Low Analyte Concentrations Research Articles

Rapid SERS Detection of Botulinum Neurotoxin Type A.

Surface-enhanced Raman scattering (SERS) is a powerful technique for decoding of 2-5-component mixes of analytes. Low concentrations of analytes and complex biological media are usually non-decodable with SERS. Recognition molecules, such as antibodies and aptamers, provide an opportunity for a specific binding of ultra-low contents of analyte dissolved in complex biological media. Different approaches have been proposed to provide changes in SERS intensity of an external label upon binding of ultra-low contents of the analytes. In this paper, we propose a SERS-based sensor for the rapid and sensitive detection of botulinum toxin type A. The silver nanoisland SERS substrate was functionalized using an aptamer conjugated with a Raman label. The binding of the target affects the orientation of the label, providing changes in an analytical signal. This trick allowed detecting botulinum toxin type A in a one-stage manner without additional staining with a monotonous dose dependence and a limit of detection of 2.4 ng/mL. The proposed sensor architecture is consistent with the multiarray detection systems for multiplex analyses.

Neonicotinoids: An overview of the newest sample preparation procedures of environmental, biological and food matrices

An overview of the actual state-of-the-art sample treatment procedures for trace and ultra-trace determination of neonicotinoids (NQs) in environmental, food and biological samples is presented. Analytical procedures for NQs, sometimes together with their metabolites/by-products, and NQ-related new generation pesticides have been considered, focusing on sample preparation, which is essential in most cases due to both the complexity of the matrices and low concentrations of residual analytes. Different strategies have been practised for analytes extraction/preconcentration/cleanup, entailing various techniques as solid-liquid extraction (SLE), liquid-liquid extraction (LLE), offline and online solid-phase extraction (SPE), dispersive SPE (d-SPE), magnetic SPE (MSPE), solid-phase microextraction (SPME) and the quick, easy, cheap, effective, rugged and safe (QuEChERS) approach. New solid phases, including inorganic and carbon-based materials, have been prepared, also starting from “green” precursors, and successfully employed in the sample treatment step before hyphenated liquid chromatography or direct electrochemical detection. As well, new environmentally friendly solvents have been evaluated for LLE. The most recent studies (2022-present), around 50 papers, have been reviewed by matrix type, briefly described and commented concerning extraction efficiency, matrix effect, sensitivity, ease of application and sample throughput. Advantages and drawbacks were highlighted, as well as new achievements paving the way for further improvements.

Collaborative validation of the quantification method for 57 allergens in ready to inject fragrance samples

AbstractThis publication presents the collaborative work conducted in CEN/TC 347/WG4 ‘Fragrance and colophony’ working group to evaluate the robustness of a GC–MS analytical methodology to quantify the extended list of 57 fragrance allergens in ‘ready to inject’ samples. The intention of this group was to provide an updated and consensual standard for the quantification of the extended allergen list. The analytical approach was based on the similar methodology than the original one developed for the 26 allergens, using a universal GC–MS simple quadrupole instrument. A ring test was performed among the different end‐user experiences of the CEN group to test the method on a representative fragrance matrix. Accuracy profile method was used to validate the analytical method within the 2–240 mg/kg concentration range. Thanks to the use of commercial standard mixes and dedicated automation of decisional tree for data treatment, 89% of tolerance intervals of targeted allergens were included into the acceptance limits in the whole 2–240 mg/kg concentration range. The objective of this article was to provide some complementary data to implement the NF EN 16274 standard in an analytical laboratory and to highlight the inherent difficulties linked to extended data treatment and complexity for the recovery of low concentration analytes and potential coelutions in a wide diversity of ready‐to inject samples.

The Cleanroom-Free, Cheap, and Rapid Fabrication of Nanoelectrodes with Low zM Limits of Detection.

Nanoscale electrodes have been a topic of intense research for many decades. Their enhanced sensitivities, born out of an improved signal-to-noise ratio as electrode dimensions decrease, make them ideal for the development of low-concentration analyte sensors. However, to date, nanoelectrode fabrication has typically required expensive equipment and exhaustive, time-consuming fabrication methods that have rendered them unsuitable for widespread use and commercialization. Herein, a method of nanoband electrode fabrication using low cost materials and equipment commonly found in research laboratories around the world is reported. The materials' cost to produce each nanoband is less than €0.01 and fabrication of a batch takes less than 1 h. The devices can be made of flexible plastics and their designs can be quickly and easily iterated. Facile methods of combining these nanobands into powerful devices, such as complete three-electrode systems, are also displayed. As a proof of concept, the electrodes are functionalized for the detection of a DNA sequence specific to SARS-CoV-2 and found to display single molecule sensitivity.

