Biolayer-Interferometry-Guided Functionalization of Screen-Printed Graphene for Label-Free Electrochemical Virus Detection.

Additive manufacturing holds promise for rapid prototyping and low-cost production of biosensors for diverse pathogens. Among additive manufacturing methods, screen printing is particularly desirable for high-throughput production of sensing platforms. However, this technique needs to be combined with carefully formulated inks, rapid postprocessing, and selective functionalization to meet all requirements for high-performance biosensing applications. Here, we present screen-printed graphene electrodes that are processed with thermal annealing to achieve high surface area and electrical conductivity for sensitive biodetection via electrochemical impedance spectroscopy. As a proof-of-concept, this biosensing platform is utilized for electrochemical detection of SARS-CoV-2. To ensure reliable specificity in the presence of multiple variants, biolayer interferometry (BLI) is used as a label-free and dynamic screening method to identify optimal antibodies for concurrent affinity to the Spike S1 proteins of Delta, Omicron, and Wild Type SARS-CoV-2 variants while maintaining low affinity to competing pathogens such as Influenza H1N1. The BLI-identified antibodies are robustly bound to the graphene electrode surface via oxygen moieties that are introduced during the thermal annealing process. The resulting electrochemical immunosensors achieve superior metrics including rapid detection (55 s readout following 15 min of incubation), low limits of detection (approaching 500 ag/mL for the Omicron variant), and high selectivity toward multiple variants. Importantly, the sensors perform well on clinical saliva samples detecting as few as 103 copies/mL of SARS-CoV-2 Omicron, following CDC protocols. The combination of the screen-printed graphene sensing platform and effective antibody selection using BLI can be generalized to a wide range of point-of-care immunosensors.

Wearable Flexible Perspiration Biosensors Using Laser-Induced Graphene and Polymeric Tape Microfluidics.

Wearable biosensors promise real-time measurements of chemicals in human sweat, with the potential for dramatic improvements in medical diagnostics and athletic performance through continuous metabolite and electrolyte monitoring. However, sweat sensing is still in its infancy, and questions remain about whether sweat can be used for medical purposes. Wearable sensors are focused on proof-of-concept designs that are not scalable for multisubject trials, which could elucidate the utility of sweat sensing for health monitoring. Moreover, many wearable sensors do not include the microfluidics necessary to protect and channel consistent and clean sweat volumes to the sensor surface or are not designed to be disposable to prevent sensor biofouling and inaccuracies due to repeated use. Hence, there is a need to produce low-cost and single-use wearable sensors with integrated microfluidics to ensure reliable sweat sensing. Herein, we demonstrate the convergence of laser-induced graphene (LIG) based sensors with soft tape polymeric microfluidics to quantify both sweat metabolites (glucose and lactate) and electrolytes (sodium) for potential hydration and fatigue monitoring. Distinct LIG-electrodes were functionalized with glucose oxidase and lactate oxidase for selective sensing of glucose and lactate across physiological ranges found in sweat with sensitivities of 26.2 and 2.47 × 10-3 µA mM-1 cm-2, detection limits of 8 and 220 µM, and linear response ranges of 0-1 mM and 0-32 mM, respectively. LIG-electrodes functionalized with a sodium-ion-selective membrane displayed Nernstian sensitivity of 58.8 mV decade-1 and a linear response over the physiological range in sweat (10-100 mM). The sensors were tested in a simulated sweating skin microfluidic system and on-body during cycling tests in a multisubject trial. Results demonstrate the utility of LIG integrated with microfluidics for real-time, continuous measurements of biological analytes in sweat and help pave the way for the development of personalized wearable diagnostic tools.

Carbon dots using a household cleaning liquid as a dopant for iron detection in hydroponic systems.

Iron (Fe) is a required micronutrient in plants for the production of chlorophyll and transport of oxygen. A commonly used surrogate for measuring nutrient levels is the measurement of electrical conductivity or total dissolved solids, but this technique is not selective towards any particular dissolved ion. In this study, using a conventional microwave, fluorescent carbon dots (CDs) are produced from glucose and a household cleaning product and applied towards monitoring dissolved ferric iron levels in hydroponic systems through fluorescent quenching. The produced particles have an average size of 3.19 ± 0.76 nm with a relatively high degree of oxygen surface groups. When using an excitation of 405 nm, a broad emission peak is centered at approximately 500 nm. A limit-of-detection of 0.196 ± 0.067 ppm (3.51 ± 1.21 µM) with minimal interference from common heavy metal quenchers and ions found in hydroponic systems was determined. Butterhead lettuce was grown while discretely monitoring iron levels via the CDs for three separate weeks of growth. The CDs displayed a non-significant difference (p > 0.05) in performance when compared to a standard method. These results along with a simple and relatively low-cost production method make the CDs in this study a promising tool for monitoring iron levels in hydroponic systems.

