iGEM 2022 — Team asij-tokyo

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## NOTEBOOK

# August 24, 2022

Time: 7:00
Member: Ms. Crissy
What: Aptamer Resuspension
**S2.2: GCAGTTGATCCTTTGGATACCCTGG**

1. Before open the container, always briefly SPIN DOWN for the first time after delivery to avoid loss of the aptamer pellet. Briefly centrifuge the aptamer tube to ensure the dried aptamer pellet is at the bottom

2. Dissolve the stock aptamers completely to the desired stock concentration with buffers by shaking. Use buffer shown above. The recommended diluent volume is 100ul. The concentration depending on your application and the yield of the resulting product. Resuspend the aptamer pellet in Aptamer Resuspension Buffer using the volume indicated in the Packing Slip that was included in your aptamer shipment. Quickly spin the sample down (>1000 x g for a few seconds)

3. Aliquot and store stock aptamers at -20°C to -70°C until you use it. Make a stock solution and working aliquots which should be thawed relatively infrequently.

4. Before every use, perform Heating & cooling (H&C) step for the PROPER FOLDING of aptamer structure in buffer including 1~5mM MgCl2 2. Please heat aptamers in proper buffer solution at 95 C for 5 min (in PCR machine), and then leave the tubes on the bench for 15 min.

5. Prior to use, dilute the aptamer to 10 - 100x working concentration in buffer shown above. Heat the aptamer solution to 90-95°C for 5 minutes

6. Allow the aptamer solution to cool to room temperature (~15 minutes). Aptamers are conformationally stable for at least one week at room temperature unless the salt or temperature is significantly changed (falls below 0°C or rises above 30°C).

7. The aptamer can now be further diluted into the final working buffer for your specific application, or Application Buffer. Please use 100 mM NaCl, 5 mM MgCl2, pH 7.2 in Tris or Hepes buffer.

# August 24, 2022

Time: 8:00-17:00
Member: Kai
Members: Kai, Koharu, Annmarie, Rui, Hana
What: FRET verification through various protocols
 diluting aptamer and biomarker solutions to the intended concentrations
 test for aptamer-biomarker binding through detecting fluorescence with the 96 wellplate reader

# Protocols:

**Protocol 1:**

1. Follow Resuspend protocol for aptasensor probes.
2. Make sure solution is suspended in correct buffer.
3. Prepare standard biomarker solution of biomarker using phosphate buffer solution, from a range of 0 to 100 nM in 10 nM increments (so 0 nM, 10 nM, 20 nM…).
4. Add biomarker to aptamer solution in a 9:1 ratio in 96-well plate (so for every 9 microliters of biomarker solution, add 1 microliter of aptamer solution). Aptamer solution should be 1000 nM Total volume should be 10 µL. Calculate final concentrations of aptamer and biomarker in solution using M1V1 = M2V2. Record in table.
5. Let incubate for 30 minutes. Remember to label each well with the concentration of biomarkers used.
6. Place under 96 well plate reader and measure fluorescence. Use fluorescence corresponding to the aptamer probe being used: excited at 627nm, emitted at 660-720. Record in table.
**Result: There was no statistical difference between the fluorescence values between wells.**

**Protocol 2:**

1. Follow Resuspend protocol for aptasensor probes.
2. Make sure solution is suspended in correct buffer.
3. Prepare standard biomarker solution of biomarker using phosphate buffer solution, from a range of 0 to 100 nM in 10 nM increments (so 0 nM, 10 nM, 20 nM…).
4. Add biomarker to aptamer solution in a 9:1 ratio in 96-well plate (so for every 90 microliters of biomarker solution, add 10 microliter of aptamer solution). Aptamer solution should be 1000 nM Total volume should be **100 µL**. Calculate final concentrations of aptamer and biomarker in solution using M1V1 = M2V2. Record in table.
5. Let incubate for 30 minutes. Remember to label each well with the concentration of biomarkers used.
6. Place under 96 well plate reader and measure fluorescence. Use fluorescence corresponding to the aptamer probe being used: excited at 627nm, emitted at 660-720. Record in table.
**Result: There was no statistical difference between the fluorescence values between wells.**

**Protocol 3:**

1. Follow Resuspend protocol for aptasensor probes.
2. Make sure solution is suspended in correct buffer.
3. Prepare standard biomarker solution of biomarker using phosphate buffer solution, from a range of 0 to 100 nM in 10 nM increments (so 0 nM, 10 nM, 20 nM…).
4. Add biomarker to aptamer solution in a 9:1 ratio in 96-well plate (so for every 180 microliters of biomarker solution, add 20 microliter of aptamer solution). Aptamer solution should be 1000 nM Total volume should be **200 µL**. Calculate final concentrations of aptamer and biomarker in solution using M1V1 = M2V2. Record in table.
5. Let incubate for 30 minutes. Remember to label each well with the concentration of biomarkers used.
6. Place under 96 well plate reader and measure fluorescence. Use fluorescence corresponding to the aptamer probe being used: excited at 627nm, emitted at 660-720. Record in table.
**Result: There was no statistical difference between the fluorescence values between wells.**

**Protocol 4:**

1. Add 50µL of aptamer solution at 1000µM into 96 wellplate
2. Add 50µL of biomarker solution at 1000µM into 96 wellplate
3. Place under 96 well plate reader and measure fluorescence. Use fluorescence corresponding to the aptamer probe being used: excited at 475nm, emitted at 500-550
**Result: There was no statistical difference between the fluorescence values between wells.**

The first round of tests were unsuccessful. Maybe the fluorescence value that we are detecting is incorrect. Also, it could be an issue with the way we resuspended the aptamers.

# August 25, 2022

Time: 7:00
Member: Ms. Crissy
What: Aptamer Resuspension
**S2.2: GCAGTTGATCCTTTGGATACCCTGG**

1. Before open the container, always briefly SPIN DOWN for the first time after delivery to avoid loss of the aptamer pellet. Briefly centrifuge the aptamer tube to ensure the dried aptamer pellet is at the bottom

2. Dissolve the stock aptamers completely to the desired stock concentration with buffers by shaking. Use buffer shown above. The recommended diluent volume is 100ul. The concentration depending on your application and the yield of the resulting product. Resuspend the aptamer pellet in Aptamer Resuspension Buffer using the volume indicated in the Packing Slip that was included in your aptamer shipment. Quickly spin the sample down (>1000 x g for a few seconds)

3. Aliquot and store stock aptamers at -20°C to -70°C until you use it. Make a stock solution and working aliquots which should be thawed relatively infrequently.

