Immobilization Of Nucleic Acids Research Articles

Itaconic Acid Cross-Linked Biomolecule Immobilization Approach on Amine-Functionalized Silica Nanoparticles for Highly Sensitive Enzyme-Linked Immunosorbent Assay (ELISA).

Biomolecule immobilization on nanomaterials is attractive for biosensors since it enables the capture of a higher concentration of bioreceptor units while also serving as a transduction element. The technique could enhance the accuracy, specificity, and sensitivity of the analytical measurements of biomolecules. However, it was found that the limitation in chemically binding biomolecules on nanoparticle surfaces could only cross-link between the C-terminal and N-terminal. Here, we report the facile one-step synthesis of amine-functionalized silica nanoparticles (AFSNPs). (3-Aminopropyl)triethoxysilane was used as a precursor to modify the functional surface of nanoparticles via the Stöber process. The biomolecules were immobilized to the AFSNPs through itaconic acid, a novel cross-linker that binds between the N-terminal and N-terminal and potentially improves proteins and nucleic acid immobilization onto the nanoparticle surface. The newly developed immobilization approach on AFSNPs for biomolecular detection enhanced the efficiency of ELISA, resulting in increased sensitivity. It might also be easily used to identify different pathogens for clinical diagnostics.

Integrated Three-Dimensional Microdevice with a Modified Surface for Enhanced DNA Separation from Biological Samples.

Functional interfaces and devices for rapid adsorption and immobilization of nucleic acids (NAs) are significant for relevant bioengineering applications. Herein, a microdevice with poly(acrylic acid) (PAA) photosensitive resin was integrated by three-dimensional (3D) printing, named DPAA for short. Precise microscale structures and abundant surface carboxyl functional groups were fabricated for fast and high-throughput deoxyribonucleic acid (DNA) separation. Surface modification was then done using polydopamine (PDA) and poly(ethylene glycol) (PEG) to obtain modified poly(acrylic acid) (PAA)-based devices DPDA-PAA and DPEG-PAA rich in amino and hydroxyl groups, respectively. The fabricated device DPAA possessed superior printing accuracy (40-50 μm). Functionalization of amino and hydroxyl was successful, and the modified devices DPDA-PAA and DPEG-PAA maintained a high thermal stability like DPAA. Surface potential analysis and molecular dynamics simulation indicated that the affinity for DNA was in the order of DPDA-PAA > DPEG-PAA > DPAA. Further DNA separation experiments confirmed the high throughput and high selectivity of DNA separation performance, consistent with the predicted affinity results. DPDA-PAA showed relatively the highest DNA extraction yield, while DPEG-PAA was the worst. An acidic binding system is more favorable for DNA separation and recovery. DPDA-PAA showed significantly better DNA extraction performance than DPAA in a weakly acidic environment (pH 5.0-7.0), and the average DNA yield of the first elution was 2.16 times that of DPAA. This work validates the possibility of modification on integrated 3D microdevices to improve their DNA separation efficiency effectively. It also provides a new direction for the rational design and functionalization of bioengineering separators based on nonmagnetic methods. It may pave a new path for the highly efficient polymerase chain reaction diagnosis.

Equipment-free, salt-mediated immobilization of nucleic acids for nucleic acid lateral flow assays

