Quantitative analysis of protein adsorption via atomic force microscopy and surface plasmon resonance

Surface properties have a significant influence on the performance of biomedical devices. The influence of surface chemistry on the amount and distribution of adsorbed proteins has been evaluated by a combination of atomic force microscopy (AFM) and surface plasmon resonance (SPR). Adsorption of albumin, fibrinogen, and fibronectin was analyzed under static and dynamic conditions, employing self-assembled monolayers (SAMs) as model surfaces. AFM was performed in tapping mode with antibody-modified tips. Phase-contrast images showed protein distribution on SAMs and phase-shift entity provided information on protein conformation. SPR analysis revealed substrate-specific dynamics in each system investigated. When multi-protein solutions and diluted human plasma interacted with SAMs, SPR data suggested that surface chemistry governs the equilibrium composition of the protein layer.     

A case study on biological activity in a surface-bound multicomponent system: the biotin-streptavidin-peroxidase system

The adsorption of multiple protein layers on biotinylated organic surfaces has been characterized using surface plasmon resonance (SPR) and atomic force microscopy (AFM). Diffusion-limited loading of the biotinylated self-assembled monolayers (SAMs) ensures a precise control of the streptavidin surface density. For the subsequent interaction with biotinylated peroxidase, SPR data hint at a streptavidin density dependent orientation during peroxidase adsorption. Microcontact printed well-defined two-dimensional patterned surfaces of biotinylated organothiols and protein-resistant OEG-thiols allow an in-situ differentiation of specific and nonspecific adsorption (e.g., mono- vs multilayer adsorption). Additionally, the very important issue of biological activity of surface-bound enzymes is addressed by comparing the enzyme activities in solution with that for surface-bound species.     

Protein adsorption on oligo(ethylene glycol)-terminated alkanethiolate self-assembled monolayers: The molecular basis for nonfouling behavior

A study of protein resistance of oligo(ethylene glycol) (OEG), HS(CH2)11(OCH2CH2)nOH (n = 2, 4, and 6), self-assembled monolayers (SAMs) on Au(111) surfaces is presented here. Hydroxyl-terminated OEG-SAMs are chosen to avoid the hydrophobic effect observed with methyl-terminated OEG-SAMs, particularly at high packing densities. The structure of the OEG-SAM surfaces is controlled by adjusting the assembly solvent. These SAMs were characterized by X-ray photoelectron spectroscopy (XPS) and atomic force microscopy (AFM). Protein adsorption on these surfaces was investigated by surface plasmon resonance (SPR). OEG-SAMs assembled from mixed ethanol and water solutions show higher packing density on gold than those from pure ethanol solution. For EG2OH- and EG4OH-SAMs, proteins (i.e., fibrinogen and lysozyme) adsorb more on the densely packed SAMs prepared from mixed ethanol and water solutions, while EG6OH-SAMs generally resist protein adsorption regardless of the assembly solvent used.     

Protein interactions with model chromatographic stationary phases constructed using self-assembled monolayers

Model surfaces representative of chromatographic stationary phases were developed by immobilising an homologous series (C2-C18) of n-alkylthiols, mixed monolayers of C4/C18 and thioalkanes with alcohol, carboxylic acid, amino and sulphonic acid terminal groups onto a flat, silver-coated glass surface using self-assembled monolayer (SAM) chemistry. The processes of adsorption and desorption of serum albumins onto the monolayer surfaces was monitored in real-time using surface plasmon resonance (SPR). Alkyl-terminated SAMs all showed a strong adsorption of bovine serum albumin which was largely independent of alkyl chain length, the ratio of mixed C4/C18 SAMs or the solution pH/ionic strength. The adsorption of human serum albumin to carboxylic and amine terminated SAMs was shown to be predominantly via non-electrostatic interactions (hydrophobic or hydrogen bonding). However, sulphonic acid terminated SAMs showed almost exclusively electrostatic interactions with human serum albumin. This preliminary work using self-assembled monolayer chemistry confirms the usefulness of well characterised SAMs surfaces for investigating protein adsorption and desorption onto/from model chromatography surfaces and gives some guidance for selecting appropriate functionalities to develop better surfaces for chromatography and electrophoresis.     

