An in situ measurement of extracellular cysteamine, homocysteine, and cysteine concentrations in organotypic hippocampal slice cultures by integration of electroosmotic sampling and microfluidic analysis

Abstract

We demonstrate an all-electric sampling/derivatization/separation/detection system for the quantitation of thiols in tissue cultures. Extracellular fluid collected from rat organotypic hippocampal slice cultures (OHSCs) by electroosmotic flow through an 11 cm (length) × 50 μm (i.d.) sampling capillary is introduced to a simple microfluidic chip for derivatization, continuous flow-gated injection, separation, and detection. With the help of a fluorogenic, thiol-specific reagent, ThioGlo-1, we have successfully separated and detected the extracellular levels of free reduced cysteamine, homocysteine, and cysteine from OHSCs within 25 s in a 23 mm separation channel with a confocal laser-induced fluorescence (LIF) detector. Attention to the conductivities of the fluids being transported is required for successful flow-gated injections. When the sample conductivity is much higher than the run buffer conductivities, the electroosmotic velocities are such that there is less fluid coming by electroosmosis into the cross from the sample/reagent channel than is leaving by electroosmosis into the separation and waste channels. The resulting decrease in the internal fluid pressure in the injection cross pulls flow from the gated channel. This process may completely shut down the gated injection. Using a glycylglycine buffer with physiological osmolarity but only 62% of physiological conductivity and augmenting the conductivity of the run buffers solved this problem. Quantitation is by standard additions. Concentrations of cysteamine, homocysteine, and cysteine in the extracellular space of OHSCs are 10.6 ± 1.0 nM (n = 70), 0.18 ± 0.01 μM (n = 53), and 11.1 ± 1.2 μM (n = 70), respectively. This is the first in situ quantitative estimation of endogenous cysteamine in brain tissue. Extracellular levels of homocysteine and cysteine are comparable with other reported values.

Figure 1

Layout of the microfluidic chip for online EO sampling and microfluidic analysis.

Figure 2

Viability assessmentfor the OHSCs. A1 (fluorescence) and A2 (bright field) are for the 100% death control. B1 and B2 are for a live control, which was handled in the same way as the experimental OHSCs, except that it was neither sampled nor exposed to ggACSF. Image C1 and C2 are foran OHSCplaced on the same culture insert as the sampled OHSC (D1/D2) in a Petri dishcontaining the ggACSF. The electroosmotic sampling was carried out by applying +3000 V at the Petri dish and GND at R4 for 10 min. The arrows indicate the location where the sampling capillary tip was positioned.

Figure 3

Effect of buffer conductivity on the flow-gatedinjection. 200 V, 200 V and −4500 V were applied to R1, R5 (no capillary was connected) and R3, respectively. R2 was switched between “GND” (0.5 s) and “floating” (14.5 s) continuously, while R4 was disabled. R1 was filled with 3.4 μM ThioGlo-1 in 20 mM Tris-HCl (pH=7.50). (A) R2, R3, and R5 have 20 mM Tris-HCl (pH=7.50). (B) R2 and R3 as in (A), R5 has ACSF (pH=7.40). (C) R2/R3 has 20 mM Tris-HCl containing 60 mM NaCl (pH=7.50),R5 same as (B).

Figure 4

Concentrations, conductivities, and velocities during injection.Snapshots of the ThioGlo-1 concentration (A1-4), the buffer conductivity (B1-4) and the velocity of the fluid (C1-4)after 0.05 s of sample injection are for different R5 conductivities.The concentration profiles of the ThioGlo-1 at 0.025 s in the separationstep are plotted against the length of the separation channel (D1). The peak concentration of ThioGlo-1 is plotted against the R5 conductivity (D2).

Figure 5

Dependence of relative Thioglo-1 concentrations at the end of the reaction channel on the presence (red/circles) or absence(black/squares) the auxiliary channel. Voltages are at the Petri dish (sample).

Figure 6

Electropherogram of analytes from EO sampling of OHSCs. EO sampling from an OHSC with only ggACSF in the Petri dish (a), or with 99.22 nM of cysteamine (b), 65.81 μM of Cysteine (c), 574.8 nM of homocysteine (d) added in ggACSF. Cysteamine (CSH), homocysteine (Hcy), cysteine (Cys) and glycylglycine (Gly-Gly) peaks marked on the plot are all ThioGlo-1 derivatized peaks. Separation conditions are described in experimental section.