Nanoparticle Labels Research Articles

Optical single-particle detection in nanoholes towards simple parallel detection of molecular binding events

A novel scheme for the optical detection of a few or even single gold nanoparticle labels is introduced. It utilizes sub-wavelength holes in a chromium layer on a glass substrate, where a bioaffinity reaction could take place and the outcome (regarding particle label binding) can be monitored using optical means. Experiments in combination with simulations demonstrate that the presence of particles in such a chromium hole can be simply detected by using the color information of images by a charge couple device (CCD) camera without the need for additional spectroscopy setups. The presence of gold nanoparticles leads to a detectable red-shift in the images of the respective nanohole, which is the sensing principle of the sensor.

Facile Synthesis and Biosensing Application of Hybrid Zinc Nanoparticles

Hybrid zinc nanoparticles were synthesized by adding thioglycolic acid (TGA) into a ZnO-particle synthesis procedure. Compared to the ZnO particles prepared without TGA, the hybrid nanoparticles are markedly different in their morphology, chemical composition, and growth kinetics. Moreover, they display colloidal stability and appropriate surface properties for bioconjugation. To demonstrate their biosensing application, the hybrid nanoparticles were conjugated and applied as biolabels or signal transducers in a sandwich immunoassay for mouse IgG. The immunoassay fluorescence signal was obtained by releasing zinc ions from these nanoparticle labels and further incubating the released zinc ions with zinc-sensitive fluorescence indicator Fluozin-3. The immunoassay presents a dynamic detection range from 10 pM to 1 nM.

Highly Sensitive Detection of Target Biomolecules on Cell Surface Using Gold Nanoparticle Conjugated with Aptamer Probe

A method of gold nanoparticle (Au NP) labeling with backscattered electron (BE) imaging of field emission scanning electron microscopy (FE-SEM) was applied for specific detection of target biomolecules on a cell surface. A single-stranded DNA aptamer, which specifically binds to the target molecule on a human acute lymphoblastic leukemia cell, was conjugated with a 20 nm Au NP and used as a probe to label its target molecule on the cell. The Au NP probe was incubated with the cell, and the interaction was confirmed using BE imaging of FE-SEM through direct counting of the number of Au NPs attached on the target cell surface. Specific Au NP-aptamer probes were observed on a single cell surface and their spatial distributions including submicron-order localizations were also clearly visualized, whereas the nonspecific aptamer probes were not observed on it. The aptamer probe can be potentially dislodged from the cell surface with treatment of nucleases, indicating that Au NP-conjugated aptamer probes can be used as sensitive and reversible probes to label target biomolecules on cells.

Highly Sensitive Detection of Target Biomolecules on Cell Surface Using Gold Nanoparticle Conjugated with Aptamer Probe

A method of gold nanoparticle (Au NP) labeling with backscattered electron (BE) imaging of field emission scanning electron microscopy (FE-SEM) was applied for specific detection of target biomolecules on a cell surface. A single-stranded DNA aptamer, which specifically binds to the target molecule on a human acute lymphoblastic leukemia cell, was conjugated with a 20 nm Au NP and used as a probe to label its target molecule on the cell. The Au NP probe was incubated with the cell, and the interaction was confirmed using BE imaging of FE-SEM through direct counting of the number of Au NPs attached on the target cell surface. Specific Au NP-aptamer probes were observed on a single cell surface and their spatial distributions including submicron-order localizations were also clearly visualized, whereas the nonspecific aptamer probes were not observed on it. The aptamer probe can be potentially dislodged from the cell surface with treatment of nucleases, indicating that Au NP-conjugated aptamer probes can be used as sensitive and reversible probes to label target biomolecules on cells.

Sensitive ECL sensor for sequence-specific DNA from Mycobacterium tuberculosis based on N-(aminobutyl)-N-ethylisoluminol functionalized gold nanoparticles labeling

A novel sensor was developed for highly sensitive detection of the sequence-specific DNA of Mycobacterium tuberculosis (MTB) by using N-(aminobutyl)-N-(ethylisoluminol) (ABEI) functionalized gold nanoparticles (ABEI-AuNPs) as labels and using 81 bp segment derived from the MTB specific insertion sequence IS6110 as the target strand. Owing to the novel ECL labels and amplification of gold nanoparticles and biotin–streptavidin system, extremely high sensitivity for ss-DNA could be achieved with a low detection limit of 3.3 × 10−16 M and a wide linear dynamic range of 1.0 × 10−15–1.0 × 10−12 M. The DNA sensor also showed good precision, good stability, low detection limit for the detection of sequence-specific DNA from MTB in serum samples, which might be of application potential in clinical diagnosis.

