Discrimination of α-Thrombin and γ-Thrombin Using Aptamer-Functionalized Nanopore Sensing.

Protein detection and identification at the single-molecule level are major challenges in many biotechnological fields. Solid-state nanopores have raised attention as label-free biosensors with high sensitivity. Here, we use solid-state nanopore sensing to discriminate two closely related proteins, α-thrombin and γ-thrombin. We show that aptamer functionalization improves protein discrimination thanks to a significant difference in the relative current blockade amplitude. To enhance discrimination, we postprocessed the signals using machine learning and training algorithms and we were able to reach an accuracy of 98.8% using seven features and ensemble methods.

Bipolar Electrochemiluminescence Imaging: A Way to Investigate the Passivation of Silicon Surfaces.

This work depicts the original combination of electrochemiluminescence (ECL) and bipolar electrochemistry (BPE) to map in real-time the oxidation of silicon in microchannels. We fabricated model silicon-PDMS microfluidic chips, optionally containing a restriction, and monitored the evolution of the surface reactivity using ECL. BPE was used to remotely promote ECL at the silicon surface inside microfluidic channels. The effects of the fluidic design, the applied potential and the resistance of the channel (controlled by the fluidic configuration) on the silicon polarization and oxide formation were investigated. A potential difference down to 6 V was sufficient to induce ECL, which is two orders of magnitude less than in classical BPE configurations. Increasing the resistance of the channel led to an increase in the current passing through the silicon and boosted the intensity of ECL signals. Finally, the possibility of achieving electrochemical reactions at predetermined locations on the microfluidic chip was investigated using a patterning of the silicon oxide surface by etched micrometric squares. This ECL imaging approach opens exciting perspectives for the precise understanding and implementation of electrochemical functionalization on passivating materials. In addition, it may help the development and the design of fully integrated microfluidic biochips paving the way for development of original bioanalytical applications.

Sensing with Nanopores and Aptamers: A Way Forward.

In the 90s, the development of a novel single molecule technique based on nanopore sensing emerged. Preliminary improvements were based on the molecular or biological engineering of protein nanopores along with the use of nanotechnologies developed in the context of microelectronics. Since the last decade, the convergence between those two worlds has allowed for biomimetic approaches. In this respect, the combination of nanopores with aptamers, single-stranded oligonucleotides specifically selected towards molecular or cellular targets from an in vitro method, gained a lot of interest with potential applications for the single molecule detection and recognition in various domains like health, environment or security. The recent developments performed by combining nanopores and aptamers are highlighted in this review and some perspectives are drawn.

Polarization Induced Electro-Functionalization of Pore Walls: A Contactless Technology.

This review summarizes recent advances in micro- and nanopore technologies with a focus on the functionalization of pores using a promising method named contactless electro-functionalization (CLEF). CLEF enables the localized grafting of electroactive entities onto the inner wall of a micro- or nano-sized pore in a solid-state silicon/silicon oxide membrane. A voltage or electrical current applied across the pore induces the surface functionalization by electroactive entities exclusively on the inside pore wall, which is a significant improvement over existing methods. CLEF's mechanism is based on the polarization of a sandwich-like silicon/silicon oxide membrane, creating electronic pathways between the core silicon and the electrolyte. Correlation between numerical simulations and experiments have validated this hypothesis. CLEF-induced micro- and nanopores functionalized with antibodies or oligonucleotides were successfully used for the detection and identification of cells and are promising sensitive biosensors. This technology could soon be successfully applied to planar configurations of pores, such as restrictions in microfluidic channels.

Enhanced Bipolar Electrochemistry at Solid-State Micropores: Demonstration by Wireless Electrochemiluminescence Imaging.

Bipolar electrochemistry (BPE) is a powerful method based on the wireless polarization of a conductive object that induces the asymmetric electroactivity at its two extremities. A key physical limitation of BPE is the size of the conductive object because the shorter the object, the larger is the potential necessary for sufficient polarization. Micrometric and nanometric objects are thus extremely difficult to address by BPE due to the very high potentials required, in the order of tens of kV or more. Herein, the synergetic actions of BPE and of planar micropores integrated in a microfluidic device lead to the spatial confinement of the potential drop at the level of the solid-state micropore, and thus to a locally enhanced polarization of a bipolar electrode. Electrochemiluminescence (ECL) is emitted in half of the electroactive micropore and reveals the asymmetric polarization in this spatial restriction. Micrometric deoxidized silicon electrodes located in the micropore are polarized at a very low potential (7 V), which is more than 2 orders of magnitude lower compared to the classic bipolar configurations. This behavior is intrinsically associated with the unique properties of the micropores, where the sharp potential drop is focused. The presented approach offers exciting perspectives for BPE of micro/nano-objects, such as dynamic BPE with objects passing through the pores or wireless ECL-emitting micropores.

Carbohydrates as new probes for the identification of closely related Escherichia coli strains using surface plasmon resonance imaging.

Prevention of foodborne diseases depends highly on our ability to control rapidly and accurately a possible contamination of food. So far, standard procedures for bacterial detection require time-consuming bacterial cultures on plates before the pathogens can be detected and identified. We present here an innovative biochip, based on direct differential carbohydrate recognitions of five closely related Escherichia coli strains, including the enterohemorragic E. coli O157:H7. Our device relies on efficient grafting of simple carbohydrates on a gold surface and on the monitoring of their interactions with bacteria during their culture using surface plasmon resonance imaging. We show that each of the bacteria interacts in a different way with the carbohydrate chip. This allows the detection and discrimination of the tested bacterial strains in less than 10 h from an initial bacterial concentration of 10(2) CFU·mL(-1). This is an improvement over previously described systems in terms of cost, easiness to use, and stability. Easily conceived and easily regenerated, this tool is promising for the future of food safety.

Oligosaccharide biosensor for direct monitoring of enzymatic activities using QCM-D.

Enzymatic modification of saccharidic biomass is a subject of intensive research with potential applications in plant or human health, design of biomaterials and biofuel production. Bioengineering and metagenomics provide access to libraries of glycoside hydrolases but the biochemical characterization of these enzymes remains challenging, requiring fastidious colorimetric tests in discontinuous assays. Here, we describe a highly sensitive carbohydrate biosensor for the detection and characterization of glycoside hydrolases. Immobilization of oligosaccharides was achieved using copper-catalyzed azide-alkyne cycloaddition of maltoheptaose-modified probes onto self-assembled monolayers bearing azide reactive groups. This biosensor allowed detection of glycoside hydrolase activities at the picomolar level using quartz-crystal microbalance with dissipation monitoring (QCM-D). To our knowledge, this protocol provides the best performance to date for the detection of glycoside hydrolase activities. For each enzyme tested, we could determine the kinetic constant from the QCM-D data, and derive conclusions that correlated well with those of standard colorimetric tests. This opens the way to a new generation of rapid and direct tests characterizing functionally carbohydrate-active enzymes.