Development of a lipase-based optical assay for detection of DNA

Article abstract of DOI:10.1039/c0ob01165g

A lipase-based assay for detection of specific DNA sequences has been developed. Lipase from Candida antarctica was conjugated to DNA and captured on magnetic beads in a sandwich assay, in which the binding was dependent on the presence of a specific target DNA. For amplification and to generate a detectable readout the captured lipase was applied to an optical assay that takes advantage of the enzymatic activity of lipase. The assay applies p-nitrophenol octanoate (NPO) as the substrate and in the presence of lipase the ester is hydrolyzed to p-nitrophenolate which has a strong absorbance at 405 nm. The method provides detection a detection limit of 200 fmol target DNA and it was able to distinguish single base mismatches from the fully complementary target.

Full text of DOI:10.1039/c0ob01165g

View Article Online / Journal Homepage / Table of Contents for this issue
Organic &
Biomolecular
Chemistry
Dynamic Article Links
Cite this: Org. Biomol. Chem., 2011, 9, 6352
www.rsc.org/obc
PAPER
Development of a lipase-based optical assay for detection of DNA†
Suttiporn Pinijsuwan,a,b Stepan Shipovskov,c Werasak Surareungchai,b Elena E. Ferapontovaa,c and
Kurt V. Gothelf*a
Received 13th December 2010, Accepted 3rd June 2011
DOI: 10.1039/c0ob01165g
A lipase-based assay for detection of specific DNA sequences has been developed. Lipase from Candida
antarctica was conjugated to DNA and captured on magnetic beads in a sandwich assay, in which the
binding was dependent on the presence of a specific target DNA. For amplification and to generate a
detectable readout the captured lipase was applied to an optical assay that takes advantage of the
enzymatic activity of lipase. The assay applies p-nitrophenol octanoate (NPO) as the substrate and in
the presence of lipase the ester is hydrolyzed to p-nitrophenolate which has a strong absorbance at 405
nm. The method provides detection a detection limit of 200 fmol target DNA and it was able to
distinguish single base mismatches from the fully complementary target.
Specific and reliable assays for DNA detection are becoming
increasingly important for applications in clinical diagnosis, food
safety and environments. This has spurred an intensive search
for new sensitive, selective, simple and rapid methods. During
the past decade, different approaches to DNA detection using
various readout systems have been discussed. These methods
have mainly included optical/colorimetric techniques,1–6 surface
plasmon resonance,7,8 microcantilever,9,10 and electrochemical
detection.11–14
Enzymes have successfully been used for colorimetric detection
of analytes providing both recognition, and amplification of
the binding event with a detectable readout. In DNA anal-
ysis, enzyme-labelling is widely used for the development, of
e.g. electrochemical15,16 and optical17 assays. Commonly used
enzymes include horseradish peroxidase,18 glucose oxidase,19
galactosidase,20 alkaline phosphatase.16 They are usually coupled
to target or reporter DNA strand by avidin–biotin binding
technology or chemical coupling.19
lipase was applied as the enzymatic label in an assay used for the
electrochemical detection of DNA.23
Here we have developed a different lipase-based assay for
detection of DNA providing an optical readout (Fig. 1 and 2).
For the capture of lipase on magnetic beads (MBs)24–27 in the
presence of target DNA, we applied a magnetic bead (MB)-DNA-
lipase sandwich assay familiar with the assay described above. For
signal amplification and optical readout we have employed the
lipase induced cleavage of p-nitrophenyl esters (NP esters). The
combination of this lipase NP ester assay with the MB-DNA-
lipase capture sandwich assay has enabled us to develop a new
and versatile detection method for DNA.
p-Nitrophenyl octanoate (NPO), is an ester which is hydrolyzed
by the lipase enzyme to form octanoate and p-nitrophenolate
(pKaH = 7.08) (Fig. 1a). The yellow colour of the p-nitrophenolate
formed in the reaction allows for the detection of the lipase activity.
