A M Klibanov

University of Missouri, Columbia, Missouri, United States

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Publications (277)1316.02 Total impact

  • Harris Liu, Igor Elkin, Jianzhu Chen, Alexander M Klibanov
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    ABSTRACT: A number of N-alkylated polyethylenimines (PEIs) were covalently attached to glass-slide surfaces, and their virucidal efficacies against three different strains of influenza viruses were examined quantitatively. The anti-influenza activities of the modified surfaces varied widely, with the most potent, immobilized N,N-hexyl,methyl-PEI and N,N-dodecyl,methyl-PEI, reducing the viral titer by over three logs (i.e., > 99.9%). While the virucidal activities of the glass surfaces derivatized with N-alkylated PEIs displayed no discernible correlation with such surface properties as hydrophobicity, charge, protein affinity, roughness, adhesive interactions, and polymer-chain extension lengths, they exhibited a marginal correlation with the surface density of quaternary ammonium group as titrated by means of fluorescein binding. However, this correlation markedly improved (to the correlation coefficient of 0.97 with a two-tailed p value of 0.044) when the titration was instead carried out using a macromolecular conjugate, the dye coupled to the protein lysozyme, suggesting that the critical determinant of the virucidal activity is the density of the immobilized quaternary ammonium groups accessible to influenza virions.
    Biomacromolecules 12/2014; · 5.37 Impact Factor
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    ABSTRACT: The currently used multistep chemical synthesis for making surfaces antimicrobial by attaching to them hydrophobic polycations is replaced herein by an aerosol-assisted plasma deposition procedure. To this end, N,N-hexyl,methyl-PEI (HMPEI) is directly plasma-coated onto a glass surface. The resultant immobilized HMPEI coating has been thoroughly characterized and shown to be robust, bactericidal against Escherichia coli, and virucidal against human influenza virus.
    Applied biochemistry and biotechnology 10/2013; · 1.94 Impact Factor
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    ABSTRACT: Previously, polymer-attached zanamivir had been found to inhibit influenza A viruses in vitro far better than did small-molecule zanamivir (1) itself. The aim of this study was to identify in vitro-using the plaque reduction assay-a highly potent 1-polymer conjugate, and subsequently test its antiviral efficacy in vivo. By examining the structure-activity relationship of 1-polymer conjugates in the plaque assay, we have determined that the most potent inhibitor against several representative influenza virus strains has a neutral high-molecular-weight backbone and a short alkyl linker. We have examined this optimal polymeric inhibitor for efficacy and immunogenicity in the mouse and ferret models of infection. 1 attached to poly-L-glutamine is an effective therapeutic for established influenza infection in ferrets, reducing viral titers up to 30-fold for 6 days. There is also up to a 190-fold reduction in viral load when the drug is used as a combined prophylactic/therapeutic in mice. Additionally, we see no evidence that the drug conjugate stimulates an immune response in mice upon repeat administration. 1 attached to a neutral high-molecular-weight backbone through a short alkyl linker drastically reduced both in vitro and in vivo titers compared to those observed with 1 itself. Thus, further development of this polymeric zanamivir for the mitigation of influenza infection seems warranted.
    Pharmaceutical Research 09/2013; · 4.74 Impact Factor
  • Alyssa M Larson, Jianzhu Chen, Alexander M Klibanov
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    ABSTRACT: By attaching multiple copies of the influenza M2 ion channel inhibitors amantadine (1) and rimantadine (2) to polymeric chains, we endeavored to recover their potency in inhibiting drug-resistant influenza viruses. Depending on loading densities, as well as the nature of the drug, the polymer, and the spacer arm, polymer-conjugated drugs were up to 30-fold more potent inhibitors of drug-resistant strains than their monomeric parents. In particular, a 20% loading density and a short linker group on the negatively charged poly-l-glutamate resulted in one of the most potent inhibitors for 2's conjugates against drug-resistant influenza strains. Although full recovery of the inhibitory action against drug-resistant strains was not achieved, this study may be a step toward salvaging anti-influenza drugs that are no longer effective. © 2013 Wiley Periodicals, Inc. and the American Pharmacists Association J Pharm Sci.
