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.

Detection of Listeria monocytogenes in foods with a textile organic electrochemical transistor biosensor.

Foods contaminated by pathogens are responsible for foodborne diseases which have socioeconomic impacts. Many approaches have been extensively investigated to obtain specific and sensitive methods to detect pathogens in food, but they are often not easy to perform and require trained personnel. This work aims to propose a textile organic electrochemical transistor-based (OECT) biosensor to detect L. monocytogenes in food samples. The analyses were performed with culture-based methods, Listeria Precis™ method, PCR, and our textile OECT biosensor which used poly(3,4-ethylenedioxythiophene) (PEDOT):polystyrene sulfonate (PSS) (PEDOT:PSS) for doping the organic channel. Atomic force microscopy (AFM) was used to obtain topographic maps of the gold gate. The electrochemical activity on gate electrodes was measured and related to the concentration of DNA extracted from samples and hybridized to the specific capture probe immobilized onto the gold surface of the gate. This assay reached a limit of detection of 1.05 ng/µL, corresponding to 0.56 pM of L. monocytogenes ATCC 7644, and allowed the specific and rapid detection of L. monocytogenes in the analyzed samples. KEYPOINTS: • Textile organic electrochemical transistors functionalized with a specific DNA probe • AFM topographic and surface potential maps of a functionalized gold gate surface • Comparison between the Listeria monocytogenes Precis™ method and an OECT biosensor.

Enrichment Free qPCR for Rapid Identification and Quantification of Campylobacter jejuni, C. coli, C. lari, and C. upsaliensis in Chicken Meat Samples by a New Couple of Primers.

Campylobacter is the main cause of bacterial foodborne disease and poultry meat is the principal source of human infections. Rapid methods for Campylobacter detection are urgently needed to decrease high bacterial prevalence in poultry products. In this study, we developed new primers, CampyPFw and CampyPRv, that target the 16S-23S rRNA genes of Campylobacter jejuni, C. coli, C. lari and C. upsaliensis. The primers were tested on positive and negative reference strains in pure cultures and in inoculated poultry meat samples before their application in real-time PCR (qPCR) protocol for analyzing chicken meat samples. In parallel, the samples were tested by using the ISO 10272-1:2006 method. The qPCR protocol based on CampyPFw and CampyPRv showed good sensitivity, with the limit of detection of 4.6 × 102 cells/mL in chicken samples without enrichment steps.

Advances in Nanomaterials-Based Electrochemical Biosensors for Foodborne Pathogen Detection.

Electrochemical biosensors utilizing nanomaterials have received widespread attention in pathogen detection and monitoring. Here, the potential of different nanomaterials and electrochemical technologies is reviewed for the development of novel diagnostic devices for the detection of foodborne pathogens and their biomarkers. The overview covers basic electrochemical methods and means for electrode functionalization, utilization of nanomaterials that include quantum dots, gold, silver and magnetic nanoparticles, carbon nanomaterials (carbon and graphene quantum dots, carbon nanotubes, graphene and reduced graphene oxide, graphene nanoplatelets, laser-induced graphene), metal oxides (nanoparticles, 2D and 3D nanostructures) and other 2D nanomaterials. Moreover, the current and future landscape of synergic effects of nanocomposites combining different nanomaterials is provided to illustrate how the limitations of traditional technologies can be overcome to design rapid, ultrasensitive, specific and affordable biosensors.

Highly sensitive detection of Campylobacter spp. In chicken meat using a silica nanoparticle enhanced dot blot DNA biosensor.

Paper-based DNA biosensors are powerful tools in point-of-care diagnostics since they are affordable, portable, user-friendly, rapid and robust. However, their sensitivity is not always as high as required to enable DNA quantification. To improve the response of standard dot blots, we have applied a new enhancement strategy that increases the sensitivity of assays based on the use of biotinylated silica-nanoparticles (biotin-Si-NPs). After immobilization of a genomic Campylobacter DNA onto a paper membrane, and addition of a biotinylated-DNA detection probe, hybridization was evidenced using streptavidin-conjugated to horseradish peroxidase (HRP) in the presence of luminol and H2O2. Replacement of the single biotin by the biotin-Si-NPs boosted on average a 30 fold chemiluminescent read-out of the biosensor. Characterization of biotin-Si-NPs onto a paper with immobilized DNA was done using a scanning electron microscope. A limit of detection of 3 pg/µL of DNA, similar to the available qPCR kits, is achieved, but it is cheaper, easier and avoids inhibition of DNA polymerase by molecules from the food matrices. We demonstrated that the new dot blot coupled to biotin-Si-NPs successfully detected Campylobacter from naturally contaminated chicken meat, without needing a PCR step. Hence, such an enhanced dot blot paves the path to the development of a portable and multiplex paper based platform for point-of-care screening of chicken carcasses for Campylobacter.

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.

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.

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.