Desorption Of DNA Research Articles

Comparison of multilocus sequence typing, RAPD, and MALDI-TOF mass spectrometry for typing of β-lactam–resistant Klebsiella pneumoniae strains

Extended spectrum of β-lactam (ESBL) resistance of Klebsiella pneumoniae has become an increasing problem in hospital infections. Typing of isolates is important to establish the intrahospital surveillance of resistant clones. In this study, the discriminatory potential of randomly amplified polymorphic DNA and matrix-assisted laser desorption ionization–time of flight (MALDI-TOF) analyses were compared with multilocus sequence typing (MLST) by using 17 β-lactam–resistant K. pneumoniae isolates of different genotypes. MLST alleles were distributed in 8 sequence types (STs). Among ESBL strains of the same ST, the presence of different β-lactamase genes was common. RAPD band patterns also revealed 8 types that corresponded to MLST-defined genotypes in 15 out of 17 cases. MALDI-TOF analysis could differentiate 5 clusters of strains. The results of this work show that RAPD may be usable as a rapid screening method for the intrahospital surveillance of K. pneumoniae, allowing a discrimination of clonally related strains. MALDI-TOF-based typing was not strongly corresponding to genotyping and warrants further investigation.

Use of magnetic polyaniline/maghemite nanocomposite for DNA retrieval from aqueous solutions

We demonstrated that the magnetic polyaniline/maghemite nanocomposite (Pani/γ-Fe2O3 MNC) is an efficient agent for retrieval of pure double stranded deoxyribonucleic acid (dsDNA) chains from aqueous solutions. The dsDNA chains used in the retrieval experiments were of sodium salt of Salmon Sperm DNA. Based on λ=260nm absorption measurements, we have employed UV–Vis spectroscopy to estimate the concentration of DNA present in solutions, before and after the interaction with the MNC. The best results corresponded to a maximum amount of 75.2mg of DNA absorbed per gram of MNC reached within only 10min of joint exposure into the aqueous solution. After magnetic separation of the fully DNA-loaded Pani/γ-Fe2O3 MNC, we achieved essentially complete DNA desorption by appropriate changes in the pH of the solution. We have shown that it is possible to recycle the use of these MNC in several adsorption–desorption cycles. By comparing the present results to those of other DNA retrieval systems reported in the literature, we argued that the Pani/γ-Fe2O3 MNC here described represent a promising low-cost material for use as a fast, simple and efficient method of DNA separation and concentration.

Processing of abasic site damaged lesions by APE1 enzyme on DNA adsorbed over normal and organomodified clay

The efficiency of the apurinic/apyrimidinic endonuclease (APE1) DNA repair enzyme in the processing of abasic site DNA damage lesions at precise location in DNA oligomer duplexes that are adsorbed on clay surfaces was evaluated. Three different forms of clay namely montmorillonite, quaternary ammonium salt modified montmorillonite and its boiled counterpart i.e. partially devoid of organic moiety were used for a comparative study of adsorption, desorption and DNA repair efficiency on their surfaces. The interaction between the DNA and the clay was analysed by X-ray diffraction, Atomic force microscopy, UV–Vis spectroscopy and Infrared spectroscopy. The abasic site cleavage efficiency of APE1 enzyme was quantitatively evaluated by polyacrylamide gel electrophoresis. Apart from the difference in the DNA adsorption or desorption capacity of the various forms of clay, substantial variation in the repair efficiency of abasic sites initiated by the APE1 enzyme on the clay surfaces was observed. The incision efficiency of APE1 enzyme at abasic sites was found to be greatly diminished, when the DNA was adsorbed over organomodified montmorillonite. The reduced repair activity indicates an important role of the pendant surfactant groups on the clay surfaces in directing APE1 mediated cleavage of abasic site DNA damage lesions.

A wormlike chain model of forced desorption of a polymer adsorbed on an attractive wall

Forced desorption of a semiflexible polymer chain on a solid substrate is theoretically investigated. The pulling force versus displacement is studied for varying adsorption energy ε and persistence length P. It is found that the relationships between pulling force and cantilever displacement show a series of characteristic force spikes at different persistence lengths P. These force spikes become more pronounced but the average magnitude of this force decreases as P grows. Our results are of relevance to the forced desorption of DNA on an attractive wall in single-molecule pulling experiments.

