Lactate Sensor Research Articles

A Dual‐Functional Lactate Sensor Based on Silver Nanoparticle‐coated Carbon Dots

Herein, a silver nanoparticle‐coated carbon dots (Ag‐CDs) was fabricated to detect lactate using colorimetric and fluorometric methods. When the Ag‐CDs were decomposed by H2O2 produced from the lactate oxidase (LOx) catalytic reaction, the color of the Ag‐CDs solution changed from yellowish to transparent, and the fluorescence of the quenched CDs was simultaneously recovered. Silver nanoparticles (Ag NPs) decomposition was confirmed by X‐ray photoelectron spectroscopy, revealing an increase in AgN bond peaks due to ionization of Ag NPs. The Ag‐CDs sensor could recognize lactate specifically, even in the presence of numerous potentially interfering biomarkers. The fabricated sensor displayed good sensitivity and selectivity, with a low detection limit of 0.6 μM and 10 μM for H2O2 and lactate sensing, respectively. Finally, the Ag‐CDs sensor could be applied to determination of lactate to intracellular imaging in 4T1 cells using confocal microscopy.

A novel device for detecting anaerobic threshold using sweat lactate during exercise

The lactate threshold (LT1), which is defined as the first rise in lactate concentration during incremental exercise, has not been non-invasively and conveniently determined in a clinical setting. We aimed to visualize changes in lactate concentration in sweat during exercise using our wearable lactate sensor and investigate the relationship between the lactate threshold (LT1) and ventilatory threshold (VT1). Twenty-three healthy subjects and 42 patients with cardiovascular diseases (CVDs) were enrolled. During exercise, the dynamic changes in lactate values in sweat were visualized in real-time with a sharp continuous increase up to volitional exhaustion and a gradual decrease during the recovery period. The LT1 in sweat was well correlated with the LT1 in blood and the VT1 (r = 0.92 and 0.71, respectively). In addition, the Bland–Altman plot described no bias between the mean values (mean differences: − 4.5 and 2.5 W, respectively). Continuous monitoring of lactate concentrations during exercise can provide additional information for detecting the VT1.

MWCNTs/PEDOT: PSS Composite as Guiding Layer on Screen-Printed Carbon Electrode for Linear Range Lactate Detection

The emerging field of nanomaterials could be utilized in biosensors for addressing challenging applications due to its abundant strategic properties. Herein, a composite of multi-walled carbon nanotubes (MWCNTs) and poly (3,4-ethylene dioxythiophene)-poly(styrene sulfonate) (PEDOT: PSS) was synthesized, and used as guiding layer on screen printed carbon electrode (SPCE) for lactate detection. Lactate plays a substantial role in health care applications. Fundamental characteristics of the composite modified SPCE were studied using FE-SEM, EDS, and Raman spectroscopy and sensor performance was analyzed by electrochemical methods. AFM was used to thoroughly study the screen-printed carbon electrode and as-deposited MWCNT/PEDOT:PSS composite film topography. Various parameters were optimized to achieve the best performance of lactate sensor. The developed sensor provided a wide linear range response (R2 = 0.97) from 1 mM to 10 mM for buffer samples with 35.224 μA mM−1 sensitivity. The proposed sensor was applied for to detect lactate in cancer (MCF-7) cells media. A calculated LOD 4.0 ± 5 μM (S/N) was achieved and the results showed a linear response up to 7 mM. As a result, the advanced approach could be applied for the detection of a range of metabolites using respective enzymes. This approach could open on-line lactate detection in organ-on-a-chip applications.

Nanowire‐Based Soft Wearable Human–Machine Interfaces for Future Virtual and Augmented Reality Applications

AbstractA virtual world has now become a reality as augmented reality (AR) and virtual reality (VR) technology become commercially available. Similar to how humans interact with the physical world, AR and VR systems rely on human–machine interface (HMI) sensors to interact with the virtual world. Currently, this is achieved via state of‐the‐art wearable visual and auditory tools that are rigid, bulky, and burdensome, thereby causing discomfort during practical application. To this end, a skin sensory interface has the potential to serve as the next‐generation AR/VR technology because skin‐like wearable sensors have advantages in that they can be ultrathin, ultra‐soft, conformal, and imperceptible, which provides the ultimate comfort and immersive experience for users. In this progress report, nanowire‐based soft wearable HMI sensors including acoustic, strain, pressure sensors, and physiological sensors are reviewed that may be adopted as skin sensory inputs in future AR/VR systems. Further, nanowire‐based soft contact lenses, haptic force, and thermal and vibration actuators are covered as potential means of feedback for future AR/VR systems. Considering the possible effects of the virtual world on human health, skin‐like wearable artery pulses, glucose, and lactate sensors are also described, which may enable imperceptible health monitoring during future AR/VR practices.

