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  • Open access
  • 103 Reads
Improving the analytical performance of weak aptamers: DNA isothermal amplification approaches

Cancer diagnosis based on serum biomarkers, so-called liquid biopsy, is the most desirable but also the most challenging approach to diagnose cancer. This method relies on the presence of specific biomarkers in the accessible body fluids of cancer patients. However, many of these biomarkers are glycoproteins found in low concentration levels in a high protein content medium, the serum, thus hindering their detection. For this reason, biomarker detection requires strategies to boost sensitivity. Nucleic acid-based receptors such as aptamers are especially appropriate for this task because they can be easily manipulated with molecular biology tools such as polymerases and ligases, so they can be integrated in analytical schemes forbidden to other receptors like antibodies.

Affinity characterization is an essential but time-consuming task to develop reliable aptamers for tumor biomarker detection and is not always thoroughly addressed. For neutrophil gelatinase-associated lipocalin (NGAL), a potential biomarker of pancreatic cancer, two DNA aptamers were described with very different affinity. Likewise, another pair of DNA aptamers was developed with very different affinity for alpha-fetoprotein (AFP), a biomarker of hepatocellular carcinoma (HCC). In this work, we estimate the dissociation constant of these aptamers by means of a direct assay on magnetic beads modified with biomarker and electrochemical detection on screen-printed carbon electrodes (SPCE). In order to improve the performance of these aptamers, we proposed the isothermal amplification of the aptamers for both biomarkers by rolling circle amplification (RCA). In the case of AFP aptamers, we also tried terminal deoxynucleotidyl transferase (TdT), a template-independent amplification. Both DNA amplifications improved the sensitivity and the apparent binding constants of the aptamers for the two cancer biomarkers. Nevertheless, this improvement depends on the true affinity of the binding pair, which ultimately limits their analytical usefulness.

  • Open access
  • 114 Reads
Intracellular sensing by a survivin molecular beacon coupled to PMMA nanoparticles in human cancer cells

In this work biocompatible polymethylmethacrylate nanoparticles (PMMA-NPs) were used as carrier of a molecular beacon (MB) for sensing survivin mRNA in cancer cells. MBs are oligonucleotide sequences generating a fluorescent signal when they hybridize with their target. They constitute potential theranostic agents as they can act at the same time as sensors, able to detect endogenous nucleic acids, and as drug, by silencing the target mRNA. NPs offer numerous advantages over conventional drug delivery approaches, such as the possibility of multiple functionalization for improving the imaging, diagnosis and targeted therapy. In particular, PMMA-NPs used in this study consist of a hydrophobic PMMA core covalently functionalized with fluorescein and an external hydrophilic shell decorated with primary amine groups and quaternary ammonium salts.

The aim of the work was to evaluate by confocal microscopy and fluorescence measurements: a) the ability of PMMA-NPs to promote, in human A549 cancer cells, the internalization of a MB specific for survivin mRNA; b) the involvement of endocytosis in the NP uptake; c) the NP fate at different times of cell incubation to verify their localization in lysosomes; d) the MB localization on the Endoplasmic Reticulum (ER) where the target mRNA is located.

The results obtained demonstrated: a) PMMA-NPs efficiently promote the MB internalization generating a specific fluorescent signal in the presence of survivin mRNA expression; b) the involvement of endocytosis in the NP uptake; c) the NP localization in lysosomes at different times of cell incubation and their subsequent release in the cell culture medium; d) the MB fluorescence localization in proximity of the ER where the target mRNA is presumably located.

  • Open access
  • 83 Reads
Single drop detection of furfural in wine by an SPR sensor based on molecularly imprinted polymer as biomimetic receptor

A surface plasmon resonance (SPR) platform, based on a D-shaped plastic optical fiber (POF), combined with a biomimetic receptor, i.e. a molecularly imprinted polymer (MIP), has been proposed by our research group. It is an easy to use and cheap device for chemical sensing of substances of interest in different fields, as health, environment and industry. The possibility of performing single drop measurements is a further favourable aspect for practical applications. We proposed the use of the SPR-MIP sensor for food industry controls, i.e. the analysis of 2-furaldheide (2-FAL) in fermented beverages as wine. The platform is based on a multimode POF with a characteristic D-shaped sensing region, which makes it possible to perform the measurement in a drop simply deposited over the flat surface. It presents, on the exposed POF core, a multilayer configuration with a buffer layer (a photoresist of high refractive index, 1.5 μm thick), a thin metal film (gold, 60 nm thick) and a MIP layer as a specific chemical receptor. The MIP was built up in situ, dropping over the gold layer the prepolymeric mixture and allowing polymerization. In the SPR here described the resonance wavelength is determined exploiting a white light source and a spectrometer. The sorption curve in aqueous solution, at constant pH, was modelled by the Langmuir equation, with a constant response for concentrations higher than 1 mg/L, corresponding to the saturation of the receptor. The affinity constant, Kaff, in water is 4.8 L/mg. The LOD in water was 0.03 mg/L, but a somewhat higher value was determined in a white wine, due to the effect of the complex matrix on Kaff. Despite the slightly higher LOD, the sensor is suitable for 2-FAL analysis in wines.

