Search Results (25)

Early, rapid, and accurate detection is essential for clinical management and epidemiological control of acute respiratory infections caused by pathogens. Traditional testing methods such as microbial culture, serological testing, and PCR are restrictive in terms of operation and logistics and are therefore not easily used in point-of-care settings. The CRISPR/Cas system is an adaptive prokaryotic immune system composed of clustered regularly interspaced short palindromic repeats and their associated proteins, which has been used as a nucleic acid diagnostic platform with programmable sequence-specific target recognition and signal-amplifying collateral cleavage activity. Existing reviews have mostly focused on the classification of Cas enzymes or amplification strategies; in this review, a pathogen-centric approach was taken, covering viral pathogens (SARS-CoV-2, influenza virus, RSV, HAdV and VZV), bacterial pathogens (Mycobacterium tuberculosis, Streptococcus pneumoniae, Mycoplasma pneumoniae and Staphylococcus aureus) and fungal pathogens (Aspergillus fumigatus and Pneumocystis jirovecii). Key technological advances, such as isothermal amplification coupling, single-vessel integrated reaction designs, amplification-free digital detection, and electrochemical biosensor integration, are evaluated for Cas9-, Cas12-, and Cas13-based systems, with their mechanistic bases outlined. The current challenges that hinder clinical translation, such as sample matrix interference, multiple signal cross-talk, crRNA off-target effects, and the lack of large-scale validation studies, are critically assessed. At the same time, future pathways for portable, integrated, and inexpensive diagnostic platforms are suggested. Full article

In this study, the nonlinear and non-monotonic behavior of amperometric bienzyme biosensors employing an enzymatic trigger reaction is investigated analytically and computationally using a two-compartment model comprising an enzymatic layer and an outer diffusion layer. The trigger enzymatic reaction is coupled with a cyclic electrochemical–enzymatic conversion (CEC) process. The model is formulated as a system of reaction–diffusion equations incorporating nonlinear Michaelis–Menten kinetics and interlayer partitioning effects. Exact steady-state analytical solutions for substrate and product concentrations, as well as for the output current, are obtained for specific cases of first- and zero-order reaction kinetics. At the transition conditions, biosensor performance is further analyzed numerically using the finite difference method. The CEC biosensor exhibits the highest signal gain when the first enzyme has low activity and the second enzyme has high activity; however, under these conditions, the response time is the longest. When the first enzyme possesses a higher substrate affinity (lower Michaelis constant) than the second, the biosensor demonstrates severalfold higher current and gain compared to the reverse configuration under identical diffusion limitations. Furthermore, increasing external mass transport resistance or interfacial partitioning can enhance the apparent signal gain. Full article

A new approach in amperometric enzyme electrodes production based on all-electrochemically assisted procedures will be described. Enzyme (glucose oxidase) immobilization was performed by in situ co-crosslinking of enzyme molecules through electrophoretic protein deposition, assuring enzyme immobilization exclusively onto the transducer surface (Pt electrode). Analogously, the poor selectivity of the transducer was dramatically improved by the electrosynthesis of non-conducting polymers with built-in permselectivity, permitting the formation of a thin permselective film onto the transducer surface, able to reject common interferents usually found in real samples. Since both approaches required a proper and distinct electrochemical perturbation (a pulsed current sequence for electrophoretic protein deposition and cyclic voltammetry for the electrosynthesis of non-conducting polymers), an appropriate coupling of the two all-electrochemical approaches was assured by a thorough study of the likely combinations of the electrosynthesis of permselective polymers with enzyme immobilization by electrophoretic protein deposition and by the use of several electrosynthesized polymers. For each investigated combination and for each polymer, the analytical performances and the rejection capabilities of the resulting biosensor were acquired so to gain information about their sensing abilities eventually in real sample analysis. This study shows that the proper coupling of the two all-electrochemical approaches and the appropriate choice of the electrosynthesized, permselective polymer permits the easy fabrication of novel glucose oxidase biosensors with good analytical performance and low bias in glucose measurement from typical interferent in serum. This novel approach, resembling classical electroplating procedures, is expected to allow all the advantages expected from such procedures like an easy preparation biosensor, a bi-dimensional control of enzyme immobilization and thickness, interferent- and fouling-free transduction of the electrodic sensor and, last but not the least, possibility of miniaturization of the biosensing device. Full article

