CN106324066B - A method of digitlization unimolecule Electrochemical Detection alkaline phosphatase - Google Patents
A method of digitlization unimolecule Electrochemical Detection alkaline phosphatase Download PDFInfo
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- CN106324066B CN106324066B CN201610643262.9A CN201610643262A CN106324066B CN 106324066 B CN106324066 B CN 106324066B CN 201610643262 A CN201610643262 A CN 201610643262A CN 106324066 B CN106324066 B CN 106324066B
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- G—PHYSICS
- 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
- G01N27/416—Systems
- G01N27/42—Measuring deposition or liberation of materials from an electrolyte; Coulometry, i.e. measuring coulomb-equivalent of material in an electrolyte
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- G—PHYSICS
- 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
- G01N27/416—Systems
- G01N27/48—Systems using polarography, i.e. measuring changes in current under a slowly-varying voltage
Abstract
The invention discloses a kind of methods of digitlization unimolecule Electrochemical Detection alkaline phosphatase.Single alkaline phosphatase（ALP）Its substrate is catalyzed to phosphoramidic acid phenol（p‑APP）Generate the para-aminophenol of reproducibility（p‑AP）, p AP can be immediately by Ag+It is reduced into Ag0Microelectrode surface is deposited to, electric signal is obtained finally by metal dissolving voltammetry.The present invention constructs nanogold microelectrode array independent of each other using soft lithography, is used for the high-throughput parallel laboratory test of digital assay.By diluting enzyme concentration and control reaction solution volume, ensure at most to contain single enzyme molecule in each microelectrode, to realize the Single Molecule Detection of alkaline phosphatase.By the combination of digital assay, the detection limit of ALP drops to 1 aM from 50 aM, is effectively improved the sensitivity and reliability of method, and is used successfully to the detection of ALP in liver cancer cells this complex systems.This method has wide practical use in the physicochemical properties, single biomolecule detection and single cell analysis for probing into molecule.
Description
Technical field
The present invention relates to a kind of methods of digitlization unimolecule Electrochemical Detection alkaline phosphatase, belong to analysis detection neck
Domain.
Background technology
With the development of science and technology, people have been deep into unicellular, single molecules level to life and natural exploration,
Great challenge is brought to conventional analysis method.Traditional experiment is all using molecule aggregate as target, and what is obtained is substance
Average behavior, often mask many important informations.Accurately, individual molecule, especially DNA and protein etc. are delicately detected
Biomolecule can bring new breakthrough in biology and clinical medicine.In addition, Single Molecule Detection can disclose the heterogeneous of molecule
Property, the physicochemical characteristics of intermolecular interaction and individual molecule.Microscopy is optically and electrically three kinds in Single Molecule Detection
Main method.
Currently, unimolecule electrochemistry (SEM) has been achieved for larger progress, it is broadly divided into two classes：(1) the single electricity of detection
The electric current generated after bioactive molecule continuous oxidation-reduction cycle.For example, using single ferrocene molecule in ultra micro point electrode and
Redox cycle between basal electrode realizes Single Molecule Detection.Although this method can directly detect electric signal, turn without signal
It changes, but the difficult and multiple redox cycle for preparing of the uncertainty of Brownian movement, two nano gap working electrodes
It is required that limiting its application in biochemistry.(2) redox state for regulating and controlling individual molecule, in conjunction with fluorescent technique.For example, sharp
There is hyperfluorescence signal in oxidation state with molecule, and almost unstressed configuration realizes Single Molecule Detection in reduction-state.Pass through spectrum electrification
It learns, single redox event can realize highly sensitive detection, but only Some redox molecule is glimmering with what be can be changed
Luminous intensity.Generally speaking, these methods mainly probe into SME from concept, methodology, and the integrity problem of SME is its reality
A challenge in.Currently, many amplifications using nano particle and enzyme are come the sensitivity of improvement method, reachable aM is horizontal,
Possibility is provided for Single Molecule Detection.But the shortage of reliable and accurate quantitative approach hinders SEM in complex system
Using.
