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Label-free, single-molecule sensing is anideal candidate for biomedical applications that rely on the detection of low copy numbers in small volumes and potentially complex biofluids. Among them, solid-state nanopores can be engineered to detect single molecules of charged analytes when they are electrically driven through the nanometer-sized aperture. When successfully applied to nucleic acid sensing, fast transport in the range of 10-100 nucleotides per nanosecond often precludes the use of standard nanopores for the detection of the smallest fragments. Herein, hydrogel-filled nanopores (HFN) are reported that combine quartz nanopipettes with biocompatible chemical poly(vinyl) alcohol hydrogels engineered in-house. Hydrogels were modified physically or chemically to finely tune, in a predictable manner, the transport of specific molecules. Controlling the hydrogel mesh size and chemical composition allowed us to slow DNA transport by 4 orders of magnitude and to detect fragments as small as 100 base pairs (bp) with nanopores larger than 20 nm at an ionic strength comparable to physiological conditions. Considering the emergence of cell-free nucleic acids as blood biomarkers for cancer diagnostics or prenatal testing, the successful sensing and size profiling of DNA fragments ranging from 100 bp to >1 kbp long under physiological conditions demonstrates the potential of HFNs as a new generation of powerful and easily tunable molecular diagnostics tools.
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High-throughput profiling/sensing of nucleic acids has recently emerged as a highly promising strategy for the early diagnosis and improved prognosis of a broad range of pathologies, most notably cancer. Among the potential biomarker candidates, microRNAs (miRNAs), a class of non-coding RNAs of 19-25 nucleotides in length, are of particular interest due to their role in the post-transcriptional regulation of gene expression. Developing miRNA sensing technologies that are quantitative, ultrasensitive and highly specific has proven very challenging because of their small size, low natural abundance and the high degree of sequence similarity among family members. When compared to optical based methods, electrochemical sensors offer many advantages in terms of sensitivity and scalability. This non-comprehensive review aims to break-down and highlight some of the most promising strategies for electrochemical sensing of microRNA biomarkers.
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Técnicas Electroquímicas/métodos , MicroARNs/análisis , Técnicas Biosensibles/métodos , Humanos , Neoplasias/diagnósticoRESUMEN
There is a growing realization, especially within the diagnostic and therapeutic community, that the amount of information enclosed in a single molecule can not only enable a better understanding of biophysical pathways, but also offer exceptional value for early stage biomarker detection of disease onset. To this end, numerous single molecule strategies have been proposed, and in terms of label-free routes, nanopore sensing has emerged as one of the most promising methods. However, being able to finely control molecular transport in terms of transport rate, resolution, and signal-to-noise ratio (SNR) is essential to take full advantage of the technology benefits. Here we propose a novel solution to these challenges based on a method that allows biomolecules to be individually confined into a zeptoliter nanoscale droplet bridging two adjacent nanopores (nanobridge) with a 20 nm separation. Molecules that undergo confinement in the nanobridge are slowed down by up to 3 orders of magnitude compared to conventional nanopores. This leads to a dramatic improvement in the SNR, resolution, sensitivity, and limit of detection. The strategy implemented is universal and as highlighted in this manuscript can be used for the detection of dsDNA, RNA, ssDNA, and proteins.
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The exponential improvements made in DNA sequencing technologies, together with the rapidly declining associated costs, has increasingly led to the expansion of the field of personalised genomic medicine. Changes in the sequence or copy number of specific deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) molecules represent key signatures for the diagnosis, prognosis, classification and monitoring of a broad range of pathologies, most notably cancer. Technologies that can detect these changes require analytical tools that can detect DNA or RNA with high sensitivity and high specificity. Sensing based on bioorthogonal oligonucleotide-templated reactions (OTRs) has been recognised as an elegant strategy that satisfies these criteria and was successfully used for the quantitative detection of nucleic acids both in vitro and in vivo. Herein, we will focus on recent efforts to implement bioorthogonal OTRs into clinically useful biosensors using probes immobilised on or embedded in customised materials and platforms.