Surface plasmon resonance spectroscopy-based high-throughput screening of ligands for use in affinity and displacement chromatography

We describe an approach that uses surface plasmon resonance (SPR) spectroscopy and self-assembled monolayers (SAMs) for the high-throughput screening of ligands for use in displacement and affinity chromatographic processes. We identified a set of commercially available organic amines and allowed them to react with SAMs presenting interchain carboxylic anhydride groups; the resulting surfaces presented ligands of interest in a background of carboxylic acid groups. We used SPR spectroscopy to determine the extent of adsorption of two model proteinslysozyme and cytochrome conto these "multimodal" surfaces and to select promising "affinity" ligands for further characterization. The attachment of selected ligands to UltraLink Biosupport resulted in beads with a significantly greater affinity for lysozyme than for cytochrome c that would be suitable for use in affinity chromatographic processes. Furthermore, we also used the screens to design "affinity displacers"small molecules that selectively retain lysozyme on chromatographic resins, while displacing cytochrome c. The combination of SPR spectroscopy and SAMs represents a powerful technique for identifying novel ligands that enable the purification of complex protein mixtures.     

Monolayers of 3-mercaptopropyl-amino acid to reduce the nonspecific adsorption of serum proteins on the surface of biosensors

Monolayers prepared with polar or ionic amino acids with short side chains have a reduced nonspecific adsorption of serum proteins compared to that of hydrophobic amino acids and organic monolayers immobilized on the gold surface of surface plasmon resonance (SPR) biosensors. Proteins contained in biological samples adsorb on most surfaces, which in the case of biosensors causes a nonspecific response that hinders the quantification of biomarkers in these biological samples. To circumvent this problem, self-assembled monolayers (SAM) of N-3-mercaptopropyl-amino acids (3-MPA-amino acids) were prepared from 19 natural amino acids. These SAM were investigated to limit the nonspecific adsorption of proteins contained in biological fluids and to immobilize molecular receptors (i.e., antibodies) that are necessary in the construction of biosensors. SPR and Ge attenuated total reflection (GATR) FTIR spectroscopy were employed to characterize the formation of the amino acid SAMs. Monolayers of 3-MPA-amino acids densely packed on the surface of the SPR biosensors result in a surface concentration of approximately 10 (15) molecules/cm (2). SPR also quantifies the surface concentration of serum proteins nonspecifically adsorbed on 3-MPA-amino acids following the exposure of the biosensor to undiluted bovine serum. The concentration of nonspecifically bound proteins ranged from approximately 400 ng/cm (2) with polar and ionic amino acids to approximately 800 ng/cm (2) with amino acids of increased hydrophobicity. The nonspecific adsorption of serum proteins on the 3-MPA-amino acids increases in the following order: Asp < Asn < Ser < Met < Glu < Gln < Thr < Gly < His < Cys < Arg < Phe < Trp < Val < Pro < Ile < Leu < Ala < Tyr. The analysis of the adsorption and desorption curves for serum proteins on the SPR sensorgram has demonstrated the strong irreversibility of the protein adsorption on each surface. The effective hydrophilicity of the SAMs was measured from the contact angle with a saline buffer and has demonstrated that surfaces minimizing the contact angle with PBS performed better in serum. The antibody for beta-lactamase was immobilized on a 3-MPA-glycine SAM, and beta-lactamase was detected in the nanomolar range. The presence of beta-lactamase is an indicator of antibiotic resistance.     

Strong resistance of oligo(phosphorylcholine) self-assembled monolayers to protein adsorption

In this work, we demonstrate the strong resistance of oligo(phosphorylcholine) (OPC) self-assembled monolayers (SAMs) to protein adsorption and cell adhesion. OPC SAMs were characterized using X-ray photoelectron spectroscopy (XPS), and protein adsorption was measured using a surface plasmon resonance (SPR) sensor. Results are compared with those of phosphorylcholine (PC) SAMs. Despite the existence of negative charge on OPC SAMs and the simple synthesis procedure of OPC thiols, OPC SAMs resist protein adsorption as effectively as or better than PC SAMs formed from highly purified PC thiols. The ease of their preparation and the effectiveness of their function make OPC SAMs an attractive alternative for creating nonfouling surfaces.     

