Detection Of Oligonucleotides Research Articles

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.

Development of a microfluidic biosensor for the diagnostics and typing of <i>Mycobacterium Tuberculosis</i>

Background. Despite on the general trend towards decreasing the incidence of newly diagnosed active forms of tuberculosis, the situation with spreading of this disease in Russian Federation remains extremely tense. At the same time, the diagnosis is carried out according to the standard scheme, which takes about a month; another month takes test formulation for drug sensitivity. Thus, the development of new methods for diagnostics and typing of mycobacteria, as well as practice implementation of these developments is an urgent direction. Modern developments in the field of microfluidic technologies open up great opportunities in this direction. Aim. Development of a method for identification and typing of Mycobacterium tuberculosis using a label-free biosensor on surface waves in a one-dimensional photonic crystal (PC SM biosensor). Methods. Oligonucleotide probes were selected and synthesized as DNA targets for M. tuberculosis typing. The photonic crystal surface was modified with aqueous solutions of (3-aminopropyl)triethoxysilane, Leuconostoc mesenteroides dextrans and bovine serum albumin. Experiments were carried out using a PC SM biosensor. Results. Sequences of detecting oligonucleotide probes were selected for spoligotyping of M. tuberculosis on the PC SM biosensor. Modification of their 3'-ends was carried out in order to create extended single-stranded regions that are not subject to the formation of secondary structures and facilitate hybridization with a single-stranded DNA target. Several series of experimental modifications of the PC surface were carried out by using L. mesenteroides dextrans with different functional groups (including detection of the modification results real time) with simultaneous registration of the increment layer size and volume refractive index of the mixture, which excludes the use of a reference cell. Other experiments were carried out to detect the specific binding of biotinylated oligonucleotide probes to the modified PC surface. Conclusions. A technique for the design of probes was developed and a model system of oligonucleotides for the detection of single-stranded DNA using a PC biosensor was proposed. The developed technique of modification of the PC surface with dextrans from L. mesenteroides, which allows to increase the sensitivity of detection of oligonucleotides using the PC SM biosensor. This approach will further expand the panel of diagnostic probes, including identification of resistance markers.

Small Oligonucleotides Detection in Three-Dimensional Polymer Network of DNA-PEG Hydrogels.

The control of the three-dimensional (3D) polymer network structure is important for permselective materials when specific biomolecule detection is needed. Here we investigate conditions to obtain a tailored hydrogel network that combines both molecular filtering and molecular capture capabilities for biosensing applications. Along this line, short oligonucleotide detection in a displacement assay is set within PEGDA hydrogels synthetized by UV radical photopolymerization. To provide insights on the molecular filter capability, diffusion studies of several probes (sulforhodamine G and dextrans) with different hydrodynamic radii were carried out using NMR technique. Moreover, fluorometric analyses of hybridization of DNA oligonucleotides inside PEGDA hydrogels shed light on the mechanisms of recognition in 3D, highlighting that mesh size and crowding effect greatly impact the hybridization mechanism on a polymer network. Finally, we found the best probe density and diffusion transport conditions to allow the specific oligonucleotide capture and detection inside PEGDA hydrogels for oligonucleotide detection and the filtering out of higher molecular weight molecules.

A strip of lateral flow gene assay using gold nanoparticles for point-of-care diagnosis of African swine fever virus in limited environment.

Recombinase polymerase amplification (RPA) was combined with lateral flow to develop a gold nanoparticles test strip for point-of-care diagnosis of African swine fever virus (ASFV), which is called lateral flow gene assay (LFGA). Common diagnostic techniques, including polymerase chain reaction (PCR) and immunochromatography, are time-consuming and labor-intensive, and generally require costly instruments. For improvement, this assay used tailed primers to produce DNA duplexes with a single-stranded tail at one end which can hybridize with a gold nanoparticle (AuNP)-labeled oligonucleotide detection probe. And then, biotin attached to the other end of the product bound to streptavidin, which previously fixed to the test line. Therefore, there would form a sandwich structure, and gold nanoparticles labeled on the detection probe would show a red band on the test line of strip. With the low reaction temperature (37~42°C) and short reaction time (30min), LFGA can specifically identify ASFV in blood samples infected with ASFV and classical swine fever virus (CSFV), and the LOD was 102 copies/μL, which was comparable to that of agarose gel electrophoresis. In addition, blood samples infected with ASFV and CSFV were tested, and it was found that the LFGA can specifically identify ASFV DNA. In conclusion, LFGA achieves visual observation of the product after rapid RPA amplification and does not require any expensive instruments during the entire process, which is very helpful for early diagnosis of ASFV. Combined recombinase polymerase amplification (RPA) with lateral flow, we developed a gold nanoparticles test strip for point-of-care diagnosis of African swine fever virus. The upstream primers of RPA were modified with biotin, and the downstream primers were modified with a C3 spacer and an oligonucleotide tail that can be hybridized to a gold nanoparticle-labeled oligonucleotide detection probe. On the strip, the test line and control line were sprayed with streptavidin and an oligonucleotide control probe. In the presence of positive products, RPA products can form a sandwich structure on the test line. Therefore, two red lines will be displayed both on the test line and control line. When there is no positive product, only the control line is shown in red. Its low reaction temperature (37~42°C) and short time of amplification and detection (30min) make ASFV realizing point-of-care diagnosis in limited environment.

