Total Internal Reflection Fluorescence Spectroscopy Research Articles

Ellipsometry and TIRF studies of adsorption processes in parenteral drug delivery

Ellipsometry and total internal reflection fluorescence spectroscopy(TIRF) were used for investigating adsorption processes of relevance forparenteral administration of colloidal drug carriers. Emphasis is put ondiscussing the effects of both protein and surface properties on theadsorption of serum proteins at phospholipid and other surfaces.Furthermore, the adsorption from multicomponent protein systems, such asblood, is addressed, and both competitive and associative adsorptionphenomena discussed. The correlation between effects of the drug carriersurface properties on the serum protein adsorption and the circulation timeand tissue distribution of colloidal drug carriers is also addressed.Finally, the potential of ellipsometry in another adsorption process ofmajor importance for phagocytosis, i.e., the adsorption of colloidalparticles to macroscopic or mesoscopic surfaces, is indicated byinvestigations of the adsorption of oil-in-water emulsion droplets atsilica.

Laser implantation of fluorescent molecules into polymer films

A new method to implant molecules into polymers by nanosecond pulse laser irradiation has been developed on the basis of studies of laser ablation. Fluorescent molecules were implanted into polymer films having different glass transition temperature. The distribution of fluorescent molecules in an implanted layer was obtained by using total internal reflection fluorescence spectroscopy.

Adsorption of Complement Protein C3 at Polymer Surfaces

The adsorption of C3 at poly(methyl methacrylate) (PMMA) and poly(styrene) (PS) surfaces was investigated within situellipsometry and compared to that at (hydrophilic and negatively charged) silica and (hydrophobic) methylated silica. The adsorption of C3 at PMMA was higher than that at PS, while the adsorbed layer thickness was the same for the two surfaces. For both PMMA and PS the adsorbed layer thickness (10 ± 2 nm) corresponds rather closely to that of end-on oriented C3 molecules. The adsorption of C3 at PMMA and PS was found to be intermediate between that at silica and methylated silica, although the adsorbed layer thickness was similar for all surfaces. The competitive adsorption among C3, human serum albumin (HSA), and factor B was investigated with ellipsometry and total internal reflection fluorescence spectroscopy (TIRF). Addition of HSA after C3 preadsorption resulted in fractional C3 desorption for both PMMA and PS. Factor B deposition at PS after preadsorption of C3 and blocking with HSA was found to be largely due to specific binding to C3/C3b, while in the case of PMMA, factor B was largely accumulated through passive (displacement) adsorption.

Effects of Hydrophilization and Immobilization on the Interfacial Behavior of Immunoglobulins

The adsorption and immobilization of rabbit anti-human immunoglobulin (rabbit IgG), as well as the effects of rinsing with buffer and addition of bovine serum albumin (BSA) or human IgG on the amount and reactivity of bound rabbit IgG, were investigated with ellipsometry, total internal reflection fluorescence spectroscopy (TIRF), and enzyme immuno assay (EIA). It was found that although rabbit IgG readily adsorbs at hydrophobic hexamethyldisiloxane (HMDSO) plasma polymer surfaces, a substantial fraction of the adsorbed protein molecules is desorbed upon rinsing with buffer. BSA was found to adsorb readily at the surfaces obtained after rinsing, although also this protein desorbed to a large extent (about 60%) upon further rinsing with buffer. The adsorption of BSA causes a further reduction in the amount of rabbit IgG adsorbed. Immobilization of rabbit IgG to acrylic acid (AA) plasma polymer surfaces, achieved by covalent coupling via a strongly adsorbed PEG–PEI copolymer, was found to overcome the problem of the desorption of rabbit IgG upon rinsing with buffer or addition of BSA. Furthermore, nonspecific adsorption was virtually absent after immobilization. However, covalently bound rabbit IgG reacted strongly with human IgG, as observed by ellipsometry, TIRF, and EIA. The immobilization of rabbit IgG to hydrophilized surfaces was found to facilitate the interpretation of EIA results.

A Realistic Diffusion Model for Ultrathin Polyelectrolyte Films

In order to obtain information on transport processes in ultrathin films of polyelectrolytes, we studied the time dependence of the fluorescence of a dye (FITC) attached to a polymer and embedded in a defined, variable depth inside the film by total internal reflection fluorescence (TIRF) spectroscopy. Adding a rhodamine solution to the outer aqueous phase, we observe FITC quenching due to energy transfer and from this determine surface concentration and permeability of rhodamine. We derive a diffusion coefficient on the order of 10-15 cm2/s. This value can be manipulated by changing the film structure. So, the diffusion coefficient varies by nearly 2 orders of magnitude going along the surface normal. Studying the paramagnetic quenching by the smaller spin label TEMPOL, we observe that the diffusion coefficient is more than a factor of 200 larger for this molecule.

