In situ AP/MALDI-MS characterization of anchored matrix metalloproteinases

Several different procedures are available for the immobilization of proteins on solid supports, as many advantages derive from this approach, such as the possibility to develop new protein solid-state assays. Enzymes that are anchored on gold surfaces can interact with several different molecules in a tag-free environment, opening the way to surface plasmon resonance (SPR) investigations. Nevertheless, it is often important to know the identity of the affinity-retained analyte, and mass spectrometric analysis, via its unique molecular mass identification, represents a very valuable complementary method. There are many pieces of evidence to suggest that matrix metalloproteinases (MMPs) are involved in normal and pathological processes, including embryogenesis, wound healing, inflammation, arthritis and cancer, but presumably also exhibiting other functions. The search for new inhibitors of MMPs has prompted research towards the development of new solid-state assays for the rapid evaluation of MMP activity. We have already reported the possibility of measuring the activity of MMP-1 anchored on solid support by coupling SPR with ESI-MS analysis. In this work, we show the in situ atmospheric pressure (AP) MALDI-MS characterization of MMPs anchored on a gold chip with known surface coverage. The study extends the MS analysis to different proteins, and sequence coverage is reported for different digestion and MS procedures.     

A new methodology for monitoring the activity of cdMMP-12 anchored and freeze-dried on Au (111)

Matrix metalloproteinases (MMPs) are cell-secreted soluble and membrane-tethered enzymes that are capable of degrading extracellular matrix proteins, but also can process a number of bioactive molecules. They are involved in the cleavage of cell surface receptors, but are also thought to play a major role on cell behaviors as well as in diverse physiological and pathological processes, including embryonic development, wound repair, inflammatory diseases, and cancer. For these reasons, it is obvious that a control over MMPs activity is highly desirable. Consequently, the frantic search for new inhibitors has been coupled to the development of high-throughput methods able to rapidly screen the effect of possible MMP inhibitors on the activity of these enzymes. In this scenario, solid-state—based methods play a major role because of their compatibility with array formats that are able to extract more information from smaller sample volumes and offer some important advantages that are not available in the standard solution assays. In this work, the catalytic domain of MMP-12 was immobilized on a gold substrate and the surface coverage was measured by FT-SPR experiments. A new experimental procedure was developed to freeze-dry the anchored molecules and their activity was measured by ESI-MS. The kinetics parameters obtained for the immobilized enzyme are in good accordance with those reported for similar systems in solution. Inhibition of the immobilized molecules was also carried out, demonstrating the applicability of the method for rapid screening of MMP inhibitors.     

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.     

Oriented binding of the His6-tagged carboxyl-tail of the L-type Ca2+ channel alpha1-subunit to a new NTA-functionalized self-assembled monolayer

Oriented stable binding of functional proteins on surfaces is of fundamental interest for receptor/ligand studies in atomic force microscopy (AFM) and surface plasmon resonance (SPR) experiments. Here we have chosen the His6-tagged carboxyl-tail (C-tail) of the alpha1c-subunit of the L-type Ca2+ channel and calmodulin (CaM) as its cognitive partner as a model system to develop a new functional surface. Covalently attached self-assembled monolayers on ultraflat gold containing NTA-thiols to which the His6-tagged C-tail was bound and thiols with triethylene-glycol groups as matrix-thiols represented the system of choice. The topography of this surface was characterized using AFM; its ability to bind C-tail proteins oriented and stable was confirmed by SPR measurements and by complementary force spectroscopy experiments with a CaM4-construct covalently attached to the tip. The developed anchoring strategy can now be used to study receptor/ligand interactions in general applying force spectroscopy and SPR on His6-tagged proteins oriented immobilized onto this new NTA-functionalized self-assembled monolayer.     

Binding enhancement of antigen-functionalized PEGylated gold nanoparticles onto antibody-immobilized surface by increasing the functionalized antigen using alpha-sulfanyl-omega-amino-PEG

We established a technique for constructing PEGylated gold nanoparticles (GNP) that have small compounds with almost complete functionalities on their surfaces using a newly synthesized hetero-telechelic poly(ethylene glycol) (PEG), and their association/dissociation behavior on an antibody-immobilized surface was evaluated using a surface plasmon resonance (SPR) sensor.     

