RESEARCH ACTIVITIES 1. Fundamental Dynamic Studies with Neutrals 2. Absorption Spectroscopy 3. Mass Spectrometry 4. Capillary Electrophoresis 5. Microfluidics and SPR Imaging 6. Biosensors 7. Single-Molecule Fluorescence Spectroscopy 8. Supercritical Fluids

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 my lab manager, Dr. David

Leahy, or any other members of my group to

discuss any project.

On page 20 is a list of all members of the Zare

group as of September 1, 2007 and information on

how to contact them. On pages 22 and 23 are floor

plans of the offices and the labs in S.G. Mudd. On

page 24 is the floor plan of our lab on the second

floor of the East wing at the J.H. Clark Center,

which is across the street from Mudd. The last

pages show maps of the Stanford campus and its

vicinity.

Do enjoy your visit!

INSIDE THIS GUIDE

Table of Contents…………………………………….2

Research Activities…………………………………..3

Selected Recent Publications…………………..18

Group Members……………………..……………..20

Group Member Photos…………………………...21

Floor Plans of Offices and Labs……………….22

Maps of Stanford Campus………………..…….25

GUIDE TO THE ZARELAB September 2007 Department of Chemistry, Stanford University

1

2

TABLE OF CONTENTS

I. Fundamental Dynamic Studies with Neutrals 3

Reaction Dynamics

Noah Goldberg, Jianyang Zhang, Dan Miller, Nate Bartlett 3

II. Absorption Spectroscopy 5

Rayleigh Scattering Measurements Using Cavity-Ring Down Spectroscopy

Douglas Kuramoto 5

Measurement of Volatile Organic Compounds (VOC) Using Cavity-Ring Down Spectroscopy (CRDS)

Vijay Surla 6

Ultra-violet Thermal Lensing

Fang Yu, Alex Kachanov 7

III. Mass Spectrometry 8 Two-Step Laser Mass Spectrometry of Terrestrial and Extraterrestrial Materials

Maegan Spencer, Matthew R. Hammond 8

Hadamard Transform Time-of-Flight Mass Spectrometry

Ignacio Zuleta, Oh Kyu Yoon, Matthew Robbins, Griffin Barbula 9

Phosphopeptide Enrichment From Peptide Mixtures For Zirconium Phosphonate Nanoparticles

Songyun Xu, Harvey Cohen 10

IV. Capillary Electrophoresis 11

Photopolymerized Sol-Gel as Chromatographic Media and Chemical Reactors

Maria T. Dulay 11

V. Microfluidics and Surface Plasmon Resonance Imaging 12 Developing Microfluidic Chips for Coupling Capillary Electrophoresis With Matrix-Assisted Laser

Desorption Ionization Mass Spectrometry

Yiqi Luo 12

Combining Surface Plasmon Resonance Imaging With Microfluidic Chips: A New Way to do

Immunoassays

Yiqi Luo, Fang Yu 13

VI. Biosensors 14 Use of Nanopores as Resistive-Pulse Biosensors

David Altman, Mozzi Etemadi 14

VII. Single-Molecule Fluorescence Spectroscopy 15

Single-Molecule Fluorescence Spectroscopy

Samuel Kim, Eric Hall 15

Single-Cell Analysis on a Microfluidic Platform

Eric Hall, Samuel Kim, Adam Knepp 16

VIII. Supercritical Fluids 17

Nanoparticle Formation and Encapsulation Using Supercritical Fluids

Gunilla B. Jacobson Andrews 17

Selected Recent Publications 18

Zare Group Members 19

Zare Group Photos 20

Floor Plans of Offices and Labs 21

Maps of the Stanford Campus 24

3

REACTION DYNAMICS

Noah Goldberg, Jianyang Zhang, Dan Miller, Nate Bartlett

Chemistry attempts to understand how atoms and molecules interact with each other and how they

combine to make new substances. The breaking and forming of molecular bonds is fundamental to this process.

Our goal is to obtain a detailed microscopic understanding of chemical transformations (specifically how bonds

break and form) by investigating simple, bimolecular chemical reactions in the gas phase. This medium allows

for the precise specification of the initial reagent conditions, isolation of a single chemical event, and quantum-

state-specific detection of the products. In this way, we gain insight into the complex molecular dance called

chemistry.

We co-expand a photolytic precursor AX, and a reactant BC through a single nozzle into the extraction

region of a Wiley-McLaren time-of-flight mass spectrometer. There, the precursor is photolyzed by a tunable,

polarized laser, producing fast A atoms with a well-defined speed and spatial distribution. After a single

collision with BC, the products of interest are state-selectively ionized via (2+1) resonance-enhanced

multiphoton ionization (REMPI) using a second laser and detected by a pair of microchannel plates. The signal

is analyzed using the photoloc (photo-initiated reaction analyzed by the law of cosines) technique to determine

the scattering angle distribution (differential cross section) in the center-of-mass frame, or integrated to obtain

the product state distributions.

H+H2 Reaction Dynamics

The simplest of all bimolecular reactions, the H + H2 reaction has been studied since the dawn of

modern quantum mechanics. The Zarelab has contributed a great deal to these studies over the last twenty years

and was among the first labs to provide experimental results sufficiently refined to compare with accurate

quantum mechanical calculations. Using the photoloc technique (photoinitiated reaction analyzed with the law

of cosines), we continue this tradition by providing state-to-state differential cross sections (DCSs) for both the

reactively and inelastically scattered products.

