---

Magnetic Nanoprobes

---

CHARACTERISATION OF PROCURED NANOPARTICLES

Q) Do the magnetic nanoparticles have a stable carboxylic group, and do they disperse well in the medium?

Analysis of the zeta potential
This experiment was done to verify that our nanoparticles had stable carboxyl groups. After we received the graph from the zeta analyzer, we were able to see that

• The graph had a single peak: This showed that our particles were well dispersed in the medium. Well-dispersed particles will provide more accurate zeta potential values.
• The zeta potential was -21 millivolts. The negative zeta potential proved that the carboxyl groups were indeed present in the nanoparticles. The negative value is caused due to the deprotonation of the carboxyl group in aqueous solution.
• The negative value of less than -20 mV proves that the carboxyl groups are stable.

Q) What is the size of the nanoparticles?

TEM analysis: The TEM image given below shows that the nanoparticles we procured were, on average, about 12 nanometers in diameter.

CONFIRMATION OF PROBE FORMATION AND MIDNA BINDING

Q) Are the sequences actually binding to form the probe? If so, can the target miDNA bind to this probe?

Native PAGE: Native Polyacrylamide gel electrophoresis ensures that the structure of the probe remains intact. This experiment can verify if our probes are actually being formed and also if our miDNA is binding to the probe. The bands in the native page given are in the following order from the left:
Sequence A (2), sequence B (3), sequence C (4), Sequence A+B (5), Sequence A+B+C (incubated for 1 hour (1:1:3 ratio) to form the probe) (6), Probe + miDNA (7) Here,

• The bands of sequence A and B are at the same level. These sequences have the same number of nucleotides.
• The A+B lane is higher than the A and B lanes, but the band is smeared. This is because A and B are only partially complementary, and all of them will not bind and form a stable structure.
• The lane which has A+B+C (which forms the probe) has just one band, which is higher than the A+B band, indicating that these three sequences are being bound together by complementarity and are forming a stable structure.
• Now, we can also see that the band in the lane of probe + miDNA is slightly higher than the band in the lane with just the probe. Indicating that the miDNA is indeed binding to the probe. The difference is only small because the miDNA is of just 22 nucleotide length.

FLUORESCENCE-BASED EXPERIMENTS USING MICROPLATE READER.

For all the following experiments,

• The percentage change in fluorescence intensity was calculated by
  Percentage change in fluorescence intensity = ((FI of the sample - FI of the control)/ FI of the control)*100
  Where FI = fluorescence intensity
• The control used in all the experiments has an equal amount of probe and buffer without the miDNA present to make the volume constant.
• For all the experiments, the incubation time was 1 hour.

Q) Did the miDNA binding actually cause the fluorophore quencher pair to move away? Fluorescence intensity change upon adding the miDNA to the probe, before binding the probe to the nanoparticle. We tried both excitation and emission scans to see what the maximum change that we could detect was.
From these experiments, we could see that:

• The maximum percentage change observed was 19 percentage.
• This was mainly because of the large background noise that was present as there was unbound A or B sequence, which had fluorophore attached to it present in the mixture. These could only be removed after binding the probes to the nanoparticle.
• It still showed that there was an increase in fluorescence intensity on the binding of miDNA in both 124 and 132 probes, proving that the binding of miDNA is causing the loop to open and separate the fluorophore quencher-pair

Q) Did the binding of the probe to the magnetic nanoparticle to form the nanoprobe actually affect the readings?

Fluorescence intensity change on adding the miDNA to the Nanoprobe (after attachment) To test this, we first measured the fluorescence intensity change of the new nanoprobes. We conducted an emission scan with a constant excitation of 490 nm and an emission range of 500 nm to 600 nm.

