Self-assembled Monolayer Surface Research Articles

Use of a Branched Linker for Enhanced Biosensing Properties in IgG Detection from Mixed Chinese Hamster Ovary Cell Cultures.

Tris(2-aminoethyl)-amine (TREN), a branched amine, was coupled to planar surfaces of alkanethiol self-assembled monolayers (SAMs) to increase the grafting density of IgG-binding peptide (HWRGWV or HWRGWVG) on gold surfaces. One of the three primary amine pendant groups of TREN anchors onto the SAM, while the other two are available for grafting with the C-termini of the peptide. The ellipsometric peptide density on the SAM-branched amine was 1.24 molecules nm-2. The surfaces carrying the peptides were investigated via surface plasmon resonance (SPR) to quantify the adsorption of IgG and showed maximum binding capacity, Qm of 4.45 mg m-2, and dissociation constant, Kd of 8.7 × 10-7 M. Real-time dynamic adsorption data was used to determine adsorption rate constants, ka values, and the values were dependent on IgG concentration. IgG binding from complex mixtures of Chinese hamster ovary supernatant (CHO) was investigated and regeneration studies were carried out. Compared to the unbranched amine-based surfaces, the branched amines increased the overall sensitivity and selectivity for IgG adsorption from complex mixtures. Regeneration of the branched amine-based surfaces was achieved with 0.1 M NaOH, with less than 10% decline in peptide activity after 12 cycles of regeneration-binding.

Nanoparticle Assembling through Click Chemistry Directed by Mixed SAMs for Magnetic Applications

“Click” chemistry, used to promote nanoparticle assemblies, is a powerful strategy which has emerged very recently to control the spatial arrangement of nanoparticles onto surfaces. Such a strategy may be of high interest for applications such as magnetic recording media or magnetic sensors which are based on the fine control of the collective properties of nanoparticles. Nevertheless, self-assembly driven by clickable functional groups still remains to be understood. Mixed self-assembled monolayers (SAMs) of alkane–thiol molecules were used to control the spatial arrangement of nanoparticles onto gold substrates. This approach was combined with click chemistry in order to control the immobilization of nanoparticles on selective areas through specific copper catalyzed alkyne–azide cycloaddition (CuAAC) reaction. Mixed SAMs consist of co-adsorbed 11-(undec-1-ynyl)thiol (S-CC) and 12-(dodecane)thiol (S-CH3) molecules. The variation of the molar ratio between both molecules resulted in significant modulation of the structure of nanoparticle assemblies. The spatial arrangement of nanoparticles revealed the very complex structure of alkyne/methylene terminated mixed SAMs. Alkyne terminal groups could not be only studied by the usual characterization surface techniques such as PM-IRRAS and XPS. Therefore, azido-terminated nanoparticles acted as probing agents to determine the spatial distribution of alkyne groups at the surface of mixed SAMs. This approach was combined with scanning tunneling microscopy (STM) and DFT calculations to get a deeper insight into the structure of mixed SAMs of S-CC and S-CH3 molecules. Gold substrate topography, chemical affinity of molecules, intermolecular interactions and length of alkyl chains were found to be critical parameters that rule the SAM structure.

Direct stimulation of cellular immune response via TLR2 signaling triggered by contact with hybrid glyco-biointerfaces composed of chitohexaose and cellohexaose.

Recognition of carbohydrates by cell receptors activates various cell signaling processes. In this report, hybrid self-assembled monolayers (SAMs) of chitohexaose (βGlcNAc6) and cellohexaose (βGlc6) are prepared to induce a highly sensitive, glyco-density-dependent inflammatory response by HEK293 cells expressing toll-like receptor 2 (TLR2), which recognizes oligo-βGlcNAc moieties. Thiosemicarbazide-labeled βGlcNAc6 and βGlc6, as a bio-signal inducer and a spacer molecule, respectively, were immobilized on a gold substrate via spontaneous chemisorption to control the βGlcNAc6 density on the SAM surface. The βGlcNAc6 density ranged from 0 to 0.65 chains nm-2. The inflammatory response of HEK293 cells varied as a function of the surface glyco-density, and the hybrid SAM with 0.146 chains nm-2 of βGlcNAc6 induced a stronger response than pure βGlcNAc6-SAM with 0.654 chains nm-2. Thus, direct activation of TLR2-mediated cellular stimulation triggered by as-designed glyco-biointerfaces was possible through a mutual interaction between the fittingly βGlcNAc6 moieties assembled on hybrid glyco-SAMs and TLR2 on the surface of HEK293 cells.

