CN106324066A - Method for detecting alkaline phosphatase through digital single-molecule electrochemistry - Google Patents

Method for detecting alkaline phosphatase through digital single-molecule electrochemistry Download PDF

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CN106324066A
CN106324066A CN201610643262.9A CN201610643262A CN106324066A CN 106324066 A CN106324066 A CN 106324066A CN 201610643262 A CN201610643262 A CN 201610643262A CN 106324066 A CN106324066 A CN 106324066A
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unimolecule
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molecule
alp
alkali phosphatase
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张志凌
伍珍
庞代文
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Wuhan University WHU
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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
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Abstract

The invention discloses a method for detecting alkaline phosphatase through digital single-molecule electrochemistry. The method comprises the steps that single alkaline phosphatase (ALP) catalyzes a substrate p-amino phenol phosphate (a-APP) of single alkaline phosphatase (ALP) to generate reductive p-aminophenol (p-AP), p-AP can immediately reduce Ag<+> into Ag<0> to be deposited on the surface of microelectrode, and an electrical signal is obtained through metal stripping voltammetry finally. According to the method, independent nanometer micro-electrode arrays are constructed by means of soft lithography and used for a digital analyzing high-throughput parallel experiment. By diluting enzyme concentration and controlling the volume reaction liquid, it is guaranteed that each microelectrode at most contains a single enzyme molecule, and therefore single molecule detection of alkaline phosphatase is achieved. By combining digital analysis, the detection limit of ALP is reduced to 1 aM from 50 aM, the sensitivity and reliability of the method are effectively improved, and the method is successfully used for ALP detection of a complex system of hepatoma carcinoma cells. The method has a wide application prospect in exploration of the physicochemical property of molecules, single biomolecule detection and single cell analysis.

Description

A kind of method of digitized unimolecule Electrochemical Detection alkali phosphatase
Technical field
A kind of method that the present invention relates to digitized unimolecule Electrochemical Detection alkali phosphatase, belongs to analysis detection neck Territory.
Background technology
Along with the development of science and technology, people have been deep into unicellular, single molecules level to life and natural exploration, Bring challenge greatly to conventional analysis method.Traditional experiment is all with molecule aggregate as target, and obtain is material Average behavior, often mask many important informations.Detect individual molecule, particularly DNA and protein etc. accurately, delicately Biomolecule, can bring new breakthrough in biology and clinical medicine.Additionally, Single Molecule Detection can disclose the heterogeneous of molecule Property, intermolecular interaction and the physicochemical characteristics of individual molecule.Microscopy, optically and electrically it it is in Single Molecule Detection three kinds Main method.
At present, unimolecule electrochemistry (SEM) has been achieved for bigger progress, is broadly divided into two classes: (1) detects single electricity The electric current produced after the circulation of bioactive molecule continuous oxidation-reduction.Such as, utilize single ferrocene molecule at ultra micro point electrode and Redox cycle between basal electrode realizes Single Molecule Detection.Although the method can directly detect the signal of telecommunication, it is not necessary to signal turns Change, but the difficult and multiple redox cycle prepared of the uncertainty of Brownian movement, two nano gap working electrodes Require to limit its application in biochemistry.(2) redox state of individual molecule, combined with fluorescent technology are regulated and controled.Such as, profit In oxidation state, there is hyperfluorescence signal with molecule, and realize Single Molecule Detection at reduction-state almost without fluorescence.By spectrum electrification Learning, single oxidoreduction event just can realize highly sensitive detection, but only Some redox molecule has variable glimmering Light intensity.Generally speaking, these methods mainly probe into the integrity problem of SME, SME from concept, methodology is that it is actual A challenge in application.At present, many utilize the amplification of nano-particle and enzyme to improve the sensitivity of method, up to aM level, Possibility is provided for Single Molecule Detection.But, reliably, accurately the shortage of quantitative approach hinder SEM in complex system Application.
Here, a kind of method that we have proposed digitized unimolecule Electrochemical Detection (dSMED) alkali phosphatase.With Enzyme product direct oxidation signal is compared, and enzymatic metallization can be by signal boost about 100 times, it is provided that a kind of efficient signal amplifies plan Slightly, possibility is provided for unimolecule Electrochemical Detection.In digital assay, output signal reads as " 0 " or " 1 ", no longer examines Considering concrete signal intensity, wherein " 0 " represents driftlessness thing molecule, and " 1 " represents and has target molecule.Public based on Poisson distribution Formula, target concentration can obtain simply by the probability calculation of " 0 ".The signal fluctuation that can be effectively prevented from individual molecule brings Impact, be suitable for the detection of small probability event in living things system, improve SME reliability in actual applications and accurately Property.
