Sortase A-catalyzed site-specific coimmobilization on microparticles via streptavidin

A microparticle surface was designed by the unique method incorporating streptavidin-biotin affinity and sortase A (SrtA)-catalyzed transpeptidation. Leucine-proline-glutamate-threonine-glycine-tagged streptavidin (Stav-LPETG)was immobilized on the surface using streptavidin-biotin affinity, and GGGGG-tagged red fluorescent protein (Gly5-RFP) was conjugated with SrtA. Biotinylated fluorescein isothiocyanate (biotin-FITC) was then bound to residual biotin-binding sites in Stav-LPETG. The resulting particles had RFP and FITC immobilized on the surface via Stav-LPETG, and RFP- and FITC-associated fluorescence was observed using fluorescence microscopy. Finally, GGG-tagged glucose oxidase and biotinylated horseradish peroxidase were immobilized on the microparticle surface, resulting in a functional particle capable of detecting glucose. This particle can be repeatedly used and is more sensitive in detecting glucose than particles prepared using chemical modification. Our method provides a simple strategy for site-specific coimmobilization on molecular surfaces and expands the use of protein hybrid devices.     

A new protein conformation indicator based on biarsenical fluorescein with an extended benzoic acid moiety.

We demonstrate herein a new protein conformation indicator based on biarsenical fluorescein with an extended benzoic acid moiety. The present indicator is reactive to a genetically introduced tetracysteine motif (Cys-Cys-Xaa-Xaa-Cys-Cys, where Xaa is a noncysteine amino acid) of proteins. Compared to the original biarsenical fluorescein (FlAsH) and the biarsenical Nile red analogue (BArNile), the present indicator exhibited larger fluorescence intensity changes in response to Ca(2+)-induced conformational rearrangements of calmodulin. A calculation of the highest occupied molecular orbital (HOMO) level of the benzoic acid moiety of the indicator molecule supports possible involvement of a photoinduced electron transfer (PET) process. These results indicate that the present indicator is useful for sensitive detection of protein conformational changes.     

Immunosensor based on fluorescence quenching matrix of the conducting polymer polypyrrole

In this study, the combination of autofluorescent proteins and fluorescence quenching polymers was shown to be a design which can increase the selectivity and sensitivity of immunosensors. With this objective, the conducting polymer polypyrrole (Ppy) was used as a matrix for immobilization of proteins, which enables biological recognition of the analyte, and as a fluorescence quencher, which increases the selectivity of fluorescence-based detection. In this study, bovine leukemia virus proteins gp51 were immobilized within the Ppy matrix and formed a polymeric layer with affinity for antibodies against protein gp51 (anti-gp51). The anti-gp51 antibodies are present at high levels in the blood serum of cattle infected by bovine leukemia virus. Secondary antibodies labeled with horseradish peroxidase (HRP) were used as specific fluorescent probes for detection of a particular target, because the fluorescence of HRP was readily detectable at the required sensitivity. The Ppy was used as fluorescent background, because its fluorescence was almost undetectable when excited by near UV light at 325 nm. Moreover the Ppy quenched the fluorescence of some fluorescent agents including fluorescein-5(6)-isothiocyanate (fluorescein), rhodamine B, and HRP by almost 100% when these fluorescent agents were adsorbed on the surface of Ppy. It is predicted that Ppy-induced fluorescence quenching could be used in the design of immunosensors to increase selectivity and sensitivity.     

Molecular organization of hydrophobic molecules and co-adsorbed water in SBA-15 ordered mesoporous silica material

The purpose of this study was to improve our understanding of the molecular organization of hydrophobic guest molecules in the presence of co-adsorbed water inside SBA-15 ordered mesoporous silica material. Understanding this adsorption competition is essential in the development of applications of controlled adsorption and desorption. The poorly water soluble drug compound itraconazole and the fluorescent probe Nile red were selected for the study. The interaction between itraconazole and SBA-15 was investigated using FT-IR, (1)H MAS NMR and (29)Si MAS NMR spectroscopy, by determination of adsorption isotherms and release kinetics in simulated gastric fluid. The distribution and migration of the hydrophobic fluorescent probe Nile red was visualized in situ using confocal fluorescence microscopy. For both molecules, there was a pronounced influence of the co-adsorbed water on adsorption, hydrophobic aggregation and migration in SBA-15 pores. These insights contribute to the development of practical methods for loading ordered mesoporous silica materials with hydrophobic molecules.     

