Mapping bacteria on filter membranes, an innovative SERS approach
Graphical abstract
Introduction
The safety of drinking water is essential to human well-being. However, in some developing countries, people do not have access to clean, sanitized drinking water in which the existence of pathogenic bacteria pose a great threat of bacterial transmitted diseases. According to the world health organization, the mortality of water-associated diseases exceeds 5 million people per year with more than 50% categorized as microbial intestinal infections (Cabral, 2010). Microbial contamination in drinking water is mainly associated with wastewater discharges in freshwater and coastal seawater, which serves as a major source of fecal microorganisms, including pathogens. Some of the pathogens responsible for the main bacterial infections in water are Salmonella, Vibrio and Escherichia coli. The United States Environmental Protection Agency (EPA) has set the standards to monitor fecal indicator bacteria such as E. coli to indicate the presence of pathogenic microorganisms caused by recent fecal contamination or unsanitary processing, and the maximum contaminant level goals are zero in most cases (Edition, 2006). However, there are no standard techniques established by the EPA to detect particularly low levels (below 101 CFU/ml) of indicator bacteria (U.S. EPA., 2014). Standard plate count has been the most developed and widely used method for pathogen detection, however it usually take days to produce a result, and not able to isolate viable but nonculturable organisms (Gunasekera et al., 2000). New and advanced technologies have been developed for the rapid detection of foodborne pathogens aiming at overcoming disadvantages associated with traditional microbiological detection techniques. Those techniques mainly include spectroscopic methods, e.g. Raman spectroscopy, Infrared spectroscopy (Chu et al., 2008; Rodriguez-Saona et al., 2001), polymerase chain reaction (PCR) (Ashimoto et al., 1996; Josephson et al., 1993) and a variety of sensor-based methods including biosensors (e.g DNA and antibodies) (Baeumner, 2003; Mao et al., 2006), chemical sensors (Su et al., 2013) and impedance sensors (Yang et al., 2004). These methods offer sensitive and specific detection of various microbial targets. Unfortunately when dealing with low concentration of bacteria in large volume of sample, pre-enrichment is often needed before applying these methods (Gracias and McKillip, 2004).
The objective of this study is to develop a rapid method that can directly detect low concentration of bacteria cells without enrichment. This method integrates filtration technique with surface enhanced Raman spectroscopic (SERS) mapping technique to realize rapid screening and quantification of bacteria cells on a filter membrane. A filter membrane can provide a threshold for the dimension of substances to be filtrated, in other words, to eliminate small molecules of interference, as well as to concentrate matter of interest. Filtration technique followed by membrane culturing is a conventional approach for separating and detecting coliforms in environmental water samples and liquid food with low particulate matters (Slanetz and Bartley, 1957; Weinbauer et al., 1998). SERS is a rapidly growing analytical technique for detection of low concentration of analytes. The chemical signature of the analyte can be tremendously enhanced by gold and silver nanoparticles. SERS mapping technique is an advanced chemical imaging technique, by which hundreds of SERS spectra can be automatically collected at every pixel of the defined area, and then integrated to generate artificial color images based on the intensity of a designated peak. Previously, we applied SERS mapping to detect and differentiate between several bacteria strains deposited on silver dendrites, as well as using 4-mercaptophenylboronic acid (4-mpba) modified silver dendrites to capture and detect Salmonella from skimmed milk (Wang et al., 2016, Wang et al., 2017). 4-mpba can interact bacterial peptidoglycan in cell wall and gives off strong and distinct SERS signals (Su et al., 2013; Wang et al., 2015).
In this study, we used a simple syringe filter to catch bacteria on a filter membrane, then 4-mpba was integrated to interact with bacteria and give off strong SERS signals for the ease of identification. E. coli was used as a model bacterium to develop the method. Different membrane types and ways of integrating 4-mpba molecules with bacteria were evaluated. The sensitivity and quantification capability of the method was also determined and compared with the membrane culturing method.
Section snippets
Materials
Citrate coated gold nanoparticles (Au NPs) with particle size of 50 nm and 20 mg/l concentration were purchased form Nanopartz (Loveland, CO, USA). 4-mercaptophenylboronic acid and sunset yellow FCF was purchased from Sigma-Aldrich Co. LLC (St. Louis, MO, USA), ammonium bicarbonate and glucose was from Fisher Scientific (Fair Lawn, NJ, U.S.A). Durapore PVDF filter membranes with 0.1 μm pore size were from EMD Millipore Inc. (Billerica, MA, USA). Nitrocellulose membrane filters with 0.2 μm pore
Determination of the optimum membrane type
Two types of membrane (PVDF and nitrocellulose) were compared for the performance using the filtration method. To make 4-mpba signals representative of bacteria, there should be no indicator signals in the absence of bacteria, i.e. negative controls. As shown in Fig. 1c and d, SERS spectra indicated that some of the 4-mpba molecules were retained on PVDF membrane even after the washing step, which displayed the characteristic peak at 1072 cm−1. This is possibly due to the hydrophilic coating on
Conclusion
By using the 4 mpba molecules, we were able to obtain SERS mapping results of bacterial cells directly from the filter membrane with consistent and sensitive signals. The percent of positive points in the mappings could be utilized to estimate the number of total bacteria cells present in the sample. Although, this method may not be able to differentiate between viable and non-viable cells, the ability to estimate initial numbers of cell contamination is also important for some applications.
