Protein resistant or “non‐fouling” surfaces are of great interest for a variety of biomedical and biotechnology applications. This article briefly reviews the development of protein resistant surfaces, followed by recent research on a new methodology to fabricate non‐fouling surfaces by surface‐initiated polymerization. We show that polymer brushes synthesized by surface‐initiated polymerization that present short oligo(ethylene glycol) side chains are exceptionally resistant to protein adsorption and cell adhesion. The importance of the protein and cell resistance conferred by these polymer brushes is illustrated by their use as substrates for the fabrication of antibody microarrays that exhibit femtomolar limits of detection in complex fluids such as serum and blood with relaxed requirements for intermediate wash steps. This example highlights the important point that the reduction in background noise afforded by protein‐resistant surfaces can greatly simplify the development of ultrasensitive heterogeneous, surface‐based clinical and proteomic assays with increased sensitivity and utility.

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We describe the incorporation of multiple fluorophores into a single stranded DNA (ssDNA) chain using terminal deoxynucleotidyl transferase (TdT), a template-independent DNA polymerase that catalyzes the sequential addition of deoxynucleotides (dNTPs) at the 3'-OH group of an oligonucleotide primer; we term this methodology surface initiated enzymatic polymerization (SIEP) of DNA. We found that long (>1 Kb) ssDNA homopolymer can be grown by SIEP, and that the length of the ssDNA product is determined by the monomer to oligonucleotide initiator ratio. We observed efficient initiation (≥50%) and narrow polydispersity of the extended product when fluorescently labeled nucleotides are incorporated. TdT's ability to incorporate fluorescent dNTPs into a ssDNA chain was characterized by examining the effect of the molar ratios of fluorescent dNTP to natural dNTP on the degree of fluorophore incorporation and the length of the polymerized DNA strand. These experiments allowed us to optimize the polymerization conditions to incorporate up to ~50 fluorescent Cy3-labeled dNTPs per kilobase into a ssDNA chain. With the goal of using TdT as an on-chip labeling method, we also quantified TdT mediated signal amplification on the surface by immobilizing ssDNA oligonucleotide initiators on a glass surface followed by SIEP of DNA. The incorporation of multiple fluorophores into the extended DNA chain by SIEP translated to a ~45 fold signal amplification compared to the incorporation of a single fluorophore. SIEP was then employed to detect hybridization of DNA, by the posthybridization, on-chip polymerization of fluorescently labeled ssDNA that was grown from the 3'-OH of target strands that hybridized to DNA probes that were printed on a surface. A dose-response curve for detection of DNA hybridization by SIEP was generated, with a ~1 pM limit of detection and a linear dynamic range of 2 logs.

Interpretation of time-of-flight secondary ion mass spectroscopy (TOF-SIMS) images from rough samples such as particles, fibers, or biological specimens can be problematic because the images are influenced not only by the sample chemistry but also by topographical features. In this article we have investigated the influence of spherical and cylindrical features on total ion yields, relative ion yields, and feature shape. TOF-SIMS images of Pluronic coated fibers and polystyrene spheres were collected using both triple focusing time and reflectron geometry instruments and a 25keV Ga+ primary ion source. The fibers and spheres were analyzed on both conducting and insulating substrates to assess the importance of field effects. Trends in the images have been explored using principal components analysis and Poisson and multinomial mixture models. The T2 test was employed to assess the statistical significance of results. The results identify three important topographic effects. The size and shape of features can be distorted as a result of the incidence angle of the primary ion beam. Additionally, both the absolute and relative intensities of ion peaks vary as a result of topography. In regions where the primary ion beam impacted the sample at a glancing angle, the relative intensity of molecular fragments characteristic of the Pluronic surfactant was up to three times higher than in regions where the beam impacted the sample at a normal angle. Comparison of results from conducting and insulating samples suggests that changes in the relative ion yields resulted primarily from differences in the incidence angle of the primary ion beam while changes in the total ion yield are influenced by both the incidence angle and distortion of the electric field by the particle. This study documents that topographic features can influence not only the absolute intensity of ion peaks but can also alter peak ratios in a statistically significant manner. In this light, a greater degree of caution is recommended when interpreting TOF-SIMS images from topographically complex samples.

The development of truly scalable, multiplexed optical microarrays requires a detection platform capable of simultaneous detection of multiple independent signals in real-time. We present a technique we term dual order snapshot spectroscopic imaging (DOSSI) and demonstrate that it can be effectively used to collect spectrally-resolved images of a full field of view of sparsely located spots in real-time. Resonant peaks of plasmonic gold nanoparticles were tracked as a function of their surrounding refractive index. Measurement uncertainty analysis indicated that the spectral resolution of DOSSI in the described configuration is approximately 0.95 nm. Further, real-time measurements by DOSSI allowed discrimination between optically identical nanoparticles that were functionalized with two homologous small molecule ligands that bound to the same protein, albeit with different affinity, based purely on their different molecular interaction kinetics– a feat not possible with slower raster-type hyperspectral imaging systems, or other darkfield optical detection systems that solely rely on end-point measurements. Kinetic measurements of plasmon bands by DOSSI can be performed with a relatively simple optical system, thereby opening up the possibility of developing low-cost detectors for arrayed plasmonic diagnostics.