In this study, a novel solid lift-off method using an ingeniously designed micropore array as a shadow mask was proposed. Efficient and precise control of cell alignment and spreading simultaneously was realized via an ingenious composite-confining structure design, with large micropores (capture pores) in central areas and small micropores (spreading pores) in surrounding areas dominating capture/alignment and adhesion/spreading, respectively.
High-throughput (2.4−3.2×104/cm2) cell patterning were achieved with high efficiencies (86.2 ± 3.2%, 56.7 ± 9.4% and 51.1 ± 4.0% for single-cell, double-cell, and triple-cell, respectively). Precise control of spreading and application toward regulating cell skeleton and cell-cell junction distribution was investigated and verified using murine skeletal muscle myoblasts. To our knowledge, this is the first report to demonstrate highly efficient and controllable multi-cell alignment and adhesion/spreading simultaneously via a simple single-step solid lift-off operation. Notably, this study successfully fills a gap in the literature and promotes the effective and reproducible application of cell patterning methodologies. This work will attract extensive interests in the fields of both basic cellular mechanism research and applied medicine including organ-on-a-chip, tissue engineering, etc.
This work realizes a rapid liquid biopsy of lung cancer by separation and detection of exfoliated tumor cells from bronchoalveolar lavage fluid (BALF) with a dual-layer PERFECT (precise, efficient, rapid, flexible, easy-to-operate, controllable and thin) filter system. A preliminary clinical trial enrolling 33 patients has been performed to verify the efficacy of the developed system.
The novel system possesses the following breakthroughs and advantages.
This system enables a short time-to-result (<30 min), benefiting from the handling ability of large-volume clinical BALF samples (6-18 mL) of complex contents at a high filtration throughput (54.6% BALF specimens were filtrated within 3 min, and others could be finished completely within 10 min).
A high detection sensitivity (80.0%) has been demonstrated, significantly higher than that from the conventional cytocentrifuge (45%). The sensitivity of this platform is neither interfered by the variations of turbidity of the BALF samples, nor associated with the types of lung cancer.
This system is compatible with various downstream analysis platforms such as immunofluorescence/immunocytochemistry staining, FISH test, PCR, sequencing and even the AI-based automatic diagnosis, which will fulfill both clinical practices and advanced mechanism studies.
The handiness in system set-up, ease of operation, wide adaptability and readiness for integration with related systems, will facilitate the developed system to find powerful applications in clinical practices, such as efficient separation and detection of rare tumor cells, fungi and bacteria from blood, urine, sputum etc.
Tingyu Li#, Yaoping Liu#, Wei Zhang, Jixin Zhang, Yan Xiong, Ligong Nie, Haichao Li* and Wei Wang*, A rapid liquid biopsy of lung cancer by separation and detection of exfoliated tumor cells from bronchoalveolar lavage fluid with a dual-layer “PERFECT” filter system, Theranostics, 2020; 10(14): 6517-6529.
1. Tingyu and Yaoping’s work about PVA-Functionalized filter for efficient cell recovery and release
This work reports a Polyvinyl Alcohol (PVA)-functionalized filter based on an easy spin-coating process for effective cell capture and release. The PVA-functionalized filter (10-μm-diagonal-micorpore) realized a high recovery rate (82.7±6.4%) of cell separation from a large-volume liquid sample (105 A549 cells spiked in 5 mL PBS) by a gravity-driven filtration in a few seconds. And an easily-operated and rapid (soaking in PBS for 3 minutes) cell release was realized at a high release rate (96.2±8.5%), along with a high cell viability (92.7±0.7% verified via the trypan blue staining), which is critical for further downstream analysis. These results demonstrate the proposed PVA-functionalized filter will find promising applicability in target cell separation for liquid biopsy.
