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This review summarizes the stepwise strategy and key points for magnetic beads (MBs)-based aptamer selection which is suitable for isolating aptamers against small and large molecules via systematic evolution of ligands by exponential enrichment (SELEX). Particularities, if any, are discussed according to the target size. Examples targeting small molecules (<1000 Da) such as xenobiotics, toxins, pesticides, herbicides, illegal additives, hormones, and large targets such as proteins (biomarkers, pathogens) are discussed and presented in tabular formats. Of special interest are the latest advances in more efficient alternatives, which are based on novel instrumentation, materials or microelectronics, such as fluorescence MBs-SELEX or microfluidic chip system-assisted MBs-SELEX. Limitations and perspectives of MBs-SELEX are also reviewed. Taken together, this review aims to provide practical insights into MBs-SELEX technologies and their ability to screen multiple potential aptamers against targets from small to large molecules., 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.)

Wearable sensors would revolutionize healthcare and personalized medicine by providing individuals with continuous and real-time data about their bodies and environments. Their integration into everyday life has the potential to enhance well-being, improve healthcare outcomes, and offer new opportunities for research. Capacitive sensors technology has great potential to enrich wearable devices, extending their use to more accurate physiological indicators. On the basis of capacitive sensors developed so far to monitor physical parameters, and taking into account the advances in capacitive biosensors, this work discusses the benefits of this type of transduction to design wearables for the monitoring of biomolecules. Moreover, it provides insights into the challenges that must be overcome to take advantage of capacitive transduction in wearable sensors for health., (© 2023. The Author(s).)

The shortage of specific glycan recognition reagents has proven a significant hurdle in the development of assays to detect altered glycoforms associated with cancer. Here, a carbohydrate-binding aptamer originally selected against the glycan moiety of prostate-specific antigen (PSA) is used as a lectin-mimicking reagent. As a first proof-of-principle, this aptamer has been applied to develop a sandwich-type electrochemical biosensor for the detection of the serum amyloid P (SAP) component, a glycosylated protein whose increased sialylation has been associated with pancreatic cancer. The assay combines a specific antibody for this potential tumor biomarker and the aptamer as capture and detection receptors, respectively. Two oriented antibody immobilization approaches, protein A-based and boronic ester-based attachment to self-assembled monolayers built onto gold surfaces, were comparatively evaluated, the latter being able to circumvent the unwanted interaction between the aptamer and the glycans on the electrode-attached antibody. The resulting biosensing platform allows the detection of the SAP glycoprotein at levels of nanograms per milliliter with a reproducibility value lower than 20%, both in aqueous buffer and in serum. This work represents a proof-of-concept of a promiscuous ligand of proteins with high levels of sialylated glycans typically produced by cancer cells.

A major societal challenge is the development of the necessary tools for early diagnosis of diseases such as cancer and sepsis. Consequently, there is a concerted push to develop low-cost and non-invasive methods of analysis with high sensitivity and selectivity. A notable trend is the development of highly sensitive methods that are not only amenable for point-of-care (POC) testing, but also for wearable devices allowing continuous monitoring of biomarkers. In this context, a non-invasive test for the detection of a promising biomarker, the protein Interleukin-6 (IL-6), could represent a significant advance in the clinical management of cancer, in monitoring the chemotherapy response, or for prompt diagnosis of sepsis. This work reports a capacitive electrochemical impedance spectroscopy sensing platform tailored towards POC detection and treatment monitoring in human serum. The specific recognition of IL-6 was achieved employing gold surfaces modified with an anti-IL6 nanobody (anti-IL-6 VHH) or a specific IL-6 aptamer. In the first system, the anti-IL-6 VHH was covalently attached to the gold surface using a binary self-assembled-monolayer (SAM) of 6-mercapto-1-hexanol (MCH) and 11-mercaptoundecanoic acid. In the second system, the aptamer was chemisorbed onto the surface in a mixed SAM layer with MCH. The analytical performance for each label-free sensor was evaluated in buffer and 10% human serum samples and then compared. The results of this work were generated using a low-cost, thin film eight-channel gold sensor array produced on a flexible substrate providing useful information on the future design of POC and wearable impedance biomarker detection platforms., (© 2023. The Author(s).)

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 H 2 O 2 /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., (© 2022. The Author(s).)

The extracellular matrix (ECM) plays an essential role in tumor progression and invasion through its continuous remodeling. The growth of most carcinomas is associated with an excessive collagen deposition that provides the proper environment for tumor development and chemoresistance. The α1 chain of a minor human collagen, type XI, is overexpressed in some tumor stroma, but not found in normal stroma. To test the clinical utility of this collagen as a cancer biomarker, specific receptors are needed. Available antibodies do not show enough selectivity or are directed toward the propeptide region that is cleaved when the protein is released to the ECM. Here we show the selection of an aptamer for the specific C-telopeptide region using a 16-mer peptide as the target for the SELEX. The aptamer selected with a K d of ∼25 nM was able to capture the collagen XI from cell lysates. It was also used for target detection in a mixed antibody-aptamer sandwich assay showing it can be useful for diagnostic purposes in biological fluids., 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 © 2021 The Authors. Published by Elsevier B.V. All rights reserved.)

The role of the extracellular matrix (ECM) remodeling in tumorigenesis and metastasis is becoming increasingly clear. Cancer development requires that tumor cells recruit a tumor microenvironment permissive for further tumor growth. This is a dynamic process that takes place by a cross-talk between tumor cells and ECM. As a consequence, molecules derived from the ECM changes associated to cancer are released into the bloodstream, representing potential biomarkers of tumor development. This article highlights the importance of developing and improving bioanalytical methods for the detection of ECM remodeling-derived components, as a step forward to translate the basic knowledge about cancer progression into the clinical practice., (© 2021. Springer-Verlag GmbH Germany, part of Springer Nature.)

