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Micropatterning of Aptamer Beacons to Create Cytokine-Sensing Surfaces.

Tuleuova N, Revzin A - Cell Mol Bioeng (2010)

Bottom Line: Cytokine molecules were expected to displace quenching strands of the beacon, disrupting FRET effect and resulting in fluorescence signal.Subsequent incubation with quencher-carrying complementary strands resulted in formation of DNA duplex and caused quenching of fluorescence due to FRET effect.In the future, we plan to co-localize aptamer beacons and cells on micropatterned surfaces in order to monitor in real-time cytokine secretion from immune cells in microwells.

View Article: PubMed Central - PubMed

ABSTRACT
Aptamer beacons are DNA or RNA probes that bind proteins or small molecules of interest and emit signal directly upon interaction with the target analyte. This paper describes micropatterning of aptamer beacons for detection of IFN-γ-an important inflammatory cytokine. The beacon consisted of a fluorophore-labeled aptamer strand hybridized with a shorter, quencher-carrying complementary strand. Cytokine molecules were expected to displace quenching strands of the beacon, disrupting FRET effect and resulting in fluorescence signal. The glass substrate was first micropatterned with poly(ethylene glycol) (PEG) hydrogel microwells (35 μm diameter individual wells) so as to define sites for attachment of beacon molecules. PEG microwell arrays were then incubated with avidin followed by biotin-aptamer-fluorophore constructs. Subsequent incubation with quencher-carrying complementary strands resulted in formation of DNA duplex and caused quenching of fluorescence due to FRET effect. When exposed to IFN-γ, microwells changed fluorescence from low (quencher hybridized with fluorophore-carrying strand) to high (quenching strand displaced by cytokine molecules). The fluorescence signal was confined to microwells, was changing in real-time and was dependent on the concentration of IFN-γ. In the future, we plan to co-localize aptamer beacons and cells on micropatterned surfaces in order to monitor in real-time cytokine secretion from immune cells in microwells.

No MeSH data available.


(a) Design of a microfluidic device used in cytokine detection studies. The device was fabricated in PDMS and contained two reaction chambers that were secured on top of microwell arrays by vacuum suction applied through a web of auxiliary channels. This PDMS attachment approach did not compromise non-fouling or sensing components of micropatterned surfaces. (b–c) 35 μm diameter wells with immobilized aptamer molecules before (b) and after (c) injection of quencher-labeled complementary strands into a microfluidic device. Loss of fluorescence in part (c) was attributed to formation of DNA duplex. (d) Fluorescence intensity measurements show 5 fold decrease in fluorescence after incubation of microwells with quenching DNA strands
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Fig4: (a) Design of a microfluidic device used in cytokine detection studies. The device was fabricated in PDMS and contained two reaction chambers that were secured on top of microwell arrays by vacuum suction applied through a web of auxiliary channels. This PDMS attachment approach did not compromise non-fouling or sensing components of micropatterned surfaces. (b–c) 35 μm diameter wells with immobilized aptamer molecules before (b) and after (c) injection of quencher-labeled complementary strands into a microfluidic device. Loss of fluorescence in part (c) was attributed to formation of DNA duplex. (d) Fluorescence intensity measurements show 5 fold decrease in fluorescence after incubation of microwells with quenching DNA strands

Mentions: In order to precisely control reagent exchange during IFN-γ detection experiments, glass slides with hydrogel microwells were outfitted with PDMS microfluidic channels and mounted on a fluorescence microscope. The design of the microfluidic device, first proposed by Schaff et al.23 and shown in Fig. 4a, allowed to reversibly seal PDMS fluid conduits on top of the hydrogel microwell arrays using vacuum suction. This obviated the need to expose micropatterned surfaces to oxygen plasma treatment and allowed to retain intact non-fouling and protein-modified domains on the surface.Figure 4


Micropatterning of Aptamer Beacons to Create Cytokine-Sensing Surfaces.

Tuleuova N, Revzin A - Cell Mol Bioeng (2010)

(a) Design of a microfluidic device used in cytokine detection studies. The device was fabricated in PDMS and contained two reaction chambers that were secured on top of microwell arrays by vacuum suction applied through a web of auxiliary channels. This PDMS attachment approach did not compromise non-fouling or sensing components of micropatterned surfaces. (b–c) 35 μm diameter wells with immobilized aptamer molecules before (b) and after (c) injection of quencher-labeled complementary strands into a microfluidic device. Loss of fluorescence in part (c) was attributed to formation of DNA duplex. (d) Fluorescence intensity measurements show 5 fold decrease in fluorescence after incubation of microwells with quenching DNA strands
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2991185&req=5

Fig4: (a) Design of a microfluidic device used in cytokine detection studies. The device was fabricated in PDMS and contained two reaction chambers that were secured on top of microwell arrays by vacuum suction applied through a web of auxiliary channels. This PDMS attachment approach did not compromise non-fouling or sensing components of micropatterned surfaces. (b–c) 35 μm diameter wells with immobilized aptamer molecules before (b) and after (c) injection of quencher-labeled complementary strands into a microfluidic device. Loss of fluorescence in part (c) was attributed to formation of DNA duplex. (d) Fluorescence intensity measurements show 5 fold decrease in fluorescence after incubation of microwells with quenching DNA strands
Mentions: In order to precisely control reagent exchange during IFN-γ detection experiments, glass slides with hydrogel microwells were outfitted with PDMS microfluidic channels and mounted on a fluorescence microscope. The design of the microfluidic device, first proposed by Schaff et al.23 and shown in Fig. 4a, allowed to reversibly seal PDMS fluid conduits on top of the hydrogel microwell arrays using vacuum suction. This obviated the need to expose micropatterned surfaces to oxygen plasma treatment and allowed to retain intact non-fouling and protein-modified domains on the surface.Figure 4

Bottom Line: Cytokine molecules were expected to displace quenching strands of the beacon, disrupting FRET effect and resulting in fluorescence signal.Subsequent incubation with quencher-carrying complementary strands resulted in formation of DNA duplex and caused quenching of fluorescence due to FRET effect.In the future, we plan to co-localize aptamer beacons and cells on micropatterned surfaces in order to monitor in real-time cytokine secretion from immune cells in microwells.

View Article: PubMed Central - PubMed

ABSTRACT
Aptamer beacons are DNA or RNA probes that bind proteins or small molecules of interest and emit signal directly upon interaction with the target analyte. This paper describes micropatterning of aptamer beacons for detection of IFN-γ-an important inflammatory cytokine. The beacon consisted of a fluorophore-labeled aptamer strand hybridized with a shorter, quencher-carrying complementary strand. Cytokine molecules were expected to displace quenching strands of the beacon, disrupting FRET effect and resulting in fluorescence signal. The glass substrate was first micropatterned with poly(ethylene glycol) (PEG) hydrogel microwells (35 μm diameter individual wells) so as to define sites for attachment of beacon molecules. PEG microwell arrays were then incubated with avidin followed by biotin-aptamer-fluorophore constructs. Subsequent incubation with quencher-carrying complementary strands resulted in formation of DNA duplex and caused quenching of fluorescence due to FRET effect. When exposed to IFN-γ, microwells changed fluorescence from low (quencher hybridized with fluorophore-carrying strand) to high (quenching strand displaced by cytokine molecules). The fluorescence signal was confined to microwells, was changing in real-time and was dependent on the concentration of IFN-γ. In the future, we plan to co-localize aptamer beacons and cells on micropatterned surfaces in order to monitor in real-time cytokine secretion from immune cells in microwells.

No MeSH data available.