Blank (BA)

For the detection, MNPs are placed in an aqueous solution containing an electrolyte with a known electrochemical response. SWV technique can explore quickly and efficiently the work electrode surface occupation. Since the MNPs are small particles composed of a dielectric material, MNPs can trigger various electrochemical processes in the solution, such as hindering ion diffusion, accumulating charges on their surfaces, or filling the surface region of the blank working electrode. These effects impact the impedance and SWV measurements. While all these mechanisms can contribute to quantifying MNPs in water, other small materials present in the water can also affect the results to some extent.

Deposited - Na₂SO₄

As an initial approach to exploring electrochemical sensing of MNPs, a solution of Na₂SO₄ was tested, which was reported to be effective for detecting microplastics.

First, EIS measurements were conducted with the blank sensor using a solution containing 0.5 M of Na₂SO₄. The typical Na₂SO₄ curves in the Bode plot are shown in Figure 2.

At higher frequencies, the impedance phase is below 45°, and the magnitude remains constant relative to the frequency, characteristics of a more resistive behavior. In the mid-to-low frequency range, the phase exceeds 45°, and the magnitude starts varying linearly with frequency, indicating a more capacitive behavior of the solution.

Based on the analysis of the electrolyte’s behavior, the circuit shown in Figure 3 was chosen to fit the EIS data. In this circuit, Na₂SO₄ solution is modeled as a resistor R_s and the interface between the work electrode and the solution is represented by a parallel combination of a capacitor C_surf and a resistor R_surf, mathematically capturing the capacitive and resistive electrochemical processes occurring in the surface. It is expected that the presence of microplastics at the interface between the working electrode and the electrochemical solution will result in changes in the measured values of R_surf and C_surf.

To verify the relationship between the concentration of MNPs and the electrochemical EIS response, each sensor was rinsed with 1 mL of deionized water, and PSNBs (1000 nm) were progressively added at concentrations of 0.01, 0.1, and 1 mg/L. At each stage, the samples were incubated for 10 minutes, measured in Na₂SO₄, and then rinsed with 2 mL of deionized water. The obtained values for R_surf and C_surf were normalized against the blank (Na₂SO₄) and evaluated in relation to the PS (polystyrene) concentration, as shown in Figure 4.

The results of the equivalent circuit fitting using impedance spectroscopy in Na₂SO₄ solution yielded unsatisfactory R² values of 0.22 and 0.25, indicating a poor correlation between the modeled and experimental data. Additionally, the reproducibility was compromised causing excessively high deviations in the measurements. Essentially, we conclude that this approach was too simple to detect microplastics effectively.

Deposited - K₃[Fe(CN)₆]/K₄[Fe(CN)₆]

The K₃[Fe(CN)₆]/K₄[Fe(CN)₆ probe is widely used in electrochemical detection experiments due to its well-established oxidation and reduction processes in the literature. Additionally, the concentration of microplastics was evaluated using square wave voltammetry (SWV), a simple, fast method that demonstrated high reproducibility. This makes the results and conclusions obtained reproducible and valuable for future iGEM teams or other projects aiming to detect microplastics.

Due to the redox processes occurring with the K₃[Fe(CN)₆]/K₄[Fe(CN)₆ probe, SWV measurements show a current peak at a specific applied potential, where the maximum current (I_Max) is related to the electroactive area of the working electrode. The PS concentration is thus connected to the surface coverage of the working electrode, and consequently to the measured I_Max. To explore this relationship, samples of PSNBs (polystyrene nanobeads) (1000 nm) were progressively analyzed at concentrations of 0.01, 0.1, 1, 10, and 100 mg/L. At each stage, the samples were incubated for 10 minutes and washed with 2 mL of deionized water. The results for the analytical curve are shown in Figure 5.

While this approach yielded slightly better outcomes compared to the impedance spectroscopy method, the results remained inadequate, with R² values (~ 0.76) still falling short of acceptable levels (> 0.9). By depositing the MNPs in the surface and using SWV, there is a poor linearity and sensitivity. Thus, they are not expected to enable the determination of MNPs in real-world samples.

Bind (BI)

In the Bind (BI) methodology, the sensor is pre-modified by binding the target protein on the work electrode. After this process, the sample of micro- and nanoplastics (MNPs) dispersed in the electrolyte is brought into contact with the electrodes. The specific interaction between the plastic particles and the protein can produce electrochemical results that determine the concentration of the MNPs with minimal interference from other potential materials in the medium.

Drop Casting - BaCBM2

To explore the effectiveness of using plastic-binding proteins for MNP sensing, the drop casting method was employed.

This protein immobilization method involves simple protein adsorption by depositing onto the substrate. SWV measurements were taken with the K₃[Fe(CN)₆]/K₄[Fe(CN)₆] probe for each clean sensor. After washing each device with 1 mL of PBS (10 mM), 5 µL of BaCBM2 protein (500 µg/mL) was added to the working electrode, followed by a 20-minute incubation.