    Journal of Pharmaceutical Sciences 07/2013; · 3.13 Impact Factor
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    ABSTRACT: PURPOSE: The aim of this study was to markedly lower the viscosities of highly concentrated protein, in particular antibody, formulations. An effective approach elaborated herein for γ-globulin and a monoclonal antibody is to replace aqueous solutions with equimolar suspensions in neat organic solvents. METHODS: Viscosities of aqueous solutions and non-aqueous suspensions of the model protein bovine γ-globulin and a murine monoclonal antibody were examined under a variety of experimental conditions. In addition, protein particle sizes were measured using dynamic light scattering and light microscopy. RESULTS: Concentrated suspensions of amorphous γ-globulin powders (up to 300 mg/mL, composed of multi-micron-sized particles) in absolute ethanol and a number of other organic solvents were found to have viscosities up to 38 times lower than the corresponding aqueous solutions. Monoclonal antibody follows the same general trend. Additionally, the higher the protein concentration and lower the temperature, the greater the viscosity benefit of a suspension over a solution. CONCLUSIONS: The viscosities of concentrated γ-globulin and monoclonal antibody suspensions in organic solvents are drastically reduced compared to the corresponding aqueous solutions; the magnitude of this reduction depends on the solvent, particularly its hydrogen-bonding properties.
    Pharmaceutical Research 03/2013; · 4.74 Impact Factor
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    ABSTRACT: The infectivity of high-titer, cell-free HIV in culture media and human milk is rapidly reduced upon exposure to polyethylene slides painted with the linear hydrophobic polycation N,N-dodecyl,methyl-polyethylenimine (DMPEI). Accompanying viral p24 protein and free viral RNA analysis of solutions exposed to DMPEI-coated surfaces suggest that virion attachment to the polycationic surface and its subsequent inactivation are the likely mechanism of this phenomenon. Biotechnol. Bioeng. © 2013 Wiley Periodicals, Inc.
    Biotechnology and Bioengineering 02/2013; · 4.16 Impact Factor
  • Daewon Park, Alyssa M Larson, Alexander M Klibanov, Yadong Wang
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    ABSTRACT: We have prepared and characterized a new polyurethane-based antimicrobial material, N,N-dodecyl,methyl-polyurethane (Quat-12-PU). It exhibits strong antiviral and antibacterial activities when coated (as an organic solution or an aqueous nanosuspension) onto surfaces and antibacterial activity when electrospun into nanofibers. Quat-12-PU surfaces are able to kill airborne Gram-positive Staphylococcus aureus and Gram-negative Escherichia coli bacteria, as well as inactivate the enveloped influenza virus (but not the non-enveloped poliovirus).
    Applied biochemistry and biotechnology 01/2013; · 1.94 Impact Factor
  • Alyssa M Larson, Alexander M Klibanov
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    ABSTRACT: Many consumer goods must be protected from bacterial and fungal colonization to ensure their integrity and safety. By making these items' packaging biocidal, the interior environment can be preserved from microbial spoilage without altering the products themselves. Herein we briefly review this concept, referred to as active packaging, and discuss existing methods for constructing active packaging systems. They are based on either packaging materials that release biocides or those that are themselves intrinsically biocidal (or biostatic), with numerous variations within each category.
