Magnetic Nanoprobes

NOTE: In our experiments, we have used miDNA instead of miRNA as they are easier to work with, and the results can hold true in miRNA systems.

Q) Can our nanoprobes bind to their respective miDNA and produce a detectable increase in fluorescence intensity?

Fluorescence intensity change on adding the miDNA to the Nanoprobe (after attachment):

We conducted an emission scan with a constant excitation of 490 nm and an emission range of 500 nm to 600 nm.

Figure 1 : Figure showing the percentage change in fluorescence intensity across different emission wavelengths for nanoprobe 132 and nanoprobe 124 bound to their respective miDNA

Here, we have a maximum percentage increase of 81 percentage indicating that:

miDNA 132 and 124 are binding to the loop region of their respective nanoprobes and causing the loop to open, which in turn separates the fluorophore quencher pair, giving a higher fluorescence intensity reading.
The chosen fluorophore quencher pair shows good quenching activity in the absence of target miDNA
There is a clear difference in the fluorescence intensity upon binding of the miDNA, proving that this method can detect and quantify miRNA in a sample.

Q) Can the probes produce detectable fluorescence intensity changes at miRNA levels present in blood?

We conducted an experiment to see if our probe could emit detectable change in fluorescence in picomolar levels of miDNA (10 picomolar), as this is the lower concentration range of our selected miRNA in blood.

Figure 2 : Figure showing the percentage change in fluorescence intensity at 532 nm wavelength emission for nanoprobe 132 and nanoprobe 124 bound to their respective miDNA present in 10 picomolar concentration.

We were able to detect the fluorescence intensity change above 15 percentage, proving that:

The nanoprobes can detect the low level of miDNA concentration present in the sample without any amplification steps.
The lower increase in fluorescence intensity when compared to samples of larger concentrations shows that there is a direct correlation between the increase in fluorescence intensity and the increase in miDNA concentration. This shows that the method can be used for quantification

It has to be noted that we detected a large change in this value when the emission wavelength was changed with a maximum of 45 percentage change in fluorescence intensity, but this was only for either one of the probes.

At the range of 530 nm to 533 nm, both the probes showed similar percentage change in fluorescence intensity with values close to 15 percentage.

Q) Are the probes specific to our miDNA?

We tested the fluorescence intensity change in 4 different samples to find this.

The samples were

Sample 1: with the 25 ul probe and 75 microliter buffer as control.
Sample 2: with 25 microliter probe and 75 microliter target miDNA.
Sample 3: with 25 microliter probe and 75 microliter miDNA with just two nucleotide differences to the target miDNA.
Sample 4: with 25 microliter probe and 75 microliter miDNA, which is completely different from the target miDNA.

All the miDNA used was of 10 nanomolar concentration.

Now, the percentage exchange in fluorescence intensity was calculated using the fluorescence intensity of control as the base value.

The graph for probe, specific to 124 is:

Figure 3 : Figure showing the percentage change in fluorescence intensity across different emission wavelengths for three different samples. Sample 2 = 124 nanoprobe + miDNA 124, sample 3 = 124 nanoprobe + miDNA 124 sequence with 2 nucleotide difference, sample 4 = 124 nanoprobe + miDNA 132.

From the graph,

The change in fluorescence intensity is close to 78 percentage for the sample with our target miDNA, proving that the experimental conditions were optimal for the binding of miDNA to happen
Both the non-target miDNA and the miDNA with just two nucleotide difference to the target miDNA sequence did not show any significant increase in fluorescence intensity. This shows that the nanoprobe 124 is highly specific to its target.

The graph for probe, specific to 132 is:

Figure 4 : Figure showing the percentage change in fluorescence intensity across different emission wavelengths for three different samples. Sample 2 = 132 nanoprobe + miDNA 132, sample 3 = 132 nanoprobe + miDNA 132 sequence with 2 nucleotide difference, sample 4 = 132 nanoprobe + miDNA 124.

The data from this experiment did not provide a reliable conclusion.

This may be due to errors in the preparation of miDNA samples or nanoprobes.

Aptasensors

Q) Does our aptamer bind to our procured biomarker?

The aptasensor mechanism only works if the procured biomarker which in our case was hydroxycortisone and serotonin (procured from Merck) displaces the cDNA strand and causes the change in conformation of aptamer that allows binding of the aptamer to the biomolecule.
- This was tested by running an ITC experiment where the sample cell held the aptamer and was titrated against the biomarker providing us with the binding affinity data.

Figure 5 : Final ITC graph of 44-mer serotonin aptamer titrated against Serotonin

Figure 6 : Final ITC graph of 85-mer cortisol aptamer titrated against Cortisol

Figure 7 : Final ITC graph of 85-mer cortisol aptamer titrated against Cortisol parameters

Q) How sensitive is our aptasensor?

