Optical Immunosensor Research Articles

Label-free immunosensor for monitoring vitellogenin as a biomarker for exogenous oestrogen compounds in amphibian species

A label-free, optical waveguide lightmode spectroscopy based immunosensor was developed for frog (Bombina orientalis) vitellogenin (Vtg) determination in biological samples as a biomarker for exogenous oestrogen compounds. Antibody against Vtg was produced in rabbits immunised with purified lipovitellin (Lpv), a precursor of Vtg, from the homogenised ovary of oriental fire-bellied toads (B. orientalis). The purified protein and Lpv/Vtg-specific serum were applied in both competitive and direct immunoassay formats using optical waveguide lightmode spectroscopy immunosensor. When measuring Vtg in direct manner, the Lpv antibody (1.76 µg mL−1) was immobilised on the sensor surface, and the linear measuring range for Vtg was 0.1–10 µg mL−1. During the competitive measurement, 100 ng mL−1 Lpv was applied for the immobilisation. The linear measuring range for Vtg was 0.5–50 ng mL−1. We studied the relative substrate specificity of the antibody, and it was concluded that the method is suitable for the sensitive and selective determination of Vtg levels in toads. Heart, liver and gonad samples from male animals were spiked with Vtg and were analysed using the newly developed method, and female toads and spawn samples were tested and compared to the calibration curve obtained by the spiked samples.

ChemInform Abstract: Chemical Sensors and Biosensors for the Detection of Melamine

AbstractReview: 173 refs.

Magnetically-actuated, bead-enhanced silicon photonic immunosensor.

Magnetic actuation has been introduced to an optical immunosensor technology resulting in improvements in both rapidity and limit of detection for an assay quantitating low concentrations of a representative protein biomarker. For purposes of demonstration, an assay was designed for monocyte chemotactic protein 1 (MCP-1), a small cytokine which regulates migration and infiltration of monocytes and macrophages, and is an emerging biomarker for several diseases. The immunosensor is based on arrays of highly multiplexed silicon photonic microring resonators. A one-step sandwich immunoassay was performed and the signal was further enhanced through a tertiary recognition event between biotinylated tracer antibodies and streptavidin-coated magnetic beads. By integrating a magnet under the sensor chip, magnetic beads were rapidly directed towards the sensor surface resulting in improved assay performance metrics. Notably, the time required in the bead binding step was reduced by a factor of 11 (4 vs 45 min), leading to an overall decrease in assay time from 73 min to 32 min. The magnetically-actuated assay also lowered the limit of detection (LOD) for MCP-1 from 124 pg mL-1 down to 57 pg mL-1. In sum, the addition of magnetic actuation into bead-enhanced sandwich assays on a silicon photonic biosensor platform might facilitate improved detection of biomarkers in point-of-care diagnostics settings.

Review: Advancements and application of immunosensors in the analysis of food contaminants

Azam MS, Rahman MRT, Lou Z, Tang Y, Raqib SM, Jothi JS. 2014. Advancements and application of immunosensors in the analysis of food contaminants. Nusantara Bioscience 6: 186-195. Immunosensors are affinity ligand-based biosensor solid-state devices in which the immunochemical reaction is coupled to a transducer. The fundamental basis of all immunosensors is the specificity of the molecular recognition of antigens by antibodies to form a stable complex. This is similar to the immunoassay methodology. Immunosensors can be categorized based on the detection principle applied. The main developments are electrochemical, optical, and microgravimetric immunosensors. In contrast to immunoassay, modern transducer technology enables the label-free detection and quantification of the immune complex. The analysis of trace substances in environmental science, pharmaceutical and food industries is a challenge since many of these applications demand a continuous monitoring mode. The use of immunosensors in these applications is most appropriate. Food chemists should take advantage of immunosensors in food and clinical diagnostics. There are many recent developments in the immunosensor field which have potential impacts. The future role of this technique in intra-laboratory, as well as bedside testing, will become even more important as the food laboratory is faced with increasing pressure to contain costs. Objective of this paper is to give a general overview of the possible application of immunosensors to the food analysis field.

