Team:Bielefeld-CeBiTec/Proof Of Concept
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When you take a look at our SAW biosensor, you will notice that biology happens only on the sensing film of the SAW biosensor. Here, antibodies or other binding molecules that specifically bind our analyte are immobilized. You can learn more about the design of our binding molecules.. The rest of the SAW chip requires a lot of state of the art electronic parts, carefully adjusted to make very sensitive measurements possible. The parts of our hardware include for example the the chip case and the electronics (PCB).
As our project is a measurement and data analysis project, having an app that saves, manages, and displays the collected data, is a key part. We created an app in python with kivy that achieves that.
We showed that relevant data sets can be inserted and by comparing the inserted data to the created graphs, displayed correctly. To learn more, visit our App page
The Printed Circuit Board (PCB) is one of the most important parts of our project, because it allows us to detect an analog signal, which indicates a possible phase shift. Thus a prediction about the ovulation can be made. To
prove the functionality of our PCB, we had to create artificial phase
shifts via the pin headers directly on the PCB. The areas marked in
red in Fig. 3 can be connected in bridges, allowing a total of 16
different time shifts (in ps) to be simulated.
In
order to cover the entire bandwidth of the DDS, we have generated the
frequencies from 50 MHz to 600 MHz in 50 MHz steps for all 16 time
shifts. We could detect the analog to digital converted voltage
values on the serial console of the Arduino IDE. The results can be
found on our hardware page.
For more detailed measurement results, all important files (such as the matlap plots, the phase shift measurement table and the Arduino code) can be downloaded via the following link. Github-Link
The
aim of our project was to determine the concentration of the cycle
hormones estradiol, progesterone and LH (luteinizing hormone). To
detect these hormones with the chip, we designed and expressed
hormone-specific binding molecules
Estradiol and progesterone complicate measurement based on mass shifts due to their small molecular mass, which is why nanobodies are needed (Learn more on our scFvs page). Using our chip, we want to detect differences in the mass of bound and unbound nanobodies. Since no nanobodies with the ability to bind our analytes exist, we decided to use nanobody grafting to construct these. For in silico grafting, one has to know the sequences of the CDRs 1 to 3 of an antibody against the target analyte. Furthermore, the functionally important framework residues of the antibody are transferred onto the acceptor scaffold to create a new nanobody.
We used ChimeraX to analyze the 3D structure of the grafted nanobodies regarding steric obstacles to antigen binding. After this control, the amino acid sequences were translated into cDNA sequences. The cDNA sequences were then optimized for GC content as well as length and amount of repeats. The modified nucleotide sequence was purchased as gene synthesis. However, this nanobody grafting process is faulty and weak binders are formed. Therefore, it is necessary to perform an affinity maturation measurement, such as phage display. The newly designed binders are expressed and tested in phage ELISAs. Learn more on our Nanobody and scFv page.
We ware able to demonstrate the functionality of our measurement components. Our work can therefore be used in future iGEM project aiming to design reliable and sensitive biosensors.
As affinity maturation method
the phage display technique, more precisely the phagemid display
technique was chosen. To induce an improvement in affinity, the
sequence of the grafted nanobodies first had to be mutated to create
different versions of the sequence. For the random mutagenesis of the
sequences the error prone PCR (epPCR) was performed. A comparison
between the two polymerases OneTaq® DNA polymerase and Taq DNA
polymerase was performed, to determine their error rates. It was
found that OneTaq® gives better epPCR results.
Therefore, we decided to continue working with this polymerase.
We succceeded in amplifying the estradiol nanobody by means of epPCR but could not amplify the progesterone nanobody with our epPCR protocol. Thus, we decided to switch to a commercial epPCR kit, which made it possible to amplify the progesterone nanobody as well.
After successful creation of a
library by random mutagenesis via epPCR, we cloned the variable
fragments into the phagemid vector pZMB0062. Subsequently, the E.
coli strain ER2738 was transformed by electroshock transformation.
