Highly-branched poly(N-isopropyl acrylamide) functionalised with pendant Nile red and chain end vancomycin for the detection of Gram-positive bacteria

Abstract

This study shows how highly branched poly(N-isopropyl acrylamide) (HB-PNIPAM) with a chain pendant solvatochromic dye (Nile red) could provide a fluorescence signal, as end groups bind to bacteria and chain segments become desolvated, indicating the presence of bacteria. Vancomycin was attached to chain ends of HB-PNIPAM or as pendant groups on linear polymers each containing Nile red. Location of the dye was varied between placement in the core of the branched polymer coil or the outer domains. Both calorimetric and fluorescence data showed that branched polymers responded to binding of both the peptide target (D-Ala-D-Aa) and bacteria in a different manner than analogous linear polymers; binding and response was more extensive in the branched variant. The fluorescence data showed that only segments located in the outer domains of branched polymers responded to binding of Gram-positive bacteria with little response when linear analogous polymer or branched polymer with the dye in the inner core was exposed to Staphylococcus aureus.

Keywords: Bacterial sensor; Diagnostic device; Polymer architecture; Solvatochromism; Specificity; Stimuli responsive.

Graphical abstract

Fig. 1

(A) Schematic diagram of a highly branched polymer with chain end ligands; (B) diagram showing polymer segments binding and desolvation of the end segments.

Fig. 2

Log molar mass distributions of one step (A) and chain extended (B) vancomycin-functional polymers and hydrodynamic radii distributions of one step (C) and chain extended (D) vancomycin-functional polymers.

Scheme 1

4-vinylbenzyl pyrrolecarbodithioate (1) based SCVP RAFT polymerisation of N-isopropyl acrylamide used to generate a HB-PNIPAM labelled Nile red (2) with post polymerization modification of the end groups by radical-radical coupling (HB-P3_COOH). The Scheme also shows how HB-P3_p was chain extended to provide a polymer with Nile red located in the inner core (HB-P5). HB-P6 and P7 were created in a similar manner by chain extending, with monomer mixtures containing 2, a core polymer (HB-P4) without pendent Nile red (P4).

Fig. 3

Peak fluorescence emission wavelength (A) and integrated emission intensity (B) of linear polymers. (● L-P0_, ■ L-P1_COOH and ▴ L-P1) compared to water, ethylene glycol and methanol.

Fig. 4

Shift in peak fluorescence emission wavelength (A) and integrated emission intensity (B) with temperature of HB-PNIPAMs, with pyrrole (HB-P3p), acid (HB-P3_COOH) and vancomycin (HB-P3) chain ends. (C) Shift of fluorescence emission wavelength of HB-P5, HB-P6 and HB-P3 with temperature. (D) Shift of fluorescence emission wavelength of HB-P7_p and HB-P7 with temperature.

Fig. 5

Micro-DSC curves for HB-PNIPAM in absence (solid lines) and presence (dashed lines) of D-Ala-D-Ala peptide in PBS. Linear polymer (L-P1) (black), HB-P3 (red), HB-P6 (green) and HB-P7 (blue).

Fig. 6

Relative fluorescence integrated intensity of (A) HB-P3 and (B) L-P1 mixed with Gram-positive and negative colonies of bacteria (♦ S. aureus, ♦ S. epidermis and ♦ E. coli), measured 5 min after mixing at 35 °C. Error bars are standard error of the mean. (C) to (F); Micrographs stained using live/dead stain®. (C) shows live (green) S, aureus and (E) shows the bacteria after being killed by exposure to isopropanol. (D) is an example of a micrograph of live bacteria after aggregation with HB-P3 and (F) shows the result of adding HB-P3 to dead bacteria. The data show that there was little or no aggregation when HB-P3 was exposed to dead bacteria but extensive aggregation with live bacteria.

Fig. 7

(A) Relative Fluorescence Integrated Intensity and (B) Wavelength shift of PNIPAM-van polymers (L-P1, HB-P3, HB-P5, HB-P6, HB-P7) exposed to increasing amounts of S. aureus relative to amount of polymer, measured 5 min post mixing at 35 °C. Error bars are standard error of the mean.