Summary of the invention
The technical problem to be solved is to provide a kind of reliable, sensitive, digitized unimolecule electrification accurately The method learning detection of alkaline phosphatase.
Technical scheme provided by the present invention is specific as follows:
A kind of method of digitized unimolecule Electrochemical Detection alkali phosphatase, comprises the following steps:
(1) using ITO slide as substrate, to be designed with the film film of n microelectrode band as mask, soft light is carried out Carve, after washing away the photoresist of exposure region, after soaking with concentrated hydrochloric acid, wash away the ito film beyond photoresist；Then photoresist is washed away, then Use piranha solution soaking, finally ITO slide is placed in chlorauric acid solution, utilize electrodeposition process to modify on ITO slide and receive Rice gold grain, obtains the ITO slide containing n nanometer gold band；Wherein, n >=10；
(2) on PDMS, beat n × m through hole, through hole ranks are alignd with nanometer gold band, utilize plasma bonding method PDMS is bonded to the ITO surface of glass slide containing n nanometer gold band, obtains nanometer gold microelectrode array；Wherein, m >=10；
(3) with the diethanolamine solution of pH=9.8, alkaline phosphatase enzymatic solution is diluted, be subsequently adding containing to phosphoramidic acid phenol And AgNO3Solution, mix homogeneously, obtain premix reactant liquor；
(4) in each aperture of nanometer gold microelectrode array, drip isopyknic premix reactant liquor, react at 37 DEG C After 30min, wash away silver ion unnecessary in aperture and to phosphoramidic acid phenol with ultra-pure water, in each aperture, then add KCl Solution, using Ag/AgCl silk as to electrode and reference electrode, carries out linear voltammetric scan, the Stripping Voltammetry signal of record silver；When Time in aperture without alkali phosphatase, the signal of telecommunication reads as 0；When in aperture at least containing an alkali phosphatase molecule, telecommunications Number read as 1；
(5) signal of telecommunication is become polychlormal balls by MATLAB Program transformation, by the number adding up dark ball and bright ball calculate 0 or The probability of 1, then calculates the concentration of premix reactant liquor alkaline phosphatase by Poisson distribution formula.
Preferably:
The photoresist that soft lithographic is used is AZ4620 photoresist.
Described piranha solution is the concentrated sulphuric acid/hydrogen peroxide mixed liquor of volume ratio 7:3.
Step (1) utilizes electrodeposition process mode of modified nano gold granule on ITO slide to be: ITO slide is placed in chlorine In auric acid solution, with Ag/AgCl as electrode, under constant potential-0.1V, stand 20 seconds.
N=10, m=50.
The concentration of diethanolamine solution is 0.5M；The concentration of premix reactant liquor alkaline phosphatase is 1-50aM.
The condition of the linear voltammetric scan of step (5) is: potential scan scope is-0.1V～0.2V, and sweep speed is 100mV/s。
In nanometer gold microelectrode array, the width of each nanometer gold band is 100 μm, adjacent two nanometer gold striation widthses For 3mm.
A diameter of 1mm of the upper through hole of PDMS.
The principle of the present invention is particularly as follows: single alkali phosphatase (ALP) is catalyzed its substrate to phosphoramidic acid phenol (p-APP) product The para-aminophenol (p-AP) of originality of surviving, p-AP can be immediately by Ag+It is reduced into Ag0Deposit to microelectrode surface, finally by gold Belong to stripping voltammetry and obtain the signal of telecommunication.The present invention utilizes soft lithography to construct 10 × 10 gold microelectrode battle arrays independent of each other Row, for the high flux parallel laboratory test of digital assay.By dilution enzyme concentration and control reactant liquor volume, it is ensured that each micro-electricity At most contain single enzyme molecule in extremely, thus realize the Single Molecule Detection of alkali 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, signal can be strengthened about 100 times by enzymatic metallization, enhances noise Ratio, beneficially unimolecule Electrochemical Detection.
