Synthesis and hybridization properties of l-oligodeoxynucleotide analogues fixed in a low anti glycosyl conformation

We have synthesized l-type enantiomers (cU and cA) of nucleoside analogues, whose glycosyl bonds are fixed in a low anti conformation (ap glycosyl conformation, [small chi][approximate] 180[degree]), and incorporated them into oligonucleotides to evaluate the hybridization ability with natural DNA and RNA sequences. Although the incorporation of the modified nucleosides into oligonucleotides decreased the hybridization ability with unmodified complementary DNA sequences, the fully-substituted 12mers (cU(12) and cA(12)) still retained the hybridization ability with the complementary unmodified DNA 12mers, regardless of their unnatural l-chirality. In contrast, cU(12) and cA(12) showed different hybridization behavior with complementary unmodified RNA 12mers. cU(12) forms a more stable duplex with rA(12) than the corresponding natural 12mer (dT(12)), whereas cA(12) cannot hybridize with rU(12). Based on the model structure of cU(12)-rA(12), we discuss these experimental results.     

Lable‐Free Electrochemical DNA Sensor Based on Gold Nanoparticles/Poly(neutral red) Modified Electrode

We present a new strategy for the label‐free electrochemical detection of DNA hybridization based on gold nanoparticles (AuNPs)/poly(neutral red) (PNR) modified electrode. Probe oligonucledotides with thiol groups at the 5‐end were covalently linked onto the surface of AuNPs/PNR modified electrode via S‐Au binding. The hybridization event was monitored by using differential pulse voltammetry (DPV) upon hybridization generates electrochemical changes at the PNR‐solution interface. A significant decrease in the peak current was observed upon hybridization of probe with complementary target ssDNA, whereas no obvious change was observed with noncomplementary target ssDNA. And the DNA sensor also showed a high selectivity for detecting one‐mismatched and three‐mismatched target ssDNA and a high sensitivity for detecting complementary target ssDNA, the detection limit is 4.2×10^?12 M for complementary target ssDNA. In addition, the DNA biosensor showed an excellent reproducibility and stability under the DNA‐hybridization conditions.     

Solution pH-regulated interfacial adsorption of diblock phosphorylcholine copolymers

Spectroscopic ellipsometry has been used to examine the pH-responsive interfacial adsorption of a series of biocompatible diblock copolymers incorporating 2-methacryloyloxyethyl phosphorylcholine-based (MPC) residues and 2-(dialkylamino)ethyl methacrylate residues, with a specific focus on 2-(diethylamino)ethyl groups (referred to as MPCm-DEAn, where m and n refer to the mean degrees of polymerization of each block) at the hydrophilic silicon oxide/water interface. For all the copolymers studied the surface excess shows only weak concentration dependence. Increasing the length of the DEA block has little effect on the dynamic or equilibrated adsorption at pH 7, indicating that the DEA block adopts a flat conformation on the silicon oxide surface at this pH. With increasing pH, however, the surface excess shows a dramatic increase, followed by a subsequent decline. The observed maximum in surface excess represents a balance between charge over-compensation of the copolymer with the oppositely charged surface and the subsequently reduced charge density of the copolymer. Variations in the observed maxima for various MPCm-DEAn diblock copolymers indicate different surface conformations at high pH. Salt addition does not affect copolymer adsorption. This behavior is attractive for biomedical applications in which the ionic strength is variable. It was also found that the preadsorbed diblock copolymers immobilized DNA from solution to an extent that is proportional to the relative charge ratio between the anionic DNA and the cationic DEA block of the copolymer.     