Dual labeled Ag@SiO2 core–shell nanoparticle based optical immunosensor for sensitive detection of E. coli

An optical nanobiosensor is presented using a fluorescent dye and anti-E. coli McAb anchored Ag@Silica core shell nanoparticles, for rapid and sensitive Escherichia coli detection in environmental samples. The synthesized dual labeled core shell (DLCS) nanoparticle shows intense fluorescence at 620nm in solution, having a narrow emission with full width at half maxima (FWHM) of 10nm, as a prerequisite to develop a sensitive detection platform for various biosensing applications. The specific E. coli was captured using an anti-E. coli antibody functionalized quartz glass, followed by a treatment with DLCS, where the photoluminescence spectroscopy was used to detect the target pathogen. The fabrication of the quartz glass based optical-immunosensor was monitored, and the results show changes in the photoluminescent patterns, which substantiate that varied species were immobilized on the surface of the antibody modified quartz glass. Consequently, the optical immunosensor demonstrated specificity and improved sensitivity, as compared to the customary methods, and was able to detect as low as 5CFU/mL. The developed DLCS based optical immunosensor was evaluated with environmental water samples, which showed acceptable precision, reproducibility and stability, and could be readily applied to the routine monitoring of pathogenic microorganisms in the environmental samples, and most importantly, demonstrate the potential of a prototype development of a simple and inexpensive diagnostic technique.

Measurement of polyphenol oxidase activity using optical waveguide lightmode spectroscopy-based immunosensor

Polyphenol oxidase (PPO) is an important quality index during food processing involving heat-treatment and sensitive determination of PPO activity has been a critical concern in the food industry. In this study, a new measurement of PPO activity exploiting an optical waveguide lightmode spectroscopy-based immunosensor is presented using a polyclonal anti-PPO antibody that was immobilized in situ to the surface of a 3-aminopropyltriethoxysilane-treated optical grating coupler activated with glutaraldehyde. When analysed with a purified PPO fraction from potato tubers, a linear relationship was found between PPO activities of 0.0005607–560.7U/mL and the sensor responses obtained. The sensor was applicable to measurement of PPO activity in real samples that were prepared from potato tubers, grapes and Kimchi cabbage, and the analytical results were compared with those obtained by a conventional colorimetric assay measuring PPO activity. When tested for long-term stability, the sensor was reusable up to 10th day after preparation.

Real time optical immunosensing with flow-through porous alumina membranes

Through the presentation of analytical data from bioassay experiments, measured by polarimetry, we demonstrate for the first time a real time immunoassay within a free standing macroporous alumina membrane. The 200nm nominal pore diameter of the membrane enables flow-through, thereby providing an ideal fluidic platform for the targeted delivery of analytes to bioreceptors immobilized on the pore walls, enabling fast sensing response times and the use of small sample volumes (<100μL). For the immunoassay, the pore walls were first coated with the functional copolymer, copoly(DMA-NAS) using a novel coupling process, before immobilization of the allergen protein, β-lactoglobulin, by spotting. The immuno-assay then proceeded with the binding of the primary and secondary antibody cognates, rabbit anti-β-lactoglobulin and anti-rabbit IgG respectively. Through the use of streptavidin coated quantum dots as refractive index signal enhancers, a noise floor for individual measurements of 3.7ng/mL (25pM) was obtained, with an overall statistical, or formal assay LOD of 33.7ng/mL (225pM), for total assay time below 1h.

Optic fiber-based immunosensor for the rapid and sensitive detection of hepatitis C virus in serum

A portable optic fiber-based immunosensor is developed to achieve rapid and sensitive hepatitis C virus detection in serum.

Synthesis and characterization of a stable, label-free optical biosensor from TiO2-coated porous silicon

A nanoscale layer of TiO2 is coated on the inner pore walls of a porous silicon (PSi) film by room-temperature infiltration of a TiO2 sol–gel precursor and firing at 500°C. The PSi:TiO2 composite films are characterized by Fourier transform infrared (FTIR), X-ray diffraction (XRD), energy dispersive X-ray spectral analysis (EDS), scanning electron microscopy (SEM) and reflective interferometric Fourier transform spectroscopy (RIFTS). The analysis indicates that TiO2 conformally coats the inner pore surfaces of the PSi film. The film displays greater aqueous stability in the pH range 2–12 relative to a PSi:SiO2 surface. A label-free optical interference immunosensor based on the TiO2-coated PSi film is demonstrated by real-time monitoring of the physical adsorption of protein A, followed by the specific binding of rabbit anti-sheep immunoglobulin (IgG) and then specific capture of sheep IgG. The time to achieve equilibrium for the physical adsorption of protein A on the surface of TiO2-coated PSi film is significantly greater than that of PSi film. The specificity of the protein A and rabbit anti-sheep IgG construct on the sensor is confirmed by tests with non-binding chicken IgG. The sensitivity of the immunosensor is shown to be 8210±170nm/refractive index unit (RIU).

