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This manuscript offers a concise overview of paper microfluidics, emphasizing its sustainable sensing applications in healthcare, environmental monitoring, and food safety. Researchers have developed innovative sensing platforms for detecting pathogens, pollutants, and contaminants by leveraging the paper's unique properties, such as biodegradability and affordability. These portable, low-cost sensors facilitate rapid diagnostics and on-site analysis, making them invaluable tools for resource-limited settings. This review discusses the fabrication techniques, principles, and applications of paper microfluidics, showcasing its potential to address pressing challenges and enhance human health and environmental sustainability. [ABSTRACT FROM AUTHOR]

In 2023, we published a research article in the Journal of Colloidal and Interface Science, based on our experimental findings and substantiating scaling arguments leading to a simple theoretical insight on the effect of red blood cell (RBC) aggregation on the wicking behaviour of a finite volume of blood as it navigates through the porous passages of a paper matrix (Laha et al., 2023). Of late, we received comments from Li (2024), which offered certain suggestions regarding the possible improvement of the capillary bundle model as considered in our article for analyzing the transport of blood through the paper pores. Herein, we provide a detailed discussion on each of the points raised by Li (2024) and rationalize our views in further details in addition to the contents already provided in our concerned article. [ABSTRACT FROM AUTHOR]

In this study, an equation for fitting the band lengths in µPADs to the concentrations/amount of analyte added to the µPAD sample area is derived. A simulation of the band formation is carried out using a discrete segment model. The detector channel is divided into equal segments with the same amount of reagent R in each segment. The sample moves into the channel in steps corresponding to segments of the same size as the detector segment. Each sample segment contains analyte A at C mole ratio to reagent R. Assuming a stoichiometric ratio of 1:1 for reaction between A and R, there will be formation of only one product band in each detector segment. By examining the number of bands (n) formed after N steps, a set of linear algebraic equations is derived to determine the number of bands (n) for any integer values of N and C. By extrapolating this result to real positive numbers, we obtain the equation L=a.CA/(b + CA), where L represents the band length, and CA represents the concentration/amount of analyte. The equation represents a rectangular hyperbola.

This manuscript offers a concise overview of paper microfluidics, emphasizing its sustainable sensing applications in healthcare, environmental monitoring, and food safety. Researchers have developed innovative sensing platforms for detecting pathogens, pollutants, and contaminants by leveraging the paper’s unique properties, such as biodegradability and affordability. These portable, low-cost sensors facilitate rapid diagnostics and on-site analysis, making them invaluable tools for resource-limited settings. This review discusses the fabrication techniques, principles, and applications of paper microfluidics, showcasing its potential to address pressing challenges and enhance human health and environmental sustainability.

The low cost, simplicity, and ease of use of paper microfluidic devices have made them valuable medical diagnostics for applications from pregnancy testing to COVID-19 screening. Meanwhile, the increasing complexity of paper-based microfluidic devices is driving the need to produce new tools and methodologies that enable more robust biological diagnostics and potential therapeutic applications. A new design framework is being used to facilitate both research and fabrication of paper-based microfluidic biological devices to accelerate the investigative process and reduce material utilization and manpower. In this work we present a methodology for this framework to dynamically place and route microfluidic components in a nondiscrete design space where fluid volume usage, surface area utilization, and the timing required to perform specified biological assays are accounted for and optimized while also accelerating the development of potentially lifesaving new 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. [ABSTRACT FROM AUTHOR]

Phage‐inducible chromosomal islands (PICIs) are a family of phage satellites that hijack phage components to facilitate their mobility and spread. Recently, these genetic constructs are repurposed as antibacterial drones, enabling a new toolbox for unorthodox applications in biotechnology. To illustrate a new suite of functions, the authors have developed a user‐friendly diagnostic system, based upon PICI transduction to selectively enrich bacteria, allowing the detection and sequential recovery of Escherichia coli and Staphylococcus aureus. The system enables high transfer rates and sensitivities in comparison with phages, with detection down to ≈50 CFU mL−1. In contrast to conventional detection strategies, which often rely on nucleic acid molecular assays, and cannot differentiate between dead and live organisms, this approach enables visual sensing of viable pathogens only, through the expression of a reporter gene encoded in the PICI. The approach extends diagnostic sensing mechanisms beyond cell‐free synthetic biology strategies, enabling new synthetic biology/biosensing toolkits. [ABSTRACT FROM AUTHOR]

[Display omitted] The complex spreading dynamics of blood on paper matrix is likely to be quantitatively altered with variations in the fractional occupancy of red blood cells in the whole blood (haematocrit). Here, we presented an apparently surprising observation that a finite volume blood drop undergoes a universal time-dependent spreading on a filter paper strip that is virtually invariant with its hematocrit level within physiologically healthy regime, though distinctively distinguishable from the spreading laws of blood plasma and water. Our hypothesis was ascertained by performing controlled wicking experiments on filter papers of different grades. Spreading of human blood samples of different haematocrit levels ranging between 15% and 51% and the plasma separated from therein were traced by combined high-speed imaging and microscopy. These experiments were complemented with a semi-analytical theory to decipher the key physics of interest. Our results unveiled the exclusive influence of the obstructing cellular aggregates in the randomly distributed hierarchically structured porous pathways and deciphered the role of the networked structures of the various plasma proteins that induced hindered diffusion. The resulting universal signatures of spontaneous dynamic spreading, delving centrally on the fractional reduction in the interlaced porous passages, provide novel design basis for paper-microfluidic kits in medical diagnostics and beyond. [ABSTRACT FROM AUTHOR]

Multiplex detection is a smart and an emerging approach in point-of-care testing as it reduces analysis time and testing cost by detecting multiple analytes or biomarkers simultaneously which are crucial for disease detection at an early stage. Application of inexpensive substrate such as paper has immense potential and matter of research interest in the area of point of care testing for multiplexed analysis as it possesses several unique advantages. This study presents the use of paper, strategies adopted to refine the design created on paper and lateral flow strips to enhance the signal, increase the sensitivity and specificity of multiplexed biosensors. An overview of different multiplexed detection studies performed using biological samples has also been reviewed along with the challenges and advantages offered by multiplexed analysis. [ABSTRACT FROM AUTHOR]

