Among the different biomolecules found in human saliva, proteins are among the most prominent. Proteins carry out many vital roles in saliva, from enzymatic catalysis to structure. Proteins also have great potential as diagnostic biomarkers, which can be easily collected using non-invasive methods. In order to catalog salivary proteins, and determine those with diagnostic potential for human disease and other health conditions, mass spectrometry-based proteomic technologies have been used over the last decade or more. Using these conventional technologies, several thousand proteins have been identified in whole saliva, with numerous studies identifying proteins with diagnostic promise for a variety of health conditions. In addition, these technologies have been used to identify proteins expressed by microbial communities in human saliva. In recent years, new mass spectrometry-based proteomics technologies have emerged that are giving researchers new tools for characterizing the proteome of whole saliva, as well as other biological sample types. Two of the most promising of these new technologies include methods for “data-independent acquisition”, which allows for large-scale hypothesis-driven proteomic discovery studies, and proteogenomics, a bioinformatics-driven approach which integrates genomic and proteomic data to characterize novel gene products missed by conventional methods. This paper will summarize the efforts of cataloging the saliva proteome to-date, including highlights of studies identifying salivary protein biomarkers of disease. In addition, the emerging technologies of data-independent acquisition and proteogenomics will be described, along with their potential in characterizing the saliva proteome for diagnostic applications.
The field of “salivary diagnostics” includes studies utilizing samples obtained from a variety of sources within the oral cavity. These samples include; whole unstimulated saliva, stimulated whole saliva, duct saliva collected directly from the parotid, submandibular/sublingual glands or minor salivary glands, swabs of the buccal mucosa, tongue or tonsils, and gingival crevicular fluid. Many publications state “we collected saliva from subjects” without fully describing the process or source of the oral fluid. Factors that need to be documented in any study include the time of day of the collection, the method used to stimulate and collect the fluid, and how much fluid is being collected and for how long. The handling of the oral fluid during and post-collection is also critical and may include addition of protease or nuclease inhibitors, centrifugation, and cold or frozen storage prior to assay. In an effort to create a standard protocol for determining a biomarker’s origin we carried out a pilot study collecting oral fluid from 5 different sites in the mouth and monitoring the concentrations of pro- and anti-inflammatory cytokines detected using MesoScaleDiscovery (MSD) electrochemiluminesence assays. Our data suggested that 3 of the cytokines are primarily derived from the submandibular gland, while 7 of the cytokines come from a source other than the major salivary glands such as the minor salivary glands or cells in the oral mucosae. Here we review the literature on monitoring biomarkers in oral samples and stress the need for determining the blood/saliva ratio when a quantitative determination is needed and suggest that the term oral diagnostic be used if the source of an analyte in the oral cavity is unknown.
Heat illness is a major source of injury for military populations in both deployed and training settings. Developing tools to help leaders enhance unit performance while reducing the risk of injury is of paramount importance to the military. Here, we review our recent systems biology approaches to heat stress in order to develop a 3-dimensional (3D) realistic thermoregulation model, identify the molecular basis and mediators of injury, and characterize associated biomarkers. We discuss the implications of our work, future directions, and the type of tools necessary to enhance force health protection in the future.
Label-free optical schemes for molecular biosensing hold a strong promise for point-of-care applications in medical research and diagnostics. Apart from diagnostic requirements in terms of sensitivity, specificity, and multiplexing capability, also other aspects such as ease of use and manufacturability have to be considered in order to pave the way to a practical implementation. We present integrated optical waveguide as well as magnetic nanoparticle based molecular biosensor concepts that address these aspects. The integrated optical waveguide devices are based on low-loss photonic wires made of silicon nitride deposited by a CMOS compatible plasma-enhanced chemical vapor deposition (PECVD) process that allows for backend integration of waveguides on optoelectronic CMOS chips. The molecular detection principle relies on evanescent wave sensing in the 0.85 μm wavelength regime by means of Mach-Zehnder interferometers, which enables on-chip integration of silicon photodiodes and, thus, the realization of system-on-chip solutions. Our nanoparticle-based approach is based on optical observation of the dynamic response of functionalized magneticcore/ noble-metal-shell nanorods (‘nanoprobes’) to an externally applied time-varying magnetic field. As target molecules specifically bind to the surface of the nanoprobes, the observed dynamics of the nanoprobes changes, and the concentration of target molecules in the sample solution can be quantified. This approach is suitable for dynamic real-time measurements and only requires minimal sample preparation, thus presenting a highly promising point-of-care diagnostic system. In this paper, we present a prototype of a diagnostic device suitable for highly automated sample analysis by our nanoparticle-based approach.
