CN108444960A - Fluorescence correlation spectroscopy detection method - Google Patents
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Abstract
The invention discloses a kind of fluorescence correlation spectroscopy detection methods, including：Probe molecule and target molecule are provided, the target molecule can be detected by fluorescence correlation spectroscopy and send out fluorescence, and with the binding domain that can be combined with the probe molecule；The solution of the probe molecule is applied to the surface of glass slide for microscope imaging, the probe molecule is made to be fixed on the surface of glass slide；Solution to be detected containing the target molecule is applied to the surface of glass slide, the target molecule and the binding domain is made to be mutually distinguishable and combine；And the variation of the fluorescence radiation of the detection target molecule.
Description
Technical field
The present invention relates to large biological molecule spectral detections and Single Molecule Detection field, more particularly to a kind of fluorescence correlation light
Spectrum detection method.
Background technology
Single molecular fluorescence technology can observe the dynamic process of biomolecule in real time in complex system, and disclose and be hidden in
Traditional ensemble average measure in important information, including the instantaneous centre in otherness and reaction process between biomolecule
State etc..The above advantage of single molecular fluorescence means makes it obtain extensive concern and application in recent years, and fast development makes
Many traditional biological questions are able to solve (W.E.Moerner, D.P.Fromm, Rev Sci with new experimental technique
Instrum 2003,74,3597-3619)。
Bioprocess such as conformation change dynamics and the time scale of interaction of molecules is distributed more widely, it is therefore desirable to have
The technology of corresponding temporal resolution and detection time scaled window.Single molecular fluorescence imaging technique is frequently with electron multiplying charge
Coupled apparatus (EMCCD) is used as detector, can be with detection time scale in a few tens of milliseconds or the dynamic process of longer time scale
(R.Roy,S.Hohng,T.Ha,Nat Methods 2008,5,507-516).New detector such as science complementary metal aoxidizes
Object semiconductor (sCMOS) camera can promote this time scale to millisecond magnitude (M.F.Juette, et al.Nat
Methods 2016,13,341-344).And another aspect fluorescence correlation spectroscopy (Fluorescence Correlation
Spectroscopy, FCS) technology is a kind of powerful unimolecule means, use avalanche mode photodiodes (APDs) conduct
Detector can capture and be happened at nanosecond other dynamic process (G.Bonnet, O.Krichevsky, A.Libchaber, P
Natl Acad Sci USA 1998,95,8602-8606).Free diffusing can occur for biomolecule in the solution, we are glimmering
Light correlation spectrum detects in volume of focus, and the time scale for the target molecule dynamic process being able to detect that is probably in sub- millisecond amount
Grade.Since the range of time scale is too narrow, the complete procedure of dynamic response cannot be detected, therefore development has wider time ruler
The new technology of degree detection range is just highly desirable.
Invention content
Based on this, it is necessary to provide a kind of fluorescence correlation spectroscopy for the time scale that can widen fluorescence correlation spectroscopy detection
Detection method.
A kind of fluorescence correlation spectroscopy detection method, including：
Probe molecule and target molecule are provided, the target molecule can be detected by fluorescence correlation spectroscopy and send out fluorescence,
And with the binding domain that can be combined with the probe molecule；
The solution of the probe molecule is applied to the surface of glass slide for microscope imaging, the probe molecule is made to fix
In the surface of glass slide；
Solution to be detected containing the target molecule is applied to the surface of glass slide, makes the target molecule and described
Binding domain is mutually distinguishable and combines；And the variation of the fluorescence radiation of the detection target molecule.
The probe molecule includes DNA, RNA, aptamer, antibody, polypeptide and albumen in one of the embodiments,
Matter.
The binding domain includes DNA, RNA, aptamer, antibody, peptide and protein in one of the embodiments,.
The combination of the target molecule and the binding domain includes nucleic acid-core in one of the embodiments,
One in acid, nucleic acid-polypeptide, nucleic acid-antibody, nucleic acid-protein, antibody-protein, antibody-polypeptide and polypeptide-protein
Kind.
