Portable Waveguide-Based Optical Biosensor.

Rapid, on-site diagnostics allow for timely intervention and response for warfighter support, environmental monitoring, and global health needs. Portable optical biosensors are being widely pursued as a means of achieving fieldable biosensing due to the potential speed and accuracy of optical detection. We recently developed the portable engineered analytic sensor with automated sampling (PEGASUS) with the goal of developing a fieldable, generalizable biosensing platform. Here, we detail the development of PEGASUS's sensing hardware and use a test-bed system of identical sensing hardware and software to demonstrate detection of a fluorescent conjugate at 1 nM through biotin-streptavidin chemistry.

A centrifugal microfluidic cross-flow filtration platform to separate serum from whole blood for the detection of amphiphilic biomarkers.

The separation of biomarkers from blood is straightforward in most molecular biology laboratories. However, separation in resource-limited settings, allowing for the successful removal of biomarkers for diagnostic applications, is not always possible. The situation is further complicated by the need to separate hydrophobic signatures such as lipids from blood. Herein, we present a microfluidic device capable of centrifugal separation of serum from blood at the point of need with a system that is compatible with biomarkers that are both hydrophilic and hydrophobic. The cross-flow filtration device separates serum from blood as efficiently as traditional methods and retains amphiphilic biomarkers in serum for detection.

Understanding the Significance of Biochemistry in the Storage, Handling, Purification, and Sampling of Amphiphilic Mycolactone.

Mycolactone, the amphiphilic macrolide toxin secreted by Mycobacterium ulcerans, plays a significant role in the pathology and manifestations of Buruli ulcer (BU). Consequently, it follows that the toxin is a suitable target for the development of diagnostics and therapeutics for this disease. Yet, several challenges have deterred such development. For one, the lipophilic nature of the toxin makes it difficult to handle and store and contributes to variability associated with laboratory experimentation and purification yields. In this manuscript, we have attempted to incorporate our understanding of the lipophilicity of mycolactone in order to define the optimal methods for the storage, handling, and purification of this toxin. We present a systematic correlation of variability associated with measurement techniques (thin-layer chromatography (TLC), mass spectrometry (MS), and UV-Vis spectrometry), storage conditions, choice of solvents, as well as the impact of each of these on toxin function as assessed by cellular cytotoxicity. We also compared natural mycolactone extracted from bacterial culture with synthesized toxins in laboratory (solvents, buffers) and physiologically relevant (serum) matrices. Our results point to the greater stability of mycolactone in organic, as well as detergent-containing, solvents, regardless of the container material (plastic, glass, or silanized tubes). They also highlight the presence of toxin in samples that may be undetectable by any one technique, suggesting that each detection approach captures different configurations of the molecule with varying specificity and sensitivity. Most importantly, our results demonstrate for the very first time that amphiphilic mycolactone associates with host lipoproteins in serum, and that this association will likely impact our ability to study, diagnose, and treat Buruli ulcers in patients.

Rapid detection of Mycobacterium tuberculosis biomarkers in a sandwich immunoassay format using a waveguide-based optical biosensor.

Early diagnosis of active tuberculosis (TB) remains an elusive challenge, especially in individuals with disseminated TB and HIV co-infection. Recent studies have shown a promise for the direct detection of pathogen-specific biomarkers such as lipoarabinomannan (LAM) for the diagnosis of TB in HIV-positive individuals. Currently, traditional immunoassay platforms that suffer from poor sensitivity and high non-specific interactions are used for the detection of such biomarkers. In this manuscript, we demonstrate the development of sandwich immunoassays for the direct detection of three TB-specific biomarkers, namely LAM, early secretory antigenic target 6 (ESAT6) and antigen 85 complex (Ag85), using a waveguide-based optical biosensor platform. Combining detection within the evanescent field of a planar optical waveguide with functional surfaces that reduce non-specific interactions allows for the ultra-sensitive and quantitative detection of biomarkers (an order of magnitude enhanced sensitivity, as compared to plate-based ELISA) in complex patient samples (urine, serum) within a short time. We also demonstrate the detection of LAM in urine from a small sample of subjects being treated for TB using this approach with excellent sensitivity and 100% corroboration with disease status. These results suggest that pathogen-specific biomarkers can be applied for the rapid and effective diagnosis of disease. It is likely that detection of a combination of biomarkers offers greater reliability of diagnosis, rather than detection of any single pathogen biomarker. NCT00341601.

Understanding the interaction of Lipoarabinomannan with membrane mimetic architectures.

Lipoarabinomannan (LAM) is a critical virulence factor in the pathogenesis of Mycobacterium tuberculosis, the causative agent of tuberculosis. LAM is secreted in urine and serum from infected patients and is being studied as a potential diagnostic indicator for the disease. Herein, we present a novel ultra-sensitive and specific detection strategy for monomeric LAM based on its amphiphilic nature and consequent interaction with supported lipid bilayers. Our strategy involves the capture of LAM on waveguides functionalized with membrane mimetic architectures, followed by detection with a fluorescently labeled polyclonal antibody. This approach offers ultra-sensitive detection of lipoarabinomannan (10 fM, within 15 min) and may be extended to other amphiphilic markers. We also show that chemical deacylation of LAM completely abrogates its association with the supported lipid bilayers. The loss of signal using the waveguide assay for deacylated LAM, as well as atomic force microscopy (AFM) images that show no change in height upon addition of deacylated LAM support this hypothesis. Mass spectrometry of chemically deacylated LAM indicates the presence of LAM-specific carbohydrate chains, which maintain antigenicity in immunoassays. Further, we have developed the first three-dimensional structural model of mannose-capped LAM that provides insights into the orientation of LAM on supported lipid bilayers.

