Trypsin Detection Using Electrochemical Reduction‐based Redox Cycling

Bulletin Korean Chem Soc

et al. 2022

The concentration of human immunoglobulin E (IgE) in blood is an indicator for diagnosing an allergy. For detecting human IgE by an electrochemical immunosensor, redox mediator molecules were modified on a gold electrode using thiolated organic molecules, which can form self-assembled monolayers on the electrode surface. Ferrocene was used as a redox mediator, and the current signal was amplified via redox cycling using 4-aminophenol. Measurements were obtained after the immobilization of the target-captured antibody, target human IgE, biotin-conjugated secondary antibody, and avidin alkaline phosphatase in a sandwich-type immunosensor configuration. The prepared immunosensor exhibited a limit of detection of 1 IU/mL and dynamic range of 3-30 IU/mL. K E Y W O R D S electrocatalytic reaction, electrochemical biosensor, ferrocene, gold (Au) electrode, human immunoglobulin E

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Immunosensor for human IgE detection using electrochemical redox cycling with ferrocene‐mixed self‐assembled monolayers modified Au electrode

Kim

Song

Bulletin Korean Chem Soc

et al. 2022

“…6,35−40 Proteolytic catalytic reactions, especially those of trypsin, can be performed in the presence of additional species involved in redox cycling. 39,40 Prostate-specific antigen (PSA) is an important cancer biomarker that is produced in prostate epithelial cells but is rarely expressed in other tissue. PSA in blood is used as a biomarker for diagnosing and monitoring prostate cancer and its recurrence since a small amount of PSA produced in the prostate is secreted into the bloodstream.…”

Section: ■ Introductionmentioning

confidence: 99%

“…Electrochemical affinity-based biosensors have been widely developed for simple, sensitive point-of-care testing. ,, Highly amplified electrochemical signals are produced in electrochemical biosensors when the rapid generation of a signaling species by a catalytic label is combined with the rapid redox cycling of the signaling species. ,− Many catalytic protease reactions are also compatible with redox cycling because the additional (bio)chemical species used for redox cycling do not influence proteolytic reaction rates. ,− Proteolytic catalytic reactions, especially those of trypsin, can be performed in the presence of additional species involved in redox cycling. , …”

Section: Introductionmentioning

confidence: 99%

Sensitive Affinity-Based Biosensor Using the Autocatalytic Activation of Trypsinogen Mutant by Trypsin with Low Self-activation

Lee

Yang

2022

ACS Appl. Bio Mater.

Self Cite

Self-propagating autocatalytic reactions of proteases that can provide high signal amplification have not been applied to affinity-based biosensors owing to the limited number of fast autocatalytic proteolytic reactions available and the selfactivation of protease proenzymes. Here, we report that a self-propagating autocatalytic reaction based on the autocatalytic activation of the trypsinogen mutant by trypsin facilitates high signal amplification and a low background level, resulting in a low detection limit for prostate-specific antigen (PSA). A commercially available trypsinogen mutant minimizes the self-activation of trypsinogen by trypsinogen. Trypsin, which is used as a catalytic label in a sandwich-type immunosensor, converts the trypsinogen mutant into trypsin; the generated trypsin then further converts the trypsinogen mutant into trypsin. The autocatalytically produced trypsin proteolytically cleaves the peptide bond of a trypsin substrate, resulting in the liberation of electrochemically active 4-aminophenol (AP). The electrochemical oxidation of AP at a modified indium tin oxide (ITO) electrode induces electrochemical−chemical redox cycling involving the ITO electrode, AP, and a reductant. The triple combination of autocatalytic activation, proteolytic cleavage, and redox cycling results in a high electrochemical signal level. The detection limit for PSA obtained using a trypsin label and trypsinogen (∼7 pg/mL) is lower than that obtained using a trypsin label alone (∼100 pg/ mL). This study demonstrated that autocatalytically activating a proenzyme is a very useful method for highly amplifying signals.

“…Proteolytic liberation of an electroactive species from a short peptide, linked with an inactive electrochemical species, enables electrochemical detection of endopeptidases such as trypsin. , However, when the proteolytic cleavage by an endopeptidase requires two consecutive specific amino acids, the cleavage of the peptide bond between two amino acids, linked with a terminal electrochemical species, does not liberate the electrochemical species. This problem can be overcome by using an externally added exopeptidase that cleaves the remaining peptide bond between the single amino acid and the terminal electrochemical species .…”

mentioning

confidence: 99%

Wash-Free Amperometric Escherichia coli Detection via Rapid and Specific Proteolytic Cleavage by Its Outer Membrane OmpT

Cho

et al. 2022

Anal. Chem.

Self Cite

Various methods have been developed for the detection of Escherichia coli (E. coli); however, they are complex and time-consuming. OmpTa cell membrane endopeptidase of E. colistrongly embedded in the outer membrane of only E. coli, exposed to external solutions, with high proteolytic activity, could be a suitable target molecule for the rapid and straightforward detection of E. coli. Herein, a wash-free, sensitive, and selective amperometric method for E. coli detection, based on rapid and specific proteolytic cleavage by OmpT, has been reported. The method involved (i) rapid proteolytic cleavage of consecutive amino acids, after cleavage by OmpT, linked to an electrochemical species (4-aminophenol, AP), by leucine aminopeptidase (LAP, an exopeptidase), (ii) affinity binding of E. coli on an electrode, and (iii) electrochemical-enzymatic (EN) redox cycling. OmpT cleaved the intermediate peptide bond of a peptide substrate containing alaninearginine-arginine-leucine-AP (−A-R-R-L-AP), forming R-L-AP, followed by the cleavage of two peptide bonds of R-L-AP sequentially by LAP, to liberate an electroactive AP. Affinity binding and EN redox cycling, in addition to rapid proteolytic cleavage by OmpT and LAP, enabled high electrochemical signal amplification. Two-sequential-cleavage was employed for the first time in protease-based detection. The calculated detection limit for E. coli cells in tap water (approximately 10 3 CFU/mL after 1 h incubation) was lower than those obtained without affinity binding and EN redox cycling. The detection method was highly selective to E. coli as OmpT is present in only E. coli. High sensitivity, selectivity, and the absence of wash steps make the developed detection method practically promising.