After learning about the increasing global problem of nanoplastic pollution, its harsh potential consequences, and the current lack of detection tools, our team has decided to develop a widely-accessible nanoplastic detection system that would open up new ways in solving this rising issue. One of the ways we envisioned our contribution to solving issues with nanoplastic pollution is to propose identification and quantification techniques that would enable rapid and frequent monitoring of the abundance of plastic nanoparticles in the surrounding environment. This is what the NanoFind project is all about.

On our journey of developing a successfully operating detection system for nanoplastics, several challenges had to be tackled:

• 1) Identification - how can we determine the specific interactions between plastic nanoparticles and the detection executing compound.
• 2) Immobilization - how can this specific interaction be tested in a contained environment.
• 3) Characterization - how can we prove that our detecting unit is bound specifically to plastic nanoparticles.

With the aim to have a good starting point for our system, we did an in-depth research on current analytical techniques used in the identification of synthetic materials. We have selected small protein molecules, peptides, as base components for the detection system. First, we have seen proof from the literature that peptides have already gained traction for solving different detection problems and have shown exceptional results in this field [1-5]. Secondly, additional fundamental research described certain peptides with the ability to attach to different types of plastics with good selectivity[6-7]. Based on the mentioned scientific literature, we chose peptides, namely liquid chromatography peak I (LCI) from a Bacillus subtilis strain A014 and tachystatin TA2 from a Japanese horseshoe crab (lot. Tachypleus tridentatus). To our advantage, it was previously shown in the literature that these peptides can be successfully synthesized and tested in a laboratory [7-8]. Also, it was shown that these peptides exhibit plastic binding abilities. Hence, we decided to use LCI and TA2 peptides as base components of our detection system.

In order to carry out the identification of plastic nanoparticles in the contained environment, a fraction of peptides had to be immobilized on the testing surface. We acknowledged that cellulose binding domains or CBDs have immense application potential in the biotechnology field [10]. Our system needed a simple and versatile immobilization unit, and cellulose proposed an ideal inert matrix for our detection system.

In our project, we chose to utilize a cellulose binding domain from Clostridium Thermocellum [9] (BBa_K4380000). The main purpose of this domain was to help immobilize plastic binding peptides LCI and TA2 on a cellulose membrane. For this, two composite parts were created: CBD-LCI fusion protein (BBa_K4380015) and CBD-TA2 fusion protein (BBa_K4380017). Peptides bound to CBDs were a crucial component of the proposed detection system.

We envisioned that the detection output could be easily measured with standard laboratory equipment. To our advantage, fluorescence based methods fulfill such requirements. For that reason, we had to include a fluorescent protein as a corresponding detection component. We chose a well-known enhanced green fluorescent protein - eGFP which allows an easy-to-interpret result to be obtained, and also enables quantification of nanoplastics in the sample.

With this detection system, we always thought about the possibility of our peptides attaching to plastic surfaces and giving false-positive results. Therefore, to create a detection system we needed a fully glass-coated material. Therefore, we decided to create a hardware device, imitating the well-known 96-well microplate. In part, this problem can be solved using a glass-coated testing surface or fully glass-based hardware solutions (See: Hardware). Besides that, experiments could not be performed using plastic equipment. Therefore small glass-based hardware devices had to be created (See: Contribution).

Our envisioned nanoplastic detection starts with an attachment of plastic-binding peptide and cellulose binding domain fusion proteins to the cellulose base. Following the immobilization, a test sample, potentially containing nanoplastic particles is delivered. Bound nanoplastics are then detected using plastic-binding peptides conjugated with eGFP. After addition of each component, the system is carefully washed to obtain a fluorescence response representing only the bound peptide-nanoplastic units.

Final concept of the "NanoFind" detection system:

We believe difficulties may arise when testing environmental samples with nanoplastics due to their extremely low concentrations. Therefore, we propose an amplification strategy that may help obtain a stronger detection signal when testing water samples. The idea for this approach is to use live bacteria that display our plastic binding peptides with a novel cell-surface display system (Parts: BBa_K4380019, BBa_K4380020). (Fig. 1).

Fig. 1. Amplification via cell-surface display technique (modified) [11].

After exposing bacteria to samples with nanoplastic and carefully washing them, bacteria, co-transformed with a compatible fluorescent protein (Parts: BBa_K4380010, BBa_K4380011) caring plasmids, would be able to amplify the signal in real time by growing and dividing.

Although using known peptides for a detection system is already a huge challenge itself, we wanted to characterize novel peptides and create peptide libraries, from which obtained plastic binding peptide variants could be used in our detection system. Therefore, we decided to work on a peptide evolution protocol, specifically designed to improve the polymer-binding peptides.

