Kinetic mechanisms in morpholino-DNA surface hybridization

Morpholinos (MOs) are DNA analogues whose uncharged nature can bring fundamental advantages to surface hybridization technologies such as DNA microarrays, by using MOs as the immobilized, or "probe", species. Advancement of MO-based diagnostics, however, is challenged by limited understanding of the surface organization of MO molecules and of how this organization impacts hybridization kinetics and thermodynamics. The present study focuses on hybridization kinetics between monolayers of MO probes and DNA targets as a function of the instantaneous extent of hybridization (i.e., duplex coverage), total probe coverage, and ionic strength. Intriguingly, these experiments reveal distinct kinetic stages, none of which are consistent with Langmuir kinetics. The initial stage, in which duplex coverage remains relatively sparse, indicates confluence of two effects: blockage of target access to unhybridized probes by previously formed duplexes and deactivation of the solid support due to consumption of probe molecules. This interpretation is consistent with a surface organization in which unhybridized MO probes localize near the solid support, underneath a layer of MO-DNA duplexes. As duplex coverage builds, provided saturation is not reached first, the initial stage can transition to an unusual regime characterized by near independence of hybridization rate on duplex coverage, followed by a prolonged approach to equilibrium. The possible origins of these more complex latter behaviors are discussed. Comparison with published data for DNA and peptide nucleic acid (PNA) probes is carried out to look for universal trends in kinetics. This comparison reveals qualitative similarities when comparable surface organization of probes is expected. In addition, MO monolayers are found capable of a broad range of reactivities that span reported values for PNA and DNA probes.     

DNA hybridization "turns on" electro-catalysis at gold electrodes

The efficiency of electro-catalysis occurring at DNA-modified gold electrodes is highly dependently on the density of DNA monolayers, as a result, DNA hybridization can "turn on" electro-catalysis by increasing the DNA surface density.     

A smart surface in a microfluidic chip for controlled protein separation

The smart surface created in a microfluidic chip has shown the capability of adsorbing and releasing proteins under electrical control. The inner surface of the chip channel was first coated by a thin layer of Au through sputtering and was subsequently modified with loosely packed self-assembled monolayers (SAMs) of thiols with terminal carboxylic or amino groups. Upon application of an external electric potential to the gold substrate, reversible conformational transformation between "bent" and "straight" states for the anchored mercapto chains could be modulated, through the electrostatic effect between the ionized terminal groups and the charged gold substrate. Thus, a hydrophobic or hydrophilic channel surface was established and could be reversibly switched electrochemically. Accordingly, the microchips prepared in this way can reversibly and selectively adsorb and release differently charged proteins under electrical control. Two model proteins, avidin and streptavidin, were demonstrated to be readily adsorbed by the smart chips under negative and positive potential, respectively. Also, more than 90 % of the adsorbed proteins could be released upon an electrical command. Furthermore, these chips were applied to the controlled separation of avidin and streptavidin mixtures with 1:1 and 1:1000 molar ratios. Under specific applied potentials, the chips adsorbed a certain protein from the mixture whereas the other protein was allowed to flow out, after which the adsorbed protein could be released by switching the applied potential. Thus, two eluted protein fractions were obtained and the separation of the two proteins was achieved. For the former mixture, each eluted fraction contained up to approximately 80-90 % avidin or streptavidin. For the latter mixture, the resulting separation efficiency indicated that the molar ratio of avidin and streptavidin could be increased from 1:1000 to about 32:1 after five run separations.     

Unimolecular rectification of monolayers of CH3C(O)S-C14H28Q(+)-3CNQ(-) and CH3C(O)S-C16H32Q(+)-3CNQ(-) organized by self-assembly, Langmuir-Blodgett, and Langmuir-Schaefer techniques

The advantage of "self-assembly" (strong covalent binding to substrates) was combined with the advantage of Langmuir-Blodgett (LB) or Langmuir-Schaefer (LS) transfer to a solid substrate (quantitative transfer of monolayers to the substrate). The electrical rectification (asymmetric conduction) by a monolayer of thioacetylalkylquinolinium tricyanoquinodimethanide was critically compared when these molecules had been transferred, by such competing techniques, onto gold electrodes, and then covered by a "cold gold" pad electrode. Unimolecular rectification was observed in the expected directions in the LB and LS monolayers. The Self-Assembled Monolayers (SAMs) were disordered; macroscopic measurements of rectification were unsuccessful for the SAMs, but successful for the down-stroke LB and LS monolayers, whose orientation and potential bonding to the Au surface should be identical to that of an ideal SAM.     

