Research Overview
(Publications)
(Programs)

 

Summary: Our research focuses on quantitative characterization of biomolecular interactions, with technological connections to diagnostics for medical and fundamental biology applications. The complexity of biological systems benefits from high-throughput approaches that allow thousands of interactions to be monitored in parallel. Such high-throughput methods are often implemented on solid supports, in which one partner in the interaction is immobilized at the surface. While this allows many bioaffinity interactions to be screened simultaneously, the interfacial environment brings significant challenges as it can bias the thermodynamic and kinetic behavior of the biomolecular interactions of interest. This is where our engineering approach enters: we wish to dissect the fundamental equilibrium and kinetic aspects of biomolecular reactions at surfaces, understand how this surface behavior relates to that in solution, elucidate the role played by the interfacial molecular organization, and apply this understanding to advance bioanalytical technologies. Our mostly experimental group has expertise in surface biomodification and characterization with methods including electrochemical techniques, X-ray photoelectron spectroscopy, ellipsometry, neutron and X-ray reflectivity, infrared spectroscopy, and imaging/optical techniques. Following are several examples of recent and ongoing work.

Research Support: National Institutes of Health, National Science Foundation.

 

Surface Hybridization Reactions

When nucleic acids hybridize at an interface, they must do so subject to a significantly more complex environment than for solution hybridization. The figure illustrates observed hybridization regimes as a function of the surface coverage of end-immobilized strands S0 and buffer concentration CB. The suppressed hybridization (SH) regime consists of sub-regimes where suppression is due to packing (SH-P) or electrostatic (SH-E) penalties. Hybridization was below detection in the NH regime, and in the Pseudo-Langmuir (PL) regime it was only weakly dependent on S0. At very low coverages, a Langmuir (L) regime in which immobilized strands do not interact is expected.

A diagram of DNA-DNA surface hybridization regimes.

Left: Comparison of morpholino and DNA molecular structures. Right: Label-free capacitive detection of nucleic acids using morpholino and DNA-derivatized sensors. Top row depicts experimental data, bottom raw shows results of Poisson-Boltzmann model calculations. VDC: readout voltage, x: extent of hybridization.

Diagnostic Applications of Morpholinos

Morpholinos are uncharged DNA mimics that recognize nucleic acids following conventional base-pairing rules. We are pursuing diagnostic applications of morpholinos for nucleic acid analysis by leveraging morpholinos' unique properties. For example, because morpholinos allow hybridization even at low salt conditions, electrostatic signatures derived from the hybridization reaction can be amplified to provide sensitive detection purely based on charge effects. One way to monitor these signatures is through interfacial capacitance Cd. We are also using morpholinos to help suppress interference from secondary structure in the analyte and to provide electric field based control over the hybridization reaction. In support of these studies, the thermodynamics of hybridization between morpholinos and nucleic acids are also being systematically explored in solution and at solid/liquid interfaces.

Analysis of Small Molecule Ligand/DNA Interactions

Interactions between small molecules and nucleic acids arise in many contexts including design of transcription regulators and DNA dyes, toxicological screens, and DNA-targeted therapy including anticancer, antibiotic, and antiviral compounds. Most DNA/ligand interactions are sensitive to the base sequence of the nucleic acid; therefore, to understand the recognition mechanisms behind the activity of a small molecule it is necessary to establish the sequence dependence. Because of minute reagent consumption and high scalability that allows characterization of thousands of interactions in a single experiment, surface-based assays could tremendously decrease the costs and labor of such measurements.

Left: Surface melting assays for thermodynamically quantifying drug binding to DNA. The plot shows shifts in the melting transition of immobilized DNA duplexes when exposed to nM to mM concentrations of netropsin, a compound that binds in the DNA minor groove.

A prototype electrochemical CMOS microarray (in collaboration with Ken Shepard, Columbia University).

Device Development

To translate research into applications it is necessary to develop technologies that are affordable and convenient to use. We are therefore interested in collaborations that bring expertise in device design as well as provide an end user's perspective. For example, we have collaborated with electrical engineers to develop biochips based on microelectronic CMOS manufacture to produce high-performance, small footprint, affordable sensor arrays capable of parallel analysis of many interactions simultaneously. Students in our group have also undertaken development of bioconjugation labels, surface biomodification chemistries, and research instrumentation.







