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A biocatalytic electrode activated by pH signals was prepared with a multilayered nanostructured interface including PQQ-dependent glucose dehydrogenase (PQQ-GDH) directly associated with the conducting support and glucose oxidase (GOx) located on the external interface. GOx was immobilized through a pH-signal-cleavable linker composed of an iminobiotin/avidin complex. In the presence of GOx, glucose was intercepted at the external interface and biocatalytically oxidized without current generation, thus keeping the electrode in its nonactive state. When the pH value was lowered from pH 7.5 to 4.5 the iminobiotin/avidin complex was cleaved and GOx was removed from the interface allowing glucose penetration to the electrode surface where it was oxidized by PQQ-GDH yielding a bioelectrocatalytic current, thus switching the electrode to its active state. This process was used to trigger a drug-mimicking release process from another connected electrode. Furthermore, the pH-switchable electrode can be activated by biochemical signals logically processed by biocatalytic systems mimicking various Boolean gates. Therefore, the developed switchable electrode can interface biomolecular computing/sensing systems with drug-release processes.

We describe in this work a novel electrochemical immunosensor design making use of carbon nanohorns (CNHs) as a scaffold for the preparation of disposable immunosensing platforms for the determination of fibrinogen (Fib). The approach involved the immobilization of Fib onto activated CNHs deposited on screen-printed carbon electrodes (SPCEs) and the implementation of an indirect competitive assay using anti-Fib labeled with horseradish peroxidase (HRP) and hydroquinone (HQ) as the redox mediator. Both CNHs and the Fib-CNHs covalent assembly were characterized by microscopic and electrochemical techniques. The different variables affecting the analytical performance of the amperometric immunosensing strategy were optimized. The calibration plot for Fib allowed a range of linearity between 0.1 and 100 μg/mL (r(2) = 0.994) and a detection limit of 58 ng/mL to be achieved. The Fib-CNHs/SPCEs exhibited an excellent storage stability of at least 42 days. The developed immunosensor provides, in general, an analytical performance better than that reported for other Fib immunosensors and commercial ELISA kits. This simple and relatively low cost immunosensor configuration permitted the sensitive and selective determination of Fib in human plasma and urine.

A novel hybrid nanomaterial was synthesised by covalent attachment of O-carboxymethylcellulose to reduced graphene oxide. Graphene oxide was first anchored with (3-aminopropyl)triethoxysilane moieties to provide reactive primary amino groups at the basal plane. Periodate-oxidised O-carboxymethylcellulose was further covalently attached to this aminated nanomaterial through reductive alkylation with NaBH4. Stable aqueous dispersions were obtained with the resulting hybrid nanomaterial, which was used to coat glassy carbon electrodes. Furthermore, the enzyme tyrosinase was covalently immobilised and the nanostructured enzyme electrode was successfully employed for the amperometric detection of catechol in the 20 nM–56 μM range. The biosensor showed excellent analytical performance with a high sensitivity of 270 mA M−1 and a low detection limit of 0.2 nM.

A novel electrochemical immunosensor for the determination of the hormone prolactin (PRL) is reported by using glassy carbon electrodes modified with a poly(pyrrolepropionic acid)/multiwalled carbon nanotubes hybrid nanomaterial as transducer. An indirect competitive assay was utilized where the antigen was covalently immobilized on the modified electrode and a fixed amount of anti-PRL labelled with alkaline phosphatase (AP) was reacted with PRL in the sample. After attachment of the remaining labelled antibody to the immobilized PRL, the affinity reaction was monitored by differential pulse voltammetry of the AP enzyme reaction product using 1-naphtyl phosphate as substrate. Upon optimization of the variables affecting the immunosensor behaviour, a calibration graph for PRL with a wide linear range between 10 −2 and 10 4 ng/mL was obtained. The calculated LOD value, 3 pg/mL resulted to be remarkably lower than those reported previously using other approaches. Moreover, excellent reproducibility of the voltammetric measurements and selectivity against other hormones was found. The immunosensor was successfully applied to the analysis of human serum and urine at the clinically relevant concentration levels.

Forensic analysis is an important branch of modern Analytical Chemistry with many legal and socially relevant implications. Biosensors can play an important role as efficient tools in this field considering their well known advantages of sensitivity, selectivity, easy functioning, affordability and capability of miniaturization and automation. This article reviews the latest advances in the use of biosensors for forensic analysis. The different methodologies for the transduction of the produced biological events are considered and the applications to forensic toxicological analysis, classified by the nature of the target analytes, as well as those related with chemical and biological weapons critically commented. The article provides several Tables where the more relevant analytical characteristics of the selected reported methods are gathered.

MicroRNAs (miRs) have emerged as important clinical biomarkers with both diagnostic and prognostic value for relevant diseases, such as cancer. MiRs pose unique challenges for detection and are currently detected by northern blotting, real-time PCR, and microarray techniques. These expensive, complicated, and time-consuming techniques are not feasible for on-site miR determination. In this study, amperometric magnetobiosensors involving RNA-binding viral protein p19 as a selective biorecognition element were developed for miR quantification. The p19-based magnetosensors were able to detect 0.4 fmol of a synthetic target and endogenous miR-21 (selected as a model for its role in a wide variety of cancers) in only 2 h in total RNA extracted from cancer cells and human breast-tumor specimens without PCR amplification and sample preprocessing. These results open up formidable perspectives for the diagnosis and prognosis of human cancers and for drug-discovery programs.

