Research

Carbon Micromachining is a carbon microfabrication technique based on the patterning of a UV sensitive epoxy resin or photoresist like SU8. SU8-50 is patterned using Photolithography to produce posts with a height of 150µ and diameter 30µ. The resulting microstructures are then pyrolysed at a temperature of 900 °C or higher. During pyrolyization, the ramp rate is maintained such that at any given time the temperature of the furnace is lower than the glass transition temperature of the crosslinked epoxy. The heat leads to the vaporization of the non-carbonaceous components of the crosslinked epoxy, with only carbon in a mixed sp2/sp3 hybridized state remaining.

Electrochemical detection/ IDEAS

Electrochemical detection was first used in the 1950s in ion chromatography and it became widespread in a short span of time. The simple procedure implementation, made it easy to detect organic and inorganic compounds as long as chemical constituents of the analyte participated in an electron exchange reaction.

In the mid 80’s, as the microfabrication industry was growing, micro-electrodes were introduced which were superior to the macro-electrodes by having a faster mass flux, higher signal to noise ratio, higher temporal and spatial resolution, etc. At the same time, different configurations of the microelectrodes such as Inter Digitated Electrode Arrays (IDEA) were introduced which were enhanced with overlapped hemispherical diffusion layers. These micro-electrodes are generally fabricated from Noble metals using lift-off process, and are defined in 2D plane.

In Bio-MEMS research group, we pioneered a new fabrication approach to make IDEA from carbon, which has a higher stability window compared to Noble metals, in the 3D realm. Our approach is to pattern the micro-electrode using a high carbon to oxygen ratio polymer such as SU-8, followed by pyrolysis. This approach results in 3D carbon based microelectrodes which are enhanced with redox cycling in which a redox species can cycle multiple times between two adjacent working electrodes before it diffuses into the bulk solution. The working electrodes are biased at reduction and oxidation potentials of the target species. This arrangement results in higher output current which ultimately improves the Limit of Detection (LOD).

Our challenge is to further optimize the geometries of the 3D carbon micro-electrodes. This requires numerical analysis of the micro-electrodes as well as driving the governing equations. We are also investigating the performance of our electrodes for end use applications such as water quality analysis, detection of heavy metals and immunoassays.

Additionally, in order to improve the performance of the carbon-IDEA we pursue two different approaches:
  1. Fabrication of higher aspect ratio electrodes: This approach requires further investigation of the correlation between the bulk concentration and output current. Higher aspect ratio electrodes may not represent a correct correlation between the bulk concentration and output current.
  2. Fabrication of suspended electrodes using electrospinning and two-photon lithography: Electrodes fabricated using this approach may suffer from smaller redox amplification; however, the capacitance is reduced due to smaller surface area which leads to higher signal to noise ratio.

Metal nanoparticle embedding into Carbon Nanofibers

Carbon has been the premiere material for most electrochemical devices because of its high thermal, mechanical and electrochemical stability and good conductivity. Recent advances in Carbon Microelectromechanical system (C-MEMS) fabrication process has addressed the challenges of machining carbon electrodes. However, the stability provided by carbon electrode is also a deterrent for their functionalization with catalysts. Many different functionalization methods for carbon have been developed over the years such as amine oxidation, diazonium reduction, nitric acid oxidation, oxygen plasma activation. However, these techniques often fall in the category for being too harsh for carbon microstructures or having poor surface coverage. Alternatively, the absorption methods have poor catalyst stability due to leeching effects. Especially, for microporous structures such as carbon nanofiber mats and cloths, maximizing the surface area coverage to fully utilize these type of electrode’s high surface area becomes even more difficult.  

Another approach that have been only explored by a few research groups, is embedding metal nanoparticles directly into the nanofibers. One of the earliest study on this type of functionalization was done by Reneker et al. who mixed an iron salt into a polyacrylonitrile (PAN) then subsequently electrospun the solution in nanofiber mats. The mats were then thermally treated in a reducing environment containing a mixture of hydrogen and nitrogen to reduce the iron salts into nanoparticles. The mats were then subsequently pyrolyzed in an inert nitrogen environment at a much higher temperature to convert the polymer fibers into carbon nanofibers. The embedded iron nanoparticles were then used as catalysts for the growth of carbon nanotubes.

