Electrochemical assay of plasmin activity and its kinetic analysis

Ohtsuka, Keiichi; Maekawa, Iwao; Waki, Michinori; Takenaka, Shigeori

doi:10.1016/j.ab.2008.11.006

Cited by 35 publications

(33 citation statements)

References 38 publications

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“…It is noteworthy that the k cat /K m values of legumain and cathepsin B are relatively low, but they have high biological significance respect to cancer diagnosis and treatment. The speed and required enzyme concentration in the electrochemical measurements can be significantly improved when this technique is used to detect other proteases with higher k cat /K m values, such as trypsin at (1.44 ± 0.1) × 10 6 M −1 s −1 , 15 α-thrombin at (1.0 ± 0.2) × 10 6 M −1 s −1 , 15 and plasmin at 6.7 × 10 5 M −1 s −1 16. …”

Section: Resultsmentioning

confidence: 99%

See 1 more Smart Citation

Electrochemical Protease Biosensor Based on Enhanced AC Voltammetry Using Carbon Nanofiber Nanoelectrode Arrays

et al. 2013

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We report an electrochemical method for measuring the activity of proteases using nanoelectrode arrays (NEAs) fabricated with vertically aligned carbon nanofibers (VACNFs). The VACNFs of ~150 nm in diameter and 3 to 5 μm in length were grown on conductive substrates and encapsulated in SiO2 matrix. After polishing and plasma etching, controlled VACNF tips are exposed to form an embedded VACNF NEA. Two types of tetrapeptides specific to cancer-mediated proteases legumain and cathepsin B are covalently attached to the exposed VACNF tip, with a ferrocene (Fc) moiety linked at the distal end. The redox signal of Fc can be measured with AC voltammetry (ACV) at ~1 kHz frequency on VACNF NEAs, showing distinct properties from macroscopic glassy carbon electrodes due to VACNF’s unique interior structure. The enhanced ACV properties enable the kinetic measurements of proteolytic cleavage of the surface-attached tetrapeptides by proteases, further validated with a fluorescence assay. The data can be analyzed with a heterogeneous Michaelis-Menten model, giving “specificity constant” kcat/Km as (4.3 ± 0.8) × 104 M−1s−1 for cathepsin B and (1.13 ± 0.38) × 104 M−1s−1 for legumain. This method could be developed as portable multiplex electronic techniques for rapid cancer diagnosis and treatment monitoring.

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Section: Resultsmentioning

confidence: 99%

“…In previous electrochemical detection of proteases including trypsin, 15 a-thrombin, 15 and plasmin, 16 normal CV measurements on gold electrodes were used. This method was found not applicable on carbon electrode.…”

Section: Resultsmentioning

confidence: 99%

Electrochemical Protease Biosensor Based on Enhanced AC Voltammetry Using Carbon Nanofiber Nanoelectrode Arrays

et al. 2013

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“…After incubation of different amounts of plasmin in FcPS‐immobilized electrode solution a significant decrease in current was noted. The kinetic parameters were found to be 10 times smaller than those of the homogeneous system, which might be documented to decrease in association rate constant of the fixed substrate on the surface and plasmin …”

Section: Non‐biological Applicationsmentioning

confidence: 85%

Miscellaneous applications of ferrocene‐based peptides/amides

Lal

Badshah

Altaf

et al. 2011

Applied Organom Chemis

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This article provides a critical review of the different applications of ferrocene-based peptides/amides in biological as well as in non-biological systems. Ferrocene-based peptides/amides find many applications in different fields such as materials science, medicine, organic synthesis, bio-organometallic and biological chemistry, asymmetric catalysis, nonlinear optics, in polymer science as redox active polymers and dendrimers, in molecular recognition as biosensors and in electrochemistry). Extensive research is being done on ferrocene-based peptides/amides but we will highlight the various applications of ferrocene-based peptides/amides for the period 2006-2010. The main factors that govern the potential biological and nonbiological applications are an electroactive core, a conjugated linker that can act as a chromophore and lower the oxidation potential of the ferrocene part, an amino acid or peptide derivative that can interact with other molecules via hydrogen bonding or any secondary bonding, and symmetric and asymmetric substitution on the ferrocene moiety.

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“…Following this, more electrochemical techniques have been employed to measure proteolysis of Fc-labeled peptides immobilized on macroscopic gold electrodes. The enzymatic properties of proteases including trypsin, thrombin and plasmin have been quantitatively analyzed with the kinetic Fc signal after adding the proteases (Adjemian et al, 2010; Ohtsuka et al, 2009). However, electrochemical detection and quantitative analyses of protease activities in more complicated samples such as whole tissue lysates have not been reported.…”

Section: Introductionmentioning

confidence: 99%

Quantitative electrochemical detection of cathepsin B activity in complex tissue lysates using enhanced AC voltammetry at carbon nanofiber nanoelectrode arrays

Swisher

Prior

Shishido

et al. 2014

Biosensors and Bioelectronics

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The proteolytic activity of a cancer-related enzyme cathepsin B is measured with alternating current voltammetry (ACV) using ferrocene (Fc) labeled tetrapeptides attached to nanoelectrode arrays (NEAs) fabricated with vertically aligned carbon nanofibers (VACNFs). This combination enables the use of high AC frequencies (~1 kHz) with enhanced electrochemical signals. The specific proteolysis of the Fc-peptide by cathepsin B produces decay in the ACV peak current versus the reaction time. The exponential component of the raw data can be extracted and defined as the “extracted proteolytic signal” which allows consistent quantitative analyses using a heterogeneous Michaelis-Menten model. A “specificity constant” kcat/KM = (3.68 ± 0.50) × 104 M−1s−1 for purified cathepsin B was obtained. The detections of cathepsin B activity in different concentrations of whole lysate of human breast tissue, tissue lysate spiked with varied concentrations of cathepsin B, and the tissue lysate after immunoprecipitation showed that there are ~13.4 nM higher cathepsin B concentration in 29.1 μg·mL−1 of whole tissue lysate than the immunoprecipitated sample. The well-defined regular VACNF NEAs by e-beam lithography show a much faster kinetics for cathepsin B proteolysis with kcat/KM = 9.2 × 104 M−1s−1. These results illustrate the potential of this technique as a portable multiplex electronic system for cancer diagnosis by rapid protease profiling of serum or blood samples.

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Electrochemical assay of plasmin activity and its kinetic analysis

Cited by 35 publications

References 38 publications

Electrochemical Protease Biosensor Based on Enhanced AC Voltammetry Using Carbon Nanofiber Nanoelectrode Arrays

Electrochemical Protease Biosensor Based on Enhanced AC Voltammetry Using Carbon Nanofiber Nanoelectrode Arrays

Miscellaneous applications of ferrocene‐based peptides/amides

Quantitative electrochemical detection of cathepsin B activity in complex tissue lysates using enhanced AC voltammetry at carbon nanofiber nanoelectrode arrays

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