DNA-conjugated polymers for self-assembled DNA chip fabrication.

We developed two DNA-conjugated polymers, one based on polyallylamine and the other on polyacrylic acid, for use in DNA chips. A 30-mer single-stranded DNA probe and thioctic acid were covalently attached to polyallylamine as sidechains. The same single-stranded DNA and 3-(pyridyldithio)propionyl hydrazide were covalently attached to polyacrylic acid as sidechains. Both DNA-conjugated polymers could be specifically immobilized onto a gold sensor substrate by a self-assembly technique. The interactions between fully matched DNA and each DNA-conjugated polymer were investigated by surface plasmon resonance. A gold surface modified with either DNA-conjugated polymer recognized fully matched DNA much better than unmatched DNA. The hybridization selectivity and efficiency of DNA-conjugated polyallylamine was optimized by adjusting the pH so as to reduce the effects of cationic polymer sidechains. The hybridization selectivity and efficiency of DNA-conjugated polymers were higher than those of a conventional immobilized thiol-based DNA. The coating of DNA-conjugated polymers reduced nonspecific adsorption of DNA by the gold substrate. DNA-conjugated polyacrylic acid was more selective toward fully matched DNA than was DNA-conjugated polyallylamine. Therefore, DNA-conjugated polymers show promise for application in novel DNA chips.     

Thermostable DNA immobilization and temperature effects on surface hybridization

Monolayer films of nucleic acids on solid supports are encountered in a range of diagnostic and bioanalytical applications. These applications often rely on elevated temperatures to improve performance; moreover, studies at elevated temperatures can provide fundamental information on layer organization and functionality. To support such applications, this study compares thermostability of oligonucleotide monolayers immobilized to gold by first coating the gold with a nanometer-thick film (an "anchor layer") of a polymercaptosiloxane, to which DNA oligonucleotides are subsequently tethered through maleimide-thiol conjugation, with thermostability of monolayers formed via widely used attachment through a terminal thiol moiety on the DNA. The temperature range covered is from 25 to 90 °C. After confirming stability of immobilization and, more importantly, retention of hybridization activity even under the harshest conditions investigated, these thermostable films are used to demonstrate measurements of (1) reversible surface melting transitions and (2) temperature dependence of competitive hybridization, when fully matched and mismatched sequences compete for binding to immobilized DNA oligonucleotides. The competitive hybridization experiments reveal a pronounced impact of temperature on rates of approach to equilibrium, with kinetic freezing into nonequilibrium states close to room temperature and rapid approach to equilibrium at elevated temperatures. Modeling of competitive surface hybridization equilibria using thermodynamic parameters derived from surface melting transitions of the individual sequences is also discussed.     

Hybridization of oligonucleotide by using DNA self-assembled monolayer

The interaction between DNA immobilized on surface and oligonucleotides at the interface is important in detection and diagnostic processes. However, it is difficult to immobilize DNA with maintaining its activity and to realize an efficient hybridization in previous methods. Here, to establish a novel DNA-functionalized surface, the DNA self-assembled monolayer (SAM) was constructed on a gold substrate using thiolated DNA composed of double-stranded (ds) and single-stranded (ss) portion. The DNA SAM was characterized by surface plasmon resonance (SPR), XPS. The hybridization of ss portion of DNA was attempted using the SAM, and in situ monitored by SPR. XPS measurement indicated that the thiolated DNA could form a stable monolayer on a gold substrate through sulfur–gold interaction. SPR measurement implied that the long axis of the DNA standing on the substrate. These results indicated formation of the DNA SAM on the substrate. Hybridization of target DNA containing a complementary sequence for the probe portion was observed by SPR. Moreover, one mismatch of oligonucleotide could be distinguished using the DNA SAM. The SPR result indicates that hybridization of target DNA and probe DNA on the DNA SAM occurs on the DNA SAM.     