Publications (Research Overview) (Programs)


R Levicky, U Koniges, N Tercero, "Diagnostic applications of morpholinos and label-free electrochemical detection of nucleic acids," ed. H. M. Moulton, J. D. Moulton, Methods in Molecular Biology, 1565, 181-190, (2017). [Abstract]

H-C Chiang, R Levicky, "Effects of chain-chain associations on hybridization in DNA brushes," Langmuir, 32, 12603-12610, (2016). [Abstract]

Y-K He, J Zhang, S Ruffin, L-N Ji, K Wang, R Levicky, X-H Xia, "An electrochemical study of the surface hybridization process of morpholino-DNA: Thermodynamics and kinetics," Electroanalysis, 28, 1647-1653, (2016). [Abstract]

D Martins, X Wei, R Levicky, Y-A Song, "Integration of multiplexed microfluidic electrokinetic concentrators with a morpholino microarray via reversible surface bonding for enhanced DNA hybridization," Analytical Chemistry, 88, 3539-3547, (2016). [Abstract]

W Qiao, H-C Chiang, H Xie, R Levicky, "Surface vs solution hybridization: Effects of salt, temperature, and probe type," Chemical Communications, 51, 17245-17248, (2015). [Abstract]

D Martins, R Levicky, Y-A Song, "Enhancing the speed of morpholino-DNA biosensor by electrokinetic concentration of DNA in a microfluidic chip," Biosens. Bioelectron., 72, 87-94, (2015). [Abstract]

K Williams, C-S Kim, JR Kim, R Levicky, "Multimodal electrochemical sensing of transcription factor-operator complexes," Analyst, 139, 1463-1471, (2014). [Abstract]

W Qiao, S Kalachikov, Y Liu, R Levicky, "Charge-neutral morpholino microarrays for nucleic acid analysis," Anal. Biochem., 434, 207-214, (2013). [Abstract]

 

A Harrison, H Binder, A Buhot, CJ Burden, E Carlon, C Gibas, LJ Gamble, A Halperin, J Hooyberghs, DP Kreil, R Levicky, PA Noble, A Ott, BM Pettitt, D Tautz, AE Pozhitkov, "Physico-chemical foundations underpinning microarray and next-generation sequencing experiments," Nucl. Acids Res., 41, 2779-2796, (2013). [Abstract]

 

K Lee, A Babajanyan, H Melikyan, C Kim, S Kim, J Kim, J-H Lee, B Friedman, R Levicky, S Kalachikov, "Label-free DNA Microarray Bioassays Using a Near-Field Scanning Microwave Microscope," Biosensors & Bioelectronics, 42, 326-331, (2013). [Abstract]

 

I Belozerova, D Ge, R Levicky, "Electrochemical Measurements of DNA Melting on Surfaces," in Nanomaterial Interfaces in Biology, eds. Paolo Bergese and Kimberly Hamad-Schifferli, Springer/Humana Press, (2013), p. 127-136. [Abstract]

 

I Belozerova, R Levicky, "Melting Thermodynamics of Reversible DNA/Ligand Complexes at Interfaces," J. Am. Chem. Soc., 134, 18667-18676, (2012). [Abstract]

 

D Ge, X Wang, K Williams, R Levicky, "Thermostable DNA Immobilization and Temperature Effects on Surface Hybridization," Langmuir, 28, 8446-8455, (2012). [Abstract]

R O'Connor, N Tercero, W Qiao, R Levicky, "Electrochemical studies of morpholino-DNA surface hybridization. ECS Trans., 35, 99-110, (2011). [Abstract]

Y Liu, D Irving, W Qiao, D Ge, R Levicky, "Kinetic Mechanisms in Morpholino-DNA Surface Hybridization," J. Am. Chem. Soc., 133, 11588-11596, (2011). [Abstract]

TCD Huang, S Paul, P Gong, R Levicky, J Kymissis, SA Amundson, KL Shepard, "Gene expression analysis with an integrated CMOS microarray by time-resolved fluorescence detection," Biosensors & Bioelectronics, 26, 2660-2665, (2011). [Abstract]

D Ge, R Levicky, "A Comparison of Five Bioconjugatable Ferrocenes for Labeling of Biomolecules," Chem. Commun., 46, 7190-7192, (2010).
[Abstract]

