Highly efficient GPCR immobilization with enhanced fouling resistance, salt tolerance, and chromatographic performance.

The feasibility of immobilized protein-based biodetection relies critically on the activity of the immobilized proteins as well as the biocompatibility of the protein surface. Although many protein immobilization strategies have been developed with satisfied detection readout signals. Non-specific interactions caused by the protein-coating surface are still of great concern since they often interfere with or affect the reliability of detection. Herein, we developed a highly efficient G protein-coupled receptor (GPCR) immobilization method by the combination of polyethylene glycol (PEG) with a self-labeling enzyme-catalyzed reaction. The immobilization relies on the covalent interaction between the fusion tag of a target GPCR (kinase domain of epidermal growth factor receptor, EGFR) and its covalent inhibitor ibrutinib, which is modified on PEGylated silica gels. Two types of GPCRs, N-methyl-D-aspartate 2 A receptor (NMDAR2A) and endothelin A receptor (ETAR), were used as examples to realize protein immobilization. The GPCR modified gels and the affinity columns packed with them have been extensively characterized, in terms of non-specific adsorptions, retention factor (k'), half peak width (W1/2), tailing factor (Tf), theoretical plates (N), and association and dissociation constants of the ligands with the receptors. The immobilized GPCRs with reduced non-specific interactions and enhanced fouling resistance, salt tolerance, and chromatographic performance were clearly observed. We believe it is the first work to introduce PEGylation in GPCR immobilization and provide comprehensive proof-of-concept studies to illustrate the improved antifouling property, salt tolerance, and chromatographic performance. This method could be generally applicable in other immobilized protein-based technology for reliable biodetection.

Conformational Changes in Surface-Immobilized Proteins Measured Using Combined Atomic Force and Fluorescence Microscopy.

Biological organisms rely on proteins to perform the majority of their functions. Most protein functions are based on their physical motions (conformational changes), which can be described as transitions between different conformational states in a multidimensional free-energy landscape. A comprehensive understanding of this free-energy landscape is therefore of paramount importance for understanding the biological functions of proteins. Protein dynamics includes both equilibrium and nonequilibrium motions, which typically exhibit a wide range of characteristic length and time scales. The relative probabilities of various conformational states in the energy landscape, the energy barriers between them, their dependence on external parameters such as force and temperature, and their connection to the protein function remain largely unknown for most proteins. In this paper, we present a multimolecule approach in which the proteins are immobilized at well-defined locations on Au substrates using an atomic force microscope (AFM)-based patterning method called nanografting. This method enables precise control over the protein location and orientation on the substrate, as well as the creation of biologically active protein ensembles that self-assemble into well-defined nanoscale regions (protein patches) on the gold substrate. We performed AFM-force compression and fluorescence experiments on these protein patches and measured the fundamental dynamical parameters such as protein stiffness, elastic modulus, and transition energies between distinct conformational states. Our results provide new insights into the processes that govern protein dynamics and its connection to protein function.

Rapid, Direct, Noninvasive Method to Determine the Amount of Immobilized Protein.

Protein immobilization is of utmost importance in many areas, where various proteins are used for selective detection of target compounds. Despite the importance given to determine the amount of immobilized protein, there is no simple method that allows direct, noninvasive detection. In this work, a method based on pH transition, occurring during change of solution ionic strength, was developed. The method utilized the ionic character of the immobilized protein while implementing biologically compatible buffers. Five different proteins, namely, glucose oxidase, horseradish peroxidase, bovine serum albumin, lysozyme, and protein A, were immobilized in different amounts on a porous polymeric matrix, and their pH transition was measured using lactate buffer of various concentrations and pH values. A linear correlation was found between the amount of immobilized protein and the amplitude of the pH transition, allowing the detection down to 2 nmol of immobilized protein. By changing the buffer concentration and pH, the sensitivity of the method could be tailored. Criteria based on the symmetry of the pH transition peak have been developed to determine if a particular measurement is within a linear range. In addition, a mathematical model was developed enabling prediction of pH transition profiles based solely on the protein amino acid sequence, the buffer pKa value(s), and the amount of immobilized protein.Hence, it can be used to design pH transition method experiments to achieve the required sensitivity for a target sample. Since the proposed method is noninvasive, it can be routinely applied during optimization of the immobilization protocol, for quality control, and also as an in-process monitoring tool.

