HYDROGEN PEROXIDE METHOD
(Cooper et al. 2000; King et al. 2007; Morris et al. 2011)
For the BioTek HT
J. Jeffrey Morris
pH 3 Buffer for Acridinium Ester Reagent
- In a clean glass beaker with a stir bar on a stir plate, add 1.5 g KH2PO4 to about 200 mL milli-Q water to dissolve.
- Add 1.05 mL phosphoric acid (H3PO4).
- Titrate to pH 3.
- Bring to 250 mL with milli-Q.
- Store at room temperature in the dark.
10X Acridinium Ester (AE) Stock
- Add 25 mL pH 3 buffer to about 175 mL milli-Q water in a clean glass beaker with a stir bar on a stir plate.
- Add 5.47 mg solid acridinium ester to beaker. Cover beaker with parafilm and stir overnight in the dark.
- Bring solution to 250 mL with milli-Q water.
- Store at 4o C in the dark. This solution is stable for at least a year under these conditions.
- Add 500 mL pH 3 buffer to about 10 mL milli-Q water in a fresh 50 mL falcon tube.
- Add 5 mL 10X AE stock to tube.
- Fill to 50 mL mark with milli-Q water. Seal tight and store in the dark at room temperature.
- Allow at least 24 hr to equilibrate prior to first use.
- The concentration of AE may be manipulated to provide higher sensitivity (more reagent) or broader range of linear response (less reagent). This concentration is ideal for measuring standard levels found in lake and ocean water (0 to 250 nM) but works well up to 500 nM (or even 1 mM with curve fitting for the standards).
2M NaCO3 Buffer
- Place approximately 200 mL milli-Q water in a clean glass beaker on a stir plate. Slowly add 53 g NaCO3 Allow to dissolve.
- Titrate to pH 11.3 using 1N HCl.
- Seal tight and store in the dark at room temperature.
- For use, pour 50 mL solution into a fresh falcon tube. Seal tight and store in the dark at room temperature.
Standardized Hydrogen Peroxide Stock
- Add 23 mL 30% HOOH to 10 mL milli-Q water and mix by vortexing.
- Clean the surface of the Take3 plate with 100% ethanol; let air dry.
- Place a 2 mL aliquot of milli-Q water on well A2 of the Take3 plate.
- Place 9 2 mL aliquots of HOOH primary stock on wells A3 through E3 of the Take3 plate.
- Open Gen5 software on plate reader computer.
- Click “Read Now” and select the program “H2O2 Standardization.prt” to measure A240 for these samples. When prompted say “OK” to export data to excel.
- The spreadsheet gives the mean and standard deviation for the measurements. Calculate the 95% confidence interval with the formula:
- The precise concentration of HOOH in this stock may be calculated using the molar extinction coefficient for HOOH of eHOOH = 38.1 L mol-1 cm-1 (Miller and Kester 1988). Simply divide the mean absorbance and 95% CI by 38.1 to give values in moles per liter.
- Filter sterilize the standardized stock using a 0.2 mm syringe filter.
- This stock is quite stable even on a multi-year time scale, as long as it remains sterile and protected from light. However, it is a good idea to make a fresh stock every 3 months or so.
0.1M Sodium Pyruvate Stock
- Place 0.55 g sodium pyruvate in 40 mL milli-Q water in a clean falcon tube and dissolve. Bring to 50 mL with milli-Q water.
- Filter sterilize using a 0.2 mm SteriFlip unit. Store in the refrigerator.
Hydrogen Peroxide Standards
- Make standards in sterilized test tubes just before your experiment and use within 3 hours.
- Prepare 10 mM HOOH stock in 10 mL sterile milli-Q water. The exact amount of HOOH standard to add to make 10 mM will vary based on the assayed concentration of the standard.
- Always mix and aerate samples thoroughly, either by vortexing (preferred) or vigorous shaking.
