CN108645905B - A method of hydrogen peroxide is detected based on solid nano hole - Google Patents
A method of hydrogen peroxide is detected based on solid nano hole Download PDFInfo
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Abstract
The present invention relates to a kind of methods based on solid nano hole detection hydrogen peroxide, by beating different nano-pores on 10-100nm ultra-thin silicon nitride film, with a variety of silanization treatments, realize nanoporous surface and inner wall differentiation modification, by silane coupled molecule by peroxidase conjugate in nano-pore inner wall, it constructs controllable, stable, unimolecule hydrogen peroxide sersor part in highly sensitive nano-pore, by the nano-pore ion current signal for parsing enzymatic reaction, the model mechanism and rule of single enzyme molecule enzymatic reaction Km and Kcat in nano-pore, with Real_time quantitative detection hydrogen peroxide.The present invention reaches nanomole or less by the controllable modification of differentiation modification and horseradish peroxidase unimolecule in nano-pore inside and outside nano-pore, the small molecule real-time sensing detection method of the specificity of foundation, precision.
Description
Technical field
The present invention relates to nano-pore sensing detections, and in particular to a kind of side based on solid nano hole detection hydrogen peroxide
Method.
Background technique
Nano-pore sensing detection technology was regarded as a kind of quick non-marked Single Molecule Detection once proposition from 1996
One of powerful technique.Nano-pore is broadly divided into solid nano hole and biological nano hole.Nano-pore having become of sensing technology
It learns and novel, important, great potential analysis detection means a kind of in biological field.It applies and is studied in DNA sequencing, single point
Son detects, and in the fields such as monomolecular chemical reaction and protein folding, has broad application prospects and huge potential value.
Nano-pore derives from the transmembrane ion channel transport phenomena of cell membrane surface earliest, and what is utilized is the general of electricresistance effect in solution
It reads.Similar concept, micron hole sensor is just put forward in the 1950s by Coulter first, so that Coulter
Counter is able to generate and develop, and becomes the instrument of existing frequently-used blood count.With the development of investigative technique, by this
Kind aperture has accomplished nanoscale, and scientist begins trying to carry out the research of molecular level, and becomes a kind of novel sensor.
Due to gene sequencing flourish, scientist begin one's study nano-pore sensing nucleic acid molecules and read nucleic acid molecules
The ability of internal information.The nineties in last century, Branton and Kasianowicz etc. are put forward for the first time nano-pore sensing detection skill
Art can be used to be sequenced, the channel protein that they successfully drive single stranded DNA electrophoresis to be located on phospholipid bilayer by one
Alpha hemolysin, and observe current blockade phenomenon, if can detecte the obstruction of different amplitudes caused by these four detections
If electric current, so that it may speculate the sequence for obtaining DNA.Therefore, nano-pore is considered as most being expected to realize that fast and low-cost DNA is surveyed
The third generation gene sequencing technology of sequence.2014, Oxford Nanopore company, Britain was proposed the commercialized nanometer of first item
Hole sequenator-MinION.The market price of MinION is 1000 dollars one at present.Britain's Oxford Nanopore company last year
It is proposed the plan on probation of MinION nano-pore sequencing instrument early stage, it was demonstrated that MinION assists genome assembly, and in standard reality
Test the potentiality of outdoor quickly sequencing microbial genome and environmental samples.The researcher of U.S.'s NASA Johnson's space center counts
It draws and MinION is taken to space in 2016, the sample of international space station is sequenced.Some researchers think that MinION can be used for i.e.
When detect (POCT), for example, quickly detection to diagnose a unknown infection, outbreak of disease such as West Africa Ebola is broken out real-time
Analysis.Roche Holding Ag also announces that it carries out strategy-driven investment, and two to nano-pore sequencing company Stratos Genomics
Company reaches cooperation agreement, prepares further exploitation single-molecule sequencing technology.In July, 2012, Stratos Genomics card
The real DNA sequencing of 36 bases, and in September, 2013, it has been improved to 210 bases rapidly.The said firm indicates that it will exploitation
The microarray dataset of a low cost, merges the speed and flux of nano-pore, and improves resolution ratio and signal-to-noise ratio, for targeting and entirely
Gene order-checking.Nano-pore sequencing company, U.S. Genia has purchased with 3.5 hundred million U.S. dollar prices in 2014 June in this year, Roche Holding Ag
Technologies；With in June, nano-pore sequencing company, the joint risk investment joint investment U.S., Roche Holding Ag Stratos
15,000,000 U.S. dollar of Genomics.Roche Holding Ag also researches and develops solid nano hole technology jointly with IBM Corporation.And Illumina and
Lifetech also is putting forth effort to develop or invest nano-pore sequencing technology.The company of Blang develops the nano-pore of a hand size
Gene sequencer, You Yigen USB data line are connect with computer, can detect the DNA in sample.This miniature instrument is in Africa
For detecting Ebola virus.This detector integrates microminiaturization, can be embedded in daily necessities, and connect with internet wireless
It connects, to continue to monitor health status.
