A Potentially Versatile Enzyme Sensor Platform: Enzyme-Loaded, Tagged, Porous Polymeric Nanocapsules.

Enzymes are essential to life and indispensable in a wide range of industries (food, pharmaceutical, medical, biosensing, etc.); however, a significant shortcoming of these fragile biological catalysts is their poor stability. To address this challenge, a variety of immobilization methods have been described to enhance the enzyme's stability. These immobilization methods generally are specific to an individual enzyme or optimal for a particular application. The aim of this study is to explore the utility of porous, indicator moiety-tagged, polymeric nanocapsules (NCs) for the encapsulation of enzymes and measurement of the enzyme's substrate. As a model enzyme, glucose oxidase (GOx) is used. The GOx enzyme-loaded, fluorophore-tagged NCs were synthesized by using self-assembled surfactant vesicle templates. To show that the biological activity of GOx is preserved during entrapment, the rate of the GOx enzyme catalyzed reaction was measured. To evaluate the protective features of the porous NCs, the encapsulated GOx enzyme activity was followed in the presence of hydrolytic enzymes. During the encapsulation of GOx and the purification of the GOx-loaded NCs, the GOx activity decayed less than 10%, and up to 30% of the encapsulated GOx activity could be retained for 3-5 days in the presence of hydrolytic enzymes. In support of the potentially unique advantages of the enzyme-loaded NCs, as a proof-of-concept example, the fluorophore-tagged, GOx-loaded NCs were used for the determination of glucose in the concentration range between 18 and 162 mg/dL and for imaging the distribution of glucose concentration in imaging experiments.

Multilayer and Surface Immobilization of EDOT-Decorated Nanocapsules.

To assess the feasibility of utilizing reagent-loaded, porous polymeric nanocapsules (NCs) for chemical and biochemical sensor design, the surfaces of the NCs were decorated with 3,4-ethylenedioxythiophene (EDOT) moieties. The pores in the capsule wall allow unhindered bidirectional diffusion of molecules smaller than the programmed pore sizes, while larger molecules are either entrapped inside or blocked from entering the interior of the nanocapsules. Here, we investigate two electrochemical deposition methods to covalently attach acrylate-based porous nanocapsules with 3,4-ethylenedioxythiophene moieties on the nanocapsule surface, i.e., EDOT-decorated NCs to the surface of an existing PEDOT film: (1) galvanostatic or bilayer deposition with supporting EDOT in the deposition solution and (2) potentiostatic deposition without supporting EDOT in the deposition solution. The distribution of the covalently attached NCs in the PEDOT films was studied by variable angle FTIR-ATR and XPS depth profiling. The galvanostatic deposition of EDOT-decorated NCs over an existing PEDOT (tetrakis(pentafluorophenyl)borate) [PEDOT(TPFPhB)] film resulted in a bilayer structure, with an interface between the NC-free and NC-loaded layers, that could be traced with variable angle FTIR-ATR measurements. In contrast, the FTIR-ATR and XPS analyses of the films deposited potentiostatically from a solution without EDOT and containing only the EDOT-decorated NCs showed small amounts of NCs in the entire cross section of the films.

Reference Electrodes with Ionic Liquid Salt Bridge: When Will These Innovative Novel Reference Electrodes Gain Broad Acceptance?

In this paper, we raise questions that researchers have to ask if they intend to replace a conventional reference electrode with an ionic liquid-based reference electrode and try to answer these questions based on our experiences and literature data. Among these questions, the most important is which ionic liquid should be used. However, beyond the chemical composition of the ionic liquid, to realize all the potential benefits of ionic-liquid based reference electrodes, there are additional, equally important considerations. Through examples we will show the importance of the (i) purity of the ionic liquid and the consequences of deviations from its stoichiometric salt composition, (ii) form of implementation of the ionic liquid-based reference electrode membrane (free-flowing salt bridge, or ionic liquid embedded in a membrane), (iii) membrane/gelling agent material and its composition, and (iv) experimental conditions (steady state or flowing conditions) under which it will be used. Finally, we recommend methods to test the performance criteria of the ionic liquid-based reference electrodes.

Generation, clearance, toxicity, and monitoring possibilities of unaccounted uremic toxins for improved dialysis prescriptions.