The duration of the probe molecule and the bonding state of the binding domain is 10ms in one of the embodiments,
~100s.
In one of the embodiments, when the combination of the target molecule and the binding domain is nucleic acid-core
When sour, the target molecule and the binding domain are combined by 9-10 base.
The step of the fixation includes in one of the embodiments,：To the surface of glass slide and the probe molecule point
Corresponding molecular modification is not carried out, and the surface of glass slide of the molecular modification and the probe molecule is made to combine.
The surface of glass slide and the combination of the probe molecule include strepto- parent in one of the embodiments,
With the affine combination of element-biotin, the addition combination of aldehyde radical-amino, the addition combination of aldehyde radical-hydrazine, sulfydryl-maleimide
Addition combination, the addition combination of epoxy-hydroxyl, the addition combination of sulfydryl-sulfydryl, Protein A-antibody constant region domains Fc
In conjunction with and the combination of Protein G-antibody constant region Fc in one kind.
The binding domain is the Partial Fragment of the target molecule itself in one of the embodiments,.
The binding domain is the segment being additionally connected on the target molecule in one of the embodiments,.
A concentration of 100pM~100 μM of the probe molecule solutions in one of the embodiments,.
In one of the embodiments, the variation of the fluorescence radiation by fluorescence resonance energy transfer, fluorescent quenching or
Person's Fluorescence Increasing is realized.
The present invention by the way that probe molecule to be fixed on to the surface of glass slide for microscope imaging, using the probe molecule and
The matching relationship of target molecule binding domain makes the probe molecule and the binding domain be mutually distinguishable and combine, and is based on the knot
The time of conjunction state can extend residence time of the target molecule in fluorescence correlation spectroscopy detection focus window, avoid institute
It states and the focus window is diffused out by molecule when dynamic change occurs for target molecule, so as to widen the dynamic detected
The time scale of process, to observe the complete dynamic changing process of target molecule.Fluorescence spectrum inspection of the present invention
Survey method can be applied to the intramolecular interaction of detection target molecule or the intermolecular phase of target molecule and other molecules
Interaction relationship.
Description of the drawings
Fig. 1 is the fluorescence correlation spectroscopy detection method flow chart of one embodiment of the invention；
Fig. 2 is the fluorescence correlation spectroscopy detection method schematic diagram of one embodiment of the invention；
Fig. 3 is the bonding state of fluorescence correlation spectroscopy the detection method middle probe molecule and binding domain of one embodiment of the invention
Duration scalogram；
Fig. 4 is the intramolecular interaction schematic diagram of one embodiment of the invention, wherein (a) is not associated with the spy of target molecule
Needle molecule, (b) fluorescence illuminated state, (c) fluorescence dark-state；
Fig. 5 is the intramolecular interaction schematic diagram of another embodiment of the present invention, wherein (a) is not associated with target molecule
Probe molecule, (b) fluorescence dark-state, (c) fluorescence illuminated state；
Fig. 6 is the intramolecular interaction schematic diagram of another embodiment of the present invention, wherein (a) is not associated with target molecule
Probe molecule, (b) fluorescence illuminated state, (c) fluorescence dark-state；
Fig. 7 is the intermolecular interaction schematic diagram of one embodiment of the invention, wherein (a) is not associated with the spy of target molecule
Needle molecule, (b) fluorescence illuminated state, (c) fluorescence dark-state；
Fig. 8 is the intermolecular interaction schematic diagram of another embodiment of the present invention, wherein (a) is not associated with target molecule
Probe molecule, (b) fluorescence illuminated state, (c) fluorescence dark-state；
Fig. 9 is the fluorescence correlation spectroscopy testing result figure of the embodiment of the present invention 1；
Figure 10 is the fluorescence correlation spectroscopy testing result figure of the embodiment of the present invention 2.
Specific implementation mode
In order to make the purpose , technical scheme and advantage of the present invention be clearer, by the following examples, it and combines attached
The fluorescence correlation spectroscopy detection method of the present invention is further elaborated in figure.It should be appreciated that described herein specific
Embodiment only to explain the present invention, is not intended to limit the present invention.
