Selective Detection of Paraquat in Adulterated and Complex Environmental Samples Using Raman Spectroelectrochemistry.

Growing demand for pesticides has created an environment prone to deceptive activities, where counterfeit or adulterated pesticide products infiltrate the market, often escaping rapid detection. This situation presents a significant challenge for sensor technology, crucial in identifying authentic pesticides and ensuring agricultural safety practices. Raman spectroscopy emerges as a powerful technique for detecting adulterants. Coupling the electrochemical techniques allows a more specific and selective detection and compound identification. In this study, we evaluate the efficacy of spectroelectrochemical measurements by coupling a potentiostat and Raman spectrograph to identify paraquat, a nonselective herbicide banned in several countries. Our findings demonstrate that applying -0.70 V during measurements yields highly selective Raman spectra, highlighting the primary vibrational bands of paraquat. Moreover, the selective Raman signal of paraquat was discernible in complex samples, including tap water, apple, and green cabbage, even in the presence of other pesticides such as diquat, acephate, and glyphosate. These results underscore the potential of this technique for reliable pesticide detection in diverse and complex matrices.

Contamination-Free Reference Electrode Using Prussian Blue for Small Oxygen Sensors.

In recent years, significant attention has been directed toward advancing compact, point-of-care testing (POCT) devices to better deliver patient care and alleviate the burden on the medical care system. Common POCTs, such as blood oxygen sensors, leverage electrochemical sensing in their design. However, conventional electrochemical devices typically use Ag/AgCl reference electrodes, which are likely to release trace amounts of silver ions that contaminate the working electrode, causing rapid deterioration of the devices. This study proposes an effective reference electrode using graphene-coated porous silica spheres (G/PSS) with embedded Prussian blue (PB), denoted PB/G/PSS, designed specifically for small oxygen sensors. PB is a redox species that is an improvement over Ag/AgCl since it is significantly less water-soluble than AgCl. Since PB is an insulator, we dispersed PB in G/PSS, well-conductive mesoporous matrices, to ensure contact between PB clusters and the electrolytes. Moreover, the monodispersed, spherically shaped PB/G/PSS is an advantageous medium for fabricating POCT devices by screen printing. In this study, the open-circuit potential of the PB/G/PSS electrode remained stable within 30 mV for 31 days. The small oxygen sensor assembled through screen printing using PB/G/PSS demonstrated stable operation for several days or more. In contrast, a similar sensor with Ag/AgCl reference electrode rapidly deteriorated within a day. This PB/G/PSS reference electrode with improved stability is expected to be an excellent alternative to the Ag/AgCl system for small electrochemical-based POCT devices.

Point-of-care testing device platform for the determination of creatinine on an enzyme@CS/PB/MXene@AuNP-based screen-printed carbon electrode.

Screen-printed carbon electrodes (SPCE) functionalized with MXene-based three-dimensional nanomaterials are reported for rapid determination of creatinine. Ti3C2TX MXene with in situ reduced AuNPs (MXene@AuNP) were used as a coreactant accelerator for efficient immobilization of enzymes. Creatinine could be oxidized by chitosan-embedded creatinine amidohydrolase, creatine amidinohydrolase, or sarcosine oxidase to generate H2O2, which could be electrochemically detected enhanced by Prussian blue (PB). The enzyme@CS/PB/MXene@AuNP/SPCE detected creatinine within the range 0.03-4.0 mM, with a limit of detection of 0.01 mM, with an average recovery of 96.8-103.7%. This indicates that the proposed biosensor is capable of detecting creatinine in a short amount of time (4 min) within a ± 5% percentage error, in contrast with the standard clinical colorimetric method. With this approach, reproducible and stable electrochemical responses could be achieved for determination of creatinine in serum, urine, or saliva. These results demonstrated its potential for deployment in resource-limited settings for early diagnosis and tracking the progression of chronic kidney disease (CKD).

Development of an amperometric biosensor for the detection of Bacillus anthracis spores.

