Reusable evanescent wave DNA biosensor for rapid, highly sensitive, and selective detection of mercury ions.

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.

Quantitative comparison of polyethylenimine formulations and adenoviral vectors in terms of intracellular gene delivery processes.

An objective of designing molecular vehicles exhibiting virus-like transgene delivery capabilities but with low toxicity and immunogenicity continues to drive synthetic vector development. As no single step within the gene delivery pathway represents the critical limiting barrier for all vector types under all circumstances, improvements in synthetic vehicle design may be aided by quantitative analysis of the contributions of each step to the overall delivery process. To our knowledge, however, synthetic and viral gene delivery methods have not yet been explicitly compared in terms of these delivery pathway steps in a quantitative manner. As a first address of this challenge, we compare here quantitative parameters characterizing intracellular gene delivery steps for an E1/E3-deleted adenoviral vector and three polyethylenimine (PEI)-based vector formulations, as well as the liposomal transfection reagent Lipofectamine and naked DNA; the cargo is a plasmid encoding the beta-galactosidase gene under a CMV promoter, and the cell host is the C3A human hepatocellular carcinoma line. The parameters were determined by applying a previously validated mathematical model to transient time-course measurements of plasmid uptake and trafficking (from whole-cell and isolated nuclei lysates, by real-time quantitative PCR), and gene expression levels, enabling discovery of those for which the adenoviral vector manifested superiority. Parameter-sensitivity analysis permitted identification of processes most critically rate-limiting for each vector. We find that the adenoviral vector advantage in delivery appears to reside partially in its import to the nuclear compartment, but that its vast superiority in transgene expression arises predominantly in our situation from postdelivery events: on the basis of per-nuclear plasmid, expression efficiency from adenovirus is superior by orders of magnitude over the PEI vectors. We find that a chemical modification of a PEI-based vector, which substantially improves its performance, appears to do so by enhancing certain trafficking rate parameters, such as binding and uptake, endosomal escape, and binding to nuclear import machinery, but leaves endosomal escape as a barrier over which transgene delivery could be most sensitively increased further for this polymer.

Non-viral gene therapy: polycation-mediated DNA delivery.

Gene therapy, i.e., the expression in cells of genetic material with therapeutic activity, holds great promise for the treatment of human diseases. A delivery vehicle (vector), of either viral or non-viral origin, must be used to carry the foreign gene into a cell. Viral vectors take advantage of the facile integration of the gene of interest into the host and high probability of its long-term expression but are plagued by safety concerns. Non-viral vectors, although less efficient at introducing and maintaining foreign gene expression, have the profound advantage of being non-pathogenic and non-immunogenic; they are the subject of this review. Polycation-DNA complexes are particularly attractive for non-viral gene therapy. To perform, they have to attach to the target cell surface, be internalized, escape from endosomes, find a way to the nucleus, and, finally, be available for transcription. The clinical usefulness of polycationic vectors depends on elucidating the role each of these steps plays in gene transfer. Recent progress in consequent rational vector improvement is highlighted by our finding of polyethylenimine derivatives more potent and yet less cytotoxic than the 25-kDa polyethylenimine (one of the most effective non-viral vectors). Such vectors could be further modified with cell-targeting ligands to enhance their utility for in vivo applications.

Inhalation delivery of proteins from ethanol suspensions.

To circumvent inherent problems associated with pulmonary administration of aqueous-solution and dry-powder protein drugs, inhalation delivery of proteins from their suspensions in absolute ethanol was explored both in vitro and in vivo. Protein suspensions in ethanol of up to 9% (wt/vol) were readily aerosolized with a commercial compressor nebulizer. Experiments with enzymic proteins revealed that nebulization caused no detectable loss of catalytic activity; furthermore, enzyme suspensions in anhydrous ethanol retained their full catalytic activity for at least 3 weeks at room temperature. With the use of Zn(2+)-insulin, conditions were elaborated that produced submicron protein particles in ethanol suspensions. The latter (insulin/EtOH) afforded respirable-size aerosol particles after nebulization. A 40-min exposure of laboratory rats to 10 mg/ml insulin/EtOH aerosols resulted in a 2-fold drop in the blood glucose level and a marked rise in the serum insulin level. The bioavailability based on estimated deposited lung dose of insulin delivered by inhalation of ethanol suspension aerosols was 33% (relative to an equivalent s.c. injection), i.e., comparable to those observed in rats after inhalation administration of dry powder and aqueous solutions of insulin. Inhalation of ethanol in a relevant amount/time frame resulted in no detectable acute toxic effects on rat lungs or airways, as reflected by the absence of statistically significant inflammatory or allergic responses, damage to the alveolar/capillary barrier, and lysed and/or damaged cells.

