Label-free Localized Surface Plasmon Resonance Research Articles

PH-Modulated Nanoarchitectonics for Enhancement of Multivalency-Induced Vesicle Shape Deformation at Receptor-Presenting Lipid Membrane Interfaces.

Multivalent ligand-receptor interactions between receptor-presenting lipid membranes and ligand-modified biological and biomimetic nanoparticles influence cellular entry and fusion processes. Environmental pH changes can drive these membrane-related interactions by affecting membrane nanomechanical properties. Quantitatively, however, the corresponding effects on high-curvature, sub-100 nm lipid vesicles are scarcely understood, especially in the multivalent binding context. Herein, we employed the label-free localized surface plasmon resonance (LSPR) sensing technique to track the multivalent attachment kinetics, shape deformation, and surface coverage of biotin ligand-functionalized, zwitterionic lipid vesicles with different ligand densities on a streptavidin receptor-coated supported lipid bilayer under varying pH conditions (4.5, 6, 7.5). Our results demonstrate that more extensive multivalent interactions caused greater vesicle shape deformation across the tested pH conditions, which affected vesicle surface packing as well. Notably, there were also pH-specific differences, i.e., a higher degree of vesicle shape deformation was triggered at a lower multivalent binding energy in pH 4.5 than in pH 6 and 7.5 conditions. These findings support that the nanomechanical properties of high-curvature lipid membranes, especially the membrane bending energy and the corresponding responsiveness to multivalent binding interactions, are sensitive to solution pH, and indicate that multivalency-induced vesicle shape deformation occurs slightly more readily in acidic pH conditions relevant to biological environments.

Silica-coated gold nanorods biofunctionalization for localized surface plasmon resonance (LSPR) biosensing

We introduce here the engineering of nanobiosensors designed from gold nanorods coated with an ultrathin layer of silica (AuNR@SiO2) and biofunctionalized with antibodies for the Localized Surface Plasmon Resonance (LSPR) biosensing of proteins. Despite the outstanding properties of AuNRs, their use for LSPR biosensing is limited due to the presence of the surfactant cetyltrimethylammonium bromide (CTAB) – mandatory for their synthesis - which forms a strongly-bounded and positively-charged bilayer at their surface and significantly complicates their bio-functionalization. When coated with a thin layer of silica, these nanomaterials exhibit an improved sensitivity to refractive index change which augurs for better analytical performances. Here, we undertook an in-depth investigation of the biofunctionalization of AuNR@SiO2via three different routes to design and test a label-free LSPR biosensor operating in solution. In the first route, we took advantage of the negatively charged external silica shell to immobilize anti-rabbit IgG antibody by electrostatic physisorption. In the second and third routes, the silica surface was reacted with thiol or aldehyde terminated silanes, subsequently utilized to covalently attach anti-rabbit IgG antibody to the surface. The resulting nanoprobes were characterized by a wide range of physical methods (TEM, XPS, DLS, ELS and UV-Visible spectroscopy) then tested for the biosensing of rabbit-IgG. The three nanobiosensors maintain an excellent colloidal stability after analyte recognition and exhibit extremely high analytical performances in terms of specificity and dynamic range, with an LoD down to 12 ng/mL.

Fabrication and measurement of a fiber optic localized surface plasmon resonance sensor chip for molecular diagnostics

For several decades, the field of molecular diagnostics has been dominated by polymerase chain reaction technology, which usually entails relatively expensive equipment, trained laboratory staff, and problems in time management. Advances in molecular diagnostics aim to provide rapid tests, miniaturized devices, increased user convenience, and high sensitivity. The real-time label-free localized surface plasmon resonance (LSPR) biosensing technique merges with a small optical fiber and is combined with a microfluidic channel. This fiber optic LSPR sensor chip has the advantages of easy handling, isolation from the external environment, and continuous transport of sample fluid. The microfluidic channel can also accelerate hybridization by supplying the target to the sensor surface in succession. The conditions for the binding of probe deoxyribonucleic acids (DNAs) on gold nanoparticles are modified. Hybridization of the immobilized probe DNA and target DNA was analyzed using the proposed sensor and the results showed a good response to DNA hybridization with a limit of detection (LOD) level up to 12.3 ng/mL (1.5 nM). The LOD was compared with other LSPR-based DNA sensors and improvement was found. The proposed sensor responded with high linearity (R2 of 0.94) as a function of change in the concentration of the target DNA. Suitable reproducibility for biosensor applications was confirmed with an average coefficient of variation of 12.5 %.

