The detection of biomarkers by means of Surface Enhanced Raman Spectroscopy (SERS) is foreseen to became a very important tool in the clinical practice because of its excellent sensitivity and potential for the simultaneous detection of multiple biomarkers. In the present paper we describe how it was possible to build a sensor for the detection of genetic biomarkers involved in acute myeloid leukemia. The assay is based on the use of a specifically designed SERS substrate made of a 2D crystal structure of polymeric pillars embedded in a gold layer. This substrate is characterized by good enhancing properties coupled with an excellent homogeneity. The SERS substrate was conjugated with DNA probes complementary to a target sequence and used in a sandwich assay with gold nanoparticles labeled with a second DNA probe and a Raman reporter. The so developed assay allowed the detection of a leukemia biomarker (WT1 gene) and an housekeeping gene with low picomolar sensitivity. At last, we optimized the assay in order to tackle one of the main limitations of SERS based assay: the loss of signal that is observed when the Raman spectra are collected in liquid. Combining a preferential functionalization on the polymeric pillars with a different height of the polymer pillars from the gold layer the assay demonstrated its effectiveness even when measured in buffer.
Surface-enhanced Raman scattering (SERS) is a potential analytical technique for the detection and identification of chemicals and biological molecules and structures in the close vicinity of metallic nanostructures. We present a novel method to fabricate tunable plasmonic nanostructures and perform a comprehensive structural and optical characterization of the structures. Spherical latex particles are uniformly deposited on glass slides and used as templates to obtain nanovoid structures on polydimethylsiloxane surfaces. The diameter and depth of the nanovoids are controlled by the size of the latex particles. The nanovoids are coated with a thin Ag layer for fabrication of uniform plasmonic nanostructures. Structural characterization of the surfaces is performed by scanning electron microscopy (SEM) and atomic force microscopy (AFM). Optical properties of these plasmonic nanostructures are evaluated via UV/Vis spectroscopy, and SERS. The sample preparation step is the key point to obtain strong and reproducible SERS spectra from the biological structures. When the colloidal suspension is used as a SERS substrate for the protein detection, the electrostatic interaction of the proteins with the nanoparticles is described by the nature of their charge status, which influences the aggregation properties such as the size and shape of the aggregates, which is critical for the SERS experiment. However, when the solid SERS substrates are fabricated, SERS signal of the proteins that are background free and independent of the protein charge. Pros and cons of using plasmonic nano colloids and nanostructures as SERS substrate will be discussed for label-free detection of proteins using SERS.
Diatoms are unicellular algae which have photonic-crystal-like biosilica frustules consisting of many pores. Each diatom frustule has a dimension around 10~20μm and can be used as a miniaturized biosensor. In this article, we demonstrate surface-enhanced Raman scattering (SERS) sensing of sub-nanoliter analyte on diatom biosilica with self-assembled silver nanoparticles (Ag NPs). An inkjet printer is used to dispense multiple ~100 pico-liter volume analyte droplets with pinpoint accuracy and precision onto each individual diatom frustule. Experimental results show up to 3x higher SERS signals of R6G on diatom compared with those from conventional colloidal SERS substrates. Furthermore, down to 10-14M R6G detection ability was also demonstrated through the inkjet printing strategy.
Lymphoma is a heterogeneous group of malignancies of the lymphoid tissue, and is prevalent worldwide affecting both children and adults with a high mortality rate. There is in dire need of accurate and noninvasive approaches for early detection of the disease. Herein, we report a facile way to fabricate silver nanoparticle based nanoprobe by incorporating the corner-stone immunotherapeutic drug Rituxan for simultaneous detection and ablation of lymphoma cells in vitro. The fabricated nanoprobe can detect CD20 positive single lymphoma cell by surface enhanced Raman scattering technique with high specificity. The engineered nanoprobe retains the same antibody property as intact drug via Antibody-Dependent Cell-mediated Cytotoxicity (ADCC) analysis. The nanoprobe efficiently eradicates lymphoma cells in vitro. By integrating the advantages of sensitive SERS detection with targeted ablation capabilities of immunotherapeutic drug through site specificity, this nanoprobe can be applied as outstanding tools in living imaging, cancer diagnosis and treatment.
