Label-Free Quantitative Detection of Protein Using
Macroporous Silicon Photonic Bandgap Biosensors
Huimin Ouyang,*,†,‡Lisa A. DeLouise,†,§Benjamin L. Miller,†,§and Philippe M. Fauchet†,‡
Center for Future Health, University of Rochester, Rochester, New York 14642, Department of Electrical and Computer
Engineering, University of Rochester, Rochester, New York 14627, and Department of Dermatology, University of Rochester,
New York 14642
silicon (pore size >100 nm) one-dimensional photonic
band gap structures that are very sensitive to refractive
index changes. In this study, we employed Tir-IBD
(translocated Intimin receptor-Intimin binding domain)
and Intimin-ECD (extracellular domain of Intimin) as the
probe and target, respectively. These two recombinant
proteins comprise the extracellular domains of two key
proteins responsible for the pathogenicity of enteropatho-
genic Escherichia coli (EPEC). The optical response of
the sensor was characterized so that the capture of
Intimin-ECD could be quantitatively determined. Our
result shows that the concentration sensitivity limit of the
sensor is currently 4 µM of Intimin-ECD. This corre-
sponds to a detection limit of approximately 130 fmol of
Intimin-ECD. We have also investigated the dependence
of the sensor performance on the Tir-IBD probe molecule
concentration and the effect of immobilization on the Tir-
IBD/Intimin-ECD equilibrium dissociation constant. A
calibration curve generated from purified Intimin-ECD
solutions was used to quantify the concentration of In-
timin-ECD in an E. coli BL21 bacterial cell lysate, and
results were validated using gel electrophoresis. This work
demonstrates for the first time that a macroporous silicon
microcavity sensor can be used to selectively and quan-
titatively detect a specific target protein with micromolar
dissociation constant (Kd) in a milieu of bacterial proteins
with minimal sample preparation.
Over the past 2 decades, the development of label-free optical
biosensors has been pursued with great interest by many
researchers. Methods including surface plasmon resonance (SPR)1
and interferometry2,3have been employed in label-free affinity
biosensors to measure the change of refractive index at the surface
of the sensor upon binding of the target molecules to the
bioreceptors. Recently, there has been interest in using photonic
band gap (PBG) structures for sensing applications.4-6The PBG
structures are highly sensitive to changes in the refractive index
of the environment because these changes occur where the
electric field is maximum.7This makes PBG structures an ideal
candidate for ultrasmall and efficient “lab-on-a-chip” devices.
Porous silicon (PSi)-based PBG structures have been investi-
gated by many groups as an optical label-free sensing platform
for chemical and biological detection.4,8-10The advantages of using
PSi include ease of fabrication and compatibility with silicon
microelectronics technology. The large internal surface area of
PSi can be chemically modified for the capture and selective
detection of different types of molecules, such as DNA,2,5,11
proteins,12-14gram-negative bacteria,9and enzymes.15PSi mem-
branes can also be released from the silicon substrate and used
for in vivo applications as a “smart bandage”16and “smart dust”.17
The nanomorphology of the porous silicon layers is critical for
biosensing applications. The pore size not only determines the
size of the molecules that can infiltrate the pores but it also impacts
the sensor sensitivity. The ultimate sensitivity performance of
porous silicon sensors with different pore sizes was recently
quantified using aminopropyltriethoxysilane (APTES) and glut-
* To whom correspondence should be addressed. Phone: 585-275-1252.
Fax: 585-275-2073. E-mail: email@example.com.
†Center for Future Health.
‡Department of Electrical and Computer Engineering.
§Department of Dermatology.
(1) Liedberg, B.; Nylander, C.; Lundstrom, I. Sens. Actuators 1983, 4, 299-
(2) Lin, V. S.; Motesharei, K.; Dancil, K. S.; Sailor, M. J.; Ghadiri, M. R.; Science
1997, 278, 840-843.
