Multivalent DNA Flowers for High-Performance Isolation, Detection, and Release of Tumor-Derived Extracellular Vesicles.

Tumor-derived extracellular vesicles (T-EVs) hold great promise for understanding cancer biology and improving cancer diagnostics and therapeutics. Herein, we developed multivalent DNA flowers (DFs) containing repeated and equidistant EpCAM aptamers for the efficient isolation of T-EVs. The multivalent aptamer chains in DFs had good flexibility to adapt to the surface morphology of T-EVs and achieved multivalent ligand-receptor interactions, thus showing enhanced isolation ability compared to monovalent aptamers. Compared with other materials for isolation of EVs, DFs were generated by rolling circle amplification (RCA) and self-assembled into microspheres in a one-pot reaction, and the recognition molecules (aptamers) were directly replicated and assembled during the RCA reaction instead of chemical modification and immobilization on the surface of solid materials. Moreover, as optically transparent biomaterials, the content of EpCAM+ EVs could be directly reflected via membrane-based hydrophobic assembly of signaling modules in DFs@EpCAM+ EVs complex, and we found that the amount of EpCAM+ EVs showed greater accuracy in cancer diagnosis than total EVs (88.3 vs 69.7%) and was also higher than the clinically commonly used marker carcinoembryonic antigen (CEA) (88.3 vs 76.7%). In addition, T-EVs could be released by lysis of DFs with the nuclease, gently and easily, keeping high intact and activity of EVs for downstream biological function studies. These results demonstrated that DFs are efficient and nondestructive tools for isolation, detection, and release of T-EVs.

Electrochemical nucleic acid hybridization biosensor based on poly(L-Aspartic acid)-modified electrode for the detection of short oligonucleotide sequences related to hepatitis C virus 1a.

This work describes, for the first time, the fabrication of poly(L-aspartic acid) (PAA) film modified pencil graphite electrode (PGE) for the detection of hepatitis C Virus 1a (HCV1a). The presence of PAA on the electrode surface can provide free carboxyl groups for covalent binding of biomolecules. The PGE surface was first coated with PAA via electropolymerization of the L-aspartic acid, and avidin was subsequently attached to the PAA modified electrode by covalent attachment. Biotinylated HCV1a probes were immobilized on avidin/PAA/PGE via avidin-biotin interaction. The morphology of PAA/PGE was examined using a scanning electron microscope. The hybridization events were monitored with square wave voltammetry using Meldola's blue (MDB). Compared to non-complementary oligonucleotide sequences, when hybridization was carried out between the probe and its synthetic targets or the synthetic polymerase chain reaction analog of HCV1a, the highest MDB signal was observed. The linear range of the biosensor was 12.5 to 100 nM and limit of detection was calculated as 8.7 nM. The biosensor exhibited favorable stability over relatively long-term storage. All these results suggest that PAA-modified electrode can be used to nucleic acid biosensor application and electropolymerization of L-aspartic acid can be considered as a good candidate for the immobilization of biomolecules.

384-Channel electrochemical sensor array chips based on hybridization-triggered switching for simultaneous oligonucleotide detection.

We investigated the feasibility of simultaneous detection of multiple environmentally- and biomedically-relevant RNA biomarker target sequences on a single newly fabricated 384-ch sensor array chip aiming at practical application. The individual sensor is composed of a photolithographically-fabricated Au/Cr-based electrode modified with peptide nucleic acid (PNA) probes. The sensor array chips showed sequence-specific responses upon hybridization of the probes with target sequences complementary to the probes in contrast to mismatch versions. The target oligonucleotides have 15-22 mer sequences from messenger RNAs for estrogen-responsive genes and microRNAs for lung cancer biomarkers. The dependence on target concentrations of sensor responses was observed by using a single chip on which experiments for detection of several target concentrations proceeded simultaneously, with the detection limit of 7.33 × 10-8 M. As more realistic samples, oligonucleotide samples amplified by PCR from a synthesized template sequence were applied to the chip. They showed sequence-specific responses, revealing the potential for fabricated sensor array chips to be utilized to analyze PCR samples. Unlike complicated and expensive chips that require nanofabrication, our sensor array chips based on glass coated with gold thin films are simple and can be fabricated from inexpensive and readily available materials.