Coupling of Silk Fibroin Nanofibrils Enzymatic Membrane with Ultra-Thin PtNPs/Graphene Film to Acquire Long and Stable On-Skin Sweat Glucose and Lactate Sensing.

The applications of enzymatic biosensors are largely limited by their relatively poor stability and short lifespan. Herein, a bio-active porous enzymatic nanofiber (PEN) membrane composed of silk fibroin nanofibrils (SFNFs) and enzymes is developed to effectively retain the enzymes in the 3D space. The 3D functional scaffolds formed by SFNFs can immobilize enzymes and provide a large surface area for molecular/ion diffusion and biochemical reactions. The PEN membrane is subsequently attached to an ultra-thin PtNPs/graphene (Pt-G) nanocomposite film to facilitate the electron transport between the enzymes and electrodes, permitting highly effective glucose and lactate sensing with long and stable performance. The as-assembled glucose and lactate sensors demonstrate high sensitivity, good cyclic reproducibility, and in particular long-term stability of up to 25 and 23.6 h, respectively. These glucose sensors have a working life that is ≈1.25-times longer than that of the best available sensors reported so far. Moreover, a wearable platform based on the sensors is developed for real-time analysis of sweat during outdoor exercising to transmit signals to a mobile handset. The high sensitivity, comfort and long-term stability of the device can benefit for long-term in-line surveillance of physiological conditions.

Continuous Sweat Lactate Sensor Based on Screen-Printed MgO-Templated Carbon with Integrated Flow System

Lactate has long been known as the indicator for distinguishing aerobic and anaerobic metabolism, and thus is a useful marker for metabolic disorders and the state of exercise [1]. Especially, the continuous monitoring of sweat lactate is a useful non-invasive method for real-time tracking the progress of exercise, e.g. of athletes, or of physical therapy patients. In biosensors for continuous monitoring, the biological recognition element, e.g. lactate oxidase (LOx) has to be immobilized on the electrode. Our group previously reported of a glucose sensor in which glucose dehydrogenase (GDH) was immobilized covalently on screen-printed MgO-templated mesoporous carbon (MgOC) modified by graft polymerization, and showed improved stability [2]. Likewise, in this study, radicals were introduced on the surface of the MgOC by electron beam irradiation. Next, glycidyl methacrylate (GMA) was polymerized on the radicalized MgOC, which resulted in epoxy groups on the surface. Ink was prepared by dispersing this poly(GMA)-MgOC (GMgOC) and a polyvinylidene difluoride (PVdF) derivative in N-methyl-2-pyrrolidone (NMP). The ink then was used for the final layer of the working electrode (WE) of a screen-printed 3-electrode strip. Next, 1,2-naphthoquinone was deposited on the WE as mediator, which is insoluble in water and thus is unlikely to dissolve into sweat during the measurement. Finally, LOx derived from Enterococcus faecium was immobilized on the WE by binding covalently to the epoxy groups. This combination of LOx immobilized on screen-printed GMgOC and naphthoquinone promises the construction of stable lactate biosensors suitable for continuous measurements.Furthermore, in this study, two other points were considered for the design and construction of the sensor device. Firstly, the sensor should be constantly supplied with a minimum amount of sweat, without the supply being interrupted. Secondly, the materials and texture of the parts of the sensor in direct contact with the skin should not be irritating. An integrated flow system can solve both points; it ensures a constant supply of sweat, and limits the material and texture in direct contact with the skin. For the construction of the flow system, first, a mold was formed on a silicon wafer using an epoxy-based negative photoresist. Next a degassed mixture of polydimethylsiloxane (PDMS) monomer and catalyst was poured over the mold and cured at 80°C for 1 hour.In first trials, the sensor and flow system were pressed together tightly onto artificial sweat glands. Leakage was prevented by constructing a connector for the sensor out of conducting adhesive tape. Potassium phosphate buffer (100 mM, pH 7.0) containing various amounts of sodium lactate were pumped through the system at 80 µL/min using a syringe pump. Figure 1a shows the response of this system to varying concentrations of lactate. The spikes at the change of concentration are due to a change in the flow when switching the syringe. The delay between the switch of the syringe and the change in response current is due to the lag time until the lactate reached the sensor. The plot of the response current vs the lactate concentration (Fig. 1b) shows a concentration dependent response up to 20 mM lactate. These results indicate that this system is suitable for measuring sweat lactate continuously. Acknowledgement: This work was partially supported by JST-ASTEP Grant Number JPMJTS1513, JSPS Grant Number 17H02162 and Private University Research Branding Project (2017-2019) from Ministry of Education, Culture, Sports, Science and Technology, and Tokyo University of Science Grant for President's Research Promotion． References [1] M.E. Payne, et al.; Sci. Rep., 9, 13720 (2019).[2] I. Shitanda, et al.; Bull. Chem. Soc. Jpn., 93, 32-36 (2020). Figure 1

