iGEM 2023 — Team unicrete


{% extends "layout.html" %}
{% block title %}Engineering Success{% endblock %}
{% block lead %}Demonstrate engineering success in a part of your project by going through at least one iteration of the
engineering design cycle.{% endblock %}
{% block page\_content %}

# Introduction

![]( https://static.igem.wiki/teams/4983/wiki/387589495-351781200525127-6555118168552605437-n.png)

Our vision is the utilization of our system in water samples collected from the field as described in the
Implementation section. However, the design of the project and the first experiments will be conducted in
lab-made deltamethrin spiked water samples whose preparation is described in the **Deltamethrin Sample
Preparation** part of the Experiments section.

In regards to the system itself, one of the most important aspects of a biosensing system is its sensitivity
and selectivity to the targeted molecule. Especially, cell-free biosensing systems lack sensitivity due to the
decrease of the signal transduction’s efficiency as the steps go on. Thus, our system contains three steps of
signal amplification to increase the overall sensitivity and minimize the concentration of Deltamethrin in the
water sample in which the pesticide is being detected.

Regarding the selectivity of the biosensor, we designed an aptamer that recognizes deltamethrin in the
analyzed water sample and modified it to transduce this signal to the following factors of the biosensing
system after the binding to deltamethrin. More specifically, the binding of dAPT (deltamethrin’s aptamer) with
deltamethrin will cause a conformational change in the structure of dAPT, rendering it able to hybridize with
the following DNA of the system called L1. This conformational change includes the release of a specific
region of dAPT, region 1, which previously was hybridized with cDAPT, a small ssDNA that forms a duplex with
dAPT in the absence of deltamethrin and gets excluded after dAPT-Deltamethrin binding. L1 molecule is a ssDNA
molecule that can form a stem-loop structure by itself. This structure breaks after dAPT-L1 hybridization,
causing the release of an L1 region, consisting of a\*b\*c\*, that was previously involved in the stem-loop
formation. With the help of this region, the L1 molecule, via the Toehold mediated strand displacement (TMSD)
mechanism, forms a dsDNA after hybridization with the L2 molecule.

Considering that only one dAPT molecule needs to bind to deltamethrin for the formation of multiple L1-L2
duplexes, L1-L2 duplex formation is the first step of signal amplification. Cas12a catalyzes the next
amplification step. In this system, Cas12a guided by its crRNA, will interact with the L1-L2 duplex and
acquire its transnuclease activity. In this manner, it can hydrolyze the next and final DNA molecule of the
system, the LINKER. LINKER is a modified ssDNA molecule that binds with a streptavidin-coated magnetic bead in
its 5’ end and a Lipase enzyme in its 3’ end. The cleavage of the LINKER molecule is the second amplification
step because just one Cas12a enzyme can hydrolyze multiple LINKER molecules, releasing many Lipase enzymes
into the supernatant. Then, each one of these Lipase enzymes will transform their substrate into a final
chromogenic product that will change the color of the supernatant. As we can understand, this is the last
signal amplification step and the final step of the biosensing system.

Obviously, a biosensing system must have both the ability to activate in response to the target’s presence but
also remain deactivated in response to the target’s absence. This is why we used magnetic beads in the
biosensing system. After the incubation of Cas12a with the aforementioned factors of the detection system, we
will concentrate all the magnetic beads into the bottom of the tube using a magnet and then, we will move the
supernatant to a new tube. If deltamethrin is present, LINKER will be cleaved as described previously and
lipase will be released into the supernatant. In this way, as we transfer the supernatant to the new tube we
will transfer Lipase as well and as a result, the color change of the new solution will be possible after
adding Lipase’s substrate. On the other hand, if deltamethrin is absent, LINKER will remain intact causing the
removal of Lipase molecules from the supernatant as they are localized to the bottom of the tube with the
magnetic beads. In this way, the new solution will not have Lipases, and thus retain its color, indicating the
absence of deltamethrin.

# Engineering Success

# CYCLE 1: DNA aptamers docking for choosing the aptamer with the best scores for deltamethrin

### **CYCLE 1A: Aptamer selection and the structural similarities between Deltamethrin and lambda-cyhalothrin**

### **Design**

The binding of the dAPT aptamer and deltamethrin is the initial step in activating the detection system. Due
to the binding, the aptamer's tertiary structure is altered and folded in a new conformation. In order to
synthesize a fully selective aptamer, we initially considered using the Capture-SELEX method based on the work
of **Yuxia Yang et al.,** 2021, but the process was time-consuming and required expertise. Thus we
contacted a company specialized in aptamers’ design, but the price was too high for any iGEM team to bear
($19,000). As a result, we considered whether we might use the aptamers' sequences provided for
lambda-cyhalothrin binding in the work of **Yuxia Yang et al., 2021.** Since lambda-cyhalothrin and
deltamethrin are both members of the pyrethroid pesticide family and share multiple structural
characteristics, we consulted Mr. Eleftheriadis and decided to test the binding capabilities of the
aforementioned aptamers with deltamethrin using the molecular docking procedure. More specifically, both of
them are pyrethroid ester pesticides and they share a common ester skeleton with S-conformation in the
asymmetric center but their difference lies on the substituents of the double bond. In deltamethrin, there are
2 bromo- groups, that are halogens but in lambda-cyhalothrin, there is one chloro- group, which is a halogen
as well, and one trifluoromethyl group. In conclusion, with the exception of the groups of the double bond
that differ between the two pesticides, their general structural conformation have many similarities.

![](https://static.igem.wiki/teams/4983/wiki/engineering/pastedgraphic-3.png)

**Figure 1.** Structure of deltamethrin

<https://en.wikipedia.org/wiki/Deltamethrin>

![](https://static.igem.wiki/teams/4983/wiki/engineering/pastedgraphic-2.png)

**Figure 2.** Structure of lambda-cyhalothrin

<https://en.wikipedia.org/wiki/Cyhalothrin>

### **Build**

As we described previously, in the process of obtaining the three-dimensional structures of the pesticides we
observed that they shared a similar structure with certain differences in their functional groups. In order to
carry out the docking procedure we needed the 3D structures of both the aptamers and the pesticides
(lambda-cyhalothrin and deltamethrin) as pdb files. Thus we retrieved the pesticides 3D structures from
Pubchem database as .xml files and converted them to .pdb files through PyMol software. Another basic element
was the determination of the three-dimensional structures of the aptamers. Therefore we reached out to the
corresponding author of the paper, Yuangen Wu **(Yuxia Yang et al., 2021)** and requested the structures.
The authors provided us with two of the thirty one 3D structures they listed in their publication, dAPT-1 and
dAPT-1-39, as we named them. While observing these 3D structures in PyMol, we noticed that the structures are
in fact created from RNA and not DNA molecules. After a series of meetings with professors specialized in
bioinformatics and structural biology, we decided to design the structures ourselves as we had yet to
elucidate the appropriate structures. Moreover, we needed to attach an extra sequence (S region, as mentioned
at " Project Design" page) to the 5′ end of the existing sequences so that the results of the docking could be
useful for later implementation into our system. The addition of the S region was necessary as the chances of
it altering the aptamer’s structure and ameliorating its affinity with Deltamethrin were high. This way, the
results of the docking would be more indicative of the actual binding capabilities of the aptamer to
Deltamethrin. Initiating the design process, we decided at first to focus on the design of the 2 sequences
with higher affinity to lambda-cyhalothrin listed by **Yuxia Yang et al., (2021),** since we expected them
to also have a sufficient affinity with our target molecule. The design of the two structures from each one of
the DNA sequences had to be conducted individually. The procedure consisted of the following: Every sequence
used, along with the additional S sequence, had to be initially imported into the UNAFold Web Server in order
to determine the appropriate 2D structure (based on those listed from Yuxia Yang et al. (2021)) and obtain the
suitable FASTA file format ( .ct in our case). Afterwards, the .ct files were uploaded to the ct2dot web
server environment from “RNAstructures” in order to convert the DNA structure into .txt files which contained
the 2D structure in BRACKET FORM that was mandatory for the next step of the process. In the final step of the
procedure the DNA sequence along with the 2D structure had to be imported into the DNA tertiary structure
prediction method tool in order to obtain the desired 3D .pdb structures. With the structures in hand, we were
able to proceed with the docking procedure.

![](https://static.igem.wiki/teams/4983/wiki/engineering/pastedgraphic-111.png)

**Figure 3.** 3D structure of l-cyhalothrin used in docking.(visualized at PyMol)

![](https://static.igem.wiki/teams/4983/wiki/engineering/pastedgraphic-112.png)

**Figure 4.** 3D structure of deltamethrin used in docking.(visualized at PyMol)

### **Test**

Initiating the docking process, we imported to the MOE (Molecular Observing Environment) software the pdb
files of the structure of the DNAs and pesticides. In its essence, the software mass-tests putative, sequence
dependent DNA structures and attempts to attach the ligand at different possible positions so as to,
ultimately, identify the five most stable receptor-ligand complexes. The algorithm employed by this software
is beyond the scope of this description. Essentially, what we tested for was the interactions of deltamethrin
and lambda-cyhalothrin with dAPT-1 and dAPT-1-39, respectively.

### **Learn**

While comparing the interaction values of the two aptamers, we observed that the binding values of
lambda-cyhalothrin are similar to the ones representing deltamethrin binding. The values listed below are
DOCKING SCORES. These scores are affinity indexes, and more specifically represent the free energy of the
complex forming while docking is taking place. More negative scores mean lower free energy of the referring
complex suggesting high stability and higher affinity between receptor and ligand. More specifically, the
values between dAPT-1, deltamethrin and lambda-cyhalothrin binding were -6.4055986 and -6.621511,
respectively. On the other hand, the binding values of dAPT-1-39 with Deltamethrin and lambda-cyhalothrin were
-5.993392 and -6.0832882, respectively. Taking the similarity of results to consideration, we concluded that
the affinity of the aptamers is not limited to lambda-cyhalothrin but seems to extend to deltamethrin as well.
However, the occurring values did not indicate, in our opinion, a sufficient affinity between neither dAPT-1
nor dAPT-1-39 with deltamethrin (as shown in our “experimental results'' page). Therefore, we concluded that
designing DNA aptamers utilizing the remaining 29 DNA selective sequences listed by Yuxia Yang et al.(2021)
and conducting a second cycle of docking using them, could potentially improve the scores and thus, help us
choose aptamers with a better affinity for deltamethrin.