Characterization of single- and double-stranded DNA on gold surfaces

Single- and double-stranded deoxy ribonucleic acid (DNA) molecules attached to self-assembled monolayers (SAMs) on gold surfaces were characterized by a number of optical and electronic spectroscopic techniques. The DNA-modified gold surfaces were prepared through the self-assembly of 6-mercapto-1-hexanol and 5'-C(6)H(12)SH -modified single-stranded DNA (ssDNA). Upon hybridization of the surface-bound probe ssDNA with its complimentary target, formation of double-stranded DNA (dsDNA) on the gold surface is observed and in a competing process, probe ssDNA is desorbed from the gold surface. The competition between hybridization of ssDNA with its complimentary target and ssDNA probe desorption from the gold surface has been investigated in this paper using X-ray photoelectron spectroscopy, chronocoulometry, fluorescence, and polarization modulation-infrared reflection absorption spectroscopy (PM-IRRAS). The formation of dsDNA on the surface was identified by PM-IRRAS by a dsDNA IR signature at approximately 1678 cm(-)(1) that was confirmed by density functional theory calculations of the nucleotides and the nucleotides' base pairs. The presence of dsDNA through the specific DNA hybridization was additionally confirmed by atomic force microscopy through colloidal gold nanoparticle labeling of the target ssDNA. Using these methods, strand loss was observed even for DNA hybridization performed at 25 degrees C for the DNA monolayers studied here consisting of attachment to the gold surfaces by single Au-S bonds. This finding has significant consequence for the application of SAM technology in the detection of oligonucleotide hybridization on gold surfaces.     

Surface electric field manipulation of the adsorption kinetics and biocatalytic properties of cytochrome c on a 3D macroporous Au electrode

Upon adsorbing on a solid-state substrate, water-soluble proteins are prone to denaturation and deterioration of their functions due to the conformation change. The surface electric field of a conductive substrate is one of the important factors that influence the character of adsorbed proteins. In this work, a 3D macroporous gold electrode has been prepared and served as the working electrode to study the influence of surface electric field on the adsorption kinetics and conformation of the adsorbed cytochrome c (cyt-c) with the help of electrochemical, in situ electrochemical IR spectroscopic, atomic force microscopic, and contact angle measurements. The external electric field creates excess surface charge which can manipulate the adsorption rate of proteins on the substrate by the enhanced electrostatic interactions between the electrode and protein patches by coupling with complementary charges. The amount of immobilized cyt-c with electrochemical activity on the 3D macroporous gold electrode showed a minimum at potential of zero charge (PZC) and it increased with increasing net excess surface charge. Higher electric field could influence the conformation and the corresponding properties such as direct electrochemistry, bioactivity, and surface character of the adsorbed cyt-c molecules. However, high external electric field leads to damage of the protein secondary structure. This study provides fundamentals for the fabrication of biomolecular devices, biosensors, and biofuel cells through electrostatic interactions. Figure Two cases are illustrated for the protein immobilized on electrode surfaces: a retention of protein structure under moderate excess surface charge, b denaturation and conformation change of proteins adsorbed at high excess surface charge, e.g., due to the higher external electric field.     

Optimization of DNA hybridization efficiency by pH-driven nanomechanical bending

The accessibility and binding affinity of DNA are two key parameters affecting the hybridization efficiency in surface-based biosensor technologies. Better accessibility will result in a higher hybridization efficiency. Often, mixed ssDNA and mercaptohexanol monolayers are used to increase the hybridization efficiency and accessibility of surface-bound oligonucleotides to complementary target DNA. Here, no mercaptohexanol monolayer was used. We demonstrate by differential microcantilever deflection measurements at different pH that the hybridization efficiency peaks between pH 7.5 and 8.5. At low pH 4.5, hydration and electrostatic forces led to tensile surface stress, implying the reduced accessibility of the bound ssDNA probe for hybridization. In contrast, at high pH 8.5, the steric interaction between neighboring ssDNA strands was decreased by higher electrostatic repulsive forces, bending the microcantilever away from the gold surface to provide more space for the target DNA. Cantilever deflection scales with pH-dependent surface hybridization efficiency because of high target DNA accessibility. Hence, by changing the pH, the hybridization efficiency is adjusted.     

电化学石英晶体微天平研究界面电场对DNA杂交的影响

采用电化学石英晶体微天平, 现场监测不同界面电场下完全匹配的靶标DNA和不完全匹配的靶标DNA分别与寡聚核苷酸探针分子杂交的过程. 结果表明, 电极表面荷正电时DNA表观杂交效率比电极表面荷负电时高, 但假阳性比较显著; 而电极表面荷负电时能有效地抑制错配杂交. 探讨了引入界面电场后探针分子取向和微观作用力对DNA杂交的影响.     

