Foldable RF Energy Harvesting System Based on Vertically Layered Metal Electrodes within a Single Sheet of Paper.

Radio frequency energy harvesting (RFEH) systems have emerged as a critical component for powering devices and replacing traditional batteries, with paper being one of the most promising substrates for use in flexible RFEH systems. However, previous paper-based electronics with optimized porosity, surface roughness, and hygroscopicity still face limitations in terms of the development of integrated foldable RFEH systems within a single sheet of paper. In the present study, a novel wax-printing control and water-based solution process are used to realize an integrated foldable RFEH system within a single sheet of paper. The proposed paper-based device includes vertically layered foldable metal electrodes, a via-hole, and stable conductive patterns with a sheet resistance of less than 1 Ω sq-1 . The proposed RFEH system exhibits an RF/DC conversion efficiency of 60% and an operating voltage of 2.1 V in 100 s at a distance of 50 mm and a transmitted power of 50 mW. The integrated RFEH system also demonstrates stable foldability, with RFEH performance maintained up to a folding angle of 150°. The single-sheet paper-based RFEH system thus has the potential for use in practical applications associated with the remote powering of wearable and Internet-of-Things devices and in paper electronics.

A paper-based point-of-care testing device for the colourimetric estimation of bilirubin in blood sample.

A paper-based colourimetric assay for the Point-of-Care Testing (PoCT) of bilirubin has been developed based on the formation of a green-coloured copper-bilirubin complex from a blue-coloured tetraamminecopper(II) sulphate complex. The reaction was studied and optimized by UV-Visible absorption spectroscopy and translated onto a paper strip. Hydrophobic circular well patterns on Whatman chromatography paper were created by wax printing. The tetraamminecopper(II) sulphate complex was drop cast and dried on the reagent zones in the wax-patterned paper. The images of reagent zones captured using a scanner were analyzed using ImageJ software. Bilirubin spiked blood serum was tested in the concentration range of 1.2 to 950 µM. The PAD exhibited sensitivities of 0.4197 a.u/µM and 0.1040 a.u/µM for concentration ranges of bilirubin 1.2 to 96 µM and 105 to 950 µM respectively and a low detection limit of 0.799 µM. The method is highly selective to bilirubin, even in the presence of other biomarkers in serum. A plasma separation membrane incorporated PAD was fabricated for the final testing and quantification of bilirubin from whole blood.

A paper-based electrochemical device for the detection of pesticides in aerosol phase inspired by nature: A flower-like origami biosensor for precision agriculture.

Pesticides are largely used at worldwide level to improve food production, fulfilling the needs of the global population which is increasing year by year. Although pesticides are beneficial for crop production, their extensive use has serious consequences for the pollution of the produced food as well as for soil and groundwaters. Indeed, it is reported that 50% of sprayed pesticides reach different destinations other than their target species, including soil, surface waters, and groundwaters. For this reason, we developed a flower-like origami paper-based device for pesticides detection in aerosol phase for precision agriculture. In detail, the paper-based electrochemical platform detects paraoxon, 2,4-dichlorophenoxyacetic acid, and glyphosate at ppb levels by measuring their inhibitory activity towards three different enzymes namely butyrylcholinesterase, alkaline phosphatase, and peroxidase enzyme, respectively. This integrated electrochemical device is composed of three office paper-based screen-printed electrodes and filter paper-based pads loaded with enzymes and enzymatic substrates. The pesticide detection is carried out by measuring through chronoamperometric technique the initial and residual enzymatic activity by using a smartphone-assisted potentiostat and evaluating the percentage of inhibition, proportional to the amount of aerosolized pesticides. This paper-based device was able to detect the three classes of pesticides in aerosol phase with limits of detection equal to 30 ppb, 10 ppb, and 2 ppb, respectively for 2,4-D, glyphosate, and paraoxon.

A Multi-Chamber Paper-Based Platform for the Detection of Amyloid ß Oligomers 42 via Copper-Enhanced Gold Immunoblotting.

