Collaborators

• S. Tannenbaum, MIT
• N.-T. Nguyen, NTU, Singapore
• C. T. Lim, NUS, Singapore
• K. Van Vliet, DMSE, MIT
• M. Dao, DMSE
• L. Griffith
• D. Lauffenburger, MIT

Graduate Students

• L. Li, Res. Asst., EECS / HST
• L. F. Cheow, Res. Asst., EECS
• A. Sarkar, Res. Asst., EECS
• S. Huang, Res. Asst., EECS
• R.-K. Kwak, Res. Asst., ME
• L. Wu, Res. Asst., BE
• H. W. Hou, Res. Asst., NUS Bioengineering (SMART)
• W. C. Lee, Res. Asst., NUS Bioengineerig (SMART)
• C. P. Lim, Res. Asst., NTU (SMART)
• T. F. Kong, Res. Asst., NTU (SMART)
• G. F. Guan, Res. Asst., NUS (SMART)

Research Staff

• Y. Song, Ph.D.
• S. Kim, Ph.D.
• H. Y. Gan, Ph. D.
• Z. Li, Ph. D. (SMART)
• C. H. Chen, Ph. D.
• Ali Bhagat, Ph. D. (SMART)
• Weng Kung Peng, Ph.D. (SMART)

Support Staff

• S. Davco, Admin. Asst.

Publications

Jeong Hoon Lee and Jongyoon Han, “Concentration-enhanced rapid detection of human chorionic gonadotropin (hCG) as a tumor marker using a nanofluidic preconcentrator,” Microfluidics and Nanofluidics, 2010, 9,973-979.

Ali Asgar S. Bhagat, Hansen Bow, Han Wei Hou, Swee Jin Tan, Chwee Teck Lim, Jongyoon Han, “Microfluidics for Cell Separation,” Medical and Biological Engineering and Computing, 2010, 48, 999-1014.

Han Wei Hou, Ali Asgar S. Bhagat, Alvin Guo Lin Chong, Pan Mao, Kevin Shyong Wei Tan, Jongyoon Han, Chwee Teck Lim, “Deformability based cell margination – A simple microfluidic design for malaria infected erythrocyte separation,” Lab on a Chip, 2010, 10, 2605 – 2613.

Diez-Silva, M., M. Dao, J. Han, C.-T. Lim, and S. Suresh, “Shape and Biomechanical Characteristics of Human Red Blood Cells in Health and Disease,” MRS Bulletin, 2010. 35(5): p. 382-388.

Li, Z.R., Liu, G.R., Hadjiconstantinou, N.G., Han, J., Wang, J.S. & Chen, Y.Z. “Dispersive Transport of Biomolecules in Periodic Energy Landscapes with application to Nanofilter Sieving Arrays,” Electrophoresis, 32, 506-517, 2011.

Jun Young Kim, Jonathan P. DeRocher, Pan Mao, Jongyoon Han, Robert E. Cohen, and Michael F. Rubner, “Formation of Nanoparticle-Containing Multilayers in Nanochannels via Layer-by-Layer Assembly,” Chemistry of Materials, 22, 6409-6415, 2010.

Dextras, P., Payer, K.R., Burg, T.P., Shen, W., Wang, Y.-C., Han, J. & Manalis, S.R., “Fabrication and Characterization of an Integrated Microsystem for Protein Preconcentration and Sensing,” Journal of Microelectromechanical Systems, 99, 1-10, 2010.

Bow, H., Pivkin, I., Diez-Silva, M., Goldfless, S.J., Dao, M., Niles, J.C., Suresh, S. & Han, J. A ., “Microfabricated deformability-based flow cytometer with application to malaria,” Lab on a Chip, 11, 1065-1073 (2011).

Lee, W.C., Bhagat, A.A.S., Sha, H., Vliet, K.J.V., Han, J. & Lim, C.T., “High-throughput cell cycle synchronization using inertial forces in spiral microchannels,” Lab Chip, 2011, 11: p. 1359-1367.

Ko, S.H., S.J. Kim, L.F. Cheow, L.D. Li, K.H. Kang, and J. Han, Massively-Parallel Concentration Device for Multiplexed Immunoassays Lab Chip, 2011. 11: p. 1351-1358.

