CN101334361A - ATP motor rotary type biosensor single molecule analysis and separation technology and method - Google Patents
ATP motor rotary type biosensor single molecule analysis and separation technology and method Download PDFInfo
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- CN101334361A CN101334361A CNA2007101180603A CN200710118060A CN101334361A CN 101334361 A CN101334361 A CN 101334361A CN A2007101180603 A CNA2007101180603 A CN A2007101180603A CN 200710118060 A CN200710118060 A CN 200710118060A CN 101334361 A CN101334361 A CN 101334361A
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Abstract
With the aid of basic researches on ATP molecular motor structures and functions, the invention further designs a full-circle biological sensor which is used for analyzing unimolecules that are taken as carrier connecting locus of Epsilon subunits, a preparation method of a plurality of CdTe semiconductor nano crystals that have pH sensitivity, water solubility and high photoluminescence efficiency, and a full-circle ATP motor sensor which is used for marking charge free bodies of ATP motor compounds in a single direction. By adopting the compound that is obtained by combining the full-circle biological sensor with the molecular motor principle and the quantum dot technique, the invention generates a unimolecule device that has novel functions, and combines biological molecule analyzing and separating functions with high resolution capability into a whole; the invention can be developed into a novel analyzing and separating technique with ultra-high resolution in physics, can be widely used in multiple fields of environment, national defense, energy and information, etc., thus having wide application value and enormous economic and social benefits.
Description
Technical field
The invention belongs to the analysis of unimolecule optical encoding and the unimolecule separation technology field thereof of nano biological ultra-high sensitive, relate to the biology sensor unimolecule design of pivoted ATP motor, the important improvement of the responsive CdTe semiconductor-quantum-point of assembling and pH mark new synthetic method, the new application of quantum dot-labeled ATP motor complex surfaces electric charge free body in optical encoding analysis and separation thereof.
Background technology
Last year, U.S. scientist Roukes etc. with unusual high frequency physical vibration principle, developed the result of study that 7000 dalton differentiate; The science magazine is made comments for this reason: think that their result " has pushed over impossible obstacle of going beyond " from then on, scientists has begun the science contest that the single dalton of a new round measures the physics instrument developing direction of direction.[Nano?letters(2006)583-586，Zeptogram-scale?nanomechanical?Mass?sensing]，Science，312(5)2006。But this technology obviously exists deficiency from application distance to be arranged under utmost point cryogenic conditions.
The main at present fluorescent apparatus of using the costliness of fluidic cell isolation technics of the analysis of biological sample and isolation technics, resolution characteristic is based on cell (30uM); The analysis of unimolecule nano particle is analyzed microRNAs with the special device streaming special device of confocal coupling microfluid channel also report Neely etal., Nat Methods 3 (2006) 41-46.; Difference is very far away but the ultrahigh resolution from present scientific research in above-mentioned all technology requires (reaching dalton differentiates).Therefore develop the quick of single molecules level, convenient, cheap super-resolution ability (reaching dalton differentiates) is analyzed with new technology of separating and new method and is become present numerous discipline development bottleneck problem.
Equally, after the F1-ATP motor unimolecule of rotation molecular motor was discovered, the reason that makes global scientist feel that favor is inspired was after the discovery according to new physical form, and the rule of the appearance of new application is all arranged.The breathtaking breakthrough of the research of molecular motor is not limited to life science; And be the physics super-resolution, separating provides brand-new thinking.
The inventor has submitted the Chinese patent application (application number: 200410098929.9 that is entitled as " micro-power biological sensor with adjustable molecular motor " on Dec 16th, 2004, Dec 16 2004 applying date, publication number CN1789425, open day on June 21st, 2006), disclose a kind of micro-power biological sensor with adjustable molecular motor, it comprises with the lower part: (1) rotation motor: F
0F
1-ATP enzyme; (2) energy converting apparatus: light reaction center and compound (RC) and ubiquinone; (3) transferred electron device: with energy converting apparatus be same system; (4) signaling molecule output unit: form by optical excitation and emitter and fluorescence probe; (5) energy resource system: by water, ATP, ADP, Phos and visible light are formed; (6) protective seam: bilayer lipid membrane is made the protective seam of position inner membrance; (7) immobilization material of timbering material and bilayer lipid membrane.This patent disclosure fluorescence probe can be the Lipids-fluorescein that is positioned at the film outside, be positioned at the fluorescein of film inboard, the molecular motor rotation is driven by the ATP hydrolysis, wherein said molecular motor rotation is to be driven by the membrane electrochemical potential difference (PD) of striding that can change, and the perhaps described membrane electrochemical potential difference (PD) of striding is by luminous energy or chemical energy conversion.In this biology sensor, load is to be connected on three β subunits of the molecular motor of biology sensor.This patent also discloses the application of described micro-power biological sensor with adjustable molecular motor in the detection of biomacromolecule, viruses molecule etc.
Summary of the invention
The present invention is the further improvement to Chinese number of patent application 200410098929.9, difference be in biology sensor of the present invention (1) adopt the novel C dTe quantum dot fluorescence probe produced by novel preparation method and (2) with different fluorescent emission wavelength wherein load be connected with the epsilon subunit of molecular motor, the single molecule analysis of really having realized the ATP motor rotary type biosensor with separate.
Particularly, the present invention relates to following every:
1. the preparation method of the water-soluble high photoluminescence efficiency CdTe quantum dot of pH responsive type, this method comprises the steps:
(1) by Te powder and NaBH
4Or KBH
4Reaction, synthetic sodium hydrogen telluride or hydrogen telluride aqueous solutions of potassium;
(2) use the synthetic nanocrystalline precursor solution of CdTe of cadmium salt or its hydrate and stabilizing agent, described cadmium salt is selected from CdNO
3And CdCl
2, described stabilizing agent selected from mercapto acetate, mercaptopropionic acid and mercapto glycerol;
(3) sodium hydrogen telluride or hydrogen telluride aqueous solutions of potassium are mixed with the nanocrystalline precursor solution of CdTe, it is nanocrystalline to add thermal synthesis CdTe;
(4) it is nanocrystalline to collect CdTe under the differential responses time, is the size CdTe quantum dot different with the fluorescent emission wavelength,
Wherein in step (2), add the photoluminescence efficiency of ammoniacal liquor with the raising product, and
Wherein the molar ratio of cadmium salt, sodium hydrogen telluride or hydrogen telluride potassium and stabilizing agent is 1: 0.1-1: 2.