Four-Dimensional Deoxyribonucleic Acid–Gold Nanoparticle Assemblies

Organization of gold nanoobjects by oligonucleotides has resulted in many three-dimensional colloidal assemblies with diverse size, shape, and complexity; nonetheless, autonomous and temporal control during formation remains challenging. In contrast, living systems temporally and spatially self-regulate formation of functional structures by internally orchestrating assembly and disassembly kinetics of dissipative biomacromolecular networks. We present a novel approach for fabricating four-dimensional gold nanostructures by adding an additional dimension: time. The dissipative character of our system is achieved using exonuclease III digestion of deoxyribonucleic acid (DNA) fuel as an energy-dissipating pathway. Temporal control over amorphous clusters composed of spherical gold nanoparticles (AuNPs) and well-defined core–satellite structures from gold nanorods (AuNRs) and AuNPs is demonstrated. Furthermore, the high specificity of DNA hybridization allowed us to demonstrate selective activation of the evolution of multiple architectures of higher complexity in a single mixture containing small and larger spherical AuNPs and AuNRs.     

基于金纳米颗粒信号放大效应电化学检测DNA聚合酶

基于金纳米颗粒(AuNPs)比表面积大、 尺寸小和能够承载大量DNA片段的特点, 建立了一种免标记、 简便、 快速检测DNA聚合酶Klenow fragment exo-(KF^-)的电化学方法. 首先将巯基化的DNA引物片段修饰在金电极上, 然后加入模板DNA链以及修饰有报告DNA链的金纳米颗粒(AuNPs-DNA), 模板DNA链能同时与DNA引物片段和修饰在AuNPs上的报告DNA链进行互补杂交形成"三明治"结构, 从而将AuNPs-DNA修饰在电极表面; 当加入电活性物质钌铵(RuHex)后, RuHex可通过静电吸附作用结合在DNA上. AuNPs上修饰的报告DNA链能够吸附大量RuHex, 导致电化学信号放大. 当加入脱氧核糖核苷三磷酸(dNTPs)以及KF^-聚合酶后, 引物片段发生延伸反应, 将与模板DNA链杂交的AuNPs-DNA竞争下来, 带走大量的RuHex, 使电信号降低, 从而实现对聚合酶的检测. 实验结果表明, 利用该方法可以检测到5 U/mL的KF^-.     

Four‐Dimensional Deoxyribonucleic Acid–Gold Nanoparticle Assemblies

Organization of gold nanoobjects by oligonucleotides has resulted in many three‐dimensional colloidal assemblies with diverse size, shape, and complexity; nonetheless, autonomous and temporal control during formation remains challenging. In contrast, living systems temporally and spatially self‐regulate formation of functional structures by internally orchestrating assembly and disassembly kinetics of dissipative biomacromolecular networks. We present a novel approach for fabricating four‐dimensional gold nanostructures by adding an additional dimension: time. The dissipative character of our system is achieved using exonuclease III digestion of deoxyribonucleic acid (DNA) fuel as an energy‐dissipating pathway. Temporal control over amorphous clusters composed of spherical gold nanoparticles (AuNPs) and well‐defined core–satellite structures from gold nanorods (AuNRs) and AuNPs is demonstrated. Furthermore, the high specificity of DNA hybridization allowed us to demonstrate selective activation of the evolution of multiple architectures of higher complexity in a single mixture containing small and larger spherical AuNPs and AuNRs.     

Electrochemical biosensors for detection of point mutation based on surface ligation reaction and oligonucleotides modified gold nanoparticles

An electrochemical method for point mutation detection based on surface ligation reaction and oligonucleotides (ODNs) modified gold nanoparticles (AuNPs) was demonstrated. Point mutation identification was achieved using Escherichia coli DNA ligase. This system for point mutation detection relied on a sandwich assay comprising capture ODN immobilized on Au electrodes, target ODN and ligation ODN. Because of the sequence-specific surface reactions of E. coli DNA ligase, the ligation ODN covalently linked to the capture ODN only in the presence of a perfectly complementary target ODN. The presence of ligation products on Au electrode was detected using chronocoulometry through hybridization with reporter ODN modified AuNPs. The use of AuNPs improved the sensitivity of chronocoulometry in this approach, a detection limit of 0.9 pM complementary ODN was obtained. For single base mismatched ODN (smODN), a negligible signal was observed. Even if the concentration ratio of complementary ODN to smODN was decreased to 1:1000, a detectable signal was observed. This work may provide a specific, sensitive and cost-efficient approach for point mutant detection.     