Abstract 13134: A Novel Device for Detecting Anaerobic Threshold Using Sweat Lactate Acid During Exercise

Background: Continuous monitoring of lactate acid (LA) levels during exercise has not been possible. We aimed to visualize real-time changes in LA levels in sweat during exercise and to investigate the relationship between the lactate threshold in sweat (sLT) and LT in blood (bLT) and the ventilatory threshold (VT). Methods: Twenty-three healthy subjects (age: median; 20 [interquartile range; 20, 21] years old) and 22 consecutive patients (age: 65 [57, 73] years old) with cardiovascular diseases (CVDs) underwent exercise tests with a RAMP protocol ergometer, simultaneously monitoring changes in the LA values in sweat using a wearable lactate sensor. The sLT was identified as the first significant increase in LA above baseline based on the graphical plots and change finder scores calculated by the Change Finder algorithm using the time-series data of the LA values during exercise. We applied the Bland-Altman method to verify similarities for each threshold. Results: During exercise, the dynamic changes in LA values in sweat were successfully visualized in real-time with a sharp continuous increase up to volitional exhaustion and a gradual decrease in the recovery period. In healthy subjects, the work rate (WR) at the sLT (WR-sLT) was substantially correlated with the WR at the bLT (r=0.90). The Bland-Altman plot described a strong agreement (the mean difference: -3.0 watt). Among 22 patients, there were 8 patients whose LA values in sweat were not measured due to a non-response in the sensor. In the remaining 13 cases, except for a patient in whom the VT could not be detected, the sLT was well correlated with bLT and VT (r=0.81 and 0.65, respectively); the mean differences were -8.3 and 3.4, and there was no bias between the mean values. Finally, the logistic regression analysis revealed that a non-response in the sensor was associated with NYHA 3/4 and low peak VO2 (Odds ratio [OR] 0.01 [95% confidence interval (CI); 0.00-0.13], OR 1.45 [95% CI; 1.09-2.20], respectively) Conclusions: Our wearable device enabled a continuous and real-time LA measurement in sweat during incremental exercise in a subset of patients with CVDs as well as healthy subjects. This can provide additional information for detecting the VT.

Non-enzymatic lactate detection by an extended-gate type organic field effect transistor

A non-enzymatic chemical sensor for lactate (LA) detection based on an extended-gate type organic field-effect transistor (OFET) is reported. The proposed device is composed of an extended-gate electrode modified by an artificial receptor phenylboronic acid derivative as the detection part and the OFET as the drive part. The mechanism of LA detection relies on the changes in transfer characteristics of the OFET upon dynamic covalent bonding with LA. After evaluating the ability of the device to detect LA, it was applied to the selectivity study. This report proved the possibility of using the OFET-based sensor device for non-enzymatic LA detection.