The early diagnosis of Alzheimer's disease (AD) remains a challenge for medical scientists worldwide, leading to a number of research efforts that focus on biosensor development for AD biomarkers. However, the application of these complicated biosensors is limited in medical diagnosis, due to the difficulties in robust sensing platform development, high costs, and the necessity for technical professionals. We successfully developed a robust straightforward manufacturing process for the fabrication of multi-chamber paper devices using the wax printing method and exploited it to detect amyloid beta 42 oligomers (AßO42, a significant biomarker of AD) using copper-enhanced gold nanoprobe colorimetric immunoblotting. Small hydrophilic reaction chambers could concentrate the target sample to the desired size to improve the sensing performance. The copper-enhanced gold nanoprobe immunoblot using the designed multi-chamber platform exhibited a highly sensitive performance with a limit of detection of 320 pg/mL by the naked eye and 23.7 pg/mL by a smartphone camera. This process from sensing manufacture to sensing conduction is simple to perform whenever medical technicians require time- and cost-savings, without complicated instruments or the need for technical professionals, making it feasible to serve as a diagnostic tool worldwide for the early monitoring of AD and scalable devices for the sensing application of various biomarkers in clinical settings.

Office Paper-Based Electrochemical Strips for Organophosphorus Pesticide Monitoring in Agricultural Soil.

Although the use of pesticides has highlighted obvious advantages on agricultural yields, intensive and widespread pesticide use raises serious environmental and health concerns. In particular, organophosphate pesticides represent >40% of the totality used in the field of agriculture, and developing countries face the issue of agricultural poisoning, also due to scarce monitoring programs. In this work, a decentralized, miniaturized, sustainable, and portable paper-based electrochemical biosensor for the quantification of organophosphorus pesticides' level has been realized. The proposed approach highlights the use of a very common paper-based substrate, namely, office paper. Office paper offers several advantages due to its nature: it allows one to print conductive strips for electrochemical connection, loading bio-hybrid nanosized probes (Prussian blue, carbon black, and butyrylcholinesterase), evaluating pesticides and reducing waste disposal compared to plastic-based strips. The portable system has been characterized by a low detection limit of 1.3 ng/mL, and accordingly to total discovered pesticide contents in EU agricultural soils, up to ca. 3 µg/mL, it can offer a valuable tool for fast monitoring. To demonstrate its effectiveness, soil and fruit vegetables have been used to perform in situ quantification. Good recovery percentages between 90 and 110% have been achieved in different matrices, highlighting to be suitable for field measurements, and a good correlation has been obtained in comparison with LC-MS analysis.

Multi-array wax paper-based platform for the pre-concentration and determination of silver ions in drinking water.

In this work, a wax-patterned chromatographic paper has been utilized as a holistic platform to 1) synthesize Prussian Blue Nanoparticles (sensing species), 2) load the reagents for the assay, 3) concentrate the sample through multistep, and 4) visualize the determination of silver ions. Waters are continuously affected by changes in the composition, thus the utilization of reagent-free analytical tools is of huge interest for smart drinking water monitoring. Herein, we report the characterization and application of a multi-array paper-based platform for the colorimetric determination of silver ions based on the conversion from Prussian Blue to its silver-based analogue, namely Ag4[Fe(CN)6]. In particular, the platform highlights the increase of sensitivity due to paper pre-concentration of sample, that can be easily adapted to the analytical necessities. Within the proposed experimental setup, Ag+ is visualized down to a detection limit of 0.9 µM, with high repeatability and satisfactory recoveries in the range comprised between 90 and 113%.

A microfluidic paper-based analytical device (µPAD) with smartphone readout for chlorpyrifos-oxon screening in human serum.

Acute intoxication incidents due to neurotoxic organophosphate (OP) insecticides are occasionally reported, related either to suicidal attempts or occupational exposure due to the misuse of protective equipment. Among them, chlorpyrifos is a compound related to great controversy, which is still authorized and easily accessible in many countries around the world. However, to screen for its exposure markers, instrumental methods are commonly applied, which cannot enable rapid monitoring at an early stage of an intoxication. Therefore, in this study, a microfluidic paper-based analytical device (µPAD) able to rapidly screen for chlorpyrifos-oxon, the toxic chlorpyrifos metabolite, in human serum was developed and fully validated. The µPAD combines wax-printed butyrylcholinesterase (BChE) paper sensors, a lab-on-a-chip (LOC) prototype injector and a smartphone as the analytical detector. In principle, the wax-printed strips with adsorbed BChE are embedded into LOC injectors able to deliver samples and reagents on-demand. A smartphone reader was used to monitor the color development on the strips providing binary qualitative results. µPAD method performance characteristics were thoroughly evaluated in terms of specificity, detection capability (CCß) and ruggedness. The developed analytical platform is rapid (results within 10 min), cost-efficient (0.70 €), potentially applicable at the point-of-need and attained a low CCß (10 µg L-1 in human serum). Finally, µPAD characteristics were critically compared to well-established methods, namely an in-house BChE microplate assay and liquid chromatography tandem mass spectrometry.

