Features of Au NPs-aptamer conjugates as a powerful competitive reagent to substitute antibody in enhancing surface plasmon resonance spectroscopy (SPR) signal for the detection of small molecule are explored for the first time. In order to evaluate the sensing ability of Au NPs-aptamer conjugates as a competitive reagent, a novel SPR sensor based on indirect competitive inhibition assay (ICIA) for the detection of adenosine is constructed by employing the competitive reaction between antiadenosine aptamer with adenosine and antiadenosine aptamer with its partial complementary ss-DNA. The partial complementary ss-DNA of antiadenosine aptamer is firstly immobilized on SPR gold film as sensing surface. When the Au NPs-antiadenosine aptamer conjugates solution is added to SPR cell in the absence of adenosine, Au NPs-antiadenosine aptamer conjugates is adsorbed to SPR sensor by the DNA hybridization reaction, and results in a large change of SPR signal. However, the change of SPR signal is decreased when the mixing solution of adenosine with Au NPs-antiadenosine aptamer conjugates is added. This is because adenosine reacts with antiadenosine aptamer in Au NPs-antiadenosine aptamer conjugates and changes its structure from ss-DNA to tertiary structure, which cannot hybridize with its partial complementary ss-DNA immobilized on SPR gold surface. Based on this principle, a SPR sensor for indirect detection of adenosine can be developed. The experimental results confirm that the SPR sensor possesses a good sensitivity and a high selectivity for adenosine, which indirectly confirms that Au NPs-aptamer conjugates is a powerful competitive reagent. More significantly, it can be used to develop other SPR sensors based on ICIA to detect different targets by changing the corresponding type of aptamer in Au NPs-aptamer conjugates. 相似文献
The use of Au/SiO(x) interfaces for the investigation of DNA hybridization using electrochemical impedance spectroscopy (EIS) and surface plasmon resonance (SPR) simultaneously is demonstrated. Standard glass chemistry was used to link single-stranded DNA (ss-DNA) on aldehyde-terminated Au/SiO(x) interfaces. The layer thickness and amount of grafted oligonucleotides (ODNs) were calculated from SPR on the basis of a multilayer system of glass/Ti/Au/SiO(x)/grafted molecule. Capacitance and resistance values of the modified interface before and after hybridization were calculated from EIS data using an equivalent circuit and allowed the affinity rate constant, K(A) = 4.07 x 10(5) M(-1), to be determined. The EIS results were comparable to those obtained by SPR hybridization kinetics recorded in parallel. 相似文献
In this work, we present an antibody array for the detection of cancer biomarker candidates by a surface plasmon resonance
(SPR) imaging sensor with polarization contrast. Responses from the SPR imaging sensor are shown to be similar to those from
a conventional spectroscopy-based SPR sensor. Antibodies are spotted onto a self-assembled monolayer (SAM) composed of oligo(ethylene
glycol) (OEG)-containing alkanethiol chains. Detection of two cancer biomarker candidates, activated leukocyte cell adhesion
molecule/CD 166 (ALCAM) and transgelin-2 (TAGLN2), is demonstrated. Limits of detection for ALCAM and TAGLN2 are established
at 6 ng/mL and 3 ng/mL, respectively, in buffer. No cross-reactivity is observed between immobilized antibodies and nonspecific
antigen. Biomarker candidates are also detected in a 10% human serum solution.
Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. 相似文献
Detection of TNT is an important environmental and security concern all over the world. We herein report the performance and
comparison of four immunoassays for rapid and label-free detection of 2,4,6-trinitrotoluene (TNT) based on surface plasmon
resonance (SPR). The immunosensor surface was constructed by immobilization of a home-made 2,4,6-trinitrophenyl–keyhole limpet
hemocyanin (TNPh–KLH) conjugate onto an SPR gold surface by simple physical adsorption within 10 min. The immunoreaction of
the TNPh–KLH conjugate with four different antibodies, namely, monoclonal anti-TNT antibody (M-TNT Ab), monoclonal anti-trinitrophenol
antibody (M-TNP Ab), polyclonal anti-trinitrophenyl antibody (P-TNPh Ab), and polyclonal anti-TNP antibody (P-TNP Ab), was
studied by SPR. The principle of indirect competitive immunoreaction was employed for quantification of TNT. Among the four
antibodies, the P-TNPh Ab prepared by our group showed highest sensitivity with a detection limit of 0.002 ng/mL (2 ppt) TNT.
