14

Bioanalytical chemistry

2. Enzymes as analytical reagents

Required reading: Sections 3.1 to 3.5.1.3 (assays), 4.1 to 4.3 (immobilized enzymes), and
7.1 to 7.3.5, 7.4 (biosensors) of Mikkelsen and Cortón, Bioanalytical Chemistry

Some objectives for this section:

• You will know the reactions catalyzed by several enzymes that are useful analytical reagents.
• You will know what a coupled enzyme assay is.
• You will have some appreciation of why one might want to immobilize a protein
• You will have an overview of the methods that one could use to immobilize a protein.
• You will be able to rationalize for yourself the advantages and disadvantages of each technique based on
your background in protein structure.
• You will understand how an immobilized enzyme can be incorporated into a simple biosensor.
• You will understand the basic principles of electrochemical biosensor design
• You will know how a glucose biosensor works
• You will have a historical perspective of how the technology has evolved over the last 40 years
• You will be familiar with some of the latest developments and trends in glucose biosensing.

Primary Source Material

• Chapter 8 of Biochemistry: Berg, Jeremy M.; Tymoczko, John L.; and Stryer, Lubert (NCBI bookshelf).
• Chapter 4 of Mikkelsen, S.R. and Corton, E., Bioanalytical Chemistry (2004) John Wiley and Sons p.
61-71.
• Gary Walsh, Proteins: Biochemistry and Biotechnology, John Wiley & Sons; 2nd edition (2002)
• http://chem.ch.huji.ac.il/~eugeniik/index.htm
• http://chem.ch.huji.ac.il/~eugeniik/electron_mediators.htm
 OMP-decarboxylase accelerates a reaction by15
a factor of greater than 1017!!
O O

HN HN
H2O, pH 7, 25 °C
O O N CO2- O O N
-O P O O t0.5 = 78 million years -O P O O
O- O-
OH OH OH OH

O O

HN HN

O O N CO2- O O N
-O P O O -O P O O
O- t0.5 = 18 ms O-
OH OH OH OH

Image source: http://www.chem.umn.edu/groups/gao/enzyme.htm

More information: http://arjournals.annualreviews.org/doi/full/10.1146/annurev.biochem.71.110601.135446

• OMP-decarboxylase turns its substrate over with a half-time of 18 ms, in a reaction that proceeds in its
absence with a half-time of 78 million years in neutral solution.
• This means that the enzyme accelerates the rate of this reaction by a factor of >1017!!
 Enzymes stabilize the transition state through16
specific binding interactions

Contacts between ODCase and a proposed

transition state analog

• A chemical reaction of substrate S to form product P goes through a transition state S‡ that has a higher
free energy than does either S or P. The double dagger denotes a thermodynamic property of the transition
state. The transition state is the most seldom occupied species along the reaction pathway because it is
the one with the highest free energy. The difference in free energy between the transition state and the
substrate is called the Gibbs free energy of activation or simply the activation energy, symbolized by ΔG‡.
• The activation-energy barrier immediately suggests how enzymes enhance reaction rate without altering
ΔG of the reaction: enzymes function to lower the activation energy, or, in other words, enzymes facilitate
the formation of the transition state.
• Because enzymes are such superb catalysts, it is tempting to ascribe to them powers that they do not
have. An enzyme cannot alter the laws of thermodynamics and consequently cannot alter the equilibrium
of a chemical reaction. This inability means that an enzyme accelerates the forward and reverse reactions
by precisely the same factor.
• Shown on the right-hand side are contacts between ODCase and a proposed transition state analog.
These contacts were observed in the x-ray crystal structure of the complex.
• The interaction of Lys-93 with the O- group of the transition state analog is relatively favourable,
foreshadowing the very great affinity that the enzyme evidently develops for the carbanion generated at
C-6 in the transition state
• Proc Natl Acad Sci U S A. 2000 February 29; 97 (5): 2011–2016
• Proc Natl Acad Sci U S A. 2000 February 29; 97 (5): 2017-2022
 17

By generating
antibodies against
small molecules that
resemble transition
states, it is possible to
generate catalytic
antibodies (abzymes)

David S. Goodsell: The Molecule of the Month appearing at the PDB

• Researchers have used the incredible functional diversity of the immune system in a clever way: to design
new enzymes.
• Enzymes work by easing molecules through a difficult chemical change. For instance, look at the Diels-
Alder reaction shown here at the bottom of the illustration. The two molecules on the left come together,
forming an unstable intermediate shown at the center in red. Then, the intermediate falls apart, releasing
sulfur dioxide and forming the desired product, shown on the right.
• Enzymes act by stabilizing the transition state and decreasing the activation barrier that has to be
overcome in order for a reaction to proceed. To make an antibody into an enzyme, we need to find an
antibody that stabilizes this transition state in a similar way. Researchers have done this by finding
antibodies that bind to a molecule that mimics the transition state, like the one shown here in green.
• These antibody-enzymes are termed catalytic antibodies. The catalytic antibody shown here, from PDB
entry 1c1e, performs the Diels-Alder condensation reaction shown in the diagram. This is significant
because this type of reaction is not performed by any natural enzymes.
• Antibodies that perform a number of other cleavage and condensation reactions have now been
developed.
 18
Enzymes used for analytical purposes:
alcohol dehydrogenase oxidizes alcohols
OH
O NH2
R R' N
H NH2 N
N O O N N
O P O P O
O O
O O

