May A.K.,Vanderbilt University |
Brady J.S.,Vanderbilt University |
Romano-Keeler J.,Vanderbilt University |
Drake W.P.,Vanderbilt University |
And 4 more authors.
Chest | Year: 2015
BACKGROUND: Ventilator-associated pneumonia (VAP) remains a common complication in critically ill surgical patients, and its diagnosis remains problematic. Exhaled breath contains aerosolized droplets that reflect the lung microbiota. We hypothesized that exhaled breath condensate fluid (EBCF) in hygroscopic condenser humidifier/heat and moisture exchanger (HCH/HME) filters would contain bacterial DNA that qualitatively and quantitatively correlate with pathogens isolated from quantitative BAL samples obtained for clinical suspicion of pneumonia. METHODS: Forty-eight adult patients who were mechanically ventilated and undergoing quantitative BAL (n = 51) for suspected pneumonia in the surgical ICU were enrolled. Per protocol, patients fulfilling VAP clinical criteria undergo quantitative BAL bacterial culture. Immediately prior to BAL, time-matched HCH/HME filters were collected for study of EBCF by real-time polymerase chain reaction. Additionally, convenience samples of serially collected filters in patients with BAL-diagnosed VAP were analyzed. RESULTS: Forty-nine of 51 time-matched EBCF/BAL fluid samples were fully concordant (concordance > 95% by κ statistic) relative to identified pathogens and strongly correlated with clinical cultures. Regression analysis of quantitative bacterial DNA in paired samples revealed a statistically significant positive correlation (r = 0.85). In a convenience sample, qualitative and quantitative polymerase chain reaction analysis of serial HCH/HME samples for bacterial DNA demonstrated an increase in load that preceded the suspicion of pneumonia. CONCLUSIONS: Bacterial DNA within EBCF demonstrates a high correlation with BAL fluid and clinical cultures. Bacterial DNA within EBCF increases prior to the suspicion of pneumonia. Further study of this novel approach may allow development of a noninvasive tool for the early diagnosis of VAP. © 2015 AMERICAN COLLEGE OF CHEST PHYSICIANS.
Agency: Department of Health and Human Services | Branch: | Program: STTR | Phase: Phase II | Award Amount: 1.44M | Year: 2013
DESCRIPTION (provided by applicant): Molecular interactions form the foundation of biology and chemistry. They are central to life itself and determine catalytic activity, cellular function, and therapeutic efficacy. The vast majority of diagnostic procedures depend on some type of specific molecular interaction. Therefore, the ability to perform pure liquid-phase molecular binding analysis at high sensitivity, without modifying the interacting species, and at physiological concentrations would be revolutionary. Yet, the tools available to quantify these interactions have limitations. Traditional methods such as the sucrose gradient technique or isothermal titrimetric calorimetry are laborious and require substantial quantities of sample to perform an assay. Fluorescence and radioactive methods are sensitive, but rely on the incorporation of signaling labels to enable detection, slowing the assay and increasing its cost. Numerous techniques, particularly the label-free methods, require surface immobilizationof one of the interacting moieties putting the species in a non-native environment. Labels and tethers can be benign, but often alter the interacting molecules and can lead to a biased result. Recently my group and others demonstrated that back-scatteringinterferometry (BSI) can be used in the academic laboratory to perform molecular interaction determinations label-free and in free-solution, with sensitivity that allows assays on small quantities of sample, at physiologically relevant concentrations. BSIis a universal sensing method that only requires the product of a reaction to refract or interact with light differently than the participating species, therefore has the potential to be widely applicable for general use as a Molecular Interaction Photometer (MIP). BSI is a prime candidate to become an MIP because it has a simple and inexpensive optical train comprised of a He-Ne laser, a microfluidic channel, and a position sensor allowing minute refractive index changes to be monitored. Measurements aremade within a microfluidic channel formed in glass, fused silica, or plastic and at physiologically relevant concentrations in complex matrices such as serum or with