CN103575934B - Single molecule force spectroscopy analysis AFM probe and substrate functional modification method - Google Patents
Single molecule force spectroscopy analysis AFM probe and substrate functional modification method Download PDFInfo
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Abstract
The probe that the object of the present invention is to provide a kind of single molecule force spectroscopy analysis atomic force microscope single molecule force spectroscopy to measure and substrate surface functional method; the PEG macromolecule that first other one end of Boc radical protection, one end is sulfydryl by the present invention is coupled in the film modified atomic force microscope probe of gold or gold substrate; secondly protein modified reagent SATA or SATP is utilized to be modified to sulfydryl the primary amine that biomolecule itself contains; finally Boc blocking group is removed; and with the biomolecular reaction of sulfhydrylation, thus by protein modified on Au probe and gold substrate.The method technique is simple, easy and simple to handle, can be implemented in the target of the Interaction Force of single molecules level research biomolecule and cell surface single molecular recognition etc.
Description
Technical field
The present invention relates to a kind of probe and substrate functional modification method, particularly relate to a kind of single molecule force spectroscopy analysis atomic force microscope probe and substrate functional modification method, belong to microcosmic scientific domain, particularly nano measurement.
Background technology
Even to this day, atomic force microscope single molecule force spectroscopy has become the instrument in having by force of research biological disperser, significant for the relation and effect in life science process of understanding biomolecular structure domain-functionalities in depth, be the direction, a forward position in current biology, chemistry, physical intersection field.Usually by one section of polyglycol (PEG) high molecular polymer chain, protein is fixed in atomic force microscope probe needle point and substrate in single molecule force spectroscopy research, thus can ensure the spatial degrees of freedom of biomolecule, and the non-specific interaction that specificity between biomolecule can be interacting at needle point and substrate from power-distance Curve figure distinguishes.Current, research mainly carries out protein in surface-functionalized modification to Atomic Force Microscopy Silicon probe and Au probe: 1) be linked on Atomic Force Microscopy Silicon detecting probe surface by difunctionalization PEG by biomolecule, the shortcoming of the method is that PEG molecule can not form fine and close PEG layer on silicon probe, thus can not avoid the non-specific interaction of needle point and substrate; 2) also there is a small amount of research, by difunctionalization PEG, biomolecule is linked to atomic force microscope Au probe on the surface, first short two ends are all linked to gold surface with the alkyl sulfhydryl of sulfydryl, thus the sulfydryl exposed is formed in gold surface, by the PEG macromolecular compound MAL-PEG-NHS of difunctionalization, biomolecule is linked in gold surface subsequently.The method advantage is that PEG layer is fine and close, and non-specific interaction is less.Shortcoming is that step is comparatively complicated, thus adds the risk of gold surface functionalization failure.Therefore build a kind of simply atomic force microscope Au probe and substrate functional method to be very important.
Summary of the invention
The object of the present invention is to provide a kind of probe for the measurement of atomic force microscope single molecule force spectroscopy and substrate surface functional method, the method step is simple, easy and simple to handle, can be implemented in the target of the single molecules level research Interaction Force of albumen and cell surface single molecular recognition etc.
Technical solution of the present invention is:
The present invention first by the other one end of one end t-butyl carbamate (Boc) radical protection be sulfydryl PEG macromolecule be coupled to gold film modified atomic force microscope probe or gold substrate on; secondly protein modified reagent N-succinimido-S-acetylthio acetic acid esters (SATA) or N-succinimide-3-acetylthiopropionate (SATP) is utilized to be modified to sulfydryl the primary amine of albumen; finally Boc blocking group is removed; and with the albumino reaction of sulfhydrylation, thus by protein modified on Au probe and gold substrate.
