The speciation of essential metal ions in biological fluids, such as blood plasma and serum, is of fundamental importance to understand the homeostasis of these elements. The activity of metal ions such as Zn²⁺ in extracellular media is thought to affect their interaction with membrane-bound transporters, and thus is critical for their cellular uptake. Previous approaches to determine “free” Zn²⁺ (i.e. the hexa-aquo ion) are based on separation by either chromatography or ultrafiltration, or on metallochromic dyes. However, both types of approach are prone to affect the relevant equilibria. These drawbacks can be circumvented with the electroanalytical technique AGNES (Absence of Gradients and Nernstian Equilibrium Stripping), since it can measure free zinc concentration without perturbing the sample speciation. Here, a Bovine Serum Albumin (BSA) + Zn synthetic mixture and Fetal Bovine Serum (FBS) are analyzed as proof of concept. Adsorption of BSA on the surface of the Hanging Mercury Drop Electrode (HMDE), despite the advantage of its renewal, is so intense that it blocks appropriate attainment of the required equilibrium, and only estimations of [Zn²⁺] can be derived. In contrast, a rotating disc electrode with a thin mercury film deposited on it (TMF-RDE) is advantageous because of its small volume and enhanced mass transfer. Protein adsorption can be prevented by covering the TMF-RDE with Nafion. A free Zn concentration [Zn²⁺] = 2.7 nmol L⁻¹ was found at pH 7.0, total Zn 20 μμmol L⁻¹ and BSA 600 μμmol L⁻¹. A sample of FBS with fixed pH 7.2 (MOPS 0.08 mol L⁻¹) yielded [Zn²⁺] = 0.25 nmol L⁻¹. This methodology opens the way to free metal concentration determinations in biological fluids.

生物体液（如血浆和血清）中基本金属离子的形态对于了解这些元素的稳态至关重要。金属离子如 Zn ²⁺在细胞外介质中的活性被认为会影响它们与膜结合转运蛋白的相互作用，因此对于它们的细胞摄取至关重要。以前测定“游离”Zn ^2+的方法（即六水合离子）基于色谱法或超滤法或金属变色染料的分离。然而，这两种方法都容易影响相关的平衡。这些缺点可以通过电分析技术 AGNES（不存在梯度和能斯特平衡剥离）来规避，因为它可以在不干扰样品形态的情况下测量游离锌浓度。在这里，分析了牛血清白蛋白 (BSA) + Zn 合成混合物和胎牛血清 (FBS) 作为概念证明。BSA 在悬挂汞滴电极 (HMDE) 表面的吸附，尽管有更新的优势，但它是如此强烈，以至于它阻碍了所需平衡的适当实现，并且仅估计 [Zn ²⁺] 可以推导出来。相比之下，具有沉积在其上的薄汞膜 (TMF-RDE) 的旋转圆盘电极是有利的，因为它体积小且质量传递增强。用 Nafion 覆盖 TMF-RDE 可以防止蛋白质吸附。在 pH 7.0、总 Zn 20 μμmol L ^-1和 BSA 600 μμmol L ^-1时，发现游离 Zn 浓度 [Zn ²⁺ ] = 2.7 nmol L ^-1。具有固定 pH 7.2 (MOPS 0.08 mol L ^-1 ) 的 FBS 样品产生 [Zn ²⁺ ] = 0.25 nmol L ^-1。该方法为生物体液中游离金属浓度的测定开辟了道路。