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ABSTRACT: Electrochemical deposition of metals represents an important approach in the bottom-up fabrication of nanostructures and microstructures. We have used this approach to generate high-performance chip-based biosensors using silicon as a platform for the generation of sensor arrays. Here, we explore the applicability of different materials to support the electrodeposition and identify the parameters that are essential for robust sensor growth. We show that inexpensive materials can be used as templates for electrodeposition, and demonstrate that these low-cost sensors exhibit clinically-relevant levels of sensitivity and specificity. In particular, we prove herein that the glass-based sensors successfully detect E. coli in urine, when present at the 100 cfu μL-1 levels found typically in samples of patients with urinary tract infections.
Lab on a Chip 03/2013; · 5.67 Impact Factor
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Rida Mourtada,
Sonali B Fonseca,
Simon P Wisnovsky,
Mark P Pereira,
Xiaoming Wang,
Rose Hurren,
Jeremy Parfitt,
Lesley Larsen,
Robin A J Smith,
Michael P Murphy,
Aaron D Schimmer, Shana O Kelley
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ABSTRACT: We have successfully delivered a reactive alkylating agent, chlorambucil (Cbl), to the mitochondria of mammalian cells. Here, we characterize the mechanism of cell death for mitochondria-targeted chlorambucil (mt-Cbl) in vitro and assess its efficacy in a xenograft mouse model of leukemia. Using a ρ° cell model, we show that mt-Cbl toxicity is not dependent on mitochondrial DNA damage. We also illustrate that re-targeting Cbl to mitochondria results in a shift in the cell death mechanism from apoptosis to necrosis, and that this behavior is a general feature of mitochondria-targeted Cbl. Despite the change in cell death mechanisms, we show that mt-Cbl is still effective in vivo and has an improved pharmacokinetic profile compared to the parent drug. These findings illustrate that mitochondrial rerouting changes the site of action of Cbl and also alters the cell death mechanism drastically without compromising in vivo efficacy. Thus, mitochondrial delivery allows the exploitation of Cbl as a promiscuous mitochondrial protein inhibitor with promising therapeutic potential.
PLoS ONE 01/2013; 8(4):e60253. · 4.09 Impact Factor
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ABSTRACT: Nucleic acid molecules can serve as robust ligands for aqueous synthesis of semiconductor nanocrystals or quantum dots (QDs). QD properties including size, morphology, dispersity, emission maximum, and quantum yield are highly dependent on the sequences and structures of nucleic acids used for the synthesis. This synthetic strategy provides a novel facile means of constructing compact, stable, and biofunctionalized QDs in one step, which is of particular interest for a variety of applications such as biosensing, bioimaging, and self-assembly. This article summarizes recent advances in nucleic acid-templated QD synthesis with an emphasis on the nucleic acids-based programing of quantum dots properties. A variety of applications based on DNA-passivated QDs are also discussed. WIREs Nanomed Nanobiotechnol 2012 doi: 10.1002/wnan.1191 For further resources related to this article, please visit the WIREs website.
Wiley Interdisciplinary Reviews Nanomedicine and Nanobiotechnology 09/2012; · 5.19 Impact Factor
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ABSTRACT: Nanomaterial-based biosensing strategies offer a number of advantages over traditional molecular diagnostic and cellular analysis techniques, including signal amplification, improved sensitivity and speed, and versatile sensing schemes that can be tailored to a desired target. In this article, we highlight a variety of nanomaterial-based sensors, and discuss the advantages of different nanomaterials compositions and probes of different biomolecular classes. Recent advances in the development of optical, electrical, or electrochemical transduction mechanisms are covered, with special regard to breakthroughs in sensitivity. The works reviewed herein emphasize the improvements that nanomaterials offer in the realm of diagnostic assays and make a solid case for further advancement with automation and multiplexing.
