CN103913447B - A kind of electrogenerated chemiluminescence imaging device and application thereof - Google Patents
A kind of electrogenerated chemiluminescence imaging device and application thereof Download PDFInfo
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Abstract
The invention discloses a kind of electrogenerated chemiluminescence imaging device, including object lens, transmission line, singl e photon detection electron multiplication C C D and electrogenerated chemiluminescence generator, described electrogenerated chemiluminescence generator includes I T O electrode, silver electrode and voltage generator, wherein, described object lens, transmission line and singl e photon detection electron multiplication C C D are sequentially connected with；The positive pole that described I T O electrode is connected with voltage generator is connected, and described silver electrode is connected with the negative pole of voltage generator.The invention allows for the application in Visual retrieval hydrogen peroxide of a kind of above-mentioned electrogenerated chemiluminescence imaging device and this device to leak on the unicellular surface of Visual retrieval the application on hydrogen peroxide and Visual retrieval cell surface molecule.The electrogenerated chemiluminescence imaging device composition of the present invention is simple, can carry out parallel quick detection, and detection flux is big, easy and simple to handle, and luminous efficiency is high.
Description
Technical field
The invention belongs to technical field of biochemical industry, more particularly to a kind of electrogenerated chemiluminescence imaging device and application thereof.
Background technology
Applied Electrochemistry technology can obtain ectochemistry component and Topically active etc. to the unicellular imaging research that carries out
Important biomolecule information, the most general microsurgical instrument is scanning electrochemical microscope (SECM)).This microscope be utilize one micro-
Electrode scans the surface of single living cells and detects the electroactive chemical species of tool.By reducing electrode size to nanoscale
The spatial resolution of scanning electrochemical microscope (SECM)) can not made to reach Nano grade.The defect of this technology is: each is thin
The necessary single independent detection of cellular surface, the flux therefore detected is severely restricted.As except scanning electrochemical microscope (SECM))
Another kind of selection, the electrochemistry imaging technique based on surface plasma resonance of development make use of the electrochemistry letter of local in the recent period
Number and surface plasma resonance signal between relation carry out Single-cell imaging.Although this new technique is by many cells
Parallel testing improve detection flux, but surface plasma resonance technology is poor due to self detection sensitivity, the most very
Difficult biomolecule to leaking in unicellular or the low abundant species of cell surface detect.
Electrogenerated chemiluminescence is a kind of high-sensitive electrochemical method.It utilizes material that electrochemical reaction excites from excited state
While returning to ground state, emit light to realize the detection to molecule.In view of whole electrode surface biomolecule produce light all
Can be received by singl e photon detection electron multiplication electronic coupled element (CCD), therefore imaging skill based on electrogenerated chemiluminescence
Art has high flux and highly sensitive advantage, the advantage possessing mesh first two unicellular electricity consumption chemical imaging technology.Existing
Electrogenerated chemiluminescence imaging has been used to the fuzzy fingerprint to electrode surface and the imaging of protein layer.State-of-the-art technology can be to micro-
The antigen imaging on ball surface.This technology is by introducing the co-reactant at concentrations up to mM rank, it is possible to micro-0.4
The region of rice produces the optical signal being able to detect that, and then spatial resolution is shifted onto submicron rank.
Although electrogenerated chemiluminescence imaging technique shows the potentiality having cell surface antigen imaging, we are by this skill
Art is used for carrying out the unicellular molecule process of leaking during imaging challenged.Because the molecular concentration discharged is the lowest,
It is typically in micro-rank of rubbing.Although in the presence of luminol, these molecules discharged are turned by its oxidase
Become can the hydrogen peroxide of electrogenerated chemiluminescence, but to realize in the region of submicron, and plus the sampling time of second rank
In the range of (temporal resolution), the biomolecule detectable optical signal of acquisition to such low concentration, or unlikely
's.