It should be noted that when element is referred to as " being fixed on " another element, it can be directly on another element
Or there may also be elements placed in the middle.When an element is considered as " connection " another element, it can be directly connected to
To another element or it may be simultaneously present centering elements.On the contrary, when element is referred to as " directly existing " another element "upper",
There is no intermediary elements.Term as used herein " vertically ", " horizontal ", "left", "right" and similar statement are
For illustrative purposes.In embodiment attached drawing it is various difference objects in convenient for enumerate explanation ratio draw, rather than press practical group
The ratio of part is drawn.
It please refers to Fig.1 and Fig. 2, the embodiment of the present invention provides a kind of fluorescence correlation spectroscopy detection method, include the following steps：
S100, provides probe molecule 2 and target molecule 3, and the target molecule 3 can be detected by fluorescence correlation spectroscopy and be sent out
Go out fluorescence, and with the binding domain 4 that can be combined with the probe molecule 2；
The solution of the probe molecule 2 is applied to 1 surface of slide for microscope imaging, makes the probe by S200
Molecule 2 is fixed on 1 surface of the slide；
S300, the solution to be detected containing the target molecule 3, which is applied to 1 surface of the slide, makes the target molecule
3 and the binding domain 4 be mutually distinguishable and combine；And
S400 detects the variation of the fluorescence radiation of the target molecule 3.
The embodiment of the present invention utilizes the spy by the way that probe molecule 2 to be fixed on to 1 surface of slide for microscope imaging
The matching relationship of 3 binding domain 4 of needle molecule 2 and target molecule, makes the probe molecule 2 and the binding domain 4 be mutually distinguishable and tie
It closes, the time based on the bonding state can extend the target molecule 3 in fluorescence correlation spectroscopy detection focus window
Residence time diffuses out the focus window when avoiding the target molecule 3 that dynamic change occurs by molecule, so as to
The time scale for widening the dynamic process detected, to observe the complete dynamic changing process of target molecule 3.The present invention
Fluorescence spectrum detection method described in embodiment can be applied to the intramolecular interaction or target point of detection target molecule 3
The intermolecular interaction relationship of son 3 and other molecules.
In the step s 100, the target molecule 3 can be the large biological molecule that itself can fluoresce, or label
Can send out the large biological molecule of fluorescence afterwards, and its fluorescence can with the variation of intramolecular or intermolecular interaction and
Variation.The variation of this fluorescence radiation can be realized by fluorescence resonance energy transfer, fluorescent quenching or Fluorescence Increasing.
The target molecule 3 has the binding domain 4 that can be combined with the probe molecule 2, and the binding domain 4 can be institute
A segment for stating target molecule 3 itself, can directly be combined with the probe molecule 2.The binding domain 4 can also be described
The segment that target molecule 3 additionally adds.If the target molecule 3 can not be combined directly with probe molecule 2, need
One section of 4 segment of the binding domain is additionally connected on the target molecule 3, to realize the target molecule 3 and the probe molecule 2
Combination.The probe molecule 2 and the binding domain 4 are combined by chemical interaction.In one embodiment, the probe
The combination of molecule 2 and the binding domain 4 can be selected from nucleic acid-nucleic acid, nucleic acid-polypeptide, nucleic acid-antibody, nucleic acid-protein
Matter, antibody-protein, antibody-polypeptide or polypeptide-protein.In one embodiment, the probe molecule 2 may include DNA,
At least one of RNA, aptamer, antibody, peptide and protein.In one embodiment, corresponding with the probe molecule 2
Binding domain 4 may include at least one of DNA, RNA, aptamer, antibody, peptide and protein.The aptamer
For oligonucleotide fragment, can specifically be combined with protein or other small-molecule substances.The polypeptide be by three or
Compound made of three or more amino acid molecular dehydrating condensations, can be combined with nucleic acid or protein by chemical action.
When the binding domain 4 is DNA, RNA or aptamer, can synthesize to obtain with described by PCR amplification or nucleic acid solid-state
The target molecule 3 of binding domain 4；When the binding domain 4 is polypeptide or protein, can synthesize to obtain first can express it is described
Then the gene of binding domain april protein-target molecule 3 gives expression to the target molecule 3 with binding domain april protein.