(3) based on digital assay, the signal of telecommunication is read as " 0 " or " 1 ", need not consider concrete current intensity, effectively Solve current fluctuation and the integrity problem of SME, ALP detection limit drops to 1aM from 50aM, and is used successfully to complex system liver The detection of ALP in cancerous cell.
Accompanying drawing explanation
Fig. 1 (A) is unimolecule Electrochemical Detection alkali phosphatase schematic diagram；Fig. 1 (B) is that the electric current under different enzyme concentration is strong Degree: (a) enzymatic metallizes；The product direct oxidation of (b) enzyme；Fig. 1 (C) be metal dissolving voltammetric signal under different enzyme concentration (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) represent current intensity under different ALP concentration (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-Circulation volt on microelectrode surface Antu；Current intensity under the different enzyme concentration of Fig. 3 (C): (a) has nanometer gold；B () is without the microelectrode array of decorated by nano-gold.
Fig. 4 (A) microelectrode array and the signal of telecommunication 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 DISTRIBUTION of lower 500 microelectrodes of 10aM enzyme concentration；Fig. 4 (C-1), Fig. 4 (C-2), Fig. 4 (C-3) are respectively Be 1,5, experiment statistics and Poisson distribution Theoretical Calculation obtain under 10aM enzyme concentration probability distribution (0,1,2,3 enzyme molecule) ratio Relatively scheme；Fig. 4 (D-1), Fig. 4 (D-2), Fig. 4 (D-3) be respectively 1,5, lower 500 signals of telecommunication of 10aM enzyme concentration are by MATLAB journey Sequence is converted to the ball of different colours.
Fig. 5 (A) is the signal of telecommunication and Ag+The linearity curve of concentration；Fig. 5 (B) is Ag0Amount, current intensity are with the change of sedimentation time Change curve；Fig. 5 (C) is initial velocity inverse and the 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 molecular number (AEM) and the relation curve (illustration being initially added enzyme concentration in each microelectrode For its corresponding linear curve)；Fig. 6 (B) is the specificity block diagram of alkali phosphatase detection.
Fig. 7 (A) is that hepatoma carcinoma cell alkaline phosphatase detects schematic diagram；Fig. 7 (B-1), Fig. 7 (B-2) are respectively Hep G2 It is the ball of different colours with 500 signals of telecommunication of MCF-7 cell by MATLAB Program transformation；Fig. 7 (C) is alkali in hepatoma carcinoma cell The specificity block diagram of acid phosphatase detection.
Detailed description of the invention
Following example are only used for further illustrating the present invention, but should not be construed as limitation of the present invention.Below with alkali As a example by acid phosphatase, method based on digitized 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 and have made microelectrode array.Use ITO slide as substrate, put On sol evenning machine, spin coating AZ4620 photoresist (forward 600rpm, 15s；After turn 2000rpm, 30s), ITO slide is placed in 75 DEG C Baking platform on toast 3min, be subsequently placed on the baking platform of 105 DEG C baking 5min.After ITO slide cools down, use that to be designed with n micro- The film film of the microelectrode array pattern of electrode strips is as mask, in 17.4mw/cm2Uv-exposure intensity under expose 40s, uses developer solution (AZ400K:H subsequently2O=1:3) ITO slide is developed, just remove dry to exposure region photoresist Ultrapure water is used after Jing.Then ITO slide is placed in concentrated hydrochloric acid immersion, washes on ITO slide in addition to photoresist other off The ito film of part, obtains the microelectrode array containing 10 100 μm width ITO bands, wherein, the spacing of each two adjacent ITO band Being about 3mm, with ultrapure water, nitrogen dries up standby.
Being poured on silicon chip by PDMS (crosslinking agent B and monomer A mass ratio are 1:10), ambient temperatare puts 30min, treats gas In bubble elimination is placed on 75 DEG C of baking ovens, heating 2h solidifies, and cuts out PDMS block, uses the card punch of a diameter of 1mm at every fritter PDMS is upper, and to make a call to 100 through holes (10 × 10, corresponding with microelectrode array) standby.
