Multi-marker Nanosensors for HIV

我们的使命是建立一个世界领先的财团，设计下一代多标记艾滋病毒智能芯片并将其商业化，以便在资源有限的环境中快速诊断、分期和监测艾滋病毒，包括地区医院、全科医生手术和发展中国家。这项大型挑战是由伦敦纳米技术中心(UCL/Imperial)的科学家、UCL/MRC医学分子病毒学中心、皇家自由医院和UCL医院的临床病毒学家、伦敦大学卫生学院NHS信托基金由卫生部资助的综合生物医学研究中心与卫生保护局和行业合作伙伴剑桥医疗创新公司、球体医疗有限公司和BionanoConsulting共同发起的一项大型多学科联合项目。我们的新型纳米诊断和监测设备建立在我们强大的多学科、企业家团队的基础上，该团队由科学家、工程师和临床医生组成，我们的团队最近在不同领域取得了一系列突破，包括血清纳米机械传感、纳米制造、纳米吸收、磁性、纳米颗粒、光学设备和新型单域骆驼抗体。我们现在寻求实施的根本步骤改变，无缝地整合了这些进步的科学前景，通过磁力驱动的力量破裂，对极低拷贝数的艾滋病毒标记进行分类和检测，最终达到单一标记水平：分类：患者的血液样本将与超顺磁性纳米颗粒混合，这些纳米颗粒已与每个标记的捕获抗体一起发挥功能。标记病毒将通过施加磁场梯度(由微型感应线圈产生)从血液中浓缩，并通过流动通过钝化柱的纳米结构阵列从裸露的纳米颗粒中分离出来，钝化柱的设计使得不同大小的颗粒遵循不同的轨迹。这种经过验证的方法的变种，由Tom Duke和USA的合作者共同开创，用于在几分钟内分离微球需要亚微米特征，这些特征可以在LCN使用电子束光刻轻松实现。它还有一个优势，那就是它将集中标志物，这将提高我们的设备灵敏度，并减少患者所需的血量。通过巧妙的标记和纳米柱梯度设计，将并行实现大小从5微米到5纳米的多标记CD4细胞、病毒粒子、抗体和p24蛋白的纳米吸附。传感：随后将使用优化的共价连接物化学，在用捕获蛋白定制的悬臂上检测标记的标记。最大的灵敏度将通过将悬臂梁的尺寸从当前的介观尺度缩小到亚微米宽度，仅相当于几个病毒粒子，以及基于磁驱动的相对未知的传感机制来实现，该机制有望将灵敏度提高许多个数量级，达到单标记水平，在那里我们可以利用随机性。我们指出，虽然这项技术很久以前就被提出了，但由于缺乏对医学相关靶点的仔细和重复测量，它还没有进入临床。读数：悬臂挠度和/或共振的变化将使用LCN首创的新型光学传输方法读取，该方法只需将悬臂视为衍射体，并使用CCD相机测量多个悬臂的弯曲。这种方法的真正优势是可以并行测量多个阵列，没有复杂的激光对准或芯片上布线程序，并且激光探测器的距离可以显著缩小。我们手持艾滋病毒多标记设备的开发最终将导致更有效地管理艾滋病毒感染，从而显著改善全球数百万艾滋病毒感染者的前景。

Our mission is to establish a world-leading consortium to engineer and commercialise the next generation of multi-marker HIV smart chips to rapidly diagnose, stage and monitor HIV in resource-limited environments, including district hospitals, GP surgeries and developing countries. This Grand Challenge is a large scale multidisciplinary joint venture between scientists at the London Centre for Nanotechnology (UCL/Imperial), clinical virologists in the UCL/MRC Centre for Medical Molecular Virology, Royal Free and UCL Hospitals, the DoH-funded Comprehensive Biomedical Research Centre at UCLH NHS Trust, in conjunction with the Health Protection Agency and industrial partners Cambridge Medical Innovations, Sphere Medical Ltd and the BionanoConsulting. Our novel nanodiagnostic & monitoring device builds on our remarkably strong multidisciplinary, entrepreneurial team of scientists, engineers and clinicians, and a series of recent breakthroughs by our team in diverse fields, including nanomechanical sensing in serum, nanofabrication, nanosorting, magnetism, nanoparticles, optical devices and novel single domain llama antibodies. The radical step change we now seek to implement, seamlessly integrates the scientific promise of these advances, to sort and sense very low copy number HIV markers, via magnetically driven force rupture, ultimately towards the single marker level:Sorting: The patient's blood sample will be mixed with superparamagnetic nanoparticles, which have been functionalised with capture antibodies specific to each marker. Tagged-viruses will be concentrated from blood by applying a magnetic field gradient (produced by a miniature induction coil) & sorted from the naked nanoparticles by flowing them through a nanostructured array of passivated pillars, designed such that particles of different size follow distinct trajectories. This variant of a proven method, pioneered by co-I Tom Duke and USA collaborators, for separation of microspheres within minutes requires submicron features which can comfortably be achieved using e-beam lithography at the LCN. It also has the advantage that it will concentrate the markers, which will enhance our device sensitivity and reduce the volume of patient blood required. Nanosorting of multi-marker CD4 cells, virions, antibodies and p24 proteins which have vastly different sizes, ranging from 5 um to 5 nm, will be achieved in parallel by clever tagging and nanopillar gradient designs.Sensing: Tagged markers will then be detected on cantilevers tailored with capture proteins, using optimised covalent linker chemistry. The maximum sensitivity will be achieved by scaling down the dimensions of the cantilever beam from the current mesoscopic scale to submicron widths, comparable to just a few virions, and a relatively unexplored sensing mechanism, based on magnetic actuation, which promises to improve the sensitivity by many orders of magnitude, towards the single marker level, where we can take advantage of stochasicity. We point out that while this technology has been proposed a long time ago, it has not found its way into the clinic because of the lack of careful and repetitive measurements on medically relevant targets. Readout: Changes in cantilever deflection and/ or resonance will be read using novel optical transmission methods pioneered at the LCN, which simply treats the cantilever as a diffractive object and measures bending of multiple cantilevers using a CCD camera. The real advantages of this approach are that multiple arrays can be measured in parallel, there is no complex laser alignment or on-chip wiring procedures, and the laser detector distance can be significantly miniaturized.The development of our handheld HIV multiple-marker device will ultimately result in more effective management of HIV infection, thereby significantly improving the prospects of millions of HIV infected people across the world.