CN104569100B - Electrochemical sensor array based on functionalization graphene - Google Patents
Electrochemical sensor array based on functionalization graphene Download PDFInfo
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Abstract
The present invention relates to a kind of electrochemical sensor array based on functionalization graphene.The electrochemical sensor array, including：Electrode, and modification is in the reduced graphene probe of stable BSA of electrode surface reduced graphene probe and calf thymus DNA functionalization；The concentration of reduced graphene the probe stable BSA is identical with the concentration of the reduced graphene probe of the calf thymus DNA functionalization.The sensor array platform, can be achieved to different cancer cells, the differentiation of same cancer cell different conditions such as resistant breast cancer cell and metastatic breast cancer cell sample.It is combined with the bionical interaction between cell and with the electrochemical method of hypersensitivity by design sensor recognition unit, significantly improve the detection performance of sensor, and the differentiation of the different type cell on 100 cellular levels can be realized, detection accuracy can reach 100%, and the test limit to the cancer cell of single type can reach individual cell level.
Description
Technical field
The present invention relates to a kind of electrochemical sensor, and in particular to a kind of electrochemical sensor based on functionalization graphene
Array.
Background technology
Although research of the whole world to cancer has put into great effort and fund of scientific research, cancer is still to cause the master of death
One of reason is wanted, and massive losses are brought to social economy.In the past few decades, although developed many advanced treatments of cancer
Method, but the early diagnosis of cancer is still the key for the treatment of of cancer.The quick diagnosis before cancer does not spread also and spread
Advancing of disease can be controlled in time with early intervention, the death rate of cancer can be not only reduced, and improve the quality of life of patient,
Health care costs can also be reduced simultaneously.The early detection of cancer is typically the analysis for tumour marker, and it is to assess cancer hair
Hair tonic exhibition and the indicative chemical substance of medication effect, are present in blood, urine or tissue, can be special
Cell, small molecule, gene, enzyme or hormone etc..At present, the work of developing cancer early stage biological label analyte detection is focused primarily upon
The following aspects：Find significantly more efficient early-stage cancer diagnostic marker thing；Liquid biological marker is found to reduce sampling pair
The invasive of patient；The accuracy of cancer diagnosis is improved with reference to a variety of efficient diagnosis indexs；Development cost performance is high, response is rapid,
Sensitivity and the high detection means of selectivity are used to detect that development of the clinical and incubation period cancer simultaneously to cancer carries out prognosis
Deng.
In the past few decades, the research in the field achieves great and substantial progress.It is various valuable
Detection method be developed, they are mainly to some intracellular markers, such as RNA or egg of cell inner expression imbalance
In vain or the marker (such as be overexpressed antibody) of cell surface carries out specific recognition detection.These methods include polymerization
PCR (PCR), enzyme-linked immunosorbent assay (ELISA), surface plasmon resonance, micro-cantilever method, biology
Imaging etc..However, screening ligands specific (the aglucon phyletic evolution technology of such as external index concentration, SELEX) and structure are special
Sensor often time-consuming and expensive, while also needing to have the specific biological marker used certain
Solution.Due to be difficult to find that a specific index can distinguish difference or specific cancer cell between cancer cell and they
Difference between transfering state cancer cell, the development in the field is severely limited.Therefore, a kind of effective inspection of development is needed badly
Platform is surveyed to distinguish different cancer cells and same cancer cell different conditions, to provide more rich examine for the early diagnosis of cancer
Disconnected information.
The content of the invention
The invention solves the problems that there is provided a kind of electrochemical sensing based on functionalization graphene for technical problem of the prior art
Device array, the electrochemical sensor array can be realized to different cancer cells, the differentiation of same cancer cell different conditions, significantly improved
The detection performance of sensor.
In order to solve the above-mentioned technical problem, technical scheme is specific as follows：
A kind of electrochemical sensor array based on functionalization graphene, including：
Electrode；
And modification is in reduced graphene the probe stable BSA of electrode surface；
And modification is in the reduced graphene probe of the calf thymus DNA functionalization of electrode surface；
The concentration of reduced graphene the probe stable BSA and the reduced graphene of the calf thymus DNA functionalization
The concentration of probe is identical.
In the above-mentioned technical solutions, the electrode is glass-carbon electrode, ITO or screen printing electrode.
In the above-mentioned technical solutions, the concentration of reduced graphene the probe stable BSA and the calf thymus DNA work(
The concentration of the reduced graphene probe of energyization is 50 μ g/mL.
In the above-mentioned technical solutions, reduced graphene the probe stable BSA is prepared by following methods：
50 μ L concentration are 1.0mg/mL graphene oxide, and 250 μ L water and 200 μ L concentration mix for 50mg/mL BSA,
The pH value of solution is transferred to rapidly 12 by the NaOH for being 1.0M with concentration, and solution is stirred 24 hours at 90 DEG C, and solution colour is by yellowish-brown
Color gradually becomes black, and product 12000rpm is cleaned with pure water after being collected by centrifugation and is re-dispersed into after three times in a certain amount of water.
