Tailoring plasmonic sensing strategies for the rapid and sensitive detection of hypochlorite in swimming water samples.

A tunable plasmonic sensor has been developed by varying the dextran content in the initially synthesized dextran-gold nanoparticle (dAuNPs) solution. A colloidal nanogold solution (dAuNPs-Sol) was initially prepared using dextran and gold salt in alkaline media by a one-pot green synthetic route. The dAuNPs-Sol was combined with varying amounts of dextran (ranging from 0.01 to 30.01%) to create a tunable probe, along with different solid formats, including tablet (dAuNPs-Tab), powder (dAuNPs-Powder), and composite (dAuNPs-Comp). Both the liquid and solid phase plasmonic probes were characterized using UV-vis spectroscopy, transmission electron microscopy (TEM) dynamic light scattering (DLS), and zeta potential analysis. The impact of dextran content in the dAuNP solution is studied in terms of surface charge and hydrodynamic size. The influence of operational treatments used to achieve solid dAuNPs probes is also explored. All plasmonic probes were employed to detect a broad range of OCl¯ concentrations (ranging from µM to mM) in water through aggregation followed by calculating a lower and upper limit of detection (LLoD, ULoD) of the proposed colorimetric sensors. Results indicate that the most sensitive detection is achieved with a lower dextran content (0.01%), which exhibits an LLoD of 50 µM. The dAuNPs-Sol sensor is selective and demonstrates real-world applicability, as confirmed by interference analysis and successful testing with various water samples. Additionally, it is found that a 20 × concentration of dextran-coated gold nanoparticles could be attained without any changes in the particle morphology. This concentration is achieved through a straightforward process that does not require the use of a centrifuge machine. This finding highlights the practicality and simplicity of the method, indicating its potential for scalable and cost-effective production of concentrated dAuNPs without compromising their structural integrity.

Tools and Techniques for Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2)/COVID-19 Detection.

The coronavirus disease 2019 (COVID-19) pandemic, caused by severe acute respiratory disease coronavirus 2 (SARS-CoV-2), has led to millions of confirmed cases and deaths worldwide. Efficient diagnostic tools are in high demand, as rapid and large-scale testing plays a pivotal role in patient management and decelerating disease spread. This paper reviews current technologies used to detect SARS-CoV-2 in clinical laboratories as well as advances made for molecular, antigen-based, and immunological point-of-care testing, including recent developments in sensor and biosensor devices. The importance of the timing and type of specimen collection is discussed, along with factors such as disease prevalence, setting, and methods. Details of the mechanisms of action of the various methodologies are presented, along with their application span and known performance characteristics. Diagnostic imaging techniques and biomarkers are also covered, with an emphasis on their use for assessing COVID-19 or monitoring disease severity or complications. While the SARS-CoV-2 literature is rapidly evolving, this review highlights topics of interest that have occurred during the pandemic and the lessons learned throughout. Exploring a broad armamentarium of techniques for detecting SARS-CoV-2 will ensure continued diagnostic support for clinicians, public health, and infection prevention and control for this pandemic and provide advice for future pandemic preparedness.

Portable, stable, and sensitive assay to detect phosphate in water with gold nanoparticles (AuNPs) and dextran tablet.

A novel and highly sensitive tablet-based colorimetric sensor is developed for the detection of phosphate (Pi) in drinking and surface water using mercaptoacetic acid-capped gold nanoparticles (MA-AuNPs). Characterization of AuNPs and MA-AuNPs was achieved by ultraviolet-visible (UV-Vis) spectroscopy, Fourier transform infrared spectroscopy (FTIR), Transmission electron microscopy (TEM) and Dynamic light scattering (DLS). The principle of this sensor is based on the aggregation and disaggregation mechanisms of AuNPs that result in a color change from blue to red due to the surface plasmon resonance effect, where europium ions (Eu3+) act as the aggregating agent. Herein, dextran is used to encapsulate the Eu3+ ions into a tablet format to make the detection system user friendly. Hence, the sensor only requires dissolving a Eu3+-dextran tablet into the water sample and subsequently adding MA-AuNPs for the colorimetric quantification of phosphate. This assay is very sensitive with a calculated detection limit of 0.3 µg L-1 and an upper detection limit of 26 µg L-1, while 10 µg L-1 is the allowable limit of Pi in drinking water. A comparative study with a conventional Hach kit confirmed the accuracy of our sensor. Also, real water samples from river, lake, and tap sources were tested to examine the sensor's applicability towards commercialization. The assay did not interfere with common ions in water, thus being Pi-specific, and the performance of the assay was stable for up to at least three weeks. Overall, our new approach provides a simple, stable, rapid, low-cost and promising device for Pi detection in water.

