(Invited) Microfluidic Dual Sensors for In Vivo Measurement of Superoxide and Glutamate
Authors: Noh Hui-Bog , Moon Jong-Min , Moon Jeon Ok , Shim Yoon-Bo 
Published: February 2020

Miniaturization and stabilization of a sensing device has been essential in the development of implantable and in vivo electrochemical sensors. Smaller electrodes provide improved spatial and temporal resolution as well as reduced tissue damage [1]. To achieve long-time stability and miniaturization, we tried to make a thin film dual sensor combined of two screen printed electrodes to form a microfluidic channel. To examine the performance of the microfluidic duel senor (MDS), we tested it to simultaneously monitor glutamate and superoxide anion (O2
•-) as a model case. Glutamate, which is a major excitatory neurotransmitter in the central nervous system and highly expressed in the mammalian body and brain [2-3].O2
•-is the most intracellular reactive oxygen species which is the primary oxygen free radical generated through the electron transport chain in mitochondria.

In the present study, O2
•- was monitored with a sensor using co-immobilization of a lipid (1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-n-dodecanylamine, DGPD) and cytochrome C-copper nanoflowers (Cyt C-CuNFs) onto a conducting polymer (CP) layer through covalent bonding (SPCE/PtNPs/DGPD/pTTBA/Cyt C-CuNFs/PEI). Otherwise, the glutamate sensor was prepared with a glutamate oxidase attached-CP layer coated on Pt nanoparticles (PtNPs) (SPCE/PtNPs/nanoCP-GlOx), where they were prepared and used as a catalyst to oxidize H2O2
generated by the enzyme reaction. Where, to immobilize the Cyt C-CuNFs and glutamate oxidase, separately, poly-[2,2′:5′,2″-terthiophene-3′-(p-benzoic acid)] (pTTBA) was formed electrochemically on the catalytic electrode surface. Male Sprague-Dawley rats (150.0 - 180.0 g) were obtained from Samtako (Osan, Korea). Animals were provided standard rat chow with free access to tap water and were maintained at a controlled temperature (23 ± 3 oC) and humidity (50 ± 20%) with a 12 h light-dark cycle. With respect to ethical issues and scientific care, the animal protocol used in this study was reviewed and approved by the Pusan National University-Institutional Animal Care and Use Committee (PNU-IACUC; Approval number PNU 2008-0541). The sensor was placed in rat liver for the measurements (inset of Figure 1(A)).

The performance of sensing device was evaluated before and after in vivo experiments, and chronoamperograms were recorded for each of the sensor at different applied potentials, simultaneously. For O2
•- detection, the applied potential was -0.40 V for monitoring the reduction current of H2O2 at the SPCE/PtNPs/DGPD/pTTBA/Cyt C-CuNFs/PEI electrode, whereas, glutamate was detected through the monitoring of oxidation current of H2O2 at a SPCE/ PtNPs/CP-GlOx micro film electrode at the applied potential of +0.45 V. Figure 1(A) shows the typical current-time plots that were obtained for O2
•- in a 0.1 M phosphate buffer solution (PBS, pH 7.0) and calibration plots for (i) before and (ii) after an in vivo experiment. Under the optimized conditions, the calibration plot showed a linear relationship with the O2
•- concentration in the range of 0.2 - 7.0 nM for before and after in vivo experiments. The relative standard deviations of O2
•- concentrations were found to be 2.5 and 4.1%, before and after an in vivo experiment. The detection limit of O2
•- was determined to be 25.0 ± 0.5 pM by SPCE/PtNPs/DGPD/pTTBA/Cyt C-CuNFs/PEI modified electrode. The amperometric response of the SPCE/PtNPs/nanoCP-GlOx sensor was carried out using various concentrations of glutamate. Figure 1(B) shows typical current-time plots for the addition of glutamate in a 0.1 M PBS (i) before and after (ii) in vivo experiments. Under the optimized conditions, the steady state currents showed a linear relationship with the glutamate concentrations in the range of 10.0 nM - 150.0 μM and 150.0 μM - 15.0 mM. The relative standard deviations at 1.0 μM glutamate concentration were found to be 2.2 and 3.6%. The detection limit of glutamate was determined to be 2.5 ± 0.5 nM by SPCE/PtNPs/nanoCP-GlOx sensor based on five-times measurement for the standard deviation of the blank noise (95% confidence level, k = 3, n = 5).

[1] Noh, H.-B.; Shim, Y.-B.; et al., Chem. Comm
., 2015, 51, 6659-6662.

[2] Rahman, Md.A.; Shim, Y.-B.; et al., Anal. Chem
., 2005, 77, 4854-4860.

[3] Rahman, Md.A.; Shim, Y.-B.; et al., Anal. Chem
., 2012, 84, 6654-6660.

Figure 1