Antibiotics are extensively used in veterinary medicine for the treatment of bacterial infections. However, their uncontrolled use can cause infiltration into milk and meat, which can cause antimicrobial resistance. Therefore, the development of sensitive and selective methods for the detection of antibiotics is an urgent need. Conventional methods such as HPLC or ELISA can detect antibiotics with high sensitivity; however, they require experienced personnel, expensive antibodies, and expensive instruments. Biosensor technology is an alternative to conventional analytical methods. A biosensor is composed of sensing layers with immobilized receptors and a transducer that convert chemical signals into measured electrical, optical, or acoustic values. DNA aptamers are relatively novel receptors that are also known as chemical antibodies. In contrast with antibodies, they are more stable and can be immobilized on various surfaces. We developed an electrochemical aptasensor based on a nanocomposite of reduced graphene oxide (rGO) and multiwalled carbon nanotubes (MWCNTs) enriched with carboxylic groups for sensitive detection of oxytetracycline (OTC). The amino-modified DNA aptamers were covalently immobilized on rGO-MWCNTs layers drop-casted on a glassy carbon electrode (GCE). Differential pulse voltammetry (DPV) in the presence of 5 mM [Fe(CN)6]^3-/4- was used for OTC detection. The limit of detection (LOD = 0.46 ng/mL) was much lower than the maximum residue limit (MRL) established for OTC by the EU (100 ng/mL). The selectivity of the sensor was demonstrated by using kanamycin, penicillin, chloramphenicol, and tetracycline antibiotics. The aptasensor was validated in 3.5 % fat milk with successful recovery.

Electrochemical biosensing has been extensively explored as a solution to cardiac troponin I (cTnI) detection. Biosensing has evolved to include signal amplification strategies such as nanoparticle-functionalised electrodes and using conductive coatings to enhance detection limits. However, these methods often introduce challenges, including signal noise, increased complexity requiring bulky equipment, intricate fabrication processes, and reduced assay reproducibility, limiting their clinical applicability. Three-dimensional printing allows for rapid and customisable electrode production, making it an ideal candidate for innovative electrode design which meets specific user needs and minimises variability in carbon electrode manufacturing. This study details various small electrodes fabricated using fused filament fabrication 3D printing [1], integrating multiwall carbon nanotubes and polylactic acid (MWCNT/PLA) as a simpler, more robust alternative. The 3D-printed electrodes were tailored for the electrochemical detection of oxidised tetramethylbenzidine (TMB_ox) using an electrochemical enzyme-linked immunosorbent assay (ELISA) for cTnI detection as formulated by previous work [2]. Electrodes of varying sizes and composite materials were fabricated and evaluated to optimise performance for TMB_ox detection. A sandwich ELISA format was used to capture and label cTnI, allowing for the indirect quantification of cTnI via chronoamperometric measurements, with absorbance-based ELISA as a comparator. This presentation will discuss how the miniaturisation and material optimisation of 3D-printed electrodes improve electrochemical ELISA sensitivity and reliability, and the prospects of integrating these technologies into a portable electrochemical ELISA format.

1. Xue, Z.; Patel, K.; Bhatia, P.; Miller, C.L.; Shergill, R.S.; Patel, B.A. 3D-Printed Microelectrodes for Biological Measurement. Anal. Chem. 2024, 96, 12701–12709, doi:10.1021/acs.analchem.4c01585.
2. Docherty, N.; Collins, L.; Pang, S.; Fu, Y.; Milne, S.; Corrigan, D. Cost-Effective Amperometric Immunosensor for Cardiac Troponin I as a Step towards Affordable Point-of-Care Diagnosis of Acute Myocardial Infarction. Sensing and Bio-Sensing Research 2025, 47, 100725, doi:10.1016/j.sbsr.2024.100725.

Lateral flow immunoassays (LFIAs) are a promising means of food quality control due to their rapidity and ease of implementation. Commonly, LFIA results are recorded based on the coloration of certain zones of test strips, in which immune complexes labeled with colored nanoparticles are formed. However, many nanoparticles have catalytic activity (are nanozymes) and can transform chromogenic substrates, enhancing coloration. Unfortunately, the choice of nanozyme size, shape modification technique, and degree of covering by immunoreagents for efficient LFIAs remain empirical.

