Optical detection enhancement in porous volumetric microfluidic capture elements using refractive index matching fluids

Abstract

Porous volumetric capture elements in microfluidic sensors are advantageous compared to planar capture surfaces due to higher reaction site density and decreased diffusion lengths that can reduce detection limits and total assay time. However a mismatch in refractive indices between the capture matrix and fluid within the porous interstices results in scattering of incident, reflected, or emitted light, significantly reducing the signal for optical detection. Here we demonstrate that perfusion of an index-matching fluid within a porous matrix minimizes scattering, thus enhancing optical signal by enabling the entire capture element volume to be probed. Signal enhancement is demonstrated for both fluorescence and absorbance detection, using porous polymer monoliths in a silica capillary and packed beds of glass beads within thermoplastic microchannels, respectively. Fluorescence signal was improved by a factor of 3.5× when measuring emission from a fluorescent compound attached directly to the polymer monolith, and up to 2.6× for a rapid 10 min direct immunoassay. When combining index matching with a silver enhancement step, a detection limit of 0.1 ng mL(-1) human IgG and a 5 log dynamic range was achieved. The demonstrated technique provides a simple method for enhancing optical sensitivity for a wide range of assays, enabling the full benefits of porous detection elements in miniaturized analytical systems to be realized.

Figure 1

Experimental process for fluorescence based direct assay. The methacrylate monolith is covalently functionalized with goat anti-rabbit IgG (a) followed by infusion of FITC tagged rabbit IgG resulting in immobilization (b). Next GMA (n=1.45) was infused to enhance the fluorescence signal (c).

Figure 2

Experimental process for direct immunoassay using transmittance based detection. (a) A packed bed of silica beads with human IgG is (b) infused with anti-human IgG attached to gold nanoparticles (AuNP) that are (c) silver enhanced to create large aggregates prior to (d) introduction of index matching sucrose solution to render the volumetric matrix transparent for optical absorbance measurements.

Figure 3

(a) An opaque monolith sheet in air (n~1) remains semi-opaque due to light scattering when water (n~1.33) is introduced. The addition of index-matching aqueous sucrose (n~1.46) significantly enhances transparency, revealing the image beneath. (b) Fluorescent intensity (I, arbitrary units) following water (I=758) and GMA monomer (I=2644) infusion through a glutaraldehyde functionalized monolith in a glass capillary.

Figure 4

Direct assay with fluorescein isothiocyanate labelled antibodies before and after infusion of GMA monomer through GMA-based monolith capture element in a glass capillary.

Figure 5

Images of a packed silica bead bed infused with (a) water and (b) 68% sucrose in water (n=1.46). (c) Measured light transmittance with varying sucrose concentration. Transmittance is defined as the ratio of average transmitted intensity of a region of interest (ROI) of the packed bed after sucrose infusion (I_index) to the background intensity (I_o) of a nearby region of the chip.

Figure 6

(a) Microfluidic chip containing silica bead detection zones, with the bead beds alternately functionalized with either 480 ng/mL or 4.8 ng/mL of human IgG. The magnified image of the packed bed is shown after selective capture of AuNP anti-human IgG conjugates and subsequent silver amplification. (b) Absorbance was measured for the regions of interest shown (dashed boxes) after silver enhancement and water rinse, and after infusion with a 68% sucrose solution for index matching. (c) Relative absorbance for a silver-enhanced direct immunoassay following infusion of index matching solution in a microfluidic silica bead bed.