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Chagas disease is an inflammatory parasitic infection caused by Trypanosoma cruzi ( T. cruzi ). Early diagnosis is crucial in guiding treatment and slowing disease progression; however, current diagnostic methods have insufficient detection limits and often require skilled technicians. Molecular tests, especially isothermal nucleic acid assays, are advantageous due to their excellent sensitivity, specificity, speed, and simplicity. Here, we optimized a colorimetric loop-mediated isothermal amplification (LAMP) assay for T. cruzi . We can detect as few as 2 genomic copies/reaction using three different T. cruzi strains. We examined selectivity using other parasitic protozoans and successfully detected T. cruzi DNA extracted from parasites in human whole blood down to 1.2 parasite equivalents/reaction. We also performed a blinded study using canine blood samples and established a 100% sensitivity, specificity, and accuracy for the colorimetric LAMP assay. Finally, we used a heated 3D printer bed and an insulated thermos cup to demonstrate that the LAMP incubation step could be performed with accessible, low-cost materials. Altogether, we have developed a high-performing assay for T. cruzi with a simple colorimetric output that would be ideal for rapid, low-cost screening at the point of use.

Testing is pivotal for early identification of disease and subsequent infection control. Pathogens' nucleic acid sequence can change due to naturally-occurring genetic drift or intentional modification. Because of the reliance on molecular assays for human, animal, and plant disease diagnosis, we must understand how nucleotide mutations affect test accuracy. Primers designed against original lineages of a pathogen may be less efficient at detecting variants with genetic changes in priming regions. Here, we made single- and multi-point mutations in priming regions of a model SARS-CoV-2 template that was used as input for a loop-mediated isothermal amplification (LAMP) assay. We found that many of the modifications impacted assay sensitivity, amplification speed, or both. Further research exploring mutations at every position in each of the eight priming regions should be conducted to evaluate trends and determine generalizability.

Introduction: The SARS-CoV-2 pandemic, and the subsequent limitations on standard diagnostics, has vastly expanded the user base of Reverse Transcription Loop-mediated isothermal Amplification (RT-LAMP) in fundamental research and development. RT-LAMP has also penetrated commercial markets, with emergency use authorizations for clinical diagnosis., Areas Covered: This review discusses the role of RT-LAMP within the context of other technologies like RT-qPCR and rapid antigen tests, progress in sample preparation strategies to enable simplified workflow for RT-LAMP directly from clinical specimens, new challenges with primer and assay design for the evolving pandemic, prominent detection modalities including colorimetric and CRISPR-mediated methods, and translational research and commercial development of RT-LAMP for clinical applications., Expert Opinion: RT-LAMP occupies a middle ground between RT-qPCR and rapid antigen tests. The simplicity approaches that of rapid antigen tests, making it suitable for point-of-care use, but the sensitivity nears that of RT-qPCR. RT-LAMP still lags RT-qPCR in fundamental understanding of the mechanism, and the interplay between sample preparation and assay performance. Industry is now beginning to address issues around scalability and usability, which could finally enable LAMP and RT-LAMP to find future widespread application as a diagnostic for other conditions, including other pathogens with pandemic potential.

Respiratory viral infections are considered a major public health threat, and breath metabolomics can provide new ways to detect and understand how specific viruses affect the human pulmonary system. In this pilot study, we characterized the metabolic composition of human breath for an early diagnosis and differentiation of influenza viral infection, as well as other types of upper respiratory viral infections. We first studied the non-specific effects of planned seasonal influenza vaccines on breath metabolites in healthy subjects after receiving the immunization. We then investigated changes in breath content from hospitalized patients with flu-like symptoms and confirmed upper respiratory viral infection. The exhaled breath was sampled using a custom-made breath condenser, and exhaled breath condensate (EBC) samples were analysed using liquid chromatography coupled to quadruplole-time-of-flight mass spectrometer (LC-qTOF). All metabolomic data was analysed using both targeted and untargeted approaches to detect specific known biomarkers from inflammatory and oxidative stress biomarkers, as well as new molecules associated with specific infections. We were able to find clear differences between breath samples collected before and after flu vaccine administration, together with potential biomarkers that are related to inflammatory processes and oxidative stress. Moreover, we were also able to discriminate samples from patients with flu-related symptoms that were diagnosed with confirmatory respiratory viral panels (RVPs). RVP positive and negative differences were identified, as well as differences between specific viruses defined. These results provide very promising information for the further study of the effect of influenza A and other viruses in human systems by using a simple and non-invasive specimen like breath., (© 2021 IOP Publishing Ltd.)

Introduction : Over the past decade, loop-mediated isothermal amplification (LAMP) technology has played an important role in molecular diagnostics. Amongst numerous nucleic acid amplification assays, LAMP stands out in terms of sample-to-answer time, sensitivity, specificity, cost, robustness, and accessibility, making it ideal for field-deployable diagnostics in resource-limited regions. Areas covered : In this review, we outline the front-end LAMP design practices for point-of-care (POC) applications, including sample handling and various signal readout methodologies. Next, we explore existing LAMP technologies that have been validated with clinical samples in the field. We summarize recent work that utilizes reverse transcription (RT) LAMP to rapidly detect SARS-CoV-2 as an alternative to standard PCR protocols. Finally, we describe challenges in translating LAMP from the benchtop to the field and opportunities for future LAMP assay development and performance reporting. Expert opinion : Despite the popularity of LAMP in the academic research community and a recent surge in interest in LAMP due to the COVID-19 pandemic, there are numerous areas for improvement in the fundamental understanding of LAMP, which are needed to elevate the field of LAMP assay development and characterization.

Background Context: The North American Spine Society's (NASS) Evidence Based Clinical Guideline for the Diagnosis and Treatment of Low Back Pain features evidence-based recommendations for diagnosing and treating adult patients with nonspecific low back pain. The guideline is intended to reflect contemporary treatment concepts for nonspecific low back pain as reflected in the highest quality clinical literature available on this subject as of February 2016., Purpose: The purpose of the guideline is to provide an evidence-based educational tool to assist spine specialists when making clinical decisions for adult patients with nonspecific low back pain. This article provides a brief summary of the evidence-based guideline recommendations for diagnosing and treating patients with this condition., Study Design: This is a guideline summary review., Methods: This guideline is the product of the Low Back Pain Work Group of NASS' Evidence-Based Clinical Guideline Development Committee. The methods used to develop this guideline are detailed in the complete guideline and technical report available on the NASS website. In brief, a multidisciplinary work group of spine care specialists convened to identify clinical questions to address in the guideline. The literature search strategy was developed in consultation with medical librarians. Upon completion of the systematic literature search, evidence relevant to the clinical questions posed in the guideline was reviewed. Work group members utilized NASS evidentiary table templates to summarize study conclusions, identify study strengths and weaknesses, and assign levels of evidence. Work group members participated in webcasts and in-person recommendation meetings to update and formulate evidence-based recommendations and incorporate expert opinion when necessary. The draft guideline was submitted to an internal and external peer review process and ultimately approved by the NASS Board of Directors., Results: Eighty-two clinical questions were addressed, and the answers are summarized in this article. The respective recommendations were graded according to the levels of evidence of the supporting literature., Conclusions: The evidence-based clinical guideline has been created using techniques of evidence-based medicine and best available evidence to aid practitioners in the diagnosis and treatment of adult patients with nonspecific low back pain. The entire guideline document, including the evidentiary tables, literature search parameters, literature attrition flowchart, suggestions for future research, and all of the references, is available electronically on the NASS website at https://www.spine.org/ResearchClinicalCare/QualityImprovement/ClinicalGuidelines.aspx., (Copyright © 2020. Published by Elsevier Inc.)

