Concentration detection of quantum dots in the visible and near-infrared range based on surface plasmon resonance sensor

We employ an optical sensor based on surface plasmon resonance (SPR) operating in the near-infrared and in the visible range to determine the concentration of CdSe/ZnS core-shell quantum dots (QDs) which are embedded in the SU8 organic films. Attenuated total reflection (ATR) measurements show that the amplitude of the shift of the resonance dip is closely related to the concentration variation of QDs in the organic films and the incident laser. The sensitivity is enhanced by 1.5-time and the detect limitation is expanded to 10⁻⁵ μmol/L in the visible range as compared to that in the near-infrared. The sensitivity enhancement and the expansion of detect limitation of the visible SPR sensor may originate from the coupling of surface plasmons to luminescence from QDs.     

Aqueous CdPbS quantum dots for near-infrared imaging

Quantum dots (QDs) are semiconducting nanocrystals that have photoluminescent (PL) properties brighter than fluorescent molecules and do not photo-bleach, ideal for in vivo imaging of diseased tissues or monitoring of biological processes. Near-infrared (NIR) fluorescent light within the window of 700-1000 nm, which is separated from the major absorption peaks of hemoglobin and water, has the potential to be detected several millimeters under the surface with minimal interference from tissue autofluorescence. Here we report the synthesis and bioimaging demonstration of a new NIR QDs system, namely, CdPbS, made by an aqueous approach with 3-mercaptopropionic acid (MPA) as the capping molecule. The aqueous-synthesized, MPA-capped CdPbS QDs exhibited an NIR emission in the range of 800-950 nm with x(i) ≥ 0.3, where x(i) denotes the initial Pb molar fraction during the synthesis. Optimal PL performance of the CdPbS QDs occurred at x(i) = 0.7, which was about 4 nm in size as determined by transmission electron microscopy, had a rock salt structure and a quantum yield of 12%. Imaging of CdPbS QDs was tested in membrane staining and transfection studies. Cells transfected with CdPbS QDs were shown to be visible underneath a slab of chicken muscle tissue of up to 0.7 mm in thickness without the use of multiple-photon microscopy.     

Near infrared surface plasmon resonance phase imaging and nanoparticle-enhanced surface plasmon resonance phase imaging for ultrasensitive protein and DNA biosensing with oligonucleotide and aptamer microarrays

The techniques of surface plasmon resonance-phase imaging (SPR-PI) and nanoparticle-enhanced SPR-PI have been implemented for the multiplexed bioaffinity detection of proteins and nucleic acids. The SPR-PI experiments utilized a near-infrared 860 nm light emitting diode (LED) light source and a wedge depolarizer to create a phase grating on a four-element single-stranded DNA (ssDNA) microarray; bioaffinity adsorption onto the various microarray elements was detected via multiplexed real time phase shift measurements. In a first set of demonstration experiments, an ssDNA aptamer microarray was used to directly detect thrombin at concentrations down to 100 pM with SPR-PI. Two different ssDNA aptamers were used in these experiments with two different Langmuir adsorption coefficients, K(A1) = 4.4 × 10(8) M(-1) and K(A2) = 1.2 × 10(8) M(-1). At concentrations below 1 nM, the equilibrium phase shifts observed upon thrombin adsorption vary linearly with concentration with a slope that is proportional to the appropriate Langmuir adsorption coefficient. The observed detection limit of 100 pM is approximately 20 times more sensitive than that observed previously with SPRI. In a second set of experiments, two short ssDNA oligonucleotides (38mers) were simultaneously detected at concentrations down to 25 fM using a three-sequence hybridization format that employed 120 nm DNA-modified silica nanoparticles to enhance the SPR-PI signal. In this first demonstration of nanoparticle-enhanced SPR-PI, the adsorbed silica nanoparticles provided a greatly enhanced phase shift upon bioaffinity adsorption due to a large increase in the real component of the interfacial refractive index from the adsorbed nanoparticle. As in the case of SPR-PI, the detection limit of 25 fM for nanoparticle-enhanced SPR-PI is approximately 20 times more sensitive than that observed previously with nanoparticle-enhanced SPRI.     

