a-versatile-automated-platform-for-micro-scale-cell-stimulation

A Versatile Automated Platform for Micro-scale Cell Stimulation Experiments

Study of cells in culture (in vitro analysis) has provided important insight into complex biological systems. Conventional methods and equipment for in ...

Sandia National Laboratories

Increasing electron demand in the reactions of G-NMBH anions with substituted phenyldimethylsulfonium ions decreases the alpha-effect for the methyl transfers toward 1.0 (zero effect). An extrapolation shows the possibility of an inverse effect (<1.0). The reactivity of G-NMBH anions correlates with SET parameters and with the known propensity of phenyldimethylsulfonium ions to accept a single electron into a sigma C-S orbital concomitant with expulsion of a CH(3) group. These correlations indicate inclusion of some SET character into the wavefunction of the S(N)2 transition state for these reactions, in agreement with the Shaik and Pross SCD model of the S(N)2 reaction.

alpha-Effect with Substituted N-Methylbenzohydroxamates and Substituted Phenyldimethylsulfonium Salts: Toward Understanding of an Intrinsic alpha-Effect.

Fused-silica capillary columns packed with 1.0-microm nonporous C18 bonded particles are evaluated with isocratic ultrahigh-pressure liquid chromatography (UHPLC). Improved UHPLC techniques have demonstrated column efficiencies as high as 730 000 plates/m and run pressures over 6800 bar (100 000 psi) for packed 10-microm-inner diameter (i.d.) columns. Columns as large as 150 microm have been tested with UHPLC and show no flow-induced heating effects on separation efficiencies. van Deemter plot analysis for column i.d.s ranging from 10 to 150 microm shows an increase in column efficiency with a decrease in column i.d. Reduced parameter analysis further illustrates a decrease in reduced parameter A term and C term values with decreasing i.d. However, reduced parameter C term values for columns evaluated with UHPLC are an order of magnitude larger than C term values for larger particles at conventional pressures. Retention factors for moderately retained compounds are observed to increase with column i.d., suggesting an increase in packing density. Highly ordered packing arrangement at the column wall is seen for packed beds extruded from large-diameter columns.

In-depth characterization of slurry packed capillary columns with 1.0-microm nonporous particles using reversed-phase isocratic ultrahigh-pressure liquid chromatography.

We demonstrate a new method for joining thermoplastic surfaces to produce microfluidic devices. The method takes advantage of the sharply defined permeation boundary of case-II diffusion to generate dimensionally controlled, activated bonding layers at the surfaces being joined. The technique is capable of producing bonds that exhibit cohesive failure, while preserving the fidelity of fine features in the bonding interface. This approach is uniquely suited to production of layered microfluidic structures, as it allows the bond-forming interface between plastic parts to be precisely manipulated at micrometre length scales. Distortions in microfluidic device channels are limited to the size scale of the permeant-swollen layer; 6 microm deep channels are routinely produced with no detectable cross-sectional distortions. Conventional thermal diffusion bonding of identical parts yields less strongly bonded microfluidic structures with increasingly severe dimensional compressions as bonding temperatures approach the thermoplastic glass-transition temperature: a preliminary rheological analysis is consistent with the observed compressions. The bond-enhancing procedure is easily integrated in standard process flows, uses inexpensive reagents, and requires no specialized equipment.

Low-distortion, high-strength bonding of thermoplastic microfluidic devices employing case-II diffusion-mediated permeant activation.