(3) Lukosz, W. Sens. Actuators, B 1995, 29, 37-50.
(4) Cunin, F.; Schmedake, T. A.; Link, J. R.; Li, Y. Y.; Koh, J.; Bhatia, S. N.;
Sailor, M. J. Nat. Mater. 2002, 1, 39-41.
(5) Chan, S.; Li, Y.; Rothberg, L. J.; Miller, B. L.; Fauchet, P. M. Mater. Sci.
Eng., C 2001, 15, 277-282.
(6) Chow, E.; Grot, A.; Mirkarimi, L. W.; Sigalas, M.; Girolami, G. Opt. Lett.
2004, 29, 1093-1095.
(7) Scheuer, J.; Green, W. M. G.; DeRose, G. A.; Yariv, A. IEEE J. Sel. Top.
Quantum Electron. 2005, 11, 476-484.
(8) Mulloni, V.; Pavesi, L. Appl. Phys. Lett. 2000, 76, 2523-2525.
(9) Chan, S.; Horner, S. R.; Fauchet, P. M.; Miller, B. L. J. Am. Chem. Soc. 2001,
(10) Chan, S.; Fauchet, P. M.; Li, Y.; Rothberg, L. J.; Miller, B. L. Phys. Status
Solidi A 2000, 182, 541-546.
(11) Archer, M.; Christophersen, M.; Fauchet, P. M. Biomed. Microdevices 2004,
(12) Ouyang, H.; Christophersen, M.; Viard, R.; Miller, B. L.; Fauchet, P. M.
Adv. Funct. Mater. 2005, 15, 1851-1859.
(13) Dancil, K. P. S.; Greiner, D. P.; Sailor, M. J. J. Am. Chem. Soc. 1999, 121,
(14) Zangooie, S.; Bjorklund, R.; Arwin, H. Thin Solid Films 1998, 313, 825-
(15) DeLouise, L. A.; Kou, P. M.; Miller, B. L. Anal. Chem. 2005, 77, 322-
(16) DeLouise, L. A.; Fauchet, P. M.; Miller, B. L.; Pentland, A. A. Adv. Mater.
2005, 17, 2199-2203.
(17) Schemedake, T. A.; Cunin, F.; Link, J. R.; Sailor, M. J. Adv. Mater. 2002,
Anal. Chem. 2007, 79, 1502-1506
Analytical Chemistry, Vol. 79, No. 4, February 15, 2007
10.1021/ac0608366 CCC: $37.00© 2007 American Chemical Society
Published on Web 01/23/2007
araldehyde.18Although the sensitivity of the biosensor decreases
as the pore size increases, large size pores (>100 nm) are useful
for the detection of macromolecules (e.g., immunoglobin) that
cannot infiltrate into devices with small pores (<30 nm). The PSi
PBG structures with pore diameters in the 20-50 nm range are
well suited for the detection of smaller molecules (<50 kDa).
Recently, we have demonstrated the fabrication of a new type of
macroporous silicon 1D PBG microcavity sensor which has pore
diameters exceeding 100 nm.12The macropores allow easy
infiltration and stacking of large size targets such as streptavidin
(68 kDa) and biotinylated IgG (150 kDa).
To further explore the capability of macroporous 1D PBG
sensors for protein detection, we investigated the design of a
sensor for label-free detection of pathogenic Escherichia coli (E.
coli). Our sensor was designed employing recombinant proteins
corresponding to the extracellular domain of Intimin and Tir
(translocated Intimin receptor), which are two proteins expressed
by the enteropathogenic (EPEC) and the enterohemerogaic
(EHEC) E. coli strains via the type III secretory pathway.19Both
proteins are essential components of this organism’s pathogenicity.
