Aptamer‐Assisted Gold Nanoparticles/PEDOT Platform for Ultrasensitive Detection of LPS

Neatly arranged gold nanoparticles (AuNPs) were directly electrodeposited on an electrochemically polymerized self‐assembled monolayer (SAM) of thiol‐functionalized 3,4‐ethylenedioxythiophene (EDOT) derivative, EDTMSHA. A thiolated single‐stranded DNA (ssDNA) aptamer with high specificity to LPS was immobilized on the AuNPs/conducting polymer composite film, serving as sensing platform for LPS detection. Electrochemical impedance spectroscopy (EIS), cyclic voltammetry (CV), scanning electron microscope (SEM), and atomic force microscopy (AFM) were utilized to characterize the modification and detection processes. The electron transfer resistance was found to have a linear relationship with LPS concentration from 0.1 pg/mL to 1 ng/mL.     

Electron transfer of plurimodified DNA SAMs

An STM-based current-voltage (I/V) investigation of deoxyribonucleic acid (DNA) 18 base pair (bp) oligonucleotide monolayers on gold is presented. Three bases of each of the immobilized and complementary strands were modified with either iodine or phenylethylene moieties. The oligonucleotides were immobilized on template stripped gold (tsg) surfaces and characterized by atomic force microscopy (AFM) and scanning tunneling microscopy (STM). AFM imaging showed that monolayers of the expected height were formed. A comparative study of normal, halogenated, and phenyl-modified DNA was made with the STM in tunneling spectroscopy (TS) mode. I/V spectroscopic measurements in the range +/-250 mV on both single- and double-stranded (ds) DNA monolayers (modified and unmodified) showed that for negative substrate bias (U(sub)) electron transfer is more efficient through a phenyl-modified monolayer than through normal or halogenated DNA. This effect was particularly clear below a threshold bias of -100 mV. For positive U(sub), unmodified ds DNA was found to conduct slightly better than the modified strands. This is presumably caused by greater order in the unmodified versus modified DNA monolayers. Modifications on the immobilized (thiolated) strand seem to improve electron transport through the DNA monolayer more than modifications on the complementary (not surface-bound) strand.     

Electroactive dipyrromethene-Cu(II) self-assembled monolayers: complexation reaction on the surface of gold electrodes

In the work presented, thiol- and COOH-terminated dipyrromethene derivatives have been applied for gold electrode modification. Dipyrromethene deposited onto a solid support, after binding Cu2+, can act as a redox active monolayer. The complexation of Cu(II) ions has been performed on the surface of gold electrodes modified with dipyrromethene. The characterization of dipyrromethene-Cu(II) self-assembled monolayers (SAMs) has been done by cyclic voltammetry (CV), wettability contact angle measurements, and atomic force microscopy (AFM). The new electroactive monolayer could be applied for the immobilization of proteins and ssDNA or for electrochemical anion sensing without redox markers in the solution.     

Fabrication of DNA nanowires by orthogonal self-assembly and DNA intercalation on a Au patterned Si/SiO2 surface

A novel Ru complex bearing both an acridine group and anchoring phosphonate groups was immobilized on a surface in order to capture double-stranded DNAs (dsDNAs) from solution. At low surface coverage, the atomic force microscopy (AFM) image revealed the "molecular dot" morphology with the height of the Ru complex ( approximately 2.5 nm) on a mica surface, indicating that four phosphonate anchor groups keep the Ru complex in an upright orientation on the surface. Using a dynamic molecular combing method, the DNA capture efficiency of the Ru complex on a mica surface was examined in terms of the effects of the number of molecular dots and surface hydrophobicity. The immobilized surface could capture DNAs; however, the optimal number of molecular dots on the surface as well as the optimal pull-up speed exist to obtain the extended dsDNAs on the surface. Applying this optimal condition to a Au-patterned Si/SiO 2 (Au/SiO 2) surface, the Au electrode was selectively covered with the Ru complex by orthogonal self-assembly of 4-mercaptbutylphosphonic acid (MBPA), followed by the formation of a Zr (4+)-phosphonate layer and the Ru complex. At the same time, the remaining SiO 2 surface was covered with octylphosphonic acid (OPA) by self-assembly. The selective immobilization of the Ru complex only on the Au electrode was identified by time-of-flight secondary-ion mass spectrometry (TOF-SIMS) imaging on the chemically modified Au/SiO 2 surface. The construction of DNA nanowires on the Au/SiO 2 patterned surface was accomplished by the molecular combing method of the selective immobilized Ru complex on Au electrodes. These interconnected nanowires between Au electrodes were used as a scaffold for the modification of Pd nanoparticles on the DNA. Furthermore, Cu metallization was achieved by electroless plating of Cu metal on a priming of Pd nanoparticles on the Pd-covered DNA nanowires. The resulting Cu nanowires showed a metallic behavior with relatively high resistance.     

