Coupled external cavity photonic crystal enhanced fluorescence.

We report a fundamentally new approach to enhance fluorescence in which surface adsorbed fluorophore-tagged biomolecules are excited on a photonic crystal surface that functions as a narrow bandwidth and tunable mirror of an external cavity laser. This scheme leads to ∼10× increase in the electromagnetic enhancement factor compared to ordinary photonic crystal enhanced fluorescence. In our experiments, the cavity automatically tunes its lasing wavelength to the resonance wavelength of the photonic crystal, ensuring optimal on-resonance coupling even in the presence of variable device parameters and variations in the density of surface-adsorbed capture molecules. We achieve ∼10(5) × improvement in the limit of detection of a fluorophore-tagged protein compared to its detection on an unpatterned glass substrate. The enhanced fluorescence signal and easy optical alignment make cavity-coupled photonic crystals a viable approach for further reducing detection limits of optically-excited light emitters that are used in biological assays.

Sensitive detection of protein and miRNA cancer biomarkers using silicon-based photonic crystals and a resonance coupling laser scanning platform.

Enhancement of the fluorescent output of surface-based fluorescence assays by performing them upon nanostructured photonic crystal (PC) surfaces has been demonstrated to increase signal intensities by >8000×. Using the multiplicative effects of optical resonant coupling to the PC in increasing the electric field intensity experienced by fluorescent labels ("enhanced excitation") and the spatially biased funneling of fluorophore emissions through coupling to PC resonances ("enhanced extraction"), PC enhanced fluorescence (PCEF) can be adapted to reduce the limits of detection of disease biomarker assays, and to reduce the size and cost of high sensitivity detection instrumentation. In this work, we demonstrate the first silicon-based PCEF detection platform for multiplexed biomarker assay. The sensor in this platform is a silicon-based PC structure, comprised of a SiO2 grating that is overcoated with a thin film of high refractive index TiO2 and is produced in a semiconductor foundry for low cost, uniform, and reproducible manufacturing. The compact detection instrument that completes this platform was designed to efficiently couple fluorescence excitation from a semiconductor laser to the resonant optical modes of the PC, resulting in elevated electric field strength that is highly concentrated within the region <100 nm from the PC surface. This instrument utilizes a cylindrically focused line to scan a microarray in <1 min. To demonstrate the capabilities of this sensor-detector platform, microspot fluorescent sandwich immunoassays using secondary antibodies labeled with Cy5 for two cancer biomarkers (TNF-α and IL-3) were performed. Biomarkers were detected at concentrations as low as 0.1 pM. In a fluorescent microarray for detection of a breast cancer miRNA biomarker miR-21, the miRNA was detectable at a concentration of 0.6 pM.

Enhanced fluorescence emission using a photonic crystal coupled to an optical cavity.

All fluorescent assays would benefit from greater signal-to-noise ratios (SNRs), which enable detection of disease biomarkers at lower concentrations for earlier disease diagnosis and detection of genes that are expressed at the lowest levels. Here, we report an approach to enhance fluorescence in which surface adsorbed fluorophore-tagged biomolecules are excited on a photonic crystal surface that is coupled to an underlying Fabry-Perot type cavity through a gold mirror reflector beneath the photonic crystal. This approach leads to 6× increase in signal-to-noise ratio of a dye labeled polypeptide compared to ordinary photonic crystal enhanced fluorescence.

Nanostructured surfaces and detection instrumentation for photonic crystal enhanced fluorescence.

Photonic crystal (PC) surfaces have been demonstrated as a compelling platform for improving the sensitivity of surface-based fluorescent assays used in disease diagnostics and life science research. PCs can be engineered to support optical resonances at specific wavelengths at which strong electromagnetic fields are utilized to enhance the intensity of surface-bound fluorophore excitation. Meanwhile, the leaky resonant modes of PCs can be used to direct emitted photons within a narrow range of angles for more efficient collection by a fluorescence detection system. The multiplicative effects of enhanced excitation combined with enhanced photon extraction combine to provide improved signal-to-noise ratios for detection of fluorescent emitters, which in turn can be used to reduce the limits of detection of low concentration analytes, such as disease biomarker proteins. Fabrication of PCs using inexpensive manufacturing methods and materials that include replica molding on plastic, nano-imprint lithography on quartz substrates result in devices that are practical for single-use disposable applications. In this review, we will describe the motivation for implementing high-sensitivity fluorescence detection in the context of molecular diagnosis and gene expression analysis though the use of PC surfaces. Recent efforts to improve the design and fabrication of PCs and their associated detection instrumentation are summarized, including the use of PCs coupled with Fabry-Perot cavities and external cavity lasers.

External cavity laser biosensor.

