Surface plasmon biosensing technology for antimicrobial susceptibility test

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Surface Plasmon Resonance Biotechnology for
Antimicrobial Susceptibility Test
How-foo Chen1, Chi-Hung Lin2,3,4, Chun-Yao Su1,
Hsin-Pai Chen5 and Ya-Ling Chiang1
1Institute of Biophotonics, National Yang Ming University, Taipei
2Institute of Microbiology & Immunology, National Yang Ming University, Taipei
3Taipei City Hospital
4Department of Surgery, Veteran General Hospital, Taipei
5Department of Medicine, National Yang-Ming University Hospital,
Yilan, Taiwan and School of Medicine, National Yang-Ming University
Taiwan
1. Introduction
Infectious diseases are a leading cause of morbidity and mortality in hospitalized patients.
This fact has placed a tremendous burden on the clinical microbiology laboratory to rapidly
diagnose the agent responsible for patient’s infection and to effectively provide therapeutic
guidance for eradication of the microorganisms. Laboratories are expected to perform these
tasks in a cost-effective and efficient manner. Two common methodologies for antimicrobial
susceptibility testing in a clinical laboratory are Kirby-Bauer disk diffusion and variations of
broth microdilution. The principle is based on the detection of bacterium reproduction
ability under the influence of antibiotics. Therefore the testing time is determined by the
doubling time of tested bacteria. These methods then usually take from one day to weeks to
complete the examination. The long incubation period is inevitable for these conventional
methods. Such a waiting period is not short for clinical doctors who urgently need the
information to adjust the therapeutic strategy. Therefore it is important to explore new
template and technology to perform an antimicrobial susceptibility test.
Surface plasmon resonance biosensing technique is well known for its characteristics of
label-free, ultra-sensitive, and real-time detection capability. Thus this technique is
considered as the candidate of the new platform. Surface plasmon polaritons (SPPs) was
first theoretically predicted by Ritchie in 1957 (Ritchie,1957) based on the analysis of surface
electromagnetic modes. The SPPs in general can be generated by electrons (Powell & Swan,
1959) or by light (Otto, 1968) under a proper excitation condition. For SPPs excited by light,
in general, the dispersion characteristic of SPPs does not allow the energy of a propagation
wave coupled into this surface mode: The spatial phase of a propagation wave is always
smaller than that of the surface mode with the same optical frequency on a dielectric-metal
interface. Thus an evanescence wave generated by a p-polarized light beam through a prism
is suggested to obtain an extra spatial phase and then excite SPPs on the other surface of the
metal layer. An alternative method to provide the additional spatial phase is through the aid

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of a grating, of which the sub-wavelength periodic structure can provide additional spatial
phase. For the past two decades, SPPs excited by light has been widely applied to the study
of biomaterial processes, which include biosensors, immunodiagnostics, and kinetic analysis
of antibody-antigen interaction (Davies, 1996; Rich & Myszka, 2005). The main application
of SPR biosensors on biomedical science is to analyze the binding dynamics between specific
antibody and antigen (Davies, 1996; Rich & Myszka, 2005; Safsten et al., 2006; Misono &
Kumar, 2005). Since the mode characteristics of SPPs depend on the refractive index of the
material within the dielectric-metal interface of about one hundred nanometers, the
refractive index of the material determines the resonance incident angle of light, the
coupling efficiency, the coupling wavelength, and the optical phase of the reflected light. All
the physical quantities can be measured by the reflected light, which is the uncoupled part
of the incident light. Therefore, a SPR system does not require fluorescence labeling and
provides real-time information with very high sensitivity (Chien & Chen, 2004). This also
guarantees a very small amount of sample needed for the detection of the refractive index
change through a SPR method.
Most of the biomedical applications of SPR focus on detection and identification of
biomolecules. Extended applications have been applied to the detection and sorting of cells
or bacteria based on the same principle (Takemoto et al., 1996). The capture of the desired
biomolecules with or without cells or bacteria attached is achieved through antibodies or
aptamers pre-coated on the metal thin film, where the SPR occurs. The enormous
applications of SPR on biomedical science using antibody-antigen affinity can be found in
Rebecca L. Rich and David G. Myszka’s Survay (Rich & Myszka, 2005). For the methods
using antibody-antigen binding, specific antibody is required and finding the specific
antibody is usually not straight forward. This is the reason that characterization of antibody
is still the main reports from utilization of SPPs. This is also an important reason that a
method utilizing antibody-antigen interaction is difficult to use for antimicrobial
susceptibility test. Different from the studies mentioned above, the method introduced in
this chapter does not require pre-coating of specific antibodies. This method is then more
versatile and can be used to detect reactions of drugs appearing on cell membranes or cell
walls. While current antimicrobial susceptible testing methods take one day or more for a
clinical laboratory to report the testing results (Poupard et al., 1994; Levinson & Jawetz,
1989), utilizing surface plasmon resonance significantly reduces the time duration to less
than or about one hour of antibiotics treatment based on our experimental study. Antibiotics
which modify or damage the cell walls of bacteria, thus, alternate the refractive index of
bacterium surfaces.
Differentiation of susceptible strains of bacteria from resistant ones by using surface
plasmon resonance (SPR) technique is discussed in this chapter. This technique detects the
refractive index change of tested bacteria subject to antibiotics treatment in real time. Instead
of detection the antimicrobial susceptibility through the cell doubling time, the SPR
biosensor technology is used to detect the biochemical change of tested bacteria. A much
shorter time to obtain the test result is achieved. Because of the feasibility of this
antimicrobial test method using surface plasmon resonance biosensors, development of new
biosensors is also very important.
Escherichia coli JM109 resistant/susceptible to ampicillin and Staphylococcus epidermidis
resistant/susceptible to tetracycline were chosen for the antimicrobial susceptibility test in
this study. Since the surface plasmon resonance is highly sensitive to the change of the