Designing surfaces that kill bacteria on contact.

Poly(4-vinyl-N-alkylpyridinium bromide) was covalently attached to glass slides to create a surface that kills airborne bacteria on contact. The antibacterial properties were assessed by spraying aqueous suspensions of bacterial cells on the surface, followed by air drying and counting the number of cells remaining viable (i.e., capable of growing colonies). Amino glass slides were acylated with acryloyl chloride, copolymerized with 4-vinylpyridine, and N-alkylated with different alkyl bromides (from propyl to hexadecyl). The resultant surfaces, depending on the alkyl group, were able to kill up to 94 +/- 4% of Staphylococcus aureus cells sprayed on them. A surface alternatively created by attaching poly(4-vinylpyridine) to a glass slide and alkylating it with hexyl bromide killed 94 +/- 3% of the deposited S. aureus cells. On surfaces modified with N-hexylated poly(4-vinylpyridine), the numbers of viable cells of another Gram-positive bacterium, Staphylococcus epidermidis, as well as of the Gram-negative bacteria Pseudomonas aeruginosa and Escherichia coli, dropped more than 100-fold compared with the original amino glass. In contrast, the number of viable bacterial cells did not decline significantly after spraying on such common materials as ceramics, plastics, metals, and wood.

Improving biomaterial properties of collagen films by chemical modification.

Films of bovine collagen were chemically modified with the goal of improving their biomaterial properties. The modified films were investigated with respect to their affinity to fibroblast and endothelial cells, as well as their antibacterial properties tested by adhesion of Staphylococcus aureus. Modifications that only change the net charge of collagen, such as acetylation, succinylation, and treatment with glutaraldehyde (all increase the negative charge), and amination with ethylenediamine (EDA), N,N-dimethyl-EDA (DMEDA), or butylamine (all increase the positive charge), did not dramatically alter the mammalian cell attachment to the film. In contrast, derivatization of collagen using methoxypoly(ethylene glycol) (PEG) diminished the attachment of fibroblasts by 98 +/- 1% and of endothelial cells by more than 99% compared to unmodified collagen. Moreover, the rate of growth of fibroblasts dropped by 97 +/- 1% and that of endothelial cells by 88 +/- 3% as a result of PEGylation of collagen. Adhesion of S. aureus cells also plummeted by 93 +/- 2% as a result of this PEGylation. With these antifouling properties, PEG-collagen may be a promising coating material for coronary stents. Subsequent derivatization of PEG-collagen with EDA or DMEDA abolished its mammalian cell-repelling ability, whereas bacterial cell repulsion was partially retained: for example, DMEDA-modified PEG-collagen exhibits up to a 5-fold lower bacterial adhesion than collagen. It is worth noting that a material that allows mammalian cell attachment but reduces bacterial adhesion could be useful as an implant or coating.

Improving enzymes by using them in organic solvents.

The technological utility of enzymes can be enhanced greatly by using them in organic solvents rather than their natural aqueous reaction media. Studies over the past 15 years have revealed not only that this change in solvent is feasible, but also that in such seemingly hostile environments enzymes can catalyse reactions impossible in water, become more stable, and exhibit new behaviour such as 'molecular memory'. Of particular importance has been the discovery that enzymatic selectivity, including substrate, stereo-, regio- and chemoselectivity, can be markedly affected, and sometimes even inverted, by the solvent. Enzyme-catalysed reactions in organic solvents, and even in supercritical fluids and the gas phase, have found numerous potential applications, some of which are already commercialized.

Peroxidase-catalyzed asymmetric sulfoxidation in organic solvents versus in water.