Optimization of printed sensors to monitor sodium, ammonium, and lactate in sweat

We describe the optimization of a flexible printed electrochemical sensing platform to monitor sodium ion (Na+), ammonium ion (NH4+), and lactate in human sweat. We used previously reported material systems and adapted them to scalable fabrication techniques. In the case of potentiometric Na+ and NH4+ sensors, ion-selective electrodes (ISEs) required minimum optimization beyond previously reported protocols, while a reference electrode had to be modified in order to achieve a stable response. We incorporated a carbon nanotube (CNT) layer between the membrane and the silver/silver chloride (Ag/AgCl) layer to act as a surface for adsorption and retention of Cl−. The resulting reference electrode showed minimal potential variation up to 0.08 mV in the solutions with Cl concentration varying from 0.1 mM to 100 mM. Increasing the ionophore content in the NH4+ ISE sensing membrane eliminated an offset in the potential readout, while incorporating CNTs into the sensing membranes had a marginal effect on the sensitivity of both Na+ and NH4+ sensors. Na+ and NH4+ sensors showed a stable near-Nernstian response with sensitivities of 60.0 ± 4.0 mV and 56.2 ± 2.3 mV, respectively, long-term stability for at least 60 min of continuous operation, and selectivity to Na+ and NH4+. For the lactate sensor, we compared the performance of the tetrathiafulvalene mediated lactate oxidase based working electrode with and without diffusion-limiting polyvinyl chloride membrane. The working electrodes with and without the membrane showed sensitivities of 3.28 ± 8 A/mM and 0.43 ± 0.11 μA/mM with a linear range up to 20 mM and 30 mM lactate, respectively.

Selective Nonenzymatic Amperometric Detection of Lactic Acid in Human Sweat Utilizing a Multi-Walled Carbon Nanotube (MWCNT)-Polypyrrole Core-Shell Nanowire.

Lactic acid plays an important role as a biochemical indicator for sports medicine and clinical diagnosis. The detection of lactic acid in sweat is a promising technique without any intrusive inconvenience or risk of infection. In this study, we present a selective nonenzymatic amperometric detection method for lactic acid in human sweat utilizing a multi-wall carbon nanotube (MWCNT)-polypyrrole core-shell nanowire. Because polypyrrole is a p-type conducting polymer, onto which anions are exclusively doped, leading to charge transfer, it offers selective detection for lactate anions at a specific potential, while being inert to the neutral and cationic species contained in human sweat. A chronoamperometric study reveals good sensing performance for lactic acid with a high sensitivity of 2.9 μA mM−1 cm−2 and detection limit of 51 μM. Furthermore, the MWCNT-polypyrrole nanowire exhibits excellent selectivity for lactic acid over interfering species, such as sodium chloride, glucose, urea, and riboflavin, which coexist with lactic acid in sweat. Finally, a nonenzymatic amperometric sensor for the selective detection of lactic acid in human sweat is demonstrated on commercial flexible electrodes. The results demonstrate the potential applications of the MWCNT-polypyrrole core-shell nanowire as a nonenzymatic amperometric lactate sensor.

Bicarbonate activates glycolysis and lactate production in corneal endothelial cells by increased pHi

Recent studies have shown that lactate coupled water flux is the underlying mechanism of the corneal endothelial pump, which is highly dependent on the presence of bicarbonate. In this study we test the hypothesis that the increased intracellular pH (pHi) caused by bicarbonate stimulates glycolytic activity and the production of lactate by endothelial cells. Primary cultures of bovine corneal endothelial cells (BCEC) were incubated in bicarbonate-free (BF) ringer, a high [HEPES] ringer, and bicarbonate-rich (BR) ringer all at pH 7.5. Lactate production and glucose consumption were greatest in BR>HEPES >BF. Similarly, pHi was greatest in BR>HEPES>BF. Increasing pHi with NH4Cl also increased lactate production in BF or BR, indicating that the increased lactate production in BR is not due to HCO3− itself. Glucose transport capacity, as measured by 2-N-(7-Nitrobenz-2-oxa-1,3-diazol-4-yl)Amino-2-Deoxyglucose (2-NBDG) uptake was unaffected by the three incubation conditions. Using Laconic, a FRET sensor for lactate, we found that intracellular [lactate] increased immediately and transiently when cells were switched from BF to BR perfusion indicating increased lactate production with subsequent matching of efflux. Moreover, induction of acute lactate influx by perfusion pulses of 10 mM lactate increased intracellular [lactate] significantly faster in BF than in BR, consistent with higher lactate production and efflux in BR. In summary, our results indicate that glycolytic flux and lactate production increase in BR due to increased pHi, consistent with the well-known pH sensitivity of phosphofructokinase, the rate limiting enzyme in glycolysis.

