CN110031517A - The preparation of compound glass nano pore and be applied to biomolecule detection - Google Patents
The preparation of compound glass nano pore and be applied to biomolecule detection Download PDFInfo
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- G01N33/48707—Physical analysis of biological material of liquid biological material by electrical means
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Abstract
The preparation method and its application in biomolecule detection that the present invention discloses a kind of compound glass nano pore, specific steps are as follows: step 1: preparing glass nano duct；Step 2: decorated phospholipid on the glass nano duct prepared in step 1；Step 3: compound glass nano pore is prepared；Step 4: go out in the modification of glass nano duct to be inserted with the interelectrode current data of the phosphatide of SWCNT by picoammeter and patch clamp measurements and record；The present invention draws glass nano duct using glass-pulling instrument, then will be inserted with the phospholipid modified in glass nano duct of single-walled carbon nanotube by the capillarity of nano-pore.Without the SWCNT of ultrasonic cut purifying for a long time, SWCNT is made to be inserted into phospholipid bilayer without using microinjection probe, method is simple.The current signal change of different large biological molecules can be measured by compound glass nano pore specificity duct, be applicable in and detect other different target molecules, there is universality, be conducive to promote the use of.
Description
Technical field
The present invention relates to nano pore detection fields, and in particular to one kind is based on single armed carbon nanotube (SWCNT) and phosphatide
The preparation method of the glass nano duct compound glass nano pore of self assembly and its application in biomolecule detection.
Background technique
Nano-pore technology is widely used in nucleic acid sequencing, protein/polypeptide as a kind of novel analyzing detecting method
The detection of the large biological molecules and metal ion such as analysis and virus, microorganism.As people are to public safety and food and medicine
The growing interest of security problems, to the detection of noxious material, higher requirements are also raised.Have in view of nano-pore analysis method
Have the advantages that highly sensitive and highly selective, many research teams are applied to the detection of noxious material, have carried out very much
Research work.Detection means at this stage is detected both for single creature molecule, and requires to use fluorescent marker
Or the reagent material of the valuableness such as biotinylation kit, at high cost, required detecting instrument is complicated, is unfavorable for popularizing.
Currently, single-walled carbon nanotube is widely used in nano pore, the diameter of single-walled carbon nanotube is about in 0.6-
Between 2nm, there is the One-dimensional System of certain rigidity and can be stabilized, it is very excellent that unique structure determines that it has
The properties such as electricity, optics, mechanics, calorifics.Compound glass nano pore based on single-walled carbon nanotube has low cost, Gao Ling
The advantages that quick and small size, therefore be extensively studied by people and be applied to field of biological detection.
Summary of the invention
The technical problem to be solved by the embodiment of the invention is that providing a kind of preparation side of compound glass nano pore
Method, this method are based on single armed carbon nanotube and phosphatide self assembly and glass nano duct, using package technique by single
Pipe is inserted in phospholipid bilayer layer surface, then is modified on glass nano duct, and compound glass nano pore is formed.
In addition the present invention also provides the application method of the compound glass nano pore in biomolecule detection.
First purpose to realize the present invention, technical solution are a kind of based on single armed carbon nanotube (SWCNT) and phosphorus
The glass nano duct of rouge self assembly passes through the preparation method of the compound glass nano pore of electric signal transition detection biomolecule,
Specific steps are as follows:
Step 1: glass nano duct is prepared；
Step 2: decorated phospholipid on the glass nano duct prepared in step 1；Operating method is as follows:
1. the L- α-phosphatidyl choline for taking 100mg is dissolved in the chloroform of 0.700mL, dissolution is placed on -25 DEG C
Refrigerator in it is spare；
2. taking a certain amount of phospholipid solution with liquid-transfering gun, the glass nano duct prepared is placed in solution and is repaired
Decorations；
Step 3: preparing compound glass nano pore, and operating method is as follows:
1. weighing the single-walled carbon nanotube of certain mass with assay balance；
2. weighed SWCNT is placed in the small bottom of the tube of centrifugation, using liquid-transfering gun pipette the phosphatide of certain volume in from
In heart tubule, centrifuge tube is then subjected to ultrasonic disperse, dispersion is completed spare under conditions of being placed on -25 DEG C；
3. the glass nano duct handled well in step 2 is taken out, the phosphatide for having SWCNT to suspend in 2. is then utilized
It is modified, obtains compound glass nano pore.
