Development of an Innovative Quantification Assay Based on Aptamer Sandwich and Isothermal Dumbbell Exponential Amplification.

Detecting blood biomarkers such as proteins with high sensitivity and specificity is of the utmost importance for early and reliable disease diagnosis. As molecular probes, aptamers are raising increasing interest for biosensor applications as an alternative to antibodies, which are used in classical enzyme-linked immuno-sorbent assays (ELISA). We have developed a sensitive and antibody-free molecular quantification assay that combines the specificity of aptamers and the sensitivity of the loop-mediated isothermal amplification (LAMP). For the proof-of-concept, we consider two types of biomarkers: (i) a model of oligonucleotide mimicking nucleic acid targets and (ii) the thrombin involved in the complex coagulation cascade as a model protein for which two relevant aptamers form a stable sandwich. The assay protocol is based on a few successive steps, similar to sandwich ELISA. First, aptamer-coated magnetic beads are added to the sample to specifically capture the targets. Then, the sandwich complex is formed by adding the second aptamer. This secondary aptamer is integrated in a larger oligonucleotide dumbbell sequence designed for LAMP detection using only two primers. After a proper rinsing step, the isothermal dumbbell exponential amplification is performed to detect and quantify a low amount of targets (limit of detection ∼ 1 pM for the oligonucleotide and ∼100 pM for thrombin). This study demonstrates that our innovative aptamero-LAMP assay could be relevant for the detection of different types of biomarkers and their quantification at physiological levels. This may also pave the way for antibody-free molecular assays.

Discrimination of deletion to point cytokine mutants based on an array of cross-reactive receptors mimicking protein recognition by heparan sulfate.

Differential sensing of proteins based on cross-reactive arrays and pattern recognition is a promising technique for the detection and identification of proteins. In this study, a rational biomimetic strategy has been used to prepare sensing materials capable of discriminating structurally similar proteins, such as deletion and point mutants of a cytokine, by mimicking the biological properties of heparan sulfate (HS). Using the self-assembly of two disaccharides, lactose and sulfated lactose at various ratios on the surface of a chip, an array of combinatorial cross-reactive receptors has been prepared. Coupling with surface plasmon resonance imaging (SPRi), the obtained cross-reactive array is very efficient for protein sensing. It is able to detect HS binding proteins (HSbps) such as IFNγ at nanomolar concentrations. Moreover, such a system is capable of discriminating between IFNγ and its mutants with good selectivity.

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.

Enhancing the sensitivity of plasmonic optical fiber sensors by analyzing the distribution of the optical modes intensity.

Improving the sensitivity of plasmonic optical fiber sensors constitutes a major challenge as it could significantly enhance their sensing capabilities for the label-free detection of biomolecular interactions or chemical compounds. While many efforts focus on developing more sensitive structures, we present here how the sensitivity of a sensor can be significantly enhanced by improving the light analysis. Contrary to the common approach where the global intensity of the light coming from the core is averaged, our approach is based on the full analysis of the retro-reflected intensity distribution that evolves with the refractive index of the medium being analyzed. Thanks to this original and simple approach, the refractive index sensitivity of a plasmonic optical fiber sensor used in reflection mode was enhanced by a factor of 25 compared to the standard method. The reported approach opens exciting perspectives for improving the remote detection as well as for developing new sensing strategies.

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.

Bio-Inspired Strategies for Improving the Selectivity and Sensitivity of Artificial Noses: A Review.

Artificial noses are broad-spectrum multisensors dedicated to the detection of volatile organic compounds (VOCs). Despite great recent progress, they still suffer from a lack of sensitivity and selectivity. We will review, in a systemic way, the biomimetic strategies for improving these performance criteria, including the design of sensing materials, their immobilization on the sensing surface, the sampling of VOCs, the choice of a transduction method, and the data processing. This reflection could help address new applications in domains where high-performance artificial noses are required such as public security and safety, environment, industry, or healthcare.

Multiplexed Remote SPR Detection of Biological Interactions through Optical Fiber Bundles.