The sensitivity of an aptasensor is its ability to detect small changes in the concentration of the target molecule, or biomarker. To test the sensitivity of the aptasensor, we used the ideal aptasensor that had been determined in previous experiments. This means that we had already optimized the aptasensor for the detection of our biomarker. Next, we measured the increase in fluorescence obtained upon adding varying concentrations of our biomarker. By measuring the increase in fluorescence, we can determine the sensitivity of the aptasensor. The higher the sensitivity, the lower the concentration of biomarkers that can be detected.

Figure 8 : Sensitivity of serotonin aptasensor

Sensitivity of serotonin aptasensor:

Serotonin concentration (ng/ml)	0	10	50	100	150	200	250	300	350	400
Fold change wrt to aptasensor + 0ng/ml	1	1.216	1.277	1.288	1.237	1.253	1.238	1.260	1.303	1.392

These data points were taken after 60 mins incubation of the aptasensor and serotonin. The fold change in fluorescence is calculated by dividing the fluorescence of the aptasensor with serotonin by the fluorescence of the aptasensor without serotonin. This allows us to compare the fluorescence of the aptasensor at different serotonin concentrations.The fact that the fluorescence increases linearly with increasing concentrations of serotonin is important because it means that the aptasensor can be used to quantify serotonin levels, although we didn’t expect an increase in fluorescence upon adding milliQ to our aptasensor the increase observed upon addition of varying concentration of serotonin showed a greater fluorescence reading. Hence we took this into consideration and indicate the fold change wrt to aptasensor + 0ng/ml therefore we can reliably correspond the fold change to a particular serotonin concentration mimicking human physiological levels present in the human serum.

Figure 9 : Change in fluorescence over time upon addition of varying concentration of serotonin.

From this graph we can see that the controls such as milli Q show decrease in fluorescence as time increases but the aptasensor plus cortisol for varying concentrations shows a similar trend of increase in fluorescence as time increases this shows that the aptasensor, this trend is visible in multiple technical replicates. This shows that the aptasensor loses its cDNA strand which causes its increase in fluorescence due to loss of the FRET system.

Sensitivity of cortisol aptasensor:

Figure 10 : Sensitivity of cortisol aptasensor

The sensitivity tests were done using cDNA 2 and 85-mer cortisol aptamer to form the ideal aptasensor since both cDNA 1 and cDNA 2 showed similar trends for the formation of aptasensor. From the result shown we can derive that the cortisol aptasensor shows higher sensitivity from the range of 50ng/ml to 200 ng/ml which lies well in the range of human physiological serum levels of cortisol.

Cortisol concentration (ng/ml)	0	10	50	100	150	200	250	300	350	400
Fold change wrt to aptasensor + 0ng/ml	1	1.03	1.07	1.10	1.15	1.16	1.05	1.06	1.03	1.05

Figure 11 : Change in fluorescence over time upon addition of varying concentration of cortisol with cortisol aptasensor.

The graph shows that the fluorescence of the controls (aptasensor and milliQ) decreases over time, but the fluorescence of the aptasensor with varying concentrations of cortisol increases over time. This suggests that the aptasensor loses its cDNA strand, which causes an increase in fluorescence due to the loss of the FRET system. This trend is visible in multiple technical replicates, which confirms that it is not a one-off event.

The sensitivity of an aptasensor is its ability to detect small changes in the concentration of the target molecule, or biomarker.We wanted to validate our chosen cortisol aptasensor formed using cDNA 2 by checking the sensitivity of the truncated cortisol aptasensor.

Figure 12 : Truncated cortisol aptasensor sensitivity

Figure 13 : Change in fluorescence over time upon addition of varying concentration of cortisol with truncated cortisol aptasensor.

The truncated cortisol aptasensor as expected showed poor sensitivity over the range of 50-300 ng/mL, which is the range of cortisol concentrations that are physiologically relevant in human serum. This means that the truncated aptasensor would not be able to reliably detect cortisol levels in human serum.

Q) How specific are our aptasensors?

Specificity of our aptasensors:

Figure 14 : Specificity of serotonin aptasensor

From this experiment we aim to find out the specificity of our serotonin aptasensor as in the effectiveness of the turn on mechanism upon interaction with the specific biomarker which in this case is serotonin. So we wanted to know if we can observe an increase in fluorescence when the serotonin aptasensor was placed with various other biomarkers such as Cortisol, miDNA 132, miDNA 124 and we also wanted to see the the effectiveness of the aptasensor in a mixture of biomarkers:

The fluorescence difference shown in the above result is fluorescence measured at time t = 90 mins minus the fluorescence at time t = 0 mins. We observed that the serotonin aptasesnor showed a greater fluorescence difference when placed in addition with serotonin biomarker and the mixture of biomarker when compared to its difference shown in presence of other biomarkers. Hence this proves that our serotonin aptasensor is specific in nature. An even greater difference is observed in the biomarker mix data but this is expected because the excess fluorescence could be due the background fluorescence seen in the presence of other biomarkers as shown in the graph