Characterization of Ages Produced from CELLS Using SECM-ELISA
Authors: Kasai Shigenobu , Takahashi Yutaro , Sato Teruya , Hosshi Hiroyoshi 
Published: February 2020

1. Introduction
Advanced glycation end products (AGEs) are substances produced in the body by nonenzymatic reaction of reducing sugars and amino groups of proteins. They can be a factor in ageing and attracts attention in recent years due to its association in diabetes complications and arteriosclerosis1). There are few cases where the amount of AGEs produced at the cell level was measured. In our current study, we present our results of electrochemical activity evaluation of competitive ELISA using SECM for the purpose of highly sensitive detection of AGEs produced at the cellular level2).
2. Experimental
2.1 Fabrication of wells for AGEs detection by the competitive method
Competition method well formation was performed in the following steps [steps 1-8].
(1) Mix 10 ug/mL of AGE conjugate and 1Xconjugate diluent at a 1: 1 ratio and take 40uL.
(2) After incubating at 4 degrees for 24 hours, remove AGE conjugate coating solution and wash twice with phosphate buffer saline (PBS) [Sigma Aldrich, USA].
(3) Add 50 uL of assay diluent and block for 1 hour at room temperature (RT).
The process up to this point was regarded as a well for detecting AGEs.
(4) After removing the blocking agent, 20uL of THP-1 cell suspension was added.
(5) Following incubating at room temperature for 10 minutes, add 20uL of 1000-fold diluted Anti-AGE antibody.
(6) Further, incubate for 1 hour at RT, remove the solution and wash 3 times with buffer
(7) Add 40uL of 1000-fold diluted secondary antibody-HRP conjugate.
(8) 3X washing with buffer [as in step 6].
2.2 AGEs production evaluation of THP-1 cells by SECM-ELISA method
Experiments were conducted using a PBS (pH 7.4) mixed with 4 mM FcCH2OH and 2 mM H2O2 as a measurement solution. An Au electrode (d= 1.6 mm) was used as a working electrode, Pt wire was used as a counter electrode, an Ag/AgCl electrode was used as a reference electrode, and the applied voltage was held at +0.05 V vs. Ag/ AgCl. The HRP activity was characterized by scanning the Au electrode at a scan rate of 10 um/s in the direction of Z-axis 500 um and measuring the reduction current of Fc+CH2OH　(Fig.1). Using the SECM-ELISA method, the difference between the initial current value and the peak current value was set ΔI and AGEs produced from THP-1 cells were characterized by three average peak current values.
3. Results and Discussion
At THP-1 cell count 1.5 × 103 cells/ 20 uL, the average peak current value was observed to be 4.8 nA which was found to be at 4.0 nA for a cell density of 1.5 × 104 cells/20uL. It was recorded to further decrease to 3.1nA for a cell density of 1.5 × 105 cells/20uL. It can be observed that the current value was decreased with the increase in the number of THP-1 cells. In addition, the results of the AGE amount produced from THP-1 cells from the calibration curve of the AGE-BSA standard are shown in Fig. 2. An increase in the amount of AGEs produced as the number of THP-1 cells increased was observed.
References
1)A. Goldin, et al., Basic Science for Clinicians, Circulation. 114, 597-605 (2006).
2)S. Kasai, et al., Analytica Chimica Acta. 566, 55-59 (2006).

Figure 1