Label-free biomarker sensing in undiluted serum with suspended microchannel resonators

Improved methods are needed for routine, inexpensive monitoring of biomarkers that could facilitate earlier detection and characterization of cancer. Suspended microchannel resonators (SMRs) are highly sensitive, batch-fabricated microcantilevers with embedded microchannels that can directly quantify adsorbed mass via changes in resonant frequency. As in other label-free detection methods, biomolecular measurements in complex media such as serum are challenging due to high background signals from nonspecific binding. In this report, we demonstrate that carboxybetaine-derived polymers developed to adsorb directly onto SMR SiO[subscript 2] surfaces act as ultralow fouling and functionalizable surface coatings. Coupled with a reference microcantilever, this approach enables detection of activated leukocyte cell adhesion molecule (ALCAM), a model cancer biomarker, in undiluted serum with a limit of detection of 10 ng/mL.National Cancer Institute (U.S.) (contract R01CA119402)SAIC-Frederick (contract 28XS119)National Institutes of Health (U.S.). Biotechnology Training Fellowshi

Achieving One‐Step Surface Coating of Highly Hydrophilic Poly(Carboxybetaine Methacrylate) Polymers on Hydrophobic and Hydrophilic Surfaces

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/108623/1/admi201400071.pd

Velocity-space sensitivity of the time-of-flight neutron spectrometer at JET

The velocity-space sensitivities of fast-ion diagnostics are often described by so-called weight functions. Recently, we formulated weight functions showing the velocity-space sensitivity of the often dominant beam-target part of neutron energy spectra. These weight functions for neutron emission spectrometry (NES) are independent of the particular NES diagnostic. Here we apply these NES weight functions to the time-of-flight spectrometer TOFOR at JET. By taking the instrumental response function of TOFOR into account, we calculate time-of-flight NES weight functions that enable us to directly determine the velocity-space sensitivity of a given part of a measured time-of-flight spectrum from TOFOR

Relationship of edge localized mode burst times with divertor flux loop signal phase in JET

A phase relationship is identified between sequential edge localized modes (ELMs) occurrence times in a set of H-mode tokamak plasmas to the voltage measured in full flux azimuthal loops in the divertor region. We focus on plasmas in the Joint European Torus where a steady H-mode is sustained over several seconds, during which ELMs are observed in the Be II emission at the divertor. The ELMs analysed arise from intrinsic ELMing, in that there is no deliberate intent to control the ELMing process by external means. We use ELM timings derived from the Be II signal to perform direct time domain analysis of the full flux loop VLD2 and VLD3 signals, which provide a high cadence global measurement proportional to the voltage induced by changes in poloidal magnetic flux. Specifically, we examine how the time interval between pairs of successive ELMs is linked to the time-evolving phase of the full flux loop signals. Each ELM produces a clear early pulse in the full flux loop signals, whose peak time is used to condition our analysis. The arrival time of the following ELM, relative to this pulse, is found to fall into one of two categories: (i) prompt ELMs, which are directly paced by the initial response seen in the flux loop signals; and (ii) all other ELMs, which occur after the initial response of the full flux loop signals has decayed in amplitude. The times at which ELMs in category (ii) occur, relative to the first ELM of the pair, are clustered at times when the instantaneous phase of the full flux loop signal is close to its value at the time of the first ELM