We have extended the principle of optical tweezers as a noninvasive technique to actively sort hydrodynamically focused cells based on their fluorescence signal in a microfluidic device. This micro fluorescence-activated cell sorter (microFACS) uses an infrared laser to laterally deflect cells into a collection channel. Green-labeled macrophages were sorted from a 40/60 ratio mixture at a throughput of 22 cells/s over 30 min achieving a 93% sorting purity and a 60% recovery yield. To rule out potential photoinduced cell damage during optical deflection, we investigated the response of mouse macrophage to brief exposures (<4 ms) of focused 1064-nm laser light (9.6 W at the sample). We found no significant difference in viability, cell proliferation, activation state, and functionality between infrared-exposed and unexposed cells. Activation state was measured by the phosphorylation of ERK and nuclear translocation of NF-kappaB, while functionality was assessed in a similar manner, but after a lipopolysaccharide challenge. To demonstrate the selective nature of optical sorting, we isolated a subpopulation of macrophages highly infected with the fluorescently labeled pathogen Francisella tularensis subsp. novicida. A total of 10,738 infected cells were sorted at a throughput of 11 cells/s with 93% purity and 39% recovery.

Microfluidic-based cell sorting of Francisella tularensis infected macrophages using optical forces.

To enable several on-chip cell handling operations in a fused-silica substrate, small shallow micropores are radially embedded in larger deeper microchannels using an adaptation of single-level isotropic wet etching. By varying the distance between features on the photolithographic mask (mask distance), we can precisely control the overlap between two etch fronts and create a zero-thickness semi-elliptical micropore (e.g. 20 microm wide, 6 microm deep). Geometrical models derived from a hemispherical etch front show that micropore width and depth can be expressed as a function of mask distance and etch depth. These models are experimentally validated at different etch depths (25.03 and 29.78 microm) and for different configurations (point-to-point and point-to-edge). Good reproducibility confirms the validity of this approach to fabricate micropores with a desired size. To illustrate the wide range of cell handling operations enabled by micropores, we present three on-chip functionalities: continuous-flow particle concentration, immobilization of single cells, and picoliter droplet generation. (1) Using pressure differentials, particles are concentrated by removing the carrier fluid successively through a series of 44 shunts terminated by 31 microm wide, 5 microm deep micropores. Theoretical values for the concentration factor determined by a flow circuit model in conjunction with finite volume modeling are experimentally validated. (2) Flowing macrophages are individually trapped in 20 microm wide, 6 microm deep micropores by hydrodynamic confinement. The translocation of transcription factor NF-kappaB into the nucleus upon lipopolysaccharide stimulation is imaged by fluorescence microscopy. (3) Picoliter-sized droplets are generated at a 20 microm wide, 7 microm deep micropore T-junction in an oil stream for the encapsulation of individual E. coli bacteria cells.

Isotropically etched radial micropore for cell concentration, immobilization, and picodroplet generation.

Biologically functional cationic phospholipid-gold nanoplasmonic carriers have been designed to simultaneously exhibit carrier capabilities, demonstrate improved colloidal stability, and show no cytotoxicity under physiological conditions. Cargo, such as RNA, DNA, proteins, or drugs, can be adsorbed onto or incorporated into the cationic phospholipid bilayer membrane. These carriers are able to retain their unique nanoscale optical properties under physiological conditions, making them particularly useful in a wide range of imaging, therapeutic, and gene delivery applications that utilize selective nanoplasmonic properties.

Biologically functional cationic phospholipid-gold nanoplasmonic carriers of RNA.

There is significant interest in developing on-chip DNA hybridization assays to leverage the advantages of lab-on-a-chip systems, which include smaller sample and reagent volumes, faster processing speeds, and greater opportunities for large-scale integration. While much research has explored ways to integrate DNA microarrays on-chip, little work has been done to incorporate hybridization with existing microscale separation platforms. We present the first separation of single-stranded and double-stranded oligonucleotides in a nanofluidic device. We couple this separation with free-solution hybridization to develop a simple, electrokinetic technique that detects DNA hybridization without sample labeling. The technique is used both to detect target DNA sequences and to quantitatively measure hybridization kinetics. To demonstrate the method, we measured the second order reaction coefficient of complementary 20-mer oligonucleotides as a function of sodium ion concentration, which ranged from 0.0048 mol(-1).sec(-1) at 5 mM sodium to 0.42 mol(-1).sec(-1) at 50 mM. We also distinguished between a pair of complementary oligonucleotides and a pair with a single nucleotide mismatch, observing a two-fold difference in hybridization rate. Additionally, we observed a relative change in the mobility of single-stranded and double-stranded DNA with increasing sodium concentration, suggesting that our device may provide a useful platform for studying biomolecule transport in nanochannels.

