ECIS, xCELLigence and CellZScope are commercially available instruments, able to measure the impedance of cellular monolayers continuously and with high precision. The small currents used allow for label-free, real-time monitoring of the cells in a non-invasive manner. Despite the widespread use of these systems individually, direct comparisons between the systems has not been published.

In this paper, we wanted to compare the temporal sensitivity and resolution of each system. More specifically, we aimed to determine whether the instrument’s impedance measurements could detect the presence of an endothelial monolayer and reliably separate this measurement into basolateral adhesion and paracellular barrier resistances.

The total impedance measurements for all three systems were relatively similar, and very similar for the ECIS and xCELLigence systems. ECIS data can be modelled into Rb (paracellular-barrier) and Ra (basolateral adhesion). This revealed that the xCELLigence measurements were a hybrid of Rb and Ra, as the instrument was unable to discriminate between the two factors.

To compare the sensitivity of the instruments, responses to the inflammatory cytokines TNF and IL1 were measured. All three instruments showed transient decreases followed by prolonged increases in impedance. Although xCELLigence could detect these changes, it was unable to attribute this data to various cellular processes. This reduced both the sensitivity and information value of this instrument. Although both the other instruments are able to reveal processes from the modelled data, ECIS had a higher sensitivity to stimulation than the CellZScope, particularly at the lower seeding densities, which made responses harder to detect. Despite this limitation, the CellZScope is the only instrument that measures transendothelial electrical resistance across Transwells, where both the apical and basolateral compartments can be stimulated or sampled. This work demonstrates that instruments must be carefully selected to ensure they are appropriate for the experiments conducted.

Imprinted polymers define robust and artificial materials able to mimic recognition processes of such analytes, such as proteins, small molecules, or ions. The process results in the selective formation of ion-sized imprinted cavities, which are complementary to a specific template in terms of its functional groups. These materials can be easily applied to identify, monitor and remove the target ions in water environment. In this view, the ion imprinted polymers (IIPs) can be described. Their synthesis can be carried out both chemically and electrochemically. The latter leads to the preparation of imprinted films, which are compatible in conjunction with transducers in sensor development. Very few works report the electrochemical synthesis of ion imprinted polymers and their application as sensors for metal ion detection. With this regard, we propose the synthesis, optimisation, characterisation and subsequent application of an electrosynthesised IIPs for the electrochemical detection of cadmium (II) in water. The proposed sensor was prepared by electropolymerisation of 4-aminophenylcarboxylic acid (4-APA) monomer in presence of Cd(II) ions, which acts as the template. The screen printed graphite electrodes (SPGE) were used as transducer during sensor development, whereas the cyclic voltammetry (CV) and differential pulse voltammetry (DPV) were selected as the electrochemical methods for the synthesis and Cd(II) ions sensing, respectively. The incubation of the developed sensor in EDTA 500 mM involved into remove the template and the formation of specific recognition cavities into the polymer. A multivariate optimisation was employed for studying the effect of four independent parameters on electrochemical performances of the sensor. The electrochemical characterisation of sensors was performed in ferrocyanide-ferricyanide redox couple and in KCl 0.1 M, the latter revealing redox properties from the polymeric film. The performances of sensors and the control (NIP) was observed in sodium acetate buffer (50 mM, pH = 5) over the Cd(II) concentration range 10 – 500 nM.