4. Before every use, perform Heating & cooling (H&C) step for the PROPER FOLDING of aptamer structure in buffer including 1~5mM MgCl2 2. Please heat aptamers in proper buffer solution at 95 C for 5 min (in PCR machine), and then leave the tubes on the bench for 15 min.

5. Prior to use, dilute the aptamer to 10 - 100x working concentration in buffer shown above. Heat the aptamer solution to 90-95°C for 5 minutes

6. Allow the aptamer solution to cool to room temperature (~15 minutes). Aptamers are conformationally stable for at least one week at room temperature unless the salt or temperature is significantly changed (falls below 0°C or rises above 30°C).

7. The aptamer can now be further diluted into the final working buffer for your specific application, or Application Buffer. Please use 100 mM NaCl, 5 mM MgCl2, pH 7.2 in Tris or Hepes buffer.

# August 25, 2022

Time: 8:00-17:00
Member: Kai
Members: Kai, Koharu, Annmarie, Rui, Hana
What: FRET verification through various protocols, diluting aptamer and biomarker solutions to the intended concentrations
 test for aptamer-biomarker binding through detecting fluorescence with the 96 wellplate reader
 (We realized yesterday that we forgot to use the buffer used for the aptamer probes to dilute with)

# Protocols:

**Protocol 5:**

1. Follow Resuspend protocol for aptasensor probes.
2. Make sure solution is suspended in correct buffer.
3. Prepare standard biomarker solution of biomarker using aptamer buffer solution, from a range of 0 to 100 nM in 10 nM increments (so 0 nM, 10 nM, 20 nM…).
4. Add biomarker to aptamer solution in a 9:1 ratio in 96-well plate (so for every 90 microliters of biomarker solution, add 10 microliter of aptamer solution). Aptamer solution should be 1000 nM Total volume should be **100 µL**. Calculate final concentrations of aptamer and biomarker in solution using M1V1 = M2V2. Record in table.
5. Let incubate for 30 minutes. Remember to label each well with the concentration of biomarkers used.
6. Place under 96 well plate reader and measure fluorescence. Use fluorescence corresponding to the aptamer probe being used: excited at 627nm, emitted at 660-720. Record in table.
**Result: There was no statistical difference between the fluorescence values between wells.**

**Protocol 6:**

1. Follow Resuspend protocol for aptasensor probes.
2. Make sure solution is suspended in correct buffer.
3. Prepare standard biomarker solution of biomarker using aptamer buffer solution, from a range of 0 to 100 nM in 10 nM increments (so 0 nM, 10 nM, 20 nM…).
4. Add biomarker to aptamer solution in a 9:1 ratio in 96-well plate (so for every 90 microliters of biomarker solution, add 10 microliter of aptamer solution). Aptamer solution should be 1000 nM Total volume should be **200 µL**. Calculate final concentrations of aptamer and biomarker in solution using M1V1 = M2V2. Record in table.
5. Let incubate for 30 minutes. Remember to label each well with the concentration of biomarkers used.
6. Place under 96 well plate reader and measure fluorescence. Use fluorescence corresponding to the aptamer probe being used: excited at 627nm, emitted at 660-720. Record in table.
**Result: There was no statistical difference between the fluorescence values between wells.**

**Protocol 7:**

1. Add 100µL of aptamer solution at 1000µM into 96 wellplate into each well A1-A12.
2. Add 100µL of biomarker solution at 1000µM into 96 wellplate into well A1
3. Perform serial dilution with 100µL from A1 to A11.
4. Let incubate for 30 minutes.
5. Place under 96 well plate reader and measure fluorescence. Use fluorescence corresponding to the aptamer probe being used: excited at 627nm, emitted at 660-720. Record in table.
**Result: There was no statistical difference between the fluorescence values between wells; however, the fluorescence values were abnormally high when set at a different frequency: excited at 475nm, emitted at 580-640.**

**Protocol 8:**

1. Add 100µL of aptamer solution at 1000µM into 96 wellplate into each well A1-A3.
2. Let incubate for 30 minutes.
3. Place under 96 well plate reader and measure fluorescence. Use fluorescence corresponding to the aptamer probe being used: excited at 475nm, emitted at 580-640. Record in table.
**Result: The fluorescence values were abnormally high when set at this frequency. We believe this to be attributed with the possibility that the manufacturer that we ordered these aptamer probes forgot to have the black hole quencher on one side, therefore, we will reorder the aptamer probes.**

# September 23, 2022

Time: 15:00-17:00
Member: Kai
Members: Kai, Julia
What: FRET verification through serial dilution
 Our reordered aptamers finally came
 Diluting aptamer and biomarker solutions to the intended concentrations
 Test for aptamer-biomarker binding through detecting fluorescence with the 96 wellplate reader
 We realized yesterday that we forgot to use the buffer used for the aptamer probes to dilute with

### Protocol:

1. Add 100µL of aptamer solution at 1000µM into 96 wellplate into each well A1-A12.
2. Add 100µL of biomarker solution at 1000µM into 96 wellplate into well A1
3. Perform serial dilution with 100µL from A1 to A11.
4. Let incubate for 30 minutes.
5. Place under 96 well plate reader and measure fluorescence. Use fluorescence corresponding to the aptamer probe being used: excited at 475nm, emitted at 580-640. Record in table.
**Result: Our experiment was successful and we were able to confirm our detection mechanism through FRET, as the fluorescence of our wells followed the concentrations of aptamer-biomarker concentrations. This means that our aptamers successfully bound to our biomarkers, fluorescing.**

![](https://static.igem.wiki/teams/4334/wiki/img-0507.jpg)

# September 28, 2022

Time: 16:00-17:00
Member: Koharu
Members: Koharu, Shun
What: ELISA Assay

### Protocol:

1: Coat 96 plate wells with 100 μl of the appropriate coating antibody, at a concentration between 1-10 μg/ml in coating buffer. Cover the plate and incubate overnight at 4°C

# September 29, 2022

Time: 16:00-17:00
Member: Kai
Members: Kai, Shun, JonJon
ELISA Assay

### Protocol:

2.Wash the plate 3 times in wash buffer.
3. Coat 96 plate wells with 100 μl of the appropriate coating antibody, at a concentration between 1-10 μg/ml in coating buffer. Cover the plate and incubate overnight at 4°C
3. Perform serial dilution with 100µL from A1 to A11.