This study describes the development of catalytic surface immobilizing dopamine via cross-linking or tyrosinase through covalent bonds on an electrografted screen-printed carbon electrode with a 4-nitrobenzenediazonium ion. A simple electrochemical reduction approach was used to covalently graft aryldiazonium ions onto the surface of commercial electrodes. After functionalization with aminophenyl groups, dopamine, an important neurotransmitter, was immobilized by imine bond formation using glutaraldehyde as a bifunctional cross-linking molecule. The presence of immobilized dopamine was confirmed by cyclic voltammetry following the electrochemical response of the hydroquinone/quinone redox process from catechol functionalities on the surface, which are responsible for the catalytic activity. In addition, the surface was also characterized by cyclic voltammetry using the redox probe, [Fe(CN)₆]^3−/4−, obtaining a signal approximately 14 times higher than that of a bare electrode, achieving a dynamic concentration range spanning three orders of magnitude. Remarkable sensitivity was also obtained by combining the electrografting, in situ diazotation, to generate grafted aryl diazonium ions on the surface, and coupling reaction to anchor the tyrosinase enzyme to the electrode surface. The response of the TYR-biosensor towards catechol, using the redox probe as mediator, was 10 times higher than that obtained with the dopamine modified catalytic surface. These modified surfaces offer promising alternatives for the voltammetric quantification of catechol in environmental fields. Full article

Electrophoretic deposition is a powerful tool for depositing materials onto a substrate by using an electric field; its application in biotechnological areas, namely, electrophoretic protein deposition (EPD), is the most promising for, e.g., fabricating novel amperometric biosensors. Unfortunately, EPD suffers from several drawbacks due to coupled parasite electrochemical processes damaging the deposit; moreover, the nature of the deposition process, the deposit, and its stability are still controversial and unknown. The present research presents a deep investigation of the EPD processes conducted by using several electroanalytical techniques and an electrochemical quartz crystal microbalance (EQCM); notably, EPD was used here as a novel tool for performing an electrophoretically assisted, classical enzyme immobilization technique like co-crosslinking, thus permitting the immobilization of the desired protein in situ, i.e., exclusively onto the deposition electrode. An electrochemical study permitted the acquisition of useful insights about electrophoresis processes as well as solvent discharge and gas evolution at the deposition electrode; further, the use of appropriate current or potential pulse sequences, as investigated and improved in this study, together with fine-tuned chemical conditions, allowed the optimization of this novel EPD approach. Moreover, an EQCM study gave useful insights into the kinetics of the process, permitting a quantitative estimate of the deposit. Full article

Antibiotics have emerged as ground-breaking medications for the treatment of infectious diseases, but due to the excessive use of antibiotics, some drugs have developed resistance to microorganisms. Because of their structural complexity, most antibiotics are excreted unchanged, polluting the water, soil, and natural resources. Additionally, food items are being polluted through the widespread use of antibiotics in animal feed. The normal concentrations of antibiotics in environmental samples typically vary from ng to g/L. Antibiotic residues in excess of these values can pose major risks the development of illnesses and infections/diseases. According to estimates, 300 million people will die prematurely in the next three decades (by 2050), and the WHO has proclaimed “antibiotic resistance” to be a severe economic and sociological hazard to public health. Several antibiotics have been recognised as possible environmental pollutants (EMA) and their detection in various matrices such as food, milk, and environmental samples is being investigated. Currently, chromatographic techniques coupled with different detectors (e.g., HPLC, LC-MS) are typically used for antibiotic analysis. Other screening methods include optical methods, ELISA, electrophoresis, biosensors, etc. To minimise the problems associated with antibiotics (i.e., the development of AMR) and the currently available analytical methods, electrochemical platforms have been investigated, and can provide a cost-effective, rapid and portable alternative. Despite the significant progress in this field, further developments are necessary to advance electrochemical sensors, e.g., through the use of multi-functional nanomaterials and advanced (bio)materials to ensure efficient detection, sensitivity, portability, and reliability. This review summarises the use of electrochemical biosensors for the detection of antibiotics in milk/milk products and presents a brief introduction to antibiotics and AMR followed by developments in the field of electrochemical biosensors based on (i) immunosensor, (ii) aptamer (iii) MIP, (iv) enzyme, (v) whole-cell and (vi) direct electrochemical approaches. The role of nanomaterials and sensor fabrication is discussed wherever necessary. Finally, the review discusses the challenges encountered and future perspectives. This review can serve as an insightful source of information, enhancing the awareness of the role of electrochemical biosensors in providing information for the preservation of the health of the public, of animals, and of our environment, globally. Full article

