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In 2022, the outbreak of the monkeypox virus occurred in many non-endemic countries, and the World Health Organization (WHO) assessed that this outbreak was "atypical". The establishment of a rapid and effective assay that can be used for the early diagnosis of monkeypox virus infection is crucial for outbreak prevention and control. In this study, the monkeypox virus A29 protein and the homologous vaccinia virus A27 protein and cowpox virus 162 protein were expressed in Escherichia coli BL21 for screening. We synthesized the monkeypox virus A2917–49 peptide as the immunogen and obtained 25 monoclonal antibodies (mAbs) against the A29 protein using mouse hybridoma techniques. Then an immunochromatographic test strip method for detecting A29 was established. The strips utilizing mAb-7C5 and 5D8 showed the best sensitivity and lowest limit of detection: 50 pg mL−1 for purified A29 and specificity tests showed that the strips did not cross-react with other orthopox viruses (vaccinia virus or cowpox virus) as well as common respiratory pathogens (SARS-CoV-2, influenza A and influenza B). Therefore, this method can be used for early and rapid diagnosis of monkeypox virus infection by antigen detection. [ABSTRACT FROM AUTHOR]

This communication describes a simple method for printing aqueous solutions with millimeter-scale patterns on a variety of substrates using an easily fabricated, paper-based microfluidic device (a paper-based “stamp”) as a contact printing device. The device is made from inexpensive materials, and it is easily assembled by hand; this method is thus accessible to a wide range of laboratories and budgets. A single device was used to print over 2500 spots in less than three minutes at a density of 16 spots per square centimeter. This method provides a new tool to pattern biochemicals—reagents, antigens, proteins, and DNA—on planar substrates. The accuracy of the volume of fluid delivered in simple paper-to-paper printing is low, and although the pattern transfer is rapid, it is better suited for qualitative than accurate, quantitative work. By patterning the paper to which the transfer occurs using wax printing or an equivalent technique, accuracy increases substantially., Chemistry and Chemical Biology

A disposable paper-based bipolar electrode (BPE) was reported for the first time for the sensitive electrochemiluminescence detection of a prostate specific antigen (PSA). [ABSTRACT FROM AUTHOR]

Cardiovascular disease is one of the leading causes of premature death worldwide, and the determination of C-reactive protein (CRP) from human serum is of vital importance for the diagnosis of the disease. For this study, we have developed an electrochemical immunosensor based on onion-like carbon@polyacrylonitrile (OLC-PAN) for the detection of CRP antigens. This was accomplished by immobilizing CRP antibodies on a modified glassy carbon electrode (GCE). Several electrochemical techniques such as cyclic voltammetry (CV), square wave voltammetry (SWV), and electrochemical impedance spectroscopy (EIS) were employed to evaluate the electrochemical detection of the CRP antigen. This ultrasensitive method for CRP antigen detection exhibited a very good logarithmic plot from −4.52 to −12.05 g mL−1 and a limit of detection (LOD) of 0.9 fg mL−1. The high selectivity, sensitivity, and stability of the developed electrochemical immunosensor would facilitate miniaturization for point-of-care applications and the efficient diagnosis of cardiovascular diseases. [ABSTRACT FROM AUTHOR]

The cytokeratin-19-fragment antigen (CYFRA 21-1), a soluble cell fragment, which is liberated into the blood during the metaphase of apoptosis, is one of the characteristic biomarkers of non-small cell lung cancer (NSCLC). In this paper, the copolymer poly(ε-caprolactone)-b-poly(ethylene oxide) (Dox–PCL–PEO) produced by ring-opening polymerization (ROP) is applied to a label-free electrochemical immunoassay for CYFRA 21-1. First, under the catalysis of tetrabutylammonium fluoride (TBAF), 4-(2-trimethylsilylethynyl) benzoic acid (TEB) was removed from the trimethylsilyl group and fixed on the surface of the electrode through Au–C. Then, the initial antibody (Ab1) was attached to the TEB to specifically recognize CYFRA 21-1. After that, the copolymer Dox–PCL–PEO was connected to the secondary antibody (Ab2) as a signal amplifying unit. Finally, Ab2–Dox–PCL–PEO specifically recognized CYFRA 21-1 to form an antibody–antigen–antibody sandwich structure. Therefore, we finally developed a complete electrochemical impedance immunoassay for the detection of CYFRA 21-1. Under the optimum experimental conditions, the detection method had a high sensitivity, and a wide linear range (100 fg mL−1–500 ng mL−1) with a lower limit of detection (LOD) of 0.021 pg mL−1, which is approximately 5.83 × 10−16 M What's more, it also showed high sensitivity, good selectivity and anti-interference in actual samples. Thus, there will be a good chance of early diagnosis of other tumor markers and biomolecules. [ABSTRACT FROM AUTHOR]

In vitro display technologies such as yeast display have been instrumental in developing the selection of new antibodies, antibody fragments or nanobodies that bind to a specific target, with affinity towards the target being the main factor that influences selection outcome. However, the roles of mechanical forces are being increasingly recognized as a crucial factor in the regulation and activation of effector cell function. It would thus be of interest to isolate binders behaving optimally under the influence of mechanical forces. We developed a microfluidic assay allowing the selection of yeast displaying nanobodies through antigen-specific immobilization on a surface under controlled hydrodynamic flow. This approach enabled enrichment of model yeast mixtures using tunable antigen density and applied force. This new force-based selection method opens the possibility of selecting binders by relying on both their affinity and force resistance, with implications for the design of more efficient immunotherapeutics. [ABSTRACT FROM AUTHOR]

