CN105510411B - The detection method of single cancer cell is realized in interaction based on cell and microelectrode - Google Patents
The detection method of single cancer cell is realized in interaction based on cell and microelectrode Download PDFInfo
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Abstract
The present invention relates to the detection method that single cancer cell is realized in a kind of interaction based on cell and microelectrode, it is to be combined using electrochemically active molecules by the specific aptamers of cancer cell with cancer cell, make aptamers DNA and the specific identification of cell surface protein for indicating electrochemically active molecules, electrochemical method is recycled to be detected, electrochemical signals can be produced when the cancer cell in electrolyte and microelectrode collide, due to the effective area very little of microelectrode, when cell and electrode collide, and adhere at the electrode surface, by the interaction for studying cell and microelectrode, realize the research of single cell and the identification of single cancer cell, the method of the present invention is easy to operate, high sensitivity, selectivity is good, it can be directed to the early diagnosis of cancer.
Description
Technical field
It is more particularly to a kind of that cell and micro- electricity are studied by electrochemical method the invention belongs to cancer cell detection technique field
The collision behavior of pole, realizes the detection method of single cell, and then realizes the early diagnosis of cancer.
Background technology
Cell electrochemistry is be combined with each other based on electrochemical principle, experimental method and cell, Protocols in Molecular Biology, to thin
Born of the same parents are analyzed and are characterized, and research or the charged ion of analog study cell or the characteristics of motion of electroactive particle energy transmission, take off
Show the recent studies on field that eucaryotic cell structure-functional relationship and exogenous molecules influence cell function.Electrochemical Detection cancer at present
Cell, is one layer of nanogold, then modifying DNA, then the combination by cell-specific, according to electrode modification will to be plated on glass-carbon electrode
It is front and rear, K in electrolytic cell4Fe(CN)6/K3Fe(CN)6The change of current signal detect cell.Modified on electrode surface
DNA is substantial amounts of, then with reference to cell be also very much, this method generally can not realize the detection to individual cells.
The content of the invention
In order to overcome the shortcomings of present in cancer cell detection technique of the prior art, the present invention provides a kind of selectivity
Good, high sensitivity and the detection and identification for realizing single cancer cell, the interaction realization based on cell and microelectrode are single
The detection method of one cancer cell.
Technical solution comprises the steps of used by the present invention realizes above-mentioned purpose：
(1) by electrochemically active molecules or adulterate electrochemically active molecules nano-particle with being capable of specific identification cancer cell
Aptamers DNA pass through Covalent bonding together；
(2) after cell to be checked is centrifuged, and the aptamers DNA of the combination electrochemically active molecules of step (1) is cultivated at 4 DEG C
After 2h, centrifuge, washing, be dispersed in spare in the phosphate buffer that concentration is 0.01mol/L, pH=7.4, make to indicate electrochemistry work
Property molecule aptamers DNA and the specific identification of cell surface protein；
(3) using in electrochemical workstation three-electrode system carry out Electrochemical Detection, using gold microelectrode as working electrode,
It is that reference electrode, the phosphate buffer of 0.01mol/L pH=7.4 are electrolyte to electrode, Ag/AgCl electrodes that platinum electrode, which is, will
Cell infusion to be checked after step (2) processing is into electrolyte, cell to be checked free movement in electrolytic cell, and single is to be checked thin
Born of the same parents collide with microelectrode surface, and when adhering in its surface, the electroactive substance on cell to be checked can occur
Oxidation reaction, then produce electrochemical signals, you can it is cancer cell to determine the single cell to be checked；If no electrochemical signals production
It is raw, then illustrate the cell non-cancerous cells.
Above-mentioned electrochemically active molecules are ferrocene small molecule, methylene blue or connection ruthenium pyridine etc..
Above-mentioned aptamers DNA is AS1411 or Sgc8c, and wherein the sequence of AS1411 is NH2-
The sequence of GGTGGTGGTGGTTGTGGTGGTGGTGG, Sgc8c are NH2-ATC TAA CTG CTG CGC CGC CGG GAA
AAT ACT GTA CGG TTA GA。
Above-mentioned nano-particle is elemental metals nano-particle or inorganic compound nano-particle or composite nanoparticle, described
Metal nanoparticle is gold, platinum, nickel or Nano silver grain, and the composite nanoparticle is gold/platinum composite nanoparticle or gold/bis-
Silica composite nanoparticle, the inorganic compound nano-particle are Nano particles of silicon dioxide or carbon quantum dot nano-particle
Or alkene C is strangled again60Nano-particle.