Nano-pore technology is in addition to other than gene sequencing field illustrates huge attraction and potential, nano-pore is as one
Kind is quick, high-throughput, highly sensitive monomolecular sensor has been widely used in individual molecule detection field and has played its height
Precision, the advantage of non-marked.Such as the detection of heavy metal ion, the glucose content detection of small molecule quantitative detection and urine disease].
In recent years, more and more researchers start to notice that nano-pore is a kind of nano-scale dimension limit space, letter
Number easily be detected, can also be used as a kind of high-precision, non-marked monitoring means.Moreover, in nano-pore detection, it is sensitive to amplify
In addition small size makes sample that femtomole concentration only be needed to can be carried out detecting to characteristic, can be used for analyzing after filtering but not into
Biomolecule sample in the physiological solution of one step processing, it is quickly small that the micro-/amount of receiving of nanometer pore single-molecule technology is detected as building
Molecular method quantification detection provides splendid micromation platform.
Hydrogen peroxide (hydrogen peroxide) is a kind of strong oxidizer, clinical in pharmacy, and environment is dug up mine, weaving, food manufacturing
Industry etc. is widely applied, in addition, hydrogen peroxide is a kind of important chemical substance in biosystem, has strong cell toxicant
Property.More seriously hydrogen peroxide may result in cancer and neurodegenerative disease, such as A Erci in Intramitochondrial accumulation
Alzheimer disease, Parkinson's disease and Huntington's chorea disease.
However, the method for traditional detection hydrogen peroxide is usually using spectroscopic methodology, chemoluminescence method, amperometric titration, electricity
Chemical method etc..Traditional spectrum and chemiluminescence method is usually time-consuming, they need expensive reagent (such as: fluorescent marker)
And equipment.In addition, many electrochemical techniques detect the direct oxidation molecule of hydrogen peroxide dependence using Pt electrode.Due to its choosing
Selecting property is poor, and sensitivity is low and electrode fouling.In view of construct a kind of integrated nanometer hole unimolecule real-time, quickly micro-/the amount of receiving hydrogen peroxide
Monitoring means is China's food industry and food safety urgent problem to be solved.Traditional hydrogen peroxide sensor, mostly from more
The level of macroscopic view is studied, and the dynamics that acquired results parameter is often difficult to embody the enzyme-to-substrate reaction of molecular level is special
Therefore sign constructs a kind of method of integrated unimolecule horseradish peroxidase sensor to Real_time quantitative detection hydrogen peroxide, shows
It obtains particularly important.
Summary of the invention
It is peppery it is an object of the invention to construct a kind of integrated unimolecule in order to solve the above-mentioned problems in the prior art
Method of the root peroxidase sensor to Real_time quantitative detection hydrogen peroxide.
To achieve the goals above, the technical solution adopted in the present invention is as follows: one kind was detected based on solid nano hole
The method of hydrogen oxide, specifically includes the following steps: it is a kind of based on solid nano hole detection hydrogen peroxide method, specifically include with
Lower step:
The preparation of step A. nano-pore: nano-pore is beaten on the ultra-thin silicon nitride film of 10-100nm；
The modification of step B. nano-pore and the building of unimolecule enzyme sensor: with a variety of silanization treatments, nano-pore table is realized
The stable assembling of single horseradish peroxidase will be fixed on by face and inner wall differentiation modification by silane coupled molecule
Nano-pore hole inner wall obtains nanopore sensor；
Step C. substrate specificity sensing detection: reaction system is added in various concentration hydrogen peroxide, analyzes current characteristic
Signal draws concentration of hydrogen peroxide standard curve, utilizes the standard curve Real_time quantitative detection hydrogen peroxide.
In the step A, using FEI Strata 201FIB system, with the accelerating potential bombardment 10-100nm's of 30kV
Ultra-thin silicon nitride film surface beats nano-pore.
