Addition of biological functionality to poly(epsilon-caprolactone) films

Biodegradable polyesters such as poly(epsilon-caprolactone) (PCL) have a number of biomedical applications; however, their usage is often limited by a lack of biological functionality. In this paper, a PCL-based polymer containing pendent groups activated by 4-nitrophenyl chloroformate (NPC) and reactive toward primary amines has been cast into thin films. The reactivity of the films toward poly(l-lysine) and the cell adhesion peptide, GRGDS, was assessed, and their cell adhesive capabilities were characterized. ATR-FTIR analysis found that NPC functional groups were present on the surface of the cast film, and the synthesis, conjugation, and visualization of a fluorescent molecule on these films further demonstrated the success of this functionalization methodology. The immersion of these films into a solution of either poly(l-lysine) (PLL) or GRGDS in PBS (pH 7.4) and subsequent 3T3 fibroblast adhesion studies demonstrated significant improvement in cell adhesion and spreading over films cast from unmodified PCL. This investigation has shown that this novel NPC-containing polymer can be utilized in many applications where increased cellular adhesion is required, or the coupling of specific molecules to polymer surfaces is of interest.     

Affinity separation using an Fv antibody fragment-"smart" polymer conjugate

Poly(N-isopropylacrylamide), or PNIPAAm, is considered a "smart" polymer because it sharply precipitates when heated above a critical temperature, about 32 degrees C in water, and redissolves when cooled. Conjugates made of PNIPAAm and IgG antibodies also exhibit the same critical temperature behavior. Interestingly, antigens that are complexed with these conjugates can also be phase-separated along with the conjugates. In this work, we conjugated PNIPAAm for the first time to the immunoglobulin Fv fragment, the smallest fragment of an antibody that still retains the antigenic affinity of the whole antibody. For our studies, we used an Fv fragment that strongly binds hen egg white lysozyme (HEL). The purified Fv fragment-polymer conjugate precipitated at the same temperature as did the pure polymer. After addition of the conjugate to a mixture containing HEL and after thermal separation of the conjugate at 37 degrees C, the amount of HEL in solution was reduced by as much as 80%. We were able to demonstrate the reversibility of the separation through three cycles of precipitation and dissolution. It was also possible to recover free HEL by thermal separation of the conjugate in the presence of an eluant, 50 mM diethylamine. The conjugate can then be recycled for second use. In conclusion, immunoseparations can be performed using smart polymer conjugates made with just the variable domains of an antibody. Unlike whole antibodies, fragments of antibodies can be produced in Escherichia coli, allowing easier genetic engineering of the antibody and tailoring of the conjugate.     

Simultaneous detection of six biohazardous agents using a planar waveguide array biosensor

Recently, we demonstrated that an array biosensor could be used with cocktails of fluorescent antibodies to perform three assays simultaneously on a single substrate, and that multiple samples could be analyzed in parallel. We extend this technology to demonstrate the simultaneous analysis of six samples for six different hazardous analytes, including both bacteria and protein toxins. The level of antibody cross-reactivity is explored, revealing a possible common epitope in two of the toxins. A panel of environmental interferents was added to the samples; these interferents neither prevented the detection of the analytes nor caused false-positive responses.     

Thermal stability of the helical structure of type IV collagen within basement membranes in situ: determination with a conformation-dependent monoclonal antibody

To examine the thermal stability of the helical structure of type IV collagen within basement membranes in situ, we have employed indirect immunofluorescence histochemistry performed at progressively higher temperatures using a conformation-dependent antibody, IV-IA8. We previously observed by competition enzyme-linked immunosorbent assay that, in neutral solution, the helical epitope to which this antibody binds undergoes thermal denaturation over the range of 37-40 degrees C. In the present study, we have reacted unfixed cryostat tissue sections with this antibody at successively higher temperatures. We have operationally defined denaturation as the point at which type IV- specific fluorescence is no longer detectable. Under these conditions, the in situ denaturation temperature of this epitope in most basement membranes is 50-55 degrees C. In capillaries and some other small blood vessels the fluorescent signal is still clearly detectable at 60 degrees C, the highest temperature at which we can confidently use this technique. We conclude that the stability of the helical structure of type IV collagen within a basement membrane is considerably greater than it is in solution, and that conformation-dependent monoclonal antibodies can be useful probes for investigations of molecular structure in situ.     