native membrane- proteins. Yet the current academic embodiment of BSI is not commerciallyviable. Tedious alignment methods, immature transduction schemes, poorly refined sample handling and introduction methods, and performance limitations due to environmental noise sensitivity all impede the wide dissemination and adoption of BSI in the lifescience and drug discovery communities. Under Phase I of this STTR grant we met our milestones demonstrating a two-channel BSI instrument with a pipette-driven injection method and a fringe detection algorithm that simplified fringe selection and alignment. Here we propose to build on these results, while capitalizing on two new innovations to transform our academic laboratory BSI into the MIP instrument we call NanoBIND. Under this STTR Phase II, we propose the completion of four aims to refine BSI through research development and technology transfer, facilitate commercialization by Molecular Sensing Inc. and allow the subsequent broad dissemination in the research community. In Aim 1 we further simplify the optical train, while retaining the advantages ofperforming a simultaneous sample-reference determination. Aim 2 implements a sample introduction method that is user friendly, minimizes the potential for contamination and constrains volumes to lt1?L. An improved algorithm enhances sensitivity, enables electronic fringe selection and alignment and addresses non-specific interactions (at cell wall) to improve assay reproducibility in Aim 3. And in Aim 4 ?- prototype version BSI systems will be constructed and used, external to Vanderbilt and MSI, to demonstrate that BSI gives meaningful and quantitative binding affinity values (from ?M to pM) and that it can be used to screen for molecular interactions in complex matrices such as serum and cell-free media, as well as DMSO. Upon completion of these Aims, three identical prototypes will have been constructed and evaluated for field utility. Feedback from these laboratories and users will provide the formal framework for refining the design under Phase III commercial deployment. PUBLIC HEALTH RELEVANCE PUBLIC HEALTH RELEVANCE: The process of determining if two or more molecules specifically interacted forms the foundation of the chemical, biological, pharmaceutical, and medical sciences. However, sensitive bench-top methods to monitor such interactions, in particular those that are label-free and do not involve surface immobilization, are unavailable. To fulfill this need, we propose to develop a low-cost and easy to use molecular interaction photometer, based on back-scattering interferometry, whichallows a wide variety of molecular interaction studies to be performed in minutes at the lab bench by modestly skilled practitioners.
Vanderbilt University and Molecular Sensing, Inc. | Date: 2012-01-10
This invention provides an interferometric detection device configured to maintain a temperature of a sensing area to within 20 m C. of a first target temperature and to maintain a temperature of the medium within 500 m C. of a second target temperature The device can do so under conditions in which ambient temperature changes from 0.1 C. to 5 C. over 5 minutes.
Molecular Sensing, Inc. and Vanderbilt University | Date: 2010-01-08
This invention provides a device and method for collection and analysis of heterogeneous samples in a single sample container by back scattering interferometry. The sample container is configured to allow collection of a heterogeneous sample, such as blood, from a subject, separation of insoluble materials, such as blood cells by, for example, centrifugation, and mounting on a back scattering interferometer for analysis. In certain embodiments the container is a capillary tube and the interferometer comprises a mounting to hold the capillary tube in position for analysis. The device and method allow point-of-care analysis of samples.
Vanderbilt University and Molecular Sensing, Inc. | Date: 2013-05-02
This invention provides methods and systems for detecting interaction between members of a binding pair. The method involves associating one member of the binding pair with a nanoparticle and detecting the interaction between the two molecules by back-scattering interferometry.
Setif P.,CEA Saclay Nuclear Research Center |
Harris N.,Molecular Sensing, Inc. |
Lagoutte B.,CEA Saclay Nuclear Research Center |
Dotson S.,Molecular Sensing, Inc. |
Weinberger S.R.,Molecular Sensing, Inc.
