CN105510411A - Method for single cancer cell detection based on cell-microelectrode interaction - Google Patents
Method for single cancer cell detection based on cell-microelectrode interaction Download PDFInfo
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Abstract
The invention relates to a method for single cancer cell detection based on cell-microelectrode interaction. Through bonding of an electrochemical active molecule and cancer cells based on a specific aptamer of the cancer cell, the aptamer DNA labeled through the electrochemical active molecule realizes cell surface protein specific identification, and then an electrochemical detection process is carried out, wherein cancer cells in an electrolyte collide a microelectrode, an electrochemical signal is produced. Because of a small effective area of the microelectrode, when the cell collides the electrolyte and adheres to the surface of the electrolyte, through research on interaction between the cells and microelectrode, single cell research and single cancer cell identification are realized. The method has simple operation processes, high sensitivity and good selectivity and can be used for early diagnosis of cancers.
Description
Technical field
The invention belongs to cancer cell detection technique field, particularly a kind of collision behavior being studied cell and microelectrode by electrochemical method, is realized the detection method of single cell, and then realizes the early diagnosis of cancer.
Background technology
Cell galvanochemistry be combined with each other based on electrochemical principle, experimental technique and cell, Protocols in Molecular Biology, cell is analyzed and characterizes, the characteristics of motion of research or the charged ion of modeling effort cell or the transmission of electroactive particle energy, discloses a recent studies on field of eucaryotic cell structure-functional relationship and the impact of exogenous molecules cellular function.Current Electrochemical Detection cancer cell glass-carbon electrode will plate one deck nm of gold, then modifying DNA, then by the combination of cell-specific, before and after electrode modification, K in electrolytic cell
4fe (CN)
6/ K
3fe (CN)
6the change of current signal detect cell.The DNA that electrode surface is modified is a large amount of, and the cell so combined also is a lot, and the method can not realize the detection to individual cells usually.
Summary of the invention
In order to overcome the deficiency existing for cancer cell detection technique of the prior art, the invention provides a kind of selectivity good, highly sensitive and realize the detection and indentification of single cancer cell, the interaction based on cell and microelectrode realizes the detection method of single cancer cell.
The present invention realizes the technical scheme that above-mentioned purpose adopts and is made up of following steps:
(1) by the nano particle of electrochemical activity molecule or doping electrochemical activity molecule with can the aptamers DNA of specific identification cancer cell be combined by covalent bond;
(2) by after cell centrifugation to be checked, after cultivating 2h with the aptamers DNA of the combined with electrochemical bioactive molecule of step (1) at 4 DEG C, centrifugal, washing, it is for subsequent use in the phosphate buffer of 0.01mol/L, pH=7.4 for being dispersed in concentration, makes the aptamers DNA and the specific identification of cell surface protein that indicate electrochemical activity molecule;
(3) three-electrode system in electrochemical workstation is utilized to carry out Electrochemical Detection, take gold microelectrode as working electrode, platinum electrode is to electrode, Ag/AgCl electrode is contrast electrode, the phosphate buffer of 0.01mol/LpH=7.4 is electrolytic solution, cell infusion to be checked after step (2) being processed is in electrolytic solution, cell to be checked free movement in electrolytic cell, single cell to be checked and microelectrode surface collide, and when adhering in its surface, can oxidation reaction be there is in the electroactive substance on cell to be checked, then produce electrochemical signals, can determine that this single cell to be checked is cancer cell, if produce without electrochemical signals, then this cell non-cancerous cells is described.
Above-mentioned electrochemical activity molecule is ferrocene Small molecular, methylene blue or connection ruthenium pyridine etc.
Above-mentioned aptamers DNA is AS1411 or Sgc8c, and wherein the sequence of AS1411 is NH
2the sequence of-GGTGGTGGTGGTTGTGGTGGTGGTGG, Sgc8c is NH
2-ATCTAACTGCTGCGCCGCCGGGAAAATACTGTACGGTTAGA.
