A fast, ultrasensitive SERS immunoassay to detect SARS-CoV-2 in saliva.

The COVID-19 pandemic has emphasized the need for accurate, rapid, point-of-care diagnostics to control disease transmission. We have developed a simple, ultrasensitive single-particle surface-enhanced Raman spectroscopy (SERS) immunoassay to detect the SARS-CoV-2 spike protein in saliva. This assay relies on the use of single chain Fv (scFv) recombinant antibody expressed in E. coli to bind the SARS-CoV-2 spike protein. Recombinant scFv labeled with a SERS-active dye in solution is mixed with unlabeled scFv conjugated to gold-coated magnetic nanoparticles and a sample to be tested. In the presence of the SARS-CoV-2 spike protein, immunocomplexes form and concentrate the labeled scFv close to the gold surface of the nanoparticles, causing an increased SERS signal. The assay detects inactivated SARS-CoV-2 virus and spike protein in saliva at concentrations of 1.94 × 103 genomes mL-1 and 4.7 fg mL-1, respectively, making this direct detection antigen test only 2-3 times less sensitive than some qRT-PCR tests. All tested SARS-CoV-2 spike proteins, including those from alpha, beta, gamma, delta, and omicron variants, were detected without recognition of the closely related SARS and MERS spike proteins. This 30 min, no-wash assay requires only mixing, a magnetic separation step, and signal measurements using a hand-held, battery-powered Raman spectrometer, making this assay ideal for ultrasensitive detection of the SARS-CoV-2 virus at the point-of-care.

Rapid, Point-of-Care scFv-SERS Assay for Femtogram Level Detection of SARS-CoV-2.

Rapid, sensitive, on-site identification of SARS-CoV-2 infections is an important tool in the control and management of COVID-19. We have developed a surface-enhanced Raman scattering (SERS) immunoassay for highly sensitive detection of SARS-CoV-2. Single-chain Fv (scFv) recombinant antibody fragments that bind the SARS-CoV-2 spike protein were isolated by biopanning a human scFv library. ScFvs were conjugated to magnetic nanoparticles and SERS nanotags, followed by immunocomplex formation and detection of the SARS-CoV-2 spike protein with a limit of detection of 257 fg/mL in 30 min in viral transport medium. The assay also detected B.1.1.7 ("alpha"), B.1.351 ("beta"), and B.1.617.2 ("delta") spike proteins, while no cross-reactivity was observed with the common human coronavirus HKU1 spike protein. Inactivated whole SARS-CoV-2 virus was detected at 4.1 × 104 genomes/mL, which was 10-100-fold lower than virus loads typical of infectious individuals. The assay exhibited higher sensitivity for SARS-CoV-2 than commercial lateral flow assays, was compatible with viral transport media and saliva, enabled rapid pivoting to detect new virus variants, and facilitated highly sensitive, point-of-care diagnosis of COVID-19 in clinical and public health settings.

Detection of Multiple Pathogens in Serum Using Silica-Encapsulated Nanotags in a Surface-Enhanced Raman Scattering-Based Immunoassay.

A robust immunoassay based on surface-enhanced Raman scattering (SERS) has been developed to simultaneously detect trace quantities of multiple pathogenic antigens from West Nile virus, Rift Valley fever virus, and Yersinia pestis in fetal bovine serum. Antigens were detected by capture with silica-encapsulated nanotags and magnetic nanoparticles conjugated with polyclonal antibodies. The magnetic pull-down resulted in aggregation of the immune complexes, and the silica-encapsulated nanotags provided distinct spectra corresponding to each antigen captured. The limit of detection was ∼10 pg/mL in 20% fetal bovine serum, a significant improvement over previous studies in terms of sensitivity, level of multiplexing, and medium complexity. This highly sensitive multiplex immunoassay platform provides a promising method to detect various antigens directly in crude serum samples without the tedious process of sample preparation, which is desirable for on-site diagnostic testing and real-time disease monitoring.

The development of surface-enhanced Raman scattering as a detection modality for portable in vitro diagnostics: progress and challenges.

