Towards Multiplexed Bacteria Detection by Enzyme Responsive Hydrogels

Abstract

In this work, patterned β‐GUS sensing chitosan hydrogels functionalized with three different colorimetric substrates were fabricated for the multiplexed detection of the enzyme β‐glucuronidase (β‐GUS), which is secreted by >98% of all known Escherichia coli (E. coli) strains. The immobilization of fluorogenic and chromogenic substrates in specified areas allows a spatially resolved readout of the corresponding colorimetric signal. The apparent initial rate of the β‐GUS induced cleavage of the reporter moieties of chitosan films functionalized with the chromogenic substrate 4‐nitrophenyl‐β‐D‐glucuronide (PNPG), the chromogenic substrate 5‐bromo‐4‐chloro‐3‐indolyl‐β‐D‐glucuronide (X‐Gluc) and the fluorogenic substrate 4‐methylumbelliferyl‐β‐D‐glucuronide (MUG) were analyzed spectroscopically and confirmed a detectable reaction within less than 60 min. Likewise the released dyes were observed in the patterns owing to different colors by naked eye detection under appropriate illumination in less than 80 min. Hence the presence of a characteristic enzyme secreted by E. coli bacteria was successfully detected by three independent sensing moieties, which is important to reduce false positives by introducing redundancy. Patterned enzyme sensing chitosan hydrogels, which are functionalized with different substrates, open the possibility for multiplexed bacteria detection and in the long run also the identification of different bacteria strains.

Quantitative E. coli Enzyme Detection in Reporter Hydrogel-Coated Paper Using a Smartphone Camera

“…The previously reported chitosan sensing hydrogel approach was first adapted to a paper substrate format [ 16 ]. The modification of filter paper with chitosan as well as the subsequent functionalization with reporter moieties was thoroughly characterized with surface analytical methods and field-emission scanning electron microscopy (FESEM).…”

“…Optical biosensors that rely on the detection of absorbed ultraviolet–visible (UV–Vis) light [ 14 , 15 , 16 , 17 , 24 , 39 , 40 , 41 , 42 ] possess usually lower sensitivity than those based on fluorescence [ 13 , 16 , 18 , 19 ]. This is also true for UV–Vis detection of indigo derivatives [ 14 , 17 , 39 ].…”

“…Chitosan-g-X-Gluc paper substrate was prepared using EDC/NHS chemistry according to the reference [ 16 ]. Briefly, 4.5 mM of X-Gluc was prepared in PBS (pH 7.4), followed by addition of EDC (6.7 mol/mol of X-Gluc) and NHS (6.7 mol/mol of X-Gluc).…”

“…Hence it is not surprising that a multitude of approaches with novel sensing materials has been reported in recent years. These approaches rely on, among other things, sensing nanoparticles [ 9 , 10 ], nanocapsules [ 11 , 12 ] and hydrogels [ 6 , 13 , 14 , 15 , 16 , 17 , 18 ]. The adaptation of such bacteria detection approaches with demonstrated feasibility to POC-compatible formats for application in remote settings without controlled temperature and reliable electricity is another challenge.…”

“…Hence a promising strategy is the combination of feasible bacteria detection approaches with a paper-based carrier system that was investigated here for the E. coli marker enzyme β-glucuronidase (ß-Gus) and the non-pathogenic E. coli Mach1 lab strain as a model organism ( Figure 1 ) with smartphone-based detection, readout and quantitative data analysis. Previously, chitosan hydrogels equipped with different fluorogenic or chromogenic substrates were shown to rapidly, selectively and sensitively detect and differentiate among different strains of bacteria with LOD for the enzymes in several cases <1 nM after 1 h observation time [ 16 , 29 ]. As we report here, chitosan-based autonomously self-reporting hydrogels were successfully adapted to this paper carrier-based format to detect and quantify concentrations of bacterial enzymes.…”