Bioinert surface to protein adsorption with higher generation of dendrimer SAMs

Interactions between proteins and biomaterial surfaces correlate with many important phenomena in biological systems. Such interactions have been used to develop various artificial biomaterials and applications, in which regulation of non-specific protein adsorption has been achieved with bioinert properties. In this research, we investigated the protein adsorption behavior of polymer brushes of dendrimer self-assembled monolayers (SAMs) with other generations. The surface adsorption properties of proteins with different pI values were examined on gold substrates modified with poly(amidoamine) dendrimer SAMs. The amount of fibrinogen adsorption was greater than that of lysozyme, potentially because of the surface electric charge. However, as the generations increased, protein adsorption decreased regardless of the surface charge, suggesting that protein adsorption was also affected by density of terminal group.     

Correlation between desorption force measured by atomic force microscopy and adsorption free energy measured by surface plasmon resonance spectroscopy for peptide-surface interactions

Surface plasmon resonance (SPR) spectroscopy is a useful technique for thermodynamically characterizing peptide-surface interactions; however, its usefulness is limited to the types of surfaces that can readily be formed as thin layers on the nanometer scale on metallic biosensor substrates. Atomic force microscopy (AFM), on the other hand, can be used with any microscopically flat surface, thus making it more versatile for studying peptide-surface interactions. AFM, however, has the drawback of data interpretation due to questions regarding peptide-to-probe-tip density. This problem could be overcome if results from a standardized AFM method could be correlated with SPR results for a similar set of peptide-surface interactions so that AFM studies using the standardized method could be extended to characterize peptide-surface interactions for surfaces that are not amenable for characterization by SPR. In this article, we present the development and application of an AFM method to measure adsorption forces for host-guest peptides sequence on surfaces consisting of alkanethiol self-assembled monolayers (SAMs) with different functionality. The results from these studies show that a linear correlation exists between these data and the adsorption free energy (ΔG(o)(ads)) values associated with a similar set of peptide-surface systems available from SPR measurements. These methods will be extremely useful to characterize thermodynamically the adsorption behavior for peptides on a much broader range of surfaces than can be used with SPR to provide information related to understanding protein adsorption behavior to these surfaces and to provide an experimental database that can be used for the evaluation, modification, and validation of force field parameters that are needed to represent protein adsorption behavior accurately for molecular simulations.     

Effect of surface composition on the adsorption of photosystem I onto alkanethiolate self-assembled monolayers on gold

We have used self-assembled monolayers (SAMs) prepared from omega-terminated alkanethiols on gold to generate model surfaces and examine the effect of surface composition on the adsorption of Photosystem I (PSI), stabilized in aqueous solution by Triton X-100. Triton-stabilized PSI adsorbs to high-energy surfaces prepared from HO- and HO2C-terminated alkanethiols but does not adsorb to low-energy surfaces. The inhibition of PSI adsorption at low-energy surfaces is consistent with the presence of a layer of Triton X-100 that adsorbs atop the hydrophobic SAM and presents a protein-resistant poly(ethylene glycol) (PEG) surface. While the presence of the PEG surface prevents the adsorption of PSI, the displacement of the inhibiting layer of Triton X-100 by dodecanol, a more active surfactant, greatly enhances the adsorption of PSI. This inhibiting effect by Triton X-100 can be extended to other protein systems such as bovine serum albumin.     

Surface grafted sulfobetaine polymers via atom transfer radical polymerization as superlow fouling coatings

One of the sulfobetaine methacrylate (SBMA) monomers, N-(3-sulfopropyl)-N-(methacryloxyethyl)-N,N-dimethylammonium betaine, was polymerized onto initiator-covered gold surfaces using atom transfer radical polymerization (ATRP) to form uniform polymer brushes. Self-assembled monolayers (SAMs) with ATRP initiators were characterized by X-ray photoelectron spectroscopy (XPS) and atomic force microscopy (AFM). The thickness of grafted poly(SBMA) films was measured by ellipsometry. Fibrinogen adsorption on poly(SBMA) grafted surfaces was measured with a surface plasmon resonance (SPR) sensor. Two approaches were compared to graft ATRP initiators onto gold surfaces for surface polymerization and subsequent protein adsorption on these polymer grafted surfaces. The first was to prepare a SAM from omega-mercaptoundecyl bromoisobutyrate onto a gold surface. Superlow fouling surfaces with well-controlled poly(SBMA) brushes were achieved using this approach (e.g., fibrinogen adsorption <0.3 ng/cm2). The second approach was to react bromoisobutyryl bromide with a hydroxyl-terminated SAM on a gold surface. Although protein adsorption decreased as the density of surface initiators increased, the surface prepared using the second approach was not able to achieve as low protein adsorption as the first approach. Key parameters to achieve superlow fouling surfaces were studied and discussed.     

