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Microfluidic-based biosensors have been developed for their precise automatic reaction control. However, these biosensors require external devices that are difficult to transport and use. To overcome this disadvantage, our group made an easy-to-use, cheap, and light pumpless three-dimensional photo paper–based microfluidic analytical device (3D-μPAD; weight: 1.5 g). Unlike conventional paper-based microfluidic analytical devices, the 3D-μPAD can be used to control fluid flow in a 3D manner, thus allowing sophisticated multi-step reaction control. This device can control fluid flow speed and direction accurately using only the capillary-driven flow without an external device like a pump. The flow speed is controlled by the width of the microfluidic channel and its surface property. In addition, fluid speed control and 3D-bridge structure enable the control of fluid flow direction. Using these methods, multi-step enzyme-linked immunosorbent assay (ELISA) can be done automatically in sequence by injecting solutions (sample, washing, and enzyme’s substrate) at the same time in the 3D-μPAD. All the steps can be performed in 14 min, and data can be analyzed immediately. To test this device, thioredoxin-1 (Trx-1), a biomarker of breast cancer, is used as the target. In the 3D-μPAD, it can detect 0–200 ng/mL of Trx-1, and the prepared 3D-μPAD Trx-1 sensor displays excellent selectivity. Moreover, by analyzing the concentration of Trx-1 in real patients and healthy individuals’ blood serum samples using the 3D-μPAD, and comparing results to ELISA, it can be confirmed that the 3D-μPAD is a good tool for cancer diagnosis.

Regulating the fluid flow in microfluidic devices enables a wide range of assay protocols for analytical applications. A programmable, photo-paper-based microfluidic device fabricated by using a method of cutting and laminating, followed by printing, is reported. The flow distance of fluid in the photo-paper-based channel was linearly proportional to time. By printing silver nanoparticle (AgNP) and poly[4,5-difluoro-2,2-bis(trifluoromethyl)-1,3-dioxole-co-tetrafluoroethylene] (PTFE) patterns on the surface of a photo-paper-based channel, we were able to either increase or decrease the fluid flow in the fabricated microfluidic devices, while maintaining the linearity in the flow distance-time relation. In comparison to the speed of fluid flow in a pristine channel, by using hydrophilic AgNP patterns, we were able to increase the speed in the channel by up to 15 times while we were able to slow the speed by a factor of 3 when using hydrophobic PTFE dots. We then further demonstrated a single-step protocol for detecting glucose and a multi-step protocol for detecting methyl paraoxon (MPO) with our methods in photo-paper-based microfluidic devices. This approach can lead to improved fluid handling techniques to achieve a wide range of complex, but programmable, assays without the need for any additional auxiliary devices for automated operation.

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.

Microfluidic-based biosensors have been developed for their precise automatic reaction control. However, these biosensors require external devices that are difficult to transport and use. To overcome this disadvantage, our group made an easy-to-use, cheap, and light pumpless three-dimensional photo paper-based microfluidic analytical device (3D-μPAD; weight: 1.5 g). Unlike conventional paper-based microfluidic analytical devices, the 3D-μPAD can be used to control fluid flow in a 3D manner, thus allowing sophisticated multi-step reaction control. This device can control fluid flow speed and direction accurately using only the capillary-driven flow without an external device like a pump. The flow speed is controlled by the width of the microfluidic channel and its surface property. In addition, fluid speed control and 3D-bridge structure enable the control of fluid flow direction. Using these methods, multi-step enzyme-linked immunosorbent assay (ELISA) can be done automatically in sequence by injecting solutions (sample, washing, and enzyme's substrate) at the same time in the 3D-μPAD. All the steps can be performed in 14 min, and data can be analyzed immediately. To test this device, thioredoxin-1 (Trx-1), a biomarker of breast cancer, is used as the target. In the 3D-μPAD, it can detect 0-200 ng/mL of Trx-1, and the prepared 3D-μPAD Trx-1 sensor displays excellent selectivity. Moreover, by analyzing the concentration of Trx-1 in real patients and healthy individuals' blood serum samples using the 3D-μPAD, and comparing results to ELISA, it can be confirmed that the 3D-μPAD is a good tool for cancer diagnosis.

