Fostering the next generation of molecular diagnostics: 3D Microfluidic RNA-sensing hydrogel biosensors for clinical analysis of miRNA biomarkers

科学动机与生物医学和临床需求。高度保守的小的非编码microRNAs（miRNAs）通过以组织特异性方式调节其靶mRNA的翻译效率来调节基因表达，从而影响细胞生理学。在无细胞血清中鉴定的循环miRNA被认为是一系列人类疾病的非侵入性生物标志物。它们独特的表达谱代表了神经退行性疾病、心血管疾病、自身免疫性疾病和各种类型癌症的指示性分子特征。因此，特异性miRNA表达谱的检测可以用作许多重要疾病的早期检测的生物标志物。检测血清中游离循环miRNA表达水平的变化可用于区分患病患者和健康个体，并且其样品收集的非侵入性性质使其成为用于人类患者的理想生物标志物。然而，缺乏标准化的技术用于在临床样品中原位定性和定量测量miRNA水平（由于它们的小尺寸和高序列同源性）是将miRNA生物标志物推进到临床的显著瓶颈。因此，该领域的主要挑战是开发一种高灵敏度的技术，用于快速，稳健和可靠地筛选miRNA生物标志物，以供未来在临床和医疗保健中的应用。项目概要。我们最近开发了新的肽基寡核苷酸水凝胶生物传感器用于检测疾病相关的RNA序列。我们已经在原理验证水平上证明了它们在皮摩尔浓度下感知完美匹配的生物靶标并将其与单核苷酸错配区分开来的能力5。通过与曼彻斯特BIOGEL有限公司的密切合作，我们现在的目标是通过制造基于我们的肽-水凝胶生物传感器的3D RNA传感微流控设备来开发下一代分子诊断，这可以更好地适应生物和临床样品的复杂性，并允许同时筛选多个临床样品。通过在化学生物学，材料科学，结构生物学，生物聚合物化学和建筑学的界面上使用综合的跨学科方法，招募的博士生将开发基于肽-寡核苷酸水凝胶的3D微流控诊断平台，以实现直接从临床样品中快速，准确和可重复地检测miRNA，用于人类疾病的早期诊断。这个协调的跨学科项目将在化学生物学（EB），合成和分析化学（HA），肽和聚合物化学（AS），材料科学（AM）和分子诊断学（EB）的接口进行。该项目的这种广度将有助于招募的博士生发展一系列跨学科技能，并鼓励学生欣赏协作和协调的多学科方法在解决生物医学和临床重大挑战方面的价值。学生将在学术界和工业界（曼彻斯特生物凝胶有限公司）之间的接口工作，通过将新的学术发现转化为商业成功来获得知识转移的宝贵经验。曼彻斯特BIOGEL有限公司的工业实习将使学生接触到中小企业业务的激烈商业环境，并严格承诺项目交付和预算目标。这种培训将支持在学术界或工业界（例如大型制药公司或小型创新公司）的一系列职业选择，包括化学生物学，纳米技术和纳米医学，材料和分析科学以及分子诊断学。具有这些领域技能的毕业生很少;因此，这次培训将为招聘的博士生提供坚实的平台，并促进他们进一步的职业加速和发展

SCIENTIFIC MOTIVATION AND BIOMEDICAL AND CLINICAL NEED. Highly conserved small non-coding microRNAs (miRNAs) regulate gene expression by modulating translational efficacy of their target mRNAs in a tissue-specific manner, thus affecting cellular physiology. Circulating miRNAs identified in cell-free serum are recognised as non-invasive biomarkers for a range of human diseases. Their unique expression profiles represent indicative molecular signatures for neurodegenerative, cardiovascular, autoimmune diseases, and various types of cancers. The detection of specific miRNA expression profiles could therefore be used as biomarkers for the early detection of many important diseases. Detection of changes in the expression levels of free-circulating miRNAs in the blood serum can be used to distinguish diseased patients from healthy individuals, and the non-invasive nature of their sample collection makes them ideal biomarkers to employ for human patients. However, the lack of standardised technologies for qualitative and quantitative measurement of miRNA levels in situ in clinical samples (due to their small size and high sequence homology) is a significant bottleneck in advancing miRNA biomarkers to the clinic. The main challenge in this area is therefore to develop a highly-sensitive technology for the rapid, robust and reliable screening of miRNA biomarkers for future application in clinic and healthcare. PROJECT OUTLINE. We have recently developed novel peptidyl-oligonucleotide hydrogel biosensors for detection of disease relevant RNA sequences. We have demonstrated at a proof-of-principle level their ability to sense a perfect-match bio-target at picomolar concentrations and discriminate it from single-nucleotide mismatches5. In close collaboration with Manchester BIOGEL Ltd, we now aim to develop a next generation of molecular diagnostics by fabricating a 3D RNA-sensing microfluidic devices based on our peptide-hydrogel biosensor, which could better suit the complexity of biological and clinical samples and allow screening of multiple clinical samples simultaneously. By using an integrative cross-disciplinary approach at the interface of chemical biology, material sciences, structural biology, biopolymer chemistry and architecture, the recruited PhD student will develop a 3D microfluidic diagnostic platform based on peptide-oligonucleotide hydrogels to enable rapid, accurate and reproducible detection of miRNAs directly from clinical samples for early-stage diagnosis of human diseases.TRAINING PROVIDED BY UoM. This coordinated cross-disciplinary project will be carried out at the interface of chemical biology (EB), synthetic and analytical chemistry (HA), peptide and polymer chemistry (AS), material sciences (AM) and molecular diagnostics (EB). This breadth in the project will help the recruited PhD student to develop a range of interdisciplinary skills and encourage the student to appreciate the value of collaborative and coordinated multidisciplinary approaches for addressing biomedical and clinical grand challenges. The student will be working at the interface between academia and industry (Manchester BIOGEL Ltd) to obtain invaluable experience in knowledge transfer by turning new academic discoveries into commercial success. The Industrial placement at Manchester BIOGEL Ltd will expose the student to the intense commercial environment of the SME business with strict commitments to project delivery and budgetary targets. Such training will support progression into a range of career options within academia or industry (e.g. Big Pharma or small innovative companies), including chemical biology, nanotechnology and nano-medicine, material and analytical sciences and molecular diagnostics. Graduates with skills-sets spanning these areas are rare; therefore, this training will provide a solid platform for the recruited PhD student and promote their further career acceleration and development