Abstract Phage‐inducible chromosomal islands (PICIs) are a family of phage satellites that hijack phage components to facilitate their mobility and spread. Recently, these genetic constructs are repurposed as antibacterial drones, enabling a new toolbox for unorthodox applications in biotechnology. To illustrate a new suite of functions, the authors have developed a user‐friendly diagnostic system, based upon PICI transduction to selectively enrich bacteria, allowing the detection and sequential recovery of Escherichia coli and Staphylococcus aureus. The system enables high transfer rates and sensitivities in comparison with phages, with detection down to ≈50 CFU mL−1. In contrast to conventional detection strategies, which often rely on nucleic acid molecular assays, and cannot differentiate between dead and live organisms, this approach enables visual sensing of viable pathogens only, through the expression of a reporter gene encoded in the PICI. The approach extends diagnostic sensing mechanisms beyond cell‐free synthetic biology strategies, enabling new synthetic biology/biosensing toolkits.

Introduction: Regular and rapid large-scale screening for pathogens is crucial for controlling pandemics like Coronavirus Disease 2019 (COVID-19). In this study, we present the development of a digital point-of-care testing (POCT) system utilizing microfluidic paper-based analytical devices (μPADs) for the detection of SARS-CoV-2 gene fragments. The system incorporates temperature tuning and fluorescent detection components, along with intelligent and autonomous image acquisition and self-recognition programs.Methods: The developed POCT system is based on the nucleic acid amplification test (NAAT), a well-established molecular biology technique for detecting and amplifying nucleic acids. We successfully detected artificially synthesized SARS-CoV-2 gene fragments, namely ORF1ab gene, N gene, and E gene, with minimal reagent consumption of only 2.2 μL per readout, representing a mere 11% of the requirements of conventional in-tube methods. The power dissipation of the system was low, at 6.4 W.Results: Our testing results demonstrated that the proposed approach achieved a limit of detection of 1000 copies/mL, which is equivalent to detecting 1 copy or a single RNA template per reaction. By employing standard curve analysis, the quantity of the target templates can be accurately determined.Conclusion: The developed digital POCT system shows great promise for rapid and reliable detection of SARS-CoV-2 gene fragments, offering a cost-effective and efficient solution for controlling pandemics. Its compatibility with other diagnostic techniques and low reagent consumption make it a viable option to enhance healthcare in resource-limited areas.

This paper reports a simple, disposable, and wearable patch inspired by lateral flow assay (LFA) that can continuously and non-invasively sample sweat for colorimetric sensing of potassium (K+). K+ is an important biomarker for healthy cardiovascular and neuromuscular functions of the body. Current techniques for K+ sensing in sweat commonly use ion-selective electrodes that require complex fabrication steps, signal drift issues, and active perspiration for continuous operation. Here, the sensor uses passive osmotic-capillary principles to collect sweat from the skin without the need of external power for sampling. A skin-mounted hydrogel patch equilibrated with glucose and glycerin solutions can extract fluid from the skin at rest because of the high osmotic strength of the hydrogel compared to sweat. The gel delivers fluid to a commercial colorimetric assay via a paper strip. The assay contains the reagent dipicrylamine which is selective to K+. On-body human trials prove that these patches function with low sweat volumes (∼2–3 µL) and measure K+ concentration in sweat extracted both during exercise and rest. The sweat potassium concentration is independent of the sweat rate and its magnitude matches that of blood. Such economical patches could open opportunities for at-home or in-field point-of-care (POC) diagnostics. • Developed and tested a simple, inexpensive, zero-power, wearable skin patch for potassium sensing in sweat. • The patch uses passive osmotic-capillary principles and paper microfluidics to collect, and transport sweat. • Potassium in sweat is detected by colorimetric techniques. • These patches can measure potassium concentration from human subjects during moderate exercise and rest. • Such economical patches could open opportunities for at-home or in-field point-of-care (POC) diagnostics. [ABSTRACT FROM AUTHOR]

Inflammation caused by viral or bacterial infection is a major threat to human health globally. Blood C-reactive protein (CRP) has been proven to be a sensitive indicator for the occurrence and development of inflammation. Furthermore, a tiny change of blood CRP concentration may portend chronic diseases; therefore, high-sensitivity CRP (hs-CRP) detection in a quantitative, rapid, user-friendly, and low-cost manner is highly demanded. In this paper, we developed a europium-chelate polystyrene microsphere (EuPSM)-based lateral flow immunoassay (LFIA) integrating with a benchtop fluorescence analyzer for hs-CRP detection. The optimization of the EuPSM-based LFIA was implemented through adjusting the antibody density on EuPSM from 100% to 60% of the saturated density. Finally, the limit of detection of 0.76 pg/mL and detection range of 0.025–250 ng/mL were obtained. Moreover, the clinical application capability of the proposed platform was validated through detecting CRP in clinical serum samples, showing high consistency with the results obtained from the clinical standard method. Hence, the proposed EuPSM-based LFIA has been verified to be well suitable for hs-CRP detection, while also showing great applicability for sensitively and rapidly detecting other biomarkers. [ABSTRACT FROM AUTHOR]

Paper‐based microfluidics emerges as an innovative platform for constructing miniaturized electrochemical devices, which mainly benefit from the spontaneous capillary action of paper. Nevertheless, the capillary‐driven flow dynamics on paper are determined exclusively by the intrinsic properties of paper and fluidics, thus lacking the controllability that conventional pump‐based microfluidics can provide. Herein, an approach to regulating the capillary flow on paper is introduced by conjugating the outlets of microfluidic channels with a photothermal module for water evaporation. The capillary flow rate on paper can be handily regulated from 4 to 37 µL min−1 under controllable illumination conditions. As a proof‐of‐concept, prototypical paper‐based microfluidic fuel cells integrated with the photothermal module are constructed. Their peak power density can be boosted from 0.3 up to 2.1 mW cm−2 under the simulated sunlight irradiation. The influence of capillary flow rate on the fuel cell performance is further validated using multiphysics simulations. The present work not only provides a practically feasible method to boost the performance of paper‐based microfluidic fuel cells using solar energy, but also opens a new avenue for modulating the performance of paper‐based microfluidics, which has long been a challenge in this field. [ABSTRACT FROM AUTHOR]