Kinetic-Diffusive-Convective Adsorption in TIRF Flow Cells

A quantitative theory of kinetic-diffusive-convective adsorption in the flow cell for total internal reflection fluorescence spectroscopy is developed for arbitrary adsorption isotherms. The effect of the convective transport is estimated in an analytical form for the short-time and long-time approximations. The boundary condition for the rate of adsorption at the interface (y = 0) is derived for kinetics-diffusion-convection controlled adsorption (Vx ≠ 0) in the flow cell, taking into account both the normal and the axial fluxes in the flow cell. The adsorption process in the flow cell for short times (t - tm → 0) is controlled by adsorption kinetics or by diffusive transfer when the rates of adsorption and desorption are infinite, and for long times (t → ∞) is controlled by diffusive and convective transfer. The simple formulae are derived to calculate from the experimental data the adsorption isotherm and the diffusion coefficient.

Variable-angle ultraviolet total-internal-reflection fluorescence spectroscopy using a white excitation light source

An apparatus for variable-angle ultraviolet total internal reflection fluorescence (TIRF) spectroscopy is described. TIRF spectroscopy is an analytical technique based on the excitation of fluorescence from a sample by means of an exponentially decaying surface energy wave. The limited penetration depth of the excitation volume results in excitation of fluorescence only near the sample/substrate interface. The angular spectrum makes TIRF spectroscopy suitable for obtaining quantitative information on the depth profile of a sample. In the instrument described here, precise positioning of incident optics with correction of the chromatic aberration due to the xenon light source enables the measurement of the excitation spectrum. The system provides the fluorescence and excitation spectra for study of the physical and chemical properties of thin-film structures. The incident- and observation-angle dependences of those spectra give important information on the depth profiles of the fluorophores in the samples. The performance of the instrument is demonstrated with thin polymer films containing a fluorescent probe of pyrene. Pyrene monomer is found to form the nonuniform depth profile different from that of pyrene excimer.

Competitive protein adsorption at phospholipid surfaces

The interfacial exchange processes between human serum albumin (HSA) and fibrinogen on different surfaces was investigated with in situ ellipsometry and total internal reflectance fluorescence (TIRF) spectroscopy. With ellipsometry, it was found that the total absorbed amount on silica on the addition of fibrinogen after the preadsorption of HSA was quite similar to that obtained without HSA preadsorption. From TIRF, it was concluded that the preadsorbed HSA is displaced, although not completely, on the addition of fibrinogen. On the other hand, preadsorbed HSA effectively blocked further adsorption of fibrinogen and IgG on hydrophobic surfaces such as methylated silica. Furthermore, the competitive adsorption of HSA and fibrinogen on two phospholipid surfaces, i.e. phosphatidylcholine (PC) and phosphatidic acid (PA), was investigated. It was found that on PA, fibrinogen adsorbs extensively even after the preadsorption of HSA. This, however, is achieved with essentially no displacement of the preadsorbed HSA. For PC, finally, the fibrinogen adsorption is much lower than that of HSA, and fibrinogen is able neither to coadsorb with HSA nor to displace the preadsorbed protein.

The role of a single end group in poly(ethylene oxide) adsorption on colloidal and film polystyrene: complimentary sedimentation and total internal reflectance fluorescence studies

This work examines “apparent equilibrium” and kinetic features of adsorbing homopolymers with a modification at one end of each chain to enhance chain-end adsorption. The end groups combined fluorescent and hydrophobic units so that polymers could be easily assayed and label-induced artifacts avoided. A series of end-modified poly(ethylene oxide)s (PEOs) of constant backbone length (molecular weight 100 000) but with varied end-group hydrophobicity were synthesized and their adsorption on polystyrene (PS) latex and films examined. Isotherms generated by the centrifugation of PS latex revealed that hydrophobically modified PEO adsorbed up to five times more than unmodified PEO. Total internal reflectance fluorescence (TIRF) spectroscopy revealed faster accumulation of hydrophobe-containing PEO compared with less hydrophobic samples. All layers, however, appeared equally stable to washing in pure solvent. Hydrophobe-containing PEOs could incorporate into or displace chains from preadsorbed layers of less hydrophobic PEO, but a preadsorbed layer of end-hydrophobic PEO excluded chains from a less hydrophobic sample. The impact of the small hydrophobic end groups on surface coverages and kinetic features was greater than expected for brush-forming polymers and PEO-surfactant systems. The enhanced end-group effects were attributed to the adsorption of the main backbone, a feature lacking in many previous polymer brush or surfactant studies. A comparison of surface coverages measured by TIRF spectroscopy and centrifugation methods revealed inaccuracies in the internal TIRF calibrations. The centrifugation method was thought to give a more accurate determination of the surface excess, while TIRF spectroscopy is better suited to kinetic and dynamic studies.