After incubation, the devices were washed with 2 mL of PBS, and SWV measurements were taken again to define the blank. A decrease in current after protein deposition indicates the proteins were likely fixed to the surface. Each sensor was then washed again with 1 mL of PBS, and PSNBs (1000 nm) were progressively added at concentrations of 0.01, 0.1, 1, 10, and 100 mg/mL. The current from each SWV measurement was normalized relative to the blank to create an analytical curve based on the microplastic concentration.

By using the BaCBM2 protein’s interaction with the MNPs, the results became significantly better, with R² going from 0.76 to 0.94 just by adding the protein. This already allows biosensing of microplastics. However, there is room for improvement, especially because the standard deviation between measurements is quite large, making the concentration values uncertain.

Self-Assembled Monolayers (SAMs) - BaCBM2

To ensure BaCBM2 immobilization and amplify the measured signal, the modification of the working electrode with three different self-assembled monolayers (SAMs) was tested: L-cysteine, cysteamine, and graphene oxide.

First, SWV measurements were taken using the K₃[Fe(CN)₆]/K₄[Fe(CN)₆] (5 mM) probe on the clean sensors. Afterward, the sensors were rinsed with 1 mL of PBS to allow for SAM deposition. Each modification (L-cysteine, cysteamine, and graphene oxide) was performed on 4 different sensors. For this, 5 µL of each solution was added to the sensors at concentrations of 50 mM, 50 mM, and 2 mg/mL, respectively, for L-cysteine, cysteamine, and graphene oxide. The samples were then incubated at 4°C for 15 hours. After incubation, the sensors were washed with 2 mL of PBS per device, and SWV analysis was conducted with K₃[Fe(CN)₆]/K₄[Fe(CN)₆] (5 mM).

Following this, the devices were rinsed with 1 mL of PBS, and 5 µL of BaCBM2 protein solution (150 µg/mL) was added. The sensors were incubated with EDC:NHS (16.6:33.33 mM) for 45 minutes at 37°C. After incubation, each device was washed with 2 mL of PBS, and SWV measurements were taken again.

Next, the samples were rinsed with 1 mL of PBS before adding 5 µL of SuperBlock solution, which was incubated for 90 minutes. After this stage, each device was washed with 2 mL of PBS, and new SWV measurements were taken as a blank.

Finally, each sensor was washed again with 1 mL of PBS, and PSNBs (1000 nm) were progressively added at concentrations of 5, 10, 50, and 100 mg/L. At each step, the samples were incubated for 20 minutes and washed with 2 mL of PBS. The current obtained from each SWV measurement was normalized against the blank to create an analytical curve for the microplastic concentration, shown in Figure 8.

Based on initial tests, we concluded that the best method (R² > 0.85) was using cysteamine to bind the protein to the gold surface. By repeating the experiment with optimized parameters, we obtained an analytical curve with R² of 0.99, showing high precision in detecting even low concentrations of microplastic.

Bulk (BU)

In the Bulk (BU) methodology, MNPs are initially placed in a solution containing a high concentration of target proteins. After a certain period, the surface of the particles is coated with these proteins, forming protein coronas (Model), thus, the interaction process occurs in the solution bulk. After this process, part of this solution is deposited specifically onto the working electrode. Following the immobilization of these MNP-protein complexes, an electrolyte is added to establish contact between the three electrodes.

For the experimental tests of the bulk methodology, the BaCBM2 protein (150 µg/mL) and PSNBs (0.01, 0.1, and 1 mg/mL) were first mixed in PBS (10 mM) using a Microtube Rotating Mixer for 10 minutes to allow the formation of protein coronas. Following this, SWV measurements were performed using clean sensors. Next, a sample containing only 150 µg/mL of BaCBM2 in PBS, which was also shaken for 10 minutes, was incubated for 10 minutes to measure the blank by SWV, where a decrease of 26% in the peak current was observed. The four concentrations of PSNBs were then progressively incubated on the sensor. After each incubation, the sensor was washed with 2 mL of PBS (10 mM), followed by SWV measurement. The resulting analytical curve is presented in Figure 10.

Statistical Comparison of Detection Methods

Based on the results obtained for each detection method tested, it is possible to evaluate them in terms of several figures of merit. For our analysis, we evaluated, in addition to the R² metric, the mean standard deviation (σ) and the sensitivity (S), shown in equations 1, 2, and 3, where the space of the N_i analyzed concentrations is represented by i and the N_j replicates at each concentration are given by j.

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Therefore, based on the calculated values for the metrics, the linearity (R² normalized relative to the methods), the precision ((-1) * āσ normalized relative to the methods), and the sensitivity (S normalized relative to the methods) were analyzed in a more didactic manner. Thus, Figure 11 provides a comparative analysis of the detection methods.

