TITLE: Antioxidant activity by Deoxyribose assay: in vitro protocol
AUTHORS: Adrieli Sachett, Matheus Gallas-Lopes, Greicy M M Conterato, Ana Herrmann, Angelo Piato

[DESCRIPTION]
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[STEPS]
?. [Preparing the reagents]
The first step is to prepare the reagents to be used in this protocol;
?. [Preparing the reagents]
KH2PO4-KOH buffer :    1.1.1 Weigh  of monobasic potassium phosphate (KH2PO4) in a beaker of appropriate size;    1.1.2 Dissolve the salt with  of ultrapure water;    1.1.3 Transfer the solution to a  volumetric flask;    1.1.4 Using ultrapure water, complete the solution's volume to reach ;    1.1.5 Weigh  of potassium hydroxide (KOH) in a beaker of appropriate size;    1.1.6 Dissolve the salt with  of ultrapure water;    1.1.7 Transfer the solution to a  volumetric flask;    1.1.8 Mix both solutions slowly in a  beaker following the steps below;Transfer  of the monobasic potassium phosphate (KH2PO4) solution to the beaker;Use a pH sensor to evaluate your solution. Expected conditions:;            If the pH of your solution is lower than 7.4 adjust the pH adding drops of the potassium hydroxide (KOH) solution;            If the pH of your solution is above 7.4 adjust the pH adding drops of the monobasic potassium phosphate (KH2PO4) solution;After adjusting the pH of this initial solution, proceed to add, slowly, drops of both buffer solutions (monobasic potassium phosphate and potassium hydroxide). Use Pasteur pipettes to add the solutions. Mix your solutions using a pH sensor, making sure the mix of both buffers is always at ;      1.1.9 Store this solution in an amber flask at ;
0.68043 g
90 mL
100 mL
100 mL
0.280053 g
90 mL
100 mL
250 mL
50 mL
8 °C
?. [Preparing the reagents]
Deoxyribose :     1.2.1 Weigh  of deoxyribose;    1.2.2 Transfer the deoxyribose to a beaker of appropriate size;    1.2.3 Add  of ultrapure water to the beaker to dissolve the salt;    1.2.4 Transfer your solution to a  volumetric flask;    1.2.5 Using ultrapure water, complete the solution's volume to reach ;
0.080478 g
9 mL
10 mL
10 mL
?. [Preparing the reagents]
Iron(III) chloride hexahydrate (FeCl3.6H2O) :     1.3.1 Weigh  of ferric chloride Iron(III) chloride hexahydrate;    1.3.2 Transfer the Iron(III) chloride hexahydrate to a beaker of appropriate size;    1.3.3 Add  of ultrapure water to the beaker to dissolve the salt;    1.3.4 Transfer your solution to a  volumetric flask;    1.3.5 Using ultrapure water, complete the solution's volume to reach ;
0.013515 g
40 mL
50 mL
50 mL
?. [Preparing the reagents]
Ethylenediaminetetraacetic acid (EDTA) :    1.4.1 Weigh  of EDTA;    1.4.2 Transfer the EDTA to a beaker of appropriate size;    1.4.3 Add  of ultrapure water to the beaker to dissolve the salt;    1.4.4 Transfer your solution to a  volumetric flask;    1.4.5 Using ultrapure water, complete the solution's volume to reach ;
0.01519648 g
40 mL
50 mL
50 mL
?. [Preparing the reagents]
Ascorbic acid :     1.5.1 Weigh  of ascorbic acid;    1.5.2 Transfer the ascorbic acid to a beaker of appropriate size;    1.5.3 Add  of ultrapure water to the beaker to dissolve the salt;    1.5.4 Transfer your solution to a  volumetric flask;    1.5.5 Using ultrapure water, complete the solution's volume to reach ;
0.017612 g
40 mL
50 mL
50 mL
?. [Preparing the reagents]
Thiobarbituric acid (TBA) :     1.6.1 Weigh  of TBA;    1.6.2 Transfer the ascorbic acid to a beaker of appropriate size;    1.6.3 Add  of ultrapure water to the beaker to dissolve the salt;    1.6.4 Transfer your solution to a  volumetric flask;    1.6.5 Using ultrapure water, complete the solution's volume to reach ;
0.1 g
9 mL
10 mL
10 mL
?. [Preparing the reagents]
Hydrochloric acid (HCl) :     1.7.1 Using a micropipette collect  of HCl ;     1.7.2 Transfer the HCl solution to a  volumetric flask;    1.7.3 Using ultrapure water, complete the solution's volume to reach ;
6.757 mL
10 mL
10 mL
?. [Preparing the reagents]
Hydrogen peroxide (H2O2) solution: Follow the steps described in section 2 of this protocol to first standardize and then prepare a hydrogen peroxide solution. This reagent should be prepared on the day of the biochemical assay, it should not be stored for later use;
?. [Standardization of H202 solution]
Every 30 days, or whenever a new bottle of hydrogen peroxide is opened, the absorbance of the concentrated solution should be checked.