Oligonucleotide hybridization and free-solution electrokinetic separation in a nanofluidic device.

Rapid and specific characterization of bacterial endospores is dependent on the ability to rupture the cell wall to enable analysis of the intracellular components. In particular, bacterial spores from the bacillus genus are inherently robust and very difficult to lyze or solubilize. Standard protocols for spore inactivation include chemical treatment, sonication, pressure, and thermal lysis. Although these protocols are effective for the inactivation of these agents, they are less well suited for sample preparation for analysis using proteomic and genomic approaches. To overcome this difficulty, we have designed a simple capillary device to perform thermal lysis of bacterial spores. Using this device, we were able to super heat (195 degrees C) an ethylene glycol lysis buffer to perform rapid flow-through rupture and solubilization of bacterial endospores. We demonstrated that the lysates from this preparation method are compatible with CGE as well as DNA amplification analysis. We further demonstrated the flow-through lysing device could be directly coupled to a miniaturized electrophoresis instrument for integrated sample preparation and analysis. In this arrangement, we were enabled to perform sample lysis, fluorescent dye labeling, and protein electrophoresis analysis of bacterial spores in less than 10 min. The described sample preparation device is rapid, simple, inexpensive, and easily integratable with various microfluidic devices.

An ultra-high temperature flow-through capillary device for bacterial spore lysis.

Next-generation sequencing (NGS) technology is a promising tool for identifying and characterizing unknown pathogens, but its usefulness in time-critical biodefense and public health applications is currently limited by the lack of fast, efficient, and reliable automated DNA sample preparation methods. To address this limitation, we are developing a digital microfluidic (DMF) platform to function as a fluid distribution hub, enabling the integration of multiple subsystem modules into an automated NGS library sample preparation system. A novel capillary interface enables highly repeatable transfer of liquid between the DMF device and the external fluidic modules, allowing both continuous-flow and droplet-based sample manipulations to be performed in one integrated system. Here, we highlight the utility of the DMF hub platform and capillary interface for automating two key operations in the NGS sample preparation workflow. Using an in-line contactless conductivity detector in conjunction with the capillary interface, we demonstrate closed-loop automated fraction collection of target analytes from a continuous-flow sample stream into droplets on the DMF device. Buffer exchange and sample cleanup, the most repeated steps in NGS library preparation, are also demonstrated on the DMF platform using a magnetic bead assay and achieving an average DNA recovery efficiency of 80%Â±4.8%.

Automated digital microfluidic sample preparation for next-generation DNA sequencing.

Digital microfluidics (DMF) has recently emerged as a popular technology for a wide range of applications. In DMF, nanoliter to microliter droplets containing samples and reagents can be manipulated to carry out a range of discrete fluidic operations simply by applying a series of electrical potentials to an array of patterned electrodes coated with a hydrophobic insulator. DMF is distinct from microchannel-based fluidics as it allows for precise control over multiple reagent phases (liquids and solids) in heterogeneous systems with no need for complex networks of connections, microvalves, or pumps. In this review, we discuss the most recent developments in this technology with particular attention to the potential benefits and outstanding challenges for applications in chemistry, biology, and medicine.

Digital microfluidics: a versatile tool for applications in chemistry, biology and medicine.

