Sandwich Surface Plasmon Resonance Research Articles

A surface plasmon resonance biosensor in conjunction with a DNA aptamer-antibody bioreceptor pair for heterogeneous nuclear ribonucleoprotein A1 concentrations in colorectal cancer plasma solutions

A new DNA aptamer and antibody pair was incorporated into surface plasmon resonance (SPR) sensing platform to detect heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1) in plasma at clinically relevant native concentrations for the diagnosis of colorectal cancer (CRC). SPR detection of hnRNP A1 was realized via formation of the surface sandwich complex of aptamer/hnRNP A1/anti-hnRNP A; the specific adsorption of hnRNP A1 onto a gold chip surface modified with a DNA aptamer followed by the adsorption of anti-hnRNP A1. Changes in the refractive unit (RU) with respect to the hnRNP A1 concentration in buffer solutions were monitored at a fixed anti-hnRNP A1 concentration of 90 nM, resulting in a dynamic range of 0.1–10 nM of hnRNP A1. The surface sandwich SPR biosensor was further applied to the direct analysis of undiluted human normal and pooled CRC patient plasma solutions. Our plasma analysis results were compared to those obtained with a commercial enzyme-linked immunosorbent assay kit.

Synthesis of Fe₃C@C from Pyrolysis of Fe₃O₄-Lignin Clusters and Its Application for Quick and Sensitive Detection of PrPSc through a Sandwich SPR Detection Assay.

The prion protein (PrPSc) has drawn widespread attention due to its pathological potential to cause prion diseases. Herein, we successfully synthesized Fe3C@C by carbonizing Fe3O4-lignin clusters, which were prepared through a facile hydrogen bonding interaction between ≡Fe-OH and hydroxyl groups of lignin. Our in-depth investigation confirmed that the composites were Fe3C@C core/shell particles. We constructed a novel sandwich surface plasmon resonance (SPR) detection assay for sensitive PrPSc detection, utilizing bare gold surface and aptamer-modified Fe3C@C (Fe3C@C-aptamer). Due to the highly specific affinity of Fe3C@C-aptamer towards PrPSc, the sandwich type SPR sensor exhibited excellent analytical performance towards the discrimination and quantitation of PrPSc. A good linear relationship was obtained between the SPR responses and the logarithm of PrPSc concentrations over a range of 0.1–200 ng/mL. The detection sensitivity for PrPSc was improved by ~10 fold compared with the SPR direct detection format. The required detection time was only 20 min. The specificity of the present biosensor was also confirmed by PrPC and other reagents as controls. This proposed approach could also be used to isolate and detect other highly pathogenic biomolecules with similar structural characteristics by altering the corresponding aptamer in the Fe3C@C conjugates.

Fabrication of Magnetic Conjugation Clusters via Intermolecular Assembling for Ultrasensitive Surface Plasmon Resonance (SPR) Detection in a Wide Range of Concentrations.

Herein, a novel sandwich surface plasmon resonance (SPR) detection assay, which utilizes prion disease-associated isoform (PrPSc) conjugating magnetic nanoparticle clusters (nanoparticle-organic clusters, NOCs) as signal amplification reagents, is constructed for the ultrasensitive detection of PrPSc. Due to the highly specific affinity of aptamer-Fe3O4 nanoparticles (AMNPs) toward PrPSc and the intermolecular assembly behaviors among PrPSc, PrPSc conjugating magnetic nanoparticle clusters were obtained after the incubation of AMNPs and PrPSc and the subsequent concentration processes in an external magnetic field. The conjugation clusters were further injected into the SPR cuvette and captured by the gold sensing film via the Au-S bonding interaction, inducing intense SPR responses. Meanwhile, a traditional sandwich SPR detection format using a gold/PrPSc/AMNPs amplification mode was conducted for the detection of PrPSc as comparison. The results reveal that the synthesized NOCs permitted a 215-fold increase of the SPR signal, while the sandwich format permitted only a 65-fold increase. Moreover, a lower detection limit (1 × 10-4 ng/mL) and a wider quantitation range (1 × 10-4-1 × 105 ng/mL) were demonstrated. The formation of the conjugation clusters and the capture of these clusters were confirmed by high-resolution AFM imaging and molecular simulations. This conjugation-cluster-induced signal amplification strategy has great potential for the detection of small analytes with similar structural characteristics in trace level concentrations with high selectivity and sensitivity by altering the corresponding aptamer labeled to magnetic particles.

