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This work reports the development and application of a disposable amperometric sensor built on magnetic microcarriers coupled to an Express PCR strategy to amplify a specific DNA fragment of the chloroplast trnH-psbA. The procedure involves the selective capture of a 68-mer synthetic target DNA (or unmodified PCR products) through sandwich hybridization with RNA capture probe-modified streptavidin MBs and RNA signaling probes, labeled using antibodies specific to the heteroduplexes and secondary antibodies tagged with horseradish peroxidase. Amperometric measurements were performed on screen-printed electrodes using the H 2 O 2 /hydroquinone system. Achieving a LOD of 3 pM for the synthetic target, it was possible to detect 2.5 pg of peanut DNA and around 10 mg kg -1 of peanut in binary mixtures (defatted peanut flours prepared in spelt wheat). However, the detectability decreased between 10 and 1000 times in processed samples depending on the treatment. The Express PCR-bioplatform was applied to the detection of peanut traces in foodstuff., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

This work presents the first bioplatform described to date for the determination of galactose-α-1,3-galactose (α-Gal), a non-primate mammalian oligosaccharide responsible for almost all cases of red meat allergy. The bioplatform is based on the implementation of an indirect competitive immunoassay and enzymatic labeling with the enzyme horseradish peroxidase (HRP) built on the surface of magnetic microparticles (MBs) and amperometric transduction on screen-printed carbon electrodes (SPCEs) using the H 2 O 2 /hydroquinone (HQ) system. The target α-Gal competed with biotinylated α-Gal immobilized on the surface of neutravidin-modified MBs for the limited immunorecognition sites of a detection antibody enzymatically labeled with an HRP-conjugated secondary antibody. The resulting magnetic immunoconjugates were trapped on the surface of the SPCE working electrode and amperometric transduction was performed, providing a cathodic current variation inversely proportional to the concentration of α-Gal in the analyzed sample. The developed biotool was optimized, characterized and applied with satisfactory results to the determination of the target allergen in different samples of raw and processed meats., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Detecting overexpression of cancer biomarkers is an excellent tool for diagnostic/prognostic and follow-up of patients with cancer or their response to treatment. This work illustrates the relevance of interrogating the levels of T-cell immunoglobulin and mucin domain 1 (TIM-1) protein as a diagnostic/prognostic biomarker of high-prevalence breast and lung cancers by using an amperometric disposable magnetic microparticles-assisted immunoplatform. The developed method integrates the inherent advantages of carboxylic acid-functionalized magnetic beads (HOOC-MBs) as pre-concentrator support and the amperometric transduction at screen-printed carbon electrodes (SPCEs). The immunoplatform involves a sandwich-type immunoassay assembled on HOOC-MBs through the specific capture/labeling of TIM-1 using capture antibodies and horseradish peroxidase (HRP)-conjugated biotinylated detection antibodies as biorecognition elements. The magnetic immunoconjugates were confined onto the working electrode (WE) surface of the SPCEs for amperometric detection using the hydroquinone/hydrogen peroxide/HRP (HQ/H 2 O 2 /HRP) redox system. The method allows the selective detection of TIM-1 protein over the 87-7500 pg mL -1 concentration range in only 45 min, with a limit of detection of 26 pg mL -1 . The developed bioplatform was successfully applied to the analysis of breast and lung cancer cell extracts, providing the first quantitative results of the target glycoprotein in these types of samples., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

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 NaIO 4 , 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, polyaldehyde-dextran 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., (Copyright © 2022 The Authors. Published by Elsevier B.V. All rights reserved.)

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 (H 2 O 2 /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., (Copyright © 2022. Published by Elsevier B.V.)

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 H 2 O 2 /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., (Copyright © 2022 The Authors. Published by Elsevier B.V. All rights reserved.)

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., (Copyright © 2021 Elsevier B.V. All rights reserved.)

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., (Copyright © 2020 Elsevier B.V. All rights reserved.)

