Detection Of Oligonucleotides Research Articles

High-Throughput Screening for Oligonucleotide Detection by ADE-OPI-MS.

Oligonucleotides represent a class of shorter DNA or RNA nucleic acid polymers extensively applied in the biomedical field. Despite progress in detecting and analyzing oligonucleotides, high-throughput analysis of the samples remains challenging. In this work, a high-throughput analysis method for oligonucleotide analysis was developed based on acoustic droplet ejection-open port interface-mass spectrometry (ADE-OPI-MS) technology. This approach was applied to determine the enzymatic activity of terminal deoxynucleotide transferase (TdT) for DNA synthesis, with a rate of 3 s/sample, which enhanced single-sample analysis efficiency approximately 60-fold over the previous gel analysis. After testing approximately 10,000 TdT mutants, we obtained three new variants with higher catalytic activities. Finally, by integrating these mutants, the catalytic activity of TdT was improved about 4 times compared to the starting mutant. Our results successfully established a high-throughput screening method for oligonucleotide analysis, which not only provides a foundation to engineer highly efficient TdT for ab initio synthesis of DNA but also paves the way for the potential application of oligonucleotide analysis in biomedical fields.

Non-Targeted Detection of Synthetic Oligonucleotides in Equine Serum Using Liquid Chromatography-High-Resolution Mass Spectrometry.

There is great concern in equine sport over the potential use of pharmaceutical agents capable of editing the genome or modifying the expression of gene products. Synthetic oligonucleotides are short, single-stranded polynucleotides that represent a class of agents capable of modifying gene expression products with a high potential for abuse in horseracing. As these substances are not covered by most routine anti-doping analytical approaches, they represent an entire class of compounds that are not readily detectable. The nucleotide sequence for each oligonucleotide is highly specific, which makes targeted analysis for these agents problematic. Accordingly, we have developed a non-targeted approach to detect the presence of specific product ions that are not naturally present in ribonucleic acids. Briefly, serum samples were extracted using solid-phase extraction with a mixed-mode cartridge following the disruption of protein interactions to isolate the oligonucleotides. Following the elution and concentration steps, chromatographic separation was achieved utilizing reversed-phase liquid chromatography. Following an introduction to a Thermo Q Exactive HF mass spectrometer using electrospray ionization, analytes were detected utilizing a combination of full-scan, parallel reaction monitoring and all ion fragmentation scan modes. The limits of detection were determined along with the accuracy, precision, stability, recovery, and matrix effects using a representative 13mer oligonucleotide. Following method optimization using the 13mer oligonucleotide, the method was applied to successfully detect the presence of specific product ions in three unique oligonucleotide sequences targeting equine-specific transcripts.

Critical Issues on the Surface Functionalization of Plasmonic Au-Ag/TiO2 Thin Films with Thiolated Oligonucleotide-Based Biorecognition Elements.

This work reports on the surface functionalization of a nanomaterial supporting localized surface plasmon resonances (LSPRs) with (synthetic) thiolated oligonucleotide-based biorecognition elements, envisaging the development of selective LSPR-based DNA biosensors. The LSPR thin-film transducers are composed of noble metal nanoparticles (NPs) embedded in a TiO2 dielectric matrix, produced cost-effectively and sustainably by magnetron sputtering. The study focused on the immobilization kinetics of thiolated oligonucleotide probes as biorecognition elements, followed by the evaluation of hybridization events with the target probe. The interaction between the thiolated oligonucleotide probe and the transducer's surface was assessed by monitoring the LSPR signal with successive additions of probe solution through a microfluidic device. The device was specifically designed and fabricated for this work and adapted to a high-resolution LSPR spectroscopy system with portable characteristics. Benefiting from the synergetic characteristics of Ag and Au in the form of bimetallic nanoparticles, the Au-Ag/TiO2 thin film proved to be more sensitive to thiolated oligonucleotide binding events. Despite the successful surface functionalization with the biorecognition element, the detection of complementary oligonucleotides revealed electrostatic repulsion and steric hindrance, which hindered hybridization with the target oligonucleotide. This study points to an effect that is still poorly described in the literature and affects the design of LSPR biosensors based on nanoplasmonic thin films.

