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Direct detection of protein biomarkers in human fluids using site-specific antibody immobilization strategies.

Soler M, Estevez MC, Alvarez M, Otte MA, Sepulveda B, Lechuga LM - Sensors (Basel) (2014)

Bottom Line: Special effort has been focused on avoiding antibody manipulation, preventing nonspecific adsorption and obtaining a robust biosurface with regeneration capabilities.ProLinker™-based approach has demonstrated to fulfill those crucial requirements and, in combination with PEG-derivative compounds, has shown encouraging results for direct detection in biological fluids, such as pure urine or diluted serum.Furthermore, we have implemented the ProLinker™ strategy to a novel nanoplasmonic-based biosensor resulting in promising advantages for its application in clinical and biomedical diagnosis.

View Article: PubMed Central - PubMed

Affiliation: Nanobiosensors and Bioanalytical Applications Group, Institut Català de Nanociència i Nanotecnologia (ICN2), CSIC & CIBER-BBN, Bellaterra, Barcelona 08193, Spain. maria.soler@cin2.es.

ABSTRACT
Design of an optimal surface biofunctionalization still remains an important challenge for the application of biosensors in clinical practice and therapeutic follow-up. Optical biosensors offer real-time monitoring and highly sensitive label-free analysis, along with great potential to be transferred to portable devices. When applied in direct immunoassays, their analytical features depend strongly on the antibody immobilization strategy. A strategy for correct immobilization of antibodies based on the use of ProLinker™ has been evaluated and optimized in terms of sensitivity, selectivity, stability and reproducibility. Special effort has been focused on avoiding antibody manipulation, preventing nonspecific adsorption and obtaining a robust biosurface with regeneration capabilities. ProLinker™-based approach has demonstrated to fulfill those crucial requirements and, in combination with PEG-derivative compounds, has shown encouraging results for direct detection in biological fluids, such as pure urine or diluted serum. Furthermore, we have implemented the ProLinker™ strategy to a novel nanoplasmonic-based biosensor resulting in promising advantages for its application in clinical and biomedical diagnosis.

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(a) Comparison of antibody immobilizations at different antibody concentrations (5, 10, 20, 50, 100 μg/mL) with different strategies. Grey: covalent strategy; Green: Protein G strategy ([Protein G] = 50 μg/mL); Purple: ProLinker™ strategy; (b) Antigen detection performed with hCG/anti-hCG for covalent strategy (black), Protein G strategy (green) and ProLinker™ strategy (purple). Concentration of anti-hCG antibody was 10 μg/mL in all cases. Dashed lines represent adsorption of nonspecific proteins onto antibody functionalized surfaces for covalent strategy (black), Protein G strategy (green) and ProLinker™ strategy (purple). Blue dotted line indicates additional control for the ProLinker™ strategy, based on the detection of hCG onto a nonspecific antibody (10 μg/mL) immobilized over ProLinker™ layer (same experimental conditions as with specific antibody).
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f2-sensors-14-02239: (a) Comparison of antibody immobilizations at different antibody concentrations (5, 10, 20, 50, 100 μg/mL) with different strategies. Grey: covalent strategy; Green: Protein G strategy ([Protein G] = 50 μg/mL); Purple: ProLinker™ strategy; (b) Antigen detection performed with hCG/anti-hCG for covalent strategy (black), Protein G strategy (green) and ProLinker™ strategy (purple). Concentration of anti-hCG antibody was 10 μg/mL in all cases. Dashed lines represent adsorption of nonspecific proteins onto antibody functionalized surfaces for covalent strategy (black), Protein G strategy (green) and ProLinker™ strategy (purple). Blue dotted line indicates additional control for the ProLinker™ strategy, based on the detection of hCG onto a nonspecific antibody (10 μg/mL) immobilized over ProLinker™ layer (same experimental conditions as with specific antibody).

Mentions: Three different antibody-immobilization strategies were compared: Prolinker™ strategy (see Figure 1), covalent binding of the antibody, and Protein-G-based coupling. Human chorionic gonadotropin (hCG) hormone and its respective specific monoclonal antibody (mAb anti-hCG) were initially used as the model pair to carry out the study. First, different antibody concentrations, between 5 and 100 μg/mL were tested in order to compare the coupling efficiency. As can be observed in Figure 2a, Protein G and ProLinker™ strategies showed higher signal response, being the ProLinker™ 10 times higher than the covalent method using the same concentration of antibody, which indicates a significant higher antibody binding efficiency.


