Quantitative methods for spatially resolved adsorption/desorption measurements in real time by surface plasmon resonance microscopy

A simple method for converting local reflectivity changes measured in surface plasmon resonance (SPR) microscopy to effective adlayer thicknesses and absolute surface coverages of adsorbed species is presented. For a range of high-contrast angles near the SPR resonance where the local metal surface's reflectivity changes linearly with angle, the change in reflectivity at fixed angle is proportional to the change in effective refractive index (eta(eff)) near the surface. This change in eta(eff) can be converted to absolute adsorbate coverage using methods developed for quantitative SPR spectroscopy. A measurement of the change in reflectivity due to changes in refractive index of bulk solutions, i.e., percent reflectivity change per refractive index unit (RIU), is the only calibration required. Application of this method is demonstrated for protein adsorption onto protein/DNA arrays on gold from aqueous solution using an SPR microscope operating at 633 nm. A detection limit of 0.072% change in absolute reflectivity is found for simultaneous measurements of all 200 microm x 200 microm areas within the 24-mm(2) light beam with 1-s time averaging. This corresponds to a change in effective refractive index of 1.8 x 10(-5) and a detection limit for protein adsorption of 1.2 ng/cm(2) (approximately 0.5 pg in a 200-microm spot). The linear dynamic range is Deltaeta(eff) = approximately 0.011 RIU or approximately 720 ng/cm(2) of adsorbed protein. Using a nearby spot as a reference channel, one can correct for instrumental drift and changes in refractive index of the solutions in the flow cell.     

Surface plasmon resonance sensor based on polarization interferometry and angle modulation

A surface plasmon resonance (SPR) sensing technique based on polarization interferometry and angle modulation is presented. Its sensitivity is not a direct function of variation of reflection intensity, nor of phase shift. Rather, it is a function of the complex reflection coefficient. A three times standard deviation detection limit of 5.1 x 10(-7) refractive index units in a 2 Hz bandwidth is obtained with our experimental setup. A theoretical analysis shows that this technique can provide a wide linear measurement range. Moreover, the sensitivity is insensitive to the thickness of gold films over approximately 5 nm. This SPR sensing technique is suitable for physical, chemical, and biological research.     

Polymeric dual-slab waveguide interferometer for biochemical sensing applications

A polymer based dual-slab waveguide Young's interferometer was demonstrated for biochemical sensing. Evanescent field is utilized for probing the binding events of biomolecules on the waveguide surface. Refractive index sensing in analyte and protein adsorption on the sensing surface were investigated with glucose de-ionized water solution and bovine serum albumin, immunoglobulin G solutions in phosphate buffered saline buffer. A detection limit of 10(-5) RIU and 4 pg/mm(2) was achieved for homogeneous and surface sensing, respectively. Also, the influence of water absorption inside the polymeric device on the measurement stability was evaluated. The results indicate that the waveguide polymer sensor fabricated with the spin coating technique can achieve a satisfactory sensitivity for homogeneous refractive index sensing and, as well, for monitoring molecular binding events on the surface.     

Miniaturization of a homogeneous fluorescence immunoassay based on energy transfer using nanotiter plates as high-density sample carriers.

The miniaturization of a homogeneous competitive immunoassay to a final assay volume of 70 nL is described. As the sample carrier, disposable plastic nanotiter plates (NTP) with dimensions of 2 x 2 cm2 containing 25 x 25 wells, corresponding to approximately 15,000 wells on a traditional 96-well microtiter plate footprint, were used. Sample handling was accomplished by a piezoelectrically actuated micropipet. To reduce evaporation while pipetting the assays, the NTP was handled in a closed humid chamber and cooled to the point of condensation. To avoid washing steps, a homogeneous assay was developed that was based on energy-transfer (ET). As a model system, an antibody-based assay for the detection of the environmentally relevant compound, simazine, in drinking water was chosen. Antibodies were labeled with the long-wavelength-excitable sulfoindocyanine dye Cy5 (donor), and a tracer was synthesized by labeling BSA with a triazine derivative and the acceptor dye Cy5.5. At low analyte concentrations, the tracer was preferably bound to the antibody binding sites. As a result of the close proximity of Cy5.5 and Cy5, an efficient quenching of the Cy5 fluorescence occurred. Higher analyte concentrations led to a progressive binding of the analyte to the antibody binding sites. The increased Cy5 fluorescence was determined by using a scanning laser-induced fluorescence detector. The limit of detection (LOD), using an antibody concentration of 20 nM, was 0.32 microg/L, or 1.11 x 10(-16) mol of simazine. In comparison, the LOD of the 96-well microtiter-plate-based ET immunoassay (micro-ETIA) was 0.15 microg/L, or 1.87 x 10(-13) mol. The LOD of the optimized micro-ETIA at 1 nM IgG, was 0.01 microg/L.     

