Wax Barriers Research Articles

A Novel Equipment-Free Paper-Based Fluorometric Method for the Analytical Determination of Quinine in Soft Drink Samples.

A simple, equipment-free, direct fluorometric method, employing paper-based analytical devices (PADs) as sensors, for the selective determination of quinine (QN) is described herein. The suggested analytical method exploits the fluorescence emission of QN without any chemical reaction after the appropriate pH adjustment with nitric acid, at room temperature, on the surface of a paper device with the application of a UV lamp at 365 nm. The devices crafted had a low cost and were manufactured with chromatographic paper and wax barriers, and the analytical protocol followed was extremely easy for the analyst and required no laboratory instrumentation. According to the methodology, the user must place the sample on the detection area of the paper and read with a smartphone the fluorescence emitted by the QN molecules. Many chemical parameters were optimized, and a study of interfering ions present in soft drink samples was carried out. Additionally, the chemical stability of these paper devices was considered in various maintenance conditions with good results. The detection limit calculated as 3.3 S/N was 3.6 mg L-1, and the precision of the method was satisfactory, being from 3.1% (intra-day) to 8.8% (inter-day). Soft drink samples were successfully analyzed and compared with a fluorescence method.

Development of a Paper-Based Analytical Method for the Colorimetric Determination of Calcium in Saliva Samples

A novel, rapid, and facile method for the colorimetric determination of calcium using micro-analytical paper-based devices (μ-PADs) was developed. The proposed analytical method utilizes the color differences developing, after the addition of calcium, on the surface of the devices because of the complexation reaction of calcium with Methylthymol Blue (MTB) at room temperature, in alkaline pH. The devices were manufactured with chromatographic paper, using wax barriers, and the analytical protocol was easily implemented without the need of any experimental apparatus except for a simple imaging device. The user must regulate the pH, add the solutions on the paper, and measure the color intensity of the formed Ca(II)-MTB complex with a flatbed scanner. The experimental conditions for optimum color development, the possible interfering substances, and the reliability of the paper devices in different preserving conditions were optimized, with satisfactory results. The method exhibited acceptable detection limits (2.9 mg L-1) with sufficiently good precision, which varied from 4.2% (intra-day) to 6.4% (inter-day). Saliva samples from healthy volunteers were successfully analyzed, and the calcium levels were calculated in the range of 30.71 to 84.15 mg L-1.

Fabrication of microtiter plate on paper using 96-well plates for wax stamping.

The online version contains supplementary material available at 10.1007/s10404-022-02606-3.

Fabrication of paper-based microfluidic devices using a 3D printer and a commercially-available wax filament

In this work, we developed an alternative manufacturing paper-based microfluidics method through 3D printing and wax filament. Microfluidic paper-based analytical devices (µPADs) are low-cost and easy-to-manufacture tools used for various chemical and biological analyses and studies. Paper-based microfluidics with wax has been limited as the manufacturers have discontinued most wax printing equipment. We aim to develop a low-cost and accessible manufacturing method that can replace conventional wax-on paper-based microfluidic manufacturing methods. Using highly available commercial 3D printing technology and wax filament, we could create hydrophobic wax barriers on the surface of different paper types. The properties and limits of this manufacturing method were characterized. Moreover, using this paper-based microfluidic manufacturing method, we were able to measure dopamine electrochemically using µPAD as a passive flow-based method in concentrations as low as 1 nM using injections as small as 15 µL.

