Voravee P. Hoven

Surface-quaternized chitosan particles as an alternative and effective organic antibacterial material

Taking advantage of the large surface area that is covered with permanent positive charges of quaternary ammonium entities, this research aimed to develop environmentally friendly, organic antibacterial material from quaternized chitosan particles that may be applicable for biomedical devices, health and textile industries. The particles were formulated by ionic crosslinking of chitosan with tripolyphosphate followed by quaternization under heterogeneous conditions, via either direct methylation or reductive N-alkylation with a selected aldehyde followed by methylation. Sub-micron, spherical and positively charged quaternized chitosan particles were formed, as determined by (1)H NMR, FT-IR, PCS and TEM analysis. Antibacterial activity tests, performed by viable cell (colony) counts, suggested that all quaternized chitosan particles exhibited superior antibacterial activity against the model Gram-positive bacteria, Staphylococcus aureus, as compared to the native chitosan particles at neutral pH. Only some quaternized chitosan particles, especially those having a high charge density and bearing large alkyl substituent groups, were capable of suppressing the growth of the model Gram-negative bacteria, Escherichia coli. The inhibitory efficiency of the quaternized chitosan particles was quantified in terms of the minimum inhibitory concentration (MIC). Damaging impact of the quaternized chitosan particles on the bacteria was also qualitatively determined by microscopic observation of the bacterial morphology.

Electrospun mat of tyrosine-derived polycarbonate fibers for potential use as tissue scaffolding material

Desaminotyrosyl-tyrosine ethyl ester (DTE) and desaminotyrosyl-tyrosine (DT) were used as monomers in the synthesis of two tyrosine-derived polycarbonates: the slow degrading homopolymer poly(DTE carbonate) and the fast degrading co-polymer poly(DTE-co-20%DT carbonate). Ultrafine fibers of these polymers were successfully fabricated using an electrospinning process. The effects of some solution and process parameters (i.e., polymer concentration, electrostatic field strength and solvent system) on morphological appearance and diameters of the obtained fibers were investigated by scanning electron microscopy (SEM). Smooth fibers were obtained at high enough solution concentrations (i.e., 15 and 20% (w/v)). The average fiber diameter was found to increase with increasing polymer concentration and applied electrostatic field strength. The electrospinnability of poly(DTE-co-20%DT carbonate) in dichloromethane was enhanced when methanol was used as the co-solvent. In all of the conditions investigated, the average diameter of the obtained smooth fibers ranged between 1.9 and 5.8 μm. A qualitative assessment of an as-spun mat of poly(DTE carbonate) fibers as a tissue scaffolding material showed that three different cultured cell lines appeared to adhere and propagate well within the scaffold. For poly(DTE carbonate) exceptionally high cell densities could be achieved after 10 days of cell culture.

Comparison of DNA, aminoethylglycyl PNA and pyrrolidinyl PNA as probes for detection of DNA hybridization using surface plasmon resonance technique

Pyrrolidinyl peptide nucleic acid bearing a D-prolyl-2-aminocyclopentanecarboxylic acid backbone (acpcPNA) has been evaluated as a new sensing probe for detection of DNA hybridization. In this study, the biotinylated acpcPNA was immobilized on surface plasmon resonance (SPR) sensor chips via biotin-streptavidin interactions for solid-phase DNA hybridization. A critical comparison between acpcPNA, DNA and conventional peptide nucleic acid (aegPNA) probes of the same sequence was made by means of SPR on various important aspects. These include the effect of ionic strength on hybridization efficiency, the specificity to detect the mismatch(es) in target DNAs, the direction of binding (parallel or antiparallel) to target DNAs, and the effect of target DNA concentration on hybridization efficiency. Results indicated that the immobilized acpcPNA probe possesses distinct hybridization properties relative to aegPNA (and/or DNA) counterparts, including a higher single-base mismatch sensitivity, antiparallel selectivity and low ionic strength dependence of target hybridization. These properties substantiate the acpcPNA applicability as sensor probes for clinical and diagnostic applications. With a proper selection of regeneration conditions (10 mM NaOH, 2 min exposure), the sensor can be reused for multiple cycles of hybridization with as little as 1.3% loss in hybridization activity per regeneration cycle.

Thiolated pyrrolidinyl peptide nucleic acids for the detection of DNA hybridization using surface plasmon resonance.

