L-Lactate Electrochemical Biosensor Based on an Integrated Supramolecular Architecture of Multiwalled Carbon Nanotubes Functionalized with Avidin and a Recombinant Biotinylated Lactate Oxidase.

L-Lactate is an important bioanalyte in the food industry, biotechnology, and human healthcare. In this work, we report the development of a new L-lactate electrochemical biosensor based on the use of multiwalled carbon nanotubes non-covalently functionalized with avidin (MWCNT-Av) deposited at glassy carbon electrodes (GCEs) as anchoring sites for the bioaffinity-based immobilization of a new recombinant biotinylated lactate oxidase (bLOx) produced in Escherichia coli through in vivo biotinylation. The specific binding of MWCNT-Av to bLOx was characterized by amperometry, surface plasmon resonance (SPR), and electrochemical impedance spectroscopy (EIS). The amperometric detection of L-lactate was performed at -0.100 V, with a linear range between 100 and 700 µM, a detection limit of 33 µM, and a quantification limit of 100 µM. The proposed biosensor (GCE/MWCNT-Av/bLOx) showed a reproducibility of 6.0% and it was successfully used for determining L-lactate in food and enriched serum samples.

Fentogram electrochemical detection of HIV RNA based on graphene quantum dots and gold nanoparticles.

This work reports the construction of an HIV-specific genosensor through the modification of carbon screen-printed electrodes (CSPE) with graphene quantum dots decorated with L-cysteine and gold nanoparticles (cys-GQDs/AuNps). Cys-GQDs were characterized by FT-IR and UV-vis spectra and electronic properties of the modified electrodes were evaluated by cyclic voltammetry and electrochemical impedance spectroscopy. The modification of the electrode surface with cys-GQDs and AuNps increased the electrochemical performance of the electrode, improving the electron transfer of the anionic redox probe [Fe(CN)6]3-/4- on the electrochemical platform. When compared to the bare surface, the modified electrode showed a 1.7 times increase in effective electrode area and a 29 times decrease in charge transfer resistance. The genosensor response was performed by differential pulse voltammetry, monitoring the current response of the anionic redox probe, confirmed with real genomic RNA samples, making it possible to detect 1 fg/mL. In addition, the genosensor maintained its response for 60 days at room temperature. This new genosensor platform for early detection of HIV, based on the modification of the electrode surface with cys-GQDs and AuNps, discriminates between HIV-negative and positive samples, showing a low detection limit, as well as good specificity and stability, which are relevant properties for commercial application of biosensors.

Electrochemical Paper-Based Nanobiosensor for Rapid and Sensitive Detection of Monkeypox Virus.

Monkeypox virus (MPXV) infection was classified as a public health emergency of international concern by the World Health Organization (WHO) in 2022, being transmitted between humans by large respiratory droplets, in contact with skin lesions, fomites, and sexually. Currently, there are no available accessible and simple-to-use diagnostic tests that accurately detect MPXV antigens for decentralized and frequent testing. Here, we report an electrochemical biosensor to detect MPXV antigens in saliva and plasma samples within 15 min using accessible materials. The electrochemical system was manufactured onto a paper substrate engraved by a CO2 laser machine, modified with gold nanostructures (AuNS) and a monoclonal antibody, enabling sensitive detection of A29 viral protein. The diagnostic test is based on the use of electrochemical impedance spectroscopy (EIS) and can be run by a miniaturized potentiostat connected to a smartphone. The impedimetric biosensing method presented excellent analytical parameters, enabling the detection of A29 glycoprotein in the concentration ranging from 1 × 10-14 to 1 × 10-7 g mL-1, with a limit of detection (LOD) of 3.0 × 10-16 g mL-1. Furthermore, it enabled the detection of MPXV antigens in the concentration ranging from 1 × 10-1 to 1 × 104 PFU mL-1, with an LOD of 7.8 × 10-3 PFU mL-1. Importantly, no cross-reactivity was observed when our device was tested in the presence of other poxvirus and nonpoxvirus strains, indicating the adequate selectivity of our nanobiosensor for MPXV detection. Collectively, the nanobiosensor presents high greenness metrics associated with the use of a reproducible and large-scale fabrication method, an accessible and sustainable paper substrate, and a low volume of sample (2.5 µL), which could facilitate frequent testing of MPXV at point-of-care (POC).