Avian leukemia is an infectious tumorous disease of chickens caused by subgroup A of the avian leukemia virus (ALV-A), which mainly causes long-term viremia, slow growth, immune suppression, decreased production performance, multi-tissue tumors, and even death. The infection rate of this disease is very high in chicken herds in China, causing huge economic losses to the poultry industry every year. We successfully expressed the specific antigen protein of ALV (P27) through recombinant protein technology and screened a pair of highly sensitive monoclonal antibodies (mAbs) through mouse immunity, cell fusion, and antibody pairing. Based on this pair of antibodies, we established a dual antibody sandwich ELISA and gold nanoparticle immunochromatographic strip (AuNP-ICS) detection method. In addition, the parameters of the dual antibody sandwich ELISA and AuNP-ICS were optimized under different reaction conditions, which resulted in the minimum detection limits of 0.2 ng mL−1 and 1.53 ng ml−1, respectively. Commonly available ELISA and AuNP-ICS products on the market were compared, and we found that our established immune rapid chromatography had higher sensitivity. This established AuNP-ICS had no cross-reactivity with Influenza A (H1N1), Influenza A (H9N2), respiratory syncytial virus (RSV), varicella-zoster virus (VZV), Listeria monocytogenes listeriolysin (LLO), and Staphylococcal enterotoxin SED or SEC. Finally, the established AuNP-ICS was used to analyze 35 egg samples, and the results showed 5 positive samples and 30 negative samples. The AuNP-ICS rapid detection method established by our group had good specificity, high sensitivity, and convenience, and could be applied to the clinical sample detection of ALV-A. [ABSTRACT FROM AUTHOR]

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]

Agglutination is an antigen-antibody reaction with visible expression of aggregation of the antigens and their corresponding antibodies. Applications extend to the identification of acute bacterial infection, hemagglutination, such as blood grouping, and diagnostic immunology. Our finger-powered agglutination lab chip with external CMOS image sensing was developed to support a platform for inexpensive, rapid point-of-care (POC) testing applications related to agglutination effects. In this paper, blood grouping (ABO and Rh grouping) was utilized to demonstrate the function of our finger-powered agglutination lab chip with CMOS image sensing. Blood antibodies were preloaded into the antibody reaction chamber in the lab chip. The blood sample was pushed through the antibody reaction chamber using finger-powered pressure actuation to initiate a hemagglutination reaction to identify the blood type at the on-chip detection area using our homemade CMOS image sensing mini-system. Finger-powered actuation without the need for external electrical pumping is excellent for low-cost POC applications, but the pumping liquid volume per finger push is hard to control. In our finger-powered agglutination lab chip with CMOS image sensing, we minimized the effects of different finger push depths and achieved robust performance for the test results with different push depths. The driving sample volume per finger push is about 0.79 mm 3 . For different chips and different pushes, the driven sample volume per finger push was observed to vary in the range of 0.64 to 1.18 mm 3 . The red blood cells were separated from the plasma on-chip after the whole blood sample was finger pumped and before the red blood cells reached the antibody chamber via an embedded plasma-separation membrane. Our homemade CMOS image mini-system robustly read and identified the agglutination results on our agglutination lab chip.

Enzymes are used to amplify signals for detection of antigen proteins in biological samples. However, the enzymes conventionally used for this purpose have limitations, such as the presence of the same (i.e., endogenous) activity in human cells and difficulty in simultaneous use of multiple enzymes because of differences in their required reaction conditions. In this report, we identify an enzyme that can overcome these problems: β- D -galacturonidase (GalUAase) from Eisenbergiella tayi. GalUAase activity was confirmed to be absent from human cells. The substrate of GalUAase, galacturonic acid, is highly hydrophilic because of its anionic carboxylate group; high substrate hydrophilicity is an ideal characteristic for the substrate of an enzyme used for detection because it decreases nonspecific adsorption to biological samples. We show that E. tayi GalUAase could be used in the detection of antigen proteins on live human cells with lower background signal than the conventionally used enzyme β- D -galactosidase. The combinatorial use of GalUAase with β- D -galactosidase enabled simultaneous detection of two antigens on live cells. [ABSTRACT FROM AUTHOR]

Membrane antigens are phenotypic signatures of cells used for distinguishing various subpopulations and, therefore, are of great interest for diagnosis of diseases and monitoring of patients in hematology and oncology. Existing methods to measure antigen expression of a target subpopulation in blood samples require labor-intensive lysis of contaminating cells and subsequent analysis with complex and bulky instruments in specialized laboratories. To address this long-standing limitation in clinical cytometry, we introduce a microchip-based technique that can directly measure surface expression of target cells in hematological samples. Our microchip isolates an immunomagnetically-labeled target cell population from the contaminating background in whole blood and then utilizes the differential responses of target cells to on-chip magnetic manipulation to estimate their antigen expression. Moreover, manipulating cells with chip-sized permanent magnets and performing quantitative measurements via an on-chip electrical sensor network allows the assay to be performed in a portable platform with no reliance on laboratory infrastructure. Using our technique, we could successfully measure expressions of the CD45 antigen that is commonly expressed by white blood cells, as well as CD34 that is expressed by scarce hematopoietic progenitor cells, which constitutes only ∼0.0001% of all blood cells, directly from whole blood. With our technology, flow cytometry can potentially become a rapid bedside or at-home testing method that is available around the clock in environments where this invaluable assay with proven clinical utility is currently either outsourced or not even accessible. [ABSTRACT FROM AUTHOR]