Sub-Nanomolar Detection of Oligonucleotides Using Molecular Beacons Immobilized on Lightguiding Nanowires.

The detection of oligonucleotides is a central step in many biomedical investigations. The most commonly used methods for detecting oligonucleotides often require concentration and amplification before detection. Therefore, developing detection methods with a direct read-out would be beneficial. Although commonly used for the detection of amplified oligonucleotides, fluorescent molecular beacons have been proposed for such direct detection. However, the reported limits of detection using molecular beacons are relatively high, ranging from 100 nM to a few µM, primarily limited by the beacon fluorescence background. In this study, we enhanced the relative signal contrast between hybridized and non-hybridized states of the beacons by immobilizing them on lightguiding nanowires. Upon hybridization to a complementary oligonucleotide, the fluorescence from the surface-bound beacon becomes coupled in the lightguiding nanowire core and is re-emitted at the nanowire tip in a narrower cone of light compared with the standard 4π emission. Prior knowledge of the nanowire positions allows for the continuous monitoring of fluorescence signals from each nanowire, which effectively facilitates the discrimination of signals arising from hybridization events against background signals. This resulted in improved signal-to-background and signal-to-noise ratios, which allowed for the direct detection of oligonucleotides at a concentration as low as 0.1 nM.

Photonic Crystal Surface Mode Real-Time Imaging of RAD51 DNA Repair Protein Interaction with the ssDNA Substrate.

Photonic crystals (PCs) are promising tools for label-free sensing in drug discovery screening, diagnostics, and analysis of ligand-receptor interactions. Imaging of PC surface modes has emerged as a novel approach to the detection of multiple binding events at the sensor surface. PC surface modification and decoration with recognition units yield an interface providing the highly sensitive detection of cancer biomarkers, antibodies, and oligonucleotides. The RAD51 protein plays a central role in DNA repair via the homologous recombination pathway. This recombinase is essential for the genome stability and its overexpression is often correlated with aggressive cancer. RAD51 is therefore a potential target in the therapeutic strategy for cancer. Here, we report the designing of a PC-based array sensor for real-time monitoring of oligonucleotide-RAD51 recruitment by means of surface mode imaging and validation of the concept of this approach. Our data demonstrate that the designed biosensor ensures the highly sensitive multiplexed analysis of association-dissociation events and detection of the biomarker of DNA damage using a microfluidic PC array. The obtained results highlight the potential of the developed technique for testing the functionality of candidate drugs, discovering new molecular targets and drug entities. This paves the way to further adaption and bioanalytical use of the biosensor for high-content screening to identify new DNA repair inhibitor drugs targeting the RAD51 nucleoprotein filament or to discover new molecular targets.

Self-Aligning Nanojunctions for Integrated Single-Molecule Circuits.

Robust, high-yield integration of nanoscale components such as graphene nanoribbons, nanoparticles, or single-molecules with conventional electronic circuits has proven to be challenging. This difficulty arises because the contacts to these nanoscale devices must be precisely fabricated with angstrom-level resolution to make reliable connections, and at manufacturing scales this cannot be achieved with even the highest-resolution lithographic tools. Here we introduce an approach that circumvents this issue by precisely creating nanometer-scale gaps between metallic carbon electrodes by using a self-aligning, solution-phase process, which allows facile integration with conventional electronic systems with yields approaching 50%. The electrode separation is controlled by covalently binding metallic single-walled carbon nanotube (mCNT) electrodes to individual DNA duplexes to create mCNT-DNA-mCNT nanojunctions, where the gap is precisely matched to the DNA length. These junctions are then integrated with top-down lithographic techniques to create single-molecule circuits that have electronic properties dominated by the DNA in the junction, have reproducible conductance values with low dispersion, and are stable and robust enough to be utilized as active, high-specificity electronic biosensors for dynamic single-molecule detection of specific oligonucleotides, such as those related to the SARS-CoV-2 genome. This scalable approach for high-yield integration of nanometer-scale devices will enable opportunities for manufacturing of hybrid electronic systems for a wide range of applications.

