Surface plasmon resonance (SPR) sensors have become valuable analytical sensors for biomolecule detection. While SPR is heralded with high sensitivity, label-free and real-time detection, nonspecific adsorption and detection of ultralow concentrations remain issues. Nonspecific adsorption can be minimized using adequate surface chemistry. For example, we have employed peptide monolayers to reduce nonspecific adsorption of crude serum or cell lysate. It is important to uncover the nature of molecules nonspecifically adsorbing to surfaces in these biofluids, to further improve understanding of the nonspecific adsorption processes. Mass spectrometry (MS) provides a complementary tool to SPR to identify biomolecule adsorbed to surface. Trypsic digestion of the proteins adsorbed to surfaces led to identification of characteristic peptides from the proteins involved in nonspecific adsorption. Nonspecific adsorption in crude cell lysate results mainly from lipids, as confirmed with SPR and MS but proteins were observed on some surfaces. In another application of SPR and MS, imaging SPR can be used in combination to imaging MS to image tissue sections. Thin sections of mouse liver were inserted in the fluidic chamber of a SPRi instrument and proteins were transferred to the SPRi chip. The SPR chip was then imaged using MALDI imaging MS to identify the biomolecules that were transferred to the SPRi chip.
The anti-cancer drug, methotrexate (MTX) as a strong inhibitor of human dihydrofolate reductase (hDHFR) has been
studied in localized surface plasmon resonance (LSPR) and surface plasmon resonance (SPR) competitive binding assays
with folic acid stabilized gold nanoparticles (FA AuNP). The latter with a diameter of 15 nm were prepared in a simple
step with sequential characterization using UV-Vis, FTIR, and Raman. A LSPR competitive binding assay between
different concentrations of MTX and FA AuNP for hDHFR in solution was designed to quantify MTX by using UV-Vis
spectroscopy. Sensitivity of the assay was optimized with respect to both concentrations of the enzyme and FA. The
detection and quantification of spiked MTX was demonstrated in phosphate buffer saline and in fetal bovine serum
accompanied by solid-phase extraction treatment of the serum. In addition, this assay could also provide as a screening
tool for potential inhibitors of hDHFR. In another perspective, MTX was measured in a competitive binding assay with
FA AuNP for histidine-tagged hDHFR immobilized on a SPR sensitive surface. In this case, FA AuNP offer a secondary
amplification of the analytical response which is indirectly proportional to the concentration of MTX. This alternative
approach could contribute to the realization of direct detection of MTX in complex biological fluids. A comparison of
characteristics and analytical parameters such as sensitivity, dynamic range and limit of detection between the LSPR and
SPR sensing platforms will also be presented. Both assays offer potential in tackling real biological samples for the
purpose of monitoring and validating anti-cancer drug levels in human serum during chemotherapy.
The plasmonic properties of metallic nanoparticles and macroscopic Au film have been thoroughly investigated for
the development of biosensors based on surface plasmon resonance (SPR). Nanoparticle based localized surface
plasmon resonance (LSPR) is a technique extremely sensitive to molecular adsorbate, whilst conventional SPR
based on the Kretschmann configuration (macroscopic smooth Au film) is especially sensitive to bulk refractive
index. SPR currently provides the best RI resolution, a measure typically used for comparison of the potential of
plasmonic sensor. A technique that could combine high bulk refractive index resolution and high sensitivity to
molecular adsorbate would increase the scope of SPR-based technique by providing lower detection limits. A
potential solution may exploit micro-structured Au films. However, the plasmonic properties of micropatterned
metallic films are still relatively unknown. We have undertaken the study of the plasmonic properties from Au film
with features on the order of 1 to 3 μm. Microtriangle and microhole arrays were fabricated by modified nanosphere
lithography, consisting of a polymer microsphere mask deposited in a close-packed hexagonal monolayer, etched by
oxygen plasma. Etch time controls the diameter of the microhole and the initial microsphere diameter sets the
periodicity. Investigation of the SPR properties in the Kretschmann configuration was undertaken using a SPR with
a dove prism and a multi-wavelength scanning angle SPR. The sensitivity of SPR with microhole arrays exhibits an
improvement by a factor of 3 in comparison to SPR using a smooth Au film. This is accomplished by tuning the
angle to near 73 degrees (with a BK7 glass prism). Moreover, the sensitivity to the immobilization of an antibody
was improved by at least a factor of 4 as demonstrated with the kinetics of immobilization for IgY, without
employing secondary amplification techniques. No modification to the instrumentation is required and microhole
arrays improve resolution of the SPR response.
