Microsieves – microengineered membranes – have been introduced in microfiltration technology as a new generation of inorganic membranes. The thin membranes are made of silicon nitride (SixN4), which gives the membranes outstanding features, such as chemical inertness and high mechanical strength. Microsieves have very well-defined pore size and pore shape, with an extremely homogeneous size distribution and high porosity. As a result, high-flux performance and excellent selectivity may be achieved. However, biofouling issues exert limitations on the application of microsieves in filtration and diagnostics. Surface functionalization was found to be a feasible way to minimize biofouling, but also to achieve biorecognition in microbiological applications. The aim of this thesis is to improve microsieve performance in biological applications by means of surface functionalization with organic coatings for protein repellence and selective capture of microorganisms.
In this thesis, SixN4 surfaces were functionalized with organic monolayers via stable Si C and N-C linkages. Coatings to render SixN4 surfaces protein repellent were studied in depth by two approaches: grafting of ethylene oxide monolayers onto the surface (Chapter 2); and grafting of zwitterionic polymers from the surface (Chapter 3). UV induced surface modification with oligo(ethylene oxide) chains with three (EO3) and six (EO6) units and the detailed characterization of these modified surfaces are described in Chapter 2. Successful attachment of EO3 and EO6 on SixN4 surfaces was achieved. The modified surfaces exhibit excellent protein repellence in bovine serum albumin (BSA) solution (~ 94%), but only moderate (~ 80%) protein repulsion was observed in fibrinogen (FIB) solution. This observation motivated the study towards grafting zwitterionic polymer brushes from SixN4 surfaces for improved protein repellence. A new method to grow zwitterionic polymers from monolayers containing tertiary bromides, via atom transfer radical polymerization (ATRP) was developed. The zwitterionic polymer coated surfaces showed excellent protein repellence in FIB solution (> 99%), while exhibiting very stable performance in PBS during one week, i.e., unchanged thickness, no hydrolysis of the polymers occurred and protein repellence in FIB solution remained constant.
The use of microsieves as detection platform for microorganisms was explored in Chapter 4. Microorganisms can be caught by microsieves whose pore sizes are smaller than the microorganisms while allowing an easy flow-through of other components. However, detection capacity of microsieves is severely hampered by fouling issues. To avoid this problem, the use of microsieves with pore sizes larger than the microorganisms, in combination with immobilized antibodies was investigated in Chapter 4. Anti Salmonella antibodies were immobilized onto epoxide monolayers on microsieve surfaces by reaction with the primary amines present in the antibody. The antibody-coated microsieves showed excellent detection of Salmonella with high sensitivity and selectivity, significantly improving detection efficiency in crude biological samples, and reducing analysis times.
The capture efficiency of Salmonella in milk samples was, however, found to be lower than that achieved in buffered solution. Most likely, this is due to nonspecific adsorption of milk proteins on the antibody-coated microsieves. In addition, the use of a blocking solution before incubation with microorganism solution remained an essential step in order to avoid the occurrence of interfering background fluorescence. In order to minimize these problems, the incorporation of antibodies on top of protein-repellent zwitterionic polymers coated on SixN4 surfaces was studied in Chapter 5. Anti-Salmonella antibodies were immobilized on zwitterionic polymer brushes coated SixN4 surfaces through the bromide moieties retained at the end of the polymer chain after ATRP. Antibody-functionalized zwitterionic polymers adsorbed only minimal amounts of FIB, indicating excellent protein repellence of the modified surfaces. Moreover, anti-Salmonella antibodies immobilized onto zwitterionic surfaces exhibit highly selective capture and improved sensitivity, as compared to antibodies on epoxide coated surfaces. This achievement offers a new approach that enables highly sensitive and selective detection of microorganism, while minimizing nonspecific adsorption of proteins that are not of interest.
In Chapter 6, an overview is given of the most important findings presented in the thesis. Recommendations, as well as additional ideas on how to bring this research into industrial application are discussed.