Affinity monolith chromatography: a review of principles and recent analytical applications
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Affinity monolith chromatography (AMC) is a type of liquid chromatography that uses a monolithic support and a biologically related binding agent as a stationary phase. AMC is a powerful method for the selective separation, analysis, or study of specific target compounds in a sample. This review discusses the basic principles of AMC and recent developments and applications of this method, with particular emphasis being given to work that has appeared in the last 5 years. Various materials that have been used to prepare columns for AMC are examined, including organic monoliths, silica monoliths, agarose monoliths, and cryogels. These supports have been used in AMC for formats that have ranged from traditional columns to disks, microcolumns, and capillaries. Many binding agents have also been employed in AMC, such as antibodies, enzymes, proteins, lectins, immobilized metal ions, and dyes. Some applications that have been reported with these binding agents in AMC are bioaffinity chromatography, immunoaffinity chromatography or immunoextraction, immobilized-metal-ion affinity chromatography, dye–ligand affinity chromatography, chiral separations, and biointeraction studies. Examples are presented from fields that include analytical chemistry, pharmaceutical analysis, clinical testing, and biotechnology. Current trends and possible directions in AMC are also discussed.
KeywordsAffinity monolith chromatography Monolithic supports Affinity chromatography Bioaffinity chromatography Immunoaffinity chromatography Immobilized-metal-ion affinity chromatography Dye–ligand affinity chromatography Biointeraction chromatography
Affinity chromatography is an important separation approach for separating or analyzing specific target compounds in samples or for studying biological interactions. This method is a type of liquid chromatography that makes use of the selective and reversible binding that is present in many biological systems, such as the binding of an antibody with an antigen or the coupling of an enzyme with a substrate. Affinity chromatography utilizes these interactions by placing one of the binding partners within a column as the stationary phase and applying to this column the complementary partner, or target analyte [1, 2, 3, 4].
Affinity columns can be used alone or in combination with other techniques for the selective isolation or analysis of the target [1, 2, 3, 4]. In addition, experiments can be conducted to obtain information on the stoichiometry, thermodynamics, and kinetics of the interaction that is occurring between the target and immobilized binding agent [1, 3, 5, 6, 7, 8]. These features continue to make affinity chromatography a popular method and an area of ongoing research in fields that range from the isolation of proteins, enzymes, and antibodies to biochemical and medical research, molecular biology, pharmaceutical or clinical testing, biotechnology, environmental analysis, and biophysical measurements [1, 2, 3, 4, 5, 6, 7, 8].
Because of the selective binding of affinity columns, it is not surprising that a growing number of applications have made use of these columns in biochemical separations and analytical chemistry. This work can be done by using binding agents that have been placed onto low-performance supports or by using supports that are designed for high-performance liquid chromatography (HPLC) [1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19]. Although most past work with affinity chromatography made use of particle-based supports [3, 4, 8, 19], there has been increasing interest in conducting affinity-based separations with monolithic supports. This combination has led to a method referred to as affinity monolith chromatography (AMC) [10, 13, 14, 15].
The initial development of AMC and applications in this field up to 2007–2008 have been discussed in previous reviews [10, 15]. Related articles have reviewed the use of AMC with carbohydrate-based monoliths  or in the specific area of bioaffinity chromatography . This review will build on these previous reports by focusing on more recent developments and applications of all AMC methods, with a particular emphasis being given to reports that have appeared in the last 5 years (i.e., since 2008). To aid in the understanding of the applications or developments of this method and possible directions in this field, the basic principles of AMC will also be discussed, along with the types of supports and binding agents that have recently been used with this approach for chemical and biochemical analysis.
Principles of affinity chromatography
The on/off elution scheme shown in Fig. 1 is often used in a situation in which there are strong and highly selective interactions between the target and the immobilized binding agent. For instance, this mode of affinity chromatography is commonly employed in cases where the immobilized binding agent is an antibody and the target is its antigen, a system for which the association equilibrium constant is often greater than 106 M-1 under physiological conditions . The strong interactions in such a system require an elution buffer that can later be passed through the column to disrupt the interactions between the target and the binding agent and to elute the retained target from the column. As mentioned earlier, this may involve a general change in the mobile phase (e.g., an alteration in pH, ionic strength, polarity, or temperature), giving a technique known as “nonspecific elution.” Alternatively, the elution buffer may contain an additive that competes with the target or immobilized binding agent for their sites of interaction, giving an approach referred to as “biospecific elution” [3, 20]. It is also possible with systems that have weaker binding to sometimes perform affinity chromatography by using isocratic conditions. This approach, referred to as “weak affinity chromatography,” employs the same mobile phase for elution as is used during application of the target [20, 21, 22, 23]. Isocratic conditions can be used if the target binds to the immobilized agent with moderate-to-weak affinity, as occurs for an interaction that has an association equilibrium constant that is less than or equal to approximately 106 M-1 .