Abstract 6317: 3D multicellular coculture platform enables rapid engineering of tumor-homing bacteria

Abstract Engineering tumor-homing bacteria to locally deliver therapeutics is an emerging approach for next-generation living therapy for cancer. However, the lack of physiologically-relevant in vitro testing environments to rapidly and quantitatively characterize complex multicellular interactions of bacteria and cancer cells is a major bottleneck for clinical translation. We recently developed a 3D multicellular coculture platform that grow various bacteria within the necrotic core of multicellular spheroids, recapitulating selective bacterial growth in solid tumor in vivo. Tumor spheroids were generated from CT26 colorectal cell lines in a round-bottom low-adhesion 96 well plate. Bacteria were inoculated into the media containing tumor spheroids and gentamicin, a poorly diffusible antimicrobial agent, were added to confine bacterial growth to spheroid cores. Time-lapse microscopy was utilized to track growth dynamics and spatial profiles. To validate efficacy, mice with hind flank tumors were treated with engineered bacteria and measured for the therapeutic effect on tumor size. The multicellular coculture platform allowed for parallel and continuous culture of bacteria in tumor spheroids. Notably, bacteria stably grew within the spheroid necrotic cores, representing tumor colonization in animals and patients. Leveraging the high throughput nature of the platform, we screened a library of ten cytotoxic molecules delivered by clinically relevant tumor-homing S. typhimurium. We validated similarities in efficacies using a syngeneic mouse model and identified novel therapeutic candidates that reduced tumor growth significantly. Additionally, we utilized the tumor spheroid to construct bacterial biosensors. As tumor microenvironment such as oxygen, pH, and metabolite gradients are recapitulated in the 3D spheroid setting, we tested hypoxia, acid and lactate sensors and confirmed their activity in tumor environment. Constructing an AND-gate logic circuit for bacterial growth using synthetic biology tools, we rapidly tested the bacterial containment capability to the spheroid cores. This strain demonstrated a 100-fold reduction in off-target colonization compared to the wildtype strain in an animal model. Rapid and quantitative characterization of dynamical bacteria-cancer cell interactions can predict therapeutic performance more accurately. We hope this scalable approach serves to accelerate future clinical development of bacterial applications for cancer therapy. Citation Format: Tetsuhiro Harimoto, Zakary Singer, Oscar Velazquez, Joanna Zhang, Samuel Castro, Taylor Hinchliffe, William Mather, Tal Danino. 3D multicellular coculture platform enables rapid engineering of tumor-homing bacteria [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6317.

Evaluation of an Anti-Thrombotic Continuous Lactate and Blood Pressure Monitoring Catheter in an In Vivo Piglet Model undergoing Open-Heart Surgery with Cardiopulmonary Bypass.