Adsorption of DNA on biomimetic apatites: Toward the understanding of the role of bone and tooth mineral on the preservation of ancient DNA

In order to shed some light on DNA preservation over time in skeletal remains from a physicochemical viewpoint, adsorption and desorption of DNA on a well characterized synthetic apatite mimicking bone and dentin biominerals were studied. Batch adsorption experiments have been carried out to determine the effect of contact time (kinetics), DNA concentration (isotherms) and environmentally relevant factors such as temperature, ionic strength and pH on the adsorption behavior. The analogy of the nanocrystalline carbonated apatite used in this work with biological apatite was first demonstrated by XRD, FTIR, and chemical analyses. Then, DNA adsorption kinetics was fitted with the pseudo-first order, pseudo-second order, Elovich, Ritchie and double exponential models. The best results were achieved with the Elovich kinetic model. The adsorption isotherms of partially sheared calf thymus DNA conformed satisfactorily to Temkin's equation which is often used to describe heterogeneous adsorption behavior involving polyelectrolytes. For the first time, the irreversibility of DNA adsorption toward dilution and significant phosphate-promoted DNA desorption were evidenced, suggesting that a concomitant ion exchange process between phosphate anionic groups of DNA backbone and labile non-apatitic hydrogenphosphate ions potentially released from the hydrated layer of apatite crystals. This work should prove helpful for a better understanding of diagenetic processes related to DNA preservation in calcified tissues.

Programmable ion-sensitive transistor interfaces. II. Biomolecular sensing and manipulation

The chemoreceptive neuron metal-oxide-semiconductor transistor described in the preceding paper is further used to monitor the adsorption and interaction of DNA molecules and subsequently manipulate the adsorbed biomolecules with injected static charge. Adsorption of DNA molecules onto poly-L-lysine-coated sensing gates (SGs) modulates the floating gate (FG) potential ψ(O), which is reflected as a threshold voltage shift measured from the control gate (CG) V(th_CG). The asymmetric capacitive coupling between the CG and SG to the FG results in V(th_CG) amplification. The electric field in the SG oxide E(SG_ox) is fundamentally different when we drive the current readout with V(CG) and V(ref) (i.e., the potential applied to the CG and reference electrode, respectively). The V(CG)-driven readout induces a larger E(SG_ox), leading to a larger V(th_CG) shift when DNA is present. Simulation studies indicate that the counterion screening within the DNA membrane is responsible for this effect. The DNA manipulation mechanism is enabled by tunneling electrons (program) or holes (erase) onto FGs to produce repulsive or attractive forces. Programming leads to repulsion and eventual desorption of DNA, while erasing reestablishes adsorption. We further show that injected holes or electrons prior to DNA addition either aids or disrupts the immobilization process, which can be used for addressable sensor interfaces. To further substantiate DNA manipulation, we used impedance spectroscopy with a split ac-dc technique to reveal the net interface impedance before and after charge injection.

Synthesis and properties of magnetic sorbents based on transition metal ferrites

We have developed and optimized a procedure for the synthesis of magnetically controlled nano-composites based on transition metal oxides (MFe2O4, M = Fe2+, Ni2+, and Co2+) with a biocompatible (SiO2)x coating. The relationship between the structure, composition, and magnetic properties of the nanocomposites has been examined. We have studied adsorption interactions of immunoglobulins (Ig) and DNA with the surface layer of the magnetically controlled nanocomposites. The amount of Ig adsorption was determined to be ∼0.5 mg for 100 mg of the adsorbents, and that of DNA adsorption, 0.55–0.6 mg for 100 mg of the adsorbents. Our results demonstrate complete Ig and DNA desorption from the surface of the nanocomposites.

Controlling the adsorption and desorption of double-stranded DNA on functionalized carbon nanotube surface

We demonstrated controlled adsorption and desorption of double-stranded DNA (dsDNA) on single-walled carbon nanotube (SWNT) surface functionalized with polyethyleneglycol (PEG SWNT). First, when dsDNA molecules were mixed with the PEG SWNT solution, the DNA molecules spontaneously adsorbed onto the PEG SWNT surface and formed dsDNA–PEG SWNT conjugates without sonication. Next, we succeeded in detaching the dsDNA adsorbed on PEG SWNT by annealing at 95°C for 30min. These results were confirmed using atomic force microscopy, agarose gel electrophoresis, and micro-Raman spectroscopy. In contrast, when we used the usual SWNT produced by the high-pressure carbon monoxide method (HiPco SWNT), the DNA molecules were fragmented during the adsorption process as sonication was necessary for the hybridization of DNA–SWNT conjugates. Furthermore, detachment of DNA molecules from HiPco SWNT by annealing was impossible. Our method may be useful for developing DNA devices using SWNTs as substrates when it is combined with previously established various biochemical techniques.

