# Protoporphyrin IX Based All-Solid-State Ion-Selective Electrodes for Choline Determination In Vitro
## 1 Introduction  
The neurotransmitter choline (Ch) plays a crucial role in brain and body functions. As the crucial precursor of acetylcholine, it is directly involved in the synthesis of acetylcholine in the brain, with the function of modulating attentional capacities, memory, learning and normal cognition. The cholinergic system dysfunction increases disease risk and age-related cognitive degradation such as Alzheimer's disease [1; 2; 3; 4]. The brain Ch concentration is also a quantitative marker of ischemia and infarct expansion, which can be applied to predict tissue prognosis better [5]. In addition, the Ch in plasma is associated with organ protection, as well as cardiovascular and cerebrovascular conditions. The growing concentration of Ch in plasma may lead to a detrimental cardiometabolic risk factor, while Ch deficiency increases the risk of several cancers [6]. In addition, Ch is essential for fetal development as a critical source of nutrition. Under a higher maternal status of Ch and other nutrition, immunity to the effects of maternal immune activation is strengthened, which is increasingly recognized as a latent risk factor for various neurological disorders [7; 8]. However, the physiological concentrations of Ch are close to micromode, for example, 0.5-4.0 \(\upmu\)M in human cerebrospinal fluid. When stimulated, the Ch concentration could increase to more than 30 \(\upmu\)M [9; 10]. Therefore, a rapid and sensitive method for Ch determination in biological fluids is vital and necessary to promote fundamental neuroscience research and monitor early-stage diagnoses. Recently, various Ch measurement strategies have been reported, including fluorescence and colorimetric methods [11; 12], chemiluminescence [13; 14], liquid chromatography [15], and capillary electrophoresis [16]. Although these methods have significantsensitivity and selectivity, the requirement of complicated procedures and devices, professional operations, and pre-treatments of samples restrict their application in real-time detection [17]. In comparison with those above techniques, electrochemical analysis has been rapidly developed in recent years with the advantages of fast response time, less sample consumption, portability, convenient operation, and good sensitivity [18]. As one of the most popular methods in electrochemical analysis, the amperometric sensors provide quality characteristics such as relatively wide linear range, low detection limit, high sensitivity and selectivity. However, they suffer from the significant interference of redox products due to the requirement of redox reactions to generate current characteristics [19; 20]. Moreover, the lack of an electroactive group in Ch causes insensitivity to electric potential. Therefore, enzymes for catalyzation in amperometric detection are necessary [21]. As a result, the immobilization of enzymes on the substrate and maintaining enzyme activities are also non-negligible barriers for solving the problems of storage and long-term stability of the sensors [22]. With their advantages including easy storage, low power consumption, and no need for enzymes and redox potential, several potentiometric ion-selective electrodes (ISEs) for analyses in actual samples have been developed in recent years [23; 24]. However, the amount of Ch ISEs is still inadequate. An ion-selective Ch electrode based on a membrane containing a valinomycin derivative for the determination of a cholinergic agent showed excellent sensitivity from \(2\times 10^{-8}\)-\(10^{-4}\) M [25], and a novel Ch ISE for concentration analyses in milk powder and infant formulas exhibited a low detection limit of 0.061 \(\upmu\)M [26]. Nevertheless, the excellent properties relied on the internal filling solution containing a high concentration of Ch bromide or hydrophilic (water-soluble) p-sulfonated calixarene, increasing the difficulty to miniaturize and store.