96孔板ORAC抗氧化快速监测方案
参考文献：
Tao Wu, Jun Yan, Ronghua Liu, Massimo F. Marcone, Haji Akber Aisa, Rong Tsao,
Optimization of microwave-assisted extraction of phenolics from potato and its downstream waste using orthogonal array design,Food Chemistry,, 2012 , 133 (4) :1292-1298
Reagents
1) Phosphate buffer (75 mM, pH 7.4): NaH2PO4 (Mw119.98): 1.71 g Na2HPO4 (Mw.141.96): 8.62 g
Dissolve in ~ 900 mL dH2O, adjust pH to 7.4 using HCl or NaOH. Add more dH2O to final volume 1L
2) Fluorescein stock solution (144.65×10-3 mM): weigh 4.8mg of fluorescein in a 2 mL mirofuge tube, dissolve in 1 mL MeOH and transfer into a 100 mL volumetric flask. Rinse the tube 3 times with 1 mL MeOH and quantitatively transfer into the volumetric flask. Add 75 mM phosphate buffer to 100 mL mark. Wrap with foil and store at 5 oC (good to 6 months).
Fluorescein working solution (8.68×10-5mM): Add 9 ?L of stock to 14.991 mL of 75mM phosphate buffer (pH 7.4). The diluted solution was made fresh daily. 3) Trolox standard stock solution (0.02M): 25 mg of Trolox is dissolved in 5 mL MeOH.
Trolox working solution: 50 ?L of stock solution was diluted with 9.95ml of 75mM phosphate buffer (pH 7.4). Final concentration was 100μM.
4) AAPH solution: 0.414g of AAPH was dissolved in 10ml of 75mM phosphate buffer (pH 7.4) to a final concentration of 153mM and made fresh daily. 5) Sample preparation: dilute the samples with phosphate buffer (you will need to try to find out the appropriate dilutions for your samples, could be 10-, 100- and 1000-fold). If samples are not soluble in buffer, use the ORAC-Lipophilic protocol. Procedure:
1) Blank wells received 25 μl of buffer .
2) Standard wells received 25 μl of Trolox dilution (6.25, 12.5, 25.0, 50.0, 100μM). 3) Sample wells received 25 μl of appropriately diluted samples. 4) 150μl of 8.68×10-5mM fluorescein working solution was added into all experimental wells
5) The plate was allowed to equilibrate by incubating for a minimum of 30 min in the Synergy HT Multi-Detection Microplate Reader at 37°C.
6) Reactions were initiated by the addition of 25 μl of AAPH reagent followed by shaking at maximum intensity for 10 seconds. The fluorescence was then mointored kinetically with data taken every minute. Operation of a Synergy HT Multi-Detection Microplate Reader
1. Open windows and turn on instrument. Click Gen5, create a protocol.
1) Define the procedure: Temperature 37°C, start kinetic ( run
time 60 min, interval 1min, Read: λem 485/20, λex 528/20, Delay 15~30min, Shake 10sec ) 2) Plate layout: Type, ID Prefix, Conc.
3) Data Reduction: Gen 5 automatically creates two steps: the
blank subtraction transformation and well analysis for MaxV. Cllick Well Analysis
? Enter a label e.g. AUC (NET AUC) ? Select Formula and enter
(R1/R1)+(R2/R1)+(R3/R1)+....+(R61/R1) ? Click OK
Click Transformation
? Enter a label e.g. AUC or (NET AUC) ? Select Formula and enter X or (X-BLK) ? Click OK
Click Curve Analysis
? Data In: X-Axis Data (Plate Layout Settings). Y-Axis Data (AUC or NET AUC Formula Result [485/20, 528/20] )
? Click OK
Save the protocol
Click protocol, select a protocol. You can run it in an experiment
2. Click preheat , Select on
3. When the temperature reach to 37 °C, put the plate in and click read, enter plate
name.
4. After reading, save the experiment name. Click plate name
Matrix: layout, data, kinetic curve Graph: standard curve, sample curve,
Statistics: Results AUC, NET AUC, TROLOX Concentration. Calculated the sample slop is Trolox equivalent concentration.
The ORAC procedure used an automated plate reader
(KC4, Bio Tek, USA) with 96-well plates (Prior et al., 2003). Analyses were conducted in phosphate buffer pH 7.4 at 37 1C. Peroxyl radical was generated using 2, 2’-azobis (2-amidino-propane) dihydrochloride which was prepared fresh for each run. Fluorescein was used as the substrate. Fluorescence conditions were as follows: excitation at 485nm and emission at 520nm. The standard curve was linear between 0 and 50 mM Trolox. Results are expressed as mM TE/g fresh mass.
Human serum: suggested dilution: 100 to 200-fold Rat serum: actual dilution: 200-fold.
Equipment: Fluorescence microplate reader (FLx800, Bio-Tek Instruments Inc., Winooski, VT, USA).