A droplet-based microfluidic platform for rapid immobilization of quantum dots on individual magnetic microbeads

This is a post-peer-review, pre-copyedit version of an article published in Microfluidics and Nanufluidics. The final authenticated version is available online at: https://doi.org/10.1007/s10404-018-2085-xQuantum dots (QDs) provide opportunities for the development of bioassays, biosensors, and drug delivery strategies. Decoration of the surface of QDs offers unique functions such as resistance to non-specific adsorption, selective binding to target molecules, and cellular uptake. The quality of decoration has substantial impact on the functionality of modified QDs. Single-phase microfluidic devices have been demonstrated for decorating QDs with biological molecules. The device substrate can serve as a solid-phase reaction platform, with a limitation being difficulty in the realization of reproducible decoration at high density of coverage of QDs. Magnetic beads (MBs) have been explored as an alternative form of solid-phase reaction platform for decorating QDs. As one example, controlled decoration to achieve unusually high density can be realized by first coating MBs with QDs, followed by the addition of molecules such as DNA oligonucleotides. Uniformity and high density of coatings on QDs have been obtained using MBs for solid-phase reactions in bulk solution, with the further advantage that the MBs offer simplification of procedural steps such as purification. This study explores the use of a droplet microfluidic platform to achieve solid-phase decoration of MBs with QDs, offering control of local reaction conditions beyond that available in bulk solution reactions. A microchannel network with a two-junction in-series configuration was designed and optimized to co-encapsulate one single 1 A mu m MB and many QDs into individual droplets. The microdroplet became the reaction vessel, and enhanced conjugation through the confined environment and fast mixing. A high density of QDs was coated onto the surface of single MB even when using a low concentration of QDs. This approach quickly produced decorated MBs, and significantly reduced QD waste, ameliorating the need to remove excess QDs. The methodology offers a degree of precision to control conjugation processes that cannot be attained in bulk synthesis methods. The proposed droplet microfluidic design can be widely adopted for nanomaterial synthesis using solid-phase assays.Natural Sciences and Engineering Research Council of Canada: STPGP 479222-2015Canada Research Chair

High Efficacy and Drug Synergy of HDAC6-Selective Inhibitor NN-429 in Natural Killer (NK)/T-Cell Lymphoma

NK/T-cell lymphoma (NKTCL) and γδ T-cell non-Hodgkin lymphomas (γδ T-NHL) are highly aggressive lymphomas that lack rationally designed therapies and rely on repurposed chemotherapeutics from other hematological cancers. Histone deacetylases (HDACs) have been targeted in a range of malignancies, including T-cell lymphomas. This study represents exploratory findings of HDAC6 inhibition in NKTCL and γδ T-NHL through a second-generation inhibitor NN-429. With nanomolar in vitro HDAC6 potency and high in vitro and in cellulo selectivity for HDAC6, NN-429 also exhibited long residence time and improved pharmacokinetic properties in contrast to older generation inhibitors. Following unique selective cytotoxicity towards γδ T-NHL and NKTCL, NN-429 demonstrated a synergistic relationship with the clinical agent etoposide and potential synergies with doxorubicin, cytarabine, and SNS-032 in these disease models, opening an avenue for combination treatment strategies

Development of HDAC Inhibitors Exhibiting Therapeutic Potential in T-Cell Prolymphocytic Leukemia

Epigenetic targeting has emerged as an efficacious therapy for hematological cancers. The rare and incurable T-cell prolymphocytic leukemia (T-PLL) is known for its aggressive clinical course. Current epigenetic agents such as histone deacetylase (HDAC) inhibitors are increasingly used for targeted therapy. Through a structure-activity relationship (SAR) study, we developed an HDAC6 inhibitor KT-531, which exhibited higher potency in T-PLL compared to other hematological cancers. KT-531 displayed strong HDAC6 inhibitory potency and selectivity, on-target biological activity, and a safe therapeutic window in nontransformed cell lines. In primary T-PLL patient cells, where HDAC6 was found to be overexpressed, KT-531 exhibited strong biological responses, and safety in healthy donor samples. Notably, combination studies in T-PLL patient samples demonstrated KT-531 synergizes with approved cancer drugs, bendamustine, idasanutlin, and venetoclax. Our work suggests HDAC inhibition in T-PLL could afford sufficient therapeutic windows to achieve durable remission either as standalone or in combination with targeted drugs.Peer reviewe

Gold Nanoparticle-Enhanced Detection of Single Nucleotide Polymorphisms in the NanoBioArray Chip