Selective interactions of concanavalin A at lipid membranes on the surface of an optical fiber

An optical configuration was developed for sampling fluorescence coupled into an optical fiber from evanescent wave excitation of fluorescent materials at a lipid membrane on a quartz fiber surface. Selective interactions of pyrene-labelled concanavalin A located on a phosphatidyl choline-cholesterol lipid membrane with fluorescein isothiocyanate-labelled dextran in bulk aqueous solution were monitored by the intrinsic fluorescence sensing configuration. Monosialoganglioside, G(M1), was employed as a receptor in a phospholipid membrane on an optical fiber for selective measurement of pyrene-labelled concanavalin A in solution. Quantitative measurement was hindered by non-selective adsorption of cancanavalin A, but the potential for use of a lipid membrane in a fluorometric biosensor was established.     

Fluorescent,magnetic dual-responsive molecularly imprinted polymers for the selective detection of moxidectin in animal samples

A simple and effective approach is presented to fabricate fluorescent and magnetic dual-responsive molecularly imprinted polymers for selective recognition of moxidectin. Magnetic gelatin was prepared to provide not only easy magnetic separation, but also significant amino functionalities, allowing surface modification with fluorescein isothiocyanate isomer I and acrylic acid (AA). Based on the double bond provided by AA, molecularly imprinted polymers could be directly coated onto the surface of modified magnetic gelatin. The dual-responsive imprinting polymers feature a high adsorption capacity (87.1 mg g^?1), selective fluorescence response toward moxidectin (imprinting factor = 3.6), rapid magnetic separation, and good reproducibility. Subsequently, the dual-responsive composites were successfully applied as sorbents for selective determination of moxidectin. Upon optimization, a linear range of 10–1000 ng mL^?1 and a detection limit of 6 ng mL^?1 were achieved. The results agree well with those obtained by the classic HPLC–FLD method (r = 0.9935). This developed strategy may find its potential application in rapid, simple, sensitive, and selective determinations of target molecules in complex samples.     

光纤荧光免疫分析系统的研制及应用

将光纤传感技术、荧光分析与免疫分析技术结合,自行设计了Y型分叉光纤、光强可调的光纤固定架及性能优良的荧光测定池,建立了新型光纤荧光免疫系统。系统性能可靠,操作简便,既可进行普通的荧光分析,又可进行光纤荧光免疫分析。将系统用于临床血清标本中肺炎支原体抗体的测试,结果与荧光显微镜法和ELISA法结果一致。该新型光纤荧光免疫系统在临床、环保等领域有广泛的应用前景。     

Preliminary assessment of the modification of polystyrene‐divinylbenzene resin with lipid‐tethered ligands for selective separations

Functionalized lipid tethered ligands use physical adsorption to anchor reactive head groups to hydrophobic supports. We previously demonstrated the use of these species adsorbed onto polypropylene capillary‐channeled polymer fibers. The general use of lipid tethered ligands on other hydrophobic chromatographic supports is demonstrated here for polystyrene‐divinylbenzene. Evaluation of ligand adsorption conditions was performed using a fluorescein isocyanate head group to quantify the extent of loading by UV‐Vis absorbance and by fluorescence microscopy. Selective protein capture was demonstrated by the detection of Texas Red labeled streptavidin (using fluorescence microscopy imaging, with quantification assessed through the depletion of solution‐phase protein using UV‐Vis absorbance. A second demonstration of the coupling involved an iminodiacetic acid head group lipid tethered ligand to capture the cationic dye, methylene blue. Two common means of alleviating nonspecific binding, adsorption in detergent media and use of a bovine serum albumin block, were evaluated. The first was found to cause release of the ligands, while the second was nominally effective. Indeed, the lipid tethered ligands itself may be most effective at impeding nonspecific binding. While further optimization and chromatographic evaluation is required, the general viability of this ligand immobilization method onto this common polymer support is demonstrated.     

