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.

Photonic crystal enhanced microscopy for imaging of live cell adhesion.

A form of microscopy that utilizes a photonic crystal biosensor surface as a substrate for cell attachment enables label-free, quantitative, submicron resolution, time-resolved imaging of cell-surface interactions without cytotoxic staining agents or temporally-unstable fluorophores. Other forms of microscopy do not provide this direct measurement of live cell-surface attachment localization and strength that includes unique, dynamic morphological signatures critical to the investigation of important biological phenomena such as stem cell differentiation, chemotaxis, apoptosis, and metastasis. Here, we introduce Photonic Crystal Enhanced Microscopy (PCEM), and apply it to the study of murine dental stem cells to image the evolution of cell attachment and morphology during chemotaxis and drug-induced apoptosis. PCEM provides rich, dynamic information about the evolution of cell-surface attachment profiles over biologically relevant time-scales. Critically, this method retains the ability to monitor cell behavior with spatial resolution sufficient for observing both attachment footprints of filopodial extensions and intracellular attachment strength gradients.

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.

Line-scanning detection instrument for photonic crystal enhanced fluorescence.

A laser line-scanning instrument was developed to optimize the near-field enhancement capability of a one-dimensional photonic crystal (PC) for excitation of surface-bound fluorophores. The excitation laser beam is shaped into an 8 µm × 1 mm line that is focused along the direction of the PC grating, while remaining collimated perpendicular to the grating. Such a beam configuration offers high excitation power density while simultaneously providing high resonant coupling efficiency from the laser to the PC surface. Using a panel of 21 immunofluorescence assays on the PC surface in a microarray format, the approach achieves an enhancement factor as high as 90-fold between on-resonance and off-resonance illumination. The instrument provides a capability for sensitive and inexpensive analysis of cancer biomarkers in clinical applications.

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.

Label-free imaging of cell attachment with photonic crystal enhanced microscopy.

We introduce photonic crystal enhanced microscopy (PCEM) as a label-free biosensor imaging technique capable of measuring cell surface attachment and attachment modulation. The approach uses a photonic crystal optical resonator surface incorporated into conventional microplate wells and a microscope-based detection instrument that measures shifts in the resonant coupling conditions caused by localized changes in dielectric permittivity at the cell-sensor interface. Four model systems are demonstrated for studying cancer cells, primary cardiac muscle cells, and stem cells. First, HepG2/C3 hepatic carcinoma cells were cultured and observed via PCEM in order to characterize cell adhesion in the context of growth and locomotion. Second, Panc-1 pancreatic cancer cells were used to verify that cell attachment density decreases in response to staurosporine, a drug that induces apoptosis. Third, we used PCEM to confirm the influence of integrin-mediated signaling on primary neonatal cardiomyocyte growth and development. Rounded cardiomyocytes consistently showed decreased cell attachment density as recorded via PCEM, while spreading cells exhibited greater attachment strength as well as increased contractility. Finally, PCEM was used to monitor the morphological changes and extracellular matrix remodeling of porcine adipose-derived stem cells subjected to a forced differentiation protocol. Each of these experiments yielded information regarding cell attachment density without the use of potentially cytotoxic labels, enabling study of the same cells for up to several days.

Application of photonic crystal enhanced fluorescence to cancer biomarker microarrays.

We report on the use of photonic crystal surfaces as a high-sensitivity platform for detection of a panel of cancer biomarkers in a protein microarray format. The photonic crystal surface is designed to provide an optical resonance at the excitation wavelength of cyanine-5 (Cy5), thus providing an increase in fluorescent intensity for Cy5-labeled analytes measured with a confocal microarray scanner, compared to a glass surface. The sandwich enzyme-linked immunosorbent assay (ELISA) is undertaken on a microarray platform to undertake a simultaneous, multiplex analysis of 24 antigens on a single chip. Our results show that the resonant excitation effect increases the signal-to-noise ratio by 3.8- to 6.6-fold, resulting in a decrease in detection limits of 6-89%, with the exact enhancement dependent upon the antibody-antigen interaction. Dose-response characterization of the photonic crystal antibody microarrays shows the capability to detect common cancer biomarkers in the <2 pg/mL concentration range within a mixed sample.

Photobleaching on photonic crystal enhanced fluorescence surfaces.

The effect of resonant fluorescent enhancement from a photonic crystal surface upon the fluorescent photobleaching rate of Cyanine-5 labeled protein has been investigated. We show that the enhanced excitation mechanism for photonic crystal enhanced fluorescence, in which the device surface resonantly couples light from an excitation laser, accelerates photobleaching in proportion to the coupling efficiency of the laser to the photonic crystal. We also show that the enhanced extraction mechanism, in which the photonic crystal directs emitted photons approximately normal to the surface, does not play a role in the rate of photobleaching. We show that the photobleaching rate of dye molecules on the photonic crystal surface is accelerated by 30x compared to an ordinary glass surface, but substantial signal gain is still evident, even after extended periods of continuous illumination at the resonant condition.

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.

A detection instrument for enhanced-fluorescence and label-free imaging on photonic crystal surfaces.

We report on the design and demonstration of an optical imaging system capable of exciting surface-bound fluorophores within the resonant evanescent electric field of a photonic crystal surface and gathering fluorescence emission that is directed toward the imaging objective by the photonic crystal. The system also has the ability to quantify shifts in the local resonance angle induced by the adsorption of biomolecules on the photonic crystal surface for label-free biomolecular imaging. With these two capabilities combined within a single detection system, we demonstrate label-free images self-registered to enhanced fluorescence images with 328x more sensitive fluorescence detection relative to a glass surface. This technique is applied to a DNA microarray where label-free quantification of immobilized capture DNA enables improved quality control and subsequent enhanced fluorescence detection of dye-tagged hybridized DNA yields 3x more genes to be detected versus commercially available microarray substrates.