Direct detection of protein biomarkers in human fluids using site-specific antibody immobilization strategies.

Soler M, Estevez MC, Alvarez M, Otte MA, Sepulveda B, Lechuga LM - Sensors (Basel) (2014)

(a) Comparison of antibody immobilizations at different antibody concentrations (5, 10, 20, 50, 100 μg/mL) with different strategies. Grey: covalent strategy; Green: Protein G strategy ([Protein G] = 50 μg/mL); Purple: ProLinker™ strategy; (b) Antigen detection performed with hCG/anti-hCG for covalent strategy (black), Protein G strategy (green) and ProLinker™ strategy (purple). Concentration of anti-hCG antibody was 10 μg/mL in all cases. Dashed lines represent adsorption of nonspecific proteins onto antibody functionalized surfaces for covalent strategy (black), Protein G strategy (green) and ProLinker™ strategy (purple). Blue dotted line indicates additional control for the ProLinker™ strategy, based on the detection of hCG onto a nonspecific antibody (10 μg/mL) immobilized over ProLinker™ layer (same experimental conditions as with specific antibody).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3958245&req=5

f2-sensors-14-02239: (a) Comparison of antibody immobilizations at different antibody concentrations (5, 10, 20, 50, 100 μg/mL) with different strategies. Grey: covalent strategy; Green: Protein G strategy ([Protein G] = 50 μg/mL); Purple: ProLinker™ strategy; (b) Antigen detection performed with hCG/anti-hCG for covalent strategy (black), Protein G strategy (green) and ProLinker™ strategy (purple). Concentration of anti-hCG antibody was 10 μg/mL in all cases. Dashed lines represent adsorption of nonspecific proteins onto antibody functionalized surfaces for covalent strategy (black), Protein G strategy (green) and ProLinker™ strategy (purple). Blue dotted line indicates additional control for the ProLinker™ strategy, based on the detection of hCG onto a nonspecific antibody (10 μg/mL) immobilized over ProLinker™ layer (same experimental conditions as with specific antibody).
Mentions: Three different antibody-immobilization strategies were compared: Prolinker™ strategy (see Figure 1), covalent binding of the antibody, and Protein-G-based coupling. Human chorionic gonadotropin (hCG) hormone and its respective specific monoclonal antibody (mAb anti-hCG) were initially used as the model pair to carry out the study. First, different antibody concentrations, between 5 and 100 μg/mL were tested in order to compare the coupling efficiency. As can be observed in Figure 2a, Protein G and ProLinker™ strategies showed higher signal response, being the ProLinker™ 10 times higher than the covalent method using the same concentration of antibody, which indicates a significant higher antibody binding efficiency.

Bottom Line: Special effort has been focused on avoiding antibody manipulation, preventing nonspecific adsorption and obtaining a robust biosurface with regeneration capabilities.ProLinker™-based approach has demonstrated to fulfill those crucial requirements and, in combination with PEG-derivative compounds, has shown encouraging results for direct detection in biological fluids, such as pure urine or diluted serum.Furthermore, we have implemented the ProLinker™ strategy to a novel nanoplasmonic-based biosensor resulting in promising advantages for its application in clinical and biomedical diagnosis.

View Article: PubMed Central - PubMed

Affiliation: Nanobiosensors and Bioanalytical Applications Group, Institut Català de Nanociència i Nanotecnologia (ICN2), CSIC & CIBER-BBN, Bellaterra, Barcelona 08193, Spain. maria.soler@cin2.es.

ABSTRACT
Design of an optimal surface biofunctionalization still remains an important challenge for the application of biosensors in clinical practice and therapeutic follow-up. Optical biosensors offer real-time monitoring and highly sensitive label-free analysis, along with great potential to be transferred to portable devices. When applied in direct immunoassays, their analytical features depend strongly on the antibody immobilization strategy. A strategy for correct immobilization of antibodies based on the use of ProLinker™ has been evaluated and optimized in terms of sensitivity, selectivity, stability and reproducibility. Special effort has been focused on avoiding antibody manipulation, preventing nonspecific adsorption and obtaining a robust biosurface with regeneration capabilities. ProLinker™-based approach has demonstrated to fulfill those crucial requirements and, in combination with PEG-derivative compounds, has shown encouraging results for direct detection in biological fluids, such as pure urine or diluted serum. Furthermore, we have implemented the ProLinker™ strategy to a novel nanoplasmonic-based biosensor resulting in promising advantages for its application in clinical and biomedical diagnosis.

Show MeSH