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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.

This research was funded by the Spanish Government (project RTI-2018–095756-B-I00 ). R.S.S. thanks the Spanish Government for a PhD fellowship ( MCIU-20-PRE2019–087618 ).

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.

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.

Prostate-specific Antigen (PSA) is the biomarker that is used for prostate cancer (PCa) detection, although its lack of specificity results in a high rate of false-positives and many unnecessary biopsies. Therefore, there is a need for more specific cancer biomarkers for PCa. Recent studies have shown that the aberrant glycosylation of proteins is a common feature of the presence of cancer. In the case of prostate cancer, there are changes in core-fucose and sialic acids in the glycan structure of PSA. In this work, we describe two different strategies to direct the selection of aptamers toward the glycans of PSA. From these strategies, we identified two aptamers (PSA-1 and PSAG-1) that bind to the glycan structure of PSA with high affinity. Both aptamers were applied in the design of electrochemical aptasensors, in sandwich and direct formats, in order to detect the changes in the glycosylation of PSA. The sensors responded to different levels of PSA in serum, and they showed higher potential to discriminate clinically-meaningful PCa than the ELISA (Enzyme-linked immunosorbent assay) test used in hospitals (reducing the number of false positives), although validation on more samples is needed.

Affinity characterization is an essential but time-consuming task to develop reliable aptamers for tumor biomarker detection and is not always thoroughly addressed. For neutrophil gelatinase-associated lipocalin (NGAL), a potential biomarker of pancreatic cancer, two DNA aptamers were described with very different affinity. Likewise, another pair of DNA aptamers was developed with very different affinity for alpha-fetoprotein (AFP), a biomarker of hepatocellular carcinoma. In this work, we estimated the dissociation constant of these aptamers by means of a direct assay on magnetic beads modified with biomarker and electrochemical detection on screen-printed carbon electrodes. In order to improve the performance of these aptamers, we proposed the isothermal amplification of the aptamers for both biomarkers by rolling circle amplification (RCA). In the case of AFP aptamers, we also tried terminal deoxynucleotidyl transferase (TdT), a template-independent amplification. Both DNA amplifications improved the sensitivity and the apparent binding constants of the aptamers for the two cancer biomarkers. Nevertheless, this improvement depends on the true affinity of the binding pair, which ultimately limits their analytical usefulness.

A sandwich genoassay for the detection of PCA3, a nucleic acid biomarker overexpressed in the urine of prostate cancer patients, has been developed by using the enzyme alkaline phosphatase (ALP) as a tracer of the hybrid generated onto the surface of magnetic particles. ALP converts D-glucose-1-phosphate into D-glucose, which is quantified with a personal glucose meter. The resulting methodology allows the reliable detection of PCA3 at low picomolar levels, thus fostering massive screening of prostate cancer.

In this work, two sensing approaches for the specific quantification of the Salmonella genome are comparatively evaluated. These devices successfully integrate solid-phase isothermal HDA or RPA amplification in electrochemical platforms and support efficient detection of small amounts of the bacterial genome without thermal cycling, while using simple equipment. The detectability of the RPA-based sensing platform (105 genomes) was surpassed by that of the HDA-based one (10 genomes), though it could be enhanced by primer design optimization. This, together with a low operation temperature compatible with conventional surface chemistry and faster amplification, makes the RPA-based electrochemical platform a promising device for point-of-need analysis.

Characterization of extracellular matrix (ECM) is becoming more and more important to decipher cancer progression. Constant remodeling results in ECM components degradation or unusual ECM accumulation that releases short fragments to the body fluids. These fragments might be potential cancer biomarkers but to detect them specific receptors are needed. In response to this demand, we present the first electrochemical aptamer-based competitive assay for the minor collagen XI, dysregulated in several carcinomas. It was performed on magnetic beads using enzymatic labeling. First, we selected the most appropriate tag for the aptamer (biotin or 6-carboxyfluorescein). The former yielded higher currents by chronoamperometry and it was used for the competitive assay. The collagen fragment, a 16mer peptide used as the target, was detected from 52 to 1000 nM with an RSD of about 5%. The LOD of the assay was estimated as 24 nM (44 ng/mL). The performance of the assay in serum diluted 1:2 was equivalent to the assay in PBS. The detection of α1 chain of human collagen XI was also possible in cell lysates and confirmed by aptacytofluorescence, which is promising as a new tool to validate this fragment as a cancer biomarker.

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.

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 Kd 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.

Hybridization-based electrochemical DNA sensors have become a methodological mainstay in a wide variety of fields. Among the possible assay formats, sandwich hybridization is the most powerful one. It is so called because the target sequence to be measured is hybridized between two complementary sequences. The analytical performance of this class of sensors is influenced by many construction and operational parameters, which need to be optimized. In this work, we comparatively analyze the analytical performance of different sandwich hybridization sensors, constructed on gold surfaces modified by chemisorption. We find that the structures of the designed probes as well as the topography of the gold support and the nature of the monolayer are key parameters affecting the sensitivity and reproducibility of the genosensors. Other important operational parameters are also described. The results of this study could serve as a guide in the design of genosensors for future applications.