Quick and Sensitive Detection of Prion Disease-Associated Isoform (PrP<sup>Sc</sup>) Using a Novel Gold Surface/PrP<sup>Sc</sup>/Gold Nanoparticles Sandwich SPR Detection Assay

Prion protein has drawn great attention due to its pathological potential to prion diseases. Discriminate and detection of the trace quantities PrPSc is an important measure for prion disease diagnosis at the presymptomatic stage. In this study, we developed a novel sandwich surface plasmon resonance (SPR) assay for the detection of PrPSc, involving bare gold surfaces and bare gold nanoparticles. PrPSc can be captured by the SPR sensing surface via a surface assisted coupling reaction between its intra-molecular disulfide bond and the gold atoms, while PrPC cannot bind to the gold surface strongly. The gold nanoparticles were proved to amplify the SPR detection signals via the coupling of their localized surface plasmon (LSP) with the propagating plasmon on the SPR gold surface. Our results confirmed that the bare SPR gold surface successfully captured the PrPSc from the solution with a LOD of 0.5ng/mL and a linear detection range from 0.5ng/mL to 500ng/mL. Injecting the gold nanoparticles after PrPSc yielded a dramatic enhancement of signal, with a lower LOD of 0.001ng/mL and a linear detection range from 0.001ng/mL to 10ng/mL. The gold nanoparticles permitted 4 to 322-fold increase of the signals. The required detection time was controlled in 15 min. PrPC, cys-protein G and their mixtures with PrPSc, were also detected via this sandwich SPR detection assay. Atomic force microscope (AFM) was used to evaluate the surface morphology of the SPR gold substrate after the detection. All the obtained results suggested that this novel SPR sandwich detection assay in our work was efficient, sensitive and specific for the detection of trace PrPSc

Quick and sensitive SPR detection of prion disease-associated isoform (PrPSc) based on its self-assembling behavior on bare gold film and specific interactions with aptamer-graphene oxide (AGO)

Herein, we constructed a novel sandwich surface plasmon resonance (SPR) detection assay for sensitive prion disease-associated isoform (PrPSc) detection, utilizing bare gold film and apatamer-graphene oxide (AGO). Due to the self-assembling behavior of PrPSc on gold surface, the non-modified gold surface can be directly used as sensing surface for the quick detection, for the purpose to avoid the interference from the traditional, complex and changeable probe-modified sensing surface. And due to the highly specific affinity of AGO towards PrPSc, the sandwich type SPR sensor exhibits excellent analytical performance towards the discrimination and quantitation of PrPSc. A good linear relationship was obtained between SPR responses and the logarithm of PrPSc concentrations over a range of 0.001–1ng/mL. The detection sensitivity for PrPSc was improved by ∼156 orders of AGO compared with SPR direct detection format. Besides, morphological changes of the sensing film surfaces were investigated by high resolution AFM imaging, confirming the capture of PrPSc molecules and their further specific recognition by AGO. The specificity of the present biosensor was also investigated by PrPC and other regents as controls. By compared with other reported methods, the AGO enhanced sandwich SPR assay was confirmed to be efficient, sensitive, and with wide working range.