This perspective provides an overview of the diverse surface-enhanced Raman scattering (SERS)-based sensor platforms that have been developed for in vitro diagnostic applications. To provide focus, protein and nucleic acid detection assays based on the principle of extrinsic SERS sensing are emphasized, as well as their potential for translation to fully integrated point-of-care (POC) test platforms. The development of intrinsic SERS sensors, which are predicated on the direct detection of analytes by laser excitation, entails unique opportunities and challenges deserving of their own attention. As the robust sensing of disease pathogens and cancers in both clinical facilities and limited resource settings is the targeted objective of many next-generation biosensors, the majority of the research progress summarized here centers on SERS sensors developed for the rapid, sensitive and selective detection of disease-causing pathogens and biomarkers. In our effort to communicate a realistic assessment of the progress that has been made and the challenges that lie ahead, we avoid an overtly optimistic appraisal of the current status of SERS diagnostics that does not tacitly acknowledge the difficulties inherent in aligning SERS-based technologies alongside ELISA and PCR technologies as a complementary method for bioanalyte detection possessing unique advantages.

Surface-enhanced Raman scattering (SERS) detection of multiple viral antigens using magnetic capture of SERS-active nanoparticles.

A highly sensitive immunoassay based on surface-enhanced Raman scattering (SERS) spectroscopy has been developed for multiplex detection of surface envelope and capsid antigens of the viral zoonotic pathogens West Nile virus (WNV) and Rift Valley fever virus (RVFV). Detection was mediated by antibody recognition using Raman reporter-coated Au nanoparticles (GNPs) and paramagnetic nanoparticles (PMPs) conjugated with polyclonal antibodies specific for each antigen target, followed by 785nm laser excitation of magnetically concentrated GNP/antigen/PMP complexes. The discrimination of WNV and RVFV antigen detection in mixed Raman spectra was achieved by SERS enhancement of Raman spectra specific for the Raman reporter dyes Infrared 792 (IR-792) and Nile Blue (NB), respectively. Assay reactions containing dilutions of both target antigens yielded a reduction in the intensification of IR-792 and NB signature spectrum peaks and provided a conservative limit of detection of ∼5fg/ml for assays conducted in phosphate buffered saline buffer (PBS) and ∼25pg/ml for assays containing PBS spiked with fetal bovine serum. Based on the inherent simplicity of the assay, magnetic capture-based SERS assays afford promise as a biosensor platform that provides high-level multiplex detection sensitivity and can be adapted for portable diagnostic applications in limited resource settings.

Surface-enhanced Raman scattering detection of DNAs derived from virus genomes using Au-coated paramagnetic nanoparticles.

A magnetic capture-based, surface-enhanced Raman scattering (SERS) assay for DNA detection has been developed which utilizes Au-coated paramagnetic nanoparticles (Au@PMPs) as both a SERS substrate and effective bioseparation reagent for the selective removal of target DNAs from solution. Hybridization reactions contained two target DNAs, sequence complementary reporter probes conjugated with spectrally distinct Raman dyes distinct for each target, and Au@PMPs conjugated with sequence complementary capture probes. In this case, target DNAs were derived from the RNA genomes of the Rift Valley Fever virus (RVFV) or West Nile virus (WNV). The hybridization reactions were incubated for a short period and then concentrated within the focus beam of an interrogating laser by magnetic pull-down. The attendant SERS response of each individually captured DNA provided a limit of detection sensitivity in the range 20-100 nM. X-ray diffraction and UV-vis analysis validated both the desired surface plasmon resonance properties and bimetallic composition of synthesized Au@PMPs, and UV-vis spectroscopy confirmed conjugation of the Raman dye compounds malachite green (MG) and erythrosin B (EB) with the RVFV and WNV reporter probes, respectively. Finally, hybridization reactions assembled for multiplexed detection of both targets yielded mixed MG/EB spectra and clearly differentiated peaks which facilitate the quantitative detection of each DNA target. On the basis of the simple design of a single-particle DNA detection assay, the opportunity is provided to develop magnetic capture-based SERS assays that are easily assembled and adapted for high-level multiplex detection using low-cost Raman instrumentation.

Enzyme nanoparticle fabrication: magnetic nanoparticle synthesis and enzyme immobilization.

Immobilized enzymes are drawing significant attention for potential commercial applications as biocatalysts by reducing operational expenses and by increasing process utilization of the enzymes. Typically, immobilized enzymes have greater thermal and operational stability at various pH values, ionic strengths and are more resistant to denaturation that the soluble native form of the enzyme. Also, immobilized enzymes can be recycled by utilizing the physical or chemical properties of the supporting material. Magnetic nanoparticles provide advantages as the supporting material for immobilized enzymes over competing materials such as: higher surface area that allows for greater enzyme loading, lower mass transfer resistance, less fouling effect, and selective, nonchemical separation from the reaction mixture by an applied a magnetic field. Various surface modifications of magnetic nanoparticles, such as silanization, carbodiimide activation, and PEG or PVA spacing, aid in the binding of single or multienzyme systems to the particles, while cross-linking using glutaraldehyde can also stabilize the attached enzymes.

