A method for detecting a target molecule that may be present in a test sample, the method comprising: (a) contacting a test sample with an aptamer comprising a 5-position pyrimidine modification and having a specific affinity for a target molecule, wherein an aptamer affinity complex is formed by the interaction of an aptamer with its target molecule, if said target molecule is present in said test sample, wherein an aptamer affinity complex is a non-covalent complex formed by the interaction of an aptamer with its target molecule; (b) after aptamer affinity complex formation in step (a) exposing said test sample to a condition that kinetically challenges components of said test sample, wherein the kinetic challenge comprises (i) introducing a competitor molecule to the test sample, or (ii) the capture of aptamer affinity complexes on a solid support followed by washing with competitor molecules present in the wash solution, wherein the competitor molecule is selected from an oligonucleotide, a polyanion, a polydextran or dNTPs, or (iii) diluting the test sample in order to enrich for an aptamer affinity complex from a set of complexes that includes an aptamer affinity complex and non-specific complexes, the dilution thereby reducing aptamer non-target complexes compared to aptamer target complexes; (c) detecting and/or quantifying the aptamer of the aptamer affinity complex or the aptamer affinity complex partitioned from the remainder of the test sample. (a) contacting a test sample with an aptamer comprising a 5-position pyrimidine modification and having a specific affinity for a target molecule, wherein an aptamer affinity complex is formed by the interaction of an aptamer with its target molecule, if said target molecule is present in said test sample, wherein an aptamer affinity complex is a non-covalent complex formed by the interaction of an aptamer with its target molecule; (b) after aptamer affinity complex formation in step (a) exposing said test sample to a condition that kinetically challenges components of said test sample, wherein the kinetic challenge comprises (i) introducing a competitor molecule to the test sample, or (ii) the capture of aptamer affinity complexes on a solid support followed by washing with competitor molecules present in the wash solution, wherein the competitor molecule is selected from an oligonucleotide, a polyanion, a polydextran or dNTPs, or (iii) diluting the test sample in order to enrich for an aptamer affinity complex from a set of complexes that includes an aptamer affinity complex and non-specific complexes, the dilution thereby reducing aptamer non-target complexes compared to aptamer target complexes; (i) introducing a competitor molecule to the test sample, or (ii) the capture of aptamer affinity complexes on a solid support followed by washing with competitor molecules present in the wash solution, wherein the competitor molecule is selected from an oligonucleotide, a polyanion, a polydextran or dNTPs, or (iii) diluting the test sample in order to enrich for an aptamer affinity complex from a set of complexes that includes an aptamer affinity complex and non-specific complexes, the dilution thereby reducing aptamer non-target complexes compared to aptamer target complexes; (c) detecting and/or quantifying the aptamer of the aptamer affinity complex or the aptamer affinity complex partitioned from the remainder of the test sample.