Nanoparticle-based electrochemiluminescence immunosensor with enhanced sensitivity for cardiac troponin I using N-(aminobutyl)-N-(ethylisoluminol)-functionalized gold nanoparticles as labels

A novel nanoparticle-based electrochemiluminescence (ECL) immunosensor was designed for highly sensitive and selective detection of human cardiac troponin I (cTnI), an important Acute Myocardial Infarction (AMI)-related biomarker, by using N-(aminobutyl)-N-(ethylisoluminol)-functionalized gold nanoparticles (ABEI-AuNPs) as labels. ABEI-AuNPs were successfully synthesized via a simple seed growth method. A great number of luminescence molecules ABEI as stabilizers were coated on the surface of the AuNPs, which exhibited better ECL activities than previously reported luminol functionalized gold nanoparticles. ABEI-AuNPs were used as new ECL labels to build bio-probes by conjugation with secondary antibodies, which showed good ECL activity, immunological activity, and stability. Another kind of AuNPs functionalized with streptavidin was modified on the electrode surface for biotinylated antibodies capture through the specific interaction of biotin/streptavidin and enhancing the electrical connectivity. By combining with the novel ECL labels and amplification of AuNPs and biotin-streptavidin system, a high sensitive sandwich-type electrochemiluminescence immunoassay was developed for detecting human cTnI with a low detection limit of 2 pg/mL. The immunosensor showed good precision, acceptable stability and reproducibility and could be used for the detection of cTnI in real samples, which was of great potential application in clinical analysis. Importantly, the sensitive detection would have far more diagnostic value than would absolute measurements during the early stage of AMI.     

Synthesis, characterization, and bioavailability of mannosylated shell cross-linked nanoparticles

Saccharide-functionalized shell cross-linked (SCK) polymer micelles designed as polyvalent nanoscaffolds for selective interactions with receptors on Gram negative bacteria were constructed from mixed micelles composed of poly(acrylic acid-b-methyl acrylate) and mannosylated poly(acrylic acid-b-methyl acrylate). The mannose unit was conjugated to the hydrophilic chain terminus of the amphiphilic diblock copolymer precursor, from which the SCK nanoparticles were derived, by the growth of the diblock copolymer from a mannoside functionalized atom transfer radical polymerization (ATRP) initiator. Mixed micelle formation between the amphiphilic diblock copolymer and mannosylated amphiphilic diblock copolymer was followed by condensation-based cross-linking between the acrylic acid residues present in the periphery of the polymer micelles to afford SCK nanoparticles. SCKs presenting variable numbers of mannose functionalities were prepared from mixed micelles of controlled stoichiometric ratios of mannosylated and nonmannosylated diblock copolymers. The polymer micelles and SCKs were characterized by dynamic light scattering (DLS), electrophoretic light scattering, atomic force microscopy (AFM), transmission electron microscopy (TEM), and analytical ultracentrifugation (AU). Surface availability and bioactivity of the mannose units were evaluated by interactions of the nanostructures with the model lectin Concanavalin A via DLS studies, with red blood cells (rabbit) via agglutination inhibition assays and with bacterial cells (E. coli) via TEM imaging.     

Silica nanoparticles for the layer-by-layer assembly of fully electro-active cytochrome c multilayers

Background

Gold nanoparticles (AuNPs) scatter light intensely at or near their surface plasmon wavelength region. Using AuNPs coupled with dynamic light scattering (DLS) detection, we developed a facile nanoparticle immunoassay for serum protein biomarker detection and analysis. A serum sample was first mixed with a citrate-protected AuNP solution. Proteins from the serum were adsorbed to the AuNPs to form a protein corona on the nanoparticle surface. An antibody solution was then added to the assay solution to analyze the target proteins of interest that are present in the protein corona. The protein corona formation and the subsequent binding of antibody to the target proteins in the protein corona were detected by DLS.