- ALWAYS use ASW of the same composition as your samples to make your standards.
|Tube||Standard||0.1M Pyruvate||10 mM HOOH||Sterile ASW|
|A||500 nM||0 mL||250 mL||4.75 mL|
|B||250 nM||0 mL||125 mL||4.9 mL|
|C||125 nM||0 mL||62.5 mL||4.9 mL|
|D||62.5 nM||0 mL||31.3 mL||5 mL|
|E||31.25 nM||0 mL||15.6 mL||5 mL|
|F||15.625 nM||0 mL||7.8 mL||5 mL|
|G||0 nM||0 mL||0 mL||5 mL|
|H||Blank||50 mL||0 mL||5 mL|
- DI Water wash: for everyday cleaning. Add 40 mL of Milli-Q water to a 50 mL falcon tubes.
- 1N HCl: For cleaning after long periods of inactivity or after field work. Add 4 mL concentrated HCl to 44 mL milli-Q water in a 50 mL falcon tube clearly labeled “1N HCl”.
- 1N NaOH: For cleaning after long periods of inactivity or after field work. Add 1.6 g NaOH solid to 50 mL milli-Q water in a 50 mL falcon tube clearly labeled “1N NaOH”. Dissolve by gentle inversion.
- Turn on plate reader and open the Gen5 2.07
- Select “Instrument Control” and “Incubate”. Make sure the “Off” box is checked.
- Allow temperature to equilibrate for at least 1 hr after either turning on the machine or changing the “Incubate” setting prior to analysis. The acridinium reaction as well as PMT response is temperature sensitive, so it is important for the temperature inside the machine to equilibrate prior to reading.
- Prepare HOOH standards (see section I).
- Shake 2M NaCO3 reagent to oxygenate. Strap onto right injector (injector 1). Make sure intake tube extends to bottom of reagent tube.
- Repeat step 5, with AE reagent. Strap onto left injector (injector 2).
- “Prime” injectors. This involves pulling some solution through the tubes to make sure reagent is available for injection into first assay well.
- Under “Instrument Control”, click “Prime/Dispense”.
- Set “Dispenser” to 1 and “Volume” to 1000 m
- Click “Prime”
- The machine will request you to insert the priming tray, which is a plastic reservoir the size of a 96-well plate. Put the plate in and click OK. The machine will then prime itself.
- Set “Dispenser” to 2 and repeat the previous steps to prime the second injector.
- Click “Close”.
Experiment setup and execution:
- Load white, flat-bottom 96-well plates (Costar 3912 or equivalent) with samples and standards. Black plates are acceptable but not ideal.
- Load 200 mL per well.
- Load standards A-H in columns both BEFORE and AFTER your samples.
- Load duplicate wells with each of your unknown samples.
- If you have leftover wells after loading samples, load more replicates of standards E and F to improve standard curve for low-concentration samples.
- On the computer in the Gen5 software, click “Read Now” and select the “Acridinium Ester HOOH.prt” Protocol to open.
- Select the wells where you plan to load your plate and click “OK”.
- Insert Assay plate and click “OK”. The machine will now read the plate by injecting first 50 mL NaCO3 reagent and then 50 mL AE reagent, followed by monitoring luminescence for 1 s.
- After the assay is over, click “OK” to save the data and again to export the data to Excel.
- If you want to run another plate in the same file, click the green “Play” button in the toolbar, select the wells you want to assay, and repeat steps 12-13.
- When you are finished reading samples for the session, remove reagent tubes from magnetic stands. Make sure to place the right cap back on each tube; one drop of carbonate buffer added to the acridinium ester reagent will completely ruin it.
- Place DI wash tube on right stand.
- Go to “Instrument Control” and select “Prime/Dispense”.
- Set “Dispenser” to 1 and “Volume” to 5000 m Click “Prime” and insert priming tray when prompted.
- Move DI wash tube to left injector and repeat step 18, but with “Dispenser” set to 2.
- Rinse priming tray with DI water and let dry outside of plate reader. Close Gen5 software and plate reader tray.
