Guide to the Zarelab

GUIDE TO THE ZARELAB

July 2010 Department of Chemistry, Stanford University

Welcome to the ZARELAB.

This booklet has been prepared to make your visit with us more rewarding by presenting a survey of our recent research activities. Each section was written by those members pursuing the work described therein.

Please feel free to ask the members of Dick Zare and the rocket test. my group to discuss any project. INSIDE THIS GUIDE On page 16 is a list of all members of the Zare group as of July 23, 2010 and Table of Contents 2 information on how to contact them. On Research Activities 3 pages 18 through 20 are floor plans of Reaction Dynamics offices and labs in the Mudd Building and Absorption Spectroscopy the Clark Center. The last pages show Mass Spectroscopy Microfluidics maps of the Stanford campus and its Nanoparticles vicinity. Selected Recent Publications 13

Do enjoy your visit! Group Members 16 Floor Plans of Offices and Labs 18 Maps of Stanford Campus & Vicinity 21

TABLE OF CONTENTS

Reaction Dynamics 3 State-to-State Reaction Dynamics Nate Bartlett, Justin Jankunas, Tapas Goswami, Nandini Mukherjee Absorption Spectroscopy 4 Measurement of Carbon Isotope Ratios Using Cavity Ring-Down Spectroscopy Doug Kuramoto Mass Spectrometry 5 Two-step Laser Mass Spectrometry of Terrestrial and Extraterrestrial Materials Amy Morrow, Hassan Sabbah

6 Hadamard Transform Time-of-Flight Mass Spectrometry Griffin Barbula, Richard Perry, Konstantin Chingin 7 Unfolding Reaction Pathways in Liquids on the Millisecond Time Scale Using Desorption Electrospray Ionization Richard H. Perry, Konstantin Chingin, Griffin Barbula Microfluidics 8 Single-cell Analysis on a Microfluidic Platform Eric Hall, Peng Guo, Romana Schirhagl 9 Development of a Cell Sorter Based on Integration of Porous Membranes into Layered Microfluidic Devices Romana Schirhagl Nanoparticles 10 Nanoparticle Formation Using Supercritical Fluid Technology Gunilla B. Jacobson, Tatsiana Lobovkina, Jun Ge, Peng Guo, Thomas Cahill 11 Encapsulation of Water-Soluble Compounds in Polymer Nanoparticles by Supercritical Carbon Dioxide Antisolvent Precipitation Jun Ge, Gunilla B. Jacobson, Tatsiana Lobovkina 12 Lipid Nanoparticles for Sustained Drug Delivery Tatsiana Lobovkina 13 Nanopore-Injection Method for Generating Organic Nanoparticles Peng Guo

Selected Publications 14 Zare Group Contact List 16 Office/Lab Floor Plans 18 Maps of Stanford & Vicinity 21

2 STATE-TO-STATE REACTION DYNAMICS Nate Bartlett, Justinas Jankunas, Tapas Goswami, Nandini Mukherjee

Using a home-built 3D ion imaging experimental apparatus1 we were previously able to study reactive and inelastic scattering in an H + D2 reaction to give HD (v′=1,3, j′) or D2 (v′=1-4, j′), respectively. The products of interest were state-selectively ionized using [2+1] resonance-enhanced multiphoton ionization (REMPI). The resulting ions were accelerated toward a multichannel plate (MCP) coupled to a delay-line anode which measures the 3D velocity distribution of the reaction products which can then be converted to a differential cross section (DCS) using the PhotoLOC (photoinitiated reaction analyzed with the law of cosines) technique developed in the Zarelab. The experimental results on reactive scattering in an H + D2 → HD (v′=1, j′) + D reaction agreed well with the theoretical predictions.2 As the rotational excitation of HD increased the DCS shifted from backward to sideward scattering, again in an agreement with the theoretical predictions. H + D2 → HD (v′=3, j′) + D reaction exhibited an even richer dynamics.3 In particular, time-delayed forward scattering was attributed to a glory effect that resulted from a near- and far-side quantum interference. Inelastic scattering experiments also revealed a few interesting features.4 It was shown, for example, that the translational H atom energy was transformed into a vibrational D2 energy mainly via a bond elongation with forward scattered products rather than bond compression and backward scattered products. Additionally, it is expected that the spatial distribution of the rotational angular momentum J of collision products is not isotropic. In other words the M-states of the reaction products are expected to be populated unequally. We are actively working towards the goal of measuring DCS and product state distributions M-state selectively. By selecting the initial and final states of the REMPI process, as well as the polarization of light used, one can detect molecules M-state selectively. This technique has been successfully demonstrated recently 5,7 for pure samples of H2, HD, and D2 in our lab. However, it turns out that the weak line strengths of the required REMPI lines and the relatively small reaction cross section of the H + D2 reaction make this technique extremely difficult to apply to reaction products. There is another way to detect molecules M-selectively which we are currently exploring. In the presence of a strong electric field, the rotational levels of molecules are shifted and split into |M|-resolved components due to the Stark effect. Commercial Nd:YAG lasers can readily produce the fields required to produce this splitting. By syncing a “Stark” laser pulse with our product ionization laser we hope to obtain the first M - resolved DCS for the H + D2 reaction. Our section is also constantly exploring other interesting and exciting paths. For example we recently finished recording product branching ratios for the D + DBr → D2 + Br/Br* reaction, where Br* represents spin-orbit excited bromine.6 We found that as expected, there is negligible product formation through the non- adiabatic reaction pathways. We are also excited to return to working on polyatomic systems with the H + neopentane (C(CH3)4) reaction. We encourage you to read more about these exciting experiments below!