The invention provides a kind of single molecule force spectroscopy analysis atomic force microscope probe and substrate functional modification method, it is characterized in that, comprise the following steps:
The first step: clean the gold-plated atomic force microscope probe of needle point and gold substrate in chloroform, probe and gold substrate, dry under a nitrogen, UV ozone is cleaned, and subsequently with ultrapure water cleaning, chloroform cleans, dry under nitrogen;
Second step: the Au probe cleaned up and gold substrate are put into lucifuge reaction in carbamic acid tertiary Ding Zhi – Ju Yi bis-Chun – sulfydryl (Boc-PEG-3000-SH) and Jia Yang Ji – Ju Yi bis-Chun – sulfydryl (mPEG-2000-SH) mixed liquor that chloroform is solvent;
3rd step: add protein modified reagent N-succinimido-S-acetylthio acetic acid esters (SATA) in protein solution, or N-succinimide-3-acetylthiopropionate (SATP), and react a period of time;
4th step: in the protein solution that the 3rd step obtains, add in de-acetyl damping fluid and react, the reaction time is 0.5 ~ 3 hour, utilizes desalting column to slough salt in protein solution subsequently;
5th step: get Au probe that second step obtains and gold substrate is put in trifluoroacetic acid, ultrapure water cleans;
6th step: Au probe and gold substrate put into crosslinking chemical 4-(N-maleimidomethyl) cyclohexane-1-carboxylic acid succinimide ester (SMCC), or in 4-(N-maleimidomehyl) cyclohexane-1-carboxylic acid sulfonic group succinimide ester sodium salt (Sulfo-SMCC) solution, reaction a period of time, ultrapure water cleans;
7th step: Au probe and gold substrate are placed to the protein solution that the 4th step process obtains, reaction, ultrapure water cleans, and to insert in phosphate (PBS) damping fluid 4 degree and saves backup.
The needle surface of described atomic force microscope probe has plated the substrate of one deck gold film as functional modification.
In polyglycol mixed liquor described in second step, Boc-PEG-3000-SH molar ratio is 0.5 ~ 10 ﹪, and total Polyethylene glycol is 1 ~ 10mM, and the reaction time is 12 ~ 24 hours.
Protein modified reagent N described in 3rd step-succinimido-S-acetylthio acetic acid esters, or N-succinimide-3-acetylthiopropionate is first dissolved in dimethyl formamide, or dimethyl sulfoxide (DMSO), join again subsequently in protein solution, the mol ratio of itself and albumen is 10:1 ~ 250:1, and the reaction time is 0.5 ~ 3 hour.
Described in 4th step, de-acetyl damping fluid is 0.5M azanol, and 25mM ethylenediamine tetraacetic acid (EDTA), PBS buffer system, pH is 7.2 ~ 7.5.
The trifluoroacetic acid reaction time described in 5th step is 5 ~ 10 minutes.
Crosslinking chemical 4-(N-maleimidomethyl) cyclohexane-1-carboxylic acid succinimide ester (SMCC) described in 6th step, or 4-(N-maleimidomehyl) cyclohexane-1-carboxylic acid sulfonic group succinimide ester sodium salt (Sulfo-SMCC) is first dissolved in dimethyl formamide or dimethyl sulfoxide (DMSO), or first Sulfo-SMCC is dissolved in ultrapure water, again SMCC or Sulfo-SMCC solution is joined in PBS buffer system subsequently, concentration is 0.1 ~ 2 mg/mL, and the reaction time is 15 ~ 45 minutes.
Protein concentration described in 7th step is 1 ~ 20 μ g/mL, and the amount of reaction is 50 ~ 300 μ L, and the reaction time is 0.5 ~ 3 hour.
The method technique is simple, easy and simple to handle, can be implemented in the target of the Interaction Force of single molecules level research biomolecule and cell surface single molecular recognition etc.
Accompanying drawing explanation
Fig. 1 is Atom force microscope Au probe of the present invention and gold substrate functional modification schematic diagram
Fig. 2 tests the unimolecule force curve obtained in the embodiment of the present invention 1.