Micro-patterned thin films interrogated in the Kretschmann configuration of SPR could extend the detection range to
lower concentrations and small biomolecules due to a greater sensitivity. This was achieved with the same
instrumentation and analysis methodologies developed for SPR with continuous films. The plasmonic properties of
micro-patterned thin films composed of various layers of Ag and Au were investigated to find an optimal structure for
biosensing application. The analytical parameters and biosensing performances were also evaluated for analysis of
biological samples. Au microhole arrays of 3.2 μm periodicity and 1.6 μm hole diameter were prepared using a modified
nanosphere lithography (NSL) technique. These microstructures showed optimal plasmonic properties for biosensing
applications as they exhibit a 50% increase in sensitivity to refractive index changes compared to continuous thin films
of the same thickness. Moreover, microhole arrays presented a faster response time to refractive index changes while
analytical parameters such as the resolution and the noise in biosensing measurement were comparable to continuous
films. When combined to the appropriate surface chemistry, a greater SPR response was measured for proteins using
microhole arrays. Although microhole arrays required an additional preparation step, a cleaning step using oxygen
plasma allowed multiple measurements with the same metallic surface with great repeatability. Hence, microhole arrays
proved to be a simple approach to improve current SPR biosensing technique. Further investigations to understand the
plasmonic properties of microhole were performed using an angle scanning SPR instrument.
The plasmonic nature of discontinuous thin films with micro-patterned structures such as triangles and hole arrays
present distinct optical properties with a Kretschmann surface plasmon resonance (SPR) instrument. Au microstructures
were prepared with a modified nanospheres lithography (NSL) method using 3.2 μm spheres which gives 1.8 μm
triangles and hole arrays with hole diameter ranging from 2.5 to 0.5 μm. The sensitivity to refractive index in thin film is
increased by up to 45% with using microhole arrays instead of continuous film. A transition in the microstructure aspect
from triangles to hole arrays with large hole diameter affects the spectral aspect of the SPR active band. Triangles
present a characteristic broad transmission maximum band while in hole arrays, a broad and weak absorption band first
appears for large holes, which sharpens and increases in intensity as the hole diameter decreases. Moreover, the SPR
penetration depth is tuned between 230 and 30 nm as the microstructure aspect shifts from continuous film, to small hole
arrays and to isolated triangles. Thus, these new plasmonic properties were observed in microhole arrays excited in
Kretschmann SPR configuration, which are spectrally similar to continuous film. These can significantly improve the
existing SPR sensing methods.
This proceeding summarizes the optical properties of plasmonic structures from nanoscale to macroscale. Of particular
interest, Au triangles and hole arrays of near micron size exhibit concomitantly surface plasmon resonance (SPR) and
localized surface plasmon resonance (LSPR) optical properties in the Vis-NIR region, resulting in excellent optical
properties for biosensing. In transmission spectroscopy, 15 nm nanoparticles absorbs at λ = 525 nm, nanotriangles of >
200 nm edge length absorbs at λ > 600 nm while nanohole arrays exhibit a more complex spectrum including absorption
and enhanced optical transmission (EOT) features. Nanohole arrays are also sensitive to refractive index (RI) change and
it can be optimized by tailoring the hole diameter and the periodicity. Au triangles ranging from nano (200 nm) to
micron size (1.5 μm) are active in LSPR with an absorption peak that redshifts with the increasing aspect ratio of the
structure. In total internal reflection (TIR) experiment, Au triangles with an edge length of 500 nm or greater present an
absorption peak at λ = 800 nm. Also, triangles of 700, 950 and 1800 nm have a maximum transmission around λ= 650
nm that is highly sensitive to refraction index (RI) variations. This absorption peak is attributed to propagating SPR,
similarly to the optical phenomenon occurring on a smooth Au film as used in the Kretschmann configuration of SPR.
