We report a one-pot, closed-vessel enzymatic assay that eliminates carryover contamination while preserving robust DNA amplification in loop-mediated isothermal amplification (LAMP), providing ...reliable and rapid detection of target DNA in contaminated samples.
The functional properties of colloidal materials can be tailored by tuning the shape of their constituent particles. Unfortunately, a reliable, general methodology for purifying colloidal materials ...solely based on shape is still lacking. Here we exploit the single-particle analysis and sorting capabilities of the fluorescence-activated cell sorting (FACS) instrument, a commonly used tool in biomedical research, and demonstrate the ability to separate mixtures of synthetic microparticles based solely on their shape with high purity. We achieve this by simultaneously obtaining four independent optical scattering signals from the FACS instrument to create shape-specific 'scattering signatures' that can be used for particle classification and sorting. We demonstrate that these four-dimensional signatures can overcome the confounding effects of particle orientation on shape-based characterization. Using this strategy, robust discrimination of particles differing only slightly in shape and an efficient selection of desired shapes from mixtures comprising particles of diverse sizes and materials is demonstrated.
We report the development of an optimized 50‐color spectral flow cytometry panel designed for the in‐depth analysis of the immune system in human blood and tissues, with the goal of maximizing the ...amount of information that can be collected using currently available flow cytometry platforms. We established and tested this panel using peripheral blood mononuclear cells (PBMCs), but included CD45 to enable its future use for the analysis of human tissue samples. The panel contains lineage markers for all major immune cell subsets, and an extensive set of phenotyping markers focused on the activation and differentiation status of the T cell and dendritic cell (DC) compartment. We outline the biological insight that can be gained from the simultaneous measurement of such a large number of proteins and propose that this approach provides a unique opportunity for the comprehensive exploration of the immune status in human samples with a limited number of cells. Of note, we tested the panel to be compatible with cell sorting for further downstream applications. Furthermore, to facilitate the wide‐spread implementation of such a panel across different cohorts and samples, we established a trimmed‐down 45‐color version which can be used with different spectral cytometry platforms. Finally, to generate this panel, we utilized not only existing panel design guidelines, but also developed new metrics to systematically identify the optimal combination of 50 fluorochromes and evaluate fluorochrome‐specific resolution in the context of a 50‐color unmixing matrix.
Biologic drugs are typically manufactured in mammalian host cells, and it is critical from a drug safety and efficacy perspective to detect and remove host cell proteins (HCPs) during production. ...This is currently achieved with sets of polyclonal antibodies (pAbs), but these suffer from critical shortcomings because their composition is inherently undefined, and they cannot detect nonimmunogenic HCPs. In this work, we report a high-throughput screening and array-based binding characterization strategy that we employed to generate a set of aptamers that overcomes these limitations to achieve sensitive, broad-spectrum detection of HCPs from the widely used Chinese hamster ovary (CHO) cell line. We identified a set of 32 DNA aptamers that achieve better sensitivity than a commercial pAb reagent set and can detect a comparable number of HCPs over a broad range of isoelectric points and sizes. Importantly, these aptamers detect multiple contaminants that are known to be responsible for therapeutic antibody degradation and toxicity in patients. Because HCP aptamer reagents are sequence-defined and chemically synthesized, we believe they may enable safer production of biologic drugs, and this strategy should be broadly applicable for the generation of HCP detection reagents for other cell lines.
The field of microfluidics has enabled the development of powerful tools for analyzing and manipulating phenomena at the micro- and nano-scales, ranging from chemical analysis of biological samples ...to controlled synthesis of colloidal materials. In this dissertation we explore four unique platforms for real-time microfluidic measurement, analysis, and control systems with applications at the intersection of biomedicine and materials engineering. First, we show that a real-time biosensor can be used to perform closed-loop control of drug concentrations in the bloodstream of live animals. Second, we show that a commercially available cell-sorting instrument can be used to sort heterogeneous suspensions of synthetic microparticles based on shape using optical scattering measurements, resulting in monodisperse microparticle suspensions with well-defined morphology. Third, we report preliminary results for an image-based cell and microparticle sorter capable of sorting objects using two-dimensional high-speed microscopy and real-time image analysis. Finally, we report a contamination-resistant microfluidic assay for quantitative genetic detection based on real-time loop-mediated isothermal amplification, improving the robustness of point-of-care pathogen detection techniques.
This work presents the first fully wireless implant system capable of continuous monitoring of therapeutic drugs in vivo. Electrochemical readout using square-wave voltammetry (SWV) is employed to ...measure the changes in the drug concentration using redox-labeled structure-switching aptamers. Ultrasound (US) powering and data transmission are employed in the implant for miniaturization, large tissue depth, and high available power. We demonstrate continuous and real-time detection in the human whole blood. Implemented in 65-nm CMOS, the entire implant system operates at 6.64 mW, and measures 140mm 3 and 0.24g.
