Essays in Biochemistry (2016) 60 59–68
DOI: 10.1042/EBC20150007
Bioconjugation and stabilisation of biomolecules in
biosensors
Susana Liébana and Guido A. Drago
Applied Enzyme Technology Ltd, Gwent Group Ltd, Monmouth House, Mamhilad Park, Pontypool NP4 OHZ, U.K.
Correspondence: Susana Liébana (susana.liebana.girona@gmail.com)
Suitable bioconjugation strategies and stabilisation of biomolecules on electrodes is essen-
tial for the development of novel and commercially viable biosensors. In the present review,
the functional groups that comprise the selectable targets for practical bioconjugation meth-
ods are discussed. We focus on describing the most common immobilisation techniques
used in biosensor construction, which are classified into irreversible and reversible meth-
ods. Concerning the stability of proteins, the two main types of stability may be defined as
(i) storage or shelf stability, and (ii) operational stability. Both types of stability are explained,
as well as the introduction of an electrophoretic technique for predicting protein–polymer
interactions. In addition, solution and dry stabilisation as well as stabilisation using the co-
valent immobilisation of proteins are discussed including possible factors that influence sta-
bility. Finally, the integration of nanomaterials, such as magnetic particles, with protein im-
mobilisation is discussed in relation to protein stability studies.
Introduction
Different biosensors rely on different biomolecules such as nucleic acids, antibodies, cells or enzymes,
which can work as both bioreceptors and signalling molecules or labels. The immobilisation and stabili-
sation of the bioreceptor on to the transducer of the biosensor are both of great importance. Biomolecules
are immobilised not only on the transducer to act as a bioreceptor, but also on other surfaces such as
magnetic, gold or latex particles, and recently in a wide variety of nanomaterials that enhance the analyt-
ical performance of the biosensors. Among the wide range of strategies dealing with the immobilisation
of biomolecules, in the present review we will focus on the immobilisation of active biological receptors
on electrode surfaces for electrochemical biosensors. A detailed discussion of the advantages and disad-
vantages of the irreversible (covalent binding, cross-linking and entrapment or micro-encapsulation) and
reversible (adsorption, bioaffinity, chelation or metal binding and formation of disulfide bonds) immobili-
sation methods will be presented. Moreover, we will address the importance of the storage and operational
stability of the biomolecules involved in the biosensing event and discuss the factors that influence protein
stability.
Immobilisation methods on electrode
surfaces
New types of transducers have been developed for use as biosensors, the most popular being op-
Version of Record published tical, electrochemical and mass-based transduction methods. As analytical systems, electrochemical-
30 June 2016 based transduction devices are robust, easy to use, portable and inexpensive [1]. Many electrode
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Figure 1. Irreversible immobilisation methods
Schematic representation of some irreversible methods: (a) covalent binding through primary amines, (b) cross-linking using carbodi-imide,
and (c) entrapment on beads or fibres and micro-encapsulation.
materials such as glassy carbon, carbon paste, graphite composites, carbon/graphite formulations, carbon nanotubes,
graphene and gold among others are used in electrochemical biosensors. Screen-printed electrodes (SPEs) are widely
used as the measuring element due to easy and reproducible fabrication at both laboratory scale and in mass pro-
duction [2,3]. Several types of SPEs, functionalised or not, are now commercially available (e.g. Gwent Group Ltd)
and many laboratories have their own facilities for in-house production. However, in addition to the configura-
tion of the electrode and materials being crucial, so is the immobilisation of the bioreceptor on to the electrode
surface.
Often when working with biological molecules like proteins, an immobilisation method optimised for one
protein may need to be adjusted to take into consideration the unique properties of another protein. For in-
stance, it may be simple to conjugate or modify highly soluble proteins that have a high degree of conforma-
tional stability. However, similar reactions carried out on hydrophobic membrane proteins or insoluble pep-
tide sequences will often require changes to the reaction conditions, which will affect the same conjugation
process.
The recent advances in bioconjugation techniques are widely described in the literature (reviewed in [4]). The
different strategies can be classified according to various levels of selectivity and difficulty, ranging from random
methods (e.g. adsorption) to more advanced techniques based on protein engineering used to facilitate directional
immobilisation (e.g. bio-orthogonal chemistries and SpyTag/SpyCatcher) [5–7]. In this review, we focus on the most
common immobilisation techniques used for biosensor construction, which can be classified into two broad cat-
egories: irreversible (Figure 1) and reversible (Figure 2) methods. The advantages and disadvantages of the main
methods are summarised in Table 1 [8,9].