Lastly, nanohole and microhole arrays spectra, measured in TIR, are a composite of both triangles (LSPR) and thin film
The development of monolayer chemistry based on amino acid and short peptides decreases significantly the nonspecific
adsorption from biological samples such as serum. Nonspecific adsorption of proteins onto the surface of biosensors
currently limits the applicability of many biosensing techniques in real biological samples. In order to minimize this
problem, a methodology to immobilize short peptides on surface plasmon resonance (SPR) biosensors was developed
using a short chain alkyl thiol monolayer derived with the selected peptides. The chain length of the alkane thiol linking
the amino acid to the gold surface influences the physico-chemical properties of the layer and the amount of
nonspecifically adsorbed proteins. Varying the composition of the monolayer with peptides formed from the natural
amino acids investigates the physico-chemical properties required to minimize nonspecific adsorption of serum. It was
observed from monolayers of single amino acids that the composition of the side chain of the amino acid greatly
influences the resistance to nonspecific adsorption, with more polar, ionic and small chains resulting in an improved
performance in biological samples. Building peptides of different lengths resulted in a further decrease of the amount of
nonspecifically bound proteins from serum. Leaving the terminal carboxylic acid end of the peptide unreacted provides
an anchoring point for a molecular receptor in the design of a biosensor. Biosensing will be demonstrated with a model
system of β-lactamase.
Surface Plasmon Resonance (SPR) spectroscopy is a potentially valuable tool for measuring protein-protein interactions and protein levels in vitro and in vivo. Fiber-optic based SPR allows for rapid quantitative measurement of disease markers such as the truncated (exons 1-7) survival of motor neuron (SMN) protein. Unlike micro fluidics-based SPR systems, sample loss is eliminated in fiber-optic SPR and the small size of the fiber optic probes (400μm or smaller) facilitates the potential for use in vivo. Recombinant SMN protein overexpressed in E. Coli as well as native SMN from cultured HeLa cells has been successfully measured using fiber-optic SPR.
Detection of multiple biologically relevant molecules was accomplished at sub-ng/mL levels in highly fouling media using fiber- optic based surface plasmon resonance sensors. Myocardial infarction markers, myoglobin and cTnI, were quantified in full serum with limits of detection below 1 ng/mL. Biologically relevant levels are between 15-30 ng/mL and 1-5 ng/mL for myoglobin and cTnI respectively. Cytokines involved in chronic wound healing, Interleukin 1, Interleukin 6, and tumor necrosis factor α, were detected at around 1 ng/mL in cell culture media. Preliminary results in monitoring these cytokines in cell cultures expressing the cytokines were obtained. The protein diagnostic of spinal muscular atrophy, survival motor neuron protein, was quantified from cell lysate. To obtain such results in complex media, the sensor's stability to non-specific protein adsorption had to be optimized. A layer of the N-hydroxysuccinimide ester of 16-mercaptohexadecanoic acid is attached to the sensor. This layer optimizes the antibody attachment to the sensor while minimizing the non-specific signal from serum proteins.
Multiple layers were attached to the gold surface of surface plasmon resonance (SPR) sensor to maximize the antibody loading and the specific signal of an antigen, while minimizing non-specific signal from full serum proteins. A three-fold improvement of the specific signal from myoglobin and a three-fold decrease of non-specific signal from serum were observed using the N-hydroxysuccinimide ester of 16-mercaptohexadecanoic acid (NHS-MHA) compared to the currently commercially available carboxymethylated dextran. Self-assembled monolayers were attached to the gold surface. 2-mercaptoacetic acid, 3-mercaptopropionic acid, 4,4'-dithiodibutyric acid, 11- mercaptoundecanoic acid, and 16-mercaptohexadecanoic acid were investigated. The covalent attachment of the layers was monitored using SPR and GATR-FTIR. Antibodies to human myoglobin were covalently attached to the sensor using EDC / NHS chemistry and detection of 25 ng/mL myoglobin solution was monitored for the specific signal. Exposing the sensors with the layers to full bovine serum, protein concentration of 72 mg/mL, monitored non-specific signal. NHS-MHA was used to quantify proteins cell culture media with limits of detection below 1 ng/mL.
