Replicating DNA in micro-sized convection cells

Diagram of DNA

Fig. 1 (from left to right): Illustration of polymerase chain reaction (PCR) process; DNA replication via the PCR in a convection cell. Thermocycling is executed in a cylindrical reactor geometry of height h and diameter d by imposing a temperature gradient between the top and bottom surfaces; Cylindrical transparent reactors with various geometries (d = 1–2 mm, h = 1–2 cm) are clamped between upper and lower heating plates

Microfluidics is an incredibly versatile field encompassing a host of disciplines including engineering, biotechnology, physics, chemistry, and microelectronics. One area where miniaturization has proven to be particularly impactful involves analysis of minute quantities of DNA, specifically as a molecular diagnostic technique to detect infectious and pathogenic diseases. Here, a major challenge lies in the design of portable instrumentation used to perform a key step in the analysis. This step, the polymerase chain reaction (PCR), consists of a sequence of thermally activated biochemical processes that selectively replicate well-defined sub regions within a longer DNA strand. The PCR is incredibly efficient (the number of DNA copies increases exponentially with each cycle; 2N after N cycles) and is straightforward to perform. Typically, a reagent mixture containing template DNA, primers, dNTPs, thermostable Taq polymerase enzyme, and buffering agents is dispensed into plastic reaction tubes or multiwell plates that are then inserted into a programmable thermocycling machine. This instrument has a single function: to repeatedly heat and cool the reagent mixture through 30 – 40 cycles between temperatures corresponding to denaturation of the double-stranded target DNA, annealing of primers to complimentary locations on the denatured single-stranded fragments, and enzyme catalyzed extension to synthesize the complimentary strands. However, these thermocycling machines are bulky, slow (30 – 60 minutes for 30 cycles), expensive and power hungry - limiting their application to a laboratory setting and not in the field where they are needed most.

We are working to actuate PCR by harnessing microscale convective flows such as those initiated by the buoyancy driven instability that arises when a microfluidic enclosure is heated from below (like the circulatory flow in lava lamps). By applying a static temperature gradient across an appropriately designed reactor geometry, a continuous circulatory flow can be established that will repeatedly transport PCR reagents through temperature zones associated with each stage of the reaction (Fig. 1). Thermocycling can therefore be actuated in a pseudo-isothermal manner by simply holding two opposing surfaces at fixed temperatures, eliminating the need to repeatedly heat and cool the instrument. This convective format significantly reduces the device complexity by using low-powered heaters and portable microfluidic convection cells.

The Protégé undergraduate involved in this project – depending on the student’s skill set and interest - will explore one of the different aspects of convective PCR setup, including (i) 3D printing and analyzing different reactor geometries (cylindrical and looped geometries), (ii) smartphone-based optical detection of PCR products (developing smartphone based image analysis app) and (iii) computational fluid dynamics (CFD) modeling of microscale convective flows (iv) implementing efficient integrated heaters (as opposed to a heat block) for improved thermal management.

Director

Headshot of Aashish Priye

Aashish Priye

Assistant Professor, CEAS - Chemical & Env Eng

693 Rhodes Hall

513-556-3261

Our research is primarily curiosity driven, where we take an engineering and physics based approach to explore biophysical processes occurring at the micro-scale. We then harness these principles towards applications pertaining to global health, bioengineering, and educational outreach. An overview of projects are described below:

Physics of Micro-scale Flows
Fluid flow arising due to thermal gradients (thermal instability driven convective flows) is quite ubiquitous in nature (oceanic currents, cloud formation, etc.) but they can exhibit unique characteristics at the micro-scale, capable of greatly accelerating biomolecular transport and reactions. We use computational tools (Computational Fluid Dynamics) and novel experimental setups (automated microfluidic systems) to study these flow states and evaluate the conditions under which they can be harnessed to actuate biomolecular transport and assembly, accelerate DNA replication and separate cells (based on their shape and size).

Point-of-Care Detection for Global Health
The recent disease outbreaks have exposed some key limitations facing current infectious disease management strategies, particularly when applied in remote underdeveloped areas. Existing approaches are highly resource intensive, relying on dispatching specially trained personnel to isolated locations where biological samples are collected and returned to dedicated laboratories for analysis. A need therefore exists for inexpensive and robust tools that can be broadly deployed to accelerate diagnosis, enable pinpoint delivery of therapeutics, and provide real-time data to better inform decision making. We engineer simple and portable diagnostic tools (such as smartphone based DNA analyzer and lab on a drone) that can be deployed and operated outside the laboratory to address global challenges of healthcare, environmental sampling, agriculture and science outreach. Projects under this area are quite multidisciplinary and collaborative in nature.

Functional Microfluidics
Microfluidics enables large-scale automation in chemical and biological sciences, suggesting the possibility of numerous experiments performed rapidly and in parallel while consuming little reagents. This has led to the emergence of the so-called lab on chip systems, making significant strides in diverse areas ranging from grand challenges such as water purification to fundamental research such as genetic analysis. Despite significant advances, few roadblocks has hindered microfluidic systems from replacing convectional bench-top analytical tools and widely penetrate the point of care in low resource settings where they are needed most. We aim to create the next generation of microfluidic devices using rapid fabrication techniques (3D printing, micro-milling and laser cutting) that would drastically simplify the prototyping and assembly processes of microfluidics systems.

We have a few positions open for passionate postdoctoral, graduate (prospective PhD/Master’s applicants) and undergraduate students. Our research is quite multidisciplinary, involving researchers from a wide range of background including engineering (chemical, mechanical, biomedical, electrical and bioengineering), applied physics, biophysics, material science and applied mathematics. Along with frequently publishing our research, we actively explore platforms to commercialize the technologies that are developed in our lab.

If you are interested in joining our lab, please send a copy of recent CV, a brief summary of your projects and a short statement of your research interests to priyeah@uc.edu.