Stenotrophomonas maltophilia is a Gram-negative opportunistic pathogen that can infect the lungs of people with cystic fibrosis (CF). The highly viscous mucus in the CF lung, expectorated as sputum, serves as the primary nutrient source for microbes colonizing this site and induces virulence-associated phenotypes and gene expression in several CF pathogens. Here, we characterized the transcriptional responses of three S. maltophilia strains during exposure to synthetic CF sputum medium (SCFM2) to gain insight into how this organism interacts with the host in the CF lung. These efforts led to the identification of 881 transcripts differentially expressed by all three strains, many of which reflect the metabolic pathways used by S. maltophilia in sputum, as well as altered stress responses. The latter correlated with increased resistance to peroxide exposure after pregrowth in SCFM2 for two of the strains. We also compared the SCFM2 transcriptomes of two S. maltophilia CF isolates to that of the acute infection strain, S. maltophilia K279a, allowing us to identify CF isolate-specific signatures in differential gene expression. The expression of genes from the accessory genomes was also differentially altered in response to SCFM2. Finally, a number of biofilm-associated genes were differentially induced in SCFM2, particularly in K279a, which corresponded to increased aggregation and biofilm formation in this strain relative to both CF strains. Collectively, this work details the response of S. maltophilia to an environment that mimics important aspects of the CF lung, identifying potential survival strategies and metabolic pathways used by S. maltophilia during infections. IMPORTANCE Stenotrophomonas maltophilia is an important infecting bacterium in the airways of people with cystic fibrosis (CF). However, compared to the other CF pathogens, S. maltophilia has been relatively understudied. The significance of our research is to provide insight into the global transcriptomic changes of S. maltophilia in response to a medium that was designed to mimic important aspects of the CF lung. This study elucidates the overall metabolic changes that occur when S. maltophilia encounters the CF lung and generates a road map of candidate genes to test using in vitro and in vivo models of CF., (Copyright © 2019 American Society for Microbiology.)

Staphylococcus aureus is a major human pathogen of the skin. The global burden of diabetes is high, with S. aureus being a major complication of diabetic wound infections. We investigated how the diabetic environment influences S. aureus skin infection and observed an increased susceptibility to infection in mouse models of both type I and type II diabetes. A dual gene expression approach was taken to investigate transcriptional alterations in both the host and bacterium after infection. While analysis of the host response revealed only minor changes between infected control and diabetic mice, we observed that S. aureus isolated from diabetic mice had significant increases in the levels of genes associated with translation and posttranslational modification and chaperones and reductions in the levels of genes associated with amino acid transport and metabolism. One family of genes upregulated in S. aureus isolated from diabetic lesions encoded the Clp proteases, associated with the misfolded protein response. The Clp proteases were found to be partially glucose regulated as well as influencing the hemolytic activity of S. aureus Strains lacking the Clp proteases ClpX, ClpC, and ClpP were significantly attenuated in our animal model of skin infection, with significant reductions observed in dermonecrosis and bacterial burden. In particular, mutations in clpP and clpX were significantly attenuated and remained attenuated in both normal and diabetic mice. Our data suggest that the diabetic environment also causes changes to occur in invading pathogens, and one of these virulence determinants is the Clp protease system., (Copyright © 2019 American Society for Microbiology.)

The discovery of the RNA-guided DNA nuclease CRISPR-Cas9 has enabled the targeted editing of genomes from diverse organisms, but the permanent and inheritable nature of genome modification also poses immense risks. The potential for accidental exposure, malicious use, or undesirable persistence of Cas9 therapeutics and off-target genome effects highlight the need for detection assays. Here we report a centrifugal microfluidic platform for the measurement of both Cas9 protein levels and nuclease activity. Because Cas9 from many bacterial species have been adapted for biotechnology applications, we developed the capability to detect Cas9 from the widely-used S. pyogenes , as well as S. aureus , N. meningitides, and S. thermophilus using commercially-available antibodies. Further, we show that the phage-derived anti-CRISPR protein AcrIIC1, which binds to Cas9 from several species, can be used as a capture reagent to broaden the species range of detection. As genome modification generally requires Cas9 nuclease activity, a fluorescence-based sedimentation nuclease assay was also incorporated to allow the sensitive and simultaneous measurement of both Cas9 protein and activity in a single biological sample.

Smartphones have shown promise as an enabling technology for portable and distributed point-of-care diagnostic tests. The CMOS camera sensor can be used for detecting optical signals, including fluorescence for applications such as isothermal nucleic acid amplification tests. However, such analysis is typically limited mostly to end point detection of single targets. Here we present a smartphone-based image analysis pipeline that utilizes the CIE xyY (chromaticity-luminance) color space to measure the luminance (in lieu of RGB intensities) of fluorescent signals arising from nucleic acid amplification targets, with a discrimination sensitivity (ratio between the positive to negative signals), which is an order of magnitude more than traditional RGB intensity based analysis. Furthermore, the chromaticity part of the analysis enables reliable multiplexed detection of different targets labeled with spectrally separated fluorophores. We apply this chromaticity-luminance formulation to simultaneously detect Zika and chikungunya viral RNA via end point RT-LAMP (Reverse transcription Loop-Mediated isothermal amplification). We also show real time LAMP detection of Neisseria gonorrhoeae samples down to a copy number of 3.5 copies per 10 μL of reaction volume in our smartphone-operated portable LAMP box. Our chromaticity-luminance analysis is readily adaptable to other types of multiplexed fluorescence measurements using a smartphone camera.

Loop-mediated isothermal amplification (LAMP), coupled with reverse transcription (RT), has become a popular technique for detection of viral RNA due to several desirable characteristics for use in point-of-care or low-resource settings. The large number of primers in LAMP (six per target) leads to an increased likelihood of primer dimer interactions, and the inner primers in particular are prone to formation of stable hairpin structures due to their length (typically 40-45 bases). Although primer dimers and hairpin structures are known features to avoid in nucleic acid amplification techniques, there is little quantitative information in literature regarding the impact of these structures on LAMP or RT-LAMP assays. In this study, we examine the impact of primer dimers and hairpins on previously published primer sets for dengue virus and yellow fever virus. We demonstrate that minor changes to the primers to eliminate amplifiable primer dimers and hairpins improves the performance of the assays when monitored in real time with intercalating dyes, and when monitoring a fluorescent endpoint using the QUASR technique. We also discuss the thermodynamic implications of these minor changes on the overall stability of amplifiable secondary structures, and we present a single thermodynamic parameter that can be correlated to the probability of non-specific amplification associated with LAMP primers.

