Development of localized surface plasmon resonance biosensors for the detection of Brettanomyces bruxellensis in wine

Incident light interacting with noble-metal nanoparticles with smaller sizes than the wavelength of the incident light induces localized surface plasmon resonance (LSPR). In this work a gold nanostructured surface was used for the immobilization of a 5\u2032 end Thiol modified DNA probe to develop a LSPR nanobiosensor for the detection of the spoiler wine yeast Brettanomyces bruxellensis. Gold was evaporated to obtain a gold thickness of 4 nm. DNA (2 \u3bcL) from the target microorganism and the negative control at various concentrations were used to test the specificity and sensitivity of the LSPR technique. Changes in the optical properties of the nanoparticles due to DNA-probe binding are reflected in the shift of LSPR extinction maximum (\u3bbmax). The results obtained using as target microorganism B. bruxellensis, and as negative control Saccharomyces cerevisiae demonstrated the specificity of both the DNA-probe and the protocol. The LSPR spectrophotometry technique detects 0.1 ng/\u3bcL DNA target confirming the possibility to utilize this system for the detection of pathogen microorganisms present in low amount in food and beverage samples. \ua9 2015 Elsevier B.V. All rights reserved

Light-Up Split Broccoli Aptamer as a Versatile Tool for RNA Assembly Monitoring in Cell-Free TX-TL Systems, Hybrid RNA/DNA Origami Tagging and DNA Biosensing

Binary light-up aptamers are intriguing and emerging tools with potential in different fields. Herein, we demonstrate the versatility of a split Broccoli aptamer system able to turn on the fluorescence signal only in the presence of a complementary sequence. First, an RNA three-way junction harbouring the split system is assembled in an E. coli-based cell-free TX-TL system where the folding of the functional aptamer is demonstrated. Then, the same strategy is introduced into a ‘bio-orthogonal’ hybrid RNA/DNA rectangle origami characterized by atomic force microscopy: the activation of the split system through the origami self-assembly is demonstrated. Finally, our system is successfully used to detect the femtomoles of a Campylobacter spp. DNA target sequence. Potential applications of our system include the real-time monitoring of the self-assembly of nucleic-acid-based devices in vivo and of the intracellular delivery of therapeutic nanostructures, as well as the in vitro and in vivo detection of different DNA/RNA targets

Point-of-Need DNA Testing for Detection of Foodborne Pathogenic Bacteria

Foodborne pathogenic bacteria present a crucial food safety issue. Conventional diagnostic methods are time-consuming and can be only performed on previously produced food. The advancing field of point-of-need diagnostic devices integrating molecular methods, biosensors, microfluidics, and nanomaterials offers new avenues for swift, low-cost detection of pathogens with high sensitivity and specificity. These analyses and screening of food items can be performed during all phases of production. This review presents major developments achieved in recent years in point-of-need diagnostics in land-based sector and sheds light on current challenges in achieving wider acceptance of portable devices in the food industry. Particular emphasis is placed on methods for testing nucleic acids, protocols for portable nucleic acid extraction and amplification, as well as on the means for low-cost detection and read-out signal amplification

Electrochemical and Optical Biosensors for the Detection of Campylobacter and Listeria: An Update Look

Foodborne safety has aroused tremendous research interest in recent years because of a global public health problem. The rapid and precise detection of foodborne pathogens can reduce significantly infection diseases and save lives by the early initiation of an effective treatment. This review highlights current advances in the development of biosensors for detection of Campylobacter spp. and Listeria monocytogenes that are the most common causes of zoonosis. The consumption of pathogen contaminated food is responsible for humans hospitalization and death. The attention focused on the recognition elements such as antibodies (Ab), DNA probes and aptamers able to recognize cells, amplicons, and specific genes from different samples like bacteria, food, environment and clinical samples. Moreover, the review focused on two main signal-transducing mechanisms, i.e., electrochemical, measuring an amperometric, potentiometric and impedimetric signal; and optical, measuring a light signal by OLED (Organic Light Emitting Diode), SPR (Surface Plasmon Resonance), and Optical fiber. We expect that high-performance of devices being developed through basic research will find extensive applications in environmental monitoring, biomedical diagnostics, and food safety

Development and Evaluation of qPCR Detection Method and Zn-MgO/Alginate Active Packaging for Controlling Listeria monocytogenes Contamination in Cold-Smoked Salmon

