Aß-Amyloid Fibrils Are Self-Triggered by the Interfacial Lipid Environment and Low Peptide Content.

We studied the surface properties of Aß(1-40) amyloid peptides mixed with 1-palmitoyl-2-oleoyl-phosphatidylcholine (POPC) (liquid state) or 1,2-disteraoyl-phosphatidylcholine (DSPC) (solid state) phospholipids by using nanostructured lipid/peptide films (Langmuir monolayers). Pure Aß(1-40) amyloid peptides form insoluble monolayers without forming fibril-like structures. In a lipid environment [phospholipid/Aß(1-40) peptide mixtures], we observed that both miscibility and stability of the films depend on the peptide content. At low Aß(1-40) amyloid peptide proportion (from 2.5 to 10% of peptide area proportion), we observed the formation of a fibril-like structure when mixed only with POPC lipids. The stability acquired by these mixed films is within 20-35 mN·m-1 compatible with the equivalent surface pressure postulated for natural biomembranes. Fibrils are clearly evidenced directly from the monolayers by using Brewster angle microscopy. The so-called nanostructured fibrils are thioflavin T positive when observed by fluorescence microscopy. The amyloid fibril network at the surface was also evidenced by atomic force microscopy when the films are transferred onto a mica support. Aß(1-40) amyloid mixed with the solid DSPC lipid showed an immiscible behavior in all peptide proportions without fibril formation. We postulated that the amyloid fibrillogenesis at the membrane can be dynamically nano-self-triggered at the surface by the quality of the interfacial environment, that is, the physical state of the water-lipid interface and the relative content of amyloid protein present at the interface.

Plasmon-assisted delivery of single nano-objects in an optical hot spot.

Fully exploiting the capability of nano-optics to enhance light-matter interaction on the nanoscale is conditioned by bringing the nano-object to interrogate within the minuscule volume where the field is concentrated. There currently exists several approaches to control the immobilization of nano-objects but they all involve a cumbersome delivery step and require prior knowledge of the "hot spot" location. Herein, we present a novel technique in which the enhanced local field in the hot spot is the driving mechanism that triggers the binding of proteins via three-photon absorption. This way, we demonstrate exclusive immobilization of nanoscale amounts of bovine serum albumin molecules into the nanometer-sized gap of plasmonic dimers. The immobilized proteins can then act as a scaffold to subsequently attach an additional nanoscale object such as a molecule or a nanocrystal. This universal technique is envisioned to benefit a wide range of nano-optical functionalities including biosensing, enhanced spectroscopy like surface-enhanced Raman spectroscopy or surface-enhanced infrared absorption spectroscopy, as well as quantum optics.

Formation of gel and solid phases in acrylic cuvettes upon exposure to DMSO, oxygen and light: implications for fluorescence spectroscopy.

We here report the formation of a turbid-gel phase in acrylic cuvettes upon exposure to pure Dimethyl Sulfoxide (DMSO) at room temperature. The observed phenomenon occurred over a 10 h to 14 h incubation in the presence of environmental oxygen. After the turbid gel was removed from the cuvette, it became a white solid exhibiting unique emission behavior. The formation of the turbid-gel phase was accelerated upon exposure to UV 295 LED pulses of light from 6 h to 8 h. Surprisingly, subsequent exposure of the white solid to a few microliters of pure DMSO and vortexing resulted in its transformation into a transparent gel state in just a few minutes, eventually acquiring transparent and liquid properties. Additionally, the white-solid phase can load other molecules, such as Resveratrol and Quercetin, leading to shifts in the respective emission spectra compared with the same molecule in liquid and pure DMSO. These novel findings highlight the interaction between UV photons, oxygen, DMSO and Acrylic, and potentially distort fluorescence spectroscopy experiments.

Photophysics, photochemistry and energetics of UV light induced disulphide bridge disruption in apo-α-lactalbumin.