In the above-mentioned technical solutions, the reduced graphene probe of the calf thymus DNA functionalization is by following method systems
It is standby：
Calf thymus DNA first obtains single-stranded DNA in 1-2 hours in 95 DEG C of heating；
The single-stranded DNA that the graphene oxide that 10mL concentration is 0.5mg/mL is 2mg/mL with 10mL concentration is mixed, and adds 8
μ L 85wt% hydrazine hydrate, mixed solution is stirred 1 hour at 100 DEG C, product is collected by centrifugation cleaned with pure water after redisperse to one
In quantitative water.
The beneficial effects of the invention are as follows：
The electrochemical sensor array based on functionalization graphene that the present invention is provided, the sensor array platform, can be real
Now to different cancer cells, the area of same cancer cell different conditions such as resistant breast cancer cell and metastatic breast cancer cell sample
Point.Design sensor recognition unit (i.e. functionalization graphene probe) bionical interaction between cell and by itself and superelevation
The electrochemical method of sensitivity is combined, and significantly improves the detection performance of sensor.In addition, the sensor array built can be with
The differentiation of the different type cell on 100 cellular levels is realized, detection accuracy can reach 100%, to single type
The test limit of cancer cell can reach individual cell level.The Electrochemical Detection platform based on functionalization graphene that the present invention is reported
New method and thinking is provided for the clinical diagnosis of cancer.
The advantage and performance summary for the electrochemical sensor array based on functionalization graphene that the present invention is provided are as follows：
(1) sensitivity is high, and the test limit to the cancer cell of single type can reach individual cell level；
(2) electrochemical sensor array can reach 100% cell differentiation accuracy, using the teaching of the invention it is possible to provide abundant diagnosis letter
Breath；
(3) diagnosable tumor class is more, and toxic side effect is small；
(4) detection cycle is short, and controllability is strong；
(5) instrument is simple, and result judgement is directly perceived.
Brief description of the drawings
The present invention is described in further detail with reference to the accompanying drawings and detailed description.
Fig. 1 is modified electrode BSA/CCG and DNA/CCG electrochemical impedance Nyquist spectrogram.
Fig. 2 is differentiation of the electrochemical sensor array of the present invention to 5 kinds of normal cells and cancer cell.
Fig. 3 is differentiation of the electrochemical sensor array of the present invention to 5 kinds of cancer cells.
The electrochemical sensor array that Fig. 4 is the present invention is thin for MCF-7 breast cancer cells and its metastatic and drug resistance
The detection of born of the same parents.
Fig. 5 is detection of the electrochemical sensor array of the present invention to various concentrations cancer cell.
Fig. 6 is the linear relationship between MCF-7 cell concentrations and Assessing parameters.
Embodiment
The present invention invention thought be：
The design and synthesis for the electrochemical sensor array that the present invention is provided：Cell membrane is main by amphipathic phosphatide, egg
White matter and carbohydrate composition.The cell of difference in functionality, phosphatide, albumen and the sugared composition of its cell membrane surface also can be different.
Different types of cell membrane is by its unique physiochemical properties for constituting, showing cell membrane surface, such as electric charge, hydrophilic and hydrophobic
It would also vary from.The difference of this physiochemical properties can be made a distinction with " chemical nose " method, because this method master
Different responses are produced dependent on the selectively acting between a variety of recognition units and target cell in sensor array
Signal.Therefore, we construct an electrochemical sensor array using the graphene of two kinds of biomolecule functionalization and are used for not
The differentiation of same type cell.Have the interaction of bionical objects system between the probe unit and cell of preparation, such as protein-protein and
Interaction between protein-nucleic acid.Functionalization graphene composite nano materials are modified electrode surface and visited as identification first
Pin.When cell is incubated in the electrode surface modified, functionalization graphene probe can with the phosphatide of cell membrane surface, albumen and
Different degrees of electrostatic interaction and hydrophobic effect occurs for carbohydrate.Cell can be fixed on electrode surface, Jin Erzhuan by these active forces
It is melted into overdelicate electrochemical impedance signal.Because different graphene probes electric charge in itself and hydrophilic and hydrophobic are different, they with
There is the difference on chemical interactions during different cell surface effects, it combines the sensor array constituted and can be considered for difference
" selectivity " probe of cell.A specific received signals fingerprint can be all produced after each type of cell and sensor array effect
Pattern, can realize the classification and discrimination of cell.
The present invention is described in detail below in conjunction with the accompanying drawings.