Blood lactate and blood pressure measurements are important predictors of life-threatening complications after infant open-heart surgeries requiring cardiopulmonary bypass (CPB). We have developed an intravascular nitric oxide (NO)-releasing 5-Fr catheter that contains a lactate sensor for continuous in-blood lactate monitoring and a dedicated lumen for third-party pressure sensor attachment. This device has antimicrobial and antithrombotic properties and can be implanted intravascularly. The importance of this design is its ability to inhibit thrombosis, due to the slow release of NO through the surface of the catheter and around the electrochemical lactate sensors, to allow continuous data acquisition for more than 48 h. An in vivo study was performed using six piglets undergoing open-heart surgery with CPB and cardioplegic arrest, in order to mimic intra-operative conditions for infants undergoing cardiac surgery with CPB. In each study of 3 h, two 5-Fr NO-releasing lactate and blood-pressure monitoring catheters were implanted in the femoral vessels (arteries and veins) and the CPB circuitry to monitor changing lactate levels and blood pressures during and immediately after aortic cross-clamp removal and separation from CBP. Electrical signals continuously acquired through the sensors were processed and displayed on the device’s display and via Bluetooth to a computer in real-time with the use of a two-point in vivo calibration against blood gas results. The study results show that lactate levels measured from those sensors implanted in the CPB circuit during CPB were comparable to those acquired by arterial blood gas measurements, whereas lactate levels measured from sensors implanted in the femoral artery were closely correlated with those acquired intermittently by blood gas prior to CPB initiation, but not during CPB. Blood pressure sensors attached to one lumen of the device displayed accurate blood pressure readings compared to those measured using an FDA approved pressure sensor already on the market. We recommend that the sensor be implanted in the CPB’s circuit to continuously monitor lactate during CPB, and implanted in the femoral arteries or jugular veins to monitor lactate before and after CPB. Blood pressures dramatically drop during CPB due to lower blood flow into the lower body, and we suspect that the femoral arteries are likely collapsing or constricting on the implanted catheter and disrupting the sensor-to-blood contact. This study shows that the device is able to accurately and continuously monitor lactate levels during CPB and potentially prevent post-surgery complications in infants.

A Flexible 3D Organic Preamplifier for a Lactate Sensor.

Organic transistors are promising platforms for wearable biosensors. However, the strategies to improve signal amplification have yet to be determined, particularly regarding biosensors that generate very weak signals. In this study, an organic voltage amplifier is presented for a lactate sensor on flexible plastic foil. The preamplifier is based on a 3D complementary inverter, which is achieved by vertically stacking complementary transistors with a shared gate between them. The shared gate is extended and functionalized with a lactate oxidase enzyme to detect lactate. The sensing device successfully detects the lactate concentration in the human sweat range (20-60mm) with high sensitivity (6.82mV mm-1 ) due to high gain of its amplification. The 3D integration process is cost-effective as it is solution-processable and doubles the number of transistors per unit area. The device presented in this study would pave the way for the development of high-gain noninvasive sweat lactate sensors that can be wearable.

Facile and Low-Cost Fabrication of a Thread/Paper-Based Wearable System for Simultaneous Detection of Lactate and pH in Human Sweat

Wearable devices have received tremendous interests in human sweat analysis in the past few years. However, the widely used polymeric substrates and the layer-by-layer stacking structures greatly influence the cost-efficiency, conformability and breathability of the devices, further hindering their practical applications. Herein, we report a facile and low-cost strategy for the fabrication of a skin-friendly thread/paper-based wearable system consisting of a sweat reservoir and a multi-sensing component for simultaneous in situ analysis of sweat pH and lactate. In the system, hydrophilic silk thread serves as the micro-channel to guide the liquid flow. Filter papers were functionalized to prepare colorimetric sensors for lactate and pH. The smartphone-based quantitative analysis shows that the sensors are sensitive and reliable. Although pH may interfere the lactate detection, the pH detected simultaneously could be employed to correct the measured data for the achievement of a precise lactate level. After being integrated with a hydrophobic arm guard, the system was successfully used for the on-body measurement of pH and lactate in the sweats secreted from the volunteers. This low-cost, easy-to-fabricate, light-weight and flexible thread/paper-based microfluidic sensing device may hold great potentials as a wearable system in human sweat analysis and point-of-care diagnostics.

Monitoring with In Vivo Electrochemical Sensors: Navigating the Complexities of Blood and Tissue Reactivity.

The disruptive action of an acute or critical illness is frequently manifest through rapid biochemical changes that may require continuous monitoring. Within these changes, resides trend information of predictive value, including responsiveness to therapy. In contrast to physical variables, biochemical parameters monitored on a continuous basis are a largely untapped resource because of the lack of clinically usable monitoring systems. This is despite the huge testing repertoire opening up in recent years in relation to discrete biochemical measurements. Electrochemical sensors offer one of the few routes to obtaining continuous readout and, moreover, as implantable devices information referable to specific tissue locations. This review focuses on new biological insights that have been secured through in vivo electrochemical sensors. In addition, the challenges of operating in a reactive, biological, sample matrix are highlighted. Specific attention is given to the choreographed host rejection response, as evidenced in blood and tissue, and how this limits both sensor life time and reliability of operation. Examples will be based around ion, O2, glucose, and lactate sensors, because of the fundamental importance of this group to acute health care.