To answer to food industry requests to monitor the presence of L. monocytogenes in cold-smoked salmon samples and to extend their shelf-life, a qPCR protocol for the detection of L. monocytogenes, and an antibacterial active packaging reinforced with zinc magnesium oxide nanoparticles (Zn-MgO NPs) were developed. The qPCR allowed the sensitive and easy detection of L. monocytogenes in naturally contaminated samples, with specificity in full agreement with the standard methods. The halo diffusion study indicated a high antibacterial efficiency of 1 mg/mL Zn-MgO NPs against L. monocytogenes, while the flow cytometry showed only moderate cytotoxicity of the nanoparticles towards mammalian cells at a concentration above 1 mg/mL. Thus, the novel active packaging was developed by using 1 mg/mL of Zn-MgO NPs to reinforce the alginate film. Cold-smoked salmon samples inoculated with L. monocytogenes and air-packed with the Zn-MgO NPs-alginate nanobiocomposite film showed no bacterial proliferation at 4 °C during 4 days. In the same condition, L. monocytogenes growth in control contaminated samples packed with alginate film alone. Our results suggest that Zn-MgO nanoparticles can extend the shelf-life of cold-smoked salmon samples

PROBE FOR THE DETECTION OF BACTERIAL INFECTIONS

The present invention relates to a nucleotide probe specific for Campylobacter jejuni, Campylobacter coli, Campylobacter lari and Campylobacter upsaliensis, its uses, and methods for detecting said microorganisms in a sample of interest, and kits comprising said probe and suitable reagents

METHOD AND SYSTEM FOR DETERMINING THE PRESENCE AND/OR AMOUNT OF AT LEAST ONE ANALYTE THAT MAY BE CONTAINED IN A SAMPLE

The present invention relates to a method for determining the presence and/or amount of at least one analyte (T) that may be contained in a sample. The method comprises: (a) a recognition step during which said sample (E) is placed in the presence of at least one primary probe (1), in order to form, if necessary, a primary complex (C1), (b) a development step during which at least one developing reagent is added, capable of forming, starting from the primary probe (1) of said primary complex (C1), a secondary complex (C2), said at least one developing reagent comprising: - at least one amplification probe (3), of the core (31) - shell (32) type, wherein said shell (32) comprises several molecules of an affinity tag (321), and - a tracer (21) for converting the secondary complex (C2) into a visible and/or measurable signal, said tracer (21) being bound or intended to bind with the affinity tag (321) of said at least one amplification probe (3), so that in said secondary complex (C2) at least some of the molecules of said affinity tag (321) of said at least one amplification probe (3) are bound with said tracer (21)

Development and application of molecular probes

The development of DNA probes started from 1950's for diagnostic purposes and it is still 16 growing. DNA probes are applied in several fields such as food, medical, veterinary, environment 17 and security, with the aim of prevention, diagnosis and treatment. The use of DNA probes permits 18 microorganism identification, including pathogen detection, and their quantification when used in 19 specific systems. Various techniques obtained success by the utilization of specific DNA probes, that 20 allowed the obtainment of rapid and specific results. From PCR, qPCR and blotting techniques that 21 were first used in well equipped laboratories to biosensors such as fiber optic, surface plasmon 22 resonance (SPR), electrochemical, and quartz crystal microbalance (QCM) biosensors that use 23 different transduction systems

Optimization of the protocol for the Brettanomyces bruxellensis detection using LSPR phenomenon with AuNPs.

The yeast Brettanomyces bruxellensis, is well-known in the oenological field for being the cause of significant defects in wine called \u201cBrett character\u201d. B. bruxellensis is responsible for contamination of cellars that lead to consequent economic losses. Classical plate methods and molecular techniques such as PCR, nested-PCR, RFLP, FISH and dot blot are used for its detection. Plate techniques are timeconsuming, PCR bases on enzyme acitivties, thus it can fail due to the presenvce of polyphenols, and hybridization based techniques cannot reach high sensitive. Localized Surface Plasmon Resonance (LSPR) has been used to build a LSPR label-free optical biosensor in connection with gold nanoparticle substrate (Au NPs). The aim of this work was to test several protocols to optimize the best one that may become an important part of the routine analysis using B. bruxellensis like positive control and Saccharomyces cerevisiae as negative control. Buffer, hybridization time and probe concentration were optimized to give high sensitivity

Electrochemical and Optical Biosensors for the Detection of Campylobacter and Listeria: An Update Look

Foodborne safety has aroused tremendous research interest in recent years because of a global public health problem. The rapid and precise detection of foodborne pathogens can reduce significantly infection diseases and save lives by the early initiation of an effective treatment. This review highlights current advances in the development of biosensors for detection ofCampylobacter spp. and Listeria monocytogenes that are the most common causes of zoonosis. The consumption of pathogen contaminated food is responsible for humans hospitalization and death. The attention focused on the recognition elements such as antibodies (Ab), DNA probes and aptamers able to recognize cells, amplicons, and specific genes from different samples like bacteria, food, environment and clinical samples. Moreover, the review focused on two main signal-transducing mechanisms, i.e., electrochemical, measuring an amperometric, potentiometric and impedimetric signal; and optical, measuring a light signal by OLED (Organic Light Emitting Diode), SPR (Surface Plasmon Resonance), and Optical fiber. We expect that high-performance of devices being developed through basic research will find extensive applications in environmental monitoring, biomedical diagnostics, and food safet