Ali Asgar. S. Bhagat, Han Wei Hou,  Leon D. Li, Chwee Teck Lim, and Jongyoon Han, “Pinched flow coupled shear modulated inertial microfluidics for high throughput rare blood cell separation,” Lab on a Chip, accepted for publication, 2011.

Song, Y.-A., A. Rabie, R. Sarpeshkar, S. Lin, and J. Han, “Hybrid Electrical and Chemical Stimulation of Nerve via In-Situ Harvesting of Neurotransmitters,”  Plastic and Reconstructive Surgery, 2010. 125(6): p. Supp. 137.

Bhagat, A.A.S., H.W. Hou, S. Huang, C.T. Lim, and J. Han, “High-throughput circulating tumor cells (CTCS) isolation using inertial forces,” in MicroTAS 2010. 2010: Groningen, The Netherlands. p. 1391-1393.

Hou, H.W., A.A.S. Bhagat, P. Mao, J. Han, and C.T. Lim, “Deformability based cell margination for malarial infected red blood cell enrichment,” in MicroTAS 2010. 2010: Groningen, The Netherlands. p. 1370-1372.

Sha Huang, Hansen Bow, Monica Diez-Silva, Jongyoon Han, “Applying a microfluidic ‘deformability cytometry’ to measure stiffness of malaria-infected red blood cells at body and febrile temperatures,” in MicroTAS 2010, Groningen, The Netherlands, p. 259-261

Kwak, R., S.J. Kim, and J. Han, “Continuous-flow biomolecule concentrator by ion concentration polarization,” in MicroTAS 2010. 2010: Groningen, The Netherlands. p. 887-889.

Lee, W.C., A.A.S. Bhagat, S. Huang, K.J.V. Vliet, J. Han, and C.T. Lim, “Cell cycle synchronization of stem cells using inertial microfluidics,” in 14th International Conference on Miniaturized Systems for Chemistry and Life Sciences (MicroTAS 2010). 2010: Groningen, The Netherlands. p. 208-210.

We present an integrated microfluidic probe that captures the contents of selected single adherent cells from standard tissue culture platforms and directly measures specific protein kinase activities in the captured lysate using either a fluorimetric assay in a small isolated chamber or a concentration-enhanced mobility-shift assay in an integrated nanofluidic concentrator. We demonstrate the use of the probe by measuring kinase activity in a single human hepatocellular carcinoma (HepG2) cell.

Traditional cellular assays measure average properties of 10³-10⁶ of cells, missing differences (e.g., drug responses) between individual cells in supposedly homogenous populations that have consequences for treatment of diseases ^[1] . Recent microfluidic or traditional tools ^[2] have studied genetic differences between single cells using nucleic acid amplification. These tools fail to capture important non-genetic sources of heterogeneity that create unique proteomes in different cells. Direct measurement of protein activities from single cells remains difficult due to limited assay sensitivity. In addition, difficulties in interfacing with adherent cells in a standard culture have led to the use of cell suspensions in microfluidic single cell assays ^[2] .

The integrated device (Figure 1) reported here interfaces with standard tissue culture plates using a microfluidic probe ^[3] that creates a limited, tunable lysis zone at its tip by simultaneously dispensing and collecting lysis agents and lyses and collects contents of selected single cells from adherent cell populations. The captured cytosol is mixed with assay reagents and flowed into a small reaction chamber, which is isolated for observation, using pneumatic micro-valves. The integrated ion-selective hydrogel-based nanofluidic concentrator ^[4] is then used to trap/concentrate the proteins/reaction products in the mixture to yield very high kinase assay sensitivity ^[5] , sufficient to probe proteins from single cells. This single cell detection platform is agnostic to specific sensing chemistry, so other biochemical assays can also be implemented with minimal modification.

1. M. Niepel, S. L. Spencer, and P. K. Sorger, “Non-genetic cell-to-cell variability and the consequences for pharmacology,” Current Opinion in Chemical Biology, vol. 13, pp. 556-561, Dec. 2009.
2. M. Leslie, “The Power of One,” Science, vol. 331, no. 6013, pp. 24-26, Jan. 2011.
3. D. Juncker, H. Schmid, and E. Delamarche, “Multipurpose microfluidic probe,”  Nature Materials, vol. 4, pp. 622-628, July 2005.
4. Y.-C Wang, A. L. Stevens, and J. Han, “Million-fold preconcentration of proteins and peptides by nanofluidic filter,” Analytical Chemistry, vol. 77,  no. 44, pp. 4293-4299, June 2005.
5. J. H. Lee, B. D. Cosgrove, D. A. Lauffenburger, and J. Han, “Microfluidic concentration-cnhanced cellular kinase activity assay,” Journal of the American Chemical Society, vol. 131, no. 30, pp. 10340-10341, July 2009.