Herein, we have proposed a kind of methods of digitlization unimolecule Electrochemical Detection (dSMED) alkaline phosphatase.With
Enzyme product direct oxidation signal is compared, and enzymatic metallization can be about 100 times by signal boost, provides a kind of efficient signal amplification plan
Slightly, possibility is provided for unimolecule Electrochemical Detection.In digital assay, output signal reads as " 0 " or " 1 ", no longer examines
Consider concrete signal intensity, wherein " 0 " is represented without target molecule, and " 1 " representative has target molecule.Based on Poisson distribution public affairs
Formula, target concentration can be obtained simply by the probability calculation of " 0 ".The signal fluctuation that individual molecule can be effectively prevented from is brought
Influence, be suitable for the detection of small probability event in biosystem, improve the reliabilities of SME in practical applications and accurate
Property.
Invention content
Technical problem to be solved by the present invention lies in provide a kind of reliable, sensitive, accurate digitlization unimolecule electrification
The method for learning detection of alkaline phosphatase.
Technical solution provided by the present invention is specific as follows：
A method of digitlization unimolecule Electrochemical Detection alkaline phosphatase includes the following steps：
(1) using ITO slides as substrate, the film film to be designed with n microelectrode band carries out soft light as mask
It carves, after the photoresist for washing away exposure region, the ito film other than photoresist is washed away after being impregnated with concentrated hydrochloric acid；Then photoresist is washed away, then
It is impregnated with piranha solution, finally ITO slides is placed in chlorauric acid solution, is modified and is received on ITO slides using electrodeposition process
Rice gold particle, obtains the ITO slides containing n nanogold band；Wherein, n >=10；
(2) n × m through-hole is beaten on PDMS, and through-hole ranks are aligned with nanogold band, utilize plasma bonding method
PDMS is bonded to the ITO surface of glass slide containing n nanogold band, obtains nanogold microelectrode array；Wherein, m >=10；
(3) alkaline phosphatase enzyme solutions are diluted with the diethanolamine solution of pH=9.8, is then added containing to phosphoramidic acid phenol
And AgNO3Solution, be uniformly mixed, obtain premix reaction solution；
(4) isometric premix reaction solution is added dropwise into each aperture of nanogold microelectrode array, is reacted at 37 DEG C
After 30min, washes away silver ion extra in aperture with ultra-pure water and to phosphoramidic acid phenol, KCl then is added into each aperture
Solution carries out linear voltammetric scan using Ag/AgCl as to electrode and reference electrode, records the Stripping Voltammetry signal of silver；When
When being free of alkaline phosphatase in aperture, electric signal reads as 0；When at least containing an alkaline phosphatase enzyme molecule in aperture, telecommunications
Number read as 1；
(5) by electric signal by MATLAB Program transformations at polychlormal balls, the number by counting dark ball and bright ball calculate 0 or
Then 1 probability calculates the concentration of premix reaction solution alkaline phosphatase by Poisson distribution formula.
Preferably：
Photoresist used in soft lithographic is AZ4620 photoresists.
The piranha solution is volume ratio 7:3 concentrated sulfuric acid/dioxygen water mixed liquid.
Step (1) by electrodeposition process on ITO slides modified nano gold particle in the way of be：ITO slides are placed in chlorine
In auric acid solution, using Ag/AgCl as electrode, 20 seconds are stood at constant potential -0.1V.
N=10, m=50.
A concentration of 0.5M of diethanolamine solution；Premix a concentration of 1-50aM of reaction solution alkaline phosphatase.
The condition of the linear voltammetric scan of step (4) is：Potential scan ranging from -0.1V~0.2V, sweep speed are
100mV/s。
The width of each nanogold band is 100 μm in nanogold microelectrode array, two neighboring nanogold band spacing
For 3mm.
A diameter of 1mm of the upper through-holes of PDMS.