  • Open access
  • 97 Reads
Systematic Derivation of New Fields of Application for Innovative Bio-based Odour Sensors with Transfected Cells and Analysis of Economic Potentials

In this paper specific market potentials for innovative odour sensors with transfected cells are identified. A novel cell-based platform technology for odor sensors is described and related to existing competitive technologies regarding their specific properties, advantages and disadvantages. This classification enables an assessment of the specific market potential for this new technology and shows future perspectives that result from the properties. The new technology enables the development of new fields of application for odor sensors, the economic potential of which has not yet been adequately investigated. The respective fulfillment of defined requirements is therefore evaluated by means of selected technical criteria in order to determine performance profiles for each technology. Further, existing fields of application for odour sensors are identified and new ones are systematically derived. For the selection of application fields, the importance of the defined criteria is compared, which leads to specific requirement profiles. The comparison of performance and requirement profiles provides information about specific fields of application for cell-based odour sensors and their economic potential is estimated. The technology assessment was carried out with the involvement of numerous experts from German research in the field of odor and taste sensorics as well as technology development for this new type of cell-based sensors.

  • Open access
  • 97 Reads
Comparison of nanosized markers in lateral flow immunoassay of antibiotic lincomycin

Improving the sensitivity of the competitive lateral flow immunoassay (LFIA) is important, given the increasing demands for the monitoring of chemical contaminants in food. The choice of nanosized marker is an essential task for improving the LFIA sensitivity. In this study, a CdSe/ZnS quantum dot (QD)-based LFIA combined with a portable reader was developed for rapid and quantitative detection of an antibiotic lincomycin (LIN). The performance of the proposed fluorescence LFIA was compared to the conventional gold nanoparticle (AuNP)-based LFIA realized with the same immunoreagents. The visual cutoff values were 10 ng/mL for AuNP-based LFIA and 20 ng/mL for QD-based LFIA. Furthermore, the instrumental limits of detection have been shown to be comparable for both nanosized markers and amounted to 0.4 ng/mL for AuNPs and 0.2 ng/mL for QDs, respectively. According to the results obtained, both LFIAs may be used for rapid, cost-effective, on-site testing of antibiotics, in particular LIN. However, the QD-based LFIA exhibits lowest limit of detection with the least immunoreagent consumption, that makes it economically beneficial.

  • Open access
  • 78 Reads
Precollaborative validation of an amperometric immunosensor for Salmonella
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Salmonella is considered an important public health issue and one of the main causes of outbreaks involving several food matrices. The method of Salmonella detection recommended is cultural and it is laborious, presents a high consumption of material, and requires about five days for presumptive results. Immunosensor is an interesting tool for microbiologic analysis that has shown promising and rapid results, although many devices have their performance evaluated only under buffering conditions and few achieve the validation stage. The objective was to perform a precollaborative validation of an electrochemical immunosensor assembled on screen-printed electrodes for the detection of Salmonella sp. in milk. The antibodies were immobilized on the surface by cysteamine self-assembled-monolayer (SAM). The sandwich-type amperometric immunosensor was evaluated for contaminated raw and whole UHT milk and compared to performance with a gold standard reference method (BAM) for microbiological analysis according to AOAC recommendations for a single laboratory. The immunosensor had a qualitative performance and a binary response (positive/ negative) was used based on a cut off established from current electric obtained for the absence of the pathogen. There was no significant difference for the results of the biosensor and the reference method, in the absence and the levels of 101 UFC mL-1 and 103 UFC mL-1 of Salmonella Typhimurium for the two types of milk. This result indicates the efficiency of the biosensor in detecting the pathogen into a complex matrix.

  • Open access
  • 73 Reads
Highly multiplexed label-free imaging sensor for accurate quantification of small molecule binding kinetics

Investigating the binding kinetics of small molecule analytes to larger ligands, such as proteins and antibodies, is a compelling task for the field of drug and biomarker development, as well as the food industry and agro-biotechnology. In 2019, the FDA approved 48 new drugs, 73% of which belong to the category of small molecules, with a molecular weight (MW) below 1KDa. On the same level, most of the fungi-produced toxins that are commonly known to affect crops also fit this description. Here, we improve the limit of detection of the Interferometric Reflectance Imaging Sensor (IRIS), a label-free, highly-multiplexed biosensor, to perform real-time affinity measurement of small molecules binding to immobilized antibodies in a microarray format. As the analytes bind to the surface probes, the biomass accumulation on the surface is quantified by measuring the optical reflectance from the layered Si/SiO2 chip through the solution, in a common-path interferometer configuration. As a proof of concept, label-free detection of biotin molecules binding to immobilized streptavidin probes is performed, achieving 1pg/mm2 sensitivity through signal averaging in a shot noise limited operation. Furthermore, we apply the optimized sensor to the screening of a 20-multiplexed antibody chip (MW~150kDa ligands) against Fumonisin B1 (MW = 721.8Da), one of the most prevalent mycotoxins found in many cereal grains such as corn and wheat. The simultaneously recorded binding curves of the toxin to the multiplexed sensor yield a signal-to-noise ratio of ≈8 when noise reduction methods of spatial and temporal averaging are utilized.

