Peptide probe for rapid and specific detection of amyloid aggregation

Abstract

A method for use of a peptide probe that generates fluorescence signals rapidly upon recognition of various Aβ aggregates without significant perturbation of samples. The present peptide probes display an increase in fluorescence signals upon coincubation with Aβ oligomers, but neither monomeric/dimeric species nor fibrils. The detection can occur within an hour or two without any additional sample preparation and incubation steps.

1 . A method for the rapid and specific detection of amyloid (Aβ) aggregation comprising:
 a) producing a peptide probe by lyophilizing a peptide, solubilizing the peptide with hexafluoroisopropanol, lyophilizing the peptide; solubilizing the peptide with dimethyl sulfoxide, and then diluting the peptide in dimethyl sulfoxide; and 
 b) measuring fluorescence signals generated by the peptide probe, 
 whereby, a presence of fluorescence signals indicates the detection of Aβ aggregates. 
 
     
     
         2 . The method as claimed in  claim 1 , wherein the peptide probe generates fluorescence signals rapidly upon recognition of various Aβ aggregates without significant perturbation of samples. 
     
     
         3 . The method as claimed in  claim 1 , wherein the peptide probe displays an increase in fluorescence signals upon coincubation with Aβ oligomers, but not with monomeric/dimeric species or fibrils. 
     
     
         4 . The method as claimed in  claim 1 , wherein the detection of Aβ aggregates occurs within two hours without any additional sample preparation and incubation steps. 
     
     
         5 . The method as claimed in  claim 1 , wherein the peptide probe generates different levels of fluorescence signals upon recognition of distinct Aβ assemblies through its conformational change. 
     
     
         6 . The method as claimed in  claim 5 , wherein the peptide probe contains the N-terminus, HCD and the C-terminus of Aβ, and the signal domain, which replaces the linker region of Aβ. 
     
     
         7 . The method as claimed in  claim 6 , wherein the signal domain is responsible for conformation-dependent fluorescence of a nontoxic, membrane-permeable biarsenical dye, FlAsH. 
     
     
         8 . The method as claimed in  claim 7 , wherein the peptide probe displays an increase in FlAsH fluorescence intensity when mixed with Aβ oligomers, but not when mixed with monomers/dimers or fibrils. 
     
     
         9 . The method as claimed in  claim 1 , wherein the peptide probe modulates the fluorescence signals through association with Aβ species. 
     
     
         10 . The method as claimed in  claim 9 , wherein the Aβ species are oligomers. 
     
     
         11 . The method as claimed in  claim 10 , wherein the peptide probe contains the N-terminus (D1-K16), HCD (L17-A21) and the C-terminus (I31-V40) of Aβ, and the signal domain. 
     
     
         12 . The method as claimed in  claim 1 , wherein the peptide is PG46. 
     
     
         13 . The method as claimed in  claim 1 , wherein the peptide is PG38. 
     
     
         14 . A method for the rapid and specific detection of amyloid (Aβ) aggregation comprising the steps of:
 a) producing a peptide probe by:
 i) lyophilizing a peptide; 
 ii) solubilizing the lyophilized peptide of step a) with hexafluoroisopropanol at 1 mg peptide/2 ml HFIP for 3 hr; 
 iii) aliquoting the solubilized peptide of step b) into 20 vials of 0.05 mg peptide each; 
 iv) lyophilizing the solubilized peptide of step c) in hexafluoroisopropanol; 
 v) solubilizing the lyophilized peptide of step d) with dimethyl sulfoxide containing 10 mM 2-mercaptoethanol at 5 mg peptide/1 ml dimethyl sulfoxide (>>1 mM PG46) for 1 hr; and then 
 vi) diluting the peptide of step e) in dimethyl sulfoxide by 100-fold into aqueous buffers containing Aβ; and 
 
 b) measuring fluorescence signals generated by the peptide probe, 
 whereby, a presence of fluorescence signals indicates the detection of Aβ aggregates. 
 
     
     
         15 . The method as claimed in  claim 14 , wherein the peptide is PG46. 
     
     
         16 . The method as claimed in  claim 14 , wherein the peptide is PG38.