Conceptually innovative fluorophores for functional bioimaging.

Fluorophore chemistry is at the forefront of bioimaging, revolutionizing the visualization of biological processes with unparalleled precision. From the serendipitous discovery of mauveine in 1856 to cutting-edge fluorophore engineering, this field has undergone transformative evolution. Today, the synergy of chemistry, biology, and imaging technologies has produced diverse, specialized fluorophores that enhance brightness, photostability, and targeting capabilities. This review delves into the history and innovation of fluorescent probes, showcasing their pivotal role in advancing our understanding of cellular dynamics and disease mechanisms. We highlight groundbreaking molecules and their applications, envisioning future breakthroughs that promise to redefine biomedical research and diagnostics.

Red and near-infrared light-activated photoelectrochemical nanobiosensors for biomedical target detection.

Photoelectrochemical (PEC) nanobiosensors integrate molecular (bio)recognition elements with semiconductor/plasmonic photoactive nanomaterials to produce measurable signals after light-induced reactions. Recent advancements in PEC nanobiosensors, using light-matter interactions, have significantly improved sensitivity, specificity, and signal-to-noise ratio in detecting (bio)analytes. Tunable nanomaterials activated by a wide spectral radiation window coupled to electrochemical transduction platforms have further improved detection by stabilizing and amplifying electrical signals. This work reviews PEC biosensors based on nanomaterials like metal oxides, carbon nitrides, quantum dots, and transition metal chalcogenides (TMCs), showing their superior optoelectronic properties and analytical performance for the detection of clinically relevant biomarkers. Furthermore, it highlights the innovative role of red light and NIR-activated PEC nanobiosensors in enhancing charge transfer processes, protecting them from biomolecule photodamage in vitro and in vivo applications. Overall, advances in PEC detection systems have the potential to revolutionize rapid and accurate measurements in clinical diagnostic applications. Their integration into miniaturized devices also supports the development of portable, easy-to-use diagnostic tools, facilitating point-of-care (POC) testing solutions and real-time monitoring.

Microfluid biosensor for detection of hpv in patient scraping samples: Determining E6 and E7 oncogenes.

E6 and E7 oncogenes are pivotal in the carcinogenic transformation in HPV infections and efficient diagnostic methods can ensure the detection and differentiation of HPV genotype. This study describes the development and validation of an electrochemical, label-free genosensor coupled with a microfluidic system for detecting the E6 and E7 oncogenes in cervical scraping samples. The nanostructuring employed was based on a cysteine and graphene quantum dots layer that provides functional groups, surface area, and interesting electrochemical properties. Biorecognition tests with cervical scraping samples showed differentiation in the voltammetric response. Low-risk HPV exhibited a lower biorecognition response, reflected in ΔI% values of 82.33 % ± 0.29 for HPV06 and 80.65 % ± 0.68 for HPV11 at a dilution of 1:100. Meanwhile, high-risk, HPV16 and HPV18, demonstrated ΔI% values of 96.65 % ± 1.27 and 93 % ± 0.026, respectively, at the same dilution. Therefore, the biorecognition intensity followed the order: HPV16 >HPV18 >HPV06 >HPV11. The limit of detection and the limit of quantification of E6E7 microfluidic LOC-Genosensor was 26 fM, and 79.6 fM. Consequently, the E6E7 biosensor is a valuable alternative for clinical HPV diagnosis, capable of detecting the potential for oncogenic progression even in the early stages of infection.

The Tunable Parameters of Graphene-Based Biosensors.