  • Open access
  • 137 Reads
Microfluidic impedance biosensor chip with DNA-based self-assembled monolayers for label-free detection of cardiac biomarker troponin I
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A microfluidic chip for electrochemical impedance spectroscopy (EIS) is presented as biosensor for the detection of cardiac troponin I (cTnI), which is one of the most specific diagnostic serum biomarkers for myocardial infarction. Impedimetric biosensors enable the detection of a variety of analytes, including small molecules, proteins, and cells. As analyte detection is direct and label-free, they allow fast detection of biomarkers, which is essential in the diagnosis of cardiac infarctions to promote a positive outcome. The EIS chip presented here consists of a microscope glass slide serving as base plate, sputtered electrodes, and a polydimethylsiloxane (PDMS) microchannel. The electrode design mainly consists of a working electrode and a counter electrode made of gold. A silver reference electrode can be included, if required. Protocols for electrode functionalization were developed considering a low initial impedance in addition to analyte-specific binding by corresponding antibodies and reduction of non-specific protein adsorption to prevent false-positive signals. Reagents tested for self-assembled monolayers (SAM) on gold electrodes included hydrocarbons with thiol groups and thiolated oligonucleotides. The optimized coating used thiolated single-strand DNA (ssDNA) and 1,4-benzenedithiol on the working electrode and 1,4-benzenedithiol on the counter electrode. After hybridization with corresponding ssDNA carrying an amino group, the reaction with glutaric anhydride led to carboxyl groups, on which anti-cTnI antibody was covalently coupled using carbodiimide chemistry. The PDMS microchannel was bonded on the glass slide with the functionalized electrodes, and the completed EIS chip was connected to the readout system. Sampling with human serum albumin (HSA), 1000 ng/mL, led to negligible signal changes, while sampling with cTnI, 1 ng/mL, led to a significant signal shift in the Nyquist plot. Sampling and measurement took only a few minutes. The results were promising regarding a future cost-effective biosensor array chip for the rapid detection of clinically relevant biomarkers in real samples.

  • Open access
  • 95 Reads
Polymeric magnetic microparticles as electrochemical immunosensing platforms
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Magnetic microparticles (MMPs) have been notably used as platforms in biosensing. Due to their magnetic behavior, they simplify purification and separation procedures, reducing time of analysis They also allow sample preconcentration, minimizing matrix effects, what is of key relevance for applications using real samples. Even though there is a great number of commercially available MMPs, their performance is not always reliable. In this work we propose the synthesis of novel polymeric MMPs for their use as electrochemical immunosensing platforms. Initially, magnetic nanoparticles of a diameter of 12 ± 2 nm and a saturation magnetization of 70 emu/g were synthesized and characterized. Then, they were encapsulated in a polymeric matrix of poly(lactic-co-glycolic) acid (PLGA), generating MMPs with a diameter of 90 ± 17 nm. Later, MMPs were functionalized with polyethyleneimine (PEI) as an intermediate step for the immobilization of affinity proteins or antibodies, necessary in electrochemical immunosensing. This would allow the obtaining of MMPs comparable to the commercially available ones but possessing higher saturation magnetization. The use of such MMPs could facilitate the detection of analytes of interest in diagnostics, among other applications.

  • Open access
  • 206 Reads
Fully graphene-based electrode platforms for biosensing applications
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A simple, fast, and reproducible analyte detection using low amounts of solution is nowadays possible thanks to the presence on the market of disposable screen-printed electrodes: they are widely employed in academic research as well as in industrial applications, as they are suited for a wide range of purposes. They are available in different geometries, on flexible or solid supports and with working electrodes consisting of conventional and non-conventional materials; in particular, different nanosized materials can be included in the printed ink or added as a coating afterwards. Among the variety of nanomaterials proposed to impart effectiveness to the sensor response, graphene derivatives are increasingly exploited in electrochemical biosensing, since their nanosized dimensions allow a great number of oxidized residues to be well exposed to the surrounding environment; these moieties are responsible for the activation of electrocatalytic processes toward several species, i.e. a decrease of the oxidation or reduction potentials of the analyte with respect to pristine carbon electrodes. To fully exploit the electrocatalytic performance of this nanosized material, we developed flexible, self-standing three-electrode cells entirely made of graphene. They were realised from pristine graphene paper (G-paper), a flexible, electrically conductive, paper-like material which has a large surface area, high porosity and can be easily obtained in different shapes. We demonstrated that G-paper electrodes can be successfully employed in electrochemical sensing, thanks to electrochemical tests with a benchmark redox species. In addition, their electrocatalytic properties were studied towards the detection of the two main products of enzymatic reactions, namely nicotinamide adenine dinucleotide (NADH), a co-factor for many dehydrogenase-type enzymes, and of H2O2, a product of many oxidase-based enzymatic reactions. These tests demonstrate the possible advantages in the use of these new devices with respect to those present on the market.