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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.


Process flow diagram for micropatterning hydrogel microwells and for immobilizing beacon molecules inside the microwells
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Fig2: Process flow diagram for micropatterning hydrogel microwells and for immobilizing beacon molecules inside the microwells

Mentions: Prior to PEG gel immobilization, the glass substrates were modified using a silane coupling agent. The substrates were exposed to O2 plasma for 3 min at 300 W, placed into N2 filled glove bag and immersed in 2 mM solution of (3-acryloxypropyl) trichlorosilane in toluene. The silane self-assembly was allowed to proceed for 1 h under N2 purge, after which the substrates were removed, rinsed in toluene and dried using N2 gas. The substrates were then placed in an oven for 3 h at 100 °C to cure the silane layer. This silanization procedure has been used by us previously for anchoring of the PEG hydrogel microstructures to glass substrates. Figure 2 illustrates the process flow for the fabrication of PEG hydrogel micropatterns. PEG hydrogel patterns were fabricated from the precursor solution of PEG-DA (MW 575) and 2% (w/v) photoinitiator, DMPA. This prepolymer solution was spin-coated at 800 rpm for 4 s onto a standard 3 × 1 in. glass slide and then exposed to UV light at 65 mW/cm2 for 15 s to convert liquid prepolymer into cross-linked hydrogel. Unpolymerized PEG solution was removed by development in DI water for 5 min. This process resulted in formation of 35 μm diameter PEG hydrogel wells on glass.Figure 2


Micropatterning of Aptamer Beacons to Create Cytokine-Sensing Surfaces.

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

Process flow diagram for micropatterning hydrogel microwells and for immobilizing beacon molecules inside the microwells
© Copyright Policy
Related In: Results  -  Collection

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

Fig2: Process flow diagram for micropatterning hydrogel microwells and for immobilizing beacon molecules inside the microwells
Mentions: Prior to PEG gel immobilization, the glass substrates were modified using a silane coupling agent. The substrates were exposed to O2 plasma for 3 min at 300 W, placed into N2 filled glove bag and immersed in 2 mM solution of (3-acryloxypropyl) trichlorosilane in toluene. The silane self-assembly was allowed to proceed for 1 h under N2 purge, after which the substrates were removed, rinsed in toluene and dried using N2 gas. The substrates were then placed in an oven for 3 h at 100 °C to cure the silane layer. This silanization procedure has been used by us previously for anchoring of the PEG hydrogel microstructures to glass substrates. Figure 2 illustrates the process flow for the fabrication of PEG hydrogel micropatterns. PEG hydrogel patterns were fabricated from the precursor solution of PEG-DA (MW 575) and 2% (w/v) photoinitiator, DMPA. This prepolymer solution was spin-coated at 800 rpm for 4 s onto a standard 3 × 1 in. glass slide and then exposed to UV light at 65 mW/cm2 for 15 s to convert liquid prepolymer into cross-linked hydrogel. Unpolymerized PEG solution was removed by development in DI water for 5 min. This process resulted in formation of 35 μm diameter PEG hydrogel wells on glass.Figure 2

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.