Team:EPFL/Results/Aptamers
Microfluidics results in a nutshell
- Thrombin aptamers 1, 2 and 3 bind α-Human Thrombin in buffer
- Thrombin aptamers 1 and 2 can detect α-Human Thrombin in a sandwich assay in buffer
- Thrombin aptamers 1 and 3 can detect α-Human Thrombin in a sandwich assay in buffer
- Thrombin can be detected in Human serum using both thrombin aptamers 1 and 3
- PDGF aptamers 1 and 2 can detect PDGF in a sandwich assay in buffer
Thrombin aptamer binding test
We tested the affinity of our Thrombin aptamer 1 (Cy5) and our Thrombin aptamer 2 (Cy3) with biotinylated human thrombin. This experiment was done on a MITOMI 768 chip flowing biotinylated human thrombin and then the two aptamers in different parts of the chip in order to see whether they bind with thrombin. The affinity is measured by the relative fluorescence in the different channels (Cy3 and Cy5). As a negative control, both Cy3 and Cy5 labeled DNA oligos with a random sequence were flown instead of the aptamer in half of the chip to make sure thrombin does not have non-specific binding.
A fluorescent scan of the microfluidic chip after wash resulted in a fluorescent signal in both Cy5 and Cy3 channels (Fig. 1a and 1b) in the button area for the aptamers and no signal for the control as expected.
Finding the relative Kds of our aptamers with thrombin in terms of RFU
We spotted on an epoxy-coated glass slide different concentrations of Cy5-labeled Thrombin aptamer 1 and Cy3-labeled Thrombin aptamer 2, ranging from 2 μM to 0.2 μM, for MITOMI experiments. Cy3 and Cy5 labeled DNA oligos were again used as negative controls. Both aptamers were resuspended and incubated for 1 hour to obtain a saturation binding curve. As expected, the fluorescent signal increases with aptamer concentration.
After data analysis, we obtain two hyperbolic saturation binding curve
(Fig. 2a and 2b).
The best statistical model was found to be a simple one-binding site model ($E_{max}$ model):
$y_i = \frac{B \times x_i}{K_d + x_i} + \epsilon_i$ with $i \in \{ 1,...,N\} $.
$x_i$ denotes the aptamer concentration in solution, $y_i$ the aptamer-protein complex
concentration and $N$ the number of observations. The residual errors are assumed
to be identically and independently distributed $\epsilon_i \sim N(0, \sigma^2)$ .
Sandwich assay with our Thrombin aptamers 1 and 2 in buffer
The Thrombin aptamer 1 is now biotinylated rather than Cy5 labeled, enabling it to be fixed to the surface. To begin the biotinylated aptamer 1 was flown, then human thrombin in half of the chip and finally the whole chip was covered with Cy3 thrombin aptamer 2. As expected, a signal in the button area was observed in the region containing containing the first aptamer, thrombin and the second Cy3 labeled aptamer. Washing steps were done with PBS and Aptamer Binding buffer (ABB, cf. protocols).
A fluorescent scan of the microfluidic chip resulted in a fluorescent signal in the upper part of the chip where Thrombin was flown (Fig. 3).
Sandwich assay with our Thrombin aptamers 1 and 3 in buffer
The Thrombin aptamer 3 is a modified version of the Thrombin aptamer 2 containing the short trigger sequence for the toehold. We tested if this aptamer could still form a sandwich complex with human thrombin and the biotinylated Thrombin aptamer 1. Briefly, the biotinylated Thrombin aptamer 1 was first flown, then human thrombin in the top half of the chip and finally the Cy3-labeled Thrombin aptamer 3 in the whole chip.
As expected, no conformational change is impacting the modified aptamer to bind to human thrombin (Fig. 4).
Sandwich assay with our Thrombin aptamers 1 and 3 in human serum
We tested whether our aptamer pair could form a sandwich with thrombin in human serum. The thrombin aptamer 1 which is biotinylated is flown first and thereby fixed to the surface. Human serum was then flown in half of the chip and finally the whole chip was covered with the Cy3-labeled thrombin aptamer 2. Washing steps were performed with a buffer (PBS + 2% BSA + 0.01% Tween 20). As expected, a signal in the button area was observed in the region containing human serum.
Sandwich assay with our PDGF aptamer 1 and 2 in buffer
We tested whether our PDGF aptamer 1 and 2 pair could form a sandwich with PDGF-BB. Briefly, the first biotinylated PDGF aptamer 1 was flown, then PDGF-BB in half of the chip and finally the whole chip was covered with Cy3-labeled PDGF aptamer 2. As expected, a signal in the button area was observed in the region containing Cy3 labeled aptamer. Washing steps were done with PBS and aptamer Binding buffer (ABB, cf. protocols) A fluorescent scan of the microfluidic chip resulted in a fluorescent signal in the upper part of the chip where PDGF was flown (Fig. 6).
