Professional Documents
Culture Documents
Carbohydrate Biosensors
Raz Jelinek* and Sofiya Kolusheva
Department of Chemistry and Staedler Minerva Center for Mesoscopic Macromolecular Engineering, Ben Gurion University of the Negev,
Beersheva 84105, Israel
Contents
1. Introduction 5987
2. Detection and Analysis of Carbohydrates in 5988
Biological Systems
2.1 Identification of Carbohydrates and 5988
Carbohydrate Derivatives
2.1.1 General Procedure 5988
2.1.2 Heparin Detection 5992
2.1.3 Carbohydrate Structures 5994
2.2 Lectin-Based Biosensors 5995
2.3 Glycoprotein and Glycosylation Biosensors 5997
2.4 Pathogen and Cancer-Detection Assays 5999
2.4.1 Pathogen Identification 5999
2.4.2 LPS Biosensors 6002 Raz Jelinek was born in 1964 in Beersheva, Israel. He obtained his B.Sc.
2.4.3 Cancer Diagnostics 6003 in chemistry (summa cum laude) at the Hebrew University, Jerusalem,
2.5 Carbohydrate Nanobiosensors 6003 Israel, in 1988 and went on to get his Ph.D. in chemistry at the University
2.6 Miscellaneous Carbohydrate Bioassays 6004 of California, Berkeley, in 1993, where he did research under the guidance
of Alex Pines on solid-state NMR of zeolites and molecular sieves. During
3. Carbohydrate Components in Biosensors 6004 his postdoctorate at the University of Pennsylvania, he switched to
3.1 Carbohydrate Recognition Elements 6004 structural biology, doing NMR on peptides in membrane environments.
3.2 Carbohydrate Scaffolds 6006 In 1996, Raz returned to Beersheva to take a position at Ben Gurion
3.3 Biosensors Utilizing ProteinCarbohydrate 6007 University, where he is conducting research on biosensor development
and biomimetic cellular systems.
Interactions
3.4 Carbohydrates in SPR 6010
4. Concluding Remarks 6011
5. Abbreviations 6012
6. References 6012
1. Introduction
Carbohydrates (denoted also oligosaccharides or
polysaccharides) constitute a large and diverse class
of compounds present in varied materials and have
major roles in applications in chemistry, biology,
materials science, and related fields. In the context
of biological systems, in particular, carbohydrate
research has emerged as the new frontier for
elucidating fundamental biochemical processes and Sofiya Kolusheva received her M.Sc. degree in biology from Tashkent
for identifying new pharmaceutical substances. Be- State University (Uzbekistan) in 1989 (magna cum laude) and her Ph.D.
side nucleic acids and proteins, carbohydrates appear degree in biology from the Institute of Bioorganic Chemistry (Tashkent,
to play critical roles in determining biological func- Uzbekistan) in 1994. She then took a postdoctoral fellowship at the Ben
tions and affecting wide-ranging physiological pro- Gurion University of the Negev (Beersheva, Israel) in 1997. She is currently
a Senior Researcher in the Biophysical Laboratory, Ilse Katz Center for
cesses, thus, their study and characterization have Nano- and Mesoscience and Technology, Ben Gurion University of the
become increasingly important. Negev. Her current scientific interests focus on supramolecular poly-
This review aims to provide a comprehensive diacetylene/lipid assembles and their application as membrane bio-
overview of recent scientific activity pertaining to sensors.
parts of the biological recognition systems. We have of nanotechnology in carbohydrate biosensor re-
particularly tried to discuss in depth several topics search.
that we believe define the current status of the The second part of the review summarizes biosen-
carbohydrate biosensors field and point to possible sors and bioassays intended not to detect carbohy-
future avenues. We have not attempted to cover all drates but in which the carbohydrates constitute
aspects of carbohydrate chemistry and biology, car- essential components in the biosensor design, either
bohydrate-detection methods, or issues concerning as recognition elements or as the building blocks
molecular processes involving carbohydrates; these within the sensor template. We discuss biosensor
aspects are broad and prolific fields of study, and the schemes that employ specific biomolecular interac-
reader is referred to relevant literature.1 We have tions such as lectin-carbohydrate affinities, carbo-
also not discussed here the highly technologically and hydrates as substrates in enzymatic processing re-
commercially important field of glucose sensing, an actions, and solid carbohydrate matrixes incorporated
active area of research because of the profound health within sensing devices. A subsection is devoted to the
effects of aberrant glucose levels in diabetes, as large body of experimental work utilizing surface
glucose is technically a monosaccharide rather than plasmon resonance (SPR) biosensors that have been
a carbohydrate. We do, however, include a description widely used in recent years for studying interactions
of monosaccharide biosensors, where such devices and molecular recognition processes involving car-
represent important concepts in biosensor designs, bohydrates.
for example, biosensors employing carbohydrate-
lectin recognition. Similarly, this review does not 2. Detection and Analysis of Carbohydrates in
address the large body of commercially oriented Biological Systems
literature (i.e., patents) related to polysaccharide
biosensors. Overall, we tried to limit the scope of this 2.1 Identification of Carbohydrates and
review to more recent published reports, rather than Carbohydrate Derivatives
providing a historical perspective of the field. Related
reviews on the subject have appeared in the litera- 2.1.1 General Procedure
ture in the past.2 The primary requirements facing new biosensor
Biosensors are generally defined as multifunctional technologies include maximization of the sensitivity,
assemblies composed of matrix-bound bioactive sub- selectivity, and reproducibility within the experimen-
stances responsible for the specific recognition of the tal setup. These issues have been predominant in
species of interest, which are directly coupled to a biosensor design and construction. In that regard, the
physicochemical transducer supplying the output complexity of carbohydrate structures and the diver-
signal. In this review, however, the term biosensor sity of their chemical properties and molecular
has been used in a somewhat broader sense, includ- context pose particular and significant bioanalytical
ing systems that can be characterized as biochemical challenges. These have led to the development of a
assays. Because of space and scope considerations, large number of biosensors and bioassays for carbo-
we have not provided here a complete description of hydrate identification and analysis using spectro-
all biological assays in which carbohydrates have scopic, biochemical, or electrochemical methods.
been directly or indirectly involved; we have focused The use of enzymatic digestion has been among the
instead on assemblies in which carbohydrates con- first and most common assay approaches for carbo-
stitute the critical or central sensing components and hydrate analysis.3-7 In such techniques, carbohydrate
discussed in more depth systems which define or detection generally relies on enzymatic catalysis of
represent special and novel functions of their carbo- saccharide substrates by immobilized glycoenzymes.3-5
hydrate constituents. Similarly, only representative Because the detected signal in enzyme-based biosen-
publications were cited in the text when we discuss sors originates from the reaction products of the
assay systems that are widely applied. enzyme action, a critical requirement in such sensors
is the maintenance and optimization of the biological
The review is divided into two sections. In the first activity of the enzyme. This could be particularly
part, we discuss schemes for detection of carbohy- demanding because in most sensing applications and
drates, where the sensors are designed to detect the devices the enzymes have to be immobilized on solid
sugar molecules by themselves or as parts of larger supports.3 Immobilization of glycosylated enzymes
biological or chemical entities (for example, glycolip- through binding to lectins has been reported.3-5 This
ids and glycoproteins). Subsections focus on the approach has certain advantages over surface bind-
significance of lectin-carbohydrate interactions in ing of the enzymes using means of covalent linkage,
biosensor design (section 2.2) and the analysis of particularly because the latter technique might in-
carbohydrate derivatives such as lipopolysacharides terfere with the stability and biological viability of
(LPS) and other glycolipids and glycoproteins for the enzyme. Furthermore, the high lectin-carbohy-
toxin, pathogen, and cancer detection (section 2.4). drate affinity constituting the basis for the im-
Certain overlap exists among the subtopics; for mobilization procedure contributes to the stability of
example, some pathogen-detection schemes utilize the biosensor assembly and its resistance to varied
carbohydrate-lectin recognition. A subsection was external degrading factors, such as heat or chemical/
devoted to the emerging field of polysaccharide nano- biological denaturation.
biosensors (section 2.5), recognizing the contribu- Frequently, the output signals produced by enzyme-
tions and unique scientific and technological potential based-detection methods are relatively low, and
Carbohydrate Biosensors Chemical Reviews, 2004, Vol. 104, No. 12 5989
amplification of the sensor response is necessary. Varied electrochemical methods have been applied
Magnification of enzymatic signals has been achieved for carbohydrate detection in biological and pharma-
through multiple glycoenzyme layering.5 Such lectin- ceutical samples. A major impetus for development
based immobilization methods have opened the way of electrochemical approaches as compared to other
to assemblies with higher glycoenzyme affinities and bioanalytical techniques has been the observation
better load factors. Varied synthetic developments that carbohydrates do not generally contain intrinsic
have focused on identification of solid matrixes that chromophores (neither fluorescent nor emitting in the
facilitated repetitive layering of glycoenzymes and UV-visible range). Recent advances in the design
lectins.3-5 Such multiple bioaffinity layering exhib- and application of electrochemistry in saccharide
ited superior analytical capabilities compared to assays were extensively reviewed.11 Two electro-
other glycoenzyme-immobilization approaches.3-5 chemical carbohydrate-detection strategies, in par-
Some technical challenges, however, are inherent in ticular, have been thoroughly explored: enzyme-
detection schemes utilizing multiple glycoenzyme based electrodes and direct oxidation at electrode
layering using immobilized lectins, primarily the surfaces, mostly employed in postseparation analysis
need for several preparative steps while retaining the in liquid chromatography or capillary electrophoresis
catalytic activities of the enzymes in the solid- schemes.