The principal strength of the confocal microscope for biological imaging lies its ability to detect only light that emerges at close to the focal plane, eliminating light originating from different focal planes. We discuss how this confocal property has considerable advantage in the detection of surface plasmons, since it defines the path of the detected radiation, thus greatly improving the lateral resolution and also the measurement precision. In this paper we show how a spatial light modulator in the back focal plane allows one to generate a whole range of new imaging properties that confer great flexibility on the system. The technique allows one to measure surface wave velocity, surface wave attenuation and perform rapid single shot measurement and effect common path operation.
The resonance wavelength of collective surface plasmon polariton in a chain of 50 nm gold nanoparticles has been calculated and compared to experimental data. The distance between the nanoparticles in a chain was varied from 100 nm to 1000 nm, and the polarization of the incident light was gradually changed from parallel to perpendicular relative to the axis connecting the nanoparticles in the chain. The calculations explicitly included the near-, middle-, and far-field dipole coupling between the nanoparticles. The numerical results matched the experimental data with less than 2% error. Arrays of noble metal nanoparticles are of interest for plasmonics, nanooptics, photovoltaics, and biochemical applications. They are widely used as biosensors and molecular rulers. Over the last decade, interest has turned towards the localized surface plasmon resonance (LSPR) in single-nanoparticle sensors. Benefits of such an approach include simplicity (it does not require momentum-matching geometry), versatility on the nanoscale level, and the possibility of single-molecule detection. While single-nanoparticle sensors offer a better sensitivity down to a single protein-receptor binding, a high degree of sensor miniaturization tends to result in a worse detection limit because of limited surface coverage. A solution to this problem might be the use arrays of nanoplasmonic sensors, each of which is capable of resolving single protein binding events. Present study provides a background for bio-sensing, waveguiding, and molecular ruler applications.
Hospital-acquired bacterial infections are frequently associated with the pathogenic biofilms on surfaces of devices and instruments used in medical procedures. The utilization of thermal plasmonic agents is an innovative approach for sterilizing hospital equipment and for in vivo therapeutic treatment of bacterial infection. A photothermal inactivation technique via array of nanoporous gold disks (NPGDs) has been developed by irradiating near infrared (NIR) light onto deposited bacterial cells (Escherichia coli, Bacillus subtilis, Exiguobacterium AT1B) on the surface of metal nanostructure. The physical and photothermal properties of the NPGD substrate were investigated using topographical scanning electron microscopy (SEM) and thermographic infrared imaging. Bacterial viability studies on NPGD substrates irradiated with and without NIR light were evaluated using a fluorescence-based two-component stain assay. The results show that the heat generated from the NPGD substrate promotes high cell death counts (~100%) at short exposure durations (<25 s) even for thermally-resistant bacterial strains. The photothermal effects on NPGD substrate can lead to point-of-care applications.
We introduce a high resolution scanning surface plasmon microscope for long term imaging of living adherent mouse myoblast cells. The coupling of a high numerical aperture objective lens with a fibered heterodyne interferometer provides both enhanced sensitivity and long term stability. This microscope takes advantage of the plasmon resonance excitation and the amplification of the electromagnetic field in near-field distance to the gold coated coverslip. This plasmon enhanced evanescent wave microscopy is particularly attractive for the study of cell adhesion and motility since it can be operated without staining of the biological sample. We show that this microscope allows very long-term imaging of living samples, and that it can capture and follow the temporal deformation of C2C12 myoblast cell protusions (lamellipodia), during their migration on a at surface.
