Advances in Microfluidics and Fluorescence Detection for Homogeneous Immunoassays

September 9, 2019

In biology and medical research in areas such as the study of insulin, achieving greater temporal resolution and lower detection limits is critical. Christopher Easley, of Auburn University, and the winner of 2019 American Electrophoresis Society (AES) Mid-Career Award, is working to address this challenge.

In biology and medical research in areas such as the study of insulin, achieving greater temporal resolution and lower detection limits is critical. Christopher Easley, of Auburn University, and the winner of 2019 American Electrophoresis Society (AES) Mid-Career Award, is working to address this challenge.

One step (mix-and-read) thermofluorimetric analysis (TFA) methods have been developed to measure both small molecule second messengers, such as cyclic adenosine monophosphate (cAMP), and downstream cell-secreted hormones (protein analytes), such as insulin. When TFA is combined with differential thermal analysis of DNA melting, the discrimination of background and signal can be accomplished without any physical separation (1). By integrating an antibody–oligonucleotide based homogeneous immunoassay on-chip, high resolution temporal sampling into droplets has been combined with separation-free quantification of insulin secretion from single islets of Langerhans using direct optical readout from the droplets. Quantitative assays of glucose-stimulated insulin secretion were demonstrated at 15-second temporal resolution and detection limits as low as 10 amol per droplet (2) and these methods have subsequently been improved to 4 second resolution and applied to adipose tissue sampling. Christopher Easley, the C. Harry Knowles Professor of the Department of Chemistry and Biochemistry at Auburn University,  has developed microfluidic and fluorescence approaches to measure individual cells or groups of cells for small molecules and hormone levels yielding temporal resolution of seconds and amols per droplet detection. Easley has received the 2019 American Electrophoresis Society (AES) Mid-Career Award, to be presented at the SciX 2019 conference in October. He recently talked with us about his work

You have published multiple papers on the use of microfluidics and thermofluorimetric analysis (TFA) for a variety of applications and using different types of instrumentation. What has prompted you to investigate such a wide variety of problems using this approach? What is unique or novel about your approach to the uses and applications of microfluidics and fluorescence?

I’ll skip the standard elevator speech on microfluidics and just say that we agree with others that the platform is ideal for studying cellular systems. With that, our approach is to do our best to keep the sampling and analysis integrated onto a single chip, and optical readout is one great way to do that. Since fluorescence is highly sensitive and can be selectively targeted to the analyte of choice, we have chosen to develop small-volume, mix-and-read fluorescence assays that are compatible with microfluidic sampling. While most of our on-chip assays are ultimately isothermal, we use thermofluorimetric analysis (TFA) with a standard quantitative polymerase chain reaction (qPCR) instrument to carefully study assay properties during development. This way, we can develop assays that work best at 37 °C on-chip, where our cells and tissue are happy.

Would you explain for our readers the differences between thermofluorimetric analysis (TFA) and traditional room temperature fluorescence (RTF) that most spectroscopists are familiar with? What are the advantages of using TFA for analysis?

As I mentioned, we use TFA to study our new assays during development. Because we exploit DNA-directed assays-for example, assays with antibody-oligonucleotide (Ab-oligo) conjugates-we hypothesized and later confirmed that TFA could be used to understand the purity of the Ab-oligos and their target-dependent properties. A nice benefit here is that all a user needs is a standard qPCR instrument, and an added bonus is that the differential analysis permits background and autofluorescence removal. With that, we showed that a qPCR instrument’s software for DNA melting curves could be sort of hijacked for assaying proteins or small molecules in human serum samples or in cell lysates, effectively removing autofluorescence interferences.

You have developed a multiplexed fluidic micro-chopper device, which you refer to as the “μChopper.” In this work you developed microfluidic valves to include both automation and droplet phase-locking capabilities. The resulting micro-analytical tool is capable of complex analytical interrogation modes on sub-microliter sample volumes while also leveraging drastic noise rejection via lock-in detection. This multichannel μChopper is designed for dynamic analyses at small volume scales (3). How did you design this tool, and what are its advantages?

Our idea leveraged analytical concepts developed decades ago with optical beam choppers. We were already planning to use microfluidic droplet generators to take advantage of small-volume and high-resolution sampling from cells, and we realized that the droplets could be viewed as fluidic sample choppers. Instead of chopping a light beam, we are just chopping the fluidic sample, and that’s it. The idea works well, particularly for eliminating 1/f (where f = frequency) noise with our homogeneous immunoassays, and we can drop optical detection limits by more than 50-fold. Our most recent work in this area showed that on-chip valves can automate the process while providing even better control over the phase and frequency of droplets. We can now use valves to automate analytical methods within droplets, such as standard addition, continuous calibration, and so forth. Because it is generally simple but very useful, it’s been fun for me to watch this project develop over the years.

From your perspective what are the most exciting developments in microfluidics when combined with spectroscopy over the past five years (in terms of both sample handling, data analysis, and instrumentation)?

Limiting myself to three, I will go with the following: droplet microfluidics, 3D-printed devices, and paper fluidics. Fortunately, there are some great speakers lined up for my award session at SciX who work in these areas, so I’m very much looking forward to that session.

