Product Details

ISBN-13: 9780854041299
Publisher: Royal Society of Chemistry, The
Publication date: 12/28/2008
Pages: 332
Product dimensions: 6.14(w) x 9.21(h) x 0.90(d)

About the Author

SÚverine Le Gac got her Ph.D. at the University of Sciences and Technologies of Lille (France) in 2004 on the topic of microfluidic systems for mass spectrometry analysis and proteomics applications. Since 2005 she has been working in BIOS, The Lab-on-a-Chip group led by Pr. Van den Berg at the University of Twente. Her current research focuses on microfluidics applications of cell analysis. Since January 2008 she has been appointed as assistant professor in the ame group to lead the research topic "from cell on a chip towards lab-in-a-cell applications. Albert van den Berg leads The Lab-on-a-Chip Group at the University of Twente. His current research interests focus on microanalysis systems and nanosensors, nanofluidics and single cells on chips. He received the Simon Stevin Master award from the Dutch Technical Science foundation (STW) in 2002 and, in the following year, was appointed captain of the Nanofluidics Flagship within the national nanotechnology program, Nanoned.

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Miniaturization and Mass Spectrometry

By Séverine Le Gac, Albert van den Berg

The Royal Society of Chemistry

Copyright © 2009 Royal Society of Chemistry
All rights reserved.
ISBN: 978-0-85404-129-9




BIOS the Lab-on-a-Chip Group, University of Twente, PO Box 217, 7500 AE Enschede, The Netherlands

The development of miniaturized analytical or chemical processing systems for both biological and chemical applications is a fast growing field because such systems enable the performance of a series of successive operations at scales which are not easily handled by human experimenters. A key challenge arising from this continuous system miniaturization towards the micrometer scale, or even smaller, lies in the ability to sensitively detect low molecular concentrations in reduced sample volumes. Additionally, such analytical systems must be coupled to microfluidic devices with minimal loss of analytes and information. The last issue is the scalability of the detection technique, as the detection is performed on small sample sizes. The ideal technique for microfluidic detection would therefore present an enhanced sensitivity upon downscaling. The dream of the users in the (bio)chemical field would be a fully integrated and portable device that includes (micro)systems for sample handling, preparation and detection. Conventional detection systems are still bulky instruments, resulting in the paradox of coupling a smaller and smaller analytical or processing device to room-sized instrumentation for the detection.

On-chip detection firstly relied on optical techniques, such as ultraviolet (UV) absorbance, fluorescence or laser-induced fluorescence (LIF). The latter technique in particular has a sensitivity in the (sub)micromolar range which is suitable for microfluidic applications. Besides optical techniques, electrical-based techniques are also widely used for on-chip detection due to their sensitivity, e.g. detection based on conductivity, electrochemistry, electro-chemiluminescence, etc. The main advantage of these techniques is that they are fully integrated on the microdevice via the introduction of electrodes; they do not rely on the use of complex and bulky instrumentation as is the case for optical techniques. More exotic techniques are also used in combination with microfluidics, such as nuclear magnetic resonance (NMR) and Raman spectroscopy. These techniques are less popular but are currently developing at a rapid rate. Since the late 1990s mass spectrometry (MS) has also been used for the detection stage for microfluidic processing systems; this combination is particularly striking if one considers the size of a mass spectrometer compared to that of a microchip! MS has rapidly replaced other techniques due to its very high sensitivity and other advantages such as a high selectivity compared to optical-based techniques, for instance. Consequently, it turned out that MS analysis could also benefit from the use of microfluidic systems for sample preparation prior to analysis. As a consequence, the field of microfluidics and MS has been rapidly growing with the appearance of dedicated products within the last decade.

In the first part of this introductory chapter, we briefly introduce the technique of mass spectrometry as well as two ionization methods, namely ESI (electrospray ionization) and MALDI (matrix-assisted laser desorption ionization), commonly used for the analysis of biological/biochemical samples or for organic chemistry purposes. The second part highlights the advantages brought by the miniaturization and coupling of microfabricated devices to MS, and how this marriage benefits both on-chip detection and the MS analysis. The third part of this chapter focuses on the different approaches adopted for coupling microfabricated systems to ESI-MS or MALDI-MS and on the miniaturization of the mass spectrometer itself. In the final part different fields of applications of miniaturization for MS analysis are presented. Moreover, the different technological developments and applications that are treated in greater detail in separate chapters in this book about miniaturization and mass spectrometry are reviewed.

