Polyclonal aptamer libraries as binding entities on a graphene FET based biosensor for the discrimination of apo- and holo-retinol binding protein 4

Abstract

Oligonucleotide DNA aptamers represent an emergently important class of binding entities towards as different analytes as small molecules or even whole cells. Without the canonical isolation of individual aptamers following...

A Polyclonal Aptamer Library for the Specific Binding of the Gut Bacterium Roseburia intestinalis in Mixtures with Other Gut Microbiome Bacteria and Human Stool Samples

“…The polyclonal libraries presented in this study originating from the FluCell-SELEX against R. intestinalis were characterized and found to increase their specificity up to the final round, 7_2. As described for the libraries from previous projects [ 31 , 35 , 36 , 40 ], this library Ri 7_2 was suitable for the labeling of R. intestinalis in fluorescence-based microtiter plate assays and for fluorescence microscopy. The measurements were also performed as microbiome samples derived from the stool samples of two human probands and compared to measurements of sequence abundance via NGS.…”

“…This may not only open the opportunity to develop novel sub-genus detection assays for R. intestinalis, but also in turn, with additional dedicated libraries, for other Roseburia species. We have recently developed a graphene field-effect transistor-based sensor chip for the quantification and differentiation of a biological marker involving polyclonal aptamer libraries [ 40 ]. This technology, in combination with the R. intestinalis polyclonal aptamer library as a binding entity on such a chip, may allow in the near future the development of sensitive and cost-efficient monitoring devices for the rapid and easy supervision of e.g., probiotic supplementation studies with R. intestinalis, and also in a generalized form for all other health-relevant gut bacteria.…”

“…Specific aptamers usually originate from libraries of single-stranded RNA or single-stranded DNA fragments obtained by the systematic evolution of ligands by exponential enrichment (SELEX) technology. The aptamers can be evolved towards a high specificity and affinity for target molecules, such as proteins, amino acids, viruses, pathogens and higher cells and tissues [ 31 , 33 , 34 , 35 , 36 , 37 , 38 , 39 , 40 ]. Initial random libraries consist of 10 12 –10 16 single-stranded deoxyribonucleic acid (ssDNA) molecules and each sequence contains fixed primer binding sites and a random region in the center of the molecule.…”

“…We were able to not only demonstrate the evolution of binding specificity by following the increase in fluorescence along the SELEX rounds, but also show that the resulting libraries (called “polyclonal” or “focused” libraries) could outperform the individual aptamers isolated from these libraries after traditional aptamer characterization via Next Generation Sequencing (NGS) and comprehensive bioinformatic analyses [ 35 , 36 ]. The use of such polyclonal libraries represents a considerable simplification of the generation of specific binding entities for biological assays and for the construction of electronic biosensors, and has proved its potential in sensors for the quantification of isoforms of a health-relevant blood protein [ 40 ]. To omit all of the sequencing and analysis efforts represents another advantage of already using polyclonal libraries without the isolation of individual aptamers, regarding the possible speed of the technological development of functional and specific binding entities; this then allows a fast reaction in case of emerging health-threats, such as novel pathogens, to equip health systems with adequate sensing techniques.…”

A Polyclonal SELEX Aptamer Library Allows Differentiation of Candida albicans, C. auris and C. parapsilosis Cells from Human Dermal Fibroblasts

“…This directed selection process has continuously been improved and modified to create more powerful, robust and specialized strategies that allow the selection of aptamers with specific binding properties for different targets in numerous applications. Not only can they be used in various fields such as biomarker discovery [ 12 ], imaging agents [ 13 ], diagnostics [ 14 ], drug delivery [ 15 ] or as pharmaceutical compounds in molecular therapy [ 16 , 17 ], but they are also used as specific affinity molecules in the construction of binding units in technical devices [ 18 ] such as electronic biosensors [ 5 , 19 ], where mainly antibodies or their derivatives have been used [ 20 , 21 ]. Therefore, based on Cell-SELEX technology, the FluCell-SELEX has been developed in order to evolve aptamers to target intact living cells without knowing the exact membrane targets in advance [ 7 , 8 , 9 ].…”