The duration of the probe molecule 2 and the bonding state of the binding domain 4 is related with the combination.It is different
The probe molecule 2 of type and the binding ability of the binding domain 4 are different.In one embodiment, the probe molecule 2
When being combined in a manner of nucleic acid-nucleic acid with the binding domain 4, the base number of the duration and the binding domain 4, base kind
Salinity is related in class and solution, salinity in base number, base type or solution that can be by adjusting the combination
To adjust the duration of the bonding state.Base number is more, and the duration is longer, and the GC ratios in base are got over
Height, the duration is longer, and salinity is higher, and the duration is longer.The duration can be 10ms~100s.
Referring to Fig. 3, in one embodiment, the target molecule 3 can form the combination of 9-10 base with the probe molecule 2,
The sequence of the probe molecule 2 is 5 '-TTTTTTATAATGGATGG, and the sequence of the binding domain 4 of the target molecule 3 is 5 '-
GATCCATTAT can form the instantaneous combination probably in 1s~10s time scales at this time.NaCl salinity is different, the knot
It is different to close the time.
In step s 200, further include that corresponding molecular modification is carried out to the probe molecule 2 and 1 surface of the slide
Step.When the solution of the probe molecule 2 is applied to 1 surface of slide, 2 energy of the probe molecule after modification
Enough 1 surfaces of the slide with by modification, which react to each other, generates chemical bond or physical absorption, and is immobly bound to the glass
1 surface of piece.In one embodiment, the surface of glass slide and the secure bond mode of the probe molecule include that strepto- is affine
The affine combination of element-biotin, the addition combination of aldehyde radical-amino, the addition combination of aldehyde radical-hydrazine, sulfydryl-maleimide plus
At combination, the addition combination of epoxy-hydroxyl, the addition combination of sulfydryl-sulfydryl, Protein A-antibody constant region domains Fc knot
One kind in conjunction and the combination of Protein G-antibody constant region Fc.In one embodiment, 1 surface strepto- of the slide is affine
Element modification, the biotin modification of the probe molecule 2 will be described using the affine combination of the Streptavidin and biotin
Probe is fixed on 1 surface of the slide.A concentration of 100pM~100 μM of probe molecule 2 described in 2 solution of the probe molecule,
Specific concentration can be adjusted according to actual needs, obtain different surface-probe density.
Can be that the solution to be detected containing the target molecule 3 is applied to 1 table of the slide in step S300
Face.For reduce the volatilization of the solution to be detected and slow down the mobility for stating solution to be detected in one embodiment can
It is carried out in such a way that the interlayer by the slide 1 described in two panels injects the detection solution.The combination of the target molecule 3
Domain 4 and be fixed on 1 surface of the slide probe molecule 2 since structure or molecular composition are mutually matched, can be mutually distinguishable, lead to
It crosses the modes such as intermolecular chemical bond, affine addition, nucleic acid hybridization, intermolecular interaction to be combined with each other, the chemical bond can
Think hydrogen bond, ionic bond, covalent bond and metallic bond.
In step S400, it is detected by detecting the variation of fluorescence radiation of the target molecule 3, collects fluorescence light
Sub-information obtains fluorescence correlation spectroscopy curve, and the fluorescence correlation spectroscopy curve, which is further analyzed, can obtain intramolecular
Or the dynamic information of intermolecular interaction.Fluorescence correlation spectroscopy detection method described in the embodiment of the present invention is by passing through
It states probe molecule 2 to combine the binding domain 4 of the target molecule 3, can extend institute using the duration of the bonding state
Residence time of the target molecule 3 in fluorescence correlation spectroscopy detection focus window is stated, avoids the target molecule 3 that dynamic occurs and becomes
The focus window is diffused out by molecule when change, so as to widen the time scale of the dynamic process detected, in institute
State the complete dynamic information that target molecule 3 is obtained on fluorescence correlation spectroscopy curve.