In a specific embodiments of the present invention, the preparation method of the water-soluble high photoluminescence efficiency CdTe quantum dot of a kind of pH responsive type is provided, it is characterized in that comprising the steps: that (1) is by Te powder and NaBH
4Or KBH
4Reaction, synthetic sodium hydrogen telluride or hydrogen telluride aqueous solutions of potassium: with 0.2-2mmolTe powder and 100-1000mg KBH
4Or NaBH
4Put into the reaction bulb of 1-10ml deionized water, be communicated with the hydrogen that is produced to get rid of with atmosphere in the system, reacted 0.5-8 hour, when solution becomes water white transparency, be the aqueous solution of synthetic KHTe or NaHTe, standby; (2) with synthetic nanocrystalline precursor solutions of CdTe of stabilizing agent such as cadmium salt or its hydrate and mercaptoacetic acid, mercaptopropionic acid, mercapto glycerols: in three neck reaction bulbs, add the 200-2000ml deionized water, add stabilizing agents such as the cadmium salt of 0.5-20mmol or its hydrate and mercaptoacetic acid, mercaptopropionic acid, mercapto glycerol then, make its dissolving, wherein cadmium salt; KHTe or NaHTe; The mol ratio of stabilizing agents such as mercaptoacetic acid, mercaptopropionic acid, mercapto glycerol is 1: 0.1-1: 2, logical high pure nitrogen 15-40 minute, add the photoluminescence efficiency that proper ammonia improves product in the experimentation, obtain the quantum dot (quantam dot) of the less stabilized illumination of size simultaneously.Adjust pH 7.0-12.0, the promptly synthetic nanocrystalline precursor solution of CdTe, standby; (3) it is nanocrystalline to add thermal synthesis CdTe: the aqueous solution of KHTe or NaHTe is poured in the three neck reaction bulbs of the nanocrystalline precursor solution of CdTe, made it to be heated to 60-100 ℃, synthetic CdTe is nanocrystalline, connects condenser pipe and makes solvent refluxing, constantly stirs; (4) it is nanocrystalline to obtain CdTe: in reaction bulb, within the synthetic reaction 5 minutes-2 hours, collect the synthetic reaction product, it is nanocrystalline to be CdTe.Nanocrystalline at the CdTe that different time is collected, be the CdTe quantum dot of the various glow colors of different size and fluorescent emission wavelength, its particle size is 2nm-4nm, its fluorescence emission spectrum peak is 500nm-680nm, its fluorescence intensity is than the obvious high 30%-50% of other synthetic method, the difference of this method and other method is: add proper ammonia in reaction system, can improve fluorescence quantum yield.The quantum dot that this method obtains has the pH sensitive natur, and with the linear characteristics that reduce of the reducing of pH (pH scope from 9 to 6), this makes us this quantum dot can be advantageously applied to the molecular motor system.In another embodiment of the inventive method, add the Zn ion to improve the quantum dot fluorescence quantum yield.
2. the CdTe quantum dot by the preparation of above 1 method.
3. improved unimolecule ATP motor rotary type biosensor is characterized in that:
The probe of its use is according to above 2 described CdTe quantum dots.The method of probe mark is that those of ordinary skills are known, for example as described in the CN1789425.
4. according to above 3 described unimolecule ATP motor rotary type biosensors, wherein with F
0F
1The epsilon subunit of-ATP enzyme is as the connection site of load.The method of attachment of epsilon subunit and load is known in the art, and identical with the method for attachment of describing in CN1789425 about the β subunit.
5. according to above 3 or 4 described unimolecule ATP motor rotary type biosensors, wherein said CdTe quantum dot is that the form as the surface charge free body exists.
In a specific embodiments of the present invention, provide the surface charge free body of the coding techniques of fluorescence quantum.Material with quantum dot replaces chemical dye to use at rotary immunosensor, its special card comprises the steps: that the folk prescription of (1) quantum dot is to the outside technology of the double-deck phosphatidase body of mark (bilayer lipid membrane), at (0.1mM, Tricine (three (methylol) methylglycine pH 6.5) system is cultivated 15 fens kinds, quantum dot is externally removed with centrifugal method, be the quantum dot chromatophore; After further the immune recognition system of assembling connected then, it was synthetic to start the chromatophorous ATP of quantum dot with ADP, and at this moment, proton is from the inner outwards transhipment of double-deck phosphatidase body, and the pH value of external systems increases, and the quantum dot fluorescence response increases fluorescence intensity; The ATP of molecular motor is synthetic to be taken place simultaneously with the proton transhipment, and its signal can be turned to low by the signal amplitude of quantum dot fluorescence by height, thereby finishes the function of identification.
Above we seen respectively different fluorescence quantum folk prescriptions to mark to chromatophore, this moment the proton translocation process, this quantum dot folk prescription forms a surface charge free body to mark to chromatophore, this surface charge free body possesses to be drawn and the charge characteristic that keeps inner proton transhipment degree, and can keep relatively independent, with the mutual interference mutually of contiguous another surface charge free body seldom, can make this surface charge free body keep stability for a long time, therefore our other application of expanding to of application of samples charge characteristic easily, as the mensuration of encoding, use electrophoresis fluid analysis etc.
6. according to above 3 or 4 described unimolecule ATP motor rotary type biosensor, wherein F
0F
1The epsilon subunit of-ATP enzyme and the antibodies of this epsilon subunit, and the antibody of described epsilon subunit further combines with biomolecule by biotin-chain enzyme antibiotin-biotin.The method of attachment of epsilon subunit is known in the art, and identical with the method for attachment of describing in CN1789425 about the β subunit.
7. according to above 6 described unimolecule ATP motor rotary type biosensors, wherein said biomolecule is the antibody of DNA, little RNA or viruses molecule specific antigen.In CN1789425, described the ATP motor rotary type biosensor has been used to detect avian influenza virus and clenobuterol hydrochloride micromolecule etc., will describe in an embodiment below the present invention unimolecule ATP motor rotary type biosensor of the present invention is used to detect little RNA (mRNA) molecule.