With dehydrated alcohol, the above-mentioned remaining photoresist of ITO surface of glass slide is washed away completely, and ((V is (dense with piranha solution Sulphuric acid): V (hydrogen peroxide)=7:3)) soak 2min.ITO slide is placed in chlorauric acid solution, constant potential-0.1V (vs.Ag/ AgCl) stand 20 seconds under, use electro-deposition method modified nano gold granule on ITO slide, obtain containing 10 nanometer gold bands The ITO slide of (bandwidth is 100 μm).Finally the PDMS block having 100 holes is bonded to containing 10 nanometers by plasma The ITO surface of glass slide of gold bar band, through hole aligns with nanometer gold pillar location, obtains 10 × 10 nanometer gold microelectrodes independent of each other Array.
2, the detection of single alkali phosphatase molecule
First with pH 9.8 0.5M diethanolamine solution, ALP is diluted to 20aM, then with containing 1mM p-APP, 100 μMs AgNO3Solution mixing, obtain premix reactant liquor；Take out 0.5 μ L premix reactant liquor respectively and be added drop-wise to nanometer gold microelectrode array Each aperture in.By ALP concentration in control premix reactant liquor and the addition volume in each aperture, control in each aperture About contain single enzyme molecule.After reacting 30min at 37 DEG C, after silver ion unnecessary in washing away aperture with ultra-pure water and substrate, add Enter 0.5M KCl solution 5 μ L, using Ag/AgCl silk as to electrode and reference electrode, in the range of-0.1V to 0.2V, 100mV/s Carry out linear voltammetric scan (LSV), obtain the Stripping Voltammetry signal of silver.Every 5 microelectrode arrays (totally 500 microelectrodes, it is possible to Microelectrode array with 10 × 50) carry out a parallel laboratory test, the electric current of different lower 500 microelectrodes of enzyme concentration of record divides respectively Cloth, and the probability distribution (containing 0,1,2 or multiple enzyme molecules) of microelectrode experiment obtained and Poisson distribution Theoretical Calculation tie Fruit is compared, and confirms reliability and accuracy that digitized unimolecule enzyme detects.
In order to investigate the metallized amplification efficiency of enzymatic, the 5 μ L diethanolamine solutions taking different enzyme concentration respectively (contain 1mM p-APP) in microelectrode array surface, after being placed at 37 DEG C reaction 30min, obtained the catalysate p-AP of ALP by LSV At the oxidation signal of electrode surface, metallized to oxidation signal and the enzymatic of p-AP silver dissolution signal is compared.
3, the highly sensitive detection of alkali phosphatase in hepatoma carcinoma cell
First about 10000 hepatoma carcinoma cell are collected in test tube, by Ultrasound Instrument by after cell breakage, centrifugal under 10000rpm 10min, takes supernatant, after suitably diluting with diethanolamine, take respectively 0.5 μ L of supernatant liquid (containing 1mM p-APP, 100 μMs AgNO3) carry out enzymatic metallization reaction in 500 microelectrode surfaces.After reacting 30min at 37 DEG C, 500 microelectrode signals of telecommunication Read as " 0 " (without enzyme molecule) or " 1 " (at least containing an enzyme molecule).Based on Poisson distribution and digital assay, hepatocarcinoma is thin Intracellular ALP concentration can obtain by reading as the probability calculation of " 0 ".
Interpretation of result:
1, the Single Molecule Detection that digitized amplifies
At present, Single Molecule Detection achieves greater advance, but the very small amount electric charge of individual molecule to be detected is highly difficult 's.Need efficient signal to amplify strategy to go to improve electron transfer number (1.6fA ≈ 10 in the unit interval4Individual electronics is per second).This In, the efficient signal of digitized (EIM) amplifies strategy, it is achieved that single alkali phosphatase Molecular Detection, such as Fig. 1 (A).ALP urges Changing its substrate and phosphoramidic acid phenol (p-APP) produces the para-aminophenol (p-AP) of reproducibility, p-AP can be immediately by Ag+It is reduced into Ag0Deposit to microelectrode surface.Reason is p-AP and Ag+Half wave potential (versus NHE) be 0.097V and 0.799V respectively, By suitable sedimentation time, substantial amounts of Ag0Deposit to electrode surface, finally in KCl solution, pass through linear sweep voltammetry (LSV) silver dissolution signal is obtained.Compared with the direct oxidation signal of enzyme product p-AP, signal can be strengthened about 100 times by EIM, as Fig. 1 (B).Its main cause is: the combination of (1) EIM and LSV can the dual amplification signal of telecommunication, improve signal to noise ratio；(2) ALP can be quick The reaction of catalytic substrate molecule, and corresponding deposition of silver process；(3) EIM can effectively suppress the substrate diffusion from electrode surface, rich Collect a large amount of Ag0At electrode surface；(4) microelectrode array having modified nanometer gold can improve EIM reaction, and nanogold particle can Seed as deposition of silver, it is to avoid the deposition of silver impact on enzymatic activity.Such as Fig. 1 (C), as ALP concentration as little as 0.05fM, still may be used Record stable current signal, provide possibility for unimolecule Electrochemical Detection.
