Viruses represent a continual threat to humans through a number of mechanisms, which include disease, bioterrorism, and destruction of both plant and animal food resources. Many contemporary techniques used for the detection of viruses and viral infections suffer from limitations such as the need for extensive sample preparation or the lengthy window between infection and measurable immune response, for serological methods. In order to develop a method that is fast, cost-effective, and features reduced sample preparation compared to many other virus detection methods, we report the application of silicon photonic microring resonators for the direct, label-free detection of intact viruses in both purified samples as well as in a complex, real-world analytical matrix. As a model system, we demonstrate the quantitative detection of Bean pod mottle virus, a pathogen of great agricultural importance, with a limit of detection of 10 ng/mL. By simply grinding a small amount of leaf sample in buffer with a mortar and pestle, infected leaves can be identified over a healthy control with a total analysis time of less than 45 min. Given the inherent scalability and multiplexing capability of the semiconductor-based technology, we feel that silicon photonic microring resonators are well-positioned as a promising analytical tool for a number of viral detection applications.
Materials and methods
Unless otherwise specified, reagents were obtained from Sigma–Aldrich (St. Louis, MO) and used as received. Monoclonal antibodies to BPMV (CAB 46400), SMV (CAB 33300), Alfalfa mosaic virus (AMV; CAB 87601), Tobacco ringspot virus (TRSV; CAB 64000), and a BPMV ELISA kit were purchased from Agdia (Elkhart, IN). The 3-N-((6-(N′-isopropylidenehydrazino))nicotinamide)propyltriethoxysilane (HyNic silane) and succinimidyl 4-formyl benzoate (S-4FB) were purchased from Solulink (San Diego, CA). Dulbecco’s phosphate buffered saline (PBS), was reconstituted in deionized water and the pH adjusted to either 7.4 or 6.0 with 1 M HCl or 1 M NaOH. BSA–PBS buffer consisted of 0.1 mg/mL bovine serum albumin (BSA) in pH 7.4 PBS. The surface blocking buffer consisted of 2% BSA in pH 7.4 PBS. Zeba spin filter columns were purchased from Pierce (Rockford, IL).
Purified BPMV was isolated from infected leaf samples soybean cultivar Williams 82 infected with BPMV isolate WP2 as described Ghabrial et al. (1977). Leaves were collected from age-matched healthy and BPMV-infected Williams 82 soybean at 2 weeks after inoculation.
Instrumentation and microring sensor array substrates
The instrumentation utilized to measure shifts in microring resonance wavelengths and sensor substrates were designed in collaboration with and acquired from Genalyte, Inc. (San Diego, CA), and were described previously (Iqbal et al., 2010; Washburn et al., 2009). Briefly, sensor chips, each having an array of 32 individually addressable microring resonators accessed by a linear waveguides with terminal diffractive grating couplers, were fabricated on silicon-on-insulator wafers. The entire surface of the substrate was uniformly coated with a perfluoropolymer and annular openings were created over 24 of the microrings via reactive ion etching, allowing solution to come into contact with the those sensor elements. The remaining 8 microrings remain occluded and are used as thermal controls, as they are not affected by chemical or biomolecular binding events.
Sensor substrates were loaded into a previously described microfluidic cartridge and light from a tunable external cavity laser (center wavelength 1560nm) coupled into the input grating coupler accessing a single microring. The laser wavelength was then swept through a 12 nm spectral window and resonances determined as negative attenuations in light intensity outcoupled through the output grating coupler. This process was repeated for the entire array of 32 resonators, enabling near real-time measurement of shifts in resonance wavelength. Solutions were flowed across the sensor array as directed by the microfluidic gasket under the control of a syringe pump.