Label-Free Localized Surface Plasmon Resonance Biosensor Used to Detect Serum Interleukin-10 in Patients with Endometrial Cancer

Label-Free Localized Surface Plasmon Resonance Biosensor Used to Detect Serum Interleukin-10 in Patients with Endometrial Cancer

Label-free biomechanical nanosensor based on LSPR for biological applications

A label-free localized surface plasmon resonance (LSPR)-based biosensor exploiting gold nanorods (GNRs) is proposed and demonstrated. For this aim, 35 ± 5 nm long and 20 ± 4 thick GNRs spaced by a few nanometers thick polyelectrolytes (PE) from a gold thin film was analyzed and synthesized. The morphology of the GNRs, the plasmon properties of GNRs, swelling of PE layers and the wettability of the surfaces were characterized by transmission and scanning electron microscopy, spectroscopic reflectivity and contact angle measurements, respectively. Indeed, when immersed in a phosphate buffer saline solution, the GNRs-PE-gold system shows an optical shift of the LSPR wavelength. This shift was found to correspond to a vertical swelling of about 2 nm, demonstrating the extreme sensitivity of the biosensor. Finally, we show that LSPR measurements can be used to detect dynamic resonance changes in response to both thickness and buffer solution, while the hydrophobic behavior of the surface can be exploited for reducing the number of liquid analytes in clinical biosensing application.

Composite-Scattering Plasmonic Nanoprobes for Label-Free, Quantitative Biomolecular Sensing.

Biosensing based on localized surface plasmon resonance (LSPR) relies on concentrating light to a nanometeric spot and leads to a highly enhanced electromagnetic field near the metal nanostructure. Here, a design of plasmonic nanostructures based on rationally structured metal-dielectric combinations is presented, called composite scattering probes (CSPs), to generate an integrated multimodal biosensing platform featuring LSPR and surface-enhanced Raman spectroscopy (SERS). Specifically, CSP configurations are proposed, which have several prominent resonance peaks enabling higher tunability and sensitivity for self-referenced multiplexed analyte sensing. Using electron-beam evaporation and thermal dewetting, large-area, uniform, and tunable CSPs are fabricated, which are suitable for label-free LSPR and SERS measurements. The CSP prototypes are used to demonstrate refractive index sensing and molecular analysis using albumin as a model analyte. By using partial least squares on recorded absorption profiles, differentiation of subtle changes in refractive index (as low as 0.001) in the CSP milieu is demonstrated. Additionally, CSPs facilitate complementary untargeted plasmon-enhanced Raman measurements from the sample's compositional contributors. With further refinement, it is envisioned that the method may lead to a sensitive, versatile, and tunable platform for quantitative concentration determination and molecular fingerprinting, particularly where limited a priori information of the sample is available.

Label-free localized surface plasmon resonance biosensor composed of multi-functional DNA 3 way junction on hollow Au spike-like nanoparticles (HAuSN) for avian influenza virus detection