Functionalised, photostable, fluorescent polystyrene nanoparticles of narrow size-distribution

Fluorescent nanoparticles continue to be of wide interest, as they have many advantages over single fluorescent molecules for biological imaging and sensing applications, such as increased fluorescence intensity and reduced photobleaching. In the following work, styrene was copolymerised with a newly synthesised, fluorescein-based, vinylic crosslinking monomer, by emulsion polymerisation, to create a series of different sized fluorescent nanoparticles (35-100 nm), each of narrow size-distribution. The particles were found to be highly fluorescent and with lower photobleaching compared to fluorescein isothiocyanate (FITC), offering an attractive alternative. The fluorescence excitation and emission spectra were recorded, being similar to fluorescein, but with interesting variation in the excitation spectra. The particles also have a wide range of potential uses, such as examining particle uptake activity of a macrophage cell line, also demonstrated. The nanoparticles were coated with albumin to provide functionality for potential conjugation to biological targeting agents.     

Using bifunctional polymers presenting vancomycin and fluorescein groups to direct anti-fluorescein antibodies to self-assembled monolayers presenting d-alanine-d-alanine groups

This paper describes the synthesis of bifunctional polyacrylamides containing pendant vancomycin (Van) and fluorescein groups, and the use of these polymers to direct antibodies against fluorescein to self-assembled monolayers (SAMs) presenting d-alanine-d-alanine (dAdA) groups. These polymers bind biospecifically to these SAMs via interactions between the dAdA and Van groups and serve as a molecular bridge between the anti-fluorescein antibodies and the SAM. The binding events were characterized using surface plasmon resonance spectroscopy and fluorescence microscopy. The paper demonstrates that polyvalent, biospecific, noncovalent interactions between a polymer and a surface can be used to tailor the properties of the surface in molecular recognition. It also represents a first step toward the design of polymers that direct arbitrarily chosen antibodies to the surfaces of cells.     

Functionalization of solid surfaces with thermoresponsive protein-resistant films

Pulsed plasma polymerization of N-isopropylacrylamide leads to the deposition of thermoresponsive films. The reversible (switching) behavior of these poly(N-isopropylacrylamide) surfaces has been exemplified by screening the adsorption of fibrinogen and fluorescein isothiocyanate labeled bovine serum albumin proteins by surface plasmon resonance (SPR) and fluorescence microscopy at low and elevated temperatures.     

Whole-column fluorescence-imaged capillary electrophoresis

An axially illuminating whole-column fluorescence imaging capillary electrophoresis (CE) experimental setup was developed. A 6 cm long Teflon tube with an inside diameter (ID) of 42 microm was used as separation column. Excitation light of 488 nm from Ar+ laser was introduced to one end of the separation column by an optical fiber. The excitation light propagated inside the separation column by total internal reflection, since the refractive index of the buffer solution was larger than that of the Teflon tube. The fluorescence from the whole separation column was imaged with a charge-coupled device (CCD) camera. Fluorescent compounds such as fluorescein isothiocyanate (FITC), 5-carboxyfluorescein, and FITC-labeled protein were used to test the basic performance of the experimental setup. Experimental results illustrate that the whole-column-fluorescence imaging CE is a fast and sensitive separation method for fluorescent compounds and fluorescent-labeled proteins. Furthermore, it could be used for simple, fast, and easy comparisons of the resistance to photodegradation for various fluorescent compounds.     

Core‐shell type dually fluorescent polymer nanoparticles for ratiometric pH‐sensing

The synthesis and pH‐sensing properties of fluorescent polymer nanoparticles (NPs) in the 20 nm diameter range with a sensitive dye covalently attached to the particle surface and a reference dye entrapped within the particle core are presented. Fluorescein‐functionalized NPs were readily obtained by conjugation of fluorescein isothiocyanate (FITC) to amine‐coated crosslinked polystyrene‐based nanoparticles prepared by microemulsion polymerization followed by postfunctionalization. This all water‐based method gave access to stable aqueous suspensions of pH‐sensing fluorescent NPs. The encapsulation of the insensitive reference fluorescent dye (1,9‐diphenylanthracene, DPA) was then conveniently achieved by soaking leading to dual fluorescent NPs containing about 20 DPA and 55 fluorescein, as deduced from spectroscopic analyses. This core‐shell type architecture maximizes the interactions of the sensing dye with the medium while protecting the reference dye. The variations of the ratio of the fluorescence emission intensities of the sensitive dye (fluorescein) to the reference dye (DPA) with pH show that the dual fluorescent NPs act as a ratiometric pH sensor with a measuring range between pH 4 and pH 8. This pH nanosensor was found to be fast, fully reversible, and robust without any leaching of dye over a long period of time. © 2008 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 46: 6206–6213, 2008     