Results

Using this simple assay, we discovered multiple molecular aberrations associated with prostate cancer from both mice and human blood serum samples. From the mice serum study, we observed difference in the size of the protein corona and mouse IgG level between different mice groups (i.e., mice with aggressive or less aggressive prostate cancer, and normal healthy controls). Furthermore, it was found from both the mice model and the human serum sample study that the level of vascular endothelial growth factor (VEGF, a protein that is associated with tumor angiogenesis) adsorbed to the AuNPs is decreased in cancer samples compared to non-cancerous or less malignant cancer samples.

Conclusion

The molecular aberrations observed from this study may become new biomarkers for prostate cancer detection. The nanoparticle immunoassay reported here can be used as a convenient and general tool to screen and analyze serum proteins and to discover new biomarkers associated with cancer and other human diseases.     

Size- and coating-dependent uptake of polymer-coated gold nanoparticles in primary human dermal microvascular endothelial cells

A library-orientated approach is used to gain understanding of the interactions of well-defined nanoparticles with primary human endothelial cells, which are a key component of the vasculature. Fifteen sequentially modified gold nanoparticles (AuNPs) based on three different core sizes (18, 35, 65 nm) and five polymeric coatings were prepared. The synthetic methodology ensured homogeneity across each series of particles to allow sequential investigation of the chemical features on cellular interactions. The toxicity of these nanoparticles, their uptake behavior in primary human dermal microvascular endothelial cells (HDMECs), and quantification of uptake were all investigated. The results of our studies indicated that high concentrations of gold nanoparticles (250 μg/mL) were nontoxic and that the number of internalized nanoparticles was related to nanoparticle size and surface chemistry. In summary, the positive-charged ethanediamine-coated AuNPs were internalized to a greater extent than the negative- or neutral-charged AuNPs. Moreover, differences in the amounts of internalized AuNPs could be shown for the three neutral-charged AuNPs, whereas the uptake of hydroxypropylamine-coated particles was preferred compared with glucosamine-coated or PEGylated AuNPs. Hydroxypropylamine-coated AuNPs were found to be the most efficient neutral-charged particles in overcoming the endothelial cell barrier and entering the cell.     

Polymerase chain reaction of nanoparticle-bound primers

Using one or two primers respectively bound to the surface of Au nanoparticles (AuNPs) or magnetic nanoparticles (MNPs), polymerase chain reaction (PCR) based on nanoparticles was systemically studied, agarose gel electrophoresis and atomic force microscopy (AFM) were respectively used to detect and observe the PCR product. The results obtained indicated that with either one or two primers respectively bound to the nanoparticle surface, PCR can proceed successfully under optimized condition and is subject to certain rules, consequently a symmetric PCR technique and an asymmetric PCR technique based on nanoparticles have been developed. A kind of nanostructured aggregates can be constructed by a symmetric PCR using two nanoparticle-bound primers.     

Tailored Aggregate-Free Au Nanoparticle Decorations with Sharp Plasmonic Peaks on a U-Type Optical Fiber Sensor by Nanosecond Laser Irradiation

A novel experimental methodology is presented for fabricating U-shaped optical fiber probes decorated with aggregate-free Au nanoparticles exhibiting sharp localized surface plasmon resonance (LSPR) spectra. The U-type tip is coated with gold nanoparticles (AuNPs) using a simple and time-efficient dip-coating procedure, without initially taking any care to prevent the formation of nanoparticle aggregates in the coated area. In a second step, the coating was irradiated with a few tens of laser pulses of 5-ns duration at 532 nm with intensities in the range of 2–14 MW/cm², leading to the formation of aggregate-free LSPR optical fiber probes. The process was monitored and controlled in real time through the changes induced into the fiber’s extinction spectra by the laser irradiation, and the coated fibers were characterized by electron microscopy. The proposed methodology resulted into the fabrication of U-type optical fiber probes coated with AuNPs exhibiting a sharp plasmon peak, which is a perquisite for their application as sensing devices.     