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Técnicas Biosensibles , ADN/química , Técnicas Analíticas Microfluídicas , Técnicas de Amplificación de Ácido Nucleico , Oligonucleótidos/química , ADN/genéticaRESUMEN
Oligonucleotide-templated reactions are valuable tools for nucleic acid sensing both inâ vitro and inâ vivo. They are typically carried out under conditions that make any reaction in the absence of template highly unfavorable (most commonly by using a low concentration of reactants), which has a negative impact on the detection sensitivity. Herein, we report a novel platform for fluorogenic oligonucleotide-templated reactions between peptide nucleic acid probes embedded within permeable agarose and alginate hydrogels. We demonstrate that under conditions of restricted mobility (that is, limited diffusion), non-specific interactions between probes are prevented, thus leading to lower background signals. When applied to nucleic acid sensing, this accounts for a significant increase in sensitivity (that is, lower limit of detection). Optical nucleic acid sensors based on fluorogenic peptide nucleic acid probes embedded in permeable, physically crosslinked, alginate beads were also engineered and proved capable of detecting DNA concentrations as low as 100â pm.
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Fluorescencia , Colorantes Fluorescentes/química , Hidrogeles/química , Oligonucleótidos/análisis , Difusión , Estructura Molecular , Tamaño de la Partícula , Propiedades de SuperficieRESUMEN
Highly abundant in cells, microRNAs (or miRs) play a key role as regulators of gene expression. A proportion of them are also detectable in biofluids making them ideal noninvasive biomarkers for pathologies in which miR levels are aberrantly expressed, such as cancer. Peptide nucleic acids (PNAs) are engineered uncharged oligonucleotide analogues capable of hybridizing to complementary nucleic acids with high affinity and high specificity. Herein, novel PNA-based fluorogenic biosensors have been designed and synthesized that target miR biomarkers for prostate cancer (PCa). The sensing strategy is based on oligonucleotide-templated reactions where the only miR of interest serves as a matrix to catalyze an otherwise highly unfavorable fluorogenic reaction. Validated in vitro using synthetic RNAs, these newly developed biosensors were then shown to detect endogenous concentrations of miR in human blood samples without the need for any amplification step and with minimal sample processing. This low-cost, quantitative, and versatile sensing technology has been technically validated using gold-standard RT-qPCR. Compared to RT-qPCR however, this enzyme-free, isothermal blood test is amenable to incorporation into low-cost portable devices and could therefore be suitable for widespread public screening.
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Biomarcadores/sangre , Técnicas Biosensibles , MicroARN Circulante/sangre , Colorantes Fluorescentes/química , Sondas de Ácido Nucleico/química , Ácidos Nucleicos de Péptidos/química , Neoplasias de la Próstata/diagnóstico , MicroARN Circulante/genética , MicroARN Circulante/metabolismo , Cumarinas/química , Humanos , Masculino , Hibridación de Ácido Nucleico , Sondas de Ácido Nucleico/metabolismo , Oligonucleótidos/química , Ácidos Nucleicos de Péptidos/síntesis química , Polimorfismo de Nucleótido Simple , Neoplasias de la Próstata/genética , Reacción en Cadena en Tiempo Real de la PolimerasaRESUMEN
A conceptually new light-up nucleic acid fluorescent probe resulting from the conjugation of a coumarin to a naphthalene diimide exhibits a single wavelength emission at 498â nm when free in solution and an additional red/NIR emission when bound to G-quadruplex DNA. The light-up response centred at 666â nm is highly specific for quadruplex DNA when compared to duplex DNA or to RNA quadruplexes.
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Cumarinas/química , ADN/química , Colorantes Fluorescentes/química , Imidas/química , Naftalenos/química , ARN/química , ADN/metabolismo , G-Cuádruplex , Imidas/metabolismo , Luz , Naftalenos/metabolismo , ARN/metabolismo , Espectrometría de FluorescenciaRESUMEN
A bimolecular synthetic reaction (imine synthesis) was performed compartmentalized in micrometer-diameter emulsion droplets. The apparent equilibrium constant (Keq) and apparent forward rate constant (k1) were both inversely proportional to the droplet radius. The results are explained by a noncatalytic reaction-adsorption model in which reactants adsorb to the droplet interface with relatively low binding energies of a few kBT, react and diffuse back to the bulk. Reaction thermodynamics is therefore modified by compartmentalization at the mesoscale--without confinement on the molecular scale--leading to a universal mechanism for improving unfavorable reactions.
