Nanozymes on paper: Transforming microfluidic devices for portable biosensing.

Paper-based microchannels are important components in paper-based microfluidic devices. Paper-based microchannels are usually manufactured by creating lines with hydrophobic patterns enclosing an area of hydrophilic paper inside. Among the many techniques for making hydrophobic lines, the printing method in general and inkjet printing in particular have become more common because of their advantages in terms of material saving, high precision, low-cost, etc. In this paper, the electrohydrodynamic (EHD) inkjet printing method, which has been studied and developed in recent years, has been used to drop glycol ether solvent-based ink onto a nitrocellulose paper. The results of the field emission scanning electron microscope (FE-SEM) show that the solvent based ink could dissolve through membrane and form a transparent hydrophobic layer on the created space. The width of the barrier lines can be controlled from about 200 μm to 260 μm, which is quite small when compared to other manufacturing methods. Based on this fabrication process, paper-based microfluidic analytical devices have been designed and fabricated for use in analytical biochemistry assays. Human chorionic gonadotropin (hCG) was selected as the biological target for this assay. Initial results have shown that the target for hCG can be quantitatively determined by these devices with a coefficient of determination (R2) of approximately 0.99. Therefore, EHD inkjet printing technology can be used for the manufacture of paper-based microfluidic analytical devices (μPADs) that may be applied in the future for analytical biochemistry assays.

ABSTRACT. Two easy-to-use, rapid, and inexpensive colorimetric-based assays for the detection of active constituent in nasal decongestant phenylephrine hydrochloride (PHP), in solution and their respective pharmaceutical preparations were reported. The assay principle depends on the diazotized sulfadimidine (DSDM) coupling with PHP active pharmaceutical compound under alkaline conditions to produce a colored azo-dye product. A UV/VIS spectrophotometry method was utilized to monitor the color absorbance change at 460 nm, with a linear calibration graph in the concentration range of 1-20 mg L-1 and a limit of detection of 0.22 mg L-1 and limit of quantification of 0.72 mg L-1. A comparison was done using a paper-based microfluidic analytical device (µPADs) and color intensity change was measured using a Samsung note 9 camera to capture digital images and Image J software to analyze the color intensity development. A linear trend was observed above approximately 10 mg L-1. Thus, paper-based microfluidic analytical devices (µPADs) can be used as an alternative platform to conventional methods to perform a semi-quantitative PHP drug analysis. The proposed method was applied for the determination of PHP in commercial pharmaceutical preparations. KEY WORDS: Phenylephrine hydrochloride, Spectrophotometry, Paper-based microfluidic analytical devices (µPADs), Pharmaceutical preparations, Colorimetric detection Bull. Chem. Soc. Ethiop. 2022, 36(4), 727-736. DOI: https://dx.doi.org/10.4314/bcse.v36i4.1

Point-of-care testing (POCT) shows its significant importance in academic and social affairs (Jansen et al. 1998; Wu et al. 1999). From commercialized products to laboratory prototypes, the creative atmosphere in this research field has maintained its vitality and drawn increasing attention from researchers in related areas. There have been many inventions that have changed the practice of medicine at the point of care in either rural or developed areas, and one prominent candidate for POCT is paper-based microfluidic analytical devices, also called Chip-on-a-Paper, or paper-based POC.

Paper-based microfluidic analytical devices (¼PAD) have great potential for use in a variety of areas, from environmental monitoring to clinical and bedside testing. Such devices are generally designed to be disposable, cost effective, practical and user-friendly. In this study, a smartphone based ¼PAD was developed which can be used in high sensitive quantitative analysis of H2O2 in transparent liquids such as water. Solid ink technology is used in the production of ¼PADs. The H2O2 concentration dependent color change (colorimetric) used in the measurement was catalyzed by horseradish peroxidase (HRP). In addition, the effect of chitosan on the measurement sensitivity was investigated. The resulting color changes were recorded in a dark environment with a special apparatus integrated smartphone camera and the color changes were digitized by an algorithm developed in MATLAB. The results showed that the produced ¼PADs can successfully be used to quantitatively analyze H2O2 between X and Y, and the chitosan significantly increases the sensitivity.

