Commercial Reference Electrode Research Articles

Chemical and Biological Sensor Capsules for Real-Time Measurement of Cell Properties in Bioreactors

Introduction Real-time bioprocess monitoring and control is needed for the scalable production and deployment of cancer cell therapies at reasonable cost. For example, CAR-T cell therapy shows promise as an effective treatment for cancer with high (70-90%) remission rates.1 However, the cost of these treatments is too expensive for their mass adoption.2 One reason for the steep price is the difficulty scaling production while ensuring delivery of an efficacious therapy to the patient. One factor preventing scalability is the lack of effective process analytical technologies. As a potential solution to this issue, we have developed a fully integrated, wireless, 3D-printed sensor capsule to be used for multiplexed sensing of critical quality attributes (CQAs), namely pH, glucose, lactate, and IL-2 concentrations within cell bioreactors. CQAs are used not only as metrics for cell viability, but also as determinants of a cell’s ability to deliver efficacious treatment. Unlike current process monitoring technology, capsule sensors are buoyant and can be propelled using impellers within the bioreactor. This allows for measurements uniformly inside the bioreactor. To our knowledge, this is the first time that 3D-printed, wireless sensor capsules have been developed for use in cell bioreactors for cancer cell therapy. Methods The capsule consists of electrochemical sensors as well as data acquisition and wireless transmission electronics integrated into a capsule made of a biocompatible polymer (Figure 1). The sensors for glucose, lactate, pH, and IL-2 along with Ag on-chip pseudo-reference electrode (pseudo-RE) were fabricated using standard lithographic techniques on silicon substrates. The reference electrode consisted of a thin-film of Ag that was later coated with polyvinyl butyral (PVB) to improve stability.3 IL-2 and pH sensing was achieved using gold electrodes. A pH-sensitive Al2O3 or HfO2 layer is deposited through atomic layer deposition on the pH sensor to achieve pH-sensitivity. Glucose and lactate sensing was achieved amperometrically using enzymes, glucose or lactase oxidase respectively, immobilized on a Pt electrode biased to 0.7 V. Detection could also be achieved potentiometrically by using the pH sensor to detect changes in pH caused by conversion of the enzyme product, H2O2, to H+.Figure 1 shows the capsule and sensor design. The capsule was 3D printed using a Stratasys Connex-350 and was designed to be fully insulated from solution except for an opening on the top revealing the sensors. The sensor chip is installed within the cap of the capsule which can be screwed off, but with wired connection to the electronics housed in the body of the capsule. This allows for simple replacement of the sensor chip while retaining the capsule components for reuse. Results and Conclusions Before testing within the capsule, the sensors were tested using microfluidics. Figure 2 shows the performance of the Ag/PVB pseudo-RE. The pseudo-RE shows a stable potential in different pH buffer solutions and similar sensitivity results when compared to a commercial reference electrode for pH sensing. By having a stable microfabricated pseudoRE, the sensing components will not limit miniaturization of the capsule. Figure 3 shows the response of the pH, lactate, and glucose sensors at different concentrations. The pH sensor shows high sensitivity (~53 mV/pH) over the range of pH 3 to 7. Glucose and lactate sensing was tested in 1X PBS. The glucose sensor shows a linear response to glucose concentrations from 1-6 mM with saturation at ~8 mM validating the use of the sensor within cell culture media.4 The lactate sensor shows sensitivity in the concentration range of 1-5 mM with saturation occurring around ~6 mM. The division of T cells requires aerobic glycolysis where glucose is converted to lactate. However, not all glucose is expected to be converted. The lactate concentration expected to be produced is ~1 mM.5 Selectivity towards IL-2 is achieved using anti-IL2 antibodies. Figure 4a shows the functionalization scheme for detection of IL-2. A thiol self-assembled monolayer (SAM) is attached to the gold electrode and then modified for amine conjugation using NHS/EDC. Anti-IL2 can then bind to the SAM and unbound sites are blocked with ethanolamine. Figure 4b reveals antibody attachment occurs optimally at pH 5. Surface plasmon resonance shows successful attachment of 10 ug/mL of IL-2 to the functionalized gold sensor. The next step is to demonstrate IL-2 sensing potentiometrically. The capsule’s wireless sensing capability was validated using pH sensing in a beaker. The pH sensing results in Figure 5b were obtained wirelessly from the capsule and were nearly identical to the results measured using our microfluidic system and Keithley-4200 lab instrument (Fig. 5a). This work has successfully demonstrated wireless sensing using a 3D-printed sensor capsule and functionality of electrochemical sensors for multiple CQAs for cell growth in bioreactors. These sensor capsules can enable scalability of the cell manufacturing process while ensuring delivery of an efficacious treatment. Future work will focus on capsule sensor validation in cell growth media and monitoring of cell growth in bioreactors.

