Antibody immobilization using pneumatic spray: Comparison with the avidin–biotin bridge immobilization method

Figueroa, Jhon; Magaña, Sonia; Lim, Daniel V.; Schlaf, R.

doi:10.1016/j.jim.2012.08.007

Cited by 4 publications

(3 citation statements)

References 37 publications

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“…Physical adsorption gives low control over the orientation of the Abs, even though immobilization of Abs using a pneumatic nebulizer is fast and reproducible. 51 Zhao et al 52 studied how solution pH, salt concentration and surface chemistry affect Ab adsorption onto silica surfaces. The salt concentration and pH did inuence the amount of adsorbed Ab and analyte binding, but did not inuence the orientation of immobilized Abs.…”

Section: Non-covalent Immobilizationmentioning

confidence: 99%

Antibody orientation on biosensor surfaces: a minireview

Trilling¹,

Beekwilder²,

Zuilhof

2013

Analyst

365

309

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Detection elements play a key role in analyte recognition in biosensors. Therefore, detection elements with high analyte specificity and binding strength are required. While antibodies (Abs) have been increasingly used as detection elements in biosensors, a key challenge remains - the immobilization on the biosensor surface. This minireview highlights recent approaches to immobilize and study Abs on surfaces. We first introduce Ab species used as detection elements, and discuss techniques recently used to elucidate Ab orientation by determination of layer thickness or surface topology. Then, several immobilization methods will be presented: non-covalent and covalent surface attachment, yielding oriented or random coupled Abs. Finally, protein modification methods applicable for oriented Ab immobilization are reviewed with an eye to future application.

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Section: Non-covalent Immobilizationmentioning

confidence: 99%

Antibody orientation on biosensor surfaces: a minireview

Trilling¹,

Beekwilder²,

Zuilhof

2013

Analyst

365

309

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“…coli O157:H7 using the avidin–biotin bridge method and its immobilization using pneumatic spray on modified glass slides reported no detection for a solution concentration of ∼10 5 cells/mL. This study cites 0% and 40% detection at 10 6 cells/mL, and 60% and 100% detection at 10 7 cells/mL for pneumatic spray and avidin–biotin bridge slides, respectively . Although the exact quantification was not reported, it can be concluded from their microscopy images that the density of the immobilized cells captured from 10 7 CFU/mL corresponded to ∼120 cells per 140 × 140 μm 2 area, which is slightly lower than the cell densities observed in this study350–400 cells per 140 × 200 μm 2 (Figure ) captured from a suspension of 10 7 CFU/mL.…”

Section: Results and Discussionmentioning

confidence: 71%

“…This study cites 0% and 40% detection at 10 6 cells/mL, and 60% and 100% detection at 10 7 cells/mL for pneumatic spray and avidin−biotin bridge slides, respectively. 30 Although the exact quantification was not reported, it can be concluded from their microscopy images that the density of the immobilized cells captured from 10 7 CFU/mL corresponded to ∼120 cells per 140 × 140 μm 2 area, which is slightly lower than the cell densities observed in this study350−400 cells per 140 × 200 μm 2 (Figure 5) captured from a suspension of 10 7 CFU/mL. When scaled, the work reported by ref 30 predicts ∼0.6 cells/10 μm 2 as compared to 1.2 cells/10 μm 2 in this work.…”

Section: ■ Results and Discussionmentioning

confidence: 99%

Sharp Transition in the Immunoimmobilization of E. coli O157:H7

et al. 2014

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This work focuses on immobilization of living enterohemorrhagic Escherichia coli O157:H7 on a gold surface as a function of the concentration of antibody tethered to the surface in the physiological environment of the organisms. Experiments are conducted using antibodies raised against bacterial surface lipopolysaccharides (LPS) tethered to gold-coated silicon wafers at surface concentrations spanning a range from submonolayers of antibodies to full coverage, an estimated 1 antibody per ∼100 nm(2). A careful optimization of surface chemistry is conducted to obtain the most efficient tethering of the antibodies to the surface. The mechanism of immobilizing the bacteria is antibody-antigen interactions between the tethered antibodies on the surface and the bacterial surface LPS firmly attached to the bacteria. This type of attachment is known as immunoimmobilization. The experiments suggest no noticeable bacterial attachment until the surface antibody concentration reaches ∼70% of a full monolayer of coverage. Above this critical antibody density, a sharp increase in immunoimmobilized bacteria is observed as they populate nearly 80% to 100% of the available surface area, reaching ∼1.2 cells/10 μm(2). This sharp increase in population is tentatively explained in terms of the minimum number of antibody-antigen interactions required per bacterium to immobilize the cell. This critical number is estimated to be ∼6000-8000 antibodies per bacterium (having a 1 μm(2) footprint on the surface) under the assumption that a full monolayer of antibodies is about 1 antibody per ∼100 nm(2). However, the large majority of the 6000-8000 antibodies are not expected to participate in antibody-antigen interactions, in that the loose LPS in solution will saturate many of these antibodies before bacteria have a chance to interact with them. Furthermore, the geometric considerations will further restrict the majority of the active antibodies from interacting with the surface antigens of the cell, reducing its effective contact area with the antibodies considerably.

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