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Química Orgánica/métodos , Iminas/síntesis química , Modelos Químicos , Adsorción , Aldehídos/química , Aminas/química , Química Orgánica/instrumentación , Difusión , Iminas/química , Cinética , Propiedades de Superficie , TermodinámicaRESUMEN
Sensing technologies based on Peptide Nucleic Acids (PNAs) and oligonucleotide-templated chemistry are perfectly suited for biomedical applications (e.g., diagnosis, prognosis and stratification of diseases) and could compete well with more traditional amplification technologies using expensive dual-labelled oligonucleotide probes. PNAs can be easily synthesised and functionalised, are more stable and are more responsive to point-mutations than their DNA counterpart. For these reasons, fluorogenic PNAs represent an interesting alternative to DNA-based molecular beacons for sensing applications in a cell-free environment, where cellular uptake is not required.
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Tecnología Biomédica , Sondas de ADN , Oligonucleótidos/química , Ácidos Nucleicos de Péptidos/química , Sondas de ADN/química , FluorescenciaRESUMEN
Nucleic acid sensors based on a peptide nucleic acid (PNA) probe have seen a surge in interest since their discovery in the 1990s, and after the patent protecting them expired in 2013. The appeal of PNA as capture and/or sensing probes as an alternative to standard DNA or RNA oligonucleotides originates from their superior chemical stability and affinity for complementary oligonucleotides, as well as their increased responsiveness to single base mismatches. The implementation of PNA probes onto optical and electrochemical sensors has showed great promise although progress has been hampered by issues mostly associated with surface chemistry, probe accessibility and non-specific binding. Herein, we report on a systematic comparison between various PNA immobilisation strategies on carbon substrates based on both covalent and non-covalent chemistries. Besides the use of standard electrochemical techniques to characterise the extent of surface modification, the ability of immobilised PNAs to engage in chemical interactions with freely diffusing molecules was also investigated. Using original chemical tags, this study provides a unique insight into the impact of immobilisation chemistries on PNA's (bio)availability. Rapid immobilisation of biotinylated PNA oligomers on screen-printed carbon electrode (SPCE) coated with adsorbed polystreptavidin (pSA) demonstrated highest efficiency and ease in the preparation process. An original nucleic acid sensor using this immobilisation chemistry is reported that is based on a sandwich assay between a surface bound PNA capture probe and a freely diffusing electrochemically active PNA sensing probe.
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Técnicas Biosensibles , Carbono , Técnicas Electroquímicas , Ácidos Nucleicos Inmovilizados , Ácidos Nucleicos de Péptidos , Ácidos Nucleicos de Péptidos/química , Técnicas Biosensibles/métodos , Carbono/química , Ácidos Nucleicos Inmovilizados/química , Técnicas Electroquímicas/métodos , Biomarcadores/análisis , Biomarcadores/química , Humanos , Propiedades de Superficie , Electrodos , Hibridación de Ácido Nucleico , ADN/químicaRESUMEN
Droplet-based microfluidics is a powerful technique allowing ultra-high-throughput screening of large libraries of enzymes or microorganisms for the selection of the most efficient variants. Most applications in droplet microfluidic screening systems use fluorogenic substrates to measure enzymatic activities with fluorescence readout. It is important, however, that there is little or no fluorophore exchange between droplets, a condition not met with most commonly employed substrates. Here we report the synthesis of fluorogenic substrates for glycosidases based on a sulfonated 7-hydroxycoumarin scaffold. We found that the presence of the sulfonate group effectively prevents leakage of the coumarin from droplets, no exchange of the sulfonated coumarins being detected over 24 h at 30 °C. The fluorescence properties of these substrates were characterized over a wide pH range, and their specificity was studied on a panel of relevant glycosidases (cellulases and xylanases) in microtiter plates. Finally, the ß-d-cellobioside-6,8-difluoro-7-hydroxycoumarin-4-methanesulfonate substrate was used to assay cellobiohydrolase activity on model bacterial strains (Escherichia coli and Bacillus subtilis) in a droplet-based microfluidic format. These new substrates can be used to assay glycosidase activities in a wide pH range (4-11) and with incubation times of up to 24 h in droplet-based microfluidic systems.
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Widespread in nature, oligonucleotide-templated reactions of phosphodiester bond formation have inspired chemists who are now applying this elegant strategy to the catalysis of a broad range of otherwise inefficient reactions. This review highlights the increasing diversity of chemical reactions that can be efficiently catalysed by an oligonucleotide template, using Watson-Crick base-pairing to bring both reagents in close enough proximity to react, thus increasing significantly their effective molarity. The applications of this elegant concept for nucleic acid sensing and controlled organic synthesis will also be discussed.