A simple programmable contact printing method using ballpoint pens with silver nanoparticle (AgNP) and carbon nanotube (CNT) ink and a digital plotter were developed for quick patterning of electrodes on paper. This printing method enables sequential and programmable printing with two different inks and with ink consisting of high viscosity materials and is amenable to reproducibility of printed electrodes and customized designs. With this printing method, AgNP and CNT patterns with low electrical resistance and high density of the material can be printed. Using these AgNP and CNT inks, we fabricated disposable electrochemical sensors (ECSs) on paper. The ECSs were successfully used to detect glucose at various concentrations from 0 to 15 mM. The characteristics of the printed AgNP and CNT patterns, such as the printing resolution, surface morphology, and electrical properties, were also studied. The proposed contact printing method opens an avenue for printing paper-based electronics and devices.

Summary Microbial rhodopsin is a retinal protein that functions as an ion pump, channel, and sensory transducer, as well as a light sensor, as in biosensors and biochips. Tara76 rhodopsin is a typical proton-pumping rhodopsin that exhibits strong stability against extreme pH, detergent, temperature, salt stress, and dehydration stress and even under dual and triple conditions. Tara76 rhodopsin has a thermal stability approximately 20 times higher than that of thermal rhodopsin at 80°C and is even stable at 85°C. Tara76 rhodopsin is also stable at pH 0.02 to 13 and exhibits strong resistance in detergent, including Triton X-100 and SDS. We tested the current flow that electrical current flow across dried proteins on the paper at high temperatures using an electrode device, which was measured stably from 25°C up to 120°C. These properties suggest that this Tara76 rhodopsin is suitable for many applications in the fields of bioengineering and biotechnology., Graphical abstract, Highlights • Tara76 rhodopsin showed strong stability against pH, temperature, detergents, dried, and salt • Tara76 rhodopsin is stable under dual and triple stress conditions • Electrical properties were measured at high temperature using the membrane protein • The hydrophobicity of helix E and the Schiff base influence the stability of rhodopsin, Microbiology; Biotechnology; Bioelectronics; Biophysics

The production of energetic crystalized micro-patterns by using one-step printing has become a recent trend in energetic materials engineering. We report a direct ink writing (DIW) approach in which micro-scale energetic composites composed of 1,3,5-trinitro-1,3,5-triazinane (RDX) crystals in selected ink formulations of a cellulose acetate butyrate (CAB) matrix are produced based on a direct phase transformation from organic, solvent-based, all-liquid ink. Using the formulated RDX ink and the DIW method, we printed crystalized RDX micro-patterns of various sizes and shapes on silicon wafers. The crystalized RDX micro-patterns contained single crystals on pristine Si wafers while the micro-patterns containing dendrite crystals were produced on UV-ozone (UVO)-treated Si wafers. The printing method and the formulated all-liquid ink make up a simple route for designing and printing energetic micro-patterns for micro-electromechanical systems.

Microfluidic paper-based analytical devices (µPADs) have become promising tools offering various analytical applications for chemical and biological assays at the point-of-care (POC). Compared to traditional microfluidic devices, µPADs offer notable advantages; they are cost-effective, easily fabricated, disposable, and portable. Because of our better understanding and advanced engineering of µPADs, multistep assays, high detection sensitivity, and rapid result readout have become possible, and recently developed µPADs have gained extensive interest in parallel analyses to detect biomarkers of interest. In this review, we focus on recent developments in order to achieve µPADs with high-throughput capability. We discuss existing fabrication techniques and designs, and we introduce and discuss current detection methods and their applications to multiplexed detection assays in relation to clinical diagnosis, drug analysis and screening, environmental monitoring, and food and beverage quality control. A summary with future perspectives for µPADs is also presented.