Synthesis and characterization of enzyme-magnetic nanoparticle complexes: effect of size on activity and recovery.

The influence of particle size on the activity and recycling capabilities of enzyme conjugated magnetic nanoparticles was studied. Co-precipitation and oxidation of Fe(OH)(2) methods were used to fabricate three different sizes of magnetic nanoparticles (5 nm, 26 nm and 51 nm). Glucose oxidase was covalently bound to the magnetic nanoparticles by modifying the surfaces with 3-(aminopropyl)triethoxysilane (APTES) and a common protein crosslinking agent, glutaraldehyde. Analysis by Transmission Electron Microscopy (TEM) showed that the morphology of the magnetic nanoparticles to be spherical and sizes agreed with results of the Brunauer, Emmett, and Teller (BET) method. Magnetic strength of the nanoparticles was analyzed by magnetometry and found to be 49 emu g(-1) (5 nm), 73 emu g(-1) (26 nm), and 85 emu g(-1) (51 nm). X-ray photoelectron spectroscopy (XPS) confirmed each step of the magnetic nanoparticle surface modification and successful glucose oxidase binding. The immobilized enzymes retained 15-23% of the native GOx activity. Recycling stability studies showed approximately 20% of activity loss for the large (51 nm) and medium (26 nm) size glucose oxidase-magnetic nanoparticle (GOx-MNP) bioconjugate and about 96% activity loss for the smallest GOx-MNP bioconjugate (5 nm) after ten cycles. The bioconjugates demonstrated equivalent total product conversions as a single reaction of an equivalent amount of the native enzyme after the 5th cycle for the 26 nm nanoparticles and the 7th cycle for the 51 nm nanoparticles.

Surface-enhanced Raman scattering detection of DNA derived from the west nile virus genome using magnetic capture of Raman-active gold nanoparticles.

A model paramagnetic nanoparticle (MNP) assay is demonstrated for surface-enhanced Raman scattering (SERS) detection of DNA oligonucleotides derived from the West Nile virus (WNV) genome. Detection is based on the capture of WNV target sequences by hybridization with complementary oligonucleotide probes covalently linked to fabricated MNPs and Raman reporter tag-conjugated gold nanoparticles (GNPs) and the subsequent removal of GNP-WNV target sequence-MNP hybridization complexes from solution by an externally applied magnetic source. Laser excitation of the pelleted material provided a signature SERS spectrum which is diagnostic for the reporter, 5,5'-dithiobis(succinimidy-2-nitrobenzoate) (DSNB), and restricted to hybridization reactions containing WNV target sequences. Hybridizations containing dilutions of the target oligonucleotide were characterized by a reduction in the intensification of the spectral peaks accorded to the SERS signaling of DSNB, and the limit of detection for target sequence in buffer was 10 pM. Due to the short hybridization times required to conduct the assay and ease with which reproducible Raman spectra can be acquired, the assay is amenable to adaptation within a portable, user-friendly Raman detection platform for nucleic acids.

A versatile SERS-based immunoassay for immunoglobulin detection using antigen-coated gold nanoparticles and malachite green-conjugated protein A/G.

A surface enhanced Raman scattering (SERS) immunoassay for antibody detection in serum is described in the present work. The developed assay is conducted in solution and utilizes Au nanoparticles coated with the envelope (E) protein of West Nile Virus (WNV) as the SERS-active substrate and malachite green (MG)-conjugated protein A/G (MG-pA/G) as a bi-functional Raman tag/antibody binding reporter. Upon incubation of these reagents with serum collected from rabbits inoculated with E antigen, laser interrogation of the sandwiched immunocomplex revealed a SERS signaling response diagnostic for MG. The intensification of signature spectral peaks is shown to be proportionate to the concentration of added serum and the limit of antibody detection is 2 ng/ml of serum. To assess assay performance relative to more a traditional immunoassay, indirect enzyme-linked immunosorbent assays conducted using the same concentrations of reagents were found to be >400-fold less sensitive. Quartz crystal microbalance with dissipation (QCM-D) monitoring of immunocomplex film deposition on solid Au surfaces also confirmed the formation of antigen-antibody-protein A/G trilayers and provided quantitative measurements of film thickness which likely position MG within the sensing distance of laser-elicited, enhanced electromagnetic fields. The sensitivity and inherent versatility of the assay, which is provided by the binding of pA/G to a broad spectrum of immunoglobulins in different mammalian species, suggest that it could be developed as an alternative immunoassay format to the ELISA.

