Seeing the invisible with microfluidic devices

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  • Is it really possible to make a lab, the size of a microchip, that can identify and profile cells so rapidly it could give a near-instant diagnosis? With current developments in microfluidics technology, pioneered by Dr Stefan H. Bossmann and Dr Christopher Culbertson at Kansas State University, it might well be. In their highly collaborative project, they have been integrating fibre optics onto microfluidic devices that will allow for single-cell detection and the potential diagnosis of diseases in these cells.

    The laboratory is the traditional home of the scientific experiment. A biology lab is often like a cell nursery, where the cells are lovingly nurtured in conditions optimal for their growth. A chemistry lab might be filled with glassware, where humans or machines mix together various liquids to synthesise new compounds or drugs. But what happens when you take a lab and shrink it down to the microscale, making it so small you could carry it around in your pocket?

    It’s a little difficult to squeeze a graduate student into a laboratory that can fit onto a microchip. However, it turns out that scaling down the size of the lab is not just a great space saver; it can actually result in faster reaction times for creating chemicals or quicker assays for identifying and analysing chemicals and cells. This isn’t due to human inefficiency either but comes from some of the weird and wonderful effects involved in microfluidics.

    A. 3D chip rendering showing the various chip components. B. When an intact cell passes under the fibre, components in it are excited by light emitted from the fibre and then light emitted from the cell is detected through the fibre. C. Image of intersection where cells moving vertically are lysed and the lysed components of the cell are injected into the right horizontal channel. D. The cell lysate is detected under this arm of the fibre optic.

    Microfluidics is all about controlling the flow of liquids on the micro-scale. Much like an engineer might build a series of dams and dykes to steer the course of a river, in microfluidics tiny channels are etched onto circuit boards, smaller than a human fingernail, through which miniscule volumes of chemicals and other liquids can flow. One use of this technology is for everyday inkjet printers, where the channels help carefully control where the ink is sprayed in the printing process. These channels can also be merged, allowing two separate chemicals to mix and react, which is why some of these microfluidic devices are sometimes known as the ‘lab-on-a-chip’.

    Scaling down the size of the lab is not just a great space saver; it can actually result in faster reaction times.

    Dr Stefan H. Bossmann and Dr Christopher Culbertson at Kansas State University are very excited by some of the possibilities that microfluidics and the lab-on-a-chip offers. In their highly interdisciplinary and collaborative project, their teams are working together to develop this technology into a miniature analysis lab. Their technology will, for the first time, offer profiling sufficiently rapid that it could be used to monitor a patient’s response to disease treatments. Such a device would also have the unprecedented ability to perform other kinds of cell analysis, in combination with the team’s work to design and synthesise new markers to help report cell activities, such as proteolytic profiles for early cancer detection.

    Laser light from the microscope objective under the chip is focused into both the microfluidic channel and the fibre optic.

    Seeing cells
    Microfluidic devices are inherently well-suited to looking at biological processes, as the micrometre channel size conveniently corresponds to the size of cells. Most cells are between 1 – 100 micrometres, with a human hair being approximately 60 micrometres thick. This means that the channels can not only be used to provide a highly controlled environment for cell growth, that is often more effective than a human-scale lab, but also to separate out different cells of different sizes.

    Light emanating from the other side of the fibre optic is used
    to excite intact cells and collect the fluorescence from these
    cells which is relayed back to the microscope objective where
    it is collected and sent to a detector.

    Many diseases are diagnosed by visually looking for deformations or abnormalities in the cell structure. For example, one of the diagnostic tests for leukaemia involves inspection under a microscope of the white blood cells taken from the patient’s bone marrow. The size and shape of the cells can indicate whether the cells are immature and not fully developed, otherwise known as lymphoblasts. Very high proportions of lymphoblasts in the bone marrow cells is one indicator of certain types of leukaemia.

    However, with the multitude of sizes and types of cell, it can be hard to differentiate them with a microscope alone. This is why it is common to use stains, which colour only specific kinds of cell, or other kinds of chemical markers that bind selectively to a cell target, and if they are illuminated and excited with a laser, the cell then glows brightly in a particular colour that acts as a flag for a certain cell type.

    Drs Bossmann and Culbertson have already succeeded at using optical fibres to integrate this light detection technology onto their lab-on-a-chip. What is unique about their design and project is off-chip placement of the optical fibre bridge: this means the chip design is not further complicated by the inclusion of the fibre. One of the big challenges with microfluidic devices is in their manufacture; making components on such small scales is difficult to do reliably and inexpensively so this is a key advantage of their design.

    The device could be able to offer sufficiently rapid profiling to monitor a patient’s response to disease triggers.

    Two 3D printed micromanipulators are used to align the fibre with both the channels and the laser light emitted from the microscope objective.

