Gold nanoparticles based chemiluminescent resonance energy transfer for immunoassay of alpha fetoprotein cancer marker

In this paper, we report a new strategy of chemiluminescence resonance energy transfer (CRET) by using gold nanoparticles (AuNPs) as efficient long-range energy acceptor in sandwich immunoassays. In the design of CRET system, we chose the highly sensitive chemiluminescence (CL) reaction of luminol and hydrogen peroxide catalysed by horseradish peroxidase (HRP) because the CL spectrum of luminol (λ(max) 425 nm) partially overlaps with the visible absorption bands of AuNPs. On the basis of CRET strategy, we developed a sandwich immunoassay of alpha fetoprotein (AFP) cancer marker. In immunoassay, two antibodies (anti-AFP-1 and anti-AFP-2) were conjugated to AuNPs and horseradish peroxidase (HRP), respectively. The sandwich-type immunoreactions between the AFP (antigen) and the two different antibodies bridged the donors (luminol) and acceptors (AuNPs), which led to the occurrence of CRET from luminol to AuNPs upon chemiluminescent reaction. We observed that the quenching of chemiluminescence signal depended linearly on the AFP concentration within a range of concentration from 5 to 70 ng mL(-1) and the detection limit of AFP was 2.5 ng mL(-1). Our method was successfully applied for determination of AFP levels in sera from cancer patients, and the results were in good agreement with ELISA assays. This approach is expected to be extended to other assay designs, that is, using other antibodies, analytes, chemiluminescent substance, and even other metallic nanoparticles.     

A new dual immunoassay for tumor markers based on chemiluminescence signal amplification by magnetic mesoporous silica and enzyme modified gold nanoparticles

A sensitive dual immunoassay was proposed for the determination of carcinoembryonic antigen (CEA) and α-fetoprotein (AFP) based on signal amplification. Monoclonal antibodies immobilized on magnetic mesoporous silica particles (Fe(3)O(4)/SiO(2)) were prepared as the primary probe. Horseradish peroxidase (HRP) labeled antibodies co-coated with HRP on gold nanoparticles (AuNPs) were used as the secondary probe to achieve signal amplification. HRP tags were retained in the flow cells after a sandwich immunoassay. By controlling two switches on the two channels, chemiluminescent substrates were injected orderly man way, and then signals for CEA and AFP were sequentially detected by HRP-luminol-H(2)O(2). Due to the increased amount of HRP on AuNPs and the increased amount of monoclonal antibodies on Fe(3)O(4)/SiO(2), the signals were largely amplified. Under the optimal conditions, CEA and AFP could be detected in the linear ranges of 1.0 - 80 and 1.0 - 75 ng mL(-1) with detection limits of 0.25 and 0.5 ng mL(-1), respectively.     

Polystyrene microspheres based sandwich immunosensor using CdTe nanoparticles amplification and ultrasensitive flow-injection chemiluminescence detection

In this paper we propose a specific sandwich immunoassay method for human-immunoglobulin G (HIgG). This immunoassay protocol takes advantage of sandwich binding of primary and secondary antibodies for increased specificity. Polystyrene microspheres (PS) serve as immobilizing support, site for sandwich immunoassay and then subsequently used for chemiluminescence (CL) detections. In this sandwich immunoassay, PS microspheres were modified with the primary anti-HIgG (Ab₁) via electrostatic interaction, while CdTe nanoparticles (CdTeNPs) were modified with horseradish peroxidase labeled anti-HIgG (Ab₂) via covalent binding. Antigen HIgG (Ag) was specifically captured by the first and secondary antibody and form sandwich immunoassay format. Combination of the remarkable sensitivity of CL method and the use of CdTe NPs as anti-HIgG–HRP carrier for the enzymatic signal amplification, provide a linear response range of HIgG from 0.01 to 300 ng mL⁻¹ with an extremely low detection limit of 0.3 pg mL⁻¹. This immunoassay system has many desirable merits including sensitivity, accuracy, and little required instrumentation. The assay results were compared with enzyme-linked immunosorbent assay (ELISA), and showed relatively good reliability. Significantly the new protocol may become quite promising technique for protein immune-detection as well as DNA analysis and other biological analyses.     