Circulating Tumor Cells Adhesion: Application in Biosensors.

The early and non-invasive diagnosis of tumor diseases has been widely investigated by the scientific community focusing on the development of sensors/biomarkers that act as a way of recognizing the adhesion of circulating tumor cells (CTCs). As a challenge in this area, strategies for CTCs capture and enrichment currently require improvements in the sensors/biomarker's selectivity. This can be achieved by understanding the biological recognition factors for different cancer cell lines and also by understanding the interaction between surface parameters and the affinity between macromolecules and the cell surface. To overcome some of these concerns, electrochemical sensors have been used as precise, fast-response, and low-cost transduction platforms for application in cytosensors. Additionally, distinct materials, geometries, and technologies have been investigated to improve the sensitivity and specificity properties of the support electrode that will transform biochemical events into electrical signals. This review identifies novel approaches regarding the application of different specific biomarkers (CD44, Integrins, and EpCAm) for capturing CTCs. These biomarkers can be applied in electrochemical biosensors as a cytodetection strategy for diagnosis of cancerous diseases.

Detection of Acetaminophen in Groundwater by Laccase-Based Amperometric Biosensors Using MoS2 Modified Carbon Paper Electrodes.

The use of enzyme-based biosensors for the detection and quantification of analytes of interest such as contaminants of emerging concern, including over-the-counter medication, provides an attractive alternative compared to more established techniques. However, their direct application to real environmental matrices is still under investigation due to the various drawbacks in their implementation. Here, we report the development of bioelectrodes using laccase enzymes immobilized onto carbon paper electrodes modified with nanostructured molybdenum disulfide (MoS2). The laccase enzymes were two isoforms (LacI and LacII) produced and purified from the fungus Pycnoporus sanguineus CS43 that is native to Mexico. A commercial purified enzyme from the fungus Trametes versicolor (TvL) was also evaluated to compare their performance. The developed bioelectrodes were used in the biosensing of acetaminophen, a drug widely used to relieve fever and pain, and of which there is recent concern about its effect on the environment after its final disposal. The use of MoS2 as a transducer modifier was evaluated, and it was found that the best detection was achieved using a concentration of 1 mg/mL. Moreover, it was found that the laccase with the best biosensing efficiency was LacII, which achieved an LOD of 0.2 µM and a sensitivity of 0.108 µA/µM cm2 in the buffer matrix. Moreover, the performance of the bioelectrodes in a composite groundwater sample from Northeast Mexico was analyzed, achieving an LOD of 0.5 µM and a sensitivity of 0.015 µA/µM cm2. The LOD values found are among the lowest reported for biosensors based on the use of oxidoreductase enzymes, while the sensitivity is the highest currently reported.

Development of a Redox-Polymer-Based Electrochemical Glucose Biosensor Suitable for Integration in Microfluidic 3D Cell Culture Systems.

The inclusion of online, in situ biosensors in microfluidic cell cultures is important to monitor and characterize a physiologically mimicking environment. This work presents the performance of second-generation electrochemical enzymatic biosensors to detect glucose in cell culture media. Glutaraldehyde and ethylene glycol diglycidyl ether (EGDGE) were tested as cross-linkers to immobilize glucose oxidase and an osmium-modified redox polymer on the surface of carbon electrodes. Tests employing screen printed electrodes showed adequate performance in a Roswell Park Memorial Institute (RPMI-1640) media spiked with fetal bovine serum (FBS). Comparable first-generation sensors were shown to be heavily affected by complex biological media. This difference is explained in terms of the respective charge transfer mechanisms. Under the tested conditions, electron hopping between Os redox centers was less vulnerable than H2O2 diffusion to biofouling by the substances present in the cell culture matrix. By employing pencil leads as electrodes, the incorporation of these electrodes in a polydimethylsiloxane (PDMS) microfluidic channel was achieved simply and at a low cost. Under flow conditions, electrodes fabricated using EGDGE presented the best performance with a limit of detection of 0.5 mM, a linear range up to 10 mM, and a sensitivity of 4.69 µA mM-1 cm-2.

Integrating machine learning and electrochemistry to develop a glucose biosensor assembled with Ganoderma applanatum lectin.

