In-Situ Detection of Saliva Cortisol with Cu-MOF Catalyst Integrated-Antibody Based on Portable E-Eye

Introduction Cortisol, as a steroid hormone secreted by the human body, is a typical stress marker. It plays an important role in regulating blood pressure, blood sugar, carbohydrate metabolism and other physiological processes. Among them, measuring saliva cortisol seems to be advantageous because it is a relatively easy and non-invasive method to extract saliva samples, and saliva can be conveniently processed and stored. Under normal circumstances, the body can well control the secretion of cortisol and regulate the content of cortisol in the blood. However, under greater physical and psychological stress, a high level of cortisol would be induced and kept for a long time, which will lead to the blood sugar, increased appetite, increased weight, and extreme fatigue, etc. The traditional methods for cortisol detection have biochemical analysis, radioimmunoassays, which is based on large-scale equipment and difficult to real-time monitoring. Therefore, the development of rapid, on-situ and sensitive cortisol detection products has important national defense strategic significance. In this study, the Cu-MOF catalyst integrated-antibody was used for cortisol in-situ colorimetric immunoassay based on the smartphone-based portable electric eye (E-eye) (Fig.1). As a metal-organic framework, Cu-MOF can conjugate with antibody without complicated operations, and then specifically recognize cortisol. As a catalase, Cu-MOF can not only catalyze the coloration of TMB, but also amplify the signal, and also protect the antibody from long-term high temperature. Based on the indirect competition method, the cortisol in the samples competes with the cortisol-BSA coated on the microtiter plate to bind the cortisol antibody. And then the conjugation of MOF-antibody (Cu-MOF@IgG) will bind with the antibody, which could catalyze the H2O2 and TMB substrates to blue color and amplify the signal, which could be quantitative recognition by portable E-eye. The degree of blue is inversely proportional to the cortisol content. Based on the portable E-eye, the proposed method processes the features of rapid, simple, high sensitivity and high throughout, which could achieve cortisol in-situ detection in practical applications. Preparation of Cu-MOF The Cu-MOF was fabricated by mixing 7.8mM CuCl2 aqueous and 31.2mM 4,4’-bipyridine equally with 2h gentle stirring under room temperature. The blue precipitates were obtained by centrifugation and washed by ultrapure water. Then it was re-dissolved in 500µl pure water for further use. The catalytic activity of Cu-MOF was verified by adding Cu-MOF to the H2O2 and TMB mixture. Under the successful preparation of Cu-MOF, the mixed solution will turn blue due to the catalase activity of Cu-MOF. The pH of the system was adjusted by NaAC/HAc buffer. The morphology (Fig.2(a) and (b)) and the catalase activity (Fig.2(c)) of Cu-MOF materials were characterized by transmission electron microscope and microplate reader. Preparation of Cu-MOF@IgG The Cu-MOF@IgG materials were formed via the following two steps: (1) 100µl of 20mg/L IgG was mixed with the 2mL of 7.8mM CuCl2 aqueous at 4 ℃ for 10 min. (2) Then 2mL of 31.2mM 4,4’-bipyridine was added to the above mixture with 12h continuously stirring at 4 ℃. Also, the 4,4’-bipyridine above was prepared by dissolving 0.0487g of 4,4’-bipyridine powder in 10 mL ultrapure and water mixture with equal volume. (3) The product obtained from the above reaction was thoroughly centrifuged and washed thrice with ultrapure water, and then re-dissolved in 500ul pure water. The prepared Cu-MOF@IgG are stored at 4 ℃ for further use. The verification of the combination of Cu-MOF and IgG was as follows: First, it has been verified that the material of Cu-MOF has no absorption to the microtiter plate (Fig. 2(d)). The obtained Cu-MOF@IgG solution was diluted with carbonate buffer, and then coated on a microtiter plate. After incubating for 12 h at 4℃, the microtiter plate was washed by PBST (0.01 M PBS + 0.01% Tween) four times to remove excess free Cu-MOF and IgG. Then the mixture of H2O2 and TMB was added to the coated microtiter plate. After incubating of 20 min at 37℃, the color of the solution would be reserved for the successful preparation of Cu-MOF@IgG (Fig.2(e)). Cortisol detection based on Cu-MOF@IgG The cortisol-BSA was diluted to 2.7μg/mL with carbonate buffer (pH = 9.6), and then coated on a microtiter plate with a volume of 100μL per well. After incubating overnight at 4°C, the coated plate was washed with PBST four times to remove the excess free primary antigen. Then the free binding sites were sealed with 2% BSA solution for 2h at 37℃ to prevent the nonspecific binding. After washing with PBST four times, the cortisol standard and cortisol MAb was added to the plate and incubate at 37°C for 1 h. Then 100µl of Cu-MOF@IgG was added to binding with the coated cortisol MAb. After incubating at 37℃ for 2h, the unbound Cu-MOF@IgG was removed by washing with PBST four times. Then the mixture of H2O2 and TMB was added to wells and incubate for 20 min at 37°C. Finally, the absorbance of wells at 650nm was detected by the microplate reader. Also, the color changes could be detected by the homemade portable E-eye. As shown in Fig. 2(f), the detection limit of cortisol in the saliva is 0.12µg/L with the detection range of 0.88-1000µg/L. Figure 1