Browsing by Author "Cao, Zhenning"

Now showing 1 - 8 of 8
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    Detecting intracellular translocation of native proteins quantitatively at the single cell level
    Cao, Zhenning; Geng, Shuo; Li, Liwu; Lu, Chang (The Royal Society of Chemistry, 2014-04-07)
    The intracellular localization and movement (i.e. translocation) of proteins are critically correlated with the functions and activation states of these proteins. Simple and accessible detection methods that can rapidly screen a large cell population with single cell resolution have been seriously lacking. In this report, we demonstrate a simple protocol for detecting translocation of native proteins using a common flow cytometer which detects fluorescence intensity without imaging. We sequentially conducted chemical release of cytosolic proteins and fluorescence immunostaining of a targeted protein. The detected fluorescence intensity of cells was shown to be quantitatively correlated to the cytosolic/nuclear localization of the protein. We used our approach to detect the translocation of native NF-_B (an important transcription factor) at its native expression level and examine the temporal dynamics in the process. The incorporation of fluorescence immunostaining makes our approach compatible with the analysis of cell samples from lab animals and patients. Our method will dramatically lower the technological hurdle for studying subcellular localization of proteins.
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    Diffusion-based Microfluidic PCR for "One-pot" Analysis of Cells
    Ma, Sai; Loufakis, Despina N.; Cao, Zhenning; Chang, Yiwen; Achenie, Luke E. K.; Lu, Chang (The Royal Society of Chemistry, 2014-05-28)
    Genetic analysis starting with cell samples often requires multi-step processing including cell lysis, DNA isolation/purification, and polymerase chain reaction (PCR) based assays. When conducted on a microfluidic platform, the compatibility among various steps often demands a complicated procedure and a complex device structure. Here we present a microfluidic device that permits a “one-pot” strategy for multi-step PCR analysis starting from cells. Taking advantage of the diffusivity difference, we replace the smaller molecules in the reaction chamber by diffusion while retaining DNA molecules inside. This simple scheme effectively removes reagents from the previous step to avoid interference and thus permits multi-step processing in the same reaction chamber. Our approach shows high efficiency for PCR and potential for a wide range of genetic analysis including assays based on single cells.
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    Electroporation-based delivery of cell-penetrating peptide conjugates of peptide nucleic acids for antisense inhibition of intracellular bacteria
    Ma, Sai; Schroeder, Betsy; Sun, Chen; Loufakis, Despina N.; Cao, Zhenning; Sriranganathan, Nammalwar; Lu, Chang (The Royal Society of Chemistry, 2014-08-14)
    Cell penetrating peptides (CPPs) have been used for a myriad of cellular delivery applications and were recently explored for delivery of antisense agents such as peptide nucleic acids (PNAs) for bacterial inhibition. Although these molecular systems (i.e. CPP–PNAs) have shown ability to inhibit growth of bacterial cultures in vitro, they show limited effectiveness in killing encapsulated intracellular bacteria in mammalian cells such as macrophages, presumably due to difficulty involved in the endosomal escape of the reagents. In this report, we show that electroporation delivery dramatically increases the bioavailability of CPP–PNAs to kill Salmonella enterica serovar Typhimurium LT2 inside macrophages. Electroporation delivers the molecules without involving endocytosis and greatly increases the antisense effect. The decrease in the average number of Salmonella per macrophage under a 1200 V cm_1 and 5 ms pulse was a factor of 9 higher than that without electroporation (in an experiment with a multiplicity of infection of 2 : 1). Our results suggest that electroporation is an effective approach for a wide range of applications involving CPP-based delivery. The microfluidic format will allow convenient functional screening and testing of PNA-based reagents for antisense applications.
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    Electroporation-delivered fluorescent protein biosensors for probing molecular activities in cells without genetic encoding
    Sun, Chen; Ouyang, Mingxing; Cao, Zhenning; Ma, Sai; Alqublan, Hamzeh; Sriranganathan, Nammalwar; Wang, Yingxiao; Lu, Chang (The Royal Society of Chemistry, 2014-08-08)
    Fluorescent protein biosensors are typically implemented via genetic encoding which makes the examination of scarce cell samples impractical. By directly delivering the protein form of the biosensor into cells using electroporation, we detected intracellular molecular activity with the sample size down to ~100 cells with high spatiotemporal resolution.