Influence of cell adhesive molecules attached onto PEG-lipid-modified fluid surfaces on cell adhesion.

The involvement of intercellular interactions in various biological events indicates the importance of studying cell-cell interactions using fluid model surfaces. Here, we propose a fluid surface composed of a self-assembled monolayer (SAM) and poly(ethylene glycol)-conjugated phospholipid (PEG-lipid) derivatives, which can be an alternative to supported lipid membranes. The modification of SAM surfaces with PEG-lipids carrying functional peptides resulted in the formation of the fluid surfaces with different mobility, which was quantitatively determined by quartz crystal microbalance with dissipation (QCM-D) and fluorescence recovery after photobleaching (FRAP). Different types of fluid surfaces with calculated diffusion coefficients between 0.9 ± 0.25 and 0.16 ± 0.03 μm2/sec for PEG-lipids derivatives were fabricated, onto which arginylglycylaspartate (RGD) peptides were immobilized for cell adhesion, and compared to solid surfaces with the same surface density of RGD peptides. The fluid surfaces revealed that cell adhesions of epithelial cells (MCF-10 A) and human umbilical vein endothelial cells (HUVEC) could not be established on the surfaces with higher fluidity, while cells could adhere onto surfaces with lower fluidity, where the lateral diffusion of PEG-lipids was approximately 20 times lower, and solid surfaces. Interestingly, cells that adhered onto the surface with lower fluidity proliferated at a normal rate while maintaining their round morphology, which was a different shape from that observed on solid surfaces. Thus, the scaffold fluidity greatly influenced cell adhesion behaviors, demonstrating that it is an important parameter for designing novel biomimetic scaffolds for biomedical applications.

Investigating the Effect of Two-Point Surface Attachment on Enzyme Stability and Activity.

Immobilization on solid supports provides an effective way to improve enzyme stability and simplify downstream processing for biotechnological applications, which has been widely used in research and in applications. However, surface immobilization may disrupt enzyme structure due to interactions between the enzyme and the supporting substrate, leading to a loss of the enzyme catalytic efficiency and stability. Here, we use a model enzyme, nitroreductase (NfsB), to demonstrate that engineered variants with two strategically positioned surface-tethering sites exhibit improved enzyme stability when covalently immobilized onto a surface. Tethering sites were designed based on molecular dynamics (MD) simulations, and enzyme variants containing cysteinyl residues at these positions were expressed, purified, and immobilized on maleimide-terminated self-assembled monolayer (SAM) surfaces. Sum frequency generation (SFG) vibrational spectroscopy and attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopy were used to deduce the NfsB enzyme orientations, which were found to be consistent with those predicted from the MD simulations. Thermal stability analyses demonstrated that NfsB variants immobilized through two tethering sites exhibited generally improved thermal stability compared with enzymes tethered at only one position. For example, NfsB enzyme chemically immobilized via positions 423 and 111 exhibits at least 60% stability increase compared to chemically immobilized NfsB mutant via a single site. This research develops a generally applicable and systematic approach using a combination of simulation and experimental methods to rationally select protein immobilization sites for the optimization of surface-immobilized enzyme activity and stability.

Fifteen Nanometer Resolved Patterns in Selective Area Atomic Layer Deposition-Defectivity Reduction by Monolayer Design.

Selective area atomic layer deposition (SA-ALD) offers the potential toreplace a lithography step and provide a significant advantage to mitigate pattern errors and relax design rules in semiconductor fabrication. One class of materials that shows promise to enable this selective deposition process are self-assembled monolayers (SAMs). In an effort to more completely understand the ability of these materials to function as barriers for ALD processes and their failure mechanism, a series of SAM derivatives were synthesized and their structure-property relationship explored. These materials incorporate different side group functionalities and were evaluated in the deposition of a sacrificial etch mask. Monolayers with weak supramolecular interactions between components (for example, van der Waals) were found to direct a selective deposition, though they exhibit significant defectivity at and below 100 nm feature sizes. The incorporation of stronger noncovalent supramolecular interacting groups in the monolayer design, such as hydrogen bonding units or pi-pi interactions, did not produce an added benefit over the weaker interacting components. Incorporation of reactive moieties in the monolayer component that enabled the polymerization of an SAM surface, however, provided a more effective barrier, greatly reducing the number and types of defects observed in the selectively deposited ALD film. These reactive monolayers enabled the selective deposition of a film with critical dimensions as low as 15 nm. It was also found that the selectively deposited film functioned as an effective barrier for isotropic etch chemistries, allowing the selective removal of a metal without affecting the surrounding surface. This work enables selective area ALD as a technology through (1) the development of a material that dramatically reduces defectivity and (2) the demonstrated use of the selectively deposited film as an etch mask and its subsequent removal under mild conditions.

