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Introduction

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In the scientific world, measurement serves as the fundamental bridge between the conceptual design and tangible results. It enables us to understand, quantify and compare the intricacies of biological systems. Comparisons are the foundation upon which the progress in this field is built, and it allows us to assess the efficiency and reliability of your work. The reliability of data is the bedrock of scientific research, empowering researchers to make informed decisions, confidently predict outcomes, and even to enhance the credibility of iGEM projects such as ours. Ensuring the accuracy and reproducibility of measurements is crucial. Replicates and controls, the two pillars of scientific rigor, allow us to confirm statistical significance and to provide a reference point to differentiate experimental and external factors. They ensure that measurements are as close to the objective scientific truth as possible and offer a baseline for comparison. Understanding the nuances of measurement is essential for driving meaningful advancements in synthetic biology, whether you're a seasoned iGEMer or new.
To get detailed analysis of our results mentioned below, check out our proof of concepts and results pages

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Magnetic Nanoprobes

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Formation of the probe:

Our probe consists of three different sequences, namely, sequences A, B and C, which, when incubated together, would bind by complementarity to form the probe. The ideal ratio of sequences A:B:C is 1:1:3 respectively. Consider A, B and C of the same concentration X in moles per litre. The solution to make the probe would be to have them in a 1:1:3 volume ratio. Here, the number of A or B will be about the same as the number of probes in the final solution. The final concentration of the probe = X/6, as the solution is getting diluted six times. This calculation is used when making the probes for running Native PAGE.

Native PAGE (Polyacrylamide Gel Electrophoresis)

The probes were prepared by using 10 uM concentration aliquots of all the sequence

• Seq A = 15 ul
• Seq B = 15 ul
• Seq C = 60 ul

The final concentration of probe = 15/3 = 1.667 uM The final volume = 90 ul This solution is incubated at 48 degrees for 2 min and then at room temperature for 1 hour. Since the native PAGE would preserve the structure of the probe, the size of the probe cannot be accurately measured using a ladder. Proper controls were used for this. The order of sample in each well from the left is Sequence A, Sequence B, Sequence C, Sequence A+B, probe (A+B+C), probe + miDNA

• A and B are of the same length and have similar structures; hence, they have moved equal distances in the gel
• A+B acts as a control.
• A+B was chosen as the control because sequence C is one-third the nucleotide length of A and B, so a band higher than A+B can only be formed if all three sequences bind together to form the stable probe structure.
• The band of probe + miDNA being higher than just the probe proves that the miDNA is binding to the probe.

Fluorescence intensity-based experiments using a microplate reader

• An equal volume of all samples was taken to avoid variation in data because of the difference in volume
• In experiments involving multiple tests on the same type of sample, the sample used was always prepared as one batch to minimise variation due to errors during preparation.
• A control was present in all experiments. The control had an equal amount of probes as all the other samples and had an equal volume of buffer without miDNA in it. This ensured that
  a) The volume was the same as other samples
  b) Any change in fluorescence intensity caused by the buffer can be detected and that variability can be accounted for.
• For all the experiments in the nanoprobe, an emission scan was performed instead of measuring the intensity at one particular wavelength. This allowed us to see the whole range of excitation and emission.
  The excitation was kept constant at 490 nm
  The emission wavelength was collected in the range of 500-600 nm
• The results for all these experiments are plotted as percentage changes in fluorescence intensity, but the values obtained from the experiment were as fluorescence intensity. This was converted into percentage changes in fluorescence intensity with the help of the control used. This conversion was made as it was a better way to present and interpret the data.

The following equation was used to calculate the percentage change in fluorescence intensity: Percentage change in FI =((FI of sample - FI of control)/FI of control)*100 For example, the plot for the fluorescence intensity scan before and after the binding of the probe would be:

The percentage change in intensity representation would be:

This representation gives us a better understanding of the difference in fluorescence intensity measured across different wavelengths.

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Aptasensors

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Hybridization time graph experiments:

• iGEM Interlab studies have shown that the reproducibility of fluorescence measurements is a major challenge, due to a variety of factors, including differences in instrumentation, sample preparation, and data analysis. This can lead to significant variations in the results obtained from different laboratories, which can hinder the advancement of scientific research and the development of new technologies.
• To assess the reproducibility of our data and to address the challenge of reproducibility in FRET aptasensor development, we measured the fluorescence readings to determine the hybridization time required for formation of aptasensor using both a microplate reader and a fluorolog spectrofluorometer and as shown in the results page and the graphs we observe a similar trend in both the plate reader and the fluorolog which shows our data is reproducible across instruments .
• We used Ft/ Fo values for plotting our data instead of absolute values because Ft/Fo are normalized to the initial fluorescence value (Fo), which makes them less prone to errors and variations in sample concentrations, excitation intensity and other factors. Since they are not affected by concentrations of the fluorophore, we can use these to compare fluorescence intensities of two different samples with more accurate and reliable results.
• For measuring fluorescence intensities we measure arbitrary units cause there is no actual unit for it; having Ft/Fo ratio helps in accounting for this variation and makes sure that the data is consistent with different samples and machines.
• To determine the hybridization time required for the formation of the cortisol aptasensor complex between the aptamer and cDNA1, we performed a time-course experiment by measuring fluorescence quenching in a plate reader (TECAN M plex plate reader). We took multiple reads per well, to reduce errors due to instrumental noise, data processing, or simple variation and to increase precision of the data.
• We included three controls in the experiment: (1) TE buffer alone, (2) aptamer alone, and (3) cDNA1 alone. We observed negligible quenching for cDNA1 alone, and very low quenching for aptamer alone and TE buffer alone. In contrast, the aptamer and cDNA1 combination showed significantly high fluorescence quenching, which increased over time as the hybridization reaction progressed.