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Figure 2. Reversible immobilisation methods
Schematic representation of some reversible methods: (a) adsorption, physical and ionic binding, (b) bioaffinity with strept(avidin)/biotin and
Protein A/G, (c) chelation or metal binding, and (d) disulfide bonds.
Table 1. Characteristics of different immobilisation methods
Immobilisation method Interaction Advantages Disadvantages
Covalent binding Irreversible Stability Cost
High binding strength
Cross-linking Irreversible Stability Diffusion limitation
High binding strength Cross-linker toxicity
Entrapment Irreversible Stable to changes in pH or ionic Limited by mass transfer
strength
Adsorption Reversible Simple Less reproducible
Fast Random orientation
Low cost Desorption following change in ionic
strength or pH
Bioaffinity Reversible Good orientation Cost
High specificity
High selectivity
High functionality
Well-controlled
Chelation or metal binding Reversible Simplicity Less reproducibility
Disulfide bonds Reversible Good orientation Cost
High sensitivity Need for linkers
Well-ordered
Stable bond
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Irreversible immobilisation methods
The immobilisation is irreversible when the bioreceptor attached to the support cannot be detached without de-
stroying either the biological activity of the biomolecule or the support. The most common methods of irreversible
immobilisation are covalent binding, cross-linking and entrapment or micro-encapsulation.
Covalent binding
The immobilisation of bioreceptors by methods based on the formation of covalent bonds are among the most widely
used. Due to the stable nature of the bonds formed between the biomolecule and support, the bioreceptor is not
released into the solution upon use. However, in order to achieve high levels of bound protein activity, the active
area of the biomolecule must not be compromised by the covalent linkage chemistry to the support. For instance,
the amino acid residues essential for catalytic activity or the recognition area of antibodies must not be hindered or
blocked; this may prove a difficult requirement to fulfil in some cases. A wide variety of reactions have been developed
depending on the functional groups available on the target. Despite the complexity of the biomolecule structure, only
a small number of functional groups comprise selectable targets for practical bioconjugation methods. In fact, just
five chemical targets account for the vast majority of chemical modification techniques:
• Primary amines (–NH 2 ). This group exists at the N-terminus of each polypeptide chain and in the side chain of
lysine (Lys, K) residues. In physiological conditions, primary amines are positively charged and usually outward-
facing on the surface of proteins, thus they are normally accessible for conjugation without denaturing the protein
structure. Primary amines can be targeted using several kinds of conjugation chemistries. The most specific and
efficient reagents are those that use the N-hydroxysuccinimidyl ester (NHS ester) reactive group.
• Carboxygroups (–COOH). This group exists at the C-terminus of each polypeptide chain and in the side chains
of aspartic acid (Asp, D) and glutamic acid (Glu, E). Like primary amines, carboxygroups are usually available
on the protein surface.
• Thiols (–SH). This group exists in the side chain of cysteine (Cys, C). Often, as part of a protein’s secondary or
tertiary structure, cysteines are joined together between their side chains via disulfide bonds (–S–S–). These must
be reduced to thiols to make them available for binding. Reagents that are activated with maleimide or iodoacetyl
groups are the most effective for thiol-directed conjugation.
• Carbonyls (–CHO). Ketone or aldehyde groups can be created in glycoproteins by oxidising the polysaccharide
post-translational modifications (glycosylation) with sodium meta-periodate.
• Carbohydrates (sugars). Glycosylation occurs primarily in the constant fragment (Fc) region of antibodies
(IgG). Component sugars in these polysaccharide moieties that contain cis-diols can be oxidised to create ac-
tive aldehydes (–CHO) for coupling. Labelling carbohydrates requires more steps than labelling amines because
the carbohydrates must first be oxidised to create reactive aldehydes; however, the strategy generally results in
antibody conjugates with high activity due to the location of the carbohydrate moieties. Aldehyde-activated (ox-
idised) sugars can be reacted directly to primary amines through reductive amination (mentioned above) or to
reagents that have been activated with hydrazide groups.