### **CYCLE 1B: Multiple aptamer sequences monitoring for deltamethrin binding**

### **Design**

In order to conduct the second cycle of the docking process, we needed to design the .pdb structures of the
remaining twenty-nine aptamers.

### **Build**

The design process for the remaining twentynine DNA sequences was conducted as mentioned before in the “
Build “ section of **DNA APTAMERS DOCKING CYCLE 1A.**

![](https://static.igem.wiki/teams/4983/wiki/engineering/pastedgraphic-4.png)

**Figure 5.** dAPT-15 2D structure (dG= -4,23 kcal mol-1)

![](https://static.igem.wiki/teams/4983/wiki/engineering/pastedgraphic-5.png)

**Figure 6.** dAPT-11 2D structure (dG= -6,22 kcal mol-1)

![](https://static.igem.wiki/teams/4983/wiki/engineering/pastedgraphic-81.png)

**Figure 7.** dAPT-11 3D(.pdb) structure used for docking

![](https://static.igem.wiki/teams/4983/wiki/engineering/pastedgraphic-6.png)

**Figure 8.** dAPT-15 3D(.pdb) structure used for docking.

### **Build**

The same testing procedure was conducted as in **CYCLE 1A.** Every aptamer (from **dAPT-2** to
**dAPT-30**) was imported separately with each ligand and the final result was the docking score of the
interaction between them.

### **Learn**

After completing CYCLE 1B of the docking procedure, and analyzing the results, we concluded that the affinity
of the remaining twenty-nine aptamer sequences extends to deltamethrin, besides l-cyhalothrin (according to
the similarity of the values between the interaction of the two pesticides with the aptamers). Comparing the
docking values between all the aptamers we chose two candidate aptamer sequences with the highest values,
dAPT-15 and dAPT-11. Their values were the highest amongst the values of the 29 remaining aptamers and were
comparable to the values by which Yuxia Yang et al., 2021 selected their aptamer for lambda-cyhalothrin
detection. More specifically, the scores between dAPT-11, deltamethrin and lambda-cyhalothrin binding were
-6.9090571 and -6.5811286, respectively. Similarly the values between dAPT-15, deltamethrin and
lambda-cyhalothrin binding were -6.7261815 and -6.9343119, respectively. We observed that dAPT-11 bound with
higher affinity to the target molecule; however, we had to validate these results in the laboratory before we
could proceed to the final selection of aptamer In order to do so, we conducted a QCM-Biosensor experimental
procedure which is presented in detail below.

# CYCLE 2: Determining the aptamer with the highest affinity for deltamethrin through acoustic Quartz Crystal Microbalance (QCM) biosensor experiments

### **Design**

Following the computational docking of the aptamers, a comparative analysis was conducted to assess the
binding energies of each aptamer in relation to their interaction with deltamethrin (delta). Subsequently, two
aptamers exhibiting the most favorable and stable binding energies were selected, signifying their potential
for enhanced interaction with deltamethrin. In light of this computational data and under the guidance of Dr.
Gizeli's expertise, the decision was made to proceed with experimental validation using a Quartz Crystal
Microbalance (QCM) biosensor. The primary objective was to empirically ascertain whether deltamethrin
effectively binds to the selected aptamers and, furthermore, to identify which of the two aptamers yielded the
most robust signal. To elucidate this process further, it is imperative to provide a concise explanation of
the operational principles underlying the QCM biosensor. In the analytical procedures, an alternating voltage
is applied to the surface of the piezoelectric material through attached electrodes, and various physical
properties of the crystal are meticulously gauged. The fundamental physical quantity under scrutiny is the
frequency of oscillation. As per the foundational law of energy conservation in physics, the energy preserved
within these oscillations remains constant. Consequently, the introduction of additional mass onto the
oscillating piezoelectric material results in a deceleration of the oscillatory process. Conversely, the
removal of this mass from the piezoelectric material prompts an acceleration of the oscillations.
Mathematically, the relationship between the oscillation frequency (∆f) and the mass (∆m) affixed to the
surface of the piezoelectric material, considering the material's density (ρ), shear modulus (μ), and active
surface area (A), is elegantly expressed by Sauerbrey in the following equation: ∆f = -2f02∆m / A(ρμ)1/2.

![](https://static.igem.wiki/teams/4983/wiki/engineering/pastedgraphic-7.png)

**Figure 9: General principle of piezoelectric biosensors.** A piezoelectric sensor
is depicted as the gold cylinder while the bound mass as the green cylinder.

In most cases of QCM fabrication, the electrodes made on a quartz are composed from two metals. The
underlayer serves for making a good mechanical and conductive contact between the electrode itself and quartz
while the upper layer should be conductive as well as chemically stable to prevent oxidation and also suitable
for connecting of the QCM into an electrical circuit (soldering or mechanical contact).

![](https://static.igem.wiki/teams/4983/wiki/engineering/pastedgraphic-8.png)

**Figure 10: Appearance of a QCM sensor.**A 5 MHz sensor based on a 14 mm quartz
circle with gold 5mm wide electrodes on the opposite sites is depicted here.

The apparatus employed in our study is denoted as a Q-Sense instrument, equipped with four distinct gold
chips/sensors. This configuration allowed for the execution of distinct reactions on each individual sensor,
resulting in unique elemental compositions being present on each chip during every experimental iteration.

### **Build**

The experimental protocol proceeded as follows: Subsequent to the installation of gold sensors within the
Q-SENSE instrument and the configuration of the Q- sense software, a series of diverse solutions were
systematically introduced. The initial solution comprised neutravidin, chosen for its binding affinity with
the gold chip surface. Following this, a PBS (Phosphate-Buffered Saline) buffer solution was applied to
effectively rinse away any unbound neutravidin. Importantly, this buffer rinse step was consistently performed
after the introduction of each subsequent solution. Subsequently, the aptamer, which had been procured in a
biotinylated form at the 3' end, was dissolved in deionized water (dH2O) and utilized at a concentration of 5
pmoles. It is noteworthy that the biotin moieties within the aptamers were intended to bind specifically with
the neutravidin molecules, thereby facilitating the positioning of the aptamers atop the neutravidin layer
immobilized on the gold chip. With the exception of a control sensor (S), wherein no aptamer solution was
introduced, all other sensors received the aptamer-containing solution. Following this step, a PBS buffer
rinse was administered once more. The introduction of deltamethrin was executed at a concentration of 0.05
milligrams per milliliter (mg/mL) and following the complete washing of the system with buffer, a fresh
deltamethrin solution was introduced, this time at a concentration of 0.5 mg/mL.

### **TEST**

# **Cycle 2.1: Detection of deltamethrin binding with aptamer dAPT15**

### **Build**

Initially, it is important to note that deltamethrin exhibits considerable hydrophobicity, rendering it
poorly soluble in aqueous environments. In accordance with prior research and comparative literature analysis,
our approach commenced with the dissolution of deltamethrin in absolute ethanol. Subsequently, the solution
was subjected to a controlled temperature of 37 οC to augment solubility, thereby enhancing both dissolution
kinetics and the resultant solution's optical clarity. Another step we designed involved injecting a chip with
an amount of deltamethrin immediately after the neutravidin step to confirm that there is no non-specific
binding between the two.

### **Test**

Timeline
PBS buffer pH 7.4 flow rate 50μl/min
30:05 S1-S4 neutravidin 0,2 mg/mL
34:23 S1-S4 buffer
2:08:19 s1 0,05 mg/ml deltamethrin
2:12:30 s1 buffer
2:24:06 s2 5pmol dAPT15, s1 control
2:27:52 s2 buffer (stopped flow accidentally)
2:39:01 start flow again
2:57:53 s1-s2 0,05 mg/mL deltamethrin
3:01:41 Buffer
3:14:10 s1-s2 0,5mg/mL deltamethrin
3:18:18 Buffer

### **Learn**

Using ethanol alone as a solvent for deltamethrin didn't work as well as expected. We noticed that our
signals weren't satisfactory, and deltamethrin didn't fully dissolve, making the solution cloudy. So, we
looked into a different approach mentioned in scientific literature. We mixed equal parts of dimethyl
sulfoxide (DMSO) and pure ethanol to dissolve the deltamethrin. Furthermore, we confirmed that there is no
non-specific binding between delta and neutravidin while we observed a very strong signal at the 5 pmol
concentration of the aptamer.

### **Cycle 2.2: Detection of Deltamethrin binding with aptamer dAPT15- change of the solution**

### **Build**

After the unsuccessful dissolution of delta in pure ethanol we decided to try another alternative. Based on
the literature, DMSO is also considered a solvent for this pesticide. So we dissolved the delta in a DMSO-pure
ethanol solution with a ratio of 1:1. The final concentration of delta in the solution was 1mg/mL and with
successive dilutions we created solutions of 0.5 and 0.05mg/mL delta. Which were re-diluted 100X and 10X
respectively with PBS. In addition, a new step added was that before the introduction of delta, we tested a
mixture we created which contained DMSO, pure ethanol and PBS with a ratio of 0.5:0.5:99 respectively. This
was done to confirm that the signals we might get would not be due to non-specific binding of the solvents.
Also, we used two different concentrations of aptamer dAPT15, 2.5pmol and 5pmol .

### **Test**

Timeline
PBS buffer pH 7.4, flow rate 50μl/min
20:22 S1-S4 Neutravidin 0.2 mg/mL
25:19 Buffer PBS
1:10:24 dAPT15 S1 Control, S2/S4 2,5pmol, S3 5pmol, 200μL each
1:14:48 Buffer PBS
2:15:19 1:100 buffer (0.5% EtOH:0.5 DMSO:99%PBS)
2:20:24 Buffer PBS
2:32:22 0,05 mg/mL (0.5% EtOH : 0.5%DMSO:99%PBS) deltamethrin
2:37:30 Buffer PBS
2:48:46 1:10 buffer (5% EtOH:5% DMSO:90%PBS)
2:54:09 Buffer PBS
3:05:51 0,5mg/mL (0.5% EtOH : 0.5%DMSO:99%PBS) deltamethrin
3:10:57 Buffer

### **Learn**

The change of the solvents resulted in much better outcomes. Deltamethrin dissolved more effectively in
DMSO-etOH (1:1), and there were no pesticide particles suspended in the solution, even without heating.
Furthermore using 2 different concentrations of delta and dAPT15, we observed that at the lower concentration
the sensor did not give any noticeable signal. Therefore, in subsequent experiments we continued with the
highest concentration of 0.5 mg/mL of delta and 5 pmol concentration for the aptamer dAPT15.

