CN109030597A - Hollow Nano needle-graphene composite material sensor and its application - Google Patents
Hollow Nano needle-graphene composite material sensor and its application Download PDFInfo
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- CN109030597A CN109030597A CN201810635797.0A CN201810635797A CN109030597A CN 109030597 A CN109030597 A CN 109030597A CN 201810635797 A CN201810635797 A CN 201810635797A CN 109030597 A CN109030597 A CN 109030597A
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- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
- G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
- G01N27/28—Electrolytic cell components
- G01N27/30—Electrodes, e.g. test electrodes; Half-cells
- G01N27/327—Biochemical electrodes, e.g. electrical or mechanical details for in vitro measurements
- G01N27/3275—Sensing specific biomolecules, e.g. nucleic acid strands, based on an electrode surface reaction
- G01N27/3278—Sensing specific biomolecules, e.g. nucleic acid strands, based on an electrode surface reaction involving nanosized elements, e.g. nanogaps or nanoparticles
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
- G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
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- G01N27/30—Electrodes, e.g. test electrodes; Half-cells
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Abstract
The present invention provides a kind of hollow Nano needle-graphene composite material sensors comprising: polycarbonate membrane, the hollow Nano needle positioned at the polycarbonate membrane one side and the hydridization graphene combination electrode positioned at the polycarbonate membrane another side.Sensor of the invention has good biocompatibility, high sensitivity, can effectively detect the biomolecule in living cells.
Description
Technical field
The invention belongs to field of nanometer material technology, and in particular to a kind of hollow Nano needle-graphene composite material sensor and
Its application in terms of detecting intracellular matter.
Background technique
With treatment from Environmental safety supervision to diagnosing human disease, Measurement for Biotechnique is wide to be applied to human lives.
Especially diagnosing human disease field specifically includes that human gene detects, disease detection and treatment and drug delivery etc..Its
In, cell level is detected as the focus of scientific researcher in disease detection, therefore, how the real-time intracellular object of accurate detection
Matter becomes current scientific research problem to be solved.
Composite material combines the advantage of each composition material, compensates for the deficiency of single material, has widened composite wood significantly
The application field of material becomes the new hot spot of scientific research field.Especially to the research of nanocomposite, even more from nanostructure
Nanocomposite properties are substantially increased, each research field such as biology, material, physics, chemistry is widely used in.Its
In, the excellent sensitivity of nanocomposite, good repetition is utilized in application of the nanocomposite in biological detection
The advantages such as property, environment friendly, make biological detection more facilitation, safe, hommization, and then simplify and increase biological inspection
The approach and means of survey.Wherein, composite electrode is for detecting intracellular matter, since its detection mode is easy, rapid, repetition
Property it is high, become many scientific researcher preferred embodiments.
But most nanocomposite can only monitor the molecule that cell is secreted, or the cell extract being digested,
The biomolecule in living cells can not be detected.Therefore, how nanocomposite real-time detection intracellular matter is utilized, simultaneously
Damaging cells do not become present scientific research hot spot.
Currently, intracellular detection is mostly to fall cell dissolution, dissolved cell liquid is extracted, detects object in cell liquid
Matter；Or certain secretagogues matter is added in cell culture fluid to promote to cultivate cell secretion target substance, to complete cell
Outer detection.Cell detection approach includes: Electrochemical Detection, fluorescence detection, Raman detection etc..The above detection mode is mostly disposable
Detection, and there is damage cell, the deficiencies of detection accuracy, it is difficult to accomplish do not destroying cell normal physiological function and active premise
On, in situ detection intracellular matter, intracellular matter can non-invasively be extracted by also lacking one kind, and be able to achieve in situ identified
With the integrated device of analysis.
Hollow Nano needle can study the function of many biosystems, from cell, biological fluid, to tissue and organism.
Currently, hollow Nano needle is mainly used in Intracellular drug release and intracellular matter extracts and transmission.With traditional probe
It compares, hollow Nano needle, which is applied to cell, has lower toxicity and interference, can effectively perceive intracellular environment.
