Kinetics of Antigen Binding to Arrays of Antibodies in Different Sized Spots

Sapsford, Kim E.; Liron, Zvi; Shubin, Yura S.; Ligler, Frances S.

doi:10.1021/ac015554e

Cited by 84 publications

(61 citation statements)

References 25 publications

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“…2. 34,35 In addition, the NRL Array Biosensor is capable of performing real-time binding analyses and has been used to determine kinetic and affinity constants for both specific, 36 semi-selective, 37 and non-specific 38 binding interactions. The NRL Array Biosensor has recently been used in the development of multiplexed assays for screening of human sera for antibodies directed against bacterial and viral antigens, including SEB, tetanus toxin, diphtheria toxin and hepatitis B.…”

Section: Antibody-based Detectionmentioning

confidence: 99%

The Array Biosensor: Portable, Automated Systems

et al. 2007

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Section: Antibody-based Detectionmentioning

confidence: 99%

The Array Biosensor: Portable, Automated Systems

et al. 2007

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“…We show that kinetic data can be extracted from the real-time binding curves of the particles on each surface. This tool could be used to investigate surface binding kinetics, which are another key parameter in diagnostic devices [19,20]. …”

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confidence: 99%

TIRF microscopy as a screening method for non-specific binding on surfaces

Charlton

Gubala

Gandhiraman

et al. 2011

Journal of Colloid and Interface Science

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We report a method for studying nanoparticle-biosensor surface interactions based on Total Internal Reflection Fluorescence (TIRF) Microscopy. We demonstrate that this simple technique allows for high throughput screening of non-specific adsorption (NSA) of nanoparticles on surfaces of different chemical composition. Binding events between fluorescent nanoparticles and functionalized Zeonor¨ surfaces are observed in real-time, giving a measure of the attractive or repulsive properties of the surface and the kinetics of the interaction. Three types of coatings have been studied: one containing a polymerized aminosilane network with terminal ÐNH 2 groups, a second film with a high density of Ð COOH surface groups and the third with sterically restraining branched poly(ethylene)glycol (PEG) functionality. TIRF microscopy revealed that the NSA of nanoparticles with negative surface charge on such modified coatings decreased in the following order ÐNH 2 > -branched PEG > -COOH. The surface specificity of the technique also allows discrimination of the degree of NSA of the same surface at different pH. KeywordsTotal Internal Reflection, Fluorescence, Microscopy, Non-Specific Binding, SurfaceParticle Interaction IntroductionCurrent research in biomedical diagnostics is directed at inexpensive, highly sensitive, miniaturized devices, in which usually disposable substrates or chips are used. Such devices are required to measure analytes in microliter volume samples, typically at picomolar level. In fluorescence-based assays, the signal can be greatly enhanced with the use of dye doped nanoparticles as fluorescent labels [1][2][3][4]. However, in a successful diagnostics device, signal-to-noise ratio is paramount to its performance and effectively controls the sensitivity. Controlling the noise and background contribution, dictated by non-specific binding of the non-analyte constituents of the sample remains very challenging. The chemical composition of the substrate surface and its interactions with the active surface area of the dye-doped nanoparticles, therefore, play a key role in the performance of diagnostic platform. In this work, we focus on surface functionalization of the substrate ZeonorÒ, a cyclo-olefin polymer (COP), which is characterized by ease of injection molding for biochip fabrication and relatively low autofluorescence, which is an important factor for fluorescence-based applications including TIRF microscopy.Total Internal Reflection Fluorescence (TIRF) microscopy [5] is presented here as a very useful and fast method to compare non-specific adsorption of detection molecules on different surfaces. This method allowed for rapid screening of surfaces to find their ability to prevent non-specific binding. Total internal reflection occurs an any interface between materials of different refractive index inside the material of higher refractive index provided the angle of incidence of the light is above the critical angle defined as:where n 1 is the refractive index of the high refractive index mat...

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“…However, long incubation times are required to reach complete antigen-antibody reactions due to the long diffusion times of the antigen molecules toward the antibody as the rate-limiting step in these systems, resulting in slow binding kinetics and compromised assay sensitivity and dynamic range. [5][6][7][8] In this study, we developed a rapid, precise and simple antigen permeation immunoassay based on the positive polyelectrolyte multilayers (PEMs)-modified membrane filter (cellulose acetate, …”

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Polyelectrolyte multilayers-modified membrane filter for rapid immunoassay: protein condensation by centrifugal permeation

Shen

Watanabe

Akashi

2010

Polym J

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We demonstrated that positive polyelectrolyte multilayers (PEMs) have great potential for conventional enzyme-linked immunosorbent assay (ELISA) systems because of the induction protein enrichment on their surface. In this study, we developed a novel simple molecular detection system using a PEMs-modified cellulose acetate (CA) membrane filter (PEMs-CA), which achieved rapid detection without compromising sensitivity as compared with conventional ELISA systems. This rapid detection was carried out by the centrifugal permeation of the antigen solution, thus allowing the local condensation of the antigen molecule in the proximity of the primary antibody-enriched PEMs-CA membrane, which overcame molecular diffusion as the time-limiting factor as compared with conventional ELISA systems. Hence, on the permeation system, the incubation time required for the antigen-antibody reaction corresponded to just the permeation time, which was 1/20 shorten than the conventional ELISA system under optimized conditions for the centrifugal forces. Moreover, the calibration curve of PEMs-CA had wide range of concentration from 0.02 to 5 lg ml À1 and larger change in signal as compared with the bare CA membrane. We concluded that this centrifugal permeation system should be further developed for rapid, precise and simple systems in various immunosensors using PEMs-modified membrane filters. Keywords: centrifugal condensation; ELISA; membrane filter; permeation detection; polyelectrolyte multilayers INTRODUCTIONSince immunoassays were first developed in the 1950s, antibodies and other affinity reagents have been developed to improve these assays in terms of specificity and sensitivity. 1 Moreover, the fundamental technique for using various precise immunosensors, electrochemical, optical and microgravimetric immunosensors has evolved significantly from the early devices that appeared in the late 1980s to the more sophisticated, integrated systems developed more recently. 2 Economic pressures in the health-care system mean that attempts have long been made to reconcile the elements of the tension triangle: 'fast-goodcheap' . Thus far, however, it has only been possible to satisfy only two of these elements. 3,4 Disposable immunotests (qualitative and quantitative), such as enzyme-linked immunosorbent assay (ELISA), retain the gold standard for protein detection and quantification. However, long incubation times are required to reach complete antigen-antibody reactions due to the long diffusion times of the antigen molecules toward the antibody as the rate-limiting step in these systems, resulting in slow binding kinetics and compromised assay sensitivity and dynamic range. [5][6][7][8] In this study, we developed a rapid, precise and simple antigen permeation immunoassay based on the positive polyelectrolyte multilayers (PEMs)-modified membrane filter (cellulose acetate,

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Kinetics of Antigen Binding to Arrays of Antibodies in Different Sized Spots

Cited by 84 publications

References 25 publications

The Array Biosensor: Portable, Automated Systems

The Array Biosensor: Portable, Automated Systems

TIRF microscopy as a screening method for non-specific binding on surfaces

Polyelectrolyte multilayers-modified membrane filter for rapid immunoassay: protein condensation by centrifugal permeation

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