# October 5, 2022

Time: 10:30-16:30
Member: Annmarie
Members: Annmarie, Kai, Koharu
ELISA Assay

### Protocol:

4. Wash the plate 3 times in wash buffer.
5. Add 150 μl of blocking solution to each well. Incubate for 1 hour at 37°C. Wash 4 times in wash buffer.

# October 6, 2022

Time: 15:00-16:00
Member: Kai
Members: Kai, Rui, Sara
What: Shelf-llife Test and Specificity Test

### Protocol:

1. Add 50 μl of aptamer solution at 1000µM into 96 wellplate into each well B1-B12.
2. Add 50 μl of biomarker solution at 1000µM into 96 wellplate into each well B1-B12.
3. Perform serial dilution with 50µL from B1 to B11.
4. Place under 96 well plate reader and measure fluorescence. Use fluorescence corresponding to the aptamer probe being used: excited at 475nm, emitted at 580-640. Record in table.
**Result: There was a statistical difference between the fluorescence values of the varying concentrations of biomarker solutions. This confirms that the aptamer probes still successfully bind to biomarkers and fluoresce after 2 weeks in the refrigerator.**

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## ENGINEERING SUCCESS

# Introduction

![](https://static.igem.wiki/teams/4334/wiki/engineering-success-cycle.jpg)

The second year of the ASIJ iGEM team project focused on expanding the aptamer-based detection method that
the 2021 team focused on through using fluorescent levels to detect Mucin-1 concentration in the FRET
assay.

Throughout the project, we actively engaged with the DBTL (Design, Build, Test, Learn) engineering cycle to
refine and learn more about the proposed detection method. Our team was faced with different challenges and
results along the way, but this helped to gather more information on the feasibility of our proposed
detection method.

# Design

### Biomarkers for Breast Cancer

A variety of biomarkers are associated with breast cancer, such as CA 15.3, MUCIN 1, and CA 27.29. These
biomarkers are present in blood and saliva. Although no protein has yet been recommended as a satisfactory
biomarker for the early detection of breast cancer, the three proteins mentioned above are being proposed as
potential candidates. Therefore, we have sought to construct a proof-of-concept using these three biomarkers, in
the hopes that future advances in the early detection of breast cancer may use the same technology and
techniques as we do here. Furthermore, the proteins shown here have established reference ranges in literature,
which makes them promising targets for future breast cancer diagnosis.

### Aptamers

Aptamers are single-stranded oligonucleotides that can bind tightly and specifically to almost any molecule.
Also known as synthetic antibodies, these short strands of DNA hold great promise as alternatives for
traditional antibodies, and have the potential to be used in a variety of clinical settings.

At the beginning of this year, we settled on using a homogeneous immunoassay to fulfill our goal of a simple kit
that can be easily administered in a clinical setting, offering a suitable alternative to costly and
uncomfortable mammograms. Initially, we were intent on adapting lateral flow assays in pursuit of this goal;
however, technical challenges and the relative sensitivity required to measure small fluctuations in biomarker
concentration indicated that this was not the best course of action. Therefore, we instead turned to highly
sensitive homogeneous immunoassays, such as the AlphaLISA test from Perkin-Elmer for inspiration.

We settled on using a homogeneous immunoassay due to the fact that such an assay does not require the
time-intensive washing steps required by a traditional heterogeneous immunoassay, such as ELISA or DELFIA.
Considering that this test kit is meant to be as convenient as possible, removing the long periods of time
needed for incubation and washing greatly streamlines the procedure and makes it realistic for this to be
administered at home or in a clinical setting. Furthermore, we decided to use aptamers instead of traditional
antibodies due to their superior stability and ease of synthesis, thus allowing this proof-of-concept to be more
feasibly implemented in the future. In addition, aptamers exhibit complicated structural changes upon binding
with the substrate (in this case, the biomarker in question), which we were further able to exploit to create a
light-switching fluorescent probe that fluoresce when bound to the biomarker of interest. One can then determine
the original concentration of the biomarker by referencing a standard curve, similar to how one would quantify
the data of an ELISA or Bradford assay. Furthermore, this form of liquid biopsy has been shown through
experiments to offer satisfactory measurements and data.

For more information, please see our Human Practices page.

### Selection of Aptamers from Literature

We reviewed the papers of Ferreira et al and Agnihotri et al to find aptamers and their binding affinities for
Mucin 1 and CA 15.3 respectively. Both papers indicated that said aptamers were highly selective for the
biomarkers of interest, and the sequences were relatively short and easy to synthesize de novo. However, there
currently seems to be no published research regarding synthetic antibodies for CA 27.29. Therefore, we selected
Mucin 1 and CA 15.3, as well as their corresponding aptamers, for this proof of concept.

### Design of Aptamer Probe

For the design of the probe, we consulted with Dr. Kazunori Ikebukuro of Tokyo University of Agriculture and
Technology and made extensive use of the aptamer modeling software UNAFold to design our probes.

Our probes were based on the FRET principle and fluorescence quenching using donor-acceptor pairs and heavily
inspired by similar research by Yang et al and Wu et al, who designed FRET-based aptasensors for the detection
of PDGF and Helicobacter pylori respectively. In both instances, the fluorophore-labeled aptamer resulted in
detection that was highly specific and did not require a long incubation period, unlike ELISA or other
immunoassays of similar specificity and sensitivity. And in both groups, fluorescence upon binding with the
substrate was achieved by a conformational change of the aptamer, which changed the distance between the
fluorophore and the quencher attached to the ends of the probe and resulted in a weakening of the FRET effect
and increased fluorescence. The final fluorescence was then quantified using a well-plate reader under
excitation and compared to a standard curve derived from standard solutions of the biomarker/cell of
interest.