Interaction provided by the present invention based on cell and microelectrode realizes that the detection method of single cancer cell is profit
Combined with electrochemically active molecules by the specific aptamers of cancer cell with cancer cell, make the adaptation for indicating electrochemically active molecules
Body DNA and the specific identification of cell surface protein, recycle electrochemical method to be detected, when the cancer cell in electrolyte with
Microelectrode can produce electrochemical signals when colliding, due to the effective area very little of microelectrode, when cell and electrode touch
Hit, and adhere at the electrode surface, by studying the interaction of cell and microelectrode, realize the research of single cell and single
The identification of cancer cell, method of the invention is easy to operate, high sensitivity, selectivity it is good, the early diagnosis of cancer can be directed to.
Brief description of the drawings
Fig. 1 is the current-vs-time relation curve of the collision of the cell and microelectrode of ferrocene connection, and straight line represents acellular
Current-vs-time relation curve, curve table are shown with the current-vs-time relation curve of cell.
Fig. 2 is the nano-particle TEM figures of the silica of amidized doping connection ruthenium pyridine.
Fig. 3 is that (current potential is under constant potential for the cell of the Nano particles of silicon dioxide connection for being modified with doping connection ruthenium pyridine
1.1V) the current-vs-time relation curve collided with microelectrode.
Embodiment
Technical scheme is further described in conjunction with embodiment and experiment, attached drawing, but the present invention is not
It is only limitted to following implementation situations.
Embodiment 1
The electrochemically active molecules selection ferrocene small molecule of the present embodiment, the aptamers DNA choosings of specific identification cancer cell
Select AS1411 (NH2- GGTGGTGGTGGTTGTGGTGGTGGTGG), the method for detecting single cancer cell is made of following step：
(1) by ferrocene small molecule and aptamers the AS1411 ((NH for being capable of specific identification Hela cells2-
GGTGGTGGTGGTTGTGGTGGTGGTGG) by Covalent bonding together, it is specifically：
(1.1) the ferrocene small molecule of activated carboxyl
Take ferrocenecarboxylic acid (Fc-COOH) 102.5mg of purifying to be dissolved in 7mL dichloromethane (abbreviation DCM), add matter
Amount is than being 5:2 EDC (N '-(ethylcarbonyl group imino group)-N, -1,3 diaminourea chloride of N- dimethylpropanes)/NHS (N- hydroxyls
Succimide) in, at room temperature, mixture persistently stirred in nitrogen atmosphere 17 it is small when, after the reaction was complete, mixture water
Wash 3 times, each water consumption is 8mL, and water is mutually extracted with the DCM of 15mL, and organic phase is by MgSO4It is dry, mixture filtering, evaporation,
Intermediate product Fc-NHS is generated, makes it easier to combine with amidized DNA.
(1.2) the aptamers AS1411 of ferrocene and specific identification Hela cells after activating passes through Covalent bonding together.
Take intermediate product to be dissolved in the sodium bicarbonate solution that 50 μ L concentration are 0.5mol/L, pH=8.5, add the two of 10 μ L
Methylformamide, adds the aptamers AS1411 of 10 μ L, 200 μm of ol/L, under the conditions of 4 DEG C of lucifuges, persistently stir 4h so that
The reaction was complete, product is dissolved in the phosphate buffer that concentration is 0.01mol/L, pH=7.4, in case using.
(2) after cell to be checked is centrifuged with conventional method add step (1.2) phosphate buffer in, make cell to be checked with
2h is cultivated at 4 DEG C with reference to the aptamers DNA of electrochemically active molecules, after conventional centrifugal, washing, it is 0.01mol/ to be dispersed in concentration
L, it is spare in the phosphate buffer of pH=7.4, due to DNA can with the specific combination of cell surface protein, so electrochemistry thing
Matter ferrocene energy significant notation is in cell surface.
