Annual Review of Analytical Chemistry - Volume 2, 2009

Editor's Note Every year, the Editorial Committee of each Annual Reviews series asks an internationally distinguished researcher to prepare a prefatory article for the volume being planned. It was an easy choice for us to seek a contribution from John B. Fenn (b. 1917), who since 1994 has been a professor of analytical chemistry at Virginia Commonwealth University in Richmond, Virginia. Few people have achieved the originality and the impact that John Fenn has shown in his development of new instruments for chemical analysis. Anyone using supersonic jet expansions or electrospray ionization owes a huge debt to this special individual, who was awarded the Nobel Prize in Chemistry in 2002. A full account of the development of electrospray ionization may be found in Fenn's 2002 article in the Journal of Biomolecular Techniques (see Related Resources at the end of the interview).

What follows is a transcript of an interview that Fenn's colleague Professor Samy El-Shall kindly conducted on our behalf. This interview captures the spirit and the imagination of this singular individual. It also shows that the path to discovery is seldom straight and narrow.

-Richard N. Zare

Due to its simplicity, versatility, and straightforward interpretation into absolute concentrations, molecular absorbance detection is widely used in liquid-phase analytical chemistry. Because this method is inherently less sensitive than zero-background techniques such as fluorescence detection, alternative, more sensitive measurement principles are being explored. This review discusses one of these: cavity ring-down spectroscopy (CRDS). Advantages of this technique include its long measurement pathlength and its insensitivity to light-source-intensity fluctuations. CRDS is already a well-established technique in the gas phase, so we focus on two new modes: liquid-phase CRDS and evanescent-wave (EW)-CRDS. Applications of liquid-phase CRDS in analytical chemistry focus on improving the sensitivity of absorbance detection in liquid chromatography. Currently, EW-CRDS is still in early stages: It is used to study basic interactions between molecules and silica surfaces. However, in the future this method may be used to develop, for instance, biosensors with high specificity.

The scanning tunneling microscope (STM) has revolutionized our ability to explore and manipulate atomic-scale solid surfaces. In addition to its unparalleled spatial power, the STM can study dynamical processes, such as molecular conformational changes, by recording current traces as a function of time. It can also be employed to measure the physical properties of molecules or nanostructures down to the atomic scale. Combining STM imaging with measurement of current–voltage (I–V) characteristics [i.e., scanning tunneling spectroscopy (STS)] at similar resolution makes it possible to obtain a detailed map of the electronic structure of a surface. For many years, STM lacked chemical specificity; however, the recent development of STM–IETS (inelastic electron tunneling spectroscopy) has allowed us to measure the vibrational spectrum of a single molecule. This review introduces and illustrates these recent developments with a few simple scholarly examples.

Nanoparticle sensors have been developed for real-time imaging and dynamic monitoring, both in live cells and in vivo, of molecular and ionic components, constructs, forces, and dynamics observed during biological, chemical, and physical processes. With their biocompatible small size and inert matrix, nanoparticle sensors have been successfully applied to noninvasive real-time measurements of analytes and fields in cells and in rodents, with spatial, temporal, physical, and chemical resolution. This review describes the diverse designs of nanoparticle sensors for ions and small molecules, physical fields, and biological features, as well as the characterization, properties, and applications of these nanosensors to in vitro and in vivo measurements. Their floating as well as localization abilities in biological media are captured by the acronym PEBBLE: photonic explorer for bioanalysis with biologically localized embedding.

Recent research trends in biosensing have been geared toward developing bioanalytical devices that are label free, small in size, and portable and that can operate in a rapid manner. The performance of these devices has been dramatically improved through the advent of new materials and micro-/nanofabrication technologies. This is especially true for micro-/nanosized cantilever sensors, which undergo a change in mechanical properties upon the specific binding of biomolecules. In this review, we introduce the basic principles of cantilever biosensors in static and dynamic modes. We also summarize a range of approaches to cantilever design, fabrication, and instrumentation according to their applications. More specifically, we describe cantilever-based detections of proteins, DNA molecules, bacteria, and viruses and discuss current challenges related to the targets’ biophysical characteristics.

Micellar electrokinetic chromatography (MEKC), a separation mode of capillary electrophoresis (CE), has enabled the separation of electrically neutral analytes. MEKC can be performed by adding an ionic micelle to the running solution of CE without modifying the instrument. Its separation principle is based on the differential migration of the ionic micelles and the bulk running buffer under electrophoresis conditions and on the interaction between the analyte and the micelle. Hence, MEKC's separation principle is similar to that of chromatography. MEKC is a useful technique particularly for the separation of small molecules, both neutral and charged, and yields high-efficiency separation in a short time with minimum amounts of sample and reagents. To improve the concentration sensitivity of detection, several on-line sample preconcentration techniques such as sweeping have been developed.

