DEVELOPMENT OF TAGLESS BIOSENSORS F0R DETECTING THE
PRESENCE OF PATHOGENS
SHORT TITLE: TAGLESS BIOSENSORS
JING–YIN CHEN*,1, JOSEPH R. KNAB1, SHUJI YE, YUNFEN HE2 AND
ANDREA G. MARKELZ3
1
State University of New York at Buffalo, Department of Physics, 239
Fronczak Hall, Buffalo, NY, USA 14260
1
Second address…
ABSTRACT. The vibrational modes corresponding to protein tertiary structural motion
lay in the far infrared or terahertz frequency range. These collective large scale motions
depend on global structure and thus will necessarily be perturbed by ligand binding
events. We discuss the use of terahertz dielectric spectroscopy to measure these
vibrational modes and the sensitivity of the technique to changes in protein
conformation, oxidation state and environment. A challenge of applying this sensitivity
as a spectroscopic assay for ligand binding is the sensitivity of the technique to both
bulk water and water bound to the protein. This sensitivity can entirely obscure the
signal from the protein or protein-ligand complex itself, thus necessitating sophisticated
sample preparation making the technique impractical for industrial applications. We
discuss methods to overcome this background and demonstrate how terahertz
spectroscopy can be used to quickly assay protein binding for proteomics and
pharmaceutical research.
Keywords: biosensors, terahertz spectroscopy, far-infrared, lysozyme, tri-N-acetyl-Dglucosamine, biomolecular sensing, ligand binding
1. Introduction
A key component to pharmaceutical development is the determination of
protein-protein and protein-ligand binding. Rapid optical assays to determine
this binding could have a significant impact, however not all binding events
Terahertz Frequency Detection and Identification of Materials and Objects
© 2007 Springer. Printed in the Netherlands
124
TAGLESS BIOSENSORS
*
To whom correspondence should be sent: J.Y. Chen, State University of New York at Buffalo,
Department of Physics, 239 Fronczak Hall, Buffalo, NY, USA 14260; email jchen9@buffalo.edu
will result in changes to UV/Vis absorbance or fluorescence since binding may
occur at a site remote from the optically active region. In the case of mid
infrared vibrational spectroscopy, while binding at any given site will locally
change the MIR vibrational modes involving on the order of 5-10 atoms, this
local change may have little effect on the dense overlapping modes in the amide
I and II regions. Global collective vibrational modes which involve large scale
structural motions on the other hand would be affected by any binding event
within the molecule. These vibrational modes associated with protein tertiary
structure lie in the far infrared or terahertz frequency range (0.03 – 6 THz, 1 –
200 cm-1). Methods of measuring these low frequency modes include inelastic
neutron scattering1 (INS) and terahertz dielectric spectroscopy2-5. Recently there
have been several reports of sensing biomolecular binding using these
techniques. For example, INS measurements demonstrated this binding
sensitivity in the case of binding of methotrexate with dihydrofolate reductase
which indicating an increase in the vibrational density of states at these
frequencies1. INS however is not a technique that is amenable to rapid parallel
assays for drug discovery.
2. Methodology
2.1 Subtitle-1
Hen egg white lysozyme (HEWL) lyophilized power (Sigma Aldrich L6876) is
dissolved in trizma buffer (pH 7.0, 0.05 M) and the final concentration of the
lysozyme solution is 200 mg/ml. Tri-N-Acetyl-D-glucosamine (N, N’, N”Triacetylchitotriose; 3NAG) lyophilized power is purchased from Toronto
Research Chemicals, Inc. (T735000). The HEWL+3NAG binding solution is
made by dissolving 3NAG powder into the 200 mg/ml lysozyme solution and
the molar ratio of lysozyme to 3NAG is 1 to 1. The solution sample cell for the
transmission measurements is composed of two brass plates sandwiching two
quartz windows (Spectrocell Inc.) with a ~ 250 m spacer10. The top plate of
the cell has two identical apertures respectively for the sample and reference.
About 14 µl of the protein solution is pipetted to fill the bottom aperture and the
upper aperture is left empty as a reference. The entire measurement was
performed for two different sets of the lysozyme and HEWL+3NAG binding
solutions and results were found to be excellent agreement.
