Eleonora Petryayeva

Quantum Dots in Bioanalysis: A Review of Applications Across Various Platforms for Fluorescence Spectroscopy and Imaging

Semiconductor quantum dots (QDs) are brightly luminescent nanoparticles that have found numerous applications in bioanalysis and bioimaging. In this review, we highlight recent developments in these areas in the context of specific methods for fluorescence spectroscopy and imaging. Following a primer on the structure, properties, and biofunctionalization of QDs, we describe select examples of how QDs have been used in combination with steady-state or time-resolved spectroscopic techniques to develop a variety of assays, bioprobes, and biosensors that function via changes in QD photoluminescence intensity, polarization, or lifetime. Some special attention is paid to the use of Forster resonance energy transfer-type methods in bioanalysis, including those based on bioluminescence and chemiluminescence. Direct chemiluminescence, electrochemiluminescence, and charge transfer quenching are similarly discussed. We further describe the combination of QDs and flow cytometry, including traditional cellular analyses and spectrally encoded barcode-based assay technologies, before turning our attention to enhanced fluorescence techniques based on photonic crystals or plasmon coupling. Finally, we survey the use of QDs across different platforms for biological fluorescence imaging, including epifluorescence, confocal, and two-photon excitation microscopy; single particle tracking and fluorescence correlation spectroscopy; super-resolution imaging; near-field scanning optical microscopy; and fluorescence lifetime imaging microscopy. In each of the above-mentioned platforms, QDs provide the brightness needed for highly sensitive detection, the photostability needed for tracking dynamic processes, or the multiplexing capacity needed to elucidate complex systems. There is a clear synergy between advances in QD materials and spectroscopy and imaging techniques, as both must be applied in concert to achieve their full potential.

Multiplexed Homogeneous Assays of Proteolytic Activity Using a Smartphone and Quantum Dots

Semiconductor quantum dot (QD) bioconjugates, with their unique and highly advantageous physicochemical and optical properties, have been extensively utilized as probes for bioanalysis and continue to generate widespread interest for these applications. An important consideration for expanding the utility of QDs and making their use routine is to make assays with QDs more accessible for laboratories that do not specialize in nanomaterials. Here, we show that digital color imaging of QD photoluminescence (PL) with a smartphone camera is a viable, easily accessible readout platform for quantitative, multiplexed, and real-time bioanalyses. Red-, green-, and blue-emitting CdSeS/ZnS QDs were conjugated with peptides that were labeled with a deep-red fluorescent dye, Alexa Fluor 647, and the dark quenchers, QSY9 and QSY35, respectively, to generate Förster resonance energy transfer (FRET) pairs sensitive to proteolytic activity. Changes in QD PL caused by the activity of picomolar to nanomolar concentrations of protease were detected as changes in the red-green-blue (RGB) channel intensities in digital color images. Importantly, measurements of replicate samples made with smartphone imaging and a sophisticated fluorescence plate reader yielded the same quantitative results, including initial proteolytic rates and specificity constants. Homogeneous two-plex and three-plex assays for the activity of trypsin, chymotrypsin, and enterokinase were demonstrated with RGB imaging. Given the ubiquity of smartphones, this work largely removes any instrumental impediments to the adoption of QDs as routine tools for bioanalysis in research laboratories and is a critical step toward the use of QDs for point-of-care diagnostics. This work also adds to the growing utility of smartphones in analytical methods by enabling multiplexed fluorimetric assays within a single sample volume and across multiple samples in parallel.

Proteolytic assays on quantum-dot-modified paper substrates using simple optical readout platforms.

Paper-based assays are a promising diagnostic format for point-of-care applications, field deployment, and other low-resource settings. To date, the majority of efforts to integrate nanomaterials with paper-based assays have utilized gold nanoparticles. Here, we show that semiconductor quantum dots (QDs), in combination with Förster resonance energy transfer (FRET), are also suitable nanomaterials for developing paper-based assays. Paper fibers were chemically modified with thiol ligands to immobilize CdSeS/ZnS QDs, the QDs were self-assembled with dye-labeled peptides to generate efficient FRET, and steady-state and fluorescence lifetime imaging microscopy (FLIM) were used for characterization. Peptides were selected as substrates for three different proteases and a series of kinetic assays for proteolytic activity was carried out, including multiplexed assays and pro-enzyme activation assays. Quantitative results were obtained within 5-60 min at levels as low as 1-2 nM of protease. These assays were possible using simple optical readout platforms that did not negate the low cost, ease of use, and overall accessibility advantages of paper-based assays. A violet light-emitting diode (LED) excitation source and color imaging with either a digital camera, consumer webcam, or smartphone camera were sufficient for analysis on the basis of a red/green color intensity ratio. At most, a universal serial bus (USB) connection to a computer was required and the instrumentation cost orders of magnitude less than that typically utilized for in vitro bioanalyses with QDs. This work demonstrates that QDs are valuable probes for developing a new generation of paper-based diagnostics.

