Developing New Techniques And Materials To Use In Biosensors For Point-Of-Care Applications
Biosensor technology is a rapidly expanding field of study in which tedious culturing techniques are being replaced by assays that use biorecognition elements such as antibodies and nucleic acids to detect biological entities. Biosensors have useful applications in areas such as food safety, water quality, clinical analysis, and defense again bioterrorism. Bench-top macro scale detection assays have limitations that restrict them to laboratory settings and require them to be performed by highly-trained personnel. Consequently, there has been a strong emphasis on developing technology that is portable and easy-to-use to enable its use in point-of-care and resource-limited settings. Thus, the concept of a micro total analysis system ([MICRO SIGN]TAS) in which all aspects of the biological assay are contained within a single device is very attractive. Benefits of [MICRO SIGN]TASs over their macro scale counterparts, aside from portability and increased ease-of-use, include smaller sample sizes, reduced reagent consumption, decreased assay time, negligible contamination, and potential automation. Nucleic acid detection within [MICRO SIGN]TASs is a commonly used method for the detection of cells and other microorganisms, as well as genomic analyses. A critical step in these assays is nucleic acid isolation within the microfluidic device. Miniaturizing nucleic acid isolation has led to the discovery of novel isolation techniques. Specific application and assay parameters determine desired characteristics of an optimal nucleic acid purification technique. Relevant parameters include sample type and size, device material and fabrication technologies available, as well as the pre- and post-isolation processes. The main nucleic acid isolation processes used within microfluidic devices are silica-based surfaces, functionalized paramagnetic beads, oligonucleotide-modified polymer surfaces, pH-dependent charged surfaces, aluminum oxide membranes, and liquid-phase isolation. A common process that follows isolation is nucleic acid amplification, and integrating both steps within the same device is key to developing a complete [MICRO SIGN]TAS. Nucleic acid sequence-based amplification (NASBA) is an isothermal amplification technique of which the primary advantage over the standard polymerase chain reaction (PCR) is the elimination of necessary thermal cycles. In this research, nucleic acid isolation and NASBA were integrated within the same simple microchannel to realize highly sensitive detection of very low concentrations of messenger RNA (mRNA). The microchannels were fabricated simply and inexpensively from poly(methyl methacrylate) (PMMA) using hot-embossing and UV/ozone-assisted thermal bonding. Unique surface chemistry modifications, involving the immobilization of polyamidoamine (PAMAM) dendrimers and subsequent covalent attachment of thymidine oligonucleotide probes to the dendrimer periphery, were used to develop a surface to facilitate the capture of mRNA from Cryptosporidium parvum (C. parvum) oocyst lysate, while remaining a suitable surface for NASBA. Using this very simple device, successful mRNA isolation and NASBA-based amplification from as few as 30 C. parvum oocysts was achieved. An emerging area of point-of-care biosensor technology is that of paper-based sensors, and specifically, the lateral flow assay (LFA) has been very well-established. The main advantages of these types of sensors are that they are inexpensive, small, portable, disposable, easy-to-use, and require no external equipment or power source due to their capillary wicking ability. Traditionally, these biosensors are fabricated from cellulose-based fibers, which have fixed properties and limited chemical modification ability. Here, electrospun nanofibers have been presented as a new material for LFAs, since their properties are highly controllable and there are numerous materials from which the nanofibers can be made, giving countless surface modification possibilities. Poly(lactic acid) (PLA)-based nanofibers were optimized and incorporated into LFAs. Initial experiments demonstrated a successful one-step assay in which streptavidin-coated sulforhodamine B (SRB)-encapsulating liposomes were captured by anti-streptavidin antibodies adsorbed onto the nanofiber surface. Subsequently, an enzymatic sandwich immunoassay was developed for Escherichia coli (E. coli), and a limit of detection of 1.9x104 cells was achieved. Finally, functional polymers were used to demonstrate that the notorious problem of non-specific binding can be eliminated through the use of anti-fouling block copolymers. Functionalized electrospun nanofibers can thus be used to enhance paper-based assays and develop highly sensitive and specific biosensors possessing many significant advantages compared to traditional assays. Concluding from the microfluidic and LFA research presented, point-of-care biosensors can be developed in a variety of formats, each having their own benefits and limitations. By catering the characteristics of the assay to the parameters surrounding its application, an ideal, reliable biosensor can be realized.
Baeumner, Antje J
Agricultural and Biological Engineering
M.S., Agricultural and Biological Engineering
Master of Science
dissertation or thesis