Therefore, long-term, reversible interaction can be carried out with cell: in addition to detecting DNA, RNA, protein molecular and other biological into the cell
Molecule to cell etiologic diagnosis and can also pass through hollow Nano needle to intracellular delivery drug etc..And hollow Nano needle is
Nano-grade size convenient for being assembled into extensive, oldered array, and then monitors the biological function of maxicell group.
Graphene has extraordinary biocompatibility, and all the time, composite is in medical domain
(as: in biological detection, gene/drug transmission, bio-imaging, targeted drug treatment, bio-sensing and treatment of cancer)
Using causing huge concern.
Summary of the invention
It is a kind of with good bio-compatible it is an object of the invention to aiming at the above technical problems to be solved, provide
Property, high sensitivity, the hollow Nano needle-graphene composite material sensor that can effectively detect biomolecule in living cells.
A further object of the present invention is to provide a kind of method that can effectively detect the biomolecule in living cells.
In order to realize the above goal of the invention, the present invention provides following technical schemes:
A kind of hollow Nano needle-graphene composite material sensor comprising: polycarbonate membrane is located at the poly- carbonic acid
The hollow Nano needle of ester film one side and hydridization graphene combination electrode positioned at the polycarbonate membrane another side.
Preferably, the polycarbonate membrane has even aperture.It is highly preferred that the pore size is
Preferably, the hollow Nano needle is aluminium oxide hollow Nano needle.
The present invention also provides the methods for preparing the hollow Nano needle-graphene composite material sensor comprising with
Lower step:
(1) one side is prepared in conjunction with plasma etching method using atomic layer deposition method is the poly- of aluminium oxide hollow Nano needle
Carbonic ester film；
(2) hydridization graphene combination electrode is prepared in the another side of polycarbonate membrane.
The present invention also provides biology point of the hollow Nano needle-graphene composite material sensor in detection cell
The application of sub- aspect.
Intracellular life is detected using the hollow Nano needle-graphene composite material sensor the present invention also provides a kind of
The method of object molecule comprising following steps: hollow Nano needle is assisted to puncture cell membrane, intracellular biology point using external pressurized
Son is by hollow Nano needle tract scattering and permeating, and the hydridization graphene combination electrode at the nanoneedle back side detects, to realize inspection
Survey the biomolecule in cell.
The hollow Nano needle-graphene composite material sensor detection intracellular biological molecule side is utilized according to above-mentioned
Method further includes steps of
By the hollow Nano needle-hydridization graphene combination electrode be fixed on dimethyl silicone polymer prepare it is micro-fluidic
Bottom device is then attached on indium oxide tin glass, cell to be detected is cultivated inside the micro-fluidic device 1-2 days；?
Plus current potential is determined between the indium oxide tin glass and the cell, the cell electroporation is realized, to obtain intracellular object
Matter, after cell membrane electrical breakdown, the intracellular matter is flowed out by the hollow Nano needle, is reached and is located at the polycarbonate membrane
The hydridization graphene combination electrode of another side, to realize the detection of intracellular biological molecule.
The present invention explores the micro-nano technologies condition such as atomic layer deposition and plasma etching, prepares hollow Nano needle.It is empty
The activity of cell is related after heart nanoneedle size is pierced to the extraction efficiency of biomolecule and cell membrane.Hollow receive can be optimized
Rice needle size so that reaching can not only make biomolecule have enough diffusivitys, but also will not cause the purpose of damage to cell.It applies
Making alive size, length of time are that nanoneedle is inserted through cell membrane and keeps the key factor of cell activity.By optimizing voltage item
Part can be such that cell membrane is in for a long time and puncture state by hollow Nano syringe needle, realize to the enough of intracellular biological molecule
Under the premise of extraction, cell activity and normal function are kept, to realize below in several days in different time points to same cell
Carry out the extraction and detection of biomolecule.Aluminium oxide hollow Nano needle set has good biocompatibility, be suitably applied towards
The drug release of cell or biological detection application.