The development of sensitive methods for in situ detection of biomarkers is a real challenge to bring medical diagnosis a step forward. The proof-of-concept of a remote multiplexed biomolecular interaction detection through a plasmonic optical fiber bundle is demonstrated here. The strategy relies on a fiber optic biosensor designed from a 300 µm diameter bundle composed of 6000 individual optical fibers. When appropriately etched and metallized, each optical fiber exhibits specific plasmonic properties. The surface plasmon resonance phenomenon occurring at the surface of each fiber enables to measure biomolecular interactions, through the changes of the retro-reflected light intensity due to light/plasmon coupling variations. The functionalization of the microstructured bundle by multiple protein probes was performed using new polymeric 3D-printed microcantilevers. Such soft cantilevers allow for immobilizing the probes in micro spots, without damaging the optical microstructures nor the gold layer. We show here the potential of this device to perform the multiplexed detection of two different antibodies with limits of detection down to a few tenths of nanomoles per liter. This tool, adapted for multiparametric, real-time, and label free monitoring is minimally invasive and could then provide a useful platform for in vivo targeted molecular analysis.

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.

Highly parallel remote SPR detection of DNA hybridization by micropillar optical arrays.

Remote detection by surface plasmon resonance (SPR) is demonstrated through microstructured optical arrays of conical nanotips or micropillars. Both geometries were fabricated by controlled wet chemical etching of bundles comprising several thousands of individual optical fibers. Their surface was coated by a thin gold layer in order to confer SPR properties. The sensitivity and resolution of both shapes were evaluated as a function of global optical index changes in remote detection mode performed by imaging through the etched optical fiber bundle itself. With optimized geometry of micropillar arrays, resolution was increased up to 10-4 refractive index units. The gold-coated micropillar arrays were functionalized with DNA and were able to monitor remotely the kinetics of DNA hybridization with complementary strands. We demonstrate for the first time highly parallel remote SPR detection of DNA via microstructured optical arrays. The obtained SPR sensitivity combined with the remote intrinsic properties of the optical fiber bundles should find promising applications in biosensing, remote SPR imaging, a lab-on-fiber platform dedicated to biomolecular analysis, and in vivo endoscopic diagnosis. Graphical abstract We present a single fabrication step to structure simultaneously all the individual cores of an optical fiber bundle composed of thousands of fibers. The resulting sensor is optimized for reflection mode (compatible with in vivo applications) and is used to perform for the first time highly parallel remote SPR detection of DNA via several thousands of individual optical fiber SPR sensors paving the way for multiplexed biological detection.

Highly-Selective Optoelectronic Nose Based on Surface Plasmon Resonance Imaging for Sensing Volatile Organic Compounds.

Monitoring volatile organic compounds (VOCs) is an important issue, but difficult to achieve on a large scale and on the field using conventional analytical methods. Electronic noses (eNs), as promising alternatives, are still compromised by their performances due to the fact that most of them rely on a very limited number of sensors and use databases devoid of kinetic information. To narrow the performance gap between human and electronic noses, we developed a novel optoelectronic nose, which features a large sensor microarray that enables multiplexed monitoring of binding events in real-time with a temporal response. For the first time, surface plasmon resonance imaging is demonstrated as a promising novel analytical tool for VOC detection in the gas phase. By combining it with cross-reactive sensor microarrays, the obtained optoelectronic nose shows a remarkably high selectivity, capable of discriminating between homologous VOCs differing by only a single carbon atom. In addition, the optoelectronic nose has good repeatability and stability. Finally, the preliminary assays using VOC binary and ternary mixtures show that it is also very efficient for the analysis of more complex samples, opening up the exciting perspective of applying it to "real-world" samples in diverse domains.

Linear Chain Formation of Split-Aptamer Dimers on Surfaces Triggered by Adenosine.

The detection of small molecules impacts various fields; however, their small size and low concentration are usually the cause of limitations in their detection. Thus, the need for biosensors with appropriate probes and signal amplification strategies is required. Aptamers are appropriate probes selected specifically against small targets such as adenosine. The possibility to split aptamers in parts led to original amplification strategies based on sandwich assays. By combining the self-assembling of oligonucleotide dimers with split-aptamer dangling ends and a surface plasmon resonance imaging technique, we developed an original amplification approach based on linear chain formation in the presence of the adenosine target. In this article, on the basis of sequence engineering, we analyzed its performance and the effect of the probe grafting density on the length of the chains formed at the surface of the biosensor.