Selective DNA detection at Zeptomole level based on coulometric measurement of gold nanoparticle-mediated electron transfer across a self-assembled monolayer

A selective DNA sensing with zeptomole detection level is developed based on coulometric measurement of gold nanoparticle (AuNPs)-mediated electron transfer (ET) across a self-assembled monolayer on the gold electrode. After immobilization of a thiolated hairpin-structured DNA probe, an alkanethiol monolayer was self-assembled on the resultant electrode to block [Fe(CN)6 ]-3-/4in a solution from accessing the electrode. In the presence of DNA target, hybridization between the DNA probe and the DNA target breaks the stem duplex of DNA probe. Consequently, stem moiety at the 3′-end of the DNA probes was removed from the electrode surface and made available for hybridization with the reporter DNA-AuNPs conjugates (reporter DNA-AuNPs). The thiolated reporter DNA matches the stem moiety at the 3′-end of the DNA probe. AuNPs were then enlarged by immersing the electrode in a growth solution containing HAuCl 4 and H2O2 after the reporter DNA-AuNPs bound onto the electrode surface. The enlarged AuNPs on the electrode restored the ET between the electrode and the [Fe(CN)6]3 -/4- , as a result, amplified signals were achieved for DNA target detection using the coulometric measurement of Fe(CN)6 3- electro-reduction by prolonging the electrolysis time. The quantities of ET on the DNA sensor increased with the increase in DNA target concentration through a linear range of 3.0 fM to 1.0 pM when electrolysis time was set to 300 s, and the detection limit was 1.0 fM. Correspondingly, thousands of DNA (zeptomole) copies were detected in 10L samples. Furthermore, the DNA sensor showed excellent differentiation ability for single-base mismatch.     

Hybridization of DNA at the surface of phospholipid monolayers. Effect of orientation of oligonucleotide chains

We studied the properties of lipid monolayers formed at the air-water interface composed of dioleoylphosphatidylcholine (DOPC) with incorporated short (19-mer) oligonucleotides. These oligonucleotides were modified by oleylamine at both (3' and 5') terminals or only at one (3') terminal. Interaction of single-stranded (19-mer) oligonucleotides without oleylamine with DOPC monolayers resulted only in slight increase of surface pressure and the area per phospholipid molecule, while more substantial and significant increase of these values were observed following incorporation of oligonucelotides modified by oleylamine. This influence is similar for both types of oligonucleotide modifications. However, considerable differences in changes of monolayer properties took place after hybridization with complementary oligonucleotides. The hybridization of oligonucleotides with the DNA modified by oleic acid at both 3' and 5' terminals at the surface of lipid monolayer resulted in further increase of surface pressure and in the increase of the area per phospholipid molecule, while decrease of both the surface pressure and the area per phospholipid molecules were observed for hybridization with DNA modified by oleic acid at 3' terminal. It is possible that in latter case, the hybridization caused the loss of hybridized molecules from monolayers. Interaction of noncomplementary chains with DOPC monolayers with incorporated oleyl acid-modified DNA also influenced the properties of monolayers, but the effect was weaker in comparison with that observed for complementary chains.     