HO OH HO OH
NAD+

H H O NH2
NH2 N N

N O O N N
O P O P O
O O
O O
O
HO OH HO OH
NADH
R R'

Goodsell: PDB molecule of the month

• Alcohol dehydrogenase has proven to be one of the more useful enzymes for bioanalytical applications.
When might you want to detect the presence of alcohols?
• There are many other examples of enzymes that can be used either directly or indirectly as diagnostic
reagents [from Table 9.2 of Gary Walsh, Proteins: Biochemistry and Biotechnology, John Wiley & Sons;
2nd edition (2002)]. Here are a few:
• Arginase: determination of L-arginine levels in plasma and urine
• Cholesterol esterase: determination of serum cholesterol levels
• Creatine kinase: diagnosis of cardiac and skeletal malfunction
• Glycerol-3-phosphate dehydrogenase: determination of serum triglycerides
• Uricase: determination of uric acid
 Enzymes used for analytical purposes: 19
Glucose oxidase catalyzes glucose oxidation
OH
O
HO
HO OH
OH
β-D-glucose O
N
HN
O2
O N N
R
FAD (oxidized)

O
H
N
HN H2O2
O N N
OH H R
O
HO FADH2 (reduced)
HO
OH O

D-glucono-1,5-lactone

http://www-biol.paisley.ac.uk/marco/enzyme_electrode/chapter3/chapter3_page1.htm

• As we will see, glucose oxidase has proven to be one of the most useful enzymes known. It is the basis for
glucose biosensors that are used for monitoring blood glucose levels of people with diabetes.
 Enzymes used for analytical purposes: 20
Horseradish peroxidase catalyzes the oxidation
of a wide variety of substrates
H2O2
+ 2 H+
+ 2 e-
substance X (reduced)

heme

substance X (oxidized)
X
2 H2O

Biochemistry. 1998 Jun 2;37(22):8054-60.

• This ability of horse radish peroxidase to oxidize a wide variety of substrates means that it is a very useful
biotechnological tool.
• This will be further illustrated on the next slide.
 21
Amplex Red:
a fluorogenic peroxidase substrate

H 2O

O2

A two-enzyme coupled assay for

detection of glucose

Image source: http://www.probes.com/handbook/

Anal Biochem. 1997 Nov 15;253(2):162-8

• In this two enzyme ‘coupled assay’, glucose oxidase is first oxidizing glucose with formation of
gluconolactone and hydrogen peroxide. From an analytical point of view we still need to detect the
formation of the product somehow. This detection is done by adding horseradish peroxidase and a
chromogenic or fluorogenic substrate such as commercially available Amplex Red. There are a wide
variety of substrates available for horseradish peroxidase.
• Horseradish peroxidase reduces the hydrogen peroxide generated by glucose oxidase and
simultaneously oxidizes amplex red to the brightly red fluorescent dye resorufin. The amount of resorufin
generated can be quantified by absorbance or fluorescence spectroscopy.
• A key reference regarding the mechanism of Amplex Red: Gorris and Walt, J. AM. CHEM. SOC. 2009,
131, 6277–6282. The authors provide evidence that the mechanism proceeds via two steps. In the first
step a 1 electron oxidation of Amplex Red occurs to give the radical (centered on a phenol oxygen). In the
second step there is a dismutation reaction in which two radicals react to form resorufin and regenerate
one molecule of Amplex Red.
 The PiPer phosphate Assay 22
Kit from Molecular Probes: a
fluorescence-based assay for
free phosphate

O2
A three-enzyme coupled assay for
H 2O
detection of free phosphate

Image source: http://www.probes.com/handbook/

• Coupled assays do need to be limited to 2 enzymes. A nice example of a 3 enzyme coupled assay is the
PiPer Phosphate Assay Kit from Molecular Probes.
• In the presence of inorganic phosphate, maltose phosphorylase converts maltose to glucose-1-phosphate
and glucose. Then, glucose oxidase converts the glucose to gluconolactone and H2O2.
• Horseradish peroxidase reduces the hydrogen peroxide generated by glucose oxidase and
simultaneously oxidizes amplex red to the brightly red fluorescent dye resorufin.
• The resulting increase in fluorescence is proportional to the amount of Pi in the sample. All of the other
components would be added in excess such that they are not limiting in terms of the overall conversion
from inorganic phosphate to resorufin.
• A key reference regarding the mechanism of Amplex Red: Gorris and Walt, J. AM. CHEM. SOC. 2009,
131, 6277–6282. The authors provide evidence that the mechanism proceeds via two steps. In the first
step a 1 electron oxidation of Amplex Red occurs to give the radical (centered on a phenol oxygen). In the
second step there is a dismutation reaction in which two radicals react to form resorufin and regenerate
one molecule of Amplex Red.
 23
Coupled assays with
immobilized enzymes
are used in glucose-
sensing ‘dipsticks’

Gary Walsh, Proteins: Biochemistry and Biotechnology, John Wiley & Sons; 2nd edition (2002)

