Pullulan encapsulation of labile biomolecules to give stable bioassay tablets

Article abstract of DOI:10.1002/anie.201403222

A simple and inexpensive method is reported for the long-term stabilization of enzymes and other unstable reagents in premeasured quantities in water-soluble tablets (cast, not compressed) made with pullulan, a nonionic polysaccharide that forms an oxygen impermeable solid upon drying. The pullulan tablets dissolve in aqueous solutions in seconds, thereby facilitating the easy execution of bioassays at remote sites with no need for special reagent handling and liquid pipetting. This approach is modular in nature, thus allowing the creation of individual tablets for enzymes and their substrates. Proof-of-principle demonstrations include a Taq polymerase tablet for DNA amplification through PCR and a pesticide assay kit consisting of separate tablets for acetylcholinesterase and its chromogenic substrate, indoxyl acetate, both of which are highly unstable. The encapsulated reagents remain stable at room temperature for months, thus enabling the room-temperature shipping and storage of bioassay components.

Full text of DOI:10.1002/anie.201403222

Angewandte
Chemie
DOI: 10.1002/anie.201403222
Bioassays
Pullulan Encapsulation of Labile Biomolecules to Give Stable Bioassay
Tablets**
Sana Jahanshahi-Anbuhi, Kevin Pennings, Vincent Leung, Meng Liu, Carmen Carrasquilla,
Balamurali Kannan, Yingfu Li, Robert Pelton, John D. Brennan,* and Carlos D. M. Filipe*
Abstract: A simple and inexpensive method is reported for the
long-term stabilization of enzymes and other unstable reagents
in premeasured quantities in water-soluble tablets (cast, not
compressed) made with pullulan, a nonionic polysaccharide
that forms an oxygen impermeable solid upon drying. The
pullulan tablets dissolve in aqueous solutions in seconds,
thereby facilitating the easy execution of bioassays at remote
sites with no need for special reagent handling and liquid
pipetting. This approach is modular in nature, thus allowing
the creation of individual tablets for enzymes and their
substrates. Proof-of-principle demonstrations include a Taq
polymerase tablet for DNA amplification through PCR and
a pesticide assay kit consisting of separate tablets for acetyl-
cholinesterase and its chromogenic substrate, indoxyl acetate,
both of which are highly unstable. The encapsulated reagents
remain stable at room temperature for months, thus enabling
the room-temperature shipping and storage of bioassay
components.
thawing can result in significant loss of activity, which often
leads to less reliable test results. These problems make
running such assays in resource-limited settings a significant
challenge.
One approach to address this problem is to place the assay
reagents into a tablet or capsule, which both provides
premeasured quantities of reagents and allows the addition
of preservatives that can prolong the shelf life of the reagents.
Such an approach is used in several commercial assay kits that
use nonbiological reagents but has thus far not been extended
to biological agents like enzymes, which are often the key
[3]
components of bioassays. This is in part due to a lack of
a suitable material that can meet the following three
conditions: 1) it allows the encapsulation of biomolecules in
a form suitable for shipping; 2) it provides outstanding
protection for entrapped biomolecules against thermal dena-
turation and chemical modification during shipping and
[
4]
storage; and 3) it is readily soluble in aqueous solution,
allows the release of the encapsulated molecules, and does not
[
5]
A
lmost all bioassays make use of bioreagents (such as
interfere with the assay itself. In this work we report on the
use of pullulan to create bioassay tablets (cast, not com-
pressed) that meet these three conditions.
enzymes and small-molecule substrates) that are labile to
various degrees and require special shipping and storage. The
instability of these molecules can arise from either thermal
Pullulan is a natural polysaccharide produced by the
[
1]
[6]
denaturation or chemical modification, such as oxidation or
fungus Aureobasidium pullulans. It readily dissolves in
[
2]
[6a,b,7]
hydrolysis. Because of these issues, they often have to be
shipped on dry ice with special packaging, which is costly.
These reagents also have to be stored in bulk in refrigerators
or freezers to minimize loss of activity, but they must be
retrieved, thawed, and aliquoted for intended tests that are
often performed at room temperature. Repeated freezing and
water but resolidifies into films upon drying.
The film-
forming property of pullulan has been utilized in some unique
applications in the pharmaceutical and food industries, such
[
6b,8]
as breath fresheners and food additives.
Recent studies
have found that pullulan coatings applied to food packaging
can act as oxygen barriers to prolong the shelf life of various
[
7,9]
foods. In addition, pullulan has been shown to preserve the
[
8]
[
*] S. Jahanshahi-Anbuhi, K. Pennings, V. Leung, Dr. M. Liu,
C. Carrasquilla, Dr. B. Kannan, Prof. Dr. Y. Li, Prof. Dr. R. Pelton,