Wavelength-scanning surface plasmon resonance imaging

A new surface plasmon resonance (SPR) imaging technique was proposed. After measurements were conducted at varying wavelengths, the wavelength affording the minimum brightness (SPR wavelength) was determined at each pixel of the image. A two-dimensional map of the SPR wavelength could be converted to a thickness profile by use of a nonlinear calibration curve, which was obtained by Fresnel calculation. An array of protein thin layers on a gold film was evaluated in air to present the layers' surface structure in nanometer scale.     

Enzymatically amplified surface plasmon resonance imaging method using RNase H and RNA microarrays for the ultrasensitive detection of nucleic acids

A novel surface enzymatic amplification method that utilizes RNA microarrays in conjunction with the enzyme RNase H is developed for the ultrasensitve detection and analysis of target DNA molecules. The enzyme RNase H is shown to selectively and repeatedly destroy RNA from RNA-DNA heteroduplexes on gold surfaces; when used in conjunction with the label-free technique of surface plasmon resonance imaging, multiple DNA targets can be detected at a concentration of 10 fM on a single chip. In addition, this method is utilized for the sequence-specific detection of the TSPY gene in both purified and unpurified PCR products. Finally, in a series of kinetics measurements, the initial rate of hydrolysis is shown to depend directly on the surface concentration of DNA-RNA heteroduplexes.     

Quantitative analyses of absorption-sensitive surface plasmon resonance near-infrared spectra

Surface plasmon resonance near-infrared (SPR-NIR) spectroscopy provides 10-100 times absorption enhancement compared with the absorption in the corresponding attenuated total reflection (ATR) NIR spectra. However, analysis of the enhanced SPR-NIR spectra is not straightforward because of the substantial contribution from SPR. This paper proposes two analysis methods for concentration-dependent changes of SPR-NIR spectra from a viewpoint of change in absorption intensity. One is based on rapid scans of the SPR-NIR spectra with a fixed incident angle, and the other is based on multi-angle sequential scans. A concentration of methanol in water has successfully been determined by both methods. From the measurement of the light intensity within an absorption band of water (5230-5120 cm(-1)) at a fixed incident angle, the concentration was calibrated to an accuracy of 0.02 wt. %. In the latter multi-angle method, it has been proved that computed bottom ridges of the envelope curve of the SPR-NIR spectra are not only enhanced 30 times compared with the corresponding ATR-NIR spectra, but are also equivalent to the conventional transmittance NIR spectra in quality. The bottom ridges allow us to analyze SPR-NIR spectra in the same manner as conventional spectral analyses based on Beer's law.     

Long-range surface plasmon resonance imaging for bioaffinity sensors

A novel bioaffinity sensor based on surface plasmon resonance (SPR) imaging measurements of a multiple-layered structure that supports the generation of long-range surface plasmons (LRSPs) at the water-metal interface is reported. LRSPs possess longer surface propagation lengths, higher electric field strengths, and sharper angular resonance curves than conventional surface plasmons. LRSPR imaging is a version of SPR imaging that requires a symmetric dielectric arrangement around the gold thin film. This arrangement is created using an SF10 prism/Cytop/gold/water multilayer film structure where Cytop is an amorphous fluoropolymer with a refractive index very close to that of water. LRSPR imaging experiments are performed at a fixed incident angle and lead to an enhanced response for the detection of surface binding interactions. As an example, the hybridization adsorption of a 16-mer single-stranded DNA (ssDNA) onto a two-component ssDNA array was monitored with LRSPR imaging. The ssDNA array was created using a new fabrication technology appropriate for the LRSPR multilayers.     

