General concept of proposed protocol for intracellular ROS detection

The procedure used in this work to detect intracellular ROS by fluorescent probes is based on the general mechanism of recognized ROS-probes23, and more specifically of DCFH-DA. In the present study three novel achievements will be developed and demonstrated to permit detecting significant intracellular ROS levels in solar irradiated bacteria in water. These are: (i) lab-hydrolysis of DCFH-DA prior to bacterial incubation, instead of natural hydrolysis by esterases in bacteria cells, which will guarantee perfect and faster interaction between DCFH and intracellular ROS; (ii) determination of optimal DCFH-DA concentration for a significant and reliable measurement of fluorescent signal for E. coli in water, and (iii) optimization of incubation time for the reaction of DCFH and intracellular E. coli-ROS to obtain time-stable response of fluorescent signal (flow cytometry and fluorescent microcopy).

Photo-chemical stability of DCFH-DA

Previous DCFH-DA protocols proposed an incubation period with cells23, then they are exposed to the oxidative stress under assessment. In the case of bacteria exposed to solar radiation, the oxidative stress is produced by continuous income of solar UV photons over bacteria. Initially, exposure of DCFH-DA to solar radiation was considered, and therefore its photo-chemical stability was tested.

Absorbance spectrum of irradiated DCFH-DA (without bacteria) was measured at different times of solar exposure, from 0 to 90 min (Fig. 2). The absorbance increase in the range of 400–500 nm, suggested the formation of photo-transformation compounds. Similar observation was published by Chignell and Sik27, they measured an increase of DCF fluorescence signal in cell-free samples containing DCF, DCFH and horseradish peroxidase when irradiated by UV-A. This can be attributed to the photo-reduction of DCF, involving (DCF)* and DFC•− generation1,3. The formation of these photo-products could also alter the ROS levels due to the high reactivity of DFC•− with oxygen, generating O 2 •− and consequently H 2 O 2 . Hence, given the photo-transformation of DCFH-DA under sunlight, the addition of DCFH-DA to E. coli should be done only after solar exposure of bacteria in water. Although some researchers used the probe prior to cellular oxidative stress28,29, alternatively the probe might be used after if ROS generation is an UV-photo induced process, as reported for cyanobacterium Anabaena sp. under UV-B30,31.

Figure 2 DCFH-DA photo-degradation. Absorbance spectrum of a 50 μM DCFH-DA solution at 0, 15, 30, 45, 60 and 90 min of solar radiation (30 W m−2 UV-A). Full size image

Hydrolysis of DCFH-DA

When bacterial cells are loaded with DCFH-DA, diacetate groups are hydrolyzed by endogenous esterase to generate DCFH. However, under irradiation, it is expected that many intracellular molecules are modified or even inactivated1,32,33. It is also assumed that esterase may be altered by solar radiation, and therefore it may be working with lower efficiency than in unaltered cells. Therefore, solar irradiated cells could be affected at esterase level, so that proper ROS detection by DCFH-DA is not optimal. To avoid this, an alternative chemical hydrolysis procedure is proposed to load the cells with DCFH instead of DCFH-DA. The advantage of pre-hydrolysis is not only to avoid potential esterases degradation, but also to permit ROS reaction with hydrolyzed DCFH-DA immediately after added to bacteria, avoiding longer periods of intracellular reactions.

Chemical hydrolysis of DCFH-DA is induced to remove diacetate groups from the molecule. Hydrolysis of DCFH-DA was done following the protocol described elsewhere34. Briefly, 0.01 N of NaOH was added to 50 μM of DCFH-DA and incubated 30 minutes in dark. Then, the probe was neutralized with PBS 0.1 M to obtain the hydrolyzed form, DCFH.

DCFH-H 2 O 2 reaction: concentration dependence

Reaction of DCFH with ROS to produce DCF was measured by absorbance and fluorescence signals. In this case, H 2 O 2 was used as a positive control of ROS. H 2 O 2 was chosen due to its capability to react with DCFH and to penetrate easily into bacteria cells35. Furthermore, H 2 O 2 is commonly accepted as one of the ROS that are generated inside bacteria under light stress36. Reaction between 10 μM DCFH and H 2 O 2 at different concentrations (0.01 to 1 mM) was done in the absence of bacteria in dark. These concentrations were selected to ensure good measurement of absorbance and fluorescent signals in the spectrophotometer and fluorimeter respectively, and to check if the hydrolyzed probe was reactive to H 2 O 2 . Further experimental work will optimize concentrations of the probe to measure ROS at intracellular levels in E. coli. Figure 3 shows that DCF formation is H 2 O 2 -concentration dependent; the higher H 2 O 2 concentration the higher DCF concentration was observed for both, absorbance and fluorescence peak values. This confirms that hydrolyzed DCFH by NaOH can be used as a good indicator of H 2 O 2 .

