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

Abstract

Easy and reliable identification of pathogenic species such as yeasts, emerging as problematic microbes originating from the genus Candida, is a task in the management and treatment of infections, especially in hospitals and other healthcare environments. Aptamers are seizing an already indispensable role in different sensing applications as binding entities with almost arbitrarily tunable specificities and optimizable affinities. Here, we describe a polyclonal SELEX library that not only can specifically recognize and fluorescently label Candida cells, but is also capable to differentiate C. albicans, C. auris and C. parapsilosis cells in flow-cytometry, fluorometric microtiter plate assays and fluorescence microscopy from human cells, exemplified here by human dermal fibroblasts. This offers the opportunity to develop diagnostic tools based on this library. Moreover, these specific and robust affinity molecules could also serve in the future as potent binding entities on biomaterials and as constituents of technical devices and will thus open avenues for the development of cost-effective and easily accessible next generations of electronic biosensors in clinical diagnostics and novel materials for the specific removal of pathogenic cells from human bio-samples.

Polyclonal Aptamer Libraries from a FluRoot-SELEX for the Specific Labeling of the Apical and Elongation/Differentiation Zones of Arabidopsis thaliana Roots

“…Fluorescence microscopy provided the opportunity to visualize and analyze the specific target binding sites of both the final aptamer libraries. Therefore, 5 pmol from each aptamer library was activated according to the prior description and incubated with either the root center or tip for 60 min at room temperature in darkened conditions on a rotator [ 10 , 12 , 13 , 14 , 15 , 16 , 17 ]. Thereafter, the root zone segments were washed two times using 100 µL DPBS and were prepared on a slide with a glass cover.…”

“…Nowadays, adapted SELEX processes such as Capture-SELEX or FluMag-SELEX enable the selection of aptamers for small solute molecules [ 6 ], chemical compounds such as metal ions [ 7 ], as well as proteins and their cofactors [ 8 , 9 , 10 ]. To obtain aptamers that are specific towards whole cells and certain microorganisms, Cell-SELEX and FluCell-SELEX have been developed to target intact living cells such as the pathogenic bacterium Pseudomonas aeruginosa or different mammalian cells, such as cancer cells, without knowing the exact membrane target in advance [ 11 , 12 , 13 , 14 , 15 , 16 , 17 ]. Even against complex consortia of cells such as tumors, specialized technologies allow for the selection of highly specific aptamers to work on pathological tissues [ 18 , 19 ].…”

Robust Fluorometric Aptamer Assay for Direct and Rapid Detection of Clinical Isolates of Candida spec.

“…However, what most of these approaches have in common is that they require additional technical components like different types of (nano)particles or they rely on sandwich-type assay principles [ 49 ]. In contrast, more simple assays have been suggested; these were based on the direct labeling of the intended target with enriched (also known as polyclonal) aptamer libraries or individual aptamers without the need for secondary binding molecules or enzyme-mediated signal amplification [ 47 , 48 , 49 , 50 , 51 , 52 , 53 ].…”

“…With C. albicans , C. auris , and C. parapsilosis as target cells for a whole-cell SELEX process (Systematic Evolution of Ligands by EXponential enrichment) [ 32 , 33 ], we developed an enriched aptamer library against this class of important human pathogens in a previous study [ 50 ]. This library was already sufficient to label these Candida species with fluorescence and allowed fungal cells to be distinguished from human dermal fibroblast (HDF) cells via fluorescence microscopy in a skin early infection model [ 50 ].…”

“…With C. albicans , C. auris , and C. parapsilosis as target cells for a whole-cell SELEX process (Systematic Evolution of Ligands by EXponential enrichment) [ 32 , 33 ], we developed an enriched aptamer library against this class of important human pathogens in a previous study [ 50 ]. This library was already sufficient to label these Candida species with fluorescence and allowed fungal cells to be distinguished from human dermal fibroblast (HDF) cells via fluorescence microscopy in a skin early infection model [ 50 ]. However, the intensity of the unspecific background signals obtained with the HDF cells in a fluorometric suspension assay suggested a considerable potential to improve the sensitivity of such an enriched SELEX library while simultaneously enhancing the specificity for Candida spec.…”