**1. Introduction**

Foodborne pathogens have become a major public health issue, increasingly attracting concern around the world. The WHO reported that almost 10% of the world population falls ill annually due to the consumption of foods and water contaminated by pathogenic microorganisms [1]. *Salmonella* is one of the main foodborne pathogens, which passes through the entire food supply chains from livestock farming to food processing, then to food logistics, and finally to retail. The clinical manifestations of *Salmonella* poisoning mainly include acute onset of fever, abdominal pain, diarrhea, nausea, and sometimes vomiting. Therefore, early screening of *Salmonella* and other common foodborne pathogens is vital to ensure food safety in food supply chains.

Ion selective electrode (ISE) is a type of electrochemical sensor that converts the activity of specific ions in a solution into the potential to determine the ion concentration [2,3]. Various ISEs have been developed and widely used in environmental monitoring [4–6], biochemical analysis [7–10], food

safety [11,12], and other applications [13,14] due to their unique advantages, such as low cost, simple operation, small size, rapid response, and good selectivity. In addition, ISEs do not require external electrical excitement during electrochemical measurements; thus they are the most straightforward tool compared to other electrochemical methods [15,16]. ISEs are generally used to measure the specific ions in a static solution, but seldom under continuous-flow conditions. Recently, dynamic potentiometry, which was first introduced by Calvo et al. [17], has gained popularity. Some studies on the dynamic behaviors of ISEs were reported in developing new kinetic potentiometric methods [18]. Cuartero et al. reported a dynamical potentiometric ISE using 18-crown-6 as ionophore for continuous-flow detection of multiple ions in binary mixtures [19]. Thus, combining ISE-based potentiometric methods with the immune methods is showing promise for the development of novel assays using some specific ions as a signal.

In recent years, fast development of various nanomaterials has opened up new possibilities for biosensing signal amplification. Among them, protein-inorganic hybrid nanoflowers have been demonstrated to be able to maintain or even increase the activity of the proteins and effectively amplify the detection signals [20–22]. Ge et al. first proposed the coprecipitation method to synthesize hybrid organic-inorganic nanoflowers using copper ions as an inorganic component and proteins as an organic component. They successfully used laccase as the model protein to create the nanoflowers for detection of epinephrine [23]. Ye et al. developed Con A-invertase-CaHPO4 hybrid nanoflowers to enhance the activity of the immobilized invertase and used them with a personal glucose meter for sensitive detection of *Escherichia coli* O157:H7 [24]. Ismail et al. reported the synthesis of hybrid nanoflowers using iron ions (Fe2+) and horseradish peroxidase (HRP) and demonstrated that they increased the catalytic activity of HRP more than five-fold [25]. Therefore, the introduction of nanoflowers might further improve the sensitivity of the potentiometric assays.

In this study, we explore a new enzyme-free assay to detect *S.* Typhimurium using protein-inorganic hybrid nanoflowers for effective amplification of biological signals, magnetic nanobeads for immunomagnetic separation of target bacteria, and fluidic chip with the calcium ion selective electrode for continuous-flow detection of calcium ions. The objectives of this study were to: (1) develop a new enzyme-free, potentiometric assay for rapid and sensitive detection of *S.* Typhimurium, and (2) experimentally evaluate the sensitivity and applicability of the assay. As shown in Scheme 1, prior to testing, the streptavidin-modified magnetic nanobeads were conjugated with the biotinylated monoclonal antibodies against *Salmonella* to form the immune magnetic nanobeads through streptavidin-biotin binding, and calcium hydrophosphate crystals were conjugated with the polyclonal antibodies against *Salmonella* to synthesize the immune calcium nanoflowers through coprecipitation method. First, the immune magnetic nanobeads were used to separate the target bacteria from the sample to form the magnetic nanobeads-bacteria complexes (magnetic bacteria) that were concentrated in a smaller volume of phosphate buffered saline (PBS). Then, the immune calcium nanoflowers were conjugated with the magnetic bacteria to form the magnetic nanobeads-bacteria-nanoflower complexes (nanoflower conjugated bacteria). Successively, hydrogen chloride was used to release calcium ions from nanoflower conjugated bacteria. After magnetic separation, the supernatant was finally injected to the fluidic chip for continuous-flow detection of calcium ions to determine the concentration of the target bacteria.

**Scheme 1.** Schematic of the proposed assay for sensitive detection of *Salmonella* Typhimurium.

#### **2. Materials and Methods**