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Oligonucleótidos/química , Emparejamiento Base , Catálisis , Fluorescencia , Estructura Molecular , Ácidos Nucleicos/síntesis química , Ácidos Nucleicos/química , Compuestos Organometálicos/síntesis química , Compuestos Organometálicos/química , Péptidos/síntesis química , Péptidos/química , Moldes GenéticosRESUMEN
Following the recent pandemic and with the emergence of cell-free nucleic acids in liquid biopsies as promising biomarkers for a broad range of pathologies, there is an increasing demand for a new generation of nucleic acid tests, with a particular focus on cost-effective, highly sensitive and specific biosensors. Easily miniaturized electrochemical sensors show the greatest promise and most typically rely on the chemical functionalization of conductive materials or electrodes with sequence-specific hybridization probes made of standard oligonucleotides (DNA or RNA) or synthetic analogues (e.g. Peptide Nucleic Acids or PNAs). The robustness of such sensors is mostly influenced by the ability to control the density and orientation of the probe at the surface of the electrode, making the chemistry used for this immobilization a key parameter. This exhaustive review will cover the various strategies to immobilize nucleic acid probes onto different solid electrode materials. Both physical and chemical immobilization techniques will be presented. Their applicability to specific electrode materials and surfaces will also be discussed as well as strategies for passivation of the electrode surface as a way of preventing electrode fouling and reducing nonspecific binding.
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Técnicas Biosensibles , Ácidos Nucleicos , Ácidos Nucleicos de Péptidos , Técnicas Biosensibles/métodos , ADN/genética , Oligonucleótidos , Electrodos , Técnicas Electroquímicas , Hibridación de Ácido NucleicoRESUMEN
An approach to make chemical diversity space more manageable is to search for smaller molecules, or fragments, and then combine or elaborate these fragments. Dynamic Combinatorial Chemistry (DCC) is a powerful approach whereby a number of molecular elements each with binding potential can be reversibly combined via covalent or noncovalent linkages to generate a dynamic library of products under thermodynamic equilibrium. Once a target molecule has been added, the distribution of products can be shifted to favor products that bind to the target. Thus the approach can be employed to identify products that selectively recognize the target. Although the size of the repertoire of reversible covalent reactions suitable for DCC has increased significantly over the past 5-10 years, the discovery of new reactions that satisfy all the criteria of reversibility and biocompatibility remains an exciting challenge for chemists. Increasing the number of chemical reactions will enable the engineering of larger and more diverse DCLs, which remains a key step toward a broader use of DCC. In this review, we aim to provide a nonexhaustive list of reversible covalent reactions that are compatible with the concept of DCC, focusing mainly on the most recent examples that were reported in the literature in the past 5 years.
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The synthesis of a geometrically constrained and near-planar hexacyclic acridinium cyanine dye 9 is reported. When compared to its unlocked and non-fluorescent monomethine cyanine dye analogue 3, this photostable dye emits in the green area of the spectrum with a remarkable quantum yield close to unity in organic solvents and above 0.5 in water. A detailed steady-state and time-resolved spectroscopic study revealed that dye 9 forms emissive aggregates in water, which are responsible for a red-shifted and broadened emission band and longer emission lifetime, τ≈33 compared to 6.5-7.0 ns for the monomeric dye. Dye 9 also binds strongly to DNA (both duplex and quadruplex) in its monomeric form and is very efficiently taken up by cells, in which it accumulates primarily into the nucleus.
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Acridinas/química , Carbocianinas/química , ADN/química , Colorantes Fluorescentes/química , Solventes/química , Luz , Estructura Molecular , AguaRESUMEN
Correction for 'On-chip miRNA extraction platforms: recent technological advances and implications for next generation point-of-care nucleic acid tests' by Loukia Petrou et al., Lab Chip, 2022, 22, 463-475, https://doi.org/10.1039/D1LC00868D.