For the reactive channel, we have measured HD(v'=1, j') scattering angle distributions for collision

energies in the range 1.48 – 1.94 eV. These experiments agree nearly perfectly with fully converged quantum

mechanical calculations. Products with low rotational excitation are predominantly back scattered, and as j'

HBr/D2 or

Cl2/CH4/He

HBr/D2 or

Cl2/CH4/He

4

increases the distribution shifts toward side scattering. For most product quantum states the DCS depends very

weakly on the collision energy. These observations are consistent with the expectation that most reactive

collisions involve a direct recoil mechanism. For HD(v'=1, j'=2) we observe a second peak which grows as the

collision energy increases. This peak is believed to originate from one or more indirect mechanisms involving

scattering from the conical intersection.

Nonreactive collisions can transfer large amounts of energy into D2 vibration; each quantum of vibration

is roughly equal in energy to the reaction barrier on the minimum energy path. We have studied D2(v'=1–3, j')

products over the collision energy range 1.58 – 1.94 eV. Surprisingly, we observe that in most cases the DCS is

essentially independent of the product vibrational state! For D2(v'=3, j'≤4) a second peak appears in the DCS.

This peak shifts toward side scattering for lower j'—the opposite of the behavior seen for the major peak.

Neither peak appears to depend strongly on the collision energy. By comparing our experimental results to

quasi-classical trajectory calculations, we have tentatively attributed the second peak to trajectories that recross

the barrier multiple times.

These recent results demonstrate that there is still much to learn about this fundamental chemical

system. We see evidence for indirect reaction mechanisms that stray far from the minimum energy path, and at

high collision energies these mechanisms become important enough to compete effectively with the well known

direct recoil mechanism. More work is needed to assign specific mechanisms to individual peaks in the DCS,

and to explain the interesting behavior seen for inelastic collisions that transfer large amounts of energy into D2

vibration.

Polyatomic Reaction Dynamics

Our section has a long history of studying molecular photodissociation, atom + diatom scattering, non-

adiabatic effects, and polyatomic reactions. Currently, we are focusing on studying atom + polyatom scattering;

specifically, investigating the effect of vibrational and translational energy on the Cl + CH4/CHD3 reaction cross

section. For these experiments the collision energy of the reactants is tuned by varying the photolysis

wavelength of Cl2 while methane is excited to the ν=1 state using our tunable IR laser source. We have studied

the reaction at a variety of collision energies between 0.15-0.3 eV. By operating the IR laser at half the

repetition rate of the other lasers the excited state reaction is readily compared to that on the ground state.

Our preliminary results suggest that translation, if anything, is more effective than vibration in

promoting the reaction. This exciting result is in disagreement with the well-known Polanyi rules which state

that for atom + diatom reactions with an early barrier translation is more effective at promoting the reaction and

vice versa for those with a late barrier. Our results provide evidence that these simple rules cannot be extended

to the more complicated atom + polyatom systems; however, more studies will be needed to draw such

conclusions. One of our future goals is to investigate the effects of vibrational and translational energy on the H

+ C2H6 reaction and the H + C3H4 reaction.

5

RAYLEIGH SCATTERING MEASUREMENTS USING CAVITY RING-DOWN SPECTROSCOPY

Doug Kuramoto

Cavity ring-down spectroscopy (CRDS) is an ultrasensitive absorption technique that is capable of

measuring absorption changes of 10-10

cm-1

. In the simplest form of CRDS, two highly reflective mirrors face

one another to form an optical cavity. A laser pulse enters the cavity through the back of one mirror and

oscillates back and forth inside the cavity, leaking out a small amount of light. The rate constant for the

exponential decay of the light intensity depends upon all losses of light within the optical cavity. These losses

include mirror transmissions, absorption by the chemical sample, and reflection and scattering caused by the

sample and any optics within the cavity.

In most CRDS experiments, the absorbance of the sample is determined to measure a trace amount of a

species or to resolve a weak absorption peak that is below the detection limit of traditional absorption

techniques. We are interested in using CRDS to look at losses caused by the sample other than absorption,

more specifically losses caused by Rayleigh scattering. Much of the theory of Rayleigh scattering was

developed over 100 years ago. However, it has been difficult to make direct measurements in the laboratory

owing to the small cross section. The extended path length of CRDS makes it possible to measure the total loss

caused by atoms or molecules in the gas phase within the cavity. By operating in regions where there are no

absorption peaks, the total loss observed is caused primarily by Rayleigh scattering from which the Rayleigh

scattering cross section can be determined.

A three-mirror cavity in the ring configuration is currently being developed (Figure 1). A benefit of this

configuration is that it directs the reflection from the injected light away from the laser, eliminating the need for

an isolator. Another benefit of this configuration is that it provides for a small amount of optical feedback to

the laser that can be used to spectrally narrow the diode laser. This allows for greater efficiency in exciting the

cavity and helps to ensure single mode excitation. Once this instrument is set up, determinations of the

Rayleigh scattering cross section of molecules in the gas phase should be possible.

Figure 1. Three Mirror Cavity Ring-Down Spectroscopy Cavity

6

MEASUREMENT OF VOLATILE ORGANIC COMPOUNDS (VOC) USING CAVITY RING-DOWN

SPECTROSCOPY (CRDS)

Vijay Surla

Volatile organic compounds (VOCs) are organic chemical compounds that have high enough vapor

pressures under normal conditions to significantly vaporize and enter the atmosphere. VOCs include a wide

variety of carbon-based molecules, such as aldehydes, ketones, and other hydrocarbons. Such VOCs contribute

to both water and air pollution and may have short- and long-term adverse health effects. VOCs are emitted by a

wide array of products numbering in the thousands and it has been found that the concentration of VOCs in

indoor air is greater than in outdoor air.