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.

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.

Electrochemical impedance spectroscopy is a powerful electrochemical technique for studying electrode–solution interfaces with applications in different fields such as energy (batteries), corrosion, and bioanalysis. When applied to report biomolecular recognition events (antigen–antibody, DNA hybridization, aptamer–ligand), it simplifies the operational procedure, although parallel tests for ensuring the specificity of the detection are compulsory. This experiment proposes the development of a label-free sensor for the detection of neomycin B based on a displacement bioassay using faradaic impedance spectroscopy, showing the usefulness of this technique in bioanalysis.

The reduced cofactor nicotinamide adenine dinucleotide (NADH) participates in hundreds of in vivo enzymatic reactions, so monitoring its generation or consumption allows the determination of many substrates or enzymes relevant in clinical or food analysis. This experiment proposes the preparation of easily renewable electrode surfaces modified with a precursor of the electrocatalyst that is straightforward generated by cyclic voltammetry. Amperometric detection is simple, fast, and sensitive enough to reach nM levels of NADH.

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., Binary recognition of the glycoprotein prostate specific antigen by aptamers: a tool for detecting aberrant glycosylation associated with cancer.

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.

This experiment proposes the construction of an amperometric ethanol biosensor to verify if the composition of several nonalcohol beers complies with the food legislation in force. The design is based on the immobilization of the enzyme alcohol dehydrogenase along with the coenzyme NAD+ and the nucleoside adenosine, acting as a precursor of the catalyst for β-nicotinamide adenine dinucleotide (NADH) oxidation, in a carbon paste electrode. An electropolymerized polyphenylenediamine film is employed to protect the sensor surface and avoid the solubilization of the different biocomponents. The current response, obtained after the oxidation of the reduced form of NADH electrocatalyst generated in the enzymatic process, is directly proportional to ethanol concentration in the sample.

This pilot project is part of the innovation project PINN -19-A-026 to be implemented at the University of Oviedo.

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.

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.

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.

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.

This work was financially supported by the Spanish Ministerio de Economía y Competitividad (Project No.CTQ2015-63567-R), and co-financed by FEDER funds.

Highly sensitive detection of cancer biomarkers in blood is key not only to find cancer at an early stage but also to help clinicians to decide the best treatment plan and to find how well treatment is working. To quantify the small changes in clinically validated biomarkers associated with carcinogenesis both selective receptors and signal amplification strategies of the recognition event between the receptor and the biomarker are highly in demand. This report covers the most recent developments in the integration of aptamer-based recognition of blood-circulating cancer biomarkers and isothermal nucleic acid amplification platforms with electrochemical readout, highlighting the potential of these novel tools, and the challenges to translate these assays to the clinical practice.

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

The integration of nanomaterials in the field of (bio)sensors has allowed developing strategies with improved analytical performance. In this work, ultrasmall core-shell Fe3O4@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 Fe3O4 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 Fe3O4@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., This work received financial support from the European Union (FEDER funds through COMPETE), National Funds (FCT, Fundação para a Ciência e a Tecnologia) through project UID/QUI/50006/2013 and Regional Funds (Principado de Asturias government through Project FC15-GRUPIN14-025), and cofinanced by FEDER funds. A.P. and M.F.B. are grateful to FCT grants SFRH/BD/97995/2013 and SFRH/BPD/78845/2011, financed by POPH–QREN–Tipologia 4.1–Formação Avançada, subsidized by Fundo Social Europeu and Ministério da Ciência, Tecnologia e Ensino Superior. C.P. thanks FCT for the FCT Investigator contract IF/01080/2015.

Real-time PCR is the gold standard technique for the unambiguous detection of sequence-specific fragments of pathogen DNA in many fields but it does not always fulfill the extremely low detectability legislation demands. Herein we demonstrate that rationally designed genosensors can rival or even supersede it when combined with previous amplification strategies even isothermal ones. Among the latter, helicase-dependent amplification (HDA) is a simple and powerful DNA amplification using helicase instead of temperature cycles to unwind DNA; it follows an amplification scheme very similar to PCR, which could facilitate its widespread adoption for point-of-need applications. Unfortunately, the primer design for HDA is more restrictive than for PCR. We show that it is possible to adapt PCR primers to isothermal amplification of a DNA sequence specific for Salmonella. We design an electrochemical DNA sensor, based on a sandwich hybridization assay on the surface of indium-tin oxide electrodes, which allows monitoring both PCR and HDA amplicons. The resulting sequence-specific detection strategy not only increased the selectivity of the detection but also its sensitivity, improving real-time PCR in two-fold and a hundred-fold when combined with HDA and PCR, respectively, achieving the single molecule detection.

