Cell Separations and Sorting

This document is the Accepted Manuscript version of a Published Work that appeared in final form in Analytical Chemistry, copyright © American Chemical Society after peer review and technical editing by the publisher. To access the final edited and published work see https://doi.org/10.1021/acs.analchem.9b05357.NIBIB Grant P41-EB020594COBRE Grant 5P20GM13042

Time-Delayed Integration–Spectral Flow Cytometer (TDI-SFC) for Low-Abundance-Cell Immunophenotyping

This document is the Accepted Manuscript version of a Published Work that appeared in final form in Analytical Chemistry, copyright © American Chemical Society after peer review and technical editing by the publisher. To access the final edited and published work see http://doi.org/10.1021/acs.analchem.9b00021.We describe a unique flow cytometer (TDI-SFC) for the immunophenotyping of low-abundance cells, particularly when cell counts are sample-limited and operationally difficult for analysis by fluorescence microscopy (>100 cells) or multiparameter flow cytometry (MFC, <10 000 cells). TDI-SFC combines the high spectral resolution of spectral flow cytometry (SFC) with a CCD operated in time-delayed integration (TDI) for improved duty cycle and sensitivity. Cells were focused with a 1D-sheathing microfluidic device, and fluorescence emission generated from a 488 nm laser was collected by epi-illumination and dispersed along one axis of a CCD by a spectrograph. Along the other axis, the CCD’s shift rate was clocked at a rate that closely matched the cells’ velocity through the field of view. This TDI-SFC format allowed the CCD shutter to remain open during signal acquisition, providing a duty cycle ∼100% and assurance that ∼95% cells were interrogated. We used fluorescent beads to optimize synchronization of TDI clocking with the sheathed-cell velocity and to improve sensitivity via the excitation intensity, epi-illumination numerical aperture, and integration time. TDI achieved integrated signals of 106 counts at a signal-to-noise ratio (SNR) of 610 for beads corresponding to a load of 4 × 105 antibodies. We also evaluated multiplexing capabilities by spectral deconvolution and undertook a proof-of-concept application to immunophenotype low-abundance cells; the demonstration consisted of immunophenotyping a model cell line, in this case SUP-B15 cells representing B-cell acute lymphoblastic leukemia (B-ALL). The B-ALL cell line was stained against a leukemic marker (terminal deoxynucleotidyl transferase, TdT), and we successfully used spectral unmixing to discriminate TdT(+) cells from TdT(−) cells even at low cell counts (∼100 cells). The TDI-SFC could potentially be used in any application requiring the immunophenotyping of low-abundance cells, such as in monitoring measurable residual disease in acute leukemias following affinity enrichment of circulating leukemia cells from peripheral blood

The Intramolecular Loss of Fluorescence by Lysine Derivatized with Naphthalenedialdehyde

This is the publisher's version, also available electronically from http://www.opticsinfobase.org/as/abstract.cfm?URI=as-44-5-858.Derivatization of primary amines such as amino acids and peptides with naphthalenedialdehyde (NDA) in the presence of cyanide ion yields cyanobenzo[f]-isoindole (CBI) adducts that are highly fluorescent. However, the fluorescence is seriously quenched with amines that possess more than one primary amine site, as is the case with lysine. Although it was found that the adsorption of CBI2-lysine on a solid substrate restored the fluorescence, the reason for the solution quenching, with respect to results for mono-derivatized amines, was investigated. The experiments to probe the quenching were based on the assumption that the mechanism responsible for quenching involved a charge-transfer (CT) excited state. Thus, it was found that the solvent properties of viscosity and polarity affected the lifetime and quantum yield of fluorescence in a manner consistent with the proposed mechanism

Complete plastic nanofluidic devices for DNA analysis via direct imprinting with polymer stamps

Development of all polymer-based nanofluidic devices using replication technologies, which is a prerequisite for providing devices for a larger user base, is hampered by undesired substrate deformation associated with the replication of multi-scale structures. Therefore, most nanofluidic devices have been fabricated in glass-like substrates or in a polymer resist layer coated on a substrate. This letter presents a rapid, high fidelity direct imprinting process to build polymer nanofluidic devices in a single step. Undesired substrate deformation during imprinting was significantly reduced through the use of a polymer stamp made from a UV-curable resin. The integrity of the enclosed all polymer-based nanofluidic system was verified by a fluorescein filling experiment and translocation/ stretching of lambda-DNA molecules through the nanochannels. It was also found that the funnel-like design of the nanochannel inlet significantly improved the entrance of DNA molecules into nanochannels compared to an abrupt nanochannel/microfluidic network interface.close131

Development of an Automated Digestion and Droplet Deposition Microfluidic Chip for MALDI-TOF MS

An automated proteolytic digestion bioreactor and droplet deposition system was constructed with a plastic microfluidic device for off-line interfacing to matrix assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). The microfluidic chips were fabricated in poly(methyl methacrylate) (PMMA), using a micromilling machine and incorporated a bioreactor, which was 100 μm wide, 100 μm deep, and possessed a 4 cm effective channel length (400 nL volume). The chip was operated by pressure-driven flow and mounted on a robotic fraction collector system. The PMMA bioreactor contained surface immobilized trypsin, which was covalently attached to the UV-modified PMMA surface using coupling reagents N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC) and hydroxysulfosuccinimide (sulfo-NHS). The digested peptides were mixed with a MALDI matrix on-chip and deposited as discrete spots on MALDI targets. The bioreactor provided efficient digestion of a test protein, cytochrome c, at a flow rate of 1 μL/min, producing a reaction time of ∼24 s to give adequate sequence coverage for protein identification. Other proteins were also evaluated using this solid-phase bioreactor. The efficiency of digestion was evaluated by monitoring the sequence coverage, which was 64%, 35%, 58%, and 47% for cytochrome c, bovine serum albumin (BSA), myoglobin, and phosphorylase b, respectively. © 2008 American Society for Mass Spectrometry