Disposable amperometric magnetosensors, involving a mixture of modified-magnetic beads (MBs), for the multiplex screening of cephalosporins (CPHs), sulfonamides (SAs) and tetracyclines (TCs) antibiotic residues in milk are reported for the first time in this work. The multiplexed detection relies on the use of a mixture of target specific modified magnetic beads (MBs) and application of direct competitive assays using horseradish peroxidase (HRP)-labeled tracers. The amperometric responses measured at −0.20 V vs . the Ag pseudo-reference electrode of screen-printed carbon electrodes (SPCE) upon the addition of H 2 O 2 in the presence of hydroquinone (HQ) as redox mediator, were used to monitor the extent of the different affinity reactions. The developed methodology, involving a simple and short pretreatment, allowed discrimination between no contaminated UHT and raw milk samples and samples containing antibiotic residues at the maximum residue limits (MRLs). The usefulness of the multiplexed magnetosensor was demonstrated by analyzing spiked milk samples in only 5 min. The results demonstrated that a clear discrimination of milk samples contaminated with antibiotics at their MRL level or their mixtures, allowing the identification of milk not complying with current legislation. These features make the developed methodology a promising alternative in the development of user-friendly devices for on-site analysis to ensure quality control for dairy products.

Nowadays there is a growing interest in the de- velopment of devices for the fast and reliable detection and quantification of biomarkers associated with cardio- vascular diseases (CVDs). The main objective is not only an early diagnosis of these diseases but also predicting the risk that an individual can suffer CVD. Electrochemi- cal biosensors can play a key role in the development of low cost, portable and rapid sensing platforms for this purpose. This review discusses critically the state of the art in the development, applications and potential suita- bility of electrochemical biosensors for the determination of cardiovascular biomarkers as well as the required char- acteristics that these sensors should fulfill to be finally ac- cepted in the clinical community.

A novel inorganic–organic hybrid nanomaterial was prepared by anchoring (3-glycidyloxypropyl)trimethoxysilane at the surface of graphene oxide, further cross-linking with polyamidoamine G-4 dendrimer, and final decoration with platinum nanoparticles. A glassy carbon electrode was coated with this hybrid nanomaterial and then used as support for the covalent immobilization of glucose oxidase. This enzyme electrode was employed to construct a mediatorless glucose amperometric biosensor. The resulting biosensor exhibited good electrocatalytical activity for the oxidation of the H 2 O 2 produced by the enzyme catalyzed reaction, being able to detect glucose when poised at +400 mV. The biosensor showed a wide linear response to glucose ranging from 10 μM to 8.1 mM, high sensitivity of 24.6 mA/M cm 2 and low detection limit of 0.8 μM. The biosensor was successfully tested for the quantification of glucose in samples of commercial soft drinks.

The use of scanning electrochemical microscopy (SECM) for the qualitative and quantitative determination of sulfapyridine (SPY) in milk is described. A direct competitive immunoassay was performed involving an antibiotic horseradish peroxidase (HRP)-labeled analog and using selective capture antibodies immobilized on the surface of Protein G-modified glassy carbon plates. SECM detection was accomplished by means of the sample generator/tip collector (GC) mode involving the reduction of benzoquinone (BQ) generated upon the HRP-catalyzed oxidation of hydroquinone (HQ) at the modified substrate surface in the presence of H2O2. The detection limit for SPY in milk samples was as low as 0.13 ng mL−1.

In the last fifteen years, the nucleic acid biosensors and delivery area has seen a breakthrough due to the interrelation between the recognition of nucleic acid’s high specificity, the great sensitivity of electrochemical and optical transduction and the unprecedented opportunities imparted by nanotechnology. Advances in this area have demonstrated that the assembly of nanoscaled materials allows the performance enhancement, particularly in terms of sensitivity and response time, of functional nucleic acids’ biosensing and delivery to a level suitable for the construction of point-of-care diagnostic tools. Consequently, this has propelled detection methods using nanomaterials to the vanguard of the biosensing and delivery research fields. This review overviews the striking advancement in functional nanomaterials’ assisted biosensing and delivery of nucleic acids. We highlight the advantages demonstrated by selected well-known and rising star functional nanomaterials (metallic, magnetic and Janus nanomaterials) focusing on the literature produced in the past five years.

This work describes a new electroanalytical device for the simultaneous and reliable determination of two fertility relevant hormones (luteinizing hormone, LH, and progesterone, P4) in saliva. The device is constructed using a custom designed and field-portable potentiostat where dual disposable immunosensing platform are connected. The immunosensors are based on sandwich-type and competitive immunoassays implemented onto magnetic microbeads (MBs) functionalized with Neutravidin and Protein G for the determination of LH and P4, respectively. Amperometric detection performed at −0.20 V vs the Ag pseudo-reference electrode using the H2O2/hydroquinone (HQ) system was employed as the transduction technique after placing the MBs with immunocomplexes for each target hormone on the appropriate working electrode of screen-printed dual carbon electrodes (SPdCEs). The method exhibits high sensitivity and selectivity for the target hormones providing detection limits of 1.7 pg mL−1 and 0.10 mIU mL−1 for P4 and LH, respectively, with a 1 h test time. The applicability of the method was confirmed by determining both hormones in saliva samples from different volunteers providing results comparable to those obtained using amperometric transduction with a commercial potentiostat and with ELISA methodologies involving the same immunoreagents for each target hormone.

Novel core–shell Fe3O4@poly(dopamine) magnetic nanoparticles were prepared through an in situ self-polymerization method. The hybrid nanomaterial showed an average core diameter of 11 ± 3 nm and a polymer thin film thickness of 1.8 ± 0.2 nm. The core–shell nanoparticles were employed as solid supports for the covalent immobilization of horseradish peroxidase (HRP), and the resulting biofunctionalized magnetic nanoparticles were employed to construct an amperometric biosensor for H2O2. The enzyme biosensor showed a high sensitivity of 442.14 mA M−1 cm−2, a low limit of detection of 182 nM, a wide linear range from 6.0 × 10−7 to 8.0 × 10−4 M and high stability for 1 month.