• CLINISTIX™ sticks contain the enzyme glucose oxidase immobilized onto the paper pad at the end of the
stick. This enzyme oxidizes glucose to yield gluconic acid and hydrogen peroxide. Peroxidase is also
immobilized on the paper pad and it uses the hydrogen peroxide to oxidize a colored dye to a form with a
different color (or no color).
• The degree of color change is proportional to the amount of glucose. The color can either be estimated by
eye or, more quantitatively, using a simple colorimeter. Why not use fluorescence in this case
• Question: On most of the slides, you mentioned glucose oxidase converts the glucose to gluconolactone.
But here you said the product of this reaction is gluconic acid. Although lactone can be opened to form a
gluconic acid, but it requires acid or base as catalyst. I'm quite confused about this.
• Answer: To the best of my knowledge, the product of the enzymatic reaction is gluconolactone. Water will
add to the gluconolactone to give gluconic acid. If you were asked what the product of the enzyme
reaction is, the correct answer is gluconolactone. 
 24
Soluble vs. immobilized enzymes

soluble enzyme immobilized enzyme

OH O2 OH H2O2 OH O2 OH H2O2
O O O O
HO HO HO HO
HO OH HO HO OH HO
OH OH O OH OH O

linker
stationary phase

• Soluble enzymes are extremely useful analytical reagents but there are some disadvantages with their
use:
• Generally not reused or recycled
• Activity decreases with time (due to oxidation, denaturing, sticking to glass, etc.)
• A solution is to use immobilized enzymes. The enzyme is somehow incorporated onto (or into) the
stationary phase and then substrate is introduced in the mobile buffer phase which passes over the
enzyme.
• Can often be reused many times
• More amenable to automation/robotic handling
• Greater stability over time
 25
Overview of immobilization methods E

E E
Non-covalent
receptor-mediated
methods
a. E
E

Chemical (covalent)
methods

b. c.

Physical methods

d. e. f.
Mikkelsen, S.R. and Corton, E., Bioanalytical Chemistry (2004)

• a. Receptor-mediated. Non-covalent immobilization based on specific protein-ligand interactions (I.e.

avidin-biotin) with an immobilized small molecule or protein binding partner.
Chemical. Chemical methods involve the formation of a covalent bond between the protein and the stationary
phase. Chemical methods can be further classified as either non-polymerizing or cross-linking.
• b. ‘Non-polymerizing’ means that covalent bonds are only formed between the enzyme and the stationary
phase.
• c. Crosslinking means that covalent bonds are formed enzyme-stationary phase and also enzyme-
enzyme.
Physical. Physical methods do not change the chemical structure of the enzyme because no new covalent
bonds are formed. These methods can be sub-classified as adsorption, entrapment, and encapsulation.
• d. Adsorption can be thought of as relatively non-specific ‘sticking’ to the stationary phase.
• e. Entrapment means that the enzyme is trapped in a cross-linked polymer but there are no covalent
bonds to the enzyme itself. Entrapment is accomplished by performing the polymerization reaction in the
presence of the enzyme.
• f. Encapsulation means that the enzyme is separated from the bulk solution by a semipermeable
membrane. Because the enzyme is big and the substrate is small, the substrate (and product) can diffuse
across the membrane but the enzyme can not.
 26
Methods for protein immobilization:
non-covalent receptor mediated

E E E

E
E

Matthew A. Cooper, Nature Reviews Drug Discovery 1, 515 - 528.

• Biotin- or streptavidin-presenting surfaces: These can be used to capture biotinylated-receptors . The

multiple biotin-binding sites of streptavidin on each face of the molecule allow biotinylated ligands to be
crosslinked by the streptavidin 'double adaptor'. This method is highly efficient and leads to stable
complexes, but is effectively irreversible. It is commonly used to immobilize 5'-biotinylated
oligonucleotides.
• Monoclonal antibodies. These can be covalently attached to a solid support by means of amine coupling.
Epitope-tagged or fusion proteins can then be directly and reversibly coupled to the surface through the
antibody–antigen interaction. Commonly used tags include, for example, glutathione S-transferase, herpes
simplex virus glycoprotein D epiptope, FLAG epitope and 6-His.
• Metal-coordinating groups. Groups such as iminodiacetic acid (IDA) and nitrilotriacetic acid (NTA) have
been widely used for direct immobilization of 6-His- and 10-His-tagged receptors. The moderate affinity of
the chelate–Ni2+–histidine ternary interaction means that there is sometimes considerable decay in the
level of immobilized receptor. For this reason, anti-6-His monoclonal antibodies are often used to enable
stable, oriented immobilization of His-tagged receptors.
 27
The ultimate protein-ligand interaction:
avidin + biotin
4 avidin
monomers

