Two-photon excitation microfluorometer for multiplexed single-step bioaffinity assays

Abstract

A new type of instrumentation for single-step bioaffinity assays and microvolume fluorometry is presented. The concept is based on the use of two-photon excitation by a low-cost near-infrared laser and individual observation of bioactive fluorescent microparticles. The applicability of the instrument is demonstrated by a microparticle based multiplexed bioaffinity assay where several fluorescent markers are simultaneously excited. This instrument can be applied in the growing fields of drug discovery, in life science research, and in routine laboratory diagnostics.

Fluorescent nanoparticles as labels for immunometric assay of C-reactive protein using two-photon excitation assay technology

“…The instrument used in this study was a modiWcation of the system that was recently described in detail by Soini et al [32,33]. The modiWed instrument included x-y positioning mechanics to enable measurement from wells of standard microtitration plates, an autofocus function to accurately position the laser beam focus within a sample in a well of a microtitration plate, and a long working distance objective to facilitate reading from microwells of diVerent bottom thicknesses.…”

Methicillin-Resistant Staphylococcus aureus Screening by Online Immunometric Monitoring of Bacterial Growth under Selective Pressure

“…Carboxyl-modified microparticles (diameter, 3 m; high acid content, hydrophilic; part 7300-3420) made of cross-linked polystyrene were purchased from Seradyn (Indianapolis, IN). Arctic Diagnostics Ltd. (Turku, Finland) provided the fluorescent labeling reagent dipyrrylmethene-BF 2 530 (BF530) (18). Cefoxitin, sodium salt (949 g/mg; catalog no.…”

“…A combination of several different monoclonal antibodies could be used to improve the assay performance in case a sufficient sensitivity is not achieved with reagents prepared with any single antibody. Such assays are readily supported by TPX plate readers (18). In the future development of the assay, we would prefer to use better-characterized high-affinity monoclonal antibodies, such as those used in new rapid identification assays, which have proven highly sensitive and specific (e.g., 98.2% and 98.9%, respectively, for the Slidex Staph Plus assay [bioMerieux, Marcyl'Etoile, France]) for the identification of S. aureus (20).…”

Rapid Method for Detection of Influenza A and B Virus Antigens by Use of a Two-Photon Excitation Assay Technique and Dry-Chemistry Reagents

“…Fluorescence from the surface of the individual microspheres is measured separation free, directly from the reaction mixture, using the ArcDia TPX detection technique (29). The optical configuration of the fluorometer (15) and the physical phenomena related to the measurement technique were described previously (30).…”

“…The new technique is based on a separation-free bioaffinity assay technique, ArcDia TPX, and the use of dry-chemistry reagents. The assay technique employs microspheres as a solid-phase reaction carrier, fluorescent antibody conjugates, and the detection of two-photon excited fluorescence from individual microspheres (10,30,41). The technique allows quantitative separation-free bioaffinity assays from a volume of a few microliters in subpicomolar sensitivity (15,29).…”