The desorption of ribonuclease A from charge density gradient surfaces studied by spatially-resolved total internal reflection fluorescence

A quaternary amine surface gradient was prepared on fused silica by a three-step surface modification process. The gradient surface displayed a transition of surface charges along the gradient dimension from a net negative surface charge of silica to a net positive surface charge at the quaternary amine end. The gradient surface was characterized by X-ray photoelectron spectroscopy, ellipsometry, colloidal gold decoration, and dynamic contact angle measurements. It displayed an increased adhesion of negatively charged gold particles towards the quaternary amine end. The water contact angles also increased with the increased surface density of aminopropylsilyl groups. The desorption of ribonuclease A labeled with fluorescein-5-isothiocyanate (FITC-RNase) from the quaternary amine gradient surface was measured using spatially resolved total internal reflection fluorescence (TIRF) spectroscopy. The experimental FITC-RNase desorption results fitted exceptionally well to a two adsorbed protein populations model. A tentative assignment of the two adsorbed protein populations is proposed based on the effect of the ionic strength of the desorbing buffer. The faster desorption population interacted primarily with the quaternary amine gradient surface sites through electrostatic interactions. The slower desorption population interacted with the surface sites via hydrophobic and possibly some electrostatic interactions.

Osteonectin in matrix remodeling. A plasminogen-osteonectin-collagen complex.

Osteonectin is an adhesive glycoprotein synthesized constitutively by osteoblasts, endothelial cells, and megakaryocytes. Bone-derived and platelet-derived osteonectins differ in their electrophoretic mobility and carbohydrate content, and each displays different affinities for collagen matrices. Both types of osteonectin bind to plasminogen (Kd(app), of 4.7 +/- 1.0 x 10(-8) M for bone osteonectin and 1.2 +/- 0.1 x 10(-7) M for platelet osteonectin). The osteonectin-plasminogen interaction is inhibited by alpha 2-antiplasmin and epsilon-aminocaproic acid, suggesting that the interaction is mediated through the kringle 1 region of plasminogen. Both osteonectins enhance the rate of plasmin generation by tissue-type plasminogen activator to approximately the same extent as fibrinogen. Equilibrium binding measurements conducted using total internal reflection fluorescence spectroscopy indicate that plasminogen binds to collagen in the presence of bone osteonectin (Kd = 1.30 +/- 0.1 x 10(-7) M). No binding of plasminogen to collagen matrix was detected in the presence of platelet osteonectin or in the absence of bone osteonectin. Bone osteonectin-dependent binding of plasminogen to collagen matrix is reversed by the addition of epsilon-aminocaproic acid. The ability of both types of osteonectin to bind to and influence plasminogen activation and of bone osteonectin to anchor plasminogen on collagen matrices suggests that osteonectin may play a role in directing extracellular matrix proteolysis.

Salt effects on a proton transfer reaction of excited 1-naphthol in a solid/liquid interface layer: A picosecond time-resolved total internal reflection fluorescence study

Analyses of the excited-state proton transfer reaction of 1-naphthol in sapphire/water interface layers have been performed using fluorescence decay data obtained by a picosecond time-resolved total internal reflection fluorescence spectroscopy. The rate constant of the excited-state proton transfer reaction, k 1, of 1-naphthol in an interface layer was reduced as compared to that in a bulk aqueous solution. Addition of NaCl to aqueous solution of 1-naphthol resulted in a decrease in k 1 for both in the interface layer and in the bulk solution, suggesting that a proton acceptor from excited 1-naphthol is not a single water molecule but a water cluster and that water clusters in an interface layer are also destroyed by NaCl.

