Fabrication of antibody functionalized silk fibroin micro-well arrays using reactive inkjet printing for circulating tumour cell capture

癌症是由细胞异常生长引起的一组疾病，并有可能扩散到身体的其他部位。它一直是全世界人类健康的主要威胁，造成820万人死亡（2012年）。在英国，每年有30万新的癌症患者，有200多种癌症，其中乳腺癌、肺癌、结肠癌和前列腺癌是最常见的类型。有些癌症如前列腺癌没有早期症状，这使得预后极其困难。因此，早期发现癌症的有效工具是非常需要的，可以大大增加成功治疗的机会或延长生命。循环肿瘤细胞（CTCs）是从原发肿瘤脱落的细胞种子，在血液中循环，随后可以生长成其他肿瘤。ctc的分离和鉴定已被证明对癌症预后有效。然而，由于血液中ctc的丰度非常低（每毫升含有数百万白细胞和红细胞的血液中只有几到几十个ctc），这项任务极具挑战性。目前，CellSearch是唯一一个FDA批准的CTC捕获设备，需要特定的设备来进行分析，并且不能区分不同类型的癌症。此外，过滤膜上捕获的ctc需要转移到细胞培养板上进行计数/表征。因此，迫切需要开发高效的高通量筛选设备/方法，能够在一次检测中筛查多种癌症，并且具有优异的灵敏度。该研究设想了这一具有挑战性的任务和迫切的需求，寻求利用新兴的增材制造技术（反应性喷墨打印，RIJ）和FDA批准的生物材料（再生丝素，RSF）来制造抗体功能化的微孔阵列，用于捕获和分选ctc。RIJ的优势不仅在于计算机辅助设计（CAD），可以在预定位置精确输送1 -12升的墨水，从而制造复杂的微阵列，而且还可以通过不同的打印头交替输送不同的抗体墨水，从而使不同的微孔具有不同的癌症生物标志物抗体。该装置不仅可以有效地捕获罕见的ctc，还可以区分不同类型的癌细胞，为有效的癌症诊断提供了一个有前途的工具。该项目还扩展了使用新兴增材制造技术的知识和技能，以具有成本效益的方式制造高价值产品。因此，该项目与EPSRC的医疗技术（新型诊断工具）和未来制造业的优先主题保持一致，并将对癌症诊断和医疗器械制造业产生重大影响。

Cancer is a group of diseases caused by the abnormal growth of cells and can potentially spread to other parts of the body. It has been the major threat to human health worldwide, counting for 8.2 million deaths (2012). In UK, there are 0.3 million new cancer patients every year with more than 200 types of cancers of which breast, lung, colon and prostate cancers are the most common types. Some of the cancers such as prostate cancer have no early symptoms making the prognosis extremely difficult. Therefore, effective tools for early detection of cancer are highly desired and can greatly increase the chances for successful treatment or extend the lifetime. Circulating tumour cells (CTCs) are cell seeds that have shed from a primary tumour and circulate in the blood stream and can subsequent growth into additional tumours. Isolation and characterization of CTCs have been proved to be effective in cancer prognosis. However, this task is extremely challenging due to the significant low abundance of CTCs in blood (a few to tens in a millilitre of blood which contains millions of white and red blood cells). Currently, CellSearch is the only FDA approved CTC capture device that require specific equipment to perform the analysis and are not able to discriminate from different types of cancers. Furthermore, the captured CTCs on the filter membrane need to be transferred to a cell culture plate for enumeration/characterization. Hence, there is an urgent need to develop efficient high throughput screen devices/methods that can screen multiple cancers in one test with excellent sensitivity.The proposed research, envisaging this challenging task and urgent needs, seeks to harness the emerging additive manufacturing technology (reactive inkjet printing, RIJ) and the FDA approved biomaterial (regenerated silk fibroin, RSF) for the fabrication of antibody functionalized micro-well arrays for the capture and sorting of CTCs. The advantages of RIJ are not only the computer assisted design (CAD) that offers the precise delivery of pico-litres (1e-12 litre) of ink at predetermined locations allowing the fabrication of complex micro arrays but also the alternate delivery of different antibody inks (through different print heads) that allows the functionalization of different micro-wells with different cancer biomarker antibodies. The fabricated devices will not only allow us to efficiently capture the rare CTCs but also allow us to discriminate different types of cancer cells, providing a promising tool for efficient cancer diagnosis. The project also extends the knowledge and skills of employing emerging additive manufacturing technology for cost-effective manufacturing of high value products. Therefore, this project firmly aligns with EPSRC priority themes of Healthcare technologies (novel diagnostic tools) and Manufacturing the future and will have significant impact to cancer diagnosis and medical device manufacturing industry.