Detection of alkaline phosphatase using surface-enhanced raman spectroscopy

Article abstract of DOI:10.1021/ac0522106

A new approach was developed to detect the activity of alkaline phosphatase (ALP) enzyme at ultralow concentrations using a surface-enhanced Raman scattering (SERS) technique. The approach is based on the use of gold nanoparticles as a SERS material whereas 5-bromo-4-chloro-3-indolyl phosphate (BCIP) is used as a substrate of ALP. The enzymatic hydrolysis of BCIP led to the formation of indigo dye derivatives, which were found to be highly SERS active. For the first time, we were able to detect ALP at a concentration of ～4 × 10^-15 M or at single-molecule levels when ALP was incubated with BCIP for 1 h in the Tris-HCl buffer. The same technique also was successfully employed to detect surface-immobilized avidin, and a detection limit of 10 ng/mL was achieved. This new technique allows the detection of both free and labeled ALP as a Raman probe in enzyme immunoassays, immunoblotting, and DNA hybridization assays at ultralow concentrations.

Full text of DOI:10.1021/ac0522106

Anal. Chem. 2006, 78, 3379-3384
Detection of Alkaline Phosphatase Using
Surface-Enhanced Raman Spectroscopy
†
‡
,‡
Chuanmin Ruan, Wei Wang, and Baohua Gu*
Oak Ridge Institute for Science and Education, Oak Ridge, Tennessee 37831, and Environmental Sciences Division,
Oak Ridge National Laboratory, Oak Ridge, Tennessee 37831
A new approach was developed to detect the activity of
alkaline phosphatase (ALP) enzyme at ultralow concentra-
tions using a surface-enhanced Raman scattering (SERS)
technique. The approach is based on the use of gold
nanoparticles as a SERS material whereas 5-bromo-4-
chloro-3-indolyl phosphate (BCIP) is used as a substrate
of ALP. The enzymatic hydrolysis of BCIP led to the
formation of indigo dye derivatives, which were found to
be highly SERS active. For the first time, we were able to
technique is also used to quantify levels of ALP present in sample
solutions or on various substrate surfaces.
Raman spectroscopy has been an invaluable technique in
studies of various chemical and biological systems and has
become widely accepted as an analytical characterization meth-
odology.12 Raman spectroscopy can give narrow characteristic
bands and the detailed fingerprint of the target molecule in a
mixed sample without tedious separation steps. Furthermore,
Raman spectra can be collected directly in aqueous solution
because of a weak Raman scattering from water molecules. On
the other hand, normal Raman spectroscopy is also known to be
insensitive because of its inherently small Raman scattering cross
sections exhibited by most molecules.13 In recent years, however,
surface-enhanced Raman scattering (SERS) has overcome the
weakness of insensitivity for normal Raman spectroscopy and
become one of the most sensitive techniques capable of detecting
single molecules or single nanoparticles.14 For example, SERS has
been used extensively in studies of signal transduction mecha-
-
15
detect ALP at a concentration of ∼4 × 10
M or at
single-molecule levels when ALP was incubated with
BCIP for 1 h in the Tris-HCl buffer. The same technique
also was successfully employed to detect surface-im-
mobilized avidin, and a detection limit of 10 ng/mL was
achieved. This new technique allows the detection of both
free and labeled ALP as a Raman probe in enzyme
immunoassays, immunoblotting, and DNA hybridization
assays at ultralow concentrations.
1
5-18
nisms in biological and chemical sensing applications.
One
Alkaline phosphatase (ALP) is one of the most commonly used
biomarkers in enzyme immunoassays, gene assays, histochemical
staining, and related affinity sensing methods for monitoring
proteins, nucleic acids, drugs, enzymes, and other analytes.1 ALP
is also an enzyme found in human serum and assayed in routine
clinical analysis, because ALP is an indicator of hepatobiliary and
of these applications takes advantage of the binding properties of
antibody and antigen molecules (or DNA strands) and uses metal
nanoparticles coated with Raman-active chromophores as tags to
-5
19-23
detect antigen molecules or genes.
SERS also has been used
2
4
to detect the activity of hydrolases at ultralow concentrations.
(9) Fenoll, J.; Jourquin, G.; Kauffmann, J. M. Talanta 2002, 56, 1021-1026.
6
bone disorder. Therefore, needs exist to detect ALP sensitively
(10) Kim, H. J.; Kwak, J. J. Electroanal. Chem. 2005, 577, 243-248.