Competitive Adsorption at Hydrophobic Surfaces from Binary Protein Systems

The adsorption of some model proteins, human serum albumin (HSA), IgG, and fibrinogen, at silica made hydrophobic by either methylation or plasma deposition of HMDSO (hexamethyldisiloxane) was investigated with in situ ellipsometry and TIRF (total internal reflection fluorescence spectroscopy). Ellipsometry experiments of the simultaneous adsorption of HSA and either IgG or fibrinogen revealed a preferential adsorption of the latter proteins. This is seen not only from the total adsorbed amount, but also from the adsorbed layer thickness and from the buildup of the adsorbed layer. However, in sequential adsorption experiments, where HSA was first allowed to adsorb, followed by rinsing and addition of either IgG or fibrinogen, the additional adsorption is quite limited. Consequently, when HSA is allowed to adsorb on its own at hydrophobic surfaces, it is not removed by either IgG or fibrinogen to any larger extent. Furthermore, preadsorbed HSA was not exchanged by HSA added after adsorption and rinsing, implying that the mechanism behind this effect is an "irreversible" adsorption of HSA at hydrophobic surfaces, possibly originating from a surface-induced conformational change of HSA at these surfaces. Analogous findings were obtained with both methylated and HMDSO-treated surfaces, using both ellipsometry and TIRF.

Aggregation of pyrene in poly(alkyl methacrylate) films revealed by time-resolved total internal reflection fluorescence spectroscopy

Fluorescence spectra and decay curves of pyrene in poly(alkyl methacrylate) (methyl, n-propyl, isobutyl and cyclohexyl) films cast on sapphire substrates were measured under total internal reflection and normal excitation conditions, and were compared with each other. On the basis of the intensity ratio of the excimer to the monomer fluorescence and the fluorescence decay curve under the normal condition, the degree of pyrene aggregation in these films is discussed in terms of the solubility parameter, chain flexibility and hydrophobicity of the host polymers. Both the difference in the degree of pyrene aggregation and the difference in micropolarity around pyrene molecules between the vicinity of the sapphire/polymer interface and the bulk were found to be quite common for these polymer films cast on sapphire plates, and are discussed in terms of the interaction between substrate surface and polymers. The effect of tacticity of poly(methyl methacrylate) on the degree of the pyrene aggregation and the effect of labelling with pyrene were also investigated.

Human serum albumin adsorption onto octadecyldimethylsilyl-silica gradient surface

The effects of long-range electrostatic repulsion and short-range hydrophobic attraction on human serum albumin adsorption were studied as a function of the surface density of octadecyldimethylsilyl chains (C18) on silica. A C18 surface density gradient was prepared on fused silica plates. The water contact angles increased smoothly from 12° to 105° in the 12.5 mm long gradient region. The maximal fractional surface coverage of the C18 chains was calculated to be 0.92. Fluorescein-5-isothiocyanate-labeled human serum albumin (FITC-HSA) adsorption from dilute buffer solution onto the C18 gradient surface was measured using spatially resolved total internal reflection fluorescence (TIRF) spectroscopy. FITC-HSA adsorbed progressively more onto the surfaces with higher hydrophobicity. When the fractional surface coverage of C18 chains was larger than 0.42 the adsorption saturation of FITC-HSA leveled off. The adsorption kinetics of FITC-HSA changed from a transport-limited process at moderate-to-high C18 surface coverages to an adsorption-limited process at lower C18 surface coverages. The kinetics of FITC-HSA adsorption, including the convective-diffusive transport of protein to the surface, were modeled as a simple binding process with a single forward and reverse rate. The apparent binding constant, derived from the initial forward and reverse binding rates, depended more strongly on the C18 surface coverage in 0.165 M buffer than in 0.025 M buffer.

Competitive protein adsorption at radio frequency plasma polymer surfaces

The competitive adsorption of human serum ablumin (HSA), IgG and fibrinogen (Fgn) at radio frequency plasma polymer surfaces was studied with in situ ellipsometry and TIRF (total internal reflection fluorescence spectroscopy). While both IgG and Fgn adsorbed preferentially over HSA at hydrophobic surfaces, such as P-HMDSO (monomer: hexamethyldisiloxane), preadsorption of the latter protein dramatically reduced the adsorption of the two former. Furthermore, the preadsorbed HSA is not exchanged by HSA added after preadsorption, i.e., the HSA “blocking” is due to an irreversible adsorption of this protein, presumably involving conformational changes of HSA on adsorption. Different plasma polymer surfaces behave differently regarding competitive protein adsorption. For example, at P-HMDSO, the adsorption from all mixtures investigated is substantial. Furthermore, HSA and IgG dominate the adsorption from a ternary mixture corresponding to 1/100 plasma, although Fgn is still present at the surface. At P-AA (monomer: acrylic acid), on the other hand, the adsorption is lower, except for the IgG/Fgn binary mixture, and Fgn and HSA dominate the adsorption from a ternary mixture corresponding to 1/100 plasma, while IgG is depleted from the surface.

