Localized surface plasmon resonance based gold nanobiosensor: Determination of thyroid stimulating hormone

An immunoassay method based on the peak shift of the localized surface plasmon resonance (LSPR) absorption maxima has been developed for the determination of the thyroid stimulating hormone (TSH) in human blood serum. The anti-TSH antibody was adsorbed on the synthesized gold nanoparticles by electrostatic forces. The efficiency of the nanobiosensor was improved by optimizing the factors affecting the probe construction such as the pH and the antibody to gold nanoparticles ratio. Dynamic light scattering was applied for the characterization of the constructed probe. The amount of peak shift of the LSPR absorption maxima was selected as the basis for determination of TSH antigen. The linear dynamic range of 0.4–12.5 mIU L⁻¹ and the calibration sensitivity of 1.71 L mIU⁻¹ were obtained. The human control serum sample was analyzed for TSH by constructed nanobiosensor and the acceptable results were obtained.     

Metal-enhanced fluorescence of tryptophan residues in proteins: Application toward label-free bioassays

The detection of submonolayers of proteins based on native fluorescence is a potentially valuable approach for label-free detection. We have examined the possibility of using silver nanostructures to increase the emission of tryptophan residues in proteins. Fluorescence spectra, intensities, and lifetimes of multilayers and submonolayers of proteins deposited on the surfaces of silver island films were measured. Increased fluorescence intensities from two- to three-fold and similar decreases in lifetimes were observed in the presence of the silver nanoparticles compared with the proteins on the surface of the bare quartz. The observed spectral effects of silver nanoparticles on tryptophan fluorescence indicates the possibility for the design of analytical tools for the detection of proteins without traditional labeling by extrinsic fluorophores.     

Colloidal Synthesis of Plasmonic Metallic Nanoparticles

Solutions of Ag and Au nanoparticles are strongly colored because of localized surface plasmon resonance in the UV/visible spectral region. The optical properties of these nanoparticles may be tuned to suit the needs of the application. This article summarizes our work in recent years on the solution synthesis of nanoparticles with tunable optical properties. The systems of interest include zero-dimensional bimetallic Ag–Au nanoparticles with different structures, one-, two-, and three-dimensional anisotropic monometallic Ag or Au nanoparticles. All of these nanosystems were prepared from colloidal synthesis through simple changes in the synthesis conditions. This is a demonstration of the versatility of colloidal synthesis as a convenient scalable technique for tuning the properties of metallic nanoparticles. Zhang, Tan, and Xie contributed equally to this article     

Surface plasmon resonance imaging on a microchip for detection of DNA-modified gold nanoparticles deposited onto the surface in a non-cross-linking configuration

We report theoretical predictions and experimental observations of the reduced detection volume with the use of surface-plasmon-coupled emission (SPCE). The effective fluorescence volume (detection volume) in SPCE experiments depends on two near-field factors: the depth of evanescent wave excitation and a distance-dependent coupling of excited fluorophores to the surface plasmons. With direct excitation of the sample (reverse Kretschmann excitation) the detection volume is restricted only by the distance-dependent coupling of the excitation to the surface plasmons. However, with the excitation through the glass prism at surface plasmon resonance angle (Kretschmann configuration), the detection volume is a product of evanescent wave penetration depth and distance-dependent coupling. In addition, the detection volume is further reduced by a metal quenching of excited fluorophores at a close proximity (below 10nm). The height of the detected volume size is 40-70nm, depending on the orientation of the excited dipoles. We show that, by using the Kretschmann configuration in a microscope with a high-numerical-aperture objective (1.45) together with confocal detection, the detection volume can be reduced to 1-2attoL. The strong dependence of the coupling to the surface plasmons on the orientation of excited dipoles can be used to study the small conformational changes of macromolecules.     

Optically responsive nanoparticle layers for the label-free analysis of biospecific interactions in array formats

A novel nanocomposite surface is prepared by coating surface-adsorbed dielectric colloidal particles with a contiguous layer of gold nanoparticles. The resulting surface shows pronounced optical extinction in reflection with the extinction peaks located in the UV–Vis and NIR region of the electromagnetic spectrum. The peak positions of these maxima change very sensitively with the adsorption of organic molecules onto the surface. For the adsorption of a monolayer of octadecanethiol, we observe a peak shift of 55 nm on average, which is about five times that of established label-free sensing methods based on propagating and localized surface plasmons. In a first proof-of-principle experiment, the interaction of peptides with specific antibodies has been detected without labeling by means of a fiber-optical set-up with microscopic lateral resolution. To avoid crosstalk in high-density arrays, the optically responsive surface areas can be locally separated on a micro- or even nanometer scale. Accordingly, the newly developed optically responsive surfaces are well suited for integration into high-density peptide or DNA arrays as demanded in genomics, proteomics, and biomedical research in general.     