Continuous 295 nm excitation of whey protein bovine apo-α-lactalbumin (apo-bLA) results in an increase of tryptophan fluorescence emission intensity, in a progressive red-shift of tryptophan fluorescence emission, and breakage of disulphide bridges (SS), yielding free thiol groups. The increase in fluorescence emission intensity upon continuous UV-excitation is correlated with the increase in concentration of free thiol groups in apo-bLA. UV-excitation and consequent SS breakage induce conformational changes on apo-bLA molecules, which after prolonged illumination display molten globule spectral features. The rate of tryptophan fluorescence emission intensity increase at 340 nm with excitation time increases with temperature in the interval 9.3-29.9°C. The temperature-dependent 340 nm emission kinetic traces were fitted by a 1st order reaction model. Native apo-bLA molecules with intact SS bonds and low tryptophan emission intensity are gradually converted upon excitation into apo-bLA molecules with disrupted SS, molten-globule-like conformation, high tryptophan emission intensity and red-shifted tryptophan emission. Experimental Ahrrenius activation energy was 21.8 ± 2.3 kJ x mol(-1). Data suggests that tryptophan photoionization from the S(1) state is the likely pathway leading to photolysis of SS in apo-bLA. Photoionization mechanism(s) of tryptophan in proteins and in solution and the activation energy of tryptophan photoionization from S(1) leading to SS disruption in proteins are discussed. The observations present in this paper raise concern regarding UV-light pasteurization of milk products. Though UV-light pasteurization is a faster and cheaper method than traditional thermal denaturation, it may also lead to loss of structure and functionality of milk proteins.

Melittin-solid phospholipid mixed films trigger amyloid-like nano-fibril arrangements at air-water interface.

We used the Langmuir monolayers technique to study the surface properties of melittin toxin mixed with either liquid-condensed DSPC or liquid-expanded POPC phospholipids. Pure melittin peptide forms stable insoluble monolayers at the air-water interface without interacting with Thioflavin T (Th-T), a sensitive probe to detect protein amyloid formation. When melittin peptide is mixed with DSPC lipid at 50 % of peptide area proportion at the surface, we observed the formation of fibril-like structures detected by Brewster angle microscopy (BAM), but they were not observable with POPC. The nano-structures in the melittin-DSPC mixtures became Th-T positive labeling when the arrangement was observed with fluorescence microscopy. In this condition, Th-T undergoes an unexpected shift in the typical emission wavelength of this amyloid marker when a 2D fluorescence analysis is conducted. Even when reflectivity analysis of BAM imaging evidenced that these structures would correspond to the DSPC lipid component of the mixture, the interpretation of ATR-FTIR and Th-T data suggested that both components were involved in a new lipid-peptide rearrangement. These nano-fibril arrangements were also evidenced by scanning electron and atomic force microscopy when the films were transferred to a mica support. The fibril formation was not detected when melittin was mixed with the liquid-expanded POPC lipid. We postulated that DSPC lipids can dynamically trigger the process of amyloid-like nano-arrangement formation at the interface. This process is favored by the relative peptide content, the quality of the interfacial environment, and the physical state of the lipid at the surface.

Photonic immobilization of BSA for nanobiomedical applications: creation of high density microarrays and superparamagnetic bioconjugates.

Light assisted molecular immobilization has been used for the first time to engineer covalent bioconjugates of superparamagnetic nanoparticles and proteins. The technology involves disulfide bridge disruption upon UV excitation of nearby aromatic residues. The close spatial proximity of aromatic residues and disulfide bridges is a conserved structural feature in proteins. The created thiol groups bind thiol reactive surfaces leading to oriented covalent protein immobilization. We have immobilized a model carrier protein, bovine serum albumin, onto Fe(3)O(4)@Au core-shell nanoparticles as well as arrayed it onto optically flat thiol reactive surfaces. This new immobilization technology allows for ultra high dense packing of different bio-molecules on a surface, allowing the creation of multi-potent functionalized active new biosensor materials, biomarkers identification and the development of nanoparticles based novel drug delivery system.

Activation energy of light induced isomerization of resveratrol.

