Indian Journal of Chemistry
Vol. 47A, December 2008, pp. 1804-1808
Notes
Surface plasmon resonance based biosensor
for label free detection of cholesterol
Rakhee Gehlota, Kavita Sharmaa, Manoth Mathewb &
Sunita Kumbhata, *
a
Biosensor Laboratory, Department of Chemistry,
J.N.V. University, Jodhpur 342 033, India
b
Material Development Group, Defense Laboratory,
Jodhpur 342 011, India
Email: [email protected]
Received 2 July 2008; revised 12 November 2008
A surface plasmon resonance based biosensor has been
proposed for a simple, label-free, highly selective and sensitive
detection of cholesterol employing the flavoenzyme, cholesterol
oxidase, as the sensing element. The immobilization of cholesterol
oxidase conjugated with bovine serum albumin on to the gold
sensor chip has been accomplished by covalent binding through
an activated self-assembled 11-mercaptoundecanoic acid
monolayers. The surface morphology of the biosensor has been
recorded using atomic force microscopy. Sensitivity of
1.41mo/mg dl-1 of cholesterol has been achieved using the
biosensor. The biosensor is highly specific for cholesterol and
shows no significant interference from potent interferents such as
ascorbic acid and uric acid.
IPC Code: Int. Cl.8 A61B5/145; G01N33/92; H01L31/14
Sensitive and selective detection of cholesterol is
significant owing to its importance in the human
physiological system. Apart from being an essential
structural constituent of biological membranes, tissues
and intracellular organelles, cholesterol is a precursor
to various steroid hormones. Although the negative
feedback regulatory system in mammals1 keeps the
biosynthesis of cholesterol and fatty acids within the
strict limits of optimum concentration, changing
lifestyle has led to elevated levels of blood cholesterol
contributing to cardiovascular diseases, cerebral
thrombosis and arteriosclerosis2.
The enzymatic analyses as well as the majority of
biosensors reported till now exploit the catalytic
activity of cholesterol oxidase3 wherein the electrons
released in oxidase catalyzed biochemical oxidation
of cholesterol are taken up by the molecular oxygen
to produce stoichiometrically equivalent amount
of hydrogen peroxide (H2O2). The H2O2 produced
is used up to oxidize a chromophore, like,
4-aminopyrine, o-diansidine, 4-aminophenazone, with
the help of an additional bio-reagent, horse radish
peroxidase (HRP). The colour intensity of oxidized
chromophore is proportional (indirectly) to the
concentration of substrate. Alternatively, the resultant
H2O2 is allowed to take part in an electrochemical
reaction in presence4,5 or absence6,7 of a charge
transfer mediator at a chemically and/or
biochemically modified sensor/electrode surface. The
resulting current is proportional (indirectly) to the
amount of cholesterol. Arya et al.8 have reviewed
recent advances in cholesterol biosensor, wherein a
number of elegant strategies have been reported for
chemically modified electrode/sensor surface using
conducting polymers, nanomaterial, sol-gel, hydrogels and self-assembled monolayers with mediators
such as ferrocene derivatives, ferrocyanide, thionin,
pyroquinoline quinine, etc., to improve the selectivity
of the biosensors. However, the desired practicability
in achieving the selectivity for cholesterol biosensor is
yet to be achieved.
Surface plasmon resonance (SPR) based optical
transducers detects the changes in the refractive index
with the change in the mass concentration close to a
metal surface in real time without labeling9-12. These
transducers are now well established for monitoring
enzyme catalyzed transformation for clinically
important analyte, viz., cholesterol13,14 and
glucose15,16,
antibody-antigen based immunoreactions for haemoglobin17 and insulin18, and
receptor-ligand interactions for dopamine19,20. The
binding between immobilized biological recognition
element and the analyte brings changes in the
refractive index at the sensor surface, leading to the
change in SPR angle, which can be monitored in real
time. The magnitude of the change in SPR signal is
directly proportional to the mass bound to the surface,
even at nanogram levels, in complex biological
samples.