Improving the instrumental resolution of sensors based on localized surface plasmon resonance

The colorimetric variations induced upon changes in interfacial refractive index of nanoscale noble metal structures exhibiting localized surface plasmon resonance (LSPR) provides a convenient means of label-free, affinity-based detection of biomolecular recognition reactions. However, despite being similar in nature to conventional SPR, LSPR has so far suffered from significantly lower data quality in terms of its signal-to-noise ratio (S/N) in typical biomolecular recognition analysis. In this work, generic data analysis algorithms and a simple experimental setup that provide a S/N upon protein binding that is comparable to that of state-of-the art SPR systems are presented. Specifically, it is demonstrated how temporal variations (rate approximately 0.5 Hz) in parameters proportional to the resonance peak position can be recorded simultaneously, yielding a peak position precision of <5 x 10(-4) nm and an extinction noise level of <5 x 10(-6) absorbance units (Abs). This, in turn, is shown to provide a S/N of approximately 2000 (equivalent to a detection limit of <0.1 ng/cm(2)) for typical protein binding reactions. Furthermore, the importance of utilizing changes in both peak position and magnitude is highlighted by comparing different LSPR active noble metal architectures that respond differently to bulk and interfacial refractive index changes.     

Ultrahigh molar mass component detected in ethylhydroxyethyl cellulose by asymmetrical flow field-flow fractionation coupled to multiangle light scattering.

Asymmetrical flow field-flow fractionation (flow FFF) was connected to multiangle light scattering (MALS) and refractive index (RI) detectors for characterization of the molar mass distribution and molecular radius of a cellulose derivative, ethylhydroxyethyl cellulose (EHEC). Experimental conditions were optimized to allow study of a wide range of molar mass including even ultrahigh molar mass (UHM) components. The weight-average molar mass was 3.1 x 10(5) g x mol(-1) representing a very broad range (of molar mass) from 4.0 x 10(4) to 10(7) g x mol(-1), which corresponds to from <20 to 200 nm rms radius. The light scattering signal showed the presence of an UHM component, possibly an aggregate of extreme size, i.e., approximately 10(8) g x mol(-1) with a hydrodynamic diameter of 0.35 microm. Careful choice of the pore size in in-line filters is necessary in order to minimize MALS detector noise without removing the UHM component. Flow FFF-MALS-RI was demonstrated to be uniquely suited to detect the presence of UHM components.     

Determination of the refractive indices of layers in a multilayer stack by a guided-wave technique

The m-lines technique is used to measure the refractive indices and thicknesses of layers embedded in a multilayer stack. The multilayer considered is deposited by ion plating. Its formula is silica-H-L-H-L-H-air, where H and L denote Ta(2)O(5) and SiO(2)lambda/4 layers, respectively, with lambda = 514.5 nm. Measurements indicate that the refractive index of Ta(2)O(5) is 5 x 10(-3) greater when the layer is close to air than when the layer is inside the coating and that the Ta(2)O(5) is slightly more birefringent.     

Design and characteristics of refractive index sensor based on thinned and microstructure fiber Bragg grating

A refractive index sensor based on the thinned and microstructure fiber Bragg grating (ThMs-FBG) was proposed and realized as a chemical sensing. The numerical simulation for the reflectance spectrum of the ThMs-FBG was calculated and the phase shift down-peak could be observed from the reflectance spectrum. Many factors influencing the reflectance spectrum were considered in detail for simulation, including the etched depth, length, and position. The sandwich-solution etching method was utilized to realize the microstructure of the ThMs-FBG, and the photographs of the microstructure were obtained. Experimental results demonstrated that the reflectance spectrum, phase shift down-peak wavelength, and reflected optical intensity of the ThMs-FBG all depended on the surrounding refractive index. However, only the down-peak wavelength of the ThMs-FBG changed with the surrounding temperature. Under the condition that the length and cladding diameter of the ThMs-FBG microstructure were 800 and 14 mum, respectively, and the position of the microstructure of the ThMs-FBG is in the middle of grating region, the refractive index sensitivity of the ThMs-FBG was 0.79 nm/refractive index unit with the wide range of 1.33-1.457 and a high resolution of 1.2 x 10(-3). The temperature sensitivity was 0.0103 nm/ degrees C, which was approximately equal to that of common FBG.     