1.1 Brief Introduction to MS Techniques and the ESI and MALDI Ionization Techniques

Mass spectrometry is an analysis technique that detects substances as a function of their molecular weight, or, more precisely, that detects substances as ions as a function of their mass-to-charge ratio (m/z). The analysis starts with the ionization of the molecules, which are subsequently separated in an analyzer according to their size (m/z ratio) before they reach the detector. A mass spectrum is composed of a series of peaks at given m/z values, indicating the presence of ionic species characterized by these mass-to-charge ratio values.

The key part of the connection between microfabricated/microfluidic devices and a mass spectrometer is the ionization of the analyte, as molecules are introduced as ions for the analysis. Subsequently, they must be ionized on the chip or at the outlet of the chip to be detected. Ionization is achieved using many different techniques, depending on the molecule properties. However, we will focus here on two ionization techniques, ESI and MALDI. These two techniques prevail nowadays in the field of MS analysis and they consist of the two main ionization methods used in combination with microfluidic analysis, although two other techniques, atmospheric pressure chemical ionization (APCI) and atmospheric pressure photoionization (APPI), have also recently been reported for on-chip detection.

MALDI and ESI ionization techniques are known as soft ionization techniques: they are suitable for the ionization and the analysis of large molecules (MW > 1 kDa) such as polymers, proteins, peptides, nucleic acids and poly- or oligosaccharides without fragmenting them. During the ionization process, the molecules acquire enough energy to be transferred to the gas phase, but the amount of energy remains low enough not to induce any fragmentation of rearrangement of the molecules. MALDI and ESI are the two mostly used ionization techniques because of their compatibility with the analysis of large molecules, and especially biomolecules, and their routine sensitivity in the low picomole down to the femtomole range. This popularity of ESI and MALDI has mainly been caused by the explosion of analytical needs in the fields of biology and biochemistry. The discovery of these two soft ionization techniques in the mid-1980s is strongly linked to the prosperity of MS, and the recent growth of proteomics analysis marks in particular the golden age of the techniques of ESI-MS and MALDI-MS. As a consequence, the discovery of both ESI and MALDI was rewarded in 2002 with the Nobel prize to their inventors, Fenn and Tanaka, respectively.

More recent technological developments in the field of MS have strengthened this prosperity and the potential of MS as an analysis tool for complex samples, "real-world" biological samples as well as complex synthetic mixtures. For instance, coupling MS to a separation technique such as capillary electrophoresis (CE) or liquid chromatography (LC) enables one to reduce the sample complexity and to successively analyze the species present therein. Also, tandem (MS2), or even MSn, helps to elucidate the structure of substances and the sequence of biopolymers such as peptides.

These points, the potential of MS and the recent developments in the field of MS account for the increasing popularity of (ESI- and MALDI)-MS and explains why MS has naturally been associated with the recent miniaturization trend and the recent appearance of microfabricated and microfluidic systems.

1.1.1 ESI Technique

Electrospray ionization was first reported by Fenn in 1984, and further developed in 1988. This technique relies on the generation of a spray from a liquid upon application of a high voltage. Typically, the sample to be analyzed is introduced in a liquid phase in a capillary. A strong electric field is created (high voltage of several kilovolts) between the liquid and a counter electrode (i.e. inlet of the mass spectrometer) placed some centimeters in front of the capillary. Upon application of the electric field, the liquid breaks into a gas of highly charged droplets whose size depends on different parameters such as the capillary inner diameter, the flow rate of the liquid and the applied voltage. During transport, the droplets evolve to the ultimate stage of ions in the gas phase. The solvent (typically, an organic solvent such as methanol, ethanol or acetonitrile) evaporates, and the droplets thereby shrink until they reach the so-called Rayleigh limit where surface tension exactly compensates electrical forces, as expressed by

q = ze = 8π(ε0γr)1/2 (1.1)

where q is the droplet charge, r its radius, γ the surface tension and ε0 the permittivity of vacuum. Beyond this limit, the equilibrium is lost and droplets break into smaller droplets that undergo the same process. This cycle proceeds until the stage of ions in the gas phase is reached, and these ions in the gas phase enter the mass spectrometer to be analyzed.