SPR imaging for label-free multiplexed analyses of DNA N-glycosylase interactions with damaged DNA duplexes

Base excision repair (BER) is the major mechanism for the correction of damaged nucleobases resulting from the alkylation and oxidation of DNA. The first step in the BER pathway consists of excision of the abnormal base by several specific DNA N-glycosylases. A decrease in BER activity was found to be related to an increased risk of carcinogenesis and aging. To investigate BER activities we set up a new device for DNA repair analysis based on surface plasmon resonance imaging (SPRi). Oligonucleotides bearing an abnormal nucleoside, namely 8-oxo-7,8-dihydro-2'-deoxyguanosine and (5'S)-5',8-cyclopurine-2'-deoxynucleoside, were grafted by a pyrrole electro-copolymerization process on a glass prism coated with a gold layer. The latter label-free DNA sensor chip permits the detection of N-glycosylase/AP-lyase activity as well as the binding of repair proteins to DNA damage without cleavage activity. Thus, the Fapy DNA N-glycosylase (Fpg) protein is shown as expected to bind and then cleave its natural substrate, namely 8-oxo-7,8-dihydro-guanine, together with the resulting abasic site. Using the current SPR imaging-based DNA array we observed an original binding activity of Fpg towards the (5'S)-5',8-cyclodAdenosine residue. These results altogether show that SPR imaging may be used to simultaneously and specifically detect recognition and excision of several damaged DNA nucleobases, and constitutes an interesting technique to screen inhibitors of DNA repair proteins.     

Preparation and characterization of thin films of SiO(x) on gold substrates for surface plasmon resonance studies

We report here on the fabrication and characterization of stable thin films of amorphous silica (SiO(x)) deposited on glass slides coated with a 5 nm adhesion layer of titanium and 50 nm of gold, using the plasma-enhanced chemical vapor deposition (PECVD) technique. The resulting surfaces were characterized using atomic force microscopy (AFM), ellipsometry, contact angle measurements, and surface plasmon resonance (SPR). AFM analysis indicates that homogeneous films of silica with low roughness were formed on the gold surface. The deposited silica films showed excellent stability in different solvents and in piranha solution. There was no significant variation in the thickness or in the SPR signal after these harsh treatments. The Au/SiO(x) interfaces were investigated for their potential applications as new surface plasmon resonance sensor chips. Silica films with thicknesses up to 40 nm allowed visualization of the surface plasmon effect, while thicker films resulted in the loss of the SPR characteristics. SPR allowed further the determination of the silica thickness and was compared to ellipsometric results. Chemical treatment of the SiO(x) film with piranha solution led to the generation of silanol surface groups that have been coupled with a trichlorosilane.     

Direct detection of genomic DNA by enzymatically amplified SPR imaging measurements of RNA microarrays

A novel surface enzymatic reaction scheme that amplifies the optical response of RNA microarrays to the binding of complementary DNA is developed for the direct detection and analysis of genomic DNA. The enzyme RNase H is shown to selectively and repeatedly destroy RNA from DNA-RNA heteroduplexes on gold surfaces; when used in conjunction with the label-free technique of surface plasmon resonance (SPR) imaging, DNA oligonucleotides can be detected at a concentration of 1 fM. This enzymatically amplified SPR imaging methodology is then utilized to detect and identify the presence of the TSPY gene in human genomic DNA without PCR amplification.     