Isomerization of trans-stilbenes is known to be induced by light. The two isomers have distinct absorption, fluorescence excitation and emission spectra. Resveratrol, 3,4',5-trihydroxystilbene, is a member of the stilbene family. The interest of the scientific community in resveratrol has increased over the last years due to its biomedical properties. Whereas there is a growing confidence that trans-resveratrol is non-toxic, very little is known about the pharmacology of cis-resveratrol. Of this very reason there is considerable interest in knowing the energetics of the trans-cis conversion. Cis-resveratrol is characterized by a large fluorescence quantum yield when compared to trans-resveratrol. In the present paper we report a detailed analysis of the spectral changes induced in trans-resveratrol upon 260 nm excitation for different time periods. Spectral changes have been monitored with UV-visible absorption and steady-state fluorescence spectroscopy at pH 4 at 20, 25, 30, 35, 40, 45 and 50 °C. Continuous 260 nm excitation induces a blue shift in the absorption and fluorescence excitation spectra of resveratrol and a 14 nm blue shift in its fluorescence emission. The photoisomerization yield is reported as a function of 260 nm excitation time. 330 min continuous excitation led to ~60% isomerization yield. The kinetics of trans-cis isomerization has been monitored following the increase in fluorescence quantum yield upon continuous 260 nm excitation of trans-resveratrol. The study was carried out at the above mentioned temperatures in order to obtain the Arrhenius activation energy of photoisomerization. Activation energy and pre-exponential factor were 3.7 ± 0.3 kcal.mol(-1) and 10.6 ± 1.6 s(-1), respectively. The activation energy is comparable with previously reported values for the photoisomerization of other stilbenes.

Towards nanoscale biomedical devices in medicine: biofunctional and spectroscopic characterization of superparamagnetic nanoparticles.

Medical interest in nanotechnology originates from a belief that nanoscale therapeutic devices can be constructed and directed towards its target inside the human body. Such nanodevices can be engineered by coupling superparamagnetic nanoparticle to biomedically active proteins. We hereby report the immobilization of a PhEst, a S-formylglutathione hydrolase from the psychrophilic P. haloplanktis TAC125 onto the gold coated surface of modified superparamagnetic core-shell nanoparticles (Fe(3)O(4)@Au). The synthesis of the nanoparticles is also reported. S-formylglutathione hydrolases constitute a family of ubiquitous enzymes which play a key role in formaldehyde detoxification both in prokaryotes and eukaryotes. PhEst was originally annotated as a putative feruloyl esterase, an enzyme that releases ferulic acid (an antioxidant reactive towards free radicals such as reactive oxygen species) from polysaccharides esters. Dynamic light scattering, scanning electron microscopy with energy dispersive X-ray spectroscopy, UV-visible absorption spectroscopy, fluorescence spectroscopy, magnetic separation technique and enzyme catalytic assay confirmed the chemical composition of the gold covered superparamagnetic nanoparticles, the binding and activity of the enzyme onto the nanoparticles. Activity data in U/ml confirmed that the immobilized enzyme is approximately 2 times more active than the free enzyme in solution. Such particles can be directed with external magnetic fields for bio-separation and focused towards a medical target for therapeutical as well as bio-sensor applications.

Stitching together a nm thick peptide-based semiconductor sheet using UV light.

Langmuir monolayer allows for a two-dimensional nano-scale organization of amphiphilic molecules. We have adapted this technique to measure lateral and transverse conductivity in confined peptide nanosheets for the first time. We reported that two retro-isomers amphipathic peptides form stable monolayers showing a semiconductor-like behavior. Both peptides exhibit the same hydrophobicity and surface stability. They differ in the lateral conductivity and current-voltage due to the asymmetric peptide bond backbone orientation at the interface. Both peptides contain several tyrosines allowing the lateral crosslinking in neighboring molecules induced by UVB. UVB-light induces changes in the lateral conductivity and current-voltage behavior as well as monolayer heterogeneity monitored by Brewster Angle Microscopy. The semiconductor properties depend on the peptide bond backbone orientation and tyrosine crosslinking. Our results indicate that one may design extended nano-sheets with particular electric properties, reminiscent of semiconductors. We propose to exploit such properties for biosensing and neural interfaces.

Characterization of Insulin Dimers by Top-Down Mass Spectrometry.

