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The glycosylation status of proteins is increasingly used as biomarker to improve the reliability in the diagnosis and prognosis of diseases as relevant as cancer. This feeds the need for tools that allow its simple and reliable analysis and are compatible with applicability in the clinic. With this objective in mind, this work reports the first bioelectronic immunoplatforms described to date for the determination of glycosylated haptoglobin (Hp) and the simultaneous determination of total and glycosylated Hp. The bioelectronic immunoplatform is based on the implementation of non-competitive bioassays using two different antibodies or an antibody and a lectin on the surface of commercial magnetic microcarriers. The resulting bioconjugates are labeled with the horseradish peroxidase (HRP) enzyme, and after their magnetic capture on disposable electroplatforms, the amperometric transduction using the H2O2/hydroquinone (HQ) system allows the single or multiple detection. The developed immunoplatform achieves limits of detection (LODs) of 0.07 and 0.46 ng mL-1 for total and glycosylated Hp in buffer solution, respectively. The immunoplatform allows accurate determination using simple and relatively short protocols (approx. 75 min) of total and glycosylated Hp in the secretomes of in vitro-cultured colorectal cancer (CRC) cells with different metastatic potentials, which is not feasible, due to lack of sensitivity, by means of some commercial ELISA kits and Western blot methodology. Funding Open Access funding provided thanks to the CRUE-CSIC agreement with Springer Nature. Spanish Ministerio de Ciencia e Innovación (PID2019-103899RB-I00 and RTI2018-095756-B-I00), AES-ISCIII Program co-founded by FEDER funds (PI17CIII/00045 and PI20CIII/00019 grants), TRANSNANOAVANSENS-CM Program from the Comunidad de Madrid (Grant S2018/NMT-4349). Sí

Potassium ion channels are expressed on the cell membranes, implicated in wide variety of cell functions and intimately linked to cancer cell behaviors. This work reports the first bioplatform described to date allowing simple and rapid detection of ion channel activity and the effect of their inhibitors in cancer cells. The methodology involves interrogation of the channel of interest from cells specifically captured on magnetic immunoconjugates using specific detection antibodies that are labeled with horseradish peroxidase enzyme. The channel activity is reflected by an amperometric signal transduction of the resulting magnetic bioconjugates onto screen-printed carbon electrodes. The bioplatform feasibility was proven for the detection of the Kv channels in U87 human glioblastoma cells and their blocking by scorpion venom KAaH1 and KAaH2 peptides. The obtained results confirm the high sensitivity (detection of 5 U87 cells⋅mL

This work reports the first amperometric biosensor for the simultaneous determination of the single or total content of the most relevant human immunoglobulin isotypes (hIgs) of anti-dsDNA antibodies, dsDNA-hIgG, dsDNA-hIgM, dsDNA-hIgA and dsDNA-three hIgs, which are considered relevant biomarkers in prevalent autoimmune diseases such as systemic lupus erythematosus (SLE) as well as of interest in neurodegenerative diseases such as Alzheimer's disease (AD). The bioplatform involves the use of neutravidin-functionalized magnetic microparticles (NA-MBs) modified with a laboratory-prepared biotinylated human double-stranded DNA (b-dsDNA) for the efficient capture of specific autoantibodies that are enzymatically labeled with horseradish peroxidase (HRP) enzyme using specific secondary antibodies for each isotype or a mixture of secondary antibodies for the total content of the three isotypes. Transduction was performed by amperometry (-0.20 V vs. the Ag pseudo-reference electrode) using the H2O2/hydroquinone (HQ) system after trapping the resulting magnetic bioconjugates on each of the four working electrodes of a disposable quadruple transduction platform (SP4CEs). The bioplatform demonstrated attractive operational characteristics for clinical application and was employed to determine the individual or total hIgs classes in serum from healthy individuals and from patients diagnosed with SLE and AD. The target concentrations in AD patients are provided for the first time in this work. In addition, the results for SLE patients and control individuals agree with those obtained by applying ELISA tests as well as with the clinical ranges reported by other authors, using individual detection methodologies restricted to centralized settings or clinical laboratories. The financial support of PID2019-103899RB-I00 and PID2021-122457OB-I00 (Spanish Ministerio de Ciencia e Innovación) Research Projects, PI17CIII/00045 and PI20CIII/00019 Grants from the AESISCIII Program co-founded by FEDER funds and the TRANSNANOAVANSENS-CM Program from the Comunidad de Madrid (Grant S2018/NMT-4349) are gratefully acknowledged. B.A. acknowledges predoctoral contracts from the Spanish Ministerio de Ciencia, Innovación y Universidades (PRE2019-087596). M.G-A. acknowledges the postdoctoral contract Margarita Salas for the requalification of the Spanish University System. A.M-C. was supported by a FPU predoctoral contract supported by the Spanish Ministerio de Educación, Cultura y Deporte. Sí

Pancreatic ductal adenocarcinoma (PDAC) is the solid tumor with the worst prognosis, representing today the third cause of cancer-related deaths in developed countries and expected to be the second in 2030. Today, CA19-9 remains the only clinically used marker for management of PDAC (FDA-approved as a disease moni- toring marker). This work reports a disposable amperometric immunoplatform for the determination of CA19-9. The immunoplatform skilfully combines the advantages of magnetic microsupports (MBs) for implementation of the immunoassay and amperometric transduction on screen-printed carbon electrodes (SPCEs). The method involves the preparation of sándwich immunocomplexes enzymatically labeled with the enzyme horseradish peroxidase (HRP) on the MBs and uses a detection antibody conjugated to HRP. Once the HRP- labeled sandwich immunocomplexes-bearing MBs were trapped on the SPCE surface, the variation of the catho- dic current was measured in the presence of H2O2 and hydroquinone (HQ), which was directly proportional to the concentration of CA19-9. Under the optimized experimental conditions, the immunoplatform allowed the amperometric determination of CA19-9 standards over the 5.0 to 500 U mL−1 concentration range, with a limit of detection (LOD) value of 1.5 U mL−1 in 1 h. The method exhibits good reproducibility and selectivity and the magnetic immunoconjugates shows a good storage stability. The immunoplatform was applied to the deter- mination of CA19-9 in serum samples of a medium-sized cohort (22 individuals) of healthy subjects and patients diagnosed with PDAC. The obtained results demonstrated the immunoplatform ability to discriminateboth types of individuals within 1 h after sample dilution. The developed immunoplatform represents an improvement in terms of cost, applicability and accessibility compared to the ELISA-based techniques currently used in the clinic.