The prostate specific antigen (PSA) test is the gold standard for the screening of prostate cancer (PCa), despite its limited clinical specificity. Long noncoding RNAs are released from the tumor tissue to the urine and show great potential for improving specificity in PCa diagnosis. This work reports on a sandwich-type hybridization assay to detect both the urinary biomarker prostate cancer antigen 3 (PCA3) and an endogenous control, the PSA mRNA. Multiple fluorescein-tagged hybridization assistant probes are used to promote the selective capture of this long noncoding RNA, and sensitivity by incorporating multiple redox enzymes per target molecule, after addition of antifluorescein Fab fragment-peroxidase conjugate. This strategy alleviates the problems associated with the low natural abundance of this marker, its large size, and complex secondary structure. The individual genosensors exhibit good sensitivity (2.48 ± 0.01 μA nM -1 and 6.4 ± 0.3 μA nM -1 for PCA3 and PSA, respectively), with wide linear ranges (from 25 pM to 10 nM for PCA3 and 1 nM for PSA), and detection limits in the low picomolar range (4.4 pM and 1.5 pM for PCA3 and PSA, respectively). This analytical performance is retained in the dual configuration without significant cross-talk, despite using the same enzyme label. The usefulness of this dual platform was demonstrated by analyzing RNA extracts from the prostate cancer cell line LNCaP and from urine samples of prostate cancer patients., (Copyright © 2021 The Author(s). Published by Elsevier B.V. All rights reserved.)

Prostate specific antigen (PSA) is the common biomarker for prostate cancer (PCa). However, its lack of specificity to differentiate PCa from benign prostate disorders stimulates the search for alternative cancer biomarkers to improve the clinical management of the patients. Different studies have described changes in the core-fucosylation level of PSA between PCa patients and healthy controls. To exploit these findings, we have adapted an impedimetric aptamer-based sensor to the dual recognition of PSA. Two different aptamers, PSAG-1 and anti-PSA, are immobilized onto two adjacent nanostructured gold electrodes. The direct binding from diluted serum samples of specific glycosylated-PSA to the first sensor and total PSA to the second one leads to changes in the charge transfer resistance, which correlate to the amount of glycosylated and total PSA in the sample. The sensors are able to measure PSA in serum with a dynamic range between 0.26 and 62.5 ng/mL (PSAG-1) and from 0.64 to 62.5 ng/mL (anti-PSA), with a reproducibility of 5.4 %. The final output of the proposed platform is the ratio between PSAG-1 reactive PSA and total PSA, defined as the glycan score. The glycan score was tested in serum samples from patients with different pathologies, showing excellent correlation between the measured score and the known diagnosis of the patients. Hence this dual aptamer-based impedimetric biosensor could be used as a minimally invasive method for the diagnosis of prostate cancer., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Enzyme-linked immunosorbent assays are currently the most popular methods to quantify gluten in foods. Unfortunately, the antibodies used as specific receptors in such methods are not compatible with the usual solvents for the extraction of gluten proteins. In consequence, commercial tests require a high dilution of the sample after the extraction, increasing the limit of quantification and decreasing convenience. In this work, we have rationally truncated an aptamer capable of recognizing gliadin in a deep eutectic solvent (DES). The truncated aptamer is a 19-nucleotides-long DNA that minimizes self-hybridization, allowing the development of an electrochemical sandwich-based sensor for the quantification of gluten in the DES ethaline. The sensor incorporates two identical biotin-labeled truncated aptamers, one of which is immobilized on a carbon screen-printed electrode and the other reports the binding of gliadin after incubation in streptavidin-peroxidase. This sensor can detect gliadin in DES, with a dynamic range between 1 and 100 μg/L and an intra-assay coefficient of variation of 11%. This analytical performance allows the quantification of 20 μg of gluten/kg of food when 1 g of food is extracted with 10 mL of ethaline. We demonstrate the ability of this method to achieve the measurement of gluten in food samples, after the extraction with pure ethaline. The assay is useful for the analysis of residual gluten levels in foods, thus facilitating the evaluation of any potential health risk associated with the consumption of such food by people with celiac disease or other gluten-related disorders., (Copyright © 2020 Elsevier B.V. All rights reserved.)

A personal glucose meter (PGM)-based method for quantitative detection of a urinary nucleic acid biomarker in prostate cancer screening, the so-called PCA3, is reported herein. A sandwich-type genoassay is conducted on magnetic beads to collect the target from the sample by specific hybridization, making the assay appropriate for PCA3 detection in biological fluids. The success of the method hinges on the use of alkaline phosphatase (ALP) to link the amount of nucleic acid biomarker to the generation of glucose. In particular, specifically attached ALP molecules hydrolyze D-glucose-1-phosphate into D-glucose, thus enabling the amplification of the recorded signal on the personal glucose meter. The developed genoassay exhibits good sensitivity (3.3 ± 0.2 mg glucose dL -1 pM -1 ) for PCA3, with a dynamic range of 5 to 100 pM and a quantification limit of 5 pM. Likewise, it facilitates point-of-care testing of nucleic acid biomarkers by using off-the-shelf PGM instead of complex instrumentation involved in traditional laboratory-based tests.

The tunability of SELEX procedure is an essential feature to supply bioaffinity receptors (aptamers) almost on demand for analytical and therapeutic purposes. This longstanding ambition is, however, not straightforward. Non-invasive cancer diagnosis, so called liquid biopsy, requires collection of body fluids with minimal or no sample pretreatment. In those raw matrices, aptamers must recognize minute amounts of biomarkers that are not unique entities but large sets of variants evolving with the disease stage. The susceptibility of aptasensors to assay conditions has driven the selection of aptamers to natural environments to ensure their optimum performance in clinical samples. We present herein a compilation of the SELEX procedures in natural milieus. By revising the electrochemical aptasensors applied to clinical samples for cancer diagnosis and tracing back to the original SELEX we analyze whether aptamers raised using these SELEX strategies are being incorporated to the diagnostic devices and how aptasensors are finding their way to a market dominated by antibody-based assays., 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 © 2020 Elsevier B.V. All rights reserved.)