INTRODUCTION: Due to most likely use of Bacillus anthracis in biological terrorism agents, the rapid and sensitive detection of its spores is crucial in both taking prophylactic measures and proper treatment. This study aimed to develop an amperometric electrochemical immunosensor for the detection of B. anthracis spores. METHODS: A new amperometric biosensor was designed using a combination of magnetic beads and multiplex screen-printed electrodes. This method measures changes in current intensity resulting from oxidation and reduction in the working electrode directly to spore concentrations. RESULTS: A standard curve was formed to test the number of live spores between 2 × 102-2 × 104 spores/ml concentrations. LOD and LOQ values were found to be 92 and 272 spores/ml, respectively. No cross-reactions were seen for Bacillus subtilis, Bacillus cereus and Bacillus thuringiencis spores. CONCLUSIONS: It is shown that the designed Anthrax immunosensor has high sensitivity and selectivity with rapid detection results.

Ionic liquid-reinforced Hydroxyapatite@nano-TiO2 as a green platform for Immuno-electrochemical sensing applications.

In contemporary society, developing dependable point-of-care (POC) biosensors for the timely detection of cancer markers is crucial. Among various sensor types, screen-printed electrode (SPE)-based sensors, particularly electrochemical ones, stand out as promising candidates for POC applications. Despite ongoing efforts to create numerous SPE-based sensors, there is a continuous pursuit to enhance their sensitivity and analytical capabilities. This study presents an advanced electrochemical sensor designed to sensitively detect the hepatocellular carcinoma (HCC) marker Alpha-fetoprotein (AFP) in saliva. The sensor employs a gold SPE modified with hydroxyapatite, TiO2 nanoparticles, 1-butyl-3-methylimidazolium bis(trifluoromethyl sulfonyl)imide ionic liquid (IL), and AFP monoclonal antibodies. After thorough characterization and optimization using cyclic voltammetry (CV) and differential pulse voltammetry (DPV), the biosensor exhibited a broad detection range (0.01-400 ng/mL), a low limit of detection (LOD) at 0.058 ng/mL, and demonstrated high selectivity, repeatability, reproducibility, and stability. Furthermore, when tested with spiked human saliva samples, the biosensor displayed excellent recovery and robustness, showcasing its potential for noninvasive and POC diagnosis of HCC. In an environmentally conscious evaluation, the biosensor's greenness was assessed using the AGREE metric, yielding a high score of 0.85. This score indicates the biosensor's alignment with the principles of green analytical chemistry, underlining its eco-friendly attributes. This innovative electrochemical sensor contributes to the ongoing efforts for efficient and reliable POC diagnostic tools and aligns with a broader commitment to developing environmentally friendly solutions.

Highly efficient electrocatalytic oxidation of levodopa as a Parkinson therapeutic drug based on modified screen-printed electrode.

The current study presents the creation of a straightforward and sensitive sensor based on ZnO/Co3O4 nanocomposite modified screen-printed electrode (ZnO/Co3O4NC/SPE) for levodopa determination. At ZnO/Co3O4NC/SPE, an oxidative peak for levodopa solution in pH 6.0 phosphate buffer solution (PBS) were seen that were both more resolved and more enhanced. Levodopa was measured using differential pulse voltammetry (DPV), which showed an excellent linear range (0.001-800.0 µM) and detection limit (0.81 nM). The presence of interference did not affect the electrochemical response of levodopa at ZnO/Co3O4NC/SPE, demonstrating high selectivity. Levodopa in a real samples have been successfully detected using the manufactured sensor.

Multiplex antibiotic susceptibility testing of urinary tract infections using an electrochemical lab-on-a-chip.

Urinary tract infections (UTIs) represent the most prevalent type of outpatient infection, with significant adverse health and economic burdens. Current culture-based antibiotic susceptibility testing can take up to 72 h resulting in ineffective prescription of broad-spectrum antibiotics, poor clinical outcomes and development of further antibiotic resistance. We report an electrochemical lab-on-a-chip (LOC) for testing samples against seven clinically-relevant antibiotics. The LOC contained eight chambers, each housing an antibiotic-loaded hydrogel (cephalexin, ceftriaxone, colistin, gentamicin, piperacillin, trimethoprim, vancomycin) or antibiotic-free control, alongside a resazurin bulk-modified screen-printed electrode for electrochemical detection of metabolically active bacteria using differential pulse voltammetry. Antibiotic susceptibility in simulated UTI samples or donated human urine with either Escherichia coli or Klebsiella pneumoniae could be established within 85 min. Incorporating electrochemical detection onto a LOC provides an inexpensive, simple method for the sensitive determination of antibiotic susceptibility that is significantly faster than using a culture-based approach.