In the step B, a variety of silanization treatments are one of methyl-monosilane, trifluoro or amino silane, carboxy-silane
Or it is a variety of.
In the step B, using comb-like graft copolymer by modifying in nanoporous surface by the way of covalently or non-covalently,
With screen nano internal surface of hole, institute is electrically charged.
In the step B, using silane coupled molecule amino silane, 1- (3- dimethylamino-propyl) -3- ethyl carbon two is sub-
Amine hydrochlorate, n-hydroxysuccinimide realize fixation and the Site selective assembly of nano-pore inner wall peroxidase molecules.
In the step B, before chemical modification, Si3N4 chip is cleaned into 30min in 90 DEG C of Piranha solution,
To generate hydroxyl on the surface Si3N4,3h is activated in methyl alcohol with 3-APTES, with Isosorbide-5-Nitrae-phenylene diisothio-cyanate two
5h is handled in methyl sulfoxide, is washed, the horseradish peroxidase of Isosorbide-5-Nitrae-phenylene diisothio-cyanate and 1mg/ml point after washing
Primary amine group present in sub- HRP carries out covalent coupling reaction and the stable assembling of horseradish peroxidase is fixed on nano-pore hole
In wall, modified channel is thoroughly washed after 12-24h with buffer solution, both obtains nanopore sensor.
In the step C, by the analysis to characteristic current signal, the characteristic signal generated by enzymatic reaction is determined,
Measure the detectable limit of substrate.
Nano-pore detection hydrogen peroxide detection limit of the present invention and standard curve, the range of linearity is in 5-15nmol/L, work
Curve: Y=-183.431X+3580.726, coefficient R ^2=0.986, detection limit reach 10pmol/L.
Compared with prior art, the beneficial effects of the present invention are:
1. the present invention by differentiation modification inside and outside nano-pore and horseradish peroxidase unimolecule in nano-pore can
Control modification, establishes a kind of small molecule real-time sensing detection method of completely new specificity, and precision reaches nanomole or less.Benefit
With the quick of nano-pore sensing detection, non-marked, highly sensitive feature, single enzyme molecule enzyme kinetic analysis and its influence are had studied
Factor, stable, the easily designed processing advantage modified with materialization in conjunction with solid nano hole construct controllable, stable, height
Unimolecule hydrogen peroxide sersor part in the nano-pore of sensitivity.
2. nano-pore detects hydrogen peroxide detection limit and standard curve, the range of linearity is in 5-15nmol/L, working curve:
Y=-183.431X+3580.726, coefficient R ^2=0.986, detection limit can reach 10pmol/L.
3. when the nanopore sensor detection hydrogen peroxide constructed through the invention, there are when hydrogen peroxide, one can be caused
Determine the current reduction in degree, and when the concentration of hydrogen peroxide is stepped up, ionic current is gradually reduced, reduction from
The concentration growth of sub- electricity amplitude and hydrogen peroxide is in a linear relationship, shows constructed nanopore sensor to hydrogen peroxide catalyzed
Reaction have the function of forward direction.
4. also can be by means of different to protein, aptamers, biological Avidin in silicon nitride nano hole of the invention
Equal realizations are covalently fixed, in the hope of studying intermolecular interaction in finer degree, between such as protein, protein
Interaction between nucleic acid and between ligand receptor.Realize real-time monitoring on single molecules level, it can be whereby with new hand
Section carries out Depth Study to the microcosmic the fluctuation phenomenon of the mechanism of action biomolecule and large biological molecule conformation, and provides pair
A kind of certain new ways of small molecule quantitative detection, detection limit receive/femtomole grade.
Detailed description of the invention
Fig. 1 nano-pore image and I-V curve；
Fig. 2 (A) enzyme immobilization step schematic diagram；(B) oxydasis reduction generates ABTS+ schematic diagram；
Fig. 3 nano-pore phenogram；
It (A) include 1.5mM in 0.1mol/L KCl I-V curve and 0.1mol/L KCl before and after HRP functionalization
ABT and hydrogen peroxide；
(B) I-V curve of the 0.1mol/L KCl comprising 1.5mM ABTS after HRP functionalization；
(C) I-V curve of the 0.1mol/L KCl comprising 1.5mM ABTS and hydrogen peroxide after HRP functionalization；
(D) unmodified nano-pore includes 1.5mM ABTS and peroxidating in 0.1mol/L KCl and 0.1mol/L KCl
The I-V curve of hydrogen；
Fig. 4 enzyme functionalized nano hole sensor reproducibility and working curve diagram；
(A) influence of the difference pH to the nano-pore of HRP functionalization；
(B) reproducibility of the nano-pore detection system of HPR functionalization；
(C) the nano-pore detection system of HPR functionalization includes 1.5mM ABTS and various concentration mistake in 0.1mol/L KCl
The I-V curve of hydrogen oxide；
(D) linear fit of various concentration hydrogen peroxide and ionic current.
