research
Back to Research Listing
Nanofluidic
Biomolecular Preconcentration and Concentration-Enhanced Assays
Executive Summary
Sample preparation is one of the bottlenecks in molecular detection and
analysis. During the past decades, significant progress has been made
both in binding assays (immunoassays) and mass spectrometry (MS). However,
issues related to limited sample capacity and low abundance target
create challenges in fully utilizing the power of these new analysis
platform. In general, only high-abundance species of a given sample
could be detected, while reliable analysis of low-abundance targets
is still challenging. To address these problems, our group has sought
ways to efficiently concentrate biomolecules in order to enhance the
detection sensitivity for both large and small sample volumes. The
nanofluidic electrokinetic concentration devices serve as;
- Ideal world-to-microchip coupling system: It can collect biomolecules
from ~µL fluidic samples (addressable by pipettes) and concentrate
them into ~nL plug (addressable by microfluidics).
- Generic sensitivity enhancement scheme for many biochemical assays:
Wide variety of biochemical assays can be enhanced simply by collecting
the reactants and / or target molecules.
• Yong-Ak Song, Rohat Melik, Amr N. Rabie, Ahmed M. S. Ibrahim, David Moses, Ara Tan, Jongyoon Han & Samuel J. Lin, "Electrochemical activation and inhibition of neuromuscular systems through modulation of ion concentrations with ion-selective membranes," Nature Materials, (doi:10.1038/nmat3146) online published, 2011.
Conventional functional electrical stimulation (FES) aims to restore functional motor activity of patients with disabilities resulting from spinal cord injury or neurological disorders. However, FES-related intervention in neurological diseases lacks an effective, implantable method that suppresses unwanted nerve signals. We have developed an electrochemical method to activate and inhibit a nerve by electrically modulating ion concentrations in situ along the nerve. Using an ion-selective membrane (ISM) to achieve different excitability states of the nerve, we observe either a reduction of electrical threshold by up to approximately 40%, or voluntary, reversible inhibition of nerve signal propagation. This low-threshold electrochemical-stimulation method is applicable in current implantable neuroprosthetic devices, whereas the on-demand nerve blocking mechanism could offer an effective clinical intervention for chronic disease states caused by uncontrolled nerve activation, such as epilepsy and chronic pain syndromes. |
 |
• Rhokyun Kwak, Sung Jae Kim and Jongyoon Han, "Continuous-flow Biomolecule and Cell Concentrator by Ion Concentration Polarization," Analytical Chemistry, 83, 7348-7355, 2011.
We present a novel continuous-flow nanofluidic biomolecule / cell concentrator, utilizing ion concentration polarization (ICP) phenomenon. The device has one main microchannel which bifurcates into two channels, one for narrow, concentrated stream and the other for wider but target-free stream. A nanojunction (cation-selective material (Nafion®)) is patterned along the tilted concentrated channel. Application of an electric field generates the ICP zone near the nanojunction, so that biomolecules and cells are guided into the narrow, concentrated channel by hydrodynamic force. Once biomolecules from the main channel are continuously streamed out to the concentrated channel, one can achieve a continuous flow of same sample solution but with higher concentrations up to 100 fold. By controlling hydrodynamic resistance of main and concentrated channel, the concentration factors can be adjusted. We demonstrated the continuous-flow concentration with various targets, such as bacteria (fluorescein sodium salt, recombinant green fluorescence protein (rGFP), red blood cells (RBCs), and Escherichia coli (E. coli)). Specially, fluorescein isothiocyanate (FITC) conjugated lectin from Lens culinaris (lentil) (FITC-lectin) was tested on the different buffer conditions to clarify the effect of polarities of target sample. This system is ideally suited for a generic concentration front-end for a wide variety of biosensors, with minimal integration-related complications. |
 |
• Lih Feng Cheow and Jongyoon Han, "Continuous Signal Enhancement for Sensitive Aptamer Affinity Probe Electrophoresis Assay Using Electrokinetic Concentration," Analytical Chemistry, 2011, accepted for publication.
We describe an electrokinetic concentration-enhanced aptamer affinity probe electrophoresis assay to achieve highly sensitive and quantitative detection of protein targets in a microfluidic device. The key weaknesses of aptamer as a binding agent (weak binding strength/ fast target dissociation) were counteracted by continuous injection of fresh sample while band-broadening phenomena were minimized due to self-focusing effects. With 30 minutes of continuous signal enhancement, we can detect 4.4pM of human immunoglobulin E (IgE) and 9 pM of human immunodifficiency virus 1 reverse transcriptase (HIV-1 RT), which is among the lowest limit of detection (LOD) reported. IgE was detected in serum sample with LOD of 39 pM due to nonspecific interactions between aptamers and serum proteins. The method presented in this paper also has broad applicability to improve sensitivities of various other mobility shift assays. |
 |
• Chia-Hung Chen, Aniruddh Sakar, Yong-Ak Song, Miles A. Miller, Sung Jae Kim, Linda G. Griffith, Douglas A. Lauffenburger and Jongyoon Han, "Enhancing Protease Activity Assay in Droplet-Based Microfluidics Using a Biomolecule Concentrator," JACS,
133, 10368-10371, 2011
We introduce an integrated microfluidic device consisting of a biomolecule concentrator and a microdroplet generator that enhances the limited sensitivity of low-abundance enzyme assays by locally increasing the concentration of biomolecules before encapsulating them into droplet microreactors. We used this platform to detect ultra low levels of matrix metalloproteinases (MMPs) directly in diluted cellular supernatant and showed that it significantly (~10-fold) reduced the time required to complete the assay and the sample volume used.
