Multi-marker Nanosensors for HIV

我们的使命是建立一个世界领先的联盟，设计和商业化下一代多标记艾滋病毒智能芯片，以快速诊断，阶段和监测艾滋病毒在资源有限的环境，包括地区医院，全科医生手术和发展中国家。这个大挑战是伦敦纳米技术中心的科学家之间的一个大规模的多学科合资企业（UCL/Imperial），UCL/MRC医学分子病毒学中心、皇家免费医院和UCL医院的临床病毒学家，UCLH NHS信托基金的卫生部资助的综合生物医学研究中心，与健康保护局和工业合作伙伴剑桥医学创新，Sphere Medical Ltd和Bionano Consulting。我们的新型纳米诊断和监测设备建立在我们非常强大的多学科创业团队的科学家，工程师和临床医生，以及我们的团队最近在不同领域的一系列突破，包括血清中的纳米机械传感，纳米纤维，纳米分选，磁性，纳米颗粒，光学设备和新型单域骆驼抗体。我们现在寻求实现的根本性步骤变化，无缝集成了这些进步的科学承诺，通过磁驱动力破裂来分选和检测非常低拷贝数的HIV标记，最终达到单一标记水平：分选：患者的血液样本将与超顺磁性纳米颗粒混合，这些纳米颗粒已被针对每个标记的捕获抗体功能化。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，并通过使其流过钝化柱的纳米结构阵列从裸露的纳米颗粒中分选出来，该阵列被设计为使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体由共同I Tom杜克和美国合作者开创，用于在几分钟内分离微球，需要亚微米特征，这可以在LCN使用电子束光刻轻松实现。它还有一个优点，即它可以浓缩标记物，这将提高我们的设备灵敏度并减少所需的患者血液量。通过巧妙的标记和纳米柱梯度设计，将同时实现多标记CD 4细胞、病毒体、抗体和p24蛋白的纳米分选，这些细胞的大小差异很大，从5 μ m到5 nm不等。传感：标记的标记物将在使用捕获蛋白定制的杠杆上检测，使用优化的共价连接化学。最大的灵敏度将通过缩小尺寸的悬臂梁从目前的介观尺度到亚微米的宽度，相当于只有几个病毒粒子，和一个相对未开发的传感机制，基于磁致动，这有望提高灵敏度的许多数量级，对单一的标记水平，在那里我们可以利用随机性。我们指出，虽然这项技术很早以前就被提出，但由于缺乏对医学相关目标的仔细和重复测量，它还没有进入临床。读数：悬臂梁偏转和/或共振的变化将使用LCN开创的新颖的光学传输方法读取，该方法简单地将悬臂梁视为衍射物体，并使用CCD相机测量多个悬臂梁的弯曲。这种方法的真实的优势在于可以并行测量多个阵列，无需复杂的激光对准或片上布线过程，并且激光探测器的距离可以显著缩小。我们的手持式HIV多标记物设备的开发最终将导致更有效的HIV感染管理，从而显著改善全球数百万HIV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.