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Over the last few years, the microfluidics phenomenon coupled with the Internet of Things (IoT) using innovative nano-functional materials has been recognized as a sustainable and economical tool for point-of-care testing (POCT) of various pathogens influencing human health. The sensors based on these phenomena aim to be designed for cost-effectiveness, make it handy, environment-friendly, and get an accurate, easy, and rapid response. Considering the burgeoning importance of analytical devices in the healthcare domain, this review paper is based on the gist of sensing aspects of the microfabricated paper-based analytical devices (μPADs). The article discusses the various used design methodologies and fabrication approaches and elucidates the recently reported surface modification strategies, detection mechanisms viz., colorimetric, electrochemical, fluorescence, electrochemiluminescence, etc. In a nutshell, this article summarizes the state-of-the-art research work carried out over the nano functionalized paper-based analytical devices and associated challenges/solutions in the point of care testing domain.

The clinical importance of blood group (BG) antigens is related to their ability to induce immune antibodies that can cause hemolysis. Yet, ABO and D (Rh) are still considered to be the key antigens for healthy blood transfusion and secondary antigens are the next priority. Serological typing is the most widely used typing method. Rapid and accurate blood grouping plays an important role in some clinical conditions, rather than conventional techniques. Hence, developing a simple and economical model for rapid blood grouping would facilitate these tests. In recent decades, paper-based microfluidics such as μPADs has gained much interest in wide application areas such as point-of-care diagnostic. In this study, we evaluated μPADs that are performed for blood grouping and its recent progress. A comprehensive literature search was performed using databases including PUBMED, SCOPUS, Web of Science and Google Scholar. Keywords were blood grouping or typing, paper analytical device, rapid test, etc. After investigation of search results, 16 papers from 2010 to 2020 were included. Further information in detail was classified in Table 1. Generally, two principles for blood typing μPADs are introduced. The lateral chromatographic flow method and the vertical flow-through method that detects BG in a visual-based manner. To detect results with acceptable clarity many factors and challenges like paper, blood sample, buffer, Ab and RBC interaction and also μPADs stability need to be considered, which are discussed. In conclusion, the simplicity, stability, cheapness, portability and biocompatibility of μPADs for blood grouping confirming its utility and also they have the capability to robust, universal blood-grouping platform. Table 1 Summary of blood grouping tests using paper-based analytical devices Antigens Type of diagnosis Validation method Sample No Accuracy Action time Paper type Stability Sample dilution Buffer Ref A, B, Rh Forward volunteers records 5 - - Whatman No. 4 - 1/2 PBS* (Khan et al. 2010) A, B, Rh Forward gel assay test and conventional slide test 100 100% 1 min Whatman No. 4 and Kleeenex paper towel 7 Days in 4 °C 1/1 NSS (Al-Tamimi et al. 2012) A, B, Rh Forward gel card assay 99 100% 20 Sec + Washing Kleeenex paper towel - 1/1 NSS (Li et al. 2012) A, B, Rh Forward - - - - Kleeenex paper towel - 45/100 PSS (Li et al. 2013) A, B, Rh Forward gel card assay 98 100% 1.5 min Kleeenex paper towel - 85/100 PBS (Guan et al. 2014b) C, E, c, e, K, Jka, Jkb, M, N, S, P1, and Lea Forward gel card assay 266 100% - Kleeenex paper towel - 1/1 NSS (Li et al. 2014b) A, B, Rh Forward and Reverse conventional slide test 96 ≈ 91% 10 min Whatman No. 1 21 Days in 4 °C 1/2 NSS (Noiphung et al. 2015) C, c, E, e, K, k, Fya, Fyb, Jka, Jkb, M, N, S and s, P1, Lea and Leb Forward - 478 - - Kleeenex paper towel - 1/1 NSS, PBS (Then et al. 2015) A, B Forward and Reverse conventional slide test 76 100% 5-8 min Whatman No. 4 38 Days in 4 °C 1/4, 1/1 NSS (Songjaroen and Laiwattanapaisal 2016) D, K Forward volunteers records 210 - 7.5 min Kleenex paper towel - 1/1 NSS (Yeow et al. 2016) A, B, c, e, D, C, E, M, N, S, s, P1, Jka, Jkb, Lea, Leb, Fya, and Fyb Forward and Reverse gel card assay 3550 ≈100% 30 s Fiber glass and cotton linter 180 Days in 25 °C 45/100, 1/1 PBS (Zhang et al. 2017) A, B Forward conventional slide test 598 100% 3 min Whatman No. 113 14 Day in 4 °C 1/1 NSS (Songjaroen et al. 2018) A, B, Rh Forward conventional slide test - - 30 Sec + Washing Unrefined sisal paper - 1/2 NSS (Casals-Terré et al. 2019) A, B, Rh Forward - - - - Whatman No.1 - 1/1 NSS (Ansari et al. 2020) ABORh Forward and Reverse conventional slide test - 100% Unrefined Eucalyptus papers - 1/2 NSS, PBS (Casals-Terré et al. 2020) A, B, Rh Forward - - - 30 Sec + Washing Whatman No. 4 modified with chitosan ≥ 100 days in 25 °C 1/1 NSS (Parween et al. 2020)