8. multiple monomolecular method in the test sample, wherein:
(1) will be respectively applied for and detect described multiple monomolecular different biological molecules and load on the epsilon subunit according to the ATP motor of the biology sensor of any one in above 3 to 7;
(2) use the biology sensor that obtains according in the different CdTe quantum dot difference markers step (1) of above 2 fluorescent emission wavelength;
(3) biology sensor that will obtain in step (2) contacts with testing sample;
(4) the ATP motor that drives described biology sensor rotates, the relatively biology sensor and the fluorescence intensity of the biology sensor that does not contact with testing sample under different fluorescent emission wavelength of acquisition in (3) are if wherein fluorescence intensity changes and has the unimolecule of the biomolecule correspondence of load on the described biology sensor in the interpret sample.
The several method of the ATP motor rotation that drives described biology sensor is disclosed in CN1789425, for example (a) changes mechanical energy with chemical energy (as: ATP is hydrolyzed into ADP and phosphorus gives off energy simultaneously, or stride membranous son transhipment, ADP and phosphorus synthesize ATP); (b) convert electronic energy (formation transmembrane potential difference) to luminous energy; (c) drive the rotation of molecular motor with external force.
Analysis and isolation technics that epsilon subunit links together as the unimolecule rotary type biosensor of the connection site of load and (2).
As the sensor of load (should be called half rotary) basic difference is arranged as the research of load and β subunit with epsilon subunit; And the F1F0-ATP motor sensor of the target site that the epsilon subunit that the present invention proposes connects as the identification molecule should be called pivoted sensor (Fig. 1).
(a) analytical technology: (1) is to the analysis of microRNAs (small fragment RNA) super-resolution ability, the analysis of (2) optical encoding.
(b) isolation technics:
Above-mentioned proposition rotary type biosensor, start 10 fens kinds of rotation with ATP after, from intracellular transport to the extracellular, the surface charge of rotary type biosensor is changed on proton, use the electrophoretic techniques can release surface electric charge free body again.This technology will be used the fluidic cell isolation technics always and electrophoresis separating method combines, and can exempt the fluorescent apparatus of fluidic cell isolation technics costliness; With the sensor of rotation molecular motor technology, develop into novel single molecule analysis and separation technology as unimolecule identification.
9. according to above 8 method, the antibody of the wherein antibodies of the epsilon subunit of biology sensor and this epsilon subunit, and described epsilon subunit further combines with biomolecule by biotin-chain enzyme antibiotin-biotin.
10. according to above 9 method, wherein said biomolecule is the antibody of DNA, little RNA or viruses molecule specific antigen.
The present invention is with the biology sensor of pivoted molecular motor principle and compound after technology of quantum dots combines, produced to have a kind of brand-new function unimolecule device; This device possess to biomolecule carry out the analysis of high resolution capacity, the function of separation is an one; Be expected to develop into super-resolution in the physics, separate new technology is provided.Can be widely used in various fields such as environment, national defence, the energy, information, using value and huge economy and society benefit are extremely widely arranged.
Detailed Description Of The Invention
By means of the structure of molecular motor, the progress of function and principle has proposed F1F0-ATP motor pivoted biological sensor new ideas and the new technology of the epsilon subunit of molecular motor F1 as the target site of identification molecule connection; Provide the responsive water-soluble high photoluminescence efficiency CdTe semiconductor-quantum-point of a kind of pH the preparation method and with the molecular motor folk prescription to mark.Content of the present invention has two impressive progresses than previous invention: (1) at first in the selection of F1 epsilon subunit as the target site that sets the molecule connection, becomes unit point identification with former β subunit three site identification turns; The β subunit has been played the part of the push-and-pull motor pattern and should be called partly rotary with the sensor that F0 rotates the composition of coupling mutually in the structure function of F1F0-ATP molecular motor; And the epsilon subunit that the present invention proposes should be called pivoted as the F1F0-ATP motor sensor that sets the target site that molecule connects; Because the mode of motion at the F1-epsilon subunit is and F0 rotation coupling mutually, owing to connect the difference in site, three site identification turns are become unit point identification, the mode of motion of epsilon subunit and F0 rotation coupling mutually, therefore being called the F1F0-ATP motor sensor should be called pivoted.In view of above-mentioned important difference, biology sensor of the present invention can be analyzed individual molecule.(2) in the labelling technique of quantum dot, still do not replace fluorescent dye, but found that the surface charge free body of the flag F 1F0-complex of fluorescence quantum exists, for the range of application that further expands molecular motor as, optical encoding is measured the proton transhipment; Different rotary bodies can carry out unimolecule to be separated.(3) important improvement of the responsive water-soluble high photoluminescence efficiency CdTe semiconductor nano synthetic method of pH.
