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.     

Enhanced fluorescence emission from quantum dots on a photonic crystal surface

Colloidal quantum dots display a wide range of novel optical properties that could prove useful for many applications in photonics. Here, we report the enhancement of fluorescence emission from colloidal quantum dots on the surface of two-dimensional photonic crystal slabs. The enhancement is due to a combination of high-intensity near fields and strong coherent scattering effects, which we attribute to leaky eigenmodes of the photonic crystal. By fabricating two-dimensional photonic crystal slabs that operate at visible wavelengths and engineering their leaky modes so that they overlap with the absorption and emission wavelengths of the quantum dots, we demonstrate that the fluorescence intensity can be enhanced by a factor of up to 108 compared with quantum dots on an unpatterned surface.     

Two-dimensional photonic crystal surfactant detection

We developed a novel two-dimensional (2-D) crystalline colloidal array photonic crystal sensing material for the visual detection of amphiphilic molecules in water. A close-packed polystyrene 2-D array monolayer was embedded in a poly(N-isopropylacrylamide) (PNIPAAm)-based hydrogel film. These 2-D photonic crystals placed on a mirror show intense diffraction that enables them to be used for visual determination of analytes. Binding of surfactant molecules attaches ions to the sensor that swells the PNIPAAm-based hydrogel. The resulting increase in particle spacing red shifts the 2-D diffracted light. Incorporation of more hydrophobic monomers increases the sensitivity to surfactants.     

Probing biomolecular interactions with dual polarization interferometry: real-time and label-free coralyne detection by use of homoadenine DNA oligonucleotide

We incorporated the specific recognition of adenine-rich singled-stranded DNA (ssDNA) into dual polarization interferometry (DPI) measurements for direct, selective, and sensitive detection of the small molecule coralyne, and we simultaneously employed the real-time and label-free technique for detailed investigation of the interaction between coralyne and adenine-rich ssDNA. Data from UV-visible spectroscopy, circular dichroism (CD) spectroscopy, and DNA melting firmly confirmed that 48-mer homoadenine ssDNA oligonucleotide (A(48)) had highly specific recognition for coralyne, whereas 48-mer homothymine ssDNA oligonucleotide (T(48)) as the control had no such recognition. The immobilization of ssDNA (A(48) or T(48)) on a silicon oxynitride chip could be achieved through a preadsorbed poly(ethylenimine) (PEI) layer. Mass, thickness, and refractive index (RI) changes resolved by DPI during the whole process of ssDNA immobilization suggested that most ssDNA molecules were likely to lie on the PEI surface mainly in the form of a flat monolayer and insert themselves partly into the PEI layer. Qualitative and quantitative analysis of mass, thickness, and RI changes in A(48)/PEI layer upon addition of different concentrations of coralyne revealed that A(48) most likely underwent a conformational change from single-stranded to double-stranded structure. By evaluation of the binding curves from changes in mass, the association rate constant (k(a)), dissociation rate constant (k(d)), and association constant (K(A)) between coralyne and A(48) were determined to be 4.95 × 10(3) M(-1) s(-1), 0.031 s(-1), and 1.6 × 10(5) M(-1), respectively. Good linear correlations between coralyne concentrations ranging from 0.5 to 12 μM and three parameters (mass, thickness, and RI) resolved by the response to coralyne binding were obtained. The detection limits were 0.22 μM for mass calibration, 0.14 μM for thickness calibration, and 0.32 μM for RI calibration. The high selectivity of the biosensor to coralyne at the A(48)/PEI interface was successfully confirmed by using the other two interfaces (T(48)/PEI and PEI) and three typical intercalators (ethidium bromide, daunomycin, and methylene blue). It is expected that the biosensing platform may be extended to simultaneously detect and characterize the interactions of a variety of target molecules with functional DNA molecules with high sensitivity.     

Gap modes of one-dimensional photonic crystal surface waves

The finite-difference time-domain method is employed for the analysis of coupling of the surface modes of two truncated one-dimensional photonic crystals separated by a gap. The wave vector, field distributions, and existence conditions of the coupled surface modes are investigated. The wave vector of symmetric gap modes increases with decreasing gap width, while that of antisymmetric modes decreases-exactly opposite of the situation for surface plasmons on metallic half-spaces separated by a dielectric gap. Photonic crystal gap modes could easily and effectively be used as nondissipating gap-mode waveguides.     

Miniature tapered photonic crystal fiber interferometer with enhanced sensitivity by acid microdroplets etching

We fabricate a miniature tapered photonic crystal fiber (PCF) interferometer with enhanced sensitivity by acid microdroplets etching. This method is very simple and cost effective, avoiding elongating the PCF, moving and refixing the device during etching, and measuring. The refractive index sensing properties with different PCF diameters are investigated both theoretically and experimentally. The tapering velocity can be controlled by the microdroplet size and position. The sensitivity greatly increases (five times, 750?nm/RIU) and the size decreases after slightly tapering the PCF. The device keeps low temperature dependence before and after tapering. More uniformly and thinly tapered PCFs can be realized with higher sensitivity (～100 times) by optimizing the etching process.     