The assembly of the prothrombinase complex on adherent platelets.

Prothrombinase complex assembly, in real time, on platelets adherent to immobilized von Willebrand Factor (vWf) was examined by total internal reflection fluorescence spectroscopy (TIRFS). Electron microscopy showed that the platelets adhered to vWf in a largely unactivated state and could be activated by thrombin. Antibody binding to glycoprotein (GP) Ib and functional GPIIb-IIIa receptor molecules on adherent platelet membranes monitored by TIRFS also indicated that platelets adhered in a largely unactivated state. Maximal expression of the receptor form of GPIIb-IIIa detected by antibody binding was seen only after thrombin stimulation of the adherent platelets. Antibody binding to GPIb was detected on adherent platelets. A reduction in antibody binding was observed after thrombin stimulation of the platelets, indicating a change in GPIb as a consequence of thrombin stimulation of the platelets. The binding of the protein components of the prothrombinase complex to adherent and thrombin-stimulated adherent platelets was then studied individually. Factor Va bound to adherent and thrombin-stimulated adherent platelets was then studied individually. Factor Va bound to adherent and thrombin-stimulated adherent platelets with an estimated Kd of 58 nmol/L. Minimal factor Xa binding was observed on adherent platelets before thrombin stimulation. Factor Xa binding was, however, readily observed on thrombin-stimulated adherent platelets. This factor Xa binding was not saturable, and no Kd value could be estimated. Direct measurement of prothrombinase complex assembly was demonstrated by using an energy transfer phenomenon between fluorescein-labeled factor Va and rhodamine-labeled factor Xa. Prothrombinase complex assembly was observed on both adherent and thrombin-stimulated adherent platelets. The estimated Kd for the factor Va/factor Xa interaction was 4 nmol/L. TIRFS demonstrated that adherent platelets have the ability to support prothrombinase complex assembly, as shown by a direct energy transfer reaction between fluorescently labeled factors Va and Xa.

Inhomogeneous Aggregation of a Merocyanine Dye at the Solid/Liquid Interface Layer. A Picosecond Time-Resolved Total Internal Reflection Fluorescence Study

Abstract Fluorescence spectra and their rise and decay dynamics of a merocyanine dye in the interface layer have been studied as a function of penetration depth of the evanescent wave intensity by total internal reflection fluorescence spectroscopy. The aggregation of the dye was enhanced in the interface layer of up to ≈ 100 nm thickness compared to the bulk solution.

Analysis of ordered arrays of adsorbed lysozyme by scanning tunneling microscopy

Scanning tunneling microscopy (STM) has been used to observe lysozyme at a graphite surface directly in order to gain mechanistic information about the molecular events involved in protein adsorption. The experiments were performed using an insulated tip in an aqueous protein solution, allowing the time course of the adsorption process to be followed, including the evolution of ordered arrays. Ordered arrays of protein molecules were observed, with lattice spacings that varied with bulk protein concentration and salt strength. Fourier analysis was used to determine the average cell dimensions of an array. From the observed lattice spacings, it was possible to estimate the surface coverage of the protein, and thus, by varying the conditions, adsorption isotherms could be obtained. These isotherms compare well with adsorption isotherms measured using total internal reflectance fluorescence (TIRF) spectroscopy on a hydrophobic surface. Since the protein is charged and the electrolyte has an effect on the isotherms, electrostatics are a likely controlling factor. Molecular electrostatics computations were thus used to investigate the possible origins of the lattice structure, and they suggest that favorable intermolecular interactions among adsorbed molecules are consistent with hydrophobically dominated protein-surface interactions.

Excited-state proton transfer of 1-naphthol in liquid—solid interface layers. Picosecond time-resolved total internal reflection fluorescence study

Picosecond time-resolved total internal reflection fluorescence spectroscopy was applied to analyze the proton transfer reaction of 1-naphthol in water—sapphire interface layers. The rate constant of the proton transfer reaction from excited neutral species became slow in the interface layer as compared with that in the bulk aqueous solution. The anomaly observed up to the penetration depth of 100 nm was interpreted in terms of rotational fluctuations of water aggregates in the interface layer.