Ochratoxin A immunoassay with surface plasmon resonance registration: Lowering limit of detection by the use of colloidal gold immunoconjugates

An immunoanalytical system was developed for the determination of ochratoxin A with the use of a surface plasmon resonance (SPR) sensor amplified by the anti-species antibody-colloidal gold particle (CGP) conjugate. The use of the binding of immune complexes to the CGP-anti-species antibody conjugate leads to the SPR signal amplification by a factor of more than 10 and results in the 60 pg/mL limit of detection of ochratoxin A with an assay time of 30 min. These characteristics are superior to those obtained both in the conventional enzyme immunoassay with the use of the same reagents and the SPR assay with unmodified antibodies and specific antibodies conjugated to colloidal gold.     

Detection of sulphamethazine with an optical biosensor and anti-idiotypic antibodies

We describe the development of an assay for the detection of sulphamethazine in animal urine with a surface plasmon resonance (SPR) biosensor. In order to obtain a general assay that can easily be transferred to other veterinary drug residues, a monoclonal antibody against sulphamethazine and a corresponding anti-idiotypic antibody were used. The assay had a lower detection limit of 5 μg/l which is well below the maximum residue limits (MRL) of 100 μg/l and can be used for screening purposes in animal urine. The binding reaction between the antibodies as occurring during sensor application was characterized with respect to avidity and kinetic properties.     

Study on design and application of fully automatic miniature surface plasmon resonance concentration analyzer

A fully automatic miniature surface plasmon resonance (SPR) concentration analyzer having high performance and low cost and developed using a Spreeta™ sensor was designed for field applications and concentration analysis. As in the case of Biacore™ instruments, the automatic sampling system of this device can introduce air segments between the sample/regeneration solution and buffer solution in the pipeline, which effectively prevents mixing of the solutions. A temperature sensor (AD 590) and temperature compensation method are used, which make the device insensitive to temperature fluctuations. A real-time data-smoothing algorithm for the SPR detection data is adopted; this can reduce the noise level to 5 × 10⁻⁷ RIU (refractive index units). The noise level of the sensorgram is 3.5% of the original level. Two types of self-prepared sensing chips—SMX-BSA (bovine serum albumin coated with sulfamethoxazole) and SMX-CM5 (carboxymethyl dextran coated with sulfamethoxazole)—are used to analyze the concentrations of sulfamethoxazole (SMX) standard solutions. Each chip's SMX calibration curve is established within the measurement range of 0-2000 ng/ml, and both limits of detection (LOD) are 2 ng/ml. One cycle of assay time is less than 15 min.     

Ultrasensitive immunosensing of tuberculosis CFP-10 based on SPR spectroscopy

An antigen (Ag), CFP-10, found in tissue fluids of tuberculosis (TB) patients may be an ultimate candidate for use as a sensitive TB marker with a sensing method for early simplified diagnosis of TB. In this study, chemical and optical optimizations were carried out using novel immuno-materials for establishment of a self-assembled surface plasmon resonance (SPR) optical immunosensor system for detection of CFP-10, which is valuable for pre-clinical work, prior to conduct of massive clinical observations. For creation of a simple sensing interface, a monoclonal antibody (anti-CFP-10) was immobilized directly on a gold surface, followed by blocking with cystamine. Orientation and accessibility of anti-CFP-10 were assessed by the selective binding of CFP-10. Recent results indicate that the reusability of the sensor chip adopting the cystamine method was found to be preferable to other immobilization methods. A linear relationship was well correlated between SPR angle shift and CFP concentrations in the range from 100 ng mL⁻¹ to 1 μg mL⁻¹. Modification of the SPR chip with antibody provides a simple experimental platform for investigation of isolated proteins under experimental conditions resembling those of their native environment.     

Highly sensitive label-free immunosensor for ochratoxin A based on functionalized magnetic nanoparticles and EIS/SPR detection

A label-free immunosensor for the detection of ochratoxin A (OTA) based on use of magnetic nanoparticles (MNPs) was developed. A gold electrode was modified using bovine serum albumin conjugate with a glutaraldehyde-thiolamine linker, creating a layer that prevents non-specific binding of OTA on gold. The OTA antibodies were attached to MNPs using the carbodiimide chemistry and afterwards were immobilized on the modified gold electrode using a strong magnetic field. Cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS) and surface plasmon resonance (SPR) were used to characterize each step in immunosensor development. The impedance variation due to the specific antibody-OTA interaction was correlated with the OTA concentration in the samples. The increase in electron-transfer resistance values was proportional to the concentration of OTA on a linear range between 0.01 and 5 ng/mL, with a detection limit of 0.01 ng/mL. SPR measurements showed a larger response range (1-50 ng/mL) with a detection limit of 0.94 ng/mL. Analytical results were in accordance with standard ELISA test kit.     