The fluorescence correlation spectroscopy detection method can be applied to the intramolecular or intermolecular mutual of analysis target molecule 3
Effect.The target molecule 3 to be detected, which is the fluorescent molecular that itself can be shone or target molecule 3 itself, to shine,
It can shine after the label of luminescent dye molecule 5.When the intramolecular of the target molecule 3 or it is intermolecular interact when
When the structure of the target molecule 3 or property being caused to change, the luminance of target molecule 3 can also occur mutually to strain therewith
Change, the intramolecular of the target molecule 3 is characterized by the variation of the fluorescence radiation state or intermolecular interacts
Molecular dynamics information.2 He of probe molecule described in the fluorescence correlation spectroscopy detection method due to the embodiment of the present invention
The combination of the target molecule 3 can widen the time scale of the dynamic process of detection focus window, so as to capture mesh
Mark the intramolecular of molecule 3 or the complete dynamic changing process of intermolecular interaction.
It can be to the flicker dynamic behavior of fluorescent dye self-molecules present using the fluorescence correlation spectroscopy detection method
Intramolecular interaction is characterized.Referring to Fig. 4, the target molecule 3 to be detected contains in one of the embodiments,
The binding domain 4 that can be combined with the probe molecule 2, and luminescent dye molecule 5 is carried out to the target molecule 3 and is marked, it is described
The target molecule 3 that luminescent dye molecule 5 marks can be cut between fluorescence illuminated state (b) and fluorescence dark-state (c) two states
It changes.The combination of the probe molecule 2 and the target molecule 3 extends the time scale of the dynamic process of detection focus window,
Therefore the intramolecular phase interaction of the flicker dynamic behavior of the fluorescent dye self-molecules present in broader time scale can be captured
Dynamic changing process obtains the flicker dynamic behavior information of more complete fluorescent molecular.
The intramolecular interaction that intra-molecular structure is converted can be carried out using the fluorescence correlation spectroscopy detection method
Characterization.Referring to Fig. 5, in one embodiment, utilizing the conversion of fluorescent quenching technique study target dna molecule hairpin structure.It is logical
Cross the conversion of the hairpin structure and non-hairpin structure of the different luminances characterization target dna molecule structure of luminescent dye molecule 5.
There are the target molecule 3 variable domains 7, the variable domains 7 can turn between hairpin structure and non-hairpin structure
It changes.Mark fluorescent dye molecule 5 and fluorescence quencher molecule 6 are distinguished in the variable domains 7 of target molecule 3, when the mesh
When marking molecule 3 by the formation hairpin structure of variable domains 7, the luminescent dye molecule 5 and the fluorescence quencher molecule 6 connect
Closely, fluorescent quenching at this time, the luminescent dye molecule 5 are in fluorescence dark-state (b)；When hairpin structure is opened, the fluorescence is quenched
The molecule 6 that goes out can be in fluorescence illuminated state (c), can send out at this time far from the luminescent dye molecule 5, the luminescent dye molecule 5
Fluorescence.The conversion of the hairpin structure and non-hairpin structure of the target molecule 3 is characterized by fluorescence radiation state.Please refer to figure
6, in another embodiment, pass through catalytic fluorescence method or fluorescence resonance energy transfer method goal in research DNA molecular hairpin structure
Conversion.The target molecule 3 has variable domains 7, and the variable domains 7 can be in hairpin structure and non-hairpin structure
Between convert.Distinguish mark fluorescent dye molecule 5 and fluorescence resonance energy transfer in the variable domains 7 of target molecule 3
In donor molecule 8, or mark fluorescent dye molecule 5 and Fluorescence Increasing molecule respectively, when the target molecule 3 pass through it is variable
When structural domain 7 forms hairpin structure, the luminescent dye molecule 5 and the donor molecule 8 be close or the luminescent dye molecule
5 and Fluorescence Increasing molecule it is close, the luminescent dye molecule 5 is entered due to fluorescence resonance energy transfer or Fluorescence Increasing at this time
Fluorescence illuminated state (b)；When hairpin structure is opened, the donor molecule 8 or the Fluorescence Increasing molecule can be contaminated far from the fluorescence
Expect molecule 5, fluorescent molecular is in fluorescence dark-state (c) at this time.When conversion between hairpin structure and non-hairpin structure needs longer
Between, for there is the transformation of the hairpin structure of physiological function generally in the magnitude of millisecond to second.Due to the target molecule 3 with it is described
Probe molecule 2 is combined with continuing for certain time, the time scale of the dynamic process of detection focus window is extended, described
In duration, the different luminances of the luminescent dye molecule 5 in time scale wide as possible are captured using microscope, are captured
The dynamic changing process of DNA molecular hairpin structure and the conversion of non-hairpin structure in broader time scale obtains more complete
The conversion multidate information of DNA molecular hairpin structure.
