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A sandwich-like strategy for the label-free detection of oligonucleotides by surface plasmon fluorescence spectroscopy (SPFS).

ABSTRACT: For the detection of oligonucleotides a sandwich-like detection strategy has been developed by which the background fluorescence is significantly lowered in comparison with surface-bound molecular beacons. Surface bound optical molecular beacons are DNA hairpin structures comprising a stem and a loop. The end of the stem is modified with a fluorophore and a thiol anchor for chemisorption on gold surfaces. In the closed state the fluorophore is in close proximity to the gold surface, and most of the fluorescence is quenched. After hybridization with a target the hairpin opens, the fluorophore and surface become separated, and the fluorescence drastically increases. Using this detection method the sensitivity is limited by the difference in the fluorescence intensity in the closed and open state. As the background fluorescence is mainly caused by non-quenched fluorophores, a strategy to reduce the background fluorescence is to cut the beacon in two halves. First a thiolated ssDNA capture probe strand (first half) is chemisorbed to a gold surface together with relatively short thiol spacers. Next the target is hybridized by one end to the surface-anchored capture probe and by the other to a fluorophore-labeled reporter probe DNA (second half). The signal readout is done by surface plasmon fluorescence spectroscopy (SPFS). Using this detection strategy the background fluorescence can be significantly lowered, and the detection limit is lowered by more than one order of magnitude. The detection of a target takes only a few minutes and the sensor chips can be used for multiple detection steps without a significant decrease in performance.

SUBMITTER: Su Q

PROVIDER: S-EPMC5166564 | biostudies-literature | 2016 Oct

REPOSITORIES: biostudies-literature

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A sandwich-like strategy for the label-free detection of oligonucleotides by surface plasmon fluorescence spectroscopy (SPFS).

Su Qiang Q Nöll Gilbert G

The Analyst 20160803 20

For the detection of oligonucleotides a sandwich-like detection strategy has been developed by which the background fluorescence is significantly lowered in comparison with surface-bound molecular beacons. Surface bound optical molecular beacons are DNA hairpin structures comprising a stem and a loop. The end of the stem is modified with a fluorophore and a thiol anchor for chemisorption on gold surfaces. In the closed state the fluorophore is in close proximity to the gold surface, and most of ...[more]

PMID: 27484040

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Project description:In the last decade, researchers have been searching for innovative platforms, methods, and techniques able to address recurring problems with the current cancer detection methods. Early disease detection, fast results, point-of-care sensing, and cost are among the most prevalent issues that need further exploration in this field. Herein, studies are focused on overcoming these problems by developing an electrochemical device able to detect telomerase as a cancer biomarker. Electrochemical platforms and techniques are more appealing for cancer detection, offering lower costs than the established cancer detection methods, high sensitivity inherent to the technique, rapid signal processing, and their capacity of being miniaturized. Therefore, Au interdigital electrodes and electrochemical impedance spectroscopy were used to detect telomerase activity in acute T cell leukemia. Different cancer cell concentrations were evaluated, and a detection limit of 1.9 × 105 cells/mL was obtained. X-ray photoelectron spectroscopy was used to characterize the telomerase substrate (TS) DNA probe self-assembled monolayer on gold electrode surfaces. Atomic force microscopy displayed three-dimensional images of the surface to establish a height difference of 9.0 nm between the bare electrode and TS-modified Au electrodes. The TS probe is rich in guanines, thus forming secondary structures known as G-quadruplex that can be triggered with a fluorescence probe. Confocal microscopy fluorescence images showed the formation of DNA G-quadruplex because of TS elongation by telomerase on the Au electrode surface. Moreover, electrodes exposed to telomerase containing 2',3'-dideoxyguanosine-5'-triphosphate (ddGTP) did not exhibit high fluorescence, as ddGTP is a telomerase inhibitor, thus making this device suitable for telomerase inhibitors capacity studies. The electrochemical method and Au microchip device may be developed as a biosensor for a point-of-care medical device.

Dataset Information

A sandwich-like strategy for the label-free detection of oligonucleotides by surface plasmon fluorescence spectroscopy (SPFS).

Publications

A sandwich-like strategy for the label-free detection of oligonucleotides by surface plasmon fluorescence spectroscopy (SPFS).

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