We present a novel microfluidic device for size-based nucleic acid (NA) fractionation using isotachophoresis (ITP) and an ionic spacer. Our rapid-prototyped laser-cut plastic device has easily modifiable channel dimensions, can process up to 10 μL of sample, and contains an in-line extraction reservoir for minimally-disruptive manual collection of size-fractionated NAs. We designed custom buffering reservoirs using 1 mL pipette tips to provide high buffering capacity and prevent bubbles from entering the microfluidic channels. We demonstrated the utility of the device by implementing a proof-of-concept assay in which NAs were preconcentrated (via ITP) and then segregated by size (using the ionic spacer and sieving matrix) to generate two separate fractions, the first comprised of small (<50 nt) NA, and the second comprised of NAs of all sizes. Through this approach, we demonstrated size-based fractionation of both DNA and RNA samples (a mixture of synthetic ssDNA molecules, and a commercially-available RNA molecular weight standard, respectively). Our results indicate that this simple, rapid (≤10 min), and label-free approach is a promising and cost-effective alternative to the commercially-available size-selection kits currently on the market. We discuss the design and features of the device, as well as challenges which must be met in the future in order to further improve its performance and utility.

Current multiplexed diagnostics for Zika, dengue, and chikungunya viruses are situated outside the intersection of affordability, high performance, and suitability for use at the point-of-care in resource-limited settings. Consequently, insufficient diagnostic capabilities are a key limitation facing current Zika outbreak management strategies. Here we demonstrate highly sensitive and specific detection of Zika, chikungunya, and dengue viruses by coupling reverse-transcription loop-mediated isothermal amplification (RT-LAMP) with our recently developed quenching of unincorporated amplification signal reporters (QUASR) technique. We conduct reactions in a simple, inexpensive and portable "LAMP box" supplemented with a consumer class smartphone. The entire assembly can be powered by a 5 V USB source such as a USB power bank or solar panel. Our smartphone employs a novel algorithm utilizing chromaticity to analyze fluorescence signals, which improves the discrimination of positive/negative signals by 5-fold when compared to detection with traditional RGB intensity sensors or the naked eye. The ability to detect ZIKV directly from crude human sample matrices (blood, urine, and saliva) demonstrates our device's utility for widespread clinical deployment. Together, these advances enable our system to host the key components necessary to expand the use of nucleic acid amplification-based detection assays towards point-of-care settings where they are needed most.

When analyzing pathogen transcriptomes during the infection of host cells, the signal-to-background (pathogen-to-host) ratio of nucleic acids (NA) in infected samples is very small. Despite the advancements in next-generation sequencing, the minute amount of pathogen NA makes standard RNA-seq library preps inadequate for effective gene-level analysis of the pathogen in cases with low bacterial loads. In order to provide a more complete picture of the pathogen transcriptome during an infection, we developed a novel pathogen enrichment technique, which can enrich for transcripts from any cultivable bacteria or virus, using common, readily available laboratory equipment and reagents. To evenly enrich for pathogen transcripts, we generate biotinylated pathogen-targeted capture probes in an enzymatic process using the entire genome of the pathogen as a template. The capture probes are hybridized to a strand-specific cDNA library generated from an RNA sample. The biotinylated probes are captured on a monomeric avidin resin in a miniature spin column, and enriched pathogen-specific cDNA is eluted following a series of washes. To test this method, we performed an in vitro time-course infection using Klebsiella pneumoniae to infect murine macrophage cells. K. pneumoniae transcript enrichment efficiency was evaluated using RNA-seq. Bacterial transcripts were enriched up to ~400-fold, and allowed the recovery of transcripts from ~2000-3600 genes not observed in untreated control samples. These additional transcripts revealed interesting aspects of K. pneumoniae biology including the expression of putative virulence factors and the expression of several genes responsible for antibiotic resistance even in the absence of drugs., Competing Interests: The authors have declared that no competing interests exist. The authors are current or former employees of Sandia National Laboratories. The specific roles of these authors are articulated in the ‘author contributions’ section. Sandia National Laboratories did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. The authors declare no competing interests related to their employment with Sandia National Laboratories. This does not alter our adherence to PLOS ONE policies on sharing data and materials. Author ZWB is presently an employee of 10X Genomics. The authors attest that 10X Genomics had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. The authors declare no competing interests related to ZWB’s present employment with 10X Genomics.

Check valves are often essential components in microfluidic devices, enabling automated sample processing for diagnostics at the point of care. However, there is an unmet need for a check valve design that is compatible with rigid thermoplastic devices during all stages of development-from initial prototyping with a laser cutter to final production with injection molding. Here, we present simple designs for a passive, normally closed check valve that is manufactured from commonly available materials with a CO 2 laser and readily integrated into prototype and production thermoplastic devices. The check valve consists of a thermoplastic planar spring and a soft elastomeric pad that act together to seal against fluid backflow. The valve's cracking pressure can be tuned by modifying the spring's planar geometry and thickness. Seal integrity is improved with the addition of a raised annular boss beneath the elastomeric pad. To demonstrate the valve's usefulness, we employ these valves to create a finger-operated on-chip reagent reservoir and a finger-actuated pneumatic pump. We also apply this check valve to passively seal a device to enable portable detection of RNA from West Nile virus in a laser-cut device.

The emergence of Zika virus (ZIKV) infections in Latin America and Southeast Asia has created an urgent need for new, simple, yet sensitive, diagnostic tests. We highlight recent work using paper-based sensors coupled with CRISPR/Cas9 to detect ZIKV RNA as a new approach to achieve rapid development and deployment of field-ready diagnostics for emerging infectious diseases., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Multiple displacement amplification (MDA) is a widely used technique for amplification of DNA from samples containing limited amounts of DNA (e.g., uncultivable microbes or clinical samples) before whole genome sequencing. Despite its advantages of high yield and fidelity, it suffers from high amplification bias and non-specific amplification when amplifying sub-nanogram of template DNA. Here, we present a microfluidic digital droplet MDA (ddMDA) technique where partitioning of the template DNA into thousands of sub-nanoliter droplets, each containing a small number of DNA fragments, greatly reduces the competition among DNA fragments for primers and polymerase thereby greatly reducing amplification bias. Consequently, the ddMDA approach enabled a more uniform coverage of amplification over the entire length of the genome, with significantly lower bias and non-specific amplification than conventional MDA. For a sample containing 0.1 pg/μL of E. coli DNA (equivalent of ~3/1000 of an E. coli genome per droplet), ddMDA achieves a 65-fold increase in coverage in de novo assembly, and more than 20-fold increase in specificity (percentage of reads mapping to E. coli) compared to the conventional tube MDA. ddMDA offers a powerful method useful for many applications including medical diagnostics, forensics, and environmental microbiology.

Reverse-transcription-loop-mediated isothermal amplification (RT-LAMP) has frequently been proposed as an enabling technology for simplified diagnostic tests for RNA viruses. However, common detection techniques used for LAMP and RT-LAMP have drawbacks, including poor discrimination capability, inability to multiplex targets, high rates of false positives, and (in some cases) the requirement of opening reaction tubes postamplification. Here, we present a simple technique that allows closed-tube, target-specific detection, based on inclusion of a dye-labeled primer that is incorporated into a target-specific amplicon if the target is present. A short, complementary quencher hybridizes to unincorporated primer upon cooling down at the end of the reaction, thereby quenching fluorescence of any unincorporated primer. Our technique, which we term QUASR (for quenching of unincorporated amplification signal reporters, read "quasar"), does not significantly reduce the amplification efficiency or sensitivity of RT-LAMP. Equipped with a simple LED excitation source and a colored plastic gel filter, the naked eye or a camera can easily discriminate between positive and negative QUASR reactions, which produce a difference in signal of approximately 10:1 without background subtraction. We demonstrate that QUASR detection is compatible with complex sample matrices such as human blood, using a novel LAMP primer set for bacteriophage MS2 (a model RNA virus particle). Furthermore, we demonstrate single-tube duplex detection of West Nile virus (WNV) and chikungunya virus (CHIKV) RNA.