Specific embodiment
The present invention passes through the nano-pore ion current signal of parsing enzymatic reaction, single enzyme molecule enzymatic reaction in nano-pore
The model mechanism and rule of Km and Kcat constructs a kind of integrated unimolecule enzyme nanopore sensor to Real_time quantitative detection mistake
Hydrogen oxide, specifically includes the following steps:
1. the preparation of nano-pore
The all solid state silicon nitride film technical study for preparing high-insulativity, successfully achieves the multiple film of 30~100nm
Thick ultra-thin silicon nitride film.By the optimization of technique, the 10-100nm ultra-thin silicon nitride film that meets punching requirement will be obtained；Pass through
To the optimization for focusing electron beam or ion beam processing condition in TEM, FIB, HIM system of processing platform, multi-thickness, hole are prepared
Diameter is with draw ratio, the nano-pore of shape.
2. the modification of nano-pore and the building of unimolecule enzyme sensor
Nanoporous surface and inner wall are realized with a variety of silanization treatments (methyl-monosilane, silicofluoroform, carboxy-silane etc.)
Differentiation modification.By silane coupled molecule by peroxidase conjugate in nano-pore inner wall.Such as comb-like graft copolymerization
Object can be modified such as poly-L-lysine-graft-poly (ethylene glycol) by way of covalently or non-covalently
In nanoporous surface, with screen nano internal surface of hole, institute is electrically charged.Using silane coupled molecule such as amino silane, EDC (1- (3-
Dimethylamino-propyl) -3- ethyl-carbodiimide hydrochloride), NHS (n-hydroxysuccinimide) realizes the peroxidating of nano-pore inner wall
The fixation of object enzyme molecule and Site selective assembly.
The characterization of committed step: nano-pore morphosis is characterized by SEM, TEM, AFM etc., Contact-angle measurement
Instrument, voltammetric current curve, SEM, TEM etc. are characterized (the hydrophilic modification of such as carboxy-silane, silicofluoroform to nano-pore chemical modification
Hydrophobic modification etc.).The speed that hole endoperoxides object enzyme molecule combines, quantity are judged using current signal variation in nano-pore.
3. substrate specificity sensing detection
Reaction system is added in various concentration hydrogen peroxide, current signature is analyzed, draws concentration of hydrogen peroxide standard
Curve, detectable limit of the measurement sensor to hydrogen peroxide.
Hydrogen peroxide catalyzed reaction equation is as follows:
HRP(Fe4+=O) Porp+HA → HRP (Fe3+)Porp+A·+H2O (3)
Using 2,2'- amino-two (3- ethyl-benzothiazole sulfonate moiety -6) ammonium salt (ABTS), in the presence of hydrogen peroxide
Under, the substrate of product Compound I. porphyrin radical cation compound that horseradish peroxidase HRP is oxidized from reduction-state
Molecule receives a compound Compound II being electronically generated.Then, compound II passes through the transmitting quilt of an electronics
It is reduced into resting enzyme, ABTS+ product is generated using ABTS as substrate, utilizes the electric signal in reaction process
Variation, detects the reaction rate of enzyme.
The stable assembling of single horseradish peroxidase is fixed in nano-pore hole wall, by characteristic current signal
Analysis determines the characteristic signal generated by enzymatic reaction, measures the detectable limit of substrate in conjunction with related mathematics physics model；
Substrate, substrate analogue, the difference of the current signatures such as competition substrate are distinguished, and carries out enzyme kinetic analysis ginseng with characteristic signal
Several measurements.