Magneto-plamonic nanoparticles enhanced surface plasmon resonance TB sensor based on recombinant gold binding antibody

The magneto-plamonic nanoparticles (MPNs) have been employed to enhance the signal of surface plasmon resonance (SPR) spectroscopy to gain new insight into MPN and gold film SPR interaction. Three different morphologies MPNs (sphere, short spiky and long spiky) of Fe3O4@Au nanoparticles with good dispersion were synthesized and characterized carefully. A sandwich SPR immunosensor was constructed by immobilizing gold binding anti-CFP-10 (Ab1) onto a gold chip surface via Au-S bond firstly. Gold binding anti-CFP-10 (Ab2) captured on MPN surface was utilized to amplify the SPR signals specifically. Compared with spiky MPNs, the structure of spherical MPN, which concentrates the electric charge density and immobilize more Abs on its surface coverage, could enhance the electronic coupling effect significantly. Attribute to the super paramagnetism of MPNs, a facile solution route was fabricated to capture and separate analyte from the real sample by outside extend magnetic field. Implementation of MPNs results in 30-fold enlargement of the SPR signal at the limit of detection. To this end, an immunoassay is carried out that couples the specificity of antibody-antigen interactions with the high sensitivity based on spherical MPNs signal enhancement SPR.

Sensitive detection of carcinoembryonic antigen using surface plasmon resonance biosensor with gold nanoparticles signal amplification

A new method for real-time detection of carcinoembryonic antigen (CEA) in human serum with high sensitivity and selectivity using surface plasmon resonance (SPR) biosensor was developed. Two kinds of antibodies were used to recognize CEA at different epitopes with high affinity and specificity. Gold nanoparticles (GNPs) modified with streptavidin (SA) were used to further enhance signal specifically via biotin–streptavidin interaction. The binding capacity of the streptavidin-modified gold nanoparticles (SA–GNPs) for ligand biotin was quantified by titration with biotin (5-fluorescein) conjugate to be 10.54 biotin binding sites per 100nm2. The developed GNPs enhanced sandwich SPR biosensor successfully fulfilled the sensitive detection of CEA in the range of 1–60ng/mL with a detection limit of 1.0ng/mL. Compared to the direct assay format, sandwich format without GNPs and SA–GNPs enhanced sandwich format led to 4.2-fold and 13.8-fold in the sensitivity, respectively. This sensor also showed good selectivity for CEA in the interference study. The results demonstrated that the proposed method could provide a high sensitivity and selectivity in the detection of CEA and offer a promising alternative for cancer biomarker than traditional clinical examinations.

Fe3O4@Au nanoparticles as a means of signal enhancement in surface plasmon resonance spectroscopy for thrombin detection

The core-shell gold capped magnetic nanoparticles (GMPs) have been receiving increasing attention because of its optical and magnetic properties. In order to evaluate the ability of GMPs as a means of signal enhancement in surface plasmon resonance (SPR) spectroscopy, a sandwich SPR sensor is constructed by using thrombin as a model analyte. In this design, thrombin is captured by thrombin aptamer 1 (Apt1) and sensitively detected through addition of GMPs-Apt2 conjugates. Compared with gold nanoparticles (GNPs) and magnetic nanoparticles (MNPs), GMPs-Apt2 conjugates result in a significant SPR angle increase, which is mainly attributed by the larger mass and higher refractive index of the GMP nanoparticles. Therefore, the detection limit can be achieved as low as 0.1nM. Since GMP may have high stability and biocompatibility, it can be a useful sandwich element for sensor fabrication and an excellent amplification reagent for SPR measurement. This work may also afford a new model to improve the sensitivity and selectivity of SPR biosensors in protein detection and disease diagnosis.

Label-free surface plasmon resonance cytosensor for breast cancer cell detection based on nano-conjugation of monodisperse magnetic nanoparticle and folic acid

Accurate diagnosis of breast cancer cells is important on determining the stage of the disease and designing the suitable therapies. Herein, we report a sensitive biosensor based on surface plasmon resonance (SPR) spectroscopy using magnetic nanoparticle (MNP) and a two-marker recognition system. While the target breast cancer cells (MCF-7) are captured by MUC1 aptamer on the surface selectively, folic acid conjugated monodisperse MNP is used as the second selective binding reagent to form a sandwich SPR assay. The signal of SPR was enhanced significantly. This enhancement was mainly attributed by the larger mass and higher refractive index of the MNP nanoparticles located in the evanescent field. The detection limit can be achieved as low as 500 cellsmL−1. This work may afford a new model to improve the sensitivity and selectivity of SPR biosensors for large-size analytes.