In unimolecule electrochemistry, background is the problem that have to consider.Such as Fig. 2 (A), at ALP Yu p-APP (a), AgNO3With ALP (b), or AgNO3In the presence of p-APP (c), almost without current signal, show under p-APP or ALP individualism Will not be by Ag+It is reduced into Ag0.Only work as AgNO3, ALP and p-APP simultaneously in the presence of, just obtain the strongest dissolution signal, and bright Aobvious higher than background signal, indicate feasibility and the specificity of unimolecule Electrochemical Detection ALP.
But, when ALP concentration dilution to single molecules level, one more or less enzyme molecule, meeting in each microelectrode Amplification efficiency and the signal of telecommunication to enzyme bring considerable influence.Additionally, the intermolecular heterogeneity of enzyme affects the steady of electric current further Qualitative, limit unimolecule electrochemistry accuracy in actual applications and reliability.Such as Fig. 2 (B), when ALP concentration is less than During 0.05fM, there is big fluctuation in the change of electric current, and only some microelectrode obtains measurable current signal.
2, the characteristic of microelectrode array
The advantages such as size little, background is low, highly sensitive, response is fast, the spatial and temporal resolution height owing to microelectrode, the most extensively General in unicellular and Single Molecule Detection.Here, in order to realize high flux parallel laboratory test in digitized single molecule analysis, logical Cross soft lithography and construct a kind of 10 × 10 uniform microelectrode arrays independent of each other, active.Because gold can induce silver Deposition, uses gold microelectrode to carry out deposition of silver as detecting electrode, can not only accelerate the reduction reaction of silver, and be conducive to silver heavy Amass detecting electrode surface and carry out stripping volt ampere analysis.Utilize simple electro-deposition, in ITO surface of glass slide in chlorauric acid solution It is prepared for having modified the microelectrode array of nanometer gold.It is scanned by Electronic Speculum (SEM) figure 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 Electroactive ratio is more uniform, it is adaptable to high flux parallel laboratory test.Additionally, with the ITO microelectrode ratio of unmodified nanometer gold, modified and received The signal of telecommunication can be strengthened about 3 times (Fig. 3 (C)), beneficially unimolecule enzyme Electrochemical Detection by the microelectrode of meter Jin.
3, combine digital assay and improve the reliability of unimolecule electrochemistry
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 " (without enzyme molecule) or " 1 " (at least containing an enzyme molecule), does not consider further that the concrete intensity of electric current, and And target concentration can be obtained by the probability calculation of " 0 ", such as Fig. 4 (A).In time without enzyme, divided by the signal of telecommunication of 500 microelectrodes Cloth, records background current about 2nA.Under low ALP concentration, the impact caused in order to avoid current fluctuation, the signal of telecommunication is read as " 0 " Or " 1 ".The enzymatic solution of dilution is added in microelectrode at random, meeting random distribution 0,1,2 or multiple enzyme molecule in each microelectrode, And its corresponding probability can be by Poisson distribution formula:Being calculated, wherein x represents molecular number, λ table Show average enzyme molecular number in each microelectrode.When enzyme concentration is 1,5 and during 10aM, and λ is respectively 0.05,0.25 and 0.5.Such as, When λ=1, it is e containing 0 or 1 enzyme molecule probability-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.