Chemically Immobilized Antimicrobial Peptide on Polymer and Self-Assembled Monolayer Substrates

Surfaces with chemically immobilized antimicrobial peptides have been shown to have great potential in various applications such as biosensors and antimicrobial coatings. This research investigated the chemical immobilization of a cecropin-melittin hybrid antimicrobial peptide on two different surfaces, a polymer surface prepared by chemical vapor deposition (CVD) polymerization and a self-assembled monolayer surface. We probed the structure of immobilized peptides using spectroscopic methods and correlated such structural information to the measured antimicrobial activity. We found that the hybrid peptide adopts an α-helical structure after immobilization onto both surfaces. As we have shown previously for another α-helical peptide, MSI-78, immobilized on a SAM, we found that the α-helical hybrid peptide lies down when it contacts bacteria. This study shows that the antimicrobial activity of the surface-immobilized peptides on the two substrates can be well explained by the spectroscopically measured peptide structural data. In addition, it was found that the polymer-based antimicrobial peptide coating is more stable. This is likely due to the fact that the SAM prepared using silane may be degraded after several days whereas the polymer prepared by CVD polymerization is more stable than the SAM, leading to a more stable antimicrobial coating.

Cytochrome c electrochemistry on peptide self-assembled monolayers

A short chain peptide is covalently attached to an evaporated gold electrode to form a biocompatible self-assembled monolayer (SAM) surface. The peptide is designed to optimize the interfacial interaction of horse heart cytochrome c (cytc) through the electrostatic interaction of the carboxylate group on the terminal amino acid glutamate and the amine on lysine in cytc. Cytc exhibits quasi-reversible voltammetry and is stable on the time scale of the experiments. Marcus theory is used to calculate the electron transfer rates, which are ~5 times faster for cytc/peptide/Au compared to cytc/11-mercaptoundecanoic acid/Au SAMs of similar chain length. The formal potentials also shift for the cytc/peptide system ~20 mV more positive than cytc adsorbed to 11-mercaptoundecanoic acid-SAM/Au electrodes.

Electrochemical and Surface-Plasmon Correlation of a Serum-Autoantibody Immunoassay with Binding Insights: Graphenyl Surface versus Mercapto-Monolayer Surface.

We present here the correlation of picomolar affinities between surface-plasmon and electrochemical immunoassays for the binding of serum glutamic acid decarboxylase 65 autoantibody (GADA), a biomarker of type 1 diabetes (T1D), to its antigen GAD-65. Carboxylated (∼5.0%)-graphene-modified immunoassembly on a gold surface-plasmon chip or on an electrochemical array provided significantly larger binding affinity, higher sensitivity, and lower detection limits than a self-assembled monolayer surface of mercaptopropionic acid (MPA). Estimation of the relative surface -COOH groups by covalent tagging of an electroactive aminoferrocene showed that the graphenyl surface displayed a greater number of -COOH groups (9-fold) than the MPA surface. X-ray-photoelectron-spectroscopy analysis showed more C-O and C═O functionalities on the graphene-COOH surface than on the MPA surface. The graphene-COOH coating on gold exhibited ∼5.5-fold enhancement of plasmon signals compared with a similar coating on a plain glass surface. In summary, this article provides a quantitative comparison of carboxylated graphene with a mercapto-monolayer immunoassembly. Additionally, we propose that the binding-constant value can be useful as a quality-control checkpoint for reproducible and reliable production of large-scale biosensors for clinical bioassays.

A Computational Comparison of Soft Landing of α-Helical vs Globular Peptides.

The effect of secondary structure on the soft landing process is investigated through direct dynamics simulations of AcA7K and AcKA7 colliding with a fluorinated, organic self-assembled monolayer (FSAM) surface. The α-helical (AcA7K) and globular (AcKA7) peptides each exhibited a similar probability of soft landing with normal incidence at all collision energies considered. Rapid conformational changes were quantified through the calculation of the time dependent, conformational entropy production that took place during the collision events, which is consistent with the prior structural measurements made by Laskin and co-workers on these systems. AcA7K produces more entropy during the collisions than AcKA7.