Conflict of interest
The authors declare no competing financial interest.
Acknowledgment
This study is funded by USDA-NIFA2015-67021-22993. Acknowledgment is dedicated to Zhiyun Zhang for his help in obtaining the SEM pictures.
References (19)
- et al.
Viable, but non-culturable, state of a green fluorescence protein-tagged environmental isolate of Salmonella typhi in groundwater and pond water
FEMS Microbiol. Lett.
(1999) - et al.
A nanoparticle amplification based quartz crystal microbalance DNA sensor for detection of Escherichia coli O157:H7
Biosens. Bioelectron.
(2006) - et al.
Rapid concentration detection and differentiation of bacteria in skimmed milk using surface enhanced Raman scattering mapping on 4-mercaptophenylboronic acid functionalized silver dendrites
Anal. Bioanal. Chem.
(2017) - et al.
Interdigitated microelectrode (IME) impedance sensor for the detection of viable Salmonella typhimurium
Biosens. Bioelectron.
(2004) - et al.
Polymerase chain reaction detection of 8 putative periodontal pathogens in subgingival plaque of gingivitis and advanced periodontitis lesions
Oral Microbiol. Immunol.
(1996) Biosensors for environmental pollutants and food contaminants
Anal. Bioanal. Chem.
(2003)Water microbiology. Bacterial pathogens and water
Int. J. Environ. Res. Public Health
(2010)- et al.
Silver nanorod arrays as a surface-enhanced Raman scattering substrate for foodborne pathogenic bacteria detection
Appl. Spectrosc.
(2008) Bacteria indicators of potential pathogens
Cited by (22)
Assessment of three SERS approaches for studying E. Coli O157:H7 susceptibility to ampicillin
2022, Spectrochimica Acta - Part A: Molecular and Biomolecular SpectroscopyCitation Excerpt :Studies that have utilized Raman and SERS techniques for detecting antibiotic resistant bacteria typically utilize one specific analytical procedure to characterize different organisms or responses to different antibiotics [23,26–28], and the SERS procedures used in these studies involve simply placing a drop of the bacterial sample on a substrate surface [23,27]. While these previous studies have demonstrated that a range of different bacteria species and antibiotic resistance mechanisms can be identified with conventional SERS methods, there are also a number of less conventional SERS techniques for bacterial analysis that could potentially be used to detect antibiotic resistant organisms, such as the analysis of the extracellular matrix liquid or bacterial lysate [29,38] and SERS filter mapping [30]. There are significant knowledge gaps regarding how effective these novel techniques would be for characterizing antibiotic resistant bacteria, and how they compare to the conventional SERS procedures in this application.
Trends in the bacterial recognition patterns used in surface enhanced Raman spectroscopy
2021, TrAC - Trends in Analytical ChemistryCitation Excerpt :It will allow microbial communities to be exploited in industrial and municipal wastewater treatments and management, such as bioaugmentation, to improve efficiency and effectiveness. There are many pre-treatment and amplification practices reported in the literature [28–30,36,41,42,45,46,50,52,53,55,59,63–67,69,72,77–103] and their key findings have been reported in Table 1. A schematic illustration for each of the platforms mentioned above has been presented in Fig. 2.
An innovative filtration based Raman mapping technique for the size characterization of anatase titanium dioxide nanoparticles
2021, TalantaCitation Excerpt :The objective of this study is to develop and evaluate a filtration-based Raman mapping technique for the size characterization of anatase TiO2-NPs. Raman mapping is an advanced chemical imaging technique where thousands of spectra are collected for every pixel of a defined sample area and integrated to generate the colorimetric image for a designated peak of an analyte [25,26]. This approach provides an advantage over point measurement technique, where the spectra are collected at points scattered throughout the sample which may result in larger variation in signal intensities.
Recent advance in SERS techniques for food safety and quality analysis: a brief review
2019, Current Opinion in Food ScienceCitation Excerpt :The authors reported successful detection of melamine from whole milk samples, with LOD and LOQ of 0.012 mmol/L and 0.039 mmol/L, respectively. Our group developed a filtration-assisted methodology to enhance detection of analytes using SERS [27,28]. In brief, the samples were casted onto filter membrane together with colloidal AgNPs for SERS analysis.