Development of a Novel Phagomagnetic-Assisted Isothermal DNA Amplification System for Endpoint Electrochemical Detection of Listeria monocytogenes.

The hitherto implemented Listeria monocytogenes detection techniques are cumbersome or require expensive non-portable instrumentation, hindering their transposition into on-time surveillance systems. The current work proposes a novel integrated system resorting to loop-mediated isothermal amplification (LAMP), assisted by a bacteriophage P100-magnetic platform, coupled to an endpoint electrochemical technique, towards L. monocytogenes expeditious detection. Molybdophosphate-based optimization of the bacterial phagomagnetic separation protocol allowed the determination of the optimal parameters for its execution (pH 7, 25 °C, 32 µg of magnetic particles; 60.6% of specific capture efficiency). The novel LAMP method targeting prfA was highly specific, accomplishing 100% inclusivity (for 61 L. monocytogenes strains) and 100% exclusivity (towards 42 non-target Gram-positive and Gram-negative bacteria). As a proof-of-concept, the developed scheme was successfully validated in pasteurized milk spiked with L. monocytogenes. The phagomagnetic-based approach succeeded in the selective bacterial capture and ensuing lysis, triggering Listeria DNA leakage, which was efficiently LAMP amplified. Methylene blue-based electrochemical detection of LAMP amplicons was accomplished in 20 min with remarkable analytical sensitivity (1 CFU mL-1). Hence, the combined system presented an outstanding performance and robustness, providing a 2.5 h-swift, portable, cost-efficient detection scheme for decentralized on-field application.

Electrochemical immunosensor towards invasion-associated protein p60: An alternative strategy for Listeria monocytogenes screening in food.

This work reports the development of an electrochemical immunosensor for rapid, specific and decentralized detection of the invasion-associated protein p60 secreted by Listeria monocytogenes, a life-threatening foodborne pathogen. A disposable screen-printed electrode was used as transducer surface and monoclonal and polyclonal antibodies that specifically recognize Listeria monocytogenes p60 protein and Listeria spp. p60 proteins, respectively, were used as the sandwich immuno-pair. The reaction was detected with the aid of an additional secondary antibody conjugated with the enzyme reporter (alkaline phosphatase) and using 3-indoxyl phosphate/silver ions as the mixture substrate. The analytical signal was acquired through the voltammetric stripping of the enzymatically deposited silver, which was directly correlated to p60 concentration in the sample. In optimized conditions, a limit of detection and quantification of 1.5 ng mL-1 and 5.1 ng mL-1 were achieved, respectively, in a useful time (<3 h). As proof-of-concept, the proposed immunosensor was successfully applied to spiked milk samples, demonstrating to be a suitable device for further use in real sample detection of Listeria monocytogenes in food products.

Development of a disposable paper-based potentiometric immunosensor for real-time detection of a foodborne pathogen.

This work reports a new paper-based sensing platform and its application in a label-free potentiometric immunosensor for Salmonella typhimurium detection based on the blocking surface principle. A paper-based strip electrode was integrated with a filter paper pad which acted as a reservoir of the internal solution. The design offers a convenient platform for antibody immobilization and sampling, proving also that is a simple and affordable methodology to control an ionic flux through a polymer membrane. Two different immunosensing interfaces were assembled on the developed paper-strip electrode. The simplest interface relied on direct conjugation of the antibody to the polymer membrane and the second one resorted to an intermediate layer of a polyamidoamine dendrimer, with an ethylenediamine core from the fourth generation. Electrochemical impedance spectroscopy was used to assess the successive interface modification steps and the resulting analytical performance of both immunosensors was compared. For such, the potential shift derived from the blocking effect of the ionic flux caused by antigen-antibody conjugation was correlated with the logarithm of the Salmonella typhimurium concentration in the sample. In optimized conditions, a limit of detection of 5 cells mL-1 was achieved. As a proof-of-concept, the proposed method was applied to apple juice samples, demonstrating to be a suitable prototype to be used in real scenarios in useful time (<1 h assay).

In situ formation of gold nanoparticles in polymer inclusion membrane: Application as platform in a label-free potentiometric immunosensor for Salmonella typhimurium detection.

Polymeric ion selective electrodes are highly sensitive to changes in zero current ion flow and this offers a route to signal amplification in label-free potentiometric immunosensors. In this work, a label-free potentiometric immunosensor toward Salmonella typhimurium (ST) assembled in a home-made pipette-tip electrode is described. The signal-output amplification was implemented on a gold nanoparticle polymer inclusion membrane (AuNPs-PIM) which was used as sensing platform and for antibody immobilization. Additionally, a marker ion was used to detect the antibody-antigen binding event at the electrode surface. The immunosensor construction was performed in several steps: i) gold salt ions extraction in PVC membrane; ii) AuNPs formation using Na2EDTA as reduction agent; iii) antibody anti-Salmonella conjugation on AuNPs-PIM in pipette-tip electrodes. The potential shift observed in potentiometric measurements was derived simply from the blocking effect in the ionic flux caused by antigen-antibody conjugation, without no extra steps, mimetizing the ion-channel sensors. A detection limit of 6 cells mL-1 was attained. As proof-of-concept, recovery studies were performed in spiked commercial apple juice samples with success. Due to the simplicity of use, the appealing cost of equipment and sensor production and being able to provide a quick analytical response (less than 1 h for a complete assay, including sample preparation for analysis), this scheme represents a good prototype device for the detection of foodborne pathogens like ST or other immune-responsive bacteria.

Electrochemical biosensors for Salmonella: State of the art and challenges in food safety assessment.

According to the recent statistics, Salmonella is still an important public health issue in the whole world. Legislated reference methods, based on counting plate methods, are sensitive enough but are inadequate as an effective emergency response tool, and are far from a rapid device, simple to use out of lab. An overview of the commercially available rapid methods for Salmonella detection is provided along with a critical discussion of their limitations, benefits and potential use in a real context. The distinguished potentialities of electrochemical biosensors for the development of rapid devices are highlighted. The state-of-art and the newest technologic approaches in electrochemical biosensors for Salmonella detection are presented and a critical analysis of the literature is made in an attempt to identify the current challenges towards a complete solution for Salmonella detection in microbial food control based on electrochemical biosensors.