Robust oxidase mimicking activity of protamine-stabilized platinum nanoparticles units and applied for colorimetric sensor of trypsin and inhibitor

Abstract

In this study, we developed a simple, sensitive and label-free colorimetric sensor for trypsin and its inhibitor using oxidase mimicking activity of protamine-stabilized platinum nanoparticles units (Pro-PtNPs units). Protamine is designed to stabilize PtNPs and served as the substrate of trypsin hydrolysis. The results show that protamine combines several PtNPs together to form active units, which have stronger oxidase mimicking activity than other PtNPs. In addition, the Michaelis constant of the Pro-PtNPs units is smaller than negatively charged citrate capped PtNPs, suggesting that the positively charged Pro-PtNPs units have better affinity with 3,3′,5,5′-tetramethylbenzidine (TMB), which is inconsistent with previous reports. The protamine stabilizer could be hydrolyzed in the presence of trypsin, and the Pro-PtNPs units would be decomposed and aggregated, resulting in a decrease of catalytic activity of Pro-PtNPs units. The color of the solution would be reduced according to the decrease of catalytic activity, so the catalytic activity is linearly related to UV absorbance. The linear range of trypsin detected by this new method is 0.06 μg/mL˜0.6 μg/mL, and the detection limit down to 0.03 μg/mL. Pro-PtNPs units have been successfully applied to the determination of serum trypsin and screening of inhibitors.

Introduction

Proteases plays a key role in some important physiological and pathological processes, so it is related to the occurrence of many diseases. They can hydrolyze the peptide bonds and dissolve some proteins involved in life activities [1,2]. As such a protease, trypsin, which belongs to serine protease, is produced by the pancreas. It specifically hydrolyzes peptide bonds formed by the lysine or arginine at the C-terminal side [3]. It has been found that the content of trypsin in serum or urine is related to many important diseases, such as pancreatitis, cancer and other pathological changes [4]. Trypsin inhibitor is a serine protease inhibitor, which can prevent the premature activation of trypsinogen and protect the pancreatic tissue from trypsin damage. Moreover, the protease inhibitors screening is important for the development of potential drugs [5]. Therefore, it is of great significance to develop a simpler, more convenient and safer method for the trypsin determination in serum or urine and inhibitors screening.

Various techniques for the detection and screening of trypsin inhibitors have been reported, including enzyme-linked immunosorbent assay (ELISA) [6], colorimetry [7], fluorescence [8], chemiluminescence [9], and electrochemical methods [10,11]. Among these methods, colorimetry can be directly observed with the naked eye or accurately quantified by ultraviolet spectrophotometer. It is the simplest, cheapest and widely used method. In the traditional ELISA, natural horseradish peroxidase (HRP) is used as a marker for colorimetry [12]. However, natural enzymes are proteins that not only require very strict reaction conditions, such as pH and temperature, but are prone to denaturation and deactivation, resulting in high cost of use. Therefore, many studies have devoted to developing a stable, cheap and efficient natural enzyme substitute. Tremendous efforts have been made to develop enzyme mimetics, which can not only replace natural enzymes, but also more stable than natural enzymes, greatly reducing costs. As a new type of artificial enzymes, nanomaterial-based enzyme mimics have been widely studied owing to their superior properties such as easy preparation, low cost, high operational stability, and facile modification [13]. Different nanomaterials have been synthesized in previous studies, including carbon-based nanomaterials [14], metal-based nanomaterials [[15], [16], [17]], metal alloy-based nanomaterials [18], metal origanic frame based nanomaterials [9] and others. Nanomaterials with enzymatic activity are called nanozymes. Nanozymes play an important role in colorimetric analysis by catalyzing the oxidation of some chromogenic substrates such as TMB to form colored substances. There have been many reports on mimic peroxidase. For example, graphene oxide [19], Carboxyl-modified gold NPs [20], CuO NPs [21] and other materials were reported to have peroxidase-like activity. Hydrogen peroxide (H₂O₂) is usually introduced as an electron acceptor in their oxidation process. However, H₂O₂ is very easy to decompose and has strict environmental requirements. Moreover, it is well known that H₂O₂ is a strong oxidant, which might damage some analytes and is also detrimental to its practical application [22]. The development of nanozymes with higher catalytic activity, safer, lower cost and lower environmental requirements is the focus of current research. Platinum nanomaterials (PtNPs) have also been extensively studied as nanozymes. Almost all studies on analytical applications of PtNPs-related nanozymes have focused on their peroxidase-mimic activity [23]. While colorimetric methods based on the oxidase mimics of PtNPs have rarely been reported. Unlike peroxidase mimics, oxidase mimics can catalyze the oxidation of some organic substrates without H₂O₂. This catalytic process avoids the involvement of destructive H₂O₂. The distinct advantage makes oxidase mimics an attractive alternative catalytic probes for designing colorimetric sensor with operational simplicity, good biocompatibility, and high reliability.

Herein, we report a novel label-free colorimetric sensor for trypsin and screening of inhibitor based on the oxidase mimetics of the protamine-stabilized PtNPs. Protamine, rich in arginine, has been designed as both the stabilizer of PtNPs and the substrate of trypsin. Protamine is easily hydrolyzed by trypsin [24]. The design principle is illustrated in Scheme 1. Pro-PtNPs units were synthesized by using protamine and sodium borohydride as stabilizer and reducing agent, respectively. The protamine as stabilizer lost its function because it was hydrolyzed by trypsin, and the Pro-PtNPs units would be decomposed and aggregated, which results in an obvious decrease in catalytic activity. In this study, Pro-PtNPs units with robust oxidase-mimicking activity are used to detect trypsin and inhibitor, not only to overcome the shortcomings of unstable natural enzyme, but also to overcome the need of introducing H₂O₂ to mimic peroxidase.