Small surface, big effects, and big challenges: toward understanding enzymatic activity at the inorganic nanoparticle–substrate interface

Enzymes are important biomarkers for molecular diagnostics and targets for the action of drugs. In turn, inorganic nanoparticles (NPs) are of interest as materials for biological assays, biosensors, cellular and in vivo imaging probes, and vectors for drug delivery and theranostics. So how does an enzyme interact with a NP, and what are the outcomes of multivalent conjugation of its substrate to a NP? This invited feature article addresses the current state of the art in answering this question. Using gold nanoparticles (Au NPs) and semiconductor quantum dots (QDs) as illustrative materials, we discuss aspects of enzyme structure–function and the properties of NP interfaces and surface chemistry that determine enzyme–NP interactions. These aspects render the substrate-on-NP configurations far more complex and heterogeneous than the conventional turnover of discrete substrate molecules in bulk solution. Special attention is also given to the limitations of a standard kinetic analysis of the enzymatic turnover of these configurations, the need for a well-defined model of turnover, and whether a “hopping” model can account for behaviors such as the apparent acceleration of enzyme activity. A detailed and predictive understanding of how enzymes turn over multivalent NP-substrate conjugates will require a convergence of many concepts and tools from biochemistry, materials, and interface science. In turn, this understanding will help to enable rational, optimized, and value-added designs of NP bioconjugates for biomedical and clinical applications

Comparison of semiconducting polymer dots and semiconductor quantum dots for smartphone-based fluorescence assays

Fluorescent nanoparticles have transformative potential for smartphone-based point-of-need diagnostics because an optimal material can reduce the technical burden to meet assay performance requirements. Semiconductor quantum dots (QDs) are a now well-established example of such a material. Semiconducting polymer dots (Pdots) and conjugated-polymer nanoparticles (CPNs) are emerging materials that bring the advantages of being bright, easy to synthesize, and metal-free when compared with QDs, but they frequently present the trade-off of spectrally broad emission and less well-defined surface chemistry. Here, we compare these two classes of nanoparticles in the context of a “bare bones” device that uses a smartphone for all-in-one excitation and imaging of fluorescence. The greater per-particle brightness of Pdots provides orders of magnitude better imaging sensitivity versus QDs, and this advantage translates to a model lateral flow assay. Our data suggest that Pdots will support multicolor imaging on a smartphone in an optimized assay, although QDs are likely superior for this purpose. These pros and cons lead to discussion of how physicochemical differences between QDs and Pdots may influence assay performance beyond differences in optical properties. Overall, Pdots have great potential for enabling smartphone-based fluorescence assays with high sensitivity and low detection limits

Optimizing Two-Color Semiconductor Nanocrystal Immunoassays in Single Well Microtiter Plate Formats

The simultaneous detection of two analytes, chicken IgY (IgG) and Staphylococcal enterotoxin B (SEB), in the single well of a 96-well plate is demonstrated using luminescent semiconductor quantum dot nanocrystal (NC) tracers. The NC-labeled antibodies were prepared via sulfhydryl-reactive chemistry using a facile protocol that took <3 h. Dose response curves for each target were evaluated in a single immunoassay format and compared to Cy5, a fluorophore commonly used in fluorescent immunoassays, and found to be equivalent. Immunoassays were then performed in a duplex format, demonstrating multiplex detection in a single well with limits of detection equivalent to the single assay format: 9.8 ng/mL chicken IgG and 7.8 ng/mL SEB

Terbium to Quantum Dot FRET Bioconjugates for Clinical Diagnostics: Influence of Human Plasma on Optical and Assembly Properties

Förster resonance energy transfer (FRET) from luminescent terbium complexes (LTC) as donors to semiconductor quantum dots (QDs) as acceptors allows extraordinary large FRET efficiencies due to the long Förster distances afforded. Moreover, time-gated detection permits an efficient suppression of autofluorescent background leading to sub-picomolar detection limits even within multiplexed detection formats. These characteristics make FRET-systems with LTC and QDs excellent candidates for clinical diagnostics. So far, such proofs of principle for highly sensitive multiplexed biosensing have only been performed under optimized buffer conditions and interactions between real-life clinical media such as human serum or plasma and LTC-QD-FRET-systems have not yet been taken into account. Here we present an extensive spectroscopic analysis of absorption, excitation and emission spectra along with the luminescence decay times of both the single components as well as the assembled FRET-systems in TRIS-buffer, TRIS-buffer with 2% bovine serum albumin, and fresh human plasma. Moreover, we evaluated homogeneous LTC-QD FRET assays in QD conjugates assembled with either the well-known, specific biotin-streptavidin biological interaction or, alternatively, the metal-affinity coordination of histidine to zinc. In the case of conjugates assembled with biotin-streptavidin no significant interference with the optical and binding properties occurs whereas the histidine-zinc system appears to be affected by human plasma

Article Interfacial Chemistry and the Design of Solid-Phase Nucleic Acid Hybridization Assays Using Immobilized Quantum Dots as Donors in Fluorescence Resonance Energy Transfer

sensor

Interfacial Chemistry and the Design of Solid-Phase Nucleic Acid Hybridization Assays Using Immobilized Quantum Dots as Donors in Fluorescence Resonance Energy Transfer

