On-Site and Daily Stress-Measurement Using Gold-Linked Electrochemical Immuno Assay for IgA Determination
Authors: Osaki Shuto , Wakida Shin-ichi , Saito Masato , Tamiya Eiichi 
Published: May 2020

[Introduction]
Mental stress is closely connected with our physical and mental wellness. In particular, depression may be associated with stress. Therefore, stress measurement can contribute to assess our lifestyle and increase our quality of life. However, objective stress-measurement is not established. To establish objective stress-measurement, an individual must indicate the stress level by obtaining numerous data continuously throughout the day. Therefore, the measurement method which can be suitable for this condition is needed. In this paper, we detect the secretory Immunoglobulin A (sIgA), which is the candidate of salivary stress-markers, with original electrochemical immunoassay: Gold-Linked Electrochemical Immuno Assay (GLEIA). This biosensor is based on a sandwich-type immunosensor, and adopt the electrochemical method to detect the reduction peak from Au nanoparticles linked to the secondary antibody. GLEIA is convenient and cost-effective that only requires low sample volume (10 µL). In addition, all devices that GLEIA needs are minimal such as note PC and portable potentiostat and printed electrode. Therefore, GLEIA method shows various advantages suitable for stress measurement as compared with other sIgA detection methods. In this paper, we aim to the on-site and continuous measurement of salivary IgA in our daily life, to determine the various concentration of sIgA in standard solution and artificial saliva using GLEIA.
[Experimental]
Primary Anti-IgA Antibody solution was dropped onto the working electrode for immobilizing the antibody by physical adsorption. After, the Au nanoparticle solution was mixed with the phosphate buffer (50 mM, pH=8.3). The anti-IgA to dissolve the 5 mM of phosphate buffer add the Au nanoparticle solution. For the preparing of secondary antibody modified Au nanoparticle, the antibody was conjugated with Au nanoparticle under the pH=8.3 condition and kept for 10 min at room temperature. Then, 10% BSA in PBS buffer and 1% PEG in PBS buffer were added in preparing Au solution. Finally, the concentration was adjusted for OD520=6.0 by centrifuge and exclude the supernatant. The Au Anti-IgA conjugate was diluted by 3 times with 50 w/v of trehalose (OD520=2) and dripping 5µL of this solution in the immunomodule. Then the immunomodule was dried in vacuum condition for 5 minutes. Dried immunomodule was stored at 4 ℃ until use. Different concentrations of the IgA antigen solution (0, 10, 60, 150, 300 ng/mL) were made by diluting in PBS. For the detection of Antibody-Antigen reaction, the IgA solutions were mixed in prepared immunomodule, After the 1 minute, and 1.4 µL of the solution was placed on immunosensor to incubate for 1 hour, after rinsing with PBS, and eliminate the PBS solution with the air gun. The direct redox reaction was performed in 2M KCl solution (20 µL) covering the entire three-electrode zone at room temperature.
[Results and Discussion]
We obtained the linear response to relate the concentration of sIgA (10-300 ng/mL) in D-PBS buffer with the artificial-saliva which includes salivary inorganic salt and typically glycoprotein (mucin). Figure 1 shows the differential pulse voltammogram of GLEIA with IgA in artificial saliva (a) and calibration curve (b). In addition, the calibration curve shows the linear range within 10-300 ng/mL and the correlation coefficient (R2) of the calibration curve for this relationship was 0.963. The peak current intensity is decreased from in standard solution. It may prevent the Antigen-Antibody reaction from artificial saliva which pH is nearly 8.5. Generally, the antibody shows the best affinity in pH= 6.5~8.4 condition. However, these results show that GLEIA is suitable for salivary IgA detection because the salivary pH is mainly neutral. Furthermore, we evaluate the selectivity of the immunosensor by comparing it with the reduction peak between IgA= 300ng/mL or 0 ng/mL and interference protein such as α-amylase, human serum albumin, immunoglobulin G (IgG), lysozyme and mucin. We acknowledge GLEIA shows the signal increasing within the existence of interference proteins. This may be the affinity of antibody and non-specific reaction between antibody and the proteins. More importantly, we recognize that the reduction peak is increasing with the concentration of sIgA in the existence of interference proteins. Therefore, GLEIA can be applied for stress measurement using salivary sIgA that can be used in daily life anywhere and anytime whenever necessary. In the future, we try to detect the sIgA in real saliva for on-site stress measurement using GLEIA and to integrate the various immunosensors for stress-markers in saliva.nd anytime whenever necessary. In the future, we try to detect the sIgA in real saliva for on-site stress measurement using GLEIA and to integrate the various immunosensors for stress-markers in saliva.

Figure 1