Pseudo-reference Electrode Research Articles

Compact analytical flow system for the simultaneous determination of l-lactic and l-malic in red wines

During the malolactic fermentation of red wines, l-malic acid is mainly converted to l-lactic acid. Both acids should be precisely measured during the entire process to guarantee the quality of the final wine, thus making real-time monitoring approaches of great importance in the winemaking industry. Traditional analytical methods based on laboratory procedures are currently applied and cannot be deployed on-site. In this work, we report on the design and development of a bi-parametric compact analytical flow system integrating two electrochemical biosensors that could be potentially applied in this scenario. The developed flow-system will allow for the first time the simultaneous measurement of both acids in real scenarios at the real-time and in remote way. Miniaturized thin-film platinum four-electrode chips are fabricated on silicon substrates by standard photolithographic techniques and further implemented in a polymeric fluidic structure. This includes a 15 µL flow cell together with the required fluidic channels for sample and reagent fluid management. The four-electrode chip includes counter and pseudo-reference electrodes together with two working electrodes. These are sequentially modified with electropolymerized polypyrrole membranes that entrap the specific receptors for selectively detecting both target analytes. The analytical performance of both biosensors is studied by chronoamperometry, showing a linear range from 5 × 10−6 to 1 × 10−4 M (LOD of 3.2 ± 0.3 × 10−6 M) and from 1 × 10−7 to 1 × 10−6 M (LOD of 6.7 ± 0.2 × 10−8 M) for the l-lactate and the l-malate, respectively. Both biosensors show long-term stability, retaining more than the 90% of their initial sensitivity after more than 30 days, this being a prerequisite for monitoring the whole process of the malolactic fermentation of the red wines (time between 20 and 40 days). The flow system performance is assessed with several wine samples collected during the malolactic fermentation process of three red wines, showing an excellent agreement with the results obtained with the standard method.

Ultra stable, inkjet-printed pseudo reference electrodes for lab-on-chip integrated electrochemical biosensors

Lab-on-Chip technology comprises one of the most promising technologies enabling the widespread adoption of Point-of-Care testing in routine clinical practice. However, until now advances in Lab-on-Chip have not been translated to the anticipated degree to commercialized tools, with integrated device mass manufacturing cost still not at a competitive level for several key clinical applications. Lab-on-PCB is currently considered as a candidate technology addressing this issue, owing to its intuitive compatibility with electronics, seamless integration of electrochemical biosensors and the extensive experience regarding industrial manufacturing processes. Inkjet-printing in particular is a compatible fabrication method, widening the range of electronic materials available and thus enabling seamlessly integrated ultrasensitive electronic detection. To this end, in this work stable pseudo-reference electrodes are fabricated for the first time by means of commercial inkjet-printing on a PCB-integrated electrochemical biosensing platform. SEM and XPS analysis are employed to characterize the electrodes’ structure and composition and identify any special characteristics, compared to published work on alternative substrates. Additionally, this paper analyzes integrated reference electrodes from a new perspective, focusing mainly on their characteristics in real-life operation: chemical sintering as opposed to high budget thermal one, stability under continuous flow, pH dependency and bias stress effects on electrode instability, a parameter often overlooked in electrochemical biosensors.

Electrochemical detection of ascorbic acid in artificial sweat using a\xa0flexible alginate/CuO-modified electrode

A flexible sensor is presented for electrochemical detection of ascorbic acid in sweat based on single-step modified gold microelectrodes. The modification consists of electrodeposition of alginate membrane with trapped CuO nanoparticles. The electrodes are fabricated at a thin polyimide support and the soft nature of the membrane can withstand mechanical stress beyond requirements for skin monitoring. After characterization of the membrane via optical and scanning electron microscopy and cyclic voltammetry, the oxidative properties of CuO are exploited toward ascorbic acid for amperometric measurement at micromolar levels in neutral buffer and acidic artificial sweat, at ultralow applied potential (− 5 mV vs. Au pseudo-reference electrode). Alternatively, measurement of the horizontal shift of redox peaks by cyclic voltammetry is also possible. Obtaining a limit of detection of 1.97 μM, sensitivity of 0.103 V log (μM)−1 of peak shift, and linear range of 10–150 μM, the effect of possible interfering species present in sweat is minimized, with no observable cross-reaction, thus maintaining a high degree of selectivity despite the absence of enzymes in the fabrication scheme. With a lateral flow approach for sample delivery, repeated measurements show recovery in few seconds, with relative standard deviation of about 20%, which can serve to detect increased loss or absence of vitamin, and yet be improved in future by optimized device designs. This sensor is envisioned as a promising component of wearable devices for e.g. non-invasive monitoring of micronutrient loss through sweat, comprising features of light weight, low cost, and easy fabrication needed for such application.Graphical Schematic depiction of the cyclic voltammetry signal change as the sweat flows over the sensor surface.