A Versatile Electronic Tongue Based on Surface Plasmon Resonance Imaging and Cross-Reactive Sensor Arrays-A Mini-Review.

Nowadays, there is a strong demand for the development of new analytical devices with novel performances to improve the quality of our daily lives. In this context, multisensor systems such as electronic tongues (eTs) have emerged as promising alternatives. Recently, we have developed a new versatile eT system by coupling surface plasmon resonance imaging (SPRi) with cross-reactive sensor arrays. In order to largely simplify the preparation of sensing materials with a great diversity, an innovative combinatorial approach was proposed by combining and mixing a small number of easily accessible molecules displaying different physicochemical properties. The obtained eT was able to generate 2D continuous evolution profile (CEP) and 3D continuous evolution landscape (CEL), which is also called 3D image, with valuable kinetic information, for the discrimination and classification of samples. Here, diverse applications of such a versatile eT have been summarized. It is not only effective for pure protein analysis, capable of differentiating protein isoforms such as chemokines CXCL12α and CXCL12γ, but can also be generalized for the analysis of complex mixtures, such as milk samples, with promising potential for monitoring the deterioration of milk.

On the use of aptamer microarrays as a platform for the exploration of human prothrombin/thrombin conversion.

Microarrays are particular biosensors with multiple grafted probes that are generally used for parallel and simultaneous detection of various targets. In this study, we used microarrays with aptamer probes in order to follow up the different biomolecular interactions of a single enzyme, the thrombin protein, involved in the complex coagulation cascade. More precisely, thanks to label-free surface plasmon resonance imaging, we were able to monitor in real time an important step in the firing of the coagulation cascade in situ-the enzymatic transformation of prothrombin into thrombin, catalyzed by factor Xa. We were also able to appraise the influence of other biochemical factors and their corresponding inhibiting or enhancing behaviors on thrombin activation. Our study opens the door for the development of a complete microarray-based platform not only for the whole coagulation cascade analysis but also for novel drug screening assays in pharmacology.

Physico-chemical foundations underpinning microarray and next-generation sequencing experiments.

Hybridization of nucleic acids on solid surfaces is a key process involved in high-throughput technologies such as microarrays and, in some cases, next-generation sequencing (NGS). A physical understanding of the hybridization process helps to determine the accuracy of these technologies. The goal of a widespread research program is to develop reliable transformations between the raw signals reported by the technologies and individual molecular concentrations from an ensemble of nucleic acids. This research has inputs from many areas, from bioinformatics and biostatistics, to theoretical and experimental biochemistry and biophysics, to computer simulations. A group of leading researchers met in Ploen Germany in 2011 to discuss present knowledge and limitations of our physico-chemical understanding of high-throughput nucleic acid technologies. This meeting inspired us to write this summary, which provides an overview of the state-of-the-art approaches based on physico-chemical foundation to modeling of the nucleic acids hybridization process on solid surfaces. In addition, practical application of current knowledge is emphasized.

Optical fiber biosensors toward in vivo detection.

This review takes stock of the various optical fiber-based biosensors that could be used for in vivo applications. We discuss the characteristics that biosensors must have to be suitable for such applications and the corresponding transduction modes. In particular, we focus on optical fiber biosensors based on fluorescence, evanescent wave, plasmonics, interferometry, and Raman phenomenon. The operational principles, implemented solutions, and performances are described and debated. The different sensing configurations, such as the side- and tip-based fiber biosensors, are illustrated, and their adaptation for in vivo measurements is discussed. The required implementation of multiplexed biosensing on optical fibers is shown. In particular, the use of multi-fiber assemblies, one of the most optimal configurations for multiplexed detection, is discussed. Different possibilities for multiple localized functionalizations on optical fibers are presented. A final section is devoted to the practical in vivo use of fiber-based biosensors, covering regulatory, sterilization, and packaging aspects. Finally, the trends and required improvements in this promising and emerging field are analyzed and discussed.

Investigation of the Affinity of Aptamers for Bacteria by Surface Plasmon Resonance Imaging Using Nanosomes.