    Annual Review of Chemical and Biomolecular Engineering 01/2013; 4:171-86. · 7.51 Impact Factor
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    ABSTRACT: This study examined the effects of BMP7 gene transfer on corneal wound healing and fibrosis inhibition in vivo using a rabbit model. Corneal haze in rabbits was produced with the excimer laser performing -9 diopters photorefractive keratectomy. BMP7 gene was introduced into rabbit keratocytes by polyethylimine-conjugated gold nanoparticles (PEI2-GNPs) transfection solution single 5-minute topical application on the eye. Corneal haze and ocular health in live animals was gauged with stereo- and slit-lamp biomicroscopy. The levels of fibrosis [α-smooth muscle actin (αSMA), F-actin and fibronectin], immune reaction (CD11b and F4/80), keratocyte apoptosis (TUNEL), calcification (alizarin red, vonKossa and osteocalcin), and delivered-BMP7 gene expression in corneal tissues were quantified with immunofluorescence, western blotting and/or real-time PCR. Human corneal fibroblasts (HCF) and in vitro experiments were used to characterize the molecular mechanism mediating BMP7's anti-fibrosis effects. PEI2-GNPs showed substantial BMP7 gene delivery into rabbit keratocytes in vivo (2×10(4) gene copies/ug DNA). Localized BMP7 gene therapy showed a significant corneal haze decrease (1.68±0.31 compared to 3.2±0.43 in control corneas; p<0.05) in Fantes grading scale. Immunostaining and immunoblot analyses detected significantly reduced levels of αSMA (46±5% p<0.001) and fibronectin proteins (48±5% p<0.01). TUNEL, CD11b, and F4/80 assays revealed that BMP7 gene therapy is nonimmunogenic and nontoxic for the cornea. Furthermore, alizarin red, vonKossa and osteocalcin analyses revealed that localized PEI2-GNP-mediated BMP7 gene transfer in rabbit cornea does not cause calcification or osteoblast recruitment. Immunofluorescence of BMP7-transefected HCFs showed significantly increased pSmad-1/5/8 nuclear localization (>88%; p<0.0001), and immunoblotting of BMP7-transefected HCFs grown in the presence of TGFβ demonstrated significantly enhanced pSmad-1/5/8 (95%; p<0.001) and Smad6 (53%, p<0.001), and decreased αSMA (78%; p<0.001) protein levels. These results suggest that localized BMP7 gene delivery in rabbit cornea modulates wound healing and inhibits fibrosis in vivo by counter balancing TGFβ1-mediated profibrotic Smad signaling.
    PLoS ONE 01/2013; 8(6):e66434. · 3.53 Impact Factor
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    ABSTRACT: Covalently conjugating multiple copies of the drug zanamivir (ZA; the active ingredient in Relenza) via a flexible linker to poly-L-glutamine (PGN) enhances the anti-influenza virus activity by orders of magnitude. In this study, we investigated the mechanisms of this phenomenon. Like ZA itself, the PGN-attached drug (PGN-ZA) binds specifically to viral neuraminidase and inhibits both its enzymatic activity and the release of newly synthesized virions from infected cells. Unlike monomeric ZA, however, PGN-ZA also synergistically inhibits early stages of influenza virus infection, thus contributing to the markedly increased antiviral potency. This inhibition is not caused by a direct virucidal effect, aggregation of viruses, or inhibition of viral attachment to target cells and the subsequent endocytosis; rather, it is a result of interference with intracellular trafficking of the endocytosed viruses and the subsequent virus-endosome fusion. These findings both rationalize the great anti-influenza potency of PGN-ZA and reveal that attaching ZA to a polymeric chain confers a unique mechanism of antiviral action potentially useful for minimizing drug resistance.
    Proceedings of the National Academy of Sciences 11/2012; · 9.81 Impact Factor
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    ABSTRACT: The utility of plasmid DNA as an immunogen has been limited by its weak immunogenicity. In the present study, we evaluated the ability of a family of linear polyethylenimine (PEI) polymers, complexed to plasmid DNA, to augment DNA expression in vivo and to enhance antigen-specific adaptive immune responses. We showed that four of five structurally different PEIs that we evaluated increased in vivo DNA expression 20- to 400-fold, and enhanced DNA-induced epitope-specific CD8+ T-cell responses 10- to 25-fold in BALB/c and C57BL/6J mice respectively, when delivered intravenously. Functional studies of the PEI-DNA-induced CD8+ T-cell responses demonstrated that formulation of DNA with PEI was associated with increased numbers of cells secreting type I cytokines. In addition, PEI-DNA complexes improved antigen-specific T(H) 1-helper cell and humoral responses. Most importantly, the PEI-DNA complexes elicited memory cellular responses, capable of rapid expansion and accelerated clearance of a lethal dose of recombinant Listeria monocytogenes. Lastly, we identified physical properties of PEI-DNA complexes that are associated with enhanced DNA-elicited immunogenicity. These findings demonstrate that PEI polymers can play an important role in the development of DNA-based vaccines in the setting of infectious disease prevention and cancer therapy.