While basic concepts of paper-based microfluidic analytical devices (μPADs) have appeared considerably, elemental technology development is worth working from different specialized points of view. Herein, we show that 2,2,6,6-tetramethylpiperidine-1-oxyl radical (TEMPO)-oxidized cellulose nanofiber (TOCN) can be reasonably used as a module of μPADs; we adopted a biochemical reaction of indigo formation by oxidation of indoxyl acetate (IDA) via its hydrolysis with acetylcholinesterase (AChE) as an example. First, we demonstrate that TOCN stably stores unstable AChE and IDA when dried and acts as a biochemical reaction field when wet. The thixotropic nature of TOCN aqueous dispersion gave inkjet printability, which will facilitate the μPADs production. The exchange of substances in the wet TOCN network took more time than in solution, but this can be read as control of reaction kinetics. Finally, we constructed semiquantitative μPADs for an organic phosphorus pesticide. We illustrate that by mounting TOCN, various functions can be incorporated on one sheet of paper to lead to the extension of design flexibility and universal use for μPADs.

Low-cost paper-based analytical devices are the latest generation of portable lab-on-chip designs that offers an innovative platform for the on/off-site analysis (biosensing) of target analytes, especially in rural and remote areas. Recently, microfluidic paper-based analytical devices (μPADs) have attained significant recognition owing to their exciting fundamental features such as: ease of fabrication, rapid operation, and precise interpretations. The incorporation of enzymes with paper-based analytical devices significantly improves analytical performance while exhibiting excellent chemical and storage stability. In addition to that, these devices are highly compact, portable, easy-to-use, and do not require any additional sophisticated equipment for the detection and quantification of target analytes. This review provides a holistic insight into design, fabrication, and enzyme immobilization strategies for the development of enzyme-μPADs, which enables them to be widely implemented for in-field analysis. It also highlights the recent application of enzyme-μPADs in the area of: biomedical, food safety, and environmental monitoring while exploring the mechanisms of detection involved. Further, in order to improve the accuracy of analysis, researchers have designed a smartphone-based scanning tool for multi-variant point-of-care devices, which is summarized in the latter part of the review. Finally, the future perspectives and outlook of major challenges associated with enzyme-μPADs are discussed with their possible solutions. The development of enzyme integrated μPADs will open a new avenue as an exceptional analytical tool to explore various applications. HIGHLIGHTS Enzyme embedded paper-based analytical devices are a revolution in the field of biosensing. The design, fabrication, and enzyme immobilization on μPADs have been comprehensively discussed. The application of enzyme-μPADs food safety, environmental monitoring, and clinical diagnostic have been reviewed. Smartphones can be used as an on-site, user-friendly, and compact next-gen scanning tool for biosensing.

Fabrication techniques for microfluidic paper-based analytical devices and their applications for biological testing: A review.

Electrochromatographic separations of three metal complexes were achieved through observing the visualization evolution process of moving chelation boundaries (MCBs) on microfluidic paper-based analytical devices (μPADs). A simplified photolithography was developed to pattern the μPADs within 5–10 min with three steps only, manually gelatinize, expose and develop, through thinning SU-8 2150 with trichloromethane. With a cutting method, many bubbles were clearly observed in the single-component MCB system on the μPADs, but were not found with this photolithography method under the same voltages. It was demonstrated that this photolithography for patterning narrow paper channels rejected Joule heating more effectively than the cutting paper method did. It was observed simultaneously that the apparent distances of electrophoretic and electroosmotic migrations of the blue [Cu-EDTA]2− chelation boundary increased with increasing electrical field strength and width of the paper channels, respectively. The higher the Cu2+ concentrations were, the slower the electrophoretic migrations of the blue boundary were, and the faster the electroosmotic migrations of the blue boundary were on the μPADs with this photolithography. The separations and concentrations of the coloured zones, pink [Co–EDTA]2−, blue [Cu–EDTA]2− and yellow [Fe–EDTA]2−, were clearly observed in the applied electric fields on the μPADs. The studies benefit extending the application of μPADs as low-cost, disposable analysis tools and deepen understanding of the comprehensive mechanisms concerning MCBs, partition adsorption and electroosmotic flow on μPADs.