N Tercero, K Wang, R Levicky, "Capacitive monitoring of morpholino-DNA surface hybridization: Experimental and theoretical analysis," Langmuir, 26, 14351-14358, (2010).[Abstract]

P Gong, K Wang, Y Liu, K Shepard, R Levicky, "Molecular mechanisms in morpholino-DNA surface hybridization," J. Am. Chem. Soc., 132, 9663-9671, (2010). [Abstract]

D Irving, P Gong, R Levicky, "DNA surface hybridization: Comparison of theory and experiment," J. Phys Chem. B, 114, 7631-7640, (2010). [Abstract]

S Khapli, JR Kim, JK Montclare, R Levicky, M Porfiri, S Sofou, "Frozen cyclohexane-in-water emulsion as a sacrificial template for the synthesis of multilayered polyelectrolyte microcapsules," Langmuir, 25, 9728-9733, (2009). [Abstract]

TCD Huang, S Sorgenfrei, P Gong, R Levicky, KL Shepard, "A 0.18-micron CMOS array sensor for integrated time-resolved fluorescence detection," IEEE J. Solid State Circuits, 44, 1644-1654, (2009). [Abstract]

N Tercero, K Wang, P Gong, R Levicky, "Morpholino monolayers: Preparation and label-free DNA analysis by surface hybridization," J. Am. Chem. Soc., 131, 4953-4961, (2009). [Abstract]

K Wang, RA Zangmeister, R Levicky, "Equilibrium electrostatics of responsive polyelectrolyte monolayers," J. Am. Chem. Soc., 131, 318-326, (2009). [Abstract]

PM Levine, P Gong, R Levicky, KL Shepard, "Real-time, multiplexed electrochemical DNA detection using an active complementary metal-oxide-semiconductor biosensor array with integrated sensor electronics," Biosensors & Bioelectronics, 24, 1995-2001, (2009). [Abstract]

P Gong, R Levicky, "DNA surface hybridization regimes," Proc. Natl. Acad. Sci. USA, 105, 5301-5306, (2008). [Abstract]

RG Bozic, AC West, R Levicky, "Square wave voltammetric detection of 2,4,6-trinitrotoluene and 2,4-dinitrotoluene on a gold electrode modified with self-assembled monolayers," Sensors & Actuators B, doi:10.1016/j.snb.2008.03.017, (2008). [Abstract]

G Shen, N Tercero, MA Gaspar, B Varughese, K Shepard, R Levicky, "Charging behavior of single-stranded DNA polyelectrolyte brushes," J. Am. Chem. Soc., 128, 8427-8433, (2006). [Abstract]

G Patounakis, K Shepard, R Levicky, "Active CMOS array sensor for time-resolved fluorescence detection," IEEE J. Solid State Circuits, 41, 2521-2530, (2006). [Abstract]

B Friedman, MA Gaspar, S Kalachikov, K Lee, R Levicky, G Shen, H Yoo, "Sensitive, label-free DNA diagnostics based on near-field microwave imaging," J. Am. Chem. Soc., 127, 9666-9667, (2005). [Abstract]

S Kim, H Yoo, K Lee, B Friedman, MA Gaspar, R Levicky, "Distance control for a near-field scanning microwave microscope in liquid using a quartz tuning fork," Appl. Phys. Lett., 86, art. 153506, (2005). [Abstract]

R Levicky, A Horgan, "Physicochemical perspectives on DNA microarray and biosensor technologies," Trends Biotechnol., 23, 143-149, (2005). [Abstract]

G Shen, MA Gaspar, R Levicky, "X-ray photoelectron spectroscopy and infrared spectroscopy study of maleimide-activated supports for immobilization of oligodeoxyribonucleotides," Nucleic Acids Res., 32, 5973-5980 (2004). [Abstract]

P Johnson, R Levicky, "X-ray photoelectron spectroscopy and differential capacitance study of thiol-functional polysiloxane films on gold supports," Langmuir, 20, 9621-9627 (2004). [Abstract]

P Johnson, MA Gaspar, R Levicky, " Polymer-anchored DNA gene monolayers," J. Am. Chem. Soc., 126, 9910-9911 (2004). [Abstract]

Z Liu, R Levicky, "Formation of nanoparticle rings on heterogeneous soft surfaces," Nanotechnology, 15, 1483-1488 (2004). [Abstract]

G Shen, A Horgan, R Levicky, "Reaction of N-phenyl maleimide with aminosilane monolayers," Colloids Surf. B - Biointerfaces, 35, 59-65 (2004). [Abstract]

Z Liu, R Levicky, "Ring structures from nanoparticles and other nanoscale building blocks," in Dekker Encyclopedia of Nanoscience and Nanotechnology, eds. James A. Schwarz, Cristian I Contescu, and Karol Putyera; Marcel Dekker Inc, New York, 2004, vol 5. 3281-3288.