RNA Protection is Effectively Achieved by Pullulan Film Formation.

RNA is a functionally versatile polymer but suffers from susceptibility to spontaneous and RNase-catalyzed degradation. This vulnerability makes it difficult to preserve RNA for extended periods of time, thus limiting its use in various contexts, including practical applications as functional nucleic acids. Here we present a simple method to preserve RNA by pullulan (a complex sugar produced by Aureobasidium pullulans fungus) film formation. This strategy can markedly suppress both spontaneous and RNase degradation. Importantly, the pullulan film readily dissolves in aqueous solution, thus allowing retrieval of fully functional RNA species. In order to illustrate the advantage of this protective method in a practical application, we engineered a simple paper sensor containing a bacteria-detecting RNA-cleaving DNAzyme. This detection capability of the device was unchanged after storage at room temperature for six months.

Target-Induced and Equipment-Free DNA Amplification with a Simple Paper Device.

We report on a paper device capable of carrying out target-induced rolling circle amplification (RCA) to produce massive DNA amplicons that can be easily visualized. Interestingly, we observed that RCA was more proficient on paper than in solution, which we attribute to a significantly higher localized concentration of immobilized DNA. Furthermore, we have successfully engineered a fully functional paper device for sensitive DNA or microRNA detection via printing of all RCA-enabling molecules within a polymeric sugar film formed from pullulan, which was integrated with the paper device. This encapsulation not only stabilizes the entrapped reagents at room temperature but also enables colorimetric bioassays with minimal steps.

Printed paper sensors for serum lactate dehydrogenase using pullulan-based inks to immobilize reagents.

In this study, a paper-based point-of-care (POC) colorimetric biosensor was developed for the detection of lactate dehydrogenase in serum using a nonporous, oxygen impermeable reversibly gelling polysaccharide material based on pullulan. The pullulan could be printed onto paper surfaces along with all required assay reagents, providing a means for high-stability immobilization of all reagents on paper. Serum containing lactate dehydrogenase (LDH) was directly spotted on to the pullulan-coated bioactive paper and provided quantitative colorimetric data that was comparable to that obtained with a conventional plate-reader method. The paper strip was found to be highly stable and could be stored at 4 °C for at least 10 weeks with no loss in performance, as compared to a complete loss in performance within 1 day when the reagents were printed without the stabilizing polysaccharide. The ease of fabrication coupled with the high stability of the printed reagents provides a facile platform for easily manufactured POC sensors.

Pullulan encapsulation of labile biomolecules to give stable bioassay tablets.

A simple and inexpensive method is reported for the long-term stabilization of enzymes and other unstable reagents in premeasured quantities in water-soluble tablets (cast, not compressed) made with pullulan, a nonionic polysaccharide that forms an oxygen impermeable solid upon drying. The pullulan tablets dissolve in aqueous solutions in seconds, thereby facilitating the easy execution of bioassays at remote sites with no need for special reagent handling and liquid pipetting. This approach is modular in nature, thus allowing the creation of individual tablets for enzymes and their substrates. Proof-of-principle demonstrations include a Taq polymerase tablet for DNA amplification through PCR and a pesticide assay kit consisting of separate tablets for acetylcholinesterase and its chromogenic substrate, indoxyl acetate, both of which are highly unstable. The encapsulated reagents remain stable at room temperature for months, thus enabling the room-temperature shipping and storage of bioassay components.

User-friendly and ultra-stable all-inclusive gold tablets for cysteamine detection.

To date, a range of nanozymes has been reported for their enzyme-mimicking catalytic activity such as solution-based sensors. However, in remote areas, the need for portable, cost-effective, and one-pot prepared sensors is obvious. In this study, we report the development of a highly stable and sensitive gold tablet-based sensor for cysteamine quantification in human serum samples. The sensor is produced in two steps: synthesis of a pullulan-stabilized gold nanoparticle solution (pAuNP-Solution) using a pullulan polymer as a reducing, stabilizing, and encapsulating agent and then, casting the pAuNP-Solution into a pullulan gold nanoparticle tablet (pAuNP-Tablet) by a pipetting method. The tablet was characterized by UV-vis, DLS, FTIR, TEM, and AFM analyses. The pAuNP-tablet exhibited a high peroxidase-mimetic activity via a TMB-H2O2 system. The presence of cysteamine in the system introduced two types of inhibition which were dependent on the cysteamine concentration. By determining Michaelis-Menten's kinetic parameters, we gained mechanistic insights into the catalytic inhibition process. Based on the catalytic inhibition capability of cysteamine, the limit of detection (LoD) was calculated to be 69.04 and 82.9 µM in buffer and human serum samples, respectively. Finally, real human serum samples were tested, demonstrating the applicability of the pAuNP-Tablet for real-world applications. The % R values in human serum samples were in the range of 91-105% with % RSD less than 2% for all replicas. The stability tests over 16 months revealed the ultra-stable properties of the pAuNP-Tablet. Overall, with a simple fabrication method and a novel employed technique, this study contributes to the advancement of tablet-based sensors and helps in cysteamine detection in clinical settings.

