Anthony J. Tavares

Beyond labels: A review of the application of quantum dots as integrated components of assays, bioprobes, and biosensors utilizing optical transduction

A comprehensive review of the development of assays, bioprobes, and biosensors using quantum dots (QDs) as integrated components is presented. In contrast to a QD that is selectively introduced as a label, an integrated QD is one that is present in a system throughout a bioanalysis, and simultaneously has a role in transduction and as a scaffold for biorecognition. Through a diverse array of coatings and bioconjugation strategies, it is possible to use QDs as a scaffold for biorecognition events. The modulation of QD luminescence provides the opportunity for the transduction of these events via fluorescence resonance energy transfer (FRET), bioluminescence resonance energy transfer (BRET), charge transfer quenching, and electrochemiluminescence (ECL). An overview of the basic concepts and principles underlying the use of QDs with each of these transduction methods is provided, along with many examples of their application in biological sensing. The latter include: the detection of small molecules using enzyme-linked methods, or using aptamers as affinity probes; the detection of proteins via immunoassays or aptamers; nucleic acid hybridization assays; and assays for protease or nuclease activity. Strategies for multiplexed detection are highlighted among these examples. Although the majority of developments to date have been in vitro, QD-based methods for ex vivo biological sensing are emerging. Some special attention is given to the development of solid-phase assays, which offer certain advantages over their solution-phase counterparts.

Quantum dots as contrast agents for in vivo tumor imaging: progress and issues

AbstractQuantum dots (QDs) have shown promise as imaging agents in cancer, heart disease, and gene therapy research. This review focuses on the design of QDs, and modification using peptides and proteins for mediated targeting of tissues for fluorescence imaging of tumors in vivo. Recent examples from the literature are used to illustrate the potential of QDs as effective imaging agents. The distribution and ultimate fate of QDs in vivo is considered, and considerations of designs that minimize potential toxicity are presented. FigureAngiogenic blood vessel

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.

On-Chip Transduction of Nucleic Acid Hybridization Using Spatial Profiles of Immobilized Quantum Dots and Fluorescence Resonance Energy Transfer

The glass surface of a glass-polydimethylsiloxane (PDMS) microfluidic channel was modified to develop a solid-phase assay for quantitative determination of nucleic acids. Electroosmotic flow (EOF) within channels was used to deliver and immobilize semiconductor quantum dots (QDs), and electrophoresis was used to decorate the QDs with oligonucleotide probe sequences. These processes took only minutes to complete. The QDs served as energy donors in fluorescence resonance energy transfer (FRET) for transduction of nucleic acid hybridization. Electrokinetic injection of fluorescent dye (Cy3) labeled oligonucleotide target into a microfluidic channel and subsequent hybridization (within minutes) provided the proximity for FRET, with emission from Cy3 being the analytical signal. The quantification of target concentration was achieved by measurement of the spatial length of coverage by target along a channel. Detection of femtomole quantities of target was possible with a dynamic range spanning an order of magnitude. The assay provided excellent resistance to nonspecific interactions of DNA. Further selectivity of the assay was achieved using 20% formamide, which allowed discrimination between a fully complementary target and a 3 base pair mismatch target at a contrast ratio of 4:1.

Biosensing with quantum dots: a microfluidic approach.

Semiconductor quantum dots (QDs) have served as the basis for signal development in a variety of biosensing technologies and in applications using bioprobes. The use of QDs as physical platforms to develop biosensors and bioprobes has attracted considerable interest. This is largely due to the unique optical properties of QDs that make them excellent choices as donors in fluorescence resonance energy transfer (FRET) and well suited for optical multiplexing. The large majority of QD-based bioprobe and biosensing technologies that have been described operate in bulk solution environments, where selective binding events at the surface of QDs are often associated with relatively long periods to reach a steady-state signal. An alternative approach to the design of biosensor architectures may be provided by a microfluidic system (MFS). A MFS is able to integrate chemical and biological processes into a single platform and allows for manipulation of flow conditions to achieve, by sample transport and mixing, reaction rates that are not entirely diffusion controlled. Integrating assays in a MFS provides numerous additional advantages, which include the use of very small amounts of reagents and samples, possible sample processing before detection, ultra-high sensitivity, high throughput, short analysis time, and in situ monitoring. Herein, a comprehensive review is provided that addresses the key concepts and applications of QD-based microfluidic biosensors with an added emphasis on how this combination of technologies provides for innovations in bioassay designs. Examples from the literature are used to highlight the many advantages of biosensing in a MFS and illustrate the versatility that such a platform offers in the design strategy.

