A vacuum-assisted, highly parallelized microfluidic array for performing multi-step digital assays


Advanced microfluidic-based digital technology that precisely manipulates nanoliter-sized droplets to perform complex laboratory protocols. This innovation enables the elimination of most manual steps has significantly transformed the conventional methods for single-molecule and single-cell quantification, resulting in higher throughput, sensitivity and accuracy. The microfluidic devices can compartmentalize numerous single molecules or single cells to generate binary readouts for absolute quantification. Based on the compartmentalization methods, these microfluidic platforms can be divided into two, namely, droplet microfluidics that discretizes aqueous solution into numerous water-in-oil emulsions and arrays microfluidic that use microfluidic arrays to generate nanoliter-sized liquid used in digital assays. Although these two approaches are commonly used in various digital assays, they are limited to one-step reactions, making them unsuitable for biochemical assays requiring a multi-step and multi-reagent reaction.

To this end, significant research efforts have been devoted to developing microfluidic platforms, such as SlipChip, printed-based platforms, to perform multi-step reactions and multi-reagent additions. Nevertheless, these techniques have two major drawbacks that limit their practical application. The first is the high engineering complexity involved in their design and development, and the second is their limited analytical throughput capabilities. Thus, there is an urgent need for facile microfluidic platforms for performing parallelized digital biochemical assays with multi-step and multi-reagent capabilities for high-throughput detection and analysis of thousands of single cells and molecules.

Herein, a team of researchers from Johns Hopkins University: Jiumei Hu, Liben Chen, Dr. Pengfei Zhang, Kuangwen Hsieh, Hui Li and Professor Tza-Huei “Jeff” Wang in collaboration with Professor Samuel Yang from Stanford University developed a feasible and high-density microfluid device for multi-reagent and multi-step digital assays. The outlet-free microarray device comprised multi-level bifurcated microchannels connected to 4096 dead-end microchambers. Most importantly, the device utilized a vacuum-assisted loading mechanism driven by the PDMS permeability. Their work is currently published in the journal, Lab on a Chip.

The research team designed the multi-step repetitive liquid sample loading by attaching an external vacuum onto the newly designed suction layer to create a negative pressure to generate the desirable liquid driving force continuously. The high device uniformity was demonstrated by sequentially loading the liquid reagents/samples until all the microchambers were filled. The device utility was improved by sealing the top of the gas-permeable PDMS using a newly developed thermosetting oil-based coverslip to prevent evaporation of high-temperature based assays. Finally, to validate the feasibility and effectiveness of the proposed approach, a digital polymerase chain reaction (PCR) detection for single-cell methicillin-resistant Staphylococcus aureus was carried out using a three-step loading involving single-cell distribution, cell lysis and buffer digitization.

The authors showed that the bifurcated microchannels connected to the dead-end chamber facilitated the partitioning of the liquid reagents, allowing for the implementation of thousands of parallel reactions. It also supported the efficient bacteria lysis through the multi-loading workflow to facilitate highly-sensitive single-cell detection. As a result, the proposed microfluidic device achieved a highly parallelized single-molecule and single-cell detection with multiple loading steps and with high sensitivity, high throughput and high accuracy that was otherwise difficult to obtain using current existing microarrays. Compared with the conventional multi-step loading platforms, the proposed device was more facile and it achieved high-throughput digital assays across 4096 microchambers. Furthermore, it was worth noting that the number of microchambers and the loading steps could be scaled up.

In summary, Johns Hopkins University researchers successfully developed a PDMS-based vacuum-driven microfluidic array capable of performing highly parallelized multi-step and multi-reagent digital biological assays. The device was very flexible in performing digital assays that require multi-step and multi-reagent reactions for both single-molecule and single-cell analysis with high sensitivity and throughput. It also showed that single bacterial cells could be detected and precisely quantified. In a statement to Medicine Innovates, Professor Tza-Huei Wang explained that the new device is versatile and its application scope could be expanded to other applications such enzymatic reactions.

About the author

Dr. Tza-Huei “Jeff” Wang is Louis M. Sardella Professor in Whiting School of Engineering at Johns Hopkins University. His research focuses on the development of new technologies for molecular analysis and biomedical research via advances in micro- and nano-scale sciences. He has contributed to developments in single-molecule fluorescence spectroscopy, microfluidics, and nano-biosensors for genetic and epigenetic biomarker-based diagnostics of cancer, infectious disease, and an array of other diseases. Dr. Wang is an inventor of 25 patents and has authored 170 peer-reviewed articles and delivered 130 invited talks. He is a Fellow of the American Institute for Medical and Biological Engineering (AIMBE), American Society of Mechanical Engineering (ASME), Institute of Electrical and Electronics Engineers (IEEE), and Royal Society of Chemistry (ROC).

About the author

Dr. Liben Chen is a Research Scientist in the Department of Mechanical Engineering at the Johns Hopkins University Whiting School of Engineering. His research focuses on developing point-of-care tests for the diagnosis of infectious diseases. Dr. Chen holds 4 patents and has authored over 70 peer-reviewed articles.


Hu, J., Chen, L., Zhang, P., Hsieh, K., Li, H., Yang, S., & Wang, T. (2021). A vacuum-assisted, highly parallelized microfluidic array for performing multi-step digital assaysLab On a Chip, 21(23), 4716-4724.

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