br Lastly the bPEI MGO was decorated
Lastly, the bPEI-MGO was decorated with PB nanoparticles to ex-hibit excellent peroxidase activity. The synthesis process was as follows: 10 mL of PB precursor solution that contained 5 mM of FeCl3 and 5 mM of K3[Fe(CN)6] was prepared. After the pH value was adjusted to 1.5, the solution was mixed with preprepared bPEI-MGO at room tem-perature for 2 h. Under the acidic environment, the Fe3+ could be re-duced to Fe2+ by GO, which immediately reacted with Fe(CN)63- to
form PB nanoparticles on the surface of the bPEI-MGO to obtain PMGO (Fig. S1).
2.3. Preparation of self-linkable PMGO
The proposed signal amplification strategy was accomplished through a self-linking reaction of three types of PMGO: PMGO-1, PMGO-2, PMGO-3. All Ifenprodil hemitartrate were first modified with sulfenyl groups by reacting with excessive Traut's reagent in darkness for 1 h for further use. The PMGO-1 was synthesized as a capture probe to bind onto the ApoA1 captured on the biochipApoA1. The AbApoA1 and mouse IgG were simultaneously conjugated on the PMGO; 1 mL of PMGO (20 μg/mL) was reacted with 33 µL of sulfosuccinimidyl 4-[N-mal-eimidomethyl] cyclohexane-1-carboxylate (sulfo-SMCC; 2 mg/mL) in darkness for 3 h and then reacted with thiol-functional AbApoA1 and mouse IgG for another 2 h in darkness to obtain PMGO-1. The PMGO-2 and PMGO-3 were synthesized as signal-amplification probes to self-link to PMGO-1 and PMGO-2; 1 mL of PMGO (20 μg/mL) was reacted with 33 µL of sulfo-SMCC (2 mg/mL) in darkness for 3 h, and then re-acted with thiol-functional rabbit anti-mouse IgG antibody (rabbit host) for another 2 h in darkness to obtain PMGO-2. Subsequently, 1 mL of PMGO (20 μg/mL) was reacted with 33 µL of sulfo-SMCC (2 mg/mL) in darkness for 3 h, and then reacted with thiol-functional goat anti-rabbit IgG antibody (goat host) for another 2 h in darkness to obtain PMGO-3. The obtained PMGO-1, PMGO-2, and PMGO-3 were blocked with 1% BSA to prevent nonspecific binding.
2.4. Colorimetric immunosensor for ApoA1 detection
For the detection process, various concentrations of ApoA1, ranging from 0.05 ng/mL to 100 ng/mL, were added into a tube containing biochipApoA1 and incubated for 30 min at room temperature. The ApoA1 was captured by AbApoA1 because of the high aﬃnity between them and the surface of the chips, which were gently washed three times with TBST. Subsequently, equal volumes (50 µL) of PMGO-1, PMGO-2, and PMGO-3 were added into the tube together and incubated for 30 min After washing with TBST, 100 µL of H2O2 + TMB as a de-veloper was added into the tube for 3 min at room temperature, and then the oxidation was stopped by addition of HCl to show an ab-sorption peak at 450 nm (Fig. S1). The produced yellow color of the solution became increasingly deep with increasing ApoA1 concentra-tions, and the A450 nm was linearly proportional to the concentration of ApoA1. All experiments were conducted in sextuplicate for precise calculations to develop a standard protocol.
2.5. Interference tests
An outstanding immunosensor should possess not only specificity but also selectivity. Therefore, interference assays were conducted on our immunosensor. A variety of substances, such as urea (250 µg/mL), ascorbic acid (AA; 370 µg/mL), bovine serum albumin (BSA; 5%), fetal bovine serum (FBS, 10%), vascular endothelial growth factor (VEGF, 100 ng/mL), carbohydrate antigen 19–9 (CA19-9; 100 ng/mL), mucin 1 (MUC1; 100 ng/mL), and FXYD domain‑containing ion transport reg-ulator 3 (FXYD3; 100 ng/mL), were employed. First, the interferences or ApoA1 were added into the tube containing biochipApoA1, and the reaction was allowed to proceed for 30 min at room temperature. After the covering was rinsed gently with DI-H2O three times, the washed biochipApoA1 was treated with 150 µL of self-linkable PMGOs (PMGO-1 + PMGO-2 + PMGO-3) for another 30 min, developing through the addition of 100 µL of H2O2 + TMB solution for 3 min at room tem-perature and then the addition of HCl to stop the reaction. The A450 nm was recorded to determine the interference level using a SpectraMax M2 (Molecular Devices Co., California, USA).