Distinguishing DNA by Analog-to-Digital-like Conversion by Using Optofluidic Lasers
Distinguishing DNA by Analog-to-Digital-like Conversion by Using Optofluidic Lasers
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DOI：
10.1002/anie.201107381
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发表时间：
2012-01-01
影响因子：
16.6
通讯作者：
Fan, Xudong
中科院分区：
文献类型：
--
作者：
Sun, Yuze;Fan, Xudong
区分靶DNA与具有单碱基错配的对应物为疾病诊断、个性化医疗和基础生物化学研究提供了关键信息。[1-3]在传统的基于荧光的检测中，将样品置于比色皿中，并使用DNA探针与靶DNA杂交并产生荧光信号。然而，由于靶和具有单碱基错配的链之间对DNA探针的结合亲和力的微小差异，所得荧光之间的区分比几乎是1，[4-8]这使得难以从错配的DNA链的池中直接和选择性地检测靶DNA。[9，10]在此，我们描述了一种使用光流体激光器的用于DNA的高度特异性腔内检测的系统。这种类型的激光器是一种新兴技术，它协同集成了染料激光器和微流体技术，用于小型化激光源，易于样品输送和极小的样品体积。[11-13]在我们的检测系统中，DNA样品和探针被并入作为激光增益介质的一部分。受激激光发射，而不是荧光（即，自发发射），被用作传感信号，以实现类似于模拟到数字的转换，这显著放大了目标和其单碱基错配对应物之间的小的固有热力学差异。一个完全匹配（PM）的DNA，一个单碱基错配（SM）的DNA，和一个分子信标（MB）探针被用作一个模型系统。进行了理论分析，以阐明潜在的腔内检测原理。然后，在PMDNA和SMDNA之间实验性地实现了240：1的辨别比（即，R= IPM/ISM，其中IPM和ISM分别是由PM DNA和SM DNA产生的光强度），这相对于基于荧光的方法增加了超过两个数量级。提出了从SM DNA池中以1：50的浓度比选择性检测PM DNA。该系统还可以在缓冲液和血清中区分更复杂的DNA序列，例如乳腺癌序列和含有单点突变的相应序列。MB是一种DNA探针，具有茎环结构和染料以及连接到序列两端的猝灭剂（图1a）。[10 PM DNA和SM DNA都能够与MB杂交。因此，一部分MB打开并产生荧光。这种基于荧光的检测（参见支持信息中第IA节的详细分析）可被视为“模拟”检测，其中PM DNA和SM DNA之间的微小热力学差异导致荧光信号的微小差异。支持信息中的图S1显示了PM DNA和SM DNA的荧光示例，其具有低区分率。在我们的腔内DNA检测系统中，一个光流环谐振腔（OFRR）被用作激光腔。如图1b所示，OFRR由一片玻璃毛细管组成，其横截面形成环形谐振器，并支持具有极高Q因子（> 107）的循环光学谐振模式。光模的倏逝场延伸到纤芯中，并与OFRR内表面附近的增益介质相互作用，从而为激射提供光反馈。当放置在OFRR中时，MB成为激光增益介质，其通过与感兴趣的DNA分子的杂交由MB的构象状态调制。虽然在约束力上有很小的差异...
Distinguishing a target DNA from a counterpart that has a single base mismatch provides critical information for disease diagnosis, personalized medicine, and basic biochemical research.[1–3] In traditional, fluorescence-based detection, samples are placed in a cuvette, and a DNA probe is used to hybridize with the target DNA and generate a fluorescent signal. However, because of the small difference in the binding affinity for the DNA probe between the target and the strand with a single base mismatch, the discrimination ratio between the resulting fluorescence is almost unity,[4–8] which makes it difficult to directly and selectively detect the target DNA from a pool of mismatched DNA strands.[9, 10] Herein, we describe a system for the highly specific intracavity detection of DNA that uses an optofluidic laser. This type of laser is an emerging technology that synergistically integrates a dye laser and microfluidics for miniaturized laser sources, easy sample delivery, and extremely small sample volumes.[11–13] In our detection system, DNA samples and probes are incorporated as part of the laser gain medium. Stimulated laser emission, rather than fluorescence (that is, spontaneous emission), is employed as the sensing signal to achieve conversion that is similar to analog-to-digital, which significantly amplifies the small intrinsic thermodynamic difference between the target and its single base mismatched counterpart. A perfectly matched (PM) DNA, a single base mismatched (SM) DNA, and a molecular beacon (MB) probe were used as a model system. A theoretical analysis was performed to elucidate the underlying intracavity detection principle. Then, a discrimination ratio (that is, R= IPM/ISM, where IPM and ISM is the light intensity generated by PM DNA and SM DNA, respectively) of 240: 1 was achieved experimentally between PMDNA and SMDNA, which is an increase of over two orders of magnitude relative to the fluorescence-based method. The selective detection of PM DNA from a pool of SM DNA at a concentration ratio of 1: 50 is presented. This system can also distinguish more complicated DNA sequences, such as a breast cancer sequence from a corresponding sequence that contains a single point mutation, in both buffer and serum. An MB is a DNA probe with a stem-loop structure and a dye as well as a quencher attached to each end of the sequence (Figure 1a).[10, 14–17] Both PM DNA and SM DNA are able to hybridize with the MB. Consequently, a fraction of MBs open and generate fluorescence. This fluorescencebased detection (see the detailed analysis in Section IA in theSupporting Information) can be regarded as “analog” detection, in which a small thermodynamic difference between PM DNA and SM DNA results in a small difference in the fluorescence signal. Figure S1 in the Supporting Information shows an example of the fluorescence from PM DNA and SM DNA, which has a low discrimination ratio. In our intracavity DNA detection system, an optofluidic ring resonator (OFRR) is used as the laser cavity. As illustrated in Figure 1b, the OFRR consists of a piece of glass capillary in which the cross-section forms the ring resonator and supports the circulating optical resonant mode with an extremely high Q factor (> 107).[18–20] The evanescent field of the optical mode extends into the core and interacts with the gain medium near the inner surface of the OFRR, thus providing the optical feedback for lasing. When placed in the OFRR, the MB becomes the laser gain medium, which is modulated by the conformational state of the MB through the hybridization with the DNA molecules of interest. Although a small difference in binding …