Monitoring protein distributions based on patterns generated by protein adsorption behavior in a microfluidic channel

We report a unique monitoring technique of protein distributions based on distinctive patterns generated by protein adsorption behavior on a solid surface in a microfluidic channel. Bare gold and COOH-modified self-assembled monolayer (SAM) sensing surfaces were pre-adsorbed with one of four different proteins: lysozyme, albumin, transferrin, or IgG. Each surface provides a thermodynamically governed platform for immobilizing proteins and generates analyte-specific response patterns. Each surface has its own thermodynamic energy governing pre-adsorbed protein behaviors, so that sample proteins react with the pre-adsorbed ones to different extents depending on their sizes, isoelectric points (pI), and characteristics of the sensing surfaces. Modified surfaces were mounted and monitored in real time using surface plasmon resonance (SPR). Buffer-prepared sample matrices (α1-antitrypsin, haptoglobin, C-reactive protein (CRP), and IgM) characterized protein response patterns. Each surface generated distinctive patterns based on individual SPR angle shifts. We classified each sample with 95% accuracy using linear discriminant analysis (LDA). Our method also discriminated between different concentrations of CRP in the cocktail sample, detecting concentrations as low as 1 nM with 91.7% accuracy. This technique may be integrated with a microfluidic lab-on-a-chip system and monitor the distribution of a specific group of proteins in human serum.     

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.     

Arg–Cys and Arg–cysteamine adsorbed on gold and the G-protein–adsorbate interaction

The dipeptide, Arg–Cys, and the related molecule, Arg–cysteamine, are adsorbed to gold surfaces and the monolayers are characterized. Chemical binding and electronic structure of the monolayers are obtained by X-ray photoelectron spectroscopy (XPS). Strong molecular binding of the adsorbates to gold surface through the sulfur atom is attained. Orientation of the adsorbates on gold is studied using infrared reflection absorption spectroscopy (IRAS). Arg–Cys is interpreted to be adsorbed on gold in a compact configuration. The Arg–cysteamine molecule is adsorbed on gold with the main molecular axis perpendicular to the surface. Interaction of G-protein with the adsorbates was studied using the surface plasmon resonance (SPR) technique. It is believed that arginine has a major role in G-protein recognition since the G-protein-coupled receptor (GPCR) ₂A has an arginine-rich region in the G-protein-binding part of the third intracellular loop.     

Chemically immobilized T4-bacteriophage for specific Escherichia coli detection using surface plasmon resonance

A bioassay platform using T4 bacteriophage (T4) as the specific receptor and surface plasmon resonance (SPR) as the transduction technique has been developed for the detection of Escherichia coli K12 bacteria. The T4 phages have been covalently immobilized onto gold surfaces using a self-assembled monolayer of dithiobis(succinimidyl propionate) (DTSP). Substrates of BSA/EA-T4/DTSP/Au prepared using different T4 phage concentrations have been characterized using scanning electron microscopy (SEM). The studies reveal that the use of DTSP results in a uniform binding of T4 phages onto the surface. The SPR analysis demonstrates that these BSA/EA-T4/DTSP/Au interfaces can detect the E. coli K12 with high specificity against non-host E. coli NP10 and NP30. Results of SEM and SPR studies indicate that the maximum host bacterial capture is obtained when 1.5 × 10(11) pfu ml(-1) concentration of T4 phages was used for immobilization. The surface of these chemically anchored phage substrates can be regenerated for repeated detection of E. coli K12 and can be used for detection in 7 × 10(2) to 7 × 10(8) cfu ml(-1) range. The results of these studies have implications for the development of online bioassays for the detection of various food and water borne pathogens using the inherent selectivity of bacteriophage recognition.     

Spin-coated methacrylic acid copolymer thin films for covalent immobilization of small molecules on surface plasmon resonance substrates

Hydrophilic copolymers of methacrylic acid and ethylene glycol dimethacrylate are simply and rapidly prepared as thin films by spin-coating on gold-coated glass slides with a concurrent photo-crosslinking step. Coating techniques were optimized for use on gold surfaces both separately and as part of surface plasmon resonance (SPR) sensor chips. The population of carboxylic acid functional groups as binding sites in the polymer matrix, as reflected in the corresponding hydrophilicity, could be easily adjusted through changes to the stoichiometric ratio of the monomers, allowing for good control of immobilization capacity. The polymers used adhered to the gold surfaces both with and without use of thiol moieties. Coating thickness was measured by ellipsometry and coatings of 30–40 nm thickness were routinely achieved on gold-coated slides. This dimension is dependent on the spin speed and the viscosity of the polymerization mixture applied. The polymers were further characterized by contact angle measurements and infrared spectroscopy before being applied to immobilization of the steroid cortisol in a BIAcore SPR instrument, where binding to a monoclonal antibody was studied and the surface coatings optimized for maximum specific binding capacity. Optimized surfaces could be regenerated and re-used, and have potential applications as immobilization matrices in plasmonic biosensors with a very rapid coating technique.     