Detecting specific protein glycoforms is attracting particular attention due to its potential to improve the performance of current cancer biomarkers. Although natural receptors such as lectins and antibodies have served as powerful tools for the detection of protein-bound glycans, the development of effective receptors able to integrate in the recognition both the glycan and peptide moieties is still challenging. Here we report a method for selecting aptamers toward the glycosylation site of a protein. It allows identification of an aptamer that binds with nM affinity to prostate-specific antigen, discriminating it from proteins with a similar glycosylation pattern. We also computationally predict the structure of the selected aptamer and characterize its complex with the glycoprotein by docking and molecular dynamics calculations, further supporting the binary recognition event. This study opens a new route for the identification of aptamers for the binary recognition of glycoproteins, useful for diagnostic and therapeutic applications., Competing Interests: There are no conflicts to declare., (This journal is © The Royal Society of Chemistry.)

Affinity characterization is essential to develop reliable aptamers for tumor biomarker detection. For alpha-fetoprotein (AFP), a biomarker of hepatocellular carcinoma (HCC), two DNA aptamers were described with very different affinity. In this work, we estimate the dissociation constant of both of them by means of a direct assay on magnetic beads modified with AFP and electrochemical detection on carbon screen-printed electrodes (SPCE). Unlike previous works, both aptamers showed similar dissociation constant (K d ) values, in the subµM range. In order to improve the performance of these aptamers, we proposed the isothermal amplification of the aptamers by both terminal deoxynucleotidyl transferase (TdT) and rolling circle amplification (RCA). Both DNA amplifications improved the sensitivity and also the apparent binding constants from 713 nM to 189 nM for the short aptamer and from 526 nM to 32 nM for the long aptamer. This improvement depends on the true affinity of the binding pair, which ultimately limits the analytical usefulness.

Latest technological advancement has tremendously expanded the knowledge on the composition of body fluids and the cancer-associated changes, which has fueled the replacement of invasive biopsies with liquid biopsies by using appropriate specific receptors. DNA emerges as a versatile analytical reagent in electrochemical devices for hybridization-based or aptamer-based recognition of all kind of biomarkers. In this mini review, we briefly introduce the current affordable targets (tumor-derived nucleic acids, circulating tumor cells and exosomes) in body fluids, and then we provide an overview of selected electrochemical methods already applied in clinical samples by dividing them into three large categories according to sample type: red (blood), yellow (urine), and white (saliva and sweat) diagnostics. This review focuses on the hurdles of the complex matrices rather than a comprehensive and detailed revision of the format schemes of DNA-based electrochemical sensing. This diverse perspective compiles some challenges that are often forgotten and critically underlines real sample analysis or clinical validation assays. Finally, the needs and trends to reach the market are briefly outlined., (Copyright © 2020 Miranda-Castro, Palchetti and de-los-Santos-Álvarez.)

The cultivation of genetically modified organisms (GMO) continues to expand worldwide. Still, many consumers express concerns about the use of GMO in food or feed, and many countries have legislated on labelling systems to indicate the presence of GMO in commercial products. To deal with the increased number of GMO events and to address related regulations, alternative detection methods for GMO inspection are required. In this work, a genosensor based on Surface Plasmon Resonance under continuous flow was developed for the detection and quantification of a genetically modified soybean (event GTS 40-3-2). In a single chip, the simultaneous detection of the event-specific and the taxon-specific samples were achieved, whose detection limits were 20 pM and 16 pM, respectively. The reproducibility was 1.4%, which supports the use of the chip as a reliable and cost-effective alternative to other DNA-based techniques. The results indicate that the proposed method is a versatile tool for GMO quantification in food and feed samples., Competing Interests: The authors have declared that no competing interests exist.

This work addresses a technological advance applied to the construction of a magnetogenoassay with electrochemical transduction for the maize taxon-specific (HMGA gene) detection using gold-coated magnetic nanoparticles as nanosized platform. Superparamagnetic core-shell Fe 3 O 4 @Au nanoparticles (10.4 ± 1.7 nm) were used to assemble the genoassay through the covalent immobilization of HMGA DNA probes onto carboxylated self-assembled monolayers at the nanoparticles surface. A hybridization reaction using sandwich format was selected to prevent inefficient hybridization connected with stable secondary DNA structures using also fluorescein isothiocyanate as DNA signaling tag. The labelling of the hybridization reaction with enzymes allowed the chronoamperometric measurement of the peroxidase activity linked to the nanoplatform located on gold surface. Using this electrochemical magnetogenoassay a linear concentration range from 0.5 to 5 nM and a LOD of 90 pM with a RSD <1.2% was calculated. Certified maize was evaluated without further purification after PCR amplification. This work highlights the efficacy of the electrochemical magnetogenoassay for the HMGA detection, showing its potential as alternative procedure for the verification of the compliance of the legislation., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Despite having been underappreciated in favor of their protein-coding counterparts for a long time, long noncoding RNAs (lncRNAs) have emerged as functional molecules, which defy the central dogma of molecular biology, with clear implications in cancer. Altered expression levels of some of these large transcripts in human body fluids have been related to different cancer conditions that turns them into potential noninvasive cancer biomarkers. In this review, a brief discussion about the importance and current challenges in the determination of lncRNAs associated to cancer is provided. Different electrochemical nucleic acid-based strategies for lncRNAs detection are critically described. Future perspectives and remaining challenges for the practical implementation of these methodologies in clinical medicine are also discussed.