Gold Nanoparticles-Based Colorimetric Assays for Environmental Monitoring and Food Safety Evaluation.

Recent years have witnessed an exponential increase in the research on gold nanoparticles (AuNPs)-based colorimetric sensors to revolutionize point-of-use sensing devices. Hence, this review is compiled focused on current progress in the design and performance parameters of AuNPs-based sensors. The review begins with the characteristics of AuNPs, followed by a brief explanation of synthesis and functionalization methods. Then, the mechanisms of AuNPs-based sensors are comprehensively explained in two broad categories based on the surface plasmon resonance (SPR) characteristics of AuNPs and their peroxidase-like catalytic properties (nanozyme). SPR-based colorimetric sensors further categorize into aggregation, anti-aggregation, etching, growth-mediated, and accumulation-based methods depending on their sensing mechanisms. On the other hand, peroxidase activity-based colorimetric sensors are divided into two methods based on the expression or inhibition of peroxidase-like activity. Next, the analytes in environmental and food samples are classified as inorganic, organic, and biological pollutants, and recent progress in detection of these analytes are reviewed in detail. Finally, conclusions are provided, and future directions are highlighted. Improving the sensitivity, reproducibility, multiplexing capabilities, and cost-effectiveness for colorimetric detection of various analytes in environment and food matrices will have significant impact on fast testing of hazardous substances, hence reducing the pollution load in environment as well as rendering food contamination to ensure food safety.

Tablet-Based Sensor: A Stable and User-Friendly Tool for Point-of-Care Detection of Glucose in Urine.

The colorimetric detection of glucose in urine through enzymatic reactions offers a low-cost and non-invasive method to aid in diabetes management. Nonetheless, the vulnerability of enzymes to environmental conditions, particularly elevated temperatures, and their activity loss pose significant challenges for transportation and storage. In this work, we developed a stable and portable tablet sensor as a user-friendly platform for glucose monitoring. This innovative device encapsulates glucose oxidase and horseradish peroxidase enzymes with dextran, transforming them into solid tablets and ensuring enhanced stability and practicality. The enzymatic tablet-based sensor detected glucose in urine samples within 5 min, using 3,3',5,5'-tetramethylbenzidine (TMB) as the indicator. The tablet sensor exhibited responsive performance within the clinically relevant range of 0-6 mM glucose, with a limit of detection of 0.013 mM. Furthermore, the tablets detected glucose in spiked real human urine samples, without pre-processing, with high precision. Additionally, with regard to thermal stability, the enzyme tablets better maintained their activity at an elevated temperature as high as 60 °C compared to the solution-phase enzymes, demonstrating the enhanced stability of the enzymes under harsh conditions. The availability of these stable and portable tablet sensors will greatly ease the transportation and application of glucose sensors, enhancing the accessibility of glucose monitoring, particularly in resource-limited settings.

Gold Tablets: Gold Nanoparticles Encapsulated into Dextran Tablets and Their pH-Responsive Behavior as an Easy-to-Use Platform for Multipurpose Applications.