Method development and quantification of all B vitamins and selected biosynthetic precursors in winter and spring samples from the North Sea and de novo synthesized by Vibrio campbellii

The simultaneous analysis of B vitamins and biosynthetic precursors in seawater is of great interest as it gives a better insight into the fluctuations of their availability for marine organisms and thus allows us to better understand the nature of interactions between prototrophic and auxotrophic organisms. The analysis of these micronutrients is challenging, as they are usually present in the marine environment in minuscule quantities only. Additionally, some of them are light- and heat-sensitive which complicates their handling. To meet these circumstances, the presented sample work-up method for seawater started with a freeze-drying step for volume reduction of the sample. This shortened the duration of the subsequent solid phase extraction and resulted in an increase in recovery. When comparing three solid phase extraction materials (C18, HLB, PPL), PPL was found to be the most suitable. This method was applied for the quantification of all eleven B vitamins and six precursors in waters from the southern North Sea taken in winter and spring. Quantification was performed by LC-MS. The winter samples showed higher concentrations for nearly all analytes, in contrast to the uptake of most nutrients at the beginning of a potential algal bloom in spring and low analyte concentrations in springtime waters. In addition, the marine bacterium Vibrio campbellii, prototrophic for most B vitamins, was cultivated without the addition of B vitamins in order to measure them intra- and extracellularly during growth. In most cases, intracellular concentrations per cell decreased towards the end of the exponential growth phase, while extracellular concentrations increased. Extracellular B vitamin and precursor concentration measurements show that V. campbellii is in exchange with its environment and thereby possibly enables growth of B vitamin auxotrophs in the environment.

A novel approach of 19F NMR based sensor

Reliable and precise methods are to be applied ranging from biological field to diagnosis and quality control to identify target analytes in samples. Nuclear magnetic resonance (NMR) is a strongly recommended process that is suitable for collecting data multidimensionally at molecular and atomic levels. Information obtained from the NMR technique provides molecular structures, molecular interactions, dynamic and diffusional properties of molecules. Overlapping of NMR signals of each component makes it difficult to interpret data and low sensitivity makes it unfitting for many samples with a low concentration of analytes. These limitations can be rectified by adding heteronuclear specially designed sensors, inventive transduction mechanism and strong pulse sequences. 19F NMR tool is extensively accepted method to detect the interactions among a variety of biological substrates with 19F-isotopic species. This review includes the detection of biomolecules, mainly enzymes and enzymatic reactions based on sensor applicability.

Synthesis of dendritic urethane acrylates for fabricating a robust honeycomb-like structure acting for SERS detection

In this study, novel dendritic urethane acrylate polymers were developed for the fabrication of robust honeycomb-like surfaces after exposure to a UV-crosslinking process. Through iterative synthesis based on a highly selective intermediate of 4-isocyanate-4′-(3,3-dimethyl-2,4-dioxo-azetidine)-diphenylmethane, poly(urea/malonamide) dendrons bearing peripheral methyl methacrylate end groups with well-defined structures were realized. A coating solution was prepared by the combined use of 30–70 wt% UA dendron acting as dendritic diluents and dendritic UA copolymers acting as amphiphiles that can fabricate honeycomb-like surfaces via a breath figure self-assembly process using water droplets as templates. The resulting film can be cured under UV light to achieve a robust honeycomb-like structure with a hexagonal structure exhibiting solvent-resistant properties in ethanol. Consequently, this solvent-resistant porous array can act as a substrate for the detection of vanishingly low concentrations of analytes that are aqueous insoluble via surface-enhanced Raman scattering analysis.

Laconic Review on Capillary Electrophoresis-Mass Spectrometry in Separation of Proteins

Abstract: The separation technique known as electrophoresis is made possible by the ability of liquid molecules to travel through an electric field. Early in the 1980s, it was discovered that considerable separation efficiencies could be obtained by passing a voltage across a capillary. In the late 1980s and early 1990s, commercial capillary electrophoresis (CE) first became accessible. Mass spectrometry is a powerful tool for identifying unknowns, studying molecular structure, and investigating the fundamental principles of chemistry. The most efficient technique combines high-resolution separations by CE with high-sensitivity and selectivity detections by MS, which is typical for bioanalytical applications and targets or thoroughly high throughput studies of low-concentration analytes frequently occurring in complicated matrices. The combination of capillary electrophoresis with mass spectrometry, or CE-MS, has emerged as an effective standard technique for the analysis of a wide variety of analytes. CEMS, a proteomic technology that combines chromatographic techniques with MS to increase the resolving power, is a well-liked technique for proteome analysis.