For our project, we based the design of the probe upon this concept, and designed two different probes for the
detection of Mucin 1 and CA 15.3. Using UNAFold, we were able to predict the conformational change of the
aptamers upon binding with our biomarkers, and were able to select one aptamer for each protein that changed in
such a way as to significantly weaken the FRET effect between the donor-acceptor pair attached to the ends upon
binding, but not when suspended unbound.

We envision this project to be used in a clinical setting, where multiple samples may be tested at once (and
thus lowering the cost).

For a more detailed discussion, please refer to the Modeling page.

### Principles of FRET (Forster Resonance Energy Transfer)

For our project this year, we decided to employ the use of a light-switching aptamer probe, which will change
shape and fluoresce under electromagnetic excitation when bound to the biomarker of interest. To that end, we
have decided to make use of the Förster Resonance Energy Transfer principle, which allows for non-radiative
energy transfers between two molecules, commonly known collectively as a donor-acceptor pair. Normally, FRET is
often used in elucidating the structure of cellular components due to the effect’s extreme sensitivity to
distance; beyond an angstrom distance of 100 Å, the interactions between the donor and acceptor greatly
decrease. Therefore, monitoring FRET interactions is a useful way to gauge the distance between said donor and
acceptor.

### Donor-Acceptor Pairs

FRET is often used in the context of elucidating protein structure. This is done through the use of what are
known as donor-acceptor pairs, which are molecules which exhibit FRET when within close proximity to each other.
A subset of this general phenomenon involves a fluorophore and a quencher (instead of two fluorophores), whereby
FRET is shown by the decrease in fluorescence by the fluorophore when in close proximity to the quencher,
instead of the emission of light from the fluorophore that is not being excited. In our case, we are using a
quencher and a fluorophore, which means that FRET interactions will decrease fluorescence of the fluorophore,
while decreased FRET interactions will increase the fluorescence of the fluorophore. Below, we will outline the
specific design for each probe.

# Build

### Design of Aptamer Probe

For our ssDNA aptasensor for the detection of Mucin 1, we settled on the donor-acceptor pair BHQ1-Cy5. Normally,
Cy5 (being a dye), is excited and fluoresces under near-infrared light. However, this donor-acceptor pair
dictates that within the distance in which FRET is effective, the BHQ1 quencher will “eat up” the fluorescence
of the Cy5 dye, due to their overlapping absorbance spectrums. Therefore, when attached to the ends of our
aptamer, the FRET interaction will take place and the fluorescence of Cy5 will be quenched, due to the fact that
the aptamer forms a hairpin shape and the distance between Cy5 and BHQ1 is less than 100 Å as a result of
Watson-Crick base pairing. However, when the aptamer is bound to the biomarker, the aptamer is predicted to
change shape and separate the ends. This increases the distance between the Cy5 dye and BHQ1 beyond 100 Å, which
results in a drastic weakening of the FRET effect and the fluorescence of Cy5 under near-infrared light as a
result. Thus, the change in fluorescence/conformation of the aptamer indicates whether it is bound to Mucin 1 or
not. The higher the concentration of biomarker, the more aptamer will fluorescence, thus leading to an increase
in measured fluorescence.

![](https://static.igem.wiki/teams/4334/wiki/project4.png)

**DNA Aptamer Probe using FAM and Guanine Nucleotides**
For our dsDNA aptasensor for the detection of CA 15.3, we settled on the donor-acceptor pair FAM-guanine. We
chose this because guanine is easier to synthesize and attach to our aptamer than other synthetic quenchers, and
FAM is also widely used. Furthermore, the FAM-guanine pair is excited by UV light, which is different from our
single-stranded probe. Therefore, this design allows us to test a different wavelength and see if it is more
effective.

Our dsDNA aptasensor relies on a strand displacement assay, similar to the ones used by XMU China in their 2018
project. After designing a partial complementary strand to our aptamer (please see the Modeling page for more
details), we attached a FAM group to the 5’ end of our complementary strand, which would be naturally quenched
by the guanine present on the 3’ end of the aptamer. As the complementary strand pairs with the aptamer in a
Watson-Crick base pairing scheme, the distance between the FAM group and the guanine is less than 100 Å, which
is enough for the FRET effect to be significant. However, when the partial complementary strand is displaced by
the biomarker, the distance between FAM and guanine drastically increases, which decreases FRET efficiency and
results in fluorescence of the FAM group under UV light.

As a further test, we also attempted to design a double stranded probe with Cyanine 3 dye and BHQ2 quencher in
place of FAM and guanine respectively.

![](https://static.igem.wiki/teams/4334/wiki/project5.png)

### Parts

We have performed an extensive literature review of aptamers for Mucin 1 and CA 15.3 (our two biomarkers of
interest) and have added the sequences to the registry, along with pertinent information from the UNAFold DNA
modeling software various tests we conducted in our lab.

# Test

In order to test our aptamer probes, we first resuspended our aptamer probes into their buffer solutions. As
an initial test, we resuspended the Mucin 1 S2.2 ssDNA aptasensor, and did not resuspend the CA15.3 Clone 2
and 4 dsDNA aptasensor.

We then performed our protocol to verify the FRET interactions between our aptamer probes and our Mucin 1
biomarker. Without the presence of the Mucin 1 biomarker, the aptamer probes should not fluoresce and we
should get fluorescence values close to that of pure buffer solutions when we put it under the 96 wellplate
reader. This is because the aptamer is still in its folded shape, and only opens up when it binds to the
Mucin 1 biomarker. This causes the FRET phenomenon where the excited Cy5 dyes transfer their energy to the
Black hole quenchers because they are close together. With the presence of the Mucin 1 biomarker, the
aptamer probes should fluoresce, and we should be able to get a fluorescence reading under the 96 wellplate
reader because the ends of the aptamer probes are too far for the FRET phenomenon to occur.