The objective of the invention is to realize by following technical scheme:
(1) the responsive water-soluble high photoluminescence efficiency CdTe semiconductor nano synthesis phase of pH is raw materials used is cadmium salt, ion-type tellurium source, sulfydryl micromolecule, NH
3Micromolecule.Cadmium salt can be CdNO
3, CdCl
2Deng; Ion-type tellurium source can be NaHTe etc.; The sulfydryl micromolecule can be mercaptoacetic acid, mercaptopropionic acid, mercapto glycerol etc.Cadmium salt, ion-type tellurium source, the micromolecular molar ratio of sulfydryl are 1: 0.1-1: 2.Add the photoluminescence efficiency that proper ammonia improves product in the experimentation, obtain the quantum dot of the less stabilized illumination of size simultaneously.The reaction heating-up temperature is 60-100 ℃ a mode, and the difference of heat time heating time can obtain the CdTe of different glow colors nanocrystalline (be also referred to as nanocrystal in this area, or quantum dot).The preparation method may further comprise the steps:
1.. by Te powder and NaBH
4Or KBH
4Reaction, synthetic sodium hydrogen telluride or hydrogen telluride aqueous solutions of potassium: with 0.2-2mmolTe powder and 100-1000m gKBH
4Or NaBH
4Put into the reaction bulb of 1-10ml deionized water, be communicated with the hydrogen that is produced to get rid of with atmosphere in the system, reacted 0.5-8 hour, when solution becomes water white transparency, be the aqueous solution of synthetic KHTe or NaHTe, standby;
2.. with synthetic nanocrystalline precursor solutions of CdTe of stabilizing agent such as cadmium salt or its hydrate and mercaptoacetic acid, mercaptopropionic acid, mercapto glycerols: in three neck reaction bulbs, add the 200-2000ml deionized water, add stabilizing agents such as the cadmium salt of 0.5-20mmol or its hydrate and mercaptoacetic acid, mercaptopropionic acid, mercapto glycerol then, make its dissolving, wherein cadmium salt; KHTe or NaHTe; The mol ratio of stabilizing agents such as mercaptoacetic acid, mercaptopropionic acid, mercapto glycerol is 1: 0.1-1: 2, and logical high pure nitrogen 15-40 minute, add the photoluminescence efficiency that proper ammonia improves product in the experimentation, obtain the quantum dot of the less stabilized illumination of size simultaneously.Adjust pH 7.0-12.0, the promptly synthetic nanocrystalline precursor solution of CdTe, standby;
3.. it is nanocrystalline to add thermal synthesis CdTe: the aqueous solution of KHTe or NaHTe is poured in the three neck reaction bulbs of the nanocrystalline precursor solution of CdTe, made it to be heated to 60-100 ℃, synthetic CdTe is nanocrystalline, connects condenser pipe and makes solvent refluxing, constantly stirs;
4.. it is nanocrystalline to obtain CdTe: in reaction bulb, within the synthetic reaction 5 minutes-2 hours, get the synthetic reaction product, it is nanocrystalline to be CdTe.Nanocrystalline at the CdTe that different time is collected, be the CdTe quantum dot of the various glow colors of different size and fluorescent emission wavelength, its particle size is 2nm-4nm, its fluorescence emission spectrum peak is 530nm-590nm.
In order to improve the water-soluble quantum dot fluorescence quantum yield.Be 10 with 20mL concentration in the test
-5M quantum dot aqueous solution and 20mL concentration are 10
-3The M zinc nitrate solution mixes, and reacts 12 hours.With 20mL concentration is 10
-5The quantum dot aqueous solution is mixed with 20mL water.The contrast of both fluorescence spectrums is found that enhancing has taken place fluorescence intensity, and the ratio of enhancing is up to from 60% to 120%.This quantum dot has the pH sensitive natur, and its fluorescence intensity is with (the pH scope is from 9-6) the linear characteristics that reduce that reduce of pH, and this makes us this quantum dot can be advantageously applied to the molecular motor system.
(2) molecular motor biology sensor of the present invention comprises the following:
(1) rotation motor: F
0F
1-ATP enzyme;
(2) energy conversion technique (ATP is synthetic, ADP and phosphorus, driven in rotation motor)
(3) signaling molecule output unit: form by optical excitation and emitter and fluorescence probe;
(4) energy resource system: by water, ATP, ADP, Phos is formed;
(5) protective seam: bilayer lipid membrane is as the functional protection layer of inner membrance;
(6) immobilization material of timbering material and bilayer lipid membrane.
(7) (preparation of the antibody of epsilon subunit and purification process are well known by persons skilled in the art to the antibody of epsilon subunit, for example referring to Biochem Biophys Res Commun 347 (2006) 752-757, Review of polyclonal antibody production procedures inmammals andpoultry, ILARJ.37 (1995) 93-118)
The structure of above system is well known by persons skilled in the art, for example referring to CN1789425.
In a preferred embodiment of the invention, above-mentioned fluorescence probe is a quantum dot fluorescence.It can folk prescription to the outside of ground mark to film, it changes insensitive to environment pH, and responsive to inner proton transhipment, utilizes our coding techniques that proposed semiconductor-quantum-point of this character to be used for the new technology of proton transhipment.Since semiconductor-quantum-point be marked at the lipase external body, make this technology not only can expand to optical encoding, can utilize simultaneously the surface charge free body of its F1F0-complex to exist and the relevant principle of molecular motor rotation, the binding soln electrophoretic techniques can be separated by unimolecule.
(8) fluorometric assay of proton transhipment:
In one embodiment of the invention, the molecular motor rotation is synthetic by striding membrane electrochemical potential difference (PD) driving ATP.
By with F
0F
1The epsilon subunit of-ATP enzyme combines with the antibody of this epsilon subunit (first antibody), alternatively further by identification body (specific recognition) the mir-145 probe sequence of chain enzyme antibiotin with biotin (biotin) mark: 5 '-GGGGAUUCCUGGAAACUGC-biotin is connected, and carries out specific recognition by the microRNAs in identification body and the cell pyrolysis liquid.
(9) fluid analysis of the surface charge free body existence of the flag F 1F0-complex of proton transhipment quantum dot
At microfluidic device (Bio-MEMS, biophysics institute, list of references: Technologies andApplications, edited by wanjun Wang, Steven, A.Soper, press, CRC, 2006, the logical direct current (1.2V) of going up of runner positive and negative electrode p168): the surface charge free body flowing velocity of the flag F 1F0-complex of difference observation of quantum point, the fluid velocity of the synthetic group of ATP are less than the synthetic group of no ATP, how much relevant with its rotational speed the surface charge that the F1F0-complex is described is, and can separate with electrophoresis method.
The present invention also provides the above-mentioned application of Methods for Coding biology sensor in the detection of little RNA (microRNAs).
Description of drawings
Fig. 1 is F
0F
1The main composition of-ATP molecular motor;
The comparison of the biology sensor that the biology sensor that three sites of F1-β subunit connect is connected with F1-epsilon subunit single-point.
