Surface Binding Events Research Articles

(Invited) Single-Walled Carbon Nanotube Functionalization Strategies for Monitoring Enzymatic Activity and Inhibition

Semiconducting single-walled carbon nanotubes (SWCNTs) fluoresce in the near-infrared range, which overlaps with the transparency window of biological samples, and they do not photobleach or blink. Moreover, they benefit from biocompatibility and the large surface area available for functionalization. Using tailored surface functionalization, SWCNTs can be rendered optical nanosensors, such that surface binding events or changes in the local proximity of the nanotubes translate to a modulation of the emitted fluorescence. This approach was successfully used to demonstrate the detection of small molecules, volatiles, bacteria, microRNA, proteins, metals, self-assembly processes, and active processes in vivo. We will discuss different strategies for monitoring enzymatic activity and inhibition using SWCNT-based sensors, including the incorporation of the enzyme’s target bond within a synthetic molecular complex used for suspending the SWCNTs, or by the detection of the product of the enzyme hydrolytic activity. We will also highlight recent applications of these techniques in clinically relevant samples. These findings not only showcase the potential of near-infrared fluorescent SWCNT in tracking enzymatic activity but also underscore their promising role in advancing biomedical research and applications.

Can classical surface plasmon resonance advance via the coupling to other analytical approaches?

For nearly 40 years, surface plasmon resonance (SPR) analysis has been used to better understand the binding interaction strength between surface immobilized bioreceptors and the analytes of interest. The advantage of surface plasmon resonance, over other affinity sensing approaches such as Western blots and ELISAs approaches, resides in its possibility to reveal binding kinetics in a label-free manner. The concept of surface plasmon resonance has in addition been widely employed for the development of biosensors capitalizing on its direct assay format, short response times, simple sample treatments along with multiplexed sensing possibilities. To this must be added the possibility to reach high sensitivity due to the capability of surface plasmon resonance to detect very small changes in refractive index at the sensing interfaces in particular for analytes of larger size such as cells (e.g., bacteria), proteins, peptides and oligonucleotides. Challenges inherent to all affinity approaches call for further research and include non-specific surface binding events, mass transportation restrictions, steric hindrance, and the risk of data misinterpretation in case of lack of selective analyte binding. This opinion article is devoted to outlining the different approaches proposed to address these challenges by e.g., coupling with fluorescence read out, electrochemical sensing, mass spectroscopy analysis and more recently to integrate lateral flow concepts into surface plasmon resonance. Other plasmonic methods such as localized surface plasmon resonance (LSPR), surface enhanced Raman spectroscopy (SERS) will not be considered in detail, as such techniques have nowadays their own standing.

Trends in Nanophotonics‐Enabled Optofluidic Biosensors

AbstractOptofluidic sensors integrate photonics with micro/nanofluidics to realize compact devices for the label‐free detection of molecules and the real‐time monitoring of dynamic surface binding events with high specificity, ultrahigh sensitivity, low detection limit, and multiplexing capability. Nanophotonic structures composed of metallic and/or dielectric building blocks excel at focusing light into ultrasmall volumes, creating enhanced electromagnetic near‐fields ideal for amplifying the molecular signal readout. Furthermore, fluidic control on small length scales enables precise tailoring of the spatial overlap between the electromagnetic hotspots and the analytes, boosting light‐matter interaction, and can be utilized to integrate advanced functionalities for the pre‐treatment of samples in real‐world‐use cases, such as purification, separation, or dilution. In this review, the authors highlight current trends in nanophotonics‐enabled optofluidic biosensors for applications in the life sciences while providing a detailed perspective on how these approaches can synergistically amplify the optical signal readout and achieve real‐time dynamic monitoring, which is crucial in biomedical assays and clinical diagnostics.

Water-dispersible graphene/amphiphilic pyrene derivative nanocomposite: High AuNPs loading capacity for CEA electrochemical immunosensing

In this work, a water-dispersible graphene/amphiphilic pyrene derivative nanocomposite has been synthesized for electrochemical immunosensing carcinoembryonic antigen (CEA). Water-soluble 4-armed poly(ethylene glycol)-NH2 is immobilized on graphene through noncovalent functionalization with the assistance of pyrene as an anchor group. The noncovalent functionalization strategy can significantly improve the hydrophilicity of graphene and loading capacity of gold nanoparticles (AuNPs) on graphene, resulting in efficient immobilization of antibodies on the nanocomposite. The morphology and conductivity of the as-prepared nanocomposites were characterized by scanning electron microscopy, Fourier transform infrared and UV–vis spectroscopy, cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). CEA monoclonal antibody (anti-CEA) is immobilized on AuNPs via gold-thiol chemistry to construct the electrochemical immunosensing platform, and the surface binding events between anti-CEA and CEA is achieved by monitoring the change of the electrochemical impedance response. Under optimum conditions, the modified electrode exhibits a linear impedance response to CEA in a wide range of 0.1–1000ngmL−1 (R2=0.9959) with low detection limit of 0.06ngmL−1 (S/N=3).