Human chorionic gonadotropin (HCG) has been amongst of essential part of medical diagnostics in gynaecology and oncology since its discovery. Application of gold nanoparticles in detecting HCG is due to gold nanoparticles possess extraordinary optical and physical properties, making them invaluable in various prominent aspects, including its synthesis, stabilization, and functionalization as a sensor. In addition, microfluidic analytical device, a platform for point-of-care testing and numerous applications in various field, can be applied for detection of HCG using diverse types of detection methods. From the literatures, combination of gold nanoparticles and microfluidic analytical device can be assembled to obtain a rapid, simple and user-friendly analytical site for detection of HCG.

Paper-based microfluidic devices have many advantages such as low cost, flexibility, light weight and easy disposability. Especially, since they can intrinsically generate capillary-driven flow (no pumps are needed), paper-based microfluidic devices are widely used in analytical or diagnostic platforms. Along with advancements in microfluidic paper-based analytical devices (μPADs), energy generation using paper materials has received significant attention. In this study, environment-friendly and flexible paper-based energy harvesting with a simple configuration is demonstrated by using the principle of reverse electrodialysis (RED). RED is a promising clean energy generation method, which converts Gibbs free energy into electricity by salinity gradients without discharging any pollutants. However, the power efficiency in a conventional RED device is limited by the essential requirement of active pumping for providing high and low concentration electrolytes. Capillary pumping from the proposed paper-based RED can save this waste of energy, and moreover, the flexible device is realized with cost effective materials and a simple fabrication step, and is environmentally friendly. By thoughtful analysis of voltage-current experiments and capillary flow rates in paper channels, the optimized channel width interfacing with a selective membrane is determined as 2 mm and the maximum power and power density are achieved as 55 nW and 275 nW cm(-2), respectively. 25.8% of the generated maximum power is successfully saved by realizing the pumpless RED system. This paper-based RED device can be integrated directly with μPADs as a practical application.

Iodine is an essential micronutrient for humans due to its fundamental role in the biosynthesis of thyroid hormones. As a key parameter to assess health conditions, iodine intake needs to be monitored to ascertain and prevent iodine deficiency. Iodine is available from various food sources (such as seaweed, fish, and seafood, among others) and dietary supplements (multivitamins or mineral supplements). In this work, a microfluidic paper-based analytical device (μPAD) to quantify iodide in seaweed and dietary supplements is described. The developed μPAD is a small microfluidic device that emerges as quite relevant in terms of its analytical capacity. The quantification of iodide is based on the oxidation of 3,3',5,5'-tetramethylbenzidine (TMB) by hydrogen peroxide in the presence of iodine, which acts as the catalyst to produce the blue form of TMB. Additionally, powder silica was used to intensify and uniformize the colour of the obtained product. Following optimization, the developed μPAD enabled iodide quantification within the range of 10-100 µM, with a detection limit of 3 µM, and was successfully applied to seaweeds and dietary supplements. The device represents a valuable tool for point-of-care analysis, can be used by untrained personnel at home, and is easily disposable, low-cost, and user-friendly.

Lab-on-paper technologies, also known as paper-based analytical devices (PADs), have received increasing attention in the last years, and nowadays, their use has spread to virtually every application area, i.e., medical diagnostic, food safety, environmental monitoring, etc. Advantages inherent to on-field detection, which include avoiding sampling, sample preparation and conventional instrumentation in central labs, are undoubtedly driving many developments in this area. Heavy metals represent an important group of environmental pollutants that require strict controls due to the threat they pose to ecosystems and human health. In this overview, the development of PADs for Hg monitoring, which is considered the most toxic metal in the environment, is addressed. The main emphasis is placed on recognition elements (i.e., organic chromophores/fluorophores, plasmonic nanoparticles, inorganic quantum dots, carbon quantum dots, metal nanoclusters, etc.) employed to provide suitable selectivity and sensitivity. The performance of both microfluidic paper-based analytical devices and paper-based sensors using signal readout by colorimetry and luminescence will be discussed.

