Label-free fluorescent enzymatic assay of citrate synthase by CoA–Au(I) co-ordination polymer and its application in a multi-enzyme logic gate cascade

Highlights

• Generating RNA-mimicking and SYBR GREEN II stainable coordination polymer of CoA-Au(I) CP in situ.

• Basing on CoA-Au(I) CP, a novel label-free fluorescent-on assay of CS and its inhibitor was developed.

• This CoA-Au(I) CP based method is homogeneous, sensitive, facile, cost-effective, and quick.

• A biomimetic AND-AND-AND-cascaded logic gate was further established in one-pot to process chemical information.

Abstract

Citrate synthase (CS) is one of the key metabolic enzymes in the Krebs tricarboxylic acid (TCA) cycle. It regulates energy generation in mitochondrial respiration by catalysing the reaction between oxaloacetic acid (OAA) and acetyl coenzyme A (Ac-CoA) to generate citrate and coenzyme A (CoA). CS has been shown to be a biomarker of neurological diseases and various kinds of cancers. Here, a label-free fluorescent assay has been developed for homogeneously detecting CS and its inhibitor based on the in situ generation of CoA–Au(I) co-ordination polymer (CP) and the fluorescence signal-on by SYBR Green II-stained CoA–Au(I) CP. Because of the unique property of the CoA–Au(I) CP, this CS activity assay method could achieve excellent selectivity and sensitivity, with a linear range from 0.0033 U/μL to 0.264 U/μL and a limit of detection to be 0.00165 U/μL. Meanwhile, this assay method has advantages of being facile and cost effective with quick detection. Moreover, based on this method, a biomimetic logic system was established by rationally exploiting the cascade enzymatic interactions in TCA cycle for chemical information processing. In the TCA cycle-derived logic system, an AND–AND–AND-cascaded gate was rigorously operated step by step in one pot, and is outputted by a label-free fluorescent signal with visualized readout.

Introduction

The regulation of cellular metabolism is the fundamental basis of life activities, thus the key enzymes or regulatory factors of metabolic pathways have important potential implications in biochemical pathological and biomedical research (He et al., 2000; Bodner et al., 1986; Garber et al., 2004;). The Krebs tricarboxylic acid (TCA) cycle is the most crucial component of cellular respiration. It takes place in mitochondria and is associated with oxidative phosphorylation to generate the majority of adenosine triphosphate (>90%) (Russell et al., 1997; Remington et al., 1992). The key regulatory enzyme of the TCA cycle is citrate synthase (CS), which stands as an important rate-limiting enzyme in the first step of the TCA cycle. It has been demonstrated that TCA is perturbed in many human disease states and the aberrant CS activity is closely related to various kinds of cancers (Schlichtholz et al., 2005; Bayley et al., 2010). Hence, developing a biosensor to probe CS activity is necessary for CS-targeted biochemical research, disease diagnosis, and drug discovery.

In general, CS catalyzes the condensation reaction of the two-carbon acetate ion residue from acetyl coenzyme A (Ac-CoA) and a molecule of four-carbon oxaloacetate ion (OAA) to form the six-carbon citrate ion and product of coenzyme A (CoA) (Mukherjee et al., 1976). Traditional methods for assaying CS activity are based on mass spectrometry (MS) (Chen et al., 2011), carbon-13 NMR spectroscopy (Jones et al., 1993), antibody-based (Marrache et al., 2014) and product-indicated assay kit (Morgunov et al., 1998) for indirectly detecting the co-products, citric acid or CoA. Although achieving nice performance, these methods are discontinuous, heterogeneous, complicated, or time-consuming, which suffers from multi-step separation or surface immobilization processes, hazardous reagents, tedious operation, and expensive labelling. Thus, a label-free and homogeneous method for facile, cost-effective, and quickly readable assay of CS activity is highly desirable. Notably, conventional CoA-indicated bioassays only focus on the detection of the thiol group of CoA, which is less specific. Hence, precise measurement of CS activity in real biological samples requires biosensing strategies with high selectivity to CoA.

Biomolecular computing aims at executing the Boolean logic functions using biochemical instead of electronic methods (Shao et al., 2002, Saghatelian et al., 2003; Ashkenasy et al., 2004; De Silva and Uchiyama, 2007; Elbaz et al., 2010.), which shows great significance in logic sensing (Zhou et al., 2012), logic bio-regulation (Miyamoto et al., 2012.), and intelligent diagnostics (You et al., 2014). The key point of biomolecular computing is to find suitable biomolecular toolkits processing the input information for decision-making, and executing the readable outputs. The catalytic functions of enzymes are promising toolkits for constructing bio-logic systems (Miyamoto et al., 2012.), and the abundant kinds of enzymes provide adequate computational complexity for logic circuits. Especially, metabolic enzymes are always cascaded to construct logic gate circuits mimicking metabolism pathway. Katz's pioneering works presented several typical representatives of metabolic enzyme-based logic systems relying on the logic control of cascade reactions among glucose oxidase, horseradish peroxidase, dehydrogenase and so on (Strack et al., 2008a, Strack et al., 2008b, Tokarev et al., 2009, Zhou et al., 2009, Privman et al., 2009; Pita et al., 2008; Niazov et al., 2006). Currently, the types of enzymes involved in biological systems are still restricted because the cross-reactivity among the enzyme-catalyzed reactions always limits the integratability and density of computations (Strack et al., 2009). The Krebs TCA cycle is a natural metabolism pathway with cascade reactions of CS and seven other enzymes, which is a promising starting point for designing bio-logic circuits with various inputted-biomolecules and less cross-reactivity in the chemical reactions. However, attempts to build a bio-mimetic logic system derived from TCA and CS are scarce.

Herein, we report a novel label-free fluorescent bioassay for probing CS activity based on in situ synthesis of nucleic acid-mimicking CoA–gold ion co-ordination polymers (CPs). CS catalyses the formation of citrate ion and CoA, and CoA is further exploited to generate CoA–Au(I) CPs. The CoA–Au(I) CP is a RNA-like nanostructure with –Au(I)–thiol– repeated segments as the backbone and multiple adenine bases as the side groups. This structure is analogous to single-stranded poly A nucleic acid and can also be stained by RNA-specific fluorogenic dye, causing the emission of fluorescence. Dependent on this CoA–Au(I) CP-caused fluorescent switch, this method enables the detection of CS activity assay with high selectivity and sensitivity. In addition, due to its in situ generation, this method is a facile, rapid, and continuous assay method for homogeneous detection of CS activity and its inhibitor. Unlike some conventional CS assays, our assay is label-free, antibody-free, and cost efficient, avoiding the sophisticated signal–probe preparation and modification. Moreover, because of the key role of the CS-catalysing reaction in the TCA cycle, this CoA–Au(I) CP-based assay could be further exploited to build a TCA cycle-derived bio-logic system. This biomimetic logic system was performed in one pot with operations carried out step by step, which clearly demonstrated the concatenation of enzymatic reactions. It presents a new type of enzymatic logic system which holds great potential in developing multi-target intelligent biosensors and high-density biocomputing circuits.