In view of the adverse effects especially to the indoor air, there is a need to obtain simple, accurate, real-

time measurement of VOCs in order to better understand the health impact of human exposure to VOCs. The

current work proposes to build an instrument based on Cavity Ring-Down Spectroscopy (CRDS) to measure

VOCs. CRDS has been clearly established over the past decade as a robust, real-time, highly sensitive and

selective technology for measuring trace gas concentrations. Such an instrument could then be placed in the

field to continuously monitor and report VOC levels in targeted locations.

In the current work, the VOC under consideration is ethanol. The approach is to build a front end device

that will convert ethanol into CO2 and H2O by catalytic oxidation as shown in Fig 1. CRDS is then used to

measure the concentration of CO2 in the outlet stream from which we can trace backwards to obtain the

concentration of ethanol. Picarro Inc. has built a CRDS analyzer in the past to measure both CO2 and H2O with

very high sensitivity and will be used as the detector in the proposed work.

Figure 1. Approach: (in steps) 1. Catalytically convert ethanol into CO2 and H20 2.Use CRDS analyzer as a

detector to measure CO2 and H20. 3. Trace backwards to measure the concentration of ethanol from

the measured CO2 concentration.

CO2 + H2O

Reactor

Catalytic converter C2H5OH + Air

Mixing Chamber Detection (CRDS)

7

0.0

5.0x10-5

1.0x10-4

1.5x10-4

2.0x10-4

2.5x10-4

(+)

(+)

(+)(+)

(-)

(-)

(-)

(-)

(-)

(++)

(++)

(++)

(++) experimental expected

TL

sig

na

l (d

I/I

0)

ULTRA-VIOLET THERMAL LENSING

Fang Yu, Alex Kachanov

In recent years, thermal lensing (TL) technology, as ancient as the laser technology, has been

reconsidered as a detection technology to meet sensitivity and miniaturization requirements. TL detects a local

change of the refractive index induced by local heating that results from optical absorption of a laser beam

(excitation) focused within this small volume of sample. To achieve better sensitivity, a separate laser (probe) is

used to probe the lensing effect. The sensitivity TL can offer has been demonstrated to be extraordinary. In a

side-by-side comparison [1] reported by us, TL shows 140 times better sensitivity than a state-of-art commercial

UV-Vis detector. In some special cases, TL can even detect single molecules.

Most of the reported TL works are still lingering in the visible wavelength range. For wider TL

applications, a UV-excitation laser is desired for detection of various ‘colorless’ chemicals and biomolecules

without labeling. We have built a TL setup using a Q-switched ND-YAG laser (λ=1064 nm) with subsequent

frequency quadrupling crystal set that yields an output wavelength of 266 nm. The direct detection of non-

aromatic amino acids was tested. In excitation power dependence studies, we discovered that two-photon

absorption dominates the UV TL signal from all of the non-aromatic amino acids and photobleaching results in

discrepancies between the experimental signal and the expected signal from quadratic extrapolation.

Figure 1. Comparison between experimental thermal lensing signal (light gray) and expected thermal

lensing signal (dark gray) based on a quadratic fit of the excitation power dependent curve of 13

non-aromatic amino acids. The vulnerability to photobleaching of each amino acid is indicated

above, ranked by negative (-), weak positive (+), and strongly positive (++), respectively.

1. F. Li, A. A. Kachanov, and R. N. Zare, "Detection of Separated Analytes in Subnanoliter Volumes Using

Coaxial Thermal Lensing," Anal. Chem. 79, 5264-5271 (2007).

8

TWO-STEP LASER MASS SPECTROMETRY OF TERRESTRIAL AND EXTRATERRESTRIAL

MATERIALS

Maegan Spencer, Matthew R. Hammond

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 through a number of different research projects. These include:

Meteoritics: Analysis of polycyclic aromatic hydrocarbons (PAHs) in meteorites, meteoritic acid residues,

and interplanetary dust particles (IDPs). The spatial mapping ability of µL2MS gives us the ability to correlate

PAHs with meteoritic structural features, yielding insight into conditions present during solar system formation.

Similarities and differences among meteoritic classes have also been further elucidated through the

characterization of meteoritic PAH ratios. Laser-based mass spectrometry methods are also being used to study

the insoluble organic matter (IOM) of carbonaceous chondrites.

Cometary PAH Chemistry: Characterization of PAHs in cometary particles and IDPs captured by the

NASA Stardust mission, which returned cometary coma particles from Wild2 to Earth in January 2006.

Comparison of cometary PAHs with our knowledge of PAHs in meteorites will provide more information on

the presolar nebula.

Abiotic Organic Synthesis: Study of the formation and chemical reactivity of PAHs in various Earth and

space environments. These include simulations of the atmospheric chemistry of Titan, analysis of interstellar ice

analogs, investigation of hydrothermal vent water from Kamchatka, a volcanic region of Siberia, and

transformations of organic molecules under high-pressure conditions. With these studies we hope to gain an

understanding of PAH abiotic synthesis/reactivity in order to learn more about the origin of complex organics

on Earth and other planets.

Soil Remediation: µL2MS detection of PAHs and polychlorinated biphenyls (PCBs) in contaminated marine

sediments provides information about where these toxic, persistent, and bioaccumulative compounds are located

and can be used to compliment soil remediation projects.

