Rapid and accurate detection of Escherichia coli growth by fluorescent pH-sensitive organic nanoparticles for high-throughput screening applications
Introduction
Rapid detection of bacterial growth is important for medical diagnostics to identify bacterial contamination in medical settings (Barker et al., 2010, Palavecino et al., 2006, Washington, 1996) and in the food industry in order to prevent contamination of food, air, and water (Farris et al., 2008, Ko et al., 2010, Wang et al., 2012).
The classic techniques used to detect bacterial growth rely on plating the sample on agar and counting of colonies after 37–48 h or more; however this is time-consuming and requires large amounts of samples and materials and may miss most types of bacteria (Lazcka et al., 2007, Nayak et al., 2009, Pourciel-Gouzy et al., 2004). Alternative methods have been developed to reduce the amount of time and sample necessary for a reliable measurement, such as the polymerase chain reaction (PCR). Although while PCR and its variants can detect bacteria within a relatively short time (Gopinath et al., 2014, Noble and Weisberg, 2005), issues like primer design and PCR-inhibitory effect of complex food matrices, in addition to its high cost, make PCR detection difficult to be a routine procedure (Gunaydin et al., 2007). Another effective method is enzyme-linked immunoabsorbent assay (ELISA). This technology uses an antigen-antibody reaction to detect unique microorganism. Different technologies have been developed to enhance ELISA measurements (Chunglok et al., 2011, Jain et al., 2012, Seo et al., 2015). These methods are highly sensitive and provide specific detections. However, this approach requires considerable amounts of expensive reactants such as antibodies and a high level of antigen for reliable detection (Mouffouk et al., 2011). Microfluidic devices are also being developed for the rapid detection of small amounts of bacteria (Chang et al., 2015), however they require relatively expensive laboratory instruments and experienced operators. Biosensing methods based on nanostructures have shown great potential for bacteria detection, such as gold nanoparticle for Escherichia coli detection (Hassan et al., 2015), magnetic nanoparticles system (Wan et al., 2014), and fluorescent silica nanoparticles (Chen et al., 2015). For these nanostructures, the potential toxicity effect on bacteria is an important issue to be considered (Adams et al., 2006, Wang et al., 2011). Polymeric organic nanoparticles, however, provide a promising platform to solve this problem. Moreover, different functional molecules can easily be immobilized on their surface to enhance their selectivity and sensitivity.
The growth of bacteria is often associated with a decrease in the pH of the growth medium due to a release of acidic metabolites such as acetic acid, lactic acid and CO2 (John et al., 2003, Young et al., 2004). Different kinds of pH sensors have therefore been used to measure the growth of bacteria based on this principle (Agayn and Walt, 1993, Badugu et al., 2008, Wang et al., 2014; Wang et al., 2013). Unfortunately, the useful life-span of these sensors is limited due to photobleaching and instability in a complex medium. More importantly, some dyes were developed for single assay-based measurements and are often unsuitable for real-time detection applications or to accommodate a large number of samples for high-throughput screening.
While high-throughput screening of bacterial growth has been conducted extensively to search for novel compounds with antibacterial activity (Zlitni et al., 2009), such efforts have been limited by the sensitivity and accuracy of bacterial resistance detection. Traditional optical density based (OD) measurements are limited by cells forming chains, clumps, filaments, or aggregates and are difficult to perform in complex growth media which can result in light scattering or absorption and interfere the detection by OD (Chalova et al., 2003, Gasol et al., 1999). Optical density was found to be linearly correlated with culture biomass between 0.01 and 0.3, but there might be variations depending on the growth phase and at higher densities the OD deviates from linearity (Martens-Habbena and Sass, 2006). Furthermore, OD measurements cannot distinguish between live, but slow growing, and dead cells. Screening of bacterial metabolism signatures can provide unambiguous evidence of the existence of live bacteria (Huang et al., 2011) and can offer a more reliable and sensitive assay for the presence of live microorganisms and the detection of antibiotic resistant strains.
In this work, we report the synthesis and full characterization of two highly water-soluble, biocompatible, stable and bright fluoresceinamine-based pH-sensitive nanoparticles. The addition of these nanoparticles to a growth medium containing bacteria allows for a rapid, accurate, reproducible and highly sensitive detection of bacterial growth.
Compared with the existing methods the one described here is based on nanoparticles that are more sensitive and more stable in common growth media, are easily synthesized at a low cost and can be used in any laboratory without additional expensive instruments or experienced operators. Moreover, these particles allow for continuous monitoring for real-time detection over long time scales and can be used for screening a large number of samples for high-throughput applications.
Section snippets
Materials
Fluoresceinamine, isomer I (Sigma-Aldrich, FA), 1-ethyl–3–(3-dimethylaminopropyl) carbodiimide hydrochloride (>98%, Fluka, EDC), ethanolamine (>99%, Sigma-Aldrich, EtA), acrylic acid (99%, Sigma-Aldrich, AA), poly(ethylene oxide) methyl ether acrylate (Mn=480 g/mol, Sigma-Aldrich, PEOA), 4,4′-azobis(4-cyanopentanoic acid) (Sigma-Aldrich, ACPA), citric acid (99.8%, Carlo Erba), sodium phosphate dibasic dihydrate (>99.5%, Sigma-Aldrich), 2-methyl-2-[(dodecylsulfanylthiocarbonyl)sulfanyl] propanoic
Synthesis of Fluoresceininamine (FA) pH-sensitive nanoparticles
Fluoresceininamine was selected as a pH-sensitive molecule because it has an intense absorption band in the visible. Even though it can adopt four different forms (dianion, anion, neutral and cation) depending on the pH, it is only highly fluorescent in its dianionic form (Sjöback et al., 1995). Furthermore, the pKa of the anion/dianion couple is about 6.4, which is suitable to measure changes in pH in biological media. The grafting of amino-functionalized molecules on carboxylic groups is a
Conclusions
The novel pH-sensitive nanoparticles described herein have distinct features that make them suitable for the development of biosensors including brightness, stability in growth medium, non-toxicity, high water solubility, high sensitivity, and ease of bioconjugation and incorporation into a biocompatible matrix. They can rapidly and accurately detect bacterial growth by detecting the change of pH resulting from cellular metabolism. Due to the brightness of these nanoparticles, they could be
Acknowledgments
Financial support for this work was provided by the CNRS (Centre National de la Recherche Scientifique) of France – Interface Physique Chimie Biologie Soutien à la prise de risque – and by Institut d’Alembert (FR 3242 CNRS – ENS de Cachan). A PhD grant for Y.S. (Contrat Doctoral n° 01/10/2012 6466) was provided by the MENESR (Ministère de l’Education Nationale, de l’Enseignement Supérieur et de la Recherche) of France.
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2022, Enzyme and Microbial TechnologyCitation Excerpt :Methods based on gene analysis are rarely applied for real-time monitors development due to high cost of bioreagents as well as laborious and time-consuming multistep bioanalytical procedures [1]. In practice, the conventional methods based on detection of optical density, like turbidimetry [3,4], and dynamic light scattering [5] or the use of fluorescent probes [6] are much more popular. It needs to be highlighted that almost all of these methods are focused on the determination of bacteria population number, concentration or biomass, but not on its viability manifested by metabolic (biocatalytic) activity.
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Present address: ISMO, CNRS UMR 8214, Université Paris-Sud, Avenue Jean Perrin, 91400 Orsay, France.