Pathogenic bacterial contamination in food is a public health concern. It represents a health and safety consumer risk that could cause several diseases and even death. Currently, the food industry uses culture-based assays to determine the presence of pathogens as a gold standard method. Although this method is highly accurate, it is often time-consuming and expensive.^[1,2] In this regard, the development of biosensing platforms results as an alternative for the reduction of time and cost of pathogenic bacteria detection in food.^[2] In this work, we report the development of a single-step bacterial detection platform based on graphene oxide. Non-radiative energy transfer between graphene oxide coated microplates (GOMs) and photoluminescence bioprobes (PLBs) is presented in absence of the target analyte, but in presence of analyte, PLBs exhibit strong photoluminescence due to the distance between GOMs and PLBs. These PLBs are a quantum dot- antibody complex, thereby resulting as a biorecognition and interrogation element. Escherichia coli was used as model analyte. In optimal conditions, the bacterial detection platform reached a limit of detection around 2 CFU mL^-1 in 30 minutes, enabling a fast and sensitive alternative for bacterial detection. The biosensing platform was also used to test food industry samples achieving a qualitative response, that allows determining the presence of E. coli during the first 45 minutes of the assay. This biosensing strategy potentially offers a low-cost and quick option for the food industry to assure the quality of the product and consumer safety.^[3]

References

[1] M. Majdinasab, A. Hayat, J. L. Marty, TrAC Trends Anal. Chem. 2018, 107, 60.

[2] Y. Wang, T. V. Duncan, Food Biotechnol. • Plant Biotechnol. 2017, 44, 74.

[3] M. D. Avila-Huerta, E. J. Ortiz-Riaño, D. L. Mancera-Zapata, E. Morales-Narváez, Anal. Chem. 2020, DOI 10.1021/acs.analchem.0c02200.

Nowadays immunoassays are used to detect chemical or biological species; therefore, they are an essential tool in a wide range of applications such as drug development, clinical diagnostics, environmental monitoring or food quality control [1]. However, conventional immunoassays, such as ELISA (Enzyme-Linked ImmunoSorbent Assay), require several procedures such as blocking, separations and washing steps. Thus, it takes at least 6 hours to get the respective results. Besides, it involves two antibodies and a sensing surface attaching and labelling the biochemical target (analyte). Fluorescence Resonance Energy Transfer (FRET) is a very useful phenomena to improve immunosensing sensitivity and avoid cumbersome procedures due to its simplicity. Graphene and its derivatives have been used as acceptors in FRET due to their wide absorption spectra, which make them outstanding quenchers of fluorescence. With this in mind, we developed a novel and single-step biosensing platform based on fluorescence quenching caused by graphene oxide, which was used for the detection of two analytes: H-IgG (which is a type and also the most common antibody found in human blood circulation) and PSA (prostate specific antigen). A single antibody conjugated with a fluorophore (FITC for H-IgG detection and quantum dots for PSA detection) is used in the capture and detection processes. When the analyte and the antibody (conjugated with the fluorophore) are added, a kinetic analysis is performed throughout 2 hours with real-time interrogation of the respective fluorescence intensity, observing that the higher analyte concentration, the less quenching of fluorescence of the immunosensing probe (antibody-fluorophore immunocomplex) due to the low affinity and the relatively long distance between GOµWs (microwells plate coated with Graphene Oxide) and immunosensing probe [2].

References

[1] In Introduction to Biophotonics, John Wiley & Sons, Ltd, 2004, pp. 311–356.

[2] E. Ortiz-Riaño, M. Avila-Huerta, D. Mancera-Zapata, E. Morales-Narváez, Biosensors and Bioelectronics 2020, 165, 112319.

