Surface immobilization strategies for the development of electrochemical nucleic acid sensors.

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.

Correction: On-chip miRNA extraction platforms: recent technological advances and implications for next generation point-of-care nucleic acid tests.

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.

Technologies for Size-Based Analysis of Circulating Cell-Free DNA: Limitations and Clinical Implementation.

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.

On-chip miRNA extraction platforms: recent technological advances and implications for next generation point-of-care nucleic acid tests.

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.

Single-molecule amplification-free multiplexed detection of circulating microRNA cancer biomarkers from serum.

MicroRNAs (miRNAs) play essential roles in post-transcriptional gene expression and are also found freely circulating in bodily fluids such as blood. Dysregulated miRNA signatures have been associated with many diseases including cancer, and miRNA profiling from liquid biopsies offers a promising strategy for cancer diagnosis, prognosis and monitoring. Here, we develop size-encoded molecular probes that can be used for simultaneous electro-optical nanopore sensing of miRNAs, allowing for ultrasensitive, sequence-specific and multiplexed detection directly in unprocessed human serum, in sample volumes as small as 0.1 µl. We show that this approach allows for femtomolar sensitivity and single-base mismatch selectivity. We demonstrate the ability to simultaneously monitor miRNAs (miR-141-3p and miR-375-3p) from prostate cancer patients with active disease and in remission. This technology can pave the way for next generation of minimally invasive diagnostic and companion diagnostic tests for cancer.

Length-Dependent, Single-Molecule Analysis of Short Double-Stranded DNA Fragments through Hydrogel-Filled Nanopores: A Potential Tool for Size Profiling Cell-Free DNA.

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.

Nucleic acid sensing via electrochemical oligonucleotide-templated reactions.

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.

Correction: Oligonucleotide-templated lateral flow assays for amplification-free sensing of circulating microRNAs.

Correction for 'Oligonucleotide-templated lateral flow assays for amplification-free sensing of circulating microRNAs' by Suraj Pavagada et al., Chem. Commun., 2019, 55, 12451-12454.

Oligonucleotide-templated lateral flow assays for amplification-free sensing of circulating microRNAs.

Herein we demonstrate the first example of oligonucleotide-templated reaction (OTR) performed on paper, using lateral flow to capture and concentrate specific nucleic acid biomarkers on a test line. Quantitative analysis, using a low-cost benchtop fluorescence reader showed very high specificity down to the single nucleotide level and proved sensitive enough for amplification-free, on-chip, detection of endogenous concentrations of miR-150-5p, a recently identified predictive blood biomarker for preterm birth.

Hydrogel-Coated Microneedle Arrays for Minimally Invasive Sampling and Sensing of Specific Circulating Nucleic Acids from Skin Interstitial Fluid.

Minimally invasive technologies that can sample and detect cell-free nucleic acid biomarkers from liquid biopsies have recently emerged as clinically useful for early diagnosis of a broad range of pathologies, including cancer. Although blood has so far been the most commonly interrogated bodily fluid, skin interstitial fluid has been mostly overlooked despite containing the same broad variety of molecular biomarkers originating from cells and surrounding blood capillaries. Emerging technologies to sample this fluid in a pain-free and minimally-invasive manner often take the form of microneedle patches. Herein, we developed microneedles that are coated with an alginate-peptide nucleic acid hybrid material for sequence-specific sampling, isolation, and detection of nucleic acid biomarkers from skin interstitial fluid. Characterized by fast sampling kinetics and large sampling capacity (∼6.5 µL in 2 min), this platform technology also enables the detection of specific nucleic acid biomarkers either on the patch itself or in solution after light-triggered release from the hydrogel. Considering the emergence of cell-free nucleic acids in bodily fluids as clinically informative biomarkers, platform technologies that can detect them in an automated and minimally invasive fashion have great potential for personalized diagnosis and longitudinal monitoring of patient-specific disease progression.

Platforms for Bioorthogonal Oligonucleotide-templated Reactions: Translating Concepts into Devices.

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.

Molecular methods in electrochemical microRNA detection.

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.

Chemically Modified Hydrogel-Filled Nanopores: A Tunable Platform for Single-Molecule Sensing.

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.

Single Molecule Trapping and Sensing Using Dual Nanopores Separated by a Zeptoliter Nanobridge.

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.

Subnanomolar Detection of Oligonucleotides through Templated Fluorogenic Reaction in Hydrogels: Controlling Diffusion to Improve Sensitivity.

Oligonucleotide-templated reactions are valuable tools for nucleic acid sensing both in vitro and in 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 100 pm.

Amplification-Free Detection of Circulating microRNA Biomarkers from Body Fluids Based on Fluorogenic Oligonucleotide-Templated Reaction between Engineered Peptide Nucleic Acid Probes: Application to Prostate Cancer Diagnosis.

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.

Red/NIR G-Quadruplex Sensing, Harvesting Blue Light by a Coumarin-Naphthalene Diimide Dyad.

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 498 nm when free in solution and an additional red/NIR emission when bound to G-quadruplex DNA. The light-up response centred at 666 nm is highly specific for quadruplex DNA when compared to duplex DNA or to RNA quadruplexes.

Oligonucleotide-templated reactions based on Peptide Nucleic Acid (PNA) probes: concept and biomedical applications.

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.

Enhanced chemical synthesis at soft interfaces: a universal reaction-adsorption mechanism in microcompartments.

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.

Synthesis of squaraine dyes under mild conditions: applications for labelling and sensing of biomolecules.

We report the synthesis of squaraine dyes under mild conditions by carbodiimide activation of squaric acid or semi-squaraine dyes. Despite low yields when the reaction was carried out in solution, these conditions were successfully applied to efficient peptide labelling on resin and nucleic acid sensing in solution.