Multiple internal reflection fluorescence spectrometry

Harrick, N. J.; Loeb, George I.

doi:10.1021/ac60326a001

Cited by 76 publications

(23 citation statements)

References 4 publications

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“…The total internal reflection fluorescence (TIRF) concept was originally patented by Hirschfeld (1 ) and applied to protein adsorption by Harrick and Loeb (2). Since then TIRF has been utilized in a limited number of investigations to study the adsorption of extrinsic fluor-labeled plasma proteins on quartz (3,4), hapten-protein conjugates (5), and polydimethylsiloxane films (6).…”

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confidence: 99%

Probing Protein Adsorption: Total Internal Reflection Intrinsic Fluorescence

Wagenen

Rockhold

Andrade

1982

Advances in Chemistry

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Interfacial intrinsic fluorescence induced by evanescent wave total internal reflection was developed to study protein ad sorption at solid-aqueous buffer solution interfaces. The tech nique has a number of advantages over conventional meth odologies for the study of adsorption including (1) continuous, real-time sampling with 0.1-s resolution; (2) in situ sensing; (3) application to biomedically relevant, flat, low surface area samples; (4) quantitation of the amount adsorbed calculated on the basis of an internal standard; and (5) ability to obtain fluorescence emission spectra of intrinsic tryptophan moieties that are sensitive to local microenvironmental changes pro duced during the protein adsorption process. These advan tages are illustrated for bovine serum albumin and γ-globulins adsorbed on hydrophilic quartz. A n understanding of protein adsorption behavior is applicable in numer ous fields including blood-synthetic materials interfaces, macromolecular-membrane interactions, receptor interactions, enzyme engineering, ad hesion, and protein separation on chromatographic supports. Many methods have evolved to study interfacial adsorption, but no single independent method seems adequate. The ideal technique should produce quantitative, real-time, in situ data concerning the amount, activity, and conformation of proteins adsorbed on well-characterized surfaces. All adsorption techniques are approximations to this optimum. Protein solution depletion via adsorption on finely divided substrates is quantitative, but applicability to low surface area materials of biomedical relevance is often minimal. Adsorption of radiolabeled macromolecules is In Biomaterials: Interfacial Phenomena and Applications; Cooper, S., et al.; Advances in Chemistry; American Chemical Society: Washington, DC, 1982.352 BIOMATERIALS: INTERFACIAL PHENOMENA AND APPLICATIONS quantitative on low surface area substrates; however, the presence of an extrinsic label may alter protein physical properties and subsequent adsorp tion behavior. Automated ellipsometry can, in principle, provide in situ, real-time information on film thickness and refractive index, but the minute differences in substrate, film, and buffer refractive indices often preclude this approach. Multiple internal reflection infrared spectroscopy is complicated by strong water signals that obscure protein amide bands and, while Fourier transform analysis seems promising, the interpretation remains difficult and the equipment is expensive. Interfacial protein fluorescence induced by internal reflection evanes cent wave excitation offers a number of advantages over conventional adsorp tion techniques. The total internal reflection fluorescence (TIRF) concept was originally patented by Hirschfeld (1 ) and applied to protein adsorption by Harrick and Loeb (2). Since then TIRF has been utilized in a limited number of investigations to study the adsorption of extrinsic fluor-labeled plasma proteins on quartz (3, 4), hapten-protein conjugates (5), and polydimethylsiloxane films (6). O...

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confidence: 99%

Probing Protein Adsorption: Total Internal Reflection Intrinsic Fluorescence

Wagenen

Rockhold

Andrade

1982

Advances in Chemistry

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“…The total mass of adsorbed BSA approaches 67 ng (268 ng/ cm 2 ). In the literature, the shape of BSA molecules has been described as spherical [94,95] with diameter 6.0 to 6.9 nm or as a prolate spheroid [96 -99] with major axis from 9.6 to 14.2 nm and minor axis from 3.8 to 7.0 nm. With minimized water content, the following compact BSA dimensions can be assumed: 4 Â 9.6 Â 4 nm (rod-like shape) as a reference point for BSA adsorption on solid surfaces.…”

Section: Eqcn Adsorption Transients and Qci Measurementsmentioning

confidence: 99%

Effect of Albumin on Underpotential Lead Deposition and Stripping on Ag‐RDE

Hepel

2005

Electroanalysis

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The effects of adsorption of bovine serum albumin (BSA) on the electrodeposition and stripping of Pb-adatoms at a Ag-RDE in acetate buffer solutions have been studied. The adsorbed BSA molecules have been found to inhibit the lead adsorption process. A decrease in the apparent charge transfer coefficient a for Pb-adatom desorption from 0.39 to 0.34 in the presence of adsorbed BSA molecules has been attributed to the electric field screening by multiply charged BSA in the adsorption film, since no major lateral interactions between Pb and BSA have been observed. The kinetics of BSA adsorption on a Ag-RDE was followed by Pb-adatom probe technique in the range of low BSA coverage, 0 < q BSA < 0.4. The adsorption (k 1 ) and desorption (k À1 ) rate constants were determined as follows:The equilibrium constant for BSA adsorption on Ag electrode from 3 Â 10 À8 M BSA solution, also determined from the Pb-adatom probe method, was: K ads ¼ 2.17 Â 10 7 L/mol and the corresponding Gibbs free energy of adsorption: DG8 ads ¼ À 9.99 kcal/mol (À 41.85 kJ/mol). The formation of BSA films on Ag electrodes has been confirmed by EQCN mass transients and AFM imaging. It follows from these experiments that BSA molecules adsorb in horizontal orientation and form a film on Ag with 4 nm thickness.

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“…Hirschfeld (2) and Harrick and Loeb ( 13) showed that fluorophores near an interface could be selectively excited by the evanescent wave propagating at the surface of a waveguide. This principle of total internal reflection of fluorescence was employed by Kronick and Little in a homogenous immunoassay (14,15).…”

Section: Configurations For Fiberoptic Biosensorsmentioning

confidence: 99%

“…In particular, the improvements in sensitivity, cost, automatability, and variety of solidstate detectors, such as PIN photodiodes, microchannel plate electron multipliers, photodiode arrays, and charge-coupled devices, argue that solid-state detectors may supplant photomultiplier tubes in many fiberoptic sensors. Fiber technology has been underutilized in the fiberoptic biosensors described in the literature; in this sense, little progress has been made since the developments of Harrick and Loeb (13). Recently, tapered fibers have been shown to improve the sensitivity of absorbance sensors (48), and have displayed encouraging results with waveguide-binding sensors (29).…”

Section: Approaches For Improved Fiberoptic Biosensorsmentioning

confidence: 99%