolithography steps are such as
Under：
Photoresist is evenly applied to potsherd surface by 1.1 according to pre-designed flow cell runner pattern, is formed thick
The mask that degree is 550 μm；
1.2 be 248nm with wavelength the above-mentioned potsherd for covering mask of ultraviolet light, illumination power be 30J/cm2,
Light application time is 160 seconds；
Above-mentioned potsherd is heated to 570 DEG C of heat treatments for carrying out 7 minutes by 1.3 removal masks；
1.4 after above-mentioned potsherd naturally cools to room temperature, are performed etching with hydrofluoric acid solution, finally wash residual
Object obtains flow channel layer.
(2) it takes the quartz slide that one piece of thickness is 800 μm as substrate, substrate surface is modified, surface is made to carry
A large amount of imide groups fix 5 ' the terminal modified primers for having carboxyl, then Random Load on it by " click " reaction on it
Surface carries the zinc selenide quantum dot of carboxyl；
(3) it takes the quartz slide that one piece of thickness is 400 μm as cover board, it is small in ends to beat size, position and runner on it
The identical aperture in hole；
(4) above-mentioned flow channel layer, substrate, cover board are cleaned with oxygen plasma, the bonding of polyurethane is then utilized to make
It is fitted together with pressing, forms single-molecule sequencing chip.
The single-molecule sequencing chip is composed by three layers, and the flow cell of formation includes 15 runners, between adjacent channels
It is 3.5mm away from the width for 3mm, each runner, length 8cm, depth is 250 μm, and both ends are respectively equipped with an aperture, wherein
One is fluid inlet, another is fluid outlet.The zinc selenide quantum dot loaded on chip can be to mesh in sequencing procedure
Mark DNA unimolecules are positioned.And because the fluorescence property of zinc selenide quantum dot is extremely stablized, swash even across repeated multiple times
Hair, is also not easy to be quenched, so can get reliable and stable location information.
Embodiment 7
A kind of single-molecule sequencing chip designs and prepares method, includes the following steps：
(1) it is 400 μm of potsherd to take one piece of thickness, is formed on its surface runner using photoetching process, photolithography steps are such as
Under：
Photoresist is evenly applied to potsherd surface by 1.1 according to pre-designed flow cell runner pattern, is formed thick
The mask that degree is 550 μm；
1.2 be 248nm with wavelength the above-mentioned potsherd for covering mask of ultraviolet light, illumination power be 30J/cm2,
Light application time is 160 seconds；
Above-mentioned potsherd is heated to 570 DEG C of heat treatments for carrying out 7 minutes by 1.3 removal masks；
1.4 after above-mentioned potsherd naturally cools to room temperature, are performed etching with hydrofluoric acid solution, finally wash residual
Object obtains flow channel layer.
(2) it takes the quartz slide that one piece of thickness is 800 μm as substrate, substrate surface is modified, surface is made to carry
A large amount of imide groups fix 5 ' the terminal modified primers for having carboxyl, then Random Load on it by " click " reaction on it
Surface carries the ZnS quantum dots of carboxyl；
(3) it takes the quartz slide that one piece of thickness is 400 μm as cover board, it is small in ends to beat size, position and runner on it
The identical aperture in hole；
(4) above-mentioned flow channel layer, substrate, cover board are cleaned with oxygen plasma, the bonding of polyurethane is then utilized to make
It is fitted together with pressing, forms single-molecule sequencing chip.
The single-molecule sequencing chip is composed by three layers, and the flow cell of formation includes 15 runners, between adjacent channels
It is 3.5mm away from the width for 3mm, each runner, length 8cm, depth is 250 μm, and both ends are respectively equipped with an aperture, wherein
One is fluid inlet, another is fluid outlet.The ZnS quantum dots loaded on chip can be to mesh in sequencing procedure
Mark DNA unimolecules are positioned.And because the fluorescence property of ZnS quantum dots is extremely stablized, swash even across repeated multiple times
Hair, is also not easy to be quenched, so can get reliable and stable location information.
