Microbiosensor fabrication by polydimethylsiloxane stamping for combined sensing of glucose and choline.

High performance microprobes for combined sensing of glucose and choline were fabricated using microcontact printing (μCP) to transfer choline oxidase (ChOx) and glucose oxidase (GOx) onto targeted sites on microelectrode arrays (MEAs). Most electroenzymatic sensing sites on MEAs for neuroscience applications are created by manual enzyme deposition, which becomes problematic when the array feature size is less than or equal to ∼100 μm. The μCP process used here relies on use of soft lithography to create features on a polydimethylsiloxane (PDMS) microstamp that correspond to the dimensions and array locations of targeted, microscale sites on a MEA. Precise alignment of the stamp with the MEA is also required to transfer enzyme only onto the specified microelectrode(s). The dual sensor fabrication process began with polyphenylenediamine (PPD) electrodeposition on all Pt microelectrodes to block common interferents (e.g., ascorbic acid and dopamine) found in brain extracellular fluid. Next, a chitosan film was electrodeposited to serve as an adhesive layer. The two enzymes, ChOx and GOx, were transferred onto different microelectrodes of 2 × 2 arrays using two different PDMS stamps and a microscope for stamp alignment. Using constant potential amperometry, the combined sensing microprobe was confirmed to have high sensitivity for choline and glucose (286 and 117 μA mM cm-2, respectively) accompanied by low detection limits (1 and 3 μM, respectively) and rapid response times (≤2 s). This work demonstrates the use of μCP for facile creation of multianalyte sensing microprobes by targeted deposition of enzymes onto preselected sites of a microelectrode array.