References: 1. K. Koszinowski, N. T. Goldberg, A. E. Pomerantz, and R. N. Zare, J. Chem. Phys. 125, 133503 (2006) 2. K. Koszinowski, N. T. Goldberg, J. Zhang, R. N. Zare, F. Bouakline, and S. Althorpe, J. Chem. Phys. 127, 124315 (2007) 3. N. T. Goldberg, J. Zhang, D. J. Miller, and R. N. Zare, J. Phys. Chem. A 112, 9366 (2008) 4. N. T. Goldberg, J. Zhang, K. Koszinowski, F. Bouakline, S. Althorpe, and R. N. Zare, PNAS 105, 18194 (2008) 5. N. C. M. Bartlett, D. J. Miller, R. N. Zare, D. Sofikitis, T. P. Rakitzis, and A. J. Alexander, J. Chem. Phys. 129, 084312 (2008) 6. J. Zhang, J. Jankunas, N. C.-M. Bartlett, N. T. Goldberg, and R. N. Zare, J. Chem. Phys. 132, 084301 (2010) 7. N. C.-M. Bartlett, J. Jankunas, R. N. Zare, and J. A. Harrison, Phys. Chem. Chem. Phys. (in press, 2010).

3 MEASUREMENT OF CARBON ISOTOPE RATIOS USING CAVITY RING-DOWN SPECTROSCOPY Doug Kuramoto

The measurement of isotopes ratios, such as 13C/12C, is important in chemistry and other fields, such as geology, since it provides useful information on formation and transport processes. The most common method used to measure isotope ratios is the isotope ratio mass spectrometer (IRMS), which is bulky, requires user expertise, and is costly. The measurement of isotope ratios can also be done through absorption spectroscopy using cavity ring-down spectroscopy (CRDS).1 We have developed an instrument capable of measuring carbon isotope ratios in organic samples (Fig. 1A). The sample is injected into a gas chromatograph (GC) where it is separated into its components. The effluent is passed through a catalytic combustor (C) consisting of platinum and oxidized nickel wires in a ceramic tube heated to 1150 ºC, which completely oxidizes the carbon in the sample to carbon dioxide. The combustion products are fed into a CRDS instrument to measure the concentrations of 12C16O16O and 13C16O16O. We refer to our setup as GC-C-CRDS. A mixture of methane, ethane, and propane injected in our instrument can be separated, oxidized, and measured as shown in Fig. 1B. The chromatographic peaks are used to determine the ratio of 13C/12C in the sample. The current instrument can measure isotope ratios with a precision of less than one part per thousand and an accuracy of less than a few parts per thousand. Our instrument does not currently match the capabilities of instruments based on IRMS, but we expect future improvements to make our instrument an attractive alternative to IRMS. Despite the present limitations, the performance is sufficient for certain applications such as those in the oil industry. We are currently measuring isotope ratios of hydrocarbon gasses produced from petroleum sources which can provide useful information for the characterization of an oil reservoir.

Figure 1. (A) The GC-C-CRDS instrument. The sample is separated using gas chromatography and combusted to produce carbon dioxide and then the isotopic carbon dioxide concentrations are measured using cavity ring-down spectroscopy. (B) Chromatograms of 12C16O16O and 13C16O16O produced from a mixture of methane, ethane, and propane.

References: 1 R. N. Zare, D. S. Kuramoto, C. Haase, S. M. Tan, E. R. Crosson, and N. M. R. Saad, Proc. Natl. Acad. Sci. (US) 106, 10928-10932 (2009).

4 TWO-STEP LASER MASS SPECTROMETRY OF TERRESTRIAL AND EXTRATERRESTRIAL MATERIALS Amy Morrow, Hassan Sabbah

Microprobe laser-desorption laser-ionization mass spectrometry (μL2MS) is a powerful and versatile microanalytical technique that is used to study organic molecules in situ in a wide range of terrestrial and extraterrestrial materials. The combination of focused laser-assisted thermal desorption and ultrasensitive laser ionization provides sensitivity, selectivity, and spatial resolution capabilities that are unmatched by traditional methods of analysis. Over the past decade, this laboratory has developed and applied the μL2MS technique in a number of different research projects. Some areas that we are currently focusing on are:  Instrument Development: To enhance the analytical ability of the μL2MS technique we are actively pursuing instrument developments. Our plans include: the addition of a camera to visualize desorption and development of the neutral plume; installation of a single photon ionization source to allow detection of PAHs without dependence on resonant absorption; and application of soft ion landing techniques to attempt to separate complex mixtures of species by mass.

 Stability of Organic Compounds Trapped in Aerogel: This study aims to further our understanding of the potential damaging effects of UV and proton radiation on compounds in both captured particles and innate organic compounds in low-density silica aerogel. Aerogel was a success on the NASA Stardust Mission and may be used for future particle-capture missions as well, making this a timely study.

 Meteoritics: Analysis of PAHs in meteorites, meteoritic acid residues and interplanetary dust particles (IDPs). Currently, the μL2MS is involved in investigating the aromatic hydrocarbon contents of the meteorite DaG 476 and fragments of the asteroid 2008TC3, otherwise known as Almahata Sitta.