4 biotin HN NH

O
S
O

free avidin tetramer avidin/biotin complex

• Avidin is a tetrameric glycoprotein isolated from chicken egg white. The ability of avidin to bind biotin with
exceptionally high affinity (Kd ~ 10-15 M) has been the basis for its exploitation as a molecular tool in
biotechnological, diagnostic and therapeutic applications, collectively known as avidin-biotin technology.
This interaction is one of the tightest non-covalent interactions known between proteins and their ligands
and the overall stability approaches that of a single covalent bond. Biotin is a small water-soluble vitamin
(otherwise known as vitamin H).
• The avidin tetramer consists of four identical subunits, all bearing 128 amino acids and possessing one
biotin-binding site. The protein is basically charged (pI ~ 10.5), and each of its monomers possesses eight
arginine and nine lysine residues.
• The polypeptide chain of avidin also contains a glycosylation site at residue Asn-17. The carbohydrate
moiety accounts for about 10% of the molecular mass of avidin and exhibits extensive glycan
microheterogeneity.
• Despite the utility of chicken avidin in the many applications of avidin-biotin technology, there are some
drawbacks associated with its use. Its high pI and the presence of carbohydrate can cause non-specific
binding to extraneous material in certain applications, and these properties, therefore, hinder its use. Due
to these difficulties, streptavidin, a non-glycosylated and neutrally charged bacterial counterpart of avidin,
has virtually replaced avidin in these applications, even though avidin contains more lysine residues for
potential attachment of probes, is more hydrophilic, and is considerably more abundant and cheaper than
streptavidin. (from FEBS Letters Volume 467, Issue 1 , 4 February 2000, Pages 31-36)
• Commercially available ‘Neutravidin’ (Pierce) is avidin which has had the carbohydrate portion removed
enzymatically (glycosidase) and has been chemically treated to lower the isoelectric point. I have not
been able to figure out what the chemical treatment is - it may be proprietary information. However, we
can guess that the chemical treatment is either the blocking of arginines or the addition of more negative
charges.
 28
Streptavidin has largely replaced avidin in
biotechnological applications

Streptomyces:
a bacteria that forms filamentous
structures similar to those formed
by fungus

• Streptavidin is a close homolog of avidin produced by the bacteria Streptomyces avidinii. The dissociation
constant (Kd) of the complex between streptavidin and biotin is similar to that of avidin and biotin,
• Streptavidin is also one of the most stable proteins known. For example, it can maintain its functional
structure at high temperatures, extremes of pH, and in the presence of high concentrations of denaturants
and organic solvents. This protein also has exceptional stability against proteolysis. These unique
properties of streptavidin, along with the ability of biotin to be incorporated easily into various biological
materials, allow streptavidin to serve as a versatile, powerful affinity tag in a variety of biological
applications. In particular, streptavidin is one of the most frequently used proteins in clinical diagnostics.
• It is interesting to note that streptavidin is not Streptomycetes only claim to fame. These bacteria are
masterful chemists and produce the majority of antibiotics applied in human and veterinary medicine and
agriculture, as well as anti-parasitic agents, herbicides, pharmacologically active metabolites (e.g.
immuno-suppressants) and several enzymes important in the food and other industries. [From Sano et al.
Journal of Chromatography B, 715 (1998) 85-91.]
 29
Adding a biotin tag to a macromolecule:
1. Chemically reactive biotin labels
O

HN NH

SH S-linker S
O
Protein of Protein of
interest interest

Thiol-specific
O
biotinylation HN NH

reagents S
O

Biotinylation and Haptenylation Reagents http://www.probes.com/handbook/sections/0402.html

 30
Adding a biotin tag to a macromolecule:
2. The Avitag
molecular
biology
Protein of Protein of
interest interest

C-term GLNDIFEAQKIEWHE

biotin
ligase O
H
N
S
N
H

Protein of
O
interest

GLNDIFEAQKIEWHE

HN NH

S
O

Home page for the Avitag: http://www.avidity.com/

• The Biotin AviTag sequence is a peptide, just 15 residues long, that is recognized as a substrate by biotin
ligase. In the presence of ATP, the ligase specifically attaches biotin to a specific lysine residue in this
sequence. Can you propose a mechanism? Sounds like it would be similar to the same basic mechanism
we have seen several times now: biotin-ligase activates biotin to form a biotinyl 5' adenylate and then
transfers the biotin to biotin-accepting proteins.
• Using vectors developed by Avidity, the Biotin AviTag can be genetically fused to a much bigger protein.
This feature effectively allows any protein that has been cloned to be tagged with a biotin molecule.
• The Biotin AviTag system affords several major advantages over the chemical labelling of proteins with
biotin: Because the biotinylation is performed enzymatically, the reaction conditions are very gentle and
the labelling is highly specific. Either in vivo or in vitro biotinylation of proteins is possible.
 31
The Streptag: low affinity interaction with
streptavidin

Protein of
interest

C-term

molecular Strep-tag II (Kd = 13 µM)

biology SBP tag (Kd = 2.5 nM)"

Protein of
interest

AWRHPQFGG-C-term

Original publications
http://www.lrz-muenchen.de/%7EBiologische-Chemie/Publikationen/Publikationen.html
http://genetics.mgh.harvard.edu/szostakweb/publications/framecontent/pubcontent.html

• The Strep-tag is a "tailor-made", 8 amino acid strepavidin binding sequence.

• This sequence was found through the systematic screening of random peptide libraries in order to identify
a peptide binding sequence with optimal affinity tag properties. It turns out that phage display was not
used for the original discovery of this peptide, but it is a suitable and practical method that could have
been used. Phage display of random peptides with selection for binding to streptavidin reliably results in
the enrichment for peptide sequences that contain the HPQ sequence. This tripeptide binds in the biotin
binding site.
• The Strep-tag is not a replacement for biotin since it is much lower affinity. However, it does allow one to
reversibly bind proteins to the many different resins and surfaces that are available with streptavidin
coatings. For example, it can be used as an affinity tag for protein purification using columns with
immobilized streptavidin. Highly specific elution of the bound protein from the column can be achieved
through the addition of biotin or related compounds using a physiological buffer, ie. no high salt or
extreme pH are required for chromatography.
 32
Just think of the possibilities!!