Advanced electrochemical detection of nitrogenous bases, synthetic oligonucleotides, and single-stranded DNA through flow injection analysis and catalytic oxidation on Prussian Blue

The effect of Prussian Blue (PB, ferric hexacyanoferrate) on oxidation of free nucleobases, synthetic oligonucleotides, single- and double-stranded DNA (ssDNA and dsDNA) was evaluated by cyclic voltammetry (CV) and flow injection analysis (FIA) on carbon screen printed electrodes, both bare (SPE) and PB modified (SPE/PB). It has been found that electrocatalytic oxidation of nucleobases, namely guanine, adenine, thymine, and cytosine, or nucleobase residues takes place via electrochemically generated Berlin Green (BG), fully oxidized form of PB. The constant potential FIA allowed one to register electrooxidation of all DNA nitrogenous bases at 0.95 V and three nitrogenous bases (except for cytosine) at 0.70 V on SPE and SPE/PB. The modification of electrode surface with PB resulted in a shift of oxidation potential to less positive values and enhanced (up to 8 times) detection sensitivity for DNA nucleobases. The pronounced catalytic effect of electrogenerated BG on their oxidation has also been observed for synthetic oligonucleotides and degraded ssDNA of natural origin. By fractionating ssDNA, it has been found that the main contributor to the total oxidation signal is the fraction of lowest molecular weight (< 3 kDa). At the same time, no oxidation signal was observed for large molecular weight native dsDNA in the potential range from 0 to 1 V on bare SPE and SPE/PB in both CV and FIA measurement modes. Therefore, the advanced electrochemical detection by FIA with SPE/PB was achieved for all nucleobases, synthetic oligonucleotides, and ssDNA of natural origin. Moreover, the obtained results expand the contemporary understanding of DNA electrochemistry.

Raman Spectroscopy-Based Quality Control of "Silicon-On-Insulator" Nanowire Chips for the Detection of Brain Cancer-Associated MicroRNA in Plasma.

Application of micro-Raman spectroscopy for the monitoring of quality of nanowire sensor chips fabrication has been demonstrated. Nanowire chips have been fabricated on the basis of «silicon-on-insulator» (SOI) structures (SOI-NW chips). The fabrication of SOI-NW chips was performed by optical litography with gas-phase etching. The so-fabricated SOI-NW chips are intended for highly sensitive detection of brain cancer biomarkers in humans. In our present study, two series of experiments have been conducted. In the first experimental series, detection of a synthetic DNA oligonucleotide (oDNA) analogue of brain cancer-associated microRNA miRNA-363 in purified buffer solution has been performed in order to demonstrate the high detection sensitivity. The second experimental series has been performed in order to reveal miRNA-363 itself in real human plasma samples. To provide detection biospecificity, the SOI-NW chip surface was modified by covalent immobilization of probe oligonucleotides (oDNA probes) complementary to the target biomolecules. Using the SOI-NW sensor chips proposed herein, the concentration detection limit of the target biomolecules at the level of 3.3 × 10−17 M has been demonstrated. Thus, the approach employing the SOI-NW chips proposed herein represents an attractive tool in biomedical practice, aimed at the early revelation of oncological diseases in humans.

Optical Monitoring of the Production Quality of Si-Nanoribbon Chips Intended for the Detection of ASD-Associated Oligonucleotides.

Gas-phase etching and optical lithography were employed for the fabrication of a silicon nanoribbon chip (Si-NR chip). The quality of the so-fabricated silicon nanoribbons (Si-NRs) was monitored by optical Raman scattering spectroscopy. It was demonstrated that the structures of the Si-NRs were virtually defect-free, meaning they could be used for highly sensitive detection of biological macromolecules. The Si-NR chips were then used for the highly sensitive nanoelectronics detection of DNA oligonucleotides (oDNAs), which represent synthetic analogs of 106a-5p microRNA (miR-106a-5p), associated with the development of autism spectrum disorders in children. The specificity of the analysis was attained by the sensitization of the Si-NR chip sur-face by covalent immobilization of oDNA probes, whose nucleotide sequence was complementary to the known sequence of miR-106a-5p. The use of the Si-NR chip was demonstrated to al-low for the rapid label-free real-time detection of oDNA at ultra-low (~10−17 M) concentrations.