Dynamics of complex formation between biological and luminescent conjugated polyelectrolytes--a surface plasmon resonance study

A water-soluble polythiophene, POWT, with zwitterionic peptide like side chains possess good characteristics for biosensor applications. The zwitterionic side chains of the polymer can couple to biomolecules via electrostatic and hydrogen bonding. This creates possibilities to imprint biomolecules to spin-coated polymer films with maintained functionality, and use the resulting matrix as a biosensor. Polymer-biomolecular interaction studies done with surface plasmon resonance (SPR) reveal a well performing sensor matrix with high affinity for DNA hybridizations as well as for protein detection. The responses are distinct and very specific. A directional dependence of antibodies binding to POWT layer has also been observed. The polymer films have also been characterized by optical methods. Emission and absorption measurements in different buffer systems confirm that the polymer matrix can undergo structural and conformational changes on surfaces. The dielectric function in the interval 300-800 nm of POWT is reported, based on variable angle spectroscopic ellipsometry. This modeling reveals that a considerable amount of water is included in the material. The polymer layer possesses the characteristics needed for biochip applications and micro array techniques.     

Light microscope identification of murine B and T cells by means of functional polymeric microspheres.

Covalent bonding of purified antibodies to polymeric microspheres of 0.4 to 0.8 μm diameter yields conjugates which can be used to label lymphocytes in the light microscope. Nonadherent microspheres can be separated by means of a discontinuous density gradient and quantitative measurements of adherent microsphere distributions can be made through examination of Wright's stained dry mounts or through fluorescent microscopic examination of cells in suspension.In general the distributions of adherent microspheres on mouse splenic and thymic lymphocytes in direct or indirect labelling assays show good agreement with results obtained from fluorescent antibody techniques. In comparison to fluorescent antibody the use of these antibody-microsphere conjugates has the advantage of allowing direct correlations between the surface antigens of cells and their histologie morphology.     

Near real-time biosensor-based detection of 2,4-dinitrophenol

A fluorescent biosensor assay has been developed for near real-time detection of 2,4-dinitrophenol (DNP). The assay was based on fluorescent detection principles that allow for the analysis of antibody/antigen interactions in solution using the KinExA immunoassay instrument. Our KinExA consisted of a capillary flow observation cell containing a microporous screen that maintains a compact capture antigen-coated bead bed. The bead bed was comprised of polymethylmethacrylate (PMMA) beads coated with dinitrophenol-human serum albumin (DNP-HSA) conjugate. Phosphate buffered saline (PBS) solutions, containing various concentrations of free DNP, were incubated for 30 min with mouse anti-DNP monoclonal antibody to equilibrium. Solutions containing the DNP-monoclonal antibody complex and possible excess free antibodies were then passed over DNP-HSA labeled beads. The free monoclonal anti-DNP antibody, if available, was then bound to the DNP-HSA fixed on the beads. The system was then flushed with excess PBS to remove unbound reactants in the bead bed. The beads were then subjected to brief contact with PBS solutions containing goat anti-mouse fluorescein isothiocyanate (FITC)-labeled secondary antibody, once again, followed by a short PBS flush. The fluorescence was recorded during the addition of the FITC labeled secondary antibody to the bead bed through the final PBS flushing with the KinExA. The amount of DNP detected could then be determined from the fluorescent slopes that were generated or by the remaining fluorescence that was retained on the beads after final PBS flushing of the system. This assay has been able to detect a minimum of 5 ng/ml of DNP in solution and can be adapted for other analytes of interest simply by changing the capture antigen and antibody pairs.     

