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.