supported environments. Historically, enzyme-based electrochemical detec-
The multiple glycoenzyme-layering technique fur- tion strategies for carbohydrates were developed
ther necessitates careful selection of the solid sup- because direct analyses of saccharide compounds
ports. The appropriate matrixes should be sufficiently were traditionally hampered by the unfavorable
reactive to allow derivatization with the lectin, have redox properties of many sugars.11 In the most basic
to exhibit relatively large and accessible surface area, amperometric enzyme electrode, glucose was oxidized
and should not interfere with the catalytic domains within an immobilized layer of glucose oxidase and
of the immobilized glycoenzyme layers.5 Biospecific then determined at a conducting platinum or carbon
sorbent matrixes were reported.3-5 Importantly, from electrodes by measuring the current resulting either
a biosensing point of view, lectin-based multilayering from oxidation of hydrogen peroxide or reduction of
methods do not dictate which detection schemes are diatomic oxygen consumed by the enzymatic reac-
to be used for measuring the enzymatic activity. tion.12 Overall, the underlying concept of enzymatic
Thus, different sensing methods based on enzymatic electrodes for carbohydrate analysis involves the
digestion in lectin-based multilayer environments highly specific conversion of mostly monosaccharide
analytes into more conveniently oxidized species
were described in the literature, including flow-
(such as H2O2).
microcalorimetry, in which changes in heat capacity
induced by the catalytic action of a glycoenzyme were Immobilization of carbohydrate-digesting enzymes
recorded.5 Other methods employed coupling between onto electrode surfaces without impairing their func-
several enzymatic processes that produce spectro- tionalities and mediation of the electron transfer to
scopically detected species.7 the electrode surface have been among the practical
impediments for implementation of enzyme-based
Signal amplification inherent in the multilayering electrochemical techniques. Accordingly, a number of
approach was employed toward achieving sensor electrochemical biosensing approaches have utilized
prototypes that could identify carbohydrates within direct oxidation of carbohydrates at electrode sur-
complex mixtures. An important consequence of this faces. Many of these techniques require for the
property is the feasibility of miniaturization within oxidation to occur electrochemical potential condi-
devices based on multienzyme assemblies. Technical tions for which many electrode materials are inad-
advances in this field have led to fabrication of equate.11 Accordingly, a critical issue in such appli-
microfabricated biosensors containing lectin-bound cations has been the proper selection of electrode
glycoenzyme layers coupled to silicon chips.6 Such composition.13 Most direct-oxidation detection schemes
achievements could open the way to diverse biosens- have combined electrochemical processing of the
ing applications, such as the fabrication of flow carbohydrates with liquid ion-exchange chromatog-
channels within the biosensor chip.6 raphy for compound separation.14,15
Immobilization of sugar-digesting enzymes within Complex carbohydrates or multicomponent mix-
miniaturized biosensor devices has been achieved by tures pose particular challenges for application of
other methods. An enzyme-based disaccharide mi- electrochemical detection methods. In such systems,
crodetector sensor prototype included enzyme-deriva- the issue of selectivity and/or separation often has
tized agarose beads placed within wells etched on a to be addressed in parallel with the actual detection
silicon chip. Miniaturization in this kind of device process. Varied methods have been developed for
allows the simultaneous analysis of carbohydrate achieving these goals, roughly divided into two main
mixtures.7 Practical advantages of enzyme-based approaches: the first relies on the actual selectivity
biosensor chips include the very low sample volumes of the chemical/biological component recognizing or
required (often in the range of nanoliters), the reacting with the carbohydrate to be analyzed (such
feasibility for presentation of different enzymes on a as the enzyme for which the carbohydrate is the
single chip, which facilitates parallel analysis of substrate); the second group of detection schemes
complex oligosaccharides or multicomponent systems, combines the electrochemical analysis with separa-
translated into significant cost reduction, and the tion techniques such as capillary electrophoresis (CE)
availability of mass production.6-10 or liquid chromatography (LC).11 Development of
5990 Chemical Reviews, 2004, Vol. 104, No. 12 Jelinek and Kolusheva
separation methods for carbohydrates is particularly of the larger molecule (a prerequisite for separation
important, because it has been found that, unlike and analysis), as well as affect the elution of the
proteins or other macromolecules, a large size or high components. These constraints naturally pose chal-
molecular weight does no significantly impair the lenges to the successful use of this methodology.
capability of electrochemical methods to accurately Other fluorescence-based assays were developed not
detect the molecule.11 only for identification of individual oligosaccharides
Coupling of electrochemical detection to CE has but also to characterize biochemical processes in
attracted interest in recent years because of the which carbohydrates participate. A fluorescence-
power of the technique to resolve and identify car- labeling technique has been introduced to study the
bohydrates in complex mixtures.16,17 Actual analysis gelation properties and cell-wall localization of algi-
of the carbohydrates is similar to other electrochemi- nate, the major cell-wall carbohydrate of brown
cal methods in which the redox reactions take place algae.23 Specifically, the fluorescent dye fluorescein
at metal electrode surfaces, while the function of CE was conjugated to short polygluronate chains and
is separation of the compounds within the mixtures. used to target the gelling subunits of the carbohy-
However, a specific technical problem that has to be drate. The method allowed rapid labeling and probing
surmounted in such devices concerns the electronic of distinct cellular regions from varied algae sources.
separation between the electrophoretic and electro- Several carbohydrate-detection schemes based on
chemical processes. This is due to the fact that boronic acid were reported in the literature, often
current leakage between the two electrical circuits utilizing fluorescent tags attached to the boronic acid
has to be avoided and minimized, because the detec- moieties.24-27 The three primary building blocks
tion potential is usually much smaller than the comprising such photoinduced electron transfer (PET)
capillary electrohoresis voltages.11 biosensors are the fluorophore, the carbohydrate
Several techniques were described in the literature receptor, and a molecular spacer separating them.27
that similarly rely on compound separation but use In particular, saccharide detection achieved with the
detection schemes other than electrochemistry, for use of these molecular assemblies rely on the reactiv-
example, UV absorbance.18 A carbohydrate-detection ity of boronic acid with vicinal cis-diols of carbohy-
technique employed in conjunction with a separation drates.24,27 Boronic acid PET biosensors exhibit no-
method was denoted polarized photometric detection table advantages and disadvantages. On one hand,
(PPD).19 The sensor apparatus of the PPD unit the criteria for molecular design allow significant
consisted of placing two light polarizers at opposite flexibility in determining saccharide ligand binding
sides of a conventional UV-vis spectrophotometer thorough shape selectivity, chiral recognition, allo-
flow cell. This arrangement allowed the measure- steric discrimination, and other factors.27 On the
ment of optical rotation of chiral compounds through other hand, functionality of the biosensor generally
the change in absorbance. Despite its crude mecha- requires high pH environments to produce ionization
nism, application of PPD was claimed to achieve of the boronic acid units (yielding boronate anions),
extremely high detection sensitivity for oligosaccha- a feature that limits the usefulness of such assays.
rides through the different rotations exerted by the Varied boronic-acid-based biosensor designs in-
molecules.19 cluded polymer hydrogels coupled to pendant boronic
Even though carbohydrate detection schemes that acid units.24 Such biosensors conform to the classic
are combined with compound separation are gener- biosensor design in that the recognition event be-
ally satisfactory in achieving high-sensitivity com- tween the sensor (the hydrogel-boronic acid conju-
pound identification, their main drawback is the gate) and the carbohydrate analyte gives rise to a
ultimate dependence upon the separation technique detectable physical change in the system, a shift of
for efficient application. Thus, many of the generic the visible wavelength of light diffracted by the
prototypes and published experimental data re- hydrogel.24 Swelling of the hydrogel (responsible for
quired, to some degree, prior knowledge of the type the change in the diffraction wavelength) is induced
of oligosaccharide mixture to be analyzed. Accord- in the sensor assembly by the increased osmotic
ingly, the majority of reported differential-elution/ pressure occurring from the decrease of pKa of the
detection methodologies have been applied toward boronic acid following binding to the carbohydrate.
analysis of simple sugars. This carbohydrate-detection scheme is simple, robust,
Fluorescence spectroscopy has had an important and quite sensitive (lower than 50 M carbohydrate
contribution to development of carbohydrate biosen- analyte detected).24 The system was demonstrated
sors, mostly through the use of fluorescent labels.20-22 primarily for detection of simple sugars, such as
Such carbohydrate fingerprinting techniques usu- glucose, although conceptually, it could be generally
ally consist of several stages. The analysis includes applied for more complex carbohydrates. Similar
attachment of nonspecific fluorescent tags that bind biosensor constructs utilizing boronic acid derivatives
to monosaccharide building blocks within the sugar consisted of a fluorophore and boronic acid attached
molecule, breaking the larger saccharide into smaller to an amine moiety.25,26 When a saccharide analyte
fluorescent-tagged units mostly by enzymatic diges- binds to the boronic acid, the boron atom becomes
tion and application of separation procedures (for more acidic, leading to an enhanced Lewis acid-base
example, liquid chromatography) for complete as- interaction with the amine nitrogen. This reduces the
signment.20,22 The coupling of the carbohydrate ana- interaction of the nitrogen lone pair with the fluoro-
lyte with additional molecular entities (the fluores- phore, thus suppressing the PET process and in-
cent tags) might interfere with both the fragmentation creasing the fluorescence.