Controlled Temporal Release of Serum Albumin Immobilized on Gold Nanoparticles.

Proteins adsorbed to gold nanoparticles (AuNPs) form bioconjugates and are critical to many emerging technologies for drug delivery, diagnostics, therapies, and other biomedical applications. A thorough understanding of the interaction between the immobilized protein and AuNP is essential for the bioconjugate to perform as designed. Here, we explore a correlation between the number of solvent-accessible thiol groups on a protein and the protein desorption rate from the AuNP surface in the presence of a competing protein. The chemical modification of human serum albumin (HSA) was carried out to install additional free thiols using Traut's reagent and create a library of HSA analogues by tailoring the molar excess of the Traut's reagent. We pre-adsorbed HSA variants onto the AuNP surface, and the resulting bioconjugates were then exposed to IgG antibody, and protein exchange was monitored as a function of time. We found that the rate of HSA displacement from the AuNP correlated with the experimentally measured number of accessible free thiol groups. Additionally, bioconjugates were synthesized using thiolated analogues of bovine serum albumin (BSA) and suspended in serum as a model for a complex sample matrix. Similarly, desorption rates with serum proteins were modulated with solvent-accessible thiols on the immobilized protein. These results further highlight the key role of Au-S bonds in the formation of protein-AuNP conjugates and provide a pathway to systematically control the number of free thiols on a protein, enabling the controlled release of protein from the surface of AuNP.

Effect of microchannel geometry and linker molecules on surface immobilization efficiency of proteins in microfluidic devices.

One of the key metrics in the design of biosensors is the presence of an effective capture layer. Surface-immobilized proteins (especially as a part of antibody-antigen combinations) are the most commonly used capture ligands in biosensors. The surface coverage of these proteins in flow-based biosensors are affected by both the linker chemistry used to attach them as well as the microchannel geometry. We used streptavidin as a model protein to compare glutaraldehyde, EDC-NHS, sulfo-SMCC and sulfo-NHS-biotin as linkers inside straight, serpentine and square-wave microchannel geometries. We found that straight microchannels achieve the highest degree of protein immobilization compared to serpentine and square-wave microchannels, irrespective of the linker chemistry used. We also showed that for a given microchannel geometry, sulfo-NHS-biotin leads to the highest immobilization of streptavidin among all the linkers.

Strategy based on multiplexed brush architectures for regulating the spatiotemporal immobilization of biomolecules.

Functional surfaces that enable both spatial and temporal control of biomolecules immobilization have attracted enormous attention for various fields including smart biointerface materials, high-throughput bioarrays, and fundamental research in the biosciences. Here, a flexible and promising method was presented for regulating the spatiotemporal arrangement of multiple biomolecules by constructing the topographically and chemically diverse polymer brushes patterned surfaces. A series of polymer brushes patterned surfaces, including antifouling brushes patterned surface, epoxy-presenting brushes patterned surface without and with antifouling background layer, were fabricated to control the spatial distribution of protein and cell adhesion through specific and nonspecific means. The fluorescence measurements demonstrated the effectiveness of spatially regulating the density of surface-immobilized protein through controlling the areal thickness of the poly (glycidyl methacrylate) (PGMA) brush patterns, leading to various complex patterns featuring well-defined biomolecule concentration gradients. Furthermore, a multiplexed surface bearing epoxy groups and azido groups with various areal densities was fabricated for regulating the spatiotemporal arrangement of different proteins, enabling binary biomolecules patterns with higher degrees of functionality and complexity. The presented strategy for the spatiotemporal control of biomolecules immobilization would boost the development of dynamic and multifunctional biosystems.

Controlled co-immobilisation of proteins via 4'-phosphopantetheine-mediated site-selective covalent linkage.