 Petroleomics: Recently, this instrument has been applied to the ongoing controversy over the study of dominant molecular architecture in asphaltenes, a fraction of heavy oil consisting of highly polar and aromatic molecules. Currently, we are testing the ability of this technique to detect a wide variety of synthetic asphaltene model compounds falling in to two main classes of architecture: island-type and archipelago-type.

5 HADAMARD TRANSFORM TIME-OF-FLIGHT MASS SPECTROMETRY Griffin Barbula, Richard Perry, Konstantin Chingin

Because time-of-flight mass spectrometry (TOFMS) involves a pulsed detection method, efficient detection of continuous ion sources remains a challenge. Increases in duty cycle (the fraction of ions that are detected) usually come at the expense of mass resolution and/or mass range. In an effort to decouple these figures of merit, our lab has developed a novel form of TOFMS that offers a 100% duty cycle over a wide mass range. Briefly, in this method ions entering the MS are rapidly switched between two detection states using a known sequence. Because the modulation sequence is based on Hadamard matrices, we have termed this method Hadamard transforms time-of-flight mass spectrometry (HT-TOFMS). Rapid modulation results in multiple ion packets that simultaneously move through the drift region and interpenetrate one another as they fly. In contrast, in a traditional TOFMS experiment a single ion packet moves through the drift region and is detected before a new packet is introduced. In HT-TOFMS, the acquired signal is the time-shifted superposition of all the packets’ mass spectra which can be decoded using knowledge of the applied modulation sequence. Because the modulation scheme allows us to detect more ions per unit time when compared to traditional, on-axis TOFMS, HT-TOF produces mass spectra with increased signal-to-noise properties, permits greater detection sensitivity, or enables faster spectral acquisition. Some areas of active research are:  Imaging TOF: Because 100% duty cycle work requires a two anode detector, we have worked to expand our research using arbitrary position detection systems. We currently employ multichannel plate detectors with delay line anodes in acquisition of our HT-TOF data.  Stopped-Flow HT-TOFMS Kinetics: Because HT-TOF is a 50 or 100% duty cycle technique, more ions are collected within a given time window than with traditional TOFMS. This signal advantage can in turn be used to acquire more statistically significant spectra in a given time period. HT-TOFMS has the potential to push into the millisecond regime of kinetics where other modern MS is limited to seconds in full scan mode. Trypsin catalyzed hydrolysis kinetics of unlabeled polypeptide systems has been studied.  Coupling to Chromatographic and Electrophoretic Separations: The continuous nature and high spectral acquisition rate of HT-TOFMS make it an ideal detector for separation techniques, particularly those which produce time-narrow peaks.  DESI: Desorption electrospray ionization (DESI), an ambient pressure ionization technique, has shown promise for high sample throughput. By coupling DESI to HT-TOFMS, the sampling rate of DESI was tested in a regime not accessible by other MS techniques with lower spectral acquisition rates. DESI was shown to be capable of achieving sample rates in excess of 100 samples/s. Selected Publications: 1. Trapp, O.; Kimmel, J. R.; Yoon, O. K.; Zuleta, I. A.; Fernandez, F. M.; Zare, R. N. Continuous Two-Channel Time-of-Flight Mass Spectrometric Detection of Electrosprayed Ions. Angew. Chem. Int. Ed. 2004, 43, 6541-6544. 2. Kimmel, J. R.; Yoon, O. K.; Zuleta, I. A.; Trapp, O.; Zare, R. N. Peak Height Precision in Hadamard Transform Time-of-Flight Mass Spectra. J. Am. Soc. Mass Spectrom. 2005, 16, 1117-1130. 3. Yoon, O. K.; Zuleta, I. A.; Robbins, M. D.; Zare, R. N. Duty Cycle and Modulation Efficiency of Two-Channel Hadamard Transform Time-of-Flight Mass Spectrometry. J. Am. Soc. Mass Spectrom., 2005, 16, 1888-1901. 4. Yoon, O. K.; Zuleta, I. A.; Robbins, M. D.; Barbula, G. K.; Zare, R. N. Simple Template-Based Method to Produce Bradbury- Nielsen Gates. J. Am. Soc. Mass Spectrom. 2007, 18,1901-1908.. 5. Zuleta, I. A.; Barbula, G. K.; Robbins, M. D.; Yoon, O. K.; Zare, R. N. Micromachined Bradbury-Nielsen Gates. Anal. Chem. 2007, 79 9160-9165. 6. Robbins, M. D.; Yoon, O. K.; Zuleta, I. A.; Barbula, G. K.; Zare, R. N. Computer-Controlled, Variable-Frequency Power Supply for Driving Multipole Ion Guides. Rev. Sci. Inst. 2008, 79, 034702. 7. Yoon, O. K.; Robbins, M. D.; Zuleta, I. A.; Barbula, G. K.; Zare, R. N. Continuous Time-of-Flight Ion Imaging: Application to Fragmentation. Anal. Chem. 2008, 80, 8299. 8. Barbula, G. K.; Robbins, M. D.; Yoon, O. K.; Zuleta, I. A.; Zare, R. N. Desorption Electrospray Ionization: Achieving Rapid Sampling Rates. Anal. Chem. 2009, 81, 9035–9040.