The given work presents an application of different nanozymes for sensitive LFIAs for antibiotics in meat products. This is in high demand due to the numerous negative consequences of antibiotics entering the human body with food and the expansion of the list of antibiotics requiring extensive monitoring to protect consumer health.

A comparison was made for mono-, bi-, and tri-component nanoparticles of noble metals (gold, silver, and platinum) of different sizes and shapes as peroxidase-like nanozymes. The advantages of nanoparticles with a branched surface obtained by two-stage synthesis, namely, the formation of spherical gold nanoparticle cores and their partial surface modification with platinum, as catalysts were shown. Changes in the catalytic and antigen-binding activity of nanozyme–antibody complexes with varying surface densities of immobilized antibodies were considered. The limits of detection (LODs) for nanozyme-based LFIAs were reduced by tens of times compared to traditional LFIAs using spherical gold nanoparticles (AuNPs). For example, in the case of tetracycline, a 7.6-fold reduction was demonstrated using AuNPs as nanozymes instead of their direct photometry. Working with Au@Pt core–shell nanozymes led to a 20-30-fold reduction in LODs for chloramphenicol, tylosin, and tetracycline. The LFIAs were conducted by following the developed accelerated (15-20 min) protocols of sample preparation for raw and finished meat products.

This research was financially supported by the Russian Science Foundation under grant 24-16-00273.

Bacterial lipopolysaccharides (LPSs) are important indicators of bacterial infection in organisms or food contamination. They can be therefore used for medical diagnostics as well as for the detection of microbiological contamination in food and dairy products. We performed a comparative analysis of different techniques to isolate LPSs from Salmonella enterica serotype typhimurium (S. typhi). Different stages of the isolation method were applied to receive LPSs and the lipid component responsible for LPS toxicity called Lipid A was separated. As receptors for LPS detection, we used the lectin concanavalin A (ConA) or DNA aptamers immobilized at the gold layers of the quartz crystal modified by 11-mercaptoundecanoic acid. Using carbodiimide chemistry, the covalent immobilization of ConA or amino-modified DNA aptamers was possible. The interaction of LPSs with the sensing surface was studied by quartz crystal microbalance with dissipation monitoring (QCM-D). We have shown that DNA aptamers that specifically bind to lipid A in LPSs from S. typhi more strongly decrease the resonant frequency and increase the dissipation in comparison with ConA layers. Significant changes in the resonant frequency were observed already at 0.3 ng/mL of LPSs. The specificity of the interaction was confirmed by using LPSs isolated from other bacteria such as E. coli. We also used an extract of the bacterial membranes from Gram-positive bacteria Listeria monocytogenes that do not contain LPSs. In this case, no significant changes in frequency and dissipation were observed. Thus, the QCM-D is a sensitive tool for the detection of LPSs using DNA aptamers with high sensitivity and selectivity.

Acknowledgments. This work was funded under the European Union’s Horizon 2020 research and innovation program through the Marie Skłodowska-Curie grant agreement No. 101007299 (T.H.) and the Science Agency VEGA, project No. 1/0445/23 (T.H.).

Carbon electrode materials have the unique advantages of being low-cost, being easily manufactured, and exhibiting good electronic conductivity and high thermal and chemical stability [1]. Carbon exhibits a good choice for solid screen-printed electrodes that can be readily utilized in electrochemical biosensing. In particular, within this work, the surface of carbon screen-printed electrodes with a diameter of 2mm was modified with Au nanoparticles via electrodeposition for the immobilization of aptamers [2,3]. For this purpose, Au-thiolated aptamers were selected, and the route of chemisorption was followed, to allow the formation of a covalent bond with the Au nanoparticles. After the aptamer was immobilized, the system was used for the detection of tetracycline (TET). In particular, a concentration of 1μM aptamer was incubated on the Au-sensitized carbon working electrode (WE), followed by 0.1mM of Mercaptohexanol (MCH), and these were left overnight. Following that, different concentrations (5nM to 1000nM) of TET were used. Differential pulse voltammetry was performed using a Ag/AgCl reference and a Pt wire as a counter. The results yielded a limit of detection of the order of 10^-9nM. It can be noted that the aptasensor developed in this study can potentially be used for the detection of tetracycline in pharmaceutical preparations, drinking water, and contaminated food samples such as milk.