Electrochemical Detection of H2O2 Released from Prostate Cancer Cells Using Pt Nanoparticle-Decorated rGO–CNT Nanocomposite-Modified Screen-Printed Carbon Electrodes

In this study, we fabricated platinum nanoparticles (PtNP)-decorated, porous reduced graphene oxide (rGO)–carbon nanotube (CNT) nanocomposites on a PtNP-deposited screen-printed carbon electrode (PtNP/rGO–CNT/PtNP/SPCE) for detection of hydrogen peroxide (H2O2), which is released from prostate cancer cells LNCaP. The PtNP/rGO–CNT/PtNP/SPCE was fabricated by a simple electrochemical deposition and co-reduction method. In addition, the amperometric response of the PtNP/rGO–CNT/PtNP/SPCE electrode was evaluated through consecutive additions of H2O2 at an applied potential of 0.2 V (vs. Ag pseudo-reference electrode). As a result, the prepared PtNP/rGO–CNT/PtNP/SPCE showed good electrocatalytic activity toward H2O2 compared to bare SPCE, rGO–CNT/SPCE, PtNP/SPCE, and rGO–CNT/PtNP/SPCE. In addition, the PtNP/rGO–CNT/PtNP/SPCE electrode exhibited a sensitivity of 206 μA mM−1·cm−2 to H2O2 in a linear range of 25 to 1000 μM (R2 = 0.99). Moreover, the PtNP/rGO–CNT/PtNP/SPCE electrode was less sensitive to common interfering substances, such as ascorbic acid, uric acid, and glucose than H2O2. Finally, real-time monitoring of H2O2 released from LNCaP cells was successfully performed by this electrode. Therefore, we expect that the PtNP/rGO–CNT/PtNP/SPCE can be utilized as a promising electrochemical sensor for practical nonenzymatic detection of H2O2 in live cells or clinical analysis.

A graphenic and potentiometric sensor for monitoring the growth of bacterial biofilms

Biofilm development on surfaces represents one of the most challenging issues in the medical field. The irreversibility of the biofilm developmental process, and the peculiar characteristics of biofilms (including the low permeability to antibacterial compounds), make biofilm associated infections difficult to treat and prone to relapse. Thanks to the short response time, low-cost, easy fabrication and potential for miniaturization, electrochemical sensors are expected to play an important role in monitoring biofilm formation. Here, we describe the use of a potentiometric sensor based on functionalized reduced graphene oxide for monitoring the formation and growth of biofilms from three bacterial species (Escherichia coli, Pseudomonas aeruginosa and Staphylococcus aureus) in liquid and semisolid culture media. The Open Circuit Potential (OCP) between an Ag/AgCl pseudo reference electrode and a working electrode, both screen-printed on a flexible polyethylene terephthalate film, was measured during the growth of bacteria. The OCP decreased over time as bacteria grew on the sensor surface exhibiting a negative correlation.

Electrochemical detection of fentanyl with screen-printed carbon electrodes using square-wave adsorptive stripping voltammetry for forensic applications

Square-wave adsorptive stripping voltammetry (SWAdSV) is proposed as a fast, simple, and sensitive approach toward the detection, identification, and semi-quantitation of fentanyl in seized drug samples using screen-printed carbon electrodes. Electrochemical oxidation of fentanyl resulted in the formation of two anodic peaks, one at 0.75 V (peak I) and the second at 0.88 V (peak II) versus a Ag/AgCl pseudo reference electrode. Voltammetric measurements were conducted under optimized experimental conditions using fentanyl standards ranging from 0.076 μg/mL to 6.9 μg/mL, which resulted in a limit of detection of 0.037 μg/mL. Reproducibility was assessed as the average percent relative standard deviation of the slopes of calibration curves and was 2.6% for oxidation peak I and 7.4% for oxidation peak II in cell. Detection capabilities were assessed in a 5-mL cell and in a 100 microliter drop. Interference studies were conducted with cocaine, methamphetamine, quinine, caffeine, and acetaminophen. The mechanism for the electrochemical oxidation of fentanyl to norfentanyl is also described. Accuracy and suitability of the method for seized drug analysis was assessed using 11 simulated samples. Suitability of the method for future analysis of fentanyl in oral fluid was assessed.