Many
Anti-biotin conjugates
Anti-(strept)avidin
Anti-strep-tag

Biotin aptamer
Streptavidin aptamer

• biotin beads • Reactive labels

many • (Strept)avidin beads • Coated surfaces:
+ magnetic • chips, slides, microplates
beads + fluorescence • The list goes on….
+ other properties

http://www.chembio.uoguelph.ca/educmat/chm730/k730.htm

• The biotin/(strept)avidin system has been employed in so many different types of applications that you
can use it to link together basically any two molecules of interest.
• This slide is supposed to just hit some highlights but there many, many possibilities.
 33
Methods for protein immobilization:
covalent non-polymerizing

E E

E E

E E
E E

E
E

E
E

Many other methods available but they almost always involve a thiol or a primary amine.
Matthew A. Cooper, Nature Reviews Drug Discovery 1, 515 - 528
EDC coupling chemistry: http://chem.ch.huji.ac.il/~eugeniik/edc.htm

• The goal of the covalent non-polymerizing immobilization strategy is to link the protein directly to the
surface using chemistry. Typically this is done through the formation of a amide bonds between amines
and carboxylic acids or thioether bonds with good electrophiles and free thiols (the only good nucleophile
in proteins)
• It is quite common to start with a surface that presents either free amines or free carboxylic acids for
derivatization.
a. To link proteins to a surface that presents carboxylic acid groups, the acid moieties can be made
reactive towards amines using water-soluble EDC/NHS mediated activation. The resultant reactive NHS
ester can then be coupled directly with available amino moieties of a protein (lysine or N-terminus) to form
a stable amide linkage. Further derivatization with sulphydryl-reactive reagents allows reaction with free
surface thiols (cysteine) to form a reversible disulphide linkage. In a similar manner, stable thioether bonds
can be formed using maleimide coupling reagents, such as sulpho-SMCC and GMBS. The surface can
also be derivatized with cystamine to effect coupling with disulphide-activated receptors. Alternatively,
treatment with hydrazine followed by a reductive amination allows coupling with aldheydes. The aldehyde
groups could be formed by mild oxidation of any cis-diols (E would have to be glycosylated, obviously) that
are present.
b. Amino-presenting surfaces can be treated with commercially available bifunctional linking reagents to
effect coupling with free amino or sulphydryl groups on the receptor
 34
Methods for protein immobilization:
covalent cross-linking
Diazonium salt is most
Glutaraldehyde reactive towards Tyr
(most common)

Isothiocyanate is Isocyanate is amine

amine reactive at reactive at room
elevated temperature
temperatures

Mikkelsen, S.R. and Corton, E., Bioanalytical Chemistry (2004)

• Covalent cross-linking is a poorly controlled method so would only be used in cases where it is not
important to retain a high percentage of enzyme activity and where the enzyme is abundant.
• During the cross-linking reaction bonds will form between the enzyme and the stationary phase but also
between multiple copies of the enzyme.
• The enzyme polymerization will generally result in lower enzymatic activity than the more controlled
methods of receptor-mediated immobilization and nonpolymerizing covalent attachment. There are
several reasons for this:
• The polymeric network of enzyme molecules will obscure or covalently modify active sites of the
enzyme.
• The rate of substrate and product diffusion will be decreased.
• The tertiary structure of the enzyme may be disrupted
• Question: to what extent is it necessary that we be able to draw chemical structures and reactions from all of
the sections of this course?
• Answer: The structures of molecules talked about in class are not that important, though the methods of
linking things together is quite important.
 35
Methods for protein immobilization:
simple adsorption

pH < pI pH > pI
++ -
-
+ + -
++

-
- - - + + +
-

-
-
+ +- - + +

-
- - + + + +
-- - ++ +

- -
+ + -
++
+

-
-- ++ +

-
-

-
++

-
-
Cation anion
exchange exchange
resin resin

http://www.lsbu.ac.uk/biology/enztech/immethod.html

• Adsorption of enzymes onto insoluble supports is a very simple method of wide applicability and capable
of high enzyme loading (about one gram per gram of matrix).
• Simply mixing the enzyme with a suitable adsorbent, under appropriate conditions of pH and ionic
strength, followed, after a sufficient incubation period, by washing off loosely bound and unbound enzyme
will produce the immobilized enzyme in a directly usable form. The driving force causing this binding is
usually due to a combination of hydrophobic effects and the formation of several salt bridges per enzyme
molecule.
• Examples of suitable adsorbents are ion-exchange matrices, porous carbon, clays, hydrous metal oxides,
glasses and polymeric aromatic resins.
• Ion-exchange matrices, although more expensive than these other supports, may be used economically
due to the ease with which they may be regenerated when their bound enzyme has come to the end of its
active life; a process which may simply involve washing off the used enzyme with concentrated salt
solutions and re-suspending the ion exchanger in a solution of active enzyme.
 36
Methods for protein immobilization:
entrapment and encapsulation

+ acrylamide + bisacrylamide + K2S2O8

enzyme

polymerization

The simplest form of

encapsulation is
separate the enzyme
from the bulk solution
with a semipermeable
membrane (just like in
dialysis)
cross-linked
polyacrylamide gel http://www.lsbu.ac.uk/biology/enztech/immethod.html

