Microfluidic technology, also known as lab-on-a-chip, enables the fabrication of low-cost, user-friendly, and portable detection devices. Microfluidic chips can be utilized for detecting biological and chemical analytes in various liquid samples, including water or biofluids such as urine, blood, and sweat. The specific and quantitative detection of ions has garnered increased attention in recent years due to their potential harm to environmental and human health. Inorganic ions are special chemicals that hold positive or negative charges with relatively small molecular weights. Among the various types of microfluidic platforms, paper-based systems are favored as simple analytical tools that rely on the generation of hydrophilic–hydrophobic contrast on filter paper. In this study, a paper-based microfluidic device was developed as an analytical tool for quantifying several ions, such as iron (Fe³⁺). The reaction spot was created by simply melting a wax crayon to form hydrophobic barriers that define hydrophilic zones. After spotting Fe³⁺ samples and potassium thiocyanate (KSCN) as a detection reagent on the reaction zone, an immediate and obvious color change was observed with different ion concentrations ranging between 50 and 500 ppm. While the naked-eye detection of color change was easy at high concentrations, quantifying ion concentrations in samples required the use of a smartphone camera. The captured images were then analyzed using ImageJ software. The developed paper-based microfluidic device exhibited good performance in quantifying Fe³⁺ ions in samples. Indeed, this simple platform is easy to store and transport, and allows the transportation of aqueous solutions without the need for external pumping or a power supply.

Introduction. About 90% of blood serotonin is stored in secretory granules of platelets and is released by exocytosis. Serotonin is a biogenic amine and the mechanisms for its accumulation in secretory granules and exocytosis are similar to some neurotransmitters. As it is an electroactive molecule, its release can be detected via amperometry. Thus, platelets are an easily accessible human cell model to study exocytosis. Methods. We have optimized boron-doped diamond (BDD) on multielectrode array (MEA) systems that allow the detection of amperometric recordings from the quantum release of serotonin from human platelets. Exocytotic events are detected as transient oxidation currents. Our initial experiments were carried out with microelectrode devices on silicone matrices (BDD-on-silicon MEAs)¹. We introduce here a new transparent material which allows microscopy observations: a BDD-on-quartz MEA. Results. BDD-on-quartz MEA devices exhibit excellent electrochemical properties similar to BDD-on-silicon MEAs¹. We present the amperometric data obtained from unloaded platelets and after loading the platelets with 10 µM serotonin for 2 h, as well as a comparative study of the quantum and kinetic characteristics of amperometrical spikes obtained with both MEA chips. Conclusions. We demonstrate the effectiveness of BDD-on-quartz MEAs as biosensors for the amperometrical measurement of serotonin exocytosis from human platelets. References. ¹ González Brito, R; Montenegro, P; Méndez, A; Carabelli, V; Tomagra, G; Shabgahi, R.E.; Pasquarelli, A.; Borges, R. Multielectrode Arrays as a Means to Study Exocytosis in Human Platelets. Biosensors 2023, 13, 86. https://doi.org/10.3390/bios13010086.

Aflatoxin M1 (AFM1) is the hydroxylated form of Aflatoxin B1 (AFB1) and is expelled in the milk of both humans and animals following the consumption of AFB1-contaminated food. AFM1 has been categorized as a Group 1 carcinogen by the International Agency for Research on Cancer. Consequently, the European Commission has established a maximum allowable concentration of 50 pg/mL for AFM1 in dairy products and milk. Here, a rapid and sensitive approach for detecting AFM1 in bovine milk is presented. The analytical setup comprises a broad-band white LED, a spectrophotometer, and a silicon photonic probe, all interconnected by a bifurcated optical fiber [1]. Additionally, a laptop powers the system and facilitates signal monitoring through specialized software. The silicon photonic probe is equipped with two Mach–Zehnder interferometers: one functionalized with AFM1-bovine serum albumin conjugate, and the other with bovine serum albumin to serve as a blank. The analysis involves immersing the probe directly into a mixture of anti-AFM1 antibodies and the sample, followed by sequential immersion into biotinylated anti-rabbit IgG antibody and streptavidin solutions. The entire assay process takes 12 minutes, and the limit of detection in undiluted milk is 20 pg/mL, below the EU maximum allowable limit of 50 pg/mL. The assay demonstrates accuracy, with %recovery values ranging from 87.5 to 112%, and repeatability, with intra/inter-assay coefficients of variation below 7.6%. Given its analytical performance and compact instrumentation, the proposed immunosensor proves to be an ideal solution for precise on-site determination of AFM1 in milk samples.