1. β/epsilon subunit antibody, 2. biotin; 3. chain enzyme antibiotin; 4. biotin, 5. biotin-nucleic acid recognizing body; 6. quantum dot fluorescence, 7. chromatophore (Chromatophores)-F1F0-ATP;
Fig. 2. the absorption spectrum of typical C dTe quantum dot, excitation spectrum and photoluminescence spectrum;
Fig. 3. zinc ion is to the influence of quantum dot fluorescence quantum yield;
Figure A:(A) zinc ion strengthens 70% for the quantum dot in 559 nanometers; (B) luminous quantum dot in 559 nanometers,
Figure B:(A) zinc ion strengthens 80% for the quantum dot in 590 nanometers; (B) luminous quantum dot in 590 nanometers;
Fig. 4. the relation that quantum dot light emitting intensity changes with the pH value
The quantum dot of curve (a) 559 nanometers is at 50mM, Tricine, the relation that middle intensity changes with the pH value;
The quantum dot of quantum dot-labeled thing 559 nanometers of curve (b) chromatophore (Chromatophores) ATP--at 50mM, Tricine, the relation that middle intensity changes with the pH value;
The quantum dot of quantum dot-labeled thing 590 nanometers of curve (c) chromatophore (Chromatophores) ATP--at 50mM, Tricine, the relation that middle intensity changes with the pH value.
Fig. 5. quantum dot is adsorbed on the chromatophore surface.Add ADP and synthesize damping fluid and start.
A: chromatophore does not add ADP, in contrast group
B: chromatophore adds ADP again and starts after adding DCCD
C: chromatophore adds ADP and starts.
We find out that clearly in the C group, the quantum dot fluorescence intensity that is marked on the chromatophore outside is strengthened greatly.
Fig. 6. to the mensuration of microRNAs
Biology sensor is fixed on the fine little plain film front surface (the straight warp of 5mm) of nitric acid in 0.45 μ M aperture, the microRNAs probe of 3 ' mark biotin (5 '-GGGGAUUCCUGGAAACUGC-3 ') is attached to the F that is positioned at the chromatophore surface by ε antibody-biotin-streptavidin
0F
1On-ATPase the epsilon subunit.Per step condition of contact is incubated at room 30 minutes.Utilize hypotonic method with cell MCF7 (teacher Chen Jianwen of biophysics institute of the Chinese Academy of Sciences is so kind as to give) cracking, thereby microRNAs is discharged in the solution.Each test sample adds 100ul lysate, with predetermined fixed hatched 5 minutes under the fine little plain film room temperature of nitric acid of biology sensor, microRNA is combined with probe.Measure putting into special-purpose luminoscope (BPCL-II) after the synthetic damping fluid adding of the ATP sample startup synthetic reaction then.From top to bottom, number of cells respectively: 0,10
610
5, 10
4, 10
3, 10
2, be F under different load
0F
1-molecular motor rotational speed changes;
Fig. 7 quantum dot and chromatophore free body surface charge characteristics.
From figure can illustrate different fluorescence quantum folk prescriptions to mark to chromatophore, this moment, the proton translocation process was not disturbed mutually with contiguous another surperficial free body.The explanation fluorescence quantum folk prescription on the left side divides other experimental result to mark to chromatophore, and (from left to right be respectively: (a), (b), (c), (d), the contrast among the figure refers to use respectively a kind of other experimental result of quantum dot-labeled branch with contrast RNA; And encode, coding RNA represents respectively with two kinds of quantum dot-labeled materials in same solution system, measure simultaneously with optics coding techniques (promptly under different excitation wavelengths)), wherein (a) is not connected with RNA for the 559mn mark; (b) be the 590mn mark and connect, measure respectively with RNA; (c) with (d) with (a) and (b) same, measure simultaneously at 559mn and 590mn but mix to excite respectively with 470nm; We see that (559mn 590mn) is marked on behind the chromatophore surface and they is mixed again, and they can independently detect object separately with two kinds of quantum dots.The The above results explanation coding of application of samples is easily measured.
Fig. 8 is with the separating resulting of electrophoresis microfluidic device to the surface charge free body of F1F0-complex.
There is the stability of surface charge in free body in the electrophoresis system
After figure (A) chromatophore adds DCCD, add ADP again and start electrophoretic velocity;
Figure (B) chromatophore adds ADP and starts electrophoretic velocity.
Free body electrophoresis direction illustrates that from positive polar motion the electric charge on chromatophore-quantum dot surface is electronegativity, when proton the transhipment, is the negative back that fetters by the electric charge on quantum dot surface and reduces electronegativity outside film in film, slows down electrophoretic velocity.
Embodiment
The present invention is described in further detail below in conjunction with embodiment, is that the present invention is limited but should not be construed as.
The preparation of embodiment 1:CdTe quantum dot
Unless otherwise noted, all chemical reagent of this paper are all available from Beijing chemical reagents corporation.With 0.5mmol Te powder and 40mg NaBH
4Put into the reaction bulb of 1ml deionized water, be communicated with the hydrogen that is produced to get rid of with atmosphere in the system, reacted 3 hours, when solution becomes water white transparency, be the aqueous solution of synthetic NaHTe, standby; (2) CdNO
3Synthesize the nanocrystalline precursor solution of CdTe with mercaptoacetic acid: in three neck reaction bulbs, add the 100ml deionized water, add the CdNO of 0.5mmol then
3Make its dissolving, wherein CdNO with mercaptoacetic acid
3, NaHTe, mercaptoacetic acid mol ratio be 1: 1: 2, logical high pure nitrogen 15 minutes adds ammoniacal liquor (weight ratio is 28%) in the experimentation, adjust pH 8.5, the promptly synthetic nanocrystalline precursor solution of CdTe, standby; (3) it is nanocrystalline to add thermal synthesis CdTe: with pouring in the three neck reaction bulbs of the nanocrystalline precursor solution of CdTe, make it to be heated to 100 ℃, synthetic CdTe is nanocrystalline, connects condenser pipe and makes solvent refluxing, constantly stirs; (4) it is nanocrystalline to obtain CdTe: in reaction bulb, synthetic reaction 1 hour is collected the synthetic reaction product, and it is nanocrystalline to be CdTe.Its particle size is 2nm, and its fluorescence emission spectrum peak is that (assay method is with reference to Gao, M.Y. for 560nm; Kirstein, S.;
H.; Rogach, A.L.; Kornowski, A.; Eychm ü ller, A.; Weller, H.J.Phys.Chem.B 1998,102,8360-8363.), (seeing accompanying drawing 2).
