Nanoplasmonic platform for multiparametric and highthroughput biosensing

The advances in proteomics and genomics have led to discover a lot of biomarkers that can potentially be used as diagnostic and prognostic indicators of diseases. Simultaneously, a huge research effort has been invested in developing biosensors that could monitor the interaction of biological materials. Such sensors are required to be fast, real time, label free and highly sensitive to the appropriate biomarker. One of the possible solutions is surface plasmon resonance imaging (iSPR) biosensor. Nevertheless, some important milestones still need to be reached for a successful application. Indeed, iSPR instruments commercially available have a poor sensitivity to low biomarker concentration and they are quite expensive. In this paper, we show a compact instrument, called Imaging NanoplasmonicsTM (iNPx), designed to overcome these limits. A nanostructured interface is introduced to increase the sensitivity of different immunoassay reactions and with the use of a very low volume of material. Then, as a proof of principle, we report an example of specific application for the monitoring of the interaction between some variants of FLAG peptides with the monoclonal antibody Anti-FLAG. The proposed platform allows extreme versatility for multiplexed diagnostic and/or food quality applications

Typical current versus time traces of ODN-coated PS microparticles passing throughout an ODN-functionalized micropore.

<p>A. Translocation of PS-ncODN. B. Capture of PS-cODN. The bias potential across the micropore was 10 mV.</p

Silicon micropore chip.

<p><b>A.</b> A photograph of the silicon micropore chip. <b>B.</b> Cross-section diagram of the pyramidal opening and the micropore in the silicon chip. A thermally grown silica layer covers the entire chip surface and the pore wall. <b>C.</b> Scanning electron microscopy image of the micropore. <b>D.</b> Optical transmission microscopy image of a micropore.</p

Selective capture of polystyrene (PS) microparticles in functionalized micropores.

<p><b>A.</b> Schematic illustration of the specific interaction between ODN probe-functionalized micropore wall and the cODN target-functionalized PS particles. <b>B.</b> Schematic representation of the focusing planes for image acquisition by optical transmission microscopy. <b>C.</b> Photographs of ODN probe-modified micropores after incubation with cODN (pores 1 and 2) or ncODN (pores 3 and 4) target-functionalized particles by focusing the microscope objective on microparticles settled on the micropore membrane (blue-framed images) or inside the micropores (red-framed images).</p

Selective capture of B or T lymphocytes from primary splenocyte samples using specific antibody-functionalized micropores.

<p>Only the T lymphocytes are fluorescently labeled. A. Schematic illustration of micropore functionalization with antibodies targeting cell surface proteins. B. Transmission and fluorescence microscopy images of cells captured in antibody-functionalized micropores, and stacks of the images. The white dashed circles in the fluorescence images indicate the position of the micropore wall.</p

Selective individual primary cell capture using locally bio-functionalized micropores.

BACKGROUND: Solid-state micropores have been widely employed for 6 decades to recognize and size flowing unlabeled cells. However, the resistive-pulse technique presents limitations when the cells to be differentiated have overlapping dimension ranges such as B and T lymphocytes. An alternative approach would be to specifically capture cells by solid-state micropores. Here, the inner wall of 15-µm pores made in 10 µm-thick silicon membranes was covered with antibodies specific to cell surface proteins of B or T lymphocytes. The selective trapping of individual unlabeled cells in a bio-functionalized micropore makes them recognizable just using optical microscopy. METHODOLOGY/PRINCIPAL FINDINGS: We locally deposited oligodeoxynucleotide (ODN) and ODN-conjugated antibody probes on the inner wall of the micropores by forming thin films of polypyrrole-ODN copolymers using contactless electro-functionalization. The trapping capabilities of the bio-functionalized micropores were validated using optical microscopy and the resistive-pulse technique by selectively capturing polystyrene microbeads coated with complementary ODN. B or T lymphocytes from a mouse splenocyte suspension were specifically immobilized on micropore walls functionalized with complementary ODN-conjugated antibodies targeting cell surface proteins. CONCLUSIONS/SIGNIFICANCE: The results showed that locally bio-functionalized micropores can isolate target cells from a suspension during their translocation throughout the pore, including among cells of similar dimensions in complex mixtures