Highly Sensitive Detection For Chymotrypsin Using Peptide Based Sensors

Chymotrypsin is a typical serine proteinase,the digestive enzyme of vertebrate.It involves in many biological processes and plays a major role in various human diseases.The level of chymotrypsin in umbilical cord blood of newborns with cystic fibrosis is about 2.2 to 7.5 times than the healthy ones.And the content of chymotrypsin in adults with acute pancreatitis can reach 35 times than normal reference value.So reliable and sensitive methods in chymotrypsin measurement is quite important for pancreas function test.Peptide has the same chemical essence to protein,and is easy to modify,which also with qualities of stability,specificity,versatility,etc.Thus,it is the ideal research target in biosensor detection.In this study,we construct optical biosensors to detect chymotrypsin,thereby achieving highly sensitive detection of chymotrypsin.(1)Detection of chymotrypsin based on graphene oxide(GO)and peptide sequence covalently binding to gold nanoparticles(Au NPs).The Au NPs are covalently bound to the thiol group at one end of the peptide.After adding GO,the complex is adsorbed on the surface of GO.Then the fluorescence of pyrene at one end of the peptide is quenched by fluorescence resonance energy transfer(FRET).Chymotrypsin is added to cleave specific sites of the peptide,and fluorescence is restored.After adding NaBr,the peptides adsorbed on the surface of Au NPs are removed,which makes Au NPs and sulfhydryl groups bind faster and more stable,strengthens the stability of the system,and improves the detection sensitivity.The limits of detection(LOD)are respectively 10.669 pg/mL and 7.394 pg/mL.(2)Detection of chymotrypsin based on thiol-modified graphene oxide(GO)and gold nanoparticles(Au NPs)combined with peptide sequence.GO-SH is synthesized by thiolation to synthesize GO-NH2 nanomaterials.After Au NPs are added,the later two are covalently combined to form an Au-S bond.Then adding the peptide sequence,sulfhydryl at one side of peptide binds to Au NPs,absorbance changes.After adding chymotrypsin,the peptide is cleaved and separated from the GO surface,and the absorbance changed.The limit of detection is 1.428 pg/mL.After adding a functional peptide optimization experiment,the peptide can resist the non-specific adsorption of charged proteins on the interface of biosensor,which improves the detection sensitivity and reduces the detection limit to 0.254 pg/mL.(3)Detection of chymotrypsin based on the peptide sequence labeled with gold nanoclusters(Au NCs)and graphene oxide modified with NHS.One side of peptide sulfhydryl combines self-made Au NCs luminescent material by the method of covalently bound to make the peptide fluorescent.NHS-PEG4-DBCO and GO-N3 undergo click reaction to make GO-NHS material,which can be used for fluorescence quenching.After mixing the two,the other side of peptide amino radical group can covalently bind with the surface of GO-NHS,causing fluorescence to be quenched.After adding chymotrypsin,the peptide chain is specifically cleaved.This keep the fluorescence away from the surface of GO-NHS,the fluorescence is restored,and the limit of detection is 2.014 pg/mL.(4)Detection of chymotrypsin based on peptide and composite nanomaterials(PCN@GO@Au NPs and PCN@Au NPs)labeled with copper cluster nanoparticles(Cu NCs).The peptide contains functional peptide and chymotrypsin-specific cleavage peptide.The amino at one side of peptide is covalently bound to the surface of the selfmade Cu NCs through NHS/EDC.The other side of peptide sulfhydryl can covalently bind to the surface of composite nanomaterials,and the fluorescence is quenched by the composite nanomaterials using FRET.After adding chymotrypsin,the specific site of the peptide chain is cleaved.Therefore,the light-emitting end is away from the surface of the composite nanomaterial,and the fluorescence is restored.The detection limit of the sensor using PCN@GO@Au NPs is significantly lower than that of the sensor using PCN@Au NPs,which is reduced from 9.574 pg/mL to 3.913 pg/mL.This method removes non-specific adsorption and provides experimental and theoretical basis for detecting chymotrypsin based on composite nanomaterials.In this study,a few optical biosensors for detecting chymotrypsin are developed to achieve highly sensitive detection of chymotrypsin.