The dissociation constant (Kd) of Tir-IBD (translocated Intimin
receptor-Intimin binding domain) and Intimin-ECD (Intimin
extracellular domain) is about 0.3 µM,20which is considerably
weaker than in the biotin-streptavidin model system used in our
previous work.12Hence, the Tir-IBD/Intimin-ECD system will
enable us to determine the impact of the increased off-rate on
detection sensitivity and the quantitative analytical performance
of this device. Sensors were prepared by immobilizing the
extracellular portion of Tir-IBD in the macroporous silicon
microcavity as the probe molecule (the bioreceptor). The Tir-
IBD-functionalized sensors were tested to determine the limit of
Intimin-ECD protein detection in solution. We also investigated
the utility of the sensor for quantitative analysis, the dependence
of the sensor performance on the probe molecule concentration,
and the ability of the sensor to selectively and quantitatively detect
Intimin-ECD expression in the supernatant of an E. coli bacterial
(Bl21) cell lysate relative to a control (JM109) cell lysate.
Porous Silicon Microcavity Preparation. The fabrication of
macroporous silicon microcavities has been described in detail
elsewhere.12Macroporous silicon 1D PBG microcavities were
electrochemically synthesized from n-type silicon wafers with 0.01
ohm-cm resistivity (from SEH Inc.) using an electrolyte solution
containing 5.5% hydrofluoric acid (HF), 94% H2O, and 0.5%
surfactant (polyoxyalkylene alkyl ether from Wako Chemicals
U.S.A. Inc.). The etching area of each sensor was approximately
150 mm2. During the microcavity formation, the etching current
density j alternated between 40 and 34 mA/cm2to form multilayer
microcavities consisting of layers with distinct porosities. A very
low porosity contrast was chosen (∼80% vs ∼70%) in order to keep
the pores as large as possible throughout the entire structure.
The microcavities consisted of two eight-period Bragg mirrors and
a defect layer of a half-wavelength optical thickness. Cross-
sectional and top view scanning electron micrographs (SEM) of
a microcavity sensor are shown in Figure 1a-c . The average
pore diameter was approximately 120 nm, and the total thickness
of the sensor was ∼5 µm. The optical reflectance spectra of the
porous silicon microcavities were taken using an Ocean Optics
HR 2000 spectrometer (550-950 nm) with a reflection probe
R200-7 and an Ocean Optics LS-1 tungsten halogen light source.
The illumination spot size was approximately 1 mm2.
After anodization, the microcavities were thermally oxidized
at 900 °C for 3 min to form a silica-like internal surface. The
spectrum blue shifted by approximately 100 nm after the oxidation,
as part of the silicon was converted into SiO2, which has a lower
Protein Preparation. Details of the purification and quanti-
fication of the proteins has been described previously by Horner
et al.20Independent overnight cultures in LB (Luria-Bertani)
broth medium of the transformed E. coli BL21-expressing 6×His-
Tir-IBD (∼10 kDa) and 6×His-Intimin-ECD (32 kDa) were
grown at 37 °C to OD 0.6 (measured at 595 nm) and induced
with 1 mM IPTG overnight at room temperature. Tir-IBD and
Intimin-ECD were purified using Amersham Biosciences-HiTrap
chelating columns and dialyzed in HEPES buffer (20 mM HEPES,
150 mM NaCl, pH 7.5). The concentrations of the purified proteins
were determined by OD at 280 nm using molar extinction
coefficients (?Tir) 705, ?intimin) 36 960).
Sensor Functionalization. The probe molecule (Tir-IBD)
was immobilized on the porous silicon matrix using standard
aminopropyltriethoxysilane (APTES) and glutaraldehyde coupling
chemistry. Each sensor was treated with 50 µL of 2% 3-aminopro-
pyltriethoxysilane (Gelest Inc.) in 48% methanol and 50% H2O for
20 min. The silane is rapidly hydrolyzed in water, which facilitates
the condensation reaction to surface hydroxyl group on the PSi
surface. The sensors were then rinsed with water and baked in
an oven at 100 °C for 10 min. Following the silane treatment, 50
µL of 2.5% glutaraldehyde (Sigma) solution in 20 mM HEPES
(18) Ouyang, H.; Striemer, C. C.; Fauchet, P. M. Appl. Phys. Lett. 2006, 88,
(19) Zaharik, M. L.; Gruenheid, S.; Perrin, A. J.; Finlay, B. B. Int. J. Med. Microbiol.