Peroxidase-catalyzed asymmetric sulfoxidations, while synthetically attractive, suffer from relatively low reaction rates due to poor substrate solubilities in water and from appreciable spontaneous oxidation of substrates (especially aryl alkyl sulfides) with H(2)O(2). In this work, we found that both of these shortcomings could be alleviated by switching from aqueous solutions to certain nearly anhydrous (99.7%) organic solvents as sulfoxidation reaction media. The rates of spontaneous oxidation of the model prochiral substrate thioanisole in several organic solvents were observed to be some 100- to 1000-fold slower than in water. In addition, the rates of asymmetric sulfoxidation of thioanisole in isopropyl alcohol and in methanol catalyzed by horseradish peroxidase (HRP) were determined to be tens to hundreds of times faster than in water under otherwise identical conditions. This dramatic activation is due to a much higher substrate solubility in organic solvents than in water and occurs even though the intrinsic reactivity of HRP in isopropyl alcohol and in methanol is hundreds of times lower than in water. Sulfoxidation of thioanisole catalyzed by four other hemoproteins (soybean peroxidase, myoglobin, hemoglobin, and cytochrome c) is also much faster in isopropyl alcohol than in water.

Improving lipase enantioselectivity in organic solvents by forming substrate salts with chiral agents.

We recently demonstrated (J Am Chem Soc 121:3334-3340, 1999) that enzymatic enantioselectivity in organic solvents can be markedly enhanced by temporarily enlarging the substrate via salt formation. In the present study, this approach was expanded by finding that, in addition to its size, the stereochemistry of the counterion can greatly affect the enantioselectivity enhancement. For example, the enantioselectivity [E = (k(cat)/K(M))(S)/(k(cat)/K(M))(R)] of crystalline Pseudomonas cepacia lipase in the propanolysis of phenylalanine methyl ester (PheOMe) in anhydrous acetonitrile was found to be 5.8 +/- 0.6; the E value doubled when PheOMe's salt with S mandelic acid was used as a substrate instead of the free ester, and rose sevenfold with R mandelic acid as a Bronsted-Lowry acid. Similar effects were observed with other bulky, but not petite, counterions. The greatest enantioselectivity enhancement was afforded by 10-camphorsulfonic acid: the E value increased to 18 +/- 2 for a salt with its R enantiomer and jumped to 53 +/- 4 for the S. These effects, also observed in other organic solvents, were explained by means of structure-based molecular modeling of the lipase-bound transition states of the substrate enantiomers and their diastereomeric salts.

Calorimetric evidence for a native-like conformation of hen egg-white lysozyme dissolved in glycerol.

Hen egg-white lysozyme, lyophilized from aqueous solutions of different pH (from pH 2.5 to 10.0) and then dissolved in water and in anhydrous glycerol, has been studied by high-sensitivity differential scanning microcalorimetry over the temperature range from 10 to 150 degrees C. All lysozyme samples exhibit a cooperative conformational transition in both solvents occurring between 10 and 100 degrees C. The transition temperatures in glycerol are similar to those in water at the corresponding pHs. The transition enthalpies in glycerol are substantially lower than in water but follow similar pH dependences. The transition heat capacity increment in glycerol does not depend on the pH and is 1.25+/-0.31 kJ mol(-1) K(-1), which is less than one fifth of that in water (6. 72+/-0.23 kJ mol(-1) K(-1)). The thermal transition in glycerol is reversible and equilibrium, as demonstrated for the pH 8.0 sample, and follows the classical two-state mechanism. In contrast to lysozyme in water, the protein dissolved in glycerol undergoes an additional, irreversible cooperative transition with a marginal endothermic heat effect at temperatures of 120-130 degrees C. The transition temperature of this second transition increases with the heating rate which is characteristic of kinetically controlled processes. Thermodynamic analysis of the calorimetric data reveals that the stability of the folded conformation of lysozyme in glycerol is similar to that in water at 20-80 degrees C but exceeds it at lower and higher temperatures. It is hypothesized that the thermal unfolding in glycerol follows the scheme: N ifho-MG-->U, where N is a native-like conformation, ho-MG is a highly ordered molten globule state, and U is the unfolded state of the protein.

Structural stability of DNA in nonaqueous solvents.