Employment of 1-Methoxy-5-Ethyl Phenazinium Ethyl Sulfate as a Stable Electron Mediator in Flavin Oxidoreductases-Based Sensors.

In this paper, a novel electron mediator, 1-methoxy-5-ethyl phenazinium ethyl sulfate (mPES), was introduced as a versatile mediator for disposable enzyme sensor strips, employing representative flavin oxidoreductases, lactate oxidase (LOx), glucose dehydrogenase (GDH), and fructosyl peptide oxidase (FPOx). A disposable lactate enzyme sensor with oxygen insensitive Aerococcus viridans-derived engineered LOx (AvLOx), with A96L mutant as the enzyme, was constructed. The constructed lactate sensor exhibited a high sensitivity (0.73 ± 0.12 μA/mM) and wide linear range (0–50 mM lactate), showings that mPES functions as an effective mediator for AvLOx. Employing mPES as mediator allowed this amperometric lactate sensor to be operated at a relatively low potential of +0.2 V to 0 V vs. Ag/AgCl, thus avoiding interference from uric acid and acetaminophen. The lactate sensors were adequately stable for at least 48 days of storage at 25 °C. These results indicated that mPES can be replaced with 1-methoxy-5-methyl phenazinium methyl sulfate (mPMS), which we previously reported as the best mediator for AvLOx-based lactate sensors. Furthermore, this study revealed that mPES can be used as an effective electron mediator for the enzyme sensors employing representative flavin oxidoreductases, GDH-based glucose sensors, and FPOx-based hemoglobin A1c (HbA1c) sensors.

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.

3D Printed Hydrogel-Based Biosensors for Wearable Applications

The ability to manufacture sensors rapidly and cost-effectively is essential for creating devices that would benefit healthcare, medical diagnostics, environmental monitoring and process industries. Because of the inherent complexity of sensing devices, new fabrication methods are needed to create sensors that could be fabricated in large scale and with high efficiency. Additive manufacturing techniques like 3D printing and 3D bioprinting enable printing of different biomaterials into intricate 3D architectures which could serve as sensing platforms. This presentation will describe the development and optimization of 3D printed hydrogel-based (bio)sensors with incorporated receptor molecules and transduction interfaces. In order to 3D print these sensors, an extrusion-based 3D bioprinter, and a computer-aided designing software were used along with a novel bio-ink formulation, which contains enzymes, catalytic and photoactive properties. The optimum hydrogel composite provided excellent mechanical properties to the printed biosensors enabling them to be used in a wide range of wearable applications. Fabrication and performance characteristics of two types of sensors will be presented: a lactate sensor for measuring physiological activity in sweat and a UV sensor for measuring harmful UV radiation exposure. The proposed sensors are reagent-less and highly portable making them ideal for use as wearable devices and printed biocatalytic platforms for portable bioelectronics. Examples of applications of these additively manufactured sensors for monitoring different analytes will be discussed. Figure 1

An Inkjet-Printed Flexible Non-Enzymatic Lactate Sensor for Clinical Blood Plasma Test

In this letter, we demonstrate a fully inkjet-printed electrochemical lactate sensor. The sensor is inkjet-printed on a flexible substrate, Kapton film, where silver nanoparticles (Ag NPs) are printed as conductive electrodes. The working and reference electrodes of the sensor are Ag NPs/NiO NPs/Polyurethane (PU) and Ag/AgCl(s)/PU layers respectively. The inkjet-printed PU film can not only prevent the annihilation of the Ag NPs resulting from the dissolution of Ag and the formation of AgCl in the phosphate-buffered saline (PBS) but also stabilize the lactic acid measurements by maintaining a potential variant within 2% at different lactate concentrations. The as-printed sensor exhibits a linear sensitivity of 8.86nA/mM/mm <sup xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">2</sup> (R=99.6%) in the range from 0.6 to 2.2 mM. Clinical blood plasma testing results show that the trends of lactic acid change in patients measured by the sensor are the same as the ones measured using the existing detection method, colorimetric assays, in a tertiary university-affiliated hospital.