In Escherichia coli, acyl carrier protein (ACP) is posttranslationally converted into its active holo-ACP form via covalent linkage of 4'-phosphopantetheine (4'-PP) to residue serine-36. We found that the long flexible 4'-PP arm could react chemoselectively with the iodoacetyl group introduced on solid supports with high efficiency under mild conditions. Based on this finding, we developed site-selective immobilisation of proteins via the active holo-ACP fusion tag, independently of the physicochemical properties of the protein of interest. Furthermore, the molecular ratios of co-immobilised proteins can be manipulated because the tethering process is predominantly directed by the molar concentrations of diverse holo-ACP fusions during co-immobilisation. Conveniently tuning the molecular ratios of co-immobilised proteins allows their cooperation, leading to a highly productive multi-protein co-immobilisation system. Kinetic studies of enzymes demonstrated that α-amylase (Amy) and methyl parathion hydrolase (MPH) immobilised via active tag holo-ACP had higher catalytic efficiency (kcat/Km) in comparison with their corresponding counterparts immobilised via the sulfhydryl groups (-SH) of these proteins. The immobilised holo-ACP-Amy also presented higher thermostability compared with free Amy. The enhanced α-amylase thermostability upon immobilisation via holo-ACP renders it more suitable for industrial application.

Structurally Altered, Not Wild-Type, Pentameric C-Reactive Protein Inhibits Formation of Amyloid-ß Fibrils.

The structure of wild-type pentameric C-reactive protein (CRP) is stabilized by two calcium ions that are required for the binding of CRP to its ligand phosphocholine. CRP in its structurally altered pentameric conformations also binds to proteins that are denatured and aggregated by immobilization on microtiter plates; however, the identity of the ligand on immobilized proteins remains unknown. We tested the hypotheses that immobilization of proteins generated an amyloid-like structure and that amyloid-like structure was the ligand for structurally altered pentameric CRP. We found that the Abs to amyloid-ß peptide 1-42 (Aß) reacted with immobilized proteins, indicating that some immobilized proteins express an Aß epitope. Accordingly, four different CRP mutants capable of binding to immobilized proteins were constructed, and their binding to fluid-phase Aß was determined. All CRP mutants bound to fluid-phase Aß, suggesting that Aß is a ligand for structurally altered pentameric CRP. In addition, the interaction between CRP mutants and Aß prevented the formation of Aß fibrils. The growth of Aß fibrils was also halted when CRP mutants were added to growing fibrils. Biochemical analyses of CRP mutants revealed altered topology of the Ca2+-binding site, suggesting a role of this region of CRP in binding to Aß. Combined with previous reports that structurally altered pentameric CRP is generated in vivo, we conclude that CRP is a dual pattern recognition molecule and an antiamyloidogenic protein. These findings have implications for Alzheimer's and other neurodegenerative diseases caused by amyloidosis and for the diseases caused by the deposition of otherwise fluid-phase proteins.

Immunospecific analysis of in vitro and ex vivo surface-immobilized protein complex.

Biomaterials used for blood contacting devices are inherently thrombogenic. Antithrombotic agents can be used as surface modifiers on biomaterials to reduce thrombus formation on the surface and to maintain device efficacy. For quality control and to assess the effectiveness of immobilization strategies, it is necessary to quantify the surface-immobilized antithrombotic agent directly. There are limited methods that allow direct quantification on device surfaces such as catheters. In this study, an enzyme immunoassay (EIA) has been developed to measure the density of a synthetic antithrombin-heparin (ATH) covalent complex immobilized on a catheter surface. The distribution of the immobilized ATH was further characterized by an immunohistochemical assay. This analyte-specific EIA is relatively simple and has high throughput, thus providing a tool for quantitative analysis of biomaterial surface modifications. These methods may be further modified to evaluate plasma proteins adsorbed and immobilized on various biomaterial surfaces of complex shapes, with a range of bioactive functionalities, as well as to assess conformational changes of proteins using specific antibodies.

Single-molecule analysis of specificity and multivalency in binding of short linear substrate motifs to the APC/C.