Reference Electrodes for Electrochemical Sensors Based on Redox Couples Immobilized within Nafion Films.

The immobilization of both components of a redox couple within films of the sulfonated tetrafluoroethylene polymer, Nafion, on the surface of carbon electrodes is reported. In particular, cationic and/or water-insoluble species are used to develop stable and robust reference electrodes by adjustment and optimization of both the film thickness and the level of doping of the film, as an alternative to using standard commercial reference electrodes. Different redox couples were evaluated: methyl viologen dication/cation (MV2+/MV+), tris(2,2'-bipyridyl)ruthenium(II)/ruthenium(III) (Ru(bpy)32+/Ru(bpy)33+), ferrocene/ferrocenium (Fc/Fc+), and poly(vinylferrocene)/poly(vinylferrocenium) (PVFc/PVFc+). These reference electrodes demonstrate a good stability over 10 days and 1000 successive scans; in particular, Nafion-confined Fc/Fc+PF6- was shown to display the best performance. This type of reference electrode, as a solid-state electrolyte-free reference electrode, has several advantages, including full retention of the redox species within the films together with ease of both fabrication and miniaturization.

Instantaneously usable screen-printed silver/silver sulfate reference electrode with long-term stability

We developed an instantaneously usable screen-printed silver/silver sulfate (Ag/Ag2SO4) reference electrode with long-term stability. The reference electrode was composed of a Ag layer, a Ag/Ag2SO4 layer, an inner electrolyte layer, and a liquid junction layer printed successively on a polyimide substrate. The liquid junction and inner electrolyte layers were prepared using a silica gel–poly(vinylidene difluoride) ink. The open-circuit potentials in different electrolyte solutions were compared to that measured using a commercial reference electrode. The fabricated reference electrode stabilized the potential shift within 30 min and gave 437 mV vs. saturated KCl/Ag/AgCl measured in a 1 M KNO3 solution; the result was in good agreement with the theoretical value. Furthermore, the fabricated reference electrode remained stable for 20 days. This Ag/Ag2SO4 reference electrode showed potential for application in measurement systems where contamination by chloride ions is not desirable.

Design and Finite Element Model of a Microfluidic Platform with Removable Electrodes for Electrochemical Analysis

A microfluidic platform for hydrodynamic electrochemical analysis was developed, consisting of a poly(methyl methacrylate) chip and three removable electrodes, each housed in 1/16” OD polyether ether ketone tube which can be removed independently for polishing or replacement. The working electrode was a 100-μm diameter Pt microdisk, located flush with the upper face of a 150 μm × 20 μm × 3 cm microchannel, smaller than previously reported for these types of removable electrodes. A commercial leak-less reference electrode was utilized, and a coiled platinum wire was the counter electrode. The platform was evaluated electrochemically by oxidizing a potassium ferrocyanide solution at the working electrode, and a typical limiting current behavior was observed after running linear sweep voltammetry and chronoamperometry, with flow rates 1−6 μL/min. While microdisk channel electrodes have been simulated before using a finite difference method in an ideal 3D geometry, here we predict the limiting current using finite elements in COMSOL Multiphysics 5.3a, which allowed us to easily explore variations in the microchannel geometry that have not previously been considered in the literature. Experimental and simulated currents showed the same trend but differed by 41% in simulations of the ideal geometry, which improved when channel and electrode imperfections were included.