Fungal lectins have enormous biotechnological potential, but limited knowledge about their biochemical and biophysical features prevents their proper use. Herein, we report an innovative alternative to use Ganoderma applanatum lectin (GAL) as a glucose biorecognition element, after identifying the ideal electroanalytical conditions by machine learning studies performed with a homologous agglutinin from the same macrofungus. The research revealed that GAL has moderate resistance to pH (4-8) and temperature (20-60 °C) variations, but its hemagglutinating activity (376.5 HU mg-1 GAL at 20 °C) was better conserved under physiological conditions. Integrating electrochemical data and semi-empirical molecular modeling, biocompatible and electrostatically favorable conditions were found to immobilize the lectin on Prussian blue-modified glassy carbon electrode, after thermal activation of the metal-complex film. The glucose dose-response relationship obtained with the developed biosensor, defined as GAL/ta-PB/GCE, showed a typical Hill equation correlation, suggesting electrodic interactions represented by a sigmoidal mathematical function. GAL/ta-PB/GCE achieved remarkable electroanalytical performance, with emphasis on the detection limit (10.2 pM) and sensitivity (0.012 µA µM-1cm-2). The biosensor was successfully used to quantify glucose in pharmaceutical formulations, reiterating that the association of theoretical and experimental information drives important advances in bioelectrochemical studies.

Detection of COVID-19-related biomarkers by electrochemical biosensors and potential for diagnosis, prognosis, and prediction of the course of the disease in the context of personalized medicine.

As a more efficient and effective way to address disease diagnosis and intervention, cutting-edge technologies, devices, therapeutic approaches, and practices have emerged within the personalized medicine concept depending on the particular patient's biology and the molecular basis of the disease. Personalized medicine is expected to play a pivotal role in assessing disease risk or predicting response to treatment, understanding a person's health status, and, therefore, health care decision-making. This work discusses electrochemical biosensors for monitoring multiparametric biomarkers at different molecular levels and their potential to elucidate the health status of an individual in a personalized manner. In particular, and as an illustration, we discuss several aspects of the infection produced by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) as a current health care concern worldwide. This includes SARS-CoV-2 structure, mechanism of infection, biomarkers, and electrochemical biosensors most commonly explored for diagnostics, prognostics, and potentially assessing the risk of complications in patients in the context of personalized medicine. Finally, some concluding remarks and perspectives hint at the use of electrochemical biosensors in the frame of other cutting-edge converging/emerging technologies toward the inauguration of a new paradigm of personalized medicine.

Electrochemical Biosensor for SARS-CoV-2 cDNA Detection Using AuPs-Modified 3D-Printed Graphene Electrodes.

A low-cost and disposable graphene polylactic (G-PLA) 3D-printed electrode modified with gold particles (AuPs) was explored to detect the cDNA of SARS-CoV-2 and creatinine, a potential biomarker for COVID-19. For that, a simple, non-enzymatic electrochemical sensor, based on a Au-modified G-PLA platform was applied. The AuPs deposited on the electrode were involved in a complexation reaction with creatinine, resulting in a decrease in the analytical response, and thus providing a fast and simple electroanalytical device. Physicochemical characterizations were performed by SEM, EIS, FTIR, and cyclic voltammetry. Square wave voltammetry was employed for the creatinine detection, and the sensor presented a linear response with a detection limit of 0.016 mmol L-1. Finally, a biosensor for the detection of SARS-CoV-2 was developed based on the immobilization of a capture sequence of the viral cDNA upon the Au-modified 3D-printed electrode. The concentration, immobilization time, and hybridization time were evaluated in presence of the DNA target, resulting in a biosensor with rapid and low-cost analysis, capable of sensing the cDNA of the virus with a good limit of detection (0.30 µmol L-1), and high sensitivity (0.583 µA µmol-1 L). Reproducible results were obtained (RSD = 1.14%, n = 3), attesting to the potentiality of 3D-printed platforms for the production of biosensors.

Acetylcholinesterase Biosensor Based on Functionalized Renewable Carbon Platform for Detection of Carbaryl in Food.