Instrument Development: In order to enhance the analytical ability of the µL2MS technique we are actively

pursuing instrument development and characterization. Our main focus is the addition of gas chromatography

between desorption and ionization steps, allowing for a more comprehensive and capable analytical technique.

9

HADAMARD TRANSFORM TIME-OF-FLIGHT MASS SPECTROMETRY

Ignacio Zuleta, Oh Kyu Yoon, Matthew Robbins, Griffin Barbula

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 that our research is focusing on are:

• Tandem HT-TOFMS: Traditional tandem mass spectrometry involves fragmentation of a single mass-selected ion

species with the goal of exploring the structure of that ion. We are developing methods to modulate the

fragmentation process with the goal of simultaneously measuring the fragments from multiple analytes.

• New Ion Gating Devices and Ion Optics: In HT-TOFMS, Bradbury-Nielson gates are used for modulation of the

ion beam. We continue to develop techniques for macro- and microfabrication of these devices and test their

applicability for our method.

• 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 phosphor

screen and delay line anodes in acquisition of our HT-TOF data.

• 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.

• 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 that produce

time-narrow peaks. We are currently building equipment for high temperature HPLC to take advantage of this

property.

• Environmental Mass Spectrometry: We are working on a long-term collaborative effort to implement the previous

advances in portable ion mobility-HT-TOF instruments as well as in developing atmospheric pressure ion sources

that will be amenable to direct analysis of seawater.

10

2000 3000 4000

0

20

40

60

2000 3000 4000

0

75

150

A: direct analysis of casein digest mixture 1:1 BSA

***

*

**

inte

nsity

**** *

**

*

*

**

*

*

**

B: ZrP enrichment of casein digest mixture 1:1 BSA

PHOSPHOPEPTIDE ENRICHMENT FROM PEPTIDE MIXTURES FOR ZIRCONIUM

PHOSPHONATE NANOPARTICLES

Songyun Xu, Harvey Cohen

Protein phosphorylation is among the most important post-translational modifications (PTM) events in

eukaryote cells, which touches the primary biological processes of cell division, growth, migration,

differentiation, and intercellular communication.1-3

Mass spectrometry (MS) such as matrix-assisted laser

adsorption/ionization (MALDI) and electrospray ionization (ESI) has become the predominant means of

identifying phosphorylation sites in native and recombinant protein.

A new phosphopeptide enrichment method using zirconium phosphonate nanoparticles has been

developed. The high specificity of this approach was demonstrated by isolation of phosphopeptides from

complex peptide mixture digested from standard phosphopreotein alpha-casein and beta-casein and

nonphosphopretein bovine serum albumin. Figure 1A shows that the direct analysis of alpha-casein and beta-

casein and BSA digest solution. From the mass spectrum, It can be seen that ion signal of phosphopetide is

weak compared to nonphosphopeptide ion signal. However, when using zirconium phosphonate nanoparticle to

pretreat casein and BSA digest mixture, it is clear that all phosphopeptides from phosphoprotein can be detected

(as shown figure 1B).

Figure 1. Mass Spectra of casein and BSA digest solution (A) direct analysis, (B) using zirconium phosphonate

nanoparticle to selectively bind the phosphopeptide.

1. M.J. Hubbard, P. Cohen, Trends Biochem. Sci. 172-177 (1993).

2. T. Pawson, J.D. Scott, Trends Biochem. Sci., 286-290 (2005).

3. T. Hunter, Cell, 113-127 (2000).

11

PHOTOPOLYMERIZED SOL-GEL AS CHROMATOGRAPHIC MEDIA AND CHEMICAL

REACTORS

Maria T. Dulay

Our work focuses on the use of porous photopolymerized sol-gel (PSG) materials to create

chromatographic media for separations as well as on-line chemical reactors. We developed a synthetic method

based on sol-gel chemistry (hydrolysis and condensation reactions) and photochemistry for the preparation of a

photopolymerized sol-gel (PSG) monolith. The methacrylate group of a trialkoxysilane reagent is

photoactivated to produce PSG in a 5-min reaction. The light source can be UV or visible. Our sol-gel

technique includes template-based processing where the silicate matrix is assembled around a suitable template

or porogen to form cavities of a specific size and shape within the cross-linked host. With the presence of free

silanol groups on the PSG monolith a variety of different functional groups can be covalently grafted to the

monolith surface, allowing us to tailor the selectivity of the monolith.

We have had considerable success in recent years with the use of PSG materials for separation and

preconcentration of dilute mixtures of analytes. The on-line preconcentration feature of our macroporous PSG

monolith offers an alternative to existing sample enrichment schemes. While the typical injected sample

volume ranges from 1 nL to 30 nL, the PSG monolith allows for injection volumes up to 10 µL because of high

mass transfer and high convective flow in the monolith structure, allowing up to 1000-fold preconcentration of

dilute test samples. We are currently working on creating a protein and peptide concentrator on-line with

capillary electrophoresis.

The PSG monolith can be thought of as a building block for the preparation of different on-column

chemical devices, including enzyme microreactors, immunoaffinity materials through the bonding of an

antibody, and catalytic reactors for organic transformations. These devices are formed in a capillary column

downstream from separation techniques such as capillary electrophoresis and capillary electrochromatography.

We have demonstrated the use of trypsin-immobilized PSG materials for on-line enzymatic digestions with

2000 times enhancement in the digestion rate of an artificial substrate as compared to the bulk solution rate.

