Sensitive detection of multiplex toxins using antibody microarray

Using a newly developed fluorescent nanoparticle (NP) that gives rise to a high-intensity and stable fluorescent light, a sensitive antibody (Ab) microarray assay system has been developed for specific detection of bioterrorism agents, as exemplified by ricin, cholera toxin (CT), and staphylococcal enterotoxin B (SEB). The Ab microarray uses a sandwich format that consists of capture Abs, analytes (toxins), biotinylated detection Abs, and avidin-conjugated NP. In all three cases, polyclonal Abs (pAbs) displayed superiority over monoclonal antibodies (mAbs) in capturing toxins on microarray slides even when the pAbs and mAbs had similar affinity as determined by enzyme-linked immunosorbent assay (ELISA). The detection system was successfully used to detect toxins spiked in milk, apple cider, and blood samples. We were able to detect ricin at 100 pg/ml in buffer and at 1 ng/ml in spiked apple cider or milk, whereas CT and SEB were detected at 10 pg/ml in buffer and 100 pg/ml in spiked apple cider or milk. High specificities were also demonstrated in the detection of mixed toxin samples with similar sensitivities. The matrix effect of blood samples on the detection of mixed toxins seems to be minimal when the toxin concentration is at or above 100 ng/ml. The current study highlights the significant role of pAb and NP in increasing selectivity and sensitivity of toxin detection in a microarray format.     

Sensitive immunoassay of Listeria monocytogenes with highly fluorescent bioconjugated silica nanoparticles probe

In this paper, a sensitive immunoassay method was proposed for Listeria monocytogenes detection by using highly fluorescent bioconjugated nanoparticles probe. (FITC-IgG)-doped fluorescent silica nanoparticles (fsNPs) firstly were synthesized by a microemulsion method and characterized by TEM and fluorescent spectra. Then the prepared fsNPs were conjugated with polyclonal rabbit anti-L. monocytogenes antibody (pAb) and used as indicator probe. A sandwich-type immune affinity reaction between polyclonal rabbit anti-L. monocytogenes antibody coated onto microplate wells, target bacteria and the fsNPs-antibody conjugates subsequently was conducted to detect target L. monocytogenes and assemble the indicator probe onto the wells. The target L. monocytogenes was measured by the fluorescent signals of the assembled indicator probes. Under the optimal conditions, the calibration graph of fluorescent intensity is proportional to the amount of target bacteria over the range of 50-10,320 CFU/mL with a detection limit of 50 CFU/mL. The proposed method has been successfully applied to detect L. monocytogenes in food samples offering the advantages of sensitivity, simplicity, and stability.     

Sensitive detection of the redox state of copper proteins using fluorescence

The blue copper protein azurin from Pseudomonas aeruginosa has been covalently labelled with the fluorescing dye Cy5. The optical spectrum of the azurin changes markedly with its redox state. These changes are reflected in the fluorescence intensity of the dye through fluorescence resonance energy transfer (FRET). This provides a sensitive way to monitor biological redox events. The method shown to work in the nanomolar range of protein concentrations, can be easily extended into the sub-nanomolar regime and holds promise for single-molecule detection.     

Quantum dot immunoassays in renewable surface column and 96-well plate formats for the fluorescence detection of botulinum neurotoxin using high-affinity antibodies

A fluorescence sandwich immunoassay using high-affinity antibodies and quantum dot (QD) reporters has been developed for detection of botulinum neurotoxin serotype A (BoNT/A) using a nontoxic recombinant fragment of the holotoxin (BoNT/A-H_C-fragment) as a structurally valid simulant for the full toxin molecule. The antibodies used, AR4 and RAZ1, bind to nonoverlapping epitopes present on both the full toxin and on the recombinant fragment. In one format, the immunoassay is carried out in a 96-well plate with detection in a standard plate reader using AR4 as the capture antibody and QD-coupled RAZ1 as the reporter. Detection to 31 pM with a total incubation time of 3 h was demonstrated. In a second format, the AR4 capture antibody was coupled to Sepharose beads, and the reactions were carried out in microcentrifuge tubes with an incubation time of 1 h. The beads were subsequently captured and concentrated in a rotating rod “renewable surface” flow cell equipped with a fiber optic system for fluorescence measurements. In PBS buffer, the BoNT/A-H_C-fragment was detected to concentrations as low as 5 pM using the fluidic measurement approach.     