Step 4: the interelectrode electricity that the phosphatide of SWCNT is inserted in the modification of glass nano duct is measured by picoammeter
Flow data simultaneously records；
A kind of glass nano duct based on single armed carbon nanotube (SWCNT) and phosphatide self assembly of the invention passes through electric signal
The preparation method of the compound glass nano pore of transition detection biomolecule, further, the glass tube in the step 1 1.
Specification be overall diameter: 1.2mm；Interior diameter: 0.6mm；Length: 10mm；Glass nano duct draw conditions in step 1 4.
Parameter is about 1.58 μm by the nano aperture that SEM is characterized as shown in following table one.
The draw conditions parameter list in one glass nano duct of table
A kind of glass nano duct based on single armed carbon nanotube (SWCNT) and phosphatide self assembly of the invention passes through electric signal
The preparation method of the compound glass nano pore of transition detection biomolecule, further, the phosphorus 2. taken out in the step 2
The volume of rouge is about 20 μ L, moditied processing time 2-3min.
A kind of glass nano duct based on single armed carbon nanotube (SWCNT) and phosphatide self assembly of the invention passes through electric signal
The preparation method of the compound glass nano pore of transition detection biomolecule, it is further, 1. used in the step 3
SWCNT is purity>95% obtained using mobile catalysis method, and diameter<2nm, length is 5-30 μm, and weighs the quality of SWCNT
Range is 0.10mg-0.40mg.
A kind of glass nano duct based on single armed carbon nanotube (SWCNT) and phosphatide self assembly of the invention passes through electric signal
The preparation method of the compound glass nano pore of transition detection biomolecule, further, at the ultrasound in the step 3 4.
It manages and handles time 10-15min in room temperature；The volume of the phosphatide pipetted is about 20 μ L.
A kind of glass nano duct based on single armed carbon nanotube (SWCNT) and phosphatide self assembly of the invention passes through electric signal
The preparation method of the compound glass nano pore of transition detection biomolecule, further, the step 4 are measured by pico-ampere
The residence time for measuring interelectrode current data or biomolecule distinguishes.
Innovation Mechanism of the invention is: single-walled carbon nanotube and phosphatide is utilized, what wherein single-walled carbon nanotube was formed receives
Metre hole pattern, duct is complete and itself possesses brilliant physical property, nano-grade size and chemical generality；Phospholipid bilayer
Layer has the characteristics such as Biofunctional, biocompatibility, chemical stability, which combines inorganic material and biological material
The advantages of material.The one-dimensional linear material that SWCNT is most popular as current research, has many abnormal mechanics, electricity and chemistry
Performance, thus be to construct the best one-dimensional linear material of electrochemica biological single-walled carbon nanotube.
The beneficial effects of the present invention are embodied in:
Passed through 1. the present invention provides one kind based on the glass nano duct of single armed carbon nanotube (SWCNT) and phosphatide self assembly
The preparation method of the compound glass nano pore of electric signal transition detection biomolecule draws certain specification using glass-pulling instrument
Glass nano duct, then by ultrasound method make single-walled carbon nanotube be inserted into phosphatide in, then will be inserted with phosphatide
Glass is carbon nano tube modified to forming compound glass nano duct on glass nano duct.It is purified for a long time without ultrasonic cut
SWCNT, make SWCNT be inserted into phospholipid bilayer without using microinjection probe, method is simple.It can be by compound
Glass nano duct specificity duct measures the current signal change of different large biological molecules, is suitable for detecting other not
Same target molecule has universality, more conducively promotes the use of.The nano-pore pattern of single-walled carbon nanotube formation, duct are complete
And itself possess brilliant physical property, nano-grade size and chemical generality；Phospholipid bilayer have Biofunctional,
The characteristics such as biocompatibility, chemical stability construct the glass based on single armed carbon nanotube (SWCNT) and phosphatide self assembly
Nano pore.