Development of a Paper-Based Analytical Method for the Selective Colorimetric Determination of Bismuth in Water Samples

A novel, direct and simple colorimetric method employing μicroanalytical paper-based devices (μ-PADs) for the selective determination of bismuth is described. The suggested method exploits the colorimetric variation of bismuth after its rapid reaction with methyl thymol blue (MTB) in an acidic medium (pH ranging between 0.7 and 3.0), modified with nitric acid, on the surface of a paper device at room temperature. The devices are low cost, composed of chromatographic paper and wax barriers and the analytical protocol is easily applicable with minimal technical expertise and without the need for experimental apparatus. The user must add a test sample and read the color intensity of the colored Bi(III)–MTB complex formed at the sensing area using a simple imaging device such as a flatbed scanner. Various chemical variables, such as HNO3 and MTB concentration, reaction time, ionic strength, detection zone size and photo-capture detector are optimized. A study of interfering ions such as K+, Na+, Ca2+, Mg2+, Cl−, SO42− and HCO3− was also conducted. The stability of the paper devices is also studied in different maintenance conditions with particularly satisfactory results, rendering the method suitable for field analysis. The detection limits are as low as 3.0 mg L−1 with very satisfactory precision, ranging from 4.0% (intra-day) to 5.5% (inter-day). Natural water samples are successfully analyzed, and bismuth percentage recoveries were calculated in the range of 82.8 to 115.4%.

Development of an equipment free paper based fluorimetric method for the selective determination of histidine in human urine samples

A direct fluorimetric method, employing μicro-analytical paper-based devices (μ-PADs) for the selective determination of histidine (HIS) is described. The suggested method exploits the fluorescence emission of histidine after its rapid reaction with o-phthalaldehyde (OPA) at a basic medium (pH = 10) on the surface of a paper device with the application of a UV lamp at 354 nm. The devices are inexpensive and are composed of chromatographic paper and wax barriers. The analytical protocol is easily applicable with minimal technical expertise and without the need of expensive experimental apparatus. The user has to add a test sample, illuminate the device with a UV lamp, and read the fluorescence of the sensing area using a simple imaging device such as a cell-phone camera. The method is free from common interferences likely to affect the measurement of histidine and is selective among all other amino acids. This analytical procedure was optimized and validated, paying special attention to its intended application. The detection limits are as low as 1.8 μM with very satisfactory precision ranging from 6.4% (intra-day) to 8.9% (inter-day). Random urine samples from adult volunteers (n = 5) were successfully analyzed and HIS content ranged between 260 and 1114 μmol L−1 with percentage recoveries in the range of 78.2 and 124.6%.

Patterned Dried Blood Spot Cards for the Improved Sampling of Whole Blood.

Dried blood spot (DBS) cards perform many functions for sampling blood that is intended for subsequent laboratory analysis, which include: (i) obviating the need for a phlebotomist by using fingersticks, (ii) enhancing the stability of analytes at ambient or elevated environmental conditions, and (iii) simplifying the transportation of samples without a cold chain. However, a significant drawback of standard DBS cards is the potential for sampling bias due to unrestricted filling caused by the hematocrit of blood, which often limits quantitative or reproducible measurements. Alternative microsampling technologies have minimized or eliminated this bias by restricting blood distribution, but these approaches deviate from clinical protocols and present a barrier to broad adoption. Herein, we describe a patterned dried blood spot (pDBS) card that uses wax barriers to control the flow and restrict the distribution of blood to provide enhanced sampling. These patterned cards reproducibly fill four replicate extraction zones independent of the hematocrit effect. We demonstrate a 3-fold improvement in accuracy for the quantitation of hemoglobin using pDBS cards compared to unpatterned cards. Patterned cards also facilitate the near quantitative recovery (ca. 95%) of sodium with no evidence of a statistically significant difference between dried and liquid blood samples. Similarly, the recovery of select amino acids was conserved in comparison to a recent report with improved intercard precision. We anticipate that this approach presents a viable method for preparing and storing samples of blood in limited resource settings while maintaining current clinical protocols for processing and analyzing dried blood spots.

Barrier-Free Microfluidic Paper Analytical Devices for Multiplex Colorimetric Detection of Analytes.