Embodiment
Embodiment 1:
Au probe functional modification: clean the gold-plated atomic force microscope probe of needle point (Au probe) in chloroform, dry under a nitrogen, UV ozone cleaning 10min, subsequently with ultrapure water cleaning, chloroform cleans, dry under nitrogen; The Au probe cleaned up and gold substrate are put into reaction 12h in the 1 mM Boc-PEG-3000-SH and mPEG-2000-SH mixed liquor (molar ratio is 0.5:99.5) that chloroform is solvent, join in trifluoroacetic acid subsequently and react 5min, ultrapure water cleans, crosslinking aid S ulfo-SMCC is dissolved in ultrapure water, concentration is 5mg/mL, be diluted to 0.5mg/mL with PBS, get 100 μ L and Au probe reacts 30min; SATA is dissolved in dimethyl formamide, concentration is 1mg/mL, get 1.5 μ L SATA solution and be added drop-wise to 100 μ L 1mg/mL monoclonal anti sheep IgG, reaction 2h, gets 3 μ L subsequently and takes off acetyl damping fluid (0.5M azanol, 25mM EDTA, PBS buffer system, pH7.2-7.5) be added drop-wise in 30 μ L monoclonal anti sheep IgG, through desalting column desalination, utilize ultraviolet absorption spectrum to measure protein concentration; Subsequently the monoclonal anti sheep IgG after the Au probe of Sulf-SMCC process and 100 μ L 1 μ g/mL desalinations is reacted 2h, ultrapure water cleans, and to insert in PBS damping fluid 4 degree and saves backup.
Gold substrate functional modification: clean gold substrate in chloroform, dry under a nitrogen, UV ozone cleaning 10min, subsequently with ultrapure water cleaning, chloroform cleans, dry under nitrogen; The gold substrate cleaned up is put into reaction 12h in the 1 mM Boc-PEG-3000-SH and mPEG-2000-SH mixed liquor (molar ratio is 0.5:99.5) that chloroform is solvent, join in trifluoroacetic acid subsequently and react 5min, ultrapure water cleans, crosslinking aid S ulfo-SMCC is dissolved in ultrapure water, concentration is 5mg/mL, be diluted to 0.5mg/mL with PBS, get 100 μ L and Au probe reacts 30min; SATA is dissolved in dimethyl formamide, concentration is 1mg/mL, get 1.5 μ L SATA solution and be added drop-wise to 100 μ L 1mg/mL polyclone sheep IgG, reaction 2h, gets 3 μ L subsequently and takes off acetyl damping fluid (0.5M azanol, 25mM EDTA, PBS buffer system, pH7.2-7.5) be added drop-wise in 30 μ L polyclone sheep IgG, through desalting column desalination, utilize ultraviolet absorption spectrum to measure protein concentration; Subsequently the polyclone sheep IgG after the gold substrate of Sulf-SMCC process and 100 μ L 1 μ g/mL desalinations is reacted 2h, ultrapure water cleans, and to insert in PBS damping fluid 4 degree and saves backup.
The single molecule force spectroscopy experimental result that this experiment obtains is that Multimode Nanoscope III (VEECO/Bruker, Inc., Santa Barbara, CA, USA) completes, and scanatron is E type.AFM probe used is the TR400PB Au probe (elasticity coefficient is 0.09 N/m) of Olympus company.During this experiment, temperature controls at about 25 DEG C.
Embodiment 2:
Au probe functional modification: clean the gold-plated atomic force microscope probe of needle point (Au probe) in chloroform, dry under a nitrogen, UV ozone cleaning 10min, subsequently with ultrapure water cleaning, chloroform cleans, dry under nitrogen; The Au probe cleaned up and gold substrate are put into reaction 12h in the 1 mM Boc-PEG-3000-SH and mPEG-2000-SH mixed liquor (molar ratio is 1:99) that chloroform is solvent, join in trifluoroacetic acid subsequently and react 5min, ultrapure water cleans, crosslinking aid S ulfo-SMCC is dissolved in ultrapure water, concentration is 5mg/mL, be diluted to 0.5mg/mL with PBS, get 100 μ L and Au probe reacts 30min; SATA is dissolved in dimethyl formamide, concentration is 1mg/mL, get 1.5 μ L SATA solution and be added drop-wise to 100 μ L 1mg/mL monoclonal anti sheep IgG, reaction 2h, gets 3 μ L subsequently and takes off acetyl damping fluid (0.5M azanol, 25mM EDTA, PBS buffer system, pH7.2-7.5) be added drop-wise in 30 μ L monoclonal anti sheep IgG, through desalting column desalination, utilize ultraviolet absorption spectrum to measure protein concentration; Subsequently the monoclonal anti sheep IgG after the Au probe of Sulf-SMCC process and 100 μ L 2 μ g/mL desalinations is reacted 2h, ultrapure water cleans, and to insert in PBS damping fluid 4 degree and saves backup.
