Innovative approaches in bioelectronics and biosensing platforms are of upmost importance to manage emergency situations such as the COVID-19 pandemic. Considering that many biomarker quantification approaches rely on either photometric or electrochemical determination without offering very often continuous monitoring to prevent critical events in the healthcare setting, the development of advanced interfacing strategies is noteworthy to improve patient safety. Regarding acute respiratory syndrome symptoms, peripheral oxygen levels (SpO₂) tend to steadily decrease upon aggravation. Notwithstanding, both SpO₂ and heartrate (HR) tend to dynamically shift according to patient health status. In this context, we investigated the use of MAX30102 photometric biosensing module coupled to personalized ESP32-based webserver to continuously gather and process SpO₂ and HR data from users (e.g. bedridden patients). Moreover, a user-friendly graphic interface was designed and implemented to better showcase biomarker levels for medical personnel and an anatomical case was 3D printed in thermoplastic polyester (polylactic acid - PLA). Results showcased that the data retrieved from MAX30102 photometric biosensor was similar to that of a standard pulse oximeter, while ESP32 webserver was able to gather, process and graphically display data in real time both in smartphones running Android and personal computers running Windows operating systems (IOS and Linux were not tested in this report). Although ESP32 showcased limitations regarding processing power, the implemented features herein described worked soundly. In conclusion, we showcased how biomarkers such as SpO₂ and HR can be continuously monitored and wirelessly interfaced to computers and smartphones through a low-cost microcontroller such as ESP32. For future investigations, we plan on implementing advanced internet of things and artificial intelligence tools to allow the herein described device to continuously evaluate patient status and predict patient critical events.

Paper-based bipolar electrochemical (P-BPEs) assays have attracted a lot of attention due to their low preparation cost, self-pumping property of paper, being portable. The dominant reporting method in the introduced P-BPEs is electrochemiluminescence (ECL). ECL limits the infield application of this type of paper-based sensor as they need expensive photon counting compartment. Therefore, the developing of a colorimetric method instead of ECL can be a promising feature. In the present study, the electrodeposition of Prussian Blue (PB) on the paper fibers has been introduced which has been used directly as a colorimetric method for the reporting cell of P-BPE. This disposable P-BPE biosensor is used for determination of glucose. The closed bipolar electrochemical cell is fabricated on a small part of paper using a laser printing-based process for paper hydrophobization. The bipolar and driving electrodes are provided by pressing the writing pencil HB on the paper. The mechanism of sensing of glucose is oxidation of the analyte in the sensing cell using glucose oxidase followed by reduction of the produced H₂O₂ by application of an external potential (10.0 V). This causes the oxidation of K₄Fe(CN)₆ in the presence of Fe(II) ions and subsequent formation of PB particles in the reporting cell. The intensity of the blue color in the reporting cell is used as a visual and colorimetric signal that can be digitally read using a scanner of digital camera. The parameters affecting the performance of the device were optimized using experimental design and chemometrics modeling. The P-BPE represents a very wide response range that extends from 0.1 mmol.L^-1 to 4.0 mol.L^-1 in the case of hydrogen peroxide, and from 0.1 to 50 mmol.L^-1 in the case of glucose. The limit of detections for hydrogen peroxide and glucose are 4.9 μmol.L^-1 and 70 μmol.L^-1 respectively.

