Magnetic multiwalled carbon nanotubes as nanocarrier tags for sensitive determination of fetuin in saliva

This paper reports the development and performance of an electrochemical immunosensor using magnetic multiwalled carbon nanotubes (m-MWCNTs) as nanocarrier tags for the determination of human fetuin A (HFA), a relevant biomarker of obesity, insulin resistance, and type-2 diabetes as well as for pancreatic and liver cancers and inflammatory processes. Screen-printed carbon electrodes were grafted with p-aminobezoic acid and streptavidin was covalently immobilized on the electrode surface. A biotinylated capture antibody was immobilized through streptavidin-biotin interaction and a sandwich assay configuration was implemented using m-MWCNTs conjugated with HRP and anti-HFA antibodies as the detection label. The determination of HFA was accomplished by measuring the current produced by the electrochemical reduction of benzoquinone at -200 mV upon addition of H2O2 as HRP substrate. The prepared m-MWCNTs were characterized by SEM, TEM, XRD and EDS. All the steps involved in the immunosensor preparation were monitored by electrochemical impedance spectroscopy and cyclic voltammetry. A linear calibration plot for HFA was found between 20 and 2000 pg/mL with a LOD value of 16 pg/mL. This performance is notably better than that reported for an ELISA kit and a chronoimpedimetric immunosensor. The favorable contribution of m-MWCNTs in comparison with MWCNTs without incorporated magnetic particles to this excellent analytical performance is also highlighted. The immunosensor selectivity against other proteins and potentially interfering compounds was excellent. In addition, the usefulness of the immunosensor was demonstrated by the analysis of HFA in saliva with minimal sample treatment

Janus particles for (bio)sensing

This review article sheds useful insight in the use of Janus nanoparticles for (bio)sensing in connection with optical and electrochemical transduction. After a brief introduction of the main properties, types and fabrication strategies of Janus nanoparticles, selected applications for their use in electrochemical and optical biosensing are critically discussed. Highlighted examples illustrate the great versatility and interesting possibilities offered by these smart multifunctional nanoparticles for (bio)sensing of relevant analytes operating both in static and dynamic modes. Progress made so far demonstrate their suitability for designing single- or multiplexed (bio)sensing strategies for target analytes of different nature (organic and inorganic compounds, proteins, cells and oligomers) with relevance in clinical (H2O2, glucose, cholesterol, CEA, human IgG, propranolol, bacterial and tumor cells) and environmental (lead and organophosphorous nerve agents) fields. Key future challenges and envisioned opportunities of the use of Janus nanoparticles in the (bio)sensing field are also discussed

Electrochemical Genosensing of Circulating Biomarkers

Management and prognosis of diseases requires the measurement in non- or minimally invasively collected samples of specific circulating biomarkers, consisting of any measurable or observable factors in patients that indicate normal or disease-related biological processes or responses to therapy. Therefore, on-site, fast and accurate determination of these low abundance circulating biomarkers in scarcely treated body fluids is of great interest for health monitoring and biological applications. In this field, electrochemical DNA sensors (or genosensors) have demonstrated to be interesting alternatives to more complex conventional strategies. Currently, electrochemical genosensors are considered very promising analytical tools for this purpose due to their fast response, low cost, high sensitivity, compatibility with microfabrication technology and simple operation mode which makes them compatible with point-of-care (POC) testing. In this review, the relevance and current challenges of the determination of circulating biomarkers related to relevant diseases (cancer, bacterial and viral infections and neurodegenerative diseases) are briefly discussed. An overview of the electrochemical nucleic acid–based strategies developed in the last five years for this purpose is given to show to both familiar and non-expert readers the great potential of these methodologies for circulating biomarker determination. After highlighting the main features of the reported electrochemical genosensing strategies through the critical discussion of selected examples, a conclusions section points out the still existing challenges and future directions in this field

Multiplexed Electrochemical Immunosensors for Clinical Biomarkers

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

Biodetection Techniques for Quantification of Chemokines

Chemokines are a class of cytokine whose special properties, together with their involvement and relevant role in various diseases, make them a restricted group of biomarkers suitable for diagnosis and monitoring. Despite their importance, biodetection techniques dedicated to the selective determination of one or more chemokines are very scarce. For some years now, the critical diagnosis of inflammatory diseases by detecting both cytokine and chemokine biomarkers, has had a strong impact on the development of multiple detection platforms. However, it would be desirable to implement methodologies with a higher degree of selectivity for chemokines, in order to provide more precise information. In addition, better development of biosensor technology applied to this specific field would make it possible to address the main challenges of detection methods for several diseases with a high incidence in the population, avoiding high costs and low sensitivity. Taking this into account, this review aims to present the state of the art of chemokine biodetection techniques and emphasize the role of these systems in the prevention, monitoring and treatment of various diseases associated with chemokines as a starting point for future developments that are also analyzed throughout the article.Depto. de Química AnalíticaFac. de Ciencias QuímicasTRUEMinisterio de Ciencia, Innovación y UniversidadesComunidad de Madridpu