COMPARISON OF MAGNETIC NANOPARTICLES AND MICROPLATES AS CARRIERS IN HETEROGENEOUS CHEMILUMINESCENT ASSAY OF MICRORNA-141 USING CATALYTIC HAIRPIN ASSEMBLY

Comparison of two types of carriers (magnetic nanoparticles and microplate) for the detection of oligonucleotides is carried out. It was shown that the value of analytical parameters in chemiluminescent heterogeneous assay of microRNA-141 using an isothermal reaction of catalytic hairpin assembly did not depend on the choice of the carrier.

Dispersion-enhancing surface treatment of AuNPs for a reduced probe loading and detection limit using t-SPR detection.

COVID-19 has shown that a highly specific and rapid diagnostic system is a necessity. A spectral imaging-based surface plasmon resonance (SPRi) platform with an integrated microfluidic biosensor to detect oligonucleotide sequences has been proposed to be a promising alternative for infectious diseases due to its safe and straightforward use. Approaches to reduce the DNA probe loading onto gold nanoparticles with various types of polyethylene glycol (PEG) were explored. Here, we demonstrated the stability of functionalised gold nanoparticles with unmodified PEG whilst lowering the probe loading density. The system was evaluated by performing the detection of a mimicking COVID-19 target sequence, single point-mutation sequence and fully mismatch sequence. Highly specific binding of the mimicking COVID-19 target sequence was observed and analysed by the spectral imaging SPR approach. Our work has demonstrated the potential of a controlled probe density using unmodified PEG as an especially promising functionalisation strategy in SPR spectral imaging assays.

Narrower Nanoribbon Biosensors Fabricated by Chemical Lift-off Lithography Show Higher Sensitivity.

Wafer-scale nanoribbon field-effect transistor (FET) biosensors fabricated by straightforward top-down processes are demonstrated as sensing platforms with high sensitivity to a broad range of biological targets. Nanoribbons with 350 nm widths (700 nm pitch) were patterned by chemical lift-off lithography using high-throughput, low-cost commercial digital versatile disks (DVDs) as masters. Lift-off lithography was also used to pattern ribbons with 2 μm or 20 μm widths (4 or 40 μm pitches, respectively) using masters fabricated by photolithography. For all widths, highly aligned, quasi-one-dimensional (1D) ribbon arrays were produced over centimeter length scales by sputtering to deposit 20 nm thin-film In2O3 as the semiconductor. Compared to 20 μm wide microribbons, FET sensors with 350 nm wide nanoribbons showed higher sensitivity to pH over a broad range (pH 5 to 10). Nanoribbon FETs functionalized with a serotonin-specific aptamer demonstrated larger responses to equimolar serotonin in high ionic strength buffer than those of microribbon FETs. Field-effect transistors with 350 nm wide nanoribbons functionalized with single-stranded DNA showed greater sensitivity to detecting complementary DNA hybridization vs 20 μm microribbon FETs. In all, we illustrate facile fabrication and use of large-area, uniform In2O3 nanoribbon FETs for ion, small-molecule, and oligonucleotide detection where higher surface-to-volume ratios translate to better detection sensitivities.

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.

A Course of Hands-On Nanopore Experiments for Undergraduates: Single-Molecule Detection with Portable Electrochemical Instruments

The prompt introduction of single-molecule science into undergraduates’ curricula is an urgent but challenging task. However, the research experiments are often too complicated and the instruments are too expensive to be widely transferred into a compulsory course of undergraduate students. Herein, we introduce a recently developed nanopore technique into an accessible laboratory course for students with the advantages of simple operation, high repeatability, and low cost. The nanopore course serves to help undergraduates in practice to understand single-molecule behaviors, think at a single molecule level, and gradually develop single-molecule solutions for scientific issues. The learning activities for undergraduate students include setup of the instrument, fabrication of a biological nanopore on an artificial lipid bilayer membrane, detection of individual oligonucleotides, and data analysis. An experiment example is single-molecule detection of the model DNA sample poly(dA)n (n = 4, 5) using the aerolysin nanopore. As our developed nanopore sensing system is simple, portable, and easy to handle, the experiment course could be implemented and popularized in undergraduates’ laboratory education. For the undergraduate students who have taken general chemistry and/or biochemistry courses, this hands-on experience of nanopore experiments would benefit them with a better understanding of the chemical world at a single-molecule level and further using single-molecule tools in various applications including DNA and/or protein sequencing.