The target DNA is detected in a sandwich assay where it hybridizes
with a DNA strand on magnetic beads and to a DNA strand
conjugated to lipase. Application of the MB-DNA-lipase to a
NPO solution results in enzymatic hydrolysis of the ester. The rate
of hydrolysis can be directly correlated with the amount of lipase
captured on the beads and thereby also with the amount of target
DNA present in the assay.
The esterase activities of lipase can be assayed by UV-Vis
spectroscopy by monitoring the absorption of p-nitrophenolate
(NP) which is produced during the enzymatic ester bond hy-
drolysis in the NP esters. The catalytic activity of lipases in-
creases with increasing hydrophobicity and in particular with
the chain length of the alcohol moiety.28,29 However, the solu-
bility of the substrate decreases with increasing hydrophobicity.
Thus, to avoid turbidity and maintain a high lipase activity, p-
nitrophenyl octanoate (NPO) was chosen as the substrate. The
hydrolysis reaction of NPO was first investigated at different
concentrations of NPO to reveal a linear correlation between
the NPO concentration and the absorbance (See ESI, Fig. S1†).
Lipases are important enzymes that catalyze the hydrolysis
and formation of a wide variety of esters. They are pivotal in
nature and have industrial applications in dairy, fat and oil, food,
pharmaceutical, cosmetic, detergents, and textile industries, and
in the manufacture of fine chemicals synthesis and polymeric
materials.21,22 Because of their great application and economic
importance, numerous lipases are available. In one prior example
aCenter for DNA Nanotechnology Department of Chemistry and iNANO,
Aarhus University, Langelandsgade 140, DK-8000, Aarhus C, Denmark.
E-mail: kvg@chem.au.dk; Fax: +45 86196199; Tel: +4589423907
bSchool of Bioresources and Technology, and Biological Engineering Pro-
gram, King Mongkut’s University of Technology Thonburi, Bangkhuntien
Campus, Bangkhuntien-Chaitalay Rd, Bangkok, 10150, Thailand
cInterdisciplinary Nanoscience Center (iNANO), Aarhus University, Ny
Munkegade 1521, DK-8000, Aarhus C, Denmark
† Electronic supplementary information (ESI) available: Fig. S1 and S2.
See DOI: 10.1039/c0ob01165g
6352 | Org. Biomol. Chem., 2011, 9, 6352–6356
This journal is
The Royal Society of Chemistry 2011
©

View Article Online
Fig. 1 Capture of the target DNA by the MB-DNA-lipase sandwich hybridization.
sequence was linked to the MBs via the streptavidin biotin linking.
Then the target DNA in the selected concentration was added
to the MB-capture probe conjugate. This was followed by the
addition of a biotin-labelled reporter DNA sequence to form the
DNA sandwich. The capture and reporter sequences each form
an 18 bp duplex with the 42 nt target. The hybridization assay
is designed with a 6 nt single stranded gap in the middle of the
sandwich to increase the flexibility of the system and to avoid
stacking between the two duplexes, since this would decrease the
single point mismatch selectivity of the assay.
Fig. 2 Lipase-catalysed generation of p-nitrophenylate absorbing at
405 nm.
After removal of the sandwich-MBs from the excess of reporter
probes streptavidin was added. After another round of purification
the biotin labelled lipase was added to the sandwich and a final
separation step was performed. During these addition/separation
cycles all additives except the target strand were in excess and thus
it is the quantity of the target DNA that determines the amount of
lipase linked to the magnetic beads. Several addition/separation
steps were required, but since the interaction relies on the very
efficient and high yielding DNA hybridization and streptavidin–
biotin interaction high efficiency and specificity are expected.
Furthermore, the separation procedures are easily performed by
removal of the solution from the magnetic beads.
We have investigated the relation between the incubation time
of the MB-lipase and the absorption at 405 nm. For these initial
experiments the target DNA was fixed at 2 pmol. As shown in Fig.
3, absorption was measured as a function of time for every hour
from 1 to 26 h.
The absorption increased gradually during the incubation time.