In the present study, we fabricated a label-free avian influenza (AIV H5N1) detection biosensor composed of a multi-functional DNA 3 way-Junction (3 W J) on a hollow Au spike-like nanoparticle (hAuSN) using a localized surface plasmon resonance (LSPR) method. To construct the multi-functional DNA (MF-DNA) as a bioprobe, the 3 W J was introduced. The proposed AIV detection bioprobe should contain three functionalities: target recognition, signal amplification, and connection to substrate. To achieve this goal, each piece of the DNA 3 W J was tailored to a hemagglutinin (HA) binding aptamer, FAM dye and thiol group, respectively. The assembly of each DNA 3 W J functional fragment was then confirmed by TBM-Native PAGE. Moreover, the hAuSN was immobilized on the indium-tin-oxide (ITO) substrate for LSPR measurement. The DNA 3 W J was immobilized onto the hAuSN electrode through the thiol-group of DNA 3 W J. The fabricated DNA 3 W J/hAuSN heterolayer on the ITO substrate was investigated by field emission scanning electron microscopy (FE-SEM) and atomic force microscopy (AFM). LSPR experiments were conducted to confirm HA protein binding to the DNA 3 W J/ hAuSN -modified electrode. The proposed biosensor can detected the HA protein in PBS buffer (LOD: 1 pM) as well as in the diluted chicken serum (LOD: 1 pM). The present study details a label-free, simple fabrication method consisted of DNA 3 W J/ hAuSN heterolayer that uses easy-to-tailor elements to detect not only AIV but also various viruses detection platform easily.

In Situ Detection and Imaging of Telomerase Activity in Cancer Cell Lines via Disassembly of Plasmonic Core-Satellites Nanostructured Probe.

The label-free localized surface plasmon resonance (LSPR) detection technique has been identified as a powerful means for in situ investigation of biological processes and localized chemical reactions at single particle level with high spatial and temporal resolution. Herein, a core-satellites assembled nanostructure of Au50@Au13 was designed for in situ detection and intracellular imaging of telomerase activity by combining plasmonic resonance Rayleigh scattering spectroscopy with dark-field microscope (DFM). The Au50@Au13 was fabricated by using 50 nm gold nanoparticles (Au50) as core and 13 nm gold nanoparticles (Au13) as satellites, both of them were functionalized with single chain DNA and gathered proximity through the highly specific DNA hybridization with a nicked hairpin DNA (O1) containing a telomerase substrate (TS) primer as linker. In the presence of telomerase, the telomeric repeated sequence of (TTAGGG)n extended at the 3'-end of O1 would hybridized with its complementary sequences at 5'-ends. This led the telomerase extension product of O1 be folded to form a rigid hairpin structure. As a result, the Au50@Au13 was disassembled with the releasing of O1 and Au13-S from Au50-L, which dramatically decreased the plasmon coupling effect. The remarkable LSPR spectral shift was observed accompanied by a detectable color change from orange to green with the increase of telomerase activity at single particle level with a detection limit of 1.3 × 10-13 IU. The ability of Au50@Au13 for in situ imaging intracellular telomerase activity, distinguishing cancer cells from normal cells, in situ monitoring the variation of cellular telomerase activity after treated with drugs were also demonstrated.

Dielectric functionalization for differential phase detecting localized surface plasmon resonance biosensor

In this paper, a novel dielectric functionalization approach is introduced for differential phase detecting localized surface plasmon resonance (LSPR) immunoassay biosensor, which exhibits high sensitivity. The plasmonic “hot spots” at the nanoscale dielectric gaps among self-assembly gold nanoislands were first functionalized. These “hot spots” are believed to be highly sensitive to localized refractive index change and the phase response induced by the binding event at these functionalized “hot spots” is monitored by our common-path spectral interferometer during biosensing. Its performance was compared with that from conventional gold functionalization protocol. It was found that the dielectric functionalization approach attained limit of detection of the same order as that from conventional gold functionalization, i.e. 1pM of human IgG antigen. The novel protocol was also applied to detection of the antigen in serum samples. To our knowledge, these are two orders of magnitude improvement over existing label-free SPR biosensors. In view of the myriad of emerging plasmonic materials in the foreseeable future, the dielectric functionalization approach would provide a convenient protocol for label-free LSPR biosensing, as it would be generally applicable to any plasmonic material on glass substrate.

A Label-free Technique for the Spatio-temporal Imaging of Single Cell Secretions.