2002, 291, 593-603.
(20) Horner, S. R.; Mace, C. R.; Rothberg, L. J.; Miller, B. L. Biosens. Bioelectron.
2006, 21, 1659-1663.
Figure 1. (a and b) Cross-sectional SEM images of a macroporous
silicon microcavity with pore size approximately 120 nm. (c) Top view
SEM image of a macroporous silicon microcavity. (d) Reflectance
spectrum of a macroporous silicon microcavity, showing the reflec-
tance dip near 830 nm.
Analytical Chemistry, Vol. 79, No. 4, February 15, 2007
buffer (pH 7.3) was applied to each sensor for 30 min. Sensors
were rinsed with HEPES buffer and dried in a stream of nitrogen.
Next, a series of sensors were fabricated by applying 50 µL of
Tir-IBD with concentrations ranging between 0 and 1 mM.
Sensors were exposed to the Tir-IBD solution for 1 h. To prevent
nonspecific attachment of Intimin-ECD to unreacted glutaralde-
hyde sites, each sensor was exposed to 50 µL of 1 M glycine
methyl ester for 1 h (pH 5). After this treatment, each sensor
was rinsed and soaked in HEPES buffer for 20 min and dried with
nitrogen flow before exposure to the target solution.
Target Incubation, Protein Supernatant, and Controls. For
target incubation, 50 µL of the Intimin-ECD solution (5-60 µM)
was applied to the sensor for 1 h, and then rinsed/soaked with
HEPES buffer for 1 ∼ 2 h and dried with nitrogen flow before
the optical measurement. This step was key to minimizing false
positive signal from trapping of nonspecifically bound protein in
the porous matrix. To determine the selectivity of the sensor to
Intimin-ECD, independent overnight cultures of E. coli cells
expressing Intimin-ECD and JM109 (stratagene) which does not
express Intimin-ECD were grown up, centrifuged, lysed, and then
resuspended in HEPES buffer (pH ) 7.5). The supernatant
solution was filtered through a 0.45 µm filter before exposure to
the sensor. The presence or absence of Intimin-ECD in the
protein supernatants from the Intimin-ECD-expressing E. coli
cell line and JM109 was verified using SDS-PAGE gel electro-
phoresis. With the use of the Bio-Rad Protein Assay, the total
concentration of protein in the BL21 cell lysate was determined
to be 2.4 mg/mL for the Intimin-ECD-expressing strain and 2.2
mg/mL for JM109.
RESULTS AND DISCUSSIONS
A PSi 1D BPG microcavity contains a defect (symmetry
breaking) layer sandwiched between two Bragg mirrors. The
optical reflectance spectrum of a microcavity is characterized by
narrow resonances that are very sensitive to the effective optical
thickness of each layer. When the sensor is exposed to the target,
the binding of target species inside the pores increases the
effective refractive index of the pores and causes a red shift of
the resonance. The total amount of red shift is linearly related to
the amount of analyte captured by the sensor. Details of the sensor
sensing principle, design, and optimization were described else-
where.21,22Simulations show that for a microcavity with layers of
80% and 70% porosity, the sensor has a wavelength shift sensitivity
of ∆λ/∆n ∼500 nm/RI, where ∆λ is the shift of the resonance
and ∆n is the change of effective refractive index of the pores.15,18
Assuming a detection system able of resolving a shift of 0.5 nm,
the minimum ∆n that can be detected is 1 × 10-3, which is
equivalent to an internal surface areal mass of ∼50 pg/mm2for
sensors with a 100 nm average pore diameter.18
A series of sensors derivatized with APTES and glutaraldehyde
were exposed to Tir-IBD solutions with different concentrations
from 0 to 1 mM. As shown in Figure 2, the red shift of the
spectrum increases as the concentration of Tir-IBD increases.
