Europium Ion-Based Magnetic-Trapping and Fluorescence-Sensing Method for Detection of Pathogenic Bacteria

Europium ions (Eu3+) have been utilized as a fluorescence-sensing probe for a variety of analytes, including tetracycline (TC). When Eu3+ is chelated with TC, its fluorescence can be greatly enhanced. Moreover, Eu3+ possesses 6 unpaired electrons in its f orbital, which makes it paramagnetic. Being a hard acid, Eu3+ can chelate with hard bases, such as oxygen-containing functional groups (e.g., phosphates and carboxylates), present on the cell surface of pathogenic bacteria. Due to these properties, in this study, Eu3+ was explored as a magnetic-trapping and sensing probe against pathogenic bacteria present in complex samples. Eu3+ was used as a magnetic probe to trap bacteria such as Staphylococcus aureus, Escherichia coli, Enterococcus faecalis, Acinetobacter baumannii, Bacillus cereus, and Pseudomonas aeruginosa. The addition of TC facilitated the easy detection of magnetic Eu3+–bacterium conjugates through fluorescence spectroscopy, with a detection limit of approximately ∼104 CFU mL–1. Additionally, matrix-assisted laser desorption/ionization mass spectrometry was employed to differentiate bacteria tapped by our magnetic probes.


Figure S1 .
Figure S1.Examination of the magnetic Eu 3+ -bacterium conjugates.Photographs of the (A) blank sample (0.39 mL) containing Tris buffer at pH 8 only and the bacterial samples (0.39 mL, 0.2 mg mL -1 ) including (B) P. aeruginosa, (C) E. faecalis, (D) B. cereus, (E) E. coli J96, and (F) A. baumannii prepared in Tris buffer (pH 8) obtained with the addition of Eu 3+ (75 mM, 10 μL) followed by microwave-heating (power: 180 W) for 2.25 min and magnetic isolation by placing an external neodymium magnet (~4000 G).The photographs were taken under room light.The red oval indicates the location of the magnetic Eu 3+ -bacterium conjugates.

Figure S3 .
Figure S3.Examination of the optimal pH for enhancing fluorescence intensity derived from Eu 3+ by TC.Representative fluorescence spectra (ex= 394 nm) of the samples containing Eu 3+ (2 mM) obtained without (black) and with (blue) adding TC (13 µM) at pH (A) 5, (B) 6, (C) 7, (D) 8, (E) 8.5, and (F) 9 after vortex-mixing for 1 h.Three replicates were conducted.(G) Bar graphs of the summarized results derived from Panels (A)-(F).Ii and I0 stand for the fluorescence intensity at 616 nm of the black and blue spectra, respectively.

Figure S4 .
Figure S4.Optimization of the concentration of the added bleach.Fluorescence spectra (ex= 394 nm) of the samples containing S. aureus (0.02 mg mL -1 , 0.39 mL) obtained by using Eu 3+ (75 mM, 10 μL) as the trapping probe followed by magnetic isolation and the addition of bleach with different concentrations under microwave-heating (power: 180 W, 1.75 min) and added with TC (30 μM, 30 μL) incubated under microwave-heating (power: 180 W, 1.75 min).

Figure S6 .
Figure S6.Examination of different model bacteria.Fluorescence spectra of the bacterial samples including (A) S. aureus, (B) P. aeruginosa, (C) E. faecalis, (D) B. cereus,(E) E. coli J96, (F) A. baumannii, and (G) blank obtained before (blue) and after (black) bleach treatment and the addition of TC.The concentrations of all the bacteria in the samples were 0.2 mg mL - 1 .All the samples (0.39 mL) were added with Eu 3+ (75 mM, 10 μL) followed by microwaveheating (power: 180 W) for 2.25 min and magnetic isolation by placing an external neodymium magnet (~4000 G) followed by rinse steps.Bleach (0.25%, 10 μL) was added to the individual samples with the final concentration of 6  10 -3 % and incubated under microwave-heating (power: 180 W) for 1.75 min.TC (30 μM, 30 μL) was added to the individual samples and incubated under microwave-heating (power: 180 W) for 1.75 min in the water bath of 2 mL.

Figure S7 .
Figure S7.Examination of the optimal binding pH of Eu 3+ toward S. aureus.Representative fluorescence spectra (ex= 394 nm) of the supernatants derived from the samples (0.39 mL) containing Eu 3+ (10 mM) obtained before and after incubated with S. aureus (0.2 mg mL -1 ) at pH (A) 5, (B) 6, (C) 7, (D) 8, and (E) 9 followed by centrifugation (3000 rpm, 5 min).Three replicates were conducted for different pH conditions.(F) Bar graphs of the summarized results of Panels (A)-(E) obtained from different pH values (n= 3) in terms of binding capacity of Eu 3+ onto S. aureus.Ii and I0 stand for the fluorescence intensity at 616 nm of the black and blue spectra, respectively.

Figure S10 .
Figure S10.Calibration curve obtained by plotting the fluorescence intensity at the wavelength of 616 nm of the fluorescence spectra shown in Figure S9 versus the concentration of S. aureus.

Figure S12 .
Figure S12.(A) Examination of precision and accuracy.(B) Fluorescence spectra of the sample containing S. aureus (150 ng mL -1 ) obtained after treated by our developed method for 30 runs (6 runs per day for 5 days).

Table S1 .
Conversion of OD600 of 1 with the concentration and CFU mL -1 (n= 4)

Table S2 .
Evaluation of the accuracy and the precision of the developed method.

Table S3 .
List of comparisons between existing methods and our work.