Colorimetric detection of pathogenic bacteria using platinum-coated magnetic nanoparticle clusters and magnetophoretic chromatography
Graphical abstract
Introduction
Prevention of food poisoning requires intense inspection of all food served in homes and restaurants. Considering the short time interval between meals, on-site inspection should be completed within 1 h. Therefore, conventional cultivation techniques that require cell culture times of a few days are not suitable for on-site inspection [1]. Various methods that do not require cultivation have been developed, including those that use polymerase chain reaction [2], [3], microcantilever [4], [5], surface plasmon resonance [6], [7], quartz crystal microbalances [8], [9], surface acoustic waves [10], electrochemical methods [11], [12], fluorescence [13], [14] and surface-enhanced Raman scattering [15], [16]. Although these techniques can determine the presence of pathogenic bacteria within a few hours, they require experienced technicians and expensive equipments.
In contrast to techniques that require analytical instruments, the use of antibody-functionalized magnetic nanoparticles with a size sorting method realized the rapid and cost-effective detection of pathogenic bacteria. After bacteria–magnetic nanoparticle complexes and free magnetic particles were magnetically separated from a sample solution, the nanoparticle-containing solution was poured onto a filter membrane. This simple process produced selective enrichment of the complexes on the membrane, which enabled identification of the presence of bacteria with the naked eye [17]. However, the inefficient separation of free magnetic nanoparticles by using a filter membrane resulted in high background noise and degraded detection sensitivity.
This problem can be addressed by adopting magnetophoretic chromatography that uses a liquid-type filter [18]. A solution containing free magnetic nanoparticles and bacteria–magnetic nanoparticle complexes is gently poured onto a viscous polymer solution to form two liquid layers. The solutions do not mix during the experimental time scale due to the difference in their viscosities. When a magnet is placed below the container containing the solutions, the magnetic nanoparticles are attracted toward the magnet. Only the bacteria–magnetic nanoparticle complexes pass through the interface and reach the bottom of the container; the free magnetic particles are trapped at the interface between the solutions. Although magnetophoretic chromatography has many advantages such as low cost, ease of use and short assay time, its sensitivity must be improved to reduce the number of false-negative errors.
In this study, we improved the limit of detection of the magnetophoretic chromatography process by adopting platinum-coated magnetic nanoparticle clusters (Pt/MNCs). The Pt nanoparticles have strong catalytic activity that can oxidize organic molecules [19] and can induce color change in a solution that contains organic molecules such as 3,3′,5,5′-tetramethylbenzidine (TMB). Half-fragments of monoclonal E. coli antibodies were functionalized to Pt/MNCs and used to capture E. coli bacteria in milk. Magnetophoretic chromatography was used to separate the Pt/MNC-EC complexes from free Pt/MNCs, then the complexes were added to a TMB solution and the resulting color change of the solution was identified with the naked eye. The limit of detection was 10 cfu mL−1 and total assay time was 30 min.
Section snippets
Materials
Iron(III) chloride hexahydrate, polyacrylamide, urea, sodium citrate, ethylenediaminetetra-acetic acid (EDTA), cysteamine hydrochloride (2-mercaptoethylamine, MEA), potassium tetrachloroplatinate (K2PtCl4), sodium borohydride (NaBH4), TMB, sodium acetate (NaAc), citric acid, sodium phosphate monobasic (NaH2PO4), sodium phosphate dibasic (Na2HPO4), sodium chloride (NaCl), Tween-20 (T20), and poly(ethylene glycol) (PEG, MW = 8000 g mol−1) were purchased from Aldrich (St. Louis, MO) and used without
Characterization of Pt/MNCs
Fig. 1a and b shows scanning electron microscopy (SEM) images of the MNCs and Pt/MNCs, respectively. The average sizes of the MNCs and Pt/MNCs were measured to be ∼150 nm with narrow size distributions. The small bright spots in SEM images of Pt/MNCs correspond to Pt nanoparticles with the average size of 16 ± 5 nm. The TEM image and elemental mappings of Pt/MNC confirms the presence of Pt nanoparticles on the surfaces of MNCs. Addition of MNCs to a TMB solution did not induce color changes, but
Conclusion
We have developed a novel assay which combines magnetophoretic chromatography and catalytic oxidation of TMB by platinum, and used this method to enable detection of E. coli in milk with the naked eye. Magnetophoretic chromatography was used to separate the Pt/MNC-EC complexes from the free Pt/MNCs; the catalytic platinum nanoparticles on the Pt/MNC-EC complexes oxidized TMB to induce color changes in the solution. Although the experimental set-up requires only a permanent magnet and a
Acknowledgements
This research was supported by a grant from Ministry of Food and Drug Safety (10162MFDS995) and the Public Welfare & Safety Research Program through the National Research Foundation of Korea (NRF), funded by the Ministry of Science, ICT, and Future Planning (NRF-2012M3A2A1051679) in 2014.
