Surface coverage and structure of mixed DNA/alkylthiol monolayers on gold: characterization by XPS, NEXAFS, and fluorescence intensity measurements

Self-assembly of thiol-terminated single-stranded DNA (HS-ssDNA) on gold has served as an important model system for DNA immobilization at surfaces. Here, we report a detailed study of the surface composition and structure of mixed self-assembled DNA monolayers containing a short alkylthiol surface diluent [11-mercapto-1-undecanol (MCU)] on gold supports. These mixed DNA monolayers were studied with X-ray photoelectron spectroscopy (XPS), near-edge X-ray absorption fine structure spectroscopy (NEXAFS), and fluorescence intensity measurements. XPS results on sequentially adsorbed DNA/MCU monolayers on gold indicated that adsorbed MCU molecules first incorporate into the HS-ssDNA monolayer and, upon longer MCU exposures, displace adsorbed HS-ssDNA molecules from the surface. Thus, HS-ssDNA surface coverage steadily decreased with MCU exposure time. Polarization-dependent NEXAFS and fluorescence results both show changes in signals consistent with changes in DNA orientation after only 30 min of MCU exposure. NEXAFS polarization dependence (followed by monitoring the N 1s --> pi* transition) of the mixed DNA monolayers indicated that the DNA nucleotide base ring structures are oriented more parallel to the gold surface compared to DNA bases in pure HS-ssDNA monolayers. This indicates that HS-ssDNA oligomers reorient toward a more-upright position upon MCU incorporation. Fluorescence intensity results using end-labeled DNA probes on gold show little observable fluorescence on pure HS-ssDNA monolayers, likely due to substrate quenching effects between the fluorophore and the gold. MCU diluent incorporation into HS-ssDNA monolayers initially increases DNA fluorescence signal by densifying the chemisorbed monolayer, prompting an upright orientation of the DNA, and moving the terminal fluorophore away from the substrate. Immobilized DNA probe density and DNA target hybridization in these mixed DNA monolayers, as well as effects of MCU diluent on DNA hybridization in complex milieu (i.e., serum) were characterized by surface plasmon resonance (SPR) and (32)P-radiometric assays and reported in a related study (Gong, P.; Lee, C.-Y.; Gamble, L. J.; Castner, D. G.; Grainger, D. W. Anal. Chem. 2006, 78, 3326-3334.).     

Microspotting streptavidin and double-stranded DNA arrays on gold for high-throughput studies of protein-DNA interactions by surface plasmon resonance microscopy

We present two strategies for microspotting 10 x 12 arrays of double-stranded DNAs (dsDNAs) onto a gold-coated glass slide for high-throughput studies of protein-DNA interactions by surface plasmon resonance (SPR) microscopy. Both methods use streptavidin (SA) as a linker layer between a biotin-containing mixed self-assembled monolayer (SAM) and biotinylated dsDNAs to produce arrays with high packing density. The primary mixed SAM is produced from biotin- and oligo(ethylene glycol)-terminated thiols bonded as thiolates onto the gold surface. In the first method, a robotic microspotter is used to deliver nanoliter droplets of dsDNA solution onto a uniform layer of this SA ( approximately 2 x 10(12) SA/cm(2)). SPR microscopy shows a density of (5-6) x 10(11) dsDNA/cm(2) (0.2-0.3 dsDNA/SA) in the array elements. The second method uses instead a microspotted array of this SA linker layer, onto which the microspots of dsDNA are added with spatial registry. SPR microscopy before addition of the dsDNA shows a SA coverage of 2 x 10(12) SA/cm(2) within the spots and a dsDNA density of 8.5 +/- 3.5 x 10(11) dsDNA/cm(2) (0.3-0.7 dsDNA/SA, depending on the length of dsDNA) after dsDNA spotting. We demonstrate the ability to simultaneously monitor protein binding with the SPR microscope in many 200-microm spots with 1-s time resolution and sensitivity to <1 pg of protein.     