The versatility of sol-gel chemistry enables us to generate a wide range of silica and organosilica materials with controlled structure, composition, morphology and porosity. These materials’ hosting and recognition properties, as well as their wide-open structures containing many easily accessible active sites, make them particularly attractive for analytical purposes. In this review, we summarize the importance of silica sol-gels in analytical chemistry by providing examples from the separation sciences, optical and electrochemical sensors, molecular imprinting, and biosensors. Recent work suggests that manipulating the structure and composition of these materials at different scales (from molecular to macromolecular states and/or from micro- to meso- and/or macroporous levels) promises to generate chemical and biochemical sensing devices with improved selectivity and sensitivity.

The role of ionic liquids (ILs) in analytical chemistry is increasing substantially every year. A decade ago there were but a handful of papers in this area of research that were considered curiosities at best. Today, those publications are recognized as seminal articles that gave rise to one of the most rapidly expanding areas of research in chemical analysis. In this review, we briefly highlight early work involving ILs and discuss the most recent advances in separations, mass spectrometry, spectroscopy, and electroanalytical chemistry. Many of the most important advances in these fields depend on the development of new, often unique ILs and multifunctional ILs. A better understanding of the chemical and physical properties of ILs is also essential.

Since the advent of soft ionization methods, mass spectrometry (MS) has found widespread application in the life sciences. Mass is now known to be a critical parameter for characterization of biomolecules and their complexes; it is also a useful parameter to characterize bioparticles such as viruses and cells. However, because of the genetic diversity of these entities, it is necessary to measure their masses individually and to obtain the corresponding mean masses and mass distributions. Here, I review recent technological developments that enable mass measurement of ultrahigh-mass biomolecules and bioparticles at the single-ion level. Some representative examples include cryodetection time-of-flight MS of single-megadalton protein ions, Millikan-type mass measurements of single viruses in a cylindrical ion trap, and charge-detection quadrupole ion trap MS of single whole cells. I also discuss the promises and challenges of these new technologies in real-world applications.

We discuss miniaturization in mass spectrometry in terms of the mass analyzer, the mass spectrometer, and the total analytical system. Mass analyzer miniaturization has focused on ion traps. Decreases in mass analyzer size facilitate reduction of the sizes of the other components of a miniature mass spectrometer, especially the radio frequency electronics and vacuum system. Appropriate sample introduction systems are needed for performance optimization. The criteria by which a miniature mass spectrometer is judged include adequate performance in the traditional areas of resolution, detection limits, and specificity; ruggedness; reliability; and fully autonomous operation. Our discussion of the total analytical system emphasizes the removal of the bottleneck of sample preparation and suggests a solution to the combination of sampling, preconcentration, and ionization: ambient ionization methods. We also describe current miniature mass spectrometers.

Analytical reactions in which short DNA strands are used in combination with DNA ligases have proven useful for measuring, decoding, and locating most classes of macromolecules. Given the need to accumulate large amounts of precise molecular information from biological systems in research and in diagnostics, ligation reactions will continue to offer valuable strategies for advanced analytical reactions. Here, we provide a basis for further development of methods by reviewing the history of analytical ligation reactions, discussing the properties of ligation reactions that render them suitable for engineering novel assays, describing a wide range of successful ligase-based assays, and briefly considering future directions.

Aptamers are ligand-binding nucleic acids whose affinities and selectivities can rival those of antibodies. They have been adapted to analytical applications not only as alternatives to antibodies, but as unique reagents in their own right. In particular, aptamers can be readily site-specifically modified during chemical or enzymatic synthesis to incorporate particular reporters, linkers, or other moieties. Also, aptamer secondary structures can be engineered to undergo analyte-dependent conformational changes, which, in concert with the ability to specifically place chemical agents, opens up a wealth of possible signal transduction schemas, irrespective of whether the detection modality is optical, electrochemical, or mass based. Finally, because aptamers are nucleic acids, they are readily adapted to sequence- (and hence signal-) amplification methods. However, application of aptamers without a basic knowledge of their biochemistry or technical requirements can cause serious analytical difficulties.