Cross-linking
Cross-linking is the process of chemically joining two or more molecules by a covalent bond. Cross-linking reagents
(or cross-linkers) are molecules that contain two or more reactive ends capable of chemically attaching to specific
functional groups (primary amines, thiols, etc.) on proteins or other biomolecules. Cross-linkers are also commonly
used to modify nucleic acids, drugs and solid surfaces. The same chemistry is applied to amino acid and nucleic
acid surface modification and labelling. There are several technical handbooks available, which detail the chemical
reactivity and the molecular properties of cross-linkers (e.g. [10]).
Entrapment or micro-encapsulation
The entrapment method is based on the occlusion of a biomolecule, mostly enzymes, within a polymeric network
that allows the substrate and products to pass through but retains the enzyme. This method differs from the coupling
methods described above, in that the enzyme is not bound to the support or membrane. There are different approaches
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to entrapping enzymes such as gel or fibre entrapping and micro-encapsulation. The practical use of these methods
is limited by mass transfer limitations through membranes or gels.
Reversible immobilisation methods
Reversibly immobilised biomolecules can be detached from the support under gentle conditions. The use of reversible
methods for bioreceptor immobilisation is highly attractive, mostly for economic reasons. After using the support, it
can be regenerated and re-loaded with fresh bioreceptor. The most common methods of reversible immobilisation
are adsorption, bioaffinity, chelation or metal binding and the formation of disulfide bonds.
Adsorption
The simplest immobilisation method is non-specific adsorption, which is mainly based on physical adsorption or
ionic binding. In physical adsorption, the bioreceptors are attached to the surface through hydrogen bonding, van
der Waals forces or hydrophobic interactions, whereas in ionic binding, the enzymes are bound through salt linkages.
The nature of the forces involved in non-covalent immobilisation results in a process that can be reversed by changing
the conditions that influence the strength of the interaction (e.g. pH, ionic strength, temperature or polarity of the
solvent). Immobilisation by adsorption is a mild easy to perform process and usually preserves the functionality of
the biomolecule [11]. The limitations of the adsorption mechanism are the random orientation and weak attachment,
which produce desorption and poor reproducibility [12].
Bioaffinity
The principle of affinity between complementary biomolecules such as lectin–sugar, antibody–antigen and biotin–
avidin has been applied to biomolecule immobilisation [13,14]. The remarkable selectivity of the interaction is a
major benefit of the method. However, the procedure often requires the covalent binding of a costly affinity ligand
(e.g. antibody or lectin) to the support. The most established procedures are the (strept)avidin–biotin interaction and
the use of Protein A or G for antibody immobilisation:
• (Strept)avidin–biotin interaction. Avidin is a glycoprotein found in egg whites that contains four identical sub-
units. Each subunit contains one binding site for biotin, or vitamin H, and one oligosaccharide modification. The
tetrameric protein is highly basic, having an isoelectric point (pI) of about 10. The biotin interaction with avidin
is among the strongest noncovalent affinities known, exhibiting a dissociation constant of about 1.3×10 − 15 M.
The only disadvantage of using avidin is its tendency to bind non-specifically with components other than biotin
due to its high pI and carbohydrate content. Streptavidin is a similar biotin-binding protein to avidin, but it is
of bacterial origin and originates from Streptomyces avidinii. The primary structure of streptavidin is consid-
erably different from that of avidin. This variation in the amino acid sequence results in a much lower pI for
streptavidin (pI 5–6). The strength of the noncovalent (strept)avidin–biotin interaction along with its resistance
to break down makes it extraordinarily useful in the bioconjugate chemistry of any biomolecule [15].
• Protein A and G. This bioconjugation is mainly used for the immobilisation of antibodies. Protein A is derived
from Staphylococcus aureus and Protein G is from Streptococcus species. Both have binding sites for the Fc
of mammalian immunoglobulins (IgG), which ensures good orientation of the antibody after conjugation. The
affinity of these proteins for IgG varies with the animal species. Protein G has a higher affinity for rat, goat, sheep
and bovine IgG, as well as for mouse IgG1 and human IgG3. Protein A has a higher affinity for cat and guinea
pig IgG. In addition to IgG Fc-binding sites, native Protein G contains binding sites for albumin, which can lead
to non-specific staining. This problem is addressed by creating recombinant forms of the protein.
