Utilization Of Antibody-Conjugated Gold Nanoparticles, Dynamic Light Scattering And Sers In Influenza Virus Detection

Influenza A H3N2, H1N1, and influenza B viruses primarily cause winter illness in humans, leading to significant morbidity and mortality in the population of the very young, the elderly, and people with chronic disease. In addition to the regular seasonal epidemics of influenza, influenza pandemics associated with the emergence of new influenza A strains are threatening due to high levels of mortality, social disruption, and economic losses. These novel strains are not affected by the human immunity developed to older strains of influenza, therefore can spread readily and infect a vast number of people. The most recent flu pandemic outbreak was in 2009, in which pandemic swine influenza A H1N1 was transmitted. Thus, an initiative to prevent human infections with new strains of influenza A virus with pandemic potential has been supported by the government and become a focus of many laboratories. The first step in any preventative measures is early detection. Therefore, it is essential to develop a detection platform that is capable of simultaneous multiplexing and exploitable for point-of-care (POC) analysis. Virus culture, nucleic acid testing, and immunoassays are primary detection approaches to confirm acute human influenza virus infection. Nucleic acid testing has great sensitivity and specificity to subtype influenza strains, and high capacity for multiplexed detection. However, it is time and labor intensive, and expensive. Virus isolation is slow, costly, and not feasible for routine diagnostic testing. Immunoassays, in contrast, are known for availability, low-cost, accuracy, and versatility, and therefore have become a centerpiece in diagnostics. Among a number of analytical detection techniques developed for immunoassays, SERS (surface enhanced Raman spectroscopy) biosensing utilizing antibody-conjugated gold nanoparticles (Ab-AuNPs) is a promising virus detection technique providing high sensitivity (down to single molecule detection) and multiplexing (distinction of different strains of a single virus type). Herein a simple, rapid, sensitive AuNP-based immunoassay was developed to quantitatively detect influenza A virus, utilizing dynamic light scattering (DLS) and surface enhanced Raman spectroscopy (SERS). The assay platform was established based on the principle of homogeneous format. Antigen-specific antibodies (Abs) were attached to the surface of gold nanoparticles (AuNPs), rendering the biospecificity for the detection. AuNPs serve as a signal generator or label. A biological sample containing targeted analytes was mixed with Ab-conjugated AuNPs (or AuNP probes); aggregation of nanoparticle was induced in the presence of the analyte(s). The antibody molecules on the particle surface recognized and bound to the analyte via the key-lock like mechanism, cross-linking AuNPs together to form aggregates. The quantification of antigen became the matter of detecting aggregation. The reaction happened in a timely fashion, oftentimes in a few minutes owing to the fast solution phase kinetics. No washing was required; therefore, time and labor were remarkably saved relative to heterogeneous assays. When utilizing this platform, alteration of different antigen-specific antibodies can perform detection of different antigen analytes individually (singleplexing). The combination of multiple types of AuNP probes in one assay allows simultaneously multiplexed detection. In order to ensure the robustness of the assay, optimization for each stage of the platform design was thoroughly studied. The optimal conditions for maintaining the stability of the gold nanoparticles coated with monoclonal antibodies (mAbs) were investigated by varying pH, conjugation chemistry, mAbs concentrations, and blocking reagents. DLS is exploited to monitor the conjugation of the antibodies on AuNPs and verify the aggregate formation of the antigen-induced AuNP probes based on hydrodynamic diameter measurements. The DLS-based immunoassay has been demonstrated as an excellent rapid screening method to evaluate the specificity and affinity of antibody-antigen binding. Comparing to a conventional method for antibody screening (i.e. ELISA), a DLS assay requires only 30 min while it takes 24 h to perform an ELISA. To address the urgent need for multiplexed detection, we have slightly modified the DLS assay to develop a SERS-based homogeneous immunoassay. Namely, Raman reporters and antibody were co-immobilized on the AuNPs to construct ERLs (extrinsic Raman labels). Raman reporters provide distinctive and amplified signal for detection. In order to detect multiple analytes, multiple types of ERLs were separately prepared; each type was a unique combination of one antigen-specific antibody and one Raman reporter. The ERLs were then mixed together and added to the sample. Aggregation was induced upon the introduction of the antigen to the suspension of ERLs on the order of minutes. ERLs of the same type were cross-linked via the antigen specific to the antibody conjugated to the very type of ERLs. The nonspecific ERLs remained unreacted if their antigens were not present in the sample. Once aggregation occurred, the SERS signals provided by the Raman reporters on the reacted ERLs were turned on. AuNPs in the aggregating state were in proximity to each other and created small gaps between them. Raman reporters once trapped in those gaps generated signal for detection. In theory, SERS analysis can be performed in solution but in reality poor plasmonic coupling between antibody-modified AuNP limits the SERS enhancement. However, dehydration of the aggregates reduces interparticle spacing to yield higher SERS signals. Therefore, separation of aggregated ERLs on a well-defined nanoporous membrane was applied to intensify the signal. The conditions for optimal filtration process have been investigated. Preliminary data have shown progress made toward a fully developed configuration for a portable multiplexed, sensitive, and rapid POC detection platform