DNA aptamer folding on gold nanoparticles: from colloid chemistry to biosensors

We have investigated the effect of the folding of DNA aptamers on the colloidal stability of gold nanoparticles (AuNPs) to which an aptamer is tethered. On the basis of the studies of two different aptamers (adenosine aptamer and K+ aptamer), we discovered a unique colloidal stabilization effect associated with aptamer folding: AuNPs to which folded aptamer structures are attached are more stable toward salt-induced aggregation than those tethered to unfolded aptamers. This colloidal stabilization effect is more significant when a DNA spacer was incorporated between AuNP and the aptamer or when lower aptamer surface graft densities were used. The conformation that aptamers adopt on the surface appears to be a key factor that determines the relative stability of different AuNPs. Dynamic light scattering experiments revealed that the sizes of AuNPs modified with folded aptamers were larger than those of AuNPs modified with unfolded (but largely collapsed) aptamers in salt solution. From both the electrostatic and steric stabilization points of view, the folded aptamers that are more extended from the surface have a higher stabilization effect on AuNP than the unfolded aptamers. On the basis of this unique phenomenon, colorimetric biosensors have been developed for the detection of adenosine, K+, adenosine deaminase, and its inhibitors. Moreover, distinct AuNP aggregation and redispersion stages can be readily operated by controlling aptamer folding and unfolding states with the addition of adenosine and adenosine deaminase.     

Thiazole orange and Cy3: improvement of fluorescent DNA probes with use of short range electron transfer

Thiazole orange was synthetically incorporated into oligonucleotides by using the corresponding phosphoramidite as the building block for automated DNA synthesis. Due to the covalent fixation of the TO dye as a DNA base surrogate, the TO-modified oligonucleotides do not exhibit a significant increase of fluorescence upon hybridization with the counterstrand. However, if 5-nitroindole (NI) is present as a second artificial DNA base (two base pairs away from the TO dye) a fluorescence increase upon DNA hybridization can be observed. That suggests that a short-range photoinduced electron transfer causes the fluorescence quenching in the single strand. The latter result represents a concept that can be transferred to the commercially available Cy3 label. It enables the Cy3 fluorophore to display the DNA hybridization by a fluorescence increase that is normally not observed with this dye.     

Transfer of Two‐Dimensional Oligonucleotide Patterns onto Stereocontrolled Plasmonic Nanostructures through DNA‐Origami‐Based Nanoimprinting Lithography

The precise functionalization of self‐assembled nanostructures with spatial and stereocontrol is a major objective of nanotechnology and holds great promise for many applications. Herein, the nanoscale addressability of DNA origami was exploited to develop a precise copy‐machine‐like platform that can transfer two‐dimensional oligonucleotide patterns onto the surface of gold nanoparticles (AuNPs) through a deliberately designed toehold‐initiated DNA displacement reaction. This strategy of DNA‐origami‐based nanoimprinting lithography (DONIL) demonstrates high precision in controlling the valence and valence angles of AuNPs. These DNA‐decorated AuNPs act as precursors in the construction of discrete AuNP clusters with desired chirality.     

Biocompatible core-shell nanoparticle-based surface-enhanced Raman scattering probes for detection of DNA related to HIV gene using silica-coated magnetic nanoparticles as separation tools

A novel, highly selective DNA hybridization assay has been developed based on surface-enhanced Raman scattering (SERS) for DNA sequences related to HIV. This strategy employs the Ag/SiO₂ core-shell nanoparticle-based Raman tags and the amino group modified silica-coated magnetic nanoparticles as immobilization matrix and separation tool. The hybridization reaction was performed between Raman tags functionalized with 3′-amino-labeled oligonucleotides as detection probes and the amino group modified silica-coated magnetic nanoparticles functionalized with 5′-amino-labeled oligonucleotides as capture probes. The Raman spectra of Raman tags can be used to monitor the presence of target oligonucleotides. The utilization of silica-coated magnetic nanoparticles not only avoided time-consuming washing, but also amplified the signal of hybridization assay. Additionally, the results of control experiments show that no or very low signal would be obtained if the hybridization assay is conducted in the presence of DNA sequences other than complementary oligonucleotides related to HIV gene such as non-complementary oligonucleotides, four bases mismatch oligonucleotides, two bases mismatch oligonucleotides and even single base mismatch oligonucleotides. It was demonstrated that the method developed in this work has high selectivity and sensitivity for DNA detection related to HIV gene.     

