Yafang Tan

Smartphone fluorescence spectroscopy

We demonstrate the first use of smartphone spectrophotometry for readout of fluorescence-based biological assays. We evaluated the smartphone fluorimeter in the context of a fluorescent molecular beacon (MB) assay for detection of specific nucleic acid sequences in a liquid test sample and compared performance against a conventional laboratory fluorimeter. The capability of distinguishing a one-point mismatch is also demonstrated by detecting single-base mutation in target nucleic acids. Our approach offers a route toward portable biomolecular assays for viral/bacterial pathogens, disease biomarkers, and toxins.

Smartphone Fluorescence Spectroscopy

We demonstrate the first use of smartphone spectrophotometry for readout of fluorescence-based biological assays. We evaluated the smartphone fluorimeter in the context of a fluorescent molecular beacon (MB) assay for detection of a specific nucleic acid sequences in a liquid test sample. The capability of distinguishing a one-point mismatch is also demonstrated by detecting single-base mutation in target nucleic acids. Our approach offers a route towards portable biomolecular assays for viral/bacterial pathogens, disease biomarkers, and toxins.

Large-area organic distributed feedback laser fabricated by nanoreplica molding and horizontal dipping

The fabrication of visible wavelength vertically emitting distributed feedback (DFB) lasers with a subwavelength grating fabricated by a replica molding process and an active polymer layer printed by a horizontal dipping process is reported. The combined techniques enable the organic DFB laser to be uniformly fabricated over large surface areas upon a flexible plastic substrate, with an approach that is compatible with roll-based manufacturing. Using a fixed grating period and depth, DFB laser output wavelength is controlled over a 35 nm range through manipulation of the waveguide layer thickness, which is controlled by the speed of the horizontal dipping process. We also demonstrate that the active area of the structure may be photolithographically patterned to create dense arrays of discrete DFB lasers.

Plastic-Based Distributed Feedback Laser Biosensors in Microplate Format

A process that combines polymer nanoreplica molding with horizontal dipping was used to fabricate large area (~ 3 × 5 inch2) distributed feedback laser biosensors (DFBLB) on flexible plastic substrates, which were subsequently incorporated into standard format 96-well microplates. A room temperature nanoreplica molding process was used to create subwavelength periodic grating structures, while a horizontal dipping process was used to apply a ~ 300 nm, dye-doped polymer film. In this work, the DFBLB emission wavelength, used to characterize the device uniformity, demonstrated a coefficient of variation (CV) of 0.41% over the fabricated device area, representing a thickness standard deviation of only ~ 35 nm for the horizontal dipping process. The fabricated sensors were further characterized for sensitivity uniformity by measuring the bulk refractive index of the media exposed to the sensor surface and by measuring adsorption of biomolecular layers. An assay for detection of the cytokine Tumor Necrosis Factor-alpha (TNF-α) was used to demonstrate the operation of the sensor in the context of label-free detection of a disease biomarker. The demonstrated capability represents an important step towards roll-to-roll manufacturability for this biosensor that simultaneously incorporates high sensitivity with excellent wavelength shift resolution, and adaptability to the microplate format that is ubiquitous in pharmaceutical research.

Enhanced live cell imaging via photonic crystal enhanced fluorescence microscopy

We demonstrate photonic crystal enhanced fluorescence (PCEF) microscopy as a surface-specific fluorescence imaging technique to study the adhesion of live cells by visualizing variations in cell-substrate gap distance. This approach utilizes a photonic crystal surface incorporated into a standard microscope slide as the substrate for cell adhesion, and a microscope integrated with a custom illumination source as the detection instrument. When illuminated with a monochromatic light source, angle-specific optical resonances supported by the photonic crystal enable efficient excitation of surface-confined and amplified electromagnetic fields when excited at an on-resonance condition, while no field enhancement occurs when the same photonic crystal is illuminated in an off-resonance state. By mapping the fluorescence enhancement factor for fluorophore-tagged cellular components between on- and off-resonance states and comparing the results to numerical calculations, the vertical distance of labelled cellular components from the photonic crystal substrate can be estimated, providing critical and quantitative information regarding the spatial distribution of the specific components of cells attaching to a surface. As an initial demonstration of the concept, 3T3 fibroblast cells were grown on fibronectin-coated photonic crystals with fluorophore-labelled plasma membrane or nucleus. We demonstrate that PCEF microscopy is capable of providing information about the spatial distribution of cell-surface interactions at the single-cell level that is not available from other existing forms of microscopy, and that the approach is amenable to large fields of view, without the need for coupling prisms, coupling fluids, or special microscope objectives.

High sensitivity automated multiplexed immunoassays using photonic crystal enhanced fluorescence microfluidic system.