As the world has been facing several deadly virus crises, including Zika virus disease, Ebola virus disease, severe acute respiratory syndrome (SARS), Middle East respiratory syndrome (MERS), and Coronavirus disease 2019 (COVID-19), lateral flow assays (LFAs), which require minimal equipment for point-of-care of viral infectious diseases, are garnering much attention. Accordingly, there is an increasing demand to reduce the time and cost required for manufacturing LFAs. The current study introduces an equipment-free method of salt-mediated immobilization of nucleic acids (SAIoNs) for LFAs. Compared to general DNA immobilization methods such as streptavidin–biotin, UV-irradiation, and heat treatment, our method does not require special equipment (e.g., centrifuge, UV-crosslinker, heating device); therefore, it can be applied in a resource-limited environment with reduced production costs. The immobilization process was streamlined and completed within 30 min. Our method improved the color intensity signal approximately 14 times compared to the method without using SAIoNs and exhibited reproducibility with the long-term storage stability. The proposed method can be used to detect practical targets (e.g., SARS-CoV-2) and facilitates highly sensitive and selective detection of target nucleic acids with multiplexing capability and without any cross-reactivity. This novel immobilization strategy provides a basis for easily and inexpensively developing nucleic acid LFAs combined with various types of nucleic acid amplification.

Non-Langmuir Kinetics of DNA Surface Hybridization

Hybridization of complementary single strands of DNA represents a very effective natural molecular recognition process widely exploited for diagnostic, biotechnology, and nanotechnology applications. A common approach relies on the immobilization on a surface of single-stranded DNA probes that bind complementary targets in solution. However, despite the deep knowledge on DNA interactions in bulk solution, the modeling of the same interactions on a surface are still challenging and perceived as strongly system dependent. Here, we show that a two-dimensional analysis of the kinetics of hybridization, performed at different target concentrations and probe surface densities by a label-free optical biosensor, reveals peculiar features inconsistent with an ideal Langmuir-like behavior. We propose a simple non-Langmuir kinetic model accounting for an enhanced electrostatic repulsion originating from the surface immobilization of nucleic acids and for steric hindrance close to full hybridization of the surface probes. The analysis of the kinetic data by the model enables quantifying the repulsive potential at the surface, as well as retrieving the kinetic parameters of isolated probes. We show that the strength and the kinetics of hybridization at large probe density can be improved by a three-dimensional immobilization strategy of probe strands with a double-stranded linker.

The estimation of quantitative parameters of oligonucleotides immobilization on mica surface

Immobilization of nucleic acids on the surface of various materials is increasingly being used in research and some practical applications. Currently, the DNA chip technology is rapidly developing. The basis of the immobilization process can be both physical adsorption and chemisorption. A useful way to control the immobilization of nucleic acids on a surface is to use atomic force microscopy. It allows you to investigate the topography of the surface by its direct imaging with high resolution. Usually, to fix the DNA on the surface of mica are used cations which mediate the interaction between the mica surface and the DNA molecules. In our work we have developed a method for estimation of quantitative parameter of immobilization of oligonucleotides is their degree of aggregation depending on the fixation conditions on the surface of mica. The results on study of aggregation of oligonucleotides immobilized on mica surface will be presented. The single oligonucleotides molecules have been imaged clearly, whereas their surface areas have been calculated and calibration curve has been plotted.

Attomolar electrochemical detection of the BCR/ABL fusion gene based on an amplifying self-signal metal nanoparticle-conducting polymer hybrid composite

In the last ten years, conjugated polymers started to be used in the immobilization of nucleic acids via non-covalent interactions. In the present study, we describe the construction and use of an electrochemical DNA biosensor based on a nanostructured polyaniline-gold composite, specifically developed for the detection of the BCR/ABL chimeric oncogene. This chromosome translocation is used as a biomarker to confirm the clinical diagnosis of both chronic myelogenous leukemia (CML) and acute lymphocytic leukemia (ALL). The working principle of the biosensor rests on measuring the conductivity resulting from the non-covalent interactions between the hybrid nanocomposite and the DNA probe. The nanostructured platform exhibits a large surface area that enhances the conductivity. Positive cases, which result from the hybridization between DNA probe and targeted gene, induce changes in the amperometric current and in the charge transfer resistance (RCT) responses. Atomic force microscopy (AFM) images showed changes in the genosensor surface after exposure to cDNA sample of patient with leukemia, evidencing the hybridization process. This new hybrid sensing-platform displayed high specificity and selectivity, and its detection limit is estimated to be as low as 69.4 aM. The biosensor showed excellent analytical performance for the detection of the BCR/ABL oncogene in clinical samples of patients with leukemia. Hence, this electrochemical sensor appears as a simple and attractive tool for the molecular diagnosis of the BCR/ABL oncogene even in early-stage cases of leukemia and for the monitoring of minimum levels of residual disease.

