The employment of plasmonic nanostructures for Surface Enhanced Raman Scattering (SERS) based biosensing has been extensively explored, especially in the framework of early cancer diagnosis, which can benefit from the label-free nature and high sensitivity of SERS in a multiplexing approach. In this work, we discuss the detection of miRNA222, an important cancer biomarker, on silver decorated porous silicon-PDMS membranes integrated in elastomeric multichamber microfluidic chips, investigating the effectiveness of different analysis configurations for the determination of miRNAs in cell extracts. A bioassay based on a two-step hybridization procedure was developed (Fig.1a). According to this functionalization protocol, a DNA probe, complementary to the target sequence, is divided in two shorter strands (half1 and half2). Half1 is immobilized on the Ag nanoparticles (NPs), in order to specifically capture the target miRNA, then, half2, modified with a Raman label at the 5’ end, is incubated for the sensitive and label-free detection by SERS. After an accurate optimization of the protocol, its specificity for the target miRNA222 was confirmed by the analysis of mixtures of several miRNAs, even at high concentration, that simulate the effect of interfering sequences in real samples. In addition, a calibration curve was obtained, showing a decrease of the limit of detection for miRNA222, which was lowered of more than two orders of magnitude in comparison to the standard one-step hybridization protocol. Thanks to the new procedure, the analysis of cell extracts in the SERS microfluidic chip enabled the specific identification of the miRNA222 in the complex biological matrix. In order to further improve the sensitivity, different analysis configurations were finally investigated. The effect of different Raman dyes (Cyanine 5/3 or Rhodamine 6G) and of their labelling position (3’ or 5’) were studied. A significant boost of the SERS signal for the labelling at the 3’ end (close to the NPs surface) compared to the 5’ (far from the surface) was verified, due to a more efficient electromagnetic enhancement. The combination of this improved sensitivity with the high specificity of the assay paves the way to the effective application of these SERS chips in the early detection of tumor markers.

Microfluidic SERS chips for the selective detection of miRNAs in biological matrixes / Novara, Chiara; Chiado', Alessandro; Paccotti, Niccolo'; Condorelli, Gerolama; de Franciscis, Vittorio; Geobaldo, Francesco; Rivolo, Paola; Giorgis, Fabrizio. - T:(2017), pp. 26-26. ((Intervento presentato al convegno Plasmonica 2017 tenutosi a Lecce nel 5-7/07/2017.

Microfluidic SERS chips for the selective detection of miRNAs in biological matrixes.

Chiara Novara;Alessandro Chiadò;Niccolò Paccotti;Francesco Geobaldo;Paola Rivolo;Fabrizio Giorgis
2017

Abstract

The employment of plasmonic nanostructures for Surface Enhanced Raman Scattering (SERS) based biosensing has been extensively explored, especially in the framework of early cancer diagnosis, which can benefit from the label-free nature and high sensitivity of SERS in a multiplexing approach. In this work, we discuss the detection of miRNA222, an important cancer biomarker, on silver decorated porous silicon-PDMS membranes integrated in elastomeric multichamber microfluidic chips, investigating the effectiveness of different analysis configurations for the determination of miRNAs in cell extracts. A bioassay based on a two-step hybridization procedure was developed (Fig.1a). According to this functionalization protocol, a DNA probe, complementary to the target sequence, is divided in two shorter strands (half1 and half2). Half1 is immobilized on the Ag nanoparticles (NPs), in order to specifically capture the target miRNA, then, half2, modified with a Raman label at the 5’ end, is incubated for the sensitive and label-free detection by SERS. After an accurate optimization of the protocol, its specificity for the target miRNA222 was confirmed by the analysis of mixtures of several miRNAs, even at high concentration, that simulate the effect of interfering sequences in real samples. In addition, a calibration curve was obtained, showing a decrease of the limit of detection for miRNA222, which was lowered of more than two orders of magnitude in comparison to the standard one-step hybridization protocol. Thanks to the new procedure, the analysis of cell extracts in the SERS microfluidic chip enabled the specific identification of the miRNA222 in the complex biological matrix. In order to further improve the sensitivity, different analysis configurations were finally investigated. The effect of different Raman dyes (Cyanine 5/3 or Rhodamine 6G) and of their labelling position (3’ or 5’) were studied. A significant boost of the SERS signal for the labelling at the 3’ end (close to the NPs surface) compared to the 5’ (far from the surface) was verified, due to a more efficient electromagnetic enhancement. The combination of this improved sensitivity with the high specificity of the assay paves the way to the effective application of these SERS chips in the early detection of tumor markers.
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11583/2749051
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