Passed through 2. the present invention provides one kind based on the glass nano duct of single armed carbon nanotube (SWCNT) and phosphatide self assembly
The preparation method of the compound glass nano pore of electric signal transition detection biomolecule, using single-walled carbon nanotube as nano pore,
Since carbon nanotube itself has certain conductive capability, the variation of biomolecule size can be delicately detected, and then by certainly
Electric signal conversion is realized in the change of body conductance, further improves sensitivity and the Monitoring lower-cut of single-walled carbon nanotube, and is wrapped up
The phosphatide of single-walled carbon nanotube has excellent biological nature again.Therefore the compound glass nano pore obtained examines biomolecule
Measuring tool has the advantages that high sensitivity, analysis speed is fast, instrument is simple, at low cost, portable and be able to achieve real-time detection in situ.
3. it then can be modified on road glass nano duct by being inserted into the single-walled carbon nanotube of different quality in phosphatide,
This is suitable for detecting other different target molecules, does not need the reagent material for using the valuableness such as fluorescent marker and biotinylation kit
Material has universality, more conducively promotes the use of.
Passed through 4. the present invention provides one kind based on the glass nano duct of single armed carbon nanotube (SWCNT) and phosphatide self assembly
The preparation method of the compound glass nano pore of electric signal transition detection biomolecule, constructs compound glass nano pore, can
To realize that the repetition detection of device uses, to same sample, multiple and different target molecules can be detected respectively.
5. other features and advantages of the present invention will be illustrated in the following description, and partly becomes from specification
It obtains it is clear that understand through the implementation of the invention.The main object of the present invention and further advantage can by specification,
Specifically noted scheme is achieved and obtained in claims.
Detailed description of the invention
In order to more clearly explain the embodiment of the invention or the technical proposal in the existing technology, to embodiment or will show below
There is attached drawing needed in technical description to be briefly described, it should be apparent that, the accompanying drawings in the following description is only this
Some embodiments of invention, for those of ordinary skill in the art, without any creative labor, according to
These attached drawings obtain other attached drawings and still fall within scope of the invention.
Fig. 1 provides a kind of glass nano duct for being based on single armed carbon nanotube (SWCNT) and phosphatide self assembly for the present invention
Pass through the preparation flow figure of the compound glass nano pore of electric signal transition detection biomolecule；
Fig. 2 is biomolecule detection devices figure；
Fig. 3 is that picoammeter detects respectively by glass nano duct, and group loads onto the glass nano duct of phosphatide, and group is loaded onto slotting
Enter the current graph of the compound glass nano pore of the phosphatide of different quality SWCNT；
Fig. 4 is that picoammeter detects the compound glass nano-pore for loading onto the phosphatide of insertion different quality SWCNT by group respectively
The current data in road compares figure.
Specific embodiment
To make the object, technical solutions and advantages of the present invention clearer, the present invention is made into one below in conjunction with attached drawing
Step ground detailed description.
Step 1: glass nano duct is prepared；Operating method is as follows:
1. taking a specification is overall diameter: 1.2mm；Interior diameter: 0.6mm；Length: the glass tube of 10mm is spare；
2. glass tube is placed in the concentrated sulfuric acid (H that volume ratio is 1:12SO4), hydrogen peroxide (H2O2) in mixed solution, ultrasound
15min or so.With high purity water, ethyl alcohol rinse, after place it in it is spare in high purity water；
3. being gone out clean glass pipe clamp with tweezers, rinsed with ethyl alcohol, and with being dried with nitrogen, it is spare；
4. glass tube to be drawn to the glass nano for being drawn into appropriate bore diameter on instrument in the glass tube for being provided with certain parameter again
Duct (shown in parameter table one as described above), is about 1.58 μm by the nano aperture that SEM is characterized.
Step 2: decorated phospholipid on the glass nano duct prepared in step 1；Operating method is as follows:
1. the L- α-phosphatidyl choline for taking 100mg is dissolved in the chloroform of 0.700mL, dissolution is placed on -25 DEG C
Refrigerator in it is spare；
2. taking the phospholipid solution of about 20 μ L with liquid-transfering gun, the glass nano duct prepared is placed in 2-3 min in solution
Glass nano duct is modified using capillarity；
Step 3: preparing compound glass nano pore, and operating method is as follows:
1. weighing mass gradient with assay balance is 0.10mg, 0.20mg, 0.30mg, the single-walled carbon nanotube of 0.40mg；
2. weighed SWCNT is placed in the small bottom of the tube of centrifugation, the phosphatide for pipetting 20 μ L using liquid-transfering gun is small in being centrifuged
Guan Zhong, then by centrifuge tube, ultrasonic disperse handles time 10-15min at room temperature, and dispersion is completed to be placed on -25 DEG C of item
It is spare under part；
3. the glass nano duct handled well in step 2 is taken out, the phosphatide for having SWCNT to suspend in 2. is then utilized
Modification 2-3min is carried out, compound glass nano pore is obtained.