Toward point-of-care diagnostics with consumer electronic devices: the expanding role of nanoparticles

There is a critical need for point-of-care (POC) diagnostics in health care and a parallel need for similar point-of-need (PON) diagnostics in other sectors. Such technology could have a profoundly positive impact on health, wellness and quality-of-life in both the developed and developing worlds. This very active area of research is converging with another very active area of research—the biomedical applications of nanotechnology—with exciting outcomes. In this review, we describe how nanoparticles facilitate the use of mass-produced consumer electronic devices for POC/PON diagnostic applications. We first highlight the growing need for POC diagnostics; provide a brief overview of clinical tests, biomarkers and lateral flow assays; describe the amenability of consumer electronic devices to POC/PON diagnostics; and summarize the attractive properties of nanoparticle materials in these contexts. Devices of interest include cell phones, smartphones, wearable technology, other CMOS imaging devices, scanners, optical drives/disc players, and strip readers. We continue to describe how nanoparticles can enable and enhance the readout of diagnostic assays with these consumer electronic devices using illustrative examples from the literature. The most utilized nanoparticles include gold nanoparticles, carbon nanoparticles, quantum dots, upconversion nanoparticles, polymer or silica nanoparticle composites with other materials, and viral nanoparticles. Given that assays combining the foregoing nanoparticles with consumer electronic devices have almost exclusively utilized optical readout, we further assess the potential for developing nanoparticle-based electrochemical assays with readout through either a smartphone or personal blood glucose meter (for non-glucose biomarkers). The review concludes with our perspective on future research and development in this area, including the role nanoparticles may play in facilitating the emergence of the smartphone as a leading personal health care device.

Near-infrared-triggered anticancer drug release from upconverting nanoparticles.

Targeted drug delivery using functional nanoparticles has provided new strategies for improving therapeutic efficacy while concurrently minimizing toxicity. Photodynamic therapy is an approach that offers control of drug delivery by use of an external photon source to allow active therapeutic release to a target area. Upconverting nanoparticles (UCNPs) have potential to operate as integral components of photodynamic therapeutic platforms based on the resonant absorption of near-infrared (NIR) radiation and emission at shorter wavelengths. NIR radiation is minimally absorbed and scattered by biological tissues, and the NIR excitation of UCNPs can generate anti-Stokes emission in the ultraviolet-visible wavelength range at intensities that can be used to trigger cleavage of bonds linking therapeutics at the nanoparticle interface. Herein, we describe an investigation of photocleavage at the surface of UCNPs to release the chemotherapeutic 5-fluorouracil (5-FU). Core-shell UCNPs composed of a β-NaYF4: 4.95% Yb, 0.08% Tm core and a β-NaYF4 shell were coated with o-phosphorylethanolamine ligands and coupled to an o-nitrobenzyl (ONB) derivative of 5-FU. NIR excitation of the UCNPs resulted in photoluminescence (PL) emission bands centered at 365, 455, and 485 nm. The UV-blue PL was in resonance with the absorption band of the ONB-FU derivative resulting in photocleavage and subsequent release of the 5-FU drug from the UCNPs for these in vitro studies. The release of 5-FU was complete in <14 min using a NIR laser source centered at 980 nm that operated at a power of <100 mW. The efficiency of triggered release was as high as 77% of the total ONB-FU conjugate, while the rate of drug release could be tuned with the laser power output. This work provides an important first step in the development of a UCNP platform capable of targeted chemotherapy.

Quantum Dot and Gold Nanoparticle Immobilization for Biosensing Applications using Multidentate Imidazole Surface Ligands

A facile approach for modification of solid substrates with multidentate imidazole ligands was developed for immobilization of high densities of quantum dots (QDs) that were capped with hydrophilic thiol-based ligands, and for immobilization of noble metal nanoparticles. Imidazole polymer was synthesized using poly(acrylic acid) as a backbone, and grafted on amine functionalized substrate in a two-step approach. The polymer-modified surface was characterized using ellipsometry, water contact angle, and X-ray photoelectron spectroscopy. Fluorescence spectroscopy and scanning electron microscopy were used to evaluate nanoparticle immobilization. Homogeneous, high density (ca. 5 × 10(11) cm(-2)) QD films formed via self-assembly were obtained within 4-6 h. Similarly, the imidazole polymer was also shown to be effective for immobilization of gold nanoparticles as a uniform film. By making use of the pH-sensitive affinity of the imidazole rings to zinc on the surface of QDs, it was possible to achieve regeneration of functional ligands suitable for subsequent immobilization of new QDs. Immobilized QDs were used as a platform for bioconjugation with oligonucleotides and peptides. The transduction of nucleic acid hybridization and enzyme activity using QDs as energy donors in interfacial fluorescence resonance energy transfer (FRET) indicated that the immobilization strategy preserved the functional properties of the QDs. The multidentate imidazole ligands used for QD immobilization offer the highest denticity of binding in comparison to the currently available approaches without compromise in their optical properties and ability to interact with biomolecules in solution.