Screen-Printed Electrodes-A Promising Tool for Antineoplastic Drug Detection (Cisplatin and Bleomycin) in Biological Samples.

Cancer remains one of the leading causes for death worldwide. Palliative chemotherapy is vital for certain cancer patients, highlighting the critical need for treatment monitoring tools to prevent drug accumulation and mitigate the risk of high toxicity. Therefore, our aim was to evaluate the potential of screen-printed electrodes for the development of sensitive and accurate biosensors for the detection/quantification of antineoplastic drugs. To this purpose, we developed a cisplatin sensor. By functionalizing the gold electrode with human serum albumin and by collecting the electrochemical signal obtained in a H2O2 solution, through voltammetry measurements, we were able to correlate the current measured at 430 mV with the concentration of cisplatin present in human serum samples, with a correlation coefficient of R2 = 0.99. Also, a bleomycin biosensor was developed and proven functional, but further optimization steps were employed in order to improve the accuracy. The developed biosensors have a detection range of 0.0006-43.2 mg/mL for cisplatin and 0.23-7.56 µg/mL for bleomycin in the serum samples. Our preliminary results show that these biosensors can facilitate the real-time monitoring of cisplatin and bleomycin serum levels, allowing healthcare professionals to tailor treatment strategies based on individual patient responses.

Electroactive Molecularly Imprinted Polymer Nanoparticles (eMIPs) for Label-free Detection of Glucose: Toward Wearable Monitoring.

The diagnosis of diabetes mellitus (DM) affecting 537 million adults worldwide relies on invasive and costly enzymatic methods that have limited stability. Electroactive polypyrrole (PPy)-based molecularly imprinted polymer nanoparticles (eMIPs) have been developed that rival the affinity of enzymes whilst being low-cost, highly robust, and facile to produce. By drop-casting eMIPs onto low-cost disposable screen-printed electrodes (SPEs), sensors have been manufactured that can electrochemically detect glucose in a wide dynamic range (1 µm-10 mm) with a limit of detection (LOD) of 26 nm. The eMIPs sensors exhibit no cross reactivity to similar compounds and negligible glucose binding to non-imprinted polymeric nanoparticles (eNIPs). Measurements of serum samples of diabetic patients demonstrate excellent correlation (>0.93) between these eMIPs sensor and the current gold standard Roche blood analyzer test. Finally, the eMIPs sensors are highly durable and reproducible (storage >12 months), showcasing excellent robustness and thermal and chemical stability. Proof-of-application is provided via measuring glucose using these eMIPs sensor in a two-electrode configuration in spiked artificial interstitial fluid (AISF), highlighting its potential for non-invasive wearable monitoring. Due to the versatility of the eMIPs that can be adapted to virtually any target, this platform technology holds high promise for sustainable healthcare applications via providing rapid detection, low-cost, and inherent robustness.

Modified Gold Screen-Printed Electrodes for the Determination of Heavy Metals.

Screen-printed electrodes (SPEs) are reliable, portable, affordable, and versatile electrochemical platforms for the real-time analytical monitoring of emerging analytes in the environmental, clinical, and agricultural fields. The aim of this study was to evaluate the electrochemical behavior of gold screen-printed electrodes (SPGEs) modified with molecules containing amino (Tr-N) or α-aminophosphonate (Tr-P) groups for the selective and sensitive detection of the toxic metal ions Pb2+ and Hg2+ in aqueous samples. After optimizing the analytical parameters (conditioning potential and time, deposition potential and time, pH and concentration of the supporting electrolyte), anodic square wave stripping voltammetry (SWASV) was used to evaluate and compare the electrochemical performance of bare or modified electrodes for the detection of Hg2+ and Pb2+, either alone or in their mixtures in the concentration range between 1 nM and 10 nM. A significative improvement in the detection ability of Pb2+ ions was recorded for the amino-functionalized gold sensor SPGE-N, while the presence of a phosphonate moiety in SPGE-P led to greater sensitivity towards Hg2+ ions. The developed sensors allow the detection of Pb2+ and Hg2+ with a limit of detection (LOD) of 0.41 nM and 35 pM, respectively, below the legal limits for these heavy metal ions in drinking water or food, while the sensitivity was 5.84 µA nM-1cm-2 and 10 µA nM-1cm-2, respectively, for Pb2+ and Hg2+. The reported results are promising for the development of advanced devices for the in situ and cost-effective monitoring of heavy metals, even in trace amounts, in water resources.