Gold substrate functional modification: clean gold substrate in chloroform, dry under a nitrogen, UV ozone cleaning 10min, subsequently with ultrapure water cleaning, chloroform cleans, dry under nitrogen; The gold substrate cleaned up is put into reaction 12h in the 1 mM Boc-PEG-3000-SH and mPEG-2000-SH mixed liquor (molar ratio is 1:99) that chloroform is solvent, join in trifluoroacetic acid subsequently and react 5min, ultrapure water cleans, crosslinking aid S ulfo-SMCC is dissolved in ultrapure water, concentration is 5mg/mL, be diluted to 0.5mg/mL with PBS, get 100 μ L and Au probe reacts 30min; SATA is dissolved in dimethyl formamide, concentration is 1mg/mL, get 1.5 μ L SATA solution and be added drop-wise to 100 μ L 1mg/mL polyclone sheep IgG, reaction 2h, gets 3 μ L subsequently and takes off acetyl damping fluid (0.5M azanol, 25mM EDTA, PBS buffer system, pH7.2-7.5) be added drop-wise in 30 μ L polyclone sheep IgG, through desalting column desalination, utilize ultraviolet absorption spectrum to measure protein concentration; Subsequently the polyclone sheep IgG after the gold substrate of Sulf-SMCC process and 100 μ L 2 μ g/mL desalinations is reacted 2h, ultrapure water cleans, and to insert in PBS damping fluid 4 degree and saves backup.
The single molecule force spectroscopy experimental result that this experiment obtains is that Multimode Nanoscope III (VEECO/Bruker, Inc., Santa Barbara, CA, USA) completes, and scanatron is E type.AFM probe used is the TR400PB Au probe (elasticity coefficient is 0.09 N/m) of Olympus company.During this experiment, temperature controls at about 25 DEG C.
Embodiment 3:
Au probe functional modification: clean the gold-plated atomic force microscope probe of needle point (Au probe) in chloroform, dry under a nitrogen, UV ozone cleaning 10min, subsequently with ultrapure water cleaning, chloroform cleans, dry under nitrogen; The Au probe cleaned up and gold substrate are put into reaction 12h in the 1 mM Boc-PEG-3000-SH and mPEG-2000-SH mixed liquor (molar ratio is 5:95) that chloroform is solvent, join in trifluoroacetic acid subsequently and react 5min, ultrapure water cleans, crosslinking aid S ulfo-SMCC is dissolved in ultrapure water, concentration is 5mg/mL, be diluted to 0.5mg/mL with PBS, get 100 μ L and Au probe reacts 30min; SATA is dissolved in dimethyl formamide, concentration is 1mg/mL, get 1.5 μ L SATA solution and be added drop-wise to 100 μ L 1mg/mL monoclonal anti sheep IgG, reaction 2h, gets 3 μ L subsequently and takes off acetyl damping fluid (0.5M azanol, 25mM EDTA, PBS buffer system, pH7.2-7.5) be added drop-wise in 30 μ L monoclonal anti sheep IgG, through desalting column desalination, utilize ultraviolet absorption spectrum to measure protein concentration; Subsequently the monoclonal anti sheep IgG after the Au probe of Sulf-SMCC process and 100 μ L 5 μ g/mL desalinations is reacted 2h, ultrapure water cleans, and to insert in PBS damping fluid 4 degree and saves backup.
