The ability to monitor neuronal processes linked to complex behaviors is very important for study of neurological disorders and abnormal behaviors. The study of neurological disorders on a chemical level requires sensitive and fast techniques to monitor the release of neurotransmitters in near-real time in vivo. The establishment of relationships between behaviors and neurotransmitter release events will help us better decipher complex systems in the brain. This information can be obtained from near-real-time neurochemical sensing. Our lab has successfully fabricated glutamate (Glut) microbiosensors which can selectively detect glutamate in the presence of the electroactive interferents, dopamine (DA) and ascorbic acid (AA), with fast response time (~1 s), high selectivity and low detection limit. However, the enzyme transfer and immobilization steps, which are the key steps in the fabrication of our electroenzymatic glutamate biosensors, formerly were achieved by manually depositing a bovine serum albumin (BSA)/glutaraldehyde (GAH)/glutamate oxidase mixture onto the surface of the electrodes. Although the GAH crosslinking enzyme immobilization method has been validated both in vivo and in vitro, the manual deposition procedure causes problems such as inconsistent enzyme layer thickness, which results in inconsistent sensor performance. In addition, if each microelectrode on a microelectrode array (MEA) format probe requires a different enzyme coating, manual enzyme immobilization will be difficult due to the spacing between each microelectrode (each electrode is ~40 μm apart). Thus, a non-manual method is needed to transfer enzyme to select microelectrode sites with high consistency and spatial resolution.
Microcontact printing (μCP) has been used to directly pattern arrays of proteins on silicon or glass substrates without compromising the activity of the proteins. An elastomeric polydimethylsiloxane (PDMS) stamp is covered with a solution of target protein for inking. This results in deposition of a layer of protein on the surface of the stamp. After removing excess liquid and drying, the protein can be transferred to a target substrate by stamping. The μCP approach allows reliable feature replication with dimensions down to about 500 nm. To test the feasibility of this technique for transferring enzyme to electrodes, PDMS stamping of enzyme onto disk electrode surfaces was attempted. Glucose oxidase (GOx) was chosen as a model enzyme for this work. BSA and GOx were deposited together on a PDMS stamp, transferred to an electrode surface and then crosslinked by GAH vapor treatment. The biosensor exhibited excellent performance characteristics including a linear range up to 2 mM with sensitivity of 29.4 � 1.3 μA mM-1 cm-2 and detection limit of 4.3 � 1.7 μM (S/N = 3) as well as a rapid response time of ~2 s. In comparison to those previously described, this glucose biosensor exhibits an excellent combination of high sensitivity, low detection limit, rapid response time, and good selectivity.
For the fabrication of microbiosensors, a microscale stamp was created by casting the polymer on a silicon mold that previously was micromachined. The stamp was used for
transferring glucose oxidase and choline oxidase to selected microelectrode sites on the same microprobe. As a result, a dual sensing microprobe (glucose and choline) was fabricated and tested in vitro. The dual sensor we fabricated showed high sensitivity for choline and glucose (286 and 117 μA/mM cm2, respectively) accompanied by a low detection limit (3 and 1 μA respectively). The work presented here shows the prospect for fabricating a microelectrode array for multiple neurotransmitter sensing and high throughput enzyme deposition, which inevitably leads to the potential development of a high performance neurotransmitter sensor.
Another route to improve sensor performance is through an increase in effective electrode surface area. A platinum black (Pt black) deposit is often used for enlarging the effective electrode surface area. The nanoscopic Pt particles making up the deposit also often show improved catalytic behavior resulting in potential reduction for electrooxidations. However, practical applicability of Pt black is limited as a consequence of its low mechanical stability. In addition, Pt black has been reported to cause a cytotoxic reaction in vivo, as traces of lead used in the electrolyte during fabrication elute from the deposit. An alternative nanostructured Pt coating was reported recently that results in impedance reduction at small electrodes. In comparison to Pt black, all chemicals used in the alternate deposition process are non-cytotoxic. The Pt nanograss was electrodeposited onto the microelectrode surface using an aqueous solution of H2PtCl6 and formic acid. According to SEM images, Pt black was irregularly attached to the surface rather than grown from the surface while Pt nanograss showed a more uniform pillar-like structure. A choline biosensor was developed by incorporating Pt nanograss deposition which results in working potential reduction (from 0.7 V to 0.45 V). This nanostructured platinum coating was coated on both working electrode and on-probe reference electrode and characterized by scanning electron microscope (SEM). In this work, polyphenylenediamine (PPD) and Nafion were coated on electrode surface
sequentially as permselective polymers. With the developed choline sensor testing at physiological concentrations, the performance of the biosensors is quite consistent. By using external Ag/AgCl reference electrode (0.45V), the detection limit was ~2 μM with sensitivity of ~196 μA/mM cm2 and response time of 3-5s. With an on-probe reference electrode (0.35 V), the detection limit was ~3 μM with the sensitivity ~123 μA/mM cm2 and response time of 3-5s. Also, no responses from the electroactive interferents, such as ascorbic acid (AA), dopamine (DA), DOPA (a DA catabolite) or DOPAC (a DA precursor), over their respective physiological concentration ranges, were detected. Therefore, Pt nanograss will be an excellent substitute for Pt black for Pt surface modification with non-cytotoxic properties, which will be beneficial for in vivo applications.