Background Context: The North American Spine Society's (NASS) Evidence-Based Clinical Guideline for the Diagnosis and Treatment of Degenerative Lumbar Spondylolisthesis features evidence-based recommendations for diagnosing and treating degenerative lumbar spondylolisthesis. The guideline updates the 2008 guideline on this topic and is intended to reflect contemporary treatment concepts for symptomatic degenerative lumbar spondylolisthesis as reflected in the highest quality clinical literature available on this subject as of May 2013. The NASS guideline on this topic is the only guideline on degenerative lumbar spondylolisthesis included in the Agency for Healthcare Research and Quality's National Guideline Clearinghouse (NGC)., Purpose: The purpose of this guideline is to provide an evidence-based educational tool to assist spine specialists when making clinical decisions for patients with degenerative lumbar spondylolisthesis. This article provides a brief summary of the evidence-based guideline recommendations for diagnosing and treating patients with this condition., Study Design: A systematic review of clinical studies relevant to degenerative spondylolisthesis was carried out., Methods: This NASS spondyolisthesis guideline is the product of the Degenerative Lumbar Spondylolisthesis Work Group of NASS' Evidence-Based Guideline Development Committee. The methods used to develop this guideline are detailed in the complete guideline and technical report available on the NASS website. In brief, a multidisciplinary work group of spine care specialists convened to identify clinical questions to address in the guideline. The literature search strategy was developed in consultation with medical librarians. Upon completion of the systematic literature search, evidence relevant to the clinical questions posed in the guideline was reviewed. Work group members used the NASS evidentiary table templates to summarize study conclusions, identify study strengths and weaknesses, and assign levels of evidence. Work group members participated in webcasts and in-person recommendation meetings to update and formulate evidence-based recommendations and incorporate expert opinion when necessary. The draft guidelines were submitted to an internal peer review process and ultimately approved by the NASS Board of Directors. Upon publication, the Degenerative Lumbar Spondylolisthesis guideline was accepted into the NGC and will be updated approximately every 5 years., Results: Twenty-seven clinical questions were addressed in this guideline update, including 15 clinical questions from the original guideline and 12 new clinical questions. The respective recommendations were graded by strength of the supporting literature, which was stratified by levels of evidence. Twenty-one new or updated recommendations or consensus statements were issued and 13 recommendations or consensus statements were maintained from the original guideline., Conclusions: The clinical guideline was created using the techniques of evidence-based medicine and best available evidence to aid practitioners in the care of patients with degenerative lumbar spondylolisthesis. The entire guideline document, including the evidentiary tables, literature search parameters, literature attrition flow chart, suggestions for future research, and all of the references, is available electronically on the NASS website at https://www.spine.org/Pages/ResearchClinicalCare/QualityImprovement/ClinicalGuidelines.aspx and will remain updated on a timely schedule., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Collection of mosquitoes and testing for vector-borne viruses is a key surveillance activity that directly influences the vector control efforts of public health agencies, including determining when and where to apply insecticides. Vector control districts in California routinely monitor for three human pathogenic viruses including West Nile virus (WNV), Western equine encephalitis virus (WEEV), and St. Louis encephalitis virus (SLEV). Reverse transcription quantitative polymerase chain reaction (RT-qPCR) offers highly sensitive and specific detection of these three viruses in a single multiplex reaction, but this technique requires costly, specialized equipment that is generally only available in centralized public health laboratories. We report the use of reverse transcription loop-mediated isothermal amplification (RT-LAMP) to detect WNV, WEEV, and SLEV RNA extracted from pooled mosquito samples collected in California, including novel primer sets for specific detection of WEEV and SLEV, targeting the nonstructural protein 4 (nsP4) gene of WEEV and the 3' untranslated region (3'-UTR) of SLEV. Our WEEV and SLEV RT-LAMP primers allowed detection of <0.1 PFU/reaction of their respective targets in <30 minutes, and exhibited high specificity without cross reactivity when tested against a panel of alphaviruses and flaviviruses. Furthermore, the SLEV primers do not cross-react with WNV, despite both viruses being closely related members of the Japanese encephalitis virus complex. The SLEV and WEEV primers can also be combined in a single RT-LAMP reaction, with discrimination between amplicons by melt curve analysis. Although RT-qPCR is approximately one order of magnitude more sensitive than RT-LAMP for all three targets, the RT-LAMP technique is less instrumentally intensive than RT-qPCR and provides a more cost-effective method of vector-borne virus surveillance.

Yersinia enterocolitica is typically considered an extracellular pathogen; however, during the course of an infection, a significant number of bacteria are stably maintained within host cell vacuoles. Little is known about this population and the role it plays during an infection. To address this question and to elucidate the spatially and temporally dynamic gene expression patterns of Y. enterocolitica biovar 1B through the course of an in vitro infection, transcriptome sequencing and differential gene expression analysis of bacteria infecting murine macrophage cells were performed under four distinct conditions. Bacteria were first grown in a nutrient-rich medium at 26 °C to establish a baseline of gene expression that is unrelated to infection. The transcriptomes of these bacteria were then compared to bacteria grown in a conditioned cell culture medium at 37 °C to identify genes that were differentially expressed in response to the increased temperature and medium but not in response to host cells. Infections were then performed, and the transcriptomes of bacteria found on the extracellular surface and intracellular compartments were analyzed individually. The upregulated genes revealed potential roles for a variety of systems in promoting intracellular virulence, including the Ysa type III secretion system, the Yts2 type II secretion system, and the Tad pilus. It was further determined that mutants of each of these systems had decreased virulence while infecting macrophages. Overall, these results reveal the complete set of genes expressed by Y. enterocolitica in response to infection and provide the groundwork for future virulence studies., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Digital microfluidics (DMF) is a powerful technique for sample preparation and analysis for a broad range of biological and chemical applications. In many cases, it is desirable to carry out DMF on an open surface, such that the matrix surrounding the droplets is ambient air. However, the utility of the air-matrix DMF format has been severely limited by problems with droplet evaporation, especially when the droplet-based biochemical reactions require high temperatures for long periods of time. We present a simple solution for managing evaporation in air-matrix DMF: just-in-time replenishment of the reaction volume using droplets of solvent. We demonstrate that this solution enables DMF-mediated execution of several different biochemical reactions (RNA fragmentation, first-strand cDNA synthesis, and PCR) over a range of temperatures (4-95 °C) and incubation times (up to 1 h or more) without use of oil, humidifying chambers, or off-chip heating modules. Reaction volumes and temperatures were maintained roughly constant over the course of each experiment, such that the reaction kinetics and products generated by the air-matrix DMF device were comparable to those of conventional benchscale reactions. This simple yet effective solution for evaporation management is an important advance in developing air-matrix DMF for a wide variety of new, high-impact applications, particularly in the biomedical sciences.