We have established a cholesterol oxidase-bovine
serum albumin (ChOx-BSA) conjugate based sensing
for a SPR based biosensor for sensitive and selective
detection of cholesterol.
Experimental
The SPR instrument, (model Springle, EcoChemie, Autolab Instruments, Netherlands) equipped
NOTES
with an open cuvette system (20-100 µl of sample
volume) and functioning based on Kretchmann
configuration was used. Gold coated BK7 type
microscopic glass plates (25 mm dia.) supplied by
Eco-Chemie, Autolab Instruments, Netherlands, were
used as SPR sensor chips. At each sensor chip four
sites were made available to study molecular
interaction by changing the position of the sensor
chip. All experiments were carried out at 25°C. All
injections were of 50 µl and after completion of every
injection, the flow was switched back to carrier
buffer, phosphate buffer saline automatically
throughout the course of the experiment. A scanning
probe microscope, model NT-MDT Solver PRO,
(NT-MDT, Russia) with a silicon probe operated in
tapping mode was used for topographic imaging
of conjugate immobilized on to the self-assembled
11-MUA monolayer modified gold coated glass chips.
Cholesterol oxidase microbial, ChOx (C1235100UN, recombinant expressed in E. coli, 3.13 mg
solid, 160 units/mg protein, 55 kDa) was reconstituted
in 1000 µl of phosphate buffer saline (PBS), pH 7.
Bovine Serum albumin (A2153-50 g, 66 kDa),
N-hydroxysuccinimide
(NHS),
N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide
hydrochloride
(EDC), ethanolamine, and 11-mercaptoundecanoic
acid (11-MUA) were sourced from Sigma-Aldrich,
St. Louis, USA. Other chemicals were of analytical
grade from Merck, India. A stock solution of bovine
serum albumin (BSA, 2 gm/dl) was prepared in
phosphate buffer saline (PBS), pH 7 (0.01M disodium
hydrogen phosphate and 0.01M potassium dihydrogen
phosphate containing 0.8% NaCl and 0.02% KCl in
deionised water). Standard solution of cholesterol
(200 mg/dl) from Ecoline cholesterol kit (E. Merck)
was used and further dilutions were made with PBS.
To get the ChOx–BSA conjugate, the stock
solution of cholesterol oxidase was further diluted
with PBS to get an enzyme solution of activity
100 units/mg. Different dilutions/proportions of ChOx
and BSA were mixed and allowed to incubate for
15 minutes at room temperature and their binding at
SAM modified SPR gold chip was monitored
by observing the change in SPR angle. The optimum
composition used in the present work was 1:3 v/v
cholesterol oxidase (10 units /ml): BSA (20 mg/ml).
Fabrication of biosensor and cholesterol assay
The SPR gold sensor chip cleaned by
ultrasonication in ethanol was immersed overnight in
1805
1 mM 11-MUA)/ethanol to allow self-assembled
monolayer (SAM) formation. The SAM modified
gold sensor disc was attached to the prism of the SPR
instrument using a matching liquid (refractive index =
1.515). After setting up the self-assembled 11-MUA
monolayer modified gold chip on to the SPR
instrument, a brief cleaning-cum-stabilization step
with a buffer of low salt concentration (0.01M sodium
acetate) was initiated for ~5 minutes. The next step
was in situ activation of SAM by immobilizing NHSEDC reagent (1:1 aqueous solution of 100 mM NHS
and 400 mM EDC) followed by immobilization of
ChOx-BSA conjugate. An optimum interaction time
for in situ activation of SAM and for immobilization
of ChOx-BSA conjugate over activated SAM was
found to be 5 min and 10 min respectively. The
unbound reactive ester groups were deactivated by a
brief flow of 1 M aqueous ethanolamine solution
(pH 8) over the sensor surface to prevent non-specific
binding. Once the sensor surface was enriched with
covalently anchored ChOx-BSA, the analyte
cholesterol was injected over the sensor surface. The
enzyme-substrate interaction between immobilized
ChOx and cholesterol (association phase) was studied
for different time periods. An optimum interaction
time of ~15 min was found to be satisfactory to get a
stable SPR signal for successful interaction and
binding of reaction product on to the sensor. Every
association phase was followed by a brief flow of
carrier buffer for ~120 s buffer to wash out
unreacted/loosely bound species (dissociation phase).