Chelation or metal binding
This method is known as ‘metal link immobilisation’. The metal salt or hydroxide, mainly titanium and zirconium salt,
is precipitated and bound by co-ordination with nucleophilic groups on the surface (e.g. cellulose-, chitin-, alginic
acid- and silica-based carriers) by heating or neutralisation. Due to steric factors, not all of the metal binding sites
are occupied, therefore some of the positions remain free to interact with groups from the biomolecule. The method
is quite simple, but the adsorption sites are not uniform and the metal ion leakage is significant, which leads to a
lack of reproducibility. In order to improve the control of the formation of the adsorption sites, chelator ligands such
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as ethylenediaminetetra-acetic acid (EDTA) can be immobilised on the solid supports by means of stable covalent
bonds. Elution of the bound proteins can be easily achieved by competition with soluble ligands or by decreasing the
pH.
Disulfide bonds
These methods are unique because, even though a stable covalent bond is formed between the support and biorecep-
tor, it can be broken by reaction with a suitable agent such as dithiothreitol (DTT) under mild conditions. Addition-
ally, because the reactivity of the thiol groups can be modulated via pH alteration, the activity yield of the methods
involving disulfide bond formation is usually high, provided that an appropriate thiol-reactive adsorbent with high
specificity is used [16].
Protein stabilisation
The stabilisation of proteins is of great importance in many applications, and particularly in biosensor development.
Stabilisation is known as the ability of a protein to retain its structural conformation or its activity when subjected
to physical or chemical manipulation. The two main types of stability may be defined as (i) storage or shelf stability,
and (ii) operational stability. The first relates to the retention of protein activity over time when stored as a dehy-
drated preparation, a solution or immobilised on to a surface. The second generally relates to the retention of activity
when in use. The retention of the biological activity of the biomolecule involved in the biorecognition of an analyte
is paramount, and this depends on retention of the biological structure. In most cases, the actual mechanism of sta-
bilisation remains to be fully understood [17]. Protein engineering can be a useful tool for increasing the stability of
certain enzymes [18] and antibodies [19]. However, this is only possible so long as structural data are available for
the protein under examination. Protein stability is also evaluated using freeze-drying and spray-drying [20–22]. In
this section, we will focus on the stabilisation achieved by the use of additives to modify the microenvironment of the
protein under investigation and the covalent immobilisation of proteins on to transducers.
Dry and solution stability of proteins
Most of the publications on enzyme stabilisation are focused on the effect of additives on protein stability showing that
it has been the most popular method of enzyme stabilisation [23]. The most important parameter in the promotion of
structural integrity and thus stability of a protein is to retain the surface water activity. The components of a stabiliser
formulation are normally made up of a combination of polyalcohols and polyelectrolytes. The polyalcohols include
sugars and sugar alcohols that modify the water environment surrounding a protein, thus replacing and competing
with free water within the system. This modified hydration shell confers protection to the protein, maintaining a 3D
structure and biological activity, and enables long-term storage of biological materials both in solution and in the de-
hydrated state. Polyelectrolytes include numerous polymers of different charge and structure that form electrostatic
interactions with proteins. As a result, large protein–polyelectrolyte complexes are formed, which retain full biologi-
cal activity. Where polyelectrolytes and polyalcohols are combined, a synergistic effect is usually observed. Ratios of
polyelectrolyte to polyalcohol are extremely important in the overall stabilisation of proteins. The buffer type, pH,
ionic strength, concentration and ratio of stabilisers to protein/enzyme all play crucial roles in protein stabilisation
both in the dry state and in solution. Some additives such as metal ions are directly related to enzyme structure and
as such are not strictly surface interactions. The addition of dilute solutions of metal salts (e.g. magnesium or cal-
cium) often stabilise proteins to a high degree and act synergistically with polyelectrolyte combinations. The addition
of polyelectrolytes to solutions of proteins promotes the formation of soluble protein–polyelectrolyte complexes by
electrostatic interaction. Polyhydroxyl compounds are then able to penetrate the structure more effectively, leading
to enhanced stabilisation.
