Mehdi Javanmard

Electrical detection of biomarkers using bioactivated microfluidic channels

Current methods used for analyzing biomarkers involve expensive and time consuming techniques like the Sandwich ELISA which require lengthy incubation times, high reagent costs, and bulky optical equipment. We have developed a technique involving the use of a micro-channel with integrated electrodes, functionalized with receptors specific to target biomarkers. We have applied our biochip to the rapid electrical detection and quantification of target protein biomarkers using protein functionalized micro-channels. We successfully demonstrate detection of anti-hCG antibody, at a concentration of 1 ng ml(-1) and a dynamic range of three orders of magnitude, in less than one hour. We envision the use of this technique in a handheld device for multiplex high throughput analysis using an array of micro-channels for probing various protein biomarkers in clinically relevant samples such as human serum for cancer detection.

Digital microfluidic assay for protein detection

Significance We developed a novel proteomic platform that can easily be translated into a protein biomarker diagnostic. This platform integrates microfluidic technology with electrical impedance sensing and embodies a unique two-chamber architecture in which the capture/reaction and detection steps are physically separated from one another. This two-chamber design is a deviation from traditional proteomic biosensors and offers the potential for greater sensitivity as the capture/reaction and detection steps can be independently optimized. We demonstrate the implementation and feasibility of this platform in assaying both protein abundance, using the human cytokine interleukin 6, and activity, using Abelson tyrosine kinase. Global studies of the human proteome have revealed a plethora of putative protein biomarkers. However, their application for early disease detection remains at a standstill without suitable methods to realize their utility in the clinical setting. There thus continues to be tremendous interest in developing new technology for sensitive protein detection that is both low in cost and carries a small footprint to be able to be used at the point of care. The current gold standard method for protein biomarker detection is the ELISA, which measures protein abundance using bulky fluorescent scanners that lack portability. Here, we present a digital microfluidic platform for protein biomarker detection that is low in cost compared with standard optical detection methods, without any compromise in sensitivity. This platform furthermore makes use of simple electronics, enabling its translation into a portable handheld device, and has been developed in a manner that can easily be adapted to assay different types of proteomic biomarkers. We demonstrate its utility in quantifying not only protein abundance, but also activity. Interleukin-6 abundance could be assayed from concentrations as low as 50 pM (an order of magnitude lower than that detectable by a comparable laboratory designed ELISA) using less than 5 μL of sample, and Abelson tyrosine kinase activity was detectable in samples containing 100 pM of kinase.

Microfluidic diagnostic tool for the developing world: contactless impedance flow cytometry

In this work, we demonstrate a novel and cost-effective approach to implement a disposable microfluidic contactless impedance cytometer. Conventional methods for single cell impedance cytometry use microfabricated electrodes in direct contact with the buffer to measure changes of its electrical impedance when cells pass through the applied electric field. However, this approach requires expensive microfabrication of electrodes, and also, the fabricated electrodes cannot be reused without thorough and time-consuming cleaning process. Here, we introduce a novel approach to allow for single cell impedance cytometry using electrodes that can be reused, without the need for microfabrication of the electrodes. This disposable device can be potentially inserted onto a printed circuit board (PCB) which has a non-disposable, yet inexpensive, electronic reading apparatus. This significantly reduces the manufacturing costs, making it suitable for low resource settings, such as point-of-care testing in the developing countries.

Label-free electronic probing of nucleic acids and proteins at the nanoscale using the nanoneedle biosensor

Detection of proteins and nucleic acids is dominantly performed using optical fluorescence based techniques, which are more costly and timely than electrical detection due to the need for expensive and bulky optical equipment and the process of fluorescent tagging. In this paper, we discuss our study of the electrical properties of nucleic acids and proteins at the nanoscale using a nanoelectronic probe we have developed, which we refer to as the Nanoneedle biosensor. The nanoneedle consists of four thin film layers: a conductive layer at the bottom acting as an electrode, an oxide layer on top, and another conductive layer on top of that, with a protective oxide above. The presence of proteins and nucleic acids near the tip results in a decrease in impedance across the sensing electrodes. There are three basic mechanisms behind the electrical response of DNA and protein molecules in solution under an applied alternating electrical field. The first change stems from modulation of the relative permittivity at the interface. The second mechanism is the formation and relaxation of the induced dipole moment. The third mechanism is the tunneling of electrons through the biomolecules. The results presented in this paper can be extended to develop low cost point-of-care diagnostic assays for the clinical setting.

Use of negative dielectrophoresis for selective elution of protein-bound particles.