Electrochemical Magnetic Immunosensors for the Determination of Ceruloplasmin

Electrochemical immunosensors for ceruloplasmin (Cp) are reported for the first time. Two configurations involving magnetic beads (MBs) functionalized with Protein A or Streptavidin for immobilization of Cp antibodies were compared, using competitive immunoassay with synthesized alkaline phosphatase-Cp conjugate. Upon capturing MBs-immunoconjugates onto screen-printed carbon electrodes, quantification of Cp was accomplished by DPV measurement of 1-naphthol generated after 1-naphthylphosphate addition. Linear ranges of calibration curves and detection limits were 0.1–1000 µg/mL and 0.040 µg/mL (Protein A-MBs), and 0.025–20 µg/mL and 0.018 µg/mL (Strept-MBs). Good results were obtained in the determination of Cp in spiked human serum samples

Electrochemical Immunosensor for Simultaneous Determination of Emerging Autoimmune Disease Biomarkers in Human Serum †

Rheumatoid arthritis is an autoimmune disorder characterized by persistent erosive synovitis, systemic inflammation and the presence of autoantibodies, which play an important role in inducing inflammation and joint damage, releasing pro-inflammatory cytokines from monocytes and macrophages [1,2]. Likewise, neutrophil activating protein-2 (CXCL7) is a platelet-derived growth factor belonging to the CXC chemokine subfamily, which is expressed in serum, synovial fluid and synovial tissue of patients developing rheumatoid arthritis during the first twelve weeks, being useful to reflect local pathological changes [3]. Besides, matrix metalloproteinase-3 (MMP-3), which is induced by inflammatory cytokines such as interleukin-1 (IL-1) and tumor necrosis factor alpha (TNF-α) in rheumatoid synovium, degrades several extracellular matrix components of cartilage and plays central roles in rheumatoid joint destruction [4]. Therefore, monitoring serum CXCL7 and MMP-3 levels is useful for predicting the disease activity in rheumatoid arthritis. In this work, the construction and analytical performance of a dual electrochemical platform for the simultaneous determination of CXCL7 and MMP-3 is described. After the optimization of experimental variables involved in the preparation and implementation of the biosensor, the analytical usefulness of the developed configuration was demonstrated by its application to the determination of these biomarkers in serum samples from healthy individuals and patients with rheumatoid arthritis. To carry out the simultaneous determination of CXCL7 and MMP3 in human serum, just a fifty-fold sample dilution in PBS of pH 7.4 was required. In addition, the results obtained using the dual immunosensor were compared with those provided by the respective ELISA immunoassays, yielding no significant differences between the two methods. It is important to highlight that reagents consumption, four times smaller using the dual immunosensor than that required in the ELISA protocol, and an assay time of 2 h 50 min versus almost 5 h, counted in both cases after incubation of the capture antibody, are advantageous features of the dual immunosensor [5].Depto. de Química AnalíticaFac. de Ciencias QuímicasTRUEMinisterio de Ciencia, Innovación y UniversidadesComunidad de Madridpu

Anti-double stranded DNA antibodies: Electrochemical isotyping in autoimmune and neurological diseases

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.Depto. de Química AnalíticaFac. de Ciencias QuímicasTRUESpanish Ministerio de Ciencia e InnovaciónAES-ISCIIIComunidad de Madridpu

Anti-double stranded DNA antibodies: Electrochemical isotyping in autoimmune and neurological diseases

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.S

Electrochemical immunosensor for simultaneous determination of interleukin-1 beta and tumor necrosis factor alpha in serum and saliva using dual screen printed electrodes modified with functionalized double–walled carbon nanotubes

Dual screen-printed carbon electrodes modified with 4-carboxyphenyl-functionalized double-walled carbon nanotubes (HOOC-Phe-DWCNTs/SPCEs) have been used as scaffolds for the preparation of electrochemical immunosensors for the simultaneous determination of the cytokines Interleukin-1b (IL-1b) and factor necrosis tumor a (TNF-a). IL-1b. Capture antibodies were immobilized onto HOOC-Phe DWCNTs/SPCEs in an oriented form making using the commercial polymeric coating Mix&Go™. Sandwich type immunoassays with amperometric signal amplification through the use of poly-HRPstreptavidin conjugates and H2O2 as HRP substrate and hydroquinone as redox mediator were implemented. Upon optimization of the experimental variables affecting the immunosensor performance, the dual immunosensor allows ranges of linearity extending between 0.5 and 100 pg/mL and from 1 to 200 pg/mL for IL-1b and TNF-a, respectively, these ranges being adequate for the determination of the cytokines in clinical samples. The achieved limits of detection were 0.38 pg/mL (IL-1b) and 0.85 pg/mL (TNF-a). In addition, the dual immunosensor exhibits excellent reproducibility of the measurements, storage stability of the anti-IL-Phe-DWCNTs/SPCE and anti-TNF-Phe-DWCNTs/SPCE conjugates, and selectivity as well as negligible cross-talking. The dual immunosensor was applied to the simultaneous determination of IL-1b and TNF-a in human serum spiked at clinically relevant concentration levels and in real saliva samples