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.

Carbendazim is a systemic, broad-spectrum benzimidazole-type fungicide effective against fungi that compromise the safety/quality of food products (fruits, vegetables, field crops, etc.). Though carbendazim constitutes a major environmental pollutant, hazardous for humans and animals and has been banned in most of European Union and USA, many countries still permit its production and use. Reliable determination of carbendazim levels in water, soil and food samples is therefore necessary to assure compliance with national/European regulations concerning MRLs and consequently minimize health risks for living organisms. In this work, an optical label-free white light reflectance spectroscopy (WLRS) biosensor for fast and sensitive determination of carbendazim is presented. The sensor employs an anti-carbendazim polyclonal antibody developed in-house against a cocktail of commercially available benzimidazole derivatives. The transducer employed is a Si chip with a 1-μm thick thermal SiO₂ on top, and is transformed to biosensing element through immobilization of a suitable benzimidazole-conjugate on the SiO₂ surface, while the determination is based on the competitive immunoassay format. For the assay, a mixture of the antibody with the calibrators or the samples is pumped over the chip surface followed by reaction with secondary biotinylated antibody and streptavidin. The WLRS biosensing platform allows for the label-free, real-time monitoring of biomolecular interactions carried out onto the SiO₂/Si chip by transforming the shift in the reflected interference spectrum caused by the immunoreaction to effective biomolecular adlayer thickness. After optimizing the assay parameters, the sensor was capable of real-time sensitive detection of carbendazim in buffer with LoD: 20 ng/mL, within 28 min total analysis time. The intra- and inter-assay CVs were ≤6.9% and ≤9.4%, respectively. The excellent analytical characteristics and short analysis time combined with its small size render the proposed WLRS biosensor ideal for future point-of-need determination of carbendazim in food and environmental samples.

Currently numerous studies are aimed at developing non-invasive technologies for analyzing blood glucose concentration. This opportunity will allow for the prevention and early diagnosis of the population to identify violations of carbohydrate metabolism, which will lead to a decrease in the amount of diabetes mellitus, as well as save huge costs associated with consumables. However, despite the intensive development of non-invasive blood glucose meters, this technology still does not exist. The complexity of the task is due to the huge number of factors affecting the reading of a non-invasive glucometer, which must be optimized to obtain an effective device. First of all, this is due to the presence of a protective skin and muscle cover of a human. As a rule, the skin and parameters of the internal environment introduce significant errors in the measured data. The authors of the project see an original solution to this problem in the study of the so-called near-field effect. The near field of the emitter penetrates deep enough since it does not experience significant absorption in the conductive medium. This will maximize the signal-to-noise ratio.

The work demonstrates a model of a near-field sensor, which has a high penetration of electromagnetic waves into highly absorbing media at a small sensor size (the diameter is 25 mm, the thickness is 0.76 mm). Based on the experimentally obtained data of the dielectric constant for glucose concentrations of 1.2-10 mmol/l, the reflected signal is shown during mathematical modeling of the sensor in the frequency range 0.5-5 GHz. The influence of other biological tissues (dermis, epidermis, fat layer, muscles) and their influence on the near field of the sensor and the received data are also demonstrated.

Cancer is associated with aberrant glycosylation and each type is characterized by a distinct change in the glycan structure. Therefore, analysis of tumor-linked glycan alterations is proposed as a valuable tool for cancer diagnosis. The glycosylation profile is currently characterized by long and complicated protocols, based on final MS detection or the use of lectins. However, these procedures cannot be applied for routine analysis in early detection of cancer and there is a clear need for new methods. Aptamers can be tailored to specifically detect glycosylation and their use as synthetic receptors appears to be a promising approach to replace the current methods . In the case of prostate cancer, it has been recently described that there is a variation in the fucose and the sialic acid of Prostate Specific Antigen (PSA).

We present two strategies to direct the selection of aptamers towards the glycans of PSA. The SELEX procedure is based on counter-selection steps against recombinant PSA and the use of lectins. Aptamers in the last cycle are cloned, sequenced and classified into families using bioinformatic tools. The most abundant aptamers are characterized by electrochemical binding assays. Two aptamers were obtained with different binding characteristics: ability to recognize glycans of any glycoprotein and to specifically recognize the glycans of PSA due to the interaction with amino acids of the protein.