Cancer diagnosis based on serum biomarkers requires receptors of extreme sensitivity and selectivity. Tunability of aptamer selection makes them ideal for that challenge. However, aptamer characterization is a time-consuming task, not always thoroughly addressed, leading to suboptimal aptamer performance. In this work, we report on the affinity characterization and potential usage of two aptamers against a candidate cancer biomarker, the neutrophil gelatinase-associated lipocalin (NGAL). Electrochemical sandwich assays on Au electrodes and SPR experiments showed a restricted capture ability of one of the aptamers (LCN2-4) and a small detectability of the other (LCN2-2). Interestingly, a truncated version of the signaling aptamer LCN2-2 selectively binds to NGAL covalently linked to magnetic beads due to high local protein concentration. The functional affinity of this aptamer is enhanced by three-orders of magnitude using rolling circle amplification (RCA), completed in only 15 min, followed by hybridization with short complementary fluorescein-tag probes, enzyme labeling and chronoamperometric measurement. Microscale thermophoresis experiments show a poor affinity for the protein in solution, which urges the importance of a full and in-depth characterization of aptamers to be used as diagnostic reagents., (Copyright © 2019 Elsevier B.V. All rights reserved.)

The development of chemical sensors capable of detecting the specific glycosylation patterns of proteins offers a powerful mean for the early detection of cancer. Unfortunately, this strategy is scarcely explored because receptors recognizing the glycans linked to proteins are challenging to discover. In this work, we describe a simple method for directing the selection of aptamers toward the glycan structure of the glycoproteins, with prostate-specific antigen (PSA) as a model target. Using this strategy, we identified one aptamer (PSA-1) that binds the glycan moiety of PSA with reasonable affinity (a dissociation constant of 177 ± 65 nM). Interestingly, an electrochemical sensor with a sandwich format employing the identified aptamer as a signaling receptor, provides a tool of discriminating human PSA from the unglycosylated protein, with a limit of detection of 0.66 ng/mL. The sensor responds to different levels of PSA in serum, correlating well with chemiluminescence ELISA used in hospitals even with higher potential to discriminate clinically meaningful prostate cancer. Although validation on a larger cohort is needed, this is the first demonstration of an aptamer-based sensor to detect PSA by focusing in its glycan moiety., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Herein, we show the feasibility of using deep eutectic solvents as a faster way of selecting aptamers targeting poorly water-soluble species. This unexplored concept is illustrated for gluten proteins. In this way, aptamer-based gluten detection can be performed directly in the extraction media with improved detectability. We envision deep implications for applications not only in food safety control but also in biomedicine., (© 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The integration of nanomaterials in the field of (bio)sensors has allowed developing strategies with improved analytical performance. In this work, ultrasmall core-shell Fe 3 O 4 @Au magnetic nanoparticles (MNPs) were used as the platform for the immobilization of event-specific Roundup Ready (RR) soybean and taxon-specific DNA sequences. Firstly, monodisperse Fe 3 O 4 MNPs were synthesized by thermal decomposition and subsequently coated with a gold shell through reduction of Au(III) precursor on the surface of the MNPs in the presence of an organic capping agent. This nanosupport exhibited high colloidal stability, average particle size of 10.2 ± 1.3 nm, and spherical shape. The covalent immobilization of ssDNA probe onto the Au shell of the Fe 3 O 4 @Au MNPs was achieved through a self-assembled monolayer (SAM) created from mixtures of alkane thiols (6-mercapto-1-hexanol and mercaptohexanoic acid). The influence of the thiols ratio on the electrochemical performance of the resulting electrochemical genoassays was studied, and remarkably, the best analytical performance was achieved for a pure mercaptohexanoic acid SAM. Two quantification assays were designed; one targeting an RR sequence and a second targeting a reference soybean gene, both with a sandwich format for hybridization, signaling probes labelled with fluorescein isothiocyanate (FITC), enzymatic amplification and chronoamperometric detection at screen-printed carbon electrodes (SPCE). The magnetogenoassays exhibited linear ranges from 0.1 to 10.0 nM and from 0.1 to 5.0 nM with similar detection limits of 0.02 nM and 0.05 nM for the event-specific (RR) and the taxon-specific (lectin) targets, respectively. The usefulness of the approach was demonstrated by its application to detect genetically modified organisms (GMOs) in feed and food., (Copyright © 2018 Elsevier B.V. All rights reserved.)

The reaction of para -hydroxybenzyl alcohols with ferrocene in the presence of a catalytic amount of InCl₃ provided ferrocenyl phenol derivatives, an interesting class of organometallic compounds with potential applications in medicinal chemistry. This transformation exhibited a reasonable substrate scope delivering the desired products in synthetically useful yields. Evidence of involvement of a para -quinone methide intermediate in this coupling process was also provided. Preliminary biological evaluation demonstrated that some of the ferrocene derivatives available by this methodology exhibit significant cytotoxicity against several cancer cell lines with IC 50 values within the range of 1.07⁻4.89 μM.

Advances in proteomics have fueled the search for novel cancer biomarkers with higher selectivity. Differential expression of low abundant proteins has been the usual way of finding those biomarkers. The existence of a selective receptor for each biomarker is compulsory for their use in diagnostic/prognostic assays. Antibodies are the receptors of choice in most cases although aptamers are becoming familiar because of their facile and reproducible synthesis, chemical stability as well as comparable affinity and selectivity. In recent years, it has been reported that the pattern of post-translational modifications, altered under neoplastic disease, is a better predictive biomarker than the total protein level. Among others, abnormal glycosylation is attracting great attention. Lectins and antibodies are being used for identification and detection of the carbohydrate moiety with low level of discrimination among various glycoproteins. Such level of selectivity is critical to bring next-generation biomarkers to the clinic. Aptamers that can be rationally tailored for a certain molecule domain can become the golden receptor to specifically detect aberrant glycosylation at each protein or even at each glycosylation site, providing new diagnostic tools for early detection of cancer. Graphical abstract Aptamers may specifically differentiate normal from aberrant glycoproteins.