We have developed a microfluidic platform that enables, in one experiment, monitoring of signaling events spanning multiple time-scales and cellular locations through seamless integration of cell culture, stimulation and preparation with downstream analysis. A combination of two single-cell resolution techniques-on-chip multi-color flow cytometry and fluorescence imaging provides multiplexed and orthogonal data on cellular events. Automated, microfluidic operation allows quantitatively- and temporally-precise dosing leading to fine time-resolution and improved reproducibility of measurements. The platform was used to profile the toll-like receptor (TLR4) pathway in macrophages challenged with lipopolysaccharide (LPS)-beginning with TLR4 receptor activation by LPS, through intracellular MAPK signaling, RelA/p65 translocation in real time, to TNF-Î± cytokine production, all in one small macrophage population (< 5000 cells) while using minute reagent volume (540 nL/condition). The platform is easily adaptable to many cell types including primary cells and provides a generic platform for profiling signaling pathways.

Microfluidically-unified cell culture, sample preparation, imaging and flow cytometry for measurement of cell signaling pathways with single cell resolution.

We have developed an automated quality control (QC) platform for next-generation sequencing (NGS) library characterization by integrating a droplet-based digital microfluidic (DMF) system with a capillary-based reagent delivery unit and a quantitative CE module. Using an in-plane capillary-DMF interface, a prepared sample droplet was actuated into position between the ground electrode and the inlet of the separation capillary to complete the circuit for an electrokinetic injection. Using a DNA ladder as an internal standard, the CE module with a compact LIF detector was capable of detecting dsDNA in the range of 5-100 pg/Î¼L, suitable for the amount of DNA required by the Illumina Genome Analyzer sequencing platform. This DMF-CE platform consumes tenfold less sample volume than the current Agilent BioAnalyzer QC technique, preserving precious sample while providing necessary sensitivity and accuracy for optimal sequencing performance. The ability of this microfluidic system to validate NGS library preparation was demonstrated by examining the effects of limited-cycle PCR amplification on the size distribution and the yield of Illumina-compatible libraries, demonstrating that as few as ten cycles of PCR bias the size distribution of the library toward undesirable larger fragments.

Quality control of next-generation sequencing library through an integrative digital microfluidic platform.

Second-generation sequencing (SGS) has become the preferred method for RNA transcriptome profiling of organisms and single cells. However, SGS analysis of transcriptome diversity (including protein-coding transcripts and regulatory non-coding RNAs) is inefficient unless the sample of interest is first depleted of nucleic acids derived from ribosomal RNA (rRNA), which typically account for up to 95% of total intracellular RNA content. Here we describe a novel microscale hydroxyapatite chromatography (HAC) normalization method to remove eukaryotic and prokaryotic high abundant rRNA species, thereby increasing sequence coverage depth and transcript diversity across non-rRNA populations. RNA-seq analysis of Escherichia coli K-12 and human intracellular total RNA showed that HAC-based normalization enriched for all non-ribosomal RNA species regardless of RNA transcript abundance or length when compared with untreated controls. Microcolumn HAC normalization generated rRNA-depleted cDNA libraries comparable to the well-established duplex specific nuclease (DSN) normalization and Ribo-Zero rRNA-depletion methods, thus establishing microscale HAC as an effective, cost saving, and non-destructive alternative normalization technique.

cDNA normalization by hydroxyapatite chromatography to enrich transcriptome diversity in RNA-seq applications.

To fully understand the interactions of a pathogen with its host, it is necessary to analyze the RNA transcripts of both the host and pathogen throughout the course of an infection. Although this can be accomplished relatively easily on the host side, the analysis of pathogen transcripts is complicated by the overwhelming amount of host RNA isolated from an infected sample. Even with the read depth provided by second-generation sequencing, it is extremely difficult to get enough pathogen reads for an effective gene-level analysis. In this study, we describe a novel capture-based technique and device that considerably enriches for pathogen transcripts from infected samples. This versatile method can, in principle, enrich for any pathogen in any infected sample. To test the technique's efficacy, we performed time course tissue culture infections using Rift Valley fever virus and Francisella tularensis. At each time point, RNA sequencing (RNA-Seq) was performed and the results of the treated samples were compared with untreated controls. The capture of pathogen transcripts, in all cases, led to more than an order of magnitude enrichment of pathogen reads, greatly increasing the number of genes hit, the coverage of those genes, and the depth at which each transcript was sequenced.

