Stochastic Electrochemical Cytometry of Human Platelets via a Particle Collision Approach

The quantitative analysis of human platelets is important for the diagnosis of various hematologic and cardiovascular diseases. In this article, we present a stochastic particle impact electrochemical (SPIE) approach for human platelets with fixation (F-HPs). Carboxylate-functionalized polystyrene particles (PSPs) are studied as well as a standard platform of SPIE-F-HPs. For SPIE-PSPs (or F-HPs), [Fe(CN)₆]^4- was used as the redox mediator, and electro-oxidation of [Fe(CN)₆]^4- to [Fe(CN)₆]^3- was conducted on a Pt ultramicroelectrode (UME) by applying a constant potential, where the corresponding oxidation current is mass-transfer-controlled. When PSPs (or F-HPs) are introduced into aqueous solution with [Fe(CN)₆]^4-, sudden current drops (SCDs) were observed, which resulted from the partial blockage of a Pt UME by collision of an individual PSP (or F-HP). For SPIE-PSPs (or F-HPs), we found that it is essential to enhance the migration of PSPs (F-HPs) toward a Pt UME by maximizing the steady state current associated with electro-oxidation of [Fe(CN)₆]^4-. This was accomplished by increasing its concentration to the solubility limit. We successfully measured the concentration of F-HPs dispersed in aqueous solution containing [Fe(CN)₆]^4- with a minimum detectable concentration of 0.1 fM, and the size distribution of F-HPs was also estimated from the obtained i_drop distribution based on the SPIE analysis, where i_drop stands for the magnitude of the current drop of each SCD. Lastly, we revealed that HPs without the fixation process (WF-HPs) are difficult to quantitatively analyze by SPIE because of their transient activation process, which results in changes from their spherical shape. The observed difficulty was also confirmed by finite element analysis, which shows that i_drop can be significantly increased, as an elongated WF-HP is adsorbed on the edge of an UME.