Biosensing and electrochemical properties of flavin adenine dinucleotide (FAD)-Dependent glucose dehydrogenase (GDH) fused to a gold binding peptide

In the present work, direct electron transfer (DET) based biosensing system for the determination of glucose has been fabricated by utilizing gold binding peptide (GBP) fused flavin adenine dinucleotide-dependent glucose dehydrogenase (FAD-GDH) from Burkholderia cepacia. The GBP fused FAD-GDH was immobilized on the working electrode surface of screen-printed electrode (SPE) which consists of gold working electrode, a silver pseudo-reference electrode and a platinum counter electrode, to develop the biosensing system with compact design and favorable sensing ability. The bioelectrochemical and mechanical properties of GBP fused FAD-GDH (GDH-GBP) immobilized SPE (GDH-GBP/Au) were investigated. Here, the binding affinity of GDH-GBP on Au surface, was highly increased after fusion of gold binding peptide and its uniform monolayer was formed on Au surface. In the cyclic voltammetry (CV), GDH-GBP/Au displayed significantly high oxidative peak currents corresponding to glucose oxidation which is almost c.a. 10-fold enhanced value compared with that from native GDH immobilized SPE (GDH/Au). As well, GDH-GBP/Au has shown 92.37% of current retention after successive potential scans. In the chronoamperometry, its steady-state catalytic current was monitored in various conditions. The dynamic range of GDH-GBP/Au was shown to be 3-30 mM at 30 °C and exhibits high selectivity toward glucose in whole human blood. Additionally, temperature dependency of GDH-GBP/Au on DET capability was also investigated at 30-70 °C. Considering this efficient and stable glucose sensing with simple and easy sensor fabrication, GDH-GBP based sensing platform can provide new insight for future biosensor in research fields that rely on DET.

Electrochemical monitoring of the oxidative degradation of glyphosate with UV/H2O2

A voltammetric method optimized for the sensitive and reliable study of the UV/H2O2 oxidation of glyphosate (N- (phosphonomethyl) glycine) (GPS). The cell consisted of three electrodes in a compartment, silver wire coated with silver chloride as a pseudo reference electrode, platinum electrode as working electrode and stainless steel as auxiliary electrode. GPS cyclic voltammetric analysis suggests the formation of a GPS-Pt complex on the electrode surface, with an enhanced signal in perchloric acid between 1.3 to -1.3V; sensitive to protonation of the amino group, no response observed at pH values greater than 5.00. The oxidative degradation of GPS with UV/H2O2 is evident at pH 3.0, it exhibits first order pseudo kinetics with a half-life of 40 ± 4min. Two degradative routes are estimated depending on the concentration of H2O2: at low concentrations (H2O2/GPS 1: 100 ratio), the formation of sarcosine and then glycine is favored; at H2O2/GPS 1:20 and 1:10 ratios predominate the formation of glycine.

3D-printed lab-in-a-syringe voltammetric cell based on a working electrode modified with a highly efficient Ca-MOF sorbent for the determination of Hg(II)

This work combines, for the first time, 3D-printing technology and a highly efficient metal organic framework (Ca-MOF) as an electrode modifier to produce a novel fully integrated lab-in-a-syringe device for the sensitive determination of Hg(II) by anodic stripping voltammetry. The specific Ca-MOF ([Ca(H4L)(DMA)2]·2DMA where H6L is the N,N’-bis(2,4-dicarboxyphenyl)-oxalamide and DMA is the N,N-dimethylacetamide) shows an exceptional Hg(II) sorption capability over a wide pH range and its mechanism is elucidated via spectroscopic and X-ray diffraction studies. The voltammetric lab-in-a-syringe device is fabricated through a single-step process using a dual extruder 3D printer and is composed of a vessel integrating two thermoplastic conductive electrodes (serving as the counter and pseudo-reference electrodes) and of a small detachable 3D-printed syringe loaded with a graphite paste/Ca-MOF mixture (which serves as the working electrode). After optimization of the fabrication and operational variables, a limit of detection of 0.6 μg L−1 Hg(II) was achieved, which is comparable or lower than that of existing sensors (plastic 3D-printed, gold and MOF-based electrodes). The adoption of 3D printing technology in combination with the highly efficient Ca-MOF enables the fabrication of a simple, low-cost and sensitive electrochemical sensor for Hg(II), which is suitable for on-site applications.

Electropolishing of two kinds of bronze in a deep eutectic solvent (Ethaline)

Electropolishing of bronze was investigated in this work in an electrolyte based on a deep eutectic solvent, Ethaline (a eutectic mixture of choline chloride and ethylene glycol in a 1:2 M ratio, respectively). Two kinds of bronzes were used: AMPCO® 22 alloy (containing Al and Fe as alloying components) and AMPCO® 712 alloy (containing Sn, Ni, Pb and Zn as alloying components). Electrochemical responses of bronze in Ethaline were studied by cyclic voltammetry technique. It was established that the electropolishing of both bronzes can be performed at the temperature of 25 °C and anode electrode potential of 2.5 V (vs. Ag pseudo-reference electrode). The surface leveling and dissolution of microdefects were confirmed by atomic force microscopy and scanning electron microscopy. The electrochemical polishing of the surfaces of AMPCO® 22 and AMPCO® 712 bronzes was shown to result in an improvement in their corrosion resistance. Electrolytes based on deep eutectic solvents seem to be a promising replacement for traditional corrosive and environmentally hazardous acid aqueous solutions for electropolishing of copper alloys.