Interactions of alkyl triphenyl phosphonium bromides with aqueous solutions of LM200 and JR400 polymers

The interactions of the cationic cellulose ether derivatives JR400 and the hydrophobically modified derivative LM200 with cationic alkyl triphenylphosphonium bromides, were investigated in aqueous media. Conductometric, tensiometric and fluorescence techniques were employed in this study. The presence of polymer induced surfactant aggregation and polymer bound aggregates, were detected for C₁₆ and C₁₄ triphenylphosphonium bromide surfactants with LM200. Gibbs free energy of transfer and the dielectric constant values sensed by the fluorescent probe at the micellar interface were evaluated and discussed in terms of strength of interaction between the polymers and surfactants.     

Highly efficient and non-doped red conjugated polymer dot for photostable cell imaging

By introducing a naphthothiadiazole (NT) unit as the main building block, a non-doped and red emissive conjugated polymer poly(9,9-dihexylfluorene-alt-naphthothiadiazole) (PFNT) is readily obtained through a two-step synthesis. Since the NT unit has a large twist angle with its neighboring segment, the aggregation-induced quenching (AIQ) effect of PFNT can be effectively suppressed in the condensed state. As a result, the corresponding PFNT polymer dot (Pdot) exhibits a high fluorescence quantum yield of 53.2% with peak emission at 616 nm, which is one of the most efficient red Pdots known. PFNT Pdot shows good biocompatibility and can be employed for living cell fluorescent imaging with high brightness. It also can be used for specific subcellular organelle imaging through immunofluorescence labeling. Furthermore, the PFNT Pdot demonstrates much better photostability for long-time cell fluorescence imaging than commercial red dyes. The high performances of PFNT Pdot make it a promising fluorescent probe for practical bioapplications.     

The effect of surface microtopography of poly(dimethylsiloxane) on protein adsorption, platelet and cell adhesion

Chemical homogeneous poly(dimethylsiloxane) (PDMS) surface with dot-like protrusion pattern was used to investigate the individual effect of surface microtopography on protein adsorption and subsequent biological responses. Fibrinogen (Fg) and fibronectin (Fn) were chosen as model proteins due to their effect on platelet and cell adhesion, respectively. Fg labeled with ¹²⁵I and fluorescein isothiocyanate (FITC) was used to study its adsorption on flat and patterned surfaces. Patterned surface has a 46% increase in the adsorption of Fg when compared with flat surface. However, the surface area of the patterned surface was only 8% larger than that of the flat surface. Therefore, the increase in the surface area was not the only factor responsible for the increase in protein adsorption. Clear fluorescent pattern was visualized on patterned surface, indicating that adsorbed Fg regularly distributed and adsorbed most on the flanks and valleys of the protrusions. Such distribution and local enrichment of Fg presumably caused the specific location of platelets adhered from platelet-rich plasma (PRP) and flowing whole blood (FWB) on patterned surface. Furthermore, the different combination of surface topography and pre-adsorbed Fn could influence the adhesion of L929 cells. The flat surface with pre-adsorbed Fn was the optimum substrate while the virgin patterned surface was the poor substrate in terms of L929 cells spread.     

Activity study of self-assembled proteins on nanoscale diblock copolymer templates

Novel methods for affixing functional proteins on surfaces with high areal density have the potential to promote basic biological research as well as various bioarray applications. The use of polymeric templates under carefully balanced thermodynamic conditions enables spontaneous, self-assembled protein immobilization on surfaces with spatial control on the nanometer scale. To assess the full potential of such nanometer-scale protein platforms in biosensing applications, we report for the first time the biological activity of proteins on diblock copolymer platforms. We utilized horseradish peroxidase, mushroom tyrosinase, enhanced green fluorescent protein, bovine immunoglobulin G, fluorescein isothiocyanate conjugated anti-bovine IgG, and protein G as model systems in our protein activity studies. When specific catalytic functions of HRP and MT, immobilized on selective domains of microphase-separated PS-b-PMMA, are evaluated over a long period of time, these enzymes retain their catalytic activity and stability for well over 3 months. By performing confocal fluorescence measurements of self-fluorescing proteins and interacting protein/protein systems, we have also demonstrated that the binding behavior of these proteins is unaffected by surface immobilization onto PS-b-PMMA diblock copolymer microdomains. Our polymer platforms provide highly periodic, high-density, functional, stable surface-bound proteins with spatial control on the nanometer scale. Therefore, our diblock copolymer-guided protein assembly method can be extremely beneficial for high-throughput proteomic applications.     

