# Single-Cell Patterning Based on Immunocapture and a Surface Modified Substrate
## 1 Introduction  
Miniature analyzing systems have made significant progress with the rapid development of microelectronics and biomedical technologies [1; 2]. Compared to traditional biomedical technologies, they have the advantages of high sensitivity, high throughput, rapidity, and low cost, indicating usefulness in in vitro tests and diagnoses [3; 4]. Based on micropatterning technology, one can manipulate and detect objects on the molecular level, through which the biological responses and biochemical reactions can be analyzed in more detail, thus promoting the development of modern biomedical technologies [5; 6; 7].  
Traditional cell detection methods typically involve a large number of cells, and the signal measured is the average information of such cells, neglecting the differences among different cells. Relevant biomedical research indicates that a considerable number of diseases, including leukemia, liver cancer, and Alzheimer's, are caused by a minute number of pathogenic cell subpopulation [8; 9; 10]. Such important cell information is often overlooked in traditional cell detection methods, and even leads to some missing or wrong diagnoses [11; 12]. Therefore, the heterogeneities among single-cell and small cell groups are crucial in such cases. Single-cell capture and separation for further single-cellanalysis and research is paramount to modern biomedical research and clinical diagnoses [13; 14; 15]. Combining it with different nanoprobes, we can investigate the cells' heterogeneities in real time, based on intracellular changes on the molecular level [16; 17; 18; 19].  
Cell patterning technologies can be divided into top-down methods, such as photolithography [20; 21], soft lithography [22; 23], and jet patterning [24], and bottom-up methods, such as molecular self-assembly technology [25; 26]. Other approaches were also involved in cell patterning [27; 28; 29]. Currently, micropatterning technology is typically achieved by a combination of such methods, instead of a particular method [30; 31]. Meanwhile, cell patterning can also provide new choices in relative analyzing systems, which has been an active area in recent years. In 2016, a digital microfluidic programmable stencil (dMPS) was invented for protein and cell patterning. As the authors said, patterning biomolecules and cells on substrates is usually a prerequisite for biological analysis and cell studies [32]. In 2017, a novel cell patterning method was developed for freeform micropatterning of living cells into a cell culture medium using direct inkjet printing [33]. In 2018, tape-assisted photolithographic-free microfluidic chip cell patterning was studied for a tumor metastasis study. It was pointed out that micropatterning is one of the most efficient ways to study tumor development, because it can tune the distribution of cells with spatial and temporal control [34]. Also in 2018, a single cell patterning method was proposed for high throughput subcellular toxicity assay, which reports a new single cell array based toxicity assay, in which cell responses at population level, single cell level, and subcellular level can be obtained simultaneously at high throughput [35].  
As a currently popular research topic, the enrichment and screening of circulating tumor cells (CTCs) is still a challenge, in which immune identification and acquisition is still the final step after microfluidic separations [36; 37; 38]. If it can be combined with single-cell patterning, more choices could be available in the following studies, which are favorable to achieve high-throughput analyses [39; 40; 41]. Immunocapture is not a totally new technology. However, it has not been effectively used in cell patterning. This paper provides an effective approach for single cell patterning based on immunocapture, including detailed procedures and parameters, which will be helpful for relevant applications.