Enriching pathogen transcripts from infected samples: a capture-based approach to enhanced host-pathogen RNA sequencing.

Next-generation sequencing (NGS) is emerging as a powerful tool for elucidating genetic information for a wide range of applications. Unfortunately, the surging popularity of NGS has not yet been accompanied by an improvement in automated techniques for preparing formatted sequencing libraries. To address this challenge, we have developed a prototype microfluidic system for preparing sequencer-ready DNA libraries for analysis by Illumina sequencing. Our system combines droplet-based digital microfluidic (DMF) sample handling with peripheral modules to create a fully-integrated, sample-in library-out platform. In this report, we use our automated system to prepare NGS libraries from samples of human and bacterial genomic DNA. E. coli libraries prepared on-device from 5 ng of total DNA yielded excellent sequence coverage over the entire bacterial genome, with >99% alignment to the reference genome, even genome coverage, and good quality scores. Furthermore, we produced a de novo assembly on a previously unsequenced multi-drug resistant Klebsiella pneumoniae strain BAA-2146 (KpnNDM). The new method described here is fast, robust, scalable, and automated. Our device for library preparation will assist in the integration of NGS technology into a wide variety of laboratories, including small research laboratories and clinical laboratories.

Article	Year
alpha-Effect with Substituted N-Methylbenzohydroxamates and Substituted Phenyldimethylsulfonium Salts: Toward Understanding of an Intrinsic alpha-Effect. The Journal of organic chemistry\| PubMed ID: 11671633	1997
In-depth characterization of slurry packed capillary columns with 1.0-microm nonporous particles using reversed-phase isocratic ultrahigh-pressure liquid chromatography. Analytical chemistry\| PubMed ID: 15456298	2004
Low-distortion, high-strength bonding of thermoplastic microfluidic devices employing case-II diffusion-mediated permeant activation. Lab on a chip\| PubMed ID: 18030407	2007
Microfluidic-based cell sorting of Francisella tularensis infected macrophages using optical forces. Analytical chemistry\| PubMed ID: 18510341	2008
Isotropically etched radial micropore for cell concentration, immobilization, and picodroplet generation. Lab on a chip\| PubMed ID: 19190785	2009
Biologically functional cationic phospholipid-gold nanoplasmonic carriers of RNA. Journal of the American Chemical Society\| PubMed ID: 19746908	2009
Oligonucleotide hybridization and free-solution electrokinetic separation in a nanofluidic device. Lab on a chip\| PubMed ID: 19789746	2009
An ultra-high temperature flow-through capillary device for bacterial spore lysis. Electrophoresis\| PubMed ID: 20737447	2010
Automated digital microfluidic sample preparation for next-generation DNA sequencing. Journal of laboratory automation\| PubMed ID: 22093297	2011
Digital microfluidics: a versatile tool for applications in chemistry, biology and medicine. Lab on a chip\| PubMed ID: 22699371	2012
Microfluidically-unified cell culture, sample preparation, imaging and flow cytometry for measurement of cell signaling pathways with single cell resolution. Lab on a chip\| PubMed ID: 22777012	2012
Quality control of next-generation sequencing library through an integrative digital microfluidic platform. Electrophoresis\| PubMed ID: 23135807	2012
cDNA normalization by hydroxyapatite chromatography to enrich transcriptome diversity in RNA-seq applications. BioTechniques\| PubMed ID: 23227988	2012
Enriching pathogen transcripts from infected samples: a capture-based approach to enhanced host-pathogen RNA sequencing. Analytical biochemistry\| PubMed ID: 23535274	2013
A microfluidic DNA library preparation platform for next-generation sequencing. PloS one\| PubMed ID: 23894387	2013

Kamlesh D. Patel

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