利用表面等离子激元共振生物传感器研究聚合酶与敏感表面上自组装的DNA的相互作用

利用SPR生物传感器研究了人的型DNA聚合酶与DNA模板一引物二聚体以及单链DNA的相互作用情况.同时,观察了两种抑制剂(神经酸和亚油酸)对这些相互作用的影响.结果表明,神经酸和亚油酸可以使β聚合酶与DNA二聚体的亲和力分别下降20倍和5倍,神经酸的抑制作用更加明显.利用这些方法,有助于清楚了解抑制剂对β聚合酶与DNA相互作用的影响和抗癌及抗病毒药物的研制.     

Electrochemical surface plasmon resonance measurement based on gold nanohole array fabricated by nanoimprinting technique

In this paper, we describe our development of an electrochemical surface plasmon resonance (EC-SPR) measurement device based on a bottom-filled gold nanohole array. The polymer based gold nanohole array was fabricated with a UV nanoimprint technique and electron beam gold deposition. Direct reflection mode measurement was used to monitor the SPR dip in the reflection spectra. A cyclic voltammogram was also operated by using the standard three electrodes containing working electrode having a gold nanohole array and counter and reference electrodes. The gold nanohole array was modified with an osmium-poly(vinylpyridine)-wired horseradish peroxidase (Os-gel-HRP) film, and its redox state induced by the change in potential was monitored simultaneously. The redox state of the local film was obtained simply by scanning the sample substrate stage. The substrate modified with Os-gel-HRP film was incorporated in a microfluidic chip, and then the hydrogen peroxide was determined in terms of the redox change in the Os complex mediator from the slope of the SPR dip shift. The linear relation of hydrogen peroxide from 10 to 250 μM was successfully monitored, and a high conversion efficiency was realized.     

Signal enhancement of surface plasmon resonance based on gold nanoparticle-antibody complex for immunoassay

In the immunoassay based on surface plasmon resonance (SPR) system, the signal enhancement was done by means of the conjugate of gold (Au) nanoparticle-antibody fragment. Antibody fragment was prepared for the improved immobilization based on Au-thiol interaction. Through the ellipsometric analysis on surface, the conjugation between Au and antibody fragment was performed in the oriented manner. The optimal fabrication conditions such as concentration and incubation time were determined for the constant size of the fabricated nanoparticle-antibody conjugate. Through the plot of SPR angle difference versus antigen concentration, the linear correlation was achieved, of which the detection limit was 100 fg/ml.     

Improving the sensitivity limit of surface plasmon resonance biosensors by detecting mixed interference signals

We demonstrate that the sensitivity limit of intensity-based surface plasmon resonance (SPR) biosensors can be enhanced when we combine the effects of the phase and amplitude contributions instead of detecting the amplitude variation only. Experimental results indicate that an enhancement factor of as much as 20 times is achievable, yet with no compromise in measurement dynamic range. While existing SPR biosensor systems are predominantly based on the angular scheme, which relies on detecting intensity variations associated with amplitude changes only, the proposed scheme may serve as a direct system upgrade approach for these systems. The new measurement scheme may therefore lead to a strong impact in the design of SPR biosensors.     

Surface plasmon resonance phase imaging measurements of patterned monolayers and DNA adsorption onto microarrays

