Ultrasensitive amplification-free detection of circulating miRNA via droplet-based processing of SERS tag-miRNA-magnetic nanoparticle sandwich nanocomplexes on a paper-based electrowetting-on-dielectric platform.

MicroRNAs (miRNAs) have emerged as a promising class of biomarkers for early detection of various cancers, including ovarian cancer. However, quantifying miRNAs in human blood samples is challenging owing to the issues of sensitivity and specificity. In this study, hsa-miR-200a-3p of the miR-200a sub-family, which is a biomarker of ovarian cancer, was used as the analyte to demonstrate the analytical capability of an integrated biosensing platform using an extremely sensitive surface-enhanced Raman scattering (SERS) nanotag-nanoaggregate-embedded beads (NAEBs), magnetic nanoparticles (MNPs), a pair of highly specific locked nucleic acid (LNA) probes, and a semi-automated paper-based electrowetting-on-dielectric (pEWOD) device to provide labor-less and thorough sample cleanup and recovery. A sandwich approach where NAEBs are modified by one LNA-1 probe and MNPs are modified by another LNA-2 probe was applied. Then, the target analyte miRNA-200a-3p was introduced to form a sandwich nanocomplex through hybridization with the pair of LNA probes. The pEWOD device was used to achieve short cleanup time and good recovery of the nanocomplex, bringing the total analysis time to less than 30 min. The detection limit of this approach can reach 0.26 fM through SERS detection. The versatility of this method without the need for RNA extraction from clinical samples is expected to have good potential in detecting other miRNAs.

A yceI Gene Involves in the Adaptation of Ralstonia solanacearum to Methyl Gallate and Other Stresses.

Ralstonia solanacearum is a plant-pathogenic bacterium causing plant bacterial wilt, and can be strongly inhibited by methyl gallate (MG). Our previous transcriptome sequencing of MG-treated R. solanacearum showed that the yceI gene AVT05_RS03545 of Rs-T02 was up-regulated significantly under MG stress. In this study, a deletion mutant (named DM3545) and an over-expression strain (named OE3545) for yceI were constructed to confirm this hypothesis. No significant difference was observed among the growth of wild-type strain, DM3545 and OE3545 strains without MG treatment. Mutant DM3545 showed a lower growth ability than that of the wild type and OE3545 strains under MG treatment, non-optimal temperature, or 1% NaCl. The ability of DM3545 for rhizosphere colonization was lower than that of the wild-type and OE3545 strains. The DM3545 strain showed substantially reduced virulence toward tomato plants than its wild-type and OE3545 counterpart. Moreover, DM3545 was more sensitive to MG in plants than the wild-type and OE3545 strains. These results suggest that YceI is involved in the adaptability of R. solanacearum to the presence of MG and the effect of other tested abiotic stresses. This protein is also possibly engaged in the virulence potential of R. solanacearum.

Integration of a Thermoelectric Heating Unit with Ionic Wind-Induced Droplet Centrifugation Chip to Develop Miniaturized Concentration Device for Rapid Determination of Salmonella on Food Samples Using Antibody-Functionalized SERS Tags.

When a centrifugation-enriched sample of 100 µL containing the surface-enhanced Raman scattering (SERS) tag-bound bacteria (Salmonella in this study) is siphoned onto a glass slide next to an embedded thermoelectric heating chip, such a sessile droplet is quickly evaporated. As the size of the sample droplet is significantly reduced during the heating process, ionic wind streams from a corona discharge needle, stationed above the sample, sweep across the liquid surface to produce centrifugal vortex flow. Tag-bound Salmonella in the sample are then dragged and trapped at the center of droplet bottom. Finally, when the sample is dried, unlike the "coffee ring" effect, the SERS tag-bound Salmonella is concentrated in one small spot to allow sensitive detection of a Raman signal. Compared with our previous electrohydrodynamic concentration device containing only a corona discharge needle, this thermoelectric evaporation-assisted device is more time-effective, with the time of concentrating and drying about 100 µL sample reduced from 2 h to 30 min. Hence, sample throughput can be accelerated with this device for practical use. It is also more sensitive, with SERS detection of a few cells of Salmonella in neat samples achievable. We also evaluated the feasibility of using this device to detect Salmonella in food samples without performing the culturing procedures. Having spiked a few Salmonella cells into ice cubes and lettuce leaves, we use filtration and ultracentrifugation steps to obtain enriched tag-bound Salmonella samples of 200 µL. After loading an aliquot of 100 µL of sample onto this concentration device, the SERS tag signals from samples of 100 g ice cubes containing two Salmonella cells and 20 g lettuce leaf containing 5 Salmonella cells can be successfully detected.

Development of microfluidic concentrator using ion concentration polarization mechanism to assist trapping magnetic nanoparticle-bound miRNA to detect with Raman tags.

MicroRNAs (miRNAs) are small noncoding single-stranded ribonucleic acid molecules. This type of endogenous oligonucleotide could be secreted into the circulation and exist stably. The detection of specific miRNAs released by cancer cells potentially provides a noninvasive means to achieve early diagnosis and prognosis of cancers. However, the typical concentration of miRNAs in blood is below the ultratrace level. This study uses a simple thermoplastic microfluidic concentration device based on an ion concentration polarization mechanism to perform enrichment and cleanup and Raman sensing beads to determine miRNA quantitatively. One sample solution containing target miRNA molecules having been hybridized with two nucleotide probes, where one probe is on a Raman tag of a nanoaggregate embedded bead (NAEB) and the other probe is on a magnetic nanoparticle (MNP), is first filled into the device. When an external field is applied across a cation exchange membrane stationed in the middle conduit of the device, the MNP-miRNA-NAEB complexed particles are enriched near the membrane edge of the cathode side. The concentrated complexed particles are further trapped using an external magnet to perform washing steps to remove excess noncomplexed NAEBs. When cleanup steps are accomplished, the remaining complexed particles are loaded into one detection capillary to acquire Raman signals from the sensing beads. Compared with that using a conventional magnetic trapping device, the cleanup time is shortened from nearly an hour to less than 10 min. Sample loss during the washing steps becomes more controllable, resulting in adequate standard curve linearity (R > 0.99) ranging from 1 to 100 pM.