In this paper with the aid of negative dielectrophoresis force in conjunction with shear force and at an optimal sodium hydroxide concentration we demonstrated a switchlike functionality to elute specifically bound beads from the surface. At an optimal flow rate and sodium hydroxide concentration, negative dielectrophoresis turned on results in bead detachment, whereas when negative dielectrophoresis is off, the beads remain attached. This platform offers the potential for performing a bead-based multiplexed assay where in a single channel various regions are immobilized with a different antibody, each targeting a different antigen. To develop the proof of concept and to demonstrate the switchlike functionality in eluting specifically bound beads from the surface we looked at two different protein interactions. We chose interactions that were in the same order of magnitude in strength as typical antibody-antigen interactions. The first was protein G-IgG interaction, and the second was the interaction between anti-IgG and IgG.

Targeted cell detection based on microchannel gating

Currently, microbiological techniques such as culture enrichment and various plating techniques are used for detection of pathogens. These expensive and time consuming methods can take several days. Described below is the design, fabrication, and testing of a rapid and inexpensive sensor, involving the use of microelectrodes in a microchannel, which can be used to detect single bacterial cells electrically (label-free format) in real time. As a proof of principle, we have successfully demonstrated real-time detection of target yeast cells by measuring instantaneous changes in ionic impedance. We have also demonstrated the selectivity of our sensors in responding to target cells while remaining irresponsive to nontarget cells. Using this technique, it can be possible to multiplex an array of these sensors onto a chip and probe a complex mixture for various types of bacterial cells.

Depletion of cells and abundant proteins from biological samples by enhanced dielectrophoresis

Platforms that are sensitive and specific enough to assay low-abundance protein biomarkers, in a high throughput multiplex format, within a complex biological fluid specimen, are necessary to enable protein biomarker based diagnostics for diseases such as cancer. The signal from an assay for a low-abundance protein biomarker in a biological fluid sample like blood is typically buried in a background that arises from the presence of blood cells and from high-abundance proteins that make up 90% of the assayed protein mass. We present an automated on-chip platform for the depletion of cells and highly abundant serum proteins in blood. Our platform consists of two components, the first of which is a microfluidic mixer that mixes beads containing antibodies against the highly abundant proteins in the whole blood. This complex mixture (consisting of beads, cells, and serum proteins) is then injected into the second component of our microfluidic platform, which comprises a filter trench to capture all the cells and the beads. The size-based trapping of the cells and beads into the filter trench is significantly enhanced by leveraging additional negative dielectrophoretic forces to push the micron sized particles (cells and beads which have captured the highly abundant proteins) down into the trench, allowing the serum proteins of lower abundance to flow through. In general, dielectrophoresis using bare electrodes is incapable of producing forces beyond the low piconewton range that tend to be insufficient for separation applications. However, by using electrodes passivated with atomic layer deposition, we demonstrate the application of enhanced negative DEP electrodes together with size-based flltration induced by the filter trench, to deplete 100% of the micron sized particles in the mixture.

Microfluidic force spectroscopy for characterization of biomolecular interactions with piconewton resolution

In this paper we present a scalable method based on the use of microfluidics and shear force spectroscopy which can be used for determining the affinity between molecules. Our method involves the use of functionalization of the surface of microfluidic channels with ligand molecules, and the surface of microspheres with receptor molecules. Bound beads are detached from the surface of the microchannels using pressure driven flow. The drag force required to detach the beads is used to determine the affinity of the bond holding the two molecules together. The minimum force we are able to detect is 5 pN. We have used this method to determine the binding force between protein-protein interactions and DNA base-pair interactions. We also have shown the ability of this technique to distinguish between strong and weak protein-protein interactions. Using this approach, it may be possible to multiplex an array of these functionalized channels onto a chip and probe the interactions between large varieties of biomolecules.

A Microfluidic Platform for Characterization of Protein–Protein Interactions

Traditionally, expensive and time consuming techniques such as mass spectrometry and Western Blotting have been used for characterization of protein-protein interactions. In this paper, we describe the design, fabrication, and testing of a rapid and inexpensive sensor, involving the use of microelectrodes in a microchannel, which can be used for real-time electrical detection of specific interactions between proteins. We have successfully demonstrated detection of target glycoprotein-glycoprotein interactions, antigen-antibody interactions, and glycoprotein-antigen interactions. We have also demonstrated the ability of this technique to distinguish between strong and weak interactions. Using this approach, it may be possible to multiplex an array of these sensors onto a chip and probe a complex mixture for various types of interactions involving protein molecules.

Electrical Detection of Proteins and DNA using Bioactivated Microfluidic Channels: Theoretical and Experimental Considerations

In order to detect diseases like cancer at an early stage while it still may be curable, its necessary to develop a diagnostic technique which can rapidly and inexpensively detect protein and nucleic acid biomarkers, without making any sacrifice in the sensitivity. We have developed a technique, based on the use of bioactivated microfluidic channels integrated with electrodes for electrical sensing, which can be used to detect protein biomarkers, target cells, and DNA hybridization. In this paper, we discuss the theoretical detection limits of this kind of sensor, and also discuss various experimental considerations in the electrical characterization of our device. In particular, we discuss the temperature dependence, the impedance drift, the noise sources, and various methods for optimizing the signal to noise ratio.