Disposable immunoplatforms for the simultaneous determination of biomarkers for neurodegenerative disorders using poly(amidoamine) dendrimer/gold nanoparticle nanocomposite.

Early diagnosis in primary care settings can increase access to therapies and their efficiency as well as reduce health care costs. In this context, we report in this paper the development of a disposable immunoplatform for the rapid and simultaneous determination of two protein biomarkers recently reported to be involved in the pathological process of neurodegenerative disorders (NDD), tau protein (tau), and TAR DNA-binding protein 43 (TDP-43). The methodology involves implementation of a sandwich-type immunoassay on the surface of dual screen-printed carbon electrodes (dSPCEs) electrochemically grafted with p-aminobenzoic acid (p-ABA), which allows the covalent immobilization of a gold nanoparticle-poly(amidoamine) (PAMAM) dendrimer nanocomposite (3D-Au-PAMAM). This scaffold was employed for the immobilization of the capture antibodies (CAbs). Detector antibodies labeled with horseradish peroxidase (HRP) and amperometric detection at - 0.20V (vs. Ag pseudo-reference electrode) using the H2O2/hydroquinone (HQ) system were used. The developed methodology exhibits high sensitivity and selectivity for determining the target proteins, with detection limits of 2.3 and 12.8pgmL-1 for tau and TDP-43, respectively. The simultaneous determination of tau and TDP-43 was accomplished in raw plasma samples and brain tissue extracts from healthy individuals and NDD-diagnosed patients. The analysis can be performed in just 1h using a simple one-step assay protocol and small sample amounts (5μL plasma and 2.5μg brain tissue extracts). Graphical abstract.

Flexible Iridium Oxide Based pH Sensor Integrated With Inductively Coupled Wireless Transmission System for Wearable Applications

This work presents a pH sensor platform combining the high performance of iridium oxide (IrOx) fabricated by cyclic voltammetry with inductively-coupled wireless (ICW) transmission. Data included presents flexible potentiometric pH sensors having IrOx as the sensing electrode and a cured Ag/AgCl paste as the pseudo-reference electrode; further investigations concerning performance tailoring via fabrication processes are shown. The fabricated sensors show the best performance with a probe surface area of ${1}\times {1}$ mm2, electrodeposited for 100 cyclic voltammetry (CV) sweeps, at 100 mV/s. The sensitivity of the fabricated sensor is typically in the range of 65–75 mV/pH, as tested using either pH 4–9 (six points) or 2–10 (five points) buffers. The sensors exhibiting those sensitivities in buffer solutions yielded a response from “artificial sweat” solutions differing by ~0.4–0.8 pH from a commercial glass pH electrode, while limit-of-quantification (LOQ) was measured to be ~0.04–0.08 pH. The sensing electrode shows a response time of less than 2 seconds and minimal hysteresis effects. Cationic interferences from up to 1M Na+ resulted in +3–8 mV/pH changes in sensitivity, depending on solution pH and probe, with minimal effects to LOQ. The performance under different bending conditions (0°, 30° at 55 mm radius, 45° at 37 mm, and 90° at 20 mm) of the flexible sensor probe show negligible variation. Finally, the presented sensors were integrated with an inductively coupled wireless (ICW) communication system for a demonstration of online monitoring. This sensor platform can easily be miniaturized due to a low count of necessary components and absence of onboard energy storage.

An electrochemical immunosensor using gold nanoparticles-PAMAM-nanostructured screen-printed carbon electrodes for tau protein determination in plasma and brain tissues from Alzheimer patients

This work reports a new sensitive strategy for the determination of tau protein, a hallmark of Alzheimer's disease (AD), involving a sandwich immunoassay and amperometric detection at disposable screen-printed carbon electrodes (SPCEs) modified with a gold nanoparticles-poly(amidoamine) (PAMAM) dendrimer nanocomposite (3D-Au-PAMAM) covalently immobilized onto electrografted p-aminobenzoic acid (p-ABA). The capture antibody (CAb) was immobilized by crosslinking with glutaraldehyde (GA) on the amino groups of the 3D-Au-PAMAM-p-ABA-SPCE, where tau protein was sandwiched with a secondary antibody labeled with horseradish peroxidase (HRP-DAb). Amperometry at −200 mV (vs the Ag pseudo-reference electrode) upon the addition of hydroquinone (HQ) as electron transfer mediator and H2O2 as the enzyme substrate was used to detect the immunocomplex formation. The great analytical performance of the immunosensor in terms of selectivity and low limit of detection (LOD) (1.7 pg mL−1) allowed the direct determination of the target protein in raw plasma samples and in brain tissue extracts from healthy individuals and post mortem diagnosed AD patients, using a simple and fast protocol.

