UC Riverside

AF4 is a size separation technique that is routinely used for nanoparticle analysis due to the open channel design, the gentle forces used, and separation being done in native and stable buffers. NTA is an excellent detector for AF4 due to the flow cell fluidics and the individual counting and sizing NTA performs, making it an excellent candidate to use as an online detector to allow simultaneous fractionation and characterization of samples. NTA is label free, making general detection of all NPs above a certain size possible, making this a very versatile detector that can be used for many types of nanoparticles and materials that may be separated by AF4. Chapter 2 focuses on the coupling connection and validation of the AF4-NTA system. The initial design required a split flow design. COMSOL simulations were done to determine the best strategy for splitting the eluent into multiple lines of equivalent sample. A splitter manifold was used to control pressure and flow while a syringe pump was used to control the flow rate going through the NTA. The AF4-NTA system showed great particle counting ability and consistent sizing of the different size populations of standards tested. Some drawbacks were identified with the split flow design, which was mainly band broadening from the larger dead volume of the NTA channel. The main counting ability showed very strong linearity when evaluating sequential increases in injection amounts and produces simple and attractive three-dimensional fractograms. Chapter 3 uses AF4-NTA for the analysis of non-spherical particles and soft-shelled and flexible materials like exosomes. AF4-NTA was used to separate and analyze two different aspect ratios of gold nanorods. The NTA hydrodynamic size and TEM data were used to determine the conversion factor between the NTA hydrodynamic size and the rod length. Additionally, it was shown that offline NTA had difficulty identifying the two populations but were easily identified when utilizing AF4 separations before NTA analysis. AF4-NTA was also used to analyze 2D nanomaterials and the protein corona formation around it once it was incubated in cell culture medium. AF4-NTA showed very rapid analysis of nanomaterials, allowing quick determination of aggregation and protein corona thickness. Finally, the system was tested against softer and more flexible materials, which AF4 has advantages over SEC. Good recovery, quantification, and sizing indicate the AF4-NTA is quite versatile and applicable to a wide array of sample types, matrices, sizes, shapes, and composition. Chapter 4 focuses on the development of AF4-NTA for exosome analysis. AF4-NTA showed applicability in measuring extracellular vesicles, but additional downstream analysis is needed to make use of the BioNP information. AF4 was used as a preparatory step for the downstream analysis while the offline NTA data characterizes the BioNPs collected. The AF4 recovery was compared to other common isolation techniques as well as the purity and native size investigated and showed comparable purification abilities. In-channel labeling was performed as an efficient way to wash exosomes of unbound probes, allowing simple plating and quantification of the probes. This technique removes every step of blocking, incubating, and washing because AF4 can complete this on the analyte directly during the size separation. While AF4-NTA was never ran with the final probe designs due to the COVID-19 shutdowns, it was shown that in-channel labeling and protein label quantification within the ranges of NTA is possible. AF4-NTA is versatile with broad applicability. The online NTA data saves time, sample, and allows direct relation of particle counts to downstream results. The throughput is very quick compared to individual steps of purification, characterization, and protein quantification. Some drawbacks still exist, such as band broadening from slow flow rates in NTA, AF4 dilution factor, and limited working ranges by NTA. Some of these can be further improved while some are inherent drawbacks of the individual instruments used, but AF4-NTA was shown to be an ideal candidate for general nanoparticle analysis, especially when dealing with complex matrices or mixtures of multiple size populations.