Predicting Dimensions in Microfluidic Paper Based Analytical Devices.

The main problem for the expansion of the use of microfluidic paper-based analytical devices and, thus, their mass production is their inherent lack of fluid flow control due to its uncontrolled fabrication protocols. To address this issue, the first step is the generation of uniform and reliable microfluidic channels. The most common paper microfluidic fabrication method is wax printing, which consists of two parts, printing and heating, where heating is a critical step for the fabrication of reproducible device dimensions. In order to bring paper-based devices to success, it is essential to optimize the fabrication process in order to always get a reproducible device. Therefore, the optimization of the heating process and the analysis of the parameters that could affect the final dimensions of the device, such as its shape, the width of the wax barrier and the internal area of the device, were performed. Moreover, we present a method to predict reproducible devices with controlled working areas in a simple manner.

Paper-based electrochemical peptide nucleic acid (PNA) biosensor for detection of miRNA-492: a pancreatic ductal adenocarcinoma biomarker.

Pancreatic ductal adenocarcinoma is the predominant neoplastic disease of the pancreas and it represents the fourth most frequent cause of death in cancer-related disease, with only 8% of survivors after 5-year to the diagnosis. The main issues of this type of cancer rely on fast progress (i.e. 14 months from T1 to a T4 stage), nonspecific symptoms with delay in diagnosis, and the absence of effective screening strategies. To address the lack of early diagnosis, we report a cost-effective paper-based biosensor for the detection of miRNA-492, which is recognised as a biomarker for pancreatic ductal adenocarcinoma. To design a miniaturised, sensitive, and robust paper-based platform, an electrochemical sensor was screen-printed on office paper previously wax-patterned via wax-printing technique. The paper-based sensor was then engineered with a novel and highly specific peptide nucleic acid (PNA) as the recognition element. The formation of PNA/miRNA-492 adduct was evaluated by monitoring the interaction between the positively charged ruthenium (III) hexamine with uncharged PNA and/or negatively charged PNA/miRNA-492 duplex by differential pulse voltammetry. The paper-based biosensor provided a linear range up to 100 nM, with a LOD of 6 nM. Excellent selectivity towards one- and two-base mismatches (1MM, 2MM) or scrambled (SCR) sequences was highlighted and the applicability for biomedical analyses was demonstrated, measuring miRNA-492 in undiluted serum samples.

Paper-based device for the colorimetric assay of bilirubin based on in-situ formation of gold nanoparticles.

A paper-based colorimetric assay for the determination of bilirubin has been developed. The method is based on the in-situ reduction of chloroauric acid to form gold nanoparticles. A chromatographic paper was patterned using a wax printer. Chloroauric acid was drop-cast onto the reagent zone. In the presence of bilirubin, gold(III) ions are reduced and form gold nanoparticles. This leads to a color change from yellow to purple. The intensity of the purple color (peak at 530 nm) increases with bilirubin concentration in the 5.0 to 1000 mg L-1 range. The detection limit is 1.0 mg L-1. For the quantification of bilirubin, images were captured using a digital camera, and data were processed with the help of machine learning-based supervised prediction using Random Forest classification. The method was applied to the determination of bilirubin in urine samples. The spiked urine samples exhibit more than 95% recovery. Graphical abstractSchematic representation of the paper-based colorimetric assay for the detection of bilirubin based on the in-situ formation of gold nanoparticles. A color band is generated for visual interpretation and used for the testing of bilirubin in urine.

Combining Wax Printing with Hot Embossing for the Design of Geometrically Well-Defined Microfluidic Papers.