Evaluation of a Manufacturable in-Situ Thin Film RTD Temperature Sensor for Lithium-Ion Batteries

As an in-situ temperature sensor for Li-Ion batteries, RTD’s (Resistance Temperature Detector) have a simple linear response to temperature and can accurately sense from -40 °C through thermal runaway initiation. An economical manufacturing method using a high-volume real-to-real flexible substrate is used that can combine the tab and sensors or produce sensor packages where either type integrate into a pouch cell in a similar way to the electrode tabs.Commercial 4.84 Ahr Li-ion NMC/graphite pouch cells were constructed incorporating a prototype thin film Nickel based RTD integrated tab at the center of the jelly-roll. This unified approach greatly simplifies the methods of construction avoiding the effects of around 2%1 strain due to the charge/discharge cycle. Much of the capital cost associated with adding new components to the manufacturing of Li-ion batteries are also avoided. Owing to the method of construction, the RTD has little to no impact on battery energy density which is very important to the competitive landscape.Experimental in-operando results illustrating the importance of sensor location are presented such as the case shown in Figure 1. More utilitarian and more extreme cases are also investigated comparing the in-situ vs ex-situ temperature responses to situations such as external short-circuit as well as normal operation modes. Progress on long term cycling will also be presented showing stability of sensor and any effects on battery performance.Incorporation of additional sensors are also discussed such as reference electrodes. In a recent review2 cathode materials such as LiFePO4 make a more suitable long term pseudo-reference electrode material than the much more difficult to handle Li metal alloys. A sensor package of the type described here allows for simple addition of a pseudo-reference electrode utilizing cathode material. 1 Drew Joseph Pereira et al. Meet. Abstr. 2019;MA2019-02:428 2 Rinaldo Raccichini et al. Batteries 2019, 5, 12 Figure 1

Development of a Hydrogen Contaminant Detector

Hydrogen Refueling Stations (HRSs) are being deployed at scale in California, Japan, Europe and South Korea to support the introduction of Fuel Cell Vehicles.1 The hydrogen dispensed to the fuel cell should meet SAE J2719 standards for the maximum allowable concentration of various impurities.2 While HRSs can have their fuel analyzed periodically to verify that they meet the strict fuel quality standards, it would be greatly advantageous to have an in-linemonitor that can alert the user to any potential fuel quality problems. Los Alamos National Laboratory has been developing a low-cost hydrogen contaminant detector (HCD) that can be deployed at the HRS and is capable of detecting CO and H2S impurities in the hydrogen fuel.The LANL HCD technology is based on a hydrogen pumping cell that utilizes a high loaded counter/pseudo-reference electrode and a low loaded working/sensing electrode.3 This hydrogen pumping cell, illustrated in Figure 1, consists of a Nafion® 117 membrane that is in contact with a water reservoir and two gas diffusion electrodes with wicking tabs that extend into the water reservoir. The active electrode area (≈ 1cm2) is exposed to dry hydrogen gas that first flows over the sensing electrode and then over the pseudo-reference electrode. The HCD is operated in a constant potential mode (0.1V) where the pumping current can be related to the purity of hydrogen. A voltage pulse (1.5V for 30s) is applied periodically (e.g. every 15mins) to clean up any contaminants that are adsorbed on the working electrode and reset the HCD current to the baseline.The LANL HCD technology was installed at the H2Frontier HRS in Burbank, California and operated under real-world conditions. Calibration was performed on this HCD with certified CO/H2 gas, and its sensitivity to low concentrations of CO was demonstrated (Figure 2). The data shown in Figure 2 plots the current at 0.1V at the end of each 15-minute interval, just before the application of the 1.5V pulse. The HCD baseline and response were tracked intermittently for a period of over a year and demonstrated exceptional baseline stability and sensitivity to the SAE J2719 allowable limit of 100ppb CO. An analysis of the field-testing data will be presented along with the response to low concentrations (4 - 100 ppb) of H2S in H2.The current LANL HCD operates at slightly above ambient temperature (30 °C) and at ambient pressures. Any change in the flow rate of H2 (usually 100sccm) over the HCD, or a drying of the water reservoir can result in a baseline drift. LANL is currently working on a HCD technology that can function without a humidification system and thus is immune to flow rate changes. Moreover, this system could be operated under pressure and at varying temperatures and has the potential to be deployed in-line just before the fueling nozzle. The development of membranes with sufficient protonic conductivity to operate in dry hydrogen and their sensitivity to CO in the H2 will also be presented in this talk. Acknowledgements This research is supported by the U.S. Department of Energy Fuel Cell Technologies Office, through the Safety, Codes & Standards (SCS) sub-program (Project Manager: Laura Hill).REFERENCES: 1. Hardman, G. Tall, Int. J. Hydrogen Energy, 43, 17857-17866 (2018).2. San Marchi, E. S. Hecht, I. W. Ekoto, K. M. Groth, C. LaFleur, B. P. Somerday, R. Mukundan, T. Rockward, T; J. Keller, C. W. James, Int. J. Hydrogen Energy, 42(11), 7263-7274 (2017)3. Brosha, T. Rockward, C. J. Romero, M. S. Wilson, C. Kreller and R. Mukundan, U.S Patent # 10,490,833 (2019). Figure 1