Layer-by-layer assembly of polysaccharide-based nanostructured surfaces containing polyelectrolyte complex nanoparticles.

Nanoscale chemical and topographical features have been demonstrated to influence a variety of significant responses of mammalian cells to biomaterials surfaces. Thus, an important goal for biomaterials scientists is the ability to engineer the nanoscale surface features of biologically active materials. The goal of the current work is to demonstrate that polyelectrolyte complex nanoparticles (PCNs) in polyelectrolyte multilayers (PEMs) can be combined to create surfaces with controlled nanoscale surface topography and nanoscale presentation of surface chemistry. The polysaccharides used in this work are the biomedically relevant chitosan, heparin, and hyaluronan. Nanostructured surface coatings were characterized on both modified gold substrates and tissue-culture polystyrene surfaces. PCNs were adsorbed to oppositely charged PEMs, and were also embedded within PEMs. The construction of the surface coatings was characterized by quartz crystal microbalance with dissipation (QCM-D). The surface morphology was characterized by scanning electron microscopy (SEM) and atomic force microscopy (AFM). The chemistry of the coatings was confirmed by both X-ray photoelectron spectroscopy (XPS) and polarization modulation infra-red reflection absorption spectroscopy (PM-IRRAS). Morphologically, we found that PCNs were colloidally stable and homogeneously distributed when adsorbed on or in the PEMs. Chemical analysis confirms that the PCNs adsorbed to PEMs significantly altered the surface chemistry, indicating significant surface coverage. Furthermore, the position of the PCNs normal to the surface can be adjusted by adding PEMs on top of adsorbed PCNs. Thus, PCNs can be used to introduce discrete nanoscale surface topographical features and varying surface chemistry into PEM surface coatings in a controlled way.

Interplay of anionic charge, poly(ethylene glycol), and iodinated tyrosine incorporation within tyrosine-derived polycarbonates: Effects on vascular smooth muscle cell adhesion, proliferation, and motility.

Regulation of smooth muscle cell adhesion, proliferation, and motility on biomaterials is critical to the performance of blood-contacting implants and vascular tissue engineering scaffolds. The goal of this study was to examine the underlying substrate-smooth muscle cell response relations, using a selection of polymers representative of an expansive library of multifunctional, tyrosine-derived polycarbonates. Three chemical components within the polymer structure were selectively varied through copolymerization: (1) the content of iodinated tyrosine to achieve X-ray visibility; (2) the content of poly(ethylene glycol) (PEG) to decrease protein adsorption and cell adhesivity; and (3) the content of desaminotyrosyl-tyrosine (DT), which regulates the rate of polymer degradation. Using quartz crystal microbalance with dissipation, we quantified differential serum protein adsorption behavior because of the chemical components DT, iodinated tyrosine, and PEG: increased PEG content within the polymer structure progressively decreased protein adsorption but the simultaneous presence of both DT and iodinated tyrosine reversed the effects of PEG. The complex interplay of these components was next tested on the adhesion, proliferation, and motility behavior cultured human aortic smooth muscle cells. The incorporation of PEG into the polymer reduced cell attachment, which was reversed in the presence of iodinated tyrosine. Further, we found that as little as 10% DT content was sufficient to negate the PEG effect in polymers containing iodinated tyrosine, whereas in non-iodinated polymers, the PEG effect on cell attachment was reversed. Cross-functional analysis of motility and proliferation revealed divergent substrate chemistry related cell response regimes. For instance, within the series of polymers containing both iodinated tyrosine and 10% of DT, increasing PEG levels lowered smooth muscle cell motility without a change in the rate of cell proliferation. In contrast, for non-iodinated tyrosine and 10% of DT, increasing PEG levels increased cell proliferation significantly while reducing cell motility. Clearly, the polycarbonate polymer library offers a sensitive platform to modulate cell adhesion, proliferation, and motility responses, which, in turn, may have implications for controlling vascular remodeling in vivo. Additionally, our data suggests unique biorelevant properties following the incorporation of iodinated subunits in a polymeric biomaterial as a potential platform for X-ray visible devices.

