Patent DE-10111420-A1 (DE)
Title: To detect an analyte by fluorescence correlation spectroscopy, a set gap is established between the light focusing unit and the volume of the sample, and the sample carrier is thermally insulated from the light source
IPC: G01N21/64
Filed: 2001-03-09
Abstract: A process to detect an analyte in a sample, using fluorescence correlation spectroscopy (FCS), sets the gap between the measured volume in the sample fluid and the focusing unit at the light source at \-1 mm. The sample fluid is thermally insulated from the light source and especially the optical focusing system. To detect an analyte in a sample, the gap between the measured volume and the light focusing unit is preferably 1-10 mm and especially 2-5 mm. A gas phase zone is between the carrier and the optical focusing unit. The carrier has a lens, in the light path between the measured volume and the light source or detector. The optical measurement unit has a numerical aperture of 0.5-1.2. The carrier has preferably at least 10<2> sample holders, and it has at least one temperature control element for the identification of an analyte at a variable temperature which differs from the ambient environment.  The analyte identification uses the bond of at least one identifying luminescent marking reagent. Or it uses a nucleic acid hybridizing, where one or more probes with luminescent marking bond to a target nucleic acid. The measurements are detected by a cross correlation of the signals, of which one contains at least two different luminescent markings derived from a complex of the analyte and identifying reagent(s). At least one signal from a luminescent identifying reagent is examined for the intensity of the luminescence and/or luminescence fall-off time of the reagent where it differs during bonding than when not in a bonded condition, using quench or energy transfer methods. The energy transfer is registered from at least one luminescent marker as the donor and at least one luminescent marker as the acceptor, in a complex of one or more reagents. The findings can also be through an enzyme reaction, or a nucleic acid amplification and especially with one or more thermo-cycling processes. Or a mutation or gene expression analysis can be used with nucleic acids. With nucleic acid hybridizing, measurements are taken of the temperature-related melting curve. Independent claims are also included for:  (1) an apparatus for the analysis of an analyte by FCS with a carrier (2) fitted with at least one holder (4) for a fluid sample containing the analyte (the luminescence is excited by a light source (6) e.g. a laser, and a focusing unit (8,8a), and an optical detector (12) to register the luminescence, the gap between the optical focusing unit and the measured volume (10) is \-1 mm, and the carrier is thermally insulated from the excitation light source); (2) the analysis of nucleic acid polymorphisms where a matrix is prepared, with two probes which bond at the matrix under hybridizing conditions (the probes are selected so that they bond to the matrix directly next to each other with the 3' end of one probe directly adjacent to the 5' end of the second probe, positions are selected for one and/or the other probe end to anticipate a polymorphosis at them on the matrix, the nucleotides at the selected ends of the probes are complementary to the positions on the matrix if it shows an initial given variant of the polymorphism, and is not complementary if another variant is indicated). Preferred Features: The probes are hybridized on the matrix. The hybridizing complex is processed so that the matrix and the two bonded probes to give a selective ligation of the two probe ends if the nucleotide at the 3' end of one probe and the nucleotide at the 5' end of the other probe are complementary to the positions on the matrix. It is then detected if there has been a ligation between the two probes, to establish a variant of the polymorphism on the matrix.