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 coassembled 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.

Adapting fluorescence resonance energy transfer with quantum dot donors for solid-phase hybridization assays in microtiter plate format.

Methods have been developed for the solid-phase detection of nucleic acids using mixed films of quantum dots (QDs) and oligonucleotide probes in microtiter plates. Polystyrene microwells were functionalized with multidentate imidazole ligands to immobilize QDs. Oligonucleotide hybridization was transduced using QDs as donors in fluorescence resonance energy transfer (FRET). One detection channel paired green-emitting QD donors with Cy3 acceptors and served as an internal standard. A second detection channel paired red-emitting QDs with Alexa Fluor 647 acceptors and served as the primary detection channel. A selective assay for multiple targets was demonstrated using a 96-well plate format, which combined the advantages of two-plex QD-FRET with the high-throughput capability and convenience of microtiter plates. The assay had excellent resistance to the nonspecific adsorption of DNA and discriminated between fully complementary and single base-pair mismatched sequences with a contrast ratio >2. Under optimal conditions for a single color (green QD) assay format, the limit of detection (LOD) was 4 nM, and the dynamic range was from 20 to 300 nM. In a two-color assay, the detection channel (red QD) exhibited linear response between 4 and 100 nM and a LOD of 4 nM.

A job for quantum dots: use of a smartphone and 3D-printed accessory for all-in-one excitation and imaging of photoluminescence

AbstractPoint-of-care (POC) diagnostic technologies are needed to improve global health and smartphones are a prospective platform for these technologies. While many fluorescence or photoluminescence-based smartphone assays have been reported in the literature, common shortcomings are the requirement of an excitation light source external to the smartphone and complicated integration of that excitation source with the smartphone. Here, we show that the photographic flash associated with the smartphone camera can be utilized to enable all-in-one excitation and imaging of photoluminescence (PL), thus eliminating the need for an excitation light source external to the smartphone. A simple and low-cost 3D-printed accessory was designed to create a dark environment and direct excitation light from the smartphone flash onto a sample. Multiple colors and compositions of semiconductor quantum dot (QD) were evaluated as photoluminescent materials for all-in-one smartphone excitation and imaging of PL, and these were compared with fluorescein and R-phycoerythrin (R-PE), which are widely utilized molecular and protein materials for fluorescence-based bioanalysis. The QDs were found to exhibit much better brightness and have the best potential for two-color detection. A model protein binding assay with a sub-microgram per milliliter detection limit and a Förster resonance energy transfer (FRET) assay for proteolytic activity were demonstrated, including imaging with serum as a sample matrix. In addition, FRET within tandem conjugates of a QD donor and fluorescent dye acceptor enabled smartphone detection of dye fluorescence that was otherwise unobservable without the QD to enhance its brightness. The ideal properties of photoluminescent materials for all-in-one smartphone excitation and imaging are discussed in the context of several different materials, where QDs appear to be the best overall material for this application. Graphical AbstractBioanalytical assays with a smartphone and 3D-printed accessory for imaging photoluminescence from quantum dots

Quantitative measurement of proteolytic rates with quantum dot-peptide substrate conjugates and Förster resonance energy transfer.

An important challenge in biology is the development of probes for visualizing and quantitatively tracking enzyme activity. Proteases are an important class of enzyme with value as both diagnostic and therapeutic targets. In this chapter, we describe the preparation of quantum dot (QD)-peptide substrate conjugates as probes for measuring proteolytic activity. QDs have several highly advantageous optical properties that make these materials especially well suited for applications in bioanalysis and bioimaging. Further, peptide substrates for proteases can be controllably self-assembled to QDs and this capability, in combination with Förster resonance energy transfer (FRET), enables the design of quantitative in vitro assays capable of directly reporting on proteolytic activity. We present a detailed method for the preparation, calibration, and application of such QD probes, along with methods of analysis to generate progress curves for the proteolytic digestion of substrate. Representative data are illustrated for two different proteases and two different QD-fluorescent dye FRET pairs. The general methodology is likely to be applicable with other hydrolytic enzymes in addition to proteases. Overall, the method is straightforward to implement with commercially available materials and does not require specialized expertise.