TAGLESS BIOSENSORS
125
The ligand binding for the solutions was verified by the fluorescence
measurements before terahertz transmission measurements. The fluorescence
measurements are taken by using the SLM8100 spectrofluorimeter. The
excitation wavelength for both types of solutions is 280 nm. We found the
fluorescence peak of lysozyme solutions is about 343 nm and that of
HEWL+3NAG binding solutions is 332 nm as expected for bound and unbound
HEWL11,12.
2.2 Subtitle-2
The solution sample cell is mounted inside a continuous-flow cryostat (Cryo
Industries). A silicon diode temperature sensor is located on the sample stick
adjacent to the solution cell. Liquid nitrogen is used as the cryogen and the
fluctuation at each temperature is lower than 0.01 K. Each temperature
measurement is taken at least 10 minutes after the temperature is stable. At
each temperature, measurements were repeated more than three times in order
to make sure that the sample reached the thermal equilibrium.
Terahertz time domain spectroscopy (THz-TDS) was used to determine the
terahertz dielectric response. The terahertz electric field is generated by a
hertzian dipole antenna and detected by the electro-optical detection13,14. The
bandwidth for this study is good from 0.2 THz to 1.6 THz at room temperature
and from 0.2 THz to 2.0 THz at low temperatures. A Ti-sapphire laser (65 fs,
82 MHz) is used to generate and detect terahertz electric field. The entire
terahertz spectroscopy system is continuously purged with dry nitrogen gas in a
plexiglass box prior to and during the measurements to avoid the atmospheric
water absorption. Each measurement consists of measuring the relative
transmission through the reference and sample by toggling between the two
apertures of the sample cell.
THzTDS measures the complex field transmission. We normalize our
measurements to the reference transmission yielding a net field transmission
with transmitted field amplitude, |t|, and the phase, :
t
Esample
Ereference
i
 te e
ikd ( ns 1)
e

d
2
(1)
where k is the frequency in wavenumbers (cm-1), d is the thickness of
sample, ns is the refractive index of sample, and  is the absorption
coefficient.
TAGLESS BIOSENSORS
126
3. Data & Result
The temperature dependent terahertz transmission for HEWL and trizma
buffer solutions is shown in Figure 1 for several frequencies. The temperature
dependence of the transmission of HEWL solution is relatively flat for T < 200
K, and then decreases more rapidly above 200 K. Above 273 K, the
transmission for both protein and buffer solutions dramatically drops due to the
strong absorbance for bulk liquid water. …
1.2
1.0
|t|
0.8
0.6
0.4
0.2
Lysozyme solution
0.52 THz
0.84 THz
1.17 THz
1.31 THz
Trizma Buffer
0.51 THz
0.83 THz
1.17 THz
1.32 THz
0.0
100
150
200
Temperature (K)
250
300
Figure 1 Transmission, |t| of the 200 mg/ml lysozyme and trizma buffer solution as a function of
temperature at several frequencies. The transmission of the lysozyme solution shows a dramatic
increase at 280 K at each frequency. Below 280 K, the transmission of HEWL solution increases
with the decreasing temperature. In comparison with the lysozyme solution, |t| of the trizma
buffer has a big jump at 273 K. Besides, |t| of buffer also increases with the decrease
temperature.
We now turn to the question of whether the terahertz dielectric response can
distinguish between HEWL and 3NAG+HEWL. The native state and binding of
HEWL and HEWL+3NAG solutions were verified by the fluorescence
measurements. For each sample solution, the transmission at 295 K is much
less than that at ~ 270 K. For both temperatures shown, the transmission
decreases with increasing frequency for the entire frequency range and
oscillates with a period ~ 0.8 THz.
TAGLESS BIOSENSORS
127
4. Conclusions
The terahertz dielectric response of the lysozyme and HEWL+3NAG
binding solutions was measured as a function of frequency at room temperature
and at 270 K. The terahertz transmission of the lysozyme solution at 295 K
cannot be distinguished from that of the HEWL+3NAG binding solution due to
the large contribution from the solvent. After the solvent was frozen, the
protein dominates the terahertz spectrum. …
ACKNOWLEDGEMENTS
This work was supported by the American Chemical Society (PRF 39554AC6) and the National Science Foundation (NSF CAREER PHY-0349256,
NSF IGERT DGE0114330 and NSF REU DMR-0243833).
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