Rapid naked-eye detection of Gram-positive bacteria by vancomycin-based nano-aggregation

Abstract

Development of a rapid, point-of-care assay for diagnosing bacterial infections is crucial for subsequent treatment of the patient and preventing the overuse of antibiotics. Herein, we describe a rapid, one-step colorimetric assay based on the formation of nano-aggregates using nanobeads targeting Gram-positive bacteria. Vancomycin was immobilized onto blue-colored polymeric nanobeads to induce specific and multivalent binding with the Gram-positive bacterial cell wall and subsequent agglomeration. Without any pre-processing steps, the addition of various types of Gram-positive pathogens to the nanobeads resulted in the formation of blue precipitates, which could be observed with the naked eye in ∼30 min. We also utilized a porous filter system for the assay, which allowed discrimination of Gram-positive targets with higher selectivity, and demonstrated feasibility as a simple diagnostic assay with minimal technical components. We anticipate that the nanobead aggregation assay can be potentially applied as a rapid and simple sensing platform, which can be easily miniaturized and enable point-of-care diagnosis of Gram-positive infections.

Fig. 1. Overall scheme illustrating the NB-vanco assay for detection of Gram-positive bacteria. Nanobeads functionalized with vancomycin (NB-vanco) are added with Gram-positive bacteria, which form nano-aggregates with blue color, and can be detected with the naked eye. The red dash-line indicates the hydrogen bond interaction between vancomycin and d-Ala-d-Ala subunits on the bacterial cell wall.

Fig. 2. Synthesis and characterization of NB-vanco. (a) NB-vanco is synthesized by the EDC/NHS reaction between amine groups of vancomycin and carboxyl groups on the nanobeads. (b) Zeta potential measurements of NB-vanco. Data are expressed as mean ± s.d. (c) XPS binding energy spectra of (1) unmodified NB and (2) NB-vanco.

Fig. 3. Characterization of the NB-vanco assay. (a) Images of sample tubes taken with iPad Air2 according to various assay time (2.5 × 10⁵ bacterial cells per μl). (b) Absorbance measurements of the assay solutions with different assay time. The decrease in absorbance values results from precipitation of NB-vanco with bacteria. (c) Absorbance measurements of the assay solutions at 30 min assay time. Data are presented as the mean ± s.d. (n = 5; *p < 0.01; **p < 0.001).

Fig. 4. Characterization of the nano-aggregates by microscopy. (a) Bright field images of the nano-aggregates according to various assay time (scale bar, 25 μm). (b) Fluorescence microscopy (blue: DAPI) and (c) scanning electron microscopy of the nano-aggregates (arrows: yellow – S. aureus, red – E. coli; arrowheads: yellow – NB-vanco, red – NB).

Fig. 5. Detection of different numbers of bacteria by the NB-vanco assay. (a) and (b) Absorbance measurements and images of sample tubes after performing the NB-vanco assay with different numbers of S. aureus (a) and E. coli (b) at various assay times. (c) Absorbance measurements for S. aureus at 30 min assay time. Data are presented as mean ± s.d. (n = 5; *p < 0.001).

Fig. 6. Integration of the NB-vanco assay with the porous filter system (NB-vanco-porous filter system). (a) Workflow: bacteria are mixed with NB-vanco and applied through the porous filter (pore size, 5 μm). In the case of Gram-positive bacteria, nano-aggregates are formed which remain on the filter membrane as blue precipitates, and can be detected with the naked eye and acquired as images using a mobile camera system. (b) Images of the filter membrane after the assay with different assay times, obtained with iPad Air 2. (c) Quantification of nano-aggregate formation. Images in (b) were analyzed by the CIEL*a*b* color system with MATLAB. Aggregation signals were obtained from counting the pixels with b* below −9. Data are expressed as mean ± s.d. (n = 2).

Fig. 7. Assay results for different types of bacteria using the NB-vanco-porous filter system. (a) Images of the filter membrane after performing the assay. (b) Quantification of the nano-aggregates using the CIEL*a*b* color system by MATLAB. Aggregation signals were obtained by counting the pixels with b* below −9. Data are expressed as mean ± s.d. (n = 2).