Sensitive detection of glucagon aggregation using amyloid fibril-specific antibodies

Sensitive detection of protein aggregates is important for evaluating the quality of biopharmaceuticals and detecting misfolded proteins in several neurodegenerative diseases. However, it is challenging to detect extremely low concentrations (<10 ppm) of aggregated protein in the presence of high concentrations of soluble protein. Glucagon, a peptide hormone used in the treatment of extreme hypoglycemia, is aggregation-prone and forms amyloid fibrils. Detection of glucagon fibrils using conformation-specific antibodies is an attractive approach for identifying such aggregates during process and formulation development. Therefore, we have used yeast surface display and magnetic-activated cell sorting to sort single-chain antibody libraries to identify antibody variants with high conformational specificity for glucagon fibrils. Notably, we find several high-affinity antibodies that display excellent selectivity for glucagon fibrils, and we have integrated these antibodies into a sensitive immunoassay. Surprisingly, the sensitivity of our assay—which involves direct (nonantibody mediated) glucagon immobilization in microtiter plates—can be significantly enhanced by pretreating the microtiter plates with various types of globular proteins before glucagon immobilization. Moreover, increased total concentrations of glucagon peptide also significantly improve the sensitivity of our assay, which appears to be due to the strong seeding activity of immobilized fibrils at high glucagon concentrations. Our final assay is highly sensitive (fibril detection limit of ~0.5–1 ppm) and is >20 times more sensitive than detection using a conventional, amyloid-specific fluorescent dye (Thioflavin T). We expect that this type of sensitive immunoassay can be readily integrated into the drug development process to improve the generation of safe and potent peptide therapeutics.     

Fluorescent nanoparticles as labels for immunometric assay of C-reactive protein using two-photon excitation assay technology

We describe the use of fluorophore-doped nanoparticles as reporters in a recently developed ArcDia TPX bioaffinity assay technique. The ArcDia TPX technique is based on the use of polymer microspheres as solid-phase reaction carrier, fluorescent bioaffinity reagents, and detection of two-photon excited fluorescence. This new assay technique enables multiplexed, separation-free bioaffinity assays from microvolumes with high sensitivity. As a model analyte we chose C-reactive protein (CRP). The assay of CRP was optimized for assessment of CRP baseline levels using a nanoparticulate fluorescent reporter, 75 nm in diameter, and the assay performance was compared to that of CRP assay based on a molecular reporter of the same fluorophore core. The results show that using fluorescent nanoparticles as the reporter provides two orders of magnitude better sensitivity (87 fM) than using the molecular label, while no difference between precision profiles of the different assay types was found. The new assay method was applied for assessment of baseline levels of CRP in sera of apparently healthy individuals.     

Sensitive detection of acetylcholine based on a novel boronate intramolecular charge transfer fluorescence probe

A highly sensitive and selective fluorescence method for the detection of acetylcholine (ACh) based on enzyme-generated hydrogen peroxide (H₂O₂) and a new boronate intramolecular charge transfer (ICT) fluorescence probe, 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-N-butyl-1,8-naphthalimide (BN), was developed. This strategy involves the reaction of ACh with acetylcholinesterase (AChE) to produce choline, which is further oxidized by choline oxidase (ChOx) to obtain betaine and H₂O₂. The enzyme-generated H₂O₂ reacts with BN and results in hydrolytic deprotection of BN to generate fluorescent product (4-hydroxyl-N-butyl-1,8-naphthalimide, ON). Two consecutive linear response ranges allow determining ACh in a wide concentration range with a low detection limit of 2.7 nM (signal/noise = 3). Compared with other fluorescent probes based on the mechanism of nonspecific oxidation, this reported boronate probe has the advantage of no interference from other biologically relevant reactive oxygen species (ROS) on the detection of ACh. This study provides a new method for the detection of ACh with high selectivity and sensitivity.     