The present invention has a variety of advantages.Firstly, hollow Nano needle-hydridization graphene combination electrode that the present invention constructs is used for
Intracellular biological molecule is detected, the composition of hydridization graphene combination electrode nano material has diversification, by optimizing graphene
Based nano-material composition, can construct the senser element for detecting intracellular different biological molecules；By optimizing graphene-based nanometer
Material structure constructs the higher bimolecular sensors of sensitivity.And then building can detect a variety of biologies in individual cells simultaneously
The combination electrode of molecule realizes to the real-time monitorings of intracellular various biomolecules, and different cell different biological molecules
Detection is extracted repeatedly.Secondly, being Electrochemical Detection for the detection method of intracellular biological molecule in the present invention, in detection method
Real-time, the multimode of intracellular biological molecule can be realized in research in conjunction with other modes such as Raman detection, fluorescence detection etc.
Detection.
Technical effect of the invention: 1. the present invention uses hollow Nano needle and applied voltage combination piercing cellular plasma membrane,
Intracellular biological molecule is extracted and detected, is extracted for minimally-invasive and provides new approaches with detection intracellular biological molecule；2. this hair
It is bright to use reverse path, intracellular biomolecule is drained into the electrode surface of outside by hollow Nano needle, for sky
The further application of heart nanoneedle provides new direction；3. the present invention utilizes hollow Nano needle-electroporation technology, in different time
Point extracts detection to intracellular biological molecule, it can be achieved that detection while high-volume cell, technique are expected to as biology
Electronics and biological information research field increase theoretical and technical foundation.4. the present invention biomolecule intracellular to Different Individual into
Row distinguishes detection, explores its distinguishing to the analysis result of individual cells.And different time points are explored, same cell is carried out
Biomolecule extracts detection, to grasp intracellular dynamic change in real time.
Detailed description of the invention
Fig. 1 is to utilize electrode real-time detection cell made of hollow Nano needle-graphene composite material according to the present invention
The schematic diagram of interior biomolecule.
Fig. 2 is the SEM phenogram of hollow Nano needle according to the present invention.
Specific embodiment
Below in conjunction with the drawings and specific embodiments, the invention will be further described.It should be understood that following embodiment is only used for
Illustrate the present invention, range and is not intended to limit the present invention.For simplicity, the part steps in following embodiment or operation ginseng
Number fails to describe one by one, but it is in range known to a person skilled in the art.
Embodiment 1:
Prepare aluminium oxide tubulose hollow Nano needle: using the poly- carbon with even aperture (preferably aperture is 400nm)
Acid esters (PC) perforated membrane is that template prepares hollow Nano needle array.
Firstly, the use of ALD (atomic layer deposition) technology including that inner hole wall deposition is upper uniform in all surface of template matrix
Alumina layer: with the trimethyl aluminium [Al (CH of gas phase3)3] presoma and water vapour pulse is used as alternately to be passed through reactor, it sinks
Product aluminium oxide Al2O3Film: 1. the exposed polycarbonate porous film surface in vapor forms the hydroxyl (- OH) of monolayer
Atomic group；2. hydroxyl atomic group is reacted with the source Al molecule, so that the source Al molecule loses a methyl radical, and chemistry is formed
Key [Al (CH3)2-O-C]；3. next period for being passed through vapor, vapor and remaining methyl complete ligand reaction, first is generated
Alkane CH4And Al2O3.Entire ALD process carries out 400 periods, obtains the Al of about 30~40nm thickness2O3Film.
Then, using plasma etching method, SF is used6And CF4Gas etching 5min carves the remaining aluminium oxide of upper surface
Eating away.
Then, O is further utilized2Plasma etching etches away the substrate film of part, forms aluminium oxide tubular nanometer
Needle head structure.
The diameter of hollow Nano needle is determined by the pore size of selected trace-etching-film, in the present invention
In, the preferred aperture polycarbonate membrane that is 400nm；And the length of nanoneedle is by O2The length of time of plasma etching determines
, in the present invention, it is preferred to O2The plasma etching time is 20min, obtains hollow Nano needle of the length at about 1 μm.
The structure of hollow Nano needle is characterized using scanning electron microscope.As shown in Figure 2.