Picoinjection is a promising technique to add reagents into pre-formed emulsion droplets on chip however, it is sensitive to pressure fluctuation, making stable operation of the picoinjector challenging. We present a chip architecture using a simple pressure stabilizer for consistent and highly reproducible picoinjection in multi-step biochemical assays with droplets. Incorporation of the stabilizer immediately upstream of a picoinjector or a combination of injectors greatly reduces pressure fluctuations enabling reproducible and effective picoinjection in systems where the pressure varies actively during operation. We demonstrate the effectiveness of the pressure stabilizer for an integrated platform for on-demand encapsulation of bacterial cells followed by picoinjection of reagents for lysing the encapsulated cells. The pressure stabilizer was also used for picoinjection of multiple displacement amplification (MDA) reagents to achieve genomic DNA amplification of lysed bacterial cells.

We present a droplet-based microfluidic system for performing bioassays requiring controlled analyte encapsulation by employing highly flexible on-demand droplet generation. On-demand droplet generation and encapsulation are achieved pneumatically using a microdispensing pump connected to a constant pressure source. The system generates single droplets to the collection route only when the pump is actuated with a designated pressure level and produces two-phase parallel flow to the waste route during the stand-by state. We analyzed the effect of actuation pressure on the stability and size of droplets and optimized conditions for generation of stable droplets over a wide pressure range. By increasing the duration of pump actuation, we could either trigger a short train of identical size droplets or generate a single larger droplet. We also investigated the methodology to control droplet contents by fine-tuning flow rates or implementing a resistance bridge between the pump and main channels. We demonstrated the integrated chip for on-demand mixing between two aqueous phases in droplets and on-demand encapsulation of Escherichia coli cells. Our unique on-demand feature for selective encapsulation is particularly appropriate for bioassays with extremely dilute samples, such as pathogens in a clinical sample, since it can significantly reduce the number of empty droplets that impede droplet collection and subsequent data analysis.

Multidrug-resistant Enterobacteriaceae are emerging as a serious infectious disease challenge. These strains can accumulate many antibiotic resistance genes though horizontal transfer of genetic elements, those for β-lactamases being of particular concern. Some β-lactamases are active on a broad spectrum of β-lactams including the last-resort carbapenems. The gene for the broad-spectrum and carbapenem-active metallo-β-lactamase NDM-1 is rapidly spreading. We present the complete genome of Klebsiella pneumoniae ATCC BAA-2146, the first U.S. isolate found to encode NDM-1, and describe its repertoire of antibiotic-resistance genes and mutations, including genes for eight β-lactamases and 15 additional antibiotic-resistance enzymes. To elucidate the evolution of this rich repertoire, the mobile elements of the genome were characterized, including four plasmids with varying degrees of conservation and mosaicism and eleven chromosomal genomic islands. One island was identified by a novel phylogenomic approach, that further indicated the cps-lps polysaccharide synthesis locus, where operon translocation and fusion was noted. Unique plasmid segments and mosaic junctions were identified. Plasmid-borne blaCTX-M-15 was transposed recently to the chromosome by ISEcp1. None of the eleven full copies of IS26, the most frequent IS element in the genome, had the expected 8-bp direct repeat of the integration target sequence, suggesting that each copy underwent homologous recombination subsequent to its last transposition event. Comparative analysis likewise indicates IS26 as a frequent recombinational junction between plasmid ancestors, and also indicates a resolvase site. In one novel use of high-throughput sequencing, homologously recombinant subpopulations of the bacterial culture were detected. In a second novel use, circular transposition intermediates were detected for the novel insertion sequence ISKpn21 of the ISNCY family, suggesting that it uses the two-step transposition mechanism of IS3. Robust genome-based phylogeny showed that a unified Klebsiella cluster contains Enterobacter aerogenes and Raoultella, suggesting the latter genus should be abandoned.

Background Context: The objective of the North American Spine Society's (NASS) Evidence-Based Clinical Guideline for the Diagnosis and Treatment of Lumbar Disc Herniation with Radiculopathy is to provide evidence-based recommendations to address key clinical questions surrounding the diagnosis and treatment of lumbar disc herniation with radiculopathy. The guideline is intended to reflect contemporary treatment concepts for symptomatic lumbar disc herniation with radiculopathy as reflected in the highest quality clinical literature available on this subject as of July 2011. The goals of the guideline recommendations are to assist in delivering optimum efficacious treatment and functional recovery from this spinal disorder., Purpose: To provide an evidence-based educational tool to assist spine specialists in the diagnosis and treatment of lumbar disc herniation with radiculopathy., Study Design: Systematic review and evidence-based clinical guideline., Methods: This guideline is a product of the Lumbar Disc Herniation with Radiculopathy Work Group of NASS' Evidence-Based Guideline Development Committee. The work group consisted of multidisciplinary spine care specialists trained in the principles of evidence-based analysis. A literature search addressing each question and using a specific search protocol was performed on English-language references found in Medline, Embase (Drugs and Pharmacology), and four additional evidence-based databases to identify articles. The relevant literature was then independently rated using the NASS-adopted standardized levels of evidence. An evidentiary table was created for each of the questions. Final recommendations to answer each clinical question were developed via work group discussion, and grades were assigned to the recommendations using standardized grades of recommendation. In the absence of Level I to IV evidence, work group consensus statements have been developed using a modified nominal group technique, and these statements are clearly identified as such in the guideline., Results: Twenty-nine clinical questions were formulated and addressed, and the answers are summarized in this article. The respective recommendations were graded by strength of the supporting literature, which was stratified by levels of evidence., Conclusions: The clinical guideline has been created using the techniques of evidence-based medicine and best available evidence to aid practitioners in the care of patients with symptomatic lumbar disc herniation with radiculopathy. The entire guideline document, including the evidentiary tables, suggestions for future research, and all the references, is available electronically on the NASS Web site at http://www.spine.org/Pages/PracticePolicy/ClinicalCare/ClinicalGuidlines/Default.aspx and will remain updated on a timely schedule., (Copyright © 2014 Elsevier Inc. All rights reserved.)

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.

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/μL, 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., (© 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Rapid separation of nucleic acids by microchip electrophoresis could streamline many biological applications, but conventional chip injection strategies offer limited sample stacking, and thus limited sensitivity of detection. We demonstrate the use of photopatterned polyacrylamide membranes in a glass microfluidic device, with or without fixed negative charges, for preconcentration of double-stranded DNA prior to electrophoretic separation to enhance detection limits. We compared performance of the two membrane formulations (neutral or negatively charged) as a function of DNA fragment size, preconcentration time, and preconcentration field strength, with the intent of optimizing preconcentration performance without degrading the subsequent electrophoretic separation. Little size-dependent bias was observed for either membrane formulation when concentrating dsDNA > 100 bp in length, while the negatively charged membrane more effectively blocks passage of single-stranded oligonucleotide DNA (20-mer ssDNA). Baseline resolution of a six-band dye-labeled ladder with fragments 100-2000 bp in size was obtained in <120 s of separation time, with peak efficiencies in the range of 2000-15,000 plates/cm, and detection limits as low as 1 pM per single dye-labeled fragment. The degree of preconcentration is tunable by at least 49-fold, although the efficiency of preconcentration was found to have diminishing returns at high field and/or long times. The neutral membrane was found to be more robust than the negatively charged membrane, with approximately 2.5-fold larger peak area during the subsequent separation, and less decrease in resolution upon increasing the preconcentration field strength., (© 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