Electrochemical aptasensors been successfully applied in a number of fields, including food safety, enivormental monitoring and the health sector, providing a robust approach to the detection of a number of analytes. In particular, aptasensors have the advantage of flexible design, low immunogenicity and relative chemical/thermal stabilty. Moreover, aptamers, i.e in-vitro synthesized oligosequences, can offer a valid alternative to antibodies. In this work, we focus on electrochemical aptasensors based on semiconducting materials utilizing a mesoporous Mn:TiO₂ working electrode (WE) for the detection of tetracycline (TET). For this purpose, Mn:TiO₂ electrodes were prepared via the screen printing route, providing a low-cost approach. In particular, 5 μΜ οf the DNA aptamer with the following sequence: 5’-CCC CCG GCA GGC CAC GGC TTG GGTTGG TCC CAC TGC GCG-3’ [1,2] was used for the detection of different amounts of TET ranging from concentrations of 0.3 to 25.0 ng/ml in spiked aqueous samples. Detection was performed via differential pulse voltammetry (DPV) using a Pt wire cathode. In particular the buffer used in the experiment was Tris–HCl (20 mM, pH 7.6), 100 mM NaCl, MgCl2(2 mM), KCl (5 mM) and CaCl2(1 mM). The limit of detection was lower than the maximum residue limit required by the European Commmision for 100ng of tetracycline/mg, showing a low-cost alternative for TET detection.

Research has revealed that over 90% of duodenal ulcers and approximately 80% of gastric ulcers are linked to Helicobacter. pylori infections. Beyond ulcers, it can cause mild chronic gastric inflammation and may even lead to gastric cancer. Several diagnostic techniques have been developed to detect H. pylori, spanning bacteriological, genomic, isotope tracing, pathological, serological, and molecular methods. Despite their effectiveness, most of these methods necessitate specialized equipment and skilled personnel, limiting their application in early detection and prevention, which can have serious health and economic implications. Hence, there is an urgent need for a simple, rapid, highly specific, and cost-effective approach for detecting H. pylori. This study presents the first selection, identification, and biosensing application of the H. pylori surface protein Cag A and the development of a novel label-free electrochemical aptasensor used for signal enhancement to achieve the facile, rapid, and direct detection of the Cag A antigen using a conductive SA hydrogel as an efficient solid support on the ITO surface. The fabricated aptaprobe has a satisfactory selective recognition function toward the Cag A antigen via the DPV technique, even in the presence of a high concentration of co-existing elements. The aptasensor demonstrated an acceptable wide linear range (0.1-80 ng mL^-1), high sensitivity [22.64 mA log₁₀ (mL ng^-1) cm^-2)], a low sensing limit (0.17 ng mL^-1) and limit of quantification (0.54 ng mL^-1), and a rapid response time (∼14 min). Although clinical validation is needed to establish its clinical utility, the findings of our study are promising and encouraging.

Introduction

The development of biomolecular systems for computational tasks is a promising area in bioengineering and biosensor techniques. Using modified protein molecules as functional components of such systems enables the creation of next-generation devices based on the principles of biocomputing. In this study, we explored approaches to synthesizing dual-modified proteins with controlled surface characteristics and their potential application in implementing logical operations.

Materials and Methods

Dual-modified proteins were synthesized through the controlled sequential conjugation of test proteins, bovine serum albumin, and gelatin with low-molecular-weight compounds such as chloramphenicol and biotin, using carbodiimide chemistry. Low-molecular-weight substances were dissolved in a mixed buffer system with dimethyl sulfoxide, and their carboxyl groups were activated by a carbodiimide reagent. The activated compounds were then added to a protein solution in borate buffer, maintaining a precise molar ratio of 1:37:12 to ensure proper structural organization. This step allowed for an equilibrium distribution of surface charges, optimizing molecular interactions. The entire process was meticulously monitored using a label-free optical biosensor system based on spectral-phase interferometry (SPI). The final conjugates were diluted in a phosphate buffer and stored at 4 °C.

Results and Discussion

This study investigated the potential of using the obtained modified protein molecules to perform computational tasks, exemplified by the logical operations “YES” and “NOT.” By adding low-molecular-weight substances to the protein solution in a specific sequence during conjugation, the surface characteristics of the resulting product could be tailored. The steric properties of the surface determine the interaction pattern of the conjugate with other molecules in the solution, enabling it to reproduce specific reactions in a defined environment. These reactions, representing the output signals of a logical operator (logical “0” or “1”), were recorded using the SPI method and digitized.