Above-mentioned nano particle is elemental metals nano particle or mineral compound nano particle or composite nanoparticle, described metal nanoparticle is gold, platinum, nickel or Nano silver grain, described composite nanoparticle is gold/platinum composite nanoparticle or gold/silicon dioxide composite nanoparticle, and described mineral compound nano particle is Nano particles of silicon dioxide or carbon quantum dot nano particle or strangles alkene C again
60nano particle.
The detection method realizing single cancer cell based on cell and the interaction of microelectrode provided by the present invention utilizes electrochemical activity molecule to be combined with cancer cell by the specific aptamers of cancer cell, make the aptamers DNA and the specific identification of cell surface protein that indicate electrochemical activity molecule, recycling electrochemical method detects, electrochemical signals can be produced when the cancer cell in electrolyte and microelectrode collide, because the useful area of microelectrode is very little, when cell and electrode collide, and adhere at the electrode surface, by studying the interaction of cell and microelectrode, realize the research of single cell and the identification of single cancer cell, method of the present invention is simple to operate, highly sensitive, selectivity is good, the early diagnosis of cancer can be devoted to.
Accompanying drawing explanation
Fig. 1 is the current-vs-time relation curve of the cell of ferrocene connection and the collision of microelectrode, and straight line represents acellular current-vs-time relation curve, and curve table is shown with the current-vs-time relation curve of cell.
Fig. 2 is that the nano particle TEM of the silicon dioxide of amidized doping connection ruthenium pyridine schemes.
Fig. 3 is the cell current-vs-time relation curve that (current potential is 1.1V) and microelectrode collide under constant potential that the Nano particles of silicon dioxide being modified with doping connection ruthenium pyridine connects.
Embodiment
Now with experiment, accompanying drawing, technical scheme of the present invention is further described, but the present invention is not limited only to following enforcement situation in conjunction with the embodiments.
Embodiment 1
The electrochemical activity Molecular Selection ferrocene Small molecular of the present embodiment, the aptamers DNA of specific identification cancer cell selects AS1411 (NH
2-GGTGGTGGTGGTTGTGGTGGTGGTGG), the method detecting single cancer cell is made up of following step:
(1) by ferrocene Small molecular with can aptamers the AS1411 ((NH of specific identification Hela cell
2-GGTGGTGGTGGTTGTGGTGGTGGTGG) combined by covalent bond, specifically:
(1.1) the ferrocene Small molecular of activated carboxyl
Ferrocenecarboxylic acid (Fc-COOH) 102.5mg getting purifying is dissolved in 7mL methylene chloride (being called for short DCM), add EDC that mass ratio is 5:2 (N '-(ethylcarbonyl group imino group)-N, N-dimethylpropane-1,3 diamido chlorides) in/NHS (N-hydroxysuccinimide), at room temperature, potpourri Keep agitation 17 hours in nitrogen atmosphere, after reacting completely, potpourri washes 3 times with water, each water consumption is 8mL, the DCM of aqueous phase 15mL extracts, and organic phase is by MgSO
4drying, potpourri filters, evaporation, generates intermediate product Fc-NHS, makes it be easy to combine with amidized DNA.
(1.2) ferrocene after activation is combined by covalent bond with the aptamers AS1411 of specific identification Hela cell.
Getting intermediate product, to be dissolved in 50 μ L concentration be the sodium bicarbonate solution of 0.5mol/L, pH=8.5, add the dimethyl formamide of 10 μ L, add the aptamers AS1411 of 10 μ L200 μm ol/L again, under 4 DEG C of lucifuge conditions, Keep agitation 4h is to make to react completely, product being dissolved in concentration is in the phosphate buffer of 0.01mol/L, pH=7.4, in order to using.
(2) add in the phosphate buffer of step (1.2) after centrifugal for cell conventional method to be checked, the aptamers DNA of cell to be checked and combined with electrochemical bioactive molecule is made to cultivate 2h at 4 DEG C, after conventional centrifugal, washing, it is for subsequent use in the phosphate buffer of 0.01mol/L, pH=7.4 for being dispersed in concentration, because DNA can combination specific with cell surface protein, so electrochemical substance ferrocene energy significant notation is at cell surface.