Highly sensitive rapid chemiluminescent immunoassay using the DNAzyme label for signal amplification

A novel trace tag for chemiluminescent (CL) immunoassay was designed by using DNAzyme to functionalize antibody-labeled Au nanoparticles (AuNPs). The trace tag showed an excellent ability to catalyze the oxidation of luminol by hydrogen peroxide, leading to strong CL emission. By coupling the trace tag with a passive mixing accelerated immunoreaction system, a highly sensitive rapid flow-through CL immunoassay method was proposed. Using carcinoembryonic antigen (CEA) as a model analyte, the capture antibody for CEA was immobilized on paramagnetic microspheres, and DNAzyme-anti-CEA antibody functionalized AuNPs were prepared as trace tag. A three-dimensional helical glass tube kept at 37 °C in a water bath was used for passively mixing immunoreagents in a two-step sandwich immunoassay, with which each immunoreaction step could be finished within 150 s. With the help of a magnet, the immunocomplex could conveniently be separated from reactants. Compared with the horseradish peroxidase-based tag, the newly designed trace tag showed obvious signal amplification due to its strong catalytic ability and high loading ratio of DNAzyme on each AuNP. The proposed method showed a linear calibration range from 0.005 to 0.5 ng mL(-1) for CEA detection with a detection limit of 4.1 pg mL(-1) at a signal-to-noise ratio of 3 and acceptable detection reproducibility. The assay results of clinical serum samples were in acceptable agreement with the reference values. The designed immunoassay system with ultrahigh sensitivity provided a programmable and low-cost approach for high-throughput clinical application.     

Rapid one-step whole blood C–reactive protein magnetic permeability immunoassay with monoclonal antibody conjugated nanoparticles as superparamagnetic labels and enhanced sedimentation

A rapid (5.5 min) one-step whole blood C–reactive protein (CRP) magnetic permeability immunoassay utilizing monoclonal antibody conjugated dextran iron oxide nanoparticles (70 nm) as superparamagnetic labels and mixed fractions (1:1 ratio of 15–40 and 60 μm) of polyclonal anti-CRP conjugated silica microparticles for enhanced sedimentation is described. In this one-step assay procedure, a whole blood sample (4 μl) is applied to an assay glass vial, containing both antibody conjugates, and mixed for 30 s. The target analyte, CRP, forms a sandwich complex between the conjugated nanoparticles and microparticles, and, subsequently, the complex sediments under normal gravitation within 5 min to the bottom of the vial. The magnetic permeability increase of the sediment due to the presence of the complexed superparamagnetic nanoparticles is determined using an inductance-based transducer. Assayed patient whole blood samples were compared with the Abbott Diagnostics Architect reference method. A strong linear correlation was observed for the CRP concentration range 0–260 mg/l in whole blood (y=1.001x+0.42, R ²=0.982, n=50). The CRP assay presented showed a limit of detection of 3 mg/l and a total imprecision (coefficient of variation) of 10.5%. On the basis of our observations, we propose a rapid, one-step, CRP assay for near-patient testing.     

Label-free amperometric immunobiosensor based on a gold colloid and Prussian blue nanocomposite film modified carbon ionic liquid electrode

A novel experimental methodology based on a Prussian blue (PB) and gold nanoparticles (AuNPs) modified carbon ionic liquid electrode (CILE) was developed for use in a label-free amperometric immunosensor for the sensitive detection of human immunoglobulin G (HIgG) as a model protein. The CILE was fabricated by using the ionic liquid 1-octyl-3-methylimidazolium hexafluorophosphate as binder. Controllable electrodeposition of PB on the surface of the CILE and coating with 3-aminopropyl triethylene silane (APS) formed a film with high electronic catalytic activity and large surface area for the assembly of AuNPs and further immobilization of HIgG antibody. The electrochemistry of the formed nanocomposite biofilm was investigated by electrochemical techniques including cyclic voltammetry, differential pulse voltammetry, and electrochemical impedance spectroscopy. The HIgG concentration was measured through the decrease of amperometric responses in the corresponding specific binding of antigen and antibody. The decreased differential pulse voltammetric values were proportional to the HIgG concentration in two ranges, 0.05–1.25 ng mL⁻¹ and 1.25–40 ng mL⁻¹, with a detection limit of 0.001 ng mL⁻¹ (S/N = 3). This electrochemical immunoassay combined the specificity of the immunological reaction with the sensitivity of the AuNPs, ionic liquid, and PB amplified electrochemical detection and would therefore be valuable for clinical immunoassays.     

