Microfluidic paper-based analytical devices for simple and nondestructive durian fruit maturity assessment.

Accurately predicting durian maturity is a critically unresolved worldwide issue. Farmers currently determine durian ripeness based on their own observation and experience leading to inconsistencies in harvest timing. This reliance on human judgment often results in premature or overripe harvests, impacting fruit quality, yield, and market value. Existing technological solutions, such as sensors are often complex and require specialized expertise, hindering their adoption by farmers and consumers. Developing sensors that can accurately measure durian ripeness without damaging the fruit, are easy to use, and affordable remains a challenge. We introduce a microfluidic paper-based analytical device (µPAD) for on-site, safe matching to meet the demands of durian maturity evaluation. The µPAD automatically collected peduncle fluid without destroying the durian fruit for dual detection of total sugar and amino acid. For determining total sugar including sucrose, glucose, and fructose, several enzymatic steps were reduced to a single step of invertase for sucrose hydrolysis before total reducing sugar was measured using gold nanoparticle (AuNP) generation. Kinetics study of invertase on the µPAD showed Vmax and Km values of 1.42 mM min-1 and 2.17 mM, respectively, that agreed with the direct study of sucrose conversion. To increase device reliability, amino acid was also simultaneously measured with sugar using the simple ninhydrin test with the addition of SnCl2. The developed sensor provided LODs of 3.50, 3.10, 3.30 µM, and 0.02 mg mL-1 for glucose, fructose, sucrose, and amino acid respectively. The µPADs were able to nondestructively discriminate between the mature and immature durians, showing high linear correlation with the standard dry weight method. The development of this µPAD technology has the potential to revolutionize durian cultivation practices, reduce post-harvest losses, and enhance the overall sustainability and profitability of the durian value chain, and can be further developed for maturity tests of other fruits.

Fill, Fold, Photo: Preconcentration and Multiplex Detection of Trace Level Heavy Metals in Water.

Heavy metal contamination is an increasing global threat to human and environmental health, particularly in resource-limited areas. Traditional platforms for heavy metal detection are labor intensive and expensive and require lab facilities. While paper-based colorimetric sensors offer a simpler approach, their sensitivity limitations prevent them from meeting legislative requirements for many metals. Existing preconcentration systems, on the other hand, can achieve lower detection limits but typically focus on analyzing only one metal, making comprehensive monitoring difficult. We address these limitations by introducing a low-cost preconcentration system coupled with colorimetric analysis for the simultaneous detection of seven metal ions at low ppb levels without the need for external equipment outside a smartphone. The system achieved detection limits of 15 ppb (Ni(II)), 7 ppb (Cu(II)), 2 ppb (Fe(III)), 20 ppb (Cr(VI)), 13 ppb (Pb(II)), 26 ppb (Hg(II)), and 15 ppb (Mn(II)) with six out of seven limits of detection values falling well below EPA regulatory guidelines for drinking water. The user-friendly Fill, Fold, Photo approach eliminates complex pretreatment steps. Smartphone-based detection offers portable quantification within seconds. Employing masking strategies ensured higher selectivity for each assay on the card, while our packaging protocols enable system stability for over 4 weeks of study, facilitating mass production and deployment within a realistic time frame. To validate the sensor's performance in real-world scenarios, the sensor was tested with environmental water samples. The sensor demonstrated good recovery, ranging from 77% to 94% compared to the standard ICP-MS method. Furthermore, spike recovery analysis confirmed the sensor's accuracy, with a relative standard deviation (RSD) of less than 15%. This technology holds significant promise for future development as a convenient, portable solution for field-based monitoring of a broad spectrum of water contaminants, including pesticides, PFAS, fertilizers, and beyond.

Peptide nucleic acid probe-assisted paper-based electrochemical biosensor for multiplexed detection of respiratory viruses.