Attomolar-level detection of respiratory virus long-chain oligonucleotides based on FRET biosensor with upconversion nanoparticles and Au–Au dimer

Upconversion nanoparticles (UCNPs) are promising nanoprobes in DNA/RNA detection, such as respiratory viral RNAs, and siRNA in cancer. However, recent studies have indicated that the sensitivity of UCNP-based biosensors is restricted, ranging from picomolar to femtomolar level. Moreover, most of existing UCNP-based probes are only able to detect short-chain oligonucleotides, which are not suitable for detection of long-chain oligonucleotides in many real applications. In this work, we introduced a new UCNP-based fluorescence resonance energy transfer (FRET) nanoprobe design composed of NaGdF4:Yb3+, Er3+@NaGdF4 core-shell UCNPs (csUCNPs) linking with Au–Au dimer to detect long-chain oligonucleotides of SARS-CoV-2 N-gene. Compared with typical single gold nanoparticles (AuNPs) in FRET biosensors, our theoretical investigation shows that a stronger electromagnetic field is generated in the Au–Au dimer where the plasmon resonance can enhance FRET efficiency and increase the working distance. Thus, the synergetic effect of plasmonic resonance and FRET enables a greater quenching efficiency (QE) of Au–Au dimer to UCNPs, which leads to more remarkable upconversion luminescence (UCL) recovery for each target gene recognition. Importantly, our design significantly improved the limit of detection (LOD) to attomolar level, with a linear response ranging from 2 aM to 2 fM. Moreover, the clinical detection with inactivated SARS-CoV-2 samples was successfully performed with excellent specificity within 30 min using the developed UCNPs biosensors incorporated with Au–Au dimer. This UCNP biosensor based on Au–Au dimer strategy with ultra-sensitivity and good selectivity opens a new path for clinical diagnosis without target amplification and plays an instructive role in other virus diagnosis.

Construction of an autocatalysis-driven DNA circuit for highly efficient detection of H5N1 oligonucleotide

H5N1 is a highly pathogenic avian influenza virus (AIV) that is a significant risk to both poultry production and human health. The sensitive and reliable determination of H5N1 is thus highly desirable for early antiviral therapy and controlling the outbreak. Isothermal DNA circuits have afforded a promising tool for molecular sensing in complex biological environments, yet are always constrained by their low amplification depth and insufficient signal gain. Herein, a simple-yet-robust autocatalytic DNA circuit (ADC) was devised for highly efficient H5N1 DNA analysis through the synergistic catalysis of catalytic hairpin assembly (CHA) and rolling circle amplification (RCA). The trigger-activated RCA circuit can generate numerous repeated initiators for stimulating the CHA transducer. Meanwhile, the initiator-stimulated CHA circuit can produce the re-assembled RCA trigger to reversely motivate the RCA system. The synergistic cross-activation between the RCA and CHA amplicon can achieve progressive reaction acceleration and exponentially amplified signal gain. The ADC system is highly versatile and capable of reliably detecting H5N1 DNA in buffer and biological samples with high performance, highlighting its great promise for low-abundance biomolecule sensing and early disease diagnosis.

Photonic Crystal Surface Mode Imaging for Multiplexed Real-Time Detection of Antibodies, Oligonucleotides, and DNA Repair Proteins

Sensors based on photonic crystal (PC) surface mode imaging are promising tools for label-free drug screening and discovery, diagnostics, and analysis of ligand–receptor interactions. Imaging of PC surface modes has been demonstrated to allow simultaneous real-time detection of multiple events at the sensor surface. Here, we report the engineering of a lateral-flow microfluidic assay where PC surface mode imaging is used for multiplexed detection of biomolecular targets (antibodies, oligonucleotides, and a DNA repair protein), as well as kinetic data on their interactions obtained without additional labelling or signal amplification. Our data demonstrate the suitability of the biosensing platform designed for ultrasensitive, quick, and low-cost detection and monitoring of interactions between different biomolecules.

Anomalous DNA hybridisation kinetics on gold nanorods revealed via a dual single-molecule imaging and optoplasmonic sensing platform.