    Another unique feature of their project is creating a microfluidic device with multiple detection and excitation spots to detect the sample of interest, while still using only one laser and detector. The motivation behind this is to increase the versatility and capabilities of the device. Now with the integration of the optical fibres they can detect the intact cell before breakdown of the cell membrane as well as the components from the cell after it is lysed. Each of the excitation spots on the microchip is like a viewing window for the cell’s activities, so the greater the number of spots you have, the greater the amount of information you can obtain. And with more information, it becomes possible to better understand exactly how diseases lead to deformation and destruction of the cell.

    Counting lines
    Drs Bossmann and Culbertson want to go beyond just being able to image and identify cells. As part of this joint project, Dr Bossmann is working on the development of new biomarkers for single-cell detection. His work involves designing very bright markers, so when the cells bind a chemical marker that glows after it absorbs light from a laser, this emitted light from the cell is sufficiently intense that a single molecule in a single cell can be detected. These markers also have to be rapidly uptaken by the cell so that the detection can be done in ‘real-time’. This is important if this device will be used to reduce patient diagnosis times.

    By making it easier to see the cells, and developing highly selective markers, Drs Bossmann and Culbertson have made it possible to investigate enzymatic activity and how cytokines, small proteins that are commonly produced by cells in association with disease, behave. The more sophisticated protease detection (detection of the enzymes that break down proteins) offered by their lab-on-a-chip will make it possible to understand how the misregulation of enzyme activity leads to the development of various diseases. The large number of enzyme markers that can be monitored will allow for the detection of many possible diseases.

    The work combining optical fibres with microfluidic devices by Drs Bossmann and Culbertson will open up many new possibilities in understanding diseases at the cellular level and more tools for cell imaging and diagnosis. All of this is an important part in the development of lab-on-a-chip technology for making rapid, handheld diagnostic devices a routine part of healthcare.

    What do you most enjoy about working in a highly interdisciplinary area?
    Many of the most important discoveries in science are made at the interfaces of the various scientific disciplines. Working closely together with our graduate and undergraduate students and post-doctoral associates, we are able to focus at the boundaries between analytical and organic chemistry, molecular biology and engineering to make new and interesting discoveries that can help people and lower health care costs.

    References

    • J. Sibbitts, K. A. Sellens, S. Jia, S. A. Klasner and C. T. Culbertson, Anal. Chem., 2018, 90, 65–85
    • J. Sadeghi, D. E. W. Patabadige, A. H. Culbertson, H. Latifi and C. T. Culbertson, Lab Chip, 2017, 17, 145–155
    • D. E. W. Patabadige, J. Sadeghi, M. Kalubowilage, S. H. Bossmann, A. H. Culbertson, H. Latifi and C. T. Culbertson, Anal. Chem., 2016, 88, 9920–9925
  • Research Objectives
    The team at Kansas State University, led by Drs Stefan H. Bossmann and Christopher T. Culbertson, is developing a microfluidic device that integrates fibre optics to speed up diagnosis of multiple diseases, including some cancers.

    Funding
    NSF

    Key Partners

    • Dr Madumali Kalubowilage, Obdulia Covarrubias: design and synthesis of peptides
    • Dr Kathleen Sellens, Jay Sibbits:, design of microfluidic devices

    Collaborators

    • Prof Dr Massoud Motamedi, Bioengineering, University of Texas Medical Branch at Galveston
    • Prof Dr Allan Brasier, Institute for Clinical and Translational Research, The University of Wisconsin at Madison

    Bio
    Dr Bossmann obtained his PhD in Chemistry from the University of Saarland in Germany (1991) and then was a postdoctoral research associate at Columbia University in New York. After working as Assistant and Associate Professor of Chemical Engineering at the University of Karlsruhe, he joined Kansas State University in 2004 as Full Professor. He has more than 200 publications in refereed journals and 17 patents.

    Dr Culbertson earned his PhD in Chemistry from The University of North Carolina at Chapel Hill and then was a postdoctoral research associate and staff scientist at Oak Ridge National Laboratory. He joined Kansas State University in 2002 and has recently been promoted to Full Professor.

    Contact
    Dr Stefan Bossmann
    Professor
    Department of Chemistry
    Kansas State University
    213 CBC Building
    1212 Mid-Campus Dr North
    Manhattan, KS 66506-0401
    USA

    E: sbossman@ksu.edu
    T: +1 785 532 6817
    W:

    Dr Christopher T. Culbertson
    Professor
    Department of Chemistry
    Kansas State University
    213 CBC Building
    1212 Mid-Campus Dr North
    Manhattan, KS 66506-0401
    USA

    E: culbert@ksu.edu
    T: +1 785 532 6685
    W:

  • Seeing the invisible with microfluidic devices

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