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.

Salmonella represents one of the major causes of foodborne diseases in humans, in addition to provoking important economic losses in the agri-food sector worldwide. Therefore, the surveillance and control of this human pathogenic bacterium in foodstuffs and biological fluids are necessary in order to prevent and diagnose the disease. Molecular methods based on the detection of DNA sequences specific to pathogenic species are an appealing alternative to traditional culture-based methods that require 5 to 6 days to obtain a definitive result. Among them, and because of its easy miniaturization, electrochemical genosensors are a suitable option for decentralized genetic testing [1-2]; however, they often require a set of sample pretreatment steps before genetic DNA analysis, thus making their implementation at the point of need more difficult. Herein, we report the integration of a nucleic acid-based sensor and an isothermal DNA amplification technique, helicase-dependent amplification or HDA, onto indium tin oxide (ITO) surfaces for the detection of a DNA sequence specific for the typA gene of Salmonella. DNA amplification process occurs at 65 oC with short oligonucleotides flanking the target sequence, which act as primers. The reversed primer is covalently bound to the ITO surface through a thiol group present at its 5’ terminus, whereas forward fluorescein-tagged primer is incorporated in solution. As a result of the isothermal elongation step, fluorescein-tagged DNA duplexes are attached to the ITO surface and their enzymatic labelling is achieved via Fab fragments directed against fluorescein, conjugated with the redox enzyme alkaline phosphatase. Then, α-naphthyl phosphate is enzymatically dephosphorylated into an electroactive derivate α-naphthol whose amount, directly related to the Salmonella present in the sample, is measured by differential pulse voltammetry. This developed integrated sensing platform allows the detection of Salmonella down to 10 genomes in just over 2 hours [3], the same detection limit as that achieved by real-time PCR but without need of high-end benchtop instrumentation. Furthermore, the sensing layer built onto ITO surfaces maintains its performance even after 9 months storage, and possesses a great potential to be extended to the in-situ, fast and reliable detection of other pathogens. References: [1] D. Mabey, R.W. Peeling, A. Ustianowski and M.D. Perkins, Nat. Rev Microbiol., 2004, 2, 231-240. [2] A.S. Patterson, K. Hsieh, H.T. Soh and K.W. Plaxco, Trends Biotechnol., 2013, 31, 704-712. [3] S. Barreda-Garcia, R. Miranda-Castro, N. de-los-Santos-Alvarez, A.J. Miranda-Ordieres, M.J. Lobo-Castanon, Chem. Comm., 2017, 53, 9721-9724. Acknowledgments: This work has been supported by the Spanish Ministerio de Economia y Competitividad (CTQ2015-63567-R), the Principado de Asturias government (FC-15-GRUPIN14-025), and co-financed by FEDER funds.

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 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.

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 Fe3O4@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

A highly sensitive electroanalytical method for determination of PQQ in solution down to subpicomolar concentrations is proposed. It is based on the heterogeneous reconstitution of the PQQ-dependent glucose dehydrogenase (PQQ-GDH) through the specific binding of its pyrroloquinoline quinone (PQQ) cofactor to the apoenzyme anchored on an electrode surface. It is shown from kinetics analysis of both the enzyme catalytic responses and enzyme surface-reconstitution process (achieved by cyclic voltammetry under redox-mediated catalysis) that the selected immobilization strategy (i.e., through an avidin/biotin linkage) is well-suited to immobilize a nearly saturated apoenzyme monolayer on the electrode surface with an almost fully preserved PQQ binding properties and catalytic activity. From measurement of the overall rate constants controlling the steady-state catalytic current responses of the surface-reconstituted PQQ-GDH and determination of the PQQ equilibrium binding (Kb = 2.4 × 10(10) M(-1)) and association rate (kon = 2 × 10(6) M(-1) s(-1)) constants with the immobilized apoenzyme, the analytical performances of the method could be rationally evaluated, and the signal amplification for PQQ detection down to the picomolar levels is well-predicted. These performances outperform by several orders of magnitude the direct electrochemical detection of PQQ in solution and by 1 to 2 orders the detection limits previously achieved by UV-vis spectroscopic detection of the homogeneous PQQ-GDH reconstitution.