The lowest detection limits observed with other commercial antibodies were 0.008 ng/mL (8 ppt), 0.25 ng/mL (250 ppt), and
40 ng/mL (ppb) for M-TNT Ab, P-TNP Ab, and M-TNP Ab, respectively, in the similar assay format. The concentration of the conjugate
and the antibodies were optimized for use in the immunoassay. The response time for an immunoreaction was 36 s and a single
immunocycle could be done within 2 min, including the sensor surface regeneration using pepsin solution. In addition to the
quantification of TNT, all immunoassays were evaluated for robustness and cross-reactivity towards several TNT analogs.
相似文献
<正>A new indirect inhibitive immunoassay using surface plasmon resonance(SPR) coupled with molecularly imprinted polymer(MIP) was developed.A sulfamethoxazole(SMX) MIP coating capillary was produced and used as an in-tube solid phase extraction(SPE) device.The MIP coating formed a nanometer film on the inner wall of the capillary.The anti-SMX mono-antibody was inhibited by SMX extracted by the MIP coating in a dose-dependent manner.The calibration curve was generated by linear fit over the range of 0.04-10.00 ng/mL.The limit of detection was 0.01 ng/mL.This method has high sensitivity and can be performed automatically. 相似文献
To electrochemically measure human myeloid leukemia cells (K562 cells), we constructed a probe consisting of peptide/single-strand (ss) DNA. Ac-H6Y4C with an acetylated N-terminal of peptide was used to enhance the probe to allow electrode responses that could detect target cells. A ss-DNA was selected as the target cell recognition moiety. The probe exhibits properties that combine the functionalities of both DNA and peptides. The measurement principle is based on changes in the peak currents of the peptide moieties that are caused by interactions between the ss-DNA and target cells. The peak currents were proportional to the concentration of K 562 cells that ranged from 10 to 2,000 cells/mL with a LOD of 3 cells/mL. 相似文献
We report on the successful application of carboxyl-rich plasma polymerized (PP) films as a matrix layer for bioreceptor immobilization in surface plasmon resonance (SPR) immunosensing. Composition and chemical properties of the carboxyl-rich PP films deposited from a mixture of maleic anhydride and acetylene were investigated. Changes in the films stored in air, water, and buffer were studied and the involved chemical changes were described. Performance in SPR immunosensing was evaluated on interactions of human serum albumin (HSA) with a specific monoclonal antibody. The comparison with the mixed self-assembled monolayer of mercaptoundecanoic acid and mercaptohexanol (MUA/MCH) and one of the most widely used surfaces for SPR, the 2D and 3D carboxymethylated dextran (CMD), was presented to show the efficacy of plasma polymerized matrix layers for biosensing. The PP film-based SPR immunosensor provided a similar detection limit of HSA (100 ng/mL) as MUA/MCH- (100 ng/mL) and 3D CMD (50 ng/mL)-based sensors. However, the response levels were about twice higher in case of the PP film-based immunosensor than in case of MUA/MCH-based alternative. The PP film surfaces had similar binding capacity towards antibody as the 3D CMD layers. The response of PP film-based sensor towards HSA was comparable to 3D CMD-based sensor up to 2.5 μg/mL. For the higher concentrations (> 10 μg/mL), the response of PP film-based immunosensor was lower due to inaccessibility of active sites of the immobilized antibody inside the flat PP film surface. We have demonstrated that due to its high stability and cost-effective straightforward preparation, the carboxyl-rich PP films represent an efficient alternative to self-assembled monolayers (SAM) and dextran-based layers in label-free immunosensing.