Thiolated pyrrolidinyl peptide nucleic acids (HS-PNAs) bearing d-prolyl-2-aminocyclopentanecarboxylic acid (ACPC) backbones with different lengths and types of thiol modifiers were synthesized and then characterized by MALDI-TOF mass spectrometry. These HS-PNAs were immobilized on gold-coated glass by self-assembled monolayer (SAM) formation via S atom linkage for the detection of DNA hybridization using surface plasmon resonance (SPR). The amount and the stability of the immobilized HS-PNAs, as well as the effects of spacer and blocking thiol on DNA hybridization efficiency, were determined. SPR results indicated that the hybridization efficiency was enhanced when the distance between the PNA portion and the thiol terminal was increased and/or when blocking thiol was used following the HS-PNA immobilization. The immobilized HS-PNA could discriminate between fully complementary DNA from one or two base mismatched DNA with a relatively high degree of mismatch discrimination (>45%) in PBS buffer at 25 degrees C. The lowest DNA concentration at which reliable discrimination between fully complementary and single mismatched DNA could still occur was at about 0.2 microM, which is equivalent to 10 pmol of DNA. This research demonstrates that using these novel thiolated PNAs in combination with the SPR technique offers a direct, rapid and non-label based method that could potentially be applied for the analysis of genomic or PCR-amplified DNA in the future.

Poly(N-isopropylacrylamide)-Stabilized Gold Nanoparticles in Combination with Tricationic Branched Phenylene-Ethynylene Fluorophore for Protein Identification

Gold nanoparticles stabilized by thermoresponsive polymer, poly(N-isopropylacrylamide) (PNIPAM-AuNPs) were prepared by surface grafting of thiol-terminated PNIPAM onto citrate-stabilized AuNPs. The color change of the PNIPAM-AuNPs solution from red to blue-purple without precipitation when the solution was heated to 40 °C, above the lower critical solution temperature (LCST) of PNIPAM, indicated the thermoresponsive property of the synthesized AuNPs. PNIPAM-AuNPs were used to detect proteins by chemical nose approach based on fluorescence quenching of fluorophore by AuNPs. An array-based sensing platform for detection of six proteins, namely bovine serum albumin, lysozyme, fibrinogen, concanavalin A, hemoglobin, holo-transferrin human can be successfully developed from the PNIPAM-AuNPs having different molecular weights (4 and 8 kDa) and conformation (varied heat treatment from 25 to 40 °C) in combination with a tricationic branched phenylene-ethynylene fluorophore. From principal component analysis (PCA) followed by linear discriminant analysis (LDA), 100% accuracy of protein classification using a leave-one-out (LOO) approach can be achieved by using only two types of PNIPAM-AuNPs.

Surface-Grafted Poly(acrylic acid) Brushes as a Precursor Layer for Biosensing Applications: Effect of Graft Density and Swellability on the Detection Efficiency

Carboxyl groups along poly(acrylic acid) (PAA) brushes attached to the surface of a gold-coated substrate served as the precursor moieties for the covalent immobilization of amino-functionalized biotin or bovine serum albumin (BSA) to form a sensing probe for streptavidin (SA) or anti-BSA detection, respectively. Surface-grafted PAA brushes were obtained by acid hydrolysis of poly(tert-butyl acrylate) brushes, formerly prepared by surface-initiated atom transfer radical polymerization of tert-butyl acrylate. As determined by surface plasmon resonance, the PAA brushes immobilized with functionalized biotin or BSA probes not only showed good binding with the designated target analytes but also maintained a high resistance to nonspecific protein adsorption, especially those PAA brushes with a high surface graft density. Although the probe binding capacity can be raised as a function of the graft density of the PAA brushes or the amount of carboxyl groups along the PAA chains, the accessibility of the target analyte to the immobilized probe was limited at the high graft density of the PAA brushes. The effect was far more apparent for the BSA-anti-BSA probe-analyte pair than for the much smaller biotin-SA probe-analyte pair. The impact of the swellability of the PAA brushes, as tailored by the degree of carboxyl group activation, on both the sensing probe immobilization and analyte detection was also addressed. This investigation demonstrated that PAA brushes having a defined graft density have a promising potential as a precursor layer for biosensing applications.

Introducing surface-tethered poly(acrylic acid) brushes as 3D functional thin film for biosensing applications.

Carboxyl groups of surface-tethered poly(acrylic acid) (PAA) brushes should be able to serve as versatile moieties for a wide range of chemical modifications, including an attachment of bioactive species that can act as sensing probes for biosensors. In this research, poly(tert-butyl acrylate) (Pt-BA) brushes were prepared by surface-initiated atom transfer radical polymerization of tert-butyl acrylate. PAA brushes were then obtained after removal of the tert-butyl groups from the Pt-BA brushes by acid hydrolysis. The carboxyl group density of the PAA brushes can be varied as a function of chain length or molecular weight. The reactivity of the carboxyl groups of PAA brushes towards the immobilization of biotin, a frequently used model bioactive probe in biosensing applications, was evaluated. Qualitative determination of streptavidin (SA) binding to the biotin-attached PAA brushes was verified by fluorescence microscopy. The efficiency of the PAA brushes to act as a three dimensional (3D) precursor layer for biosensing applications was further demonstrated using surface plasmon resonance (SPR), where the biotin-attached PAA brushes showed an enhanced signal for the biospecific binding of SA in comparison with a self-assembled monolayer (SAM) of a carboxyl-terminated alkanethiol, used as a model two-dimensional (2D) conventional precursor layer. The PAA brushes showed very low non-specific interactions with two other tested proteins of a similar pI but different sizes. This desirable feature should be highly beneficial for the development of biosensors.