Solid-State Reference Electrode Based on Thin-Films of Tetrahydrofurfuryl Acrylate (pTHFA) Photopolymer

The reference electrode based on thin film of the poly-tetrahydrofurfuryl acrylate (pTHFA) prepared by photopolymers has been successfully fabricated. In this study, the electrode reference fabrication process was carried out by three methods of electrolyte filling, the first by the immersion process after the photopolymer (type I), the second method of electrolyte in doping together with the photopolymer process (type II), and the third was done as the second process followed the immersion process (type III). The type III reference electrodes exhibit characteristics as reference electrodes with test results ΔmV of the Cl ions < 10 mV in various of the Cl concentrations and pH solutions. Meanwhile, the real test of reference electrode type III as reference electrode produces a value close to the Nernstian number when paired with a Cl sensor or pH sensor. Performance testing with cyclic Voltammetry technique shows CV graphic results that are similar to CV test results with SCE commercial reference electrodes. The working electrode used in the test by cyclic Voltammetry technique is the carbon electrode and the Ag electrode. While the drift test shows the reference electrode type III is quite stable because it has drifted 1 mV/min.

Fabrication of integrated solid state electrode for extended gate-FET pH sensor

To realize the miniaturization of an extended gate field effect transistor (EGFET) based sensor, an all solid state phase electrodes are needed. This paper presents the facile fabrication of an integrated solid state electrode (ISSE) consisting a solid state silver/silver chloride (Ag/AgCl) reference electrode (RE) and ZnO nanostructures sensing electrode (SE) on a single 2 × 2 cm2 indium tin oxide (ITO) substrate. The RE and SE was fabricated by thermal evaporation (TE) and simple immersion process respectively. The ISSE was then tested by applying it to an EGFET pH sensor setup and compared with commercialized Ag/AgCl RE; and it was found that the sensor sensitivity for both electrodes are comparable (commercial RE: 52.4 mV pH−1, ISSE: 53.4 mV pH−1).

Designing and Using 3D-Printed Components That Allow Students To Fabricate Low-Cost, Adaptable, Disposable, and Reliable Ag/AgCl Reference Electrodes

A reference electrode is an essential component of all three-electrode electrochemical measurements. Common commercial reference electrodes, including the saturated calomel electrode and Ag/AgCl reference electrode, are expensive with the former containing toxic mercury. Cheaper alternatives have been proposed including Ag/AgCl references made from pipets and test tubes. However, electrodes prepared in this way are difficult to work with and are limited by the size and shape of glass casings that are available. This paper proposes an in-house manufactured Ag/AgCl reference electrode that uses some 3D-printed components in the fabrication process. This electrode is cheap to manufacture ($5 vs $60–100 CAD for the commercial reference electrode), and the design can be quickly altered due to the 3D printer’s capabilities in rapidly printing new electrode shapes to suit different analysts’ needs. The lab-made reference electrodes demonstrated stability and consistency in peak potential measurements in the cyclic voltammetry (CV) experiments. In ferricyanide CV tests, the recorded differences in anodic and cathodic peak potential (ΔEp) values for the commercial reference electrode and both lab-made electrodes were 68 ± 9%, 70 ± 12%, and 69 ± 13%, respectively. For all tests, the results were statistically comparable with those of the commercial Ag/AgCl reference electrode.

Effect of glass former (B2O3, SiO2, GeO2 and P2O5) addition to Fe2O3-Bi2O3 glass on pH responsivity