Carbohydrate Biosensors Chemical Reviews, 2004, Vol. 104, No. 12 5991
Detection methods employed in boronic-acid-based carbohydrate derivatives adsorbed onto the polymer
biosensors were not limited to fluorescence tech- framework. The key methodological requirements
niques. Indeed, the use of the chemical reaction successfully demonstrated in that report were, first,
between the saccharide and boronic acid as the the synthesis of a functional fluorescent monomer
defining feature of a biosensor facilitates the applica- displaying strong binding interactions with particular
tion of a plethora of sensing approaches that were structural elements in carbohydrates (cis-diols) and,
recently reviewed.25 Specific bioanalytical techniques second, retaining the discrimination capabilities of
included chiral saccharide recognition using circular the fluorophore and its fluorescence sensitivity inside
dichroism (CD) and liquid crystalline suspensions,25,28 the polymer framework. Gao et al. attained these
the use of colorimetric carbohydrate receptors,29,30 goals for detecting fructose with the use of a monomer-
electrochemical detection via coupling of the boronic conjugated boronic acid monomer.37
acid recognition assembly to a redox unit such as A practical weakness encountered in MIP applica-
ferrocene,31,32 and others. tions, particularly saccharide-templated materials,
Several carbohydrate-sensing schemes have been has been the low reloading capacity of the analytes.
based on recently developed chemical and biophysical To overcome this limitation, some studies proposed
techniques. Molecularly imprinted polymers (MIPs), to enhance the binding capability of the polymer
for example, have attracted an increasing interest as matrix through chemical manipulations, for example,
templates for carbohydrate-detection assays. Molec- by increasing the polarity of the polymer backbone,
ular imprinting creates recognition sites in polymers thus enabling multiple hydrogen bonding between
by using template molecules; the templates are the polymer framework and the incorporated carbo-
prepared by initiation of the polymerization pro- hydrate guest molecules.38 Specifically, the research-
cesses, while molecules of a particular analyte are ers explored the effects on saccharide rebinding of
incorporated within the solidifying material.33,34 Fol- inclusion of multiple metal cations, such as CuII2,
lowing the removal of the embedded analyte mol- within the polymer template and the use of polar
ecules, the polymer essentially becomes a porous cross linkers such as pentaerythritol within the
framework that selectively adsorbs only the analyte polymer matrix. Improved performance of the MIP
molecules within the pre shaped binding sites.35 This was indeed demonstrated for several polysaccharides,
kind of templating biosensing approach could be indicating that varied synthetic routes could be
particularly well-suited for carbohydrate detection employed to optimize the bioanalytical performance
and analysis because the imprinting procedure might of MIP-based sensors.
be able to distinguish between different functional Carbohydrate detection using whole-cell biosensors
units and/or saccharide moieties within complex has been also an active field of research in recent
carbohydrates. years. Even though the technique was so far em-
Proofs of concept for the application of MIPs for ployed almost only for detection of mono- and disac-
detection of simple sugars were described in several charides,39 it holds promise as a highly generic
publications. Quartz crystal microbalance (QCM, see approach for carbohydrate analysis in natural sam-
more in-depth description of the technique below) ples. In contrast to simple, modular carbohydrate
coated with MIPs was recently synthesized for detec- biosensors, the interest in development of cell-based
tion of sialic acid, the cell-surface receptor of influ- carbohydrate is precisely due to the intrinsic sophis-
enza virus.36 That design built upon template im- ticated, cooperative properties of whole cells. Indeed,
printing of sialic acid moieties via boronic acid- living cells are routinely engaged in converting
derivatized polymer for construction of a QCM sensor. complex substrates into smaller molecular units
The significance of this type of study lies in the through distinct metabolic pathways. Cells are also
demonstration of a MIP as viable technology for use capable to continuously repair their enzymatic cas-
in fundamental biosensor design. In fact, the repre- cades, including those involved in carbohydrate
sentative QCM-MIP sensor points to a primary digestion.39-41 Whole-cell biosensors could have ad-
criterion in the design of MIP-based biosensors, vantages over simplified carbohydrate-detection meth-
which is the choice of the detection scheme. Specif- ods such as enzyme-based sensors because cell assays
ically, one of the important issues underlying MIP generally monitor sum parameters such as toxicity
sensors is how would the sensors respond to the or oxygen uptake, rather than individual molecular
specific binding of analytes in general, carbohydrate analytes in solution.
analytes in particular, and how would the signal Held et al. constructed a microbial biosensor array
produced within the biosensor be recorded. Detection consisting of immobilized Escherichia coli bacterial
of binding interactions to the polymer template mutants lacking specific metabolic systems for indi-
through embedded fluorescence tags could be the vidual carbohydrates.40 The sensor components in-
technology of choice, albeit this approach could pose cluded an electrode for monitoring electrochemical
significant technical and synthetic challenges. Cou- potential arising from the reduction of molecular
pling between imprinted polymer technology and oxygen. The oxygen for its part was produced by E.
fluorescence-based detection of carbohydrates was coli mutants immobilized within a solid matrix and
reported in a representative study.37 In that work, was indicative of the metabolic activity of the bacte-
the researchers synthesized a fluorescent monomer ria. In particular, the bacterial mutants used were
that facilitated detection of cis-diols, which was then deficient in translational pathways for specific car-
successfully assembled into an imprinted polymer, bohydrates; thus, addition of those carbohydrates
preserving its fluorescence-sensing capabilities for resulted in increased metabolic activities and higher
5992 Chemical Reviews, 2004, Vol. 104, No. 12 Jelinek and Kolusheva
markable pH sensitivity of the interaction between hand, the technique could indeed serve as an excel-
heparin and plasma histidine-proline-rich glycopro- lent tool for initial fast analysis of cell-wall carbohy-
tein (HPRG). The extraordinary abundance of histi- drates. Combining MALDI-TOF MS with other
dine residues in the protein sequence makes it highly separation and detection methods and the construc-
sensitive to the solution pH through protonation of tion and use of relevant databases could make
the histidines. Because the heparin-binding site enzymatic fingerprinting a powerful tool for analysis
spans some of the histidines, the sensitivity of the and sequencing of complex carbohydrates.