Acknowledgements: This work was financially supported by the European Regional Development Fund of the European Union and Greek national funds through the Operational Program Competitiveness, Entrepreneurship and Innovation, under the call RESEARCH–CREATE–INNOVATE (project code: Τ2ΕΔΚ-01934 FOODSENS).

[1] M. Angelopoulou, et al., Biosens. Bioelectron. 215 (2022) 114570

This study presents a fully automated microfluidic electrochemical sensor for the detection of single-stranded DNA (ssDNA) on the ESSENCE platform. The sensor utilizes functionalized single-walled carbon nanotubes (SWCNTs) with short ssDNA strands immobilized through EDC-NHS coupling, placed between non-planar interdigitated electrodes. The detection process involves sequential flow of a background electrolyte and redox probe through the microfluidic channel before introducing the target DNA solution. The same solution is then circulated to enhance selectivity by removing non-specifically bound targets. Electrochemical impedance signals are acquired after the initial and final flow steps, utilizing changes in impedance spectra to quantify target DNA concentration. To streamline complex flow steps and eliminate manual interventions, the system integrates a fully automated fluid control system with syringe pumps, valves, and pressure sensors. Electrochemical impedance spectroscopy (EIS) data is acquired using the Analog Discovery 2 USB oscilloscope, and LabVIEW automation ensures a seamless transition from sample introduction to data acquisition. The transducer material's flow-through design enables efficient differentiation between different degrees of base pair mismatches, extending applicability to single nucleotide polymorphisms. The system exhibits high sensitivity, detecting single-stranded DNA at concentrations as low as 1 fM within a rapid 15-minute detection time. Its compact design and automated data acquisition make it a promising candidate for point-of-care biomolecule sensing, including antigens and toxins. Future applications involve functionalizing SWCNTs with relevant antibodies to enhance the platform's capabilities for detecting a diverse range of target molecules in clinical settings.

A fully electrochemical approach for fabrication and detection is employed to develop a simple, low-cost, and highly sensitive biosensor for matrix metalloproteinase 2.

Graphene oxide (GO) was deposited using the simplest technique, i.e., drop casting over the working electrodes commercially available in the form of an interdigitated electrode array. The GO monolayer uniform film was reduced using an electrochemical approach to obtain an electrochemically reduced graphene oxide (ERGO) ultra-thin film. The ERGO's conductivity and electrochemical activity were controlled by reduction process optimization, where the number of cyclic voltammetry cycles was determined to obtain a highly conductive ERGO film.

In our approach, GO was reduced in a 1x PBS solution with a voltage range of -0.4 to -1.2 V and a scanning rate of 50 mV s^-1. We observed that 20 cycles of CV scanning produce stable and highly conductive ERGO. Furthermore, the biosensor was constructed using specific anti-MMP2 aptamers, which are covalently attached to the ERGO surface by pyrene-based chemistry. Initially, we tested the sensitivity in a buffer medium, finding a limit of detection of 3.32 pg mL^-1 using electrochemical impedance spectroscopy (EIS), which is much lower than that of previously reported graphene-based devices of similar technology. Moreover, the MMP-2 biosensor showed a high specificity toward different similar proteins.

The wide range of active MMP-2 concentrations (10 pg mL^-1 to 100 ng mL^-1) opens the potential for the development of point-of-care devices for the early prediction of different diseases, with an emphasis on cancer.

This work was supported by projects funded by the European Union’s Horizon 2020 research and innovation programme NANOFACTS under grant agreement no. 952259 ( https://doi.org/10.3030/952259).