Here, we have a maximum percentage increase of 81 percentage. Comparing the results before and after attachment of the probe to the nanoparticle to form the nanoprobe:

This result clearly indicates that the nanoprobes give better fluorescence intensity change when compared to just probes. The results for the detection limit and sensitivity experiments are provided in the proof of concept page. The future works planned for further development of our project can be found here

---

Aptasensors

---

Q) How did we confirm the hybridisation of the cDNA and the aptamer to form our aptasensor?

Native PAGE (14%)

• The aim of this experiment was to detect the hybridised complex of cDNA 1 and the 85-mer aptamer.
• The protocol and the concentrations used are mentioned in the experiment and lab notebook page respectively.
• No clear indication of hybridisation could be derived from the native PAGE result, this could be due to the small size of our aptamer and cDNA. The experiment was carried out using a 16% gel and similar results were seen.



• The only band was observed for 85-mer aptamer plus cortisol ,we presume the cDNA 2 which is a 15-mer had run off the gel.



• In this experiment, we increased the concentrations of the cDNA and the Quantifiers, as well as the percentage of the gel to 16%, in order to visualise the small fragments of cDNA, such as the cDNA 2 (15-mer). However, even with these modifications, we could only observe bands corresponding to the 85-mer aptamer.

Isothermal Titration Calorimetry:

• The aim of this experiment was to detect the hybridised complex of cDNA and aptamer.
• To check duplex formation between the 44-mer serotonin aptamer and 20-mer cDNA 1:
• We prepared 0.25 micromolar concentration of the 44-mer serotonin aptamer and titrated it against 1 micromolar concentration of cDNA 1.The reference cell contained the same Milli-Q water that we used to prepare our working concentration.
• 1.5 microliters of injection were given every 80 seconds for a total of 20 injections.

• From the ITC result shown we can conclude that there is a strong association between cDNA 1 and the 44-mer serotonin aptamer. Given below are the obtained parameters from the ITC experiment:

• To check duplex formation between the 85-mer cortisol aptamer and 40-mer cDNA 1: We used the same procedure as described above, and a detailed protocol is available on the experiments page.</li>

In our experiments, we also observed a relationship between the cortisol cDNA 1 and the 85-mer aptamer. This relationship was evident in the shape of the graph, which became more sigmoid-shaped towards the end of the injections. This suggests that the two molecules are interacting with each other in a specific way.

We believe that this result could be strengthened by using a higher concentration of cDNA. This is because a higher concentration of cDNA would mean that more molecules would be available to bind to the aptamer. This would lead to more complete binding in fewer injections, and would therefore make the relationship between the two molecules more apparent. To test this hypothesis, we would need to conduct further experiments. These experiments would involve using different concentrations of cDNA and measuring the binding between the cDNA and the aptamer. If our hypothesis is correct, we expect to see more complete binding in fewer injections when a higher concentration of cDNA is used.

• To check duplex formation between the 44-mer serotonin aptamer and 12-mer cDNA 2
• We used the same procedure as described above, and a detailed protocol is available on the experiments page.
• The results of the ITC experiment show that there is no obvious interaction between cDNA 2 and the 44-mer serotonin aptamer. This result is consistent with the results of the fluorescent-based hybridization experiments shown below.

Fluorescence based experiments:

• We designed these experiments to detect the hybridization of the aptamer to the cDNA by observing the rate of quenching of the FRET signal. A successful binding event results in the quenching of the FRET signal.
• More details on the result can be found below.

Q) How did we confirm the ideal molar ratio for the formation of our aptasensors?