Embodiment
Reagent and material: APTES, Sigma；Potassium chloride (KCl), Sigma；Methanol, Sigma；Dimethyl sulfoxide, Hefei Xin Yuan
Biotechnology Co., Ltd；Horseradish peroxidase (HRP), Sigma；ABTS, 98%Sigma；Hydrogen peroxide (hydrogen peroxide),
30%SL Labor-Service；Methanol, 98%Sigma；Amino silane, 98%Sigma；
Testing equipment: field emission scanning electron microscope, Malvern particle size analyzer, assay balance, Vibration isolation of Taiwan, shielded box,
Filamentary silver, liquid-transfering gun, ultrapure water, patch-clamp,
Experimental solutions are prepared
A.1mol/L the preparation of KCl solution: weighing the KCl of 0.7458g with assay balance, and dissolution, constant volume sets the appearance of 10mL
It in measuring bottle, shakes up, and with 0.02 μm of Anotop of membrane filtration, then is diluted to 0.1mol/L KCl.
The preparation of B.piranha solution: i.e. Piranha solution, by ratio be 3:1 (volume ratio) the concentrated sulfuric acid (98%) and
Hydrogen peroxide solution (30%), is prepared in draught cupboard, and when preparation pays attention to protecting.Hydrogen peroxide solution is slowly added
Stirring, addition sequence cannot absolutely overturn into the concentrated sulfuric acid, while quickly.
C.0.5% bitoscanate prepares: weighing 0.005g and is dissolved in the dimethyl sulfoxide of 8mL simultaneously to APTES
Constant volume in 10mL volumetric flask is set, is stored in -20 DEG C of environment, for use.
Sample solution preparation: 0.001g HRP is weighed with assay balance and is diluted to 10mmol/L PBS ultimate density (1mg/
ML), it is stored in spare in 4 DEG C of refrigerators.
It is above-mentioned to prepare all solution by through Milli-Q water purification system (18.2M Ω, 25 DEG C) and by 0.02 μm of filtering
Device filters (filter paper) resulting ultrapure water preparation.
A method of hydrogen peroxide is detected based on solid nano hole, specifically includes the following steps:
(1) preparation of nano-pore: the nano-pore of silicon nitride film obtains one in the silicon substrate deposit film of 300um thickness
Independent silicon nitride film (nominal thickness of 100nm).The preparation of this film is first to deposit one layer of low stress SiNx, then make
With low-pressure chemical vapor deposition, (deposition rate of silicon wafer is 5nm/min, chamber pressure 4mbar, underlayer temperature 810
DEG C) followed by photoetching (size of the open window of photoetching is 500um × 500um), deep reaction ion etching and tetramethyl hydrogen-oxygen
Change ammonium (TMAH) etching and forms 50 μ m, 50 μm of films.(TMAH is used for silicon etching, and Si3N4 is used as etching mask, carves for TMAH
Erosion.Etch rate is about 40 μm/h, and Si/Si3N4 Etch selectivity is greater than 1000.Si3N4 (500 μ m, 500 μ is etched with DRIE
M), 5%TMAH etching is then carried out at 80 DEG C, silicon wafer is<100>).FEI Strata 201FIB system (FEI Co.,
Hillsboro or the U.S.), it is bored on the surface of Ga+ ion when current value is 1pA with the accelerating potential bombarded surface of 30kV
Hole.Under SPOT mode, the milling time is 1.5S.(Fig. 1) is imaged by FESEM in the obtained nano-pore of Si3N4 thin film chip.It is small
The radius of opening (AS) is lower than FESEM resolution ratio, and is determined according to current-voltage (I-V) curve (Fig. 1).
(2) nano-pore HRP is enzyme functionalized: as shown in Fig. 2, giving the schematic diagram of experimental procedure in fig. 2 a.In chemistry
Before modification, Si3N4 chip is cleaned into 30min in 90 DEG C of Piranha solution, to generate hydroxyl on the surface Si3N4.In
It is repaired on the surface of enzyme molecule with primary amine in the hydroxyl (- OH) of channel Surface Creation by following steps in chemical reaction process
Decorations: firstly, covering entire silicon nitride surface on the surface Si3N4,3h is activated with 3-APTES (1%V/V is in methyl alcohol) at room temperature
(Fig. 2 b).Then, 5h is handled with Isosorbide-5-Nitrae-phenylene diisothio-cyanate (0.5%W/V is in dimethyl sulfoxide) crosslinking agent, then existed
It is washed in dimethyl sulfoxide, and is washed twice in double distilled water twice.Next step is that Isosorbide-5-Nitrae-phenylene two is different
Thiocyanates and primary amine group covalent coupling present in horseradish peroxidase molecule (HRP, 1mg/ml), reaction is overnight.Most
Afterwards, modified channel is thoroughly washed with buffer solution.