Since the red shift of the spectrum is approximately linearly related
to amount of protein captured inside the microcavity, we conclude
that the total amount of proteins immobilized in the sensor
increases as the protein concentration increases. Thus, it is
possible to prepare different surface concentrations of the probe
molecule. At high Tir-IBD concentrations (∼1 mM), the red shift
saturates at ∼12 nm, which is consistent with Langmuir adsorption
isotherm behavior where all surface adsorption sites are filled and
multilayer adsorption does not occur.
With the use of our model for the quantitative analysis of the
sensor sensitivity, a 0.5 nm red shift of the microcavity corre-
sponds to the capture of ∼50 pg/mm2of protein inside the pores.18
Thus, in this case, a 12 nm red shift corresponds to ∼1.2 ng/
mm2of Tir-IBD. Assuming that the total internal surface area of
the sensor is 15 000 mm2, the total mass of Tir-IBD needed to
achieve a saturated surface coverage is ∼18 µg or 1.8 nmol for
the entire sensor with a 150 mm2top surface.
The kinetics of protein binding to surface-tethered receptors
may be impacted by steric effects. These are exacerbated in the
case of multivalent proteins.23Studies have shown that the
performance of solid-phase sensors may depend on the probe
molecule surface density.24To investigate the importance of this
effect on the binding of Intimin-ECD, we studied the sensor
response as a function of the Tir-IBD surface concentration and
Intimin solution concentration. In this experiment, four identical
sets of microcavity sensors were prepared. Each set contains six
samples that were functionalized with different amounts of Tir-
IBD as described above. Next, purified Intimin-ECD solutions
with different concentrations (5-60 µM) were applied to each set
of sensors. As shown in Figure 3, the optical red shift of the
microcavities is related to the amount of both Tir-IBD and
As expected, the amount of Intimin-ECD captured by the
sensor depends on both the immobilized Tir-IBD concentration
and the Intimin-ECD solution concentration. Figure 3 illustrates
a general trend that for a fixed Tir-IBD receptor concentration
the sensor red shift, hence the amount of Intimin-ECD captured,
increases with the Intimin-ECD solution concentration. Also, for
a fixed Intimin-ECD solution concentration, the sensor red shift
increases with the Tir-IBD receptor concentration. In the latter
case, the magnitude of the shift (amount of bound Intimin) tends
to saturate at the highest Tir-IBD coverage. In our previous work,
we had observed that the optimum surface coverage of biotin for
(21) Ouyang, H.; Fauchet, P. M. Proc. SPIE-Int. Soc. Opt. Eng. 2005, 6005,
(22) Ouyang, H.; Lee, M.; Miller, B. L.; Fauchet, P. M. Proc. SPIE-Int. Soc. Opt.
Eng. 2005, 5926, 59260J1-J11.
(23) Schuck, P. Annu. Rev. Biophys. Biomol. Struct. 1997, 26, 541-566.
(24) Jung, L. S.; Nelson, K. E.; Stayton, P. S.; Campbell, C. T. Langmuir 2000,
Figure 2. Red shift of the macroporous silicon microcavity reflec-
tance dip as a function of the Tir-IBD concentration. The total volume
of the solution applied to each sensor was 50 µL.
Analytical Chemistry, Vol. 79, No. 4, February 15, 2007
binding streptavidin is ∼50%.12A higher biotin surface coverage
resulted in a lower streptavidin capture, which was attributed to
steric effects associated with its “pocket-type” tight bonding
(mechanical lock and key) structure.24For the Tir-IBD/Intimin-
ECD system the binding pocket is “end-on” (binding sites are at
the end of the cylindrical-like molecules),25and hence steric
crowding at high Tir concentrations does not appear to impact
binding. It can be seen from Figure 3 that when the Intimin-
ECD concentration is higher than 30 µM some nonspecific binding
of Intimin-ECD is detectable at zero Tir-IBD concentration.