References (28)
- et al.
Microbial detection
Biosens. Bioelectron.
(1996) - et al.
Simultaneous detection of Listeria monocytogenes and Salmonella by multiplex PCR in cooked ham
Food Microbiol.
(2005) - et al.
Ultrasensitive detection of Vibrio cholerae O1 using microcantilever-based biosensor with dynamic force microscopy
Biosens. Bioelectron.
(2010) - et al.
Rapid and sensitive immunodetection of Listeria monocytogenes in milk using a novel piezoelectric cantilever sensor
Biosens. Bioelectron.
(2013) - et al.
Comparison of sensing strategies in SPR biosensor for rapid and sensitive enumeration of bacteria
Biosens. Bioelectron.
(2012) - et al.
SPR bacterial pathogen biosensor: the importance of fluidic conditions and probing depth
Talanta
(2014) - et al.
QCM immunosensor detection of Escherichia coli O157:H7 based on beacon immunomagnetic nanoparticles and catalytic growth of colloidal gold
Biosens. Bioelectron.
(2011) - et al.
Real-time and sensitive detection of Salmonella Typhimurium using an automated quartz crystal microbalance (QCM) instrument with nanoparticles amplification
Talanta
(2013) - et al.
A Love wave immunosensor for whole E. coli bacteria detection using an innovative two-step immobilisation approach
Biosens. Bioelectron.
(2007) - et al.
In-situ fluorescent immunomagnetic multiplex detection of foodborne pathogens in very low numbers
Biosens. Bioelectron.
(2014)
Au@Pt nanostructures as oxidase and peroxidase mimetics for use in immunoassays
Biomaterials
Fe3O4@Pt nanoparticles with enhanced peroxidase-like catalytic activity
Mater. Lett.
A colorimetric biosensor for the detection of foodborne bacteria
Sens. Actuators B
Strategies for the detection of Escherichia coli O157:H7 in foods
J. Appl. Microbiol.
Cited by (46)
A simple gradient centrifugation method for bacteria detection in skim milk
2023, Microchemical JournalMulticomponent magnetic nanoparticle engineering: the role of structure-property relationship in advanced applications
2022, Materials Today ChemistryNano-labeled materials as detection tags for signal amplification in immunochromatographic assay
2022, TrAC - Trends in Analytical ChemistryCitation Excerpt :MNPs are a new type of nano-labeling materials born at the end of the 1980s, have the large surface area and volume ratios like magnetite (Fe3O4) and hypohematite (r-Fe2O3) [63]. Compared with traditional gold nanoparticle labeling materials, the strong magnetic force of MNPs can realize the separation and enrichment of the analyte without centrifugation [64], which have been developed and applied in ICA strips, can effectively improve the concentration of the substance to be tested. Quantitative detection can also be achieved by reading the magnetic signal captured by the equipment to improve the sensitivity of the ICA [65].
A review on magnetic sensors for monitoring of hazardous pollutants in water resources
2022, Science of the Total EnvironmentRecent advances in optical biosensors for specific detection of E. coli bacteria in food and water
2022, Food ControlCitation Excerpt :The higher the amount of E. coli in the solution, the more intense the green color. The sensor enabled the detection of E. coli in the presence of 10 CFU/mL with a linear range of 10–105 CFU/mL (Kwon et al., 2015). You et al. (2018) detected E. coli with LOD 10 CFU/mL and linear range 101–103 CFU/mL in water and milk by designing a colorimetric sensor based on antibodies and gold NPs.
Nanomaterial application in bio/sensors for the detection of infectious diseases
2021, TalantaCitation Excerpt :Poly (ethylene glycol) medium caused separation Pt/MNC- E.coli O157: H7 nanoparticle from Free Pt/MNC nanoparticle. 10 CFU mL−1 E.coli O157: H7 could detect by color change (oxidation TMB with nanoparticle) [136]. More colorimetric biosensors are presented in Table 2.