Journal of the American Chemical Society | Year: 2010
The dissociation constant Kd of the photosystem I (PSI):ferredoxin complex has been measured by backscattering interferometry (BSI) with cyanobacterial PSI (350 kDa) and ferredoxin (10.5 kDa). The BSI signal, consisting of shifts for interference fringes resulting from a change in refractive index due to complex formation, was monitored as ferredoxin concentration was titrated. Kd values of 0.14-0.38 M were obtained with wild-type PSI whereas no complex was detectable with a PSI mutant containing a single mutation (R39Q) in the PsaE extrinsic subunit. These results are in quantitative agreement with previous functional determinations consisting in the detection of fast electron transfer within the complex. They provide evidence that the main contribution for the high affinity binding of ferredoxin to PSI is due to a single region of PsaE comprising arginine 39. They do not support the existence of a secondary binding site that could have escaped functional detection. © 2010 American Chemical Society.
Molecular Sensing, Inc. | Date: 2010-01-08
This invention provides methods and devices for analyzing interference patterns. The methods include fitting a Gaussian distribution to a cross correlation of two patterns from interferometric analysis of a liquid at a first and second time; identifying a positional shift of the pattern by comparing a selected value of the Gaussian distributions of the pattern at the first and second times; and determining a change in refractive index of the liquid from the positional shift. In another aspect, a method of extending the dynamic range of an interferometric data set is provided that comprises linearizing the data set, for example, using the arcsine function.
Agency: Department of Health and Human Services | Branch: | Program: SBIR | Phase: Phase II | Award Amount: 2.29M | Year: 2011
DESCRIPTION (provided by applicant): Molecular interactions form the basis of healthy metabolism as well as the manifestation of disease, and comprise the very foundation of drug treatment. Tools available to study molecular interactions in their nascent environment and physiological concentrations without chemical modification, such as surface immobilization or labeling, are limited. Current label-free technologies cannot perform homogeneous (free solution) measurements of membrane protein target interactions. Membrane proteins, which make up about 1/3 of the human proteome, interact with a wide range of biologically relevant species. A specific class of membrane proteins known as G-protein coupled receptors (GPCR) is of particular interest, as they represent the principle drug target for about 40% of all prescription pharmaceuticals and over half of the top one hundred best selling drugs. Currently, there does not exist a practical label-free means to study GPCR - lead interactions as they proceed in their native environment. Consequently, label-free approaches significantly under-serve a major drug discovery need, and as such, there exists a profound requirement for a label-free technique to support research demands in the all important GPCR and membrane protein fields. Molecular Sensing Inc.'s (MSI) Phase II SBIR proposal entitled, High Throughput, Label-free Molecular Interaction Platform for Membrane Protein Targets, leverages the achievements of our Phase I program and is directed towards producing a robust Back-Scattering Interferometry (BSI) instrument for use in drug research. In addition to refining the platform, under Phase II we will demonstrate the unique strengths and capabilities of BSI to significantly advance progress in the all important area of membrane protein drug research by capitalizing on collaborations with three world-class research environments: The Groves Laboratory at the University of California at Berkeley, the Finn Laboratory of Scripps Research Institute, and the Bornhop Laboratory at Vanderbilt University. The culmination of our Phase II program will result in the creation of a prototype research product, which the company will sell to its early access customers in translational and pharmaceutical research markets. Phase III activities will complete the product development process, making this powerful new tool accessible to laboratories worldwide. As our initial market experience of Phase I technology has taught, our commercial product will create a sustained impact to basic, translational, and drug discovery research that will positively influence healthcare through the accelerated release and development of new and powerful therapeutics and diagnostics, consistent with the mission of the National Institutes of Health. PUBLIC HEALTH RELEVANCE: High Throughput, Label-Fee Molecular Interaction Platform for Membrane Protein Targets Drugs directed against cell membrane targets comprise one of the most important classes of therapeutics and focused pharmaceutical research. Tools available to support this research lack enabling capabilities, and are a barrier to progress. Our program will result in the creation of a novel research platform that will exert a substantial and powerful impact to advance progress in membrane target drug research, yielding new and improved therapeutics that will positively impact the treatment of disease, improving healthcare while reducing its cost.
Molecular Sensing, Inc. | Date: 2013-03-15
Methods and systems for improved chemical event detection from back scattering interferometry fringe data provide sensitive detection of a chemical event by more selectively analyzing fringe shift data.