John W. Silzel

System for simultaneously conducting multiple ligand binding assays

A system for simultaneously conducting multiple ligand assays on a sample potentially containing target analytes uses as a detector a waveguide having a planar surface with a plurality of probes of known recognition to the target analytes thereon. The probes are in discrete areas on the waveguide. A sample containing target analyte is treated with a light-responsive compound such that it binds to the target analyte to form a conjugate and the conjugate is applied to the probes on the waveguide. A laser light is passed into the planar surface of the waveguide at a plurality of different locations, by causing relative movement between the waveguide and the laser light, so that evanescent waves radiate from the waveguide. Where conjugate has attached to a probe, there is emission of light different from that emitted by a probe without conjugate attached thereto.

Near IR fluorescence: instrumentation, application and pitfalls

Abstract In this paper, we describe the advantages of utilizing NIR fluors as probe molecules for assays in biological matrices. We describe the development of a sensitive fluorometer capable of sensitive measurements of these molecules and demonstrate the gains in sensitivity achieved as the system develops. A time resolution system is also described and the benefits of controlling this parameter to separate fluorescent signal from scattered signal is shown.

Near IR: new sources, new detectors, and new solutions to old problems

Use of the short-wave near infrared (NIR) region for fluorescence spectroscopy is shown to afford enhanced freedom, relative to the visible spectrum, from background signals and noise resulting from scattering and endogenous fluorescence from the sample matrix itself. The detection of 0.1 attomole (10-13 mole liter-1) of two cyanine dyes in methanol solution is demonstrated using diode laser excitation at 780 nm and CCD spectrometric detection. The fluorophores exhibit excellent photostability in this solvent even under prolonged laser illumination. Limitation of sensitivity in this system is imposed by shot noise associated with the solvent Raman signals. When the same dyes are placed in aqueous solutions containing bovine serum albumin (BSA), endogenous fluorescence from the BSA and photobleaching or photolysis of the cyanine raise the practical detection limits to 10-9 moles liter-1. Despite these difficulties, the NIR region affords signal to background ratios which are superior to those observed in similar experiments performed in the visible region.

Mass-Sensing Multianalyte Micro-Array Based Immunoassay by Direct Nir Fluorescence and Quantitative Image Analysis

It has been found that 200 micron-diameter spots of covalently immobilized analyte-specific capture reagent can significantly deplete analyte from several hundred microliters of liquid sample. The quantitative ligand binding assays which result sense the total analyte mass in a sample, rather than concentration, and provide high signal to background by concentrating analytes at the solid-liquid interface. Data from an avidin-biotin model shows the detection of 106 analyte molecules per spot. Preliminary results from a simultaneous multianalyte assay of the four human IgG subclasses validate the avidin-biotin results, and indicate sensitivity comparable to conventional ELISA methods requiring 100 times the antibody mass.

Construction of a shortwave near-infrared spectrofluorometer with diode laser source and CCD detection

Spectrofluorometers employing xenon arc lamp excitation and photomultiplier tube detectors afford sensitivity over the UV/VIS spectral region for which these instruments were designed, but suffer sensitivity limitations in the short-wave near infrared (NIR) region (800 - 1000 nm) because of their limited source energy and low detector quantum efficiency. To achieve high sensitivity in the NIR region, a 30 mW diode laser source, an imaging spectrograph, and a cryogenically cooled charge-coupled device (CCD) have been combined in a spectrofluorometer specifically designed for use in the NIR region. The diode laser source incorporates integral source filters, optics, and a beam trap, and utilizes a vertical beam geometry which provides an illuminated volume oriented conveniently for the imaging of fluorescence emissions on the entrance slit of the spectrograph. Data is presented which demonstrates that the temporal and spectral stability of the source is equal or superior to that of an arc lamp for solution-phase fluorometry. In addition to spectral information, the CCD detector provides spatial resolution of fluorescence emissions along the vertical path of the excitation beam. An absolute photometric calibration of the CCD detector, and measurement of its read noise, fixed pattern noise, and linear dynamic range is performed using the photon transfer technique of Janesick, et al. Improvement in the instrument performance by more than six decades is demonstrated by measured LOD of NIR dyes using a commercial SLM 4800 instrument and the new diode laser/CCD arrangement. Origin of the present detection limits is discussed.