A high throughput spectrophotometric method for singlet oxygen quenching

doi:10.1038/protex.2016.054

Method Article

A high throughput spectrophotometric method for singlet oxygen quenching

https://doi.org/10.1038/protex.2016.054

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An easy spectrophotometric method for evaluating quenchers of singlet oxygen is described which can be used for high throughput assessment of large number of samples in a 96-well plate format. Singlet oxygen is a higher energy state molecular oxygen species produced in various intracellular reactions, extracellular reactions by the aid of photo sensitizer, light source and/or by chemical means. In this protocol, we have developed a simple and accurate procedure for generating singlet oxygen with no side reactions at physiological pH. The second aspect of this assay is detection by p-nitroso dimethylaniline (RNO), which is an already established suitable trap with imidazole and/or its derivatives for singlet oxygen. We have synchronized the assay system by performing various combinations of reagent used and also by plotting their spectra, pH and time-dependence curve monitored spectrophotometrically at 440nm

COMMENTS

Protocol edited 29/07/2016

Ashleigh Carver, Editorial Assistant, Nature Protocols

Biochemistry

Singlet oxygen

Hypochlorous acid

Hydrogen peroxide

RNO

L-Histidine

Singlet oxygen (1O₂) is a high energy appearance of oxygen exists in the singlet state with a total quantum spin of zero¹. In 1931, it was predicted as meta-stable intermediate state in dye-sensitized photo-oxygenations². However, in 1964, it was fully established that 1O₂ was same whether generated in sensitized photo-oxygenations³, through radio frequency⁴ or chemical reactions (e.g. H₂O₂/NaOCl)⁵. It forms a crucial part in almost every field of science including biological (food oxidation, cell signalling, oxidative stress)^6-8, medical (photodynamic therapy for microbes, malaria or cancer) ^9-11, environmental (as an air pollutant due to ozone decomposition)¹², chemical (chemical production of 1O₂ and reactions involving 1O₂)¹³ and physical (the best part to explain the properties of 1O₂, especially its reactivity and stability in various solvents and how it influences numerous aspects like light-related properties with the help of quantum theory)¹⁴ sciences (Figure 1 & Figure 2).

In this protocol, we are reporting a simple high throughput procedure for singlet oxygen quenching which can be easily determined spectrophotometrically in a 96 well plate format at 440 nm. This protocol is an improved and customized version wherein the system used to generate singlet oxygen can be done chemically (equimolar H₂O₂/NaOCl) or biologically (photosensitizer and light/UV source) with RNO (p-nitroso dimethylaniline), a strong yellow color dye and imidazole or its derivatives (L-Histidine)¹⁵. The bleaching is a consequence of 1O₂ capture by the imidazole ring which results in the formation of a trans-annular peroxide intermediate complex¹⁶. In the absence of RNO, 1O₂ decomposes or rearranges into the final oxygenation product, therefore, the imidazole system along with RNO can be used as a sensitive and selective test for the presence of 1O₂ in aqueous solutions (Figure 3). Singlet oxygen can also partially bleach RNO even in the absence of imidazole derivatives. In such a case, the bleaching of RNO is strongly increased by the presence of imidazole molecules with a characteristic dependence on their concentration. The separation of the product of RNO bleaching by thin layer chromatography also serves as an additional proof of the presence of 1O₂ in the system. The imidazole with RNO technique has been applied to a number of sensitizing and non-sensitizing dyes^15-20. Various other methods have also been reported (Table 1)^19-45 but they all require highly specialized instruments, costly fluorescent/luminescent probes, and/or highly dangerous radioactive probes still being more sensitive and efficient. If the sample size is huge and has to be assessed on a large scale, our method is more useful, easy, simple, accurate and cost-effective.

• 25mM Disodium hydrogen phosphate (Na₂HPO₄, 61755005001046, Merck Ltd.)

• 25mM Sodium dihydrogen phosphate (NaH₂PO₄, 61784505001046, Merck Ltd.)

