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.

Gait analysis of the hind limb in Labrador Retrievers with and without cranial cruciate ligament disease

Cranial cruciate ligament (CCL) deficiency is the leading cause of lameness affecting the stifle joints of large breed dogs, especially Labrador Retrievers. Although CCL disease has been studied extensively, its exact pathogenesis and the primary cause leading to CCL rupture remain controversial. However, weakening secondary to repetitive microtrauma is currently believed to cause the majority of CCL instabilities diagnosed in dogs. Techniques of gait analysis have become the most productive tools to investigate normal and pathological gait in human and veterinary subjects. The inverse dynamics analysis approach models the limb as a series of connected linkages and integrates morphometric data to yield information about the net joint moment, patterns of muscle power and joint reaction forces. The results of these studies have greatly advanced our understanding of the pathogenesis of joint diseases in humans. A muscular imbalance between the hamstring and quadriceps muscles has been suggested as a cause for anterior cruciate ligament rupture in female athletes. Based on these findings, neuromuscular training programs leading to a relative risk reduction of up to 80% has been designed. In spite of the cost and morbidity associated with CCL disease and its management, very few studies have focused on the inverse dynamics gait analysis of this condition in dogs. The general goals of this research were (1) to further define gait mechanism in Labrador Retrievers with and without CCL-deficiency, (2) to identify individual dogs that are susceptible to CCL disease, and (3) to characterize their gait. The mass, location of the center of mass (COM), and mass moment of inertia of hind limb segments were calculated using a noninvasive method based on computerized tomography of normal and CCL-deficient Labrador Retrievers. Regression models were developed to determine predictive equations to estimate body segment parameters on the basis of simple morphometric measurements, providing a basis for nonterminal studies of inverse dynamics of the hind limbs in Labrador Retrievers. Kinematic, ground reaction forces (GRF) and morphometric data were combined in an inverse dynamics approach to compute hock, stifle and hip net moments, powers and joint reaction forces (JRF) while trotting in normal, CCL-deficient or sound contralateral limbs. Reductions in joint moment, power, and loads observed in CCL-deficient limbs were interpreted as modifications adopted to reduce or avoid painful mobilization of the injured stifle joint. Lameness resulting from CCL disease affected predominantly reaction forces during the braking phase and the extension during push-off. Kinetics also identified a greater joint moment and power of the contralateral limbs compared with normal, particularly of the stifle extensor muscles group, which may correlate with the lameness observed, but also with the predisposition of contralateral limbs to CCL deficiency in dogs. For the first time, surface EMG patterns of major hind limb muscles during trotting gait of healthy Labrador Retrievers were characterized and compared with kinetic and kinematic data of the stifle joint. The use of surface EMG highlighted the co-contraction patterns of the muscles around the stifle joint, which were documented during transition periods between flexion and extension of the joint, but also during the flexion observed in the weight bearing phase. Identification of possible differences in EMG activation characteristics between healthy patients and dogs with or predisposed to orthopedic and neurological disease may help understanding the neuromuscular abnormality and gait mechanics of such disorders in the future. Conformation parameters, obtained from femoral and tibial radiographs, hind limb CT images, and dual-energy X-ray absorptiometry, of hind limbs predisposed to CCL deficiency were compared with the conformation parameters from hind limbs at low risk. A combination of tibial plateau angle and femoral anteversion angle measured on radiographs was determined optimal for discriminating predisposed and non-predisposed limbs for CCL disease in Labrador Retrievers using a receiver operating characteristic curve analysis method. In the future, the tibial plateau angle (TPA) and femoral anteversion angle (FAA) may be used to screen dogs suspected of being susceptible to CCL disease. Last, kinematics and kinetics across the hock, stifle and hip joints in Labrador Retrievers presumed to be at low risk based on their radiographic TPA and FAA were compared to gait data from dogs presumed to be predisposed to CCL disease for overground and treadmill trotting gait. For overground trials, extensor moment at the hock and energy generated around the hock and stifle joints were increased in predisposed limbs compared to non predisposed limbs. For treadmill trials, dogs qualified as predisposed to CCL disease held their stifle at a greater degree of flexion, extended their hock less, and generated more energy around the stifle joints while trotting on a treadmill compared with dogs at low risk. This characterization of the gait mechanics of Labrador Retrievers at low risk or predisposed to CCL disease may help developing and monitoring preventive exercise programs to decrease gastrocnemius dominance and strengthened the hamstring muscle group.