The following paper presents a construct for a paper-based device which utilizes latex as the hydrophobic material for the fabrication of its hydrophobic barrier, which was deposited onto the cellulose surface either by free-hand or stenciled drawing. This method demands the least amount of expertise and time from its use, enabling a simple and rapid fabrication experience. Several properties of the hydrophobic material were characterized, such as the hydro head and penetration rate, with the aim of assessing its robustness and stability. The presented hydrophobic barriers fabricated using this approach have a barrier width of 4 mm, a coating thickness of 208 µm, and a hydrophilic resolution of 446.5 µm. This fabrication modality boasts an excellent solvent resistance with regard to the hydrophobic barrier. These devices were employed for on-the-spot detection of Metanil Yellow, a banned food adulterant often used in curcumin and pigeon peas, within successful limits of detection (LOD) of 0.5% (w/w) and 0.25% (w/w), respectively. These results indicate the great potential this fabricated hydrophobic device has in numerous paper-based applications and other closely related domains, such as diagnostics and sensing, signalling its capacity to become commonplace in both industrial and domestic settings. [ABSTRACT FROM AUTHOR]

The urgent need for real-time and noninvasive monitoring of health-associated biochemical parameters has motivated the development of wearable sweat sensors. Existing electrochemical sensors show promise in real-time analysis of various chemical biomarkers. These sensors often rely on labels and redox probes to generate and amplify the signals for the detection and quantification of analytes with limited sensitivity. In this study, we introduce a molecularly imprinted polymer (MIP)-based biochemical sensor to quantify a molecular biomarker in sweat using electrochemical impedance spectroscopy, which eliminates the need for labels or redox probes. The molecularly imprinted biosensor can achieve sensitive and specific detection of cortisol at concentrations as low as 1 pM, 1000-fold lower than previously reported MIP cortisol sensors. We integrated multimodal electrochemical sensors with an iontophoresis sweat extraction module and paper microfluidics for real-time sweat analysis. Several parameters can be simultaneously quantified, including sweat volume, secretion rate, sodium ion, and cortisol concentration. Paper microfluidic modules not only quantify sweat volume and secretion rate but also facilitate continuous sweat analysis without user intervention. While we focus on cortisol sensing as a proof-of-concept, the molecularly imprinted wearable sensors can be extended to real-time detection of other biochemicals, such as protein biomarkers and therapeutic drugs.

Electrochemical paper-based microfluidics has attracted much attention due to the promise of transforming point-of-care diagnostics by facilitating quantitative analysis with low-cost and portable analyzers. Such devices harness capillary flow to transport samples and reagents, enabling bioassays to be executed passively. Despite exciting demonstrations of capillary-driven electrochemical tests, conventional methods for fabricating electrodes on paper impede capillary flow, limit fluidic pathways, and constrain accessible device architectures. This account reviews recent developments in paper-based electroanalytical devices and offers perspective by revisiting key milestones in lateral flow tests and paper-based microfluidics engineering. The study highlights the benefits associated with electrochemical sensing and discusses how the detection modality can be leveraged to unlock novel functionalities. Particular focus is given to electrofluidic platforms that embed electrodes into paper for enhanced biosensing applications. Together, these innovations pave the way for diagnostic technologies that offer portability, quantitative analysis, and seamless integration with digital healthcare, all without compromising the simplicity of commercially available rapid diagnostic tests., (© 2024 The Authors. Small published by Wiley‐VCH GmbH.)

This paper introduces a novel software framework to support automated development of paper-based microuidic devices. Compared to existing lab-on-a-chip technologies, paper-based microuidics difiers in terms of substrate technologies and point-of-care usage across a wide variety environmental conditions. This paper addresses the contexts in which the software can address these challenges and presents several initial case studies that demonstrate the capabilities of the framework to produce workable and usable paper microuidic devices.

This paper introduces a novel software framework to support automated development of paper-based microuidic devices. Compared to existing lab-on-a-chip technologies, paper-based microuidics difiers in terms of substrate technologies and point-of-care usage across a wide variety environmental conditions. This paper addresses the contexts in which the software can address these challenges and presents several initial case studies that demonstrate the capabilities of the framework to produce workable and usable paper microuidic devices.

Conventional techniques (e.g., culture-based method) for bacterial detection typically require a central laboratory and well-trained technicians, which may take several hours or days. However, recent developments within various disciplines of science and engineering have led to a major paradigm shift in how microorganisms can be detected. The analytical sensors which are widely used for medical applications in the literature are being extended for rapid and on-site monitoring of the bacterial pathogens in food, water and the environment. Especially, within the low-resource settings such as low and middle-income countries, due to the advantages of low cost, rapidness and potential for field-testing, their use is indispensable for sustainable development of the regions. Within this context, this paper discusses analytical methods and biosensors which can be used to ensure food safety, water quality and environmental monitoring. In brief, most of our discussion is focused on various rapid sensors including biosensors and microfluidic chips. The analytical performances such as the sensitivity, specificity and usability of these sensors, as well as a brief comparison with the conventional techniques for bacteria detection, form the core part of the discussion. Furthermore, we provide a holistic viewpoint on how future research should focus on exploring the synergy of different sensing technologies by developing an integrated multiplexed, sensitive and accurate sensors that will enable rapid detection for food safety, water and environmental monitoring.

Total bilirubin values have been used as a potential marker to pre-screen and diagnose various liver-based diseases such as jaundice, bile obstruction, liver cancer, etc. A device known as KromaHealth Kit, composed of paper and an acrylic backbone, is developed to quantify total bilirubin in human serum using image processing and machine learning technology. The biochemical assays are deposited on absorbent paper pads that act as reaction zones when serum is added. A dedicated smartphone app captures images of the colorimetric changes on the pad and converts them into quantitative values of bilirubin. The range of bilirubin concentration that can be quantified using the device ranges from 0.5 mg/dl to 7.0 mg/dl. The precision, limit of detection, interference analysis, linearity, stability, and comparison with a predicate are studied in this paper in accordance with clinical and laboratory standards institute. The results indicate that the KromaHealth Kit can be used as an inexpensive alternative to conventional bilirubin testing in clinical settings. With its level of precision, ease-of-use, long shelf-life, and short turnaround time, it will prove to be invaluable in limited-resource settings.