Another important factor to consider in any application of affinity chromatography is the type of chromatographic bed that is employed. Although this review will focus on monolithic supports, it is useful for the sake of comparison to consider also other support materials that have been used in the past for affinity chromatography. For instance, agarose and cellulose are carbohydrate supports that are frequently employed for preparative applications of this method or for the use of affinity columns for sample pretreatment [3, 18, 19]. These supports are attractive for such work because of their good stability over a wide pH range and their low nonspecific binding of biological molecules. In addition, agarose has a large pore diameter and is useful in applications involving large biological compounds [18, 19]. Some alternatives to carbohydrate-based supports are modified silica particles [4, 8, 19], which were the main materials used in HPLC-based affinity columns prior to the more recent development of monolithic supports for affinity separations [10, 13, 14, 15, 19]. The binding agent of interest is typically covalently attached to the support through various coupling techniques (e.g., amine- or sulfhydryl-based methods) [10, 15, 18]. Alternatively, the binding agent may be bound to the support through a secondary binding agent (e.g., the biospecific adsorption of antibodies to supports that contain an antibody-binding protein such as protein A or protein G) [16, 18].
The immobilized binding agent, or “affinity ligand,” is another vital factor to consider when performing any affinity chromatography method. Most of the ligands that are used in affinity chromatography and AMC are obtained from a biological source, as is the case for antibodies, enzymes, transport proteins, and lectins [1, 2, 3, 4, 5, 16, 18]. However, affinity chromatography can also use synthetic ligands such as metal-ion chelates and biomimetic dyes [24, 25, 26, 27, 28]. The type of ligand that is employed is often used to divide affinity chromatography into several subcategories. Examples of these subcategories are bioaffinity chromatography, immunoaffinity chromatography, and immobilized-metal-ion affinity chromatography (IMAC). The use of AMC in each of these areas will be examined later in this review.
Advantages of affinity monoliths
Monoliths are also available in several formats, including those based on organic polymers and silica or agarose monoliths, among others [10, 13, 15, 19, 29, 30] (see [14, 19, 29, 30, 31, 32, 33, 34, 35, 36] for more details on the pore structure and morphology of such materials). These monolithic supports generally contain relatively large, flow-through pores that allow the mobile phase to easily travel through the support. Also present are smaller side pores that provide most of the surface area for placing a stationary phase on the support, while also giving good mass transfer properties that allow an analyte to quickly reach this stationary phase for retention [19, 33]. As illustrated in Fig. 2b, an affinity monolith can provide lower plate heights and higher efficiencies than traditional HPLC-grade particulate supports , especially when used at high linear velocities and flow rates [10, 11, 15, 30]. This again results in supports that are attractive for use in high-throughput separations or fast analysis methods [10, 15].
Another advantage of monoliths is they can be prepared in a variety of formats. These formats include columns, such as those reported in Fig. 2, as well as disks, capillaries, and microchips. As a result, it has been possible to employ affinity monoliths in traditional HPLC systems, in methods combining affinity columns with other techniques (e.g., mass spectrometry), and in microanalytical systems [9, 10, 13, 15]. Monoliths have also been combined with a variety of affinity ligands, allowing the resulting supports to be used in many types of biochemical separations [9, 10, 11, 12, 13, 14, 15, 19, 29]. Examples of specific applications that have used these features of monolith supports in AMC will be discussed in the following sections.
