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.

Electrochemical sensor for tricyclic antidepressants with low nanomolar detection limit: Quantitative Determination of Amitriptyline and Nortriptyline in blood.

Amitriptyline and its metabolite, Nortriptyline are commonly used tricyclic antidepressant (TCA) drugs that are electrochemically active. In this work, the performance characteristics of a plasticized PVC membrane-coated glassy carbon (GC) electrode are described for the voltammetric quantification of Amitriptyline and Nortriptyline in whole blood. The highly lipophilic Amitriptyline and Nortriptyline preferentially partition into the plasticized PVC membrane where the free drug is oxidized on the GC electrode. The concentrations of the drugs in the membrane are orders of magnitude larger than in the sample solution, resulting in superb limit of detection (LOD) of the membrane-coated voltammetric sensor: 3 nmol/L for Amitriptyline and 20 nmol/L for Nortriptyline. Conversely, hydrophilic components of the sample solution, e.g., proteins, the protein-bound fraction of the drugs, and electrochemically active small molecules are blocked from entering the membrane, which provides exceptional selectivity for the membrane-coated sensor and feasibility for the measurements of Amitriptyline in whole blood. In this work, the concentrations of Amitriptyline and Nortriptyline were determined in whole blood using the sensor and the results of our analysis were compared to the results of the standard HPLC-MS method. Based on our experience, the one-step voltammetric methods with the membrane-coated sensor may become a real alternative to the significantly more complex HPLC-MS analysis.

Polymeric membrane-modified voltammetric sensors for lipophilic analytes with nanomolar detection limit: Key parameters influencing the response characteristics.

Thin plasticized PVC membrane-coated glassy carbon working electrodes have been used for the voltammetric measurement of highly lipophilic, electroactive drugs. Compared to conventional working electrodes, these membrane-coated electrodes exhibit remarkable detection limit and selectivity and are less prone to electrode fouling. The unique performance characteristics of these sensors are related to the large partition coefficient of the analyte in the membrane coating where it is oxidized in a non-aqueous membrane phase. To analyze the influence of the key parameters of the response of membrane-coated sensors, we derived theoretical expressions on the voltammetric response of the sensors. In our analysis we considered 1) the partition coefficient (Pmw) of the analyte between the aqueous sample and the organic membrane, 2) the membrane volume to sample volume ratio (Vm/Vw), and 3) the binding constant of constituents in the sample that bind the analyte (K). The results of our theoretical analysis have been tested through voltammetric measurement of highly lipophilic analytes with logPow values (logarithm of the partition coefficient between octanol and water) ranging between 0.3 and 7.5. By understanding of the influence of the sensor design parameters on the overall sensor response, these parameters can be tuned for optimized response slope, detection limit, etc., for solving specific analytical tasks.

A "Smart" Biosensor-Enabled Intravascular Catheter and Platform for Dynamic Delivery of Propofol to "Close the Loop" for Total Intravenous Anesthesia.

BACKGROUND: Target-controlled infusion anesthesia is used worldwide to provide user-defined, stable, blood concentrations of propofol for sedation and anesthesia. The drug infusion is controlled by a microprocessor that uses population-based pharmacokinetic data and patient biometrics to estimate the required infusion rate to replace losses from the blood compartment due to drug distribution and metabolism. The objective of the research was to develop and validate a method to detect and quantify propofol levels in the blood, to improve the safety of propofol use, and to demonstrate a pathway for regulatory approval for its use in the USA. METHODS: We conceptualized and prototyped a novel "smart" biosensor-enabled intravenous catheter capable of quantifying propofol at physiologic levels in the blood, in real time. The clinical embodiment of the platform is comprised of a "smart" biosensor-enabled catheter prototype, a signal generation/detection readout display, and a driving electronics software. The biosensor was validated in vitro using a variety of electrochemical methods in both static and flow systems with biofluids, including blood. RESULTS: We present data demonstrating the experimental detection and quantification of propofol at sub-micromolar concentrations using this biosensor and method. Detection of the drug is rapid and stable with negligible biofouling due to the sensor coating. It shows a linear correlation with mass spectroscopy methods. An intuitive graphical user interface was developed to: (1) detect and quantify the propofol sensor signal, (2) determine the difference between targeted and actual propofol concentration, (3) communicate the variance in real time, and (4) use the output of the controller to drive drug delivery from an in-line syringe pump. The automated delivery and maintenance of propofol levels was demonstrated in a modeled benchtop "patient" applying the known pharmacokinetics of the drug using published algorithms. CONCLUSIONS: We present a proof-of-concept and in vitro validation of accurate electrochemical quantification of propofol directly from the blood and the design and prototyping of a "smart," indwelling, biosensor-enabled catheter and demonstrate feedback hardware and software architecture permitting accurate measurement of propofol in blood in real time. The controller platform is shown to permit autonomous, "closed-loop" delivery of the drug and maintenance of user-defined propofol levels in a dynamic flow model.