The principle of the present invention is specially：Single alkaline phosphatase (ALP) is catalyzed its substrate and is produced to phosphoramidic acid phenol (p-APP)
The para-aminophenol (p-AP) for originality of surviving, p-AP can be immediately by Ag+It is reduced into Ag0Microelectrode surface is deposited to, finally by gold
Belong to stripping voltammetry and obtains electric signal.The present invention constructs 10 × 10 gold microelectrode battle arrays independent of each other using soft lithography
Row are used for the high-throughput parallel laboratory test of digital assay.By diluting enzyme concentration and control reaction solution volume, ensure each micro- electricity
At most contain single enzyme molecule in extremely, to realize the Single Molecule Detection of alkaline phosphatase.
The present invention has the following advantages and beneficial effect：
(1) digital assay is combined by the present invention with unimolecule electrochemistry (SME), is effectively improved the reliable of method
Property and accuracy.
(2) compared with enzyme product direct oxidation signal, enzymatic metallization can be about 100 times by signal enhancing, enhance noise
Than being conducive to unimolecule Electrochemical Detection.
(3) it is based on digital assay, electric signal is read as " 0 " or " 1 ", does not have to consider specific current strength, effectively
Ground solves current fluctuation and the integrity problem of SME, and ALP detection limits drop to 1aM from 50aM, and are used successfully to complex system liver
The detection of ALP in cancer cell.
Description of the drawings
Fig. 1 (A) is unimolecule Electrochemical Detection alkaline phosphatase schematic diagram；Fig. 1 (B) is that the electric current under different enzyme concentrations is strong
Degree：(a) enzymatic metallizes；(b) enzyme product direct oxidation；Fig. 1 (C) be different enzyme concentrations under metal dissolving voltammetric signal (0.05,
0.2,0.5,0.7,1fM)。
Fig. 2 (A) is the electric current stripping curve under different solutions composition：(a)ALP+p-APP；(b)AgNO3+ALP；(c)
AgNO3+p-APP；(d)AgNO3+p-APP+ALP；Fig. 2 (B) indicate under different ALP concentration current strength (0.001,0.01,
0.02,0.05,0.1,0.15,0.2fM)。
Fig. 3 (A) is the scanning electron microscope (SEM) photograph of gold microelectrode；Fig. 3 (B) is [Fe (CN)6]3-/4-Cycle on microelectrode surface lies prostrate
Antu；Current strength under Fig. 3 (C) difference enzyme concentrations：(a) there is nanogold；(b) microelectrode array without decorated by nano-gold.
Fig. 4 (A) microelectrode arrays and electric signal read as " 0 " or " 1 " schematic diagram；Fig. 4 (B-1), Fig. 4 (B-2), Fig. 4 (B-
3) be respectively 1,5, the current distributions of lower 500 microelectrodes of 10aM enzyme concentrations；Fig. 4 (C-1), Fig. 4 (C-2), Fig. 4 (C-3) are respectively
Probability distribution (the 0,1,2,3 enzyme molecule) ratio obtained for experiment statistics under 1,5,10aM enzyme concentrations and Poisson distribution theoretical calculation
Compared with figure；Fig. 4 (D-1), Fig. 4 (D-2), Fig. 4 (D-3) be respectively 1,5, lower 500 electric signals of 10aM enzyme concentrations pass through MATLAB journeys
Sequence is converted to the ball of different colours.
Fig. 5 (A) is electric signal and Ag+The linearity curve of concentration；Fig. 5 (B) is Ag0Amount, current strength with sedimentation time change
Change curve；Fig. 5 (C) is the initial velocity inverse and Ag of enzymic catalytic reaction+The linearity curve of inverse concentration；Fig. 5 (D) is different
The current-time curvel of single enzyme molecule.
Fig. 6 (A) is average enzyme molecule number (AEM) and the relation curve (illustration for being initially added enzyme concentration in each microelectrode
For its corresponding linear curve)；Fig. 6 (B) is the specific block diagram of alkaline phosphatase detection.
Fig. 7 (A) is liver cancer cells alkaline phosphatase detects schematic diagram；Fig. 7 (B-1), Fig. 7 (B-2) are respectively Hep G2
Pass through ball that MATLAB Program transformations are different colours with 500 electric signals of MCF-7 cells；Fig. 7 (C) is alkali in liver cancer cells
The specific block diagram of acid phosphatase detection.