Methylmalonic acid (MMA) plays a vital role in metabolism and energy production. It has been studied and reported as a sensitive early indicator for mild or serious Vitamin B12 deficiency. The normal range of MMA in blood among healthy people is from 0 up to 0.40 µM. Majority of MMA research was originally focused on Vitamin B12 deficiency with this small detection range. Recently, MMA has been reported to promote tumor progression due to age-induced accumulation. It was found that MMA concentration can reach as high as 80 µM in elderly people. Therefore, MMA can be a promising biomarker for cancer diagnostics, as well as a therapeutic target for cancer treatment. Clinical determination of MMA concentration in blood is normally executed by gas chromatography mass spectroscopy (GCMS) or liquid chromatography mass spectroscopy (LCMS). However, these methods require extensive sample pre-treatment, large sample volume and skilled lab personal. They are also expensive and time-consuming. Hence, we proposed an attractive and effective strategy to detect MMA with a broad linear range by a low-cost molecularly imprinted polyaniline paper sensor. The polyaniline paper strip was fabricated by a one-step solution process using MMA as the template by molecular imprinting technology. The concentration of MMA was determined by the resistance change of the paper sensor by a portable multimeter wirelessly. A calibration curve as a function of MMA concentration in aqueous solution was acquired with a correlation coefficient of 0.968. We also demonstrated detection of the added MMA in plasma with a wide concentration range of 0 to 100 µM with a limit of detection (LOD) of 0.197 µM. This low-cost disposable paper sensor has great potential in point-of-care MMA detection for cancer prognostics and diagnostics, especially in underserved communities.

Metallic nanoparticles potentially have wide practical applications in various fields ofscience and industry. In biosensorics they usually act as catalysts (nanozymes, NZ) and/or mediators in electron transfer. In our present work we describe the development of amperometric biosensors (ABSs) based on oxidases and nanoparticles of CuCe (nCuCe). nCuCe was synthesized chemically from the correspondent salts and Na₂S as a reducing agent. nCuCe, being a very active peroxidase (PO) mimetic, was used here as a hydrogen peroxide sensing platform for oxidase-based ABSs. Using glucose oxidase (GO), alcohol oxidase (AO), methylamine oxidase (AMO), L-arginine oxidase (ArgO), and nCuCe as PO-like NZ (for GO, AO, AMO and ArgO-based ABSs) and as electro-active mediator (for laccase-based ABS), the biosensors on glucose, primary alcohols, methyl amine, L-arginine and catechol, respectively, were constructed and characterized. Enzymes were isolated from the correspondent over-producing cells according to the methods, proposed by the authors earlier. The developed mono-enzyme ABSs exhibited improved analytical characteristics in comparison with the correspondent bi-enzyme ABSs, which contained natural PO. Including electrodeposit nanoporous gold (npAu) in chemosensing layer on graphite electrode (GE), allows to additionally increase sensitivity of the constructed ABSs twice due to the increased and advanced surface. As an example, the bioelectrodes, containing laccase/GE, laccase/nCuCe/GE and laccase/nCuCe/npAu/GE had the sensitivities, respectively, 2300, 5055 and 9280 A·M⁻¹·m⁻². The proposed laccase-based ABSs were successfully tested on the samples of pharmaceuticals and food products for analysis of phenolic compounds.

An important and critical characteristic for continuous monitoring using DNA biosensor is the microfluidic design. Some of the significant functions of microfluidic structures used in DNA biosensors are sample manipulation, effective and rapid reaction and ultra-low detection limit of the analyte. This study explores different designs and parameters of microfluidic microchambers. The selection of the appropriate geometrical design and control of microfluidic parameters are highly important for the optimised performance of the biosensor. Several combinations of different shapes of microchambers are designed and assessed computationally. Flow parameters such as average velocity, pressure drop, flow rates and shear parameters are critically assessed at different cross-sections within the microchamber.

Optimised design of the microchamber is selected based on optimal rinsing, minimum flow shear, avoidance of slow zones, simple geometry and homogenous distribution of the analyte target. Steady and unsteady laminar flow simulations were performed using a commercial Computational Fluid Dynamics software. A range of different geometrical shapes were assessed using parametric studies and a design optimisation analysis. 3D printing technology was used to construct and evaluate the optimised designs in combination with experimental investigations which were carried out and the optimum design was selected and characterised.