This paper presents a new approach towards the design of paper based autonomous microfluidic devices. Autonomy in the device operation is achieved through the incorporation of mechanically actuated microfluidic switches that are versatile in their design and may be configured to be simple time triggered ON or OFF switches or more complex switches that can be timed to be in multiple states (timed ON, followed by timed OFF). These switches are self-contained and require no external power for their operation, deriving their functionality solely through stored elastic energy. This paper presents the design and fabrication of these switches as fluidic analogs of electronic transistors, and their integration into microfluidic paper based circuit demonstrating their operation as a programmable paper-based microfluidic device.

Compared with traditional drug delivery methods, transdermal drug delivery has many advantages in avoiding the side effects in gastrointestinal tract, reducing the fluctuations in drug concentration, and improving patients' compliance. Among them, electrically controlled drug delivery is a promising solution. This work presents a wireless, battery-free and wearable device with electrically controlled drug delivery capability. The electronic component of the device is a flexible circuit board with a temperature sensor and a near-field communication module. With the help of smartphone, the device could wirelessly obtain energy and implement data transmission. The drug delivery component is a paper-based electrode modified with polypyrrole, in which non-steroidal anti-inflammatory drug sodium salicylate was encapsulated. The applied potential for electrically controlled drug delivery was more negative than -0.6 V. The drug release dose and release rates could be controlled by applying potentials with different amplitudes and durations through this device. It provided a minimalized wearable transdermal drug delivery platform for monitoring diseases such as gout. This wearable device shows promising potential in develop closed-loop drug delivery and monitoring systems for the treatment of various diseases.

This study aims to explore the channel patterns and the characteristic parameters of the zigzag microchannel based on microfluidic paper-based analytical devices (μPADs), in which the mixing efficiency and speed can be greatly enhanced. Better mixing of the solutions was obtained by adding a simple directing electric field to the optimized structure of the zigzag microchannel on paper-based chips instead of the traditional complex devices. A higher mixing efficiency was reached when the direct-current (DC) power supply reached 20 V. Meanwhile, a piezoelectric transducer (PZT) driver was used in the mixing experiment with the paper-based zigzag microchannel. The results show that the mixing efficiency reached a maximum value when the input voltage and frequency were 30 V and 150 Hz, respectively. These paper-based devices meet the requirements of the biochemical analysis field because they are low cost, easy to operate, and have high efficiencies, giving them good prospects for future applications. Graphical Abstract.

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.

Cystinuria, is an autosomal recessive genetic disorder involving increasingly high levels of poorly soluble cysteine in urine leading to formation of stones. Developing a facile, low-cost, point-of-care and selective sensor for diagnosis of cysteine is imperative. Accordingly, for the detection of cysteine, the present study demonstrates an inexpensive colorimetric, paper-based vertical flow plasmonic micro-well device with a two-minute turn-around time. The method encompasses the use of microbially-synthesized silver nanoparticles (AgNPs) that change from light brown / yellow to dark brown upon binding with Sulphur present in cysteine. This technique allows for visual detection up to 1 × 10−5 mM cysteine and can be easily offered as a rapid diagnostic test even at setups with minimal resources.