A dual immunosensor is reported for the simultaneous determination of two important immunity-related cytokines: BAFF (B cell activation factor) and APRIL (a proliferation-induced signal). Sandwich-type immunoassays with specific antibodies (cAbs) and a strategy for signal amplification based on labelling the detection antibodies (dAbs) with binary MoS2/MWCNTs nanostructures and using horseradish peroxidase (HRP) were implemented. Amperometric detection was carried out at screen-printed dual carbon electrodes (SPdCEs) through the hydroquinone HQ/H2O2 system. The developed dual immunosensor provided limit of detection (LOD) of 0.08 and 0.06 ng mL-1 for BAFF and APRIL, respectively, and proved to be useful for the determination of both cytokines in cancer cell lysates and serum samples from patients diagnosed with autoimmune diseases and cancer. The obtained results agreed with those found using ELISA methodologies. Open Access funding provided thanks to the CRUE-CSIC agreement with Springer Nature. Financial support was provided by Spanish Ministerio de Ciencia, Innovacion y Universidades (RTI2018-096,135-B-I00), Spanish Ministerio de Ciencia e Innovación (PID2019-103899RB-I00) Research Project, and the TRANSNANOAVANSENS-CM Program from the Comunidad de Madrid (Grant S2018/NMT-4349). Financial support of the PI17CIII/00045 and PI20CIII/00019 grants from the AES-ISCIII program was also provided to R. B. A predoctoral contract from the Spanish Ministerio de Ciencia, Innovacion y Universidades (PRE2019-087,596) was provided to B. Arévalo. A FPU predoctoral contract supported by the Spanish Ministerio de Educación, Cultura y Deporte was provided to A. M.-C. G. S.-F. is recipient of a predoctoral contract (grant number 1193818 N) supported by The Flanders Research Foundation (FWO). Sí

Early diagnosis in primary care settings can increase access to therapies and their efficiency as well as reduce health care costs. In this context, we report in this paper the development of a disposable immunoplatform for the rapid and simultaneous determination of two protein biomarkers recently reported to be involved in the pathological process of neurodegenerative disorders (NDD), tau protein (tau), and TAR DNA-binding protein 43 (TDP-43). The methodology involves implementation of a sandwich-type immunoassay on the surface of dual screen-printed carbon electrodes (dSPCEs) electrochemically grafted with p-aminobenzoic acid (p-ABA), which allows the covalent immobilization of a gold nanoparticle-poly(amidoamine) (PAMAM) dendrimer nanocomposite (3D-Au-PAMAM). This scaffold was employed for the immobilization of the capture antibodies (CAbs). Detector antibodies labeled with horseradish peroxidase (HRP) and amperometric detection at − 0.20 V (vs. Ag pseudo-reference electrode) using the H2O2/hydroquinone (HQ) system were used. The developed methodology exhibits high sensitivity and selectivity for determining the target proteins, with detection limits of 2.3 and 12.8 pg mL−1 for tau and TDP-43, respectively. The simultaneous determination of tau and TDP-43 was accomplished in raw plasma samples and brain tissue extracts from healthy individuals and NDD-diagnosed patients. The analysis can be performed in just 1 h using a simple one-step assay protocol and small sample amounts (5 μL plasma and 2.5 μg brain tissue extracts).

Magnetic nanoparticles (MNPs) can be used as antibody carriers in a wide range of immunosensing applications. The conjugation chemistry for preparing antibody-MNP bionanohybrids should assure the nanoparticle’s colloidal dispersity, directional conformation and high biofunctionality retention of attached antibodies. In this work, peroxidase (HRP) was selected as model target analyte, and stable antibody-MNP conjugates were prepared using polyaldehyde-dextrans as multivalent linkers, also to prevent nanoparticles agglomeration and steric shielding of non-specific proteins. Under the manipulation of the oxidation variables, MNP-conjugated antibody showed the highest Fab accessibility, of 1.32 μmol analyte per μmol antibody, corresponding to 139 μmol aldehyde per gram of nanocarrier (5 mM NaIO4, 4 h). Demonstrating anti-interference advantage up to 10% serum, colorimetric immunoassay gave a detection limit (LOD) of 300 ng mL− 1 , while electrochemical transduction led to a considerable (680 times) improvement, with a LOD of 0.44 ng mL− 1 . In addition, polyaldehydedextran showed priority over polycarboxylated-dextran as the multivalent antibody crosslinker for MNPs in terms of sensitivity and LOD value, while immunosensors constructed with carboxylated magnetic microbeads (HOOC-MBs) outperformed MNPs-based immunoplatforms. This work sheds light on the importance of surface chemistry (type and density of functional groups) and the dimension (nanosize vs micrometer) of magnetic carriers to conjugate antibodies with better directional orientation and improve the analytical performance of the resulting immunosensors.

Glial fibrillary acidic protein (GFAP) is a member of the intermediate filament family of proteins with increased levels in serum and cerebrospinal fluid of patients with Alzheimer disease (AD) and other neurodegenerative diseases (NDs), such as vascular dementia (VD). This work describes the first magnetic microbeads (MBs)-based electrochemical immunoplatform for GFAP determination. The platform design comprises a sandwich immunoassay implemented on the MBs surface and amperometric transduction at single-use screen-printed carbon electrodes (SPCEs). Micro-sized carboxylic acid magnetic particles (COOH-MBs) were modified with a specific capture antibody (CAb) to selectively link the target protein, which was sandwiched with a biotinylated detector antibody (btn-DAb) further conjugated with a streptavidin-peroxidase (Strep-HRP) conjugate. Amperometric transduction was performed at SPCEs upon capturing the magnetic bioconjugates on their surface and through the hydrogen peroxide/hydroquinone (H2O2/HQ) system. The immunoplatform achieved a limit of detection of 67 pg mL(-1) for the amperometric detection of standards and selectivity compatible with clinical applicability to assist in minimally invasive NDs diagnosis and prognosis. The MBs-based immunoplatform was applied with good results to determine the endogenous content of GFAP in protein brain extracts without matrix effect and using just 6.25 ng of sample per determination. Furthermore, the developed methodology was capable of differentiating between healthy subjects and patients diagnosed with VD and AD in only 2 h, providing accurate results in line with those obtained by an ELISA kit that used the same immunoreagents.

