Hybrid Capillary-Microfluidic Device for the Separation, Lysis, and Electrochemical Detection of Vesicles

Abstract

The primary method for neuronal communication involves the extracellular release of small molecules that are packaged in secretory vesicles. We have developed a platform to separate, lyse, and electrochemically measure the contents of single vesicles using a hybrid capillary-microfluidic device. This device incorporates a sheath-flow design at the outlet of the capillary for chemical lysis of vesicles and subsequent electrochemical detection. The effect of sheath-flow on analyte dispersion was characterized using confocal fluorescence microscopy and electrochemical detection. At increased flow rates, dispersion was minimized, leading to higher separation efficiencies, but lower detected amounts. Large unilamellar vesicles (diameter ∼ 200 nm), a model for secretory vesicles, were prepared by extrusion and loaded with an electroactive molecule. They were then separated and detected using the hybrid capillary-microfluidic device. Determination of size from internalized analyte concentration provides a method to characterize the liposomal suspension. These results were compared to an orthogonal size measurement using dynamic light scattering to validate the detection platform.

Prediction of Local pH Variations during Amperometric Monitoring of Vesicular Exocytotic Events at Chromaffin Cells

“…Accordingly, when the pH av value reaches its constant plateau (Figure 4 C), these equations may be rewritten as follows [Eqs. (15) and (16)] [note that we use here macroscopic fluxes, in mol s À1 , rather than microscopic ones as in Equations (3) and (4)]: In Equation (15), the last term, W(h,d,R), is a factor with dimension of a volume which needs not to be further explained here since this is a constant for a given experimental setup. The term F(n,1) is a numerical factor, depending on the frequency and on 1, which is introduced now to account for the intensity and duration of the pH spikes created by each successive event (compare Figures 4 A, C).…”

“…Even if the measured charge may not be always directly related to the whole content of mediators in the vesicle before its fusion this provides extremely important knowledge about this content. [15] Each method thus addresses an important series of elements that are generally transparent to the other two. Their ensemble should then provide a precise and completive view of the whole phenomenon, from the docking stage to the completion of release (Figure 1), whenever their information could be crossed.…”

Reconstruction of Aperture Functions during Full Fusion in Vesicular Exocytosis of Neurotransmitters

“…Indeed, several works have pointed out and convincingly established that depending on the exact conditions which prevail during vesicular exocytotic events a fraction of the total chemical messenger may remain poised inside the vesicle at the end of the process, that is, when the current value drops below the noise level. [14] Anyway, even if a fraction of species remains ultimately sequestrated inside the vesicle this does not alter the present analysis owing to the additive properties of diffusion. So, for our purpose here, it is sufficient to consider that c 0 is such as [Eq.…”

Small-Volume Analysis of Cell–Cell Signaling Molecules in the Brain

“…Another benefit of CE, especially for neurochemical applications, is that separation is based on the differential migration of charged molecules; many of the neurotransmitters and neuromodulators in the nervous system are charged over a range of pH values, and thus can be separated from the cellular milieu under typical CE conditions. CE has been successfully used for discovering new serotonin metabolites (Squires et al, 2006), and characterizing the content of single cells (Nemes et al, 2012) and single vesicles (Omiatek et al, 2009). Chiral analysis of excitatory acids from the brain by CE has been reported (Wang et al, 2011;Wagner et al, 2012).…”