Gold substrate functional modification: clean gold substrate in chloroform, dry under a nitrogen, UV ozone cleaning 10min, subsequently with ultrapure water cleaning, chloroform cleans, dry under nitrogen; The gold substrate cleaned up is put into reaction 12h in the 1 mM Boc-PEG-3000-SH and mPEG-2000-SH mixed liquor (molar ratio is 5:95) that chloroform is solvent, join in trifluoroacetic acid subsequently and react 5min, ultrapure water cleans, crosslinking aid S ulfo-SMCC is dissolved in ultrapure water, concentration is 5mg/mL, be diluted to 0.5mg/mL with PBS, get 100 μ L and Au probe reacts 30min; SATA is dissolved in dimethyl formamide, concentration is 1mg/mL, get 1.5 μ L SATA solution and be added drop-wise to 100 μ L 1mg/mL polyclone sheep IgG, reaction 2h, gets 3 μ L subsequently and takes off acetyl damping fluid (0.5M azanol, 25mM EDTA, PBS buffer system, pH7.2-7.5) be added drop-wise in 30 μ L polyclone sheep IgG, through desalting column desalination, utilize ultraviolet absorption spectrum to measure protein concentration; Subsequently the polyclone sheep IgG after the gold substrate of Sulf-SMCC process and 100 μ L 5 μ g/mL desalinations is reacted 2h, ultrapure water cleans, and to insert in PBS damping fluid 4 degree and saves backup.
The single molecule force spectroscopy experimental result that this experiment obtains is that Multimode Nanoscope III (VEECO/Bruker, Inc., Santa Barbara, CA, USA) completes, and scanatron is E type.AFM probe used is the TR400PB Au probe (elasticity coefficient is 0.09 N/m) of Olympus company.During this experiment, temperature controls at about 25 DEG C.
Embodiment 4:
Au probe functional modification: clean the gold-plated atomic force microscope probe of needle point (Au probe) in chloroform, dry under a nitrogen, UV ozone cleaning 10min, subsequently with ultrapure water cleaning, chloroform cleans, dry under nitrogen; The Au probe cleaned up and gold substrate are put into reaction 12h in the 1 mM Boc-PEG-3000-SH and mPEG-2000-SH mixed liquor (molar ratio is 2:98) that chloroform is solvent, join in trifluoroacetic acid subsequently and react 5min, ultrapure water cleans, crosslinking aid S ulfo-SMCC is dissolved in ultrapure water, concentration is 5mg/mL, be diluted to 0.5mg/mL with PBS, get 100 μ L and Au probe reacts 30min; SATA is dissolved in dimethyl formamide, concentration is 1mg/mL, get 1.5 μ L SATA solution and be added drop-wise to 100 μ L 1mg/mL polyclone sheep IgG, reaction 2h, gets 3 μ L subsequently and takes off acetyl damping fluid (0.5M azanol, 25mM EDTA, PBS buffer system, pH7.2-7.5) be added drop-wise in 30 μ L polyclone sheep IgG, through desalting column desalination, utilize ultraviolet absorption spectrum to measure protein concentration; Subsequently the polyclone sheep IgG after the Au probe of Sulf-SMCC process and 100 μ L 2 μ g/mL desalinations is reacted 2h, ultrapure water cleans, and to insert in PBS damping fluid 4 degree and saves backup.
Gold substrate functional modification: clean gold substrate in chloroform, dry under a nitrogen, UV ozone cleaning 10min, subsequently with ultrapure water cleaning, chloroform cleans, dry under nitrogen; The gold substrate cleaned up is put into reaction 12h in the 1 mM Boc-PEG-3000-SH and mPEG-2000-SH mixed liquor (molar ratio is 2:98) that chloroform is solvent, join in trifluoroacetic acid subsequently and react 5min, ultrapure water cleans, crosslinking aid S ulfo-SMCC is dissolved in ultrapure water, concentration is 5mg/mL, be diluted to 0.5mg/mL with PBS, get 100 μ L and Au probe reacts 30min; SATA is dissolved in dimethyl formamide, concentration is 1mg/mL, get 1.5 μ L SATA solution and be added drop-wise to 100 μ L 1mg/mL monoclonal anti sheep IgG, reaction 2h, gets 3 μ L subsequently and takes off acetyl damping fluid (0.5M azanol, 25mM EDTA, PBS buffer system, pH7.2-7.5) be added drop-wise in 30 μ L monoclonal anti sheep IgG, through desalting column desalination, utilize ultraviolet absorption spectrum to measure protein concentration; Subsequently the monoclonal anti sheep IgG after the gold substrate of Sulf-SMCC process and 100 μ L 2 μ g/mL desalinations is reacted 2h, ultrapure water cleans, and to insert in PBS damping fluid 4 degree and saves backup.
