“Enzymes for Biosensors – Important factors in your choice”.
Biosensors are becoming increasingly sophisticated and therefore it is increasingly
important that the enzymes that are the key part of this technology are of the
required quality.
For use in a biosensor the enzyme chosen must fulfil several important
requirements. The first key requirement is that the enzyme is suitably specific to
avoid any false signals. False signals can be caused by a general lack of specificity
of the enzyme itself or by the presence of other contaminating enzymes which can
cause interfering reactions.
In addition it is important that the enzyme is of high Specific Activity i.e. enzyme
activity per unit protein and that it is soluble at high concentrations to enable
sufficient units of activity to be deposited on the biosensor surface. It is also
important to have good batch to batch consistency.
The enzyme must have good stability at high temperature and must also be stable
and active over the required range of pH.
Other factors that should be considered when choosing an enzyme for use in
biosensor technology include the Michaelis–Menten constant (Km) of the enzyme
and its ability to transfer electrons to a range of suitable mediators.
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Specificity
The most common analyte measured using a biosensor is glucose. There are two
common classes of enzyme used in such biosensors these being glucose oxidase
(GO) and glucose dehydrogenase (GDH). These enzymes show some remarkable
differences in specificity for sugars other than glucose.
Glucose oxidase has excellent specificity, showing less than 1% activity on a range
of different sugars compared to its activity on glucose when measured at a
concentration of 30mM substrate. This would seem to make it an ideal enzyme for
use in glucose biosensors. However, in glucose oxidase test strips, oxygen acts as a
competing electron acceptor and the corresponding reaction will vary depending on
the partial pressure of oxygen (pO2) in blood samples. High pO2 is most common in
the critically ill, or in patients receiving oxygen therapy or undergoing surgery. These
patients will show erroneously low glucose levels. Low pO2 is common in neonates
or patients at high altitudes and glucose readings will be anomalously high.
Although GO is still widely used today there has been an increase in glucose
biosensors which use a variety of GDH enzymes as an alternative. However, GDH
enzymes are not as specific as GO. For example, commercially available PQQ-GDH
enzymes exhibit a high level of activity against sugars such as maltose, galactose
and xylose. Maltose is a disaccharide formed from two glucose molecules and is
found in certain immunoglobulin products. Additionally, icodextrin used in peritoneal
dialysis is metabolized to produce maltose. Galactose and xylose are found in
certain foods, herbs, and nutritional supplements, and are also used in diagnostic
tests. Biosensors incorporating PQQ-GDH should not be used in patients who may
have these sugars in their blood as they can lead to dangerously misleading glucose
results.
FAD GDH has a much improved specificity for maltose and galactose compared to
PQQ GDH but still suffers from some interference against xylose which may
preclude its use with patients who are undergoing xylose absorption tests.
Techniques such as site directed mutagenesis are increasingly being used to
attempt to produce novel FAD GDH enzymes with improved specificity to xylose.