Many applications using gold nanoparticles (AuNPs) require (i) their functionalization with a biopolymer to increase their stability and (ii) their transformation into an easy-to-handle material, which provide them with specific properties. In this research, a portable tablet platform is presented based on dextran-encapsulated gold nanoparticles (AuNPs-dTab) by a ligand exchange reaction between citrate-capped gold nanoparticles (AuNPs-Cit) and dextran. These newly fabricated tablets were characterized utilizing ultraviolet-visible spectroscopy (UV-vis), Fourier transform infrared spectroscopy-attenuated total reflectance (FTIR-ATR), transmission electron microscopy (TEM), dynamic light scattering (DLS), X-ray diffraction spectroscopy (XRD), differential scanning calorimetry (DSC), and atomic force microscopy (AFM) techniques. The results showed that dextran-capped gold nanoparticles in a tablet platform (AuNPs-dTab) were well-dispersed and highly stable for at least a year at room temperature. In addition to particle and surface characterization of AuNPs-dTab, the tablet morphology in terms of thickness, diameter, density, and opacity was also measured using 6 and 10% dextran with 2, 4 and 8 nM AuNPs-Cit. We further investigated the pH-responsive behavior of AuNPs-dTab in the presence and absence of sodium chloride. Results showed that neutral and alkaline environments were suitable to render AuNPs dispersed in a tablet, while an acidic condition controls the aggregation rate of AuNPs as confirmed by concentration-dependent aggregation phenomena. Besides the easy fabrication, these tablets were portable and low-cost (approx. 1.22 CAD per 100 tablets of a 100 µL solution of dextran-capped gold nanoparticles (AuNPs-dSol)). The biocompatible nature of dextran along with the acidic medium trigger nature of AuNPs makes our proposed tablet a potential candidate for cancer therapy due to the acidic surrounding of tumor tissues as compared to normal cells. Also, our proposed tablet approach paves the way for the fabrication of portable and easy-to-use optical sensors based on the AuNPs embedded in a natural polymeric architecture that would serve as a colorimetric recognition indicator for detecting analytes of interest.

Thermal Stabilization of Viral Vaccines in Low-Cost Sugar Films.

Most currently available vaccines, particularly live vaccines, require the cold chain, as vaccine efficacy can be significantly hampered if they are not stored in a temperature range of 2-8 °C at all times. This necessity places a tremendous financial and logistical burden on vaccination programs, particularly in the developing world. The development of thermally stable vaccines can greatly alleviate this problem and, in turn, increase vaccine accessibility worldwide. In this paper, we detail a simple and cost-effective method for stabilizing live vaccines that uses FDA-approved materials. To this end, we dried enveloped DNA (Herpes Simplex Virus type 2) and RNA (Influenza A virus) viral vaccines in a pullulan and trehalose mixture. The results of these studies showed that the live-attenuated HSV-2 vaccine retained its efficacy for at least 2 months of storage at 40 °C, while the inactivated influenza vaccine was able to retain its immunogenicity for at least 3 months of storage at 40 °C. This work presents a simple approach that allows thermo-sensitive vaccines to be converted into thermo-stable vaccines that do not require refrigeration, thus contributing to the improvement of vaccine deployment throughout the world.

A Printed Multicomponent Paper Sensor for Bacterial Detection.

We present a simple all-in-one paper-based sensor for E. coli detection using a composite ink made of a fluorogenic DNAzyme probe for bacterial recognition and signal generation, lysozyme that lyses whole bacterial cells, and pullulan/trehalose sugars that stabilize printed bioactive molecules. The paper sensor is capable of producing a fluorescence signal as a readout within 5 minutes upon contacting E. coli, can achieve a limit of detection of 100 cells/mL, in a variety of sample matrixes, without sample enrichment, and remains stable for at least 6 months when stored at ambient temperature. Therefore, this simple paper sensor provides rapid bacterial testing on site, and can be shipped and stored under ambient conditions to benefit users living in resource-limited regions.

Automating multi-step paper-based assays using integrated layering of reagents.

We describe a versatile and simple method to perform sequential reactions on paper analytical devices by stacking dry pullulan films on paper, where each film contains one or more reagents or acts as a delay layer. Exposing the films to an aqueous solution of the analyte leads to sequential dissolution of the films in a temporally controlled manner followed by diffusive mixing of the reagents, so that sequential reactions can be performed. The films can be easily arranged for lateral flow assays or for spot tests (reactions take place sequentially in the z-direction). We have tested the general feasibility of the approach using three different model systems to demonstrate different capabilities: 1) pH ramping from low to high and high to low to demonstrate timing control; 2) rapid ready-to-use two-step Simon's assays on paper for detection of drugs of abuse utilizing a 2-layer stack containing two different reagents to demonstrate the ability to perform assays in the z-direction; and 3) sequential cell lysing and colorimetric detection of an intracellular bacterial enzyme, to demonstrate the ability of the method to perform sample preparation and analysis in the form of a spot assay. Overall, these studies demonstrate the potential of stacked pullulan films as useful components to enable multi-step assays on simple paper-based devices.

Simple and ultrastable all-inclusive pullulan tablets for challenging bioassays.