DNA-DNA interaction on dendron-functionalized sol-gel silica films followed with surface plasmon fluorescence spectroscopy

Since we observed that dendron-assembled surface provided high single nucleotide polymorphism discrimination efficiency for DNA microarrays, and that the binding yield for streptavidin increased when biotin was immobilized on top of it, the nanoscale-controlled surface is examined for surface plasmon field-enhanced fluorescence spectroscopy (or SPFS). Firstly, a silica film was coated onto a gold substrate using the sol-gel technique, followed by the covalent immobilization of a layer of second-generation dendrons with a DNA catcher strand at their apex. The thickness of the inorganic interlayer (d=33 nm) was effectively suppressing fluorescence quenching. Thus, the kinetics and affinity characteristics of DNA hybridization could be investigated very sensitively by SPFS. The kinetic rate constants found for DNA hybridization on the dendron-modified surface were larger than those reported for a streptavidin-modified surface by one order of magnitude, except for dissociation rate constant for a single mismatched case. In addition, we observed that the DNA on the cone-shaped linker maintained its capability to capture DNA target strands even after extended storage at ambient conditions.     

Voltammetric determination of DNA hybridization using methylene blue and self-assembled alkanethiol monolayer on gold electrodes

An electrochemical DNA biosensor based on the recognition of single stranded DNA (ssDNA) by hybridization detection with immobilized complementary DNA oligonucleotides is presented. DNA and oligonucleotides were covalently attached through free amines on the DNA bases using N-hydroxysulfosuccinimide (NHS) and N-(3-dimethylamino)propyl-N′-ethylcarbodiimide hydrochloride (EDC) onto a carboxylate terminated alkanethiol self-assembled monolayers (SAM) preformed on a gold electrode (AuE). Differential pulse voltammetry (DPV) was used to investigate the surface coverage and molecular orientation of the immobilized DNA molecules. The covalently immobilized probe could selectively hybridize with the target DNA to form a hybrid on the surface despite the bases being attached to the SAM. The changes in the peak currents of methylene blue (MB), an electroactive label, were observed upon hybridization of probe with the target. Peak currents were found to increase in the following order: hybrid-modified AuE, mismatched hybrid-modified AuE, and the probe-modified AuE which indicates the MB signal is determined by the extent of exposed bases. Control experiments were performed using a non-complementary DNA sequence. The effect of the DNA target concentration on the hybridization signal was also studied. The interaction of MB with inosine substituted probes was investigated. Performance characteristics of the sensor are described.     

Molecular engineering of the polarity and interactions of molecular electronic switches

We have investigated and learned to control switching of oligo(phenylene ethynylene)s embedded in amide-containing alkanethiol self-assembled monolayers on Au{111}. We demonstrate bias-dependent switching of the oligo(phenylene ethynylene)s as a function of the interaction between the dipole moment of the oligo(phenylene ethynylene)s and the electric field applied between the scanning tunneling microscope tip and the substrate. We are able to invert the polarity of the switches by altering their design-inverting their dipole moments. For appropriately designed switches and matrix molecules, the conductance states are stabilized by intermolecular hydrogen bonding. These results further support the hypothesis that conductance switching in these molecules is due to hybridization changes at the molecule-substrate bonds due to tilting of the switch molecules.     

Solvent-dependent assembly of terphenyl- and quaterphenyldithiol on gold and gallium arsenide

The assembly of terphenyldithiol (TPDT) and quaterphenyldithiol (QPDT) on gold and gallium arsenide from ethanol (EtOH), tetrahydrofuran (THF), and solutions consisting of both solvents has been characterized by near-edge X-ray absorption fine structure spectroscopy. The surface coverage and the average orientation of both TPDT and QPDT on gold are solvent-independent. These molecules readily form monolayers on gold with an ensemble-average backbone tilt of 30 degrees +/- 3 degrees from the substrate normal. In sharp contrast, the assembly of TPDT and QPDT on gallium arsenide is extremely solvent-sensitive. At high ethanol fractions, both molecules form monolayers with an ensemble-average orientation that is indistinguishable from those on gold substrates. At low ethanol fractions and in pure THF, however, these molecules are disordered on gallium arsenide and the surface coverage is poor.     