Cy5 labeled antimicrobial peptides for enhanced detection of Escherichia coli O157:H7

Fluorescently labeled antimicrobial peptides were evaluated as a potential replacement of labeled antibodies in a sandwich assay for the detection of Escherichia coli O157:H7. Antimicrobial peptides naturally bind to the lipopolysaccharide component of bacterial cell walls as part of their mode of action. Because of their small size relative to antibodies peptides can bind to cell surfaces with greater density, thereby increasing the optical signal and improving sensitivity. This method combines the specificity of a capture antibody with the increased sensitivity provided by using a labeled peptide as a detection molecule. The antimicrobial peptides cecropin P1, SMAP29, and PGQ were labeled with the fluorescent dye Cy5 via maleimide linker chemistry. Preliminary screening using a whole-cell solution binding assay revealed that Cy5 cecropin P1 enhanced the detection of E. coli O157:H7 relative to a Cy5 labeled anti-E. coli O157:H7 antibody 10-fold. Detection sensitivity of antibody and peptide were also compared with a prototype immuno-magnetic bead biosensor. Detection using Cy5 cecropin P1 resulted in a 10-fold improvement in sensitivity. Correlation of peptide antimicrobial activity with detection of E. coli O157:H7 indicated that activity was not predictive of the sensitivity of the fluorescent assay.     

Array biosensor for detection of biohazards

A fluorescence-based biosensor has been developed for simultaneous analysis of multiple samples for multiple biohazardous agents. A patterned array of antibodies immobilized on the surface of a planar waveguide is used to capture antigen present in samples; bound analyte is then quantified by means of fluorescent tracer antibodies. Upon excitation of the fluorophore by a small diode laser, a CCD camera detects the pattern of fluorescent antibody:antigen complexes on the waveguide surface. Image analysis software correlates the position of fluorescent signals with the identity of the analyte. This array biosensor has been used to detect toxins, toxoids, and killed or non-pathogenic (vaccine) strains of pathogenic bacteria. Limits of detection in the mid-ng/ml range (toxins and toxoids) and in the 10(3)-10(6) cfu/ml range (bacterial analytes) were achieved with a facile 14-min off-line assay. In addition, a fluidics and imaging system has been developed which allows automated detection of staphylococcal enterotoxin B (SEB) in the low ng/ml range.     

Amperometric hydrogen peroxide biosensor with sol-gel/chitosan network-like film as immobilization matrix

A new type of sol-gel/organic hybrid composite material based on the cross-linking of natural polymer chitosan with (3-aoryloxypropyl) dimethoxymethylsilane was developed for the fabrication of an amperometric H(2)O(2) biosensor. The composite film was used to immobilize horseradish peroxidase (HRP) on a gold disk electrode. The properties of sol-gel/chitosan and sol-gel/chitosan-HRP films have been carefully characterized by atomic force microscopy and Fourier transform infrared. By using fluorescent label, a protein density on sol-gel/chitosan has been calculated to be 3.14 x 10(12) moleculescm(-2). With the aid of catechol mediator, the biosensor had a fast response of less than 2 s with linear range of 5.0 x 10(-9)-1.0 x 10(-7) mol l(-1) and a detection limit of 2 x 10(-9) mol l(-1). Its current response shows a typical Michaelis-Menten mechanism. The apparent Michaelis-Menten constant K(M)(app) is found to be 1.30 micromol l(-1). The activation energy for enzymatic reaction is calculated to be 8.22 kJ mol(-1). The biosensor retained approximately 75% of its original activity after about 60 days of storage in a phosphate buffer at 4 degrees C.     

Influence of ibuprofen as a solid-state plasticizer in eudragit® RS 30 D on the physicochemical properties of coated beads

The purpose of this study was to investigate the physicochemical properties of nonpareil beads coated with Eudragit RS 30 D containing ibuprofen as a multifunctional agent. The influence of the concentration of ibuprofen in the film coating and the effect of the coating level on drug release from coated beads was determined in pH 7.2 phosphate buffer solution. The influence of storage time at 23 degrees C and 60 degrees C on the release of ibuprofen from coated beads was also investigated. The thermal properties of the films were determined using a differential scanning calorimeter. Scanning electron microscopy was employed to image the surface morphology of the coated beads. Infrared spectroscopy was used to study the interaction of Eudragit RS 30 D and ibuprofen. Results from the dissolution studies demonstrated that increasing the amount of ibuprofen in the polymeric film reduced the rate of drug release, mainly because of a more complete coalescence of the polymeric particles of the latex dispersion. The glass transition temperature (Tg) of Eudragit RS 30 D films decreased and the surface of the coated beads became smoother as the concentration of ibuprofen was increased. Hydrogen bonding between the polymer and ibuprofen was demonstrated by Fourier transform infrared spectroscopy. No significant differences were found in drug dissolution between the coated beads stored at 23 degrees C for 12 months and those stored at 60 degrees C for 12 hours. The results of this study demonstrated that the ibuprofen plasticized the Eudragit RS 30 D. Furthermore, the dissolution rate of ibuprofen can be controlled and changes in the drug release rate can be minimized by using the drug-induced plasticization technique with this polymer.     

