Elsevier

Analytica Chimica Acta

Volume 883, 9 July 2015, Pages 61-66
Analytica Chimica Acta

Colorimetric detection of pathogenic bacteria using platinum-coated magnetic nanoparticle clusters and magnetophoretic chromatography

https://doi.org/10.1016/j.aca.2015.04.044Get rights and content

Highlights

  • Pt/MNCs and magnetophoretic chromatography were used to detect E. coli.

  • MNCs/E. coli were separated from free MNCs by magnetophoretic chromatography.

  • Color change by Pt enabled detection of 10 cfu/mL E. coli with the naked eye.

Abstract

A colorimetric method that uses platinum-coated magnetic nanoparticle clusters (Pt/MNCs) and magnetophoretic chromatography is developed to detect pathogenic bacteria. Half-fragments of monoclonal Escherichia coli O157:H7 (EC) antibodies were functionalized to Pt/MNCs and used to capture E. coli bacteria in milk. After magnetic separation of free Pt/MNCs and Pt/MNC-EC complexes from the milk, a precision pipette was used to imbibe the E. coli-containing solution, then a viscous polyethylene glycol solution. Due to difference in viscosities, the solutions separate into two liquid layers inside the pipette tip. The Pt/MNC-EC complexes were separated from the free Pt/MNCs by applying an external magnetic field, then added to a tetramethylbenzidine (TMB) solution. Catalytic oxidation of TMB by Pt produced color changes of the solution, which enabled identification of the presence of 10 cfu mL−1 E. coli bacteria with the naked eye. The total assay time including separation, binding and detection was 30 min.

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.

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