Embodiment 8
Use the single-molecule sequencing chip pair DNA template sequences to be measured (SEQ ID No.1) 5 '-described in embodiment 1
CTACGTTCGAACTACTAACTTGATGTAGCTTCGTAGTAATTTTTTTTTTTTTTTTT T TT-3 ' carry out unimolecule survey
Sequence.First the template sequence to be measured is incubated 5 minutes with the primer for being fixed on chip surface at 65 DEG C and is hybridized, is then existed
With the reversible terminator nucleotide extension primer of four kinds of different fluorescent markers, extension time it is 15 under the action of archaeal dna polymerase
Minute, temperature is 37 DEG C.The letter of sequence to be measured is can be obtained after extension by detecting the fluorescence signal of extension products
Breath.It should be noted that before fluorescence signal of the label in each base primers/template composite on sequencing reagent,
It needs to carry out exciting irradiation to the quantum dot fluorescence for being fixed on chip surface, to obtain the location information of primer/template.Fig. 4
It is single molecular fluorescence photo of the primer on sequence testing chip after once extending, sequence to be measured can be read according to fluorescence signal
Upper corresponding base is A.It is positioned using the cadmiumsulfide quantum dot of chip surface, it can be in the mistake for reading base sequence one by one
It is tracked with the monomolecular fluorescence signals of a batch DNA in journey.As a result display is recycled by 40 sequencings, that is, is read length and reached 40, own
Sequence to be measured is correct, and the serial read of mistake is not observed.Single-molecule sequencing further the result shows that, use cadmium sulfide amount
Single-molecule sequencing chip of the son point as positioning fluorescence, the sequencing reading length is up to 50, and error rate only has 0.2%.
The method that the chip carries out single-molecule sequencing, includes the following steps：
B1, template solution to be measured is added in runner by second fluid entrance, is existed with the primer for being fixed on substrate surface
It incubates 5 minutes and is hybridized at 65 DEG C, exported by second fluid and extract substrate surface residual liquid out；
B2, by second fluid entrance by the reversible terminator nucleotide of the different bases of four kinds of different fluorescent markers, polymerization
Enzyme, buffer solution mixture be added in runner, carry out extension under the action of polymerase, the time is 15 minutes, and temperature is
37℃；
After B3, extension, buffer solution for cleaning runner is injected from second fluid entrance, exports and extracts out from second fluid
Residual liquid after cleaning；
B4, using suitable wavelength exciting light exposure label(l)ing substrate surface positioning fluorescent marker, to participation prolong
The primer stretched/template composite carries out fluorescence localization, then irradiates drawing after participating in extending with the exciting light of other wavelength again
Object/template composite, the fluorescence by participating in extension products (what is detected at this time is the fluorescein marked on sequencing reagent) are believed
Number recognizable corresponding base to be measured；It should be noted that the label in each base primers/template composite is tried in sequencing
Before fluorescence signal in agent, it is required to carry out exciting irradiation to the quantum dot fluorescence for being fixed on chip surface, to be drawn
The location information of object/template；
Cleavage reagent solution is added dropwise in B5, the substrate surface in runner bottom, is reacted 5 minutes at 37 DEG C, then protection is added dropwise
The reaction was continued 3 minutes for agent solution, injects buffer solution for cleaning runner from second fluid entrance, after second fluid exports extraction cleaning
Residual liquid；
B6, above step is repeated, the sequencing to template to be measured can be completed.