Effect of Various Carbon Electrodes on MIP-Based Sensing Proteins Using Poly(Scopoletin): A Case Study of Ferritin.

Sensitivity in the sub-nanomolar concentration region is required to determine important protein biomarkers, e.g., ferritin. As a prerequisite for high sensitivity, in this paper, the affinity of the functional monomer to the macromolecular target ferritin in solution was compared with the value for the respective molecularly imprinted polymer (MIP)-based electrodes, and the influence of various surface modifications of the electrode was investigated. The analytical performance of ferritin sensing was investigated using three different carbon electrodes (screen-printed carbon electrodes, single-walled-carbon-nanotube-modified screen-printed carbon electrodes, and glassy carbon electrodes) covered with a scopoletin-based MIP layer. Regardless of the electrode type, the template molecule ferritin was mixed with the functional monomer scopoletin, and electropolymerization was conducted using multistep amperometry. All stages of MIP preparation were followed by evaluating the diffusional permeability of the redox marker ferricyanide/ferrocyanide through the polymer layer by differential pulse voltammetry. The best results were obtained with glassy carbon electrodes. The MIP sensor responded up to 0.5 µM linearly with a Kd of 0.30 µM. Similar results were also obtained in solution upon the interaction of scopoletin and ferritin using fluorescence spectroscopy, resulting in the quenching of the scopoletin signal, with a calculated Kd of 0.81 µM. Moreover, the binding of 1 µM ferritin led to 49.6% suppression, whereas human serum albumin caused 8.6% suppression.

Temperature-Sensitive Sensors Modified with Poly(N-isopropylacrylamide): Enhancing Performance through Tailored Thermoresponsiveness.

The development of temperature-sensitive sensors upgraded by poly(N-isopropylacrylamide) (PNIPAM) represents a significant stride in enhancing performance and tailoring thermoresponsiveness. In this study, an array of temperature-responsive electrochemical sensors modified with different PNIPAM-based copolymer films were fabricated via a "coating and grafting" two-step film-forming technique on screen-printed platinum electrodes (SPPEs). Chemical composition, grafting density, equilibrium swelling, surface wettability, surface morphology, amperometric response, cyclic voltammograms, and other properties were evaluated for the modified SPPEs, successively. The modified SPPEs exhibited significant changes in their properties depending on the preparation concentrations, but all the resulting sensors showed excellent stability and repeatability. The modified sensors demonstrated favorable sensitivity to hydrogen peroxide and L-ascorbic acid. Furthermore, notable temperature-induced variations in electrical signals were observed as the electrodes were subjected to temperature fluctuations above and below the lower critical solution temperature (LCST). The ability to reversibly respond to temperature variations, coupled with the tunability of PNIPAM's thermoresponsive properties, opens up new possibilities for the design of sensors that can adapt to changing environments and optimize their performance accordingly.

Cellulose acetate-coated capacitive sensor for determining carbon-cycle enzymes activity and as a microbial Indicator for soil health.

This study demonstrates cellulose acetate (CA)-coated screen-printed carbon electrodes (SPCEs) for soil microbial activity detection. A capacitive sensor design utilizes a coated CA layer for effective insulation in electrolytes, eliminating the need for additional signal protection. Optimization involved comparing spin and dip coating methods, with a one-layer 10-second dip coating identified as the best balance between quality and yield. These CA/SPCEs exhibited remarkable stability over a month, suggesting their potential for long-term use in monitoring agricultural soils. Analysis of CA/SPCE profile and thickness provided insights into surface characteristics and the impact of the CA coating on electrode roughness. ATR-FTIR analysis, along with capacitive sensing, demonstrated superior sensitivity and precision for monitoring CA film degradation compared to mechanical gauges. Chemical degradation studies suggest CA's potential immunity in near-neutral environments, while enzymatic degradation investigations revealed dominance by enzymes, particularly in the initial stages. The CA/SPCE sensor responds to both enzymatic and chemical degradation, potentially serving as an indicator of total soil microbial activity. Soil experiments explored CA/SPCE with Cap-S for microbial activity sensing. Significant differences in the long-term degradation rate were observed in mycorrhizal fungi-enriched soil compared to controls, highlighting microbial influences. This study underscores the adaptability and versatility of this technology, particularly for assessing C-cycle microbial activity in agricultural fields.