(
(
11) Ruan, C. M.; Li, Y. Talanta 2001, 54, 1095-1103.
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38A-346A.
and selectively in many diagnostic and clinical assays. Current
monitoring techniques for ALP commonly use an enzymatic
substrate of ALP, and upon reactions with ALP, products of the
3
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9932-9939.
7
substrate or its derivatives give strong chemiluminescent, colo-
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10,11
rimetric, fluorometric, or electrochemical signals.
The same
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*
To whom correspondence should be addressed. Phone: (865)-574-7286.
Ziegler, L. D. J. Phys. Chem. B 2005, 109, 312-320.
E-mail: gub1@ornl.gov.
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4357-4360.
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†
Oak Ridge Institute for Science and Education.
Oak Ridge National Laboratory.
‡
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1) Ito, S.; Yamazaki, S. I.; Kano, K.; Ikeda, T. Anal. Chim. Acta 2000, 424,
7-63.
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Chem. 1996, 42 (9), 1542-1546.
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1
0.1021/ac0522106 CCC: $33.50 © 2006 American Chemical Society
Analytical Chemistry, Vol. 78, No. 10, May 15, 2006 3379
Published on Web 04/08/2006

a color enhancer, has been widely exploited to locate enzymatic
activity on cell walls, tissues, membranes, and chromatographic
gels because it gives the intense color in the vicinity of the
enzyme.2
7-30
In comparison with these conventional assay tech-
niques, SERS enables the detection of BCIP dimer or ALP activity
directly at ultralow concentrations. Furthermore, a system based
on the biological binding reaction of avidin/ALP-biotin is
demonstrated for detecting immobilized biomolecules on the glass
surface in a way similar to those of membrane-based immunob-
lotting protocols.
EXPERIMENTAL SECTION
Chemicals and Biochemicals. Alkaline phosphatase (5.8
mg/mL) from bovine intestinal mucosa was purchased from Sigma
(No. P5521) in a solution containing 3.2 M (NH
4 2 4
) SO , 1 mM
MgCl , and 0.1 mM ZnCl . Gold(III) chloride trihydrate, BCIP,
2
2
trisodium citrate (98%), sodium borohydride (98%), (3-aminopro-
pyl)triethoxysilane (99%), and other reagent chemicals and
solvents were purchased from Aldrich (St. Louis, MO). Sodium
phosphate dibasic (GR) and sodium phosphate monohydrate (GR)
were bought from EM Science (Cherry Hill, NJ). Bovine serum
albumin, glutaric dialdehyde (25%), tris(hydroxymethyl)ami-
nomethane, and hydrogen peroxide (30%) were purchased from
J. T. Baker (Phillipsburg, N. J.). Alkaline phosphatase-conjugated
biotin (1 mg) and unconjugated avidin (10 mg) were purchased
from Rockland Immunochemicals (Gilbertsville, PA).
Solutions and Buffers. Tris-HCl (0.05 M, pH 9.8) buffer was
prepared by dissolving tris(hydroxymethyl)aminomethane in
deionized water. Phosphate buffer (PBS; 0.01 M, pH 7.2, 0.15 M
NaCl) was prepared by dissolving an equal amount of sodium
phosphate monohydrate and sodium phosphate dibasic in deion-
ized water. The pH values of buffer solutions were adjusted using
dilute NaOH or HCl.
Figure 1. (a) Structure of 5-bromo-4-chloro-3-indolyl phosphate and
its enzymatic reaction products, BCIP dimer; (b) corresponding SERS
spectra of BCIP (substrate) at 0.3 mg/mL and its reaction products.
-
10
ALP concentration 10
M, reaction time 1 h. Laser 785 nm, ∼1.5
mW. Acquisition time 10 s.