Charge reversible gold nanoparticles for high efficient absorption and desorption of DNA

Mercaptoundecylamine and mercaptoundecanoic acid co-modified Au nanoparticles were prepared by two-step ligand exchange of 6-mercaptohexanoic acid modified gold nanoparticles. Such particles terminated by appropriate ratios of the amine and carboxyl groups (R N/C) were identified to show reversible charge on their surface, which were switchable by pH of the solution. The isoelectric point (IEP) of the particles is tunable by changing the ratios of the amine and carboxyl groups on the particle surfaces. The particles can absorb DNA effectively at pH lower than the IEP driven by the direct electrostatic interactions between DNA and the particle surface. When pH of the solutions was elevated to be higher than the IEP, the absorbed DNA can be released almost completely due to the electrostatic repulsion between the particle surface and DNA. With appropriate R N/C ratios of 0.8, the absorption and desorption efficiencies of DNA were 97 and 98 %, respectively, corresponding an extraction efficiency of 95 %. Such particles with reversible surface charges allow the high efficient extraction of DNA by simply changing pH instead of by changing salt concentration in the conventional salt bridge method.

Semiquantification of ATP in Live Cells Using Nonspecific Desorption of DNA from Graphene Oxide as the Internal Reference

In this aritcle, we have developed an interesting imaging method for intracellular ATP molecules with semiquantitation. While there has been a lot of work in understanding intracellular events, very few can come close to quantitation or semiquantitation in living cells. In this work, we made an effective use of nanomaterials, graphene oxides, both as a quencher and a carrier for intracellular delivery. In addition, this graphene oxide also serves as the carrier for reference probes for fluorescent imaging. An ATP aptamer molecular beacon (AAMB) is adsorbed on graphene oxide (GO) to form a double quenching platform. The AAMB/GO spontaneously enters cells, and then AAMB is released and opened by intracellular ATP. The resulting fluorescence recovery is used to perform ATP live-cell imaging with greatly improved background and signaling. Moreover, a control ssDNA, which is released nonspecifically from GO by nontarget cellular proteins, can serve as an internal reference for ATP semiquantification inside living cells using the intensity ratio of the AAMB and control. This approach can serve as a way for intracellular delivery and quantitative analysis.

Molecular Beacon Lighting up on Graphene Oxide

A molecular beacon (MB) is comprised of a fluorophore and a quencher linked by a DNA hairpin. MBs have been widely used for homogeneous DNA detection. In addition to molecular quenchers, many nanomaterials such as graphene oxide (GO) also possess excellent quenching efficiency. Most reported fluorescent sensors relied on DNA probes physisorbed by GO, which may suffer from nonspecific probe displacement and false positive signal. In this work, we report the preparation and characterization of a MB using graphene oxide (GO) as quencher, where an amino and FAM (6-carboxyfluorescein) dual labeled DNA was covalently attached to GO via an amide linkage. A major challenge was to remove noncovalently attached probes due to strong DNA adsorption by GO. While DNA desorption was favored at low salt, high pH, high temperature, and by using organic solvents, the cDNA was required to achieve complete desorption of noncovalently linked DNA probes. The DNA adsorption energy was measured using isothermal titration calorimetry, revealing the heterogeneous nature of GO. The covalent probe has a detection limit of 2.2 nM using a sample volume of 0.05 mL. With a 2 mL sample, the detection limit can reach 150 pM. The covalent probe is highly resistant to nonspecific probe displacement and will find applications in serum and cellular samples where high probe stability is demanded.

Pd Nanowires as New Biosensing Materials for Magnified Fluorescent Detection of Nucleic Acid

The designed synthesis of new nanomaterials with controlled shape, composition, and structure is critical for tuning their physical and chemical properties, and further developing interesting analytical sensing devices. Herein, we presented that Pd nanowires (NWs) can be used as a new biosensing platform for high-sensitivity nucleic acid detection. The general sensing concept is based on the fact that Pd NWs can adsorb the fluorescently labeled single-stranded DNA probe and lead to substantial fluorescence quenching of dye, followed by specific hybridization with the complementary region of the target DNA sequence. This results in desorption of double-stranded DNA from Pd NWs surface and subsequent recovery of fluorescence. Furthermore, an amplification strategy based on Pd NWs for nucleic acid detection by using exonuclease III (Exo III) was demonstrated. The present dual-magnification sensing system combined Pd NWs with Exo III has a detection range of 1.0 nM to 2.0 μM with the detection limit of 0.3 nM (S/N = 3), which is about 20-fold higher than that of traditional unamplified homogeneous assays.