(3) three-electrode system in electrochemical workstation is utilized to carry out Electrochemical Detection, take gold microelectrode as working electrode, platinum electrode is to electrode, Ag/AgCl electrode is contrast electrode, the phosphate buffer of 0.01mol/LpH=7.4 is electrolytic solution, cell infusion to be checked after step (2) being processed is in electrolytic solution, under constant potential (current potential is 1.1V), the cell to be checked being marked with ferrocene molecule moves freely in the electrolytic solution, because the size of microelectrode is little, there is electrical charge rejection and sterically hindered between cell simultaneously, just easily realize single cell and microelectrode surface collides, when single cell to be checked and microelectrode surface collide, and when adhering in its surface, can oxidation reaction be there is in the ferrocene Small molecular on cell to be checked, produce instantaneously and increase obvious current signal, as shown in Figure 1, namely electrochemical signals is produced, can determine that this single cell to be checked is Hela cell, if produce without electrochemical signals, then illustrate that this cell is not Hela cell.
Embodiment 2
The electrochemical activity Molecular Selection connection ruthenium pyridine of the present embodiment, nano particle selects Nano particles of silicon dioxide, and the aptamers DNA of specific identification cancer cell selects AS1411 (NH
2-GGTGGTGGTGGTTGTGGTGGTGGTGG), the method detecting single cancer cell is made up of following step:
(1) by the Nano particles of silicon dioxide of doping connection ruthenium pyridine with can aptamers the AS1411 ((NH of specific identification Hela cell
2-GGTGGTGGTGGTTGTGGTGGTGGTGG) combined by covalent bond, specifically:
(1.1) at 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 detailed process is as follows: wherein TritonX-100 makes surfactant, cosurfactant made by n-hexyl alcohol, cyclohexane give continuous phase, ultrapure water is done dispersed, form the microemulsion of w/o type, at room temperature 25 DEG C, get the TritonX-100 of 1.8mL respectively, the n-hexyl alcohol of 1.8mL, the cyclohexane of 7.5mL mixes, after magnetic agitation 30min, after adding ultrapure water, solution keeps clear, add the connection ruthenium pyridine (Ru (bpy) of 50-100 μ L again
3 2+), connection ruthenium pyridine (Ru (bpy)
3 2+) can be dispersed in water environment, form stable Water-In-Oil structure, after continuing to stir 1h, add ethyl orthosilicate, ammoniacal liquor, after Keep agitation 24h, then add ethyl orthosilicate and 3-aminopropyl triethoxysilane, stir 24h again, after question response is complete, add acetone breakdown of emulsion, collected by centrifugation, use ethanol respectively, ultrapure water cyclic washing 2 or 3 times, at least place two days in a vacuum, forms the Nano particles of silicon dioxide of the doping connection ruthenium pyridine of surface amination as shown in Figure 2.
The Nano particles of silicon dioxide of the doping of surface amination connection ruthenium pyridine is added in the dimethyl sulfoxide of 12mL, in the butanedioic anhydride adding 96mg more respectively and 150 μ L triethylamines, at 40 DEG C of reaction 24h, centrifuging, 3 times are washed with ethanol, dispersion in ethanol, forms the Nano particles of silicon dioxide of the doping connection ruthenium pyridine of surface carboxyl groups.
Surface is dispersed in water its respective Zeta potential of gained respectively without any the Nano particles of silicon dioxide of functionalization, amination, carboxylated doping connection ruthenium pyridine, as shown in table 1 below.
Table 1 is the Zeta potential table of different nano particle
(1.2) Nano particles of silicon dioxide of the doping connection ruthenium pyridine of surface carboxyl groups is combined by covalent bond with the aptamers AS1411 of specific identification Hela cell.