Organic-based affinity monoliths
There are a variety of organic-based polymers that have been employed in monoliths for liquid-phase separations (e.g., see reviews in [33, 34, 35]). However, only a few of these have been used in AMC, and most work with organic-based affinity monoliths has been done by using copolymers of glycidyl methacrylate (GMA) and ethylene glycol dimethacrylate (EDMA) [9, 10, 11, 13, 15, 29, 30, 31, 37]. There are several reasons for the popularity of GMA/EDMA monoliths for use with affinity ligands. First, GMA/EDMA monoliths are commercially available [9, 10, 13, 15]. Second, these materials are relatively easy to prepare in a hydrophilic form (e.g., after conversion of the epoxy groups on GMA into a diol form) that provides low nonspecific binding for most biological agents [9, 10]. Third, there are various ways of modifying this type of monolith to make it suitable for ligand attachment. Fourth, these monoliths can be prepared with a variety of pore sizes, shapes, and surface areas. All of these features have led to the creation of AMC supports in which GMA/EDMA has been employed with numerous binding agents and separation formats [10, 13, 15].
The binding agent is often immobilized within an activated GMA/EDMA monolith by passing through this support a solution of the binding agent in an appropriate buffer [10, 15]. The final quantity of the immobilized binding agent can be adjusted by varying the concentration of the applied ligand, the immobilization method, or the buffer composition and pH [9, 10]. It is also possible to alter the relative amounts of the porogenic solvents that are used to generate the monolith. This last approach has been found to have a significant influence on the pore size and accessible surface area that is available for immobilization in GMA/EDMA monoliths. As an example, it has been shown that altering the ratio of dodecanol to cyclohexanol that is used in the polymerization mixture can have a large effect on the number of antibodies that can later be attached to GMA/EDMA monoliths for use in immunoextraction .
Ligands that have been immobilized on GMA/EDMA monoliths have included antibodies, antibody-binding proteins (e.g., protein A and protein G), lectins, avidin, l-histidine, trypsin, peptides, and serum albumins [9, 10, 11, 13, 14, 15, 41, 42, 43, 44]. A recent example involved the immobilization of trypsin on an epoxy-containing monolith to create an enzyme reactor for protein analysis . Other work used a GMA/EDMA monolith that contained immobilized monomeric avidin for binding to biotin-labeled agents. This monolith was used in combination with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry for the enrichment and analysis of biotinylated proteins and peptides . One disadvantage of using GMA/EDMA monoliths is that they tend to have a lower surface area compared with conventional particulate supports or silica monoliths; this difference, in turn, can limit the amount of ligand that can be immobilized on these supports [10, 13].
In spite of the popularity of GMA/EDMA monoliths in AMC, there have been several works that have considered and used other organic polymers for this purpose. For instance, one study used glyceryl methacrylate (GMM) in place of GMA as a copolymer with EDMA for use in immobilizing lectins . An alternative cross-linking agent to EDMA that has been used with GMA is trimethylolpropane trimethacrylate (TRIM) [13, 46], which has been recently employed in the creation of GMA/TRIM monoliths that contain HSA as a chiral stationary phase . Copolymers based on GMA and divinylbenzene (DVB) have been used for the creation of immobilized trypsin enzyme reactors  and with immobilized chelating agents plus Cu2+  or Fe3+  in IMAC. In addition, monoliths based on DVB  or EDMA  have been used to entrap metal oxide nanoparticles for the capture of phosphopeptides. The use of copolymers of 2-hydroxyethyl methacrylate, piperazine diacrylamide, and (+)-N,N-diallyltartardiamide has also been examined as an alternative to the use of GMA/EDMA monoliths in AMC [13, 53, 54].
Inorganic-based affinity monoliths
There have been several examples in which silica monoliths have been employed in AMC. Two studies used HSA and α1-acid glycoprotein (AGP) that were coupled to silica monoliths for use in chiral separations [32, 58]. Other binding agents that have been used in combination with silica monoliths are enzymes, iminodiacetic acid (for use in chelating metal ions), trypsin, and 3-(2-aminoethylamino)propyl ligands [59, 60, 61, 62]. Another recent example involved the use of silica monoliths that contained immobilized serum transport proteins to estimate the dissociation rate constants for various drugs from these binding agents .
There are several advantages in using silica monoliths for AMC. These advantages again include the good mass transfer properties and low back pressures of these materials when compared with particulate supports . In addition, commercially available silica monoliths can be modified for use in AMC [32, 58]. Another important advantage arises from the fact that silica monoliths have the same surface chemistry as silica particles, so similar immobilization methods and conditions can be employed with these two types of supports [10, 15]. Silica monoliths also suffer from the same limitations as silica particles, such as their limited pH stability [10, 19]. However, the different pore structure and morphology of silica monoliths compared with GMA/EDMA supports tends to give silica monoliths greater accessible surface areas for protein immobilization, allowing an increase in the amount of protein that can be attached within such materials [10, 32, 58].