Table 1 shows the reaction of GO and various GDH enzymes with various sugars at
30mM. The results are expressed as the % of activity compared to the activity on
glucose
Table 1
Sekisui GO
HPS300
Sekisui FAD
GDH
Competitor
FAD GDH
Competitor
NAD GDH
Competitor
PQQ GDH
L-Glucose
0.00006%
0.09%
0.09%
0.00%
0.00%
Mannitol
0.00005%
0.08%
0.16%
0.00%
0.00%
Sucrose
0.00009%
0.03%
0.12%
0.00%
0.00%
Trehalose
0.00002%
0.16%
0.12%
0.00%
0.00%
Sorbitol
0.0007%
0.06%
0.03%
0.08%
0.20%
Fructose
0.002%
0.12%
0.16%
0.30%
1.37%
Xylose
0.042%
10.76%
16.04%
3.36%
11.24%
Galactose
0.10%
0.67%
0.77%
0.51%
13.41%
Maltose
0.004%
0.02%
0.15%
0.46%
99.07%
Ribose
0.00008%
0.05%
0.12%
0.00%
6.07%
Mannose
0.26%
0.43%
0.79%
1.04%
10.85%
Lactose
0.00005%
0.07%
0.20%
0.31%
61.20%
61.20%
10.85%
99.07%
13.41%
11.24%
16.04%
10%
10.76%
Specificity as measured versus activity against glucose
9%
8%
7%
6%
5%
4%
3%
2%
1%
0%
Xylose
Galactose
Maltose
Ribose
Sekisui GO HPS300
Sekisui FAD GDH
Competitor NAD GDH
Competitor PQQ GDH
Mannose
Lactose
Competitor FAD GDH
Interference from contaminating enzymes
An example of how contaminating enzymes can cause false signals is in the case of
the presence of the enzyme cellulase in GO or GDH. Various forms of cellulose are
commonly included in inks for screen printing of biosensor elements to enhance the
rheological properties of the ink. Fungal enzymes such as GO from Aspergillus niger
are prone to contamination with cellulase. If such enzyme preparations are added to
inks, the cellulose is rapidly degraded, resulting in loss of desired rheological
properties of the ink paste and the potential for false positives due to glucose
release.
Use of a highly sensitive, viscometer based, cellulase assay has enabled Sekisui to
develop a proprietary purification process to manufacture GO products with very low
levels of cellulase.
Figure 1 shows the removal of cellulase throughout the GO HPS300 manufacturing
process as evaluated using this viscometer method. This method has also been
used to show that our other biosensor grade enzymes contain measurable levels of
cellulase contamination which might be significant in generating a false glucose
signal.
Figure 1
cPs Decrease in Viscosity in 24 Hours per 1000 Units
1,000
800
166,667
600
400
566
394
200
176
9
Step 3
Final
Product
0
Starting
material
Step 1
Step 2
Sekisui biosensor grade GO and GDH enzymes are assayed for a wide range of
contaminating activities to ensure that consistently low levels are achieved in every
batch of product.
Purity
As biosensors are becoming increasing miniaturized it is even more important than
usual that the enzyme used has a high Specific Activity and good solubility, if it is
sold as a freeze dried powder. Only a relatively small drop of enzyme can be
deposited on the surface of the biosensor and therefore it is crucial that the enzyme
can be prepared at a high enough concentration to deposit sufficient units of activity.
Batch to batch consistency in terms of both powder activity and Specific Activity are
also important. This ensures that, when changing between different lots of enzyme,
similar amounts of any additives in the powder and similar amounts of protein are
added when depositing the required amount of activity on the strip. This helps to
prevent the need to re-calibrate glucose meters for every new batch of strips.
Stability
Another factor of importance in enzymes for use in biosensors includes stability both
at high temperatures and across a range of pH values. Temperature stability is of
particular importance for application where the enzyme is dried down on the strip at
elevated temperatures and it is also important to ensure that the enzyme activity
remains at the required level throughout the shelf life of the biosensor. Datasheets
are available for Sekisui enzymes showing details of pH and temperature stability
which can aid in choosing a suitable enzyme for your application.
Km and turnover number
With highly purified enzymes the turnover number reflects the number of substrate
molecules converted in unit time by a single enzyme molecule and therefore with the
increasing drive to make biosensors with increasingly rapid response times it is
important to choose enzymes with a high turnover number.
The Michaelis-Mentent constant (Km) for a given enzyme provides an indication of
the binding strength of that enzyme to its substrate. A high Km indicates that the
enzyme binds the substrate weakly. Conversely, a low Km indicates a higher affinity
for the substrate. In general it is good to choose an enzyme with a low Km when it is
to be used in an end point assay and to choose an enzyme with a higher Km when it
is to be used in a kinetic assay.
When choosing an enzyme for use in a biosensor it is important that the Km value is
close to or higher than the top end of the measuring range.