High-molecular weight products (HMWP) are an important critical quality attribute in research and development of insulin biopharmaceuticals. We here demonstrate on two case studies of covalent insulin dimers, induced by Fe2+ incubation or ultraviolet (UV) light stress, that de novo characterization in top-down mass spectrometry (MS) workflows can identify cross-link types and sites. On the MS2 level, electron-transfer/higher-energy collision dissociation (EThcD) efficiently cleaved the interchain disulfide bonds in the dimers to reveal cross-link connectivities between chains. The combined utilization of EThcD and 213 nm ultraviolet photodissociation (UVPD) facilitated identification of the chemical composition of the cross-links. Identification of cross-link sites between chains at residue level was achievable for both dimers with MS3 analysis of MS2 fragments cleaved at the cross-link or additionally the interchain disulfide bonds. UVPD provided identification of cross-link sites in the Fe2+-induced dimer without MS3, while cross-link site identification with MS2 was not possible for the UV light-induced dimer. Thus, using varied multistage approaches, it was discovered that in the UV light-induced dimer, Tyr14 of the A-chain participated in an -O-S- cross-link in which the sulfur was derived either from Cys7 or Cys19 of the B-chain. In the Fe2+-induced dimer, Phe1 from both B-chains were cross-linked through a -CH2-. The UV chromophoric side chain of Phe1 was indicated in the cross-link, explaining why UVPD-MS2 was effective in fragmenting the cross-link and nearby backbone bonds. Our results demonstrated that higher-energy collisional dissociation (HCD), EThcD, and UVPD combined with MS3 were powerful tools for direct de novo characterization of cross-linked insulin dimers.

Biophysical properties of phenyl succinic acid derivatised hyaluronic acid.

Modification of hyaluronic acid (HA) with aryl succinic anhydrides results in new biomedical properties of HA as compared to non-modified HA, such as more efficient skin penetration, stronger binding to the skin, and the ability to blend with hydrophobic materials. In the present study, hyaluronic acid has been derivatised with the anhydride form of phenyl succinic acid (PheSA). The fluorescence of PheSA was efficiently quenched by the HA matrix. HA also acted as a singlet oxygen scavenger. Fluorescence lifetime(s) of PheSA in solution and when attached to the HA matrix has been monitored with ps resolved streak camera technology. Structural and fluorescence properties changes induced on HA-PheSA due to the presence of singlet oxygen were monitored using static light scattering (SLS), steady state fluorescence and ps time resolved fluorescence studies. SLS studies provided insight into the depolymerisation kinetics of PheSA derivatised HA matrix in the presence of singlet oxygen. Time resolved fluorescence studies grave insight into the dynamics of the reaction mechanisms induced on HA-PheSA by singlet oxygen. These studies provided insight into the medical relevance of PheSA derivatised HA: its capacity of scavenging singlet oxygen and of quenching PheSA fluorescence. These studies revealed that HA-PheSA is a strong quencher of electronic excited state PheSA and acts as a scavenger of singlet oxygen, thus medical applications of this derivatised form of HA may protect tissues and organs, such as skin, against reactive oxygen species damage.

Direct Ultraviolet Laser-Induced Reduction of Disulfide Bonds in Insulin and Vasopressin.

Ultraviolet (UV) light has been shown to induce reduction of disulfide bonds in proteins in solution. The photoreduction is proposed to be a result of electron donation from excited Tyr or Trp residues. In this work, a powerful UV femtosecond laser was used to generate photoreduced products, while the hypothesis of Tyr/Trp mediation was studied with spectroscopy and mass spectrometry. With limited irradiation times of 3 min or less at 280 nm, the laser-induced reduction in arginine vasopressin and human insulin led to significant yields of ∼3% stable reduced product. The photogenerated thiols required acidic pH for stabilization, while neutral pH primarily caused scrambling and trisulfide formation. Interestingly, there was no direct evidence that Tyr/Trp mediation was a required criterion for the photoreduction of disulfide bonds. Intermolecular electron transfer remained a possibility for insulin but was ruled out for vasopressin. We propose that an additional mechanism should be increasingly considered in UV light-induced reduction of disulfide bonds in solution, in which a single UV photon is directly absorbed by the disulfide bond.

Influence of moderate electric fields in ß-lactoglobulin thermal unfolding and interactions.

In this study, the effects of moderate electric fields during thermal denaturation of ß-lactoglobulin were examined through an in situ circular dichroism approach, complemented by intrinsic extrinsic fluorescence analysis. Results have shown that the effects of electric fields in protein unfolding were linearly dependent on the applied electric field intensity (V/cm) and increased by the use of low electric frequencies - i.e. 50 to 200 Hz. These electric effects caused significant changes on ß-lactoglobulin melting temperature, unfolded conformation and subsequent intermolecular interactions, revealed by the increase of surface hydrophobicity (ANS affinity) and higher conservation of retinol binding. The obtained data provides a clear evidence that moderate electric fields contribute to distinct folding/unfolding of ß-lactoglobulin, resulting in structural modifications. These findings are relevant for (bio)-technological applications involving electric fields processing, bringing new insights for the development of innovative strategies to control protein function and tune production of functional protein systems.