The immobilization methods that are used with silica monoliths are often based on methods that have been adapted from silica particles, and include procedures similar to many of those shown in Fig. 4 for GMA/EDMA monoliths. This list includes the epoxy method and the Schiff base method , as well as the use of hydrazide-activated silica and mild oxidation conditions for immobilizing carbohydrate-containing agents such as AGP . Another option that can sometimes be employed with small silica monoliths (e.g., filled capillaries) is to include the affinity ligand in the reaction mixture during the sol–gel process. This approach leads to the encapsulation of the ligand within the support, but can result in some ligand denaturation owing to alcoholic by-products that are released during the polymerization process . However, work has also been conducted with a protein-compatible silane for the sol–gel method that can avoid the release of these by-products and minimize such denaturation .
Other types of monoliths for AMC
Agarose has long been a popular support in affinity chromatography [1, 2, 18], so it is not surprising that research has also been conducted into making monolithic supports from agarose [14, 19]. Agarose is a polysaccharide containing repeating units of d-galactose and 3,6-anhydro-l-galactose. This material is hydrophilic and has low nonspecific binding to most proteins and biological ligands. Agarose also has good chemical stability over a broad pH range. The major limitation for agarose-based supports is their lack of high mechanical stability, which limits their use in HPLC unless some cross-linking of the support is employed . An advantage in using agarose in monoliths is that these supports are fairly easy to prepare in a variety of shapes and sizes [14, 19].
The preparation of agarose monoliths occurs through several steps. The first step involves dissolving the agarose in water and heating the solution to 90–100 °C. The agarose solution is then mixed with a water-immiscible organic solvent. This mixture is shaken to form an emulsion and poured into a mold such as a column. The monolith is allowed to cool and take the shape of the mold. The end result is a monolith that is a continuous unit and that contains flow-through pores .
Cryogels are another group of monoliths that have been used in AMC [10, 15, 66, 67, 68, 69, 70, 71, 72]. These supports can have good chemical and physical stability and have been reported to be cost-effective to use . Cryogels are gel matrices that are formed in the presence of moderately frozen solutions of monomeric or polymeric precursors. These conditions can provide interconnected macropores that allow relatively fast diffusion as analytes pass through this type of material. Cryogel monoliths can be prepared in the form of either columns or membranes . Like other types of monoliths, cryogels often allow the use of high flow rates, making it possible to pass large sample volumes through them in a short time [66, 67]. One possible limitation of a cryogel is its relatively low surface area, which could limit the amount of binding agent that can be immobilized on this support for AMC .
Monoliths in bioaffinity chromatography
Bioaffinity chromatography is a type of affinity chromatography that employs a biologically related ligand as the stationary phase . Some examples of binding agents that can be used in this method are immunoglobulin-binding proteins, enzymes, and lectins (note that antibodies and serum proteins, two other groups of biologically related binding agents, will be discussed separately in later sections) [10, 13]. Two binding agents from this group that have been employed in AMC are protein A and protein G [12, 41, 73]. These proteins are used to bind immunoglobulins and antibodies. Protein A and protein G are found on the surface of certain bacterial cells and bind to the Fc region of antibodies. Protein A is produced by Staphylococcus aureus and protein G is produced by streptococci [16, 18]. There have been a number of examples in which protein A and protein G have been used in affinity monoliths [13, 15]. Protein A has been immobilized on a GMA/EDMA monolith for the purification of IgG-class antibodies  and was used to bind IgG from microliter samples of human serum . In addition, a GMA/EDMA monolith was modified with protein G and used in combination with an anion-exchange monolith disk for the rapid analysis of IgG, transferrin, and insulin in cell cultures .