    European Journal of Immunology 08/2012; · 4.97 Impact Factor
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    Alyssa M Larson, Hyung Suk Oh, David M Knipe, Alexander M Klibanov
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    ABSTRACT: PURPOSE: To explore surface-immobilized and suspended modalities of the hydrophobic polycation N,N-dodecyl,methyl-polyethylenimine (DMPEI) for the ability to reduce viral infectivity in aqueous solutions containing herpes simplex viruses (HSVs) 1 and 2. METHODS: Surface-immobilized (coated onto surfaces) and suspended DMPEI were incubated with aqueous solutions containing HSV-1 or -2 to measure the antiviral effect of the hydrophobic polycation's formulations on HSVs. RESULTS: DMPEI coated on either polyethylene slides or male latex condoms dramatically decreases infectivity in solutions containing HSV-1 or -2. Moreover, DMPEI suspended in aqueous solution markedly reduces the infectious titer of these HSVs. CONCLUSION: Our results suggest potential uses of DMPEI for both prophylaxis (in the form of coated condoms) and treatment (as a topical suspension) for HSV infections.
    Pharmaceutical Research 07/2012; · 4.74 Impact Factor
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    ABSTRACT: Using the plaque reduction assay, relatively simple bicyclic quinone molecules, as well as multiple copies thereof covalently attached to a long polyglutamate-based polymeric chain, were examined as new inhibitors of various naturally occurring strains of influenza A virus. The polymer-conjugated inhibitors were found to have a far greater potency (for some as high as two orders of magnitude when a long spacer arm was employed) than their corresponding parent molecules against the human Wuhan influenza strain. However, such polymeric inhibitors failed to exhibit higher potency compared with their small molecule predecessors against the human Puerto Rico and avian turkey influenza strains. These observations, further explored by means of molecular modeling, reveal the previously unrecognized unpredictability of the benefits of multivalency, possibly because of poor accessibility of the viral targets to polymeric agents.
    Journal of Pharmaceutical Sciences 07/2012; 101(10):3896-905. · 3.13 Impact Factor
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    ABSTRACT: To discover, elucidate the structure-activity relationship (SAR), and explore the mechanism of action of excipients able to drastically lower the viscosities of concentrated aqueous solutions of humanized monoclonal antibodies (MAbs). Salts prepared from hydrophobic cations and anions were dissolved into humanized MAbs solutions. Viscosities of the resulting solutions were measured as a function of the nature and concentration of the salts and MAbs. Even at moderate concentrations, some of the salts prepared herein were found to reduce over 10-fold the viscosities of concentrated aqueous solutions of several MAbs at room temperature. To be potent viscosity-lowering excipients, the ionic constituents of the salts must be hydrophobic, bulky, and aliphatic. A mechanistic hypothesis explaining the observed salt effects on MAb solutions' viscosities was proposed and verified.
    Pharmaceutical Research 06/2012; 29(11):3102-9. · 4.74 Impact Factor
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    ABSTRACT: Polyelectrolyte multilayer films assembled from a hydrophobic N-alkylated polyethylenimine and a hydrophilic polyacrylate were discovered to exhibit strong antifouling, as well as antimicrobial, activities. Surfaces coated with these layer-by-layer (LbL) films, which range from 6 to 10 bilayers (up to 45 nm in thickness), adsorbed up to 20 times less protein from blood plasma than the uncoated controls. The dependence of the antifouling activity on the nature of the polycation, as well as on assembly conditions and the number of layers in the LbL films, was investigated. Changing the hydrophobicity of the polycation altered the surface composition and the resistance to protein adsorption of the LbL films. Importantly, this resistance was greater for coated surfaces with the polyanion on top; for these films, the average zeta potential pointed to a near neutral surface charge, thus, presumably minimizing their electrostatic interactions with the protein. The film surface exhibited a large contact angle hysteresis, indicating a heterogeneous topology likely due to the existence of hydrophobic-hydrophilic regions on the surface. Scanning electron micrographs of the film surface revealed the existence of nanoscale domains. We hypothesize that the existence of hydrophobic/hydrophilic nanodomains, as well as surface charge neutrality, contributes to the LbL film's resistance to protein adsorption.
