Bioaffinity Assays Using UV One-Dimensional Photonic Crystals (1DPC)

 描述（由申请人提供）：生物分子相互作用的测量对于蛋白质组学和蛋白质-蛋白质相互作用（Interactome）越来越重要。除了表面等离子体共振（SPR）之外，大多数替代方法依赖于使用与一种或多种相互作用物质共价连接的外来荧光团或探针。随着生物分子数量的增加，这些标记步骤变得越来越昂贵和复杂。 我们提出了一种新的方法，使用一维光子晶体（1DPC）和内在的蛋白质荧光的无标记测量。由于大多数蛋白质含有色氨酸，这些残基为蛋白质提供了一种质量传感器，类似于SPR，其也通过折射率的变化来感测质量。我们将在1DPC上使用布洛赫表面波（BSW）。BSW提供了高度局部化的激发和观察表面结合蛋白的机会。BSW基板将由多层紫外线传输层组成。这些结构不含金属，因此在短距离内不会发生淬火。 具体目标1。设计用于UV波长的BSW结构。并非所有1DPC都显示BSW。我们将使用传输矩阵法来模拟光学模式，并选择尺寸和尺寸。更复杂的数值模拟将被用来预测色氨酸耦合的效率和发射的方向性。 具体目标2。制造并测试用于UV波长的1DPC。合适的结构必须显示出良好的UV透射率、BSW和低的自发荧光。我们将测试三种氧化物的组合，二氧化硅（SiO2），氮化硅（Si 3 N4）和五氧化二钽（Ta 2 O 5）。将用白蛋白、链霉亲和素和抗体的模型系统测试底物。 具体目标3。BSW结构的生物学试验。这些结构将用两个重要的生物系统进行测试。第一个是p53肿瘤抑制蛋白与其负效应物之间的结合。第二个是HIV gp 120蛋白与其CD 4受体和潜在的HIV抗体的结合。 冲击该项目将产生一种新的通用方法来测量蛋白质-蛋白质相互作用。基板将是简单和廉价的。该方法可以与现有的或修改的读板器一起使用。光学器件的简单性将允许扩展到高通量测量。

 DESCRIPTION (provided by applicant): Measurements of biomolecule interactions are increasingly important for proteomics and protein-protein interactions (the Interactome). With the exception of surface plasmon resonance (SPR) most of the alternative methods rely on the use of extrinsic fluorophores or probes which are covalently linked to one or more of the interacting species. These labeling steps become increasingly costly and complex as the number of biomolecules is increased. We propose a new approach to label-free measurements using one-dimensional photonic crystals (1DPCs) and intrinsic protein fluorescence. Since most proteins contain tryptophan these residues provide a type of mass sensor for proteins, analogous to SPR which also senses mass by changes in the refractive index. We will use Bloch surface waves (BSWs) on the 1DPCs. BSWs provide opportunities for highly localized excitation and observation of surface-bound proteins. The BSW substrates will be composed of multiple layers of UV-transmitting dielectrics. These structures will not contain metals so there is no quenching at short distances. Specific Aim 1. Design BSW structures for use at UV wavelengths. Not all 1DPCs display BSW. We will use the transfer matrix method to simulate the optical modes and select the dielectrics and dimensions. More complex numerical simulations will be used to predict the efficiency of trp coupling and directionality of the emission. Specific Aim 2. Fabricae and test 1DPCs for use with UV wavelengths. Suitable structures must display good UV transmission, a BSW and low autofluorescence. We will test combinations of three dielectrics, silica (SiO2), silicon nitride (Si3N4) and tantalum pentoxide (Ta2O5). Substrates will be tested wih model systems of albumin, streptavidin and antibodies. Specific Aim 3. Biological testing of the BSW structures. The structures will be tested with two important biological systems. The first is binding between the p53 tumor suppressor protein and its negative effectors. The second is binding of the HIV gp120 protein with its CD4 receptor and with potential HIV antibodies. Impact. This project will result in a new generic method to measure protein-protein interactions. The substrates will be simple and inexpensive. The method can be used with existing or modified plate readers. The simplicity of the optics will permit the extension to high-throughput measurements.