Conclusions

This study demonstrates the potential of dual-modified proteins for use in biocomputing systems. Future work will focus on expanding the range of logical operations and optimizing the synthesis of dual-modified proteins to enhance their reproducibility and performance in biocomputing systems.

Introduction

Chloramphenicol (CAP) is a widely used antibiotic in veterinary medicine, posing a risk of contamination in animal-derived food products. Detecting CAP is crucial due to its potential health hazards, such as allergic reactions and antibiotic resistance. Immunoanalytical test systems based on monoclonal antibodies (mAbs) are commonly used to detect CAP. However, most studies focus on mAb interactions with CAP-conjugates, while the kinetics of interactions with free CAP remain poorly understood. Our study addresses this gap, for the first time evaluating the kinetic parameters of mAbs binding to both CAP-conjugates and free CAP. This dual characterization is essential for designing more effective detection systems.

Materials and Methods

To achieve this, we used a label-free biosensor system based on low-coherence interferometry. Carboxylated glass chips immobilized with bovine serum albumine (BSA-CAP) conjugates served as the sensor surface. The interactions of the sensor with monoclonal antibodies and mixtures of antibodies with free CAP were analyzed. A tailored kinetic model was applied to calculate dissociation constants and evaluate the binding dynamics.

Results and Discussion

Our results reveal the kinetic parameters for mAb interactions with both BSA-CAP conjugates and free CAP. The dissociation constants and binding dynamics provide critical insights into the affinity and specificity of mAbs under different conditions. This approach demonstrates the system's capacity to differentiate interactions with conjugated and free CAP, showcasing its applicability in developing precise and reliable CAP detection methods.

Conclusions

This study establishes a novel methodology for characterizing mAb interactions with free CAP alongside conjugated forms. By leveraging a low-coherence interferometry-based biosensor, the findings contribute to the creation of advanced, high-sensitivity detection systems for CAP, ultimately enhancing food safety monitoring.­­­­­

Cellulose is one of the most abundant biopolymers, and its many properties make it suitable for application in numerous fields, such as sensor and biosensor fabrication. It is indeed biodegradable and renewable, nontoxic, biocompatible, and widely available at low cost (https://doi.org/10.3390/chemosensors10090352). In addition, cellulose boasts different structural arrangements such as Cellulose Nanocrystals (CNCs), which are among the most promising cellulose-derived nanomaterials for applications in biosensors. In fact, CNCs can be formulated into thin films to be used as support material for the immobilization of proteins (enzymes, antibodies, etc.) (https://doi.org/10.3390/chemosensors10090352, https://doi.org/10.1016/j.sbsr.2020.100368). In this work, we exploited antibody-functionalized CNCs to develop a microfluidic paper-based analytical device (µPAD) for the detection of myeloperoxidase blood levels, which have been shown to be altered in patients with Alzheimer's disease (AD) (https://doi.org/10.5539/gjhs.v6n5p87, https://doi.org/10.1111/j.1471-4159.2004.02527.x, https://doi.org/10.1016/j.arr.2020.101130). In particular, upon their covalent functionalization with anti-myeloperoxidase antibody, CNCs were deposited onto filter paper, thus providing a uniform and stable functionalized layer. Subsequently, employing the origami format, myeloperoxidase contained in the sample was captured by the immobilized antibody and then detected, exploiting its ability to catalyse the luminol/hydrogen peroxide chemiluminescence reaction. Emitted photons were detected by employing a portable highly sensitive charge-coupled device (CCD) camera and analyzed to obtain quantitative information. A limit of detection of 5.8 ng/mL was obtained, which enables clear distinction between healthy and AD patients’ myeloperoxidase blood levels (https://doi.org/10.3233/jad-131469). In addition, the selectivity of the system was evaluated by testing the possible interference of haemoglobin, which is also able to catalyse the chemiluminescence reaction. In the future, the same approach will be used for developing origami µPADs for detecting other AD biomarkers exploiting chemiluminescence sandwich immunoassays.

This research is supported by the PRIN2022 project 2022WN89PC “Biomimetic sensing platforms for the detection of Alzheimer's disease related biomarkers”.