Expanded Multiplexing on Sensor-Constrained Microfluidic Partitioning Systems.

Microfluidics can split samples into thousands or millions of partitions, such as droplets or nanowells. Partitions capture analytes according to a Poisson distribution, and in diagnostics, the analyte concentration is commonly inferred with a closed-form solution via maximum likelihood estimation (MLE). Here, we present a new scalable approach to multiplexing analytes. We generalize MLE with microfluidic partitioning and extend our previously developed Sparse Poisson Recovery (SPoRe) inference algorithm. We also present the first in vitro demonstration of SPoRe with droplet digital PCR (ddPCR) toward infection diagnostics. Digital PCR is intrinsically highly sensitive, and SPoRe helps expand its multiplexing capacity by circumventing its channel limitations. We broadly amplify bacteria with 16S ddPCR and assign barcodes to nine pathogen genera by using five nonspecific probes. Given our two-channel ddPCR system, we measured two probes at a time in multiple groups of droplets. Although individual droplets are ambiguous in their bacterial contents, we recover the concentrations of bacteria in the sample from the pooled data. We achieve stable quantification down to approximately 200 total copies of the 16S gene per sample, enabling a suite of clinical applications given a robust upstream microbial DNA extraction procedure. We develop a new theory that generalizes the application of this framework to many realistic sensing modalities, and we prove scaling rules for system design to achieve further expanded multiplexing. The core principles demonstrated here could impact many biosensing applications with microfluidic partitioning.

Extreme Compressed Sensing of Poisson Rates from Multiple Measurements.

Compressed sensing (CS) is a signal processing technique that enables the efficient recovery of a sparse high-dimensional signal from low-dimensional measurements. In the multiple measurement vector (MMV) framework, a set of signals with the same support must be recovered from their corresponding measurements. Here, we present the first exploration of the MMV problem where signals are independently drawn from a sparse, multivariate Poisson distribution. We are primarily motivated by a suite of biosensing applications of microfluidics where analytes (such as whole cells or biomarkers) are captured in small volume partitions according to a Poisson distribution. We recover the sparse parameter vector of Poisson rates through maximum likelihood estimation with our novel Sparse Poisson Recovery (SPoRe) algorithm. SPoRe uses batch stochastic gradient ascent enabled by Monte Carlo approximations of otherwise intractable gradients. By uniquely leveraging the Poisson structure, SPoRe substantially outperforms a comprehensive set of existing and custom baseline CS algorithms. Notably, SPoRe can exhibit high performance even with one-dimensional measurements and high noise levels. This resource efficiency is not only unprecedented in the field of CS but is also particularly potent for applications in microfluidics in which the number of resolvable measurements per partition is often severely limited. We prove the identifiability property of the Poisson model under such lax conditions, analytically develop insights into system performance, and confirm these insights in simulated experiments. Our findings encourage a new approach to biosensing and are generalizable to other applications featuring spatial and temporal Poisson signals.

Development of a Novel Class of Self-Assembling dsRNA Cancer Therapeutics: A Proof-of-Concept Investigation.

Cancer has proven to be an extremely difficult challenge to treat. Several fundamental issues currently underlie cancer treatment, including differentiating self from nonself, functional coupling of the recognition and therapeutic components of various therapies, and the propensity of cancerous cells to develop resistance to common treatment modalities via evolutionary pressure. Given these limitations, there is an increasing need to develop an all-encompassing therapeutic that can uniquely target malignant cells, decouple recognition from treatment, and overcome evolutionarily driven cancer resistance. We describe herein a new class of programmable self-assembling double-stranded RNA (dsRNA)-based cancer therapeutics that uniquely targets aberrant genetic sequences and in a functionally decoupled manner, undergoes oncogenic RNA-activated displacement (ORAD), initiating a therapeutic cascade that induces apoptosis and immune activation. As a proof of concept, we show that RNA strands targeting the EWS/Fli1 fusion gene in Ewing sarcoma cells that are end blocked with phosphorothioate bonds and additionally sealed with a 2'-deoxyuridine (2'-U)-modified DNA protector can be used to induce specific and potent killing of cells containing the target oncogenic sequence but not wild type.