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    Focusing of mammalian cells under an ultrahigh pH gradient created by unidirectional electropulsation in a confined microchamber
    Loufakis, Despina N.; Cao, Zhenning; Ma, Sai; Mittelman, David; Lu, Chang (The Royal Society of Chemistry, 2014-06-09)
    The transport and manipulation of cells in microfluidic structures are often critically required in cellular analysis. Cells typically make consistent movement in a dc electric field in a single direction, due to their electrophoretic mobility or electroosmotic flow or the combination of the two. Here we demonstrate that mammalian cells focus to the middle of a closed microfluidic chamber under the application of unidirectional direct current pulses. With experimental and computational data, we show that under the pulses electrochemical reactions take place in the confined microscale space and create an ultrahigh and nonlinear pH gradient (~2 orders of magnitude higher than the ones in protein isoelectric focusing) at the middle of the chamber. The varying local pH affects the cell surface charge and the electrophoretic mobility, leading to focusing in free solution. Our approach provides a new and simple method for focusing and concentrating mammalian cells at the microscale.
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    Immunomagnetic separation of tumor initiating cells by screening two surface markers
    Sun, Chen; Hsieh, Yuan-Pang; Ma, Sai; Geng, Shuo; Cao, Zhenning; Li, Liwu; Lu, Chang (Springer Nature, 2017-01-11)
    Isolating tumor initiating cells (TICs) often requires screening of multiple surface markers, sometimes with opposite preferences. This creates a challenge for using bead-based immunomagnetic separation (IMS) that typically enriches cells based on one abundant marker. Here, we propose a new strategy that allows isolation of CD44(+)/CD24(-) TICs by IMS involving both magnetic beads coated by anti-CD44 antibody and nonmagnetic beads coated by anti-CD24 antibody (referred to as two-bead IMS). Cells enriched with our approach showed significant enhancement in TIC marker expression (examined by flow cytometry) and improved tumorsphere formation efficiency. Our method will extend the application of IMS to cell subsets characterized by multiple markers.
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    Microfluidic Engineering for Ultrasensitive Molecular Analysis of cells
    Cao, Zhenning (Virginia Tech, 2015-10-05)
    The main focus of this research was the development of microfluidic technology for ultrasensitive and fast molecular analysis of cells. Chromatin immunoprecipitation (ChIP) assay followed by next generation sequencing serves as the primary technique to characterize the genomic locations associated with histone modifications. However, conventional ChIP-seq assay requires large numbers of cells. We demonstrate a novel microfluidics-based ChIP-seq assay which dramatically reduced the required cell number. Coupled with next generation sequencing, the assay permitted the analysis of histone modifications at the whole genome from as few as ~100 cells. Using the same device, we demonstrated that MeDIP-seq with tiny amount of DNA (<5ng) generated high quality genome-wide profiles of DNA methylation. Off-chip sonication often leads to sample loss due to multiple tube transferring. In addition, conventional sonicators are not able to manipulate samples with small volume. We developed a novel microfluidic sonicator, which is able to achieve on-chip DNA/chromatin shearing into ideal fragment size (100~600bp) for both chromatin immunoprecipitation (ChIP) and methylated DNA immunoprecipitation (MeDIP). The integrated on-chip sonication followed by immunoprecipitation (IP) reaction can significantly reduce sample loss and contamination. Simple and accessible detection methods that can rapidly screen a large cell population with single cell resolution have been seriously lacking. We demonstrate a simple protocol for detecting translocation of native proteins using a common flow cytometer which detects fluorescence intensity without imaging. Using our approach, we successfully detected the translocation of native NF-kappa B (an important transcription factor) at its native expression level and examine the temporal dynamics in the process. Droplets with encapsulated beads and cells have been increasingly used for studying molecular and cellular biology. However, a mixed population of droplets with an uneven number or type of encapsulated particles is resulted and used for screening. We developed a fluorescence-activated microfluidic droplet sorter that integrated a simple deflection mechanism. By passing droplets through a narrow interrogation channel, the encapsulated particles were detected individually. The microcontroller conducted the computation to determine the number and type of encapsulated particles in each droplet and made the sorting decision. Our results showed high efficiency and accuracy for sorting and enrichment.
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    Microfluidic systems and methods for chromatin immunoprecipitation (ChIP)
    (United States Patent and Trademark Office, 2017-08-15)
    An integrated microfluidic chromatin immunoprecipitation assay dramatically improves the collection efficiency of ChIP DNA from cells. Immunoprecipitation of chromatin fragments is conducted in a microfluidic chamber with a large fraction of its volume (e.g., ˜15-40%) occupied by magnetic immunoprecipitation (IP) beads. Oscillating washing of the beads, enabled by, e.g., solenoid valves (controlled by a computer) and high pressure attached to both ends of the microfluidic chamber, effectively removes unbound chromatin and produces high-quality ChIP DNA. ChIP DNA produced by an example device generates excellent results in the subsequent DNA library preparation. The ChIP-seq (i.e., ChIP followed by next-generation sequencing) results match very well with public data generated using much larger cell sample sizes and a conventional approach.