news
ANALYTICAL CURRENTS
15
Signal intensity
Protein analysis by MS could get easier by using a microcolumn that enables sample preconcentration, cleanup, and digestion in one place. Liang Li and colleagues from the University of Alberta (Canada) show that columns filled with protein-capturing microbeads can help identify analytes ranging from classic cyctochrome c to those found in a human plasma fraction. In a typical experiment, 100 µL of a 10 nM protein sample were pumped through the microcolumn containing hydrophobic beads and then digested for 30 min. With cytochrome c, this yielded 28 peaks by MALDI MS, which represented 91% sequence coverage; bovine serum albumin produced 43 peptides for 69% coverage. Good results were also obtained with just a 5-min digestion of these proteins, although some peaks were missing. The microcolumn also simplified the cleanup of organic and inorganic salts, and, with care, detergents, such as sodium dodecyl sulfate. The procedure also worked with an E. coli extract that was fractionated by anion exchange chromatography. For a hydrophobic protein, such as bacteriorhodopsin, an additional step using CNBr before the digestion aided in getting the protein onto the beads. Finally, the researchers used their approach to examine a fraction of human plasma. Using LC electrospray ionization with MS/MS, they identified 14 polypeptides from a single-column digest. (J. Proteome Res. 2002, 1, 537–547)
Br
(a)
10
Br Br
Br
5
Br
T
T
Br Br
Br Br
0 1200
1000
1400
1600
1800
2000
2200
m/z (b) 10
Signal intensity
Microcolumn improves MS identifications
8
6
Br T
4
Br Br
2
Br Br
T
Br Br Br
Br
0 1200
1000
1400
1600
1800
2000
2200
m/z
(a) An in-column digestion of bacteriorhodopsin using CNBr and trypsin yields better mass spectra than (b) an in-solution reaction with the same protein and reactants. Br marks bacteriorhodopsin fragments; T points out trypsin peptides.
SAMs by SIMS A new method for analyzing alkanethiol self-
SIMS measurements. To quantify their data,
the competitive binding for different thiols.
assembled monolayers (SAMs) bound to gold
the authors analyzed mixed monolayers con-
Thus, the authors found that the competitive
not only quantifies the level of coverage but
sisting of one of several four-carbon thiols
adsorption probability for 1-butanethiol with
also provides insights into how steric effects
and decanethiol. Values were recorded as
respect to decanethiol is about 220 times larg-
control surface adsorption. In this proof-of-
the mole ratio of the “short” thiol versus the
er than the more sterically hindered 2-methyl-
concept paper, Joseph Gardella and Limin
10-carbon analogue. The SIMS data were plot-
1-propanethiol. The data can also be used to
Sun from the State University of New York–
ted as a straight line versus this molar ratio.
predict the molar concentration ratios needed
Buffalo converted alkanethiols to sulfonates
Changing from one four-carbon thiol to
with ozone and determined surface coverage
another—for example, 1-butanethiol to 2-
butanethiol–decanethiol SAM would need a
using negative-ion secondary ion MS (SIMS).
methyl-1-propanethiol—yielded only straight-
5:1 ratio, whereas a 2-methyl-1-propanethiol–
The authors found that a 3-min exposure
line graphs but with different slopes. The ratios
decanethiol monolayer should require a 1000:1
of the slopes, in turn, were used to measure
mixture. (Langmuir 2002, 18, 9289–9295)
to ozone yielded sufficient oxidation for their
to form a 1:1 mixed SAM; for example, a 1-
F E B R U A R Y 1 , 2 0 0 3 / A N A LY T I C A L C H E M I S T R Y
51 A
news
ANALYTICAL CURRENTS barrels become biosensors Transmembrane pores, which are the cell’s gatekeepers, have an impressive ability to admit molecules selectively. Capitalizing on this characteristic, some
A rigid-rod barrel creates a synthetic pore in a lipid bilayer. (Adapted with permission. Copyright 2002 American Association for the Advancement of Science.)
researchers have designed bioassays in which natural or synthetic pores capture target analytes, and a detection scheme measures the degree of blockage. Now, Stefan Matile and colleagues at the University of Geneva (Switzerland) measure the activities of various enzymes using a new synthetic pore and “good-old” fluorometric detection. The researchers fabricated the pores by allowing hollow cylindrical structures known as rigid-rod “ barrels” to selfassemble within lipid bilayers. These bilayers formed the walls of vesicles that held fluorescent dye molecules at concentrations high enough to allow selfquenching. The pores were blocked more completely by large substrate molecules, such as adenosine triphosphate, than by the smaller products of enzymatic reactions, such as adenosine monophosphate or inorganic phosphate. As
the enzymes converted the substrate into product, some dye leaked out of the vesicles, which reduced the amount of self-quenching and increased the fluorescent signal. In the proof-of-concept experiment, the researchers compared the activities of several mutants of the enzyme potato apyrase. A subsequent pore design yielded a more sensitive assay and could be used with various enzymes and substrates; thus, it offered a combination of selectivity (distinguishing between substrates and products) and adaptability (applicable to various substrate–enzyme combinations), the researchers say. In addition, this assay uses fluorometric detection to determine the degree of blockage of the pores instead of measurements of ion currents, which are more typical in synthetic pore systems. (Science 2002, 298, 1600–1602)
52 A
Using the COOH-terminated probes, the researchers recorded force–distance curves over 250 250 nm2 areas and adhesion maps. They found that the curves and adhesion maps were strongly influenced by pH. No adhesion was measured at neutral or alkaline pH, and multiple adhesion forces were recorded at pH ≤ 5. Three pieces of evidence show that these changes were related to differences in the ionization state of the functional groups on the cell’s surface. First, the researchers correlated the curve showing the adhesion forces as a function of pH with microelectrophoresis data and found that the pH of the largest adhesion force corresponded to the cell’s isoelectric point. Next, they treated the cells with Cu(II) ions, which reversed the cells’ surface charges at neutral pH and promoted adhesion toward the negatively charged probe. Finally, control experiments using nonionizable hydroxyl-
A N A LY T I C A L C H E M I S T R Y / F E B R U A R Y 1 , 2 0 0 3
pH 10
COO COO COO
COO
COO COO COO
pH 7
Force
Yves Dufrêne and co-workers at the Université catholique de Louvain (Belgium) use atomic force microscopy (AFM) with carboxyl-terminated probes to investigate the electrostatic properties and isoelectric point of microbial cell surfaces. Cell surface charge plays a critical role in controlling cell adhesion and aggregation. To demonstrate the approach, Dufrêne’s group investigated the wellstudied yeast S. cerevisiae, which has great potential for industrial applications such as water treatment. The researchers functionalized the surfaces of gold-coated AFM cantilevers with alkanethiol self-assembled monolayers terminated with carboxyl or hydroxyl groups. The yeast cells were immobilized by mechanical trapping in a polycarbonate membrane with pore sizes similar to the cell size, which eliminated the need for chemical treatments or drying that would cause rearrangement or denaturation of the surface molecules.
