1.1.Biological and Chemical Materials

1.1.Biological and chemical materials

Human PSA antigen, mouse PSA monoclonal antibody and biotinylated mouse PSA polyclonal antibody are purchased from Linc-Bio Science Co. Ltd. (Shanghai, China, Dynabeads® MyoneTM streptavidin C1 was purchased from Invitrogen ( Phosphate buffered saline (PBS) was purchased from Medicago AB (Uppsala, Sweden, 11-mercaptoundecanoic acid was purchased from Aladdin Chemistry Co. Ltd. (Beijing, China, 1-ethyl-3-[3-dimet-hylaminoprop-yl] carbodiimide (EDC) hydrochloride was purchased from Aladdin Chemistry Co. Ltd (Beijing, China, N-Hydroxysulfosuccinimide sodium salt (NHS) was purchased from Medpep Co. Ltd. (Shanghai, China, Albumin from bovine serum (BSA) was purchased from Via-gene pro bio Technologies Co. ltd. (Shanghai, China, Hydrochloricacid (HCl) was purchased from Sinopharm Chemical Reagent (Shanghai, China, Sodium hydroxide (NaOH) was purchased from Pinghu Chemical Reagent (Pinghu, China, In all experiments, deionized water was used.

1.2. Preparation of PSA samples

Double antibody sandwich immunoassay and streptavidin–biotin binding assay was employed for the immobilization and labeling of PSA. The PSA samples was achieved on small glass which the surface was sputtered an Au film with thickness was 300 nm.

1. Washing the Au film: the small glass with Au film was first cleaned before self-assembly in 1mol/L NaOH for 10 min followed by 1 mol/L HCl for 10 min. Then rinsed with deionized water and alcohol; finally dried with N2.

2. 11-mercaptoundecanoic acid served as self-assemble monolayer on the Au film: self-assembly on the gold film was carried out in 30 mL 11-mercaptoundecanoic acid at room temperature for 3 h and washed twice in anhydrous alcohol followed by drying.

3. Activation of 11-mercaptoundecanoic by EDC and NHS: the Au film was immersed and activated in a mixture obtained by dissolving 0.2 mol/L EDC and 0.05 mol/L NHS in phosphate buffer (pH=7.4) for 30 min. Finally, the film was washed several times with phosphate buffer (pH=7.4).

4. Immobilization of PSA monoclonal antibodies: PSA monoclonal antibodies were dissolved in phosphate buffer (pH=7.4) and the concentration was controlled at 0.5-1 mg⋅mL-1. Then, a 10 μL drop of the PSA monoclonal antibody solution was dropped on the gold film. After incubating at room temperature for 2 h, the wafer was washed twice with phosphate buffer (pH=7.4, including 1% (w/v) BSA).

5. BSA was used to block the nonspecific adsorption sites: a 100 μLBSA solution (including 1% (w/v) BSA, 0.2% (v/v) tween 20) at 4 ℃for 2 h was used for blocking. Finally, the wafer was washed twice with PBS solution (pH=7.4).

6. PSA polyclonal antibody labeled by Dynabeads with the diameter was 1μm: 100 μL of 1 mg/mL biotinylated PSA polyclonal antibody solution was mixed with 100 μL of 100 μg/mL streptavidin-functioned DynabeadsMyoneTM suspension and was incubated at 37℃for 1h. Magnetic separation to remove excess polyclonal antibody was carried out using DynaMagTM-Spin and the excess solution was pipetted out.

7. Immobilization of PSA by PSA polyclonal antibody which labeled with Dynabeads; PSA solution was diluted with PBS solution (PH=7.4) to six concentrations (0 ng/mL (BSA), 0.1 ng/mL, 0.5 ng/mL, 1 ng/mL, 5 ng/mL and 10 ng/mL). 40 μl of 10 μg⋅mL-1 streptavidin-functionalized Dynabeads conjugated with biotinylated PSA polyclonal antibody was inserted into 1 mL PSA solution for each of the test solutions mentioned above. These solutions were incubated at room temperature for 2 h followed by inserting the mixture into DynaMagTM-Spin for magnetic separation and excess supernatant was removed.

8. Capture of PSA, 40 μL of the six solutions with different concentrations of PSA was dropped on separate functionalized gold films. The films were then incubated at room temperature for 2 h, and washed twice with PBS solution (PH=7.4, including 1% BSA). Samples with carrying PSA concentrations of 0 ng/mL (BSA), 0.1 ng/mL, 0.5 ng/mL, 1 ng/mL, 5 ng/mL and 10 ng/mL were prepared for the detection experiments. A schematic illustration of double-antibody sandwich assay for PSA detection is shown in Fig. S1.

[Fig.S1]