Dendritic cells (DCs), which are immune sentinels in the peripheral tissues, play a number of roles, including patrolling for pathogens, internalising antigens, transporting antigens to the lymph nodes (LNs), interacting with T cells, and secreting cytokines. The well-coordinated migration of DCs under various immunological or inflammatory conditions is therefore essential to ensure an effective immune response. Upon maturation, DCs migrate faster and more persistently than immature DCs (iDCs), which is believed to facilitate CCR7-dependent chemotaxis. It has been reported that lipopolysaccharide-activated DCs produce IL-12 only transiently, and become resistant to further stimulation through exhaustion. However, little is known about the influence of DC exhaustion on cellular motility. Here, we studied the cellular migration of exhausted DCs in tissue-mimicked confined environments. We found that the speed of exhausted matured DCs (xmDCs) decreased significantly compared to active matured DCs (amDCs) and iDCs. In contrast, the speed fluctuation increased compared to that of amDCs and was similar to that of iDCs. In addition, the diffusivity of the xmDCs was significantly lower than that of the amDCs, which implies that DC exhaustion reduces the space exploration ability. Interestingly, CCR7-dependent chemotaxis against CCL19 in xmDCs was not considerably different from that observed in amDCs. Taken together, we report a unique intrinsic cell migration behaviour of xmDCs, which exhibit a slower, less persistent, and less diffusive random motility, which results in the DCs remaining at the site of infection, although a well-preserved CCR7-dependent chemotactic motility is maintained. [ABSTRACT FROM AUTHOR]

Therapeutic antibodies are paramount in treating a wide range of diseases, particularly in auto-immunity, inflammation and cancer, and novel antibody candidates recognizing a vast array of novel antigens are needed to expand the usefulness and applications of these powerful molecules. Microdevices play an essential role in this challenging endeavor at various stages since many general requirements of the overall process overlap nicely with the general advantages of microfluidics. Therefore, microfluidic devices are rapidly taking over various steps in the process of new candidate isolation, such as antibody characterization and discovery workflows. Such technologies can allow for vast improvements in time-lines and incorporate conservative antibody stability and characterization assays, but most prominently screenings and functional characterization within integrated workflows due to high throughput and standardized workflows. First, we aim to provide an overview of the challenges of developing new therapeutic candidates, their repertoires and requirements. Afterward, this review focuses on the discovery of antibodies using microfluidic systems, technological aspects of micro devices and small-scale antibody protein characterization and selection, as well as their integration and implementation into antibody discovery workflows. We close with future developments in microfluidic detection and antibody isolation principles and the field in general. [ABSTRACT FROM AUTHOR]

Highly stereoselective 1,3-dipolar cycloaddition allowed the synthesis of the original stable TN antigen mimic presenting a constraint on the amino acid part. Two main conformations of the GalNAc moiety were detected by molecular modelling and NMR. A ligand/protein interaction study was performed by saturation transfer difference NMR analyses using two GalNAc-specific model lectins highlighting that the constrained C-glycoside analog was recognized by these proteins as well as the TN antigen derivative. [ABSTRACT FROM AUTHOR]

The envelope glycoprotein domain III (EDIII) of dengue virus (DENV) has been recognised as the antigenic region responsible for receptor binding. In the present work, we have proposed a novel immunosensor constructed on a graphene-coated screen-printed carbon electrode (SPCE) using plant-derived EDIII as the probe antigen to target DENV IgG antibodies. The developed immunosensor demonstrated high sensitivity towards DENV IgG within a wide linear working range (125–2000 ng mL−1) under the optimised sensing conditions. The limit of detection was determined to be 22.5 ng mL−1. The immunosensor also showed high specificity towards DENV IgG, capable of differentiating DENV IgG from the antibodies of other infectious diseases including the similarly structured Zika virus (ZIKV). The ability of the immunosensor to detect dengue antibodies in serum samples was also verified by conducting tests on mouse serum samples. The proposed immunosensor was able to provide a binary (positive/negative) response towards the serum samples comparable to the conventional enzyme-linked immunosorbent assay (ELISA), indicating promising potential for realistic applications. [ABSTRACT FROM AUTHOR]

An evaluation of a rapid portable gold-nanotechnology measuring SARS-CoV-2 IgM, IgA and IgG antibody concentrations against spike 1 (S1), spike 2 (S) and nucleocapsid (N) was conducted using serum samples from 74 patients tested for SARS-CoV-2 RNA on admission to hospital, and 47 historical control patients from March 2019. 59 patients were RNA(+) and 15 were RNA(−). A serum (±) classification was derived for all three antigens and a quantitative serological profile was obtained. Serum(+) was identified in 30% (95% CI 11–48) of initially RNA(−) patients, in 36% (95% CI 17–54) of RNA(+) patients before 10 days, 77% (95% CI 67–87) between 10 and 20 days and 95% (95% CI 86–100) after 21 days. The patient-level diagnostic accuracy relative to RNA(±) after 10 days displayed 88% sensitivity (95% CI 75–95) and 75% specificity (95% CI 22–99), although specificity compared with historical controls was 100% (95%CI 91–100). This study provides robust support for further evaluation and validation of this novel technology in a clinical setting and highlights challenges inherent in assessment of serological tests for an emerging disease such as COVID-19. [ABSTRACT FROM AUTHOR]

