title: A highly sensitive and simply operated protease sensor toward point-of-care testing.
abstract: Protease sensors for point-of-care testing (POCT) require simple operation, a detection period of less than 20 minutes, and a detection limit of less than 1 ng mL(-1). However, it is difficult to meet these requirements with protease sensors that are based on proteolytic cleavage. This paper reports a highly reproducible protease sensor that allows the sensitive and simple electrochemical detection of the botulinum neurotoxin type E light chain (BoNT/E-LC), which is obtained using (i) low nonspecific adsorption, (ii) high signal-to-background ratio, and (iii) one-step solution treatment. The BoNT/E-LC detection is based on two-step proteolytic cleavage using BoNT/E-LC (endopeptidase) and l-leucine-aminopeptidase (LAP, exopeptidase). Indium-tin oxide (ITO) electrodes are modified partially with reduced graphene oxide (rGO) to increase their electrocatalytic activities. Avidin is then adsorbed on the electrodes to minimize the nonspecific adsorption of proteases. Low nonspecific adsorption allows a highly reproducible sensor response. Electrochemical-chemical (EC) redox cycling involving p-aminophenol (AP) and dithiothreitol (DTT) is performed to obtain a high signal-to-background ratio. After adding a C-terminally AP-labeled oligopeptide, DTT, and LAP simultaneously to a sample solution, no further treatment of the solution is necessary during detection. The detection limits of BoNT/E-LC in phosphate-buffered saline are 0.1 ng mL(-1) for an incubation period of 15 min and 5 fg mL(-1) for an incubation period of 4 h. The detection limit in commercial bottled water is 1 ng mL(-1) for an incubation period of 15 min. The developed sensor is selective to BoNT/E-LC among the four types of BoNTs tested. These results indicate that the protease sensor meets the requirements for POCT.
authors.affiliation: Department of Chemistry and Chemistry Institute for Functional Materials, Pusan National University, Busan 46241, Korea. hyang@pusan.ac.kr.
authors.forename: Seonhwa
authors.initials: S
authors.lastname: Park
authors.forename: Yu Mi
authors.initials: YM
authors.lastname: Shin
authors.forename: Jeongwook
authors.initials: J
authors.lastname: Seo
authors.forename: Ji-Joon
authors.initials: JJ
authors.lastname: Song
authors.forename: Haesik
authors.initials: H
authors.lastname: Yang
mesh_terms: D000327:Adsorption; D000595:Amino Acid Sequence; D000627:Aminophenols / Q000737:chemistry; D015374:Biosensing Techniques / Q000295:instrumentation / Q000379:methods*; D001905:Botulinum Toxins / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism; D004229:Dithiothreitol / Q000737:chemistry; D004563:Electrochemistry; D004566:Electrodes; D010450:Endopeptidases / Q000378:metabolism*; D020689:Exopeptidases / Q000378:metabolism*; D057230:Limit of Detection; D000067716:Point-of-Care Testing; D059748:Proteolysis; D017971:Tin Compounds / Q000737:chemistry
chemical_list: D000627:Aminophenols; D017971:Tin Compounds; C109984:indium tin oxide; D010450:Endopeptidases; D020689:Exopeptidases; D001905:Botulinum Toxins; C026729:4-aminophenol; C096326:botulinum toxin type E; D004229:Dithiothreitol