Rapid evolution of pervasive computing and body-sensing technologies propels the Internet of Health, encouraging clinical researchers to use these methods in more ecological and real-life experiments. Important applications include neuropharmacological research, behavioural neuroscience, and preventive healthcare. However, there are several challenges to overcome. We lack data management standard to integrate data from multiple devices used by one subject, thus the extent of data collection is limited to single specialized biosensor platforms. We also lack freely available, standardized and device-independent visualization and processing tools. Therefore, data harmonization, cross-modal data integration and data visualization are prohibitively cumbersome. These heterogeneities limit the scale, equity and inclusivity of the data collection process--keeping such systems available to few major industry players or wealthier academic institutes. This problem has been addressed since early 1990s by the neuroimaging community, that has taken advantage of functional and anatomical brain imaging in population neuroscience studies. Today, a wealth of knowledge exists about best practices to undertake epidemiological or lifestyle study in Alzheimer's disease or Autism, using magnetic resonance imaging (MRI) and, recently, electro- and magnetoencephalography (EEG &MEG). To integrate myriad large-scale longitudinal data generated across different modalities (e.g. positron emission tomography, EEG, MRI), on scanners from different manufacturers, and across the globe, is now common practice. Researchers are now interested incorporating ecological data from body-worn sensors, apps or interactive and immersive games. We described Sensomatrix framework as a response to this need, by inspiration from existing data management tools available in neuroimaging. Here, we provide an example of its application in serious game development, where we studied the relation between cognition, and psychophysiological stress caused by computer games in their prospective users, older adults. By drawing parallels between neuroimaging and biosensor data, we provide provide ideas for standardization of such technologies for use in large-scale studies.

Worldwide, the increasing use of fertilisers rises the risk of eutrophication, a sudden growth of algae in presence of nitrates and phosphates that leads to oxygen depletion in water with critical economic and environmental consequences. Continuous monitoring of oxygen in environmental waters could improve the early detection of eutrophication events and prevent anoxic conditions. However, online and in situ dissolved oxygen sensors are yet to be implemented due to cost, portability and power limitations. Here we propose to use a self-powered ceramic soil microbial fuel cell to detect algal growth via monitoring of dissolved oxygen in water. When immersed in an algal solution, the sensor signal follows the characteristic photosynthetic cycle with maximum day current of 0.18 ± 0.2 mA and a minimum night current of 0.06 ± 0.34 mA, which correlates with dissolved oxygen (R²=0.85 (day); R²=0.5 (night)) and algal concentration (R²=0.63). A saturated design of experiments on seven factors suggests that temperature, dissolved oxygen, nitrates and pH are most influential operational factors on the voltage output; while operating the system at maximum power point (R_ext= 2 kΩ) improves the sensor sensitivity, in the range of study. This is, to our knowledge, the first proposed MFC based biosensor for in field early detection of eutrophication events.

MicroRNAs are widely studied as circulating biomarkers for early stage diagnosis of several diseases, but their detection and quantification are currently performed through complex and time consuming procedures. Herein, we demonstrate a rapid, multiplex, homogenous detection method based on two-step amplification of the signal measured by a recent label-free optical biosensor, Reflective Phantom Interface (RPI). The specific capture with surface DNA probes is combined to mass amplification by an antibody targeting DNA-RNA hybrids and polyclonal secondary antibody, all performed without washing steps. Through this method, we achieved linear, sub-pM quantification of different miRNAs in less than 2 hours.
The RPI enabled the characterization of equilibrium and kinetics of each individual interaction involved in this multi-step process, which allowed to model and optimize the relative concentrations and the time intervals of the assay. In particular, we found that the sensitivity for a miRNA target is affected by its sequence features, like the binding affinity and the degree of self-complementarity, which indirectly regulates probe-probe interactions. The limit of detection is ultimately determined by the kinetic constant for association between the probe and the target. Therefore, the performance of the assay may be further improved by acting on the probe architecture, favouring additional stacking interactions with the miRNA.

This work reports the fabrication of an open-ended mesoporous porous silicon membrane (PSiM) for the fast and indirect detection of bacteria through their lysate. Porous Silicon (PSi) is a promising material for biosensing applications because of its tuneable optical properties and large surface area. The performance of PSi-biosensors is however hindered by the diffusion-limited infiltration of analyte, which lowers the sensitivity and increases the detection time. To overcome this limitation, Porous Silicon Membranes (PSiMs) have been fabricated using standard microfabrication techniques and previously studied for the detection of small biomolecules. In this work, PSiMs were developed for the optical detection of Bacillus cereus lysate. The protocol of detection starts with a bacterial suspension, which is incubated for 30 min at 30°C in the presence of PlyB221, an endolysin encoded by the bacteriophage Deep-Blue targeting B. cereus. In the meantime, a signal baseline is established by flowing PBS through the sensor. After the incubation, the lysate is pumped through the PSiM at a flow speed of ~15 µL/min. The PSiM is monitored optically and the detection is based on a shift in the effective optical thickness (EOT). The EOT equals 2nL, where n is the average refractive index of the PSi and L its thickness. The penetration of bacterial lysate induces an average EOT shift of ~600 nm after 1 hour, which is nearly 3 times higher than the noise level in PBS. The initial concentration of bacteria before the lysis was measured as 10⁶ CFU/ml. Not only does this biosensor enable the fast detection of bacteria, but the same technique can be adopted for other bacteria after their selective lysis.