This article introduces a novel inkjet printing method for the fabrication of a microfluidic paper-based analytical device (μPADs) with improved analytical performance for colorimetric measurements. Firstly, a hydrophobic boundary was created by wax printing on chromatography paper. Then, chitosan (CHI), 3,3',5,5'-Tetramethylbenzidine (TMB) and enzymatic mixture solvent (glucose oxidase (GOx) and horseradish peroxidase (HRP)) were sequentially printed in the sensing zone. Polyethylene glycol (PEG6000) was mixed with the bienzymatic solution to act as an enzyme stabilizer, forming the printable ink. The resulting μPADs exhibited a linear relationship between color intensity and glucose concentration from 0.0 25 mg/ml to 0 .5mg/ml. The detectable glucose concentration was in a clinically relevant range from 0.01 mg/ml to 4 mg/ml. The limit of detection (LOD) was achieved at 0.01 mg/ml. After 60-day storage under 4 °C, the color intensity at the testing zone retained over 80% of the original intensity. In addition, a smartphone application was developed for in situ colorimetric image processing, and the colorimetric analysis results were compared with those from the use of a scanner followed by processing using ImageJ. Furthermore, the development of this ink printing method also provides a point of care (POC) platform for other substances detection purposes.

Rapid, disposable, point-of-care (POC) oral fluid testing has gained considerable attention in recent years as saliva contains biomarker and components of the serum proteome that offer important information on both oral and systemic disease. Microfluidic paper-based analytical devices (μPADs) coupled with smartphone reflectance sensing systems have long been considered to be an effective POC tool for the diagnostics of biomarkers in oral fluid. However, the existing portable systems are limited by the poor color distribution in the detection area as well as not being universally applicable. Therefore, using the properties of nanomaterials to our advantage, we present a simple, universally applicable approach that features graphene oxide (GO) coated μPADs coupled with smartphone-based colorimetric detection for the direct quantification of glucose. An integrated portable system is used to implement the approach. Owing to the enhanced reagents absorptivity, reactive efficiency and homogeneity of color distribution from the deposition of GO, the glucose assay performance was improved. Also, by using a self-developed app, the glucose concentrations in physiological range can be automatically quantified. Finally, the approach is universally applicable as the modification of μPADs with GO can be achieved without the use of any linker, binder or retention aid, which avoids possible enzyme cross contamination. The system was first calibrated by standard glucose buffer solutions, and the limit of detection as well as the linear dynamic range were found to be 0.02 mM and 0~1 mM, respectively, which are appropriate for analyzing glucose concentrations in a clinically relevant range. Finally, the system was used for quantifying glucose concentrations in artificial saliva and the results obtained using our portable system showed reasonable agreement with the actual use concentrations. Thus, the utility of the system in sensitively quantifying glucose concentrations in a portable, and repeatable manner is demonstrated.

Nowadays, there is great interest in the use of microfluidic paper-based analytical devices (μPADs) for low-cost diagnostics. In most cases, the test equipment is needed. Here we report a new type of device-independent detection method based on timer-paper-based analytical devices, which can be tested by smartphone, and its application for cholesterol detection. The Quick Response code was designed as the timer component of this device. Wax printing method was employed to print the pattern on filter paper. The color and enzyme reagents have been immobilized in the hydrophilic channel to complete the colorimetric detection for cholesterol. Under laminar flow conditions of the cellulose network, the liquid volume of detection zone has been quantified by monitoring the fluid residence time on different area of the timer-paper-based devices. The precise monitoring of detection time can promote the accuracy of colorimetric detection. This is very important for the quantitative detection of paper-based analytical devices. One significant outcome of this report is that simple and accurate timer can be used for detection process self-clocking. The factors of total detection time have been investigated. The linear range of the calibration curve was 3.0 ~ 6.0 mmol L−1, with correlation coefficient of 0.9956. With the characteristic of easy to use, low cost and accurate monitoring of detection time, this kind of timer-paper-based devices can be applied to cholesterol or other substances rapid detection.

Temperature is a critical extrinsic physical parameter that determines cell fate. Hyperthermia therapy has become an efficient treatment for tumor ablation. To understand the response of tumor cells under thermal shocks, we present a paper-based photothermal array that can be conveniently coupled with commercial 96-well cell culture plates. This paper chip device was fabricated in one step using Parafilm® and Kimwipers® based on a heat lamination strategy. Liquid was completely adsorbed and confined within the cellulose fibres of hydrophilic regions. Then, Prussian blue nanoparticles (PB NPs) as the photothermal initiator were introduced into the loading wells, and thermal energy was generated via near infrared (NIR) laser irradiation. After assembling the paper device with a 96-well plate, the temperature of each well could be individually controlled by varying the loading amount of PB NPs and laser irradiation time. As a proof-of-concept study, the effects of local thermal shocks on HeLa cells were investigated using MTT cell viability assay and Live/Dead cell staining. The variation of cell viability could be monitored in situ with controllable temperature elevation. The proposed paper photothermal array loaded with thermal initiators represents an enabling tool for investigating the hyperthermia responses of biological cells. Moreover, the facile fabrication technique for paper patterning is advantageous for customizing high-throughput microfluidic paper-based analytical devices (μPADs) with extremely low cost.

