Lysozyme-encapsulated gold nanocluster-based affinity mass spectrometry for pathogenic bacteria Po-Han Chan, Song-Yi Wong, Shu-Hsuan Lin and Yu-Chie Chen* Department of Applied Chemistry, National Chiao Tung University, Hsinchu 300, Taiwan RATIONALE: Bacterial infections can be difficult to treat and can lead to irreversible damage to patients if proper treatment is not provided in time. Additionally, the emerging threat from antibiotic-resistant bacterial strains makes medical treatment even more difficult. Thus, rapid identification of infected bacterial strains is essential to assist diagnostics and medical treatment. METHODS: Lysozymes are glycoside hydrolases that can bind with peptidoglycans on bacterial cell walls. In this work, we demonstrated that lysozyme-encapsulated gold nanoclusters (lysozyme-AuNCs) with red photoluminescence can be used as affinity probes to concentrate pathogenic bacteria. After bacteria had been probed by the lysozyme-AuNCs in a sample solution, the lysozyme-AuNC-bacteria conjugates were readily spun down at a low centrifugation speed. The red emission from the AuNCs on the conjugates could be visualized with the naked eye under illumination of ultraviolet light. The bacteria in the conjugates can be identified by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) combined with principal component analysis (PCA). RESULTS: We demonstrated that pathogenic bacteria including Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, pandrug-resistant Acinetobacter baumannii, Staphylococcus aureus, Enterococcus faecalis, and vancomycin-resistant Enterococcus faecalis (VRE) can be readily concentrated by the lysozyme-AuNCs and distinguished by the results combining MALDI-MS and PCA. Additionally, the possibility of using the current approach to differentiate E. faecalis from VRE was also demonstrated. The lowest detection concentration for E. coli using the current approach is ~10 6 cells/mL. CONCLUSIONS: The results indicated that the lysozyme-AuNCs are effective affinity probes for Gram-positive and Gram-negative bacteria. By combining the results from MALDI-MS and PCA, different bacteria can be easily distinguished. The current approach can be potentially used to assist the identification of bacteria from biological fluids. Copyright © 2013 John Wiley & Sons, Ltd. Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) is a useful tool for the rapid characterization of microorganisms [1–11] such as bacteria [1–8] and fungi, [9–11] which can cause infections or food poisoning. However, bacterial samples obtained from infected biological fluids or food poisoning samples are difficult to be characterized directly by MALDI-MS because the ions generated from complex sample matrices may seriously suppress the ions generated from bacterial cells. Thus, cell culturing or sample pretreatment is generally required to obtain sufficient amounts of bacteria and minimize the number of undesirable species prior to MALDI- MS analysis. However, cell culturing is time consuming. Sample pretreatment through selective concentration of bacteria from complex samples using affinity approaches can shorten analysis time. Functional magnetic nanoparticles that can recognize bacteria have been used as probes in pretreating bacterial samples. [4–8] Non-target species can be subsequently eliminated by magnetic separation, and the isolated bacteria trapped by the functional probes can be characterized by MALDI-MS. Gold nanoclusters (Au NCs) with high water solubility, good stability, and observable photoluminescence have been extensively used in chemical and biochemical sensing. [12–23] Protein-directed synthesis of AuNCs (protein-AuNCs) has recently drawn considerable attention because of the straightforward generation of protein-encapsulated AuNCs. [12–15,24–28] In addition, the proteins on the generated AuNCs may retain their bioactivity. [13,25] Xie et al. [12] used bovine serum albumin (BSA) as the reagent to sequester and reduce Au precursors for the generation of AuNCs in aqueous tetrachloroauric acid solution. Although the protein structure on the BSA-AuNCs cannot be preserved, [12] further exploration using other types of proteins showed the feasibility of retaining protein bioactivity. [13,25] For instance, lysozymes are a family of enzymes that can catalyze the hydrolysis of peptidoglycans on bacterial cell walls. [29] Lysozyme-encapsulated AuNCs can be used to effectively inhibit the cell growth of bacteria, including antibiotic-resistant bacteria, indicating that the activity of lysozyme on lysozyme-AuNCs is retained. [13] To accelerate the generation of the lysozyme-AuNCs, microwave-heating has been used to accelerate the generation of protein-encapsulated AuNCs. [13] The cell growth of antibiotic-resistant bacteria can be effectively inhibited in the presence of lysozyme-AuNCs * Correspondence to: Y.-C. Chen, Department of Applied Chemistry, National Chiao Tung University, Hsinchu 300, Taiwan. E-mail: yuchie@mail.nctu.edu.tw Copyright © 2013 John Wiley & Sons, Ltd. Rapid Commun. Mass Spectrom. 2013, 27, 2143–2148 Research Article Received: 26 February 2013 Revised: 24 May 2013 Accepted: 2 July 2013 Published online in Wiley Online Library Rapid Commun. Mass Spectrom. 2013, 27, 2143–2148 (wileyonlinelibrary.com) DOI: 10.1002/rcm.6674 2143