Enzyme Engineering to Produce SWCNT Nanosensors as a Generalizable Platform for Biochemical Sensing

Single-walled carbon nanotubes (SWCNT) stand out as attractive candidates for near-infrared (nIR) fluorescent imaging and chemical sensing. However, to render SWCNT chemically-selective, rational design of the surface corona is required to achieve sufficient sensitivity and selectivity to the target analyte. Recently, we reported on a facile sonication-based generation of glucose oxidase (GOx)-SWCNT conjugate for glucose detection [1]. Notably, we found that the analyte response mechanism does not involve catalytic oxidation of glucose, which motivated us to develop nanosensors using catalytically inactive GOx for enhanced reversibility and biocompatibility. In this study, we study the generalizability of our approach in producing SWCNT-based nanosensors based on 1- facile direct probe tip sonication, and 2- with engineered enzymes, showing the potential applicability of the sonication-based approach as a generalizable strategy for generating rationally designed nanosensors capable of detecting and imaging various analytically-relevant biomolecules.<br/><br/>In this work, to test the generalizability of generating enzyme-SWCNT nanosensors, we generated conjugates by facile direct sonication of SWCNT with following enzymes: GOx, choline oxidase (ChOx), horseradish peroxidase (HRP), acetylcholineesterase, tyrosinase, cholesterol oxidase (CholOx), lactate oxidase, alcohol oxidase, xanthine oxidase, and galactose oxidase. Remarkably, six out of nine enzymes yielded stable suspensions of enzyme-SWCNT conjugates following probe-tip sonication, all of which successfully worked as nanosensors to detect their corresponding analytes. These nanosensors, in particular HRP-SWCNT and CholOx-SWCNT, exhibited excellent responses to their targets, with a maximum ΔF/F0 of up to 300% and 100%, respectively. H₂O₂ is known to quench SWCNT fluorescence, and H₂O₂ nanosensors previously developed with SWCNT exhibited a negative fluorescence modulation as the sensor output with some challenges towards nanosensor selectivity. We highlight that our approach enables HRP-SWCNT nanosensor generation, which provides a strong Δ<i>F</i>/<i>F</i>₀ = 300% and instantaneous turn-on response towards H₂O₂ within 2 s. Next, we sought to show that enzyme inactivation can produce SWCNT-based nanosensors that detect analytes without analyte consumption. To do so, we use ChOx as a model system to show that recombinant enzymes can be used as a generalizable approach to generating catalytically-inactive nanosensors. As expected, nanosensors prepared with mutant-ChOx exhibited responses comparable to those prepared with native ChOx, but without consuming the analyte or producing toxic byproducts such as H₂O₂. Thus, sonication-based physisorption of engineered enzymes to SWCNTs holds the potential to facilitate rapid nanosensor generation capable of detecting various biologically relevant molecules with reversibility and biocompatibility, thereby motivating their use in in vivo applications.<br/><br/>Nishitani, S., Tran, T., Puglise, A., Yang, S. & Landry, M. P. Engineered Glucose Oxidase-Carbon Nanotube Conjugates for Tissue-Translatable Glucose Nanosensors. <i>Angew. Chem. Int. Ed Engl.</i> e202311476 (2024)