Antibodies immobilized as arrays to profile protein post-translational modifications in mammalian cells

Previously, we demonstrated that antibodies printed on a solid support were able to detect protein-protein interaction in mammalian cells. Here we further developed the antibody array system for detecting proteins with various post-translational modifications in mammalian cells. In this novel approach, immunoprecipitated proteins were labeled with fluorescent dye followed by incubation over antibody arrays. Targeted proteins, captured by the antibodies immobilized on PVDF membrane or glass slide, were detected by means of near infrared fluorescent scanner or fluorescent microscopy. To demonstrate the application of the antibody arrays in protein post-translational modifications, we profiled protein tyrosine phosphorylation, ubiquitination, and acetylation in mammalian cells under different conditions. Our results indicate that antibody array technology can provide a powerful means of profiling a large number of proteins with different post-translational modifications in cells.     

Supramolecular formation of antibodies with viologen dimers: utilization for amplification of methyl viologen detection signals in surface plasmon resonance sensor

Monoclonal antibodies for 1-(carboxypentyl)-1'-methyl-4,4'-bipyridinium dichloride have been prepared. The complex formation of one of the antibodies, 10D5, with viologen dimer has been studied by a biosensor technique based on surface plasmon resonance. The dissociation constants of the complex between antibody 10D5 and methyl viologen or viologen dimer are found to be (2.0 +/- 0.2) x 10 (-7) and (1.5 +/- 0.5) x 10 (-7) M, respectively. Enhancement of response signal intensities in SPR is observed by the addition of the antibody solution to the viologen dimer-antibody complex indicating the formation of linear supramolecules between the antibody and viologen dimer. Amplification of methyl viologen sensing processes is realized by the inhibition of the complex formation between antibodies and viologen dimer-antibody complexes by methyl viologen and signal enhancement due to the supramolecular formation of the antibody and viologen dimer. The sensitivity in this system is found to be 2 orders larger than that obtained in the simple addition of methyl viologen to the antibody immobilized to the surface of the sensor chip.     

Green fluorescent antibodies: novel in vitro tools

We produced a fluorescent antibody as a single recombinant protein in Escherichia coli by fusing a red-shifted mutant of green fluorescent protein (EGFP) to a single-chain antibody variable fragment (scFv) specific for hepatitis B surface antigen (HepBsAg). GFP is a cytoplasmic protein and it was not previously known whether it would fold correctly to form a fluorescent protein in the periplasmic space of E.COLI: In this study we showed that EGFP alone or fused to the N'- and C'-termini of the scFv resulted in fusion proteins that were in fact highly fluorescent in the periplasmic space of E.COLI: cells. Further characterization revealed that the periplasmic N'-terminal EGFP-scFv fusion was the most stable form which retained the fluorescent properties of EGFP and the antigen binding properties of the native scFv; thus representing a fully functional chimeric molecule. We also demonstrated the utility of EGFP-scFv in immunofluorescence studies. The results showed positive staining of COS-7 cells transfected with HepBsAg, with comparable sensitivity to a monoclonal antibody or the scFv alone, probed with conventional fluorescein-labelled second antibodies. In this study, we developed a simple technique to produce fluorescent antibodies which can potentially be applied to any scFv. We demonstrated the utility of an EGFP-scFv fusion protein for immunofluorescence studies, but there are many biological systems to which this technology may be applied.     