Preparation of Au Colloids. Colloidal Au nanoparticles were
3
1,32
prepared according to previously published methods
with
Similar techniques have been used in enzyme immunoassays or
in the detection of an antigen-antibody complex by SERS without
using chromophore-tagged secondary antibodies.25,26 However,
despite these technological advances, relatively few studies have
examined the use of SERS in immunoassays and DNA hybridiza-
tion assays, perhaps because of the requirements of high detection
sensitivity and reproducibility. Few studies have achieved a
sensitivity of better than 10 M for Raman tags using the SERS
technique. As such, detections by fluorescence, chemilumines-
cence, and electrochemical and UV-visible absorption signals are
among the most widely used diagnostic techniques in enzyme-
linked immunosorbent assays based on enzyme amplification
reactions.
minor modifications. Briefly, all glassware used in the following
procedures was cleaned in a bath of freshly prepared 3:1 HCl
(36.5%)/HNO
Au “seed colloids” suspension was prepared by mixing 1 mL of
1% aqueous HAuCl ‚3H O with 100 mL of H O under vigorous
stirring, and at 1 min apart, 1 mL of 1% trisodium citrate and 1
mL of 0.075% NaBH in 1% trisodium citrate were added. The
3
(69%) and rinsed thoroughly in water prior to use.
4
2
2
4
-
14
reaction was allowed to continue for an additional 5 min to
complete, and the solution (with Au seed colloids) was stored at
4 °C until use. Au nanoparticles of ∼54-nm diameter were then
4 2 2
prepared by refluxing 4 mL of 1% HAuCl ‚3H O in 900 mL of H O,
followed by the addition of 1 mL of the above “seed colloid”
(25) Drachev, V. P.; Nashine, V. C.; Thoreson, M. D.; Ben-Amotz, D.; Davisson,
In this work, we report the development of a SERS-based assay
technique that allows rapid and ultrasensitive analysis of ALP at
V. J.; Shalaev, V. M. Langmuir 2005, 21, 8368-8373.
(26) Dou, X.; Yamguchi, Y.; Yamamoto, H.; Doi, S.; Ozaki, Y. J. Raman Spectrosc.
-
15
1998, 29, 739.
concentrations as low as ∼4 × 10 M using gold nanoparticles
(
27) Luo, H. S.; Cui, S.; Chen, D. W.; Liu, J. L.; Liu, Z. X. J. Histochem. Cytochem.
004, 52, 787-803.
(28) Ruan, C. M.; Yang, L. J.; Li, Y. B. Anal. Chem. 2002, 74, 4814-4820.
as a SERS substrate. For the first time, we demonstrate that
2
5
-bromo-4-chloro-3-indolyl phosphate (BCIP), a substrate of ALP,
(
(
29) Ebersole, R. C.; Ward, M. D. J. Am. Chem. Soc. 1988, 110, 8623-8628.
30) Ruan, C. M.; Zeng, K. F.; Varghese, O. K.; Grimes, C. A. Anal. Chem. 2003,
becomes highly SERS active upon exposure to active alkaline
phosphatase. Exposure of BCIP to ALP induces the hydrolysis of
the phosphate moiety of BCIP and generates an enol intermediate
compound, which is subsequently oxidized in air to insoluble blue
BCIP dimers (Figure 1a). The BCIP dimer is a member of the
class of indigo dyes, and its coupling with nitro blue tetrazolium,
7
5, 6494-6498.
(31) Olson, L. G.; Lo, Y.-S.; Beebe, T. P., Jr.; Harris, J. M. Anal. Chem. 2001,
3, 4268.
7
(
32) Grabar, K. C.; Allison, K. J.; Baker, B. E.; Bright, R. M.; Brown, K. R.;
Freeman, R. G.; Fox, A. P.; Keating, C. D.; Musick, M. D.; Natan, M. J.
Langmuir 1996, 12, 2353.
3380 Analytical Chemistry, Vol. 78, No. 10, May 15, 2006

solution and 4 mL of a 1% trisodium citrate solution. The mixture
was refluxed for 10 min at boiling temperature and allowed to
cool with continuous stirring.31 The synthesized Au colloids were
concentrated by centrifugation at 17 000 rcf for 10 min, and the
volume was reduced to ∼2 mL prior to use. The size and size
distribution of Au nanocolloids were determined by means of
dynamic light scattering using a ZetaPlus particle-size analyzer
Table 1. Assignments of Selected Raman Vibrational
Bands for Enzyme Reaction Product (BCIP Dimer)
vibrational
-1
band assignmenta
frequency, cm
1
1
620
576
ν (CdC), δ (C-H)
ν (C)C), ν (CdO)
δ (N-H), δ (C-H)
δ (C-C)
1336
1
1
1
285
225
158
(Brookhaven Instruments Corp., Holtsville, NY). Data were
δ (C-H)
collected at room temperature for every batch of Au colloids.