Prof. Dr. J. D. Brennan, Prof. Dr. C. D. M. Filipe
Departments of Chemical Engineering, Chemistry & Chemical
Biology, and Biochemistry and Biomedical Sciences
McMaster University
viability of bacteria under various storage conditions.
Given the above findings, we hypothesized that pullulan
might be suitable for producing assay tablets with encapsu-
lated enzymes or other labile molecules, and more impor-
tantly, that these tablets may not only allow the long-term
storage of unstable molecules at room temperature but may
also provide a simplified platform for carrying out bioassays
in resource-limited settings. Herein, we demonstrate the use
of pullulan to create tablets to facilitate two different
enzymatic reactions: a single-tablet system for DNA amplifi-
cation through the polymerase chain reaction (PCR) with Taq
DNA polymerase (TaqDP); and a two-tablet system for
pesticide detection, where one tablet contains acetylcholines-
terase (AChE) and the other contains indoxyl acetate (IDA,
a chromogenic substrate for AChE).
1280 Main Street West, Hamilton, ON, L8S 4M1 (Canada)
E-mail: brennanj@mcmaster.ca
filipec@mcmaster.ca
[
**] We thank the Natural Sciences and Engineering Research Council of
Canada and Sentinel Bioactive Paper Network for funding. The
authors also thank the Canada Foundation for Innovation and the
Ontario Innovation Trust for support of this work. R.P. holds the
Canada Research Chair in Interfacial Technologies. J.D.B. holds the
Canada Research Chair in Bioanalytical Chemistry and Biointerfa-
ces. We would also like to thank Dr. Clemence Sicard for her useful
consultation. We also acknowledge the McMaster Biointerfaces
Institute (BI) where part of the experiments was performed.
Our first case study involves using AChE and IDA for the
detection of malathion, a widely used organophosphate
pesticide that has been implicated in causing reduced neuro-
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.201403222.
Angew. Chem. Int. Ed. 2014, 53, 1 – 6
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Angewandte
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logical function, eventually leading to Alzheimerꢀs, ADHD,
followed by the addition of an IDA tablet (Figure 1c). If
malathion is present, the sample remains colorless or turns
faint blue (dependent on the concentration of malathion, as
discussed below). In the absence of malathion, IDA is fully
hydrolyzed by AChE and the test sample turns deep blue
(Figure 1c). Experimental details are provided in the Sup-
porting Information.
[
10]
reduced IQ, or even death.
IDA can be hydrolyzed by
AChE into hydroxyindole and acetic acid: in the presence of
oxygen, hydroxyindole spontaneously changes into blue-
[11]
colored indigo (Figure 1a). Malathion is known to be an
[12]
inhibitor of AChE and the AChE–IDA combination has
been shown to be an excellent system for the colorimetric
[
13]
detection of malathion. However, the main challenge for
The tablet system can not only be used achieve qualitative
colorimetric detection of malathion by eye, it can also provide
quantitative analysis of the pesticide concentration in a test
sample when using a smartphone and image-processing
[
13b,c,17]
software (such as ImageJ).
Figure 2 shows a plot of
the dose-dependent inhibition of AChE by malathion, with
data obtained using a smartphone. This simple method can be
used to detect malathion at levels as low as 64 nm (S/N = 3).
Figure 1. Tablet assay kit for malathion. a) Hydrolysis of IDA by AChE.
b) A photograph of the AChE and IDA pullulan tablets. c) Operational
principle of the assay kit.
Figure 2. Dose-dependent inhibition of AChE at varying concentrations
of malathion.
[
14]
this assay is the instability of both AChE and IDA. When
stored at room temperature, AChE loses its enzymatic
activity and IDA becomes oxidized within a few hours.
We next evaluated the long-term stability of both the
AChE and IDA tablets. As shown in Figure 3, AChE stored in
solution at room temperature became completely inactive
within 3 days. Similarly, IDA in solution at room temperature
lost 70% of its activity within one day and become completely
inactive within a week. In sharp contrast, both AChE and
IDA in tablet form remained fully active for at least 2 months
when stored at room temperature. In the case of IDA, the loss
[
14b,15]
After being left in air, unprotected IDA turns slightly pink as
a result of indirubin formation. There are two competing
oxidative reactions involved in the hydrolysis of IDA. One
results in the formation of the indigo dimer (the desired
product of the assay), while the other produces isatin, which
[16]
then dimerizes with indoxyl to give indirubin (pinkish).
[
16]
Therefore, fresh solutions must be prepared prior to each test,
thus making this test unsuitable for on-site applications.
Given these issues, the AChE–IDA system offers an excellent
case study for examining the utility of pullulan tablets.
We produced individual AChE–pullulan tablets and IDA–
pullulan tablets (Figure 1b) by using a simple process that
involves 1) the mixing of a pullulan solution with either an