### FRET Troubleshooting

Upon conducting our FRET experiment using the aptamer probes that came initially, we found that they did not
fluoresce properly. Firstly, they were not fluorescing at the values we gathered from literature review, yet,
that was still acceptable as part of our experimentation. However, the aptamer probes were fluorescing
regardless of the presence of the Mucin 1 biomarker. This meant that the aptamer probes were:

 A. not manufactured properly so that there was no black hole quencher, leading to the absence of the FRET
phenomenon
 B. were not folded properly so that the attached black hole quencher could not quench the energy emitted
by the dye
 C. not binding to the biomarker, and the fluorescence value should be even higher when the aptamer probes
are bound to the biomarker.

Upon the second day of experiments with newly resuspended aptamer probes, the aptamer probes did not fluoresce
properly again. We omitted possibility B as we followed proper resuspension protocols, and hypothesized that it
was possibility A. Thus, we reordered the aptamer probes, making sure that we had S2.2 aptamer probes with Cy5
dye on one end and BHQ1 on the other end. They arrived approximately a month later, and we were able to conduct
the FRET experiment again.

In our second round of FRET experiments, our aptamer probes fluoresced properly. Our advice to iGEM teams
working with aptamer probes would be to verify their FRET interactions as soon as they arrive, as they take time
to reorder and arrive.

![](https://static.igem.wiki/teams/4334/wiki/reader1.png)

For further information on our mathematical analysis of our FRET experiments, please view the Math Model
page.

### Analysis of FRET results

The Hill’s Equation is a useful equation to determine the amount of protein (biomarker) bound to the ligand
(aptamer). Since it is theoretically unlikely that all of the biomarkers will bind to the aptamers, we will need
a method to calculate the concentration of the biomarker that is **bound** to the aptamer. Only using the
standard
curve will result in an underestimation of the concentration of the biomarker. Hill’s Equation helps us
determine the original concentration of biomarker by relating the biomarker concentration with the aptamer
concentration and proportions of the pattern-biomarker complex (the dissociation constant). Although the
dissociation constant was to be obtained from the Scatchard Plot, test results were inconclusive. Therefore, the
Kd value was derived from 3D modeling through UNAFold.

![](https://static.igem.wiki/teams/4334/wiki/mm2.png)

The Hill’s Equation is shown above. [PLn] is the concentration of protein (biomarker) bound to ligand (aptamer),
[P0] is the total concentration of biomarkers, [L] is the total ligand (aptamer) concentration, and Kd is the
apparent dissociation constant. The dissociation constant (Kd) describes the ability of a compound to dissociate
or break down to its constituent components. In our case, it describes the ability of the biomarker to break
away from the aptamer.

We were able to get a total of 12 total data points with calibration on 475nm excitation peak and 580—640
emission peak. We then were able to plot the line of best fit and find the equation of the standard curve.

![](https://static.igem.wiki/teams/4334/wiki/mm4.png)

Above is the standard curve obtained from the serial dilution. The equation for the line of best fit is shown
above the graph and the vertical error bars are depicted. To determine the bound concentration of the biomarker,
the equation of the standard curve was initially rearranged in terms of the fluorescence value and then plugged
into the Hill’s Equation. The Hill’s Equation was then used with the Kd found through 3D Modeling.

![](https://static.igem.wiki/teams/4334/wiki/engeq.png)

Where F is the fluorescence value (RFU) from the standard curve, [L] is the aptamer concentration, and [PLn] is
the concentration of the aptamer-biomarker complex. Using this equation, we are able to calculate the
concentration of the biomarker bound to the aptamer.

### Specificity Test

Furthermore, we conducted a specificity test using Bovine Serum Albumin instead of our Mucin 1 Biomarker. Our
results showed that the fluorescence value did not change from pure buffer solution, meaning that the Mucin 1
S2.2 aptamer probes did not bind to the BSA. This confirms the specificity of the S2.2 aptamer probes to Mucin
1.

![](https://static.igem.wiki/teams/4334/wiki/img-0569.jpg)

### Shelf Life Test

Additionally, we conducted a shelf life test with the S2.2 aptamer probe, and we were able to verify that the
shelf life of the aptamer probe was at least two weeks.

![](https://static.igem.wiki/teams/4334/wiki/img-0567.jpg)

Due to the unexpected manufacturing with our aptamer probes, we were unable to perform tests on our CA 15.3
dsDNA aptasensors. Additionally, please see our Notebook page for more details on what we did when performing
our tests.

# Learn

### Weak RFU Value than Expected

Through our tests, we were able to learn many things about our aptamer probe. For example, we learned that
the aptamer probes had much weaker fluorescence values than we had imagined. With a 100µL solution of 500µM
aptamer solution and 500µM biomarker solution, the maximum Relative Fluorescence Unit (RFU) was 846. While
this is a measurable value, we realized that there is much more research needed to improve this system and
increase the RFU value in some way. This would be able to prevent false positives during screenings, and we
would recommend other teams to explore this topic of amplification.

### Specificity and Durability of Aptamer Probes

Furthermore, we were able to verify the specificity and durability of aptamers. Although we did know through
literature that aptamers were better in terms of specificity and durability compared to antibodies, we were able
to verify this through our specificity test and shelf life test. Our experiments build off of prior experiments
with aptamers, and is another example of the versatility of aptamers probes.

### Versatility of FRET

We also learned that the FRET mechanism works very well. In our experiments, when we had only a solution of S2.2
aptamer probes without Mucin 1 biomarkers, the aptamer probes virtually did not fluoresce, since the RFU values
were similar to that of pure buffer solution. This exemplifies the high accuracy of the aptamer probe mechanism
utilizing the FRET mechanism to make the aptamer probes only fluoresce in the presence of the desired target.

# Design

In regards to the future applications of our project, we believe that this mechanism should not be further
investigated until more concrete evidence is found on the levels of Mucin 1 biomarker patients and its
correlation to having breast cancer. We explored various previous literature conducted on breast cancer
patients, as well as literature on the detection of breast cancer through the ELISA method detecting levels
of Mucin 1 biomarker. As shown in the box and whisker chart below, literature showed that the error bars of
the standard mean error overlapped between breast cancer patients and healthy patients.

![](https://static.igem.wiki/teams/4334/wiki/engineeringchart.png)

(Werfalli, et al.)