Graphene-based surface plasmon resonance (SPR) biosensors have emerged as a promising technology for the highly sensitive and accurate detection of biomolecules. This study presents a comprehensive theoretical analysis of graphene-based SPR biosensors, focusing on configurations with single and bimetallic metallic layers. In this study, we investigated the impact of various metallic substrates, including gold and silver, and the number of graphene layers on key performance metrics: sensitivity of detection, detection accuracy, and quality factor. Our findings reveal that configurations with graphene first supported on gold exhibit superior performance, with sensitivity of detection enhancements up to 30% for ten graphene layers. In contrast, silver-supported configurations, while demonstrating high sensitivity, face challenges in maintaining detection accuracy. Additionally, reducing the thickness of metallic layers by 30% optimizes light coupling and enhances sensor performance. These insights highlight the significant potential of graphene-based SPR biosensors in achieving high sensitivity of detection and reliability, paving the way for their application in diverse biosensing technologies. Our findings pretend to motivate future research focusing on optimizing metallic layer thickness, improving the stability of silver-supported configurations, and experimentally validating the theoretical findings to further advance the development of high-performance SPR biosensors.

Upconverting Nanoparticle-based Enhanced Luminescence Lateral-Flow Assay for Urinary Biomarker Monitoring.

Development of efficient portable sensors for accurately detecting biomarkers is crucial for early disease diagnosis, yet remains a significant challenge. To address this need, we introduce the enhanced luminescence lateral-flow assay, which leverages highly luminescent upconverting nanoparticles (UCNPs) alongside a portable reader and a smartphone app. The sensor's efficiency and versatility were shown for kidney health monitoring as a proof of concept. We engineered Er3+- and Tm3+-doped UCNPs coated with multiple layers, including an undoped inert matrix shell, a mesoporous silica shell, and an outer layer of gold (UCNP@mSiO2@Au). These coatings synergistically enhance emission by over 40-fold and facilitate biomolecule conjugation, rendering UCNP@mSiO2@Au easy to use and suitable for a broad range of bioapplications. Employing these optimized nanoparticles in lateral-flow assays, we successfully detected two acute kidney injury-related biomarkers─kidney injury molecule-1 (KIM-1) and neutrophil gelatinase-associated lipocalin (NGAL)─in urine samples. Using our sensor platform, KIM-1 and NGAL can be accurately detected and quantified within the range of 0.1 to 20 ng/mL, boasting impressively low limits of detection at 0.28 and 0.23 ng/mL, respectively. Validating our approach, we analyzed clinical urine samples, achieving biomarker concentrations that closely correlated with results obtained via ELISA. Importantly, our system enables biomarker quantification in less than 15 min, underscoring the performance of our novel UCNP-based approach and its potential as reliable, rapid, and user-friendly diagnostics.

Role of Graphene in Surface Plasmon Resonance-Based Biosensors.

This work explores the transformative role of graphene in enhancing the performance of surface plasmon resonance (SPR)-based biosensors. The motivation for this review stems from the growing interest in the unique properties of graphene, such as high surface area, excellent electrical conductivity, and versatile functionalization capabilities, which offer significant potential to improve the sensitivity, specificity, and stability of SPR biosensors. This review systematically analyzes studies published between 2010 and 2023, covering key metrics of biosensor performance. The findings reveal that the integration of graphene consistently enhances sensitivity. Specificity, although less frequently reported numerically, showed promising results, with high specificity achieved at sub-nanomolar concentrations. Stability enhancements are also significant, attributed to the protective properties of graphene and improved biomolecule adsorption. Future research should focus on mechanistic insights, optimization of integration techniques, practical application testing, scalable fabrication methods, and comprehensive comparative studies. Our findings provide a foundation for future research, aiming to further optimize and harness the unique physical properties of graphene to meet the demands of sensitive, specific, stable, and rapid biosensing in various practical applications.

Detection of foodborne pathogens in contaminated food using nanomaterial-based electrochemical biosensors.

Foodborne pathogens are a grave concern for the for food, medical, environmental, and economic sectors. Their ease of transmission and resistance to treatments, such as antimicrobial agents, make them an important challenge. Food tainted with these pathogens is swiftly rejected, and if ingested, can result in severe illnesses and even fatalities. This review provides and overview of the current status of various pathogens and their metabolites transmitted through food. Despite a plethora of studies on treatments to eradicate and inhibit these pathogens, their indiscriminate use can compromise the sensory properties of food and lead to contamination. Therefore, the study of detection methods such as electrochemical biosensors has been proposed, which are devices with advantages such as simplicity, fast response, and sensitivity. However, these biosensors may also present some limitations. In this regard, it has been reported that nanomaterials with high conductivity, surface-to-volume ratio, and robustness have been observed to improve the detection of foodborne pathogens or their metabolites. Therefore, in this work, we analyze the detection of pathogens transmitted through food and their metabolites using electrochemical biosensors based on nanomaterials.

