Selective Discrimination of Key Enzymes of Pathogenic and Nonpathogenic Bacteria on Autonomously Reporting Shape-Encoded Hydrogel Patterns

Abstract

This work reports on a new approach to rapidly and selectively detect and discriminate enzymes of pathogenic from those of nonpathogenic bacteria using a patterned autonomously reporting hydrogel on a transparent support, in which the selectivity has been encoded by the pattern shape to enable facile detection by a color change at one single wavelength. In particular, enzyme-responsive chitosan hydrogel layers that report the presence of the enzymes β-glucuronidase (β-Gus) and β-galactosidase (β-Gal), produced by the nonvirulent Escherichia coli K12 and the food-borne biosafety level 3 pathogen enterohemorrhagic E. coli, respectively, via the blue color of an indigo dye were patterned by two complementary strategies. The comparison of the functionalization of patterned chitosan patches on a solid support with two chromogenic substrates on one hand and the area-selective conjugation of the substrates on the other hand showed that the two characteristic enzymes could indeed be rapidly and selectively discriminated. The limits of detection of the highly stable sensing layers for an observation time of 60 min using a spectrophotometer correspond to enzyme concentrations of β-Gus and β-Gal of ≤5 and ≤3 nM, respectively, and to ≤62 and ≤33 nM for bare eye detection in nonoptimized sensor patches. These results confirm the applicability of this approach, which is compatible with the simple measurement of optical density at one single wavelength only as well as with parallel, multiplexed detection, to differentiate the enzymes secreted by a highly pathogenic E. coli from a nonpathogenic E. coli on the basis of specifically secreted enzymes. Hence, a general approach for the rapid and selective detection of enzymes of different bacterial species for potential applications in food safety as well as point-of-care microbiological diagnostics is described.

Multiplexed detection and differentiation of bacterial enzymes and bacteria by color-encoded sensor hydrogels

“…In comparison to earlier studies, which utilized more sensitive fluorescence or UV–vis spectrometers, the LOD values of α-glucosidase and β-glucuronidase sensing hydrogel reported here, are improved by a factor of 100 [ 49 ] and 3 times [ 47 ], respectively. The LOD of β-galactosidase sensing hydrogel obtained here is similar to the one reported in the literature (≤3 nM) [ 50 ]. The details are shown in Table S1 .…”

“…Additionally, by recording the kinetics of the enzymatic reaction in the hydrogel with various concentrations of β-galactosidase ( Figure S4a ), it was found that the initial apparent reaction rate (for t ≤ 20 min) depended linearly on the initial enzyme concentration ( Figure S4b ). The apparent rate constant was equal to 0.033 (±0.003) min −1 μM −1 here, which is 10-fold larger than in our previous work [ 50 ]. This difference is likely due to the increased loading with X-Gal substrate in the hydrogel, as well as enhanced hydrogel swelling.…”

“…This approach is based on the reaction of enzymes, produced and secreted by the target bacteria, with reporter units coupled to the chitosan films [ 47 , 48 ]. Furthermore, the concept was expanded to the detection and discrimination of various bacterial enzymes and bacteria exploiting various colors afforded by different substrates [ 49 ] or differently shaped patterns exhibiting the same [ 50 ] or different [ 51 ] colors.…”

Hydrogels and Their Role in Biosensing Applications

“…This review aims to investigate, how hydrogels contribute to improving these parameters. [23] (PEG methacrylate) Dye, protein, Oligonucleotide Fluorescence Covalent, Diels-Alder cycloaddition [50,51] Chitosan Enzymes Chromogenic (indigo) Covalent, amide bond [54] Chitosan, dextran Glucose Electrochemical Electrostatic [76] Responsive Hydrogels Acrylic acid and dimethylaminoethyl methacrylate Urea Piezoresistive pressure sensor Encapsulation [90] Shape-memory DNA film pH, Ag + /cysteine Photonic crystal Hybridization [93] Poly(methyl methacrylate-co-methacrylic acid)…”

Effective detection of biocatalysts with specified activity by using a hydrogel-based colourimetric assay – β-galactosidase case study

“…This solution is applicable in medicine in the construction of so-called ‘diagnostic dressings’ designed to identify bacterial infections that occur in the wound site. In this case the mechanism is based on monitoring virulence factors, specifically the enzymes characteristic of given bacterial strains [ 26 , 28 ]. To the best of the author's knowledge, the use of hydrogel matrices with immobilized substrates for the detection of enzymes with a specified catalytic activity in aqueous solution is a novel scientific issue that has not yet been reported in the available literature.…”