Chemical Vapor Deposition of Azidoalkylsilane Monolayer Films.

N3-functionalized monolayers on silicon wafer substrates are prepared via the controlled vapor-phase deposition of 11-azidoundecyltrimethoxysilanes at reduced pressure and elevated temperature. The quality of the layer is assessed using contact angle, attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR), and ellipsometry measurements. At 60 °C, longer deposition times are needed to achieve monolayers with similar N3 density compared to depositions at 145 °C. The monolayers formed via the vapor phase are denser compared to those formed via a solvent-based deposition process. ATR-FTIR measurements confirm the incorporation of azido-alkyl chains in the monolayer and the formation of siloxane bridges with the underlying oxide at both deposition temperatures. X-ray photon spectroscopy shows that the N3 group is oriented upward in the grafted layer. Finally, the density was determined using total reflection X-ray fluorescence after a click reaction with chlorohexyne and amounts to 2.5 × 1014 N3 groups/cm2. In summary, our results demonstrate the formation of a uniform and reproducible N3-containing monolayer on silicon wafers, hereby providing a functional coating that enables click reactions at the substrate.

Real-time and label-free ring-resonator monitoring of solid-phase recombinase polymerase amplification.

In this work we present the use of a silicon-on-insulator (SOI) chip featuring an array of 64 optical ring resonators used as refractive index sensors for real-time and label-free DNA detection. Single ring functionalisation was achieved using a click reaction after precise nanolitre spotting of specific hexynyl-terminated DNA capture probes to link to an azido-silanised chip surface. To demonstrate detectability using the ring resonators and to optimise conditions for solid-phase amplification, hybridisation between short 25-mer single stranded DNA (ssDNA) fragments and a complementary capture probe immobilised on the surface of the ring resonators was carried out and detected through the shift in the resonant wavelength. Using the optimised conditions demonstrated via the solid-phase hybridisation, a 144-bp double stranded DNA (dsDNA) was then detected directly using recombinase and polymerase proteins through on-chip target amplification and solid-phase elongation of immobilised forward primers on specific rings, at a constant temperature of 37°C and in less than 60min, achieving a limit of detection of 7.8·10(-13)M (6·10(5) copies in 50µL). The use of an automatic liquid handler injection instrument connected to an integrated resealable chip interface (RCI) allowed programmable multiple injection protocols. Air plugs between different solutions were introduced to prevent intermixing and a proportional-integral-derivative (PID) temperature controller minimised temperature based drifts.

Label-free genosensor based on immobilized DNA hairpins on gold surface.

In this report, we demonstrate a label-free genosensor based on DNA hairpins coupled to gold coated sensor surfaces. The hairpin probes were labeled with a thiolated moiety for immobilization at the 5' end and with a fluorophore for signal transduction at the 3' end. In the absence of the complement, the fluorophore is quenched by energy transfer to the gold surface. Addition of the target sequence leads to the hairpin unfolding, and releases the fluorescent signal. This built-in property, using a gold film as both the immobilizing substrate and quenching agent, has the advantage of simplicity in design and ease of further integration. Our results showed that lengths of both the stem and the loop structures have significant effects on the sensor performance. Hybridization kinetics was investigated for various probe/target lengths and concentrations. An optimized hairpin probe gave a fluorescent signal increase of 39 folds after hybridization, which is much higher than the earlier reported results. A limit of detection (LOD) down to 0.3 nM for the complementary target DNA detection has been achieved. The developed sensor was further successfully applied for the detection of single-base mismatch targets, as well as for the direct detection of PCR products.