At the beginning the activities processed quite fast reaching ~0.9
after 12 h and it is not necessarily needed to extend the detection
time further. In our experiments, 4 h was chosen as the optimized
incubation time.
We have also investigated the response of the assay to different
amounts of target DNA as shown in Fig. 4. Target DNA amounts
were varied between 2 fmol to 200 pmol and the background
absorption obtained in the absence of target DNA was subtracted.
A correlation coefficient of 44.6 mmol-1 (or 8.9 mM-1) was
derived.
Next we have investigated the activity of different derivatives
of the lipase Candida antarctica using NPO as the substrate.
The concentrations of lipase of the MB were calculated based
on the binding capacity of the beads. The assay was followed
by incubating the MB-lipase enzyme in the NPO solution. The
relation between the varying concentrations of lipase and the
absorption at 405 nm is shown in Fig. S2.† However, comparison
of the activities of free lipase from Candida antarctica and lipase
immobilized on MB was not straightforward. As described by
Pencreac’h, the lipases used are enzymatically prepared and
contain other protein components.30 To overcome this problem
we have compared the activities of lipase conjugated on MBs
to lipase conjugated to DNA. It was performed and measured
by using the NPO assay as shown in Fig. S2.† The absorbance
resulting from of MB-lipase hydrolysis was 1.3 times larger than
the absorbance from the lipase-biotin hydrolysis of NPO after 4 h.
It is assumed, based on previous work that this difference is caused
by enhancement of the surface area-to-volume ratio for the MB
immobilized lipase.23
Hybridization experiments were carried out in the sandwich
type format (Fig. 1). The commercially available MBs are coated
with streptavidin and an excess of a biotin-labelled capture
This journal is
The Royal Society of Chemistry 2011
Org. Biomol. Chem., 2011, 9, 6352–6356 | 6353
©

View Article Online
Fig. 3 Time dependency of the ester digestion after incubation with
DNA-MB assembled in the presence of 2 pmol target DNA.
Fig. 5 Histogram shows peak absorbance using 100 pmol complementary
target, 100 pmol single base mismatch at MB side, 100 pmol single base
mismatch at lipase side, 100 pmol of mismatches at both sides, after
subtraction of the background absorption obtained without target DNA.
mismatch provided significantly lower signals. Surprisingly, the
double mismatch target resulted in a signal of similar magnitude.
Conclusion
A lipase-based colorimetric assay for detection of DNA was
developed. The target DNA was captured on MBs in a sandwich
assay that also binds a reporter DNA strand, linked to lipase from
Candida antarctica. Application of magnetic beads enables fast
and efficient removal of nonspecific reagents after the washing
steps.26 The binding of target DNA in the sandwich is amplified
by the enzymatic hydrolysis of p-nitrophenol octanoate (NPO)
by the lipase. Hydrolysis of NPO leads to the formation of p-
nitrophenolate which has a strong absorption at 405 nm to provide
a colorimetric readout. The method allowed detection of down to
200 fmol (or ~0.97 nM) of target DNA in the 205 mL detection
solution. The method is also highly selective and could distinguish
between fully matched target DNA and target DNA containing
single point mutations. This is the first example of the employment
of lipase for optical detection of DNA. Lipases are readily available
and furthermore no “fuel” is required for the enzymatic hydrolysis
to take place, unlike the commonly used redox enzymes. Future
development of the lipase-based assay may enable fast and direct
visual detection of low concentrations DNA.
Fig. 4 Calibration of target DNA captured by the MB sandwich assay
versus the background subtracted absorption in the absence of target DNA;
inset: 0.02–200 pmol range shown with linearized data.
At low amounts of target DNA of 2–20 fmol no significant increase
in the absorbance was observed, however, at 200 fmol a clear
increase of the absorbance was observed. A hyperbolic parabolid
curve was used to fit the data (Fig. 4). This hyperbolic graph
was depicted in a linear fit in the inset of Fig. 4. Increasing the
target DNA from 200 fmol to 200 pmol showed increases in the
absorbance that indicated a relationship between the amount of
target DNA and the absorption.