The study of the human microbiome is a multidisciplinary area ranging from the field of technology to that of personalized medicine. The possibility of using microbiota biomarkers to improve the diagnosis and monitoring of diseases (e.g., cancer), health conditions (e.g., obesity) or relevant processes (e.g., aging) has raised great expectations, also in the field of bioelectroanalytical chemistry. The well-known advantages of electrochemical biosensors—high sensitivity, fast response, and the possibility of miniaturization, together with the potential for new nanomaterials to improve their design and performance—position them as unique tools to provide a better understanding of the entities of the human microbiome and raise the prospect of huge and important developments in the coming years. This review article compiles recent applications of electrochemical (bio)sensors for monitoring microbial metabolites and disease biomarkers related to different types of human microbiome, with a special focus on the gastrointestinal microbiome. Examples of electrochemical devices applied to real samples are critically discussed, as well as challenges to be faced and where future developments are expected to go.

This work reports the first electrochemical bioplatform for the determination of soy traces in food. The bioplatform involves sandwich-type immunoassays using specific antibodies for β-conglycinin and glycinin, which are the main allergenic soy proteins, and carboxylic acid-modified magnetic microbeads. Amperometric detection at −0.20 V (vs. an Ag pseudo-reference electrode) was performed using single or dual screen-printed carbon electrodes and the H2O2/hydroquinone (HQ) system. The measured variation in the cathodic current was directly proportional to the concentration of target allergenic proteins. The developed bioplatforms exhibit a good selectivity and sensitivity providing limits of detection (LOD) values of 0.03 and 0.02 ng mL−1 for β-conglycinin and glycinin, respectively. The determination of both proteins can be carried out in only 1.5 h. The electrochemical bioplatforms allow their accurate determinations (with results statistically comparable to those provided by ELISA methodologies) in raw cookie dough and baked cookies enriched with soy flour. The results obtained confirm, in a pioneering way with electrochemical biosensors, the possibility of discriminating samples incurred with as little as 0.0005 ppm of a food allergen in model cookie extracts., The financial support of PID2019-103899RB-I00 to S.C., (Spanish Ministerio de Ciencia e Innovación) Research Project, and TRANSNANOAVANSENS-CM Program from the Comunidad de Madrid (Grant S2018/NMT-4349) to S.C. are gratefully acknowledged. S.B. acknowledges a Juan de la Cierva Incorporación contract (Ref.: IJCI-2017-31345) from the Spanish Ministerio de Economía, Industria y Competitividad.

Current molecular diagnostics of prostate cancer relies on detection of elevated levels of PSA protein in serum, but its specificity has been questioned due to its higher levels also in non-malignant prostate diseases. A long non-coding RNA biomarker, PCA3, demonstrated excellent specificity for prostate cancer, and thus has become an interesting alternative to PSA monitoring. Its detection utilizes mostly reverse transcription PCR with optical detection, making the protocol longer and more expensive. To avoid PCR, we have developed an electrochemical assay coupled with LAMP, an isothermal amplification technique showing high sensitivities at constant temperatures and shorter reaction times. We amplified PCA3 RNA as well as PSA mRNA (serving as a control), hybridized LAMP products on magnetic beads and measured them with chronoamperometry at carbon electrode chips. We show good sensitivity and specificity for both biomarkers in prostate cancer cell lines, and successful detection of PCA3 in clinical samples, i.e., urine samples from 11 prostate cancer patients and 7 healthy controls, where we obtained excellent correlation with clinical data. This is to our knowledge a first such attempt to apply electrochemistry to determine two RNA biomarkers directly in urine samples of prostate cancer patients in a minimally invasive diagnostics format.

This work reports the first amperometric immunosensor for the simultaneous determination of four fertility-related hormones in saliva: progesterone (P4), luteinizing hormone (LH), estradiol (E2), and prolactin (PRL). The immune platform involves direct competitive (P4 and E2), and sandwich (LH and PRL) assays implemented onto functionalized magnetic microbeads (MBs). The amperometric transduction was performed upon placing the MBs-immunoconjugates onto each of the four working electrodes of the SPCE array (SP4CEs) and applying a detection potential of -0.20 V (vs. Ag pseudo-reference electrode) using the H2O2/hydroquinone (HQ) system. The achieved analytical and operational characteristics of the developed multiplexed immunoplatform showed a sensitivity that allows the determination of these hormones in saliva, and an adequate selectivity to analyse complex clinical samples. The bioplatform was employed for the determination of the set of four hormones in human saliva samples collected from individuals with different hormonal profiles. The results obtained using a conventional potentiostat were compared with those provided employing a novel low-cost custom-designed and field-portable quadruple potentiostat. Similar results were found which also agreed with those obtained by applying ELISA methods for the determination of single hormones.

This paper reports a simple electrochemical strategy for the determination of microRNAs (miRNAs) using a commercial His-Tag-Zinc finger protein (His-Tag-ZFP) that binds preferably (but non-sequence specifically) RNA hybrids over ssRNAs, ssDNAs, and dsDNAs. The strategy involves the use of magnetic beads (His-Tag-Isolation-MBs) as solid support to capture the conjugate formed in homogenous solution between His-Tag-ZFP and the dsRNA homohybrid formed between the target miRNA (miR-21 selected as a model) and a biotinylated synthetic complementary RNA detector probe (b-RNA-Dp) further conjugated with a streptavidin-horseradish peroxidase (Strep-HRP) conjugate. The electrochemical detection is carried out by amperometry at disposable screen-printed carbon electrodes (SPCEs) (- 0.20 V vs Ag pseudo-reference electrode) upon magnetic capture of the resultant magnetic bioconjugates and H2O2 addition in the presence of hydroquinone (HQ). The as-prepared biosensor exhibits a dynamic concentration range from 3.0 to 100 nM and a detection limit (LOD) of 0.91 nM for miR-21 in just ~ 2 h. An acceptable discrimination was achieved between the target miRNA and other non-target nucleic acids (ssDNA, dsDNA, ssRNA, DNA-RNA, miR-122, miR-205, and single central- or terminal-base mismatched sequences). The biosensor was applied to the analysis of miR-21 from total RNA (RNAt) extracted from epithelial non-tumorigenic and adenocarcinoma breast cells without target amplification, pre-concentration, or reverse transcription steps. The versatility of the methodology due to the ZFP's non-sequence-specific binding behavior makes it easily extendable to determine any target RNA only by modifying the biotinylated detector probe.

