ABSTRACT
Rapid detection of unlabeled SARS-CoV-2 genetic target was demonstrated using a competitive displacement hybridization assay made by a nanostructured anodized alumina oxide (AAO) membrane. The assay applied the toehold-mediated strand displacement reaction. The nanoporous surface of the membrane was functionalized with a complementary pair consisting of Cy3-labeled probe and quencher-labeled nucleic acids through a chemical immobilization process. In the presence of the unlabeled SARS-CoV-2 target, the quencher-tagged strand of the immobilized probe-quencher duplex was separated from the Cy3-modifed strand. A stable probe-target duplex formed and regained a strong fluorescence signal, thus enabling real-time and label-free SARS-CoV-2 detection. Assay designs with different numbers of base pair (bp) matches were synthesized to compare their affinities. Because of the large surface of a free-standing nanoporous membrane, two orders enhancement of the fluorescence was observed, where the detection limit of the unlabeled concentration can be improved to 1 nM. The assay was miniaturized by integrating a nanoporous AAO layer onto an optical waveguide device. The detection mechanism and the sensitivity improvement of the AAO-waveguide device were illustrated from the finite difference method (FDM) simulation and the experimental results. Light-analyte interaction was further improved due to the presence of the AAO layer, which created an intermediate refractive index and enhanced the waveguide's evanescent field. Our competitive hybridization sensor is an accurate and label-free testing platform applicable to the deployment of compact and sensitive virus detection strategies.
ABSTRACT
Chip-scale SARS-CoV-2 testing was demonstrated using silicon nitride (Si3N4) nanoslot fluidic waveguides to detect a tagged oligonucleotide with a coronavirus DNA sequence. The slot waveguides were fabricated using complementary metal-oxide-semiconductor (CMOS) fabrication processes, including multiscale lithography and selective reactive ion etching (RIE), forming femtoliter fluidic channels. Finite difference method (FDM) simulation was used to calculate the optical field distribution of the waveguide mode when the waveguide sensor was excited by transverse electric (TE) and transverse magnetic (TM) polarized light. For the TE polarization, a strong optical field was created in the slot region and its field intensity was 14× stronger than the evanescent sensing field from the TM polarization. The nanoscale confinement of the optical sensing field significantly enhanced the light-analyte interaction and improved the optical sensitivity. The sensitivity enhancement was experimentally demonstrated by measuring the polarization-dependent fluorescence emission from the tagged oligonucleotide. The photonic chips consisting of femtoliter Si3N4 waveguides provide a low-cost and high throughput platform for real-time virus identification, which is critical for point-of-care (PoC) diagnostic applications.
Subject(s)
COVID-19 Testing/methods , COVID-19/diagnosis , COVID-19/virology , DNA, Viral/analysis , Nanoparticles/chemistry , SARS-CoV-2/genetics , SARS-CoV-2/isolation & purification , Silicon Compounds/chemistry , Humans , Optics and Photonics , Point-of-Care Systems , Refractometry , Semiconductors , Sensitivity and SpecificityABSTRACT
An ultra-thin and highly sensitive SARS-CoV-2 detection platform was demonstrated using a nano-porous anodic aluminum oxide (AAO) membrane. The membrane surface was functionalized to enable efficient trapping and identification of SARS-CoV-2 genomic targets through DNA-DNA and DNA-RNA hybridization. To immobilize the probe oligonucleotides on the AAO membrane, the pore surface was first coated with the linking reagents, 3-aminopropyltrimethoxysilane (APTMS) and glutaraldehyde (GA), by a compact vacuum infiltration module. After that, complementary target oligos with fluorescent modifier was pulled and infiltrated into the nano-fluidic channels formed by the AAO pores. The fluorescent signal applying the AAO membrane sensors was two orders stronger than a flat glass template. In addition, the dependence between the nano-pore size and the fluorescent intensity was evaluated. The optimized pore diameter d is 200 nm, which can accommodate the assembled oligonucleotide and aminosilane layers without blocking the AAO nano-fluidic channels. Our DNA functionalized membrane sensor is an accurate and high throughput platform supporting rapid virus tests, which is critical for population-wide diagnostic applications result in a page being rejected by search engines.
ABSTRACT
Raman spectroscopy using aluminum nitride (AlN) optical waveguides was demonstrated for organic compound analysis. The AlN waveguide device was prepared by reactive sputtering deposition and complementary-metal-oxide semiconductor (CMOS) processes. A fundamental waveguide mode was observed over a broad visible spectrum and the waveguide evanescent wave was used to excite the Raman signals of the test analytes. The performance of the waveguide sensor was characterized by measuring the Raman spectra of the benzene derivative mixtures consisting of benzene, anisole, and toluene. The compositions and concentrations were resolved by correlating the obtained Raman spectrum with the characteristic Raman peaks associated with C-C, C-H, and C-O functional groups. With the advantages of real-time detection and enhanced Raman signal intensity, the AlN waveguides provided a sensor platform for nondestructive and online chemical compound monitoring.
