Selective assembly and functionalization of miniaturized redox capacitor inside microdevices for microbial toxin and mammalian cell cytotoxicity analyses

Abstract

We report a novel strategy for bridging information transfer between electronics and biological systems within microdevices. This strategy relies on our "electrobiofabrication" toolbox that uses electrode-induced signals to assemble biopolymer films at spatially defined sites and then electrochemically "activates" the films for signal processing capabilities. Compared to conventional electrode surface modification approaches, our signal-guided assembly and activation strategy provides on-demand electrode functionalization, and greatly simplifies microfluidic sensor design and fabrication. Specifically, a chitosan film is selectively localized in a microdevice and is covalently modified with phenolic species. The redox active properties of the phenolic species enable the film to transduce molecular to electronic signals (i.e., "molectronic"). The resulting "molectronic" sensors are shown to facilitate the electrochemical analysis in real time of biomolecules, including small molecules and enzymes, to cell-based measurements such as cytotoxicity. We believe this strategy provides an alternative, simple, and promising avenue for connecting electronics to biological systems within microfluidic platforms, and eventually will enrich our abilities to study biology in a variety of contexts.

Figure 1:

(A) Setup of a standard three-electrode electrochemical cell. The BBRC film is deposited on the working electrode. The system is characterized in a solution of Ru³⁺ and Fc. (B) Cyclic voltammogram (CV) comparing standard gold electrode modified with BBRC film (Cat-Chit) and chitosan film (Chit) using chamber in (A). (C) A miniaturized three-electrode electrochemical system designed and built inside a microdevice that is enclosed in a 3D printed chip holder. (D) Schematics showing the configuration of the molectronic sensor. The three-electrode system is contained within a reaction chamber. Lower panels depict bright field (BF) and fluorescent microscopic images of the functionalized electronics. (E) CV comparing the molectronic biosensor (Cat-Chit) and the control device (Chit) using electrode system in (D). W. E.: working electrode, C. E.: counter electrode, R. E.: reference electrode.

Figure 2:

(A) Schematic illustrating the microbiological analysis using BBRC films made possible by amplifying electrochemical signals in the bacterial secretome. (B) Reductive redox-cycling between PYO^O/PYO^R and the BBRC film. (C) Oxidative redox-recycling between Fc⁺/Fc and the BBRC film. (D) Thermodynamics of electron transfer. (E) Cyclic voltammogram (CV) of PYO^O in PB with concentrations from 0 μM to 20 μM. (F) Calibration curve of PYO measurement. (G) On-chip PYO measurements within LB and conditioned media (CM) from E. coli, S. Typhimurium and P. aeruginosa overnight cultures (**, p < 0.01). Results are compared between the molectronic sensor (Cat-Chit) and the control device (Chit). Q: quinone; QH₂: catechol

Figure 3:

(A) Schematics showing that the membrane disruption of mammalian cells releases cytosolic lactate dehydrogenase (LDH) into surrounding environment. The released LDH can catalyze the added substrates (lactate & NAD⁺) and in turn charge the molectronic sensor in an analogous manner to battery. The final charge of the molectronic sensor can be measured to reflect the health state of tested cell culture. (B) Reaction catalyzed by LDH. Reductive redox-cycling between NAD⁺/NADH and the BBRC film. (C) Oxidative redox-recycling between Fc⁺/Fc and the BBRC film. (D) Thermodynamics of electron transfer. (E) Cyclic voltammogram (CV) of FC oxidation at the surface of electrodes modified with fully discharged (Q) and fully charged (QH₂) BBRC films. (F) Chronocoulometry (CC) results comparing the molectronic sensor (Cat-Chit) and the control device (Chit). (G) Calibration curve of the molectronic sensor on LDH activity measurement. Q: quinone; QH₂: catechol

Figure 4:

(A) Schematics demonstrating cytotoxicity assay steps using the molectronic sensor. (B) Microscopic results of Caco-2 cell cultures treated with various concentration of Triton X-100. Green and red fluorescent images depict live and dead cells after 120 min, respectively. Scale bar = 50 μm. BF: bright field. (C) Cytotoxicity measurements by the molectronic sensor and the commercialized cytotoxicity kit (*, p < 0.05). All results are normalized to data from the positive control, in which cell cultures are treated with 0.1% Triton X-100.

Scheme 1:

(A) Electrodeposition of chitosan film on a gold electrode. Dissolved chitosan responds to the pH change induced by a cathode initiating the formation of a three-dimensional hydrogel film on its surface. (B) Catechol grafting to the chitosan film is enabled by an anodic voltage. (C) A catechol molecule conjugated to the nucleophilic amino group of the chitosan can undergo oxidation and reduction reactions in the assembled bio-based redox capacitor (BBRC). (D) Reductive redox-cycling between biological reductants and the BBRC film. (E) Oxidative redox-recycling between Fc⁺/Fc and the BBRC film. (F) Thermodynamics of electron transfer. Q: quinone; QH₂: catechol.