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

Abstract

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.

Keywords: E-jet printing; enhanced fluorescence; homogeneous assay; photonic crystal; submicron channel.

Figure 1

(a) Schematic diagram of the device structure, comprised of a transparent glass layer, a microfluidic region and a PC surface for fluorescence enhancement. (b) SEM image of the one dimensional PC with period of 360nm and duty cycle of 36%. (c) Evanescent electric field enhancement distribution at the resonant coupling condition of the PC (d) The average enhancement of field intensity as the function of distance, d, above the top TiO₂ layer of the PC.

Figure 2

(a) Schematic diagram of the fabrication process: I. The channel pattern was e-jet printed on a silicon PC (Si-PC); II. The Si-PC was carefully aligned with a glass slide and pressed against each other by a roller to squeeze the droplets. The layout of the channel consisting of two inlets with diameters D_I=1mm (left), one observation window with a diameter D_R=2mm (middle), and one outlet with a diameter D_I=1mm (right). The total length and the width of the channel are L=13.6mm and W=0.5mm. (b) Photograph of the PC integrated into the submicron flow channel. (c) Optical image of the observation window. (d) Optical image of the printed NOA74 droplets. (e) Optical image of the continuous NOA74 film after squeezing the droplets. (f) SEM image of cross section of a completed device, showing the thickness of the channel is ~830nm.

Figure 3

Reflection spectra of a device filled with water, illuminated with a broadband light source and captured at normal incidence (black, solid) and at an incidence angle of 2.4 degrees (red, dashed). At an incidence angle of 2.4 degrees, the resonant peak occurs at a wavelength of λ=637nm.

Figure 4

(a) Schematic diagram of the detection instrumentation. (b) Fluorescence intensities obtained from 50μg/mL LD700 solution filled within the channel when excited over a range of illumination angles.

Figure 5

Results of the miR21 detection by DNA molecular beacon assay. (a) Dose response curves of mature miR21 (target, black lines) and one pair mismatched mutant miR21 (control, blue lines), obtained from PC at on-resonance (solid lines) and off-resonance (dashed lines) conditions. The results confirm the selectivity of the assay as well as the PCEF enhanced excitation effect. (b) A zoom-in plot of the fluorescence intensities for the detection of mature miR21 at on-resonance (black, solid line) and off-resonance (black, dashed line) conditions. The red lines represent the lowest detectable fluorescence at on-resonance (solid line) and off-resonance (dashed line) conditions.