Localized surface plasmon resonance biosensor using silver nanostructures fabricated by glancing angle deposition

Glancing angle deposition was used to produce approximately 150-nm-thick silver nanoparticle films, which were evaluated as localized surface plasmon resonance (LSPR) biosensors. The films have a strong extinction peak around 368 nm in air due to LSPR. As the refractive index of the surrounding environment is increased, the extinction peak red-shifts with a linear dependence. The films were functionalized with 11-amino-1-undecanethiol and rabbit immunoglobulin G (rIgG) to allow for the detection of anti-rIgG binding. Binding of biomolecules to the nanoparticle surface increases the local refractive index and results in a red-shifting of the extinction peak. The wavelength shift at varying concentrations of anti-rIgG was measured and fit to the Langmuir isotherm. This yielded approximate values for the saturation response, Delta lambda max = 29.4 +/- 0.7 nm, and the surface confined binding constant, Ka = (2.7 +/- 0.3) x 10(6) M(-1). The response to nonspecific binding was also investigated.     

Enhanced middle-infrared light transmission through Au/SiO(x)N(y)/Au aperture arrays

The enhanced middle-infrared light transmission through Au/SiO(x)N(y)/Au aperture arrays by changing the refractive index and the thickness of a dielectric layer was studied experimentally. The results indicated that the transmission spectra was highly dependent on the refractive index and the thickness of SiO(x)N(y). We found that the transmission peaks redshifted regularly along with the refractive index from 1.6 to 1.8, owing to the role of surface plasmon polaritons (SPP) coupling in the Au/SiO(x)N(y)/Au cascaded metallic structure. Simultaneously, a higher transmission efficiency and narrower transmission peak was obtained in Au/SiO2.1N0.3/Au cascaded metallic structure with small refractive index (1.6) than in Au/SiO0.6N1/Au cascaded metallic structure with large refractive index (1.8). When the thickness of SiO(x)N(y) changes from 0.2 to 0.4 microm, the shape of transmission spectra exhibits a large change. It was found that a higher transmission efficiency and narrower transmission peak was obtained in Au/SiO(x)N(y)/Au cascaded metallic structure with a thin dielectric film (0.2 microm), with the increase of SiO(x)N(y) film's thickness, the transmission peak gradually widened and disappeared finally. This effect is useful in applications of biochemical sensing and tunable integrated plasmonic devices in the middle-infrared region.     

Systematic characterization of spectral surface plasmon resonance sensors with absorbance measurement

Spectral surface plasmon resonance (SPR) sensors with absorbance measurement were prepared. Resonant wavelengths (lambda) versus effective refractive indexes of the SPR mode were measured with different media in contact with the gold layer. An investigation into the refractive-index sensitivity of the sensor at a fixed angle reveals a linear dependence of lambda(R) on the refractive index of the solution (n(c)), with Dlambda /Dn(c) = 3553.6 nm in a small range of 1.333< or = n(c) < or =1.347. It was observed that the effective refractive index slowly decreases with increasing n(c), attributable to wavelength-induced modulation of optical dielectric constant for the gold layer. Adsorption of bromothymol blue (BTB) on the gold layer leads to a redshift of Dlambda(R) = 3.7 nm, larger than Dlambda(R) = 2.5 nm induced by myoglobin (Mb) adsorption. On the basis of Fresnel equations, calculations with d approximately 1 nm and n=1.69 for BTB and d approximately 3 nm and n = 1.40 for Mb also demonstrate that the SPR band shift induced by full-monolayer adsorption of BTB is larger than that for full-monolayer Mb adsorption. The combination of both measured and calculated results suggests that the contribution of the adlayer index of refraction to the sensitivity of the sensor is greater than that of the adlayer thickness.     