One of the crucial aspects of DNA assays is their ability to
detect single point mutations in DNA and therefore the specificity
of our technique was examined. The response from 100 pmol
of fully complementary target was compared with the following:
(1) 100 pmol of complementary target DNA, (2) 100 pmol of a
target with a single base mismatch in the part of the sequence
which is complementary to the capture DNA attached to the
MBs, (3) 100 pmol of a target with a single base mismatch at
the part complementary to the reported (lipase bound) sequence,
and (4) 100 pmol of a target sequence with mismatches at
both sides. As shown in Fig. 5, the fully complementary target
provides a response that is larger than any of the sequences
containing mismatches. The sequences containing a single point
Experimental
Materials
Lipase from Candida antarctica, BiotinTagTM Micro Biotinylation
Kit, Streptavidin from Streptomyces avidinii and p-nitrophenyl
octanoate were supplied by Sigma–Aldrich. Streptavidin-coated
TM
R
magnetic beads Dynabeadsꢀ MyOne
Streptavidin T1 was
purchased from Invitrogen, USA.
An oligonucleotide capture probe with a biotin-modified 5¢-
position, target oligonucleotide and 3¢-position biotin-modified
reporter probe were synthesized by DNA Technology A S-1,
Risskov, Denmark. The DNA strands had the following sequence:
6354 | Org. Biomol. Chem., 2011, 9, 6352–6356
This journal is
The Royal Society of Chemistry 2011
©

View Article Online
DNA capture probe: 5¢-biotin-(CH2)6-TTT TTT TTT TAA
GTC GAA CGA GCT TCC-3¢
DNA target: 5¢-AAC TCA CCA GTT CGC CAC TGA CGT
GGA AGC TCG TTC GAC TTA-3¢
DNA reporter probe: 5¢-GTG GCG AAC TGG TGA GTT
TTT TTT TTT (TEG)-biotin-3¢
(TEG = triethyleneglycol)
DNA mismatch at MB side: AAC TCA CCA GTT CGC CAC
TGA CGT GGA AGC TCC TTC GAC TTA
DNA mismatch at lipase side: AAC TCA CCA CTT CGC CAC
TGA CGT GGA AGC TCG TTC GAC TTA
DNA mismatch at both sides: AAC TCA CCA CTT CGC CAC
TGA CGT GGA AGC TCC TTC GAC TTA
All the chemicals were purchased from Sigma–Aldrich and
prepared with 18.2 MX Millipore water (MilliQ) throughout the
work.
Tween 20 by using vertex system, decanted with a magnet and
the MBs resuspended in PBS solution. Then, 4-fold excess of
capture DNA probe was added into the MB solution and it was
incubated for 40 min. The MB modified capture probe was washed
3 times in PBS, decanted and re-suspended in PBS containing
MgCl2 (10 mM). The MB modified capture probe was divided to
4 Eppendorf tubes. Each of the samples was mixed with different
concentrations of target DNA from 0.1 to 100 pmol. The solution
was incubated for 60 min, vortexing the sample for 2 min every
15 min.. Then the MBs were washed 3 times with PBS and isolated
with a magnet and re-suspended in PBS. Next, it was reacted with
0.6 nmoles of the reporter probe to each MB-capture-target DNA
complex. It was incubated for 60 min vortexing the sample for
2 min every 15 min. Then it was washed 3 times with PBS and
the MBs isolated using a magnet and the beads were re-suspended
in PBS. The modified MB was mixed with a streptavidin solution
to conjugate with the lipase-biotin conjugate for 45 min (2 min
vortexing every 15 min) and the MBs were washed 3 times with
PBS and isolated using a magnet. Finally, a 4-fold excess of the
lipase-biotin conjugate was added to the MB modified DNA. It
was washed 3 times with PBS, decanted with a magnet and re-
suspended in PBS.
Apparatus
UV-visible spectra were recorded using a Bio-TEK Instruments
model mQuant spectrophotometer for wells plate. Micro well plate
(clear color/sterile) was from NUNCTM (NunclonTM D Surface).
