*3.2. Synthesis of CdSe1*−*xSx/ZnS Supraparticles*

The procedure was adapted from the literature [3,5] and carried out under ambient conditions. A 20 mg/mL solution of CdSe1−xSx/ZnS alloyed core/shell CQDs was prepared in chloroform. Approximately 100 μL of this solution was added to 450 μL of a 2.5 w% solution of poly(vinyl alcohol) (PVA) in deionised water and stirred vigorously at room temperature for 4.5 h with constant monitoring of the emulsion after the first 2 h. The reaction mixture was centrifuged at 8000 rpm for 10 min, the supernatant was discarded, and the pellet was resuspended in DI water and stored at 4 ◦C. Microsphere size distributions were measured using ImageJ software using images taken from an optical microscope equipped with a Thorlabs™ camera. SEM images were captured using a JEOL JSM-IT100 operated at 20 kV.

#### *3.3. Surface Functionalisation of SPs*

The procedure was adapted from literature to make it suitable for SPs rather than CQDs [35,51]. Approximately 200 μL of oleic acid-capped SPs (OA-SPs) were re-dispersed in 500 μL of a 3:1 water/ethanol mix. Excess MPA (500μL) was added, and the solution was stirred at room temperature overnight. The reaction mixture was centrifuged at 8000 rpm for 10 min and the precipitated MPA-SPs were washed with ethanol twice to remove unbound MPA. The MPA-SPs were air dried and then stored at 4 ◦C.

The procedure adapted from the literature [52,53]. Approximately 200 μL of MPA-SPs were activated by the addition of 30 μL of 40 mM EDC and 30 μL of 15 mM NHS with NaOH (10 mM in H2O) added to increase the pH to between 7.5 and 8 before leaving the mixture to stir for 1 h. Then, 20 μL of 10 μM TBA-15 was added and the solution was left stirring for 4 h with the pH of the reaction mixture kept at approximately pH 6.5–7.0 by the addition of 2 μL of 10 mM NaOH every 20 min. The SPs were characterised using a Malvern Zeta Potentiometer and FTIR which was obtained using a Nicolet iS5 FTIR Spectrometer.

#### *3.4. Optical Characterisation*

Approximately 10 μL samples of SPs after each functionalisation step were drop cast on glass slides and left to dry. Lasing measurements of self-assembled microlasers were obtained using a 355 nm, 5 ns pulsed Nd:YAG laser at a 10 Hz repetition rate with a beam spot area of ~2.6 × <sup>10</sup>−<sup>5</sup> cm2. The beam intensity was altered using a variable wheel neutral density attenuator and focused on the sample using a 10× magnified objective lens. An Avantes AvaSpec-2048-4-DT spectrometer was used to acquire spectral data.

#### **4. Conclusions**

In conclusion, SPs of CdSeS/ZnS CQDs were successfully modified with the thrombin binding aptamer TBA-15 and are capable of lasing post-functionalisation, with lasing functionality retained after storage in water for over 1 month. Surface functionalisation is essential to realising the potential of these self-assembled SP lasers for a myriad of applications; therefore, this proof of concept paves the way for the use of SPs in biosensing applications. While surface functionalisation could reduce the toxicity of SPs, due to the nature of toxicity of the CQDs used, these SPs would only be used for in vitro sensing, for example, in a lab-on-a-chip sensing device. Using a facile and relatively mild procedure, the oleic acid originally coating the surface of the SPs can be exchanged with MPA to create a carboxylic acid-coated surface which can undergo EDC/NHS coupling to attach TBA-15 to the surface of the SPs in a three-step modification process. Zeta potential values of −21.5 ± 7.22 mV, +24.1 ± 4.99 mV, and −22.4 ± 7.13 mV for OA-, MPA-, and TBA-SPs, respectively, confirmed the successful surface functionalisation, along with the FTIR spectra. The SPs before and after functionalisation demonstrated low lasing thresholds, with an OAand TBA-SP exhibiting thresholds as low as 4.10 ± 0.37 mJ·cm−<sup>2</sup> and 7.23 ± 0.78 mJ·cm<sup>−</sup>2, respectively. The thresholds of an OA- and TBA-SP were measured after storage in water for over 1 month and remained similar to those measured for fresh SPs, with values of 11.1 ± 1.0 mJ·cm−<sup>2</sup> and 5.63 ± 0.50 mJ·cm−<sup>2</sup> for the OA- and TBA-SP, respectively. Obtaining SPs with this surface chemistry creates a platform that is incredibly versatile because EDC/NHS coupling is a very well-known reaction that can be used to attach any molecule containing an amine group to the surface of MPA-capped SPs [36]. The stability of SPs when exposed to further procedures could be improved using a surface coating to add an extra barrier between the SPs and the surrounding environment, making it harder for the spheres to break down [46]. For example, encasing SPs in materials such as silica enabled lasing at temperatures as high as 450 K for time periods as long as 40 min [47]. Other possibilities include the modification of SPs with short polyethylene glycol (PEG) chains containing functional groups that can be used to achieve further functionalisation using different techniques [15,16]. Adding an extra layer of protection to the SPs could also improve the stability of the spheres over time; however, these SPs have already been shown to be stable when stored in water. Work is currently underway to investigate the biosensing capabilities of these SPs.

**Author Contributions:** B.K.C., C.J.E. and N.L. conceived and designed the experiments.; B.K.C. performed the experiments, with C.J.E. and I.N. contributing data for the statistical analysis and D.H.D. and C.J.E. obtaining the SEM data. B.K.C. and P.U.A. analysed the data. B.K.C. wrote the original paper and C.J.E., P.U.A. and N.L. reviewed and edited the manuscript. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by the Leverhulme Trust under the Research Leadership Award RL-2019-038.

**Data Availability Statement:** The dataset can be found at: https://doi.org/10.15129/d8b31125-2 3cb-4325-b136-38073dd6a4a6, 18 September 2023.

**Acknowledgments:** We would like to thank Paul R. Edwards for training and use of the SEM, Sian Sloan-Dennison for training and access to the zeta potentiometer, and Patrick Allen for access to the FTIR spectrometer.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


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