The optical technique of surface plasmon resonance phase imaging (SPR-PI) is implemented in a linear microarray format for real-time measurements of surface bioaffinity adsorption processes. SPR-PI measures the phase shift of p-polarized light incident at the SPR angle reflected from a gold thin film in an ATR Kretschmann geometry by creating an interference fringe image on the interface with a polarizer-quartz wedge depolarizer combination. The position of the fringe pattern in this image changes upon the adsorption of biomolecules to the gold thin film. By using a linear array of 500 μm biosensor element lines that are perpendicular to the interference fringe image, multiple bioaffinity adsorption measurements can be performed in real time. Two experiments were performed to characterize the sensitivity of the SPR-PI measurement technique: First, a ten line pattern of a self-assembled monolayer of 11-mercaptoundecamine (MUAM) was created via photopatterning to verify that multiple phase shifts could be measured simultaneously. A phase shift difference (Δφ) of Δφ = 182.08 ± 0.03° was observed for the 1.8 nm MUAM monolayer; this value agrees with the phase shift difference calculated from a combination of Fresnel equations and Jones matrices for the depolarizer. In a second demonstration experiment, the feasibility of SPR-PI for in situ bioaffinity adsorption measurements was confirmed by detecting the hybridization and adsorption of single stranded DNA (ssDNA) onto a six-component DNA line microarray patterned monolayer. Adsorption of a full DNA monolayer produced a phase shift difference of Δφ = 28.80 ± 0.03° at the SPR angle of incidence and the adsorption of the ssDNA was monitored in real time with the SPR-PI. These initial results suggest that SPR-PI should have a detection limit roughly 100 times lower than traditional intensity-based SPR imaging measurements.     

Tuned longitudinal surface plasmon resonance and third-order nonlinear optical properties of gold nanorods

In order to elucidate the relationship for third-order nonlinear optical properties of anisotropic metal nanoparticles between the incident laser wavelength and surface plasmon resonance (SPR) wavelength, gold nanorods (GNRs) with a tuned longitudinal SPR mode in frequency were prepared by seed-mediated methods with two different surfactants, cetyltrimethylammonium bromide (CTAB) and benzyldimethylammonium chloride (BDAC). The real and imaginary parts of the third-order nonlinear optical susceptibilities χ(3) were examined by near-infrared (800 nm) femtosecond Z-scan and I-scan techniques for various gold sols with SPR wavelengths of 530 nm (spheres), 800 nm (nanorods) and 1000 nm (nanorods), named as 530GNSs, 800GNRs and 1000GNRs, respectively. All the samples showed intrinsically third-order nonlinear optical refractive responses. However, as for the real part of χ(3) for one particle, 800GNRs whose plasmon peak was tuned to the incident laser wavelength exhibited a Reχ(3) value 45 times stronger than 530GNSs. More interestingly, the imaginary part of χ(3) was more greatly influenced at the tuned SPR wavelength. Here we first demonstrate that 800GNRs showed plasmon-enhanced saturable absorption (SA) due to a longitudinal SPR tuned to the incident laser wavelength.     

Simultaneous excitation of propagating and localized surface plasmon resonance in nanoporous gold membranes

Materials multifunctionality for optical sensing of adsorbates has obvious advantages-in addition to the potential for greater sensitivity, the different length scales associated with a variety of optical phenomena allow a greater variety of adsorption characteristics to be examined. Here, we show that ultrathin (approximately 100 nm) nanoporous gold membranes possess features of both planar metal films that exhibit propagating SPR excitations and nanofeatured metals that exhibit localized SPR excitations. This is the first report of such multifunctionality in an optically active metal. We give illustrative examples of using this material to probe biorecognition reactions and to probe the structure evolution of a layer-by-layer deposition of charged dendrimers. Our results are consistent with the very different lengths of the tail of the evanescent field decays associated with each of these plasmon excitation modes.     

Determination of ribonuclease H surface enzyme kinetics by surface plasmon resonance imaging and surface plasmon fluorescence spectroscopy

