Agnese Sonato

A peptide nucleic acid label-free biosensor for Mycobacterium tuberculosis DNA detection via azimuthally controlled grating-coupled SPR

Grating coupled surface plasmon resonance phenomena under azimuthal control of incident light (φ ≠ 0° GC-SPR) have recently been exploited for the development of biosensing solutions with a sensitivity similar to that of classic prism-coupled SPR sensors, with the advantage of higher miniaturization potential. Here we combined the use of φ ≠ 0° GC-SPR with the use of peptide nucleic acid (PNA) probes and a strategy for maximizing the signal-to-noise ratio for the sensitive detection of Mycobacterium tuberculosis (MT) DNA. We focused our attention on the optimization of the PNA-based sensing layer by controlling the sensing surface composition with the PNA-based probe and a poly(ethylene oxide) (PEO)-based antifouling layer. We tested the sensor response first in the presence of complementary and non-complementary oligonucleotides, and then we applied our strategy for the detection of PCR amplified samples, using the fluorescence-based microarray technology as the control. With the φ ≠ 0° GC-SPR set-up adopted, a limit of detection (LOD 0.26 pM) more than one order of magnitude lower than that obtained by the fluorescence method (LOD 8.9 pM) was observed using a complementary oligonucleotide target. Also when PCR amplicons were analysed on SPR grating surfaces, lower DNA concentrations were detectable with the SPR readout as compared to the fluorescence one, and with an experimental protocol that does not include the need to use expensive fluorophore molecules. The whole approach, involving the sensor fabrication, the sensing surface control and DNA detection, has demonstrated that φ ≠ 0° GC-SPR is a good starting point for a sensitive, versatile and scalable biosensing technique that will be further investigated in future experiments.

Label-Free Efficient and Accurate Detection of Cystic Fibrosis Causing Mutations Using an Azimuthally Rotated GC-SPR Platform

Plasmonic nanosensors are candidates for the development of new sensors with low detection limits, high sensitivity, and specificity for target detection: these characteristics are of critical importance in the screening of mutations responsible for inherited diseases. In this work, we focused our study on the detection of some of the most frequent mutations responsible for cystic fibrosis (CF) among the Italian population. For the detection of the CF mutations we adopted a recently developed and highly sensitive Grating Coupled-Surface Plasmon Resonance (GC-SPR) enhanced spectroscopy method for label-free molecular identification exploiting a conical illumination configuration. Gold sinusoidal gratings functionalized with heterobifunctional PEG were used as sensing surfaces, and the specific biodetection was achieved through the coupling with DNA hairpin probes designed for single nucleotide discrimination. Such substrates were used to test unlabeled PCR amplified homozygous wild type (wt) and heterozygous samples, deriving from clinical samples, for the screened mutations. Hybridization conditions were optimized to obtain the maximum discrimination ratio (DR) between the homozygous wild type and the heterozygous samples. SPR signals obtained from hybridizing wild type and heterozygous samples show DRs able to identify univocally the correct genotypes, as confirmed by fluorescence microarray experiments run in parallel. Furthermore, SPR genotyping was not impaired in samples containing unrelated DNA, allowing the platform to be used for the concomitant discrimination of several alleles also scalable for a high throughput screening setting.

Plasmonic Platforms for Biodetection Devices

We designed and experimentally realized the prototype for a bio-recognition device based on Grating Coupled Surface Plasmon Resonance (GCSPR) which can find a wide range of applications for detection of chemical and biological species. A sinusoidal grating (period of 500 nm and peak-to-valley height of 40 nm) was realized by Laser Interference Lithography (LIL) and replicated onto a polymeric resin film. After substrate coating with a Cr/Au film a thio-mPEO (Mw 5000) layer with a medium thickness of 4 nm was deposited onto the plasmonic surface. The SPR detection system allowed to detect the biological layer with a refractive index sensitivity of 995.2°/RIU. Our solution has been demonstrated to be a very sensitive platform for the development of a complete GCSPR biosensor.

Perfluoropolyether (PFPE) Intermediate Molds for High-Resolution Thermal Nanoimprint Lithography

Among soft lithography techniques, Thermal Nanoimprint Lithography (NIL) is a high-throughput and low-cost process that can be applied to a broad range of thermoplastic materials. By simply applying the appropriate pressure and temperature combination, it is possible to transfer a pattern from a mold surface to the chosen material. Usually, high-resolution and large-area NIL molds are difficult to fabricate and expensive. Furthermore, they are typically made of silicon or other hard materials such as nickel or quartz for preserving their functionality. Nonetheless, after a large number of imprinting cycles, they undergo degradation and become unusable. In this paper, we introduce and characterize an innovative two-step NIL process based on the use of a perfluoropolyether (PFPE) intermediate mold to replicate sub-100 nm features from a silicon mold to the final thermoplastic material. We compare PFPE elastomeric molds with molds made of the standard polydimethylsiloxane (PDMS) elastomer, which demonstrates better resolution and fidelity of the replica process. By using PFPE intermediate molds, the nanostructured masters are preserved and the throughput of the process is significantly enhanced.

Novel compact architecture for high-resolution sensing with plasmonic gratings in conical mounting

A novel compact architecture implementing grating-coupled surface plasmon resonance (GCSPR) based on polarization modulation in conical mounting is presented. In this system a plasmonic grating is azimuthally rotated in order to support the excitation of high-sensitivity surface plasmon polaritons (SPPs). At SPP resonance, a scan of the incident polarization is performed before and after the binding event and the phase term of the output trend is exploited as sensing parameter. The mechanical complexity of the SPR system is significantly reduced and a resolution down to 10-7 refractive index units is assured. In this work a numerical study of the polarization-based grating-coupled SPR technique is performed and analyzed with Chandezon’s method. Therefore an experimental test on an assembled prototype is presented and applied to the detection of binding events on the grating surface (avidin/biotin reaction, DNA/PNA probes).