Step 4: the interelectrode electricity that the phosphatide of SWCNT is inserted in the modification of glass nano duct is measured by picoammeter
Flow data simultaneously records.
As shown in Fig. 2, bipolar electrode system is constituted using two Ag/AgCl electrodes, then by compound glass nanometer obtained
Duct be assembled into as shown in the figure wherein one by Ag/AgCl electrode, two electrodes are finally placed in the electrolysis equipped with buffer
Chi Zhong carries out the detection of electric current and record to it with picoammeter respectively.Buffer be 10mM HEPES solution (PH=7.03,
0.1M NaCl), so the volume of the buffer solution pipetted is 6.0mL.
Examples 1 to 4 is prepared according to the method described above, and reaction condition is as shown in following table two:
The reaction condition of two Examples 1 to 4 of table
Detection device such as Fig. 2 of bis- electrode system of Ag/AgCl, each pair of Ag electrode must use NaClO solution before the use
Pretreatment is carried out to obtain Ag/AgCl electrode material.Two electrodes are placed in the HEPES for being 10mM equipped with 6.0mL buffer
In the electrolytic cell of solution (PH=7.03,0.1M NaCl), the detection of electric current and note are carried out to it with picoammeter and patch-clamp respectively
Record.Glass nano duct without any modification can measure its interelectrode size of current by picoammeter, use at this time
It is that the alternating voltage of+2V is detected the size of current by nano-pore.Since the aperture in glass nano duct is micron level,
Measuring is high current；Glass nano duct is handled with phospholipid solution, since biological phosphatide is by the glass nano duct of script
It seals, what is measured at this time is low current；Further received with the glass of the phospholipid solution modification of the SWCNT of insertion different quality gradient
After metre hole road, obtained current data is between low current and high current, and as quality is continuously increased electric current gradually
Become larger.It is compared by current data, if obtaining electric current between low current and high current, can prove that single wall carbon is received
Mitron is inserted into phosphatide；If electric current is low current, prove that single-walled carbon nanotube is not inserted into phospholipid bilayer.
Glass nano duct provided by the invention based on single armed carbon nanotube (SWCNT) and phosphatide self assembly passes through telecommunications
The compound glass nano pore of number transition detection biomolecule, HEPES buffer solution and compound glass nanometer between Ag/AgCl electrode
Duct connection, utilizes Na in buffer+Mobile carry out electric current transmission between nano-pore, phospholipid solution can limit a certain amount of
Na+Pass through, to obtain the data of low current variation.And it can be used as ion channel when SWCNT insertion phosphatide and delicately catch
Grasp Na+By when electric signal increase, further improve sensitivity and Monitoring lower-cut.
Specific detecting step:
The detector as closed circuit is used in conjunction in compound glass nano pore and Ag/AgCl electrode, is measured by pico-ampere
Measure its curent change.Detect examples of biomolecules: as shown in Figure 2 and Ag/ by the compound glass nano pore prepared in embodiment 2
The HEPES solution (PH=7.03,0.1M NaCl) that 6.0mL buffer is 10mM is added in AgCl electrode combination in a cell.
As Fig. 3 group loads onto the current curve of the compound glass nano pore of the phosphatide of insertion different quality SWCNT and passes through glass nano
Duct, group are loaded onto shown in the current curve comparison in the glass nano duct of phosphatide.If interelectrode electric current is high current, explanation
SWCNT has been successively inserted into formation nanoparticle channel in phosphatide.If electric electrode current is low current, illustrate SWCNT not
It is inserted into phosphatide.