A novel electrochemical immunosensor for the determination of ceruloplasmin (Cp) in human serum and urine is reported. The immunosensor configuration involves an indirect competitive immunoassay implying covalent immobilization of Cp on activated carboxylic groups at carbon nanotubes-modified screen-printed electrodes (CNTs/SPE). After Cp immobilization and reaction between the target analyte and anti-ceruloplasmin antibodies in solution, the remaining non-conjugated antibody is attached on the Cp-CNTs modified electrode. Monitoring of Cp is performed by means of a secondary antibody labeled with peroxidase (HRP-anti-IgG) and measurement of the amperometric current resulting from the addition of hydrogen peroxide in the presence of hydroquinone as the redox mediator. The experimental variables affecting the analytical performance of the immunosensor were optimized. Calibration curves for Cp provided a linear range between 0.07 and 250 μg/mL (r=0.997). The limit of detection achieved was 21 ng/mL. These analytical characteristics allow the immunosensor to be successfully used for the determination of Cp in spiked human serum and urine at various concentration levels.

A non-invasive, passive and simple to use skin surface based sensing device for determining the blood's ethanol content (BAC) by monitoring transdermal alcohol concentration (TAC) is designed and developed. The proposed prototype is based on bienzyme amperometric composite biosensors that are sensitive to the variation of ethanol concentration. The prototype correlates, through previous calibration set-up, the amperometric signal generated from ethanol in sweat with its content in blood in a short period of time. The characteristics of this sensor device permit determination of the ethanol concentration in isolated and in continuous form, giving information of the BAC of a subject either in a given moment or its evolution during long periods of time (8h). Moreover, as the measurements are performed in a biological fluid, the evaluated individual is not able to alter the result of the analysis. The maximum limit of ethanol in blood allowed by legislation is included within the linear range of the device (0.0005-0.6 g L(-1)). Moreover, the device shows higher sensitivity than the breathalyzers marketed at the moment, allowing the monitoring of the ethanol content in blood to be obtained just 5 min after ingestion of the alcoholic drink. The comparison of the obtained results using the proposed device in the analysis of 40 volunteers with those provided by the gas chromatographic reference method for determination of BAC pointed out that there were no significant differences between both methods.

A label-free dual electrochemical immunosensor was constructed for the multiplexed determination of human growth (hGH) and prolactin (PRL) hormones. The immunosensor used an electrochemical platform composed of carbon nanotube-screen printed carbon electrodes (CNT/SPCEs) modified with poly(ethylene-dioxythiophene) (PEDOT) and gold nanoparticles, on which the corresponding hGH and PRL antibodies were immobilized. The affinity reactions were monitored by measuring the decrease in the differential pulse voltammetric oxidation response of the redox probe dopamine. The experimental variables involved in the preparation of both AuNP/PEDOT/CNT/SPC modified electrodes and the dual immunosensor were optimized. The immunosensor exhibited an improved analytical performance for hGH and PRL with respect to other electrochemical immunosensor designs, showing wide ranges of linearity and low detection limits of 4.4 and 0.22 pg mL(-1), respectively. An excellent selectivity against other hormones and in the presence of ascorbic and uric acids was found. The usefulness of the dual immunosensor for the simultaneous analysis of hGH and PRL was demonstrated by analyzing human serum and saliva samples spiked with the hormones at different concentration levels.

The design, preparation and analytical performance of a novel integrated amperometric immunosensor based on the immobilization of selective capture antibodies on the surface of Protein G-modified screen-printed dual carbon electrodes (SPdCEs) for the multiplexed determination of sulfonamide and tetracycline antibiotics residues in milk is reported in this work. Protein G was covalently immobilized onto a 4-aminobenzoic acid (4-ABA) film grafted on the disposable electrode, and a direct competitive immunoassay using horseradish peroxidase (HRP)-labeled tracers was performed. The amperometric responses measured at -0.2 V vs. the silver pseudo-reference electrode of the SPdCE upon the addition of H2O2 in the presence of hydroquinone (HQ) as mediator were used to monitor the extent of the immunoreactions. The developed methodology showed very low limits of detection (in the low ppb level) for sulfonamide and tetracycline antibiotics tested in untreated milk samples, and a good selectivity against other antibiotic residues frequently detected in milk and dairy products. The usefulness of the dual immunosensor was demonstrated by analyzing spiked milk samples as well as a reference milk containing a certified oxytetracycline (OTC) content. Good recoveries were attained in an analysis time of 30 min.

A highly sensitive magnetoimmunosensor for the determination of human C-reactive protein (CRP) is described in this work. A sandwich format involving covalent immobilization of the capture antibody (antiCRP) onto carboxylic-modified magnetic beads (HOOC-MBs) activated with N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide (EDC) and N-hydroxysulfosuccinimide (sulfo-NHS), the antigen–antibody reaction and incubation of the modified MBs with a biotynilated antibody (biotin-antiCRP), is used. Furthermore, an incubation step with a Streptavidin HRP (Strp-HRP) conjugate is employed to allow monitoring of the affinity reaction. The resulting modified MBs are captured by a magnet placed under the surface of a disposable gold screen-printed electrode (Au/SPE) and the amperometric responses are measured at −0.10 V (vs. a Ag pseudo-reference electrode), upon addition of 3,3′,5,5′-tetramethylbenzidine (TMB) as electron transfer mediator and H2O2 as the enzyme substrate. The CRP magnetoimmunosensor exhibited a wide range of linearity between 0.07 and 1000 ng mL−1 with a low detection limit of (0.021 ± 0.005) ng mL−1. The magnetoimmunosensor was successfully applied to the analysis of an international standard for CRP serum.