![](https://static.igem.wiki/teams/4983/wiki/engineering/pastedgraphic-10.png)

![](https://static.igem.wiki/teams/4983/wiki/engineering/pastedgraphic-11.png)

**Figure 11: General principle of piezoelectric biosensors.** A piezoelectric
sensor
is depicted as the gold cylinder while the bound mass as the green cylinder.

![](https://static.igem.wiki/teams/4983/wiki/engineering/pastedgraphic-12.png)

**Figure 12:** Change in frequency and dissipation.

### **Cycle 2.3: Detection of Deltamethrin binding with aptamer dAPT11**

### **Build**

Following the completion of the initial two experiments and the subsequent determination of the optimal delta
solution conditions, we embarked on a parallel experimental procedure for the dAPT11 aptamer. Given our
limited knowledge regarding its binding properties with deltamethrin, we opted to explore two distinct aptamer
concentrations. Consequently, among the four available gold chips, one was designated as the control sensor,
with no DNA introduced. Two chips were designated for the introduction of DNA at a concentration of 2.5
picomoles (pmol), while the remaining chip received the aptamer at a concentration of 5 pmol. Also,
deltamethrin was introduced at two varying concentrations: 0.5 and 0.05 milligrams per milliliter (mg/mL).
These solutions had been previously prepared and stored at 4°C one week prior to the experiment.

### **Test**

The same procedure as above applied for dAPT15 was followed except that the aptamer concentrations applied to
each chip were different (2.5 pmol and 5 pmol). It's important to note that the deltamethrin solutions
utilized had been prepared one week prior to the experiment. All other parameters, encompassing delta
solvents, delta concentrations, and the operational conditions of the machinery, remained consistent and
unaltered.

Timeline
PBS buffer pH 7.4, flow rate 50 μl/min
12:28 s1-s4 neutravidin 0,2 mg/mL
17:46 s1-s4 buffer PBS pH 7,4
44:05 s1: buffer / s2+s4: 2.5pmol dAPT11 / s3: 5pmol dAPT11
49:56 S1-S4 buffer PBS
1:11:45 s1-s4 buffer 1:100 (0.5% EtOH:0.5 DMSO:99%PBS)
1:16:41 s1-s4 buffer PBS
1:33:11 s1-s4 0,05 mg/mL (0.5% EtOH : 0.5%DMSO:99%PBS) deltamethrin
1:38:16 s1-s4 buffer PBS
2:00:40 s1-s4 buffer 1:10 (0.5% EtOH:0.5 DMSO:99%PBS)
2:05:33 s1-s4 buffer PBS
2:20:01 s1-s4 0.5 mg/mL (0.5% EtOH : 0.5%DMSO:99%PBS) deltamethrin
2:28:25 s1-s4 buffer PBS

### **Learn**

From the above experiment, it was confirmed that the delta concentration that gives a signal for the dAPT11
aptamer is that of 0.5 mg/mL, while the concentration of 5 pmol seemed more suitable for the dAPT11 aptamer,
similarly to the dAPT15 aptamer. Finally, some small repeating signals were observed on sensors which piqued
our interest.

![](https://static.igem.wiki/teams/4983/wiki/engineering/pastedgraphic-13.png)
![](https://static.igem.wiki/teams/4983/wiki/engineering/pastedgraphic-14.png)

**Figure 13:** Comparison of frequency in the two concentrations

### **Cycle 2.4: Comparison of deltamethrin binding to aptamers dAPT15 and dAPT11 and the "delta fridge" test**

### **Build**

The occurrence of sporadic, yet discernible signals within certain sensors with the emergence of atypical
response curves in others, prompted an investigation into the underlying causative factors. This perturbation
was notably correlated with the utilization of pre-prepared delta solutions that had been securely stored for
one week at a refrigerated temperature of 4°C. To comprehensively address this phenomenon, we instituted an
experimental strategy that encompassed the inclusion of two distinct control groups. One control group was
subjected to a freshly prepared deltamethrin solution, while the second control group received the
refrigerated solution. Moreover, as part of the experimental design, 5 picomoles (pmol) of dAPT11 and 5 pmol
of dAPT15 were introduced alongside our sensors (representing 2 out of the total 4 sensors) to enable
comparative analysis of the outcomes.

### **Test**

Timeline 1 (exp1)
PBS buffer pH 7.4, flow rate 50 μl/min
11:38 Neutravidin 0,2 mg/mL
16:19 Buffer PBS
49:30 dAPT11: 5 pmol/ CT15 5pmol/ control fridge/ control
54:11 Buffer PBS
1:27:25 DMSO:EtOH (1:1) 10X PBS
1:31:57 Buffer PBS
1:52:26 Delta 0,5 mg/mL (S1-S4/ S3: delta fridge)
1:57:06 Buffer PBS
Timeline 2 (exp2)
50ul/min, PBS, pH= 7,4
13:13 Neutravidin 0,2mg/mL
18:09 BUFFER PBS
29:25 S1-dAPT11/S2-CONTROL/S4-dAPT15
33:30 BUFFER PBS
57:22 DMSO:EtOH (1:1) 10X PBS
1:02:01 BUFFER PBS
1:19:48 DELTA 0.5mg/mL
1:23:53 BUFFER PBS
Timeline 3 (exp3)
PBS pH=7.4, flow rate 50 μL/min
10:21 Neutravidin 0,2 mg/mL
14:16 BUFFER PBS
26:22 S1-dAPT15/ S2-dAPT11/S3-CONTROL
30:37 BUFFER
41:11 DMSO:EtOH (1:1) 10X PBS
45:34 BUFFER PBS
1:00:55 DELTA 0,5 mg/mL
1:05:02 BUFFER PBS

### **Learn**

This triplet of experiments showed us that both aptamers, when compared during the experimental procedure,
show similar binding to deltamethrin. The signals we received sometimes ranged from 160 Hz to 400 Hz, which
may be explained by the fact that the experiments were performed on different pumps. However, since the
signals in each experiment were not disproportionate to each other, we considered that we can interpret them
in the above way. Additionally, compared to the control where appreciable signals were observed, we inferred
that a proportion of the signals was due to non-specific binding of delta to either the sensor’s gold or
neutravidin since the neutravidin surface was in direct contact with the dissolved delta. Contrary to this, in
the sensors with aptamers the neutravidin is covered by the aptamers attached to it, through bonds with biotin
at the 3'end.

![](https://static.igem.wiki/teams/4983/wiki/engineering/pastedgraphic-15.png)
![](https://static.igem.wiki/teams/4983/wiki/engineering/pastedgraphic-16.png)

**Figure 14: Comparison of the ΔF between dAPT11 and dAPT15**

### **Learn**

The description provided above encapsulates an iteration of the entire design-build-test-learn cycle, while
the testing consists of 4 experimental cycles where each one helped to optimize the protocol of the next one.
Overall, the experiments were conducted to determine which aptamer would exhibit the greatest selectivity for
binding to deltamethrin. To this end, separate experiments were conducted using dAPT11 and dAPT15 on different
days and at varying concentrations. Furthermore, additional trials were executed, involving the concurrent use
of both aptamers on the same day, at identical concentrations, under uniform experimental conditions. Notably,
the results from the latter set of experiments revealed that the signals generated by the aptamers were
closely aligned with each other, distinguishing them from the control group. It became evident that dAPT11
exhibited marginally larger signals and it gave signals in the initial experiments that were more
interpretable. For instance, in an experiment where the aptamer was tested at concentrations of 2.5 pmol and 5
pmol, the signal disparity was notably more pronounced than in the case of dAPT15, where the signals at 2.5
pmol and 5 pmol were closely matched, contrary to expectations. Hence, the evidence and insights gleaned from
these experiments led us to the conclusion that, based on the aforementioned observations, dAPT11 emerged as
the preferred aptamer for integration into our system.

# CYCLE 3: DNA polyacrylamide gel for identification of duplex and hairpin structures

### **Design**

Before proceeding to building the entire system, we firstly individually tested the function of its
constituent parts. One of the most important steps regarding the sensitivity of the biosensor lies in the
hybridization of L1 and L2 molecules with the subsequent creation of a L1-L2 duplex. A necessary condition for
this reaction is firstly the hybridization between dAPT and L1. We tested the formation of the aforementioned
hybridizations through the conduction of an electrophoresis in polyacrylamide gel. The aptamer is being used
in a duplex formation dAPT/cdAPT, so the hybridization between dAPT and L1 in the absence of deltamethrin will
only be possible after the thermal denaturation of dAPT/cdAPT duplex and the hairpin structure of L1 molecule.
More specifically, the S (a, b, c) region of dAPT binds to the complementary a\* b\* c\* regions of L1 and thus
structure dAPT-L1 is created. Subsequently, inside the actual biosensing system via the Toehold mediated
strand displacement (TMSD) process, the L1 molecule hybridizes with the L2 molecule to produce a dsDNA,
through the hybridization of b, c, d\*,c\* regions of L2 to L1 b\*, c\*, d, c regions, respectively. However, in
order to run the electrophoresis we incubated only L1 and L2 molecules without the previous components of the
system. In this case, L1-L2 hybridization requires the thermal denaturation of the L1 and L2 molecules for the
breaking of the hairpin structures and for the release of the subregions that participate in the
hybridization. Because of the small size of L1 and L2 (55b and 46b respectively), a highly sensitive variation
of a DNA Polyacrylamide Gel Electrophoresis technique was used to indicate the hybridization between dAPT and
L1 and between L1 and L2.

### **CYCLE 3A: Identification of duplex and hairpin structures**

### **Build**

L1, L2 and dAPT were all placed in 1ml tubes mixed with TE buffer, according to the concentrations provided
in the table below. Additionally, we used L1, L2 as controls in separate tubes.