We demonstrate an ultra-compact on-chip spectrometer for near-infrared (NIR) spectroscopy based on narrow-band band-pass filter array. Each individual filter consists of a plasmonic metallic grating with subwavelength period and extremely narrow slits on a quartz substrate, with a polymer cover layer as the waveguide layer. A narrow-band guided-mode resonance (GMR) associated with a surface-plasmon resonance (SPR) gives rise to the narrow-band transmission spectrum. Full width at half maximum (FWHM) of fabricated filter’s spectrum is measured to be from 7 to 13 nm, and the operation bandwidth of the entire filter array covers wavelength range over 270 nm from 1510 to 1780 nm. We measure the NIR absorbance spectrum of xylene using our filter array device to demonstrate its application as a spectrometer.
Plasmonic nanoparticles have several applications ranging from catalysis to super-resolution imaging and information storage. Maximum density of optical states is confined on the nanoparticle surface, which are collectively excited by electromagnetic wave and are called surface plasmons. Using nanoparticle based plasmonic interaction with biological cells in an optical fiber integrated microfluidic chip, we show enhancement of fluorescence intensity. Signal from in situ imaging is analyzed with various controls to understand the mechanism. The present study is focused on nanoparticle interaction with cells and on optimization strategies to maximize the fluorescence enhancement at the vicinity of the nanoparticles, for important applications such as fluorescence-based biochip platforms. Result is also correlated ZnO nanoparticle effect on fluorescence enhancement, which has different optoelectronic properties compared to gold nanoparticles. Electromagnetic wave field model is employed to simulate the effect of gold and ZnO nanoparticle on cell with the assumption that the nanoparticles are a collection of discrete dipoles, which are ordered with the fluorescence molecules on cell wall. Simulation model shows enhancement of fluorescence intensity is occurred in presence of gold nanoparticles rather than ZnO nanoparticles, which is confirmed with experimental data.
In this study, we report photoacoustic (PA) measurements of gold-covered polystyrene nanoparticles (Au nanoshells). Two types of Au nanoshells were examined: 1) polystyrene core with sparsely covered Au nanoparticles, and 2) polystyrene core which were fully covered by Au nanoparticles. The fully covered Au nanoshell exhibited a broad extinction cross section (500 nm – 850 nm), which is in the first infrared optical window where light transmission is optimal for optical based studies in tissues. The optical properties were compared to numerical simulations using Mie scattering theory. Using a photoacoustic microscope, the PA signal measured from fully covered Au nanoshells was 1.27 ± 0.18 mV per fluence (mJ/cm2), which was 10x greater than the PA signal from sparsely covered Au nanoshells (0.12 ± 0.14 mV). These novel Au nanoshell nanoparticles can be used for multispectral optical and PA imaging.
Nanohole array surface plasmon resonance (SPR) sensors offer a promising platform for high-throughput label-free biosensing. Integrating nanohole arrays with group-IV semiconductor photodetectors could enable low-cost and disposable biosensors compatible to Si-based complementary metal oxide semiconductor (CMOS) technology that can be combined with integrated circuitry for continuous monitoring of biosamples and fast sensor data processing. Such an integrated biosensor could be realized by structuring a nanohole array in the contact metal layer of a photodetector. We used Fouriertransform infrared spectroscopy to investigate nanohole arrays in a 100 nm Al film deposited on top of a vertical Si-Ge photodiode structure grown by molecular beam epitaxy (MBE). We find that the presence of a protein bilayer, constitute of protein AG and Immunoglobulin G (IgG), leads to a wavelength-dependent absorptance enhancement of ~ 8 %.
Demonstrated herein is a simple method for the induction of J-aggregate formation in a colloidal solution of gold nanoparticles through the use of pseudoisocyanine (PIC) and polyvinyl sulfate. The plasmon-exciton coupling of the nanoparticle J-aggregate complex results in a split lineshape absorption spectrum with upper and lower plexcitonic branches. The use of nanoparticles with various plasmon resonances causes a shift in the upper plexcitonic band while the lower plexcitonic band remains at the same wavelength.