Optical immunosensor for quantifying C-telopeptide fragments of type II collagen as an osteoarthritis biomarker in urine

A fluorescence-based biosensor for measuring the quantity of C-telopeptide fragments of type II collagen (CTX-II) was developed as an osteoarthritis biomarker in urine. During osteoarthritis progression, joint components such as cartilage collagen are degraded by collagenase-protease and secreted into the serum as CTX-II. To detect CTX-II in urine (uCTX-II) having structural heterogeneity, conventional sandwich format assay does not provide sufficient accuracy. As an alternative, a competitive immunoassay was developed for the quantification of uCTX-II, which uses antibody-conjugated fluoro-microbeads to generate an optical signal. After preparing the biosensing surface, the uCTX-II sample and antibody-optical probe conjugates were reacted on the PEG4-EKGPDP exposed biosensing surface. The optical probes competed with the uCTX-II in the sample for binding to the immobilized PEG4-EKGPDP. This assay was able to detect uCTX-II concentrations between 200 ng/mmol (corrected value vs. creatinine) and 1,400 ng/mmol, encompassing the clinical detection ranges required for osteoarthritis diagnosis. The competitive immunoassay developed for uCTX-II detection was rapid and exhibited good correlation with conventional ELISA methods. This novel competition assay is a promising tool for the diagnosis and monitoring of osteoarthritis using urine samples.

Direct detection of orchid viruses using nanorod-based fiber optic particle plasmon resonance immunosensor

A fiber optic particle plasmon resonance (FOPPR) immunosensor is developed for label-free detection of orchid viruses that use gold nanorods (AuNRs) as the sensing material. The AuNRs are employed to create a near-infrared sensing window to solve the color interference problem of sample matrix for direct sensing of target analyte. This work cannot be achieved using gold nanospheres (AuNSs) because the signal of sample color absorption largely overlaps the signal of molecular recognition events in the visible spectrum, making the signal interpretation much more difficult. The AuNRs are immobilized on the unclad fiber core surface, and functionalized by antibodies which can specifically recognize the corresponding Cymbidium mosaic virus (CymMV) or Odontoglossum ringspot virus (ORSV) for rapid viral infection diagnosis. The refractive index resolution of the AuNR-FOPPR sensor is estimated to be 8×10−6 RIU. The limits of detection (LODs) for CymMV and ORSV in leaf saps are 48 and 42pg/mL, respectively, which are better than the LODs of 1200pg/mL for both viruses obtained by enzyme-linked immunosorbent assay (ELISA). Exploiting the AuNR-FOPPR sensing strategy not only solves the color interference problem encountered by using AuNSs, but provides faster analysis, better reproducibility, and lower detection limit than ELISA. The sensor can distinguish between healthy and infected orchids in 10min, and can further provide the quantitative analysis of infection level. It is potentially applicable to the quality control of orchid cultivation industry, but not limited to this, especially for creating special spectral sensing window for particular samples.

Sequential injection immunoassay for human bone morphogenic protein-7 using an immunoreactor immobilized with anti-human bone morphogenic protein-7 antibody–CdSe/ZnS quantum dot conjugates

The detection of human bone morphogenic protein-7 (BMP-7) was achieved using a sequential injection immunoassay (SIIA) system. The SIIA system is based on the binding between BMP-7 and anti-human BMP-7 (AbBMP7)–CdSe/ZnS quantum dot (QD) conjugates immobilized onto a glass disk or an optical fiber, using fluorescence detection at excitation and emission wavelengths of 470nm and 580nm, respectively. The AbBMP7–QD conjugates were prepared by conjugating anti-human BMP-7 antibody (AbBMP7) to hydrophilic CdSe/ZnS core/shell quantum dots (QDs). The SIIA system was fully automated using software written in the LabVIEW™ development environment. The analytical performance of the SIIA system was characterized with a number of variables such as carrier flow rate and elution buffer. Under partially optimized operating conditions, the SIIA system had a linear calibration graph at up to 10.0ngmL−1 BMP-7 (R2≥0.975) and a sample frequency of two samples per hour. The SIIA system with an optical fiber immunosensor was used to detect and quantify BMP-7 in spiked real samples obtained from a biological process with recoveries in the range of 95–102%.