A simple, efficient, and repeatable combination of wax printing and hot embossing is reported. This combination yields microfluidic channels in paper, where fluid transport driven by paper-intrinsic capillary forces takes place inside the noncompressed areas, whereas embossed and wax-bonded areas serve as hydrophobic barriers laterally confining the fluid flow. Lab-made paper sheets first coated with a hydrophobic wax were hot-embossed with a tailor-made metal stamp. Both paper-intrinsic (e.g., grammage, fiber type) and paper-extrinsic parameters (e.g., embossing force) were studied for their influence on the geometry of the embossed structures and the resulting redistribution of the wax within the paper matrix. Embossing of wax-printed paper at temperatures above the wax melting point was completed within 15 s. Cotton linters papers required higher embossing forces than eucalyptus papers, which can be explained by their different intrinsic mechanical properties. In summary, both paper-intrinsic and paper-extrinsic parameters were found to have strong impact on resolution and reproducibility of the channels. All in all, the approach yields microfluidic channels in a fast and robust and reproducible manner with comparably low constrains on the precision of manufacturing parameters, such as embossing time, force, or temperature. Most importantly, embossing greatly reduces the lateral spreading of the wax as seen with melting approaches and therefore allows for a much higher feature density than the latter.

Design considerations for reducing sample loss in microfluidic paper-based analytical devices.

The field of microfluidic paper-based analytical devices (µPADs) is most notably characterized by portable and low-cost analysis; however, struggles to achieve the high sensitivity and low detection limits needs required for many environmental applications hinder widespread adoption of this technology. Loss of analyte to the device material represents an important problem impacting sensitivity. Critically, we found that at least 50% of a Ni(II) sample is lost when being transported down a 30 mm paper channel that is representative of structures commonly found in µPADs. In this work, we report simple strategies such as adding a waste zone, enlarging the detection zone, and using an elution step to increase device performance. A µPAD combining the best performing functionalities led to a 78% increase in maximum signal and a 28% increase in sensitivity when transporting Ni(II) samples. Using the optimized µPAD also led to a 94% increase in maximum signal for Mn(II) samples showing these modifications can be applied more generally.

Characteristics of Microfluidic Paper-based Analytical Devices Fabricated by Four Different Methods.

We report on the effects of fabrication methods, photolithography, wax printing, screen printing, and craft cutting, on selected properties of microfluidic paper-based analytical devices (µPADs): cost, fabrication precision, wicking rate, and analytical accuracy. Photolithography requires numerous fabrication steps, and an oxygen plasma treatment is necessary when using an aqueous solution. Although the boundary between the hydrophobic and hydrophilic areas in the µPAD is sharpest, the obtained K-scale intensity in measuring of protein concentrations is lower than those of the devices by other methods. Wax printing offers the simplest and fastest fabrication, although solution leakage measures should be taken to improve the wicking rate and to prevent cross-contamination. Screen printing also offers easy fabrication. The screen-printed µPAD has a good wicking performance and shows a high detection intensity. Craft cutting allows automated fabrication of many µPADs at once. The craft cut µPAD has the fastest wicking rate among the four µPADs due to bare cellulose fibers. We consider that the detection intensity of this µPAD can be raised by optimizing the evaporation rate.

Wax-Assisted One-Step Enzyme-Linked Immunosorbent Assay on Lateral Flow Test Devices.

Lateral flow tests (LFTs) are widely used analytical tools characterized by portability, operator simplicity and short analysis times. A remaining challenge is their limited analytical sensitivity, which in classical immunoassay formats is overcome by enzyme-linked immunosorbent assay (ELISA) formats. The implementation of ELISA to an LFT format however, is hampered by the complexity of the procedure requiring the enzyme substrate addition after sample addition. In this work, a simple method for automation of this procedure without user interference is presented. Originally used sample pads of LFTs have been replaced by hydrophobic wax-modified filter paper-based sample pads to realize a delayed flow a pre-deposited colorimetric ELISA substrate without other alterations to the classical lateral-flow immunoassay format. The performance of the system has been characterized by visualizing flow behavior and final proof-of-concept is provided by a model mouse IgG assay, achieving a limit of detection of 15.8 ng mL-1 from just a single application of the sample solution.

Development of an automated wax-printed paper-based lateral flow device for alpha-fetoprotein enzyme-linked immunosorbent assay.