Neurotransmitters (NTs) play a crucial role in regulating the behavioral and physiological functions of the nervous system. Imbalances in the concentrations of NT have been directly linked to various neurological diseases (e.g., Parkinson’s, Huntington’s, and Alzheimer’s disease), in addition to multiple psychotic disorders such as schizophrenia, depression, dementia, and other neurodegenerative disorders. Hence, the rapid and real-time monitoring of the NTs is of utmost importance in comprehending neurological functions and identifying disorders. Among different sensing techniques, electrochemical biosensors have garnered significant interest due to their ability to deliver fast results, compatibility for miniaturization and portability, high sensitivity, and good controllability. Furthermore, the utilization of enzymes as recognition elements in biosensing design has garnered renewed attention due to their unique advantages of catalytic biorecognition coupled with simultaneous signal amplification. This review paper primarily focuses on covering the recent advances in enzymatic electrochemical biosensors for the detection of NTs, encompassing the importance of electrochemical sensors, electrode materials, and electroanalytical techniques. Moreover, we shed light on the applications of enzyme-based biosensors for NTs detection in complex matrices and in vivo monitoring. Despite the numerous advantages of enzymatic biosensors, there are still challenges that need to be addressed, which are thoroughly discussed in this paper. Finally, this review also presents an outlook on future perspectives and opportunities for the development of enzyme-based electrochemical biosensors for NTs detection. Full article

The functional significance of cytochrome P450s (CYP) enzymes is their ability to catalyze the biotransformation of xenobiotics and endogenous compounds. P450 enzymes catalyze regio- and stereoselective oxidations of C-C and C-H bonds in the presence of oxygen as a cosubstrate. Initiation of cytochrome P450 catalytic cycle needs an electron donor (NADPH, NADH cofactor) in nature or alternative artificial electron donors such as electrodes, peroxides, photo reduction, and construction of enzymatic “galvanic couple”. In our review paper, we described alternative “handmade” electron sources to support cytochrome P450 catalysis. Physical-chemical methods in relation to biomolecules are possible to convert from laboratory to industry and construct P450-bioreactors for practical application. We analyzed electrochemical reactions using modified electrodes as electron donors. Electrode/P450 systems are the most analyzed in terms of the mechanisms underlying P450-catalyzed reactions. Comparative analysis of flat 2D and nanopore 3D electrode modifiers is discussed. Solar-powered photobiocatalysis for CYP systems with photocurrents providing electrons to heme iron of CYP and photoelectrochemical biosensors are also promising alternative light-driven systems. Several examples of artificial “galvanic element” construction using Zn as an electron source for the reduction of Fe³⁺ ion of heme demonstrated potential application. The characteristics, performance, and potential applications of P450 electrochemical systems are also discussed. Full article

In this work, two human epidermal growth factor receptors, HER-1 and HER-2, were selected as biomarkers to enable the detection of breast cancer. Therefore, two biosensors were developed using gold sensor chips coupled with amperometric detection of the enzyme label horse radish peroxidase (HRP). The biosensors/immunosensors relied on indirect sandwich enzyme-linked immunosorbent assays with monoclonal antibodies (Ab) against HER-1 and HER-2 attached to the sensors to capture the biomarkers. Detection polyclonal antibodies followed by secondary anti-rabbit (for HER-1) and anti-goat (for HER-2) IgG antibody-HRP were then applied for signal generation. In buffer, the developed sensors showed limits of detections (LOD) of 1.06 ng mL⁻¹ and 0.95 ng mL⁻¹ and limits of quantification (LOQ) of 2.1 ng mL⁻¹ and 1.5 ng mL⁻¹ for HER-1 and HER-2, respectively. In 100% (undiluted) serum, LODs of 1.2 ng mL⁻¹ and 1.47 ng mL⁻¹ and LOQs of 1.5 ng mL⁻¹ and 2.1 ng mL⁻¹ were obtained for HER-1 and HER-2, respectively. Such limits of detections are within the serum clinical range for the two biomarkers. Furthermore, gold nanoparticles (AuNP) labelled with secondary anti-rabbit and anti-goat IgG antibody-HRP were then used to enhance the assay signal and increase the sensitivity. In buffers, LODs of 30 pg mL⁻¹ were seen for both sensors and LOQs of 98 pg mL⁻¹ and 35 pg mL⁻¹ were recorded for HER-1 and HER-2, respectively. For HER-2 the AuNPs biosensor was also tested in 100% serum obtaining a LOD of 50 pg mL⁻¹ and a LOQ of 80 pg mL⁻¹. The HER-2 AuNP electrochemical immunosensor showed high specificity with very low cross-reactivity to HER-1. These findings demonstrate that the two developed sensors can enable early detection as well as monitoring of disease progression with a beneficial impact on patient survival and clinical outcomes. Full article