Flash photolysis of cutinase: identification and decay kinetics of transient intermediates formed upon UV excitation of aromatic residues.

Aromatic amino acids play an important role in ultraviolet (UV)-induced photochemical reactions in proteins. In this work, we aim at gaining insight into the photochemical reactions induced by near-UV light excitation of aromatic residues that lead to breakage of disulfide bridges in our model enzyme, Fusarium solani pisi cutinase, a lipolytic enzyme. With this purpose, we acquired transient absorption data of cutinase, with supplemental experimental data on tryptophan (Trp) and lysozyme as reference molecules. We here report formation kinetics and lifetimes of transient chemical species created upon UV excitation of aromatic residues in proteins. Two proteins, lysozyme and cutinase, as well as the free amino acid Trp, were studied under acidic, neutral, and alkaline conditions. The shortest-lived species is assigned to solvated electrons (lifetimes of a few microseconds to nanoseconds), whereas the longer-lived species are assigned to aromatic neutral and ionic radicals, Trp triplet states, and radical ionic disulphide bridges. The pH-dependent lifetimes of each species are reported. Solvated electrons ejected from the side chain of free Trp residues and aromatic residues in proteins were observed 12 ns after excitation, reaching a maximum yield after approximately 40 ns. It is interesting to note that the formation kinetics of solvated electrons is not pH-dependent and is similar in the different samples. On the other hand, a clear increase of the solvated electron lifetime is observed with increasing pH. This observation is correlated with H3O+ being an electron scavenger. Prolonged UV illumination of cutinase leads to a larger concentration of solvated electrons and to greater absorption at 410 nm (assigned to disulphide electron adduct RSSR *-), with concomitant faster decay kinetics and near disappearance of the Trp* radical peak at 330 nm, indicating possible additional formation of TyrO* formed upon reaction of Trp* with Tyr residues. Prolonged UV illumination of cutinase also leads to a larger concentration of free thiol groups, known to originate from the dissociation of RSSR *-. Additional mechanisms that may lead to the near disappearance of Trp(*) are discussed. Our study provides insight into one key UV-light-induced reaction in cutinase, i.e., light-induced disruption of disulphide bridges mediated by the excitation of aromatic residues. Knowledge about the nature of the formed species and their lifetimes is important for the understanding of UV-induced reactions in humans that lead to light-induced diseases, e.g., skin cancer and cataract formation.

Photonic immobilization of high-density protein arrays using Fourier optics.

The technique of UV-light-assisted immobilization of disulfide containing proteins has been combined with the Fourier-transforming properties of lenses as well as with a simple millimeter scale feature size spatial mask. The result is a new simple and inexpensive way of creating high-density protein arrays with feature sizes down to a few hundred nanometers, which represents an improvement of tenfold over existing commercially available high-density protein arraying methods.

Printing novel molecular architectures with micrometer resolution using light.

In this paper we present a new photonic technology and demonstrate that it allows for precise immobilisation of proteins to sensor surfaces. The new technology secures spatially controlled molecular immobilisation since the immobilisation of each molecule to a support surface can be limited to the focal point of the UV laser beam, with dimensions as small as a few micrometers. We have demonstrated that we are not limited to immobilising molecules according to conventional patterns like microarrays. We can immobilise molecules on a surface with any arbitrary pattern. The different illumination/immobilisation setups presented expand the capabilities and usefulness of the new technology, since immobilisation can both be achieved with a laser system and with an affordable Xenon lamp setup. Of extreme relevance to the success of this technology is the precise knowledge of photon flux, energy flux, total number of photons per area, fluency and peak intensity. The expected resolution, taken into account the size of the focused laser beam, the precision of translation stage, and the scanner resolution of our laser scanner is in good agreement with the experimental resolution obtained. The flexibility of this new technology allows creating any patterns/ structures of molecules, with micrometer resolution, thus being of relevance for present and future nanotechnological applications.

Immobilizing biomolecules near the diffraction limit.

