- 學位論文
化學冷光探針對病原菌 β-內醯胺抗藥性的簡便和即時檢測
Facile and Point-of-care Detection of β-lactamase Mediated Resistance in Pathogenic Bacteria with a Chemiluminescent Probe
摘要
細菌感染的治療相當倚重化學療法， 而 β-內醯胺類化合物仍是現存最重要的抗生素之一。不幸的是，致病菌對 β-內醯胺類藥物的抗藥性正以驚人的速度在全世界蔓延，而細菌主要藉由產生能夠破壞該種抗生素的 β-內醯胺水解酶來獲得抗藥性。因此，早期檢測 β-內醯胺水解酶的活性將具有重要的臨床價值。在這份論文中，我們介紹了一種探針的設計和合成，該探針可在 β-內醯胺水解酶的催化下發出明亮的化學冷光。我們的探針能夠反映各種臨床分離的致病菌，如肺炎克雷伯氏菌，鮑氏不動桿菌， 綠膿桿菌，大腸桿菌和嗜麥芽窄食單胞菌的超聲波破菌液中β-內醯胺水解酶的表達量。為了進一步簡化診斷流程及樣品前處理，我們開發了利用界面活性劑的方法來有效地透化細菌外膜並釋出酵素，從而大大降低了偵測極限。為了在低成本的環境下實現快速的臨床檢測，我們利用 3D 列印製作了簡易的手機設備。藉由長時間曝光攝影，該設備能以高準確度再現在專業微量孔盤讀取儀上獲得的數據。總體而言， 這份論文展現了我們的探針在早期檢測細菌抗藥性方面的潛在優勢和應用。
並列摘要
Treatment of bacterial infections relies heavily on chemotherapy, and β-lactams have remained one of the most important antibiotics in our arsenal. Unfortunately, resistance to β-lactams in pathogenic bacteria is spreading worldwide at an alarming rate. The expression of β-lactamases, enzymes capable of destroying β-lactams, is a major mechanism of resistance. Early detection of β-lactamase activities would therefore be of great clinical value. In this work, we present the design and synthesis of a probe that emits bright chemiluminescence upon hydrolysis mediated by β-lactamases. Our probe is able to reflect the levels of β-lactamase expression in sonicated lysates of various clinically isolated pathogenic bacteria, such as K. pneumoniae, A. baumannii, P. aeruginosa, E. coli, and S. maltophilia. In order to further simplify sample preparations, we utilized adetergent-based method to effectively permeabilize bacterial cells, resulting in a detection limit down to the level of 105 CFUs. To achieve rapid point-of-care detection in a lowcost setting, we fabricated a cell phone device by 3D-printing. Through long exposure photography, the device is able to reproduce data obtained on a microplate reader with good accuracy. Overall, the results demonstrate potential advantages and applications of our probe in the early detection of bacterial resistance.