Evidence of DNA transfer through F-pilus channels during Escherichia coli conjugation

The mechanism of DNA transfer from Escherichia coli ( E. coli) Hfr donor strain AT2453 to recipient strain AB1157 during the conjugation process has been investigated by liquid atomic force microscopy (AFM). With the success of immobilizing both E. coli strains on gelatin-treated glass under aqueous solution, the F-pilus between an E. coli mating pair could be clearly imaged and dissected by an AFM probe. Another AFM probe functionalized with an anti-single-stranded DNA (ssDNA) antibody was then applied to detect transferring ssDNA. According to the AFM force spectrum, the transferring ssDNA could be detected only in the dissected area with a binding force of 109 +/- 5 pN measured. Our results provide direct evidence indicating that the DNA was transferred through the F-pilus channel between an E. coli mating pair during their conjugation.     

DNA as invisible ink for AFM nanolithography

We have used nanografting, an atomic force microscopy (AFM)-based nanolithography technique, to fabricate thiolated DNA nanostructures on gold surfaces. The tip-guided assembly offers opportunities for locally controlling the packing order, density, and thus the thickness of the DNA patterns. By selecting proper nanografting parameters, we can embed single-stranded DNA (ssDNA) patches into a background composed of the same DNA molecule prepared by self-assembly, in which the patches remain topographically (and chemically) invisible but have much improved packing order. When the complementary DNA (cDNA) is added, the thickness of the nanografted layer increases much more dramatically than that of the self-assembled layer during the hybridization process, and as a result, the pattern emerges. Interestingly, the pattern can be reversibly hidden and shown with high fidelity simply by dehybridizing and appending the cDNA repeatedly.     

Building a novel vitronectin assay by immobilization of integrin on calixarene monolayer

Membrane proteins possess significant hydrophobic domains and are likely to deplete their native activity immobilized on the solid surface relative to those occurring in a membrane environment. To investigate an efficient immobilization method, calix[4]crown-ether monolayer as an artificial protein linker system was constructed on the gold surface and characterized by Fourier transform infrared reflection absorption spectroscopy (FTIR-RAS), atomic force microscopy (AFM) and cyclic voltammetry (CV). Integrin alpha(v)beta3 was functionally immobilized onto the monolayer and the integrin-vitronectin interaction was investigated by surface plasmon resonance (SPR). It was found that calix[4]crown-ether was assembled as a monolayer on the gold surface. Orientation and accessibility of integrin alpha(v)beta3 was assessed by sensitive binding of its natural ligand, vitronectin at pg mL(-1) level. Moreover, surface coverage of integrin layer and thickness calculated through SPR curve simulation verified that integrin layer was a monolayer in activated form. In combination with the SPR method, this calix[4]crown monolayer provided a reliable and simple experimental platform for the investigation of isolated membrane proteins under experimental conditions resembling those of their native properties.     