In recent years, microfluidic paper analytical devices (μPADs) have been extensively utilized to conduct multiplex colorimetric assays. Despite their simple and user-friendly operation, the need for patterning paper with wax or other physical barriers to create flow channels makes large-scale manufacturing cumbersome. Moreover, convection of rehydrated reagents in the test zones leads to nonuniform colorimetric signals, which makes quantification difficult. To overcome these challenges, we present a device called a barrier-free μPAD (BF-μPAD) that consists of a stack of two paper membranes having different wicking rates-the top layer acting as a fluid distributing layer and the bottom layer containing reagents for colorimetric detection. Multiple analytes can be detected using this assembly without the need to pattern either layer with wax or other barriers. In one embodiment, a device is capable of delivering the sample fluid to 20 distinct dried reagent spots stored on an 8 cm × 2 cm membrane in as few as 30 s. The multiplexing feature of the BF-μPAD is demonstrated for colorimetric detection of salivary thiocyanate, protein, glucose, and nitrite. Most importantly, the device improves the limit of detection of colorimetric assays performed on conventional μPADs by more than 3.5×. To understand fluid imbibition in the paper assembly, the device geometry is modeled in COMSOL Multiphysics using the Richards equation; the results obtained provide insights into the nonintuitive flow pattern producing perfectly uniform signals in the barrier-free assembly.

Paper-Based Potentiometric Sensors for Nicotine Determination in Smokers' Sweat.

Herein, we describe for the first time, the design and fabrication of a novel nicotine paper-based sensor, in which a miniaturized paper reference electrode is integrated for potentiometric measurements. The paper-based sensors were designed using printed wax barriers to define the electrochemical cell and the sample zones. The electrodes were based on the use of the ion association complexes of the nicotinium cation (Nic) with either tetraphenylborate (TPB) or 5-nitrobarbiturate (NB) counter anions as sensing materials for nicotine recognition. A poly (3,4 ethylenedioxythiophene)/poly-(styrene sulfonate) (PEDOT/PSS) conducting polymer was used as an ion-to-electron transducer. The performance characteristics of the proposed sensors were evaluated and it revealed a rapid and stable response with a Nernstian slope of 55.2 ± 0.3 and 51.2 ± 0.6 mV/decade over the linear range of 1.0 × 10–5 to 1.0 × 10–2 M and detection limits of 6.0 and 8.0 μM for [Nic/TPB] and [Nic/NB], respectively. The sensors revealed a constant response over the pH range 3.5–6.5. The designed sensors provided a portable, inexpensive, and disposable way of measuring trace levels of nicotine coming from different cigarettes and in the collected human sweat of heavy smokers. All results were compared favorably with those obtained by the standard gas chromatographic method.

An origami electrical biosensor for multiplexed analyte detection in body fluids

We developed an affordable, highly sensitive, and specific paper-based microfluidic platform for fast multiplexed detections of important biomarkers in various body fluids, including urine, saliva, serum, and whole blood. The sensor array consisted of five individual sensing channels with various functionalities that only required a micro liter-sized sample, which was equally split into aliquots by the built-in paper microfluidics. We achieved the individual functionalizations of various bioreceptors by employing the use of wax barriers and ‘paper bridges’ in an easy and low-cost manner. Pyrene carboxylic acid-modified single-walled carbon nanotubes (PCA/SWNTs) were deposited by quantitative inkjet printing with an optimal 3-dimensional semiconductor density on a paper substrate. Multiple antibodies were immobilized onto the SWNTs surface for highly sensitive and specific field-effect transistor (FET)/chemiresistor (CR) biosensors. We explored the optimal sensing conditions for the paper-based CR biosensor to achieve high sensitivities and specificities towards the target biomarker proteins (human serum albumin (HSA) and human immunoglobulin G (HIgG)) and achieved an ultralow detectable concentration of HSA and HIgG at 1.5 pM. Besides, origami folding was employed to simplify the fabrication process further. The sensing platform described in this work was cost-effective, semi-automated, and user-friendly. It demonstrated the capability of having multiple sensing functions in one paper-based microfluidic sensing platform. It envisioned the potential of a point-of-care device with full-analysis for practical diagnostics in an ASSURED (Affordable, Sensitive, Specific, User-friendly, Rapid and robust, Equipment-free and Deliverable to end-users) fashion for a quick test of targets of interest.