It is tested by control, it was demonstrated that the reduction of electric current is actually to be derived from the change of chemical surface coefficient.Firstly, in Si3N4
Entire silicon nitride surface is covered on surface, is placed on 3h in methanol (1ml) at room temperature.Then, chip dimethyl sulfoxide
(1ml) handles 5h, is then washed in dimethyl sulfoxide, is then washed twice in ddH2O twice.Finally, with
Buffer solution carries out staying overnight processing (1ml).
(3) small molecule hydrogen peroxide (hydrogen peroxide) is examined using the nano-pore after fixed HRP as sensor
It surveys.
1. nano-pore characterizes:
Mono-cylindrical nano-pore is prepared for by symmetrical method.Nano-pore is cleaned in 90 DEG C of Piranha solution
30min generates hydroxyl (- OH) group in hole surface.And for taper nano-pore, under neutral and alkaline pH, and water-soluble
Nano-pore in liquid is substantially filled with the charge ion opposite with the fixed group on hole wall, ionogen (- OH) Xiang Kongbi
Apply negative electrical charge.After each potential difference that outside applies, the reason of observed conductance is the cation in nano-pore
Monopole solution caused by.In contrast, cylindrical nanometer hole is asymmetric without rectifying due to lacking inherent (geometry and electrostatic)
Electric current.As shown in figure 3, Fig. 3 (A) show have on hole wall-theory of the cylindrical hole of COOH group and experiment I-V it is bent
Line, to measure I-V curve in the 1mol/L KCl electrolyte solution of pH=7.6.Electrolyte concentration is enough in solution at this time
Height, the influence of-COO- group to fixed charge almost can be ignored.Therefore, the geometry of only nano-pore controls single
Ionic flux on pore membrane.
The nano-pore of 2.HRP biological functional:
Covalent linkage of the HRP enzyme molecule on hole wall is completed with methanol soluble reagent 3-APTES.Entire film is used
3-APTES activation, 3-APTES are connected with the hydroxyl for the Native Oxide silicon layer being present in silicon nitride surface.Then Isosorbide-5-Nitrae-Asia is used
Phenyl diisothio-cyanate crosslinking agent handles the chip.Finally, crosslinking agent and primary amine carry out covalent coupling on enzyme molecule surface.
The I/V characteristic (Fig. 3) of the modified hole membranes of HRP is determined under symmetrical electrolyte conditions with 0.1mol/L KCl solution.From phase
The variation of the nanometer pore conductance for the I-V curve measurement answered is detection biomolecule mobilization and supermolecule bioconjugate in hole table
Method is predominantly detected on face.In fact, the fixation of HRP causes to significantly reduce in the hole conductance of 1V: unmodified cylinder
The conductance in shape hole is 10ns, is 3.33ns after HRP is fixed.The 66% of observed ionic conductance decline is most likely from having
Imitate the reduction of nanometer pore radius.Before HRP functionalization, nano-pore used has the radius of 35.5nm, identical nanometer
Radius of the hole after HRP functionalization is 20.4nm.It and is about that 3nm × 3.5nm × 6nm size protein is compared with molecular weight
Compared with it is very high that nanometer pore radius, which reduces 15nm,.Fig. 2 give it is this reduce pore plugging reasonable dismissal: this be mainly because
Experienced three steps of molecule assembling for fixed enzyme molecule, so as to cause aperture reduction degree contain one it is fixed
HRP molecule, there are also the silanizations and crosslinking agent in assembling.
Shown in Fig. 3 (A), it can be nanometer that the pH of the HRP nanochannel system rectification I-V curve of measurement, which relies on sexual behaviour,
The research of conduit wall success immobilised enzymes provides evidence.The oxidation that immobilised enzymes in single nanopore-channel is limited in volume
The research that can play the role of in reduction reaction: immobilized HRP enzyme is containing the bis- (3- ethyl-benzothiazole -6- of 2,20- azepine
Sulfonate) (ABTS) tested as in the system of substrate using hydrogen peroxide (hydrogen peroxide) as analyte, such as Fig. 3 (B)
It is shown.Fig. 3 (C) is shown before and after hydrogen peroxide is added, there are the substrates of 0.1mol/L KCl (pH=6.5)
The transmembrane current recorded in the presence of ABTS (1.5mmol/L).In the case where no hydrogen peroxide, I-V curve be it is smooth, it is non-
Often it is similar to the curve only recorded in 0.1mol/L KCl solution.Positive electrode current caused by 0.5mmol/L hydrogen peroxide is added
It substantially reduces, also becomes more unstable.To show that curent change is occurred in the presence of HRP, ABTS and hydrogen peroxide
The cationic product of redox reaction it is outer existing.
