MEASURMENT

This year, our iGEM project is titled: "High-Value Conversion of Phosphorescent Materials– Turning Waste into Treasure". Our core objective is to depolymerize food waste and waste plastics using PETase and CGTase enzymes, generating high-value phosphorescent materials (TPA-CD), while also addressing the technical challenges of enzyme activity detection and material performance characterization.

I.CGTase Enzyme Activity Measurement

In recent years, methods for detecting cyclodextrin and the related enzyme CGTase have become increasingly diverse, with common techniques including High-Performance Liquid Chromatography (HPLC) and the phenolphthalein method. Among these, HPLC offers high precision and is suitable for complex samples but has limitations such as expensive equipment and complex operation (Duan, 2020). The phenolphthalein method, while low-cost and simple to operate, suffers from poor solution stability, high sensitivity to pH, and poor reproducibility (Goel, 1995). Furthermore, our team's experiments revealed that the inclusion complex formed in this method must be measured immediately after reaction completion; otherwise, significant data deviation occurs, leading to non-linear standard curves and compromised result reliability.

To overcome these limitations, our team is committed to developing a novel cyclodextrin detection method. The basic principle is to indirectly characterize CGTase enzyme activity by measuring the remaining cyclodextrin content in the reaction system. After systematically screening various dyes, we identified 8-Anilino-1-naphthalenesulfonic acid (ANS) as an ideal indicator. ANS, an industrial dye commonly used as a fluorescent probe for hydrophobic microenvironments in life sciences, was innovatively combined with fluorescence spectroscopy by our team to establish a new cyclodextrin detection method.

Based on the characteristic significant fluorescence enhancement when ANS binds to cyclodextrin, this method offers high sensitivity and a wide linear range, with a stable response suitable for precise quantitative analysis. Compared to the phenolphthalein method, this system is less susceptible to pH fluctuations, exhibits high solution stability, does not require immediate measurement (offering a wider experimental window), and is more convenient and reliable to operate. Additionally, the ANS reagent is low-cost, and detection only requires a standard fluorescence spectrophotometer, making it easy to implement in ordinary laboratories. Furthermore, this method shows good selectivity for common cyclodextrin types like β-CD and is less affected by common matrix interferences, demonstrating excellent anti-interference capability.

Experimental Procedure

From the experimental results

β-CD: Also exhibited a stable fluorescence enhancement effect at 510 nm, with a high goodness-of-fit for the standard curve.

γ-CD: Showed a noticeable fluorescence signal enhancement, indicating the good applicability of this method for γ-CD as well.

Furthermore, our protocol can not only measure the content of pure α-CD, β-CD, and γ-CD but can also calculate the respective contents of α-CD, β-CD, and γ-CD in a mixed cyclodextrin solution through a single measurement.

To demonstrate the feasibility of this approach, we prepared a system in sodium phosphate buffer with concentrations of α-CD = 0.333 mg/mL, β-CD = 0.833 mg/mL, γ-CD = 1.000 mg/mL (i.e., ratio 1:2.5:3). The ANS concentration was maintained at 1×10^−4 M, and the intensity at 0 mg/mL CD was set as 20.00.

At the wavelength of 510 nm, the measured Intensity was 61.02793. From Fig 10, it can be seen that α-CD, β-CD, and γ-CD do not have significant intrinsic peaks at 510 nm, so we subtract the baseline 20 (contribution from ANS). The net effect from the pure α-CD, β-CD, and γ-CD is thus 41.02793. The influence weights of α-CD, β-CD, and γ-CD on the intensity differ, as evident from the slopes of their respective standard curves. Therefore, the contribution of each CD to the intensity is proportional to its concentration and its specific slope factor.

Calculating the influence factors for α-CD, β-CD, and γ-CD from their standard curves gives approximately 2.653, 44.77, and 78.642 respectively. The total influence factor sum is 126.065. The content of α-CD, β-CD, and γ-CD is then calculated based on their proportional contribution to this total influence factor, weighted by the ratio of their individual influence factor to the total.

The calculated contents for α-CD, β-CD, γ-CD were 0.325 mg/mL, 0.813 mg/mL, and 0.975 mg/mL respectively, resulting in an error of 2.50%.

In another similar experiment, we prepared a system in sodium phosphate buffer with concentrations of α-CD = 1.000 mg/mL, β-CD = 1.000 mg/mL, γ-CD = 1.000 mg/mL (i.e., ratio 1:1:1). The ANS concentration was maintained at 1×10^−4 M, and the intensity at 0 mg/mL CD was set as 20.00

At the wavelength of 510 nm, the measured Intensity was 68.41906. Subtracting the baseline 20 (ANS contribution), the net effect from the pure CDs is 48.41906.