Reliable detection of decreasing amounts of gluten in food is the only way of ensuring the safety of all coeliac patients. Results obtained with the method of choice, immunochemical assays, are not entirely comparable and many of them are sandwich assays that cannot recognize hydrolyzed proteins. In this work, we propose a competitive electrochemical sensor based on a recently described aptamer targeting the gliadin immunodominant peptide 33-mer that triggers the coeliac disease. The sensing layer is built on the surface of a screen-printed carbon electrode (SPCE) by adsorption of streptavidin and subsequent peptide immobilization. A competition between the peptide and gluten proteins from samples for a defined concentration of biotinylated Gli 4 aptamer is established. The aptamer bound to the peptide on the surface is finally measured after enzyme labelling and chronoamperometric detection of an enzymatically obtained electrochemically active product. This method is able to detect as low as 0.113 μg L −1 of gliadin, which corresponds to 380 μg kg −1 of gluten in food, taking all dilutions and conversion factors into consideration, with a reproducibility lower than 11%. The aptasensor was applied to food samples with gluten contents above and below the legislated threshold for gluten-free labelling in the EU, obtaining good agreement with the official R5 immunochemical method.

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.

A magnetic microsphere-based hybridization assay for the electrochemical detection of pathogen-specific DNA sequences is reported. Enzyme-linked sandwich hybridization assays with different tags (biotin, fluorescein or digoxigenin) and enzyme conjugates (streptavidin-alkaline phosphatase, streptavidin-peroxidase, antifluorescein-peroxidase or antidigoxigenin-peroxidase) are comparatively evaluated. Sensitivity and limit of detection depend on both the choice of the enzyme and the tag. The highest sensitivity and lowest limit of detection were obtained when antibody Fab fragments conjugated to peroxidase were used as reporter molecule. The digoxigenin system yields the lowest detection limit, mainly due to producing much lower background signals.

A comparative evaluation of an electrochemical sandwich genoassay for pathogenic bacteria based on immobilized hairpin DNA probes and three different enzyme labels (horseradish peroxidase, alkaline phosphatase and a biomimetic peroxidase-like DNAzyme) is reported. The natural enzymes were used as streptavidin conjugates, coupled to the surface duplex by using a biotin-labeled signaling probe, whereas the DNAzyme was directly incorporated to the sequence of the signaling probe. HRP provides enhanced sensitivity although the choice of a catalytic reporter DNA sequence could simplify the assay.

The use of nucleic acid with a specific sequence and a highly ordered secondary structure such as hairpins, quadruplexes and pseudoknots as biological recognition elements and switches in biosensors is rapidly increasing because of their improved features (e.g. selectivity) when compared with the traditional linear probes. Owing to the novelty, a critical outlook of their characteristics and a compilation of the latest advances are lacking. This article describes the potential of those nucleic acids probes whose molecular recognition ability relies on a conformational change (e.g. folding/unfolding mechanism) in electrochemical sensing. It provides an overview of the toolbox of assays using these probes for genosensors and aptasensors, highlighting its performance characteristics and the prospects and challenges for biosensor design.

Human infections with Legionella pneumophila represent a public health problem. Current culture assays for surveillance and control of L. pneumophila in water are time-consuming and limited by the sensitivity, especially when samples also contain microorganisms that inhibit Legionella growth. In this work, an electrochemical method, different from real-time polymerase chain reaction (PCR) approaches, for semiquantitative evaluation of L. pneumophila is presented. A PCR assay targeting the 16S-rRNA gene of L. pneumophila giving rise to a 95-mer amplicon was established. Amplicons were hybridized to a biotin-labeled reporter sequence and then to a thiolated stem-loop structure immobilized onto gold electrodes as a reporter molecule with 1-naphthyl phosphate as a substrate. 1-Naphthol enzymatically generated was determined by differential pulse voltammetry (DPV). For a constant number of amplification cycles, results show that the voltammetric signal is related to the number of copies in the sample thus achieving a useful semiquantitative estimation of L. pneumophila. After 40 cycles of PCR amplification this methodology has a limit of detection of 10 genomes, allowing the reliable detection of 10(2) genomes of L. pneumophila as well as distinguishing 10(3) and 10(4) genomes of the pathogen, values related to corrective actions in water systems in buildings, in accordance with the legislation currently in force.

A disposable voltammetric sensor for the detection of DNA sequences specific of Legionella pneumophila is presented. The sensor is based on a sandwich-hybridization format assay, using a stem-loop DNA structure as capture probe immobilized onto a screen-printed gold electrode. The signaling-DNA probe is labeled with biotin, and streptavidin-alkaline phosphatase is used as reporter molecule. 1-Naphthol, the enzymatic hydrolysis product of anaphthyl phosphate, is voltammetrically detected. The hairpin-modified screen-printed gold electrode allows the detection of a 52-mer DNA sequence at 400 pM level. With a sample volume of 40 mL, this concentration corresponds to ca. 16 fmol of a DNA sequence specific of L. pneumophila. The analytical performance of this platform is compared with that previously obtained for a sensor constructed on a conventional disk gold electrode with the same geometry (˘ ¼ 1.6 mm) and design. A lower quantification limit is obtained with the SPE. In addition, the selectivity is improved as the disposable device shows a better discrimination between L. pneumophila and L. longbeachae or L. micdadei under identical nonstringent conditions.