Magnetic nanobead-based immunoassay for the simultaneous detection of aflatoxin B1 and ochratoxin A using upconversion nanoparticles as multicolor labels

A novel and sensitive immunoassay for the simultaneous detection of aflatoxin B₁ (AFB₁) and ochratoxin A (OTA) in food samples was developed by using artificial antigen-modified magnetic nanoparticles (MNPs) as immunosensing probes and antibody functionalized upconversion nanoparticles (UCNPs) as signal probes. NaY_0.78F₄:Yb_0.2, Tm_0.02 and NaY_0.28F₄:Yb_0.7,Er_0.02 UCNPs were prepared and functionalized, respectively, with immobilized monoclonal anti-AFB₁ antibodies and anti-OTA antibodies as signal probes. Based on a competitive immunoassay format, the detection limit for both AFB₁ and OTA under optimal conditions was as low as 0.01 ng mL⁻¹, and the effective detection range was from 0.01 to 10 ng mL⁻¹. The proposed method was successfully applied to measure AFB₁ and OTA in naturally contaminated maize samples and compared to a commercially available ELISA method. The high sensitivity and selectivity of this method is due to the magnetic separation and concentration effect of the MNPs, the high sensitivity of the UCNPs, and the different emission lines of Yb/Tm and Yb/Er doped NaYF₄ UCNPs excited by 980 nm laser. Multicolor UCNPs have the potential to be used in other applications for detecting toxins in the field of food safety and other fields.     

Sensitive detection of GFP utilizing tyramide signal amplification to overcome gene silencing

The green fluorescent protein (GFP) is among the most commonly used expression markers in biology. GFP-tagged cells have played a particularly important role in studies of cell lineage. Sensitive detection of GFP is crucially important for such studies to be successful, and problems with detection may account for discrepancies in the literature regarding the possible fate choices of stem cells. Here we describe a very sensitive technique for visualization of GFP. Using it we can detect about 90% of cells of donor origin while we could only see about 50% of these cells when we employ the methods that are in general use in other laboratories. In addition, we provide evidence that some cells permanently silence GFP expression. In the case of the progeny of bone marrow stem cells, it appears that the more distantly related they are to their precursors, the more likely it is that they will turn off the lineage marker.     

Simultaneous electrochemical detection of multiple biomarkers using gold nanoparticles decorated multiwall carbon nanotubes as signal enhancers

In this work, a novel sandwich-type electrochemical immunosensor has been developed for simultaneous detection of carcinoembryonic antigen (CEA) and α-fetoprotein (AFP) based on metal ion labels. Gold nanoparticles decorated multiwall carbon nanotubes (AuNPs@MWCNTs) were used as carriers to immobilize secondary antibodies and distinguishable electrochemical tags of Pb²⁺ and Cd²⁺ to amplify the signals. Due to the intrinsic property of high surface-to-volume ratio, the AuNPs@MWCNTs could load numerous secondary antibodies and labels. Therefore, the multiplexed immunoassay exhibited good sensitivity and selectivity. Experimental results revealed that this sandwich-type immunoassay displayed an excellent linear response, with a linear range of 0.01 to 60 ng mL^–1 for both analytes and detection limits of 3.0 pg mL^–1 for CEA and 4.5 pg mL^–1 for AFP (at a signal-to-noise ratio of 3). The method was successfully applied for the determination of AFP and CEA levels in clinical serum samples.     

Characterization of feline serum ferritin-binding proteins: the presence of a novel ferritin-binding protein as an inhibitory factor in feline ferritin immunoassay

Ferritin-binding proteins (FBPs) such as anti-ferritin antibody, α-2-macroglobulin, apolipoprotein B are expected to interact with circulating ferritin to eliminate it from circulation. However, we found that feline serum more strongly inhibits the detection of canine liver ferritin by immunoassay than its apoferritin; putative FBPs probably conceal ferritin epitopes detected by anti-ferritin antibodies. After complex formation between affinity-purified FBPs and canine liver ferritin, co-immunoprecipitates of the complex by anti-bovine spleen ferritin antibody were found to contain autoantibodies (IgG, IgM, and IgA) to ferritin by immunoblot analysis with antibodies specific for feline IgG, IgM, and IgA. On the other hand, affinity-purified samples did not show any inhibitory effect in the ferritin immunoassay. This result shows that feline serum has another FBP, which inhibits ferritin immunoassays, but not anti-ferritin autoantibody. A feline FBP was partially purified from feline serum by (NH₄)₂SO₄ fractionation (33–50%), gel filtration chromatography, and anion exchange chromatography. After binding of the partially purified sample with canine liver ferritin coupled-Sepharose gel, the FBP was separated and purified from complexes formed in a native-PAGE gel. SDS–PAGE analysis showed that the purified FBP is a homomultimer composed of 31 kDa monomeric subunits connected by intermolecular disulfide bonds. Detection of feline liver ferritin by immunoassay was inhibited by FBP in a dose-dependent manner. The purified protein molecules appeared to be conglomerate of pentraxin-like molecules by its electron micrographic appearance. These results demonstrate that feline serum contains a novel FBP as inhibitory factor of ferritin immunoassay with different molecular properties from those of other mammalian FBPs, in addition to auto-antibodies (IgG, IgM, and IgA) to ferritin.     