• Entrapment of enzymes within gels or fibres is a convenient method for use in processes involving low
molecular weight substrates and products. Amounts in excess of 1 g of enzyme per gram of gel or fibre
may be entrapped.
• Because large molecules have trouble approaching the catalytic sites of entrapped enzymes it is not
practical to entrap enzymes with high molecular weight substrates.
• The enzyme is trapped by performing a polymerization reaction with acrylamide (CH2=CH-CO-NH2) and
bisacrylamide (H2N-CO-CH=CH-CH=CH-CO-NH2) to form a gel. The polymer forms around the enzyme
and the gel pores are too small for the enzyme to diffuse out. This approach does not covalently modify
the enzyme. We will learn more about the formation of polyacrylamide gels in the section on
electrophoresis.
• Encapsulation of enzymes may be achieved by a number of quite different methods, all of which depend
on the semipermeable nature of the membrane. This must confine the enzyme whilst allowing free
passage for the reaction products and, in most configurations, the substrates.
• The simplest of these methods is achieved by placing the enzyme on one side of the semipermeable
membrane whilst the reactant and product stream is present on the other side.This is basically the same
as dialysis, though done on an analytical scale.
 37
Biosensors
a detection system that relies on a biomolecule for
molecular recognition and a transducer to produce
an observable output

although biosensors are often discussed in generic

terms, there is really only one target molecule that
drives the vast majority of research in this area:
glucose
Image source: http://www.kumetrix.com/biosensor.html
http://cmr.asm.org/content/18/4/583.full.pdf#page=1&view=FitH

• A biosensor is an analytical device that uses the exquisite molecular recognition capabilities of
biomolecules in conjunction with a transducer, such as an electrode, optical device or quartz crystal
microbalance, as the basis for analyte detection in biological systems
• in vivo: in a living organism (contrast with in vitro or ‘in glass’ analysis and ex vivo or ‘outside an
organism’).
• in situ: ‘in place’, i.e. analytes in their natural environment but not necessarily a living organism
(contrast with ex situ or ‘out of place’ analysis).
• Biosensors also offer many advantages in comparison to many conventional analytical approaches in
terms of simplicity, lower limits of detection and sensitivity. The simplicity of many biosensor formats often
allows for their use by untrained personnel such as by patients for home monitoring of, for example,
glucose within blood or urine or alternatively within a doctor's surgery - so negating the need for samples
to be returned to pathology laboratories or other centralized clinical biochemistry laboratory facilities.
• One of the greatest advantages that Biosensors frequently enjoy is their specificity due to their
exploitation of biological molecules such as enzymes or antibodies.
• Biosensors often allow for real time information to be obtained which contrasts strongly with periodic
sampling analytical intervals.
• Analyses via biosensors may frequently be performed without the need for formal training and for this
reason many human sources of error may often be eliminated.
 Blood components of diagnostic significance 38

Insulin

OH
O
glucose HO
HO
OH OH

http://endocrineweb.com/insulin.html
David S. Goodsell: The Molecule of the Month appearing at the PDB

• GLUCOSE: Glucose, formed by the digestion of carbohydrates and the conversion of glycogen by the
liver, is the primary source of energy for most cells. It is regulated by insulin, glucagon, thyroid hormone,
liver enzymes, and adrenal hormones. It is elevated in diabetes, liver disease, obesity, pancreatitis, due to
steroid medications, or during stress. Low levels may be indicative of liver disease, overproduction of
insulin, hypothyroidism, or alcoholism.
• INSULIN: Insulin is one of the most important hormones, carrying messages that describe the amount of
sugar that is available from moment to moment in the blood. Insulin is made in the pancreas and added to
the blood after meals when sugar levels are high. This signal then spreads throughout the body, to the
liver, muscles and fat cells. Insulin tells these organs to take glucose out of the blood and store it, in the
form of glycogen or fat.
• Diabetes Mellitus: When insulin function is impaired, either by damage to the pancreas or by the rigors
of aging, glucose levels in the blood rise dangerously, leading to diabetes mellitus. For people totally
deficient in insulin, such as children that develop diabetes early in life, this can be acutely dangerous.
High glucose levels lead to dehydration, as the body attempts to flush out the excess sugar in urine, and
life-threatening changes in blood pH, as the body turns to other acidic molecules for delivery of energy.
 39
The Clark-type glucose biosensor
The other thing that Leland
Clark is famous for: http://
www.youtube.com/watch?
v=2NmU7VKd3VA

Electrolytic cell

(Glucose + O2 H2O2 + gluconolactone)

(Excludes protein but is O2 permeable) Cathodic process O2 + 2H2O + 4e- 4OH-

Or Anodic process H2O2 O2 +2H+ + 2e-

Is the current generated directly or inversely proportional to the glucose concentration?