Synthesis and characterization of a novel MPEG–chitosan diblock copolymer and self-assembly of nanoparticles

In this paper, a simple and novel method based on free-radical polymerization initiated by potassium persulfate (KPS) was developed to synthesize the MPEG–chitosan diblock copolymer (MPEG–CS). The obtained MPEG–CS diblock copolymer was characterized by Fourier transform infrared (FTIR), ¹H nuclear magnetic resonance (¹H NMR), X-ray diffraction (XRD) and differential scanning calorimetry (DSC), respectively. The MPEG–CS copolymer could self-assemble into nanoparticles in aqueous solution. A typical TEM photography indicated that the well-spherical nanoparticles with diameter at about 200 nm were obtained. In vitro cell culture assay indicated that MPEG–CS nanoparticles are non-toxic and cell-compatible as the polymer concentration was smaller than 0.6 mg/ml. In conclusion, the obtained MPEG–CS nanoparticles might have great potential application in drug-delivery system.     

Targeting prostate cancer cells with PSMA inhibitor-guided gold nanoparticles

Prostate-specific membrane antigen (PSMA) is a notable biomarker for diagnostic and therapeutic applications in prostate cancer. Gold nanoparticles (AuNPs) provide an attractive nanomaterial platform for combining a variety of targeting, imaging, and cytotoxic agents into a unified device for biomedical research. In this study, we present the generation and evaluation of the first AuNP system functionalized with a small molecule phosphoramidate peptidomimetic inhibitor for the targeted delivery to PSMA-expressing prostate cancer cells. The general approach involved the conjugation of streptavidin-coated AuNPs with a biotin-linked PSMA inhibitor (CTT54) to generate PSMA-targeted AuNPs. In vitro evaluations of these targeted AuNPs were conducted to determine PSMA-mediated and time-dependent binding to PSMA-positive LNCaP cells. The PSMA-targeted AuNPs exhibited significantly higher and selective binding to LNCaP cells compared to control non-targeted AuNPs, thus demonstrating the feasibility of this approach.     

Fluorescently labeled branched polymers and thermal responsive nanoparticles for live cell imaging

Branched poly(methoxy-PEG acrylate) and thermally responsive poly(methoxy-PEG acrylate)-block-poly(N-isopropylacrylamide) are synthesized by RAFT polymerization. After reduction, these polymers are fluorescently labeled by reacting the free thiol groups with N-(5-fluoresceinyl)maleimide. As shown by DLS, the labeled copolymer poly(methoxy-PEG acrylate)-block-poly(N-isopropylacrylamide) forms nanoparticles at body temperature (37 °C) due to the presence of the thermosensitive poly(N-isopropylacrylamide). These materials were used as bioprobes for imaging HUVECs in vitro and chick embryo CAM in vivo. Both labeled polymer and nanoparticles are biocompatible and can be used as efficient fluorescent bioprobes.     

Visible-light Induced Reduction of Graphene Oxide Using Plasmonic Nanoparticle

Present work demonstrates the simple, chemical free, fast, and energy efficient method to produce reduced graphene oxide (r-GO) solution at RT using visible light irradiation with plasmonic nanoparticles. The plasmonic nanoparticle is used to improve the reduction efficiency of GO. It only takes 30 min at RT by illuminating the solutions with Xe-lamp, the r-GO solutions can be obtained by completely removing gold nanoparticles through simple centrifugation step. The spherical gold nanoparticles (AuNPs) as compared to the other nanostructures is the most suitable plasmonic nanostructure for r-GO preparation. The reduced graphene oxide prepared using visible light and AuNPs was equally qualitative as chemically reduced graphene oxide, which was supported by various analytical techniques such as UV-Vis spectroscopy, Raman spectroscopy, powder XRD and XPS. The reduced graphene oxide prepared with visible light shows excellent quenching properties over the fluorescent molecules modified on ssDNA and excellent fluorescence recovery for target DNA detection. The r-GO prepared by recycled AuNPs is found to be of same quality with that of chemically reduced r-GO. The use of visible light with plasmonic nanoparticle demonstrates the good alternative method for r-GO synthesis.     