We describe an integrated microfluidic device (μFlowFISH) capable of performing 16S rRNA fluorescence in situ hybridization (FISH) followed by flow cytometric detection for identifying bacteria in natural microbial communities. The device was used for detection of species involved in bioremediation of Cr(vi) and other metals in groundwater samples from a highly-contaminated environmental site (Hanford, WA, USA). The μFlowFISH seamlessly integrates two components: a hybridization chamber formed between two photopolymerized membranes, where cells and probes are electrophoretically loaded, incubated and washed, and a downstream cross structure for electrokinetically focusing cells into a single-file flow for flow cytometry analysis. The device is capable of analyzing a wide variety of bacteria including aerobic, facultative and anaerobic bacteria and was initially tested and validated using cultured microbes, including Escherichia coli, as well as two strains isolated from Hanford site: Desulfovibrio vulgaris strain RCH1, and Pseudomonas sp.strain RCH2 that are involved in Cr(vi) reduction and immobilization. Combined labeling and detection efficiencies of 74-97% were observed in experiments with simple mixtures of cultured cells, confirming specific labeling. Results obtained were in excellent agreement with those obtained by conventional flow cytometry confirming the accuracy of μFlowFISH. Finally, the device was used for analyzing water samples collected on different dates from the Hanford site. We were able to monitor the numbers of Pseudomonas sp. with only 100-200 cells loaded into the microchip. The μFlowFISH approach provides an automated platform for quantitative detection of microbial cells from complex samples, and is ideally suited for analysis of precious samples with low cell numbers such as those found at extreme environmental niches, bioremediation sites, and the human microbiome., (This journal is © The Royal Society of Chemistry 2011)

Directing the orientation of molecular assemblies is a key step toward creating complex hierarchical structures that yield higher order functional materials. Here, we demonstrate the directed orientation of functionalized lipid domains and protein-membrane assemblies, using an electric field.

Protein-based polymers are increasingly being used in biomaterial applications because of their ease of customization and potential monodispersity. These advantages make protein polymers excellent candidates for bioanalytical applications. Here we describe improved methods for producing drag-tags for free-solution conjugate electrophoresis (FSCE). FSCE utilizes a pure, monodisperse recombinant protein, tethered end-on to a ssDNA molecule, to enable DNA size separation in aqueous buffer. FSCE also provides a highly sensitive method to evaluate the polydispersity of a protein drag-tag and thus its suitability for bioanalytical uses. This method is able to detect slight differences in drag-tag charge or mass. We have devised an improved cloning, expression, and purification strategy that enables us to generate, for the first time, a truly monodisperse 20 kDa protein polymer and a nearly monodisperse 38 kDa protein. These newly produced proteins can be used as drag-tags to enable longer read DNA sequencing by free-solution microchannel electrophoresis.

We report a capillary-based DNA sequencing read length of 100 bases in 16 min using end-labeled free-solution conjugate electrophoresis (FSCE) with a monodisperse poly-N-substituted glycine (polypeptoid) as a synthetic drag-tag. FSCE enabled rapid separation of single-stranded (ss) DNA sequencing fragments with single-base resolution without the need for a viscous DNA separation matrix. Protein-based drag-tags previously used for FSCE sequencing, for example, streptavidin, are heterogeneous in molar mass (polydisperse); the resultant band-broadening can make it difficult to obtain the single-base resolution necessary for DNA sequencing. In this study, we synthesized and HPLC-purified a 70mer poly-N-(methoxyethyl)glycine (NMEG) drag-tag with a molar mass of - 11 kDa. The NMEG monomers that comprise this peptoid drag-tag are interesting for bioanalytical applications, because the methoxyethyl side chain's chemical structure is reminiscent of the basic monomer unit of polyethylene glycol, a highly biocompatible commercially available polymer, which, however, is not available in monodisperse preparation at an - 11 kDa molar mass. This is the first report of ssDNA separation and of four-color, base-by-base DNA sequencing by FSCE through the use of a chemically synthesized drag-tag. These results show that high-molar mass, chemically synthesized drag-tags based on the polyNMEG structure, if obtained in monodisperse preparation, would serve as ideal drag-tags and could help FSCE reach the commercially relevant read lengths of 100 bases or more.

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.

Towards designing a portable diagnostic device for detecting biological toxins in bodily fluids, we have developed microfluidic chip-based immunoassays that are rapid (< 20 minutes), require minimal sample volume (<10 microL) and have appreciable sensitivity and dynamic range (microM-pM). The microfluidic chip is being integrated with miniaturized electronics, optical elements, fluid-handling components, and data acquisition software to develop a portable, self-contained device. The device is intended for rapid, point-of-care (and, in future, point-of-incident) testing in case of an accidental or intentional exposure/intoxication to biotoxins. Detection of toxins and potential host-response markers is performed using microfluidic electrophoretic immunoassays integrated with sample preconcentration and mixing of analytes with fluorescently labeled antibodies. Preconcentration is enabled by photopolymerizing a thin, nanoporous membrane with a MW cut-off of approximately 10 kDa in the sample loading region of the chip. Polymeric gels with larger pores are located adjacent to the size exclusion membrane to perform electrophoretic separation of antibody-analyte complex and excess antibody. Measurement of the ratio of bound and unbound immune-complex using sensitive laser-induced fluorescence detection provides quantitation of analyte in the sample. We have demonstrated electrophoretic immunoassays for the biotoxins ricin, Shiga toxin I, and Staphylococcal enterotoxin B (SEB). With off-chip mixing and no sample preconcentration, the limits of detection (LOD) were 300 pM for SEB, 500 pM for Shiga toxin I, and 20 nM for ricin. With a 10 min on-chip preconcentration, the LOD for SEB is <10 pM. The portable device being developed is readily applicable to detection of proteinaceous biomarkers of many other diseases and is intended to represent the next-generation diagnostic devices capable of rapid and quantitative measurements of multiple analytes simultaneously.

We have developed a new method for the analysis of low abundant point mutations in genomic DNA using a combination of an allele-specific ligase detection reaction (LDR) with free-solution conjugate electrophoresis (FSCE) to generate and analyze the genetic products. FSCE eliminates the need for a polymer sieving matrix by conjugating chemically synthesized polyamide "drag-tags" onto the LDR primers. The additional drag of the charge-neutral drag-tag breaks the linear scaling of the charge-to-friction ratio of DNA and enables size-based separations of DNA in free solution using electrophoresis with no sieving matrix. We successfully demonstrate the conjugation of polyamide drag-tags onto a set of four LDR primers designed to probe the K-ras oncogene for mutations highly associated with colorectal cancer, the simultaneous generation of fluorescently labeled LDR/drag-tag conjugate (LDR-dt) products in a multiplexed, single-tube format with mutant:WT ratios as low as 1:100, respectively, and the single-base, high-resolution separation of all four LDR-dt products. Separations were conducted in free solution with no polymer network using both a commercial capillary array electrophoresis (CAE) system and a PMMA microchip replicated via hot-embossing with only a Tris-based running buffer containing additives to suppress the EOF. Typical analysis times for LDR-dt were 11 min using the CAE system and as low as 85 s for the PMMA microchips. With resolution comparable to traditional gel-based CAE, FSCE along with microchip electrophoresis decreased the separation time by more than a factor of 40.