The similar transmission patterns and early symptoms of respiratory viral infections, particularly severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), influenza (H1N1), and respiratory syncytial virus (RSV), pose substantial challenges in the diagnosis, therapeutic management, and handling of these infectious diseases. Multiplexed point-of-care testing for detection is urgently needed for prompt and efficient disease management. Here, we introduce an electrochemical paper-based analytical device (ePAD) platform for multiplexed and label-free detection of SARS-CoV-2, H1N1, and RSV infection using immobilized pyrrolidinyl peptide nucleic acid probes. Hybridization between the probes and viral nucleic acid targets causes changes in the electrochemical response. The resulting sensor offers high sensitivity and low detection limits of 0.12, 0.35, and 0.36 pM for SARS-CoV-2 (N gene), H1N1, and RSV, respectively, without showing any cross-reactivities. The amplification-free detection of extracted RNA from 42 nasopharyngeal swab samples was successfully demonstrated and validated against reverse-transcription polymerase chain reaction (range of cycle threshold values: 17.43-25.89). The proposed platform showed excellent clinical sensitivity (100 %) and specificity (≥97 %) to achieve excellent agreement (κ ≥ 0.914) with the standard assay, thereby demonstrating its applicability for the screening and diagnosis of these respiratory diseases.

Novel theranostic wounds dressing based on pH responsive alginate hydrogel/graphene oxide/levofloxacin modified silk.

Integrating pH sensor with controlled antibiotic release is fabricated on silk to create a theranostic wound dressing. Alginate (ALG) hydrogel and graphene oxide (GO) loaded with levofloxacin (LVX) and a pH indicator are applied to fabricate a pH-responsive theranostic wound dressing. The modified silk color changes from yellow to green in response to elevated skin pH, indicating the skin infection. The semi-quantitative analysis was conducted using ImageJ, revealing significant color changes across the wide range. At elevated pH levels, the ionization of the COOH bonds within ALG induces repulsion among the COO- groups, thereby accelerating the release of the incorporated drug compared to release under lower pH. At an infected pH of 8, ALG hydrogel triggers LVX releasing up to 135.86 ± 0.3 µg, while at a normal pH of 7, theranostic silk releases 123.13 ± 0.26 µg. Incorporating GO onto silk fibers enhances LVX loading and sustains LVX release. Furthermore, these modified silks possess antimicrobial abilities without causing irritation or allergies on the human skin. This theranostic silks represents a major step forward in smart wound care, introducing a versatile platform of smart wound care.

Stability of Protein Pharmaceuticals: Recent Advances.

There have been significant advances in the formulation and stabilization of proteins in the liquid state over the past years since our previous review. Our mechanistic understanding of protein-excipient interactions has increased, allowing one to develop formulations in a more rational fashion. The field has moved towards more complex and challenging formulations, such as high concentration formulations to allow for subcutaneous administration and co-formulation. While much of the published work has focused on mAbs, the principles appear to apply to any therapeutic protein, although mAbs clearly have some distinctive features. In this review, we first discuss chemical degradation reactions. This is followed by a section on physical instability issues. Then, more specific topics are addressed: instability induced by interactions with interfaces, predictive methods for physical stability and interplay between chemical and physical instability. The final parts are devoted to discussions how all the above impacts (co-)formulation strategies, in particular for high protein concentration solutions.'

Size-Dependent Electrochemistry of Laser-Induced Graphene Electrodes.

Laser-induced graphene (LIG) electrodes have become popular for electrochemical sensor fabrication due to their simplicity for batch production without the use of reagents. The high surface area and favorable electrocatalytic properties also enable the design of small electrochemical devices while retaining the desired electrochemical performance. In this work, we systematically investigated the effect of LIG working electrode size, from 0.8 mm to 4.0 mm diameter, on their electrochemical properties, since it has been widely assumed that the electrochemistry of LIG electrodes is independent of size above the microelectrode size regime. The background and faradaic current from cyclic voltammetry (CV) of an outer-sphere redox probe [Ru(NH3)6]3+ showed that smaller LIG electrodes had a higher electrode roughness factor and electroactive surface ratio than those of the larger electrodes. Moreover, CV of the surface-sensitive redox probes [Fe(CN)6]3- and dopamine revealed that smaller electrodes exhibited better electrocatalytic properties, with enhanced electron transfer kinetics. Scanning electron microscopy, Raman spectroscopy, and X-ray photoelectron spectroscopy showed that the physical and chemical surface structure were different at the electrode center versus the edges, so the electrochemical properties of the smaller electrodes were improved by having rougher surface and more density of the graphitic edge planes, and more oxide-containing groups, leading to better electrochemistry. The difference could be explained by the different photothermal reaction time from the laser scribing process that causes different stable carbon morphology to form on the polymer surface. Our results give a new insight on relationships between surface structure and electrochemistry of LIG electrodes and are useful for designing miniaturized electrochemical devices.

