There is a strong demand for bioanalytical techniques to rapidly detect protease activities with high sensitivity and high specificity. This study reports an activity-based electrochemical method toward this goal. Nanoelectrode arrays (NEAs) fabricated with embedded vertically aligned carbon nanofibers (VACNFs) are functionalized with specific peptide substrates containing a ferrocene (Fc) tag. The kinetic proteolysis curves are measured with continuously repeated ac voltammetry, from which the catalytic activity is derived as the inverse of the exponential decay time constant based on a heterogeneous Michaelis–Menten model. Comparison of three peptide substrates with different lengths reveals that the hexapeptide H2N─(CH2)4─CO─Pro-Leu-Arg-Phe-Gly-Ala─NH─CH2─Fc is the optimal probe for cathepsin B. The activity strongly depends on temperature and is the highest around the body temperature. With the optimized peptide substrate and measuring conditions, the limit of detection of cathepsin B activity and concentration can reach 2.49 × 10−4 s−1 and 0.32 nM, respectively. The peptide substrates show high specificity to the cognate proteases, with negligible cross-reactions among three cancer-related proteases cathepsin B, ADAM10, and ADAM17. This electrochemical method can be developed into multiplex chips for rapid profiling of protease activities in cancer diagnosis and treatment monitoring.

No abstract

Proteases are critical signaling molecules and prognostic biomarkers for many diseases including cancer. There is a strong demand for multiplex bioanalytical techniques that can rapidly detect the activity of extracellular proteases with high sensitivity and specificity. This study demonstrates an activitybased electrochemical biosensor of a 3 × 3 gold microelectrode array for the detection of cathepsin B activity in human serum diluted in a neutral buffer. Proteolysis of ferrocene-labeled peptide substrates functionalized on 200 × 200 μm microelectrodes is measured simultaneously over the nine channels by AC voltammetry. The protease activity is represented by the inverse of the exponential decay time constant (1/τ), which equals to (k cat /K M )[CB] based on the Michaelis−Menten model. An enhanced activity of the recombinant human cathepsin B (rhCB) is observed in a low-ionic-strength phosphate buffer at pH = 7.4, giving a very low limit of detection of 8.49 × 10 −4 s −1 for activity and 57.1 pM for the active rhCB concentration that is comparable to affinity-based enzyme-linked immunosorbent assay (ELISA). The cathepsin B presented in the human serum sample is validated by ELISA, which mainly detects the inactive proenzyme, while the electrochemical biosensor specifically measures the active cathepsin B and shows significantly higher decay rates when rhCB and human serum are activated. Analyses of the kinetic electrochemical measurements with spiked active cathepsin B in human serum provide further assessment of the protease activity in the complex sample. This study lays the foundation to develop the gold microelectrode array into a multiplex biosensor for rapid detection of the activity of extracellular proteases toward cancer diagnosis and treatment assessment.

Electrolysis research is typically conducted using commercially available cells with standard designs that are difficult to modify or custom-machined parts that are time-consuming to produce. Herein, we describe a method to rapidly produce lab-scale electrolysis cells that uses high-resolution, high-fidelity stereolithography 3D printing combined with electroless metal plating. Cells prepared by the printing/electroless plating method were compared to the same cells machined in Ti for CO electrolysis experiments. The printed cells showed very similar performance to the machined cells across a wide current density range (up to 250 mA cm-2) and demonstrated 24 h of stable operation in a zero-gap configuration. This work demonstrates the ability to prototype electrochemical cell designs in a fraction of the time required using conventional machining.

Apply It! This article explores the frequency, intensity, time, and type framework within the context of HIIT, the unique elements of HIIT (e.g., intensity and work-to-recovery ratio), and concludes with examples of HIIT exercise regimens. By reading this article, health and fitness professionals will be reminded of the following takeaways: • HTN develops gradually and is generally the product of lifestyle choices concerning diet and exercise. • Although HTN is routinely treated with pharmacological intervention, lifestyle intervention is a primary therapeutic option for those newly diagnosed with hypertension. • Research supports the implementation of aerobically based HIIT and MICT for inducing similar reductions in systolic blood pressure and diastolic blood pressure in adults with pre-HTN and/or HTN. • HIIT for any client must be introduced gradually — and deliberately — over time. The introduction of HIIT should start with a single, brief set of HIIT (e.g., a few minutes of HIIT) to evaluate the client’s readiness and receptivity to the approach.