Surface Science of DNA Adsorption onto Citrate-Capped Gold Nanoparticles

Single-stranded DNA can be adsorbed by citrate capped gold nanoparticles (AuNPs), resulting in increased AuNP stability, which forms the basis of a number of biochemical and analytical applications, but the fundamental interaction of this adsorption reaction remains unclear. In this study, we measured DNA adsorption kinetics, capacity, and isotherms, demonstrating that the adsorption process is governed by electrostatic forces. The charge repulsion among DNA strands and between DNA and AuNPs can be reduced by adding salt, reducing pH or by using noncharged peptide nucleic acid (PNA). Langmuir adsorption isotherms are obtained, indicating the presence of both adsorption and desorption of DNA from AuNPs. While increasing salt concentration facilitates DNA adsorption, the desorption rate is also enhanced in higher salt due to DNA compaction. DNA adsorption capacity is determined by DNA oligomer length, DNA concentration, and salt. Previous studies indicated faster adsorption of short DNA oligomers by AuNPs, we find that once adsorbed, longer DNAs are much more effective in protecting AuNPs from aggregation. DNA adsorption is also facilitated by using low pH buffers and high alcohol concentrations. A model based on electrostatic repulsion on AuNPs is proposed to rationalize the DNA adsorption/desorption behavior.

In‐line coupling SPE and CE for DNA preconcentration and separation

An in-line SPE method coupled to CE was developed for the analysis of DNA. The amino silica monolith was prepared in situ by polymerization of tetraethoxysilane and N-(β-aminoethyl)-γ-aminopropyltriethoxysilane in ethanol aqueous solution at the inlet end of a 100 μm id fused-silica capillary, and the remaining part of the capillary was used as separation channel. The procedure for this in-line SPE-CE method was constructed on the basis of investigation on operational conditions such as the introduction mode of sieving matrix, the composition of elution solvent and the elution time. Twenty millimolar ammonium hydroxide was demonstrated to be effective for DNA desorption from the monolith, and linear poly(N-isopropylacrylamide) was used as the separation matrix. The proposed method could achieve limits of detection of 0.065-0.123 ng/mL for six DNA fragments ranging 100-2000 bp. Compared with conventional CE, preconcentration factors of over 100 times were obtained. The applicability of the in-line SPE-CE method was further demonstrated by analyzing plasmid DNA from Escherichia coli crude lysate.

Adsorption and Desorption Behaviors of DNA with Magnetic Mesoporous Silica Nanoparticles

The interaction between DNA and mesopores is one of the basic concerns when mesoporous silica nanoparticle (MSN) is used as a DNA carrier. In this work, we have synthesized a type of mesoporous silica nanoparticle that has a Fe(3)O(4) inner core and mesoporous silica shell. This magnetic mesoporous silica nanoparticle (denoted as M-MSN) offers us a convenient platform to manipulate the DNA adsorption and desorption processes as it can be easily separated from solution by applying a magnetic field. The DNA adsorption behavior is studied as a function of time in chaotropic salt solution. The maximum amount of adsorbed DNA is determined as high as 121.6 mg/g. We have also developed a method to separate the DNA adsorbed onto the external surface and into the mesopores by simply changing temperature windows. The desorption results suggest that, within the whole adsorbed DNA molecules, about 89.5% has been taken up by M-MSN mesopores. Through the dynamic light scattering experiment, we have found that the hydrodynamic size for M-MSN with DNA in its mesopores is higher than the naked M-MSN. Finally, the preliminary result of the adsorption mechanism study suggests that the DNA adsorption into mesopores may generate more intermolecular hydrogen bonds than those formed on the external surface.

Study of DNA Interaction with Cobalt Ferrite Nanoparticles

Interaction of cobalt ferrite nanopowder and nucleic acid was investigated. Superparamagnetic cobalt ferrite nanoparticles (6-12 nm) were prepared by mechanochemical synthesis. Structure of the nanopowder was characterized using X-ray diffraction. It was shown that cobalt ferrite nanoparticles were associated with ssDNA and dsDNA in Tris-buffer resulting in bionanocomposite formation with mass weight relation nanoparticles: DNA 1:(0.083 +/- 0.003) and 1:(0.075 +/- 0.003) respectively. The mechanism of interaction between a DNA and cobalt ferrite nanoparticles was considered basing on the whole set of obtained data: FTIR-spectroscopy, analyzing desorption of DNA from the surface of the particles while changing the chemical content of the medium, and on the modeling interaction of specific biomolecule fragments with surface of a inorganic material. It was supposed that the linkage was based on coordination interaction of the phosphate groups and oxygen atoms heterocyclic bases of DNA with metal ions on the particle surface. These data can be used to design specific magnetic DNA-nanoparticles hybrid structures.