Comparison of the anchoring of nematic liquid crystals on self-assembled monolayers formed from semifluorinated thiols and alkanethiols

The anchoring of nematic liquid crystals on self-assembled monolayers (SAMs) formed by the chemisorption of semifluorinated thiols or alkanethiols on gold is compared and contrasted. The planar anchoring of 4-n-pentyl-4-cyanobiphenyl (5CB) observed in the past on SAMs formed from alkanethiols is also observed on SAMs formed from semifluorinated thiols. The azimuthal anchoring of 5CB, however, differs on these two types of surfaces: nematic 5CB anchored on SAMs formed from alkanethiols has a grainy appearance due to the formation of domains with sizes 10 mum whereas 5CB forms large domains ( 100 mum) with diffuse branches emerging from defects of strength 1/2 when anchored on SAMs formed from semifluorinated thiols. Mixed (two-component) SAMs formed from either short and long semifluorinated thiols or short and long alkanethiols cause homeotropic anchoring of 5CB. We discuss these results in light of the known differences in the structure of SAMs formed from alkanethiols and semifluorinated thiols, i.e. the tilt of the chains and conformational freedom (flexibility) of the chains within these SAMs.     

The Influence of Molecular Dipole Moment on the Redox-Induced Reorganization of α-Helical Peptide Self-Assembled Monolayers: An Electrochemical SPR Investigation

Self-assembled monolayers (SAMs) of ferrocene-labeled α-helical peptides were prepared on gold surfaces and studied using electrochemical surface plasmon resonance (EC-SPR). The leucine-rich peptides were synthesized with a cysteine sulfhydryl group either at the C- or N-terminus, enabling their immobilization onto gold surfaces with control of the direction of the molecular dipole moment. Two electroactive SAMs were studied, one in which all of the peptide dipole moments are oriented in the same direction (SAM1), and the other in which the peptide dipole moment of one peptide is aligned in the opposite direction to that of its surrounding peptide molecules (SAM2). Cyclic voltammetry combined with SPR measurements revealed that SAM reorientations concomitant with the oxidation of the ferrocene label were more significant in SAM2 than in SAM1. The substantially greater change in the peptide film thickness in the case of SAM2 is attributed to the electrostatic repulsion between the electrogenerated ferrocinium moiety and the positively charged gold surface. The greater permeability of SAM1 to electrolyte anions, on the other hand, appears to effectively neutralize this electrostatic repulsion. The film thickness change in SAM2 was estimated to be 0.25 ± 0.05 nm using numerical simulation. The timescale of the redox-induced SPR changes was established by chronoamperometry and time-resolved SPR measurements, followed by fitting of the SPR response to a stretched exponential function. The time constants measured for the anodic process were 16 and 6 ms for SAM1 and SAM2 respectively, indicating that the SAM thickness changes are notably fast.     

Activity-based enrichment of matrix metalloproteinases using reversible inhibitors as affinity ligands

Matrix metalloproteinases (MMPs) are zinc dependent metalloproteases characterized by the ability to cleave extracellular matrix and many other extracellular proteins. MMP activity is tightly regulated but disturbances in this regulation can contribute to various disease processes characterized by a progressive destruction of the extracellular matrix. The ability to profile classes of enzymes based on functionally related activities would greatly facilitate research about the involvement of MMPs in physiological and/or pathological states. Here we describe the characterization of an affinity sorbent using an immobilized reversible inhibitor as a stationary phase for the activity-based enrichment of MMPs from biological samples. With a ligand density of 9.8 mM and binding constant of 58 micromol/l towards MMP-12, the capturing power of the affinity sorbent was strong enough to extract MMP-12 spiked into serum with high selectivity from relatively large sample volumes. Experiments with endogenous inhibitors revealed that MMP-12 extraction is strictly activity-dependent, offering powerful means to monitor MMP activities in relation to physiological and/or pathological events by using affinity extraction as a first step in an MMP profiling method.     