A composite electrode consisted of gold nanoparticles (AuNPs), multi-walled carbon nanotubes (MWCNTs) and Teflon, to which peroxidase (HRP) and ferrocene (Fc) were incorporated as auxiliary enzyme and redox mediator, respectively, was constructed. The enzymes creatininase, creatinase and sarcosine oxidase were then co-immobilized onto the surface of the resulting HRP/Fc/AuNPs/MWCNTs/Teflon electrode for the preparation of a creatinine biosensor. Amperometry in stirred solutions using a detection potential of 0.0 V vs Ag/AgCl allowed a linear calibration plot to be obtained in the 0.003–1.0 mM creatinine concentration range with a limit of detection of 0.1 μM (S/N = 3). The apparent Michaelis-Menten constant for creatininase was KMap = 4.1 ± 0.4 mM. The developed biosensor was validated with good results by determining creatinine in human serum and correlating with the spectrophotometric Jaffe's method.

Novel label free electrochemical magnetoimmunosensors for the determination of lactoperoxidase (LPO) were developed based on the use of streptavidin-magnetic beads (Strep-MBs) and graphite-Teflon composite or screen-printed carbon electrodes (SPCEs) as electrochemical transducers. Biotinylated antiLPO antibodies were immobilized onto the Strep-MBs and a direct-type immunoassay by detecting the enzymatic activity of the captured target enzyme using amperometric detection at −0.20 V and the H2O2/hydroquinone (HQ) system was employed. The SPCEs-based magnetoimmunosensors allowed a detection limit (LOD) of 0.12 μg mL−1 and a good selectivity against nontarget proteins. Moreover, their analytical usefulness was successfully demonstrated by analyzing different types of milk samples.

Growing demand for developing ultrasensitive electrochemical bioassays has led to the design of numerous signal amplification strategies. In this context, carbon-based nanomaterials have been demonstrated to be excellent tags for greatly amplifying the transduction of recognition events and simplifying the protocols used in electrochemical biosensing. This relevant role is due to the carbon-nanomaterials’ large surface area, excellent biological compatibility and ease functionalization and, in some cases, intrinsic electrochemistry. These carbon-based nanomaterials involve well-known carbon nanotubes (CNTs) and graphene as well as the more recent use of other carbon nanoforms. This paper briefly discusses the advantages of using carbon nanostructures and their hybrid nanocomposites for amplification through tagging in electrochemical biosensing platforms and provides an updated overview of some selected examples making use of labels involving carbon nanomaterials, acting both as carriers for signal elements and as electrochemical tracers, applied to the electrochemical biosensing of relevant (bio)markers.

Nanoparticles composed of Prussian Blue, PB, and the cyanometalate structural analogues, CuFe, FeCoFe, and FeCo, are examined as inorganic clusters that mimic the functions of peroxidases. PB acts as a superior catalyst for the oxidation of dopamine to aminochrome by H2O2. The oxidation of dopamine by H2O2 in the presence of PB is 6-fold faster than in the presence of CuFe. The cluster FeCo does not catalyze the oxidation of dopamine to aminochrome. The most efficient catalyst for the generation of chemiluminescence by the oxidation of luminol by H2O2 is, however, FeCo, and PB lacks any catalytic activity toward the generation of chemiluminescence. The order of catalyzed chemiluminescence generation is FeCo ≫ CuFe > FeCoFe. The clusters PB, CuFe, FeCoFe, and FeCo mimic the functions of NADH peroxidase. The catalyzed oxidation of NADH by H2O2 to form NAD+ follows the order PB ≫ CuFe ∼ FeCoFe, FeCo. The efficient generation of chemiluminescence by the FeCo-catalyzed oxidation of luminol by H2O2 is used to develop a glucose sensor. The aerobic oxidation of glucose in the presence of glucose oxidase, GOx, yields gluconic acid and H2O2. The chemiluminescence intensities formed by the GOx-generated H2O2 relate to the concentration of glucose, thus providing a quantitative readout signal for the concentrations of glucose.

Owing to the outstanding conductivity and biocompatibility as well as numerous other fascinating properties of two-dimensional (2D)-nanomaterials, 2D-based nanohybrids have shown unparalleled superiorities in the field of electrochemical biosensors. This review highlights latest advances in electrochemical immunosensors for clinical biomarkers based on different hybrid 2D-nanomaterials. Particular attention will be given to hybrid nanostructures involving graphene and other graphene-like 2D-layered nanomaterials (GLNs). Several recent strategies for using such 2D-nanomaterial heterostructures in the development of modern immunosensors, both for tagging or modifying electrode transducers, are summarized and discussed. These hybrid nanocomposites, quite superior than their rival materials, will undoubtedly have an important impact within the near future and not only in clinical areas. Current challenges and future perspectives in this rapidly growing field are also outlined.

A sensitive and rapid method for the determination of the clinically relevant biomarker receptor tyrosine kinase AXL in serum involving amperometric disposable immunosensors is reported. The target protein was sandwiched between a specific capture antibody covalently immobilized on screen-printed carbon electrodes modified with electropolymerized poly(pyrrolepropionic acid) and a biotinylated detector antibody labeled with a streptavidin-horseradish peroxidase conjugate. The amperometric responses were measured at −0.20 V vs the Ag pseudo-reference electrode of the SPCE upon the addition of H2O2 in the presence of hydroquinone (HQ) as mediator. This integrated immunosensing platform showed a low limit of detection (337 pg mL−1), a good selectivity against other non-target serum proteins, and provided results statistically in agreement with those obtained by using a commercial ELISA kit. These attractive features together with the simplicity and easy automation and miniaturization of the required instrumentation make the developed methodology a promising alternative in the development of devices for on-site clinical analysis., The financial support of projects: Retos Colaboración RTC2015-4184-1 (cofinanced by the Ministry of Economy and Competitivity and FEDER), 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.

