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Cross-linkage urease nanoparticles: a high-efficiency signal-generation tag for portable pH meter-based electrochemical immunoassay of lipocalin-2 protein diagnostics Ai-Li Sun 1 & Qing-An Qi 2 & Li-Juan Zhi 3 Received: 28 April 2020 / Accepted: 25 July 2020 # Springer-Verlag GmbH Austria, part of Springer Nature 2020

Abstract An innovative signal-transduction tag based on cross-linked urease nanoparticles (CLENP) was designed for the development of a pH meter-based immunoassay of lipocalin-2 (LCN2). The CLENP was synthesized with a typical desolvation method using ethanol as desolvation agent, followed by functionalization with polyaspartic acid. The carboxylated CLENP were used as the signal-generation tags for the labelling of secondary antibodies via the carbodiimide coupling. Upon target LCN2 introduction, a sandwich-type immune reaction was performed between capture antibody-coated plate and the labeled secondary antibody on the CLENP. The conjugated CLENP in the microplate hydrolyzed urea into ammonia (NH4+) and carbonate (CO32−), resulting in the pH change of solution, which was determined with a handheld pH meter. The pH variation was proportional to target concentration in the sample. By monitoring the pH variation of the urea solution, the level of LCN2 at a concentration as low as 5.2 pg mL−1 was evaluated. The pH meter-based electrochemical immunoassay can be utilized for mass production of miniaturized lab-on-a-chip devices with handheld pH meter, thereby opening new opportunities for protein diagnostics and biosecurity. Keywords Electrochemical immunoassay . pH measurement . Cross-linkage urease nanoparticles . Signal-transduction tags . Enzymatic hydrolysis

Introduction A pH meter, an instrument used to measure hydrogen-ion activity (acidity or alkalinity) in solution on a scale of 0 to 14, is currently one of the most widely electric devices because of to its portable size, low cost, easy operation, and reliable quantitative results [1]. Typically, the quantitative information provided through the pH value expresses the degree of the activity of an acid or base in terms of hydrogen

* Qing-An Qi [email protected] 1

Key Laboratory of Nano-carbon Modified Film Technology Engineering (Henan Province), Department of Pharmacy, Xinxiang University, Xinxiang 453000, China

2

Department of General Surgery, The Hospital of Eighty-Third Army, Xinxiang 453000, Henan, China

3

Department of Chemistry and Chemical Engineering, Xinxiang University, Xinxiang 453000, Henan, China

ion activity [2]. The pH value of substance directly depends on the ratio of hydrogen ion (H+) and hydroxyl ion (OH−) concentration, and the relationship between two ions in a given solution is constant for a given set of conditions, either one can be determined by knowing the other [3]. One major advantage of using pH meters is that no external excitements such as current and voltage are utilized during the assay in comparison with other electrochemical detection strategies (i.e., voltammetric or impedimetric) [