Infectious diseases, mostly caused by bacteria and viruses but also a result of fungal and parasitic infection, have been one of the most important public health concerns throughout human history. The first step in combating these pathogens is to get a timely and accurate diagnosis at an affordable cost. Many kinds of diagnostics have been developed, such as pathogen culture, biochemical tests and serological tests, to help detect and fight against the causative agents of diseases. However, these diagnostic tests are generally unsatisfactory because they are not particularly sensitive and specific and are unable to deliver speedy results. Nucleic acid-based diagnostics, detecting pathogens through the identification of their genomic sequences, have shown promise to overcome the above limitations and become more widely adopted in clinical tests. Here we review some of the most popular nucleic acid-based diagnostics and focus on their adaptability and applicability to routine clinical usage. We also compare and contrast the characteristics of different types of nucleic acid-based diagnostics.

ToF-SIMS experiments were done at Surface Interface Ontario, University of Toronto. The instrument used was an ION-TOF TOFSIMS IV (Muenster, Germany), which was operated in static mode. The primary ion source was a Bin+ cluster ion primary source with a potential of 25 keV and a maximum current of 1 pA. Ion currents of 1 pA was used for Bi+ , about 0.4 pA was used for Bi3+, and about 0.05 pA was used for Bi5+. The experiments that used the same ion source were at the same current. A spot area of 500 μm × 500 μm was sampled and the spectra were obtained in high-mass resolution mode.[4] Both negative and positive ion modes were investigated and a mass range from 0 to 500 atomic mass units (amu) was scanned for collection of SIMS spectra. The raw ToF-SIMS data were processed by IonSpec Application software, version 4.5.0.0 by ION-TOF GmbH (1996–2002).

Covalently immobilized mixed films of oligonucleotide and oligomer components on glass and silicon surfaces are reported. This work has investigated how such films can improve selectivity for the detection of multiple base-pair mismatches. The intention was to introduce a "matrix isolation" effect on oligonucleotide probe molecules by surrounding the probes with oligomers, thereby reducing oligonucleotide-to-oligonucleotide and/or oligonucleotide-to-surface interactions. Thiol-functionalized oligonucleotide was coupled onto (3-aminopropyl)trimethoxysilane (APTMS) via a heterobifunctional linker, succinimidyl 4-[N-maleimidomethyl]cyclohexane-1-carboxylate (sulfo-SMCC). Using a variety of monomers such as 2-hydroxyethyl methacrylate (HEMA), oligomers were grown by surface-initiated atom transfer radical polymerization (ATRP) from a bromoisobutyryl NHS ester initiator which was immobilized onto APTMS sites that coated glass and oxidized silicon substrates. Various surface modification steps on silicon substrates were characterized by ellipsometry, wettability, atomic force microscopy, X-ray photoelectron spectroscopy, and time-of-flight secondary ion mass spectrometry. Polymerized HEMA (PHEMA) in mixture with oligonucleotide probes was evaluated for fluorescence transduction of hybridization. The presence of PHEMA was found to provide a sharper melt curve for hybrids containing both fully complementary and three base-pair mismatched targets, and this surface derivatization also minimized non-selective adsorption. The maximum increase in slope was improvement by a factor of 3-fold. An increase of up to 30% in difference of melting temperatures between fully complementary and 3 base-pair mismatched targets was achieved using PHEMA. The results suggest that the presence of oligomers dispersed among DNA hybrids can improve selectivity through what is believed to be a reduction of dispersity of interactions of probes with targets, and probes within their local environment at a surface.

Preliminary studies of mixed films composed of oligonucleotides and poly(2-hydroxyethyl methacrylate) (PHEMA) have recently been shown to enhance the selectivity for detection of 3 base-pair mismatched (3bpm) oligonucleotide targets. Evaluation of selectivity for detection of single nucleotide polymorphisms (SNP) using such mixed films has now been completed. The selectivity was quantitatively determined by considering the sharpness of melt curves and melting temperature differences (ΔTm) for fully complementary targets and SNPs. Stringency conditions were investigated, and it was determined that the selectivity was maximized when a moderate ionic strength was used (0.1–0.6M). Increases of ΔTm when using mixed films were up to 3-fold larger compared to surfaces containing only immobilized oligonucleotide probes. Concurrently, increases in sharpness of melt curves for 1bpm targets were observed to be up to 2-fold greater for mixed films. The co-immobilization of PHEMA resulted in a more homogeneous distribution of oligonucleotide probes on surfaces. Lifetime measurements of fluorescence emission from immobilized oligonucleotide probes labeled with Cy3 dye indicated the difference in microenvironment of immobilized oligonucleotides in the presence of PHEMA.

Silane coupling agents are commonly used to activate surfaces for subsequent immobilization of biomolecules. The homogeneity and surface morphology of silane films is important for controlling the structural order of immobilized single-stranded DNA probes based on oligonucleotides. The surfaces of silicon wafers and glass slides with covalently attached 3-glycidoxypropyltrimethoxysilane (GOPS) have been characterized by using angularly dependent X-ray photoelectron spectroscopy (XPS), time-of-flight secondary-ion mass spectrometry (ToF–SIMS), atomic force microscopy (AFM), scanning electron microscopy (SEM), and monochromatic and spectroscopic ellipsometry. XPS and ToF–SIMS data provided evidence of complete surface coverage by GOPS. Data from angularly resolved XPS and ellipsometry methods suggested that the GOPS films were of monolayer thickness. AFM and SEM data indicated the presence of films that consisted of nodules approximately 50–100 nm in diameter. Modeling suggested that the nodules may lead to a nanoscale structural morphology that might influence the hybridization kinetics and thermodynamics of immobilized oligonucleotides.

Biosensors and biochips can determine the presence of nucleic acid sequences in a test sample through fluorescence detection of hybridization between an immobilized nucleic acid (probe) and a nucleic acid in a test sample (target). The finding that the control of the environment of immobilized single-stranded probe molecules on fused silica surfaces can be used to tune selectivity to facilitate detection of even single base pair mismatches provides opportunities for design of novel biochips. We have typically used silane coupling agents to activate silicon and silicate surfaces for subsequent immobilization of biomolecules for development of optical biosensors. A self-assembled immobilization providing good structural order would be preferred. Studies have been done using thiol-terminated reagents for assembly of oligonucleotides on GaAs substrates. The spacing of Ga and As can be controlled across a surface, and in turn provides a template to control the density of self-assembled oliogonucleotide. Initial experimental work has begun using homogeneous GaAs surfaces, and the homogeneity and surface morphology of immobilized oligonucleotide films grown onto GaAs has been characterized by atomic force microscopy (AFM) and fluorescence methods. Cycles of hybridization and denaturation suggest that the GaAs provides a surface that is stable to loss of immobilized oligonucleotide, but that efforts to protect from non-selective adsorption are essential. Data suggested that the films were of monolayer thickness, and that it was possible to induce the presence of nodules of approximately 10-50 nm in diameter.