Microfluidic System for Detection of Viral RNA in Blood Using a Barcode Fluorescence Reporter and a Photocleavable Capture Probe

Abstract

A microfluidic sample preparation multiplexer (SPM) and assay procedure is developed to improve amplification-free detection of Ebola virus RNA from blood. While a previous prototype successfully detected viral RNA following off-chip RNA extraction from infected cells, the new device and protocol can detect Ebola virus in raw blood with clinically relevant sensitivity. The Ebola RNA is hybridized with sequence specific capture and labeling DNA probes in solution and then the complex is pulled down onto capture beads for purification and concentration. After washing, the captured RNA target is released by irradiating the photocleavable DNA capture probe with ultraviolet (UV) light. The released, labeled, and purified RNA is detected by a sensitive and compact fluorometer. Exploiting these capabilities, a detection limit of 800 attomolar (aM) is achieved without target amplification. The new SPM can run up to 80 assays in parallel using a pneumatic multiplexing architecture. Importantly, our new protocol does not require time-consuming and problematic off-chip probe conjugation and washing. This improved SPM and labeling protocol is an important step toward a useful POC device and assay.

Integration of sample preparation and analysis into an optofluidic chip for multi-target disease detection

“…Other specific labeling strategies for proteins and nucleic acids, for e.g. using single probes with multiple fluorophores such as Nanostrings 41 , can also be developed and have been previously implemented in automaton architectures like ours 36 .…”

SingleChlamydomonas reinhardi(C. reinhardtii) cell separation from bacterial cells and auto-fluorescence tracking with a nano-sieve device

“…1 , the trapped C. reinhardtii cells has a larger fringe than the cells presented in cell media, indicating that the cells were deformed by the flexible PDMS roof. Thus, our device also has potential for quantitative studies of cell mechanics by controlling the PDMS deformation (Mills et al, 2008; Huh et al, 2007; Du et al, 2017). This configuration can also be applied to study the metabolic and physiological responses of the trapped microalgae cells to the external chemical/mechanical triggering (Ferraria et al, 2019; Liu et al, 2013).…”

Perspective of Molecular Diagnosis in Healthcare: From Barcode to Pattern Recognition

“…When the target concentration is low (300 fM), the target molecules are confirmed by both excitations (red boxes). Exploiting the sequence combination, changing the sequence of the fluorescent dyes on the backbone of the barcode dye can provide genomic information of different targets, resembling traditional barcodes [20], thus allowing highly multiplexing detection of the target molecules. For example, by changing the arrangement of four different colors, multiplexing detection of up to 800× can be achieved [18].…”

“…Recently, the common barcodes are integrated with several encoding methods, such as using DNA/RNA and immune antibodies encoding to report the targets with the aid of nanoparticles or nanorods, dyes, and others as reporters [24,25]. We used DNA oligonucleotide as a linker to bind the target by hybridizing with complementary oligonucleotide, and the accompanying fluorescent dye as a reporter to transfer the information of targets [20]. Similarly, gold nanoparticles as reporters could be detected not only by spectrometry because of their function of Raman signal enhancement, but also by naked eye because of changeable color by their aggregation and dispersion [26].…”