Biomarker discovery and proteomics have become synonymous with mass spectrometry in recent years. Although this conflation is an injustice to the many essential biomolecular techniques widely used in biomarker-discovery platforms, it underscores the power and potential of contemporary mass spectrometry. Numerous novel and powerful technologies have been developed around mass spectrometry, proteomics, and biomarker discovery over the past 20 years to globally study complex proteomes (e.g., plasma). However, very few large-scale longitudinal studies have been carried out using these platforms to establish the analytical variability relative to true biological variability. The purpose of this review is not to cover exhaustively the applications of mass spectrometry to biomarker discovery, but rather to discuss the analytical methods and strategies that have been developed for mass spectrometry–based biomarker-discovery platforms and to place them in the context of the many challenges and opportunities yet to be addressed.

The ability to understand and control molecular transport is critical to numerous chemical measurement strategies, especially as they apply to mass-limited samples in nanometer-scale structures. The characteristics of nanoscale structures and devices highlighted in the examples discussed in this article include enhanced mass transport, accessing novel physical behavior, large surface-to-volume ratio, diminished background signals, and the fact that molecular characteristics can dominate the behavior of the structure. The control of nanoscale transport is physically embodied in different structures and experiments. Those structures and experiments highlighted here are featured because of their centrality (nanochannels and nanopores), their connection to more familiar macroscale phenomena (nanoelectrodes), and/or their ability to introduce control (stimulus-responsive materials) or because they represent especially interesting possibilities (stochastic sensing structures).

Forensic chemistry is unique among chemical sciences in that its research, practice, and presentation must meet the needs of both the scientific and the legal communities. As such, forensic chemistry research is applied and derivative by nature and design, and it emphasizes metrology (the science of measurement) and validation. Forensic chemistry has moved away from its analytical roots and is incorporating a broader spectrum of chemical sciences. Existing forensic practices are being revisited as the purview of forensic chemistry extends outward from drug analysis and toxicology into such diverse areas as combustion chemistry, materials science, and pattern evidence.

Analytical chemistry plays a role in the two strategies of defense against chemical or biological weapons that are discussed in this review: the detect-to-protect and the prevent-and-detect strategies. The detect-to-protect method, which is based on detection of a known chemical agent with a specific chemical sensor designed for said agent, has serious flaws. I argue that this approach should be replaced with the prevent-and-detect strategy. Such a change in the defense paradigm would require reallocation of resources, but it is necessary for effective protection of enclosed civilians from chemical and/or biological attack.

This review focuses on the chromatography research that has been carried out within industry or in close cooperation with industry and that has been reported in the scientific literature between 2006 and mid-2008. Companies in the health care sector, such as pharmaceutical and biotechnology companies, are the largest contributors. Industrial research seems to take place in an open environment in cooperation with academia, peer companies, and institutions. Industry appears ready to embrace new technologies as they emerge, but they focus strongly on making chromatography work robustly, reliably, rapidly, and automatically. “Hyphenated” systems that incorporate on-line sample-preparation techniques and mass-spectrometric detection are the rule rather than the exception. Various multidimensional separation methods are finding numerous applications. Strategies aimed at speeding up the development of new chromatographic methods remain the focus of attention. Also, there is a clear trend toward exploring chromatographic methods for parallel processing along with other strategies for high-throughput analysis.

In electrogenerated chemiluminescence, also known as electrochemiluminescence (ECL), electrochemically generated intermediates undergo a highly exergonic reaction to produce an electronically excited state that then emits light. These electron-transfer reactions are sufficiently exergonic to allow the excited states of luminophores, including polycyclic aromatic hydrocarbons and metal complexes, to be created without photoexcitation. For example, oxidation of [Ru(bpy)3]²⁺ in the presence of tripropylamine results in light emission that is analogous to the emission produced by photoexcitation. This review highlights some of the most exciting recent developments in this field, including novel ECL-generating transition metal complexes, especially ruthenium and osmium polypyridine systems; ECL-generating monolayers and thin films; the use of nanomaterials; and analytical, especially clinical, applications.

This review examines the application of polymer brush–modified flat surfaces, membranes, and beads for protein immobilization and isolation. Modification of porous substrates with brushes yields membranes that selectively bind tagged proteins to give 99% pure protein at capacities as high as 100 mg of protein per cubic centimeter of membrane. Moreover, enrichment of phosphopeptides on brush-modified matrix-assisted laser desorption/ionization (MALDI) plates allows detection and characterization of femtomole levels of phosphopeptides by MALDI mass spectrometry. Because swollen hydrophilic brushes can resist nonspecific protein adsorption while immobilizing a high density of proteins, they are attractive as substrates for protein microarrays. This review highlights the advantages of polymer brush–modified surfaces over self-assembled monolayers and identifies some research needs in this area.