Monitoring the Size and Lateral Dynamics of ErbB1 Enriched Membrane Domains through Live Cell Plasmon Coupling Microscopy

To illuminate the role of the spatial organization of the epidermal growth factor receptor (ErbB1) in signal transduction quantitative information about the receptor topography on the cell surface, ideally on living cells and in real time, are required. We demonstrate that plasmon coupling microscopy (PCM) enables to detect, size, and track individual membrane domains enriched in ErbB1 with high temporal resolution. We used a dendrimer enhanced labeling strategy to label ErbB1 receptors on epidermoid carcinoma cells (A431) with 60 nm Au nanoparticle (NP) immunolabels under physiological conditions at 37°C. The statistical analysis of the spatial NP distribution on the cell surface in the scanning electron microscope (SEM) confirmed a clustering of the NP labels consistent with a heterogeneous distribution of ErbB1 in the plasma membrane. Spectral shifts in the scattering response of clustered NPs facilitated the detection and sizing of individual NP clusters on living cells in solution in an optical microscope. We tracked the lateral diffusion of individual clusters at a frame rate of 200 frames/s while simultaneously monitoring the configurational dynamics of the clusters. Structural information about the NP clusters in their membrane confinements were obtained through analysis of the electromagnetic coupling of the co-confined NP labels through polarization resolved PCM. Our studies show that the ErbB1 receptor is enriched in membrane domains with typical diameters in the range between 60–250 nm. These membrane domains exhibit a slow lateral diffusion with a diffusion coefficient of = |0.0054±0.0064| µm2/s, which is almost an order of magnitude slower than the mean diffusion coefficient of individual NP tagged ErbB1 receptors under identical conditions.

Application of europium(III) chelates-bonded silica nanoparticle in time-resolved immunofluorometric detection assay for human thyroid stimulating hormone

Eu(III) chelate-bonded silica nanoparticle was used as a fluorescent label to develop a highly sensitive time-resolved immunofluorometric assay (TrIFA) for human thyroid stimulating hormone (hTSH). The limit of detection of the assay calculated according to the 2SD method was 0.0007mIUL−1 and became 0.003mIUL−1 when serum-based matrix was used for calibrators, indicating that this TrIFA is comparable with the most sensitive assays. The linear range was from 0.005 to 100mIUL−1 of hTSH with coefficient of variation between 1.9% and 8.3%. The correlation study using 204 blood spot samples from newborns showed that the results from this new method were coincident with that of the commercial dissociation-enhanced lanthanide fluorescence immunoassay (DELFIA) system, with a correlation coefficient of 0.938. The fluorescent nanoparticle label allows directly reading the fluorescent signal, omitting the signal development step required for the DELFIA system, and the whole procedure of this assay is fulfilled within 2h. Thus, we developed a novel, sensitive, quantitative and simple nanoparticle label-based TrIFA assay, suitable for routine application in hTSH screening of neonatal hypothyroidism.

Nanomaterials‐based electrochemical immunosensors for proteins

For this special issue on 90 years of polarography, the following personal account describes how my early research in electrochemistry and polarography in the laboratory of Prof. Petr Zuman led to a major research effort in the determination of proteins for cancer detection and monitoring. It reviews the very recent history of nanoparticle labels and multiplexed detection in protein immunosensors. It then describes our journey of discovery that has led to ultrasensitive protein immunosensors achieved by combining nanostructured electrodes with particles labeled with up to ½ million enzymes that can detect down to as little as 1 fg mL(-1) protein in diluted serum. Our most mature multiple protein detection system is a microfluidic device with eight sensors coated with 5-nm gold nanoparticles that uses off-line protein detection with heavily labeled magnetic particles. This approach has led to reliable sub pg mL(-1) detection limits for multiple proteins, provides excellent correlation with referee ELISA methods, and is currently being used for validation of panels of biomarkers for oral and prostate cancer. The article ends with a section on future perspectives.

Sensing Technique of Silver Nanoparticles as Labels for Immunoassay Using Liquid Electrode Plasma Atomic Emission Spectrometry

We report the use of liquid electrode plasma-atomic emission spectrometry (LEP-AES) in protein sensing studies employing Ag nanoparticle labeling. LEP-AES requires no plasma gas and no high-power source and is suitable for onsite portable analysis. Human chorionic gonadotropin (hCG) was used as a model target protein, and the immunoreaction in which hCG is sandwiched between two antibodies, one of which is immobilized on the microwell and the second is labeled with Ag nanoparticles, was performed. Sensing occurs at the narrow pass in the center of a quartz chip following oxidative dissolution of the Ag nanoparticles by nitric acid. hCG was analyzed in the range from 10 pg/mL to 1 ng/mL, and the detection limit for hCG was estimated at 1.3 pg/mL (22.8 fM). The proposed detection method has a wide variety of promising applications in metal-nanoparticle-labeled biomolecule detection.