Liquid mechanical behavior of mixed monolayers of amino and alkyl silanes by atomic force microscopy

The adsorption of mixed terminally aminated organosilyl compounds with long-chain n-alkyltrichlorosilanes on silica substrates has been studied by FTIR and AFM to deposit and study DNA. By optimization of deposition conditions, the mixed monolayers were found to be well organized and homogeneous. The amino group was protected to obtain a reproducible grafting and then deprotected after the film formation. In addition, atomic force microscopy (AFM) studies in both dynamical modes, amplitude modulation and frequency modulation, reveal that the layer behaves as a fluid as measured by the tip-cantilever and has a smaller characteristic time than the tip-cantilever. For three amplitudes, the experimental frequency shifts have been modeled for a fluidlike layer crossed by the tip. Finally, we show that this new fluidlike monolayer is suitable for DNA deposition and AFM studies.     

ssDNA binding reveals the atomic structure of graphene

We used AFM to investigate the interaction of polyelectrolytes such as ssDNA and dsDNA molecules with graphene as a substrate. Graphene is an appropriate substrate due to its planarity, relatively large surfaces that are detectable via an optical microscope, and straightforward identification of the number of layers. We observe that in the absence of the screening ions deposited ssDNA will bind only to the graphene and not to the SiO(2) substrate, confirming that the binding energy is mainly due to the π-π stacking interaction. Furthermore, deposited ssDNA will map the graphene underlying structure. We also quantify the π-π stacking interaction by correlating the amount of deposited DNA with the graphene layer thickness. Our findings agree with reported electrostatic force microscopy (EFM) measurements. Finally, we inspected the suitability of using a graphene as a substrate for DNA origami-based nanostructures.     

Covalently linked DNA/protein multilayered film for controlled DNA release

A stable, biocompatible single strand DNA (ssDNA)/bovine serum albumin (BSA) multilayered film for control release of DNA was fabricated on PEI-coated quartz slides, gold-evaporated plates and silicon wafers, respectively through a formaldehyde-induced, covalently linked layer-by-layer (LBL) assembly technique. The constructed film structure was well characterized by using UV-vis spectrometry, surface plasmon resonance (SPR) and atomic force microscopy (AFM). The results showed that the DNA incorporated LBL film was fabricated successfully and the amount of ssDNA and BSA in the film could be tailored simply by controlling the number of the bilayers. The control release of DNA from the film was also monitored in this study. UV-vis spectrometry, SPR and AFM measurements indicated that the release of ssDNA and amino acid was adjustable by changing the proteinase K incubation time. This biocompatible covalently assembled film demonstrates an innovative approach to engineer a DNA/protein based nanostructure for controlled DNA release, which could provide stability, controllability and flexibility superior to that of LBL film assembled by electrostatic attraction. Since the film in this work can be assembled on different substrates, it is very feasible to fabricate nanoparticle-based gene therapy systems with this new approach and to have great potential in biomedical applications.     

Hybridization with nanostructures of single-stranded DNA

Nanostructures of single-stranded DNA (ssDNA) were produced within alkanethiol self-assembled monolayers using nanografting, an atomic force microscopy (AFM) based lithography technique. Next, variations of the fabrication parameters, such as the concentration of ssDNA or lines per frame, allowed for the regulation of the density of ssDNA molecules within the nanostructures. The label-free hybridization of nanostructures, monitored using high-resolution AFM imaging, has proven to be highly selective and sensitive; as few as 50 molecules can be detected. The efficiency of the hybridization reaction at the nanometer scale highly depends on the ssDNA packing density within the nanostructures. This investigation provides a fundamental step toward sensitive DNA detection and construction of complex DNA architectures on surfaces.     

Epi-fluorescence microscopic characterization of potential-induced changes in a DOPC monolayer on a Hg drop