New Approaches for Bottom-Up Assembly of Tobacco Mosaic Virus-Derived Nucleoprotein Tubes on Defined Patterns on Silica- and Polymer-Based Substrates

The capability of some natural molecular building blocks to self-organize into defined supramolecular architectures is a versatile tool for nanotechnological applications. Their site-selective integration into a technical context, however, still poses a major challenge. RNA-directed self-assembly of tobacco mosaic virus-derived coat protein on immobilized RNA scaffolds presents a possibility to grow nucleoprotein nanotubes in place. Two new methods for their site-selective, bottom-up assembly are introduced. For this purpose, isothiocyanate alkoxysilane was used to activate oxidic surfaces for the covalent immobilization of DNA oligomers, which served as linkers for assembly-directing RNA. Patterned silanization of surfaces was achieved (1) on oxidic surfaces via dip-pen nanolithography and (2) on polymer surfaces (poly(dimethylsiloxane)) via selective oxidization by UV-light irradiation in air. Atomic force microscopy and X-ray photoelectron spectroscopy were used to characterize the surfaces. It is shown for the first time that the combination of the mentioned structuring methods and the isothiocyanate-based chemistry is appropriate (1) for the site-selective immobilization of nucleic acids and, thus, (2) for the formation of viral nanoparticles by bottom-up self-assembly after adding the corresponding coat proteins.

Oligonucleotide immobilization using 10-(carbomethoxy)decyl-dimethylchlorosilane for mRNA isolation and cDNA synthesis on a microfluidic chip

Fabrication of microfluidic systems capable of extracting and isolating nucleic acids from biological samples and preparing them for downstream applications within the same device is of interest as on-chip sample preparation reduces the time and effort expended on multi-step benchtop procedures. A microfluidic chip capable of cell lysis, nucleic acid extraction and immobilization has been developed in our laboratory. This report focuses on substrate development and chip integration of the nucleic acid immobilization platform. The immobilization region was initially developed using poly-adenylated mRNA released from normal human lymphoblastoid cells. After immobilization, the mRNA remains functional for amplification by the reverse transcription polymerase chain reaction (RT-PCR) and quantitative real-time PCR (qPCR) gene expression analyses. Detailed surface functionalization, chemical modification, and covalent oligonucleotide immobilization methods are described. Vapor and liquid deposition techniques for Pyrex functionalization by 10-(carbomethoxy)decyl-dimethylchlorosilane (CMDCS) were developed and an optimal method was determined for microfluidic chip application. CMDCS substrate depositions and modification were characterized by atomic force microscopy and contact angle goniometry, respectively. Oligonucleotide immobilizations and mRNA bindings were determined by fluorescence microscopy. Functional, selective platform integration within the microfluidic chip was confirmed by standard RT-PCR and qPCR systems.

Oligonucleotide derivatives in the hybridization analysis of nucleic acids. I. Covalent immobilization of oligonucleotide probes on nylon

The features of UV-induced immobilization of oligonucleotides on a nylon membranes and the effectiveness of enzymatic labeling of immobilized probes at heterophase detection of nucleic acids are studied. Short terminal oligothymidilate (up to 10 nt) sequences are suggested to attach to the probe via a flexible ethylene glycol based linker. The presence of such fragment enhances the intensity of immobilization and reduces UV-dependent degradation of the targeted (sequence-specific) part of the probe by reducing the dose needed for the immobilization of DNA. The optimum dose of UV-irradiation is determined to be ~0.4 J/cm(2) at the wavelength 254 nm. This dose provides high level of hybridization signal for immobilized probes with various nucleotide composition of the sequence specific moiety. The amide groups of the polyamide are shown to play the key role in the photoinduced immobilization of nucleic acids, whereas the primary amino groups in the structure of PA is not the center responsible for the covalent binding of DNA by UV-irradiation, as previously believed. Various additives in the soaking solution during the membrane of UV-dependent immobilization of probes are shown to influence its effectiveness. The use of alternative to UV-irradiation system of radical generation are shown to provide the immobilization of oligonucleotides onto the nylon membrane.