Fluorescent probe for detection of Cu2+ using core‐shell CdTe/ZnS quantum dots

Core‐shell CdTe/ZnS quantum dots capped with 3‐mercaptopropionic acid (MPA) were successfully synthesized in aqueous medium by hydrothermal synthesis. These quantum dots have advantages compared to traditional quantum dots with limited biological applications, high toxicity and tendency to aggregate. The concentration of Cu²⁺ has a significant impact on the fluorescence intensity of quantum dots (QDs), therefore, a rapid sensitive and selective fluorescence probe has been proposed for the detection of Cu²⁺ in aqueous solution. Under optimal conditions, the fluorescence intensity of CdTe/ZnS QDs was linearly proportional to the concentration of Cu²⁺ in the range from 2.5 × 10^–9 M to 17.5 × 10^–7 M with the limit of 1.5 × 10^–9 M and relative standard deviation of 0.23%. The quenching mechanism is static quenching with recoveries of 97.30–102.75%. Copyright © 2015 John Wiley & Sons, Ltd.     

The cut-off value for interleukin 34 as an additional potential inflammatory biomarker for the prediction of the risk of diabetic complications

In the present study, we have decided to evaluate whether serum interleukin 34 (IL-34) levels may have diagnostic value in predicting the risk of vascular diabetic complications. The study included 49 patients with type 2 diabetes mellitus (T2DM) and 23 high-risk group. The receiver operating characteristic (ROC) curve analysis has shown that IL-34 has more discriminatory power than C-reactive protein (CRP) for the risk of diabetic complications. The cut-off value for IL-34 was established as 91.2 pg/mL. The gist of our research was identification of IL-34 as an additional potential inflammatory biomarker for the prediction of the risk of vascular diabetic complications.     

An organic-inorganic hybrid nanostructure-functionalized electrode for electrochemical immunoassay of biomarker by using magnetic bionanolabels

We present here a fluorescence anisotropy method for the quantification of the polymerization of FtsZ, an essential protein for cytokinesis in prokaryotes whose GTP-dependent assembly initiates the formation of the divisome complex. Using Alexa 488 labeled wild-type FtsZ as a tracer, the assay allows determination of the critical concentration of FtsZ polymerization from the dependence of the measured steady-state fluorescence anisotropy on the concentration of FtsZ. The incorporation of the labeled protein into FtsZ polymers and the lack of spectral changes on assembly were independently confirmed by time-resolved fluorescence and fluorescence correlation spectroscopy. Critical concentration values determined by this new assay are compatible with those reported previously under the same conditions by other well-established methods. As a proof of principle, data on the sensitivity of the assay to changes in FtsZ assembly in response to Mg²⁺ concentration or to the presence of high concentrations of Ficoll 70 as crowding agent are shown. The proposed method is sensitive, low sample consuming, rapid, and reliable, and it can be extended to other cooperatively polymerizing systems. In addition, it can help to discover new antimicrobials that may interfere with FtsZ polymerization because it can be easily adapted to systematic screening assays.     

Synthesis of CdS nanoparticles from cadmium sulfate solutions using the extracellular polymeric substances of B. licheniformis as stabilizing agent

Mining and hydrometallurgical industries produce large amounts of hazardous metal sulfate solutions as a by-product which can be recycled and exploited to produce valuable and advanced materials. Here, for the first time, extracellular polymeric substances of Bacillus licheniformis were applied as biosurfactants to synthesize quantum dots of cadmium sulfide from pure artificial and impure industrial cadmium sulfate solutions. The bacterial biopolymers stabilized the generated crystalline nuclei as colloidal dots and prevented their further growth or agglomeration. In order to discover the composition and size distribution of the produced particles, characterization was performed by X-ray diffraction (XRD), and transmission electron microscopy (TEM). Results showed that the particles biosynthesized from the pure solution were nano-sized cubic crystals of CdS with the dimensions of 2–10 nm. The same product was also derived from the impure industrial solution. The outcomes of this study indicate the feasibility of cadmium or probably other metal recovery from industrial solutions and wastewaters in the form of valuable metal sulfide nanoparticles.     