The single molecule force spectroscopy experimental result that this experiment obtains is that Multimode Nanoscope III (VEECO/Bruker, Inc., Santa Barbara, CA, USA) completes, and scanatron is E type.AFM probe used is the TR400PB Au probe (elasticity coefficient is 0.09 N/m) of Olympus company.During this experiment, temperature controls at about 25 DEG C.
Embodiment 5:
Au probe functional modification: clean the gold-plated atomic force microscope probe of needle point (Au probe) in chloroform, dry under a nitrogen, UV ozone cleaning 10min, subsequently with ultrapure water cleaning, chloroform cleans, dry under nitrogen; The Au probe cleaned up and gold substrate are put into reaction 12h in the 1 mM Boc-PEG-3000-SH and mPEG-2000-SH mixed liquor (molar ratio is 5:95) that chloroform is solvent, join in trifluoroacetic acid subsequently and react 5min, ultrapure water cleans, crosslinking aid S ulfo-SMCC is dissolved in ultrapure water, concentration is 5mg/mL, be diluted to 0.5mg/mL with PBS, get 100 μ L and Au probe reacts 30min; SATA is dissolved in dimethyl formamide, concentration is 1mg/mL, get 1.5 μ L SATA solution and be added drop-wise to 100 μ L 1mg/mL polyclone sheep IgG, reaction 2h, gets 3 μ L subsequently and takes off acetyl damping fluid (0.5M azanol, 25mM EDTA, PBS buffer system, pH7.2-7.5) be added drop-wise in 30 μ L polyclone sheep IgG, through desalting column desalination, utilize ultraviolet absorption spectrum to measure protein concentration; Subsequently the polyclone sheep IgG after the Au probe of Sulf-SMCC process and 100 μ L 5 μ g/mL desalinations is reacted 2h, ultrapure water cleans, and to insert in PBS damping fluid 4 degree and saves backup.
Gold substrate functional modification: clean gold substrate in chloroform, dry under a nitrogen, UV ozone cleaning 10min, subsequently with ultrapure water cleaning, chloroform cleans, dry under nitrogen; The gold substrate cleaned up is put into reaction 12h in the 1 mM Boc-PEG-3000-SH and mPEG-2000-SH mixed liquor (molar ratio is 5:95) that chloroform is solvent, join in trifluoroacetic acid subsequently and react 5min, ultrapure water cleans, crosslinking aid S ulfo-SMCC is dissolved in ultrapure water, concentration is 5mg/mL, be diluted to 0.5mg/mL with PBS, get 100 μ L and Au probe reacts 30min; SATA is dissolved in dimethyl formamide, concentration is 1mg/mL, get 1.5 μ L SATA solution and be added drop-wise to 100 μ L 1mg/mL monoclonal anti sheep IgG, reaction 2h, gets 3 μ L subsequently and takes off acetyl damping fluid (0.5M azanol, 25mM EDTA, PBS buffer system, pH7.2-7.5) be added drop-wise in 30 μ L monoclonal anti sheep IgG, through desalting column desalination, utilize ultraviolet absorption spectrum to measure protein concentration; Subsequently the monoclonal anti sheep IgG after the gold substrate of Sulf-SMCC process and 100 μ L 5 μ g/mL desalinations is reacted 2h, ultrapure water cleans, and to insert in PBS damping fluid 4 degree and saves backup.
The single molecule force spectroscopy experimental result that this experiment obtains is that Multimode Nanoscope III (VEECO/Bruker, Inc., Santa Barbara, CA, USA) completes, and scanatron is E type.AFM probe used is the TR400PB Au probe (elasticity coefficient is 0.09 N/m) of Olympus company.During this experiment, temperature controls at about 25 DEG C.
