CN109709185A - A kind of the nano-pore detection device and production method of modified biological probe - Google Patents
A kind of the nano-pore detection device and production method of modified biological probe Download PDFInfo
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- CN109709185A CN109709185A CN201711013614.3A CN201711013614A CN109709185A CN 109709185 A CN109709185 A CN 109709185A CN 201711013614 A CN201711013614 A CN 201711013614A CN 109709185 A CN109709185 A CN 109709185A
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- 239000011148 porous material Substances 0.000 title claims abstract description 78
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- 238000001514 detection method Methods 0.000 title claims abstract description 53
- 238000004519 manufacturing process Methods 0.000 title claims abstract description 14
- 102000004169 proteins and genes Human genes 0.000 claims abstract description 20
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 20
- 201000010099 disease Diseases 0.000 claims abstract description 11
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 claims abstract description 11
- 239000000090 biomarker Substances 0.000 claims abstract description 6
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- 229920001184 polypeptide Polymers 0.000 claims description 5
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- 102100030684 Sphingosine-1-phosphate phosphatase 1 Human genes 0.000 claims description 3
- 101710168942 Sphingosine-1-phosphate phosphatase 1 Proteins 0.000 claims description 3
- 238000005267 amalgamation Methods 0.000 claims description 3
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- 230000012743 protein tagging Effects 0.000 claims description 3
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Abstract
The invention discloses a kind of nano-pore detection devices of modified biological probe, including device noumenon, described device body interior is provided with the first basal layer, first basal layer offers at least one micropore, along the axial direction of the micropore, it is provided with the nano pore for being modified with probe at the top of the micropore, the micropore bottom is provided with the second basal layer with protein nano hole array.When detection, the nano pore for being modified with probe can capture specific biomarkers, generate specific curent change, by whether containing object to be detected and concentration in analysis curent change, that is, samples can be analyzed, by modifying specific bioprobe to nano pore, detection range is greatly expanded, and there is very high specificity and sensitivity, can be widely applied to disease detection and analysis based on nano-pore technology.The production method of the detection device is also disclosed, production method is simple, low in cost, and obtained device is easy to carry about with one, and can detect immediately.
Description
Technical field
The invention belongs to nano-pore detection technique field, it is related to the production method of a kind of nano-pore detection device and device,
Relate in particular to a kind of nano-pore detection device and production method for being modified with bioprobe.
Background technique
Nano-pore technology is a kind of new unimolecular detection technique, and the basic principle of nano-pore technology is: nano-pore is located at
In the antiseepage film of two electrolyte liquor chambers, when voltage acts on electrolyte liquor chamber, the steady state ion electric current across hole is just formed, in aperture
The instant of ionic flux changes so that large biological molecule is distributed on film, therefore detects and make molecular recognition by the electric current in aperture
It is possibly realized.Be electrolysed indoor charged molecule sample (such as DNA and protein) hole pass in and out or duct with nanometer scale on
Specific probe when combining, generate a series of fluctuations of ion current signal, the fluctuation of these signals reflect sample perhaps
More characteristics, such as molecular size, concentration and structure.Grasp the size of nano-pore, its surface characteristic, application voltage and electrolyte
Condition, so that it may which different types of biomolecule is detected by appropriate adjustment nano-pore.In the past decade, nano-pore technology has been
It is widely used in the research in terms of DNA sequencing, disease detection, drug screening and environmental monitoring.
Nano-pore can be divided into solid nano hole and biological nano hole, and biological nano hole refers to that be present in biology intracorporal various
The nano-pore and nanochannel of various kinds, the protein channel of the nanotubes specifically formed in biological lipid film, they are
Connect biology interior and the external approach for carrying out energy, mass exchange.Solid nano hole be using physics, chemical method drilling or
The nanoscale channel for etching solid substrate to prepare.Wherein, biological nano hole is received in terms of modifiability and cost compared with solid-state
Metre hole tool has great advantage, but it has that the stability to test environment, electric current and noise requirements are relatively high.And consolidate
State nano-pore has a significant advantage at the integrated aspect of stability, electric current, noise and technique, but presently, there are be difficult to modify
Technical bottleneck, while its cost is high.
Currently, nano-pore technology has been achieved for some breakthrough progress in terms of being applied to DNA sequencing, but in disease
Sick context of detection, when facing complicated and diversified biological sample, there are no substantive breakthroughs, this is because lacking at present a kind of available
In the effective protein modified structure for capturing related disease molecule, on the other hand current nano-pore detection technique not yet can be with
High-throughput nano-pore chip combines, so that Monitoring lower-cut is still higher, detection sensitivity is insufficient, and existing nanometer
Hole detection device volume is larger, inconvenient to carry.