In this work, we present a new iSIMPLE concept (infusion Self-powered Imbibing Microfluidic Pump by Liquid Encapsulation), which requires no external power for activation nor liquid manipulation, it is easy to use while its fabrication method is extremely simple, inexpensive and suited for mass replication. The pump consists of a working liquid, which is - after finger activation - absorbed in a porous material (e.g. filter paper). The air expelled from the porous material increases the pressure in the downstream outlet channel and propels the outlet liquid (i.e. the sample) through the channel or ejects it. Here we investigated the influence of different filter papers on the iSIMPLE flow rates, achieving a wide range from 30 down to 0.07 μL/min. We also demonstrated the versatility of the iSIMPLE in terms of the liquid volume that can be manipulated (from 0.5 μL up to 150 μL) and the working pressure reaching 64 kPa, unprecedented high for a self-powered microfluidics pump. In addition, using a 34 G microneedle mounted on the iSIMPLE, we successfully injected liquids with different viscosities (from 0.93 up to 55.88 cP) both into an agarose matrix and a skin-like biological ex vivo substrate (i.e. chicken breast tissue). This work validated the compatibility of the iSIMPLE with drug delivery in a controlled way into a skin-like matrix, envisioning a whole new scenario for intradermal injections using self-contained skin patch. In addition, due to the extreme flexibility of the design and manufacturing, the iSIMPLE concept offers enormous opportunities for completely autonomous, portable and cost effective LOC devices. ispartof: BIOMEDICAL MICRODEVICES vol:20 issue:2 ispartof: location:United States status: Published online

Globally, the microbe Neisseria gonorrhoeae (NG) causes 106 million newly documented sexually transmitted infections each year. Once appropriately diagnosed, NG infections can be readily treated with antibiotics, but high-risk patients often do not return to the clinic for treatment if results are not provided at the point of care. A rapid, sensitive molecular diagnostic would help increase NG treatment and reduce the prevalence of this sexually transmitted disease. Here, we report on the design and development of a rapid, highly sensitive, paperfluidic device for point-of-care diagnosis of NG. The device integrates patient swab sample lysis, nucleic acid extraction, thermophilic helicase-dependent amplification (tHDA), an internal amplification control (NGIC), and visual lateral flow detection within an 80 min run time. Limits of NG detection for the NG/NGIC multiplex tHDA assay were determined within the device, and clinical performance was validated retroactively against qPCR-quantified patient samples in a proof-of-concept study. This paperfluidic diagnostic has a clinically relevant limit of detection of 500 NG cells per device with analytical sensitivity down to 10 NG cells per device. In triplicate testing of 40 total urethral and vaginal swab samples, the device had 95% overall sensitivity and 100% specificity, approaching current laboratory-based molecular NG diagnostics. This diagnostic platform could increase access to accurate NG diagnoses to those most in need.

Vitamin K antagonists such as warfarin are the most widely used class of oral anticoagulants. Due to a narrow therapeutic window, patients on warfarin require regular monitoring. Self-testing using point-of-care (POC) diagnostic devices is available, but cost makes this monitoring method beyond reach for many. The main objective of this research was to assess the clinical utility of a low-cost, paper-based lateral flow POC diagnostic device developed for anticoagulation monitoring without the need for a separate electronic reader. Custom-fabricated lateral flow assay (LFA) test strips comprised of a glass fiber sample pad, a nitrocellulose analytical membrane, a cellulose wicking pad, and a plastic backing card were assembled in a plastic cassette. Healthy volunteers and patients on warfarin therapy were recruited for this prospective study. For each participant, a whole blood sample was collected via fingerstick to determine: (1) international normalized ratio (INR) using the CoaguChek® XS coagulometer, (2) hematocrit by centrifugation, and (3) red blood cell (RBC) travel distance on the experimental LFA device after 240 s using digital image analysis. RBC travel distance measured on the LFA device using blood samples obtained from warfarin patients positively correlated with increasing INR value and the LFA device had the capability to statistically distinguish between healthy volunteer INR values and those for patients groups with INR ≥ 2.6. From these data, it is predicted that this low-cost, paper-based LFA device can have clinical utility for identifying anticoagulated patients taking vitamin K antagonists who are outside of the desired therapeutic efficacy window.