Secondary Electrohydrodynamic Flow Generated by Corona and Dielectric Barrier Discharges

One of the main goals of applied electrostatics engineering is to discover new perspectives in a wide range of research areas. Controlling the fluid media through electrostatic forces has brought new important scientific and industrial applications. Electric field induced flows, or electrohydrodynamics (EHD), have shown promise in the field of fluid dynamics. Although numerous EHD applications have been explored and extensively studied so far, most of the works are either experimental studies, which are not capable to explain the in depth physics of the phenomena, or detailed analytical studies, which are not time effective. The focus of this study is to provide the model that in a reasonable computational time is able to give us accurate results in different electric-fluid interactions. So, the main goals of this study is to provide a model to simulate all essential physical phenomena, applicable in different EHD systems. So, in this thesis, first, a two-dimensional numerical solver is presented for the dynamic simulation of the Dielectric Barrier Discharge (DBD) and the Corona Discharge (CD) in point to plane configuration. The simulations start with the single-species model and the different steps of the numerical technique are tested for this simplified model. The ability of the technique to model the expected physical behavior of ions and electric field is investigated. The studied physics were implemented in different geometry configurations such as wire to plane, wire to wire, and plane to plane geometries. The electrostatic field and ionic space charge density due to corona discharge were computed by numerically solving Poisson and current continuity equations, using a Finite Element method (FEM). The detailed numerical approach and simulation procedure is discussed and applied throughout the thesis. Then, the technique is applied to a more complicated model in order to address several existing EHD applications. The complicated mutual interaction between the three coexisting phenomena of electrostatic field, the charge transport, and fluid dynamics, which affect the EHD process, were taken into account in all the simulations. Calculations of the gas flow were carried out by solving the Reynolds-averaged Navier-Stokes (RANS) equations using FEM. The turbulence effect was included by using the k-ε model included in commercial COMSOL software. An additional source term was added to the gas flow equation to include the effect of the electrostatic body force. In all the simulations, the effects of different parameters on the overall performance of the system and its characteristics are investigated. In some cases, the simulation results were compared with the existing experimental data published in literature

Arsenic speciation in animal feed and feed ingredients by ICP-MS and HPLC-ICP-MS methods

Master's Thesis in Quality in the Analytical LaboratoryQAL399BJMAMN-QAL

The ingenuity of common workmen: and the invention of the computer

Since World War II, state support for scientific research has been assumed crucial to technological and economic progress. Governments accordingly spent tremendous sums to that end. Nothing epitomizes the alleged fruits of that involvement better than the electronic digital computer. The first such computer has been widely reputed to be the ENIAC, financed by the U.S. Army for the war but finished afterwards. Vastly improved computers followed, initially paid for in good share by the Federal Government of the United States, but with the private sector then dominating, both in development and use, and computers are of major significance.;Despite the supposed success of public-supported science, evidence is that computers would have evolved much the same without it but at less expense. Indeed, the foundations of modern computer theory and technology were articulated before World War II, both as a tool of applied mathematics and for information processing, and the computer was itself on the cusp of reality. Contrary to popular understanding, the ENIAC actually represented a movement backwards and a dead end.;Rather, modern computation derived more directly, for example, from the prewar work of John Vincent Atanasoff and Clifford Berry, a physics professor and graduate student, respectively, at Iowa State College (now University) in Ames, Iowa. They built the Atanasoff Berry Computer (ABC), which, although special purpose and inexpensive, heralded the efficient and elegant design of modern computers. Moreover, while no one foresaw commercialization of computers based on the ungainly and costly ENIAC, the commercial possibilities of the ABC were immediately evident, although unrealized due to war. Evidence indicates, furthermore, that the private sector was willing and able to develop computers beyond the ABC and could have done so more effectively than government, to the most sophisticated machines.;A full and inclusive history of computers suggests that Adam Smith, the eighteenth century Scottish philosopher, had it right. He believed that minimal and aloof government best served society, and that the inherent genius of citizens was itself enough to ensure the general prosperity

Revolutionary Concepts of Radiation Shielding for Human Exploration of Space

This Technical Memorandum covers revolutionary ideas for space radiation shielding that would mitigate mission costs while limiting human exposure, as studied in a workshop held at Marshall Space Flight Center at the request of NASA Headquarters. None of the revolutionary new ideas examined for the .rst time in this workshop showed clear promise. The workshop attendees felt that some previously examined concepts were de.nitely useful and should be pursued. The workshop attendees also concluded that several of the new concepts warranted further investigation to clarify their value