Summary of the invention
Goal of the invention: for solving technical problem present in prior art, the present invention proposes a kind of simple structure, receives light efficiency
Rate is high, can realize the electroluminescent chemistry detecting the biomolecule leaked in unicellular or the low abundant species of cell surface
Luminous imaging device and application thereof.
Technology contents: for realizing above-mentioned technical purpose, the present invention proposes a kind of electrogenerated chemiluminescence imaging device, including thing
Mirror, transmission line, singl e photon detection electron multiplication CCD and electrogenerated chemiluminescence generator, described electroluminescent chemistry is sent out
Optical generator includes ITO electrode silver electrode and voltage generator, wherein, described object lens, transmission line and single photon
Detection electron multiplication CCD is sequentially connected with；Described ITO electrode is connected with the positive pole of voltage generator, described silver electrode and
The negative pole of voltage generator is connected.
Wherein, described object lens are any one in 5 times of mirrors, 10 times of mirrors, 20 times of mirrors, 40 times of mirrors and 100 times of mirrors,
The N.A. of object lens is the bigger the better.Preferably, described object lens be N.A. be 20 times of mirrors of 0.50.According to spatial resolution
Requirement select suitable object lens and N.A value thereof.
The invention allows for the application in Visual retrieval hydrogen peroxide of the above-mentioned electrogenerated chemiluminescence imaging device.
Specifically, described application comprise the steps: the surface mount O at ito glass sheet as solution room,
With ito glass sheet as working electrode, silver electrode is to electrode, joins in O by luminescence reagent and hydrogen peroxide,
Periodic luminous voltage-recovery voltage is applied to ITO electrode while singl e photon detection electron multiplication CCD exposes,
Detection imaging effect.
Wherein, described luminescence reagent be described luminescence reagent be L012, luminol, N-(4-aminobutyl)-N-second
The different luminol of base, any one in N-(6-amino base) the different luminol of-N-ethyl, the different luminol of isothiocyanic acid, consumption
Being 100 μMs～500 μMs for concentration, preferably luminescence reagent is L012, and the consumption of luminescence reagent is 100 μMs～500 μMs,
It is preferably 200 μMs.
Wherein, the detection range of described hydrogen peroxide is 10～200 μMs.
Preferably, the time of singl e photon detection electron multiplication CCD exposure is two luminous voltages-recovery voltage cycle, its
In, a luminous voltage-recovery voltage cycle is: 2 ± 1s under the positive voltage of luminous voltage: 1.0 ± 0.1V；Recovery voltage:
0.5 ± 0.25s under the negative voltage of-1.0 ± 0.1V.It is further preferable that one of described periodic luminous voltage-recovery voltage
Cycle is: 2s under the positive voltage of luminous voltage: 1.0V；Recover 0.5s under the negative voltage of voltage :-1.0V.
The invention allows for the application that said apparatus leaks in hydrogen peroxide on the unicellular surface of Visual retrieval.
Specifically, the leak step of hydrogen peroxide of the unicellular surface of described Visual retrieval includes: on the surface of ito glass sheet
Stickup O is as solution room, and with ito glass sheet as working electrode, silver electrode is to electrode, by cell at solution
Cultivating in room, add luminescence reagent in solution room, being subsequently adding PMA stimulates intracellular NADPA oxidase to make
Cell surface produces hydrogen peroxide, applies periodically to ITO electrode while singl e photon detection electron multiplication CCD exposes
Luminous voltage-recovery voltage, detect imaging effect.
Wherein, described luminescence reagent is L012, luminol, N-(4-aminobutyl) the different luminol of-N-ethyl, N-(6-
Amino base) any one in the different luminol of-N-ethyl and the different luminol of isothiocyanic acid, consumption is 100 μMs～500 μMs.
Preferably luminescence reagent is L012, and the consumption of luminescence reagent is 100 μMs～500 μMs, it is therefore preferable to 200 μMs.
