Utilizing its integrated camera as a spectrometer, we demonstrate the use of a smartphone as the detection instrument for a label-free photonic crystal biosensor. A custom-designed cradle holds the smartphone in fixed alignment with optical components, allowing for accurate and repeatable measurements of shifts in the resonant wavelength of the sensor. Externally provided broadband light incident upon an entrance pinhole is subsequently collimated and linearly polarized before passing through the biosensor, which resonantly reflects only a narrow band of wavelengths. A diffraction grating spreads the remaining wavelengths over the camera's pixels to display a high resolution transmission spectrum. The photonic crystal biosensor is fabricated on a plastic substrate and attached to a standard glass microscope slide that can easily be removed and replaced within the optical path. A custom software app was developed to convert the camera images into the photonic crystal transmission spectrum in the visible wavelength range, including curve-fitting analysis that computes the photonic crystal resonant wavelength with 0.009 nm accuracy. We demonstrate the functionality of the system through detection of an immobilized protein monolayer, and selective detection of concentration-dependent antibody binding to a functionalized photonic crystal. We envision the capability for an inexpensive, handheld biosensor instrument with web connectivity to enable point-of-care sensing in environments that have not been practical previously.
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 5–90%, 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.
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 couples 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 minute. 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.
Technical variability during DNA capture probe printing remains an important obstacle to obtaining high quality data from microarray experiments. While methods that use fluorescent labels for visualizing printed arrays prior to hybridization have been presented, the ability to measure spot density using label-free techniques would provide valuable information on spot quality without altering standard microarray protocols. In this study, we present the use of a photonic crystal biosensor surface and a high resolution label-free imaging detection instrument to generate prehybridization images of spotted oligonucleotide microarrays. Spot intensity, size, level of saturation, and local background intensity were measured from these images. This information was used for the automated identification of missed spots (due to mechanical failure or sample depletion) as well as the assignment of a score that reflected the quality of each printed feature. Missed spots were identified with >95% sensitivity. Furthermore, filtering based on spot quality scores increased pairwise correlation of posthybridization spot intensity between replicate arrays, demonstrating that label-free spot quality scores captured the variability in the microarray data. This imaging modality can be applied for the quality control of printed cDNA, oligonucleotide, and protein microarrays.
BackgroundThere has been a long standing interest in the identification of medicinal plants and derived natural products for developing cancer therapeutics. Our study focuses upon pancreatic cancer, due to its high mortality rate, that is attributed in part to the lack of an effective chemotherapeutic agent. Previous reports on the use of medicinal plant extracts either alone or alongside conventional anticancer agents in the treatment of this cancer have shown promising results. This work aims to investigate the therapeutic properties of a library of medicinal plants from Bangladesh.Methods56 extracts of 44 unique medicinal plants were studied. The extracts were screened for cytotoxicity against the pancreatic adenocarcinoma cell line Panc-1, using a label-free biosensor assay. The top cytotoxic extracts identified in this screen were tested on two additional pancreatic cancer cell lines (Mia-Paca2 and Capan-1) and a fibroblast cell line (Hs68) using an MTT proliferation assay. Finally, one of the most promising extracts was studied using a caspase-3 colorimetric assay to identify induction of apoptosis.ResultsCrude extracts of Petunia punctata, Alternanthera sessilis, and Amoora chittagonga showed cytotoxicity to three cancer cell lines with IC50 values ranging between 20.3 - 31.4 μg/mL, 13.08 - 34.9 μg/mL, and 42.8 - 49.8 μg/mL, respectively. Furthermore, treatment of Panc-1 cells with Petunia punctata was shown to increase caspase-3 activity, indicating that the observed cytotoxicity was mediated via apoptosis. Only Amoora chittagonga showed low cytotoxicity to fibroblast cells with an IC50 value > 100 μg/mL.ConclusionBased upon the initial screening work reported here, further studies aimed at the identification of active components of these three extracts and the elucidation of their mechanisms as cancer therapeutics are warranted.
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