?. [Standardization of H202 solution]
First, we must calculate the molarity of the H2O2 solution in the bottle:Concentration stated in the bottle:  Molecular weight: 34.0147 g/mol1 M ------------ 1 mol ---------- 1000 mL 1 M ----- -----34.0147 g ------- 1000 mLx -------------- 350 g ---------1000 mL x =
?. [Standardization of H202 solution]
Prepare a  solution () of H2O2:    2.2.1 Follow the calculations below to determine the volume of the primary solution needed:C1 x V1 = C2 x V2 x V1 =  x V1 =     2.2.2 Using a micropipette, collect  of H2O2 and transfer to a  volumetric flask;    2.2.3 Using ultrapure water, complete the solution's volume to reach ;
10 mL
10 mL
9.72
9.72 µl
10 mL
10 mL
?. [Standardization of H202 solution]
Read the absorbance of the solution at  in a microplate reader;
240
?. [Standardization of H202 solution]
Calculate the real concentration of your sample following these steps:A standardized  H2O2 solution has an absorbance of 0.39410 mM H2O2 ---------------- 0.394 Abs                                          X          ---------     Absorbance of your sampleX = Represents the real concentration of the hydrogen peroxide sample
?. [Standardization of H202 solution]
Calculate the real concentration of the bottled solution following these steps:As X (calculated on the last step) Represents the real concentration of the hydrogen peroxide sample10 mM H2O2 ---------------- 10.29 M     X         ---------     ZZ = Real concentration of hydrogen peroxide in the bottle
?. [Standardization of H202 solution]
Prepare a  solution of H2O2: this reagent should be prepared on the day of the biochemical assay, it should not be stored for later use;    2.6.1 Follow the calculations below to determine the volume of the primary solution needed:C1 x V1 = C2 x V2Z (the real concentration determined on the last step) x V1 =  x V1 = depends on the concentration of your hydrogen peroxide solution    2.6.2 Using a micropipette, collect the determined volume of H2O2 and transfer it to a  volumetric flask;    2.6.3 Using ultrapure water, complete the solution's volume to reach ;    2.6.4 Store this solution in an amber flask at  when needed;
10 mL
10 mL
10 mL
8 °C
?. [Incubation of the samples]
To optimize the reaction, an incubation step is needed.
?. [Incubation of the samples]
Prepare  microtubes, to be used to store the samples, with the correct information. The number of microtubes depends on the number of samples. You should provide at least five replicates (n = 5) of each sample with at least one control tube per sample. You should also prepare negative and positive control samples. Perform the test at least two times to ensure the results are as correct as possible.
1.5 mL
?. [Incubation of the samples]
For each sample, fill the plastic microtubes as described below. Using a micropipette fill the tubes in this order: KH2PO4–KOH buffer + Deoxyribose + FeCl3 + EDTA + Ascorbic acid + H2O2 + Sample. Mix the solution with the pipette tip to homogenize the content; MicrotubesKH2PO4–KOH (µL)Deoxyribose (µL)FeCl3 (µL)EDTA (µLAscorbic acid (µL)H2O2 (µL)Sample (µL)Control501010101010-Sample blank50-1010101010Sample40101010101010
MicrotubesKH2PO4–KOH (µL)Deoxyribose (µL)FeCl3 (µL)EDTA (µLAscorbic acid (µL)H2O2 (µL)Sample (µL)Control501010101010-Sample blank50-1010101010Sample40101010101010
?. [Incubation of the samples]
Incubate all your samples at  for  using a dry bath;
37 °C
?. [Incubation of the samples]
Add to all of your microtubes  of TBA  and  of HCl ;
100
100
?. [Incubation of the samples]
Incubate all your samples at  for  using a dry bath;
100 °C
?. [Reading your samples]
Prepare to read the absorbance of your samples in a microplate reader;
?. [Reading your samples]
Use a conventional 96-well microplate to run your samples. Before start pipetting, each well of the microplate should be marked for sample identification.
?. [Reading your samples]
Transfer  of the content of each microtube to its corresponding well in the microplate;
250
?. [Reading your samples]
Read the absorbance of the samples at  in a microplate reader;
532
?. [Calculating data and determinig results]
Prepare to analyze the results obtained after reading the absorbance of the samples;
?. [Calculating data and determinig results]
Determine the percentage of inhibition of the hydroxyl radical:
?. [Calculating data and determinig results]
Results should be expressed as % of inhibition of the hydroxyl radical.
?. [Calculating data and determinig results]
Calculate the mean absorbance of your replicates;