Non-invasive iron deficiency diagnosis: a saliva-based approach using capillary flow microfluidics.

Iron deficiency anemia (IDA) is a condition characterized by lower-than-average iron (Fe) levels in the body, affecting a substantial number of young children and pregnant women globally. Existing diagnostic methods for IDA rely on invasive analysis of stored Fe in ferritin from blood samples, posing challenges, especially for toddlers and young children. To address this issue, saliva has been proposed as a non-invasive sample matrix for IDA diagnosis. However, conventional Fe analysis techniques often necessitate complex and costly instrumentation. This study presents the first non-invasive, saliva-based preliminary screening test for IDA using a nitrocellulose lateral flow system. In this study, we introduce a novel approach using the ferroin reaction with bathophenanthroline (Bphen) and ferrous (Fe2+) ions to quantify Fe levels in saliva. Our methodology involves a capillary flow-driven microfluidic device integrated into a lateral flow system utilizing nitrocellulose membranes. Here, we present the first instance of saliva on a nitrocellulose substrate to detect salivary Fe levels. The optimized system yielded a linear response over the 1-200 ppm range in buffer solution, with a limit of detection (LoD) of 5.6 ppm. Furthermore, the system demonstrated a linear response in pooled saliva samples across the 1-1000 ppm range, with a LoD of 55.1 ppm. These results underscore the potential of our capillary flow-driven microfluidic device as a viable non-invasive diagnostic tool for IDA, particularly in remote and resource-limited settings.

Multiplexed Capillary-Flow Driven Immunoassay for Respiratory Illnesses.

Multiplexed analysis in medical diagnostics is widely accepted as a more thorough and complete method compared to single-analyte detection. While analytical methods like polymerase chain reaction and enzyme-linked immunosorbent assay (ELISA) exist for multiplexed detection of biomarkers, they remain time-consuming and expensive. Lateral flow assays (LFAs) are an attractive option for point-of-care testing, and examples of multiplexed LFAs exist. However, these devices are limited by spatial resolution of test lines, large sample volume requirements, cross-reactivity, and poor sensitivity. Recent work has developed capillary-flow microfluidic ELISA platforms as a more sensitive alternative to LFAs; however, multiplexed detection on these types of devices has yet to be demonstrated. In the aftermath of the initial SARS-CoV-2 pandemic, the need for rapid, sensitive point-of-care devices has become ever clearer. Moving forward, devices that can distinguish between diseases with similar presenting symptoms would be the ideal home diagnostic. Here, the first example of a multiplexed capillary-flow immunoassay device for the simultaneous detection of multiple biomarkers is reported. From a single sample addition step, the reagents and washing steps required for two simultaneous ELISAs are delivered to spatially separated test strips. Visual results can be obtained in <15 min, and images captured with a smartphone can be analyzed for quantitative data. This device was used to distinguish between and quantify H1N1 hemagglutinin (HA) and SARS-CoV-2 nucleocapsid protein (N-protein). Using this device, analytical detection limits of 840 and 133 pg/mL were obtained for hemagglutinin and nucleocapsid protein, respectively. The presence of one target in the device did not increase the signal on the other test line, indicating no cross-reactivity between the assays. Additionally, simultaneous detection of both N-protein and HA was performed as well as simultaneous detection of N-protein and human C-reactive protein (CRP). Elevated levels of CRP in a patient infected with SARS-CoV-2 have been shown to correlate with more severe outcomes and a greater risk of death as well. To further expand on the simultaneous detection of two biomarkers, CRP and N-protein were detected simultaneously, and the presence of SARS-CoV-2 N-protein did not interfere with the detection of CRP when both targets were present in the sample.

Recent Trends in Nanomaterial Based Electrochemical Sensors for Drug Detection: Considering Green Assessment.