M Islam, S O'Brien, M Steigerwald, Z Liu, R Levicky, I Herman, "Addition, suppression, and inhibition in the electrophoretic deposition of nanocrystal mixture films for CdSe nanocrystals with
g-Fe2O3 and Au nanocrystals," Nano Letters, 3, 1603-1606 (2003). [Abstract]

P Johnson, R Levicky, "Polymercaptosiloxane anchor films for robust immobilization of biomolecules to gold supports," Langmuir, 19, 10288-10294 (2003). [Abstract]

L Jin, A Horgan, R Levicky, "Preparation of end-tethered DNA monolayers on siliceous surfaces using heterobifunctional crosslinkers," Langmuir, 19, 6968-6975 (2003). [Abstract]

Z Liu, K Pappacena, J Cerise, J Kim, CJ Durning, B O'Shaughnessy, R Levicky, "Organization of nanoparticles on soft polymer surfaces," Nano Letters, 2, 219-224, (2002). [Abstract]

AB Steel, R Levicky, TM Herne, MJ Tarlov, "Immobilization of nucleic acids at solid surfaces: Effect of oligonucleotide length on layer assembly," Biophys. J., 79, 975-981, (2000). [Abstract]

R Levicky, TM Herne, MJ Tarlov, SK Satija, "Using self-assembly to control the structure of DNA monolayers on gold: A neutron reflectivity study," J. Am. Chem. Soc., 120, 9787-9792, (1998). [Abstract]

R Levicky, N Koneripalli, M Tirrell, JF Ankner, H Kaiser, SK Satija, "Selectively swollen films of triblock/diblock copolymer blends: Dependence of swollen film structure on blend composition," Macromolecules, 31, 4908-4914, (1998). [Abstract]

R Levicky, N Koneripalli, M Tirrell, SK Satija, "Stratification in bidisperse polymer brushes from neutron reflectivity," Macromolecules, 31, 2616-2621, (1998). [Abstract]

R Levicky, N Koneripalli, M Tirrell, SK Satija, "Concentration profiles in densely tethered polymer brushes," Macromolecules, 31, 3731-3734 (1998). [Abstract]

N Koneripalli, R Levicky, FS Bates, MW Matsen, SK Satija, J Ankner, H Kaiser, "Ordering in blends of diblock copolymers," Macromolecules, 31, 3498-3508 (1998).

N Koneripalli, R Levicky, FS Bates, J Ankner, H Kaiser, SK Satija, "Confinement-induced morphological changes in diblock copolymer films," Langmuir, 12, 6681-6690 (1996).

N Koneripalli, N Singh, R Levicky, FS Bates, PD Gallagher, SK Satija, "Confined block copolymer thin films," Macromolecules, 28, 2897-2904 (1995).

M Tirrell, R Levicky, "End-tethered chains at liquid interfaces," Curr. Opin. Solid St. Mat. Sci., 2, 668-672 (1997). [Abstract]




Programs (Research Overview) (Publications)



Particular

"Particular" is a graphics driven game that simulates the behavior of water and ions as a function of temperature, charge, gravity and other parameters. It was written and created by Prof. Andy Nealen and Dr. Bert Buchholz, with consultation from the Levicky lab. A 64bit Windows version can be downloaded by clicking on the above link (right click to open the link, download the zipped folder after accepting any cautions, unzip the folder, and run particular.exe). (Support: National Science Foundation)

CVFit    
Documentation    
Sample Initialization File    
Sample Data File

"CVFit" fits cyclic voltammetry traces with 0, 1, or 2 surface-bound redox species (tags), and calculates the total charge associated with the oxidation/reduction of each tag. (Support: National Science Foundation)