Robust regulatory signals in the cell often depend on interactions between short linear motifs (SLiMs) and globular proteins. Many of these interactions are poorly characterized because the binding proteins cannot be produced in the amounts needed for traditional methods. To address this problem, we developed a single-molecule off-rate (SMOR) assay based on microscopy of fluorescent ligand binding to immobilized protein partners. We used it to characterize substrate binding to the Anaphase-Promoting Complex/Cyclosome (APC/C), a ubiquitin ligase that triggers chromosome segregation. We find that SLiMs in APC/C substrates (the D box and KEN box) display distinct affinities and specificities for the substrate-binding subunits of the APC/C, and we show that multiple SLiMs in a substrate generate a high-affinity multivalent interaction. The remarkably adaptable substrate-binding mechanisms of the APC/C have the potential to govern the order of substrate destruction in mitosis.

Wnt Inhibitory Factor 1 Binds to and Inhibits the Activity of Sonic Hedgehog.

The hedgehog (Hh) and Wnt pathways, crucial for the embryonic development and stem cell proliferation of Metazoa, have long been known to have similarities that argue for their common evolutionary origin. A surprising additional similarity of the two pathways came with the discovery that WIF1 proteins are involved in the regulation of both the Wnt and Hh pathways. Originally, WIF1 (Wnt Inhibitory Factor 1) was identified as a Wnt antagonist of vertebrates, but subsequent studies have shown that in Drosophila, the WIF1 ortholog serves primarily to control the distribution of Hh. In the present, work we have characterized the interaction of the human WIF1 protein with human sonic hedgehog (Shh) using Surface Plasmon Resonance spectroscopy and reporter assays monitoring the signaling activity of human Shh. Our studies have shown that human WIF1 protein binds human Shh with high affinity and inhibits its signaling activity efficiently. Our observation that the human WIF1 protein is a potent antagonist of human Shh suggests that the known tumor suppressor activity of WIF1 may not be ascribed only to its role as a Wnt inhibitor.

A Neoglycoprotein-Immobilized Fluorescent Magnetic Bead Suspension Multiplex Array for Galectin-Binding Studies.

Carbohydrate-protein conjugates have diverse applications. They have been used clinically as vaccines against bacterial infection and have been developed for high-throughput assays to elucidate the ligand specificities of glycan-binding proteins (GBPs) and antibodies. Here, we report an effective process that combines highly efficient chemoenzymatic synthesis of carbohydrates, production of carbohydrate-bovine serum albumin (glycan-BSA) conjugates using a squarate linker, and convenient immobilization of the resulting neoglycoproteins on carboxylate-coated fluorescent magnetic beads for the development of a suspension multiplex array platform. A glycan-BSA-bead array containing BSA and 50 glycan-BSA conjugates with tuned glycan valency was generated. The binding profiles of six plant lectins with binding preference towards Gal and/or GalNAc, as well as human galectin-3 and galectin-8, were readily obtained. Our results provide useful information to understand the multivalent glycan-binding properties of human galectins. The neoglycoprotein-immobilized fluorescent magnetic bead suspension multiplex array is a robust and flexible platform for rapid analysis of glycan and GBP interactions and will find broad applications.

Single-molecule conformational dynamics of a transcription factor reveals a continuum of binding modes controlling association and dissociation.

Binding and unbinding of transcription factors to DNA are kinetically controlled to regulate the transcriptional outcome. Control of the release of the transcription factor NF-κB from DNA is achieved through accelerated dissociation by the inhibitor protein IκBα. Using single-molecule FRET, we observed a continuum of conformations of NF-κB in free and DNA-bound states interconverting on the subseconds to minutes timescale, comparable to in vivo binding on the seconds timescale, suggesting that structural dynamics directly control binding kinetics. Much of the DNA-bound NF-κB is partially bound, allowing IκBα invasion to facilitate DNA dissociation. IκBα induces a locked conformation where the DNA-binding domains of NF-κB are too far apart to bind DNA, whereas a loss-of-function IκBα mutant retains the NF-κB conformational ensemble. Overall, our results suggest a novel mechanism with a continuum of binding modes for controlling association and dissociation of transcription factors.