Claims (8)
1. single molecule force spectroscopy analysis atomic force microscope probe and a substrate functional modification method, is characterized in that, comprise the following steps:
The first step: clean the gold-plated atomic force microscope probe of needle point and gold substrate in chloroform, probe and gold substrate are dry under a nitrogen, UV ozone is cleaned, and subsequently with ultrapure water cleaning, chloroform cleans, dry under nitrogen;
Second step: the Au probe cleaned up and gold substrate are put into lucifuge reaction in carbamic acid tertiary Ding Zhi – Ju Yi bis-Chun – sulfydryl (Boc-PEG-3000-SH) and Jia Yang Ji – Ju Yi bis-Chun – sulfydryl (mPEG-2000-SH) mixed liquor that chloroform is solvent;
3rd step: add protein modified reagent N-succinimido-S-acetylthio acetic acid esters (SATA) in protein solution, or N-succinimide-3-acetylthiopropionate (SATP), and react a period of time;
4th step: in the protein solution that the 3rd step obtains, add in de-acetyl damping fluid and react, the reaction time is 0.5 ~ 3 hour, utilizes desalting column to slough salt in protein solution subsequently;
5th step: get Au probe that second step obtains and gold substrate is put in trifluoroacetic acid, ultrapure water cleans;
6th step: Au probe and gold substrate put into crosslinking chemical 4-(N-maleimidomethyl) cyclohexane-1-carboxylic acid succinimide ester (SMCC), or in 4-(N-maleimidomehyl) cyclohexane-1-carboxylic acid sulfonic group succinimide ester sodium salt (Sulfo-SMCC) solution, reaction a period of time, ultrapure water cleans;
7th step: Au probe and gold substrate are placed to the protein solution that the 4th step process obtains, reaction, ultrapure water cleans, and to insert in phosphate (PBS) damping fluid 4 degree and saves backup.
2. single molecule force spectroscopy analysis atomic force microscope probe and substrate functional modification method according to claim 1, is characterized in that, the needle surface of described atomic force microscope probe has plated the substrate of one deck gold film as functional modification.
3. single molecule force spectroscopy analysis atomic force microscope probe and substrate functional modification method according to claim 1, it is characterized in that, in polyglycol mixed liquor described in second step, Boc-PEG-3000-SH molar ratio is 0.5 ~ 10 ﹪, total Polyethylene glycol is 1 ~ 10mM, and the reaction time is 12 ~ 24 hours.
4. single molecule force spectroscopy analysis atomic force microscope probe and substrate functional modification method according to claim 1, it is characterized in that, protein modified reagent N described in 3rd step-succinimido-S-acetylthio acetic acid esters, or N-succinimide-3-acetylthiopropionate is first dissolved in dimethyl formamide, or dimethyl sulfoxide (DMSO), join subsequently in protein solution, the mol ratio of itself and albumen is 10:1 ~ 250:1, and the reaction time is 0.5 ~ 3 hour again.
5. single molecule force spectroscopy analysis atomic force microscope probe and substrate functional modification method according to claim 1; it is characterized in that, described in the 4th step, de-acetyl damping fluid is 0.5M azanol, 25mM ethylenediamine tetraacetic acid (EDTA); PBS buffer system, pH is 7.2 ~ 7.5.
6. single molecule force spectroscopy analysis atomic force microscope probe and substrate functional modification method according to claim 1, it is characterized in that, the trifluoroacetic acid reaction time described in the 5th step is 5 ~ 10 minutes.
7. single molecule force spectroscopy analysis atomic force microscope probe and substrate functional modification method according to claim 1, it is characterized in that, crosslinking chemical 4-(N-maleimidomethyl) cyclohexane-1-carboxylic acid succinimide ester (SMCC) described in 6th step, or 4-(N-maleimidomehyl) cyclohexane-1-carboxylic acid sulfonic group succinimide ester sodium salt (Sulfo-SMCC) is first dissolved in dimethyl formamide or dimethyl sulfoxide (DMSO), or first Sulfo-SMCC is dissolved in ultrapure water, again SMCC or Sulfo-SMCC solution is joined in PBS buffer system subsequently, concentration is 0.1 ~ 2 mg/mL, reaction time is 15 ~ 45 minutes.
8. single molecule force spectroscopy analysis atomic force microscope probe and substrate functional modification method according to claim 1, it is characterized in that, protein concentration described in the 7th step is 1 ~ 20 μ g/mL, and the amount of reaction is 50 ~ 300 μ L, and the reaction time is 0.5 ~ 3 hour.
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