Ion-selective electrodes based on laser-induced graphene as an alternative method for nitrite monitoring.

Nitrite is an important food additive for cured meats; however, high nitrite levels pose adverse health effects to humans. Hence, monitoring nitrite concentration is critical to comply with limits imposed by regulatory agencies. Laser-induced graphene (LIG) has proven to be a scalable manufacturing alternative to produce high-performance electrochemical transducers for sensors. Herein, we expand upon initial LIG studies by fabricating hydrophilic and hydrophobic LIG that are subsequently converted into ion-selective sensors to monitor nitrite in food samples with comparable performance to the standard photometric method (Griess method). The hydrophobic LIG resulted in an ion-selective electrode with improved potential stability due partly to a decrease in the water layer between the electrode and the nitrite poly(vinyl) chloride-based ion-selective membrane. These resultant nitrite ion-selective sensors displayed Nernstian response behavior with a sensitivity of 59.5 mV dec-1, a detection limit of 0.3 ± 0.1 mg L-1 (mean ± standard deviation), and a broad linear sensing range from 10-5 to 10-1 M, which was significantly larger than currently published nitrite methods. Nitrite levels were determined directly in food extract samples of sausage, ham, and bacon for 5 min. These sensor metrics are significant as regulatory agencies limit nitrite levels up to 200 mg L-1 in finished products to reduce the potential formation of nitrosamine (carcinogenic compound). These results demonstrate the versatility of LIG as a platform for ion-selective-LIG sensors and simple, efficient, and scalable electrochemical sensing in general while demonstrating a promising alternative to monitor nitrite levels in food products ensuring regulatory compliance.

Aerosol-jet-printed graphene electrochemical immunosensors for rapid and label-free detection of SARS-CoV-2 in saliva.

Rapid, inexpensive, and easy-to-use coronavirus disease 2019 (COVID-19) home tests are key tools in addition to vaccines in the world-wide fight to eliminate national and local shutdowns. However, currently available tests for SARS-CoV-2, the virus that causes COVID-19, are too expensive, painful, and irritating, or not sufficiently sensitive for routine, accurate home testing. Herein, we employ custom-formulated graphene inks and aerosol jet printing (AJP) to create a rapid electrochemical immunosensor for direct detection of SARS-CoV-2 Spike Receptor-Binding Domain (RBD) in saliva samples acquired non-invasively. This sensor demonstrated limits of detection that are considerably lower than most commercial SARS-CoV-2 antigen tests (22.91 ± 4.72 pg/mL for Spike RBD and 110.38 ± 9.00 pg/mL for Spike S1) as well as fast response time (~30 mins), which was facilitated by the functionalization of printed graphene electrodes in a single-step with SARS-CoV-2 polyclonal antibody through the carbodiimide reaction without the need for nanoparticle functionalization or secondary antibody or metallic nanoparticle labels. This immunosensor presents a wide linear sensing range from 1 to 1000 ng/mL and does not react with other coexisting influenza viruses such as H1N1 hemagglutinin. By combining high-yield graphene ink synthesis, automated printing, high antigen selectivity, and rapid testing capability, this work offers a promising alternative to current SARS-CoV-2 antigen tests.

Fate of enteric viruses during leafy greens (romaine lettuce) production using treated municipal wastewater and AP205 bacteriophage as a surrogate.

Water reuse programs are being explored to close the gap between supply and demand for irrigation in agriculture. However, these sources could contain hazardous microbial contaminants, and pose risks to public health. This study aimed to grow and irrigate romaine lettuce with inoculated wastewater effluent to track AP205 bacteriophage prevalence through cultivation and post-harvest storage. AP205 is a bacteriophage and was used as a surrogate for enteric viruses. Low and high dosages (mean ± standard deviation) of AP205 at 4.8 ± 0.4 log PFU/mL and 6.6 ± 0.2 log PFU/mL; respectively, were prepared to examine viral load influence on contamination levels. Foliage, leachate, and soil contamination levels were directly related to AP205 concentrations in the effluent. AP205 concentrations increased throughout cultivation for foliage and leachate, suggesting bacteriophage accumulation. During post-harvest storage (14 day at 4 °C), there was a significant decrease in AP205 concentration on the foliage. Results show that wastewater effluents usage for leafy greens cultivation can pose risks to humans and additional steps are required to safely apply wastewater effluents to soils and crops.

Prevalence of Escherichia coli and Antibiotic-Resistant Bacteria During Fresh Produce Production (Romaine Lettuce) Using Municipal Wastewater Effluents.