Magnetic Nanoplatforms for Covalent Protein Immobilization Based on Spy Chemistry.

Immobilization of proteins on magnetic nanoparticles (MNPs) is an effective approach to improve protein stability and facilitate separation of immobilized proteins for repeated use. Herein, we exploited the efficient SpyTag-SpyCatcher chemistry for conjugation of functional proteins onto MNPs and established a robust magnetic-responsive nanoparticle platform for protein immobilization. To maximize the loading capacity and achieve outstanding water dispersity, the SpyTag peptide was incorporated into the surface-charged polymers of MNPs, which provided abundant active sites for Spy chemistry while maintaining excellent colloidal stability in buffer solution. Conjugation between enhanced green fluorescence protein (EGFP)-SpyCatcher-fused proteins and SpyTag-functionalized MNPs was efficient at ambient conditions without adding enzymes or chemical cross-linkers. Benefiting from the excellent water dispersity and interface compatibility, the surface Spy reaction has fast kinetics, which is comparable to that of the solution Spy reaction. No activity loss was observed on EGFP after conjugation due to the site-selective nature of Spy chemistry. The immobilization process of EGFP on MNPs was highly specific and robust, which was not affected by the presence of other proteins and detergents, such as bovine serum albumin and Tween 20. The MNP platform was demonstrated to be protective to the conjugated EGFP and significantly improved the shelf life of immobilized proteins. In addition, experiments confirmed the retained magnetophoresis of the MNP after protein loading, demonstrating fast MNP recovery under an external magnetic field. This MNP is expected to provide a versatile and modular platform to achieve effective and specific immobilization of other functional proteins, enabling easy reuse and storage.

Atomically Resolved Characterization of Optically Driven Ligand Reconfiguration on Nanoparticle Catalyst Surfaces.

Dynamic ligand layers on nanoparticle surfaces could prove to be critically important to enhance the functionality of individual materials. Such capabilities could complement the properties of the inorganic component to provide multifunctionality or the ability to be remotely actuated. Peptide-based ligands have demonstrated the ability to be remotely responsive to structural changes when adsorbed to nanoparticle surfaces via incorporation of photoswitches into their molecular structure. In this contribution, direct spectroscopic evidence of the remote actuation of a photoswitchable peptide adsorbed onto Au nanoparticles is demonstrated using X-ray absorption fine structure spectroscopic methods. From this analysis, Au-X (X = C or N) coordination numbers confirm the changes before and after photoswitching in the surface ligand conformation, which was correlated directly to variations in the catalytic application of the materials for nitrophenol reduction processes. In addition, the catalytic application of the materials was demonstrated to be significantly sensitive to the structure of the nitrophenol substrate used in the reaction, suggesting that changes in the reactivity are likely based upon the peptide conformation and substrate structure. Such results confirm that surface ligands can be remotely reconfigured on nanoparticle surfaces, providing pathways to apply such capabilities to a variety of applications beyond catalysis ranging from drug delivery to sensing.

A Simplistic Single-Step Method for Preparing Biomimetic Nanoparticles from Endogenous Biomaterials.