Human food-borne diseases have been significantly increased in the last decades, causing numerous deaths, as well as money and time loss in the agri-food sector and food supply chain worldwide. The standard analyses that are currently used for bacteria detection have significant limitations regarding cost, special facilities, highly trained staff, and a long procedural time that can be crucial for foodborne pathogens with high hospitalization and mortality rates, such as Listeria monocytogenes. Improved and accurate techniques that provide fast detection are of great importance since it is very crucial to detect pathogenic microorganisms and withdraw the contaminated products from the markets before their distribution to consumers. Aim of this study was to develop a biosensor able to perform robust and accurate detection of L. monocytogenes in various food substrates within 3 minutes. For this purpose, a cell-based biosensor technology (BERA) and a portable device developed by EMBIO Diagnostics called B.EL.D (Bio Electric Diagnostics), were used. Biosensors were created for L. monocytogenes detection using anti-Listeria monocytogenes antibodies and tests were conducted in ready-to-eat lettuce salads, milk, and halloumi cheese samples. Results indicated that the biosensor managed to differentiate samples with and without Listeria with 90%, 89% and 91% accuracy in ready-to-eat lettuce salads, milk, and halloumi samples, respectively, after a primary enrichment step. Method’s sensitivity, specificity, positive and negative predictive values ranged from 83-95%, while the limit of detection was determined to be 10² CFU mL^-1 or g^-1 in all food substrates.

Veterinary drugs could contaminate animal derived food products for human consumption. Some antibiotic residues (eg. chloramphenicol, nitrofuran metabolites) are banned in foodstuffs of animal origin (eg. milk, honey, etc.) in European Union because of toxicological risks for the consumer. Screening methods are the first stage of food control and so are essential for food safety monitoring. There is always a need to develop novel screening methods for antibiotic residues detection, preferably with the potential for the field-testing (eg. farm control, self-control). Electrochemical biosensors make it possible to develop a promising and economically interesting approach.

An innovative and cheap electrochemical method based on disposable Screen Printed Carbon Electrodes (SPCE), coupled to magnetic beads (MB), allowing the simultaneous detection of 3 families of antibiotics in milk, was published by a Spanish academic team [1]. This system was applied to develop a screening method for banned antibiotic residues in honey. We faced two major issues: firstly, the very low levels of residues to be reach (ie. Regulatory limits usually below 1 µg/kg), secondly the complexity of honey matrix. There is not a single honey matrix, but a wide variety of honeys. Honey composition and colour varies considerably depending on the botanical origin. Moreover some honey ingredients can interfere with the electrochemical detection, especially substances with antioxidant activities (eg. polyphenols).

Therefore in parallel with the optimization of the electrochemical method to reach the required sensitivity, a lot of work had to be done to improve sample extraction to reduce matrix effects. The results will be presented to the conference, discussing the advantages and drawbacks of amperometric biosensors for the screening of antibiotic residues in food products.

1. Conzuelo F, Ruiz-Valdepeñas Montiel V, Campuzano S, Gamella M, Torrente-Rodríguez RM, Reviejo AJ, Pingarrón JM. 2014. Anal. Chim. Acta. 820:32-38.

Lactate is a metabolite biomarker of tissue oxygenation and it can be used in medicine to evaluate a pathology or in sport activities to evaluate physical performance. Lactate level assessment is also important for the food industry. This acid is found in food and beverages and the concentration level can be correlated with the freshness, stability and quality of several products. In this work, we present a smartphone-based enzymatic biosensor utilizing the unique colorimetric properties of the poly(aniline-co-anthranilic acid) (ANI-co-AA) composite film coupled with lactate oxidase-horseradish peroxidase (LOx-HRP) enzymes. The enzymes are immobilized on the composite polymer film by adsorption and they catalyze a reversible redox color change of the host polymer from green to blue in the presence of L-lactate as the substrate. A smartphone was applied as color detector, for image acquisition and data handling. The free-of-charge ColorLab® application for Android OS was used to enable easy and clear display of the sensor’s response indicating remarkable changes in the optical features. The results were confirmed by spectrophotometric measurements. The developed colorimetric enzymatic biosensors were studied and optimized in relation to different experimental parameters. Moreover, the colorimetric enzymatic biosensors were applied to food and clinical analysis. It has been shown by these studies that the colorimetric biosensors are promising as quick and simple tests for handheld analysis in various fields.