Sialyl-Tn Polysaccharide A1 as an Entirely Carbohydrate Immunogen: Synthesis and Immunological Evaluation.

Sialyl Thomsen-nouveau (STn) is a tumor-associated carbohydrate antigen (TACA) that is overexpressed in a variety of carcinomas such as breast, ovarian, and colon cancer. In normal tissue, STn is not detectable, which is critical for opportunities in developing cancer immunotherapies. A novel, entirely carbohydrate, semisynthetic STn-polysaccharide (PS) A1 conjugate was prepared and evaluated in C57BL/6 mice. STn-PS A1 was combined with commercially available monophosphoryl lipid A-based adjuvant, and after immunization, ELISA indicated a strong immune response for inducing anti-STn IgM/IgG antibodies. The specificity of these antibodies was concomitantly investigated using FACS analysis, and the results indicated excellent cell surface binding events to STn-expressing cancer cell lines MCF-7 and OVCAR-5. An INF-γ ELISpot assay was conducted to further confirm a robust cellular immunity invoked by STn-PS A1. Most importantly, the raised antibodies conferred complement-dependent cellular cytotoxicity against MCF-7 and OVCAR-5 cells.

A Microfluidic Device with Continuous Ligand Gradients in Supported Lipid Bilayers to Probe Effects of Ligand Surface Density and Solution Shear Stress on Pathogen Adhesion

Studying binding interactions involving living cells requires a platform that carefully mimics the physiological parameters that govern these phenomena. Very often the amount of ligands that receptors can bind affect overall binding strength as is the case in cell adhesion. In addition, the physical environment can strongly influence these processes. This is exemplified by the effect of shear stress in catch‐bond‐mediated binding of bacteria. Traditional analysis techniques do not allow to probe these factors simultaneously. To this end, continuous ligand gradients in locked‐in supported lipid bilayers (SLBs) are prepared in a microfluidic device to control fluid flow. This platform allows for one‐pot characterization of cell surface binding events and 1) the effect of ligand density and 2) shear stress, simultaneously. The model interaction between the FimH receptor found on Escherichia coli and mannose found on the mammalian cell membrane is used to evaluate the platform. Using a single chip, specific E. coli ORN 178 adhesion (K d of 0.9 × 10−21 m), detachment and displacement are shown to depend on the mannose‐density and shear stress. For the first time, these effects are studied in a single chip device with high quality. This chip provides entry to further our understanding of other cell–cell interactions.

Quantitative analysis of insulin-like growth factor 2 receptor and insulin-like growth factor binding proteins to identify control mechanisms for insulin-like growth factor 1 receptor phosphorylation.

BackgroundThe insulin-like growth factor (IGF) system impacts cellular development by regulating proliferation, differentiation, and apoptosis, and is an attractive therapeutic target in cancer. The IGF system is complex, with two ligands (IGF1, IGF2), two receptors (IGF1R, IGF2R), and at least six high affinity IGF-binding proteins (IGFBPs) that regulate IGF ligand bioavailability. While the individual components of the IGF system are well studied, the question of how these different components integrate as a system to regulate cell behavior is less clear.ResultsTo analyze the relative importance of different mechanisms that control IGF network activity, we developed a mass-action kinetic model incorporating cell surface binding, phosphorylation, and intracellular trafficking events. The model was calibrated and validated using experimental data collected from OVCAR5, an immortalized ovarian cancer cell line. We then performed model analysis to examine the ability of IGF2R or IGFBPs to counteract phosphorylation of IGF1R, a critical step for IGF network activation. This analysis suggested that IGF2R levels would need to be 320-fold greater than IGF1R in order to decrease pIGF1R by 25 %, while IGFBP levels would need to be 390-fold greater. Analysis of The Cancer Genome Atlas (TCGA) data set suggested that this level of overexpression is unlikely for IGF2R in ovarian, breast, and colon cancer. In contrast, IGFBPs can likely reach these levels, suggesting that IGFBPs are the more critical regulator of IGF1R network activity. Levels of phosphorylated IGF1R were insensitive to changes in parameters regulating the IGF2R arm of the network.ConclusionsUsing a mass-action kinetic model, we determined that IGF2R plays a minor role in regulating the activity of IGF1R under a variety of conditions and that due to their high expression levels, IGFBPs are the dominant mechanism to regulating IGF network activation.Electronic supplementary materialThe online version of this article (doi:10.1186/s12918-016-0263-6) contains supplementary material, which is available to authorized users.