The effective rise at the end of dissociation phase with
respect to resonance angle at initiation of interactive
phase is correlated with the analyte concentration.
In the regeneration step, the surface bound enzymatic
reaction product of analyte is liberated, leaving
the sensor surface, active for the next injection.
The ChOx-BSA conjugate immobilized assay format
used in present study could be reused for multiple
analyses by flowing PBS buffer for prolonged
period.
Results and discussion
To avoid non-specific adsorption/binding of
extraneous material from the sample, a suitable
modification of sensor surface under carefully
controlled experimental conditions is a prerequisite,
especially for biomedical applications. In the
present work, a self-assembled monolayer of
11-mercaptoundecanoic acid acts as matrix for
immobilization flavoenzyme, cholesterol oxidase as
1806
INDIAN J CHEM, SEC A, DECEMBER 2008
the sensing element. The carboxyl groups of SAM
(-S(CH2)10COOH) extending out from sensor
surface is activated by EDC-NHS mixture20
and by incorporating N-hydroxy succinimide
(-S(CH)10COON(CO)2C2H4). This facilitates covalent
binding for immobilization of the sensing element.
The preliminary trials to immobilize ChOx onto a
activated sensor surface showed very poor binding.
Therefore, an attempt was made during present the
study to synthesize ChOx-BSA conjugate, similar to
enzymes being associated with serum proteins. The
immobilsation of ChOx-BSA conjugate on to the
EDC/NHS activated SAM of 11-MUA, through
amine group of BSA and carboxyl group of activated
SAM forms a stable amide bond, (-S(CH)10CONHR,
where R= ChOx-BSA conjugate) on the gold sensor
surface. Even with very efficient immobilization of
the recognition element, some reactive esters remain
unbound and needs to be deactivated to avoid nonspecific binding. Ethanolamine was used to block the
unbound reactive ester at the sensor surface
(-S(CH)10CONHC2H4O), which initially raised the
SPR angle due to change in refractive index at the
surface. However base line could be maintained by
restoring the flow of carrier buffer.
Figure 1 depicts the schematic representation of
the immobilization of ChOx-BSA conjugate over
EDC-NHS activated self-assembled 11-mercaptoundecanoic acid monolayer coated gold sensor
surface. The surface morphology of ChOx-BSA
conjugate immobilized SPR gold surface was studied
by atomic force microscopy and is shown in Fig. 2.
The AFM image indicates a homogenous, compact
and dense film of the conjugate. Thus, bovine serum
albumin acts as a carrier protein to the enzyme
cholesterol oxidase which anchors very well to the
sensor surface.
Gold shows strong affinity for sulphur and
therefore substituted thiols form SAM over the bare
gold sensor chip. In the present study, self-assembled
11-MUA layer coated SPR gold sensor chip reveals
an average rise of 197 m° (millidegree) in the
resonance angle (fig. not shown) as compared to the
Fig. 1—Schematic representation of fabrication of cholesterol
biosensor.
bare gold chip, signifying a good quality and stable
matrix of SAM over gold surface. Figure 3 shows the
sensogram recorded for the immobilisation of ChOxBSA conjugate, where position1 represents the base
line position of SAM coated gold sensor chip. SPR
response of in situ activation of SAM by EDC-NHS
mixture is seen at position 2 (Fig. 3), followed by a
brief wash with PBS (position 3, Fig. 3) to remove
unbound species from the sensor surface. The
immobilization of ChOx-BSA conjugate onto the
activated self assembled 11-MUA layer immobilized
gold surface is evidenced by a sharp rise in the
resonance angle (position 4, Fig. 3) due to appreciable
rise in refractive index of the sensor surface.