1 nN
AFM probes microbial cell surface charges
pH 5 COOH COOH COOH
COOH
COOH COOH COOH
pH 3
0
100
200
300
400
500
Surface separation (nm)
Force–distance curves between the surface of a single S. cerevisiae cell and a COOHterminated probe, which were recorded in 1 mM KNO3 solutions of varying pH.
terminated probes showed that changes in adhesion forces weren’t simply from the titration of the probe’s surface charges. (Langmuir 2002, 18, 9937–9941)
news
Beads make better microfluidic mixers Microfluidic capillaries on the light path Antonio Garcia and colleagues at Arizona State University report the first successful use of light to move water inside a capillary tube. By coating the tube with photochromic spiropyrans, they were able to move the water ~2–3 mm in ~300–1500 s within a 500-µm-diam capillary. The researchers used fluorescence spectroscopy and epifluorescence microscopy to observe the light-induced changes in the surface energy inside the glass capillary. The key to the technique is that exposure to 366-nm light isomerizes the spiropyran to a highly polar merocyanine form and drives the liquid up the capillary. Visible light reversed this effect. The resulting change in surface polarity, which the researchers call “photocapillarity”, was reflected in variations in surface wettability as measured by the water–surface contact angle. At pH 5.5, the angle reversibly changed by 11–14°. Previous attempts to move liquids by light in a capillary had been frustrated by the unfavorable hysteresis between the advancing and receding contact angles. In their design, Garcia’s team fixed the capillary vertically and connected it to a two-way valve at the top, which was exposed to the atmosphere. The bottom of the capillary was submerged in a beaker of water. The result is that the advancing contact angle in the capillary was significant, but the receding contact angle was negligible because of the relatively large diameter of the beaker. The researchers say the geometrical arrangement prevented the water from moving down the capillary when the light was switched from UV to visible. (Langmuir 2002, 18, 8062–8069)
(a) Water droplets on a photosensitive spiropyran surface after visible and UV irradiation. (b) Light-induced changes in water contact angles on coated slides. Solid lines represent slides coated with the photochromic spiropyrans; the broken line is a control.
In the macroscale world, when two fluids run together, turbulent flow quickly mixes them. In microfluidic systems, however, laminar flow predominates, and the only method for mixing fluids, even when they are in the same channel, is diffusion. Richard Crooks and Gi Hun Seong at Texas A&M University bypass this problem by using microbeads to mix fluids in a relatively short distance. In addition, the large surface area of the beads allows them to be used as reaction sites to catalyze reactions or serve as biochemical sensing devices. Diffusion between fluids is related to the surface area that they share, and placing the microbead obstacles in the flow path generates smaller streams that intermix with each other. In addition, the surface area that the two fluids share increases. (a) Schematic of the mixer with closeTo demonups of (b) the microbeads and weir. (c, d) strate their techWithout the beads, the fluorescein and nique, Crooks and buffer streams remain separate. (e–g) Seong constructed With the microbeads, the fluorescein a 100-µm-wide, and buffer streams mix. 23-µm-deep mixing channel, which was fed by two sample inlet channels. The channel included a 7- to 12-µm-deep weir, which prevented the microbeads from being carried away. Downstream, the channel returned to normal depth and split into 10 50-µm-wide channels. With no beads in the main channel, separate solutions of the dye fluorescein and a buffer flowed through the apparatus and remained apart all the way through the outlet channels; five channels held only fluorescein, five had just buffer. With 15.5-µm-diam beads placed in the main channel just before the weir, the fluorescein and buffer solutions mixed completely. Crooks and Seong constructed a second device using beads coated with glucose oxidase. Glucose and buffer solutions were run over the beads and mixed. At the same time, glucose reacted with the enzyme on the beads to form gluconic acid and peroxide. An amplex red solution was then added, and the flows were sent through a second section of beads, which were covered with horseradish peroxidase. The enzyme catalyzed a reaction to form resorufin, which fluoresces. (J. Am. Chem. Soc. 2002, 124, 13,360–13,361) F E B R U A R Y 1 , 2 0 0 3 / A N A LY T I C A L C H E M I S T R Y
53 A