Draw the CURRENT DISTRIBUTION of different lower 500 microelectrodes of enzyme concentration, such as Fig. 4 (B-1)-Fig. 4 (B-3), each enzyme concentration Under all occur in that the biggest current fluctuation.But the probability containing 1,2 or multiple enzyme molecules increases along with enzyme concentration and increases.Cause This, 500 microelectrodes can be 1.8 ± 1.0,8.0 ± 1.0,11.7 ± 1.5 by its signal of telecommunication and 15.8 ± 1.5nA is divided into four Part, respectively containing 0,1,2 and multiple enzyme molecule.Wherein 1.8nA represents background current, and 8nA represents the signal of telecommunication of single enzyme molecule. Such as Fig. 4 (C-1)-Fig. 4 (C-3), the enzyme distribution situation that experiment statistics obtains and Poisson distribution the calculated results coincide preferably, table The reliability of bright digital assay unimolecule Electrochemical Detection ALP and accuracy.In order to simplify statistic processes, 500 signals of telecommunication Polychlormal balls is become by MATLAB Program transformation, such as Fig. 4 (D-1)-Fig. 4 (D-3), can be simply by adding up dark ball and bright ball number Calculate " 0 " or the probability of " 1 ".
4, the Kinetic Characterization of single alkali phosphatase molecule
First, enough ALP, p-APP and sedimentation time ensure Ag+Fully it is reduced into Ag0, thus depict Ag+Concentration with The linearity curve of the signal of telecommunication, such as Fig. 5 (A).Under the conditions of unimolecule enzyme, the signal of telecommunication increases along with sedimentation time and increases, and electrode Surface A g0Deposition can be calculated by Fig. 5 (A) standard curve.Such as Fig. 5 (B), Ag0Deposition and electric current are with sedimentation time Change coincide preferably, shows that the signal of telecommunication comes from enzymatic and metallizes and the Stripping Voltammetry signal of metal, rather than background signal.This Outward, such as Fig. 5 (C), inverse and the Ag of response speed are depicted+The linearity curve of inverse concentration, by Lineweaver-Burk side Journey:The Michaelis constant (Km) of single ALP calculates 0.169mM, has with the 0.15mM of normal experiment There is comparability, show that single enzyme molecule still has strong catalysis activity to substrate.Finally, by different enzyme molecules with sedimentation time Curent change situation it was confirmed the intermolecular heterogeneity of single enzyme, such as Fig. 5 (D).
5, digitized unimolecule Electrochemical Detection alkali phosphatase
As x=0, formula (1) can be deformed into: λ0=-lnPX=0(3).And then, enzyme concentration can be by formula: It is calculated, wherein NARepresent Avogadro's number (6.02 × 1023mol-1), V represents reactant liquor volume.Such as Fig. 6 (A), in each microelectrode, average enzyme molecular number (AEM) increases with the increase of enzyme concentration added, and from its illustration, from The linearity curve that can obtain under the enzyme concentration of 1aM to 20aM, and detection limit as little as 1aM, it is believed that be the sensitiveest in ALP detection Method.Therefore, by the combination of digital assay, ALP detection limit is reduced to 1aM from 50aM, improves unimolecule electrochemistry Sensitivity in actual applications and reliability.
Fig. 6 (B) is with 10pM glucose, 10pM carbamide, 20fM ascorbic acid and 15fM thrombin gained as a control group To microelectrode in enzyme molecular number block diagram.As can be seen from the figure experimental group (containing 20aM ALP) is apparently higher than matched group, and When interferent concentration is higher than 3-6 the order of magnitude of object, test results is 20 times of matched group, it was demonstrated that the method has ratio Preferably specificity.
6, alkali phosphatase detection in hepatoma carcinoma cell
By the reliability of the highly sensitive detection digitized discussed further unimolecule Electrochemical Detection of ALP in complex system And sensitivity.Cell crushing instrument is utilized to extract ALP from the hepatoma carcinoma cell Hep G2 and breast cancer cell MCF-7 of cracking molten Liquid, realizes digital assay in 500 microelectrode arrays, such as Fig. 7 (A).By MATLAB program, 500 signals of telecommunication are changed Become polychlormal balls, thus the probability of " 0 " can simply by dark ball number statistical out.Such as Fig. 7 (B-1) and Fig. 7 (B-2), for MCF-7 cell, almost without bright ball, shows that ALP molecule is little, and more ALP molecule detected in Hep G2.Its reason is probably ALP is frequently as the mark in clinical diagnosis, and in serum, the rising of ALP is the most relevant with the disease such as bone, liver.Based on equation (3) and (4), in being calculated single hepatoma carcinoma cell, ALP content is about 12.1aM, shows that the method can be used successfully to living things system The detection of middle ALP.
Vanadic acid sodium (Na3VO4) it is a kind of conventional ALP inhibitor.In order to investigate digitized Single Molecule Detection at complex system In selectivity and specificity, Hep G2+Na3VO4With MCF-7 as a control group.Such as Fig. 7 (C), only Hep G2 cell is examined Measure obvious ALP molecule, show that the method has good specificity.