Nanoparticle assisted nuclear relaxation-based oligonucleotide detection.

We present a proof-of-concept "on-off" detection scheme, which uses gadolinium phthalocyanine (GdTcPc)-grafted silica nanoparticles as paramagnetic centers, capable of modifying the transverse relaxation time (T2) of water protons in solution. A DNA strand (as probe) was conjugated to the GdTcPc to act as a recognition element. In the presence of the target DNA, which was complementary to the probe, an increase in the T2 value was detected, with magnitude proportional to the target DNA concentration. The linear range was observed from 30 to 140 nM, with limit of detection of 15 nM. The developed nuclear relaxation-based detection scheme is shown to be a simple, fast and selective method to detect DNA and could be useful in point-of-care diagnostic applications.

Critical evaluation of quantification methods for oligonucleotides formulated in lipid nanoparticles.

There is a very large variety in the types of nanoparticulate lipid formulations for oligonucleotides, and remarkably, also a very large heterogeneity in the methods that are used for analyzing oligonucleotide load, encapsulation efficiency and oligonucleotide release. Furthermore, a literature survey showed that the extent to which these procedures are reported in scientific literature varies greatly, with some of them not even reporting any quantification at all. This greatly hampers the reproducibility of nanoparticle preparation between different researchers and between different laboratories, which slows down the clinical translation of such nanomedicines. In this work, a standardized extraction method from liposomes is proposed, in which potential contaminants from the carrier are removed by a simple extraction of the oligonucleotides. These extracts were then analyzed with seven commonly used methods for oligonucleotide quantification, including several absorbance based methods and the most commonly applied dye binding assay. Strikingly, differences in absolute values up to fourfold were found when the same sample was analyzed using different methods which should be taken into consideration when reports using different methods are compared. Furthermore, these results indicate that the most commonly applied method, the dye binding assay, may -without adaptations- not be suitable for short oligonucleotides like siRNAs. The found differences in quantification methods as described here underscore the need for proper documentation of methods to correctly interpret published results, which -with regard to oligonucleotide analysis- is currently lacking in many reports.

Structural analysis of small to medium-sized molecules by mass spectrometry after electron-ion fragmentation (ExD) reactions.

Electron capture dissociation (ECD) is a tandem mass spectrometry (MS/MS) method that utilizes the interaction of ions and electrons. Its unique ability to preserve labile bonds distinguishes it from conventional threshold-based MS/MS methods, the most important of which is collision-induced dissociation (CID). During the last decade, ECD has opened up several new venues in protein analyses, for example top-down sequencing, identification of post-translational modifications, and characterization of protein-protein interactions. In recent years, a number of related dissociation techniques, so-called ExD techniques, particularly electron transfer dissociation (ETD), electron detachment dissociation (EDD), electron induced dissociation (EID), and negative electron transfer dissociation (NETD), have emerged and have extended the application range of ion-electron dissociations further. Importantly, ExD techniques have been applied beyond protein analyses, which is the focus of the current paper. This short introduction describes the application of ExD to small and medium-sized molecules and reviews important applications to natural products, biomedical compounds, synthetic molecules, crude oils, and environmental toxins.

Image-based detection of oligonucleotides--a low cost alternative to spectrophotometric or fluorometric methods.

Herein, we report a sensitive and low cost image-based (photocolorimetric) method for the detection of oligonucleotides on an activated polypropylene microtest plate (APPµTP). The assay was developed on the APPµTP by covalently immobilising 20-mer amino-modified oligonucleotides. Biotin-tagged complementary target sequences were then hybridised with the immobilised oligonucleotides. Colour was developed by streptavidin-HRP conjugate and the image of the coloured assay solution was taken by a desktop scanner and analysed using colour saturation. The developed method was analysed for its detection limit, accuracy, sensitivity and interference. The linearity range was found to be 1.7-170 ng mL(-1) while the lower limit of detection and limit of quantification were 1.7 and 5.6 ng mL(-1) respectively. The method showed comparable sensitivity to fluorometric methods, and was found to be correlated to fluorescence (R(2) = 0.8081, p-value < 0.0001) and absorbance (R(2) = 0.9394, p-value < 0.0001)-based quantification. It discriminates mismatched base sequences from perfectly matched sequences efficiently. Validation of the method was carried out by detecting por A DNA of Neisseria meningitidis in bacterial meningitis samples. The por A-specific probe having a 6-carbon spacer at its 5'-NH2 terminus was immobilised covalently to the APPµTP and hybridised with different samples of biotinylated single-stranded por A DNA.