A quantitative method for acid-base titrations in paper-based devices (PADs) is described to analyze acetic acid in vinegar samples. In this work, two different types of PADs were developed: a device for individual spot testing and a microfluidic device. Digital colorimetry was used as the detection method, and the images were acquired using a smartphone and a homemade box with LED lights for controlled image acquisition. Titration curves were built with just eight points, using the R channel based on the gradual color transition from red to blue of litmus, a natural indicator. The endpoint was accurately determined by second derivative calculations. Both systems were applied to fifteen vinegar samples of different types, and good concentration results were obtained in comparison to the reference method. The proposed methodology is simple, fast, environmentally friendly, and surpasses the need for calibration curve construction. Moreover, the subjective endpoint identification is eliminated, and the method was automated to provide a high throughput workflow, suitable for quality control processes and real-time measurements. [Display omitted] • Quantitative acid-base titration method based on two types of PADs. • Smartphone-based detection of acetic acid in vinegar samples. • Color change from red to blue, and titration curve built using R channel values. • Accurate endpoint determination with second derivative calculations. • Method automation with GIMP and Excel or MATLAB for rapid and simple process control. [ABSTRACT FROM AUTHOR]

Root exudates control critical processes in the rhizosphere, retaining water, selecting for beneficial microorganisms or solubilising nutrients prior to uptake by the plant. Analysing root exudation patterns however is challenging because existing methods are often destructive and unable to resolve spatial and temporal variations in the production of root exudates. Here, we present a paper-based microfluidic device with integrated colorimetric sensors for the continuous extraction and analysis of root exudates along plant roots. The microfluidic device used standard filter paper wax printer to create channels for water to carry the exudates towards the sensors. TiO 2 nanotubes/alginate hydrogel-based sensors were used to analyse the glucose content of the root exudates of living plants. The study shows that the paper microfluidic substrate successfully extracts the released glucose from the root, and transfers it to the hydrogel-based sensor to be colorimetrically detected from independent sections of the root at different times, up to 7 days. This method was tested on two different wheat varieties Triticum aestivum (rgt Tocayo and Filon varieties), where significant differences in exudation patterns were recorded. This research demonstrates the feasibility of low-cost technological solution for high precision screening and diagnostic of the biochemical composition of root exudates. • Simple paper device allows continuous extraction of exudates along roots. • Wax printing creates microfluidic channels that transport exudates to a sensor. • Image analysis predict glucose concentration from colorimetric sensor. • Wheat varieties have different concentration of glucose in their root exudates. [ABSTRACT FROM AUTHOR]

Hypoxanthine (Hx), produced by adenosine triphosphate (ATP) metabolism, is a valuable indicator that determines the quality and degradation status of meat products and is also an important biochemical marker to certain diseases such as gout. The rapid emergence of paper-based enzyme biosensors has already revolutionized its on-site determination. But it is still limited by the complex patterning and fabrication, unstable enzyme and uneven coloration. This work aims to develop an eco-friendly method to construct engineered paper microfluidic, which seeks to produce reaction and non-reaction zones without any patterning procedure. Chito-oligosaccharide (COS), derived from shrimp shells, was used to modify nitrocellulose membranes and immobilize xanthine oxidase (XOD) and chromogenic agent of nitro blue tetrazolium chloride (NBT). After modification, micro fluids could converge into the modification area and Hx could be detected by XOD-catalyzed conversion. Due to the positively charged cationic basic properties of COS, the enzyme storage stability and the color homogeneity could be greatly strengthened through the electrostatic attraction between COS and XOD and formazan product. The detection limit (LOD) is 2.30 μM; the linear range is 0.05–0.35 mM; the complete test time can be as short as 5 min. The COS-based biosensor shows high specificity and can be used directly for Hx in complex samples such as fish and shrimp samples, and different broths. This biosensor is eco-friendly, nontechnical, economical and therefore a compelling platform for on-site or home-based detection of food freshness. [Display omitted] • A green manufacturing method for hypoxanthine enzyme sensor is proposed. • COS engineered paper microfluidics omits any patterning procedure. • The sensor has excellent detection performance, color uniformity, and stability. [ABSTRACT FROM AUTHOR]

Paper-based nucleic acid detection and diagnosis are currently gaining much interest in point-of-care (POC) applications. The major steps involved in any nucleic acid amplification testing (NAAT) based diagnostics are nucleic acid isolation, reverse transcription (RT) (in the case of RNA), amplification and detection. RT is an important step in quantifying the viral load in case of disease diagnosis as well as quantifying gene expression levels in other molecular studies. cDNA synthesis is routinely carried out using a thermal cycler, with the process requiring temperatures between 40ºC to 65ºC. Here we report for the first time an instrument-free RT, performed at room temperature on cellulose-based paper devices. cDNA synthesis on paper was confirmed by RT-PCR and Sanger sequencing of the PCR products. Purified RNA from varied sources such as cell lysate, tissue and blood were used to test the methodology. Synthetic hepatitis C virus (HCV) RNA and human blood RNA were used as proof-of-concept to demonstrate the use of these devices in diagnostic applications. Further, ready-to-use paper-based reverse transcription (PRT) devices have been developed, wherein only the RNA sample is added on the device and the cDNA can be eluted after 30 min of incubation at room temperature. The devices were found to be stable for 30 days at − 20ºC storage. The cellulose-based PRT devices are simple, time saving and user-friendly for a complete instrument-free cDNA synthesis at room temperature. [Display omitted] • A complete instrument-free reverse transcription at room temperature is demonstrated. • Reverse transcription can be performed on cellulose paper-based microfluidic device. • Ready-to-use PRT devices need only the addition of RNA and water to obtain cDNA. • The reaction is completed in 30 mins, thus time saving. [ABSTRACT FROM AUTHOR]

