Programmed Assembly of Biofunctionalized Nanowires for Biosensing Applications

Open Access
Author:
Morrow, Thomas J
Graduate Program:
Chemistry
Degree:
Doctor of Philosophy
Document Type:
Dissertation
Date of Defense:
July 08, 2010
Committee Members:
  • Christine Dolan Keating, Dissertation Advisor
  • Christine Dolan Keating, Committee Chair
  • Mary Elizabeth Williams, Committee Member
  • Philip C. Bevilacqua, Committee Member
  • Theresa Stellwag Mayer, Committee Member
Keywords:
  • Assembly
  • Nanowires
  • Dielectrophoresis
  • Photolithography
  • DNA
Abstract:
The experiments performed in this dissertation were designed to investigate a fabrication method that could ultimately be used to develop chip-based biosensors such as high-density arrays of field effect transistors (FET) or nanoelectromechanical sensors (NEMS) for eventual incorporation with integrated circuits. When combined with integrated circuits, consumers could be given direct access to these sensors. As a result,consumers could use these sensors as monitoring devices allowing people to take a more pro-active approach to their health. This fabrication method consisted of functionalizing nanowires synthesized off-chip, then positioning them to predetermined locations and integrating them with features already patterned on a chip surface. Because silicon nanowires used in FET and NEMS sensors are difficult to fabricate in high yield, the SiO<sub>2</sub> coated metal nanowires used to develop this fabrication method, were used only to mimic the native oxide that forms on Si nanowires allowing us to use the same binding chemistry on the nanowire mimics as on Si nanowires. Chapter 2 describes the positioning process and examines the selectivity of the probe DNA to fluorescently labeled target sequences following exposure to non-uniform electric fields, which were required to position the nanowires. In addition, treatments were performed on the biofunctionalized nanowires to mimic the lithographic steps the nanowires must endure when being integrated with chip electronics. These treatments included coating the nanowires with a photoresist, submerging them in a photoresist remover, and combining the two processes. Selectivity of the probe DNA for binding a fully complementary oligonucleotide as compared to a non-complementary sequence was examined following these treatments. We determined that following both the exposure to non-uniform electric fields and the lithographic treatments, the probe DNA selectively hybridized to completely complementary versus non-complementary fluorescently labeled DNA target sequences. By positioning multiple batches of nanowires each functionalized with a different single stranded probe DNA sequence to specific locations on the chip, the sensors would be more able to simultaneously detect multiple target analytes, reducing the need for high sample volumes while decreasing the time for diagnosis of diseases. In Chapter 3 this fabrication method was expanded upon by delivering three different batches of probe-functionalized nanowires to predetermined electrode gaps using the non-uniform electric field method described in Chapter 2. In addition, microwells patterned halfway into a photoresist dielectric were used to further position the nanowires along the electrode gaps. These microwells enabled uniform spacing between the nanowires and also permitted the use of the lithographic techniques described in Chapter 2. These lithographic steps were used to fabricate contacts over one end of the positioned nanowires forming singly clamped NEMS-like structures, or over both ends of the nanowires forming FET-like devices, while also integrating them in registration with features already patterned on the chip surface. Positioning and selectivity of the biofunctionalized nanowires was verified using three different DNA targets each labeled with a different fluorescent dye. Chapter 4 describes experiments performed adapting this fabrication method to develop FET-like structures. FETs are attractive as biosensors for their label-free, realtime responses to target analyte binding; high sensitivities in desalted solutions; and their electrical readout, which make them desirable candidates for incorporation with integrated circuits. To accomplish this, a number of changes were made to the fabrication method. These included patterning dual contacts over both ends of the nanowires forming source and drain electrodes, unlike the singly clamped NEMS-like structures described in Chapter 3. In addition, electrical isolation of the nanowires is required to avoid cross-talk between multiple sensing elements. In order to accomplish this, a second lithography step was used to pattern lines around the positioned nanowires. While the biofunctionalized nanowires were protected under a photoresist layer, Cl<sub>2</sub> plasma was used to etch the Au alignment electrodes in these lines. Treatments mimicking these added steps included coating the nanowires with a new photoresist and removing it with the photoresist remover used in Chapters 2 and 3. This new photoresist, Megaposit SPR 3012, was different from that used in the experiments described in Chapters 2 and 3. This new photoresist was required for the new instrument used in the process, the GCA 8000 stepper, which aided in greater accuracy and precision during the photolithographic steps. A second round of treatment with the photoresist and the remover mimicked the second lithography step needed for line patterning. To mimic the Au etch of the alignment electrodes, following the first round of treatments with the photoresist and remover, photoresist was applied a second time, then exposed to the Cl<sub>2</sub> plasma, then submerged in the photoresist remover. After it was determined that the individual steps do not adversely affect the probe DNA, FET-like devices were fabricated using biofunctionalized/SiO<sub>2</sub> coated nanowires. Selectivity of the biofunctionalized nanowires was determined by incubating the devices with target- and fluorescently-labeled tag DNA. When Au was used as the nanowire, high fluorescence intensities were observed on the assembled devices. In addition to the FET-like devices described in Chapter 4, we were also interested in developing NEMS devices for DNA/RNA biomarker detection. Chapter 5 describes the experiments performed to direct hybridization of target DNA and DNA Au conjugates to the tip of Rh nanowires. Rh nanowires have been shown to have excellent mechanical properties when used as cantilevers. The added mass at the tip of the cantilevers would have the greatest effect on the resonating properties. Au/Rh/Au multisegmented nanowires were fabricated and functionalized with probe DNA, and Au conjugates were used to verify the selective hybridization. Scanning electron microscopy (SEM) images show that the Au conjugates primarily bound to the Au segments of the nanowires. Probe coverage experiments performed on separate Au and Rh nanowires suggested that the thiolated probe was binding to both metals, but that the DNA bound to the Rh segments was less available for hybridization possibly due to multiple point attachment. The fabrication methods described in Chapters 2 through 4 show that a combination of top-down and bottom-up techniques can be used to fabricate biosensing structures such as bioFETs and NEMS, which can eventually be combined with integrated circuits. Beyond the scope of solely biosensing, this method holds the promise of integrating a variety of materials such as III-V semiconductors and graphene that in turn may revolutionize the semiconducting industry. In this work, we show that nanomaterials, specifically biofunctionalized nanowires, are attractive candidates as sensing elements in chip-based biosensors because of their unique optical, electrical, and chemical properties, in addition to their high surface-to-volume ratio. More importantly, the results described in Chapters 2 through 5 show that nanowires can be manipulated to develop these sensors and that they may be combined with integrated circuits.