Embodiment 2:
It is 1mol/L's that one layer of concentration is coated at the aluminium oxide hollow Nano needle array substrate back side prepared by embodiment 1
CuSO4Solution, after flash baking, in the same way, in coating CuSO4Continue to coat one on substrate after solution and drying
The low-concentration oxidizing graphene aqueous solution (1.25mg/mL) of layer, it is dry rapidly.It is primary to repeat above step, can be obtained with more
Hollow Nano needle-hydridization graphene combination electrode of pore structure.
Embodiment 3:
Hollow Nano needle-hydridization graphene combination electrode and cell piercing devices prepared by embodiment 2 is integrated.
Intermediate porose cell culture container (about 5 millimeters of diameter) is prepared using dimethyl silicone polymer (abbreviation PDMS).
Hollow Nano needle-hydridization graphene combination electrode is then horizontally placed at PDMS intermediate hole portion bottom end, micropin side upward, hydridization
Graphene combination electrode side is fixed on tin indium oxide (abbreviation ITO) glass.Ito glass is as Electrode Negative.The cavity PDMS
Interior culture cell 4, a platinum electrode 2 is set on cell culture fluid, and conducting wire connects external power supply and function generator.PDMS mould
Block-hollow Nano needle substrate and hydridization graphene combination electrode-ito glass use uncured PDMS glue bond.
Embodiment 4:
As shown in Figure 1, hollow Nano needle-hydridization graphene combination electrode and cell the piercing devices integration for preparation detect
The schematic diagram of intracellular matter.Wherein, 1 is Ag/AgCl reference electrode, and 2 be platinum electrode, and 3 be Graphene electrodes, and 4 be to be detected
Cell, 5 be intracellular protein molecule, 6 be hollow Nano needle, 7 be polycarbonate perforated membrane, 8 be PDMS culture hole.
Cell culture and nanometer needle-penetration cell membrane: the present invention selects attached cell Hela cell line to use as model cell
It (can also be extended for other types cell) in infrastest.Make in the culture hole bottom laying ito glass of diameter about 0.5mm
For cathode, it is connected to electrochemical workstation 9.Ag/AgCl reference electrode 1, platinum electrode 2 are also respectively connected to electrochemical workstation 9.
PDMS culture hole 8- hollow Nano needle-hydridization graphene combination electrode-ito glass device carries out at sterilizing before cultivating cell 4
Reason, then, in the internal coat poly-D-lysine of PDMS culture hole 8 to increase the adherency of cell 4, after culture 24 hours, cell 4 is pasted
Wall sprawls growth and wraps up hollow Nano needle 6.Electric pulse parameter (pulse voltage, pulse duration, arteries and veins are adjusted by function generator
Punching interval, pulse number), apply voltage between ito glass and cell 4, until cell membrane is punctured by hollow Nano needle 6, releases
Release intracellular protein molecule 5.Membrane perforation case study can be conveyed by hollow Nano needle 6 and is difficult to through cell membrane
Fluorescent molecule (such as Propidium iodide) determine to intracellular.And the survival of cell 4, then it can pass through the double-colored dye of living cells/dead cell
Reagent determines.
The present invention uses the polycarbonate substrate film (trace-etching-film) with even aperture as template, hollow to receive
Rice needle-hydridization graphene combination electrode be working electrode, silver/silver chloride electrode be reference electrode, platinum electrode be to electrode, thus
Composition three-electrode system analyzes hollow Nano needle-hydridization graphene combination electrode performance using electrochemical method.
Hollow Nano needle-hydridization graphene combination electrode that the present invention constructs assists hollow Nano needle to puncture using external pressurized
Cell membrane after cell punctures, cell is studied in different time sections activity and function, to optimize hollow Nano needle size
And applied voltage, reach cell and be in the state of wearing out for a long time, while keeping the purpose of cell activity.Intracellular biomolecule
By hollow Nano needle tract scattering and permeating, the hydridization graphene combination electrode at the nanoneedle back side is detected, to realize difference
Cell biological molecule extracts detection and different time points repeatedly, carries out biomolecule repeatedly to the same cell and extracts, inspection
Survey the dynamic change of intracellular biological molecule.