We demonstrate the first use of a non-natural, genetically engineered protein polymer drag-tag to sequence DNA fragments by end-labeled free-solution electrophoresis (ELFSE). Fluorescently labeled DNA fragments resulting from the Sanger cycle sequencing reaction were separated by free-solution capillary electrophoresis, with much higher resolution and cleaner results than previously reported for this technique. With ELFSE, size-based separation of DNA in the absence of a sieving matrix is enabled by the end-on attachment of a polymeric "drag-tag" that modifies the charge-to-friction ratio of DNA in a size-dependent fashion. Progress in ELFSE separations has previously been limited by the lack of suitable large, monodisperse drag-tags. To address this problem, we designed, constructed, cloned, expressed, and purified a non-natural, genetically engineered 127mer protein polymer for use as an ELFSE drag-tag. The Sanger cycle sequencing reaction is performed with the drag-tag covalently attached to the sequencing primer, a major advance over previous strategies for ELFSE sequencing. The electrophoretic separation is diffusion-limited, without significant adsorption of the drag-tag to capillary walls. Although the read length (at about 180 bases) is still short, our results provide evidence that larger protein polymer drag-tags, currently under development, could extend the read length of ELFSE to more competitive levels. ELFSE offers the possibility of very rapid DNA sequencing separations without any of the difficulties associated with viscous polymeric sieving networks and hence will be amenable to implementation in microchannel and chip-based electrophoresis systems.

High-throughput screening assays of native and recombinant proteins are increasingly crucial in life science research, including fields such as drug screening and enzyme engineering. These assays are typically highly parallel, and require minute amounts of purified protein per assay. To address this need, we have developed a rapid, automated microscale process for isolating specific proteins from sub-microlitre volumes of E. Coli cell lysate. Recombinant proteins are genetically tagged to drive partitioning into the PEG-rich phase of a flowing aqueous two-phase system, which removes approximately 85% of contaminating proteins, as well as unwanted nucleic acids and cell debris, on a simple microfluidic device. Inclusion of the genetic tag roughly triples recovery of the autofluorescent protein AcGFP1, and also significantly improves recovery of the enzyme glutathione S-transferase (GST), from nearly zero recovery for the wild-type enzyme, up to 40% with genetic tagging. The extraction process operates continuously, with only a single step from cell lysate to purified protein, and does not require expensive affinity reagents or troublesome chromatographic steps. The two-phase system is mild and does not disrupt protein function, as evidenced by recovery of active enzymes and functional fluorescent protein from our microfluidic process. The microfluidic aqueous two-phase extraction forms the core component of an integrated lab-on-a-chip device comprising cell culture, lysis, purification and analysis on a single device.

The conjugation of an uncharged polymer to DNA fragments makes it possible to separate DNA by free-solution electrophoresis. This end-labeled free-solution electrophoresis method has been shown to successfully separate ssDNA with single monomer resolution up to about 110 bases. It is the aim of this paper to investigate in more detail the coupled hydrodynamic and electrophoretic deformation of the ssDNA-label conjugate at fields below 400 V/cm. Our model is an extension of the theoretical approach originally developed by Stigter and Bustamante [Biophys. J. 75, 1197 (1998)] to investigate the problems of a tethered chain stretching in a hydrodynamic flow and of the electrophoretic stretch of a tethered polyelectrolyte. These two separate models are now used together since the charged DNA is "tethered" to the uncharged polymer (and vice versa), and the resulting self-consistent model is used to predict the deformation and the electrophoretic velocity for the hybrid molecule. Our theoretical and experimental results are in good qualitative agreement.

We report a new, bioconjugate approach to performing highly multiplexed single-base extension (SBE) assays, which we demonstrate by genotyping a large panel of point mutants in exons 5-9 of the p53 gene. A series of monodisperse polyamide "drag-tags" was created using both chemical and biological synthesis and used to achieve the high-resolution separation of genotyping reaction products by microchannel electrophoresis without a polymeric sieving matrix. A highly multiplexed SBE reaction was performed in which 16 unique drag-tagged primers simultaneously probe 16 p53 gene loci, with an abbreviated thermal cycling protocol of only 9 min. The drag-tagged SBE products were rapidly separated by free-solution conjugate electrophoresis (FSCE) in both capillaries and microfluidic chips with genotyping accuracy in excess of 96%. The separation requires less than 70 s in a glass microfluidic chip, or about 20 min in a commercial capillary array sequencing instrument. Compared to gel electrophoresis, FSCE offers greater freedom in the design of SBE primers by essentially decoupling the length of the primer and the electrophoretic mobility of the genotyping products. FSCE also presents new possibilities for the facile implementation of SBE on integrated microfluidic electrophoresis devices for rapid, high-throughput genetic mutation detection or SNP scoring.

In end-labeled free-solution electrophoresis (ELFSE), DNA molecules are labeled with a frictional modifier or "drag-tag", allowing their size-based electrophoretic separation in free solution. Among the interesting observations from early work with dsDNA using streptavidin as a drag-tag was that the drag induced by including a streptavidin label at both ends was significantly more than double that from a single streptavidin (Heller, C. et al.., J. Chromatogr. A 1998, 806, 113-121). This finding was assumed to be in error, and subsequent work focused on experiments in which only a single drag-tag is appended to one end of the DNA molecule. Recent theoretical work (McCormick, L. C., Slater, G. W., Electrophoresis 2005, 26, 1659-1667) has examined the contribution of end-effects to the free-solution electrophoretic mobility of charged-uncharged polymer conjugates, reopening the question of enhanced drag from placing a drag-tag at both ends. In this study, this effect is investigated experimentally, using custom-synthesized ssDNA oligonucleotides allowing the attachment of drag-tags to one or both ends, as well as dsDNA PCR products generated with primers appropriate for the attachment of drag-tags at one or both ends. A range of sizes of drag-tags are used, including synthetic polypeptoid drag-tags as well as genetically engineered protein polymer drag-tags. The enhanced drag arising from labeling both ends has been confirmed, with 6-9% additional drag for the ssDNA and 10-23% additional drag for the dsDNA arising from labeling both ends than would be expected from simply doubling the size of the drag-tag at one end. The experimental results for ssDNA labeled at both ends are compared to the predictions of the recent theory of end-effects, with reasonably good quantitative agreement. These experimental findings demonstrate the feasibility of enhancing ELFSE separations by labeling both ends of the DNA molecule, leading to greater resolving power and a wider range of applications for this technique.

The development of innovative technologies designed to reduce the cost and increase the throughput of DNA separations continues to be important for large-scale sequencing and genotyping efforts. We report research aimed at the further development of a free-solution bioconjugate method of DNA size separation by capillary electrophoresis (CE), in particular, the determination of an optimal molecular architecture for polyamide-based "drag-tags". We synthesized several branched poly(N-methoxyethyl glycine)s (poly(NMEG)s, a class of polypeptoids) as novel friction-generating entities for end-on attachment to DNA molecules. A 30-mer poly(NMEG) "backbone," comprising five evenly spaced reactive epsilon-amino groups, was synthesized on solid phase, cleaved, and purified to monodispersity by RP-HPLC. Three different comblike derivatives of this backbone molecule were created by (1) acetylating the epsilon-amino groups or (2) appending small, monodisperse NMEG oligomers (a tetramer and an octamer). Grafting of the oligo(NMEG)s was done using solution-phase amide bond formation chemistry. Once purified to total monodispersity, the three different drag-tags were studied by free-solution electrophoresis to observe the effect of branching on their hydrodynamic drag or "alpha" and hence their ability to separate DNA. Drag was found to scale linearly with total molecular weight, regardless of branch length. The octamer-branched drag-tag-DNA conjugate was used to separate ssDNA products of 50, 75, 100, and 150 bases in length by free-solution CE in less than 10 min. Hence, the use of branched or comblike drag-tags is both a feasible and an effective way to achieve high frictional drag, allowing the high-resolution separation of relatively large DNA molecules by free-solution CE without the need to synthesize very long polymers.