Embodiment 9
Use the single-molecule sequencing chip pair template sequence to be measured (SEQ ID No.2) 5 '-described in embodiment 2
CTACGTTCGAACTACTAAGCAATCCGGCAGATCGTCACTTTTTTTTTTTTTTTTTT TTT-3 ' carry out unimolecule survey
Sequence.First the sequence is incubated 5 minutes with the primer for being fixed on chip surface at 65 DEG C and is hybridized, then in the work of polymerase
With the lower reversible terminator nucleotide extension primer with four kinds of different fluorescent markers, the extension time is 15 minutes, temperature 37
℃.Then the cleavage reagent solution of 500mM is added dropwise in chip surface, is reacted 5 minutes at 37 DEG C, then the protection of 500mM is added dropwise
The reaction was continued 3 minutes for agent solution.The step of removing chip surface residual liquid, repeating first time extension, obtains second
The product of secondary extension.Each time sequence to be measured is can be obtained by detecting the fluorescence signal of extension products after extension
Information.It should be noted that before fluorescence signal of the label in each base primers/template composite on sequencing reagent,
It is required to carry out exciting irradiation to the quantum dot fluorescence for being fixed on chip surface, to obtain the location information of primer/template.Figure
5 be primer on sequence testing chip in the single molecular fluorescence photo after extending twice, can be read according to fluorescence signal to be measured
Second base is C in sequence.It is positioned using the blue-fluorescence polystyrene fluorescent microsphere of chip surface, it can be one by one
It is tracked with the monomolecular fluorescence signals of a batch DNA during reading base sequence.In the present embodiment, glimmering using polystyrene
Light microballoon is positioned, and as positioning fluorescence in extension, is positioned to template/primer complex, is then detected again
Participate in the fluorescence information of the reversible terminator extended.In this experiment, we have found very happily, position the performance of fluorescence very
Stablize, have passed through more than 50 irradiation, the fluorescence of fluorescent microsphere is still highly stable, so completely avoids the 3 of template to be measured
End label positioning fluorescein, it is final the experimental results showed that, sequencing reading length is up to 78, error rate 0.2%.It is expected that being joined by testing
Several optimization, error rate have improved space with length is read.
Embodiment 10
Use the different template sequence to be measured of the single-molecule sequencing chip pair four described in embodiment 3
5 '-CTACGTTCGAACTACTAACTTGATGTAGCTTCGTAGTAATTTTTTTTTTTTTTTTT T TT-3 ' (are waited for
Row 1, SEQ ID No.1 is sequenced),
5 '-CTACGTTCGAACTACTAATGGCCAACTTTAGGTACAGGCTTTTTTTTTTTTTTTTT TTT-3 ' (are waited for
Row 2, SEQ ID No.3 is sequenced),
5 '-CTACGTTCGAACTACTAAGCAATCCGGCAGATCGTCACTTTTTTTTTTTTTTTTTT TTT-3 ' (are waited for
Row 3, SEQ ID No.2 is sequenced),
5 '-CTACGTTCGAACTACTAAAACTGGTACAGCCAACGTCTGTTTTTTTTTTTTTTTTT TTT-3 ' (are waited for
Row 4, SEQ ID No.4 is sequenced)
Carry out single-molecule sequencing.First by four not homotactic templates and to be fixed on the primer of chip surface warm at 65 DEG C
It educates 5 minutes and is hybridized, then drawn with the reversible terminator nucleotide extension of four kinds of different fluorescent markers under the action of polymerase
Object, extension time are 15 minutes, and temperature is 37 DEG C.It is by detecting the fluorescence signal of extension products after extension
The information of sequence to be measured can be obtained.It should be noted that the label in each base primers/template composite is in sequencing reagent
On fluorescence signal before, be required to be fixed on chip surface quantum dot fluorescence carry out exciting irradiation, so as to obtain primer/
The location information of template.Fig. 6 is primer on sequence testing chip in the single molecular fluorescence photo after once extending, according to fluorescence
It is respectively A (sequence 1), C (sequence 2), T (sequence 3), G (sequences that signal, which can read corresponding base in four sequences to be measured,
4).It is positioned, can be tracked with a batch DNA unimolecules during reading base sequence one by one using CdSe quantum dots
Fluorescence signal.And because the fluorescence property of CdSe quantum dots is extremely stablized, even across repeated multiple times excitation, do not allow yet
It is easily quenched, so can get reliable and stable location information.In the present embodiment, preliminary experimental results show that sequencing reading length can
Up to 50, and error rate only has 0.2%.
Embodiment 11
Using a single-molecule sequencing chip pair DNA of embodiment 4-7 template sequences to be measured 5 '-
CTACGTTCGAACTACTAACTTGATGTAGCTTCGTAGTAATTTTTTTTTTTTTTTTT T TT-3 ' carry out unimolecule survey
Sequence, sequencing approach are identical with embodiment 8.Its result also shows result similar to Example 8.