Highly sensitive testing of nucleic acids is essential to improve the detection of pathogens, which pose a major threat for public health worldwide. Currently available molecular assays, mainly based on PCR, have a limited utility in point-of-need control or resource-limited settings. Consequently, there is a strong interest in developing cost-effective, robust, and portable platforms for early detection of these harmful microorganisms. Since its description in 2004, isothermal helicase-dependent amplification (HDA) has been successfully applied in the development of novel molecular-based technologies for rapid, sensitive, and selective detection of viruses and bacteria. In this review, we highlight relevant analytical systems using this simple nucleic acid amplification methodology that takes place at a constant temperature and that is readily compatible with microfluidic technologies. Different strategies for monitoring HDA amplification products are described. In addition, we present technological advances for integrating sample preparation, HDA amplification, and detection. Future perspectives and challenges toward point-of-need use not only for clinical diagnosis but also in food safety testing and environmental monitoring are also discussed. Graphical Abstract Expanding the analytical toolbox for the detection of DNA sequences specific of pathogens with isothermal helicase dependent amplification (HDA).

The main goal of food safety assessment is to provide reliable information on the identity and composition of food and reduce the presence of harmful components. Nowadays, there are many countries where rather than the presence of pathogens, common public concerns are focused on the presence of hidden allergens, fraudulent practices, and genetic modifications in food. Accordingly, food regulations attempt to offer a high level of protection and to guarantee transparent information to the consumers. The availability of analytical methods is essential to comply these requirements. Protein-based strategies are usually employed for this purpose, but present some limitations. Because DNA is a more stable molecule, present in most tissues, and can be amplified, there has been an increasing interest in developing DNA-based approaches (polymerase chain reaction, microarrays, and genosensors). In this regard, electrochemical genosensors may play a major role in fulfilling the needs of food industry, such as reliable, portable, and affordable devices. This work reviews the achievements of this technology applied to allergen detection, species identification, and genetically modified organisms testing. We summarized the legislative framework, current design strategies in sensor development, their analytical characteristics, and future prospects.

An on-surface isothermal helicase-dependent amplification is devised for simple, point-of-need quantification of bacterial genomes. The method relies on the enzyme-extension of a thiol-modified reverse primer anchored to indium tin oxide electrodes, which shows strikingly high thermal and storage stability. Amplification and electrochemical detection of only 10 genomes are thus performed on the same platform without thermal cycling.

Genosensing technology has mostly based on mixed self-assembled monolayers (SAMs) of thiol-modified oligonucleotides and alkanethiols on gold surfaces. However, the typical backfilling approach, which incorporates the alkanethiol in a second step, gives rise to a heterogeneous distribution of oligonucleotide probes on the surface, negatively affecting to both hybridization efficiency and surface stability. Despite aromatic thiols present a remarkably different behavior from alkanethiols, with higher rigidity and stronger intermolecular interactions, they have been scarcely explored for the fabrication of DNA sensing platforms. We have investigated different approaches involving SAMs of aromatic thiols, namely p-mercaptobenzoic acid (p-MBA) and p-aminothiophenol (p-ATP), to yield DNA sensing layers for sequence-specific detection of target oligonucleotides. The studied monolayers were evaluated by DNA surface coverage and further information was obtained by determining their functionality in a sandwich hybridization assay with enzymatic amplification of the electrochemical read-out. The insertion of thiol-oligonucleotides into p-ATP monolayers previously oxidized, and the covalent binding of amino-oligonucleotides to pure p-MBA monolayers give rise to increased storage stability and better analytical performance. The quantification of RNA from Legionella pneumophila cellular lysates was successfully performed, illustrating the usefulness of these sensing architectures for detecting pathogenic bacteria., (Copyright © 2017 Elsevier B.V. All rights reserved.)

The design of screening methods for the detection of genetically modified organisms (GMOs) in food would improve the efficiency in their control. We report here a PCR amplification method combined with a sequence-specific electrochemical genosensor for the quantification of a DNA sequence characteristic of the 35S promoter derived from the cauliflower mosaic virus (CaMV). Specifically, we employ a genosensor constructed by chemisorption of a thiolated capture probe and p -aminothiophenol gold surfaces to entrap on the sensing layer the unpurified PCR amplicons, together with a signaling probe labeled with fluorescein. The proposed test allows for the determination of a transgene copy number in both hemizygous (maize MON810 trait) and homozygous (soybean GTS40-3-2) transformed plants, and exhibits a limit of quantification of at least 0.25% for both kinds of GMO lines.

Methods for the early and sensitive detection of pathogenic bacteria suited to low-resource settings could impact diagnosis and management of diseases. Helicase-dependent isothermal amplification (HDA) is an ideal tool for this purpose, especially when combined with a sequence-specific detection method able to improve the selectivity of the assay. The implementation of this approach requires that its analytical performance is shown to be comparable with the gold standard method, polymerase chain reaction (PCR). In this study, we optimize and compare the asymmetric amplification of an 84-base-long DNA sequence specific for Mycobacterium tuberculosis by PCR and HDA, using an electrochemical genomagnetic assay for hybridization-based detection of the obtained single-stranded amplicons. The results indicate the generalizability of the magnetic platform with electrochemical detection for quantifying amplification products without previous purification. Moreover, we demonstrate that under optimal conditions the same gene can be amplified by either PCR or HDA, allowing the detection of as low as 30 copies of the target gene sequence with acceptable reproducibility. Both assays have been applied to the detection of M. tuberculosis in sputum, urine, and pleural fluid samples with comparable results. Simplicity and isothermal nature of HDA offer great potential for the development of point-of-care devices. Graphical Abstract Comparative evaluation of isothermal helicase-dependent amplification and PCR for electrochemical detection of Mycobacterium tuberculosis.