(3) using in electrochemical workstation three-electrode system carry out Electrochemical Detection, using gold microelectrode as working electrode,
It is that reference electrode, the phosphate buffer of 0.01mol/L pH=7.4 are electrolyte to electrode, Ag/AgCl electrodes that platinum electrode, which is, will
Cell infusion to be checked after step (2) processing under constant potential (current potential 1.1V), is marked with ferrocene molecule into electrolyte
Cell to be checked move freely in the electrolytic solution, since the size of microelectrode is small, while there are electrical charge rejection and sky between cell
Between steric hindrance, be readily achieved single cell and collide with microelectrode surface, when single cell to be checked and microelectrode surface are sent out
Raw collision, and when adhering in its surface, oxidation reaction can occur for the ferrocene small molecule on cell to be checked, and moment produces increasing
Big obvious current signal, as shown in Figure 1, producing electrochemical signals, it may be determined that the single cell to be checked is Hela cells；
If no electrochemical signals produce, it is Hela cells to illustrate that the cell is not.
Embodiment 2
The electrochemically active molecules selection connection ruthenium pyridine of the present embodiment, nano-particle is to select Nano particles of silicon dioxide,
The aptamers DNA selection AS1411 (NH of specific identification cancer cell2- GGTGGTGGTGGTTGTGGTGGTGGTGG), detection is single
The method of cancer cell is made of following step：
(1) Nano particles of silicon dioxide of connection ruthenium pyridine will be adulterated and be capable of the aptamers of specific identification Hela cells
AS1411((NH2- GGTGGTGGTGGTTGTGGTGGTGGTGG) by Covalent bonding together, be specifically：
(1.1) in Nano particles of silicon dioxide (RuSNPs) surface functionalization of doping connection ruthenium pyridine
With the Nano particles of silicon dioxide (RuSNPs) of the method synthesizing blender connection ruthenium pyridine of reverse micro emulsion, its specific mistake
Journey is as follows：Wherein Triton X-100 make surfactant, and n-hexyl alcohol makees cosurfactant, and hexamethylene makees continuous phase, ultrapure
Water makees dispersiveness, forms the microemulsion of w/o type, at 25 DEG C of room temperature, is taking Triton X-100, the 1.8mL of 1.8mL respectively just
Hexanol, the hexamethylene of 7.5mL are uniformly mixed, and after magnetic agitation 30min, after adding ultra-pure water, solution keeps clear, then adds
Enter the connection ruthenium pyridine (Ru (bpy) of 50-100 μ L3 2+), connection ruthenium pyridine (Ru (bpy)3 2+) can be dispersed in water environment, formed and stablized
Water-In-Oil structure, continue stir 1h after, add ethyl orthosilicate, ammonium hydroxide, persistently stir 24h after, add ethyl orthosilicate
With 3- aminopropyl triethoxysilanes, 24h is stirred for, after complete reaction, acetone demulsification is added, is collected by centrifugation, uses second respectively
Alcohol, ultra-pure water are washed 2 or 3 times, at least placed two days in a vacuum repeatedly, form the two of the doping connection ruthenium pyridine of surface amination
Silicon oxide nanoparticle is as shown in Figure 2.
The Nano particles of silicon dioxide of the doping connection ruthenium pyridine of surface amination is added in the dimethyl sulfoxide of 12mL, then is divided
It Jia Ru not react 24h at 40 DEG C, centrifuge, washed 3 times, be dispersed in ethanol in the butanedioic anhydride and 150 μ L triethylamines of 96mg
In ethanol, the Nano particles of silicon dioxide of the doping connection ruthenium pyridine of surface carboxyl groups is formed.
Surface is not had to the Nano particles of silicon dioxide for adulterating connection ruthenium pyridine of any function dough, amination, carboxylated
Its respective Zeta potential obtained by being dispersed in water respectively, it is as shown in table 1 below.
Table 1 is the Zeta potential table of different nano-particles
(1.2) Nano particles of silicon dioxide of doping connection ruthenium pyridine of surface carboxyl groups and fitting for specific identification Hela cells
Ligand AS1411 passes through Covalent bonding together.
