Electrochemical Disposable Biosensor to Monitor Dabigatran in Point-of-Care Anticoagulation Therapy

Figures

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  Scheme 1. Photo of the disposable sensor (left) together with a schematic representation of the enzymatic reaction followed by an electrochemical signal amplification inside the disposable co-facing gold electrode.

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  Figure 1. Cyclic voltammograms of 1.0 mM Tos-Gly-Pro-Arg-ACP in 20 mM Tris buffer (pH 8.8) (a) in the absence and (b–f) presence of 0.10 µM trypsin obtained with the use of the disposable co-facing gold electrodes. Voltammograms (b–f) were obtained within 4 min after loading the mixture onto the disposable electrode. Scan rate: 0.05 V s−1; T: 37 ± 0.2 °C.

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  Figure 2. (A–D) Example of chronoamperograms obtained after background subtraction (A) in buffer solutions of pH (a) 6, (b) 7, (c) 8, (d) 9, (e) 10, and (f) 11, containing 0.50 mM Tos-Gly-Pro-Arg-ACP, 20 mM [Fe(EDTA)]−1, and 0.10 µM trypsin; (B) in 20 mM Tris (50 mM NaCl, 1.0 mM CaCl2) buffer (pH 8.8) solution containing 0.10 µM trypsin, 20 mM [Fe(EDTA)]−1, and (a) 0.05, (b) 0.10, (c) 0.20, (d) 0.30, (e) 0.40, and (f) 0.50 mM Tos-Gly-Pro-Arg-ACP. (C) in buffer solutions of pH (a) 6, (b) 7, (c) 8, (d) 9, (e) 10, containing 0.50 mM Tos-Gly-Pro-Arg-ACP, 20 mM [Fe(EDTA)]−1, and 1.0 µM thrombin; (D) in 20 mM Tris (50 mM NaCl, 1.0 mM CaCl2) buffer (pH 7.4) solution containing 1.0 µM thrombin, 20 mM [Fe(EDTA)]−1, and (a) 0.05, (b) 0.10, (c) 0.20, (d) 0.30, (e) 0.40, and (f) 0.50 mM Tos-Gly-Pro-Arg-ACP. (E,F) Variation of the mean current extracted from chronoamperograms at 60 s (error bar = ±1 SD; n = 3) as a function of (E) pH and (F) the substrate (Tos-Gly-Pro-Arg-ACP, [S]) concentration for (a) trypsin and (b) thrombin using the disposable co-facing gold electrodes and the conditions mentioned in (A–D). Eapp = 0.4 V; T: 37 ± 0.2 °C.

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  Figure 3. (A) Chronoamperograms after background subtraction of ten-fold diluted plasma in 20 mM Tris buffer (pH 7.4) solution containing 0.30 mM Tos-Gly-Pro-Arg-ACP, 20 mM [Fe(EDTA)]−1, 0.22 µM thrombin and (a) 0.0, (b) 15, (c) 30, and (d) 140 ng mL−1 DAB. (B) First 16 s of CA shown in A and the respective simulations (◯) using Equation (1). Simulation parameters: A = 0.11 cm2, DACP = 1.16 × 10−5 cm2 s−1, z = 2, L = 95 µm. (C) Correlation between the change in the reaction rate constant (obtained from (B)) and the natural logarithm of the inhibitor concentration. (D) Correlation between the change in current magnitude extracted from the CAs at 60 s and the natural logarithm of DAB concentration. Error bars = ±1 SD; T = 22 ± 1 °C.

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  Figure 4. (A) Chronoamperograms obtained with the trypsin sensor to demonstrate the activity of this enzyme in different media: (a) Tris buffer (pH 8.8) solution; (b) plasma solution containing 0.1 µM trypsin, 0.07 mM Tos-Gly-Pro-Arg-ACP, and 20 mM [Fe(EDTA)]−1; (c) CA of the filtered plasma containing 0.10 µM trypsin, 0.07 mM Tos-Gly-Pro-Arg-ACP, and 20 mM [Fe(EDTA)]−1; and (d) CA obtained in a Tris buffer (pH 8.8) solution containing 0.10 µM trypsin, 0.07 mM Tos-Gly-Pro-Arg-ACP, and 20 mM [Fe(EDTA)]−1. Eapp = 0.4 V; T: 37 ± 0.2 °C. (B) CA after background subtraction for filtered plasma samples containing 0.07 mM Tos-Gly-Pro-Arg-ACP, 20 mM [Fe(EDTA)]−1, 0.10 µM trypsin and (a) 0, (b) 70, (c) 280, and (d) 400 ng mL−1 DAB. (C,D) Correlation between the change in current magnitude (C) and the change in the reaction rate constant (D) extracted from the CAs presented in (B).