Multilayer film assembled from charged derivatives of chitosan: physical characteristics and biological responses.

Polyelectrolyte multilayer films were successfully assembled from each of the three charged derivatives of chitosan; N-[(2-hydroxyl-3-trimethylammonium)propyl]chitosan chloride (HTACC), N-succinyl chitosan (SCC) and N-sulfofurfuryl chitosan (SFC), paired with one of the two oppositely charged polyelectrolytes, poly(acrylic acid) (PAA) and poly(allylamine hydrochloride) (PAH) on surface-treated poly(ethylene terephthalate) (treated PET) substrates by alternate layer-by-layer adsorption. Surface coverage and wettability of the multilayer films were determined by AFM and water contact angle measurements, respectively. Analysis by quartz crystal balance with dissipation (QCM-D) has suggested that all multilayer films are relatively rigid and have a high water content associated within their structures, accounting for up to 85-90% (w/w) for films having 7-10 layers. In vitro cytocompatibility tests for the fibroblast-like L929 cell line revealed a slight dependency for cell adhesion and proliferation on the outermost layer. The multilayer film containing HTACC exhibited moderate antibacterial activity against E. coli and S. aureus. Bearing negative charges, the multilayer films terminating with SFC and having at least 10 layers were capable of suppressing the adsorption of plasma proteins and platelet adhesion at a comparable level to the multilayer film assembled from heparin, a well-known antithrombogenic polymer.

Patterned Poly(acrylic acid) Brushes Containing Gold Nanoparticles for Peptide Detection by Surface-Assisted Laser Desorption/Ionization Mass Spectrometry.

Patterned poly(acrylic acid) (PAA) brushes was successfully generated via photolithography and surface-initiated reversible addition-fragmentation chain transfer (RAFT) polymerization of acrylic acid as verified by water contact angle measurements and FT-IR analysis. The carboxyl groups of PAA brushes can act as reducing moieties for in situ synthesis of gold nanoparticles (AuNPs), without the use of additional reducing agent. The formation of AuNPs was confirmed by transmission electron microscopy and X-ray photoelectron spectroscopy. The glass surface-modified by PAA brushes and immobilized with AuNPs (AuNPs-PAA) can be used as a substrate for SALDI-MS analysis, which is capable of detecting both small peptides having m/z ≤ 600 (glutathione) and large peptides having m/z ≥ 1000 (bradykinin, ICNKQDCPILE) without the interference from matrix signal suggesting that AuNPs were stably trapped within the PAA brushes and the carboxyl groups of PAA can serve as internal proton source. By employing AuNPs as the capture probe, the AuNPs-PAA substrate can selectively identify thiol-containing peptides from the peptide mixtures with LOD as low as 0.1 and 0.05 nM for glutathione and ICNKQDCPILE, respectively. An ability to selectively detect ICNKQDCPILE in a diluted human serum is also demonstrated. The patterned format together with its high sensitivity and selectivity render this newly developed substrate a potential platform for high-throughput analysis of other biomarkers, especially those with low molecular weight in complex biological samples.

Surface plasmon resonance study of PNA interactions with double-stranded DNA

Peptide nucleic acid (PNA) is a well known DNA analogue bearing a N-(2-aminoethyl)glycine backbone (aegPNA). This molecule is able to not only form a duplex with single stranded (ss) nucleic acids but also higher-order (i.e., three- and four-stranded) complexes with double-stranded (ds) DNA in a sequence specific manner. Here, the application of surface plasmon resonance (SPR) to study the binding of PNA to dsDNA is reported for the first time. SPR protocols were developed to verify the sequence rules and conditions for binding (pH and ionic strength) of homopyrimidine and homopurine aegPNAs to dsDNA, for which the solution phase behaviors are known, allowing a direct comparison. Then, using real-time SPR measurements, the hybridization efficiency, binding direction (antiparallel and parallel direction), sequence-dependent binding modes of the PNA to dsDNA (triplex formation and duplex invasion) and the binding kinetics associated with the binding mode were all ascertained. These SPR protocols were then further applied to study the dsDNA binding properties of a new conformationally rigid PNA bearing a D-prolyl-2-aminocyclopentanecarboxylic acid (ACPC) backbone (acpcPNA), which revealed that acpcPNA cannot form higher-order complexes with dsDNA through either triplex formation or duplex invasion. The SPR technique is thus shown to be a powerful technique for studying higher-order nucleic acid complexes. The binding behaviors of aegPNA obtained from the SPR analysis in the solid-liquid phase measurement correlate well with those in the literature derived from solution phase measurements.