It is 5 by mass ratio in the Nano particles of silicon dioxide of the doping connection ruthenium pyridine of surface carboxyl groups:2 EDC (N '-
(ethylcarbonyl group imino group)-N, N- dimethylpropane -1,3 diaminourea chloride), and NHS (N- hydroxysuccinimides) additions
To the phosphate buffer PBS of 100mmol/L, pH=6 of 400 μ L, then, then the nano-particle of 30 8 μm of ol/L of μ L is separately added into
With the PBS buffer of 470 μ L, final total volume is 1300 μ L, after mixture ultrasound 5min, prevents nano-particle from reuniting, will
Mixture culture is to darkroom 1h, it is therefore an objective to activates the COOH group of nanoparticle surface, centrifuges, supernatant abandons, and precipitation washes two
It is secondary, in sodium bicarbonate solution (pH=9) solution for precipitating the 50mM for being dispersed in 200 μ L, then add a certain amount of amidized
The ratio 1 of the quality of AS1411, nano-particle and AS1411:200, sediment ultrasound 5min form solution, and solution is trained in darkroom
18h is supported, and is constantly stirred, it is therefore an objective to which the nano-particle of carboxylated is combined with amidized AS1411, and centrifugation, ultrasound, disperses
In 0.01M phosphate buffers.
(1.2) Nano particles of silicon dioxide and specific identification Hela cells of the doping connection ruthenium pyridine of surface functionalization
Aptamers AS1411 passes through Covalent bonding together.
Take intermediate product to be dissolved in the sodium bicarbonate solution that 50 μ L concentration are 0.5mol/L, pH=8.5, add the two of 10 μ L
Methylformamide, adds the aptamers AS1411 of 10 μ L, 200 μm of ol/L, under the conditions of 4 DEG C of lucifuges, persistently stir 4h so that
The reaction was complete, product is dissolved in the phosphate buffer that concentration is 0.01mol/L, pH=7.4.
(2) after cell to be checked is centrifuged with conventional method add step (1.2) phosphate buffer in, make cell to be checked with
2h is cultivated at 4 DEG C with reference to the Nano particles of silicon dioxide of the doping connection ruthenium pyridine of aptamers DNA, after conventional centrifugal, washing, is disperseed
It is spare in the phosphate buffer that concentration is 0.01mol/L, pH=7.4, since DNA can be with the specific knot of cell surface protein
Close, so electrochemically active molecules join ruthenium pyridine energy significant notation in cell surface, make to indicate the aptamers DNA of connection ruthenium pyridine with
The specific identification of cell surface protein to be checked.
(3) using in electrochemical workstation three-electrode system carry out Electrochemical Detection, using gold microelectrode as working electrode,
It is that reference electrode, the phosphate buffer of 0.01mol/L pH=7.4 are electrolyte to electrode, Ag/AgCl electrodes that platinum electrode, which is, will
Cell infusion to be checked after step (2) processing is into electrolyte, under constant potential, is marked with the silica nanometer of connection ruthenium pyridine
The cell to be checked of particle moves freely in the electrolytic solution, and when adhering in its surface, the Ru on cell to be checked in nano-particle
(bpy)3 2+Oxidation reaction can occur, moment produces the current signal significantly increased, many due to being adulterated in a nano-particle
Connection ruthenium Pyridine Molecules, and a cell can combine substantial amounts of nano-particle, so the electroactive small molecule on cell surface
Quite a lot of, current signal is obvious, as shown in figure 3, up to 10-11~10-6A, you can determine that the single cell to be checked is thin for Hela
Born of the same parents；If no electrochemical signals produce, it is Hela cells to illustrate that the cell is not.
Found through Experimental comparison, when common electro-chemical activity small molecule reacts on the electrode, electric current is one constant
Value, if but be marked with the cell of electrochemically active molecules and electrode collide reaction when, having a discontinuous moment increases
Big current signal produces, and obvious wave crest can be produced on current-vs-time relation curve, therefore, can be according to electrochemical signals
Judge whether cell to be measured is cancer cell that aptamers are capable of specific recognition, so as to fulfill the detection of single cancer cell.
Embodiment 3
The electrochemically active molecules of the present embodiment can be replaced with methylene blue, it can pass through covalent bond and aptamers
With reference to it is rear with cell to be checked further combined with, so that cell to be checked be marked, others operation is same as Example 1,
And oxidation reaction can occur under appropriate current potential for methylene blue, therefore, its detection result is same as Example 1.
Embodiment 4
Aptamers DNA in above-described embodiment 1~3 can also use Sgc8c (NH2-ATC TAA CTG CTG CGC CGC
CGG GAA AAT ACT GTA CGG TTA GA) to replace, it can correspond to detection human lymphoid's cancer cell, specific operation
It is identical with embodiment 1,2 correspondences.
