Exploiting pH-Regulated Dimer-Tetramer Transformation of Concanavalin A to Develop Colorimetric Biosensing of Bacteria

Abstract

Gold nanoparticles (AuNPs) aggregation-based colorimetric biosensing remains a challenge for bacteria due to their large size. Here we propose a novel colorimetric biosensor for rapid detection of Escherichia coli O157:H7 (E. coli O157:H7) in milk samples based on pH-regulated transformation of dimer/tetramer of Concanavalin A (Con A) and the Con A-glycosyl recognition. Briefly, antibody-modified magnetic nanoparticles was used to capture and concentrate E. coli O157:H7 and then to label with Con A; pH adjusted to 5 was then applied to dissociate Con A tetramer to release dimer, which was collected and re-formed tetramer at pH of 7 to cause the aggregation of dextran-modified AuNPs. The interesting pH-dependent conformation-transformation behavior of Con A innovated the design of the release from the bacteria surface and then the reconstruction of Con A. Therefore, we realized the sensitive colorimetric biosensing of bacteria, which are much larger than AuNPs that is generally not suitable for this kind of method. The proposed biosensor exhibited a limit of detection down to 41 CFU/mL, short assay time (~95 min) and satisfactory specificity. The biosensor also worked well for the detection in milk sample, and may provide a universal concept for the design of colorimetric biosensors for bacteria and virus.

Figure 1

Illustration of the procedures and mechanism of the colorimetric biosensor.

Figure 2

UV-vis spectra of dextran@AuNPs before (curve 1) and after (curve 2) mixing with Con A in PBS, pH 7.4, or mixed with Final-solution without bacteria (curve 3) or with 10⁵ CFU/mL E. coli O157:H7 (curve 4), or mixed with 10⁵ CFU/mL E. coli O157:H7 but without ConA (curve 5).

Figure 3

(A) UV-vis spectra of dextran@AuNPs mixed with different concentration of Con A, 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 μg/mL. (B) The absorption ratio in 635 nm and 525 nm for the concentration of Con A.

Figure 4

TEM imaging of EAbs modified MNPs coupled into bacterial cell surface (A); dextran@AuNPs before (B) and after (C) coupled with released Con A in the Final-solution.

Figure 5

(A) UV-vis spectra of dextran@AuNPs with released Con A in different concentration of E. coli O157:H7. Inset is the photos of dextran@AuNPs mixed with released Con A at the concentration of 0, 10², 10³, 10⁴, 10⁵, 10⁶, 10⁷ CFU/mL bacteria. (B) Linear relationship between the absorption change ratio A ₆₃₅/A ₅₂₅ and the concentration of E. coli (N = 3).

Figure 6

(A) UV-vis spectra of dextran@AuNPs with released Con A toward the same concentration (10⁷ CFU/mL) of E. coli O157:H7, S. Typhimurium, or L. monocytogenes. (B) Histogram of absorption ratio at 635 nm and 525 nm with different kind of pathogenic bacteria. Duncan’s multiple range test, alpha < 0.05; **p < 0.001.