Synthesis of novel bispyrene diamines and their application as ratiometric fluorescent probes for detection of DNA

Fluorescent DNA probes with 1,6-hexanediyl as the linker between two pyrenes, phenylpyrenes or phenylethynyl pyrene fluorophores were synthesized (Py-1, Py-2 and Py-3) and their interactions with DNA were studied by UV–vis absorption spectra, fluorescence spectra and viscosity measurements. The probes show red-shifted emission compared with pyrene (up to 20 nm). We found the interaction of these probes with DNA can be either intercalation or groove binding. Ratiometric fluorometry (ratio of the monomer and excimer emission intensity versus concentration of DNA) was achieved with these probes for DNA quantification (with limit of detection, LOD, up to 0.1 μg/mL). We also found that the undesired oxygen sensitivity of the emission intensity of pyrene fluorophore can be greatly suppressed by extending the π-conjugation framework of pyrene (the I_Ar/I_air value is decreased from 8.10 for pyrene to less than 2.20 for the DNA probes described herein).     

Objective assessment of stress levels and health status using routinely measured clinical laboratory parameters as biomarkers

Observed stress intensity was estimated using a scoring system from 0?100. Health status was estimated using the readily available laboratory measurements of C-reactive protein, neutrophil count, and fasting plasma glucose. We found that the stress score determined was linked to patient health status. Further studies are indicated.     

A chronocoulometric aptasensor based on gold nanoparticles as a signal amplification strategy for detection of thrombin

A sensitive chronocoulometric aptasensor for the detection of thrombin has been developed based on gold nanoparticle amplification. The functional gold nanoparticles, loaded with link DNA (LDNA) and report DNA (RDNA), were immobilized on an electrode by thrombin aptamers performing as a recognition element and capture probe. LDNA was complementary to the thrombin aptamers and RDNA was noncomplementary, but could combine with [Ru(NH₃)₆]³⁺ (RuHex) cations. Electrochemical signals obtained by RuHex that bound quantitatively to the negatively charged phosphate backbone of DNA via electrostatic interactions were measured by chronocoulometry. In the presence of thrombin, the combination of thrombin and thrombin aptamers and the release of the functional gold nanoparticles could induce a significant decrease in chronocoulometric signal. The incorporation of gold nanoparticles in the chronocoulometric aptasensor significantly enhanced the sensitivity. The performance of the aptasensor was further increased by the optimization of the surface density of aptamers. Under optimum conditions, the chronocoulometric aptasensor exhibited a wide linear response range of 0.1–18.5 nM with a detection limit of 30 pM. The results demonstrated that this nanoparticle-based amplification strategy offers a simple and effective approach to detect thrombin.     

Sensitive detection of sequence similarity using combinatorial pattern discovery: a challenging study of two distantly related protein families

We investigate the performance of combinatorial pattern discovery to detect remote sequence similarities in terms of both biological accuracy and computational efficiency for a pair of distantly related families, as a case study. The two families represent the cupredoxins and multicopper oxidases, both containing blue copper-binding domains. These families present a challenging case due to low sequence similarity, different local structure, and variable sequence conservation at their copper-binding active sites. In this study, we investigate a new approach for automatically identifying weak sequence similarities that is based on combinatorial pattern discovery. We compare its performance with a traditional, HMM-based scheme and obtain estimates for sensitivity and specificity of the two approaches. Our analysis suggests that pattern discovery methods can be substantially more sensitive in detecting remote protein relationships while at the same time guaranteeing high specificity.     

Synthesis,characterization and optical studies of highly luminescent ZnS nanoparticles associated with hypromellose matrix as a green and novel stabilizer

ZnS nanoparticles stabilized by a carbohydrate‐based matrix, hypromellose (hydroxypropyl methylcellulose) were prepared via a wet chemical method. The nanocomposite was characterized by X‐ray diffraction, transmission electon microscopy and Fourier transform infrared spectroscopy. X‐Ray diffraction patterns revealed a zinc blende structure. Thermogravimetric analysis suggested that polymer attached to the surface decomposes at 700 °C. Absorption measurements were carried out and calculation of the diameter polydispersity index (DPI) suggests the formation of monodisperse nanoparticles. The optical properties of the as‐prepared samples were studied by UV/vis spectroscopy and steady‐state photoluminescence (PL) spectroscopy. The PL studies indicate the applicability of these nanoparticles as biocompatible sensors or luminescence markers in future. Copyright © 2013 John Wiley & Sons, Ltd.