With the increase in cancer risk, early immunodiagnosis is of great significance for timely therapy. In this work, a DNA-mediated immunosensor for the highly sensitive detection of prostate specific antigen (PSA) is proposed, which is mainly based on a portable personal glucose meter (PGM). Gold nanoparticles (AuNPs) functionalized with PSA detection antibodies and DNA primers are introduced. When the target of the PSA is present, rolling circle amplification (RCA) reactions on AuNPs are triggered and numerous repeated RCA products hybridize with the DNA-conjugated invertase; thus the signal of the PGM is generated and the PSA is quantified indirectly. With the use of a portable PGM, our method realizes a linear detection range of 0.003–50 ng mL−1, with a low detection limit of 0.1 pg mL−1, which is comparable to that of the traditional methods using expensive apparatus. Besides, the analysis of clinical human serum samples is performed to investigate its good practicability. This simple, low-cost, and miniaturized immunosensor is promising for the point-of-care testing of cancer markers. [ABSTRACT FROM AUTHOR]

Immunoassays analyzing interactions between antigens and antibodies can be affected by capillary action together with binding affinity. This paper studies contact-angle changes of bacterial suspensions on antibody immobilized surfaces. The capillary action and the dried pattern of the bacterial suspensions are analyzed and correlated with specific- and nonspecific bindings between bacteria and antibodies. [ABSTRACT FROM AUTHOR]

Membrane antigens control cell function by regulating biochemical interactions and hence are routinely used as diagnostic and prognostic targets in biomedicine. Fluorescent labeling and subsequent optical interrogation of cell membrane antigens, while highly effective, limit expression profiling to centralized facilities that can afford and operate complex instrumentation. Here, we introduce a cytometry technique that computes surface expression of immunomagnetically labeled cells by electrically tracking their trajectory under a magnetic field gradient on a microfluidic chip with a throughput of ＞500 cells per min. In addition to enabling the creation of a frugal cytometry platform, this immunomagnetic cell manipulation-based measurement approach allows direct expression profiling of target subpopulations from non-purified samples. We applied our technology to measure epithelial cell adhesion molecule expression on human breast cancer cells. Once calibrated, surface expression and size measurements match remarkably well with fluorescence-based measurements from a commercial flow cytometer. Quantitative measurements of biochemical and biophysical cell characteristics with a disposable cytometer have the potential to impact point of care testing of clinical samples particularly in resource limited settings. [ABSTRACT FROM AUTHOR]

The interaction between incident light and surface electrons in conductive nanoparticles produces localized plasmon oscillations with a resonant frequency that strongly depends on the composition, size, geometry, and dielectric environment. Hybrid heterostructure materials combining two or more materials in one structure represent a powerful way to achieve unique properties and multifunctionality compared to those of the individual nanoparticle components. Hybrid gold nanorods and gold nanoclusters (GNR/AuNCs) heterostructures prepared by intimate integration of GNRs with AuNCs exhibit both localized surface plasmon resonance (LSPR) property and peroxidase-like activity. It is found that the catalytic activity of the AuNC/GNR heterostructure could be remarkably enhanced by LSPR induced by photon–plasmon coupling in the visible to near-infrared (NIR) region. Meanwhile, the catalytic activity of enzyme-like AuNC/GNRs may be regulated by immunoreactions to realize specific recognition of a target analyte. Accordingly, a fast colorimetric assay within 5 min for the detection of prostate specific antigen (PSA) was developed based on a AuNC/GNRs heterostructure mask regulated by the target molecule under photon-plasmon coupling. The color intensity is inversely proportional to the PSA concentration, and quantitative analysis may be achieved in a range of 10 and 200 pg mL−1. This sensor was practically applied to detect PSA levels in prostate cancer serum samples and the determined values agreed well with those measured by the hospital using standard methods. This indicates that the AuNC/GNRs heterostructure-based assay has high accuracy for the analysis of practical samples. Moreover, the new method has the advantages of very fast determination and low sample volume requirements. [ABSTRACT FROM AUTHOR]

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.