Our success with trypsin-PSG materials for capillary columns is the basis for our current work that involves the

creation of enzyme microreactors in plastic pipette tips.

More generally, we want to expand the use of the PSG material for on-line organic reactions. The PSG

monolith can be used to entrap catalytic materials or the monolith can be used as a support matrix for chemicals

that can effect a chemical reaction. For example, a PSG material with covalently bound amine groups has the

potential to be used in Knoevenagel condensation reactions at room temperature with downstream

chromatographic or electrophoretic separation of the products. A PSG chemical reactor will allow us to

combine synthesis, separation, and detection of products in one step in our capillary system. The advantages of

such a device include small volumes of reagents and starting materials and an increase in the number of

different reactions that can be run. In addition, there is some evidence to suggest that immobilization of a

catalyst can lead to improvements in the efficiency and activity of the catalyst.

12

DEVELOPING MICROFLUIDIC CHIPS FOR COUPLING CAPILLARY ELECTROPHORESIS

WITH MATRIX-ASSISTED LASER DESORPTION IONIZATION-MASS SPECTROMETRY

Yiqi Luo

Recently, mass spectrometry (MS) has attracted a lot of attention as a novel tool for increasingly expanding

applications on peptide and protein analysis. As one of the most widely used MS mode, matrix-assisted laser

desorption ionization-mass spectrometry (MALDI-MS) shows excellent

accuracy, high sensitivity and distinguished stability. However, MALDI-

MS also has some drawbacks, such as (1) the signals of analytes is

reduced in salty environment and (2) the signal of some analytes is

suppressed by other analytes in a multi-component sample due to

unequal absorbance to laser energy. Therefore, sample preparation such

as desalting and separating components in complex samples.

As a powerful chromatographic technology, capillary

electrophoresis (CE) especially free-solution CE is rarely reported to be

coupled with MALDI-MS. The main reason is that the motion of

analytes is driven by an electric field applied in the capillary, which

gives difficulties to interface CE separation with depositing analytes on

MALDI sample plates that is usually implemented by a pressure-driven

flow of analytes and bulk solution. Therefore, it is necessary to

fractionate separated analytes before transferring them out of the

capillary when serially coupling CE and MALDI-MS. By using a

microfluidic chip with active actuators, fractionation and

transferring of analytes after CE separation can be achieved, so

that CE and MALDI-MS may be coupled in microfluidic chips

for microscale analysis.

To develop a microfluidic chip prototype for this purpose,

a row of fractionation valves is positioned right above the main

channel (the longest horizontal channel used as the capillary

for CE separation), as shown in Figure 1. After sample loading

in the double-T junction located to the left, CE separation is

carried out by applying voltage across the main channel. The

fractionation valves close immediately after the CE separation

is accomplished. Then the microfluidic pump (three parallel

horizontal valve series) actuates to pump the fractions of

separated sample into reservoirs through the side channels

connecting with the main channel for transferring separated

sample out of the microfluidic chip for MALDI-MS analysis.

The procedure is shown in Figure 2 where food colorings are

used to visualize the manipulation steps. Applications of the

system are under investigation.

Figure 1. Layout and photograph

of the microfluidic chip designed

for coupling CE and MALDI-MS.

Figure 2. (a)-(f) The procedure of CE

separation, fractionation of the separated

sample and pumping the fractions into

reservoirs.

(a) (b)

(c) (d)

(e) (f)

13

COMBINING SURFACE PLASMON RESONANCE IMAGING WITH MICROFLUIDIC CHIPS:

A NEW WAY TO DO IMMUNOASSAYS

Yiqi Luo, Fang Yu

Immunoassays, a type of methods for identifying and determining biomolecules through antibody-antigen

recognition and binding, have become one of the most powerful technologies in current biomedical research and

diagnostics. Regular immunoassays, such as the widely used enzyme-linked immunosorbent assay (ELISA) and

fluorescent immunoassay, are carried out in sub-milliliter volumes without sample replenishment and rely on

molecular labels to generate signals during measurements, which are usually colorimetric or fluorescent. The

ELISA measurement often takes hours to complete. This

long time is needed both for the multiple steps involved

and the need for each step to reach equilibrium.

Moreover, sample consumption is at the sub-milliliter

level, which for rare samples can present an important

limitation.

To develop an immunoassay system for more rapid

analysis and for lower sample consumption, we combine

a microfluidic chip, made of polydimethylsiloxane

(PDMS) and an array of thin gold spots, with a surface

plasmon resonance (SPR) imaging system. This concept

offers significant advantages: (1) the microfluidic chip

provides nanoliter volume sample loading channels and

reaction chambers, which reduces the sample consumption;

(2) the large surface area-to-volume ratio in the

microfluidic chip significantly enhances the efficiency of

antibody-antigen binding; and (3) SPR, which senses

refractive index change caused by molecules binding to a

metal surface, provides label-free detection for the

immuno-species, which eliminates additional reaction and

washing steps. Thus, a clear read-out of an antibody-

antigen reaction can be obtained typically in half an hour,

compared to several hours for standard ELISA. The

relatively large field of view of the SPR imaging system

and the easy-to-use microfabrication method for PDMS

(soft lithography) should facilitate further improvement of

the system throughput. Clinical applications of the system

are under investigation, with an extra step of signal

amplification by nanoparticle-labeled secondary antibody.

(a)

(b) (c)

Figure 2. (a) Background corrected end-point

SPR image of the microfluidic chip obtained

after anti-biotin antibody binding to

biotinylated BSA covered gold spots. (b)

Kinetic curves of anti-biotin antibody binding

to pure biotinylated BSA covered gold spots.