• Before we try to find the ideal molar ratio, we need to determine the lowest concentration of aptamer that is detectable in our fluorolog fluorospectrometer: The chosen starting concentration of 0.25 micromolar for the aptamer was derived by running various concentrations ranging from 0.1 micromolar to 0.5 micromolar concentrations,we wanted find the minimal concentration of aptamer that gives a considerable/detectable fluorescence level, attached below are the fluorescence emission scan ranging from 495 nm to 700 nm of the various molar concentrations:

Concentration (micromolar)	0.1	0.25	0.5
Fluorescence obtained from fluorolog at 517 nm	77490	689130	4852050

• We used the same starting concentration of 0.25 micromolar for the 85-mer aptamer and the 44-mer serotonin aptamer, but we used a starting concentration of 0.5 micromolar for the truncated cortisol aptamer. We determined this starting concentration using the same method as described above.
• Finding the ideal molar ratio is important for obtaining the most sensitive aptasensor. Now that we have determined the starting molar concentration for the aptamers, we need to determine the ideal molar ratio for forming our aptasensor. The ideal molar ratio can be measured by observing the difference in fluorescence intensity at t=0 (before binding) and t=60 mins (after binding).
• The ideal molar ratio will vary depending on the aptamer and target molecule being used. However, a good starting point is to use a molar ratio of 1:1. This means that there will be an equal number of aptamer molecules and target molecules in the solution. Once you have determined the ideal molar ratio, you can use it to prepare a solution for your aptasensor. This solution will be the most sensitive for detecting the target molecule.



• For 85-mer cortisol aptamer and cDNA 1 the ideal molar ratio was observed to be 1:3 and 1:5 as seen from the graph



• For the 85-mer cortisol aptamer and cDNA 2 the ideal molar ratio was observed to be 1:3



• For the truncated aptamer and cDNA the ideal molar ratio was decided to be 1:2 because similar fluorescence differences were seen for 1:4 and 1:5.



• The ideal molar ratio of 44-mer serotonin aptamer to cDNA 1 serotonin was observed to be 1:3

Q) How did we confirm the ideal hybridization time for formation of aptasensors?

• The ideal hybridization time is determined by plotting the Ft/F0 values against time, using data from the fluorospectrometer and plate reader. This data can not only help us determine the ideal hybridization time, but it can also show us the quenching efficiency of the FRET system (fluorophore-quencher pair) for different molar ratios.
• Formation of cortisol aptasensor from 85-mer aptamer and cDNA 1



• The data points in the graph were obtained from a Fluorolog fluorospectrometer. From the graph, we can estimate that the ideal hybridization time is around 50-60 minutes, because the graph plateaus around that time. This means that maximum quenching of the donor fluorophore is achieved within 30 minutes of the hybridization reaction.

This data was also replicated using a 1:5 molar ratio of the 85-mer aptamer to cDNA 1 complex. From this, we can conclude that a 1:3 molar ratio is much more effective in forming the duplex due to the shorter hybridization time required to reach the plateau level of Ft/F0.

• This formation experiment of cortisol aptasensor with varying molar ratios ranging from 1:1 to 1:5 was performed using the microplate reader, The importance of this experiment is discussed in the measurements page. A major change in this experiment is the use of T.E. buffer to prepare the oligos aliquots instead of milliQ which was the resuspension media for the previous experiment. From this graph we were able to reproduce the ideal hybridisation time of 60 mins as seen from the fluorolog data. Upon examining the quenching outcomes, it is evident that the 1:3 molar ratio exhibits the highest quenching, approximately 45%. This finding aligns with our previous molar ratio results. The elevated quenching capability, rising from 30% as indicated in the fluorolog data to 45%, could potentially be attributed to the use of T.E buffer in lieu of milliQ. The need for using various controls in this experiment is discussed in the measurement page.

• Formation of cortisol aptasensor from 85-mer aptamer and cDNA 2:



• The graph shows the results of a fluorescence experiment to measure the hybridization time of an 85-mer aptamer to cDNA 2. The hybridization time is the amount of time it takes for the aptamer and cDNA 2 to bind to each other completely. The graph shows that the ideal hybridization time is around 50-60 minutes for both the 1:3 and 1:5 molar ratios. Maximum quenching of the fluorophore is achieved within 30 minutes of the hybridization reaction, for both the 1:3 and 1:5 molar ratios. This experiment shows that the ideal hybridization time is the same for both the 1:3 and 1:5 molar ratios. This suggests that the molar ratio of the aptamer to cDNA 2 does not significantly affect the hybridization time.
• This graph shows the hybridisation time and the quenching capacity showing similar trends across both the cDNA that was designed for the 85-mer aptamer sequence.
• Formation of cortisol aptasensor from truncated aptamer and cDNA:

• From this graph we can see that no plateau has been reached even after running the experiment for 90 mins and the maximum quenching efficiency observed is around 12% which is seen by 1:2 molar ratio again aiding our result obtained from the molar ratio graph.
• Comparing the various possible cortisol aptasensors to determine the best cDNA aptamer pair:



• The engineering page provides a detailed explanation of the DBTL cycle used to develop this aptasensor. The graph shows that the truncated cDNA and aptamer complex does not produce a very efficient FRET system. This is likely because the truncated cDNA is shorter and has fewer binding sites for the aptamer. As a result, there are fewer FRET pairs formed, and the quenching efficiency is lower. Additionally, the graph shows that cDNA 1 and cDNA 2 produce similar results in terms of hybridization time and quenching efficiency, regardless of whether they are complexed with the 85-mer aptamer. This suggests that the binding affinity of the aptamer for cDNA 1 and cDNA 2 is similar. The overall results of this experiment suggest that the truncated cDNA and aptamer complex is not a very good candidate for developing an aptasensor. The quenching efficiency is too low, and the hybridization time is too long. cDNA 1 and cDNA 2 along with the 85-mer aptamer are better candidates for developing an aptasensor.
• Formation of serotonin aptasensor from 44-mer aptamer and serotonin cDNA 1:



• We performed this experiment to determine the ideal molar ratio and hybridisation time for forming a serotonin aptasensor. We used a microplate reader to measure the fluorescence intensity of the aptasensor at different molar ratios of aptamer to cDNA. We performed multiple technical replicates and 3 experimental replicates to ensure the accuracy of our results. The ideal hybridization time for the serotonin aptasensor is observed to be around 50-60 minutes, which is similar to the ideal hybridization time for the cDNA cortisol aptamer complex. The quenching efficiency of the serotonin aptasensor is around 30% at a 1:3 molar ratio of serotonin to aptamer. This is consistent with the prediction that a 1:3 molar ratio is ideal for forming the serotonin aptasensor. The lowest quenching efficiency was observed at a 1:1 molar ratio of serotonin to aptamer, which was around 15%. The quenching data is supported by the ITC report, which shows that there is high affinity between the cDNA 1 and serotonin aptamer.

• Formation of serotonin aptasensor from 44-mer aptamer and serotonin cDNA 2:
















• From the above graphs we can observe that there is an increase in fluorescence upon apparent hybridisation of the cDNA 2 and serotonin aptamer oligos for all molar ratios. This trend was also observed in the replicate data, which is plotted on the same graphs. Multiple technical replicates were also performed, as shown by the standard deviations plotted with both the first and replicate data points. This data is further justified by the ITC plot we obtained for cDNA 2 and serotonin aptamer which showed us that there was no significant association between cDNA 2 and serotonin aptamer.

Q) How sensitive is our aptasensor? 

• Sensitivity tests included using the ideal aptasensor determined by the previous experiments and measuring the increase in fluorescence obtained upon addition of varying concentration of our biomarker mimicking the human physiological value.
• In conclusion we measured fold change with respect to aptasensor + 0ng/ml for the serotonin and cortisol aptasensor and for the serotonin aptamer, we could observe a linear relationship with increase in serotonin concentration but we found the cortisol aptasensor to show higher sensitivity with 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.
• The analysis of the results will be discussed in length in our proof of concept page.

Q) How specific is our aptasensor?