To sum up, the present invention is a kind of glass nano duct for being based on single armed carbon nanotube (SWCNT) and phosphatide self assembly, is led to
The preparation method for crossing the compound glass nano pore of electric signal transition detection biomolecule successfully constructs compound glass nano-pore
Road, by can be to DNA using the composite Nano duct, RNA, protein, the large biological molecules such as metal ion be detected.By
It is about 0.5-2nm in the diameter that single-walled carbon nanotube has, when having target molecule by single-walled carbon nanotube, is examined using picoammeter
The current data of survey can carry out certain differentiation effect to specific biomolecule, thus have higher sensitivity.Have simultaneously
Have the advantages that analyze that speed is fast, instrument is simple, at low cost, portable and be able to achieve real-time detection in situ.
The above disclosure is only the preferred embodiments of the present invention, cannot limit the right model of the present invention with this certainly
It encloses, therefore equivalent changes made in accordance with the claims of the present invention, is still within the scope of the present invention.
Claims (8)
1. a kind of preparation method of compound glass nano pore, it is characterised in that the following steps are included:
(1) glass nano duct is prepared；
(2) decorated phospholipid on the glass nano duct prepared in step (1)；
(3) it prepares compound glass nano pore: single-walled carbon nanotube and phosphatide being subjected to ultrasonic Centrifugal dispersion and mixed, single wall is obtained
The phosphatide of carbon nanotube suspended dispersed, then the glass nano duct by step (2) processing utilizes the single-walled carbon nanotube
The phosphatide of suspended dispersed is modified, and compound glass nano pore is obtained.
2. the preparation method of compound glass nano pore according to claim 1, which is characterized in that in the step (1)
Glass nano duct be prepared by the following method:
(1.1) glass material is placed in volume ratio is the concentrated sulfuric acid of 1:1, in mixed solution of hydrogen peroxide, then ultrasonic treatment is used
High purity water, ethyl alcohol rinse, after place it in it is spare in high purity water；
(1.2) glass material that will be clean, is rinsed with ethyl alcohol, and with being dried with nitrogen, spare；
(1.3) again by glass material in the glass nano duct for being drawn into suitable aperture.
3. the preparation method of compound glass nano pore according to claim 1, it is characterised in that:
The glass material of step (1.1) uses glass tube in the step (1), and specification is overall diameter: 1.2mm；Interior diameter:
0.6mm；Length: 10mm；The nano aperture in the glass nano duct of the preparation is 1.58 μm.
4. the preparation method of compound glass nano pore according to claim 1, it is characterised in that:
Specific step is as follows for the step (2):
(2.1) the L- α-phosphatidyl choline for taking 100mg is dissolved in the chloroform of 0.700mL, and dissolution is placed on -25 DEG C
In refrigerator, it is spare to obtain phospholipid solution；
(2.2) the glass nano duct prepared is placed in the phospholipid solution and is modified, moditied processing time 2-
3min。
5. the preparation method of compound glass nano pore according to claim 1, it is characterised in that: in the step (3)
Single-walled carbon nanotube used is purity>95% obtained using mobile catalysis method, and diameter<2nm, length is 5-30 μm, and is claimed
The mass range for taking single-walled carbon nanotube is 0.10mg-0.40mg.
6. the preparation method of compound glass nano pore according to claim 5, it is characterised in that: in the step (3)
Ultrasonic treatment in room temperature handle time 10-15min；The volume of the phosphatide pipetted is 20 μ L.
7. compound glass nano pore prepared by a kind of preparation method as described in one of claim 1-6 is in biomolecule detection
Middle application.
8. application according to claim 7, it is characterised in that: by compound glass nano pore and the Ag/AgCl electricity
The detector as closed circuit is used in conjunction in pole, measures its curent change by picoammeter, to obtain corresponding biomolecule to be measured
Current data.
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| CN111272851B (en) * | 2020-02-25 | 2021-07-27 | 华东师范大学 | Glass nanopore sensor for detecting OH and preparation and application thereof |
| CN111487307A (en) * | 2020-05-07 | 2020-08-04 | 温州大学 | Method for detecting protein based on borosilicate glass nanopores modified by metal organic framework material |
| CN111487307B (en) * | 2020-05-07 | 2023-02-10 | 温州大学 | Method for detecting protein based on borosilicate glass nano-pore channel modified by metal organic framework material |
| CN113138223A (en) * | 2021-04-23 | 2021-07-20 | 华东理工大学 | Single-molecule detection method for perfluoro and polyfluoroalkyl pollutants |
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