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

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.

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

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

QCM BIOSENSOR

Introduction

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.

Figure 4. Change in frequency ΔF

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

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.

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.

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.

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.

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.

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.

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.

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
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