Coupled solid phase extraction and microparticle-based stability and purity-indicating immunosensor for the determination of recombinant human myelin basic protein in transgenic milk

An optical immunosensor was developed and validated on the surface of microparticles for the determination of a biopharmaceutical protein. The recombinant human myelin basic protein (rhMBP) was produced in milk of transgenic cows as a His-tagged fusion protein. Previous work indicated exclusive association of rhMBP with milk casein micelles that hindered direct determination of the protein in milk. In this work, a solid phase extraction using a cation exchange matrix was developed in order to liberate rhMBP from casein micelles. A sandwich-type immunoassay was then used for in-process monitoring of the full-length protein in the presence of major milk proteins. The assay was successfully employed for detection of ultra-traces of rhMBP (LOD=6.04ngmL−1≈0.3nmolL−1) and for quantitative determination over a wide concentration range (10.00–10,000.00ngmL−1). The assay was able also to detect the rhMBP in the presence of its human counterpart that lacks the His-tag. The high sensitivity along with the ability of the assay to determine the full length protein enabled monitoring of the stability of rhMBP. The testing protocol is particularly useful for intrinsically unstructured proteins that are extremely sensitive to proteolysis and lack a traceable enzymatic activity. This immunosensor provides a specific, ultrasensitive high throughput tool for in-process monitoring in biopharmaceutical industry.

Enzyme-Mediated Growing Au Nanoparticles for Electrochemical and Optical Immunosensors: Signal-on and Signal-off

Enzyme-Mediated Growing Au Nanoparticles for Electrochemical and Optical Immunosensors: Signal-on and Signal-off

Preparation of metallocarbonyl–gold-antibody bioconjugates for mid-IR optical immunosensing

Metallocarbonyl-labeled gold nanoparticles (displaying specific and intense absorption bands assigned to the stretching vibrations of the carbonyl ligands in IR spectroscopy) were derivatized with an anti-mouse IgG antibody to provide IR reporter immunoprobes that were characterized by several analytical techniques like ELISA, IR and transmission electron microscopy (TEM). These immunoprobes were able to detect a mouse monoclonal anti-staphylococcal enterotoxin A (SEA) antibody chemisorbed to a planar gold-coated glass sensor. The formation of the immune complex between metallocarbonyl–gold nanoparticle-antibody bioconjugate and anti-SEA antibody was assessed by mid-IR surface and atomic force microscopy (AFM) analysis.

Highly specific fiber optic immunosensor coupled with immunomagnetic separation for detection of low levels of Listeria monocytogenes and L. ivanovii

BackgroundImmunomagnetic separation (IMS) and immunoassays are widely used for pathogen detection. However, novel technology platforms with highly selective antibodies are essential to improve detection sensitivity, specificity and performance. In this study, monoclonal antibodies (MAbs) against Internalin A (InlA) and p30 were generated and used on paramagnetic beads of varying diameters for concentration, as well as on fiber-optic sensor for detection.ResultsAnti-InlA MAb-2D12 (IgG2a subclass) was specific for Listeria monocytogenes and L. ivanovii, and p30-specific MAb-3F8 (IgM) was specific for the genus Listeria. At all bacterial concentrations (103–108 CFU/mL) tested in the IMS assay; the 1-μm diameter MyOne beads had significantly higher capture efficiency (P < 0.05) than the 2.8-μm diameter M-280 beads with both antibodies. The highest capture efficiency for MyOne-2D12 (49.2% for 105 CFU/mL) was significantly higher (P < 0.05) than that of MyOne-3F8 (16.6 %) and Dynabeads anti-Listeria antibody (9 %). Furthermore, capture efficiency for MyOne-2D12 was highly specific for L. monocytogenes and L. ivanovii. Subsequently, we captured L. monocytogenes by MyOne-2D12 and MyOne-3F8 from hotdogs inoculated with mono- or co-cultures of L. monocytogenes and L. innocua (10–40 CFU/g), enriched for 18 h and detected by fiber-optic sensor and confirmed by plating, light-scattering, and qPCR assays. The detection limit for L. monocytogenes and L. ivanovii by the fiber-optic immunosensor was 3 × 102 CFU/mL using MAb-2D12 as capture and reporter antibody. Selective media plating, light-scattering, and qPCR assays confirmed the IMS and fiber-optic results.ConclusionsIMS coupled with a fiber-optic sensor using anti-InlA MAb is highly specific for L. monocytogenes and L. ivanovii and enabled detection of these pathogens at low levels from buffer or food.

