Ajax应用安全研究

1、afm spectroscopyme 382: micro/nano science and engineeringravi agrawal, kevin lee, and deepak ponnavolureport submitted: 03/12/05presented to professorabstractin this paper, we discuss the use of an atomic force microscope (afm) to do spectroscopy. spectroscopy of dna, proteins and other biomolecule

2、s are important in order to characterize the biomolecule as well as determine its properties. some major considerations for spectroscopy include the use of an afm in liquids for use with biomolecules and the functionalization of substrate and the tip for alignment and selectivity. potential applicat

3、ions for force spectroscopy include characterization of single molecules under tensile or torsional load, cell-cell interactions, and interaction of surfaces with molecules etc. a huge amount of research is being done in this area to exploit the resolution obtained through afms in these kinds of app

4、lications.contents1. introduction22. working principle23. using afm in aqueous medium34. functionalization of substrate45. tip parameters and top functionalization66. applications and developments107. conclusion111. introductionspectroscopy is typically used in the physical and analytical chemistry

5、for the indentification of substances. involving the use of the atomic force microscope, force spectroscopy is a dynamic analytical technique which allows the study of mechanical properties of polymer molecules and chemical bonds- force spectroscopy measures the behavior of a molecule under stretchi

6、ng or torsional forces, and unlike most other forms of spectroscopy does not measure the matterradiation interaction.2. working principleafm spectroscopy is based on the simple principle of attaching the tip of the cantilever to one end of the biomolecules to be analyzed, the other end being held by

7、 the substrate. the cantilever is then moved using a piezocontroller to exert a tensile load on the molecule. using the force mode of afm, force characteristic curves for the biomolecules can be obtained in this manner. fig. 2shows a schematic view of the process-sfm can tileverligand/receptor inter

8、actionsprotein unfoldingligandfig. 2.1 schematic view of single molecule force spectroscopy using ligand-receptor binding3< using afm in aqueous mediumatomic force microscopy (afm) is a method in which a probe scans the surface of a material with a sharp tip in order to clearly image the features

9、 of a sample surface within the diameter located at the free end of a cantilever is brought close to the desired sample surface. the forces between the tip and the sample surface cause the cantilever to deflect, in which a detector measures and records data and generates a map of surface topography.

10、 the ability of afm, as a non destructive method, to produce measurements of the adhesion forces acting between the tip and the surface allows information about the binding energy in the subnanonewton range to be obtained.in the biological field, afm is employed to examine biomolecules. this however

11、 requires a substrate liquid to be used as biomolecules must be kept and experimented with in a liquid environment. this requiem for biological measurements produces many problems for the typical afm procedure0 for instance, the large oscillation amplitude on the order of 10 to100a used to attain su

12、itable signal-to-noise ratio makes interpretation of the force curve very difficult. in order to prevent this, optimum oscillation amplitude can be reduced by using a stiffer force sensor compared to the conventional. furthermore, the q-value required for tapping afm measurements is hardly implement

13、ed due to the viscosity of the liquid. the reduction of the viscous effect can be considered as:d/2 d/,where m is the effective mass of the oscillator, b is the viscous constant and co()is given by k = mo)o2- the resonance occurs whenand the q-value of the system is given byfrom the q-value expressi

14、on, it is implied that a large mass is favorable in order to attain a large q-value. typically most sensors used in tapping afm have small thickness typically less than 10 pm, but by increasing the thickness relative to the length and width it is possible to have both a large k and q value without s

15、ignificantly increasing the damping also, another problem in tapping afm in a liquid arises from the cone angle of about 30 to 70°，which superimposes a large van der waals force upon the measured force curve4. functionalization of substratewhen using biopolymers, even a very minor exposure to a

16、ir could result in contamination due to picking up of hydrocarbons from the air; hence this increases our need to use a substrate. the biopolymers are normally dissolved in aqueous solution and then deposited onto the substrate. compared with the size of the sample molecule the substrate has to be f