A gel-based solid-phase amplification and its application for SNP typing and sequencing on-chip

As conventional solid-phase amplification (SPA) on a two-dimensional slide has a low amplification capacity due to a limited amount of immobilized primers, we propose a three-dimensional SPA by immobilizing primers in hydrogel attached to a slide. One of the PCR primers, modified with an acrylamide group at the 5'-terminal, was copolymerized with both polyacrylamide gel and an acryl-modified glass slide, resulting in a high amplification capacity. The immobilization process was carried out by adding the catalysis reagent N,N,N',N'-tetramethylethylenediamine (TEMED) volatilized in vacuum, with uniform sample-concentration and gel-viscosity in the course of one-step nucleic acid immobilization. The porous structure of polyacrylamide gel, which allows PCR reagents such as Taq DNA polymerase, primers, dNTPs and DNA templates to freely enter the gel matrix, provides a homogeneous solution-mimicking environment for SPA on the interface or the inside of gel pads. Based on gel-based SPA, genotypes of different samples were accurately discriminated by either dual-color fluorescence hybridization or BAMPER (Bioluminometric Assay coupled with Modified Primer Extension Reactions). Pyrosequencing was also successfully carried out on SPA products. As the linkage between DNA molecules and gel is very strong, SPA products immobilized on gel pads could be reused several times if extended strands were removed by electrophoresis. Thus, the gel-based SPA provides a powerful tool for directly using on-chip amplicons for parallel detection.

Improved Multi-Stranded Nucleic Acid Immobilization Technique: Enhanced Double-Stranded, Triple-Stranded and Four-Stranded DNA Microarray Platform

Extended abstract of a paper presented at Microscopy and Microanalysis 2008 in Albuquerque, New Mexico, USA, August 3 – August 7, 2008

Single probe nucleic acid immobilization on chemically modified single protein by controlling ionic strength and pH

In an effort toward determining the feasibility of single molecule analysis, we describe a case whereby the binding of one biotinylated DNA to one streptavidin molecule via electrostatic interactions was controlled by altering in pH 4.0–9.0 and 0.16 of the ion strength. The quantitative analysis of immobilized probe ssDNA was realized in real-time via a quartz crystal microbalance (QCM) and electrochemical (EC) measurement in the range 100 pM to 50 μM of probe oligonucleotide concentration. The variation amount of biotinylated ssDNA immobilized on the streptavidin-modified surface at pH 7.5 was about 0.16 pmol, giving a ratio of streptavidin to biotinylated ssDNA of about 1:1.1. On the other hand, at pH 4.9, it was immobilized about 0.29 pmol. From the shape of the Langmuir plot and QCM, the immobilization efficiency of biotinylated DNA via streptavidin at pH 4.9 was approximately twofold that at pH 7.5. In view points of the reaction velocity, it was increased with decreasing buffer solution pH, indicating a strong interaction of negatively charged probe DNA with the positively charged streptavidin. And also the EC response value of Δ I/ I streptavidin for the immobilized biotinylated ssDNA in pH 4.9 was about 49%, while the corresponding value for the pH 7.5 was approximately 34%. As DNA molecules possess negative charges, electrostatic repulsion occurred between streptavidin and biotinylated ssDNA at pH 7.5. At pH 4.9, the attraction between the biotinylated ssDNA and streptavidin resulted in increased adsorption which has an isoelectric point of about 5.9. It was deduced that the binding of biotinylated ssDNA to one or two of the four binding sites of streptavidin can be controlled by adjusting the pH-controlled electrostatic interaction.