Integration of Faradaic electrochemical impedance spectroscopy into a scalable surface plasmon biosensor for in tandem detection.

We present an integrated label-free biosensor based on surface plasmon resonance (SPR) and Faradaic electrochemical impedance spectroscopy (f-EIS) sensing modalities, for the simultaneous detection of biological analytes. Analyte detection is based on the angular spectroscopy of surface plasmon resonance and the extraction of charge transfer resistance values from reduction-oxidation reactions at the gold surface, as responses to functionalized surface binding events. To collocate the measurement areas and fully integrate the modalities, holographically exposed thin-film gold SPR-transducer gratings are patterned into coplanar electrodes for tandem impedance sensing. Mutual non-interference between plasmonic and electrochemical measurement processes is shown, and using our scalable and compact detection system, we experimentally demonstrate biotinylated surface capture of neutravidin concentrations as low as 10 nM detection, with a 5.5 nM limit of detection.

Dual resonance approach to decoupling surface and bulk attributes in photonic crystal biosensor

A sub-wavelength grating-based photonic crystal sensor is designed to excite two spectrally and spatially different guided mode resonances that have distinctive electric field distributions. We present and validate the uni-polarized dual resonance approach to separating bulk index perturbations from surface-binding events in a single measurement by monitoring the resonance wavelength shifts. This self-referencing method will reduce errors in the measurement of biomolecule binding events on sensor surfaces in a perturbed environmental background.

Electrochemical immunosensors for effective evaluation of amyloid-beta modulators on oligomeric and fibrillar aggregation processes.

A novel electrochemical immunosensor fabricated from gold compact disc electrodes was designed for rapid evaluation of aggregation processes that lead to the formation of oligomeric and fibrillar states of amyloid-beta(1-42) (Aβ(1-42)) during Alzheimer's disease. Conformation-specific antibodies were immobilized on the surface of the gold electrode using a 3,3'-dithiobis (sulfosuccinimidyl) propionate (DTSSP) linker. Surface binding events were analyzed by electrochemical impedance spectroscopy (EIS) in which the formation of an antigen-antibody complex was quantified as a function of charge transfer resistance using a [Fe(CN)6](3-/4-) redox probe. The effectiveness of novel sym-triazine-derived aggregation modulators (TAE-1, TAE-2) to reduce the population of toxic oligomers was evaluated. Aβ fibril formation was validated by thioflavin T (ThT) fluorescence, whereas oligomer formation was investigated by MALDI. Antigen detection by EIS was further supported by immuno dot blot assays for oligomeric and fibrillar components. Docking simulations of the aggregation modulators TAE-1 and TAE-2 with Aβ(1-42) fibrils performed using Autodock Vina suggest a mechanism for the improved aggregation inhibition observed for TAE-2. The results demonstrate the utility and convenience of impedance immunosensing as an analytical tool for rapid and comprehensive evaluation of effective Aβ aggregation modulating agents.

Silica Nanowire Arrays for Diffraction‐Based Bioaffinity Sensing

Arrays of electrodeposited silica nanowires (SiO2 NWs) have been fabricated over large areas (cm(2)) on fluoropolymer thin films attached to glass substrates by a combination of photolithography and electrochemically triggered sol-gel nanoscale deposition. Optical and scanning electron microscopy (SEM) measurements revealed that the SiO2 NW arrays had an average spacing of ten micrometers and an average width of 700 nm with a significant grain structure that was a result of the sol-gel deposition process. The optical diffraction properties at 633 nm of the SiO2 NW arrays were characterized when placed in contact with solutions by using a prism-coupled total internal reflection geometry; quantification of changes in these diffraction properties was applied in various sensing applications. Bulk refractive index sensing by using the SiO2 NW grating was demonstrated with a sensitivity of 1.30×10(-5) RIU. Toposelectively chemically modified SiO2 NW arrays were used for diffraction biosensing measurements of surface binding events, such as the electrostatic adsorption of gold nanoparticles and the bioaffinity adsorption of streptavidin onto a biotin monolayer. Finally, the application of the SiO2 NW arrays for practical medical-diagnostic applications was demonstrated by monitoring the diffraction of SiO2 NW arrays functionalized with a single-stranded (ss)DNA aptamer to detect human α-thrombin from solutions at sub-pathologic nanomolar concentrations.