Thus, we determined that our method may not provide the most accurate assessment of who requires further
screening due to risk of false positives—and alarming potential patients is a situation we’d like to avoid. For
this reason, we propose to implement our screening method to detect diseases where any presence of biomarkers is
an indication for the necessity of treatment (including hepatitis B virus). When it comes to breast cancer
applications, we propose to apply this method in the future where technology for the identification of Mucin 1
biomarker concentrations in patients with breast cancer have improved.

Moreover, in the broad scope of the implications of our system, we believe that teams in the future can build
off of our ssDNA and dsDNA aptamer probe designs, utilizing the donor-acceptor pairs of BHQ1 and Cy5, and BHQ2
and Cy3 dye.

# References

1. “28.2: Base Pairing in DNA - the Watson-Crick Model.” Chemistry LibreTexts, Libretexts, 26 Aug. 2015,
   https://chem.libretexts.org/Bookshelves/Organic\_Chemistry/Organic\_Chemistry\_(McMurry)/28%3A\_Biomolecules\_-\_Nucleic\_Acids/28.02%3A\_Base\_Pairing\_in\_DNA\_-\_The\_Watson-Crick\_Model.
2. AlphaLISA Immunoassays. https://www.perkinelmer.com/category/immunoassays-alpha. Accessed 7 Oct. 2022.
3. An Introduction to Fluorescence Resonance Energy Transfer (FRET) Technology and Its Application in
   Bioscience.
   https://www.biotek.com/resources/white-papers/an-introduction-to-fluorescence-resonance-energy-transfer-fret-technology-and-its-application-in-bioscience/.
   Accessed 7 Oct. 2022.
4. Bajar, Bryce T., et al. “A Guide to Fluorescent Protein FRET Pairs.” Sensors , vol. 16, no. 9, Sept.
   2016, https://doi.org/10.3390/s16091488.
5. Duffy, Michael J., et al. “Biomarkers in Breast Cancer: Where Are We and Where Are We Going?” Advances
   in Clinical Chemistry, vol. 71, June 2015, pp. 1–23.
6. Ferreira, C. S. M., et al. “DNA Aptamers That Bind to MUC1 Tumour Marker: Design and Characterization of
   MUC1-Binding Single-Stranded DNA Aptamers.” Tumour Biology: The Journal of the International Society for
   Oncodevelopmental Biology and Medicine, vol. 27, no. 6, Oct. 2006, pp. 289–301.
7. Huang, Lei, et al. “Aptamer-Based Lateral Flow Assay on-Site Biosensors.” Biosensors & Bioelectronics,
   vol. 186, Apr. 2021, p. 113279.
8. Kazarian, Anna, et al. “Testing Breast Cancer Serum Biomarkers for Early Detection and Prognosis in
   Pre-Diagnosis Samples.” British Journal of Cancer, vol. 116, no. 4, Feb. 2017, pp. 501–08.
9. Keefe, Anthony D., et al. “Aptamers as Therapeutics.” Nature Reviews. Drug Discovery, vol. 9, no. 7,
   July 2010, pp. 537–50.
10. Kim, Mi-Gyeong, et al. “Double Stranded Aptamer-Anchored Reduced Graphene Oxide as Target-Specific Nano
    Detector.” Biomaterials, vol. 35, no. 9, Mar. 2014, pp. 2999–3004.
11. Li, Jiawei, et al. “Non-Invasive Biomarkers for Early Detection of Breast Cancer.” Cancers, vol. 12, no.
    10, Sept. 2020, https://doi.org/10.3390/cancers12102767.
12. Safer, Daniel, et al. “What pH Buffer Solution Best Replicates Physiological Conditions?” ResearchGate,
    31 Dec. 1994,
    https://www.researchgate.net/post/What\_pH\_buffer\_solution\_best\_replicates\_physiological\_conditions.
13. Team:XMU-China/hardware - 2018.Igem.Org. https://2018.igem.org/Team:XMU-China/Hardware. Accessed 7 Oct.
    2022.
14. Wu, Hangjie, et al. “Rapid Detection of Helicobacter Pylori by the Naked Eye Using DNA Aptamers.” ACS
    Omega, vol. 6, no. 5, Feb. 2021, pp. 3771–79.
15. Yang, Chaoyong James, et al. “Light-Switching Excimer Probes for Rapid Protein Monitoring in Complex
    Biological Fluids.” Proceedings of the National Academy of Sciences of the United States of America,
    vol. 102, no. 48, Nov. 2005, pp. 17278–83.
16. Agnihotri, Nandita P., Shikha Dubey, and Mangla Bhide. 2014. “Design and Characterization of DNA Aptamer
    for Breast Tumor Marker by an Advantageous Method.” International Journal of Innovative Research in
    Science, Engineering and Technology 03 (10): 16642–48.
17. “Aptamer-Handling-Best-Practices\_July-2018.pdf.” n.d.
    https://www.basepairbio.com/wp-content/uploads/2015/04/Aptamer-Handling-Best-Practices\_July-2018.pdf.
18. aptamer., Of. n.d. “General Handling Guide for Aptamers.” Accessed October 12, 2022.
    https://resources.amsbio.com/Guide/General%20handling%20guide%20for%20aptamers\_2021\_v2.pdf.
19. Osterman, and Biosciences. n.d. “The Next Step in Near Infrared Fluorescence: IRDye QC-1 Dark Quencher.”
    Energy Transfer Assays. Analytical Biochemistry.
    https://web.archive.org/web/20110713204326if\_/http://biosupport.licor.com/docs/QC-1DarkQuencher\_v5.pdf.
20. “Team:XMU-China.” n.d. Accessed October 12, 2022. https://2018.igem.org/Team:XMU-China.
21. Agnihotri, Nandita P., Shikha Dubey, and Mangla Bhide. 2014. “Design and Characterization of DNA Aptamer
    for Breast Tumor Marker by an Advantageous Method.” International Journal of Innovative Research in
    Science, Engineering and Technology 03 (10): 16642–48.
22. Werfalli, Sumeia G. n.d. “SALIVARY MUCINS IN PATIENTS WITH BURNING MOUTH SYNDROME.” Accessed October 12,
    2022.