We demonstrate a platform that integrates photonic crystal enhanced fluorescence (PCEF) detection of a surface-based microspot fluorescent assay with a microfluidic cartridge to achieve simultaneous goals of high analytic sensitivity (single digit pg/mL), high selectivity, low sample volume, and assay automation. The PC surface, designed to provide optical resonances for the excitation wavelength and emission wavelength of Cyanines 5 (Cy5), was used to amplify the fluorescence signal intensity measured from a multiplexed biomarker microarray. The assay system is comprised of a plastic microfluidic cartridge for holding the PC and an assay automation system that provides a leak-free fluid interface during introduction of a sequence of fluids under computer control. Through the use of the assay automation system and the PC embedded within the microfluidic cartridge, we demonstrate pg/mL-level limits of detection by performing representative biomarker assays for interleukin 3 (IL3) and Tumor Necrosis Factor (TNF-α). The results are consistent with limits of detection achieved without the use of the microfluidic device with the exception that coefficients of variability from spot-to-spot are substantially lower than those obtained by performing assays with manual manipulation of assay liquids. The systems capabilities are compatible with the goal of diagnostic instruments for point-of-care settings.

Photonic crystal enhancement of a homogeneous fluorescent assay using submicron fluid channels fabricated by E-jet patterning

We demonstrate the enhancement of a liquid-based homogenous fluorescence assay using the resonant electric fields from a photonic crystal (PC) surface. Because evanescent fields are confined to the liquid volume nearest to the photonic crystal, we developed a simple approach for integrating a PC fabricated on a silicon substrate within a fluid channel with submicron height, using electrohydrodynamic jet (e-jet) printing of a light-curable epoxy adhesive to define the fluid channel pattern. The PC is excited by a custom-designed compact instrument that illuminates the PC with collimated light that precisely matches the resonant coupling condition when the PC is covered with aqueous media. Using a molecular beacon nucleic acid fluorescence resonant energy transfer (FRET) probe for a specific miRNA sequence, we demonstrate an 8× enhancement of the fluorescence emission signal, compared to performing the same assay without exciting resonance in the PC detecting a miRNA sequence at a concentration of 62 nM from a liquid volume of only ∼20 nL. The approach may be utilized for any liquid-based fluorescence assay for applications in point-of-care diagnostics, environmental monitoring, or pathogen detection.

Distributed Feedback Laser Biosensor Noise Reduction

We report on a signal processing approach that enables detection of lasing wavelength shifts as small as Δλ~1.5pm from a distributed feedback laser biosensor (DFBLB) fabricated upon a plastic substrate and incorporated into microplates.

Application of photonic crystal enhanced fluorescence to detection of low serum concentrations of human IgE antibodies specific for a purified cat allergen (Fel D1).

We demonstrate the detection of low concentrations of allergen-specific Immunoglobulin E (IgE) in human sera using a Photonic Crystal Enhanced Fluorescence (PCEF) microarray platform. The Photonic Crystal (PC) surface, designed to provide optical resonances for the excitation wavelength and emission wavelength of Cy5, was used to amplify the fluorescence signal intensity measured from a multiplexed allergen microarray. Surface-based sandwich immunoassays were used to detect and quantify specific IgE antibodies against a highly purified cat allergen (Fel d1). A comparison of the lowest detectable concentration of IgE measured by the PC microarray system and a commercially available clinical analyzer demonstrated that the PCEF microarray system provides higher sensitivity. The PCEF system was able to detect low concentrations of specific IgE (~0.02 kU/L), which is 5-17-fold more sensitive than the commercially available FDA-approved analyzers. In preliminary experiments using multi-allergen arrays, we demonstrate selective simultaneous detection of IgE antibodies to multiple allergens.

Direct detection of transcription factors in cotyledons during seedling development using sensitive silicon-substrate photonic crystal protein arrays.

Transcription factors in seedling cotyledons are quantified using novel silicon photonic crystal protein arrays and their levels are correlated with transcript abundances. Transcription factors control important gene networks, altering the expression of a wide variety of genes, including those of agronomic importance, despite often being expressed at low levels. Detecting transcription factor proteins is difficult, because current high-throughput methods may not be sensitive enough. One-dimensional, silicon-substrate photonic crystal (PC) arrays provide an alternative substrate for printing multiplexed protein microarrays that have greater sensitivity through an increased signal-to-noise ratio of the fluorescent signal compared with performing the same assay upon a traditional aminosilanized glass surface. As a model system to test proof of concept of the silicon-substrate PC arrays to directly detect rare proteins in crude plant extracts, we selected representatives of four different transcription factor families (zinc finger GATA, basic helix-loop-helix, BTF3/NAC [for basic transcription factor of the NAC family], and YABBY) that have increasing transcript levels during the stages of seedling cotyledon development. Antibodies to synthetic peptides representing the transcription factors were printed on both glass slides and silicon-substrate PC slides along with antibodies to abundant cotyledon proteins, seed lectin, and Kunitz trypsin inhibitor. The silicon-substrate PC arrays proved more sensitive than those performed on glass slides, detecting rare proteins that were below background on the glass slides. The zinc finger transcription factor was detected on the PC arrays in crude extracts of all stages of the seedling cotyledons, whereas YABBY seemed to be at the lower limit of their sensitivity. Interestingly, the basic helix-loop-helix and NAC proteins showed developmental profiles consistent with their transcript patterns, indicating proof of concept for detecting these low-abundance proteins in crude extracts.