博士 / 國立陽明大學 / 放射醫學科學研究所 / 95 / A novel optical heterodyne surface plasma wave biosensor (OHSPWB) with a Zeeman laser is proposed. Two surface plasma waves are excited simultaneously by two correlated P-polarized waves in a SPR device with the Kretschmann configuration. Two reflected P waves are optically heterodyned such that the magnitude of the heterodyned signal is proportional to the multiplication of two attenuated reflected P waves. This results in a more sensitive detection of the refractive index and the thickness of sample layer. Then the detection sensitivity and the dynamic range based on this amplitude-sensitive method are enhanced. In addition, lock-in amplifier capable of narrowband detection is used to get rid of the influence of background noise. Moreover, it is known that the sensitivity of intensity-based detection, in which the incident angle is fixed, is higher than that of conventional resonance unit detection, which traces the dip in the SPR curve. In the experiments, 0.01wt.% sucrose solution was detected and a 100pg/ml mouse IgG corresponding to a refractive index change of RIU (refractive index unit) and 7fg/mm2 of surface coverage is achieved. A multi-channel OHSPWB using a micro-fluidic system purchased from Biacore can detect 0.01wt.% sucrose solution and 100pg/ml mouse IgG as well.
Due to the elliptical polarization and nonorthogonality of Zeeman laser, it induces decorrelation between paired SPWs partially and then obviously results in a degradation of detection sensitivity because of a lowering on signal to noise ratio of the detected heterodyne signal. Therefore, a common-path polarized heterodyne interferometer is integrated into SPR device with the Kretschmann configuration to construct a novel paired surface plasma waves biosensor (PSPWB), then the common phase noise including the background phase noise and the frequency noise are inherently cancelled out. In addition, an amplitude ratio algorithm is introduced into this paired SPW biosensor whereby the excess noise of laser intensity fluctuation can be effectively reduced. The experimental results correctly verify the properties of a PSPWB that the detection sensitivity is able to measure a 0.001wt.% sucrose-water solution and 0.01wt.% glycerin solution. This result confirms the detection sensitivity of 10pg/ml mouse IgG/anti-IgG interaction up in real time which corresponds to refractive index change of RIU and 0.7fg/mm2 of surface coverage successfully. In order to further enhance the detection sensitivity of PSPWB colloid gold (Au) nanoparticles of 20nm diameter are used in PSPWB to further enhance the detection sensitivity. In this experiment, the sensitivity at 0.33pg/ml of Au-nanoparticle conjugated protein A (Au-PA) interacting with mouse IgG was detected successfully. There is a 6-fold enhancement by this arrangement compared with the experimental results without Au nanoparticles.
β-amyloid protein (Aβ) is proposed as the principle pathogenetic cause of Alzheimer’s disease (AD). Substantial evidence indicates that aggregation of Aβ protein resulting in neuronal toxicity probably play a causative role in the etiology of AD. In the brain from AD and normal aging (NA), three main soluble Aβ species has been identified including the full-length Aβ1-42 and two N-terminal truncated peptides AβPE3-42 and AβPE11-42. The soluble aggregates present in AD show a composition weighting differing from that in NA. Therefore, the developed two-frequency optical heterodyne surface plasma wave biosensor was used to detect the polymerization behavior of the two mixtures and the three individual Aβ species.
Both of the homogeneous and heterogeneous aggregation assays of the three individual soluble Aβ oligomers show that the order of aggregation rate was as follows, AβPE11-42> AβPE3-42> Aβ1-42. These results indicate that the loss of N-terminal charged polar amino acids have significant effect on the conformational character and depositability of these three oligomers. From thioflavin T fluorimetric assay, it also confirms that AβPE11-42 oligomer has a highest aggregation rate and Aβ1-42 is the lowest.
The aggregation rates between the three Aβ species and mixture NA and AD show as follows, AβPE3-42> AβPE11-42> Aβ1-42. This is owing to that both two mixtures NA and AD have a higher selectivity to aggregate with AβPE3-42 rather than with AβPE11-42. To aggregate with these three Aβ species, mixture AD capable of a higher aggregation rate than mixture NA also demonstrates that higher composition weighting of AβPE3-42 in mixture AD has a important role to increase aggregation rate.
Sulforhodamine B (SRB) cell toxicity assay of the three Aβ species shows that Aβ1-42 and AβPE11-42 has a similar cell toxicity and AβPE3-42 is lesser toxic than the other two isoforms. Besides, the cell toxicity of mixture AD is much higher than the three individual Aβoligomers and mixture NA is much lower than that. These results imply that these three Aβ species are capable of synergetic effect to enhance or depress cell toxicity in mixture AD and NA depending upon their composition weighting of mixture.
AβPE3-42 shows a minor cytotoxicity, but it is the main composition of mixture AD and it shows a highest aggregation rate with both mixtures. Moreover AβPE3-42 is more protease resistant than untruncated Aβto accumulate in brain resulting in membrane impairment and cell necrosis and apoptosis. These findings suggest that formation of N-terminal truncated peptides play an important role to enhance aggregation and cell toxicity and to expedite the progression and onset of the disease.
| Identifer | oai:union.ndltd.org:TW/095YM005605004 |
| Date | January 2007 |
| Creators | Wu, Hsieh-Ting, 吳學鼎 |
| Contributors | Chien Chou, 周晟 |
| Source Sets | National Digital Library of Theses and Dissertations in Taiwan |
| Language | zh-TW |
| Detected Language | English |
| Type | 學位論文 ; thesis |
| Format | 134 |
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