Graphene oxide and dextran capped gold nanoparticles based surface plasmon resonance sensor for sensitive detection of concanavalin A

Carbohydrate–protein interactions mediate the important physiological and pathophysiological processes in living organism. Their study has attracted great attention due to its importance in understanding these biological processes and in fabricating biosensors for diagnostics and drug development. Here, by using concanavalin A (ConA) as a model protein, a novel surface plasmon resonance (SPR) sensor was developed for sensitive detection ConA. In this sensing platform, dextran (Dex) capped gold nanoparticles (Dex-Au NPs) were initially synthesized in one-pot and utilized as amplification reagent. After deposition of graphene oxide (GO) on the SPR gold film, phenoxy-derivatized dextran (DexP) was assembled onto the GO-modified gold chip surface through π–π interaction. The resultant GO/DexP sensing interface could specifically capture ConA which could further react with Dex-Au NPs through the specific interaction between ConA and Dex, forming a sandwich configuration. The morphologies and the electrochemistry of the formed sensing surface were investigated by using scanning electron microscopy and electrochemical techniques including electrochemical impedance spectroscopy and cyclic voltammogram. Owing to the high surface area of GO and the excellent amplification of Dex-Au NPs, the developed sandwich SPR sensor successfully fulfilled the sensitive detection of ConA in the range of 1.0–20.0μgmL−1 with a detection limit of 0.39μgmL−1. Compared to the direct assay format, the prepared sandwich SPR sensor led to an improvement of 28.7-fold in the sensitivity. The results demonstrated that the proposed method might provide a new direction in designing high-performance SPR biosensors for sensitive and selective detection of a wide spectrum of biomolecules.

Sensitive detection of tuberculosis using nanoparticle-enhanced surface plasmon resonance

We show that the antigen CFP-10 (found in tissue fluids of tuberculosis patients) can be used as a marker protein in a surface-plasmon resonance (SPR) based method for early and simplified diagnosis of tuberculosis. A sandwich SPR immunosensor was constructed by immobilizing the CFP-10 antibody on a self-assembled monolayer on a gold surface, this followed by blocking it with bovine serum albumin. Following exposure of the sensor surface to a sample containing CFP-10, secondary antibody immobilized on nickel oxide nanoparticles are injected which causes a large SPR signal change. The method has a dynamic range from 0.1 to around 150 ng per mL of CFP-10, and a detection limit as low as 0.1 ng per mL. This is assumed to be due to the high amplification power of the NiO nanoparticles.

Molecular Basis for the High Degree of Antigenic Cross-Reactivity between Hepatitis B Virus Capsids (HBcAg) and Dimeric Capsid-Related Protein (HBeAg): Insights into the Enigmatic Nature of the e-Antigen

The hepatitis B virus core gene codes for two closely related antigens: a 21-kDa protein that forms dimers that assemble as multimegadalton capsids, and a 17-kDa protein that also forms dimers but that do not assemble. The proteins, respectively referred to as core antigen (HBcAg) and e-antigen (HBeAg), share a sequence of 149 residues but have different amino- and carboxy-termini. Their structural and serological relationship has long been unclear. With insights gained from recent structural studies on immune complexes of the capsids, the relationship was reassessed using recombinant forms of the antigens and a panel of monoclonal antibodies (mAbs) commonly believed to discriminate between core and e-antigen. Surface plasmon resonance (SPR) was used to measure the affinities, in contrast to previous studies that used more error-prone and less sensitive plate-type assays. Four of the six mAbs did not discriminate between core and e-antigen, nor did they discriminate between e-antigen and dimers of dissociated core antigen capsids. One mAb (3120) was specific for assembled capsids and one (e6) was specific for unassembled dimers. Epitope valency of the e-antigen was also studied, using a sandwich SPR assay where e-antigen was captured with one mAb and probed with a second. The e-antigen is often considered to be a monomeric protein on the basis of monovalent reactivity with antibody pairs specific for either an alpha or beta epitope (in a prior nomenclature for e-antigen specificity). This model, however, is incorrect, because recombinant e-antigen is a stable dimer and its apparent monovalency is due to steric blockage. This was proven by the formation of a 2:1 Fab e6-e-antigen complex. These results suggest new approaches for the isolation of the authentic e-antigen, its biological assay, and its stabilization as an immune complex for structural studies.