protein to heparin association could be fine-tuned Enzyme digestion was also used in a high-through-
through controlling the pH. The researchers further put assay by which Arabidopsis thaliana stems were
demonstrated that heparin binding to HPRG was hydrolyzed with driselase or trifluoroacetic acid
highly dependent on metal ions; little binding of (TFA).70 Specifically, driselase, a mixture of fungal
HPRG to heparin was detected at physiological pH enzymes, hydrolyzes cellulose (to glucose) and all of
in the absence of metals, but the interaction was the major matrix carbohydrates, while TFA hydro-
promoted by nanomolar concentrations of zinc and lyzes the matrix carbohydrates but not cellulose to
copper.64 Indeed, the frequently encountered high monosaccharides. The application of the two sub-
affinities between particular protein classes (such as stances together yielded a carbohydrate profile of the
lectins, see below) and their carbohydrate ligands cell wall, facilitating, for example, identification of
(heparin or others) has been thoroughly exploited for mutants with differing compositions of cellulose,
carbohydrate analysis. xyloglucan, or xylan.70
2.1.3 Carbohydrate Structures Enzymatic digestion and electrochemical detection
of the enzymatic cleavage products have been widely
Deciphering the organization and order of the utilized for determination of oligosaccharide struc-
monosaccharide units within oligosaccharides poses tures.71-73 The chemical profiles of carbohydrate
as one of the most formidable analytical challenges moieties expressed on several glycopeptides were de-
in glycobiology. Varied approaches and generic tech- termined by enzymatic desialylation and deglycosy-
niques were applied to facilitate accurate analysis of lation combined with analytical separation.73 Another
the individual monomers in complex carbohydrates.65 representative report described identification and
Gel electrophoresis methodologies were modified for analysis of carbohydrates by using an enzyme array/
extraction, separation, and analysis of bacterial cell- amperometric-detection scheme.72 The technique could
surface (capsular) polysaccharides.66 Enzymatic pro- decipher structures of complex carbohydrates by
cessing of carbohydrates and glycoconjugates has direct quantification of monosaccharides released by
been frequently used for determination of carbohy- enzymatic reactions (carried out within the enzyme
drate structures and sequences because of the overall array) through pulsed amperometric detection at a
accuracy of the technique and the requirement of gold electrode, rather than determination of the
small sample quantities.67 Recent studies have con- uncleaved carbohydrate moieties. The enzyme array
centrated on the integration of advanced separation electrochemical detection method does not require
and detection methods for achieving fast and ac- any separation or prior labeling of oligosaccharides.72
curate oligosaccharide sequencing. Simultaneous de- However, this method faces several limitations. First,
tection by UV absorbance and electrospray ionization- the ultimate resolution power of the sensor is deter-
mass spectrometry (ESI-MS), for example, provide mined by the size and diversity of the enzyme array,
important structural information on the oligosaccha- and one could anticipate a situation when similar
ride components of mixtures.68 oligosaccharides would produce nondistinguishable
Detailed structural analysis of bacterial capsular cleavage products. Moreover, correct interpretation
carbohydrates has been achieved by enzymatic of the sensor output depends on the assumption that
fingerprinting procedures combining high-perfor- the tested carbohydrates are pure, rather than
mance anion-exchange/pulsed-amperometric detec- complex mixtures. The use of an array setup, how-
tion liquid chromatography, fluorophore-assisted car- ever, is promising in that it opens the way to high-
bohydrate electrophoresis, and matrix-assisted laser- throughput screening applications and the inclusion
desorption ionization time-of-flight (MALDI-TOF) of database analysis as an integral part of the
mass spectrometry (MS).69 This carbohydrate profil- biosensor usage. Array-inspired bioanalytical meth-
ing technique made possible rapid identification of ods in which enzymatic digestion was coupled to
plant-cell-wall mutants and was proposed as a viable fluorescence detection of specific attached markers
alternative for more cumbersome genetic or biochemi- were applied for carbohydrate structural analysis.20-22
cal phenotyping methods.69 Specifically, Lerouxel et Other bioanalytical techniques were developed to
al. explored the advantages and disadvantages of elucidate carbohydrate sequences. Nuclear magnetic
application of the bioanalytical techniques for the resonance (NMR) spectroscopy has been highly useful
capsular oligosaccharide analysis, particularly in for determination of carbohydrate and glycoconjugate
terms of speed, reliability, and accuracy. The re- sequences, conformations, and dynamics.74,75 CD
searchers asserted that MALDI-TOF MS offers an spectroscopy is another important bioanalytical tech-
efficient and rapid method for carbohydrate analy- nique that was applied for analysis of oligosaccharide
sis.69 This claim could be somewhat problematic secondary structures and conformational dynamics.76
because of the fact that prior knowledge of specific Similarly, MS was also applied for obtaining struc-
carbohydrate components is necessary for the cor- tural information on oligosaccharides.77 The use of
rect interpretation of MALDI-TOF MS. On the other permethylation combined with gas chromatography-
Carbohydrate Biosensors Chemical Reviews, 2004, Vol. 104, No. 12 5995
mass spectrometry (GC-MS) for linkage and se- eties and saccharides can thus be complemented by
quence analysis of oligosaccharides was reviewed.78 analytical methods such as MS and enzymatic diges-
SPR was also successfully applied to glycoconjugate tion for complete structural analysis. In reality, the
analysis (see detailed discussion in section 3.4 below). limitations of neoglycolipids for generic biosensor
A generic and elegant methodology for carbohy- applications can be traced to their origin in synthetic
drate biosensor design has been the construction of organic chemistry. For example, one has to verify the
neoglycolipids. These new molecular composites, sufficient yields of the lipid-coupling reactions, as
based on the coupling of oligosaccharides to lipid well as the efficient immobilization of the neogly-
residues, constitute a chemical-synthesis route for colipid products onto the solid matrixes, prior to
deciphering carbohydrate sequences and structures. putative application as carbohydrate-detection de-
The attachment of hydrophobic lipid moieties to vices. In addition, the technique generally requires
carbohydrates opens the way for applications of several preparative and analysis steps that limit its
versatile immobilization methods.79,80 There are sev- applicability in faster biosensing uses.
eral important advantages of the neoglycolipid ap- While neoglycolipids are created synthetically,
proach for biosensor purposes. First, neoglycolipids studying carbohydrate structures and properties
contain preselected single lipid residues rather than within naturally occurring glycoconjugate entities,
the heterogeneity of acyl chains encountered in such as glycoproteins or glycolipids, is often critical
natural glycolipids, which often adds to the complex- for understanding the biological functions of such
ity of analysis of saccharide derivatives from natural assemblies. Evaluation of carbohydrate organization
sources. Another inherent strength of neoglycolipid- and structures within aggregates of collagen, an
based assays is the selective reactivity of different abundant fibrous protein localized in various tissues,
carbohydrates in heterogeneous mixtures following has been carried out by photometric measurements
their chemical derivatization, facilitating their sepa- of textural birefringence.82 That research has shown
ration through varied analytical means. In addition, that the extent of optical retardations because of
surface display of carbohydrates immobilized through birefringence was indicative of the ordering conferred
their lipid chains is well-suited to probing directly to collagen fibers by the attached carbohydrate
the biological roles of oligosaccharide sequences as moieties. The birefringence measurements exposed
antigens, ligands, or other recognition elements, thus the important role played by collagen-bound carbo-
providing valuable information on the glycome, the hydrate molecules in the ordered aggregation of
entire spectrum of glycans produced by the cell. collagen fibers and subsequent attachment of other
Furthermore, neoglycolipids are particularly adapt- structured macromolecules to the fibers.
able for modern microarray applications for high-
throughput evaluation of the specificities of oligosac- 2.2 Lectin-Based Biosensors
charide-recognizing proteins (see below). Lectins constitute a broad family of proteins in-
In an extension of the original neoglycolipid con- volved in diverse biological processes, occasionally
cept, chemical derivatization techniques utilizing having potent toxic properties.83-85 Lectins generally
fluorescent glycoconjugates were developed to deci- exhibit strong binding to specific carbohydrate moi-
pher carbohydrate components in complex mixtures, eties (glycans), and this property has been exten-
particularly focusing on ligand discovery within sively exploited as a basis for biosensor design.
varied mixtures of neutral and acidic oligosaccha- Furthermore, particular structural profiles of glycans
rides.81 The important advantage of this approach is and their recognition by lectins have been attributed
that it adds to the detection capabilities for employing to disease progression, making analysis of saccha-
neoglycolipids, which by themselves do not contain ride-lectin binding processes important as a diag-
chromophores other than the saccharides. Further nostic tool.86 Glucose biosensor designs, for example,
strength of the technique is the analysis of carbohy- have frequently utilized the specificity and high
drates through fluorescence emitted directly from the affinity of different lectins to this monosaccharide.
saccharide-coupled fluorophore (rather than indirect Varied detection methods based on lectin-glucose
detection of fluorescent substances that bind to the recognition have been reported in the literature,
neoglycolipid). A recent report described conjugation including electrochemical detection of the monosac-
of an aminolipid 1,2-dihexadecyl-sn-glycero-3-phos- charides via immobilization of lectins on electrode
phoethanolamine (DHPE) and the fluorescent label surfaces,87 and glucose-sensing based on the competi-
anthracene.81 This reagent is highly fluorescent and tive reversible binding of a mobile fluorophore-labeled
can form neoglycolipids by reaction with diverse lectin concanavalin-A (con A) to immobile pendant
oligosaccharides through reductive amination. Such glucose moieties within Sephadex beads.88 Lectins
conjugates can be resolved by thin-layer chromatog- also exhibit high potential in peripheral biotechnol-
raphy (TLC) and high-performance liquid chroma- ogy industries, such as food safety; their unique
tography (HPLC) and quantified either spectroscopi- recognition properties are finding promising applica-
cally or through scanning densitometry. tions in detecting microorganisms and carbohydrate
Overall, neoglycolipid technology offers a compre- additives in foods. Reported data suggest that the use
hensive carbohydrate characterization approach, of certain lectins may provide a simple and rapid
whereby an oligosaccharide ligand population is alternative to traditional methods of bacterial analy-
detected and isolated through selective chemical sis and screening.89
derivatization. The construction of new and discrete The high affinity of lectins to saccharide units has
chemical entities containing hydrophobic lipid moi- been attributed to multivalency and spatial organiza-
5996 Chemical Reviews, 2004, Vol. 104, No. 12 Jelinek and Kolusheva
body immobilized on the sensor surface. Other ap- with glycopeptides and glycolipids and contribution
proaches utilized more conventional constituents for of the carbohydrate moieties to gp120 interactions
saccharide recognition and binding such as lectins were evaluated with bioanalytical techniques such
(section 2.2) for assaying glycoprotein composition as enzyme-linked immunosorbent assay (ELISA).135
and glycosylation. Several reviews summarize lectin Assays measuring the effect of glycosylation on the
overlay assays in which the sugar moieties were immunoreactivity of glycoprotein hormones were also
initially detached from the protein residues by en- evaluated.136 Different techniques have been devel-
zyme digestion procedures.124,125 The effectiveness oped to determine hemoglobin glycosylation, believed
and clinical potential of lectin-based assays for study- to provide an accurate index of long-term blood
ing subtle changes in serum protein glycosylation, glucose control in diabetes mellitus, including ion-
particularly associated with disease onset, have been exchange chromatography, electrophoresis, isoelectric
reviewed.126 focusing, thiobarbituric acid colorimetry, and affinity
An intriguing technique for creating potential chromatography.137,138
glycoprotein sensors based on Langmuir-Blodgett Protein glycosylation by chemically modified oli-
films of fullerene-glycodendron conjugates was de- gosaccharides (oligosaccharide tags) could become
scribed by Cadullo et al.127 The authors constructed a useful tool for investigating protein and peptide
monolayers at the air-water interface that were targeting in cellular processes. Analysis of glycosy-
comprised of fullerene-dendrimers covalently at- lation patterns of glycopeptide enzyme substrates
tached to glycodendron headgroups. The noteworthy was carried out by glucosylation of a set of the glycan
achievement of the researchers was the prevention substrates in vitro, followed by determination of
of fullerene aggregation within the monolayers, ac- glucose composition by MS.139 Synthesis of maleim-
complished by optimization of the hydrophilic/hydro- ide-activated carbohydrates as site-specific tags for
phobic structure of the fullerene-dendrimer conju- peptides and proteins was also reported.140 This work
gates. The absence of aggregation and consequent built upon the high reactivity of maleimide with thiol
display of the carbohydrate units at the film surface groups, making possible attachment of maleimide-
could be potentially applied to glycoprotein detection. activated mono- and polysaccharides to cysteine-
Interactions between viral envelope glycoproteins containing peptides. Even though technically this
and host cells play fundamental roles in viral pen- method essentially creates artificial glycopeptides,
etration into cells and viral pathogenesis.128,129 Ac- tagging peptides with different saccharide moieties
cordingly, studying the molecular recognition and could be useful for detection of carbohydrate-recogni-
interactions between cellular receptors and viral tion sites and carbohydrate receptors on cell surfaces.