The mechanical properties of tissues and cells have important implications on their differentiated state, functions and responses to injury. Altered cell deformability is both a cause of and biomarker for potentially severe diseases, such as cancer, sickle cell anemia and malaria. In the past, several techniques have been developed to measure single-cell deformability including micropipette aspiration, atomic force microscopy, and optical tweezers. However, many of these measurements assess only static cell deformations which often fail to reflect in vivo situation when cells are in microcirculation. Additionally, the low throughput of the techniques limits sampling size per experiment, which may potentially lead to misrepresentation of population-wide trait. Therefore, we aim to develop a microfluidic device which measures cell dynamic deformability with high sensitivity and high throughput.

In this project, the relation between cell dynamic deformability and disease state is aimed to be established for several representative cell lines including human erythrocytes, breast cancer cells, and mesenchymal stem cells. The impact of microenvironmental controls such as temperature fluctuation and drug treatment on the deformability of malaria infected cells is also investigated.

Aptamers are emerging as popular alternatives to antibodies as affinity probes in immunoassays. From a point-of-care diagnostics standpoint, aptamers have an advantage over antibodies since they are stable over a wide range of conditions and can be chemically synthesized at low cost. Affinity probe capillary electrophoresis (CE) ^{[1] [2]} is a promising platform with which to perform aptamer-based biomarker detection as it features fast homogeneous reaction kinetics and requires only one affinity probe species, although sensitivity is still limited due to band dispersion, complex dissociation and lack of amplification reaction.

We have previously demonstrated microfabricated nanofluidic preconcentration devices that can continuously accumulate a charged biomolecule species at a specified location ^{[3] [4] [5]} . In this work, we showed that these devices can also efficiently separate biomolecules with different mobilities by focusing them at different locations. This phenomenon lends itself well to aptamer affinity probe CE, where aptamers undergo a significant mobility shift upon binding to larger target proteins. The important advantage of this scheme compared to conventional CE is that aptamer-protein dissociation and band broadening effects are counteracted by electrokinetic focusing. By simultaneously focusing and separating free aptamers from aptamer-protein complex in this device, we can obtain highly sensitive and quantitative measurement of target biomarkers using aptamers.

With this scheme, we showed enhanced detection sensitivity for IgE and HIV-1 RT in simple buffer solution. The limits of detection obtained (4.5 pM for IgE and 9 pM for HIV-1 RT) are among the lowest reported in the literature. The limit of detection for IgE in 10% serum was 10-fold higher due to nonspecific interactions between aptamers and serum proteins. Due to the simple readout for this assay, multiple samples can be assayed in parallel. As the assay is driven by gravitational flow, uses low voltages (30 V), and does not require multiple processing steps, it is well-suited towards low-cost point-of-care analysis.

1. I. German, D. D. Buchanan, and R. T. Kennedy, “Aptamers as ligands in affinity probe capillary electrophoresis,” Anal. Chem., vol. 70, pp. 4540-4545, 1998.
2. H. Zhang, X. F. Li, and X. C. Le, “Tunable aptamer capillary electrophoresis and its application to protein analysis,” J. Am. Chem. Soc., vol. 130, pp. 34-35, 2008.
3. Y. C. Wang, A. L. Stevens, and J. Han, “Million-fold preconcentration of proteins and peptides by nanofluidic filter,” Anal. Chem., vol. 77, pp. 4293-4299, 2005.
4. Y. C. Wang and J. Han, “Pre-binding dynamic range and sensitivity enhancement for immuno-sensors using nanofluidic preconcentrator,” Lab on a Chip, vol. 8, pp. 392, 2008.
5. L. F. Cheow, S. H. Ko, S. J. Kim, K. H. Kang, and J. Han, “Increasing the sensitivity of enzyme-linked immunosorbent assay using multiplexed electrokinetic concentrator,” Anal. Chem., vol. 82, pp. 3383-3388, 2010.