Rapid Colorimetric Detection of Bacterial Species through the Capture of Gold Nanoparticles by Chimeric Phages

Abstract

Rapid, inexpensive, and sensitive detection of bacterial pathogens is an important goal for several aspects of human health and safety. We present a simple strategy for detecting a variety of bacterial species based on the interaction between bacterial cells and the viruses that infect them (phages). We engineer phage M13 to display the receptor-binding protein from a phage that naturally targets the desired bacteria. Thiolation of the engineered phages allows the binding of gold nanoparticles, which aggregate on the phages and act as a signal amplifier, resulting in a visible color change due to alteration of surface plasmon resonance properties. We demonstrate the detection of two strains of Escherichia coli, the human pathogens Pseudomonas aeruginosa and Vibrio cholerae, and two strains of the plant pathogen Xanthomonas campestris. The assay can detect ∼100 cells with no cross-reactivity found among the Gram-negative bacterial species tested here. The assay can be performed in less than an hour and is robust to different media, including seawater and human serum. This strategy combines highly evolved biological materials with the optical properties of gold nanoparticles to achieve the simple, sensitive, and specific detection of bacterial species.

Keywords: bacteria; biosensor; detection; gold nanoparticles; phage display.

Figure 1

Scheme for chimeric phage detection of bacterial species. (a) M13 phage (gray) is engineered to express a foreign receptor-binding protein (blue circle) fused to the minor coat protein pIII, and the chimeric phage is thiolated (yellow) through EDC chemistry. (b) Thiolated chimeric phages are added to media containing bacteria (blue rectangle) and may attach to the cells. Centrifugation separates cell-phage complexes from free phage. The pellet is resuspended in solution with gold nanoparticles (red), whose aggregation on the thiolated phage produces a color change (purple).

Figure 2

Preparation of thiolated phage capsids and AuNPs. (a) ATR-FTIR of purified thiolated M13KE phage indicates gain of S–H stretching (2550 cm^–1) and C–S stretching (659 cm^–1) signals. Shown are cysteamine (black), phage before modification (red), and phage after modification (blue). Representative TEM images of wild-type M13KE phage (b) before and (c) after thiolation indicate little change in gross morphology. (d) TEM image of AuNPs shows homogeneous particles of ∼4 nm diameter.

Figure 3

Detection of E. coli with thiolated M13KE and AuNPs. (a, c) Digital photos and (b, d) UV–vis spectra are shown. From left to right in panel a, samples contain AuNPs and: no bacteria or phages (black line in panel b), unmodified M13KE with 10⁶ CFU E. coli (red line in panel b), and thiolated M13KE with E. coli at 10², 10⁴, and 10⁶ CFU (blue, magenta, and green lines, respectively, in panel b). From left to right in panel c, samples contain AuNPs and no bacteria or phages (black line in panel d), unmodified M13KE phage and ∼1 or ∼10 CFU of E. coli (red or blue lines in panel d, respectively), and thiolated M13KE phage and ∼1 or ∼10 CFU of E. coli (magenta or green lines in panel d, respectively).

Figure 4

Detection of E. coli ER2738 in (a, b) seawater or (c, d) human serum. (a, c) Digital photos and (b, d) UV–vis spectra are shown. From left to right in panels a and c, samples contain AuNPs and no bacteria or phages (black lines in panels b and d), unmodified M13KE with 10⁶ CFU E. coli (red lines in b and d), and thiolated M13KE with E. coli at 10², 10⁴, and 10⁶ CFU (blue, magenta, and green lines, respectively, in panels b and d).

Figure 5

Detection of several bacterial species in relevant media: (a, b) V. cholerae 0395 in seawater, (c, d) X. campestris (pv campestris) in tap water, (e, f) X. campestris (pv vesicatoria) in tap water, (g, h) P. aeruginosa in tap water, and (i, j) human serum and (k, l) E. coli (I⁺) in tap water. The corresponding chimeric phage (Table 1) was used in each case. Shown are digital photographs (left) and UV–vis spectra (right). Left column: from left to right, samples contain AuNPs and no bacteria or phages (black line in right column), unmodified phage with 10⁶ CFU host bacteria (red line in right column), and thiolated phage with host bacteria at 10², 10⁴, and 10⁶ CFU (blue, magenta, and green lines, respectively, in the right column).

Figure 6

Specificity of bacterial detection. Absorption spectra of (a) M13KE, (b) M13-g3p(CTXϕ), (c) M13-g3p(If1), (d) M13-g3p(Pf1), (e) M13-g3p(ϕLf), and (f) M13-g3p(ϕXv) when incubated with different bacterial species and AuNPs. Bacterial species shown are E. coli (F⁺) (red), V. cholerae 0395 (blue), E. coli (I⁺) (magenta), P. aeruginosa (orange), X. campestris (pv campestris) (gray), and X. campestris (pv vesicatoria) (green). The spectrum of AuNPs alone (dotted black line) is also shown.