The electrochemical sensor array based on functionalization graphene that the present invention is provided, including：Electrode, and modify in electrode
Reduced graphene the probe stable BSA on surface and the reduced graphene probe of calf thymus DNA functionalization；The BSA is stable
Reduced graphene probe concentration it is identical with the concentration of the reduced graphene probe of the calf thymus DNA functionalization, preferably
Concentration is 50 μ g/mL；The electrode is glass-carbon electrode, ITO or screen printing electrode.The electrochemical sensor array is by following sides
It is prepared by method：
(1) preparation of reduced graphene (BSA/CCG) probe stable BSA：
50 μ L concentration are 1.0mg/mL graphene oxide, and 250 μ L water and 200 μ L concentration mix for 50mg/mL BSA,
The pH value of solution is transferred to rapidly 12 by the NaOH for being 1.0M with concentration, and solution is stirred 24 hours at 90 DEG C, and solution colour is by yellowish-brown
Color gradually becomes black, and product 12000rpm is cleaned with pure water after being collected by centrifugation and is re-dispersed into after three times in a certain amount of water.
(2) preparation of reduced graphene (DNA/CCG) probe of calf thymus DNA (ctDNA) functionalization：
Calf thymus DNA first obtains single-stranded DNA in 1-2 hours in 95 DEG C of heating；
The single-stranded DNA that the graphene oxide that 10mL concentration is 0.5mg/mL is 2mg/mL with 10mL concentration is mixed, and adds 8
μ L 85wt% hydrazine hydrate, mixed solution is stirred 1 hour at 100 DEG C, product is collected by centrifugation cleaned with pure water after redisperse to one
In quantitative water.
Fig. 1 is modified electrode BSA/CCG and DNA/CCG electrochemical impedance Nyquist spectrogram, spectrogram reflection electrode
Surface electronic transmits situation, because the graphene probe of biomolecule functionalization itself has certain electrochemical impedance signal, because
This as background signal, deducts the signal from the sample signal measured.
(3) preparation of electrochemical sensor array：
A diameter of 3mm glass-carbon electrode in particle diameter is respectively first to be beaten on 1.0,0.3 and 0.05 μm of aluminum oxide polishing powder end
Mill, electrode is cleaned by ultrasonic 3 minutes in ultra-pure water after often step polishing.The reduction graphite of BSA stabilizations prepared by 10 μ L steps (1)
The reduced graphene probe dispersant liquid drop of calf thymus DNA functionalization prepared by alkene probe and 10 μ L steps (2) is added to pretreatment
The electrode surface crossed, at room temperature drying for standby.Described glass-carbon electrode may be replaced by ITO or screen printing electrode.
Cell is detected with the electrochemical sensor array based on functionalization graphene of preparation：
(1) cell culture and processing：
Cell is cultivated in the culture mediums of RPMI 1640, and 10% hyclone, penicillin and streptomysin are contained in culture medium
(100 μ g/mL) is in moist 5%CO2In 37 DEG C incubation.After culture 72 hours, 1000rpm is centrifuged 5 minutes and is collected cell, is used
PBS is cleaned twice.Cell is resuspended in PBS (pH 7.2) cushioning liquid, Petroff-Hausser cell counts are used
Device is counted to cell.
(2) cell is detected：
10 μ L are added dropwise on the electrochemical sensor array surface based on functionalization graphene and contain the molten of 100 or so cells
Liquid, 37 DEG C are incubated 30 minutes.After the careful cleaning electrode of buffer solution, electrode is placed in electrolyte and measures impedance.Using traditional
Three electrode test systems：Glass-carbon electrode of the auxiliary to electrode platinum filament Pt, reference electrode Ag/AgCl and by modification is used as work electricity
Pole, with K containing 10mM3[Fe(CN)6]/K4[Fe(CN)6](1:1) and 1M KCl 100mM phosphate buffers (PBS) as branch
Hold electrolyte.The test frequency scope of impedance spectrum is 10-2-105Hz, the sinusoidal amplitude voltage of application is 5mV.There to be cell
In the presence of the impedance that measures deduct the impedance of independent graphene modified electrode and obtain electrochemical response data.Every kind of cell is all parallel
Measurement 5 times, the data of gained carry out linear discriminant analysis (LDA).
Application Example 1
Distinguish normal cell and cancer cell：
We have selected following 5 kinds of cell lines to be tested：Pheochromocytoma on mouse fibroblast cell NIH-3T3, mouse kidney
Cell PC-12, human embryonic kidney cells HEK-293T, breast cancer cell MCF-7 and human lung adenocarcinoma cell.In order to confirm that electrochemistry is passed
Sensor array has really to this 5 kinds of cells distinguishes effect, and we are with graphene sensor array to the electricity of different type cell
Chemical impedance is measured, and impedance data is handled with linear discriminant analysis method.This statistical analysis technique be by
Data carry out linear combination and reach the purpose for distinguishing two or more analyte.The scatter diagram drawn in electrochemical response
In, each point represents Whole Response of the individual cells to sensor array, and different cell aggregations form 5 groups of nonoverlapping data
Point (as shown in Figure 2), illustrates that this simple electrochemical sensor array being made up of two kinds of functionalization graphene probes can be realized
The differentiation of different type cancer cell, detection accuracy can reach 100%.
