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The immobilization of antibodies on solid-phase materials has been used in many areas such as purification, diagnostic immunoassays and immunosensors. Problems associated with the loss of biological activity of the antibodies upon immobilization have been noticed in many cases. One of the main reasons for such loss is attributed to the random orientation of the asymmetric macromolecules on support surfaces. In this paper, the approaches for achieving oriented coupling of antibodies to increase the antigen binding capacity are reviewed. Some issues such as steric hindrance caused by neighbouring antibody molecules, the distance between an antibody and the support surface and the use of antibody fragments are dealt with. Some applications of the oriented immobilized antibodies in immunoassays and immunosensors are examined.

Paper diagnostics have successfully been employed to detect the presence of antigens or small molecules in clinical samples through immunoassays; however, the detection of many disease targets relies on the much higher sensitivity and specificity achieved via nucleic acid amplification tests (NAAT). The steps involved in NAAT have recently begun to be explored in paper matrices, and our group, among others, has reported on paper-based extraction, amplification, and detection of DNA and RNA targets. Here, we integrate these paper-based NAAT steps into a single paperfluidic chip in a modular, foldable system that allows for fully integrated fluidic handling from sample to result. We showcase the functionality of the chip by combining nucleic acid isolation, isothermal amplification, and lateral flow detection of human papillomavirus (HPV) 16 DNA directly from crude cervical specimens in less than 1 hour for rapid, early detection of cervical cancer. The chip is made entirely of paper and adhesive sheets, making it low-cost, portable, and disposable, and offering the potential for a point-of-care molecular diagnostic platform even in remote and resource-limited settings. [ABSTRACT FROM AUTHOR]

A single-step, easy-to-use, and fast capillary-type immunoassay device composed of a polyethylene glycol (PEG) coating containing two kinds of antibody-reagents, including an antibody–graphene oxide conjugate and fluorescently labelled antibody, was developed in this study. The working principle involved the spontaneous dissolution of the PEG coating, diffusion of reagents, and subsequent immunoreaction, triggered by the capillary action-mediated introduction of a sample solution. In a sample solution containing the target antigen, two types of antibody reagents form a sandwich-type antigen–antibody complex and fluorescence quenching takes place via fluorescence resonance energy transfer between the labelled fluorescent molecules and graphene oxide. Antigen concentration can be measured based on the decrease in fluorescence intensity. An antigen concentration-dependent response was obtained for the model target protein sample (human IgG, 0.2–10 μg mL−1). The present method can shorten the reaction time to within 1 min (approximately 40 s), while conventional methods using the same reagents require reaction times of approximately 20 min because of the large reaction scale. The proposed method is one of the fastest immunoassays ever reported. Finally, the present device was used to measure human IgG in diluted serum samples to demonstrate that this method can be used for fast medical diagnosis. [ABSTRACT FROM AUTHOR]

We report a novel wash-free magnetic immunoassay technique for prostate-specific antigen (PSA) that uses a surface-enhanced Raman scattering (SERS)-based microdroplet sensor. The magnetic bar embedded in a droplet-based microfluidic system segregates the free and bound SERS tags by splitting the droplets into two smaller parts. The presence of PSA targets leads more SERS tags to immunocomplex in one droplet so that fewer SERS tags remain in another supernatant solution droplet. Thus, SERS signal measurement enables the quantitative evaluation of PSA markers. This approach can provide a rapid and sensitive assay that is applicable for PSA cancer markers in serum without any washing. Specifically, SERS signals were measured at 174 droplets per minute and averaged for quantitative evaluation of PSA. The limit of detection (LOD) determined by our SERS-based microdroplet sensor was estimated to be below 0.1 ng mL−1, which is significantly below the clinical cut-off value for the diagnosis of prostate cancer. In addition, because the entire assay can be carried out automatically, only a minimal amount of sample is needed. Accordingly, the approach is expected to be useful as a potential clinical tool for the early diagnosis of prostate cancer. [ABSTRACT FROM AUTHOR]