Enzymes are another class of binding agents that can be used in AMC, as well as in related applications such as enzyme reactors and enzyme-based biosensors [14, 43, 75, 76, 77, 78, 79]. For instance, dihydrofolate reductase has been placed in an affinity monolith for screening potential inhibitors of this enzyme . The use of affinity monoliths for enzyme reactors has been of particular interest [13, 15]. This is because the good mass transfer properties, low back pressures, and reasonable surface areas of these materials have made it possible to obtain high digestion efficiencies and good immobilization yields for enzymes . Two recent examples involved the use of silica monoliths that contained immobilized trypsin for the digestion of proteins [76, 77]. This type of bioreactor has been coupled online with liquid chromatography–electrospray ionization tandem mass spectrometry for the analysis and identification of proteins [76, 77]. Another study used trypsin immobilized within a monolith based on GMA/DVB and placed into pipette tips. This support was then used for the microwave-assisted tryptic digestion of proteins within only a few minutes . Other reports have used trypsin with GMA/EDMA monoliths  and various enzymes within agarose-based monoliths .
Monoliths in immunoaffinity chromatography
Affinity columns that make use of immobilized antibodies or related agents produce a special type of bioaffinity chromatography that is known as immunoaffinity chromatography [17, 80]. Many immunoaffinity chromatography methods have been developed for the isolation and purification of hormones, enzymes, peptides, viruses, and other biologically relevant substances [17, 80, 81, 82, 83, 84, 85, 86]. Immunoextraction is a special type of immunoaffinity chromatography in which an affinity column is used to isolate compounds from a sample prior to analysis by a second method [17, 80].
Monoliths in IMAC
IMAC is a type of affinity chromatography that makes use of the specific interactions that can occur between immobilized metal ions and targets such as amino acids, peptides, proteins, and nucleic acids . The immobilized metal ions frequently consist of Co2+, Zn2+, or Ni2+ ions, among others. Iminodiacetic acid (IDA), nitrilotriacetate, carboxymethylated aspartic acid, and tris(carboxymethyl)ethylenediamine are chelating agents that have been used to immobilize metal ions in columns for traditional IMAC [15, 25]. Of these, the most common chelating agent that has been used in combination with monoliths is IDA. Amino acid residues on proteins and peptides that are able to bind to the immobilized metal ions are generally residues such as histidine, tryptophan, and cysteine, although other types of amino acids or modified residues may also be involved .
Several types of monoliths have been employed in IMAC, ranging from silica monoliths to polymethacrylate-based supports and cryogels [49, 50, 60, 91, 92]. One previous application used a silica monolith capillary column that was functionalized with IDA and Fe3+. This capillary column was used to capture phosphopeptides from samples of α-casein . Another application employed cryogel monoliths and used N-methacryloyl-l-histidine methyl ester as a chelating agent for Cu2+ ions. This monolith was used for the rapid purification of cytochrome c from a rat liver homogenate . A separate study involved a commercial GMA/EDMA disk that was coupled with IDA–Cu2+ for use in plasmid DNA purification .
A number of recent articles have explored an interesting variation of IMAC known as metal oxide affinity chromatography, in which metal oxide particles are used as ligands for the isolation of metal-binding analytes (e.g., targets containing phosphate groups) . In one such article, it was reported that titanium dioxide and zirconium dioxide microparticles and nanoparticles were placed within highly porous poly(divinylbenzene) monoliths and were tested for use in the isolation of phosphopeptides obtained from digests of α-casein, β-casein, and phosphorylated extracellular-signal-regulated kinase 1 . Titanium dioxide particles have also been placed within monoliths prepared with EDMA and through emulsion photopolymerization for the isolation of phosphopeptides from digests of α-casein and β-casein . Another study used a coating of iron oxide particles on GMA/EDMA monoliths that had been modified to contain quaternary amine groups, with the resulting support then being used to extract phosphopeptides from digests of α-casein and β-casein as model systems .
Monoliths in dye–ligand affinity chromatography
Dye–ligand affinity chromatography is a type of affinity chromatography that employs a synthetic dye as the immobilized binding agent. There are several advantages in using dyes as the stationary phase for an affinity column, including their good stability, ease of preparation, low cost, and high binding capacity . Often these binding agents are based on triazine dyes such as Cibracron Blue 3GA. There have been several studies in which these dyes were used in combination with monolithic supports such as agarose and cryogel-based monoliths [15, 97, 98]. One study used superporous agarose beds containing Cibracron Blue 3GA for the purification of lactate dehydrogenase from a crude bovine heart extract . Another study used Cibracron Blue 3GA immobilized on a poly(hydroxyethyl methacrylate) cryogel for the rapid removal of high-abundance proteins such as albumin from human serum (see Fig. 7) .