A chemiluminescent metalloimmunoassay based on silver deposition on colloidal gold labels

A sensitive chemiluminescent (CL) immunoassay of human immunoglobulin (IgG) which combined the inherent high sensitivity of CL analysis with the dramatic signal amplification of silver precipitation on colloidal gold tags was developed. First, the sandwich-type complex was formed in this protocol by the primary antibody immobilized on the polystyrene wells, the analyte in the sample, and the secondary antibody labeled with colloidal gold. Second, the colloidal gold was treated by an Ag(+) reduction solution, which resulted in the catalytic precipitation of silver on the surface of colloidal gold. Third, a large number of Ag(+) were oxidatively released in HNO(3) solution from the silver metal anchored on the sandwich-type complexes and then the human IgG was indirectly determined by a sensitive combined CL reaction of Ag(+)-K(2)S(2)O(8)-Mn(2+)- H(3)PO(4)-luminol. The chemiluminescence intensity depends linearly on the logarithm of the concentration of human IgG over the range of 0.02-50ngml(-1) and detection limit (3sigma) is 0.005ngml(-1) (i.e., approximately 3x10(-14)M, 3amol in 100-mul sample). This assay has been successfully applied to the determination of human IgG in human serum samples and showed great potential for numerous applications in immunoassay.     

Label-free and homogeneous DNA hybridization detection using gold nanoparticles-based chemiluiminescence system

We find that the catalytic activity of gold nanoparticles (GNPs) on luminol-H₂O₂ chemiluminescence (CL) system is greatly enhanced after it is aggregated by 0.5 M NaCl. We use this observation to design a CL detection of DNA hybridization. It is based on that the single- and double-stranded oligonucleotides have different propensities to adsorb on GNPs in colloidal solution, and the hybridization occurred between the probe DNA and target DNA can result in aggregation of the GNPs, producing strong CL emission. In the assay, no covalent functionalization of the GNPs, the probe, or the target DNA is required. The assay, including hybridization and detection, occurs in homogenous solution. The detection limit of target DNA (3σ) was estimated to be as low as 1.1 fM. The sensitivity was increased more than 6 orders of magnitude over that of GNPs-based colorimetric method. The present CL method for DNA hybridization detection offers the advantages of being simple, cheap, rapid and sensitive.     

Label-free immunosensor based on gold nanoparticle silver enhancement

A label-free immunosensor for the sensitive detection of human immunoglobulin G (IgG) was prepared based on gold nanoparticle-silver enhancement detection with a simple charge-coupled device (CCD) detector. The gold nanoparticles, which were used as nuclei for the deposit of metallic silver and also for the adsorption of antibodies, were immobilized into wells of a 9-well chip. With the addition of silver enhancement buffer, metallic silver will deposit onto gold nanoparticles, causing darkness that can be optically measured by the CCD camera and quantified using ImageJ software. When antibody was immobilized onto the gold nanoparticles and antigen was captured, the formed immunocomplex resulted in a decrease of the darkness and the intensity of the darkness was in line with IgG concentrations from 0.05 to 10 ng/ml. The CCD detector is simple and portable, and the reported method has many desirable merits such as sensitivity and accuracy, making it a promising technique for protein detection.     

One-step homogeneous non-stripping chemiluminescence metal immunoassay based on catalytic activity of gold nanoparticles

The catalytic activity of gold nanoparticles (AuNPs) on a luminol–H₂O₂ chemiluminescence (CL) system is found to be greatly enhanced after its crosslinking aggregation induced by immunoreaction. Based on this observation, a one-step homogeneous non-stripping CL metalloimmunoassay was designed. In the presence of corresponding antigen (Ag), the immunoreaction caused the aggregation of antibody (Ab)-modified AuNPs, and these crosslinking aggregated AuNPs could catalyze luminol–H₂O₂ CL reaction to produce a much stronger CL signal than dispersed Ab-modified AuNPs. The assay, including immunoreaction and detection, can be accomplished in homogeneous solution. In the assay, no tedious and strict stripping of metal nanoparticles, difficult synthesis of labels, multiple steps of immunoreactions and washings, and complicated magnetic separation process were required. The detection limit of human immunoglobulin G (IgG, 3σ) was estimated to be as low as 3.2 × 10⁻¹¹ g ml⁻¹. The sensitivity was increased by two orders of magnitude over that of other AuNP-based CL immunoassay. The current CL metalloimmunoassay offers the advantages of being simple, cheap, rapid, and sensitive.     