Long-Range Modulations of Electric Fields in Proteins

Electrostatic interactions are essential for controlling the protein structure and function. Whereas so far experimental and theoretical efforts focused on the effect of local electrostatics, this work aims at elucidating the long-range modulation of electric fields in proteins upon binding to charged surfaces. The study is based on cytochrome c (Cytc) variants carrying nitrile reporters for the vibrational Stark effect that are incorporated into the protein via genetic engineering and chemical modification. The Cytc variants were thoroughly characterized with respect to possible structural perturbations due to labeling. For the proteins in solution, the relative hydrogen bond occupancy and the calculated electric fields, both obtained from molecular dynamics (MD) simulations, and the experimental nitrile stretching frequencies were used to develop a relationship for separating hydrogen-bonding and non-hydrogen-bonding electric field effects. This relationship provides an excellent description for the stable Cytc variants in solution. For the proteins bound to Au electrodes coated with charged self-assembled monolayers (SAMs), the underlying MD simulations can only account for the electric field changes Δ Eads due to the formation of the electrostatic SAM-Cytc complexes but not for the additional contribution, Δ Eint, representing the consequences of the potential drops over the electrode/SAM/protein interfaces. Both Δ Eads and Δ Eint, determined at distances between 20 and 30 Å with respect to the SAM surface, are comparable in magnitude to the non-hydrogen-bonding electric field in the unbound protein. This long-range modulation of the internal electric field may be of functional relevance for proteins in complexes with partner proteins (Δ Eads) and attached to membranes (Δ Eads + Δ Eint).

Origin of Hydrophilic Surface Functionalization-Induced Thermal Conductance Enhancement across Solid-Water Interfaces.

Thermal transport across solid-water interfaces is critical for a wide range of applications such as solar thermal evaporation, nanoparticle-assisted hyperthermia therapeutics, and nanofluids. Surface functionalization using self-assembled monolayers (SAMs) to change the hydrophilicity of the solid surface is a common strategy to improve the thermal conductance of solid-water interfaces. Although it is known that hydrophilic interfaces increase the interfacial bonding, how it impacts the molecular level energy transport across the interface is still not clear. In this paper, we perform molecular dynamics simulations to calculate the thermal conductance of differently functionalized gold (Au)-water interfaces. Combining the heat flux decomposition to different interatomic interactions across interfaces and analyses of water structures close to the functionalized surfaces, we found that there is a collaborative effect from the electrostatic interactions and the Lennard-Jones (L-J) interactions (especially the repulsive part). The electrostatic interactions, which are between the polar functional groups of SAMs and water, will attract water molecules closer to the SAM surface, leading both the electrostatic and L-J interactions to have larger effective forces across the interfaces. This increases the power exchanged between solid and water atoms, enhancing the thermal energy transport. The results from this work will provide new insights to the understanding of thermal transport across solid-water interfaces.

Formation of Alkanethiol Supported Hybrid Membranes Revisited.

A phospholipid monolayer supported on an alkanethiol self-assembled monolayer (SAM) constitutes a supported hybrid membrane, a model of biological membranes optimized for electronic access through the underlying metal support surface. It is believed that phospholipids, when deposited from aqueous liposome suspension, spontaneously cover the alkanethiol-modified surface, owing to the reduction of surface free energy of the hydrophobic alkane surface exposed to the solution. However, the formation of the hybrid layer has to overcome significant energy barriers in rupturing the vesicle and "unzipping" the membrane leaflets; hence drivers of the spontaneous hybrid membrane formation are unclear. In this work, the authors studied the efficiency of the liposome deposition method to form hybrid membranes on octanethiol and hexadecanethiol SAMs in aqueous environment. Using quartz crystal microbalance to monitor the deposition process it was found that the hybrid membrane did not form spontaneously; the deposit was dominated by hemi-fused liposomes that can only be removed by applying osmotic stress. However, osmotic stress yielded a reproducible layer characterized by ≈-5Hz frequency change that is also confirmed by fluorescence microscopy imaging, irrespective of lipid concentration and the chain length of the SAMs. The frequency change is ≈20% of the frequency change expected for a tightly bound bilayer membrane, or 40% of a single leaflet, suggesting that the lipid layer is in a different conformation compared to a bilayer membrane: the acyl chains are most likely parallel to the SAM surface, likely due to strong hydrophobic interaction. Comparing these results to the literature it appears that the initial formation of hybrid membranes is inhibited by the ionic environment, while osmotic stress leads to the observed unique layer conformation.