Preparation of the lipase-biotin conjugate
Detection of lipase activity using p-nitrophenyl octanoate, NPO
Conjugation of biotin to lipase was performed by using
BiotinTagTM Micro Biotinylation Kit. Briefly, lipase from Candida
antarctica was prepared in 20 mM phosphate buffer, pH 7.0
containing 0.15 M NaCl (PBS). The solution was activated
with the labelling reagent using freshly prepared solution of bi-
otinamidohexanoic acid 3-sulfo-N-hydroxysuccinide ester (BAC-
SulfoNHS). It was incubated with gentle stirring for 40 min at
room temperature, followed by a fast gel-filtration using G-50
micro-spin columns to separate the conjugate from unreacted
or hydrolyzed reagent. The lipase-biotin conjugate was used as
labeling for magnetic beads (MB) and DNA hybridization.
The assay is based on monitoring the hydrolysis of the ester bond
in NPO to form 4-nitrophenlate (pNA), which in contrast to the
ester has a strong maximal absorbance at 405 nm.
The initial stock solution was made in DMSO with a concen-
tration of 100 mM of NPO and then it was diluted to 0.1 mM
NPO by 20 mM phosphate buffer containing 0.15 M NaCl (PBS),
and 0.005% Triton X-100 for substrate solubilization.
The reaction mixture (in a 96 well plate) consisted of 200 ml
of NPO solution and 5 ml of lipase or lipase-biotin conjugates of
known concentrations. In order to measure the p-nitrophenolate
released from the substrate, a control was prepared by adding PBS
into the NPO solution.
Preparation of the MB-lipase conjugate
MB-lipase and MB-DNA conjugates were prepared using the
same procedure as for the lipase/lipase-biotin conjugates but
they were incubated in Eppendorf tubes The activated solution
was then decanted using a magnet to retain the MBs and the
solutions were loaded into 96 wells plate. Finally, the absorbance
of the solution was monitored at 405 nm on a mQuant microplate
spectrophotometer.
For this procedure we used 1 mm diameter streptavidin-coated
MBs from Dynabeadsꢀ MyOneTM Streptavidin T1. First, 100 mL
R
of MB (10 mg mL-1) was prepared in Eppendorf tube. The tube
was placed on a strong magnet for 2 min and the supernatant was
removed by aspiration with a pipette. Then, the tube was removed
from the magnet, the MBs were washed with PBS containing 0.1%
Tween 20 and collected by the magnet 3 times and then they were
resuspended in PBS buffer. The biotinylated lipase was added to
the washed MB, with the binding capacity of 400 pmol of biotin-
conjugates per 1 mg of beads, and incubated for 30 min with
gentle stirring at room temperature. The mixture tube was placed
on string magnet, and the supernatant discharged. The MBs were
washed 3 times with PBS and resuspended in PBS buffer.
Acknowledgements
S.P. gratefully acknowledges a PhD Scholarship from the Royal
Golden Jubilee project of the Thailand Research Fund and the
Thailand Commission on Higher Education Fellowship. This
project was supported by The Danish National Research by
support to Center for DNA Nanotechnology, Aarhus University,
Denmark.
Preparation of MB-DNA conjugate
The DNA assembly was performed using streptavidin coated
MBs, a biotinylated capture DNA probe, target DNA, and a
biotinylated reporter probe which can conjugate lipase-biotin with
a streptavidin linker. First 100 mL of streptavidin coated MBs
(10 mg mL-1) were washed 3 times with PBS containing 0.1%
Notes and references
1 Y. C. Cao, R. Jin, C. S. Thaxton and C. A. Mirkin, Talanta, 2005, 67,
449.
2 S. Diercks, L. K. Medlin and K. Metfies, Harmful Algae, 2008, 7, 137.
3 V. S. Fluxa´, D. Wahler and J.-L. Reymond, Nat. Protoc., 2008, 3, 1270.
This journal is
The Royal Society of Chemistry 2011
Org. Biomol. Chem., 2011, 9, 6352–6356 | 6355
©

View Article Online
4 R. Fu, T. Li and H. G. Park, Chem. Commun., 2009, 5838.
5 S. Kumar, K. Kikon, A. Upadhyay, S. S. Kanwar and R. Gupta, Protein
Expression Purif., 2005, 41, 38.