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Morbi
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et,
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vestibulum orci. Curabitur id ex ligula. Suspendisse maximus arcu sagittis, ultricies sapien
at,
posuere sem. Cras vel dolor lobortis, vehicula metus nec, ullamcorper diam. Sed pretium nibh
lectus,
id euismod urna consectetur non. Nulla facilisi. Praesent congue magna ac neque lobortis,
vel
sagittis neque euismod. Donec venenatis augue quis lorem fermentum, id dapibus nunc
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Praesent aliquam gravida lacus, sit amet rhoncus nunc dignissim quis.

Quisque rhoncus libero quis odio ornare maximus. Vivamus quis condimentum massa. In luctus
hendrerit
dolor, quis blandit nisi porta accumsan. Nullam neque sapien, molestie non ex convallis,
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consectetur ante. Integer a, **gravida enim sed**, molestie sem. Nam tincidunt
sapien
et
justo cursus, ut placerat sem sagittis. Cras maximus purus augue, ut cursus nunc
pellentesque
ut.
Fusce commodo libero et egestas rutrum. Cras ac justo lorem. Maecenas tempus ut leo sit amet
rutrum.

Morbi blandit cursus diam id ultrices. Vestibulum a vulputate justo. Quisque volutpat
finibus
efficitur. Ut ultricies nibh non arcu suscipit dapibus. Phasellus interdum bibendum nibh sed
tincidunt. Pellentesque in felis metus. Phasellus euismod consequat dignissim. Proin in
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tortor. Quisque sagittis facilisis tortor nec commodo. Integer condimentum sagittis luctus.
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iaculis.
In
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tortor
cursus mauris, sit amet dignissim risus ante vel justo. Sed fermentum erat id nibh interdum
sollicitudin. Vestibulum tempor metus ac libero maximus semper. Donec sed ligula neque.
Morbi
sagittis orci odio, eget blandit urna molestie hendrerit. Fusce orci tellus, iaculis at
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convallis in tortor. Curabitur finibus orci tortor, eget euismod lectus imperdiet quis.

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vel
vestibulum orci. Curabitur id ex ligula. Suspendisse maximus arcu sagittis, ultricies sapien
at,
posuere sem. Cras vel dolor lobortis, vehicula metus nec, ullamcorper diam. Sed pretium nibh
lectus,
id euismod urna consectetur non. Nulla facilisi. Praesent congue magna ac neque lobortis,
vel
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pharetra.
Praesent aliquam gravida lacus, sit amet rhoncus nunc dignissim quis.

Quisque rhoncus libero quis odio ornare maximus. Vivamus quis condimentum massa. In luctus
hendrerit
dolor, quis blandit nisi porta accumsan. Nullam neque sapien, molestie non ex convallis,
ornare
consectetur ante. Integer a, **gravida enim sed**, molestie sem. Nam tincidunt
sapien
et
justo cursus, ut placerat sem sagittis. Cras maximus purus augue, ut cursus nunc
pellentesque
ut.
Fusce commodo libero et egestas rutrum. Cras ac justo lorem. Maecenas tempus ut leo sit amet
rutrum.

Morbi blandit cursus diam id ultrices. Vestibulum a vulputate justo. Quisque volutpat
finibus
efficitur. Ut ultricies nibh non arcu suscipit dapibus. Phasellus interdum bibendum nibh sed
tincidunt. Pellentesque in felis metus. Phasellus euismod consequat dignissim. Proin in
efficitur
tortor. Quisque sagittis facilisis tortor nec commodo. Integer condimentum sagittis luctus.
Phasellus congue rhoncus eros, euismod efficitur neque. Etiam sagittis vitae ligula non
iaculis.
In
volutpat sollicitudin risus nec venenatis. Donec elementum, sem eu ornare bibendum, enim
tortor
cursus mauris, sit amet dignissim risus ante vel justo. Sed fermentum erat id nibh interdum
sollicitudin. Vestibulum tempor metus ac libero maximus semper. Donec sed ligula neque.
Morbi
sagittis orci odio, eget blandit urna molestie hendrerit. Fusce orci tellus, iaculis at
pretium
et,
convallis in tortor. Curabitur finibus orci tortor, eget euismod lectus imperdiet quis.

# Place Holder 4

Lorem ipsum dolor sit amet, consectetur adipiscing elit. Aliquam venenatis ex nibh, quis
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lectus hendrerit vitae. Nulla non ex suscipit, fringilla enim gravida, mollis ante. Nullam
vel
vestibulum orci. Curabitur id ex ligula. Suspendisse maximus arcu sagittis, ultricies sapien
at,
posuere sem. Cras vel dolor lobortis, vehicula metus nec, ullamcorper diam. Sed pretium nibh
lectus,
id euismod urna consectetur non. Nulla facilisi. Praesent congue magna ac neque lobortis,
vel
sagittis neque euismod. Donec venenatis augue quis lorem fermentum, id dapibus nunc
pharetra.
Praesent aliquam gravida lacus, sit amet rhoncus nunc dignissim quis.

Quisque rhoncus libero quis odio ornare maximus. Vivamus quis condimentum massa. In luctus
hendrerit
dolor, quis blandit nisi porta accumsan. Nullam neque sapien, molestie non ex convallis,
ornare
consectetur ante. Integer a, **gravida enim sed**, molestie sem. Nam tincidunt
sapien
et
justo cursus, ut placerat sem sagittis. Cras maximus purus augue, ut cursus nunc
pellentesque
ut.
Fusce commodo libero et egestas rutrum. Cras ac justo lorem. Maecenas tempus ut leo sit amet
rutrum.

Morbi blandit cursus diam id ultrices. Vestibulum a vulputate justo. Quisque volutpat
finibus
efficitur. Ut ultricies nibh non arcu suscipit dapibus. Phasellus interdum bibendum nibh sed
tincidunt. Pellentesque in felis metus. Phasellus euismod consequat dignissim. Proin in
efficitur
tortor. Quisque sagittis facilisis tortor nec commodo. Integer condimentum sagittis luctus.
Phasellus congue rhoncus eros, euismod efficitur neque. Etiam sagittis vitae ligula non
iaculis.
In
volutpat sollicitudin risus nec venenatis. Donec elementum, sem eu ornare bibendum, enim
tortor
cursus mauris, sit amet dignissim risus ante vel justo. Sed fermentum erat id nibh interdum
sollicitudin. Vestibulum tempor metus ac libero maximus semper. Donec sed ligula neque.
Morbi
sagittis orci odio, eget blandit urna molestie hendrerit. Fusce orci tellus, iaculis at
pretium
et,
convallis in tortor. Curabitur finibus orci tortor, eget euismod lectus imperdiet quis.

