Norman D. Brault

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(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.

Controlled Hierarchical Architecture in Surface-initiated Zwitterionic Polymer Brushes with Structurally Regulated Functionalities

Hierarchical polymer films with structurally regulated functionalities are achieved by integrating 2D and 3D structures to enable ultralow nonspecific protein binding and high loading of molecular recognition elements, such as antibodies.

Reversibly switchable polymer with cationic/zwitterionic/anionic behavior through synergistic protonation and deprotonation

A polymer capable of fully and reversibly switching throughout the entire charge regime is highly desirable for many applications such as drug and gene delivery, controlled ion and molecular transport and tunable filtration membranes. It is essential that for biologically relevant applications the polymer needs to be nonfouling. However, conventional nonfouling zwitterionic polymers have a permanently positive quaternary nitrogen center, making it impossible to switch between charges. Here, we present a rationally designed polymer with a tertiary amine and a carboxylic acid, which is capable of reversibly switching among three distinct charged states, viz., cationic, zwitterionic and anionic, and importantly maintaining the zwitterionic state under physiological pH conditions. Oppositely charged proteins adsorbed on a charged surface selectively can be completely removed by switching the surface to the zwitterionic state. We have also found that these two moieties (i.e., a tertiary amine and a carboxylate moiety) stimulate each other synergistically to achieve a strongly zwitterionic state under physiological conditions and to resist non-specific protein adsorption from undiluted blood plasma and serum when they are close to each other.

Cellulose Paper Sensors Modified with Zwitterionic Poly(carboxybetaine) for Sensing and Detection in Complex Media

Poly(carboxybetaine) (PCB) functionalized cellulose paper was used as a paper-based microfluidic device. The results showed that the PCB modified paper sensor was able to achieve (a) more rapid and sensitive glucose detection from undiluted human serum compared to bare cellulose and (b) specific antigen detection via covalently immobilized antibodies.

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/cm(2) 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.

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

It is highly desirable to develop a universal nonfouling coating via a simple one-step dip-coating method. Developing such a universal coating method for a hydrophilic polymer onto a variety of surfaces with hydrophobic and hydrophilic properties is very challenging. This work demonstrates a versatile and simple method to attach zwitterionic poly(carboxybetaine methacrylate) (PCB), one of the most hydrophilic polymers, onto both hydrophobic and hydrophilic surfaces to render them nonfouling. This is achieved by the coating of a catechol chain end carboxybetaine methacrylate polymer (DOPA-PCB) assisted by dopamine. The coating process was carried out in water. Water miscible solvents such as methanol and tetrahydrofuran (THF) are added to the coatings if surface wettability is an issue, as for certain hydrophobic surfaces. This versatile coating method was applied to several types of surfaces such as polypropylene (PP), polydimethyl siloxane (PDMS), Teflon, polystyrene (PS), polymethylmethacrylate (PMMA), polyvinyl chloride (PVC) and also on metal oxides such as silicon dioxide.

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/cm(2), 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.

Internal Architecture of Zwitterionic Polymer Brushes Regulates Nonfouling Properties

In this work, we study how film thickness and chain packing density affect the protein-resistant properties of polymer brushes in complex media. Polymer brushes based on dual-functional poly(carboxybetaine acrylamide) (pCB) were prepared via surface-initiated photoiniferter-mediated polymerization. By adjusting UV radiation time and solvent polarity, pCB films with different thicknesses can be achieved and characterized using an ellipsometer. The packing density of pCB polymer chains is directly related to the swelling ratio of swollen to collapsed film thicknesses. Results showed that the dry film thickness alone, used often in the literature, is not sufficient to correlate with nonfouling properties and the chain packing density must be considered for the design of nonfouling surface coatings.

Zwitterionic polymer-modified silicon microring resonators for label-free biosensing in undiluted human plasma

A widely acknowledged goal in personalized medicine is to radically reduce the costs of highly parallelized, small fluid volume, point-of-care and home-based diagnostics. Recently, there has been a surge of interest in using complementary metal-oxide-semiconductor (CMOS)-compatible silicon photonic circuits for biosensing, with the promise of producing chip-scale integrated devices containing thousands of orthogonal sensors, at minimal cost on a per-chip basis. A central challenge in biosensor translation is to engineer devices that are both sensitive and specific to a target analyte within unprocessed biological fluids. Despite advances in the sensitivity of silicon photonic biosensors, poor biological specificity at the sensor surface remains a significant factor limiting assay performance in complex media (i.e. whole blood, plasma, serum) due to the non-specific adsorption of proteins and other biomolecules. Here, we chemically modify the surface of silicon microring resonator biosensors for the label-free detection of an analyte in undiluted human plasma. This work highlights the first application of a non-fouling zwitterionic surface coating to enable silicon photonic-based label-free detection of a protein analyte at clinically relevant sensitivities in undiluted human plasma.

Simple and robust approach for passivating and functionalizing surfaces for use in complex media.

Pluronic is a popular triblock copolymer used as a surfactant to introduce hydrophilic coatings onto many different types of material surfaces, from engineering to biomedical applications. Unfortunately, this is limited in its ability to resist fouling from complex media (i.e., blood) and leaves the surface hard for further modification. Herein, we report a simple, yet robust approach for passivating and functionalizing surfaces based on zwitterionic poly(carboxybetaine) (PCB) based triblock copolymer, which can be directly applied to surfaces to prevent nonspecific protein adsorption from undiluted blood plasma, and to provide additional functionalities needed for the attachment of biomolecules. Several hydrophobic surfaces including polydimethylsiloxane, silanized silica, and self-assembled monolayers are tested to demonstrate its applicability to a wide range of systems. This approach provides a robust, convenient, and effective surface modification method for real-world applications from simple surface passivation to specific targeting in complex media.