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Development of Indirect Competitive Immuno-Assay Method Using SPR Detection for Rapid and Highly Sensitive Measurement of Salivary Cortisol Levels.

Tahara Y, Huang Z, Kiritoshi T, Onodera T, Toko K - Front Bioeng Biotechnol (2014)

Bottom Line: The surface of an Au chip was modified with PEG6-COOH aromatic dialkanethiol self-assembled monolayers and hydrocortisone 3-(O-carboxymethyl) oxime (hydrocortisone 3-CMO) as a cortisol analog.A detection limit of 38 ppt range with a measurement range of 10 ppt-100 ppb was accomplished without the incubation of a mixing solution consisting of standard cortisol and an anti-cortisol antibody, and the time for quantification of cortisol concentration was 8 min from the sample injection.It was found that the results obtained by the cortisol immuno-assay had a good correlation with those obtained by ELISA assay (R = 0.96).

View Article: PubMed Central - PubMed

Affiliation: Graduate School of Information Science and Electrical Engineering, Kyushu University , Fukuoka , Japan.

ABSTRACT
The monitoring of salivary cortisol as a key biomarker of an individual's stress response has been increasingly focused on. This paper describes the development of a novel cortisol immuno-assay method based on an indirect competitive method using a commercially available surface plasmon resonance instrument. The surface of an Au chip was modified with PEG6-COOH aromatic dialkanethiol self-assembled monolayers and hydrocortisone 3-(O-carboxymethyl) oxime (hydrocortisone 3-CMO) as a cortisol analog. A detection limit of 38 ppt range with a measurement range of 10 ppt-100 ppb was accomplished without the incubation of a mixing solution consisting of standard cortisol and an anti-cortisol antibody, and the time for quantification of cortisol concentration was 8 min from the sample injection. We experimentally compared our immuno-assay with a commercialized salivary cortisol enzyme-linked immunosorbent assay (ELISA) kit using human saliva samples. It was found that the results obtained by the cortisol immuno-assay had a good correlation with those obtained by ELISA assay (R = 0.96). Our findings indicate the potential utility of the cortisol immuno-assay for measurements of human salivary cortisol levels.

No MeSH data available.


Fabrication procedure of the sensor chip modified with cortisol analog.
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Figure 1: Fabrication procedure of the sensor chip modified with cortisol analog.

Mentions: As a preliminary cleaning step, an Au sensor chip covered with a 50-nm-thick layer of unmodified gold was first ultrasonically cleaned in acetone for 10 min, ethanol for 2 min, and 2-propanol for 2 min. Subsequently, the sensor surface was cleaned in standard clean solution (a mixture of ammonia solution, hydrogen peroxide solution, and pure water with a ratio of 1:1:5) heated to 90°C for 20 min. Modification of the sensor chip surface was performed as follows (Figure 1): the sensing surface of the Au sensor chip was immersed in 1 mM PEG6-COOH (Lahiri et al., 1999) in ethanol for 24 h to form SAMs on the sensor surface. After cleaning with ethanol by ultrasonication, a mixture solution consisting of 0.2 M EDC in water and 50 mM NHS in water was added dropwise onto the sensor surface and incubated for 30 min to activate the terminal carboxyl group. Next, ethylenediamine in the pH 8.5 borate buffer was added dropwise onto the sensor surface and incubated for 30 min to induce amine coupling by covering the terminal carboxyl group of the SAM into an amino group. Simultaneously, 10 mM hydrocortisone 3-CMO in DMF with a carboxyl group as a cortisol analog, 0.4 M EDC in water, and 0.1 M NHS in DMF were mixed with a volume ratio of 1:1:1 and incubated for 60 min to activate the carboxyl group of the cortisol analog. The mixed solution was added dropwise onto the sensor surface and incubated for 60 min. Finally, the cortisol analog was bound to ethylenediamine immobilized on the film surface as a result of amine coupling.


