GHz Frequency Microfluidics-Integrated CMOS Platform for Label-Free Cellular Dielectric Spectroscopy

Label-free cell differentiation and sorting based on intrinsic physical properties of cells are essential techniques in cellular biology and medical research, which moves away from traditional methods that often require chemical dyes, antibodies, or genetic modification to identify and sort specific cell populations. This label-free approach leverages the inherent physical characteristics of cells such as size, density, electrical properties, and deformability to distinguish between different cell types. This methodology not only reduces potential perturbations to the cells but also simplifies the sorting process and can be more cost-effective and faster than conventional techniques. Microfluidics can play a pivotal role in label-free cell sorting, using channels smaller than a hair’s width to manipulate and sort cells based on size and deformability. Cells can be sorted when they pass through these channels based on how easily they deform, allowing for separation without the need for labels. Dielectrophoresis is another label-free technique that employs non-uniform electric fields to induce movement in particles depending on their dielectric properties. Cells with different dielectric properties can be separated effectively using dielectrophoresis, providing a powerful tool for label-free cell sorting. Further research to advance label-free cell sorting methods will make the technology become even more precise, efficient, and integrated into various research and clinical workflows. The development of new materials and microfluidic designs, alongside improvements in computational methods for data analysis, will further enhance the capabilities of label-free cell sorting, opening up new avenues for biological discovery and therapeutic development. To bridge the gap between traditional cytometry techniques and the need for high-throughput, detailed cellular analysis without the requirement for external labels that may alter cell behavior, a study published in Lab on Chip and conducted by Professor Jun-Chau Chien, Ali Ameri, Erh-Chia Yeh, Alison N Killilea, Professor Mekhail Anwar, and Professor Ali Niknejad from the University of California at Berkeley, the researchers developed microfluidics-integrated, label-free flow cytometry-on-a-CMOS platform, which operates at GHz frequencies for the characterization of cellular dielectric properties. The experiments were designed to demonstrate the platform’s ability to penetrate the cellular membrane with high-frequency electric fields, allowing for direct probing of intracellular permittivity, a crucial advancement over traditional MHz impedance cytometry methods. The heart of their innovative approach is in its employment of GHz frequencies for impedance cytometry, as opposed to the more common MHz frequencies. This shift to higher frequencies enables direct probing of intracellular permittivity by allowing the electric fields to penetrate the cellular membrane, a crucial step forward since the internal properties of cells hold valuable information for distinguishing cell types, states, and health. By overcoming the challenge of detection at such high frequencies through the use of on-chip oscillator-based sensors, the team has enabled the sensitive, direct measurement of cell dielectric properties, a capability not feasibly attainable with lower-frequency systems. The complexity of the system’s design are noteworthy. It incorporated an array of on-chip LC oscillators operating at several different frequencies from 6 to 30 GHz, alongside a phase-detection technique known as injection locking for sensitive dielectric detection. This design enabled simultaneous frequency generation, electrode excitation, and signal detection using a small silicon area and also significantly enhances the sensitivity of the spectrometer.

The team evaluated the spectrometer’s sensitivity by testing its ability to detect capacitance changes with a resolution of less than 1 aFrms or a 5 ppm frequency shift, at a noise filtering bandwidth of 100 kHz. This was crucial for validating the platform’s capability to differentiate minute variations in cellular properties. They assessed the throughput by measuring the system’s ability to analyze over 1,000 cells per second, providing a quantitative basis for its high-throughput capability. The measured pulses induced by the passage of the cells across the CMOS sensing area shows a signal-to-noise ratio exceeding 28 dB. This marked a significant advancement over traditional methods utilizing bulky microwave instrumentations, and enabled more precise and faster cellular analysis. Moreover, they developed a novel epoxy-molding technique to address the size mismatch between microfluidic components and CMOS, facilitating seamless integration at low cost. It is worth mentioning the work also incorporated 3-D hydrodynamic focusing microfluidics, allowing for precise manipulation of the cell position within the fluidic channel to ensure consistent and repeatable data. They used the platform to characterize four different cell lines, including two breast cell lines (MCF-10A and MDA-MB-231) and two leukocyte cell lines (K-562 and THP-1). The dielectric properties were measured at selected GHz frequencies (6.5, 11, 17.5, and 30 GHz) to explore the unique dielectric properties of each cell type. Measurements were normalized at the higher frequencies to the 6.5 GHz data to account for size-independent dielectric opacity, which facilitated a clearer differentiation between cell lines based on their dielectric properties. The designed platform revealed distinguishable dielectric properties between the tested cell lines. Notably, differences in dielectric opacity at 17.5 GHz indicated distinct permittivity dispersions between normal and highly metastatic breast cells, which suggests potential applications in cancer diagnostics. The findings showed that cells’ dielectric properties at GHz frequencies could provide new biomarkers for cellular differentiation, which offers advantage over previously unattainable with lower frequency methods, suggesting potential applications in cancer diagnosis and research.

In conclusion, the study conducted by Professor Jun-Chau Chien and colleagues represents a pioneering effort in the field of cytometry. The successful development of a microfluidics-integrated, label-free flow cytometry-on-a-CMOS platform operating at GHz frequencies that overcomes existing limitations in cellular analysis and also heralds a new chapter in the exploration of cellular properties. Importantly, the team utilizes modern semiconductor technology to significantly miniaturize the detecting electronics into a millimeter-sized CMOS chip. These CMOS chips, especially when mass-produced, is extremely low cost. Yet, with innovative circuit and system engineering, the detection circuits can outperform those bulky and expensive benchtop instrument, all in a handheld platform, applicable to the point-of-care applications. Through deep understanding of both the biological and technical challenges, this work significantly enhances our ability to analyze cells in their native state, paving the way for groundbreaking advancements in medical research and diagnostic technologies. The findings underscore the significance of exploring cellular dielectric properties at higher frequencies for improved cell characterization, opening new avenues for research and medical diagnostics.