Figure 3 DCF concentration is dependent on H 2 O 2 concentration. (a) Absorbance spectrum and (b) fluorescence emission signal (excitation: 497 nm) of the formed DCF product from the reaction between DCFH (10 μM) and several H 2 O 2 concentrations. Full size image

In order to determine the saturation concentration of DCFH for a certain amount of H 2 O 2 , and to avoid overload of DCFH which may give undesired fluorescence background signal, several reactions of DCFH and H 2 O 2 , at variable concentrations of both, were carried out in absence of bacteria and measured with the fluorimeter. Concentrations of DCFH were 1, 5, 10, 15, and 20 μM with different concentrations of H 2 O 2 , 0.01, 0.1, 0.2, 0.3, 0.5 and 1 mM. At view of first results, 1, 15 and 20 μM-DCFH were discarded, as the signal was too low for 1 μM, while it was unstable at 15 and 20 μM, probably caused by the effect of ambient light and oxygen. Two replicates of reactions at 5 and 10 μM of DCFH with all concentrations of H 2 O 2 were done, and resulted highly reproducible (confidence level > 99%). Average values are shown in Fig. 4.

Figure 4 Effect of DCFH concentration in fluorescence measurements. Maximum fluorescence emission signal (excitation/emission: 497/520 nm) of DCF product formed from the reaction between 5 and 10 μM (chemically hydrolyzed) DCFH with H 2 O 2 (0.01, 0.1, 0.2, 0.3, 0.5, and 1 mM). Full size image

At low H 2 O 2 concentrations, the fluorescence signal for both, 5 and 10 μM of DCFH, led to very similar values, while at higher H 2 O 2 concentrations (>0.3 mM) 10 μM of DCFH showed higher values than for 5 μM, being 10 μM more precise to detect differences in fluorescence measurements. Due to the stability of the measurements at all H 2 O 2 concentrations tested, both DCFH concentrations (5 and 10 μM) could be a good option to determine the internal ROS in E. coli. In this work, 10 μM of DCFH was selected for further experiments to detect fluorescence signal caused by intracellular generated ROS in solar exposed E. coli. Although E. coli intracellular ROS values are expected to be very low as compared to this previous calibration; for instance 0.01 μM H 2 O 2 intracellular concentration was reported37, it is more accurate to work in excess of DCFH by the following reasons, (i) DCFH is expected to react with not only H 2 O 2 but with all ROS generated after the stress-generating process, and (ii) the addition of the DCFH is done to samples with a high bacterial concentration 106 CFU mL−1 and not for a single cell. Measurements of DCFH in bacterial suspensions will be done with the following protocol, which was also validated for flow cytometry, as explained below.

DCFH - E. coli incubation

According to the literature, cells incubation time at 37 °C in presence of DCFH-DA should range between 30 and 60 minutes, to permit the diffusion of probes inside cells and the hydrolysis by endogenous esterase38,39. Nevertheless, these incubation times for samples containing solar irradiated E. coli may lead to modifications of physiological state of the cells, including ROS levels, due to metabolic activities during the incubation period. Therefore, a large incubation period could lead to false-positive or higher signal detection by an increment of ROS concentration that doesn’t represent the real state of the cells under evaluation. For this reason, this work is proposing the use of hydrolyzed DCFH-DA to DCFH as a tool that will also permit decreasing incubation times with bacteria, as esterase internal step is skipped.