envelope glycoproteins showing receptor-binding ac- Diverse glycosylation processes occur on cell sur-
tivity are of great importance both for understanding faces, and elucidating cellular carbohydrate expres-
the molecular basis of virus entry, as well as for sion and glycosylation pathways is essential for
developing antiviral drugs and diagnostic tools. Ber- understanding varied cellular events.92,141 Elegant
tucci et al. have used an optical biosensor to study biochemical techniques were developed for probing
the binding of recombinant glycoproteins of herpes oligosaccharide compositions and carbohydrate pro-
simplex virus (HSV) to an immobilized recombinant cesses at cell surfaces. Bertozzi and others have
form of the human cellular receptor for HSV.130 The expanded upon the concept of chemical glycobiology
mode of action of the biosensor was based on detec- as a generic approach for deciphering biochemical
tion of changes in the refractive index close to the processes in which carbohydrates constitute central
sensor surface, which was dependent upon the mass components and for studying structure-function
of the adsorbed species. The resonant mirror technol- relationships involving surface-expressed oligosac-
ogy utilized in the research represents a class of charides.92,142 The approach, which was also denoted
biosensor technologies that essentially detect binding metabolic oligosaccharide engineering involves
events and biomolecular interactions in real time. chemical modification of specific saccharide units.
The strengths of the resonant mirror biosensor are These unnatural carbohydrates could then be incor-
mainly traced to the increased sensitivity (nano- to porated into various cell compartments and locations
microgram range for glycoproteins), the short time via the biosynthetic machinery of the cell.142 In
required to perform the experiment (less than an particular, it was shown that interference with
hour), and the fact that there is no need for additional biochemical and metabolic pathways contributing to
labeling of the analytes.131 The biosensor could be oligosaccharide biosynthesis could shed light on the
regenerated after measurements through washing of progression and significance of such processes.92,143
the bound species, although some decrease of the Chemical intervention in biochemical processes
reproducibility of the results was observed after occurring at cellular levels has other important
repeated use.130 features. The method allows, for example, insertion
Envelope glycoproteins of the HIV, in particular of varied reactive functional groups and labels onto
gp41 and gp120, have been implicated in viral entry the cell; some studies demonstrated incorporation of
to various cell types.132,133 Glycosylation of these two glycoconjugates containing sensor probes into the cell
proteins is believed to play an important role in their wall, facilitating analysis of distinct reactions and
antigenicity and cell-surface interactions, and specific transformations involving the carbohydrate mol-
assays were developed to decipher the structure and ecules.143 Charter et al. showed that unnatural sialic
molecular interactions of the carbohydrates attached acid analogue containing levulinoyl moieties can be
to these glycoproteins.134 The association of gp120 incorporated into neuronal cell surfaces. The ketone
Carbohydrate Biosensors Chemical Reviews, 2004, Vol. 104, No. 12 5999
group within levulinoyl could then be used for cell detection are based on the use of antibodies specific
imaging using biotin, facilitating insight into meta- to enzymes or other proteins expressed by the mi-
bolic pathways involving adhesion molecules (con- croorganism to be examined.149 Such methods, how-
taining sialic acid) on the cell surface.143 ever, often require prior knowledge of the identity of
Biosynthetic construction of unnatural saccharide the pathogenic species to be analyzed. The search for
assemblies in surfaces of living cells could aid explo- rapid, low-cost diagnostic pathogen techniques has
ration of complex processes involving carbohydrates also focused on the use of oligosaccharides, which
and contribute to the search for inhibitors, agonists, constitute primary molecular components and mark-
and antagonists to various carbohydrate and glyco- ers on pathogen surfaces. The diversity and broad
conjugate receptors. Predetermined and controlled knowledge base regarding surface-displayed carbo-
modification of cell-surface glycans might lead to hydrates could aid the design of diagnostic tests for
promising diagnostic applications, particularly be- specific bacteria. Rapid agglutination assays have
cause varied diseases are associated with altered cell been routinely used for detection of microorganisms
glycosylation patterns (see section 2.4.3 below). A through binding of their surface carbohydrates to
possible metabolic carbohydrate engineering ap- varied external substances, such as antibodies and
proach can be conceived for discrimination of tumor receptors. The latex agglutination test (LAT), for
cells through their altered surface glycan expres- example, utilizes latex beads coated with polyclonal
sion.142,144 Additionally important in term of carbo- antibodies against the capsular carbohydrate of
hydrate biosensor development, the ability to chemi- particular bacteria. Aggregation of the beads can be
cally modify glycoproteins on cell surfaces could open observed via the solution turbidity, indicating the
the way for molecular or whole-cell imaging and high- presence of bacteria. The technique facilitated, for
throughput screening in proteomics, glycomics, and example, identification of mycoplasma in an early
cellomics applications. development stage within farm animals.150
The compositions and structural features of car- Optimization and enhancement of conventional
bohydrates expressed on cell surfaces have been agglutination tests were reported. Application of
employed as a tool for cell visualization and physi- ultrasonic standing waves in conjunction with im-
ological research. Cytochemical methods have been munoagglutination has significantly enhanced the
applied to probe the localization and distribution of speed and sensitivity of the assay.151,152 In that
glycoproteins expressed on cell surfaces by utilizing diagnostic technique, the researchers suspended
the targeting of specific carbohydrate moieties by antibody-coated microparticles in an acoustic field,
lectins or antibodies.91,145 Researchers utilized both physically promoting interactions between the anti-
lectins that bind specifically to terminal disaccharides bodies and sugar antigens and accelerating formation
as well as monoclonal antibodies against carbohy- of aggregates. Using the ultrasound-enhanced ag-
drate epitopes.91 Comparative staining based on these glutination procedure, more than a 50-fold increase
molecular systems differentiated and partially char- in sensitivity was observed for bacterial carbohy-
acterized several glycoconjugates in various sites and drates, approaching the detection levels obtained by
allowed evaluation of the relationship between chemi- the polymerase chain reaction (PCR).
cal heterogeneity and neural speciation. Other immuno-based techniques, such as the widely
Advanced high-sensitivity MS approaches have used ELISA, were applied for pathogen detection by
been increasingly used for deciphering glycoprotein employing cell-displayed (capsular) carbohydrates.
structures. MS has been capable to elucidate the While some assays were designed to detect the
primary structures of highly complex glycoprotein capsular carbohydrates themselves, most ELISA
mixtures, and the technique could provide an insight applications utilize the carbohydrates within the
into post-translational protein modification processes sensor framework as recognition elements designed
in particular and structural glycobiology in general.146 to bind to carbohydrate-specific antibodies.153 Pub-
Recent technical advances in MS, specifically fast lished ELISA methods employing saccharide-anti-
atom bombardment (FAB), ESI, and MALDI consid- body binding have mostly used carbohydrate immo-
erably increased the analytical capabilities of the bilization onto the solid support, while variations
technology to analyze complex carbohydrates and exist regarding the immobilization procedures. Among
glycoconjugates. For an in-depth discussion of the the methods summarized were biotinylation of the
subject, the reader is referred to a recent compre- carbohydrates,153 conjugating to poly-L-lysine polypep-
hensive review.146 tide for coating the microtiter plates,154 and others.