In this study, a novel wax-printed paper-based lateral flow device has been developed as an alternative approach for an automated and one-step enzyme-linked immunosorbent assay (ELISA). The design pattern consisted of a non-delayed channel, a wax-delayed channel, a test zone and a control zone. This system was easily fabricated on a nitrocellulose membrane using a wax-printing method and then baked in an oven at 100°C for 1min. The four barriers of the wax-delayed channel could delay the flow time for 11s compared to the flow time of the non-delayed channel. To use the device under optimal conditions, alpha-fetoprotein (AFP) was detected at a limit of detection of 1ngmL-1 and assessed with the naked eye within 10min. A colorimetric intensity was also measured using a smart phone and computer software at a linear range of 0.1-100ngmL-1 with a good correlation. Furthermore, the proposed device was successfully applied to detect AFP in human serum. Therefore, the wax-printing demonstrates a user-friendly, easy and quick method for the fabrication of the device, which could be used as a one-step, portable, disposable, low-cost, simple, instrument-free and point-of-care device for the automated ELISA.

Improving FoRe: A New Inlet Design for Filtering Samples through Individual Microarray Spots.

In this publication we present an improvement to our previously introduced vertical flow microarray, the FoRe array, which capitalizes on the fusion of immunofiltration and densely packed micron test sites. Filtering samples through individual microarray spots allows us to rapidly analyze dilute samples with high-throughput and high signal-to-noise. Unlike other flowthrough microarrays, in the FoRe design samples are injected into micron channels and sequentially exposed to different targets. This arrangement makes it possible to increase the sensitivity of the microarray by simply increasing the sample volume or to rapidly reconcentrate samples after preprocessing steps dilute the analyte. Here we present a new inlet system which allows us to increase the analyzed sample volume without compromising the micron spot size and dense layout. We combined this with a model assay to demonstrate that the device is sensitive to the amount of antigen, and as a result, sample volume directly correlates to sensitivity. We introduced a simple technique for analysis of blood, which previously clogged the nanometer-sized pores, requiring only microliter volumes expected from an infant heel prick. A drop of blood is mixed with buffer to separate the plasma before reconcentrating the sample on the microarray spot. We demonstrated the success of this procedure by spiking TNF-α into blood and achieved a limit of detection of 18 pM. Compared to traditional protein microarrays, the FoRe array is still inexpensive, customizable, and simple to use, and thanks to these improvements has a broad range of applications from small animal studies to environmental monitoring.

A paper-based nanomodified electrochemical biosensor for ethanol detection in beers.

Herein, we report the first example of a paper-based screen-printed biosensor for the detection of ethanol in beer samples. Common office paper was adopted to fabricate the analytical device. The properties of this paper-based screen-printed electrode (SPE) were investigated by cyclic voltammetry, electrochemical impedance spectroscopy, and scanning electron microscopy, and they were compared with the well-established polyester-based SPEs as well. Paper demonstrated similar properties when compared with polyester, highlighting suitability towards its utilization in sensor development, with the advantages of low cost and simple disposal by incineration. A nanocomposite formed by Carbon Black (CB) and Prussian Blue nanoparticles (PBNPs), namely CB/PBNPs, was utilized as an electrocatalyst to detect the hydrogen peroxide generated by the enzymatic reaction between alcohol oxidase (AOx) and ethanol. After optimizing the analytical parameters, such as pH, enzyme, concentration, and working potential, the developed biosensor allowed a facile quantification of ethanol up to 10 mM (0.058 %vol), with a sensitivity of 9.13 µA/mM cm2 (1574 µA/%vol cm2) and a detection limit equal to 0.52 mM (0.003%vol). These satisfactory performances rendered the realized paper-based biosensor reliable over the analysis of ethanol contained in four different types of beers, including Pilsner, Weiss, Lager, and alcohol-free. The proposed manufacturing approach offers an affordable and sustainable tool for food quality control and for the realization of different electrochemical sensors and biosensors as well.