DNA adsorption and desorption on mica surface studied by atomic force microscopy

The adsorption of DNA molecules on mica surface and the following desorption of DNA molecules at ethanol–mica interface were studied using atomic force microscopy. By changing DNA concentration, different morphologies on mica surface have been observed. A very uniform and orderly monolayer of DNA molecules was constructed on the mica surface with a DNA concentration of 30 ng/μL. When the samples were immersed into ethanol for about 15 min, various desorption degree of DNA from mica (0–99%) was achieved. It was found that with the increase of DNA concentration, the desorption degree of DNA from the mica at ethanol–mica interface decreased. And when the uniform and orderly DNA monolayers were formed on the mica surface, almost no DNA molecule desorbed from the mica surface in this process. The results indicated that the uniform and orderly DNA monolayer is one of the most stable DNA structures formed on the mica surface. In addition, we have studied the structure change of DNA molecules after desorbed from the mica surface with atomic force microscopy, and found that the desorption might be ascribed to the ethanol-induced DNA condensation.

Electrostatic interaction effect for human DNA separation with functionalized mesoporous silicas

This work describes the development of highly efficient human DNA separation with functionalized mesoporous silica (FMS) materials. To demonstrate the electrostatic interaction effect between the target DNA molecules and FMS, three aminofunctionality types comprised of a mono-, a di-, and a tri-amine functional group were introduced on the inner surfaces of mesoporous silica particles. Systematic characterization of the synthesized materials was achieved by solid-state 29Si and 13C-NMR techniques, BET, FT-IR, and XPS. The DNA separation efficiency was explored via the function of the amino-group number, the amount used, and the added NaCl concentration. The DNA adsorption yields were high in terms of the use of triaminofunctionalized FMS at the 10 ng/L level, and the DNA desorption efficiency showed the optimum level at over 3.0 M NaCl concentration. The use of FMS in a DNA separation process provides numerous advantages over the conventional silica-based process.

Adsorption and Desorption of DNA on Graphene Oxide Studied by Fluorescently Labeled Oligonucleotides

Being the newest member of the carbon materials family, graphene possesses many unique physical properties resulting is a wide range of applications. Recently, it was discovered that graphene oxide can effectively adsorb DNA, and at the same time, it can completely quench adsorbed fluorophores. These properties make it possible to prepare DNA-based optical sensors using graphene oxide. While practical analytical applications are being demonstrated, the fundamental understanding of binding between graphene oxide and DNA in solution received relatively less attention. In this work, we report that the adsorption of 12-, 18-, 24-, and 36-mer single-stranded DNA on graphene oxide is affected by several factors. For example, shorter DNAs are adsorbed more rapidly and bind more tightly to the surface of graphene. The adsorption is favored by a lower pH and a higher ionic strength. The presence of organic solvents such as ethanol can either increase or decrease adsorption depending on the ionic strength of the solution. By adding the cDNA, close to 100% desorption of the absorbed DNA on graphene can be achieved. On the other hand, if temperature is increased, only a small percentage of DNA is desorbed. Further, the adsorbed DNA can also be exchanged by free DNA in solution. These findings are important for further understanding of the interactions between DNA and graphene and for the optimization of DNA and graphene-based devices and sensors.

Nanoscale Repulsive and Attractive Forces on Transistors - a Study of DNA Interaction with Non Volatile Charge

Label-free DNA detection by transistors is shown to have a tremendous potential to detect hybridization events and dielectric properties of adsorbed membranes within minutes. We report on an integrated-circuit detection scheme to monitor DNA-DNA interaction with high temporal resolution and novel features of manipulating the adsorbed DNA through long range in situ repulsive electrostatic forces. The floating-gate MOS transistor [1] can also be used as a detector in impedimetric mode by applying independent dc and ac biases from the control gate and the solution, respectively. This technique is shown to accurately measure changes in capacitance at the DNA-transistor interface as the operating point is held constant through the control gate. The output is a strong function of frequency. The transient IV and impedance spectroscopy then probe the surface adsorption, hybridization and molecular make up of the target DNA. In addition by using the property of fowler nordheim (FN) tunneling we can tunnel charges in and out of a floating gate. The stored charge creates an in situ refreshable mechanism at the interface that leads to DNA desorption. Experiments are underway to demonstrate addressable sensor pixel arrays. [1]. Krishna Jayant, Shantanu R. Rajwade, Lois Pollack and Edwin C.Kan, “Controlled Adsorption and Desorption on CMOS- Towards a Bi-Directional Bioelectronic Interface”, Biosensors (2010), World Congress on Biosensors and Bioelectronics, Glasgow, Scotland, UK 26-28thMay 2010. P31.081