Current opinion in chemical biology 07/2012; 16(3-4):415-21. · 8.30 Impact Factor
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ABSTRACT: Diagnostic technologies that can provide the simultaneous detection of nucleic acids for gene expression, proteins for host response and small molecules for profiling the human metabolome will have a significant advantage in providing comprehensive patient monitoring. Molecular sensors that report changes in the electrostatics of a sensor's surface on analyte binding have shown unprecedented sensitivity in the detection of charged biomolecules, but do not lend themselves to the detection of small molecules, which do not carry significant charge. Here, we introduce the neutralizer displacement assay that allows charge-based sensing to be applied to any class of molecule irrespective of the analyte charge. The neutralizer displacement assay starts with an aptamer probe bound to a neutralizer. When analyte binding occurs the neutralizer is displaced, which results in a dramatic change in the surface charge for all types of analytes. We have tested the sensitivity, speed and specificity of this system in the detection of a panel of molecules: (deoxy)ribonucleic acid, ribonucleic acid, cocaine, adenosine triphosphate and thrombin.
Nature Chemistry 06/2012; · 20.52 Impact Factor
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ABSTRACT: Mitochondrially targeted agents have the capacity to be both vehicles for the delivery of bioactive agents and mitochondrial disrupters and show promise for the treatment of various diseases. Engineering these agents to specifically accumulate or disrupt the mitochondrion is challenging, as there is a fine line between characteristics of the molecules that accomplish each task. Here, we assess the physicochemical properties governing mitochondrial matrix accumulation or membrane disruption caused by mitochondria-penetrating peptides. Increases in peptide length and hydrophobicity were uncovered as the dominant factors in deriving membrane disruptive activity. Shorter, less hydrophobic peptides did not disrupt the mitochondrial membrane, but rather accumulated in the mitochondrial matrix without interfering with cellular activity. These shorter peptides, however, can trigger cytochrome c release through activation of the permeability transition pore complex (PTPC), but only at very high concentrations. This study illustrates that the activity of a mitochondria-localizing agent can be controlled through alterations in peptide hydrophobicity and dosing concentrations.
ChemBioChem 02/2012; 13(3):476-85. · 3.94 Impact Factor
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ABSTRACT: Diagnostic technologies that can provide the simultaneous detection of nucleic acids for gene expression, proteins for host response and small molecules for profiling the human metabolome will have a significant advantage in providing comprehensive patient monitoring. Molecular sensors that report changes in the electrostatics of a sensor's surface on analyte binding have shown unprecedented sensitivity in the detection of charged biomolecules, but do not lend themselves to the detection of small molecules, which do not carry significant charge. Here, we introduce the neutralizer displacement assay that allows charge-based sensing to be applied to any class of molecule irrespective of the analyte charge. The neutralizer displacement assay starts with an aptamer probe bound to a neutralizer. When analyte binding occurs the neutralizer is displaced, which results in a dramatic change in the surface charge for all types of analytes. We have tested the sensitivity, speed and specificity of this system in the detection of a panel of molecules: (deoxy)ribonucleic acid, ribonucleic acid, cocaine, adenosine triphosphate and thrombin.
Nature Chemistry 01/2012; 4(8):642-8. · 20.52 Impact Factor
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ABSTRACT: An important goal for improved diagnosis and management of infectious disease is the development of rapid and accurate technologies for the decentralized detection of bacterial pathogens. Most current clinical methods that identify bacterial strains require time-consuming culture of the sample or procedures involving the polymerase chain reaction. Neither of these approaches has enabled testing at the point-of-need because of the requirement for skilled technicians and laboratory facilities. Here, we demonstrate the performance of an effective, integrated platform for the rapid detection of bacteria that combines a universal bacterial lysis approach and a sensitive nanostructured electrochemical biosensor. The lysis is rapid, is effective at releasing intercellular RNA from bacterial samples, and can be performed in a simple, cost-effective device integrated with an analysis chip. The platform was directly challenged with these unpurified lysates in buffer and urine. We successfully detected the presence of bacteria with high sensitivity and specificity and achieved a sample-to-answer turnaround time of 30 min. We have met the clinically relevant detection limit of 1 cfu/μL, indicating that uncultured samples can be analyzed. This advance will greatly reduce time to successful detection from days to minutes.
Analytical Chemistry 12/2011; 84(1):21-5. · 5.86 Impact Factor
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Chemistry 09/2011; 17(44):12257-61. · 5.93 Impact Factor
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ABSTRACT: To create a new class of mitochondria-penetrating peptides (MPPs) that would facilitate drug delivery into the organelle through the inclusion of delocalized lipophilic cations (DLCs) in the peptide sequence.