Small molecule hydrogen peroxide (hydrogen peroxide) is detected using the nano-pore after fixed HRP as sensor,
Using ammonium salt (ABTS) as the enlarge-effect object of small molecule hydrogen peroxide, it is expected that generating redox products ABTS+, more directly
Display hydrogen peroxide testing result.HRP nano-pore detection hydrogen peroxide exists in ABTS, 0.1mol/L KCl and only
Curent change record is as shown in Fig. 3 (D) in 0.1mol/L KCl.It can be seen from the figure that when there is no that hydrogen peroxide is added, I-
V curve and do not change substantially before, as shown in Fig. 3 (B).But as shown in Fig. 3 (C), electric current is had occurred substantially
It reduces, and apparent I-V curve becomes tortuous and shape and differs widely before, becomes no longer smooth, this is to be added
The phenomenon that occurring after 0.5mmol/L hydrogen peroxide.At the same time, with the nano-pore in similarly sized aperture, but not by HRP
Modification, to detect hydrogen peroxide curent change in ABTS, 0.1mol/L KCl and 0.1mol/L KCl, and is recorded.Hair
Existing I-V curve does not change substantially, including in the case of no addition hydrogen peroxide and addition 0.5mmol/L hydrogen peroxide, knot
Fruit is the same.By above-mentioned it is demonstrated experimentally that obtaining only HRP, ABTS and hydrogen peroxide three while in nano-pore,
Can cause the reduction such as figure electric current and the change of I-V curve shape, thus show enzymatic it is desirable that redox
Reaction generates desired product ABTS+.
3. enzyme functionalized nano hole sensor reproducibility and working curve:
Influence of the pH to the rectification characteristic of the nano-pore after modification for changing solution can be with from figure as shown in Fig. 4 (A)
Find out, change pH 2~9, be not too big on the nano-pore I-V curve shape influence after HRP modification, but has a little variation,
Show that job stability of the nano-pore at different pH is in eligible state.
There are two the more important indexs of constructed nano-pore detection system: reproducibility and sensitivity.By multiple
It measures bent in the I-V of the hydrogen peroxide there are 0.1mol/L KCl, 1.5mmol/L ABTS and in the case where be not present simultaneously
Line.The measurement process is to need every time by manual operation, changes solution type and concentration in experimental provision successively to realize
's.Fig. 4 (B) is the reproducibility of the nano-pore detection system ionic current under 1000mV voltage, it is found that electric current stabilization,
It changes very little.The above result shows that: performance of the constructed nanopore sensor in reproducibility is qualified, this nano-pore
Sensor has preferable reproducibility, and identical nanochannel can be used for multiple sensing experiment.Only need a nano-pore can
It is actually detected for several times with the completion of high quality.
I-V curve shown in Fig. 4 (C) is to measure our constructed HRP nano-pore detection systems respectively in 0.1mol/L
In the state of KCl, drawn after 1.5mmol/L ABTS and various concentration hydrogen peroxide is added.As seen from the figure, work as peroxidating
When the concentration of hydrogen is gradually increased, ionic current size is gradually reduced, and reduced ion current magnitude and hydrogen peroxide
Concentration be it is in a linear relationship, as shown in Fig. 4 (D).5~15nmol/L of the hydrogen peroxide range of linearity, obtained standard are bent
Line: Y=-183.431X+3580.726, coefficient R ^2=0.986, detection limit is minimum to have reached 10pM.
Claims (7)
1. a kind of method based on solid nano hole detection hydrogen peroxide, it is characterised in that: specifically includes the following steps:
The preparation of step A. nano-pore: nano-pore is beaten on the ultra-thin silicon nitride film of 10-100 nm；
The modification of step B. nano-pore and unimolecule enzyme sensor building: use a variety of silanization treatments, realize nanoporous surface with
Inner wall differentiation modification, a variety of silanization treatments are methyl-monosilane, trifluoro or amino silane, a variety of in carboxy-silane；
The stable assembling of single horseradish peroxidase is fixed on nano-pore hole inner wall by silane coupled molecule and obtains nano-pore biography
Sensor；
Step C. substrate specificity sensing detection: being added reaction system for various concentration hydrogen peroxide, analyze current signature,
Concentration of hydrogen peroxide standard curve is drawn, the standard curve Real_time quantitative detection hydrogen peroxide is utilized；In the step A, use
201 FIB system of FEI Strata beats nanometer with the ultra-thin silicon nitride film surface of the accelerating potential bombardment 10-100 nm of 30 kV
Hole.