Preferably, the time of singl e photon detection electron multiplication CCD exposure is two luminous voltages-recovery voltage cycle, its
In, a luminous voltage-recovery voltage cycle is: 2 ± 1s under the positive voltage of luminous voltage: 1.0 ± 0.1V；Recovery voltage:
0.5 ± 0.25s under the negative voltage of-1.0 ± 0.1V.It is further preferable that one of described periodic luminous voltage-recovery voltage
Cycle is: 2s under the positive voltage of luminous voltage: 1.0V；Recover 0.5s under the negative voltage of voltage :-1.0V.
The invention allows for said apparatus application on Visual retrieval cell surface molecule.
Specifically, comprise the steps: the surface mount O at ito glass sheet as solution room, with ITO glass
Glass sheet is working electrode, and silver electrode is to electrode, is cultivated by cell in solution room, adds luminescence reagent in solution room,
Described testing molecule is converted into hydrogen peroxide by the oxidase utilizing the testing molecule of cell surface corresponding, at singl e photon detection electricity
Apply periodic luminous voltage-recovery voltage to ITO electrode while son multiplication CCD exposure, detect imaging effect.
Wherein, described luminescence reagent is L012, luminol, N-(4-aminobutyl) the different luminol of-N-ethyl, N-(6-
Amino base) any one in the different luminol of-N-ethyl and the different luminol of isothiocyanic acid, consumption is 100 μMs～500 μMs.
Preferably luminescence reagent is LO12, and the consumption of luminescence reagent is 100 μMs～500 μMs, it is therefore preferable to 200 μMs.
Preferably, the time of singl e photon detection electron multiplication CCD exposure is two luminous voltages-recovery voltage cycle, its
In, a luminous voltage-recovery voltage cycle is: 2 ± 1s under the positive voltage of luminous voltage: 1.0 ± 0.1V；Recovery voltage:
0.5 ± 0.25s under the negative voltage of-1.0 ± 0.1V.It is further preferable that one of described periodic luminous voltage-recovery voltage
Cycle is: 2s under the positive voltage of luminous voltage: 1.0V；Recover 0.5s under the negative voltage of voltage :-1.0V.
Beneficial effect: compared with prior art, the present invention has the following advantages:
(1) the electrogenerated chemiluminescence imaging device composition of the present invention is simple, receives light rate high, can carry out parallel quick detection,
And the spatial resolution of detection can be adjusted by object lens multiplying power and singl e photon detection electron multiplication CCD, and pass through
Current potential regulation promotes luminous efficiency, obtains detectable signal, meanwhile, directly can will cultivate the ITO electrode of cell
Be positioned on microscope stage detection, need not modified electrode further, therefore can easily this electrogenerated chemiluminescence be sent out
Light analysis technology is used for biological study, and can obtain the illuminated message of ITO surface all cells simultaneously thus increase inspection
The flux surveyed, significant for Single cell analysis.
(2) by the luminous voltage of loading cycle during electrochemiluminescdetection detection and recovery voltage, permissible
Avoiding being continuously applied high pressure on electrode can cause electrode to inactivate, and improves luminous efficiency.
(3) the electrogenerated chemiluminescence formation method of the present invention can detect the hydrogen peroxide of low concentration effectively, and has height
Electroluminescent chemical method degree of accuracy, it is possible to detect leaking of unicellular surface hydrogen peroxide, such that it is able to be under different conditions
The hydrogen peroxide of cell release the evidence that its submicron existed is distributed is provided.
(4) the electrogenerated chemiluminescence formation method of the present invention can be the distribution letter that pharmaceutical analysis provides activation Membrane cholesterol
Breath, provides more biological information by cell activation cholesterol is imaged as cellular cholesterol transportation research.