Specific implementation mode
Following embodiment is only used for further illustrating the present invention, but should not be construed as limiting the invention.Below with alkali
For acid phosphatase, the method based on digitlization unimolecule Electrochemical Detection enzyme is described in detail.
Embodiment 1
1, the preparation of microelectrode array
We combine the micro-processing method of soft lithographic to make microelectrode array.Using ITO slides as substrate, set
In on sol evenning machine, spin coating AZ4620 photoresists (forward 600rpm, 15s；After turn 2000rpm, 30s), ITO slides are placed in 75 DEG C
Baking platform on toast 3min, be subsequently placed on 105 DEG C of baking platform and toast 5min.It is micro- using being designed with n after the cooling of ITO slides
The film film of the microelectrode array pattern of electrode strips is as mask, in 17.4mw/cm2Uv-exposure intensity under expose
40s then uses developer solution (AZ400K:H2O=1：3) develop to ITO slides, done until exposure region photoresist just removes
Ultrapure water is used after net.Then ITO slides are placed in concentrated hydrochloric acid and are impregnated, wash off on ITO slides in addition to photoresist other
Partial ito film obtains the microelectrode array containing 10 100 μm wide ITO bands, wherein the spacing of the adjacent ITO bands of each two
About 3mm, with ultrapure water, nitrogen drying is spare.
By PDMS, (crosslinking agent B is 1 with monomer A mass ratios:10) it is poured on silicon chip, places 30min at room temperature, wait for gas
Bubble elimination is placed on heating 2h in 75 DEG C of baking ovens and is cured, and PDMS blocks is cut out, using the card punch of a diameter of 1mm in every fritter
It is spare that 100 through-holes (10 × 10, corresponding with microelectrode array) are made a call on PDMS.
The above-mentioned remaining photoresist of ITO surface of glass slide is washed away completely with absolute ethyl alcohol, piranha solution is used in combination, and ((V is (dense
Sulfuric acid)：V (hydrogen peroxide)=7:3) 2min) is impregnated.ITO slides are placed in chlorauric acid solution, constant potential -0.1V (vs.Ag/
AgCl 20 seconds are stood under), using electro-deposition method on ITO slides modified nano gold particle, obtain contain 10 nanogold bands
The ITO slides of (bandwidth is 100 μm).Finally the PDMS blocks for there are 100 holes are bonded to by plasma containing 10 nanometers
The ITO surface of glass slide of gold bar band, through-hole are aligned with nanogold pillar location, obtain 10 × 10 independent of each other nanometers of gold microelectrodes
Array.
2, the detection of single alkaline phosphatase enzyme molecule
ALP is first diluted to 20aM with 9.8 0.5M diethanolamine solutions of pH, then with p-APP containing 1mM, 100 μM
AgNO3Solution mixing, obtain premix reaction solution；0.5 μ L premix reaction solutions are taken out respectively is added drop-wise to nanogold microelectrode array
Each aperture in.The addition volume in reaction solution in ALP concentration and each aperture is premixed by control, is controlled in each aperture
About contain single enzyme molecule.After reacting 30min at 37 DEG C, after silver ion and substrate extra in aperture are washed away with ultra-pure water, add
Enter 5 μ L of 0.5M KCl solution, using Ag/AgCl as to electrode and reference electrode, within the scope of -0.1V to 0.2V, 100mV/s
Linear voltammetric scan (LSV) is carried out, the Stripping Voltammetry signal of silver is obtained.(totally 500 microelectrodes, also may be used every 5 microelectrode arrays
Microelectrode array with 10 × 50) parallel laboratory test is carried out, the electric current point of different lower 500 microelectrodes of enzyme concentration is recorded respectively
Cloth, and the probability distribution (containing 0,1,2 or multiple enzyme molecules) and Poisson distribution theoretical calculation knot for the microelectrode that experiment is obtained
Fruit is compared, to confirm the reliability and accuracy of digitlization unimolecule enzyme detection.