The resulting clones were screened by colony PCR for the integration
of the insert into the vector. The majority of ER2738 clones carried
the vector with the insert. We checked the mutation frequency by
Illumina MiSeq sequencing and found out, that the kit had a
lowermutation frequency compared tothe “selfmade” approach which
was using the protocol from McCullum et al. from 2010. Nevertheless
we chose the kit, because with this kit both nanobody fragments could
be amplified and a library could be created.
Proof of concept
When you take a look at our SAW biosensor, you will notice that biology happens only on the sensing film of the SAW biosensor. Here, antibodies or other binding molecules that specifically bind our analyte are immobilized. You can learn more about the design of our binding molecules.. The rest of the SAW chip requires a lot of state of the art electronic parts, carefully adjusted to make very sensitive measurements possible. The parts of our hardware include for example the the chip case and the electronics (PCB).
Fig. 1 - SAW sensor components. Quartz piezoelectric crystal, IDTs (interdigital transducer) are built on the crystal, the sensing film is located between the IDTs, the SAW Chip is inserted in a chip case which establish the connection to the electronics, the electronics are connected to the app to evaluate the data.
App
We showed that relevant data sets can be inserted and by comparing the inserted data to the created graphs, displayed correctly. To learn more, visit our App page
Fig. 2 - Graph showing the in the entry.json (check the data here) saved data.
Printed Circuit Board
Fig. 3 - Printed Circuit Board visualized in the Altium Designer developement environment. This view allows the developer to edit the path of the tracks as well as the position of the various components.
Fig. 4 - PCB Pinheader Marked which could be connected with bridges to create an artificial time shift.
For more detailed measurement results, all important files (such as the matlap plots, the phase shift measurement table and the Arduino code) can be downloaded via the following link. Github-Link
Antibodies - The magic on the sensing film
Estradiol and progesterone complicate measurement based on mass shifts due to their small molecular mass, which is why nanobodies are needed (Learn more on our scFvs page). Using our chip, we want to detect differences in the mass of bound and unbound nanobodies. Since no nanobodies with the ability to bind our analytes exist, we decided to use nanobody grafting to construct these. For in silico grafting, one has to know the sequences of the CDRs 1 to 3 of an antibody against the target analyte. Furthermore, the functionally important framework residues of the antibody are transferred onto the acceptor scaffold to create a new nanobody.
We used ChimeraX to analyze the 3D structure of the grafted nanobodies regarding steric obstacles to antigen binding. After this control, the amino acid sequences were translated into cDNA sequences. The cDNA sequences were then optimized for GC content as well as length and amount of repeats. The modified nucleotide sequence was purchased as gene synthesis. However, this nanobody grafting process is faulty and weak binders are formed. Therefore, it is necessary to perform an affinity maturation measurement, such as phage display. The newly designed binders are expressed and tested in phage ELISAs. Learn more on our Nanobody and scFv page.
We ware able to demonstrate the functionality of our measurement components. Our work can therefore be used in future iGEM project aiming to design reliable and sensitive biosensors.
Fig. 3 - Grafting process of nanobodies.
Ep PCR
We succceeded in amplifying the estradiol nanobody by means of epPCR but could not amplify the progesterone nanobody with our epPCR protocol. Thus, we decided to switch to a commercial epPCR kit, which made it possible to amplify the progesterone nanobody as well.
Cloning
Phage ELISA
The difficult situation with COVID-19 (reduced time in the lab, less people allowed to work at the same time in less time, companies that refuse sponsorship or cooperation, etc.) resulted in only a preliminary test for binding affinity by phage ELISA with our original nanobody grafts. The results showed that the first grafts of the two antibodies did not bind their respective antigens or bound them poorly. Therefore, the specificity of the antibodies has to be increased further by appropriate affinity maturation.
Fig. 4 - Schematic overview of the phage ELISA. If nanobody-fusion protein expressing phages bind the via streptavidin-biotin immobilized antigen, the phages can be detected after washing by the enzymatic reaction catalyzed by the horseradish peroxidase (HRP). HRP is conjugated to an antibody targeting protein VIII (pVIII) from phage M13. The strength of the signal correlates to the number of binding phages: The more phages bind, the stronger the signal.