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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) Immobilization protocol for gold nanodisks substrates: (i) ProLinker™ layer formation; (ii) Antibody immobilization and blocking step with PLL-PEG; and (iii) Specific antigen detection; (b) Scanning electron microscopy (SEM) images of gold nanodisks fabricated by hole-mask colloidal lithography (c) CRP detection curves performed with the nanoplasmonic biosensor at different concentrations of antibody immobilized (10, 20, 50 μg/mL) with ProLinker™ strategy; (d) Calibration curves for CRP detection performed on gold film (orange) and gold nanodisks nanoplasmonic (blue). Antibody concentration was 20 μg/mL and PLL-PEG was employed as blocking agent for both substrates. Dashed lines represent linear fit of linear region; (e) Nonspecific adsorption study of serum at different concentrations (10%, 25%, 50%, 100%) using different buffers in flow (PBST 0.5% and HBB) performed for both sensor substrates: SPR gold film (orange) and LSPR gold nanodisks (blue).
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f6-sensors-14-02239: (a) Immobilization protocol for gold nanodisks substrates: (i) ProLinker™ layer formation; (ii) Antibody immobilization and blocking step with PLL-PEG; and (iii) Specific antigen detection; (b) Scanning electron microscopy (SEM) images of gold nanodisks fabricated by hole-mask colloidal lithography (c) CRP detection curves performed with the nanoplasmonic biosensor at different concentrations of antibody immobilized (10, 20, 50 μg/mL) with ProLinker™ strategy; (d) Calibration curves for CRP detection performed on gold film (orange) and gold nanodisks nanoplasmonic (blue). Antibody concentration was 20 μg/mL and PLL-PEG was employed as blocking agent for both substrates. Dashed lines represent linear fit of linear region; (e) Nonspecific adsorption study of serum at different concentrations (10%, 25%, 50%, 100%) using different buffers in flow (PBST 0.5% and HBB) performed for both sensor substrates: SPR gold film (orange) and LSPR gold nanodisks (blue).

Mentions: The Prolinker™ strategy was evaluated on a gold nanodisks surface. We employed a homemade nanoplasmonic biosensor based on the LSPR of gold nanodisks when illuminated at a fixed angle of incidence (70°) [15]. Tracking the displacements of λLSPR caused by changes in the RI of the surrounding medium, enables monitoring biological events that take place on the gold nanodisks surface. By changing the sensor surface and the polarization of the light (i.e., TM for gold film and TE for gold nanodisks), this biosensing device allows working in both SPR and LSPR configurations. Hence, a reliable comparison between both biosensing approaches could be performed. We have implemented the ProLinker™ strategy to functionalize nanoplasmonic substrates based on the initial results obtained with plain gold films in SPR (see Figure 6a). Several antibody concentrations were evaluated (10, 20 and 50 μg/mL). The antigen detection curves obtained with the different antibody concentrations showed increasing signals when higher amount of receptor was immobilized (Figure 6c). We selected an antibody concentration ([Antibody] = 20 μg/mL) to continue with the study. Calibration curves performed under same conditions (same antibody concentration and PLL-PEG as blocking agent) for both SPR and LSPR biosensing schemes were carried out (see Figure 6d) reaching LODs of LOD(SPR) = 30.8 ng/mL (R2 = 0.9859) and LOD(LSPR) = 16.2 ng/mL (R2 = 0.9950).


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) Immobilization protocol for gold nanodisks substrates: (i) ProLinker™ layer formation; (ii) Antibody immobilization and blocking step with PLL-PEG; and (iii) Specific antigen detection; (b) Scanning electron microscopy (SEM) images of gold nanodisks fabricated by hole-mask colloidal lithography (c) CRP detection curves performed with the nanoplasmonic biosensor at different concentrations of antibody immobilized (10, 20, 50 μg/mL) with ProLinker™ strategy; (d) Calibration curves for CRP detection performed on gold film (orange) and gold nanodisks nanoplasmonic (blue). Antibody concentration was 20 μg/mL and PLL-PEG was employed as blocking agent for both substrates. Dashed lines represent linear fit of linear region; (e) Nonspecific adsorption study of serum at different concentrations (10%, 25%, 50%, 100%) using different buffers in flow (PBST 0.5% and HBB) performed for both sensor substrates: SPR gold film (orange) and LSPR gold nanodisks (blue).
© Copyright Policy
Related In: Results  -  Collection

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

f6-sensors-14-02239: (a) Immobilization protocol for gold nanodisks substrates: (i) ProLinker™ layer formation; (ii) Antibody immobilization and blocking step with PLL-PEG; and (iii) Specific antigen detection; (b) Scanning electron microscopy (SEM) images of gold nanodisks fabricated by hole-mask colloidal lithography (c) CRP detection curves performed with the nanoplasmonic biosensor at different concentrations of antibody immobilized (10, 20, 50 μg/mL) with ProLinker™ strategy; (d) Calibration curves for CRP detection performed on gold film (orange) and gold nanodisks nanoplasmonic (blue). Antibody concentration was 20 μg/mL and PLL-PEG was employed as blocking agent for both substrates. Dashed lines represent linear fit of linear region; (e) Nonspecific adsorption study of serum at different concentrations (10%, 25%, 50%, 100%) using different buffers in flow (PBST 0.5% and HBB) performed for both sensor substrates: SPR gold film (orange) and LSPR gold nanodisks (blue).
Mentions: The Prolinker™ strategy was evaluated on a gold nanodisks surface. We employed a homemade nanoplasmonic biosensor based on the LSPR of gold nanodisks when illuminated at a fixed angle of incidence (70°) [15]. Tracking the displacements of λLSPR caused by changes in the RI of the surrounding medium, enables monitoring biological events that take place on the gold nanodisks surface. By changing the sensor surface and the polarization of the light (i.e., TM for gold film and TE for gold nanodisks), this biosensing device allows working in both SPR and LSPR configurations. Hence, a reliable comparison between both biosensing approaches could be performed. We have implemented the ProLinker™ strategy to functionalize nanoplasmonic substrates based on the initial results obtained with plain gold films in SPR (see Figure 6a). Several antibody concentrations were evaluated (10, 20 and 50 μg/mL). The antigen detection curves obtained with the different antibody concentrations showed increasing signals when higher amount of receptor was immobilized (Figure 6c). We selected an antibody concentration ([Antibody] = 20 μg/mL) to continue with the study. Calibration curves performed under same conditions (same antibody concentration and PLL-PEG as blocking agent) for both SPR and LSPR biosensing schemes were carried out (see Figure 6d) reaching LODs of LOD(SPR) = 30.8 ng/mL (R2 = 0.9859) and LOD(LSPR) = 16.2 ng/mL (R2 = 0.9950).

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
Related in: MedlinePlus