The cell-SELEX method enables efficient selection of aptamers that bind whole bacterial cells. However, after selection, it is difficult to determine their binding affinities using common screening methods because of the large size of the bacteria. Here we propose a simple surface plasmon resonance imaging method (SPRi) for aptamer characterization using bacterial membrane vesicles, called nanosomes, instead of whole cells. Nanosomes were obtained from membrane fragments after mechanical cell disruption in order to preserve the external surface epitopes of the bacterium used for their production. The study was conducted on Bacillus cereus (B. cereus), a Gram-positive bacterium commonly found in soil, rice, vegetables, and dairy products. Four aptamers and one negative control were initially grafted onto a biochip. The binding of B. cereus cells and nanosomes to immobilized aptamers was then compared. The use of nanosomes instead of cells provided a 30-fold amplification of the SPRi signal, thus allowing the selection of aptamers with higher affinities. Aptamer SP15 was found to be the most sensitive and selective for B. cereus ATCC14579 nanosomes. It was then truncated into three new sequences (SP15M, SP15S1, and SP15S2) to reduce its size while preserving the binding site. Fitting the results of the SPRi signal for B. cereus nanosomes showed a similar trend for SP15 and SP15M, and a slightly higher apparent association rate constant kon for SP15S2, which is the truncation with a high probability of a G-quadruplex structure. These observations were confirmed on nanosomes from B. cereus ATCC14579 grown in milk and from the clinical strain B. cereus J066. The developed method was validated using fluorescence microscopy on whole B. cereus cells and the SP15M aptamer labeled with a rhodamine. This study showed that nanosomes can successfully mimic the bacterial membrane with great potential for facilitating the screening of specific ligands for bacteria.

Advances in Amplification Methods for Biosensors.

Today, there is a rapidly growing demand for sensitive and selective biosensors in various domains, including environmental monitoring such as (waste)water control, detection of pollution for personal/public safety, agricultural/food safety and quality control, veterinary and medical diagnostics, etc [...].

Recent Advances on Peptide-Based Biosensors and Electronic Noses for Foodborne Pathogen Detection.

Foodborne pathogens present a serious issue around the world due to the remarkably high number of illnesses they cause every year. In an effort to narrow the gap between monitoring needs and currently implemented classical detection methodologies, the last decades have seen an increased development of highly accurate and reliable biosensors. Peptides as recognition biomolecules have been explored to develop biosensors that combine simple sample preparation and enhanced detection of bacterial pathogens in food. This review first focuses on the selection strategies for the design and screening of sensitive peptide bioreceptors, such as the isolation of natural antimicrobial peptides (AMPs) from living organisms, the screening of peptides by phage display and the use of in silico tools. Subsequently, an overview on the state-of-the-art techniques in the development of peptide-based biosensors for foodborne pathogen detection based on various transduction systems was given. Additionally, limitations in classical detection strategies have led to the development of innovative approaches for food monitoring, such as electronic noses, as promising alternatives. The use of peptide receptors in electronic noses is a growing field and the recent advances of such systems for foodborne pathogen detection are presented. All these biosensors and electronic noses are promising alternatives for the pathogen detection with high sensitivity, low cost and rapid response, and some of them are potential portable devices for on-site analyses.

Rapid prototyping of a polymer MEMS droplet dispenser by laser-assisted 3D printing.

In this work, we introduce a polymer version of a previously developed silicon MEMS drop deposition tool for surface functionalization that consists of a microcantilever integrating an open fluidic channel and a reservoir. The device is fabricated by laser stereolithography, which offers the advantages of low-cost and fast prototyping. Additionally, thanks to the ability to process multiple materials, a magnetic base is incorporated into the cantilever for convenient handling and attachment to the holder of a robotized stage used for spotting. Droplets with diameters ranging from ∼50 µm to ∼300 µm are printed upon direct contact of the cantilever tip with the surface to pattern. Liquid loading is achieved by fully immersing the cantilever into a reservoir drop, where a single load results in the deposition of more than 200 droplets. The influences of the size and shape of the cantilever tip and the reservoir on the printing outcome are studied. As a proof-of-concept of the biofunctionalization capability of this 3D printed droplet dispenser, microarrays of oligonucleotides and antibodies displaying high specificity and no cross-contamination are fabricated, and droplets are deposited at the tip of an optical fiber bundle.