Analytical Currents: Analyzing tissues without the stain - Analytical

Analytical Currents: Analyzing tissues without the stain. Anal. Chem. , 2003, 75 (1), pp 11 A–11 A. DOI: 10.1021/ac0312209. Publication Date (Web): ...
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ANALYTICAL CURRENTS Large-scale integration for microfluidics Current efforts in microfluidics primarily recreate one laboratory activity at a time in the microscale. If multiple laboratory functions could be integrated instead, one could build a system akin to the integrated circuit, making possible complex functions that were previously cumbersome or unachievable. Stephen R. Quake, Todd Thorsen, and Sebastian J. Maerkl at the California Institute of Technology introduce a microfluidic multiplexor containing arrays of binary valve patterns that can be controlled with a minimum number of inputs. The researchers used this technique to construct a fluid version of the dynamic random access memory (dRAM) used in computers and a fluidic comparator, similar to integrated circuit operational amplifiers. The multiplexor is key to both fluidic chips. It contains monolithic, leak-proof, scalable microvalves and multiplexed addressing and control of these valves. The multiplexor is made from two poly(dimethylsiloxane) (PDMS) layers: a control layer, which contains channels that regulate the valves, and a flow layer, where channels conduct samples and purge fluid.

device compares the Where the control 0 1 2 3 4 5 6 7 input with a reference, channels cross the 0 and if the input exceeds flow channels, a thin 1 the reference, the comPDMS membrane, parator generates a high which acts like a 1 output, thus amplifying hydraulic valve, is 0 the original signal; if formed. Pressurizing 1 not, the output remains the control channels 0 low. The integrated syscan actuate valves. The tem was constructed pressure required for with 2056 microvalves each valve depends on that could load 2 difthe dimensions of the ferent reagents and control channel and is The multiplexor: Vertical lines are move them together or provided by steel pins flow channels, horizontal lines are apart in 256 subnanothat are connected to control channels, and wide areas liter reaction chambers. computer-controlled are valves. Valves with x’s are closed. The researchers testsolenoids. (Adapted with permission. Copyright ed the device with an The dRAM system 2002 American Association for the E. coli that produced an is composed of 1000 Advancement of Science.) enzyme that converted independent coma substrate into a fluorescent product. The partments and 3574 microvalves. To microorganism was distributed unevenly demonstrate system function, the central among several compartments. When the memory storage chambers were loaded substrate was supplied to all of the comwith a dye, and then individual champartments, only those containing the E. bers were purged with water leaving the coli yielded an amplified fluorescent signal, dye to spell out “CIT”. whereas the rest remained at the reference The fluidic comparator is similar in level. (Science 2002, 298, 580–584) function to an operational amplifier. The

Analyzing tissues without the stain Richard Caprioli and colleagues at Vander-

tured cells using the MALDI instrument’s

than 100 ppm below m/z 30,000 on the LCM

bilt University introduce an optimized tech-

camera system, which helped them get an

film with internal calibration.

nique to isolate and analyze healthy and

accurate location of the cells and increased

diseased tissues for laser capture microdis-

the spectral quality, peak number, and in-

noted more than 40 peaks that significantly

section (LCM) and MALDI MS without stain-

tensity. They also compared the spectra of

differed in intensity between invasive mam-

ing the tissues.

stained and unstained mouse liver tissue

mary carcinoma and normal epithelia. They

The researchers examined mouse liver

With human breast tissue, the researchers

and discovered that the stains actually in-

also determined that the method could be

tissue, mouse colon crypts, and human

terfered with the quality of the MS spectra.

used to obtain abundant signals from 10 or

breast tissue. They deposited subnanoliter

By eliminating staining, Caprioli and his col-

fewer LCM-captured cells. (J. Am. Soc. Mass

volumes of the matrix solution onto the cap-

leagues obtained a mass accuracy better

Spectrom. 2002, 13, 1292–1297)

J A N 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

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ANALYTICAL CURRENTS Rapid MS identification of intact microorganisms In an effort to combat bioterrorism, Zhong-Ping Yao, Carlos Afonso, and Catherine Fenselau at the University of Maryland–College Park introduce a simple, swift new method for identifying intact microorganisms that doesn’t require a special search algorithm or database. The researchers use a brief onslide proteolytic digestion followed by MALDI MS/MS and conventional database searching. The goal was to rapidly obtain enough peptide ions for MS/MS without isolating or fractionating the microorganisms. Yao and his group used enterobacteriophage MS2 as the model microorganism. To digest the viral proteins, they deposited a 1-µL MS2 suspension on the MALDI probe tip, allowed the suspension to dry, and then added trypsin for a 20-min digestion. They analyzed the sample using MALDI FT-ion cyclotron resonance (FT-ICR)

MS with sustained off-resonance irradiation collision-induced dissociation. Next, to hunt down the microorganism, they used four fragment ions from the MS/MS spectrum and the precursor ion at m/z 1753.957. (Yao and his colleagues point out that the precursor peak was the dominant one in the spectrum.) The database search results suggested that those ions were enough to identify the microorganism. When the database was searched using a mass tolerance of ±0.01 Da, only three candidates—the coat proteins from enterobacteriophages MS2, R17, and F2—were found. When a mass tolerance of ±1 Da was used instead, the three coat proteins ranked first in the search results with scores of 44, and other candidates scored below 33. The researchers say their method can be used with lower-resolution instruments and applied to various MS techniques, such as MALDI quadrupole time

Refining DIY nanostructures poly(dimethylsiloxane) (PDMS) masks,

(DIY) approach can appreciate the efforts

the researchers formed arrays of rings

of George Whitesides’ laboratory at Har-

with 30- to 40-nm line widths. Specifical-

vard University over the past few years.

ly, the structures were formed by expos-

This group is one of a few that have devel-

ing a photoresist to UV light through masks

oped protocols for fabricating micro- and

that bore 2-µm lines spaced by 2 µm. The

nanoscale structures using simple, inex-

mask was then rotated 90°, and the pho-

pensive equipment. Now, Whitesides and

toresist was exposed again. This process

colleagues push the envelope further and

produced masters with arrays of 100- to

make structures with critical dimensions

400-nm-diam posts. The masters were

as small as 30 nm. Using their method, it is

subsequently used to create new PDMS

also possible to pattern structures over

masks from which the final polymer de-

areas of several square centimeters,

vices were fabricated. The researchers

whereas methods such as X-ray photoli-

used a similar protocol to make arrays of

2 thography cover areas of only 0.01–1 mm .