**Table 1.** Samples loaded in the wells.

| TUBE/QUANTITY | L1 | L2 | dAPT> | TE Buffer (ph 8) | Loading buffer 6X |
| --- | --- | --- | --- | --- | --- |
| 1. L1 | 3μl | - | - | 6μl | 3μl |
| 2. L2 | - | 3μl | - | 6μl | 3μl |
| 3. L1-L2 | 3μl | 3μl | - | 6μl | 3μl |
| 4. dAPT-L1 | 3μl | - | 3μl | 6μl | 3μl |

The four mixtures were incubated in a water bath at 94oC for 4 minutes so that the double-stranded DNAs were
denatured into single strands. Hybridization was achieved by naturally reducing the temperature of the water
until it approached room temperature. Following, the samples were spinned down and were ready to be loaded
into the Polyacrylamide Gel.
The concentration of the polyacrylamide matrix used for a gel is a crucial factor that influences how DNA
fragments are separated during DNA electrophoresis. Based on the small size range of the DNA fragments L1 and
L2, we chose a 12% acrylamide gel as it is the optimal choice for a 25-150 (bp) resolution. 4.8 ml 30%
Acrylamide (29:1), 4.8 ml H20, 200μl APS, 2.4 ML 1X TBE, 10 μl TEMED and DNA Safe Stain were mixed for the
gel. We added 3 μl of the 6X loading buffer into each tube. Each gel well was fully filled with the entire
solution amount of each tube. In order to prevent the system’s overheating as we increased the voltage after
the first 15 minutes, we had to place the tray on ice.

### **Test**

The electrophoresis was completed after 30 minutes and the pictures of the gel were captured using UV
radiation. Αlthough the bands for L1 and L2 respectively were depicted as expected, with L1 being higher
(closer to the negative electrode) than L2 due to slightly larger size, the band that would theoretically
indicate the hybridization of L1 and L2 was slightly higher than the band of L2, but lower (closer to the
positive electrode) than the corresponding band of L1. In the column in which well, we placed L1 with dAPT
there was only one band with similar mobility to the one of L1-L2.

### **Learn**

The above results were not the expected ones. Generally, if a DNA sample containing various DNA fragments is
loaded and run through the gel, a pattern where smaller DNA fragments with higher mobility that move farther
down the gel than larger fragments will be observed. That is why we expected that the band of the hybridized
L1 and L2 would be higher than the non-hybridized bands of L1 and L2. The same result was expected in the
column in which we run the 4th mixture (dAPT-L1) due to the dAPT-L1 duplex formation. In our attempt to
explain the observed results, we initially hypothesized that no hybridization took place in any of the
columns. In this case, however, in each column there should have been multiple clearly visible bands that
correspond to the molecules placed in each well. For instance, two bands, one for each DNA, should have
appeared in the well where L1 and L2 were loaded together. Therefore, in order to explain the depicted bands
we consulted Prof. Spilianakis, an expert in the field of Molecular Biology. Mr. Spilianakis explained that
the bands might actually indicate hybridization between L1-L2 and dAPT-L1, contrary to what we initially
thought. He noted that the separation of DNAs in a DNA PAGE depends not only on the molecular weight and size
of the DNA molecules but also on their structure. In our case, L1 and L2 molecules adopt a hairpin structure
by themselves which can lead to lower mobility than the estimated one based solely on their size. Furthermore,
the L1-L2 duplex is completely linear due to the complete hybridization of the 2 molecules. A linear structure
could be in fact responsible for the higher mobility of L1-L2 duplex than the estimated mobility based solely
on its size. As a result, the L1-L2 duplex as well as the dAPT-L1 duplex could have an intermediate mobility
compared to the mobility of L1 and L2 molecules. In order to reinforce this explanation, we asked him how it
is possible to not see any other band even if hybridization actually occurs. We thought that as a reaction,
hybridization can not have a 100% efficiency, meaning that there should be some molecules of L1 and L2 that
don’t participate in the binding and thus creating bands in the column with the same mobility of L1 and L2
columns, respectively. He replied that due to the higher melting temperature of the L1-L2 duplex, this
confirmation will occur much faster than the hairpin structures of L1 and L2 in the same high temperatures.
This means that a single L1 or L2 molecule, even if it’s not hybridized at first, due to its lower TM will be
linear at high temperature and will be more likely hybridized until the temperature reaches the room’s
temperature than remain as a single strand and adopt its hairpin structure. Finally, he advised to use a gel
with 8% polyacrylamide. An 8% gel has larger pores compared to a 12% gel and can possibly allow the movement
of the DNA molecules to be mostly affected by their size and not their structure, as a hairpin structured
ssDNA will have no problem moving through the large pores of an 8% gel.

### **CYCLE 3B: Polyacrylamide gel concentration correction for more size-based separation by differential mobility**

### **Build**

L1, L2 and DAPT were all placed in tubes of 1000μl mixed with TE buffer as in the previous experiment,
according to the combinations of the table below. We used L1, L2 as controls in separate tubes.

**Table 2:** Samples loaded in the wells during CYCLE 3B

| TUBE/QUANTITY | L1 | L2 | dAPT> | TE Buffer (ph 8) | Loading buffer 6X |
| --- | --- | --- | --- | --- | --- |
| 1. L1 | 3μl | - | - | 6μl | 2.5μl |
| 2. L2 | - | 3μl | - | 6μl | 2.5μl |
| 3. L1-L2 | 3μl | 3μl | - | 6μl | 2.5μl |
| 4. dAPT-L1 | 3μl | - | 3μl | 6μl | 2.5μl |

The procedure of incubation was the same. However the gel mixture was at 8% polyacrylamide concentration and
not 12%, as Mr. Spilianakis advised. This time 3.2ml 30% Acrylamide (29:1), 6.4ml H20, 200μl APS, 2.4 mL 1X
TBE, 10 μl TEMED and DNA Safe Stain were mixed. Additionally, the amount of loading buffer used was reduced
(as indicated by the table above) in order to avoid the dark-appearing smears and enhance the sharpness of the
bands.

### **Test**

The occurring bands were exactly the same as the ones that occurred in the first experiment.

### **Learn**

After the repetition of the same experiment with 8% gel and reduced amount of dye, the replicable results
have led us to adopt the Prof Spilianakis’ hypothesis that the hybridization of L1 and L2 and DAPT with L1 was
successful, as described in the Learn Section of **CYCLE 3A.**

# CYCLE 4: Streptavidin coated magnetic beads (SA-MABs) synthesis procedure

### **Design**

Every detection system should be able to turn on and off in response to the presence or absence of the
targeted molecule. Our Wet Lab team, after thorough research, found that using streptavidin coated magnetic
beads (SA-MBs) to achieve this responsiveness, is ideal in our case. The SA-MBs were integrated in the system
in such a way in which they can remove lipase, the chromogenic enzyme that depict by its function
deltamethrin's presence, from the solution. To do so, they must be linked with lipase in a way in which they
can also be separated if deltamethrin is present. The LINKER DNA molecule is responsible for connecting the
magnetic beads with Lipase and the same molecule gets hydrolyzed if deltamethrin is present. In this way,
SA-MBs and Lipase are separated. One of the easiest ways to connect a streptavidin coated magnetic bead with a
DNA molecule is through the biotin-streptavidin interaction. For this reason, we designed our LINKER molecule
and the magnetic beads to contain biotin in the 5’ end and be streptavidin-coated, respectively.

![](https://static.igem.wiki/teams/4983/wiki/engineering/pastedgraphic-15.png)
![](https://static.igem.wiki/teams/4983/wiki/engineering/pastedgraphic-16.png)

**Figure 14:** Streptavidin Coated Magnetic Beads.

### **Build**

We followed a modified version of the protocol provided by Peijun et al. (2012), as described in detail in
the Experiments section. The adjustment of the protocol was based on many factors, such as the reagents and
equipment readily available in the lab as well as the desired outcome of the synthesis. The first step was the
synthesis of the magnetic beads that are in fact magnetite aggregates. To create them, we mixed 4.46g FeCl2
and 1.6g FeCl3 and diluted them in dH2O. The aggregation of magnetite molecules requires alkaline conditions
at a pH of around 10, so after stirring, we added NH4OH dropwise . However, a neutral pH is necessary for
streptavidin binding to the magnetic beads. Therefore, after their formation, we repeatedly washed the mixture
to lower the pH back to neutral (pH around 7). Then, 10ml of the stock solution of magnetic beads was mixed
with 37μl of streptavidin and stirred overnight at 4oC. The next day, the mixture was washed to remove any
excess unreacted streptavidin molecules.

![](https://static.igem.wiki/teams/4983/wiki/engineering/pastedgraphic-17.png)
![](https://static.igem.wiki/teams/4983/wiki/engineering/pastedgraphic-18.png)

**Figure 15:** The solution color after mixing FeCl2 and FeCl3 (left). The change of
the color after adding the first drops of NH4OH due to the formation of magnetite aggregates (right).

### **Test**

After conducting the experiment once, we realized that our magnetic beads had lost almost all their magnetic
properties because there was no significant impact on the mixture after close contact with a magnet. Their
color remained black, so there was no problem with the magnetic beads alone, because the black color only
occurs if magnetite aggregates are present that are by default magnetic.

![](https://static.igem.wiki/teams/4983/wiki/engineering/pastedgraphic-19.png)
![](https://static.igem.wiki/teams/4983/wiki/engineering/pastedgraphic-20.png)

**Figure 16:** Magnetic beads solution just after putting on top of a magnet (left).
Localization of magnetite aggregates in the bottom of the solution due to magnetic forces by the magnet
(right).

### **Learn Stage**

We hypothesized two reasons for this magnetic loss. Firstly, streptavidin causes this deficit after coating
the aggregates. This might seem logical, but the literature has mentioned several times that streptavidin
coating can not change magnetic properties by itself. Another possible cause could be the high concentration
of the magnetic beads used in the incubation and the high volumes of both the magnetic beads and the
streptavidin that were used. We noticed that after fractionating a small quantity of the stock of magnetic
beads, which was then diluted with dH2O, the occurring solution was almost instantly cleared after putting it
next to a magnet due to the localization of the beads in the tube’s wall next to the magnet. This effect was
weak in the stock solution. Therefore, we made two dilutions of the stock solution, one at 1/20 and another at
1/100 after mixing 50μl and 10μl of the stock solution with 950μl and 990μl of dH2O, respectively. Then, we
added only 2μl of streptavidin to both diluted solutions and followed the same protocol.

