Simultaneous, multiplex quantification of protease activities using a gold microelectrode array

Anderson, Morgan J.; Song, Yang; Fan, Huafang; Wright, Jestin Gage; Ren, Zhaoyang; Hua, Duy H.; Koehne, Jessica E.; Meyyappan, M.; Li, Jun

doi:10.1016/j.bios.2020.112330

Cited by 10 publications

(21 citation statements)

References 37 publications

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“…All electrochemical protease detection measurements in this study used an in-house synthesized hexapeptide attached with a ferrocene (Fc) redox tag, i.e., H 2 N-(CH 2 ) 4 -CO-Pro-Leu-Ala-Phe-Val-Ala-NH-CH 2 -Fc, as the substrate (referred to as “peptide-Fc” in this study). This peptide substrate was selected from about 30 synthesized peptides (named H-15 in our previous study) and has shown the highest proteolytic activity by cathepsin B. It was specifically cleaved by cathepsin B between Leu and Ala residues as determined by HPLC-MS .…”

Section: Methodsmentioning

confidence: 99%

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Quantitative Detection of Cathepsin B Activity in Neutral pH Buffers Using Gold Microelectrode Arrays: Toward Direct Multiplex Analyses of Extracellular Proteases in Human Serum

et al. 2021

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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.

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Section: Methodsmentioning

confidence: 99%

“…Recently, we have fabricated a 3 × 3 gold MEA and demonstrated its capability for multiplexed detection of cathepsin B activity using three specific peptide substrates functionalized on separate microelectrodes . Highly consistent proteolysis results have been obtained over nine independent channels simultaneously.…”

mentioning

confidence: 99%

Quantitative Detection of Cathepsin B Activity in Neutral pH Buffers Using Gold Microelectrode Arrays: Toward Direct Multiplex Analyses of Extracellular Proteases in Human Serum

et al. 2021

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“…In fact, mechanical probe stations, beside complexity, cost, and large dimensions, generally have very few test points [14]. Macroscopic connectors (e.g., spring-loaded pins) typically require contact pads with lateral dimensions in the millimeter range [9,[15][16][17][18][19][20], thus limiting the number of pads, and can easily damage delicate devices (e.g., thin metals deposited on elastic polymers such as PDMS [3,15,16,21]). The number of wires and pads can be reduced by sharing the ground terminal or by simplifying structures used for characterizing mismatch [22], but these approaches still require interconnects, which are invasive (i.e., conductors connecting to the materials under test must be added), increase area and opacity (in case of transparent substrates and opaque conductors), can prevent full characterization of the materials under test (interconnects are not measurable), waste materials (in case of additive manufacturing) with related issues (environmental, economic), and require more complex or time-consuming fabrication (in case of serial manufacturing).…”

Section: Introductionmentioning

confidence: 99%

Twin-Wire Networks for Zero Interconnect, High-Density 4-Wire Electrical Characterizations of Materials

et al. 2022

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Four-wire measurements have been introduced by Lord Kelvin in 1861 and have since become the standard technique for characterizing small resistances and impedances. However, high-density 4-wire measurements are generally complex, time-consuming, and inefficient because of constraints on interconnects, pads, external wires, and mechanical contacts, thus reducing reproducibility, statistical significance, and throughput. Here, we introduce, systematically design, analyze, and experimentally validate zero interconnect networks interfaced to external instrumentation by couples of twin wire. 3D-printed holders with magnets, interconnects, nonadhesive layers, and spacers can effortlessly establish excellent electrical connections with tunable or minimum contact forces and enable accurate measurements even for delicate devices, such as thin metals on soft polymers. As an example, we measured all the resistances of a twin-wire 29-resistor network made of silver-nanoparticle ink printed on polyimide, paper, or photo paper, including during sintering or temperature calibration, resulting in an unprecedentedly easy and accurate characterization of both resistivity and its temperature coefficient. The theoretical framework and experimental strategies reported here represent a breakthrough toward zero interconnect, simple, and efficient high-density 4-wire characterizations, can be generalized to other 4-wire measurements (impedances, sensors) and can open the way to more statistically meaningful and reproducible analyses of materials, high-throughput measurements, and minimally invasive characterizations of biomaterials.