Hence, to achieve maximum Intimin-ECD detection sensitivity
with minimum nonspecific binding, the sensors used in our
quantitative analytical detection studies described below were
functionalized with a saturated coverage of Tir-IBD by treating
each sensor with 50 µL of 1 mM Tir-IBD.
We conducted simulations to investigate the effect of im-
mobilization on the equilibrium dissociation constant Kdfor Tir-
IBD and Intimin-ECD using the following equation:
where [Inttotal] is the solution concentration of Intimin-ECD which
varied from 0 to 60 µM, [Intbound] is the amount of Intimin-ECD
captured by the sensor, and [Tirtotal] is the surface-immobilized
concentration of Tir-IBD which we estimated from the red shift
in Figure 2. It can be seen from the equation that for a given
value of Kd, [Intbound] can be plotted as a function of [Tirtotal]. In
our simulation, the maximum Tir concentration was set at 36 µM
(1.8 nmol/50 µL). The amount of Intimin captured by the sensor
() ([Intbound])(50 µL)) was calculated using different Kdvalues.
The best fit solutions were obtained with Kdset to be 1 × 10-4
M. These simulation results are plotted on a secondary x- and
y-axis as shown in Figure 3. Although this analysis provides a
crude estimation of Kd, it nevertheless yields a value consistent
with the expected much weaker association for immobilized probe
receptors compared to that of free Tir-IBD and Intimin-ECD in
solution. The increase of Kdby 2 orders of magnitude (from 0.3
× 10-6to 1 × 10-4M) may reflect steric factors that alter the
binding pocket or a kinetic effect due to pH or concentration
gradient within the porous structure.
An Intimin calibration curve using recombinant protein control
solutions is shown in Figure 4. The sensor red shift is plotted as
a function of the target concentration. The concentration sensitivity
limit of the sensor is currently 4 µM of Intimin-ECD. Although
the current concentration detection limit of the sensor is higher
(less sensitive) than that of traditional proteomics techniques, such
as silver staining in 2D-PAGE or ELISA, this label-free optical
sensing method shows great potential due to its ease of fabrication
and operation. This technology offers the potential to be developed
for rapid, point-of-care diagnostic or laboratory sample screening
applications where ultrasensitive detection (subpicomolar) sen-
sitivity is not a concern. Using existing microelectronic techniques,
one could easily shrink down the size scale of the sensor and
integrate it into a microfluidic system to build a lab-on-a-chip
device. This integration would reduce the total volume of sample
required for the test. We estimate that our current detection limit
corresponds to a mass sensitivity of ∼600 ng/sensor by assuming
that 10% of the protein in the 50 µL solution that was applied to
the sensor was captured by the PSi matrix (∼1 µL). The total
internal surface area of the sensor is ∼15 000 mm2; thus, the areal
mass sensitivity is ∼50 pg/mm2, which is consistent with the
theoretical estimation.18Since the spot size of the measurement
is approximately 1 mm2, the amount of Intimin-ECD that
contributed to the red shift is approximately 130 fmol.
To demonstrate selectivity, a 2 × 2 sensor array was designed.