Embodiment 5
Nano particles of silicon dioxide in above-described embodiment 2 can also be replaced with metal nanoparticles such as gold, platinum, nickel or silver
Change, or can be replaced with composite nanoparticles such as gold/platinum, gold/silica composite nanoparticles, carbon quantum can also be used
Point strangles alkene C again60Replaced Deng other inorganic compound nano-particles, other detecting steps are same as Example 2.
Screening, the centrifugation extraction of cell and the functional group of electrochemically active molecules of aptamers in the various embodiments described above
Change operation and the selection of nano-particle etc. and belong to routine techniques, corresponding processing method can be selected according to practical application,
Therefore, do not illustrate one by one in detail in the present specification.
Claims (7)
1. the detection method of single cancer cell is realized in a kind of interaction based on cell and microelectrode, it is characterised in that by following
Step forms：
(1) by the nano-particle of electrochemically active molecules or doping electrochemically active molecules with being capable of the suitable of specific identification cancer cell
Ligand DNA passes through Covalent bonding together；
(2) after cell to be checked is centrifuged, and the aptamers DNA of the combination electrochemically active molecules of step (1) cultivates 2h at 4 DEG C
Afterwards, centrifuge, washing, be dispersed in concentration be 0.01mol/L, pH=7.4 phosphate buffer in it is spare, make to indicate electro-chemical activity
The aptamers DNA of molecule and the specific identification of cell surface protein；
(3) Electrochemical Detection is carried out using the three-electrode system in electrochemical workstation, using gold microelectrode as working electrode, platinum electricity
Extremely it is that reference electrode, the phosphate buffer of 0.01mol/L pH=7.4 are electrolyte to electrode, Ag/AgCl electrodes, by step
(2) handle after cell infusion to be checked into electrolyte, cell to be checked free movement in electrolytic cell, single cell to be checked with
Microelectrode surface collides, and when adhering in its surface, the electroactive substance on cell to be checked can aoxidize
Reaction, then produce electrochemical signals, you can it is cancer cell to determine the single cell to be checked；If no electrochemical signals produce,
Illustrate the cell non-cancerous cells.
2. the detection method of single cancer cell is realized in the interaction according to claim 1 based on cell and microelectrode,
It is characterized in that：The electrochemically active molecules are ferrocene small molecule, methylene blue or connection ruthenium pyridine.
3. the detection method of single cancer cell is realized in the interaction according to claim 1 based on cell and microelectrode,
It is characterized in that：The aptamers DNA is AS1411 or Sgc8c, and wherein the sequence of AS1411 is NH2-
The sequence of GGTGGTGGTGGTTGTGGTGGTGGTGG, Sgc8c are NH2-ATC TAA CTG CTG CGC CGC CGG GAA
AAT ACT GTA CGG TTA GA。
4. the detection method of single cancer cell is realized in the interaction according to claim 1 based on cell and microelectrode,
It is characterized in that：The nano-particle is elemental metals nano-particle or inorganic compound nano-particle or composite nanoparticle.
5. the detection method of single cancer cell is realized in the interaction according to claim 4 based on cell and microelectrode,
It is characterized in that：The metal nanoparticle is gold, platinum, nickel or Nano silver grain.
6. the detection method of single cancer cell is realized in the interaction according to claim 4 based on cell and microelectrode,
It is characterized in that：The composite nanoparticle is gold/platinum composite nanoparticle or gold/silica composite nanoparticle.
7. the detection method of single cancer cell is realized in the interaction according to claim 4 based on cell and microelectrode,
It is characterized in that：The inorganic compound nano-particle is Nano particles of silicon dioxide or carbon quantum dot nano-particle or multiple Le
Alkene C60Nano-particle.
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| CN110018089B (en) * | 2019-03-11 | 2020-11-03 | 中南大学 | High-sensitivity multifunctional electrochemical detection method based on single-particle collision |
| CN113720893A (en) * | 2021-09-01 | 2021-11-30 | 致慧医疗科技(上海)有限公司 | Method for evaluating malignancy degree of tumor based on surface charge intensity of cancer cells |
| CN115032257A (en) * | 2022-05-30 | 2022-09-09 | 杭州电子科技大学 | Method for determining the concentration of gram-negative bacteria in a solution using an electrochemical method |
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