Force Spectroscopy To Study The Surface Properties And Mechanical Behavior Of Serum Albumin In The Presence Of Dugs By AFM

Proteins participate in most of the life activities based on the structure and biomechanical properties.On the one hand,proteins functions work through the interaction between proteins or proteins and other molecules in body fluids.On the other hand,the function of the corresponding system is changed by the surface characteristics and mechanical behaviors.Therefore,it is of great significance to study the surface properties and mechanical behaviors of proteins for deeper understanding of their functions.Solution environment will affect the mechanical behavior of proteins.BSA was chosen as the test protein while AGs,QLs,MLs,and TCs as the test drugs.Exploring the effect of these antibiotics on the mechanical action of bovine serum albumin（BSA）and investigating the relationship between the properties of the antibiotics and the mechanical behavior of BSA in the drug environment conduce to the establishment of a method of drug analysis based on mechanical measurements,guidance of discovery of new drug targets and drug screening.Atomic force microscopy（AFM）has high advantages in protein imaging and mechanical measurement.Thus,this article focused on the effect of antibiotics on surface properties and mechanical behavior of BSA by AFM.It mainly carried out the following aspects of works:（1）Self-assembled BSA layers were characterized after preparation by chemical modification.16-Mercaptoalkanoic Acid（MHA）was fixed to the golden surface with Au-S bond,and successively activated by N-Hydroxysuccinimide（NHS）and 1-Ethyl-3-（3-Dimethylaminopropyl）–Carbodiimide Hydrochloride（EDC）.Afterwards BSA was covalently linked to the end of MHA via amide bond,forming the BSA layer with stability and smoothness.Characterization of bare Au,MHA layers and BSA layers were performed by AFM imaging,contact angle（CA）,X-ray photoelectron spectroscopy（XPS）,and Grazing incidence X-ray diffraction（GIXRD）tests,respectively.The results were outlined below:（1）AFM result shows differences of the size and distribution of particles.The average heights were 2.474,3.141 and 5.375 nm,respectively,and increasing heights of 0.667 nm and 2.234 nm accord with the molecule length of MHA and BSA.（2）CA results show that MHA and BSA layers are hydrophilic accompanied with hydrophilicity disparity while Au layer is hydrophobic.（3）The 2θdegrees of MHA layers and BSA layers obtained from GIXRD were obviously smaller than the bare Au surface and distributed in the range of 0-15°.However,the profiles and distributions of the diffraction peaks of the MHA and BSA layers were different.（4）In addition,the full spectra and binding energy spectra of the three surfaces were consistent with either Au4f,S2p,or N1s,respectively.（2）The effects of AGs,QLs,MLs and TCs on the adhesion of BSA were investigated by AFM.Adhesion force(₍（6（9））was obtained from the pull-off event characterized by Force-distance（F-d）curve.The Gauss fitted maximum value in distribution was used to characterize the adhesion force.Compared with the control group,₍(6（9） in drug solutions（10μM）integrally decreasein order of TCs<QLs<MLs<AGs and₍(6（9） differed among similar antibiotics.₍(6（9） at antibiotics in different concentrations were calculated and decomposed into specific force(₄))and non-specific force().₄)decreased and decrease rate saturated with drug concentration whilevalue hold inapparent variation in AGs solution.By contrary,₄)value stabilized whiledecreased with drug concentration and saturated at 10μM in QLs and MLs.Besides,₄)stabilized as well asin TCs,revealing no relevance to concentration.These drugs can affect adhesion of BSA in different ways,which might attribute to the binding force between BSA and drugs.It is concluded that the binding force between BSA and AGs is hydrogen bond while QLs and MLs as van der Waals or hydrophobic interaction and TCs as electrostatic interaction.（3）The effects of different drugs on the surface properties of BSA layers were measured by AFM imaging and FFM mode with bare tip.The surface properties of BSA were quantified through multiple parameters.The results are shown as follows:（1）BSA layers appeared aggregation and lateral diffusion on account of the weakness of hydration of protein after addition of antibiotics,showing greater smoothness.Bothand₍（6） decreased in varing degree without obvious regularity.（2）According to the elastic contact mode of multiple rough peaks,curve of friction force()versus applied normal force()was investigated in varied solutions.It is observed that protein layer would appear lateral rupture event when load force exceeded the payload.Based on von Mises Yield Criterion,the depression depthδand yield strengthderived show an indistinct distinction.It indicates that the surface strength of BSA is not related to drugs,but only to the elastic properties itself.Therefore,the tip was functionalized to study the friction between BSA layers.（4）After functionalization,the measurement error caused by multi-molecular events can be reduced by diluting the density of BSA.P/C mixed layers were formed by mixing NDM and BSA onto gold surface by covalent bond,hence the inert group CH₃was added to generate mixed protein layer with different densities（P:C=1:0,1:1,1:2）.The curve ofversuswas measured using the FFM mode.The results show lateral rupture on the surface and similar trend ofwith increasingof mixed layers.For different protein layers,the limit load and friction coefficient has declined as the density increased,indicating that the mixed SAMs has superior friction performance.Theoretically,the result is attributed to the higher disorders and molecular mobility in outer layers.On the one hand,the rupture event of protein layers become inapparent when the density continually decreased.On the other hand,higher density leads to greater loss of protein layer during the scanning process of the probe.Synthetically speaking,the mixed layers with P:C=1:1 has suitable measurement performance in the follow-up works.（5）FFM was used to study the tribological behavior of P/C（1:1）layers in different solutions.The optimalset as 1n N through the-curve with real-time observation of topography,in which BSA layers would maintain complete condition and friction characteristics.between functionalized tip and substrate was measured under the constant load of 1 n N.Compared to the control,in varied antibiotic solutions decreased in order of TCs<QLs<MLs<AGs.differed in similar antibiotics and show linearity with₍(6（9） between BSA layers.It is concluded that drug molecules would affect the friction,which may be related to decrease of the adhesion force between BSA layers.（6）The combination and formation between BSA and drugs was determined by multi-spectroscopy（UV,fluorescence,Raman）and molecular docking.The results are as follows:（1）Four kinds already combined to BSA with different binding site and mode.（2）Surface Enhanced Raman Scattering（SERS）spectra show that the relative content of secondary structure is basically same as the BSA with no drugs addition.It is inferred that BSA changed in level of tertiary structure after combination with drugs.Since the change of tertiaty structure of proteins depends on the secondary bonds,the results are consistent with AFM measurements.（3）Surflex-dock was employed to estimate the combination and formation between BSA and drugs.Herein,the crystal structure of BSA in complex with naproxen was used for molecular docking.It is observed that the predominant force for BSA-AGs complex is the hydrogen bonding,while BSA-QLs,BSA-MLs and BSA-TCs are hydrophobic effect,van der Waals force and electrostatic force,respectively.The results are consistent with the analysis from AFM measurements,which comfirms the molecular mechanism of the effect of anbiotics on mechanical behavior of BSA.（7）The correlation of AFM results,molecular docking and drug parameters show that in the same kind of drugs,the interaction between BSA molecules in drug environment is related to the chemical and physical properties of the drug.Molecular weight,spatial structure and number of hydrogen bond donors of AGs are related to the interaction forces between BSA molecules as well as molecular weight,surface reach area and van der Waals area of MLs,and the hydrophobicity of QLs,which reveals the reason for the differences of adhesion and friction force in the similar drugs.In summary,the effects of exogenous drugs on the surface properties and mechanical behavior of proteins using AFM and the molecular mechanism of the effects was in-depth studied,which is of great significance for the subsequent establishment of drug analysis method based on mechanical determination.