Toward a solid-phase nucleic acid hybridization assay within microfluidic channels using immobilized quantum dots as donors in fluorescence resonance energy transfer

AbstractThe optical properties and surface area of quantum dots (QDs) have made them an attractive platform for the development of nucleic acid biosensors based on fluorescence resonance energy transfer (FRET). Solid-phase assays based on FRET using mixtures of immobilized QD–oligonucleotide conjugates (QD biosensors) have been developed. The typical challenges associated with solid-phase detection strategies include non-specific adsorption, slow kinetics of hybridization, and sample manipulation. The new work herein has considered the immobilization of QD biosensors onto the surfaces of microfluidic channels in order to address these challenges. Microfluidic flow can be used to dynamically control stringency by adjustment of the potential in an electrokinetic-based microfluidics environment. The shearing force, Joule heating, and the competition between electroosmotic and electrophoretic mobilities allow the optimization of hybridization conditions, convective delivery of target to the channel surface to speed hybridization, amelioration of adsorption, and regeneration of the sensing surface. Microfluidic flow can also be used to deliver (for immobilization) and remove QD biosensors. QDs that were conjugated with two different oligonucleotide sequences were used to demonstrate feasibility. One oligonucleotide sequence on the QD was available as a linker for immobilization via hybridization with complementary oligonucleotides located on a glass surface within a microfluidic channel. A second oligonucleotide sequence on the QD served as a probe to transduce hybridization with target nucleic acid in a sample solution. A Cy3 label on the target was excited by FRET using green-emitting CdSe/ZnS QD donors and provided an analytical signal to explore this detection strategy. The immobilized QDs could be removed under denaturing conditions by disrupting the duplex that was used as the surface linker and thus allowed a new layer of QD biosensors to be re-coated within the channel for re-use of the microfluidic chip. FigureSchematic view of detection of nucleic acid hybridization within microfluidic channels using immobilized quantum dots as donors in fluorescence resonance energy transfer

On-chip multiplexed solid-phase nucleic acid hybridization assay using spatial profiles of immobilized quantum dots and fluorescence resonance energy transfer.

A microfluidic based solid-phase assay for the multiplexed detection of nucleic acid hybridization using quantum dot (QD) mediated fluorescence resonance energy transfer (FRET) is described herein. The glass surface of hybrid glass-polydimethylsiloxane (PDMS) microfluidic channels was chemically modified to assemble the biorecognition interface. Multiplexing was demonstrated using a detection system that was comprised of two colors of immobilized semi-conductor QDs and two different oligonucleotide probe sequences. Green-emitting and red-emitting QDs were paired with Cy3 and Alexa Fluor 647 (A647) labeled oligonucleotides, respectively. The QDs served as energy donors for the transduction of dye labeled oligonucleotide targets. The in-channel assembly of the biorecognition interface and the subsequent introduction of oligonucleotide targets was accomplished within minutes using a combination of electroosmotic flow and electrophoretic force. The concurrent quantification of femtomole quantities of two target sequences was possible by measuring the spatial coverage of FRET sensitized emission along the length of the channel. In previous reports, multiplexed QD-FRET hybridization assays that employed a ratiometric method for quantification had challenges associated with lower analytical sensitivity arising from both donor and acceptor dilution that resulted in reduced energy transfer pathways as compared to single-color hybridization assays. Herein, a spatial method for quantification that is based on in-channel QD-FRET profiles provided higher analytical sensitivity in the multiplexed assay format as compared to single-color hybridization assays. The selectivity of the multiplexed hybridization assays was demonstrated by discrimination between a fully-complementary sequence and a 3 base pair sequence at a contrast ratio of 8 to 1.