Composite Ag/AgCl/KCl Screen-Printed Reference Electrode

The drive for automation in multiple industries has generated unmet demand for low-cost, disposable sensors with short response times for routine electrochemical sensing. Glass ion selective electrodes containing a liquid electrolyte have remained the standard for decades, despite their fragile, bulky nature and regular maintenance requirements restricting their suitability for long-term in-situ monitoring. Advances in printing technologies have enabled solid thick film electrodes to emerge as a possible solution to these challenges.Screen printing can be used to mass-produce units of simple, modifiable, and reproducible sensors using minimal materials, increasing portability and reducing fabrication costs. Traditional glass electrode materials are not suitable for printed fabrication, so new materials and designs are required to deliver reliable and robust printed sensors for a variety of industries. While electrochemical sensors can be used to monitor key aqueous environmental indicators, including pH, heavy metals, and oxidation-reduction potential, they often require a stable reference electrode to gain meaningful measurements. Many previous printed electrochemical sensors have relied on a pseudo-reference electrode containing no stabilising electrolyte, however any resultant potential variation can affect measurement accuracy in unknown solutions.In this study, a new screen-printed "true" reference electrode was developed based on Ag/AgCl and a solid composite KCl electrolyte. The electrode lifetime was tested in a 0.1 M K2SO4 solution, and open circuit potential (OCP) stability was tested in H2SO4, KOH, alkali cations (Li+, K+, Na+), halogen anions (F-, Cl-, Br-, I-), KMnO4, H2O2, ascorbic acid, KCl, and standard pH buffers (pH 4, 7, 10). The initial stabilisation and water permeation was monitored via electrochemical impedance spectroscopy (EIS).The observed electrode lifetime in 0.1 M K2SO4 was up to 39 days, with less than 5 mV deviation from the theoretical value (-45 mV vs SCE) and a shelf life of over 1 year. According to the EIS and OCP data, the electrode typically stabilised within 2 hours. In the stability tests, the electrode potential did not deviate more than 10 mV from the theoretical value in the presence of KOH, alkali cations, halogen anions, ascorbic acid, KCl, and standard pH buffers. The electrode was also stable in H2O2 at concentrations up to 0.1 M. On average, the electrode potential was 12 mV below the theoretical value in 10% H2SO4 and was significantly below the theoretical value in 0.1 M KMnO4.The results of this study suggest that this screen-printed reference electrode is suitable for use in electroanalytical systems in most tested chemical environments. The relatively long lifetime and stability of the electrode allows it to be used in a variety of applications, including environmental monitoring, laboratory measurements, and industrial process monitoring, as part of a printed sensor system.

Laser Induced Porous 3D-Graphene/MnO2 Electrodes Embedded in a Paper-Based Microfluidic Device for Point-of-Care Testing of Neurotransmitters in Finger-Prick Blood