Paper-Based Microfluidics Immunoassay for Detection of Canine Distemper Virus

ABSTRACT Paper-based devices present low-cost and are versatile, making them very attractive for clinical analysis. To manufacture those devices wax patterns are printed on paper surface and upon heating the wax permeates through the entire thickness of the paper, creating hydrophobic barriers that delimit test areas. Antibodies produced in rabbits against canine distemper virus (CDV) were physically adsorbed on the surface of gold nanoparticles (AuNPs) and incubated with CDV viral antigens, forming the immunocomplex. Anti-CDV antibodies were immobilized into the microchannels by physical adsorption, forming the test region. The test solution containing conjugated AuNPs was applied at the bottom of the microchannel and it was eluted with a phosphate buffer solution 0.01 M pH 7.4. When the solution containing the AuNPs reached the test zone the recognition of antigens contained on the immunocomplex occurred with the consequent development of a red line, which represents a positive outcome for the test. This method demonstrated the success of physical immobilization of antibodies on AuNPs and the physical immobilization of antibodies on cellulose's surface. This colorimetric assay brings simplicity and versatility to clinical analyses, presenting potential for CDV diagnosis.

Development of a microfluidic paper-based analytical device for the determination of salivary aldehydes.

A low cost, disposable and easy to use microfluidic paper-based analytical device (µPAD) was developed for simple and non-invasive determination of total aldehydes in saliva with a potential to be used in epidemiological studies to assess oral cancer risk. The µPAD is based on the colour reaction between aldehydes (e.g. acetaldehyde, formaldehyde), 3-methyl-2-benzothiazolinone hydrazone (MBTH) and iron(III) to form an intense blue coloured formazan dye. The newly developed µPAD has a 3D design with two overlapping paper layers. The first layer comprises 15 circular detection zones (8 mm in diameter), each impregnated with 8 µL of MBTH, while the second layer contains 15 reagent zones (4 mm in diameter). Two µL of iron(III) chloride are added to each one of the second layer zones after the addition of sample to the detection zones in the first layer. All hydrophilic zones of the µPAD are defined by wax printing using a commercial wax printer. Due to the 2-step nature of the analytical reaction, the two paper layers are separated by a cellulose acetate interleaving sheet to allow for the reaction between the aldehydes in the saliva sample with MBTH to proceed first with the formation of an azine, followed by a blue coloured reaction between the azine and the oxidized by iron(III) form of MBTH, produced after the removal of the interleaving sheet. After obtaining a high resolution image of the detection side zone of the device using a flatbed scanner, the intensity of the blue colour within each detection zone is measured with Image J software. Under optimal conditions, the µPAD is characterised by a working range of 20.4-114.0 µM, limit of detection of 6.1 µM, and repeatability, expressed as RSD, of less than 12.7% (n = 5). There is no statistically significant difference at the 95% confidence level between the results obtained by the µPAD and the reference method (Student's t-test: 0.090 < 0.38). The optimized µPAD is stable for more than 41 days when stored in a freezer (≤-20 °C).

Simultaneous forward and reverse ABO blood group typing using a paper-based device and barcode-like interpretation.

A new platform of a paper-based analytical device (PAD) for simultaneous forward and reverse ABO blood group typing has been reported. This platform can overcome the discrepancy results as influenced by the individual haematocrit. The test and the control of non-haemagglutination on each channel were performed in parallel. The PAD was fabricated by printing six parallel channels with wax onto Whatman No. 4 filter paper. An LF1 blood separation membrane was used for the separation of plasma from whole blood for reverse grouping. The blood group was identified by haemagglutination of the corresponding antigen-antibody. For forward grouping, Anti-A, -B and -A,B were treated on the test line of PAD, and inactivated Anti-A, -B and -A,B were immobilized on the control line. For reverse grouping, 30% standard A-cells, B- and O- were added to the test channel after plasma separation, and O-cells were used as a control. Then, 0.9% normal saline (NSS) containing 1% Tween-20 was bi-functionally used for dilution of the blood sample and elution of the non-agglutinated RBCs within the channels. The distance of agglutinated RBCs in each test line was compared with the distance of non-agglutinated RBCs in the parallel control line. The forward and reverse patterns of blood groups A, B, AB and O were a barcode-like chart in which the results can be visually analysed. The PAD has excellent reproducibility when 10 replications of the A, B, AB or O blood groups were performed. The results of both forward and reverse grouping were highly correlated with conventional methods compared with the slide method and tube method, respectively (n = 76). Thus, this ABO typing PAD holds great potential for future applications in blood typing point-of-care testing.