Materials and methods
Microfluidic analytical devices with optical detection are considered one of the best options for developing robust and sensitive lab-on-chip devices. Key features for diagnostic and prognostic usefulness of such devices are high sensitivity and specificity, ability to provide quantitative information and multiplexing capability. On the other hand, one of the main challenges for real life applications and commercial exploitation is full integration, which includes the open issue of coupling the lab-on-chip with a portable and miniaturized detector .
The use of CL detection in a disposable microfluidic chip with integrated array of a:Si-H photosensors has been previously shown to provide excellent analytical performance in enzyme assays and immunoassays applications, along with detector on-chip integration, optimal optical coupling, device compactness and reduced power consumption and memory occupancy .
In the present work, the performance of a portable analytical device based on a disposable microfluidic cartridge integrated with an array of 30 a-Si:H photosensors for CL detection of viral DNA was evaluated. In particular a microfluidic reaction chip was designed comprising a PDMS microfluidic layer coupled with a glass slide on which genotype specific B19 capture oligonucleotide probes had been arrayed. The reaction chip was then integrated with the array of photosensors, aligning each photosensor with one oligonucleotide probe spot in order to monitor luminescent signals. Both the reaction chip and the a-Si:H photosensors are inexpensive to manufacture and suitable for the development of portable analytical devices. The design of the reaction chip combined with the array of photosensors make it possible to detect simultaneously three B19 DNA in a unique analysis. Moreover the specificity of the oligoprobes and the sensitivity of photosensors allowed an accurate quantification of the target analyte. In fact, target detectability was 0.07nmolL for the three different B19 DNA genotypes which is comparable to the LOD of 0.1nmolL obtained both for the Phos-tag step of B19 amplified products in conventional assay formats (e.g., PCR-ELISA methods) or exploiting other portable detection systems (e.g., CCD camera) . Moreover the photosensors showed low instrumental noise levels, good reproducibility and negligible crosstalk between adjacent sensors.
Financial support was provided by the Italian Ministry of Instruction, University and Research (MIUR): PRIN 2010-2011 project: prot. 20108ZSRTR “ARTEMIDE (Autonomous Real Time Embedded Multi-analyte Integrated Detection Environment): a fully integrated lab-on-chip for early diagnosis of viral infections”.
Biosensors for the direct detection of biomolecules at the single molecule level would be highly desirable for a range of diagnostic applications, especially if these bioanalytes could be delivered sequentially through a nanopore. So far, metallic nanostructures have been shown to provide an enhancement factor of 1014 with a large cross-sectional area of 10cm2/molecule using surface enhanced Raman scattering approaches . The rational design of metallic nanostructures for this application, also known as plasmonic substrates or devices, for sensing applications is well established ; the functional characteristics of these devices are based upon the behaviour of plasmons at the interface of a metal-dielectric medium. For metallic nanostructures, the electrons in the metal are excited and oscillate within the metal core near the interface with a surrounding dielectric material; these collective electron excitations are known as surface plasmon polaritons.
The potential of nanostructured metallic structures for optical applications has been demonstrated for (i) biosensing applications , (ii) surface enhanced Raman spectroscopy (SERS) , (iii) guiding and manipulating the light , (iv) sub-diffraction limited imaging  and (v) trapping of micro/nano-sized particles . In order to apply the nanostructures for these applications, the structure dimensions and geometry have to be appropriate for (i) tuning of the plasmon resonance coupling, (ii) the near field enhancement, (iii) the confinement in sub-wavelength region coupling, (iv) enhanced evanescent waves and (v) the near- and far-field enhancement,  respectively to each application. Moreover it is essential that there are high levels of local-electric field intensity at locations within the sensor where the plasmon enhancement of signal from the transducer will be most beneficial — this is especially true for sensing applications where the analyte must be within the region of highest electric field (E-field) intensity. Various metallic nanostructures have been proposed for sensing using a range of different shapes and geometries , materials [10,11] and fabrication methods , notably for the optimization of SERS measurements  tip-enhanced Raman scattering, (TERS)  and fluorescent enhancement . Most of the nanostructures studied so far are multi-scale nanoparticles and nanocrystals. The nanoparticles can be classified as 1-dimensional , 2-dimensional and 3-dimensional nanoparticles i.e., nanorods, nanocrescent and nanopores respectively. More recently planar plasmonic substrates have been developed for ‘nanofocusing of plasmons’ [17–22] and SERS [23,24].
Materials and methods