•  To check the specificity of our aptasensor we conducted a biomarker mix test where we tested the specificity of our serotonin aptasensor by checking it’s rise in fluorescence upon addition of various different biomarkers other than serotonin and a mixture of all the biomarkers: the theory benign if our aptasensor is specific the increase in fluorescence upon addition of other biomarkers other than serotonin should show reduced increase in fluorescence compared to the increase obtained when bound to serotonin, this increase should also be visible when the aptasensor in exposed to the mixture of biomarkers.
• In conclusion 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.
• The analysis of these results will be discussed in detail in the proof of concept page

---

Gsα Protein

---

Our Initial plans for Gsα protein included expression, purification and quantification by aptasensors. We intended to synthesize an aptamer for Gsα protein using the SELEX (Systematic Evolution of Ligands by Exponential Enrichment ) method to select the aptamers that binds specific to the Gsα Protein.  Unfortunately, due to time and budget constraints we were unable to procure and work with the aptamers. The cloning experiments performed by us are documented here and our future protocols regarding purification and formation of aptasensors is further elaborated on the Implementation page. The Gsα Gblock was procured through IDT.

Q) How did we confirm the amplification of Gsα protein?

We did a PCR reaction for the amplification of the Gsα Gblock. This PCR reaction was run at 57 degrees celsius and there was no band observed. While consequent troubleshooting, the annealing temperature was manually calculated and obtained to be around 63 degrees celsius. In order to confirm the amplification of the Gsα Gblock, we ran a gradient PCR. The optimum temperature for amplification was found to be at 62.5 degrees celsius. 

• We did not visualize the ChemiDoc imaging system since we did not want to compromise the quality of our elute DNA. It was visualised in a UV transilluminator.
• Using the gradient function of the universal block, a gradient of 54 to 63°C was set. This was achieved by setting a 9 degree C gradient across 12 heating blocks. The following test parameters were selected: denaturation 95,30 s, annealing 55°C -63°C, 20 s, elongation 68°C, 150s, Taq-Polymerase 1.25units.
• Tubes H,I,J,K,L was annealed at 55.1, 57.5,60.5,62.4,63.1 degree celsius respectively. Positive control (1.5kB dna fragment) was annealed at 55.1 degrees.
• The bands were found to be most visible in lanes K and L. This implied that the correct annealing temperature lies in the range of 62-63 degrees. 
• PCR amplification was performed again with the right annealing temperature

Q) How did we confirm restriction digestion?

After overnight restriction digestion, we ran an agar gel with the Gsα Gblock and PET 19b Plasmid. We used two positive controls, a gene fragment of 1.5kb and our amplified Gsα Gblock (1.2kB). However, while we observed bands for the restriction-digested insert and plasmid, we were not able to observe the band for positive control.

• We did not visualize the ChemiDoc imaging system since we did not want to compromise the quality of our elute DNA. It was visualised in a UV transilluminator.
• Second band was observed to be around 1kb which confirms the formation of restriction digested insert of 1198bp size
• Third band is observed between 5kB and 6kB bands of the ladder which confirms the presence of restriction digested vector. An undigested plasmid would have given three different bands for nicked, linear and supercoiled. Since we observed only one band here, we assume that most of the pET19B(5.7kB) is succesfully restriction digested.
• Bands are not visible for the positive controls. This might be because 

1. Compromised gel integrity on one half of the gel only - wells might have collapsed
2. Contamination of samples might hinder the migration of fragments during agarose gel electrophoresis.
3. Improper mixing of loading dye and sample

After restriction digestion we were able to get the fragments of insert and vector. So in order to introduce our desirable DNA fragment into our vector for cloning and transformation, ligation had to be done to join the insert and vector fragment. We set up a ligation reaction with a reverse and backward primer and a T4 DNA ligase. It did not yield a desirable result.  When further transformation experiments were carried out, we found 2 colonies. A master plate was made and a colony PCR was performed. This again did not work. The possible reasons for ligation PCR to not give desirable results could be due to the machine being faulty. Due to time constraint, we were unable to proceed forward with troubleshooting the protocol and further experiments for the Gsα Gblock.