Journal: Scientific Reports

Article Title: A multiparametric fluorescence assay for screening aptamer–protein interactions based on microbeads

doi: 10.1038/s41598-022-06817-0

Figure Lengend Snippet: Principle of the multiplexed aptamer binding assay using VideoScan technology. (1) Dye/size-encoded microbead populations presenting different proteins on their surfaces are mixed (2) incubated with a fluorescence-labelled aptamer (3) dissolved in a binding buffer of choice. After removing unbound aptamers by washing, the microbead suspension was transferred into a cavity of a 96 well plate (4). The microbeads were allowed to settle down forming a microbead chip on the transparent bottom (5). A fluorescence microscope (6) is used to take pictures of the microbead chip (7). Imaging software analyses the pictures, recognises and counts microbeads, measures their surface fluorescence intensity and groups them into populations (8). Finally, for each population the referenced mean fluorescence intensity (rMFI) per microbead population is calculated (9). (10) Shows a 96-well microtiter plate placed into the VideoScan system.

Article Snippet: All measurements of aptamer-target interactions were done with our in-house developed fully automatised multispectral inverse fluorescence microscopy platform, called VideoScan , commercialised as Calaidoscan 100 (CS100) at Attomol GmbH (Germany) (Fig. ).

Techniques: Binding Assay, Incubation, Fluorescence, Suspension, Microscopy, Imaging, Software

Journal: Scientific Reports

Article Title: A multiparametric fluorescence assay for screening aptamer–protein interactions based on microbeads

doi: 10.1038/s41598-022-06817-0

Figure Lengend Snippet: Influence of ionic strength on aptamer binding. Microbeads coupled with ( A ) IFNγ, ( B ) thrombin, ( C ) streptavidin and ( D ) Pf LDH were incubated with their fluorescence-labelled aptamers in the presence of varying NaCl concentrations (0–1000 mM). The binding of the aptamer to its target was measured by quantifying the surface fluorescence of the microbeads using VideoScan technology (mean values, n = 2). The half-maximal value (ED 50 in mM) was calculated after fitting non-linear models (IFNγ-apta: EXD.2; T2-apta: LL.2; T1-apta, SA-apta, Pf LDH-apta: LL.3).

Article Snippet: All measurements of aptamer-target interactions were done with our in-house developed fully automatised multispectral inverse fluorescence microscopy platform, called VideoScan , commercialised as Calaidoscan 100 (CS100) at Attomol GmbH (Germany) (Fig. ).

Techniques: Binding Assay, Incubation, Fluorescence

Journal: Scientific Reports

Article Title: A multiparametric fluorescence assay for screening aptamer–protein interactions based on microbeads

doi: 10.1038/s41598-022-06817-0

Figure Lengend Snippet: Influence of pH value on aptamer binding. Microbeads coupled with ( A ) IFNγ, ( B ) thrombin, ( C ) streptavidin and ( D ) Pf LDH were incubated with their fluorescence-labeled aptamer in binding buffers (= corresponding selection buffer) of varying pH values. ( E ) Binding capacity of Pf LDH-apta to non-target proteins immobilised on fluorescence-labelled microbeads under varying pH values. The binding of all aptamers to their targets were measured by quantifying the surface fluorescence of the microbeads using VideoScan technology. All data are represented as mean ± SD (n = 4).

Article Snippet: All measurements of aptamer-target interactions were done with our in-house developed fully automatised multispectral inverse fluorescence microscopy platform, called VideoScan , commercialised as Calaidoscan 100 (CS100) at Attomol GmbH (Germany) (Fig. ).

Techniques: Binding Assay, Incubation, Fluorescence, Labeling, Selection