Accompanying drawing explanation
Fig. 1 is the schematic diagram of the electrogenerated chemiluminescence imaging device of the present invention；
Fig. 2 is combination voltage membrane type and the impact on hydrogen peroxide luminous detection of the constant voltage membrane type；
Fig. 3 is different electric voltage frequencies and the impact on imaging signal to noise ratio of the different time of exposure；
Fig. 4 is the Visual retrieval of hydrogen peroxide in solution, and wherein, A is adhesive tape overlay area and the exposed area of ITO electrode
The photograph via bright field on the border in territory；B is the illuminated diagram after adding 200 μMs of L012；C for add 200 μMs of L012 and
The illuminated diagram of 10 μMs of hydrogen peroxide；D is the difference pseudocolour picture of C and B figure；E be variable concentrations hydrogen peroxide and luminescence strong
Graph of a relation between degree；
Fig. 5 is the Visual retrieval that cell surface hydrogen peroxide leaks, and wherein, A is the Hela cytological map in ITO electrode
Photograph via bright field；B is the illuminated diagram adding 200 μMs of L012；C is the illuminated diagram that cell stimulates through PMA；D is
Luminous difference diagram between figure B and C；E is photograph via bright field and the stacking chart of difference photo；F is that cell leaks hydrogen peroxide
Pseudocolour picture；
Fig. 6 is the detection schematic diagram that the Hela cell in ITO electrode adds PBS, and wherein, A is photograph via bright field；
B is the luminescence imaging figure adding 200 μMs of L012；C is the luminous one-tenth adding PBS (10mM) in 200 μMs of L012
As figure；D is figure B and the luminescence imaging difference diagram of figure C；Wherein, brightness regulation has arrived optimal visibility degree；
Fig. 7 is ACAT repressed Hela cell detection image in the presence of 200 μMs of L012 in ITO electrode, its
Middle A is photograph via bright field；B is the luminous difference diagram before and after addition cholesterol oxidase；C is the stacking chart of figure A and B；
D is the pseudocolour picture of the distribution of the activation Membrane cholesterol of two cells chosen；E is that ACAT and HMGCoA is common
The repressed cell luminous difference diagram before and after adding cholesterol oxidase；F be the repressed cell of ACAT and
The common repressed luminescence of cell intensity histograms of ACAT, HMGCoA；Wherein, the deviation in figure is figure B and figure E
The standard deviation of the luminous intensity of middle cell compartment；
Fig. 8 is that in ITO electrode, ACAT, HMGCoA the most repressed Hela cell adds 200 μMs of L012
Detection image, wherein, A is photograph via bright field；B is difference diagram luminous before and after adding 1U/ml cholesterol oxidase；C is
Light field figure and the stacking chart of difference diagram.
Detailed description of the invention
The schematic diagram of the electrogenerated chemiluminescence imaging device of the present invention is as it is shown in figure 1, include object lens, transmission line, list
Photon detection electron multiplication CCD and electrogenerated chemiluminescence generator, described electrogenerated chemiluminescence generator includes ITO electricity
Pole, silver electrode and voltage generator, wherein, object lens, transmission line and singl e photon detection electron multiplication CCD are successively
Connecting, described ITO electrode is connected with the positive pole of voltage generator, and silver electrode is connected with the negative pole of voltage generator.Under
Stating in each embodiment, unless otherwise indicated, the diameter as the O of solution room that each ITO electrode is pasted is equal
For 1cm, the amount of solution of detection is 600 μ L.
The Visual retrieval of hydrogen peroxide in embodiment 1 solution.
(1) determination of testing conditions.