We synthesized two novel amino acids featuring DLCs and incorporated them into peptides. Systematic studies were conducted to compare peptides containing these residues to those with natural cationic amino acids. Diastereomers were compared to determine the most advantageous arrangement for these peptides. Peptide lipophilicity, cellular uptake and mitochondrial specificity were compared for a variety of peptides.
Synthetic DLC residues were found to increase mitochondrial localization of MPPs due to higher overall hydrophobicity. MPP stereochemistry was important for cellular uptake rather than subcellular localization. This study reaffirmed the importance of uniform overall charge distribution for mitochondrial specificity.
DLCs can be incorporated into synthetic peptides and facilitate mitochondrial drug delivery. Lipophilicity and charge distribution must be carefully balanced to ensure localization within mitochondria.
Pharmaceutical Research 08/2011; 28(11):2808-19. · 4.09 Impact Factor
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ABSTRACT: The difficulty of accessing the mitochondrial matrix has limited the targeting of therapeutics to this organelle. Here, we report, to our knowledge, the first successful delivery of an active DNA alkylating agent--chlorambucil--to mitochondria, and describe unexpected features that result from rerouting this drug within the cell. Mitochondrial targeting of this agent dramatically potentiates its activity, and promotes apoptotic cell death in a variety of cancer cell lines and patient samples. This retention of activity is observed even in cells with resistance to chlorambucil or disabled apoptotic triggering.
Chemistry & biology 04/2011; 18(4):445-53. · 6.52 Impact Factor
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Angewandte Chemie International Edition 04/2011; 50(18):4137-41. · 13.45 Impact Factor
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ABSTRACT: Detection of biomolecules at low abundances is crucial to the rapid diagnosis of disease. Impressive sensitivities, typically measured with small model analytes, have been obtained with a variety of nano- and microscale sensors. A remaining challenge, however, is the rapid detection of large native biomolecules in real biological samples. Here we develop and investigate a sensor system that directly addresses the source of this challenge: the slow diffusion of large biomolecules traveling through solution to fixed sensors, and inefficient complexation of target molecules with immobilized probes. We engineer arrayed sensors on two distinct length scales: a ∼100 μm length scale commensurable with the distance bacterial mRNA can travel in the 30 min sample-to-answer duration urgently required in point-of-need diagnostic applications; and the nanometer length scale we prove necessary for efficient target capture. We challenge the specificity of our hierarchical nanotextured microsensors using crude bacterial lysates and document the first electronic chip to sense trace levels of bacteria in under 30 min.
ACS Nano 03/2011; 5(4):3360-6. · 10.77 Impact Factor
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ABSTRACT: Multiplexed assays that can measure protein biomarkers and internal standards are highly desirable given the potential to reduce false positives and negatives. We report here the use of a chip-based platform that achieves multiplexed immunosensing of the ovarian cancer biomarker CA-125 without the need for covalent labeling or sandwich complexes. The sensor chips allow the straightforward comparison of detectors of different sizes, and we used this feature to scan the microscale size regime for the best sensor size and optimize the limit of detection exhibited down to 0.1 U/mL. The assay has a straightforward design, with readout being performed in a single step involving the introduction of a noncovalently attached redox reporter group. The detection system reported exhibits excellent specificity, with analysis of a specific cancer biomarker, CA-125, performed in human serum and whole blood. The multiplexing of the system allows the analysis of the biomarker to be performed in parallel with an abundant serum protein for internal calibration.
Analytical Chemistry 02/2011; 83(4):1167-72. · 5.86 Impact Factor
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ABSTRACT: The number of antimicrobial agents available for use in humans is limited by the difficulty of discovering chemical agents with selective toxicity to bacterial targets. Numerous small molecule inhibitors have potential as antimicrobial agents, yet their use has been prevented by high levels of toxic cross-reactivity in human cells. For example, methotrexate (Mtx) is an effective antimetabolite that exerts its effects by inhibiting DHFR. It is a potent antibacterial when accumulated intracellularly, but toxicity in human cells limits clinical utility in infectious disease treatment. Here, we describe peptide conjugates of Mtx that are sequestered into the mitochondria of human cells (mt-Mtx). This alteration in localization of Mtx, which directs it away from its enzyme target, decreases its toxicity in human cells by a factor of 10(3). Mt-Mtx, however, maintains activity against a variety of pathogenic gram-positive organisms, including methicillin-resistant Staphylococcus aureus (MRSA). The results from this proof-of-principle study describe a novel methodology for augmenting the antibacterial efficacy of drugs amenable to peptide conjugation while simultaneously decreasing their toxicity to the host organism.
