Warkiani Majid Ebrahimi, Tay Andy Kah Ping, Khoo Bee Luan, Xiaofeng Xu, Han Jongyoon, Lim Chwee Teck
BioSystems and Micromechanics (BioSyM) IRG, Singapore-MIT Alliance for Research and Technology (SMART) Centre, Singapore.
Lab Chip. 2015 Feb 21;15(4):1101-9. doi: 10.1039/c4lc01058b.
Diagnosis of malaria at the early stage of infection is challenging due to the difficulty in detecting low abundance parasites from blood. Molecular methods such as real-time polymerase chain reaction (qPCR) can be especially useful for detecting low parasitemia levels due to their high sensitivity and their ability to recognize different malarial species and strains. Unfortunately, the accuracy of qPCR-based malaria detection can be compromised by many factors, including the limited specificity of primers, presence of PCR inhibitors in blood serum and DNA contamination from nucleated blood cells. Here, we use a label-free, shear-modulated inertial microfluidic system to enrich malaria parasites from blood so as to facilitate a more reliable and specific PCR-based malaria detection. This technique capitalizes on cell focusing behaviors in high aspect ratio microchannels coupled with pinched flow dynamics to isolate ring-stage malaria parasites from lysed blood containing white blood cells (WBCs). In this high aspect ratio (ratio of the channel height to the width) platform, the high shear rate along the channel width causes the dispersed WBCs at the inlet to migrate and align into two streams near the channel sidewalls while the malaria parasites remain unfocused. Sensitive detection of parasites at spiked densities ranging from 10(3) to 10(4)Plasmodium falciparum parasites per mL (~2-10 per μL) has been demonstrated; they have also been quantified in whole blood using qPCR. This is approximately 100-fold more sensitive than the gold standard conventional microscopy analysis of thick blood smears. The simplicity of this device makes it ideal for integration with an automatic system for ultra-fast and accurate detection despite low levels of parasitemia. It can also help in malaria screening and elimination efforts.
由于难以从血液中检测到低丰度的疟原虫,疟疾感染早期的诊断具有挑战性。实时聚合酶链反应(qPCR)等分子方法因其高灵敏度以及识别不同疟原虫种类和菌株的能力,对于检测低疟原虫血症水平特别有用。不幸的是,基于qPCR的疟疾检测的准确性可能会受到许多因素的影响,包括引物特异性有限、血清中存在PCR抑制剂以及有核血细胞的DNA污染。在这里,我们使用一种无标记、剪切调制的惯性微流控系统从血液中富集疟原虫,以便促进更可靠、更特异的基于PCR的疟疾检测。该技术利用高纵横比微通道中的细胞聚焦行为以及收缩流动动力学,从含有白细胞(WBC)的裂解血液中分离出环状期疟原虫。在这个高纵横比(通道高度与宽度之比)的平台上,沿通道宽度方向的高剪切速率使入口处分散的白细胞迁移并在通道侧壁附近排列成两股流,而疟原虫仍未聚焦。已经证明能够灵敏地检测每毫升10³至10⁴个恶性疟原虫寄生虫的加标密度(约每微升2至十 个);它们也已通过qPCR在全血中进行了定量。这比厚血涂片的金标准传统显微镜分析灵敏度高约100倍。该设备的简单性使其非常适合与自动系统集成,以实现超快速、准确的检测,尽管疟原虫血症水平较低。它还可以有助于疟疾筛查和消除工作。