Fluorescence energy transfer-based multiplexed hybridization assay using gold nanoparticles and quantum dot conjugates on photonic crystal beads

A multiplexed assay strategy was developed for the detection of nucleic acid hybridization. It is based on fluorescence resonance energy transfer (FRET) between gold nanoparticles (AuNPs) and multi-sized quantum dots (QDs) deposited on the surface of silica photonic crystal beads (SPCBs). The SPCBs were first coated with a three-layer primer film formed by the alternating adsorption of poly(allylamine hydrochloride) and poly(sodium 4-styrensulfonate). Probe DNA sequences were then covalently attached to the carboxy groups at the surface of the QD-coated SPCBs. On addition of DNA-AuNPs and hybridization, the fluorescence of the donor QDs is quenched because of the close proximity of the AuNPs. However, the addition of target DNA causes a recovery of the fluorescence of the QD-coated SPCBs, thus enabling the quantitative assay of hybridized DNA. Compared to fluorescent dyes acting as acceptors, the use of AuNPs results in much higher quenching efficiency. The multiplexed assay displays a wide linear range, high sensitivity, and very little cross-reactivity. This work, where such SPCBs are used for the first time in a FRET assay, is deemed to present a new and viable approach towards high-throughput multiplexed gene assays.

Ensembles of nanoelectrodes modified with gold nanoparticles: characterization and application to DNA-hybridization detection

A new method to increase the active area (A _act) of nanoelectrode ensembles (NEEs) is described. To this aim, gold nanoparticles (AuNPs) are immobilized onto the surface of NEEs using cysteamine as a cross-linker able to bind the AuNPs to the heads of the nanoelectrodes to obtain the so-called AuNPs-NEEs. The analysis of the cyclic voltammograms recorded in pure supporting electrolyte showed that the presence of the nanoparticles reflects in an, approximately, ten-times increase in the electrochemically active area of the ensemble. The measurement of the amount of electroactive polyoxometalates, which can be adsorbed on the gold surface of NEEs vs. AuNPs-NEEs, confirmed a significant increase of active area for the latter. These evidences indicate that there is a good electronic connection between the AuNPs and the underlying nanoelectrodes. The possibility to exploit AuNPs-NEEs for biosensing application was tested for the case of DNA-hybridization detection. After immobilization on the gold surface of AuNPs-NEEs of a thiolated single-stranded DNA, the hybridization with complementary sequences labeled with glucose oxidase (GOx) was performed. The detection of the hybridization was achieved by adding to the electrolyte solution the GOx substrate (i.e., glucose) and a suitable redox mediator, namely the (ferrocenylmethyl) trimethylammonium (FA⁺) cation; when the hybridization occurs, an electrocatalytic increase of the oxidation current of FA⁺ is recorded. Comparison of electrocatalytic current recorded at DNA modified NEEs and AuNPs-NEEs indicate, for the latter, a significant increase in sensitivity in the detection of the DNA-hybridization event.     

Surface-induced phase transition of asymmetric diblock copolymer in selective solvents

Surface-induced phase transition of asymmetric diblock copolymer in selective solvents is first theoretically investigated by using the real-space version of self-consistent field theory (SCFT). By varying the distance between two parallel hard surfaces (or the film thickness) W and the block copolymer concentration f(p), several morphologies are predicted and the phase diagram is constructed. Self-assembly morphologies of the diblock copolymer in dilute solution are found to change significantly with different film thickness. In confined systems, stable morphologies found in the bulk solution become unstable due to the loss of polymer conformation entropy. We find that in a very dilute block copolymer solution, phase separation can be induced through polymer depletion as the solution becomes more confined. Our findings provide an interesting starting point for a renewed effort in both experimental and theoretical investigations of confined block copolymer solutions.     