For the purpose of point of care (POC), a disposable polymer-molding prism with two parabolic side surfaces is employed for the ultra compact SPRI biosensor. A compact SPRI biosensor downsized to a form factor of 20 cm *15 cm*5 cm with extremely high sensitivity and large dynamic range is proposed in this study. With the cost effective and disposable polymer-molding prism design, the cross contamination between samples can be avoided. In this demonstration, we integrated the CCD detection system and multichannel fluidic system into this device that allows users to quickly screen various samples simultaneously.
Scattering cross-section of metal nanoparticles is enhanced due to Localized Surface Plasmons Resonance (LSPR) permitting the observation of single metal nanoparticles as small as 40 nm using dark-field microscopy. Single particle resolved measurements allow the study of reactions happening on the nanoparticle surface involving an ultra-low number of reactant molecules to understand stochastic effects in reactive systems. Here we report a method to enhance the intensity of resonantly scattered light by using appropriately designed substrates. Specifically, we show that by using a multi-layer dielectric substrate with its high reflectance window spanning the LSPR resonance position, one can increase the intensity of scattered light by nearly an order of magnitude. We took three substrates namely Silicon, glass and the multilayer dielectric mirror. Disk shaped gold nanostructures with sizes ranging from 80 nm – 300 nm were fabricated using electron beam lithography on all three substrates. Sizes of individual nanostructures were determined by atomic force microscopy (AFM) and the dark-field image of each nanostructure was taken with an optical microscope. It was observed that the intensity of light scattered by single nanparticles was roughly an order magnitude larger than that from Silicon and glass substrates. We used a numerical scheme based on Discrete Dipole Approximation to computationally validate our results. The numerical results matched the experiments quite well. The substrate enhanced scattering signal will useful to improve the signal to noise ratio in single particle resolved measurements.
Since surface-enhanced Raman spectroscopy (SERS) makes it possible to enhance weak Raman signals which represent molecular own vibrational transition as a fingerprint, it has gotten much attention in the field of biosensor. Although SERS can detect specific molecules with high sensitivity and selectivity, it is still difficult to fabricate efficient SERS substrates, align ‘hot-spot’ with a detection site, and increase reproducibility for molecular sensing. Here, we converged plasmonic trapping with conventional SERS in order to overcome these drawbacks. As plasmonic trapping is to move nano particles toward the desired position by electric field gradient, we could trap gold nano particles (GNPs) onto a raw bowtie substrate and fabricate self-aligned hot-spots by using plasmonic trapping, which is directly contributed to enhancing weak signals by shortening structure-to-structure distances. Also, since a united laser was used to trap GNPs and to detect target molecules at the same time, it was possible to directly obtain Raman signal on the self-aligned hotspots. To further verify our technique, we also conducted numerical analysis for electric field distribution and trapping force by using finite element method and the results were well matched with the experimental data. This increases low reproducibility of SERS and as a result, we could repetitively obtain same results.
Surface-enhanced Raman scattering (SERS) is a powerful technique used for characterization of biological and nonbiological molecules and structures. Since plasmonic properties of the nanomaterials is one of the most important factor influencing SERS activity, tunable plasmonic properties (wavelength of the surface plasmons and magnitude of the electromagnetic field generated on the surface) of SERS substrates are crucial in SERS studies. SERS enhancement can be maximized by controlling of plasmonic properties of the nanomaterials. In this study, a novel approach to fabricate tunable plasmonic 3D nanostructures based on combination of soft lithography and nanosphere lithography is studied. Spherical latex particles having different diameters are uniformly deposited on glass slides with convective assembly method. The experimental parameters for the convective assembly are optimized by changing of latex spheres concentration, stage velocity and latex particles volume placed between to two glass slides that staying with a certain angle to each other. Afterwards, polydimethylsiloxane (PDMS) elastomer is poured on the deposited latex particles and cured to obtain nanovoids on the PDMS surfaces. The diameter and depth of the nanovoids on the PDMS surface are controlled by the size of the latex particles. Finally, fabricated nanovoid template on the PDMS surfaces are filled with the silver coating to obtain plasmonic 3D nanostructures. Characterization of the fabricated surfaces is performed by scanning electron microscopy (SEM) and atomic force microscopy (AFM). SERS performance of fabricated 3D plasmonic nanostructures will be evaluated using Raman reporter molecules.
