5.1 Summary 215 5. Summary and Conclusion 5.1. Summary Nanoparticles are being developed for a multitude of applications in the fields of biomed- icine and reproductive biology to date. An appealing fabrication method is the pulsed la- ser ablation in liquids (PLAL), which enables the production of gold nanoparticles and also their in situ bioconjugation with biomolecules on the timescale of minutes. A crucial drawback of the PLAL process is generally that there is a mis- match between an efficient production yield and the maintenance of optimal conditions for the fabrication of functional nanobioconjugates. For a long time, only a maximum nanobioconjugate yield of approxi- mately 11 µg min-1 had been achieved using femtosecond-pulsed LAL that resulted in nearly 100 % integrity preservation of biomolecules.[39] That was the basis for the development of this thesis. To enhance the nanoparticle yield, it was studied whether longer pulse duration could increase the ablated gold mass per time. In fact, us- ing picosecond pulses for PLAL instead of femtosecond pulses, the nanobioconjugate yield was significantly increased by a factor of 15 to 168 µg min-1. Moreover, the pro- duced nanobioconjugates featured nearly 100 % integrity preservation when fabricated with strictly defined process parameters. Interestingly, the nanoparticle concentration could be further increased by the post-processing techniques of ultrafiltration and solvent evaporation. A maximum concentration factor of 2-3 was reached with ultrafiltration. The up-concentrated nanobioconjugates were functional; however the efficiency of ultrafiltra- tion was highly dependent on the material of the filter membrane. Moreover, high particle losses of approximately 40 % had to be accepted. Conversely, the concentration increase of nanobiohybrids by solvent evaporation was highly efficient by a factor of 13. However, because of long processing times at room temperature the risk of biomolecule inactiva- tion is quite high and should be considered carefully. In summary, the adoption of ps-PLAL for AuNP and AuNP bioconjugate fabrication could allow for competitiveness of the PLAL technique on the NP fabrication market, especially if it is combined with an additional post-processing step of ultrafiltration or solvent evaporation, yielding mg mL-1 concentration scale. When starting the work on this thesis, there had been no comprehensive guideline for the laser-based fabrication of gold nanoparticle bioconju- gates, especially regarding the specific demand on structure-function relationship. During the thesis workout, it turned out to be a particular challenge to include all relevant process parameters because of their di- versity. Moreover, the parameters did not only influence the conjugation process but they © Springer Fachmedien Wiesbaden 2016 A. Barchanski, Laser-Generated Functional Nanoparticle Bioconjugates, DOI 10.1007/978-3-658-13515-7 216 5 Summary and Conclusion could also amplify or erase the benefit and function of each other. However, four consid- eration areas were subdivided for the discussion of the process parameters. (I) The effects of nanoparticles’ intrinsic parameters were studied on the examples of particle size and surface charge. The methods of in situ photofragmentation and ex situ centrifugation were successfully applied to modify the particle size distribution and to separate distinct particle size classes. Moreover, the fabrication of AuNPs with ps-PLAL was found to generate partially oxidized surfaces (~ 5 % of atoms) with an Au+ configuration. Compared to other studies, the extent of surface oxidation seems to be strongly dependent on the laser parameters such as pulse length, pulse energy, repetition rate, fluence and wavelength. (II) Choice of binding stability and functional group, of ligand amount and ligand charge and of the surrounding medium. The solvent for ablation should provide optimal conditions for the electro- static stability of nanoparticles and biomolecules and it should allow for the dilution or transfer of nanobioconjugates into biological relevant media. For optimal binding stability, the covalent attachment of ligands with a thiol or disulfide function should be aimed. There was no difference for the conjugation by thiol or disul- fide function determined. However, the molecule structure should be considered careful- ly, because spontaneous fragmentation/dissolution of AuNPs by electron transfer from electron-donor-containing moieties could occur. To achieve optimal nanobioconjugate formation and functionality without precipitation or multilayer formation, the adopted ligand concentration should be within a concentration window that is defined by two thresholds termed minimum ligand concentration and maximum ligand