The outstanding properties of metal nanoclusters, stabilized with different scaffolds i.e. proteins, nucleic acids, polymers and dendrimers, enabled their applications in a wide range of fields [1]. The recent advances in the fabrication and synthesis of nanoclusters have revolutionized the design of biosensors leading to significant improvement in the selective and sensitive determination of several targets. In particular, in recent years, copper nanoclusters (CuNCs) have attracted more attention mainly for the unique fluorescent properties as well as large Stokes shifts, low toxicity, and high biocompatibility [2]. The high photoluminescent features of CuNCs provide the high sensitive target detection even in complex biological matrices. For these reasons, in this work, we exploited the specific template-targeted CuNCs growth for the sensitive and accurate determination of human serum albumin (HSA) in urine and human serum. HSA is the most abundant protein in plasma acting as a carrier for many key biological molecules such as hormones, fatty acids and steroids and it provides to the maintenance of the oncotic blood pressure. The concentration of HSA in body fluids greatly influences the state of health of the patients. Taking into account these considerations, quantitative detection of human serum albumin plays a key role in early diagnosis of serious pathological conditions like as albuminuria and albuminemia. Here, we present a CuNCs-based assay in which copper nanoclusters were used as fluorescent signal indicators to detect serum albumin in complex biological matrix.

1. Cao, Q.; Li, J.; Wang, E. Recent advances in the synthesis and application of copper nanomaterials based on various DNA scaffolds. Biosens. Bioelectron. 2019, 132, 333–342.
2. Moghadam, F. M.; Rahaie, M. A signal-on nanobiosensor for VEGF 165 detection based on supraparticle copper nanoclusters formed on bivalent aptamer. Biosens. Bioelectron. 2019, 132, 186–195.

Currently, there is a lack of low-cost, prompt and robust bioanalytical methods to detect small peptide hormones (e.g. gonadorelin, buserelin, leuprorelin, etc.) in the routine anti-doping protocol. These peptides are improperly used by male athletes to improve their sports performances by stimulating the endogenous secretion of testosterone in the bloodstream via the hypothalamic-pituitary-gonadal axis. For this reason, low molecular weight peptide hormones (< 2000 Da) were banned by the World Anti-Doping Agency (WADA) and represent a new frontier in antidoping research. Our study aimed to design a molecularly imprinted polymer (MIP)-based assay to detect gonadorelin (pGlu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH₂) levels in urine samples. The process of molecular imprinting involves the synthesis of a polynorepinephrine (PNE) network structure with imprinted template binding sites. The interaction between gonadorelin and the “tailor-made” synthetic receptor has been investigated and optimized through a surface plasmon resonance (SPR) platform. Afterward, a competitive biomimetic assay was set by employing biotinylated gonadorelin tethered to streptavidin as a signal-enhancer competitor molecule. This type of assay is suitable for the detection of small molecules that lack multiple epitopes. Encouraging results were recorded for gonadorelin in buffer and spiked artificial urine samples (_av%recovery = 96.30 %) in the low ppb range, perfectly in line with the Minimum Required Level (MRPL = 2 ppb) at which all WADA-accredited laboratories must operate in routine daily operations.

Moreover, we intend to provide a strategy to detect gonadorelin which can be easily miniaturized to set-up a sensing device for in-situ athletes’ monitoring.