In most cases, the biological activity of the protein, enzyme or antibody is used as the main parameter to determine
the stabilisation effect. When no simple method is available to directly measure biological activity, other techniques
can be used to determine any molecular and structural modifications, such as gel electrophoresis, circular dichro-
ism, fluorescence and turbidimetric measurements. Gel electrophoresis is normally used to examine the interactions
between proteins and polymers and can be used to predict specific formulations, which lead to improved protein
stability. Due to the extremely large size of the polymers, interaction between the polymer and protein is detected
as the retardation of the enzyme in the gel matrix. This technique also helps in the determination of the affinity of
polymer binding, allowing the prediction of the amount of polymer needed and subsequently reducing the cost of
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stabilisation considerably. However, in most cases, simple activity tests or immunoassays are sufficient to evaluate the
remaining activity [17].
Stabilisation by protein immobilisation
The covalent immobilisation of proteins onto transducers is considered another way of stabilising the biomolecule
involved in the biosensing event. Each protein examined is unique, so in most cases what works for one type of protein
rarely works for another. In addition, the problem of stability can be complicated during the immobilisation process,
which can result in a stable protein but with vastly reduced residual activity. Of the methods described above, the
most common immobilisation method used on a pre-activated carbon transducer utilises covalent coupling to the
amino groups. In some enzymes, such as acetylcholinesterase [24], most of the amino groups are situated on the
back face opposite to the active site, therefore this methodology ensures retaining the activity after immobilisation.
Covalent immobilisation of other enzymes such as glucose oxidase has been described in many cases. The results show
that the immobilised complex is more stable than the native immobilised enzyme. Stabilisation of the immobilised
enzyme with polyelectrolyte combinations shows a distinct difference from that of the soluble enzyme dehydrated
from solution. The orientation of the enzyme on to the surface of the transducer might explain this effect [25,26].
The advantages of having enzymes attached to surfaces have been exploited by living cells for as long as life has
existed. In fact, there is experimental evidence to suggest that the immobilised state might be the most common state
for enzymes in their natural environment. The attachment of enzymes to an appropriate surface ensures that they
remain at the site where their activity is required. This immobilisation enhances the protein concentration at the
proper location and it may also protect the enzyme from being destroyed. Multimolecular assembly depends upon
the combination of weak non-covalent forces, hydrophobic interactions and covalent bonds (e.g. disulfide bridges)
[27,28]. All of these different forces have been exploited in the development of immobilised enzymes.
One of the main problems associated with the use of immobilised enzymes is the loss of catalytic activity. This is
probably due to the immobilisation site blocking access to the substrate-binding site of the protein resulting in the
observed loss of enzyme activity. There are several strategies to avoid these steric problems. The careful choice of
enzyme residues involved in the immobilisation, and the use of hydrophilic and inert spacer arms can reduce steric
hindrance dramatically [29].
More recently, other immobilisation strategies have been adopted. These include the use of magnetic particles
(MPs), which is attracting interest due to the intrinsic advantages of the material. MPs have been commercially avail-
able for many years (e.g. BioMag R
, Dynabeads R
, AdembeadsR
and MiltenyiR
) and are widely used in laboratories
to extract desired biological components, such as cells, organelles or DNA, from a fluid. In recent years, the mag-
netic properties of MPs have also been used as labels [30] and bioreceptor platforms in biosensing [31]. As shown
in Figure 3 (left), they consist of an inorganic core of iron oxide (magnetite (Fe3 O4 ), maghemite (Fe2 O3 ) or other
insoluble ferrites) coated with a polymer to confer stability (e.g. polystyrene, dextran, polyacrylic acid or silica), with
added functional groups (e.g. amino and carboxylic acids) to make subsequent conjugations easy. Hence, iron ox-
ide particles can carry diverse ligands, such as peptides, small molecules, proteins, antibodies and nucleic acids. In
particular, this material is attractive for its use in immunomagnetic separation where antibodies are conjugated to
the particles allowing the capture and orientation of the antigen on the surface of the electrode [32]. An example of
solution stability for antibody-modified MPs is showed in Figure 3 (right). The antibody-modified MPs were stored
in a number of stabiliser formulations at 32 ◦ C in the solution state. The different MPs were processed over several
days by magneto-immunoassay to detect prostate-specific antigen (PSA). Agglomeration of particles in the differ-
ent solutions caused variability; however, the results showed that the Q2030317P4 stabiliser retained at least 85 % of
the original antibody activity after 3 months of storage at this temperature. Meanwhile the unstabilised version lost
complete binding ability within 1 month.