17、lat and easy to prepareto strengthen the adsorption (between sample and substrate), functionalized surfaces can be introduced. the simplest method is to treat the surface with certain molecules, such as poly-l- lysine or poly-l-arginine, in order to change the charge behavior of the surface. therefo

18、re the adsorption can be improved or modified we can also cross-link groups to the surface. so far, many techniques have been used for afm imaging in solutions one such technique is based on silanizing a solid surface with 3-aminopropyltriethoxysilane (aptes), which protonates at neutral ph. the sil

19、ane group in aptes is highly reactive and affects the surface by forming covalent bonds with surface atoms. something else that has been done is introducing a crosslinking group at the amino end of aptes on a glass surface, n-5-azido2- nitrobenzoyloxysucciniinide (anbnos). the azide group, upon ultr

20、aviolet irradiation, can make non-specific covalent bonds to proteins on contact. since the functionalized surface becomes hydrophobic and soluble proteins do not come close enough to be cross-linked, a squeezing pressure of 10-500 atm must be used to force macromolecules to come within reach of the

21、 azide group. in another technique, an ultraflat au (111) surface is used as a substrate for n- hydroxysuccinimide terminated self-assembled monolayers of dithio-bis(succinidylundecanoate). this monolayer readily reacts with amino groups, covalently linking the protein to the substrate.41 procedures

22、 for some of these techniques4.1.1 binding of biological molecules to a solid substratebiomolecules and supramolecular can be bound to assemblies such as: bacteriophage t4 polyheads eucariotic intermediate filaments hpi layer of deinococcus radioduransfor the covalent binding of samples to the modif

23、ied glass surface, the aptes-anbnos- covered cover slips are squeezed between two glass discs (borosilicate safety sight glass; diameter = 12 cm, thickness = 2 cm) at a pressure of 100 to 5000 n/cm to bring the hydrophilic biological structures into close contact with the hydrophobic cross-linker. c

24、ovalent coupling of the samples is induced by activating the azide with ultraviolet (uv) irradiation at 366 nm (sylvania f8t5) at a distance of 10 cm for 3 min. the extent of the reaction is determined from the change in the absorption band of anb-nos at 312 nm. cover slips are rinsed thoroughly wit

25、h water to remove excess protein and stored in water or buffer.another procedure that can be used: ten microliters of e-pha lectin (2 mg/ml in phosphate- buffered saline pbs buffer: 0.145 m nacl and 0.005 m nah2po4/na2hpo4, ph at 7.4) are compressed between tow anbnos-coated glass cover slips under

26、irradiation at 302 nm (8 watt) at 10 cm from the light source for 3 min to bring the hydrophilic lectin in close contact with the hydrophobic anbnos. completion of light-activated crosslinking is then confirmed spectrophotometrically. the cover slips are rinsed with pbs five times then stored in pbs

27、.4.1.2 dna adsorption to aptes mica modified mica strips are immersed into dna in tris/hcl buffer (ph =7) (0.01 m tris/hcl, 0.010-0.020 m nacl, 0.005 m edta) and incubated at room temperature for between 1 and 2 h. concentration of dna was varied between 0.01 and 0.1 pg/ml. after the adsorption stag

28、e had been completed, the samples were rinsed with deionized water, blotted at the edge and vacuum- dried.fig. 4.1.1 afm reveals a change in dna conformation during unbinding of the protein-dna complex. dna fragments 390 nm long, containing the expg promoter region at one end，were imaged in buffer s

29、olution in the presence of expg(his)6 proteins. when the protein (red arrow) breaks away (time between images: 9 min), the curvature of the dna binding region changes.<2)4.1.3 protein adsorption protein adsorption is a net result of various complex interactions between and within all components,