Lateral flow assay modified with time-delay wax barriers as a sensitivity and signal enhancement strategy

The ease of use, low cost and quick operation of lateral flow assays (LFA) have made them some of the most common point of care biosensors in a variety of fields. However, their generally low sensitivity has limited their use for more challenging applications, where the detection of low analytic concentrations is required. Here we propose the use of soluble wax barriers to selectively and temporarily accumulate the target and label nanoparticles on top of the test line (TL). This extended internal incubation step promotes the formation of the immune-complex, generating a 51.7-fold sensitivity enhancement, considering the limit of quantification, and up to 96% signal enhancement compared to the conventional LFA for Human IgG (H-IgG) detection.

Wax-printed well pads and colorimetric LAMP detection of ApxIA toxin gene

Actinobacillus pleuropneumoniae (A. pleuropneumoniae) produces hemolytic cytotoxins, ApxI, ApxII, ApxIII, and ApxIV, causing a huge loss to the swine industry. We developed the PadLysis protocol for extracting genomic DNA (gDNA) for the LAMP—LeucoCrystal Violet (LCV) colorimetric detection. A paper-based well pad for gDNA extraction was fabricated using a wax printing method. Furthermore, the LAMP amplicons were detected in situ with colorimetric dye, LCV. Wax-printed well pads (WPWPs) have hydrophobic wax barriers by printing a well pattern that allows the establishment of simple and equipment-free PadLysis protocol consisting of the three steps: (1) 2 min lysis, (2) 2 min washing, and (3) 1 min drying. This proposed platform allowed direct LAMP amplification for A. pleuropneumoniae field isolates. We optimized LCV chemical components to enable the accurate LAMP signal readouts. The overall assay takes up to 45 min including 5 min PadLysis, 40 min LAMP amplification, and LCV signal observation with the naked eye. Our WPWPs–LAMP–LCV approach can be used as a rapid screening tool for A. pleuropneumoniae pleuropneumonia (APP) caused by ApxIA toxin in future outbreak areas.

Roll-to-roll wax transfer for rapid and batch fabrication of paper-based microfluidics

This study proposed a novel method for the rapid fabrication of paper-based microfluidic devices using a roll-to-roll wax transfer method. In this approach, a custom-made device was designed and fabricated for the direct roll-to-roll wax transfer from commercially available wax ribbon to the surface of paper substrate. After wax transfer, a thermal reflow process was conducted to enable the melt wax penetration through the whole thickness of the hydrophilic paper substrate to form hydrophobic wax barriers. Compared with the conventional approaches for paper-based microfluidics (wax printing, photolithography, and inkjet printing), the proposed fabrication technique for paper-based microfluidic devices is suitable for the batch fabrication of paper-based microfluidic chips at high production speed. Additional advantages including simple fabrication process, low material and fabrication cost, low-energy consumption, and without the requirement of cleanroom environment or facilities. The paper-based microfluidics fabricated with the proposed roll-to-roll wax transfer method is widely adaptable for microfluidic applications in biological and chemical fields.

Simultaneous quantification of multiple biomarkers on a self-calibrating microfluidic paper-based analytic device

In this study, we developed a point-of-care assay platform with simultaneous detection and self-calibration capabilities for multiple targets based on a microfluidic paper-based analytical device (μPAD). This system is easily manufactured using a wax printing method on chromatographic paper. The design pattern consists of a zone of detection and a calibrant zone for controlled loading using wax barriers with different thicknesses. We showed the utility and applicability of this approach by a proof-of-concept study for two clinically important markers: glucose and lactate. With the naked eye, the results could be fully distinguished and recorded to evaluate the analytical performance with a flatbed scanner. The detection limits of glucose and lactate were 0.3125 mM and 0.2975 mM, respectively, and simultaneous detection was possible from a small sample (0.4 μL) with high sensitivity. Furthermore, this device has a self-calibration function, which minimizes the influence of environmental conditions (i.e., ambient light intensity, temperature, humidity, and pressure). Therefore, the developed multiplex paper-based device is promising for clinical multianalyte point-of-care testing since it is easy to manufacture, cost-effective, user-friendly, and highly sensitive.