Differential surface plasmon resonance imaging for high-throughput bioanalyses

A new imaging technique for high-throughput surface plasmon resonance (SPR) measurements is described. It is the application of a CCD camera for simultaneous processing of two images at two different wavelengths provided by two laser diodes. The two lasers are brought to resonance by tuning of the angle of incidence so that the detection power and the dynamic range are optimized for the wavelength pair selected. Applying a special differential processing of the two images, SPR measurements can be performed near the shot noise limit taking into account the number of CCD pixels involved. It is shown that the detection limit of imaging methods can be improved significantly if the working point is set near to the reflection minimum instead of choosing the angle with the steepest slope of the reflection curve. The technique is demonstrated by simultaneous measurement of hybridization reactions of three different types of thiolated oligonucleotides in 30 small areas set by a commercial spotter. A noise level of 1.5 x 10(-6) refractive index units (RIU) was obtained for single, 500 x 500 microm2 reaction areas. The noise level was about 6 x 10(-7) RIU when five areas were taken into account. The present arrangement and the particular spotter applied would allow simultaneous measurements of up to 400 binding reactions with a noise level of about 1.5 x 10(-6) RIU.     

Peptide-labeled near-infrared quantum dots for imaging tumor vasculature in living subjects

We report the in vivo targeting and imaging of tumor vasculature using arginine-glycine-aspartic acid (RGD) peptide-labeled quantum dots (QDs). Athymic nude mice bearing subcutaneous U87MG human glioblastoma tumors were administered QD705-RGD intravenously. The tumor fluorescence intensity reached maximum at 6 h postinjection with good contrast. The results reported here open up new perspectives for integrin-targeted near-infrared optical imaging and may aid in cancer detection and management including imaging-guided surgery.     

Sensing characteristics of NIR localized surface plasmon resonances in gold nanorings for application as ultrasensitive biosensors

The optical responses of 75-150 nm diameter gold nanorings to changes in local refractive index have been quantified by near-infrared extinction spectroscopy and compared to DDA calculations and an analytical approach. The "bulk" refractive index sensitivities of gold nanorings are substantially (>5 times) larger than those of nanodisks with similar diameters. Nanorings retain a significantly larger sensitivity than nanodisks at the same spectral position, demonstrating a clear shape dependence that may correlate to a systematic difference in the influence of the dielectric substrate. The nanoring bulk refractive index sensitivity scales linearly with plasmon peak position. The spectral sensitivity to thin films of alkanethiols gave a shift of approximately 5.2 nm/CH2 unit while bulk sensitivities as high as 880 nm/RIU were observed, the highest such reported sensitivities. Both bulk and thin dielectric film sensitivities correlated well with theory. Real-time label-free monitoring of protein binding via molecular recognition was demonstrated.     

Determination of surface selection rule of surface plasmon resonance near-infrared spectroscopy by using a Langmuir-Blodgett film

Near-infrared (NIR) absorption spectra of a cadmium arachidate Langmuir-Blodgett (LB) film were measured by surface plasmon resonance near-infrared spectroscopy (SPR-NIRS) based on the Kretschmann configuration with a 18.8-nm gold film. An NIR spectrum enhanced severalfold was obtained as a top ridge of the SPR-NIR spectra measured at different incident angles by using the principle of absorption-sensitive SPR. In order to determine the surface selection rule of SPR-NIRS, the enhanced NIR absorption spectrum of the LB film was compared to an unenhanced one without the gold film and to a normal incidence transmission spectrum. Moreover, a pair of out-of-plane (OP) and in-plane (IP) spectra were obtained by multiangle infrared spectroscopy analysis from a series of oblique incidence transmission measurements in the NIR region. It became obvious that the salient feature of the enhanced NIR absorption spectrum, i.e., the top ridge of the SPR-NIR spectra is almost equivalent to that of the OP spectrum. On the other hand, the unenhanced spectrum showed IP modes. These experimental results were well explained by calculation of the mean-square electric field based on the Fresnel formula.     

Fiber-optic surface plasmon resonance sensors in the near-infrared spectral region