The technique of ESI gives rise to multi-charged species as long as there are several protonation sites on the analytes. A typical ESI mass spectrum presents a group of peaks for one analyte species, corresponding to the different charge states with a Gaussian distribution. One advantage of this is that the analyzer and detector of the mass spectrometer work with a reduced mass range as ions are analyzed and detected as a function of m/z and not of their molecular weight. Mass spectra are of course more difficult to interpret compared to MALDI mass spectra, although many deconvolution techniques are now routinely available to transform raw data into reconstructed spectra presenting peaks for mono-charged species. A limitation of the technique of ESI, especially compared to MALDI, is the low analysis throughput and its tedious and cumbersome preparation with the manual introduction of the sample in the capillary source. ESI vs. nanoESI

There are now two commonly defined regimes for electrospray analysis. These regimes are distinguished by the inner diameter of the capillary source, the liquid flow rate and the applied ionization voltage, and as already mentioned above these three parameters dictate the size of the generated droplets. These two regimes are

• classical ESI with a capillary inner diameter of ca 100 µm, a flow rate of 1–20 µL min-1 and ionization voltage of 3–4 kV; and

• nanoESI with a capillary inner diameter of ca 1–10 µm, a flow rate below 1 µL min-1 and ionization voltage of 1 kV.

The miniaturization of the technique started in 1994 with the description of microESI by the group of Caprioli. A further step towards miniaturization was reported by Wilm and Mann in the 1990s with the development of nanoESI. The miniaturization of the technique is driven by the improvement it offers. ESI is sensitive only to the concentration of species and not to the total amount of analytes; as a consequence, its miniaturization does not affect the sensitivity of the analysis but rather enhances it, as explained below.

Working with smaller sources appears to be an advantage as it leads to the formation of smaller droplets, and this in turn gives a number of improvements. The increased surface-to-volume ratio of the droplets brings two main advantages: it favors evaporation phenomena allowing for using a higher relative amount of water in solution; and it yields an increased surface charge density, which promotes coulombic fission of the droplets. The evaporation–fission cycle becomes more efficient and gives more ions in the gas phase, and this leads all together to an increase of the ionization yield, from 10-9 for classical ESI up to 10-4 for nanoESI. This is illustrated by Equation (1.2) that gives the charge concentration as a function of the droplet size (radius): the smaller the droplet, the higher the charge concentration, and the higher the ionization probability for a molecule in the droplet:

q/V = 3(ε0γ/2r3)1/2 (1.2)

where q/V represents the charge volume concentration in a droplet, r the radius of a droplet and γ the surface tension. Moreover, this increased charge concentration limits ion suppression phenomena; the competition between molecules to acquire a charge is lower, and as droplets are smaller there are also obviously fewer analytes per droplet. One particular consequence is a higher tolerance of the analysis to the presence of salts or other solution contamination; this is of great interest for the technique of ESI where the presence of salts usually fully hinders the process of ionization.

Additionally, miniaturization of the sources also leads to a decrease of the sample flow rate and the use of a lower ionization voltage. Ionization conditions are smoother, and the ionization source can be placed closer to the mass spectrometer inlet. Consequently, not only more ions are formed, but also more ions enter the mass spectrometer for their analysis.

The enhancement in performance brought about by nanoESI compared to classical ESI is crucial for some fields of applications, such as bioanalysis, and especially proteomics. Miniaturizing ESI provides increased sensitivity of the analysis and enables its application for the analysis of complex samples with a wide range of analyte concentrations. When working with nanoESI the probability of ionizing molecules is higher, and this is of great importance for the analysis of complex real-world samples where some compounds are present as traces.

As ESI works on a continuous flow of liquid, it has quickly been coupled to LC or other liquid-phase separation techniques as an alternative to optical detection. Mass spectrometry gives more information on the eluted compound, and the resulting hyphenated technique enables one to decrease the complexity of samples before their analysis by MS. High performance liquid chromatography (HPLC) is coupled to conventional ESI-MS while nanoLC is connected to nanoESI-MS for a better match in the flow-rate values.

1.1.2 MALDI Technique

Matrix-assisted laser desorption ionization has simultaneously been developed by Karas and Hillenkamp in Germany and by Tanaka et al. in Japan in 1985. With this technique molecules are ionized via laser irradiation of the sample and with the help of other small organic molecules, called the matrix. The matrix strongly absorbs the light of the laser and transfers it together with a charge, mostly a proton, to the analytes. Thereby, analytes reach the gas phase as ions that are ultimately analyzed by the mass spectrometer.