Materials used for fabricating biomedical devices, such as implantable biosensors need to possess appropriate physical, chemical and biological properties, depending on specific circumstances. Among a variety of materials commonly used as biosensing platforms, cellulose and its derivatives have gained considerable attention. Due to the promising physical and biological characteristics, as well as chemical structure, cellulose has demonstrated to be a versatile material, affording a high-quality platform for accomplishing the immobilization process of biologically active molecules into biosensors.

To promote the sufficiency of cellulose in biosensing, several researchers have investigated pathways to enhance cellulose properties to meet biosensing requirements. Research on cellules and its derivatives as biosensors is developing rapidly through the innovation and improvement of raw materials, chemical synthesis and methods of preparation and formulations, with more than 500 organizations around the world are currently involved in the patent activity and filing concerning cellulose-based biosensors. This trend is justified by the several advantages that cellulose offer for biosensing and biomedical applications. This is also evident from the elevation in the number of patent applications filed each year worldwide in research and development of this area.

This study in the form of patent analysis presents the state of the art by introducing what has been patented in relation to cellulose-based biosensors between 2010 and 2020. Furthermore, a detailed analysis of the patentability has been provided by determining publication years, classifications, inventors, applicants, owners, and jurisdictions. Finally, this work which gives an analysis of the past, present and future trends lead to various recommendations that could help one to plan and innovate research strategy.

This work continues our research into developing novel biosensing technologies for early prostate cancer (PCa) diagnostics. The existing PCa diagnostics based on PSA detection (prostate cancer antigene) in blood serum often yield controversial outcomes and require improvement. However, the long non-coded RNA transcript PCA3 overexpressed in PCa patients’ urine is an ideal biomarker for PCa diagnosis. Thus, recent research mainly focuses on developing biosensors for the detection of PCA3. One of the most promising directions in this research is aptamers as bio-receptors for PCA3. We demonstrated the earlier great potential of electrochemical sensors exploiting aptamer labelled with redox group ferrocene. In this work, we use a 227 nt RNA-based aptamer labelled with methylene blue, which offers a higher affinity to PCA3 than commonly used DNA-based aptamers. Also, conditions for immobilisation of aptamers on gold electrodes were optimised, such as different gold electrodes used (interdigitated electrodes and three-electrode assemblies), the electrode’s roughness and polished surfaces, and the concentration of immobilised aptamers. Initial tests were carried out using cyclic voltammograms(CV) measurements and showed a correlation between oxidation/reductions peaks intensities and the concentration of PCA3. This work proved the main concept of the proposed apta-sensing, e.g. the changes of aptamer secondary structure during binding the target (PCA3). In addition, the redox label comes closer to the electrode surface, thus increasing the charge transfer. The lowest recorded concentration of PCA3 in CV measurements was 0.1 nM which was sufficient for medical diagnostics. However, the main focus was on the use of electrochemical impedance spectroscopy (EIS), which offered even high sensitivity in PCA3 detection down to the pM level. The results obtained are very encouraging and constitute a major step towards developing a simple, reliable, and cost-effective diagnostic tool for the early detection of prostate cancer.

Bacterial contamination in food presents real and valid danger for human health. Every year tons of food need to be thrown out and it is estimated billions of people get sick from food poisoning, leading to deaths in some hundreds of thousand cases (mostly in children). Most of the bacterial contaminations can be traced to about 20-30 pathogenic bacteria. In our work we focused on the detection of Escherichia coli and Listeria monocytogenes with optical and acoustic methods. In both methods we used specific DNA aptamers as receptors. Aptamers are single stranded DNA or RNA that in correct environment folds into structures specifically binding to a target on bacteria with K_d around 1-10 nM. For optical method it is possible to modify gold nanoparticles (AuNp) with aptamer (if it is modified with thiol group for example) and it is then possible to see the interaction of AuNp with bacteria on change of the absorbance spectrum. Aptamers also electrostatically interact with the AuNp without specific binding and it is possible to increase the stability of AuNp to increase of ionic strength by salt with aptamer being present. The incubation of such solution with bacteria then removes the protecting aptamer and after addition of salts to a solution of gold nanoparticles it is possible to measure their rate of aggregation as a function of bacterial concentration present in a sample. Another way to exploit optical system to measure the binding of bacteria to aptamers is to use white light reflectometry to measure change in thickness on a silicon chip modified with aminylated aptamer through silica chemistry. Lastly it is possible to modify the gold electrode of quartz TSM crystal with thiolated or aminylated aptamer and measure frequency change and dissipation change after bacteria binding. It seems that the interaction of bacteria with the surface is not strictly mass based, and it is therefore advantageous to perform analysis of change of viscoelastic properties on the surface. We will present the comparative analysis of the sensitivity of optical and acoustics methods for detection of bacterial pathogens.