|
 |
• Aniruddh Sarkar and Jongyoon Han, "Non-Linear and Linear Enhancement of Enzymatic Reaction Kinetics using a Biomolecule Concentrator," Lab on a Chip,
11(15), 2569-2576, 2011
We investigate concentration-enhanced enzyme activity assays in nanofluidic biomolecule concentrator chips which can be used to detect and study very low abundance enzymes from cell lysates and other low volume, low concentration samples. A mathematical model is developed for a mode of operation of the assay in which enzyme and substrate are concentrated together into a plug on chip which results in a non-linear enhancement of the reaction rate. Two reaction phases, an initial quadratic enzyme-limited phase and a later, linear substrate-limited phase, are predicted and then verified with experiments. It is determined that, in most practical situations, the reaction eventually enters a substrate-limited phase, therefore mitigating the concern for non-specific reactions of biosensor substrates with off-target enzymes in such assays. We also use this mode to demonstrate a multiplexed concentration-enhanced enzyme activity assay. We then propose and demonstrate a new device and mode of operation, in which only the enzyme is concentrated and then mixed with a fixed amount of substrate in an adjacent picoliter-scale reaction chamber. This mode results in a linear enhancement of the reaction rate and can be used to perform mechanistic studies on low abundance enzymes after concentrating them into a plug on chip.
|
 |
• Sung Hee Ko*, Sung Jae Kim*, Lih Feng Cheow, Leon D. Li, Kwan Hyoung Kang** and Jongyoon Han**, "Massive-Parallel Concentration Device for Multiplexed Immonoassays," Lab on a Chip, 11(7), 1351-1358, 2011.
A massively parallel nanofluidic concentration device array for multiplexed and high-throughput
biomolecule detection is demonstrated. By optimizing the microchannel/nanojunction design and
channel conductivity, an array of up to 128 nanofluidic concentration devices were fabricated.
Operation of the entire array requires only one inlet and one outlet reservoir, with the application of
a single operational voltage bias across them. Concentration efficiencies of the devices were found to be
uniform within the array, within 5% error. Alternatively, concentration speed in each channel can be
individually tuned by controlling the length of the inlet microchannel and thus controlling the flow rate
based on change of the tangential electric field. This allows immuno-binding reactions at different
concentration ranges to be performed in parallel. Using multiplexed, successive-concentration
enhanced detection in the device, we have shown that the dynamic range and reliability of the
immunoassay can be significantly increased.
|
 |
• Lih Feng Cheow, Sung Hee Ko, Sung Jae Kim, Kwan Hyoung Kang and Jongyoon Han, “Increase of Sensitivity of ELISA using Multiplexed Electrokinetic Preconcentrator,” Analytical Chemistry, 2010, 82(8), 3383-3388.
We developed a novel method to increase the sensitivity of standard Enzyme-Linked Immunosorbent Assay (ELISA) using a multiplexed electrokinetic concentration chip. The poly(dimethylsiloxane) (PDMS) molecular concentrator1 was used to trap and collect charged fluorescent product of target-bound enzyme turnover reaction of ELISA that occurred in a standard 96 well plate. Detection sensitivities of both Prostate Specific Antigen (PSA) and CA 19-9 (a human pancreatic and gastrointestinal cancer marker) ELISAs in serum are enhanced ~100 fold with a low CV of <17%. We also integrated this method with an on-chip bead-based ELISA that lends itself toward a fully-automated on-chip diagnostic device. Detection sensitivity of microfluidic bead-based CA 19-9 ELISA in serum is enhanced ~65 fold compared to the results without electrokinetic accumulation step. This chip can be directly applied to enhance the readout sensitivity of a wide range of existing ELISA kits at concentrations below the current detection limit.
|
 |
• Vincent Liu, Yong-Ak Song and Jongyoon Han, "Capillary-valve-based fabrication of ion-selective membrane junction for electrokinetic sample preconcentration in PDMS chip," Lab on a Chip, 2010, 10, 1485-1490.