In this thesis, we report the use of gold nanoparticles (AuNPs) to enhance the detection of single nucleotide polymorphism (SNPs) in the NanoBioArray (NBA) chip. A combination of gold nanoparticles (AuNPs) and nucleic acids has recently been used in many biosensing applications. However, there is a poor fundamental understanding of how gold nanoparticle surfaces influence the DNA hybridization process. Our kinetic analysis shows that in the presence of AuNP-ssDNA interactions, mechanisms of DNA hybridization and dehybridization are altered. Our proposed mechanisms include a shift of the rate-limiting step of hybridization from mismatch-insensitive to the mismatch-sensitive zipping step. Furthermore, the binding of gold nanoparticles to the single-stranded DNA segments (commonly known as bubbles) in the mismatched (MM) duplex DNAs, destabilize the duplexes and accelerates the dehybridization process. We employ these alterations in mechanisms, both of which disfavor the formation of MM duplexes, to enhance the detection of SNPs in the NBA chip. In this technique, we load the target DNAs on the surface of AuNPs (i.e. AuNP targets) and then introduce them to the surface-immobilized probes for DNA hybridization. Our results show that AuNP targets, in contrast to the targets free in the solution (free targets), were able to discriminate between the perfectly matched (PM) probes and the mismatched (MM) ones. Using AuNP targets, we developed a room-temperature method for detection of SNPs in the KRAS gene codon 12 in the NBA chip. Then, a novel wash method based on AuNPs was developed to preserve the DNA hybridization signals in CD-NBA chip while discriminating MM duplexes from PM duplexes. In this method, AuNPs are suspended in the wash buffers to preferentially destabilize the MM duplexes, in presence of the PM duplexes. Enjoying this targeted mechanism, AuNP wash method enhances specificity without compromising signal intensity. This method is simple and compatible with multiplexed DNA hybridization settings. The findings in this thesis can be used to enhance the reliability of DNA biosensors (e.g. DNA microarrays) and might lead to new applications in DNA biosensing

Enhanced Immunoassay Using a Rotating Paper Platform for Quantitative Determination of Low Abundance Protein Biomarkers

The changing concentrations of circulating protein biomarkers have been correlated with a variety of diseases. Quantitative bioassays capable of sensitive and specific determination of protein biomarkers at low levels can be essential for therapeutic treatments that can improve outcomes for patients. Herein, we describe the investigation of a rotating paper device (RPD) for quantitative determination of targeted proteins at the fM concentration level. The RPD consists of two circular papers each separately supported with a plastic disc. Protein detection is conducted via enhanced immunoassay using amplification in a sequential workflow, which includes a sandwich immunoassay in the upper paper and a signal amplification reaction in the lower paper. The sandwich immunoassay is conducted using biobarcode nanoparticles (BNPs) and results in the release of reporter oligonucleotides from BNPs. These oligonucleotides are transferred to the bottom paper, where they engage in a target recycling methodology that leads to the production of a colorimetric signal. The assay was evaluated for quantitation of interleukin-6 (IL-6), a cytokine biomarker in serum. A limit of detection of 63 fM and a dynamic range of 200 fM-8 pM was observed for the assay. The specificity of the assay was successfully verified against several common protein biomarkers.Natural Sciences and Engineering Research Council of Canad

Rapid Immobilization of Oligonucleotides at High Density on Semiconductor Quantum Dots and Gold Nanoparticles

Oligonucleotide-coated nanoparticles (NPs) have been used in numerous applications such as bioassays, as intracellular probes and for drug delivery. One challenge that is confronted in the preparation of oligonucleotide-NP conjugates derives from surface charge, as nanoparticles are often stabilized and made water soluble with a coating of negatively charged capping ligands. Therefore, an electrostatic repulsion is present when attempting to conjugate oligonucleotides. The result is that the conjugation can be a slow process, sometimes requiring 1-2 days to equilibrate at the highest surface density. The effect is compounded by electrostatic repulsion between neighboring oligonucleotide strands on the NP surfaces, which tends to lower the surface density. Herein, we report a novel method that enables conjugation in less than 1 min with a surface density of oligonucleotides up to the theoretical physical limit of occupancy. Negatively charged NPs are first adsorbed onto the surface of positively charged magnetic beads (MBs) to create MB-NP conjugates. Oligonucleotides are subsequently electrostatically adsorbed onto the MB surfaces when added to a suspension of MB-NP conjugates. This creates an oligonucleotide concentration 105 to 106 greater than in bulk solution in the vicinity of the nanoparticles, resulting in the promotion of the kinetics by over 1000 fold, and achieving the maximum density possible for the conjugation reaction.Natural Sciences and Engineering Research Council of Canad

Cancer biomarker determination by resonance energy transfer using functional fluorescent nanoprobes

ManuscriptThe development of bioanalytical methods that provide early detection of the presence of cancer by sensitive and specific determination of biomarkers such as small biomolecules, nucleic acids, proteins, enzymes, and even whole cells are essential to improve opportunity for improved patient treatment and to diminish the rate of cancer mortality. Förster resonance energy transfer (FRET) methods have been increasingly used to develop bioassays that offer speed, selectivity and low detection levels with practicality that is appropriate for providing point-of-care measurements for screening. The unique optical and photophysical properties of fluorescent nanoparticles such as semiconductor quantum dots (QDs), upconversion nanoparticles (UCNPs), graphene quantum dots (GQDs) and other materials have been reported to operate as efficient donors and/or acceptors for replacement of fluorescent organic dye molecules in various FRET-based assays. This review is focused on the recent progress that has been made in the development of nanoparticle-based FRET bioassays, and considers nanoparticle synthesis, design of optical properties, conjugation chemistry and approaches to fluorescence detection that provide for selective and sensitive quantification of cancer biomarkers.Natural Sciences and Engineering Research Council of Canad