Cytotoxicity,intracellular localization and exocytosis of citrate capped and PEG functionalized gold nanoparticles in human hepatocyte and kidney cells

Surface-modified gold nanoparticles (AuNPs) are nanomaterials that hold promise in drug delivery applications. In this study, the cytotoxicity, uptake, intracellular localization, and the exocytosis of citrate-stabilized (Cit-AuNP) and polyethylene glycol (PEG)-modified gold nanoparticles with the carboxyl (COOH) terminal functional group were assessed in human embryonic kidney (HEK 293) and the human caucasian hepatocytes carcinoma (Hep G2) cell systems, representing two major accumulation sites for AuNPs. The zeta (ζ)-potential measurements confirmed the negative surface charge of the AuNPs in water and in cell growth medium. The transmission electron microscopy confirmed the size and morphology of the AuNPs. Both types of AuNPs were shown to induce cytotoxic effects in cells. The Hep G2 cells were more sensitive cell type, with the COOH-PEG-AuNPs inducing the highest toxicity at higher concentrations. Dark field microscopy and TEM images revealed that the AuNPs were internalized in cells, mostly as agglomerates. TEM micrographs further revealed that the AuNPs were confined as agglomerates inside vesicle-like compartments, likely to be endosomal and lysosomal structures as well as in the cytosol, mostly as individual particles. The AuNPs were shown to remain in cellular compartments for up to 3 weeks, but thereafter, clearance of the gold nanoparticles from the cells by exocytosis was evident. The results presented in this study may therefore give an indication on the fate of AuNPs on long-term exposure to cells and may also assist in safety evaluation of AuNPs.     

Ultrasensitive electrochemical detection of Bacillus thuringiensis transgenic sequence based on in situ Ag nanoparticles aggregates induced by biotin-streptavidin system

A novel electrochemical biosensor was developed for detecting short DNA oligonucleotide of Bacillus thuringiensis (Bt) transgenic sequence based on Ag nanoparticle aggregates. To fabricate this DNA biosensor, the thiol-modified capture DNA (cDNA) was first anchored on gold (Au) electrode, and then the target DNA (tDNA) was hybridized with the immobilized cDNA. Subsequently, the probe DNA (pDNA) functionalized by biotinylated Ag nanoparticle was associated with the fixed tDNA, and the single Ag nanoparticle label was obtained (cited as SAg label). Finally, dissociative biotinylated Ag nanoparticle was bound to the resultant biotinylated SAg label assembled on Au electrode by virtue of bridge molecule streptavidin (SA) through biotin-SA specific interaction, which could lead to in situ aggregate of Ag nanoparticles on Au electrode and induce a novel tag including multiple Ag nanoparticles (cited as MAg tag). The novel tag exhibited excellent electroactive property in the solid-state Ag/AgCl process and was successfully applied to Bt transgenic sequence assay. A detection limit of 10 fM was achieved, which was improved by three orders of magnitude as compared to the SAg label. Furthermore, this novel DNA biosensor demonstrated a good selectivity towards tDNA.     

A piezoelectric immunosensor for specific capture and enrichment of viable pathogens by quartz crystal microbalance sensor, followed by detection with antibody-functionalized gold nanoparticles

A sensitive bacteria enrichment and detection system for viable Escherichia coli O157:H7 was developed using a piezoelectric biosensor-quartz crystal microbalance (QCM) with antibody-functionalized gold nanoparticles (AuNPs) used as detection verifiers and amplifiers. In the circulating-flow QCM system, capture antibodies for E. coli O157:H7 were first immobilized onto the QCM chip. The sample containing E. coli O157:H7 was circulated through the system in the presence of 10ml of brain heart infusion (BHI) broth for 18h. The cells of E. coli O157:H7 specifically captured and enriched on the chip surface of the QCM were identified by QCM frequency changes. Listeria monocytogenes and Salmonella Typhimurium were used as negative controls. After bacterial enrichment, detection antibody-functionalized AuNPs were added to enhance the changes in detection signal. The use of BHI enrichment further enhanced the sensitivity of the developed system, achieving a detection limit of 0-1log CFU/ml or g. The real-time monitoring method for viable E. coli O157:H7 developed in this study can be used to enrich and detect viable cells simultaneously within 24h. The unique advantages of the system developed offer great potential in the microbial analysis of food samples in routine settings.     

