Patent EP-2269724-A1 (EP)
Inventor: PAWLAK MICHAEL
IPC: G01N33/543
Filed: 2005-10-29
Abstract: Detection of analytes in biological samples with a complex composition, comprises applying diluted/undiluted samples at discrete places on a solid carrier/after prior application of an adhesive mediation layer on the solid carrier, contacting an array of discrete measuring ranges with a first-, second- and third solution containing binding reagents as specific binding partners for the analytes and optionally in the presence of detection reagents, localized measuring of first-, second- and third optical signals, and recording and comparing of optical signals. The detection of analytes in biological samples with a complex composition, comprises applying diluted/undiluted samples at discrete places on a solid carrier/after prior application of an adhesive mediation layer on the solid carrier, contacting an array of discrete measuring ranges with a first-, second- and third solution containing binding reagents as specific binding partners for the analytes and optionally in the presence of detection reagents, localized measuring of first-, second- and third optical signals, and recording and comparing of optical signals. The binding- and detection reagents are applied at the same time or sequentially. The samples are lysates of cell populations, cell extracts, body fluids and components of body fluids. The measuring range represents a material of less than 1 nL. The detection reagents comprise a polyclonal/monoclonal anti-bodies and antibody fragments, nucleic acids and nucleic acid derivatives and their derivatives with artificial bases, biotin, avidin, streptavidin and neutravidin, and also mass label and/or luminescence label. The labels bind/deposit themselves respectively on/to the binding reagents, on/to the detection reagents or on/to the complexes between the analytes. The binding reagents and optional detection reagents, and/or compounds and/or materials, are pre-incubated with one another and the solutions are brought in contact with the arrays of measuring ranges. A multiplicity of different analytes is detected in a multiplicity of arrays of discrete measuring ranges by addition of the arrays of measuring ranges with different binding reagents. The number of different analytes is equal to the number of various detection reagents, which differ from one another in the excitation and/or emission wavelength of a luminescence. The different binding reagents are applied for the detection of every analyte. A known/different concentrations of compounds are added to the applied material as standards, which are analogous to the analytes. The number of measuring ranges and the extent of the known different concentrations are sufficient to produce a calibration curve to determine the unknown quantity concentrations of the analytes by means of the addition step of a first solution. Many analogous arrays of measuring ranges are arranged on a solid carrier in which the same positions of measuring ranges in different arrays with respect to arrangement in rows and columns. The addition of the samples, measurement- and recordings of the signal take place on different analogous arrays of measuring ranges. The first optical signal produced by non-specific interaction with the reagents is determined from the difference between the optical signals measured after the addition of the second/third solution and the first solution. The concentration/quantity of the analyte is determined from the difference between the measured optical signal and the optical signal produced by non-specific interaction. In the method, less than 10% differences of the concentration of analyte are determined in different samples. The ranges between the measuring ranges are passivated for the minimization of non-specific binding of reagents, in which non-binding chemically neutral components are applied between the spatially separated measuring ranges with respect to the mentioned binding reagents and/or detection reagents. The solid carrier is optically transparent to an irradiated excitation light/measuring light and has microscope plates, micro-titer plates, nano-titer plates, filters, membranes, micro-structured carriers, and a single- or multi-layered optical wave guide that is continuous or divided into discrete wave-guiding ranges. The excitation or measuring light of polychromatic or monochromatic sources of light is led to measuring ranges of the arrays and optical signals from the measuring ranges and/or changes or differences of the optical signals from the measuring ranges are measured in a localized manner and recorded. Independent claims are included for: (1) a micro array for quantitative regulation of analytes in samples of biological origin; and (2) a procedure for the quantitative regulation of analytes in samples of biological origin.