A novel electrochemical disposable nucleic acid biosensor for simple, rapid, and specific detection of adulterations with horsemeat is reported in this work. The biosensing platform involves immobilization of a 40-mer RNA probe specific for a characteristic fragment of the mitochondrial DNA D-loop region of horse onto the surface of magnetic microcarriers. In addition, signal amplification was accomplished by using a commercial antibody specific to RNA/ DNA duplexes and a bacterial protein conjugated with a horseradish peroxidase homopolymer (ProtA-HRP40). Amperometric detection at −0.20 V vs Ag pseudoreference electrode was carried out at disposable screen-printed carbon electrodes. The methodology achieved a limit of detection (LOD) of 0.12 pM (3.0 attomoles) for the synthetic target and showed ability to discriminate between raw beef and horsemeat using just 50 ng of total extracted mitochondrial DNA (∼16 660 bp in length) without previous fragmentation. The biosensor also allowed discrimination between 100% raw beef and beef meat samples spiked with only 0.5% (w/w) horse meat (levels established by the European Commission) using raw mitochondrial lysates without DNA extraction or polymerase chain reaction (PCR) amplification in just 75 min. These interesting features made the developed methodology an extremely interesting tool for beef meat screening, and it can be easily adapted to the determination of other meat adulterations by selection of the appropriate specific fragments of the mitochondrial DNA region and capture probes.

New amperometric immune-scaffolds for the rapid, sensitive and reliable determination of [...]

Electrochemical sandwich immunoassay strategies involving the use of carboxyl-functionalized magnetic microbeads (cMBs) and magnetic nanoparticles (cMNPs) have been evaluated and compared. The proteolytically cleaved soluble tyrosine kinase receptor sAXL was used as the target analyte. Antibodies against AXL were covalently immobilized on cMBs or cMNPs. Immunobinding of AXL was detected by means of a secondary biotinylated antibody and a streptavidin-horseradish peroxidase conjugate. The electrochemical transduction was accomplished by capturing the cMBs or cMNPs bearing the immunoconjugates onto screen-printed carbon electrodes (SPCEs) by using a small magnet. The amperometric response was measured at −0.20 V (vs the silver pseudo-reference electrode of the SPCE) upon the addition of H2O2 in the presence of hydroquinone as the redox mediator. The calibration plots for AXL extended up to 7.5 ng mL−1 when cMBs were used for the preparation of the immunosensor and up to 40 ng mL−1 in the case of using cMNPs. The respective slope values were 158 (cMBs) and 43 nA mL ng−1 (cMNPs), while the achieved LODs were 74 (cMBs) and 75 pg mL−1 (cMNPs). Although the immunosensors prepared with cMBs provided a shorter range of linearity, they exhibited a 3.7-times larger sensitivity than those constructed with cMNPs. The successful application of the new strategies was demonstrated for the determination of the endogenous content of sAXL in real human serum samples (a cut-off value of 71 ng mL−1 have been established for patients with risk of heart failure). The immunosensors constructed using cMBs or cMNPs can be advanta geously compared, in terms of sensitivity and fabrication time, with the only immunosensor for AXL previously reported. In addition, these new immunosensors took approximately half time than ELISA to perform the assay., The financial support of projects: Retos Colaboración RTC-2015-4184-1 (cofinanced by the Ministry of Economy and Competitivity and FEDER Buna 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 Ministry of Economy and Competitivity

Management and prognosis of disease requires the accurate determination of specific biomarkers indicative of normal or disease-related biological processes or responses to therapy. Moreover since multiple determinations of biomarkers have demonstrated to provide more accurate information than individual determinations to assist the clinician in prognosis and diagnosis, the detection of several clinical biomarkers by using the same analytical device hold enormous potential for early detection and personalized therapy and will simplify the diagnosis providing more information in less time. In this field, electrochemical immunosensors have demonstrated to offer interesting alternatives against conventional strategies due to their simplicity, fast response, low cost, high sensitivity and compatibility with multiplexed determination, microfabrication technology and decentralized determinations, features which made them very attractive for integration in point-of-care (POC) devices. Therefore, in this review, the relevance and current challenges of multiplexed determination of clinical biomarkers are briefly introduced, and an overview of the electrochemical immunosensing platforms developed so far for this purpose is given in order to demonstrate the great potential of these methodologies. After highlighting the main features of the selected examples, the unsolved challenges and future directions in this field are also briefly discussed.

A disposable amperometric sensor using magnetic microcarriers has been designed and implemented to be used in combination with the so called Express PCR to detect the presence of hazelnut traces in foodstuffs through the detection of Cor a 9 allergen coding sequence. The developed procedure involves the use of streptavidin-modified magnetic microbeads (Strep-MBs), specific biotinylated capture and detector probes which hybridize with a specific region of the gene encoding the protein Cor a 9, and appropriate primers for PCR amplification. A 50-mer synthetic target DNA or unmodified 100-bp PCR products were selective captured via sandwich hybridization with capture probe modified MBs and biotinylated signaling probes. The resulting biotinylated hybrids were coupled with a commercial streptavidin–peroxidase (Strep-HRP) conjugate and the final modified MBs were magnetically captured onto a screen-printed carbon electrode to perform amperometric detection using the H2O2/HQ system. A LOD of 0.72�pM was obtained for the synthetic target and the applicability studies demonstrated the possibility to detect the denatured PCR amplified samples obtained using only 20�pg of genomic DNA extracted from hazelnut. RSD values obtained, below 10% in all cases, confirmed the good reliability of extraction, amplification and quantification protocols involved in the developed methodology. The strict specificity of the designed primers and selected probes for hazelnut was demonstrated by performing PCR amplification of genomic DNA extracted from different hazelnut varieties and other species of similar families (pistachio, cashew, walnut and tangerine) and analyzing the resultant amplicons by the developed electrochemical sensor. The reliable and sensitive results achieved indicate that Express PCR in conjunction with an electrochemical DNA sensor, used for the first time in this work, provides a suitable sensitive, specific, and cost-effective method for routine food allergens determinations, particularly useful for resource-limited settings. � 2017 Elsevier B.V.