The effect of glass former (constituent capable to vitrify by itself such as B2O3, SiO2, GeO2 and P2O5) addition to Fe2O3-Bi2O3 (FeBi) glass on pH responsivity (pH sensitivity and pH response time) was investigated in order to develop novel pH responsive, hydrophobic glasses. Additionally, the effect of glass composition on pH sensitivity of Fe2O3-Bi2O3 glasses was investigated in order to develop contaminant-free reference electrodes with very low pH sensitivity. A small amount of B2O3 and SiO2 drastically increased the pH sensitivity. The addition of glass formers of 20 mol% provided high pH sensitivity (>90%) in any of the glass formers. Although all glass formers increased their contact angle with water for Fe2O3-Bi2O3 glass, the addition of GeO2 was most effective. Most of the Fe2O3-Bi2O3-GeO2 (FeBiGe) glasses with 20–30mol% GeO2, which serve as a working electrode, had compatibility between high pH sensitivity and a high contact angle with water (100°). However, certain Fe2O3-Bi2O3 glasses with approximately 20 mol% Fe2O3 showed low pH sensitivity (<10%). These FeBi glasses are considered to serve as contaminant-free reference electrodes, whereas KCl aqueous solution, as a contaminant, gradually leaks in commercial reference electrodes (Ag/AgCl immersed in KCl aqueous solution). Since the novel pH complex glass electrodes, consisting of a FeBiGe working electrode and a FeBi reference electrode, resist fouling of itself and serve as contaminant-free glass electrodes, these electrodes are expected to be highly useful in biological systems and drug development.

Fabrikasi Elektroda Pembanding Ag/AgCl dengan Berbagai Membran

Ag/AgCl reference electrode has been fabricated using various types of membranes, i.e. cellulose, polyisoprene, graphite, kaolinite, and LDPE. The electrodes performance were observed by measuring 0.1 M K 4 [Fe(CN) 6 ] solution. The measurements were repeated for every 2 hours during 24 hours, then every 24 hours during 6 days, and after that every 3 days for two times using cyclic voltammetry. The voltammograms obtained of various membranes were compared to Ag/AgCl commercial reference electrode voltammogram. Validation of the results was conducted by F-test and t-test. The Ag/AgCl reference electrode using polyisoprene and LDPE membranes have a performance close to commercial electrode.

Surface and Electrical Characterization of Ag/AgCl Pseudo-Reference Electrodes Manufactured with Commercially Available PCB Technologies.

Lab-on-Chip is a technology that could potentially revolutionize medical Point-of-Care diagnostics. Considerable research effort is focused towards innovating production technologies that will make commercial upscaling financially viable. Printed circuit board manufacturing techniques offer several prospects in this field. Here, we present a novel approach to manufacturing Printed Circuit Board (PCB)-based Ag/AgCl reference electrodes, an essential component of biosensors. Our prototypes were characterized both structurally and electrically. Scanning Electron Microscopy (SEM) and X-Ray Photoelectron Spectroscopy (XPS) were employed to evaluate the electrode surface characteristics. Electrical characterization was performed to determine stability and pH dependency. Finally, we demonstrate utilization along with PCB pH sensors, as a step towards a fully integrated PCB platform, comparing performance with discrete commercial reference electrodes.

A solid-state thin-film Ag/AgCl reference electrode coated with graphene oxide and its use in a pH sensor.

In this study, we describe a novel solid-state thin-film Ag/AgCl reference electrode (SSRE) that was coated with a protective layer of graphene oxide (GO). This layer was prepared by drop casting a solution of GO on the Ag/AgCl thin film. The potential differences exhibited by the SSRE were less than 2 mV for 26 days. The cyclic voltammograms of the SSRE were almost similar to those of a commercial reference electrode, while the diffusion coefficient of Fe(CN)63− as calculated from the cathodic peaks of the SSRE was 6.48 × 10−6 cm2/s. The SSRE was used in conjunction with a laboratory-made working electrode to determine its suitability for practical use. The average pH sensitivity of this combined sensor was 58.5 mV/pH in the acid-to-base direction; the correlation coefficient was greater than 0.99. In addition, an integrated pH sensor that included the SSRE was packaged in a secure digital (SD) card and tested. The average sensitivity of the chip was 56.8 mV/pH, with the correlation coefficient being greater than 0.99. In addition, a pH sensing test was also performed by using a laboratory-made potentiometer, which showed a sensitivity of 55.4 mV/pH, with the correlation coefficient being greater than 0.99.