Microfluidic electrochemical paper-based devices (ePADS) are a promising platform for point of care (PoC) diagnostics. In this work, we present a microfluidic device for home-based detection of the neurotransmitter dopamine, which is crucial for further understanding of nervous system disorders such as Parkinson’s disease and other chronic mental illnesses including schizophrenia. Our device features a 3-electrode ePAD, where the electrodes are aligned along a wax-defined microfluidic channel, in a capillary flow device which uses Whatman filter paper as the porous wicking substrate. The working electrode was embedded directly into the Whatman filter-paper by using the paper as a precursor for laser-induced-graphene (LIG). To obtain LIG, we treat the Whatman filter in commercial flame-retardant spray and then treat with a CO2 infrared laser, this treatment produces a highly conductive, 3D porous graphene network with high surface area and electrical conductivity, directly in the paper matrix. The working and counter LIG electrodes are patterned by laser alongside the wax-defined microfluidic channel as shown in figure 1a, and a third reference electrode was defined in inkjet-printed silver (Dimatix DMP-2831) and then treated in commercial bleach to obtain an Ag/AgCl pseudo-reference electrode. The charge-transfer kinetics of the LIG surface was obtained by cyclic voltammetry (figure 1b) of the LIG electrode in a 3-electrode setup (platinum counter electrode and Ag/AgCl reference electrode in 3.0 M KCl). The LIG electrode was cycled in 0.1 M aqueous solution of KCl consisting of 5mM ferro-ferricyanide redox couple. The Randles–Sevcik analysis was used to obtain an electroactive surface area of 1.97 mm2 (2.5 times higher than the geometrical surface area) and the analysis also showed a rapid electron transport kinetic.The working electrode was modified from the pristine LIG by initially (i) chemical activation in 2 molar urea solution to introduce nitrogen-functional groups on the LIG and (ii) electrodeposition of MnO2 nanoparticles over the N-functionalised graphene to produce a sensing electrode with low background current and rapid electron transfer characteristics.Crucially for microfluidic devices to be used as a real PoC device, the patient needs to be able to simply deposit the biomarker. For neurotransmitter analysis, blood/plasma from a finger-prick enables a plausible route for sampling. Here, we show a novel blood sampling design of the ePAD cassette (holder) which enables rapid and low volume blood sampling, which is shown in figure 1c. The holder includes a hydrophilic microfluidic channel which rapidly samples 5 μL blood from a finger-prick droplet. An air vent at the channel end enables the channel to be filled to exactly 5.0 μL but not over-filled. The blood is prevented from entering the ePAD by a removable adhesive tab, which also covers a reservoir filled with running buffer (RB), (TBS + 0.05% Tween + 0.5% BSA). When the channel is filled with blood, the removal of the adhesive tab enables release of the defined volume of blood and RB into (i) a Pall Cytosep 1668 blood filtration membrane and then (ii) into the wax-patterned lateral flow channel of the ePAD device for rapid electrochemical detection of dopamine, creatinine and uric acid by square wave anodic stripping voltammetry. Figure 1

Microscale Electrochemical Platform for Temperature-Dependent Biomolecular Research and Biosensing

Introduction Detection of various types of biomolecules, as well as the measurement of biomolecular properties and how those properties change under different conditions, are important processes in applied areas such as drug discovery, biomanufacturing, disease screening, and therapeutic treatment monitoring. While many instrumental techniques characterize biomolecules in free solution, biosensors often use immobilized probes to monitor the presence and properties of biomolecular species.A miniature platform has been designed and fabricated as a prototype to perform temperature-dependent electrochemical analyses on small-volume (< 10 µL) biomolecular samples [1]. Square wave voltammetry signals have been employed to track binding to and conformational changes of immobilized forms of DNA. The fabrication methods for the platforms are scalable to array configurations which could offer an efficient and automated electronic tool for biochemical research and healthcare applications. Fabrication and Features of the Microdevices Each microdevice consists of a planar set of three surface electrodes, integrated components for temperature control and a top-surface sample cell (Figure 1). The devices are produced in wafer runs with techniques that are compatible with producing single units or multi-element arrays with individual addressability. Fabrication begins with 100 mm fused-silica wafers on which serpentine Pt films are deposited for use as both resistive microheaters and platinum resistance thermometers (PRTs). Lithography and etch-back are utilized for processing the sputter-deposited 220 nm Pt layer. A thin (~ 1 µm) high-quality PECVD SiO2 layer is then deposited over the Pt serpentine, and annealed to 800°C, to allow for effective transfer of heat while also providing defect-free electrical insulation between the microheater/PRT and top-surface electrodes. A liftoff process patterns the three planar surface electrodes (of thickness 200 nm over 20 nm Ti) in Pt for the pseudo-reference and counter electrodes, and Au for the working electrode. Data Acquisition For acquisition of temperature-dependent electrochemical profiles, PID-controlled from ~ 10°C to ~ 70°C using the calibrated PRT, two types of mounting assemblies have been employed: one that uses ambient cooling with resistive heating ramps, and one that uses a programmed commercial thermoelectric for cooling and heating [2]. Samples are prepared over the top electrodes within a PDMS containment volume. Biomolecular probes are immobilized to the Au working electrode via thiol attachment, with 6-mercaptohexanol backfill. Methylene blue tags are included on studied biomolecules to enhance the electrochemical (current) signals measured as a function of voltage (and temperature) with square wave voltammetry (Figure 2). All aspects of the electrochemical measurements and temperature control are computer automated. Results and Conclusions A number of sample systems have been used to demonstrate the capabilities of this microscale electronic technology. Single-strand DNA has been immobilized and then the melting temperature, Tm, was determined for binding of a fully complementary strand, one with a single base-pair mismatch, and one involving two mismatches. Tm shifts to lower temperatures by 5.1°C and 7.3°C for the mismatches indicate the ability to detect single nucleotide polymorphisms (SNPs) [1]. The binding of ligands (diaminazene aceturate, DMZ, and proflavine) to surface-immobilized duplex DNA have also been examined (Figure 3) to show their stabilizing influence, as Tm was increased by 11.0°C and 13.5°C for these intercalator and minor-groove binders, respectively [2]. Related measurements are also being performed on other DNA species, including poly-thymine, streptavidin aptamer and G-quadruplexes. Further development of the approach, with incorporation of microfluidics for array formats, offers the opportunity of a sensitive research and sensing tool for efficient analyses of biomolecular interactions and response to chemical and physical stressors, without interference phenomena like those that can be encountered in fluorescence-based techniques.