Paper devices are cutting‐edge platforms for creating miniaturized energy conversion, storage, and sensing devices. In such devices, co‐laminar flow and embedded capillaries not only eliminate membranes and external pumps but also allow widespread application. The fabrication, optimization, and characterization of a microfluidic paper ethanol fuel cell (MPEFC) has been demonstrated in the presented study. The MPEFC uses ethanol and sodium hydroxide as fuel and sulfuric acid as electrolytes. Numerous experiments have been conducted to improve the performance of MPEFC by optimizing various electrode types such as laser‐induced graphene (LIG), Ag nano‐ink/LIG, buckypaper, Ag nano‐ink/buckypaper, and two different 3D printed conductive electrodes. Different electrolyte concentrations (NaOH‐0.25, 0.5, 1 m) have been studied to understand the impact on ion conductivity. It is observed that the developed MPEFC with multiwalled carbon nanotube ‐buckypaper as an optimized electrode, 1 M ethanol with 0.5 m NaOH as anolyte, and 1 m H2SO4 as electrolyte delivered the best performance with a current density of 1756.2 μA cm−2, and power density of 72.62 μW cm−2, at a stable open‐circuit voltage of 235 mV with Gr 1 cellulose microchannel with parallel stacked configuration. This work demonstrates the effective enhancement of the output power with inexpensive individual MPEFC. [ABSTRACT FROM AUTHOR]

We demonstrate the use of patterned aerosol adhesives to construct both planar and nonplanar 3D paper microfluidic devices. By spraying an aerosol adhesive through a metal stencil, the overall amount of adhesive used in assembling paper microfluidic devices can be significantly reduced. We show on a simple 4-layer planar paper microfluidic device that the optimal adhesive application technique and device construction style depends heavily on desired performance characteristics. By moderately increasing the overall area of a device, it is possible to dramatically decrease the wicking time and increase device success rates while also reducing the amount of adhesive required to keep the device together. Such adhesive application also causes the adhesive to form semi-permanent bonds instead of permanent bonds between paper layers, enabling single-use devices to be non-destructively disassembled after use. Nonplanar 3D origami devices also benefit from the semi-permanent bonds during folding, as it reduces the likelihood that unrelated faces may accidently stick together. Like planar devices, nonplanar structures see reduced wicking times with patterned adhesive application vs uniformly applied adhesive.

Microfluidic devices can provide a versatile, cost-effective platform for disease diagnostics and risk assessment by quantifying biomarkers. In particular, simultaneous testing of several biomarkers can be powerful. Here, we critically review work from the previous 4 years up to February 2021 on developing microfluidic devices for multiplexed detection of biomarkers from samples. We focus on two principal approaches: electrical and optical detection methods that can distinguish and quantify biomarkers. Both electrical and spectroscopic multiplexed detection strategies are being employed to reach limits of detection below clinical sample levels. Some of the most promising strategies for point-of-care assays involve inexpensive materials such as paper-based microfluidic devices, or portable and accessible detectors such as smartphones. This review does not comprehensively cover all multiplexed microfluidic biomarker studies, but rather provides a critical evaluation of key work and suggests promising prospects for future advancement in this field. [ABSTRACT FROM AUTHOR]

The outbreak of new viral strains promotes advances in universal diagnostic techniques for detecting infectious diseases with unknown viral sequence. Long double-stranded RNA (dsRNA), a hallmark of infections, serves as a virus marker for prompt detection of viruses with unknown genomes. Here, we report on-chip paper electrophoresis for ultrafast screening of infectious diseases. Negatively charged RNAs pass through the micro and nanoscale pores of cellulose in order of size under an external electric field applied to the paper microfluidic channel. Quantitative separation of long dsRNA mimicking poly I:C was analyzed from 1.67 to 33 ng·μL−1, which is close to the viral dsRNA concentration in infected cells. This paper-based capillary electrophoresis chip (paper CE chip) can provide a new diagnostic platform for ultrafast viral disease detection at the point-of-care (POC) level. [ABSTRACT FROM AUTHOR]

Inflammation caused by viral or bacterial infection is a major threat to human health globally. Blood C-reactive protein (CRP) has been proven to be a sensitive indicator for the occurrence and development of inflammation. Furthermore, a tiny change of blood CRP concentration may portend chronic diseases; therefore, high-sensitivity CRP (hs-CRP) detection in a quantitative, rapid, user-friendly, and low-cost manner is highly demanded. In this paper, we developed a europium-chelate polystyrene microsphere (EuPSM)-based lateral flow immunoassay (LFIA) integrating with a benchtop fluorescence analyzer for hs-CRP detection. The optimization of the EuPSM-based LFIA was implemented through adjusting the antibody density on EuPSM from 100% to 60% of the saturated density. Finally, the limit of detection of 0.76 pg/mL and detection range of 0.025–250 ng/mL were obtained. Moreover, the clinical application capability of the proposed platform was validated through detecting CRP in clinical serum samples, showing high consistency with the results obtained from the clinical standard method. Hence, the proposed EuPSM-based LFIA has been verified to be well suitable for hs-CRP detection, while also showing great applicability for sensitively and rapidly detecting other biomarkers.

The development of smartphone electronics and the prevalence of quick response (QR) codes make them valuable tools for digitizing and decentralizing tests to fields and households. Now there is a growing trend for adapting them into sensing and diagnostic needs from paper microfluidics. Herein, we show a new QR-coded microfluidic paper-based analytical device (QR-coded μPAD) consisting of stacked layers of printed QR code, transparency film, and microfluidically patterned paper. It combines multichannel colorimetric sensing with QR code generation into smartphone-based multiplexed assays with the aid of gray patterns on the transparency film. The QR-coded μPAD paired with an accompanying mobile application is successfully applied to simultaneous implementation of beverage/aquatic tests and QR code functions. Our QR code conversion strategy provides the superb flexibility and compatibility for paper microfluidics of various designs. The work suggests that our versatile integration of paper microfluidics, smartphone science, and QR code uses can make rapid, interactive, and automated on-site testing and reporting, leading to opportunities to advance the development of smart paper microfluidics. [Display omitted] • A versatile QR code conversion strategy for paper microfluidics was proposed. • Stacked layer design and printed gray patterns hold the key to device fabrication. • Simultaneous implementation of analyte analysis and QR code functions in one scan. • Our QR-coded μPADs were applied into beverage tests and water quality monitoring. [ABSTRACT FROM AUTHOR]