Adsorption behavior of plasmid DNA on binary self-assembled monolayers modified gold substrates

Gold is known to have good biocompatibility because of its inert activity and the surface property can be easily tailored with self-assembled monolayers (SAMs). In previous works, gold surfaces were tailored with homogeneously mixed amine and carboxylic acid functional groups to generate surfaces with a series of isoelectronic points (IEPs). In other words, by tailoring the chemical composition in binary SAMs, different surface potentials can be obtained under controlled pH environments. To understand how the surface potentials affect the interaction at the interface, a binary-SAMs-modified Au electrode on a quartz crystal microbalance with dissipation detection (QCM-D) was used owing to the high weight sensitivity of QCM-D. In QCM-D, the frequency shift and the energy dissipation are monitored simultaneously to determine the adsorption behaviors of the plasmid DNA to surfaces of various potentials in Tris-buffered NaCl solutions of different pH. The results revealed that the plasmid DNA can be adsorbed on the SAM-modified surfaces electrostatically; thus, in general, the amount of adsorbed plasmid DNA decreased with increasing environmental pH and the decreasing ratio of the amine functional groups on the surfaces owing to weaker positive potentials on the surface. For the high amine-containing surfaces, due to the strong electrostatic attraction, denser films were observed, and thus, the apparent thickness decreased slightly. The negatively charged carboxylic acid surfaces can still adsorb the negatively charged plasmid DNA at some conditions. In other words, the electrostatic model cannot explain the adsorption behavior completely, and the induced dipole (Debye) interaction between the charged and polarizable molecules needs to be considered as well.     

Antigen binding forces of single antilysozyme Fv fragments explored by atomic force microscopy

We used atomic force microscopy (AFM) to explore the antigen binding forces of individual Fv fragments of antilysozyme antibodies (Fv). To detect single molecular recognition events, genetically engineered histidine-tagged Fv fragments were coupled onto AFM tips modified with mixed self-assembled monolayers (SAMs) of nitrilotriacetic acid- and tri(ethylene glycol)-terminated alkanethiols while lysozyme (Lyso) was covalently immobilized onto mixed SAMs of carboxyl- and hydroxyl-terminated alkanethiols. The quality of the functionalization procedure was validated using X-ray photoelectron spectroscopy (surface chemical composition), AFM imaging (surface morphology in aqueous solution), and surface plasmon resonance (SPR, specific binding in aqueous solution). AFM force-distance curves recorded at a loading rate of 5000 pN/s between Fv- and Lyso-modified surfaces yielded a distribution of unbinding forces composed of integer multiples of an elementary force quantum of approximately 50 pN that we attribute to the rupture of a single antibody-antigen pair. Injection of a solution containing free Lyso caused a dramatic reduction of adhesion probability, indicating that the measured 50 pN unbinding forces are due to the specific antibody-antigen interaction. To investigate the dynamics of the interaction, force-distance curves were recorded at various loading rates. Plots of unbinding force vs log(loading rate) revealed two distinct linear regimes with ascending slopes, indicating multiple barriers were present in the energy landscape. The kinetic off-rate constant of dissociation (k(off) approximately = 1 x 10(-3) s(-1)) obtained by extrapolating the data of the low-strength regime to zero force was in the range of the k(off) estimated by SPR.     

Reversible protein adsorption and bioadhesion on monolayers terminated with mixtures of oligo(ethylene glycol) and methyl groups

Surface-grafted, environmentally responsive polymers have shown great promise for controlling adsorption and desorption of macromolecules and cells on solid surfaces. In the paper, we demonstrate that certain mixed self-assembled monolayers (SAMs) of oligo(ethylene glycol) (OEG) and methyl-terminated alkanethiolates on gold form surfaces with switchable hydrophobicity and tendency for protein adsorption and cellular attachment. At temperatures above 32 degrees C, SAMs with a surface density of approximately 50% OEG adsorbed significant amounts of pyruvate kinase and lysozyme, whereas below this temperature, these same SAMs were resistant to the adsorption of these proteins. Furthermore, protein layers adsorbed to these SAMs above 32 degrees C were removed upon rinsing with water below this temperature. Similar results were seen for attachment and release of the marine bacterium, Cobetia marina. The change from nonresistance to adsorptive state of the SAMs was concomitant with a change in advancing water contact angle. Vibrational sum frequency generation spectroscopy suggests that the temperature-induced changes coincide with a disorder-to-partial order transition of the hydrated methylene chains of the OEG moieties within the SAMs. Mixed OEG-methyl SAMs represent both a convenient means of controlling macromolecular and cellular adsorption within the laboratory and a useful tool for relating adsorption properties to molecular structures within the SAMs.     

