SPR Oana Hosu

1) Spalare 3X 200 ul PBS

2) Masurare profil SPR dupa aptamer – inainte de MCH

3) Spalare 3X 200 ul PBS

4) Incubare 30’ cu 100 uM MCH

5) Spalare 3X 200 ul PBS

6) Masurare profil SPR dupa aptamer si MCH

7) Injectari

V inj (ul) Debit t exp

60 20 3 min
ul/min

Nr prob C (nM) V s (ul) V Pbs

(ul)
0 0 0 60
0 0 0 60
1 25 60 0
2 50 6 54
3 75 9 51
4 125 15 45
5 250 30 30
6 500 60 0
7 1000 9.4 50.6

Vs pt 1: 25 nM Vs pt 7: 2 ug/mL
Vs pt 2 - 6: 500 nM

8) Disociere

- dupa injectarea asteptam/injectam buffer – 4-5 min (de observant)

9) Regenerari

- 3 min (eventual lungim) – 60 ul NaCl 5 mM in apa (20 ul/min)

“…employing an empirical relationshipbetween reflectance angle change and amount of protein
immobi-lized (120 m°= 1 ng mm-2)

- pt regenerare:

“In addition, this aptasensor can be reused with good regeneration by simple incubation with high concentration of NaCl solution. “
After detection of target, the electrochemical aptasensor was regenerated to remove the hybridized cDNA and the bound AFB1 by a
simple incubation of NaCl solution (5 M) for 10 min and subsequent washing with ultrapure water for 3 min.

1000 ml 5 * 58.44 = 292.2 g

5 ml ……………x=1.461 g

60 ul inj = 3 min

Inject binding buffer or aptamer solution over sample flow cell (FC2) for 40 s at a flow rate of 5 μL/min.
Approximately 70 pmol (1 – 2 μg) of aptamer per run is sufficient for observing small molecule binding and
should yield aptamer capture levels of approximately 2,000 – 5,000 RU. Extend injection duration or increase
flow rate for dilute RNA samples or low molecular weight ligands to achieve higher capture levels. For protein
ligands, less aptamer needs to be captured, as protein ligands produce a much larger binding response.

70 pmol … 150 ul
X=467pmol 1000 ul = 1 ml
Y=467 nmol= 467 nM 1000 ml

Optimization of association and dissociation phase lengths and ligand concentrations tested are necessary for
accurate measurement. Association phase lengths depend on the time needed to reach equilibrium, which can
range from less than 5 s to greater than 180 s at a flow rate of 30 μL/min (1). Reaching equilibrium in the
ligand association phase is necessary for determining KD through steady-state affinity but not for determining
binding kinetics; whether equilibrium is reached may depend on the maximum injection volume of the
instrument used, which for the Biacore X100 instrument limits the injection length to 180 s at a flow rate of 30
μL/min. Dissociation phase lengths are chosen so that substantial ligand dissociation is observed, generally
with at least a 10-fold greater signal-to-noise ratio for the decrease in ligand binding response.
Inject binding buffer or ligand solution at a flow rate of 30 μL/min for a ligand association phase length relevant
for the aptamer–ligand pair being characterized.

Dissociate ligand by injecting binding buffer at a flow rate of 30 μL/min for a ligand dissociation
phase length relevant for the aptamer–ligand pair being characterized.

Solutions of 50 μL at 0.6 μM of each ssDNA were used for overnight functionalization

Functionalization of SPR bare gold chip (Au-chip, BIAcore, GE Healthcare), using the initial library
for the reference flow-cell, and the tested cycle in the working flow-cell. After passivation with 1 mM
6-mercapto-hexanol for 30′, and several washes with TE 1X (10 mM Tris-HCl pH 7.4, 1 mM EDTA),
the chip was used for kinetic analysis in the SPR machine.

several serial dilution solutions of

tobramycin (from 0.02 μM to 200 μM) were injected for 180 s at 20 μL/min, followed by a
dissociation time of 240 s at 20 μL/min and a regeneration injection of NaCl 1 M with SDS 0.2% (120
s at 5 μL/min with 120 s of stabilization).

Different concentrations of AFB1 were prepared by dilution in running buffer from 1 μM AFB1
solution in 0.3% acetonitrile, and 200 μL of solution was prepared for each concentration. The AFB1
solution (60 μL) was injected at a flow rate of 30 μL·min−1 for 120 s. The binding between AFB1 and
aptamer on SPR chip was monitored in real time. After each injection, further running for 250 s was
continued to allow the signal back to the baseline.

This shift corresponds to a 1.7 _ 10_13 mol mm_2 of surface

coverage, calculated by employing an empirical relationship
between reflectance angle change and amount of protein immobilized
(120 m_ = 1 ng mm_2). The theoretical surface coverage value
for DNA strands [16] is equal to 3.7 _ 10_13 mol mm_2, which is
2.2 times greater than the experimental value obtained.

93.44 fM/mm2 = 93.44 x 10-15 = 9.344 x10-4 moli/mm2

Bibliografie

Secventa Oana:
5′-TGG GCA CGT GTT GTC TCT CTG TGT CTC GTG CCC T-3′
Alta secventa testata SPR:
5′-GCA CGT GTT GTC TCT CTG TGT CTC GTG C-3′