Genalyte has developed a multiplex silicon photonic chip diagnostics platform (MaverickTM) for rapid detection of up to 32 biological analytes from a drop of sample in just 10 to 20 minutes. The chips are manufactured with waveguides adjacent to ring resonators, and probed with a continuously variable wavelength laser. A shift in the resonant wavelength as mass binds above the ring resonators is measured and is directly proportional to the amount of bound macromolecules. We present here the ability to multiplex the detection of hemorrhagic fever antigens in whole blood, serum, and saliva in a 16 minute assay. Our proof of concept testing of a multiplex antigencapture chip has the ability to detect Zaire Ebola (ZEBOV) recombinant soluble glycoprotein (rsGP), Marburg virus (MARV) Angola recombinant glycoprotein (rGP) and dengue nonstructural protein I (NS1). In parallel, detection of 2 malaria antigens has proven successful, but has yet to be incorporated into multiplex with the others. Each assay performs with sensitivity ranging from 1.6 ng/ml to 39 ng/ml depending on the antigen detected, and with minimal cross-reactivity.
The diverse human HLA (human leukocyte antigen) system is responsible for antigen presentation and recognition. It is essential for the immune system to maintain a stable defense line, but also is also involved in autoimmunity as well as metabolic disease. HLA-haplotype (HLA-B27), for instance, is associated with inflammatory diseases such as Bechterew's disease. The administration of the HIV drug Abacavir in combination with another HLA-haplotype (HLAB57) is associated with severe hypersensitivity reactions. Accordingly, the HLA status has to be monitored for diagnosis or prior to start of therapy. Along this line, a miniaturized microfluidic platform has been developed allowing performing the complete analytical process from “sample-in” to “answer-out” in a point-of-care environment. The main steps of the analytical cascade inside the integrated system are blood cell lysis and DNA isolation, DNA purification, real-time PCR and quantitative monitoring of the rise of a fluorescent signal appearing during the PCR based sequence amplification. All bio-analytical steps were intended to be performed inside one chip and will be actuated, controlled and monitored by a matching device. This report will show that all required processes are established and tested and all device components work well and interact with the functional modules on the chips in a harmonized fashion.
Rapid and inexpensive virus detection and quantification at the point-of-care is of paramount importance for HIV management in resource-limited settings. Here, we report on an easy-to-fabricate, cellulose paper-based microchip with printed graphene-modified electrodes for rapid detection of HIV-1 through electrical sensing. We evaluated the effect of electrode material and geometry on the performance of the microchip to detect serially diluted, electrically conductive samples. We evaluated the optimized microchip with HIVspiked samples.
In the spring of 2014, the Ebola virus (EBOV) strain Zaire caused a dramatic outbreak in several regions of West Africa. The RT-PCR and antigen capture diagnostic proved to be effective for detecting EBOV in blood and serum. In this paper, we present data of a rapid antigen capture test for the detection of VP40. The test was performed in a microfluidic chip for immunofiltration analysis. The chip integrates all necessary assay components. The analytical sensitivity of the rapid test was 8 ng/ml for recombinant VP40. In serum and whole blood samples spiked with virus culture material, the detection limit was 2.2 x 102 PFU/ml. The performance data of the rapid test (15 min) are comparable to that of the VP40 laboratory ELISA.
Assays that target DNA and RNA (xNA) are regarded as the “gold standards” in pathogen detection, surveillance, and diagnostics. However, they are often considered inappropriate for use at points-of-sampling and in low resource environments. This paper discusses innovations created by scientists at the FfAME and Firebird that promise to change this. The first is an artificially expanded genetic information system (AEGIS), a species of DNA having eight nucleotide "letters" added to the four found naturally in DNA. AEGIS nucleobases pair with geometries similar to standard Watson- Crick pairs, but with hydrogen bonding patterns different from (and orthogonal to) patterns that join the A:T and G:C pairs. Thus, AEGIS DNA allows xNA capture and amplification to have very high specificity and very low noise. A second innovation is a self-avoiding molecular recognition system (SAMRS). SAMRS is a species of DNA that behaves the opposite of AEGIS; SAMRS oligonucleotides bind with Watson-Crick complementarity to natural DNA, but not to other SAMRS oligonucleotides. A third innovation is a molecule beacon design that signals the presence of a target xNA even in complex biological mixtures. A fourth innovation is isothermal amplification of xNA targets, without PCR instruments or the skills needed to interpret their output. Here, levels of detection are as few as 30 molecules. Finally, we offer a sample preparation architecture that, as its very first step, sterilizes a sample, rendering it non-hazardous to inexperienced users. It then allows complete xNA capture and CLIA-waivable amplification.
