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The goal of this research is to develop novel biosensors for enhanced sensitivity and specificity. This will be accomplished through the development of novel sensing mechanisms and novel sensor platforms. Specifically, novel sensing mechanisms such as gold nanoparticles (AuNP) immunosensors and quantum dot protease biosensors will be investigated. For sensor platforms, liquid core waveguides and hollow glass waveguides will be investigated. It is hypothesized that these novel biosensor will be: selective (with a minimum of false positive readings), sensitive (with a very low limit of detection), reliable, portable, and inexpensive. <P>The research objectives are: Specific Objective 1: Investigate novel nanosensor architectures. In this objective, nanoscale materials, such as quantum dots (QDs) and gold nanoparticles, will be utilized as optical markers conjugated to peptide sensing element and antibody sensing elements, respectively. It is hypothesized that the fluorescent nanosensors will enhance specificity, which will allow accurate detection of analytes at very low concentrations.<P> Specific Objective 2: Fabricate novel sensor platforms. In this objective, Teflon cladded liquid core waveguides and hollow core capillary tubes will be investigated as sensor platforms. We hypothesize that sensitivity can be improved by utilizing Teflon cladded liquid core waveguides (LCWs) and/or hollow glass tubes. The purpose of this objective is to investigate sensing platforms that will be amendable to field, clinic, or home testing. Signal to noise ratios along with dB/m losses will be investigated to optimize detection sensitivity. <P>Specific Objective 3: Determine the efficacy of nanosensors in the chip based liquid core waveguides and/or hollow glass tubes. We hypothesize that interfacing the nanosensors with the sensor platforms will result in a biosensor that effectively measures extremely low concentrations of analyte.<P> We will target the analyte, PRRSv, in the development of the AuNP immunosensor. A detection limit of 2 infectious particles/ml is desirable, since it is known that 2 particles can initiate a PRRS infection. The second analyte we will target is botulinum using the QD-peptide biosensor. A detection limit of 100 particles/ml is targeted. <P>Our unique nanosensor designs, which utilize quantum dots and/or gold nanoparticles, will allow rapid detection of the targeted analyte with enhanced specificity. The nanosensors integrated with LCWs and/or capillary tubes achieve a dual use of microfluidic channels as pathways for fluid transport and as waveguides for strong light material interaction and highly efficient fluorescent light collection. This will give us tremendous advantages in terms of increased signal collection and a reduction of on-chip elements permitting overall size reduction.
Non-Technical Summary: While great strides have been made in biosensor technology, many technological problems remain that hinder their widespread use. Fast, accurate, and reproducible diagnostic tests are vital for such areas as food pathogen detection, water quality, and viral detection. This project proposes to address novel biosensor development with the goal of enhanced sensitivity and specificity. <P> Approach: To address the first objective, experiments will be performed on novel nanosensor architectures that utilize gold nanoparticles and QDs. The two nanosensors are stated below: 1. gold nanoparticle-Protein A and organic fluorophore-antibody; 2. quantum dot and organic fluorophore peptide construct. Nanosensor #1; Alexa Fluor 546/AuNP for PRRSv Detection: The AuNP acts as a quencher (acceptor); it will be utilized with the organic dye, Alexa Fluor 546 (donor). The fluorophore quencher system offers the advantage of better signal to noise information at low PRRSV concentrations. AuNP has several superior properties that make it ideal for use in energy transfer based assays. It has a broad absorption in the visible light spectrum, which allows quenching in the red region. Nanosensor #2; QD/fluorescein for Botulinum: The second sensor will consist of a blue QD that is interfaced with an organic fluorophore (fluorescein). Initially, generic peptide sequences such as H2N-Cys-dPhe-Pro-Arg-Gly-Lys (Ahx-Fluorescein)-OH, will be synthesized (via the Protein Synthesis Lab at MU). The carboxylated-QDs (purchased form Evident Technologies) will be covalently linked to the peptide via EDC/s-NHS to conjugate the carboxyl to amine groups in the peptides. Energy transfer from the QD to the fluorophore will occur initially, but after cleavage by the enzyme, the fluorophore will be removed and thus the QD will fluoresce. In solution testing: Known and unknown concentrations of the analyte will be introduced into solutions containing the nanosensors and then scanned with a spectrofluorometer. The change in fluorescence upon the addition of the analyte will be recorded. As a control, non-specific proteins (such as bovine serum albumin, BSA) will also be introduced into the solutions. ELISA and/or PCR assays will be performed to provide a comparison to the sensor response. The means and standard deviation of the resulting values from all samples will be calculated by using the General Linear Models procedure of SAS. To address the second objective, we will perform studies on liquid core waveguides (LCWs) and hollow capillary tubes. The purpose of this objective is to investigate sensing platforms that are amendable to field, home, and clinical assay testing. Initially, Teflon AF 2400 tubes will be utilized for testing as well as Teflon AF microchannels that will be microfabricated in the Center for NEMS and MEMS at MU. For the hollow capillary tubes, we will purchase silica tubes (10 microliter) from Fischer. The third objective will be addressed via the interfacing of the nanosensors with the sensor platforms. Both types of nanosensors will be interfaced to the two different platforms. Sensor Platform #1 involves the LCWs. For the Sensor Platform #2, silica capillary tubes, we will silanize the inner surface so that nanosensor #1 and nanosensor #2 will be immobilized. The following parameters will be ascertained: Limit of detection; Sensitivity; Selectivity; Response Time, Repeatibility. All sensors will be calibrated using samples of known concentrations and compared to PCR/ELISA assays.