AChE or IDA solution, 2) the casting of each mixture into
a polypropylene mold with small wells (3 mm in diameterꢁ
in performance was related to oxidation. Our data suggests
that pullulan acts as a strong barrier to oxygen, an effect that
[7,9]
is consistent with previous findings.
In theory, an antiox-
idant could be used to prevent the oxidation of IDA during
storage at room temperature. However, the antioxidant
would also inhibit the formation of indigo during the assay.
The loss of AChE activity, however, is attributed to
thermal denaturation. To further examine the role of pullulan
in stabilizing AChE, we monitored the activity of AChE as
a function of temperature. For this experiment, native
(unencapsulated) AChE and the corresponding pullulan
tablet were treated at a given temperature for 30 min,
followed by activity assessment at room temperature.
Figure 4 shows that the free AChE became completely
3
mm in depth), and 3) air-drying. Note that defined concen-
trations of AChE and IDAwere chosen to achieve a maximum
rate of color formation (see Figure S1 in the Supporting
Information). To conduct the assay, an AChE tablet is added
to the sample to allow preincubation with the pesticide,
2
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Chemie
Figure 5. TaqDP activity loss during room-temperature storage with
and without pullulan encapsulation.
noteworthy that when combined with portable PCR systems,
TaqDP–pullulan tablets should facilitate the on-site detection
of DNA.
Figure 3. Evaluation of the long-term stability of AChE and IDA tablets
stored at room temperature. Normalized activity of encapsulated AChE
in reaction with fresh IDA, encapsulated IDA in reaction with fresh
AChE, unencapsulated (native) AChE in reaction with fresh substrate,
and unencapsulated IDA in reaction with fresh AChE.
In summary, we demonstrate that pullulan is an excellent
material for producing water-soluble bioassay tablets con-
taining labile enzymes and small molecules. Most signifi-
cantly, the pullulan tablets provide exceptional protection for
the entrapped reagents against thermal denaturation or
chemical modification, thereby allowing room-temperature
storage for extended periods of time. Equally important, the
tablet strategy provides a general platform for carrying out
bioassays with minimal steps and user intervention, which is
ideal for resource-limited regions, particularly in the devel-
oping world.
Received: March 11, 2014
Published online: && &&, &&&&
Keywords: carbohydrates · encapsulation · enzymes · pullulan ·
.
stabilization
Figure 4. Loss of AChE activity as a function of temperature. Both
encapsulated AChE and native enzyme were assessed.
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Chemie
Communications
Bioassays
Locked and loaded: Pullulan encapsula-
tion enables the room-temperature ship-
ping and long-term storage of unstable
biomolecules (such as enzymes and their
substrates). The pullulan tablets dissolve
quickly in aqueous solution to release
defined premeasured amounts of the
reagent. The reagent stabilization and
ease of use afforded by this method
would be particularly advantageous for
bioassays intended for use in resource-
limited settings.
S. Jahanshahi-Anbuhi, K. Pennings,
V. Leung, M. Liu, C. Carrasquilla,
B. Kannan, Y. Li, R. Pelton, J. D. Brennan,*
C. D. M. Filipe*
&&&& — &&&&
Pullulan Encapsulation of Labile
Biomolecules to Give Stable Bioassay
Tablets
Bioassays
Bei Raumtemperatur verschicken und
lagern lassen sich instabile Biomolekꢀle
(wie Enzyme und ihre Substrate) nach
Verkapselung mit Pullulan. Die Pullulan-
Tabletten lçsen sich in wꢁssriger Lçsung
rasch auf und setzen definiert zugesetzte
Mengen des Reagens frei. Die Reagens-
stabilisierung und die einfache Hand-
habung, die diese Methode bietet, sollten
besonders vorteilhaft fꢀr Bioassays sein,
die in Situationen mit begrenzten Res-
sourcen genutzt werden.
S. Jahanshahi-Anbuhi, K. Pennings,
V. Leung, M. Liu, C. Carrasquilla,
B. Kannan, Y. Li, R. Pelton, J. D. Brennan,*
C. D. M. Filipe*
&&&& — &&&&
Pullulan Encapsulation of Labile
Biomolecules to Give Stable Bioassay
Tablets
Angew. Chem. Int. Ed. 2014, 53, 1 – 6
ꢀ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.angewandte.org
5
These are not the final page numbers!

.
Angewandte
Communications
Communications
Bioassays
Locked and loaded: Pullulan encapsula-
tion enables the room-temperature ship-
ping and long-term storage of unstable
biomolecules (such as enzymes and their
substrates). The pullulan tablets dissolve
quickly in aqueous solution to release
defined premeasured amounts of the
reagent. The reagent stabilization and
ease of use afforded by this method
would be particularly advantageous for
bioassays intended for use in resource-
limited settings.
S. Jahanshahi-Anbuhi, K. Pennings,
V. Leung, M. Liu, C. Carrasquilla,
B. Kannan, Y. Li, R. Pelton, J. D. Brennan,*
C. D. M. Filipe*
&&&& — &&&&
Pullulan Encapsulation of Labile
Biomolecules to Give Stable Bioassay
Tablets
6
www.angewandte.org
ꢀ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Angew. Chem. Int. Ed. 2014, 53, 1 – 6
These are not the final page numbers!