Viologen-SWCNT hybrids are synthesized by aryl-diazonium chemistry in the presence of isoamyl nitrite followed by condensation reaction of the resulting HOOC-PheSWCNT with 1-(3-aminoethyl)-4,4’-bipyridinium bromine and N-alkylation with 2- bromoethylamine. The V-Phe-SWCNT hybrids were characterized by using different spectroscopic techniques (FT-IR, Raman, UV-vis), TGA and Kaiser test. ViologenSWCNTs were used for the preparation of an electrochemical immunosensor for the determination of the transforming growth factor β1 (TGF-β1) cytokine considered as a reliable biomarker in several human diseases. The methodology involved preparation of VPhe-SWCNT(-HRP)-anti-TGF conjugates by covalent linkage of HRP and anti-TGF onto V-Phe-SWCNT hybrids. Biotinylated anti-TGF antibodies were immobilized onto 4- carboxyphenyl-functionalized SPCEs modified with streptavidin and a sandwich type 2 immunoassay was implemented for TGF-β1 with signal amplification using V-PheSWCNT(-HRP)-anti-TGF conjugates as carrier tags. The analytical characteristics exhibited by the as prepared immunosensor (range of linearity between 2.5 and 1000 pg mL-1 TGF-β1; detection limit of 0.95 pg mL-1 ) improve notably those reported with other previous immunosensors or ELISA kits. A great selectivity against other proteins was also found. The prepared immunosensor was validated by determining TGF-β1 in real saliva samples. Minimal sample treatment was required and the obtained results were in excellent agreement with those obtained by using a commercial ELISA kit.

The first electrochemical immunosensor for the determination of 8-isoprostane (8-iso prostaglandin F2α, ISO), one of the most reliable biomarkers of lipid peroxidation in the human body and of aging related to Alzheimer´s disease or atherosclerosis is reported in this article. Disposable screen-printed carbon electrodes modified with carboxylated carbon nanohorns (CNHs) were employed as scaffolds for covalent immobilization of a specific anti-ISO antibody). A competitive immunoassay involving ISO and HRPlabeled antigen was designed and the determination of ISO was carried out by amperometry at -200 mV using the H2O2/hydroquinone (HQ) system. Under the optimized conditions, the immunosensor provides a linear response for ISO (r2 = 0.998) extending up to 700 pg/mL, which is suitable for the determination of the target compound in human serum. The analytical performance of the immunosensor improves that claimed for ELISA kits in terms of linearity of the calibration plot, precision, with RSD values lower than 1 % , and assay time (1h 30min), and exhibits a low limit of detection, 12 pg/mL, a long storage stability (30 days), and an excellent selectivity against other proteins that may be found in human serum. The analytical utility of the developed immunosensor was demonstrated by determining ISO in two types of human serum samples: lyophilized spiked serum, and real human serum from healthy male and female individuals with good results.

Early diagnosis is often the key to successful patient treatment and survival. The identification of various disease signaling biomarkers which reliably reflect normal and disease states in humans in biological fluids explain the burgeoning research field in developing new methodologies able to determine the target biomarkers in complex biological samples with the required sensitivity and selectivity and in a simple and rapid way. The unique advantages offered by electrochemical sensors together with the availability of high affinity and specific bioreceptors and their great capabilities in terms of sensitivity and stability imparted by nanostructuring the electrode surface with different carbon nanomaterials have led to the development of new electrochemical biosensing strategies that have flourished as interesting alternatives to conventional methodologies for clinical diagnostics. This paper briefly reviews the advantages of using carbon nanostructures and their hybrid nanocomposites as electrode modifiers to construct efficient electrochemical sensing platforms for diagnosis. The review provides an updated overview of some selected examples involving attractive amplification and biosensing approaches which have been applied to the determination of relevant genetic and protein diagnostics biomarkers.

An amperometric magnetoimmunosensor for the determination of human p53 protein is described in this work using a sandwich configuration involving the covalent immobilization of a specific capture antibody onto activated carboxylic-modified magnetic beads (HOOC-MBs) and incubation of the modified MBs with a mixture of the target protein and horseradish peroxidase-labeled antibody (HRP-anti-p53). The resulting modified MBs are captured by a magnet placed under the surface of a disposable carbon screen-printed electrode (SPCE) and the amperometric responses are measured at −0.20 V (vs. an Ag pseudo-reference electrode), upon addition of hydroquinone (HQ) as a redox mediator and H2O2 as the enzyme substrate. The magnetoimmunosensing platform was successfully applied for the detection of p53 protein in different cell lysates without any matrix effect after a simple sample dilution. The results correlated accurately with those provided by a commercial ELISA kit, thus confirming the immunosensor as an attractive alternative for rapid and simple determination of this protein using portable and affordable instrumentation.

A new method for the detection of miRNAs making use of a competitive RNA/RNA hybridization configuration is described in this work. A biotinylated miRNA (biotin-miRNA) of identical sequence to that of the target miRNA is mixed with the samples to be analyzed allowing competition to be accomplished with the target miRNA for a thiolated RNA probe assembled onto a gold nanoparticles (AuNPs) modified screen-printed electrode. After labeling the hybridized biotin-miRNA with streptavidin-HRP conjugates, amperometric detection at -0.20V was carried out using the H2O2/hydroquinone (HQ) system. The decrease in the amperometric response was proportional to the concentration of model target miRNA-21 in the 100 fM to 25.0 pM range. The integrated sensor provided a very low detection limit (25 fM, 0.25 attomol in 10μL sample) for miRNA-21 without any amplification step, a complete discrimination against single nucleotide mismatched sequences under practical conditions and high storage stability. The usefulness of the developed method was demonstrated by determining the endogenous levels of the mature target miRNA in total RNA (RNAt) extracted from cancerous and non-cancerous cells.

