Chapel Hill, NC, United States
Chapel Hill, NC, United States

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Battle K.N.,Louisiana State University | Jackson J.M.,University of North Carolina at Chapel Hill | Witek M.A.,University of North Carolina at Chapel Hill | Hupert M.L.,University of North Carolina at Chapel Hill | And 6 more authors.
Analyst | Year: 2014

We present a novel microfluidic solid-phase extraction (μSPE) device for the affinity enrichment of biotinylated membrane proteins from whole cell lysates. The device offers features that address challenges currently associated with the extraction and purification of membrane proteins from whole cell lysates, including the ability to release the enriched membrane protein fraction from the extraction surface so that they are available for downstream processing. The extraction bed was fabricated in PMMA using hot embossing and was comprised of 3600 micropillars. Activation of the PMMA micropillars by UV/O3 treatment permitted generation of surface-confined carboxylic acid groups and the covalent attachment of NeutrAvidin onto the μSPE device surfaces, which was used to affinity select biotinylated MCF-7 membrane proteins directly from whole cell lysates. The inclusion of a disulfide linker within the biotin moiety permitted release of the isolated membrane proteins via DTT incubation. Very low levels (∼20 fmol) of membrane proteins could be isolated and recovered with ∼89% efficiency with a bed capacity of 1.7 pmol. Western blotting indicated no traces of cytosolic proteins in the membrane protein fraction as compared to significant contamination using a commercial detergent-based method. We highlight future avenues for enhanced extraction efficiency and increased dynamic range of the μSPE device using computational simulations of different micropillar geometries to guide future device designs. © 2014 The Royal Society of Chemistry.


Battle K.N.,Louisiana State University | Uba F.I.,University of North Carolina at Chapel Hill | Soper S.A.,Louisiana State University | Soper S.A.,University of North Carolina at Chapel Hill | And 2 more authors.
Electrophoresis | Year: 2014

The development of fully automated and high-throughput systems for proteomics is now in demand because of the need to generate new protein-based disease biomarkers. Unfortunately, it is difficult to identify protein biomarkers that are low abundant when in the presence of highly abundant proteins, especially in complex biological samples such as serum, cell lysates, and other biological fluids. Membrane proteins, which are in many cases of low abundance compared to the cytosolic proteins, have various functions and can provide insight into the state of a disease and serve as targets for new drugs making them attractive biomarker candidates. Traditionally, proteins are identified through the use of gel electrophoretic techniques, which are not always suitable for particular protein samples such as membrane proteins. Microfluidics offers the potential as a fully automated platform for the efficient and high-throughput analysis of complex samples, such as membrane proteins, and do so with performance metrics that exceed their bench-top counterparts. In recent years, there have been various improvements to microfluidics and their use for proteomic analysis as reported in the literature. Consequently, this review presents an overview of the traditional proteomic-processing pipelines for membrane proteins and insights into new technological developments with a focus on the applicability of microfluidics for the analysis of membrane proteins. Sample preparation techniques will be discussed in detail and novel interfacing strategies as it relates to MS will be highlighted. Lastly, some general conclusions and future perspectives are presented. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.


Kamande J.W.,Louisiana State University | Hupert M.L.,BioFluidica LLC | Hupert M.L.,University of North Carolina at Chapel Hill | Witek M.A.,University of North Carolina at Chapel Hill | And 12 more authors.
Analytical Chemistry | Year: 2013

In this manuscript, we discuss the development and clinical use of a thermoplastic modular microsystem for the high-throughput analysis of CTCs directly from whole blood. The modular system offers some innovative features that address challenges currently associated with many CTC platforms; it can exhaustively process 7.5 mL of blood in less than 45 min with recoveries >90%. In addition, the system automates the postselection CTC processing steps and thus, significantly reduces assay turnaround time (from selection to enumeration <1.5 h as compared to >8 h for many reported CTC platforms). The system is composed of 3 functional modules including (i) a thermoplastic CTC selection module composed of high aspect ratio (30 μm × 150 μm) channels containing anti-EpCAM antibodies that is scalable in terms of throughput by employing channel numbers ranging from 50 to 320; the channel number is user selected to accommodate the volume of blood that must be processed; (ii) an impedance sensor module for label-less CTC counting; and (iii) a staining and imaging module for the placement of released cells into a 2D array within a common imaging plane for phenotypic identification. To demonstrate the utility of this system, blood samples from patients with local resectable and metastatic pancreatic ductal adenocarcinoma (PDAC) were analyzed. We demonstrate the ability to select EpCAM positive CTCs from PDAC patients in high purity (>86%) and with excellent yields (mean = 53 CTCs per mL for metastatic PDAC patients) using our modular system. In addition, we demonstrate the ability to detect CTCs in PDAC patients with local resectable disease (mean = 11 CTCs per mL). © 2013 American Chemical Society.