Observing the hybridisation kinetics of DNA probes immobilised on plasmonic nanoparticles is key in plasmon-enhanced fluorescence detection of weak emitting species, and refractive index based single-molecule detection on optoplasmonic sensors. The role of the local field in providing plasmonic signal enhancements for single-molecule detection has been studied in great detail. Nevertheless, few studies have compared the experimental results in both techniques for single-molecule studies. Here we developed the first optical setup that integrates optoplasmonic and DNA-PAINT based detection of oligonucleotides to compare these sub-platforms and provide complementary insights into single molecule processes. We record the fluorescence and optoplasmonic sensor signals for individual, transient hybridisation events. The hybridisation events are observed in the same sample cell and over a prolonged time (i.e. towards high binding site occupancies). A decrease in the association rate over the measurement duration is reported. Our dual optoplasmonic sensing and imaging platform offers insight into the observed phenomenon, revealing that irreversible hybridisation events accumulate over detected step signals in optoplasmonic sensing. Our results point to novel physicochemical mechanisms that result in the stabilisation of DNA hybridisation on optically-excited plasmonic nanoparticles.

MicroRNA Detection at Femtomolar Concentrations with Isothermal Amplification and a Biological Nanopore.

Nanopore sensing is a powerful tool for the rapid and label-free detection of oligonucleotides, including microRNA. When moving towards actual diagnostic applications, detection of microRNA at low concentrations is one of the significant issues to be addressed. We here describe a method to detect ultra-low concentrations of microRNA using isothermal amplification and nanopore technology. Using this method, the amplified DNA from 1 fM of target microRNA can be measured by a nanopore measurement.

"Silicon-On-Insulator"-Based Biosensor for the Detection of MicroRNA Markers of Ovarian Cancer.

Ovarian cancer is a gynecological cancer characterized by a high mortality rate and tumor heterogeneity. Its early detection and primary prophylaxis are difficult to perform. Detecting biomarkers for ovarian cancer plays a pivotal role in therapy effectiveness and affects patients' survival. This study demonstrates the detection of microRNAs (miRNAs), which were reported to be associated with ovarian cancer tumorigenesis, with a nanowire biosensor based on silicon-on-insulator structures (SOI-NW biosensor). The advantages of the method proposed for miRNA detection using the SOI-NW biosensor are as follows: (1) no need for additional labeling or amplification reaction during sample preparation, and (2) real-time detection of target biomolecules. The detecting component of the biosensor is a chip with an array of 3 µm wide, 10 µm long silicon nanowires on its surface. The SOI-NW chip was fabricated using the "top-down" method, which is compatible with large-scale CMOS technology. Oligonucleotide probes (oDNA probes) carrying sequences complementary to the target miRNAs were covalently immobilized on the nanowire surface to ensure high-sensitivity biospecific sensing of the target biomolecules. The study involved two experimental series. Detection of model DNA oligonucleotides being synthetic analogs of the target miRNAs was carried out to assess the method's sensitivity. The lowest concentration of the target oligonucleotides detectable in buffer solution was 1.1 × 10-16 M. In the second experimental series, detection of miRNAs (miRNA-21, miRNA-141, and miRNA-200a) isolated from blood plasma samples collected from patients having a verified diagnosis of ovarian cancer was performed. The results of our present study represent a step towards the development of novel highly sensitive diagnostic systems for the early revelation of ovarian cancer in women.

Lifetime of glass nanopores in a PDMS chip for single-molecule sensing

SummaryNanopore sensing is an emerging technology that has many biosensing applications ranging from DNA sequencing using biological pores to biomolecular analysis using solid-state pores. Solid-state nanopores that are more stable are an attractive choice for biosensing applications. Still, biomolecule interactions with the nanopore surface reduce nanopore stability and increase usage costs. In this study, we investigated the biosensing capability for 102 quartz glass nanopores with a diameter of 11–18 nm that were fabricated using laser-assisted capillary pulling. Nanopores were assembled into multiple microfluidic chips that were repeatedly used for up to 19 weeks. We find that using vacuum storage combined with minimal washing steps improved the number of use cycles for nanopores. The single-molecule biosensing capability over repeated use cycles was demonstrated by quantitative analysis of a DNA carrier designed for detection of short single-stranded DNA oligonucleotides.

Sensitive Oligonucleotide Detection Using Resonant Coupling between Fano Resonance and Image Dipoles of Gold Nanoparticles.