In the Nano particles of silicon dioxide of the doping connection ruthenium pyridine of surface carboxyl groups, by mass ratio be 5:2 EDC (N '-(ethylcarbonyl group imino group)-N, N-dimethylpropane-1, 3 diamido chlorides), the 100mmol/L of 400 μ L is joined with NHS (N-hydroxysuccinimide), the phosphate buffer PBS of pH=6, subsequently, add the nano particle of 30 μ L8 μm ol/L and the PBS damping fluid of 470 μ L more respectively, finally total volume is 1300 μ L, after the ultrasonic 5min of potpourri, prevent nano particle from reuniting, potpourri is cultivated to darkroom 1h, object is the COOH group of activation nanoparticle surface, centrifugal, supernatant abandons, precipitation washes twice, precipitation is dispersed in sodium bicarbonate solution (pH=9) solution of the 50mM of 200 μ L, then a certain amount of amidized AS1411 is added, the ratio 1:200 of nano particle and the quality of AS1411, the ultrasonic 5min of sediment forms solution, solution cultivates 18h in darkroom, and constantly stir, object is that carboxylated nano particle is combined with amidized AS1411, centrifugal, ultrasonic, be dispersed in 0.01M phosphate buffer.
(1.2) Nano particles of silicon dioxide of the doping connection ruthenium pyridine of surface functionalization is combined by covalent bond with the aptamers AS1411 of specific identification Hela cell.
Getting intermediate product, to be dissolved in 50 μ L concentration be the sodium bicarbonate solution of 0.5mol/L, pH=8.5, add the dimethyl formamide of 10 μ L, add the aptamers AS1411 of 10 μ L200 μm ol/L again, under 4 DEG C of lucifuge conditions, Keep agitation 4h is to make to react completely, and product being dissolved in concentration is in the phosphate buffer of 0.01mol/L, pH=7.4.
(2) add in the phosphate buffer of step (1.2) after centrifugal for cell conventional method to be checked, cell to be checked is made to cultivate 2h with the Nano particles of silicon dioxide that the doping in conjunction with aptamers DNA joins ruthenium pyridine at 4 DEG C, after conventional centrifugal, washing, it is for subsequent use in the phosphate buffer of 0.01mol/L, pH=7.4 for being dispersed in concentration, because DNA can combination specific with cell surface protein, so electrochemical activity molecule connection ruthenium pyridine energy significant notation is at cell surface, make the aptamers DNA and the specific identification of cell surface protein to be checked that indicate connection ruthenium pyridine.
(3) three-electrode system in electrochemical workstation is utilized to carry out Electrochemical Detection, take gold microelectrode as working electrode, platinum electrode be to electrode, Ag/AgCl electrode be contrast electrode, the phosphate buffer of 0.01mol/LpH=7.4 is electrolytic solution, cell infusion to be checked after step (2) being processed is in electrolytic solution, under constant potential, the cell to be checked being marked with the Nano particles of silicon dioxide of connection ruthenium pyridine moves freely in the electrolytic solution, and when adhering in its surface, the Ru (bpy) on cell to be checked in nano particle
3 2+oxidation reaction can be there is, produce the current signal obviously increased instantaneously, due to the connection ruthenium Pyridine Molecules adulterating a lot in a nano particle, and a cell can in conjunction with a large amount of nano particles, so the electroactive Small molecular on cell surface is quite a lot of, current signal is obvious, as shown in Figure 3, can reach 10
-11~ 10
-6a, can determine that this single cell to be checked is Hela cell; If produce without electrochemical signals, then illustrate that this cell is not Hela cell.
Find through Experimental comparison, when common electrochemical activity Small molecular reacts on electrode, electric current is a constant value, if but being marked with the cell of electrochemical activity molecule and electrode collides when reacting, have the discontinuous current signal increased instantaneously to produce, current-vs-time relation curve can produce obvious crest, therefore, can judge that whether cell to be measured is that aptamers can the cancer cell of specific recognition according to electrochemical signals, thus realize the detection of single cancer cell.