Two samples containing Tir-IBD and two samples without Tir-
IBD were prepared. Sensors with and without Tir-IBD function-
alization were exposed separately to cell lysate (supernatants)
obtained from the BL21 E. coli cell line (expressing Intimin-ECD)
and from the JM109 E. coli cell line (not expressing Intimin-
ECD) prepared with a nearly equivalent total protein concentration
(∼2 mg/mL). A positive response should be obtained only when
Analytical Chemistry, Vol. 79, No. 4, February 15, 2007
(25) Luo, Y.; Frey, E. A.; Pfuetzner, R. A.; Creagh, A. L.; Knoechel, D. G.; Haynes,
C. A.; Finlay, B. B.; Strynadka, N. C. Nature 2000, 405, 1073.
Figure 3. Red shift of sensors that were functionalized with different
Tir-IBD concentrations after exposure to purified Intimin-ECD
solutions with different concentrations from 5 to 60 µM. Theoretical
calculation of the bound Intimin as a function of Tir concentration is
plotted in the secondary axis using a Kd∼10-4M. The agreement
between the theoretical calculation and experimental data suggests
a higher Kdfor immobilized probes Tir-IBD.
Kd)([Tirtotal] - [Intbound])([Inttotal] - [Intbound])
Figure 4. Dependence of the sensor red shift on the Intimin-ECD
solution concentration. These sensors are functionalized with 1 mM
the sensor with Tir-IBD immobilized inside the pores is exposed Download full-text
to the protein mixture containing Intimin-ECD.
A 5 nm red shift was obtained from the Tir-IBD-functionalized
sensor following exposure to the BL21 cell lysate containing
Intimin-ECD. A very small shift (<1 nm) was detected from the
Tir-IBD-functionalized sensor after exposure to the JM109 cell
lysate solution that does not contain Intimin-ECD. The sensors
prepared without Tir-IBD functionalization did not respond to
the protein mixture with and without Intimin-ECD. The results
indicate that the sensors can selectively detect Intimin-ECD from
the protein mixture. From the red shift, we can estimate the
Intimin-ECD concentration in the protein mixture with minimal
background interference (nonspecific binding) using the calibra-
tion curve shown in Figure 4. A 5 nm red shift corresponds to a
15 µM concentration of Intimin-ECD. To validate the Intimin-
ECD concentration in the cell lysate protein mixture, we used
SDS-PAGE gel electrophoresis. Pure Intimin-ECD control
solutions with known concentrations were run with the E. coli
(BL21 and JM109) cell lysate solutions (both ∼2 mg/mL total
protein), and the optical densities of the pure Intimin-ECD protein
solution bands were compared. Using the gel analysis function
in ImageJ (see the Supporting Information), we estimated the
concentration of Intimin-ECD in the supernatant to be ∼14 µM,
which is very close to the value predicted from the sensor optical
response. No Intimin-ECD band was observed in the JM109
protein cell lysate, as expected.
To conclude, we have demonstrated the use of a macroporous
silicon 1D PBG microcavity as a quantitative analytical device for
optical label-free biosensing. The molecule binding events increase
the effective refractive index of the sensor, which can be easily
detected by monitoring the shift of the optical reflectance spectrum
of the device. We have successfully functionalized the biosensors
with Tir-IBD, a protein secreted by enteropathogenic E. coli.
These protein sensors were able to selectively and quantitatively
detect purified Intimin-ECD (target) binding to Tir-IBD (recep-
tor). This work demonstrates for the first time the analytical
capability of a macroporous 1D PBG sensor using a micromolar
Kdprotein bioconjugate system for which target specificity was
validated in the testing of a complex bacterial cell lysate. In this
sample, the target was a minority component (23%) of the total
protein milieu. This is significant because our operating protocol
utilized a long wash step (1-2 h) to minimize nonspecific binding.
While this protocol compromises our lower limit of detection, the
analytical capability is retained. Future studies will investigate
methods to enhance the detection limit by exploring chemical
nonspecific blocking agents and by upgrading our optical detection
This work is supported by the National Science Foundation
through Grant BES 0427919 and the National Institutes of Health
SUPPORTING INFORMATION AVAILABLE
SDS-PAGE analysis results and calibration curve of the
Intimin concentration. This material is available free of charge
via the Internet at http://pubs.acs.org.
Received for review May 5, 2006. Accepted October 1,
Analytical Chemistry, Vol. 79, No. 4, February 15, 2007