Multiplexed detection and differentiation of the DNA strains for influenza A (H1N1 2009) using a silicon-based microfluidic system.

Pandemic influenza by the swine-origin influenza virus (H1N1 2009) has attracted considerable concern worldwide. A convenient and accurate diagnostic approach that can be deployed at the point of care, such as in a doctor's office or at an airport, is critical for disease control. Here we report the development of a silicon-based microfluidic system for subtype differentiation of the novel H1N1 2009 strain vs. the seasonal influenza A (FluA) strain. The proposed system included two functional modules: a multiplexed PCR module for amplification of nucleic acid targets and a multiplexed silicon nanowire (SiNW) module for sequence determination. The PCR module consisted of a microfluidic PCR chamber and an electrical controller to perform a multiplexed protocol that simultaneously enriched specific segments of both H1N1 and FluA strains (if present), with 10(4)-10(5) amplification efficiency. The PCR amplicon was subsequently denatured and transferred to the SiNW sensing module for a label-free, multiplexed detection. A control SiNW was implemented, for the first time, in order to eliminate background interference. The detection module demonstrated a 10× change in the magnitude of differential current when the target DNA was injected. Overall, the system achieved a sensitivity of 20-30 fg/µl for H1N1 and seasonal FluA nucleic acids in a 10 µl sample. The low sample consumption, high sensitivity and high specificity render it a potential point-of-care (POC) platform to help doctors reach a yes/no decision for infectious diseases.

Highly sensitive and reversible silicon nanowire biosensor to study nuclear hormone receptor protein and response element DNA interactions.

To thoroughly understand the role that estrogen receptors partake in regulation of gene expression, characterization of estrogen receptors (ERs) and estrogen-response elements (EREs) interactions is essential. In the work, we present a highly sensitive and reusable silicon nanowire (SiNW) biosensor to study the interactions between human ER proteins (ER, α and ß subtypes) and EREs (dsDNA). The proteins were covalently immobilized on the SiNW surface. Various EREs including wild-type, mutant and scrambled DNA sequences were then applied to the protein-functionalized SiNW surface. Due to negatively charged dsDNA, binding of the EREs to the ERs on the n-type SiNW biosensor leads to the accumulation of negative charges on the surface, thereby inducing increase in resistance. The results show that the specificity of the ERE-ERα binding is higher than that of the ERE-ERß binding, what is more, the mutant ERE reduces the binding affinity for both ERα and ERß. By applying various concentrations of wild-type ERE to the bound ERα, a very low concentration of 10 fM wild-type ERE was found to be able to bind to the ERα. The reversible association and dissociation between ERα and wt-ERE was achieved, pointing to a reusable biosensor for protein-DNA binding. Through the study, we have established the SiNW biosensor as a promising method in providing comprehensive study for hormone receptor-response element interactions.

Morpholino-functionalized silicon nanowire biosensor for sequence-specific label-free detection of DNA.

We investigated Morpholino-functionalized silicon nanowires (SiNWs) as a novel gene chip platform for the sequence-specific label-free detection of DNA. Morpholino attachment and subsequent Morpholino-DNA hybridization on silicon surface was characterized by X-ray photoelectron spectroscopy and fluorescence microscopy. The resultant Morpholino-modified surfaces showed high specificity of recognition for DNA. Subsequently, by using the same protocol, the surface of the SiNW biosensor was functionalized with Morpholino, and this was used for label-free Morpholino-DNA hybridization detection. Real-time measurements of the Morpholino-functionalized SiNW biosensor exhibited a decrease in a time-dependent conductance when complementary and mutant DNA samples were added. Furthermore, identification of fully complementary versus mismatched DNA samples was carried out by the Morpholino-functionalized SiNW biosensor. We demonstrated that DNA detection using the Morpholino-functionalized SiNW biosensor could be carried out to the hundreds of femtomolar range. The Morpholino-functionalized SiNWs show a novel biosensor for label-free and direct detection of DNA with good selectivity, and a promising application in gene expression.