Embodiment 2: zinc ion is to the influence of quantum dot fluorescence quantum yield
With 20mL concentration is 10
-5M quantum dot aqueous solution and 20mL concentration are 10
-3The M zinc nitrate solution mixes, reacted 12 hours, and be 10 with 20mL concentration
-5The quantum dot aqueous solution is mixed with 20mL water, and both fluorescence spectrum contrasts are found that enhancing has taken place fluorescence intensity, and the ratio of enhancing is relevant with the size of quantum dot, and the ratio of enhancing is up to from 60% to 120%.Further experiment shows that the reason of enhancing is a zinc ion, has all found the phenomenon that the quantum dot quantum yield strengthens under the effect of different water-soluble zinc salts.Also got rid of the influence of pH value to quantum yield simultaneously, because the zinc nitrate that adds makes the pH value reduce, quantum yield should reduce, but instead quantum yield increases, and this has just confirmed the effect that zinc ion strengthens fluorescence more.(seeing accompanying drawing 3, figure A:(A) zinc ion strengthens 70% for the quantum dot (pressing embodiment 1 described method preparation) in 559 nanometers; (B) luminous quantum dot in 559 nanometers, figure B:(A) zinc ion is in the quantum dot of 590 nanometers (press embodiment 1 described method preparation) enhancing 80%; (B) luminous quantum dot) in 590 nanometers.
Embodiment 3: the assembling of full rotation molecular motor biology sensor
As shown in Figure 1, according to CN1789425, carry out the assembling of molecular motor biology sensor.
(1) the photosynthetic compound of molecular device (Chromatophores) preparation
(R.Capsulatus I 20 is purchased the institute of microbiology from the Chinese Academy of Sciences, Chinese common micro-organisms culture presevation administrative center (CGMCC), preserving number: 1.2359) to cultivate photosynthetic bacteria.(all ingredients merchandise resources: not marked is homemade analysis net product) nutrient culture media: KH
2PO
4, 1.0g, MgCl
20.5g, CaCl
2, 0.1g, NaCl 1.0g, sodium acetate 1.0g, sodium succinate 1g, yeast extract, 1.0g, NaHCO
30.5g, peptone 0.5g (OXOID Britain), micro-1.0ml, vitamin liquid 1.0ml, distilled water 1.0L, 6.8,8 pounds of pH, autoclaving 30 minutes.Vitamin liquid composition: biotin 0.1g (import packing), nicotinic acid 0.35g, nicotinic acid thiamine (Thiamine dichloride) 0.3g, calcium pantothenate (Ca-panthothenate) 0.1g, Cobastab
12(Vitamin B
12, 0.05g, Pyridoxamine Hydrochloride (Pyridoxolium Hydrochloride, Fluka) 0.1g, distilled water 1.0L; Trace element composition: FeCl
24H
2O 1.8g; CoCl
26H
2O 0.25g; NiCl
26H
2O, 0.01g; CuCl
22H
2O0.01g, MnCl
24H
2O 0.07g; ZnCl
20.1g, B
3PO
40.5g, Na
2SeO
35H
20.01g, NaMoO
4H
2O 0.03g, distilled water 1.0L) temperature 30-35 ℃.Results: the cultivation temperature of bacterium is 28 ℃, and (4,000Lx) following cultivation is 5-7 days, the centrifugal collection of 5000g in illumination.The extraction of chromatophore (Chromatophores): sample TS damping fluid (50mM, Tricine (three (methylol) methylglycine, Amresco import packing) pH 8.0,0.25M sucrose; 5mM MgCl
2) wash once, and then add the TS damping fluid of 15ml, after the suspension, add the lysozyme (sigma packing) of 1mg/ml, in ice, hatched ultrasonic (20% amplitude 30 minutes, Cole Parmer CPX 600 Ultrasound Instrument 13# probe, Ultrasound Instrument) 10 minutes, 25, centrifugal 30 minutes of 000g, keep supernatant, again with supernatant 180,000g 4 ℃ centrifugal 90 minutes, be precipitated as chromatophore (Chromatophores).This compound is for containing a molecular motor F
0F
1-ATPase, a plurality of light conversion carriers.
(2) folk prescription of fluorescence probe is to mark
Chromatophore and the CdTe quantum dot that obtains according to the method for embodiment 1 by 1: 1 volume ratio at (0.1mM, in Tricine (three (methylol) methylglycine pH 6.5) system (30min), with quantum dot centrifugal method (10000g/30min, 4C) removal rest parts externally.In the compound-quantum dot fluorescence is as the fluorescent emission body.
(3) timbering material and bilayer lipid membrane are fixed
Solid surface (as glass) is coated with the shop with poly-D-lysine earlier, uses biotin-AC then
5-sulfo-Osu (Dojindo, Japan) use streptavidin-connection after the connection again, bilayer lipid membrane is equipped with-fat-biotin ((PE-biotin (Avidin company product), be dissolved in ethanol (1mg/ml, 2 μ l), it is middle after about 2-3 minute to join bilayer lipid membrane (20mg/ml), with 10,0000g/30min is once centrifugal), will being equipped with of the bilayer lipid membrane-sample of fat-biotin and the streptavidin of solid surface-be connected (bilayer lipid membrane chromatophore (Chromatophores) be as above natural purifying preparation) be housed.As required, (preparation is referring to Biochem Biophys Res Commun 347 (2006) 752-757 with epsilon subunit antibody, Review of polyclonal antibody production procedures inmammals and poultry, ILARJ.37 (1995) 93-118) and F
1The epsilon subunit selectivity combination of-ATP motor, the other end and biotin-Avidin-biotin (DNA discerns body) link, and have so just formed the biology sensor of an a pair of load difference 7000 molecular weight.Finish the assembling of molecular motor biology sensor of the present invention thus.
The relation that embodiment 4. quantum dot light emitting intensity change with the pH value
Utilize chromatophore (Chromatophores) the ATP-quantum dot that obtains among quantum dot (quantum dots of the quantum dot of 559 nanometers and 590 nanometers) that the method for embodiment 1 obtains and the embodiment 3 to study the relation of quantum dot light emitting intensity and pH value.The result as shown in Figure 4.