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“…As a lysosomal cysteine protease, cathepsin B (CTSB) plays a significant role in a large number of biological events in cancer cells including initiation, growth, angiogenesis, invasion, and metastasis. − It is worth mentioning that the abnormal level of CTSB is associated with cancer invasion and progression, making it an attractive biomarker in the diagnosis of several cancers such as glioblastoma, laryngeal, oral, esophageal, gastric, colorectal, prostate, pancreas, and breast cancers. − Therefore, accurate and sensitive screening of CTSB levels is of great significance for performing efficient therapy to overcome cancer at the early stage. To date, a myriad of quantitative methods have been reported that focus on the monitoring of CTSB activity, including fluorimetry, − colorimetry, electrochemistry, , bioluminescence, chemiluminescence, and so forth.…”

Section: Introductionmentioning

confidence: 99%

Nucleolin-Targeted Ratiometric Fluorescent Carbon Dots with a Remarkably Large Emission Wavelength Shift for Precise Imaging of Cathepsin B in Living Cancer Cells

Shen

Wang

et al. 2021

Anal. Chem.

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As one of the most promising biomarkers for numerous malignant tumors, accurate and reliable reporting of Cathepsin B (CTSB) activity is of great significance to achieve efficient diagnosis of cancers at an early stage and predicting metastasis. Here, we report a vigorous ratiometric fluorescent method integrating a cancer-targeting recognition moiety with a remarkably large emission wavelength shift into a single matrix to report CTSB activity sensitively and specifically. As a proof of concept, we synthesized amine-rich carbon quantum dots (CQDs) with a blue fluorescence, which offered an efficient scaffolding to covalently assemble the nucleolin-targeting recognition nucleic acid aptamer AS1411 and a CTSB-cleavable peptide substrate Gly-Arg-Arg-Gly-Lys-Gly-Gly-Cys–COOH that tethered with a near-infrared (NIR) fluorophore chlorin e6 (Ce6–GRRGKGGC, Ce6–Pep), enabling a cancer-targeting and CTSB stimulus-responsive ratiometric nanoprobe AS1411–Ce6–CQDs. Owing to the efficient fluorescence resonance energy transfer (FRET) process from the CQDs to Ce6 inside the assembly of nanoprobe, the blue fluorescence of CQDs at ∼450 nm was remarkably quenched, along with an obvious NIR fluorescence enhancement of Ce6 at ∼650 nm. After selective entry into cancer cells via nucleolin-mediated endocytosis, the overexpressed CTSB in lysosome could cleave Ce6–Pep and trigger the Ce6 moiety dissociation from AS1411–Ce6–CQDs, thus leading to the termination of FRET process, achieving the efficient ratiometric fluorescence response toward endogenous CTSB with a remarkably large emission wavelength shift of ∼200 nm from NIR to blue emission region. Notably, the nanoprobe AS1411–Ce6–CQDs exhibited an excellent specificity for ratiometric fluorescent sensing of CTSB activity with an ultralow detection limit of 0.096 ng/mL, demonstrating its promising use for early precise cancer diagnosis in the near future.

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Simultaneous, multiplex quantification of protease activities using a gold microelectrode array

Cited by 10 publications

References 37 publications

Quantitative Detection of Cathepsin B Activity in Neutral pH Buffers Using Gold Microelectrode Arrays: Toward Direct Multiplex Analyses of Extracellular Proteases in Human Serum

Quantitative Detection of Cathepsin B Activity in Neutral pH Buffers Using Gold Microelectrode Arrays: Toward Direct Multiplex Analyses of Extracellular Proteases in Human Serum

Twin-Wire Networks for Zero Interconnect, High-Density 4-Wire Electrical Characterizations of Materials

Nucleolin-Targeted Ratiometric Fluorescent Carbon Dots with a Remarkably Large Emission Wavelength Shift for Precise Imaging of Cathepsin B in Living Cancer Cells

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