The sensitivity of fiber-optic surface plasmon resonance (SPR) sensors was improved by a factor of at least thirteen for aqueous solutions by modifying the tip geometry to allow interrogation of the surface plasmon (SP) band in the near-infrared (NIR) region. This was achieved by tuning the angle at the distal end of the SPR sensor to a dual taper of 71 degrees and 19 degrees . Using a low numerical aperture (NA) fiber-optic sensor, NA = 0.12, is necessary to obtain a functional SPR sensor working in the NIR region. Theoretical simulations using the Maxwell equations demonstrated that even higher enhancement is theoretically possible while maintaining a narrow spectral feature upon the excitation of the SP bands on gold surfaces. The manufacture of the SPR sensors yields good agreement between theoretical simulations and experimental observations. To investigate the properties of these fiber-optic SPR-NIR sensors, sucrose solutions ranging from 0 to 15 x 10(-3) in mole fraction were utilized. The increased sensitivity of the fiber-optic SPR sensors, when used to monitor biomarkers, would yield lower detection limits. The smaller sensing area, compared to planar or other fiber-optic SPR sensors, combined with an improvement of the sensitivity, would yield a dramatic reduction of the absolute amount detected by biosensors.     

Parallel scan spectral surface plasmon resonance imaging

We describe a parallel scan spectral surface plasmon resonance (SPR) imaging technique. We demonstrate experimentally, with a line-shaped light illumination, that an image acquired with an area CCD detector provides both SPR wavelength information and one-dimensional spatial distribution. Thus two-dimensional distribution of the refractive index of the entire sensing plane can be obtained with a one-dimensional optical line parallel scan. The technique offers advantages of both high sensitivity and high throughput, and could have potential applications in biochips analysis.     

High-throughput immunophenotyping by surface plasmon resonance imaging

Cell binding assays on antibody arrays permit the rapid immunophenotyping of living cells. The throughput of the analysis, however, is still limited due to our inability to perform parallel and quantitative detection of cells captured on the array. To address this limitation, we employed here an imaging technique based on surface plasmon resonance (SPR). SPR has been frequently used to monitor capture of proteins on antibody microarrays, while few cases were reported for capture of cells. Antibody arrays were prepared through the photopatterning of an alkanethiol monolayer on a gold-evaporated glass plate and the subsequent immobilization of various antibodies onto 4-9 separate spots created by photopatterning. A glass slip was mounted onto the array with a thin spacer to construct a parallel-plate chamber. Leukemia cells were injected into the chamber to conduct a binding assay, while refractive index changes at the vicinity of the array surface were monitored by SPR imaging. We observed that SPR signals were intensified on specific antibody spots but not on nonspecific spots. Confocal laser scanning microscopy revealed that the observed SPR signals were attributed to cell deformations caused by multivalent interactions with immobilized antibody, which effectively elevated the refractive index of a medium phase within an evanescent field. This effect could be suitably utilized to monitor quantitatively cell binding to multiple spots from a heterogeneous cell population.     

Surface plasmon resonance near-infrared spectroscopy

Near-infrared (NIR) spectroscopy is ill-suited to microanalysis because of its low absorptivity. We have developed a highly sensitive detection method for NIR spectroscopy based on absorption-sensitive surface plasmon resonance (SPR). The newly named SPR-NIR spectroscopy, which may open the way for NIR spectroscopy in microanalysis and surface science, is realized by an attachment of the Kretschmann configuration equipped with a mechanism for fine angular adjustment of incident light. The angular sweep of incident light enables us to make a tuning of a SPR peak for an absorption band of sample medium. From the dependences of wavelength, incident angle, and thickness of a gold film on the intensity of the SPR peak, it has been found that the absorbance can be enhanced by approximately 100 times compared with the absorbance obtained without the gold film under optimum conditions. This article reports the details of the experimental setup and the characteristics of absorption-sensitive SPR in the NIR region, together with some experimental results obtained by using it.     

Nanoparticle-enhanced diffraction gratings for ultrasensitive surface plasmon biosensing

Ultrasensitive surface bioaffinity sensors are created by the adsorption of gold nanoparticles onto gold diffraction gratings. An enhanced diffraction obtained in a surface plasmon resonance geometry is observed due to the optical coupling of the planar surface plasmons in the grating to the localized surface plasmons in the gold nanoparticles. As a first example, these nanoparticle grating biosensors are employed to detect unmodified DNA at a concentration of 10 fM.     