Immunoassay for P38 MAPK using surface enhanced resonance Raman spectroscopy (SERRS)

A micro-bead sandwich assay for P38 mitogen-activated protein kinase using surface enhanced resonance Raman spectroscopy (SERRS) detection is reported. Monoclonal capture antibodies were immobilised on a solid phase of magnetic micro-beads with secondary detection using a rhodamine-labelled antibody. Quantitative SERRS detection of the secondary antibody was possible with a limit of detection of 9.5 x 10(-12) mol dm(-3). The sandwich assay was quantitative and sensitive to 6 ng ml(-1). The mechanism of the SERRS detection in the immunoassay was investigated. The addition of SERRS aggregating agents causes the dissociation of the immuno-complex from the magnetic beads. Scanning electron microscopy images indicate that the colloidal suspension rather than adsorbed silver nanoparticles on the beads provide the SERRS signals, that the aggregate size is partially controlled and that there is some inhomogeneity in the distribution of organic matter on the nanoscale.     

A nonenzymatic optical immunoassay strategy for detection of Salmonella infection based on blue silica nanoparticles

A novel nonenzymatic optical immunoassay strategy was for the first time designed and utilized for sensitive detection of antibody to Salmonella pullorum and Salmonella gallinarum (S. pullorum and S. gallinarum) in serum. The optical immunoassay strategy was based on blue silica nanoparticles (Blue-SiNps) and magnetic beads (MB). To construct such an optical immunoassay system, the Blue-SiNPs were first synthesized by inverse microemulsion method, characterized by SEM, Zeta potential and FTIR. Two nanostructures including Blue-SiNPs and MB were both functionalized with antibody against S. pullorum and S. gallinarum (anti-PG) without using enzyme labeled antibody. Anti-PG functionalized blue silica nanoparticles (IgG-Blue-SiNps) were used as signal transduction labels, while anti-PG functionalized magnetic beads (IgG-MB) were selected to separate and enrich the final sandwich immune complexes. In the process of detecting negative serum, a sandwich immunocomplex is formed between the IgG-MB and IgG-Blue-SiNPs. With the separation of the immunocomplex using an external magnetic field, the final plaque displayed bright blue color. While in the detection of infected serum, IgG-MB and anti-PG formed sandwich immunocomplexes, IgG-Blue-SiNPs were unable to bind to the limited sites of the antigen, and a light brown plaque was displayed in the bottom of microplate well. Stable results were obtained with an incubation time of 60 min at room temperature, and different colors corresponding to different results can be directly detected with naked eye. The reaction of IgG-Blue-SiNPs with S. pullorum was inhibited by 1:100 dilution of positive chicken serum. Such a simple immunoassay holds great potential as sensitive, selective and point-of-care (POC) tool for diagnosis of other biological molecules.     

Multiple detection of proteins by SERS-based immunoassay with core shell magnetic gold nanoparticles

A highly selective and sensitive surface-enhanced Raman scattering (SERS)-based immunoassay for the multiple detection of proteins has been developed. The proposed core shell magnetic gold (Au) nanoparticles allow for successful protein separation and high SERS enhancement for protein detection. To selectively detect a specific protein in a mixed protein solution, we employed the sandwich type SERS immunoassay with core shell magnetic Au nanoparticles utilizing specific antigen–antibody interactions. Based on this proposed SERS immunoassay, we can successfully detect proteins in very low concentrations (∼800 ag/mL of mouse IgG and ∼5 fg/mL of human IgG) with high reproducibility. Magnetically assisted protein separation and detection by this proposed SERS immunoassay would provide great potential for effective and sensitive multiple protein detection. This technique allows for the straightforward SERS-based bioassays for quantitative protein detections.     