High demand for food and water encourages the exploration of new water reuse programs, including treated municipal wastewater usage. However, these sources could contain high contaminant levels posing risks to public health. The objective of this study was to grow and irrigate a leafy green (romaine lettuce) with treated wastewater from a municipal wastewater treatment plant to track Escherichia coli and antibiotic-resistant microorganisms through cultivation and post-harvest storage to assess their fate and prevalence. Contamination levels found in the foliage, leachate, and soil were directly (p < 0.05) related to E. coli concentrations in the irrigation water. Wastewater concentrations from 177 to 423 CFU ml-1 resulted in 15-25% retention in the foliage. Leachate and soil presented means of 231 and 116% retention, respectively. E. coli accumulation on the foliage was observed (p < 0.05) and increased by over 400% during 14-day storage (4°C). From randomly selected E. coli colonies, in all four biomass types, 81 and 34% showed resistance to ampicillin and cephalothin, respectively. Reclaimed wastewater usage for leafy greens cultivation could pose potential health risks, especially considering the bacteria found have a high probability of being antibiotic resistance. Successful reuse of wastewater in agriculture will depend on appropriate mitigation and management strategies to guarantee an inexpensive, efficient, and safe water supply.

3D printed imaging platform for portable cell counting.

Despite having widespread application in the biomedical sciences, flow cytometers have several limitations that prevent their application to point-of-care (POC) diagnostics in resource-limited environments. 3D printing provides a cost-effective approach to improve the accessibility of POC devices in resource-limited environments. Towards this goal, we introduce a 3D-printed imaging platform (3DPIP) capable of accurately counting particles and perform fluorescence microscopy. In our 3DPIP, captured microscopic images of particle flow are processed on a custom developed particle counter code to provide a particle count. This prototype uses a machine vision-based algorithm to identify particles from captured flow images and is flexible enough to allow for labeled and label-free particle counting. Additionally, the particle counter code returns particle coordinates with respect to time which can further be used to perform particle image velocimetry. These results can help estimate forces acting on particles, and identify and sort different types of cells/particles. We evaluated the performance of this prototype by counting 10 µm polystyrene particles diluted in deionized water at different concentrations and comparing the results with a commercial Beckman-Coulter Z2 particle counter. The 3DPIP can count particle concentrations down to ∼100 particles per mL with a standard deviation of ±20 particles, which is comparable to the results obtained on a commercial particle counter. Our platform produces accurate results at flow rates up to 9 mL h-1 for concentrations below 1000 particle per mL, while 5 mL h-1 produces accurate results above this concentration limit. Aside from performing flow-through experiments, our instrument is capable of performing static experiments that are comparable to a plate reader. In this configuration, our instrument is able to count between 10 and 250 cells per image, depending on the prepared concentration of bacteria samples (Citrobacter freundii; ATCC 8090). Overall, this platform represents a first step towards the development of an affordable fully 3D printable imaging flow cytometry instrument for use in resource-limited clinical environments.

Delivery of selenium using chitosan nanoparticles: Synthesis, characterization, and antioxidant and growth effects in Nile tilapia (Orechromis niloticus).

This study aimed to elucidate the effects of selenium-loaded chitosan nanoparticles used as a dietary supplement on Nile tilapia (Oreochromis niloticus) antioxidant and growth responses. First, chitosan-based nanoparticles containing selenium (Se) were synthesized using the ionotropic gelation method and their physicochemical characteristics, controlled release profile, and antioxidant activity properties were investigated. Thereafter, the effects on glutathione peroxidase and antioxidant activities (by radical scavenging activity), growth, and whole-body composition of Nile tilapia were evaluated when they were fed with Se-loaded chitosan nanoparticles and compared with other selenium dietary supplements. Se-loaded chitosan nanoparticles showed high entrapment efficiency (87%), spherical shape, smooth surface, and broad size distribution. The controlled release of Se consisted of an initial burst followed by a gradual release over 48 h. Se-loaded nanoparticles presented significantly higher antioxidant activity compared to free Se. A 60-day feeding trial was conducted to compare the effects of supplementing different dietary Se sources, including selenomethionine (as organic source), sodium selenite (as inorganic source), and Se-loaded chitosan nanoparticles (Se-Nano and Se-Nano x1.5) on antioxidant and growth responses of Nile tilapia. A basal diet without Se supplementation was used as the control. The dietary supplementations with different Se sources (free and encapsulated selenium) lead to significant improvements in final weight and feed efficiency of Nile tilapia fingerlings. However, dietary treatments did not affect whole-body protein and lipid content. Diets containing Se-Nano and Se-Nano x1.5 were more effective than sodium selenite and selenomethionine in preventing oxidative stress and improving antioxidant activity in Nile tilapia. Overall, Se-loaded nanoparticles presented a great potential as an efficient source for delivering dietary Se to Nile tilapia, directly affecting the growth performance, feed efficiency, oxidative stress, and antioxidant activity of this species.