• 100mM Sodium hypochlorite (239305-500ml, Sigma-Aldrich)

• 100mM Hydrogen peroxide (107209, Merck Ltd.)

• 12.5 mM L-Histidine (73767-100mg, Sigma-Aldrich)

• 100µM N,N-Dimethyl-4-Nitrosoaniline [RNO] (D172405-25g, Sigma-Aldrich)

• Positive control: Rose Bengal (330000-1g, Sigma-Aldrich)

• Negative control: DABCO (D27802-25g, Sigma-Aldrich)

• Pipettes from Thermo-Fisher Scientific India Pvt. Ltd., Noida, India.

• 96 well plates from M/s Tarsons Products Pvt. Ltd., New Delhi, India.

• Microtips (10µl) from M/s Tarsons Products Pvt. Ltd.,New Delhi, India

• Microtips (100µl) from M/s Tarsons Products Pvt. Ltd.,New Delhi, India

• Microtips (1000µl) from M/s Tarsons Products Pvt. Ltd.,New Delhi, India

• Centrifuge 15 ml tube from M/s Tarsons Products Pvt. Ltd.,New Delhi, India

• Eppendorf tubes from Genaxy Scientific Solan, Himachal Pradesh, India.

• Microplate Reader from Molecular Devices Corporation, California, USA.

• pH Meter from Eutech Instruments, Navi Mumbai, India.

PROCEDURE

Reagent Preparation

Sodium Phosphate Buffer (25mM, pH 7.0)

Separate solutions of disodium hydrogen phosphate in 100ml deionized water (25mM, by dissolving 0.445g) and sodium hydrogen phosphate monobasic in 100ml deionized water (25mM, by dissolving 0.39gm) are made and then pH 7.0 is maintained by adding two solutions slowly and measuring it by pH meter.

RNO Preparation (100 µM)

It is prepared by dissolving 0.15 mg in 10ml of 25mM sodium phosphate buffer. Wrap the bottle containing RNO solution with aluminium foil.

L-Histidine (12.5mM)

Dissolve 48.5mg in 25 ml of 25mM sodium phosphate buffer.

Sodium hypochlorite (100mM)

Commercially available sodium hypochlorite (Sigma) has molarity of 0.7M. We use 100mM, so add 28.5µl in the 200µl reaction system.

Hydrogen Peroxide (100mM)

Commercially available H₂O₂ has 9.7Molarity. We prepare 1M stock solution from which we add 20µl for 200µl reaction mixture.

Sample Preparation

Positive control (Rose Bengal) and negative control (DABCO) is prepared in deionized water likewise other samples either a crude extract from plant, cell, tissue, pure samples can be prepared in their respective solvent i.e. DMSO, ethanol or water.

Methodology

Mark 96 well plate in such a way that one row in triplicate is used for L-histidine followed by another row in triplicate without L-histidine. Rows with L-Histidine contain 25µl L-histidine solution while rows without L-histidine contains same amount of buffer.
Add the test sample/standard in triplicate in both marked row of 96-well plate and keep control wells without any sample or standard.
For a standard sample, we used 100µM, 50µM, 25µM concentrations in triplicate in both L-histidine and without L-histidine rows.
For control wells (without any sample) we have added 106.5µl of 25mM sodium phosphate buffer for 200µl reaction mixture.
Add 20µl RNO in each well and keep the plate in a dark place.

CRITICAL STEP: wrap the RNO tube with aluminum foil because it is light sensitive.

Then, add 20µl of 100mM H₂O₂ in each well followed by immediate addition of 28.5µl of 100mM NaOCl.

CRITICAL STEP: Add NaOCl immediately after H2O2 (there should be no time gap) otherwise RNO dye will be bleached as it is sensitive to hypochlorous acid and this may give false positive results.