A polymer nanostructured Fabry-Perot interferometer based biosensor

A polymer nanostructured Fabry–Perot interferometer (FPI) based biosensor is reported. Different from a conventional FPI, the nanostructured FPI has a layer of Au-coated nanopores inside its cavity. The Au-coated nanostructure layer offers significant enhancement of optical transducing signals due to the localized surface plasmon resonance effect and also due to the significantly increased sensing surface area, which is up to at least two orders of magnitude larger than that of a conventional FPI-based biosensor. Using this technical platform, the immobilization of captures proteins (protein A) on the nanostructure layer and their binding with immunoglobulin G (IgG) has been monitored in real time, resulting in the shift of the interference fringes of the optical transducing signals. Current results show that the limit-of-detection of the biosensor should be lower than 10 pg/mL for IgG-protein A binding.     

Comparison of surface plasmon resonance and capacitive immunosensors for cancer antigen 125 detection in human serum samples

This paper presents a comparison between surface plasmon resonance (SPR) and capacitive immunosensors for a flow injection label-free detection of cancer antigen 125 (CA 125) in human serum. Anti-CA 125 was immobilized on gold surface through a self-assembled monolayer. Parameters affecting the responses of each system were optimized. Under optimal conditions, SPR provided a detection limit of 0.1 U ml⁻¹ while 0.05 U ml⁻¹ was obtained for the capacitive system. Linearity for SPR was between 0.1 and 40 U ml⁻¹ and 0.05–40 U ml⁻¹ for capacitive system. These immunosensors were applied to analyze CA 125 concentrations in human serum samples and compared with conventional enzyme linked fluorescent assay (ELFA). Both systems showed good agreement with ELFA (P < 0.05). Moreover, these immunosensors were very stable and provided good reproducible responses after regeneration, up to 32 times for SPR and 48 times for capacitive system with relative standard deviation lower than 4%. The SPR immunosensor provided advantages in term of fast response and real-time monitoring while capacitive immunosensor offered a sensitive and cost-effective method for CA 125 detection.     

Surface plasmon resonance imaging for real-time, label-free analysis of protein interactions with carbohydrate microarrays

Plant lectin recognition of glycans was evaluated by SPR imaging using a model array of N-biotinylated aminoethyl glycosides of β-d-glucose (negative control), α-d-mannose (conA-responsive), β-d-galactose (RCA₁₂₀-responsive) and N-acetyl-β-d-glucosamine (WGA-responsive) printed onto neutravidin-coated gold chips. Selective recognition of the cognate ligand was observed when RCA₁₂₀ was passed over the array surface. Limited or no binding was observed for the non-cognate ligands. SPR imaging of an array of 40 sialylated and unsialylated glycans established the binding preference of hSiglec7 for α2-8-linked disialic acid structures over α2-6-sialyl-LacNAcs, which in turn were recognized and bound with greater affinity than α2-3-sialyl-LacNAcs. Affinity binding data could be obtained with as little as 10–20 μg of lectin per experiment. The SPR imaging technique was also able to establish selective binding to the preferred glycan ligand when analyzing crude culture supernatant containing 10–20 μg of recombinant hSiglec7-Fc. Our results show that SPR imaging provides results that are in agreement with those obtained from fluorescence based carbohydrate arrays but with the added advantage of label-free analysis.     

A simple colorimetric DNA detection by target-induced hybridization chain reaction for isothermal signal amplification

A novel DNA detection method is presented based on a gold nanoparticle (AuNP) colorimetric assay and hybridization chain reaction (HCR). In this method, target DNA hybridized with probe DNA modified on AuNP, and triggered HCR. The resulting HCR products with a large number of negative charges significantly enhanced the stability of AuNPs, inhibiting aggregation of AuNPs at an elevated salt concentration. The approach was highly sensitive and selective. Using this enzyme-free and isothermal signal amplification method, we were able to detect target DNA at concentrations as low as 0.5 nM with the naked eye. Our method also has great potential for detecting other analytes, such as metal ions, proteins, and small molecules, if the target analytes could make HCR products attach to AuNPs.     

高通量SPRI技术与MicroRNA检测

miRNA是一种内源性小分子非编码RNA,含19～23个核苷酸,通过与mRNA的3 ′非编码区结合从而阻断翻译,调节植物或动物的基因表达,并在细胞发育过程中包括细胞裂解、增殖、凋亡及诱导疾病发挥重要的作用.因此,miRNA迅速成为生命科学研究的新领域.而检测miRNA的表达是研究miRNA功能的第一步.表面等离子体共振成像(surface plasmon resonance imaging, SPRI)技术是一种基于光学原理的传感检测技术,它利用表面等离子体共振原理,可将生物分子相互作用或生化反应的信号转化成光信号进行传感,具有无需标记、实时检测、快速、高通量检测等优点,在miRNA的检测上将显示出巨大的发展潜力和应用价值.本文对目前miRNA的检测方法,以及高通量SPRI技术在miRNA检测中的应用发展趋势做一综述.     

