Sandia journal manuscript; Not yet accepted for publication
Koh, Chung-Yan K.; Piccini, Matthew E.; Schaff, Ulrich Y.; Stanker, Larry H.; Cheng, Luisa W.; Ravichandran, Easwaran; Singh, Bal-Ram; Sommer, Greg J.; Singh, Anup K.
Multiple cases of attempted bioterrorism events using biotoxins have highlighted the urgent need for tools capable of rapid screening of suspect samples in the field (e.g., mailroom and public events). We present a portable microfluidic device capable of analyzing environmental (e.g., white powder), food (e.g., milk) and clinical (e.g., blood) samples for multiplexed detection of biotoxins. The device is rapid (<15-30 min sample-to-answer), sensitive (< 0.08 pg/mL detection limit for botulinum toxin), multiplexed (up to 64 parallel assays) and capable of analyzing small volume samples (< 20 μL total sample input). The immunoassay approach (SpinDx) is based on binding of toxins in a sample to antibody-laden capture particles followed by sedimentation of particles through a density-media in a microfluidic disk and quantification using a laser-induced fluorescence detector. A direct, blinded comparison with a gold standard ELISA revealed a 5-fold more sensitive detection limit for botulinum toxin while requiring 250-fold less sample volume and a 30 minute assay time with a near unity correlation. A key advantage of the technique is its compatibility with a variety of sample matrices with no additional sample preparation required. Ultrasensitive quantification has been demonstrated from direct analysis of multiple clinical, environmental and food samples, including white powder, whole blood, saliva, salad dressing, whole milk, peanut butter, half and half, honey, and canned meat. We believe that this device can met an urgent need in screening both potentially exposed people as well as suspicious samples in mail-rooms, airports, public sporting venues and emergency rooms. The general-purpose immunodiagnostics device can also find applications in screening of infectious and systemic diseases or serve as a lab device for conducting rapid immunoassays.
Cell signaling is a dynamic and complex process. A typical signaling pathway may begin with activation of cell surface receptors, leading to activation of a kinase cascade that culminates in induction of messenger RNA (mRNA) and noncoding microRNA (miRNA) production in the nucleus, followed by modulation of mRNA expression by miRNAs in the cytosol, and end with production of proteins in response to the signaling pathway. Signaling pathways involve proteins, miRNA, and mRNAs, along with various forms of transient posttranslational modifications, and detecting each type of signaling molecule requires categorically different sample preparation methods such as Western blotting for proteins, PCR for nucleic acids, and flow cytometry for posttranslational modifications. Since we know that cells in populations behave heterogeneously,1 especially in the cases of stem cells, cancer, and hematopoiesis, there is need for a new technology that provides capability to detect and quantify multiple categories of signaling molecules in intact single cells to provide a comprehensive view of the cell’s physiological state. In this Technology Brief, we describe our automated microfluidic platform with a portfolio of customized molecular assays that can detect nucleic acids, proteins, and posttranslational modifications in single intact cells with >95% reduction in reagent requirement in under 8 h.
Flow cytometry in combination with fluorescent in situ hybridization (flow-FISH) is a powerful technique that can be utilized to rapidly detect nucleic acids at single-cell resolution without the need for homogenization or nucleic acid extraction. Here, we describe a microfluidic-based method which enables the detection of microRNAs or miRNAs in single intact cells by flow-FISH using locked nucleic acid (LNA)-containing probes. Our method can be applied to all RNA species including mRNA and small noncoding RNA and is suitable for multiplexing with protein immunostaining in the same cell. For demonstration of our method, this chapter details the detection of miR155 and CD69 protein in PMA and ionomycin-stimulated Jurkat cells. Here, we also include instructions on how to set up a microfluidic chip sample preparation station to prepare cells for imaging and analysis on a commercial flow cytometer or a custom-built micro-flow cytometer.
A microfluidic RNA interference screening device was designed to study which genes are involved in Rift Valley Fever Virus (RVFV) infection. Spots of small interfering RNA (siRNA) are manually spotted onto a glass microscope slide, and aligned to a screening device designed to accommodate cell seeding, siRNA transfection, cell culture, virus infection and imaging analysis. This portable and disposable PDMS-based microfluidic device for RNAi screening was designed for a 96-well library of transfection against variety of gene targets. Current results show transfection of GFP-22 siRNA within the device, as compared to controls, which inhibit the expression of GFP produced by recombinant RVFV. This technique can be applied to host-pathogen interactions for highly dangerous systems in BSL-3/4 laboratories, where bulky robotic equipment is not ideal.
Uncultivable microorganisms likely play significant roles in the ecology within the human body, with subtle but important implications for human health. Focusing on the oral microbiome, we are developing a processor for targeted isolation of individual microbial cells, facilitating whole-genome analysis without the need for isolation of pure cultures. The processor consists of three microfluidic modules: identification based on 16S rRNA fluorescence in situ hybridization (FISH), fluorescence-based sorting, and encapsulation of individual selected cells into small droplets for whole-genome amplification. We present here a technique for performing microscale FISH and flow cytometry, as a prelude to single cell sorting.