Calculating the influence factors for this specific measurement context (based on the slopes and the known 1:1:1 ratio target for calculation) gave values of approximately 2.653, 17.908, and 26.214 for α-CD, β-CD, γ-CD respectively, with a total influence factor of 46.775. The content was then calculated similarly.

The calculated contents for α-CD, β-CD, γ-CD were 1.035 mg/mL, 1.035 mg/mL, and 0.987 mg/mL respectively, resulting in an average error of 3.51%.

In summary, the ANS fluorescence method developed by our team leverages the characteristic fluorescence enhancement upon binding of 8-Anilino-1-naphthalenesulfonic acid to cyclodextrins. It offers advantages including stable fluorescence signals, strong versatility (applicable to α-CD, β-CD, γ-CD), simple operation, low cost, and good reproducibility. This method can provide a more reliable and easily disseminable technical solution for enzyme activity detection.

II. Characterization of Phosphorescent Properties

In Room Temperature Phosphorescence (RTP) research, steady-state photoluminescence (PL) spectroscopy remains the most common characterization technique. It can intuitively provide the material's emission spectrum and relative brightness, making it widely used in laboratories (Li et al., 2024). However, this method cannot effectively distinguish short-lived fluorescence from long-lived phosphorescence signals, nor can it directly provide key kinetic information such as afterglow lifetime (He, 2024).

Aiming at resource-limited laboratories, our team has also explored low-cost alternative schemes. For instance, image analysis based on smartphone photography combined with the open-source software ImageJ can enable semi-quantitative assessment of sample brightness and color changes. Although this method cannot match the precision and resolution of professional equipment, it offers unique advantages in rapid feedback, sample screening, and experimental optimization. Its low cost and simple operation make it highly suitable for high-throughput screening and process optimization.

We have compiled a detailed operation manual and established a standard procedure to facilitate rapid adoption by other teams.

To optimize the phosphorescent properties of the produced PPA-CD complex and verify its potential as a supramolecular phosphorescent material, we examined its phosphorescence performance under 400 nm UV light irradiation. To determine the optimal reaction conditions, we utilized ImageJ analysis for rapid assessment. We initially used PPA powder as the sole substrate and varied the pH for qualitative experiments. After identifying a specific range, we conducted gradient experiments to find the optimal pH. Under the optimal pH, we tested different salts to determine the best environment. Subsequently, we gradually changed the substrate from PPA powder to starch, and then to rice, making it progressively closer to real food waste, to verify the stability of the phosphorescence performance of the enzymatic degradation products. For each experiment, the phosphorescence intensity (represented by Integrated Density, IntDen) of the purified product under UV light was recorded and analyzed.

As shown in the gradient experiment (Fig 14), PPA-CD exhibits significantly higher phosphorescence in alkaline pH environments. Under pH=10, the phosphorescent performance of PPA-CD was significantly stronger than under pH 7 and 8 conditions, roughly identifying the optimal pH around 10.

Under pH=10, the effect of different buffer systems on PPA-CD was tested (Fig 15, 16). The IntDen value detected when using the K2CO3 buffer system was the highest, significantly better than other tested buffer systems (Na2CO3, KOH, K2HPO3).

After determining the optimal pH and optimal buffer, the substrate was gradually refined (Fig 17), moving from PPA powder to starch, and then to rice, making the substrate increasingly similar to real food waste (i.e., lower purity, more impurities). As substrate purity decreased, the phosphorescent performance of PPA-CD did not decrease substantially, further confirming the stability of the phosphorescent performance of the degradation products.

Finally, we determined the optimal conditions to be the K2CO3 buffer system at pH=10. ImageJ analysis assisted us in efficiently completing the condition screening, significantly saving time and cost. Furthermore, we also used ImageJ to analyze the RGB values of the phosphorescent products and converted and plotted them on the CIE 1931 chromaticity diagram, achieving precise presentation of the luminescence color. The conversion formulas are as follows:

X=0.4124564 × R+0.3575761 × G+0.1804375 × B
Y=0.2126729 × R+0.7151522 × G+0.0721750 × B
Z =0.0193339 × R+0.1191920 × G+0.9503041 × B
x =X/(X+Y+Z)
y=Y/(X+Y+Z)

Our Measurement methods are characterized by standardization, high precision, low cost, ease of dissemination, and reproducibility, providing efficient and feasible measurement references for the future development of phosphorescent materials.