An individual's therapeutic drug exposure level is directly linked to corresponding clinical effects. Rapid, sensitive, inexpensive, portable and reliable devices are needed for diagnosis related to drug exposure, treatment, and prognosis of diseases. Electrochemical sensors are useful for drug monitoring due to their high sensitivity and fast response time. Also, they can be combined with portable signal read-out devices for point-of-care applications. In recent years, nanomaterials such as carbon-based, carbon-metal nanocomposites, noble nanomaterials have been widely used to modify electrode surfaces due to their outstanding features including catalytic abilities, conductivity, chemical stability, biocompatibility for development of electrochemical sensors. This review paper presents the most recent advances about nanomaterials-based electrochemical sensors including the use of green assessment approach for detection of drugs including anticancer, antiviral, anti-inflammatory, and antibiotics covering the period from 2019 to 2023. The sensor characteristics such as analyte interactions, fabrication, sensitivity, and selectivity are also discussed. In addition, the current challenges and potential future directions of the field are highlighted.

Improving design features and air bubble manipulation techniques for a single-step sandwich electrochemical ELISA incorporating commercial electrodes into capillary-flow driven immunoassay devices.

Integrating electrochemistry into capillary-flow driven immunoassay devices provides unique opportunities for quantitative point-of-care testing. Although custom electrodes can be inexpensive and are tunable, they require skilled fabrication. Here, we report the incorporation of a commercial electrode into a capillary-flow driven immunoassay (iceCaDI) device for a single end-user step sandwich electrochemical enzyme-linked immunosorbent assay (ELISA). The iceCaDI device is a pump-free portable microfluidic device with an integrated commercial screen-printed electrode and flow driven by capillary action. The iceCaDI device is composed of alternating polyester transparency film and double-sided adhesive film layers that are patterned with a laser cutter. This platform was designed to address known limitations of laminated device fabrication methods and operation. First, we developed a foldable laminated device fabrication using hinges for easy assembly and precise alignment. Second, reagent dispersing was achieved by incorporating a 1 mm wide arrow-shaped notch in the middle of the channel that trapped an air bubble and formed a baffle that facilitated reagent spreading to cover the detection area. Third, small vent holes were added to the top layer of the channels to prevent air bubbles from blocking flow. Finally, we fabricated a CRP immunosensor with a detection range of 0.625 to 10.0 µg mL-1 as a proof-of-concept to demonstrate an automatically driven sandwich electrochemical ELISA using the iceCaDI device. Three concentrations of CRP were successfully measured under flow conditions within 8 min. Our proposed device is a promising approach and a step forward in the development of point-of-care (POC) devices for techniques that traditionally require multiple user steps.

Microfluidics in environmental analysis: advancements, challenges, and future prospects for rapid and efficient monitoring.

Microfluidic devices have emerged as advantageous tools for detecting environmental contaminants due to their portability, ease of use, cost-effectiveness, and rapid response capabilities. These devices have wide-ranging applications in environmental monitoring of air, water, and soil matrices, and have also been applied to agricultural monitoring. Although several previous reviews have explored microfluidic devices' utility, this paper presents an up-to-date account of the latest advancements in this field for environmental monitoring, looking back at the past five years. In this review, we discuss devices for prominent contaminants such as heavy metals, pesticides, nutrients, microorganisms, per- and polyfluoroalkyl substances (PFAS), etc. We cover numerous detection methods (electrochemical, colorimetric, fluorescent, etc.) and critically assess the current state of microfluidic devices for environmental monitoring, highlighting both their successes and limitations. Moreover, we propose potential strategies to mitigate these limitations and offer valuable insights into future research and development directions.

Characterization of Factors Affecting Stripping Voltammetry on Thermoplastic Electrodes.

Thermoplastic carbon electrodes (TPEs) are an alternative form of carbon composite electrodes that have shown excellent electrochemical performance with applications in biological sensing. However, little has been done to apply TPEs to environmental sensing, specifically heavy metal analysis. The work here focuses on lead analysis and based on their electrochemical properties, TPEs are expected to outperform other carbon composite materials; however, despite testing multiple formulations, TPEs showed inferior performance. Detailed electrode characterization was conducted to examine the cause for poor lead sensing behavior. X-Ray photoelectron spectroscopy (XPS) was used to analyze the surface functional groups, indicating that acidic and alkaline functional groups impact lead electrodeposition. Further, scanning electron microscopy (SEM) and electrochemical characterization demonstrated that both the binder and graphite can influence the surface morphology, electroactive area, and electron kinetics.

Surface Modification of Thermoplastic Electrodes for Biosensing Applications via Copper-Catalyzed Click Chemistry.