UNFOLDING REACTION PATHWAYS IN LIQUIDS ON THE MILLISECOND TIME SCALE USING DESORPTION ELECTROSPRAY IONIZATION Richard H. Perry, Konstantin Chingin and Griffin Barbula

Identifying intermediates is essential to understanding chemical reactions. However, few analytical techniques can intercept transient species in solution on the millisecond timescale. The high speed, sensitivity and information content of mass spectrometry (MS) make it one of the preferred methods for studying chemical reactivity. A relatively new ionization method called desorption electrospray ionization (DESI) has advanced the abilities of MS by allowing chemical analyses in the open HV Reactant A N2 environment without the need for sample pretreatment In (Spray) DESI, charged microdroplets bombard a surface of interest m/z Desorption INTERMEDIATES to extract absorbed chemicals into secondary microdroplets. Electrospray Ionization (DESI) The microdroplets evaporate and enter the mass spectrometer less than 2 ms after leaving the surface. By Reactant B placing reagents in the spray and on the surface, chemical (Surface) reactions occurring in the secondary microdroplets are analyzed in real-time. So, species produced during the first Figure 1: Organometallic reaction intermediates are few milliseconds of a reaction are intercepted by DESI, detected following bombardment of a surface-bound Ru(II) providing valuable information about reaction pathways.[1] complex with charged droplets containing a ligand. The reaction studied is used to synthesize Ru(II) catalysts The current research areas involving mass containing amino alcohol ligands, which facilitate the spectrometric elucidation of reaction mechanisms include, asymmetric transfer hydrogenation of ketones and but are not limited to: imines.[1]

Mechanism of Fast Reactions  Organometallic catalysis  Hypergolic systems involving ionic liquids

New Technologies for Mass Spectrometry  High throughput screening of chemical reactivity  Intact Delivery of Air-Sensitive Catalysts to the mass spectrometer

Ionization Mechanisms  Microdroplet chemistry in DESI and electrospray ionization (ESI)  Energy transfer in DESI and ESI

References [1] R. H. Perry, M. Splendore, A. Chien, N. Davis, R. N. Zare, Angew. Chem. Int. Ed. 2010, accepted

SINGLE-CELL ANALYSIS ON A MICROFLUIDIC PLATFORM Eric Hall, Peng Guo

In the study of a biological population, how important is individuality? Are the members of the population so similar that the average behavior can describe them all, or are deviations significant enough to make this kind of description misleading? The conventional techniques in biology use a large number of cells and generate the ensemble-averaged values to describe cellular characteristics. These methods are fast and efficient ways of observation as long as the individual cells exhibit little deviation from this average behavior. However, if the deviations are significant, the large-scale ensemble averaging methods fail to give a proper picture of biological phenomena. A simple example will be the case of a bimodal distribution, where the cells with an average behavior actually represent a smaller fraction of the population. Recent advances in microfluidics opened up a new possibility in single-cell biology by providing the necessary toolkits for handling and analyzing individual cells. We believe that it is an opportune time to apply microfluidic technologies to investigate individuality of cells because important information relevant to the most pressing biological questions is very likely obfuscated by ensemble averaging techniques. Our section develops techniques for performing single-cell analysis on a microfluidic device, more commonly referred to as “lab-on-a-chip”. We have made pioneering contributions to the field, including the development of a device capable of capturing a single cell and delivering precise amounts of reagents,1 and an on-chip chemical cytometer integrated with a picoliter micropipette for cell lysis and derivatization.2 More recently, we have extended this technology to study the phycobilisome degradation process in individual cyanobacteria cells.3 There are currently two main goals in our section. The first is to develop a microfluidic device capable of capturing a large number of single cells and sustaining them in an on-chip culture for a prolonged period of time, using two layers of channels separated by a membrane of conical nanopores (Fig. 1). This will allow for time-resolved observation of a statistically significant number of single cells, an ability currently lacking in flow cytometry and traditional microscopy-based approach. The second goal is to integrate this design with an on- chip device capable of extracting and amplifying sufficient DNA from a single cell for sequencing (Fig. 2).

Fig. 1. Microfluidic channels and control Fig. 2. Cells are manipulated with microfluidic channels valves (black) facilitate the capture of single (black) and control valves (red) into chambers (blue) cells on a nanoporous membrane (red). designed to deliver specific nanoliter volumes of reagents.

References: 1. A.R. Wheeler, W.R. Throndset, R.J. Whelan, A.M. Leach, R.N. Zare, Y.H. Liao, K. Farrell, I.D. Manger, A. Daridon, Anal. Chem. 75, 3581 (2003). 2. H. Wu, A.R. Wheeler, R.N. Zare, Proc. Natl. Acad. Sci. U.S.A. 101, 12809 (2004). 3. B. Huang, H. Wu, D. Bhaya, A.R. Grossman, S. Granier, B.K. Kobilka, R.N. Zare, Science 315, 81 (2007).