δ (C-C), ν (C-C) ring
γ (C-H)
-
5
ALP Enzyme Assays. Stock solution of ALP (4.1 × 10 M)
1087
9
7
6
45
78
99
γ (C-H), δ (C-C) ring
δ (C-H), δ (C-N-C)
δ (C-C)
was diluted with Tris-HCl buffer solution (pH 9.8) to give
-
10
-15
concentrations ranging from 4.1 × 10
to 4.1 × 10
M.
Solutions of 0.05 M MgCl (100 µL) and BCIP (100 µL at 3 mg/
2
600
δ (CdC-CO-C)
mL) were subsequently added into 800 µL of ALP solutions,
respectively. The mixture was incubated at room temperature for
a
Key: ν, stretching; δ, in-plane rocking or scissoring; γ, out-of-plane
wagging or twisting.
1
h, followed by the addition of 50 µL of 1 M H
3 4
PO solution to
each vial to stop enzymatic reaction and to adjust the pH of the
-
3
solution. Finally, 100 µL of concentrated Au colloids (∼2.5 × 10
assays, 10 µL of above solutions with varying concentrations of
ALP was transferred onto the glass slides, and samples were dried
in the air for 30 min before SERS analysis.
M) was added, and 10 µL of each resulting solution was
transferred onto glass slides for SERS measurements.
ALP as a Biomarker for Avidin Assays. Small glass tubes
(
8 mm diameter × 7.5 mm long) were first thoroughly washed
33
RESULTS AND DISCUSSION
following procedures reported by Zheng et al. The washed tubes
were silanized by immersing them into 100 mL of methanol
solution containing 1% (3-aminopropyl)triethoxysilane and 0.1 mL
of 0.01 M HCl for overnight at room temperature. They were then
immersed into 25% glutaric dialdehyde solution for 1 h to obtain
aldehyde surface functionalized tubes, which were necessary to
assay avidin sorbed on glass surfaces. Avidin was diluted with
PBS buffer to concentrations ranging from 100 µg/mL to 1 ng/
mL, and deionized water was used as a control. One milliliter of
each avidin solution was then added into aldehyde functionalized
tubes and allowed to react for 2 h at room temperature.
Subsequently, 1 mL of 3% bovine albumin was added into each
tube to react for an additional 2 h to block free aldehyde functional
groups on glass surfaces, and the tubes were washed thoroughly
with water to remove excess albumin. To assay adsorbed avidin
on glass surfaces at varying concentrations, 100 µL of ALP-
conjugated biotin solution (1:1000 dilutions) was added into each
tube and allowed to react with adsorbed avidin on glass for 1 h.
Excess amounts of ALP-conjugated biotin were washed off
thoroughly with water. Finally, a Tris-HCl buffer solution (pH 9.8)
SERS Spectra of Substrate and Enzymatic Reaction
Products. SERS spectra of both BCIP and its enzymatic reaction
product, dimer of BCIP, were collected first in order to examine
the feasibility and potential interferences in detecting ALP using
BCIP as a substrate (Figure 1b). Results indicate that BCIP
substrate itself was not SERS active or gave no discernible SERS
signal except a broad peak at ∼1400 cm , which is assigned to
the background scattering from glass slides. This feature ensured
that the BCIP substrate itself was unable to complex with Au
nanoparticle surfaces and thus made it “invisible” to SERS. Such
a low background signal is essential for the development of an
assay technique for sensitive detection of ALP.
-
1
On the other hand, BCIP dimers, the enzymatic reaction
products, generated an intense SERS spectrum with the vibrational
fingerprint of the molecule clearly observable. The strong vibra-
tional bands of enzymatic reaction product adsorbed onto Au
colloids were observed at 600, 699, 778, 945, 1087, 1158, 1225,
-
1
1
285, 1336, 1529, 1576, and 1619 cm (Figure 1b). This SERS
spectrum of BCIP dimers on Au colloids is similar to the Raman
spectra of indigo dyes reported previously, except for the
2
containing 0.3 mg/mL BCIP and 0.005 M MgCl was added to
each tube and allowed to react with ALP for 30 min. The reaction
products were assayed similarly as described earlier.
-
1 34,35
enhanced Raman shift at 600 cm
.