Magnetic levitation is a technique for measuring the density and the magnetic properties of objects suspended in a paramagnetic field. We describe a novel magnetic levitation-based method that can specifically detect cell membrane-bound and soluble antigens by measurable changes in levitation height that result from the formation of antibody-coated bead and antigen complex. We demonstrate our method's ability to sensitively detect an array of membrane-bound and soluble antigens found in blood, including T-cell antigen CD3, eosinophil antigen Siglec-8, red blood cell antigens CD35 and RhD, red blood cell-bound Epstein–Barr viral particles, and soluble IL-6, and validate the results by flow cytometry and immunofluorescence microscopy performed in parallel. Additionally, employing an inexpensive, single lens, manual focus, wifi-enabled camera, we extend the portability of our method for its potential use as a point-of-care diagnostic assay. [ABSTRACT FROM AUTHOR]

Low antigenic expression of ABO subgroup system on red blood cell (RBC) is cause of discrepancy between forward and reverse blood typing in the standard agglutination technique. Neutralization agglutination is employed for verification of the detection of ABH substances in saliva. However, the neutralization technique is complicated, time-consuming and requires expertise. To overcome these drawbacks, surface plasmon resonance (SPR) imaging was developed for ABH antigen detection on RBCs and in saliva. An antibody array was designed to classify the ABO subgroups by anti-A, anti-B, and anti-H antibodies; the array was immobilized on a carboxymethyl-dextran sensor-surface. RBCs and saliva specimens from sixty-four donors were analysed by passing them over the antibody array, where the secretor status and blood group could be simultaneously identified. Consequently, the immobilized antibodies could specifically and quantitatively detect the ABH antigen on RBCs. Using the direct assay, the SPR signal of saliva detection was weaker than that of RBC detection. However, a sandwich assay with a mixture of anti-A, anti-B, and anti-H antibodies could efficiently enhance the signal. The sensor chip provided high specificity (cut-off at 100 to 175 micro refractive index units) and high precision at 0.06%–4.9% CV. The blood group results of the sixty-four donor specimens obtained by SPR agreed with the standard agglutination test with 100% accuracy. SPR could indicate different ABH antigen densities on the RBCs and nearly the same amounts of ABH substances in the saliva of strong and weak subgroups. Finally, we also demonstrated reduced assay time and fewer complications with the SPR imaging platform compared to the neutralization technique. [ABSTRACT FROM AUTHOR]

Nanomaterials are increasingly being developed for applications in biotechnology, including the delivery of therapeutic drugs and of vaccine antigens. However, there is a lack of screening systems that can rapidly assess the dynamics of nanoparticle uptake and their consequential effects on cells. Established in vitro approaches are often carried out on a single time point, rely on time-consuming bulk measurements and are based primarily on populations of cell lines. As such, these procedures provide averaged results, do not guarantee precise control over the delivery of nanoparticles to cells and cannot easily generate information about the dynamics of nanoparticle–cell interactions and/or nanoparticle-mediated compound delivery. Combining microfluidics and nanotechnology with imaging techniques, we present a microfluidic platform to monitor nanoparticle uptake and intracellular processing in real-time and at the single-cell level. As proof-of-concept application, the potential of such a system for understanding nanovaccine delivery and processing was investigated and we demonstrate controlled delivery of ovalbumin-conjugated gold nanorods to primary dendritic cells. Using time-lapse microscopy, our approach allowed monitoring of uptake and processing of nanoparticles across a range of concentrations over several hours on hundreds of single-cells. This system represents a novel application of single-cell microfluidics for nanomaterial screening, providing a general platform for studying the dynamics of cell–nanomaterial interactions and representing a cost-saving and time-effective screening tool for many nanomaterial formulations and cell types. [ABSTRACT FROM AUTHOR]

Ti4+ immobilized SiO2 graphene-like multilayer nanosheets were designed and synthesized for the ultrasensitive enrichment of phosphopeptides with the detection limit of 1.0 pg μL−1 (4 × 10−17 mol μL−1). The enrichment selectivity of the nanosheets was evaluated by capturing phosphopeptides from a peptide mixture of bovine β-casein (β-casein) and bovine serum albumin (BSA) with the molar ratio of 1 : 1000 (1.7 × 10−12 mol of β-casein and 1.7 × 10−9 mol of BSA in 100 μL). [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]

Lab-on-a-chip systems with molecular motor driven transport of analytes attached to cytoskeletal filament shuttles (actin filaments, microtubules) circumvent challenges with nanoscale liquid transport. However, the filaments have limited cargo-carrying capacity and limitations either in transportation speed (microtubules) or control over motility direction (actin). To overcome these constraints we here report incorporation of covalently attached antibodies into self-propelled actin bundles (nanocarriers) formed by cross-linking antibody conjugated actin filaments via fascin, a natural actin-bundling protein. We demonstrate high maximum antigen binding activity and propulsion by surface adsorbed myosin motors. Analyte transport capacity is tested using both protein antigens and microvesicles, a novel class of diagnostic markers. Increased incubation concentration with protein antigen in the 0.1–100 nM range (1 min) reduces the fraction of motile bundles and their velocity but maximum transportation capacity of ＞1 antigen per nm of bundle length is feasible. At sub-nanomolar protein analyte concentration, motility is very well preserved opening for orders of magnitude improved limit of detection using motor driven concentration on nanoscale sensors. Microvesicle-complexing to monoclonal antibodies on the nanocarriers compromises motility but nanocarrier aggregation via microvesicles shows unique potential in label-free detection with the aggregates themselves as non-toxic reporter elements. [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]