Monoliths in biointeraction chromatography
Biointeraction chromatography is a special type of affinity chromatography in which this method is used to study a biological interaction [7, 8]. One method that can be used for this purpose is frontal analysis. In frontal analysis, the analyte is continuously applied to a column that contains the immobilized binding agent until a characteristic breakthrough curve is formed . If the analyte and binding agent have relatively fast interactions on the timescale of the experiment, the mean position of this breakthrough curve can be used to obtain information on the association equilibrium constants and the binding capacity for the analyte on the column [6, 8]. In one recent report, affinity silica monoliths were evaluated by frontal analysis to examine interactions between the drug carbamazepine and the proteins HSA and AGP to aid in the development of chiral stationary phases based on these proteins [32, 58]. A similar study used GMA/EDMA monoliths to examine the binding of naproxen to immobilized bovine serum albumin . Frontal analysis has been further employed with mass spectrometry as a technique for screening the binding of small-molecule libraries against immobilized enzymes, as has been done with dihydrofolate reductase contained in a sol–gel in a fused-silica capillary .
Monoliths in chiral separations
Conclusions and future directions
This review has examined the basic principles and recent applications of AMC. The advantages of using monoliths over traditional affinity supports include the good efficiency and low back pressures of such materials, as well as their ability to be made in a variety of formats. A variety of materials have been used to make columns and supports for AMC. Organic monoliths based on GMA/EDMA have been particularly popular in this method. However, silica monoliths, agarose monoliths, and cryogels have also been used. These supports have been used in formats that have ranged from traditional columns to disks, microcolumns, capillaries, and microchips [10, 13, 14, 15, 47, 99, 100].
An area of growth that is expected for AMC is the modification and adaption of new types of monoliths for work with affinity ligands. This trend is expected to include the modification of existing, alternative types of organic polymers to provide a broader range of pore sizes, surface areas, and morphologies that can be used in this method. The need for supports with high accessible surface areas for ligand immobilization and the creation of suitable binding capacities or binding properties is further expected to grow as research continues into the use of AMC in miniaturized analytical systems [9, 10, 13, 15, 99, 100]. The creation of composite materials for AMC is also expected to be of interest, as is illustrated by recent work that combined organic monoliths with metal oxide particles for the isolation of phosphopeptides [51, 52, 96]. The possible use of hybrids of silica and organic monolith supports, as has been reported for other chromatographic methods [82, 101], is another combination which may create supports with unique properties for AMC.
Many types of binding agents have already been used with monolithic supports in AMC. These binding agents have included antibodies, enzymes, proteins, peptides, lectins, immobilized metal ions, and dyes. The result has been a variety of applications that have been reported for AMC that are based on methods such as bioaffinity chromatography, immunoaffinity chromatography or immunoextraction, IMAC, dye–ligand affinity chromatography, and chiral separations. However, growth in this area is expected as there are still many types of binding agents that have not yet been adapted for work with affinity monoliths. For example, the number of studies that have used AMC for dye–ligand affinity chromatography is still small considering the widespread use and importance of this separation method with traditional affinity supports in the areas of protein and enzyme purification for biochemistry and biopharmaceuticals . The same is true for the use of AMC for chiral separations, which is an important method when used with more traditional HPLC-based affinity supports for pharmaceutical and clinical analysis [7, 15, 16]. Other binding agents that may offer additional areas of growth in AMC include antibody mimics such as aptamers and molecularly imprinted polymers [13, 27, 102, 103, 104].
As alternative supports and affinity ligands are used in AMC, it is anticipated that more work will also be needed in the characterization of these supports and in better understanding the unique properties and behavior they may offer in liquid-phase separations. Such developments are already occurring in AMC as this method is being used with microscale analytical devices [9, 10, 13, 15], hybrid materials [51, 52, 96], and biointeraction chromatography [10, 32, 58, 64, 99, 100]. The combined efforts that are expected to occur in these practical and theoretical developments should help to increase the use of AMC in analytical chemistry, pharmaceutical analysis, clinical testing and research, and biotechnology, to name a few fields. Given the wide range of supports, formats, and binding agents that can already be employed in AMC, it is expected that the use of this method will continue to grow in these and other fields in the coming years.
This work was supported in part by the National Institutes of Health under grant R01 GM044931, the National Science Foundation (NSF) REU program, and the NSF/EPSCoR program under grant EPS-1004094.
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