Developing self-generated calibration curves using a capillary-driven wax-polyester lab on a chip device and thermal gates

Here we describe the development of a capillary-driven wax-polyester lab on a chip device and the concept of thermal gates to create self-generated calibration curves. The devices were fabricated by printing a wax pattern on a polyester film, and laminating two mirrored layers together. Victoria Blue R and a diluent of methanol/water (1:1) were placed in separate wells, and capillary forces drove the reagents through three additional mixing wells, resulting in respective dilutions of 50%, 25% and 12.5% of the original dye concentration (100%). A photo was taken after the capillary action ceased, and the color brightness of the four different concentrations was used for the creation of a calibration curve, resulting in a coefficient of determination of 0.99. The chip was readily adapted to include an additional channel to perform chemical reactions with KOH to increase the available detection options. The chip designs required up to three reagents to be simultaneously added, with any discrepancies in the synchronization of reagent addition leading to irreproducible results. To overcome these synchronization discrepancies, thermal gates were added by printing 0.15 mm wax barriers across the channel inlets to prevent liquid movement. These gates were simultaneously opened to allow the solutions to flow by applying heat, controlled by an Arduino-based instrument controller, through bent copper filaments positioned adjacent to the barriers for 380 milliseconds.

Laser-induced selective wax reflow for paper-based microfluidics.

This study proposes a novel method for the fabrication of paper-based microfluidic devices using laser-induced selective thermal reflow for wax penetration. A layer of wax was evenly deposited on the front side of a filter paper; then a low-cost diode laser was used to scan the designed area from the back side of the filter paper. At the laser irradiated spot, the wax was heated, melted down and penetrated through the whole thickness of the filter paper, and formed hydrophobic barriers on the hydrophilic cellulose fibers. The patterned hydrophobic wax barriers on the filter paper defined the flow path of the fluid for the paper-based microfluidic device. Compared with conventional two-step (deposit and reflow) approaches for paper-based microfluidics using wax barriers, e.g. wax printing, stamping or photolithography, the proposed fabrication protocol achieved wax patterning and reflow simultaneously, conducted during the laser scan process, and without the requirement for any sophisticated instruments or a cleanroom environment. A series of tests were also conducted for the characterization of the proposed paper-based microfluidic device fabrication technique. The fabrication technique used in this approach could have broad application potential in point-of-care diagnosis and testing, especially for applications in the developing world.

Fast and flexible strategy to produce electrochemical paper-based analytical devices using a craft cutter printer to create wax barrier and screen-printed electrodes

This paper describes a simple, low-cost, and highly flexible rapid prototyping method to construct electrochemical paper-based analytical device (ePAD) for multiplexed analyte determinations. The ePAD was composed of two electrochemical cell (EC) compartments, separated by hydrophobic barriers of wax, and screen-printed electrodes (SPEs) deposited directly over the surface of the filter paper. The ePAD was entirely constructed using an inexpensive craft cutter printer with no needed of a wax printer. The rapid prototyping method involves two steps: the deposition of the SPEs and the creation of the wax barriers. In this case, the SPEs were screen-printed on filter paper by using adhesive tape as mask by cutting the electrodes pattern with the cutter printer. Following, the wax barriers were created using stamps made of filter paper also cut with the printer and impregnated with wax. In the ePAD, each ECs containing an array of 4-working electrodes, allowing up to 4 replicates in a single measurement. Both ECs shared one counter and one reference electrodes, permitting the simultaneous multianalysis. The ePAD was successfully applied to simultaneous detection of paracetamol (PAR), caffeine (CAF), and ascorbic acid (AA) in drugs. PAR and CAF were detected in a sample using one EC and AA was detected, in a different sample, on the other EC, both with no chemical modifications in the working electrodes. Limits of detection of 0.04 mmol L−1 for PAR, 0.22 mmol L−1 for CAF, and 0.40 mmol L−1 for AA were obtained. The construction process proposed provide an easy way to implement screen-printing electrodes and wax barriers in filter paper to create electrochemical devices for fast and simultaneous multianalysis.