Characterization of the potential-induced changes of a lipid-coated Hg-0.1 M KCl interface through electrochemical techniques and newly developed in situ fluorescence microscopy is described. Fluorescence of a fluorophore-containing dioleoyl phosphatidylcholine (DOPC) layer deposited from the gas-solution interface was observed to be dependent upon the potential of the Hg surface. The largest changes occurred for potentials where the lipid layer was desorbed: the lipid moved away from the electrode surface, reducing the efficiency of metal-mediated quenching of the excited state resulting in an increase in fluorescence. Electric potential-induced changes in the morphology of the adsorbed or desorbed DOPC lipid monolayer were observed optically for the first time using this technique. The observed potential-dependent fluorescence was compared to previous studies on an octadecanol-coated Au(111) electrode. Fluorescence microscopy was also used to characterize the fusion of DOPC liposomes with a previously adsorbed DOPC layer. Large changes in fluorescence were observed for the DOPC layer after fusion with liposomes. The fusion was accomplished via potential-created defects in the adsorbed DOPC monolayer through which the liposomes interact. The integration of the liposomes into the adsorbed monolayer results in a hybrid layer in which some lipid exists further from the electrode surface, resulting in a large increase in fluorescence. Possibilities for the creation of a biomimetic adsorbed hybrid lipid layer on Hg are also discussed.     

Reaction kinetics study of short DNA strands using a maximum entropy method and nonlinear curve fitting

It is important to understand the formation of double-strand DNA (dsDNA) in a salt solution because it is one of the key reactions in life. A short cDNA strand pair was designed, and each single-strand DNA (ssDNA) was attached to a fluorescent dye that was either a donor or an acceptor of fluorescence resonance energy transfer. The fluorescence intensity was expected to change as time passed as the complementary pairs of ssDNAs formed dsDNAs. The concentration of dsDNA was theoretically calculated, and the measured data were consistent with theoretical results. The analysis of the nonlinear fitting method and the maximum entropy method detected that the reaction curve contains two major types of kinetics that likely represent the formation of dsDNA and mismatching.     

Dendrimer-functionalized self-assembled monolayers as a surface plasmon resonance sensor surface

We report here a multistep route for the immobilization of DNA and proteins on chemically modified gold substrates using fourth-generation NH(2)-terminated poly(amidoamine) dendrimers supported by an underlying amino undecanethiol (AUT) self-assembled monolayer (SAM). Bioactive ultrathin organic films were prepared via layer-by-layer self-assembly methods and characterized by fluorescence microscopy, variable angle spectroscopic ellipsometry, atomic force microscopy (AFM), X-ray photoelectron spectroscopy (XPS), and attenuated total internal reflection Fourier transform infrared spectroscopy (ATR-FTIR). The thickness of the AUT SAM base layer on the gold substrates was determined to be 1.3 nm from ellipsometry. Fluorescence microscopy and AFM measurements, in combination with analyses of the XPS/ATR-FTIR spectra, confirmed the presence of the dendrimer/biopolymer molecules on the multilayer sensor surfaces. Model proteins, including streptavidin and rabbit immunoglobulin proteins, were covalently attached to the dendrimer layer using linear cross-linking reagents. Through surface plasmon resonance measurements, we found that sensor surfaces containing a dendrimer layer displayed an increased protein immobilization capacity, compared to AUT SAM sensor surfaces without dendrimer molecules. Other SPR studies also revealed that the dendrimer-based surfaces are useful for the sensitive and specific detection of DNA-DNA interactions. Significantly, the multicomponent films displayed a high level of stability during repeated regeneration and hybridization cycles, and the kinetics of the DNA-DNA hybridization process did not appear to be influenced by surface mass transport limiting effects.     

Surface characterization of 3-glycidoxypropyltrimethoxysilane films on silicon-based substrates

Silane coupling agents are commonly used to activate surfaces for subsequent immobilization of biomolecules. The homogeneity and surface morphology of silane films is important for controlling the structural order of immobilized single-stranded DNA probes based on oligonucleotides. The surfaces of silicon wafers and glass slides with covalently attached 3-glycidoxypropyltrimethoxysilane (GOPS) have been characterized by using angularly dependent X-ray photoelectron spectroscopy (XPS), time-of-flight secondary-ion mass spectrometry (ToF–SIMS), atomic force microscopy (AFM), scanning electron microscopy (SEM), and monochromatic and spectroscopic ellipsometry. XPS and ToF–SIMS data provided evidence of complete surface coverage by GOPS. Data from angularly resolved XPS and ellipsometry methods suggested that the GOPS films were of monolayer thickness. AFM and SEM data indicated the presence of films that consisted of nodules approximately 50–100 nm in diameter. Modeling suggested that the nodules may lead to a nanoscale structural morphology that might influence the hybridization kinetics and thermodynamics of immobilized oligonucleotides.     