Embedding nanoparticles have several advantages such as better surface coverage, improved contact with the catalysts and improved stability of catalysts (no leeching). However, the process outlined previously has a few flaws. Specifically, the process has limited control on the uniformity of the nanoparticle coverage and limited control on the size and spacing between individual nanoparticles and is not compatible with all types of metal nanoparticles; a notable exception is gold because its low melting temperature would be too low to be compatible with the subsequent pyrolysis step. To improve upon these previous work, a block-co-polymer is chosen as the carbon precursor instead of PAN. Specifically, poly(styrene-co-4-pyridine) is used because its low carbonization temperatures makes it more compatible with not only a larger range of metal nanoparticles but also more compatible with smaller nanoparticles. Furthermore, the amphiphilic nature of this block-co-polymer allows for better control over the size and spacing between each nanoparticles, allowing us to optimize these parameters for improved catalysis performances.

Nano Gap

In molecular electronics, individual molecules are integrated with the rest of the circuit by positioning them in electrode gaps of the order of the molecule size. Such nanoscale gaps (nanogaps) have emerged as important experimental platforms for the electrical characterization of molecules, capturing unique transport phenomena of both organic and inorganic materials. Most nanogaps are fabricated from metal, such as gold and platinum. An important alternative to metal-interconnects are one-dimensional carbon nanotubes (CNTs) and carbon nanofibers (CNFs), which can be used to produce nanogaps by Joule heating-induced breakdown. Carbon-based devices offer several advantages over metal electrodes, including better contacts to organic molecules due to strong C-C bonding, biocompatibility, and good electrical and thermal conductivities.

Nanogap platforms are highly dependent on the structural configuration of the gap electrodes and the substrate. In the case of CNFs and CNTs in particular, it is desired that the position and orientation of the nanowire is controlled. An approach to the fabrication of carbon nanowires is based on the electrospinning-based technique known as Electro-Mechanical Spinning (EMS), where electrified jets of polymer solutions are used to produce nanofibers while maintaining control over the position of the deposited fiber.

By merging EMS with the C-MEMS photolithography process, we can fabricate devices based on suspended CNFs featuring a stable ohmic contact between the supporting carbon walls and CNFS without the need of any further processing.  Then, by precisely applying an electrical bias to the CNF, nanogap electrodes with separations of the order of 10 nm can be obtained.

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Figure 1. Nanogaps and their dependence on the downscaling of CNFs

CD Microfluidics is a sub-field of microfluidics that deals with the behavior, precise control and manipulated of fluids in the micro-domain. CD Microfluidics takes advantage of centrifugal forces for fluid propulsion. Fluidic channels and reservoirs are embedded in a CD-like plastic substrate and the whole platform is spun on a motor in order to manipulate fluids. A whole range of fluidic functions have been designed and implemented which include valving, decanting, calibration, mixing, metering, sample splitting, and separation. Our group has developed an optimized various capabilities, including valving (capillary valve, Coriolis valve, syphon valve, serial syphon valve), reciprocal mixing, separation (such as DEP on the CD).

Those fluidic functions have been combined with analytical measurement techniques, such as optical imaging, absorbance, and fluorescence spectroscopy and mass spectrometry, to make the centrifugal platform a powerful solution for medical and clinical diagnostics and high throughput screening (HTS) in drug discovery. Applications of a compact disc (CD) based centrifuge platform include two-point calibration of an optode-based ion sensor, an automated immunoassay platform, multiple parallel screening assays, and cellular-based assays.