Moreover, even after a brief desorption of the loosely
bound conjugate with PBS washing (position 5,
Fig. 3), followed by blocking of the unbound reactive
esters by ethanolamine solution (position 8, Fig. 3) to
avoid non-specific adsorption, a net rise of 264 m°
indicates that the gold surface is covered with
covalently bound ChOx-BSA conjugate and is stable
on the self assembled 11-MOU layer modified gold
sensor surface.
Estimation of cholesterol
The SPR response for the specific enzymaticsubstrate interaction at different concentrations of
cholesterol was recorded in absence of cholesterol and
also in presence of varying concentrations of
cholesterol. An initial sharp rise of resonance angle
Fig. 2—AFM image of ChOx-BSA conjugate immobilized on to
the EDC-NHS-activated self-assembled 11-MUA mono layer
coated gold chip.
NOTES
1807
examined. The SPR response corresponding to the
oxidative binding interactions of ChOx-BSA
conjugate with 50 µl injections of 100 mg/dl each of
AA, UA and cholesterol was recorded. The AA and
UA injections did not bring about any change in SPR
angle, indicating absence of any interaction of
immobilsed enzyme ChOx with AA and UA, which
subsequently get washed away with the carrier buffer
and thus bringing no rise in the SPR angle. However,
cholesterol injection brought a net rise of 118 m°.
This is understandable since flavin-adeninedinucleotide (FAD) containing flavoenzyme ChOx,
catalyses the dehydrogenation of hydroxyl group at
C3 position of sterol structure in cholesterol.
Fig. 3—Immobilization of cholesterol oxidase-BSA conjugate on
self-assembled 11-MUA layer coated gold sensor chip. [Position
1, SAM coated gold chip; Position 2, injection of 1:1 EDC-NHS
mixture; Positions 3, 5, 7, PBS flow; Position 4, injection of
ChOx-BSA conjugate; Position 6, 2nd injection of ChOx-BSA
conjugate, and, Position 8, injection of EA.].
shows a fast interaction and thereafter the steady state
suggests a stable binding of cholestenone (the enzyme
catalyzed oxidation product of cholesterol) by
physical adsorption at the sensor surface. The rise in
resonance angle increases with an increase in the
concentration of free cholesterol. The linear
regression equation for standard cholesterol solution
in the range 10-200 mg/dl was y = 1.38±0.13x
+6.45±7.5 (n=5, p=0.002, R=0.98523) and the
sensitivity of 1.41mo/mg dl-1 for cholesterol was
achieved using Au/11-MAU/EDC-NHS/ChOx-BSA
biosensor.
The pattern of SPR signal recorded with the
proposed biosensor reveals a close similarity to the
earlier reported biosensor employing 1-fluoro-2-nitro4-azidobenzene modified self-assembled monolayer
of polyhexylthiophene13 and that of octadecanethiol14
involving photochemical reaction to produce highly
reactive nitrene group for immobilization of ChOx to
the SPR sensor chip. Data show that the present
approach is simpler and faster as compared to earlier
reported sensor for estimation of cholesterol.
The biggest challenge of cholesterol biosensor is
the specificity of detection in its active biochemical
constitution. In order to evaluate the specificity of the
present biosensor, the effect of potential interferents
like ascorbic acid (AA) and uric acid (UA) was
Acknowledgement
Financial support by Department of Biotechnology,
Government of India, New Delhi (Research Project
No.BT/PR/6290/Med/14/772/2005/20-9-06)
and
advisory support of Dr. Viond Joshi, Desert Medicine
Research Centre (ICMR), Jodhpur, is great fully
acknowledged.
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