Split Aptamers Immobilized on Polymer Brushes Integrated in a Lab-on-Chip System Based on an Array of Amorphous Silicon Photosensors: A Novel Sensor Assay.

Manasa Nandimandalam,Lorenzo Iannascoli,Cesare Manetti,Francesca Costantini,Augusto Nascetti,Nicola Lovecchio,Domenico Caputo,Giampiero De Cesare

doi:10.3390/ma14237210

Abstract

Innovative materials for the integration of aptamers in Lab-on-Chip systems are important for the development of miniaturized portable devices in the field of health-care and diagnostics. Herein we highlight a general method to tailor an aptamer sequence in two subunits that are randomly immobilized into a layer of polymer brushes grown on the internal surface of microfluidic channels, optically aligned with an array of amorphous silicon photosensors for the detection of fluorescence. Our approach relies on the use of split aptamer sequences maintaining their binding affinity to the target molecule. After binding the target molecule, the fragments, separately immobilized to the brush layer, form an assembled structure that in presence of a “light switching” complex [Ru(phen)2(dppz)]2+, emit a fluorescent signal detected by the photosensors positioned underneath. The fluorescent intensity is proportional to the concentration of the target molecule. As proof of principle, we selected fragments derived from an aptamer sequence with binding affinity towards ATP. Using this assay, a limit of detection down to 0.9 µM ATP has been achieved. The sensitivity is compared with an assay where the original aptamer sequence is used. The possibility to re-use both the aptamer assays for several times is demonstrated.

Highlights

Aptamers are single standard DNA or RNA sequences that have been shown to bind non-nucleic acid target molecules with high affinity and specificity [1]
We reported the development of functionalized microfluidic channels for the detection of Ochratoxin A and adenosine triphosphate (ATP) using a Lab-onchip (LoC) in which the detection occurs through an array of amorphous silicon photosensors (a-Si:H) optically aligned with the functionalized microfluidic network [20,21,22]
Our hypothesis is that the fragments after binding ATP would assemble into a folded structure that in presence of [Ru(phen)2(dppz)]2+ would give a fluorescent signal proportional to the concentration of ATP present in solution. [Ru(phen)2(dppz)]2+ is a molecular light switching complex, which has no luminescence in aqueous solution but strong luminescence when intercalating into the non-aqueous pocket of DNA duplex [35]

Summary

Introduction

Aptamers are single standard DNA or RNA sequences that have been shown to bind non-nucleic acid target molecules with high affinity and specificity [1]. Nanoscience and nanotechnology has generated novel nanomaterials which results in aptasensor systems with many applications in the field of health-care and diagnostics [3,4,5]. Optical detection methods such as colorimetry, fluorescence, surface-enhanced Raman scattering (SERS), and surface plasmon resonance (SPR) are widely applied for signal detection in aptasensors because of their ease of use and high sensitivity [6,7,8,9]. There is no need of chemically modifying the aptamers with the fluorophore, which eliminates time-consuming, labor-intensive, and costly synthesis processes [12,13]

Methods

Results

Conclusion