Effect of buffer composition on PNA-RNA hybridization studied in the microfluidic microarray chip

Peptide nucleic acids (PNAs) are used as the probe species for detection of RNA. A microfluidic microarray (MMA) chip is used as a platform for detection of hybridizations between immobilized PNA probes and RNA targets. The RNA targets used are related to influenza A. This paper discusses the optimization of the two probe technologies used for RNA detection and investigates how the composition of the probe buffer or the content of the hybridization solution can influence the overall results. Our data shows that the PNA probe is a better choice over the DNA probe when there is low salt in the probe buffer composition. Furthermore, we have shown that the absence of salt (NaCl) in the hybridization buffer does not hinder the detection of RNAs. The results conclude that PNA probes are superior to DNA probes in term of sensitivity and adaptability, as PNA immobilization and PNA-RNA hybridization are less affected by salt content in the reaction buffers unlike DNA probes.The accepted manuscript in pdf format is listed with the files at the bottom of this page. The presentation of the authors' names and (or) special characters in the title of the manuscript may differ slightly between what is listed on this page and what is listed in the pdf file of the accepted manuscript; that in the pdf file of the accepted manuscript is what was submitted by the author

A Proposed Mechanism of the Influence of Gold Nanoparticles on DNA Hybridization

A combination of gold nanoparticles (AuNPs) and nucleic acids has been used in biosensing applications. However, there is a poor fundamental understanding of how gold nanoparticle surfaces influence the DNA hybridization process. Here, we measured the rate constants of the hybridization and dehybridization of DNA on gold nanoparticle surfaces to enable the determination of activation parameters using transition state theory. We show that the target bases need to be detached from the gold nanoparticle surfaces before zipping. This causes a shift of the rate-limiting step of hybridization to the mismatch-sensitive zipping step. Furthermore, our results propose that the binding of gold nanoparticles to the single-stranded DNA segments (commonly known as bubbles) in the duplex DNA stabilizes the bubbles and accelerates the dehybridization process. We employ the proposed mechanism of DNA hybridization/dehybridization to explain the ability of 5 nm diameter gold nanoparticles to help discriminate between single base-pair mismatched DNA molecules when performed in a NanoBioArray chip. The mechanistic insight into the DNA–gold nanoparticle hybridization/dehybridization process should lead to the development of new biosensors. A combination of gold nanoparticles (AuNPs) and nucleic acids has been used in biosensing applications. However, there is a poor fundamental understanding of how gold nanoparticle surfaces influence the DNA hybridization process. Here, we measured the rate constants of the hybridization and dehybridization of DNA on gold nanoparticle surfaces to enable the determination of activation parameters using transition state theory. We show that the target bases need to be detached from the gold nanoparticle surfaces before zipping. This causes a shift of the rate-limiting step of hybridization to the mismatch-sensitive zipping step. Furthermore, our results propose that the binding of gold nanoparticles to the single-stranded DNA segments (commonly known as bubbles) in the duplex DNA stabilizes the bubbles and accelerates the dehybridization process. We employ the proposed mechanism of DNA hybridization/dehybridization to explain the ability of 5 nm diameter gold nanoparticles to help discriminate between single base-pair mismatched DNA molecules when performed in a NanoBioArray chip. The mechanistic insight into the DNA–gold nanoparticle hybridization/dehybridization process should lead to the development of new biosensors

Inorganic Nanoparticles as Donors in Resonance Energy Transfer for Solid-Phase Bioassays and Biosensors

Bioassays for the rapid detection and quantification of specific nucleic acids, proteins and peptides are fundamental tools in many clinical settings. Traditional optical emission methods have focused on the use of molecular dyes as labels to track selective binding interactions, and as probes that are sensitive to environmental changes. Such dyes can offer good detection limits based on brightness, but typically have broad emission bands and suffer from time-dependent photobleaching. Inorganic nanoparticles such as quantum dots and upconversion nanoparticles are photo-stable over prolonged exposure to excitation radiation and tend to offer narrow emission bands, providing greater opportunity for multi-wavelength multiplexing. Importantly, in contrast to molecular dyes, nanoparticles offer substantial surface area and can serve as platforms to carry a large number of conjugated molecules. The surface chemistry of inorganic nanoparticles offers both challenges and opportunities for control of solubility and functionality for selective molecular interactions by assembly of coatings through coordination chemistry. This report reviews advances in the compositional design and methods of conjugation of inorganic quantum dots and upconversion nanoparticles, and the assembly of combinations of nanoparticles to achieve energy exchange. The interest is exploration of configurations where the modified nanoparticles can be immobilized to solid substrates for the development of bioassays and biosensors that operate by resonance energy transfer (RET).This work was sponsored by the Natural Sciences and Engineering Council of Canad