| 摘要: | 表面電漿共振生物感測器是一種具有相當高靈敏度之生物感測器，而且無需標定並能即時地分析分子間的交互作用，大大地縮短檢測的時間及提供更多的生物資訊。本研究結合表面電漿共振與外差干涉技術建構一套振幅式成對表面電漿波生物感測器，對於等效折射率變化的偵測極限高達8.4×10-9 RIU，而在偵測攝護腺特異抗原方面，於磷酸鹽緩衝液中的偵測極限約為10 fg/mL (~300 aM)左右，並且也成功地在10倍稀釋血清中量測到的情況下，即時地偵測到稀釋血清中的目標抗原。接著我們把此成對表面電漿波生物感測器結合雙流道系統，以降低系統的雜訊，並針對H1N1新型流感病毒進行量測。實驗上，此新型的雙流道成對表面電漿波生物感測系統在緩衝液中的偵測極限為30 PFU/mL，大幅超越目前臨床上常用的快篩試劑，並且此雙流道成對表面電漿波生物感測系統的偵測時間只需要約20分鐘左右，就時效性來說是很有競爭力的。最後，我們發展了一套數學模型可以計算極低濃度蛋白質結合常數(結合速率常數、解離速率常數、平衡結合常數、平衡解離常數)。實驗上，我們成功的在緩衝液甚至是稀釋血清中，且濃度在0.1 pg/mL to 1 ng/mL範圍內，量測出攝護腺特異抗原與抗體間的蛋白質結合常數。An amplitude-sensitive paired surface plasma waves biosensor (PSPWB), which is based on SPR in conjunction with an optical heterodyne technique, has been demonstrated to show a detection limit of Δneff = 8.4×10-9 RIU. In addition, the biosensor is capable of real-time detection of total prostate-specific antigen (t-PSA) in phosphate buffered saline (PBS) solution and in diluted human serum without labeling. These experimental results exhibited a linear relationship between surface plasmon resonance (SPR) signals and t-PSA concentrations over a wide range from 10 fg/mL (~300 aM) to 100 pg/mL (~3 pM) and it is without complicated operating procedures when detecting biological analytes in clinical sample. And then, we applied the developed PSPWB in a dual-channel biosensor for rapid and sensitive detection of swine-origin influenza A (H1N1) virus (S-OIV). In conjunction with the amplitude ratio method with a reference channel, the stability of PSPWB system is significantly improved experimentally such that the theoretical LOD of the dual-channel PSPWB for S-OIV is 30 PFU/mL, which was two orders of magnitude more sensitive than the commercial rapid influenza diagnostic test (RIDT) at worst. Furthermore, under in vivo condition, this experiment demonstrates that the assay successfully measured S-OIV at a concentration of 1.8×102 PFU/mL in mimic solution, which contained PBS-diluted normal human nasal mucosa. Most importantly, the assay time took less than 20 min as opposed to several hours by polymerase chain reaction (PCR)-based methods. Finally, we establish a method that can calculate the binding constants of protein-protein interactions at ultra-low concentrations based on gold nanoparticle enhancement in sandwich configuration. The association rate constant, dissociation rate constant, equilibrium association constant, and the equilibrium dissociation constant, i.e., ka, kd, KA, KD, respectively, of the kinetics of binding between t-PSA and anti-t-PSA at concentrations from 0.1 pg/mL to 1 ng/mL, were measured either in PBS or in human serum. This is the first time that the kinetic constants have been measured at such a low concentration range in a complex sample such as human serum. |
