Colorimetric screening of bacterial enzyme activity and inhibition based on the aggregation of gold nanoparticlesw

Since the antibiotic properties of penicillin were first discovered in the beginning of the last century, b-lactam antibiotics have been well developed as miracle drugs in the treatment of bacterial infections in clinics. However, increased bacterial resistance to b-lactam antibiotics has been extensively documented and has now become a serious public health threat. The most common mechanism for b-lactam antibiotic resistance is the production of a family of bacterial enzyme: b-lactamases (Blas), a type of enzymes secreted both by gram-positive and gram-negative bacteria. Among the different members of the Bla family, class A Bla is one of the most prevalent plasmid-encoded or chromosomally encoded bacterial enzymes for mediating b-lactam resistance through the efficient hydrolysis of the b-lactam ring in penicillins and cephalosporins. One practical method utilized in clinics to improve the antibiotic efficacy and combat bacterial resistance caused by these enzymes is to combine the b-lactam antibiotics with Bla inhibitors. As such, development of molecules that inhibit the Bla, and similarly, the specific methods for the highthroughput screening of efficient Bla inhibitors have become extremely crucial in clinical settings. Although some commonly used chromogenic (e.g., nitrocefin) or fluorescent (e.g. protein fusion and genotyping based on polymerase chain reaction (PCR)) assays are able to successfully perform such tasks. Other methods based on hydrogels, phage display, and dynamic light scattering have also been developed to identify the Bla inhibitors in vitro. A simple and novel method for the efficient screening of Bla inhibitors is still necessary since most of the current methods have high reliance on specific instruments, require tedious sample pre-treatment, or do not provide efficient measurements in real-time. Recently, metal nanoparticles (NPs) such as silver or gold have received much attention in bio-nanotechnology because of their unique optical and physical properties. Considerable studies have shown that silver or gold NPs can be extensively exploited to monitor specific biomolecular recognition, such as in DNA hybridization or aptamer identification, sugar or metal ions tests, and toxin, amino acids or enzyme activities detection. Herein, we present a simple and specific gold NPsbased colorimetric method, which enables for simultaneous determination of the bacterial enzyme activity in the absence and presence of enzyme inhibitors in vitro and in bacterial strains. This new method could serve as an alternative platform for efficient screening of the bacterial enzyme inhibitors with the naked eyes or a simpler 96-well microtiter plate. Scheme 1 outlines the principal design of a monomeric b-lactam substrate for Bla activity and inhibition screening. A flexible 2-(4-mercaptophenyl) acetic acid coupled 1,2-bis(2-aminoethoxy) ethane linker is connected to the 30-position of cephalosporin through iodo-thiol substitution. As an excellent leaving group, the thiol group facilitates the release of the fragment on the b-lactam ring upon enzyme hydrolysis. The free thiol terminal and positively charged amino groups in the released fragment lead to the aggregation of Au-NPs based on the Au–S bond and electrostatic interaction between the charged amino group and the citrate ions on the surface of gold nanoparticles, thus demonstrating the significant color change from red to blue. This red-shifting aggregate can be used as a colorimetric sensor to identify Bla activity in the absence and presence of the inhibitors. The efficiency of the enzyme activity inhibition can be screened based on the specific colour changes. After obtaining the precursor, the b-lactam substrate was used to determine the enzymatic activity in vitro. In a typical experiment, substrate (8.0 mM) was initially incubated with transformed TEM-1 Bla (2.0 nM) in a PBS buffer (pH 7.4) in the absence of an inhibitor for 20 min. The resulting solution was subsequently transferred into Au-NPs suspension (15 nm, 2.5 nM). As shown in Fig. 1(a), a significant colour change