Photonic crystals utilized for label-free and amplified fluorescence biodetection

Abstract

ABSTRACT Photonic crystals are fabricated on pl astic surfaces, producing narrow bandwidth resonan ces at any desired wavelength. While shifts in the resonant wavelength quantify the density of adsorbed biomaterial, the resonances also enhance the output of adsorbed fluorophores. The combined attributes of photonic crystals enable highly sensitive label-free detection and greatly amplified sensitivity of any fluorescence- based assay for applications in life science research, drug discovery, and environmental pathogen detection. Applications for label-free selective detection of viral pathogens with sensitivity of ~30 focal forming units, and detection of spore pathogens with single-unit resolution are highlighted. Amplified fluorescence, meanwhile, enables gene expression de tection of DNA at concentrations ~2 orders of magnitude lower than detection on optically passive surfaces. Keywords : Label-free biosensors, optical biosensors, fluorescence enhancement, photonic crystal Introduction The ability to efficiently scr een the biochemical interaction of poten tial pharmaceutical drug compounds with a wide array of proteins and cells before the clinical trial stage is an increasingly important capability for avoiding costly failures when the drug is introduced to animals and humans. Likewise, testing of patients’ blood or tissue samples for expression of a gene profile will become common practice to aid in decisions regarding the most promising course of treatment. Efficient and sensitive assays are required throughout life science research and patient diagnosis, but are also increasingly important for diverse applications such as environmental monitoring for viral contaminants in crop irrigation water, early identification of spores that threaten food crops, and detection of biological warfare agents for homeland security. The key attributes for acceptance of ne w technology in these fields are sensitivity (how low of a concentration of a chemical, protein, or gene may be detected), cost per test, and throughput (the number of tests that can be performed at once). Biological analytes may be most simply detected directly through their dielectric permittivity by resonant optical transducer s in what is called “label-free” detectio n, or alternatively, greater sensitivity may be obtained by attachment of a fluorescent compound to the analyte of interest, followed by excitation of the fluorophore for detection. Of the label-free methods that may be used to directly detect pathogens, optical biosensors provide a useful combination of high sensitivity, low cost, high throughput and ease of use. In general, optical biosensors are designed to produce a measurable change in some characteristic of light th at is coupled to the sensor surface. Rather than detecting mass directly, all optical biosensors rely on the dielectric permittivity of detected substances to produce a measurable signal[1]. The advantage of this approach is that a direct physical connection between the excitation source, the detection instrument, and the transducer surface itself is not required, thus circumventing the need for electrical connections to the transd ucer that must be kept separate d from liquid sample media. Pu blications in recent years have described the application of optical biosensor methods like Surface Plasmon Re sonance (SPR) and acoustic label-free technologies such as Quartz Crystal Microbalance (QCM) for detection of intact viral particles. The functionality of label-free virus detection has been demonstrated with SPR and QCM for herpes, dengue and influenza [2-5]. Though both technologies offer the advantages of label-free biosensing and have high sensitivity—single virion detection limits in the case of QCM [4], extensive application of virus detection on these platforms is yet to be achieved in medical diagnostic testing or environmental detection. This is due to a combination of factors that include sensor cost, instrumentation complexity, low assay multiplexing throughput and lack of incorporation of multiple positive/negative controls to reduce the rate of false diagnostics. In clinical and laboratory settings, ELISA, PCR and culture methods are still widely used, with ELISA being a primary choice. Yet as ide from ELISA, the use of others methods is discouraged