With this work, it is intended to propose a novel approach based on high resolution melting (HRM) analysis to detect wheat and discriminate it from other gluten-containing cereals. The method consisted of a real-time PCR assay targeting the gene encoding for the germ agglutinin isolectin A protein (Tri a 18 allergen), using the fluorescent Evagreen dye combined with HRM analysis. The results enabled wheat differentiation from other phylogenetically related cereals, namely barley, rye and oat with high level of confidence. Additionally, a quantitative real-time PCR approach was proposed, allowing detecting and quantifying wheat down to 20mg/kg in rice flour and 20pg of wheat DNA (∼1.1 DNA copies). Its application was successfully achieved in the analysis of processed foods to verify labelling compliance, being considered as a cost-effective tool for the specific detection of cereals in gluten-free foods., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Celiac disease is a lifelong autoimmune disorder triggered by foods containing gluten, the storage protein in wheat, rye, and barley. The rapidly escalating number of patients diagnosed with this disease poses a great challenge to both food industry and authorities to guarantee food safety for all. Therefore, intensive efforts are being made to establish minimal disease-eliciting doses of gluten and consequently to improve gluten-free labeling. These efforts depend to a high degree on the availability of methods capable of detecting the protein in food samples at levels as low as possible. Current analytical approaches rely on the use of antibodies as selective recognition elements. With limited sensitivity, these methods exhibit some deficiencies that compromise the accuracy of the obtained results. Aptamers provide an ideal alternative for designing biosensors for fast and selective measurement of gluten in foods. This article highlights the challenges in gluten detection, the current status of the use of aptamers for solving this problem, and what remains to be done to move these systems into commercial applications.

Electrochemical genosensors have undergone an enormous development in the last decades, but only very few have achieved a quantification of target content in highly processed food samples. The detection of allergens, and particularly gluten, is challenging because legislation establishes a threshold of 20 ppm for labeling as gluten-free but most genosensors expresses the results in DNA concentration or DNA copies. This paper describes the first attempt to correlate the genosensor response and the wheat content in real samples, even in the case of highly processed food samples. A sandwich-based format, comprising a capture probe immobilized onto the screen-printed gold electrode, and a signaling probe functionalized with fluorescein isothiocyanate (FITC), both hybridizing with the target was used. The hybridization event was electrochemically monitored by adding an anti-FITC peroxidase (antiFITC-HRP) and its substrate, tetramethylbenzidine. Binary model mixtures, as a reference material, and real samples have been analyzed. DNA from food was extracted and a fragment encoding the immunodominant peptide of α2-gliadin amplified by a tailored PCR. The sensor was able to selectively detect toxic cereals for celiac patients, such as different varieties of wheat, barley, rye and oats, from non-toxic plants. As low as 0.001% (10 mg/kg) of wheat flour in an inert matrix was reliably detected, which directly compete with the current method of choice for DNA detection, the real-time PCR. A good correlation with the official immunoassay was found in highly processed food samples., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Cultivation of genetically modified organisms (GMOs) and their use in food and feed is constantly expanding; thus, the question of informing consumers about their presence in food has proven of significant interest. The development of sensitive, rapid, robust, and reliable methods for the detection of GMOs is crucial for proper food labeling. In response, we have experimentally characterized the helicase-dependent isothermal amplification (HDA) and sequence-specific detection of a transgene from the Cauliflower Mosaic Virus 35S Promoter (CaMV35S), inserted into most transgenic plants. HDA is one of the simplest approaches for DNA amplification, emulating the bacterial replication machinery, and resembling PCR but under isothermal conditions. However, it usually suffers from a lack of selectivity, which is due to the accumulation of spurious amplification products. To improve the selectivity of HDA, which makes the detection of amplification products more reliable, we have developed an electrochemical platform targeting the central sequence of HDA copies of the transgene. A binary monolayer architecture is built onto a thin gold film where, upon the formation of perfect nucleic acid duplexes with the amplification products, these are enzyme-labeled and electrochemically transduced. The resulting combined system increases genosensor detectability up to 10(6)-fold, allowing Yes/No detection of GMOs with a limit of detection of ∼30 copies of the CaMV35S genomic DNA. A set of general utility rules in the design of genosensors for detection of HDA amplicons, which may assist in the development of point-of-care tests, is also included. The method provides a versatile tool for detecting nucleic acids with extremely low abundance not only for food safety control but also in the diagnostics and environmental control areas.

Enzyme immunoassays are currently the methods of choice for gluten control in foods labelled as gluten free, providing a mechanism for assessing food safety for consumption by coeliac and other allergic patients. However, their limitations, many of them associated to the reactivity of the different antibodies used and their degree of specificity, have prevented the establishment of a standardised method of analysis. We explore new methods for quantitatively determining gluten content in foods based on the use of two recently described aptamers, raised against a 33-mer peptide recognised as the immunodominant fragment from α2-gliadin. The assays use the target peptide immobilised onto streptavidin-coated magnetic beads in combination with a limited amount of biotin-aptamer in a competitive format, followed by streptavidin-peroxidase labelling of the aptamer that remains bound to the magnetic beads. The enzyme activity onto the beads, measured by chronoamperometry in disposable screen-printed electrodes, is inversely related to the target concentration in the test solution. We find that while the assay using the aptamer with the highest affinity towards the target (Gli 4) achieves low detection limits (~0.5 ppm) and excellent analytical performance, when challenged in samples containing the intact protein, gliadin, it fails in detecting the peptide in solution. This problem is circumvented by employing another aptamer (Gli 1), the most abundant one in the SELEX pool, as a receptor. The proposed assays allow the convenient detection of the allergen in different kinds of food samples, including heat-treated and hydrolysed ones. The obtained results correlate with those of commercially available antibody-based assays, providing an alternative for ensuring the safety and quality of nominally gluten-free foods. Graphical Abstract Electrochemical magnetoassay for gluten determination using biotin-aptamers as receptors.

