Nanobiotechnology and Bioanalysis Group, INTERFIBIO, Departament d׳Enginyeria Quimica, Universitat Rovira i Virgili, Avinguda Països Catalans 26, 43007 Tarragona, Spain.
Fraunhofer ICT-IMM, Carl-Zeiss-Strasse 18-20, 55129 Mainz, Germany.
Biosens Bioelectron. 2014 Sep 15;59:342-9. doi: 10.1016/j.bios.2014.03.024. Epub 2014 Apr 12.
Tularemia is a highly infectious zoonotic disease caused by a Gram-negative coccoid rod bacterium, Francisella tularensis. Tularemia is considered as a life-threatening potential biological warfare agent due to its high virulence, transmission, mortality and simplicity of cultivation. In the work reported here, different electrochemical immunosensor formats for the detection of whole F. tularensis bacteria were developed and their performance compared. An anti-Francisella antibody (FB11) was used for the detection that recognises the lipopolysaccharide found in the outer membrane of the bacteria. In the first approach, gold-supported self-assembled monolayers of a carboxyl terminated bipodal alkanethiol were used to covalently cross-link with the FB11 antibody. In an alternative second approach F(ab) fragments of the FB11 antibody were generated and directly chemisorbed onto the gold electrode surface. The second approach resulted in an increased capture efficiency and higher sensitivity. Detection limits of 4.5 ng/mL for the lipopolysaccharide antigen and 31 bacteria/mL for the F. tularensis bacteria were achieved. Having demonstrated the functionality of the immunosensor, an electrode array was functionalised with the antibody fragment and integrated with microfluidics and housed in a tester set-up that facilitated complete automation of the assay. The only end-user intervention is sample addition, requiring less than one-minute hands-on time. The use of the automated microfluidic set-up not only required much lower reagent volumes but also the required incubation time was considerably reduced and a notable increase of 3-fold in assay sensitivity was achieved with a total assay time from sample addition to read-out of less than 20 min.
兔热病是一种由革兰氏阴性球菌杆菌、土拉弗朗西斯菌引起的高度传染性人畜共患病。由于其高毒性、传播性、死亡率和简单的培养性，兔热病被认为是一种潜在的致命生物战剂。在本报告的工作中，开发了用于检测整个土拉弗朗西斯菌的不同电化学免疫传感器格式，并比较了它们的性能。一种抗弗朗西斯菌抗体 (FB11) 用于检测在细菌外膜中发现的脂多糖。在第一种方法中，使用金支持的羧酸末端双足烷硫醇自组装单层来与 FB11 抗体共价交联。在第二种替代方法中，生成了 FB11 抗体的 F(ab) 片段，并直接化学吸附到金电极表面。第二种方法导致捕获效率提高和灵敏度提高。实现了脂多糖抗原的检测限为 4.5ng/mL，土拉弗朗西斯菌的检测限为 31 个细菌/mL。在证明了免疫传感器的功能后，电极阵列用抗体片段功能化，并与微流控技术集成，并安装在测试器设置中，该设置方便了分析的完全自动化。唯一的最终用户干预是添加样品，需要不到一分钟的手动操作时间。自动化微流控设置的使用不仅需要更少的试剂体积，而且所需的孵育时间也大大缩短，总分析时间从添加样品到读取不到 20 分钟，检测灵敏度提高了 3 倍。
