Our the research, experiments, and protocols
Validate the affinity of aptamer for PFOA in our hands
PFOA competes with quencher strand for aptamer binding, fluorophore signal gives a readout for quencher strand occupancy at various PFOA concentrations.
Adapted from Park, J. et al.1
Part 1: Quencher binding test
Part 2: Target binding test - 50 nM uL aptamer (5 uL) 250 nM quencher (25 uL) - Fill to 100 uL - Mix aptamer and quencher - Heat at 95 for 6 min - Let cool to room temp for 30 mins in dark room to induce hybridization - Add target compound, react for 40 mins - PFOA 500, 250, 125, 62.5, 31.25, 15.625 uM - (50, 25, 12.5, 6.25, 3.125, 1.5625 uL) - Quencher: 0.13 mg = 27 nmol * (4733.4 g/mol) - Aptamer: 0.19 mg = 11.6 nmol * (16438.7 g/mol)
Quantifies the affinity of the aptamer to the progesterone. We wanted to do this because we need an aptamer that has a binding affinity for its target around the same concentration that the target is going to be found at
For example, if the binding affinity is lower than the concentration of the target it will be unlikely for the sensor to work.
Binding affinity is concentration at which half the binding sites will be occupied.
Essentially, it is a feasibility check to make sure our aptamer (just the aptamer, no electronics, DNA modifications, or quencher involved) binds to progesterone sufficiently strongly to make a sensor based on the aptamer possible.
P4G03 from Jimenez et al
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Reference: see the article by TA Instruments2.
For the ITC experiment, we created a buffer for the progesterone aptamer that has 0.15% ethanol and 99.85% PBS. The Aptamer was dissolved in this buffer to make a stock solution 481.5 µM solution. A 4.8 µM progesterone solution was created that was 0.15% ethanol and 99.85% PBS. Isothermal Calorimeter was used from TA Instruments. 350 µL was added to the cell and 50 µL was added to the titration syringe. Data was analyzed with the Nanoanlyze Software from TA Instruments.
We performed three rounds of Circular Dichroism (CD) spectroscopy to study how our progesterone-binding aptamer, P4G03, changes shape when it interacts with progesterone. Each round built on the previous one: the first helped us learn how to collect reliable data, the second confirmed measurable folding, and the third tested whether that folding was specific to progesterone or could be triggered by other steroids.
All measurements were performed on an Aviv Model 420 CD spectrometer at 37 °C using a 1 mm UV-transparent quartz cuvette. Each spectrum was collected with a 1 nm bandwidth, 4 seconds per point, and averaged over two accumulations. Data were acquired with Aviv software and analyzed in Excel. Cuvettes were rinsed and blanked with deionized water before use, and samples were allowed to equilibrate for five minutes before scanning. Results are reported in millidegrees (mdeg).
This first experiment was exploratory. We wanted to make sure we could collect usable data on our CD machine and learn what concentrations of aptamer and progesterone would produce readable spectra. We also wanted to see if any detectable difference appeared when progesterone was added, giving us an early idea of how the system might behave.
With a working concentration range established, the next step was to confirm that P4G03 actually undergoes a conformational change when binding progesterone. This experiment was designed to collect clearer data across a broader set of ratios, including one much higher ratio to test for saturation effects.
Materials and Equipment Same as Experiment 1.
After confirming that the aptamer folds when binding progesterone, we wanted to test whether this response is specific or if it also occurs with other similar steroids. We compared the CD spectra of P4G03 exposed to progesterone, dihydrotestosterone (DHT), and cholesterol.
These experiments serve as our exploration of the bio-impedimetric properties of the P4G03 aptamer. We test the bare aptasensor system - aptamer attached to gold electrode in solution, hooked up to the necessary potentiostat system for measurement - with various concentrations of progesterone, to see how much of a signal we are able to get when varying levels. See Xiao et al.3 for where we got technical guidance for this kind of experiment.
Materials:
Equipment:
Develop Aptamer solution ready for monolayer using TCEP. Place Gold Electrode in QCM/hooked up to Potentiostat. Flow aptamer solution into cell and incubate for 2 hours to form monolayer. After incubation, wash with PBS to remove extra aptamers and flow 6-mercaptohexanol into the cell. This fills in the extra holes between the aptamers, giving them stability. Incubate for 3 hours in the 6-mercaptohexanol solution. Once monolayer is formed, use the potentiostat to get a base level in PBS. Do this by running AC voltammetry sequence with step voltage of 10mV/second, sweeping from -.4 to -.1 V. Record the peak current value from the Voltage vs Current graph produced. We recorded these peak values over time. When you want to test with Progesterone, flow a progesterone solution into the cell and run the AC sweep again. When making the progesterone solution, use ethanol to make the progesterone dissolvable in PBS. Mix well to ensure minimal clumping of progesterone.
Park, J., Yang, K.-A., Choi, Y. & Choe, J. K. Novel ssDNA aptamer-based fluorescence sensor for perfluorooctanoic acid detection in water. Environ. Int. 158, 107000 (2022).
TA Instruments. Quick Start: Isothermal Titration Calorimetry (ITC). https://www.tainstruments.com/pdf/literature/MCAPN-2016-1.pdf.
Jiménez, G. C. et al. Aptamer-Based Label-Free Impedimetric Biosensor for Detection of Progesterone. ACS Publications https://pubs.acs.org/doi/pdf/10.1021/ac503639s (2015) doi:10.1021/ac503639s.
Xiao, Y., Lai, R. Y. & Plaxco, K. W. Preparation of electrode-immobilized, redox-modified oligonucleotides for electrochemical DNA and aptamer-based sensing. Nat Protoc 2, 2875–2880 (2007).