Multifunctional Zwitterionic Surface Chemistry for Applications in Complex Media

Thesis (Ph.D.)--University of Washington, 2014The realization of personalized medicine relies on the discovery of clinically relevant biomarkers as well as on the development of corresponding assays. Due to the complexity of human blood plasma and serum, the most common sources for biomarker analysis, current attempts to integrate existing biosensing assays with analyte detection has resulted in two major shortcomings: high rates of false-positives, from non-specific binding, and a lack of assay sensitivity, due to low ligand loading. Taken together, these two factors indicate that a high signal-to-noise ratio (S/N) is vital for achieving sensitive biomarker-based diagnostics. Furthermore, a single material that can (1) exhibit non-fouling properties from undiluted human blood, (2) present abundant and easily functionalizable chemical groups for ligand attachment, and (3) possesses high immobilization capacities, would offer the most promising approach to achieving this goal. Such idealities were addressed using zwitterionic poly(carboxybetaine) (pCB) surface chemistry. First, an important parameter was realized for identifying surface coatings suitable for real-world applications involving undiluted complex media. It was found that ultra low fouling properties using a thin film is possible if it is densely packed. While such prevention of non-specific adsorption is important, the detection of biomarkers also hinges on the ability to immobilize biologically active ligands all while maintaining the original ultra low fouling background noise of the surface coating. Hence, the dual-functionality of pCB, which provides both protein resistance and ligand functionalization, was then applied to protein arrays. Here, uniform spot morphology as well as excellent non-fouling properties following antibody immobilization was achieved. This enabled improvements in the sensitivity for multiplexed detection of target analytes directly from undiluted human plasma. As even the best non-fouling background combined with the highest affinity ligand would still have a limited S/N ratio due to the 2-dimensional (2-D) structure of polymer films, two efforts to improve the "signal" component were also investigated. The first method led to the development of a hierarchical architecture consisting of a thin and highly dense first layer and a loose but controlled second layer, for low fouling and high ligand loading, respectively. The second approach for improving biomarker assay performance involved taking advantage of new biosensor devices. Such novel sensor designs exhibit decreasing surface dimensions with unique geometries and enhanced theoretical sensitivities. Due to these distinct characteristics, the development of a dual-functional "graft-to" surface coating was necessary. Here, the conjugation of the adhesive molecule DOPA with pCB enabled the successful attachment to a biosensor surface while also demonstrating ultra low fouling and functionalization properties. This "graft-to" technology can be readily extended to other device platforms. Finally, while normal immobilization conditions for pCB allow for the attachment of acidic and neutrally charged ligands, two strategies for expanding the range of ligands, to include basic proteins (i.e. with high isoelectric points), which can be coupled to an ultra low fouling zwitterionic background were also investigated. It was found that the use of reversible citraconic anhydride protection enabled the coupling of the highly basic protein lysozyme to the pCB surface. The second strategy, involving a novel zwitterionic ultra low fouling material, led to an initial characterization which indicated promising results. In summary, this work represents a multifunctional zwitterionic surface chemistry readily suitable for applications in undiluted complex media

Directly Functionalizable Surface Platform for Protein Arrays in Undiluted Human Blood Plasma

Protein arrays are a high-throughput approach for proteomic profiling, vital for achieving a greater understanding of biological systems, in addition to disease diagnostics and monitoring therapeutic treatments. In this work, zwitterionic carboxybetaine polymer (pCB) coated substrates were investigated as an array surface platform to enable convenient amino-coupling chemistry on a single directly functionalizable and unblocked film for the sensitive detection of target analytes from undiluted human blood plasma. Using a surface plasmon resonance (SPR) imaging sensor, the antibody immobilization conditions which provided excellent spot morphology and the largest antigen response were determined. It was found that pCB functionalization and the corresponding antigen detection both increased with pH and antibody concentration. Additionally, immobilization only required an aqueous buffer without the need for additives to improve spot quality. The nonspecific protein adsorption to undiluted human plasma on both the antibody immobilized pCB spots and the background were found to be about 9 and 6 ng/cm², respectively. A subsequent array consisting of three antibodies spotted onto pCB revealed little cross-reactivity for antigens spiked into the undiluted plasma. The low postfunctionalized nonfouling properties combined with antibody amplification showed similar sensitivities achievable with conventional spectroscopic SPR sensors and the same pCB films, but now with high-throughput capabilities. This represents the first demonstration of low fouling properties following antibody functionalization on protein arrays from undiluted human plasma and indicates the great potential of the pCB platform for high-throughput protein analysis

Two-Layer Architecture Using Atom Transfer Radical Polymerization for Enhanced Sensing and Detection in Complex Media

A novel, two-layer hierarchical architecture based on surface-initiated atom transfer radical polymerization was investigated. It combines a thin and highly dense first layer, for nonfouling properties, with a loose second layer for high immobilization levels of active biomolecules. Sodium azide treatment, to reduce the concentration of macroinitiators on the first layer for reinitiation, and by controlling the polydispersity allowed one to achieve three polymer architectures with low, moderate, or high azide substitution. Moderate substitution enabled the highest immobilization levels with a nonfouling background. Integration with dual-functional zwitterionic poly­(carboxybetaine) made this platform suitable for applications in undiluted complex media such as blood. It was demonstrated via a surface plasmon resonance biosensor that antigen accessibility and antibody loading were greatly improved. These results indicate the two-layer strategy as a generic concept suitable for applications from diagnostics to medical coatings in order to maximize and minimize specific and nonspecific responses, respectively

Dry Film Refractive Index as an Important Parameter for Ultra-Low Fouling Surface Coatings

Here we demonstrate that the film refractive index (RI) can be an even more important parameter than film thickness for identifying nonfouling polymer films to undiluted human blood plasma and serum. The film thickness and RI are two parameters obtained from ellipsometry. Previously, film thickness has been correlated to ultra-low fouling properties. Practically, the film RI can be used to characterize polymer density but is often overlooked. By varying the water content in the surface-initiated atom transfer radical polymerization of zwitterionic carboxybetaine, a minimum of ∼1.5 RI units was necessary to achieve <5 ng/cm² of adsorption from undiluted human serum. A model of the film structure versus water content was also developed. These results point to an important parameter and simple approach for identifying surface coatings suitable for real-world applications involving complex media. Therefore, ultra-low fouling using a thin film is possible if it is densely packed