Cost-effective nanostructured thin-film solar cell with enhanced absorption

Nanostructured transparent conductive electrodes are highly interesting for efficient light management in thin-film solar cells, but they are often costly to manufacture and limited to small scales. This work reports on a low-cost and scalable bottom-up approach to fabricate nanostructured thin-film solar cells. A folded solar cell with increased optical absorber volume was deposited on honeycomb patterned zinc oxide nanostructures, fabricated in a combined process of nanosphere lithography and electrochemical deposition. The periodicity of the honeycomb pattern can be easily varied in the fabrication process, which allows structural optimization for different absorber materials. The implementation of this concept in amorphous silicon thin-film solar cells with only 100 nm absorber layer was demonstrated. The nanostructured solar cell showed approximately 10% increase in the short circuit current density compared to a cell on an optimized commercial textured reference electrode. The concept presented here is highly promising for low-cost industrial fabrication of nanostructured thin-film solar cells, since no sophisticated layer stacks or expensive techniques are required.

Analytical Quality Solid-State Composite Reference Electrode and Electrochemical Platform

A new type of all-solid-state reference electrodes based on a polymer/inorganic salt composite was designed, prepared, and characterized. Both a chemical polymerization method and an injection molding method were used for fabricating those novel electrodes. A rigorous testing procedure was used to reveal possible influence of pH, solution composition, the concentrations, and mobility of ions on the electrode response. The tests demonstrated the insensitivity of the electrodes to the matrix effects, excellent stability of the potential readings, and significantly reduced leakage of inorganic salt. It was shown that composite reference electrodes were on par or better in the performed tests than high-quality commercial reference electrodes. The reference electrodes described here are of analytical quality allowing for continuous, prolonged, and intensive usage. The performance of the reference electrode in demanding matrices such as environmental and biological samples was studied. This concept was further extended to produce the solid state electrochemical platform.

Analytical Quality Solid-State Composite Reference Electrode

The reference electrode is an indispensable part of an electrochemical measurement setup and its role is to establish a precise, stable, and reproducible potential which is independent of the sample composition [1]. Internal filling solution used in conventional reference electrodes causes many drawbacks and inconveniences [2]. There are several papers proposing alternative ways of eliminating the inner filling solutions. The reference electrodes proposed there are indeed interesting from the scientific point of view but they should be further developed to be of practical applicability. The classical reference electrodes have so far been superior in terms of continuous, prolonged, and intensive usage.A new type of all-solid-state reference electrodes based on a polymer/inorganic salt composite (SSC) was designed and characterized. Both a chemical polymerisation method [3] and an injection moulding method [4] were used for fabricating those novel electrodes.A rigorous testing procedure was used to reveal possible influence of pH, solution composition, as well as the concentrations and mobility of ions. The tests demonstrated the insensitivity of the electrodes to the matrix effects, excellent stability of the potential readings, and significantly reduced leakage of inorganic salt. It was shown that composite reference electrodes were on par or better in the performed tests than high-quality commercial reference electrodes. The SSC reference electrodes described here are of analytical quality allowing for continuous, prolonged, and intensive usage.

A liquid-junction-free reference electrode based on a PEDOT solid-contact and ionogel capping membrane

Liquid-junction-free reference electrodes have been prepared on screen printed substrates using poly-3,4-ethylenedioxythiophene (PEDOT) as solid-contact and novel ionogels as capping membrane. The chemico-physical properties of the PEDOT layer were tuned by changing the electropolymerization media and the electrodeposition technique. Electrodepositing PEDOT films potentiostatically or potentiodynamically were found to have a significant impact on the stability of the electrodes during the conditioning step. Optimization of the capping membrane formulation, e.g., acrylate monomers, ionic liquid, cross-linkers and photo-initiators, produced electrodes with properties almost equivalent with a commercial reference electrode. Thus, calibration plots of Na+ ion-selective electrodes against the optimized solid-contact ionogel reference electrodes (SCI-REs) or against a double-liquid junction Ag/AgCl electrode did not present any significant difference. Such SCI-REs may provide an effective route to the generation of future low-cost components for potentiometric sensing strips.

