ExoSERRS Amplification free direct genomic sequence analysis by optical spectroscopy

这项研究涉及检测特定的DNA序列，这些序列与特定的疾病状态有关，如囊性纤维化或指示感染的存在，如耐甲氧西林金黄色葡萄球菌（MRSA）感染。与现有的检测方法相比，该方法在速度和成本方面都有优势，因为在检测样品中的特定序列之前，它不需要增加样品中DNA的含量。该研究涉及使用一种称为表面增强共振拉曼散射（SERRS）的技术。如果特定波长的光被引导到非常小的银金属片上，即纳米颗粒，那么一些反射光的波长将发生变化。这种波长的变化与纳米颗粒表面的分子有关，并提供了可用于识别的分子指纹。这种金属被用来放大这种效应，并可用于研究单个分子。由于产生了分子的指纹谱，因此可以在不分离的情况下容易地识别混合物的组成。如果被分析的分子具有发色团，即是有色分子，则这些信号进一步增加。有色分子或标记可以附着在DNA上，使其能够产生强烈的SERRS信号。为了检测一个确定的DNA序列，使用互补序列，它将特异性杂交，从而识别所需的序列。由于添加了互补序列，因此可以对其进行修饰以掺入标记，通常是只有当互补序列结合时才能“看到”的有色分子。只有当靶DNA存在时才能看到该标记，如果不存在，则互补序列将不结合，并且将看不到信号。互补序列可以以几种不同的方式设计，以允许当它与其互补物结合时检测到信号。这里提出了一种新的方法，即一种特殊的染料标记附着到互补的DNA片段，不会给出SERRS信号。当这段DNA与靶DNA结合时，将添加一种酶，该酶将消化互补DNA，释放特殊的染料标记，然后在溶液中游离，现在能够给出SERRS信号。只有当目标DNA存在时，信号才会存在，并且可以想象，探针的性质将允许另一个探针分子与目标结合，然后被破坏，释放更多信号，等等。与依赖于目标而不是信号放大的当前方法相比，这将允许放大所获得的信号。该酶仅在特别设计的探针与靶标结合时起作用，并且当其在溶液中游离并以单链形式时不会消化探针并释放染料。这种方法的吸引力是极端灵敏度和多重能力的结合，即一次检测多个DNA靶标的能力。最终目的是实现从临床相关样品中检测特定DNA序列的无PCR检测。

The research proposed involves the detection of specific DNA sequences which will relate to a particular disease state, such as cystic fibrosis or indicate the presence of an infection, such as Methicillin-resistant Staphylococcus aureus (MRSA) infection. The method proposed offers benefits over existing detection methods in terms of speed and cost since it would not require an initial step to increase the amount of DNA in the sample before detection of the specific sequence in the sample could be carried out.The research involves the use of a technique called surface enhanced resonance Raman scattering (SERRS). If light of a particular wavelength is directed onto very small pieces of silver metal, known as nanoparticles, then some of the reflected light will have changed wavelength. This change in wavelength is related to the molecules on the nanoparticles' surface and provides a molecular fingerprint that can be used for identification. The metal is used to amplify this effect and can be used to study a single molecule. Since a fingerprint spectrum of a molecule is produced, the composition of mixtures can easily be identified without separation. These signals are further increased if the molecule being analysed has a chromophore i.e. is a coloured molecule. Coloured molecules or labels can be attached to DNA giving it the ability to generate intense SERRS signals.To detect a defined DNA sequence a complementary sequence is used which will hybridise specifically and hence recognize the desired sequence. Since the complementary sequence is added it can be modified to incorporate a label, usually a coloured molecule which will only be 'seen' when the complementary sequence has bound. This label will only be seen when the target DNA is present, if it is not present the complementary sequence will not bind and no signal will be seen. The complementary sequence can be designed in several different ways to allow a signal to be detected when it binds to its complement. Here is proposed a new method whereby a special dye label is attached to the complementary piece of DNA which will not give a SERRS signal. When this piece of DNA binds to the target DNA, an enzyme will be added which will digest the complementary DNA, releasing the special dye label which will then be free in solution and now able to give a SERRS signal. The signal will only be present when the target DNA is present and it is envisaged that the nature of the probe will allow another probe molecule to then bind to the target and then be destroyed releasing more signal and so on. This will allow for an amplification in the signal obtained, compared to current methods which rely on amplification of the target rather than the signal. This enzyme only works when the specially designed probe is bound to the target and will not digest the probe and release the dye when it is free in solution and in a single stranded form. The attraction of this approach is the combination of extreme sensitivity and the multiplexing capacity i.e. the ability to detect multiple DNA targets at once. The ultimate aim is to achieve PCR-less detection of a specific DNA sequence from a clinically relevant sample.