8 DEVELOPMENT OF A CELL SORTER BASED ON INTEGRATION OF POROUS MEMBRANES INTO LAYERED MICROFLUIDIC DEVICES Romana Schirhagl, Huibin Wei

Layered microfluidic devices integrated with porous polycarbonate or polyester membranes have been widely used for mass transport control, immunoassays, and blood cell sorting. The placement of a semi-porous membrane at the interface of two channel layers is crucial to minimize unwanted crossover of fluid flows between microchannels while allowing diffusive mixing of reagents. We introduce an alternative strategy of directly using PDMS as a porous membrane itself to fabricate monolithic microfluidic devices. In this case, the integration of a porous PDMS membrane can be completed without clogging microchannels by plasma oxidation. To prepare porous PDMS membranes, SU-8 posts with different sizes are created on a silicon wafer. A thin film of PDMS is prepared by spin coating the wafer. The resulting thin PDMS with holes is lifted cleanly off the wafer using a specially designed cured PDMS frame. This method allows varying sizes of pores on a single membrane, compared to commercially available porous membranes with a fixed pore size. This concept was successfully used to separate platelets and white and red blood cells from each other. A microfluidic chip for sorting full blood samples was generated achieving separation efficiencies of 99.7%. Even better separation was observed with polystyrene particles.1 Because leukaemia cells differ from other bone marrow cells in size, one possible application is to sort, count and further analyse leukaemia stem cells.

Figure 1: scheme of a microfluidic device for blood cell sorting with an integrated porous membrane

References: 1. H. Wei, B. Chueh, H. Wu, E. Hall, C. Li, J.-M. Lina, and R. N. Zare, “Particle Sorting Using a Porous Membrane in a Microfluidic Device,” Lab on a Chip (submitted, 2010).

9 NANOPARTICLE FORMATION USING SUPERCRITICAL FLUID TECHNOLOGY Gunilla B. Jacobson, Tatsiana Lobovkina, Jun Ge, Peng Guo, Thomas Cahill

Drug delivery via nano-materials is a blossoming field in chemistry, chemical engineering, and the emergent field of biological engineering as an alternative to methods such as constructed bacterial strains to deliver genes to tumors. Inert delivery agents such as biodegradable nanoparticles (NPs) that conceal the gene/enzyme from degradative/immunological factors are preferred. In addition, the use of NPs in drug delivery promises to overcome several obstacles to effective treatment including the poor water-solubility and short circulation half-life of many small-molecule drugs, their stability in circulation, dose-limiting cytotoxicity, sub- therapeutic concentrations at the target site, drug resistance and an inconvenient dosing schedule. We have specific interest in preparing NPs of therapeutic compounds whose size and surroundings can be controlled. Important pharmaceutical issues, such as chemical and physical stability, dissolution rate, and therapeutic performance, are often related to particle size, morphology, and surface properties. By working on the nanoscale range new drug delivery systems can be explored, as well as increased target specificity along with lower dosage requirements and therefore lower unwanted side effects. We are using supercritical carbon dioxide as an antisolvent in the preparation of our nps. When a fluid is taken above its critical temperature (Tc) and critical pressure (Pc), it exists in a condition called a supercritical fluid (SCF), as illustrated in Fig. 1. It is the possibility of controlling the solvent properties of a SCF by small changes in temperature and pressure that makes SCFs unique for the desired tight process control. Also, the high diffusivity of SCFs allows much faster diffusion into the liquid solvent and formation of supersaturation of the solute. This in turn allows for much smaller nanosized particles to be formed, as well as offering better control of the size distribution, as compared to using liquid antisolvents, or other techniques such as jet milling. We are exploring the encapsulation of the NPs by various means for the purpose of increasing their stability, or optimizing their targeting, or both. The work ranges from fundamental studies of how NPs are formed in supercritical fluids to how they can be used in pharmaceutical applications by studying their use in sustained release experiments and distribution in living organisms. For the production of NPs, we use a wide variety of techniques including supercritical fluid technology, liquid precipitation via nanopore membrane, and electro-spraying.

Fig. 1. Phase diagram of carbon dioxide.

10 ENCAPSULATION OF WATER-SOLUBLE COMPOUNDS IN POLYMER NANOPARTICLES BY SUPERCRITICAL CARBON DIOXIDE ANTISOLVENT PRECIPITATION Jun Ge, Gunilla B. Jacobson, Tatsiana Lobovkina

Many therapeutically active biological compounds, such as genetic materials and proteins, are insoluble, easily denatured or degraded in most organic solvents. To overcome these obstacles, we utilize water-in-oil microemulsions to better disperse these macromolecular biological components in organic solvent. The mechanism to prepare drug encapsulated polymer nanoparticles utilizes water-in-oil microemulsion followed by antisolvent precipitating in supercritical carbon dioxide. The formation of the water-in-dichloromethane microemulsion systems have been achieved using n- octyl β-D-glucopyranoside, which is biocompatible and degradable, as the surfactant and n-butanol as the co- surfactant. The sizes of the water droplets range from several nanometers to nearly two hundred nanometers depending on the water content. These microemulsions are stable at room temperature and appear transparent indicating dispersion. We applied the water-in-oil microemulsion to disperse water-soluble compounds in the continuous oil phase which contains the polymer (Fig. 1). After injection of the stable microemulsion into supercritical carbon dioxide, the encapsulated polymer nanoparticles are obtained in powder form. For example, tRNA (used as model system for siRNA) was dissolved in the water phase of a water-in- dichloromethane microemulsion, while the biodegradable polymer poly(lactic acid)-poly(ethylene glycol) (PLA-PEG) was dissolved in the dichloromethane phase. The tRNA encapsulated nanoparticles in powder form were prepared by precipitating the microemulsion in supercritical carbon dioxide. The observed release over weeks of tRNA from PLA-PEG nanoparticles in vitro (Fig. 2) is promising for taking the next investigative step to studying sustained release of siRNA in vivo.