Simultaneous Observation of the Orientation and Activity of Surface-Immobilized Enzymes.

Surface immobilized enzymes have been widely used in many applications such as biosensors, biochips, biofuel production, and biofuel cell construction. Many factors dictate how enzymes' structure, activity, and stability may change when immobilized, including surface functionalization, immobilization chemistry, nature of the solid support, and enzyme surface density. To better understand how immobilization affects enzyme structure and activity, we have developed a method to measure both surface-sensitive protein vibrational spectra and enzymatic activity simultaneously. To accomplish this, an optical/fluorescence microscope was incorporated into a sum frequency generation (SFG) spectrometer. Using β-glucosidase (β-Glu) as a model system, enzymes were covalently tethered to a self-assembled monolayer surface using cysteine-maleimide chemistry. Their orientations were determined by SFG spectroscopy, with a single native cysteine residue oriented toward the functionalized surface, and activity measured simultaneously using a fluorogenic substrate resorufin β-d-glucopyranoside, with a loss of activity of 53% as compared to comparable solution measurements. Measuring β-Glu activity and orientation simultaneously provides more accurate information for designing and further improving enzymatic activity of surface-bound enzymes.

Metallization of self-assembled organic monolayer surfaces by Pd nanocluster deposition

A condensation-cell-type cluster aggregation source was used to deposit Pd clusters on the self-assembled monolayers of four different types of organic molecules. Mica slides covered by Au (111) were coated with self-assembled monolayer films of n-dodecanethiol, 4-mercaptopyridine, dimethyldithiocarbamate, and diethyldithiocarbamate, and the behaviour of Pd clusters on these surfaces, as well as on the bare Au (111) surface, was characterized using scanning tunnelling microscopy and X-ray photoelectron spectroscopy. The aim of this study was to present an alternative means for the metallization of organic layers, through the use of preformed clusters that, unlike single adatoms, are less likely to penetrate the organic layer and cause unwanted interfacial layers or short circuits, and deduce its suitability for the various types of organic self-assembled monolayers. Our experimental results indicate that all of the studied self-assembled monolayers are impenetrable for Pd clusters deposited at thermal energies. Contrary to most methods of metallization, Pd cluster deposition therefore provides an efficient means of growing metal overlayers on organic self-assembled monolayers.

Visible light controls cell adhesion on a photoswitchable biointerface.

Bioactive surfaces with specific interactions with cells have been greatly interested due to their potential applications in biosensors and tissue engineering. Herein, we fabricated a dopamine contained photoswitch molecule (compound 1) which could form self-assembled monolayer (SAM) on substrates. The SAM showed a good photoswitch ability and manifested excellent fatigue resistance, which displayed its potential application as a biologically friendly surface coating. Contact angle analysis and cell experiments exhibited that the SAM surface was hydrophobic before irradiation which favored cell adhesion, while, it turned hydrophilic and induced cell unfouling or detachment after light irradiation. The uses of visible light stimulation (λex = 530 nm) and the reversible regulation on cell adhesion and detachment should open up new avenues for bioacitve surfaces in biomedical applications.

Chemical Dynamics Simulations of Thermal Desorption of Protonated Dialanine from a Perfluorinated Self-Assembled Monolayer Surface.

Classical chemical dynamics simulation results are presented for the thermal desorption kinetics and energetics of protonated dialanine ions (ala2-H+) physisorbed on/in a perfluorinated self-assembled monolayer (F-SAM) surface. Previously developed analytic potentials were used for the F-SAM and the ala2-H+/F-SAM intermolecular interaction, and the AMBER valence force field was used for ala2-H+. The activation energy, Ea = 13.2 kcal/mol, determined from the simulations is consistent with previous simulations of the ala2-H+/F-SAM binding energy. The A-factor, 7.8 × 1011 s-1, is about an order of magnitude lower than those representative of small molecule desorption from metal and semiconductor surfaces. This finding is consistent with the decreased entropies of ala2-H+ and the F-SAM upon desorption. Using the Arrhenius parameters for ala2-H+ desorption from the F-SAM, the lifetime of ala2-H+ adsorbed on the F-SAM at 300 K is 5 × 10-3 s. Larger peptide ions are expected to have longer adsorption lifetimes.