Embodiment 12
The reading length and accuracy being sequenced using the single-molecule sequencing chip of embodiment 1-7 are verified：
After 49 bases on continuously having read template sequence to be measured, the 50th extension, acquired results have been carried out
As shown in Figure 8.Fluorescence signal in template to be measured can read (bright spot), illustrate to be sequenced using the single-molecule sequencing chip
Reading length can at least reach 50.Fluorescence photo shares 44 template sequences within sweep of the eye, has carried out 50 sequencings altogether and has followed
Ring, the number for adding up mistake occur is 9 times altogether, and accuracy rate is 1-9/ (44*50)=99.6%.
Comparative example 1
The single-molecule sequencing chip of this comparative example and embodiment 12 is essentially identical, the difference is that only：In this comparative example,
The step (2) is not in sequence testing chip surface Random Load as the cadmiumsulfide quantum dot for positioning fluorescence.
The chip of comparative example 1 is used for DNA template sequences to be measured (SEQ ID No.1) 5 '-
CTACGTTCGAACTACTAACTTGATGTAGCTTCGTAGTAATTTTTTTTTTTTTTTTT T TT-3 ' carry out unimolecule survey
Sequence.As a result,：Sequencing reading length only has 20, and error rate is up to 5%.
Specific embodiments of the present invention are described above.It is to be appreciated that the invention is not limited in above-mentioned
Particular implementation, those skilled in the art can make a variety of changes or change within the scope of the claims, this not shadow
Ring the substantive content of the present invention.In the absence of conflict, the feature in embodiments herein and embodiment can arbitrary phase
Mutually combination.
Sequence table
<110>Shanghai Communications University
<120>A kind of DNA single-molecule sequencings chip and preparation method thereof
<130> DAG36019
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 59
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 1
ctacgttcga actactaact tgatgtagct tcgtagtaat tttttttttt ttttttttt 59
<210> 2
<211> 59
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 2
ctacgttcga actactaagc aatccggcag atcgtcactt tttttttttt ttttttttt 59
<210> 3
<211> 59
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 3
ctacgttcga actactaatg gccaacttta ggtacaggct tttttttttt ttttttttt 59
<210> 4
<211> 59
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 4
ctacgttcga actactaaaa ctggtacagc caacgtctgt tttttttttt ttttttttt 59
Claims (10)
1. a kind of single-molecule sequencing chip, which is characterized in that including runner plate, substrate and cover board, the runner plate is arranged in base
Between plate and cover board；Flow cell is set on the runner plate, and the flow cell includes multiple runners disposed in parallel；The circulation
Substrate is arranged in the top setting cover board in pond, the lower section of flow cell；The bottom of the runner is connected to substrate surface.
2. single-molecule sequencing chip according to claim 1, which is characterized in that the table of the substrate contacted with runner plate
Sequencing primer and positioning fluorescent marker are fixed in face.
3. according to claim 2 single-molecule sequencing chip, which is characterized in that the primer is 5 ' to repair for-N3 or 5 '-alkynyls
The primer of decorations；Positioning one kind that fluorescent marker is in fluorescence quantum, nanometer carbon dots and fluorescent microsphere.
4. single-molecule sequencing chip according to claim 3, which is characterized in that the fluorescence quantum surface carries energy
The functional group for reacting to each other with the group of substrate surface or substance or being combined with non-covalent bond；The fluorescent microsphere is diameter
The polystyrene fluorescent microsphere of 20-100nm, polystyrene fluorescent microsphere surface carries can be with the group or substance phase of substrate surface
Mutual reactance or the functional group combined with non-covalent bond.
5. according to claim 2-4 any one of them single-molecule sequencing chips, which is characterized in that fixed in the substrate surface
The method of sequencing primer and positioning fluorescent marker includes the following steps：
A1, substrate surface is activated and is modified, substrate surface is made to carry reactive functional；
A2, sequencing primer is connect with the substrate surface after modification using water-soluble difunctional connection unit；
A3, positioning fluorescent marker is connect with the substrate surface after modification using water-soluble difunctional connection unit.