In an experiment, tin indium oxide (ITO) sheet glass pastes the O of a diameter of 1cm as solution room, and
With this ito glass sheet as working electrode, select L012 as the luminescent substance under positive voltage.L012 is a kind of luminol
Analog, has more higher illumination effect than luminol in the presence of hydrogen peroxide, so the shiner under being selected as positive voltage
Matter.L012 and the hydrogen peroxide of 100 μMs, the 600 μ L altogether of 200 μMs is dripped in O.In view of L012 and
Hydrogen peroxide luminescence peak voltage on ito glass electrode is that (wherein, 1.0V is that hydrogen peroxide is at ITO electrode table to 1.0V
Face powers on the crest voltage decomposed, and the oxygen-derived free radicals then produced makes L012 luminous, so to different luminescence reagents,
Luminescence peak voltage is all similar, therefore, for other luminescence reagent, such as luminol, N-(4-aminobutyl)-N-second
The different luminol of base, the luminous voltage of N-(6-amino base) the different luminol of-N-ethyl, the different luminol of isothiocyanic acid etc. all exists
Between 0.9V～1.1V), while CCD exposes, on electrode, therefore apply 1.0V constant voltage make L012 and dioxygen
Water obtains the luminous intensity of maximum.But, electrode is continuously applied such high pressure electrode can be caused to inactivate, be unfavorable for
Luminous.In order to avoid electrode inactivates, we apply a negative voltage-1.0V on electrode so that electrode updates and recovers.
Therefore electrode voltage uses 1.0V and-1.0V translative mode to obtain more luminous signal.Each voltage change-over period
Including the 1.0V voltage of 2s, reconvert applies the-1.0V voltage of 0.5s.Compare with constant voltage mode, this combination electricity
Die pressing type is so that L012-hydrogen peroxide obtains higher illumination effect (as shown in Figure 2).This combination voltage pattern pair
The amplification of illumination effect indicates its importance for ECL imaging.Illumination effect can extending into because of time of exposure
One step is strengthened.But for Single-cell imaging, the shortest time of exposure can obtain more preferable temporal resolution.
Different electric voltage frequencies and the impact on imaging signal to noise ratio of the different time of exposure is compared in order to balance the two parameter, as
Shown in Fig. 3.Finally we have selected the time of exposure of 5s, and namely two voltage cycles are as the parameter of imaging experiment.
(2) electrogenerated chemiluminescence imaging device Visual retrieval hydrogen peroxide.
Being attached to ito glass sheet surface with an adhesive tape, ITO exposed under the conditions of utilizing making alive can be luminous, and by adhesive tape
Cover place will not luminous such phenomenon, verify and visualize inspection with the electrogenerated chemiluminescence imaging device of the present invention
It is feasible for surveying hydrogen peroxide.Fig. 4 A shows exposed glass region and the border in adhesive tape region under light field.At exposed glass
The O of the boundary stickup 1cm in region and adhesive tape region, as solution room, makes exposed glass region and adhesive tape region
Respectively account for half.In the case of without hydrogen peroxide, L012 can self-luminous under voltage effect, molten to the O type on ITO surface
The L012 of 200 μMs, record bias light of L012 under dark surrounds is added in liquid chamber.Result is as shown in Figure 4 B.
The same with intended, photographed faint luminescence at ITO exposed region, adhesive tape region is then completely black.When adding
After the hydrogen peroxide of 10 μMs, continue under dark surrounds, take pictures (Fig. 4 C).In order to measure the luminous strong of hydrogen peroxide generation
Degree, utilize software I mage J to calculate the difference between Fig. 4 B and Fig. 4 C, difference diagram with pseudo-colours dye after, as
Shown in Fig. 4 D.The reaction produced by L012 and hydrogen peroxide causes obtaining higher light intensity at ITO exposed region, it was demonstrated that
Our system can effectively observe the hydrogen peroxide of low concentration.Photograph via bright field and the tape edge shape of luminous difference diagram
Concordance explanation electrogenerated chemiluminescence imaging degree of accuracy.The region meter of a diameter of 50 μm is randomly selected in ITO region
Calculating luminous intensity, the light intensity relative deviation obtained is 2.63%.The homogeneity of the light intensity obtained ensure that detection at ITO not
Cultivate cell with region, and carry out the accuracy of imaging analysis.