Electron Transfer-Based Single Molecule Fluorescence as a Probe for Nano-Environment Dynamics

Electron transfer (ET) is one of the most important elementary processes that takes place in fundamental aspects of biology, chemistry, and physics. In this review, we discuss recent research on single molecule probes based on ET. We review some applications, including the dynamics of glass-forming systems, surface binding events, interfacial ET on semiconductors, and the external field-induced dynamics of polymers. All these examples show that the ET-induced changes of fluorescence trajectory and lifetime of single molecules can be used to sensitively probe the surrounding nano-environments.

Enhancement of field–analyte interaction at metallic nanogap arrays for sensitive localized surface plasmon resonance detection

We investigated the near-field enhancement of a localized surface plasmon resonance (LSPR) structure based on gold nanograting pairs with a nanosized gap. The results calculated by finite-difference time-domain and rigorous coupled-wave analysis methods presented that the nanogap enclosed by two neighboring nanogratings produced significant confinement and enhancement of electromagnetic fields and allowed a sensitive detection in sensing of surface binding events. Gold gratings with a narrow gap distance less than 10 nm showed enhanced refractive index sensitivity due to the intensified optical field at the nanogap, outperforming the LSPR structure with noninteracting nanogratings. Also, we analyzed the effectiveness of using an overlap integral (OI) between analyte and local plasmon field to estimate the detection sensitivity. We found a strong correlation of field-analyte OI with far-field sensor sensitivity.

Demonstration of near infrared gas sensing using gold nanodisks on functionalized silicon

In this work, we demonstrate experimentally the use of an array of gold nanodisks on functionalized silicon for chemosensing purposes. The metallic nanostructures are designed to display a very strong plasmonic resonance in the infrared regime, which results in highly sensitive sensing. Unlike usual experiments which are based on the functionalization of the metal surface, we functionalized here the silicon substrate. This silicon surface was modified chemically by buildup of an organosilane self-assembled monolayer (SAM) containing isocyanate as functional group. These groups allow for an easy surface regeneration by simple heating, thanks to the thermally reversible interaction isocyanate-analyte, which allows the cyclic use of the sensor. The technique showed a high sensitivity to surface binding events in gas and allowed the surface regeneration by heating of the sensor at 150 °C. A relative wavelength shift ∆λ(max)λ(0)=0.027 was obtained when the saturation level was reached.

Investigation of the effects of design parameters on sensitivity of surface plasmon resonance biosensors

Surface based analytical tools have gained more importance for rapid, sensitive and label-free monitoring of molecular recognition events. The surface plasmon resonance (SPR) approach has played a prominent role in real time monitoring of surface binding events. This paper first provides a brief description of the existing biosensing methods. Next, an investigation of the role of thin films of gold, silver, and aluminum for protein detection in SPR biosensors is presented. It is shown that the sensitivity, which is indicated by the shift of plasmon dip, is not linearly related to the thickness of protein but quadratic over a specific range. The approach involves plotting the reflectivity curve as a function of the angle of incidence.

Functional Nanostructured Plasmonic Materials

Plasmonic crystals fabricated with precisely controlled arrays of subwavelength metal nanostructures provide a promising platform for sensing and imaging of surface binding events with micrometer spatial resolution over large areas. Soft nanoimprint lithography provides a robust, cost-effective method for producing highly uniform plasmonic crystals of this type with predictable optical properties. The tunable multimode plasmonic resonances of these crystals and their ability for integration into lab-on-a-chip microfluidic systems can both be harnessed to achieve exceptionally high analytical sensitivities down to submonolayer levels using even a common optical microscope, circumventing numerous technical limitations of more conventional surface plasmon resonance techniques. In this article, we highlight some recent advances in this field with an emphasis on the fabrication and characterization of these integrated devices and their demonstrated applications.