Hyper-Rayleigh scattering of protein-modified gold nanoparticles

The nonlinear optical properties of protein-modified gold nanoparticles has been studied by the hyper-Rayleigh scattering (HRS) technique. HRS signals from the nanoparticles coated with goat-anti-human IgG have been obtained when pumped with a laser pulse with a wavelength of 1064 nm. The HRS signals of gold nanoparticles with IgG were larger than those of bare gold nanoparticles. This can be explained by a noncentrosymmetric effect. It was also found that the HRS signals from the IgG-coated gold nanoparticles could be greatly increased when the antigen was added due to gold nanoparticle aggregation. Our experiment found that the HRS method could produce a measurable signal with 10 microg/ml antigen added, while the colorimetric method using UV spectrum detection required 100 microg/ml of added antigen. The results show that the HRS measurement of immunogold nanoparticles could become a potential immunoassay in determining small levels of antigen in aqueous samples.     

Phase-polarisation contrast for surface plasmon resonance biosensors

A technique of phase-polarisation contrast (PPC) for the enhancement of the contrast of a surface plasmon resonance (SPR) intensity profile is proposed and experimentally realised. The technique exploits the peculiarities of light phase and polarisation behaviour under SPR. It applies to non-optimum SPR coupling conditions and enables one to lower the resonant minimum of reflected intensity nearly to zero, and hence to increase substantially the ratio of the intensity from the resonance to that at the minimum. We observed the contrast enhancement by more than one order of magnitude when we applied the PPC scheme. The PPC can be efficiently employed in commercial SPR sensors, as it significantly reduces restrictions on allowable parameters of SPR-supporting metal films and biomolecular layers immobilised on them, facilitates SPR observation, and increases the accuracy of SPR shift measurements.     

Computational design of a new hydrogen bond network and at least a 300-fold specificity switch at a protein-protein interface

The redesign of protein-protein interactions is a stringent test of our understanding of molecular recognition and specificity. Previously we engineered a modest specificity switch into the colicin E7 DNase-Im7 immunity protein complex by identifying mutations that are disruptive in the native complex, but can be compensated by mutations on the interacting partner. Here we extend the approach by systematically sampling alternate rigid body orientations to optimize the interactions in a binding mode specific manner. Using this protocol we designed a de novo hydrogen bond network at the DNase-immunity protein interface and confirmed the design with X-ray crystallographic analysis. Subsequent design of the second shell of interactions guided by insights from the crystal structure on tightly bound water molecules, conformational strain, and packing defects yielded new binding partners that exhibited specificities of at least 300-fold between the cognate and the non-cognate complexes. This multi-step approach should be applicable to the design of polar protein-protein interactions and contribute to the re-engineering of regulatory networks mediated by protein-protein interactions.     

Estrogen conjugation and antibody binding interactions in surface plasmon resonance biosensing

Thioether-linked 3-mercaptopropionic acid derivatives of 17beta-estradiol and estrone were formed at the A-ring 4-position of the steroids by substitution of their 4-bromo analogues. The carboxylic acid terminal was used to link to an oligoethylene glycol (OEG) chain of 15-atoms in length. The OEG derivative of 17beta-estradiol was then in situ immobilized on a carboxymethylated dextran-coated gold sensor surface used to detect refractive index changes upon protein binding to the surface by surface plasmon propagation in a BIAcore surface plasmon resonance (SPR) instrument. Two other estradiol-OEG derivatives with Mannich reaction linkage at the 2-position and hemisuccinate linkage at the 3-position were also immobilized on the sensor surfaces for comparison. Binding performance between these immobilized different positional conjugates and monoclonal anti-estradiol antibody, raised from a 6-position conjugate, clearly demonstrated that both 2- and 4-conjugates, not conjugated through existing functional groups, gave strong antibody bindings, whereas the 3-conjugate through an existing functional group (3-OH) gave very little binding (2% compared to the 2-conjugate). Both 2- and 4-position conjugates were then applied in a highly sensitive estradiol SPR immunoassay with secondary antibody mediated signal enhancement that gave up to a 9.5-fold signal enhancement of primary antibody binding, and a detection limit of 25 pg/mL was achieved for a rapid and convenient flow-through immunoassay of estradiol.