Many works utilize products isolated from nature as capping agents to functionalize gold nanoparticles for targeting and therapeutic applications. Some of the most advanced of these strategies utilize complex multicomponent biomaterials, such as whole cell-membranes, for nanoparticle functionalization strategies for evading or initializing immune response as well as for targeting. Strategies like these, wherein whole cell membrane is utilized for functionalization, take advantage of the complexity of the protein-lipid content and organization, which cells normally use for communication and interaction (instilling these capacities to nanoparticle vectors). Many approaches for achieving this in functionalizing the surface of nanoparticles rely on multistep processes, which necessitate the addition and then removal of synthetic molecules, heating, or pH modifications. These processes can have deleterious modifying effects on the functionalizing biomolecules, resulting in loss of product and time during each purification step, as well as potentially changing the biomolecule functionality toward a nondesirable effect. Here, we describe methods for forming gold nanoparticles at room temperature in a single step, functionalized with proteins, using nicotinamide adenine dinucleotide (NADH). This process enables formation of nanoparticles that can be functionalized by individual proteins (demonstrated with FBS) or whole cells membrane (extracted from B16F10 cells). This work is derivative from observations found in the literature by us and others, that mammalian cells are capable of producing gold nanoparticles from ionic gold without the supplementation of chemical species. The products of this single-step synthesis described herein have been optimized to maintain biomolecule integrity and so that there are no further purification steps required. To characterize the nanoparticles in terms of their shape, size, surface functionality, and biomolecule integrity throughout development, we employed light-based spectroscopy techniques, molecular modeling, electron microscopy, light scattering, and gel electrophoresis techniques. In order to compare the optimized biomolecule-functionalized nanoparticles against current standards (which require synthetic linkers, heating, or pH manipulation), we employed metabolic and live/dead assays as well as light-based microscopy/spectroscopy in vitro. In comparing our synthetic process against others for forming gold nanoparticles functionalized with complex biomolecule components (whole-cell membrane), we found that this process had superior particle internalization. Our strategy has similar outlets for application to these other works, however, because this process is entirely reliant on endogenous biomaterials and has additional potential.

A Synergistic New Approach Toward Enhanced Antibacterial Efficacy via Antimicrobial Peptide Immobilization on a Nitric Oxide-Releasing Surface.

Despite technological advancement, nosocomial infections are prevalent due to the rise of antibiotic resistance. A combinatorial approach with multimechanistic antibacterial activity is desired for an effective antibacterial medical device surface strategy. In this study, an antimicrobial peptide, nisin, is immobilized onto biomimetic nitric oxide (NO)-releasing medical-grade silicone rubber (SR) via mussel-inspired polydopamine (PDA) as a bonding agent to reduce the risk of infection. Immobilization of nisin on NO-releasing SR (SR-SNAP-Nisin) and the surface characteristics were characterized by Fourier transform infrared spectroscopy and scanning electron microscopy with energy-dispersive X-ray spectroscopy and contact angle measurements. The NO release profile (7 days) and diffusion of SNAP from SR-SNAP-Nisin were quantified using chemiluminescence-based nitric oxide analyzers and UV-vis spectroscopy, respectively. Nisin quantification showed a greater affinity of nisin immobilization toward SNAP-doped SR. Matrix-assisted laser desorption/ionization mass spectrometry analysis on surface nisin leaching for 120 h under physiological conditions demonstrated the stability of nisin immobilization on PDA coatings. SR-SNAP-Nisin shows versatile in vitro anti-infection efficacy against Gram-negative Escherichia coli and Gram-positive Staphylococcus aureus in the planktonic and adhered states. Furthermore, the combination of NO and nisin has a superior ability to impair biofilm formation on polymer surfaces. SR-SNAP-Nisin leachates did not elicit cytotoxicity toward mouse fibroblast cells and human umbilical vein endothelial cells, indicating the biocompatibility of the material in vitro. The preventative and therapeutic potential of SR-SNAP-Nisin dictated by two bioactive agents may offer a promising antibacterial surface strategy.

Glycan Array Evaluation of Synthetic Epitopes between the Capsular Polysaccharides from Streptococcus pneumoniae 19F and 19A.

Vaccination represents the most effective way to prevent invasive pneumococcal diseases. The glycoconjugate vaccines licensed so far are obtained from capsular polysaccharides (CPSs) of the most virulent serotypes. Protection is largely limited to the specific vaccine serotypes, and the continuous need for broader coverage to control the outbreak of emerging serotypes is pushing the development of new vaccine candidates. Indeed, the development of efficacious vaccine formulation is complicated by the high number of bacterial serotypes with different CPSs. In this context, to simplify vaccine composition, we propose the design of new saccharide fragments containing chemical structures shared by different serotypes as cross-reactive and potentially cross-protective common antigens. In particular, we focused on Streptococcus pneumoniae (Sp) 19A and 19F. The CPS repeating units of Sp 19F and 19A are very similar and share a common structure, the disaccharide ManNAc-ß-(1→4)-Glc (A-B). Herein, we describe the synthesis of a small library of compounds containing different combinations of the common 19F/19A disaccharide. The six new compounds were tested with a glycan array to evaluate their recognition by antibodies in reference group 19 antisera and factor reference antisera (reacting against 19F or 19A). The disaccharide A-B, phosphorylated at the upstream end, emerged as a hit from the glycan array screening because it is strongly recognized by the group 19 antisera and by the 19F and 19A factor antisera, with similar intensity compared with the CPSs used as controls. Our data give a strong indication that the phosphorylated disaccharide A-B can be considered a common epitope among different Sp 19 serotypes.