120 100 80 60 SEDS

c.r.% 40 1 20 2 0 3

0 5 10 15 20 25 t/days Fig. 1. Preparation of nanoparticles from Fig. 2. Cumulative release of tRNA from PLA- microemulsions using supercritical antisolvent PEG nanoparticles in PBS pH 7.4 buffer at 37 precipitation. The polymer is solubilized in the oC. 1: 5.1wt% tRNA loading; 2: 2.3wt% tRNA continuous oil phase, while water-soluble drugs are loading; 3: 1.2wt% tRNA loading (sample 1 and solubilized in the dispersed aqueous phase. The 2 were subjected to measurements without acronym SEDS refers to Solution Enhanced washing, while sample 3 was washed with PBS Dispersion by Supercritical fluids. to remove unencapsulated tRNA prior to the timed release measurement).

11 LIPID NANOPARTICLES FOR SUSTAINED DRUG DELIVERY Tatsiana Lobovkina

Methods of synthesizing lipid-based nanoparticles are rare and thus provide novel approaches for delivery of important therapeutic agents such as small interfering RNA (siRNA). siRNA is a class of double- stranded RNA molecules, 20-23 nucleotides long, which is used in development of nucleic acid-based therapeutics. It has been shown that cationic liposomes, complexed with oligonucleotides, are essential for efficient oligonucleotide delivery in vitro. These complexes are easy to prepare by combining aqueous solutions of liposomes and oligonucleotides.[1,2] These complexes show low efficiency in vivo, largely due to non-specific interactions (e.g. blood components), aggregations and short circulation time. Here, we propose an alternative approach for synthesizing lipid-based nanoparticles. Instead of mixing liposome and oligonucleotide aqueous solutions, we are preparing a hydrophobic ion pair consisting from cationic lipid (e.g. DOTAP) and siRNA. Complexation is achieved by extracting the siRNA (dissolved in the aqueous phase) into the organic solvent (containing the cationic lipid). This complex is held together by electrostatic interactions between positively charged lipid head group and negatively charged phosphate group of siRNA. We have demonstrated that the interaction is strong enough to provide sustained release of siRNA in aqueous solution (PBS) over two weeks. In addition, we are incorporating a cationic lipid/siRNA complex into solid lipid nanoparticles (SLN). SLN are particles made from lipids (e.g., tristearin) that remain solid at body temperature. SLNs combine the advantages of both the polymer-based formulations and the liposomal carriers. Because solid lipid nanoparticles are physically stable at body temperature, they protect incorporated drugs from degradation, and thus act as excellent carriers for controlled drug release. SLNs have an excellent biocompatibility and biodegradability, and are successfully used in parenteral, oral, ocular, nasal, and topical applications.[3]

References: 1. Barron, L.G., L.S. Uyechi, and F.C. Szoka, Cationic lipids are essential for gene delivery mediated by intravenous administration of lipoplexes. Gene Therapy, 1999. 6(6): p. 1179-1183. 2. Schroeder, A., et al., Lipid-based nanotherapeutics for siRNA delivery. Journal of Internal Medicine. 267(1): p. 921. 3. Wissing, S.A., O. Kayser, and R.H. Muller, Solid lipid nanoparticles for parenteral drug delivery. Advanced Drug Delivery Reviews, 2004. 56(9): p. 1257-1272.

12 NANOPORE-INJECTION METHOD FOR GENERATING ORGANIC NANOPARTICLES Peng Guo

The sustained and targeted release of pharmaceuticals is of great importance and has the potential to dramatically improve the quality of life for those struck with illness. This potential is challenging, but one approach is to create drug-loaded nanoparticles synthesized from biodegradable polymers. Our lab recently developed a facile strategy, based on the use of a nanoporous membrane, which separates two liquid reservoirs. By pumping one liquid into the other, across the membrane, nanoparticles at or near the exits of the membrane are generated. The experimental device is composed of a nanoporous membrane, which separates two solutions; the feed solution and receiver solution (Fig. 1). The biopolymer is chosen on two criteria: (1) soluble in the feed solution and (2) insoluble in the receiver solution. Then the feed solution is forced under pressure through the pores of the membrane into the receiver solution. Upon exiting the nanoporous membrane, the solubility of the biopolymer changes and it precipitates in the receiver solution. The size of the biopolymer nanoparticles are roughly controlled by both the size of the nanopore size and the flow rate. An example of this technique utilizing poly(D,L-lactic-co-glycolic acid)-block-poly(ethylene glycol) (PLGA-PEG) is illustrated in Fig. 2. This method of flowing liquid through a nanoporous membrane provides a general mechanism for incorporating guest molecules within the host nanoparticles. We believe that many other biodegradable polymer systems can be loaded with different organic compounds, which suggests the practical use of this technique in preparing pharmaceuticals in nanoparticle form for drug delivery.

Fig. 1. Method for producing organic Fig. 2. Typical scanning electron microscope (SEM) image nanoparticles though a nanoporous membrane. of PLGA-PEG nanoparticles. About 130 nm nanoparticles with a narrow size distribution.

Reference: 1. Guo, P.; Martin, C. R.; Zhao, Y.; Ge, J. And Zare, R. N. Nano Lett., 2010, 10, 2202-2206.