Ultrafast and highly sensitive detection of SARS-CoV-2 spike protein by field-effect transistor graphene-based biosensors.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), etiological agent for the coronavirus disease 2019 (COVID-19), has resulted in over 775 million global infections. Early diagnosis remains pivotal for effective epidemiological surveillance despite the availability of vaccines. Antigen-based assays are advantageous for early COVID-19 detection due to their simplicity, cost-effectiveness, and suitability for point-of-care testing (PoCT). This study introduces a graphene field-effect transistor-based biosensor designed for high sensitivity and rapid response to the SARS-CoV-2 spike protein. By functionalizing graphene with monoclonal antibodies and applying short-duration gate voltage pulses, we achieve selective detection of the viral spike protein in human serum within 100 µs and at concentrations as low as 1 fg ml-1, equivalent to 8 antigen molecules perµl of blood. Furthermore, the biosensor estimates spike protein concentrations in serum from COVID-19 patients. Our platform demonstrates potential for next-generation PoCT antigen assays, promising fast and sensitive diagnostics for COVID-19 and other infectious diseases.

CRISPR-based electrochemical biosensors: an alternative for point-of-care diagnostics?

The combination of CRISPR technology and electrochemical sensors has sparked a paradigm shift in the landscape of point-of-care (POC) diagnostics. This review explores the dynamic convergence between CRISPR and electrochemical sensing, elucidating their roles in rapid and precise biosensing platforms. CRISPR, renowned for its remarkable precision in genome editing and programmability capability, has found a novel application in conjunction with electrochemical sensors, promising highly sensitive and specific detection of nucleic acids and biomarkers associated with diverse diseases. This article navigates through fundamental principles, research developments, and applications of CRISPR-based electrochemical sensors, highlighting their potential to revolutionize healthcare accessibility and patient outcomes. In addition, some key points and challenges regarding applying CRISPR-powered electrochemical sensors in real POC settings are presented. By discussing recent advancements and challenges in this interdisciplinary field, this review evaluates the potential of these innovative sensors as an alternative for decentralized, rapid, and accurate POC testing, offering some insights into their applications across clinical scenarios and their impact on the future of diagnostics.

Utilizing COVID-19 as a Model for Diagnostics Using an Electrochemical Sensor.

This paper reports a rapid and sensitive sensor for the detection and quantification of the COVID-19 N-protein (N-PROT) via an electrochemical mechanism. Single-frequency electrochemical impedance spectroscopy was used as a transduction method for real-time measurement of the N-PROT in an immunosensor system based on gold-conjugate-modified carbon screen-printed electrodes (Cov-Ag-SPE). The system presents high selectivity attained through an optimal stimulation signal composed of a 0.0 V DC potential and 10 mV RMS-1 AC signal at 100 Hz over 300 s. The Cov-Ag-SPE showed a log response toward N-PROT detection at concentrations from 1.0 ng mL-1 to 10.0 µg mL-1, with a 0.977 correlation coefficient for the phase (θ) variation. An ML-based approach could be created using some aspects observed from the positive and negative samples; hence, it was possible to classify 252 samples, reaching 83.0, 96.2 and 91.3% sensitivity, specificity, and accuracy, respectively, with confidence intervals (CI) ranging from 73.0 to 100.0%. Because impedance spectroscopy measurements can be performed with low-cost portable instruments, the immunosensor proposed here can be applied in point-of-care diagnostics for mass testing, even in places with limited resources, as an alternative to the common diagnostics methods.

Revolutionizing food safety with electrochemical biosensors for rapid and portable pathogen detection.

