Luke V. Schneider

High-sensitivity immunoassay using a novel upconverting phosphor reporter

A new class of reporter label, consisting of rare earth elements embedded in a crystalline particle, has been developed for in vitro diagnostic applications. These unique labels upconvert low energy (IR) radiation to high energy light by a multiphoton absorption process and subsequent phosphorescence emission. As a result, upconverting phosphors can be visualized with no biological background or autofluorescence signal. In addition, phosphors have narrow absorption and emission bands, making them ideal for simultaneous multianalyte test. The crystalline nature of the phosphors makes them insensitive to environmental conditions, with essentially infinite shelf life and no photobleaching at the irradiances used for excitation. We have covalently coupled (Y0.86Yb0.08Er0.06)6O2S phosphor labels to antibody probes to create a reporter reagent that can be excited by 980 nm radiation from a diode laser and detected by a modified spectrofluorimeter. Target analyte sensitivities of approximately 10 ng/mL to Staphylococcal enterotoxin B have been demonstrated using a sandwich assay in a magnetic bead or capillary wick formats in a non-optimized assay system. These results are directly applicable to the development of assays that can be performed on microfabricated biochips or in microflow channels.

Detection of DNA using upconverting phosphor reporter probes

We have developed a nucleic acid detection assay using an upconverting phosphor DNA reagent as a reporter probe for the detection of target single-stranded DNA molecules. Upconverting phosphors allow the detection of DNA against zero background signal because low energy IR light is absorbed by a two-photon process and remitted as a visible photon, eliminating the presence of autofluorescence. The spectral properties of upconverting phosphors, which emit in the blue, red, or green region of the spectrum, are not sensitive to solvent conditions or to temperature and can therefore be used in a variety of buffers for the qualitative and quantitative detection of biological molecules. Short DNA oligonucleotides were covalently attached to the surface of submicron size phosphor particles and used to detect M13mp18(+) DNA targets captured by magnetic beads labeled with a capture probe specific to the target. This system allowed the detection on the order of femtomoles of M13mp18(+) strand DNA. Buffer conditions were optimized for the use of the phosphor reporters. The phosphor-labeled probes are stable in aqueous solution for 2 weeks and have a lyophilized shelf life of at least one month.

Compact upconverting phosphor detection system for wick assays

We describe an integrated detection system based on upconverting phosphor particles bound to capture sites on the inside surfaces of rectangular wick capillaries. This deice can be used with either antibody or nucleic acid to detect specific micro-organisms. The system uses a high- power, 980 nm, semiconductor diode laser to illuminate 200 X 300 X 20 micrometers capture surfaces. The rectangular capillary wicks are held in a tray that is inserted into the detection system, positioning the capture surface at the object plane of the optical system. Phosphorescent light emitted from the capture surface is collected by a high numerical aperture microscope objective and directed through a series of filters onto either a CCD camera or a photomultiplier. A combination of band-reject filters attenuates the 980 nm laser excitation light and its harmonic at 490 nm, and a tunable liquid crystal filter provides for rapid scanning from 400 to 750 nm. The data acquisition and control is controlled by a laptop PC with a custom GUI interface developed using LabWindows/CVI. The system can detect a single phosphor particle bound to a capture surface.

Method for Recovery and Immunoaffinity Enrichment of Membrane Proteins Illustrated with Metastatic Ovarian Cancer Tissues

Integral membrane proteins play key biological roles in cell signaling, transport, and pathogen invasion. However, quantitative clinical assays for this critical class of proteins remain elusive and are generally limited to serum-soluble extracellular fragments. Furthermore, classic proteomic approaches to membrane protein analysis typically involve proteolytic digestion of the soluble pieces, resulting in separation of intra- and extracellular segments and significant informational loss. In this paper, we describe the development of a new method for the quantitative extraction of intact integral membrane proteins (including GPCRs) from solid metastatic ovarian tumors using pressure cycling technology in combination with a new (ProteoSolve-TD) buffer system. This new extraction buffer is compatible with immunoaffinity methods (e.g., ELISA and immunoaffinity chromatography), as well as conventional proteomic techniques (e.g., 2D gels, western blots). We demonstrate near quantitative recovery of membrane proteins EDG2, EDG4, FASLG, KDR, and LAMP-3 by western blots. We have also adapted commercial ELISAs for serum-soluble membrane protein fragments (e.g., sVEGFR2) to measure the tissue titers of their transmembrane progenitors. Finally, we demonstrate the compatibility of the new buffers with immunoaffinity enrichment/mass spectrometric characterization of tissue proteins.

Design and manufacture of capillary wicks for ultrasensitive detection of antigenic and nucleic acid analytes

Using upconverting phosphor reporter probes to detect antigenic and nucleic acid analytes in environmental samples required the design and manufacture of a novel sampling and assay device that exploits the unique characteristics of the upconverting phosphors. The absence of natural materials that upconvert energy make single particle detection possible with these reporters. Ultrasensitive detection and quantitation of analytes is governed by the area of the capture surface, the density of the capture probe,s as well as the specific upconversion efficiency of the phosphor label. The optimization of a flow channel and capture surface for single phosphor particle detection is a complex function of the volume of sample that is drawn through the channel, the probability that this sample volume contains an analyte particle, the fraction of analytes contained in the sampled volume that bind to the capture site, and the optical dimensions of the capture site. The result of this optimization using theoretical diffusion models for submicron upconverting phosphor particles was a rectangular flow channel with a 200 X 300 micrometers capture surface. Capture surfaces of this size have been prepared using photo-directed synthesis methods inside glass capillary wicks that have been subsequently used for the phosphor- based assays.