1,721,024 research outputs found
Immunomagnetic bead-based cell concentration microdevice for dilute pathogen detection
No full textA cell concentration microdevice for immunomagnetic pathogen isolation from a dilute sample is presented. Cells are driven by integrated on-chip pumps through a fluidized bed of immobilized immunomagnetic beads. Off-chip polymerase chain reaction and capillary electrophoretic analysis are used to determine capture efficiencies of E. coli and to optimize the system. Beads are immobilized after each split in a bifurcated channel system to ensure a balanced distribution of beads in all the capture channels. The addition of a pumping flutter step to repeatedly drive sample through the bead bed was found to enhance capture. Capture efficiencies of 70% and a limit of detection of 2 cfu/mu L were achieved; specific capture of E. coli at a concentration of 100 cfu/mu L in a 100-fold background of S. aureus is shown. This capture/concentration system is an important step in overcoming the macro-to-micro interface challenge in the development of microdevices for pathogen detection.the NIH (#U01AI056472
Multiplex mini Y short tandem repeat haplotyping using fluorescence energy transfer labeled primers
No full textA fluorescence energy transfer (ET) cassette labeled mini Y short tandem repeat (STR) genotyping system is presented. A capillary electrophoretic (CE) microdevice with a cross-injector design is used to determine the fluorescence intensities of ET and single dye-labeled STR amplicons, demonstrating a 2-12 fold higher fluorescence signal of ET cassette labels than the corresponding single dye-labeled ones. Eleven extended haplotype mini Y-STRs using ET cassette labeled primers are constructed, and sensitivity and mixed sample studies are performed. Due to the improved spectroscopic properties of ET labels, multiplex mini Y-STR typing is successful with as low as 30 pg of genomic male DNA, and in the high background of female DNA. These results indicate the practical advantage of ET cassette labels for low copy number and poor-quality DNA STR genotyping to be applied for criminal investigations, paternity testing, and evolutionary studies
A 77 K Cold Stage for Raman Microprobes and Optical Microscopy
No full textA 77‐K cold stage has been developed for spectroscopic measurements in an optical microscope. This stage eliminates fogging of the optical windows observed in an earlier design by adding vacuum jackets around the liquid nitrogen transfer lines and by maximizing the sample‐to‐environment distance without sacrificing optical imaging power. Increased maneuverability of the cold stage is achieved by using flexible stainless‐steel liquid‐nitrogen transfer lines. In our application, the sample in the cold stage is illuminated with a focused laser beam and vibrational resonance Raman scattering spectra are recorded. We have used this Raman microprobe system to measure the Raman scattering from a variety of individual rod photoreceptor cells. This work has provided new information about the mechanism of wavelength regulation in color vision
Integrated portable polymerase chain reaction-capillary electrophoresis microsystem for rapid forensic short tandem repeat typing
No full textA portable forensic genetic analysis system consisting of a microfluidic device for amplification and separation of short tandem repeat (STR) fragments as well as an instrument for chip operation and four-color fluorescence detection has been developed. The microdevice performs polymerase chain reaction (PCR) in a 160-nL chamber and capillary electrophoresis (CE) in a 7-cm-long separation channel. The instrumental design integrates PCR thermal cycling, electrophoretic separation, pneumatic valve fluidic control, and four-color laser excited fluorescence detection. A quadruplex Y-chromosome STR typing system consisting of amelogenin and three Y STR loci (DYS390, DYS393, DYS439) was developed and used for validation studies. The multiplex amplification of these 4 loci with 35 PCR cycles followed by CE separation and 4-color fluorescence detection was completed in 1.5 h. All the amplicons can be detected with a limit of detection of 20 copies of male standard DNA in the reactor. Real-world forensic analyses of oral swab and human bone extracts from case evidence were also successfully performed. Mixture analysis demonstrated that a balanced profile can be obtained even at a male-to-female template ratio of 1:10. The successful development and operation of this portable PCR-CE system establishes the feasibility of rapid point-of-analysis DNA typing of forensic casework, of mass disaster samples or of individuals at a security checkpoint.Grant 2004-DN-BX-K216 awarded by the NAtional Institute of Justic
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Microchip Technology for High-Throughput Cancer Pathology
Full text linkNovel methodologies for the molecular detection of human cancer have been advanced using microfabricated capillary electrophoresis devices as analytical platforms. These techniques enable direct and quantitative characterization of the unique biomolecular signature inherent to individual cancers and, through reduction of sample usage and analysis time, further the goal of routine genetic screening in the clinical setting.The majority of the work detailed here applies a comparative sequencing technique known as Polymorphism Ratio Sequencing (PRS) to cancer detection with a focus on mutations in the mitochondrial DNA (mtDNA). First, a rigorous optimization of sample processing protocols was undertaken to improve PRS separations on a 96-lane microfabricated sequencing device. A modification of electrokinetic injection conditions has increased capillary success rates to nearly 100%, while the introduction of dynamic coatings and automated data processing has decreased analysis time by 75%. These optimized conditions were validated through a complete