Ultra Sensitive Bioaffinity Assay for Micro Volumes

Abstract

An ultrasensitive separation free micro volume bioaffinity assay method is presented. The concept is based on the use of two-photon excited fluorescence and polystyrene microspheres as bioaffinity binding solid phase. Measurements are performed by observing individual microspheres in the diffraction limited focal volume of twophoton excitation.The method is suitable for measuring bioaffinity binding assays directly from the reaction volume without separation and washing steps. Depending on the sensitivity requirements the assay can be scaled. Scaling is achieved by adjusting the amount of solid phase microspheres for the assay. The scalability is demonstrated by measuring standard curves for an immunoassay. As a model assay we chose the human alpha-fetoprotein (AFP) immunometric assay. Operating ranges from 10 pg/ml up to 20 ng/ml and 100 pg/ml up to 400 ng/ml were achieved.

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

“…One approach is to reduce the number of coated microspheres in the reaction mixture [6]. This method, however, leads inevitably to increased measurement times, assuming a constant assay precision.…”

“…The detection of individual microspheres is based on anti-Stokes' emission and nonlinear property of two-photon excited Xuorescence. According to this, a near-infrared laser, generating pulses at 1064 nm, is focused in the reaction suspension and Xuorescence emission from individual microspheres is recorded at visible range of spectrum [1,5,6]. In contrast to most assay techniques, the signal intensity provided by the ArcDia TPX technique is independent of assay volume.…”

“…This unique property allows one to decrease the assay volume without compromises in the assay performance. It has recently been demonstrated that the assay technique allows immunometric assays of protein antigens with picomolar [1,7] or even subpicomolar [6] detection limit, with dynamic range of 3-4 orders of magnitude, and with average precision of 5%. However, this detection limit is not suYcient to cover all markers of clinical or biological interest; higher sensitivity is required, for example, for the assay of thyroid-stimulating hormone or cardiac Troponin I.…”

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

“…These microspheres work as a solid-phase reaction carrier for the immunocomplex formation. 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 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). The applicability of the technique for the detection of serum antigens (10,15) and antibodies (17), for the detection of antigens bound on the cell surface (32), for competitive binding assays (33), and for recognition of nucleic acid sequences (22,35) has been demonstrated.…”