Exposure of therapeutic and diagnostic medical devices to biological fluids is often accompanied by interfacial adsorption of proteins, cells, and microorganisms. Biofouling of surfaces can lead to compromised device performance or increased cost and in some cases may be life-threatening to the patient. Although numerous antifouling polymer coatings have enjoyed short-term success in preventing protein and cell adsorption on surfaces, none have proven ideal for conferring long-term biofouling resistance. Here we describe a new biomimetic antifouling N-substituted glycine polymer (peptoid) containing a C-terminal peptide anchor derived from residues found in mussel adhesive proteins for robust attachment of the polymer onto surfaces. The methoxyethyl side chain of the peptoid portion of the polymer was chosen for its chemical resemblance to the repeat unit of the known antifouling polymer poly(ethylene glycol) (PEG), whereas the composition of the 5-mer anchoring peptide was chosen to directly mimic the DOPA- and Lys-rich sequence of a known mussel adhesive protein. Surfaces modified with this biomimetic peptide-peptoid conjugate exhibited dramatic reduction of serum protein adsorption and resistance to mammalian cell attachment for over 5 months in an in vitro assay. These new synthetic peptide based antifouling polymers may provide long-term control of surface biofouling in the physiologic, marine, and industrial environments.

We demonstrate the feasibility of end-labeled free-solution electrophoresis (ELFSE) separation of DNA using genetically engineered protein polymers as drag-tags. Protein polymers are promising candidates for ELFSE drag-tags because their sequences and lengths are controllable not only to generate monodisperse polymers with high frictional drag, but also to meet other drag-tag requirements for high-resolution separations by microchannel electrophoresis. A series of repetitive polypeptides was designed, expressed in Escherichia coli, and purified. By performing an end-on conjugation of the protein polymers to a fluorescently labeled DNA oligomer (22 bases) and analyzing the electrophoretic mobilities of the conjugate molecules by free-solution capillary electrophoresis (CE), effects of the size and charge of the protein polymer drag-tags were investigated. In addition, the electrophoretic behavior of bioconjugates comprising relatively long DNA fragments (108 and 208 bases) and attached to uncharged drag-tags was observed, by conjugating fluorescently labeled polymerase chain reaction (PCR) products to charge-neutral protein polymers, and analyzing via CE. We calculated the amount of friction generated by the various drag-tags, and estimated the potential read-lengths that could be obtained if these drag-tags were used for DNA sequencing in our current system. The results of these studies indicate that larger and uncharged drag-tags will have the best DNA-resolving capability for ELFSE separations, and that theoretically, up to 233 DNA bases could be sequenced using one of the protein polymer drag-tags we produced, which is electrostatically neutral with a chain length of 337 amino acids. We also show that denatured (unfolded) polypeptide chains impose much greater frictional drag per unit molecular weight than folded proteins, such as streptavidin, which has been used as a drag-tag before.

DNA is a free-draining polymer. This subtle but "unfortunate" property of highly charged polyelectrolytes makes it impossible to separate nucleic acids by free-flow electrophoresis. This is why one must typically use a sieving matrix, such as a gel or an entangled polymer solution, in order to obtain some electrophoretic size separation. An alternative approach consists of breaking the charge to friction balance of free-draining DNA molecules. This can be achieved by labeling the DNA with a large, uncharged molecule (essentially a hydrodynamic parachute, which we also call a drag-tag) prior to electrophoresis; the resulting methodology is called end-labeled free-solution electrophoresis (ELFSE). In this article, we review the development of ELFSE over the last decade. In particular, we examine the theoretical concepts used to predict the ultimate performance of ELFSE for single-stranded (ssDNA) sequencing, the experimental results showing that ELFSE can indeed overcome the free-draining issue raised above, and the technological advances that are needed to speed the development of competitive ELFSE-based sequencing and separation technologies. Finally, we also review the reverse process, called free-solution conjugate electrophoresis (FSCE), wherein uncharged polymers of different sizes can be analyzed using a short DNA molecule as an electrophoretic engine.

TEMPLE UNIVERSITY'S NEUROSURGERY program has had a colorful and distinguished history since its creation in 1929. It has always functioned under challenging circumstances with limited resources but with a strong sense of mission. It was one of the 20 neurosurgical training programs in existence when the American Board of Neurosurgery was founded in 1940. Over the past 64 years, the program has trained approximately the same number of neurosurgeons, many of whom have contributed significantly to our specialty. Some of the advances pioneered in part at Temple include clinical hypothermia (Fay), the biplanar stereoscopic angiographic unit (Chamberlain), human stereotactic surgery (Spiegel and Wycis), lumboperitoneal shunts (Scott), posterior lumbar interbody fusion (Lin), microsurgery for acoustic tumors (Buchheit), and new pharmacological approaches to neuroprotectors (Strauss and Narayan). The Temple neurosurgery program has survived many challenges in the past and will no doubt weather the current financial and medicolegal challenges that confront the neurosurgical community in Philadelphia. It remains a strong clinical program that serves an otherwise underserved community and attracts patients beyond its geographic area because of its strong clinical reputation and the excellence of its clinical faculty and residents.

We describe a novel method for the determination of glutamine deamidation in a long protein polymer via bioconjugate capillary electrophoresis. Since the current best technique for detection of glutamine (or asparagine) deamidation is mass spectrometry, it is practically impossible to precisely detect the degree of deamidation (i.e., how many residues are deamidated in a polypeptide) in a large protein containing a significant number of glutamine (or asparagine) residues, because the mass difference between native and deamidated residues is just 1 atomic mass unit. However, by covalently attaching polydisperse protein polymers (337 residues) to a monodisperse DNA oligomer (22 bases), the degree of glutamine deamidation, which could not be determined accurately by mass spectrometry, was resolved by free-solution capillary electrophoresis. Electrophoretic separations were carried out after different durations of exposure of the protein to a cyanogen bromide cleavage reaction mixture, which is a general treatment for the purpose of removing an oligopeptide affinity purification tag from fusion proteins. For protein polymers with increasing extents of deamidation, the electromotive force of DNA + polypeptide conjugate molecules increases due to the introduced negative charge of deamidated glutamic acid residues, and consequently CE analysis reveals increasing differences in the electrophoretic mobilities of conjugate molecules, which qualitatively shows the degree of deamidation. Peak analysis of the electropherograms enables quantitative determination of the first four deamidations in a protein polymer. A first-order rate constant of 0.018 h(-1) was determined for the deamidation of a single glutamine residue in the protein polymer during the cyanogen bromide cleavage reaction.