Single step and mask-free 3D wax printing of microfluidic paper-based analytical devices for glucose and nitrite assays

Microfluidic paper-based analytical devices (μPADs) have recently emerged as a simple, portable, user-friendly, and affordable alternative to more instrument-intensive analytical approaches for point-of-care testing (POCT), food safety analysis, and environmental monitoring. However, most of the existing methods for the fabrication of μPADs still face a great challenge because of different trade-offs among cost, convenience, and the pattern resolution. In this work, we report a facile one-step approach to prepare a μPAD using an affordable, easy-to-build 3D printer to generate patterns of solid wax on laboratory filter paper. The presented wax printing method did not require the use of predesigned masks and an external heat source to form complete hydrophobic wax barrier through the use of a custom-made extruder. The results revealed a strong linear relationship (R2 = 0.985) between the nominal and the printed widths of the wax barriers. The achievable resolution of the wax barrier printed on filter paper was 468 ± 72 µm, which was lower than previously reported minimum barrier feature sizes achieved by wax printing and other wax patterning techniques, such as stamping and screen-printing. The analytical utility of the fabricated μPADs was evaluated for colorimetric nitrite and glucose detection in artificial solutions. It was found that the fabricated μPADs provided adequate accuracy and reproducibility for quantitative determination of nitrite and glucose within concentration ranges relevant to the disease detection in human saliva and urine. The wax printing approach reported here provides a simple, rapid, and cost-effective fabrication method for paper-based microfluidics and may bring benefits to medical diagnostics in the developing world.

Versatile and Robust Integrated Sensors To Locally Assess Humidity Changes in Fully Enclosed Paper-Based Devices.

The synergic combination of materials and interfaces to create novel functional devices is a crucial approach for various applications, including low-cost paper-based point-of-care systems. In this work, we demonstrate the implementation of surface-modified polypyrrole (PPy) structures, monolithically integrated into a three-dimensional multilayered paper-based microfluidic device, to locally assess humidity changes. The fabrication and integration of the system include the deterministic incorporation of PPy into the paper-based structure by gas-phase polymerization and the modification of the polymer properties to allow local humidity monitoring. The functionalization of PPy changes both the wettability and the chemical composition of the interface, what is of fundamental importance for the sensor's operation. The PPy structure has excellent mechanical stability, enduring at least 600 bending cycles, what is of relevance on flexible electronics. The electrical resistance correlates with the local relative humidity (RH) inside of the sealed microfluidic system, and the sensor response is fully reversible. The integrated system capable of locally monitoring the RH allowed us to verify that inside the microfluidic channel, water molecules can diffuse across the wax barriers-a possibility disregarded so far. Our results attest that RH variations of 5-10% can affect the flow of extended channels (>5 cm) even when they are fully enclosed.

Selective Distance-Based K+ Quantification on Paper-Based Microfluidics.

In this study, paper-based microfluidic devices (μPADs) capable of K+ quantification in aqueous samples, as well as in human serum, using both colorimetric and distance-based methods are described. A lipophilic phase containing potassium ionophore I (valinomycin) was utilized to achieve highly selective quantification of K+ in the presence of Na+, Li+, and Mg2+ ions. Successful addition of a suspended lipophilic phase to a wax printed paper-based device is described and offers a solution to current approaches that rely on organic solvents, which damage wax barriers. The approach provides an avenue for future alkali/alkaline quantification utilizing μPADs. Colorimetric spot tests allowed for K+ quantification from 0.1-5.0 mM using only 3.00 μL of sample solution. Selective distance-based quantification required small sample volumes (6.00 μL) and gave responses sensitive enough to distinguish between 1.0 and 2.5 mM of sample K+. μPADs using distance-based methods were also capable of differentiating between 4.3 and 6.9 mM K+ in human serum samples. Distance-based methods required no digital analysis, electronic hardware, or pumps; any steps required for quantification could be carried out using the naked eye.