An inexpensive, customizable microscopy system for the automated quantification and characterization of multiple adherent cell types.

Cell quantification assays are essential components of most biological and clinical labs. However, many currently available quantification assays, including flow cytometry and commercial cell counting systems, suffer from unique drawbacks that limit their overall efficacy. In order to address the shortcomings of traditional quantification assays, we have designed a robust, low-cost, automated microscopy-based cytometer that quantifies individual cells in a multiwell plate using tools readily available in most labs. Plating and subsequent quantification of various dilution series using the automated microscopy-based cytometer demonstrates the single-cell sensitivity, near-perfect R2 accuracy, and greater than 5-log dynamic range of our system. Further, the microscopy-based cytometer is capable of obtaining absolute counts of multiple cell types in one well as part of a co-culture setup. To demonstrate this ability, we recreated an experiment that assesses the tumoricidal properties of primed macrophages on co-cultured tumor cells as a proof-of-principle test. The results of the experiment reveal that primed macrophages display enhanced cytotoxicity toward tumor cells while simultaneously losing the ability to proliferate, an example of a dynamic interplay between two cell populations that our microscopy-based cytometer is successfully able to elucidate.

A mechanistic investigation exploring the differential transfection efficiencies between the easy-to-transfect SK-BR3 and difficult-to-transfect CT26 cell lines.

BACKGROUND: Gold-polyamidoamine (AuPAMAM) has previously been shown to successfully transfect cells with high efficiency. However, we have observed that certain cell types are more amenable to Au-PAMAM transfection than others. Here we utilized two representative cell lines-a "difficult to transfect" CT26 cell line and an "easy to transfect" SK-BR3 cell line-and attempted to determine the underlying mechanism for differential transfection in both cell types. Using a commonly established poly-cationic polymer similar to PAMAM (polyethyleneimine, or PEI), we additionally sought to quantify the relative transfection efficiencies of each vector in CT26 and SK-BR3 cells, in the hopes of elucidating any mechanistic differences that may exist between the two transfection vectors. RESULTS: A comparative time course analysis of green fluorescent protein reporter-gene expression and DNA uptake was conducted to quantitatively compare PEI- and AuPAMAM-mediated transfection in CT26 and SK-BR3, while flow cytometry and confocal microscopy were used to determine the contribution of cellular uptake, endosomal escape, and cytoplasmic transport to the overall gene delivery process. Results from the time course analysis and flow cytometry studies revealed that initial complex uptake and cytoplasmic trafficking to the nucleus are likely the two main factors limiting CT26 transfectability. CONCLUSIONS: The cell type-dependent uptake and intracellular transport mechanisms impacting gene therapy remain largely unexplored and present a major hurdle in the application-specific design and efficiency of gene delivery vectors. This systematic investigation offers insights into the intracellular mechanistic processes that may account for cell-to-cell differences, as well as vector-to-vector differences, in gene transfectability.

Universal microbial diagnostics using random DNA probes.

Early identification of pathogens is essential for limiting development of therapy-resistant pathogens and mitigating infectious disease outbreaks. Most bacterial detection schemes use target-specific probes to differentiate pathogen species, creating time and cost inefficiencies in identifying newly discovered organisms. We present a novel universal microbial diagnostics (UMD) platform to screen for microbial organisms in an infectious sample, using a small number of random DNA probes that are agnostic to the target DNA sequences. Our platform leverages the theory of sparse signal recovery (compressive sensing) to identify the composition of a microbial sample that potentially contains novel or mutant species. We validated the UMD platform in vitro using five random probes to recover 11 pathogenic bacteria. We further demonstrated in silico that UMD can be generalized to screen for common human pathogens in different taxonomy levels. UMD's unorthodox sensing approach opens the door to more efficient and universal molecular diagnostics.

Changes in Optical Properties of Plasmonic Nanoparticles in Cellular Environments are Modulated by Nanoparticle PEGylation and Serum Conditions.