(c) End-point calibration curves of the density

of bound anti-biotin antibody versus its

working concentration.

Figure 1. Layout and photograph of the

microfluidic chip designed for coupling with

SPR imaging system.

14

USE OF NANOPORES AS RESISTIVE-PULSE BIOSENSORS

David Altman, Mozzi Etemadi

In a resistive-pulse sensor, a pore is placed between two electrolyte solutions. Application of a potential

difference on either side of the pore results in passage of an ion current through it. When an analyte of

comparable size passes through the pore, the ion current is transiently blocked, resulting in a corresponding dip

in current (see the figure below). The frequency and structure of this dip yields information about the

concentration, size, and electrical properties of the analyte. For a comprehensive discussion of resistive-pulse

sensing, see the review by Bayley et al.1

In collaboration with Dr. Henry White’s lab at the University of Utah, we are designing a resistive pulse

sensor that utilizes conical nanopores manufactured at the end of narrow glass capillaries. 2

We are studying

how the impedance of the pore is altered as analytes of varying sizes and electrical properties are detected. As a

long-term goal, we will use this sensor to discern the identities of cells passing through the pore by their

characteristic impedances. Such a detector can differentiate cell types in a varied population of cells, and could

thus be useful as a means of detecting pathological cells in a sample.

Cartoon depiction of a resistive pulse sensor. LEFT: Application of a potential difference

on either side of a pore in an electrolyte (represented by the charges) results in an ion

current across the pore (the green arrow). RIGHT: When an analyte (orange hexagon)

passes through the pore, the current decreases transiently.

1. H. Bayley and C.R. Martin, Chem. Rev. 100, 2575-2594 (2000).

2. B. Zhang, J. Galusha, P.G. Shiozawa, G. Wang, A.J. Bergern, R.M. Jones, R.J. White, E.N. Ervin, C.C. Cauley,

H.S. White, Anal. Chem. 79, 4778-4787 (2007).

15

Single-molecule spectroscopy of proteins. (A) Molecules diffusing at the laser focus. (B) 2-species

model fits the PCH data obtained from the receptor labeled with tetramethylrhodamine. (C) spFRET

histogram (top) and a model of a bovine rhodopsin reconstituted into HDL (bottom, from ref. 3).

SINGLE MOLECULE FLUORESCENCE SPECTROSCOPY

Samuel Kim, Eric Hall

Why bother to look at single molecules? Every chemistry student is taught to think of matter as being

composed of fundamental building blocks, i.e., molecules. Our understanding of chemical reactions comes

almost exclusively, however, from experiments on many molecules, where only average properties are

measured. It is only by looking at the behavior of a system molecule by molecule that we no longer measure

just the average behavior, but can study the full range of a property present in a sample. The same principle

applies to the single cell experiments. For example, if one were interested in studying the heights of people in a

crowd, the bulk behavior would yield one average height. It would be possible to gain a greater insight by

looking at the crowd person by person. By studying the distribution of heights, a deeper understanding can be

achieved. In this example, perhaps there are two subgroups of height (e.g., men being on average taller than

women). In the same way, examining a chemical system at the level of its individual components (in this case,

molecules) also provides a deeper insight into many of the problems tackled by science today.

We are currently developing a number of single-molecule fluorescence techniques and applying them to

solve biological questions.

• One fluorophore: photon-counting histograms (PCH). PCH is a method of fluorescence fluctuation

spectroscopy that is sensitive to the change in molecular brightness. By using this method, the

conformational dynamics of cytochrome c has been resolved.1 We are currently developing a combined

approach utilizing both the separation power of on-chip capillary electrophoresis and the single-

molecule sensitivity of PCH.

• Two fluorophores: single-pair fluorescence resonance-energy transfer (spFRET). Drug-induced

conformational change of β2 adrenergic receptor in detergent micelles was monitored using ensemble

FRET.2 With spFRET technique, conformations with different structures, thus different FRET

efficiencies, can be resolved. Current research focuses on the application of spFRET technique to the

receptor molecule reconstituted into a high-density lipoprotein (HDL) particle, which mimics the cell

membrane.3

(A) (B) (C)

1. T.D. Perroud, M.P. Bokoch, R.N. Zare, Proc. Natl. Acad. Sci, U.S.A. 102, 17570 (2005).

2. S. Granier, S. Kim, A.M. Shafer, V.R.P. Ratnala, J.J. Fung, R.N. Zare, B.K. Kobilka, J. Biol. Chem. 282, 13895

(2007).

3. M.R. Whorton et al, Proc. Natl. Acad. Sci. U.S.A. 104, 7682 (2007).

16

SINGLE-CELL ANALYSIS ON A MICROFLUIDIC PLATFORM

Eric Hall, Samuel Kim, Adam Knepp

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 (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

further improve the chemical cytometry technique developed in our lab (Fig. 2) by increasing its throughput and

optimizing the time-intensive cell lysis procedure.

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. 1010, 12809 (2004).

3. B. Huang, H. Wu, D. Bhaya, A.R. Grossman, S. Granier, B.K. Kobilka, R.N. Zare, Science 315, 81 (2007).

Figure 1. Microfluidic cell culture

array. Figure 2. On-chip chemical content analysis.

17

ENCAPSULATED NANOPARTICLES FOR TARGETED DRUG DELIVERY

Gunilla B. Jacobson Andrews

The Supercritical Fluids section develops nanoparticles for pharmaceutical applications, and specifically

to prepare particles whose size and surroundings can be precisely 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 reducing any unwanted side effects.