It confirms that indigo dye
derivatives were formed during the enzymatic reaction. The
assignments of selected Raman bands for BCIP dimers are listed
Surface-Enhanced Raman Scattering Measurements. Ra-
man spectra were obtained through a Renishaw micro-Raman
system equipped with a 300-mW near-infrared diode laser at a
wavelength of 785 nm for excitation (Renishaw Inc., New Mills,
U.K.). The laser beam was set in position through a Leica Imaging
Microscope objective (50×) at a lateral spatial resolution of ∼2
µm on the sample. A charge-coupled device array detector was
used to achieve signal detection from a 1200 grooves/mm grating
light path controlled by Renishaw WiRE software and analyzed
by Galactic GRAMS software. An intensity of <1% of the laser
power was used at the exit of the 50× objective. For enzyme
in Table 1.3
4,36-38
These assignments of Raman shifts were
referenced from Raman spectra of indigo dye derivatives in the
literature because no SERS spectra of BCIP dimers are available.
-
1
The strongest Raman shift at 600 cm was assigned to the
CdCsCOsC bending vibration.34 The fact that this band was
significantly enhanced in the presence of Au colloids may be
(
34) Karapanayiotis, T.; Villar, S. E. J.; Bowen, R. D.; Edwards, H. G. M. Analyst
004, 129, 613-618.
(35) Chiavari, G.; Fabbri, D.; Prati, S.; Zoppi, A. Chromatographia 2005, 61, 403-
08.
36) Shadi, I. T.; Chowdhry, B. Z.; Snowden, M. J.; Withnall, R. Chem. Commun.
004, 12, 1436-1447.
2
4
(
2
(
33) Zheng, J. W.; Zhu, Z. H.; Chen, H. F. Liu, Z. F. Langmuir 2000, 16, 4409-
412.
(37) Min, E. S.; Nam, S. I.; Lee, M. S. Bull. Chem. Soc. Jpn. 2002, 75, 677-680.
(38) Brysbaert, A.; Vandenabeele, P. J. Raman Spectrosc. 2004, 35, 686-693.
4
Analytical Chemistry, Vol. 78, No. 10, May 15, 2006 3381

Figure 2. Time-dependent SERS spectra of alkaline phosphatase-
catalyzed hydrolysis of BCIP. The initial BCIP concentration was 0.4
mg/mL. ALP enzyme was at 4.1 × 10-10 M. (a) Background without
ALP; (b) 1, (c) 5, (d) 10, (e) 30, and (f) 45 min. Laser 785 nm, ∼1.5
mW. Acquisition time 10 s.
Figure 3. Peak intensity of the Raman band at 600 cm-1 as a
function of incubation time. The initial BCIP concentration was 0.4
-10
mg/mL. ALP enzyme was at 4.1 × 10
M. Each data point
represents an average of 5-7 measurements (error bar is the
standard deviation).
attributed to its adsorption onto Au colloids. These observations
demonstrate that BCIP dimer released from the enzymatic
reaction is SERS active, and the Raman shift at 600 cm-1 can be
used as a characteristic peak to quantify ALP and the dynamics
of these enzymatic reactions.
observed in the absence of either ALP or BCIP even at an
incubation time of 45 min (controls), indicating that the nonen-
zymatic hydrolysis of BCIP is negligible, and the enzymatic
reaction is the sole route for observed SERS signal of indigo dyes.
ALP Assay. Quantification of ALP in samples was carried out
by recording SERS spectra of ALP at different concentrations
Quantification of ALP. Kinetics of Enzymatic Hydrolysis of
BCIP. The above technique was subsequently used to detect the
kinetics of enzymatic hydrolysis of BCIP. Experiments were
-10
-15
ranging from 4.1 × 10 to 4.1 × 10 M after incubation with
BCIP in pH 9.8 Tris-HCl buffer containing 0.005 M MgCl for 1 h
Figure 4). Again, intensities of the characteristic Raman band at
2
-
10
performed at a fixed concentration of ALP (4.1 × 10 M) and
(
6
BCIP (0.4 mg/mL) in 1 mL of Tris-HCl buffer solution containing
-1
00 cm were used to quantify the concentrations of ALP in
0
.005 M MgCl
SERS spectra of samples were recorded after mixing with 50 µL
of 1 M H PO and 100 µL of Au colloids. Results (Figure 2) showed
2
at room temperature. At different time intervals,
samples, and a linear relationship was observed (Figure 5). Note
that each data point in Figure 5 represents an average of five to
seven measurements across the SERS specimen. Variations in
signal intensities of different sample spots were typically <20%,
shown as error bars in the plot. This is in contrast to previous
3
4
a consistently increased intensity of SERS spectra with time, which
is indicative of efficient enzymatic turnover of the substrate leading
to a continuous buildup in the concentration of indigo dye. After
40,41
findings that SERS detection usually relies on a few “hot spots”,
1
-min reaction, the reaction product was already detectable by
and we found that this methodology is quite reproducible.