Development and optimization of surface-enhancement Raman scattering-based lateral flow immunoassays for the ultrasensitive detection of cardiac troponin I

Cardiac Troponin I is measurable using point-of-care testing (POCT) and lateral flow assay (LFA) for diagnosing acute myocardial infarction (AMI) in a clinical method that combines the advantages of cost-effectiveness, rapid detection, and ease of use. However, the limitation of LFA is that it hinders the diagnosis of patients with a low analyte concentration, necessitating additional blood tests. Surface-enhancement Raman scattering (SERS)-based LFA can provide the quantitative assay with high sensitivity. This study reports a straightforward process for developing SERS-based LFA that detected 0.02 ng/mL analyte in human serum compared to the 1.56 ng/mL analyte detected using optical intensity measurement in LFA. To ensure highly sensitive detection, the size (30 to 100 nm) of Au nanoparticle (NP) in SERS tags was optimized in SERS-based LFA, and a stronger SERS intensity was achieved using 50 nm AuNP SERS tags. The reaction time and LFA buffer components were determined, and laser wavelength and power were investigated. By optimizing these parameters for SERS-based LFA, the sensitivity of SERS was 78-fold higher than that of optical intensity measurement. This procedure could be beneficial for developing a SERS-based LFA to detect various biomarkers with high sensitivity for potential POCT applications.

Recent Advances in Two-Dimensional MXene-Based Electrochemical Biosensors for Sweat Analysis.

Sweat, a biofluid secreted naturally from the eccrine glands of the human body, is rich in several electrolytes, metabolites, biomolecules, and even xenobiotics that enter the body through other means. Recent studies indicate a high correlation between the analytes' concentrations in the sweat and the blood, opening up sweat as a medium for disease diagnosis and other general health monitoring applications. However, low concentration of analytes in sweat is a significant limitation, requiring high-performing sensors for this application. Electrochemical sensors, due to their high sensitivity, low cost, and miniaturization, play a crucial role in realizing the potential of sweat as a key sensing medium. MXenes, recently developed anisotropic two-dimensional atomic-layered nanomaterials composed of early transition metal carbides or nitrides, are currently being explored as a material of choice for electrochemical sensors. Their large surface area, tunable electrical properties, excellent mechanical strength, good dispersibility, and biocompatibility make them attractive for bio-electrochemical sensing platforms. This review presents the recent progress made in MXene-based bio-electrochemical sensors such as wearable, implantable, and microfluidic sensors and their applications in disease diagnosis and developing point-of-care sensing platforms. Finally, the paper discusses the challenges and limitations of MXenes as a material of choice in bio-electrochemical sensors and future perspectives on this exciting material for sweat-sensing applications.

Microsphere-assisted enhanced photothermal lens detection integrated with digital holographic microscopy for 3D particle sensing and thermal diffusivity measurement

In this paper, we show that by extending the concept of “microsphere-assisted imaging” into thermal lens (TL) detection, enhancement of photothermal detection is achieved. Furthermore, by integration of TL detection and digital holographic microscopy (DHM), we provide simultaneous measurement of photothermal properties as well as complementary imaging of the sample. Simultaneous acquisition of photothermal properties and imaging is essential for characterization of a variety of samples in biochemistry, material science, and process technologies. TL is a sensitive methodology to detect low concentrations of analytes in low-loss samples, and DHM in the transmission mode is an effective technique for label-free, non-contact, and real-time imaging and measurement of phase objects, such as biomaterials. We demonstrate the usefulness of the TL-DHM integrated system by applying it for acquisition of photothermal maps of samples as well as obtaining their morphometrical information. Additionally, we measure the thermal diffusivity of polydimethylsiloxane because of the focusing effect of polystyrene microspheres. The system has the potential to be applied for a variety of objects including bio-samples and may be proposed as a bench-top characterization device.

Evaluation and classification of fentanyl-related compounds using EC-SERS and machine learning.

