Bio-sample detection on paper-based devices with inkjet printer-sprayed reagents

Paper-based analytical devices have been substantially developed in recent decades. Many fabrication techniques for paper-based analytical devices have been demonstrated and reported. Herein, we report a relatively rapid, simple, and inexpensive method for fabricating paper-based analytical devices using parafilm hot pressing. We studied and optimized the effect of the key fabrication parameters, namely pressure, temperature, and pressing time. We discerned the optimal conditions, including a pressure of 3.8 MPa, temperature of 80 °C, and 3 min of pressing time, with the smallest hydrophobic barrier size (821 µm) being governed by laminate mask and parafilm dispersal from pressure and heat. Physical and biochemical properties were evaluated to substantiate the paper functionality for analytical devices. The wicking speed in the fabricated paper strips was slightly lower than that of non-processed paper, resulting from a reduced paper pore size after hot pressing. A colorimetric immunological assay was performed to demonstrate the protein binding capacity of the paper-based device after exposure to pressure and heat from the fabrication. Moreover, mixing in a two-dimensional paper-based device and flowing in a three-dimensional counterpart were thoroughly investigated, demonstrating that the paper devices from this fabrication process are potentially applicable as analytical devices for biomolecule detection. Fast, easy, and inexpensive parafilm hot press fabrication presents an opportunity for researchers to develop paper-based analytical devices in resource-limited environments.

Enclosed paper-based analytical devices: Concept, variety, and outlook

Paraquat is a highly toxic herbicide. Paraquat poisoning is often fatal and is an important public health threat in many places. The quick identification and timely initiation of treatment based on timely analysis of the paraquat concentration in urine/serum could improve the prognosis for patients. However, current paraquat concentration measurements are time-consuming and difficult to implement due to the expensive and bulky equipment required. To address these practical challenges, paper-based devices have emerged as alternative diagnostic tools for improving point-of-care testing. In this study, we demonstrate the successful use of a paper-based analytical device for the accurate detection of urine paraquat concentration. The developed paper-based analytical device employs colorimetric paraquat concentration measurements. The R2 value for the urine paraquat standard curve was 0.9989, with a dynamic range of 0–100 ppm. The limit of detection was 3.01 ppm. Two other optical-based approaches, Spectrochip and NanoDrop, were used for comparison. The results suggest that the developed paper-based analytical device is comparable to other colorimetric measurements, as determined by Bland–Altman analysis. The device was clinically validated using urine from six paraquat-poisoned patients. The results prove that the developed paper-based analytical device is accurate, easy-to-use, and efficient for urine paraquat concentration measurement, and may enable physicians to improve clinical management.

In this work, indoxyl-glucoside was used as the substrate to develop a cost-effective, paper-based analytical device for the fluorescent and colorimetric dual-mode detection of β-glucosidase activity through a smartphone. The β-glucosidase can hydrolyze the colorless substrate indoxyl-glucoside to release indoxyl, which will be self-oxidized to generate green products in the presence of oxygen. Meanwhile, the green products emit bright blue-green fluorescence under ultraviolet–visible light irradiation at 365 nm. Fluorescent or colorimetric images were obtained by a smartphone, and the red-green-blue channels were analyzed by the Adobe Photoshop to quantify the β-glucosidase activity. Under the optimum conditions, the relative fluorescent and colorimetric signals have a good linear relationship with the activity of β-glucosidase, in the range of 0.01–1.00 U/mL and 0.25–5.00 U/mL, and the limits of detection are 0.005 U/mL and 0.0668 U/mL, respectively. The activities of β-glucosidase in a crude almond sample measured by the fluorescent and colorimetric methods were 23.62 ± 0.53 U/mL and 23.86 ± 0.25 U/mL, respectively. In addition, the spiked recoveries of normal human serum and crude almond samples were between 87.5% and 118.0%. In short, the paper-based device, combined with a smartphone, can provide a simple, environmentally friendly, and low-cost method for the fluorescent and colorimetric dual-mode detection of β-glucosidase activity.

To transform from reactive to proactive healthcare, there is an increasing need for low-cost and portable assays to continuously perform health measurements. The paper-based analytical devices could be a potential fit for this need. To miniaturize the multiplex paper-based microfluidic analytical devices and minimize reagent use, a fabrication method with high resolution along with low fabrication cost should be developed. Here, we present an approach that uses a desktop pen plotter and a high-resolution technical pen for plotting high-resolution patterns to fabricate miniaturized paper-based microfluidic devices with hundreds of detection zones to conduct different assays. In order to create a functional multiplex paper-based analytical device, the hydrophobic solution is patterned on the cellulose paper and the reagents are deposited in the patterned detection zones using the technical pens. We demonstrated the effect of paper substrate thickness on the resolution of patterns by investigating the resolution of patterns on a chromatography paper with altered effective thickness. As the characteristics of the cellulose paper substrate such as thickness, resolution, and homogeneity of pore structure affect the obtained patterning resolution, we used regenerated cellulose paper to fabricate detection zones with a diameter as small as 0.8 mm. Moreover, in order to fabricate a miniaturized multiplex paper-based device, we optimized packing of the detection zones. We also showed the capability of the presented method for fabrication of 3D paper-based microfluidic devices with hundreds of detection zones for conducting colorimetric assays.