Enzymatic electrochemical biosensors play an important role in the agri-food sector due to the need to develop sustainable, low-cost, and easy-to-use analytical devices. Such biosensors can be used to monitor pathogens, endocrine disruptors, and pesticides, such as carbaryl, widely used in many crops. The use of renewable carbon (RC) sources, provided from biomass pyrolysis has been often applied in the fabrication of such sensors. This material is a great candidate for biosensor fabrication due to the presence of surface functional groups, porosity, and moderate surface area. This work describes the functionalization of RC material through an acid treatment with a sulfonitric solution HNO3/H2SO4 (1:3) and the resulting material was characterized by scanning electron microscopy. The obtained RC functionalized (RCF) and the acetylcholinesterase enzyme (AChE) were applied in the construction of the electrochemical biosensor on glassy carbon (GC) electrode and used to detect carbaryl in apple samples. The GC/RCF/AChE biosensor was able to detect the carbaryl pesticide from 5.0 to 30.0 nmol L-1, displaying a LOD of 4.5 nmol L-1. The detection of carbaryl in apple samples presented recoveries between 102.5 to 118.6% through the standard addition method. The proposed biosensor is a promising renewable tool for food safety.

Rapid Detection of Anti-SARS-CoV-2 Antibodies with a Screen-Printed Electrode Modified with a Spike Glycoprotein Epitope.

BACKGROUND: The coronavirus disease of 2019 (COVID-19) is caused by an infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). It was recognized in late 2019 and has since spread worldwide, leading to a pandemic with unprecedented health and financial consequences. There remains an enormous demand for new diagnostic methods that can deliver fast, low-cost, and easy-to-use confirmation of a SARS-CoV-2 infection. We have developed an affordable electrochemical biosensor for the rapid detection of serological immunoglobulin G (IgG) antibody in sera against the spike protein. MATERIALS AND METHODS: A previously identified linear B-cell epitope (EP) specific to the SARS-CoV-2 spike glycoprotein and recognized by IgG in patient sera was selected for the target molecule. After synthesis, the EP was immobilized onto the surface of the working electrode of a commercially available screen-printed electrode (SPE). The capture of SARS-CoV-2-specific IgGs allowed the formation of an immunocomplex that was measured by square-wave voltammetry from its generation of hydroquinone (HQ). RESULTS: An evaluation of the performance of the EP-based biosensor presented a selectivity and specificity for COVID-19 of 93% and 100%, respectively. No cross-reaction was observed to antibodies against other diseases that included Chagas disease, Chikungunya, Leishmaniosis, and Dengue. Differentiation of infected and non-infected individuals was possible even at a high dilution factor that decreased the required sample volumes to a few microliters. CONCLUSION: The final device proved suitable for diagnosing COVID-19 by assaying actual serum samples, and the results displayed good agreement with the molecular biology diagnoses. The flexibility to conjugate other EPs to SPEs suggests that this technology could be rapidly adapted to diagnose new variants of SARS-CoV-2 or other pathogens.

Wearable electrochemical biosensors to measure biomarkers with complex blood-to-sweat partition such as proteins and hormones.

Smart electronic devices based on micro-controllers, also referred to as fashion electronics, have raised wearable technology. These devices may process physiological information to facilitate the wearer's immediate biofeedback in close contact with the body surface. Standard market wearable devices detect observable features as gestures or skin conductivity. In contrast, the technology based on electrochemical biosensors requires a biomarker in close contact with both a biorecognition element and an electrode surface, where electron transfer phenomena occur. The noninvasiveness is pivotal for wearable technology; thus, one of the most common target tissues for real-time monitoring is the skin. Noninvasive biosensors formats may not be available for all analytes, such as several proteins and hormones, especially when devices are installed cutaneously to measure in the sweat. Processes like cutaneous transcytosis, the paracellular cell-cell unions, or even reuptake highly regulate the solutes content of the sweat. This review discusses recent advances on wearable devices based on electrochemical biosensors for biomarkers with a complex blood-to-sweat partition like proteins and some hormones, considering the commented release regulation mechanisms to the sweat. It highlights the challenges of wearable epidermal biosensors (WEBs) design and the possible solutions. Finally, it charts the path of future developments in the WEBs arena in converging/emerging digital technologies.

Electrochemical Biosensor for Sensitive Detection of Hepatitis B in Human Plasma.

