Rapid Development of New Protein Biosensors Utilizing Peptides Obtained via Phage Display 英文参考文献.docVIP

RapidDevelopmentofNewProteinBiosensorsUtilizing PeptidesObtainedviaPhageDisplay JunWu1¤a,JongPilPark1¤b,KevinDooley1,DonaldM.Cropek2,AlanC.West1,ScottBanta1* 1DepartmentofChemicalEngineering,ColumbiaUniversity,NewYork,NewYork,UnitedStatesofAmerica,2UnitedStatesArmyEngineerResearchandDevelopment Center,ConstructionEngineeringResearchLaboratory(CERL),Champaign,Illinois,UnitedStatesofAmerica Abstract Thereisaconsistentdemandfornewbiosensorsforthedetectionofproteintargets,andasystematicmethodfortherapid development of new sensors is needed. Here we present a platform where short unstructured peptides that bind to a desired target are selected using M13 phage display. The selected peptides are then chemically synthesized and immobilized on gold, allowing for detection of the target using electrochemical techniques such as electrochemical impedance spectroscopy (EIS). A quartz crystal microbalance (QCM) is also used as a diagnostic tool during biosensor development.Wedemonstratetheutilityofthisapproachbycreatinganovelpeptide-basedelectrochemicalbiosensorfor the enzyme alanine aminotransferase (ALT), a well-known biomarker of hepatotoxicity. Biopanning of the M13 phage displaylibraryoverimmobilizedALT,ledtotherapididentificationofanewpeptide(ALT5-8)withanaminoacidsequence of WHWRNPDFWYLK. Phage particles expressing this peptide exhibited nanomolar affinity for immobilized ALT (Kd,app=85620nM).ThenewlyidentifiedALT5-8peptidewasthenchemicallysynthesizedwithaC-terminalcysteinefor goldimmobilization.Theperformanceofthegold-immobilizedpeptideswasstudiedwithcyclicvoltammetry(CV),QCM, andEIS.UsingQCM,thesensitivityforALTdetectionwas8.960.9Hz/(mg/mL)andthelimitofdetection(LOD)was60ng/ mL. Using EIS measurements, the sensitivity was 142612 impedance percentage change %/(mg/mL) and the LOD was 92ng/mL.Inbothcases,theLODwasbelowthetypicalconcentrationofALTinhumanblood.AlthoughbothQCMandEIS produced similar LODs, EIS is preferable due to a larger linear dynamic range. Using QCM, t