6. This automatized standard procedure (using a commercially
available kit solution) is used to passivate the chip, avoiding
any unspecific binding of analytes of the biosurface.
7. The procedure to analyze three successive steps for polymerase
reaction is not usual and deviates from the pre-programmed
assay in the switchBUILD software. For this reason, we have
chosen to insert three successive injections, each starting
directly after the previous one, without regenerating the chip.
For this purpose, three successive dilutions have to be defined
manually, and the “Regeneration” option should be
unchecked. Instead of filling the tubes with three different
dilutions of polymerase (as requested in the predefined parameters), the first dilution tube will contain one concentration
of polymerase diluted with the 1Â Reaction Buffer, the second
will contain the same concentration of polymerase and 250 μM
of each dNTP, and the third dilution tube will contain 1Â
Reaction Buffer supplemented with 0.1 mg/mL heparin.
8. Static mode means that a constant voltage (À0.1 V in our
conditions) is applied on the chip, maintaining the nanolever
at a constant angle in the 1Â Reaction Buffer. Any event
affecting this angle and/or the distance of the fluorophore
with the quenching surface of the biochip (protein binding,
polymerization, dissociation) will be an interpretable signal for
the experiment.
9. Completion of association, polymerization, and dissociation
steps are dependent of kinetics parameters of the considered
polymerase, concentration, and temperature. Typically, for the
HIV-1 RT polymerase at 25
C, these three steps are set to 5, 4,
3, and 2 min for the concentrations of 0.15, 0.45, 1.35, and
4.05 nM, respectively.
10. Flow rate is automatically set from the estimated k ON value set
in the “predicted interaction value” tool implemented in the
switchSENSE software. For HIV-1 RT polymerase (k ON ¼ 1.3
E+7 M
À1 s
À1 at 25
C), flow rate is automatically set to 50 μL/
min for all the steps.
11. This automatized standard procedure removes analytes and
ligands from the surface and defines conditions suitable for
chip storage.
12. The switchCONTROL software takes the simplicity of switchBUILD scripts and converts them into advanced commands
for the unattended operation of the DRX instrument.
152
Guillaume Bec and Eric Ennifar
available kit solution) is used to passivate the chip, avoiding
any unspecific binding of analytes of the biosurface.
7. The procedure to analyze three successive steps for polymerase
reaction is not usual and deviates from the pre-programmed
assay in the switchBUILD software. For this reason, we have
chosen to insert three successive injections, each starting
directly after the previous one, without regenerating the chip.
For this purpose, three successive dilutions have to be defined
manually, and the “Regeneration” option should be
unchecked. Instead of filling the tubes with three different
dilutions of polymerase (as requested in the predefined parameters), the first dilution tube will contain one concentration
of polymerase diluted with the 1Â Reaction Buffer, the second
will contain the same concentration of polymerase and 250 μM
of each dNTP, and the third dilution tube will contain 1Â
Reaction Buffer supplemented with 0.1 mg/mL heparin.
8. Static mode means that a constant voltage (À0.1 V in our
conditions) is applied on the chip, maintaining the nanolever
at a constant angle in the 1Â Reaction Buffer. Any event
affecting this angle and/or the distance of the fluorophore
with the quenching surface of the biochip (protein binding,
polymerization, dissociation) will be an interpretable signal for
the experiment.
9. Completion of association, polymerization, and dissociation
steps are dependent of kinetics parameters of the considered
polymerase, concentration, and temperature. Typically, for the
HIV-1 RT polymerase at 25
C, these three steps are set to 5, 4,
3, and 2 min for the concentrations of 0.15, 0.45, 1.35, and
4.05 nM, respectively.
10. Flow rate is automatically set from the estimated k ON value set
in the “predicted interaction value” tool implemented in the
switchSENSE software. For HIV-1 RT polymerase (k ON ¼ 1.3
E+7 M
À1 s
À1 at 25
C), flow rate is automatically set to 50 μL/
min for all the steps.
11. This automatized standard procedure removes analytes and
ligands from the surface and defines conditions suitable for
chip storage.
12. The switchCONTROL software takes the simplicity of switchBUILD scripts and converts them into advanced commands
for the unattended operation of the DRX instrument.
152
Guillaume Bec and Eric Ennifar