On average, the SERS intensities increased as the enzyme
concentration increased, suggesting that more indigo dye was
produced in the presence of higher concentrations of ALP enzyme.
This linear relationship holds over 5 orders of magnitude
-
1
measuring the Raman band at 600 cm . The peak intensity and
area of this Raman band increased consistently, and detailed
vibrational fingerprints of indigo dye molecules became clearer
with an increased reaction time. A plot of the peak intensity at
-
1
6
00 cm as a function of time (Figure 3) indicated a first-order
-11
-15
concentrations of ALP (from 4.1 × 10
to 4.1 × 10
M).
2
or exponential decay of BCIP substrate (R ) 0.98). This
observation is in agreement with that of Ashrafi et al., who
reported the initial zero-order reaction kinetics of ALP with
p-nitrophenyl phosphate disodium as an enzymatic substrate
However, the relationship became nonlinear at higher concentra-
tions of enzyme, which may be attributed to limited surface
coverage of indigo dyes on Au colloids. Because of low back-
-1
ground interferences, the characteristic Raman band at 600 cm
3
9
within the first 25 min. No significant changes in Raman
could be clearly discerned even at an ALP concentration of 4.1 ×
intensities were observed after ∼30 min of the reaction (Figure
-15
1
0
M (after incubation with BCIP for 1 h). This concentration
3
), suggesting that the maximum coverage of indigo dyes on Au
is more than three times the standard deviation of background
noise and can thus be regarded as the detection limit of free ALP
nanoparticles could have been achieved under the given experi-
mental conditions. On the other hand, no Raman bands were
(40) Kneipp, K.; Wang, Y.; Kneipp, H.; Perelman, L. T.; Itzkan, I.; Dasari, R.;
(
39) Ashrafi, S. H.; Naqvi, S. N. H.; Qadri, M. A. H. Ohio J. Sci. 1969, 69, 183-
91.
Feld, M. S. Phys. Rev. Lett. 1997, 78, 1667.
(41) Nie, S.; Emory, S. R. Science 1997, 275, 1102.
1
3382 Analytical Chemistry, Vol. 78, No. 10, May 15, 2006

Figure 4. SERS spectra of enzyme reaction products at different
ALP concentrations: (a) background without ALP; and (b) 4.1 ×
Figure 6. SERS spectra of ALP reaction products corresponding
to varying avidin concentrations, which were immobilized on glass
surfaces. (a) Background; (b) 1, (c) 10, and (d) 100 ng/mL; (e) 1 µg/
mL. Laser 785 nm, ∼1.5 mW. Acquisition time 10 s.
-
-
15
11
-14
-13
-12
1
1
7
0
, (c) 4.1 × 10 , (d) 4.1 × 10 , (e) 4.1 × 10 , and (f) 4.1 ×
0
M. BCIP concentration 0.3 mg/mL. Reaction time 1 h. Laser
85 nm, ∼ 1.5 mW. Acquisition time 10 s.
assays prompted us to examine a biological binding reaction
between avidin (or streptavidin) and biotin by using ALP as a
biotin conjugate. Avidin, found in the whites of chicken eggs, is
a glycoprotein with a molecular mass of 68 kDa. The interaction
between avidin and biotin results in the strongest affinity (dis-
-
15
sociation constant, K
d
∼ 10 M) known between a ligand and a
protein.37 The reaction also is highly specific and has resulted in
the avidin-biotin system being extensively used in biological
applications such as affinity-based separation and diagnostic
assays.44 For the first time, we introduce a SERS-based methodol-
ogy to detect immobilized avidin (on glass surfaces) using ALP
conjugated biotin as a probe. ALP bound on the glass surface
through avidin-biotin reaction can be detected by catalytic
hydrolysis of BCIP, and the reaction products of ALP are detected
by SERS. Figure 6 shows the results of the SERS-based determi-
nation of immobilized avidin on glass tubes. Clearly, intensities
of Raman spectra increased consistently with the concentration
of immobilized avidin, and a linear relationship between peak
-
1
intensities at 600 cm and avidin concentrations was observed
Figure 5. ALP assay by measuring peak intensities of the SERS
in the range from 10 ng/mL to 1 µg/mL (Figure 6c). At 1 ng/mL
-
1
band at 600 cm (shown in Figure 4). Each data point represents
an average of 5-7 measurements (error bar is the standard devia-
tion).