Dual signal amplification of surface plasmon resonance imaging for sensitive immunoassay of tumor marker

Surface plasmon resonance imaging (SPRi) is an intriguing technique for immunoassay with the inherent advantages of being high throughput, real time, and label free, but its sensitivity needs essential improvement for practical applications. Here, we report a dual signal amplification strategy using functional gold nanoparticles (AuNPs) followed by on-chip atom transfer radical polymerization (ATRP) for sensitive SPRi immunoassay of tumor biomarker in human serum. The AuNPs are grafted with an initiator of ATRP as well as a recognition antibody, where the antibody directs the specific binding of functional AuNPs onto the SPRi sensing surface to form immunocomplexes for first signal amplification and the initiator allows for on-chip ATRP of 2-hydroxyethyl methacrylate (HEMA) from the AuNPs to further enhance the SPRi signal. High sensitivity and broad dynamic range are achieved with this dual signal amplification strategy for detection of a model tumor marker, α-fetoprotein (AFP), in 10% human serum.     

Porous silicon affinity chips for biomarker detection by MALDI-TOF-MS

B-type natriuretic peptide (BNP) is an important biomarker in early diagnosis of congestive heart failure. Many efforts have been made previously to evaluate the BNP level in human plasma. We developed a porous silicon (PSi) affinity chip to detect BNP present at low concentrations in human plasma by matrix assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS) directly. The PSi surface immobilized with antibodies captured and concentrated BNP through antibody-antigen interaction specifically and sensitively. A detection limit as low as 10 pg/mL BNP in human plasma was demonstrated by mass analysis. This effective on-chip recognition, enrichment, and detection strategy could be employed in identification of biomarkers in complex body fluids in diagnoses.     

A novel bioassay for the monitoring of carcinoembryonic antigen in human biofluid using polymeric interface and immunosensing method

Carcinoembryonic antigen (CEA) is a member of a family of cell surface glycoproteins. Recognition of CEA is needed to monitor the physiological status of the patient for treatment and also it is important to assess the severity of the disease. In this work, we reported a novel sandwich‐type electrochemical immunosensor based on gold nanoparticles functionalized cysteamine‐glutaraldehyde (AuNPs‐CysA‐GA) and it successfully designed to detection of the CEA biomarker in a human plasma sample. The AuNPs‐CysA‐GA provides a large surface area for the effective immobilization of CEA antibody, as well as it ascertains the bioactivity and stability of immobilized CEA antigens. Biotinylated‐anti‐CEA antibody (Ab1) was immobilized on the surface of glassy carbon electrode (GCE) modified AuNPs‐CysA‐GA. Also, secondary antibody (HRP‐Ab2) was costed immobilized to complete the sandwich part of immunosensor. Field emission scanning electron microscope (FE‐SEM and EDS), was employed to monitor the sensor fabrication procedure. The immunosensor was used for the detection of CEA using differential pulse voltammetry (DPVs) technique. The proposed interface led to enhancement of accessible surface area for immobilizing high amount of anti‐CEA antibody, increasing electrical conductivity, boosting stability, and biocompatibility. Finally, the low limit of quantitation (LLOQ) of the proposed immunosensor was obtained as 7 ng/mL with the linear range of 0.001‐5 μg/L. The proposed immunoassay was successfully applied for the monitoring of the CEA in unprocessed human plasma samples. Obtained results paved that the proposed bioassay can be used as a novel bioassay for the clinical diagnosis of cancer based on CEA monitoring.     

Triply triggered doxorubicin release from supramolecular nanocontainers

The synthesis of a supramolecular double hydrophilic block copolymer (DHBC) held together by cucurbit[8]uril (CB[8]) ternary complexation and its subsequent self-assembly into micelles is described. This system is responsive to multiple external triggers including temperature, pH and the addition of a competitive guest. The supramolecular block copolymer assembly consists of poly(N-isopropylacrylamide) (PNIPAAm) as a thermoresponsive block and poly(dimethylaminoethylmethacrylate) (PDMAEMA) as a pH-responsive block. Moreover, encapsulation and controlled drug release was demonstrated with this system using the chemotherapeutic drug doxorubicin (DOX). This triple stimuli-responsive DHBC micelle system represents an evolution over conventional double stimuli-responsive covalent diblock copolymer systems and displayed a significant reduction in the viability of HeLa cells upon triggered release of DOX from the supramolecular micellar nanocontainers.     