When plasmonic nanoparticles (NPs) are internalized by cells and agglomerate within intracellular vesicles, their optical spectra can shift and broaden as a result of plasmonic coupling of NPs in close proximity to one another. For such optical changes to be accounted for in the design of plasmonic NPs for light-based biomedical applications, quantitative design relationships between designable factors and spectral shifts need to be established. Here we begin building such a framework by investigating how functionalization of gold NPs (AuNPs) with biocompatible poly(ethylene) glycol (PEG), and the serum conditions in which the NPs are introduced to cells impact the optical changes exhibited by NPs in a cellular context. Utilizing darkfield hyperspectral imaging, we find that PEGylation decreases the spectral shifting and spectral broadening experienced by 100 nm AuNPs following uptake by Sk-Br-3 cells, but up to a 33 ± 12 nm shift in the spectral peak wavelength can still occur. The serum protein-containing biological medium also modulates the spectral changes experienced by cell-exposed NPs through the formation of a protein corona on the surface of NPs that mediates NP interactions with cells: PEGylated AuNPs exposed to cells in serum-free conditions experience greater spectral shifts than in serum-containing environments. Moreover, increased concentrations of serum (10, 25, or 50 %) result in the formation of smaller intracellular NP clusters and correspondingly reduced spectral shifts after 5 and 10 h NP-cell exposure. However, after 24 h, NP cluster size and spectral shifts are comparable and become independent of serum concentration. By elucidating the impact of PEGylation and serum concentration on the spectral changes experienced by plasmonic NPs in cells, this study provides a foundation for the optical engineering of plasmonic NPs for use in biomedical environments.

Systematically probing the bottom-up synthesis of AuPAMAM conjugates for enhanced transfection efficiency.

BACKGROUND: Gold nanoparticles (AuNPs) have shown great promise as scaffolds for gene therapy vectors due to their attractive physiochemical properties which include biocompatibility, ease of functionalization via the nearly covalent gold-sulfur dative bond, and surface plasmon optical properties. Previously, we synthesized stable AuNP-polyamidoamine (AuPAMAM) conjugates and showed their success in vitro as non-viral gene delivery vectors. RESULTS: In this study, we systematically perturbed each component of the AuPAMAM conjugates and analyzed the resulting effect on transfection efficiency. Due to the modular, bottom-up nature of the AuPAMAM synthesis, we were able to probe each step of the fabrication process. The relationship between each conjugation parameter and the function of the final vector were investigated. More than fourfold enhanced transfection efficiency was achieved by modifying the PAMAM concentration, PAMAM core chemistry, PAMAM terminus chemistry, and self-assembled monolayer composition of the AuPAMAM conjugates. CONCLUSIONS: This work suggest that AuPAMAM synthesis platform is a promising non-viral gene therapy approach and highlights the importance of inspecting the role of each individual constituent in all nanotechnology hybrid materials.

Quantifying spectral changes experienced by plasmonic nanoparticles in a cellular environment to inform biomedical nanoparticle design.

Metal nanoparticles (NPs) scatter and absorb light in precise, designable ways, making them agile candidates for a variety of biomedical applications. When NPs are introduced to a physiological environment and interact with cells, their physicochemical properties can change as proteins adsorb on their surface and they agglomerate within intracellular endosomal vesicles. Since the plasmonic properties of metal NPs are dependent on their geometry and local environment, these physicochemical changes may alter the NPs' plasmonic properties, on which applications such as plasmonic photothermal therapy and photonic gene circuits are based. Here we systematically study and quantify how metal NPs' optical spectra change upon introduction to a cellular environment in which NPs agglomerate within endosomal vesicles. Using darkfield hyperspectral imaging, we measure changes in the peak wavelength, broadening, and distribution of 100-nm spherical gold NPs' optical spectra following introduction to human breast adenocarcinoma Sk-Br-3 cells as a function of NP exposure dose and time. On a cellular level, spectra shift up to 78.6 ± 23.5 nm after 24 h of NP exposure. Importantly, spectra broaden with time, achieving a spectral width of 105.9 ± 11.7 nm at 95% of the spectrum's maximum intensity after 24 h. On an individual intracellular NP cluster (NPC) level, spectra also show significant shifting, broadening, and heterogeneity after 24 h. Cellular transmission electron microscopy (TEM) and electromagnetic simulations of NPCs support the trends in spectral changes we measured. These quantitative data can help guide the design of metal NPs introduced to cellular environments in plasmonic NP-mediated biomedical technologies.