mtDNA sequence comparison of two unrelated individuals, uncovering 44 confirmed germline variations, eight of which were undetected in early PRS experiments. Further analysis of paired tumor and blood mtDNA from six individuals with lung and bladder cancer revealed three heteroplasmic somatic variants while uncovering 18 erroneous mutations identified in previous microarray analysis.To establish potential clinical relevance, PRS was independently applied to a mitochondrial D-loop analysis of fourteen bladder cancer patients. A total of 21 somatic variations were identified, with seven patients harboring at least one mutation. Fifteen of these mutations were heteroplasmic, often occurring at low levels or problematic base locations inaccessible to conventional technologies. Where available, matched urine mtDNA was found to contain abundant populations of the mutant genotype, establishing the potential use of bodily fluids for noninvasive screening.Finally, an integrated microdevice capable of PRS extension followed by inline purification and electrophoretic separation is presented. This device makes use of dual on-chip thermal cyclers and orthogonal Sanger extension primers to generate a complete set of PRS fragments prior to oligonucleotide-based capture and injection. Further integration with upstream sample processing steps, including single-cell capture and PCR amplification, is proposed, providing the framework for real-time mutant quantitation in microbiopsies, ultimately enabling full clinical integration
High-performance forensic DNA profiling using fluorescence energy transfer primers and a 96-lane microfabricated capillary array
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Time Resolved Structural Studies of Photochemical Processes Using Resonance Raman
Full text linkI have used femtosecond time resolved resonance Raman spectroscopy to probe vibrational dynamics during ultrafast photochemical reactions in synthetic and biological materials, revealing nuclear motions that play key roles in the photoactivity of these systems. These results expose mechanistically significant structural details important for advancing our fundamental understanding of photochemical processes and our ability to design improved photoactive materials. In performing these studies I have improved the capabilities of tunable femtosecond stimulated Raman spectroscopy (FSRS) by developing high-throughput detection techniques and by redesigning the optical layout of the instrument for improved performance, tune-ability, and time-resolution.My initial studies were performed on a synthetic photoactive system relevant for electron transfer. The iron(II) complex, [Fe(tren(py)3)]2+, is a spin-crossover compound that undergoes an ultrafast ∆S = 2 transition upon excitation of its metal-to-ligand charge transfer band at ~ 560 nm. Using time resolved FSRS I was able to record the vibrational dynamics of this intersystem crossing during the 5 ps following actinic initiation of the photochemistry. Analysis of the time resolved vibrational spectra show that the spin-crossover process takes place in < 200 fs, and is intimately associated with the expansion of iron-ligand bonds, providing important temporal and structural characterization of the photoreactivity of this compound.I have also completed a study of the structural dynamics of the biological photoreceptor photoactive yellow protein (PYP). Upon absorption of light, the PYP chromophore, para-hydroxy-cinnamic acid, undergoes trans-cis isomerization in < 3 ps defining the primary photochemistry of the PYP photocycle. The constraints on the chromophore imposed by the protein binding pocket suggest an isomerization mechanism that involves the out-of-plane rotation of the chromophore's C9=O carbonyl. Using FSRS I was able to acquire vibrational spectra of the PYP chromophore from 0 fs to 300 ps following photoexcitation, recording the dynamics of the C9=O out-of-plane vibration during the initiation of the PYP photocycle for the first time. Following excitation, these data show structural evidence for a ~ 150 fs charge shift in the chromophore excited state preceding isomerization. The frequency of the C9=O out of plane vibration downshifts in ~ 800 fs as the excited chromophore decays to the early cissoid photocycle intermediate, I0, confirming the key role of carbonyl motion for entering the active photocycle. Following formation of I0, the structure of the chromophore is highly distorted. The relaxation of this distortion is likely a key driving force for the continuation of the PYP photocycle.The results presented in this work provide important benchmarks for the two systems studied, as well as laying the groundwork for future mechanistic studies on a variety of photochemical systems. The technique and methodology presented may be applied to the development of synthetic photoactive materials in a straightforward way, providing chemists with a direct means of identifying structural elements that promote the desired photoactivity. These experiments demonstrate the value of time resolved structural characterization in developing a clear understanding of ultrafast chemical processes, in addition to demonstrating FSRS' potential as a valuable tool for revealing the relationship between structure and function in photochemical systems
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Microfluidic technologies for quantitative single cell analysis
