Ultrasensitive detection and identification of fluorescent molecules by FCS: Impact for immunobiology

An experimental application of fluorescence correlation spectroscopy is pre sented for the detection and identification of fluorophores and auto-Abs in solution. The recording time is between 2 and 60 sec. Because the actual n umber of molecules in the unit volume (confocal detection volume of about 1 fl) is integer or zero, the fluorescence generated by the molecules is dis continuous when single-molecule sensitivity is achieved. We first show that the observable probability, N, to find a single fluorescent molecule in th e very tiny space element of the unit volume is Poisson-distributed below a critical bulk concentration c*. The measured probability means we have tra ced, for example, 5 x 10(10) fluorophore molecules per ml of bulk solution. The probability is related to the average frequency, C, that the volume of detection contains a single fluorescent molecule and to the concentration, c, of the bulk solution. The analytical sensitivity of an assay is calcula ted from the average frequency C. In the Goodpasture experiment, we determi ned as analytical sensitivity a probability of 99.1% of identifying one sin gle immune complex. Under these conditions, a single molecule event is prov en. There exist no instrumental assumptions of our approach on which the ex periment itself, the theoretical background, or the conclusion are based. O ur results open up a broad field for analytics and diagnostics in solution, especially in immunology.