Microdrop analysis of a bead-based immunoassay

The progress to electrochemical detection of a microbead-based immunoassay in small volumes has led to a reduced assay time and lower detection limits. Three electrochemical techniques are described for an immunoassay with detection in a microdrop. The techniques are amperometric detection with a rotating disk electrode (RDE), a microelectrode, and an interdigitated array (IDA) electrode. An enzyme-labeled sandwich immunoassay with mouse IgG as the model analyte is used to demonstrate the three techniques. The microbead assay is carried out in a test tube using a magnet to control bead collection. Once the immunocomplex is formed on the microbead, the beads are transferred to a microdrop where the enzyme, either alkaline phosphatase or β-galactosidase, generates 4-aminophenol (PAP). PAP is oxidized at the electrode with an applied potential of +290 mV vs. Ag/AgCl. For all three techniques, the upper limit of the dynamic range was 1000 ng/ml mouse IgG, and the detection limits were: 50 ng/ml for the RDE, 40 ng/ml for the microelectrode, and 26 ng/ml for the IDA electrode.     

Synthesis-based approach toward direct sandwich immunoassay for ciguatoxin CTX3C

Ciguatoxins are the major causative toxins of ciguatera seafood poisoning. Limited availability of ciguatoxins has hampered the development of a reliable and specific immunoassay for detecting these toxins in contaminated fish. Monoclonal antibodies (mAbs) specific against both ends of ciguatoxin CTX3C were prepared by immunization of mice with protein conjugates of rationally designed synthetic haptens, 3 and 4, in place of the natural toxin. Haptenic groups that possess a surface area larger than 400 A(2) were required to produce mAbs that can bind strongly to CTX3C itself. A direct sandwich enzyme-linked immunosorbent assay (ELISA) using these mAbs was established to detect CTX3C at the ppb level with no cross-reactivity against other related marine toxins, including brevetoxin A, brevetoxin B, okadaic acid, or maitotoxin.     

Gold nanoparticles decorated carbon fiber mat as a novel sensing platform for sensitive detection of Hg(II)

The three-dimensional fibril-like carbon fiber mat electrode (CFME) decorated with Au nanoparticles (AuNPs) was employed to construct Hg(II) sensing platform for the first time. The highly porous feature of CFME combining the high affinity of AuNPs for mercury endowed the sensing platform with high sensitivity and good reproducibility. Under optimal conditions, the prepared AuNPs/CFME was capable of sensing Hg(II) with a detection limit of 0.1 μg L^− 1 (S/N = 3) using differential pulse anodic stripping voltammetry (DPASV). Finally, the AuNPs/CFME was successfully demonstrated for the determination of Hg(II) in real water samples with satisfactory results.     

Catechin mediated green synthesis of Au nanoparticles: Experimental and theoretical approaches to the determination HOMO-LUMO energy gap and reactivity indexes for the (+)-epicatechin (2S, 3S)

This work suggests a green method for synthesizing Au nanoparticles (AuNPs) using the aqueous extract of Salix aegyptiaca extract. The mechanism of green synthesized AuNPs was examined by molecular electrostatic potential (MEP) calculations. AuNPs were characterized with different techniques such as Ultraviolet–visible spectroscopy (UV–vis), Fourier-transform infrared spectroscopy (FT-IR) spectroscopy, X-ray diffraction (XRD), and Transmission electron microscopy (TEM). Electrochemical investigation of modified glassy carbon electrode using AuNPs (AuNPs/GCE) shows that the electronic transmission rate between the modified electrode and [Fe (CN)₆]^3?/4? increased. Process of oxidation, energy gap, and chemical reactivity indexes of the (+)-epicatechin (2S,3S) were investigated using electrochemical techniques (cyclic voltammetry (CV) and differential pulse voltammetry (DPV) as well as UV–Visible spectroscopy and compared with quantum mechanical calculations. DPV and CV were used to obtain HOMO energies of the (+)-epicatechin (2S,3S), an optical energy gap was obtained from the UV–Vis spectroscopy. Frontier molecular orbitals analysis (FMO) and reactivity indexes such as chemical hardness (?), electrophilicity (?), electronic chemical potential (μ), electron acceptor power (?⁺), electron donor power (?^?) were determined with functional theory (DFT) calculations. In summary, the HOMO energy obtained from the experimental analyses (E_HOMO (from DPV) = -5.24 eV, and E_HOMO (from CV) = -5.28 eV) has a relative agreement with the HOMO energy calculated by B3LYP/6–31 g (d, p) including the solvent effect (water) (E_HOMO (from B3LYP) = -5.75 eV). Also, UV–Vis spectroscopy gives the bandgap energy equal to 4.31 eV, while the 4.13 eV is calculated by TD-DFT-b3lyp/6–31 + g(d).     