A novel electrochemical approach for determination of miRNAs involving a sandwich hybridization assay onto streptavidin-magnetic beads (Strep-MBs), hybridization chain reaction (HCR) amplification and amperometric detection at disposable screen-printed carbon electrodes is reported. Using miRNA-21 as the target analyte, a dynamic linear range from 0.2 to 5.0nM with a 60pM (1.5fmol in 25μL) detection limit was obtained. The achieved sensitivity is 24-fold higher than a non-HCR amplification approach involving conventional sandwich type assay onto MBs. Moreover, the whole assay time lasted 1h 45min which is remarkably shorter than other reported methodologies. The methodology exhibited full selectivity against other non-complementary miRNAs as well as an acceptable discrimination between homologous miRNA family members. The applicability of this novel approach was demonstrated by determining mature miRNA-21 in total RNA (RNAt) extracted from tumor cells and human tissues.

A new automatic bioanalyzer for L-malic acid using an integrated amperometric biosensor as detector is reported for the first time in this work. The biosensor is constructed by gold film sputtering deposition on a stainless steel disk electrode and co-immobilization of the enzymes malate dehydrogenase (MDH) and diaphorase (DP) together with the redox mediator tetrathiafulvalene (TTF) by means of dialysis membrane. The analytical performance of the biosensor was evaluated when it was used as amperometric detector in three different analytical methodologies: stirred solutions, semiautomatic FIA system and automatic bioanalyzer. The bienzyme biosensor exhibited great analytical performance in terms of sensitivity, selectivity and reproducibility of the measurements and its usefulness was demonstrated by analyzing wine reference materials with certified content of L-malic acid. The attractive analytical and operational characteristics demonstrated by the automatic bioanalyzer make it a promising simple, rapid and field-based tool for routine wine and fruit control.

A novel label-free electrochemical strategy for nucleic acid detection was developed by using gold electrodes coated with mesoporous silica thin films as sensing interface. The biosensing approach relies on the covalent attachment of a capture DNA probe on the surface of the silica nanopores and further hybridization with its complementary target oligonucleotide sequence, causing a diffusion hindering of an Fe(CN)6 (3-/4-) electrochemical probe through the nanochannels of the mesoporous film. This DNA-mesoporous silica thin film-modified electrodes allowed sensitive (91.7 A/M) and rapid (45 min) detection of low nanomolar levels of synthetic target DNA (25 fmol) and were successfully employed to quantify the endogenous content of Escherichia coli 16S ribosomal RNA (rRNA) directly in raw bacterial lysate samples without isolation or purification steps. Moreover, the 1-month stability demonstrated by these biosensing devices enables their advanced preparation and storage, as desired for practical real-life applications. Graphical abstract Mesoporous silica thin films as scaffolds for the development of novel label-free electrochemical genosensors to perform selective, sensitive and rapid detection of target oligonucleotide sequences. Application towards E. coli determination.

Two different D-dimer disposable amperometric immunosensing designs based on indirect competitive or sandwich formats and the use of carboxylic acid-modified magnetic beads (COOH-MBs) and screen-printed carbon electrodes (SPCEs) have been developed and compared. In both approaches, the resulting modified MBs were magnetically captured on the surface of a SPCE which was used as the transducer for the electrochemical detection at −0.20 V upon addition of H2O2, and hydroquinone (HQ). Both configurations exhibited linear ranges of clinical usefulness and detection limits quite below the clinical threshold (0.5 µg mL−1 D-dimer). The sandwich configuration has been successfully tested with serum samples.

This work demonstrates the design and fabrication of an all cyclo-olefin polymer based microfluidic device capable of capturing magnetic beads and performing electrochemical detection in a series of gold electrodes. The size of chip is of a microscope slide and features six independent measuring cells for multianalyte detection purposes. The aim of this work is to show that rapid prototyping techniques can be instrumental in the development of novel bioassays, particularly in clinical diagnosis applications. We show the successful determination of troponin-T, a cardiac disease marker, in the clinically relevant range of 0.05-1.0 ng/mL. This methodology achieves a detection limit of 0.017 ng/mL in PBS solutions, and is capable of detecting less than 1 ng/mL in a 1:50 human serum dilution.

A novel nanocomposite electrode material constituted of gold nanoparticles (AuNPs), multi-walled carbon nanotubes (MWCNTs) and n-octylpyridinium hexafluorophosphate (OPPF6) ionic liquid was prepared and checked for the development of electrochemical (bio)sensing devices. AuNPs/MWCNTs/OPPF6 paste electrodes with micrometer dimensions (500 μm, i.d.) were constructed and applied to the determination of cortisol and androsterone hormones. Regarding cortisol determination, the microsized paste electrode was used to detect 1-naphtol generated upon addition of 1-naphthyl phosphate as enzyme substrate in the competitive immunoassay between alkaline phosphatase-labelled cortisol and cortisol. Squarewave voltammetry allowed determining the hormone within the 0.1- to 10-ng/mL linear range (r = 0.990) with a detection limit of 15 pg/mL and a EC50 value of 0.46 ± 0.06 ng/mL cortisol. The method was applied to the determination of cortisol in urine and serum samples containing a certified cortisol content. Moreover, a microsized enzyme biosensor prepared by bulk modification of the AuNPs/MWCNTs/OPPF6 electrode with the enzyme 3α-hydroxysteroid dehydrogenase was used for the determination of androsterone through the amperometric detection of reduced nicotinamide adenine dinucleotide. A calibration plot with a linear range between 0.1 and 120 μg/mL (r = 0.993) and a limit of detection of 89 ng/mL were obtained. The biosensor was applied to the analysis of human serum spiked with androsterone at the 250 ng/mL concentration level.