Directed immobilization of nucleic acids at ultramicroelectrodes using a novel electro-deposited polymer

A two-step method for the directed immobilization of nucleic acids at ultramicroelectrodes with micron-size dimensions is described. The approach is based on the immobilization of streptavidin at the surface of carbon or noble metal electrodes within a novel electro-deposited polymer, formed by electropolymerization of the natural compound scopoletin (7-hydroxy-6-methoxy-coumarin) at potentials between 0.4 and 0.7 V vs. Ag/AgCl. Biotin-tagged nucleic acids or proteins are immobilized on top of the modified electrodes in a second step. The new method has some advantages compared to classical electropolymerization approaches (e.g. polypyrrole, polyphenol) , because the growing polymer is highly hydrophilic, resulting in efficient incorporation of streptavidin and a high biotin binding capacity of 6 pmol cm −2. The polymer film seems to be non-conductive but shows good swelling properties in aqueous solutions. The feasibility of the method for the electro-directed biochemical modification of individual microelectrodes has been demonstrated by sequential immobilization of two different single strand oligonucleotides onto interdigitated ultramicroelectrodes. The resulting miniature DNA probe was used for single base mutation detection with two synthetic targets (fluorescence-labeled 17-mer oligomers) by evaluating the fluorescence patterns after hybridisation with the immobilised DNA probes. The new method is useful for the production of microelectrode based DNA chips and for the electro-directed immobilisation of biomolecules at microelectrode structures with high spatial resolution and yield.

Dendrislides, dendrichips: a simple chemical functionalization of glass slides with phosphorus dendrimers as an effective means for the preparation of biochips

Materials with tailored surface properties are useful for diverse applications, in particular for adsorption and/or covalent immobilization of various biomolecules such as nucleic acids and proteins. One of the main challenges is to design a reactive surface chemistry for stable binding of biomolecules on the support, which in addition keeps away the biomolecules from the support to reduce interference. We report here a novel support functionalization that is based on the formation of a reactive layer, covalently grafted on glass slides onto which amino-modified DNA probes were covalently fixed. The layer is composed of spherical home-made neutral phosphorus dendrimers containing a large number of aldehyde functions at their periphery. The method of preparation of these dendrimer-activated glass slides (so-called dendrislides) is performed in a few steps and results in stable activated supports, since aldehyde dendrimers are stable compounds. After immobilization of nucleic acids the so-called dendrichip products were investigated by means of hybridization experiments using complementary fluorescent labelled-oligonucleotide targets. Our results indicate that this novel grafting technology leads to surfaces with a high binding capacity for amino-modified oligonucleotides compared to commercially available aldehyde slides and with a detection limit of 1 pM labelled targets. Since links between the surface, the dendrimers and the nucleic acids are covalent, the dendrichips could be re-used up to 10 times without significant change in the fluorescence signal intensity. Finally, the performance of dendrichips in detection of a single base mutation was demonstrated.

Immobilization of Nucleic Acids at Solid Surfaces: Effect of Oligonucleotide Length on Layer Assembly

This report investigates the effect of DNA length and the presence of an anchoring group on the assembly of presynthesized oligonucleotides at a gold surface. The work seeks to advance fundamental insight into issues that impact the structure and behavior of surface-immobilized DNA layers, as in, for instance, DNA microarray and biosensor devices. The present study contrasts immobilization of single-stranded DNA (ssDNA) containing a terminal, 5′ hexanethiol anchoring group with that of unfunctionalized oligonucleotides for lengths from 8 to 48 bases. Qualitatively, the results indicate that the thiol anchoring group strongly enhances oligonucleotide immobilization, but that the enhancement is reduced for longer strand lengths. Interestingly, examination of the probe coverage as a function of strand length suggests that adsorbed thiol-ssDNA oligonucleotides shorter than 24 bases tend to organize in end-tethered, highly extended configurations for which the long-term surface coverage is largely independent of oligonucleotide length. For strands longer than 24 bases, the surface coverage begins to decrease notably with probe length. The decrease is consistent with a less ordered arrangement of the DNA chains, presumably reflecting increasingly polymeric behavior.