Advantages of the microfluidic CD
  • Multiplex
  • Automation
  • Simple
  • Compatible with wide range of samples
  • High throughput
  • Mass Producible
  • Fast Development (Rapid Prototyping)
  • Low Cost (< $1/disc)

Extreme Point of Care Diagnostics

In developing countries like India, there is an uneven distribution of doctors and specialists with most access to healthcare being concentrated in urban regions. There is a lack of basic amenities like electricity and clean water required to operate a full-fledged hospital creating a dearth of access to primary health care for the rural population. The Primary Health care system is responsible for tests such as Blood Pressure, pre and post-natal care, ECG and so on. Owing to the lack of facilities and skilled manpower, these set ups are heavily overworked. Therefore, there is an accumulation of health issues amongst the rural population forcing them to resort to secondary and tertiary healthcare which is expensive and forces the patient to travel a lot to obtain these facilities. This dictates a need for a more robust primary healthcare with a focus on point of care diagnostic devices which can function in these extreme environments.

Our research focusses on this problem and ultimately aims to develop CD Microfluidic Technology for Extreme POC (Point of Care) that could potentially replace the need for a laboratory facility in these regions by providing reliable, inexpensive and portable diagnostic tools.

IDEAs on CDs

Interdigitated Electrode Arrays (IDEAs) allow to electrochemically detect species that can undergo a redox reactions and analytes that can be linked to such species. IDEAs consist of an electrochemical setup consisting of interdigitated working electrodes which are biased at different potentials, a counter and a reference electrode. A redox species can cycle multiple times between these working electrodes before it diffuses out into the bulk, amplifying the output current and hence allowing for a lower limit of detection.

The BioMEMs group pioneered a manufacturing approach which allows for fabrication of carbon IDEAs at much cheaper cost and with viewer manufacturing steps than conventional approaches. The carbon precursor used is a negative photoresist which through pyrolisis is turned into volumetrically smaller carbon structures that are biocompatible and have a wider stability window than e.g. gold electrodes.

In order to obtain higher amplification factors our group pursues two main approaches for manufacturing IDEAs. These are increasing the aspect ratio of the IDEAs and fabricating suspended IDEAs in order to increase surface area for redox cycling. The approaches used are UV-Photolithography, Electrospinning and Two-Photon-Photolithography.

To utilize IDEAs as a sensor for affordable point of care diagnostics we are integrating them onto CD. Possible applications go towards water analysis, detection of heavy metals and immunoassays.

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Figure 1. Left: Species redox-cycling between adjacent interdigitated beams of working electrodes; Right: Principle sketch of Generator and Collector electrodes

Droplet PCR on Disc

Droplet microfluidics is a subset of the microfluidic field aimed at producing emulsions/droplets on a micron scale through a combination of fine channel geometry and immiscible fluids. Using junctions created by unique channel configurations, one fluid (i.e oil)  helps to shear off droplets of the other immiscible fluid (i.e. water) that comes into contact with it. Droplets generated through a microfluidic device can be used for a variety of applications including digital PCR, drug delivery, cell population characterization, and more broadly multiplexing of diagnostic assays.

Droplets can be generated on lateral flow devices with common geometries including the T-junction, co-flow, and flow focusing. Step emulsification is yet another droplet generation method that has more recently been investigated. Schuler al developed a variation of this method of generating droplets on a CD platform by integrating the capillary and centrifugal forces with the principle of immiscible fluids.

By expanding current droplet generation designs on the disc, the CD team is developing a sample-to-answer assay on the CD aimed at integrating droplet generation with DNA lysis and extraction and digital PCR to detect specific bacteria in wastewater samples.

 UTI Detection Assay

Antibiotic resistance is a global healthcare crisis that is forcing physicians to treat common infectious diseases with ever more potent antibiotics.  New diagnostic strategies are urgently needed for rapid pathogen identification (ID) and antimicrobial susceptibility testing (AST) to guide antibiotic selection for patients with urinary tract infection (UTI). Rapid and accurate assays that MD. David Haake from UCLA med school developed have readout times of 30 minutes for ID and 150 minutes for AST, fast enough to impact initial antibiotic selection. However, these assays require labor work by trained person, which is not capable for near-to-care situation.

Low cost centrifugal disk (CD) platform has been developed as a low cost medical detection device. Centrifugal microfluidics takes advantage of the unique forces generated on a rotating platform to manipulate biological fluids in complex sample-to-answer assays. A simple motor generates a centrifugal force which acts as a pump to propel fluid through chambers and channels milled into the disc. This mechanism allows the CD to be completely enclosed, with no external pumps or tubing required. Furthermore, automation of the assay significantly reduces the time for sample-to-answer and simplifies the user experience. It is ideally suitable for the identification and antimicrobial susceptibility test.

