Application Cases

Introduction

Since its emergence in the 1960s, conventional flow cytometry (FCM) has been widely adopted in cell biology, immunology, oncology and related fields, benefiting from its high precision, multi-parameter analysis and high throughput. However, its drawbacks—including costly instrumentation, high reagent consumption, low cell viability, high risk of cross-contamination and complex operation—have become increasingly pronounced.Microfluidic flow cytometry (MFCM) is recognized as an effective alternative with advantages of miniaturization, low cost and high cell viability. By integrating cell focusing, fluorescence detection and sorting functions on a microfluidic chip, it offers a more powerful platform for single-cell analysis, cancer cell sorting and drug development. Nonetheless, current MFCM systems still fail to strike a sound balance among sorting throughput, cell viability, sorting efficiency and cost.Existing technical solutions all face distinct limitations: dielectrophoresis-based high-resolution single-cell printing is constrained by a throughput of 100 cells/s; surface acoustic wave-based image sorting systems depend on expensive high-speed cameras; sorters based on pulsed laser-activated bubbles require costly optical systems with rigorous calibration; and piezoelectric actuator-driven μFACS is limited by switching speed, achieving a maximum throughput of 1500 cells/s, while systems configured with high-sampling-rate FPGAs and PMTs are complex and prohibitively expensive.Therefore, developing a microfluidic cell sorting system featuring low cost, high throughput, high precision and high cell viability to meet the urgent demand for an economical and high-performance alternative to FCM in both scientific research and clinical practice has emerged as a key breakthrough direction in this field.

This work falls under the application‑oriented sub‑branch of cell sorting and flow cytometry within the broader field of microfluidics. Its core connections to microfluidics are reflected as follows: it inherits the general advantages of microfluidic technology, including precise fluid manipulation within micrometer‑scale channels, integration of multi‑functional units (focusing, detection, sorting), miniaturization, and low reagent consumption. Meanwhile, it overcomes the technical bottleneck of conventional microfluidic sorting systems, which struggle to balance throughput, precision and cost. By adopting a photon‑incremental counting‑based fluorescence detection method, the required sampling frequency is reduced from the conventional 50 kHz to 1 kHz, and the data volume is decreased by 50‑fold, which greatly simplifies system architecture and lowers hardware costs. Employing an electrostatic‑induction‑based droplet pre‑charging sorting mechanism, a sorting efficiency of 90.7 % and cell viability of 94.3 % are achieved at a droplet generation frequency of 1316 Hz. In addition, the fully‑enclosed droplet‑encapsulated chip design effectively isolates external contamination and preserves cell viability.Rather than subverting the fundamental principles of microfluidics, this study introduces innovations in detection methodology (photon‑incremental counting instead of continuous analog‑signal sampling) and sorting mechanism (electrostatic‑induction droplet charging replacing complex actuation approaches such as piezoelectricity, dielectrophoresis and surface acoustic waves). It delivers a practical, low‑cost, high‑performance and easily‑integrated pathway for microfluidic flow cytometry, advancing microfluidic technology from laboratory‑scale research toward routine biomedical applications in resource‑limited settings, including single‑cell cloning, drug screening and point‑of‑care diagnosis.

Research Direction

Photon‑incremental Counting‑based Fluorescence Detection Method,Electrostatic‑induction‑based Droplet Pre‑charging Sorting Mechanism,Verification of High‑throughput Single‑cell Droplet Encapsulation and Sorting,Droplet Manipulation and Sorting Technology for Microfluidic Chips.

Experimental objective

Droplets are charged via an electrostatic‑induction mechanism, and deflected for sorting by means of a non‑uniform electric field. The charging signal is generated by a signal generator, amplified by the ATA‑2161 power amplifier, and then applied to the charging electrode to accumulate electric charges on droplet surfaces. Under the electric field produced by the deflection electrodes, charged droplets are deflected directionally toward the target collection channel. Experiments were carried out to analyze the effects of factors including droplet generation frequency, charging voltage and pulse width on sorting accuracy, and to verify the system’s high‑viability and high‑efficiency performance in droplet sorting.

Testing equipment

Precision Syringe Pump (LSP02‑3B),Inverted Fluorescence Microscope (XDS‑600C),LED Light Source (LED‑D1‑470 nm),Photon‑counting PMT (M111),DAQ Data Acquisition Card (USB2872A‑D),Workstation (AcutEye2 M‑1000),Signal Generator (Agilent 33250A),Signal Amplifier (Aigtek ATA2161),High‑voltage DC Power Supply (DW‑N303‑1ACH2, DW‑P303‑1ACH2),Oxygen Plasma Cleaner (Harrick Scientific),Hot Plate (LabTech),High‑speed Camera,Benchtop Centrifuge

Experimental process

In this experiment, a droplet‑charging sorting test platform was constructed using a signal generator, an ATA‑2161 power amplifier, a high‑voltage DC power supply, and a high‑speed camera. The signal generator outputs charging pulse signals, which are amplified by the ATA‑2161 power amplifier and applied to the charging electrodes to realize charge accumulation on droplets. The high‑voltage DC power supply generates a deflection electric field to guide charged droplets to deflect directionally into target channels. A high‑speed camera monitors droplet generation and the sorting process, while the fluorescence detection system is employed to evaluate sorting accuracy and cell viability, so as to verify the high efficiency and high cell viability of the system.

Figure1 Schematic of the constructed microfluidic flow cell detection and sorting system.

Figure2  experimental setup.

Experimental results

Experimental results demonstrate that the electrostatic‑induction sorting system based on the ATA‑2161 power amplifier achieves high‑precision droplet manipulation through timing‑sequence regulation and electric‑field optimization. The width of the charging pulse is set to an integer multiple (n = 1‑10) of the droplet generation time (0.625 ms), which enables modulation of the number of sorted droplets per trigger (4/6/8/10). Benefiting from timing‑sequence calibration, parameter matching and structural optimization, the system delivers sub‑millisecond‑level sorting control capability, offering a reliable solution for the precise manipulation of high‑viability cells and microspheres.

Figure3 (a) Experimental results showing the effect of varying positive voltage values on the deflection of uncharged droplets while maintaining a  constant negative voltage of −500 V. (b) Experimental results showing the effect of varying charging voltage values on droplet deflection while  maintaining a constant positive voltage of 700 V and a constant negative voltage of −500 V. (c–f) Precise deflection of (c) 4, (d) 6, (e) 8 and (f) 10 droplets simultaneously. (g) Time plot of droplet deflection corresponding to charging every 10 ms (starting from the first deflected droplet). (h) Diagram of the charging deflection process for a single droplet. 

The effectiveness of the amplifier in this experiment

1. Amplify the low‑voltage charging pulses output by the signal generator to the required voltage (e.g., 250 V) to realize electrostatic‑induction charging of droplets.

2. Adjust the charging voltage by tuning the amplification factor, so as to precisely modulate the surface charge quantity of droplets and vary their deflection amplitude.

3. Combined with the setting of charging pulse width, achieve selective charging and deflection of a specific number of droplets to complete on‑demand sorting.

4. Rapidly respond to high‑frequency signals to adapt to droplet generation frequencies up to 1600 Hz and guarantee the stability of high‑speed sorting.

Application fields

Single‑cell Cloning and Single‑cell Analysis,Drug Screening and Pharmacodynamic Evaluation,Circulating Tumor Cell (CTC) Detection and Liquid Biopsy,Immunology and Cell Therapy,Regenerative Medicine and Stem Cell Research