Microsphere-based rolling circle amplification microarray for the detection of DNA and proteins in a single assay.
Microsphere-based rolling circle amplification microarray for the detection of DNA and proteins in a single assay.
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DOI：
10.1021/ac900694y
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发表时间：
2009-07-15
影响因子：
7.4
通讯作者：
Walt, David R.
中科院分区：
文献类型：
--
作者：
Konry, Tania;Hayman, Ryan B.;Walt, David R.
我们描述了一个高密度的微阵列，同时检测蛋白质和DNA在一个单一的测试。在该系统中，滚环扩增（RCA）被用作用于蛋白质和核酸检测的信号扩增方法。用合成DNA和纯化的重组蛋白分析物测试微球传感器。靶DNA序列是从编码流感嗜血杆菌分型和非分型菌株的外膜蛋白P6（OMP-P6）的高度保守基因设计的。选择促炎介质IL-6和IL-8作为靶蛋白。首先将捕获抗体固定在荧光编码的微球上。然后将微球加载到成像光纤束的蚀刻微孔中。使用生物素标记的二抗对靶蛋白IL-6和IL-8进行夹心测定。然后通过抗生物素蛋白桥将生物素化的捕获DNA探针连接至检测抗体。随后将与靶序列互补的挂锁探针与捕获探针杂交。在靶序列存在的情况下，连接挂锁探针，并将该环状序列用于RCA。RCA后，将多个荧光标记的信号探针与每个扩增的序列杂交，并使用落射荧光显微镜对微阵列成像。通过该测定，蛋白质和靶DNA的检测限分别低至10 fM和1 pM。除了使用RCA在单一测试中检测蛋白质和DNA的新方法之外，与类似的基于微球的测定相比，IL-8和IL-6的检测限提高了三个数量级。
We describe a high-density microarray for simultaneous detection of proteins and DNA in a single test. In this system, Rolling Circle Amplification (RCA) was used as a signal amplification method for both protein and nucleic acid detection. The microsphere sensors were tested with synthetic DNA and purified recombinant protein analytes. The target DNA sequence was designed from a highly conserved gene that encodes the outer membrane protein P6 (OMP-P6) of both typeable and non-typeable strains of Haemophilus influenzae. The proinflammatory mediators IL-6 and IL-8 were selected as target proteins. Capture antibodies were first immobilized on fluorescently-encoded microspheres. The microspheres were then loaded into the etched microwells of an imaging optical fiber bundle. A sandwich assay was performed for target proteins IL-6 and IL-8 using biotin-labeled secondary antibodies. Biotinylated capture DNA probes were then attached to the detection antibodies via an avidin bridge. A padlock probe, complementary to the target sequence, was subsequently hybridized to the capture probe. In the presence of the target sequence, the padlock probe was ligated and this circular sequence was used for RCA. Following RCA, multiple fluorescently-labeled signal probes were hybridized to each amplified sequence and the microarray was imaged using an epi-fluorescence microscope. With this assay, detection limits down to 10 fM and 1 pM were achieved for proteins and target DNA, respectively. In addition to this new approach for detecting both protein and DNA in a single test using RCA, the limit of detection for IL-8 and IL-6 was improved by three orders of magnitude compared to similar microsphere-based assays.
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影响因子：
14.9
作者：
HENDRICKSON, ER;TRUBY, TMH;EBERSOLE, RC
通讯作者：
EBERSOLE, RC
影响因子：
14.2
作者：
MARINI, M;VITTORI, E;MATTOLI, S
通讯作者：
MATTOLI, S
影响因子：
56.9
作者：
RUZICKA, V;MARZ, W;GROSS, W
通讯作者：
GROSS, W
影响因子：
7.8
作者：
Czarnik, AW
通讯作者：
Czarnik, AW
影响因子：
3.9
作者：
Favis, R;Huang, JM;Barany, F
通讯作者：
Barany, F