The spatial resolution of electrogenerated chemiluminescence imaging device of the present invention is controlled by object lens and CCD.Although having plurality
The high magnification object lens in value aperture (N.A.) can increase receipts light efficiency, more high magnification and each pixel can be caused to present
Actual size is the least, so that the light intensity that each pixel obtains is reduced to below detection limit.In this detection system, thing
Mirror be N.A be that 20 times of mirrors of 0.50 are as optimum selection.Meanwhile, in order to collect weak light, have employed low resolution
The singl e photon detection electron multiplication CCD (EM CCD) of (512 × 512).Due to CCD each pixel 16 μm, thing
Mirror is 20 times of amplifications, so the spatial resolution being calculated detection system is 0.8 μm.This imaging device can pass through
Increase object lens multiplying power and realize the image quality of 0.4 μm, however it is necessary that by prolonging exposure time or increase hydrogen peroxide dense
Degree obtains.Needing low detection limit and short exposure time in view of us, the multiplying power of object lens is still fixed on 20 times,
The spatial resolution of 0.8 acquired μm is also suitable for single cell analysis.
(3) concentration of hydrogen peroxide and the combination voltage impact on ECL imaging.
In order to detect the relation between hydrogen peroxide concentration and luminous intensity, the concentration of fixing L012 is 200 μMs, in detection
System constantly raises hydrogen peroxide concentration and completes imaging process.After having deducted the bias light not having hydrogen peroxide,
Luminous intensity is different, as shown in Figure 4 E according to the concentration difference of hydrogen peroxide.Between luminous intensity and hydrogen peroxide concentration
Positive correlation shows that the light intensity of image can reflect the hydrogen peroxide concentration of local, and minimum visible hydrogen peroxide concentration is
10μM.As a comparison, under conditions of applying the constant voltage of 1.0v on electrode, minimum visible hydrogen peroxide concentration is 50 μMs.
The improvement of this visual detection limit determines the importance using combination voltage for ECL imaging.
Leak the Visual retrieval of hydrogen peroxide on embodiment 2 unicellular surface.
The unicellular hydrogen peroxide imaging that leaks is the solution room cultivated on ito glass sheet by 100-5000 Hela cell
In, then with crotonyl alcohol .-12-myristate-13-acetas (PMA, the Phorbol of 10 μ l20ng/ml
12-myristate-13-acetate, is called for short phorbol exters, buys from sigma-aldrich, and article No. is P8139) stimulate generation double
Oxygen water.It has been reported that PMA can stimulate intracellular NADPA oxidase to make the dioxygen water yield accumulate, thus cause
Hydrogen peroxide leaks.According to identical image-forming step, the light field figure under L012 existence condition and Background (figure are obtained
5A and 5B).In illuminated diagram, it is diffused into electrode surface owing to cell attachment hinders L012 at electrode surface, from
And it is rendered as dark intensity.After cell discharges hydrogen peroxide after PMA stimulates, take pictures at once, obtain one and send out
Light figure (Fig. 5 C).By by Fig. 5 C background correction Fig. 5 B, obtain cell in figure and brighten (Fig. 5 D).Luminescence of cell
Should not be to be contacted generation with L012, because in cell week from stacking chart by the oxygen-derived free radicals being diffused on cell
Enclose not luminescence phenomenon (Fig. 5 E).After eliminating free radical diffusion and causing luminous probability, bottom cell
There is L012 is exactly the only explanation to this phenomenon.For attached cell, the fold of cell membrane is believed at the bottom of cell
Portion and supporter surface form small space, thus cause L012 existence between cell and electrode.Through thorn
After Jiing, the hydrogen peroxide of cell lower surface release produces free radical under voltage effect, and with cell bottom L012
React, produce luminescence.Use software I mageJ, by the calculating to each pixel light intensity, with reference in Fig. 4 E
Light intensity signal, the light intensity that cell surface photographed is converted into the concentration of hydrogen peroxide at 10～30 μMs by labelling different colours
(Fig. 5 F).This result demonstrates the inhomogeneities that hydrogen peroxide leaks.