# CYCLE 5: Conjugation procedure and crosslinking verification

### **Design**

If the biosensoxawdr detects deltamethrin in the water sample, we will observe a color change in the
supernatant. We decided to use an enzyme that will change the color of the water to be immense and easily
observable. More specifically, we used Lipase which converts p-nitrophenyl ester to 4-nitrophenol which is the
substance that will give the supernatant its yellow color. Furthermore, LINKER will be linked to Lipase in its
3’ end and to the magnetic bead in its 5’ end. If the water contains deltamethrin, LINKER will get hydrolyzed,
and by using a magnet, the magnetic bead and a part of the LINKER will get precipitated in the bottom of the
tube. However, Lipase and the rest of the LINKER will remain in the supernatant. The supernatant will then be
moved to a new tube and after the addition of p-nitrophenyl its color will change. However, if deltamethrin is
absent, LINKER will remain intact. As a result, after removing the magnetic beads with the magnet, we will
also remove Lipase because it is attached to the bead via the LINKER. As a result, the transferred aliquot of
the supernatant will retain its color after the addition of Lipase substrate.

### **Build**

The formation of an amide and a thioether bond allows the conjugation of LINKER with Lipase. Before the
linkage, both lipase and LINKER should get activated. An important step is mixing the DNA, sodium phosphate
buffer with pH 5,5 and TCEP. After 1 hour at room temperature, they get centrifuged, the 3' Thiol Modifier C3
S-S attachment of LINKER will break and the required thiol group will be formed.
For the activation of Lipase, a purification protocol is followed in order for some salts to be removed and
the lipase to be condensed. More specifically, the lipase is washed and then the supernatant is purified 3
times in the centrifuge using Amicon-10k filters and Buffer A solution (pH of 7.3). Then, Lipase is mixed on
the shaker with sulfo-SMCC in a buffer containing sodium phosphate buffer and NaCl for 1 hour at ambient
conditions. Then, the solution is centrifuged, the excess of insoluble SMCC is discarded, and the supernatant
is purified using Amicon filters.
For the conjugation of LINKER-Enzyme, the LINKER with the activated thiol group is mixed and incubated with
the activated enzyme for 48 hours at room temperature.
Sulfo-SMCC is applied for specific bioconjugates via one- or two-step crosslinking reactions. More
specifically, SMCC is an amine-to-sulfhydryl crosslinker that contains NHS-ester and maleimide reactive groups
at opposite ends of a medium-length cyclohexane spacer arm and it can react towards amino and sulfhydryl
groups. From the one side, the ester-group side, will be conjugated with the enzyme, via the formation of a
covalent amide bond. The ester will be converted into an amide by an aminolysis reaction with the N-Terminal
peptide. We followed the protocol provided by (reference) as described in detail in the Experiments section.
Besides amine-reactive compounds, those having chemical groups that form bonds with sulfhydryls (–SH) are the
most common cross-linkers and modification reagents. The maleimide group reacts specifically with sulfhydryl
groups when the pH of the reaction mixture is between 6.5 and 7.5; the result is formation of a stable
thioether linkage that is not reversible. So, the Linker that now has an available thiol group, will be also
conjugated with the SMCC but from the maleimide side (the opposite side).

![](https://static.igem.wiki/teams/4983/wiki/engineering/pastedgraphic-21.png)

**Figure 17:** Two-step reaction for conjugating LINKER and enzyme with Sulfo-SMCC.

Finally, the process of making the trimeric complex requires the additional preparation of the
streptavidin-coated magnetic beads. Firstly, the beads are resuspended using Buffer A solution as described in
the Experiments Section. The interaction between streptavidin and biotin allows SA-MBs to connect to the
LINKER-Lipase complex, completing the cross-linkage process. Therefore, the LINKER-Lipase complex is mixed
with the solution of streptavidin-coated magnetic beads and remains in the shaker for half an hour at room
temperature. After those steps, the complex consisting of MBs-LINKER-Lipase is formed.

![](https://static.igem.wiki/teams/4983/wiki/engineering/pastedgraphic-22.png)

**Figure 18:** SA-MBs interaction with biotinylated dsDNA

### **Test**

During the above experiments we kept 10 µl of the enzyme used to ensure the enzyme's functionality by
observing a color change after the addition of its substrate. After mixing this amount of enzyme with 10μl of
its substrate, a color change was observed as the supernatant changed from transparent to yellowish; thus
confirming that the enzyme was indeed functional. Subsequently, after joining the magnetic beads with the
LINKER-Lipase complex, we also wanted to confirm that the formation of the trimeric complex was adequately
completed. So, in the solution containing these three parts, we used a magnet to pull this triple complex to
the wall and took 10μl of supernatant to see if it would cause a color change when it's being added to the
substrate solution. We expected no color change in substrate solution as the beads and Lipase would be pulled
to the wall in contact with the magnet resulting in the complete removal of Lipase from the supernatant.
Indeed, 10 µl of the supernatant were mixed with an equivalent amount of substrate and no color change was
observed indicating the successful crosslinking between SA-MBs-LINKER-Lipase.

### **Learn**

From the conduction of the above cycle, we verified the successful crosslinking between the 3 factors which
was a necessary step before proceeding to the next part of the system.

![](https://static.igem.wiki/teams/4983/wiki/engineering/pastedgraphic-23.png)

**Figure 19:** The left tube contains the supernatant of the crosslinking solution
mixed with Lipase’s substrate. It can be clearly seen that the substrate solution retained its color.

![](https://static.igem.wiki/teams/4983/wiki/engineering/pastedgraphic-23.png)

**Figure 17:** The left tube contains the LINKER-Lipase solution mixed with the
substrate solution. The color change of the solution indicates Lipase’s functionality.

# CYCLE 6: Biosensor implementation into deltamethrin-spiked samples and the characterization of its functionality

### **Design**

During all the previous steps, our objective was to see if the individual elements of the experiment were
working properly. For example, we started by choosing the most selective aptamer via the Docking procedure and
the QCM biosensor, we checked the hybridizations of dAPT-L1 and L1-L2 molecules via PAGE and verified the
binding of the magnetic beads with lipase by observing the color change of the supernatant solution. The only
part of the system that could not be determined whether it was working or not was the activation of Cas12a.
Since all the confirmatory steps were carried out, the final experiment was the actual detection of the
pesticide in lab-made deltamethrin-spiked samples to test the functionality of the biosensing system as a
whole.

### **Build**

For the preparation of deltamethrin samples, a stock solution of 5 mg/ml was initially formed which would be
further diluted during the preparation of the total system solutions. 20 µl of deltamethrin stock sample was
mixed with dAPT, L1 and L2 DNA molecules and diluted in Tris-HCl and deionised water to a final volume of 200
µl and a final concentration of 0.5 mg/ml. We set deltamethrin concentration to 0.5mg/ml in the
deltamethrin-dAPT solution as this concentration was indicated as the ideal one for dAPT-delta interaction by
the QCM experiments.
Afterwards, Cas12a enzyme sample was prepared. Cas12a is responsible for the release of lipase into the
supernatant caused by the hydrolysis of LINKER and the subsequent change in color of the substrate solution.
More specifically, 10μl of 10X NEB buffer r2.1 were mixed with 75μl of 1μM Cas12a and 2μl of 100μM crRNA.
Finally, dAPT-deltamethrin, Cas12a and SA-MBs-LINKER-Lipase solutions were mixed and incubated. This would
allow the interaction of particular factors such as L1-L2 hybridization and the induction of several reactions
such as the hydrolysis of LINKER by Cas12a.
In the last part of the detection system described above we use a magnet to attach the magnetic beads to the
walls so that they can be separated from the rest of the solution leaving lipase in the supernatant.
Afterwards, the supernatant was transferred in a new eppendorf and 10μl of it were added in 10μl substrate
solution.

### **Test**

After the incubation of the aforementioned solutions, we performed magnetic separation to the mixture causing
the localization of the SA-MBs to the tube’s wall next to the magnet. As we have described in the design
section of this cycle, we implemented the biosensor to a deltamethrin-spiked water sample. This means that we
expected a positive result from the sensor which would be depicted in a color change of the substrate solution
after adding 10μl of supernatant. However, no color change was observed.

### **Learn**

The fact that there was no color change in the substrate’s solution implies the absence of Lipase in the
supernatant. This in turn means that Lipase was also localized with the SA-MBs during the magnetic separation
stage which could only be possible if LINKER remained intact. As can be easily understood, Cas12a for some
reason was not activated resulting in the non-hydrolysis of LINKER.
The inability for Cas12a activation could in fact lie on several preceding steps of the system. However, as
mentioned above, after a series of experiments in QCM, we have seen that the aptamer can indeed bind with
deltamethrin and that L1-L2 is most likely hybridized as established during the DNA polyacrylamide gel
electrophoresis procedure. Additionally, we didn't only confirm that lipase was functional but also that the
SA-MBs-LINKER-Lipase binding was successful. The successful formation of this trimeric complex rules out the
possibility of problems occurring during the system signal transduction after the hydrolytic action of Cas12a.
As a result, we concluded that the most probable reason for the non-activation of Cas12a was its insufficient
interaction with crRNA which is necessary for Cas12a normal function. In general, RNA molecules are easily
degradable due to their physicochemical properties but also the function of RNases. Therefore, it is highly
possible that during the handling of crRNA it got damaged and rendered non-functional. Another possible cause
could be the inhibition of L1-L2 duplex formation as the dAPT-Deltamethrin solution contained small amount of
DMSO and ethanol which are strong organic solvents that can interfere with hydrogen bond formation.

{% endblock %}

{% extends "layout.html" %}
{% block title %}Experiments{% endblock %}
{% block lead %}Describe the research, experiments, and protocols you used in your iGEM project.{% endblock %}
{% block page\_content %}

* [DNA Aptamers Docking](#docking)
* [QCM Biosensor](#qcm)
* [DNA PAGE](#dna)
* [SA-MBs SYNTHESIS](#strep)
* [LINKER PREPARATION](#linker)
* [LIPASE PREPARATION](#lipase)
* [LINKER-LIPASE CROSSLINKING](#linker-lipase)
* [SA-MBs-LINKER-LIPASE CROSSLINKING](#cross)
* [DELTAMETHRIN SAMPLE PREPARATION](#preparation)
* [DELTAMETHRIN-dAPT INTERACTION](#interaction)
* [Cas12a SOLUTION PREPARATION](#solution)
* [dAPT-DELTAMETHRIN, Cas12a AND SA-MBs-LINKER-LIPASE SOLUTIONS MIXTURE AND INCUBATION](#formation)
* [NEGATIVE CONTROL AND BIOSENSOR OUTPUT](#control)
* [MAGNETIC SEPARATION AND BIOSENSOR OUTPUT](#magnetic)
* [References](#references)

# DNA APTAMERS DOCKING

### **Introduction**

The selection of an appropriate aptamer was the first part needed for our biosensor to be functional. The
structures of the candidate aptamer sequences for deltamethrin and lambda-cyhalothrin binding were uploaded
into the MOE (Molecular Operating Environment) program in order to compare the selectivity of each aptamer to
the two ligands.