Foodborne disease caused by foodborne pathogens is a very important issue in food safety. Therefore, the rapid screening and sensitive detection of foodborne pathogens is of great significance for ensuring food safety. At present, many research works have reported the application of biosensors and signal amplification technologies to achieve the rapid and sensitive detection of pathogenic bacteria. Thus, this review summarized the use of biosensors coupled with signal amplification technology for the detection of pathogenic bacteria, including (1) the development, concept, and principle of biosensors; (2) types of biosensors, such as electrochemical biosensors, optical biosensors, microfluidic biosensors, and so on; and (3) different kinds of signal amplification technologies applied in biosensors, such as enzyme catalysis, nucleic acid chain reaction, biotin-streptavidin, click chemistry, cascade reaction, nanomaterials, and so on. In addition, the challenges and future trends for pathogenic bacteria based on biosensor and signal amplification technology were also discussed and summarized. Full article

Enzymatic biosensors enjoy commercial success and are the subject of continued research efforts to widen their range of practical application. For these biosensors to reach their full potential, their selectivity challenges need to be addressed by comprehensive, solid approaches. This review discusses the status of enzymatic biosensors in achieving accurate and selective measurements via direct biocatalytic and inhibition-based detection, with a focus on electrochemical enzyme biosensors. Examples of practical solutions for tackling the activity and selectivity problems and preventing interferences from co-existing electroactive compounds in the samples are provided such as the use of permselective membranes, sentinel sensors and coupled multi-enzyme systems. The effect of activators, inhibitors or enzymatic substrates are also addressed by coupled enzymatic reactions and multi-sensor arrays combined with data interpretation via chemometrics. In addition to these more traditional approaches, the review discusses some ingenious recent approaches, detailing also on possible solutions involving the use of nanomaterials to ensuring the biosensors’ selectivity. Overall, the examples presented illustrate the various tools available when developing enzyme biosensors for new applications and stress the necessity to more comprehensively investigate their selectivity and validate the biosensors versus standard analytical methods. Full article

Harmful algal blooms (HABs) are more frequent as climate changes and tropical toxic species move northward, especially along the Iberian Peninsula, a rich aquaculture area. Monitoring programs, detecting the presence of toxic algae before they bloom, are of paramount importance to protect ecosystems, aquaculture, human health and local economies. Rapid, reliable species identification methods using molecular barcodes coupled to biosensor detection tools have received increasing attention as an alternative to the legally required but impractical microscopic counting-based techniques. Our electrochemical detection system has improved, moving from conventional sandwich hybridization protocols using different redox mediators and signal probes with different labels to a novel strategy involving the recognition of RNA heteroduplexes by antibodies further labelled with bacterial antibody binding proteins conjugated with multiple enzyme molecules. Each change has increased sensitivity. A 150-fold signal increase has been produced with our newest protocol using magnetic microbeads (MBs) and amperometric detection at screen-printed carbon electrodes (SPCEs) to detect the target RNA of toxic species. We can detect as few as 10 cells L⁻¹ for some species by using a fast (~2 h), simple (PCR-free) and cheap methodology (~2 EUR/determination) that will allow this methodology to be integrated into easy-to-use portable systems. Full article