Based upon advances in gene sequencing and construction, it is now possible to identify specific genes or sequences thereof for gene delivery applications. Recombinant adenovirus serotype-5 (Ad5) viral vectors have been utilized in the settings of gene therapy, vaccination, and immunotherapy but have encountered clinical challenges because they are recognized as foreign entities to the host. This recognition leads to an immunologic clearance of the vector that contains the inserted gene of interest and prevents effective immunization(s). We have reported on a new Ad5-based viral vector technology that can be utilized as an immunization modality to induce immune responses even in the presence of Ad5 vector immunity. We have reported successful immunization and immunotherapy results to infectious diseases and cancers. This improved recombinant viral platform (Ad5 [E1-, E2b-]) can now be utilized in the development of multiple vaccines and immunotherapies.
Solid-state nanopores have gained much attention as a bioanalytical platform. By virtue of their tunable nanoscale dimensions, nanopore sensors can a spatial resolution that spans a wide range of biological species from a single-molecule to a single virus or microorganism. Several groups have already used solid-state nanopores for tag-free detection of viruses. However, no one has reported use of nanopores to capture a single virus for further interrogation by the electric field inside nanopores. In this paper we will report detection of single HIV-1 particle with solid-state nanopores and demonstrate the ability to trap a single HIV-1 particle on top of a nanopore and force it to squeeze through the pore using an electric field.
Current methods of rapid detection of influenza are based on detection of the nucleic acids or antigens of influenza viruses. Since influenza viruses constantly mutate leading to appearance of new strains or variants of viruses, these detection methods are susceptible to genetic changes in influenza viruses. Type A and B influenza viruses contain neuraminidase, an essential enzyme for virus replication which enables progeny influenza viruses leave the host cells to infect new cells. Here we describe an assay method, the homogeneous biochemiluminescent assay (HBA), for rapid detection of influenza by detecting viral neuraminidase activity. The assay mimics the light production process of a firefly: a viral neuraminidase specific substrate containing a luciferin moiety is cleaved in the presence of influenza virus to release luciferin, which becomes a substrate to firefly luciferase in a light production system. All reagents can be formulated in a single reaction mix so that the assay involves only one manual step, i.e., sample addition. Presence of Type A or B influenza virus in the sample leads to production of strong, stable and easily detectable light signal, which lasts for hours. Thus, this influenza virus assay is suitable for use in point-of-care settings.
Highly evolved bacteria living within immobile marine animals are being targeted as a source of antitumor pharmaceuticals. This paper describes 2 electo-optical sensor systems developed for identifying species of tunicates and actinobacteria that live within them. Two stages of identification include 1) a benthic survey apparatus to locate species and 2) a laboratory housed cell analysis platform used to classify their bacterial micro-biome. Marine Optics Sampling- There are over 3000 species of Tunicates that thrive in diverse habitats. We use a system of cameras, GPS and the GPS/photo integration application on a PC laptop to compile a time / location stamp for each image taken during the dive survey. A shape-map of x/y coordinates of photos are stored for later identification and sampling. Flow Cytometers/cell sorters housed at The Medical University of South Carolina and The University of Maryland have been modified to produce low-noise, high signal wave forms used for bacteria analysis. We strive to describe salient contrasts between these two fundamentally different sensor systems. Accents are placed on analog transducers and initial step sensing systems and output.
Global Earth Observation Systems of Systems (GEOSS) are bringing vital societal benefits to people around the globe. In this research article, we engage undergraduate students in the exciting area of space exploration to improve the health of millions of people globally. The goal of the proposed research is to place students in a learning environment where they will develop their problem solving skills in the context of a world crisis (e.g., malaria). Malaria remains one of the greatest threats to public health, particularly in developing countries. The World Health Organization has estimated that over one million die of Malaria each year, with more than 80% of these found in Sub-Saharan Africa. The mosquitoes transmit malaria. They breed in the areas of shallow surface water that are suitable to the mosquito and parasite development. These environmental factors can be detected with satellite imagery, which provide high spatial and temporal coverage of the earth's surface. We investigate on moisture, thermal and vegetation stress indicators developed from NOAA operational environmental satellite data. Using these indicators and collected epidemiological data, it is possible to produce a forecast system that can predict the risk of malaria for a particular geographical area with up to four months lead time. This valuable lead time information provides an opportunity for decision makers to deploy the necessary preventive measures (spraying, treated net distribution, storing medications and etc) in threatened areas with maximum effectiveness. The main objective of the proposed research is to study the effect of ecology on human health and application of NOAA satellite data for early detection of malaria.