Multiple analytical techniques for the screening of fentanyl-related compounds exist. High discriminatory methods such as GC-MS and LC-MS are expensive, time-consuming, and less amenable to onsite analysis. Raman spectroscopy provides a rapid, inexpensive alternative. Raman variants such as electrochemical surface-enhanced Raman scattering (EC-SERS) can provide signal enhancements with 1010 magnitudes, allowing for the detection of low-concentration analytes, otherwise undetected using conventional Raman. Library search algorithms embedded in instruments utilizing SERS may suffer from accuracy when multicomponent mixtures involving fentanyl derivatives are analyzed. The complexing of machine learning techniques to Raman spectra demonstrates an improvement in the discrimination of drugs even when present in multicomponent mixtures of various ratios. Additionally, these algorithms are capable of identifying spectral features difficult to detect by manual comparisons. Therefore, the goal of this study was to evaluate fentanyl-related compounds and other drugs of abuse using EC-SERS and to process the acquired data using machine learning-convolutional neural networks (CNN). The CNN was created using Keras v 2.4.0 with Tensorflow v 2.9.1 backend. In-house binary mixtures and authentic adjudicated case samples were used to evaluate the created machine-learning models. The overall accuracy of the model was 98.4 ± 0.1% after 10-fold cross-validation. The correct identification for the in-house binary mixtures was 92%, while the authentic case samples were 85%. The high accuracies achieved in this study demonstrate the advantage of using machine learning to process spectral data when screening seized drug materials comprised of multiple components.

Paramagnetic Relaxation Agents for Enhancing Temporal Resolution and Sensitivity in Multinuclear FlowNMR Spectroscopy.

Sensitivity in FlowNMR spectroscopy for reaction monitoring often suffers from low levels of pre-magnetisation due to limited residence times of the sample in the magnetic field. While this in-flow effect is tolerable for high sensitivity nuclei such as 1 H and 19 F, it significantly reduces the signal-to-noise ratio in 31 P and 13 C spectra, making FlowNMR impractical for low sensititvity nuclei at low concentrations. Paramagnetic relaxation agents (PRAs), which enhance polarisation and spin-lattice relaxation, could eliminate the adverse in-flow effect and improve the signal-to-noise ratio. Herein, [Co(acac)3 ], [Mn(acac)3 ], [Fe(acac)3 ], [Cr(acac)3 ], [Ni(acac)2 ]3, [Gd(tmhd)3 ] and [Cr(tmhd)3 ] are investigated for their effectiveness in improving signal intensity per unit time in FlowNMR applications under the additional constraint of chemical inertness towards catalytically active transition metal complexes. High-spin Cr(III) acetylacetonates emerged as the most effective compounds, successfully reducing 31 P T1 values four- to five-fold at PRA concentrations as low as 10 mM without causing adverse line broadening. Whereas [Cr(acac)3 ] showed signs of chemical reactivity with a mixture of triphenylphosphine, triphenylphosphine oxide and triphenylphosphate over the course of several hours at 80° C, the bulkier [Cr(tmhd)3 ] was stable and equally effective as a PRA under these conditions. Compatibility with a range of representative transition metal complexes often used in homogeneous catalysis has been investigated, and application of [Cr(tmhd)3 ] in significantly improving 1 H and 31 P{1 H} FlowNMR data quality in a Rh-catalysed hydroformylation reaction has been demonstrated. With the PRA added, 13 C relaxation times were reduced more than six-fold, allowing quantitative reaction monitoring of substrate consumption and product formation by 13 C{1 H} FlowNMR spectroscopy at natural abundance.

Ultrasensitive Terahertz Biodetection Enabled by Quasi-BIC-Based Metasensors.

Advanced sensing devices, highly sensitive, and reliable in detecting ultralow concentrations of circulating biomarkers, are extremely desirable and hold great promise for early diagnostics and real-time progression monitoring of diseases. Nowadays, the most commonly used clinical methods for diagnosing biomarkers suffer from complicated procedures and being time consumption. Here, a chip-based portable ultra-sensitive THz metasensor is reported by exploring quasi-bound states in the continuum (quasi-BICs) and demonstrate its capability for sensing low-concentration analytes. The designed metasensor is made of the designed split-ring resonator metasurface which supports magnetic dipole quasi-BIC combining functionalized gold nanoparticles (AuNPs) conjugated with the specific antibody. Attributed to the strong near-field enhancement near the surface of the microstructure enabled by the quasi-BICs, light-analyte interactions are greatly enhanced, and thus the device's sensitivity is boosted significantly. The system sensitivity slope is up to 674 GHz/RIU, allowing for repeatable resolving detecting ultralow concentration of C-reactive protein (CRP) and Serum Amyloid A (SAA), respectively, down to 1 pM. The results touch a range that cannot be achieved by ordinary immunological assays alone, offering a novel non-destructive and rapid trace measured approach for next-generation biomedical quantitative detectionsystems.