Synthesis of a quantum nanocrystal-gold nanoshell complex for near-infrared generated fluorescence and photothermal decay of luminescence.

Multifunction nanoparticle complexes have previously been developed to aid physicians in both diagnosis and treatment of cancerous tissue. Here, we designed a nanoparticle complex structure that consists of a plasmonically active hollow gold nanoshell core surrounded by photoluminescent quantum nanocrystals (QNs) in the form of PbS encapsulated by a silica layer. There are three main design variables including HGN synthesis and optical tuning, formation of the silica layer on the hollow gold nanoshell surface, and fabrication and photoluminescence tuning of PbS quantum nanocrystals. The hollow gold nanoshells were deliberately designed to function in the optical regimes that maximize tissue transmissivity (800 nm) and minimize tissue absorption (1100 nm). Secondly, several chemical ligands were tested such as (3-mercaptopropyl)trimethoxysilane and mercaptoundecanoic acid for controlled growth of the silica layer. Last, PbS QNs were synthesized and optimized with various capping agents, where the nanocrystals excited at the same wavelength were used to activate the photothermal properties of the hollow gold nanoshells. Upon irradiation of the complex with a lower power 800 nm laser, the nanocrystals luminesce at 1100 nm. At ablative temperatures the intrinsic luminescent properties of the QNs are altered and the luminescent output is significantly reduced (>70%). While this paper focuses on synthesis and optimization of the QN-HGN complex, in the future we believe that this novel particle complex design may have the potential to serve as a triple theranostic agent, which will aid satellite tumor localization, photothermal treatment, and ablative confirmation.

Variations in optical coherence tomography resolution and uniformity: a multi-system performance comparison.

Point spread function (PSF) phantoms based on unstructured distributions of sub-resolution particles in a transparent matrix have been demonstrated as a useful tool for evaluating resolution and its spatial variation across image volumes in optical coherence tomography (OCT) systems. Measurements based on PSF phantoms have the potential to become a standard test method for consistent, objective and quantitative inter-comparison of OCT system performance. Towards this end, we have evaluated three PSF phantoms and investigated their ability to compare the performance of four OCT systems. The phantoms are based on 260-nm-diameter gold nanoshells, 400-nm-diameter iron oxide particles and 1.5-micron-diameter silica particles. The OCT systems included spectral-domain and swept source systems in free-beam geometries as well as a time-domain system in both free-beam and fiberoptic probe geometries. Results indicated that iron oxide particles and gold nanoshells were most effective for measuring spatial variations in the magnitude and shape of PSFs across the image volume. The intensity of individual particles was also used to evaluate spatial variations in signal intensity uniformity. Significant system-to-system differences in resolution and signal intensity and their spatial variation were readily quantified. The phantoms proved useful for identification and characterization of irregularities such as astigmatism. Our multi-system results provide evidence of the practical utility of PSF-phantom-based test methods for quantitative inter-comparison of OCT system resolution and signal uniformity.

Encapsulated Fe3O4 /Ag complexed cores in hollow gold nanoshells for enhanced theranostic magnetic resonance imaging and photothermal therapy.

Designed and fabrication of a novel magnetic hollow gold nanoshell complexes that incorporates iron oxide nanoparticles in the hollow interior. The combined effect of the smaller IONPs improved the overall magnetic properties of the design and MRI contrast capability. The overall complex could be synthesized in the range of 60-80 nm in diameter while still having a plasmonic peak in the near infrared region.

Optimization of PAMAM-gold nanoparticle conjugation for gene therapy.