In order to investigate the amplification efficiency of enzymatic metallization, 5 μ L diethanolamine solutions of different enzyme concentrations is taken (to contain respectively
1mM p-APP) in microelectrode array surface, it is placed at 37 DEG C after reacting 30min, the catalysate p-AP of ALP is obtained by LSV
In the oxidation signal of electrode surface, the silver dissolution signal that oxidation signal and the enzymatic of p-AP metallize is compared.
3, in liver cancer cells alkaline phosphatase highly sensitive detection
About 10000 liver cancer cells are collected first in test tube, with Ultrasound Instrument by after clasmatosis, are centrifuged under 10000rpm
10min takes supernatant, after suitably being diluted with diethanol amine, take respectively 0.5 μ L supernatants (containing 1mM p-APP, 100 μM
AgNO3) in 500 microelectrode surfaces progress enzymatic metallization reactions.After 30min being reacted at 37 DEG C, 500 microelectrode electric signals
It reads as " 0 " (being free of enzyme molecule) or " 1 " (at least containing an enzyme molecule).Based on Poisson distribution and digital assay, liver cancer is thin
Intracellular ALP concentration can be obtained by reading as the probability calculation of " 0 ".
Interpretation of result：
1, the Single Molecule Detection of digitlization amplification
Currently, Single Molecule Detection achieves greater advance, but to detect that the minute quantity charge of individual molecule is highly difficult
's.Efficient signal amplification strategy is needed to go to improve electron transfer number (1.6fA ≈ 10 in the unit interval4A electronics is per second).This
In, the efficient signal amplification strategy of digitlization (EIM) realizes single alkaline phosphatase Molecular Detection, such as Fig. 1 (A).ALP is urged
Change the para-aminophenol (p-AP) that its substrate generates phosphoramidic acid phenol (p-APP) reproducibility, p-AP can be immediately by Ag+It is reduced into
Ag0Deposit to microelectrode surface.The reason is that p-AP and Ag+Half wave potential (versus NHE) be 0.097V and 0.799V respectively,
By suitable sedimentation time, a large amount of Ag0Electrode surface is deposited to, finally passes through linear sweep voltammetry in KCl solution
(LSV) silver dissolution signal is obtained.Compared with the direct oxidation signal of enzyme product p-AP, EIM can be about 100 times by signal enhancing, such as
Fig. 1 (B).Its main cause is：(1) combination of EIM and LSV can dual amplification electric signal, improve signal-to-noise ratio；(2) ALP can be quick
It is catalyzed the reaction of substrate molecule, and corresponding deposition of silver process；(3) EIM can effectively inhibit diffusion of the substrate from electrode surface, rich
Collect a large amount of Ag0In electrode surface；(4) EIM reactions can be improved in the microelectrode array for having modified nanogold, and nanogold particle can
As the seed of deposition of silver, influence of the deposition of silver to enzymatic activity is avoided.As Fig. 1 (C) still may be used when ALP concentration is down to 0.05fM
Stable current signal is measured, possibility is provided for unimolecule Electrochemical Detection.
In unimolecule electrochemistry, background is to have to consider the problems of.Such as Fig. 2 (A), in ALP and p-APP (a),
AgNO3With ALP (b) or AgNO3In the presence of p-APP (c), almost no signal current, shows under p-APP or ALP individualisms
It will not be by Ag+It is reduced into Ag0.Only work as AgNO3, ALP and p-APP be when existing simultaneously, just obtain very strong dissolution signal, and bright
It is aobvious to be higher than background signal, show the feasibility and specificity of unimolecule Electrochemical Detection ALP.
However, when ALP concentration dilutions are to single molecules level, one more or less enzyme molecule in each microelectrode, meeting
Larger impact is brought to the amplification efficiency and electric signal of enzyme.In addition, the heterogeneity between enzyme molecule further influences the steady of electric current
It is qualitative, limit the accuracy and reliability of unimolecule electrochemistry in practical applications.Such as Fig. 2 (B), when ALP concentration is less than
When 0.05fM, there is big fluctuation in the variation of electric current, and only some microelectrode obtains measurable current signal.