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.

Plasticized PVC Membrane Modified Electrodes: Voltammetry of Highly Hydrophobic Compounds.

In the last 50 years, plasticized polyvinyl chloride (PVC) membranes have gained unique importance in chemical sensor development. Originally, these membranes separated two solutions in conventional ion-selective electrodes. Later, the same membranes were applied over a variety of supporting electrodes and used in both potentiometric and voltammetric measurements of ions and electrically charged molecules. The focus of this paper is to demonstrate the utility of the plasticized PVC membrane modified working electrode for the voltammetric measurement of highly lipophilic molecules. The plasticized PVC membrane prevents electrode fouling, extends the detection limit of the voltammetric methods to sub-micromolar concentrations, and minimizes interference by electrochemically active hydrophilic analytes.

Kinetic Description of the Membrane-Solution Interface for Ion-Selective Electrodes.

The theoretical models for ISEs almost exclusively assume thermodynamic equilibrium at the membrane/solution-phase boundary. In this report, we present a new, congruent model which combines first-order reaction kinetics of ion-exchange at the phase boundary and diffusional mass transport in the adjoining phases in the continuity equation. The influence of the rate constant in the new kinetic model has significant impact on the predicted transients corresponding to instantaneous change in the sample solution composition. The simulated transients generated with the new model coincide with the transients recorded in common potentiometric experiments, e.g., with transients recorded upon step change in the primary or interfering ion concentrations. The simulated transients also align well with previously published transients representing special cases of potentiometry (e.g., super-Nernstian response, non-Nernstian responses in the presence of highly interfering ions). The implementation of the kinetic model for simulating the transients in the water layer test also resulted in a better agreement with the experiments compared to the previous models.

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.

Voltammetric determination of diffusion coefficients in polymer membranes.

The diffusion-controlled transport of ions and molecules through polymer membranes utilized in chemical and biosensors is often the key factor determining the response characteristics of these sensors. In this paper, a simple voltammetric method is described for the determination of diffusion coefficients of redox molecules in resistive polymer membranes using a planar electrochemical cell (PEC) with a 5 µm radius carbon fiber as working electrode. In the proposed method, the diffusion coefficients are assessed from the scan rate dependence of the peak (or limiting) currents in linear sweep or cyclic voltammograms. The accuracy of the method is estimated through simulations using the method of Nicholson and Shain describing the quantitative relationship between the measured peak current (ip) and the square root of the scan rate (v1/2). The proposed method has been used for measuring the diffusion coefficients of ferrocene derivatives in highly resistive aqueous solutions, organic solvents, plasticizers, and plasticized PVC membranes. The measured diffusion coefficients are in agreement with theoretical models and previously reported values.

A Feedback Control Approach to Organic Drug Infusions Using Electrochemical Measurement.

GOAL: Target-controlled infusion of anesthesia is a closed-loop automated drug delivery method with a computer-aided control. Our goal is to design and test an automated drug infusion platform for propofol delivery in total intravenous anesthesia (TIVA) administration. METHODS: In the proposed method, a dilution chamber with first-order exponential decay characteristics was used to model the pharmacodynamics decay of a drug. The dilution chamber was connected to a flow system through an electrochemical cell containing an organic film-coated glassy carbon electrode as working electrode. To set up the feedback-controlled delivery platform and optimize its parameters, ferrocene methanol was used as a proxy of the propofol. The output signal of the sensor was connected to a PI controller, which prompted a syringe pump for feedback-controlled drug infusion. RESULTS: The result is a bench-top drug infusion platform to automate the delivery of a propofol based on the measurement of concentration with an organic film-coated voltammetric sensor. CONCLUSION: To evaluate the performance characteristics of the infusion platform, the propofol concentration in the dilution chamber was monitored with the organic film-coated glassy carbon electrode and the difference between the set and measured concentrations was assessed. The feasibility of measurement-based feedback-controlled propofol delivery is demonstrated and confirmed. SIGNIFICANCE: This platform will contribute to high-performance TIVA application of intravenous propofol anesthesia.