The development of efficient and biocompatible non-viral vectors for gene therapy remains a great challenge, and exploiting the properties of both nanoparticle carriers and cationic polymers is an attractive approach. In this work, we have developed gold nanoparticle (AuNP) polyamidoamine (PAMAM) conjugates for use as non-viral transfection agents. AuPAMAM conjugates were prepared by crosslinking PAMAM dendrimers to carboxylic-terminated AuNPs via EDC and sulfo-NHS chemistry. EDC and sulfo-NHS have been utilized widely and in numerous applications such as amino acid coupling; however, their use in the coupling of PAMAM dendrimers to AuNPs presents new challenges to form effective and stable constructs for delivery that have not yet been examined. Enhanced colloidal stability and DNA condensation ability was established by probing two critical synthetic parameters: the reaction rate of the PAMAM crosslinking step, and the amine to carboxyl ratio. Based on this work, increasing the amine to carboxyl ratio during conjugation of PAMAM onto AuNPs yielded the optimal vector with respect to colloidal stability and transfection efficiency in vitro. AuPAMAM conjugates present attractive candidates for non-viral gene delivery due to their commercial availability, ease of fabrication and scale-up, high yield, high transfection efficiency and low cytotoxicity.

Elimination of metastatic melanoma using gold nanoshell-enabled photothermal therapy and adoptive T cell transfer.

Ablative treatments such as photothermal therapy (PTT) are attractive anticancer strategies because they debulk accessible tumor sites while simultaneously priming antitumor immune responses. However, the immune response following thermal ablation is often insufficient to treat metastatic disease. Here we demonstrate that PTT induces the expression of proinflammatory cytokines and chemokines and promotes the maturation of dendritic cells within tumor-draining lymph nodes, thereby priming antitumor T cell responses. Unexpectedly, however, these immunomodulatory effects were not beneficial to overall antitumor immunity. We found that PTT promoted the infiltration of secondary tumor sites by CD11b(+)Ly-6G/C(+) myeloid-derived suppressor cells, consequently failing to slow the growth of poorly immunogenic B16-F10 tumors and enhancing the growth of distant lung metastases. To exploit the beneficial effects of PTT activity against local tumors and on antitumor immunity whilst avoiding the adverse consequences, we adoptively transferred gp100-specific pmel T cells following PTT. The combination of local control by PTT and systemic antitumor immune reactivity provided by adoptively transferred T cells prevented primary tumor recurrence post-ablation, inhibited tumor growth at distant sites, and abrogated the outgrowth of lung metastases. Hence, the combination of PTT and systemic immunotherapy prevented the adverse effects of PTT on metastatic tumor growth and optimized overall tumor control.

Interactions of liposome carriers with infectious fungal hyphae reveals the role of ß-glucans.

Relatively little is known about how liposomal formulations modulate drug delivery to fungal pathogens. We compared patterns of hyphal cell wall binding for empty rhodmine-labeled liposomes and the clinically available amphotericin B-containing liposomal formulation (AmBisome) in Aspergillus fumigatus and Candida albicans. Following 0.5 h of coincubation with A. fumigatus , empty liposomes concentrated primarily in fungal septae along at the surface of the cell wall, suggesting that liposome uptake is concentrated in areas of the cell wall where linear glucan is exposed on the cell surface, which was confirmed by aniline blue staining. Consistent with this hypothesis, pretreatment of liposomes with soluble linear glucan (laminarin) decreased liposome binding in both Aspergillus and Candida fungal hyphae, while growth of Aspergillus hyphae in the presence of an agent that increases fungal cell wall surface exposure of linear ß-glucans without cell death (caspofungin) increased liposome uptake throughout the Aspergillus fungal cell wall. Increasing the polyethylene glycol (PEG) concentration in liposomes from 0 to 30% significantly increased fungal uptake of liposomes that was only modestly attenuated when fungal cells were incubated in serum concentrations ranging from 10 to 100%. The presence of ß-glucans on the fungal hyphae cell walls of Aspergillus fumigatus is one of the factors responsible for mediating the binding of liposome carriers to the hyphae and could explain possible synergy reported between liposomal amphotericin B and echinocanins.

Characterizing the point spread function of retinal OCT devices with a model eye-based phantom.

