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- Banerjee, Indradumna, et al.
(author)
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Microfluidic centrifugation assisted precipitation based DNA quantification
- 2019
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In: Lab on a Chip. - : Royal Society of Chemistry. - 1473-0197 .- 1473-0189. ; 19:9, s. 1657-1664
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Journal article (peer-reviewed)abstract
- Nucleic acid amplification methods are increasingly being used to detect trace quantities of DNA in samples for various diagnostic applications. However, quantifying the amount of DNA from such methods often requires time consuming purification, washing or labeling steps. Here, we report a novel microfluidic centrifugation assisted precipitation (mu CAP) method for single-step DNA quantification. The method is based on formation of a visible precipitate, which can be quantified, when an intercalating dye (GelRed) is added to the DNA sample and centrifuged for a few seconds. We describe the mechanism leading to the precipitation phenomenon. We utilize centrifugal microfluidics to precisely control the formation of the visible and quantifiable mass. Using a standard CMOS sensor for imaging, we report a detection limit of 45 ng mu l(-1). Furthermore, using an integrated lab-on-DVD platform we recently developed, the detection limit is lowered to 10 ng mu l(-1), which is comparable to those of current commercially available instruments for DNA quantification. As a proof of principle, we demonstrate the quantification of LAMP products for a HIV-1B type genome containing plasmid on the lab-on-DVD platform. The simple DNA quantification system could facilitate advanced point of care molecular diagnostics.
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| 2. |
- Zhang, Wang, 1988-
(author)
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Development of novel molecular and microfluidics tools for identification and characterization of latent HIV-1 reservoir
- 2019
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Doctoral thesis (other academic/artistic)abstract
- The existence of latent HIV-1 reservoir (LR) in all HIV-1 infected patients serves as a major obstacle to completely cure HIV-1 infection. However, up to now there is still no available assay that provides an accurate measurement of the reservoir size. This thesis aims to address this challenge from different aspects with several novel technologies, using both molecular and microfluidics-based tools. To find a proper tool to identify the latent HIV-1 reservoir, in Paper I and II, LIPS assay, RNAflow, and RNAscope assay were optimized and evaluated for indirect and direct detection of latent HIV-1 reservoir. The results indicated the LIPS method might not be sufficient for latent HIV-1 reservoir detection, although it has been proposed to quantify the latent HIV-1 reservoir indirectly. Furthermore, the optimized RNAscope technique performed better than RNAflow for transcription and translation competent latent HIV-1 reservoir identification. The RNAscope was also found to be independent of the HIV-1 subtype and can be applied to patient samples at single cell level. As there are currently no available surface biomarkers for latent HIV-1 reservoir, in Paper III, transcriptomics and proteomics-based analysis method for high-throughput selection of potential biomarker were established and applied to different patient groups. Twelve membrane protein-coding genes were identified as downregulated in the patient group who were hypothesized to have lower latent reservoir. These proteins might have the potential to be used as surface biomarkers for latent HIV-1 reservoir. CD4+ T cells, monocyte/macrophages, and natural killer cells are believed to be the primary source for HIV-1 reservoirs in peripheral blood. In paper IV, a microfluidic chip was developed to simultaneously isolate these three mononuclear leukocyte cell types directly from whole blood. The microfluidic method reduces the sample volume requirement and is a promising tool for latent HIV-1 reservoir study. Together, though further improvement and clinical verification are necessary, the work in this thesis has contributed to the advancement of latent HIV-1 reservoir characterization and may facilitate future development of the latent HIV-1 reservoir targeting and clearance methods with the ultimate goal – to cure HIV-1 infection.
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