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.

Propofol detection and quantification in human blood: the promise of feedback controlled, closed-loop anesthesia.

The performance of a membrane-coated voltammetric sensor for propofol (2,6-diisopropylphenol) has been characterized in long term monitoring experiments using an automated flow analytical system (AFAS) and by analyzing human serum and whole blood samples by standard addition. It is shown that the signal of the membrane-coated electrochemical sensor for propofol is not influenced by the components of the pharmaceutical formulation of propofol (propofol injectable emulsion). The current values recorded with the electrochemical propofol sensor in buffer solutions and human serum samples spiked with propofol injectable emulsion showed excellent correlation with the peak heights recorded with an UV-Vis detector during the HPLC analysis of these samples (R(2) = 0.997 in PBS and R(2) = 0.975 in human serum). However, the determination of propofol using the electrochemical method is simpler, faster and has a better detection limit (0.08 ± 0.05 µM) than the HPLC method (0.4 ± 0.2 µM). As a first step towards feedback controlled closed-loop anesthesia, the membrane-coated electrochemical sensor has been implemented onto surface of an intravenous catheter. The response characteristics of the membrane-coated carbon fiber electrode on the catheter surface were very similar to those seen using a macroelectrode.

Facile directed assembly of hollow polymer nanocapsules within spontaneously formed catanionic surfactant vesicles.

Surfactant vesicles containing monomers in the interior of the bilayer were used to template hollow polymer nanocapsules. This study investigated the formation of surfactant/monomer assemblies by two loading methods, concurrent loading and diffusion loading. The assembly process and the resulting aggregates were investigated with dynamic light scattering, small angle neutron scattering, and small-angle X-ray scattering. Acrylic monomers formed vesicles with a mixture of cationic and anionic surfactants in a broad range of surfactant ratios. Regions with predominant formation of vesicles were broader for compositions containing acrylic monomers compared with blank surfactants. This observation supports the stabilization of the vesicular structure by acrylic monomers. Diffusion loading produced monomer-loaded vesicles unless vesicles were composed from surfactants at the ratios close to the boundary of a vesicular phase region on a phase diagram. Both concurrent-loaded and diffusion-loaded surfactant/monomer vesicles produced hollow polymer nanocapsules upon the polymerization of monomers in the bilayer followed by removal of surfactant scaffolds.

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.

Preliminary investigation of crosslinked chitosan sponges for tailorable drug delivery and infection control.

Local versus systemic antibiotic delivery may be an effective strategy for treating musculoskeletal infections, especially when antibiotic-resistant bacteria are present. Lyophilized uncrosslinked, genipin crosslinked, and genipin crosslinked with poly(N-isopropylacrylamide) (PNIPAM) chitosan sponges were analyzed for their in vitro degradation rate, chemical crosslinking, antibiotic uptake, elution, biologic activity, and cytotoxicity. These evaluations were pursued to determine if crosslinking with genipin could be used to create a tailorable point of care loaded sponge for local infection control. Crosslinking the chitosan sponges decreased degradation in phosphate-buffered saline from 4.48 ± 2.28 wt % remaining of the uncrosslinked sponges to 78.82 ± 1.15 and 73.87 ± 1.27 wt % remaining at week 1 for the genipin and PNIPAM/genipin crosslinked sponges, respectively. The PNIPAM/genipin crosslinked sponges exhibited the most sustained release of biologically active antibiotics, with an average antibiotic release 63% higher than uncrosslinked and 37% higher than genipin crosslinked sponges, after 96 h. No significant cytotoxic effects from sponges or eluates were exhibited with NIH 3T3 fibroblasts. These preliminary results indicate that genipin crosslinked chitosan sponges, with or without PNIPAM, have potential as local delivery systems for adjunctive therapy for infection control, especially when longer degradation periods and higher antibiotic elutions are desired.