Introduction: Maintaining the balance of urine pH and urine metabolite concentrations is vital for a good health, with abnormal levels indicating various conditions such as infection, kidney disease, or metabolic disorders. Hence, this work aims to create a biosensor using field-effect transistors (Bio-FET) to measure urine pH and detect multiple metabolites, starting with Immunoglobulin G (IgG).

Methods: Our Bio-FET system comprises a microfabricated gold electrode, working as an Extended Gate (EG) and a readout circuit for signal detection and amplification. pH tests were conducted with six distinct solutions (10.34, 8.60, 7.40, 7.00, 4.00, and 2.91) applied onto the EG surface, resulting in I_ds – V_ref transfer curves performed within the linear region of a MOSFET, allowing us to create a calibration curve (I_ds – pH). For the IgG detection, an Anti-IgG was covalently bound to a mixed 11-Mercaptoundecanoic acid (MUA) and 11-Mercapto-1-undecanol (MUD) (ratio 1:2) Self-Assembled Monolayer (SAM) on the gold electrodes. Each step of the surface functionalization was characterized using ellipsometry. Preliminary tests were conducted to the Bio-FET response when exposed to two distinct solutions: (1) phosphate-buffered saline (PBS) solution at pH 7.40; and (2) PBS solution spiked with IgG (0.01 mg/mL).

Results: Regarding pH tests, the results showed a linear relationship, wherein elevated pH values were associated with increased current values, with a corresponding sensitivity of 2.20 µA/pH. The binding of target IgG with the specific antibodies immobilized on the EG surface triggered a positive vertical shift of the PBS-IgG solution transfer curve when compared to the one obtained when the PBS solution alone was used.

Conclusions: The Bio-FET system shows a good response both for pH measurement and IgG detection. However, regarding IgG detection, further tests using different PBS-IgG solutions are required to evaluate the limit-of-detection and linear range of the Bio-FET system as an IgG sensor.

Chemiluminescence (CL) is a widely used detection technique in biosensing due to its high sensitivity, simplicity of analysis and low cost. These characteristics make it commonly used to quantify hydrogen peroxide (H₂O₂) by carrying out its reaction with luminol in the presence of the horseradish peroxidase enzyme (HRP). Hydrogen peroxide is a clinically relevant biomarker since it appears to be involved in numerous pathologies including diabetes, cancer, Parkinson’s disease, Alzheimer’s disease and cardiovascular and neurodegenerative disorders. In addition, H₂O₂ is a reaction product of various oxidases such as glucose oxidase ¹ and, therefore, can be used to indirectly quantify the substrates of these enzymes. Hydrogels are hydrophilic, highly water-swellable polymer networks that have become of great interest in the development of biosensors, owing to their high biocompatibility and ability to incorporate foreign substances while preserving a benign environment for biosensing. Due to the 3D porous structure of hydrogels, which increases the surface area of these materials, it is possible to load large amounts of recognition bioelements while maintaining the native structure of the biomolecules². A CL guanosine-derived hydrogel was prepared via the simultaneous incorporation of luminol and hemin. The self-assembled hydrogel consisted of K⁺-stabilized hemin/G-quadruplex structures, showing significant peroxidase-like activity to the H₂O₂-mediated oxidation of luminol³. After adding H₂O₂, the generated CL signal lasts for several minutes and is intense enough to be captured via smartphone’s CMOS camera. As a proof of principle, this biomaterial was also used for the indirect detection of glucose in artificial serum samples after the incorporation of glucose oxidase into the hydrogel. The sensor showed a linear response with an estimated limit of detection of 50 µmol L^-1 (equivalent to 5 nmol of glucose)⁴.

References

[1] Calabria et al. Analytical Biochemistry (2020) 600:113760.

[2] Buenger et al. Progress in Polymer Science (2012) 37.12: 1678-1719

[3] Ye et al. Talanta (2021) 230:122351.

