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.