13 SOME SELECTED RECENT PUBLICATIONS

FUNDAMENTAL REACTION DYNAMICS STUDIES N. C.-M. Bartlett, J. Jankunas, R. N. Zare, and J. A. Harrison, "Time-Dependent Depolarization of Aligned D2 Caused By Hyperfine Coupling," Phys. Chem. Chem. Phys. (in press, 2010). N. Mukherjee and R. N. Zare, "Preparation of Polarized Molecules Using Coherent Infrared Multicolor Ladder Excitation," J. Chem. Phys. 132, 154302-1-9 (2010). N. T. Goldberg, J. Zhang, K. Koszinowski, F. Bouakline, S. C. Althorpe, and R. N. Zare, "Vibrationally Inelastic H + D2 Collisions Are Forward Scattered," Proc. Natl. Acad. Sci. USA 105, 18194-18199 (2008). N. C. M. Bartlett, D. J. Miller, A. J. Alexander, D. Sofikitis, T. P. Rakitzis, and R. N. Zare, "Time- Dependent Depolarization of Aligned HD Molecules," Phys. Chem. Chem. Phys. 11, 142-147 (2008). S. J. Greaves, E. Wrede, N. T. Goldberg, J. Zhang, D. J. Miller, and R. N. Zare, "Vibrational Excitation Through Tug-of-War Inelastic Collisions," Nature 454, 88-91 (2008). J. A. Beswick and R. N. Zare, "On the Quantum and Quasiclassical Angular Distributions of Photofragments," J. Chem. Phys. 129, 164315-1-9 (2008). K. Koszinowski, N. T. Goldberg, J. Zhang, R. N. Zare, F. Bouakline, and S. C. Althorpe, “Differential cross section for the H + D2  HD (v = 1, j = 2, 6, 10) + D reaction as a function of collision energy,” J. Chem. Phys. 127, 124315 (2007). M. R. Martin, D. J. A. Brown, A. S. Chiou, and R. N. Zare, “Reaction of Cl with CD4 excited to the second C-D stretching overtone,” J. Chem. Phys. 126, 44315-44316 (2007). ABSORPTION SPECTROSCOPY Thermal Lensing F. Yu, A. A. Kachanov, A. Wainright, and R. N. Zare, "Ultraviolet Thermal Lensing Detection of Amino Acids," J. Chromatography A 1216, 3423-3430 (2009). Cavity Ring-down Spectroscopy R. N. Zare, D. S. Kuramoto, C. Haase, S. M. Tan, E. R. Crosson, and N. M. R. Saad, "High-Precision Optical Measurements of 13C/12C Isotope Ratios in Organic Compounds at Natural Abundance," Proc. Natl. Acad. Sci. (US) 106, 10928-10932 (2009). MASS SPECTROMETRY Two-Step Laser Mass Spectrometry of Terrestrial and Extraterrestrial Materials H. Sabbah, A. L. Morrow, A. E. Pomerantz, O. C. Mullins, X. Tan, M. R. Gray, K. Azyat, R. R. Tykwinski, and R. N. Zare, "Comparing Laser Desorption Laser Ionization Mass Spectra of Asphaltenes and Model Compounds," Energy Fuels (in press, 2010). H. Sabbah, A. L. Morrow, P. Jenniskens, M. Shaddad, and R. N. Zare, "Polycyclic aromatic hydrocarbons in asteroid 2008 TC3: dispersion of organic compounds inside asteroids," Meteoritics & Planetary Science (in press, 2010). A. Pomerantz, M. Hammond, A. Morrow, and R. N. Zare, "Asphaltene Molecular Mass Distribution Determined by Two-Step Laser Mass Spectrometry," Energy & Fuels 23, 1162-1168 (2009). A. E. Pomerantz, M. R. Hammond, A. L. Morrow, O. C. Mullins, and R. N. Zare, "Two-Step Laser Mass Spectrometry of Asphaltenes," J. Am. Chem. Soc. 130, 7216-7217 (2008). M. K. Spencer, S. J. Clemett, S. A. Sandford, D. S. McKay, and R. N. Zare, "Organic Compound Alteration during Hypervelocity Collection of Carbonaceous Materials in Aerogel," Meteoritics and Planetary Science 44, 15-24 (2009). Hadamard Transform Time-of-Flight Mass Spectrometry G. K. Barbula, M. D. Robbins, O. K. Yoon, I. Zuleta, and R. N. Zare, "Desorption Electrospray Ionization: Achieving Rapid Sampling Rates," Anal. Chem. 81, 9035–9040 (2009). O. K. Yoon, M. Robbins, I. Zuleta, G. Barbula, and R. N. Zare, "Continuous Time-of-Flight Ion Imaging: Application to Fragmentation," Anal. Chem. 80, 8299-8306 (2008). MICROFLUIDICS AND SINGLE-MOLECULE SPECTROSCOPY 14 S. Kim and R. N. Zare, "Microfluidic Platforms for Single-Cell Analysis," Annu. Rev. Biomed. Eng. 12, 187-201 (2010). Y. Luo and R. N. Zare, "Perforated Membrane Method for Fabricating Three-Dimensional Polydimethylsiloxane Microfluidic Devices," Lab on a Chip 8, 1688-1694 (2008). S. Granier, S. Kim, A. M. Shafer, V. R. P. Ratnala, J. J. Fung, R. N. Zare, and B. K. Kobilka, “Structure and conformational changes in the C-terminal domain of the beta 2-adrenoceptor: insights from fluorescence resonance energy transfer studies,” J. Biol. Chem. 282, 13895 (2007). B. Huang, H. Wu, D. Bhaya, A. R. Grossman, S. Granier, B. K. Kobilka, and R. N. Zare, “Counting low-copy-number proteins in a single cell,” Science 315 (2007). H. K. Wu, B. Huang, and R. N. Zare, “Generation of complex, static solution gradients in microfluidic channels,” J. Am. Chem. Soc. 128, 4194-4195 (2006). B. Huang, H. K. Wu, S. Kim, B. K. Kobilka, and R. N. Zare, “Phospholipid biotinylation of polydimethylsiloxane (PDMS) for protein immobilization,” Lab on a Chip 6, 369-373 (2006). NANOPARTICLE SYNTHESIS, CHARACTERIZATION AND APPLICATION G. B. Jacobson, E. Gonzalez-Gonzalez, R. Spitler, R. Shinde, D. Leake, R. L. Kaspar, C. H. Contag, and R. N. Zare, "Biodegradable Nanoparticles with Sustained Release of Functional siRNA in Skin," J. Pharm. Sci. (in press, 2010). P. Guo, C. R. Martin, Y. Zhao, J. Ge, and R. N. Zare, "General Method for Producing Organic Nanoparticles Using Nanoporous Membranes," Nano Lett. 10, 2202-2206 (2010). G. B. Jacobson, R. Shinde, R. L. McCullough, N. J. Chen, A. Creasman, A. Beyene, R. P. Hickerson, C. Quan, C. Turner, R. L. Kaspar, C. H. Contag, and R. N. Zare, "Nanoparticle Formation of Organic Compounds with Retained Biological Activity," J. Pharm. Sci. 99, 2750-2755 (2010). G. B. J. Andrews, R. Shinde, C. H. Contag, and R. N. Zare, "Drugs Dispersed in Polymer Nanoparticles for Sustained Release," Angew. Chem. Int. Ed. 47, 7880-7882 (2008).