Microfluidic paper-based analytical devices (μPADs) are simple but powerful analytical tools that are gaining significant recent attention due to their many advantages over more traditional monitoring tools. These include being inexpensive, portable, pump-free, and having the ability to store reagents. One major limitation of these devices is slow flow rates, which are controlled by capillary action in the hydrophilic pores of cellulosic paper. Recent investigations have advanced the flow rates in μPADs through the generation of a gap or channel between two closely spaced paper sheets. This multilayered format has opened up μPADs to new applications and detection schemes, where large gap sizes (>300 μm) provide at least 169× faster flow rates than single-layer μPADs, but do not conform to established mathematical models for fluid transport in porous materials, such as the classic Lucas-Washburn equation. In the present study, experimental investigations and analytical modeling are applied to elucidate the driving forces behind the rapid flow rates in these devices. We investigate a range of hypotheses for the systems fluid dynamics and establish a theoretical model to predict the flow rate in multilayered μPADs that takes into account viscous dissipation within the paper. Device orientation, sample addition method, and the gap height are found to be critical concerns when modeling the imbibition in multilayered devices.

A simple double-layered three-dimensional (3D) microfluidic paper-based analytical device (μPAD) was designed for the simultaneous determination of six metal ions-Fe(III), Ni(II), Cr(VI), Cu(II), Al(III), and Zn(II)-for the first time. The 3D μPAD was composed of two paper layers: a top pretreatment layer and a bottom colorimetric detection layer. The sample solution added to the central sample reservoir of the 3D μPAD could be automatically divided into eight flow pathways and be automatically pretreated while flowing through the pretreatment zones located in the microfluidic channels, and automatically carried out the chromogenic reactions after reaching the detection zones. Random diffusion of the chromogenic reagents was effectively prevented by transport of the pretreated sample solution to the detection zones through 3D microfluidic channels with an L-type circuitous flow route design, resulting in highly increased color uniformity and reproducibility. Combined with use of a flat LED lamp as an upward lighting source and a smartphone as a convenient detector, improved color perception, highly enhanced sensitivity, and an extended detection range were obtained. Finally, the double-layered 3D μPAD was applied to the multiplexed determination of the six metal ions in mixtures and environmental samples with satisfactory results. Detection limits as low as 0.2, 0.3, 0.1, 0.03, 0.08, and 0.04 mg/L for Fe(III), Ni(II), Cr(VI), Cu(II), Al(III), and Zn(II) detection, respectively, were achieved, which are about one order of magnitude lower than obtained with previously reported μPADs for the detection of metal ions. The present 3D μPAD is simple, fast, selective, sensitive, and user-friendly, and holds great application potential for multiplexed on-site analysis.

In this work, two microfluidic paper-based analytical devices (μPADs) were developed for the quantification of urea and NHx in human saliva to aid in the diagnosis/monitoring of chronic kidney disease (CKD). The NHx determination was based on the conversion of ammonium to ammonia, followed by its diffusion through a hydrophobic membrane and then the color change of bromothymol blue (BTB) indicator. In the urea determination, prior to the ammonium conversion and BTB color change, the enzymatic conversion of urea into ammonium was produced, using urease. Several optimization studies were carried out to attain a quantification range of 0.10–5.0 mM with 0.032 mM limit of detection for the NHx μPAD, and a determination range of 0.16–5.0 mM with 0.049 mM limit of detection for the urea μPAD. The method accuracy was assessed, and the measurements obtained with NHx μPAD were compared with the ones obtained from an ammonia ion selective electrode; while the measurements of the urea μPAD were compared with the ones obtained from a commercially available kit. There were no statistically significant differences between methods, proving that both NHx and urea μPAD were effective on-hand tools for CKD monitoring in saliva. To evaluate their functionality as point-of-care devices, stability studies were also performed and revealed that both NHx and urea μPAD were stable when stored in a vacuum for 2 and 1 month, respectively. After the sample introduction, the NHx μPAD could be scanned within the first 2 h and the urea μPAD within 1 h.