From Holme and Peck Analytical Biochemistry 3rd edition p 168
Cunningham, Introduction to bioanalytical sensors, p. 81

• An electrode is a conductor used to make contact with a nonmetallic part of a circuit. An electrode in an
electrochemical cell is referred to as either an anode or a cathode. The anode is defined as the electrode
at which oxidation occurs, and the cathode is defined as the electrode at which reduction occurs. Each
electrode may become either the anode or the cathode depending on the type of reaction occurring in the
cell. In an electrolytic cell (as opposed to voltaic or galvanic), the current which flows between the
electrodes depends not only on the voltage that is applied but also on the electrical properties of the
solution.
• Leland C. Clark had the ingenious idea of placing very close to the surface of the platinum electrode (by
trapping it physically against the electrode with a piece of dialysis membrane) an enzyme that reacted
with oxygen.
• He reasoned that he could follow the activity of the enzyme by following the changes in the oxygen
concentration around it, thus a chemosensor became a biosensor. Based on this experience and
addressing his desire to expand the range of analytes that could be measured in the body, he made a
landmark address in 1962 at a New York Academy of Sciences symposium in which he described how "to
make electrochemical sensors (pH, polarographic, potentiometric or conductometric) more intelligent" by
adding "enzyme transducers as membrane enclosed sandwiches".
• The concept was illustrated by an experiment in which glucose oxidase was entrapped at a Clark oxygen
electrode using dialysis membrane. The decrease in measured oxygen concentration was proportional to
glucose concentration. In the published paper (Clark, L.C. Jnr. Ann. NY Acad. Sci. 102, 29-45, 1962),
Clark and Lyons coined the term enzyme electrode. Clark's ideas became commercial reality in 1975 with
the successful re-launch (first launch 1973) of the Yellow Springs Instrument Company (Ohio) glucose
analyser based on the amperometric detection of hydrogen peroxide. This was the first of many
biosensor-based laboratory analysers to be built by companies around the world.
 40
Yellow Springs glucose
biosensor technology

http://www.ysi.com/extranet/BTKL.nsf/447554deba0f52f2852569f500696b21/0f7ccb69a6ee82cf852569e70047cd17!OpenDocument

• Most fundamentally, the Clark electrode detects hydrogen peroxide H2O2. It consists of platinum metal
coated with glucose oxidase, an enzyme extracted from cows, and a semi-permeable membrane.
Basically, the enzyme breaks down glucose forming hydrogen peroxide as a by-product. As the peroxide
reacts with the metal it produces a current that is proportional to the concentration of glucose in the blood.
The current is then converted into a glucose concentration.
• Yellow Springs Instruments (YSI) continues to make Clark-type glucose biosensors.
• An enzyme specific for the substrate of interest is immobilized between two membrane layers,
polycarbonate and cellulose acetate. The substrate is oxidized as it enters the enzyme layer, producing
hydrogen peroxide, which passes through cellulose acetate to a platinum electrode where the hydrogen
peroxide is oxidized. The resulting current is proportional to the concentration of the substrate.
• YSI membranes contain three layers. The first layer, porous polycarbonate, limits the diffusion of the
substrate into the second layer (enzyme), preventing the reaction from becoming enzyme-limited. The
third layer, cellulose acetate, permits only small molecules, such as hydrogen peroxide, to reach the
electrode, eliminating many electrochemically-active compounds that could interfere with the
measurement.
• H2O2 is oxidized at the platinum anode, producing electrons. The electron flow is proportional to the H2O2
concentration and, therefore, to the concentration of the substrate.
 41
Improved glucose
sensors using an
electron-relay
mediator to help
get electrons from
the FAD to the
surface

Enzyme ‘wiring’

http://chem.ch.huji.ac.il/~eugeniik/
electron_mediators.htm

• The covalent attachment of electron-relay units at the protein periphery as well as inner sites, yields short
inter-relay electron-transfer distances. Electron ‘hopping’ or tunneling between the periphery and the
active site enables electrical communication between the redox enzyme and its environment. The
simplest systems of this kind involve electron relay-functionalized enzymes diffusionally communicating
with electrodes, but more complex assemblies include immobilized enzymes on electrodes as integrated
assemblies.
• One way of establishing an electrical contact between the redox-center of an enzyme and its environment
is to synthetically extend the cofactor such that it reaches out of the enzyme and into the bulk solvent. In
the first step, the FAD-redox centers of glucose oxidase is removed to yield the apo-enzyme.
• The amino-functionalized semisynthetic N6-(2-aminoethyl)-FAD (23) was covalently linked to (6-
ferrocenemethylamino) hexanoic acid (14), then the bifunctional redox-active ferrocene-FAD cofactor (24)
was reconstituted into apo-GOx or apo-AOx.
• As shown in the figure, the ferrocene group acts as an electron-relay that electrically contacts the FAD
center with the electrode surface.
• A simliar approach that uses gold nanoparticles rather than ferrocene units has been reported (Xiao et al.
"Plugging into Enzymes": Nanowiring of Redox Enzymes by a Gold Nanoparticle, Science, Vol 299, Issue
5614, 1877-1881, 21 March 2003)
 42
At home finger sticks and continuous in vivo
glucose monitoring
Accu-chek sensor from Roche Diagnostics Continuous Glucose Monitoring
System from MiniMed Inc

Glucose Biosensors: 40 Years of Advances and Challenges Joseph Wang

http://www.usc.edu/dept/engineering/illumin/vol3issue1/glucose_sensing/index1.html

• How is the glucose concentration of the blood actually found?