Nucleic-acid immobilization, recognition and detection at chronopotentiometric DNA chips

Wide-scale DNA testing requires the development of fast, small, easy-to-use biosensing devices. Various synthetic oligonucleotides and DNA have thus been immobilized onto microfabricated thick-film carbon transducers for performing several new nucleic-acid assay protocols. These include hybridization detection of nucleic acid sequences, determination of small molecules (drugs, pollutants) based on their collection into the dsDNA layer or via monitoring their effect upon the intrinsic DNA oxidation signal, and direct adsorptive stripping measurementsof ultratrace levels of nucleic acids. Transduction of these DNA recognition processes is accomplished by a new highly-sensitive constant-current stripping chronopotentiometric operation. Comparison to traditional electrodes indicates that the biosensing performance is not compromised by the use of mass-producible disposable transducers. Such thick-film DNA biosensors have been coupled to a compact, user-friendly, hand-held analyzer. Applicability for the detection of sequences from M. tuberculosis and HIV-1 DNAs is illustrated. Such activity in the author's laboratory, aimed at developing DNA-coated screen-printed electrodes, is reviewed.

Multilayer films containing immobilized nucleic acids. Their structure and possibilities in biosensor applications

Langmiur-Blodgett (LB) and film technologies based on electrostatic attraction self-assembly (SA) are shown to be useful for immobilization of nucleic acids (DNA, polynucleotides) onto solid supports in sensor devices. The nucleic acids were immobilized in complexes with cationic surfactants (for LB) and polycations (for SA). Infrared spectral studies showed that DNA unfolds in multilayer LB films with octadecylamine and conserves its double helical structure in the LB films with dioctadecyldimethylammonium and in the SA films with polyallylamine, polyethylenimine and poly-L-lysine. Atomic groups and the types of interactions determining the complex formation of these films have been identified. The hydration of LB and SA films was studied to find out binding sites of water molecules and to evaluate the flexibility of nucleic acid compounds in the multilayer films. The possibilities of biosensor applications of these LB and SA films were monitored on binding of specific reagents for DNA by DNA-containing films and mononucleotides by a complementary single-stranded polynucleotide immobilized on a positively charged solid support.

Critical evaluation of membrane supports for use in quantitative hybridizations

The quantification of 16S rRNA by oligonucleotide probe hybridization was investigated with MagnaGraph (Micron Separation, Inc. [MSI]), Magna Charge (MSI), Magna (MSI), Immobilon-N (Millipore Corporation), and Nytran (Schleicher & Schuell, Inc.) membranes as supports for nucleic acid immobilization. The levels of detectability provided by the Magna Charge and Immobilon-N membranes were 20 to 50 times better than those obtained with the MagnaGraph, Magna, and Nytran membranes. The variability of the signal response for individual membranes ranged from 10 to 50%, with the Magna Charge and Immobilon-N membranes demonstrating the lowest variability.

4806631 Immobilization of nucleic acids on solvolyzed nylon supports: Robert J Carrico, Robert Hatch, William L Patterson assigned to Miles Inc

4806631 Immobilization of nucleic acids on solvolyzed nylon supports: Robert J Carrico, Robert Hatch, William L Patterson assigned to Miles Inc

4818681 Fast and specific immobilization of nucleic acids to solid supports: Nanibhushan Dattagupta assigned to Molecular Diagnostics Inc

4818681 Fast and specific immobilization of nucleic acids to solid supports: Nanibhushan Dattagupta assigned to Molecular Diagnostics Inc