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

Abstract

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.

Keywords: Bacteria detection; Bacterial enzyme; Colorimetric substrates; Multiplexed biosensors; Reporter hydrogels.

Graphical abstract

Fig. 1

Schematic of color-encoded sensor hydrogel for multiplexed bacterial enzyme differentiation and bacteria detection with the listed bacterial strains. a) Chemical structures of colorimetric substrates and hydrogel matrices as well as chemical modification of hydrogel in circular area with colorimetric substrates via N-(3-dimethylaminopropyl)-N-ethylcarbodiimide (EDC)/N-hydroxy succinimide (NHS) chemistry. b) The release of the dyes, which are characterized by different colors, is caused by cleavage reactions catalyzed by the corresponding target enzymes secreted from S. aureus RN4220, MRSA N315, EHEC E32511 and E. coli DH5α, respectively. c), d) and e) Schematic of release of specific dyes that occurs exclusively in presence of the target enzyme.

Fig. 2

a) Fluorescence spectra (measured in a fluorescence spectrometer) of 4-MU released during the enzymatic reaction in α-glucosidase sensing hydrogel (MUD-g-NSC) on silicon. ([MUD]_mod = 2.5 mM; [α-Glucosidase] = 0.2 μM, λ_ex = 325 nm, measurement repeat interval: 3 min). b) Plot of I_F at λ_max = 376 nm of MUD and at λ_max = 450 nm of 4-MU in panel a) versus time. c) UV–vis spectra (microplate reader) of dimerized indigo during the enzymatic reaction in the β-galactosidase sensing hydrogels in a transparent 96-well plate ([X-Gal]_mod = 2.5 mM, [β-Galactosidase] = 0.2 μM, measurement repeat interval: 21 min). d) Plot of absorbance at λ_max = 615 nm of dimerized indigo in panel c) versus time. e) UV–vis spectra (microplate reader) of released 4-NP during the enzymatic reaction in the β-glucuronidase sensing hydrogels in a transparent 96-well plate ([PNPG]_mod = 10 mM; [β-Glucuronidase] = 0.2 μM, measurement repeat interval: 12.5 min). f) Plot of absorbance at λ_max = 400 nm of released 4-NP in panel e) versus time. The arrows indicate the temporal changes in the spectra.

Fig. 3

Plots of the LOD versus time estimated for a) α-glucosidase in α-glucosidase sensing hydrogel, b) β-galactosidase in β-galactosidase-sensing hydrogel, and c) β-glucuronidase in β-glucuronidase-sensing hydrogel (microplate reader, 96-well plate, [MUD]_mod = 2.5 mM, [X-Gal]_mod = 2.5 mM, [PNPG]_mod = 10 mM, 25 °C). The data were fitted with: a) LOD _{(α-glucosidase)} = 11.9 nM min t⁻¹; b) LOD _{(β-galactosidase)} = 272.7 nM min t⁻¹; c) LOD _{(β-glucuronidase)} = 205.9 nM min t⁻¹ (error bars: standard deviation, n = 3).

Fig. 4

Photograph of the color-encoded enzyme sensing hydrogels after enzymatic reactions. The photo was taken after a reaction time of 110 min at 25 °C, under UV illumination (hand-held lamp, ${\lambda }_{ex}$ = 365 nm, left) on a black background as well as white light (right) on a white background. Circularly patterned chitosan was modified with MUD, X-Gal or PNPG substrates with the help of PDMS mask. ([MUD]_mod = 2.5 mM, [X-Gal] _mod = 2.5 mM, [PNPG] _mod = 10 mM). PBS was added in the 1st row. A mixed enzyme solution (final concentration of each enzyme is corresponding to 0.2 μM) was applied in each pattern in the 2nd row. Individual solutions of α-Glucosidase, β-Galactosidase and β-Glucuronidase (all 0.2 μM) were added in the 3rd to 5th row, respectively (the length and thickness of the square PDMS mask are about 16 mm and 2.8 mm, respectively; 70 μL of the enzyme solution was applied into each circular area).