Single-cell Multiplexed Fluorescence Imaging to Visualize Viral Nucleic Acids and Proteins and Monitor HIV, HTLV, HBV, HCV, Zika Virus, and Influenza Infection.

Capturing the dynamic replication and assembly processes of viruses has been hindered by the lack of robust in situ hybridization (ISH) technologies that enable sensitive and simultaneous labeling of viral nucleic acid and protein. Conventional DNA fluorescence in situ hybridization (FISH) methods are often not compatible with immunostaining. We have therefore developed an imaging approach, MICDDRP (multiplex immunofluorescent cell-based detection of DNA, RNA and protein), which enables simultaneous single-cell visualization of DNA, RNA, and protein. Compared to conventional DNA FISH, MICDDRP utilizes branched DNA (bDNA) ISH technology, which dramatically improves oligonucleotide probe sensitivity and detection. Small modifications of MICDDRP enable imaging of viral proteins concomitantly with nucleic acids (RNA or DNA) of different strandedness. We have applied these protocols to study the life cycles of multiple viral pathogens, including human immunodeficiency virus (HIV)-1, human T-lymphotropic virus (HTLV)-1, hepatitis B virus (HBV), hepatitis C virus (HCV), Zika virus (ZKV), and influenza A virus (IAV). We demonstrated that we can efficiently label viral nucleic acids and proteins across a diverse range of viruses. These studies can provide us with improved mechanistic understanding of multiple viral systems, and in addition, serve as a template for application of multiplexed fluorescence imaging of DNA, RNA, and protein across a broad spectrum of cellular systems.

SOI-Nanowire Biosensor for the Detection of Glioma-Associated miRNAs in Plasma

Herein, we report the development of a highly sensitive nanotechnology-based system—silicon-on-insulator nanowire biosensor for the revelation of microRNAs (miRNAs), associated with the development of glioma in the human. In this system, a sensor chip, bearing an array of silicon nanowire structures, is employed. The sensor chip is fabricated using a top-down technology. In our experiments reported herein, we demonstrated the detection of DNA oligonucleotide (oDNA), which represents a synthetic analogue of microRNA-363 associated with the development of glioma. To provide biospecific detection of the target oligonucleotides, the surface of the nanowire structures is modified with oligonucleotide probes; the latter are complementary to the target ones. The concentration limit of the target oligonucleotide detection, attained using our nanowire biosensor, is at the level of DL~10−17 M. The revelation of the elevated level of glioma-associated miRNA in plasma is also demonstrated.

Recognition of Single‐Point Mutation Using a Biological Nanopore

AbstractHere the recognition of a single‐point mutation in oligonucleotides is described by using nanopore measurements. The translocation behavior of a series of mutated DNA strands, hybridized with a complementary DNA probe, is analyzed via blocking current and unzipping time. Discernment of the mutation position at the single nucleotide level is achieved by analysis of a 2D graph of the bootstrapped translocation data. The proposed approach provides a useful tool for the mutation detection of oligonucleotides secreted from tumor cells and is applicable in simple and label‐free diagnoses as a nanopore liquid biopsy.

Rapid and label-free electrochemical DNA biosensor based on a facile one-step electrochemical synthesis of rGO-PPy-(L-Cys)-AuNPs nanocomposite for the HTLV-1 oligonucleotide detection.

Human T cell leukemia virus type 1 (HTLV-1) as the first human retrovirus is currently a serious endemic health challenge. Despite the use of assorted molecular or serological assays for HTLV-1 detection, there are several limitations due to the lack of a confirmatory test that may affect the accuracy of the results. Herein, a novel label-free biosensor for the detection of HTLV-1 Tax gene has been reported. An electrochemical facile ecofriendly synthesis method has been demonstrated based on a synthesis of nanocomposite of reduced graphene oxide, polypyrrole, and gold nanoparticles (rGO-PPy-(l-Cys)-AuNPs) deposited on the surface of screen-printed carbon electrode. Electrochemical techniques were used to characterize and study the electrochemical behavior of the rGO-PPy-(l-Cys)-AuNPs, which exhibited a stable reference peak at 0.21V associated with hybridization forms by applying the differential pulse voltammetry. The designed DNA biosensor presented a wide linear range from 0.1fM to 100µM and a low detection limit of 20 atto-molar. The proposed biosensor presented in this study provides outstanding selectivity, sensitivity, repeatability, and reproducibility.