Varied techniques have been developed to facilitate
2.4 Pathogen and Cancer-Detection Assays rapid detection of pathogen-displayed carbohydrates
2.4.1 Pathogen Identification that could also be applied in field conditions at high
sensitivity. A fluorescence polarization assay (FPA)
Development of biosensors and rapid detection kits was successfully applied for serological diagnosis of
for microorganisms such as E. coli, Salmonella ty- brucellosis in cattle and other farm animals through
phimurium, and others are highly desirable because antibody binding of the capsular carbohydrate epitopes
of the adverse and often devastating health effects of several Brucella strains.155 The FPA technology is
of pathogen infection.147 In recent years, diverse based on the rotational differences between a solu-
techniques have been introduced aiming to detect bilized fluorescent-labeled free antigen and the an-
pathogens in shorter times and with maximal poten- tigen molecule bound to its antibody. In principle, a
tial sensitivity.148 Varied techniques for pathogen small molecule will rotate randomly at a rapid rate,
6000 Chemical Reviews, 2004, Vol. 104, No. 12 Jelinek and Kolusheva
carbohydrate analytes.127 Binding was achieved The AFM methodology can further identify indi-
through deposition of ordered Langmuir monolayers vidual carbohydrate molecules in solution, contribut-
of the bucky-ball conjugated with glycodendron head- ing to its bioanalytical applicability. For example, the
groups at the air-water interface. The films could capability of AFM to distinguish among chair-twist-
be further transferred to solid quartz surfaces, point- boat conformational transitions of the pyranose ring
ing to their potential applications in biosensor design. within different R-(1,4)-linked carbohydrates could
Atomic force microscopy (AFM) has been a major serve as a nanomechanical fingerprinting of differ-
driving force in nanotechnology research and devel- ent oligosaccharides.220 AFM was also used to evalu-
opment. AFM is conceptually similar to the way old ate minute differences in the forces between carbo-
long-play records were read by the stylus of a hydrate moieties on bacterial cell walls and biopolymer
phonograph, where the AFM tip acts like a stylus surfaces, pointing to its use as a tool for bacterial
capable to image molecules and atoms on solid detection.221
surfaces.214 Among the most widespread applications
of AFM in carbohydrate research has been imaging 2.6 Miscellaneous Carbohydrate Bioassays
of single carbohydrate molecules and surface char- A large number of bioanalytical techniques are
acterization of oligosaccharide assemblies.213,214 An used for routine carbohydrate analysis. Detection
example of the practical application of AFM was its methods of carbohydrates in food products, particu-
use for evaluation of the structure and texture of food larly fruit, have been reviewed.222 The majority of
carbohydrates.215 saccharide analysis schemes in food processing com-
AFM has been explored as a tool for varied bio- bine compound separation, mostly chromatography,
sensor-related applications, such as determination of and detection. GC has been popular for carbohydrate
carbohydrate heterogeneity on bacterial surfaces216 analysis because it has the advantage of speed,
or the observation of a nonhomogeneous distribution although this technique generally requires carbohy-
of specific oligosaccharide units on the surface of drate prederivatization.223 TLC is relatively inexpen-
yeast cells through derivatization of the AFM tip with sive; however, it lacks in resolution and quantifica-
lectins.217 Such studies illustrate both the capabilities tion information.224,225 Other techniques have been
as well as the significant hurdles for application of used, primarily HPLC using polar and nonpolar
single-molecule imaging and force measurements in columns,226 anion-exchange columns,222 or cation-
biosensors. On one hand, the atomic-level resolution exchange columns.227,228
of AFM could provide unique carbohydrate imaging Theoretical analyses have been employed in con-
fingerprinting for bacterial and other cellular sur- junction with carbohydrate biosensor studies. Fractal
faces. One can conceive, in principle, the construction analysis was used to characterize the binding kinetics
of an AFM image database for bacterial surfaces that between cell-surface receptors (such as bacterial-
might be used for rapid pathogen identification. displayed oligosaccharides) and external soluble ana-
Further contributions could be envisaged from inte- lytes.229 Such theoretical treatments could be of use
gration of computer-aided image analysis into AFM- in interpreting oligosaccharide-binding data and for
biosensor applications. However, the particular optimization of sensor performance.
strength of AFM as a single-molecule-imaging tech-
nique rather than characterizing large-population 3. Carbohydrate Components in Biosensors
ensembles could raise formidable difficulties in using
this method for sufficiently fast and reliable detec- 3.1 Carbohydrate Recognition Elements
tion. For example, the carbohydrate heterogeneity Carbohydrates often constitute fundamental parts
exposed by Camesano and Abu-Lail216 could make within biosensor devices, either comprising the rec-
any interpretation of AFM images of unknown patho- ognition elements or as scaffold components of the
gens inconclusive and highly complex. Furthermore, sensor matrixes. Such applications take advantage
the very high sensitivity of AFM to environmental of two important (and unrelated) properties of car-
factors, such as temperature, salt types, and concen- bohydrates. The first is the participation of numerous
trations, etc., might lead to impracticality as a bio- oligosaccharides in molecular recognition phenom-
sensing method. ena, which could make them ideal for targeting
The capability of AFM to resolve chemical and specific analytes. Another oft-encountered charac-
physical events involving single molecules has led to teristic of molecular framework arrays constructed
exploration of other potential biosensor applications. from saccharide assemblies is their stability and
AFM was used for characterizing structural proper- rigidity, making them attractive components in bio-
ties of a single xanthan molecule on a solid surface.218 sensor design.
AFM was also employed for detection of bacterially Films composed of synthetic saccharide derivatives
secreted carbohydrates in river sediments.219 A novel for potential biosensor applications have been con-
saccharide force fingerprinting technique, based on structed.230 That study presented a detailed physi-
the single-molecule-imaging capabilities of AFM was cochemical characterization of the organization and
reported.124 The method has built upon the variability cooperative properties of lipo/glycohomopolymer and
of force-induced conformational transitions of the random lipo/glycocopolymer monolayers assembled at
pyranose ring, which are also dependent upon the the air-water interface. The researchers have fur-
glycosidic linkages in the molecules. These transi- ther proposed utilization of the molecular recognition
tions yield characteristic force-spectrum fingerprints properties of such films in carbohydrate-based bio-
for specific carbohydrates.124 sensor designs.230 Similar surface-deposited films of
Carbohydrate Biosensors Chemical Reviews, 2004, Vol. 104, No. 12 6005
tran coatings were applied to flat silicon wafer hurdle to such biosensor applications has been an
surfaces to be used in potential sensor devices.256 insufficient adsorption of the cells to the saccharide
Other representative biosensor applications of dex- template.269
tran include its use as a substrate for -cyclodextrin Novel uses for carbohydrates as templating agents
immobilization in immunosensors,254 as a material were reported in the framework of molecular-im-
used for functionalization of novel carbon nanotubes printing technology.270,271 Shi et al. described a
in electronic sensors,257 and for enzyme immobiliza- template-imprinted matrix for protein recognition in
tion.258 Several schemes utilized fluorescently labeled which the protein-binding sites were molded by a
dextran. Dextran labeled with fluoroscein isothiocy- disaccharide framework.270 The carbohydrate mol-
anate (FITC) was used as a framework for a glucose ecules were particularly important in that setup,
biosensor using the FRET technique.259 Dextran was providing added synthetic flexibility and analyte
co-entrapped with a hydrolytic enzyme in sol-gel specificity. This experimental achievement is note-
films developed for pH sensing.260 Fluorescently worthy because it points to a generic synthetic
labeled dextran was deployed in conjunction with the pathway for constructing molecularly imprinted pro-
lectin con A in a hydrogel arrangement for glucose tein biosensors, a highly challenging goal in recent
sensing,102 employed as a surface-functionalizing years.
agent facilitating antibody immobilization in chemolu- Several studies have addressed theoretical aspects
minescent immunosensors,261,262 and as the constitu- pertaining to carbohydrate-containing biosensors.
ent of a coating layer in surface acoustic waveguide Griesser et al. investigated the interfacial forces
(SAW) biosensors.263 between carbohydrate surfaces and adsorbed pro-
Cyclodextrins, macrocyclic carbohydrates with non- teins.237 When theoretical predictions and experi-
polar internal cavities that participate in numerous mental approaches such as X-ray photoelectron spec-
chemical systems and applications, have been also troscopy (XPS), MS, and AFM are combined, the
widely used in biosensor design.264 These inclusion researchers have established key parameters respon-
compounds have generally appeared in sensor schemes sible for the resistance of particular polymer coatings
as framework elements facilitating immobilization of to the adsorption of proteins, an important feature
other molecular species that are essential for the of varied biosensor arrangements.
functionality of the sensor. A representative cyclo-
dextrin-based biosensor was described by David et 3.3 Biosensors Utilizing ProteinCarbohydrate
al., constructing an immunosensor by grafting amino- Interactions
-cyclodextrin onto functionalized gold surfaces.254 Molecular recognition and interactions between
The incorporation of additional dextran-derivatized carbohydrates and proteins play key roles in many
adamantyl groups (adamantane derivatives being the biochemical processes. The participation of specific
common ligand of cyclodextrins) enabled the coupling oligosaccharide sequences in protein targeting and
of antibodies as the biological recognition elements folding and in propagating infection and inflamma-
within the biosensor. Other cyclodextrin-templated tion processes through interactions with receptors
biosensors were reported, including cross-linked cy- and antibodies have become increasingly apparent.1
clodextrin films within dopamine biosensors265 and Studying such interactions is also desirable for
-cyclodextrin derivates impregnated in graphite development of therapeutic substances that would
paste for enzyme immobilization in amperometric mimic or interfere with the recognition process.
