A rapid bioassay for single bacterial cell quantitation using bioconjugated nanoparticles

Abstract

The rapid and sensitive determination of pathogenic bacteria is extremely important in biotechnology, medical diagnosis, and the current fight against bioterrorism. Current methods either lack ultrasensitivity or take a long time for analysis. Here, we report a bioconjugated nanoparticle-based bioassay for in situ pathogen quantification down to single bacterium within 20 min. The bioconjugated nanoparticle provides an extremely high fluorescent signal for bioanalysis and can be easily incorporated with biorecognition molecules, such as antibody. The antibody-conjugated nanoparticles can readily and specifically identify a variety of bacterium, such as Escherichia coli O157:H7, through antibody-antigen interaction and recognition. The single-bacterium-detection capability within 20 min has been confirmed by the plate-counting method and realized by using two independent optical techniques. The two detection methods correlated extremely well. Furthermore, we were able to detect multiple bacterial samples with high throughput by using a 384-well microplate format. To show the usefulness of this assay, we have accurately detected 1-400 E. coli O157 bacterial cells in spiked ground beef samples. Our results demonstrate the potential for a broad application of bioconjugated nanoparticles in practical biotechnological and medical applications in various biodetection systems. The ultimate power of integrating bionanotechnology into complex biological systems will emerge as a revolutionary tool for ultrasensitive detection of disease markers and infectious agents.

Fig. 1.

Fluorescent nanoparticles. Transmission electron microscope image of RuBpy-doped silica nanoparticles before bioconjugation. Each of these nanoparticles contains tens of thousands of dye molecules inside, emits strong fluorescence signal, has excellent photostability, and can be used for easy and effective biomolecule conjugation for biorecognition.

Fig. 2.

Images of bacterial cells. (A) Scanning electron microscope image of E. coli O157:H7 cell incubated with antibody-conjugated nanoparticles. (B) Scanning electron microscope image of E. coli DH5α cell (negative control) incubated with nanoparticles conjugated with antibody for E. coli O157:H7. (C) Fluorescence image of E. coli O157:H7 after incubation with antibody-conjugated nanoparticles. The fluorescence intensity is strong, enabling single-bacterium cell identification in aqueous solution.

Fig. 3.

Single-bacterium quantitation. Comparison of single bacterium detection with the plate-counting method [a golden standard for bacteria counting (22)] vs. the spectrofluorometer method with antibody-conjugated nanoparticles. In both experiments, 21 samples of 25 bacteria were used. The variation in counted number of bacteria is caused by the sampling nature of bacteria-containing solutions, not the detection methods.

Fig. 4.

A laboratory-made flow cytometer was used to detect single bacterium. Detection of different concentrations of bacteria after incubation with antibody-conjugated nanoparticles (NP) was done with a laboratory-made flow cytometer. The trace was recorded under different experimental conditions as those described in the keys.

Fig. 5.

Single-bacterium detection with beef sample. Detection of E. coli O157:H7 in spiked ground beef was done with the plate-counting method and the antibody-conjugated, nanoparticle (NP)-based method. Bacteria in the range of 1–400 cells per sample were detected. The two methods had linear correlation, with an R value of 0.99. Total detection time for the beef sample was ≈20 min for the antibody-conjugated, nanoparticle-based method, and that for the plate-counting method was >1 day.