Total internal reflection fluorescence: a technique for examining interactions of macromolecules with solid surfaces

Lok, Brent K; Cheng, Yu-Ling; Robertson, Channing R.

doi:10.1016/0021-9797(83)90316-8

Cited by 165 publications

(79 citation statements)

References 6 publications

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“…The initial adsorption rate, 0.7 mg/ m 2 Á s, is much larger than that for RNase A adsorption and is consistent with the rate predicted by the Levêque equation for transport-limited adsorption rates (Lok et al, 1983a(Lok et al, , 1983b for these conditions. The calculation was based upon the PEG diffusion coefficient measured by dynamic light scattering.…”

Section: Adsorption To a Fresh Plg Surfacesupporting

confidence: 68%

“…Figure 4 shows the adsorption kinetics for a 1.5-mM solution of unmodified RNase A, undergoing laminar flow at a wall shear rate of 4 s À1 over a fresh PLG surface. The measured initial rate of RNase A adsorption, 0.125 mg/m 2 Á s, is 90% smaller than the 1.25 mg/m 2 Á s rate predicted by the Levêque equation for the initial transport-limited adsorption rate for solutions undergoing laminar slit flow (Lok et al, 1983a(Lok et al, , 1983b under the current conditions. Thus, the RNase A adsorption kinetics were not transport-limited but were limited instead by the intrinsic adsorption kinetics of the protein on the PLG surface (surface-limited adsorption).…”

Section: Adsorption To a Fresh Plg Surfacementioning

confidence: 69%

“…The FITC labeling procedure for RNase A (Robeson, 1995) and the basic principles of TIRF (Buijs and Hlady, 1997;Lok et al, 1983b;Roth and Lenhoff, 1995) have been described elsewhere. The average labeling ratio of RNase A in stock solution was 0.4 AE 0.1 fluorescein molecules per RNase A molecule, expressed as the mean AE the standard deviation of multiple labeling experiments, as determined by the ratio of the optical absorbance at 500 and 278 nm.…”

Section: Exchangeability Of Adsorbed Proteinmentioning

confidence: 99%

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Adsorption of poly(ethylene glycol)‐modified ribonuclease A to a poly(lactide‐co‐glycolide) surface

Daly¹,

Przybycien²,

Tilton³

2005

Biotech & Bioengineering

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Protein adsorption is a source of variability in the release profiles of therapeutic proteins from biodegradable microspheres. We employ optical reflectometry and total internal reflection fluorescence to explore the extent and kinetics of ribonuclease A (RNase A) adsorption to spin-cast films of poly(lactide-co-glycolide) (PLG) and, in particular, to determine how covalent grafting of polyethylene glycol (PEG) to RNase A affects adsorption. Adsorption kinetics on PLG surfaces are surface-limited for RNase A but transport-limited for unconjugated PEG homopolymers and for PEG-modified RNase A, indicating that PEG anchors the conjugates to the surface during the transport-limited regime. PEG modification of RNase A decreases the total number of adsorbed molecules per unit area but increases the areal surface coverage because the grafted PEG chains exclude additional surface area. Total internal reflection fluorescence-based exchange measurements show that there is no exchange between adsorbed and solution-phase protein molecules. This indicates an unusually tenacious adsorption. Streaming current measurements indicate that the zeta potential of the PLG surface becomes increasingly negative as the film is exposed to water for several weeks, as expected. Aging of the PLG surface results in increased adsorption of unmodified RNase A but decreased adsorption of unconjugated PEG homopolymers and of PEG-RNase A conjugates, relative to the extent of adsorption on freshly prepared PLG surfaces. Adsorption results correlate well with an increase in the rate, total extent and preservation of bioactivity of RNase A released from PLG microspheres for the PEG-modified version of RNase A.

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Section: Adsorption To a Fresh Plg Surfacesupporting

confidence: 68%

Section: Adsorption To a Fresh Plg Surfacementioning

confidence: 69%

Section: Exchangeability Of Adsorbed Proteinmentioning

confidence: 99%

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Adsorption of poly(ethylene glycol)‐modified ribonuclease A to a poly(lactide‐co‐glycolide) surface

Daly¹,

Przybycien²,

Tilton³

2005

Biotech & Bioengineering

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“…While the allylamine is a primary amine, having the monomer structure of CH 2 ==CHCH 2 NH 2 , the resulting plasma polymer does not reproduce the monomer chemistry. Differentiation between primary, secondary and tertiary forms of the amine groups on the pp.AAm surface, which have close pK a values [45], is not possible from the data obtained in this study, as the nitrogen is combined in the films as imines and amides as well as the variety of amine forms [31].…”

Section: Plasma-polymerized Surfacesmentioning

confidence: 69%

Studies of electroosmotic flow and the effects of protein adsorption in plasma‐polymerized microchannel surfaces

2009

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This paper presents a study of EOF properties of plasma-polymerized microchannel surfaces and the effects of protein (fibrinogen and lysozyme) adsorption on the EOF behavior of the surface-modified microchannels. Three plasma polymer surfaces, i.e. tetraglyme, acrylic acid and allylamine, are tested. Results indicate EOF suppression in all plasma-coated channels compared with the uncoated glass microchannel surfaces. The EOF behaviors of the modified microchannels after exposure to protein solutions are also investigated and show that even low levels of protein adsorption can significantly influence EOF behavior, and in some cases, result in the reversal of flow. The results also highlight that EOF measurement can be used as a method for detecting the presence of proteins within microchannels at low surface coverage (<1 ng/cm(2) on glass). Critically, the results illustrate that the non-fouling tetraglyme plasma polymer is able to sustain EOF. Comparison of the plasma-polymerized surfaces with conventionally grafted polyelectrolyte surfaces demonstrates the stabilities of the plasma polymer films, enabling multiple EOF runs over 3 days without deterioration in performance. The results of this study clearly demonstrate that plasma polymers enable the surface chemistry of microfluidic devices to be tailored for specific applications. Critically, the deposition of the non-fouling tetraglyme coating enables stable EOF to be induced in the presence of protein.

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“…36 ' 37 Other roles for TIRFS technology include the examination of protein adsorption at solid-liquid interfaces 38 and the adsorption of proteins on polymer films. 39 Under the experimental conditions chosen, there is selective excitation of bound fluorescent probes at the interface without significant excitation of fluorescent molecules in the bulk solution. 33 The binding measurements can also be carried out in a continuous manner, in real time, under equilibrium conditions.…”

Section: The Assembly Of the Prothrombinase Complex On Adherent Platementioning

confidence: 99%

The assembly of the prothrombinase complex on adherent platelets.

Swords

Mann

1993

Arterioscler Thromb

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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 with an estimated K a of 58 nmoI/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 K d 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 K A 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. (Arterioscler Thromb.

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Total internal reflection fluorescence: a technique for examining interactions of macromolecules with solid surfaces

Cited by 165 publications

References 6 publications

Adsorption of poly(ethylene glycol)‐modified ribonuclease A to a poly(lactide‐co‐glycolide) surface

Adsorption of poly(ethylene glycol)‐modified ribonuclease A to a poly(lactide‐co‐glycolide) surface

Studies of electroosmotic flow and the effects of protein adsorption in plasma‐polymerized microchannel surfaces

The assembly of the prothrombinase complex on adherent platelets.

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