-1
avidin (Figure 6b), the peak intensity at 600 cm generated no
significant differences from the background. A small band at 600
-1
cm was observed in the background, suggesting the nonspecific
adsorption of ALP on the modified glass surface. Such a nonspe-
cific adsorption is quite common and often difficult to eliminate
by SERS. It is comparable with or better than some commonly
used techniques, such as electrochemical assay (with a calculated
4
5
in most immunoassays or biological binding assays. In this
approach, although 3% bovine serum albumin was used to block
nonspecific adsorption of biotin conjugated ALP, the nonspecific
adsorption still could be detected due to a high sensitivity of SERS
for indigo dye derivatives. We therefore claim a detection limit at
detection limit of 10-14 M)42 and enzyme biosensors (with a
-
14
43
detection limit of 6.7 × 10 M.
ALP as a Biomarker for Avidin Detection. The importance
of ALP as a biomarker in immunoassays and DNA hybridization
(
(
42) Authier, L.; Schollhorn, B.; Limoges, B. Electroanalysis 1998, 10, 1255-
259.
43) Serra, B.; Morales, M. D.; Reviejo, A. J.; Hall, E. H.; Pingarron, J. M. Anal.
Biochem. 2005, 336, 289-294.
(44) Weber, P. C.; Ohlendorf, D. H.; Wendoloski, J. J.; Salemme, F. R. Science
1989, 243, 85-88.
(45) Masarik, M.; Kizek, R.; Kramer, K. J.; Billova, S.; Brazdova, M.; Vacek, J.;
1
Bailey, M.; Jelen, F.; Howard, J. A. Anal. Chem. 2003, 75, 2663-2669.
Analytical Chemistry, Vol. 78, No. 10, May 15, 2006 3383

∼
10 ng/mL avidin in this study, but an even lower detection limit
of the actual sample with an estimated volume at picoliters to
femtoliters.24 This translates that we are likely to be sampling only
reactions arising from tens or a few hundreds of enzyme
molecules, at most. In other words, the technique allows the
detection at single-molecule levels and thus has attractive pos-
sibilities with respect to moving toward in vivo monitoring of
enzyme reactions. This same technique also was successfully
employed to detect surface-immobilized avidin through ALP-
avidin-biotin binding reactions. Therefore, apart from its potential
usage as a SERS probe in studies of enzyme activity in living cells
and gene diagnosis, the technique could be used as a new
immunoassay tool (i.e., SERS immunoassay) with improved
sensitivity in real-time analysis.
could be achieved with further development of the methodology.
Our results demonstrate that the SERS technique could potentially
be used as a sensitive tool to detect surface-immobilized avidin
and other biological agents by using conjugated ALP as a probe.
The major advantages of using SERS to detect these biological
agents are its high sensitivity, specificity, versatility (either in
solids or liquid for many bioagents), and short analytical time.
CONCLUSIONS
A new SERS-based approach was developed to detect the
activity of ALP at ultralow concentrations or single-molecule levels.
ALP was detected through the hydrolysis of BCIP to form indigo
dye of BCIP dimers, which were subsequently adsorbed on
surface-modified gold nanoparticles as a SERS substrate. The
ACKNOWLEDGMENT
-
15
This research was supported in part by the Laboratory Directed
detection limit of ALP was determined to be ∼10 M upon 1-h
Research and Development SEED Program of Oak Ridge National
Laboratory, managed by UT-Battelle, LLC for the U.S. Department
of Energy under DE-AC05-00OR22725.
incubation in basic Tris-HCl solution containing MgCl . The
2
technique is equivalent to or better than the most commonly used
fluorescence assay techniques with a greatly reduced analytical
time. It is also worth drawing attention to the sampling setup of
the Raman system, in which the exciting light is focused onto
the sample by a 50× microscope objective and the SERS spectra
collected at 180°. The lens thus interrogates a small proportion
Received for review December 14, 2005. Accepted March
20, 2006.
AC0522106
3384 Analytical Chemistry, Vol. 78, No. 10, May 15, 2006