The quantum dot of curve (a) 559 nanometers is at 50mM, Tricine, the relation that middle intensity changes with the pH value;
The quantum dot of quantum dot-labeled thing 559 nanometers of curve (b) chromatophore (Chromatophores) ATP--at 50mM, Tricine, the relation that middle intensity changes with the pH value;
The quantum dot of quantum dot-labeled thing 590 nanometers of curve (c) chromatophore (Chromatophores) ATP--at 50mM, Tricine, the relation that middle intensity changes with the pH value.
Embodiment 5: the synthetic research of fluorescence quantum response ATP
Chromatophore-quantum dot is in that (10mM, 10min in Tricine (three (methylol) methylglycine pH 6.5) system removes quantum dot externally with centrifugal method, remaining by centrifugal (10000g/30min, 4 ℃) flush away.For adding quantum dot fluorescence in the above-mentioned compound as intracellular fluorescent emission body, the interface that chromatophore-quantum dot is fixing, excitation wavelength: 470nm; Emission wavelength is respectively at 559/590nm; Under 37 ℃ of conditions, observe respectively when adding ATP synthetic system (2mM ADP and phosphorus), it is synthetic to observe startup ATP, and the fluorescence signal of quantum dot is along with the change kinetics process of time.(A: chromatophore does not add ADP to the result, in contrast group as shown in Figure 5; B: chromatophore adds ADP again and starts after adding DCCD; C: chromatophore adds ADP and starts).As DCCD (50 μ M) (N, N '-dicyclohexylcarbodiimide, F0-ATPase proton inhibitor, available from Sigma) add, cultivate 30min, when adding ATP synthetic system (2mM ADP and phosphorus), it is synthetic to observe startup ATP, its fluorescence signal is not along with its fluorescence intensity of variation of time obviously increases, illustrated the fluorescence quantum signal response be with the ATP building-up process in the proton transhipment take place simultaneously.
Embodiment 6: to the mensuration of little RNA
After the biology sensor of above embodiment 3 preparations is fixed on the fine little plain film of the nitric acid surface in 0.45 μ M aperture, be attached to the F that is positioned at the Chromatophores surface by ε antibody-biotin-streptavidin
0F
1On-ATPase the epsilon subunit, connect the microRNAs specific probe of 3 ' mark biotin then.After the cracked solution of the different extension rates of MCF7 added the biology sensor microRNAs detection architecture that fixes, add the synthetic damping fluid of ATP and start reaction.
Particularly, biology sensor is fixed on the fine little plain film front surface (5mm diameter) of nitric acid in 0.45 μ M aperture, the microRNAs probe of 3 ' mark biotin (5 '-GGGGAUUCCUGGAAACUGC-3 ') is attached to the F that is positioned at the chromatophore surface by ε antibody-biotin-streptavidin
0F
1On-ATPase the epsilon subunit.Per step condition of contact is incubated at room 30 minutes.Utilize hypotonic method with human breast cancer cell strain MCF7 (ATCC HTB22) lysis, thereby microRNAs is discharged in the solution.Each test sample adds the 100ul lysate (ddH that does not contain the RNA enzyme that contains the RNA enzyme inhibitor
2O), with predetermined fixed hatched 5 minutes under the fine little plain film room temperature of nitric acid of biology sensor, microRNA is combined with probe.Then with ATP synthetic damping fluid (ADP 4mM buffer, 0.1mM Tricine, 5mM MgCl
2, 5mMK
2HPO
4, and 10% glycerol, pH 8.0) add sample start put into special-purpose luminoscope (BPCL-II, assay method is referring to Zhang Zhonglun, biological chemistry is made progress with biophysics, 2000, (2)) after the synthetic reaction) measure.From top to bottom, cell concentration respectively: 0,10
610
5, 10
4, 10
3, 10
2(individual) is F under different load
0F
1-molecular motor rotational speed changes.The result as shown in Figure 6.
Embodiment 7: the proof of surface charge free body
(1) the fluorescence system exists: the surface charge free body of the coding techniques of quantum dot.Above we seen respectively different fluorescence quantum folk prescriptions to mark to chromatophore, this moment the proton translocation process, this quantum dot folk prescription forms a surface charge free body to mark to chromatophore, does not disturb mutually with contiguous another surface charge free body.Because the stability of this surface charge free body, so we can use the coding mensuration of various product easily.
Measurement result:
From Fig. 7 can illustrate different fluorescence quantum folk prescriptions to mark to chromatophore, this moment, the proton translocation process was not disturbed mutually with contiguous another surperficial free body.The explanation fluorescence quantum folk prescription on the left side divides other experimental result to mark to chromatophore: (a) be not connected with RNA as described in embodiment 6 for the quantum dot-labeled of 559nm; (b) be the quantum dot-labeled of 590nm and as described in embodiment 6, be connected, measure respectively with RNA; (c) with (d) with (a) and (b) same, measure simultaneously at 559mn and 590mn but mix to excite respectively with 470nm; We see that (559mn 590mn) is marked at behind the chromatophores surface and they is mixed again, and they can independently detect object separately with two kinds of quantum dots.The The above results explanation coding of application of samples is easily measured.
Annotate: the contrast among Fig. 7 refers to only with a kind of quantum dot-labeled experimental result with contrast RNA; And encode, coding RNA represents respectively with two kinds of quantum dot-labeled materials in same solution system, measure simultaneously with the optics coding techniques.
(2) stability that has the surface charge free body in the electrophoresis system.