6. single-molecule sequencing chip according to claim 1 or 2, which is characterized in that the middle part of the runner is cuboid
Type, both ends are tapered, and first fluid entrance and first fluid outlet is respectively set in both ends；
On the cover board position corresponding with first fluid entrance and first fluid outlet be respectively arranged with second fluid entrance and
Second fluid exports, and the first fluid entrance is connected to second fluid entrance, and second fluid outlet connects with second fluid outlet
It is logical；
The runner quantity is 2-16, and each width of flow path is 2-8mm, length 5-10cm, the spacing of adjacent channels
For 2-5mm.
7. single-molecule sequencing chip according to claim 1, which is characterized in that the thickness of the substrate is 500-1000 μ
The thickness of m, the cover board are 100-500 μm；
The material of the runner plate is one kind in silicon chip, glass or ceramics；The material of the substrate and cover board is quartz slide
Or high borosilicate slide.
8. single-molecule sequencing chip according to claim 1, which is characterized in that the runner is prepared using photoetching process, tool
Body includes the following steps：
S1, photoresist is evenly applied to by flow field plate surfaces according to runner pattern, forms mask；
S2, the runner plate that mask is covered using ultraviolet light, are then removed mask, are heat-treated；
S3, it after being cooled to room temperature the runner plate after heat treatment, performs etching, then wash residue, that is, forms runner.
9. a kind of preparation method of single-molecule sequencing chip according to claim 1, which is characterized in that including following step
Suddenly：
After runner plate, substrate and cover board are cleaned, it is pressed to get the unimolecule by assembling using adhesive in order
Sequence testing chip.
10. a kind of method carrying out single-molecule sequencing using chip described in claim 1, which is characterized in that including following step
Suddenly：
B1, template solution to be measured is added in runner by second fluid entrance, and is fixed on the primer of substrate surface at 65 DEG C
Lower incubate 5 minutes is hybridized, and is exported by second fluid and is extracted substrate surface residual liquid out；
B2, by second fluid entrance by the reversible terminator nucleotides of fluorescein-labeled different bases, polymerase, buffer solution
Mixture is added in runner, and extension is carried out under the action of polymerase, and the time is 15 minutes, and temperature is 37 DEG C；
After B3, extension, buffer solution for cleaning runner is injected from second fluid entrance, extraction cleaning is exported from second fluid
Residual liquid afterwards；
B4, using suitable wavelength exciting light exposure label(l)ing substrate surface positioning fluorescent marker, to participate in extend
Primer/template composite carries out fluorescence localization, then irradiates primer/mould after participating in extending with the exciting light of other wavelength again
Plate compound, the recognizable corresponding base to be measured of fluorescence signal by participating in extension products；
Cleavage reagent solution is added dropwise in B5, the substrate surface in runner bottom, is reacted 5 minutes at 37 DEG C, then that protective agent is added dropwise is molten
The reaction was continued 3 minutes for liquid, injects buffer solution for cleaning runner from second fluid entrance, is remained after exporting extraction cleaning from second fluid
Liquid；
B6, above step is repeated, the sequencing to template to be measured can be completed.
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| CN109852679A (en) * | 2019-03-01 | 2019-06-07 | 赛纳生物科技(北京)有限公司 | A kind of gene sequencing chip recognition methods |
| CN109852679B (en) * | 2019-03-01 | 2022-08-26 | 赛纳生物科技(北京)有限公司 | Gene sequencing chip identification method |
| CN110092592A (en) * | 2019-04-17 | 2019-08-06 | 华南理工大学 | A kind of activation alkynes modification of surfaces glass material and surface modifying method for biomolecule fixation |
| WO2021018080A1 (en) * | 2019-07-31 | 2021-02-04 | 齐鲁工业大学 | Method for preparing terminal base flow fluorescent sequencing microspheres and use thereof |
| CN112195227A (en) * | 2020-09-18 | 2021-01-08 | 赛纳生物科技(北京)有限公司 | Preparation method of gene sequencing substrate |
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