2. Yaoping and Meixuan’s work about Kirigami-inspired mesh for rare cell recovery
This work proposes a Kirigami-inspired mesh to achieve an effective recovery of rare cell from whole blood. The shape and size of the micropores were well programmed via careful design of the geometric size of the Kirigami structure and accurate control of the strain loading of stretch. Two different shaped (diamond and hexagon) micropore arrays were successfully realized through the stretching operation. The applicability in rare tumor target retrieval was verified and the recovery rate of rare spiked cancer cell from undiluted whole blood reachedupto81.2±15.2%(67%strainfordiamond-shapedmicropore) at a high throughput (>18mL/(min∙cm2)).
3. Tingting and Yaoping’s work about a circulating filtration system for cell recovery
Filtration has been widely known as a promising approach to effectively achieve rare target cell recovery from large-volume samples at a high throughput for liquid biopsy. This paper proposes a circulating filtration system to achieve the recovery of rare target cells at a high efficiency along with a high purity.
4. Wenbo and Yaoping’s work about rapid separation and detection of rare fungi spores from whole blood based on a dual-layer micropore array filtration
This work reports a novel dual-layer filtration system including two membranes with different micropore diameters for rapid separation of fungi spores from undiluted whole blood. The upper membrane is for the depletion of white blood cells (WBCs) and the lower filtration membrane is for the recovery of targeted spores while depleting red blood cells (RBCs). The results demonstrated a rapid spore separation with high recovery rates (82.47%±10.90% from undiluted whole blood with rare spiked spores as targets), along with low blood cell contamination. The proposed system will be a promising point-of-care detection (POCD) tool for fungemia.
5. Yaoping’s work about large-scale nanopore array based on a cost-effective shrinkage process for nanosized target separation
This work proposes a cost-effective shrinkage-based process for large-scale Parylene-C nanopore array fabrication from a prepared micropore array. The diameter of nanopore was well controlled via tuning the shrinkage thickness. Large-scale (>1 cm2) and uniformly-distributed nanopore arrays with different diameters (100‒900 nm) and only a small thickness (<8 μm) are successfully achieved. The prepared nanopore array was packaged with a home-designed gadget for nanoparticle recovery (104 in 1 mL PBS) through a filtration driven by a centrifugation (200 rpm@1 min). A good performance in nanoparticle recovery is well demonstrated, which indicates the promising applicability in exosome separation for liquid biopsy.
Congratulations! Yaoping’s work “A high-throughput liquid biopsy platform based on a 2.5D micropore-arrayed filtration membrane” is featured in a recent issue of Lab on a Chip! This technique will enable a powerful system for practical clinical applications of liquid biopsy.
Cover Art – A high-throughput liquid biopsy platform with a high recovery rate and a high cell viability is realized based on a 2.5D micropore-arrayed filtration membrane.
This article is part of the themed collection: Personalised Medicine: Liquid Biopsy.
Lingqian Zhang, Xiaolong Rao, Yudan Pi, Xiaofan Zhao, Yuanjian Wu, Haida Li, Yechang Guo and Han Xu graduated from Wangwei Group. We held the farewell party on July 8, 2018.
Group photo of the graduation partyGraduates and prof. Wei Wang at the party. From left to right: Yechang Guo, Yuanjian Wu, Yudan Pi (front), Haida Li, prof. Wei Wang, Lingqian Zhang, Xiaolong Rao, Han Xu.From left to right: Han Xu, Haida Li, Lingqian Zhang, prof. Wei Wang, Yudan Pi, Yuanjian Wu, Yechang GuoGroup photo of our members
To the graduates:
there is life after graduation.
And put forth your best effort in everything you do.
Dissertation defense meeting for Ph.D. candidates from EECS, Peking University was held in Micro/Namo Electronics Building on May 31st, 2018. Lingqian Zhang directed by prof. Zhihong Li and prof. Wei Wang has passed her dissertation defense.
Defense meeting committee includes Prof. Shanhong Xia as chairman, Prof. Chengjun Huang, Prof. Chen Zhang, Prof. Dacheng Zhang, Prof. Wengang Wu, Prof. Zhihong Li and Prof. Wei Wang as secretary.