2, the characteristic of microelectrode array
The advantages such as size small, background is low, high sensitivity, fast, the spatial and temporal resolution height of response of microelectrode are attributed to, extensively
It is general to be used in unicellular and Single Molecule Detection.Herein, in order to realize high-throughput parallel laboratory test in digitlization single molecule analysis, lead to
It crosses soft lithography and constructs a kind of 10 × 10 independent of each other, active uniform microelectrode arrays.Because gold can induce silver
Deposition carries out deposition of silver as detecting electrode using gold microelectrode, can not only accelerate the reduction reaction of silver, and it is heavy to be conducive to silver
Product carries out stripping volt ampere analysis to detecting electrode surface.Using simple electro-deposition, in ITO surface of glass slide in chlorauric acid solution
It is prepared for having modified the microelectrode array of nanogold.Electronic Speculum (SEM) figure is scanned by it may be clearly seen that surface of glass slide is successfully repaiied
The gold nano grain (Fig. 3 (A)) of decorations.In 0.5mM [Fe (CN)6]3-/4-In cyclic voltammetric such as Fig. 3 (B), it is seen that between microelectrode
It is electroactive than more uniform, be suitable for high-throughput parallel laboratory test.In addition, the ITO microelectrode ratios with unmodified nanogold, have modified and have received
Electric signal can be enhanced about 3 times (Fig. 3 (C)) by the microelectrode of meter Jin, be conducive to unimolecule enzyme Electrochemical Detection.
3, the reliability of unimolecule electrochemistry is improved in conjunction with digital assay
The combination of digital assay can efficiently solve current fluctuation and the integrity problem of unimolecule electrochemistry, due to electricity
Signal is read as " 0 " (being free of enzyme molecule) or " 1 " (at least containing an enzyme molecule), does not consider further that the specific intensity of electric current, and
And target concentration can be obtained by the probability calculation of " 0 ", such as Fig. 4 (A).When without enzyme, pass through the electric signal point of 500 microelectrodes
Cloth measures background current about 2nA.Under low ALP concentration, in order to avoid influence, electric signal are read as " 0 " caused by current fluctuation
Or " 1 ".Diluted enzyme solutions are added in microelectrode at random, meeting 0,1,2 or multiple enzyme molecule of random distribution in each microelectrode,
And its corresponding probability can be by Poisson distribution formula：It is calculated, wherein x represents molecular number, λ tables
Show average enzyme molecule number in each microelectrode.When enzyme concentration is 1,5 and 10aM, λ is respectively 0.05,0.25 and 0.5.For example,
It is e containing 0 or 1 enzyme molecule probability as λ=1-1=0.368；It is e containing 2 enzyme molecule probability-1/ 2=0.184；Containing 3 enzymes
Molecule probability is e-1/ 6=0.08.
The current distribution for drawing different lower 500 microelectrodes of enzyme concentration, such as Fig. 4 (B-1)-Fig. 4 (B-3), each enzyme concentration
Under all there is prodigious current fluctuation.But the probability containing 1,2 or multiple enzyme molecules increases as enzyme concentration increases.Cause
This, 500 microelectrodes can be that 1.8 ± 1.0,8.0 ± 1.0,11.7 ± 1.5 and 15.8 ± 1.5nA is divided into four by its electric signal
Part contains 0,1,2 and multiple enzyme molecules respectively.Wherein 1.8nA represents background current, and 8nA indicates the electric signal of single enzyme molecule.
Such as Fig. 4 (C-1)-Fig. 4 (C-3), the enzyme distribution situation that experiment statistics obtain is coincide preferably with Poisson distribution the calculated results, table
The reliability and accuracy of bright digital assay unimolecule Electrochemical Detection ALP.In order to simplify statistic processes, 500 electric signals
It can be simply by the dark ball of statistics and bright ball number such as Fig. 4 (D-1)-Fig. 4 (D-3) at polychlormal balls by MATLAB Program transformations
To calculate the probability of " 0 " or " 1 ".