In this work, we report the construction of a novel electrochemical device for molecular diagnosis of hepatitis B virus in the blood plasma of infected patients, using graphite electrodes functionalized with poly(4-aminophenol) and sensitized with a specific DNA probe. The recognition of genomic DNA was evaluated by electrochemical techniques (DPV and EIS) and scanning electron microscopy. The genosensor was efficient in detecting genomic DNA with a linear range from 1.176 to 4.825 µg mL-1 and detection limit of 35.69 ng mL-1 (4.63 IU ml-1 or 25.93 copies.ml-1), which is better than the 10.00 IU ml-1 limit of reference method, real-time PCR, used in point of care. EIS analysis shows that the genosensor resistance increased exponentially with the concentration of the genomic DNA target. This novel platform has advantages to its applicability in real samples, such as good sensitivity, selectivity, low sample volume, and fast assay time (36 min), thus interesting for application in the diagnosis of hepatitis B virus in blood plasma. Also, the ease of synthesis of the low-cost polymer by electrosynthesis directly on the electrode surface allows the translation of the platform to portable devices.

Self-Enriching Electrospun Biosensors for Impedimetric Sensing of Zika Virus.

Zika virus (ZIKV) infection is associated with the Guillain-Barré syndrome, and when non-vector congenital transmission occurs, fetal brain abnormalities are expected. After ZIKV infection, the blood, breast milk, and other body fluids contain low viral loads. Their detection is challenging as it requires the processing of larger input volumes of the clinical samples. Pre-enrichment is a valuable strategy to increase the analyte concentration. Therefore, the authors propose the use of a hierarchal composite polyaniline-(electrospun nanofiber) hydrogel mat (ENM) for the simultaneous enrichment and impedimetric sensing of ZIKV viral particles. The electrospinning conditions of polyvinyl alcohol and alginate, including blend formulation, were optimized through a factorial design. Disintegration and gelatinization were controlled via cross-linking to improve the hydrogel properties. Hierarchization was achieved by in situ chemical deposition of conductive polyaniline. The carboxyl groups of the ENM were used for the covalent immobilization of anti-ZIKV polyclonal antibodies used in the specific recognition of ZIKV within the medium of Vero cell culture. The specific capture and desorption of virions were studied at different pHs. ENMs were characterized by scanning electron microscopy and FTIR. Atomic force microscopy along with UV-vis and electrochemical impedance spectroscopies was used to monitor the antibody immobilization, ZIKV capture, and elution processes. Our results show that 14.2 mg (0.25 cm3) of ENM can capture 38.7 ± 2.5 µg of ZIKV with a desorption rate of 99.97% (38.29 ± 2.7 µg ZIKV), which is reusable for at least three times. Therefore, the capture capacity (micrograms of ZIKV captured per milligram of ENM) of polyaniline-hierarchized mats was 2.72 µg ZIKV/mg. The impedance LOD value was determined to be 2.76 µg of ZIKV particles (approximately 6.6 × 103 PFU/mL). As a result, we present a fast small-scale purification system that can simultaneously monitor ZIKV electrochemically and optically.

Biosensing Membrane Base on Ferulic Acid and Glucose Oxidase for an Amperometric Glucose Biosensor.

A biosensing membrane base on ferulic acid and glucose oxidase is synthesized onto a carbon paste electrode by electropolymerization via cyclic voltammetry in aqueous media at neutral pH at a single step. The developed biosensors exhibit a linear response from 0.082 to 34 mM glucose concentration, with a coefficient of determination R2 equal to 0.997. The biosensors display a sensitivity of 1.1 µAmM-1 cm-2, a detection limit of 0.025 mM, and 0.082 mM as glucose quantification limit. The studies reveal stable, repeatable, and reproducible biosensors response. The results indicate that the novel poly-ferulic acid membrane synthesized by electropolymerization is a promising method for glucose oxidase immobilization towards the development of glucose biosensors. The developed glucose biosensors exhibit a broader linear glucose response than other polymer-based glucose biosensors.

Detecting cancer cells with a highly sensitive LbL-based biosensor.

Early diagnosis of cancer is crucial for therapeutic methods to be more effective and to decrease the mortality rate due to this disease. Current diagnostic methods include imaging techniques that require expensive equipment and specialized personnel, making it difficult to apply them to many patients. To overcome these limitations, many biosensors have been developed to monitor cancer biomarkers. Here, we report on the electrochemical biosensor for selective detection of tumor cells using a simple and low-cost methodology. Layer-by-layer (LbL) self-assembly was used to modify indium tin oxide (ITO) electrodes with alternating layers of polyallylamine hydrochloride (PAH) and folic acid (FA), which binds to overexpressed folate receptors alpha (FRα) in tumor cells. The LbL-based biosensor showed high sensitivity in detecting cervical cancer cells (HeLa cells) using cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). A linear dependence with the logarithm cell concentration was observed and excellent detection limits were found, 4 cells mL-1 and 19 cells mL-1 for EIS and CV measurements, respectively. The developed biosensor also presented great reproducibility (RSD = 1.7%) and repeatability (RSD = 1.8%). The selectivity was confirmed after the biosensor interaction with healthy cells (HMEC cells), which did not produce significant changes in the electrochemical signals. Furthermore, it was demonstrated that selective detection of tumor cells occurs via an interaction with FA. The LbL-based biosensor provides a simple, accurate, and cost-effective platform to be applied in the early diagnosis of cancer.