Analytical Protocol: The Standard Curve Method
Broadly, this method produces one master set of standards by adding standardized amounts of HOOH to ASW. These standards are added to the first and last “columns” of the assay plate and are used to construct a standard curve for interpolating the HOOH concentrations of each assay well. A “true zero” where all HOOH has been removed with pyruvate is used as a blank. Two mechanical replicates are performed for each sample, and 40 samples can be assayed per 96-well plates. The loading scheme is as follows:
|A||500 nM||Samples 1-8||Samples 9-16||Samples 17-24||Samples 25-32||Samples 33-40||500 nM|
|B||250 nM||250 nM|
|C||125 nM||125 nM|
|D||62.5 nM||62.5 nM|
|E||31.25 nM||31.25 nM|
|F||15.625 nM||15.625 nM|
|G||0 nM||0 nM|
These standards (prepared in ASW) are ideal for most ecologically-relevant applications. As the standard curve loses linearity above 500 nM, it is better to dilute more concentrated samples rather than use higher standards. The HOOH concentration of the sample is calculated by simply creating a regression line using the standards and applying it to each sample. A typical limit of detection (defined as three times the standard error of the blank) for this Protocol is about 10 nM.
To calculate HOOH, first subtract the average luminescence of the pyruvate blank wells from ALL well measurements. Use blanked readings for subsequent calculations.
Calculate Standard Curve using “linest” function in Excel, gathering the standard deviations of the slope and y-intercept. Note: HOOH values of standards A-G are “y-values” and luminescence readings are “x-values”. Note that linest is an array formula, which must be entered by selecting a 2×3 cell array, typing in the formula
=linest(y-values, x-values, 1, 1)
and hitting ctrl+shift+enter. The output should look like this:
|SLOPE (b)||Y-INTERCEPT (a)|
|Std. error of b||Std. error of a|
|R2||Std. error of y estimate (s)|
Note that a is the concentration of HOOH in your untreated ASW (usually around 50 nM).
Compute the [HOOH] for each well from its luminescence, x, correcting for dilution factor (d), if any:
If desired, you can calculate the 95% confidence interval of this value using the excel code:
= s * tinv(.05,n-2)
where “n” is the number of values used to make the standard curve.
Rules of thumb for loading plates:
- Each sample should be loaded at least twice to provide mechanical replicates that will be averaged later.
- The total sample volume must be 200 mL, including dilution water and/or standard additions.
- For samples exceeding 500 nM HOOH (the approximate limit of linearity for the AE response), dilution is required. A good way of doing this is to add sample to an amount of ASW sufficient to achieve the desired dilution. This may be done directly in each sample well, using a multichannel pipettor to add the dilution blank. If more dilution than this is required, it may be done using a similar method on a separate 96-well plate to facilitate sequential 10-fold dilutions. Don’t forget to correct for the amount of HOOH in the ASW itself!
- Using a P200 pipettor to load samples gives more reproducible results than using a P1000. (Or a P20 is better than a P100 if doing dilutions). Try to avoid bubbles and splashing (don’t go to the second plunger stop on the pipettor) and try not to get sample stuck on the edges of the wells.
- If the samples need to remain sterile, make sure not to take both replicate aliquots using the same tip, as the 96-well plate is not sterile and you will contaminate your samples otherwise! My usual experimental method is to withdraw a 500 mL + aliquot into a separate sterile tube from which I do subsequent HOOH analysis (and whatever else), thus minimally contacting the experimental solution and reducing the risk of contamination. Note that a single bacterium producing catalase can completely destroy 1 mM HOOH in a few days, so cleanliness is of utmost importance in studying HOOH over longer periods.
Cooper, W. J., J. K. Moegling, R. J. Kieber, and J. J. Kiddle. 2000. A chemiluminescence method for the analysis of H2O2 in natural waters. Mar. Chem. 70: 191-200.
King, D. W. and others 2007. Flow injection analysis of H2O2 in natural waters using acridinium ester chemiluminescence: method development and optimization using a kinetic model. Anal. Chem. 79: 4169-4176.
Miller, W. L., and D. R. Kester. 1988. Hydrogen peroxide measurement in seawater by (p-hydroxyphenyl)acetic acid dimerization. Anal. Chem. 60: 2711-2715.
Morris, J. J., Z. I. Johnson, M. J. Szul, M. Keller, and E. R. Zinser. 2011. Dependence of the cyanobacterium Prochlorococcus on hydrogen peroxide scavenging microbes for growth at the ocean’s surface. PLoS ONE 6: e16805.