An ultrasensitive electrochemical immunosensor for the detection of cluster of differentiation 146 antigen (CD146) based on TiO2 colloidal sphere laden Au/Pd nanoparticles (Au/Pd@TiO2) was developed. In this work, reduced graphene oxide-tetraethylene pentamine (rGO-TEPA) was applied as an electrode modifying material to modify the surface of a glassy carbon electrode (GCE). Au/Pd@TiO2 was used as the secondary-antibody (Ab2) label for the fabrication of the immunosensor. Amperometric response of the immunosensor for electrocatalytic reduction of hydrogen peroxide (H2O2) was recorded. Electrochemical impedance spectroscopy (EIS) proved that fabrication of the immunosensor was successful. The anti-CD146 primary antibody (Ab1) was immobilized on the rGO-TEPA modified GCE by a cross-linking reagent of glutaraldehyde (GA). With Ab1 immobilized onto the rGO-TEPA modified GCE and Ab2 linked with Au/Pd@TiO2, the immunosensor displayed a wide linear range (0.0050–20 ng mL−1), a low detection limit (1.6 pg mL−1), good reproducibility, good selectivity and acceptable stability. The designed sensing strategy may provide a potential application in the detection of other tumor markers. [ABSTRACT FROM AUTHOR]

An ultra-sensitive gas phase biosensor/tracer/bio-sniffer is an emerging technology platform designed to provide real-time information on air-borne analytes, or those in liquids, through classical headspace analysis. The desired bio-sniffer measures gaseous 17α– ethinylestradiol (ETED) as frequency changes on a quartz crystal microbalance (QCM), which is a result of the interactions of liquid sample components in the headspace (ETED and water) with a biorecognition layer. The latter was constructed by immobilization of polyclonal antiserum against a phenolic A-ring of estrogenic receptors through protein A. The QCM response exhibited stretched exponential kinetics of negative frequency shifts with reversible and “irreversible” components of mass uptake onto the sensor surface in static headspace conditions when exposed to water solutions of ETED over the sensor working range, from 10−10 to 10−17 g L−1. It was shown that the variations in the QCM response characteristics are due to the change of the water-binding capacity of the sensing layer induced by protein transformations initiated by the binding of ETED molecules. This result is well correlated with the natural physiological function of estrogens in controlling the homeostasis of body fluids in living beings. [ABSTRACT FROM AUTHOR]

SANSSERIFWe used the microarray technology to develop chips containing a comprehensive set of p/SANSSERIFroteins and peptides covering the proteome of HIV-1 clade C, which is the HIV-1 subtype that causes the majority of infections worldwide. We demonstrate that the HIV microarray allows simultaneous, sensitive and specific detection of antibody responses for the major immunoglobulin classes (IgG, IgA, IgM, IgE) and subclasses (IgG1–4) with minute amounts of serum samples towards a large number of HIV antigens and peptides. Furthermore, we show that the HIV chip can be used for the monitoring of antibody responses during the course of the disease and during treatment. The HIV microarray should be useful to study antibody responses to multiple HIV antigens and epitopes in HIV-infected patients to explore pathomechanisms of the disease, for diagnosis and for monitoring of treatment and of vaccine trials. [ABSTRACT FROM AUTHOR]

Herein, we demonstrate the protocol of a label-free photoelectrochemical (PEC) immunoassay on the basis of ordered mesoporous carbon (CMK-3) and water-soluble CdSe@ZnS core-shell quantum dots (QDs) coupled with a biospecific interaction for the ultrasensitive detection of human immunoglobulin (antigen, H-IgG) as a model protein. The CMK-3 was dispersed with chitosan (CS-CMK-3), which contains a large amount of amino groups (-NH2), and the CdSe@ZnS QDs were treated with thioglycolic acid, which contains carboxylic acid groups (-COOH). The layer-by-layer assembling of CdSe@ZnS QDs and CS-CMK-3 achieved through the covalent bonding of -COOH and -NH2 was employed as a photoactive antibody (Ab) immobilization matrix. Improved sensitivity was achieved through the synergistic effect of the excellent electrical conductivity and high specific surface area of CMK-3, as well as the high photon-to-electron conversion efficiency of CdSe@ZnS QDs. The photoexcitation of a CMK-3/CdSe@ZnS QDs-modified ITO electrode potentiostated at 0 V (vs. Ag/AgCl) under white light led to a stable anodic photocurrent. To perform the immunoassay, anti-human immunoglobulin (antibody, anti-H-IgG) was conjugated onto the CdSe@ZnS QDs-modified electrode by using EDC-NHS coupling reactions between -COOH of CdSe@ZnS QDs and -NH2 of the antibody. The concentrations of H-IgG were measured through the decrease in photocurrent intensity resulting from the increase in steric hindrance due to the formation of the immunocomplex. Under the optimal conditions, a linear relationship between photocurrent decrease and H-IgG concentration was obtained in the range 10 pg mL-1-100 ng mL-1 with a detection limit of 5 pg mL-1. This strategy opens a simple perspective for the application of mesoporous conductive material and core-shell QDs as light-harvesting architecture, which might be of great significance in PEC bioanalysis in the future. [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]