4, the Kinetic Characterization of single alkaline phosphatase enzyme molecule
First, enough ALP, p-APP and sedimentation time ensure Ag+Fully it is reduced into Ag0, to depict Ag+Concentration with
The linearity curve of electric signal, such as Fig. 5 (A).Under the conditions of unimolecule enzyme, electric signal increases as sedimentation time increases, and electrode
Surface A g0Deposition can be calculated by Fig. 5 (A) standard curve.Such as Fig. 5 (B), Ag0Deposition is with electric current with sedimentation time
Variation coincide preferably, shows that electric signal comes from the Stripping Voltammetry signal rather than background signal of enzymatic metallization and metal.This
Outside, such as Fig. 5 (C), the inverse and Ag of reaction speed are depicted+The linearity curve of inverse concentration, by the side Lineweaver-Burk
Journey：The Michaelis constant (Km) of single ALP calculates to obtain 0.169mM, has with the 0.15mM of routine experiment
There is comparativity, shows that single enzyme molecule still has strong catalytic activity to substrate.Finally, by different enzyme molecules with sedimentation time
Curent change situation, it was confirmed that the heterogeneity between single enzyme molecule, such as Fig. 5 (D).
5, unimolecule Electrochemical Detection alkaline phosphatase is digitized
As x=0, formula (1) can be deformed into：λ00=-lnPX=0(3).In turn, enzyme concentration can be by formula：
(4) it is calculated, wherein NAIndicate Avgadro constant (6.02 × 1023mol-1), V indicates reaction solution volume.Such as Fig. 6 (A),
Average enzyme molecule number (AEM) increases with the increase of the enzyme concentration of addition in each microelectrode, and by its illustration it is found that from 1aM
The linearity curve that can be got well under to the enzyme concentration of 20aM, and detection limit is down to 1aM, it is believed that it is most sensitive in ALP detections
Method.Therefore, by the combination of digital assay, ALP detection limits are reduced to 1aM from 50aM, improve unimolecule electrochemistry and exist
Sensitivity in practical application and reliability.
Fig. 6 (B) is to use 10pM glucose, 10pM urea, 20fM ascorbic acid and 15fM fibrin ferments gained as a control group
To microelectrode in enzyme molecule number block diagram.As can be seen from the figure experimental group (ALP containing 20aM) is apparently higher than control group, and
When interferent concentration be higher than the 3-6 order of magnitude of object when, test results are 20 times of control group, it was demonstrated that this method with than
Preferable specificity.
6, alkaline phosphatase detects in liver cancer cells
The reliability of digitlization unimolecule Electrochemical Detection is further discussed by the highly sensitive detection of ALP in complex system
And sensitivity.It is molten that from the liver cancer cells Hep G2 and breast cancer cell MCF-7 of cracking ALP is extracted using cell crushing instrument
Liquid realizes digital assay in 500 microelectrode arrays, such as Fig. 7 (A).500 electric signals are converted by MATLAB programs
At polychlormal balls, can simply be come out by dark ball number statistical to the probability of " 0 ".Such as Fig. 7 (B-1) and Fig. 7 (B-2), for
MCF-7 cells show that ALP molecules are seldom almost without bright ball, and more ALP molecules are detected in Hep G2.Its reason may be
ALP frequently as the marker in clinical diagnosis, and in serum ALP raising it is often related with the diseases such as bone, liver.Based on equation
(3) and (4), it is about 12.1aM that ALP contents in single liver cancer cells, which are calculated, shows that this method can be used successfully to biosystem
The detection of middle ALP.
Sodium vanadate (Na3VO4) it is a kind of common ALP inhibitor.In order to investigate digitlization Single Molecule Detection in complex system
In selectivity and specificity, Hep G2+Na3VO4As a control group with MCF-7.Such as Fig. 7 (C), only examined in Hep G2 cells
Apparent ALP molecules are measured, show that this method has good specificity.