# Place Holder 5

Lorem ipsum dolor sit amet, consectetur adipiscing elit. Aliquam venenatis ex nibh, quis
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lectus hendrerit vitae. Nulla non ex suscipit, fringilla enim gravida, mollis ante. Nullam
vel
vestibulum orci. Curabitur id ex ligula. Suspendisse maximus arcu sagittis, ultricies sapien
at,
posuere sem. Cras vel dolor lobortis, vehicula metus nec, ullamcorper diam. Sed pretium nibh
lectus,
id euismod urna consectetur non. Nulla facilisi. Praesent congue magna ac neque lobortis,
vel
sagittis neque euismod. Donec venenatis augue quis lorem fermentum, id dapibus nunc
pharetra.
Praesent aliquam gravida lacus, sit amet rhoncus nunc dignissim quis.

Quisque rhoncus libero quis odio ornare maximus. Vivamus quis condimentum massa. In luctus
hendrerit
dolor, quis blandit nisi porta accumsan. Nullam neque sapien, molestie non ex convallis,
ornare
consectetur ante. Integer a, **gravida enim sed**, molestie sem. Nam tincidunt
sapien
et
justo cursus, ut placerat sem sagittis. Cras maximus purus augue, ut cursus nunc
pellentesque
ut.
Fusce commodo libero et egestas rutrum. Cras ac justo lorem. Maecenas tempus ut leo sit amet
rutrum.

Morbi blandit cursus diam id ultrices. Vestibulum a vulputate justo. Quisque volutpat
finibus
efficitur. Ut ultricies nibh non arcu suscipit dapibus. Phasellus interdum bibendum nibh sed
tincidunt. Pellentesque in felis metus. Phasellus euismod consequat dignissim. Proin in
efficitur
tortor. Quisque sagittis facilisis tortor nec commodo. Integer condimentum sagittis luctus.
Phasellus congue rhoncus eros, euismod efficitur neque. Etiam sagittis vitae ligula non
iaculis.
In
volutpat sollicitudin risus nec venenatis. Donec elementum, sem eu ornare bibendum, enim
tortor
cursus mauris, sit amet dignissim risus ante vel justo. Sed fermentum erat id nibh interdum
sollicitudin. Vestibulum tempor metus ac libero maximus semper. Donec sed ligula neque.
Morbi
sagittis orci odio, eget blandit urna molestie hendrerit. Fusce orci tellus, iaculis at
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## RESULTS

# ELONA Assay and Experimental Determination of Dissociation Constant

We followed established protocols for ELONA, an ELISA assay modified to accommodate aptamers, in order to
determine the dissociation constant. We used a protocol modified from Sang et al to determine the Kd value
using the Hill equation, a Scatchard plot, and non-linear regression. However, the results were inconclusive
so we decided to find our Kd value through the 3D modeling software, UNAFold.

# Aptamer Resuspension and Characterization

We followed standard aptamer resuspension protocols provided by the manufacturer, and were able to
successfully resuspend our aptamers in the buffers used in the original papers. More information on this is
provided in our lab notebook.

We were able to conduct a shelf life test on the aptamers by constructing a standard curve (results shown in
next section), leaving the aptamer solution in the refrigerator for two weeks before testing the
concentration of various standardized biomarker samples with known concentrations using the aptamer probes
without resuspending or reheating them. The results below exemplify that the aptamer probes still
successfully fluoresce 2 weeks after resuspension with fluorescence values close to their relative biomarker
concentrations.

![](https://static.igem.wiki/teams/4334/wiki/reader5.png)

The results show that the aptamers have reasonable shelf life, even when they are not resuspended, if
handled properly and kept in the dark using tinfoil. Therefore, we believe that this highlights practicality
of our design in a clinical setting, where the time needed to prepare this assay is drastically shorter than
traditional immunoassays. Furthermore, we have also demonstrated the superior durability of aptamers over
antibodies, thus also making a strong case for the feasibility of this technology.

We were also able to test the specificity of our aptamer probes to our biomarkers using a solution with BSA.
The results are shown below. For more information, please refer to our lab notebook.

![](https://static.igem.wiki/teams/4334/wiki/reader4.png)

Overall, we have shown that our probe is extremely sensitive and able to function well, even in complex
environments.

# Fluorescence of ssDNA Mucin 1 Aptasensor

We suspended the Mucin 1 aptasensors in the presence and absence of Mucin 1, and used a 96 well plate reader
to excite the probe and monitor its fluorescence. Mucin aptamer S2.2 was used. The resulting data is shown
below. As one can see, the presence of Mucin 1 greatly increased the fluorescence of the DNA probe solution,
thus indicating that our design was successful.

![](https://static.igem.wiki/teams/4334/wiki/reader6.png)

# Fluorescence in Standard Solutions

We added aptamer solutions to standard solutions of their respective biomarkers (Mucin 1) in a buffer
solution, then excited them using a 96 well-plate reader to find the fluorescence of the aptamer probes in
different concentrations of biomarker. Using this data, we were able to create standardized fluorescence vs.
concentration curves for the aptamer probes. Then, using mathematical manipulation (see the Modeling page
for more information), we were able to devise a simple method to calculate the total concentration of
biomarkers in the original solution from the fluorescence of the solution by devising a computer algorithm
based on the Hill equation.

We were able to determine the limit-of-detection and sensitivity of our aptasensors by measuring the
fluorescence in decreasing concentrations of their respective biomarkers and assessing the range at which
the decrease in fluorescence became negligible. The results are shown below. Furthermore, the error between
the measured concentrations (using the protocols detailed in the lab notebook and using the program
described on the Modeling page) and actual concentrations of standard solutions are shown below,
demonstrating that our aptasensor can assess the concentrations of biomarkers in solution with relative
accuracy.

![](https://static.igem.wiki/teams/4334/wiki/reader1.png)

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