A proof-of-concept for a label-free and reagentless optical biosensing platform based on nanostructured porous silicon (PSi) and aptamers is presented in this work. Aptamers are oligonucleotides (single-stranded DNA or RNA) that can bind their targets with high affinity and specificity, making them excellent recognition elements for biosensor design. Here we describe the fabrication and characterization of aptamer-conjugated PSi biosensors, where a previously characterized his-tag binding aptamer (6H7) is used as model system. Exposure of the aptamer-functionalized PSi to the target proteins as well as to complex fluids (i.e., bacteria lysates containing target proteins) results in robust and well-defined changes in the PSi optical interference spectrum, ascribed to specific aptamer-protein binding events occurring within the nanoscale pores, monitored in real time. The biosensors show exceptional stability and can be easily regenerated by a short rinsing step for multiple biosensing analyses. This proof-of-concept study demonstrates the possibility of designing highly stable and specific label-free optical PSi biosensors, employing aptamers as capture probes, holding immense potential for application in detection of a broad range of targets, in a simple yet reliable manner.

One of the basic operations in microfluidic systems for biological and chemical applications is the rapid mixing of different fluids. However, flow profiles in microfluidic systems are laminar, which means molecular diffusion is the only mixing effect. Therefore, mixing structures are crucial to enable more efficient mixing in shorter times. Since traditional microfabrication methods remain laborious and expensive, 3D printing has emerged as a potential alternative for the fabrication of microfluidic devices. In this work, five different passive micromixers known from literature are redesigned in comparable dimensions and manufactured using high‐definition MultiJet 3D printing. Their mixing performance is evaluated experimentally, using sodium hydroxide and phenolphthalein solutions, and numerically via computational fluid dynamics. Both experimental and numerical analysis results show that HC and Tesla‐like mixers achieve complete mixing after 0.99 s and 0.78 s, respectively, at the highest flow rate (Reynolds number (Re) = 37.04). In comparison, Caterpillar mixers exhibit a lower mixing rate with complete mixing after 1.46 s and 1.9 s. Furthermore, the HC mixer achieves very good mixing performances over all flow rates (Re = 3.7 to 37.04), while other mixers show improved mixing only at higher flow rates.

In this work, we report digital loop-mediated isothermal amplification (LAMP) or reverse-transcription LAMP (RT-LAMP) on a commercial membrane, without the need for complex chip fabrication or use of specialized equipment. Due to the pore size distribution, the theoretical error for digital LAMP on these membranes was analyzed, using a combination of Random Distribution Model and Multivolume Theory. A facile peel-off process was developed for effective droplet formation on the commercial track-etched polycarbonate (PCTE) membrane. Each pore functions as an individual nanoreactor for single DNA amplification. Absolute quantification of bacteria genomic DNA was realized with a dynamic range from 11 to 1.1 × 105 copies/μL. One-step digital RT-LAMP was also successfully performed on the membrane for the quantification of MS2 virus in wastewater. With the introduction of new probes, the positive pores can be easily distinguished from negative ones with 100 times difference in fluorescence intensities. Finally, the cost of a disposable membrane is less than $0.10/piece, which, to the best of our knowledge, is the most inexpensive way to perform digital LAMP. The membrane system offers opportunities for point-of-care users or common laboratories to perform digital quantification, single cell analysis, or other bioassays in an inexpensive, flexible, and simplified way.