The surface plasmon resonance (SPR)-based sensor has been widely used for biodetection. One of the attractive roles is the gold nanostructure with Fano resonance. Its sharp resonant profile takes advantage of the high figure of merit (FoM) in high-sensitivity detection. However, it is still difficult to detect small molecules at low concentrations due to the extremely low refractive index changes on the metallic surface. We propose using the coupling of image dipoles of gold nanoparticles (AuNPs) and Fano resonance of periodic capped gold nanoslits (CGNs) for sensitive small-molecule detections. The coupling mechanism was verified by three-dimensional finite-difference time-domain calculations and experiments. AuNPs on CGN form image dimer assemblies and induce image dipole with resonance wavelengths ranging from 730 to 550 nm. The surface plasmon polaritons (SPPs) interact with the image dipole of the AuNP on the CGNs and then scatter out through the periodic gold caps. The experimental results show that the peak intensity of grating resonance is decreased by the effect of image dipole and exhibits the maximum intensity change when the Fano resonance matches the resonance of image dipole. The 50 nm AuNPs can be detected with a surface density of less than one particle/μm2 by using the intensity change as the signal. With the resonant coupling between Fano resonance and image dipole extinction, the oligonucleotide with a molecular weight of 5.5 kDa can be detected at a concentration of 100 fM. The resonant coupling dramatically pushes the sensitivity boundary, and we report the limit of detection (LOD) to be 3 orders of magnitude lower than that of the prism-based SPR. This study provides a promising and efficient method for detecting low concentrations of small molecules such as aptamers, miRNA, mRNA, and peptides.

Sensitive Quantification of MicroRNA in Blood through Multi‐amplification Toehold‐Mediated DNA‐Strand‐Displacement Paper‐Spray Mass Spectrometry (TSD‐PS MS)

AbstractAccurate quantification of disease‐signature microRNAs (miRNAs) in biomedical samples is in high demand for clinical diagnosis but still challenging because of the low abundance of miRNAs and complicating interferences in the milieus. Here, we report a multi‐amplification strategy based on paper‐spray mass spectrometry (PS MS) for the analysis of miRNAs in blood. A toehold‐mediated DNA‐strand‐displacement reaction (TSD) is employed to amplify the signal chain and to ensure the specificity. The signal chain is then cleaved by UV light to release signal molecules for detection. Moreover, the paper spray method can efficiently filter out interfering substances in the blood and further enhance the sensitivity of detection. This concept is successfully demonstrated in the prototypical detection of the cancer biomarker miRNA‐141 in blood and serum. The proposed TSD‐PS MS approach provides an efficient method for the sensitive detection of oligonucleotides at low concentration in complicated milieus.

Sensitive Quantification of MicroRNA in Blood through Multi-amplification Toehold-Mediated DNA-Strand-Displacement Paper-Spray Mass Spectrometry (TSD-PS MS).

Accurate quantification of disease-signature microRNAs (miRNAs) in biomedical samples is in high demand for clinical diagnosis but still challenging because of the low abundance of miRNAs and complicating interferences in the milieus. Here, we report a multi-amplification strategy based on paper-spray mass spectrometry (PS MS) for the analysis of miRNAs in blood. A toehold-mediated DNA-strand-displacement reaction (TSD) is employed to amplify the signal chain and to ensure the specificity. The signal chain is then cleaved by UV light to release signal molecules for detection. Moreover, the paper spray method can efficiently filter out interfering substances in the blood and further enhance the sensitivity of detection. This concept is successfully demonstrated in the prototypical detection of the cancer biomarker miRNA-141 in blood and serum. The proposed TSD-PS MS approach provides an efficient method for the sensitive detection of oligonucleotides at low concentration in complicated milieus.

