Multiplex single-cell droplet PCR with machine learning for detection of high-risk human papillomaviruses.

High-risk human papillomavirus (HPV) testing can significantly decline the incidence and mortality of cervical cancer. Microfluidic technology provides an effective method for accurate detection of high-risk HPV by utilizing multiplex single-cell droplet polymerase chain reaction (PCR). However, current strategies are limited by low-integration microfluidic chip, complex reagent system, expensive detection equipment and time-consuming droplet identification. Here, we developed a novel multiplex droplet PCR method that directly detected high-risk HPV sequences in single cells. A multiplex microfluidic chip integrating four flow-focusing structures was designed for one-step and parallel droplet preparation. Using single-cell droplet PCR, multi-target sequences were detected simultaneously based on a monochromatic fluorescence signal. We applied machine learning to automatically identify the large populations of single-cell droplets with 97% accuracy. HPV16, 18 and 45 sequences were sensitively detected without cross-contamination in mixed CaSki and Hela cells. The approach enables rapid and reliable detection of multi-target sequences in single cells, making it powerful for investigating cellular heterogeneity related to cancer diagnosis and treatment.

40 GHz high-efficiency Michelson interferometer modulator on a silicon-rich nitride and thin-film lithium niobate hybrid platform.

We propose and demonstrate a Michelson interferometer modulator with integrated Bragg reflectors on a silicon-rich nitride-thin-film lithium niobate hybrid platform. High-reflectivity Bragg reflectors are placed at the ends of both arms, which double the electro-optic (E-O) interaction length and reduce the velocity mismatch between the microwave and optical wave. The presented Michelson interferometer modulator achieves a measured half-wave voltage length product as low as 1.06 V cm and high-speed modulation up to 70 Gbps. A 3-dB E-O bandwidth beyond 40 GHz is also achieved, which is, to the best of our knowledge, the highest modulation bandwidth of Michelson interferometer modulators.

Portable and Battery-Powered PCR Device for DNA Amplification and Fluorescence Detection.

Polymerase chain reaction (PCR) is a technique for nucleic acid amplification, which has been widely used in molecular biology. Owing to the limitations such as large size, high power consumption, and complicated operation, PCR is only used in hospitals or research institutions. To meet the requirements of portable applications, we developed a fast, battery-powered, portable device for PCR amplification and end-point detection. The device consisted of a PCR thermal control system, PCR reaction chip, and fluorescence detection system. The PCR thermal control system was formed by a thermal control chip and external drive circuits. Thin-film heaters and resistance temperature detectors (RTDs) were fabricated on the thermal control chip and were regulated with external drive circuits. The average heating rate was 32 °C/s and the average cooling rate was 7.5 °C/s. The disposable reaction chips were fabricated using a silicon substrate, silicone rubber, and quartz plate. The fluorescence detection system consisted a complementary metal-oxide-semiconductor (CMOS) camera, an LED, and mirror units. The device was driven by a 24 V Li-ion battery. We amplified HPV16E6 genomic DNA using our device and achieved satisfactory results.

Fluorescence quantification of intracellular materials at the single-cell level by an integrated dual-well array microfluidic device.

We present an integrated microfluidic device for quantifying intracellular materials at the single-cell level. This device combines a dual-well structure and a microfluidic control system. The dual-well structure includes capture wells (20 µm in diameter) for trapping a single cell and reaction wells (200 µm in diameter) for confining reagents. The control system enables a programmable procedure for single-cell analysis. This device achieves highly efficient trapping of single cells, overcoming the Poisson distribution, while affording sufficient biochemical reagents for each isolated reactor. We successfully utilized the presented device to monitor the catalytic interaction between intracellular alkaline phosphatase enzyme and a fluorogenic substrate and to quantify the intracellular glucose concentration of a single K562 cell based on an external standard method. The results demonstrate the feasibility and convenience of our dual-well array microfluidic device as a practical single-cell research tool.

A novel dual-well array chip for efficiently trapping single-cell in large isolated micro-well without complicated accessory equipment.

Conventional cell-sized well arrays have advantages of high occupancy, simple operation, and low cost for capturing single-cells. However, they have insufficient space for including reagents required for cell treatment or analysis, which restricts the wide application of cell-sized well arrays as a single-cell research tool alone. Here, we present a novel dual-well array chip, which integrates capture-wells (20 µm in diameter) with reaction-wells (100 µm in diameter) and describe a flow method for convenient single-cell analysis requiring neither complicated infra-structure nor high expenditure, while enabling highly efficient single cell trapping (75.8%) with only 11.3% multi-cells. Briefly, the cells are first loaded into the dual-wells by gravity and then multi-cells in the reaction-wells are washed out by phosphate buffer saline. Next, biochemical reagents are loaded into reaction-wells using the scraping method and the chip is packed as a sandwich structure. We thereby successfully measured intracellular ß-galactosidase activity of K562 cells at the single-cell level. We also used computational simulations to illustrate the working principle of dual-well structure and found out a relationship between the wall shear stress distribution and the aspect ratio of the dual-well array chip which provides theoretical guidance for designing multi-wells chip for convenient single-cell analysis. Our work produced the first dual-well chip that can simultaneously provide a high occupancy rate for single cells and sufficient space for reagents, as well as being low in cost and simple to operate. We believe that the feasibility and convenience of our method will enhance its use as a practical single-cell research tool.

Polarization-independent directional coupler and polarization beam splitter based on asymmetric cross-slot waveguides.

The polarization dependence of a directional coupler (DC) based on asymmetric cross-slot waveguides is investigated. Due to structural birefringence, the coupling behaviors of the quasi-TE and quasi-TM modes in the DC vary with the waveguide geometry. A polarization-independent directional coupler (PIDC) and polarization beam splitter (PBS) are proposed by tailoring the ratio of the coupling length for quasi-TE and quasi-TM modes. The simulated results show that the coupling lengths of the designed PIDC and PBS are 8 and 28.25 µm, respectively. Both the PIDC and PBS show an insertion loss (IL) <0.7 dB on a bandwidth over 100 nm. The extinction ratios are ∼20 dB for PIDC and ∼14 dB for PBS. The fabrication-error tolerance of the practical devices is also discussed. In this study, we employ a commercial software tool for finite difference eigenmode and three-dimensional finite difference time domain methods to perform the numerical simulations.

An open-pattern droplet-in-oil planar array for single cell analysis based on sequential inkjet printing technology.

Cellular heterogeneity represents a fundamental principle of cell biology for which a readily available single-cell research tool is urgently required. Here, we present a novel method combining cell-sized well arrays with sequential inkjet printing. Briefly, K562 cells with phosphate buffer saline buffer were captured at high efficiency (74.5%) in a cell-sized well as a "primary droplet" and sealed using fluorinated oil. Then, piezoelectric inkjet printing technology was adapted to precisely inject the cell lysis buffer and the fluorogenic substrate, fluorescein-di-ß-D-galactopyranoside, as a "secondary droplet" to penetrate the sealing oil and fuse with the "primary droplet." We thereby successfully measured the intracellular ß-galactosidase activity of K562 cells at the single-cell level. Our method allows, for the first time, the ability to simultaneously accommodate the high occupancy rate of single cells and sequential addition of reagents while retaining an open structure. We believe that the feasibility and flexibility of our method will enhance its use as a universal single-cell research tool as well as accelerate the adoption of inkjet printing in the study of cellular heterogeneity.