Purification and preconcentration of genomic DNA from whole cell lysates using photoactivated polycarbonate (PPC) microfluidic chips

We discuss the use of a photoactivated polycarbonate (PPC) microfluidic chip for the solid-phase, reversible immobilization (SPRI) and purification of genomic DNA (gDNA) from whole cell lysates. The surface of polycarbonate was activated by UV radiation resulting in a photo-oxidation reaction, which produced a channel surface containing carboxylate groups. The gDNA was selectively captured on this photoactivated surface in an immobilization buffer, which consisted of 3% polyethylene glycol, 0.4 M NaCl and 70% ethanol. The methodology reported herein is similar to conventional SPRI in that surface-confined carboxylate groups are used for the selective immobilization of DNA; however, no magnetic beads or a magnetic field are required. As observed by UV spectroscopy, a load of ∼7.6 ± 1.6 µg/ml of gDNA was immobilized onto the PPC bed. The recovery of DNA following purification was estimated to be 85 ± 5%. The immobilization and purification assay using this PPC microchip could be performed within ∼25 min as follows: (i) DNA immobilization ∼6 min, (ii) chip washout with ethanol 10 min, and (iii) drying and gDNA desorption ∼6 min. The PPC microchip could also be used for subsequent assays with no substantial loss in recovery, no observable carryover and no need for ‘reactivation’ of the PC surface with UV light

Solid-phase XRN1 reactions for RNA cleavage: application in single-molecule sequencing

Modifications in RNA are numerous (∼170) and in higher numbers compared to DNA (∼5) making the ability to sequence an RNA molecule to identify these modifications highly tenuous using next generation sequencing (NGS). The ability to immobilize an exoribonuclease enzyme, such as XRN1, to a solid support while maintaining its activity and capability to cleave both the canonical and modified ribonucleotides from an intact RNA molecule can be a viable approach for single-molecule RNA sequencing. In this study, we report an enzymatic reactor consisting of covalently attached XRN1 to a solid support as the groundwork for a novel RNA exosequencing technique. The covalent attachment of XRN1 to a plastic solid support was achieved using EDC/NHS coupling chemistry. Studies showed that the solid-phase digestion efficiency of model RNAs was 87.6 ± 2.8%, while the XRN1 solution-phase digestion for the same model was 78.3 ± 4.4%. The ability of immobilized XRN1 to digest methylated RNA containing m6A and m5C ribonucleotides was also demonstrated. The processivity and clipping rate of immobilized XRN1 secured using single-molecule fluorescence measurements of a single RNA transcript demonstrated a clipping rate of 26 ± 5 nt s−1 and a processivity of >10.5 kb at 25°C

Thermoplastic nanofluidic devices for biomedical applications

This review presents an overview of recent advancements in the fabrication, surface modification and applications of thermoplastic nanofluidic devices

Simple room temperature bonding of thermoplastics and poly(dimethylsiloxane)

We describe a simple and versatile method for bonding thermoplastics to elastomeric polydimethylsiloxane (PDMS) at room temperature. The bonding of various thermoplastics including polycarbonate (PC), cyclic olefin copolymer (COC), polymethylmethacrylate (PMMA), and polystyrene (PS), to PDMS has been demonstrated at room temperature. An irreversible bonding was formed instantaneously when the thermoplastics, activated by oxygen plasma followed by aminopropyltriethoxysilane modification, were brought into contact with the plasma treated PDMS. The surface modified thermoplastics were characterized by water contact angle measurements and Xray photoelectron spectroscopy. The tensile strength of the bonded hybrid devices fabricated with PC, COC, PMMA, and PS was found to be 430, 432, 385, and 388 kPa, respectively. The assembled devices showed high burst resistance at a maximum channel pressure achievable by an in-house built syringe pump, 528 kPa. Furthermore, they displayed very high hydrolytic stability; no significant change was observed even after the storage in water at 37 degrees C over a period of three weeks. In addition, this thermoplastic-to-PDMS bonding technique has been successfully employed to fabricate a relatively large sized device. For example, a lab-on-a-disc with a diameter of 12 cm showed no leakage when it spins for centrifugal fluidic pumping at a very high rotating speed of 6000 rpm.close443

A Simple Micromilled Microfluidic Impedance Cytometer with Vertical Parallel Electrodes for Cell Viability Analysis

Microfluidic impedance cytometry has been demonstrated as an effective platform for single cell analysis, taking advantage of microfabricated features and dielectric cell sensing methods. In this study, we present a simple microfluidic device to improve the sensitivity, accuracy, and throughput of single suspension cell viability analysis using vertical sidewall electrodes fabricated by a widely accessible negative manufacturing method. A microchannel milled through a 75 µm platinum wire, which was embedded into poly-methyl-methacrylate (PMMA), created a pair of parallel vertical sidewall platinum electrodes. Jurkat cells were interrogated in a custom low-conductivity buffer (1.2 ± 0.04 mS/cm) to reduce current leakage and increase device sensitivity. Confirmed by live/dead staining and electron microscopy, a single optimum excitation frequency of 2 MHz was identified at which live and dead cells were discriminated based on the disruption in the cell membrane associated with cell death. At this frequency, live cells were found to exhibit changes in the impedance phase with no appreciable change in magnitude, while dead cells displayed the opposite behavior. Correlated with video microscopy, a computational algorithm was created that could identify cell detection events and determine cell viability status by application of a mathematical correlation method