Chronoamperometric Ammonium Ion Detection in Water via Conductive Polymers and Gold Nanoparticles.

Monitoring of ammonium ion levels in water is essential due to its significant impact on environmental and human health. This work aims to fabricate and characterize sensitive, real-time, low-cost, and portable amperometric sensors for low NH4+ concentrations in water. Two strategies were conducted by cyclic voltammetry (CV): electrodeposition of Au nanoparticles on a commercial polyaniline/C electrode (Au/PANI/C), and CV of electropolymerized polyaniline on a commercial carbon electrode (Au/PANIep/C). Au NPs increase the electrical conductivity of PANI and its ability to transfer charges during electrochemical reactions. The electrode performances were tested in a concentration range from 0.35 µM to 7 µM in NH4+ solution. The results show that the Au/PANI/C electrode performs well for high NH4+ concentrations (0.34 µM LoD) and worsens for low NH4+ concentrations (0.01 µM LoD). A reverse performance occurs for the electrode Au/PANIep/C, with a 0.03 µM LoD at low NH4+ concentration and 0.07 µM LoD at high NH4+ concentration. The electrodes exhibit a good reproducibility, with a maximum RSD of 3.68% for Au/PANI/C and 5.94% for Au/PANIep/C. In addition, the results of the repeatability tests show that the electrochemical reaction of sensing is fully reversible, leaving the electrode ready for a new detection event.

One-pot electrochemical detection of foodborne pathogen based on in situ nucleic acid amplification and wash-free assay.

A signal amplification electrochemical biosensor chip was developed to integrate loop-mediated isothermal amplification (LAMP) based on in situ nucleic acid amplification and methyl blue (MB) serving as the hybridization redox indicator for sensitive and selective foodborne pathogen detection without a washing step. The electrochemical biosensor chip was designed by a screen-printed carbon electrode modified with gold nanoparticles (Au NPs) and covered with polydimethylsiloxane membrane to form a microcell. The primers of the target were immobilized on the Au NPs by covalent attachment for in situ amplification. The electroactive MB was used as the electrochemical signal reporter and embedded into the double-stranded DNA (dsDNA) amplicons generated by LAMP. Differential pulse voltammetry was introduced to survey the dsDNA hybridization with MB, which differentiates the specifically electrode-unbound and -bound labels without a washing step. Pyrene as the back-filling agent can further improve response signaling by reducing non-specific adsorption. This method is operationally simple, specific, and effective. The biosensor showed a detection linear range of 102-107 CFU mL-1 with the limit of detection of 17.7 CFU mL-1 within 40 min. This method showed promise for on-site testing of foodborne pathogens and could be integrated into an all-in-one device.

A renewable screen-printed electrode based on magnetic NiCo@N-doped carbon nanotubes derived from Co-MOF for ractopamine detection.

A renewable electrochemical screen-printed electrode (SPE) is proposed based on magnetic bamboo-like nitrogen-carbon (N-C) nanotubes loaded with nickel-cobalt alloy (NiCo) nanoparticles (NiCo@N-CNTs) for the determination of ractopamine (RAC). During the preparation of NiCo@N-CNTs, Co-MOF-67 (ZIF-67) was firstly synthesized, and then blended with dicyandiamide and nickel acetate, followed by a one-step pyrolysis procedure to prepare NiCo@N-doped carbon nanotubes. The surface morphology, structure, and chemical composition of NiCo@N-CNTs were characterized by SEM, TEM, XRD, XPS, and EDS. The electrocatalytic and electrochemical behavior of NiCo@N-CNTs were investigated by cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). The results demonstrated that NiCo@N-CNTs possessed remarkable conductivity and electrocatalysis to the oxidation of ractopamine (RAC). By using screen-printed electrode (SPE), NiCo@N-CNTs, and a designed base support, a magnetic RAC sensor (NiCo@N-CNTs/SPE) was successfully constructed. It presented a detection linear range of 0.05-80 µM with a detection limit of 12 nM (S/N = 3). It also exhibited good sensitivity, reproducibility, and practicability in spiked real pork samples. Since the adhesion of NiCo/N-CNTs on SPE was controlled by magnet, the NiCo@N-CNTs was easily detached from the SPE surface by magnetism and thus displayed excellent renewability. This work broadened insights into portable devices for on-site and real-time analysis.