6 B. Li, Y. Du, T. Li and S. Dong, Anal. Chim. Acta, 2009, 651, 234.
7 J. B. Mandir, M. R. Lockett, M. F. Phillips, H. T. Allawi, V. I. Lyamichev
and L. M. Smith, Anal. Chem., 2009, 81, 8949.
18 G. Liu, Y. Wan, V. Gau, J. Zhang, L. Wang, S. Song and C. Fan, J. Am.
Chem. Soc., 2008, 130, 6820.
19 J. Hajdukiewicz, S. Boland, P. Kavanagh and D. Leech, Biosens.
Bioelectron., 2010, 25, 1037.
20 Z. Li, R. B. Hayman and D. R. Walt, J. Am. Chem. Soc., 2008, 130,
12622.
8 J. Pollet, F. Delport, K. P. F. Janssen, K. Jans, G. Maes, H. Pfeiffer, M.
Wevers and J. Lammertyn, Biosens. Bioelectron., 2009, 25, 864.
9 K. M. Hansen and T. Thundat, Methods, 2005, 37, 57.
10 J. Mertens, C. Rogero, M. Calleja, D. Ramos, J. A. Mart´ın-Gago, C.
Briones and J. Tamayo, Nat. Nanotechnol., 2008, 3, 301.
11 K. J. Odenthal and J. J. Gooding, Analyst, 2007, 132, 603.
12 E. E. Ferapontova, Curr. Anal. Chem., 2011, 7, 51.
13 E. Farjami, L. Clima, K. V. Gothelf and E. E. Ferapontova, Anal.
Chem., 2011, 83, 1594.
21 R. D. Schmid and R. Verger, Angew. Chem., Int. Ed., 1998, 37,
1608.
22 S. Shipovskov, Biotechnol. Prog., 2008, 24, 1262.
23 E. E. Ferapontova, M. N. Hansen, A. M. Saunders, S. Shipovskov, D.
S. Sutherland and K. V. Gothelf, Chem. Commun., 2010, 46, 1836.
24 G. Aragay, J. Pons, J. Ros and A. Merkoci, Langmuir, 2010, 26, 10165.
25 F. Berti, S. Laschi, I. Palchetti, J. S. Rossier, F. Reymond, M. Mascini
and G. Marrazza, Talanta, 2009, 77, 971.
26 O. A. Loaiza, S. Campuzano, M. Pedrero, M. I. Pividori, P. Garcia and
J. M. Pingarron, Anal. Chem., 2008, 80, 8239.
14 J. Zhang, R. Lao, S. Song, Z. Yan and C Fan, Anal. Chem., 2008, 80,
9029.
27 J. Wang, G. D. Liu and A. Merkoci, J. Am. Chem. Soc., 2003, 125,
15 X. Mao, J. Jiang, X. Xu, X. Chu, Y. Luo, G. Shen and R. Yu, Biosens.
3214.
Bioelectron., 2008, 23, 1555.
28 P. Helisto and T. Korpela, Enzyme Microb. Technol., 1998, 23, 113.
29 T. Vorderwiibecke, K. Kieslich and H. Erdmann, Enzyme Microb.
Technol., 1992, 14, 631.
16 X. Zhang, L. Li, L. Li, J. Chen, G. Zou, Z. Si and W. Jin, Anal. Chem.,
2009, 81, 1826.
17 J. Li, S. Song, X. Liu, L. Wang, D. Pan, Q. Huang, Y. Zhao and C. Fan,
Adv. Mater., 2008, 20, 497.
30 G. Pencreac’h, M. Leullier and J. C. Baratti, Biotechnol. Bioeng., 1997,
56, 181.
6356 | Org. Biomol. Chem., 2011, 9, 6352–6356
This journal is
The Royal Society of Chemistry 2011
©