Application Example 2
Distinguish different cancer cells：
With human cancer cell of the sensor array to 5 types：A549 (lung), HeLa (cervix), HepG2 (liver),
K562 (leukaemia), MCF-7 (mammary gland) have carried out detection and distinguished.Five kinds of cancer cells show that completely different electrochemical impedance rings
Induction signal, two variation factors that linear discriminant analysis is obtained are respectively 68.66% and 31.34%, as shown in Figure 3.In electrification
Learn in response scatter diagram, different types of cell aggregation forms the data group of five non-overlapping copies, show the electrochemistry of the present invention
Sensor array can distinguish this five kinds of cancer cells completely according to the physiochemical properties difference of cell surface.
Application Example 3
Distinguish breast cancer cell, resistant breast cancer cell and metastatic breast cancer cell：
We selected three-type-person's breast cancer cell be used for investigate the sensor that builds distinguishing same cancer cell difference shape
The application of state, they are MCF-7 breast cancer cells, MDA-MB-231 metastatic breast cancers cell, MF-7/ADR drug resistances breast respectively
Adenocarcinoma cell.Three kinds of cancer cells show completely different electrochemical impedance response signal, obtain two of linear discriminant analysis
Variation factor is respectively 63.63% and 36.37%, as shown in Figure 4.In electrochemical response scatter diagram, different types of cell
Aggregation forms the data group of three non-overlapping copies, shows that the electrochemical sensor array of the present invention can be according to the life of cell surface
Physicochemical nature difference distinguishes three kinds of cancer cells completely.
Application Example 4
Cancer cell is quantitatively detected：
Clinical cancer diagnosis application is not required nothing more than carries out qualitative detection to cancer cell, while also needing to quantitative detection.We
The electrochemical impedance of the cancer cell of various concentrations by model determination of MCF-7 cells.As a result show, the cell of various concentrations gathers
Collection forms the data group (Fig. 5) of non-overlapping copies, (Fig. 6) linear between Assessing parameters and concentration, electrochemistry of the invention
Sensor array can realize the cancer cell detection of individual cell level.
Obviously, above-described embodiment is only intended to clearly illustrate example, and the not restriction to embodiment.It is right
For those of ordinary skill in the art, can also make on the basis of the above description it is other it is various forms of change or
Change.There is no necessity and possibility to exhaust all the enbodiments.And the obvious change thus extended out or
Among changing still in the protection domain of the invention.
Claims (5)
1. a kind of electrochemical sensor array based on functionalization graphene, it is characterised in that including：
Electrode；
And modification is in reduced graphene the probe stable BSA of electrode surface；
And modification is in the reduced graphene probe of the calf thymus DNA functionalization of electrode surface；
The concentration of reduced graphene the probe stable BSA and the reduced graphene probe of the calf thymus DNA functionalization
Concentration it is identical.
2. the electrochemical sensor array according to claim 1 based on functionalization graphene, it is characterised in that the electricity
Extremely glass-carbon electrode, ITO or screen printing electrode.
3. the electrochemical sensor array according to claim 1 based on functionalization graphene, it is characterised in that described
The concentration and the concentration of the reduced graphene probe of the calf thymus DNA functionalization of reduced graphene probe stable BSA are equal
For 50 μ g/mL.
4. the electrochemical sensor array according to claim 1 based on functionalization graphene, it is characterised in that described
Reduced graphene probe stable BSA is prepared by following methods：
50 μ L concentration are 1.0mg/mL graphene oxide, and 250 μ L water and 200 μ L concentration mix for 50mg/mL BSA, with dense
Spend the NaOH for 1.0M and the pH value of solution be transferred to rapidly 12, solution is stirred 24 hours at 90 DEG C, solution colour by yellowish-brown by
Black is faded to, product 12000rpm is cleaned with pure water after being collected by centrifugation and is re-dispersed into after three times in a certain amount of water.
5. the electrochemical sensor array according to claim 1 based on functionalization graphene, it is characterised in that described small
The reduced graphene probe of bovine chest gland DNA functionalization is prepared by following methods：
Calf thymus DNA first obtains single-stranded DNA in 1-2 hours in 95 DEG C of heating；
The single-stranded DNA that the graphene oxide that 10mL concentration is 0.5mg/mL is 2mg/mL with 10mL concentration is mixed, and adds 8 μ L
85wt% hydrazine hydrate, mixed solution 100 DEG C stir 1 hour, product is collected by centrifugation cleaned with pure water after redisperse to necessarily
In the water of amount.
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