Intermolecular interaction can be characterized using the fluorescence spectrum detection method.Referring to Fig. 7, one
In embodiment, the confession in target molecule 3 and the effect difference mark fluorescent of molecule 3 ' dye molecule 5 and fluorescence resonance energy transfer
Body molecule 8, when target molecule 3 and effect molecule 3 ' can interact, when can approach and combine, the fluorescent dye
Molecule 5 and the donor molecule 8 are close to each other so that the luminescent dye molecule 5 is in fluorescence illuminated state (b)；When target molecule 3
It can not interact with effect molecule 3 ', the luminescent dye molecule 5 and the donor molecule 8 can not be close, make
It obtains the luminescent dye molecule 5 and is in fluorescence dark-state (c).Change characterization target molecule 3 and effect molecule 3 ' by luminance
It can interact.Referring to Fig. 8, in another embodiment, in 3 mark fluorescent dye molecule 5 of target molecule and fluorescence
Donor molecule 8 in Resonance energy transfer, the target molecule 3 in initial configuration, such as hairpin structure, when fluorescence dye
Expect that molecule 5 and the donor molecule 8 apart from relatively closely, can occur fluorescence resonance energy transfer and make the luminescent dye molecule
5 are in fluorescence illuminated state (b).When being combined when acting on molecule 3 ' and interaction can occur with the target molecule 3, it can make institute
The initial configuration for stating target molecule 3 changes, such as becomes non-hairpin structure, and makes the luminescent dye molecule 5
With the donor molecule 8 apart from becoming remote, fluorescence resonance energy transfer can not occur and the luminescent dye molecule 5 is in fluorescence
Dark-state (c).In the embodiment of above-mentioned Fig. 7 and Fig. 8, acts on molecule 3 ' and the interaction dynamics process of target molecule 3 is general
Across long time range, usually from sub- millisecond to the magnitude of second.Due to the target molecule 3 and the probe molecule 2
Be combined with continuing for certain time, extend detection focus window dynamic process time scale, in the duration
It is interior, the different luminances of the luminescent dye molecule 5 in time scale wide as possible are captured using microscope, when capturing broader
Between target molecule 3 in scale intermolecular interaction dynamic changing process, obtain more detailed intermolecular interaction
Multidate information.
Embodiment 1 carries out the fluorescence radiation of ATTO655 luminescent dye molecules 5 using the fluorescence correlation spectroscopy detection method
Flicker dynamics research.
ATTO655 luminescent dye molecules 5 can flicker between fluorescent state and non-fluorescence state, and reducing agent and oxidant are such as
The presence of ascorbic acid (AA) and methyl viologen (MV) can accelerate this process.With ATTO655 luminescent dye molecules 5 different dense
For flicker dynamics under the conditions of degree AA and MV, referring to Fig. 9, the fluorescence spectrum detection method described in the present embodiment can be real
Now from 1.1ms to 168ms the intramolecular dynamic process of time scale detection.
Specific experiment is as follows：
Slide 1 suitable for microscope imaging passes through aminosilane reagents (1ml aminopropyltriethoxywerene werenes, 5ml second
Acid and 94ml methanol) it handles overnight at room temperature, then reacted 3 hours in the sodium bicarbonate of 0.1M with polyethylene glycol (PEG).