Mechanism of mercury detection based on interaction of single-strand DNA and hybridized DNA with gold nanoparticles

Mechanisms of interaction of single-strand DNA and hybridized DNA on gold nanoparticles in the presence of Hg²⁺ was studied in this work. Recently the detection of Hg²⁺ using unmodified gold nanoparticles (AuNPs) combined with DNA is becoming a promising technique with the advantages of simplicity, cost-effectiveness and high sensitivity. However, few studies focused on the interaction of ssDNA and hybridized DNA on AuNPs to date. In the present work, we compared the interactions of different DNA probes on AuNPs using both absorption and fluorescence detection. It was found that there were only small partial dsDNA dissociated from the surface of AuNPs after hybridization in the presence of Hg²⁺. Moreover, we found that the aggregated AuNPs/DNA system tended to be dispersed again with increasing Hg²⁺ concentration up to 250 μM. Based on these results, the mechanisms of mercury detection based on interaction between DNA-conjugated gold nanoparticles were investigated. Positively charged dsDNA could bind to the surface of AuNPs and dominate the electrostatic interactions and consequently aggregation of the AuNPs/DNA system.     

Monte Carlo Simulations of Copolymer Adsorption at Planar Chemically Patterned Surfaces: Effect of Interfacial Interaction

Summary: Monte Carlo simulation utilizing the bond fluctuation model in conjunction with single and configurational biased Monte Carlo moves is used to study the adsorption of diblock (A‐block‐B) and alternating (A‐alt‐B) copolymers at flat, chemically heterogeneous surfaces comprising C and D domains. The main objective of this work is to address the effect of the strength of attraction between the adsorbing surface domains, D, and the copolymer adsorbing segments, B, on the copolymer's ability to recognize the chemical pattern on the surface. The results of our simulations reveal that both block and alternating copolymers have the ability to recognize the surface motif and transcribe it into the bulk material. The extent to which diblock copolymers transfer the chemical pattern from the surface to the bulk is relatively unaffected when the attractive B‐D potential is increased beyond a certain critical value. This behavior stems from the brush‐like conformation adopted by the diblock copolymer at the substrate. In contrast to the diblock copolymer, the adsorption of the alternating copolymer is influenced by the strength of the attraction between the copolymer's adsorbing segments and the adsorbing domains on the surface. Since the B segments are distributed evenly along the backbone, the alternating copolymers are more likely to adopt conformations in which the whole chain is “zipped” to the surface. The resultant entropic frustration is then alleviated through an increased formation of loops with little change to their length. Such conformational changes endow the alternating copolymer with the ability to invert the substrate pattern as the distance away from the surface is increased.

     

Hybridization induced ion-barrier effect for the label-free and sensitive electrochemical sensing of Hepatocellular Carcinoma biomarker of miRNA-122

A label-free and sensitive electrochemical biosensing strategy for a hepatocellular carcinoma biomarker of miRNA-122 has been proposed based on hybridization induced ion-barrier effect on the electroactive sensing interface.First,a bifunctional electroactive electrode with the nanocomposite of Prussian blue(PB) and gold nanoparticles(AuNPs) was prepared through a two-step electrodeposition process.The PB endows the electrode excellent K~+-dependent voltammetric signal and the AuNPs act as the matrix for the self-assembly immobilization of the thiolated probe DNA.Upon specific hybridization of probe DNA with the target miRNA-122,the formed double duplex induced the ion-barrier effect,which blocked the diffusion of the K~+ from the bulk solution to the electrode surface.As a result,the voltammetric signal of the PB on the electrode was surpressed,and thus the target miRNA-122 was monitored.The sensing assay showed that the miRNA-122 could be analyzed in the concentration range from 0.1 fmol/L to 1.0 nmol/L,with a detection limit of 0.021 fmol/L.The practical applicability of the biosensor was also verified by the spiking serum assay.