Surface potential switching by metal ion complexation/decomplexation using bipyridinethiolate monolayers on gold

Surface potential switching on gold(111) surfaces is induced by complexation/decomplexation reactions of a bipyridine (BP) derivative and palladium(II) chloride, as observed by Kelvin probe force microscopy (KFM). On the basis of the theoretical predictions, a 4-(5-phenylethynyl-2,2'-bipyridine-5'-yl-ethynyl)benzenethiol (PhBP) derivative was synthesized and used as an active monolayer to catch transition metal ions. By using the microcontact printing (CP) technique, micron-size patterned PhBP monolayers, which act as effective hosts to coordinate palladium(II) chloride, were prepared on gold(111) surfaces. The KFM signal decreases by complexation of the Pd(II) chloride in PhBP monolayers and is recovered by removal of Pd ions using an ethylenediamine solution, as confirmed by X-ray photoelectron spectroscopy. This process is reversible, indicating that the surface potential switching is realized by complexation/decomplexation of Pd(II). A CP PhBP monolayer, when it detects the target palladium ion, shows sensitivity for the picomolar level detection judged from surface potential changes in KFM measurements. The dipole moment estimated by the surface potentials is much smaller than the calculated value, indicating that mechanisms for the reduction of the surface dipole moment exist in real monolayers prepared by the CP method.     

自组装DNA吸附取向的表面增强拉曼光谱法研究

对金基体上自组装寡聚核苷酸探针杂交前后进行电化学非现场及现场表面增强拉曼光谱(SERS)研究.非现场SERS研究表明,杂交形成的dsDNA在基体表面以A型和B型两种构象同时存在,杂交过程可能伴随DNA链在基体表面吸附取向的变化.根据现场SERS研究结果可知,ssDNA及dsDNA的大多数SERS谱带强度随电极电位正移而降低,尤其是归属于碱基A的两种面外振动模式,谱带变化更为明显.利用SERS表面选择定则判断出随着电极电位由负向正变化,ssDNA及dsDNA螺旋吸附取向由垂直吸附向平躺吸附于金基体表面变化.     

Studies of surface-enhanced Raman scattering of C60 Langmuir-Blodgett film on a new substrate

A new substrate of "gold nano-particle/silver nano-rod/ITO surface" was obtained by electrodeposition. Surface-enhanced Raman scattering (SERS) spectrum of high quality of C60 and stearic acid (SA) mixed Langmuir-Blodgett (LB) film shifted onto the new substrate was reported for the first time according to our knowledge. The results show that the substrate of "gold nano-particle/silver nano-rod/ITO surface" is very effective and active for C60 LB film. Furthermore, the C60 molecules are oriented on pentagons of C60 on the substrate. It is difficult to separate the electromagnetic and chemical mechanisms to the great enhancement of the Raman signal. On the one hand, the gold nano-particles grown on silver nano-rod surface perform an important action for magnifying the surface local electric field through the resonant excitation of surface plasma. And the needle-like rod may further magnify the local electric field because of lightning rod effect. On the other hand, charge transfer factor may not be neglected.     

Morphology of dry solid-supported protein monolayers dependent on the substrate and protein surface properties

The morphologies of dry MrgA protein monolayers on different solid substrates prepared by a three-step procedure (adsorption from an incubation solution, rinsing to remove excess salt and protein, and drying) were investigated using atomic force microscopy. MrgA is a dodecameric iron-storage protein which can form hexagonal, two-dimensional (2D) crystalline monolayers on hydrophilic surfaces at low supersaturation. The formation of such two-dimensional crystals is heavily dependent on the pH and the salinity of the incubation solution as well as on the surface properties. Correlation of surface coverage with substrate charge, ionic strength, and pH indicates the dominance of electrostatic effects in adsorption, with the balance shifting between intermolecular repulsion and protein-substrate attraction. Close to the isoelectric point (pI) of MrgA, adsorption to the surface and the formation of 2D crystals are favored. By preparation of self-assembled monolayers of thiols with different end groups on template-stripped gold, the surface properties can be varied easily from high to very low protein affinity. The resulting patterns of the crystalline protein structures are novel and could be a starting point for further scientific study, e.g., solid-supported cocrystallization with DNA, and indeed developments with technological applications, such as mesostructured deposition of MrgA-caged nanoparticles.     

Imaging photoelectron transmission through self-assembled monolayers: the work-function of alkanethiols coated gold

In this paper, we present a new approach for studying the electronic properties of self-assembled monolayers and their interaction with a conductive substrate, the low-energy photoelectron imaging spectroscopy (LEPIS). LEPIS relies on imaging of photoelectrons ejected from a conductive substrate and subsequently transmitted through organic monolayers. Using this method, we measure the relative work-function of alkanethiols of different length on gold substrate, and we are able to follow the changes occurring when the surface coverage is varied. We also computed the work-function of model alkanethiols using a plane-wave density functional theory approach, in order to demonstrate the correlation between changes in the work-function with the monolayer organization and density.