Inter-cellular communication is an integral part of a complex system that helps in maintaining basic cellular activities. As a result, the malfunctioning of such signaling can lead to many disorders. To understand cell-to-cell signaling, it is essential to study the spatial and temporal nature of the secreted molecules from the cell without disturbing the local environment. Various assays have been developed to study protein secretion, however, these methods are typically based on fluorescent probes which disrupt the relevant signaling pathways. To overcome this limitation, a label-free technique is required.In this paper, we describe the fabrication and application of a label-free localized surface plasmon resonance imaging (LSPRi) technology capable of detecting protein secretions from a single cell. The plasmonic nanostructures are lithographically patterned onto a standard glass coverslip and can be excited using visible light on commercially available light microscopes. Only a small fraction of the coverslip is covered by the nanostructures and hence this technique is well suited for combining common techniques such as fluorescence and bright-field imaging.A multidisciplinary approach is used in this protocol which incorporates sensor nanofabrication and subsequent biofunctionalization, binding kinetics characterization of ligand and analyte, the integration of the chip and live cells, and the analysis of the measured signal. As a whole, this technology enables a general label-free approach towards mapping cellular secretions and correlating them with the responses of nearby cells.

A Label-free Technique for the Spatio-temporal Imaging of Single Cell Secretions

Inter-cellular communication is an integral part of a complex system that helps in maintaining basic cellular activities. As a result, the malfunctioning of such signaling can lead to many disorders. To understand cell-to-cell signaling, it is essential to study the spatial and temporal nature of the secreted molecules from the cell without disturbing the local environment. Various assays have been developed to study protein secretion, however, these methods are typically based on fluorescent probes which disrupt the relevant signaling pathways. To overcome this limitation, a label-free technique is required. In this paper, we describe the fabrication and application of a label-free localized surface plasmon resonance imaging (LSPRi) technology capable of detecting protein secretions from a single cell. The plasmonic nanostructures are lithographically patterned onto a standard glass coverslip and can be excited using visible light on commercially available light microscopes. Only a small fraction of the coverslip is covered by the nanostructures and hence this technique is well suited for combining common techniques such as fluorescence and bright-field imaging. A multidisciplinary approach is used in this protocol which incorporates sensor nanofabrication and subsequent biofunctionalization, binding kinetics characterization of ligand and analyte, the integration of the chip and live cells, and the analysis of the measured signal. As a whole, this technology enables a general label-free approach towards mapping cellular secretions and correlating them with the responses of nearby cells.

Integrated Nanoplasmonic Sensing for Cellular Functional Immunoanalysis Using Human Blood

Localized surface plasmon resonance (LSPR) nanoplasmonic effects allow for label-free, real-time detection of biomolecule binding events on a nanostructured metallic surface with simple optics and sensing tunability. Despite numerous reports on LSPR bionanosensing in the past, no study thus far has applied the technique for a cytokine secretion assay using clinically relevant immune cells from human blood. Cytokine secretion assays, a technique to quantify intercellular-signaling proteins secreted by blood immune cells, allow determination of the functional response of the donor’s immune cells, thus providing valuable information about the immune status of the donor. However, implementation of LSPR bionanosensing in cellular functional immunoanalysis based on a cytokine secretion assay poses major challenges primarily owing to its limited sensitivity and a lack of sufficient sample handling capability. In this paper, we have developed a label-free LSPR biosensing technique to detect cell-secreted tumor necrosis factor (TNF)-α cytokines in clinical blood samples. Our approach integrates LSPR bionanosensors in an optofluidic platform that permits trapping and stimulation of target immune cells in a microfluidic chamber with optical access for subsequent cytokine detection. The on-chip spatial confinement of the cells is the key to rapidly increasing a cytokine concentration high enough for detection by the LSPR setup, thereby allowing the assay time and sample volume to be significantly reduced. We have successfully applied this approach first to THP-1 cells and then later to CD45 cells isolated directly from human blood. Our LSPR optofluidics device allows for detection of TNF-α secreted from cells as few as 1000, which translates into a nearly 100 times decrease in sample volume than conventional cytokine secretion assay techniques require. We achieved cellular functional immunoanalysis with a minimal blood sample volume (3 μL) and a total assay time 3 times shorter than that of the conventional enzyme-linked immunosorbent assay (ELISA).

