Development of Nano-Micro Fused LSPR Chip for In Situ Single-Cell Secretion Analysis.

Single-cell analysis has become increasingly important in uncovering cell heterogeneity, which has great implications in medicine and biology for a deep understanding of cell characteristics. Owing to its significance, it is vital to create novel devices that can reveal special or unique cells. In this work, we developed a single-cell secretion detection chip consisting of microwells that can trap single cells. Each well is surrounded by Au nanopillars capable of localized surface plasmon resonance (LSPR) measurement. Using microfabrication and nanofabrication techniques, Au nanopillar and microwell structures were fabricated on a COP film. The Au nanopillar was modified with IL-6 antibodies for the direct detection of single-cell secreted IL-6 via LSPR absorbance peak shift. Specific IL-6 detection was successfully demonstrated using a null and IL-6 oversecreting Jurkat cell. A high single-cell trapping efficiency of over 80% was also achieved. Overall, the development of this single-cell secretion detection chip with a simple LSPR measurement setup represents a significant development in the field of cell biology and immunology, providing researchers with a powerful tool for studying individual cells and their secreted cytokines, and is useful for point-of-care testing (POCT) diagnostics.

Linking antigen specific T-cell dynamics in a microfluidic chip to single cell transcription patterns.

A new non-invasive screening profile has been realized that can aid in determining T-cell activation state at single-cell level. Production of activated T-cells with good specificity and stable proliferation is greatly beneficial for advancing adoptive immunotherapy as innate immunological cells are not effective in recognizing and eliminating cancer as expected. The screening method is realized by relating intracellular Ca2+ intensity and motility of T-cells interacting with APC (Antigen Presenting Cells) in a microfluidic chip. The system is tested using APC pulsed with OVA257-264 peptide and its modified affinities (N4, Q4, T4 and V4), and the T-cells from OT-1 mice. In addition, single cell RNA sequencing reveals the activation states of the cells and the clusters from the derived profiles can be indicative of the T-cell activation state. The presented system here can be versatile for a comprehensive application to proceed with T-cell-based immunotherapy and screen the antigen-specific T-cells with excellent efficiency and high proliferation.

Deskilled and Rapid Drug-Resistant Gene Detection by Centrifugal Force-Assisted Thermal Convection PCR Device.

Here we report the improved Cyclo olefin polymer (COP) microfluidic chip and polymerase chain reaction (PCR) amplification system for point-of-care testing (POCT) in rapid detection of Carbapenem-resistant Enterobacteriaceae (CRE). The PCR solution and thermal cycling is controlled by the relative gravitational acceleration (7G) only and is expected to pose minimal problem in operation by non-expert users. Detection is based on identifying the presence of carbapenemase encoding gene through the corresponding fluorescence signal after amplification. For preliminary tests, the device has been demonstrated to detect blaIMP-6 from patients stool samples. From the prepared samples, 96.4 fg/µL was detected with good certainty within 15 min (~106 thermocycles,) which is significantly faster than the conventional culture plate method. Moreover, the device is expected to detect other target genes in parallel as determination of the presence of blaNDM-1 and blaOXA-23 from control samples has also been demonstrated. With the rising threat of drug-resistant bacteria in global healthcare, this technology can greatly aid the health sector by enabling the appropriate use of antibiotics, accelerating the treatment of carriers, and suppressing the spread.

Au-Capped Nanopillar Immobilized with a Length-Controlled Glycopolymer for Immune-Related Protein Detection.

A Au-capped nanopillar chip was prepared using nanoimprint lithography (NIL) and Au sputtering onto a cyclo-olefin polymer film. The Au surface of the chip exerting localized surface plasmon resonance (LSPR) phenomena was immobilized with a glycopolymer for the detection of cytokines. The glycopolymers were synthesized by reversible addition-fragmentation chain transfer (RAFT) polymerization for controlled polymer chain length, and thiol-terminated glycopolymers with chain lengths of 20-, 100-, and 200-mers were designed. The thickness of the biomolecular layer on the Au surface was controlled by changing the polymer chain length of the immobilized glycopolymer, and the absorption of proteins onto the Au surface was detected by the shift of absorbance peak wavelength. The value of absorbance peak wavelength shift by protein adsorption increased as the glycopolymer layer thickness became thinner. This difference in LSPR signal response was remarkable for cytokine recognition compared to larger proteins. It was shown that controlling the biomolecular layer thickness was effective for the detection of small proteins, and our research suggested the usefulness of the controlled glycopolymer surface as a molecular recognition material for cytokine detection.

Real-Time Monitoring and Detection of Single-Cell Level Cytokine Secretion Using LSPR Technology.

Cytokine secretion researches have been a main focus of studies among the scientists in the recent decades for its outstanding contribution to clinical diagnostics. Localized surface plasmon resonance (LSPR) technology is one of the conventional methods utilized to analyze these issues, as it could provide fast, label-free and real-time monitoring of biomolecule binding events. However, numerous LSPR-based biosensors in the past are usually utilized to monitor the average performance of cell groups rather than single cells. Meanwhile, the complicated sensor structures will lead to the fabrication and economic budget problems. Thus, in this paper, we report a simple synergistic integration of the cell trapping of microwell chip and gold-capped nanopillar-structured cyclo-olefin-polymer (COP) film for single cell level Interleukin 6 (IL-6) detection. Here, in-situ cytokine secreted from the trapped cell can be directly observed and analyzed through the peak red-shift in the transmittance spectrum. The fabricated device also shows the potential to conduct the real-time monitoring which would greatly help us identify the viability and biological variation of the tested single cell.

Centrifugation-Controlled Thermal Convection and Its Application to Rapid Microfluidic Polymerase Chain Reaction Devices.

Here, we report the developed cyclo olefin polymer (COP) microfluidic chip on a fabricated rotating heater stage that utilizes centrifugation-assisted thermal cycle in a ring-structured microchannel for polymerase chain reaction (PCR). The PCR solution could be driven by thermal convection and continuously exchanged high/low temperatures in a ring structured microchannel without the use of typical syringe pump. More importantly, the flow rate was controlled by the relative gravitational acceleration only. The platform enables amplification within 10 min at 5G and has a detection limit of 70.5 pg/channel DNA concentration (ß-actin, 295 bp). The current rotating system is capable of testing four different samples in parallel. The microfluidic chip can be preloaded with the PCR premix solution for on-site utility, and, with all of the features integrated to the system, the test can be conducted without the need for specialized laboratory and trained laboratory staff. In addition, this innovative chemical reaction technique has the potential to be utilized in other micromixing applications.