The development of small surface plasmon resonance (SPR) sensors to detect biological markers for myocardial ischemia (MI), spinal muscular atrophy (SMA), and wound healing was achieved at low ng/mL and in less than 10 minutes. The markers of interest for MIs are myoglobin (MG) and cardiac Troponin I (cTnI). The limits of detection for these markers are respectively 600 pg/mL and 1.4 ng/mL in saline solution. To study SMA, the level of survival motor neuron protein (SMN) was investigated. A limit of detection of 990 pg/mL was achieved for the detection of SMN. The interactions of SMN with MG decreased the signal for both SMN and MG. Interleukin 6 and tumor necrosis factor alpha (TNFa) were investigated to monitor wound healing. The sensor's performance in more complex solutions, e.g.: serum, showed a large non-specific signal. Modifying the support on which the antibodies are attached improved the sensor's stability in serum by a factor of 5. To achieve this non-specific binding (NSB) reduction, different polysaccharides, biocompatible polymers and short chain thiols were investigated.
Surface Plasmon Resonance (SPR) spectroscopy offers many potential industrial applications. SPR sensors are suitable to monitor liquid and gas phase mixtures. The use of fiber-optic SPR sensors enables the possibility of remote sensing in real-time. The sensors can be made as small as 45mm long using 200um optical fibers. Measurement of organic vapors and salinity are demonstrated using the SPR sensors. The mixing dynamics are easily accessible using SPR sensors. The mixing of hexanes and isopropanol in static solution was monitored in real time. Another important application is the analysis of the excess dielectric properties for various binary mixtures using a SPR sensor. Binary mixtures with similar refractive index were measured. Strong deviations from ideality are seen using SPR to monitor the dielectric properties. SPR sensors can be integrated to production lines to monitor the extend of products or compounds inline.
An in vitro sensor to detect cardiac markers at less than 2ng/mL and in less than 5 minutes has been achieved. This fiber optic based Surface Plasmon Resonance (SPR) sensor is being applied to detect myoglobin and cardiac Troponin I. Early detection of the onset of myocardial infarction (MI) will greatly enhance the patient care. Myoglobin (MG) and cardiac Troponin I (cTnI) are two biological markers released after a MI. The detection at biologically relevant levels can be diagnostic of MI. Antibodies specific to the antigen of interest are attached to a carboxymethylated dextran (CM-dextran) gold SPR surface. With the method developed, the detection limit for MG is lower than 5ng/mL when detected at 25°C. For cTnI, a detection limit of less than 2ng/mL was achieved in the preliminary tests. For MG, the reaction conditions for antibody attachment of pH 5-6 and a temperature of 37°C gave the highest sensitivity to MG. At pH 6 and 37°C reaction conditions for the antibody attachment were optimal for cTnI. The CM-dextran chain length also influences the antibody loading on the surface: longer CM-dextran chain gives a higher sensitivity to cTnI. The sensor fouls when in contact with serum. Replacing CM-dextran with different biocompatible polymers greatly improve the sensor performance in serum.
Analytical instruments capable of detecting nerve agents in battlefield conditions where speed, accuracy and ease of operation are a must in today's military. Fast detection and decontamination of nerve agents in very low concentrations is the primary focus of our research. The method presented here focuses on optimizing polymer stabilized sensing elements on the surface of SPR fiber-optic probes. A number of polymers & polymer supported metal complexes capable of reversibly binding to the species of interest & which have robust operation in hostile environments are incorporated with the fiber optic sensing elements. An optical technique, such as Surface Plasmon Resonance (SPR), better suited to rapid data collection without sample pretreatment is employed. The approach using polymer-based optical fibers with off-the-shelf SPR system components has been tested for the detection of Pinacolyl methylphosphonate (PMP), a simulant for nerve agent Soman. Surface initiated polymeric sensors have higher sensitivity toward detecting PMP than bulk-polymerized sensors.