The stability of surface charge free body is as the application of optical encoding, but can be in the electrophoresis system existence and the application of stability.Use following proof:
It is synthetic that the quantum dot-labeled thing of chromatophore (Chromatophores) ATP-adds the synthetic liquid startup of ATP ATP, behind the 37 ℃/10min, be added in tiselius apparatus (Bio-MEMS in the microfluid of plane, referring to Technologies and Applications, edited by wanjun Wang, Steven, A.Soper, press, CRC, 2006, p168) in, under fluorescent microscope, observe the flow direction and the mobile flowing velocity of the quantum dot-labeled thing of chromatophore ATP-, and it is synthetic in contrast not start ATP, the result shows: the quantum dot-labeled thing of ATP-surface is negative charge, and the movement velocity that starts the rotation group is slower than contrast (DCCD).Illustrated that this surface charge free body separates and uses in the electrophoresis system.Fig. 8 has shown with the separating resulting of electrophoresis microfluidic device to the surface charge free body of F1F0-complex.After figure (A) chromatophore adds DCCD, add ADP again and start electrophoretic velocity; Figure (B) chromatophore adds ADP and starts electrophoretic velocity.Free body electrophoresis direction illustrates that from positive polar motion the electric charge on chromatophore-quantum dot surface is electronegativity, when proton the transhipment, is the negative back that fetters by the electric charge on quantum dot surface and reduces electronegativity outside film in film, slows down electrophoretic velocity.
Claims (10)
1. the preparation method of the water-soluble high photoluminescence efficiency CdTe quantum dot of pH responsive type, this method comprises the steps:
(1) by Te powder and NaBH
4Or KBH
4Reaction, synthetic sodium hydrogen telluride or hydrogen telluride aqueous solutions of potassium;
(2) use the synthetic nanocrystalline precursor solution of CdTe of cadmium salt or its hydrate and stabilizing agent, described cadmium salt is selected from CdNO
3And CdCl
2, described stabilizing agent selected from mercapto acetate, mercaptopropionic acid and mercapto glycerol;
(3) sodium hydrogen telluride or hydrogen telluride aqueous solutions of potassium are mixed with the nanocrystalline precursor solution of CdTe, it is nanocrystalline to add thermal synthesis CdTe;
(4) it is nanocrystalline to collect CdTe under the differential responses time, is the size CdTe quantum dot different with the fluorescent emission wavelength,
Wherein in step (2), add the photoluminescence efficiency of ammoniacal liquor with the raising product, and
Wherein the molar ratio of cadmium salt, sodium hydrogen telluride or hydrogen telluride potassium and stabilizing agent is 1: 0.1-1: 2.
2. add the Zn ion to improve the quantum dot fluorescence quantum yield according to the process of claim 1 wherein.
3. pass through the CdTe quantum dot of the method preparation of claim 1.
4. improved unimolecule ATP motor rotary type biosensor is characterized in that:
The probe of its use is a CdTe quantum dot according to claim 3.
5. unimolecule ATP motor rotary type biosensor according to claim 4 is wherein with F
0F
1The epsilon subunit of-ATP enzyme is as the connection site of load.
6. according to claim 4 or 5 described unimolecule ATP motor rotary type biosensors, wherein said CdTe quantum dot is that the form as the surface charge free body exists.
7. according to claim 4 or 5 described unimolecule ATP motor rotary type biosensor, wherein F
0F
1The epsilon subunit of-ATP enzyme and the antibodies of this epsilon subunit, and the antibody of described epsilon subunit further combines with biomolecule by biotin-chain enzyme antibiotin-biotin.
8. unimolecule ATP motor rotary type biosensor according to claim 7, wherein said biomolecule are the antibody of DNA, little RNA or viruses molecule specific antigen.
9. multiple monomolecular method in the test sample, wherein:
(1) will be respectively applied for and detect described multiple monomolecular different biological molecules and load on the epsilon subunit according to the ATP motor of the biology sensor of any one in the claim 4 to 8;
(2) with the biology sensor that obtains in the different CdTe quantum dot difference markers step (1) of fluorescent emission wavelength according to claim 3;
(3) biology sensor that will obtain in step (2) contacts with testing sample;
(4) the ATP motor that drives described biology sensor rotates, the relatively biology sensor and the fluorescence intensity of the biology sensor that does not contact with testing sample under different fluorescent emission wavelength of acquisition in (3) are if wherein fluorescence intensity changes and has the corresponding unimolecule of biomolecule of load on the described biology sensor in the interpret sample.
10. according to the method for claim 9, the antibodies of the epsilon subunit of biology sensor and this epsilon subunit wherein, and the antibody of described epsilon subunit further combines with biomolecule by biotin-chain enzyme antibiotin-biotin, and preferred described biomolecule is the antibody of DNA, little RNA or viruses molecule specific antigen.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102135543A (en) * | 2010-01-27 | 2011-07-27 | 中国科学院生物物理研究所 | Real-time and sensitive novel method for detecting biomacromolecule and preparation method for kit |
| CN102221610A (en) * | 2010-04-16 | 2011-10-19 | 北京市理化分析测试中心 | Method for rapidly detecting pathogenic bacteria at high efficiency, biological dependent sensor and preparation method thereof |
| CN102798715A (en) * | 2011-05-24 | 2012-11-28 | 中国科学院生物物理研究所 | Construction and detection method of biomolecular motor resonance sensor |
| CN103364460A (en) * | 2013-02-05 | 2013-10-23 | 国家纳米科学中心 | Friction-nanogenerator-based molecular sensor |
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2007
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102135543A (en) * | 2010-01-27 | 2011-07-27 | 中国科学院生物物理研究所 | Real-time and sensitive novel method for detecting biomacromolecule and preparation method for kit |
| CN102221610A (en) * | 2010-04-16 | 2011-10-19 | 北京市理化分析测试中心 | Method for rapidly detecting pathogenic bacteria at high efficiency, biological dependent sensor and preparation method thereof |
| CN102221610B (en) * | 2010-04-16 | 2013-08-14 | 北京市理化分析测试中心 | Method for rapidly detecting pathogenic bacteria at high efficiency, biological dependent sensor and preparation method thereof |
| CN102798715A (en) * | 2011-05-24 | 2012-11-28 | 中国科学院生物物理研究所 | Construction and detection method of biomolecular motor resonance sensor |
| CN102798715B (en) * | 2011-05-24 | 2014-11-05 | 中国科学院生物物理研究所 | Construction and detection method of biomolecular motor resonance sensor |
| CN103364460A (en) * | 2013-02-05 | 2013-10-23 | 国家纳米科学中心 | Friction-nanogenerator-based molecular sensor |
| CN103364460B (en) * | 2013-02-05 | 2015-11-18 | 北京纳米能源与系统研究所 | A kind of molecule sensor based on friction nanometer power generator |
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