Fast and sensitive detection of ochratoxin A in red wine by nanoparticle-enhanced SPR

Herein, we present a fast and sensitive biosensor for detection of Ochratoxin A (OTA) in a red wine that utilizes gold nanoparticle-enhanced surface plasmon resonance (SPR). By combining an indirect competitive inhibition immunoassay and signal enhancement by secondary antibodies conjugated with gold nanoparticles (AuNPs), highly sensitive detection of low molecular weight compounds (such as OTA) was achieved. The reported biosensor allowed for OTA detection at concentrations as low as 0.75 ng mL⁻¹ and its limit of detection was improved by more than one order of magnitude to 0.068 ng mL⁻¹ by applying AuNPs as a signal enhancer. The study investigates the interplay of size of AuNPs and affinity of recognition elements affecting the efficiency of the signal amplification strategy based on AuNP. Furthermore, we observed that the presence of polyphenolic compounds in wine samples strongly interferes with the affinity binding on the surface. To overcome this limitation, a simple pre-treatment of the wine sample with the binding agent poly(vinylpyrrolidone) (PVP) was successfully applied.     

Fluorescence polarization immunoassay for rapid screening of ochratoxin A in red wine

A fluorescence polarization (FP) immunoassay, based on a monoclonal antibody and an ochratoxin A (OTA)-fluorescein tracer, has been developed for rapid screening of OTA in red wine. Wine samples were diluted with methanol and passed through aminopropyl solid-phase extraction columns prior to the FP assay. Average recoveries from samples spiked with OTA at levels of 2.0 and 5.0 ng/mL were 79% with RDS of 11% (n = 6). The limit of detection of the FP immunoassay was 0.7 ng/mL OTA, and the whole analysis was performed in less than 10 min. The assay was tested on 154 red wine samples (naturally contaminated or spiked at level ranging from 0.1 to 5.0 ng/mL) and compared with an high-performance liquid chromatography/immunoaffinity column clean-up method, showing a good correlation (r = 0.9222). Their compliance with the European regulation (2.0 ng/mL OTA maximum permitted level) was correctly assessed for 70% of the analyzed samples of red wine, whereas confirmatory analyses were required for the remaining ones with OTA levels close to the regulatory limit. No false-negative or positive results were observed using the FP immunoassay. The proposed FP assay is a useful screening method for OTA in red wines, when high throughput is required, that could also be used for white and rosé wines, which are known to contain less interfering compounds such as polyphenols.     

Ultra-sensitive immunosensor for detection of hepatitis B surface antigen using multi-functionalized gold nanoparticles

The signal amplification for analytical purposes has considerable potential in detecting trace levels of analytes for clinical, security or environmental applications. In the present report a strategy based on a sandwich type immunoassay system was designed for the detection of hepatitis B surface antigen which exploits the specific affinity interaction between streptavidin and biotin recognition systems. The method involves the specific coupling of multi-functionalized gold nanoparticles (bearing biotin and luminol molecules) to the streptavidin modified by secondary antibody. The chemiluminescent signal is produced by the gold nanoparticles in the presence of HAuCl₄ as catalyst and hydrogen peroxide as oxidant. The immunosensor was able to detect hepatitis B surface antigen in the linear concentration range from 1.7 to 1920 pg mL⁻¹ and the detection limit of 0.358 pg mL⁻¹, at signal/noise = 3.     

Magnetic porous carbon derived from Co‐doped metal–organic frameworks for the magnetic solid‐phase extraction of endocrine disrupting chemicals

Metal–organic frameworks‐5 (MOF‐5) was explored as a template to prepare porous carbon due to its high surface area, large pore volume, and permanent nanoscale porosity. Magnetic porous carbon, Co@MOF‐5‐C, was fabricated by the one‐step direct carbonization of Co‐doped MOF‐5. After carbonization, the magnetic cobalt nanoparticles are well dispersed in the porous carbon matrix, and Co@MOF‐5‐C displays strong magnetism (with the saturation magnetization intensity of 70.17emu/g), high‐specific surface area, and large pore volume. To evaluate its extraction performance, the Co@MOF‐5‐C was applied as an adsorbent for the magnetic solid‐phase extraction of endocrine disrupting chemicals, followed by their analysis with high‐performance liquid chromatography. The developed method exhibits a good linear response in the range of 0.5–100 ng/mL for pond water and 1.0–100 ng/mL for juice samples. The limits of detection (S/N  = 3) for the analytes were in the range of 0.1–0.2 ng/mL.     