Foodborne diseases remain a worldwide concern, despite the advances made in sanitation, pathogen surveillance and food safety management systems. The methods routinely applied for detecting pathogens in foods are time consuming, labor intensive and usually require trained and qualified individuals. The objective of this review was to highlight the use of biosensors, with a focus on the electrochemical devices, as promising alternatives for detecting foodborne pathogens. These biosensors present high speed for obtaining results, with the possibility of evaluating foods in real time, at low cost, ease of use, in addition to being compact and portable. These aspects are considered advantageous and suitable for use in food safety management systems. This work also shows some limitations for the application of biosensors, and we present perspectives with the development and use of nanomaterials.

Single-Domain Antibody-Gold Nanoparticle Bioconjugates as Immunosensors for the Detection of Hantaviruses.

INTRODUCTION: Hantavirus, a zoonotic pathogen, causes severe syndromes like hemorrhagic fever with renal syndrome (HFRS), sometimes fatal in humans. Considering the importance of detecting the hantavirus antigen, the construction of an immunosensor is essential. The structural and functional characteristics of camelid nanobodies (VHHs) encourage their application in the areas of nanobiotechnology, therapeutics, diagnostics, and basic research. Therefore, this study aimed to standardize stable bioconjugates using gold nanoparticles (AuNPs) and VHHs, in order to develop immunobiosensors for the diagnosis of hantavirus infection. METHODS: Immobilized metal affinity chromatography (IMAC) was performed to obtain purified recombinant anti-hantavirus nucleocapsid nanobodies (anti-prNΔ85 VHH), while AuNPs were synthesized for bioconjugation. UV-visible spectrophotometry and transmission electron microscopy (TEM) analysis were employed to characterize AuNPs. RESULTS: The bioconjugation stability parameters (VHH-AuNPs), analyzed by spectrophotometry, showed that the ideal pH value and VHH concentration were obtained at 7.4 and 50 µg/mL, respectively, after addition of 1 M NaCl, which induces AuNP aggregation. TEM performed before and after bioconjugation showed uniform, homogeneous, well-dispersed, and spherical AuNPs with an average diameter of ~ 14 ± 0.57 nm. Furthermore, high-resolution images revealed a thin white halo on the surface of the AuNPs, indicating the coating of the AuNPs with protein. A biosensor simulation test (dot blot-like [DB-like]) was performed in stationary phase to verify the binding and detection limits of the recombinant nucleocapsid protein from the Araucária hantavirus strain (prN∆85). DISCUSSION: Using AuNPs/VHH bioconjugates, a specific interaction was detected between 5 and 10 min of reaction in a dose-dependent manner. It was observed that this test was sensitive enough to detect prNΔ85 at concentrations up to 25 ng/µL. Considering that nanostructured biological systems such as antibodies conjugated with AuNPs are useful tools for the development of chemical and biological sensors, the stability of the bioconjugate indicates proficiency in detecting antigens. The experimental results obtained will be used in a future immunospot assay or lateral flow immunochromatography analysis for hantavirus detection.

Portable T-2 toxin biosensor based on target-responsive DNA hydrogel using water column height as readout.

T-2 toxin, a hazardous mycotoxin often present in cereals and products based on cereals, poses a substantial risk to humans and animals due to its high toxicity. The development of uncomplicated, quick and highly sensitive methods for detecting T-2 toxin is imperative. In this work, a portable sensing system was constructed using water column height as a readout device in combination with a controlled release system, which allows for an accurate quantitative analysis of T-2 toxin without the need for expensive instrumentation or skilled technicians. Hyaluronic acid (HA) hydrogel was constructed by double cross-linked DNA/aptamer hybrids with polyethyleneimine (PEI) and embedded with platinum nanoparticles (Pt NPs). The aptamer specifically bound to T-2 toxin in its presence, resulting in the disruption of the hydrogel and subsequent release of the Pt NPs. These Pt NPs were later mixed with a solution of H2O2 in a confined reaction flask, leading to the decomposition of H2O2 into O2. A glass capillary tube containing a column of red water had been inserted into the cap of the reaction flask, and the low solubility of O2 led to an increase in pressure within the reaction unit, causing the red water column to rise. There is a good linear correlation between the height of the capillary liquid level and the T-2 toxin concentration in the range of 20 ng/mL to 6 µg/mL. The system has been successfully used to detect T-2 toxin in samples of barley tea and corn.