The excellent capabilities demonstrated over the last few years by electrochemical affinity biosensors should be largely attributed to their coupling with particular nanostructures including dendrimers, DNA-based nanoskeletons, molecular imprinted polymers, metal-organic frameworks, nanozymes and magnetic and mesoporous silica nanoparticles. This review article aims to give, by highlighting representative methods reported in the last 5 years, an updated and general overview of the main improvements that the use of such well-ordered nanomaterials as electrode modifiers or advanced labels confer to electrochemical affinity biosensors in terms of sensitivity, selectivity, stability, conductivity and biocompatibility focused on food and environmental applications, less covered in the literature than clinics. A wide variety of bioreceptors (antibodies, DNAs, aptamers, lectins, mast cells, DNAzymes), affinity reactions (single, sandwich, competitive and displacement) and detection strategies (label-free or label-based using mainly natural but also artificial enzymes), whose performance is substantially improved when used in conjunction with nanostructured systems, are critically discussed together with the great diversity of molecular targets that nanostructured affinity biosensors are able to quantify using quite simple protocols in a wide variety of matrices and with the sensitivity required by legislation. The large number of possibilities and the versatility of these approaches, the main challenges to face in order to achieve other pursued capabilities (development of antifouling, continuous operation, wash-, calibration- and reagents-free devices, regulatory or Association of Official Analytical Chemists, AOAC, approval) and decisive future actions to achieve the commercialization and acceptance of these devices in our daily routine are also noted at the end. Full article

A novel nanobiosensor was constructed with graphene oxide (GO) sheets coupled to pear extract-based green-synthesised silver nanoparticles (Ag-NPs) to which cytochrome P450-2D6 (CYP2D6) enzyme was attached. The biosensor was applied in the electrochemical detection of the tuberculosis (TB) treatment drugs, ethambutol (EMB) and pyrazinamide (PZA). The surface morphology of the green-synthesised nanocomposites was studied by performing High-Resolution Transmission Electron Microscopy (HR-TEM) and High-Resolution Scanning Electron Microscopy (HR-SEM). Fourier Transform Infrared Spectroscopy (FTIR) and Raman Spectroscopy were used for structural analysis, while Ultraviolet Visible (UV-Vis) Spectroscopy was used in the optical characterisation of the nanocomposite material. Electrochemical studies on glassy carbon electrode (GCE), which were done by Cyclic Voltammetry (CV), showed that the GO|Ag-NPs||GCE electrode was highly conductive, and thereby indicating its suitability as a platform for nanobiosensor development. The non-toxic and low-cost green GO|Ag-NPs|CYP2D6||GCE nanobiosensor was used to determine EMB and PZA. The very low limit of detection (LOD) values of the biosensor for EMB (0.2962 × 10⁻² nM, S/N = 3) and PZA (0.897 × 10⁻² nM, S/N = 3) demonstrate that the green nanobiosensor is more sensitive than other biosensors reported for EMB and PZA. Full article

The genus Yersinia contains three well-recognized human pathogens, including Y. enterocolitica, Y. pestis, and Y. pseudotuberculosis. Various domesticated and wild animals carry Yersinia in their intestines. Spread to individuals arises from eating food or water contaminated by infected human or animal faeces. Interaction with infected pets and domestic stock may also lead to infection. Yersinia is able to multiply at temperatures found in normal refrigerators; hence, a large number of the bacteria may be present if meat is kept without freezing. Yersinia is also rarely transmitted by blood transfusion, because it is able to multiply in stored blood products. Infection with Yersinia can cause yersiniosis, a serious bacterial infection associated with fever, abdominal pain and cramps, diarrhea, joint pain, and symptoms similar to appendicitis in older children and adults. This paper describes a novel immunosensor approach using graphene quantum dots (GQDs) as enzyme mimics in an electrochemical sensor set up to provide an efficient diagnostic method for Y. enterecolitica. The optimum assay conditions were initially determined and the developed immunosensor was subsequently used for the detection of the bacterium in milk and human serum. The GQD-immunosensor enabled the quantification of Y. enterocolitica in a wide concentration range with a high sensitivity (LOD_milk = 5 cfu mL⁻¹ and LOD_serum = 30 cfu mL⁻¹) and specificity. The developed method can be used for any pathogenic bacteria detection for clinical and food samples without pre-sample treatment. Offering a very rapid, specific and sensitive detection with a label-free system, the GQD-based immunosensor can be coupled with many electrochemical biosensors. Full article