Droplet-based microfluidics has already impacted our understanding of biology, with advancements such as digital assays, drug screening, high throughput single-cell genetics or epigenetics, and high resolution cell sampling. Bob Kennedy (ANACHEM Award winner at SciX this year) has introduced new sample-handling methods to interface separations, droplets, and mass spectrometry. New instruments such as kilohertz droplet sorters and mergers (David Weitz, Adam Abate, Ryan Bailey) are allowing novel methods to be developed. Our group strives to make an impact in high throughput sampling for dynamic cell and tissue analysis. A number of companies such as Elveflow, Raindance Technologies, and Bio-Rad have also made significant investments in the area.

Recent advancements in 3D-printed microdevices are also very exciting. Nordin and Woolley have pioneered the high-resolution printing side of things. Michael Breadmore, Dana Spence, Scott Martin, and others have proven a variety of applications of 3D-printed devices. Earlier, I had my doubts that the printers would ever be good enough to generate usable microdevices, but I was obviously wrong!

A variety of microfluidic paper-based analytical devices (µPADs) have been developed recently, and these have exciting implications in point-of-care analysis or in low-resource settings. The Whitesides group continues to make strong impacts here. Chuck Henry’s group has made a big splash here as well. I don’t really work in this area, but I have enjoyed following the advancements.

What are some major gaps in knowledge in the fundamental physics of micro-volumes of fluids for the theory of microfluidics that you would like to see more research and development time devoted to?

There have been some excellent papers and books devoted to the physics involved in droplet microfluidics over the last 15 years or so. On the other hand, I do think there is still plenty of room to study the fundamentals of droplets and their applications from a perspective in information theory. Droplets can be considered as digital packets of information, yet there are a variety of classes of information that can be carried within droplets, not just “on” or “off” voltages like in digital circuitry. I would be very interested to see a digital-style language developed for droplet fluidics, and I think this could open new applications for all of us that were previously not envisioned.

What have been your greatest challenges in scientific discovery over your career? What is your general approach to problem solving in your scientific work?

That’s a good question, and my best answer is the following: time management. The opportunities for scientific discovery are endless, and I have been blessed to have great students and team members working with me so far. Because of my excitement for various projects, I feel that I have had the tendency to overcommit and dilute my efforts. Fortunately, I love what I do, and I am committed to continue learning and improving my approaches.

With respect to my problem-solving approach, I obviously go with the scientific method, but some important mottos in my lab are to trust the data (whether it confirms your hypothesis or not), avoid over-interpretation, confirm the results with replication, consider using different analytical techniques for confirmation, spend time reflecting on results, and collaborate with others.

What do you anticipate will be your next major area of research?

I’m really excited about the latest electrochemical methods that we’ve developed. Although I haven’t mentioned this area of our work here so far, we have built on our knowledge of free-solution, DNA-driven assays, and we have applied these ideas to electrode surfaces. It’s been fun to experiment and observe the differences and similarities between these modes of analysis. We’ve developed an electrochemical proximity assay (ECPA) that is a highly sensitive, generalizable immunoassay or aptamer-based assay for proteins. We also recently developed an on-electrode DNA nanostructure that can be used to detect small molecules, peptides, proteins, or antibodies with the same platform. We even built some custom instruments for differential current measurements and for thermally scanning the electrodes-analogous to TFA in free solution. The readers can refer to our latest papers (4,5) and stay tuned for some upcoming work in the literature. We’re definitely excited to merge this work with our microfluidic sampling systems to study cellular dynamics, but we can also apply these electrochemical methods toward point-of-care clinical applications.


  • J. Hu and C.J. Easley, Homogeneous Assays of Second Messenger Signaling and Hormone Secretion Using Thermofluorimetric Methods That Minimize Calibration Burden, Anal. Chem.89(16), 8517–8523 (2017).

  • X. Li, J. Hu, and C.J. Easley, Automated microfluidic droplet sampling with integrated, mix-and-read immunoassays to resolve endocrine tissue secretion dynamics, Lab on a Chip18(19), 2926–2935 (2018).

  • J.T. Negou, J. Hu, X. Li, and C.J. Easley, Advancement of analytical modes in a multichannel, microfluidic droplet-based sample chopper employing phase-locked detection, Anal. Methods10(28), 3436–3443 (2018).

  • J. Hu, Y. Yu, J.C. Brooks, L.A. Godwin, S. Somasundaram, F. Torabinejad, J. Kim, C. Shannon, and C.J. Easley, A Reusable Electrochemical Proximity Assay for Highly Selective, Real-Time Protein Quantitation in Biological Matrices, J. Am. Chem. Soc.136(23), 8467–8474 (2014).

  • S. Somasundaram, M.D. Holtan, and C.J. Easley, Understanding signal and background in a thermally resolved, single-branched DNA assay using square wave voltammetry, Ana. Chem.90(5), 3584–3591 (2018).

Christopher J. Easley, PhD is the C. Harry Knowles Professor in the Department of Chemistry and Biochemistry at Auburn University, USA. He is the 2019 AES Mid-Career Award winner and is an Associate Editor at Analytical Methods (Royal Society of Chemistry). He was formally an NIH Postdoctoral Fellow at Vanderbilt Medical Center (2006-08), he received his PhD in Analytical Chemistry from the University of Virginia, and his Bachelor of Science degree in Chemistry from Mississippi State University.