Summary: Paper microfluidic devices are innovative platforms for building miniaturized devices for energy harvesting, energy storage, and sensing applications. Such devices are reported to be used as membraneless microfluidic fuel cells to power portable electronic in a pump‐less manner by leveraging the embedded capillaries and co‐laminar flow. This paper demonstrates fabrication and characterization of the high‐performance metal‐free aluminum‐air microfluidic paper fuel cell (AAMPFC). Herein, aluminum foil acts as an anode, buckypaper with built‐in multiwalled carbon nanotubes (MWCNTs) acts as a cathode and sodium hydroxide (NaOH) works as an electrolyte. The novel AAMPFC is analyzed with various electrolyte concentrations, and maximum output voltage, current density, and power density are recorded. This easy‐to‐fabricate AAMPFC platform, costing less than US$ 0.5, is further optimized to achieve peak energies by stacking four AAMPFCs (in series and parallel) with various concentrations of electrolyte (NaOH). With parallel configuration, maximum open‐circuit potential (OCP), peak current (mA/cm2), and power density (mW/cm2) of 1.32 V, 50.67 mA/cm2 and 22.80 mW/cm2, respectively, are observed with optimized electrolyte, while with series stacked configuration, a stable OCP of 4.10 V is measured. In a series configuration, AAMPFC generated sufficient power to powering a scientific calculator with LCD display for 3 days and 11 LEDs for 10 hours continuously. This research work provides a significant method to enhance the output power with inexpensive parameters (buckypaper, single paper microchannel) to power portable electronic devices. Further, the performance can be enhanced by investigating AAMPFC with various grades of aluminum foil in combination with different grades of porous cellulose papers. [ABSTRACT FROM AUTHOR]

The use of pesticides in agriculture field remains a serious issue related to public health. This necessitates the need to develop a low cost/portable, sensitive and selective bio-sensing platform for detection of pesticides in food chain. In this work a low-cost biosensing platform for ultrasensitive detection of chlorpyrifos (CPF) is developed. Electrochemical micro Paper Analytical Device ($\text{E}\mu $ PAD) is fabricated by conventional screen-printing technology. Metal Organic Framework (Z1200 MOF) having zinc metal and imidazole ligand is used as a transducing element which facilitates biocompatible matrix for Acetylcholinesterase (AChE) enzyme immobilization. The limit of detection for CPF is found be 3 ng/L with sensitivity 0.521 $\text{k}\Omega $ /ng $\text{L}^{-1}$ /mm $^{2}$. The sensor required $100\mu \text{L}$ of reagent and was tested with a linear concentration range of 10 ng/L to 1000 ng/L with response time of 5s. The sensor is further integrated with portable electronics based on Arduino microcontroller and Artificial Intelligence (AI) which provided economical, portable and user-friendly sensing platform. The stability of the sensor was 30 days. The developed sensor was also tested with real samples and the sensor response is in agreement with conventional technique. [ABSTRACT FROM AUTHOR]

We hereby show that root systems adapt to a spatially discontinuous pattern of water availability even when the gradients of water potential across them are vanishingly small. A paper microfluidic approach allowed us to expose the entire root system of Brassica rapa plants to a square array of water sources, separated by dry areas. Gradients in the concentration of water vapor across the root system were as small as 10-4·mM·m-1 (~4 orders of magnitude smaller than in conventional hydrotropism assays). Despite such minuscule gradients (which greatly limit the possible influence of the well-understood gradient-driven hydrotropic response), our results show that 1) individual roots as well as the root system as a whole adapt to the pattern of water availability to maximize access to water, and that 2) this adaptation increases as water sources become more rare. These results suggest that either plant roots are more sensitive to water gradients than humanmade water sensors by 3-5 orders of magnitude, or they might have developed, like other organisms, mechanisms forwater foraging that allow them to findwater in the absence of an external gradient in water potential. [ABSTRACT FROM AUTHOR]

Increased levels of extracellular nicotinamide phosphoribosyltransferase (eNAMPT) are increasingly recognized as a highly useful biomarker of inflammatory disease and disease severity. In preclinical animal studies, a monoclonal antibody that neutralizes eNAMPT has been generated to successfully reduce the extent of inflammatory cascade activation. Thus, the rapid detection of eNAMPT concentration in plasma samples at the point of care (POC) would be of great utility in assessing the benefit of administering an anti-eNAMPT therapeutic. To determine the feasibility of this POC test, we conducted a particle immunoagglutination assay on a paper microfluidic platform and quantified its extent with a flow rate measurement in less than 1 min. A smartphone and cloud-based Google Colab were used to analyze the flow rates automatically. A horizontal flow model and an immunoagglutination binding model were evaluated to optimize the detection time, sample dilution, and particle concentration. This assay successfully detected eNAMPT in both human whole blood and plasma samples (diluted to 10 and 1%), with the limit of detection of 1-20 pg/mL (equivalent to 0.1-0.2 ng/mL in undiluted blood and plasma) and a linear range of 5-40 pg/mL. Furthermore, the smartphone POC assay distinguished clinical samples with low, mid, and high eNAMPT concentrations. Together, these results indicate this POC assay, which utilizes low-cost materials, time-effective methods, and a straightforward immunoassay (without surface immobilization), may reliably allow rapid determination of eNAMPT blood/plasma levels to advantage patient stratification in clinical trials and guide ALT-100 mAb therapeutic decision-making., (© The Author(s) 2024. Published by Oxford University Press on behalf of National Academy of Sciences.)

Abstract Application of optically superior, tunable fluorescent nanotechnologies have long been demonstrated throughout many chemical and biological sensing applications. Combined with microfluidics technologies, i.e. on lab-on-a-chip platforms, such fluorescent nanotechnologies have often enabled extreme sensitivity, sometimes down to single molecule level. Within recent years there has been a peak interest in translating fluorescent nanotechnology onto paper-based platforms for chemical and biological sensing, as a simple, low-cost, disposable alternative to conventional silicone-based microfluidic substrates. On the other hand, smartphone integration as an optical detection system as well as user interface and data processing component has been widely attempted, serving as a gateway to on-board quantitative processing, enhanced mobility, and interconnectivity with informational networks. Smartphone sensing can be integrated to these paper-based fluorogenic assays towards demonstrating extreme sensitivity as well as ease-of-use and low-cost. However, with these emerging technologies there are always technical limitations that must be addressed; for example, paper’s autofluorescence that perturbs fluorogenic sensing; smartphone flash’s limitations in fluorescent excitation; smartphone camera’s limitations in detecting narrow-band fluorescent emission, etc. In this review, physical optical setups, digital enhancement algorithms, and various fluorescent measurement techniques are discussed and pinpointed as areas of opportunities to further improve paper-based fluorogenic optical sensing with smartphones.