SERS and advanced chemometrics – Utilization of Siamese neural network for picomolar identification of beta-lactam antibiotics resistance gene fragment

The enormous development and expansion of antibiotic-resistant bacterial strains impel the intensive search for new methods for fast and reliable detection of antibiotic susceptibility markers. Here, we combined DNA-targeted surface functionalization, surface-enhanced Raman spectroscopy (SERS) measurements, and subsequent spectra processing by decision system (DS) for detection of a specific oligonucleotide (ODN) sequence identical to a fragment of blaNDM-1 gene, responsible for β-lactam antibiotic resistance. The SERS signal was measured on plasmonic gold grating, functionalized with capture ODN, ensuring the binding of corresponded ODNs. Designed DS consists of a Siamese neural network (SNN) coupled with robust statistics and Bayes decision theory. The proposed approach allows manipulation with complex multicomponent samples and predefine the desired detection level of confidence and errors, automatically determining the number of required spectra and samples. In constant to commonly used classification-type SNN, our method was applied to analyze samples with compositions previously “unknown” to DS. The detection of targeted ODN was performed with ≥99% level of confidence up to 3 × 10−12 M limit on the background of 10−10 M concentration of similar but not targeted ODNs.

Quantification of Antisense Oligonucleotides by Splint Ligation and Quantitative Polymerase Chain Reaction.

Reliable detection and quantification of antisense oligonucleotides (ASOs) in experimental and clinical specimens are essential to understand the biological function of novel oligonucleotide-based therapeutics. In this study, we describe a method to detect and quantify ASOs in biological samples, whereby the ASO acts as a splint to direct the ligation of complementary probes and quantitative real-time PCR was used to monitor ligation products. Low levels of 2′-O-methoxyethyl (2′-O-MOE) gapmer ASO in serum, liver, kidney, lung, heart, muscle, and brain tissues can be detected over a 6-log linear range for detection using this method. This method allows quantification of various types of chemically modified ASOs, including phosphorothioate linkage, 2′-O-methyl, 2′-O-MOE, and locked nucleic acid, as well as siRNAs. This method does not require probe modifications, and can be performed using standard laboratory equipment; making it a fast, sensitive, and reliable technique that can be widely applied. This detection method may find potential applications in detection of therapeutic oligonucleotides in biological samples.

Nanoparticle-Assisted Nuclear Relaxation-Based Detection of Oligonucleotides.

Nuclear relaxation-based techniques rely on the capability of paramagnetic center to affect the transverse relaxation time (T2) of the water protons. These changes are sensitive to the microenvironment of the paramagnetic center, which can be used to detect a variety of targets. In this work, we present an "on-off" oligonucleotide detection scheme in aqueous solutions, which uses gadolinium phthalocyanine (GdTcPc)-grafted silica nanoparticles as the paramagnetic centers. A probe oligonucleotide strand was conjugated to the GdTcPc to act as a recognition element. In the presence of the target oligonucleotide, which was complementary to the probe, an increase in the ΔT2 value, which was measured by subtracting the characteristic T2 value of the background solution from that of the sample using a benchtop relaxometer, was observed. The magnitude of this increase was proportional to the target oligonucleotide concentration. A linear range was obtained from 30 to 140nM, with a detection limit of 15nM. The developed nuclear relaxation-based detection scheme is shown to be a simple, fast, and selective method to detect oligonucleotide and could be useful in point-of-care diagnostic applications.

Hydrogel Microparticles for Fluorescence Detection of miRNA in Mix-Read Bioassay.

Herein we describe the development of a mix-read bioassay based on a three-dimensional (3D) poly ethylene glycol—(PEG)-hydrogel microparticles for the detection of oligonucleotides in complex media. The key steps of hydrogels synthesis and molecular recognition in a 3D polymer network are elucidated. The design of the DNA probes and their density in polymer network were opportunely optimized. Furthermore, the diffusion into the polymer was tuned adjusting the polymer concentration and consequently the characteristic mesh size. Upon parameters optimization, 3D-PEG-hydrogels were synthetized in a microfluidic system and provided with fluorescent probe. Target detection occurred by double strand displacement assay associated to fluorescence depletion within the hydrogel microparticle. Proposed 3D-PEG-hydrogel microparticles were designed for miR-143-3p detection. Results showed 3D-hydrogel microparticles with working range comprise between 10−6–10−12 M, had limit of detection of 30 pM and good specificity. Moreover, due to the anti-fouling properties of PEG-hydrogel, the target detection occurred in human serum with performance comparable to that in buffer. Due to the approach versatility, such design could be easily adapted to other short oligonucleotides detection.