The use of quantum dots (QDs) as donors in fluorescence resonance energy transfer (FRET) offer several advantages for the development of multiplexed solid-phase QD-FRET nucleic acid hybridization assays. Designs for multiplexing have been demonstrated, but important challenges remain in the optimization of these systems. In this work, we identify several strategies based on the design of interfacial chemistry for improving sensitivity, obtaining lower limits of detection (LOD) and enabling the regeneration and reuse of solid-phase QD-FRET hybridization assays. FRET-sensitized emission from acceptor dyes associated with hybridization events at immobilized QD donors provides the analytical signal in these assays. The minimization of active sensing area reduces background from QD donor PL and allows the resolution of smaller amounts of acceptor emission, thus lowering the LOD. The association of multiple acceptor dyes with each hybridization event can enhance FRET efficiency, thereby improving sensitivity. Many previous studies have used interfacial protein layers to generate selectivity; however, transient destabilization of these layers is shown to prevent efficient regeneration. To this end, we report a protein-free interfacial chemistry and demonstrate the specific detection of as little as 2 pmol of target, as well as an improved capacity for regeneration

More than a light switch: engineering unconventional fluorescent configurations for biological sensing

Fluorescence is a powerful and sensitive tool in biological detection, used widely for cellular imaging and in vitro molecular diagnostics. Over time, three prominent conventions have emerged in the design of fluorescent biosensors: a sensor is ideally specific for its target, only one fluorescence signal turns on or off in response to the target, and each target requires its own sensor and signal combination. These are conventions but not requirements, and sensors that break with one or more of these conventions can offer new capabilities and advantages. Here, we review “unconventional” fluorescent sensor configurations based on fluorescent dyes, proteins, and nanomaterials such as quantum dots and metal nanoclusters. These configurations include multifluorophore Förster resonance energy transfer (FRET) networks, temporal multiplexing, photonic logic, and cross-reactive arrays or “noses”. The more complex but carefully engineered biorecognition and fluorescence signaling modalities in unconventional designs are richer in information, afford greater multiplexing capacity, and are potentially better suited to the analysis of complex biological samples, interactions, processes, and diseases. We conclude with a short perspective on the future of unconventional fluorescent sensors and encourage researchers to imagine sensing beyond the metaphorical light bulb and light switch combination

Toward clinical applications of smartphone spectroscopy and imaging

This chapter focuses on optical smartphone platforms that are under development and have potential to impact clinical medicine. It aims to revisit how a smartphone camera can be used for optical measurements, including spectroscopy, and how the use of peripherals enables the creation of spectroscopic devices that can measure clinical samples. Smartphone-based imaging systems and spectrometers utilize the camera chip of the smartphone as an optical detector for measurements of light intensity and color. The chapter discusses types of clinical samples, the biomarker content to be analyzed by smartphone spectroscopy and imaging. The distinct advantage to creating a smartphone imaging or spectrometric analysis platform is portability and reduction in instrumentation cost. Smartphone microscopy has been developed, with many commercial devices now available for bright-field imaging. Smartphone cameras can be readily manipulated to read and even perform complete clinical assays for a wide variety of biomarkers in multiple different formats, in both resource-limited and near-patient settings

A Quantum Dot-Based Concentric FRET Configuration for the Parallel Detection of Protease Activity and Concentration

Protease expression, activity, and inhibition play crucial roles in a multitude of biological processes; however, these three aspects of their function are difficult for any one bioanalytical probe to measure. To help address this challenge, we report a multifunctional concentric Förster resonance energy transfer (FRET) configuration that combines two modes of biorecognition using aptamers and peptide substrates co-assembled to a central semiconductor quantum dot (QD). The aptamer is sensitive to the concentration of protease and the peptide is sensitive to its hydrolytic activity. The role of the QD is to serve as a nanoscale scaffold and initial donor for energy transfer with both Cyanine 3 (Cy3) and Alexa Fluor 647 (A647) fluorescent dyes associated with the aptamer and peptide, respectively. Using thrombin as a model protease, we show that a ratiometric analysis of the emission from the QD, Cy3, and A647 permits discrimination between thrombin and thrombin-like activity, and distinguishes between active, reversibly inhibited, and irreversibly inhibited thrombin. Reliable quantitative results were obtained from a kinetic analysis of the changes in FRET. This concentric FRET format, which capitalizes on both the physical and optical properties of QDs, should be adaptable to other protease targets for which both peptide substrates and binding aptamers are known. It is thus expected to become valuable a tool for the real-time analysis of protease activity and regulation.Chemistry, Department ofScience, Faculty ofReviewedFacult

Concentric Förster Resonance Energy Transfer Imaging

Concentric Förster resonance energy transfer (cFRET) configurations based on semiconductor quantum dots (QDs) are promising probes for biological sensing because they offer multiplexing capability in a single vector with robust ratiometric detection by exploiting a network of FRET pathways. To expand the scope and utility of cFRET probes, it is necessary to develop and validate cFRET imaging methodology. In this technical note, we present such a methodology using a protease-sensitive cFRET configuration that comprises a green-emitting QD, Alexa Fluor 555 (A555), and Alexa Fluor 647 (A647). Photoluminescence (PL) images were acquired with three filter-based emission channels to permit measurement of A555/QD and A647/QD PL ratios. With reference to calibration samples, these PL ratios were used to calculate quantitative progress curves for proteolytic activity in regions of interest in the acquired images. Importantly, the imaging methodology reproduces quantitative results obtained with a monochromator-based fluorescence plate reader. Spatiotemporal resolution is demonstrated by tracking the activity of two prototypical proteases, trypsin and chymotrypsin, as they diffuse down the length of a capillary. This methodology is expected to enable the future use of cFRET probes for cellular sensing and other imaging assays