Development of optical and electrochemical immunodevices for dengue virus detection.

Dengue virus (DENV) is the most prevalent global arbovirus, exhibiting a high worldwide incidence with intensified severity of symptoms and alarming mortality rates. Faced with the limitations of diagnostic methods, an optical and electrochemical biosystem was developed for the detection of DENV genotypes 1 and 2, using cysteine (Cys), cadmium telluride (CdTe) quantum dots, and anti-DENV antibodies. Cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS), surface plasmon resonance (SPR), atomic force microscopy (AFM), and Fourier transform infrared spectroscopy (FTIR) were employed to characterize the immunosensor. The AFM and SPR results demonstrated discernible topographic and angular changes confirming the biomolecular recognition. Different concentrations of DENV-1 and DENV-2 were evaluated (0.05 × 106 to 2.0 × 106 PFU mL-1), resulting in a maximum anodic shift (ΔI%) of 263.67% ± 12.54 for DENV-1 and 63.36% ± 3.68 for DENV-2. The detection strategies exhibited a linear response to the increase in viral concentration. Excellent linear correlations, with R2 values of 0.95391 for DENV-1 and 0.97773 for DENV-2, were obtained across a broad concentration range. Data analysis demonstrated high reproducibility, displaying relative standard deviation values of 3.42% and 3.62% for Cys-CdTe-antibodyDENV-1-BSA and Cys-CdTe-antibodyDENV-2-BSA systems. The detection limits were 0.34 × 106 PFU mL-1 and 0.02 × 106 PFU mL-1, while the quantification limits were set at 1.49 × 106 PFU mL-1 and 0.06 × 106 PFU mL-1 for DENV-1 and DENV-2, respectively. Therefore, the biosensing apparatus demonstrates analytical effectiveness in viral screening and can be considered an innovative solution for early dengue diagnosis, contributing to global public health.

Point-of-Care Electrochemical Immunosensor Applied against Nosocomial Infection: Staphylococcus aureus Detection in Human Hand Skin.

Staphylococcus aureus is an important pathogen that causes nosocomial infections, resulting in unacceptable morbidity and mortality rates. In this work, we proposed the construction of a nanostructured ZnO-based electrochemical immunosensor for qualitative and semiquantitative detection of S. aureus using simple methods for growing zinc oxide nanorods (ZnO NRs) on a sensor board and immobilizing the anti-S. aureus antibody on ZnO NRs through cystamine and glutaraldehyde. The immunosensor detected S. aureus in the 103-107 colony-forming unit (CFU) mL-1 range and showed a limit of detection (LoD) around 0.792 × 103 CFU mL-1. Beyond a satisfactory LoD, the developed immunosensor presented other advantages, such as high versatility for point-of-care assays and a suitable selective factor that admits the detection of the S. aureus concentration range in human hand skin after washing. Moreover, the immunosensor showed the potential to be an excellent device to control nosocomial infection by detecting the presence of S. aureus in human hand skin.

Detection of Mtb and NTM: preclinical validation of a new asymmetric PCR-binary deoxyribozyme sensor assay.