Cu(I)-catalyzed 1,3-dipolar cycloaddition (CuAAC), also known as click chemistry, has been demonstrated to be highly robust while providing versatile surface chemistry. One specific application is biosensor fabrication. Recently, we developed thermoplastic electrodes (TPEs) as an alternative to traditional carbon composite electrodes in terms of cost, performance, and robustness. However, their applications in biosensing are currently limited due to a lack of facile methods for electrode modification. Here, we demonstrate the feasibility of using CuAAC following the diazonium grafting of TPEs to take advantage of two powerful technologies for developing a customizable and versatile biosensing platform. After a stepwise characterization of the electrode modification procedures was performed, electrodes were modified with model affinity reagents. Streptavidin and streptavidin-conjugated IgG antibodies were successfully immobilized on the TPE surface, as confirmed by electrochemical impedance spectroscopy and X-ray photoelectron spectroscopy.

Maximizing flow rate in single paper layer, rapid flow microfluidic paper-based analytical devices.

Small, single-layer microfluidic paper-based analytical devices (µPADs) offer potential for a range of point-of-care applications; however, they have been limited to low flow rates. Here, we investigate the role of laser cutting paper channels in maximizing flow rate in small profile devices with limited fluid volumes. We demonstrate that branching, laser-cut grooves can provide a 59.23-73.98% improvement in flow rate over a single cut, and a 435% increase over paper alone. These design considerations can be applied to more complex microfluidic devices with the aim of increasing the flow rate, and could be used in stand-alone channels for self-pumping. Supplementary Information: The online version contains supplementary material available at 10.1007/s10404-023-02679-8.

Microfluidic organotypic device to test intestinal mucosal barrier permeability ex vivo.

To protect the body from external pathogens, the intestines have sophisticated epithelial and mucosal barriers. Disruptions to barrier integrity are associated with a variety of disorders such as irritable bowel disease, Crohn's disease, and celiac disease. One critical component of all barriers are collagens in the extracellular matrix. While the importance of the intestinal barrier is established, current models lack the ability to represent the complex biology that occurs at these barriers. For the current study a microfluidic device model was modified to determine the effectiveness of collagen breakdown to cause barrier disruption. Bacterial collagenase was added for 48 h to the luminal channel of a dual flow microfluidic device to examine changes in intestinal barrier integrity. Tissues exhibited dose-dependent alterations in immunoreactive collagen-1 and claudin-1, and coincident disruption of the epithelial monolayer barrier as indicated by goblet cell morphologies. This ex vivo model system offers promise for further studies exploring factors that affect gut barrier integrity and potential downstream consequences that cannot be studied in current models.

Capillary flow-driven immunoassay platform for COVID-19 antigen diagnostics.

Over the last few years, the SARS-CoV-2 pandemic has made the need for rapid, affordable diagnostics more compelling than ever. While traditional laboratory diagnostics like PCR and well-plate ELISA are sensitive and specific, they can be costly and take hours to complete. Diagnostic tests that can be used at the point-of-care or at home, like lateral flow assays (LFAs) are a simple, rapid alternative, but many commercially available LFAs have been criticized for their lack of sensitivity compared to laboratory methods like well-plate ELISAs. The Capillary-Driven Immunoassay (CaDI) device described in this work uses microfluidic channels and capillary action to passively automate the steps of a traditional well-plate ELISA for visual read out. This work builds on prior capillary-flow devices by further simplifying operation and use of colorimetric detection. Upon adding sample, an enzyme-conjugated secondary antibody, wash steps, and substrate are sequentially delivered to test and control lines on a nitrocellulose strip generating a colorimetric response. The end user can visually detect SARS-CoV-2 antigen in 15-20 min by naked eye, or results can be quantified using a smartphone and software such as ImageJ. An analytical detection limit of 83 PFU/mL for SARS-CoV-2 was determined for virus in buffer, and 222 PFU/mL for virus spiked into nasal swabs using image analysis, similar to the LODs determined by traditional well-plate ELISA. Additionally, a visual detection limit of 100 PFU/mL was determined in contrived nasal swab samples by polling 20 untrained end-users. While the CaDI device was used for detecting clinically relevant levels of SARS-CoV-2 in this study, the CaDI device can be easily adapted to other immunoassay applications by changing the reagents and antibodies.