The kinetics of the ribonuclease H (RNase H) surface hydrolysis of RNA-DNA heteroduplexes formed on DNA microarrays was studied using a combination of real-time surface plasmon resonance imaging (SPRI) and surface plasmon fluorescence spectroscopy (SPFS). Time-dependent SPRI and SPFS data at various enzyme concentrations were quantitatively analyzed using a simple model that couples diffusion, enzyme adsorption, and surface enzyme kinetics. This model is characterized by a set of three rate constants, enzyme adsorption (k(a)), enzyme desorption (k(d)), enzyme catalysis (k(cat)), and one dimensionless diffusion parameter (beta). Values of k(a) = 3.15 (+/-0.20) x 10(6) M(-1).s(-1), k(d) = 0.10 (+/-0.05) s(-1), and k(cat) = 0.95 (+/-0.10) s(-1) were determined from fitting all of the SPRI and SPFS data sets. One of the most interesting kinetic parameters is the surface RNase H hydrolysis reaction rate constant (k(cat)), which was found to be approximately 10 times slower than that observed in solution, but approximately 100 times faster than that recently observed for the exonuclease III surface hydrolysis of double-stranded DNA microarrays (k(cat) = 0.009 s(-1)). Moreover, the surface coverage of the intermediate enzyme-substrate complex (ES) was found to be extremely small during the course of the reaction because k(cat) is much larger than the product of k(a) and the bulk enzyme concentration.     

Enzymatically amplified surface plasmon resonance imaging detection of DNA by exonuclease III digestion of DNA microarrays

This paper describes a novel approach utilizing the enzyme exonuclease III in conjunction with 3'-terminated DNA microarrays for the amplified detection of single-stranded DNA (ssDNA) with surface plasmon resonance (SPR) imaging. When ExoIII and target DNA are simultaneously introduced to a 3'-terminated ssDNA microarray, hybridization adsorption of the target ssDNA leads to the direction-dependent ExoIII hydrolysis of probe ssDNA strands and the release of the intact target ssDNA back into the solution. Readsorption of the target ssDNA to another probe creates a repeated hydrolysis process that results over time in a significant negative change in SPR imaging signal. Experiments are presented that demonstrate the direction-dependent surface enzyme reaction of ExoIII with double-stranded DNA as well as this new enzymatically amplified SPR imaging process with a 16-mer target ssDNA detection limit of 10-100 pM. This is a 10(2)-10(3) improvement on previously reported measurements of SPR imaging detection of ssDNA based solely on hybridization adsorption without enzymatic amplification.     

Optical properties and instrumental performance of thin gold films near the surface plasmon resonance

The optical properties of very thin gold films have been evaluated by Fresnel analysis, with optical boundary conditions pertaining to the surface plasmon resonance (SPR) at the gold-water interface. The experimental SPR characteristic was evaluated in the angular interrogation mode. Film morphology was characterized by high resolution transmission electron microscopy. The magnitude of the resonance, i.e., the SPR signal, sensitively depends on, and is affected by film thickness and morphology. A sharply defined thickness of 55 ± 5 nm is required, to achieve optimum SPR excitation conditions, and instrumental sensitivity. With decreasing film thickness, below 40 nm, the resonance angle starts to shift to larger values. A substantial increase of the intrinsic resonance broadening parameter is observed below 70 nm, associated with an increasingly asymmetric SPR line shape. A similar effect occurs in the presence of a very thin chromium adhesion layer. Surface roughness and film thickness modulations determine the experimentally observed line broadening parameter. Instrumental noise levels largely depend on accuracy and quality at which the resonance angle can be determined. Substantial improvement and instrumental sub-pixel resolution is achievable by optimum fitting routines, accounting for drastic noise reduction and improved instrumental sensitivity, up to two orders of magnitude over the inherent geometric sensor pixel resolution.     

Ellipsometric detection of gases with the surface plasmon resonance effect on gold top-coated with sensitive layers

An ellipsometric gas sensor based on the surface Plasmon resonance (SPR) effect of ~ 43 nm thick gold layers was investigated. To protect the gold layer from contamination and to improve the detection limits, the gold layers were top-coated with 5-6 nm thick layers of organic a-C:H or with inorganic metal oxides TiO₂ or ZrO₂. The additional layers increased the long-term stability, whereas the metal oxide layers protect better than a-C:H. Furthermore, the additional layers decreased the detection limits by one order of magnitude in case of a-C:H and two orders of magnitude in case of the oxides. It could be shown that the detection limits also depend on the kind of preparation (sol-gel process or physical vapour deposition) of the additional layers.     

