| 1. |
- Lee, Sujin, et al.
(författare)
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Aptamer/ISET-MS: A New Affinity-Based MALDI Technique for Improved Detection of Biomarkers
- 2014
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Ingår i: Analytical Chemistry. - : American Chemical Society (ACS). - 1520-6882 .- 0003-2700. ; 86:15, s. 7627-7634
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Tidskriftsartikel (refereegranskat)abstract
- With the rapid progress in the development of new clinical biomarkers there is an unmet need of fast and sensitive multiplex analysis methods for disease specific protein monitoring. Immunoaffinity extraction integrated with matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) analysis offers a route to rapid and sensitive protein analysis and potentially multiplex biomarker analysis. In this study, the previously reported integrated selective enrichment target (ISET)-MALDI-MS analysis was implemented with ssDNA aptamer functionalized microbeads to address the specific capturing of thrombin in complex samples. The main objective for using an aptamer as the capturing ligand was to avoid the inherently high background components, which are produced during the digestion step following the target extraction when antibodies are used. By applying a thrombin specific aptamer linked to ISET-MALDI-MS detection, a proof of concept of antibody fragment background reduction in the ISET-MALDI-MS readout is presented. Detection sensitivity was significantly increased compared to the corresponding system based on antibody-specific binding as the aptamer ligand does not induce any interfering background residues from the antibodies. The limit of detection for thrombin was 10 fmol in buffer using the aptamer/ISET-MALDI-MS configuration as confirmed by MS/MS fragmentation. The aptamer/ISET-MALDI-MS platform also displayed a limit of detection of 10 fmol for thrombin in five different human serum samples (1/10 diluted), demonstrating the applicability of the aptamer/ISET-MALDI-MS analysis in clinical samples.
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| 2. |
- Park, Jeewoong, et al.
(författare)
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Acousto-microfluidics for screening of ssDNA aptamer
- 2016
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Ingår i: Scientific Reports. - : Springer Science and Business Media LLC. - 2045-2322. ; 6
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Tidskriftsartikel (refereegranskat)abstract
- We demonstrate a new screening method for obtaining a prostate-specific antigen (PSA) binding aptamer based on an acoustofluidic separation (acoustophoreis) technique. Since acoustophoresis provides simultaneous washing and separation in a continuous flow mode, we efficiently obtained a PSA binding aptamer that shows high affinity without any additional washing step, which is necessary in other screening methods. In addition, next-generation sequencing (NGS) was applied to accelerate the identification of the screened ssDNA pool, improving the selecting process of the aptamer candidate based on the frequency ranking of the sequences. After the 8 th round of the acoustophoretic systematic evolution of ligands by exponential enrichment (SELEX) and following sequence analysis with NGS, 7 PSA binding ssDNA aptamer-candidates were obtained and characterized with surface plasmon resonance (SPR) for affinity and specificity. As a result of the new SELEX method with PSA as the model target protein, the best PSA binding aptamer showed specific binding to PSA with a dissociation constant (K d) of 0.7 nM.
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