The effects of an immiscible, lubricating polydimethylsiloxane fluid, referred to as silicone oil, on the static deformation and on the dynamic motion of a water drop on paper induced by electrowetting were investigated. The deformation of a drop on a hydrophobic film of amorphous fluoropolymers top-coated with less hydrophobic silicone oil was much more predictable, reversible and reproducible than on the uncoated surface. In the dynamic tribological experiment for a sliding drop along an inclined surface, a significant decrease in the friction coefficient, with an unexpected dependency of the contact area, was observed. Based on the curve fitting analysis, the shear stress and the net friction force were estimated quantitatively. Because of the tribological effect and the reduced shear friction force of the oil film, the static and the dynamic electrowetting states of the water drop were enhanced.

Recent advanced paper-based microfluidic devices provide an alternative technology for the detection of biomarkers by using affordable and portable devices for point-of-care testing (POCT). Programmable paper-based microfluidic devices enable a wide range of biomarker detection with high sensitivity and automation for single- and multi-step assays because they provide better control for manipulating fluid samples. In this review, we examine the advances in programmable microfluidics, i.e., paper-based continuous-flow microfluidic (p-CMF) devices and paper-based digital microfluidic (p-DMF) devices, for biomarker detection. First, we discuss the methods used to fabricate these two types of paper-based microfluidic devices and the strategies for programming fluid delivery and for droplet manipulation. Next, we discuss the use of these programmable paper-based devices for the single- and multi-step detection of biomarkers. Finally, we present the current limitations of paper-based microfluidics for biomarker detection and the outlook for their development.

A spoof fingerprint was fabricated on paper and applied for a spoofing attack to unlock a smartphone on which a capacitive array of sensors had been embedded with a fingerprint recognition algorithm. Using an inkjet printer with an ink made of carbon nanotubes (CNTs), we printed a spoof fingerprint having an electrical and geometric pattern of ridges and furrows comparable to that of the real fingerprint. With this printed spoof fingerprint, we were able to unlock a smartphone successfully; this was due to the good quality of the printed CNT material, which provided electrical conductivities and structural patterns similar to those of the real fingerprint. This result confirms that inkjet-printing CNTs to fabricate a spoof fingerprint on paper is an easy, simple spoofing route from the real fingerprint and suggests a new method for outputting the physical ridges and furrows on a two-dimensional plane.

In order to fabricate a digital microfluidic (DMF) chip, which requires a patterned array of electrodes coated with a dielectric film, we explored two simple methods: Ballpoint pen printing to generate the electrodes, and wrapping of a dielectric plastic film to coat the electrodes. For precise and programmable printing of the patterned electrodes, we used a digital plotter with a ballpoint pen filled with a silver nanoparticle (AgNP) ink. Instead of using conventional material deposition methods, such as chemical vapor deposition, printing, and spin coating, for fabricating the thin dielectric layer, we used a simple method in which we prepared a thin dielectric layer using pre-made linear, low-density polyethylene (LLDPE) plastic (17-&mu, m thick) by simple wrapping. We then sealed it tightly with thin silicone oil layers so that it could be used as a DMF chip. Such a treated dielectric layer showed good electrowetting performance for a sessile drop without contact angle hysteresis under an applied voltage of less than 170 V. By using this straightforward fabrication method, we quickly and affordably fabricated a paper-based DMF chip and demonstrated the digital electrofluidic actuation and manipulation of drops.

The printing and modular fabrication of a paper-based active microfluidic lab on a chip implemented with electrochemical sensors (ECSs) is developed and integrated on a portable electrical control system. The electrodes of a chip plate for active electrowetting actuation of digital drops and an ECS for multiple analysis assays are fabricated by affordable printing techniques. For enhanced sensitivity of the sensor, the working electrode is modified through the electrochemical method, namely by reducing graphene with voltammetry and coating gold nanoparticles by amperometry. Detachable sensor and absorber modules are assembled modularly on an open chip plate, forming various novel hybridized open–closed chip formats. By varying the coupled or decoupled sensor modules, excellent detection of three diagnostic biological molecules is demonstrated (glucose, dopamine, and uric acid in human serum). With a newly designed portable power supply and wireless control system, the active paper-based chip platform can be utilized as an advanced point-of-care device for multiple assays in digital microfluidics.