Patent
Hoffmann-La Roche, University of North Carolina at Chapel Hill and Biofluidica Inc | Date: 2013-11-08

This invention provides methods and compositions for capturing circulating tumor cells (CTCs) as well as various divergent CTC phenotypes using seprase-specific affinity reagents. Methods of analyzing CTCs and assessing their metastatic potential in vivo and in vitro are also disclosed.


Hupert M.L.,University of North Carolina at Chapel Hill | Hupert M.L.,BioFluidica Inc. | Jackson J.M.,University of North Carolina at Chapel Hill | Wang H.,University of North Carolina at Chapel Hill | And 7 more authors.
Microsystem Technologies | Year: 2014

Microsystem-based technologies are providing new opportunities in the area of in vitro diagnostics due to their ability to provide process automation enabling point-of-care operation. As an example, microsystems used for the isolation and analysis of circulating tumor cells (CTCs) from complex, heterogeneous samples in an automated fashion with improved recoveries and selectivity are providing new opportunities for this important biomarker. Unfortunately, many of the existing microfluidic systems lack the throughput capabilities and/or are too expensive to manufacture to warrant their widespread use in clinical testing scenarios. Here, we describe a disposable, all-polymer, microfluidic system for the high-throughput (HT) isolation of CTCs directly from whole blood inputs. The device employs an array of high aspect ratio (HAR), parallel, sinusoidal microchannels (25 × 150 μm; W × D; AR = 6.0) with walls covalently decorated with anti-EpCAM antibodies to provide affinity-based isolation of CTCs. Channel width, which is similar to an average CTC diameter (10-20 μm), plays a critical role in maximizing the probability of cell/wall interactions and allows for achieving high CTC recovery. The extended channel depth allows for increased throughput at the optimized flow velocity (2 mm/s in a microchannel); maximizes cell recovery, and prevents clogging of the microfluidic channels during blood processing. Fluidic addressing of the microchannel array with a minimal device footprint is provided by large cross-sectional area feed and exit channels poised orthogonal to the network of the sinusoidal capillary channels (so-called Z-geometry). Computational modeling was used to confirm uniform addressing of the channels in the isolation bed. Devices with various numbers of parallel microchannels ranging from 50 to 320 have been successfully constructed. Cyclic olefin copolymer (COC) was chosen as the substrate material due to its superior properties during UV-activation of the HAR microchannels surfaces prior to antibody attachment. Operation of the HT-CTC device has been validated by isolation of CTCs directly from blood secured from patients with metastatic prostate cancer. High CTC sample purities (low number of contaminating white blood cells) allowed for direct lysis and molecular profiling of isolated CTCs. © 2013 Springer-Verlag Berlin Heidelberg.


Grant
Agency: Department of Health and Human Services | Branch: National Institutes of Health | Program: SBIR | Phase: Phase I | Award Amount: 399.68K | Year: 2015