We present a new concept for rapid and fully portable prostate specific antigen (PSA) measurements, termed “lab-in-a-briefcase”, which integrates an affordable microfluidic ELISA platform utilising a melt-extruded fluoropolymer microcapillary film (MCF) containing an array of 10 200 μm internal diameter capillaries, a disposable multi-syringe aspirator (MSA), a sample tray pre-loaded with all of the required immunoassay reagents, and a portable film scanner for colorimetric signal digital quantification. Each MSA can perform 10 replicate microfluidic immunoassays on 8 samples, allowing 80 measurements to be made in less than 15 minutes based on semi-automated operation, without the need of additional fluid handling equipment. The assay was optimised for the measurement of a clinically relevant range of PSA of 0.9 to 60.0 ng ml−1 in 15 minutes with CVs on the order of 5% based on intra-assay variability when read using a consumer flatbed film scanner. The PSA assay performance in the MSA remained robust in undiluted or 1 : 2 diluted human serum or whole blood, and the matrix effect could simply be overcome by extending sample incubation times. The PSA “lab-in-a-briefcase” is particularly suited to a low-resource health setting, where diagnostic labs and automated immunoassay systems are not accessible, by allowing PSA measurement outside the laboratory using affordable equipment. [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]

The core requirements for point-of-care (POC) diagnostics necessitate low-cost, portability, easily integrated sample preparation, and quick measurement time. Frequency-shift based magnetic sensing is a measurement technique utilizing a complementary metal-oxide-semiconductor (CMOS) integrated-circuit (IC) chip for magnetic label detection. The sensing scheme leverages the low-cost manufacturing of IC chips while demonstrating the potential for multiplexing capabilities. In this article, we present modifications to this scheme for POC viability. We introduce a handheld reusable reader and a disposable open-well cartridge for the detection of nucleic acids and antigens. The diagnostic system utilizes a novel “magnetic freezing” technique to reduce measurement time, obviates baseline measurement before or during biological assay, and reduces sensor noise. We utilize these enhancements for the room temperature, amplification-free detection of a 31 base-pair DNA oligomer and the interferon-γ (IFN-γ) protein. We have demonstrated reliable measurements down to 100 pM for the DNA assay and 1 pM for the protein. [ABSTRACT FROM AUTHOR]

The activation of lymphocytes occurs when they are exposed to viruses or other foreign antigens. The aim of this work was to verify if Raman spectroscopy can be used to screen the activation of lymphocytes during viral infection. There are distinct peaks that reveal differences between activated and intact cells. The most important marker of the lymphocyte activation process is the prominent 521 cm−1 disulfide band which marks the immunoglobulin formation. The up shift of the S–S mode from the broad band centered at 510 cm−1 of human normal immunoglobulin to a single band at 521 cm−1 of human B cells indicates a selection of the optimal geometry of the disulphide bridges to bind to a foreign antigen. Polarization data is used to detect the structural alteration between domain fragments. Differences in other band intensities may be due to different protein compositions in both the investigated forms. B cell activation causes the change of the intracellular cytoplasm composition due to the secretion of immunoglobulins during the fighting of the infection. [ABSTRACT FROM AUTHOR]

This work presents an AC electrokinetic impedance sensing method that is capable of detecting specific interactions between macromolecules such as antigen–antibody binding. Serum samples were added to the surface of interdigitated electrodes that had been coated with bacterial antigens. After applying an AC signal of 100 mV at a specific frequency continuously, the electrodes' impedance change was recorded and used to determine the occurrence and level of antibody binding to the antigen. Our theoretical analysis indicated that with this AC signal, the target macromolecules will experience a sufficiently strong attraction force towards the electrode surface for acceleration of the binding process. Using this method, 11 human tuberculosis and 10 bovine tuberculosis serum samples were tested. The results were consistent with those obtained by a conventional ELISA method. The limit of detection of the impedance sensing method was estimated to be better than 10 ng mL−1. In summary, we demonstrate that AC electrokinetic impedance sensing can be used for rapid and sensitive detection of specific antibodies in serum samples. This method may form a basis for development of a point of care diagnostic device for human and bovine tuberculosis. [ABSTRACT FROM AUTHOR]

Phosphatidylinositol mannosides (PIMs) have been investigated as lipidic antigens for a new subunit tuberculosis vaccine. A non-natural diacylated phosphatidylinositol mannoside (Ac2PIM2) was designed and synthesized by mimicking the natural PIM6 processing procedure in dentritic cells. This synthetic Ac2PIM2 was achieved from α-methyl D -glucopyranoside 1 in 17 steps in 2.5% overall yield. A key feature of the strategy was extending the use of the chiral myo-inositol building block A to the O-2 and O-6 positions of the inositol unit to allow for introducing the mannose building blocks B1 and B2, and to the O-1 position for the phosphoglycerol building block C. Building block A, being a flexible core unit, may facilitate future access to other higher-order PIM analogues. A preliminary antigenic study showed that the synthetic PIM epitope (Ac2PIM2) was significantly more active than natural Ac2PIM2, which indicated that the synthetic Ac2PIM2 can be strongly immunoactive and may be developed as a potential vaccine. [ABSTRACT FROM AUTHOR]

Zirconium-89 is currently being used in numerous clinical trials involving monoclonal antibodies and positron emission tomography. This report describes a revised strategy that reduces preparation time while increasing the specific activity of clinically relevant immuno-PET agents. Additionally, it demonstrates that n-acetyl-l-cysteine acts as a superior radioprotective agent that improves long-term stability without compromising antigen affinity in vivo. [ABSTRACT FROM AUTHOR]

