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Background: The demand for point-of-care testing (POCT) devices has rapidly grown since they offer immediate test results with ease of use, makingthem suitable for home self-testing patients and caretakers. However, the POCT development has faced the challenges of increased cost and limited resources. Therefore, the paper substrate as a low-cost material has been employed to develop a cost-effective POCT device, known as “Microfluidic paper-based analytical devices (μPADs)”. This device is gaining attention as a promising tool for medicinal diagnostic applications owing to its unique features of simple fabrication, low cost, enabling manipulation flow (capillarydriven flow), the ability to store reagents, and accommodating multistep assay requirements. Objective: This review comprehensively examines the fabrication methods and device designs (2D/3D configuration) and their advantages and disadvantages, focusing on updated μPADs applications for motif identification. Methods: The evolution of paper-based devices, starting from the traditional devices of dipstick and lateral flow assay (LFA) with μPADs, has been described. Patterned structure fabrication of each technique has been compared among the equipment used, benefits, and drawbacks. Microfluidic device designs, including 2D and 3D configurations, have been introduced as well as their modifications. Various designs of μPADs have been integrated with many powerful detection methods such as colorimetry, electrochemistry, fluorescence, chemiluminescence, electrochemiluminescence, and SER-based sensors for medicinal diagnosis applications. Conclusion: The μPADs potential to deal with commercialization in terms of the state-of-the-art of μPADs in medicinal diagnosis has been discussed. A great prototype, which is currently in a reallife application breakthrough, has been updated.

A novel platform of microfluidic paper-based analytical devices (μPADs) for dual detection of bisphenol A (BPA), a model analyte, was fabricated using an electronic digital plotter to create the stacked layer of μPADs and generate the lateral-flow channel without using an external pump. Two detection techniques, including electrochemical detection and laser desorption ionization mass spectrometric detection (LDI-MS), were used complementarily to improve the precision in the detection of BPA. The fluid sample was delivered to both detection zones by the capillary action, automatically generated from the fabricated microfluidic device. For an electrochemical sensor, two ballpoint pens filled with silver nanoparticles (AgNPs) and multiwall carbon nanotube (MWCNT) ink were used to print onto the paper with a contact printing method using a digital plotter. To further improve the sensitivity, zinc oxide (ZnO) was used to modify both electrochemical and LDI-MS detection zones. For BPA detection, high electrocatalytic properties and strong UV absorption of ZnO promote the electron transfer in the electrochemical sensor and ionization efficiency in LDI-MS with low interferences compared with a conventional organic matrix. Under optimal conditions, this platform showed a dual detection capability for BPA with a detection limit of 0.35 μM for electrochemical detection and with an ultralow detection limit of 0.01 pM for LDI-MS. This novel platform might be very useful for trace analyses requiring high precision detection of various analytes.

Nanomaterials have been explored as alternative matrices in MALDI-MS to overcome some of the limitations of conventional matrices. Recently, we demonstrated a new means by which nanomaterials can improve peptide ionization and detection in MALDI-MS analyses by exploiting the tendency of nanomaterials to form "coffee rings" upon drying from liquids. In the current work, we investigate how nanomaterial size and composition affect the signal enhancement of peptides through the coffee-ring effect. From studies of eight different types of nanomaterials ranging in size and composition, we find that most nanomaterials can provide signal enhancement ranging from 2- to 10-fold for individual peptides, as long as a coffee ring is formed. However, when a mixture of peptides is present in a sample, the signal enhancement is the greatest for peptides whose net charge is complementary to the nanomaterial's surface charge. These results suggest that careful design of NM surface properties could allow for selective, enhanced MALDI-MS detection of specific peptides in complex mixtures.

A label-free electrochemical immunosensor based on polyaniline (PANI) micellar electrode was firstly fabricated for direct AMH detection. To control the size regularity of PANI, a micelle-based method using ammonium peroxydisulfate (APS) as a reducing agent was employed in the polymerization process. The Anti-AMH antibodies were readily immobilized onto PANI via peptide bond to enhance the sensor specificity and sensitivity. This sensor was applied for the detection of AMH, an ovarian response indicator in female related to residual eggs during a woman's monthly cycle. The sensor performances were systematically investigated by differential pulse voltammetry. The anodic peak current decreases with the increase of AMH concentration owing to blocking of electron transfer by AMH. Under the optimal conditions, this sensor offers high sensitivity with a low detection limit of 0.1 ng mL−1 and a wide linear range of 0.1–4 ng mL−1, which is sensitive enough to indicate the ability to produce eggs during a woman's monthly cycle. Furthermore, this system requires lower sample volume (5 μL), while offers the simple fabrication with low cost and no synthetic challenge and faster analysis compared with a standard ELISA. Ultimately, this sensor was successfully applied for the detection of AMH in human serum with satisfactory results. Thus, it might be an alternative tool for AMH screening in clinical setting.

Zinc Oxide (ZnO) nanorods were synthesized by using zinc acetate as a precursor via thermal decomposition method and then used to prepare ZnO@G nanocomposite by facile room-temperature approach using a colloidal coagulation effect. The as prepared ZnO@G nanocomposite was applied to modify the working electrode surface in an electrochemical sensor of Cd2+ and Pb2+for the first time. The morphologies of ZnO and ZnO@G nanocomposite were characterized by transmission electron microscopy, that exhibits the nanorod shape with an average diameter of 89 ± 18 nm, uniformly dispersed and intercalated between each G layer. Furthermore, the important factors affecting on the sensor sensitivity, including ZnO@G ratio, ZnO@G concentration, Bi3+ concentration and electrochemical parameters were systematically investigated using square-wave anodic stripping voltammetry. Under the optimal conditions, a linear range was found to be 10–200 μg L−1 and the detection limits were 0.6 and 0.8 μg L-1 for Cd2+ and Pb2+, respectively. Eventually, it was successfully used for the simultaneous detection of Cd2+ and Pb2+ in real wastewater samples and the results corresponded well with a standard inductively coupled plasma-optical emission spectroscopy.

A novel label-free electrochemical immunosensor for neutrophil gelatinase-associated lipocalin (NGAL) detection has been developed. The immunosensor has been constructed by immobilization of NGAL capture antibodies to electropolymerized aniline deposited on top of an electrosprayed graphene/polyaniline (G/PANI) modified screen printed carbon electrode. Electrospraying of G/PANI increases the electrode surface area while electropolymerization of aniline increases the number of amino groups (-NH2) for antibody immobilization. The factors affecting the sensor sensitivity (i.e. aniline concentration, scan number and scan rate of electropolymerization) have been optimized. In a prior report, Kannan et al. reported a broad oxidation peak in cyclic voltammetry upon the binding between NGAL with its antibody. In this study, a dramatic increase (58-fold) in the oxidation current upon the binding between NGAL and its antibody is obtained when compared to an unmodified electrode, verifying a substantial improvement in the electrochemical sensitivity of this system. Under optimal conditions, this system exhibits high sensitivity with a limit of detection (LOD) of 21.1ngmL-1, wide linearity (50-500ngmL-1) and high specificity toward NGAL detection from small samples (10μL). As an example application, the sensor is tested for the detection of NGAL in human urine, and the results correspond well with the values obtained from a standard ELISA. Compared to the ELISA method, our system requires less analysis time (≤30min/sample), less sample and less operating cost.