SERS detection of indirect viral DNA capture using colloidal gold and methylene blue as a Raman label.

An indirect capture model assay using colloidal Au nanoparticles is demonstrated for surface enhanced Raman scattering (SERS) spectroscopy detection of DNA. The sequence targeted for capture was derived from the West Nile Virus (WNV) RNA genome and selected on the basis of exhibiting minimal secondary structure formation. Upon incubation with colloidal Au, hybridization complexes containing the WNV target sequence, a complementary capture oligonucleotide conjugated to a strong tethering group and a complementary reporter oligonucleotide conjugated to methylene blue (MB), a Raman label, anchors the resultant ternary complex to Au nanoparticles and positions MB within the required sensing distance for SERS enhancement. The subsequent elicitation of surface enhanced plasmon resonance by laser excitation provides a spectral peak signature profile that is capture-specific and characteristic of the Raman spectrum for MB. Detection sensitivity is in the submicromolar range and was shown to be highest for thiol, and less so for amino, modifications at the 5' terminus of the capture oligonucleotide. Finally, using Quartz Crystal Microbalance-Dissipation as a tool for modeling ternary complex binding to Au surfaces, quantitative measurements of surface mass coverage on Au plated sensor crystals established a positive correlation between levels of ternary complex adsorption and their correspondent levels of SERS signal intensification. Adapted to a compact Raman spectrometer, which is designed for analyte detection in capillary tubes, this assay provides a rapid, mobile and cost effective alternative to expensive spectroscopic instrumentation, which is often restricted to analytical laboratories.

Polysaccharide-based polyelectrolyte complex nanoparticles from chitosan, heparin, and hyaluronan.

The formation of polyelectrolyte complex nanoparticles (PCN) was investigated at different charge mixing ratios for the chitosan-heparin (chi-hep) and chitosan-hyaluronan (chi-ha) polycation-polyanion pairs. The range of 0.08-19.2 for charge mixing ratio (n(+)/n(-)) was examined. The one-shot addition of polycation and polyanion solutions used for the formation of the PCN permitted formation of both cationic and anionic particles from both polysaccharide pairs. The influence of the charge mixing ratio on the size and zeta potential of the particles was investigated. The morphology and stability of the particles when adsorbed to surfaces was studied by scanning electron microscopy (SEM). For most conditions studied, colloidally stable, nonstoichiometric PCN were formed in solution. However, PCN formation was inhibited by flocculation at charge mixing ratios near 1. When adsorbed to surfaces and dried, some formulations resulted in discrete nanoparticles, while others partially or completely aggregated or coalesced, leading to different surface morphologies.

Synthesis of degradable functional poly(ethylene glycol) analogs as versatile drug delivery carriers.

Poly(ethylene glycol) (PEG) is widely used as a water soluble carrier for polymer-drug conjugates. Herein, we report degradable linear PEG analogs (DPEGs) carrying multifunctional groups. The DPEGs were synthesized by a Michael addition based condensation polymerization of dithiols and PEG diacrylates (PEGDA) or dimethacrylates (PEGDMA). They were stable at pH 7.4 but quickly degraded at pH 6.0 and 5.0. Thus, DPEGs could be used as drug carriers without concern for their retention in the body. DPEGs could be made to carry such functional groups as terminal thiol or (meth)acrylate and pendant hydroxyl groups. The functional groups were used for conjugation of drugs and targeting groups. This new type of PEG analog will be useful for drug delivery and the PEGylation of biomolecules and colloidal particles.

Quantitative biorelevant profiling of material microstructure within 3D porous scaffolds via multiphoton fluorescence microscopy.

This study presents a novel approach, based on fluorescence multiphoton microscopy (MPM), to image and quantitatively characterize the microstructure and cell-substrate interactions within microporous scaffold substrates fabricated from synthetic biodegradable polymers. Using fluorescently dyed scaffolds fabricated from poly(DTE carbonate)/poly(DTO carbonate) blends of varying porosity and complementary green fluorescent protein-engineered fibroblasts, we reconstructed the three-dimensional distribution of the microporous and macroporous regions in 3D scaffolds, as well as cellular morphological patterns. The porosity, pore size and distribution, strut size, pore interconnectivity, and orientation of both macroscale and microscale pores of 3D scaffolds were effectively quantified and validated using complementary imaging techniques. Compared to other scaffold characterizing techniques such as confocal imaging and scanning electron microscopy (SEM), MPM enables the acquisition of images from scaffold thicknesses greater than a hundred microns with high signal-to-noise ratio, reduced bulk photobleaching, and the elimination of the need for deconvolution. In our study, the morphology and cytoskeletal organization of cells within the scaffold interior could be tracked with high resolution within the limits of penetration of MPM. Thus, MPM affords a promising integrated platform for imaging cell-material interactions within the interior of polymeric biomaterials.