### **Materials and Methods used**

* Deltamethrin structure (.pdb file)
* lambda-cyhalothrin structure (.pdb file)
* DNA APTAMERS 1-30 structures (.pdb files)
* [The UNAFold Web Server ( DNA
  Folding Form),](http://www.unafold.org/mfold/applications/dna-folding-form.php) used to obtain aptamer 2D structures
* [ct2dot Web Server by "
  RNAstructure Web Server",](https://rna.urmc.rochester.edu/RNAstructureWeb/Servers/ct2dot/ct2dot.html) used to obtain the aptamer 2D structure as bracket form
* [3dRNA/DNA Web Server (an RNA and DNA tertiary structure
  prediction method) by Xiao lab,](http://biophy.hust.edu.cn/new/3dRNA) used to obtain the .pdb file formats
* MOE (Molecular Operating Environment) Software

### **Procedure**

For both pesticides and aptamers, we created their structure as .pdb files, as described thoroughly in the
according Engineering Success Section and uploaded them to the program (MOE). Then, we decided which part of
the aptamer's three-dimensional structure should be used in the docking procedure and observe how it
interacted with each pesticide. In order to choose the most suitable ones, we determined the top five
configurations after docking that showed the strongest interactions between each aptamer and pesticides. The
best-rated aptamers (those with the highest negative G values) for lambda-cyhalothrin and deltamethrin were
used for the following stage.

### **Results:**

**Table 1.** The results from the five ( 1ST-5TH) most stable conformations of aptamer
**dAPT-1**  and **dAPT-1-39** that docked Deltamethrin are depicted in the table below.

| Deltamethrin | 1ST | 2ND | 3RD | 4RTH | 5TH |
| --- | --- | --- | --- | --- | --- |
| dAPT-1 | -6.4055986 | -6.1100316 | -6.1075654 | -6.0320368 | -6.0149779 |
| dAPT-1-39 | -5.993392 | -5.9848695 | -5.8813043 | -5.8759136 | -5.7243261 |

**Table 2.** The corresponding results of the five most stable conformation of
aptamers
**dAPT-1**  and **dAPT-1-39** were docked with l-cyhalothrin are depicted in the table below.

| lambda- cyhalothrin | 1ST | 2ND | 3RD | 4RTH | 5TH |
| --- | --- | --- | --- | --- | --- |
| dAPT-1 | -6.621511 | -6.522243 | -6.3864374 | -6.2075047 | -6.1549258 |
| dAPT-1-39 | -6.0832882 | -6.0449562 | -6.0015407 | -5.9946046 | -5.9796228 |

As described in the Engineering Success section, the aptamers with the highest scores in binding deltamethrin
and lambda-cyhalothrin were **dAPT-11** and **dAPT-15.** The images below indicate the binding values of
**dAPT-11** and
**dAPT-15** to deltamethrin in rows 1 to 5 and the binding values to lambda-cyhalothrin in rows 6 to 10.

![](https://static.igem.wiki/teams/4983/wiki/experiments/pastedgraphic-1.png)

**Figure 1.** Docking scores for **dAPT-11** (left).

![](https://static.igem.wiki/teams/4983/wiki/experiments/pastedgraphic-2.png)

**Figure 2.** Docking scores for **dAPT-15** (left).

# QCM BIOSENSOR

### **Introduction**

![](https://static.igem.wiki/teams/4983/wiki/experiments/pastedgraphic-4.png)

Quartz crystal microbalance (QCM) systems have proven to be a formidable biosensing platform in the
context of detecting pesticides. Through the utilization of specific aptamers, these QCM-based biosensors
offer a highly sensitive and precise means of detecting deltamethrin and check the binding with our
different aptamers dAPT11 and dAPT15. Their label-free mechanism allows for real-time monitoring of the
binding interactions between deltamethrin and the tailored aptamers, making them a valuable tool in
pesticide detection and analysis.

**Figure 3:** Technical Note forQCM-based biosensors

### **Materials and Methods used**

* PBS buffer ( pH 7.4, 10 mM)
* Neutravidin 0.2 mg/mL
* DNA aptamers dAPT11/ dAPT15, 5pmol (each)
* Deltamethrin in different concentrations ( 0.05 mg/mL and 0.5 mg/mL)
* Buffer D ( 5% DMSO : 5% EtOH : 90% PBS)

### **Biosensor Conditions**

* Temperature 25 oC
* Flow rate 50 μL/ min
* F= 5 MHz

### **Dissolution of Deltamethrin**

* An one-ml solution containing 1:1 DMSO-EtOH was prepared.
* 5 mg Delta was added to 1mL DMSO- EtOH solution and two new solutions with concentrations of 0.5 and
  0.05 mg/mL were created by successive dilutions.

### **Cleaning protocol after the experimental procedure**

* 1st wash Hellmanex (0.5 mL/ min)
* 2nd wash dH2O (0.5 mL/ min)
* 3rd wash EtOH (0.5 mL/ min)
* Air flow until the tubes are dry (1 mL/ min)

### **Procedure**

1. Gold chips are immersed in Hellmanex for a duration of 45 minutes.
2. Subsequently, the chips undergo two sequential washes, alternating between deionized water (dH2O) and 100%
   ethanol.
3. The chips are then subjected to nitrogen drying.
4. Placing the dried chips onto a slide, they are exposed to UV-ozone for 30 minutes to ensure surface
   sterilization.
5. To prepare the phosphate-buffered saline (PBS) solution, it is first filtered through 0.2 µm filters.
6. The biosensor is initiated following the conditions outlined above.
7. The flow is initiated with PBS at a rate of 0.5 mL/min for a duration of 10 minutes.
8. Subsequently, a systematic procedure is enacted within the biosensor where distinct solutions are
   introduced into the tubing system, each followed by a thorough PBS wash.
9. Open QCM Q−1 software was used to monitor resonance frequency (Hz), dissipation, and temperature (°C)
10. In further detail, the wash steps are executed in the following sequence:
    1. Buffer PBS
    2. Neutravidin
    3. Buffer PBS
    4. Aptamers
    5. Buffer PBS
    6. Dimethyl sulfoxide 1:1 (DMSO)- Ethanol (EtOH) 10Χ PBS buffer without the presence of deltamethrin
       (delta) (in our experiments 100μL DMSO-EtOH were dissolved in 900 μL PBS).
    7. Buffer PBS
    8. Buffer containing varying concentrations of deltamethrin (0.05 and 0.5 mg/mL)
11. 11. Finally, the cleaning protocol described above is followed.

### **Results**

The experiments performed in QCM were 6, the first 3 were different from each other and when we optimized the
experimental protocol there was a final triplet of experiments. In this triplet, both aptamers and delta were
placed at the same time ( for each sensor) so that we could compare the signals and conclude which showed the
highest affinity to deltamethrin. Below are presented the results in graphs, which show the comparable signals
of dAPT 11 and dAPT15 in terms of the change in frequency ΔF and the change in dissipation ΔD. As explained in
more detail in Engineering Success, a negative change in frequency means an increase in the mass found on the
sensor.

![](https://static.igem.wiki/teams/4983/wiki/experiments/pastedgraphic-5.png)

**Figure 4.** Change in frequency ΔF

![](https://static.igem.wiki/teams/4983/wiki/experiments/pastedgraphic-6.png)

**Figure 5.** Change in dissipation ΔD

The graphs were randomly selected and belong to the last experiment. What we observe is the negative shift in
frequency which is almost the same in both aptamers suggesting that the aptamers could show the same
selectivity for the pesticide. Overall, in all experiments we had signals, while in few these signals were not
fully interpreted, which indicates the success of the experimental process.

# DNA POLYACRYLAMIDE GEL ELECTROPHORESIS (DNA PAGE)

DNA Polyacrylamide gel electrophoresis (PAGE) is a common method for separating DNA fragments based on their
size and structure. Given that L1 and L2 are DNAs of small length, we used PAGE to verify the assembly of the
hairpin structures for L1 and L2 molecules and observe the L1-L2 duplex as well as the L1-dAPT duplex. In
order to proceed with the electrophoresis, a hybridization between the molecules L1-L2 and DAPT-L1 had to be
carried out first.

### **Hybridization**

### **Equipment used**

* Incubator

### **Materials used**

* L1
* L2
* dAPT
* TE Buffer (pH 8)

### **Procedure**

1. 3 μl of each DNA sample is mixed with 6μl TE buffer in 1000 µl tubes.
2. The tubes are placed in a water bath, adjusted to a temperature of 94oC.
3. The machine is turned off and the water is left to reach room temperature.
4. The tubes are removed from the water and subjected to spin down.

### **Electrophoresis**

### **Equipment used**

* Gel Electrophoresis Apparatus
* Power Supply
* Gel Casting System
* Pipettes of 10 μM and the Tips of the respective size
* UV Transilluminator
* Gel Documentation System

### **Materials used**

* Acrylamide:bisacrylamide (29:1) (30% w/v)
* H2O
* Ammonium persulfate (10% w/v)
* 1x TBE electrophoresis buffer
* 10% TEMED
* DNA Safe Stain
* Loading Buffer 6X (Dye)
* L1 DNA solution
* L2 DNA solution
* dAPT DNA solution

### **Procedure**

1. The glass plates and spacers of the gel caster are rinsed with deionized water and ethanol and are set
   aside to dry, so that no air bubbles are formed in the gel.
2. A 8% polyacrylamide gel solution is prepared to make a 12ml minigel of 2mm thickness. 3.2ml 30% Acrylamide
   (29:1), 6.4ml H2O, 200μl APS, 2.4ml 1X TBE, 10μl TEMED, and DNA Safe Stain are mixed.
3. The gel is inserted into vertical glass plates with spacers in the gel caster. The appropriate combs of 15
   positions are inserted into the gel, slightly higher than the top of the glass, and clamped in place with
   bulldog paper clips.
4. The acrylamide is set to polymerize for 30-60 minutes at room temperature.
5. After the spilled gel has been carefully cleaned from the back of white plates, the gel is ready to be
   removed from the gel caster and inserted into the gelbox. Running buffer 1X TBE is added, and the combs are
   removed from the polymerized gel.
6. DNA samples are mixed with different amounts of gel loading buffer (dye), and the mixture is loaded into
   the wells using a micropipette.
7. The electrodes are connected to a power pack, the power is turned on, and the electrophoresis begins to
   run.
8. The gel runs at a voltage of 80V for 15 minutes. The procedure continues with an increase of 10V (100V)
   for 15 minutes more, until the marker dyes have migrated the desired distance.
9. After 30 minutes, the electric power is turned off, the leads are disconnected, and the electrophoresis
   buffer is discarded from the reservoirs. The glass plates are detached and laid on the bench. After using a
   spacer or plastic wedge to lift a corner of the upper glass plate, the gel remains attached to the lower
   (white) plate, and the upper plate is pulled smoothly away. The spacers are removed, and the gel is exposed
   to a UV Transilluminator in a Gel Documentation System, which takes a photo. The photo is processed by the
   application Photo Imager.