Embodiment 3
The electrochemical activity molecule of the present embodiment can be replaced with methylene blue, its can be combined with aptamers by covalent bond after with cell to be checked further combined with, thus cell to be checked is marked, other operation is all identical with embodiment 1, and can oxidation reaction be there is in methylene blue under suitable current potential, therefore, its Detection results is identical with embodiment 1.
Embodiment 4
Aptamers DNA in above-described embodiment 1 ~ 3 can also use Sgc8c (NH
2-ATCTAACTGCTGCGCCGCCGGGAAAATACTGTACGGTTAGA) to replace, it can corresponding human body lymphocytic cancer cell, concrete operation and embodiment 1,2 corresponding identical.
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, or can replace with composite nanoparticles such as gold/platinum, gold/silicon dioxide composite nanoparticles, by carbon quantum dot or alkene C can also be strangled again
60mineral compound nano particle Deng other is replaced, and other detecting step is identical with embodiment 2.
Choosing etc. of the functionalization operation of the screening of the aptamers in the various embodiments described above, the centrifugal extraction of cell and electrochemical activity molecule and nano particle all belongs to routine techniques, corresponding disposal route can be selected according to practical application, therefore, do not illustrate one by one in detail in this manual.
Claims (7)
1. realize a detection method for single cancer cell based on the interaction of cell and microelectrode, it is characterized in that being made up of following steps:
(1) by the nano particle of electrochemical activity molecule or doping electrochemical activity molecule with can the aptamers DNA of specific identification cancer cell be combined by covalent bond;
(2) by after cell centrifugation to be checked, after cultivating 2h with the aptamers DNA of the combined with electrochemical bioactive molecule of step (1) at 4 DEG C, centrifugal, washing, it is for subsequent use in the phosphate buffer of 0.01mol/L, pH=7.4 for being dispersed in concentration, makes the aptamers DNA and the specific identification of cell surface protein that indicate electrochemical activity molecule;
(3) three-electrode system in electrochemical workstation is utilized to carry out Electrochemical Detection, take gold microelectrode as working electrode, platinum electrode is to electrode, Ag/AgCl electrode is contrast electrode, the phosphate buffer of 0.01mol/LpH=7.4 is electrolytic solution, cell infusion to be checked after step (2) being processed is in electrolytic solution, cell to be checked free movement in electrolytic cell, single cell to be checked and microelectrode surface collide, and when adhering in its surface, can oxidation reaction be there is in the electroactive substance on cell to be checked, then produce electrochemical signals, can determine that this single cell to be checked is cancer cell, if produce without electrochemical signals, then this cell non-cancerous cells is described.
2. the interaction based on cell and microelectrode according to claim 1 realizes the detection method of single cancer cell, it is characterized in that: described electrochemical activity molecule is ferrocene Small molecular, methylene blue or connection ruthenium pyridine.
3. the interaction based on cell and microelectrode according to claim 1 realizes the detection method of single cancer cell, and it is characterized in that: described aptamers DNA is AS1411 or Sgc8c, wherein the sequence of AS1411 is NH
2the sequence of-GGTGGTGGTGGTTGTGGTGGTGGTGG, Sgc8c is NH
2-ATCTAACTGCTGCGCCGCCGGGAAAATACTGTACGGTTAGA.
4. the interaction based on cell and microelectrode according to claim 1 realizes the detection method of single cancer cell, it is characterized in that: described nano particle is elemental metals nano particle or mineral compound nano particle or composite nanoparticle.
5. the interaction based on cell and microelectrode according to claim 4 realizes the detection method of single cancer cell, it is characterized in that: described metal nanoparticle is gold, platinum, nickel or Nano silver grain.
6. the interaction based on cell and microelectrode according to claim 4 realizes the detection method of single cancer cell, it is characterized in that: described composite nanoparticle is gold/platinum composite nanoparticle or gold/silicon dioxide composite nanoparticle.
7. the interaction based on cell and microelectrode according to claim 4 realizes the detection method of single cancer cell, it is characterized in that: described mineral compound nano particle is Nano particles of silicon dioxide or carbon quantum dot nano particle or strangles alkene C again
60nano particle.
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