In the past, protein analysis has been performed in a singleplex format1
, analyzing one protein at a time, which can be a slow and time consuming process when one wants to analyze a complex biological system. For applications such as systems biology2
, biomarker discovery3
, drug discovery4
, high throughput multiplex analysis of proteins simultaneously is necessary. In systems biology, in order to gain a complete understanding of various metabolic pathways, the study of protein-protein interactions5
, protein-glycan interactions6
, protein-small molecule interactions7
, protein-nucleic acid interactions8
, nucleic acid-nucleic acid9
, and even protein-cellular interactions10
is necessary. There are various ways of performing these assays, including mass spectrometry and microarray technology. Each approach has its' own advantages and disadvantages. Mass spectrometry allows for very high throughput analysis without the need for any surface immobilization techniques, yet is expensive and can be time consuming. Array format assays allow for high throughput detection as well, however require surface immobilization, however are less time consuming and less expensive. Microarrays generally require expensive fluorescent scanners. In general, sensitivity of the assay is limited by the dynamic range and background fluorescence of the non-targeted interactions. In both mass spectrometry and microarray technology the cost of the detector is high. Fluorescent microarrays require an excitation source and a detector which scans the entire surface of the array and detects the optical signal point by point.
Microfluidics technology offers the advantage of greater sensitivity and reduction in sample volume and the amount of reagents used11
which is of great importance in protein-protein interaction studies, where the cost and time required for protein purification is high.
Here we aim to capitalize on the advantages offered by microfluidics and microarray technology. This would allow for the minimal use of reagents and high throughput analysis at the same time. Additionally, one way to reduce the cost of the detector is to perform the detection at a single point, rather than requiring scanning across the whole surface of the array.
Here we envision performing a bead-based multiplexed assay for analyzing protein-protein interactions where in a single channel an array of proteins to be studied is patterned. Similar to a protein array, the idea is to study the interaction of that array of proteins against a single protein (). For multiplexed analysis, we need to selectively elute the specifically-bound beads from each individual region one at a time for further downstream quantification and analysis. With this format, the detection of the beads will occur downstream, simply requiring a single detector for the whole system, without the need for scanning the whole surface. The challenge lies in finding a method suitable for selectively eluting beads from a desired element of the array with minimal disturbance to beads attached to other elements of the array. Effectively this requires a smart surface.
Bead-based multiplexed assay. Applying voltage V2 turns nDEP on, resulting in elution of beads from the surface of the second set of interdigitated electrodes.
The ability to manipulate the flow of fluids and bioparticles in an integrated micro total analysis system (microTAS) continues to be a challenging problem. In order to develop a true high throughput microfluidic bioanalysis platform, an important requirement is the ability to independently control the movement of fluids and various other bioparticles in an addressable manner, similar to the ability to control the movement of current in an integrated electronic circuit. To date, several well established methods have been developed, each with its' own advantages and disadvantages, including on-chip pneumatic valves 12
, various electrokinetic methods 13
, and also magnetic manipulation 14, 15
. Each of the above listed methods may be appropriate for a set of applications, while being inappropriate for others. On chip pneumatic microfluidic valves for example are very versatile and rapid and have proven quite successful, however the disadvantage is that the requirement for a large number of tubes for controlling the flow required can become a plumber's nightmare as the number of control channels becomes large. Thus, these types of valves may not be suitable for clinical applications requiring low cost handheld devices.
For our multiplexed bead-based assay, we propose the use of negative dielectrophoresis (nDEP) in conjunction with shear force, which can provide the switch-like behavior necessary to achieve this goal. At an optimal flow rate, nDEP turned on results in bead detachment, whereas when nDEP is off, the beads remain attached. Once the beads have been eluted, they can be quantified and analyzed. At this point, the viability of the proteins on the bead is no longer important, since the goal is to quantify the beads.
Over last few years the dielectrophoresis (DEP) has found use in cell capture, microfluidic pre-concentration, cell trapping, and biomolecular analysis. Computer-aided design tools are now utilized to model the electric field gradients generated by various electrode designs. Moreover, the advances in fabrication technology resulted in development of sophisticated electrode designs and microfluidic devices that are amenable to biomedical applications. Fabrication of microfluidic devices in PDMS (polydimethylsiloxane) by soft lithography offered a faster and less expensive method to create microfluidic devices 16
. Recently, DEP devices that can be deployed for manipulation and separation of biological particles were fabricated using rapid and low cost microfabrication technologies 17
Several groups have developed and characterized DEP-based platforms geared towards characterization and manipulation of biological particles and interactions. The holding force on a single-particle in dielectrophoretic trap was determined 18
. Besides, the holding forces for DEP traps on an array of interdigitated electrodes were characterized and modeled 19
, and the relationship of the geometry of the design and the magnitude of the DEP force was analyzed 20
Previously, dielectrophoretic crossover measurements and analysis were used to characterize the dielectric properties of antibody-coated submicrometer Latex spheres 21
. Also, the use of DEP in an integrated dielectrophoretic chip was reported for continuous filtering, focusing, sorting, trapping, and detection of the bioparticles 22
. In addition, based on nDEP, generated by an array of interdigitated electrodes, a rapid and separation-free assay was performed 23
. Similarly, using nDEP, in an interdigitated microarray electrode, different cell types were patterned, without any special pretreatment of a culture slide 24
. Baek et al. 25
developed an array of interdigitated electrodes to quantify chemical and biological interactions. Gadish et al. 26
used a combination of interdigitated electrodes and a chaotic mixer to achieve high throughout particle concentration and capture system. Such efforts on interdigitated electrodes illustrate the convenience of utilizing this form of geometry for a DEP system to manipulate and characterize biological particles. Thus, we chose this geometry to illustrate the microfluidic switch-like functionality of our DEP-based platform for the purpose of this work.