PMA is substituted by the phosphate buffered solution (PBS) of pH7.4 so that cell will not be subject in matched group is tested
Stimulate.Its imaging picture is as shown in Figure 6.Illuminated diagram after having deducted background is displayed without any light and strengthens situation appearance
At cell compartment.Cellular control unit does not has any hydrogen peroxide to discharge, so as expected, these cells do not have luminescence.
It can be the cell release under different conditions that the dependency that luminous intensity and hydrogen peroxide leak shows that our experimental technique is
Hydrogen peroxide provide its exist submicron distribution foundation.
With scanning electrochemical microscope (SECM)), cell hydrogen peroxide imaging is compared, use electrogenerated chemiluminescence formation method to have device
Simply with can be with the advantage of parallel quick detection.For electrogenerated chemiluminescence imaging, the ITO electrode cultivating cell is placed
Detecting on microscope stage, electrode need not modify further.This simple method can promote electrogenerated chemiluminescence
Analytical technology is used for biological study.Meanwhile, the illuminated message that can simultaneously obtain ITO surface all cells increases detection
Flux, this is the most significant for Single cell analysis.The luminous signal of 6 cells show for average intensity
Standard deviation be 10.0%, this reflects that the fluctuation of the dioxygen water yield that different cell leaks is less.
The electrogenerated chemiluminescence imaging of the unicellular surface molecular of embodiment 3.
Except the hydrogen peroxide of cell release being carried out imaging, the electrogenerated chemiluminescence formation method of the present invention can also profit
Convert them into hydrogen peroxide with the oxidase that the molecule of cell surface is corresponding, thus realize the one-tenth to cell surface molecule
Picture.In order to prove can be to cell surface molecule imaging, we have selected cholesterol as template molecule, and detection cell membrane is lived
Change the distribution of cholesterol molecule.Activation cholesterol has higher chemical potential (fleeing from trend), and it is to cell membrane cholesterol
Transport has important function.Although cell membrane cholesterol can utilize fluorescence cholesterol analog or flipin to become
Picture, but be because these fluorescent probes and cannot be distinguished by activation cholesterol and disactivation cholesterol, therefore cell surface activation gallbladder
Sterin is never successfully imaged.And the electrogenerated chemiluminescence method of the present invention can utilize activation cholesterol and cholesterol oxidation
Enzyme reaction generates this characteristic of hydrogen peroxide, by the method for ECL imaging, activation cholesterol is carried out imaging.In experiment,
First cell processed in the culture fluid containing sandoz58035 (buying in sigma-aldrich, article No. is S9318)
Night, by suppression acyl-coenzyme a-cholesterol acyltransferase (acyl-coA/cholesterol acyltransferase) (ACAT)
Albumen, it is achieved more cholesterol accumulates in cell membrane, thus increases the activity of cholesterol.When cell is exposed to gallbladder admittedly
In alcohol oxidase and L012 environment, the two material all can diffuse in the slight void bottom cell.When cholesterol oxygen
After changing enzyme and activation cholesterol reaction generation hydrogen peroxide, apply voltage and will realize electrogenerated chemiluminescence imaging.Light field and adding
Enter the emission difference figure after cholesterol oxidase respectively such as Fig. 7 A, shown in 7B.From the point of view of Fig. 7 B, cell surface goes out
Now brighter than surrounding light intensity, shows it may be seen that the activating cell Membrane cholesterol of cell surface.From adding puppet
Colored Fig. 7 C and partial enlarged drawing (Fig. 7 D) are upper it can be seen that the activation cholesterol distribution of cell surface exists certain
Unevenness.