Lactate is an essential biomarker for determining the health of the muscles and oxidative stress levels in the human body. However, most of the currently available sweat lactate monitoring devices require external power, cannot measure lactate under low sweat rates (such as in humans at rest), and do not provide adequate information about the relationship between sweat and blood lactate levels. Here, we discuss the on-skin operation of our recently developed wearable sweat sampling patch. The patch combines osmosis (using hydrogel discs) and capillary action (using paper microfluidic channel) for long-term sweat withdrawal and management. When subjects are at rest, the hydrogel disc can withdraw fluid from the skin via osmosis and deliver it to the paper. The lactate amount in the fluid is determined using a colorimetric assay. During active sweating (e.g., exercise), the paper can harvest sweat even in the absence of the hydrogel patch. The captured fluid contains lactate, which we quantify using a colorimetric assay. The measurements show the that the total number of moles of lactate in sweat is correlated to sweat rate. Lactate concentrations in sweat and blood correlate well only during high-intensity exercise. Hence, sweat appears to be a suitable biofluid for lactate quantification. Overall, this wearable patch holds the potential of providing a comprehensive analysis of sweat lactate trends in the human body.

Investigation of the potential adverse effects of chemicals and drugs is essential during the drug development process. In vitro cell model systems have been developed over the past years towards such toxicity investigation. 96-well plate is the common platform for screening drug toxicity due to its simplicity. However, this platform only offers 2D cell culture environment and lacks the flow of solutions, which fails to provide the suitable environment for the cells to adequately metabolize the drugs, for the media to replenish, and for the metabolites and wastes to be removed. Microfluidic chips populated with human or animal cells, known as organ-on-a-chip (OOC), can reconcile many issues of in vitro cell models, such as the lack of extracellular matrix and flow as well as the species difference. However, OOC can be complicated to fabricate and operate. To bridge this gap, we utilized paper as a primary substrate for OOC, considering its fibrous structure that can mimic natural extracellular matrix, as well as a syringe pump and filter that are commonly available in most laboratories. Paper microfluidic model was designed and fabricated by wax printing on nitrocellulose paper, seeded and proliferated with liver cells (primary rat hepatocytes and HepG2 cells), and two paper substrates were stacked together to complete the paper model. To this paper-based liver cell model, the following drugs were added: Phenacetin (pain reliever and fever reducer), Bupropion (antidepressant), Dextromethorphan (antidepressant), and phosphate-buffered saline (PBS) as a control, all under a physiologically relevant flow rate. The combination of these drugs with Fluconazole (antifungal drug) was also investigated. Cell count, cell morphology, protein production, and urea secretion after drug treatment confirmed that the model successfully predicted toxicity within 40 minutes. This simple, paper-based liver cell model provided enhanced and faster cell response to drug toxicity and showed comparable or better behavior than the cells cultured in conventional 2D in vitro models. [ABSTRACT FROM AUTHOR]

A simple paper-based microfluidic device, fabricated on a highly porous coating consisting of functionalised calcium carbonate (FCC) pigment and a microfibrillated cellulose (MFC) and/or polyvinyl alcohol (PVOH) binder, was developed for glucose detection. Both the hydrophobic patterns, consisting of alkyl ketene dimer (AKD), and the enzyme ink, consisting of glucose oxidase (GOx), horseradish peroxidase (HRP) and an indicator containing 4-aminoantipyrine (4-AAP), and sodium 3,5-dichloro-2-hydroxy-benzenesulphonic acid (DHBS) were inkjet-printed. Multiple coating formulations were tested using two indicators, potassium iodide (KI) and 4-AAP/DHBS, to find the optimal formulation with regard to detection sensitivity and assay stability. Higher binder concentrations increased the enzyme activity, especially in the case of PVOH. Two coatings, containing either solely MFC or both MFC and PVOH were ultimately chosen for the glucose assay experiments. For the assays, a 42 nl volume of buffered enzyme solution, containing a total of 5.04 mU GOx and approximately 1.01 mU HRP, in combination with 4 mM 4-AAP and 8 mM DHBS, was printed, and a 1 µl glucose solution, made up over a range of concentrations, was applied onto the samples to evaluate the response. A good linearity was achieved between detection and glucose concentration between 0.1 and 0.6 mM with both coatings. The assays remained stable for four weeks when stored at − 20 °C. The results show that the functional coated substrates offer a viable alternative to cellulose-based substrates for microfluidic applications enabling the use of small reagent and sample volumes. [ABSTRACT FROM AUTHOR]

Green/sustainable cellulose paper-based platforms at point-of-care testing have received much interest in biomedical fields at resource-limited settings. In such platforms, biomolecules would have to be immobilized onto paper substrate to achieve detection. Therefore, effective molecule immobilization is critical for the performance of paper-based testing, especially the detection sensitivity. Although various methods have been developed to improve the detection sensitivity, the immobilization capacity of biomolecules has not been considered yet. In this study, we developed a facile method for modifying paper materials by incorporating chitosan into NC membrane, which has positive effect on the pore size, porosity, and surface groups of NC membrane, hence, enhancing the immobilization capacity of biomolecules on paper substrate. We demonstrated the effectiveness of the above mentioned approach by using nucleic acid lateral flow assay (NALFAs) as a model paper-based platform; in comparison with the unmodified NALFAs, the results showed ten-fold increase in the detection sensitivity for HBV. We envision that this facile modification method will have a great potential for developing other highly sensitive paper-based detecting platforms. [ABSTRACT FROM AUTHOR]