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Circulating microRNAs (or miRNAs) in bodily fluids, are increasingly being highlighted as promising diagnostic and predictive biomarkers for a broad range of pathologies. Although nucleic acid sensors have been developed that can detect minute concentrations of biomarkers with high sensitivity and sequence specificity, their robustness is often compromised by sample collection and processing prior to analysis. Such steps either (i) involve complex, multi-step procedures and toxic chemicals unsuitable for incorporation into portable devices or (ii) are inefficient and non-standardised therefore affecting the reliability/reproducibility of the test. The development of point-of-care nucleic acid tests based on the detection of miRNAs is therefore highly dependent on the development of an automated, on-chip, sample processing platform that would enable extraction or pre-purification of the biological specimen prior to reaching the sensing platform. In this review we categorise and critically discuss the most promising technologies that have been developed to facilitate the transition of nucleic acid tests based on miRNA detection from bench to bedside.
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MicroARNs , Ácidos Nucleicos , MicroARNs/análisis , Ácidos Nucleicos/análisis , Análisis de Secuencia por Matrices de Oligonucleótidos , Sistemas de Atención de Punto , Reproducibilidad de los ResultadosRESUMEN
Prostate cancer is the second most common malignancy in men worldwide, and incidence is likely to rise in the next decade. The current screening options have limitations and have been shown to result in over-treatment of clinically insignificant disease. New biomarkers and technologies to detect them are therefore needed to better diagnose and stratify patients in primary care. Circulating cell-free DNA (ccfDNA) has gained interest as a potential minimally invasive biomarker, detectable in many bodily fluids (such as blood, urine, and cerebral spinal fluid) and reflecting the mutational landscape in tumors. More recently, the size distribution of ccfDNA fragments has also gained interest as a specific biomarker, where differences in size distribution have been observed between healthy volunteers and cancer patients, resulting in the new field of fragmentomics. Analysis of ccfDNA sizes provides avenues for alternative analytical technologies but commercial options are currently limited. Most focus on mutation detection and are subject to several biases that may affect size distribution. Here, we discuss the available technologies and identify major issues and considerations that may affect their implementation as a clinically useful test based on ccfDNA size profiling.
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Ácidos Nucleicos Libres de Células , Neoplasias de la Próstata , Biomarcadores , Ácidos Nucleicos Libres de Células/genética , Humanos , Masculino , MutaciónRESUMEN
Fast sampling followed by sequence-independent sensing and length-dependent detection of short double-stranded DNA fragments, the size of those found in blood and other bodily fluids, is achieved using engineered molecular sensors, dubbed hydrogel-filled nanopores (HFNs). Fragments as short as 100 base pairs were blindly sampled and concentrated at the tip of an HFN before reversing the applied potential to detect and distinguish individual molecules based on fragment length as they translocate out of the nanopore. A remarkable 16-fold increase in the signal-to-noise ratio was observed in the eject configuration compared to the load configuration, enabling the resolution of fragments with a size difference of 50 nucleotides in length. This fast and versatile technology offers great tunability for both sampling and detection. While increasing sampling time leads to an increase in the local DNA concentration at the tip prior to detection, a linear correlation between the peak current and DNA fragment size enables good resolution of fragments up to 250 bp long.
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Ácidos Nucleicos Libres de Células/análisis , ADN/análisis , Hidrogeles/química , Nanoporos , Nanotecnología/métodos , Imagen Individual de Molécula/métodos , Ácidos Nucleicos Libres de Células/química , ADN/químicaRESUMEN
Short single-stranded nucleic acids as found in a variety of bodily fluids have recently emerged as minimally invasive biomarkers for a broad range of pathologies, most notably cancer. Because of their small size, low natural abundance and high sequence homology between family members they are challenging to detect using standard technologies suitable for use at the point-of-care. Herein we report the design, engineering and testing of a novel sensing strategy: electrochemically active molecular probes based on peptide nucleic acid (PNA) scaffolds for the detection of single-stranded oligonucleotides, in particular microRNAs (or miRs). As a proof-of-principle, a wide range of probes were designed and tested to detect miR-141, a known diagnostic biomarker for prostate cancer. Optimal quantitative sensing of miR-141 was achieved via the first example of an electrochemical oligonucleotide-templated reaction (EOTR), whereby two PNA probes - functionalized with an aniline and a 1,4-catechol respectively - preferentially react with each other upon simultaneous hybridization to the same RNA target strand, serving here as a template. Quantitative, electrochemical detection of the product of this bio-orthogonal reaction showed direct correlation between adduct formation and miR-141 concentration. Coupling the specificity of OTR with the speed and sensitivity of electrochemical sensing delivers EOTRs as a promising new technique for fast, low-cost, quantitative and sequence-specific detection of short nucleic acids from liquid biopsies.