Fig. 5

Detection of enzymes in bacterial suspensions with enzyme sensing hydrogels in a microplate reader format at 25 °C (1st row: detection of S. aureus RN4220 (~8 × 10⁹ CFU/mL) and MRSA N315 (~2 × 10⁹ CFU/mL), 2nd and 3rd rows: detection of EHEC E32511 (~6 × 10⁸ CFU/mL), and E. coli DH5α (~3 × 10⁹ CFU/mL), respectively. 1st, 2nd and 3rd columns: Measurements with α-glucosidase, β-galactosidase, and β-glucuronidase sensing hydrogels, respectively): a) I_F at maximum emission λ_max = 450 nm versus reaction time of α-glucosidase sensing hydrogel incubated with suspensions of S. aureus RN4220 and MRSA N315 (biological triplicates). b) and c) Calibrated UV–vis spectra of released indigo and 4-NP in β-galactosidase and β-glucuronidase sensing hydrogels, respectively, incubated with suspension of S. aureus RN4220 and MRSA N315; (spectra were corrected according to the spectra of the corresponding dye in Fig. 2) (biological duplicates). d) Fluorescence spectra of the neat cultured EHEC E32511 suspension (i.e. using a hydrogel free well) as well as of the released 4-MU in a α-glucosidase sensing hydrogel incubated with EHEC E32511 (technical replicates). e) Absorbance at λ = 615 nm versus time for the reaction of the β-galactosidase sensing hydrogel in EHEC E32511 as well as the growth curve of pure cultured bacterial suspension in the absence of hydrogel. Kinetics measurement repeat interval: 1.5 min. Insets of e) UV–vis spectra of pure EHEC E32511 suspension and released indigo after 20 h of enzymatic reaction of the hydrogel in the EHEC E32511 (biological duplicates). f) UV–vis spectrum of released 4-NP in β-glucuronidase sensing hydrogel in EHEC E32511 suspension (technical replicates). g) I_F at λ_max = 450 nm versus reaction time of α-glucosidase sensing hydrogel incubated with E. coli DH5α (biological triplicates). h) UV–vis spectrum of released indigo in β-galactosidase sensing hydrogel in E. coli DH5α (biological duplicates). i) Absorbance at λ_max = 400 nm versus time for the reaction of hydrogel in E. coli DH5α. Inset of i) Absorbance at wavelength λ = 600 nm versus time for the reaction of β-glucuronidase sensing hydrogel in E. coli DH5α. Kinetics measurement repeat interval: 10 min (biological triplicates). All spectra in b, c, d, f, h) were acquired after 24 h reaction of the hydrogel in bacterial suspension. Baseline subtraction: Kinetics measurements in a, g, i) I_F or absorbance of corresponding bacteria suspension cultured in sensing hydrogel-free wells; Kinetics measurements in e) Absorbance or UV–Vis spectra of LB for hydrogel free wells as well as absorbance or UV–Vis spectra of sensor hydrogels incubated in LB; Spectra measurement in f, h) UV–vis spectra of corresponding bacteria suspension cultured in sensing hydrogel free wells; spectra measurement in d) and insets of e) Fluorescence/UV-spectra of LB in sensing hydrogel filled well or sensing hydrogel free well for the curve of bacterial suspensions in hydrogel as well as bacterial suspensions in hydrogel free well, respectively. (100 μL of LB or bacterial suspensions, for a, d, g) [MUD]_mod = 2.5 mM, λ_ex = 365 nm b, e, h) [X-Gal]_mod = 2.5 mM c, f, i) [PNPG]_mod = 10 mM). The vertical arrows in a, g, i) point out the time that was required to be able to detect the released dye at the determined LOD by bare eye. Hydrogel free well means the well contained only bacteria suspension.