Peg6000

Polyethylene glycol (PEG) precipitation was carried out as previously described (8 (link)). In brief, 100μL of serum was added to the same volume of 25% cold PEG 6000 (Sinopharm chemical reagent Beijing Co., Ltd. #20161104) and mixed vigorously, followed by centrifugation at 3,000×g for 30 min. Both serum insulin levels (FINS before PEG, also called direct FINS) and the insulin levels in the supernatant (FINS after PEG, also called free FINS) after PEG precipitation were measured on an electrochemiluminescence immunoassay analyzer (Cobas e601, Roche Diagnostic, Germany). As the manufacturer had indicated, the FINS measured by this kit showed no cross-reactivity with insulin aspart, insulin lispro, and insulin glargine (9 , 10 (link)). The PEG/precipitated insulin ratio (PPIR), which represents the proportion of antibody-bound insulin, was calculated as (FINS before PEG – FINS after PEG)/FINS before PEG. Due to dilution bias during sample processing, the final result for subjects without IAs is often negative. The inter-assay and within-assay coefficient of variations (CVs) for the FINS after PEG precipitation were 2.79% and 2.16%, respectively.

GO solution (1 mg/mL) was purchased from Suzhou TANFENG Graphene Technology Co., Ltd. (Suzhou, China). Phosphate buffered saline (PBS, pH 7.4) and PEG-6000 were purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). EDOT and chloroplatinic acid hexahydrate were purchased from Cool Chemical Technology (Beijing, China) Co., Ltd. Agar powder was purchased from Beijing Chembase Co., Ltd. (Beijing, China). The laser diodes (445 nm, 80 mW) were purchased from SINOSEMIC Co., Ltd. (Jinan, China). Anisotropic conductive films (ACF) were purchased from HITACHI, Ltd. (Tokyo, Japan). Scanning electron microscopy (SEM) analysis was performed using a high vacuum scanning electron microscope (ULTRA55, Zeiss, Germany). The electrochemical measurements and modifications were conducted with an electrochemical workstation (CHI660E, CH instrument). Noise signals were recorded using an RHD2000 Evaluation System (Intan Technology, LA, CA, USA).