Tethered DNA scaffolds on optical sensor platforms for detection of hipO gene from Campylobacter jejuni

DNA biosensors have gained increased attention over traditional diagnostic methods due to their fast and responsive operation and cost-effective design. The specificity of DNA biosensors relies on single-stranded oligonucleotide probes immobilized to a transduction platform. Here, we report the development of biosensors to detect the hippuricase gene (hipO) from Campylobacter jejuni using direct covalent coupling of thiol- and biotin-labeled single-stranded DNA (ssDNA) on both surface plasmon resonance (SPR) and diffraction optics technology (DOT, dotLab) transduction platforms. This is the first known report of the dotLab to detect targeted DNA. Application of 6-mercapto-1-hexanol as a spacer thiol for SPR gold surface created a self-assembled monolayer that removed unbound ssDNA and minimized non-specific detection. The detection limit of SPR sensors was shown to be 2.5 nM DNA while dotLab sensors demonstrated a slightly decreased detection limit of 5.0 nM (0.005 μM). It was possible to reuse the SPR sensor due to the negligible changes in sensor sensitivity (∼9.7 × 10⁻⁷ ΔRU) and minimal damage to immobilized probes following use, whereas dotLab sensors could not be reused. Results indicated feasibility of optical biosensors for rapid and sensitive detection of the hipO gene of Campylobacter jejuni using specific ssDNA as a probe.     

Detection of EGFR on living human gastric cancer BGC823 cells using surface plasmon resonance phase sensing

Label-free and real-time information acquisition of molecular phenotype and its function on living cells plays a significant role in disease diagnosis and drug development. In this paper, SPR phase sensing was applied to monitor the interactions between EGFR antibody, EGFR1, and membrane proteins EGFR on living human gastric cancer BGC823 cells. When 50 μg/mL EGFR1 was added onto the fixed cells chip and the living cells chip, a significant difference in the binding amount could be observed from the immunofluorescence images. Quantitative results were obtained by following SPR detection, which were 722 RU and 438 RU, respectively. On the same living cells chip, SPR detection also showed markedly different results of cellular responses when it was stimulated by EGFR1 at different concentrations, such as adhesion and/or morphology variation, revealing the EGFR1's cytotoxic effect on the BGC823 cells. The results demonstrate SPR phase sensing is capable of real-time detection of molecular interactions and cellular responses on living cells, and suggest that further studies on the mechanism and the technique may allow SPR sensing become a powerful tool not only for the basic research of cell biology, but also for medical diagnosis and drug development.     

Development of sulfhydrylated antibody functionalized microcantilever immunosensor for taxol

An immobilization method using a sulfhydrylated monoclonal antibody (mAb) on the gold surface of a microcantilever immunosensor was developed for label-free detection of taxol. The sulfhydrylated anti-taxol mAb was synthesized. Then it was immobilized on the gold surface of the microcantilever. The deflection of the microcantilever corresponding to different taxol concentrations was real time monitored by optical lever technique. The activity of the anti-taxol mAb before and after sulfhydrylation, and the binding of the sulfhydrylated anti-taxol mAb on the gold surface of the microcantilever were evaluated by non-competitive enzyme-linked immunosorbent assay (ELISA). The limit of detection is 1 ng/mL and the method was used for detection of taxol in human plasma sample.     

Full-field surface plasmon resonance sensor for high resolution refractive index measurement using common-path heterodyne interferometer

This study presents a full-field surface plasmon resonance (SPR) sensor induced by attenuated total reflection (ATR)-couple for liquid refractive index measurement. The system adopts a common-path heterodyne interferometer to measure the phase difference between P- and S-wave after passing through the SPR sensor. In order to realize the full-field measurement, it adopts a three-frame integrating-bucket method. The experimental results show great consistency profile between single point and full-field liquid refractive index measurement from 1.330 to 1.340 RIU. It shows that the best sensitivity and resolution of a single pixel in charge couple device (CCD) for liquid refractive measurement are 3.3 × 10⁴ (deg/RIU) and 3.53 × 10⁻⁶ (RIU), respectively. As compared with traditional single-point method, the proposed method with a regular CCD has no degradation. Therefore, the system has many applications in chemistry and biology.     