enantioselective drug biosensors.266 Various approaches have been introduced to probe
Other carbohydrates have been used as substrates carbohydrate-protein binding and to utilize such
in gel constructs for detection of reactant species in recognition events in the action mechanism of bio-
the mobile phase. Carboxymethyl (CM)-curdlan, a sensors. However, elucidation and understanding of
carbohydrate linked with a chromatic dye, was as- the bioactive domains within oligosaccharides and
sembled within polyacrylamide gels for facilitating their protein-binding properties pose distinct bioana-
rapid colorimetric detection of glucanases.267 Beside lytical and chemical challenges.
applications in which carbohydrates have been di- From the standpoint of biosensor design, protein-
rectly involved in biochemical reactions, saccharides carbohydrate binding has been employed as a plat-
have been incorporated in sensor assemblies as form for extraction and analysis of varied proteins.
chemically inert species, albeit essential to the func- In most of these applications, the biosensor operation
tionality of the systems. Brinkman et al., for example, relies on immobilization of carbohydrate species,
reported on the construction of hydrogels comprised which generally function as the recognition elements,
of poly(vinyl alcohol) and heparin.268 On one hand, followed by generation of measurable signals induced
the cross-linked assembly was shown to resist non- by association with their complementary macromol-
specific protein permeation, an important require- ecules. Construction of carbohydrate-modified recog-
ment for biosensor design but, on the other hand, nition surfaces is synthetically demanding because
facilitated slow release of the incorporated heparin, of the structural complexity of oligosaccharides.
thus pointing to potential biosensor applications. Distinct problems have been encountered because of
Saccharide derivatives were also examined for their the multiplicity of hydroxyl groups that might make
ability to form solid gels for cell-based biosensors.269 specific binding difficult, as well as the requirement
OConnor et al. examined the entrapment of neuronal of appropriate linker systems to facilitate display and
cells in a three-dimensional matrix constructed from access to the immobilized oligosaccharides.250 The two
a novel sugar poly(acrylate) hydrogel.269 A significant most common carbohydrate immobilization tech-
6008 Chemical Reviews, 2004, Vol. 104, No. 12 Jelinek and Kolusheva
niques employed in such sensors exploit the high facilitate rapid evaluation of protein-saccharide
affinity of the biotin-avidin pair272 or the deposition interactions. Carbohydrate chips for evaluation of
of alkane thiolate monolayers on gold surfaces.273 lectin binding and glycoenzyme substrate specificities
Biosensor technologies based on surface immobi- were prepared by saccharide immobilization onto
lization of oligosaccharides have to address critical SAMs of cyclopentadiene conjugates via the Diels-
technical and fundamental issues. A primary require- Alder reaction275 or through coupling of the carbo-
ment concerns the feasibility of attaching the gener- hydrates to thiol moieties.276 The functionalized
ally hydrophilic carbohydrate molecules to solid monolayers in those studies contained chemical enti-
transducer surfaces. In that regard, most surface- ties such as benzoquinone275 or maleimide276 for
layering strategies use hydrophobic chemical interac- covalently bonding the carbohydrate derivatives but
tions. Consequently, many sugar immobilization also displayed ethylene glycol for minimization of
methods require chemical modification of the sac- nonspecific protein attachment to the surface. Such
charide molecular units. Such chemical treatments, surface engineering strategies might find uses in
however, should not interfere or adversely affect the sensor applications for analysis of complex carbohy-
biological properties of the examined carbohydrates, drate structures. However, the ultimate utility of
in particular, molecular recognition by soluble mac- such biochip designs would most likely depend on
romolecules. Furthermore, any proposed biosensor the detection method to be used, its sensitivity,
design has to exhibit high sensitivity and sufficient reproducibility, and technical limitations. For ex-
versatility for allowing detection of a wide range of ample, fluorescence microscopy could provide high
proteins and other biomolecular analytes. sensitivity and spatial resolution; however, this
A recent development with potentially significant technique might incur problems of bleaching, back-
implications for glycobiology research in general and ground signals, and surface regeneration.
studying carbohydrate-protein interactions in par- Progress in carbohydrate array research has been
ticular has been the fabrication of carbohydrate also achieved through the creation of microarrays of
arrays as a tool for rapid analysis of sugar-binding neoglycolipids and their display on solid surfaces.277
events and carbohydrate interactions. Examples for Neoglycolipids, comprised of oligosaccharides chemi-
such applications include array carbohydrates that cally conjugated to lipids, can be readily immobilized
are first immobilized on pretreated surfaces, followed on solid matrixes through their hydrophobic lipid
by addition of fluorescently labeled carbohydrate- residues, thus facilitating the surface display of the
binding proteins; binding occurrence can then be carbohydrate molecules for rapid screening of binding
monitored by fluorescence spectroscopy.274 The chal- interactions.81 Immobilized neoglycolipid assemblies
lenges for wide applications of such methodologies, could achieve higher avidity of protein analytes
however, are mostly synthetic, i.e., the construction because of lipid clustering and surface oligomeric
of diverse enough, analyte-accessible immobilized organizations of the oligosaccharides.81 The microar-
carbohydrate arrays. rays constructed by Fukui et al.277 contained neogly-
An elegant and important demonstration of non- colipids prepared from diverse physiological and
covalent immobilization of a carbohydrate antigen synthetic sources (including extracts from whole
array on glass surfaces was recently reported.26 The organs). That exploratory and potentially ground-
researchers assembled dextran polymers produced by breaking study demonstrated that carbohydrate-
Lactobacillaceae bacteria on nitrocellulose-coated recognizing proteins bound their ligands not only
glass slides and examined binding of anti-dextran within arrays of homogeneous oligosaccharides but
antibodies to the slides using fluorescence scanning. also within mixture of heterogeneous carbohydrate
Immobilization and specific antibody-antigen bind- species. The technology could have much more gen-
ing were detected in this configuration. Glass-im- eral diagnostic appeal, as a tool for profiling carbo-
mobilized carbohydrate microarrays could have sig- hydrate-binding proteins from different sources, for
nificant diagnostic and clinical applications, including discovery of new carbohydrate-binding proteins within
rapid detection of specific antibodies in physiological cellular targets, and for large-scale analysis of protein-
solutions and antibody profiling of such solutions, binding characteristics of the glycome.
identification of cross-reactive antibodies and anti- A recently reported screening assay for protein-
gens, and quantitative determination of carbohydrate carbohydrate recognition utilized surface immobiliza-
diversity within microorganisms. The platform de- tion of sulfated carbohydrates.278 The technique,
veloped by Wang et al. is particularly robust and denoted sulfated carbohydrates coating ELISA (SPC-
involves relatively straightforward preparative steps, ELISA) employed initial coating of sulfated carbo-
facilitating rapid analysis of complex solutions through hydrates followed by binding with different target
simple and sensitive detection schemes. Further- proteins, consequently detected by a conventional
more, this method intrinsically enables the display ELISA method. Complementing carbohydrate im-
of a large repertoire of cellular carbohydrates and mobilization and immunosorbent detection in SPC-
carbohydrate antigens on a single slide, approaching ELISA has some advantages over other frequently
the capacity to include oligosaccharides encountered applied immunosensing techniques, including its
in most common pathogens. compatibility with automation in general and high-
Varied chemical strategies were introduced for throughput screening methodologies and equipment
fabrication of carbohydrate arrays for high-through- in particular and the versatility of the technique with
put screening applications. As a parallel to the more regard to molecular-target variability and detection
widely used DNA chips, carbohydrate chips could methods.
Carbohydrate Biosensors Chemical Reviews, 2004, Vol. 104, No. 12 6009
A novel technique for the screening of carbohy- cans such as heparin and heparin derivatives has
drate-peptide interactions through phage-display been problematic because of the presence of only a
selection of peptide binding to mirror-image sugars single reducing-end amine group.283 Original methods
has been developed.279 The researchers used phage for surface immobilization of heparin were proposed,
display to identify peptides that bind to surface- including covalent attachment of heparin on an
immobilized synthetic L-type saccharide enantiomers; evanescent wave biosensor cuvette,285 binding as an
the corresponding mirror image peptides that bind albumin conjugate on a functionalized polystyrene
the D-type saccharides could then be identified through surface,286 and on a SPR biochip.283 Evanescent wave
application of SPR. The technique was demonstrated biosensors have been used for studying heparin-
for detection of saccharide binding to high-affinity protein interactions.285 Optical sensing of heparin/
antibodies. albumin thin films was used to measure modifica-
Interactions between proteins and glycolipids are tions of film thickness by the pH of the solution.287
of particular importance in carbohydrate-based bio- SPR analysis was also carried out to systematically
sensor design. The lipid moieties of glycolipids are evaluate interactions between collagens and different
generally buried within the hydrophobic membrane heparin derivatives.288
bilayer, leaving on one hand, the oligosaccharide QCM has been applied for detection of various
components exposed to the solution but, on the other biological saccharide-binding reagents. There has
hand, close enough to the bilayer surface facilitating been, however, some skepticism as to the accuracy
ligand presentation. Furthermore, the structural and applicability of the technique for analysis of
features of immobilized glycolipids might play pivotal molecular recognition, partly related to problems
roles in shaping carbohydrate-protein binding. This arising from immobilization and positioning of large
is mostly due to the observations that multivalent biomolecules on the sensor surface.52 A procedure for
interactions rather than the relatively weak monova- incorporation of R-galactose antigen on a microbal-
lent affinities are prevalent between proteins and ance surface resulting in a rigid and sensitive rec-
carbohydrates.280 Studies of protein-saccharide rec- ognition biofilm was recently described.289 In that
ognition and the effects of the membrane environ- work, SAMs of R galactose were prepared by thiol-
ment on these phenomena are in their infancy.281 The tail derivatization, allowing construction of a highly
presence of the acyl chains could be further advanta- reproducible and selective lectin sensor.
