Multiplex Immunoassays Utilizing Differential Affinity Using Aptamers Generated by MARAS.

Disease diagnosis typically requires to determine concentration of multiple biomarkers in patient serums. Here, a novel method for multiplex immunoassays is proposed and the feasibility is demonstrated. The method utilizes the differential affinity between aptamers and multiple analytes for multiplex immunoassays. During the selection, aptamers capable of binding to multiple analytes with different affinities are screened from a random oligonucleotide library using the MARAS procedure with different magnetic field conditions for different target analytes. During the detection, the same magnetic field conditions are applied to differentiate different target analytes in blind serums. The results show that the recovery rates of the spiked targets in BD buffer and blind serums are similar. Moreover, there is a minimal interference resulting from non-specific binding of molecules in serums other than the target molecules. Therefore, the use of differential affinities between aptamers and different analytes for multiplex immunoassays is proved to be feasible.

Generation of Aptamers from A Primer-Free Randomized ssDNA Library Using Magnetic-Assisted Rapid Aptamer Selection.

Aptamers are oligonucleotides that can bind to specific target molecules. Most aptamers are generated using random libraries in the standard systematic evolution of ligands by exponential enrichment (SELEX). Each random library contains oligonucleotides with a randomized central region and two fixed primer regions at both ends. The fixed primer regions are necessary for amplifying target-bound sequences by PCR. However, these extra-sequences may cause non-specific bindings, which potentially interfere with good binding for random sequences. The Magnetic-Assisted Rapid Aptamer Selection (MARAS) is a newly developed protocol for generating single-strand DNA aptamers. No repeat selection cycle is required in the protocol. This study proposes and demonstrates a method to isolate aptamers for C-reactive proteins (CRP) from a randomized ssDNA library containing no fixed sequences at 5' and 3' termini using the MARAS platform. Furthermore, the isolated primer-free aptamer was sequenced and binding affinity for CRP was analyzed. The specificity of the obtained aptamer was validated using blind serum samples. The result was consistent with monoclonal antibody-based nephelometry analysis, which indicated that a primer-free aptamer has high specificity toward targets. MARAS is a feasible platform for efficiently generating primer-free aptamers for clinical diagnoses.