This part of the project was inspired by Rübsam and colleagues (2017), who developed a peptide evolution method, called PePevo (peptide-polymer evolution protocol). According to the authors, this protocol was validated in two directed evolution campaigns for PBPs and polymers, namely peptide LCI and polypropylene (PP), peptide TA2 and polystyrene (PS). As selection pressure for improved PBPs surfactants were used: non-ionic surfactant Triton X-100 and anionic surfactant LAS. From this work, we acquired knowledge about the efficiency of the used method, peptide-polymer combinations and selection conditions. However, for simplicity measures, we used an alternative approach - error-prone PCR (epPCR) protocol and optimized conditions to our convenience, expecting similar improved PBP variants to be obtained. The idea for this approach is to use live bacteria that display our mutated variants of plastic binding peptides with a novel cell-surface display system (Parts: BBa_K4380014, BBa_K4380019, BBa_K4380020). For this, the team designed this system specific epPCR primers (BBa_K4380006, BBa_K4380007) (Fig. 1).

In our case, a high mutation rate had to be reached. Therefore, two different error prone PCR techniques were applied:

This epPCR method utilizes a mixture of triphosphates of nucleoside analogs. The performed method relies on DNA amplification in vitro with Taq polymerase in the presence of two known modified nucleotides: 8-Oxo-dGTP (8-Oxo-2`deoxyguanosine-5`-triphosphate) and dPTP (6H,8H-3,4-Dihydro-pyrimido(4,5-c)(1,2)oxazin-7-one-8-β-D-2'-deoxy-ribofuranoside-5'-triphosphate ) (Fig. 2, 3).

Fig. 2 8-Oxo-dGTP chemical structure.

Fig. 3 dPTP chemical structure.

8-oxo-(d)-GTP produced by reactive oxygen species (ROS) is incorporated into DNA/RNA and mispaired with adenine, resulting in two transition mutations (A→C and T→G) and causing replicational and transcriptional errors. Similar ROS-induced modifications are also found in 8-oxo-deoxyadenosine (8-oxo-dA). (Fig. 4).

Fig. 4 8-Oxo-dGTP mismatch mechanism

The triphosphate derivative of dP (dPTP) is an excellent substrate for Taq polymerase and it is incorporated in place of TTP and has approximately 4-fold lower efficiency, in place of dCTP [12] (Fig. 5).

Fig. 5 dPTP base pairing.

The reaction mixture, containing dPTP and 8-oxo-dGTP results in dP and 8-oxo-dG induced mutations and an extensive array of codon changes with the absence of insertions and deletions. Therefore, this method was used for high frequencies of base substitutions, which allows regulation of the mutational load via the number of DNA amplification cycles and it yields both transition and transversions, allowing us to make large mutant peptide libraries.

• It enables very high frequencies of base substitutions;
• It yields both transition and transversion mutations;
• It is capable of generating large and diverse libraries;
• It can be applied for mutant protein generation with improved or novel binding affinities;
• It allows regulation of mutational load via the number of DNA amplification cycles;

The epPCR technique used in our project is based on the protocol of Cadwell and Joyce (1992) [13]. This technique takes advantage of the inherently low fidelity of Taq DNA polymerase, which may be further decreased by the addition of Mn²⁺, increasing the Mg²⁺concentration and using unequal dNTP concentrations. The desired extent of mutation depends on the type of activity one is attempting to generate and the number of library members that can be screened.

This method utilizes a commercial kit (PickMutant™ Error-Prone PCR Kit) also based on the Cadwell and Joyce (1992) protocol [13], but it contains pre-made buffer solutions, dNTP concentration, and a protocol for generating mutations in a range of 0,6-2,0%. Although the mutation rate is quite low, we wanted to see whether a commercialized kit with slight modifications and adjustments could successfully mutate our peptides.

To better understand the behavior of nanoplastics, it is essential to conduct experiments with representative and well-characterized nanoplastics, therefore nanoplastics had to be self made in order to replicate the natural origin of plastic nanoparticles as if they were generated during weathering processes. For this reason, a custom nanoplastic top down method based on mechanical degradation was applied.

Nanoplastic making process consisted of two steps: mechanical fragmentation using a coffee grinder and size reduction from macro- and micro-sized plastic particles using a planetary ball mill machine [14]. The fragmented nanoplastics were then characterized using a complete nanoscale dedicated analytical strategy: dynamic light scattering (DLS) and atomic force microscopy (AFM).

In conclusion, the Vilnius-Lithuania iGEM 2022 team designed a novel nanoplastic detection system based on specific interactions between peptides and nanoplastic particles. In order to test, measure, quantify and evaluate our detection system, a thorough analysis of all single components of the system was performed with great accuracy. Multiple iterative engineering cycles were performed, and precise measurements at every step of the project were performed.