2. a kind of method based on solid nano hole detection hydrogen peroxide according to claim 1, it is characterised in that: described
In step B, using comb-like graft copolymer by modifying in nanoporous surface by the way of covalently or non-covalently, with screen nano hole
Inner surface institute is electrically charged.
3. a kind of method based on solid nano hole detection hydrogen peroxide according to claim 1 or 2, it is characterised in that:
In the step B, using silane coupled molecule amino silane, 1- (3- dimethylamino-propyl) -3- ethyl-carbodiimide hydrochloride,
N-hydroxysuccinimide realizes fixation and the Site selective assembly of nano-pore inner wall peroxidase molecules.
4. a kind of method based on solid nano hole detection hydrogen peroxide according to claim 3, it is characterised in that: described
In step B, before chemical modification, by Si3N4Chip cleans 30 min in 90 DEG C of Piranha solution, in Si3N4Table
Hydroxyl is generated on face, activates 3 h in methyl alcohol with 3-APTES, with Isosorbide-5-Nitrae-phenylene diisothio-cyanate in dimethyl sulfoxide
5 h are handled, washs, is deposited in the horseradish peroxidase molecule HRP of Isosorbide-5-Nitrae-phenylene diisothio-cyanate and 1mg/ml after washing
Primary amine group carry out covalent coupling and react assembling that horseradish peroxidase is stable being fixed in nano-pore hole wall, 12-
Wash modified channel thoroughly with buffer solution afterwards for 24 hours to get nanopore sensor.
5. a kind of method based on solid nano hole detection hydrogen peroxide according to claim 1 or 4, it is characterised in that:
In the step C, by the analysis to characteristic current signal, determines the characteristic signal generated by enzymatic reaction, measure substrate
Detectable limit.
6. a kind of method based on solid nano hole detection hydrogen peroxide according to claim 5, it is characterised in that: nanometer
Hydrogen peroxide detection limit and standard curve are detected in hole, and the range of linearity is in 5-15 nmol/L, working curve: Y=- 183.431
X+3580.726, coefficient R^2=0.986, detection limit reaches 10 pmol/L.
7. modifying the peroxidating of unimolecule horseradish in a kind of nano-pore obtained by any one of claim 1,2,3,5 the method
The nanopore sensor of object enzyme.
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| CN109270154A (en) * | 2018-10-29 | 2019-01-25 | 东南大学 | Based on the tetrahedral solid nano hole unimolecule protein detection method for amplifying signal of DNA and DNA tetrahedron |
| CN109358106B (en) * | 2018-11-05 | 2021-04-13 | 中国科学院重庆绿色智能技术研究院 | Polysaccharide monomolecular structure analysis method based on solid-state nanopore technology |
| CN109856227A (en) * | 2019-02-28 | 2019-06-07 | 南通大学 | A kind of method that enzyme molecule is controllably modified in solid nano hole |
| CN110132947B (en) * | 2019-06-14 | 2021-09-28 | 福州大学 | Method for regulating electrochemiluminescence signal by charge density on surface of conical micron hole |
| CN110702563B (en) * | 2019-09-18 | 2020-08-18 | 西北工业大学 | Label-free chemical biological reaction detection method |
| JP2023549796A (en) * | 2020-11-13 | 2023-11-29 | ナンジン、ユニバーシティ | Programmable Nanoreactor for Probabilistic Sensing (PNRSS) |
| CN112834757B (en) * | 2020-12-31 | 2023-02-28 | 中国科学院重庆绿色智能技术研究院 | C-reactive protein detection method based on composite solid-state nanopore monomolecular technology |
| CN114137029B (en) * | 2021-11-26 | 2024-05-14 | 复旦大学 | TA-MS/AAO heterojunction nano-channel and preparation method thereof |
| CN115078460B (en) * | 2022-02-22 | 2023-06-30 | 南京邮电大学 | Hydrogen peroxide detection reagent based on solid-state nanopore sensor and quantitative detection method thereof |
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