15 ZARELAB CONTACT LIST

Lab Member Office Phone1 Office/Lab2 EMAIL Griffin BARBULA 723-4332 M 017B [email protected] Nate BARTLETT 723-4333 M 017A [email protected] Thomas CAHILL 723-8280 C E250 [email protected] Konstantin CHINGIN 723-4333 M 017A [email protected] Jun GE 723-8280 C E250 [email protected] Tapas GOSWAMI 725-2983 M 315A [email protected] Peng GUO 723-8280 C E250 [email protected] Eric HALL 723-8280 C E250 [email protected] Gunilla JACOBSON 725-0690 C E250 [email protected] Justin JANKUNAS 725-2983 M 315 [email protected] Doug KURAMOTO 723-4333 M 017A [email protected] Tanya LOBOVKINA 723-8280 C E250 [email protected] Barbara MARCH 723-4313 M 133 [email protected] Amy MORROW 723-4318 M 317 [email protected] Nandini MUKHERJEE 723-2983 M 315A [email protected] Manik PRADHAN 723-4333 M 017A [email protected] Richard PERRY 723-4334 M 017C [email protected] Hassan SABBAH 723-4318 M 317 [email protected] Romana SCHIRHAGL 723-8280 C E250 [email protected] Dick ZARE 723-3062 M 133 [email protected] 1 Area Code: 650 2 M = S. G. Mudd building; C = J. H. Clark Center

16 THE ZARELAB

Richard Zare

Griffin Barbula Nate Bartlett Tom Cahill Kostya Chingin Jun Ge

Tapas Goswami Peng Guo Eric Hall Gunilla Jacobson Justin Jankunas

Doug Kuramoto Tanya Lobovkina Barbara March Amy Morrow Manik Pradhan

Richard Perry Nandini Mukherjee Hassan Sabbah Romana Schirhagl

17 FLOOR PLAN OF LABS AND OFFICES – S.G. MUDD BLDG. BASEMENT

Prep Room Machine 017C Office 017B Office Shop

State-to-State

Chemistry Lounge/Kitchen Area 017A Office Room 015 Room 017

Stairs

Elevator

Storage

Sub Basement

Room 053

Flammables

Cavity Ring-Down Hadamard Time-of-Flight Spectroscopy Mass Spectrometry

18 FLOOR PLANS OF LABS AND OFFICES – S.G. MUDD BLDG. 1st and 3rd FLOOR

315B Office 315A Office

H + H 2 Reaction Dynamics Two-Step Laser Mass Spectrometry

317A Office Room 317 Room 315

MUDD, 3rd FLOOR

131 Lab Manager Chemistry Department

Main Offices

133 Professor Zare

Stairs Down Elevator

Stairs Up

MUDD, 1ST FLOOR

19 FLOOR PLAN OF LABS – J.H. CLARK CENTER

Other Research

Groups

Supercritical Fluids (SAS) Suite E277

Single Molecule Suite E276

SAS

Single SAS Molecule

Single Molecule

Suite E250

Kitchen Stairs (Campus Dr.) Administrative Office

Elevator

s CLARK CENTER, EAST WING

20 STANFORD CAMPUS MAP

ZARELAB (West) ZARELAB J.H. Clark Center Mudd Chemistry Bldg 318 Campus Drive 333 Campus Drive

STANFORD VICINITY http://stanford.edu/home/visitors/vicinity.html

22 NOTES