Recent advances in nanomaterials-based electrochemical immunosensors and aptasensors for HER2 assessment in breast cancer.

Human epidermal growth factor receptor 2 (HER2) is one of the key molecular targets in breast cancer pathogenesis. Overexpression and/or amplification of HER2 in approximately 15-20% of breast cancer patients is associated with high mortality and poor prognosis. Accumulating evidence shows that accurate and sensitive detection of HER2 improves the survival outcomes for HER2-positive breast cancer patients from targeted therapies. The current methods of clinical determination of HER2 expression levels are based on slide-based assays that rely on invasively collected primary tumours. Alternatively, ELISA-based detection of the shredded HER2 extracellular domain (HER2-ECD) of has been suggested as a surrogate method for monitoring disease progress and treatment response in breast cancer patients. In the past decade, biosensors have emerged as an alternative modality for the detection of circulating HER2-ECD in human serum samples. In particular, electrochemical biosensors based on nanomaterials and antibodies and aptamers have been increasingly developed as promising tools for rapid, sensitive, and cost-effective detection of HER2-ECD. These biosensors harness the high affinity and specificity of antibodies and aptamers, and unique conductive properties, biocompatibility, large surface area, and chemical stability of nanomaterials for selective and sensitive assessment of the HER2. This review provides an overview of the recent advances in the application of nanomaterials-based immunosensors and aptasensors for detection of circulating HER2-ECD. In particular, various electrochemical techniques, detection approaches, and nanomaterials are discussed. Further, analytical figures of merit of various HER2 immunosensors and aptasensors are compared. Finally, possible challenges and potential opportunities for biosensor-based detection of HER2-ECD are discussed.

Photolithographic Patterning of FluorAcryl for Biphilic Microwell-Based Digital Bioassays and Selection of Bacteria.

FluorAcryl 3298 (FA) is a UV-curable fluoroacrylate polymer commonly employed as a chemically resistant, hydrophobic, and oleophobic coating. Here, FA was used in a cleanroom-based microstructuring process to fabricate hydrophilic-in-hydrophobic (HiH) micropatterned surfaces containing femtoliter-sized well arrays. A short protocol involving direct UV photopatterning, an etching step, and final recovery of the hydrophobic properties of the polymer produced patterned substrates with micrometer resolution. Specifically, HiH microwell arrays were obtained with a well diameter of 10 µm and various well depths ranging from 300 nm to 1 µm with high reproducibility. The 300 nm deep microdroplet array (MDA) substrates were used for digital immunoassays, which presented a limit of detection in the attomolar range. This demonstrated the chemical functionality of the hydrophilic and hydrophobic surfaces. Furthermore, the 1 µm deep wells could efficiently capture particles such as bacteria, whereas the 300 nm deep substrates or other types of flat HiH molecular monolayers could not. Capturing a mixture of bacteria expressing red- and green-fluorescent proteins, respectively, served as a model for screening and selection of specific phenotypes using FA-MDAs. Here, green-fluorescent bacteria were specifically selected by overlaying a solution of gelatin methacryloyl (GelMA) mixed with a photoinitiator and using a high-magnification objective, together with custom pinholes, in a common fluorescence microscope to cross-link the hydrogel around the bacteria of interest. In conclusion, due to the straightforward processing, versatility, and low-price, FA is an advantageous alternative to more commonly used fluorinated materials, such as CYTOP or Teflon-AF, for the fabrication of HiH microwell arrays and other biphilic microstructures.