Current EU regulations on the mandatory labeling of genetically modified organisms (GMOs) with a minimum content of 0.9% would benefit from the availability of reliable and rapid methods to detect and quantify DNA sequences specific for GMOs. Different genosensors have been developed to this aim, mainly intended for GMO screening. A remaining challenge, however, is the development of genosensing platforms for GMO quantification, which should be expressed as the number of event-specific DNA sequences per taxon-specific sequences. Here we report a simple and sensitive multiplexed electrochemical approach for the quantification of Roundup-Ready Soybean (RRS). Two DNA sequences, taxon (lectin) and event-specific (RR), are targeted via hybridization onto magnetic beads. Both sequences are simultaneously detected by performing the immobilization, hybridization and labeling steps in a single tube and parallel electrochemical readout. Hybridization is performed in a sandwich format using signaling probes labeled with fluorescein isothiocyanate (FITC) or digoxigenin (Dig), followed by dual enzymatic labeling using Fab fragments of anti-Dig and anti-FITC conjugated to peroxidase or alkaline phosphatase, respectively. Electrochemical measurement of the enzyme activity is finally performed on screen-printed carbon electrodes. The assay gave a linear range of 2-250 pM for both targets, with LOD values of 650 fM (160 amol) and 190 fM (50 amol) for the event-specific and the taxon-specific targets, respectively. Results indicate that the method could be applied for GMO quantification below the European labeling threshold level (0.9%), offering a general approach for the rapid quantification of specific GMO events in foods., (Copyright © 2015 Elsevier B.V. All rights reserved.)

A highly sensitive and robust method for the quantification of specific DNA sequences based on coupling asymmetric helicase-dependent DNA amplification to electrochemical detection is described. This method relies on the entrapment of the amplified ssDNA sequences on magnetic beads followed by a post-amplification hybridization assay to provide an added degree of specificity. As a proof-of-concept a 84-bases long sequence specific of Mycobacterium tuberculosis is amplified at 65°C, providing 3×10(6) amplification after 90 min. Using this system 0.5 aM, corresponding to 15 copies of the target gene in 50 µL of sample, can be successfully detected and reliably quantified under isothermal conditions in less than 4h. The assay has been applied to the detection of M. tuberculosis in sputum, pleural fluid and urine samples. Besides this application, the proposed assays is a powerful and general tool for molecular diagnostic that can be applied to the detection of other specific DNA sequences, taking full advantage of the plethora of genomic information now available., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Aptamers are starting to increase the reagents tool box to develop more sensitive and reliable methods for food allergens. In most of these assays, aptamers have to be modified for detection and/or immobilization purposes. To take full advantage of their affinity, which decisively influence the detectability, these modifications must be faced rationally. In this work, a recently developed aptamer for an immunotoxic peptide of gliadin associated to celiac disease is used in different configurations and modified with various markers and anchored groups to evaluate the influence of such modifications on the real affinity. The interaction in solution with the peptide is strong for a relatively small molecule (Kd = 45 ± 10 nM, 17 °C) and slightly stronger than that for the immobilized intact protein due to a cooperative binding effect. Comparatively, while only minor differences were found when the peptide or the aptamer were immobilized, labeling with a biotin resulted preferable over fluorescein (Kd = 102 ± 11 vs 208 ± 54 nM, 25 °C). These findings are of prime importance for the design of an aptamer-based analytical method for gluten quantification., (Copyright © 2015 Elsevier B.V. All rights reserved.)

High selectivity of genosensors is crucial for certain applications such as those involving species with high genetic variability. This is an unresolved problem when dealing with long target sequences that is further complicated when the target contains repetitive sequence domains. As a model for this situation, the problem of detecting gluten in food with identification of the source is studied. In order to discriminate the specific DNA sequence that encodes the wheat prolamin (gliadin) from rye and barley prolamins, the exquisite selectivity of a rationally designed hairpin capture probe is proposed and compared to a nonstructured capture probe. An electrochemical sandwich assay is proposed, involving capture probes chemisorbed on Au surfaces and biotinylated-signaling probes in combination with streptavidin-peroxidase labeling conjugates. As a result, a genosensor with similar sensitivity to that observed with linear probes but with complete specificity against closely related species was achieved. The surface-attached DNA stem-loop yields a device capable of accurately discriminating wheat DNA from rye and barley with a limit of detection of 1 nM.