A MALDI-MS analysis proceeds as follows. A solution of matrix is mixed with the sample to be analyzed. A droplet of the resulting mixture is deposited on a plate and allowed to dry; the evaporation of the solvents leads to the co-crystallization of the analytes with the matrix. Once placed under vacuum, the spots of crystal are irradiated with the laser; the laser energy is absorbed by the molecules of the matrix, and subsequently transferred to the analytes that are desorbed from the surface. Simultaneously, the analytes capture a proton from the matrix molecules and become ions. Analytes finally reach the state of ions in the gas phase after desolvation of the matrix molecules.

As for ESI, the MALDI technique depends neither on the properties of the analytes nor on the absorption properties of the molecule as energy transfer proceeds via molecules of matrix. Besides, the mass or the size of the molecules does not influence the ionization and desorption process so that it can be applied to any molecule. Contrary to ESI, analyzed species are mono-charged with MALDI; this simplifies the interpretation of mass spectra but this imposes working with a detector covering a wider mass (m/z) range.

The laser wavelength can be either in the UV or infrared range, with the former being most commonly used. Therefore, matrix molecules are aromatic compounds that can absorb UV light and present a carboxylic acid moiety for the protonation of the analytes. Matrix molecules are simultaneously detected together with the analytes, and give peaks in the low mass range, i.e. below m/z 600. Consequently, MALDI-MS analysis is often limited to compounds with a molecular weight above m/z 600. Compared to ESI, the MALDI technique has a higher tolerance to the contamination of samples, and especially to the presence of salts that strongly hinders analyte ionization in ESI-MS. On-target sample cleaning is widely used in case of a high level of contamination. Another advantage of MALDI compared to ESI is the higher throughput of the analysis and its possible automation.

While ESI works on continuous flows of samples, MALDI analysis is performed on droplets or discrete amounts of liquid. Yet, recent developments have aimed at coupling a separation step relying on liquid chromatography to MALDI-MS analysis. For that purpose, the liquid eluted from the chromatography column is deposited in a continuous and automated way on a MALDI target. Other improvements concern the MALDI target to alleviate the use of a matrix. The matrix can be covalently attached on the target plate surface to avoid its desorption together with the analytes, or targets based on porous silicon are used that does not require the addition of a matrix and that gives enhanced analysis sensitivity. Lastly, an obvious improvement for the process of ionization comes from on-target concentration of the analytes, as will be discussed later. An easy way to obtain sample concentration is to confine it on a smaller surface area. Subsequently, a major breakthrough recently was obtained with the appearance of smart targets consisting of a uniform hydrophobic area patterned with small hydrophilic spots where the sample is deposited.


Excerpted from Miniaturization and Mass Spectrometry by Séverine Le Gac, Albert van den Berg. Copyright © 2009 Royal Society of Chemistry. Excerpted by permission of The Royal Society of Chemistry.
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Table of Contents

Chapter 1. Introduction; Chapter 2. The coupling of microfabricated fluidic devices with electrospray ionization mass spectrometers; Chapter 3. A silicon-based microfabricated nozzle with integrated counterelectrode and its applications combined with mass spectrometry; Chapter 4. PDMS ESI emitter tips; Chapter 5. Microfabricated nanoelectrospray emitter tips based on a microfluidic capillary slot; Chapter 6. Microfabricated parylene electrospray tips Integrated with cyclo-olefin microchips for ESI-MS; Chapter 7. Microfluidic bioanalytical platforms with mass spectrometry detection for biomarker discovery and screening; Chapter 8. Modular microfluidics devices combining multi-dimensional separations; Applications to targeted proteomics analyses of complex cellular extracts; Chapter 9. Simple chip-based interfaces for on-line nanospray mass spectrometry; Chapter 10. On-line and off-line MALDI from a microfluidic device; Chapter 11. Microfluidic platforms enabling on-line monitoring of chemical reactions by mass spectrometry; Chapter 12. MALDI-TOF mass spectrometry and digital microfluidics for the investigation of pre-steady state enzyme kinetics; Chapter 13. Development of miniaturized MALDI time-of-flight mass spectrometers for homeland security and clinical diagnostics.

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