    Biomacromolecules 02/2012; 13(3):719-26. · 5.37 Impact Factor
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    ABSTRACT: To explore (i) the potential of polyethylenimine (PEI)-DNA nanoparticles as a vector for delivering genes into human corneal fibroblasts, and (ii) whether the nanoparticle-mediated soluble extracellular domain of the transforming growth factor-β type II receptor (sTGFβRII) gene therapy could be used to reduce myofibroblasts and fibrosis in the cornea using an in vitro model. PEI-DNA nanoparticles were prepared at a nitrogen-to-phosphate ratio of 30 by mixing linear PEI and a plasmid encoding sTGFβRII conjugated to the fragment crystallizable (Fc) portion of human immunoglobulin. The PEI-DNA polyplex formation was confirmed through gel retardation assay. Human corneal fibroblasts (HCFs) were generated from donor corneas; myofibroblasts and fibrosis were induced with TGFβ1 (1 ng/ml) stimulation employing serum-free conditions. The sTGFβRII conjugated to the Fc portion of human immunoglobulin gene was introduced into HCF using either PEI-DNA nanoparticles or Lipofectamine. Suitable negative and positive controls to compare selected nanoparticle and therapeutic gene efficiency were included. Delivered gene copies and mRNA (mRNA) expression were quantified with real-time quantitative PCR (qPCR) and protein with enzyme-linked immunosorbent assay (ELISA). The changes in fibrosis parameters were quantified by measuring fibrosis marker α-smooth muscle actin (SMA) mRNA and protein levels with qPCR, immunostaining, and immunoblotting. Cytotoxicity was determined using cellular viability, proliferation, and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay. PEI readily bound to plasmids to form nanoparticular polyplexes and exhibited much greater transfection efficiency (p<0.01) than the commercial reagent Lipofectamine. The PEI-DNA-treated cultures showed 4.5×10(4) plasmid copies/µg DNA in real-time qPCR and 7,030±87 pg/ml sTGFβRII protein in ELISA analyses, whereas Lipofectamine-transfected cultures demonstrated 1.9×10(3) gene copies/µg DNA and 1,640±100 pg/ml sTGFβRII protein during these assays. The PEI-mediated sTGFβRII delivery remarkably attenuated TGFβ1-induced transdifferentiation of corneal fibroblasts to myofibroblasts in cultures, as indicated by threefold lower levels of SMA mRNA (p<0.01) and significant inhibition of SMA protein (up to 96±3%; p<0.001 compared to no-gene-delivered cultures) in immunocytochemical staining and immunoblotting. The nanoparticle-mediated delivery of sTGFβRII showed significantly better antifibrotic effects than the Lipofectamine under similar experimental conditions. However, the inhibition of myofibroblast in HCF cultures by sTGFβRII overexpression by either method was significantly higher than the naked vector transfection. Furthermore, PEI- or Lipofectamine-mediated sTGFβRII delivery into HCF did not alter cellular proliferation or phenotype at 12 and 24 h post-treatment. Nanoparticles treated with HCF showed more than 90% cellular viability and very low cell death (2-6 TUNEL+ cells), suggesting that the tested doses of PEI-nanoparticles do not induce significant cell death. This study demonstrated that PEI-DNA nanoparticles are an attractive vector for the development of nonviral corneal gene therapy approaches and that the sTGFβRII gene delivery into keratocytes could be used to control corneal fibrosis in vivo.
    Molecular vision 01/2012; 18:2598-607. · 1.99 Impact Factor
  • Roger A Nassar, Edward P Browne, Jianzhu Chen, Alexander M Klibanov
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    ABSTRACT: Heparin covalently attached to a water-insoluble resin suspended in HIV-infected aqueous buffer or whole blood captures the virus; subsequent physical separation of the immobilized heparin reduced the viral titers by over 80 and 50%, respectively. The detoxification concept has been validated by both circulating an HIV-1 solution through a column packed with the heparin-sepharose beads and successively mixing an HIV-1 solution with fresh beads.
    Biotechnology Letters 12/2011; 34(5):853-6. · 1.85 Impact Factor
  • Thomas P Schaer, Suzanne Stewart, Bryan B Hsu, Alexander M Klibanov
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    ABSTRACT: Adhesion of microorganisms to biomaterials with subsequent formation of biofilms on such foreign bodies as orthopedic trauma hardware is a critical factor in implant-associated infections; once a biofilm has been established, its microorganisms become recalcitrant to the host's immune surveillance and markedly resistant to drugs. We have previously reported that painting with the hydrophobic polycation N,N-dodecyl,methyl-PEI (PEI = polyethylenimine) renders solid surfaces bactericidal in vitro. Herein we observe that N,N-dodecyl,methyl-PEI-derivatized titanium and stainless steel surfaces resist biofilm formation by Staphylococcus aureus compared to the untreated ones. Using imaging, microbiology-, histopathology-, and scanning electron microscopy (SEM) experiments in a clinically relevant large-animal (sheep) trauma model, we subsequently demonstrate in vivo that orthopedic fracture hardware painted with N,N-dodecyl,methyl-PEI not only prevents implant colonization with biofilm but also promotes bone healing. Functionalizing orthopedic hardware with hydrophobic polycations thus holds promise in supporting bone healing in the presence of infection in veterinary and human orthopedic patients.