The Streptavidin of the slide 1 and 0.05mg/mL of modifying PEG reacts 3 minutes.Then the probe of biotin modification is added
Molecule 2 reacts 3 minutes, after can the probe molecule 2 being fixed to PEG passivation by biotin and Streptavidin reaction
1 surface of the slide on.Concentration by adjusting the probe molecule 2 can obtain suitable surface-probe density.
Target molecule 3 is nucleic acid molecules, can introduce amino on end or certain intermediate particular bases by synthesis in solid state
(NH2) modification, the ATTO655 luminescent dye molecules 5 are connected on the nucleic acid molecules.The ATTO655 fluorescent dyes
Molecule 5 and the nucleic acid molecules are with 20:1 molar ratio mixing, reaction is overnight, then pure by ethanol precipitation at room temperature
Change the nucleic acid molecules for being connected to the ATTO655 luminescent dye molecules 5.
The nucleic acid molecules that the ATTO655 luminescent dye molecules 5 are marked in 10nM are added to be observed secure
On the slide 1 of the probe molecule 2, by 640nm excitations, the fluorescent photon within the scope of 662nm-738nm, inspection are collected
Survey device is APD, is arrived in 1.1ms referring to Fig. 9, can obtain ATTO655 luminescent dye molecules 5 by the analysis of fluorescence curve of spectrum
The fluorescence curve under different AA and MV concentration in 168ms time scales, the variation by analyzing the fluorescence curve characterize
Scintillation fluor dynamic information of the ATTO655 luminescent dye molecules 5 under different AA and MV concentration.
Embodiment 2 carries out DNA double chain Cambium periodicity research using the fluorescence correlation spectroscopy detection method.
The present embodiment combines the fluorescence correlation spectroscopy detection method and fluorescence resonance energy transfer, utilizes
3 single strand dna of target molecule that Alexa488 luminescent dye molecules 5 mark is as fluorogenic donor, Cy5 luminescent dye molecules 5
Complementary 3 ' the single strand dna of effect molecule of label is used as fluorescent receptor, 3 single stranded DNA of goal in research molecule and its complementary
Act on the dynamics that molecule 3 ' forms double-stranded DNA and the ends DNA cell breath.Referring to Fig. 10, glimmering described in the present embodiment
Light spectral method of detection can realize the big time for crossing over three orders of magnitude from 0.45ms to 3.78s for intermolecular interaction
Kinetic measurement in range scale.
Specific experiment is as follows：
Probe molecule 2 is fixed on 1 surface of slide, the fixing means of embodiment 1 is seen, repeats no more.
By in fluorescence resonance energy transfer donor molecule 8 (Alexa488) and luminescent dye molecule 5 (Cy5) link respectively
Onto corresponding two single stranded nucleic acid molecule (effect molecule 3 ' and target molecule 3) that can form complementary double-strand.Dye molecule and
Single stranded nucleic acid molecule is with 20:1 molar ratio mixing, at room temperature reaction overnight, are then purified by ethanol precipitation and are linked
The single stranded nucleic acid molecule of luminescent dye molecule 5 and the donor molecule 8.
The target of the 3 ' nucleic acid molecules of effect molecule of the Alexa488 labels of 100nM and the Cy5 labels of 10nM
3 nucleic acid molecules of molecule are added on the slide 1 for securing the probe molecule 2 to be observed, pass through the fluorogenic donor
The exciting light 488nm laser excitations of Alexa488, collect fluorescent photon of the wavelength within the scope of 662nm-738nm, and detector is
APD.During fluorescence resonance energy transfer, fluorescence resonance energy turn can be occurred by only forming the target molecule of double-strand
It moves, i.e., by 488nm laser excitations, is collected into the fluorescence of Cy5, therefore the target point that will not be marked by the Cy5 of free state
The influence of the 3 ' nucleic acid molecules of effect molecule of sub 3 nucleic acid molecules and the Alexa488 labels of free state.Referring to Fig. 10, passing through
The analysis of fluorescence curve of spectrum can obtain effect molecule 3 ' and target molecule 3 forms 7 in 0.45ms to 3.78s time scales
The fluorescence radiation change curve for the double-strand that a, 8 or 9 bases combine passes through and analyzes the variation characterization of the fluorescence curve and make
The dynamic information of double-strand and nucleic acid end cell breath is formed with molecule 3 ' and target molecule 3.