concentration. The effect of charge compensation between net-charge negative AuNPs and net-charge positive ligands should be avoided, because it induces the reduction of the interparticle distance and allows for particle agglomeration. (III) Ligand characteristics as their length, dimension, binding orientation or amphiphilic nature and the adoption of diverse ligands for bivalent functionalization and surface saturation. The chain of linear ligands should be kept short to avoid enhanced coiling and wrapping effects of the flexible ligands around the AuNP surface, which significantly limits the surface coverage. Moreover, it should be considered that the molecular size is in direct relation to a large molecular footprint and thus to the number of attachable ligands. For ligands that have active centers, a correct orientation on the nanoparticles should be ena- bled by the use of specific linker molecules. In addition, it should be considered for nu- cleotides that the insertion position of a sulphuric function at the strand end will either yield a high amount of full-length and capped failure nucleotides with the modification or 5.1 Summary 217 only a low amount of the full-length product with the modification. The fabrication of bivalent AuNP bioconjugates can be enabled with three different approaches, including the in situ conjugation with ligand A and the ex situ conjugation with ligand B, the in situ conjugation with ligand B and the ex situ conjugation with ligand A and the in situ co-conjugation with both ligands at once. All approaches will result in bivalent conjugates. However, the co-conjugation is most effective due to a single-step process. A non-functional dummy ligand might be applied to pre-saturate the particle surface in order to control the surface coverage of a functional secondary ligand. However, it may also be applied to post-saturate the particle in order to increase the nanobioconjugate sta- bility even in highly saline media. Moreover, the amphiphilic nature of the biomolecule may influence the biological functionality of the nanobioconjugates. Depending on the application, polycationic, bivalent bioconjugates should feature different properties than cationic-neutral, bivalent bioconjugates and amphiphilic bioconjugates with a twisted ani- onic-neutral-cationic composition and a high hydrophobic content. (IV) The biological functionality of nanobioconjugates is their most crucial quality. Thus, the PLAL-generated conjugates that were fabricated in the framework of this thesis were analyzed regarding functionality in various laboratory assays such as immunoblotting and they were applied for in vitro tests such as cellular uptake studies, cytotoxicity screening or the specific immunolabeling. Within all those studies, the PLAL-generated nanobioconjugates were highly functional and featured the same or even a better quality than commercial products. The transferability of an established technique e.g. from one to another materials is an important factor for the broadband-compatibility of a method. When the structure of this thesis was outlined, the laser-based in situ bioconjugation was solely used for the functionalization of gold nanoparticles with (thiolized) ligands in a single-step process. However, the adoption of the technique to other biologically relevant materials such as silicon and iron was of high interest. Within this thesis, the fabrication of silicon nanobioconjugates and magnetic, iron-based nanobioconjugates with in situ bioconjugation technique during PLAL was successfully demonstrated. The obtained nanobioconjugates were directly fab- ricated in ultrapure water and featured a high colloidal stability. This is outstanding, com- pared to the conventional biofunctionalization approaches of nanoparticles from those materials, which are generally performed in organic solvents with high amounts of stabi- lizers and which comprise complex purification procedures. However, with in situ biocon- jugation method, reasonable surface coverage values were gained, depending on the con- jugation mechanism and applied biomolecule. Moreover, the nanobioconjugates featured biological functionality for bio-imaging and magnetic manipulation applications and are highly promising for biomedical applications. 218 5 Summary and Conclusion 5.2. Conclusion and Outlook Golden bioperspective Thus far, the nanotechnology has transcended the traditional boundaries between com- mon research areas such as physics, chemistry and biology/medicine and has been charac- terized by its capacity to revolutionize nearly everything. This has made the work on this thesis highly challenging but also very exciting. This thesis deals with the complex issue of functional nanobioconjugate fabrication with in situ