Biocompatible polymers for antibody support on gold surfaces

The elimination or minimization of non-specific protein adsorption from serum is critical for the use of surface plasmon resonance (SPR) sensors for in vitro and in vivo analysis of complex biological solutions. The ultimate goals in this application are to minimize non-specific adsorption of protein and to maximize analyte signal. A reduction of the non-specific protein adsorption from serum of up to 73% compared to carboxymethylated-dextran 500 kDa (CM-dextran) was achieved following a survey of eight biocompatible polymers and 10 molecular weights of CM-dextran. These coatings minimize non-specific adsorption on the sensor while also serving as immobilization matrices for antibody fixation to the probes. Polymers including polysaccharides: CM-dextrans, CM-hyaluronic acid, hyaluronic acid, and alginic acid were investigated. Humic acid, polylactic acid, polyacrylic acid, orthopyridyldisuldfide–polyethyleneglycol–N-hydroxysuccinimide (OPSS–PEG–NHS), and a synthesized polymer; polymethacrylic-acid-co-vinyl-acetate (PMAVA) were also used. The non-specific protein adsorption reduction was measured over a 14 day period at 0 °C for each polymer. Calibration curves using some of these polymers were constructed to show the performance and low detection limit possibilities of these new antibody supports. For many of the polymers, this is the first demonstration of employment as an antibody support for an optical or surface active sensor. CM-dextran is the polymer offering the largest signal for the antigen detection. However, the biocompatible polymers demonstrate a greater stability to non-specific binding in serum. These biocompatible polymers offer different alternatives for CM-dextran.     

Effect of surface grafted polymers on the adsorption of different model proteins

Adsorption of a model protein to a surface with end-grafted polymers was studied by Monte Carlo simulations. In the model the effect on protein adsorption in the presence of end-grafted polymers was evaluated by calculating the change in free energy between an end-grafted surface and a surface without polymers. The change in free energy was calculated using statistical mechanical perturbation theory. Apart from ordinary athermal polymer-polymer and protein-polymer interactions we also study a broad selection of systems by varying the interaction between proteins and polymers and effective polymer-solvent interactions. The interactions between the molecules span an interval from -0.5 to +0.5 kT. Consequently, general features of protein adsorption to end-grafted surfaces is investigated by systematically changing properties like hydrophilicity/hydrophobicity of the polymer, protein and surface as well as grafting density, degree of polymerization and protein size. Increasing grafting density as well as degree of polymerization decreases the adsorption of protein except in systems with attractive polymer-protein interactions, where adsorption increases with increasing chain length and higher grafting density. At a critical polymer-protein interaction neither chain length nor grafting density affects the free energy of adsorption. Hydrophilic polymers were found to prevent adsorption better than hydrophobic polymers. Very small particles with radii comparable to the size of a polymer segment were, however, better excluded from the surface when using hydrophobic than hydrophilic polymers. For systems with attractive polymer-protein interaction not only the volume of the protein was shown to be of importance but also the size of the exposed surface.     

Fluorescein-based pI markers for capillary isoelectric focusing with laser-induced fluorescence detection

We prepared a series of low-molecular-mass fluorescent ampholytes with narrow pI range. These fluorescein-based ampholytes are detection compatible with argon laser-induced fluorescence (LIF) detection. The selected properties, important for their routine use as fluorescent pI markers, were examined. The pI values of new fluorescein-based pI markers were determined by capillary isoelectric focusing (CIEF) using currently available low-molecular-mass pI markers for CIEF with photometric detection. The examples of CIEF with fluorometric detection of new compounds together with fluorescein isothiocyanate (FITC) derivatized proteins are presented.