长程表面等离子体共振技术在检测大肠杆菌浓度中的应用研究

建立了一种基于长程表面等离子体共振技术检测大肠杆菌浓度的方法及系统。基于此,制备了能够产生长程表面等离子体共振效应的双层膜传感芯片,并在实验上将长程表面等离子体共振(LSPR)和传统表面等离子体共振(CSPR)两种生物传感器的性能进行了对比。结果表明LSPR生物传感器共振曲线的平均半高宽比CSPR传感器共振曲线的平均半高宽窄1.79倍,且其灵敏度是CSPR的2倍。由此,证实了基于LSPR的生物传感器对大肠杆菌浓度的改变更加敏感。此外,该方法分辨率高,试剂用量少,有效克服分界面所带来的影响,并能够对大肠杆菌进行实时检测。     

Surface plasmon resonance sensors based on uniform-waist tapered fibers in a reflective configuration

We present a configuration for surface plasmon resonance sensors based on uniform-waist tapered optical fibers and reflective elements. Once the fiber is tapered fulfilling the adiabatic criterion, a multilayer including a metallic medium is asymmetrically deposited on the uniform waist of the fiber. This feature provides the resonant excitation of multiple surface plasma waves. In addition, a mirror is produced at the fiber tip by a chemical Tollens reaction. In this way, the sensor operates in a reflective mode, more convenient for dip probes. When these sensors are spectrally interrogated, a high sensitivity of 10-4 refractive index units per nanometer is attained. These devices can be advantageously used for any kind of chemical sensing and biosensing.     

Use of radiation and hybrid modes to increase the accuracy in the determination of the refractive indices of rutile

A new method to correlate prism and substrate refractive indices is presented and applied to the determination of rutile refractive indices, both ordinary and extraordinary, at different wavelengths in the visible and the near infrared. The method exploits radiation modes and, when the sample structure allows it, hybrid modes. An accuracy of approximately 2 x 10(-4) was achieved without the need of any specific preparation of the samples. The method exhibits a good versatility since it can be exploited for the determination of the refractive index of a coupling prism when the substrate index is known and vice versa.     

Side-polished multimode fiber biosensor based on surface plasmon resonance with halogen light

A side-polished multimode fiber sensor based on surface plasmon resonance (SPR) as the transducing element with a halogen light source is proposed. The SPR fiber sensor is side polished until half the core is closed and coated with a 37 nm gold thin film by dc sputtering. The SPR curve on the optical spectrum is described by an optical spectrum analyzer and can sense a range of widths in wavelengths of SPR effects. The measurement system using the halogen light source is constructed for several real-time detections that are carried out for the measurement of the index liquid detections for the sensitivity analysis. The sensing fiber is demonstrated with a series of refractive index (RI) liquids and set for several experiments, including the stability, repeatability, and resolution calibration. The results for the halogen light source with the resolution of the measurement based on wavelength interrogation were 3 x 10(-6) refractive index units (RIUs). The SPR dip shifted in wavelength is used as a measure of the RI change at a surface, and this RI change varies directly with the number of biomolecules at the surface. The SPR dip shift in wavelength, which was hybridized at 0.1 microM of the target DNA to the probe DNA, was 8.66 nm. The all-fiber multimode SPR sensor, which has the advantages of being low cost, being disposable, having high stability and linearity, being free of labeling, and having potential for real-time detection, permit the sensor and system to be used in biochemical sensing and environmental monitoring.     

Surface plasmon resonance spectroscopic study of UV-addressable phenylalanine sensing based on a self-assembled spirooxazine derivative monolayer

Light-addressable compounds are very interesting due to the possibilities of their practical use such as optical switches and memories or variable transmission materials. For example, transportation of phenylalanine across liposomal bilayers mediated by a photoresponsive carrier like spirooxazine through electrostatic interaction between phenylalanine and spirooxazine derivative. Thus, the spirooxazine is expected to form a UV-addressable phenylalanine sensing interface.In this study, we prepared phenylalanine sensing interface of a spirooxazine derivative by self-assembly technique and evaluated interaction between a spirooxazine moiety and phenylalanine with a surface plasmon resonance (SPR). The refractive index change of monolayer caused by interaction between a spirooxazine derivative and phenylalanine led to the SPR angle shifts upon UV irradiation. The SPR angle shift increased with increasing the concentration of phenylalanine solution. These results indicated that the spirooxazine derivative self-assembled monolayer (SAM) has an application potential for UV-addressable phenylalanine sensing.     

