Microcantilever sensors for biochemical detection and analysis

Biochemical agents, including bacteria and toxins, are potentially dangerous and responsible for a wide variety of diseases. Reliable detection and characterization of small samples is necessary in order to reduce and eliminate their harmful consequences. Microcantilever sensors offer a potential alternative to the state of the art due to their small size, fast response time, and the ability to operate in air and liquid environments. At present, there are several technology limitations that inhibit application of microcantilever to biochemical detection and analysis, including difficulties in conducting temperature-sensitive experiments, material inadequacy resulting in insufficient cell capture, and poor selectivity of multiple analytes. This work aims to address several of these issues by introducing microcantilevers having integrated thermal functionality and by introducing nanocrystalline diamond as new material for microcantilevers. Microcantilevers are designed, fabricated, characterized, and used for capture and detection of cells and bacteria. The first microcantilever type described in this work is a silicon cantilever having highly uniform in-plane temperature distribution. The goal is to have 100 μm square uniformly heated area that can be used for thermal characterization of films as well as to conduct chemical reactions with small amounts of material. Fabricated cantilevers can reach above 300C while maintaining temperature uniformity of 2−4%. This is an improvement of over one order of magnitude over currently available cantilevers. The second microcantilever type is a doped single crystal silicon cantilever having a thin coating of ultrananocrystalline diamond (UNCD). The primary application of such a device is in biological testing, where diamond acts as a stable, electrically isolated reaction surface while silicon layer provides controlled heating with minimum variations in temperature. This work shows that composite cantilevers of this kind are an effective platform for temperature-sensitive biological experiments, such as heat lysing and polymerase chain reaction. The rapid heat-transfer of Si-UNCD cantilever compromised the membrane of NIH 3T3 fibroblast and lysed the cell nucleus within 30 seconds. Bacteria cells, Listeria monocytogenes V7, were shown to be captured with biotinylated heat-shock protein on UNCD surface and 90% of all viable cells exhibit membrane porosity due to high heat in 15 seconds. Lastly, a sensor made solely from UNCD diamond is fabricated with the intention of being used to detect the presence of biological species by means of an integrated piezoresistor or through frequency change monitoring. Since UNCD diamond has not been previously used in piezoresistive applications, temperature-denpendent piezoresistive coefficients and gage factors are determined first. The doped UNCD exhibits a significant piezoresistive effect with gauge factor of 7.53±0.32 and a piezoresistive coefficient of 8.12×10^−12 Pa^−1 at room temperature. The piezoresistive properties of UNCD are constant over the temperature range of 25−200C. 300 μm long cantilevers have the highest sensitivity of 0.186 m-Ohm/Ohm per μm of cantilever end deflection, which is approximately half that of similarly sized silicon cantilevers. UNCD cantilever arrays were fabricated consisting of four sixteen-cantilever arrays of length 20–90 μm in addition to an eight-cantilever array of length 120 μm. Laser doppler vibrometry (LDV) measured the cantilever resonant frequency, which ranged as 218 kHz−5.14 MHz in air and 73 kHz−3.68 MHz in water. The quality factor of the cantilever was 47−151 in air and 18−45 in water. The ability to measure frequencies of the cantilever arrays opens the possibility for detection of individual bacteria by monitoring frequency shift after cell capture.

Bioexploration of wild Alaskan berries: From field screening to functional food

Wild berries are fundamental components of traditional diet and medicine for Native American and Alaska Native tribes and contain a diverse array of phytochemicals, including anthocyanins and proanthocyanidins, with known efficacy against metabolic disorders. Bioexploration represents a new paradigm under which bioactive preparations are screened in coordination with indigenous communities, to prepare for subsequent in-depth chemical and biological analysis. The inclusive, participatory philosophical approach utilized in bioexploration has additional benefits that could be realized in seemingly disparate areas, such as education and economics. Five species of wild Alaskan berries (Vaccinium uliginosum, V. ovalifolium, Empetrum nigrum, Rubus chamaemorus, and R. spectabilis) were tested using “Screens-to-Nature” (STN), a community-participatory approach to screen for potential bioactivity, in partnership with tribal members from three geographically distinct Alaskan villages: Akutan, Seldovia, and Point Hope. Berries were subsequently evaluated via HPLC and LC-MS2, yielding significant species and location-based variation in anthocyanins (0.9-438.6 mg eq /100g fw) and proanthocyanins (73.7-625.2 mg eq /100g fw). A-type proanthocyanidin dimers through tetramers were identified in all species tested. Berries were analyzed for in vitro and in vivo activity related to diabetes and obesity. R. spectabilis samples increased preadipocyte-factor-1 levels by 82% over control, and proanthocyanidin-rich fractions from multiple species reduced lipid accumulation in 3T3-L1 adipocytes. Furthermore, extracts of V. uliginosum and E. nigrum (Point Hope) reduced serum glucose levels in C57bl/6j mice up to 45%. The same precepts of bioexploration, especially the inclusion of indigenous community perspectives and knowledge, have relevance in other areas of study, such as education and economics. Studies have established the apathetic, low-motivational environment characteristic of many introductory science laboratory classes is detrimental to student interest, learning, and continuation in scientific education. A primary means of arresting this decline and stimulating the students’ attention and excitement is via engagement in hands-on experimentation and research. Using field workshops, the STN system is investigated as to its potential as a novel participatory educational tool, using assays centered around bioexploration and bioactive plant compounds that hold the potential to offset human health conditions. This evaluation of the STN system provided ample evidence as to its ability to augment and improve science education. Furthermore, Strengths, Weaknesses, Opportunities, and Threats (SWOT) analysis was employed as a theoretical framework to review the potential benefits and hurdles associated with developing a wild Alaskan berry commodity. Synthesizing various sources of information – including logistics and harvest costs, sources of initial capital, opportunities in the current superfruit industry, and socioeconomic factors – the development of a berry commodity proves to be a complex amalgam of competing factors which would require a delicate balance before proceeding.