Airborne particulate matter (PM) pollution significantly impacts human health, but the cellular mechanisms of PM-induced toxicity remain poorly understood. A leading hypothesis on the effects of inhaled PM involves the generation of cellular oxidative stress. To investigate PM-induced oxidative stress, analytical methods have been developed to study the chemical oxidation of dithiothreitol (DTT) in the presence of PM. Although DTT readily reacts with several forms of reactive oxygen species, this molecule is not endogenously produced in biological systems. Glutathione (GSH), on the other hand, is an endogenous antioxidant that is produced throughout the body and is directly involved in combating oxidative stress in the lungs and other tissues. We report here a new method for measuring aerosol oxidative activity that uses silver nanoparticle (AgNP) aggregation coupled to glutathione (GSH) oxidation in a paper-based analytical device. In this assay, the residual reduced GSH from the oxidation of reduced GSH to its disulfide induces the aggregation of AgNPs on a paper-based analytical device, which produces a reddish-brown product. Two methods for aerosol oxidative reactivity are presented: one based on change in color intensity using a traditional paper-based techniques and one based on the length of the color product formed using a distance-based device. These methods were validated against traditional spectroscopic assays for DTT and GSH that employ Elman's reagent. No significant difference was found between the levels measured by all three GSH methods (our two paper-based devices and the traditional method) at the 95% confidence level. PM reactivity towards GSH was less than towards DTT most likely due to the difference in the oxidation potential between the two molecules.

Recent advances in microfluidic paper-based electrochemiluminescence analytical devices for point-of-care testing applications

The need for providing rapid and, possibly, on-the-spot analytical results in the case of intoxication has prompted researchers to develop rapid, sensitive, and cost-effective methods and analytical devices suitable for use in nonspecialized laboratories and at the point of need (PON). In recent years, the technology of paper-based microfluidic analytical devices (μPADs) has undergone rapid development and now provides a feasible, low-cost alternative to traditional rapid tests for detecting harmful compounds. In fact, µPADs have been developed to detect toxic molecules (arsenic, cyanide, ethanol, and nitrite), drugs, and drugs of abuse (benzodiazepines, cathinones, cocaine, fentanyl, ketamine, MDMA, morphine, synthetic cannabinoids, tetrahydrocannabinol, and xylazine), and also psychoactive substances used for drug-facilitated crimes (flunitrazepam, gamma-hydroxybutyric acid (GHB), ketamine, metamizole, midazolam, and scopolamine). The present report critically evaluates the recent developments in paper-based devices, particularly in detection methods, and how these new analytical tools have been tested in forensic and clinical toxicology, also including future perspectives on their application, such as multisensing paper-based devices, microfluidic paper-based separation, and wearable paper-based sensors.

Paper-based analytical devices (PADs) have been largely applied for detection of different health-related compounds in clinical and food samples as well as in water and pharmaceutical quality control. While the majority of paper-based analytical technologies rely on colorimetric detection due to its ease of use and seemingly simple data interpretation, this mode of detection often suffers from limited sensitivity, small linear ranges and/or high detection limits. Electrochemical paper-based analytical devices (ePADs) present a promising alternative, offering higher sensitivity and selectivity by directly measuring electrical signals produced in chemical reactions. Building on this capability, bioreagents such as enzymes and antibodies serve as essential components of ePADs, delivering outstanding specificity and sensitivity in detecting target analytes. However, bioreagents’ application is hindered by limited stability and shelf-life in paper-based devices. While commercial electrochemical sensors, typically based on durable materials like polymers or plastics, remain stable for 18-24 months, paper-based electrochemical biosensors exhibit limited stability, typically below 2 months when refrigerated at 4 ℃.This issue is intensified during shipping, when fluctuations in temperature further compromise device performance and stability.To address this challenge, this research focuses on enhancing the thermal stability of bioreagents in ePADs by incorporating stabilizing agents into the device design. The present study focuses on ePADs for glucose detection as a model analyte with well-characterized detection properties. The ePADs were fabricated as three-electrode systems using screen-printed carbon ink and evaluated through cyclic voltammetry with potassium ferri/ferrocyanide to assess the functionality and reproducibility of the devices, which demonstrated acceptable reproducibility. The results for glucose detection demonstrated the ability to detect concentrations across a range of 0–20 mM with high sensitivity, a wide linear range, and a low limit of detection. Stability tests were further conducted at temperatures ranging from room temperature to 50 °C to evaluate bioreagent performance under thermal stress.

Sensing of body fluid hormones using paper-based analytical devices

Automation Highlights from the Literature

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

A disposable dual-mode electrochemical/colorimetric paper-based analytical device for simultaneous detection of hydroquinone and mercury ion.

Viscosity measurements utilizing a fast-flow microfluidic paper-based device

Paper-based analytical device for colorimetric detection of Cu2+ in Brazilian sugarcane spirits by digital image treatment