Nanofluidic-Based Accumulation of Antigens for Miniaturized Immunoassay

Abstract

The continuous advances of Nanofluidics have been stimulating the development of novel nanostructures and strategies to accumulate very diluted analytes, for implementing a new class of high sensitivity miniaturized polymeric sensors. We take advantage of the electrokinetic properties of these structures, which allow accumulating analytes inside asymmetric microfluidic structures to implement miniaturized sensors able to detect diluted solutions down to nearly 1.2 pg/mL. In particular, exploiting polydimethylsiloxane devices, fabricated by using the junction gap breakdown technique, we concentrate antigens inside a thin microfunnel functionalized with specific antibodies to favor the interaction and, if it is the case, the recognition between antigens in solution and antibodies anchored to the surface. The transduction mechanism consists in detecting the fluorescence signal of labeled avidin when it binds to biotinylated antigens. Here, we demonstrate that exploiting these electrokinetic phenomena, typical of nanofluidic structures, we succeeded in concentrating biomolecules in correspondence of a 1 pL sensing region, a strategy that grants to the device performance comparable to standard immunoassays.

Keywords: PDMS; antibody-antigen recognition; immunoassay; immunobiosensing; miniaturized device; nanofluidic device; nanofunnel.

Figure 1

Polydimethylsiloxane (PDMS) miniaturized device: (a) schematic representation of the device consisting in a PDMS replica with two U-shaped microchannels (500 µm wide and 50 µm deep, separated by a gap 100 µm thick) sealed to a glass coverslip; (b) optical microscope caption of the cis- and trans- microchannels linked by a single funnel (highlighted by the red rectangle) (scale bar 100 µm); (c) bright-field caption of the entire funnel after the junction gap breakdown procedure (scale bar 10 µm); (d) epifluorescence microscope image of the nanoporous tip of the funnel, filled with a fluorescent solution, after the junction gap breakdown (scale bar 10 µm).

Figure 2

Antibody immobilization on PDMS: (a) scheme of the steps for functionalizing PDMS and glass with antibodies; (b) ATR-FTIR reflectance spectra acquired at different stages of the functionalization process; (c) detail of the reflectance ATR-FTIR spectra in the wavenumber range corresponding to amide I and II.

Figure 3

Accumulation experiments in devices functionalized with antibodies. (a) Current-voltage curve of a micro/nanofluidic device, filled with 1 M KCl, measured before (black squares), and after (red circles) the junction gap breakdown procedure. (b,c) Epifluorescence microscopy images of the funnel after 10 min at −10 V (b) and 10 V (c), with the cis-microchannel filled with avidin–fluorescein (12 pg/mL). (d) Sequence of epifluorescence images acquired at time intervals of 30 s during the application of a constant bias of −10 V for 10 min with the cis-microchannel filled by 30 pg/mL avidin–fluorescein.

Figure 4

Antigen-antibody recognition mechanism and biosensing experiments. (a) Positive test: target biotinylated IL10 antigens in solution recognize and bind to the probe antibodies anchored to the surface; fluorescent avidin binds to the biotin, and it is detected by fluorescence microscopy; (b) negative test: biotinylated not-matching antigens do not bind with the probe antibodies, and they are washed away, no fluorescence signal can be detected. (c) Epifluorescence microscopy images of the funnel after a positive test performed with a biotinylated IL10 concentration of 1.2 pg/mL (right) and after a negative test (left).