We use supercritical carbon dioxide as an antisolvent in the in the precipitation of particles from a liquid

cosolvent. When a fluid is taken above its critical temperature (Tc) and critical pressure (Pc), it exists in a

condition called a supercritical fluid (SCF), Fig. 1. The possibility to control the solvent properties of a SCF by

small changes in temperature and pressure makes SCFs uniquely qualified for tight process control. The high

diffusivity of SCFs allows much faster diffusion into the liquid solvent and formation of supersaturation of the

solute, which in turn allows for much smaller nanosized particles to be formed as well as control of the size

distribution.

In addition, we are exploring the encapsulation of the nanoparticles by various means for the purpose of

increasing their stability, allow for slow release, or for target specific delivery. The work ranges from

fundamental studies of polymer solubility/encapsulation in supercritical fluids to how they can be used in

pharmaceutical applications by studying their use in drug-controlled release experiments and distribution in

living organisms. Current applications include encapsulating siRNA for targeted drug delivery, using transgenic

mice to follow the fate of the particles in vivo.

Fig 1. Phase diagram of supercritical carbon dioxide.

18

SOME SELECTED RECENT PUBLICATIONS

FUNDAMENTAL DYNAMIC STUDIES WITH NEUTRALS

Reaction Dyanmics

N.T. Goldberg, J. Zhang, K. Koszinowski, D.J. Miller, and R.N. Zare, “Differential cross sections for

inelastic H + D2 collisions,” manuscript in preparation.

K. Koszinowski, N.T. Goldberg, J. Zhang, R.N. Zare, F. Bouakline, 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., in press (2007).

N.T. Goldberg, K. Koszinowski, A.E. Pomerantz, R.N. Zare, “Doppler-free ion imaging of hydrogen

molecules produced in bimolecular reactions,” Chem. Phys. Lett. 433, 439-443 (2007).

M.R. Martin, D.J.A. Brown, A.S. Chiou, R.N. Zare, “Raction of Cl with CD4 excited to the second C-D

stretching overtone,” J. Chem. Phys. 126, 44315-44316 (2007).

K. Koszinowski, N.T. Goldberg, A.E. Pomerantz, R.N. Zare, “Construction and calibration of an

instrument for three-dimensional ion imagining,” J. Chem. Phys. 125, 133503 (2006).

J.P. Camden, H.A. Bechtel, D.J.A. Brown, R.N. Zare, “Comparing reactions of H and CL with C-H

stretch-excited CHD3,” J. Chem. Phys. 124, 1-7 (2006).

MASS SPECTROMETRY

Two-Step Laser Mass Spectrometry of Terrestrial and Extraterrestrial Materials

J.E. Elsila, N.P. De Leon, P.R. Buseck, and R.N. Zare, “Alkylation of polycyclic aromatic

hydrocarbons in carbonaceous chondrites,” Geochimica et Cosmochimica Acta, 69, 1349 (2005).

S.A. Sanford, J. Aleon, C.M. O’D. Alexander, T. Araki, S. Bajt, G.A. Baratta, J. Borg, J.R. Brucato,

M.J. Burchell, H. Busemann, A. Butterworth, S.J. Clemett, G. Cody, L. Colangeli, G. Cooper, L.

D’Hendecourt, Z. Djouadi, J.P. Dworkin, G. Ferrini, H. Fleckenstein, G.J. Flynn, I.A. Franchi, M. Fries,

M.K. Gilles, D.P. Glavin, M. Gounelle, F. Grossemy, C. Jacobsen, L.P. Keller, A.L.D. Kilcoyne, J.

Leitner, G. Matrait, A. Meibom, V. Mennella, S. Mostefaoui, L.R. Nittler, M.E. Palumbo, F. Robert, A.

Rotundi, C.J. Snead, M.K. Spencer, A. Steele, T. Stephan, T. Tyliszczak, A. J. Westphal, S. Wirick, B.

Wopenka, H. Yabuta, R.N. Zare, M. Zolensky, “Organics captured from Comet Wild 2 by the Stardust

spacecraft,” Science 314, 1720-1724 (2006).

Hadamard Transform Time-of-Flight Mass Spectrometry

I.A. Zuleta, G.K. Barbula, M.D. Robbins, O.K. Yoon, R.N. Zare, “Micromachined Bradbury-Nielsen

Gates,” Anal. Chem., submitted (2007).

O.K. Yoon, I.A. Zuleta, M.D. Robbins, G.K. Barbula, R.N. Zare, “Simple template-based method to

produce Bradbury-Nielsen Gates,” J. Am. Soc. Mass Spectrom., in press (2007).

O.K. Yoon, I.A. Zuleta, J.R. Kimmel, M.D. Robbins, and R.N. Zare, “Duty cycle and modulation

efficiency of two-channel Hadamard Transform time-of-flight mass spectrometry,” J. Am. Soc. Mass

Spectrom. 16, 1888-1901(2005).

J.R. Kimmel, O.K. Yoon, I.A. Zuleta, O.Trapp, and R.N. Zare, “Peak height precision in Hadamard

Transform time-of-flight mass spectra,” J. Am. Soc. Mass Spectrom. 16, 1117-1130, (2005).

CAPILLARY ELECTROPHORESIS

N. Johannesson, E. Pearce, M. T. Dulay, R.N. Zare, J. Berquist, and K. E. Markides, “On-line

biological sample cleanup for electrospray mass spectrometry using sol-gel columns,” J. Chromatogr. B

842, 70-74 (2006).