Tuberculosis (TB) and infectious diseases caused by non-tuberculous mycobacteria (NTM) are global concerns. The development of a rapid and accurate diagnostic method, capable of detecting and identifying different mycobacteria species, is crucial. We propose a molecular approach, the BiDz-TB/NTM, based on the use of binary deoxyribozyme (BiDz) sensors for the detection of Mycobacterium tuberculosis (Mtb) and NTM of clinical interest. A panel of DNA samples was used to evaluate Mtb-BiDz, Mycobacterium abscessus/Mycobacterium chelonae-BiDz, Mycobacterium avium-BiDz, Mycobacterium intracellulare/Mycobacterium chimaera-BiDz, and Mycobacterium kansasii-BiDz sensors in terms of specificity, sensitivity, accuracy, and limit of detection. The BiDz sensors were designed to hybridize specifically with the genetic signatures of the target species. To obtain the BiDz sensor targets, amplification of a fragment containing the hypervariable region 2 of the 16S rRNA was performed, under asymmetric PCR conditions using the reverse primer designed based on linear-after-the-exponential principles. The BiDz-TB/NTM was able to correctly identify 99.6% of the samples, with 100% sensitivity and 0.99 accuracy. The individual values of specificity, sensitivity, and accuracy, obtained for each BiDz sensor, satisfied the recommendations for new diagnostic methods, with sensitivity of 100%, specificity and accuracy ranging from 98% to 100% and from 0.98 to 1.0, respectively. The limit of detection of BiDz sensors ranged from 12 genome copies (Mtb-BiDz) to 2,110 genome copies (Mkan-BiDz). The BiDz-TB/NTM platform would be able to generate results rapidly, allowing the implementation of the appropriate therapeutic regimen and, consequently, the reduction of morbidity and mortality of patients.IMPORTANCEThis article describes the development and evaluation of a new molecular platform for accurate, sensitive, and specific detection and identification of Mycobacterium tuberculosis and other mycobacteria of clinical importance. Based on BiDz sensor technology, this assay prototype is amenable to implementation at the point of care. Our data demonstrate the feasibility of combining the species specificity of BiDz sensors with the sensitivity afforded by asymmetric PCR amplification of target sequences. Preclinical validation of this assay on a large panel of clinical samples supports the further development of this diagnostic tool for the molecular detection of pathogenic mycobacteria.

Ultrasensitive Aptasensing Platform for the Detection of ß-Amyloid-42 Peptide Based on MOF Containing Bimetallic Porphyrin Graphene Oxide and Gold Nanoparticles.

The prompt detection of diseases hinges on the accessibility and the capability to identify relevant biomarkers. The integration of aptamers and the incorporation of nanomaterials into signal transducers have not only expedited but also enhanced the development of nanoaptasensors, enabling heightened sensitivity and selectivity. Here, the bimetallic nickel-cobalt-porphyrin metal-organic framework ((Ni + Cu)TPyP MOF) is regarded as an electron mediator, immobilization platform for an Alzheimer aptamer and to increase the electrochemical signal for the detection of the main biomarker of Alzheimer's disease (AD), amyloid ß (Aß-42). Furthermore, the ((Ni + Cu)TPyP MOF) was combined with reduced graphene oxide (rGO) and gold nanoparticles (AuNPs), on a gold electrode (GE) to provide an efficient interface for immobilizing aptamer strands. Concurrently, the incorporation of rGO and AuNPs imparts enhanced electrical conductivity and efficacious catalytic activity, establishing them as adept electrochemical indicators. Owing to the superior excellent electrical conductivity of rGO and AuNPs, coupled with the presence of ample mesoporous channels and numerous Ni and Cu metal sites within (Ni + Cu)TPyP MOF, this nanostructure with abundant functional groups is proficient in immobilizing a substantial quantity of aptamer. These interactions are achieved through robust π-π stacking and electrostatic interactions, alongside the high affinity between the thiol group of the aptamer and AuNPs concurrently. The as-prepared ternary (Au@(Ni + Cu)TPyP MOF/rGO) nanostructure electrode exhibited an enhancement in its electrochemically active surface area of about 7 times, compared with the bare electrode and the Aß-42 redox process is highly accelerated, so the peak currents are significantly higher than those obtained with bare GE substrate. Under the optimized conditions, the designed aptasensor had the quantitative detection of Aß-42 with a low detection limit of 48.6 fg mL-1 within the linear range of 0.05 pg mL-1 to 5 ng mL-1 by differential pulse voltammetry (DPV), accompanied by precise reproducibility, satisfactory stability (95.6% of the initial activity after 10 days), and minimal impact of interfering agents. Recorded results in human blood plasma demonstrated the high efficacy of porphyrin MOF system sensing even in the clinical matrix. The great performance of this aptasensor indicates that our new design of Au@(Ni + Cu)TPyP MOF/rGO nanostructure provides more opportunities for the detection of chemical signals in early diagnosis of Alzheimer's disease.