The Potential Use of a Thin Film Gold Electrode Modified with Laccases for the Electrochemical Detection of Pyrethroid Metabolite 3-Phenoxybenzaldehyde.

There is increasing interest in developing portable technologies to detect human health threats through hybrid materials that integrate specific bioreceptors. This work proposes an electrochemical approach for detecting 3-Phenoxybenzaldehyde (3-PBD), a biomarker for monitoring human exposure to pyrethroid pesticides. The biosensor uses laccase enzymes as an alternative recognition element by direct oxidation of 3-PBD catalysts by the enzyme onto thin-film gold electrodes. The thin-film gold electrode modified by the immobilized laccase was characterized by Fourier-transform infrared spectrometry and scanning electron microscopy. The detection method's electrochemical parameters were established, obtaining a linear range of 5 t 50 µM, the limit of detection, and quantification of 0.061 and 2.02 µM, respectively. The proposed biosensor's analytical performance meets the concentration of pyrethroids detected in natural environments, reflecting its potential as an alternative analytical tool for monitoring the pyrethroid insecticide's presence.

Enzymatic Electroanalytical Biosensor Based on Maramiellus colocasiae Fungus for Detection of Phytomarkers in Infusions and Green Tea Kombucha.

In this work, we developed an enzymatic voltammetric biosensor for the determination of catechin and gallic acid in green tea and kombucha samples. The differential pulse voltammetry (DPV) methodology was optimized regarding the amount of crude enzyme extract, incubation time in the presence of the substrates, optimal pH, reuse of the biosensor, and storage time. Samples of green tea and kombucha were purchased in local markets in the city of Goiânia-GO, Brazil. High performance liquid chromatography (HPLC) and Folin-Ciocalteu spectrophotometric techniques were performed for the comparison of the analytical methods employed. In addition, two calibration curves were made, one for catechin with a linear range from 1 to 60 µM (I = -0.152 * (catechin) - 1.846), with a detection limit of 0.12 µM and a quantification limit of 0.38 µM and one for gallic acid with a linear range from 3 to 60 µM (I = -0.0415 * (gallic acid) - 0.0572), with a detection limit of 0.14 µM and a quantification limit of 0.42 µM. The proposed biosensor was efficient in the determination of phenolic compounds in green tea.

Optimized PAH/Folic acid layer-by-layer films as an electrochemical biosensor for the detection of folate receptors.

Folate receptor alpha (FR-α) is a glycoprotein overexpressed in tumor cell surfaces, especially in gynecologic cancers, and can be used as a biomarker for diagnostics. Currently, FRα is quantified by positron emission tomography (PET) or fluorescence imaging techniques. However, these methods are costly and time-consuming. We report on the development of an electrochemical biosensor for FRα detection based on the use of nanostructured layer-by-layer (LbL) films as modified electrodes. Multilayer films were deposited on indium tin oxide (ITO) electrodes by the alternately assembling of positively charged polyallylamine hydrochloride (PAH) and negatively charged folic acid (FA), used as the biorecognition element. UV-vis and FTIR spectroscopies revealed the successful PAH and FA adsorption on ITO. Devices performance was evaluated by cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). The [PAH/FA] films presented a good reproducibility (RSD of 1.12%) and stability when stored in the Tris-HCl solution (RSD 6.7%). The biosensor electrochemical response exhibited a linear relationship with FRα concentration in the range from 10 to 40 nM. The limit of detection reached for CV and EIS measurements were 0.7 and 1.5 nM, respectively. As a proof-of-concept, we show that the devices can differenciate tumor cells from healthy cell, showing an excellent selectivity. The biosensor device based on [PAH/FA] films represents a promising strategy for a simple, rapid, and low-cost cancer diagnosis through FRα quantification on the surface of cancer cells.