EAGER:A Novel Lab-on-a-Chip Concept for Characterization of Nanovesicles based on their Dielectric Properties

本项目的目标是开发一种快速、准确的芯片上实验室设备，用于基于纳米级囊泡独特和固有的生物物理特性进行表征。该项目的直接焦点是确定小细胞分泌的囊泡(外体)的特征，这些小细胞分泌的囊泡被归类为与许多类型的疾病相关的循环生物标志物，包括癌症、糖尿病、心血管疾病、感染性疾病和神经退行性疾病。这项研究还有可能扩展到外切体以外的领域，以检测和研究包括病毒在内的其他小的膜结合囊泡。因此，这种高通量检测工具可用于广泛的医疗诊断和生物医学研究，通过向患者提供频繁、负担得起的早期测试，可能潜在地降低医疗保健成本。此外，这个多学科项目跨越了工程学、物理学和生物医学之间的传统界限，将为学生在学术培训期间提供独特的教育体验。本科生将通过与研究生密切合作，并在首席研究员的指导下，接受微纳技术和生物医学领域的培训和接触。拟议的项目还将通过YouTube频道和将在俄亥俄州西南部播出的“ThinkTV”节目，教育公众有关医学微纳米技术领域的重要性。胞外体是直径约30-120 nm的胞外囊泡，从多种细胞类型释放到胞外空间。它们由含有各种受体的脂质双层膜和Tetraspanin蛋白组成。它们还将核酸、蛋白质和脂类包裹在腔内。Exosome是有前景的生物标志物，原因有几个：1)它们在所有体液中高度丰富，因此很容易获得；2)它们的组成反映了它们的细胞起源，因此可以作为病理学指标；以及3)它们是稳定的。此外，已有研究表明，从不同细胞来源分泌的外切体，特别是致病外切体，会经历成分变化，并可能具有额外的膜受体和/或与其总电荷和偶极相关的核酸水平的升高或抑制。然而，由于缺乏可行的技术来及时可靠地分离和严格表征其独特的性质，限制了将外体用作生物标志物的工作。虽然，外体的一些生物物理性质，如大小、密度和形态已经被表征，但与其独特的组成电荷相关的介电性质还没有被研究过。建议的无标签芯片上实验室设备将在一系列硼硅酸盐微管中利用可控的电动作用力，快速捕获吸管顶端的外切体，并通过测量其阻抗来表征囊泡的独特介电特性。阻抗测量将通过嵌入在吸管尖端附近的微电极阵列进行，同时在宽频谱范围(500 kHz至50 MHz)施加交流电流(AC)。在较宽的频率范围内，交流场将极化与外体结构相关的束缚电荷和非束缚电荷。阻抗测量的差异将与胞外体独特的介电性质有关，包括它们的膜电容和胞质电导。此外，还将测量来自不同细胞来源和大小分布的外切体的阻抗，并研究它们在介电性质上的独特性。这种快速且无标签的电动装置可以进一步发展为一种诊断工具，用于对致病外切体进行初始非侵入性检测，同时保持其成分的完整性，以便进行进一步的下游分析。该奖项反映了NSF的法定使命，并通过使用基金会的智力优势和更广泛的影响审查标准进行评估，被认为值得支持。

The objective of this project is to develop a rapid and accurate lab-on-a-chip device for characterization of nano-size vesicles based on their unique and intrinsic biophysical properties. The immediate focus of this project is to characterize small cell-secreted vesicles (exosomes) which are classified as circulating biomarkers associated with many types of disease including cancer, diabetes, cardiovascular, infectious, and neurodegenerative diseases. This research also has the potential to be expanded beyond exosomes to detect and study other small membrane bound vesicles including viruses. Thus, this high throughput detection tool can be utilized in a wide range of medical diagnosis and biomedical research which could potentially reduce the cost of healthcare by providing frequent, affordable, and early testing to patients. Furthermore this multidisciplinary project crosses the traditional boundaries between engineering, physics, and biomedical sciences which will provide students with a unique educational experience during their academic training. The undergraduate students will be trained and exposed to the field of micro- nanotechnology and biomedical sciences by closely working with the graduate students and being mentored by the principal investigator. The proposed project will also educate public about the importance of the field of micro-nanotechnologies for medicine through YouTube channel and “ThinkTV” program which will be broadcasting in Southwest Ohio. Exosomes are extracellular vesicles with diameters of ~30-120 nm, released from many cell types into the extracellular space. They are composed of a lipid bilayer membrane containing various receptors and tetraspanin proteins. They also encapsulate nucleic acids, proteins, and lipids in their lumen. Exosomes are promising biomarkers for several reasons: 1) they are highly abundant in all bodily fluids and therefore easily accessible; 2) their composition reflects their cellular origins and can therefore serve as indicators of pathology; and 3) they are stable. Also, it has been shown that exosomes secreted from different cellular origins, in particular pathogenic exosomes, undergo compositional changes and could have additional membrane receptors and/or elevated or suppressed levels of nucleic acids which can be associated with their total electric charges and dipoles. However, use of exosomes as biomarkers has been hampered by the lack of workable technologies to reliably isolate and rigorously characterize their unique properties in a timely manner. Although, some of the biophysical properties of exosomes such as size, density and morphology have been characterized before, their dielectric property which is associated with their unique compositional charges has not yet been investigated. The proposed label-free lab-on-a-chip device will utilize controllable electrokinetic forces across an array of borosilicate micropipettes to rapidly entrap exosomes at the tip of the pipettes and characterize the vesicles based on their unique dielectric properties by measuring their impedance. The impedance measurement will be conducted across an array of microelectrodes embedded in close proximity to the pipettes’ tips as an alternative current (AC) is applied at a wide range of frequency spectrum (500 KHz to 50 MHz). The AC field at a wide frequency range will polarize the bound and unbound charges associate with exosomes’ structure. The difference in impedance measurements will be linked to exosomes’ unique dielectric properties which includes their membrane capacitance and cytosolic conductance. Additionally, the impedance of exosomes secreted from different cellular origins and size distribution will be measured and their uniqueness in dielectric properties will be investigated. This rapid and label-free electrokinetic device can be further evolved as a diagnostic tool for initial non-invasive detection of pathogenic exosomes while keeping their compositions intact for further downstream analysisThis award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.