Effect of removing Kupffer cells on nanoparticle tumor delivery

Significance Nanomaterials are developed for treating and diagnosing cancer, but only 0.7% (median) are delivered to a solid tumor. To address this delivery problem, we are examining each biological barrier to determine its impact on tumor delivery. Because the liver sequesters up to 70% of nanomaterials, in this study, we asked, if liver Kupffer cells were removed, what is the impact on tumor delivery? While we demonstrate that the tumor delivery increased up to 150 times, we achieved 2% for nanomaterials of different size, material, and tumor type. This suggests the need to focus on tumor pathophysiology to increase delivery efficiency, since this approach led to a greater availability of nanoparticles in the blood, but 98% did not accumulate in solid tumors. A recent metaanalysis shows that 0.7% of nanoparticles are delivered to solid tumors. This low delivery efficiency has major implications in the translation of cancer nanomedicines, as most of the nanomedicines are sequestered by nontumor cells. To improve the delivery efficiency, there is a need to investigate the quantitative contribution of each organ in blocking the transport of nanoparticles to solid tumors. Here, we hypothesize that the removal of the liver macrophages, cells that have been reported to take up the largest amount of circulating nanoparticles, would lead to a significant increase in the nanoparticle delivery efficiency to solid tumors. We were surprised to discover that the maximum achievable delivery efficiency was only 2%. In our analysis, there was a clear correlation between particle design, chemical composition, macrophage depletion, tumor pathophysiology, and tumor delivery efficiency. In many cases, we observed an 18–150 times greater delivery efficiency, but we were not able to achieve a delivery efficiency higher than 2%. The results suggest the need to look deeper at other organs such as the spleen, lymph nodes, and tumor in mediating the delivery process. Systematically mapping the contribution of each organ quantitatively will allow us to pinpoint the cause of the low tumor delivery efficiency. This, in effect, enables the generation of a rational strategy to improve the delivery efficiency of nanoparticles to solid tumors either through the engineering of multifunctional nanosystems or through manipulation of biological barriers.

Toward an on-chip multiplexed nucleic acid hybridization assay using immobilized quantum dot-oligonucleotide conjugates and fluorescence resonance energy transfer

Semiconductor quantum dots (QD) are a class of NP with photophysical properties that are ideally suited for optical multiplexing and use as donors in fluorescence resonance energy transfer (FRET). A new strategy is presented for the development of multiplexed DNA hybridization assays using immobilized QDs in a microfluidic system. Green- or red-emitting QDs were immobilized via self-assembly with a multidentate-thiol-derivatized glass slide, and subsequently conjugated with amine-terminated probe oligonucleotides using carbodiimide activation. Immobilized QD-probe conjugates were then passivated with adsorbed non-complementary oligonucleotides to achieve selectivity in microfluidic assays. Target nucleic acid sequences hybridized with QD-probe conjugates and were labeled with Cy3 or Alexa Fluor 647 as acceptor dyes for the QD donors, where FRET-sensitized dye emission provided a signal for the detection of picomolar quantities of target. The simultaneous immobilization of green- and red-emitting QDs at different ratios within a microfluidic channel was demonstrated as a step toward multiplexed assays.

Toward a hybridization assay using fluorescence resonance energy transfer and quantum dots immobilized in microfluidic channels

Quantum dots (QDs) have been widely adopted as integrated components of bioassays and biosensors. In particular, solid phase nucleic acid hybridization assays have been demonstrated to have several advantages and permit the detection of up to four DNA targets simultaneously using fluorescence resonance energy transfer (FRET). This work explores the potential for miniaturization of a solid-phase nucleic acid hybridization assay using QDs and FRET on a microfluidics platform. A method was developed for the immobilization of Streptavidin coated QDs and the preparation of QD-probe oligonucleotide conjugates within microfluidic channels using electrokinetic delivery. Proof-of-concept was demonstrated for the selective detection of target DNA using FRET-sensitized emission from a Cy3 acceptor paired with a green emitting QD donor. The microfluidic platform offered the advantages of smaller sample volumes, nearly undetectable non-specific adsorption, and hybridization within minutes. This work is an important first step toward the development of biochips that enable the multiplexed detection of nucleic acid targets.