One of the defining physicochemical features of DNA in aqueous solution is its ability to maintain a double-helical structure and for this structure to undergo a cooperative, heat-induced denaturation (melting). Herein we show that a 21-mer synthetic DNA can form and maintain such a duplex structure not only in water but even in 99% glycerol; moreover, this double-helical structure reversibly and cooperatively melts in that solvent, with a T(m) value of some 30 degrees lower than in water. Two much larger, natural DNAs, from calf thymus and salmon testes, exhibit similar behavior in glycerol. All three DNAs can also sustain a double-helical structure in 99% ethylene glycol, although its thermostability (as reflected by the melting temperature) is some 20 degrees lower than in glycerol. In contrast, no duplex structure of any of the DNAs was detected in 99% formamide, methanol, or DMSO. This solvent trend resembles that previously observed in studies of protein structure and folding and underscores the importance of hydrophobic interactions in both protein and DNA structure and stability. Our findings suggest that water may not be unique as a suitable medium not only for protein structure but also for that of nucleic acids.

A new approach to preparative enzymatic synthesis. Reprinted from Biotechnology and Bioengineering, Vol. XIX, No. 9, Pages 1351-1361.

A new approach to preparative organic synthesis in aqueous-organic systems is suggested. It is based on the idea that the enzymatic process is carried out in a biphasic system "water-water-immiscible organic solvent." Thereby the enzyme is localized in the aqueous phase-this eliminates the traditional problem of stabilizing the enzymes against inactivation by a nonaqueous solvent. Hence, in contrast to the commonly used combinations "water-water-miscible organic solvent," in the suggested system the content of water may be infinitely low. This allows one to dramatically shift the equilibrium of the reactions forming water as a reaction product (synthesis of esters and amides, polymerization of amino acids, sugars and nucleotides, dehydration reactions, etc.) toward the products. The fact that the system consists of two phases provides another very important sources for an equilibrium shift, i.e., free energies of the transfer of a reagent from one phase to the other. Equations are derived describing the dependence of the equilibrium constant in a biphasic system on the ratio of the volumes of the aqueous and nonaqueous phases and the partition coefficients of the reagents between the phases. The approach has been experimentally verified with the synthesis of N-acetyl-L-tryptophan ethyl ester from the respective alcohol and acid. Porous glass was impregnated with aqueous buffer solution of chymotrypsin and suspended in chloroform containing N-acetyl-L-tryptophan and ethanol. In water (no organic phase) the yield of the ester is about 0.01%, whereas in this biphasic system it is practically 100%. The idea is applicable to a great number of preparative enzymatic reactions.

Visual evidence of acidic environment within degrading poly(lactic-co-glycolic acid) (PLGA) microspheres.

PURPOSE: In the past decade, biodegradable polymers have become the materials of choice for a variety of biomaterials applications. In particular, poly(lactic-co-glycolic acid) (PLGA) microspheres have been extensively studied for controlled-release drug delivery. However, degradation of the polymer generates acidic monomers, and acidification of the inner polymer environment is a central issue in the development of these devices for drug delivery. METHODS: To quantitatively determine the intrapolymer acidity, we entrapped pH-sensitive fluorescent dyes (conjugated to 10,000 Da dextrans) within the microspheres and imaged them with confocal fluorescence microscopy. The technique allows visualization of the spatial and temporal distribution of pH within the degrading microspheres (1). RESULTS: Our experiments show the formation of a very acidic environment within the particles with the minimum pH as low as 1.5. CONCLUSIONS: The images show a pH gradient, with the most acidic environment at the center of the spheres and higher pH near the edges, which is characteristic of diffusion-controlled release of the acidic degradation products.

Striking activation of oxidative enzymes suspended in nonaqueous media.

The catalytic activity of four lyophilized oxidative enzymes-horseradish peroxidase, soybean peroxidase, Caldariomyces fumago chloroperoxidase, and mushroom polyphenol oxidase-is much lower when directly suspended in organic solvents containing little water than when they are introduced into the same largely nonaqueous media by first dissolving them in water and then diluting with anhydrous solvents. The lower the water content of the medium, the greater this discrepancy becomes. The mechanism of this phenomenon was found to arise from reversible denaturation of the oxidases on lyophilization: because of its conformational rigidity, the denatured enzyme exhibits very limited activity when directly suspended in largely nonaqueous media but renatures and thus yields much higher activity if first redissolved in water. Two independent means were discovered for dramatically minimizing the lyophilization-induced inactivation, both involving the addition of certain types of excipients to the aqueous enzyme solution before lyophilization. The first group of excipients consists of phenolic and aniline substrates as well as other hydrophobic compounds; these presumably bind to the hydrophobic pocket of the enzyme active site, thereby preventing its collapse during dehydration. The second group consists of general lyoprotectants such as polyols and polyethylen glycol that apparently preserve the overall enzyme structure during dehydration. The activation effects of such excipients can reach into the tens and hundreds of fold. Moreover, the activations afforded by the two excipient groups are additive, resulting in up to a complete protection against lyophilization-induced inactivation when representatives of the two are present together.