Piezomicrogravimetric and impedimetric oligonucleotide biosensors using conducting polymers of biotinylated bis(2,2'-bithien-5-yl)methane as recognition units.

A new conducting polymer of biotinylated bis(2,2'-bithien-5-yl)methane was prepared and applied as the recognition unit of two different biosensors for selective oligonucleotide determination using either electrochemical impedance spectroscopy (EIS) or piezoelectric microgravimetry (PM) for label-free analytical signal transduction. For preparation of this unit, first, a biotinylated bis(2,2'-bithien-5-yl)methane functional monomer was designed and synthesized. Then, this monomer was potentiodynamically polymerized to form films on the surface of a glassy carbon electrode (GCE) and a Au electrode of a quartz crystal resonator (QCR) for the EIS and PM transduction, respectively. On top of these films, neutravidin was irreversibly immobilized by complexing the biotin moieties of the polymer. Finally, recognizing biotinylated oligonucleotide was attached by complexing the surface-immobilized neutravidin. This layer-by-layer assembling of the poly(thiophene-biotin)-neutravidin-(biotin-oligonucleotide) recognition film served to determine the target oligonucleotide via complementary nucleobase pairing. Under optimized determination conditions, the target oligonucleotide limit of detection (LOD) was 0.5 pM and 50 nM for the EIS and PM transduction, respectively. The sensor response to the target oligonucleotide was linear with respect to logarithm of the target oligonucleotide concentration in a wide range of 0.5 pM to 30 µM and with respect to its concentration in the range of 50 to 600 nM for the EIS and PM transduction, respectively. The biosensors were appreciably selective with respect to the nucleobase mismatched oligonucleotides.

PCR synthesis of double stranded DNA labeled with 5-bromouridine. A step towards finding a bromonucleoside for clinical trials.

Incorporation of 5-bromouridine (5BrdU) into DNA makes it sensitive to UV and ionizing radiation, which opens up a prospective route for the clinical usage of 5-bromouridine and other halonucleosides. In the present work the polymerase chain reaction (PCR) protocol, which enables a long DNA fragment (resembling DNA synthesized in the cell in the presence of halonucleosides) to be completely substituted with 5BrdU, was optimized. Using HPLC coupled to enzymatic digestion, it was demonstrated that the actual amounts of native nucleosides and 5BrdU correspond very well to those calculated from the sequence of PCR products. The synthesized DNA is photosensitive to photons of 300nm. HPLC analysis demonstrated that the photolysis of labeled PCR products leads to a significant decrease in the 5BrdU signal and the simultaneous occurrence of a uridine peak. Agarose and polyacrylamide gel electrophoresis suggest that single strand breaks and cross-links are formed as a result of UV irradiation. The PCR protocol described in the current paper may be employed for labeling DNA not only with BrdU but also with other halonucleosides.

Thermodynamic and biological evaluation of a thrombin binding aptamer modified with several unlocked nucleic acid (UNA) monomers and a 2'-C-piperazino-UNA monomer.

Thrombin binding aptamer is a DNA 15-mer which forms a G-quadruplex structure and possess promising anticoagulant properties due to specific interactions with thrombin. Herein we present the influence of a single 2'-C-piperazino-UNA residue and UNA residues incorporated in several positions on thermodynamics, kinetics and biological properties of the aptamer. 2'-C-Piperazino-UNA is characterized by more efficient stabilization of quadruplex structure in comparison to regular UNA and increases thermodynamic stability of TBA by 0.28-0.44 kcal/mol in a position depending manner with retained quadruplex topology and molecularity. The presence of UNA-U in positions U3, U7, and U12 results in the highest stabilization of G-quadruplex structure (ΔΔG(37)(°)=-1.03kcal/mol). On the contrary, the largest destabilization mounting to 1.79 kcal/mol was observed when UNA residues were placed in positions U7, G8, and U9. Kinetic studies indicate no strict correlation between thermodynamic stability of modified variants and their binding affinity to thrombin. Most of the studied variants bind thrombin, albeit with decreased affinity in reference to unmodified TBA. Thrombin time assay studies indicate three variants as being as potent as TBA in fibrin clotting inhibition.