Self-assembled monolayers of dendritic polyglycerol derivatives on gold that resist the adsorption of proteins

Highly protein-resistant, self-assembled monolayers (SAMs) of dendritic polyglycerols (PGs) on gold can easily be obtained by simple chemical modification of these readily available polymers with a surface-active disulfide linker group. Several disulfide-functionalized PGs were synthesized by N,N'-dicyclohexylcarbodiimide-mediated ester coupling of thioctic acid. Monolayers of the disulfide-functionalized PG derivatives spontaneously form on a semitransparent gold surface and effectively prevent the adsorption of proteins, as demonstrated by surface plasmon resonance (SPR) kinetic measurements. A structure-activity relationship relating the polymer architecture to its ability to effectuate protein resistance has been derived from results of different surface characterization techniques (SPR, attenuated total reflectance infrared (ATR-IR), and contact-angle measurements). Dendritic PGs combine the characteristic structural features of several highly protein-resistant surfaces: a highly flexible aliphatic polyether, hydrophilic surface groups, and a highly branched architecture. PG monolayers are as protein resistant as poly(ethylene glycol) (PEG) SAMs and are significantly better than dextran-coated surfaces, which are currently used as the background for SPR spectroscopy. Due to the higher thermal and oxidative stability of the bulk PG as compared to the PEG and the easy accessibility of these materials, dendritic polyglycerols are novel and promising candidates as surface coatings for biomedical applications.     

Mechanism underlying bioinertness of self-assembled monolayers of oligo(ethyleneglycol)-terminated alkanethiols on gold: protein adsorption, platelet adhesion, and surface forces

The mechanism underlying the bioinertness of the self-assembled monolayers of oligo(ethylene glycol)-terminated alkanethiol (OEG-SAM) was investigated with protein adsorption experiments, platelet adhesion tests, and surface force measurements with an atomic force microscope (AFM). In this work, we performed systematic analysis with SAMs having various terminal groups (-OEG, -OH, -COOH, -NH(2), and -CH(3)). The results of the protein adsorption experiment by the quartz crystal microbalance (QCM) method suggested that having one EG unit and the neutrality of total charges of the terminal groups are essential for protein-resistance. In particular, QCM with energy dissipation analyses indicated that proteins absorb onto the OEG-SAM via a very weak interaction compared with other SAMs. Contrary to the protein resistance, at least three EG units as well as the charge neutrality of the SAM are found to be required for anti-platelet adhesion. When the identical SAMs were formed on both AFM probe and substrate, our force measurements revealed that only the OEG-SAMs possessing more than two EG units showed strong repulsion in the range of 4 to 6 nm. In addition, we found that the SAMs with other terminal groups did not exhibit such repulsion. The repulsion between OEG-SAMs was always observed independent of solution conditions [NaCl concentration (between 0 and 1 M) and pH (between 3 and 11)] and was not observed in solution mixed with ethanol, which disrupts the three-dimensional network of the water molecules. We therefore concluded that the repulsion originated from structured interfacial water molecules. Considering the correlation between the above results, we propose that the layer of the structured interfacial water with a thickness of 2 to 3 nm (half of the range of the repulsion observed in the surface force measurements) plays an important role in deterring proteins and platelets from adsorption or adhesion.     

Modeling protein binding and elution over a chromatographic surface probed by surface plasmon resonance

Surface plasmon resonance (SPR) spectroscopy is used as a scaled-down, analytical, pseudo-chromatography tool for analyzing protein binding and elution over an ion-exchange surface under cyclic sorption conditions. A micrometric-scale adsorption surface was produced by immobilizing a typical ion exchange ligand – diethylaminoethyl (DEAE) – onto commercially available planar gold sensor chip surfaces pre-derivatized with a self-assembled monolayer of 11-mercaptoundecanoic acid with known density. An explicit mathematical formulation is provided for the deconvolution and interpretation of the SPR sensorgrams. An adsorption rate model is proposed to describe the SPR sensorgrams for bovine serum albumin, used here as model protein, when the DEAE surface is subjected to a cyclic series of binding and elution steps. Overall, we demonstrate that the adsorption rate model is capable of quantitatively describing BSA binding and elution for protein titers from dilute conditions up to overloaded conditions and a broad range of salt concentrations.     