Summary of the invention
For this purpose, technical problem to be solved by the present invention lies in Conventional nano hole detection techniques in terms of disease detection still not
It is able to satisfy demand, Monitoring lower-cut is higher, sensitivity is insufficient, and detection device is not portable, to propose that one kind can be used for disease inspection
It surveys, Monitoring lower-cut is low, high sensitivity, the nano-pore detection device and production method of portable modified biological probe.
In order to solve the above technical problems, the technical solution of the present invention is as follows:
The present invention provides a kind of nano-pore detection device of modified biological probe comprising device noumenon, described device sheet
Body is internally provided with the first basal layer, and first basal layer offers at least one micropore, described in the axial direction along the micropore
It is provided with the nano pore for being modified with probe at the top of micropore, the micropore bottom is provided with second with protein nano hole array
Basal layer.
Preferably, upper cell cavity is formed at the top of first basal layer between described device ontology inner top surface, it is described on
Microfluidic module is provided in cell cavity.
Preferably, being provided with indicator electrode in the microfluidic module, voltage is provided with below first basal layer
Electrode, the voltage electrode are arranged in a one-to-one correspondence with the micropore.
Preferably, the voltage electrode is connected to CMOS chip.
Preferably, the aperture of the micropore is 2-200 μm, the height of the micropore is 5-500 μm；The nano-pore battle array
The aperture of nano-pore is 1-100nm in column.
Preferably, the nano pore for being modified with probe is set to third basal layer, the third basal layer is to change
Learn polymer film.
Preferably, described device bodies top is also provided with liquid flow inlet and fluid outlet.
The present invention also provides the production methods of the nano-pore detection device described in one kind comprising following steps:
S1, to nano pore modified biological probe, can specific capture disease biomarker using protein fusion method
Probe amalgamation and expression in the end or inside in protein nano duct, obtain the nano pore for being modified with probe；
S2, assemble nanometer hole detection device inject the conducting solution containing lipid or chemical multimer into device noumenon,
The lipid or chemical multimer are laid into film at the top of micropore, are then injected into and are modified with probe containing what the step S1 was obtained
Nano pore conducting solution, to get to inspection in the film for being embedded into the nano pore for being modified with probe at the top of micropore
Survey device；
Preferably, further including step S3, test sample, the biological sample for needing to analyze is injected into described device ontology,
Biological sample molecule be modified on micropore in conjunction with the nano pore of probe, generate nonspecific signal, pass through the analysis special letter
Number, analyze sample composition and concentration.
Preferably, the protein nano duct is Phi29 connector albumen duct, SPP1, T3, T4, T5, T7 phagocytosis
One of body connector albumen duct, alpha-hemolysin, MspA albumen duct；The probe be polypeptide, antibody or
One kind of antibody variable region；After the step S2 will be modified in the film at the top of the nano pore insertion micropore of probe, further include
The step of controlling direction and the size, limitation more multi-pore channel insertion of voltage.
The above technical solution of the present invention has the following advantages over the prior art:
(1) the nano-pore detection device of modified biological probe of the present invention, including device noumenon, described device ontology
It is internally provided with the first basal layer, first basal layer offers at least one micropore, described micro- along the axial direction of the micropore
It is provided with the nano pore for being modified with probe at the top of hole, the micropore bottom is provided with the second base with protein nano hole array
Bottom.When detection, the nano pore for being modified with probe can capture specific biomarkers, generate specific curent change,
It is specific by being modified to nano pore by whether containing object to be detected and concentration in analysis curent change, that is, samples can be analyzed
Bioprobe greatly expands detection range, and has very high specificity and sensitivity, can be widely applied to based on nano-pore
The disease detection and analysis of technology.
(2) the nano-pore detection device of modified biological probe of the present invention, at the top of first basal layer with it is described
Upper cell cavity is formed between device noumenon inner top surface, the upper storage reservoir is intracavitary to be provided with microfluidic module.The microfluidic module is available
In replacement conducting liquid, formed chemical polymerization object film and insertion duct, liquid it is replaceable, expand detection device apply model
It encloses, while reducing its use cost.
(3) the nano-pore detection device of modified biological probe of the present invention is provided with finger in the microfluidic module
Show electrode, be provided with voltage electrode below first basal layer, the voltage electrode is arranged in a one-to-one correspondence with the micropore, inspection
When survey, each micropore can be detected individually, greatly reduce Monitoring lower-cut, while have cheap testing cost and shorter
Detection time.