Contribution of gold nanoparticles to the signal amplification in surface plasmon resonance

Gold nanoparticle labelling has been shown to produce a remarkable improvement in sensitivity for small molecule detection based on Surface Plasmon Resonance (SPR) bio-sensing. The LSPR (localised SPR)-SPR coupling effect and size/mass-material properties associated with gold nanoparticles are the two main factors to change the SPR resonance condition and cause the enhancement. In this paper we examine the separation of these factors in the context of a classical SPR bio-interaction assay format, and consider the implications on the design of biodetection systems to maximise response. The coupling effect plays a distance dependent role, which allows it to be mapped. The dominant enhancement associated with this factor only occurs within ∼8 nm for a 20 nm gold nanoparticle and is changing by ∼7 nm wavelength shift/nm distance from the Au SPR thin film. Beyond this distance, the size/mass associated with the nanoparticle itself dominates the enhancement. This is demonstrated in a 20-mer DNA sequence sandwich detection, where the enhancement ratio between the coupling effect and the mass is ∼1.5 : 1. This simple method for deconvolution of the mass and coupling effects allows consideration of formats with LSPR nanoparticle labelling for small molecule detection and the best design of the labelling geometry.

Elimination of Tumor Cells Using Folate Receptor Targeting by Antibody‐Conjugated, Gold‐Coated Magnetite Nanoparticles in a Murine Breast Cancer Model

Background. The chemotherapeutic treatment of cancer suffers from poor specificity for targeting the tumor cells and often results in adverse effects such as systemic toxicity, damage to nontarget tissues, and development of drug‐resistant tumors in patients. Increasingly, drug nanocarriers have been explored as a way of lessening or overcoming these problems. In this study, antibody‐conjugated Au‐coated magnetite nanoparticles, in conjunction with inductive heating produced by exposure to an oscillating magnetic field (OMF), were evaluated for their effects on the viability of tumor cells in a murine model of breast cancer. Treatment effects were evaluated by light microscopy and SEM. Results. 4T1 mammary epithelial carcinoma cells overexpressing the folate receptor were targeted with an anti‐folate receptor primary antibody, followed by labeling with secondary antibody‐conjugated Au‐coated magnetite nanoparticles. In the absence of OMF exposure, nanoparticle labeling had no effect on 4T1 cell viability. However, following OMF treatment, many of the labeled 4T1 cells showed extensive membrane damage by SEM analysis, and dramatically reduced viability as assessed using a live/dead staining assay. Conclusions. These results demonstrate that Au‐coated magnetite targeted to tumor cells through binding to an overexpressed surface receptor, in the presence of an OMF, can lead to tumor cell death.

Dual signal amplification for bioassays using ion release from nanolabels and ion-activated enzyme kinetics

A dual signal amplification technique was developed for bioassays. The technique consists of zinc-ion release from ZnS nanoparticle labels and enzyme kinetics activated by the released zinc ions as cofactors. In the ion release process, each ZnS nanoparticle label liberates a high number of zinc ions by acidic dissolution. After the ion release, at appropriate pH levels, the released zinc ions are used as cofactors to trigger the enzymatic activity of carbonic anhydrase. The fluorescence produced from the activated enzyme kinetics is measured for bioassay signal quantification. A model bioassay on mouse IgG adopting this technique presents a detection limit around 0.5 pM and a detection range over at least two orders of magnitude. This technique was also successfully applied to the detection of human cardiac troponin I (cTnI) in human serum samples to demonstrate a clinical diagnosis application. The developed immunoassay is capable of distinguishing clinically critical levels of cTnI. This technique possesses a high detection resolution and offers the advantage of straightforward operation (simple preparation of ZnS nanoparticles and no enzyme immobilization).