Silicon Photonic Microring Resonators for Quantitative Cytokine Detection and T-Cell Secretion Analysis

The ability to perform multiple simultaneous protein biomarker measurements in complex media with picomolar sensitivity presents a large challenge to disease diagnostics and fundamental biological studies. Silicon photonic microring resonators represent a promising platform for real-time detection of biomolecules on account of their spectral sensitivity toward surface binding events between a target and antibody-modified microrings. For all refractive index-based sensing schemes, the mass of bound analytes, in combination with other factors such as antibody affinity and surface density, contributes to the observed signal and measurement sensitivity. Therefore, proteins that are simultaneously low in abundance and have a lower molecular weight are often challenging to detect. By employing a more massive secondary antibody to amplify the signal arising from the initial binding event, it is possible to improve both the sensitivity and the specificity of protein assays, allowing for quantitative sensing in complex sample matrices. Herein, a sandwich assay is used to detect the 15.5 kDa human cytokine interleukin-2 (IL-2) at concentrations down to 100 pg/mL (6.5 pM) and to quantitate unknown solution concentrations over a dynamic range spanning 2.5 orders of magnitude. This same sandwich assay is then used to monitor the temporal secretion profile of IL-2 from Jurkat T lymphocytes in serum-containing cell culture media in the presence of the entire Jurkat secretome. The same temporal secretion analysis is performed in parallel using a commercial ELISA, revealing similar IL-2 concentration profiles but superior precision for the microring resonator sensing platform. Furthermore, we demonstrate the generality of the sandwich assay methodology on the microring resonator platform for the analysis of any biomolecular target for which two high-affinity antibodies exist by detecting the approximately 8 kDa cytokine interleukin-8 (IL-8) with a limit of detection and dynamic range similar to that of IL-2. This work demonstrates the first application of silicon photonic microring resonators for detecting cellular secretion of cytokines and represents an important advance for the detection of protein biomarkers on an emerging analytical platform.

A biosensor based on photonic crystal surface waves with an independent registration of the liquid refractive index

A high-precision optical biosensor technique capable of independently determining the refractive index (RI) of liquids is presented. Photonic crystal surface waves were used to detect surface binding events, while an independent registration of the critical angle was used for accurate determination of the liquid RI. This technique was tested using binding of biotin molecules to a streptavidin monolayer at low and high biotin concentrations. The attained baseline noise is 5 × 1 0 − 13 m/Hz 1/2 for adlayer thickness changes and 9 × 1 0 − 8 RIU/Hz 1/2 for RI changes.

Multispectral Thin Film Biosensing and Quantitative Imaging Using 3D Plasmonic Crystals

This work provides plasmonic crystal platforms for quantitative imaging mode biosensing and multispectral immunoassays, establishing and validating both the optical and equilibrium bases for their operation. We investigated the distance-dependent refractive index sensitivity of full 3D plasmonic crystals to thin polymeric films formed using layer by layer (LbL) assembly of polyelectrolytes as a model system. LbL was also used to determine the preferred gold thickness and plasmonic crystal design rules (nanowell diameter and periodicity) for improved thin-film sensitivity, and full 3D finite-difference time-domain (FDTD) calculations were used to quantitatively model and confirm the experimentally observed thin film sensitivities. The integrated multispectral response of the crystals increases approximately linearly with film thickness for values <70 nm, which enables the use of molecular rulers with known thicknesses (such as self-assembled monolayers of alkanethiols on gold) to calibrate these optics for quantitative detection and speciation of surface binding events in a multiplexed imaging format. The utility of these sensors and multispectral analysis for applications in quantitative biosensing was further demonstrated by measuring the equilibrium response curve of an antibody/antigen pair (rabbit antigoat IgG/goat IgG) at increasing antigen concentrations. Fitting the integrated response to a Langmuir isotherm yielded a calculated binding constant on the order of approximately 10(7) M(-1), which is in agreement with the affinity constants reported in the literature for anti-IgG/IgG binding pairs and provides intrinsic detection limits of approximately 400 pM for such unamplified assays.

Evanescent-wave fluorescence microscopy using symmetric planar waveguides

We describe a new evanescent-wave fluorescence excitation method, ideally suited for imaging of biological samples. The excitation light propagates in a planar optical waveguide, consisting of a thin waveguide core sandwiched between a sample in an aqueous solution and a polymer with a matching refractive index, forming a symmetric cladding environment. This configuration offers clear advantages over other waveguide-excitation methods, such as superior image quality, wide tunability of the evanescent field penetration depth and compatibility with optical fibers. The method is well suited for cell membrane imaging on cells in culture, including cell-cell and cell-matrix interaction, monitoring of surface binding events and similar applications involving aqueous solutions.