• To further validate our results, we repeated the hybridization time experiment for the formation of the FRET aptasensor complex with cDNA1 and cDNA2 at two different concentrations, 1:3 and 1:5 in a spectrofluorometer (Fluorolog). We observed similar results to those obtained with the plate reader, indicating that the FRET aptasensor is a reliable and reproducible method for measuring the hybridization time of aptamers with different cDNA targets at different concentrations.

ITC experiments:

• Isothermal titration calorimetry (ITC) is a technique that measures the heat released or absorbed during a biomolecular binding event. This heat change is proportional to the binding affinity, which is a measure of the strength of the interaction between the two molecules. We used ITC to confirm the binding affinity of the cortisol FRET aptamer and the serotonin FRET aptamer with their respective cDNA targets, as well as the binding affinity of the aptamers with their respective biomarkers.

• Since biomarkers have a higher affinity for the aptamers than the cDNAs,it could be one the possible reason for displacing the cDNAs when they are present in the solution, the aptamers should also have a good binding affinity with their biomarkers. The fact that biomarkers have a higher affinity for aptamers than cDNAs is important for the development of aptasensor-based diagnostic tests. It ensures that the aptasensors will be able to detect biomarkers even in the presence of high levels of cDNA, such as those found in blood samples.
• Isothermal titration calorimetry (ITC) can provide a wealth of information about a biomolecular binding event, including Ka, delta H, delta S, etc. A higher Ka value suggests a stronger binding association between the two biomolecules.
• We found Ka values and other parameter which define the binding between the our aptamer and cDNA which validates our aptasensor formation and the binding between the procured biomarker and its respective aptamer

Native PAGE -

• Native polyacrylamide gel electrophoresis (PAGE) can also be used to study the binding of FRET aptamers to their cDNA targets and biomarkers. It works by separating molecules based on their size and charge.
• If the aptamer and its cDNA target (or biomarker) are bound together, they will form a complex that is larger and has a higher charge than either molecule alone. As a result, the complex will migrate slower through the gel.
• The intensity of the bands can also be used to assess the binding affinity between the aptamer and its cDNA target (or biomarker). A stronger binding affinity will result in a more intense band for the complex.

• By adding only FRET aptamer cortisol, only cDNA , and only cortisol in three wells, you can differentiate between the band sizes of the quantifiers and quantifiers and biomarkers on a native PAGE gel.

• We performed the same native PAGE experiment for both FRET aptamer-cDNA1 and FRET aptamer-cDNA2 pairs to compare the binding affinities of the two cDNAs for the FRET aptamer. By comparing the intensities of the quantifier bands on the gel, we could determine which cDNA has a higher binding affinity for the FRET aptamer.
• The FRET aptamer and cDNA1 pair produce a high intensity band on the gel, suggesting that this pair has a higher binding affinity than the FRET aptamer and cDNA2 pair.

Sensitivity experiments:

• We investigated the change in fluorescence intensity of FRET aptasensors upon the addition of different concentrations of biomarkers. We added biomarkers from 10ng/ml concentration to 400 ng/ml concentration, to match the physiological concentration of the biomarker present in plasma.
• We included four controls in the experiment: Milli-Q water, FRET aptamer only, cDNA only and Quantifier with 0ng/ml cortisol . More details about the experimental setup and data obtained can be found on the proof of concept page.
• We had multiple technical and experimental replicates for this experiment since this is one of the major experiments, and having more replicates for an experiment reduces variations and random errors in data.
• We did aptasensor sensitivity experiments for both cortisol and serotonin. For both sets of experiments, the fluorescence intensity of the controls remained constant, while the fluorescence intensity of the FRET aptasensors increased linearly with increasing concentrations of serotonin and cortisol.

• The graph shows that the fluorescence intensity of the FRET aptasensors increases linearly with increasing concentrations of cortisol and serotonin, while the fluorescence intensity of the controls relatively decreases.

Biomarker mix:

• To assess the specificity of our quantifiers for their respective biomarkers, we mixed all of the biomarkers with the quantifier at the same time.
• We had six different controls for this experiment: MQ, only FRET aptasensor, FRET aptasensor with only cortisol, FRET aptasensor with only serotonin, FRET aptasensor with only miDNA 132 and FRET aptasensor with only miDNA124.
• We added controls with quantifiers with only one biomarker to check its contribution in change in fluorescence of the final aptasensor with biomarker mix.
• We had multiple technical replicates for both FRET aptasensor cortisol and FRET aptasensor serotonin biomarker mixes.