Probing the Functionalization of Gold Surfaces and Protein Adsorption by PM‐IRRAS

The control of morphology and coating of metal surfaces is essential for a number of organic electronic devices including photovoltaic cells and sensors. In this study, we monitor the functionalization of gold surfaces with 11‐mercaptoundecanoic acid (MUA, HS(CH₂)₁₀CO₂H) and cysteamine, aiming at passivating the surfaces for application in surface plasmon resonance (SPR) biosensors. Using polarization‐modulated infrared reflection–absorption spectroscopy (PM‐IRRAS), cyclic voltammetry, atomic force microscopy and quartz crystal microbalance, we observed a time‐dependent organization process of the adsorbed MUA monolayer with alkyl chains perpendicular to the gold surface. Such optimized condition for surface passivation was obtained with a systematic search for experimental parameters leading to the lowest electrochemical signal of the functionalized gold electrode. The ability to build supramolecular architectures was also confirmed by detecting with PM‐IRRAS the adsorption of streptavidin on the MUA‐functionalized gold. As the approaches used for surface functionalization and its verification with PM‐IRRAS are generic, one may now envisage monitoring the fabrication of tailored electrodes for a variety of applications.     

Enhanced Photocurrent Generation in Self-Assembled Monolayers Formed at Plasmonic Gold Nanostructures

The effect of localized surface plasmon resonance (SPR) appearing at the surfaces of gold nanoparticles (AuPs) was successfully applied for the enhancement of photocurrents from porphyrin ( Po ) immobilized on AuPs. The electrode consisting of AuPs with different sizes (∼15 or ∼35 nm) was prepared using the AuP film formed at the liquid/liquid interface, and then Po was self-assembled on the gold surface. The SPR effect for the AuP film was verified from the attenuated total reflection SPR and absorption measurements. Photocurrents from the modified electrodes were compared with the corresponding planar electrode. Appreciable enhancement of photocurrents was observed.     

Suspended,one-side anchored,or double-side anchored nematic droplets in an isotropic medium

Nematic droplets in an isotropic liquid crystal provide an interesting geometrical environment for director alignment, depending on the type of surface used. We prepared three types of substrates with nematic-philic, nematic-phobic, and neutral surfaces. By choosing the proper surface type, we were able to selectively produce three types of droplets (suspended, one-side anchored, and double-side anchored) near the phase transition temperature. These droplets exhibited single-polar or bipolar defects depending on the anchoring status. The one-side anchored and the suspended droplets underwent anchoring transitions in one-step and in two-steps processes on cooling, respectively. A hybrid cell with two types of substrates exhibited a truncated cone-shaped droplet, and two nearby cone-shaped droplets produced a stable doule-curved surface geometry with a saddle point of – 1 defect between isotropic-nematic boundaries. The mergence of these droplets was also investigated.     

A novel Surface Plasmon Resonance enhanced Total Internal Reflection Ellipsometric application: electrochemically grafted isophthalic acid nanofilm on gold surface

The scope of this study is to modify a Surface Plasmon Resonance (SPR) sensor slide with isophthalic acid to evaluate the possible application on the detection of copper(II) ions in aqueous media by total internal reflection ellipsometry. A gold sensor surface was modified by an electrochemical diazonium reduction modification method. The modified surfaces are characterized with cyclic voltammetry (CV) and ellipsometry. Isophthalic acid monolayer modified gold slides were used for in situ detection of aqueous Cu(2+) solution with the SPR enhanced total internal reflection ellipsometry (SPRe-TIRE) technique. Layer formation, pH dependency of adsorption, sensor response of the SPRe-TIRE and isothermal kinetic parameters were examined. A high dependency on the number of CV cycles in the monolayer-multiple layer transition was observed. The suggested sensor gave a linear response over a wide range of Cu(2+) concentrations. It was also reported that adsorption on the SPRe-TIRE sensor gave Langmuir adsorption model behavior.