Bioconjugation of CdTe quantum dot for the detection of 2,4-dichlorophenoxyacetic acid by competitive fluoroimmunoassay based biosensor

Quantum dots (QD) are semiconductor fluorescent nanoparticles, which can be made use of for environmental monitoring with high sensitivity. In view of the alarming levels of pesticides and herbicides being used in agriculture practices, there is a need for their rapid, sensitive and specific detection in food and environmental samples, as pesticides and herbicides are harmful to living beings even at trace levels. Present study was carried out to develop a reliable and rapid method for analysis and detection of 2,4-D (herbicide) using cadmium telluride quantum dot nanoparticle (CdTe QD). Fluoroimmunoassay based on the fluorescent property of quantum dot was used along with immunoassay to detect 2,4-D. CdTe capped with mercaptopropionic acid, was conjugated using N-(3-dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride (EDC) and a coupling reagent like N-hydroxysuccinimide (NHS) to alkaline phosphatase (ALP) which was in turn conjugated to 2,4-D molecule. Anti 2,4-D-IgG antibodies were immobilized in an immunoreactor column using Sepharose CL-4B as an inert matrix. The detection of 2,4-D was carried out by fluoroimmunoassay-based biosensor using competitive binding between conjugated 2,4-D-ALP-CdTe and free 2,4-D with immobilized anti 2,4-D antibodies in an immunoreactor column. It was possible to detect 2,4-D upto 250pgmL(-1). Present study also emphasizes on the resonance energy transfer between ALP and CdTe QD as a result of bioconjugation, which can be used for future biosensor development based on quantum dot-biomolecular interactions.     

Atomic force spectroscopy-based study of antibody pesticide interactions for characterization of immunosensor surface

Development of immunobiosensor detector surfaces involves the immobilization of active antibodies on the capture surface without any significant loss of antigen binding activity. An atomic force microscope (AFM) was used to directly evaluate specific interactions between pesticides and antibodies on a biosensor surface. Oriented immobilization of antibodies against two herbicide molecules 2,4-dichlorophenoxyacetic acid (2,4-D) and atrazine, on gold, was carried out to create the active immunobiosensor surfaces. The adhesive forces between immobilized antibodies and their respective antigens were measured by force spectroscopy using hapten-carrier protein functionalized AFM cantilevers. Relative functional affinity (avidity) measurements of the antibodies carried out prior to immobilization, well correlated with subsequent AFM force measurement observations. Analysis showed that immobilization had not compromised the reactivity of the surface immobilized antibody molecules for antigen nor was there any change in their relative quality with respect to each other. The utility of the immunoreactive surface was further confirmed using a Surface Plasmon Resonance (SPR) based detection system. Our study indicates that AFM can be utilized as a convenient immunobiosensing tool for confirming the presence and also assessing the strength of antibody-hapten interactions on biosensor surfaces under development.     

The investigation of Protein A and Salmonella antibody adsorption onto biosensor surfaces by atomic force microscopy

The investigation of Protein A and antibody adsorption on surfaces in a biological environment is an important and fundamental step for increasing biosensor sensitivity and specificity. The atomic force microscope (AFM) is a powerful tool that is frequently used to characterize surfaces coated with a variety of molecules. We used AFM in conjunction with scanning electron microscopy to characterize the attachment of protein A and its subsequent binding to the antibody and Salmonella bacteria using a gold quartz crystal. The rms roughness of the base gold surface was determined to be approximately 1.30 nm. The average step height change between the solid gold and protein A layer was approximately 3.0 +/- 1.0 nm, while the average step height of the protein A with attached antibody was approximately 6.0 +/- 1.0 nm. We found that the antibodies did not completely cover the protein A layer, instead the attachment follows an island model. Salt crystals and water trapped under the protein A layer were also observed. The uneven adsorption of antibodies onto the biosensor surface might have led to a decrease in the sensitivity of the biosensor. The presence of salt crystals and water under the protein A layer may deteriorate the sensor specificity. In this report, we have discussed the application and characterization of protein A bound to antibodies which can be used to detect bacterial and viral pathogens.     

Peptides, antibodies, and FRET on beads in flow cytometry: A model system using fluoresceinated and biotinylated beta-endorphin.