(4) production method of the nano-pore detection device of modified biological probe of the present invention, including to protein nano
Modification particular organisms probe in duct can detect after the nano pore for being modified with bioprobe is prepared at the top of micropore,
Molecules detected can be evenly distributed at the top of each micropore, in conjunction with the protein nano duct for being modified with probe on micropore, be generated
Specific signals determine whether the concentration containing detectable substance and detectable substance, device preparation process letter by analyzing these signals
Single, production cost is low, and detection device detection range obtained is wide, specificity and high sensitivity, the generally portable chip of device
Shape structure, is easy to carry about with one, and can be detected immediately.
Detailed description of the invention
In order to make the content of the present invention more clearly understood, it below according to specific embodiments of the present invention and combines
Attached drawing, the present invention is described in further detail, wherein
Fig. 1 is the nano-pore structure of the detecting device schematic diagram of modified biological probe described in the embodiment of the present invention.
Appended drawing reference indicates in figure are as follows: 1- device noumenon；The first basal layer of 2-；3- micropore；4- is modified with the nanometer of probe
Duct；The second basal layer of 5-；6- indicator electrode；7- third basal layer；8- liquid flow inlet；9- fluid outlet；10- microfluid mould
Block.
The present invention can be implemented with many different forms, should not be construed as limited to embodiment set forth herein, on the contrary,
These embodiments are provided, so that the disclosure is thorough and complete, and will fully convey the concept of the invention to art technology
Personnel, the present invention will be defined by the claims.In the accompanying drawings, for clarity, the size of each device and opposite can be exaggerated
Size.Description of the invention and term " first " in claims and attached drawing, " second " etc. are for distinguishing similar right
As without being used to describe a particular order or precedence order.It should be understood that the data used in this way in the appropriate case can be with
It exchanges.In addition, term " includes ", " having " and their any deformation, it is intended that cover and non-exclusive include.
Specific embodiment
Embodiment
The present embodiment provides a kind of nano-pore detection devices of modified biological probe, can be used for based on nano-pore technology
Disease detection and analysis, the nano-pore detection device are as shown in Figure 1 comprising device noumenon 1, described device ontology 1 are interior
Portion is provided with the first basal layer 2, first substrate in described device ontology 1 according to the portable chip structure with accommodation space
Layer 2 is SU-8 photoresist base material, and the micropore 3 that at least one aperture is 2-200 μm is offered on first basal layer 2,
In the present embodiment, the aperture of the micropore 3 is 90 μm, and the height of micropore 3 is 5-500 μm, is 100 μm in the present embodiment, described
The quantity of micropore 3 is 500.The top of each micropore 3 is provided with the nano pore 4 for being modified with probe, the nano-pore
Road be protein nano duct, probe be can specific capture biomarker polypeptide, antibody, single-chain antibody or antibody variation
Area, the probe are set to the end or inside in the albumen duct.3 bottom of micropore is provided with the second basal layer 5, and second
Base layer material is silicon and its derivative, graphene, aluminium oxide etc., and protein nano hole battle array is provided on second basal layer 5
Column, in the protein nano hole array, the aperture of single nano-pore is 1-100nm, in the present embodiment, the aperture of the nano-pore
For 50nm, the distance between adjacent nano hole is 50-5000 μm, is 200 μm in the present embodiment.
Further, the space between 2 top of the first basal layer and the inner top surface of described device basic body 1 is formed
Cell cavity, the microporous space between the nano pore 4 for being modified with probe and second basal layer 5 forms lower cell cavity, described
Upper storage reservoir is intracavitary to be provided with microfluidic module 10, and the microfluidic module 10 is for replacing conducting liquid, in the microfluidic module
It is provided with an indicator electrode 6, voltage electrode (not shown), the voltage electrode are provided with below first basal layer 2
It is arranged in a one-to-one correspondence with the micropore 3, each 3 bottom of micropore is provided with a voltage electrode, so that each micropore can
Individually to be detected, greatly reduces Monitoring lower-cut, improves detection sensitivity.The voltage electrode is also electrically coupled to CMOS core
The software of piece, the CMOS chip and control unit connects, and the signal that will test is transmitted in the software of control unit and carries out
Analysis.
Further, the nano pore 4 for being modified with probe is set to third basal layer 7, the third basal layer 7
For chemical polymerization object film layer.The top of described device ontology 1 is also provided with liquid flow inlet 8 and fluid outlet 9.
The present embodiment also provides a kind of production method of the nano-pore detection device of modified biological probe comprising such as
Lower step:
S1, to protein nano duct modified biological probe, can specific capture disease organism mark using protein fusion method
The probe amalgamation and expression of object is remembered in the end or inside in the protein nano duct, and the probe can be can specific capture disease
Polypeptide, antibody, single-chain antibody or the antibody variable region of biomarker can such as be repaired NTA using the His label of albumen end
The probe modification of decorations obtains the nano pore 4 for being modified with probe in albumen bore end.