Full text linkMulticellular organisms, from roundworms to humans, are composed of interacting individual cells that give rise to an ensemble behavior. Most current technologies enable observation of only the population-level average and often ignore the vast degree of cell heterogeneity present even in clonal populations. Single cell resolution assays of DNA, RNA, proteins, and other biomolecules can yield insights into the complex interactions present in tissues, organs, and whole organisms. Microfluidic systems facilitate single cell analyses by leveraging the micron-scale geometry for improving sensitivity, decreasing reaction time, decreasing reagents consumption, and improving parallelization and automation for high throughput. Microfluidically-generated droplets, in particular, offer extremely high scalability of reactions and straightforward single cell manipulation. This thesis presents the development of microfluidic droplet generator designs and their application for single cell analysis. Developments in microfabricated chip design presented here have resulted in versatile droplet generation tools for a wide range of applications, while novel microfabrication techniques dramatically reduced fabrication time in commercially-viable materials. A radial micropump design increased throughput per device up to 3x106 droplets per hour, allowing us to detect via digital PCR a single pathogenic E. coli O175 in a background of 105 nonpathogenic bacteria. Developing a rapid nickel mold fabrication method has facilitated prototyping and testing of microfluidic designs in thermoplastic materials in as little as 1-3 hours. These microfabrication innovations have accelerated the pace of device development to meet the needs of single cell analysis and other applications. Microfluidic technologies are opening up research paths that so far have been difficult to pursue using conventional methods. High-throughput droplet generation is used to screen purified DNA from healthy subjects exposed to carcinogens for the lymphoma-related t(14;18) chromosomal translocation with a limit of quantitation of less than 1 mutation in 107 genomes and a dynamic range of 105. We also identify unique breakpoint sites and demonstrate the ability to quantify the relative and absolute mutation frequencies within individuals for subjects with multiple mutation events. For analysis of single cell genomes, we present a novel approach for robust DNA purification and analysis using microfluidic agarose droplet encapsulation of single cells. Agarose provides a rigid yet porous shell around cells that enables purification of whole genomes for thousands of cells in parallel without the loss of single cell fidelity. We apply this method to detecting cells containing the t(14;18) translocation and sequencing two DNA targets per cell. This is extended to 9-plex forensic profiling of single cells, thus enabling analysis of complex crime scene samples with multiple contributors or samples with excessive DNA contamination. Finally, droplets are applied to investigating multiple biological parameters per cell, including growth rate, gene expression, and alternative splicing. We perform cell culture in nanoliter droplets for fast generation and monitoring of colonies originating from single cells. Colonies are subsequently assayed for telomerase hTR RNA and hTERT mRNA expression levels and hTERT splice variants. We observe a large degree of expression level bimodality for several splice variants and significant reductions in bimodality coupled with increases in alpha splicing following exposure to sub-lethal concentrations of the anti-cancer compound curcumin. Prospects for microfluidic droplets are discussed in the context of multiparameter single cell analysis as well as applications of single cell analysis to microfluidic organs-on-a-chip. Understanding basic molecular biology mechanisms from the perspective of single cells will yield insights into behavior of multicellular populations with far-reaching scientific and clinical impacts
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High-Performance Integrated Genetic Analyzers for Forensic DNA Typing
Full text linkMicrofabrication technology offers great potential for the integration of all steps of forensic DNA typing onto a single microdevice. This integration should enable rapid, low-cost and reliable short tandem repeat (STR) analysis not only in forensic laboratories but also at crime scenes or other relevant point-of-analysis. As a first step towards making on-site STR typing possible, I developed a microdevice consisting of a 160-nL polymerase chain reaction (PCR) chamber and a 7-cm capillary electrophoresis (CE) channel and a portable instrument for its operation. A four-plex mini Y STR typing system was constructed for testing the capability of this microsystem for forensic STR typing. The successful analyses of casework and mixture samples validate the concept of forensic STR typing on a portable microfluidic system. To critically evaluate the capabilities of this portable system and the feasibility of DNA typing at a crime scene, real-time DNA analyses using a 9-plex autosomal STR typing system on a modified PCR-CE microdevice containing a co-injection structure for fragment sizing calculation were carried out at a mock crime scene. Blood stain collection, DNA extraction, STR analysis on the PCR-CE microsystem, and a DNA profile search against a mock CODIS database were successfully conducted within 6 hours of crime scene arrival. This demonstration establishes the feasibility of real-time DNA typing at a crime scene or other point-of-care situations.Finally, to achieve a total integrated analysis system for real-time STR typing, an up-front sequence-specific DNA extraction and concentration method using magnetic beads was developed and incorporated into the PCR-CE microdevice. Fragmented genomic DNA was hybridized with capture probes and immobilized onto magnetic beads via streptavidin-biotin binding in microchannels. The bead-DNA conjugates were then transported to a PCR reactor for amplification followed by inline injection using a novel capture concentration method for CE separation. This fully integrated system significantly advances the forensic DNA typing by providing a high-performance platform with sample-in-answer-out capability for real-time human identification
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