Preparation and characterization of zinc oxide nanoparticles and their sensor applications for electrochemical monitoring of nucleic acid hybridization.

In this study, ZnO nanoparticles (ZNP) of approximately 30 nm in size were synthesized by the hydrothermal method and characterized by X-ray diffraction (XRD), Braun-Emmet-Teller (BET) N2 adsorption analysis and transmission electron microscopy (TEM). ZnO nanoparticles enriched with poly(vinylferrocenium) (PVF+) modified single-use graphite electrodes were then developed for the electrochemical monitoring of nucleic acid hybridization related to the Hepatitis B Virus (HBV). Firstly, the surfaces of polymer modified and polymer-ZnO nanoparticle modified single-use pencil graphite electrodes (PGEs) were characterized using scanning electron microscopy (SEM). The electrochemical behavior of these electrodes was also investigated using differential pulse voltammetry (DPV) and electrochemical impedance spectroscopy (EIS). Subsequently, the polymer-ZnO nanoparticle modified PGEs were evaluated for the electrochemical detection of DNA based on the changes at the guanine oxidation signals. Various modifications in DNA oligonucleotides and probe concentrations were examined in order to optimize the electrochemical signals that were generated by means of nucleic acid hybridization. After the optimization studies, the sequence-selective DNA hybridization was investigated in the case of a complementary amino linked probe (target), or noncomplementary (NC) sequences, or target and mismatch (MM) mixture in the ratio of (1:1).

A regenerable flow-through affinity sensor for label-free detection of proteins and DNA.

Label-free monitoring of biomolecular reactions in real-time is of great interest since it can provide valuable information about binding kinetics and equilibrium constants. In this report, a sensor based on White Light Reflectance Spectroscopy (WLRS) is presented that is capable of real-time monitoring of biomolecular reactions taking place on top of a polymer covered silicon dioxide reflective surface. The optical set-up consists of a visible-near infrared light source, a bifurcated optical fiber and a spectrometer. The outer part of the optical fiber guides the light vertically onto the surface where the biomolecular reactions occur, whereas the reflected light is driven from the central part of the fiber to the spectrometer. A microfluidic module in combination with a pump supplies the reagents at a constant rate. The biomolecular interactions are monitored as shifts of the wavelength of the interference minimum. The proposed methodology was applied for real-time and label-free monitoring mouse gamma-globulins binding onto immobilized anti-mouse IgG antibody. Mouse gamma-globulins at concentrations down to 150pM were detected in reaction times of 1-min. Regeneration of immobilized antibody was accomplished up to seven times without loss of its activity. In addition, real-time monitoring of hybridization reaction between complementary oligonucleotides was accomplished. The proposed sensor provides a simple, fast, low cost approach for label-free monitoring of biomolecular interactions and therefore it should by suitable for a wide range of analytical applications.

Quantification of oligonucleotide containing sequence failure product: comparison of isotope dilution mass spectrometry with other quantification methods.

There is an increasing demand to develop a method for accurate quantification of DNA. Because current methods such as the ultraviolet (UV) absorption-based method are only capable of relative quantification, the quantification result depends completely on the reference material. To achieve accurate quantification of DNA, we have performed isotope dilution mass spectrometry (ID MS)-based quantification of oligonucleotides. We chose a 20-mer synthetic oligonucleotide as the analyte with a longer sequence failure product. Oligonucleotides sometimes contain sequence failure products, which are difficult to remove. It is important to quantify a target product in such mixture. After evaluating the content of the sequence failure product, the analyte spiked with stable isotopically labeled deoxynucleotide monophosphates (dNMPs) was digested by enzyme to its constituent dNMPs or deoxynucleosides, and quantified by liquid chromatography-mass spectrometry. The obtained mass fractions of the 20-mer oligonucleotide showed a good agreement with the results based on phosphate analysis by inductively coupled plasma-optical emission spectrometry and ion chromatography. UV absorption, the general method for DNA quantification, resulted in underestimation. On the other hand, the mass fraction obtained by the gravimetric method was overestimated. This study shows that the ID MS method can determine the precise mass fraction of the target oligonucleotide with the sequence failure product and possesses potential as the primary method for the certification of DNA as a reference material.

