The long-term commercial objective of this project is to develop a rapid (minutes), sensitive, inexpensive nanospring-based (NS) biological platform that does not require expensive, complex instrumentation for obtaining results due to the highly active biological surface area in a small two-dimensional footprint. Current multiplex ELISA analysis is limited to antigen- or antibody-coated beads which require laser detection systems in microfluidic channels, or electrochemilumiscent-based assays with high equipment costs and complex electronic chip antibody-capture platforms. The long-term specific aims of this project are to: 1) to determine loading capacity and biological activity of NS bound antibodies. (Phase I);2) evaluate both antigen-down and capture (sandwich) formats based on the protocols developed in Phase I;3) develop a multiplex ELISA format that is capable of performing rapid, simultaneous analyses on multiple samples;4) evaluate the potential of the nanospring platform for ""miniaturization"" of the assay;5) determine the ability of nanosprings to enhance the activity of a wide variety of other biological agents. The ability to rapidly perform multiplex ELISA assays has relevance from several different perspectives. Beyond the ability to assay for several different biomarkers which are used in disease diagnosis, monitoring disease progression or treatment procedures in humans, multiplex assays have a demonstrated need in bacterial and viral disease detection and diagnosis in veterinary medicine, and drug and toxin screening. The ability to attach other bioreactive molecules onto NS has utility in the development of smaller and more efficient bioreactors for drug and pharmaceutical production, wasterwater treatment, and tissue growth in cell culture. For Phase I we propose to use silica nanosprings as the solid- phase for the coating of capture antibodies or antigens, and to determine the maximum capacity of antibody and antigen binding, and maximum activity of these molecules as compared to currently available materials used in standard ELISA assays (i.e microtiter plates). For these studies, NS will be deposited on a support medium of glass microbeads. We will investigate both the density of nanospring deposition as a function of activity of the coated proteins. NS offer a distinct advantage over these other materials due to the dramatic increase in the surface area available for biological materials, the wide variety of materials available for coating and the ease at which these materials can be coated, the low cost of using NS. As a result of the large surface area available for binding, we anticipate that the sensitivity of the assay can be increased while reducing the footprint of the assay from a standard 96-well format to a 384- or 1536-well or array format. This ""miniaturization"" will effectively reduce the volume of sample and reagents needed thus reducing the time of the assay from hours to minutes.
Public Health Relevance: A greater available surface area afforded by nanosprings (250 fold) will facilitate the binding of multiple capture proteins and/or antibodies, allowing for a single assay test-well to be used in the detection of multiple chemical compounds (multiplex immunoassay). Tests may be completed in a shorter time (minutes rather than hours), on a smaller test platform and on the site of examination and will be invaluable in a variety of health-related applications: viral detection and vaccine development;multiple detection of pro-inflamatory cytokines in multi- symptomatic diseases (e.g. reflex sympathetic dystrophy);the detection of angiogenic cytokines in human tumor tissue (allowing for the selection of specific chemotherapeutic drugs);detection and discrimination of hepatitis B and C, and HIV type-1 viruses (common transfusion-transmitted pathogens);combined detection of Chlamydia. trachomatis and human papillomaviruses (two asymptomatic, sexually transmitted diseases);to evaluate the changes in specific biomarkers as a predictive indicator of positive-outcome chemotherapy;or to detect food pathogens and toxins prior to distribution of contaminated products, to name a few.
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