Vertical surface emitting open coupled-cavities based on photonic crystal surface modes

Two types of vertical surface emitting photonic crystal cavities based on beaming mechanism and coupled surface modes are studied. It is shown that vertical emission with a zero divergence angle and a high quality factor can be easily achieved by the back-to-back cavity design. The periodic modulation to the cavity surface alters nonradiative surface modes into radiative surface modes, and the constructive interference of the radiative waves gives rise to vertical emission and improves the quality of the output beam. A high quality factor can be attributed to the nonradiative surface mode on the cavity back whose small part of energy can be transferred into the cavity surface by coupling. The resonant property and the coupling efficiency of the cavities are investigated and optimal cavity configurations are obtained. These open coupled-cavities are good candidates of highly directional light sources.     

Directional, enhanced fluorescence from molecules near a periodic surface

We extend the research of Holland and Hall on the use of waveguide modes to enhance the fluorescent signal from a layer of molecules [Opt. Lett. 10,414 (1985)] by incorporating a grating into the basic sample structure. Our measurements show that the combination of the directionality imposed by the grating and the previously reported enhancement mechanism has the effect of increasing the intensity of the signal detected over a narrow angular range from a layer of fluorescing molecules by a factor of ~ 1000 over that from a reference sample. Simultaneously our method allows for both polarization and wavelength discrimination of the emitted radiation because of the characteristic nature of the incorporated grating.     

UV fluorescence lifetime imaging microscopy: a label-free method for detection and quantification of protein interactions

Due to the ability to detect multiple parameters simultaneously, protein microarrays have found widespread applications from basic biological research to diagnosis of diseases. Generally, readout of protein microarrays is performed by fluorescence detection using either dye-labeled detector antibodies or direct labeling of the target proteins. We developed a method for the label-free detection and quantification of proteins based on time-gated, wide-field, camera-based UV fluorescence lifetime imaging microscopy to gain lifetime information from each pixel of a sensitive CCD camera. The method relies on differences in the native fluorescence lifetime of proteins and takes advantage of binding-induced lifetime changes for the unequivocal detection and quantification of target proteins. Since fitting of the fluorescence decay for every pixel in an image using a classical exponential decay model is time-consuming and unstable at very low fluorescence intensities, we used a new, very robust and fast alternative method to generate UV fluorescence lifetime images by calculating the average lifetime of the decay for each pixel in the image stack using a model-free average decay time algorithm.To validate the method, we demonstrate the detection and quantification of p53 antibodies, a tumor marker in cancer diagnosis. Using tryptophan-containing capture peptides, we achieved a detection sensitivity for monoclonal antibodies down to the picomolar concentration range. The obtained affinity constant, Ka, of (1.4 +/- 0.6) x 10(9) M(-1), represents a typical value for antigen/antibody binding and is in agreement with values determined by traditional binding assays.     

Long path-length axial absorption detection in photonic crystal fiber

In this paper, we present a long path-length axial absorption detection method in photonic crystal fibers (PCFs). A PCF, also called a holey fiber or microstructured fiber, is an optical fiber which consists of a periodic array of very tiny and closely spaced air holes on the scale of 1 microm running through the whole length of the fiber. Here, a PCF with porous microstructures was used as a sample container for absorption detection. Light was guided by total internal reflection and propagated axially in the air holes of PCFs that were filled with the solution of the absorbing species by vacuum pumping. Excellent linearity was obtained for different sample concentrations, and high sensitivity was achieved due to the long optical path length. In addition, as the dimension of the PCF is small, the sample volume is greatly reduced. Moreover, due to its robustness, the PCF can be coiled up to keep the footprint small, making it suitable for microchip absorption detection. It can be widely used for both off-chip and on-chip detection of absorbing species, such as ions, alkaloids, and biomolecules.     

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.     

Multiplexed hybridization detection of quantum dot-conjugated DNA sequences using surface plasmon enhanced fluorescence microscopy and spectrometry

In this study, the general suitability of quantum dot (QD)-DNA conjugates for the surface plasmon enhanced fluorescence spectroscopy technique is demonstrated. Furthermore, the QD-DNA system is transferred to the platform of surface plasmon enhanced fluorescence microscopy. Using this technique together with a microarray format, in which the sensor-bound single-stranded catcher probes are organized in individual surface spots, results in a simultaneous qualitative analysis of QD-conjugated analyte DNA strands as multicolor images. A clear decomposition of different QD(x)()-DNA(y)() mixtures can be achieved for sequential, as well as mixture injections. Besides this, the study describes the successful approach of measuring spectrally resolved surface plasmon enhanced fluorescence signals derived from catcher probe hybridized QD-DNA conjugates.     