The experiment of negative control group uses statins to come in cell repressed to ACAT
The work of 3-hydroxy-3-methylglutaryl-CoA (3-hydroxy-3-methylglutaryl-coenzyme A reductase, HMGCoA)
Property suppresses, and causes reducing intracellular cholesterol biosynthesis thus the activation cholesterol that lowers on cell membrane.Luminous intensity
Difference diagram as shown in the photograph via bright field in Fig. 7 E and Fig. 8 and superposition photo.From HMGCoA and ACAT simultaneously
Be suppressed luminescence of cell figure it is known that, although intracellular cholesteryl in this kind of cell and Membrane cholesterol total amount are significantly
Reduce, but activation cholesterol still exists.Luminescence of cell repressed with ACAT is compared, HMGCoA, ACAT
It can be that pharmaceutical analysis provides activation film gallbladder solid that the weak light that the most repressed cell produces demonstrates ECL formation method
The distributed intelligence of alcohol.This is imaged as cellular cholesterol transportation research to cell activation cholesterol and provides more biology
Information.
In sum, present invention employs what cell was leaked by the ECL imaging system that a set of spatial resolution is 0.8 μm
Hydrogen peroxide and cell membrane activation cholesterol have carried out imaging.Electrode surface luminescence of cell is so that multiple cell becomes simultaneously
Picture, therefore this set of simple system has high detection flux.Follow-up study is visited being conceived to more efficient ECL luminescence
Pin increases luminous intensity to reach higher signal to noise ratio to obtain the Single-cell imaging figure of higher resolution.
Claims (2)
1. electrogenerated chemiluminescence imaging device leaks on the unicellular surface of Visual retrieval the application in hydrogen peroxide, institute
State electrogenerated chemiluminescence imaging device and include object lens, transmission line, singl e photon detection electron multiplication CCD and electroluminescentization
Learning luminous generator, described electrogenerated chemiluminescence generator includes ITO electrode, silver electrode and voltage generator, wherein,
Described object lens, transmission line and singl e photon detection electron multiplication CCD are sequentially connected with；Described ITO electrode is sent out with voltage
The positive pole of raw device is connected, and silver electrode is connected with the negative pole of voltage generator, and its application comprises the steps: at ito glass
The surface mount O of sheet is as solution room, and with ito glass sheet as working electrode, silver electrode is to electrode, will be thin
Born of the same parents cultivate in solution room, add luminescence reagent in solution room, and being subsequently adding PMA stimulates intracellular NADPA
Oxidase makes cell surface produce hydrogen peroxide, executes to ITO electrode while singl e photon detection electron multiplication CCD exposes
Adding periodic luminous voltage-recovery voltage, detect imaging effect, wherein, singl e photon detection electron multiplication CCD exposes
Time be two luminous voltages-recovery voltage cycle, wherein, a luminous voltage-recovery voltage cycle is: luminous electricity
Pressure: 2 ± 1s under the positive voltage of 1.0 ± 0.1V；Recover 0.5 ± 0.25s under the negative voltage of voltage :-1.0 ± 0.1V.
Application the most according to claim 1, it is characterised in that described luminescence reagent is L012, luminol,
N-(4-aminobutyl) the different luminol of-N-ethyl, N-(6-amino base) the different luminol of-N-ethyl and the different luminol of isothiocyanic acid
In any one, consumption is 100 μMs～500 μMs.
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| CN105717100A (en) * | 2016-04-12 | 2016-06-29 | 山东大学 | Detecting system capable of accurately acquiring ECL (electrochemiluminescence) spectral information |
| CN105928927B (en) * | 2016-04-15 | 2019-03-12 | 南京大学 | The method that electrogenerated chemiluminescence imaging method quickly analyzes unicellular interior ingredient |
| CN107764803B (en) * | 2017-09-21 | 2021-04-13 | 中山大学 | Biomarker detection method using electrochemiluminescence imaging recognition technology |
| CN111157769A (en) * | 2020-01-06 | 2020-05-15 | 广州大学 | Electrochemiluminescence imaging system and imaging method thereof |
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