Conclusions
The field of bioconjugation has advanced at incredible pace. Tens of thousands of additional publications have
appeared in biological, medical, polymer, material science and chemistry journals describing novel reactions and
reagents along with their use in a variety of bioconjugation techniques. In this review, we discussed the most typical
immobilisation techniques for active biological receptors on electrode surfaces, which could be extended to other
transducer systems such as optical, piezoelectric or calorimetric as well as nanomaterials for all types of biosensors.
The goal of the immobilisation method is maintaining biological activity while favouring, or at least not altering, the
kinetics of the biological reaction.
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Essays in Biochemistry (2016) 60 59–68
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Figure 3. Stabilisation by protein immobilisation
Schematic representation of (a) magnetic particles, (b) activated with functional groups, and (c) conjugated to biological molecules [32]; (d)
solution stability of antibody-modified magnetic particles over 3 months at 32 ◦ C using stabilisers from Applied Enzyme Technology Ltd.
Activity tested by optical magneto-immunoassay using 15 ng/ml PSA, 0.1 mg/ml anti-PSA antibody-modified magnetic particles (Abcam,
ab10184) and 1 μg/ml horseradish peroxidase (HRP)-labelled antibody (Abcam, ab24466).
The actual stabilisation factor for most biosensors seems to be a combination of structural stabilisation by immo-
bilisation on to a surface and the addition of specific stabiliser molecules. Both shelf stability and operational stability
are improved by using novel polyelectrolyte stabilisers. The methodology is relatively generic and can be adapted for
many application areas. The molecular mechanisms of stabilisation are currently under investigation by using more
sophisticated techniques such as circular dichroism, fluorescence spectroscopy, differential scanning calorimetry,
electrophoretic techniques, analytical centrifugation and electron microscopy. Data accumulated from such experi-
ments will help researchers to understand more about how proteins denature at the molecular level and ultimately
enable the stabilisation of proteins in a more predictable fashion.
The combination of immobilising and stabilising biomolecules together with the integration of micro- and nano-
structured materials within biosensing devices is providing excellent analytical performances for different applica-
tions. The need of more flexible, reliable and sensitive targeting of analytes has promoted research into the potential
of nanomaterials and their incorporation into biosensor systems. Most of the immobilisation methods described in
this review are used for the bioconjugation of biomolecules into nanomaterials such as carbon nanotubes (CNTs),
gold nanoparticles (AuNPs), MPs or quantum dots (QDs).
Research and development into biosensors is focused on designs compatible with technologies, such as screen-
printing techniques, which allow the industrial production of low-cost devices. In this context, both suitable bioconju-
gation strategies and the stabilisation of biomolecules on electrodes are essential for the development of commercially
viable biosensors.
Summary
• Biosensors rely on different biomolecules (nucleic acids, antibodies, cells or enzymes) as bioreceptors and
signalling molecules or labels. Both immobilisation and stabilisation of the bioreceptor onto the transducer
of the biosensor are of great importance.
• Immobilisation of active biological receptors on electrode surfaces for electrochemical biosensors construc-
tion. The most common immobilisation techniques used are classified in two broad categories: irreversible
methods, when the bioreceptor cannot be detached without destroying either the biological activity of the
biomolecule or the support, and reversible methods, when immobilised biomolecules can be detached from
the support under gentle conditions.
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Essays in Biochemistry (2016) 60 59–68
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• Protein stabilisation is known as the ability of a protein to retain its structural conformation or its activity
when subjected to physical or chemical manipulations. Stabilisation of proteins is achieved by the use of
additives to modify the microenvironment of the protein under investigation.
• Covalent immobilisation of proteins onto transducers is considered another way of stabilising the
biomolecule involved in the biosensing event. Immobilisation of proteins onto nanomaterials such as mag-
netic particles provides analytical advantages.
Abbreviations
MP, magnetic particle; PSA, prostate-specific antigen; SPE, screen-printed electrode.
Funding
We acknowledge funding by the European Commission Framework Programme 7 through the Marie Curie Initial Training Network
PROSENSE [grant number 317420, 2012 – 2016].
Competing Interests
The Authors declare that there are no competing interests associated with the manuscript.
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