Nowadays, materials, solvents, and techniques are being used in accordance with the principles of green analytical chemistry for the development and application of electrochemical (bio)sensors. Electrodes prepared from nontoxic materials, eco-friendly solvents and reagents, and methodologies that make it possible the reduction of the sample size and the amount of waste products are being used with the main objective of minimizing the environmental impact. These aspects are briefly discussed in this short review through addressing the state of the art of electrochemical (bio)sensors in connection with the green chemistry. The relevant aspects related to the different items are considered with some illustrative examples taken from the recent literature. With the aim of providing suitable complete information, a table summarizing the fundamentals and analytical characteristics of some configurations is included in the Supporting Information .

Nowadays, there is increasing demand to overcome the major constraints involved in cumbersome and protracted procedures used in conventional laboratory methods for molecular detection and develop methodologies suitable to near real-time, continuous, and direct monitoring of the target analyte in the analyzed sample at decentralized settings. In this context, biosensors using electrochemistry as transduction technique are particularly attractive to monitor binding-induced changes in the rigidity of a redox-tagged nucleic acid or peptide-switching probes (switch-based biosensors) or a capture antibody attached to an interrogating electrode. Sensors based on biomolecular switches, DNAs, aptamers, and peptides that reversibly shift between two or more conformations (or conformational ensembles) in response to the binding of a specific target ligand have been applied to the determination of a wide range of relevant analytes (nucleic acids, proteins, small molecules, and ions) and are rapid (responding in minutes), sensitive, reagentless, easily reusable, and less prone to fouling issues. They are capable to report in near real time the target binding event without any subsequent processing step, directly in challenging samples or continuously in flowing samples, and even in vivo using affordable and miniaturization-compatible instrumentation. Only a few redox-labeled capture antibody–based immunosensing strategies have been reported to date for the reagentless and continuous electrochemical determination of relevant analytes.

The first electrochemical immunosensor for the determination of CXCL7 (chemokine (C-X-C motif) ligand 7) autoimmune biomarker is reported in this work. Click chemistry-assisted antibodies immobilization was performed by reaction of azide functionalized-multi-walled carbon nanotubes (MWCNTs) and ethynyl-IgG onto screen-printed carbon electrodes. The capture antibodies were further immobilized onto IgG-MWCNTs conjugates. After a blocking step with casein, a sandwich immunoassay was implemented involving biotinylated detector antibodies and alkaline phosphatase (AP)-streptavidin conjugate. Differential pulse voltammetry upon addition of 1-naphthylphosphate was used as the analytical readout. A linear calibration plot between 0.5 and 600 pg mL−1 CXCL7 and a LOD value of 0.1 pg mL−1 were obtained. The usefulness of the immunosensor was demonstrated by the successful analysis of serum samples from patients with rheumatoid arthritis.

Autoantibodies (AAbs) are antibodies produced against our own cells or tissues either providing a first defense against infections or indicating the presence of pathological processes. They are not only able to inform on the evolution of diseases but also to predict some illnesses well in advance. Currently, the evaluation of the number and type of formed AAbs is employed to assess the risk, rate, severity and progression of autoimmune and cancer diseases, and to help find therapies to prevent or mitigate the impact of these illnesses. Conventional methods for the determination of AAbs generally suffer from low sensitivity, time-consuming and laborious methodologies, and need specialized technicians and well-equipped labs. Consequently, seeking new methodologies for the rapid and low-cost screening of AAbs is of great interest for global health because it would shorten the delay between sample collection and diagnosis and improve the introduction of modern diagnostics into the developing world. Electrochemical biosensors are considered as a promising alternative to conventional techniques for the determination of clinical biomarkers due to their simplicity of use, low cost, high sensitivity, multiplexing abilities or compatibility with microfabrication and point of care testing. This review is focused on the critical discussion of selected electrochemical biosensors described to date for the determination of AAbs related to autoimmune diseases and several types of cancer. An overview pointing out future directions in this field is also provided.

Metastasis is responsible for about 90% of cancer-associated deaths. In the context of solid tumors, the low oxygen concentration in the tumor microenvironment (hypoxia) is one of the key factors contributing to metastasis. Tumor cells adapt to these conditions by overexpressing certain proteins such as programmed death ligand 1 (PD-L1) and hypoxia-inducible factor 1 alpha (HIF-1α). However, the determination of these tumor hypoxia markers that can be used to follow-up tumor progression and improve the efficiency of therapies has been scarcely addressed using electrochemical biosensors. In this work, we report the first electrochemical bioplatform for the determination of PD-L1 as well as the first one allowing its simultaneous determination with HIF-1α. The target proteins were captured and enzymatically labeled on magnetic microbeads and amperometric detection was undertaken on the surface of screen-printed dual carbon electrodes using the hydrogen peroxide/peroxidase/hydroquinone system. Sandwich immunoassays were implemented for both the HIF-1α and PD-L1 sensors and the analytical characteristics were evaluated providing LOD values of 86 and 279 pg mL−1 for the amperometric determination of PD-L1 and HIF-1α standards, respectively. The developed electrochemical immunoplatforms are competitive versus the only electrochemical immunosensor reported for the determination of HIF-1α and the “gold standard” ELISA methodology for the single determination of both proteins in terms of assay time, compatibility with the simultaneous determination of both proteins making their use suitable for untrained users at the point of attention. The dual amperometric immunosensor was applied to the simultaneous determination of HIF-1α and PD-L1 in cancer cell lysates. The analyses lasted only 2 h and just 0.5 μg of the sample was required.

his work reports the first electrochemicalimmunoassay involving magnetic microbeads (MBs) forthe determination of galectin-3 (Gal-3), a -galactosidase-binding lectin that acts as mediator of heart failure (HF).MBs-captured sandwich-type immune complexes andamperometric detection at disposable screen-printed car-bon electrodes were used. The immunoplatform showed adetection limit of 8.3 pgmL 1, good reproducibility, andexcellent selectivity. The endogenous concentration ofGal-3 in human plasma from HF patients was determinedwith results in agreement with those obtained usingELISA. The multiplexing feasibility of the developedimmunoplatform was demonstrated for the simultaneousdetermination of Gal-3 and N-terminal pro-brain natriu-retic peptide (NT-proBNP), This work is part of the POSITION-II project funded bythe ECSEL Joint Undertaking under grant number Ecsel-783132-Position-II-2017-IA; www.position-2.eu, andPID2019-103899RB-I00 (Ministerio de Ciencia e Innova-ción), and of the NANOCARDIOFLEX Project (RetosColaboración RTC-2015-4184-1, cofinanced by the Minis-terio de Economía y Competitividad and FEDER “unamanera de hacer Europa”). The financial support of theRTI2018-096135-B-I00 (Ministerio de Ciencia, Innovacióny Universidades) and PID2019-103899RB-I00 (Ministeriode Ciencia e Innovación) and Research Projects and theTRANSNANOAVANSENS-CM Program from the Co-munidad de Madrid (Grant S2018/NMT-4349) are alsogratefully acknowledged. S.V.P acknowledges the doctor-al fellowship from the Fundación Carolina