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.

(Invited) Agriculture 4.0: Development of Chemical and Biochemical Smart Sensors Systems for Sustainable Food Production

Introduction With the global population expected to grow to over 9.6 billion by 2050 it is a projected that a 50-60 % increase in food production will be required. In addition, the increasing wealth of the BRIC (Brazil, Russia, India and China) countries is driving the transition from traditional to more westernized high protein diets. A key challenge then, going forward, will be to close the food gap. This must be achieved against the backdrop of climate change & desertification, labor shortages and competition for energy, land & resources. It is clear then, that addressing this challenge will require the development of more efficient and sustainable food production techniques and processes [1]. Digitizing the entire food chain using smart sensor systems would help enable such an approach. This convergence between the Internet of Things (IoT) and the agri-food industry requires sensor systems and technologies that provide real time data to producers and processers; required for rapid, but informed, decision making. To this end, we are using microelectronic fabrication approaches to develop advanced end-to-end chemical and bio-chemical nanosensor sensor systems specifically tailored to the needs of both producers and processors [2]. These sensor systems are being designed to be used by non-specialists and be rugged enough for field deployment [3]. Sensor Development On-chip devices containing six gold microband electrodes, a gold counter and a platinum pseudo-reference electrodes were fabricated using standard nanofabrication processes on Si/SiO2 substrates as described in detail previously [4]. A custom built Teflon cell permit analysis of small sample volumes (~50 µL), see Fig 1. Gold contact pads were configured on-chip to permit electrical connection to the potentiostat via a microSD port. Chips could be easily plugged in/out enabling rapid analysis of multiple samples. Optical micrographs were acquired using a calibrated microscope (Axioskop II, Carl Zeiss Ltd.) with a charge-coupled detector camera (CCD; DEI-750, Optronics). Electrochemical experiments were carried out using an Autolab Potentiostat/ Galvanostat PGSTAT128N (Metrohm Ltd, Utrecht, The Netherlands) controlled by the Autolab NOVA software. All experiments employed a standard three-electrode cell configuration using a single gold nanoband as the working electrode, versus the on-chip gold counter and platinum pseudo-reference electrodes,. In this study, microband sensors were applied to the detection of Bovine IgG and anti-Fasciola antibodies in serum samples. On-chip microband electrodes were first cleaned using a mixed solvent clean process (acetone, isopropyl alcohol and DI water) for 15 minutes. Cyclic voltammetry was then employed for electropolymerisation of o-aminobenzoic acid (o-ABA, 50 mM in 0.5 M H2SO4) to create a carboxylic terminated polymer layer at the gold electrode surface. A fresh NHS/EDC mixture was prepared in 10 mM acetate buffer, pH 4.0 and subsequently deposited onto the polymerised electrodes, for approximately 30 min, to activate the surface. Concerning IgG, activated electrodes were modified with 1 ng/ml anti-Bovine IgG in 10 mM sodium acetate buffer, pH 4.0 (AB). Anti-IgG was incubated on the electrode for 15 min.. Following deposition of these capture biomolecules, unbound active-sites on all electrodes were blocked with ethanolamine (1 M) for 20 minutes. A secondary blocking of 1 % NANNY care® goat based milk in PBS was used for the IgG assay, for a comparison. Results and Conclusions Electrochemical Impedance Spectroscopy (EIS) techniques allows for the monitoring of the increase of the Rct signal and was used to interrogate antigen binding. The reduction of the electron transfer causes an increase in the Rct as seen in Figure 2. Following exposure to igG antibodies an increase in impedance was observed. A semi-logarithmic relationship exists between the concentration of the target and the change in Rct. This correlates with the trend seen in the ELISA calibration plot. The Nyquist plot shows a much larger change in signal between each concentration of Bo IgG than the CV and thus EIS is widely used for evaluating immunosensors such as these.