BACKGROUND: Particulate surfaces such as beads are routinely used as platforms for molecular assembly for fundamental and practical applications in flow cytometry. Molecular assembly is transduced as the direct analysis of fluorescence, or as a result of fluorescence resonance energy transfer. Binding of fluorescent ligands to beads sometimes alters their emission yield relative to the unbound ligands. Characterizing the physical basis of factors that regulate the fluorescence yield of bound fluorophores (on beads) is a necessary step toward their rational use as mediators of numerous fluorescence based applications. METHODS: We have examined the binding between two biotinylated and fluoresceinated beta-endorphin peptides and commercial streptavidin beads using flow cytometric analysis. We have analyzed the assembly between a specific monoclonal antibody and an endorphin peptide in solution using resonance energy transfer and compared the results on beads in flow cytometry using steady-state and time-resolved fluorescence. RESULTS: We have defined conditions for binding biotinylated and fluoresceinated endorphin peptides to beads. These measurements suggest that the peptide structure can influence both the intensity of fluorescence and the mode of peptide binding on the bead surface. We have defined conditions for binding antibody to the bead using biotinylated protein A. We compared and contrasted the interactions between the fluoresceinated endorphin peptide and the rhodamine- labeled antibody. In solution we measure a K(d) of <38 nM by resonance energy transfer and on beads 22 nM. DISCUSSION: Some issues important to the modular assembly of a fluorescence resonance energy transfer (FRET) based sensing scheme have been resolved. The affinity of peptides used herein is a function of their solubility in water, and the emission intensity of the bound species depends on the separation distance between the fluorescein and the biotin moiety. This is due to the quasi-specific quenching interaction between the fluorescein and a proximal binding pocket of streptavidin. Detection of antibodies in solution and on beads either by FRET or capture of fluorescent ligands by dark antibodies subsequently enables the determination of K(d) values, which indicate agreement between solution and flow cytometric determinations.     

Multi-analyte assay for triazines using cross-reactive antibodies and neural networks

A biosensor system based on total internal reflectance fluorescence (TIRF) was used to discriminate a mixture of the triazines atrazine and simazine. Only cross-reactive antibodies were available for these two analytes. The biosensor is fully automated and can be regenerated allowing several hundreds of measurements without any user input. Even a remote control for online monitoring in the field is possible. The multivariate calibration of the sensor signal was performed using artificial neural networks, as the relationship between the sensor signals and the concentration of the analytes is highly non-linear. For the development of a multi-analyte immunoassay consisting of two polyclonal antibodies with cross-reactivity to atrazine and simazine and different derivatives immobilised on the transducer surface, the binding characteristics between these substances like binding capacity and cross-reactivity were characterised. The examination of three different measurement procedures showed that a two-step measurement using only one antibody per step allows a quantification of both analytes in a mixture with limits of detection of 0.2 microg/l for atrazine and 0.3 microg/l for simazine. The biosensor is suitable for online monitoring in the field and remote control is possible.     

Development of a Cryptosporidium oocyst assay using an automated fiber optic-based biosensor

An intestinal protozoan parasite, Cryptosporidium parvum, is a major cause of waterborne gastrointestinal disease worldwide. Detection of Cryptosporidium oocysts in potable water is a high priority for the water treatment industry to reduce potential outbreaks among the consumer populace. Anti-Cryptosporidium oocyst polyclonal and monoclonal antibodies were tested as capture and detection reagents for use in a fiber optic biosensor assay for the detection of Cryptosporidium oocysts. Antibodies were validated using enzyme-linked immunosorbent assays, flow cytometry, Western blotting and fluorescent microscopy. Oocysts could be detected at a concentration of 10⁵ oocysts/ml when the polyclonal antibodies were used as the capture and detection reagents. When oocysts were boiled prior to detection, a ten-fold increase in sensitivity was achieved using the polyclonal antibody. Western blotting and immunofluorescence revealed that both the monoclonal and polyclonal antibodies recognize a large (>300 kDa) molecular weight mucin-like antigen present on the surface of the oocyst wall. The polyclonal antibody also reacted with a small (105 kDa) molecular weight antigen that was present in boiled samples of oocysts. Preliminary steps to design an in-line biosensor assay system have shown that oocysts would have to be concentrated from water samples and heat treated to allow detection by a biosensor assay.