S2, assemble nanometer hole detection device are injected from liquid flow inlet 8 into device noumenon 1 containing lipid or chemical poly
The conducting solution of body, chemical multimer or matrix material can tile film forming behind the top of micropore 3, obtain third basal layer 7,
Signal can be detected by electrode and the CMOS chip and inspection software of connection；It is then injected into and is repaired containing what step S1 was obtained
It is decorated with the conducting solution of the nano pore 4 of probe, when the protein nano duct for being individually modified with probe is embedded into third basal layer 7
After, more ducts are limited by the direction and size that control voltage and are embedded in, insertion is modified with probe at the top of each micropore 3
Protein nano duct after i.e. obtain detection device, can start detect or save.
The biological sample for needing to analyze is injected described device ontology 1, biological sample by liquid flow inlet 8 by S3, test sample
The nano pore 4 that probe is modified on molecule and micropore 3 is combined, and nonspecific signal is generated, by analyzing the nonspecific signal, analysis
Sample composition and concentration.
Wherein, protein nano duct is Phi29 connector albumen duct, SPP1, T3, T4, T5, T7 bacteriophage connector
One of albumen duct, alpha-hemolysin, MspA albumen duct；The probe is polypeptide, antibody or antibody variation
The one kind in area.
Obviously, the above embodiments are merely examples for clarifying the description, and does not limit the embodiments.It is right
For those of ordinary skill in the art, can also make on the basis of the above description it is other it is various forms of variation or
It changes.There is no necessity and possibility to exhaust all the enbodiments.And it is extended from this it is obvious variation or
It changes still within the protection scope of the invention.
Claims (10)
1. a kind of nano-pore detection device of modified biological probe, which is characterized in that including device noumenon, in described device ontology
Portion is provided with the first basal layer, and first basal layer offers at least one micropore, is provided with and is modified at the top of the micropore
The nano pore of probe, the micropore bottom are provided with the second basal layer with protein nano hole array.
2. the nano-pore detection device of modified biological probe according to claim 1, which is characterized in that first substrate
Upper cell cavity is formed between layer top and described device ontology inner top surface, the upper storage reservoir is intracavitary to be provided with microfluidic module.
3. the nano-pore detection device of modified biological probe according to claim 2, which is characterized in that the microfluid mould
It is provided with indicator electrode in block, voltage electrode, the voltage electrode and the micropore one are provided with below first basal layer
One is correspondingly arranged.
4. the nano-pore detection device of modified biological probe according to claim 3, which is characterized in that the voltage electrode
It is connected to CMOS chip.
5. the nano-pore detection device of modified biological probe according to claim 1-4, which is characterized in that described
The aperture of micropore is 2-200 μm, and the height of the micropore is 5-500 μm；The aperture of nano-pore is 1- in the nanohole array
100nm。
6. the nano-pore detection device of modified biological probe according to claim 5, which is characterized in that described to be modified with spy
The nano pore of needle is set to third basal layer, and the third basal layer is chemical polymerization object film layer or lipid film layer.
7. the nano-pore detection device of modified biological probe according to claim 6, which is characterized in that described device ontology
Top is also provided with liquid flow inlet and fluid outlet.
8. a kind of production method of such as described in any item nano-pore detection devices of claim 1-7, which is characterized in that including such as
Lower step:
S1, to nano pore modified biological probe, using protein fusion method by can specific capture disease biomarker spy
Needle amalgamation and expression obtains the nano pore for being modified with probe in the end or inside in protein nano duct；
S2, assemble nanometer hole detection device inject the conducting solution containing lipid or chemical multimer into device noumenon, described
Lipid or chemical multimer are laid into film at the top of micropore, are then injected into and are modified with receiving for probe containing what the step S1 was obtained
The conducting solution in metre hole road, to get to detection dress in the film for being embedded into the nano pore for being modified with probe at the top of micropore
It sets.
9. the production method of nano-pore detection device according to claim 8, which is characterized in that further include step S3, inspection
The biological sample for needing to analyze is injected described device ontology, is modified with probe on biological sample molecule and micropore by sample
Nano pore combines, and generates nonspecific signal, by analyzing the nonspecific signal, analyzes sample composition and concentration.
10. the production method of nano-pore detection device according to claim 8, which is characterized in that the protein nano hole
Road be Phi29 connector albumen duct, SPP1, T3, T4, T5, T7 bacteriophage connector albumen duct, alpha-hemolysin,
One of MspA albumen duct；The probe is one kind of polypeptide, antibody or antibody variable region；The step S2 will be modified
It further include direction and the size, limitation more multi-pore channel for controlling voltage after having in the film at the top of the nano pore insertion micropore of probe
The step of insertion.
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