Lectin-Mimicking Aptamer as a Generic Glycan Receptor for Sensitive Detection of Glycoproteins Associated with Cancer.

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.

Capacitive spectroscopy as transduction mechanism for wearable biosensors: opportunities and challenges.

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.

Comparing nanobody and aptamer-based capacitive sensing for detection of interleukin-6 (IL-6) at physiologically relevant levels.

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.

First bioelectronic immunoplatform for quantitative secretomic analysis of total and metastasis-driven glycosylated haptoglobin.

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 H2O2/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.

Bioanalytical methods for circulating extracellular matrix-related proteins: new opportunities in cancer diagnosis.

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.

New Uses for the Personal Glucose Meter: Detection of Nucleic Acid Biomarkers for Prostate Cancer Screening.

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.

Long noncoding RNAs: from genomic junk to rising stars in the early detection of cancer.

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.

Post-translational modifications in tumor biomarkers: the next challenge for aptamers?

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.

Helicase-dependent isothermal amplification: a novel tool in the development of molecular-based analytical systems for rapid pathogen detection.

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).

Carbon nanotube modified glassy carbon electrode for electrochemical oxidation of alkylphenol ethoxylate.

Electrochemical oxidation of an emerging pollutant, 2-(4-methylphenoxy)ethanol (MPET), from water has been studied by cyclic voltammetry (CV). Multiwall carbon nanotubes glassy carbon electrodes (MWCNT-GCE) were used as working electrode due to their extraordinary properties. The oxidation process is irreversible, since no reduction peaks were observed in the reverse scan. The electrocatalytic effect of MWCNT was confirmed as the oxidation peak intensity increases in comparison to bare-GCE. The effect of functional groups on MWCNT was also studied by MWCNT functionalized with NH2 (MWCNT-NH2) and COOH (MWCNT-COOH) groups. The oxidation peak current decreases in the following order: MWCNT > MWCNT-NH2 > MWCNT-COOH. Taking into account the normalized peak current, MWCNT-NH2 exhibits the best results due to its strong interaction with MPET. Under optimal conditions (pH = 5.0 and volume of MWCNT = 10 µL), degradation was studied for MWCNT-GCE and MWCNT-NH2-GCE. A complete MPET removal was observed using MWCNT-GCE after four CV cycles, for a volume/area (V/A) ratio equal to 19. In the case of MWCNT-NH2-GCE, the maximum MPET removal was close to 90% for V/A = 37, higher than that obtained for MWCNT-GCE at the same conditions (≈80%). In both cases, no organic by-products were detected.

A Quantitative PCR-Electrochemical Genosensor Test for the Screening of Biotech Crops.

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.

Targeting helicase-dependent amplification products with an electrochemical genosensor for reliable and sensitive screening of genetically modified organisms.

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.

Sensitive gluten determination in gluten-free foods by an electrochemical aptamer-based assay.

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.

Hairpin-based DNA electrochemical sensor for selective detection of a repetitive and structured target codifying a gliadin fragment.

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.

A review on magnetic beads-based SELEX technologies: Applications from small to large target molecules.

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.

Current developments of SELEX technologies and prospects in the aptamer selection with clinical applications.

Aptamers are single-stranded oligonucleotide sequences capable of binding to specific ligands with high affinity. In this manner, they are like antibodies but have advantages such as lower manufacturing costs, lower immunogenicity, fewer batch-to-batch differences, a longer shelf life, high tolerance to different molecular milieus, and a greater number of potential targets. Due to their special features, they have been used in drug delivery, biosensor technology, therapy, and diagnostics. The methodology that allowed its production was the "Systematic Evolution of Ligands by Exponential enrichment" (SELEX). Unfortunately, the traditional protocol is time-consuming and laborious. Therefore, numerous variants with considerable optimization steps have been developed, nonetheless, there are still challenges to achieving real applications in the clinical field. Among them, are control of in vivo activities, fast renal filtration, degradation by nucleases and toxicity testing. This review focuses on current technologies based on SELEX, the critical factors for successful aptamer selection, and its upcoming biomedical and biotechnological applications.

Aptamer-based analysis: a promising alternative for food safety control.

Ensuring food safety is nowadays a top priority of authorities and professional players in the food supply chain. One of the key challenges to determine the safety of food and guarantee a high level of consumer protection is the availability of fast, sensitive and reliable analytical methods to identify specific hazards associated to food before they become a health problem. The limitations of existing methods have encouraged the development of new technologies, among them biosensors. Success in biosensor design depends largely on the development of novel receptors with enhanced affinity to the target, while being stable and economical. Aptamers fulfill these characteristics, and thus have surfaced as promising alternatives to natural receptors. This Review describes analytical strategies developed so far using aptamers for the control of pathogens, allergens, adulterants, toxins and other forbidden contaminants to ensure food safety. The main progresses to date are presented, highlighting potential prospects for the future.