Non-enzymatic biosensor based on F,S-doped carbon dots/copper nanoarchitecture applied in the simultaneous electrochemical determination of NADH, dopamine, and uric acid in plasma.

This work presents the synthesis and characterization of an innovative F,S-doped carbon dots/CuONPs hybrid nanostructure obtained by a direct mixture between F,S-doped carbon dots obtained electrochemically and copper nitrate alcoholic solution. The hybrid nanostructures synthesized were characterized by absorption spectroscopy in the Ultraviolet region (UV-vis), high-resolution transmission electron microscopy (HRTEM), X-ray photoelectron spectroscopy (XPS), and different electrochemical techniques. The fluoride and sulfur-doped carbon dots/CuONPs nanostructures were used to prepare a non-enzymatic biosensor on a printed carbon electrode, exhibiting excellent electrocatalytic activity for the simultaneous determination of NADH, dopamine, and uric acid in the presence of ascorbic acid with a detection limit of 20, 80, and 400 nmol L-1, respectively. The non-enzymatic biosensors were also used to determine NADH, dopamine, and uric acid in plasma, and they did not suffer significant interference from each other.

Electrochemical microfluidic immunosensor with graphene-decorated gold nanoporous for T-2 mycotoxin detection.

T-2 is one of the most potent cytotoxic food-borne mycotoxins. In this work, we have developed and characterized an electrochemical microfluidic immunosensor for T-2 toxin quantification in wheat germ samples. T-2 toxin detection was carried out using a competitive immunoassay method based on monoclonal anti-T-2 antibodies immobilized on the poly(methyl methacrylate) (PMMA) microfluidic central channel. The platinum wire working electrode at the end of the channel was in situ modified by a single-step electrodeposition procedure with reduced graphene oxide (rGO)-nanoporous gold (NPG). T-2 toxin in the sample was allowed to compete with T-2-horseradish peroxidase (HRP) conjugated for the specific recognizing sites of immobilized anti-T-2 monoclonal antibodies. The HRP, in the presence of hydrogen peroxide (H2O2), catalyzes the oxidation of 4-tert-butylcatechol (4-TBC), whose back electrochemical reduction was detected on the nanostructured electrode at -0.15 V. Thus, at low T-2 concentrations in the sample, more enzymatically conjugated T-2 will bind to the capture antibodies, and, therefore, a higher current is expected. The detection limits found for electrochemical immunosensor, and commercial ELISA procedure were 0.10 µg kg-1 and 10 µg kg-1, and the intra- and inter-assay coefficients of variation were below 5.35% and 6.87%, respectively. Finally, our microfluidic immunosensor to T-2 toxin will significantly contribute to faster, direct, and secure in situ analysis in agricultural samples.

Tyrosinase-based nanobiosensor for environmental monitoring of hormones in river water.

This study explores the application of a tyrosinase cantilever nanobiosensor for detecting 17ß-estradiol and estrone in typical water systems. The physical-chemical parameters of water were evaluated within the Tigre River micro-basin in Erechim, RS, to determine water potability for urban populations. Water clarity, conductivity, and pH levels were essential markers, adhering to recognized standards for water quality and human consumption. The cantilever nanobiosensor demonstrated strong sensitivity and a broad linear range, with a limit of detection (<0.00051 ppb) surpassing other enzymatic biosensors and covering a range of 0.0001-100 ppb. The real water sample quality investigated in relation to contamination with 17ß-estradiol and estrone by nanobiosensor showed values below the LOD for both compounds. Recovery studies demonstrated the reliability of the nanobiosensor. Selectivity tests indicated minimal interference from structurally similar substances. This study validates the nanobiosensor's potential for environmental monitoring and hormone detection, aligning with standard practices.