A novel screen-printed microfluidic paper-based analytical device with all-carbon electrode-enabled electrochemical assay (SP-ACE-EC-μPAD) has been developed. The fabrication of these devices involved wax screen-printing, which was simple, low-cost and energy-efficient. The working, counter and reference electrodes were screen-printed using carbon ink on the patterned paper devices. Different wax screen-printing processes were examined and optimized, which led to an improved method with a shorter heating time (~5 s) and a lower heating temperature (75 °C). Different printing screens were examined, with a 300-mesh polyester screen yielding the highest quality wax screen-prints. The carbon electrodes were screen-printed on the μPADs and then examined using cyclic voltammetry. The analytical performance of the SP-ACE-EC-μPADs for the detection of glucose and uric acid in standard solutions was investigated. The results were reproducible, with a linear relationship [R(2) = 0.9987 (glucose) or 0.9997 (uric acid)] within the concentration range of interest, and with detection limits as low as 0.35 mM (glucose) and 0.08 mM (uric acid). To determine the clinical utility of the μPADs, chronoamperometry was used to analyze glucose and uric acid in real urine samples using the standard addition method. Our devices were able to detect the analytes of interest in complex real-world biological samples, and have the potential for use in a wide variety of applications.

Devices based on rapid, paper-based, isothermal nucleic acid amplification techniques have recently emerged with the potential to fill a growing need for highly sensitive point-of-care diagnostics throughout the world. As this field develops, such devices will require optimized materials that promote amplification and sample preparation. Herein, we systematically investigated isothermal nucleic acid amplification in materials currently used in rapid diagnostics (cellulose paper, glass fiber, and nitrocellulose) and two additional porous membranes with upstream sample preparation capabilities (polyethersulfone and polycarbonate). We compared amplification efficiency from four separate DNA and RNA targets (Bordetella pertussis, Chlamydia trachomatis, Neisseria gonorrhoeae, and Influenza A H1N1) within these materials using two different isothermal amplification schemes, helicase dependent amplification (tHDA) and loop-mediated isothermal amplification (LAMP), and traditional PCR. We found that the current paper-based diagnostic membranes inhibited nucleic acid amplification when compared to membrane-free controls; however, polyethersulfone allowed for efficient amplification in both LAMP and tHDA reactions. Further, observing the performance of traditional PCR amplification within these membranes was not predicative of their effects on in situ LAMP and tHDA. Polyethersulfone is a new material for paper-based nucleic acid amplification, yet provides an optimal support for rapid molecular diagnostics for point-of-care applications.

Inspired by the paper platforms for 3-D cell culture, a paper-based microfluidic device containing drug concentration gradient was designed and constructed for investigating cell response to drugs based on high throughput analysis. This drug gradient generator was applied to generate concentration gradients of doxorubicin (DOX) as the model drug. HeLa cells encapsulated in collagen hydrogel were incubated in the device reservoirs to evaluate the cell viability based on the controlled release of DOX spatially. It was demonstrated that drug diffusion through the paper fibers created a gradient of drug concentration, which influenced cell viability. This drug screening platform has a great opportunity to be applied for drug discovery and diagnostic studies with simultaneous and parallel tests of drugs under various gradient concentrations.

We developed a simple method to achieve semiquantitative detection of an amphiphilic biosample through measuring the length of flow on a microfluidic analytical device (μPAD) based on paper. When an amphiphilic sample was dripped into a straight microchannel defined with a printed wax barrier (hydrophobic) on filter paper (hydrophilic), the length of flow was affected by the reciprocal effect between the sample, the filter-paper channel and the wax barrier. The flow length decreased with increasing concentration of an amphiphilic sample because of adsorption of the sample on the hydrophobic barrier. Measurement of the flow length enabled a determination of the concentration of the amphiphilic sample. The several tested samples included surfactants (Tween 20 and Triton X-100), oligonucleotides (DNA), bovine serum albumin (BSA), human albumin, nitrite, glucose and low-density lipoprotein (LDL). The results show that the measurement of the flow length determined directly the concentration of an amphiphilic sample, whereas a non-amphiphilic sample was not amenable to this method. The proposed method features the advantages of small cost, simplicity, convenience, directness, rapidity (