A dynamic micromotor-based immunoassay, exemplified by cortisol detection, based on the use of tubular micromotors functionalized with a specific antibody is described. The use of antibody-functionalized micromotors offers huge acceleration of both direct and competitive cortisol immunoassays, along with greatly enhanced sensitivity of direct and competitive immunoassays. The dramatically improved speed and sensitivity reflect the greatly increased likelihood of antibody-cortisol contacts and fluid mixing associated with the dynamic movement of these microtube motors and corresponding bubble generation that lead to a highly efficient and rapid recognition process. Rapid naked-eye detection of cortisol in the sample is achieved in connection to use of horseradish peroxidase (HRP) tag and TMB/H 2 O 2 system. Key parameters of the competitive immunoassay (e.g., incubation time and reaction volume) were optimized. This fast visual micromotor-based sensing approach enables "on the move" specific detection of the target cortisol down to 0.1μgmL -1 in just 2min, using ultrasmall (50µL) sample volumes., (Copyright © 2017 Elsevier B.V. All rights reserved.)

While high storage stability of sequence-selective DNA biosensors is crucial towards their routine applications, commonly used electrochemical hybridization biosensors are characterized with limited storage stability. In this article we demonstrate that recently developed ternary thiolated monolayers impart dramatic improvement in the storage stability of DNA electrochemical biosensors. In particular, highly stable multicomponent interfaces are prepared by co-immobilizing the thiolated capture probe (SHCP) with 1,6-hexanedithiol (HDT) on gold substrates, followed by the incorporation of 6-mercapto-1-hexanol (MCH) diluent. The resulting (SHCP/HDT+MCH) DNA hybridization recognition platform offers substantially higher storage stability compared to conventional binary (SHCP+MCH) monolayers. The (SHCP/HDT+MCH) ternary monolayers maintain their initial signal (S)-to-noise (N) ratio (S/N) over a prolonged 3 months period upon storage at 4 °C, compared to the rapid sensitivity loss observed using the common binary interfaces. This attractive stability performance promises the convenient usage of pre-prepared electrodes after prolonged time storage without any treatment. Such dramatic improvements in the storage stability have been achieved through a rational optimization of the concentration ratio of the SHCP and the other components of the ternary SAM. The improved storage stability of SHCP/HDT+MCH interfaces observed at higher concentrations of SHCP is attributed to a hindered displacement of SHCP by MCH in the resulting compact layers. The ability to design highly stable nucleic acid interfaces using common chemicals obviates the need of using specialized expensive reagents., (Copyright © 2012 Elsevier B.V. All rights reserved.)

The development of rapid, low-cost and reliable diagnostic methods is crucial for the identification and treatment of many diseases. Screen-printed gold electrodes (Au/SPEs), coated with a ternary monolayer interface, involving hexanedithiol (HDT), a specific thiolated capture probe (SHCP), and 6-mercapto-1 hexanol (MCH) (SHCP/HDT/MCH) are shown here to offer direct and sensitive detection of nucleic acid hybridization events in untreated raw biological samples (serum, urine and crude bacterial lysate solutions). The composition of the ternary monolayer was modified and tailored to the surface of the Au/SPE. The resulting SHCP/HDT/MCH monolayer has demonstrated to be extremely useful for enhancing the performance of disposable nucleic acid sensors based on screen-printed electrodes. Compared to common SHCP/MCH binary interfaces, the new ternary self-assembled monolayer (SAM) resulted in a 10-fold improvement in the signal (S)-to-noise (N) ratio (S/N) for 1 nM target DNA. The SHCP/HDT/MCH-modified Au/SPEs allowed the direct quantification of the target DNA down to 25 pM (0.25 fmol) and 100 pM (1 fmol) in undiluted/untreated serum and urine samples, respectively, and of 16S rRNA Escherichia coli (E. coli) corresponding to 3000 CFU μL(-1) in raw cell lysate samples. The new SAM-coated screen-printed electrodes also displayed favorable non-fouling properties after a 24h exposure to raw human serum and urine samples, offering great promise as cost-effective nucleic acid sensors for a wide range of decentralized genetic tests., (Copyright © 2011 Elsevier B.V. All rights reserved.)