An sp2 and sp3 Hybrid Nanocrystalline Carbon Film Electrode for Anodic Stripping Voltammetry and Its Application for Electrochemical Immunoassay

A hybridized nanocrystalline carbon film electrode consisting of sp(2) and sp(3) bonds was investigated to reveal the reduction properties of Cd(2+) and for application as a highly sensitive and reliable electrochemical immunoassay. Conductive nanocrystalline carbon film consisting of about 60% sp(2) and 40% sp(3) bonds was fabricated using electron cyclotron resonance (ECR) sputtering equipment, and then the Cd(2+) concentrations were measured with an ECR sputtered carbon (ECR nano-carbon) electrode by employing an anodic stripping voltammetry (ASV) technique. The preconcentrated Cd was analyzed with Kelvin probe force microscopy and energy dispersive X-ray spectroscopy while observing the morphology change with an atomic force microscope and a scanning electron microscope. The preconcentrated Cd on the ECR nano-carbon electrode was revealed to be a thin sheet structure, which was significantly different from the Cd on a conventional carbon material that grows with a coralloid structure. The background current during an ASV measurement maintains a low level equivalent to that found with boron-doped diamond because the surface of the ECR nano-carbon is robust and angstrom-level flat. The carbon-electrode performance for ASV was improved by controlling its structure at a nanometer scale without any metal doping or coating. Finally, the ECR nano-carbon was used for biomolecular determination by electrochemical immunoassay with a CdSe nanoparticle label. Electrochemical immunoassay results were successfully obtained with the ECR nano-carbon, and they correlated well with fluorescence results obtained for CdSe nanoparticles.

Sensitive electrochemical immunosensor for α-synuclein based on dual signal amplification using PAMAM dendrimer-encapsulated Au and enhanced gold nanoparticle labels

A novel electrochemical immunosensor for sensitive detection of α-synuclein (α-SYN), a very important neuronal protein, has been developed based on dual signal amplification strategy. Herein, G4-polyamidoamine dendrimer-encapsulated Au nanoparticles (PAMAM-Au nanocomposites) were covalently bound on the poly- o-aminobenzoic acid (poly- o-ABA), which was initially electropolymerized on the electrode surface to perform abundant carboxyl groups. The formed immunosensor platform, PAMAM-Au, was proved to provide numerous amino groups to allow highly dense immobilization of antigen, and facilitate the improvement of electrochemical responses as well. Subsequently, the enhanced gold nanoparticle labels ({HRP-Ab 2-GNPs}) were fabricated by immobilizing horseradish peroxidase-secondary antibody (HRP-Ab 2) on the surface of gold nanoparticles (GNPs). After an immunoassay process, the {HRP-Ab 2-GNPs} labels were introduced onto the electrode surface, and produced an electrocatalytic response by reduction of hydrogen peroxide (H 2O 2) in the presence of enzymatically oxidized thionine. On the basis of the dual signal amplification of PAMAM-Au and {HRP-Ab 2-GNPs} labels, the designed immunosensor displayed an excellent analytical performance with high sensitivity and stability. This developed strategy was successfully proved as a simple, cost-effective method, and could be easily extended to other protein analysis schemes.

Single-molecule protein arrays enabled by scanning probe block copolymer lithography

The ability to control the placement of individual protein molecules on surfaces could enable advances in a wide range of areas, from the development of nanoscale biomolecular devices to fundamental studies in cell biology. Such control, however, remains a challenge in nanobiotechnology due to the limitations of current lithographic techniques. Herein we report an approach that combines scanning probe block copolymer lithography with site-selective immobilization strategies to create arrays of proteins down to the single-molecule level with arbitrary pattern control. Scanning probe block copolymer lithography was used to synthesize individual sub-10-nm single crystal gold nanoparticles that can act as scaffolds for the adsorption of functionalized alkylthiol monolayers, which facilitate the immobilization of specific proteins. The number of protein molecules that adsorb onto the nanoparticles is dependent upon particle size; when the particle size approaches the dimensions of a protein molecule, each particle can support a single protein. This was demonstrated with both gold nanoparticle and quantum dot labeling coupled with transmission electron microscopy imaging experiments. The immobilized proteins remain bioactive, as evidenced by enzymatic assays and antigen-antibody binding experiments. Importantly, this approach to generate single-biomolecule arrays is, in principle, applicable to many parallelized cantilever and cantilever-free scanning probe molecular printing methods.

Sensitive detection of cardiac biomarker using ZnS nanoparticles as novel signal transducers

C-reactive protein (CRP), a 115 kDa pentameric protein, is one of the important cardiac biomarkers that are indicative of coronary heart events. Sensitive detection of CRP in human serum is critical for the diagnosis of coronary heart disease. This work presents a sensitive sandwich immunoassay for the detection of CRP in human serum using zinc sulfide (ZnS) nanoparticles as novel fluorescence signal transducers. In this assay, monoclonal anti-CRP antibodies are used to capture CRP in human serum, and then the captured CRPs are incubated with biotinylated monoclonal anti-CRP and Neutravidin coated ZnS nanoparticle to form sandwich immunocomplexes. Quantification of CRP occurs when zinc ions released from ZnS nanoparticle labels are mixed with zinc-ion sensitive fluorescence indicator Fluozin-3 for fluorescence generation. The developed assay presents a detection limit around 10 pM and a detection range with more than two orders of magnitude.