Journal of the American Chemical Society 02/2011; 133(10):3260-3. · 9.91 Impact Factor
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ABSTRACT: The electronic and optical properties of colloidal quantum dots, including the wavelengths of light that they can absorb and emit, depend on the size of the quantum dots. These properties have been exploited in a number of applications including optical detection, solar energy harvesting and biological research. Here, we report the self-assembly of quantum dot complexes using cadmium telluride nanocrystals capped with specific sequences of DNA. Quantum dots with between one and five DNA-based binding sites are synthesized and then used as building blocks to create a variety of rationally designed assemblies, including cross-shaped complexes containing three different types of dots. The structure of the complexes is confirmed with transmission electron microscopy, and photophysical studies are used to quantify energy transfer among the constituent components. Through changes in pH, the conformation of the complexes can also be reversibly switched, turning on and off the transfer of energy between the constituent quantum dots.
Nature Nanotechnology 01/2011; 6(8):485-90. · 27.27 Impact Factor
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Sumaiya Sharmeen,
Marko Skrtic,
Mahadeo A Sukhai,
Rose Hurren,
Marcela Gronda,
Xiaoming Wang,
Sonali B Fonseca,
Hong Sun,
Tabitha E Wood,
Richard Ward,
Mark D Minden,
Robert A Batey,
Alessandro Datti,
Jeff Wrana, Shana O Kelley,
Aaron D Schimmer
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ABSTRACT: To identify known drugs with previously unrecognized anticancer activity, we compiled and screened a library of such compounds to identify agents cytotoxic to leukemia cells. From these screens, we identified ivermectin, a derivative of avermectin B1 that is licensed for the treatment of the parasitic infections, strongyloidiasis and onchocerciasis, but is also effective against other worm infestations. As a potential antileukemic agent, ivermectin induced cell death at low micromolar concentrations in acute myeloid leukemia cell lines and primary patient samples preferentially over normal hematopoietic cells. Ivermectin also delayed tumor growth in 3 independent mouse models of leukemia at concentrations that appear pharmacologically achievable. As an antiparasitic, ivermectin binds and activates chloride ion channels in nematodes, so we tested the effects of ivermectin on chloride flux in leukemia cells. Ivermectin increased intracellular chloride ion concentrations and cell size in leukemia cells. Chloride influx was accompanied by plasma membrane hyperpolarization, but did not change mitochondrial membrane potential. Ivermectin also increased reactive oxygen species generation that was functionally important for ivermectin-induced cell death. Finally, ivermectin synergized with cytarabine and daunorubicin that also increase reactive oxygen species production. Thus, given its known toxicology and pharmacology, ivermectin could be rapidly advanced into clinical trial for leukemia.
Blood 11/2010; 116(18):3593-603. · 9.90 Impact Factor
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ABSTRACT: The detection of biologically important molecules such as proteins and nucleic acids is of high and growing interest in the diagnosis of diseases from cancer to infectious and cardiovascular disease. The use of nanostructures to enhance sensitivity in biomolecular detection has now been reported in a broad range of assays. Here we provide direct evidence that the display of nucleic acid probe molecules on a nanostructured surface dramatically enhances hybridization efficiency compared to the case of the same probe molecules tethered on a smoother surface. Another factor expected to influence hybridization is the density of the probe monolayer. Remarkably, we find herein that the effect of nanostructuring dominates over probe density: the benefits of a high degree of nanostructuring can more than overcome the influence of dense probe packing. The results obtained herein give guidance to the development of high-performance biosensors for medical and environmental applications.
Analytical Chemistry 07/2010; 82(14):5928-31. · 5.86 Impact Factor
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Journal of the American Chemical Society 06/2010; · 9.91 Impact Factor
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Nature Material 01/2010; · 32.84 Impact Factor