In the last decades, nanomaterials have been extensively used in biosensors design to enhance their electrochemical performance in terms of sensitivity, reproducibility and oftentimes selectivity. Polymers, nanoparticles, and carbon based-materials have attracted a lot of attention in this field. Three different electrochemical nano-platforms for pharmaceutical applications are described.

First, an electrochemical non-enzymatic sensor for glucose based on 3D copper nanostructures with Ni foams as promotor of the enhanced nanoporous morphology was developed. Detection of glucose was performed by means of chronoamperometry (0.55V) and the sensor was successfully tested in the presence of the designated target even in the presence of common interference agents found in biological samples.

A polycarboxipyrol based platform was developed by electrodiposing carboxylic pyrol (PyCOOH) at screen printed electrodes (SPCE) by means of CV from a mixture of PyCOOH/Polivinilpirolidone/LiClO₄ solution in order to obtaind to obatin cone-like polycarboxipyrol shapes. The application of the platform was proven when a Folic acid oxidation peak (-0.55V) was triggered by Differential Pulse Voltammetry (DPV) from bulk solution and pharmaceutical tablets solution at the polymer-based electrode surface.

A hybrid composite material containing polyaniline (PANI) and bimetallic nanoparticles (Au-PtNPs) was synthesized at SPCE by means of electrochemical procedures as cyclic voltammetry (CV:-0.4-0.8V, 10 cycles, 50 mV/s from an aniline 2.5mM solution) and chronoamperometry (+0.2V for 130s from a HAuCl₄/H₂PtCl₆ mixture solution). The sensor was succesfuly applied for arsenic detection.

Acknowledgements

This work was supported by grants of the Romanian National Authority for Scientific Research and Innovation, CCCDI-UEFISCDI, within PNCDI-III, ERANET-RUS-PLUS-PLASMON-ELECTROLIGHT/46/2018 and INTELMAT/39/2018 projects. Gheorghe Melinte thanks: UMF internal grant number 2462/37/17.01.2020.

Lysozyme is an enzyme present in multiple organisms where it plays various vital roles. One of the most important relies on its antibacterial activity, being also called the body’s own antibiotic. Despite its proven utility, lysozyme can potentially trigger allergic reactions in sensitive individuals, even in trace amounts, thus the need of continue monitoring of lysozyme in products rich in lysozyme like wine or egg white is of high importance [1].

In this work, an electrochemical aptasensor was designed for the flow analysis of lysozyme. Firstly, polylysine, polydopamine and poly(lysine-co-dopamine) were electrodeposited at screen printed carbon electrodes (SPCE) in order to obtain a more structured platform with higher electroactive area. The best architecture was further chosen for sensor development. Next, gold nanostructures (nanoleaves) were electrodeposited from a mixture of HAuCl₄ and PEG 6000 solution for enhanced electrocatalytic effect and to serve as immobilization platform for the thiolated aptamer [2]. All platforms were electrochemically and morphologically characterized.

For lysozyme detection, the best platform in terms of electronic conductivity was selected for the 1^st aptamer immobilization within the thiol group from its 3’-end, followed by a blocking step of the remaining free sites of the gold nanoleaves with 6-mercaptohexanol. Next, after the lysozyme solution was dropped casted on the electrode surface and the aptamer-target reaction was performed, a 2^nd aptamer, labelled with biotin, bounds also the lysozyme to obtain a sandwich assay. Further, streptavidin-alkaline phosphatase (ALP) reacted with the biotin bounds to the 2^nd aptamer. For lysozyme quantification, ALP oxidised the substrate, 1-naphtil phosphate and the resulting signal was registered using differential pulse voltammetry.

References

[1] A. Vasilescu, Q, Wang, M. Li, R. Boukherroub, S. Szunerits, Chemosensors, 2016, 4(2), 10

[2] M. Negahdary, H. Heli, Talanta, 2019, 198, 510-517.

Acknowledgements

Ghorghe Melinte thanks: UMF “Iuliu Hațieganu” Cluj-Napoca, for internal grant no. 2462/37/17.01.2020