Simulation of Prostate Specific Antigen (PSA) ISFET Sensor with Different Nanostructures

It is crucial to have early detection of prostate cancer as it is the third most common cancer in men and Prostate Specific Antigen (PSA) is a protein-based biomarker commonly used to achieve the purpose. Besides, ISFET integrated with nanostructures such as nanowire and nanosphere has been the focus of research for disease diagnosis. Simulations are carried out in Biosensor Lab to investigate the performance of ISFET with planar, nanowire and nanosphere structures in detecting PSA. Using nanostructured ISFET in disease diagnosis is demonstrated by comparing the performance with the surface-to-volume (S/V) ratio. The nanosphere biosensor with the highest S/V ratio showed the lowest settling time when there was a low analyte concentration. Its settling time is 788 times lower than planar and almost 3 times lower than nanowire ISFET. In addition, the time needed for a planar biosensor to capture M of PSA is 5 times slower than a nanowire biosensor and nanosphere biosensor. In signal-to-noise ratio (SNR), nanosphere ISFET exhibits the highest value compared to other structures. These results indicate that a higher S/V ratio contributed to better performance in detecting PSA.

Spatially Addressable Multiplex Biodetection by Calibrated Micro/Nanostructured Surfaces

A challenge of any biosensing technology is the detection of very low concentrations of analytes. The fluorescence interference contrast (FLIC) technique improves the fluorescence-based sensitivity by selectively amplifying, or suppressing, the emission of a fluorophore-labeled biomolecule immobilized on a transparent layer placed on top of a mirror basal surface. The standing wave of the reflected emission light means that the height of the transparent layer operates as a surface-embedded optical filter for the fluorescence signal. FLIC extreme sensitivity to wavelength is also its main problem: small, e.g., 10 nm range, variations of the vertical position of the fluorophore can translate in unwanted suppression of the detection signal. Herein, we introduce the concept of quasi-circular lenticular microstructured domes operating as continuous-mode optical filters, generating fluorescent concentric rings, with diameters determined by the wavelengths of the fluorescence light, in turn modulated by FLIC. The critical component of the lenticular structures was the shallow sloping side wall, which allowed the simultaneous separation of fluorescent patterns for virtually any fluorophore wavelength. Purposefully designed microstructures with either stepwise or continuous-slope dome geometries were fabricated to modulate the intensity and the lateral position of a fluorescence signal. The simulation of FLIC effects induced by the lenticular microstructures was confirmed by the measurement of the fluorescence profile for three fluorescent dyes, as well as high-resolution fluorescence scanning using stimulated emission depletion (STED) microscopy. The high sensitivity of the spatially addressable FLIC technology was further validated on a diagnostically important target, i.e., the receptor-binding domain (RBD) of the SARS-Cov2 via the detection of RBD:anti-S1-antibody.

Challenges in the Analytical Preparation of a Biological Matrix in Analyses of Endocrine-Disrupting Bisphenols

Endocrine-disrupting chemicals (EDCs) are xenobiotics presented in a variety of everyday products that may disrupt the normal activity of hormones. Exposure to bisphenol A as EDC at trace and ultra-trace levels is associated with adverse health effects, and children are recognized as the most vulnerable group to EDCs exposure. In this review, a summary is presented of up-to-date sample preparation methods and instrumental techniques applied for the detection and quantification of bisphenol A and its structural analogues in various biological matrices. Biological matrices such as blood, cell-free blood products, urine, saliva, breast milk, cordial blood, amniotic and semen fluids, as well as sweat and hair, are very complex; therefore, the detection and later quantification of bisphenols at low levels present a real analytical challenge. The most popular analytical approaches include gas and liquid chromatography coupled with mass spectrometry, and their enhanced reliability and sensitivity finally allow the separation and detection of bisphenols in biological samples, even as ultra-traces. Liquid/liquid extraction (LLE) and solid-phase extraction (SPE) are still the most common methods for their extraction from biological matrices. However, many modern and environmentally safe microextraction techniques are currently under development. The complexity of biological matrices and low concentrations of analytes are the main issues for the limited identification, as well as understanding the adverse health effects caused by chronical and ubiquitous exposure to bisphenols and its analogues.

