Innovative Hydrophobic Valve Allows Complex Liquid Manipulations in a Self-Powered Channel-Based Microfluidic Device.

We present an innovative, simple, and versatile hydrophobic valve enabling all-important complex liquid manipulations on self-powered, channel-based microfluidic devices and as such being extremely valuable for the design of highly demanding point-of-care (POC) platforms. The presented hydrophobic valve is made of filter paper treated with a fluorinated compound (i.e., Aquapel) and shows both superhydrophobic properties (contact angle up to 155°) and high resistance to liquid pressure (up to 9 kPa), while retaining gas permeability and utter fabrication simplicity. Whereas this valve can be integrated in any channel-based system and can be used both as a vent, to delay liquid displacement on chip, or as a barrier, to stop the liquid flow in a certain direction, in this work we demonstrate some of its capacities by combining it with our in house developed self-powered SIMPLE and iSIMPLE platforms. First, we integrated it with the infusion iSIMPLE pump, thus generating completely fail-proof activation regardless of how the operator is actuating the system. Second, we used hydrophobic valves as both barrier and vent in the same microfluidic chip, which allowed the combination of two SIMPLE pumps for splitting one sample in two parallel channels. This attribute is fundamental for achieving multiplex analysis on completely autonomous microfluidic platforms. Finally, we achieved an unprecedented liquid manipulation for a self-powered microfluidic platform, namely, shuttling of liquid, after a single user activation by combining for the first time SIMPLE and iSIMPLE with the developed hydrophobic vent and barrier, all in a single chip. These results convincingly demonstrated that the developed hydrophobic valve combined with SIMPLE/iSIMPLE presents an essential building block for an ideal POC system, which is self-powered, inexpensive, and robust and can perform complex bioassays upon a single user activation.

Teflon-on-Glass Molding Enables High-Throughput Fabrication of Hydrophilic-in-Hydrophobic Microwells for Bead-Based Digital Bioassays.

In recent years, Teflon-on-glass microwells have been successfully implemented in bead-based digital bioassays for the sensitive detection of single target molecules. Their hydrophilic-in-hydrophobic (HIH) nature enables the isolation and analysis of individual beads, carrying the target molecules, which can be further manipulated accurately through optical tweezer (OT) setups. However, these Teflon HIH-microwell platforms are conventionally fabricated through a complex, time-consuming and labor-intensive dry lift-off procedure which involves a series of major steps, limiting the up-scaling potential of these platforms. Alternative Teflon-based microwell fabrication methods have been extensively explored in literature but they preclude the generation of hydrophobic wells with hydrophilic bottom, thereby hampering the bioassay performance. Here, we present a new Teflon-on-glass molding method for the high throughput fabrication of hydrophilic-in-hydrophobic (HIH) microwell arrays, able to empower bead-based digital bioassays. Microwells 2.95 µm in depth and 3.86 µm in diameter were obtained to host individual beads. In these microwell arrays, sealing of reagents was demonstrated with an efficiency of 100% and seeding of superparamagnetic beads was achieved with an efficiency of 99.6%. The proposed method requires half as many steps when compared to the traditional dry lift-off process, is freely scalable and has the potential to be implemented in different bead-based bioassay applications.

Sub-femtomolar detection of DNA and discrimination of mutant strands using microwell-array assisted digital enzyme-linked oligonucleotide assay.

Detection methods that do not rely on the amplification of DNA but can reach sensitivity, specificity and throughput of gold standard methods, such as qPCR, have been extensively explored in recent years. Here, we present a hydrophilic-in-hydrophobic (HIH)-microwell array platform that empowers a panel of different amplification-free DNA bioassays: digital enzyme-linked oligonucleotide assay (ELONA), ligation-assisted (LA) digital ELONA and so-called 'analog' bioassays. We developed all three bioassays by using magnetic beads for capturing DNA target, followed by hybridization of enzyme-labelled detection probes and sealing of the built complexes into the femtoliter HIH microwells to achieve the fluorescent readout of single DNA molecules. With the optimized digital ELONA bioassay, we successfully detected 97 and 200 nt-long ssDNA molecules down to 68 and 92 aM, respectively, demonstrating extremely high sensitivity of the bioassay and its flexibility towards targets of different lengths. Importantly, we also proved that the same bioassay concept was suited to detect substantially higher concentrations of ssDNA (up to picomolar levels) by quantifying the total fluorescent intensity rather than counting fluorescent events for digital quantification. Finally, we advanced this concept towards LA digital ELONA capable of differentiating wildtype strands from those carrying single-point mutations even when the former were constituting only 1% of the DNA mixture and were present at 2 fM concentration. In conclusion, the developed platform showed remarkably high sensitivity, specificity and versatility for amplification-free detection of DNA and as such can be valuable for numerous applications in medical diagnostics, gene analysis, food safety and environmental monitoring.

Digital ELISA for the quantification of attomolar concentrations of Alzheimer's disease biomarker protein Tau in biological samples.

The close correlation between Tau pathology and Alzheimer's disease (AD) progression makes this protein a suitable biomarker for diagnosis and monitoring of the disorder evolution. However, the use of Tau in diagnostics has been hampered, as it currently requires collection of cerebrospinal fluid (CSF), which is an invasive clinical procedure. Although measuring Tau-levels in blood plasma would be favorable, the concentrations are below the detection limit of a conventional ELISA. In this work, we developed a digital ELISA for the quantification of attomolar protein Tau concentrations in both buffer and biological samples. Individual Tau molecules were first captured on the surface of magnetic particles using in-house developed antibodies and subsequently isolated into the femtoliter-sized wells of a 2 × 2 mm2 microwell array. Combination of high-affinity antibodies, optimal assay conditions and a digital quantification approach resulted in a 24 ±â€¯7 aM limit of detection (LOD) in buffer samples. Additionally, a dynamic range of 6 orders of magnitude was achieved by combining the digital readout with an analogue approach, allowing quantification from attomolar to picomolar levels of Tau using the same platform. This proves the compatibility of the presented assay with the wide range of Tau concentrations encountered in different biological samples. Next, the developed digital assay was applied to detect total Tau levels in spiked blood plasma. A similar LOD (55 ±â€¯29 aM) was obtained compared to the buffer samples, which was 5000-fold more sensitive than commercially available ELISAs and even outperformed previously reported digital assays with 10-fold increase in sensitivity. Finally, the performance of the developed digital ELISA was assessed by quantifying protein Tau in three clinical CSF samples. Here, a high correlation (i.e. Pearson coefficient of 0.99) was found between the measured percentage of active particles and the reference protein Tau values. The presented digital ELISA technology has great capacity in unlocking the potential of Tau as biomarker for early AD diagnosis.

Digital Microfluidics Assisted Sealing of Individual Magnetic Particles in Femtoliter-Sized Reaction Wells for Single-Molecule Detection.

Digital microfluidics has emerged in the last years as a promising liquid handling technology for a variety of applications. Here, we describe in detail how to build up an electrowetting-on-dielectric-based digital microfluidic chip with unique advantages for performing single-molecule detection. We illustrate how superparamagnetic particles can be printed with very high loading efficiency (over 98 %) and single-particle resolution in the microwell array patterned in the Teflon-AF® surface of the grounding plate of the chip. Finally, the potential of the device for its application to single-molecule detection is demonstrated by the ultrasensitive detection of the biotinylated enzyme ß-Galactosidase captured on streptavidin-coated particles in the described platform.