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refractive index of cells near the cell-metal interface, ampicillin as the antibiotic inhibiting
the synthesis of cell walls was used for the examination of Escherichia coli JM109. This is
designed for the measurement of direct effect of antibiotics on cells. Different from
ampicillin, tetracycline works as an inhibitor of protein synthesis. The influence of
tetracycline on cell walls and cell membranes is then indirect. Therefore, Staphylococcus
epidermidis used as another type of bacteria susceptible/resistant to tetracycline was used for
the measurement of indirect effect of antibiotics on cells.
2. Devices and methods
The detection principle can be realized on the detection of biochemical change of bacteria
subject to antibiotics through the detection of their refractive index. This change on the
refractive index of bacteria is achieved by an SPR biosensor. A chemical treatment of Poly-L-
Lysine on the surface of the Au thin film in the SPR biosensor is used to trap bacteria. The
Poly-L-Lysine layer does not provide specfic binding to select specific bacterium strain so
that a pre-purification to select tested bacteria is required for the test. After the tested
bacterium strain is trapped on the Poly-L-Lysine layer, antibiotic is appled to examine the
antimicroial susceptibility.
2.1 Surface plasmon resonance biosensor
The experimental setup for the examination of drug resistance of the bacteria is shown in
Fig. 1(a). The setup is the combination of the two parts: one is for the excitation of the
surface plasmon and the other is the flow cell chamber. For the excitation of the surface
plasmon, a Helium-Neon laser is used as the light source to provide the laser beam with
wavelength 632.8 nm. Since surface plasmon can only be excited by p-polarized light, a
polarized beam splitter is used to separate the p-polarized and s-polarized light. The s-
polarized light is used as the normalization factor to eliminate the deterioration of
measurement accuracy caused by the laser instability. After the polarized beam splitter, the
p-polarized light is injected onto the Au thin film through a prism to generate surface
plasmon. The required phase matching condition to excite the surface plasmon is provided
by the proper incident angle and the prism, which provides an extra spatial phase along the
gold film surface through its refractive index of the prism. Matching oil is applied between
the prism and the glass substrate coated with the Au thin film to avoid occurrence of
multiple reflection between the prism and the glass slide. The excitation efficiency of the
surface plasmon by the p-polarized laser beam is measured through the silicon
photodetector which receives the reflected p-polarized beam from the Au thin layer. When
the surface plasmon resonance angle is reached, the energy of injected laser beam was
transformed into the surface plasmon polaritons. Thus, the laser beam reflected from the Au
layer reaches minimum. The photocurrent generated from the photodetector is amplified
and transformed into a voltage signal via 16-bit A/D converter（Adventech PCI-1716）.
The intensity, normalized to the intensity of the s-polarized beam, of the reflected p-
polarized beam as a function of the incident angle is obtained by the computer. Incident
angle was controlled by a motorized rotation stage through a controller. The other arm that
is for receiving reflection was controlled accordingly by another rotation stage to measure
the power of the reflected beam. The resolution of the system on the change of refractive
index of the dielectrics is
1.4 10

refractive index unit (RIU), which corresponds to the
value of the SPR angle shift as 0.00867 degree.
4

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(a)

(b)
Fig. 1. SPR biosensor used for the experiment. (a) The configuration of SPR biosensor used
in the study. The SPPs was excited by 632.8nm He-Ne laser. A polarizer is used to enhance
the extinction of the laser beam polarization. A polarized bean splitter (BS) direct the s-
polzaried light into a detector for normalization of laser intensity fluctuation. The p-
polarized light is used to excite SPPs. The reflectance of the light is direct to the second
detector for measurement of resonance angle, and thus measure the refractive index change
of bacteria subject to antibiotics; (b) Picture of the home-made SPR biosensor. The solid red
line indicates the laser beam.

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2.2 Cell chamber
A flow cell chamber was constructed on the SPR system described above to provide the
bacteria for testing, DI water for washing, and the antibiotics for the examination of drug
resistance. An O-ring is attached to the chamber to prevent the liquid leakage. A thermister
of 10KΩ is used to monitor the temperature of the chamber and a TE cooler is used to
control the temperature by receiving the temperature information from the thermister. The
temperature of the cell chamber was controlled with the fluctuation less than 0.1 oC, which
is achieved by a temperature controller usually used for controlling the temperature of laser
diodes. As is depicted in Fig 2, the target bacteria are first injected into the chamber through
the flow channel and attach on the gold film by the adhesion of the Poly-L-lysine.
Antibiotics are then added to test if the cell walls or membranes are affected.
2.3 Bacterium adhesive coating
Poly-L-Lysine has been demonstrated as an effective tissue adhesive for use in various
biochemistry procedures. Poly-L-Lysine solution is diluted with deionized water prior to the
coating procedure. The flat glass deposited with Au thin film was immersed in poly-L-lysine
solution (concentration = 200 ug/ml) for from a couple of hours to 24 hours to interact with
Au thin film as the preparation of the biochips. Different time intervals provide different
adhesion of Poly-L-Lysine to the bacteria and antibiotics. After incubation, cells can be
immobilized on the Au-coated glass.

detecto
Gold film
O-ring
Flow in Flow out
Bacteria
Poly-L-lysine


Fig. 2. Schematic illustration of the SPR device and the mechanisms of the experiment
2.4 Bacterium preparation
Preparation of Escherichia coli resistant to ampicillin Penicillin is called β-lactam drugs. An
intact ting structure of β-lactam ring is essential for antibacterial activity; cleavage of the
ring by penicillinases （β-lactamase）inactivates the drug (Levinson & Jawetz, 1989;
laser