Development of optical immunosensors for detection of proteins in serum

The detection of proteins in biological samples such as blood, serum or plasma by biosensors is very challenging due to the complex nature of the matrix, which contains a high level of many interfering compounds. Here we show the application of a novel polymeric immobilisation matrix that helps in the detection of specific protein analytes in biological samples by surface plasmon resonance (SPR) immunosensors. This polymer matrix contains thioacetal functional groups included in the network, and these groups do not require any further activation in order to react with proteins, making it attractive for sensor fabrication. The protein prostate specific antigen (PSA) was selected as a model target analyte. A sandwich format with two primary antibodies recognising different parts (epitopes) of the analyte was used for the detection of PSA in serum. The efficiency of the reduction of non-specific binding achieved with novel polymer was compared with those of other techniques such as coating of sensor surface with polyethylene glycol (PEG), use of charged hydrophilic aspartic acid and surfactants such as Tween20. The detection limit of the polymer based immunosensor was 0.1ngml−1 for free form PSA (f-PSA) in buffer and 5ngml−1 in 20% serum. This is an improvement compared with similar devices reported on literature, indicating the potential of the immunosensor developed here for the analysis of real samples.

Biosensors in food processing.

Optical based sensing systems that measure luminescence, fluorescence, reflectance and absorbance, etc., are some of the areas of applications of optical immunosensors. Immunological methods rely on specific binding of an antibody (monoclonal, polyclonal or engineered) to an antigen. Detection of specific microorganisms and microbial toxins requires immobilization of specific antibodies onto a given transducer that can produce signal upon attachment of typical microbe/microbial toxins. Inherent features of immunosensors such as specificity, sensitivity, speed, ease and on-site analysis can be made use for various applications. Safety of food and environment has been a major concern of food technologists and health scientists in recent years. There exists a strong need for rapid and sensitive detection of different components of foods and beverages along with the food borne and water borne pathogens, toxins and pesticide residues with high specificity. Biosensors present attractive, efficient alternative techniques by providing quick and reliable performances. There is a very good potential for application of biosensors for monitoring food quality and safety in food and bioprocessing industries in India.

Optical immunosensor using carbon nanotubes coated with a photovoltaic polymer

In this work, an on-chip optical immunosensor using an individually assembled carbon nanotube (CNT) coated with a photovoltaic polymer has been proposed, developed, characterized, and applied for the detection of cardiac biomarkers. An individual CNT was self-assembled on a nickel (Ni)-patterned electrode by magnetically attracting the residual iron catalyst at one end of the CNT. After the CNT self-assembled electrode was prepared, it was coated with a photovoltaic polymer to implement a CNT photodetector. Under an incident light, the photovoltaic polymer generated electrons that changed the conductivity of the CNT. The CNT photodetector was finally insulated with parylene to prevent interruptions of charged molecules in a sample solution, such as non-specifically bound proteins and various ions. Chemiluminescent immunoassay was directly performed on the CNT photodetector for an on-chip detection of cardiac troponin T (cTnT) with a detection limit of 12pg/mL. High sensitivity and reliable selectivity have been achieved through the use of on-chip measurement of chemiluminescent light by the CNT photodetector. As a result, the developed device is envisaged as a new platform for optical immunosensing using the individually self-assembled CNT for point-of-care (POC) clinical diagnostics.

Gold Nanoclusters as Signal Amplification Labels for Optical Immunosensors

A portable, low-cost optical immunosensor has been developed using environment-friendly gold nanoclusters (AuNCs) as signal amplification labels. The stable and robust film, poly(dopamine), was used to immobilize biomolecules on an indium tin oxide chip for the construction of biosensor, as confirmed by scanning electron microscopy, contact angle, and electrochemical impedance spectroscopy. Human IgG served as a model to demonstrate the performance of the proposed sensor. Through sandwiched immunoreaction, IgG could be qualitatively and quantitatively determined by colorimetry, fluorescence method, as well as eye observation according to the clinical requirement. On the basis of the dual signal amplification of AuNCs and silver enhancement, the sensitivity was improved to 5 pg/mL. Moreover, the resulting immunosensor possessed portability, high precision, excellent stability, and reproducibility, which could be used in the clinical analysis.