Enzymatically biocatalytic precipitates amplified antibody–antigen interaction for super low level immunoassay: An investigation combined surface plasmon resonance with electrochemistry

We demonstrated a simple and efficient strategy, which based on the enzymatically biocatalytic precipitates amplified antibody–antigen interaction, for improving the response signals of surface plasmon resonance (SPR) immunosensing. The antibody–antigen-alkaline phosphatase (AP) labeled secondary antibody sandwich were successfully prepared and characterized by SPR, cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). The SPR signal amplification was accomplished through probing resonance angle shift and Faradaic electron impedance of [Fe(CN) 6] 3−/4− redox pair after the enzymatically biocatalytic products precipitating on the immunosensing electrode surface. As a result, the accumulation of the enzymatically biocatalytic precipitates leads to significantly resonance angle shift and increase of electron transfer impedance of [Fe(CN) 6] 3−/4− probe. The precipitates-enhanced sandwich SPR immunoassay for mouse immunoglobulin G (m-IgG) can easily detect solution protein concentrations in the linear range of 0.02–40 ng mL −1 and with a detection limit of 200 fg mL −1, which is more than four-orders and 10 times better compared with the values using streptavidin–biotinylated protein complex and biotinylated HRP biocatalyzation amplification methods. Moreover, this method is generally applicable to other sandwich immunoassays and also can be expanded to monitor other antibody–antigen interaction for immunosensing detection at low concentrations.

Kinetic interaction analysis of human interleukin 5 receptor alpha mutants reveals a unique binding topology and charge distribution for cytokine recognition.

Human interleukin 5 receptor alpha (IL5Ralpha) comprises three fibronectin type III domains (D1, D2, and D3) in the extracellular region. Previous results have indicated that residues in the D1D2 domains are crucial for high affinity interaction with human interleukin 5 (IL5). Yet, it is the D2D3 domains that have sequence homology with the classic cytokine recognition motif that is generally assumed to be the minimum cytokine-recognizing unit. In the present study, we used kinetic interaction analysis of alanine-scanning mutational variants of IL5Ralpha to define the residues involved in IL5 recognition. Soluble forms of IL5Ralpha variants were expressed in S2 cells, selectively captured via their C-terminal V5 tag by anti-V5 tag antibody immobilized onto the sensor chip and examined for IL5 interaction by using a sandwich surface plasmon resonance biosensor method. Marked effects on the interaction kinetics were observed not only in D1 (Asp(55), Asp(56), and Glu(58)) and D2 (Lys(186) and Arg(188)) domains, but also in the D3 (Arg(297)) domain. Modeling of the tertiary structure of IL5Ralpha indicated that these binding residues fell into two clusters. The first cluster consists of D1 domain residues that form a negatively charged patch, whereas the second cluster consists of residues that form a positively charged patch at the interface of D2 and D3 domains. These results suggest that the IL5 x IL5Ralpha system adopts a unique binding topology, in which the cytokine is recognized by a D2D3 tandem domain combined with a D1 domain, to form an extended cytokine recognition interface.