~250-nm-diam wells, in which individual nanocrystals of inorganic salts were

polymer devices were molded from the

grown. (J. Am. Chem. Soc. 2002, 124,

masters that had been created using

12,112–12,133)

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Sustained off-resonance irradiation collision-induced dissociation spectrum of the m/z 1753.957 ion from enterobacteriophage MS2, obtained on a MALDI FT-ICR instrument using the new rapid identification technique. (Adapted with permission. Copyright 2002 John Wiley & Sons.)

of flight (TOF), MALDI-TOF/TOF, MALDI ion trap, and MALDI ion trap TOF. (Rapid Commun. Mass Spectrom. 2002, 16, 1953–1956)

(a)

Anyone who prefers the do-it-yourself

Drawing on earlier protocols in which

image not available for use on the Web.

A N A LY T I C A L C H E M I S T R Y / J A N U A R Y 1 , 2 0 0 3

Composite PDMS mask

2 µm

Expose to UV

Si Develop photoresist

Positive resist

100–400 nm 400 nm (b)

Master

Composite PDMS mask

400 nm 100–400 nm Expose to UV Positive or negative resist Develop photoresist

30–50 nm ~350 nm

Positive photoresist

100–250 nm 100 nm

Negative photoresist

(a) The preparation of a master device with features down to 100 nm in diam. (b) The final devices are fabricated from masks that were molded against the master.

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Nanoparticle thermometers

Glucose consumption by live cells

Although taking the temperature of everyday objects is fairly simple, it is not so easy to do for fast-moving parts, integrated circuits, or individual cells. However, a new method using luminescent nanoparticles, developed by Wei Chen, Shaopeng Wang, and Sarah Westcott at Nomadics, Inc., might solve this difficulty.

One of the most fundamental measures of cellular health and activity is the rate at which cells consume glucose. Craig Schulz and Jaromir Ruzicka from the University of Washington introduce an automated technique for measuring the glucose consumption of live cells, which could be the basis of a method for looking at a range of other relevant biological analytes.

The method uses fluorescent thermometry, in which a temperature increase makes different transition states available in a phosphor, thus changing the phosphor’s excited-state lifetime and intensity. Conventional fluorescent thermometry uses micrometer-sized crystalline semiconductor phosphors, which

Image not available for use on the Web.

limit resolution by scattering excitation and emitted light and can insulate an object, changing its temperature. Nanoparticles have already been used to measure temperatures with higher spatial resolution, but only at low temperatures. Chen and colleagues tested the fluorescence of three classes of nanoparticles—semiconductor, doped, and double-doped— between 30 and 150 °C. They found that the fluorescence of 2+ semiconductor CdTe and doped ZnS:Mn nanoparticles changed

in a reversible, linear way with a resolution of up to 0.02 °C. The 2+ 3+ double-doped ZnS:Mn ,Eu particles could be excited by two

The heart of it all. Microbeads hold live cells that react with analytes flowing past. A plug holds the beads in place but allows the analytes to flow into a detection chamber filled with reagent that is monitored by UV–vis spectroscopy through fiber-optic lines. (Adapted with permission. Copyright 2002 Royal Society of Chemistry.)

different wavelengths, which provides a possible means of controlling for other factors that might affect the temperature measurement. Some nanoparticles showed irreversibility at high temperatures, which the researchers attributed to thermal curing of the nanoparticle’s surface effects. (J. Phys. Chem. B 2002, 106,

Fluorescence intensity (cps)

11,203–11,209) 30 ∞C 40 ∞C 50 ∞C 60 ∞C 70 ∞C 80 ∞C 90 ∞C 110 ∞C 130 ∞C 150 ∞C

1.4¥106 1.2¥106 1.0¥106 8.0¥105 6.0¥105 4.0¥105 2.0¥105 0.0

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Wavelength (nm)

Fluorescence spectra at 30–150 °C of 10-nm single-doped 2+ ZnS:Mn nanoparticles excited at 360 nm.

The method is based on the microsequential injection and lab-on-valve (µSI-LOV) techniques pioneered in Ruzicka’s lab. In this case, mouse hepatocyte cells were cultured on microcarrier beads and loaded into the LOV device. In optimized experiments, only 3 µL of beads were necessary, but that small volume contained 104–105 cells. The high cell density meant that glucose was depleted from the medium in a matter of minutes. The key to the analysis system is a plug with a narrow channel through it, which the researchers describe as a nozzle. This plug held the microbeads in place, but the channel allowed a small stream of analytes to flow through and into a chamber containing reagents for an enzyme-based glucose detection scheme. A relatively fast flow rate of 30 µL/s through the plug generated a laminar flow and adequate mixing of glucose and reagents in the detection chamber, yielding an absorbance signal that was measured in 10 s. Using these optimized parameters, the researchers monitored glucose concentrations over the 0.1- to 5.6-mM range in real time. They say that their design offers rapid mixing and flow rates that can be stopped, pulsed, or even reversed. (Analyst 2002, 127, 1293–1298) J A N 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

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