    Biomaterials 11/2011; 33(5):1245-54. · 8.31 Impact Factor
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    ABSTRACT: The biocompatibility and antibacterial properties of N,N-hexyl,methyl-polyethylenimine (HMPEI) covalently attached to the Boston Keratoprosthesis (B-KPro) materials was evaluated. By means of confocal and electron microscopies, we observed that HMPEI-derivatized materials exert an inhibitory effect on biofilm formation by Staphylococcus aureus clinical isolates, as compared to the parent poly(methyl methacrylate) (PMMA) and titanium. There was no additional corneal epithelial cell cytotoxicity of HMPEI-coated PMMA compared to that of control PMMA in tissue cultures in vitro. Likewise, no toxicity or adverse reactivity was detected with HMPEI-derivatized PMMA or titanium compared to those of the control materials after intrastromal or anterior chamber implantation in rabbits in vivo.
    Biomaterials 09/2011; 32(34):8783-96. · 8.31 Impact Factor
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    F Long, C Gao, H C Shi, M He, A N Zhu, A M Klibanov, A Z Gu
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    ABSTRACT: Mercury ions (Hg(2+)) are a highly toxic and ubiquitous pollutants requiring rapid and sensitive on-site detection methods in the environment and foods. Herein, we report an envanescent wave DNA-based biosensor for rapid and very sensitive Hg(2+) detection based on a direct structure-competitive detection mode. In this system, a DNA probe covalently immobilized onto a fiber optic sensor contains a short common oligonucleotide sequences that can hybidize with a fluorescently labeled complementary DNA. The DNA probe also comprises a sequence of T-T mismatch pairs that binds with Hg(2+) to form a T-Hg(2+)-T complex by folding of the DNA segments into a hairpin structure. With a structure-competitive mode, a higher concentration of Hg(2+) leads to less fluorescence-labeled cDNA bound to the sensor surface and thus to lower fluorescence signal. The total analysis time for a single sample, including the measurement and surface regeneration, was under 6 min with a Hg(2+) detection limit of 2.1 nM. The high specificity of the sensor was demonstrated by evaluating its response to a number of potentially interfering metal ions. The sensor's surface can be regenerated with a 0.5% SDS solution (pH 1.9) over 100 times with no significant deterioration of performance. This platform is potentially applicable to detect other heavy metal ions or small-molecule analytes for which DNA/aptamers can be used as specific sensing probes.
    Biosensors & Bioelectronics 06/2011; 26(10):4018-23. · 6.45 Impact Factor

Publication Stats

10k Citations
1,316.02 Total Impact Points

Institutions

  • 2012–2013
    • University of Missouri
      • School of Medicine
      Columbia, Missouri, United States
  • 1980–2013
    • Massachusetts Institute of Technology
      • • Department of Chemistry
      • • Department of Biological Engineering
      • • Department of Chemical Engineering
      • • Division of Health Sciences and Technology
      Cambridge, MA, United States
  • 2011
    • Massachusetts Eye and Ear Infirmary
      • Department of Ophthalmology
      Boston, MA, United States
    • University of Pennsylvania
      • School of Veterinary Medicine
      Philadelphia, PA, United States
  • 2009
    • University of Seoul
      Sŏul, Seoul, South Korea
  • 2002–2005
    • Northeastern University
      • Department of Biology
      Boston, MA, United States
  • 1990
    • University of Pittsburgh
      • Chemical and Petroleum Engineering
      Pittsburgh, PA, United States
  • 1971–1979
    • Lomonosov Moscow State University
      • • Division of Chemistry
      • • Laboratory of Bioorganic Chemistry
      Moskva, Moscow, Russia
  • 1977
    • Moscow State Textile University
      Moskva, Moscow, Russia