• There are many methods, including at-home finger sticks and intravenous catheters used at hospitals.
Almost all of today's glucose sensors incorporate some form of the Clark electrode and the enzyme
glucose oxidase.
• One of the most reliable methods of continuous sensing is the Continuous Glucose Monitoring System
(CGMS) offered by MiniMed Inc.. Unlike conventional sensors that take isolated glucose readings from
the blood, the CGMS continuously detects glucose levels in interstitial fluid (the fluid which surrounds our
cells).
• This system uses a needle to insert a very narrow Clark electrode beneath the skin (usually on the
abdomen) where it encounters interstitial fluid. This electrode is worn for 72 hours before it is replaced
with a new electrode. Readings are acquired every 10 seconds and an average value is saved in a pager-
sized monitor every 5 minutes (see above figure). Every two weeks the data is downloaded onto a
physician's computer where the continuous glucose data can be viewed in graphical form. Their goal is to
"help identify periods of significant glycemic excursion which would allow the physician to suggest specific
changes in the timing and dosage of insulin infusion or injection".
 43
Continuous in vivo monitoring

www.freestylenavigator.com
 44
Long-term in vivo glucose monitoring using
fluorescent hydrogel fibers

Y. J. Heo, H. Shibata, T. Okitsu, T. Kawanishi, S. Takeuchi, Long-term in vivo glucose

monitoring using fluorescent hydrogel fibers. Proc Natl Acad Sci U S A 108, 13399 (2011).

• In this work, the authors inserted a glucose responsive fiber under the skin
• The fluorescence of the fiber reports on the concentration of glucose.
• The fiber contains a ‘glucose sensing’ unit composed of the fluorophore anthracene and two boronic acids
which serve as a binding site for sugars such as glucose
• This sensor likely operates by a photoinduced electron transfer mechanism. In the absence of glucose, an
electron from the lone pair on the nitrogen can be donated to the excited anthracene chromophore and
quench the excited state. In the presence of glucose, the conformation is distorted such that the lone pair
is no longer able to donate electrons to the anthracene, and so the quenching is relieved.
• The authors demonstrated that polyethylene glycol (PEG)-bonded polyacrylamide (PAM) hydrogel fibers
reduced inflammation relative to uncoated fibers
• This can be seen in the images of the mice ears, where the uncoated PAM fibre is red and inflamed after
31 days, but the PEG-coated PAM is not.
• PEG is a polymer with structure: HO-CH2-(CH2-O-CH2-)n-CH2-OH
• A PEG coating is widely used for reducing immune response against foreign biomolecules or objects
placed under the skin
 45
The future? Non-invasive monitoring.

GlucoWatch from
Cygnus.

Some approaches based on

spectroscopy seem to be
supported by substantial
amounts of peer-reviewed
research:
www.inlightsolutions.com
www.sensysmedical.com
www.solianis.com
An area ripe for scams! www.groveinstruments.com
Check out www.calistomedical.com. Vaporware!
(update: On Monday, February 13, 2012, status on the GLUCOBAND trademark changed to
ABANDONED - NO STATEMENT OF USE FILED (www.trademarkia.com)

• Noninvasive approaches for continuous glucose monitoring represent a promising route for obviating the
challenges of implantable devices. However, it is not at all clear to me that this will be possible any time in
the near future.
• One example of a non-invasive device that seemed to work (at least to some degree) is a wearable
glucose monitor, the GlucoWatch from Cygnus Inc., based on the coupling of reverse iontophoretic (?)
collection of glucose and biosensor functions.
• Given the obvious importance of such a device (if it were to work), there is very little reliable information
available online. I suspect that this device simply does not work well enough for widespread use.
• It seems as though the most promising non-invasive technologies involve near infrared spectroscopic
measurements obtained through the skin. Is it possible to measure glucose reliably through the skin? I
doubt it, but there are quite a few companies who seem to think it can work. Such a device will probably
not be wearable.
 46
Enzymes as analytical reagents
• Glucose oxidase catalyses the oxidation of glucose with formation of hydrogen peroxide
• Peroxidase catalyses the reduction of hydrogen peroxide and the simultaneous oxidation of another molecule
• There are chromogenic and fluorogenic substrates for peroxidase.
• For most analytical/diagnostic/industrial purposes, immobilized proteins (enzyme or antibodies) are more useful
than soluble proteins
• There are a large number of methods for immobilizing proteins but they can basically be classified as
• Non-covalent receptor mediated
• Covalent non-polymerizing or cross-linking
• Non-covalent adsorption or entrapment
• As we will see in subsequent lectures, immobilized enzymes are very important in immunoassays and in
biosensors.
• The ‘ultimate’ binding interaction is that between biotin and avidin. The Kd for this interaction is about 10-15
M.This is one of the strongest non-covalent associations known.
• Avidin/Biotin are a central tool of modern biotechnology!!
• Glucose biosensors are the original and still the dominant commercially successful biosensor technology.
• Almost all glucose biosensors rely on the enzyme glucose oxidase to oxidize glucose to gluconolactone.
• This oxidation results in the generation of electrons which can be converted into a measurable current.
• Most of the technological developments in glucose biosensors have focused on two areas
• Efficiently transferring electrons from the FAD cofactor to the electrode surface
• Making the technology easier to employ for people with diabetes (I.e. a trend towards less invasive
measurements)
• We have not discussed other biosensor designs but the principles of the glucose sensor are highly general and
almost all biosensors operate by related mechanisms.