Using U-Shaped Localized Surface Plasmon Resonance Sensors to Compensate for Nonspecific Interactions

We report a simple label-free localized surface plasmon resonance sensor that uses the multiple resonances of a U-shaped gold nanostructure to differentiate the target interaction of interest from background refractive index interference and nonspecific binding of interfering molecules. U-shaped nanostructures were fabricated using electron-beam lithography and their sensing capabilities were tested by introducing different solutions to simulate various specific and nonspecific effects. The three resonances of the nanostructure yield distinct bulk as well as specific and nonspecific surface sensitivities that allow for the differentiation of the three effects.

A label-free immunosensor for determination of salbutamol based on localized surface plasmon resonance biosensing

We developed a localized surface plasmon resonance (LSPR)-based label-free optical biosensor for detection of salbutamol (Sal). Hollow gold nanoparticles (HGNs) which deposited on transparent indium tin oxide (ITO) film coated glass was used to sensing platform. Antibody against Sal was immobilized on HGN surface to recognize the target Sal molecules. Thus, the change of LSPR peak was proportional to the concentration of Sal in the solution. The experimental results demonstrated that the LSPR immunosensor possessed a good sensitivity and a high selectivity for Sal. The detection range for Sal was from 0.05 to 0.8μg/mL with a correlation coefficient of 0.996. The biosensor was applied for the detection for Sal in spiked animal feed and pork liver samples, and the recoveries were in the range of 97-105%. Therefore, it is expected that this approach may offer a new method in designing label-free LSPR immunosensor for detection of small molecules.

DNA functionalized gold nanorods/nanoplates assembly as sensitive LSPR-based sensor for label-free detection of mercury ions

Core–satellite assembly, i.e., gold nanoplates (AuNPs) surrounded by gold nanorods (AuNRs), has been obtained by Thymine–Hg2+–Thymine (T–Hg2+–T) coordination. A novel label-free localized surface plasmon resonance (LSPR) detection of mercury ions can be performed via the core–satellite assembly. The proposed detection protocol exhibits good linear correlation between wavelength shifts of the plasmon band and concentrations of mercury ions over the range from 10nM to 0.2μM. Moreover, the detection limit (3σ) for the probes is 2.2nM, which is better than colorimetric detection via random aggregation of gold nanoparticles.

Magnetic Nanoparticle Mediated Enhancement of Localized Surface Plasmon Resonance for Ultrasensitive Bioanalytical Assay in Human Blood Plasma

We demonstrate that Fe(3)O(4) magnetic nanoparticle (MNP) can greatly enhance the localized surface plasmon resonance (LSPR) of metal nanoparticle. The high refractive index and molecular weight of the Fe(3)O(4) MNPs make them a powerful enhancer for plasmonic response to biological binding events, thereby enabling a significant improvement in the sensitivity, reliability, dynamic range, and calibration linearity for LSPR assay of small molecules in a trace amount. Rather than using fluorescence spectroscopy or magnetic resonance imaging, this study marks the first use of the label-free LSPR nanosensor for a disease biomarker in physiological solutions, providing a low cost, clinical-oriented detection. This facile and ultrasensitive nanosensor with an extremely light, robust, and low-cost instrument is attractive for miniaturization on a lab-on-a-chip system to deliver point-of-care medical diagnostics. To further evaluate the practical application of Fe(3)O(4) MNPs in the enhancement of LSPR assay, cardiac troponin I (cTnI) for myocardial infarction diagnosis was used as a model protein to be detected by a gold nanorod (GNR) bioprobe. MNP-captured cTnI molecules resulted in spectral responses up to 6-fold higher than direct cTnI adsorption on the GNR sensor. The detection limit (LOD) was lowered to ca. 30 pM for plasma samples which is 3 orders lower than a comparable study. To the best of our knowledge, this marks the lowest LOD for a real plasma protein detection based on label-free LSPR shift without complicated instrumentation. The observed LSPR sensing enhancement by Fe(3)O(4) MNPs is independent of nonspecific binding.