In this analysis, we can observe how the first method evaluated (blank) is limited in terms of linearity (R² = 0.76) and precision (σ = 12.4 %), due to the absence of a recognition element. In contrast, the bind method using protein applied by drop casting showed a significant improvement in linearity (R² = 0.94), thanks to the presence of the BaCBM2 protein as a biological recognition element. However, it still exhibited poor precision (σ = 10.88 %), likely due to variability in the electrode modifications through this method.

bind method with BaCBM2 linked to a self-assembled monolayer (SAM) of cysteamine achieved high linearity (R² = 0.99) and excellent precision (σv = 0.88 %), owing to both the biological recognition element and a more precise fabrication method. Finally, the bulk method demonstrated good linearity (R² = 0.96) and precision (σ = 4.39 %), indicating the potential of this approach. However, more tests are needed to confirm its reliability. In terms of sensitivity, the methods presented similar results.

Dissociation Constant

on the results of the electrochemical assays, we demonstrated that it is possible to estimate the dissociation constant involved in the protein-plastic interaction.

1960, the analysis of the dissociation constant has been a studied method for evaluating the affinity between molecules through a binding assay [Colquhoun, D. (2006)][Yalow, R. S. et al (1960)]. In electrochemical assays based on Square Wave Voltammetry (SWV), the measured signal often depends on the occupancy of target molecules or particles (T) on the working electrode surface. This surface coverage (Θ) is influenced by the concentration of particles in the medium ([T]), the maximum surface coverage (Θ_{Max}), and the interaction between the target and the surface, which is related to the dissociation constant (K_d). The general expression for this process can be represented by the Langmuir isotherm [Hulme, E. C. et al (2010)], as shown in Equation 4.

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Thus, the general graphs observed for this process can be analyzed in Figure 12. It illustrates a progressive surface filling with increasing target concentration. Additionally, when plotted against the logarithm of the target concentration, the graph shows an initial exponential region, followed by a linear region, and finally an asymptotic region at the end.

Langmuir isotherm approximation can be particularly useful in describing the measurements obtained from the self-assembled monolayers (SAM) in the bind (BI) methodology, as seen in the experiments conducted with the electrochemical sensor. During the incubation of PS particles on the working electrode, the interaction of particles with the affinity proteins (BaCBM2) follows the relationship described by Equation 4. As a result, surface coverage—and thus the measured signal—depends non-linearly on the concentration of PS in the medium, explaining part of the behavior observed in the obtained results.

In the second analytical curve, obtained with the cysteamine modification (Figure 8), a highly linear relationship (R² = 0.99) between the measured signal and the logarithm of PS concentration was observed. This suggests that, for this configuration, the chosen concentration range falls within the linear region of Figure 12. On one hand, this may indicate greater measurement precision30, but on the other hand, it precludes the estimation of maximum surface coverage and thus the dissociation constant.

Conversely, the first analytical curve generated with the cysteamine modification shows some saturation of the signal as log([PS]) increases (Figure 8.B), suggesting that the selected concentration range for this configuration is closer to the asymptotic region. While this may affect measurement precision, it allows for the estimation of the dissociation constant involved in the interaction between PSNBs and the surface in this system.

Thus, Equation 4 was applied to fit the data obtained from the first cysteamine modification, as shown in Figure 13.

Through this analysis, the dissociation constant (K_d) for PS particles binding to the surface of the working electrode was determined to be 15.13 mg/L. This value reflects the interaction between the anchored BaCBM2 proteins on the electrode and the plastic particles.

It is important to note that, although this value relates to the PS-protein interaction, it is influenced by various experimental factors such as temperature, stirring, incubation time, PS-gold interaction, protein-solution interaction, the reduced degrees of freedom of the protein after immobilization on the surface, among other factors. These variables complicate direct comparison with computationally predicted K_d values, for instance.

Nevertheless, despite the limitations when comparing computational data, this method of calculating the dissociation constant can be particularly useful for comparing the affinity of different proteins for PS particles, or more broadly, for any target molecule of interest.

Scanning Electron Microscopy

In order to characterize the MNPs interaction with the surface of the electrode, as well as the reusability of the sensor, Scanning Electron Microscopy (SEM) imaging was used. The SU-8 boundary, responsible for delimiting the active area of the working electrode, was characterized in Figure 14 and shown to have a thickness of 4.7 μm.

Figure 15 demonstrates the effectiveness of using Ar plasma for cleaning the gold surface. This guarantees the quality of experiments that were carried subsequently on the same sensor.

X-ray Photoelectron Spectroscopy

To determine the composition of material deposited on the surface of the sensor, the technique of X-ray Photoelectron Spectroscopy (XPS) was utilized. This characterization allows us to determine whether the SAMs and the protein adhere properly to the gold surface after washing.

First, we look at the peaks of the 4f orbital of gold atoms for reference.

Nitrogen is present on both the protein and the cysteamine, but it is more abundant on the protein because of the number of amino acids. Figure 18 is consistent with this expected result and shows both cysteamine and BaCBM2 remained fixed on the surface.

Finally, Figure 19 confirms this result by demonstrating the presence of the sulfur from the cysteamine monolayer on the surface. The drop casting also generates a sulfur peak due to the cysteine group of the protein itself, although less abundant than the SAM.