As a leading cause of cancer-related deaths in men globally, prostate cancer (PCa) demands immense attention for theranostic purposes. There is an increasing need for the development of rapid, sensitive, economical, miniaturized and multiplexable assays. Towards this goal, we present a systematic approach for the optimisation of a microfluidic sandwich immunoassay, which can be applied as a generic biosensor platform for PCa detection. Prostate specific antigen (PSA) was used as the model biomarker, and its free form was captured using commercially available antibodies and detected using chemiluminescence, both in spiked buffer and matrix solutions. Along with the optimisation of surface chemistry and microfluidic parameters, we report a bio-affinity amplification strategy based on biotin–streptavidin chemistry to bring the limits of detection for free-PSA from 21.4 ng mL−1 down to 2.7 ng mL−1, within the clinically relevant range. An estimate of the surface coverage and simulations of the interactions taking place in the microfluidic biosensor during the assay are also presented. This novel platform using a simple passive adsorption-based bio-affinity strategy, when coupled with multiplexing and integrated detection, can serve as a promising point-of-care diagnostic tool for PCa. [ABSTRACT FROM AUTHOR]

This work reports on the coupling of antibody-functionalized micromotors and microwire-tagged proteins for rapid and multiplexed immunoassays. While micromotor-induced mixing accelerates the immunoreaction, tagging the proteins with microscopic particles of different sizes and shapes allows for their multiplexed discrimination, alerting of the presence of a biological threat. [ABSTRACT FROM AUTHOR]

Mycotoxins are produced by fungi as secondary metabolites. They often multi-contaminate food and feed commodities posing a health risk to humans and animals. A fast and easy to apply multiplex screening of these commodities could be useful to detect multi-contamination. For this, we developed a semi-quantitative 6-plex immunoassay using a suspension array of paramagnetic colour-coded microspheres combined with imaging planar array detection for the mycotoxins aflatoxin B1, ochratoxin A, zearalenone, deoxynivalenol, T2-toxin, HT-2 toxin and fumonisin B1. Mycotoxin specific monoclonal antibodies were coupled to different sets of microspheres and mycotoxins conjugated to the fluorescent protein R-phycoerythrin served as reporter molecules. Competition between free mycotoxins in the sample and mixed reporter molecules for antibody binding sites on mixed microspheres created a multiplex direct inhibition immunoassay. The reagents were selected for no or low cross-interactions between the assays and cross-reactions with metabolites and possible masked forms were determined. A within-laboratory validation was carried out using blank and spiked barley samples. Furthermore, the 6-plex was used to screen available barley, and malted barley, reference materials. The validation showed very high inter and intra-day precision for all samples with a maximum relative standard deviation value of 10%. The screening assay allows easy and rapid multiplex detection of the target mycotoxins in barley according to EU legislation. With a cut off factor of 50%, based on the EU maximum levels, we were able to screen at 2 μg kg−1 for aflatoxin B1, 2.5 μg kg−1 for ochratoxin A, 625 μg kg−1 for deoxynivalenol, 50 μg kg−1 for zearalenone, 1000 μg kg−1 for fumonisin B1 and 25 μg kg−1 for T-2 toxin. Thanks to the transportable planar array system, the developed 6-plex has potential for future on-site testing. Future implementation of this method as a pre-screening tool, prior to instrumental analysis, is highly attractive since costly LC-MS/MS analysis of samples below the maximum levels can be avoided. [ABSTRACT FROM AUTHOR]

We present a novel “Lab-on-a-Disk” platform and demonstrate its capability for rapid and sensitive measurement of vascular endothelial growth factor (VEGF) intended for patients suffering from diabetic retinopathy (DR) and age-related macular degeneration (AMD). This approach combines sedimentation principles applied to microspheres under centrifugal force with signal amplification using an enzyme and a fluorogenic substrate for readout. The simple single channel per assay platform separates, washes and concentrates antibody-coated microspheres from excess label to produce a sensitive fluorogenic response proportional to the amount of VEGF in the sample. This platform has comparable sensitivity to conventional ELISA and can generate a readout within 16–18 min with no sample preparation beyond mixing assay reagents and loading on the disk. In the context of ocular diagnostics, this device has the potential to facilitate accurate dosing of anti-VEGF medications utilized to treat DR and AMD, as well as identify patients whose ocular VEGF levels are not elevated and who would therefore not benefit from standard anti-VEGF medications. [ABSTRACT FROM AUTHOR]

Recent research has looked to develop innovative, powerful and novel biofunctionalized nanoparticles, controlling and tailoring their properties in a very predictable manner to meet the needs of clinic immunoassays in the biomedical field. This minireview briefly summarizes recent advances covering the last 3 years, exploiting nanoparticle-based electrochemical, optical, mass-sensitive, colorimetric and immunodipstick assays. The enormous signal enhancement associated with the use of nanoparticles and formation of nanoparticle–antibody–antigen assemblies provide the basis for sensitive detection of disease-related proteins or biomarkers. Rather than being exhaustive, this minireview focuses on selected examples to illustrate novel concepts and promising applications. Finally, a small amount of speculation of possible future developments in nanoparticle-based immunoassays is provided. [ABSTRACT FROM AUTHOR]