Decoupling refractive index and geometric thickness from interferometric measurements of a quartz sample using a fourth-order polynomial

A Michelson interferometer setup was used to determine refractive index and thickness of a fused-quartz sample with no knowledge of either parameter. At small angles, < 10 degrees, the interferometer equation follows a fourth-order polynomial in the sample refractive index alone, effectively decoupling the sample thickness from the equation. The incident angle of the He-Ne laser beam versus fringe shift was fitted to the polynomial, and its coefficients obtained. These were used to determine refractive index to within 6 x 10(-4) of the known value with an accuracy of +/- 1.3%. Sample thickness was determined to an accuracy of +/-2.5%. Reproducibility of the rotating table was determined to be +/-2 x 10(-3) degrees.     

Measurement of the refractive index of distilled water from the near-infrared region to the ultraviolet region

By the minimum deviation method using a prism shaped cell, the absolute refractive indices of high-performance liquid chromatography distilled water were measured at the wavelengths from 1129 to 182 nm, at the temperature of 19 degrees C, 21.5 degrees C, and 24 degrees C, and then dn/dt at 21.5 degrees C was calculated. The coefficients of the four-term Sellmeier dispersion formula were determined by using the refractive indices at each temperature. As a result of the comparison of our refractive index data in the visible wavelength region with the formula by Tilton et al. at the National Bureau of Standards in 1938, both the refractive index data corresponded within 6 x 10(-6). In the UV region, the absolute refractive index at 193.39 nm calculated by the data measured nearby the wavelengths from 200 to 190 nm was 1.436517 (21.5 degrees C). The value was lower by 9 x 10(-5) or 10 x 10(-5) than the data measured by Burnett et al. at the National Institute of Standards and Technology.     

Cross-correlation of optical microcavity biosensor response with immobilized enzyme activity. Insights into biosensor sensitivity

Porous silicon multilayer structures have remarkable optical and morphological properties that can be exploited for biosensing. In particular, a high internal surface area (>100 m(2)/cm(3)) and a linear response profile to changes in the dielectric environment enable fabrication of sensitive devices and a straightforward quantitation of the optical response. These essential operating characteristics are illustrated for p+ mesoporous silicon (pore diameter 15-20 nm) optical microcavities. A series of devices were prepared to permit the immobilization of glutathione-S-transferase ( approximately 50 kDa) within the porous matrix. Enzyme activity was exploited as an indirect means to quantitate the amount of protein immobilized. Activity was positively correlated with the optical sensor response. However, at high enzyme load the activity becomes nonlinear while the microcavity response remains linear. These data were used to determine the transduction limit (minimum amount of protein required to transduce an optical response), which is reported as areal mass sensitivity ranging between 50 and 250 pg/mm(2). This value is considered in context with the dynamic range of the bulk sensitivity, defined as the magnitude of the wavelength shift per refractive index unit, which was measured as a function of microcavity design parameters. This work has uncovered key parameters that can be tuned to improve the detection limit of this sensor modality. Because of the ever increasing number of emerging new biosensor technologies, defining sensor detection limits has become an ambiguous topic and a need exists to standardize measurements and sensitivity units. For chip-based devices, it seems appropriate to report sensitivity in terms of the minimum number of grams of bound target per surface area.     

Sensitive Label-Free Immunoassay by Colorimetric Plasmonic Biosensors Using Silver Nanodome Arrays

The sensitive direct detection of biomolecules is demonstrated by a colorimetric plasmonic biosensor utilizing the surface colors of plasmonic metasurfaces named Ag nanodome arrays. The Ag nanodome arrays consist of polystyrene bead monolayers coated with Ag thin films whose surface colors are optimized by changing the size of the polystyrene beads. The bulk refractive index sensitivity of colorimetric detection evaluated using the hue angle is 590° RIU⁻¹ (RIU: refractive index unit). For selected geometry, the refractive index resolution (5.0 × 10⁻⁵ RIU) obtained by colorimetric detection surpasses that of spectroscopic detection evaluated via the dip wavelength in the reflection spectrum. The numerical simulations predict an enhanced sensing performance when the hue angle of the surface colors of the Ag nanodome arrays changes from 300° to 200°, corresponding to changes in the dip wavelength from 570 to 600 nm in the reflection spectra. Furthermore, the detection sensitivity of the biomolecules is characterized using a direct IgG immunoassay format. The detection limit of the IgG concentration is as low as 134 pM using simple colorimetric detection. The feasibility of sensitive label-free immunoassays using a colorimetric plasmonic biosensor is expected to accelerate the development of highly sensitive and reliable smartphone-based plasmonic biosensors.