Current dialysis-dosing calculations provide an incomplete assessment of blood purification. They exclude clearances of protein-bound uremic toxins (PB-UTs), such as polyamines, p-cresol sulfate, and indoxyl sulfate, relying solely on the clearance of urea as a surrogate for all molecules accumulating in patients with end-stage renal disease (ESRD). PB-UTs clear differently in dialysis but also during normal renal function. The kidney clears PB toxins via the process of secretion, whereas it clears urea through filtration. Herein, we review the clearance, accumulation, and toxicity of various UTs. We also suggest possible methods for their monitoring toward the ultimate goal of a more comprehensive dialysis prescription. A more inclusive dialysis prescription would retain the kidney-filtration surrogate, urea, and consider at least one PB toxin as a surrogate for UTs cleared through cellular secretion. A more comprehensive assessment of UTs that includes both secretion and filtration is expected to result in a better understanding of ESRD toxicity and consequently, to reduce ESRD mortality.

Medical Sensors for the Diagnosis and Management of Disease: The Physician Perspective.

The objective of this paper is to assist developers of medical sensors to better formulate the clinically relevant design criteria and required performance characteristics of their novel sensor based on an understanding of how these devices will be used by physicians. Sensor technologies play a central role in medicine, and the most critical aspect of the sensor's clinical utility relates to these design decisions. Clinically, sensors are used by health care providers to make both diagnostic and management decisions, and the sensors that aid in these decisions are evaluated by certain clinical, as well as analytical, criteria. Failure to adequately address these end-user requirements can lead to the development of sensors without clinical utility.

Solid-Contact pH Sensor without CO2 Interference with a Superhydrophobic PEDOT-C14 as Solid Contact: The Ultimate "Water Layer" Test.

The aim of this study was to find a conducting polymer-based solid contact (SC) for ion-selective electrodes (ISEs) that could become the ultimate, generally applicable SC, which in combination with all kinds of ion-selective membranes (ISMs) would match the performance characteristics of conventional ISEs. We present data collected with electrodes utilizing PEDOT-C14, a highly hydrophobic derivative of poly(3,4-ethylenedioxythiophene), PEDOT, as SC and compare its performance characteristics with PEDOT-based SC ISEs. PEDOT-C14 has not been used in SC ISEs previously. The PEDOT-C14-based solid contact (SC) ion-selective electrodes (ISEs) (H+, K+, and Na+) have outstanding performance characteristics (theoretical response slope, short equilibration time, excellent potential stability, etc.). Most importantly, PEDOT-C14-based SC pH sensors have no CO2 interference, an essential pH sensors property when aimed for whole-blood analysis. The superhydrophobic properties (water contact angle: 136 ± 5°) of the PEDOT-C14 SC prevent the detachment of the ion-selective membrane (ISM) from its SC and the accumulation of an aqueous film between the ISM and the SC. The accumulation of an aqueous film between the ISM and its SC has a detrimental effect on the sensor performance. Although there is a test for the presence of an undesirable water layer, if the conditions for this test are not selected properly, it does not provide an unambiguous answer. On the other hand, recording the potential drifts of SC electrodes with pH-sensitive membranes in samples with different CO2 levels can effectively prove the presence or absence of a water layer in a short time period.

PEDOT(PSS) as Solid Contact for Ion-Selective Electrodes: The Influence of the PEDOT(PSS) Film Thickness on the Equilibration Times.

To understand the rate determining processes during the equilibration of poly(3,4-ethylenedioxythiophene):polystyrenesulfonate-based (PEDOT(PSS)-based) solid contact (SC) ion-selective electrodes (ISEs), the surfaces of Pt, Au, and GC electrodes were coated with 0.1, 1.0, 2.0, and 4.0 µm thick galvanostatically deposited PEDOT(PSS) films. Next, potential vs time transients were recorded with these electrodes, with and without an additional potassium ion-selective membrane (ISM) coating, following their first contact with 0.1 M KCl solutions. The transients were significantly different when the multilayered sensor structures were assembled on Au or GC compared to Pt. The differences in the rate of equilibration were interpreted as a consequence of differences in the hydrophilicity of PEDOT(PSS) in contact with the substrate electrode surfaces based on X-ray photoelectron spectroscopy (XPS) and synchrotron radiation-XPS (SR-XPS) analysis of 10-100 nm thick PEDOT(PSS) films. The influence of the layer thickness of the electrochemically deposited PEDOT(PSS)-films on the hydrophilicity of these films has been documented by contact angle measurements over PEDOT(PSS)-coated Au, GC, and Pt electrode surfaces. This study demonstrates that it is possible to minimize the equilibration (conditioning) time of SC ISEs with aqueous solutions before usage by optimizing the thickness of the SC layer with a controlled ISM thickness. PEDOT(PSS)-coated Au and GC electrodes exhibit a significant negative potential drift during their equilibration in an aqueous solution. By coating the PEDOT(PSS) surface with an ISM, the negative potential drift is compensated by a positive potential drift related to the hydration of the ISM and activity changes at the PEDOT(PSS)|ISM interface. The potential drifts related to activity changes in the ISM have been determined by a novel adaptation of the "sandwich membrane" method.