Surface plasmon resonance immunosensor using Au nanoparticle for detection of TNT

In order to develop the fully integrated portable surface plasmon resonance (SPR) system for detection of explosives, the amplification strategy of SPR signal was investigated. Indirect competitive inhibition method allowed the middle-sized SPR sensor to detect trinitrotoluene (TNT) at ppt level. However, this enhanced SPR signal was not high enough to detect TNT at ppt level by a miniaturized SPR sensor. Therefore, localized surface plasmon resonance (LSPR) effect using Au nanoparticle as further signal amplification approach was used. The amplification method of indirect competitive inhibition and LSPR were combined together for fabrication of the immunosurface using Au nanoparticle. TNT detectable range of this immunosurface was from 10 ppt (10 pg/ml) to 100 ppb (100 ng/ml), which was almost comparable to that without Au nanoparticle. The observed resonance angle change due to binding monoclonal TNT antibody (M-TNT Ab) with the immunosurface modified with Au nanoparticle was amplified to four times higher than that in absence of Au nanoparticle.     

赤潮毒素大田软海绵酸表面等离子共振免疫检测方法研究

结合表面等离子体共振技术与免疫检测技术,研究和建立了一种响应速度快、免标记、低成本新型海洋赤潮毒素大田软海绵酸检测方法.基于SpreetaTM传感器构建了小型表面等离子共振免疫检测系统,采用共价偶联方法在传感器金膜表面修饰大田软海绵酸-牛血清蛋白抗原作为生物敏感膜;测得该方法相对标准偏差为1.51％ (n=12),定量...     

Sensitive electrochemical immunosensor for the detection of cancer biomarker using quantum dot functionalized graphene sheets as labels

Quantum dot (QD) functionalized graphene sheets (GS) were prepared and used as labels for the preparation of sandwich-type electrochemical immunosensors for the detection of a cancer biomarker (i.e., prostate specific antigen (PSA)). The primary anti-PSA antibody was also immobilized onto the GS. The immunosensor displayed a wide range of linear response (0.005-10 ng/mL), low detection limit (3 pg/mL), and good reproducibility, selectivity and stability. The immunosensor was used to detect PSA in patient serum samples with satisfactory results. Thus, this unique immunosensor may provide many applications in clinical diagnosis.     

Highly accurate and sensitive surface plasmon resonance sensor based on channel photonic crystal waveguides

A highly sensitive surface plasmon resonance (SPR) sensor based on channel photonic crystal waveguide (PCW) is proposed. The PCW is based on widely used lithographic and nano-fabrication compatible materials like TiO₂ and SiO₂. Gold has been used as a SPR active metal. By rigorously optimizing the different waveguide parameters, we have shown that there is significant transfer of modal power around phase-matching or resonance wavelength which has been utilized to design a compact and highly sensitive sensor for lab on chip. The ultra narrow width (∼765 pm for an interaction length of 10 mm) of surface plasmon resonance curve and sensitivity as high as 7500 nm-RIU⁻¹ will open a new window for bio-chemical sensing applications.     

Signal enhancement of surface plasmon resonance-based immunoassays for the allergen detection

A surface plasmon resonance (SPR) biosensor was used to determine the recombinant group 1 house dust mite allergen (rDer f1) in both HBS-EP buffer and fetal bovine serum (FBS). The monoclonal antibody was immobilized onto the CM5 sensor chip surface using an amine coupling method. The procedures of antibody immobilization and the subsequent primary and enhanced immunoassay were monitored in real time. The sensitivity for rDer f1 detection was remarkably improved by using intact polyclonal antibody as signal amplifying agent. Using this signal enhanced SPR immunosensor, rDer f1 in HBS-EP buffer and FBS was detected at a concentration of 15.4 and 32.1 ng/ml, respectively. The result demonstrates that SPR biosensor is a simple and reliable method for allergen detection.     

An ultra-sensitive chemiluminescence immunosensor of carcinoembryonic antigen using HRP-functionalized mesoporous silica nanoparticles as labels

A novel chemiluminescence immunosensor using horseradish peroxidase (HRP)-functionalized mesoporous silica nanoparticles (MSN) as labels was developed, which increases the sensitivity of the chemiluminescence immunoassay. The enzyme-functionalized MSN were fabricated by simultaneous coimmobilization of HRP and the carcinoembryonic antigen antibody (anti-CEA) onto the surface of MSN using 3-aminopropyltriethoxysilane (APTES) as the linkage. Because the large surface area of MSN carriers increased the amount of HRP bound per sandwiched immunoreaction, the conjugates provided a much higher signal and increased sensitivity. This is an improvement over the traditional sandwich immunoassay which often has one or two enzyme molecules per antibody. This approach was successfully demonstrated as a simple, cost-effective, specific, and potent method to detect CEA in practical samples. The analysis showed a linear response within the range of 0.1-40 ng/mL (r = 0.9912). The relative standard deviation (RSD) for 11 parallel measurements of 20 ng/mL CEA was 3.9%. The sensitivity of the immunosensor using MSN-HRP-Ab2 as labels was about 10-fold higher than that of traditional labels. These labels for immunosensors may provide many potential applications for detection of different biomolecules.     