Cover the 96-well plate with lid and wrap with aluminum foil then incubate the plate in the dark for 10 min at room temperature.
Finally, take the absorbance or kinetic reading (for 10 min) at 440nm in a microplate reader. Kinetic reading taken for 10 min with 5 sec orbital shaking, medium and read at 30-sec interval.
The graph is drawn with respect to control. Bleaching of RNO decreases the intensity of yellow color with decrease in optical density which indicates that singlet oxygen has not been quenched by the sample otherwise if there is no bleaching, it means that singlet oxygen has been quenched by the sample. Percentage of singlet oxygen inhibition by sample is calculated by
```
                       = \(control O.D-sample O.D) X100
```
IC₅₀ values can be derived using curve-fitting methods with statistical analysis.

Time line estimated for nine samples and two standards along with control.

Step 1-3 : 30 min

Step 4-8 : 30 min

Step 9-13: 90 min to 120 min

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HTML Table Header

Issue

Explanation

Solution

RNO bleaching on addition of NaOCl

RNO is sensitive to hypochlorous acid but when used with H2O2, reactions take place resulting in HCl and singlet oxygen production

Use H2O2 first then NaOCl

Stock solution of RNO decolourizes in light

RNO is light sensitive

Wrap RNO with aluminium foil

Variation in reading

Two possible reasons:

Both H2O2 and NaOCl are not added immediately after one another

Plate does not incubate in dark/ experiment not performed in dark

Use H2O2first and then immediately add NaOCl

Incubate plate in dark/experiment should be performed in a dark place

RNO bleaching gives false positive result

NaOCl and H2O2 bleach RNO on prolonged exposure

Record reading within 10 min

Kinetic reading shows saturated line

Kinetic reading start after 10-15 min, it means reaction is already completed

Kinetic reading should start within given time i.e. 10 min

Variation in kinetic graph

Sample is not properly dissolved or may be precipitated in well

Sample should be dissolved properly. Use different solvent to check their solubility and also run a vehicle control

In this protocol RNO with L-Histidine functions as a 1O₂ trap (Table 2). We have chosen different concentrations of buffer, RNO, L-Histidine and equimolar solution of NaOCl/H₂O₂ in order to find the appropriate combination of concentrations which can give least absorbance value (Table 3; Figure 4-5). In short, if this system is working properly then more absorbance means more quenching of singlet oxygen. In Figure 4, spectra of various molarities of RNO and buffer were taken and λ max was observed. By this experiment, we have established that λ max for 100, 50, 25µm of RNO was 440nm; while at other concentration it was found to be at 350nm. We have chosen 100µm RNO concentration for our further experiments as it shows maximum absorbance while there was no significant changes obtain at different molarities of buffer. We also wish to judge the best concentration of L-Histidine and NaOCl/H₂O₂ which can be used for the assay procedure. We have taken five different concentrations of L-Histidine and three different concentrations of NaOCl/H₂O₂ and tried all possible permutation and/or combinations as reflected in Figure 5. It was observed that a combination of 12.5mM of L-Histidine and 100mM of NaOCl/H₂O₂ gives a minimum optical density (absorbance), hence it was used for further experimentation.

The equimolar concentration of NaOCl/H₂O₂ generates singlet oxygen which is captured by L-Histidine forming trans-annular complex with RNO resulting in the bleaching of RNO as a consequence a decrease in the optical density (absorbance) was observed. The experiment was also performed at different pH in order to get the optimum pH and the least optical density (absorbance). At pH 7 which also represent the physiological pH (Figure 6) significant and required values were revealed. Spectral analysis was also performed with standard antioxidant in a range of 340-600nm and it was observed that 440nm was the best wavelength for all the studied antioxidant compounds (Figure 7). Time-dependent experiments revealed (Figure 8) that initial change occurs within 2 min after the reaction starts and saturation occurs after 10 min which is the endpoint.

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We are thankful to Council of Scientific and Industrial Research (CSIR), New Delhi for financial support (BSC 0121) at CSIR-Central Institute of Medicinal and Aromatic Plants, Lucknow.

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A high throughput spectrophotometric method for singlet oxygen quenching

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