The preparation of an integrated biosensor for the easy, fast, and sensitive determination of miRNAs is described based on a direct hybridization format and a label-free voltammetric detection. The biosensor involves a disposable carbon electrode substrate doubly nanostructured with reduced graphene oxide (rGO) and AuNPs modified with pyrene carboxylic acid (PCA) and 6-ferrocenylhexanethiol (Fc-SH), respectively. A synthetic amino terminated DNA capture probe was covalently immobilized on the CO2H moieties of PCA/rGO, while Fc-SH was used as a signaling molecule. Differential pulse voltammetry was employed to record the decrease in the oxidation peak current of Fc after the hybridization due to the hindering of the electron transfer upon the formation of the DNA-RNA duplex on the electrode surface. The stepwise biosensor preparation was characterized by surface and electrochemical techniques showing the role played by each biosensor component as well as the reliability of the target miRNA determination. The determination of the oncogene miRNA-21 synthetic target allowed quantification in the low femtomolar range (LOD of 5 fM) with a high discrimination of single-base mismatched sequences in a single 30-min incubation step. The bioplatform allowed the determination of the target miRNA in a small amount of total RNA extracted from breast cancer (BC) cells or directly in serum samples collected from BC patients without the need for prior extraction, purification, amplification, or reverse transcription of the genetic material and with no matrix effect. Graphical abstract.

Over the past decade, artificial nanomaterials that exhibit properties similar to those of enzymes are gaining attraction in electrochemical biosensing as highly stable and low-cost alternatives to enzymes. This review article discusses the main features of the various nanomaterials (metal oxide, metal, and carbon-based materials) explored so far to mimic different kinds of enzymes. The unprecedented opportunities imparted by these functional nanomaterials or their nanohybrids, mostly providing peroxidase-like activity, in electrochemical affinity biosensing are critically discussed mainly in connection with their use as catalytic labels or electrode surface modifiers by highlighting representative strategies reported in the past 5 years with application in the food, environmental, and biomedical fields. Apart from outlining the pros and cons of nanomaterial-based enzyme mimetics arising from the impressive development they have experienced over the last few years, current challenges and future directions for achieving their widespread use and exploiting their full potential in the development of electrochemical biosensors are discussed. Graphical abstract.

Nowadays, electrochemical biosensors are reliable analytical tools to determine a broad range of molecular analytes because of their simplicity, affordable cost, and compatibility with multiplexed and point-of-care strategies. There is an increasing demand to improve their sensitivity and selectivity, but also to provide electrochemical biosensors with important attributes such as near real-time and continuous monitoring in complex or denaturing media, or in vivo with minimal intervention to make them even more attractive and suitable for getting into the real world. Modification of biosensors surfaces with antibiofouling reagents, smart coupling with nanomaterials, and the advances experienced by folded-based biosensors have endowed bioelectroanalytical platforms with one or more of such attributes. With this background in mind, this review aims to give an updated and general overview of these technologies as well as to discuss the remarkable achievements arising from the development of electrochemical biosensors free of reagents, washing, or calibration steps, and/or with antifouling properties and the ability to perform continuous, real-time, and even in vivo operation in nearly autonomous way. The challenges to be faced and the next features that these devices may offer to continue impacting in fields closely related with essential aspects of people’s safety and health are also commented upon.

This paper describes the first biosensor reported to date for the determination of mustard seed traces. The biosensor consists of an amperometric immunosensing platform able to sensitively and selectively determine Sin a 1 content, the major allergen of yellow mustard and the most abundant protein of these seeds. The immunosensing platform exploits the coupling of magnetic microbeads (MBs) modified with sandwich-type immune complexes, comprising polyclonal and monoclonal antibodies, selective to the target protein for its capturing and detection, respectively. In addition, a HRP-conjugated secondary antibody was used for enzymatic labelling of the monoclonal antibody, and amperometric transduction was made at screen-printed carbon electrodes (SPCEs) using the hydroquinone (HQ)/H2O2 system. The electrochemical immunosensor allows the simple and fast detection (a single 1-h incubation step) of Sin a 1 with a limit of detection of 0.82 ng mL−1 (20.5 pg of protein in 25 μL of sample) with high selectivity against structurally similar non-target allergenic proteins (such as Pin p 1 from pine nut). The developed immunoplatform was successfully used for the analysis of peanut, rapeseed, cashew, pine nut and yellow mustard extracts, giving only positive response for the yellow mustard extract with a Sin a 1 content, in full agreement with that provided by conventional ELISA methodology.

This work reports the first bioplatform able to determine electrochemically 5-hydroxymethylcytosine (5-hmC) methylation events at localized sites and single-base sensitivity. The described bioplatform relies on a specific antibody (anti-5-hmC), further conjugated with commercial bioreagents loaded with multiple horseradish peroxidase (HRP) molecules, recognizing the epimark in a target DNA, captured through hybridization onto streptavidin-magnetic microbeads (Strep-MBs) modified with a complementary DNA capture probe. The electrochemical detection is performed by amperometry (-0.20 V vs Ag pseudoreference electrode) at disposable screen-printed carbon electrodes (SPCEs) in the presence of H2O2/hydroquinone (HQ) upon magnetic capture of the modified MBs onto the SPCE. The use of the commercial bioreagents ProtA-polyHRP80 and Histostar, very scarcely explored so far in electrochemical biosensors, provides high sensitivities for a synthetic target DNA sequence with a unique 5-hmC in the promoter region of MG...

