4.3. Assembly of PEC biosensor
The base Au NPs / rGO electrode was first cleaned using sulfuric acid, potassium hydroxide, and nitric acid as described previously[43]. The 5′-thiol-modified ssDNA1 was exposed to 10 µM TCEP for 10 min to avoid the generation of disulfide bonds and then diluted to 0.25 µM by addition of 10 mM Tris buffer containing 10 mM EDTA. Then, 200 µL of the 0.25 µM ssDNA1 solution was incubated onto the abovementioned electrode at room temperature for 1 h to obtain the ssDNA1/Au NPs/rGO electrode through Au–S bonding. Subsequently, the modified electrode was coated with 200 µL of 2 mM MCH for a duration of 30 minutes to block nonspecific adsorption sites and achieve a highly uniform surface. This step resulted in the formation of the MCH/ssDNA1/Au NPs/rGO electrode. Then 200 µL of PBS buffer containing ssDNA2 and 5′-amino-modified ssDNA3 with an equimolar concentration of 0.25 µM was added to the modified electrode and incubated at room temperature for 1 h to obtain an MCH/dsDNA/Au NPs/rGO electrode based on sequence complementarity. Subsequently, the CdTe/ZnS QDs suspension was stimulated by blending it with MES buffer (0.1 M, pH 6.0) consisting of 20 mM EDC and 10 mM NHS at a volume ratio of 1:9 for 1 h. Next, 200 μL of a suspension of activated CdTe/ZnS QDs was added to the modified electrode and incubated for 2 h to immobilize the CdTe/ZnS QDs via amide bonds. The obtained electrode was denoted as CdTe/ZnS QDs/MCH/dsDNA/Au NPs/rGO electrode, namely CdTe/ZnS QDs–dsDNA/Au NPs/rGO electrode. Following each step of the biosensor fabrication process, the electrode was subjected to three rinses with a washing buffer. Finally, the sensor was dried using nitrogen gas to prepare for PEC testing.