Identification of single-strand DNA by in situ scanning tunneling microscopy

We report here in situ scanning tunneling microscopy imaging of hexaammineruthenium (II)/(III) (RuHex) binding to single-strand DNA oligonucleotide with 13 bases immobilized in a mixed monolayer on a single-crystal Au(111) surface. RuHex does not bind to the other component in the monolayer, mercaptohexanol. Images before and after addition of RuHex show a strong contrast increase, suggesting that single-stranded DNA is, indeed, conducting when a redox group is bound to the strand. When compared with previous results, the data also suggest that the mechanism of domain formation of the immobilized DNA is independent of the sequence.     

Where is the sodium in self-assembled monolayers of single-stranded DNA?

Monolayers of single-stranded DNA (ssDNA) immobilized on surfaces form the basis of a number of important biotechnology applications, including DNA microarrays and biosensors. The organization of ssDNA as layer on a solid substrate allows one to investigate various properties of the DNA in a controlled manner and to use DNA for analytical applications as well as for exploring futuristic schemes for molecular electronics. It is commonly assumed that the adsorbed DNA layer contains some structural water and the cations. Here we show, based on XPS studies, that when monolayers of ssDNA are formed from sodium phosphate buffer and washed thoroughly, no Na+ signal is detected. A finite concentration of ions is observed when the DNA is made from a solution of Mg2+ ions, but it is still only a fifth of what it would be if all the phosphate ions were fully neutralized by the metal cations.     

Assessment of DNA Binding to Human Rad51 Protein by using Quartz Crystal Microbalance and Atomic Force Microscopy: Effects of ADP and BRC4‐28 Peptide Inhibitor

The interaction of human Rad51 protein (HsRad51) with single‐stranded deoxyribonucleic acid (ssDNA) was investigated by using quartz crystal microbalance (QCM) monitoring and atomic force microscopy (AFM) visualization. Gold surfaces for QCM and AFM were modified by electrografting of the in situ generated aryldiazonium salt from the sulfanilic acid to obtain the organic layer Au–ArSO₃H. The Au–ArSO₃H layer was activated by using a solution of PCl₅ in CH₂Cl₂ to give a Au–ArSO₂Cl layer. The modified surface was then used to immobilize long ssDNA molecules. The results obtained showed that the presence of adenosine diphosphate promotes the protein autoassociation rather than nucleation around DNA. In addition, when the BRC4‐28 peptide inhibitor was used, both QCM and AFM confirmed the inhibitory effect of BRC4‐28 toward HsRad51 autoassociation. Altogether these results show the suitability of this modified surface to investigate the kinetics and structure of DNA–protein interactions and for the screening of inhibitors.     

Silica-nanoparticle-based interface for the enhanced immobilization and sequence-specific detection of DNA

A biocompatible and uniform interface based on silica nanoparticles derivatized with amino groups has been constructed for the effective immobilization and sensitive sequence-specific detection of calf thymus DNA. Atomic force microscopy (AFM) and scanning electron microscopy (SEM) results showed that a monolayer of silica nanoparticles can be formed on a gold electrode under our experimental conditions using cysteine self-assembly monolayer as binder medium. Electrochemical impedance spectroscopy and X-ray photoelectron spectroscopy (XPS) verified the successful immobilization of DNA on silica-nanoparticle-modified gold electrodes. Quantitative results demonstrated that enhanced immobilization of single-strand DNA (ss-DNA) up to 1.6×10^–8 mol cm^–2 could be achieved owing to the larger surface area and the special properties of silica nanoparticles. In addition, hybridization experiments demonstrated that the immobilized ss-DNA on silica nanoparticles could specifically interact with complementary DNA in solutions.