Versatile Electrochemical Magneto-Assays: from Manual to Automatable Microfluidic approaches

In an ever-evolving world in constant need for prompt, precise, and efficacious decisions, the detection of (bio)markers has become paramount in a variety of fields, including environmental analysis, food safety, and clinical diagnostics. In the latter, for instance, the detection of particular biomarkers makes the difference between early intervention versus late diagnosis, or personalised medicine versus generic treatment. Year after year, bioanalytical chemistry experiences continually growing technological advancement in this matter. Indeed, biosensors and bioassays have largely demonstrated to be instrumental in addressing the challenges associated with such ultra-sensitive detections. Electrochemical biosensors have emerged as cost-effective and versatile tools, with minimal requirements in terms of instrumentation and excellent adaptability as portable devices apt for point-of-care analyses. Among the numerous approaches, many common biosensing strategies are based on the recognition of the desired analyte using specific biomimetic receptors, followed by the generation of a signal proportionate to the amount of analyte captured. These approaches can be synergically coupled with magnetic separation of the target analyte and enzymatic signal enhancement. In particular, the first part of this 3-year PhD program was dedicated to the assessment of a simple but flexible electrochemical magneto-assay, namely a biosensing scheme that employed superparamagnetic microbeads coated with biomimetic receptors to capture target analytes. Once immobilised onto their surface, the magnetic beads can be used to isolate the analytes from the matrix by means of a simple magnet. Then, through a second bioreceptor labelled with the enzyme alkaline phosphatase, an enzymatic signal amplification could be introduced. Indeed, when adding a suitable substrate (e.g., α-naphthyl phosphate), a vast amount of electroactive molecules can be catalytically generated in situ on the electrode surface, hence leading to a dramatic enhancement of the electrochemical signal registered. Interestingly, this approach can be considered highly versatile, since by selecting the proper biorecognition elements it is possible to virtually determine a large variety of analytes without changing the general scheme of the assay. Thus, multiple biomimetic receptors were used in this dissertation to detect diverse analytes. In Chapter II, biotinylated DNA probes were used to capture target nucleic acid molecules on streptavidin-coated magnetic beads. The strong interaction between biotin and streptavidin was harnessed to immobilise the capture probe on the beads’ surfaces. Then, a biotinylated signalling probe labelled with streptavidin-coated alkaline phosphatase was used to generate and amplify the electrochemical signal. By changing the sequences of such probes, it was possible to use the general scheme of this genomagneto-assay to analyse various nucleic acid markers of importance in diverse fields, spanning from cancer-related clinical markers (e.g., non-coding RNAs and nucleotide excision repair genes), environmental DNA (e.g., the genome of endangered species), foodborne pathogens’ genome, and antimicrobial resistance genes. After testing the applicability of the genomagneto-assay against short DNA sequences, the method was further tested on DNA amplification products (Chapter III). The gold standard polymerase chain amplification was initially considered, but soon substituted with a novel enzyme-assisted isothermal amplification technique: recombinase polymerase amplification. This technique eliminates the need for costly instruments (e.g., thermocyclers) and is able to amplify nucleic acids in twenty minutes while working at fixed low temperatures (e.g., 37 °C). Chapter IV focuses instead on the application of the magneto-assay for the detection of a particular cancer-related protein marker using bicyclic peptides as the main biorecognition elements. Chapter V further pushes the versatility of the magneto-assay by challenging it on the detection of more complex analytes: extracellular vesicles. Combinations of aptamers and antibodies were used for this analysis. Once that the performances and the flexibility of the magneto-assay were assessed, the second main objective of the PhD project was pursued. The analytical protocol required to run the abovementioned analyses comprises a series of very repetitive and tedious steps that expert personnel have to carry out manually by pipetting reagents in/from plastic vials. Thus, efforts were dedicated to transforming this procedure into a practical and automatable microfluidic approach that could be performed inside a small card-based platform with limited human intervention (Chapter VI). More specifically, magnetic beads functionalised with a capture molecule were loaded inside a microchannel engraved on poly(methyl methacrylate). A permanent magnet ensured the beads in position, while all assay solutions required for the magneto-assay were fluxed by using a peristaltic pump. Once that the capture, hybridisation, and labelling reactions occurred, all in a continuous-controlled microfluidic flow, the beads could be collected for the electrochemical readout. After multiple optimisations to maximise the efficiency of each step, this workflow was applied to the detection of isothermally amplified sulfonamide resistance genes from <0.3 µL total DNA extracted from cells of Escherichia coli. The whole analysis could be completed in around 1 h. Limits of detection in the picomolar range with synthetic DNA sequences were obtained. Amplicons diluted up to ≥500-fold could be detected under the conditions investigated. The next step was to work on the full automation of the system by using software-controlled mechanisms remotely controlled with a computer. The following elements were implemented: a computerised vessel mixer, an automatic reagent selector, motor-controlled magnets, and a computerised peristaltic pump. The final platform will be enclosed in a suitable case to make it portable, allowing transport to remote locations for on-site analyses. Promising preliminary results in the detection of DNA were obtained, but further optimisations and adjustments to the system must still be introduced. Sustainability was also considered in this project. With the idea of substituting plastic-based screen-printed carbon electrodes with more sustainable solutions, electrodes obtained from waste materials were considered. In particular, Chapter VII reports on sensors produced with a carbonaceous paste obtained from sewage sludges. After promising proof-of-concept applications on agri-food samples, such electrodes were preliminary tested on the analysis of DNA samples processed through the microfluidic system, obtaining encouraging results. Lastly, during the period of international mobility to the University of Oviedo (Spain), a self-reporter folding-based aptasensor against the prostate-specific antigen was studied (Chapter VIII). Setups with gold electrodes and multi-potentiostats were explored, together with software tools to handle high-volume electrochemical data in a faster and more automatable way. Overall, the results obtained suggest the possibility of applying the proposed automated system to the analysis of target DNA sequences and other analytes and biomarkers. The desired ultimate goal is to deliver a fully autonomous system that, once configured, can perform the magneto-assay described by simply pressing a button.