Equipment‐free paper‐based devices with quantitative readout have gained considerable interests in recent years, due to the features of remarkable cheapness, prominent portability, and excellent user friendliness. These equipment‐free strategies have been demonstrated to be able to detect a variety of analytes with good performance, such as metal ions, nucleic acids, and cells, thus showing great potential for point‐of‐care testing in resource‐limited settings. In this review, the latest advances of equipment‐free quantitative readout strategies in paper‐based point‐of‐care testing are summarized, including distance‐based strategy, counting‐based strategy, time‐based strategy, and text‐based strategy. Each type of equipment‐free strategy is sequentially introduced and discussed. Finally, conclusions and perspectives are discussed. [ABSTRACT FROM AUTHOR]

Advancements in health care monitoring demand a rapid, accurate and reliable early diagnosis of "Heart Attack" (acute myocardial infarction) with an objective to develop a cost-effective, rapid and label-free point of care diagnostic test kit for the detection of cardiac troponin I (cTnI) on paper-based multi-frequency impedimetric transducers. Paper based sensing platforms were developed by integrating carboxyl group functionalized multi-walled carbon nanotubes (MWCNT) with antibodies of cardiac troponin I (anti-cTnI) biomarker and was characterized using Electrochemical Impedance Spectroscopy (EIS). Various concentrations of cTnI with anti cTnI were studied as a function of impedance change. The suitability of the proposed immunosensor is demonstrated by spiking cTnI in blood serum samples. The limit of detection (LoD) and sensitivity of the proposed sensor was determined to be 0.05 ng/mL and 1.85 mΩ/ng/mL respectively, with a response time of ~1 min. The shelf life of the fabricated sensor was nearly 30 days. The rapid response, very low detection limit, and cost effectiveness offer a portable platform to detect cTnI in blood serum samples. The proposed immunosensor, therefore, offers an affordable healthcare diagnostic platform in resource limited areas. [ABSTRACT FROM AUTHOR]

In this paper, we present a simple yet smart electro-fluidic platform that enables automatic time control in a very affordable and simple manner. The system is based on the electric detection of a fluid front when it crosses a particular area of a paper strip. The detection can be used to trigger the sequential activation or deactivation of different electronic modules (heating of molecular diagnostics, time interval detection, or readout of test results) with an accuracy within the range of minutes. The whole system is implemented with a few number of discrete electronic components such as transistors, resistors and capacitors that, if required, can be totally fabricated using printed electronics technology. This platform opens new possible applications for paper-based point-of care (POC) diagnostic devices and enables the possibility of these devices to introduce time control functions without the need for any external instrumentation and human action. [ABSTRACT FROM AUTHOR]

The medical field and public health rely on diagnostics carried out by sensors and laboratory testing methods. However, conducting laboratory tests is a demanding task, requiring both substantial equipment and the skilled touch of experienced operators. The microfluidic field has tackled this challenge over recent years. Paper microfluidic devices have become popular recently due to their testing speed, low cost, and accessibility. They are more suitable for field operations and developing regions with limited access to centralized healthcare centers and medical laboratories since they do not require external flow control instruments and are much cheaper to make when compared to conventional microfluidic chips. However, the majority of paper-based analytical devices tend to be qualitative or semi-quantitative; consequently, there is a significant need for paper-based quantitative transduction methods, given their expanding range of applications. Here, we introduce a novel microwave paper-based metamaterial-inspired transduction method that relies on dielectric sensing. Using paper as both the microfluidic channel and the resonator substrate hugely reduces the sensing device's costs, complexity, and environmental impact. We designed the metamaterial-based transducer using computational methods and demonstrated its fabrication and operation experimentally. Dielectric sensing of multiple analytes with different permittivities is demonstrated as a proof of concept. Although the device has a non-linear response curve in a wide range of permittivity change, the response can be assumed linear in the shorter spans. The results for permittivities lower than 30 show a 2.14 MHz/RPU sensitivity. This design is a prototype demonstrating the possibility of integrating a porous media microfluidic channel and paper-based microwave resonators. The linear sensing performance illustrated by the platform indicates its potential applications in the biosensing field. [Display omitted] • A novel paper-based metamaterial-inspired transducer is introduced. • Paper serves as microfluidic channel and substrate, reducing costs and eco impact. • This unique transduction method is a new type of quantitative analysis for μ PADs. • The linear sensing concept is demonstrated using various solutions. • This approach has implications for affordable quantitative μ PADs. [ABSTRACT FROM AUTHOR]

A digital microfluidic modular interface (chip-to-chip interface) which possesses an electrode with an orifice to vertically transport core–shell droplets is presented. The electrodes were geometrically designed to promote droplet deformation and suspension. The droplets were then applied with an electrical potential for insertion into and passage through the orifice. The concepts were tested with three types of droplets at the volume of 0.75~1.5 μL, which is usually difficult to transfer through an orifice. The integration of electrowetting on dielectric (EWOD) with paper-based microfluidics was demonstrated: the droplet could be transported within 10 s. More importantly, most of the core droplet (~97%) was extracted and passed through with only minimal shell droplets accompanying it.

The development of diagnostic tools for measuring a wide spectrum of target analytes, from biomarkers to other biochemical parameters in biological fluids, has experienced a significant growth in the last decades, with a good number of such tools entering the market. Recently, a clear focus has been put on miniaturized wearable devices, which offer powerful capabilities for real-time and continuous analysis of biofluids, mainly sweat, and can be used in athletics, consumer wellness, military, and healthcare applications. Sweat is an attractive biofluid in which different biomarkers could be noninvasively measured to provide rapid information about the physical state of an individual. Wearable devices reported so far often provide discrete (single) measurements of the target analytes, most of them in the form of a yes/no qualitative response. However, quantitative biomarker analysis over certain periods of time is highly demanded for many applications such as the practice of sports or the precise control of the patient status in hospital settings. For this, a feasible combination of fluidic elements and sensor architectures has been sought. In this regard, this paper shows a concise overview of analytical tools based on the use of capillary-driven fluidics taking place on paper or fabric devices integrated with solid-state sensors fabricated by thick film technologies. The main advantages and limitations of the current technologies are pointed out together with the progress towards the development of functional devices. Those approaches reported in the last decade are examined in detail.