建立循環流動系統壓電式生物感測器用於及時檢測病原菌大腸桿菌 O157:H7及登革熱病毒

Abstract

在公共衛生的觀點上，病原菌的感染已經成為嚴重的疾病問題。 然而，在我們的日常生活中，有許多病原菌類別存在，包括致病性的病毒、細菌、黴菌、寄生蟲、海生的浮游植物及藍綠藻等。 病原菌的傳播機制不僅只有利用環境的因子如水、空氣或者是土壤，也包含食品的污染，血液的輸送或接觸的感染等。 由於在每個國家中致死性的病原菌往往伴隨著經濟的損失及人民的死亡，因此，如何預防及前期偵測病原菌的感染爆發是一重要的任務。 生物感測器為快速檢測生物分子的一新穎技術，此研究主要為發展在循環流動系統中一核酸壓電式生物感測方法用以及時偵測病原菌及病毒。 首先設計一具有30個去氧核苷酸及額外具有12個去氧單磷酸胸腺苷（dT）的專一性核酸探針以解決生物感測系統上空間的阻礙。 此具有12 dT於探針上如同一spacer，可顯著的增強雜交的效率（P < 0.05）。 結果中指出當探針分別與30 mer與104 mer的目標物雜交時，spacer增加雜交效率分別為1.4倍及2倍。 尤其當探針與較長的核酸目標物雜交時，spacer 減少了固定化的核酸探針與目標物雜交時的空間阻礙並提供雜交行為的支援。 此核酸壓電式生物感測系統也被用於從大腸桿菌O157:H7的PCR擴增的DNA產物的真實樣本上的檢測過程中所使用，其雜交效率的結果 PCR放大的雙股DNA可相當於合成的目標物 T-104AS之單股DNA。 更進一步地，此循環流動系統之壓電式生物感測器基於奈米金球的放大與驗證方法被用於一食品中病原菌大腸桿菌O157:H7的及時檢測。 延續前部分的研究，一含有12-dT及修飾硫醇且互補於目標物序列的第二探針被與奈米金球結合，並且作為像一質量的放大者及序列的檢驗者，用於放大在DNA壓電式生物感測器上頻率的改變。 在大腸桿菌O157:H7的樣品檢測方面，經由後PCR的放大後，可藉由此DNA壓電式生物感測器所測得1.2 × 10^2 CFU/ml的大腸桿菌O157:H7，並且當大腸桿菌O157:H7從10^2 至 10^6 CFU/ml時其具有線性相關。 在真實的食物樣品的檢測上，此DNA壓電式生物感測器亦可檢測出目標物。 除了病原菌的檢測，病毒的檢測是在壓電式生物感測器上的一個挑戰，考慮到病毒需要許多時間以傳統的方法進行檢測，此部分基於前面利用及時與可連至電腦的檢測成果，並結合奈米金球層疊的方法用於快速檢測病毒。 在此研究中，一對在登革熱病毒外膜蛋白的基因保留區的通用型引子對被設計用來放大其DNA片段，並且設計兩專一性探針用以識別在台灣登革熱病毒二型常見亞型。 根據先前的方法，在DNA壓電式生物感測器的表面上，第一探針與目標物進行雜交用以辨識登革熱病毒。 然後，第二探針結合奈米金球並且與目標物進行雜交，用以增加訊號值與驗證。 為了增加更多的雜交效率，第一探針被結合至不同大小粒徑的奈米金球並以層疊法引入，且與自由流動的目標物進行雜交。 接著，第二探針與目標物雜交於另一端，此經由層疊法的結構類似樹枝狀的結構。 藉由此層疊法，2.1×10^1 (PFU)/ml的登革熱病毒二型可藉由此壓電式生物感測器所測得，其介於頻率變化與病毒濃度的對數從2.1×10^6 至 2.1×10^1 PFU/ml呈線性相關。

Pathogens infections have been a serious problem on the public health aspect. There are many classes of pathogenic microorganisms, including pathogenic viruses, bacteria, fungi, parasites, marine phytoplankton, and cyanobacteria, etc. in our daily lives. The pathogen transmission mechanism use not only environment factors, ex. water, air or soil, but also the food contamination, blood transfusion or contract infection etc… At every country, the deadly pathogens are usually accompanying economy damage and life loss. Hence, how to prevent and early detect pathogen infection and outbreak is important tasks. Biosensor is a novel technology for rapid detection of biomolecules. In this study, we develop a DNA piezoelectric biosensing method for In-time detection of pathogens or viruses in a circulating flow system. First, the specific probes of a 30-mer oligonucleotide with additional 12 deoxythymidine 5'-monophosphate (12-dT) is designed to solve steric interference on the biosensor system. The addition of 12-dT to the probes as a spacer, significantly enhanced (P < 0.05) the hybridization efficiency (H%). The results indicate that the spacer enhanced the H% by 1.4- and 2-fold when the probes are hybridized with 30-mer and 104-mer targets, respectively. The spacer reduced steric interference of the support on the hybridization behavior of immobilized oligonucleotides, especially when the probes hybridized with relatively long oligonucleotide targets. The DNA piezoelectric biosensing system is also applied in the detection of PCR-amplified DNA from real samples of Escherichia coli O157:H7. The resultant H% of the PCR-amplified double-strand DNA is comparable to that of the synthetic target T-104AS, a single strand DNA. Further, a circulating-flow piezoelectric biosensor, based on an Au nanoparticle amplification and verification method, is used for real-time detection of a foodborne pathogen, E. coli O157:H7. Continuing the first part study, a second thiolated probe with 12-dT, complementary to the target sequence, is conjugated to the Au nanoparticles and used as a “mass enhancer” and “sequence verifier” to amplify the frequency change of the DNA piezoelectric biosensor. The PCR products amplifing from concentrations of 1.2 × 10^2 CFU/ml of E. coli O157:H7 are detectable by the DNA piezoelectric biosensor. A linear correlation is found when the E. coli O157:H7 detected from 102 to 106 CFU/ml. The piezoelectric biosensor is also able to detect targets from real food samples. Besides bacteria detection, virus detection is a challenge in the filed of piezoelectric biosensor. It needs a lot of time to detect viruses by traditional methods. This part of study combined the previously developed In-time and on-line work, with Au nanoparticles layer by layer method for rapid detection of virus. In this study, a pair of universal primers of dengue virus envelope gene conserve region was used to amplify cDNA fragment, and two specific probes for the identification of dengue virus type II common subtypes are developed in Taiwan. According to pervious process, first probe hybridizes with the target to identify dengue virus in DNA piezoelectric biosensor surface, then, second probe conjugates with Au nanoparticles and hybridizes with target to enhance signal and verification. In order to increase more hybridization efficiency, the layer by layer method is recommended for the first probe to conjugate to Au nanoparticles in different sizes and to hybridize with free targets. Further, the second probes are hybridized with targets at other terminals. The structure is like dendritic form via layer by layer hybridization. Following layer by layer method, as low as 2.1×10^1 plaque forming unit (PFU)/ml DENV type 2 can be detected by the DNA piezoelectric biosensor. Linear correlation between frequency change and logarithmic number of virus concentration is found for DENV from 2.1×10^6 to 2.1×10^1 PFU/ml.