Why fake death? Environmental and genetic control of tonic immobility in larval lacewings (Neuroptera: Chrysopidae).

Tonic immobility is a passive antipredator strategy employed late in the predation sequence that may decrease individual mortality in prey animals. Here, we investigate how energetic state and genetic predisposition influence antipredator decision-making in green lacewing larvae, Chrysoperla plorabunda (Fitch), using simulated predatory encounters. We demonstrate that tonic immobility is a plastic response influenced by energetic resource limitation. Larvae exposed to 1 or 2 days of food deprivation initiate tonic immobility more often and with less physical provocation than individuals fed ad libitum. Recently molted individuals exposed to food deprivation, the individuals most energetically challenged, engage in tonic immobility at a higher rate than any other group. We also find that variation in antipredator strategy between individuals is partly the result of within-population genetic variation. We estimate the propensity to enter tonic immobility to have a broad-sense heritability of 0.502. Taken together our results suggest that larval lacewings under energetic stress are more likely to engage in tonic immobility. Yet, energetic state does not explain all within-population variation, as individuals can have a genetic predisposition for tonic immobility.

Fast and easy synthesis of silver, copper, and bimetallic nanoparticles on cellulose paper assisted by ultrasound.

This work focuses on a systematic method to produce Ag, Cu, and Ag/Cu metallic nanoparticles (MNPs) in situ assisted with ultrasound on cellulose paper. By tuning the concentration of AgNO3 and CuSO4 salt precursors and ultrasound time, combined with a fixed concentration of ascorbic acid (AA) as a reducing agent, it was possible to control the size, morphology, and polydispersity of the resulting MNPs on cellulose papers. Notably, high yield and low polydispersity of MNPs and bimetallic nanoparticles are achieved by increasing the sonication time on paper samples pre-treated with salt precursors before reduction with AA. Moreover, mechanical analysis on paper samples presenting well-dispersed and distributed MNPs showed slightly decreasing values of Young's modulus compared to neat papers. The strain at break is substantially improved in papers containing solely Ag or Cu MNPs. The latter suggests that the elastic/plastic transition and deformation of papers are tuned by cellulose and MNPs interfacial interaction, as indicated by mechanical analysis. The proposed method provides insights into each factor affecting the sonochemistry in situ synthesis of MNPs on cellulose papers. In addition, it offers a straightforward alternative to scale up the production of MNPs on paper, ensuring an eco-friendly method.

Application of Formulation Principles to Stability Issues Encountered During Processing, Manufacturing, and Storage of Drug Substance and Drug Product Protein Therapeutics.

The field of formulation and stabilization of protein therapeutics has become rather extensive. However, most of the focus has been on stabilization of the final drug product. Yet, proteins experience stress and degradation through the manufacturing process, starting with fermentaition. This review describes how formulation principles can be applied to stabilize biopharmaceutical proteins during bioprocessing and manufacturing, considering each unit operation involved in prepration of the drug substance. In addition, the impact of the container on stabilty is discussed as well.

Recent Advances of Optical Biosensors in Veterinary Medicine: Moving Towards the Point of Care Applications.

While food safety issues are attracting public concern due to their detrimental effects on human health, monitoring livestock health is urgently needed to diagnose animal diseases at an early stage by applying proper treatments, controlling, and preventing outbreaks, particularly in resource- limited countries. In addition, unhealthy farms are not only a threat to livestock but also to human lives. The available diagnostic techniques for the detection of key health threats within both the food and livestock sectors require labor-intensive and time-consuming experimental procedures and sophisticated and expensive instruments. To tackle this issue, optical biosensing strategies have been incorporated into point-of-care (POC) systems, offering real-time monitoring, field-deployable, and low-cost devices, which help make on-the-spot decisions. This review aims to discuss the recent cutting-edge research on POC optical biosensing platforms for on-farm diagnosis of animal diseases and on-site detection of animal-derived food-borne contaminants, including pathogens, antibiotics, and mycotoxins. Moreover, this review briefly presents the basic knowledge of various types of optical biosensors and their development using various recent strategies, including nanomaterial combinations, to enhance their performance in POC tests. This review is expected to help scientists to understand the evolution and challenges in the development of point-of-care biosensors for the food and livestock industry, benefiting global healthcare.