Surface Plasmon Resonance Analysis of Staphylococcal Enterotoxin B in Food

Surface plasmon resonance (SPR) biosensors are electro-optical instruments used for analyzing real-time protein-protein interactions. This work evaluates an SPR biosensor (Biacore 3000) in the detection of staphylococcal enterotoxin B (SEB) in foods. A sandwich SPR immunosensor involving two antibodies was used. The capturing antibody, bound covalently to the surface of the biosensor chip, performs the initial binding of the antigen and a second antibody binds to the captured antigen. The second antibody makes antigen verification possible and amplifies the signal. Pure SEB as well as SEB in spiked foods (milk and meat) were detected with little interference from the food matrix. In the control experiments with uncontaminated food samples no significant signal was detected. The SPR biosensor assay detects SEB at ~10 ng/ml rapidly, with initial binding within 2 min. The entire measurement cycle (including washing and chip regeneration) may take 5 min using one antibody or 8 min using two antibodies. These results suggest that the SPR biosensor may be a useful tool for real-time analysis of toxin in foods.

A dimeric bispecific miniantibody combines two specificities with avidity

Bispecific antibodies extend the capabilities of nature and might be applied in immunotherapy and biotechnology. By fusing the gene of a single-chain Fv (scFv) fragment to a helical dimerization domain, followed by a second scFv fragment of different specificity, we were able to express a functional protein in E. coli, which is bispecific and has two valencies for each specificity. The dimeric bispecific (DiBi) miniantibody preserves the natural avidity of antibodies in a very small-sized molecule of only 120 kDa. The generality of the principle was shown with a scFv fragment binding the EGF-receptor (named scFv 425) in three combinations with scFv fragments either directed against CD2 (ACID2.M1), phosphorylcholine (McPC603) or fluorescein (FITC-E2). Binding was analyzed by sandwich surface plasmon resonance biosensor (BIAcore) measurements.

Mutagenesis in the C-terminal region of human interleukin 5 reveals a central patch for receptor alpha chain recognition.

Cassette mutagenesis was used to identify side chains in human interleukin 5 (hIL-5) that mediate binding to hIL-5 receptor alpha chain (hIL-5R alpha). A series of single alanine substitutions was introduced into a stretch of residues in the C-terminal region, including helix D, which previously had been implicated in receptor alpha chain recognition and which is aligned on the IL-5 surface so as to allow the topography of receptor binding residues to be examined. hIL-5 and single site mutants were expressed in COS cells, their interactions with hIL-5R alpha were measured by a sandwich surface plasmon resonance biosensor method, and their biological activities were measured by an IL-5-dependent cell proliferation assay. A pattern of mutagenesis effects was observed, with greatest impact near the interface between the two four-helix bundles of IL-5, in particular at residues Glu-110 and Trp-111, and least at the distal ends of the D helices. This pattern suggests the possibility that residues near the interface of the two four-helix bundles in hIL-5 comprise a central patch or hot spot, which constitutes an energetically important alpha chain recognition site. This hypothesis suggests a structural explanation for the 1:1 stoichiometry observed for the complex of hIL-5 with hIL-5R alpha.

An immunosensor for syphilis screening based on surface plasmon resonance

In this paper the development of a surface plasmon resonance (SPR) immunosensor for syphilis screening is described. This immunosensor is based on the detection of antibodies in serum against the causative organism Treponema pallidum. In order to achieve selectivity a recombinant Treponema pallidum membrane protein A (TmpA) was used. This antigen can react with antibodies to T. pallidum, present in serum of syphilitic patients. Reproducible results have been obtained, using a ‘sandwich SPR’ method: binding of a sandwich antibody to the treponemal antibody after serum incubation was measured in real time while the binding was taking place. The SPR results obtained from ten blind-coded sera corresponded well with classical syphilis tests ( Treponema pallidum haemagglutination assay (TPHA), fluorescent treponemal antibody-absorbed test (FTA-ABS), venereal diseases research laboratory flocculation test (VDRL) and TmpA-based enzyme-linked immunosorbent assay (TmpA-ELISA)). Preliminary experiments showed that direct measurement of serum (in the ‘one step SPRS̊) is not yet possible, probably as a result of non-uniformity of serum samples. The application of latex beads is considered to solve this problem.