Electrochemical immunosensor for salbutamol detection based on CS-Fe3O4-PAMAM-GNPs nanocomposites and HRP-MWCNTs-Ab bioconjugates for signal amplification

An ultrasensitive electrochemical immunosensor based on chitosan-iron oxide-poly(amino-amine) dendrimers-gold nanoparticles (CS-Fe₃O₄-PAMAM-GNPs) nanocomposites and horseradish peroxidase-multiwall carbon nanotubes-antibody (HRP-MWCNTs-Ab) bioconjugates was developed for the detection of salbutamol (SAL). CS-Fe₃O₄-PAMAM-GNPs nanocomposites as immobilization matrix were used to enhance the electroactivity and stability of the electrode. HRP-MWCNTs-Ab bioconjugates as label were used to improve catalytic activity for hydrogen reduction of the electrode. Under the optimized conditions, a calibration plot for SAL was obtained with a linear range between 0.11 ng/mL and 1061 ng/mL (r = 0.9984). The detection limit was 0.06 ng/mL. The immunosensor was examined in real samples for the analysis of SAL.     

Surface plasmon resonance based fiber optic sensor for the detection of low water content in ethanol

A fiber optic sensor utilizing surface plasmon resonance (SPR) has been fabricated for the detection of low content of water in ethanol. The sensor utilizes spectral interrogation technique for operation. The resonance wavelength has been found to vary linearly with water content in the range 0-10% with sensitivity of 1.149 nm per percentage of water. The results are in agreement with the refractive index variation of ethanol-water mixture. The sensor has a water resolution of 0.145% which is better than the evanescent wave absorption sensor reported for the similar study. The sensor will find application in determining the low water content in ethanol which is used as a bio-fuel and in the field of medicine and organic chemistry.     

A label-free amperometric immunosensor based on horseradish peroxidase functionalized carbon nanotubes and bilayer gold nanoparticles

In this work, a novel label-free amperometric immunosensor has been constructed for detecting α-1-fetoprotein (AFP) based on nanocomposite of horseradish peroxidase (HRP) labeled carbon nanotubes (CNTs). First, the gold nanoparticles (AuNPs) were electrodeposited on the surface of the glass carbon electrode by electrochemical reduction of gold chloride tetrahydrate (HAuCl₄) to immobilize horseradish peroxidase labeled carbon nanotubes (HRP-CNTs). Then HRP-CNTs bioconjugate was immobilized on the surface of the electrodeposited AuNPs layer by the combination of forces (coordination and electrostatic force). Subsequently, it was immersed into gold colloidal nanoparticles (GNPs) solution, which was used to immobilize antibody biomolecules (anti-AFP). Enhanced sensitivity was obtained by using bioconjugates featuring HRP labeled (HRP-CNTs), which had lager specific surface area and good electronic catalysis (current response signal) compared to carbon nanotubes. Under optimized conditions, the linear ranges were from 0.2 to 200 ng mL⁻¹ with a detection limit of 0.067 ng mL⁻¹ (at an S/N of 3). The proposed immunosenor showed good precision, acceptable stability and reproducibility and could be used for the detection AFP in normal human serum, which provided a potential alternative tool for the detection of protein in clinical diagnosis.     

ZnO modified gold disc: A new route to efficient glucose sensing

Surface of a gold disc was modified by depositing ZnO film electrochemically. AFM analysis of the film shows c-axis oriented pillar like structures grown normal to the surface. Sensor surface was prepared by immobilizing glucose oxidase (GOD) on the ZnO modified gold disc. Different concentrations of glucose (50-1000 ng/ml) were taken to monitor the sensor response. Sensor was found to be highly sensitive to low concentrations of glucose and sensitivity increases linearly in the range of 50-250 ng/ml. The high sensitivity of the ZnO modified gold disc may be attributed to the SPR induced electron transfer between the two systems (i.e. Au and ZnO). The work indicates promising application of the system as a tool for studying sensitive bio-specific interactions, with further development of highly sensitive and selective bio-molecular and chemical SPR based optical sensors.