Edible electrochemical biosensors with remarkable prolonged resistance to extreme acidic conditions are described for direct glucose sensing in gastrointestinal (GI) fluids of different pH ranges and compositions. Such direct and stable glucose monitoring is realized using carbon-paste biosensors prepared from edible materials, such as olive oil and activated charcoal, shown to protect the activity of the embedded glucose oxidase (GOx) enzyme from strongly acidic conditions. The enzymatic resistance to low-pH deactivation allowed performing direct glucose monitoring in strong acidic environments (pH 1.5) over a 90-min period, while the response of conventional screen-printed (SP) biosensors decreased significantly following 10-min incubation in the same fluid. The developed edible biosensor displayed a linear response between 2 and 10 mM glucose with sensitivity depending on the pH of the corresponding GI fluid. In addition, coating the electrode surface with pH-responsive enteric coatings (Eudragit® L100 and Eudragit® E PO), of different types and densities, allows tuning the sensor activation in gastric and intestinal fluids at specific predetermined times. The attractive characteristics and sensing performance of these edible electrochemical biosensors, along with their pH-responsive actuation, hold considerable promise for the development of ingestible devices towards the biosensing of diverse target analytes after prolonged incubation in challenging body fluids. Graphical Abstract Edible biosensors allow direct electrochemical sensing in different gastrointestinal fluids and display remarkable prolonged resistance to extreme acidic conditions.

This short article reviews critically the latest advances, current trends, potentiality and existing challenges in electrochemical sensing of circulating biomarkers for early and minimally invasive diagnosis. The extensive literature in this field clearly demonstrates that electrochemical biosensors provide very interesting features and substantial advantages over more complex conventional strategies to perform, in a simple and rapid way, single or multiplexed determination of target biomarkers at low concentrations in minimally treated liquid biopsies, meeting also the requirements of point-of-care devices and applicability at different settings. Despite the numerous challenging issues to overcome before achieving the translation of these biosensing strategies into the clinic, the potentiality demonstrated so far for the accurate electrochemical determination of biomarkers at different molecular levels for early and non-invasive diagnosis permits to foresee they will take a privileged place together with the traditional methods in the clinical routine.

A sensitive amperometric immunosensor has been prepared by immobilization of capture antibodies onto gold nanoparticles (AuNPs) grafted on a screen-printed carbon electrode (SPCE) through aryl diazonium salt chemistry using 4-aminothiophenol (AuNPs-S-Phe-SPCE). The immunosensor was designed for the accurate determination of clinically relevant levels of B-type natriuretic peptide (BNP) in human serum samples. The nanostructured electrochemical platform resulted in an ordered layer of AuNPs onto SPCEs which combined the advantages of high conductivity and improved stability of immobilized biomolecules. The resulting disposable immunosensor used a sandwich type immunoassay involving a peroxidase-labeled detector antibody. The amperometric transduction was carried out at −0.20 V (vs the Ag pseudo-reference electrode) upon the addition of hydroquinone (HQ) as electron transfer mediator and H2O2 as the enzyme substrate. The nanostructured immunosensors show a storage stability of at least 25 days, a linear range between 0.014 and 15 ng mL−1, and a LOD of 4 pg mL−1, which is 100 times lower than the established cut-off value for heart failure (HF) diagnosis. The performance of the immunosensor is advantageously compared with that provided with immunosensors prepared by grafting SPCE with p-phenylendiamine (H2N-Phe-SPCE) and attaching AuNPs by immersion into an AuNPs suspension or by electrochemical deposition, as well as with immunosensors constructed using commercial AuNPs-modified SPCEs. The developed immunosensor was applied to the successful analysis of human serum from heart failure (HF) patients upon just a 10-times dilution as sample treatment., The financial support of projects: RetosColaboración RTC-2015- 4184-1 (cofinanced by the Ministry of Economy and Competitivity and FEDER “una manera de hacer Europa”), CTQ2015-70023-R and CTQ2015-64402-C2-1-R (Spanish Ministry of Economy and Competitivity Research Projects) and S2013/MT-3029 (NANOAVANSENS Program from the Comunidad de Madrid) are gratefully acknowledged. R.M. Torrente-Rodríguez acknowledges a predoctoral contract from the Spanish Ministerio de Economía y Competitividad.

An electrochemical immunosensor for the fast, sensitive, and selective determination of salivary prolactin (PRL) is reported in this work. The developed methodology uses a sandwich-type immunoassay implemented on magnetic microparticles (MBs) functionalized with neutravidin (NA-MBs) where the capture antibody was immobilized. The resulting immunocomplexes were placed onto a screen-printed carbon electrode (SPCE) and the target hormone quantification was made by amperometry at − 0.20 V (vs. Ag pseudo-reference electrode) using the HQ/H2O2 system as electron transfer mediator and enzyme substrate. The developed immunosensor allows the determination of PRL in a linear range between 1.1 and 100 ng mL−1 (R2 = 0.996) with a limit of detection (LOD) value of 0.32 ng mL−1 PRL, and shows an excellent selectivity. The whole assay protocol takes 46 min. The immunosensor was able to determine PRL in saliva with results in agreement with those provided by ELISA method. In addition, the developed immunosensor was applied to the determination of PRL in breast milk.

This review overviews and revalues the role that the electrochemical techniques and the continuously evolving electrode designs play in the tremendous progress experienced by electrochemical biosensing. These aspects are scarcely covered in the recent literature on electrochemical biosensors and are discussed in this article through selected representative examples together with other approaches involving the use of signal amplification strategies, electrocatalytic materials, ratiometric designs or antifouling chemistries. Regarding electrodes, the continuous advances made in screen-printed, micro-, paper-based and flexible and wearable materials are critically overviewed. Concerning electrochemical techniques, the use of selected widely known techniques (electrochemical impedance spectroscopy, stripping voltammetry, and potentiometry) to successfully address different applications in response to current biosensing challenges is highlighted through their smart coupling with new experimental designs, bioreceptors and materials.

Human disease development involves a highly dynamic and interactive system of multiple layers of molecular markers (e.g. genetics, epigenetics, mRNA transcripts, proteins, and metabolites). Being aware of this, precision medicine aims to provide a detailed characterization of a disease to customize healthcare. Although high-throughput omics technologies are currently able to gather in a single experiment, large amounts of data about a specific type of molecules, it is now fully accepted that the simultaneous analysis of multiple layers of molecular markers leads to novel strategies for their early detection, prevention, and treatment of diseases. In this sense, electrochemical biosensing, also in constant evolution, shows versatility for the simultaneous monitoring of different omics layers biomarkers, using simple, affordable and short testing time devices. This article overviews, briefly and critically, through representative recent reported examples, how electrochemical biosensing can contribute and assist multiomics techniques to facilitate the advent of the eagerly awaited precision medicine as well as the challenges to face it.