[4] Calabria et al. Biosensors (2023) 13.6:650

New techniques have been developed for functionalizing surfaces with nanometer-thick organic films, useful for attaching bio-molecules in sensors and biosensors. This method involves a novel surface functionalization technique that anchors alkyl chains at low potentials. It utilizes a process where aryl radicals from dazonium salts, which typically react with surfaces, are instead redirected to create reactive aliphatic radicals.

This is achieved using the 2,6-dimethylphenyl radical, which does not bind to surfaces like other aryl radicals but instead helps in generating alkyl radicals that attach to glassy carbon surfaces, forming stable nanometer-thick alkyl films. This process is more energy-efficient than direct reduction, with a 2eV advantage.

The method also allows for creating complex, mixed bifunctional nanometer films by reacting an aromatic radical with RI or RBr on the surface. These films can trap other molecules, contributing to the field of nanomedicine, especially in targeted drug delivery using polymeric nanoparticles.

Key findings include the following:

• Transfer of iodine atoms from alkyl iodides to sterically hindered aryl radicals, bypassing surface competition;
• Durability of grafted alkyl chains, resisting harsh conditions like ultrasonication, boiling solvents, and extreme voltage changes;
• Capability to form mono- or multilayer films ranging from 1 to 5 nm;
• Comprehensive characterization using techniques like IR, XPS, electrochemistry, ellipsometry, and water contact angle measurements.

Additionally, this method can be adapted to graft nanomaterials onto non-conductive surfaces using a reducing agent in solution instead of an electrode.

The research on DNA/RNA and protein identification down to the single oligomer level has significant advances. Nanopore-inspired systems have been extensively developed for applications in genome sequencing and are being adapted for protein sequencing. The surface-enhanced Raman scattering (SERS) method has the detectability of single oligomers but includes spectral variations related to environments and conformations. The machine learning (ML) methods were successfully applied in the spectral measurements. Molecular dynamics (MD) can provide us with simulated vibrational spectra in various environment. Identification of oligomers can be done by an ML Random Forest (RF) algorithm that has shown high accuracy for the experimental SERS. We investigate the applicability of the RF algorithm to identify nucleotides by vibrational spectra in MD sensors. The ring-averaged vibrational spectra of the DNA nucleotides were used. The spectra were obtained in interaction with the system of Au nanoparticles attached to a graphene sheet with nanopore. The first step was to apply the baseline correction to the decay component of the velocity correlation function present in the MD vibrational spectra to adjust intensities because the intensity of the peaks becomes comparable with the subtracted decay component at low frequencies. The 20 points b-spline and piece-wise linear baseline corrections have been tested. The frequencies f, amplitudes I, and differences for adjacent grid points were used as training test for RF algorithm that has shown accuracy of 93-96% on the grid of some 170 spectral points. The RF algorithm identifies the methylated forms of cytosine and reproduces differences in nucleotide spectra. Still, the lower frequency part of spectra [<1000 cm^-1] is reproduced with higher validity as compared to the higher frequency part above 1000 cm^-1 for nucleotides.

Enzymes have great application prospects in environmental monitoring and catalysis because they are highly specific and sensitive. However, maintaining the activity and stability of free enzymes for a long time is a huge problem. One method to overcome these problems is the immobilization of enzymes onto carriers. Our previous work proposed immobilizing enzymes onto core–shell nanoparticles, which have suitable polymer brushes and can obtain high immobilization yields while maintaining enzyme activity. However, non-conductive polymers and nanoparticle carriers limit the possibility of further applications of enzymes, such as sensors or wearable electronic devices. In this contribution, we aim to construct conductive hybrid particle systems by introducing conductive nanoparticles while loading enzymes and studying the activity of the enzyme at this condition.

We designed two strategies: 1. Fixed metal nanoparticles on the surface of silica particles combined with grafted polymers to immobilize enzymes. 2. Fixed metal particles on the grafted polymer brushes for enzyme immobilization. We also control the thickness of the polymer brushes to find the most suitable system for carrying enzymes by adjusting the polymerization time. Our study provides a better understanding of the positioning of conductive components in hybrid particles and opens the door to novel particle-based inks for the printing of enzymatic biosensors.