Nanoscale Biomolecular Structures on Self‐Assembled Monolayers Generated from Modular Pegylated Disulfides

A solid‐phase synthetic strategy was developed that uses modular building blocks to prepare symmetric oligo(ethylene glycol)‐terminated disulfides with a variety of lengths and terminal functionalities. The modular disulfides, composed of alkyl amino groups linked by an amide group to oligoethylene chains were used to generate self‐assembled monolayers (SAMs), which were characterised to determine their applicability for biomolecular applications. X‐ray photoelectron spectroscopy (XPS) of the SAMs obtained from these molecules demonstrated improved stability towards displacement by 16‐hexadecanethiol, while surface plasmon resonance (SPR) analyses of SAMs prepared with the hydroxy‐terminated oligoethylene disulfide showed equal resistance to non‐specific protein adsorption in comparison to 11‐mercaptoundecyl tri(ethylene glycol). SAMs made from these adsorbates were amenable to nanoscale patterning by scanning near‐field photolithography (SNP), facilitating the fabrication of nanopatterned, protein‐functionalised surfaces. Such SAMs may be further developed for bionanotechnology applications such as the fabrication of nanoscale biological arrays and sensor devices.     

Lipase-catalyzed reactions at different surfaces

Starting from gold chips, we have tailor-made three surfaces by the self-assembly monolayer technique: one entirely hydrophobic, one hydrophobic with dispersed carboxyl groups, and one hydrophilic, containing hydroxyl groups. Rhizomucor miehei lipase has been adsorbed to the hydrophobic and the hydrophilic surfaces and covalently bound to the surface containing carboxyl groups. The adsorption of two substrates-capric acid (decanoic acid) and monocaprin-on the lipase-covered surfaces was monitored by the surface plasmon resonance (SPR) technique. Biocatalysis was also performed in the SPR instrument by circulating a solution of the substrate, dissolved in an 85:15 water-glycerol mixture at a(w) = 0.81, through the instrument, thus exposing the capric acid or the monocaprin to the lipase-covered surfaces. The product composition was found to depend on the type of surface used. Lipase adsorbed at the hydrophilic surface favored hydrolysis, and capric acid was the main product formed when monocaprin was used as substrate. Lipase adsorbed at a hydrophobic surface and, in particular, lipase covalently bound to a hydrophobic surface favored condensation. More dicaprin than capric acid was formed in experiments with monocaprin as the substrate. Reactions performed outside the SPR instrument showed that small amounts of triglyceride were also formed under these conditions. We believe that this work constitutes the first example of the SPR instrument being used for in-situ biotransformation.     

Electrochemically controlled assembly and logic gates operations of gold nanoparticle arrays

The reversible assembly of β-cyclodextrin-functionalized gold NPs (β-CD Au NPs) is studied on mixed self-assembled monolayer (SAM), formed by coadsorption of redox-active ferrocenylalkylthiols and n-alkanethiols on gold surfaces. The surface coverage and spatial distribution of the β-CD Au NPs monolayer on the gold substrate are tuned by the self-assembled monolayer composition. The binding and release of β-CD Au NPs to and from the SAMs modified surface are followed by surface plasmon resonance (SPR) spectroscopy. The redox state of the tethered ferrocene in binary SAMs controls the formation of the supramolecular interaction between ferrocene moieties and β-CD-capped Au NPs. As a result, the potential-induced uptake and release of β-CD Au NPs to and from the surface is accomplished. The competitive binding of β-CD Au NPs with guest molecules in solution shifted the equilibrium of the complexation-decomplexation process involving the supramolecular interaction with the Fc-functionalized surface. The dual controlled assembly of β-CD Au NPs on the surface enabled to use two stimuli as inputs for logic gate activation; the coupling between the localized surface plasmon, associated with the Au NP, and the surface plasmon wave, associated with the thin metal surface, is implemented as readout signal for "AND" logic gate operations.