Signal Amplification in Electrochemical DNA Biosensors Using Target-Capturing DNA Origami Tiles

Electrochemical DNA (e-DNA) biosensors are feasible tools for disease monitoring, with their ability to translate hybridization events between a desired nucleic acid target and a functionalized transducer, into recordable electrical signals. Such an approach provides a powerful method of sample analysis, with a strong potential to generate a rapid time to result in response to low analyte concentrations. Here, we report a strategy for the amplification of electrochemical signals associated with DNA hybridization, by harnessing the programmability of the DNA origami method to construct a sandwich assay to boost charge transfer resistance (RCT) associated with target detection. This allowed for an improvement in the sensor limit of detection by two orders of magnitude compared to a conventional label-free e-DNA biosensor design and linearity for target concentrations between 10 pM and 1 nM without the requirement for probe labeling or enzymatic support. Additionally, this sensor design proved capable of achieving a high degree of strand selectivity in a challenging DNA-rich environment. This approach serves as a practical method for addressing strict sensitivity requirements necessary for a low-cost point-of-care device.

A deep dive into biomolecular condensates using single-droplet surface-enhanced Raman spectroscopy.

An intense research activity reveals that living cells contain (noncanonical) membraneless organelles that are formed via phase separation of intrinsically disordered proteins/regions (IDPs/IDRs) with nucleic acids and other biomolecules. These biomolecular condensates are involved in a myriad of critical cellular functions and neurodegenerative diseases. Unmasking the role of intrinsic disorder and conformational heterogeneity of IDPs/IDRs in promoting promiscuous and ephemeral interactions resulting in a liquid-like behavior of these condensates is crucial to understanding the molecular drivers of phase separation. While a host of existing microscopic and spectroscopic tools exist for studying phase separation, most of these methodologies are inadequate in illuminating the conformational heterogeneity and distribution within individual droplets. To overcome these limitations, we developed and adapted a novel and highly-sensitive methodology that combines the capabilities of vibrational spectroscopy and optical microscopy that can illuminate the unique molecular details of the polypeptide chains within the mesoscopic liquid condensed phase at the single-droplet resolution. Often low Raman scattering cross-section of proteins makes the recording of vibrational signatures under physiological conditions in aqueous solutions extremely challenging. In this direction, we utilize surface-engineered, plasmonic metal nanostructures giving rise to high electromagnetic/chemical enhancement of Raman signals even at extremely low analyte concentrations that can increase Raman scattering cross-section by several orders of magnitude, allowing single-molecule detection and characterization even at a much lower laser power. Our ultra-sensitive single-droplet SERS methodology captures the exquisite details involving conformational disorder, heterogeneity, and distribution within FUS condensates. Additionally, this method can capture some key structural details within the droplets formed in the presence of varying RNA-protein ratios and highlight RNA-mediated partial unwinding of the ordered domains in the C-terminal RBD that increases polypeptide chain disorder that can promote both homotypic and heterotypic interactions within the condensed phase.

3D-printed sensor decorated with nanomaterials by CO2 laser ablation and electrochemical treatment for non-enzymatic tyrosine detection

Thecombination of CO2 laser ablation and electrochemical surface treatments is demonstratedto improve the electrochemical performance of carbon black/polylactic acid (CB/PLA) 3D-printed electrodes through the growth of flower-like Na2O nanostructures on their surface. Scanning electron microscopy images revealed that the combination of treatments ablated the electrode's polymeric layer, exposing a porous surface where Na2O flower-like nanostructures were formed. The electrochemical performance of the fabricated electrodes was measured by the reversibility of the ferri/ferrocyanide redox couple presenting a significantly improved performance compared withelectrodes treated by only one of the steps. Electrodes treated by the combined method also showed a better electrochemical response for tyrosine oxidation. These electrodes were used as a non-enzymatic tyrosine sensor for quantification in human urine samples. Two fortified urine samples were analyzed, and the recovery values were 106 and 109%. The LOD and LOQ for tyrosine determination were 0.25 and 0.83 μmol L-1, respectively, demonstrating that the proposed devices are suitable sensors for analyses of biological samples, even at low analyte concentrations.