Analytical characterization of chiral drug-protein interactions: comparison between the optical biosensor (surface plasmon resonance) assay and the HPLC perturbation method

Two modern, fundamentally different methods were used for a detailed investigation of enantioselective drug-protein interactions, a surface plasmon resonance (SPR)-based Biacore 2000 biosensor assay and the previously validated HPLC perturbation method (HPLC-PM). This is the first time SPR has been used for this purpose. The fundamental features of the two methods were investigated, and the consequences for operation and data evaluation were addressed. With HPLC-PM, chiral data could be obtained directly from the racemic mixture, whereas a separate analysis of each pure enantiomer was required to obtain chiral data with SPR. It was shown that if chirality is not attributed in the SPR analysis, misleading average racemic binding constants will be obtained. Both drug and protein consumption were considerably higher with HPLC-PM. HPLC-PM was found to be best suited for measurements of weak affinity interactions, whereas the SPR method was best for strong interactions. With both methods, the presence of DMSO in the samples severely affected the interactions, introducing errors. The binding of the beta-blockers alprenolol and propranolol to Cel7a cellulase was used as a model system. These methods gave results that agreed quite well qualitatively, but considerable quantitative deviations were sometimes obtained.     

Gas-phase detection of trinitrotoluene utilizing a solid-phase antibody immobilized on a gold film by means of surface plasmon resonance spectroscopy

A multilayered biosensor was constructed and found to detect trinitrotoluene (TNT) in ppb concentrations in air both prior to and after detonation of TNT without use of a liquid phosphate buffered saline (PBS) superstrate. The biosensor surface was fabricated from a monoclonal antibody for TNT covalently bound to an 11,11'-dithio-bis(succinimidoylundecanoate) (DSU) self-assembled monolayer immobilized on a thin gold film bonded to a BK7 glass slide. The binding between the immobilized antibody and TNT antigen was detected using surface plasmon resonance spectroscopy (SPRS). Biosensor specificity for TNT was demonstrated with chemical homologues as well as against an unrelated explosive, RDX.     

Label-free biosensing by surface plasmon resonance of nanoparticles on glass: optimization of nanoparticle size

The unique optical properties of noble metal nanoparticles have been used to design a label-free biosensor in a chip format. In this paper, we demonstrate that the size of gold nanoparticles significantly affects the sensitivity of the biosensor. Gold nanoparticles with diameters in the range of 12-48 nm were synthesized in solution and sensor chips were fabricated by chemisorption of these nanoparticles on amine-functionalized glass. Sensors fabricated from 39-nm-diameter gold nanoparticles exhibited maximum sensitivity to the change of the bulk refractive index and the largest "analytical volume", defined as the region around the nanoparticle within which a change in refractive index causes a change in the optical properties of the immobilized nanoparticles. The detection limit for streptavidin-biotin binding of a sensor fabricated from 39-nm-diameter nanoparticles was 20-fold better than a previously reported sensor fabricated from 13-nm-diameter gold nanoparticles. We also discuss several other factors that could improve the performance of the next generation of these immobilized metal nanoparticle sensors.     

Fabrication of a surface plasmon resonance biosensor based on gold nanoparticles chemisorbed onto a 1,10-decanedithiol self-assembled monolayer

We have investigated the fabrication of surface plasmon resonance (SPR) biosensors using self-assembled monolayers (SAMs) and adsorbed gold nanoparticles. The SAM of 1,10-decanedithiol was first fabricated onto a gold substrate. Gold nanoparticles were then chemisorbed onto the SAM surface by bonding with the terminal thiol groups, forming a sensor that can be used to immobilize proteins. Bovine serum albumin (BSA) was used as a test protein in this study. Several spectroscopic and microscopic techniques were used to investigate both the SAM and the chemisorption of gold nanoparticles at the SAM surface. Our results confirm the covalent bonding of the gold nanoparticles onto the SAM. Surface plasmon resonance (SPR) was used to study both the adsorption of BSA to the SAM surface and to the gold nanoparticle-coated SAM. For SAM surfaces with adsorbed gold nanoparticles a larger SPR response to BSA than to the sensors with a bare SAM is observed.     