FTIR characterization of the secondary structure of proteins encapsulated within PLGA microspheres.

A commonly used technique for protein encapsulation in microspheres is the double-emulsion method wherein an initial water-in-oil (w/o) emulsion of protein and polymer is formed via sonication, and then a second emulsion (w/o)/w is formed by dispersion in an aqueous phase via homogenization. This approach is often used to produce microspheres of biodegradable poly(lactic-co-glycolic acid) (PLGA). The harsh processing associated with this method can cause denaturation of the encapsulated protein. Herein, we have used Fourier transform infrared (FTIR) spectroscopy to determine the secondary structures of two model proteins, bovine serum albumin (BSA) and chicken egg-white lysozyme, within PLGA microspheres. The alpha-helix content of both proteins in the microspheres was about a third lower than in the lyophilized state, indicating conformational changes upon protein entrapment within the microspheres. BSA microspheres containing the stabilizing excipient trehalose have a higher alpha-helix content than those without excipient, suggesting that trehalose partially prevents the denaturing effects incurred during processing. In addition, BSA released from microspheres is improved by incorporation of trehalose: analysis of the protein released from the microspheres indicates that there is less BSA dimer formation in the trehalose-containing microspheres than in those without trehalose.

Structure of lysozyme dissolved in neat organic solvents as assessed by NMR and CD spectroscopies.

The structure of the model protein hen egg-white lysozyme dissolved in water and in five neat organic solvents (ethylene glycol, methanol, dimethylsulfoxide (DMSO), formamide, and dimethylformamide (DMF)) has been examined by means of 1H NMR and circular dichroism (CD) spectroscopies. The NMR spectra of lysozyme reveal the lack of a defined tertiary structure in all five organic solvents, although the examination of line widths suggests the possibility of some ordered structure in ethylene glycol and in methanol. The near-UV CD spectra of the protein suggest no tertiary structure in lysozyme dissolved in DMSO, formamide, and DMF, while a distinctive (albeit less pronounced than in water) tertiary structure is seen in ethylene glycol and a drastically changed one in methanol. A highly developed secondary structure was observed by far-UV CD in ethylene glycol and methanol; interestingly, the alpha-helix content of the protein in both was greater than in water, while the beta-structure content was lower. (Solvent absorbance in the far-UV region prevents conclusions about the secondary structure in DMSO, formamide and DMF.)

Structure, thermostability, and conformational flexibility of hen egg-white lysozyme dissolved in glycerol.

Hen egg-white lysozyme dissolved in glycerol containing 1% water was studied by using CD and amide proton exchange monitored by two-dimensional 1H NMR. The far- and near-UV CD spectra of the protein showed that the secondary and tertiary structures of lysozyme in glycerol were similar to those in water. Thermal melting of lysozyme in glycerol followed by CD spectral changes indicated unfolding of the tertiary structure with a Tm of 76.0 +/- 0.2 degreesC and no appreciable loss of the secondary structure up to 85 degreesC. This is in contrast to the coincident denaturation of both tertiary and secondary structures with Tm values of 74.8 +/- 0.4 degreesC and 74.3 +/- 0.7 degreesC, respectively, under analogous conditions in water. Quenched amide proton exchange experiments revealed a greater structural protection of amide protons in glycerol than in water for a majority of the slowly exchanging protons. The results point to a highly ordered, native-like structure of lysozyme in glycerol, with the stability exceeding that in water.

Protein refolding in predominantly organic media markedly enhanced by common salts.

The refolding/reoxidation of unfolded/reduced hen egg-white lysozyme was investigated in a variety of predominantly nonaqueous media consisting of protein-dissolving organic solvents and water. It was discovered that LiCl and other common salts dramatically (up to more than 100-fold) increased the refolding yield of lysozyme in such nonaqueous systems, while reducing it in water. The mechanism of this surprising phenomenon appears to involve salt-induced suppression of nonspecific lysozyme aggregation during refolding due to an enhanced protein solubility.