DESCRIPTION provided by applicant Epithelial Ovarian Cancer EOC is the th leading cause of cancer related deaths among women in the US and is the leading gynecological cancer killer with cases reported in EOC has been called a andquot silent killerandquot because most women andgt are diagnosed at late stages of disease due to a lack of clinically sensitive and specific screening test and a lack of symptoms until late stage disease onset While the incidence rate of EOC in the US is highest among white females there exists a dramatic disparity in post diagnosis outcomes in other populations with African American women exhibiting substantially greater mortality based on year survival rates A combination of insufficient early detection strategies as well as reduced access to surgical treatment required at later stages of disease appears to contribute to the disparity Improved access to advanced screening assays and thereby earlier detection is therefore urgently needed to improve outcomes particularly in disparately effected populations Analysis of circulating tumor cells CTCs from cancer patientsandapos blood have demonstrated prognostic value in metastatic cancers but the use of CTCs for diagnosing early stage disease requires a degree of sensitivity and specificity currently unavailable in existing CTC analysis technologies An innovative CTC detection technology will emanate from this SBIR research project that will have unique capabilities suited to the early detection of EOC and potentially other cancers as well The technology uses low cost disposable plastic CTC fluidic cartridge that possess the ability to select CTCs directly from whole blood with extremely high recoveries andgt unprecedented purities andgt and can enumerate the captured CTCs using a label less approach with a simple single cell transducer micro CTC Coulter Counter C The technology provides a simple workflow with full process automation ideal for a screening test that can be utilized within resource limited settings The technology will process whole blood directly mL input and affinity select the CTCs release the CTCs from the capture surface and count them using an on chip C Unique to our platform is the ability to select different CTCs subpopulations from a single sample BioFluidica has tested its capture system using a novel CTC isolation antigen which as the preliminary data indicates is more prevalent in early stage disease compared to the more commonly used selection target epithelial cell adhesion molecule EpCAM The ability to capture multiple stage specific CTC subpopulations has the potential to diagnose patients at early stages of disease as well as assess treatment efficacy and the potential for disease recurrence The goal of this proposal is to initiate the development of a powerful EOC screening test accelerating treatment and improving currently poor prognoses especially in disparately affected African American women Successful completion of this Phase I program will provide the impetus to move the technology into early detection scenarios for other cancers that show disparities such as breast cancer in African American women PUBLIC HEALTH RELEVANCE Epithelial ovarian cancer EOC exhibits the th highest mortality rate amongst women in the US While its incidence rate is higher among white women epidemiological data reports a significant reduction in the year survival rates for African American women While new screening assays are broadly required to detect early stage disease an automated cost effective and accurate test has the best chance of improving the disparate mortality rates in various populations This project is focused on developing a transformative technology that can serve as a screening test which isolates circulating tumor cells CTCs from blood samples to diagnose early stage EOC appropriate for ethnic groups with EOC health disparities


Grant
Agency: Department of Health and Human Services | Branch: National Institutes of Health | Program: SBIR | Phase: Phase II | Award Amount: 1.50M | Year: 2015

DESCRIPTION provided by applicant Multiple myeloma MM is a plasma cell malignancy and comprises about of malignant tumors and of all hematopoietic neoplasms According to the American Cancer Society in an estimated new cases were diagnosed in the U S with the estimated deaths being Monoclonal gammopathy of undetermined significance MGUS is associated with a per year risk of progression to overt malignancy most commonly MM and is found in approximately of individuals over with the prevalence increasing with age Without a clear diagnostic test to monitor MGUS malignancy progression a range of factors are currently monitored including changes in serum free light chains ratio elevated serum M protein level and high bone marrow plasma cell percentage Recent studies have demonstrated that circulating multiple myeloma cells CMMCs secured from bone marrow biopsies measured by immuno uorescence microscopy or ow cytometry are useful for diagnosis prognosis and measuring therapeutic response for MM However securing a bone marrow biopsy is painful to the patient prohibiting frequent testing required for monitoring disease progression drug response and or recurrence Development of novel instrumentation and techniques for CMMC selection and analysis directly from peripheral blood is essential to improve the management of patients with MM or its early disease stages Building on extensive preliminary data secured from a Phase I type study this Phase II project is focused on the development of an integrated system that uses a disposable fluidic cartridge and the associated instrument peripherals for the rapid and fully automated processing of CMMCs directly from whole blood obviating the need for bone marrow biopsies and thus allowing for more frequent testing especially those with MGUS The instrument will be able to select CMMCs at andgt recovery and andgt purity release the selected cells and electronically enumerate them and prepare them for FISH analysis or immunophenotyping The system can also off load the selected CMMCs for molecular profiling such as searching for KRAS mutations Operation of the instrument will be automated with an overall sample to answer processing time of h The system will be subjected to both internal validation by BioFluidica staff as well as external validation by collaborators at the University of North Carolina at Chapel Hill While MM will be used as a case study for clinical performance verification of the instrument it can easily be utilized in different cancer related diseases by programming in the correct selection antibody to search for the relevant blood borne biological cells PUBLIC HEALTH RELEVANCE Building on BioFluidicaandapos s Inc experiences for analyzing circulating tumor cells CTCs this project will generate an innovative system for the analysis of circulating multiple myeloma cells CMMCs directly from peripheral blood for all stages of the disease including the early monoclonal gammopathy of undetermined significance MGUS to symptomatic multiple myeloma The project outcome will be an automated system for the analysis of CMMCs including enumeration molecular profiling and fluorescence in situ hybridization with translation into clinical settings for securing data pertinent to future FDA clearance and or implementation in CLIA laboratories enabling oncologists to obtain valuable diagnostic information currently not available on multiple myeloma

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