In this paper, we report a novel method for fabricating ion-selective membranes in poly(dimethylsiloxane) (PDMS)/glass-based microfluidic preconcentrators. Based on the concept of capillary valves, this fabrication method involves filling a lithographically patterned junction between two microchannels with an ion-selective material such as Nafion resin; subsequent curing results in a high aspect-ratio membrane for use in electrokinetic sample preconcentration. To demonstrate the concentration performance of this high-aspect-ratio, ion-selective membrane, we integrated the preconcentrator with a surface-based immunoassay for R-Phycoerythrin (RPE). Using a 1xPBS buffer system, the preconcentrator-enhanced immunoassay showed an approximately 100x improvement in sensitivity within 30 minutes. This is the first time that an electrokinetic microfluidic preconcentrator has been used in high ionic strength buffer solutions to enhance the sensitivity of surface-based immunoassay. |
 |
• Lee, J.H., Cosgrove B., Lauffenburger A.D. and Han J., "Microfluidic concentration-enhanced cellular kinase activity assay," Journal of the American Chemical Society, 131, 10340-10341 (2009).
This paper reported a simple, disposable PDMS micro/nanofluidic preconcentration chip for in vitro concentration-enhanced cell kinase assays. A 25-fold increase in reaction velocity and 65-fold enhancement in sensitivity could be achieved by utilizing the preconcentration (electrokinetic trapping) directly from cell lysate (1 mM ATP) samples, In addition, the assay time is shortened to less than 10 min with sample volume only from 5 cells. This opens up a possibility of a true single cell kinase activity assay. In addition, the assay was done using cellular lysate, confirming that concentration-enhanced assays can be done.
|
 |
• Lee, J. H., Song, Y.-A., Tannenbaum, S. and Han, J. "Increase of Reaction
Rate and Sensitivity of Low-Abundance Enzyme Assay using Micro/Nanofluidic
Preconcentration Chip," Analytical Chemistry, 80, 3198-3204 (2008).
This paper reported a novel method to increase the reaction rate and
the sensitivity of low-abundance enzyme assay by concentrating the enzyme
and the substrate in a disposable poly (dimethylsiloxane) (PDMS) preconcentration
chip. Trypsin is used as the model enzyme and BODIPY FL casein is used
as the fluorogenic substrate. The reaction time required to turn over
substrates at 1 ng/mL was only 10 min, which demonstrates a significantly
higher reaction rate through the increase in both enzyme and substrate
concentrations. Trypsin activity can be measured down to a concentration
level of 10 pg/mL, which is a 100 fold enhancement in sensitivity.
• Wang, Y.C., and Han, J. "Pre-binding dynamic range and sensitivity
enhancement for immuno-sensors using nanofluidic preconcentrator," Lab
on a Chip, 8, 392-394 (2008).
A highly efficient and flexible way to enhance immunoassay detection
sensitivity and binding kinetics is demonstrated using a nanofluidic
based electrokinetic preconcentrator. The device is a microfluidic integration
of charge-based biomolecule concentrator and a bead-based immunoassay.
With a 30 min preconcentration, the immunoassay sensitivity is enhanced
by more than 500 fold from higher 50 pM to the sub 100 fM range. By adjusting
the preconcentration time, a broader dynamic range of detection is obtained
for a given bead-based assay.
• Schoch, R.B., Cheow, L.F., and Han, J., "Electrical Detection of Fast
Reaction Kinetics in Nanochannels with an Induced Flow," Nanoletters,
7, 3895-3900 (2007).
Diffusion-limited binding can be overcome by performing the detection
of analytes in nanochannels with an applied convective flow through them
to enhance mass transport. By monitoring the nanochannel impedance, low
analyte concentrations have been detected electrically in nanofluidic
channels within response times of 1−2 h. At high-flow velocities, the
presented method of reaction kinetics enhancement is potentially limited
by force-induced dissociations of the receptor−ligand bonds.
• Wang, Y.-C., Stevens, A. and Han, J. "Million-fold preconcentration
of proteins and peptides by nanofluidic filter," Analytical Chemistry
77, 4293-4299 (2005).
We have developed a nanofluidic preconcentrator that can concentrate
biomolecular samples up to million fold. Due to the electrical double
layer overlapping, sub 100 nm nanochannels have preferential transfer
over counterions (or counterion current). As a result, a well known phenomenon
called ion concentration polarization can be observed. By coupling a
tangential field across the ion depletion zone, we can have a fast accumulation
of charged molecules in front of it. In short, this device collects charged
biomolecules based on two features: (i) the energy barrier for charged
biomolecules generated by the induced space charge layer near the nanofluidic
filter; (ii) a faster nonlinear electroosmotic flow for sample deliveries.
We are able to achieve more than a million fold enhancement factor in
30 mins. The preconcentration factors and collection speed are close
to those of the PCR for nucleic acids, which is an essential step for
many genomics researches.
References
|
