PREPARATION OF BILIRUBIN STANDARD SOLUTIONS
Bilirubin (BR) solutions can be prepared in amber glass vials or in aluminum-coated screw cap tubes (Table 1).
Weigh a few mg of the dry BR powder in an Eppendorf tube on a microbalance.
Add an appropriate volume of DMSO to obtain a 5 mM solution and vortex.
Store 20 µL aliquots of this solution in Eppendorf tubes at -20 °C.
Stability: 4 months at -20 °C.
Dilute 10 µL Solution A in 4990 µL PBS to obtain a 10 µM solution and vortex.
Dilute 500 µL Solution B1 in 4500 µL PBS to obtain a 1 µM solution and vortex.
Stability: <10 min. Use and analyze it immediately after preparation.
Dilute 10 µL Solution A in 4990 µL PSB-BSA 4 g·L-1.
Dilute 500 µL Solution B2 in 4500 µL PBS to obtain a 1 µM solution and vortex.
Stability: 2 h at T = 25°C; wait at least 30 min before using this solution.
Standard Solutions C1
Dilute serial volumes of Solution B1 (1 and 10 µM) in PBS to a final volume of 5 mL.
The volumes are given in Table 1.
Stability: Use and analyse immediately after preparation (within 5 minutes).
Standard Solutions C2
Dilute serial volumes of Solution B2 (1 and 10 µM) in PBS-BSA 0.4 g·L-1 to a final volume of 5 mL.
The volumes are given in Table 2.
Stability: Use and analyse the solution within 1 hour of preparation.
QUALITY CONTROL OF BILIRUBIN SOLUTIONS
1) UV-VIS Spectroscopy
o Add 3 mL Solution B1 or Solution B2 to a quartz cuvette (l = 1 cm).
o Prepare a set of n = 4 samples.
o Use PBS or PBS-BSA as blank.
o Record UV-VIS spectra in the range 350 < λ < 600 nm and find Amax = 440 nm and 465 nm for Solution B1 and B2, respectively.
o BR solutions should be considered accurate if the absorbance value is:
§ Solution B1: 0.446 (± 0.05) at λmax = 440 nm
§ Solution B2: 0.636 (± 0.05) at λmax = 465 nm
2) HUG-based Fluorometric assay 8
o Transfer 200 µL of Standard Solution C1 or C2 to a black 96-well plate containing 10 µL of 1 g/L (or 16.6 µM) HUG (final volume = 210 µL; final concentration HUG = 0.79 µM).
o Fill 5 wells for each BR concentration.
o Use 200 µL of the solvent of either C1 or C2 solutions (PBS or PBS-BSAdil).
o Incubate the covered plate at room temperature (T = 25 °C).
o Reaction time:
1 hour for Standard Solution C1
2 hours for Standard Solution C2.
o Instrumental settings: λex = 485 nm, λem = 528 nm, T = 25 °C, gain 100, reading height 2.50 mm.
3) Range of applicability
o 7< pH <10
o The same calibration curve is obtained at T = 25°C or 37°C
o BSA concentration does not affect the stability and solubility of BR in standard solution or the kinetics of binding with HUG in a wide range of concentrations (0.4 - 40 g·L-1).
4) Data Analysis
o Calculate the mean ± standard deviation for each BR concentration (n = 5).
o Fit the data of standard curves by linear regression analysis.