Minute changes in composition of polymer substrates produce amplified differences in cell adhesion and motility via optimal ligand conditioning.

We explored the interplay between substratum chemistry of polymeric materials and surface-adsorbed ligand concentration (human plasma fibronectin) in the control of cell adhesion and cell motility. We found that small changes in the chemical composition of a polymeric substratum had different effects on cellular motility--depending on the concentration of preadsorbed fibronectin. We used two tyrosine-derived polyarylates, poly(DTD diglycolate) and poly(DTD glutarate), as substrata for the seeding of NIH-3T3 fibroblasts. The only compositional difference between the two test polymers was that one single oxygen atom in the polymer backbone of poly(DTD diglycolate) had been substituted by a methylene group in the backbone of poly(DTD glutarate), The two polymers had closely matched hydrophobicity and physical properties. Flat, spin-coated surfaces of these polymers were pretreated with different concentrations of human plasma fibronectin (0-20 microg/ml). After seeding with NIH-3T3 fibroblasts, we examined the adhesion and motility behavior of these cells. We found that NIH-3T3 fibroblasts migrated significantly faster on poly(DTD diglycolate), but only when the polymer surfaces were pretreated with intermediate concentrations of fibronectin. Only at these intermediate levels of ligand conditioning, did the presence of an extra oxygen atom in the backbone of poly(DTD diglycolate) relative to poly(DTD glutarate) (i) alter the overall organization/concentration of the fibronectin; (ii) weaken cell attachment strength and inhibited excessive cell spreading; and (iii) promote cell motility kinetics. These findings indicate that the biological effect of minute changes in substratum chemistry is critically dependent on the level of surface-adsorbed cell-binding ligands.

X-ray photoelectron spectroscopy and differential capacitance study of thiol-functional polysiloxane films on gold supports.

Polymeric molecules containing multiple thiol groups (polythiols) provide tenacious attachment to metal surfaces such as gold. Polythiol films are also well suited for subsequent derivatization with biomacromolecules through remnant free thiol groups of the film. In this study, 1-3 nm thick layers of a commercial polythiol, poly((mercaptopropyl)methylsiloxane) (PMPMS), are investigated with X-ray photoelectron spectroscopy (XPS) and electrochemical impedance spectroscopy. XPS is used to reveal the surface coverage of thiolate-Au bonds between the polythiol and the metal support, which is found to be approximately 30% lower than that in alkanethiol self-assembled monolayers. The surface density of thiolate-Au bonds did not depend on film thickness provided sufficient PMPMS material was present. Differential capacitance measurements show that the effective dielectric barrier presented by PMPMS films under aqueous environments corresponds closely to their physical thickness, with even approximately 1 nm films remaining impermeable to electrolyte species. Modification of the films with an oligoethylene glycol compound was also examined, in anticipation of future applications in label-free, impedance-based biomolecular diagnostics.

Polymer-anchored DNA gene monolayers.

Monolayers of DNA chains of polymeric dimensions, considered here to be longer than approximately 100 nucleotides, are widely encountered in biomolecular diagnostics as well as present for a model system for investigating behavior of polyelectrolytes at interfaces. A major challenge in advancing such applications is assembling the DNA on the surface in a controlled way. Although covalent immobilization is expected to produce optimal stability, the multitude of potential reactive sites along the contour of long DNA molecules requires that any chemical transformations be strictly site-specific to preserve control over attachment geometry and function. A synthetic approach to fabricating monolayers of DNA genes on gold using polymeric anchor (adhesion) films is presented that (i) possesses stringent site-specificity of surface-attachment, (ii) exhibits excellent stability to elevated temperatures, allowing denaturation of duplex chains at 90 degrees C without loss of surface-linked strands, and (iii) achieves surface coverages suitable for investigating multichain polyelectrolyte behavior in regimes of strong interchain interactions.