### **Results**

![](https://static.igem.wiki/teams/4983/wiki/experiments/pastedgraphic-7.png)

**Figure 6.** The bands depicted in the polyacrylamide gel after the first cycle of
Electrophoresis as they are described in the Learn Section of Cycle 3A of the Engineering Success.

![](https://static.igem.wiki/teams/4983/wiki/experiments/pastedgraphic-8.png)

**Figure 7.** The bands depicted in the polyacrylamide gel after the second cycle
of Electrophoresis as they are described in the Learn Section of Cycle 3B of the Engineering Success.

# STREPTAVIDIN COATED MAGNETIC BEADS SYNTHESIS

The streptavidin coated magnetic beads were synthesized in the lab. The main redox reaction which took place
for the formation of magnetic beads was:
2Fe3+(aq) + Fe2+(aq) + 8OH- ⇒ Fe3O4(s) +
4H2O
The formation of magnetite aggregates (Fe3O4 (s)) occurs under alkaline conditions which are induced by the
addition of NH4OH, after which, beads are mixed with streptavidin in order to form the desired complex.

### **Materials and Methods used**

* FeCl2
* FeCl3
* Streptavidin
* dH2O
* Conical flask
* Magnets
* Stirrer
* Cold Room
* NH4OH
* Weigher

### **Procedure**

1. 4.46g of FeCl3 and 1.6g of FeCl2 were diluted in 80 ml of dH2O and stirred for 30 minutes at room
   temperature.
2. 10 ml of NH4OH (29% w/w) were added dropwise to the mixture.
3. Stirring for another 10 minutes at room temperature.
4. The mixture was heated until it reached 90°C and stirred for another 90 minutes.
5. Repeated washes of the formatted magnetite beads with dH2O until the pH of the solution was back to normal
   (pH=7).
6. After the final wash, the beads were redispersed in 50 ml dH2O. This solution was used as a stock.
7. 50μl and 10μl from the stock were transferred to 2 new eppendorf tubes and were diluted with the addition
   of 950μl and 990μl in order to make 1/20 and 1/100 dilutions, respectively.
8. 2μl of Streptavidin solution were added to each tube, and the mixture was stirred overnight at 4°C.
9. Finally, the streptavidin coated magnetic beads were repeatedly washed and redispersed in 1.5 ml dH2O.
10. Storage of streptavidin coated magnetic beads at room temperature.
11. The verification of the completed synthesis of SA-MBs came through imaging with a Scanning Electron
    Microscope (SEM) and Energy-dispersive X-Ray (EDX) analysis.

### **Results**

Firstly, through SEM microscopy we verified the completed synthesis of the Magnetic Beads. The captured image
was the following.

![](https://static.igem.wiki/teams/4983/wiki/experiments/pastedgraphic-9.png)

**Figure 8.** SA-MBs observation using SEM.

Afterwards, we investigated the chemical composition of the magnetic beads through EDX analysis. The way EDX
analysis works is that the electron beam hits the inner shell of an atom, knocking off an electron from the
shell, while leaving a positively charged electron hole. When the electron is displaced, it attracts another
electron from an outer shell to fill the vacancy. As the electron moves from the outer higher-energy to the
inner lower-energy shell of the atom, this energy difference can be released in the form of an X-ray. The
energy of this X-ray is unique to the specific element and transition.

![](https://static.igem.wiki/teams/4983/wiki/experiments/pastedgraphic-10.png)

**Figure 9.** EDX analysis explanation.

The results of the EDX analysis are depicted in a diagram in which Y axis represents the count of the photons
emitted by the atoms of the beads and the X axis represents the Energy of each captured photon which, as
described previously, characterizes the atom from which the photon was produced.

![](https://static.igem.wiki/teams/4983/wiki/experiments/pastedgraphic-11.png)

**Figure 10.** EDX analysis results.

Each peak in the diagram is due to the existence of a specific atom in the examined sample. There are clearly
many peaks which correlate to the Fe and O atoms, which are obviously abundant in the sample as the beads
themselves are in fact magnetite aggregates (Fe3O4) which are mostly composed of O and Fe atoms. The high peak
which correlates to the Si atom is formed because the beads were placed on top of a glass plane when they were
analyzed in SEM. Lastly, we can also see a peak correlating to the C atom which is most likely formed by the C
atoms of the Streptavidin molecules thus indicating the successful coating.

# LINKER PREPARATION

### **Introduction**

LINKER DNA is an intermediate in connecting Lipase with streptavidin coated magnetic beads (SA-MBs). But
firstly, it must go through a preparation phase to be attached onto the enzyme and the SA-MBs. An important
step is mixing LINKER and TCEP, which will break the 3' Thiol Modifier C3 S-S attachment of LINKER thus
creating the required thiol group for the LINKER to bind onto the enzyme by a disulfide bond.

### **Materials and Methods used**

* LINKER DNA
* ddH2O
* Centrifuge
* Amicon-10k filters
* TCEP
* Buffer A
* Sodium phosphate buffer, pH=5.5

### **Composition of Sodium phosphate buffer, pH=5.5**

1. Prepare 800 mL of distilled water in a suitable container.
2. Add 20.214 g of Sodium Phosphate Dibasic Heptahydrate to the solution (mw: 268.07 g/mol).
3. Add 3.394 g of Sodium Phosphate Monobasic Monohydrate to the solution (mw: 137.99 g/mol).
4. Adjust the solution to the final desired pH using HCl or NaOH.
5. Add distilled water until the volume is 1 L.

### **Composition of Buffer A**

* 0.1M NaCl
* 0.1M sodium phosphate buffer

### **Procedure**

1. 40μl of DNA3 with a concentration of 1mM, 5μl of sodium phosphate buffer with pH 5.5 and a concentration
   of 1M, and 4μl of 25mM TCEP are mixed and kept at room temperature for 1 hour. For the preparation of 25mM
   TCEP, 1.4mg of TCEP was dissolved in 0.2 ml of buffer A.
2. The above mixture is purified 3 times in the centrifuge using Amicon-10k filters and Buffer A solution (pH
   of 7.3).

# LIPASE PREPARATION

### **Introduction**

Lipase (Biolipasa R) is the last part of the detection system and is the chromogenic enzyme which ultimately
changes the color of the solution under deltamethrin presence. Lipase, as previously described, is connected
to LINKER. Like LINKER, Lipase must undergo a preparation phase to be able to participate in this connection.
In this preparation phase Lipase is mixed with sulfo-SMCC, a known reagent for its ability to create specific
bioconjugates via one- or two-step crosslinking reactions.

### **Materials and Methods used**

* Sulfo-SMCC
* Lipase
* Amicon-10k filters
* Vortex
* Shaker
* Buffer A

### **Composition of Buffer A**

* 0.1M NaCl
* 0.1M sodium phosphate buffer

### **Procedure**

1. 743μl of Lipase 175μM is mixed with 500μl of Buffer A, and 1.15mg of sulfo-SMCC is added to the occurring
   solution.
2. The mixture is vortexed for 5 minutes and then left on the shaker for 1 hour at room temperature.
3. The solution is centrifuged, and the excess insoluble sulfo-SMCC is discarded.
4. The supernatant is purified 3 times in the centrifuge using Amicon-10k filters and Buffer A solution (pH
   of 7.3).

# LINKER-LIPASE CROSSLINKING

### **Introduction**

By connecting Lipase to LINKER we can ensure that Lipase is left in the supernatant after magnetic separation
only if Deltamethrin is present. As previously described, Lipase will be crosslinked with LINKER with the help
of sulfo-SMCC.

### **Materials and Methods used**

* TCEP preincubated LINKER
* sulfo-SMCC activated Lipase
* Centrifuge
* Amicon-10k filters
* Buffer A

### **Composition of Buffer A**

* 0.1M NaCl
* 0.1M sodium phosphate buffer

### **Procedure**

1. TCEP preincubated LINKER and sulfo-SMCC activated Lipase are mixed and incubated for 48 hours at room
   temperature.
2. For the removal of non-reacted LINKER, the mixture is purified 3 times in the centrifuge using Amicon-10k
   filters and Buffer A solution (pH of 7.3).

# SA-MBS-LINKER-LIPASE CROSSLINKING

### **Introduction**

SA-MBs after their synthesis should be prepared and resuspended in the same buffer (Buffer A) in which the
LINKER linked Lipase was resuspended, as described previously. The process itself is simple and is achieved by
streptavidin-biotin interaction.

### **Materials and Methods used**

* Streptavidin coated-magnetic beads (SA-MBs)
* LINKER-Lipase solution
* dH2O
* Buffer A
* Stirrer

### **Composition of Buffer A**

* 0.1M NaCl
* 0.1M sodium phosphate buffer

### **Procedure**

1. 10μl of the 1/20 dilution of SA-MBs were mixed with 90μl of Buffer A solution.
2. 100μl of LINKER-Lipase solution were mixed with the above SA-MBs solution and stirred for 30 minutes at
   room temperature.

### **Results**

The results of the synthesis were depicted by Lipase function. As described in the Engineering Success
section, Lipase converts p-nitrophenyl ester to the chromogenic product 4-nitrophenol which changes the color
of the solution to yellow. However, before we proceeded to the crosslinking process we tested Lipase activity,
which was already linked with LINKER. The test consisted of mixing 10μl Lipase solution with 10μl
p-nitrophenyl ester solution. Almost immediately, the color of the solution turned bright yellow indicating
Lipase’s function.

