“…With increasing reaction time, the color became more intense. Qualitatively similar observation in E. coli suspension indicates the presence of the enzyme also in the LB medium ( Figure 4 ), in agreement with previously published work [ 29 ]. The quantitative analysis of the photographs and the enzymatic reaction kinetics is key for an application of related sensors in the future.…”
Section: Discussionsupporting
confidence: 92%
“…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.…”
There is a growing demand for rapid and sensitive detection approaches for pathogenic bacteria that can be applied by non-specialists in non-laboratory field settings. Here, the detection of the typical E. coli enzyme β-glucuronidase using a chitosan-based sensing hydrogel-coated paper sensor and the detailed analysis of the reaction kinetics, as detected by a smartphone camera, is reported. The chromogenic reporter unit affords an intense blue color in a two-step reaction, which was analyzed using a modified Michaelis–Menten approach. This generalizable approach can be used to determine the limit of detection and comprises an invaluable tool to characterize the performance of lab-in-a-phone type approaches. For the particular system analyzed, the ratio of reaction rate and equilibrium constants of the enzyme–substrate complex are 0.3 and 0.9 pM−1h−1 for β-glucuronidase in phosphate buffered saline and lysogeny broth, respectively. The minimal degree of substrate conversion for detection of the indigo pigment formed during the reaction is 0.15, while the minimal time required for detection in this particular system is ~2 h at an enzyme concentration of 100 nM. Therefore, this approach is applicable for quantitative lab-in-a-phone based point of care detection systems that are based on enzymatic substrate conversion via bacterial enzymes.
“…With increasing reaction time, the color became more intense. Qualitatively similar observation in E. coli suspension indicates the presence of the enzyme also in the LB medium ( Figure 4 ), in agreement with previously published work [ 29 ]. The quantitative analysis of the photographs and the enzymatic reaction kinetics is key for an application of related sensors in the future.…”
Section: Discussionsupporting
confidence: 92%
“…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.…”
There is a growing demand for rapid and sensitive detection approaches for pathogenic bacteria that can be applied by non-specialists in non-laboratory field settings. Here, the detection of the typical E. coli enzyme β-glucuronidase using a chitosan-based sensing hydrogel-coated paper sensor and the detailed analysis of the reaction kinetics, as detected by a smartphone camera, is reported. The chromogenic reporter unit affords an intense blue color in a two-step reaction, which was analyzed using a modified Michaelis–Menten approach. This generalizable approach can be used to determine the limit of detection and comprises an invaluable tool to characterize the performance of lab-in-a-phone type approaches. For the particular system analyzed, the ratio of reaction rate and equilibrium constants of the enzyme–substrate complex are 0.3 and 0.9 pM−1h−1 for β-glucuronidase in phosphate buffered saline and lysogeny broth, respectively. The minimal degree of substrate conversion for detection of the indigo pigment formed during the reaction is 0.15, while the minimal time required for detection in this particular system is ~2 h at an enzyme concentration of 100 nM. Therefore, this approach is applicable for quantitative lab-in-a-phone based point of care detection systems that are based on enzymatic substrate conversion via bacterial enzymes.
“…The details are shown in Table S1 . Additionally, the values for the LOD for the detection of the enzymes at an observation time of 60 min are much lower than the enzyme concentrations in overnight cultured bacteria suspension, which are about 7–85 μM of α-glucosidase in S. aureus [ 49 ], 60 nM of β-glucuronidase in E. coli [ 61 ], and about 7 nM of β-galactosidase in EHEC [ 67 ]. Fig.…”
Section: Resultsmentioning
confidence: 99%
“…certain EHEC and non EHEC E . coli strains, which was previously shown with an autonomously reporting chitosan hydrogel film sensor [ 67 ].…”
We report on the fabrication and characterization of color-encoded chitosan hydrogels for the rapid, sensitive and specific detection of bacterial enzymes as well as the selective detection of a set of tested bacteria through characteristic enzyme reactions. These patterned sensor hydrogels are functionalized with three different colorimetric enzyme substrates affording the multiplexed detection and differentiation of α-glucosidase, β-galactosidase and β-glucuronidase. The limits of detection of the hydrogels for an observation time of 60 min using a conventional microplate reader correspond to concentrations of 0.2, 3.4 and 4.5 nM of these enzymes, respectively. Based on their different enzyme expression patterns,
Staphylococcus aureus
strain RN4220, methicillin-resistant
S
.
aureus
(MRSA) strain N315, both producing α-glucosidase, but not β-glucuronidase and β-galactosidase,
Escherichia coli
strain DH5α, producing β-glucuronidase and α-glucosidase, but not β-galactosidase, and the enterohemorrhagic
E
.
coli
(EHEC) strain E32511, producing β-galactosidase, but none of the other two enzymes, can be reliably and rapidly distinguished from each other. These results confirm the applicability of enzyme sensing hydrogels for the detection and discrimination of specific enzymes to facilitate differentiation of bacterial strains. Patterned hydrogels thus possess the potential to be further refined as detection units of a multiplexed format to identify certain bacteria for future application in point-of-care microbiological diagnostics in food safety and medical settings.
“…In addition, it was shown that this approach can be expanded to application in selective assays, e.g. to differentiate Staphylococcus aureus from Pseudomonas aeruginosa (with clear medical relevance) or to differentiate the S‐1 level E. coli ( K12 ) from the S‐3 level pathogenic E. coli EHEC (with implications for food safety) …”
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.
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