Our group has previously shown that biomolecules containing disulfide bridges in close proximity to aromatic residues can be immobilized, through covalent bonds, onto thiol derivatized surfaces upon UV excitation of the aromatic residue(s). We have also previously shown that our new technology can be used to print arrays of biomolecules and to immobilize biomolecules according to any specific pattern on a planar substrates with micrometer scale resolution. In this paper we show that we can immobilize proteins according to diffraction patterns of UV light. We also show that the feature size of the immobilized patterns can be as small as the diffraction limit for the excitation light, and that the immobilized patterns correspond to the diffraction pattern used to generate it. The flexibility of this new technology will in principle make it possible to create any pattern of biomolecules onto a substrate, which can be generated by a UV diffraction pattern. Such patterns can have sub-micron feature sizes and could therefore be of great relevance for present and future nanotechnological applications.

Characterization of Ultraviolet Photoreactions in Therapeutic Peptides by Femtosecond Laser Catalysis and Mass Spectrometry.

Peptides and proteins have diverse ultraviolet (UV) photoreaction pathways that can be activated by the energy of the UV photons absorbed. Simple light sources such as lamps are conventionally used to study these photoreactions in solution. This work provides a proof of concept that femtosecond laser technology can function as a highly potent UV source in rapidly conducting UV photostability studies of peptides. Correspondingly, sufficient quantities of photoproducts were generated in 1 min or less, allowing for identification of known and new photomodifications in the therapeutic peptides somatostatin-14 and arginine vasopressin. Identical photoproducts were also generated with a conventional continuous source. The major modifications included N-formylkynurenine, a cross-link between Trp and Phe, a Tyr product with an NH3 loss, and disruption of an unstable disulfide bond into a complex mixture of a trisulfide bond and multiple scrambled dimeric products. In conclusion, femtosecond lasers are extremely useful to drive fast UV-induced reactions for high throughput screening of photostability and modifications in amino acid polymers.

The repeated 36 amino acid motif of Chlamydia trachomatis Hc2 protein binds to the major groove of DNA.

The gram-negative, obligate intracellular human pathogen, Chlamydia trachomatis has a bi-phasic developmental cycle. The histone H1-like C. trachomatis DNA binding protein, Hc2, is produced late during the developmental cycle when the dividing reticulate body transforms into the smaller, metabolically inactive elementary body. Together with Hc1, the two proteins compact the chlamydial chromosome and arrest replication and transcription. Hc2 is heterogeneous in length due to variation in the number of lysine rich pentamers. Six pentamers and one hexamer constitute a 36 amino acid long repetitive unit that, in spite of variations, is unique for Chlamydiaceae. Using synthetic peptides, the DNA-binding capacity of the 36 amino acid peptide and that of a randomized peptide was analyzed. Both peptides bound and compacted plasmid DNA, however, electron microscopy of peptide/DNA complexes showed major differences in the resulting aggregated structures. Fluorescence spectroscopy was used to analyze the binding. After complexing plasmid DNA with each of three different intercalating dyes, increasing amounts of peptides were added and fluorescence spectroscopy performed. The major groove binder, methyl green, was displaced by both peptides at low concentrations, while the minor groove binder, Hoechts, and the intercalating dye, Ethidium Bromide, were displaced only at high concentrations of peptides.

Photonic activation of disulfide bridges achieves oriented protein immobilization on biosensor surfaces.

Photonic induced immobilization is a novel technology that results in spatially oriented and spatially localized covalent coupling of biomolecules onto thiol-reactive surfaces. Immobilization using this technology has been achieved for a wide selection of proteins, such as hydrolytic enzymes (lipases/esterases, lysozyme), proteases (human plasminogen), alkaline phosphatase, immunoglobulins' Fab fragment (e.g., antibody against PSA [prostate specific antigen]), Major Histocompability Complex class I protein, pepsin, and trypsin. The reaction mechanism behind the reported new technology involves "photonic activation of disulfide bridges," i.e., light-induced breakage of disulfide bridges in proteins upon UV illumination of nearby aromatic amino acids, resulting in the formation of free, reactive thiol groups that will form covalent bonds with thiol-reactive surfaces (see Fig. 1). Interestingly, the spatial proximity of aromatic residues and disulfide bridges in proteins has been preserved throughout molecular evolution. The new photonic-induced method for immobilization of proteins preserves the native structural and functional properties of the immobilized protein, avoiding the use of one or more chemical/thermal steps. This technology allows for the creation of spatially oriented as well as spatially defined multiprotein/DNA high-density sensor arrays with spot size of 1 microm or less, and has clear potential for biomedical, bioelectronic, nanotechnology, and therapeutic applications.