Surface-enhanced Raman scattering (SERS) is an emerging technique for the detection and identification of biological structures. SERS is based on immunoassay methods are mostly used for the specific detection and identification of bacteria. In this study, SERS substrates are developed with deposition of synthesized spherical 13 nm gold nanoparticles (AuNPs) and 50 nm silver nanoparticles (AgNPs) on regular glass slides with convective assembly method for SERS based immunoassay for the detection and identification of bacteria. The synthesized NPs are characterized by UV-vis absorption spectroscopy, dynamic light scattering (DLS) and atomic force microscopy (AFM). Colloidal suspensions are concentrated by centrifugation to obtain thin films by the deposition of NPs on a regular glass slide with the convective assembly. The experimental parameters for the convective assembly are optimized by changing of NP concentration, stage velocity and NPs volume dropped between two glass slides. Structural characterization of thin films is performed by AFM and SEM. SERS is also used for the optical characterization of the prepared thin films of NPs. In this study, 4- aminothiophenol (4-ATP) is used as probe molecules to evaluate SERS activity of the thin films depending on the type and concentration of NPs. The results demonstrate that, SERS performances of the thin films are dependent on not only the type of NPs but also it depends on the concentration of NPs which forms thin films. The thin film having highest SERS activity could be used for the SERS-based immunoassays for the detection and identification of bacteria.
The controlled assembly of plasmonic nanoparticles by a molecular binding event has emerged as a simple yet sensitive methodology for protein detection. Metallic nanoparticles (NPs) coated with functionalized aptamers can be utilized as biosensors by monitoring changes in particle optical properties, such as the LSPR shift and enhancement of the SERS spectra, in the presence of a target protein. Herein we test this method using two modified aptamers selected for the protein biomarker interleukin 6, an indicator of the dengue fever virus and other diseases including certain types of cancers, diabetes, and even arthritis. IL6 works by inducing an immunological response within the body that can be either anti-inflammatory or pro-inflammatory. The results show that the average hydrodynamic diameter of the NPs as measured by Dynamic Light Scattering was ~42 nm. After conjugation of the aptamers, the peak absorbance of the AgNPs shifted from 404 to 408 nm indicating a surface modification of the NPs due to the presence of the aptamer. Lastly, preliminary results were obtained showing an increase in SERS intensity occurs when the IL-6 protein was introduced to the conjugate solution but the assay will still need to be optimized in order for it to be able to monitor varying concentration changes within and across the desired range.
Locally amplified near-fields can be induced with nanostructures within a sub-diffraction-limited volume, which is useful for biomedical imaging and sensing applications. Employment of field localization in the biomedical applications where the pulsed light is used necessitates the spatial and temporal characteristics of fields near nanostructures. We considered the gold nano-post arrays of three different shapes to localize the near-fields which are circular, rhombic, and triangular. They were modeled to be located on an ITO film and a quartz substrate with periods changing from 300 to 900 nm by 200 nm. Their size changes from 50 to 250 nm which corresponds to the radius for the case of circular nanoposts and the distance between the center and the vertices for equilateral rhombic and triangular nanoposts. Numerical calculation of near-fields at the top of nanoposts was performed with finite difference time domain method when the Gaussian pulses at center wavelengths of 532, 633, and 850 nm were normally incident. Near-fields localization occurred mainly at vertices of the nanoposts, which makes the triangular nanoposts of primary interest with an observation of the strongest field intensity within a diffraction limited field-of-view. The observed fields on the triangular vertices were enhanced by 7.85, 51.54, and 7268 when the center wavelengths were 532, 633, and 850 nm respectively. Their temporal peaks were delayed by 2.05, 4.03, and 14.49 fs, which indicates the correlation between field enhancement and time delay associated with electron damping process. It was shown that with rhombic and triangular nanoposts fields can be localized below 10 nm on vertices and their signal-to-noise ratio increased with a larger period.