Chemical-selective real-time laser precision control of biomolecules

生物分子的化学选择性实时激光精密控制

• 批准号: 10693950
• 负责人: Chi Zhang
• 金额: $ 36.95万
• 依托单位: PURDUE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-09-01 至 2027-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10693950
• 关键词: Ablation; Affect; Behavior Control; Biological; Cells; Chemicals; Classification; Color; Consumption; Detection; Electronics; Fluorescence; Gene Silencing; Genetic; Image; Incubated; Ion Channel; Laboratories; Lasers; Light; Location; Logic; Manuals; Methods; Molecular Target; Neurons; Optics; Organelles; Output; Pharmaceutical Preparations; Proteins; Radiation; Resolution; Sampling; Scanning; Signal Transduction; Site; Speed; System; Techniques; Technology; Time; Transfection; absorption; biological systems; controlled release; design; digital; enzyme activity; improved; inhibitor; interest; laser tweezer; neuroregulation; optic tweezer; optogenetics; portability; prototype; small molecule inhibitor; submicron

PROJECT SUMMARY The capability to precisely control behaviors of biomolecules in living cells is a challenging task. Current methods can be classified into chemical-based and laser-based approaches. For example, small molecule inhibitors or activators can be introduced into the biological system for manipulating enzyme activities. However, it is impossible to control the interaction locations with high precision, which poses off-target effects. Protein control using genetic methods such as gene silencing or editing might selectively impact a targeted protein, but requires transfection and incubation, and cannot be performed in real-time. Optical techniques such as optical tweezers can manipulate small targets at the laser focus, but can only interact with a few targets at a time. Current laser manipulation and ablation methods usually require a pre-acquired image together with a manual selection of target locations on samples. This method is not only time-consuming but also unsuitable to apply to highly dynamic living biological samples. Optogenetic methods can control neuron functions using light radiation and light-sensitive ion channels, but only at the single-cell level. There’s no existing technology that can select molecular targets in cells and control only these targets at sub-micron resolution in real-time. In this application, we develop a real-time precision opto-control (RPOC) platform that can selectively and precisely control biomolecules only at the desired interaction site using lasers. RPOC is based on a high-speed laser scanning system. First, during the laser scanning, an optical signal is generated at a specific pixel from the target molecule. Then, this optical signal will be compared with a preset threshold and to send out an electronic signal to control an acousto-optic modulator which is used as a fast switch to couple another laser beam to interact with the same pixel. The optical signal detection, processing, and laser control happen within 20 ns, much faster than the pixel dwell time. Digital logic circuits will also be designed with the comparator circuits to control the interaction laser beam based on the logic output from multiple signal channels. We will use photo- switchable proteins and design photo-convertible inhibitors and activators to demonstrate precision control of enzyme activities on site. Furthermore, we will use multiple continuous-wave lasers and acousto-optic tunable filters to design a portable and multicolor RPOC that can operate outside an optical lab. RPOC can accurately control and manipulate biomolecules in real-time without affecting other biomolecules in the system. It is highly chemical selective since the optical signal can be selected from fluorescence, Raman, or absorption signals. It will allow biologists to control and interrogate only the biomolecules of interest during laser scanning without affecting other parts of the sample with sub-micron precision. RPOC would be widely applied to study enzyme activities in cells, understand organelle interactions, improve controlled-release of drugs, and perform precision neuromodulation.

项目摘要 精确控制活细胞生物分子行为的能力是一项挑战任务。当前方法 可以分类为基于化学和激光的方法。例如，小分子抑制剂或 激活剂可以引入以操纵酶活性的生物系统中。但是，是 无法以高精度来控制相互作用位置，从而定位靶向效果。蛋白质控制 使用基因沉默或编辑等遗传方法可能会选择性地影响靶向蛋白质，但需要 转染和孵育，不能实时执行。光学镊子等光学技术 可以在激光焦点下操纵小目标，但一次只能与几个目标相互作用。当前激光 操纵和消融方法通常需要预先获得的图像以及手动选择 样品上的目标位置。这种方法不仅是耗时的，而且不适用于高度应用 动态生物生物样品。光遗传学方法可以使用光辐射控制神经元功能，并且 光敏离子通道，但仅在单细胞水平上。没有现有的技术可以选择 细胞中的分子靶标，仅在实时分辨率下以亚微米分辨率控制这些靶标。 在此应用程序中，我们开发了一个实时的精度OPT-Control（RPOC）平台，可以选择性地和 精确控制生物分子仅在所需的相互作用位点使用激光器。 RPOC基于高速 激光扫描系统。首先，在激光扫描期间，从特定像素中生成光学信号 靶分子。然后，将将此光学信号与预设阈值进行比较并发送电子信号 信号控制声学调制器，该调制器被用作快速开关，将另一个激光束搭配到 与同一像素相互作用。光信号检测，处理和激光控制发生在20 ns之内， 比像素居住时间快得多。数字逻辑电路也将使用比较电路进行设计 根据来自多个信号通道的逻辑输出来控制相互作用激光束。我们将使用照片 - 可切换蛋白质和设计光转化抑制剂和激活剂，以证明对 现场的酶活性。此外，我们将使用多个连续的波激光器和声音可调 滤波器设计可以在光学实验室外运行的便携式和多色RPOC。 RPOC可以准确 实时控制和操纵生物分子，而不会影响系统中的其他生物分子。这是高度的 化学选择性，因为可以从荧光，拉曼或滥用信号中选择光信号。它 将允许生物学家在无需激光扫描过程中控制和询问感兴趣的生物分子而无需 用亚微米精度影响样品的其他部分。 RPOC将被广泛应用于研究酶 细胞中的活动，了解细胞器相互作用，改善药物的受控释放并执行精度 神经调节。