Exciplex detection: application of (i) novel detector systems and (ii) software for signal extraction from noise

近年来的重大科学进步之一是越来越多地使用基于DNA样本的信息来帮助日常生活的许多方面做出决策。直接明显的例子是DNA的法医用途，或医疗用途，如诊断疾病或潜在病原体的方法。最近，随着人类基因组DNA序列的测定，许多类似的基因组测定应该有助于改善或健康和安全。有大量的方法来检测DNA样本的特定区域，例如从患者或潜在的致病生物体中检测，这取决于我们检测称为荧光的光发射的能力。当然，目的是在任何测定中使用尽可能少的材料，因此，我们正在寻找方法来最小化DNA分析方法所具有的任何背景荧光。在曼彻斯特大学，最近开发了一种新的方法，将两个没有任何本征荧光的分子聚集在一起，形成待检测的特定DNA序列。探测分子必须在空间中非常精确地排列，才能成功地发出荧光。探测器分子的这种正确排列实际上是由DNA靶标序列本身强制执行的。该系统的背景荧光小于1%(这可以与其他目前通常具有大于60%的背景的DNA荧光探针进行比较)。通过仔细设计这两个探针分子的化学结构，该系统只有在找到正确的DNA序列时才能发出强烈的荧光。如果样品序列中甚至有一个DNA碱基不正确，就无法检测到荧光发射。这个新项目利用莱斯特空间中心科学家和爱丁堡大学天体物理学家的投入，扩大了这些目标组装的激基复合体检测DNA序列的范围。自2001年以来，莱斯特大学空间研究中心和生物系与欧空局/欧空局空间科学系一道，一直在调查为空间天文学开发的探测器在生命科学和医学的光学荧光测量中的应用。他们对超导隧道结(STJ)的研究导致了一种基于超导隧道结(STJ)的扫描仪，以取代当前类型的探测器(基于CCD和光电倍增管(PMT))，用于读出微阵列或基因芯片，用于细胞成像、蛋白质阵列、流式细胞仪和许多其他应用。与硅CCD和传统光电倍增管(PMT)相比，STJ具有至少100倍的灵敏度优势，同时在逐个光子的基础上独特地测量荧光强度的光谱形式。这一新系统将应用于DNA激基复合体，不仅允许更灵敏的测量，而且还可以提供不同于以前在此背景下使用的仪器的检测系统。上述检测方法都与荧光光的颜色强度有关。然而，荧光还有一个额外的性质--它发生在非常短的(纳秒)，但离散和可测量的时间段。这些DNA激基复合体的时间依赖性模式相当复杂，这种复杂性使得它有可能作为未知样本中存在或不存在这种激基复合体的唯一标志。确定时间事件的独特模式是宇宙学中的一个常见问题，因此爱丁堡大学的天文学小组将应用他们的专业数学方法来开发新的方法来检测激基复合体时间信号。使用这种新方法的检测很可能会允许该系统用于非常低的浓度-可能低于仅基于荧光颜色感觉的使用。

One of the major scientific advances of recent years has been the inreasing use of information based on DNA samples to help in decision-making in many aspects of everyday life. Immediately obvious examples are the forensic uses of DNA, or medical uses, such as methods for diagnosing disease or potential pathogens. The determination of DNA sequence of the human genome recently has been followed by many similar genome deteminations that should serve to improve or health and safety. A huge number of methods to detect particular regions of a DNA sample, such as from a patient or a potential disease-causing organism, depend on our ability to detect light emission called fluorescence. Naturally, the aim is use as little material as possible in any detemination and for this reason we are seeking ways to minimise any background fluorescence that the DNA analysis method possesses. At Manchester University a new method has recently been developed in which two molecules that do not have any intrinsic fluorescence are brought together on the particular sequence of DNA that is to be detected. The detection molecules have to be very precisely arranged in space for successful fluorescence emission. This correct arrangement of the detector molecules is actually enforced by the DNA target sequence itself. The background fluorescence in this system is less than 1% (this can be compared with other current fluorescence probes for DNA that typically have backgrounds of greater than 60%). By careful design of the chemical structures of these two probe molecules, the system is only able to emit strong fluorescence when exactly the correct DNA sequence has been found. If even a single DNA base is incorrect in the sample sequence, the fluorescence emission is not detectable. This new project extends the scope of these target-assembled exciplex detection of DNA sequences using input of Leicester Space Centre scientists and Edinburgh University astrophysicists. Since 2001 the University of Leicester Space Research Centre (SRC) and Department of Biology, together with the Space Science Department at ESA/ESTEC, have been investigating the application of detectors developed for space astronomy to optical fluorescence measurements in the life sciences and medicine. Their work with superconducting tunnel junctions (STJs) has led to an STJ-based 'scanner' to replace the current types of detectors (CCD- and photomultiplier tube (PMT)-based) for the readout of microarrays or gene chips, for cellular imaging, protein arrays, flow cytometry and many other applications. The STJ offers sensitivity advantages of at least 100 times compared to a silicon CCD and conventional photomultiplier tubes (PMTs) while uniquely measuring the spectral form of fluorescence intensity on a photon-by-photon basis. This new system wil be applied to the DNA exciplexes and allow not only more sensitive measurment, but also a detection system that provides different information from instruments previously used in this context. The above detection methods have concerned themselves with the intensity of the colour of the fluorescence light. However, fluorescence has an additional property - it takes place over a very short (nanosecond), but discrete and measurable, time period. The pattern of the time dependence for these DNA exciplexes is rather complex and this very complexity allows its potential use as a unique badge of the presence or otherwise of such an exciplex in an unknown sample. The detemination of unique patterns of timed events is a common problem in cosmology and so the astronomy groups at Edinburgh University are going to apply their specialist mathematical methods to developing new ways to detect the exciplex time signarure. It is likely that detection using this novel approach will allow the system to be used for very low concentratins - possibly lower than could ever be used based on fluorescence colour insensity alone.