A surface acoustic wave biosensor concept with low flow cell volumes for label-free detection

Special surface acoustic wave (SAW) devices using horizontally polarized surface shear waves can be operated in water. They allow an easy detection of molecules with biological relevance (e.g., proteins) via direct detection of the adsorbed mass. The transducer structures of conventional SAW devices are usually connected to the electronics by bond wires. In consequence, flow cell volumes can hardly be designed smaller than 50 microL. A new type of SAW device that is coupled capacitively with the electronics enables the reduction of flow cell volumes down to 60 nL, which decreases sample consumption and reduces the length of the measurement cycles down to a few minutes. To create an immunosensor, the SAW devices first are coated with a thin parylene layer for creating a sensor surface that is chemically homogeneous. Then OptoDex, a dextran containing both photoactive and functional groups is immobilized photochemically. Finally, antibodies are coupled via conventional EDC/NHS chemistry. The technique has been used to monitor urease binding at anti-urease-coated SAW devices in real time and with good resolution. Because of the simple sensor handling and the economical sample use, the new SAW device is particularly suitable for the design of an array.     

Nanoarray-based biomolecular detection using individual Au nanoparticles with minimized localized surface plasmon resonance variations

Here, we present a mean to expand the use of individual metallic nanoparticles to two-dimensional plasmonic nanoarrays. An optical detection platform to track down localized surface plasmon resonance (LSPR) signals of individual nanoparticles on substrates was built for the application of plasmonic nanoarrays. A pseudoimage of nanoparticles on a substrate was reconstructed from their scattering spectra obtained by scanning a user-defined area. The spectral and spatial resolutions of the system were also discussed in detail. Most importantly, we present a method to normalize the localized surface plasmon resonance from geometrically different nanoparticles. After normalization, plasmonic responses from different particles become highly consistent, creating well-fitted dose-response curves of both surrounding refractive index changes and receptor-analyte binding to the surface of individual nanoparticles. Finally, the proof-of-concept system for plasmonic nanoarray detection is demonstrated by the measurement of the aptamer-thrombin binding event.     

Reactive photonic film for label-free and selective sensing of cyanide

Three-dimensional ordered inverse-opal films bearing a reactive trifluoroacetyl group are successfully constructed. Through the specific reaction between cyanide and trifluoroacetyl, the photonic films can selectively detect sub-micromolar levels of cyanide by distinct structural color change. Labeled molecules are not necessary for the sensing mechanism.     

Silica colloidal crystals for enhanced fluorescence detection in microarrays

Silica colloidal crystals were investigated for their potential as high surface area materials to enhance sensitivity over planar surfaces for microarrays using fluorescence detection. A relation was derived showing how crystal thickness and transmission, as well as colloid size, combine to determine the optically accessible surface area for enhancing sensitivity. Experimentally, crystals of 250-nm colloids were prepared with thicknesses determined by SEM to be 1.6, 4.2, and 11.0 microm. The material was sintered at 1000 degrees C to make it durable without affecting the crystalline structure, as confirmed by SEM. UV/visible spectrometry showed the depth of penetration (1/e) to be 8.4 microm at 488 nm for these materials. Fluorescein-labeled streptavidin and biotin were used as a model ligand-receptor pair. For the fluorescence measurements, biotin was covalently bonded to the silica surfaces, and the fluorescence was detected from the captured streptavidin-fluorescein. The observed fluorescence enhancement agreed well with the theory developed here. Compared to a planar surface, the colloidal crystal of 11.0 microm in thickness enhanced the fluorescence by nearly a factor of 80, with only a 0.3% increase in fluorescence background.     

Fabrication of glass photonic crystal fibers with a die-cast process

We demonstrate a novel method for the fabrication of glass photonic crystal fibers (PCFs) with a die-cast process. SF6 glass is used as the material for PCFs, and the die is made of heat-resisting alloy steel, whose inner structure matches the PCF's structure. The die is put vertically in the vessel with SF6 glass, and the vacuum hose is attached to the top of the die. The die and glass are put in the furnace to heat at 870 K. The die is slowly filled with the softening glass under vacuum conduction until it is full. It is kept in the furnace to anneal at a rate of 20 K/h to remove the thermal stress that could lead to cracks. The outer tube of the die is taken apart when its temperature is close to room temperature, and the fused glass bundle is etched in an acidic solution to remove the heat-resisting alloy steel rods. Thus, the etched bundle is ready to use as a PCF preform. The PCF is observed in the generation of a supercontinuum, with the flat plateau in the spectrum of the output emission stretching from 400 to 1400 nm by experimental measurement. The transmission loss is 0.2-0.3 dB/m at wavelengths of 420-900 nm.     

Enhanced backscattering for infrared detection using photonic crystal based flat lens

An n=-1 flat lens based on photonic crystal semiconductor technology is evaluated for infrared detection purposes. The idea consists in exploiting the backscattered waves of an incident plane wave impinging on a target placed in the focal region of a flat lens. It is shown that subwavelength detection of micronic dielectric targets can be obtained at 1.55?μm using the double focus of reflected waves induced by negative refraction. Complex relations among the intrinsic nature, the shape and size of the target, and detection efficiency are interpreted in terms of target eigenmode excitation. Reflectivity is modulated by the intrinsic mode nature, transverse, circular, or longitudinal, with an enhancement of the detection sensitivity in the case of whispering-gallery modes. It is believed that such a study paves the way to the definition of original noninvasive infrared sensors.