M.T. Dulay, Q.J. Baca, and R.N. Zare, “Enhanced proteolytic activity of covalently bonded enzymes in

photopolymerized sol-gel,” Anal. Chem. 77, 4604-4610 (2005).

19

M.Kato, K. Sakai-Kato, H.M. Jin, K. Kubota, H. Miyano, T. Toyo’oka, M.T. Dulay, and R.N. Zare,

“Integration of on-line protein digestion, peptide separation, and protein identification using pepsin-

coated photopolymerized sol-gel monolithic column,” Anal. Chem. 76(7), 1986-1902 (2004).

SURFACE-PLASMON RESONANCE IMAGING

B. Huang, F. Yu, R.N. Zare, “Surface plasmon resonance imaging using a high numerical aperture

microscope objective,” Anal. Chem. 79, 2979-2983 (2007).

SINGLE-MOLECULE FLUORESCENCE SPECTROSCOPY

T.D. Perroud, M.P. Bokoch, R.N. Zare, “Cytochrome c conformations resolved by the photon-counting

histogram: watching the alkaline transition with single-molecule sensitivity,” Proc. Natl. Acad. Sci.

U.S.A. 102, 17570 (2005).

S. Granier, S. Kim, A.M. Shafer, V.R.P. Ratnala, J.J. Fung, R.N. Zare, 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, Rn.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).

20

ZARELAB CONTACT LIST

Lab Member Office Phone1 Office/Lab

2 EMAIL

David ALTMAN 723-4318 M 317 daltman@stanford.edu

Griffin BARBULA 723-4332 M 017B barbula@stanford.edu

Nate BARTLETT 723-4333 M 017A nbartlet@stanford.edu

Maria DULAY 723-8280 C E250 mdulay@stanford.edu

Noah GOLDBERG 725-2983 M 315A ntg@stanford.edu

Eric HALL 723-8280 M E250 erichall@stanford.edu

Matthew HAMMOND 723-4318 M 317 mrhammon@stanford.edu

Gunilla JACOBSON 725-0690 C E250 gunilla@stanford.edu

Samuel KIM 723-8280 C E250 slkim@stanford.edu

Doug KURAMOTO 723-4333 M O17A kuramoto@stanford.edu

Dave LEAHY 723-4393 M 131 djleahy@stanford.edu

Yiqi LUO 723-4333 M 017A rubenluo@stanford.edu

Barbara MARCH 723-4313 M 133 march1@stanford.edu

Dan MILLER 725-2980 M 315A dmiller2@stanford.edu

Matt ROBBINS 723-4398 M 017B mdr@stanford.edu

Maegan SPENCER 723-4318 M 317 mspencer@stanford.edu

Vijay SURLA 723-4334 M 017C vksurla@stanford.edu

Songyun XU 723-4334 M 017C syxu@stanford.edu

Oh Kyu YOON 723-4332 M 017B okyoon@stanford.edu

Fang YU 723-4334 M 017C fangy@stanford.edu

Dick ZARE 723-3062 M 133 zare@stanford.edu

Jianyang ZHANG 725-2983 M 315A jyzhang6@stanford.edu

Ignacio ZULETA 723-4398 M 017B izuleta@stanford.edu 1 Area Code: 650

2 M = S.G. Mudd building; C = J.H. Clark Center

21

David Altman Nate Bartlett Maria Dulay Noah Goldberg

Eric Hall Matt Hammond Gunilla Jacobson Samuel Kim Doug Kuramoto

David Leahy Yiqi Luo Barbara March Dan Miller Matt Robbins

Maegan Spencer Vijay Surla Songyun Xu Oh Kyu Yoon Fang Yu

Richard Zare Jianyang Zhang Ignacio Zuleta

Griffin Barbula

22

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

MUDD BASEMENT

Elevator

Stairs

Storage

Phospho-

peptide

Enrichment

Prep Room Machine

Shop

017C Office 017B Office

State-to-State

Chemistry

Lounge/Kitchen Area

017

017A Office

Sub Basement

Flammables

Electrospray Mass Spectrometry Cavity Ring-Down Spectroscopy

Absorption Spectroscopy

Thermal

Lensing

23

FLOOR PLANS OF LABS AND OFFICES – S.G. MUDD BLDG.

MUDD, 3rd

FLOOR

315A Office 315B Office

317A Office

Biosensors

Two-Step Laser Mass Spectrometry

Room 317 Room 315

H + H2

Reaction Dynamics

Chemistry Department

Main Offices

131

Lab Manager

133

Professor Zare

Elevator

Stairs

Down

Stairs

Up

MUDD, 1ST

FLOOR

24

FLOOR PLAN OF LABS – J.H. CLARK CENTER

CLARK CENTER, EAST WING

Capillary Electrophoresis (CE)

Capillary

Electrochromatography

(CEC)

SAS

Supercritical Fluids (SAS)

Suite E277

CE/CEC

Suite E276

Suite E250

Administrative Office

Elevator

s

Kitchen Stairs (Campus Dr.)

Other

Research

Groups

Single

Molecule

Single Molecule

SAS CE

CEC

25

STANFORD VICINITY

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

26

STANFORD CAMPUS MAP

Visitormap.pdf

ZARELAB

Mudd Chemistry Bldg

333 Campus Drive

ZARELAB (West)

J.H. Clark Center

318 Campus Drive