We employ the selected aptamers and an aptamer previously described for the development of electrochemical aptasensors, based on sandwich or direct label-free assays. Both aptasensors respond to different levels of PSA in human serum and are applied to the analysis of serum samples. The results point to the detection of glycans as an alternative for the detection of prostate cancer, with potential to improve clinical outcomes of current PSA tests, which would reduce the number of unnecessary biopsies.

Liquid biopsy has emerged as a potential supplement of traditional biopsy for early cancer diagnosis. It consists in monitoring the level of tumour biomarkers present in appropriate bodily fluids. Consequently, the development of non-invasive, easy-to-use and inexpensive methods for the detection of biomarkers is highly demanded for improving diagnosis and minimizing the mortality of cancer. In this context, electrochemical platforms have demonstrated great potential due to their high sensitivity, low cost, fast response as well as compatibility with point-of-care (POC) testing, what contributes to decentralize the analyses and favours the implementation of screening studies. Among them, personal glucose meter (PGM) employed daily in the measurement of blood glucose is the most used analytical method worldwide. Taking advantage of this mature technology, PGM has been recently applied to quantitate a wide range of non-glucose analytes by establishing a relationship between their recognition and glucose generation. This strategy is of particular interest for monitoring tumour biomarkers since PGMs are already approved by international agencies for application to clinical practice, thus paving the way for the launch of the new device to the market.

Thus motivated, we describe the development of a hybridization assay for the detection of prostate cancer antigen-3 or PCA3, a urinary RNA biomarker for prostate cancer diagnosis. Specifically, a sandwich-type genoassay has been implemented onto magnetic microparticles functionalized with streptavidin in combination with a PGM and alkaline phosphatase (ALP) as a glucose-generating enzyme for signal transduction. Moreover, in order to boost the method sensitivity, two fluorescein-tagged reporting probes has been designed for incorporation of two antifluorescein-ALP conjugates per target analyte. This enzyme catalyses the conversion of glucose-1-phosphate into glucose, whose concentration is subsequently determined with a glucometer. The resulting portable approach allows the reliable detection of PCA3 at picomolar levels.

There is an increasing demand for rapid and sensitive methods for the quantification of bacterial pathogen in very diverse areas, such as clinical diagnosis, environmental monitoring and food safety. Molecular methods, based on the amplification of a specific sequence of the bacterium genome by the polymerase chain reaction (PCR) are widely used. Unfortunately, these methods are usually limited to centralized laboratories. To meet the ASSURED criteria outlined by the WHO [1] for the design of a test useful in point-of-need detection, the use of isothermal nucleic acid amplification methods could be an excellent alternative to PCR.
Here, we present two different electrochemical platforms for Salmonella genome quantification. Both platforms integrate an isothermal amplification process with chronoamperometric detection. Specifically, we compare the signaling and analytical performance of two miniaturized DNA sensing devices fabricated using either indium-tin oxide surfaces in combination with helicase dependent amplification (HDA) [2] or gold surfaces and recombinase polymerase amplification (RPA) [3]. These devices support efficient detection of small amounts of the Salmonella genome on the same platform without thermal cycling while using simple equipment.
References
[1] Wu, G.; Zaman, M.H. Bulletin of the World Health Organization. 2012, 90, 914-920.
[2] Barreda-García S., Miranda-Castro R., de-los-Santos-Álvarez N., Miranda-Ordieres A.J., Lobo-Castañón M. J. Chem. Commun 2017, 53, 9721-9724.
[3] Sánchez-Salcedo R., Miranda-Castro R., de-los-Santos-Álvarez N., Lobo-Castañón M.J. ChemElectroChem 2019, 6, 793-800.
Acknowledgments: This research was funded by the Spanish Government (project RTI-2018-095756-B-I00) and Principado de Asturias Government (IDI2018-000217), co-financed by FEDER funds.