geous for immobilization of the carbohydrate recogni- Carbohydrate-protein binding has an additional
tion elements within varied hydrophobic surfaces in advantage when utilized in biosensor design. This is
potential membrane-mimic biosensor designs. The due to the fact that one of the most important
creation of surface patterns of glycolipid targets and criterion for efficient, reversible surface immobiliza-
biosensor arrays282 would be a natural extension of tion of biomolecules in sensor devices is whether such
the immobilization capabilities. molecules retain their biological functions. This issue
Pathogen detection is an important field in which is particularly important in biosensors based on
glycolipid-carbohydrate interactions could be of par- enzymatic reactions.290 The optimal design should
ticular importance. The interactions between gan- permit high affinity of the enzyme to the surface to
gliosides and CTs have been widely studied and avoid loss; however, the attachment should not be
included in biosensor designs, in many instances too strong as to not allow enzyme elution and
using surface immobilization of GM1 (see section regeneration.291 Chemical or physical adsorption
2.4.1, above). A multiarray evanescent wave biosen- techniques are often inadequate for such require-
sor for detection of CT was described in which ments, and biospecific methods are also problematic.
gangliosides immobilized at discrete locations on the For example, binding based on the avidin-biotin
surface of an optical waveguide.282 Rapid and easy system is too strong (binding constant Kass in the
detection of the fluorescent-labeled CT or tracer order of 1015), and antibody-hapten association,
antibodies was achieved using the same technique.157 while in the correct binding-strength range, is highly
Other examples for the use of the CT-GM1 recogni- dependent upon the immuno system and solution
tion pair in biosensor design are described above (see conditions. Lectin-carbohydrate binding (Kass ) 106-
section 3.3, pathogen detection). The binding between 107), on the other hand, offers a practical route for
globotriaosylceramide (Gb3) and E. coli verotoxins reversible immobilization of enzymes and recognition
could similarly constitute the core of diverse bacterial elements in biosensors.292 Koneke et al. demonstrated
detection schemes.281 the use of con A for reversible immobilization of
Heparin-protein binding constitutes the basis of glucoenzymes within a fluoride ion-sensitive field-
varied peptide and protein bioassays. A range of effect transistor (FET).291 Enzyme-reloading in the
techniques has exploited surface expression and the biosensor assembly was achieved through removal of
selective protein-binding properties of heparin and the lectin-bound glucoenzymes by elution with soluble
its derivatives in biosensor devices and as vehicles mannosides.291
for diverse detection schemes. Heparin biochips Carbohydrate-lectin binding is central to other
were constructed for applications in techniques such biosensor designs. Galanina et al. have synthesized
as SPR to measure the extent of heparin-protein and compared radioactively and fluorescently labeled
interactions.283,284 In such applications, it was ob- carbohydrate conjugates for detection of cell-ex-
served that the biosensor response was often affected pressed lectins.293 The technique was based on the
by the method of heparin immobilization on the solid coupling of the saccharide moiety to a soluble poly-
surface.284 Covalent attachment of glycosaminogly- acrylamide spacer, onto which were attached the
6010 Chemical Reviews, 2004, Vol. 104, No. 12 Jelinek and Kolusheva
carbohydrates, for example, the in situ analysis of achieved thorough attachment of the biotinylated
dextran monolayer degradation by dextranase.312 saccharide molecules to streptavidin-coated sensor
Carbohydrate-lectin interactions have been fre- surfaces. The protein affinities to LPS in such assays
quently employed as a basis for SPR biosensor were evaluated through the changes in mass close
applications. These approaches were aided by the to the sensor surface.
broad knowledge base regarding the pool of saccha- While most oligosaccharide immobilization tech-
ride ligands attracted to various lectins. Examples niques have been based on the avidin-biotin high-
of such SPR applications include steady-state and affinity system, other methods were reported. Cat-
kinetic analyses of lectin binding to oligosaccharides imel et al. described antibody detection by SPR,
and glycopeptides,115,313 analysis of lectin interactions which was carried out through direct immobilization
with C and O glycosides linked to a carboxymethyl of gangliosides onto the sensor surface by hydropho-
dextran layer on the SPR sensor surface,314 carbo- bic interactions.327 The advantage of this type of
hydrate-binding activity and specificity of a lectin approach was the forestalling of chemical derivati-
extracted from bulbs of spring crocus,315 structural zation of the saccharide molecules or of the sensor
basis for the unusual carbohydrate-binding specificity surface, leading to simplification of biosensor con-
of jacalin, the seed lectin from jack fruit (Artocarpus struction. In a different modification of the SPR
integrifolia),316 and others. Lectin-glycolipid binding sensor chip, complete vesicles containing ganglioside
formed the basis of surface immobilization procedures GM1 were surface-immobilized, deriving affinity and
in miniaturized SPR sensors.317 kinetic information upon binding of CT.163
SPR was used for detection of carbohydrates in Other chemical methods were introduced to im-
physiological solutions at high sensitivities.318 A SPR mobilize and display carbohydrates and glycoconju-
sensor for heparin featured a surface that was coated gates on SPR biosensor surfaces. Stein et al. reported
either with protamine or polyethylene imine. Impor- on modifying a carboxymethyldextran surface to
tantly, the degrees of heparin affinities were depend- couple the lipid-anchored contact site A (csA) of a
ent upon the receptor species coating the surface in homophilic adhesion glycoprotein of the bacterium
each case. The sensor performance was also found Dictyostelium discoideum.328 The carboxy groups in
to be affected by incubation time, heparin dilution, the derivatized layer were modified to enable hydro-
and the presence of other components in the analyte phobic binding of the glycoprotein via its lipid anchor
solution. Nonspecific adsorption had to be addition- to the dextran matrix. Alternatively, the researchers
ally overcome by optimization of the experimental employed covalent binding through a perfluorophen-
conditions, overall indicating that the intrinsic high ylazide-derived hydrophobic cross linker. Titration
sensitivity of the SPR technique could also pose experiments verified that the bound csA molecules
problems for carbohydrate-binding analysis. Aside reacted with antibodies that recognize either the
from detection of carbohydrates in varied solution native or the denatured glycoprotein; thus, they most
environments, heparin and its derivatives have been likely adopt a native state in the sensor surface
employed as recognition elements in SPR biosensors. environments.328
SPR was used to evaluate heparin binding to chemo- In addition to detection of carbohydrate-binding
kines, a process believed to be central to chemokine species, SPR has been used for studying various
functionality.319 The relative degree of avidin binding parameters contributing to such interactions. SPR
to heparin and its derivatives was also evaluated has been applied, for example, to assess modulation
using SPR biosensors.320 Heparin and heparan sul- by pH, divalent cations, and polyamines on the high-
fate were used as substrates for studying membrane affinity binding of antibodies to polysialic acid (PSA)
interactions and host entry of HSV.321 Another ap- expressed on the vertebrate neural cell adhesion
plication employed heparin-modified gold surfaces for molecule (NCAM).329 In such experiments, the sen-
analysis of low-density lipoproteins (LDL).322 sitivity of the optical signal generated by the sensor
response facilitated identification of slight changes
SPR has been additionally used for determination in the binding events.
of glycosylation changes in proteins. SPR analysis of
glycoproteins has been generally achieved through
immobilization of the proteins on the sensor surface 4. Concluding Remarks
by using antibodies and identification of carbohydrate The increasing awareness of the biological impor-
epitopes through binding of specific lectins.323 It was tance of oligosaccharide derivatives and growing
also reported that modifications of the affinity be- interest in glycobiology applications have clearly
tween the biosensor-immobilized proteins in the cell- become a major driving force toward development of
culture supernatant and added lectins allowed analy- new techniques for carbohydrate characterization.
sis of glycoprotein concentrations and changes in This review summarized the large body of recent
protein glycosylation.323 experimental work dedicated to construction of bio-
Evaluation of protein binding to surface-immobi- sensors and bioassays designed to detect and analyze
lized LPS was carried out using specially designed carbohydrates and glycoconjugates, and sensors uti-
SPR biosensors, because of the importance of LPS lizing carbohydrates for detection of other soluble
constituents in affecting protein binding to varied cell biomolecules.
surfaces.324,325 Biosensor chip surfaces derivatized The complexity and high variability of carbohy-
with different quantities of LPS were used for deter- drate structures have often placed formidable barri-
mination of peptide- and protein-binding constants.326 ers toward their practical applications; however,
Immobilization of LPS in these sensor chips was these properties might as well open new avenues to
6012 Chemical Reviews, 2004, Vol. 104, No. 12 Jelinek and Kolusheva
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