A sensitive and selective method of immunoaffinity monolithic capillary microextraction (CME) based on a sandwich immunoreaction with quantum dots (QDs) labels coupled with micro-concentric nebulizer inductively coupled plasma mass spectrometry (MCN-ICP-MS) was proposed for the determination of antigen human IgG in human serum. Aminopropyltriethoxysilane (APTES)-silica hybrid monolithic capillary which was prepared by room temperature ionic liquid (RTIL)-mediated sol–gel method was immobilized with primary goat-anti-human antibody. After a complete immunoreaction among primary antibody, IgG antigen and secondary antibody labeled with CdSe QDs in monolithic capillary, the concentration of IgG was quantified by MCN-ICP-MS simultaneous analysis of Cd and Se released from captured QDs labels with an acid-dissolution (pH 2.0) step. With consumption of only 50 μL sample solution, the established method presented a limit of detection of 0.058 μg L−1based on the Cd signal and 0.097 μg L−1based on the Se signal for human IgG antigen, with the relative standard deviations (RSDs) of 7.2% based on the Cd signal and 13% based on the Se signal (chumanIgGantigen= 5 μg L−1, n= 7), respectively. The response of CME-MCN-ICP-MS method for human IgG was linear over a dynamic range from 0.2 to 10 μg L−1based on the Cd signal and from 0.5 to 10 μg L−1based on the Se signal, respectively. The proposed method was successfully applied for the determination of IgG in real human serum with good recoveries. For validation, the same sample was also analyzed by the commonly used clinical method of immunoturbidimetry, and the analytical result obtained by the proposed method is in good agreement with that the result obtained by immunoturbidimetry. By using CdSe QDs labels, the quantification of human IgG antigen could be performed by ICP-MS determination of both Cd and Se, and the analytical results for human IgG antigen could be validated in one run. The developed method was fast, sensitive, selective, versatile and could be easily extended to other protein quantification schemes as well as in DNA analysis. [ABSTRACT FROM AUTHOR]

A new strategy was described for amplifying the response of the antigen–antibody sensing processes by functionalizing SiO2nanoparticles labeled secondary antibodies based on a sandwich immunoassay in this work. At first, the multi-walled carbon nanotubes (CNTs) were individually dispersed in the aqueous bovine serum albumin (BSA) to obtain BSA molecules coated CNTs (BSA–CNTs). Then the gold colloids (nano-Au) were absorbed on the BSA–CNTs surface by the amido and disulfide groups of BSA. Later, a functionalized gold/carbon nanotube composite nanohybrid (DpAu/nano-Au/BSA–CNTs) modified electrode was developed by electrochemical deposition of Au3+onto nano-Au/BSA–MWNTs surface. Thus, a sensitive immunosensor for carbohydrate antigen 19-9 (CA19-9) has been constructed by further employment of Nafion coated SiO2nanoparticles labeled secondary antibody (SiO2–Ab2) for the signal amplification. More importantly, the loading of SiO2–Ab2can not only cause the construction of the dielectric antigen–antibody immunocomplex layer but also introduce the insulated Nafion coated SiO2nanoparticles which demonstrate the relatively high resistance, resulting in a strong detection signal. The proposed sensing strategy provides a wide linear dynamic range from 0.15 to 150 U mL−1with a low detection limit of 0.06 U mL−1at 3 times the background noise. Moreover, the extremely high stability of the functionalized gold/carbon nanotube composite nanohybrid monolayer allows the designed biosensing interface to obtain a good stability and long-term life. [ABSTRACT FROM AUTHOR]

Gold quantum dots (AuQDs) were synthesized and electrostatically conjugated to goat-derived anti-human IgG for the purpose of detecting human IgG in solution over a broad range of concentrations. The system is able to detect human IgG by linear fluorescence quenching over a micromolar to nanomolar concentration range. We have demonstrated the specificity and a wide dynamic range of the proposed immunoassay. The quenching is a result of competitive surface quenching of the AuQDs. Characterization, details of the immunoassay, and the quenching mechanism, are discussed. [ABSTRACT FROM AUTHOR]

We found that the formylbenzenediazonium (FBD)-grafted organic layers can be degraded by treating with an aqueous solution of salt and Tween. An electrochemical immunoassay was carried out on the degraded surface, which allowed highly sensitive detection of antigen mouse IgG. [ABSTRACT FROM AUTHOR]