Each technical characteristic of embodiment described above can be combined arbitrarily, to keep description succinct, not to above-mentioned reality
It applies all possible combination of each technical characteristic in example to be all described, as long as however, the combination of these technical characteristics is not deposited
In contradiction, it is all considered to be the range of this specification record.
Several embodiments of the invention above described embodiment only expresses, the description thereof is more specific and detailed, but simultaneously
Cannot the limitation to the scope of the claims of the present invention therefore be interpreted as.It should be pointed out that for those of ordinary skill in the art
For, without departing from the inventive concept of the premise, various modifications and improvements can be made, these belong to the guarantor of the present invention
Protect range.Therefore, the protection domain of patent of the present invention should be determined by the appended claims.
Claims (12)
1. a kind of fluorescence correlation spectroscopy detection method, including：
Probe molecule and target molecule are provided, the target molecule can be detected by fluorescence correlation spectroscopy and send out fluorescence, and
With the binding domain that can be combined with the probe molecule；
The solution of the probe molecule is applied to the surface of glass slide for microscope imaging, the probe molecule is made to be fixed on institute
State surface of glass slide；
Solution to be detected containing the target molecule is applied to the surface of glass slide, makes the target molecule and the combination
Domain is mutually distinguishable and combines；And the variation of the fluorescence radiation of the detection target molecule.
2. fluorescence correlation spectroscopy detection method according to claim 1, which is characterized in that the probe molecule include DNA,
RNA, aptamer, antibody, peptide and protein.
3. fluorescence correlation spectroscopy detection method according to claim 1, which is characterized in that the binding domain include DNA,
RNA, aptamer, antibody, peptide and protein.
4. fluorescence correlation spectroscopy detection method according to claim 1, which is characterized in that the target molecule and the knot
Close domain the combination include nucleic acid-nucleic acid, nucleic acid-polypeptide, nucleic acid-antibody, nucleic acid-protein, antibody-protein,
One kind in antibody-polypeptide and polypeptide-protein.
5. fluorescence correlation spectroscopy detection method according to claim 1, which is characterized in that the probe molecule and the knot
The duration for closing the bonding state in domain is 10ms~100s.
6. fluorescence correlation spectroscopy detection method according to claim 1, which is characterized in that when the target molecule and described
When the combination of binding domain is nucleic acid-nucleic acid, the target molecule and the binding domain are combined by 9-10 base.
7. fluorescence correlation spectroscopy detection method according to claim 1, which is characterized in that the step of the fixation includes：
Corresponding molecular modification is carried out respectively to the surface of glass slide and the probe molecule, makes the slide of the molecular modification
Surface and the probe molecule combine.
8. fluorescence correlation spectroscopy detection method according to claim 7, which is characterized in that the surface of glass slide and the spy
The combination of needle molecule includes the affine combination of Streptavidin-biotin, the addition combination of aldehyde radical-amino, aldehyde radical-
The addition combination of hydrazine, the addition combination of sulfydryl-maleimide, the addition combination of epoxy-hydroxyl, sulfydryl-sulfydryl
One kind in the combination of addition combination, the combination of Protein A-antibody constant region domains Fc and Protein G-antibody constant region Fc.
9. fluorescence correlation spectroscopy detection method according to claim 1, which is characterized in that the binding domain is the target
The Partial Fragment of molecule itself.
10. fluorescence correlation spectroscopy detection method according to claim 1, which is characterized in that the binding domain is additionally to connect
The segment being connected on the target molecule.
11. fluorescence correlation spectroscopy detection method according to claim 1, which is characterized in that the probe molecule solutions
A concentration of 100pM~100 μM.
12. fluorescence correlation spectroscopy detection method according to claim 1, which is characterized in that the variation of the fluorescence radiation
It is realized by fluorescence resonance energy transfer, fluorescent quenching or Fluorescence Increasing.
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