Simultaneous detection of transgenic DNA by surface plasmon resonance imaging with potential application to gene doping detection

Surface plasmon resonance imaging (SPRi) was used as the transduction principle for the development of optical-based sensing for transgenes detection in human cell lines. The objective was to develop a multianalyte, label-free, and real-time approach for DNA sequences that are identified as markers of transgenosis events. The strategy exploits SPRi sensing to detect the transgenic event by targeting selected marker sequences, which are present on shuttle vector backbone used to carry out the transfection of human embryonic kidney (HEK) cell lines. Here, we identified DNA sequences belonging to the Cytomegalovirus promoter and the Enhanced Green Fluorescent Protein gene. System development is discussed in terms of probe efficiency and influence of secondary structures on biorecognition reaction on sensor; moreover, optimization of PCR samples pretreatment was carried out to allow hybridization on biosensor, together with an approach to increase SPRi signals by in situ mass enhancement. Real-time PCR was also employed as reference technique for marker sequences detection on human HEK cells. We can foresee that the developed system may have potential applications in the field of antidoping research focused on the so-called gene doping.     

Investigation of adsorption behavior of bisphenol A on well fabricated organic surfaces using surface plasmon resonance spectroscopy

Molecular adsorption of bisphenol A (BPA) on three types of self-assembled monolayers with different functionalities, such as -CH3, -SH, and -COOH, was examined using surface plasmon resonance (SPR) spectroscopy. BPA molecules in an aqueous solution were easily adsorbed onto a hydrophobic surface compared to a hydrophilic surface. Sorption behavior of BPA into poly(2-methoxyethyl acrylate) (PMEA) layer, which is known as a biocompatible polymer, was also investigated. Sorption and desorption dynamics of BPA into PMEA were found to be very rapid and quite reversible. The swelling of PMEA by sorption of BPA results in the change in SPR angle and allows one to quantify the BPA concentration below 100 ppm. In addition, the transport mechanism of BPA within the membrane of organ can be inferred by the experimental results.     

Structure and DNA hybridization properties of mixed nucleic acid/maleimide-ethylene glycol monolayers

The surface structure and DNA hybridization performance of thiolated single-strand DNA (HS-ssDNA) covalently attached to a maleimide-ethylene glycol disulfide (MEG) monolayer on gold have been investigated. Monolayer immobilization chemistry and surface coverage of reactive ssDNA probes were studied by X-ray photoelectron spectroscopy and time-of-flight secondary ion mass spectrometry. Orientation of the ssDNA probes was determined by near-edge X-ray absorption fine structure (NEXAFS). Target DNA hybridization on the DNA-MEG probe surfaces was measured by surface plasmon resonance (SPR) to demonstrate the utility of these probe surfaces for detection of DNA targets from both purified target DNA samples and complex biological mixtures such as blood serum. Data from complementary techniques showed that immobilized ssDNA density is strongly dependent on the spotted bulk DNA concentration and buffer ionic strength. Variation of the immobilized ssDNA density had a profound influence on the DNA probe orientation at the surface and subsequent target hybridization efficiency. With increasing surface probe density, NEXAFS polarization dependence results (followed by monitoring the N 1s --> pi* transition) indicate that the immobilized ssDNA molecules reorient toward a more upright position on the MEG monolayer. SPR assays of DNA targets from buffer and serum showed that DNA hybridization efficiency increased with decreasing surface probe density. However, target detection in serum was better on the "high-density" probe surface than on the "high-efficiency" probe surface. The amounts of target detected for both ssDNA surfaces were several orders of magnitude poorer in serum than in purified DNA samples due to nonspecific serum protein adsorption onto the sensing surface.