Equilibration Time of Solid Contact Ion-Selective Electrodes.

Papers published on ion-selective electrodes (ISEs) generally report on the performance characteristics of these devices after long, extensive conditioning. Conditioning refers to the equilibration of the ion-selective electrode in an aqueous solution before the measurement of the sample. The requirement for long and repeated conditioning is a significant burden in a variety of applications, for example, single-use sensors aimed for in vivo or field applications and solid contact (SC) ISEs, which were developed to provide simple, mass-produced sensors that have the potential to be implemented without calibration and extensive conditioning. In this study we recorded the potential of SC K(+), Na(+), and H(+) ISEs as a function of time following their first contact with an aqueous electrolyte solution and used these transients to determine their equilibration times. The SC electrodes were built on Au, Pt, and glassy carbon (GC) substrates using galvanostatically deposited conductive polymer (PEDOT(PSS(-)), poly(3,4-ethylenedioxythiophene) polystyrenesulfonate) as ion-to-electron transducer (solid internal contact) between the ion-selective membrane and the substrate. The SC electrodes built on GC and Au had significantly shorter equilibration times (between 5 and 13 min) than the SC electrodes built on Pt substrates (>60 min). Such significant differences in the equilibration times of SC ISEs built on different substrate electrodes are reported here for the first time. These unexpected findings suggest that the interface between the conductive polymer and the electron-conducting substrate (EC) has significant influence on the long-term dynamic behavior of SC ISEs.

A tutorial on the application of ion-selective electrode potentiometry: an analytical method with unique qualities, unexplored opportunities and potential pitfalls; tutorial.

Ion-selective potentiometry enjoys practical utility as a simple analytical technique to measure ionic constituents in complex samples. Advances in the field have improved the selectivity and decreased the detection limit of ion-selective electrodes (ISEs) by orders of magnitude such that trace analysis in micro and nanomolar concentrations is now possible with potentiometric sensors. This tutorial reviews the fundamental principles of ion-selective potentiometry, describes the practical considerations involved in the use of these sensors to measure real samples, and discusses the statistical evaluation of experimental results compared with alternative analytical techniques.

Measurement of sodium ion concentration in undiluted urine with cation-selective polymeric membrane electrodes after the removal of interfering compounds.

The measurement of sodium ion concentration in urine can provide diagnostic information and guide therapy. Unfortunately, neutral-carrier-based ion-selective electrodes show a large positive drift and loss in selectivity in undiluted urine. The extraction of electrically neutral lipids from the urine into the sensing membrane was suggested as the main source of the drift, loss of selectivity and the consequent incorrect concentration readings. In this work, (i) solvent-solvent extraction, (ii) membrane-immobilized solvent extraction and (iii) solid phase extraction were used to remove interfering compounds from urine samples. The "cleaned" urine samples were subsequently analyzed using a calixarene (sodium ionophore X)-based, solid-contact, sodium-selective electrode in a flow-through manifold. The solid-contact sodium sensors had excellent stability in cleaned urine and an acceptable bias compared to commercial clinical analyzers.

Microfabricated ISEs: critical comparison of inherently conducting polymer and hydrogel based inner contacts.

A rigorous side by side comparison of miniature planar potassium-selective electrodes with hydrogel and potassium hexacyanoferrate(II)/(III) doped polypyrrole (PPy/FeCN) based inner contacts is presented. The planar electrodes were manufactured by screen printing as four- and five-site arrays on ceramic substrates. These electrode arrays were incorporated into a flow-through cell, which could accommodate nine electrode sites. Two identical flow cells were connected in series and the effect of the inner contacts on the analytical performance of the respective electrodes has been critically evaluated. The time necessary to reach steady state conditions has been determined and the effect of experimental parameters (temperature, ambient light intensity, CO(2), and O(2) concentration of the sample) on the potential stability of the electrodes was analyzed. At controlled temperature, the drift of the planar potassium electrodes with hydrogel and PPy/FeCN solid contact were 0.11+/-0.02mVh(-1) and 0.03+/-0.007mVh(-1), respectively. The experimental data proved that there is no aqueous film formation between the PPy/FeCN film and the potassium-selective solvent polymeric membrane.