生物標靶結合生物機能磁性奈米粒子中磁鬆弛之研究

本論文旨在透過各種磁性物理特性分析，且自組裝變溫交流磁化率系統，用以研究「生物官能化之磁性奈米粒子(biofunctionalized magnetic nanoparticles, BMNs)」與「生物標靶 (biotargets)」結合情形。其中「生物官能化之磁性奈米粒子」即在磁性奈米粒子外塗覆上葡聚醣，而後在葡聚醣上塗覆生物官能基。此研究中所使用生物標靶蛋白為甲胎蛋白(alpha-fetaprotein, AFP)，而生物官能化之磁性奈米粒子為四氧化三鐵(Fe3O4)奈米粒子塗覆甲胎蛋白抗體(anti-alpha-fetaprotein, antiAFP)形成Fe3O4-antiAFP。因磁性奈米粒子為單一磁域的超順磁性材料，當生物分子反應結合後，將促使磁性奈米粒子聚集形成團簇，進而產生磁特性的變化，故可藉由飽和磁化強度、殘餘磁化強度、磁鬆弛時間與阻礙(塞)溫度…等物理特性來探討其結合情形。據本研究觀察，在固定濃度的antiAFP狀況下，添加不同濃度的AFP與其反應兩小時後，再取適量反應物滴於試紙進行乾燥處理，量測其飽和磁化強度(Ms) 會伴隨AFP的濃度增加而增加，利用該特性能有效鑑別AFP濃度約在2.64 ppm，然而觀察在剩餘100 Oe的磁場底下，其低磁場下磁化強度(Mlf) 鑑別AFP最小濃度將能提升至0.24 ppm。此外，利用交流磁化率系統量測其相位之去磁時間(τ)，顯示出AFP濃度的鑑別度能小於0.03 ppm。因此，我們證明了通過飽和磁化強度(Ms)、低磁場下磁化強度(Mlf)與去磁時間(τ)為可靠的方法，用於鑑別血液中特徵蛋白的含量。此外本研究還發現，因超順磁性所特有的阻礙(塞)溫度（TB）特徵，故也能被用來測定AFP濃度。其中發現粒子間所產生的平均磁各向異性會因AFP濃度減少而減少，且實驗數據與粒子間交互作用的理論模型具有良好的一致性。因此證明造成阻礙(塞)溫度(TB)改變的原因，是由於不同濃度的AFP結合狀況不同，進而產生整體平均磁各向異性的改變。綜合飽和磁化強度(Ms)、低磁場下磁化強度(Mlf)、去磁時間(τ)與阻礙(塞)溫度(TB)的結果，可歸因於粒子間交互作用與BMNs中磁矩的尼爾運動。研究過程中了解阻礙(塞)溫度可以定義為T_B=∆E⁄(k_B ln(τ_m/τ_0 ))，其中τ_m是測量時間，這意味著當測量時間(τ_m)縮短，則阻礙(塞)溫度(TB)將增加。利用MPMS測量時間通常為1-100秒的DC測量，因此開發了一套變溫單頻交流磁化率的系統，頻率達數千赫茲(kHz)。該系統成功的將阻礙(塞)溫度(TB)提高，從68 K（MPMS，DC測量）提升至約250 K（變溫交流磁化率系統，AC測量）。最後本研究預期將變溫交流磁化率系統與高溫超導干涉量子元件(high-Tc SQUID)結合。利用SQUID對磁場高靈敏度的特性，藉以區別系統背景與訊號強度的差異，即增加信噪比（SNR），預期信號變化將更顯著，藉以提升對樣品的鑑別度。

關鍵字

甲胎蛋白；磁性奈米粒子；阻礙(塞)溫度；磁鬆弛時間；交流磁化率；高溫超導干涉量子元件

並列摘要

In this thesis, we study the characteristics of the biofunctionalized magnetic nanoparticles being conjugated with biotargets by virtue of physical analysis of their magnetic features. Parts of the experiments were carried out by the homemade temperature-variable AC susceptometer. The biofunctionalized magnetic nanoparticles (BMNs) is anti-alpha-fetaprotein (antiAFP) coated onto dextran-covered iron oxide nanoparticles and is labeled as Fe3O4-antiAFP and then conjugated with AFP biotargets. Because the magnetic nanoparticle is superparamagnetic and is with a single domain, the conjugation between the BMNs will prompt the magnetic nanoparticles to form into a cluster, and the conjugation will vary their magnetic characteristics. Therefore we could explore the conjugation by investigating the saturated magnetization Ms, the low field magnetization Mlf, the demagnetization time τ and the blocking temperature TB. It is found that the saturated magnetization Ms increases as the concentration of the associated AFP is increased. However, the low field magnetization Mlf in a field of 100 Oe decreases rapidly as the concentration of AFP is below 0.24 ppm. Furthermore, the demagnetization time (τ) measured by the AC susceptometer also showed a rapid increase as the concentration of AFP is lower than 0.03 ppm. Thus, we have demonstrated a sensitive platform for detecting biomarkers by characterizing Ms, Mlf, τ, and with a sensitivity limit of 0.02-ppm AFP. It is also found that due to the characteristics of blocking temperature the superparamagnetic materials have, they can also be used to determine the concentration of AFP. The averaged magnetic anisotropy among the particles decreased with the decrease of the AFP concentration. Furthermore, the data of the measurements are in good agreement with the interparticle interactions model. It therefore proved that the variation of TB is caused by the different AFP concentration of conjugation which alter the whole averaged magnetic anisotropy of the sample. Integrating the results of the saturated magnetization Ms, the low field magnetization Mlf, the demagnetization time τ and the blocking temperature TB can be attributed to the interparticle interactions and the Néel motion of magnetic moments in BMNs. The blocking temperature can be defined as T_B=∆E⁄(k_B ln(τ_m/τ_0 )), where k_B is the Boltzmann constant, ∆E is the energy barrier to moment reversal, τ_0 is a characteristic time, and τ_m is the time of the measurement. The small value of the τ_m means that a larger TB will be obtained. In addition, a platform developed for assaying bio-molecules by using a single frequency and temperature-variable ac susceptometer has been set up. If the signal frequency of excitation coils is higher, it means that the measurement time τ_m will be much less and hence the TB will increase. The measurement time is typically 1-100 sec for DC measurements, and is reciprocal to the frequency. The platform have successfully improved the TB from about 60 K (DC measurements) to about 250 K (AC measurements). This thesis also studied AC magnetic susceptibility with a high-Tc SQUID. The method will increase the signal-to-noise ratio (abbreviated to SNR). The signal variation will become clearer to be observed.

並列關鍵字

alpha-fetaprotein ； magnetic nanoparticle ； blocking temperature ； relaxation time ； ac susceptibility ； High-Tc SQUID

參考文獻

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生物標靶結合生物機能磁性奈米粒子中磁鬆弛之研究

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