Bacteria detection in food is very important, since in the US approximately 9 million cases per year are related to foodborne illness caused by 31 pathogenic bacteria among which Salmonella spp. and Escherichia coli O157:H7. Therefore, rapid, sensitive and accurate detection methods for bacteria detection are crucial not only for consumer’s health but also for food industries. The conventional methods for identification of bacteria are based on culturing and plating and although reliable they require several days for the completion of the analysis. To shorten the analysis time ELISA- and DNA-based methods have been employed for bacteria identification but they are not appropriate for point of need applications. For this reason, recently, biosensors are gaining ground in foodborne bacteria detection. Here, we present a miniaturized immunosensor for the simultaneous, label-free, real-time determination of bacteria in milk. The transducer consists of an array of ten broad-band Mach-Zehnder interferometers (MZIs) integrated onto silicon chips along with the corresponding optical sources. For bacteria detection, the sensing areas of the MZIs were biofunctionalized with bacteria membrane antigens, prior to the assembly of the microfluidic module. For the assay, bacteria solutions were mixed with anti-bacteria-specific antibodies, pre-incubated for 30 min and run over the chip followed by running biotinylated anti-species specific antibody and streptavidin solutions. The transmission spectra of MZIs were continuously recorded by an external miniaturized spectrometer and subjected to Discrete Fourier Transform to convert spectral shifts to phase shifts. The assays were accurate and reproducible with detection limits of 5X10² CFU/mL for S. typhimurium and E. coli in milk samples. The analytical characteristics, combined with the short analysis time (~10 min) and the small chip size make the proposed biosensor ideal for on-site bacteria determination in food samples.

Acknowledgment: M. Angelopoulou was supported by the program of Industrial Scholarships of Stavros Niarchos Foundation.

Ochratoxins are a group of mycotoxins produced as secondary metabolites by several fungi of Aspergillus and Penicillium species. Ochratoxin A (OTA) is the most toxic member of the group and can be found in a large variety of highly consumed foods, such as coffee, cocoa, wine, and flour. Reliable determination of ΟΤΑ levels in food samples is therefore indispensable to assure compliance with national/European regulations concerning MRLs and consequently minimize health risks posed for living beings. In the current study, a label-free biosensor based on White Light Reflectance Spectroscopy (WLRS) for rapid and accurate determination of OTA in flour specimens is demonstrated. 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 OTA-protein conjugate on the SiO₂ surface, while the determination is based on a dedicated competitive immunoassay format. For the assay performance, a mixture of an in-house developed anti-OTA antibody with the calibrators or the samples is injected over the chip surface followed by reaction with secondary biotinylated antibody and streptavidin for signal amplification. 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 optimization, the sensor was capable of real-time detection of OTA in flour samples with LoD of 60 pg/mL, within 25 min total analysis time. The intra- and inter-assay CVs were ≤5.9% and ≤9.0%, respectively. The excellent analytical characteristics and short analysis time in combination with its small size render the proposed WLRS system ideal for the quantitative determination of minute OTA levels at the Point-of-Need for food-, environment-, and health-related purposes.