Analytical quality solid-state composite reference electrode manufactured by injection moulding

An injection moulding method was used for fabricating solid-state reference electrodes (Ag/AgCl type) based on a polymer/inorganic salt composite. In this method, a silver/silver chloride wire was placed inside a mould into which the mixture of polymer and inorganic salt was injected. The obtained solid-state composite reference electrodes were extensively tested to study the influence of different parameters such as solution composition, the concentrations and mobility of ions and pH on the potential stability of the electrodes. These experiments revealed that the composite reference electrodes are insensitive to the matrix effect, have excellent potential readings stability and considerably reduced leakage of inorganic salt. The composite reference electrodes were compared favourably to high-quality commercial reference electrodes. It was concluded that the composite reference electrodes obtained by injection moulding are of analytical quality allowing for continuous, prolonged and intensive usage.

Investigation of the electrical stability of Si-nanowire biologically sensitive field-effect transistors with embedded Ag/AgCl pseudo reference electrode

We report on the electrical stability of Si-nanowire biologically sensitive field-effect transistors (BioFETs) fabricated using conventional microfabrication technique, with an embedded Ag/AgCl pseudo-reference electrode (pRE) formed by an electrochemical method. The open-circuit potential (OCP) characteristics between the pRE and a commercial reference electrode have been measured in order to evaluate the influence of the pRE potential on the device performance. In a pH sensing mode, the fabricated pRE follows the applied potential accurately with a small offset value of below 6 mV for pH in the range of 4 to 10. The BioFET was also used for the detection of alpha fetoprotein (AFP) with a detection limit of 10 pg mL−1 and the corresponding OCP fluctuation of the pRE was less than 1.5 mV, independent of the AFP concentrations. These results suggest that the Si-NW BioFETs with the embedded Ag/AgCl pRE are very promising for reliable biosensing applications.

An analytical quality solid-state composite reference electrode

A new type of all-solid-state reference electrode was designed and characterized. The electrodes are based on a polymer/inorganic salt composite and a silver/silver chloride reference element. A rigorous testing procedure was used to reveal the possible influence of pH, solution composition, as well as the concentration and mobility of ions. The tests demonstrated the insensitivity of the electrodes to the matrix effects, excellent stability of the potential readings, and significantly reduced leakage of inorganic salt. In the performed tests the composite reference electrodes were on a par with or better than the high-quality commercial reference electrodes. The reference electrodes described here are of analytical quality allowing for continuous, prolonged, and intensive usage.

Artefacts in electrochemical impedance measurement in electrolytic solutions due to the reference electrode

Electrochemical impedance measurements performed on gas-evolving electrodes in high-conductivity solutions have shown artefacts at high frequency (HF) when using a commercial reference electrode (RE) due to its relatively high impedance. A “trick” consisting in coupling a platinum wire in series with a capacitor of 0.1 μF to the standard RE, hence making a dual RE, has been used for a long time without a clear understanding of how it works. This paper investigates HF EIS measurements in high-conductivity solutions from experiments in 1 M H 2SO 4 solutions and simulations based on the equivalent circuit of the cell and of the electrode potential measurement device. In particular, the value of the capacitor and the position of the platinum wire are considered. Curiously, while the HF artefact is removed by the dual RE, a spurious loop may appear at low frequency depending on the position of the platinum wire. This is explained from simulations based on the equivalent circuit of the cell.

Voltammetric characterization of a fully integrated, patterned single walled carbon nanotube three-electrode system on a glass substrate

A single walled carbon nanotube (SWCNT)-based three-electrode system was fully integrated on glass substrates using a standard microfabrication process and electrochemically characterized using cyclic voltammetry. O(2) plasma functionalization of the SWCNT film working electrode for achieving high sensitivity was voltammetrically optimized with respect to the plasma power and treatment time. Chlorination of a Ag thin-film was done in an acidic solution for different dip times to form a thin-film Ag/AgCl reference electrode. The Nernstian behavior of as-prepared and seven-day-aged Ag/AgCl thin-film electrodes was investigated for seeking the optimum reference electrode with long-term stability and was compared to a commercial reference electrode. A quality control evaluation and a performance assessment of the fully integrated SWCNT-transferred sensing systems were performed using cyclic voltammetry. The proposed SWCNT-based three electrode device exhibited clear electrochemistry under voltammetric conditions, and is therefore a candidate for use in all electrochemical biosensors.