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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.


Regeneration of cytokine-sensing surfaces. Hydrogel microwells containing aptamer beacons (DNA duplex) were exposed to 100 nM IFN-γ, producing fluorescence signal. Subsequently, microwells were washed with urea buffer to denature/remove IFN-γ and regenerate fluorescence of the sensing surface. In the last step, hydrogel microwells were incubated with the quenching strand to regenerate aptamer beacon (DNA duplex). This process allowed us to use the same sensing microwells multiple times with minimal loss of sensitivity
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Fig6: Regeneration of cytokine-sensing surfaces. Hydrogel microwells containing aptamer beacons (DNA duplex) were exposed to 100 nM IFN-γ, producing fluorescence signal. Subsequently, microwells were washed with urea buffer to denature/remove IFN-γ and regenerate fluorescence of the sensing surface. In the last step, hydrogel microwells were incubated with the quenching strand to regenerate aptamer beacon (DNA duplex). This process allowed us to use the same sensing microwells multiple times with minimal loss of sensitivity

Mentions: In addition to real-time analyte monitoring, aptamers offer an advantage of chemical stability. The lack of chemical stability makes storage of Ab-based assays difficult and re-use of these assays nearly impossible. In contrast, as shown in Fig. 6, aptamer-based biosensing surfaces could be reused multiple times with minimal loss in sensitivity. In these experiments, microwells with immobilized aptamer beacon (DNA duplex) were challenged with 100 nM IFN-γ as described in the previous section of this paper. Subsequently, microfluidic devices were infused with 7 M urea buffer for 5 min. This treatment was sufficient to disrupt aptamer-cytokine interactions and regenerate fluorescence of sensing surfaces. As can be seen from Fig. 6, the same micropatterned surface regenerated and challenged with 100 nM IFN-γ showed minimal cycle-to-cycle variability.Figure 6


Micropatterning of Aptamer Beacons to Create Cytokine-Sensing Surfaces.

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

Regeneration of cytokine-sensing surfaces. Hydrogel microwells containing aptamer beacons (DNA duplex) were exposed to 100 nM IFN-γ, producing fluorescence signal. Subsequently, microwells were washed with urea buffer to denature/remove IFN-γ and regenerate fluorescence of the sensing surface. In the last step, hydrogel microwells were incubated with the quenching strand to regenerate aptamer beacon (DNA duplex). This process allowed us to use the same sensing microwells multiple times with minimal loss of sensitivity
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2991185&req=5

Fig6: Regeneration of cytokine-sensing surfaces. Hydrogel microwells containing aptamer beacons (DNA duplex) were exposed to 100 nM IFN-γ, producing fluorescence signal. Subsequently, microwells were washed with urea buffer to denature/remove IFN-γ and regenerate fluorescence of the sensing surface. In the last step, hydrogel microwells were incubated with the quenching strand to regenerate aptamer beacon (DNA duplex). This process allowed us to use the same sensing microwells multiple times with minimal loss of sensitivity
Mentions: In addition to real-time analyte monitoring, aptamers offer an advantage of chemical stability. The lack of chemical stability makes storage of Ab-based assays difficult and re-use of these assays nearly impossible. In contrast, as shown in Fig. 6, aptamer-based biosensing surfaces could be reused multiple times with minimal loss in sensitivity. In these experiments, microwells with immobilized aptamer beacon (DNA duplex) were challenged with 100 nM IFN-γ as described in the previous section of this paper. Subsequently, microfluidic devices were infused with 7 M urea buffer for 5 min. This treatment was sufficient to disrupt aptamer-cytokine interactions and regenerate fluorescence of sensing surfaces. As can be seen from Fig. 6, the same micropatterned surface regenerated and challenged with 100 nM IFN-γ showed minimal cycle-to-cycle variability.Figure 6

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.