The preparation and performance of a disposable amperometric magneto-immunosensor, involving the use of a selective capture antibody immobilized on the surface of protein G-functionalized magnetic beads (ProtG-MBs) and screen-printed carbon electrodes (SPCEs), for the specific detection and quantification of tetracyclines (TCs) residues in milk is reported. A direct competitive immunoassay using a tracer with horseradish peroxidase (HRP) for the enzymatic labeling was performed. The amperometric response measured at -0.2 V vs. the silver pseudo-reference electrode of the SPCE upon the addition of H(2)O(2) in the presence of hydroquinone (HQ) as redox mediator was used as transduction signal. The developed methodology showed very low limits of detection (in the low ppb level) for 4 tetracycline antibiotics tested in untreated milk samples, and a good selectivity against other antibiotic residues frequently detected in milk and dairy products. The usefulness of the magneto-immunosensor was demonstrated by analyzing UHT whole milk samples spiked with 44 ng mL(-1) tetracycline (TC) as well as a reference milk containing a certified oxytetracycline (OTC) content. These features, together with the short analysis time (30 min), the simplicity, and easy automation and miniaturization of the required instrumentation make the developed methodology a promising alternative in the development of devices for on-site analysis.

A new layer-by-layer supramolecular approach for the construction of self-assembled nanoarchitectures of polyamidoamine (PAMAM) dendrimers and peroxidase on gold surface is reported. The methodology is based on the supramolecular self-assembly of alternated layers of adamantane-modified horseradish peroxidase and β-cyclodextrin-branched PAMAM G-5 dendrimers on a gold electrode, previously coated with β-cyclodextrin-modified cysteamine core PAMAM G-4 dendron. The formation of layer-by-layer assemblies (up to three dendrimer/peroxidase bilayers) was studied by SPR, quartz crystal microbalance, AFM and cyclic voltammetry. The analytical applicability of these architectures was evaluated by constructing a H 2 O 2 biosensor. The electroanalytical response of the biosensor towards H 2 O 2 increased with the number of enzyme layers. The bioelectrode constructed with three enzyme layers showed a low detection limit of 160 nM, a sensitivity of 602 μA/M cm 2 and retained 63% of its initial activity after 30 days of storage in wet conditions.

The preparation and performance of a disposable amperometric immunosensor, based on the use of a selective capture antibody and screen-printed carbon electrodes (SPCEs), for the specific detection and quantification of sulfonamide residues in milk is reported. The antibody was covalently immobilized onto a 4-aminobenzoic acid (4-ABA) film grafted on the disposable electrode, and a direct competitive immunoassay using a tracer with horseradish peroxidase (HRP) for the enzymatic labeling was performed. The amperometric response measured at −0.2 V vs the silver pseudo-reference electrode of the SPCE upon the addition of H2O2 in the presence of hydroquinone (HQ) as mediator was used as transduction signal. The developed methodology showed very low limits of detection (in the low ppb level) for 6 sulfonamide antibiotics tested in untreated milk samples, and a good selectivity against other families of antibiotics residues frequently detected in milk and dairy products. These features, together with the short analysis time (30 min), the simplicity, and easy automation and miniaturization of the required instrumentation make the developed methodology a promising alternative in the development of devices for on-site analysis.

The preparation and characteristics of a disposable amperometric magnetoimmunosensor, based on the use of functionalized magnetic beads (MBs) and gold screen-printed electrodes (Au/SPEs), for the specific detection and quantification of Staphylococcal protein A (ProtA) and Staphylococcus aureus (S. aureus) is reported. An antiProtA antibody was immobilized onto ProtA-modified MBs, and a competitive immunoassay involving ProtA antigen labelled with HRP was performed. The resulting modified MBs were captured by a magnetic field on the surface of tetrathiafulvalene-modified Au/SPEs and the amperometric response obtained at -0.15 V vs the silver pseudo-reference electrode of the Au/SPEs after the addition of H2O2 was used as transduction signal. The developed methodology showed very low limits of detection (1 cfu S. aureus/mL of raw milk samples), and a good selectivity against the most commonly involved foodborne pathogens originating from milk. These features, together with a short analysis time (2 h), the simplicity, and easy automation and miniaturization of the required instrumentation make the developed methodology a promising alternative in the development of devices for on-site analysis.

Nowadays, detecting the presence of bioterrorism and biohazard agents in environmental and food samples is of great concern, due to their toxicity, and because many of them are prone to be used in terrorism attacks. The use of functionalized magnetic beads (MBs) in the development of electrochemical immuno‐ and genosensors has resulted in innovative and powerful detection strategies that may be applied to environmental, food and clinical analysis. This review describes current research on the combination of functionalized MBs with electrochemical detection for the development of magnetobiosensors applied to rapid, sensitive and specific detection of bioterrorism and biohazard agents.

Enzyme biosensors for cholesterol were prepared using a transduction platform constituted of a glassy carbon electrode (GCE) modified with gold nanoparticles (AuNPs)/poly-(diallyldimethyl-ammonium chloride) (PDDA)–multi-walled carbon nanotubes (MWCNTs) nanocomposites. This modified electrode exhibited an improved response to H 2 O 2 when compared with other configurations based on CNTs. The incorporation of cholesterol oxidase (ChOx) to this electrode matrix allows the preparation of a biosensor that responded linearly to cholesterol in the 0.02–1.2 mM range with a slope of 2.23 μA/mM and a limit of detection 4.4 μM. All the variables involved in the preparation and performance of the modified electrode and the enzyme biosensor were optimized. The interferences caused by the presence of ascorbic acid and/or uric acid were minimized by coating the electrode surface with a thin film of Nafion which also enhanced the stability of the biosensor. Moreover, the possibility of preparing bienzyme ChOx–HRP biosensors using the same electrode platform was demonstrated. This allowed a detection potential as low as −50 mV to be applied using hydroquinone as mediator. With this design, the sensitivity was almost one order of magnitude higher than that achieved with the single ChOx biosensor and no interference from ascorbic acid or uric acid was apparent. Both biosensors designs were used for the determination of cholesterol in human serum samples spiked at physiological levels with recoveries ranging between 98.2% and 104%.