30、including the solid surface, the protein, the solvent and any other solutes present. interaction forces include dipole and induced dipole moments, hydrogen bond forces and electrostatic potentials. all these inter- and intramolecular forces will contribute to a decrease of the gibbs energy during ab

31、sorption.a-helical domainnoamfasciclin iilg domain . cadhertn domaindystrophin fibronectin spectrin tenascin nh cadherinfn iii domaincooh cooh cooh coohfig. 4.l2 schematic representations of several proteins attached to the substrate that are exposed to mechanical stress 5. tip functionalization and

32、 tip parameters5.1. tip functionalizationmost commonly used probe materials are silicon and silicon nitride as they can be easilymicromachined. the tip sample interactions are influenced by the material to be analyzed as wellas the ambience in which it is to be analyzed when working in liquid atmosp

33、here, theinteraction depends on the ph and the electrolyte concentration in the solution as the ambienceprovides some kind of polarity to the tip by charging it. for many biological applications, it isnecessary to have a specific surface charge on the tip or to change its hydration properties. thisc

34、an be achieved by silanization or plasma treatment. increased hydrophobicity of the tip reducesthe applied force by reducing hydration and capillary forces in air and it also prevents the wearof the tip when imaging in liquid.silanization is another way of changing the surface properties of the tip.

35、 organochloro- and organoalkoxy silanes are covalently bonded to the probe surface. (fig. 5.1.1(a)-ohor一 ohor-ohorfig 5.1.1 (a) schematic representation of silanization process, (b) functionalized cantilever for force measurement on ligand-receptor pairs/41functionalization of tips by coating them w

36、ith appropriate kind of molecules provides an opportunity to study interactions at the molecular level e.g. ligand-recptor pairs or cell-cell interactions-(fig- 5.1.1(b) chemical coating by silanes or thiols is the first step before biological functionalization biological coatings help map the distr

37、ibution of binding partners on samples as well as force measurements. different kind of forces can be investigated like forces between a receptor and a ligand, forces between molecules and cells, forces between cells.many protocols can be used to bind a protein molecule to the tip, most of which inv

38、olves the use of a “spacer" to covalently bind the protein. for example, peg, polyethylene glycol is a common spacer- a thiol group is used in between to bind this peg onto a gold coated silicon nitride tip. an amine group at the other end of the peg molecule attaches prot&ns via a covalent

39、 bond cells can also be grown on tip-less cantilevers or the cantilevers with small beads at the end. they can also be chemically attached via peg.fig. 5.1.2 (a) sketch of a sensor molecule (brown) tethered to an afm tip with a flexible peg linker. the sensor molecule can move around freely to find

40、its target molecules (blue) bound to the surface (green) (b) different ligands tethered to afm tip via flexible peg linker(10)proteinn广 h.seproteinnta52 tip parametersfundamental parameters of an afm probe are the shape of the tip and its mechanical properties like stiffness, resonance frequency and

41、 quality factor "q：5.2.1 tip shapetip sharpness determines the lateral resolution of an afm image, and therefore the image obtained is a combination of the tip shape and the sample topography. the duller the tip, the wider the topography appears 一 "tip broadening effect” when interacting w

42、ith biomolecules, the minimum possible tip radius is desired. however the sharp tips exert a huge pressure on the cells and thus can poke through the membrane destroying the cell. therefore, sometimes it is preferred to use dull tips rather than sharp tips compromising on the quality of image obtain

43、ed.if dull tips are used for biological applications, the following techniques are implemented to study the tip shape and restoring the true sample image: tip measurement using electron microscopy or field ion emission microscopy by scanning a sample whose topography is already known tip shape can a

44、lso be estimated using mathematical morphology operations 一 blind mathematical restoration. these linear mathematical operations consist in an over- and under-estimation of tip broadening effectfig* 5.2 sharpness of the tip required for biological applications (b)5.2.2 spring constantfor force measu