Prostate-specific membrane antigen (PSMA) can serve as a molecular cell surface target for the detection and treatment of prostate cancer. Near-infrared (NIR) fluorescence imaging enables highly sensitive, rapid, and non-radioactive imaging of PSMA, though specific targeting still remains a challenge because no optimized contrast agents exist. [ABSTRACT FROM AUTHOR]

Mass spectrometry-based methods offer an alternative means of determining allergens in foods. Whilst targeted methods are likely to offer the most robust approach for detection and quantification, little is known about how food processing may affect the behaviour of peptide targets. A systematic study has been undertaken to investigate the effects of thermal processing (boiling, roasting, frying) on the behaviour of a suite of peanut peptide targets representing the major clinically-relevant allergens. Initially the effect of thermal processing on protein extractability was investigated and a mass spectrometry-compatible buffer identified comprising 50 mM Tris-HCl, pH 8.8 containing 50 mM dithiothreitol and 0.04% (w/v) acid labile detergent which was able to extract 45–100% of protein from raw, boiled, roasted and fried peanuts using sonication at 60 °C. Eight peptide targets were identified including two peptides from each cupin allergen, Ara h1 and Ara h3 and four peptides from the prolamin superfamily allergens Ara h2, 6 and 7. AQUA peptide standards were synthesised and used to undertake multiple-reaction monitoring experiments, giving assay sensitivities of 0.1–30 amoles of peptide on-column (3 : 1 signal : noise), calculated limits of quantification between 96–1343 amoles of peptide on-column and a linear dynamic range of 4–5 orders of magnitude. Absolute quantification of individual peanut allergens in thermally processed samples showed that peptide targets in the cupin allergens were more prone to processing-induced effects than those from Ara h2, 6 and 7. Targets flanked by arginine residues showed greater thermostability. Identification of processing-stable targets, coupled with more efficient extraction procedures and a wide dynamic range, shows that targeted mass spectrometry methods have great potential as an additional method for quantifying peanut allergens in complex food matrices. [ABSTRACT FROM AUTHOR]

Whilst recent advances in nanotechnology have yielded many new biosensing capabilities, innovative biological attachment and detection modalities remain relatively underdeveloped. Bi-specific antibodies (bsAbs) – which exhibit binding capability for two separate targets – offer an inherent advantage over conventional antibody reagents by significantly simplifying sensor surface preparation. Herein, we report the deployment of bsAbs for simultaneous attachment to a polymer-coated transducer and label-free, electrochemical (EC) detection of target antigens. [ABSTRACT FROM AUTHOR]

An ultrasensitive analytical method was developed to determine prostate specific antigen (PSA) tagged with TiO2 nanoparticles (NPs) using inductively coupled plasma mass spectrometry (ICP-MS). The synthesized TiO2 NPs (19.8 nm) were modified with monoclonal antibody, IgG, for tagging on the PSA through the antigen–antibody reaction. For the sample pretreatment, magnetic NPs (MNPs) were also synthesized and modified for the collection of PSA in a serum sample. The sandwiched complexes thereby obtained were collected using a permanent magnet in order to remove any remaining unbound particles. The MNPs were then eliminated after the cleavage of antigen–antibody bindings by treatment with 0.33 M nitric acid. The concentration of the TiO2 NPs remaining in the solution was determined by ICP-MS with a flow injection system and a 10 μL injection loop. The PSA concentration was determined by measuring the 49Ti signal of the tagged TiO2 NPs. The obtained limit of detection for PSA in serum was 1.16 fg mL−1, which was more than 3.44 × 106 times lower than the current PSA levels for cancer diagnosis. [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]

Protein-conjugated gold nanoparticles (AuNPs) have been extensively explored for the development of many novel protein assays. In this article, we demonstrate that nanoparticle tracking analysis (NTA) can be used as a rapid and simple analytical tool to monitor bioconjugation and to study protein–protein interactions. The adsorption of protein A onto gold nanoparticles was analyzed using NTA. The conjugation resulted in a measurable increase in hydrodynamic radius that correlated with protein A concentration, allowing conditions for complete conjugation to be elucidated. NTA was then used to investigate the binding of mouse IgG to protein A-conjugated AuNPs and the Ka was measured as 2.00 × 107 M−1. Furthermore, an assay for the detection of mouse IgG was developed using NTA to detect the binding to antibody–AuNP conjugates. This assay provided a detection limit of 3.2 ng mL−1; however, the formation of aggregates resulting from the use of a polyclonal antibody and multiple binding sites on the antigen prevented the determination of binding affinity for this antibody–antigen system. To measure the binding affinity for this antibody–antigen system the IgG antigen was conjugated to the AuNPs and NTA was used to monitor the binding of the antibody. In this configuration aggregation of conjugates was not detected and a binding affinity constant of 2.80 × 108 M−1 was measured. NTA results obtained in this work were validated by comparison to DLS. This work represents the first evaluation of NTA as an analytical tool for characterizing AuNP bioconjugates, investigating protein–protein binding, and detecting low levels of antigen in a bioassay. [ABSTRACT FROM AUTHOR]

Cancer cells are characterized by the presence of diverse antigens on their surface. The combination of these structures in a single molecule represents an attractive strategy to obtain vaccine candidates with improved immunological properties. Herein we report the synthesis of a mixture of heterotopic platforms bearing the Tn and TF carbohydrate antigens and an immunostimulating poliovirus peptide. [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]