Quantitative characterization of fluorophores in multi-component nanoprobes by single-molecule fluorescence

Multi-modal nanoparticles incorporating fluorophores are increasingly being used for medical applications. The number of fluorophores incorporated into the nanoparticles during synthesis is stochastic, leaving some nanoparticles devoid of fluorophores. Determining the number, the brightness and the photostability of the fluorophores incorporated, and the percentage of labeled nanoparticles (labeling efficiency) remains challenging. We have determined the aforementioned quantities for two synthesized multi-modal nanoparticles by exploiting the photobleaching of fluorophores at the single-molecule level using a total internal reflection fluorescence microscope. Labeling efficiency was determined by fitting the distribution of incorporated fluorophores with a statistical model and verified by independent experiments.

Catalytic effect of ReAu nanoalloy on the Te particle reaction and its application to resonance scattering spectral assay of CA125

ReAu nanoparticles with a molar ratio of 2:8 Re and Te nanoparticles were prepared by NaBH₄ reduction. In HCl medium at 65°C, ultratrace Re, Te and ReAu bimetallic nanoparticles strongly catalyzed the slow reaction between Sn(II) and Te(VI) to form Te particles, which exhibited the strongest resonance scattering (RS) peak at 782 nm. As the amount of nanocatalyst increased, the RS intensity at 782 nm (I(782 nm) ) increased linearly, and the increase in intensity ΔI(782 nm) was linear to the ReAu, Re and Te concentrations in the ranges 0.07-9.0, 0.01-4.5 and 30-1200 nM, respectively. As a model, a ReAu immunonanoprobe catalytic Te-particle resonance scattering spectral (RSS) method was established for detection of CA125, using ReAu nanoparticle labeling CA125 antibody (CA125Ab) to obtain an immunonanoprobe (ReAuCA125Ab) for CA125. In pH 7.6 citric acid-Na₂HPO₄ buffer solution, ReAuCA125Ab aggregated nonspecifically. Upon addition of CA125, the immunonanoprobe reacted with it to form ReAuCA125Ab-CA125 dispersive immunocomplex in the solution. After the centrifugation, the supernatant containing the immunocomplex was used to catalyze the reaction of Te(VI)-Sn(II) to produce the Te particles that resulted in the I(782 nm) increasing. The ΔI(782 nm) was linear to CA125 concentration (C(CA125)) in the range 0.1-240 mU/mL. The regression equation, correlation coefficient and detection limit were ΔI(782 nm) = 1.61 C(CA125) + 1.5, 0.9978 and 0.02 mU/mL, respectively. The proposed method was applied to detect CA125 in serum samples, with satisfactory results.

Stable Cellulose Nanospheres for Cellular Uptake

Pure, perfectly spherical cellulose nanoparticles with sizes of ≈80-260 nm can be prepared by dialysis starting from trimethylsilylcellulose (TMSC). The aqueous suspensions obtained are storable for several months. Subsequent covalent labeling of the cellulose nanoparticles with FITC has no influence on particle size, shape, and stability. The particles can be sterilized and suspended in biological media without structural changes. Incorporation of FITC-labeled cellulose nanoparticles into living human fibroblasts is studied using confocal LSM. In contrast to cellulose nanocrystals, fast cellular uptake is found for the nanospheres without transfection reagents or attachment of a receptor molecule. This suggests an influence of the geometry of biocompatible nanomaterials on endocytosis.

Using fluorescence measurement of zinc ions liberated from ZnS nanoparticle labels in bioassay for Escherichia coli O157:H7

In this study, an alternative approach using ZnS nanoparticle biolabels as fluorescence signal transducers is reported for the immunoassay of E. coli O157:H7 in tap water samples. Instead of measuring the fluorescence of ZnS nanoparticles in the assay, the fluorescence signal is generated through the binding of zinc ions released from nanoparticle labels with zinc-ion sensitive fluorescence indicator Fluozin-3. In the assay, ZnS nanoparticles around 50 nm in diameter were synthesized, bioconjugated, and applied for the detection of E. coli O157:H7. The assay shows a detection range over two orders of magnitude and a detection limit around 1000 colony-forming units (cfu) of E. coli O157:H7.