Early detection is often the key to successful treatment and patient survival. The presence of various disease signaling salivary biomarkers that accurately reflect normal and disease states in humans as well as the sampling benefits and easy storage and transport of saliva compared to blood are some of the reasons for being increasingly recognized as an attractive diagnostic fluid. This explains the burgeoning research field in developing new methodologies able to determine low concentrations of different levels of biomarkers in saliva in a simple and rapid way. Aiming at overcoming some limitations of conventional strategies, electrochemical affinity biosensors have demonstrated to offer interesting alternatives for salivary diagnostics. This paper reviews briefly the significance of saliva and salivary biomarkers for clinical diagnosis and therapeutic applications, and highlights recent advances, main challenges and future prospects in sensing bioaffinity platforms developed for the single or multiplexed determination of salivary biomarkers.

Proteases are involved in cancer‚ taking part in immune (dis)regulation, malignant progression and tumour growth. Recently, it has been found that expression levels of one of the members of the serine protease family, trypsin, is upregulated in human cancer cells of several organs, being considered as a specific cancer biomarker. Considering the great attention that electrochemical peptide sensors have nowadays, in this work, we propose a novel electroanalytical strategy for the determination of this important biomolecule. It implies the immobilization of a short synthetic peptide sequence, dually labelled with fluorescein isothiocyanate (FITC) and biotin, onto neutravidin-modified magnetic beads (MBs), followed by the peptide digestion with trypsin. Upon peptide disruption, the modified MBs were incubated with a specific fluorescein Fab fragment antibody labelled with horseradish peroxidase (HRP-antiFITC) and magnetically captured on the surface of a screen-printed carbon electrode (SPCE), where amperometric detection was performed using the hydroquinone (HQ)/HRP/H2O2 system. The biosensor exhibited a good reproducibility of the measurements (RSD 3.4%, n = 10), and specificity against other proteins and proteases commonly found in biological samples. This work reports the first quantitative data so far on trypsin expression in human cell lysates. The developed bioplatform was used for the direct determination of this protease in lysates from pancreatic cancer, cervix carcinoma and kidney cells in only 3 h and 30 min using low amounts (~ 0.1 μg) of raw extracts.

A disposable enzyme−free biosensing platform for the sensitive and selective voltammetric determination of miRNAs is reported. The bioplatform implies a sandwich−type hybridization configuration involving the use of two synthetic DNA probes that hybridize contiguously with the target miRNA−21. A thiolated capture probe was immobilized through thiol chemistry on disposable carbon electrodes modified with a hybrid nanomaterial composed of reduced graphene oxide (rGO) and gold nanoparticles (AuNPs). A biotinylated detection probe was conjugated with ferrocene-capped AuNPs modified with streptavidin (Fc−AuNPs−Strep) which were used as labeling nanocarriers. The extent of the hybridization event was followed by differential pulse voltammetric measurement of the Fc oxidation peak. Under the optimized conditions, the developed biosensor provides attractive characteristics for the determination of the synthetic target miRNA, with a linear range between 10 fM and 2 pM and a limit of detection (LOD) of 5 fM, fully discrimination towards a highly homologous miRNA (with just one mismatched base) and a storage stability of at least two months. The biosensor was able to determine accurately the target miRNA directly in scarcely diluted serum from breast cancer (BC) patients with no need for a previous total RNA (RNAt) extraction and in a very small amount of RNAt extracted from breast adenocarcinoma cells without the need for amplification or reverse transcription to complementary DNA.

Early detection and effective treatment are crucial to reduce the physical, emotional, and financial pressure exerted by growing cancer burden on individuals, families, communities, and health systems. Currently, it is clear that the accurate analysis of emerging cancer epigenetic and metastatic-related biomarkers at different molecular levels is envisaged as an exceptional solution for early and reliable diagnosis and the improvement of therapy efficiency through personalized treatments. Within this field, electrochemical biosensing has demonstrated to be competitive over other emerging and currently used methodologies for the determination of these biomarkers accomplishing the premises of user-friendly, multiplexing ability, simplicity, reduced costs and decentralized analysis, demanded by clinical oncology, thus priming electrochemical biosensors to spark a diagnostic revolution for cancer prediction and eradication. This review article critically discusses the main characteristics, opportunities and versatility exhibited by electrochemical biosensing, through highlighting representative examples published during the last two years, for the reliable determination of these emerging biomarkers, with great diagnostic, predictive and prognostic potential. Special attention is paid on electrochemical affinity biosensors developed for the single or multiplexed determination of methylation events, non-coding RNAs, ctDNA features and metastasis-related protein biomarkers both in liquid and solid biopsies of cancer patients. The main challenges to which further work must be addressed and the impact of these advances should have in the clinical acceptance of these emerging biomarkers are also discussed which decisively will contribute to understand the molecular basis involved in the epigenetics and metastasis of cancer and to apply more efficient personalized therapies.

Proper functionalization of electrode surfaces and/or nanomaterials plays a crucial role in the preparation of electrochemical (bio)sensors and their resulting performance. In this context, copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC) has been demonstrated to be a powerful strategy due to the high yields achieved, absence of by-products and moderate conditions required both in aqueous medium and under physiological conditions. This particular chemistry offers great potential to functionalize a wide variety of electrode surfaces, nanomaterials, metallophthalocyanines (MPcs) and polymers, thus providing electrochemical platforms with improved electrocatalytic ability and allowing the stable, reproducible and functional integration of a wide range of nanomaterials and/or different biomolecules (enzymes, antibodies, nucleic acids and peptides). Considering the rapid progress in the field, and the potential of this technology, this review paper outlines the unique features imparted by this particular reaction in the development of electrochemical sensors through the discussion of representative examples of the methods mainly reported over the last five years. Special attention has been paid to electrochemical (bio)sensors prepared using nanomaterials and applied to the determination of relevant analytes at different molecular levels. Current challenges and future directions in this field are also briefly pointed out.