Label-free sandwich type of immunosensor for hepatitis C virus core antigen based on the use of gold nanoparticles on a nanostructured metal oxide surface

We report on the construction of a label-free electrochemical immunosensor for detecting the core antigen of the hepatitis C virus (HCV core antigen). A glassy carbon electrode (GCE) was modified with a nanocomposite made from gold nanoparticles, zirconia nanoparticles and chitosan, and prepared by in situ reduction. The zirconia nanoparticles were first dispersed in chitosan solution, and then AuNPs were prepared in situ on the ZrO₂-chitosan composite. In parallel, a nanocomposite was synthesized from AuNPs, silica nanoparticles and chitosan, and conjugated to a secondary antibody. The properties of the resulting nanocomposites were investigated by UV-visible photometry and transmission electron microscopy, and the stepwise assembly process was characterized by means of cyclic voltammetry and electrochemical impedance spectroscopy. An sandwich type of immunosensor was developed which displays high sensitivity to the HCV core antigen in the concentration range between 2 and 512?ng?mL^?1, with a detection limit of 0.17?ng?mL^?1 (at S/N?=?3). This immunosensor provides an alternative approach towards the diagnosis of HCV.

Electrochemical Sensing Platforms for HER2‐ECD Breast Cancer Biomarker Detection

Screening and early diagnosis are crucial to increase the success of cancer patients’ treatments and improve the survival rate. To contribute to this success, distinct electrochemical immunosensing platforms were developed for the analysis of the ExtraCellular Domain of the Human Epidermal growth factor Receptor 2 (HER2‐ECD) through sandwich assays on nanomaterial‐modified screen‐printed carbon electrodes (SPCEs). The most promising platforms showed to be SPCEs modified with (i) gold nanoparticles (AuNPs) and (ii) multiwalled carbon nanotubes combined with AuNPs. The antibody‐antigen interaction was detected using a secondary antibody labelled with alkaline phosphatase and 3‐indoxyl phosphate and silver ions as the enzymatic substrate. The electrochemical signal of the enzymatically generated metallic silver was recorded by linear sweep voltammetry. Under the optimized conditions, linear calibration plots were obtained between 7.5 and 50 ng/mL and the total assay time was 2 h 20 min, achieving LODs of 0.16 ng/mL (SPCE‐MWCNT/AuNP) and 8.5 ng/mL (SPCE‐AuNP), which are well below the established cut‐off value of 15 ng/mL for this cancer biomarker.     

Preparation of highly hydrophilic magnetic nanoparticles with anion‐exchange ability and their application for the extraction of non‐steroidal anti‐inflammatory drugs in environmental samples

Diallyldimethylammonium chloride modified magnetic nanoparticles were synthesized by the “thiol‐ene” click chemistry reaction. Diallyldimethylammonium chloride rendered the material plenty of quaternary ammonium groups, and thus the excellent aqueous dispersibility and anion‐exchange capability. The novel material was then used as the magnetic solid‐phase extraction sorbent to extract eight non‐steroidal anti‐inflammatory drugs from water samples. Combined with high‐performance liquid chromatography and ultraviolet detection, under the optimal conditions, the developed method exhibited wide linearity ranges (1–1000, 2–1000, and 5–1000 ng/mL) with recoveries of 88.0–108.6% and low limits of detection (0.3–1.5 ng/mL). Acceptable precision was obtained with satisfactory intra‐ and inter‐day relative standard deviations of 0.4–4.4% (n = 3) and 1.1–5.5% (n = 3), respectively. Batch‐to‐batch reproducibility was acceptable with relative standard deviations <9.7%. The hydrophilic magnetic nanoparticle featured with quaternary ammonium groups showed high analytical potential for acidic analytes in environmental water samples.