Planar Interdigitated Aptasensor for Flow-Through Detection of Listeria spp. in Hydroponic Lettuce Growth Media.

Irrigation water is a primary source of fresh produce contamination by bacteria during the preharvest, particularly in hydroponic systems where the control of pests and pathogens is a major challenge. In this work, we demonstrate the development of a Listeria biosensor using platinum interdigitated microelectrodes (Pt-IME). The sensor is incorporated into a particle/sediment trap for the real-time analysis of irrigation water in a hydroponic lettuce system. We demonstrate the application of this system using a smartphone-based potentiostat for rapid on-site analysis of water quality. A detailed characterization of the electrochemical behavior was conducted in the presence/absence of DNA and Listeria spp., which was followed by calibration in various solutions with and without flow. In flow conditions (100 mL samples), the aptasensor had a sensitivity of 3.37 ± 0.21 k log-CFU-1 mL, and the LOD was 48 ± 12 CFU mL-1 with a linear range of 102 to 104 CFU mL-1. In stagnant solution with no flow, the aptasensor performance was significantly improved in buffer, vegetable broth, and hydroponic media. Sensor hysteresis ranged from 2 to 16% after rinsing in a strong basic solution (direct reuse) and was insignificant after removing the aptamer via washing in Piranha solution (reuse after adsorption with fresh aptamer). This is the first demonstration of an aptasensor used to monitor microbial water quality for hydroponic lettuce in real time using a smartphone-based acquisition system for volumes that conform with the regulatory standards. The aptasensor demonstrated a recovery of 90% and may be reused a limited number of times with minor washing steps.

Laser-Induced Graphene Electrochemical Immunosensors for Rapid and Label-Free Monitoring of Salmonella enterica in Chicken Broth.

Food-borne illnesses are a growing concern for the food industry and consumers, with millions of cases reported every year. Consequently, there is a critical need to develop rapid, sensitive, and inexpensive techniques for pathogen detection in order to mitigate this problem. However, current pathogen detection strategies mainly include time-consuming laboratory methods and highly trained personnel. Electrochemical in-field biosensors offer a rapid, low-cost alternative to laboratory techniques, but the electrodes used in these biosensors require expensive nanomaterials to increase their sensitivity, such as noble metals (e.g., platinum, gold) or carbon nanomaterials (e.g., carbon nanotubes, or graphene). Herein, we report the fabrication of a highly sensitive and label-free laser-induced graphene (LIG) electrode that is subsequently functionalized with antibodies to electrochemically quantify the food-borne pathogen Salmonella enterica serovar Typhimurium. The LIG electrodes were produced by laser induction on the polyimide film in ambient conditions and, hence, circumvent the need for high-temperature, vacuum environment, and metal seed catalysts commonly associated with graphene-based electrodes fabricated via chemical vapor deposition processes. After functionalization with Salmonella antibodies, the LIG biosensors were able to detect live Salmonella in chicken broth across a wide linear range (25 to 105 CFU mL-1) and with a low detection limit (13 ± 7 CFU mL-1; n = 3, mean ± standard deviation). These results were acquired with an average response time of 22 min without the need for sample preconcentration or redox labeling techniques. Moreover, these LIG immunosensors displayed high selectivity as demonstrated by nonsignificant response to other bacteria strains. These results demonstrate how LIG-based electrodes can be used for electrochemical immunosensing in general and, more specifically, could be used as a viable option for rapid and low-cost pathogen detection in food processing facilities before contaminated foods reach the consumer.

Development and optimization of pH-responsive PLGA-chitosan nanoparticles for triggered release of antimicrobials.

The aim of this work was to develop and optimize a pH-responsive nanoparticle based on poly(D,L-lactide-co-glycolide) (PLGA) and chitosan (CHIT) for delivery of natural antimicrobial using trans-cinnamaldehyde (TCIN) as a model compound. The optimization was performed using a central composite design and the desirability function approach. The optimized levels of variables considering all significant responses were 4% (w/w) of TCIN and 6.75% (w/w) of CHIT. After, optimized nanoparticles were produced and characterized according to their physicochemical properties and their antimicrobial activity against Salmonella Typhimurium and Staphylococcus aureus. Optimized nanoparticles characterization indicated a satisfactory TCIN encapsulation (33.20 ±â€¯0.85%), spherical shape, pH-responsive controlled release, with faster release in the presence of CHIT at low pH, and enhanced antimicrobial activity against both pathogens. TCIN encapsulation using PLGA coated with CHIT enhanced its antimicrobial activity and generated a delivery system with pH-sensitivity for controlled release with promising properties for food safety applications.