Pass through above every detection, it was demonstrated that the present invention can well solve the integrity problem of unimolecule electrochemistry, push away
Its practical application in complex system is moved, and this method is simple, high sensitivity, specificity are good, is not necessarily to complex instrument.This hair
It is bright to be generalized to other single biomolecule, single celled detection.
Claims (9)
1. a kind of method of digitlization unimolecule Electrochemical Detection alkaline phosphatase, which is characterized in that include the following steps：
(1) using ITO slides as substrate, the film film to be designed with n microelectrode band carries out soft lithographic as mask,
After the photoresist for washing away exposure region, the ito film other than photoresist is washed away after being impregnated with concentrated hydrochloric acid；Then photoresist is washed away, then is used
Piranha solution impregnates, and finally ITO slides are placed in chlorauric acid solution, nanometer is modified on ITO slides using electrodeposition process
Gold particle obtains the ITO slides containing n nanogold band；Wherein, n >=10；
(2) n × m through-hole is beaten on PDMS, and through-hole ranks are aligned with nanogold band, it will using plasma bonding method
PDMS is bonded to the ITO surface of glass slide containing n nanogold band, obtains nanogold microelectrode array；Wherein, m >=10；
(3) alkaline phosphatase enzyme solutions are diluted with the diethanolamine solution of pH=9.8, be then added containing to phosphoramidic acid phenol and
AgNO3Solution, be uniformly mixed, obtain premix reaction solution；
(4) isometric premix reaction solution is added dropwise into each aperture of nanogold microelectrode array, reacts 30min at 37 DEG C
Afterwards, it washes away silver ion extra in aperture with ultra-pure water and to phosphoramidic acid phenol, KCl solution is then added into each aperture,
Linear voltammetric scan is carried out as to electrode and reference electrode using Ag/AgCl, records the Stripping Voltammetry signal of silver；Work as aperture
In be free of alkaline phosphatase when, electric signal reads as 0；When at least containing an alkaline phosphatase enzyme molecule in aperture, electric signal is read
Go out is 1；
(5) by electric signal by MATLAB Program transformations at polychlormal balls, pass through and count the number of dark ball and bright ball and calculate 0 or 1
Then probability calculates the concentration of premix reaction solution alkaline phosphatase by Poisson distribution formula.
2. the method for digitlization unimolecule Electrochemical Detection alkaline phosphatase according to claim 1, it is characterised in that：It is soft
Photoresist used in photoetching is AZ4620 photoresists.
3. the method for digitlization unimolecule Electrochemical Detection alkaline phosphatase according to claim 1, it is characterised in that：Institute
The piranha solution stated is volume ratio 7:3 concentrated sulfuric acid/dioxygen water mixed liquid.
4. the method for digitlization unimolecule Electrochemical Detection alkaline phosphatase according to claim 1, it is characterised in that：Step
Suddenly (1) by electrodeposition process on ITO slides modified nano gold particle in the way of be：ITO slides are placed in chlorauric acid solution,
Using Ag/AgCl as electrode, 20 seconds are stood at constant potential -0.1V.
5. the method for digitlization unimolecule Electrochemical Detection alkaline phosphatase according to claim 1, it is characterised in that：n
=10, m=50.
6. the method for digitlization unimolecule Electrochemical Detection alkaline phosphatase according to claim 1, it is characterised in that：Two
A concentration of 0.5M of ethanolamine solutions；Premix a concentration of 1-50aM of reaction solution alkaline phosphatase.
7. the method for digitlization unimolecule Electrochemical Detection alkaline phosphatase according to claim 1, it is characterised in that：Step
Suddenly the condition of (4) linear voltammetric scan is：Potential scan ranging from -0.1V~0.2V, sweep speed 100mV/s.
8. the method for digitlization unimolecule Electrochemical Detection alkaline phosphatase according to claim 1, it is characterised in that：It receives
The width of each nanogold band is 100 μm in rice gold microelectrode array, and two neighboring nanogold band spacing is 3mm.
9. the method for digitlization unimolecule Electrochemical Detection alkaline phosphatase according to claim 1, it is characterised in that：
A diameter of 1mm of the upper through-holes of PDMS.
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