We have designed, fabricated, and tested a nanoparticle-embedded phantom (NEP) incorporated into a model eye in order to characterize the point spread function (PSF) of retinal optical coherence tomography (OCT) devices in three dimensions under realistic imaging conditions. The NEP comprises a sparse distribution of highly backscattering silica-gold nanoshells embedded in a transparent UV-curing epoxy. The commercially-available model eye replicates the key optical structures and focusing power of the human eye. We imaged the model eye-NEP combination with a research-grade spectral domain OCT system designed for in vivo retinal imaging and quantified the lateral and axial PSF dimensions across the field of view in the OCT images. We also imaged the model eye-NEP in a clinical OCT system. Subtle features in the PSF and its dimensions were consistent with independent measurements of lateral and axial resolution. This model eye-based phantom can provide retinal OCT device developers and users a means to rapidly, objectively, and consistently assess the PSF, a fundamental imaging performance metric.

Tunable nanostructures as photothermal theranostic agents.

The theranostic potential of several nanostructures has been discussed in the context of photothermal therapies and imaging. In the last several decades, the burden of cancer has grown rapidly, making the need for new theranostic approaches vital. Lasers have emerged as promising tools in cancer treatment, especially with the advent of photothermal therapies wherein light absorbing dyes or plasmonic gold nanoparticles are used to generate heat and achieve tumor damage. Recently, photoabsorbing nanostructures have materialized that can be employed in conjunction with lasers in the near-infrared region in order to enhance both imaging and photothermal effects. The incorporation of tunable nanostructures has resulted in improved specificity in cancer treatment. Silica-cored gold nanoshells and gold nanorods currently serve as the chief plasmonic structures for photothermal therapy. Although gold nanorods and silica-cored gold nanoshells have shown promise as therapeutic agents, over the past few years new nanostructures have emerged that offer comparable and even superior theranostic properties. In the present review, several theranostic agents and their impact on the development of more effective photothermal therapies for the treatment of cancer are discussed. These agents include hollow gold nanoshells, gold gold-sulfide nanoparticles, gold nanocages, carbon and titanium nanotubes, photothermal-based nanobubbles, polymeric nanoparticles and copper-based nanocrystals.

Gold-silver alloy nanoshells: a new candidate for nanotherapeutics and diagnostics.

We have developed novel gold-silver alloy nanoshells as magnetic resonance imaging (MRI) dual T1 (positive) and T2 (negative) contrast agents as an alternative to typical gadolinium (Gd)-based contrast agents. Specifically, we have doped iron oxide nanoparticles with Gd ions and sequestered the ions within the core by coating the nanoparticles with an alloy of gold and silver. Thus, these nanoparticles are very innovative and have the potential to overcome toxicities related to renal clearance of contrast agents such as nephrogenic systemic fibrosis. The morphology of the attained nanoparticles was characterized by XRD which demonstrated the successful incorporation of Gd(III) ions into the structure of the magnetite, with no major alterations of the spinel structure, as well as the growth of the gold-silver alloy shells. This was supported by TEM, ICP-AES, and SEM/EDS data. The nanoshells showed a saturation magnetization of 38 emu/g because of the presence of Gd ions within the crystalline structure with r1 and r2 values of 0.0119 and 0.9229 mL mg-1 s-1, respectively (Au:Ag alloy = 1:1). T1- and T2-weighted images of the nanoshells showed that these agents can both increase the surrounding water proton signals in the T1-weighted image and reduce the signal in T2-weighted images. The as-synthesized nanoparticles exhibited strong absorption in the range of 600-800 nm, their optical properties being strongly dependent upon the thickness of the gold-silver alloy shell. Thus, these nanoshells have the potential to be utilized for tumor cell ablation because of their absorption as well as an imaging agent.

Rainbow Plasmonic Nanobubbles: Synergistic Activation of Gold Nanoparticle Clusters.

The synergistic physical and biological effects of selective targeting and activation of plasmonic nanoparticles were studied for a transient vapor nanobubble mode. Simultaneous optical activation of two plasmon resonances in multi-nanoparticle clusters significantly improved the selectivity and efficacy of the nanobubble generation through and was termed "rainbow plasmonic nanobubbles." The rainbow nanobubble mechanism has been studied in water and in living cells in vitro. This mechanism provided maximal selectivity of the nanobubble generation in both models and therefore, can the therapeutic selectivity and optical contrast of gold nanoparticles in a heterogeneous physiological microenvironment at cell level.