Surfaces for tuning of oligonucleotide biosensing selectivity based on surface-initiated atom transfer radical polymerization on glass and silicon substrates.

Covalently immobilized mixed films of oligonucleotide and oligomer components on glass and silicon surfaces are reported. This work has investigated how such films can improve selectivity for the detection of multiple base-pair mismatches. The intention was to introduce a "matrix isolation" effect on oligonucleotide probe molecules by surrounding the probes with oligomers, thereby reducing oligonucleotide-to-oligonucleotide and/or oligonucleotide-to-surface interactions. Thiol-functionalized oligonucleotide was coupled onto (3-aminopropyl)trimethoxysilane (APTMS) via a heterobifunctional linker, succinimidyl 4-[N-maleimidomethyl]cyclohexane-1-carboxylate (sulfo-SMCC). Using a variety of monomers such as 2-hydroxyethyl methacrylate (HEMA), oligomers were grown by surface-initiated atom transfer radical polymerization (ATRP) from a bromoisobutyryl NHS ester initiator which was immobilized onto APTMS sites that coated glass and oxidized silicon substrates. Various surface modification steps on silicon substrates were characterized by ellipsometry, wettability, atomic force microscopy, X-ray photoelectron spectroscopy, and time-of-flight secondary ion mass spectrometry. Polymerized HEMA (PHEMA) in mixture with oligonucleotide probes was evaluated for fluorescence transduction of hybridization. The presence of PHEMA was found to provide a sharper melt curve for hybrids containing both fully complementary and three base-pair mismatched targets, and this surface derivatization also minimized non-selective adsorption. The maximum increase in slope was improvement by a factor of 3-fold. An increase of up to 30% in difference of melting temperatures between fully complementary and 3 base-pair mismatched targets was achieved using PHEMA. The results suggest that the presence of oligomers dispersed among DNA hybrids can improve selectivity through what is believed to be a reduction of dispersity of interactions of probes with targets, and probes within their local environment at a surface.

Positively charged compact quantum Dot-DNA complexes for detection of nucleic acids.

Novel QD-DNA complexes are prepared by simple electrostatic interaction between pegylated amine-functionalized CdSe/ZnS quantum dots (QDs) and DNA. The cationic nature of the amine functionality on the QD surface allows for formation of an electrostatic complex with negatively charged DNA. The presence of polyethylene glycol (PEG5000) molecules on the QD leads to enhanced stability and decreased nonspecific adsorption of DNA on the QD surface. Unlike assembly of QD-DNA based on hydrogen bonding, the present QD probes tend to be more strongly stabilized during the hybridization process by increasing the overall negative charges. In addition, the DNA loading efficiency can be modulated by changing the pH of the reaction medium. The fluorescence of the QD is quenched up to 90% by complexation with 5'-TAMRA-modified oligonucleotide (TAMRA=carboxytetramethylrhodamine) through fluorescence resonance energy transfer (FRET). With the FRET pair we selected, the R(0) value was calculated to be 5.5 nm and r is about 5 nm. This quenching of QD fluorescence is then reversed on binding of unlabeled target DNA. The maximum recovery of QD fluorescence is 60%. The QD-DNA probe (5DNA/QD) exhibits selective photoluminescence (PL) recovery in the presence of target oligonucleotide with a PL ratio of 3 for complementary versus noncomplementary. The present QD-DNA probes also show the capability to detect the synthetic 100-mer oligonucleotide derived from H5N1 influenza virus when present at concentrations as low as 200 nM in the solution.

DNA modification of live cell surface.

We report a novel approach for the attachment of DNA fragments to the surface of live cells. By using fluorescence microscopy and flow cytometry we demonstrated that our synthetic conjugates of fatty acid with oligonucleotides can be incorporated in plasma membrane and then hybridized with complementary sequences at the cell surface. Method permits to control amount of immobilized DNA on the cell surface. All procedures can be completed within minutes and do not alter cell viability. Using this approach we tethered floating myeloid HL-60 cells to adherent A431 epitheliocytes in a sequence specific fashion. Thus, this method allows rapid and simple DNA multicoding of the cell surface and, therefore, opens new opportunities in manipulating with cell-cell interactions.

Quantitative evaluation of oligonucleotide surface concentrations using polymerization-based amplification.