Surface plasmon resonance (SPR) sensors have been used for detection of various biomolecules because of their simplicity, high specificity and sensitivity, real-time detection, low cost, and no requirement of labeling. Recently, molecularly imprinted polymers that are easy to prepare, less expensive, stable, have talent for molecular recognition and also are used for creation selective binding sites for target molecule on the SPR sensors. Here, we show that preparation of cyclic citrullinated peptide antibody (anti-CCP) imprinted SPR sensor to detect CCP antibodies. For this purpose, anti-CCP/AAm pre-complex was synthesized by interacting acrylamide (AAm) monomer with anti-CCP. Then, anti-CCP imprinted (anti-CCP/PAAm) SPR sensor was obtained by reacting with anti-CCP/AAm pre-complex in the presence of the crosslinker, and initiator/activator pair. Besides this, non-imprinted (PAAm) SPR sensor was also prepared without using anti-CCP template. The SPR sensors were characterized and then adsorption-desorption studies were performed with pH 7.0 phosphate buffer (10 mM) and acetic acid (10%) with Tween 20 (1%) in pH 7.0 phosphate buffer. Selectivitiy of sensors was investigated by using immunoglobulin M (IgM) and bovine serum albumin (BSA). To determine the adsorption model of interactions between anti-CCP solutions and anti-CCP/PAAm SPR sensor, different adsorption models were performed. The calculated maximum reflection, detection limit, association and dissociation constants were 1.079 RU/mL, 0.177 RU/mL, 0.589 RU/mL and 1.697 mL/RU, respectively. Repeatability experiments of anti-CCP/PAAm SPR sensor was performed four times with adsorption-desorption-regeneration cycles without any performance losing. Results showed that anti-CCP/PAAm SPR sensor had high selectivity and sensitivity for detection of CCP antibodies. 相似文献
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.
Figure
Schematic diagram of sensor chip configuration (left) and SPR study based on amplification strategy with NiO nanoparticles (right). 相似文献
We report on the use of new biofunctionalized gold nanoparticles (bio-AuNPs) that enable a surface plasmon resonance (SPR) biosensor to detect low levels of carcinoembryonic antigen (CEA) in human blood plasma. Bio-AuNPs consist of gold nanoparticles functionalized both with (1) streptavidin, to provide high affinity for the biotinylated secondary antibody used in the second step of the CEA sandwich assay, and with (2) bovine serum albumin, to minimize the nonspecific interaction of the bio-AuNPs with complex samples (blood plasma). We demonstrate that this approach makes it possible for the SPR biosensor to detect CEA in blood plasma at concentrations as low as 0.1?ng/mL, well below normal physiological levels (approximately nanograms per milliliter). Moreover, the limit of detection achieved using this approach is better by a factor of more than 1,000 than limits of detection reported so far for CEA in blood plasma using SPR biosensors. 相似文献
Recently, several papers indicated that the surface plasmon resonance (SPR) technique was available to monitor stimulation responses of mammalian cells adhered on sensor chips. On the other hand, the newly developed two-dimensional SPR (2D-SPR) imager system can obtain 2D-images of local refractive index change on the surface of a gold thin film. From these backgrounds, we expected that the 2D-SPR imager can visualize the individual response of many mammalian cells, simultaneously. Here, we report the observation of an allergenic response of a model mast cell, rat basophilic leukaemia cell (RBL-2H3), by using the high magnification 2D-SPR imaging system after pre-sensitization with 0.1 μg mL(-1) anti-dinitrophenyl immunoglobulin E (anti-DNP IgE). The response of the cells was successfully observed as the increment of the SPR signal (reflection intensity) upon stimulation with 0.1-1000 ng mL(-1) DNP-modified bovine serum albumin (DNP-BSA). 相似文献
A homemade array surface plasmon resonance (SPR)-based imaging biosensor was used to develop sensitive and fast immunoassays to determine sulfamethoxazole (SMOZ) and sulfamethazine (SMT) in buffer. Two conjugations of sulfonamide-bovine serum albumin (BSA) were separately immobilized on two different rows of the array chip with one row as reference. The immobilization was carried out in the instrument to monitor the quantity of the conjugations immobilized. The antibody mixed with the sulfonamide in the buffer was injected over the surface of the chip to get a relative response which was inversely proportional to the concentration of the sulfonamide in the PBS buffer. Two calibration curves were constructed and the limit of detection for sufamethoxazole in buffer was 3.5 ng/mL and for sulfamethazine 0.6 ng/mL. The stability and specificity of the antibody were also studied. The monoclonal antibody did not bind with BSA. 相似文献
The surface plasmon resonance (SPR) biosensor system with dispersionless microfluidics for the direct and label-free detection