Many biodetection systems employ labile enzymes and substrates that need special care, making it hard to routinely use them for point-of-care or field applications. In this work we provide a simple solution to this challenging problem through the creation of all-inclusive pullulan assay tablets. The proposed tablet system not only enhances the long-term stability of both enzymes and organic substrates, but also simplifies the assay procedure. The enhanced stability is attributed to two factors: the restriction of the molecular motion of proteins and impermeability to molecular oxygen afforded by the tables. These tablets dissolve rapidly upon addition to testing samples, making the test very easy to perform. Using the ATP-detecting luciferase-luciferin system as an example, we show that the tablet-based assay can achieve highly sensitive detection of ATP in biological samples and that the activity of the assay tablets remains unchanged for over a month at room temperature.

Design Rules for Fluorocarbon-Free Omniphobic Solvent Barriers in Paper-Based Devices.

The utility of hydrophobic wax barriers in paper-based lateral flow and multiwell devices for containment of aqueous solvents was extended to organic solvents and challenging aqueous surfactant solutions by preparation of a three layer barrier, consisting of internal pullulan impregnated paper barriers surrounded by external wax barriers. When paper impregnated with pullulan solution dries, the polymer forms solvent blocking lenses in the paper structure. Lens formation was illustrated by forming pullulan lenses in glass capillaries. The lens shapes were less curved compared to the predictions of a model based upon minimizing surface area. For barriers on Whatman # 1 filter paper, the pullulan molecular weight must be greater than ∼70 kDa, the mass fraction of pullulan in the barrier zone must be at least 32%, and there are restrictions on the minimum width of the pullulan impregnated zone.

Tools for water quality monitoring and mapping using paper-based sensors and cell phones.

In this paper we describe a combination of paper-based sensors and a novel smart-phone application for on-site quantification of colorimetric readouts as an ultra-low cost solution to monitoring water quality. The system utilizes a paper-based analytical device (µPAD) that produces a colorimetric signal that is dependent on the concentration of a specific target; a cell phone equipped with a camera for capturing images of two µPADs - one tested with a water sample and the other tested with clean water that is used as a control; and an on-site image processing app that uses a novel algorithm for quantifying color intensity and relating this to contaminant concentration. The cell phone app utilizes a pixel counting algorithm that performs with less bias and user subjectivity than the typically used lab-based software, ImageJ. The use of a test and control strip reduces bias from variations in ambient lighting, making it possible to acquire and process images on-site. The cell phone is also able to GPS tag the location of the test, and transmit results to a newly developed website, WaterMap.ca, that displays the quantitative results from the water samples on a map. We demonstrate our approach using a previously developed µPAD that detects the presence of organophosphate pesticides based on the inhibition of immobilized acetylcholinesterase by these contaminants. The objective of this paper is to highlight the importance and potential of developing and integrated monitoring system consisting of µPADs, cell-phones and a centralized web portal for low-cost monitoring environmental contaminants at a large-scale.

Paper-based microfluidics with an erodible polymeric bridge giving controlled release and timed flow shutoff.

Water soluble pullulan films were formatted into paper-based microfluidic devices, serving as a controlled time shutoff valve. The utility of the valve was demonstrated by a one-step, fully automatic implementation of a complex pesticide assay requiring timed, sequential exposure of an immobilized enzyme layer to separate liquid streams. Pullulan film dissolution and the capillary wicking of aqueous solutions through the device were measured and modeled providing valve design criteria. The films dissolve mainly by surface erosion, meaning the film thickness mainly controls the shutoff time. This method can also provide time-dependent sequential release of reagents without compromising the simplicity and low cost of paper-based devices.

Creating fast flow channels in paper fluidic devices to control timing of sequential reactions.

This paper reports the development of a method to control the flow rate of fluids within paper-based microfluidic analytical devices. We demonstrate that by simply sandwiching paper channels between two flexible films, it is possible to accelerate the flow of water through paper by over 10-fold. The dynamics of this process are such that the height of the liquid is dependent on time to the power of 1/3. This dependence was validated using three different flexible films (with markedly different contact angles) and three different fluids (water and two silicon oils with different viscosities). These covered channels provide a low-cost method for controlling the flow rate of fluid in paper channels, and can be added following printing of reagents to control fluid flow in selected fluidic channels. Using this method, we redesigned a previously published bidirectional lateral flow pesticide sensor to allow more rapid detection of pesticides while eliminating the need to run the assay in two stages. The sensor is fabricated with sol-gel entrapped reagents (indoxyl acetate in a substrate zone and acetylcholinesterase, AChE, in a sensing zone) present in an uncovered "slow" flow channel, with a second, covered "fast" channel used to transport pesticide samples to the sensing region through a simple paper-flap valve. In this manner, pesticides reach the sensing region first to allow preincubation, followed by delivery of the substrate to generate a colorimetric signal. This format results in a uni-directional device that detects the presence of pesticides two times faster than the original bidirectional sensors.