Quantitative evaluation of minimal polynucleotide concentrations has become a critical analysis among a myriad of applications found in molecular diagnostic technology. Development of high-throughput, nonenzymatic assays that are sensitive, quantitative and yet feasible for point-of-care testing are thus beneficial for routine implementation. Here, we develop a nonenzymatic method for quantifying surface concentrations of labeled DNA targets by coupling regulated amounts of polymer growth to complementary biomolecular binding on array-based biochips. Polymer film thickness measurements in the 20-220 nm range vary logarithmically with labeled DNA surface concentrations over two orders of magnitude with a lower limit of quantitation at 60 molecules/microm(2) (approximately 10(6) target molecules). In an effort to develop this amplification method towards compatibility with fluorescence-based methods of characterization, incorporation of fluorescent nanoparticles into the polymer films is also evaluated. The resulting gains in fluorescent signal enable quantification using detection instrumentation amenable to point-of-care settings.

Analysis of oligonucleotides using capillary zone electrophoresis and electrospray mass spectrometry.

This chapter illustrates the usefulness of capillary zone electrophoresis (CZE) coupled to high-resolution electrospray ionization quadrupole time-of-flight mass spectrometry for the single-step desalting, and separation, as well as characterization of oligonucleotides in the framework of quality control after synthesis. Separation is performed using a 25 mM ammonium carbonate buffer supplemented with 0.2 mM trans-1,2-diaminocyclohexane-N, N, N', N' id (CDTA) (pH 9.7). During the electrophoretic process, sodium and potassium ions are removed from the polyanionic backbone of the oligonucleotides by exchange of these ions with ammonium ions or by chelation on CDTA, thus eliminating a sample preparation step. A sample stacking procedure used to concentrate the samples on the CZE capillary is described. After analysis, the obtained spectrum is deconvoluted to the zero charge spectrum to yield the molecular mass of the oligonucleotide. A misincorporation of one nucleotide can be detected by a difference in mass.

Double-wavelength technique for surface plasmon resonance measurements: basic concept and applications for single sensors and two-dimensional sensor arrays.

A new technique for on-line monitoring of analyte binding to sensor surfaces by surface plasmon resonance (SPR) detection is described. It is based on differential measurements using two wavelengths provided by two diode lasers. The technique is as simple and robust as the conventional SPR detection measuring the reflected radiation at fixed incidence angle, but it has the advantage of being nonsensitive to variations of the resonance width and providing essentially higher signal/noise ratios. The paper presents the first four channel prototype system for parallel 2D-monitoring at four different spots. One channel is always used as a reference to compensate temperature fluctuations and nonspecific adsorptions. Calibration with sucrose solutions revealed an absolute sensitivity of Deltan approximately 5 x 10(-6). The new technique is tested with a biotin-streptavidin binding and with hybridization/denaturation of DNA. Biotin binding to a streptavidin monolayer is detected with a signal/noise ratio of about 5, which demonstrates the high potential of the new technique for applications in drug discovery. Applications to gene analysis are tested with short oligonucleotides of the sequences used for genotyping human hepatitis C viruses. A selective response to complementary oligonucleotides is observed. The high reproducibility in subsequent cycles of hybridization/denaturation (by formamide or by heating) points out potential applications of the technique in medical diagnostics, food industry, genomics, and proteomics too.

Gram-scale synthesis and biofunctionalization of silica-coated silver nanoparticles for fast colorimetric DNA detection.

A direct silica-coating method has been developed for the gram-scale synthesis of well-dispersed Ag@SiO(2) nanoparticles. Subsequent surface functionalization via the well-established silica surface chemistry provided arching points for straightforward bioconjugation with amino-terminated oligonucleotides. Fast hybridization kinetics of the resulting robust oligo-modified Ag@SiO(2) nanoprobes with complementary target oligonucleotides render themselves very useful for the fast colorimetric DNA detection based on the sequence-specific hybridization properties of DNA. Additionally, the reliable protocols developed in this study for preparing and functionalizing Ag@SiO(2) nanoparticles can be readily extended to other silica-coated nanoparticles, which can also provide a specific platform for the covalent attachment of biomolecules such as amino-rich proteins, enzymes, or amino-terminated oligonucleotides for diverse bioapplications.