Field-matter integral overlap to estimate the sensitivity of surface plasmon resonance biosensors

We have analyzed the effectiveness of field-matter integral overlap between target index distribution and local near-fields to assess detection sensitivity of surface plasmon resonance (SPR) biosensors. The correlation of the overlap with sensitivity was clear. An overlap integral defined with lateral electric field intensity produced the highest correlation due to tangential continuity across a boundary. Among the three detection scenarios considered, the correlation for localized SPR sensing was slightly lower than that of thin film-based detection and improved with an increased fill factor in the structure. The results will be useful to maximize the optical signature created by target interactions and to produce highest sensitivity of SPR detection to variations when target or field distribution is not uniform.     

Nonregeneration protocol for surface plasmon resonance: study of high-affinity interaction with high-density biosensors

Surface plasmon resonance (SPR) has been used in determining kinetics and thermodynamics of biological interaction in the past decades. One difficulty encountered in this technology is the need for a proper regeneration, which means the removal of analytes from the bound complexes to regenerate the activity of the ligands. Regeneration is not always practical since the harsh regeneration reagents may destroy the bioactivity of the ligands. It is even more difficult for complexes with high affinity constants. In this paper, we report a nonregeneration protocol for SPR techniques in which subsequent ligand/analyte interactions can be measured without regeneration; thus ligand biological activity could be retained. Kinetics, binding models, and mathematics of this protocol are discussed in detail using rabbit IgG as the analyte and engineered recombinant antibody A10B single-chain fragment variables (scFv) as the ligand. The affinity constant of rabbit IgG binding with A10B scFv measured by using a nonregeneration protocol was (2.5 +/- 0.2) x 10(7) M(-1), which was comparable with the value determined with a conventional regeneration SPR method ((2.2 +/- 1.5) x 10(7) M(-1)) and quartz crystal microbalance (1.9 x 10(7) M(-1)). A paradigm of streptavidin-biotin binding was analyzed to validate this protocol. The affinity constant for each binding subunit of streptavidin to the immobilized biotin was determined to be (7.3 +/- 0.2) x 10(6) M(-1), which was comparable with the solution-based value of 2 x 10(7) M(-1). The nonregeneration protocol requires a relatively high ligand density on the biosensor surface so that more data points can be obtained before surface saturation. The small size of scFv enables them to be constructed in the biosensors for such purpose.     

A kinetic study of analyte-receptor binding and dissociation for surface plasmon resonance biosensors applications

A fractal analysis, which takes into account the effect of surface heterogeneity brought about by ligand immobilization on the reaction kinetics in surface plasmon resonance (SPR) biosensors, is presented. The binding and dissociation of estrogen receptors (ERs), ERa and ER/spl alpha/ and ER/spl beta/, in solution to different ligands immobilized on the SPR biosensor is analyzed within the fractal framework. The heterogeneity on the biosensor surface is made quantitative by using a single number, the fractal dimension D/sub f/. The analysis provides physical insights into the binding of these receptors to different ligands and compounds, particularly the endocrine disrupting compounds (EDCs). These EDCs have deleterious effects on humans and on wildlife. Single- and dual-fractal models were employed to fit the ER-binding data obtained from the literature. Values of the binding and dissociation rate coefficient and fractal dimensions were obtained from a regression analysis provided by Corel Quattro Pro, 8.0. Values for the affinity K/sub D/(=k/sub d//k/sub a/) were also calculated. This provides us with some extra flexibility in designing biomolecular assays. The analysis should provide further information on the mode of action and interaction of EDCs with the ERs. This would help in the design of agents and modulators against these EDCs.     

Deep UV nano-microstructuring of substrates for surface plasmon resonance imaging

In this paper, we describe wafer-scale fabrication and characterization of plasmonic chips-containing different sizes and spacings of metallic micro- and nanoline structures-using deep UV lithography. Using a high dose (25 mJ cm( - 2)) and a proper lift-off process, feature sizes as small as 25 nm are obtained. Moreover, we study the dependence of surface plasmon resonance on the angle of incidence and wavelength for different micro- and nanoline size and spacing values, yielding localized to quasi-propagative plasmonic behaviors. Rigorous coupled wave analysis (RCWA) techniques are employed to numerically confirm these experimental observations. Finally, the refractive index of media around the SPRI sensor chips is varied, showing the angulo-spectral regions of higher sensitivity for each type of structure.