With the aim of determine if shorter periods are adequate for incubation of DCFH and E. coli, preliminary experiments were done using unaltered E. coli suspensions and a fluorescence microscope, for direct counting of number of fluorescent bacteria. This will also give an idea of the baseline fluorescence signal of non-altered bacteria and its stability over time. The incubation experiments were done as follows. 10 μM of DCFH and 109 CFU mL−1 E. coli were incubated at 37 °C for different times, 5, 15, 30, and 60 min, in dark. The fluorescent bacteria were directly observed and numbered using a counting chamber of 25 μL of samples. After each observation the total amount of fluorescent bacteria per sample was determined. Figure 5(a) shows a photograph of the fluorescence cells after 15 min incubation. Cell counting was done immediately after the tested incubation times, additionally each sample was re-numbered at different post-incubation times to confirm the stability of the measurement (Fig. 5). The first count result (time 0 in Fig. 5(b)) for all incubation times (5, 15, 30, and 60 min) revealed the same concentration of fluorescent bacteria, 109 CFU mL−1, which was the expected initial concentration of alive bacteria in the suspension. These results confirm that diffusion of DCFH inside bacteria and further reaction with intracellular ROS is very fast, for 5 and 15 min incubation, and it doesn’t change over time (up to 60 min incubation). Nevertheless, longer incubation periods, 30 and 60 min, generated an increment in the number of counted florescent cells over post incubation time. This confirms the hypothesis that longer incubation times could lead to modifications in cells states and in intracellular ROS levels. Therefore, an incubation time between 5 and 15 min was found ideal for this protocol, because it gives a stable measurement over time of unmodified ROS. We selected 15 min incubation, which avoids long incubation periods and guarantee stable fluorescent measurement by successful DCFH diffusion and ROS-reaction inside cells.

Figure 5 Incubation time of E. coli cells loaded with DCFH. (a) 40x fluorescence microscope photograph of 109 CFU mL−1 E. coli sample loaded with 10 μM DCFH an incubated 15 minutes at 37 °C in dark; (b) Fluorescent E. coli cells counts with 10 μM DCFH an incubated for 5, 15, 30, and 60 min over a post incubation period (0–60 min). Full size image

Validation of DCFH protocol using flow cytometry (H 2 O 2 as positive control)

The proposed modified protocol is summarized in Fig. 6. Its validation was done by measuring the fluorescence intensity of bacteria exposed to oxidative stress of added H 2 O 2 using a flow cytometer. Bacterial suspension cells (108 CFU mL−1) were exposed for 10 minutes in dark to H 2 O 2 (0, 0.1, 0.5, 0.75, 1, and 1.5 mM). Then samples were taken and loaded with 10 μM DCFH, incubated according to the above protocol and measured in the flow cytometer. Figure 7 shows the FITC-A average value of 50 000 cells reaching detector. The fluorescence intensity increased linearly with added H 2 O 2 . This result shows that the whole protocol works without any limitation in the flow cytometer, as well as H 2 O 2 diffusion into cells was correctly happening. The calibration curve has a good linear response in the range of tested concentrations. In addition, the high resolution of the equipment is clear, as FITC-A values were lower than 100.

Figure 6 General scheme of proposed protocol for ROS detection in E. coli cells using DCFH-DA previously hydrolyzed as ROS-probe. Briefly, chemical hydrolysis of DCFH-DA to obtain DCFH, addition of DCFH to bacterial suspension, incubation period and fluorescence detection by flow cytometry. Full size image

Figure 7 Validation of the internal ROS detection protocol using H 2 O 2 as positive control. Fluorescent signal (excitation/emission: 488/530 nm) of E. coli-10 μM DCFH exposed to different concentrations of H 2 O 2 by flow cytometry. Full size image

Bacterial intracellular ROS formation during solar exposure

During solar water disinfection experiments (four replicates), samples were taken every 30 min over 4 hours. Each sample was evaluated for viable E. coli counts and ROS determination using the proposed DCFH protocol (Fig. 8). E. coli concentration as a function of solar exposure time showed the typical SODIS inactivation curve, where a small shoulder for the first 30 min followed by a close to linear decay until reaching the detection limit (not counted colonies) at 180 min. Water temperature varied from 20 to 27 °C along the experiment; solar UV-A irradiance increased from 12.7 W m−2 (t = 0 min) to 36.1 W m−2 (t = 155 min), and then remained almost constant to the end of experiment.

Figure 8 Solar water disinfection of E. coli within distilled water. Inactivation curve of E. coli ( ), UV-A irradiance ( ) during solar irradiation, normalized FITC-A ( ), reference FITC-A value for control samples ( ). Full size image