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Layer-by-layer composite films of carbon nanotubes (CNTs) within a chitosan matrix with sarcosine oxidase enzyme and capped with Nafion have been developed and optimized as a versatile 1st generation amperometric sarcosine biosensing platform that operates successfully both as an isolated sarcosine sensor as well as a functional component within a creatinine sensor. Accurate measurement of sarcosine in urine and creatinine in blood may help with early diagnosis of diseases such as prostate cancer and renal failure, respectively. In this study, each material within the film is systematically optimized toward sarcosine sensitivity, including a critical evaluation of different CNTs effect on sensing performance. Films featuring carboxylic acid–modified single–walled carbon nanotubes and strategic enzyme doping were shown to be most effective sarcosine sensing platforms, exhibiting excellent sensitivity (~0.5 μA/mM), a linear response (≤0.75 mM), fast response time (8 s), low limits of detection (~6 μM), as well as both continuous use stability (7 days) and effective shelf life (>12 days). Operation of the sarcosine sensor was demonstrated in a sarcosine-spiked urine matrix, detecting the analyte at physiologically relevant concentrations (≥20 μM) and successfully quantifying sarcosine-spiked urine samples at 20, 40, and 90 μM (n = 9, 7, and 3) with high average percent recoveries (>98%) and low relative error. The sarcosine sensing platform was also adapted to a 1st generation creatinine biosensing scheme in which the sarcosine enzymatic reaction is critical to a tri-enzymatic cascade event. The creatinine sensor yielded sensitivity of ~0.6 μA/mM, similar sensing performance parameters to the sarcosine sensor, and was effectively operated in blood serum at physiologically relevant creatinine concentrations (>1 mM). The demonstrated functionality of these sensors in their respective biological fluids at physiological concentrations of the analyte species suggests potential clinical application as diagnostic tools.

A layer-by-layer (LbL) strategy of modifying an electrode with a specific combination of polymeric and xerogel materials is used to create an effective first generation biosensor for uric acid (UA) - a clinically relevant molecule implemented in pregnancy induced hypertension (PIH) diagnosis, a condition that can lead to a serious disorder called preeclampsia. In addition to offering a new, promising sensor for UA, this study represents significant progress for amperometric biosensor development since the strategy and materials employed were successfully and readily adapted from a glucose biosensor model scheme and used for the effective detection of UA, a fundamentally different molecule compared to glucose. Specifically, each of four, functional modifying layers (outer polyurethane (PU) selective membrane, the inner selective electropolymer, and the xerogel bi-layer) are systematically investigated and tailored for UA permeability and interferent discrimination. The role of PU hydrophobicity and its UA permeability are established while the enzyme-doped and outer diffusional xerogel layers are evaluated for uricase (UOx) species/loading and silane precursor dependence, respectively. LbL systematic evaluation reveals the specific combination of hydroxyl-methyl triethoxy silane (HMTES) xerogels, a polyluminol-aniline electropolymer, and 100% hydrophilic polyurethane yields impressive uric acid sensing performance: effective sensitivity (0.8 nA/μM), linear response across physiologically relevant UA concentrations (100–700 μM), fast response times (~ 10 s), low limits of detection (

Amperometric uric acid (UA) biosensing schemes incorporating networks of alkanethiolate-protected gold nanoparticles, monolayer protected clusters (MPCs), and platinum black (Pt-B) electrode modification through the layer-by-layer construction of xerogels are investigated. MPC doping and Pt-B augmentation are implemented within hydroxymethyltriethoxysilane xerogel bilayers at platinum electrodes. The first xerogel adlayer is doped with an MPC network and houses uricase for the enzymatic reaction required for first-generation schemes. Polyluminol–aniline and polyurethane are used as selective/stabilizing interfacial layers. The sensing performance with and without Pt-B and/or MPC doping is assessed by amperometry with standardized UA injections. The use of each individual material results in an enhancement of UA sensitivity compared with analogous films without these materials. The use of Pt-B and MPC doping in concert results in a biosensor design with the highest observed UA sensitivity (0.97 μA mm−1) and fast, linear responses over physiologically relevant UA concentrations. Enhancement is attributed to Pt-B providing increased electrode surface area and integration into the xerogel for greater electronic coupling of the MPC network and more efficient reporting of H2O2 oxidation. The findings have implications for advancing clinical in vivo sensing devices that require scalability or additional biocompatibility layering, both of which would benefit from signal enhancement strategies.

Modified electrodes featuring specific adsorption platforms able to access the electrochemistry of the copper containing enzyme galactose oxidase (GaOx) were explored, including interfaces featuring nanomaterials such as nanoparticles and carbon nanotubes (CNTs). Electrodes modified with various self-assembled monolayers (SAMs) including those with attached nanoparticles or amide-coupled functionalized CNTs were examined for their ability to effectively immobilize GaOx and study the redox activity related to its copper core. While stable GaOx electrochemistry has been notoriously difficult to achieve at modified electrodes, strategically designed functionalized CNT-based interfaces, cysteamine SAM-modified electrode subsequently amide-coupled to carboxylic acid functionalized single wall CNTs, were significantly more effective with high GaOx surface adsorption along with well-defined, more reversible, stable (≥ 8 days) voltammetry and an average ET rate constant of 0.74 s-1 in spite of increased ET distance - a result attributed to effective electronic coupling at the GaOx active site. Both amperometric and fluorescence assay results suggest embedded GaOx remains active. Fundamental ET properties of GaOx may be relevant to biosensor development targeting galactosemia while the use functionalized CNT platforms for adsorption/electrochemistry of electroactive enzymes/proteins may present an approach for fundamental protein electrochemistry and their future use in both direct and indirect biosensor schemes.