Development of Indirect Competitive Immuno-Assay Method Using SPR Detection for Rapid and Highly Sensitive Measurement of Salivary Cortisol Levels.

Tahara Y, Huang Z, Kiritoshi T, Onodera T, Toko K - Front Bioeng Biotechnol (2014)

Fabrication procedure of the sensor chip modified with cortisol analog.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4126460&req=5

Figure 1: Fabrication procedure of the sensor chip modified with cortisol analog.
Mentions: As a preliminary cleaning step, an Au sensor chip covered with a 50-nm-thick layer of unmodified gold was first ultrasonically cleaned in acetone for 10 min, ethanol for 2 min, and 2-propanol for 2 min. Subsequently, the sensor surface was cleaned in standard clean solution (a mixture of ammonia solution, hydrogen peroxide solution, and pure water with a ratio of 1:1:5) heated to 90°C for 20 min. Modification of the sensor chip surface was performed as follows (Figure 1): the sensing surface of the Au sensor chip was immersed in 1 mM PEG6-COOH (Lahiri et al., 1999) in ethanol for 24 h to form SAMs on the sensor surface. After cleaning with ethanol by ultrasonication, a mixture solution consisting of 0.2 M EDC in water and 50 mM NHS in water was added dropwise onto the sensor surface and incubated for 30 min to activate the terminal carboxyl group. Next, ethylenediamine in the pH 8.5 borate buffer was added dropwise onto the sensor surface and incubated for 30 min to induce amine coupling by covering the terminal carboxyl group of the SAM into an amino group. Simultaneously, 10 mM hydrocortisone 3-CMO in DMF with a carboxyl group as a cortisol analog, 0.4 M EDC in water, and 0.1 M NHS in DMF were mixed with a volume ratio of 1:1:1 and incubated for 60 min to activate the carboxyl group of the cortisol analog. The mixed solution was added dropwise onto the sensor surface and incubated for 60 min. Finally, the cortisol analog was bound to ethylenediamine immobilized on the film surface as a result of amine coupling.

Bottom Line: The surface of an Au chip was modified with PEG6-COOH aromatic dialkanethiol self-assembled monolayers and hydrocortisone 3-(O-carboxymethyl) oxime (hydrocortisone 3-CMO) as a cortisol analog.A detection limit of 38 ppt range with a measurement range of 10 ppt-100 ppb was accomplished without the incubation of a mixing solution consisting of standard cortisol and an anti-cortisol antibody, and the time for quantification of cortisol concentration was 8 min from the sample injection.It was found that the results obtained by the cortisol immuno-assay had a good correlation with those obtained by ELISA assay (R = 0.96).

View Article: PubMed Central - PubMed

Affiliation: Graduate School of Information Science and Electrical Engineering, Kyushu University , Fukuoka , Japan.

ABSTRACT
The monitoring of salivary cortisol as a key biomarker of an individual's stress response has been increasingly focused on. This paper describes the development of a novel cortisol immuno-assay method based on an indirect competitive method using a commercially available surface plasmon resonance instrument. The surface of an Au chip was modified with PEG6-COOH aromatic dialkanethiol self-assembled monolayers and hydrocortisone 3-(O-carboxymethyl) oxime (hydrocortisone 3-CMO) as a cortisol analog. A detection limit of 38 ppt range with a measurement range of 10 ppt-100 ppb was accomplished without the incubation of a mixing solution consisting of standard cortisol and an anti-cortisol antibody, and the time for quantification of cortisol concentration was 8 min from the sample injection. We experimentally compared our immuno-assay with a commercialized salivary cortisol enzyme-linked immunosorbent assay (ELISA) kit using human saliva samples. It was found that the results obtained by the cortisol immuno-assay had a good correlation with those obtained by ELISA assay (R = 0.96). Our findings indicate the potential utility of the cortisol immuno-assay for measurements of human salivary cortisol levels.

No MeSH data available.