A shrimp tropomyosin (TPM) immunosensor has been developed and optimized to detect trace amounts of shrimp (in the ppm range), based on a combination of an amperometric transduction, magnetic particles and disposable screen-printed electrodes. The approach is based on the implementation of a sandwich immunoassay format on the surface of magnetic beads and their coupling onto disposable screen-printed electrodes to finally register the amperometric response at −200 mV vs. Ag pseudo-reference electrode, using H2O2 as enzymatic substrate and hydroquinone as redox mediator. The use of carboxyl-functionalized magnetic microbeads (MBs) and in-house made magnetic nanoparticles (MNPs) as solid supports have been evaluated and compared. Our experimental results confirm that the use of MBs, in addition to simplifying the test protocol, improves the resulting sensitivity, so they were selected for the implementation of the immunosensor. In the optimized experimental conditions, the developed immunosensor offered a LOD of 47 pg mL−1 for amperometric determination of shrimp TPM standards and great selectivity against TPM from other sources, thus allowing differentiation between crustaceans (shrimp) and mollusks (squid). Applicability studies demonstrated successful determination both in crude and cooked samples using very simple protocols. Additionally, processed foods based on fish and mollusks that could potentially include crustaceans in their composition have been analyzed using the sensor and compared to the declared ingredients. The sensitivity and specificity showed by the sensor in the analysis of heterogeneous food samples without a previous purification or enrichment stage, also outperforms existing solutions in terms of time and cost effectiveness and permits its direct and smooth implementation in the food industry for routine allergen analyses.

This paper describes the results obtained in the development of the first electrochemical immunosensor described to date for the detection of E‐cadherin (E‐cad) protein, a relevant biomarker of prognosis and metastasis in cancer, based on the use of magnetic microcarriers (MBs) and amperometric transduction at screen‐printed carbon electrodes (SPCEs). Thus, the determination of E‐cad protein involved the use of two specific antibodies against this protein (one of them labelled with HRP) in a sandwich configuration onto HOOC‐MBs. The magnetic bioconjugates were captured onto SPCEs and the amperometric transduction was performed using the H2O2/hydroquinone (HQ) system. Under optimal conditions, this bioplatform demonstrated a wide linear concentration range (0.50–25 ng mL−1) and a detection limit as low as 0.16 ng mL−1, well below the optimal cut‐off level for the E‐cad protein (defined as 10,000 ng mL−1 for soluble E‐cad levels in serum). The developed sensor also showed a good reproducibility among measurements with seven different sensors constructed in the same manner (RSD, 5.4 %), stability for more than 15 days and good specificity towards other proteins commonly found on biological samples. The applicability of this simple handling bioplatform for the direct determination of this protein in cell lysates with different metastatic potential and extracts from paraffined‐embedded human colorectal cancer tissues of different grade were also demonstrated., The financial support of the Spanish Ministerio de Economía y Competitividad (CTQ2015‐64402‐C2‐1‐R Project) and the NANOAVANSENS Program from the Comunidad de Madrid (S2013/MT‐3029 Project) are gratefully acknowledged. C.M.S.M. was supported by a contract of the S2013/MT‐3029 Project. R.B. acknowledges the financial support of the PI17CIII/00045 grant from the AES‐ISCIII program. M.G.A. was supported by a contract of the Programa Operativo de Empleo Juvenil y la Iniciativa de Empleo Juvenil (YEI) with the participation of the Consejería de Educación, Juventud y Deporte de la Comunidad de Madrid y del Fondo Social Europeo.

This paper describes a dual electrochemical immunoassay for the simultaneous determination of IL-13Rα2 and CDH-17, two biomarkers of emerging relevance in metastatic processes. The sandwich assay uses a screen-printed dual carbon electrode that was electrochemically grafted with p-aminobenzoic acid to allow the covalent immobilization of capture antibodies. A hybrid composed of graphene quantum dots (GQDs) and multiwalled carbon nanotubes (MWCNTs) act as nanocarriers for the detection antibodies and horseradish peroxidase. The use of this hybrid material considerably improves the assay (in comparison to the use of MWCNTs) due to the peroxidase mimicking activity of the GQDs. The method works at a low working potential (0.20 V vs. Ag pseudo-reference electrode) and thus is not readily interfered by unknown electroactive species. The dual immunoassay allows for the selective determination of both biomarkers with LOD values of 1.4 (IL-13sRα2) and 0.03 ng mL−1 (CDH-17). The simultaneous determination of IL-13Rα2 and CDH-17 was accomplished in lysates from breast and colorectal cancer cells with different metastatic potential, and in paraffin-embedded tumor tissues extracts from patients diagnosed with colorectal cancer at different stages. The applicability to discriminate the metastatic potential even in intact cells through the detection of both extracellular receptors has been demonstrated also. The assay can be performed within 3 h, requires small sample amounts (0.5 μg), and has a simple protocol.

Matrix metalloproteinase 9 (MMP-9) is a zinc-dependent endopeptidase that promotes angiogenesis, tumor growth, metastasis and cell invasion through the degradation of extracellular matrix. This work reports a magnetic microbeads (MBs)-based sandwich immunoassay for the amperometric determination of MMP-9 at screen-printed carbon electrodes (SPCEs). The suitable capture antibody (cAb) is immobilized onto carboxylic MBs to selectively capture the antigen which is sandwiched with a biotinylated detector antibody (biotin-dAb) further conjugated with a commercial streptavidin-horseradish peroxidase (Strep-HRP) polymer. This immunoplatform provides great analytical characteristics in terms of selectivity and sensitivity, achieving a LOD value of 2.4 pg mL−1 for standards in buffered solutions. Although this value is similar to those reported for some other approaches described so far, the method described here is simpler involving a single 30 min incubation step which makes it ideal for automation or implementation in POC devices. Moreover, the method was assayed for the accurate determination of endogenous MMP-9 in both cancer cell lysates and serum samples of patients diagnosed with different subtypes of breast cancer (BC) after a simple dilution. The results obtained show that the disposable and affordable immunoplatform developed is able not only to discriminate BC patients from healthy individuals but also to do it for the worst outcome triple negative (TNBC) subtype.

Alpha-lactalbumin (α-LA) is one of the whey proteins in cows' milk that has been identified as allergenic. In this work, we present, for the first time, a very sensitive magnetic beads (MBs)-based immunosensor for the determination of α-LA. A sandwich configuration involving selective capture and horseradish peroxidase-labeled detector antibodies was implemented on carboxylic acid-modified magnetic beads, captured magnetically under the surface of a disposable screen-printed carbon electrode for amperometric detection using the hydroquinone (HQ)/H2O2 system. The α-LA immunosensor exhibited a wide linear range (37.0-5000pg/ml), a low limit of detection (LOD, 11.0pg/ml) and noteworthy selectivity against other non-target proteins. The MBs-based immunosensing platform was applied successfully for the determination of α-LA in several varieties of milk (raw and UHT cows' milk as well as human milk) and infant formulations. The results were corroborated with those obtained using a commercial ELISA method, thereby substantiating the analytical merits of this unique method.