The zinc ion (Zn²⁺) is a common ingredient of many over-the-counter multivitamin and mineral dietary supplements. Many enzymes incorporate Zn²⁺ as a co-factor and participate in various physiological activities, such as apoptosis, DNA synthesis, gene expression, immunity, and neurotransmission. The free intracellular Zn²⁺ concentration ([Zn²⁺]_i) is maintained at a sub-nM level by Zn²⁺ transporters at the membranes of organelles and binding proteins. Our previous reports have shown that treating neurons with dopamine or Zn²⁺ elevates [Zn²⁺]_i and causes cell death. Dopamine elevates [Zn²⁺]_i with a change of hundreds of pM and activates the autophagic pathway via the D1 receptor-protein kinase A (PKA)-nitric oxide (NO) cascade in cultured cortical neurons. In addition, blocking this elevation in [Zn²⁺]_i suppresses dopamine-induced autophagosome formation (Hung et al., 2017). These findings reveal that intracellular Zn²⁺ homeostasis is important for the activation of various signaling pathways responsible for neuronal survival.

In synaptic vesicles of glutamatergic neurons, a high concentration of Zn²⁺, 100-300 μM, is coreleased with glutamate upon stimulation, thereby elevating the local extracellular Zn²⁺ concentration ([Zn²⁺]_e). Abundant extracellular Zn²⁺-chelating proteins are suggested to maintain [Zn²⁺]_e at a low sub-nM level. Extracellularly, Zn²⁺ can bind to the N-methyl-D-aspartate (NMDA) receptor and regulate the kinetics of the conjugated ion channels; Zn²⁺ can also facilitate the aggregation of amyloid β (Aβ) and enhance neurodegeneration. Some Zn²⁺-related metal ionophores, such as PBT2, are under clinical trials to verify the importance of Zn²⁺ homeostasis in neurodegenerative diseases. However, most studies adopt a high concentration of Zn²⁺ (> 100 mM), and whether physiological levels of [Zn²⁺]_e are sufficient to activate these physiological responses is not clear, as traditional biological approaches cannot accurately monitor [Zn²⁺]_e in real time.

Over the past several years, with the collaboration of Dr. Yit-Tsong Chen (Department of Chemistry, NTU) and Chii-Dong Chen (Institute of Physics, Academia Sinica), we have focused on exploring the capabilities of silicon nanowire field-effect transistors (SiNW-FETs) in detecting molecules released from neurons (Figure 1). The SiNW-FET-based biosensor is a reliable, sensitive, label-free, and real-time tool that has been widely applied to analyze the presence of biological molecules. Through modification of the surface with appropriate receptors, the device is able to recognize specific target molecules with a sensitivity at the pM level. Compared to the production and manipulation required for other similar techniques, such as Biacore, which requires an expensive optical instrument, the low-cost production and ease of manipulation of this device makes this technique ideal for biological research. Furthermore, the SiNW-FET could be applied to detect cell activities in real time. We have obtained a patent for applying this design to study biomolecular interactions (Reusable Nanowire Field-Effect Transistor System for Detecting Biomolecular Interactions, US Patent number 8420328; 2013/4/16).

To detect various molecules released from cultured cells, we modified the surface of SiNW-FET with oligonucleotide aptamers. Aptamers can form a structure to bind target molecules specifically. We have verified that dopamine and neuropeptide Y are released differentially from pheochromocytoma (PC12) cells stimulated with different concentrations of ATP (Banerjee et al., 2016); in addition, we have shown that the efflux of K⁺ from excited neurons elevates the local extracellular K⁺ concentration (Anand et al., 2017).

In this report, (Anand et al., 2018) (Figure 2), we first chemically modified the Zn²⁺-sensitive fluorophore, FluoZin-3, so it can anchor onto the SiNW-FET (FZ-3/SiNW-FET). This FZ-3/SiNW-FET has good specificity against Zn²⁺ (dissociation constant, K_d, ~12 nM) over other divalent cations. To quantify the Zn²⁺ released from excited neurons in real time, we placed a coverslip seeded with cultured embryonic cortical neurons atop an FZ-3/SiNW-FET, and the [Zn²⁺]_e increased to ~110 nM upon stimulation with α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA). Blockers against AMPA receptors (AMPARs) and exocytosis greatly suppressed the AMPA-induced elevation in [Zn²⁺]_e. In addition, a SiNW-FET modified with Aβ (Aβ/SiNW-FET) bound Zn²⁺ with a K_d of ~633 nM. Placing neurons atop the Aβ/SiNW-FET and stimulation with AMPA induced a significant change in conductance. These results reveal that neurons release Zn²⁺ from synaptic vesicles upon stimulation and that the free [Zn²⁺]_e surrounding the excited neurons is high enough to bind Aβ.

[Zn²⁺]_e homeostasis is important for the health of the brain. Understanding the [Zn²⁺]_e surrounding neurons is critical in evaluating the physiological functions of Zn²⁺. Our findings show that the [Zn²⁺]_e surrounding overexcited neurons is ~115 nM, which is already sufficient to support the formation of the Zn²⁺-Aβ complex. Considering that Aβ fibrillary aggregates are a pathogenic factor for Alzheimer’s disease, the Zn²⁺ coreleased with glutamate at the axonal terminals must be under strict regulation to avoid the formation of Aβ-Zn²⁺ complexes. In addition, this [Zn²⁺]_e level is able to bind to NMDA receptors, which have a high affinity (~ nM level) for Zn²⁺, to regulate neurotransmission. In conclusion, our SiNW-FET system is sufficiently sensitive to characterize physiological [Zn²⁺]_e in real time and examine how Zn²⁺ homeostasis modulates neuronal activities.