Copper Micro-Flowers for Electrocatalytic Sensing of Nitrate Ions in Water.

The progressive increase in nitrate's (NO3-) presence in surface and groundwater enhances environmental and human health risks. The aim of this work is the fabrication and characterization of sensitive, real-time, low-cost, and portable amperometric sensors for low NO3- concentration detection in water. Copper (Cu) micro-flowers were electrodeposited on top of carbon screen-printed electrodes (SPCEs) via cyclic voltammetry (with voltage ranging from -1.0 V to 0.0 V at a scan rate of 0.1 V s-1). The obtained sensors exhibited a high catalytic activity toward the electro-reduction in NO3-, with a sensitivity of 44.71 µA/mM. They had a limit of detection of 0.87 µM and a good dynamic linear concentration range from 0.05 to 3 mM. The results were compared to spectrophotometric analysis. In addition, the devices exhibited good stability and a maximum standard deviation (RSD) of 5% after ten measurements; reproducibility, with a maximum RSD of 4%; and repeatability after 10 measurements with the RSD at only 5.63%.

Waste DVD polycarbonate substrate for screen-printed carbon electrode modified with PVP-stabilized AuNPs for continuous free chlorine detection.

An electrochemical free chlorine sensor was developed by modifying a lab-made screen-printed carbon electrode (SPCE) with gold nanoparticles synthesized with polyvinylpyrrolidone (AuNPs-PVP). The electrode was made by screen printing carbon ink on a waste digital versatile disc (SPC-wDVD). PVP was used to stabilize AuNPs. Scanning electron microscopy showed that AuNPs aggregated without the stabilizer. The electrochemical behavior of the SPC-wDVD was evaluated by comparison with commercial SPCEs from two companies. Electrochemical characterization involved cyclic voltammetry and electrochemical impedance spectroscopy. The detection of free chlorine in water samples was continuous, facilitated by a flow-injection system. In the best condition, the developed sensor exhibited linearity from 0.25 to 3.0 and 3.0 to 500 mg L-1. The limit of detection was 0.1 mg L-1. The stability of the sensor enabled the detection of free chlorine at least 475 times with an RSD of 3.2 %. The AuNPs-PVP/SPC-wDVD was able to detect free chlorine in drinking water, tap water and swimming pool water. The agreement between the results obtained with the proposed method and the standard spectrophotometric method confirmed the precision of the developed sensor.

Fabrication of Strontium Molybdate with Functionalized Carbon Nanotubes for Electrochemical Determination of Antipyretic Drug-Acetaminophen.

In recent years, there has been a significant interest in the advancement of electrochemical sensing platforms to detect antipyretic drugs with high sensitivity and selectivity. The electrochemical determination of acetaminophen (PCT) was studied with strontium molybdate with a functionalized carbon nanotube (SrMoO4@f-CNF) nanocomposite. The SrMoO4@f-CNF nanocomposite was produced by a facial hydrothermal followed by sonochemical treatment, resulting in a significant enhancement in the PCT determination. The sonochemical process was applied to incorporate SrMoO4 nanoparticles over f-CNF, enabling a network-like structure. Moreover, the produced SrMoO4@f-CNF composite structural, morphological, and spectroscopic properties were confirmed with XRD, TEM, and XPS characterizations. The synergistic effect between SrMoO4 and f-CNF contributes to the lowering of the charge transfer resistance (Rct=85 Ω·cm2), a redox potential of Epc=0.15 V and Epa=0.30 V (vs. Ag/AgCl), and a significant limit of detection (1.2 nM) with a wide response range of 0.01-28.48 µM towards the PCT determination. The proposed SrMoO4@f-CNF sensor was studied with differential pulse voltammetry (DPV) and cyclic voltammetry (CV) techniques and demonstrated remarkable electrochemical properties with a good recovery range in real-sample analysis.

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.