Quantitative multiplex detection of pathogen biomarkers on multichannel waveguides.

No single biomarker can accurately predict disease. An ideal biodetection technology should be capable of the quantitative, reproducible, and sensitive detection of a limited suite of such molecules. To this end, we have developed a multiplex biomarker assay for protective antigen and lethal factor of the Bacillus anthracis lethal toxin using semiconductor quantum dots as the fluorescence reporters on our waveguide-based biosensor platform. The platform is extendable to a wide array of biomarkers, facilitating rapid, quantitative, sensitive, and multiplex detection, better than achievable by conventional immunoassay. Our assay allows for the sensitive (limit of detection 1 pM each), specific (minimal nonspecific binding), and rapid (15 min) detection of these biomarkers in complex biological samples (e.g., serum). To address the issue of reproducibility in measurement and to increase our sample throughput, we have incorporated multichannel waveguides capable of simultaneous multiplex detection of biomarkers in three samples in quadruplicate. In this paper, we present the design, fabrication, and development of multichannel waveguides for the simultaneous detection of lethal factor and protective antigen in serum. Evaluation of the multichannel waveguide shows an excellent concordance with single-channel data and effective, simultaneous, and reproducible measurement of lethal toxins in three samples.

Optimizing a waveguide-based sandwich immunoassay for tumor biomarkers: evaluating fluorescent labels and functional surfaces.

The sensor team at the Los Alamos National Laboratory has developed a waveguide-based optical biosensor for the detection of biomarkers associated with disease. We have previously demonstrated the application of this technology to the sensitive detection of carcinoembryonic antigen in serum and nipple aspirate fluid from breast cancer patients. In this publication, we report improvements to this technology that will facilitate transition to a point-of-care diagnostic system and/or robust research tool. The first improvement involved replacing phospholipid bilayers used for waveguide functionalization with self-assembled monolayers. These thin films are stable, specific, and robust silane-based surfaces that reduce nonspecific binding and enhance the signal to background ratio. Second, we have explored four different fluorescent labeling paradigms to determine the optimal procedure for use in the assay. Labeling the detector antibody with an organic dye (AlexaFluor 647) in the hinge region allows for unusual signal enhancement with repeat excitation (at 635 nm) in our assay format, thereby facilitating a better signal resolution at lower concentrations of the antigen. We have also labeled the detector antibody with photostable quantum dots through either the amine groups of lysine (Fc, NH) or using a histidine tag in the hinge region of the antibody (Hinge, H). Both labeling strategies allow for acceptable signal resolution, but quantum dots show much greater resistance to photobleaching than organic dyes.

Waveguide-based biosensors for pathogen detection.

Optical phenomena such as fluorescence, phosphorescence, polarization, interference and non-linearity have been extensively used for biosensing applications. Optical waveguides (both planar and fiber-optic) are comprised of a material with high permittivity/high refractive index surrounded on all sides by materials with lower refractive indices, such as a substrate and the media to be sensed. This arrangement allows coupled light to propagate through the high refractive index waveguide by total internal reflection and generates an electromagnetic wave-the evanescent field-whose amplitude decreases exponentially as the distance from the surface increases. Excitation of fluorophores within the evanescent wave allows for sensitive detection while minimizing background fluorescence from complex, "dirty" biological samples. In this review, we will describe the basic principles, advantages and disadvantages of planar optical waveguide-based biodetection technologies. This discussion will include already commercialized technologies (e.g., Corning's EPIC(®) Ô, SRU Biosystems' BIND(™), Zeptosense(®), etc.) and new technologies that are under research and development. We will also review differing assay approaches for the detection of various biomolecules, as well as the thin-film coatings that are often required for waveguide functionalization and effective detection. Finally, we will discuss reverse-symmetry waveguides, resonant waveguide grating sensors and metal-clad leaky waveguides as alternative signal transducers in optical biosensing.

Functional PEG-modified thin films for biological detection.

We report a general procedure to prepare functional organic thin films for biological assays on oxide surfaces. Silica surfaces were functionalized by self-assembly of an amine-terminated silane film using both vapor- and solution-phase deposition of 3'-aminopropylmethyldiethoxysilane (APMDES). We found that vapor-phase deposition of APMDES under reduced pressure produced the highest quality monolayer films with uniform surface coverage, as determined by atomic force microscopy (AFM), ellipsometry, and contact angle measurements. The amine-terminated films were chemically modified with a mixture of carboxylic acid-terminated poly(ethylene glycol) (PEG) chains of varying functionality. A fraction of the PEG chains (0.1-10 mol %) terminated in biotin, which produced a surface with an affinity toward streptavidin. When used in pseudo-sandwich assays on waveguide platforms for the detection of Bacillus anthracis protective antigen (PA), these functional PEG surfaces significantly reduced nonspecific binding to the waveguide surface while allowing for highly specific binding. Detection of PA was used to validate these films for sensing applications in both buffer and complex media. Ultimately, these results represent a step toward the realization of a robust, reusable, and autonomous biosensor.

Racemic and chiral lactams as potent, selective and functionally active CCR4 antagonists.

A series of racemic and chiral, nonracemic lactams that display high binding affinities, functional chemotaxis antagonism, and selectivity toward CCR4 are described. Compound 41, which provides reasonably high blood levels in mice when dosed intraperitoneally, was identified as a useful pharmacological tool to explore the role of CCR4 antagonism in animal models of allergic disease.

Discovery and SAR of trisubstituted thiazolidinones as CCR4 antagonists.

Substituted thiazolidinones were identified as CCR4 antagonists from high throughput screening. Subsequent lead optimization efforts resulted in defined structure-activity relationships and the identification of potent antagonists (compounds 90 and 91) that inhibited the chemotaxis of Th2 T-cells in vitro.