Embodiment 5:
For probing into for the self-regeneration situation after membrane perforation: 5-10 minutes after removing voltage, conveying iodate pyrrole
Pyridine fluorescent molecule.If there is Propidium iodide red fluorescence into the cell, cell membrane is still in holding state of rupture.Adjust pulse
Interval makes cell membrane close on the state of rupture more put again and cell membrane can be made by voltage breakdown to keep relatively long time, from
And intracellular more biomolecule is made to diffuse into hollow Nano needle, reach hydridization graphene combination electrode.
Embodiment 6:
The detection of intracellular biological molecule:
The present invention assists hollow Nano needle to puncture cell membrane using applying pulse voltage, and intracellular biological molecule passes through hollow
Hydridization graphene combination electrode of the nanoneedle to substrate back.Using hollow Nano needle-hydridization graphene combination electrode as work electricity
Pole, silver/silver chloride electrode are reference electrode, platinum electrode be to electrode, to form three-electrode system, using electrochemical method,
Hollow Nano needle-hydridization graphene combination electrode performance is analyzed.
The results show: into the cell there are Propidium iodide red fluorescence, intracellular biomolecule diffuses into hollow receive
Rice needle, reaches hydridization graphene combination electrode, and hollow Nano needle-hydridization graphene combination electrode property of can choose detects in solution
Biomolecule, and there is good sensitivity and repeatability.
Embodiment 7:
The minimally invasive Journal of Sex Research of cell: two days after cell electroporation, cell activity is checked.
After testing result shows cell electroporation, good cell activity is still maintained, the normal life of cell will not be damaged
It is long.
Claims (7)
1. a kind of hollow Nano needle-graphene composite material sensor, characterized by comprising: polycarbonate membrane, be located at it is described
The hollow Nano needle of polycarbonate membrane one side and hydridization graphene combination electrode positioned at the polycarbonate membrane another side.
2. hollow Nano needle-graphene composite material sensor according to claim 1, which is characterized in that the poly- carbon
Acid esters film has even aperture.
3. hollow Nano needle-graphene composite material sensor according to claim 1, which is characterized in that described hollow
Nanoneedle is aluminium oxide hollow Nano needle.
4. life of the described in any item hollow Nano needle-graphene composite material sensors of claims 1 to 3 in detection cell
Application in terms of object molecule.
5. according to claim 1 to the needle of hollow Nano described in 3-graphene composite material sensor preparation method, feature
Be the following steps are included:
(1) the poly- carbonic acid that one side is aluminium oxide hollow Nano needle is prepared in conjunction with plasma etching method using atomic layer deposition method
Ester film；
(2) hydridization graphene combination electrode is prepared in the another side of polycarbonate membrane.
6. detecting intracellular biological molecule using hollow Nano needle described in claims 1 to 3 3-graphene composite material sensor
Method, it is characterised in that the following steps are included: assisting the hollow Nano needle to puncture cell membrane, the cell using external pressurized
For interior biomolecule by the hollow Nano needle tract scattering and permeating, the hydridization graphene in hollow Nano needle back face is multiple
Composite electrode detection, to realize the biomolecule detected in the cell.
7. according to the method described in claim 6, it is characterized in that, specific steps are as follows:
Hollow Nano needle-hydridization the graphene combination electrode is fixed on to the micro-fluidic device prepared with dimethyl silicone polymer
Bottom is then attached on indium oxide tin glass, cell to be detected is cultivated inside the micro-fluidic device 1-2 days；Described
Plus current potential is determined between indium oxide tin glass and the cell, the cell electroporation is realized, to obtain the intracellular object
Matter, after cell membrane electrical breakdown, the intracellular biomolecule is flowed out by the hollow Nano needle, is reached and is located at the poly- carbon
The hydridization graphene combination electrode of acid esters film another side, to realize the detection of the intracellular biomolecule.
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| CN110907416A (en) * | 2019-11-05 | 2020-03-24 | 中山大学 | Circulating tumor cell detection device based on hollow nano needle tube electroporation system and detection method thereof |
| CN111172034A (en) * | 2020-02-21 | 2020-05-19 | 中山大学 | Adjustable voltage mode cell perforation and membrane permeation system based on nanotube array sensor |
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Application publication date: 20181218 |