This project is aimed at developing a fully automated system on CD microfluidics system to perform rapid and accurate assays from sample preparation to signal detection. The fluid sequence shown below includes sample introduction, volume definition, incubation, lysis, neutralization, hybridization, detection and wash. Between each two sections there is a valve to control the biology fluidics by different mechanisms (capillary valve, hydrophobic valve, siphon valve, laser valve etc.).

The disk is supposed to spin on a spinstand with a detection camera system and a magnetic field. The alternating magnetic field drives the metal plates in the disk moving along the radius to lysis the sample. Moreover, as the motor is precise in speed control and angular positioning, it is feasible to carry out multiple assays on a single disk.

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Figure 1. Examples of MicrobeDx compact discs with integrated assay
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Figure 2. Assembly of disc highlighting the different layers
In Partnership with MicrobeDX:http://www.microbedx.com/

While stem cell therapies have been around for over 50 years, in the past two decades there has been increasing interest and research into novel stem cell therapies for modern diseases as varied as Parkinson’s, Alzheimers, and even Depression. As a result, new centers and labs have opened up to investigate ways of growing specific stem cell lineages and special physiologies.

While significant progress has been made in understanding the chemical factors involved in the growth and differentiation of stem cells, surprisingly little is known about the effects of environmental factors such as the material properties of the substrate, electrical signals, and magnetic fields. A greater understanding and control over all of the environmental factors that cause stem cells to grow and selectively differentiate would be a major advantage in helping stem cell therapy become a viable treatment option for more diseases.

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Figure 1. Flourescence microscopy of cells grown on flat carbon electrodes with magnetic stimulation. Proliferating cells are stained with Hoechst, Phosphohistonase and KI-67. Differentiaing cells are stained with Hoechst, Tuj-1, and Tyrosine Hydroxalase

The tremendous advances in control and fabrication at the micro and nano domains has the potential to solve this problem. One of these approaches uses Micro Electro Mechanical Systems (MEMS) to create smart devices and new materials, that give scientists unprecedented control over disease diagnosis and treatment. In the area of stem cell therapy, MEMS techniques can be used to develop smart stem cell culture devices that act as a completely contained environment, mediating all of the environmental properties necessary to grow and differentiate stem cells. Advantages of using MEMS include enhanced mass transport, flexible materials that change stiffness or conductivity, the ability to fabricate 3D structures that mimic the in vivo environment, and nanostructures used as supports for cells and to mediate electron transfer. Furthermore, advanced fluid handling systems can be used to automate cell culture, stimulation, and detection in one simple to operate platform.

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Figure 2.
In the Madou lab we use MEMS techniques to develop three-dimensional carbon scaffolds that enhance differentiation of stem cells toward neuronal lineages. The effects of the material structure, conductivity, and geometry on stem cell adhesion and growth are investigated.
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Figure 3.  A stem cell electrode with an attached cloning cylinder is shown in the top left. Stem cells are seeded directly onto the carbon electrode. Contact pads are coated with silver paste. Metal clips connect the contact pads to the potentiostat. The benchtop incubator is shown with electrodes inside the petri dishes. An ambient environment of 37 C and 5% CO2 gas is mainted for optimal stem cell growth. 

Furthermore, centrifugal microfluidic systems are used to automate stem cell culture and to apply different electromagnetic fields to growing cells. The CD spins in a modified benchtop incubator, where humidity, temperature, media change, and waste removal are automated and controlled.

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Figure 4. A microfluidic disc used to culture stem cells is shown. The disc sits in a benchtop incubator and includes special fluidic chambers which deliver nutrient media to the stem cell chamber (grown on carbon electrode). Contact pads are connected to a potentiostat for electrical stimulation of stem cells during growth. A Coriolis flow switching valve allows stem cell byproducts to go to either waste chamber or a detection chamber.