Adsorption and decontamination of α-synuclein from medically and environmentally-relevant surfaces.

The assembly and accumulation of α-synuclein fibrils are implicated in the development of several neurodegenerative disorders including multiple system atrophy and Parkinson's disease. Pre-existing α-synuclein fibrils can recruit and convert soluble non-fibrillar α-synuclein to the fibrillar form similar to what is observed in prion diseases. This raises concerns regarding attachment of fibrillary α-synuclein to medical instruments and subsequent exposure of patients to α-synuclein similar to what has been observed in iatrogenic transmission of prions. Here, we evaluated adsorption and desorption of α-synuclein to two surfaces: stainless steel and a gold surface coated with a 11-Amino-1-undecanethiol hydrochloride self-assembled-monolayer (SAM) using in-situ combinatorial quartz crystal microbalance with dissipation and spectroscopic ellipsometry. α-Synuclein was found to attach to both surfaces, however, increased α-synuclein adsorption was observed onto the positively charged SAM surface compared to the stainless steel surface. Dynamic light scattering data showed that larger α-synuclein fibrils were preferentially attached to the stainless steel surface when compared with the distributions in the original α-synuclein solution and on the SAM surface. We determined that after attachment, introduction of a 1N NaOH solution could completely remove α-synuclein adsorbed on the stainless steel surface while α-synuclein was retained on the SAM surface. Our results indicate α-synuclein can bind to multiple surface types and that decontamination is surface-dependent.

Host–Guest Interaction at Molecular Interfaces: Cucurbit[7]uril as a Sensitive Probe of Structural Heterogeneity in Ferrocenyl Self-Assembled Monolayers on Gold

Herein, we combine host–guest recognition chemistry and electrochemical analysis to demonstrate that the nanometer-size, supramolecular hosts can be adapted as sensitive probes for structural heterogeneity in organized molecular assemblies on a surface. In particular, we carried out thorough cyclic voltammetric (CV) studies to evaluate the binding of cucurbit[7]uril on mixed ferrocenylundecanethiolate/n-alkanethiolate self-assembled monolayers (SAMs) on gold (FcC11S-/CmS-Au) prepared with different methods (coadsorption vs postassembly exchanges) and with varied diluting n-alkanethiols. On the basis of the distinct CV responses of CB[7]@Fc complex and free Fc on the SAM surfaces, we were able to determine the conversion ratio from Fc to CB[7]@Fc, a direct indication of its overall density and uniformity. We have shown that the FcC11S-/C8S-Au prepared by coadsorption in a binary solution with low mole fraction of FcC11SH (5%) and by exchanging preassembled C8S-Au SAM with FcC11SH for a short time (1 min) has the “ideal” structure with isolated and uniformly distributed Fc groups on the surface. In contrast, with similar Fc surface coverage, the FcC11S-/C8S-Au prepared by exchanging FcC11S-Au with C8SH for a prolonged time (20 h) has clustered and nonuniformly distributed Fc groups at the surface. While consistent with previous observations based on conventional electrochemical or microscopic studies, the present finding expands the capability of host–guest chemistry as a new tool to probe the structures of organized molecular assemblies at the nanometer scale.

Direct Mapping of Heterogeneous Surface Coverage in DNA-Functionalized Gold Surfaces with Correlated Electron and Fluorescence Microscopy

The characterization of biofunctionalized surfaces such as alkanethiol self-assembled monolayers (SAMs) on gold modified with DNA or other biomolecules is a challenging analytical problem, and access to a routine method is desirable. Despite substantial investigation from structural and mechanistic perspectives, robust and high-throughput metrology tools for SAMs remain elusive but essential for the continued development of these devices. We demonstrate that scanning electron microscopy (SEM) can provide image contrast of the molecular interface during SAM functionalization. The high-speed, large magnification range, and ease of use make this widely available technique a powerful platform for measuring the structure and composition of SAM surfaces. This increased throughput allows for a better understanding of the nonideal spatial heterogeneity characteristic of SAMs utilized in real-world conditions. SEM image contrast is characterized through the use of fluorescently labeled DNA, which enables correlative SEM and fluorescence microscopy. This allows identification of the DNA-modified regions at resolutions that approach the size of the biomolecule. The effect of electron beam irradiation dose is explored, which leads to straightforward lithographic patterning of DNA SAMs with nanometer resolution and with control over the surface coverage of specifically adsorbed DNA.