Utilizing a tunable photonic crystal resonant reflector as a mirror of an external cavity laser cavity, we demonstrate a new type of label-free optical biosensor that achieves a high quality factor through the process of stimulated emission, while at the same time providing high sensitivity and large dynamic range. The photonic crystal is fabricated inexpensively from plastic materials, and its resonant wavelength is tuned by adsorption of biomolecules on its surface. Gain for the lasing process is provided by a semiconductor optical amplifier, resulting in a simple detection instrument that operates by normally incident noncontact illumination of the photonic crystal and direct back-reflection into the amplifier. We demonstrate single-mode, biomolecule-induced tuning of the continuous-wave laser wavelength. Because the approach incorporates external optical gain that is separate from the transducer, the device represents a significant advance over previous passive optical resonator biosensors and laser-based biosensors.

Multiplexed cancer biomarker detection using quartz-based photonic crystal surfaces.

A photonic crystal (PC) surface is demonstrated as a high-sensitivity platform for detection of a panel of 21 cancer biomarker antigens using a sandwich enzyme-linked immunosorbent assay (ELISA) microarray format. A quartz-based PC structure fabricated by nanoimprint lithography, selected for its low autofluorescence, supports two independent optical resonances that simultaneously enable enhancement of fluorescence detection of biomarkers and label-free quantification of the density of antibody capture spots. A detection instrument is demonstrated that supports fluorescence and label-free imaging modalities, with the ability to optimize the fluorescence enhancement factor on a pixel-by-pixel basis throughout the microarray using an angle-scanning approach for the excitation laser that automatically compensates for variability in surface chemistry density and capture spot density. Measurements show that the angle-scanning illumination approach reduces the coefficient of variation of replicate assays by 20-99% compared to ordinary fluorescence microscopy, thus supporting reduction in limits of detectable biomarker concentration. Using the PC resonance, biomarkers in mixed samples were detectable at the lowest concentrations tested (2.1-41 pg/mL), resulting in a three-log range of quantitative detection.

Spatially selective photonic crystal enhanced fluorescence and application to background reduction for biomolecule detection assays.

By combining photonic crystal label-free biosensor imaging with photonic crystal enhanced fluorescence, it is possible to selectively enhance the fluorescence emission from regions of the PC surface based upon the density of immobilized capture molecules. A label-free image of the capture molecules enables determination of optimal coupling conditions of the laser used for fluorescence imaging of the photonic crystal surface on a pixel-by-pixel basis, allowing maximization of fluorescence enhancement factor from regions incorporating a biomolecule capture spot and minimization of background autofluorescence from areas between capture spots. This capability significantly improves the contrast of enhanced fluorescent images, and when applied to an antibody protein microarray, provides a substantial advantage over conventional fluorescence microscopy. Using the new approach, we demonstrate detection limits as low as 0.97 pg/ml for a representative protein biomarker in buffer.

Using photonic crystal enhanced fluorescence on quartz substrates to improve the sensitivity of DNA microarrays.

Gene expression analysis of low abundance genes remains difficult when DNA microarrays are performed on standard glass substrates. However, we have shown that by using photonic crystals (PC) made on quartz substrates, the fluorescence intensity of Cyanine-5 (Cy5) labeled microarray spots is greatly enhanced. In a 1-color microarray experiment studying gene expression of soybean cotyledon tissue, an average signal enhancement factor of 17.8× was observed on the PC. Furthermore, twice as many genes were detectable on these PCs as compared to glass. By improving the sensitivity of this fluorescent assay, low expression genes that were undetectable on glass were quantified on the PC.

Photonic crystal enhanced fluorescence using a quartz substrate to reduce limits of detection.

A Photonic Crystal (PC) surface fabricated upon a quartz substrate using nanoimprint lithography has been demonstrated to enhance light emission from fluorescent molecules in close proximity to the PC surface. Quartz was selected for its low autofluorescence characteristics compared to polymer-based PCs, improving the detection sensitivity and signal-to-noise ratio (SNR) of PC Enhanced Fluorescence (PCEF). Nanoimprint lithography enables economical fabrication of the subwavelength PCEF surface structure over entire 1x3 in2 quartz slides. The demonstrated PCEF surface supports a transverse magnetic (TM) resonant mode at a wavelength of λ = 632.8 nm and an incident angle of θ = 11°, which amplifies the electric field magnitude experienced by surface-bound fluorophores. Meanwhile, another TM mode at a wavelength of λ = 690 nm and incident angle of θ = 0° efficiently directs the fluorescent emission toward the detection optics. An enhancement factor as high as 7500 × was achieved for the detection of LD-700 dye spin-coated upon the PC, compared to detecting the same material on an unpatterned glass surface. The detection of spotted Alexa-647 labeled polypeptide on the PC exhibits a 330 × SNR improvement. Using dose-response characterization of deposited fluorophore-tagged protein spots, the PCEF surface demonstrated a 140 × lower limit of detection compared to a conventional glass substrate.