of a soluble vascular endothelial growth factor receptor (sVEGFR-1) is described. The detection approach takes advantage of
an affinity interaction between sVEGFR-1 and its ligand, vascular endothelial growth factor (VEGF-A), which is covalently
immobilized on the surface of the SPR sensor. The ability of the immobilized VEGF-A to specifically bind the sVEGFR-1 receptor
is demonstrated in a buffer. The detection of sVEGFR-1 in 2% human blood plasma is carried out by using the sequential injection
approach. The detection limit of 25 ng/mL is achieved. In addition, we demonstrate that the functional surface of the sensor
can be regenerated for repeated use. 相似文献
Dual polarization interferometry (DPI) is used for a detailed study of antibody immobilization with and without orientation control, using prostate specific antigen (PSA) and its antibody as model. Thiol modified DPI chips were activated by a heterobifunctional cross-linker (sulfo-GMBS). PSA antibody was either directly immobilized via covalent binding or coupled via the Fc-fragment to protein G covalently attached to the activated chip. The direct covalent binding leads to a random antibody orientation and the coupling through protein G leads to an end-on orientation. Ethanolamine (ETH) was used to block remaining active sites following the direct antibody immobilization and protein G immobilization. A homobifunctional cross-linker (BS3) was used to stabilize the antibody layer coupled on protein G. DPI provides a real-time measurement of the stepwise molecular binding processes and gives detailed geometrical and structural values of each layer, i.e., thickness, mass, and density. These values evidence the end-on orientation of closely packed antibody on protein G layer and reveal structural effects of ETH blocking/deactivation and BS3 stabilization. With the end-on immobilized antibody, PSA at 10 pg/mL can be detected by DPI through a sandwich complex that satisfies the clinical requirement (assuming <30 pg/mL as clinically safe). However, the randomly immobilized antibody failed to detect PSA at 1 ng/mL. In a parallel study using surface plasmon resonance (SPR) spectroscopy, random and end-on antibody immobilization on streptavidin-modified gold surface was evaluated to further validate the importance of antibody orientation control. With the closely packed antibody layer on protein G surface, SPR can also detect PSA at 10 pg/mL. 相似文献
A novel Zn(II) ions imprinted poly (2-hydroxyethyl Methacrylate-N-methacryloyl-(L)-histidine methyl ester) poly(HEMAH) surface plasmon resonance (SPR) nanosensor were designed for detection of Zn(II) ions in aqueous solution and artificial plasma providing a low cost, rapid and reliable results compared to other techniques such as atomic absorption spectroscopy, inductively coupled plasma-mass spectrometer, X-ray fluorescence with synchrotron radiation. Zn(II) ions imprinted nanofilm on the SPR chip surface was synthesized by bulk polymerization. Characterization of Zn(II) ions imprinted nanosensor was performed by contact angle measurement, atomic force microscopy (AFM), ellipsometry and Fourier transform infrared spectroscopy-attenuated total reflection (FTIR-ATR). Designed nanosensor was applied for selective detection of Zn(II) ions in aqueous solution within the range of 0.5–1.0?µg/mL. The limit of detection (LOD) and limit of quantification (LOQ) were calculated as 0.19 and 0.64?ng/mL, respectively. Association kinetics analysis, Scatchard, Langmuir, Freundlich, Langmuir–Freundlich, Tempkin and Dubinin-Radushkevich isotherms were analyzed to the experimental data in order to identify the adsorption behavior. The selectivity of the SPR nanosensor was examined by using competitive metal ions such as Cd(II), Cu(II), Pb(II), and Fe(II). To evaluate the imprinting effect of Zn(II) ions imprinted (MIP) and non-imprinted (NIP) nanosensor was also prepared as the control. Repeatability of the response signal was tested by four times adsorption–desorption–regeneration cycle. 相似文献
An ultrasensitive electrogenerated chemiluminescence (ECL) detection method of DNA hybridization based on single-walled carbon-nanotubes (SWNT) carrying a large number of ruthenium complex tags was developed. The probe single strand DNA (ss-DNA) and ruthenium complex were loaded at SWNT, which was taken as an ECL probe. When the capture ss-DNA with a thiol group was self-assembled onto the surface of gold electrode, and then hybridized with target ss-DNA and further hybridized with the ECL probe to form DNA sandwich conjugate, a strong ECL response was electrochemically generated. The ECL intensity was linearly related to the concentration of perfect-matched target ss-DNA in the range from 2.4 × 10−14 to 1.7 × 10−12 M with a detection limit of 9.0 × l0−15 M. The ECL signal difference permitted to discriminate the perfect-matched target ss-DNA and two-base-mismatched ss-DNA. This work demonstrates that SWNT can provide an amplification platform for carrying a large number of ECL probe and thus resulting in an ultrasensitive ECL detection of DNA hybridization. 相似文献