A signal amplification assay for HSV type 1 viral DNA detection using nanoparticles and direct acoustic profiling

• Main Authors: Hammond Richard, Uludağ Yildiz, Cooper Matthew A
• Format: Article
• Language: English
• Published: BMC 2010-02-01
• Series: Journal of Nanobiotechnology
• Online Access: http://www.jnanobiotechnology.com/content/8/1/3

<p>Abstract</p> <p>Background</p> <p>Nucleic acid based recognition of viral sequences can be used together with label-free biosensors to provide rapid, accurate confirmation of viral infection. To enhance detection sensitivity, gold nanoparticles can be employed with mass-sensitive acoustic biosensors (such as a quartz crystal microbalance) by either hybridising nanoparticle-oligonucleotide conjugates to complimentary surface-immobilised ssDNA probes on the sensor, or by using biotin-tagged target oligonucleotides bound to avidin-modified nanoparticles on the sensor. We have evaluated and refined these signal amplification assays for the detection from specific DNA sequences of Herpes Simplex Virus (HSV) type 1 and defined detection limits with a 16.5 MHz fundamental frequency thickness shear mode acoustic biosensor.</p> <p>Results</p> <p>In the study the performance of semi-homogeneous and homogeneous assay formats (suited to rapid, single step tests) were evaluated utilising different diameter gold nanoparticles at varying DNA concentrations. Mathematical models were built to understand the effects of mass transport in the flow cell, the binding kinetics of targets to nanoparticles in solution, the packing geometries of targets on the nanoparticle, the packing of nanoparticles on the sensor surface and the effect of surface shear stiffness on the response of the acoustic sensor. This lead to the selection of optimised 15 nm nanoparticles that could be used with a 6 minute total assay time to achieve a limit of detection sensitivity of 5.2 × 10^-12M. Larger diameter nanoparticles gave poorer limits of detection than smaller particles. The limit of detection was three orders of magnitude lower than that observed using a hybridisation assay without nanoparticle signal amplification.</p> <p>Conclusions</p> <p>An analytical model was developed to determine optimal nanoparticle diameter, concentration and probe density, which allowed efficient and rapid optimisation of assay parameters. Numerical analysis and subsequent associated experimental data suggests that the response of the mass sensitive biosensor system used in conjunction with captured particles was affected by i) the coupled mass of the particle, ii) the proximal contact area between the particle and the sensor surface and iii) the available capture area on the particle and binding dynamics to this capture area. The latter two effects had more impact on the detection limit of the system than any potential enhancement due to added mass from a larger nanoparticle.</p>