In the present work, the development of a genosensor for the event-specific detection of MON810 transgenic maize is proposed. Taking advantage of nanostructuration, a cost-effective three dimensional electrode was fabricated and a ternary monolayer containing a dithiol, a monothiol and the thiolated capture probe was optimized to minimize the unspecific signals. A sandwich format assay was selected as a way of precluding inefficient hybridization associated with stable secondary target structures. A comparison between the analytical performance of the Au nanostructured electrodes and commercially available screen-printed electrodes highlighted the superior performance of the nanostructured ones. Finally, the genosensor was effectively applied to detect the transgenic sequence in real samples, showing its potential for future quantitative analysis., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Electrochemical genosensors are becoming cost-effective miniaturizable alternatives to real-time PCR (RT-PCR) methods for the detection of sequence-specific DNA fragments. We report on the rapid detection of PCR amplicons without the need of purification or strand separation. A challenging target sequence for both PCR amplification and electrochemical detection allowed us to address some difficulties associated to hybridization on electrode surfaces. The target was a highly specific oligonucleotide sequence of wheat encoding the most immunogenic peptide of gliadin that triggers the immune response of celiac disease (CD), the 33-mer. With a sandwich assay format and a rational design of the capture and tagged-signaling probes the problems posed by the strong secondary structure of the target and complementary probes were alleviated. Using a binary self-assembled monolayer and enzymatic amplification, a limit of detection of 0.3 nM was obtained. The genosensor did not respond to other gluten-containing cereals such as rye and barley. Coupling to PCR to analyze wheat flour samples required tailoring both the capture and signaling probes. This is the first time that deleterious steric hindrance from long single-stranded regions adjacent to the electrode surface is reported for relatively short amplicons (less than 200 bp). The importance of the location of the recognition site within the DNA sequence is discussed. Since the selected gene fragment contains several repetitions of short sequences, a careful optimization of the PCR conditions had to be performed to circumvent the amplification of non-specific fragments from wheat flour., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Celiac disease represents a significant public health problem in large parts of the world. A major hurdle in the effective management of the disease by celiac sufferers is the sensitivity of the current available methods for assessing gluten contents in food. In response, we report a highly sensitive approach for gluten analysis using aptamers as specific receptors. Gliadins, a fraction of gluten proteins, are the main constituent responsible for triggering the disease. However, they are highly hydrophobic and large molecules, regarded as difficult targets for in vitro evolution of aptamers without nucleobase modification. We describe the successful selection of aptamers for these water insoluble prolamins that was achieved choosing the immunodominant apolar peptide from α2-gliadin as a target for selection. All aptamers evolved are able to bind the target in its native environment within the natural protein. The best nonprotein receptor is the basis for an electrochemical competitive enzyme-linked assay on magnetic particles, which allows the measurement of as low as 0.5 ppb of gliadin standard (0.5 ppm of gluten). Reference immunoassay for detecting the same target has a limit of detection of 3 ppm, 6 times less sensitive than this method. Importantly, it also displays high specificity, detecting the other three prolamins toxic for celiac patients and not showing cross-reactivity to nontoxic proteins such as maize, soya, and rice. These features make the proposed method a valuable tool for gluten detection in foods.

Establishing an efficient method for evaluating the affinity changes after post-SELEX modification of aptamers is essential for broadening the application of these oligonucleotides in biosensing. This is especially challenging when the ligand is a small molecule. Changes in affinity upon partial or total replacement of 2'-OH with 2'-OMe groups in the ribose moieties of a tobramycin binding RNA aptamer are described. The kinetic profile and binding properties of the different anti-tobramycin aptamers were measured by surface plasmon resonance (SPR) experiments through a real-time binding assay with the antibiotic covalently coupled to the gold sensor. This configuration maximizes the changes associated to the recognition event, which is otherwise undetectable. The results indicated that the modification slightly affects the binding characteristics of the parent RNA, while conferring biological stability to the aptamers against nucleases., (Copyright © 2012 Elsevier B.V. All rights reserved.)

To counteract and prevent the deleterious effect of free radicals the living organisms have developed complex endogenous and exogenous antioxidant systems. Several analytical methodologies have been proposed in order to quantify antioxidants in food, beverages and biological fluids. This paper revises the electroanalytical approaches developed for the assessment of the total or individual antioxidant capacity. Four electrochemical sensing approaches have been identified, based on the direct electrochemical detection of antioxidant at bare or chemically modified electrodes, and using enzymatic and DNA-based biosensors., (Copyright © 2011. Published by Elsevier B.V.)

An RNA aptamer is proposed as a recognition element for the detection of tobramycin in human serum. A displacement assay was developed using faradaic-electrochemical impedance spectroscopy (F-EIS) as a detection technique. Two modified aptamers, a partially (ATA) and a fully O-methylated aptamer (FATA) were evaluated and compared. The affinity constant, K(D), for both aptamers was estimated by F-EIS resulting virtually identical within the experimental error. The selectivity towards other aminoglycosides was also studied. The analytical characteristics were evaluated in aqueous solution using both aptamers and FATA was selected for human serum experiments. Using a 1:0.5 dilution of the serum, a linear range between 3 μM and 72.1 μM was obtained, which included the therapeutic range of the antibiotic., (Copyright © 2010 Elsevier B.V. All rights reserved.)

Reactive oxygen species (ROS) are produced as a consequence of normal aerobic metabolism and are able to induce DNA oxidative damage. At the cellular level, the evaluation of the protective effect of antioxidants can be achieved by examining the integrity of the DNA nucleobases using electrochemical techniques. Herein, the use of an adenine-rich oligonucleotide (dA(21)) adsorbed on carbon paste electrodes for the assessment of the antioxidant capacity is proposed. The method was based on the partial damage of a DNA layer adsorbed on the electrode surface by OH radicals generated by Fenton reaction and the subsequent electrochemical oxidation of the intact adenine bases to generate an oxidation product that was able to catalyze the oxidation of NADH. The presence of antioxidant compounds scavenged hydroxyl radicals leaving more adenines unoxidized, and thus, increasing the electrocatalytic current of NADH measured by differential pulse voltammetry (DPV). Using ascorbic acid (AA) as a model antioxidant species, the detection of as low as 50 nM of AA in aqueous solution was possible. The protection efficiency was evaluated for several antioxidant compounds. The biosensor was applied to the determination of the total antioxidant capacity (TAC) in beverages., (Copyright © 2010 Elsevier B.V. All rights reserved.)