45、rements it is crucial to know the value of spring constant. the accuracy of the force measurement is determined by the error of the spring constant and by any errors in system detecting the deflection of the cantilever. various methods which are used to determine the spring constant are: using equip

46、artition theorem to deduce the spring constant from the thermal vibration spectrum of a cantilever measuring the change in resonance frequency at different loads using geometrical dimensions or quality factor to calculate the stiffness.5.2.3 resonance frequency and quality factorin tapping mode, a c

47、antilever preferentially oscillates at a frequency close to its natural frequency which is between 900 hz & 88 khz for silicon nitride cantilevers and between 60khz & 400khz for silicon cantilevers. it is important to tune the frequency very close to the surface because there can be a freque

48、ncy shift as the tip approaches the surface.quality factor is a measure of the dissipation and it affects the scan speed and the sensitivity. high q is preferred for tapping mode to optimize sensitivity. q values are reduced significantly in water due to hydrodynamic damping and are around 1 in wate

49、r as compared to its value of 100300 in air. with a positive feedback system, the effective quality factor of over 300 can be obtained in liquid environment also, which facilitates tracking of the resonant frequency and the separation of elastic and viscous forces.5.2.4 problems of tip contamination

50、 and tip cleaningin biological applications, the tip can be easily contaminated due to detachment from the sample surface, which reduces the lateral resolution. the most popular method of cleaning is uv light treatment that produces ozone and removes organic debris other possible ways are to incubat

51、e the tips in piranha solution for 30 minutes or use plasma etching. a cleaned probe is generally hydrophilic because of the removal of oily organic contaminants.5.3. example of tip functionalization5.3.1 tip functionalization for lexa-dna moleculesi3nq tips are first cleaned by exposure to uv light

52、 for 30 min, and then salinized in nl3- (trimethoxysilyl)propyldiethylene-triamine (aldrich) by incubating at 90°c for 10 min. subsequently, the tips are washed first in ethanol and then in deionized water at 90° c for 2 h. the tips are then incubated at room temperature for 10 min with ca

53、rboxy methylamylose-10 mg (sigma) + nhs-4 mg (n-hydroxysuccinimide, aldrich) + edc-17 mg (1 - ethyl-3-3- (dimethylamino)propylcarbamide, sigma) diluted in 200 ml pbs. finally, they are washed with pbs several times. in the last step, the cantilevers are incubated either with 20 “l rec operator or ye

54、bg operator at room temperature for 1 h 30 niin.6. applications and developmentsthis technique of using afm to look at the interactions taking place at molecular or cellular level has opened up many new applications, some of which are mentioned below:6.1. measurement of adhesion forces between indiv

55、idual cells tip-less afm cantilevers can be covalently functionalized using lectin, which results in firm attachment of the cell to the cantilever. this cantilever is then moved in a controlled fashion to make contact and apply a certain pre-defined force for a pre-defined time on the target cell in

56、 a petri dish. this results in cell-cell adhesion. the cantilever is then retracted and the cell rupture forces can be measured.afig. 6.1 (a) force spectroscopy of adhesion between individual d. discoideum cells, (b) light-microscopic image of a cantilever mounted cell before being bought into conta

57、ct with another celt scale bar represents 20 (7)microns.6.2. chemical force spectroscopy on single-walled carbon nanotube paper the unique combination of properties like high electrical conductivity, low mass and high mechanical strength makes carbon nanotube very suitable for polymeric composites.

58、using afm spectroscopy, the binding forces of single molecules to the side walls of carbon nanotube can be measured it was found that affinity of terminally substituted alkanethiols decrease in the following order:nh2 > -cooh > -c=c > -oh > -f3 > -ch36.3 estimating bulk reaction kinet

59、ics and energies using force measurements on single molecule force measurements on single molecules can be related to quantities measured in bulk such as affinity, the rate constants and energies of reactions the technique works differently for different kind of reactions. in the reactions at chemical equilibrium, an applied force can produce a thermodyna