By above every detection, it was demonstrated that the present invention can solve the integrity problem of unimolecule electrochemistry well, pushes away Its actual application in complex system dynamic, and the method is simple, highly sensitive, specificity is good, it is not necessary to complex instrument.This Bright can also be generalized to other single biomolecule, single celled detection.

Claims (9)

1. the method for a digitized unimolecule Electrochemical Detection alkali phosphatase, it is characterised in that comprise the following steps:
(1) using ITO slide as substrate, to be designed with the film film of n microelectrode band as mask, soft lithographic is carried out, After washing away the photoresist of exposure region, after soaking with concentrated hydrochloric acid, wash away the ito film beyond photoresist；Then wash away photoresist, then use Piranha solution soaking, is finally placed in ITO slide in chlorauric acid solution, utilizes electrodeposition process to modify nanometer on ITO slide Gold grain, obtains the ITO slide containing n nanometer gold band；Wherein, n >=10；
(2) on PDMS, beat n × m through hole, through hole ranks are alignd with nanometer gold band, utilize plasma bonding method to incite somebody to action PDMS is bonded to the ITO surface of glass slide containing n nanometer gold band, obtains nanometer gold microelectrode array；Wherein, m >=10；
(3) with the diethanolamine solution of pH=9.8, alkaline phosphatase enzymatic solution is diluted, be subsequently adding containing to phosphoramidic acid phenol and AgNO3Solution, mix homogeneously, obtain premix reactant liquor；
(4) in each aperture of nanometer gold microelectrode array, drip isopyknic premix reactant liquor, at 37 DEG C, react 30 min After, wash away silver ion unnecessary in aperture and to phosphoramidic acid phenol with ultra-pure water, in each aperture, then add KCl solution, Using Ag/AgCl silk as to electrode and reference electrode, carry out linear voltammetric scan, the Stripping Voltammetry signal of record silver；Work as aperture In without alkali phosphatase time, the signal of telecommunication reads as 0；When in aperture at least containing an alkali phosphatase molecule, the signal of telecommunication is read Go out is 1；
(5) signal of telecommunication is become polychlormal balls by MATLAB Program transformation, calculate 0 or 1 by the number adding up dark ball and bright ball Probability, then calculates the concentration of premix reactant liquor alkaline phosphatase by Poisson distribution formula.
The method of digitized unimolecule Electrochemical Detection alkali phosphatase the most according to claim 1, it is characterised in that: soft The photoresist that photoetching is used is AZ4620 photoresist.
The method of digitized unimolecule Electrochemical Detection alkali phosphatase the most according to claim 1, it is characterised in that: institute The piranha solution stated is the concentrated sulphuric acid/hydrogen peroxide mixed liquor of volume ratio 7:3.
The method of digitized unimolecule Electrochemical Detection alkali phosphatase the most according to claim 1, it is characterised in that: step Suddenly (1) utilizes electrodeposition process mode of modified nano gold granule on ITO slide to be: be placed in chlorauric acid solution by ITO slide, With Ag/AgCl as electrode, under constant potential-0.1 V, stand 20 seconds.
The method of digitized unimolecule Electrochemical Detection alkali phosphatase the most according to claim 1, it is characterised in that: n= 10, m=50。
The method of digitized unimolecule Electrochemical Detection alkali phosphatase the most according to claim 1, it is characterised in that: two The concentration of ethanolamine solutions is 0.5 M；The concentration of premix reactant liquor alkaline phosphatase is 1-50 aM.
The method of digitized unimolecule Electrochemical Detection alkali phosphatase the most according to claim 1, it is characterised in that: step Suddenly the condition of (5) linear voltammetric scan is: potential scan scope is-0.1 V ~ 0.2 V, and sweep speed is 100 mV/s.
The method of digitized unimolecule Electrochemical Detection alkali phosphatase the most according to claim 1, it is characterised in that: receive In rice gold microelectrode array, the width of each nanometer gold band is 100 μm, and adjacent two nanometer gold striation widthses are 3 mm.
The method of digitized unimolecule Electrochemical Detection alkali phosphatase the most according to claim 1, it is characterised in that: A diameter of 1 mm of the upper through hole of PDMS.
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