ENSAM: Europium Nanoparticles for Signal enhancement of Antibody Microarrays on nanoporous silicon

To improve the sensitivity of antibody microarray assays, we developed ENSAM (Europium Nanoparticles for Signal enhancement of Antibody Microarrays). ENSAM is based on two nanomaterials. The first is polystyrene nanoparticles incorporated with europium chelate (beta-diketone) and coated with streptavidin. The multiple fluorophores incorporated into each nanoparticle should increase signal obtained from a single binding event. The second nanomaterial is array surfaces of nanoporous silicon, which creates high capacity for antibody adsorption. Two antibody microarray assays were compared: ENSAM and use of streptavidin labeled with a nine-dentate europium chelate. Analyzing biotinylated prostate-specific antigen (PSA) spiked into human female serum, ENSAM yielded a 10-fold signal enhancement compared to the streptavidin-europium chelate. Similarly, we observed around 1 order of magnitude greater sensitivity for the ENSAM assay (limit of detection < or = 0.14 ng/mL, dynamic range > 10(5)) compared to the streptavidin-europium chelate assay (limit of detection < or = 0.7 ng/mL, dynamic range > 10(4)). Analysis of a titration series showed strong linearity of ENSAM ( R2 = 0.99 by linear regression). This work demonstrates the novel utility of nanoparticles with time-resolved fluorescence for signal enhancement of antibody microarrays, requiring as low as 100-200 zmol biotinylated PSA per microarray spot. In addition, proof of principle was shown for analyzing PSA in plasma obtained from patients undergoing clinical PSA-testing.     

Shape effects of nanoparticles conjugated with cell-penetrating peptides (HIV Tat PTD) on CHO cell uptake

In order to probe the nanoparticle shape/size effect on cellular uptake, a spherical and two cylindrical nanoparticles, whose lengths were distinctively varied, were constructed by the selective cross-linking of amphiphilic block copolymer micelles. Herein, we demonstrate that, when the nanoparticles were functionalized with the protein transduction domain of human immunodeficiency virus type 1 Tat protein (HIV Tat PTD), the smaller, spherical nanoparticles had a higher rate of cell entry into Chinese hamster ovary (CHO) cells than did the larger, cylindrical nanoparticles. It was also found that nanoparticles were released after internalization and that the rate of cell exit was dependent on both the nanoparticle shape and the amount of surface-bound PTD.     

Biocompatible nanoparticles trigger rapid bacteria clustering

This study reveals an exciting phenomenon of stimulated bacteria clustering. Rapid aggregation and microbial arrest are shown to occur in Escherichia coli solutions of neutral pH when chitosan nanoparticles with positive zeta potential are added. Because chitosan nanoparticles can easily be dispersed in aqueous buffers, the rapid clustering phenomenon requires only minuscule nanoparticle concentrations and will be critical in developing new methods for extricating bacterial pathogens. This work establishes the dominant role of electrostatic attraction in bacteria‐nanoparticle interactions by varying the nanoparticle zeta potential from highly positive to strongly negative values, and by exploring concentration effects. For strongly negative nanoparticles, no clusters form, while aggregates are small and loose at intermediate conditions. In addition, optical density measurements indicate that over 90% of the suspended bacteria flocculate within seconds of being mixed with chitosan nanoparticles of a highly positive surface charge. Finally, the nanoparticles are significantly more efficient as a clustering agent compared to an equal mass of molecular chitosan in solution, as the bacteria‐nanoparticle clusters formed are substantially larger. The bacteria‐nanoparticle aggregation effect demonstrated here promises a rapid separation method for aiding pathogen detection and for flocculation of bacteria in fermentation processes. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009