Fiber Optic Particle Plasmon Resonance Biosensor for Label-Free Detection of Nucleic Acids and Its Application to HLA-B27 mRNA Detection in Patients with Ankylosing Spondylitis.

We developed a label-free, real-time, and highly sensitive nucleic acid biosensor based on fiber optic particle plasmon resonance (FOPPR). The biosensor employs a single-strand deoxyoligonucleotides (ssDNA) probe, conjugated to immobilized gold nanoparticles on the core surface of an optical fiber. We explore the steric effects on hybridization affinity and limit of detection (LOD), by using different ssDNA probe designs and surface chemistries, including diluent molecules of different lengths in mixed self-assembled monolayers, ssDNA probes of different oligonucleotide lengths, ssDNA probes in different orientations to accommodate target oligonucleotides with a hybridization region located unevenly in the strand. Based on the optimized ssDNA probe design and surface chemistry, we achieved LOD at sub-nM level, which makes detection of target oligonucleotides as low as 1 fmol possible in the 10-μL sensor chip. Additionally, the FOPPR biosensor shows a good correlation in determining HLA-B27 mRNA, in extracted blood samples from patients with ankylosing spondylitis (AS), with the clinically accepted real-time reverse transcription-polymerase chain reaction (RT-PCR) method. The results from this fundamental study should guide the design of ssDNA probe for anti-sense sensing. Further results through application to HLA-B27 mRNA detection illustrate the feasibility in detecting various nucleic acids of chemical and biological relevance.

Oligonucleotide Detection and Optical Measurement with Graphene Oxide in the Presence of Bovine Serum Albumin Enabled by Use of Surfactants and Salts

As graphene oxide-based oligonucleotide biosensors improve, there is a growing need to explore their ability to retain high sensitivity for low target concentrations in the context of biological fluids. Therefore, we innovatively combined assay milieu factors that could influence the key performance parameters of DNA hybridization and graphene oxide (GO) colloid dispersion, verifying their suitability to enhance oligonucleotide–GO interactions and biosensor performance. As a model system, we tested single-strand (ss) DNA detection in a complex solution containing bovine serum albumin (BSA) and salts with surfactants. A fluorescein conjugated 30-mer oligonucleotide ssDNA probe was combined with its complementary cDNA target, together with solute dispersed GO and either non-ionic (Triton X-100 and Tween-20) or anionic sodium dodecyl sulfate (SDS) surfactants. In this context, we compared the effect of divalent Mg2+ or monovalent Na+ salts on GO binding for the quench-based detection of specific target–probe DNA hybridization. GO biosensor strategies for quench-based DNA detection include a “turn on” enhancement of fluorescence upon target–probe interaction versus a “turn off” decreased fluorescence for the GO-bound probe. We found that the sensitive and specific detection of low concentrations of oligonucleotide target was best achieved using a strategy that involved target–probe DNA hybridization in the solution with a subsequent modified “turn-off” GO capture and the quenching of the unhybridized probe. Using carefully formulated assay procedures that prevented GO aggregation, the preferential binding and quenching of the unhybridized probe were both achieved using 0.1% BSA, 0.065% SDS and 6 mM NaCl. This resulted in the sensitive measurement of the specific target–probe complexes remaining in the solution. The fluorescein-conjugated single stranded probe (FAM–ssDNA) exhibited linearity to cDNA hybridization with concentrations in the range of 1–8 nM, with a limit of detection equivalent to 0.1 pmoles of target in 100 µL of assay mix. We highlight a general approach that may be adopted for oligonucleotide target detection within complex solutions.

Pharmacokinetic Studies of Antisense Oligonucleotides Using MALDI-TOF Mass Spectrometry.

Cardiac diseases are the most frequent causes of death in industrialized countries. Pathological remodeling of the heart muscle is caused by several etiologies such as prolonged hypertension or injuries that can lead to myocardial infarction and in serious cases also the death of the patient. The micro-RNA miR-132 has been identified as a master-switch in the development of cardiac hypertrophy and adverse remodeling. In this study, MALDI-TOF mass spectrometry (MS) was utilized to establish a robust and fast method to sensitively detect and accurately quantify anti-microRNA (antimiR) oligonucleotides in blood plasma. An antimiR oligonucleotide isolation protocol containing an ethanol precipitation step with glycogen as oligonucleotide carrier as well as a robust and reproducible MS-analysis procedure has been established. Proteinase K treatment was crucial for releasing antimiR oligonucleotides from plasma- as well as cellular proteins and reducing background derived from biological matrices. AntimiR oligonucleotide detection was achieved from samples of studies in different animal models such as mouse and pig where locked nucleic acids-(LNA)-modified antimiR oligonucleotides have been used to generate pharmacokinetic data.