![](https://static.igem.wiki/teams/4983/wiki/experiments/pastedgraphic-12.png)

**Figure 11.** Color change of the substrate solution after adding Lipase.

After the crosslinking process, we tested the completion of the conjugation between the aforementioned
factors. The crosslinking solution was next to a magnet causing the immediate localization of the magnetic
beads to the wall in contact with the magnet. The solution did not contain any Cas12a so the LINKER molecule
remained intact. If the crosslinking happened, Lipase would also be localized at the tube’s wall so the
supernatant remains without any enzyme molecules. Indeed, after transferring 10μl of the supernatant to the
substrate there was no color change, indicating the absence of Lipase in the supernatant and the successful
crosslinking process.

![](https://static.igem.wiki/teams/4983/wiki/experiments/pastedgraphic-13.png)

**Figure 12.** The left tube contains the supernatant of the crosslinking solution
mixed with Lipase’s substrate. The right tube is the one also shown in **Figure 11** which contains the
LINKER-Lipase solution mixed with the substrates solution.

# DELTAMETHRIN SAMPLE PREPARATION

### **Introduction**

Deltamethrin-spiked water samples were produced in the laboratory. The concentration of the Deltamethrin was
set to 0.5mg/ml according to the results of QCM biosensor procedure that indicated that the aforementioned
concentration of deltamethrin was the ideal for the most efficient binding with the aptamer.

### **Materials and Methods used**

* Deltamethrin
* DMSO
* Ethanol 100%
* dH2O
* Pipettes
* Weigher

### **Procedure**

1. 510μl of DMSO and 510μl of EtOH were mixed to produce the resuspension solution.
2. 5.1 mg of dried Deltamethrin were weighed and resuspended with the resuspension solution to create the
   stock solution.

# DELTAMETHRIN-dAPT INTERACTION AND L1-L2 DUPLEX FORMATION

### **Introduction**

In this part, the deltamethrin sample was mixed with the first 3 biosensing factors of the system which are
dAPT, L1 and L2 DNA molecules. As described in the Engineering Success section, deltamethrin will bind with
dAPT which will ultimately cause L1-L2 duplex formation.

### **Materials and Methods used**

* Deltamethrin stock solution
* 2μl of 100μM dAPT
* 4μl of 100μM L1
* 4μl of 100μM L2
* Tris-HCl 2X buffer solution, 20mM, pH 7.4
* dH2O

### **Composition of 10ml Tris-HCl 2X buffer solution, 40mM, pH 7.4**

1. 660μl of 3M NaCl
2. 66μl of 3M KCl
3. 200μl of 1M MgCl2
4. 800μl of 0.5M Tris-HCL with a pH of 7.4
5. 8.247 ml of dH2O

### **Procedure**

1. 95μl of Tris-HCL 2X buffer solution was mixed with 20μl Deltamethrin stock solution and 75μl dH2O.
2. 2μl of 100μM dAPT, 4μl of 100μM L1, and 4μl of 100μM L2 were added to the aforementioned deltamethrin
   solution.
3. The mixture was incubated for 50 minutes at room temperature.

# Cas12a SOLUTION PREPARATION

### **Introduction**

Cas12a enzyme is responsible for LINKER hydrolysis which will ultimately release Lipase into the supernatant,
as described in the Engineering Success section. NEB Buffer r2.1 is necessary for optimum Cas12a activity as
it provides the ideal pH and ionic concentration. Additionally, Cas12a requires the presence of a gRNA, called
crRNA in our system, by which sequence Cas12a will hydrolyze its target DNA molecule.

### **Materials and Methods used**

* 10μl of 10X NEB buffer r2.1
* 75μl of 1μΜ Cas12a
* 2μl of 100μΜ crRNA
* 13μl dH2O
* Eppendorf tube

### **Composition of 10X NEB buffer r2.1 (pH 7.9)**

1. 500 mM NaCl 100 mM Tris-HCl
2. 100 mM MgCl2
3. 1000 µg/ml Recombinant Albumin

### **Procedure**

1. 10μl 10X NEB buffer r2.1 were mixed with 75μl of 1μM Cas12a, 2μl of 100μΜ crRNA and 13μl dH2O.

# dAPT-DELTAMETHRIN, Cas12a AND SA-MBs-LINKER-LIPASE SOLUTIONS MIXTURE AND INCUBATION

### **Introduction**

In the Deltamethrin’s solution due to pesticide-aptamer interaction L1 and L2 molecules will form a dsDNA.
Additionally, Cas12a after interacting with crRNA and forming Cas12a-crRNA complex is able to bind onto any
DNA molecule that has a complementary sequence to the sequence of crRNA. Therefore, crRNA, L1 and L2 molecules
are designed in such a way that the Cas12a can bind onto L1-L2 duplex due to its hybridization with crRNA.
Cas12a is integrated into the biosensing system in order to hydrolyze LINKER DNA. This will release Lipase
into the supernatant which color will change after its mixing with substrate solution. All the aforementioned
reactions require the sequential interaction of the system’s factors which in turn requires the incubation of
the individual solutions.

### **Materials and Methods used**

* Deltamethrin-dAPT solution
* Cas12a solution
* SA-MBs-LINKER-Lipase crosslinking solution
* Pipettes

### **Procedure**

1. 200μl of Deltamethrin-dAPT solution was mixed with 100μl Cas12a solution and 100μl SA-MBs-LINKER-Lipase
   crosslinking solution.
2. The resulting solution was incubated for 90 minutes at room temperature.

# NEGATIVE CONTROL AND BIOSENSOR OUTPUT

### **Introduction**

A step of testing the biosensing system with a negative control which contains no deltamethrin is an
absolutely necessary part for characterizing the biosensor’s function and liability. In our case, the negative
control will verify that the biosensing system is not activated and thus not produce any outcome in case of
deltamethrin’s absence. Lastly, in order to produce reliable results about the negative control we have to
execute the system exactly as we executed it while analyzing the examination samples. This means that we
conduct the exact same steps using the same reagents with the same volumes and concentration as we did while
testing the examination sample.

### **Materials and Methods used**

* We conducted the same steps while using the same reagents with same volumes and concentration as described
  in all of the previous sections
* The final step of the magnetic separation and the depiction of the biosensor output are conducted as
  explained in the next section MAGNETIC SEPARATION AND BIOSENSOR OUTPUT

### **Procedure**

As we mentioned previously, the biosensing procedure remained the same. However, the negative control sample
must not contain any deltamethrin residues. For this reason, while preparing the negative sample we did not
add any deltamethrin to the 1ml 1:1 DMSO:ethanol solution.

### **Results**

After magnetic separation of the negative-dAPT-Cas12a-SA-MBs-LINKER-Lipase mixture and the transfer of 10μl
supernatant to lipase substrate solution no color change was observed. This was the expected result, as Cas12a
should have not been activated due to the fact that dAPT duplex was not denaturated and thus the L1-L2 duplex
should have never been formed. However, an expected outcome while analyzing a negative control does not always
imply that the biosensor functions properly. In order to further test our biosensor function we have to
analyze a deltamethrin-spike lab-made examination sample and expect to see a positive result.

# MAGNETIC SEPARATION AND BIOSENSOR OUTPUT

### **Introduction**

The final step of the biosensing system is the magnetic separation of the magnetic beads and the
fractionation of supernatant. Magnetic separation is an efficient way to prevent false positive results, as it
removes lipase from the supernatant thus preventing the subsequent color change of the substrate solution.

![](https://static.igem.wiki/teams/4983/wiki/experiments/pastedgraphic-14.png)

**Figure 13** Animation of the magnetic separation process.

<https://www.phenomenex.com/products/biozenspe-sample-preparation-products/biozen-magbeads>

![](https://static.igem.wiki/teams/4983/wiki/experiments/pastedgraphic-15.png)

**Figure 14** Magnetic separation process conducted in the lab.

### **Materials and Methods used**

* 10 μl of dAPT-Deltamethrin, Cas12a And SA-MBs-Linker-Lipase Mixture
* Magnet
* p-nitrophenyl ester
* Pipette

### **Procedure**

1. A magnet comes in contact with the tube containing the biosensing mixture.
2. Complete magnetic separation of the mixture, which is indicated by the clearance of the mixture due to the
   localization of the magnetic beads at the tube’s wall in contact with the magnet.
3. 10μl of the clear supernatant is transferred to the tube containing lipase’s substrate.
4. Color change observation.

### **Results**

As we mentioned in the **deltamethrin’s sample preparation** section, the examination sample contained 0.5
mg/ml
deltamethrin. Obviously, the expected result was the color change of the substrate solution as the supernatant
should contain Lipase. However, after the addition of supernatant to substrate solution, the latter retained
its color indicating the absence of Lipase in the supernatant. The fact that lipase was not contained in the
supernatant is most likely due to the non-hydrolysis of LINKER by Cas12a. However, the determination of the
cause that leads to dysfunctioning of Cas12a is very difficult as such non-activation can lie in almost all of
the previous steps of the system. All of the possible causes and our hypothesis about the unexpected result
are described thoroughly in the according Engineering success section.

# REFERENCES

1. <https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7443316/pdf/main.pdf>
2. <https://www.researchgate.net/publication/356686700_Quartz_Crystal_Microbalance_QCM_Sensing_Materials_in_Biosensors_Development>
3. <https://www.neb.com/en/products/b6002-nebuffer-r2-1#Product%20Information>
4. Chen, J., Shi, G., & Yan, C. (2023). Portable biosensor for on-site detection of kanamycin in water
   samples based on CRISPR-Cas12a and an off-the-shelf glucometer. The Science of the total environment, 872,
   162279. <https://doi.org/10.1016/j.scitotenv.2023.162279>
5. Yang, Y., Tang, Y., Wang, C., Liu, B., & Wu, Y. (2021). Selection and identification of a DNA aptamer for
   ultrasensitive and selective detection of λ-cyhalothrin residue in food. Analytica chimica acta, 1179,
   338837. <https://doi.org/10.1016/j.aca.2021.338837>
6. Gong, P., Peng, Z., Wang, Y. et al. Synthesis of streptavidin-conjugated magnetic nanoparticles for DNA
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