Mercaptoacetic acid

Firstly, the electrodes were washed ultrasonically in ethanol for 5 min and then were immersed in piranha solution (H₂O₂(v)/H₂SO₄(v) = 1/3) for 5 min to clean the surfaces. Subsequently, the electrodes were rinsed with sterile ultrapure water for 10 times and were dried with nitrogen. After dripping 2 μL mercaptoacetic acid (Sigma, USA) on the surface of each working electrode, the chip was placed in an airtight container for 1 h to form a carboxylic self-assembled monolayer. Then, the chip was rinsed with ethanol and was dried with nitrogen gently. The carboxyl groups on the surface of electrodes were activated with 0.4 M 1-ethyl-3-[3-dimethylaminopropyl] carbodiimide hydrochloride (EDC) and 0.2 M N-hydroxysuccinimide (NHS) solution prepared in a 0.1 M phosphate buffer solution (PBS, pH = 7.4) for 20 min for immobilizing antibodies. After rinsing with PBS buffer and drying with nitrogen, six kinds of antibodies (anti-CEA, anti-CA19-9, anti-H.P., anti-P53, anti-PG I, and anti-PG II) solutions were respectively dripped on the surfaces of six working electrodes and incubated at 37 °C for 3 h. Lastly, the immunological chip was obtained after incubating 0.5 % BSA (bovine serum albumin) at 37 °C for 1 h to block non-specific binding sites and rinsing with 0.01 M PBS buffer. The prepared chips were stored at 4 °C for next immunoreaction and electrochemical detection.

Graphene oxide (GO) (N002-PS) and quantum dots (QD) (QSA-490, CdSSe/ZnS core/shell QDs with amine group) were purchased from Angstron Materials (Dayton, OH, USA) and Ocean NanoTech (San Diego, CA, USA), respectively. N-isopropylacrylamide (NIPAM), azobisisobutyronitrile (AIBN), mercaptoacetic acid (MAA), chitosan (deacetylation degree = 98%, molecular weight = 1.5 × 10⁵ Da), 2-morpholinoethane sulfonic acid (MES), 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide, (MTT), 2,4,6-trinitrobenzene sulfonic acid (TNBS) and 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI), folic acid (FA), doxorubicin (DOX) hydrochloride were purchased from Sigma-Aldrich (St. Louis, MO, USA). 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) and N-hydroxysuccinimide (NHS) were obtained from Acros (Geel, Belgium). Potassium salt of D-luciferin was obtained from Gold Biotechnology, Inc. (New Taipei City, Taiwan). Hyaluronic acid (HA, sodium hyaluronate) from Streptococcus zooepidemicus with an average molecular weight of 1.8 × 10⁶ Da was purchased from Bloomage Freda Biopharm Co. (Jinan, China). Minimum Essential Medium (α-MEM, ThermoFisher Scientific, Waltham, MA, USA) and fetal bovine serum (FBS, HyClone, Logan, UT, USA) were used for cell culture.

Indium(iii) chloride, silver and copper(ii) nitrates, zinc(ii) acetate dihydrate, Na₂S·9H₂O, NH₄OH (aqueous 5.0 M solution), mercaptoacetic acid (MAA), 2-propanol were purchased from Sigma-Aldrich and Acros Organics and used without additional purification. All the solutions were prepared using deionized (DI) Milli-Q water (Millipore).