Competitive immunoassay for imidaclothiz using upconversion nanoparticles and gold nanoparticles as labels

The authors describe a sensitive and rapid upconversion fluorescence based immunoassay for the neonicotinoid insecticide imidaclothiz (IMI). Upconversion nanoparticles (UCNPs) consisting of hexagonal phase NaYF₄:Yb,Er were functionalized with amino groups and coupled to antibody against IMI. Gold nanoparticles (AuNPs) were used to label the antigen (analyte). Competitive binding of IMI and AuNPs-labeled IMI to the UCNPs-labeled antibody results in a change in the fluorescence of the UCNPs at excitation/emission wavelengths of 980/544 nm. Under optimal conditions, the concentration of IMI producing a 50% saturation of the signal (SC₅₀) is 18.9 ng mL^?1, and the limit of detection is 2.1 ng mL^?1. The method was applied to the determination of IMI in (spiked) paddy water, soil, pear, rice, apple, tomato, pakchoi and cabbage. Average recoveries range from 67.4% to 104.6%, and relative standard deviations from 1.9% to 10.3%. The results correlate well with those obtained by HPLC, the relative correlation coefficient (R²) being 0.9811.

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Graphical abstract Based on inner filter effect (IFE), a novel immunoassay for imidaclothiz (IMI) was developed by using upconversion nanoparticles (UCNPs) and gold nanoparticles (AuNPs) as labels. Competitive binding of IMI and AuNPs-labeled IMI to the UCNPs-labeled antibody results in a change in the fluorescence of the UCNPs at excitation/emission wavelengths of 980/544 nm.

     

Antibody-biotemplated HgS nanoparticles: extremely sensitive labels for atomic fluorescence spectrometric immunoassay

In this work, antibody goat anti-human IgG as a scaffold was employed for the synthesis and biofunctionalization of HgS nanoparticles (NPs) via a facile one-pot process. After a complete sandwich-type immunoreaction among primary antibody, human IgG and secondary antibody labeled with HgS NPs, a large number of mercury ions released from captured HgS NPs dissolution were quantitatively detected by chemical vapor generation atomic fluorescence spectrometry (CVG-AFS). Taking advantage of the signal amplification property of HgS NPs and the high sensitivity of CVG-AFS, the assay detected human IgG with a limit of detection (S/N = 3) of 0.6 ng mL(-1) (4.0 fmol mL(-1) or 0.4 fmol) and the response was linear over a dynamic range from 1.0 to 5.0 × 10(4) ng mL(-1) with a correlation coefficient of 0.996. A relative standard deviation (RSD) of 1.0 × 10(2) ng mL(-1) human IgG was 1.5% for within-batch (intra-assay) and 4.5% for between-batch (inter-assay). Other proteins, such as goat anti-rabbit IgG, goat anti-human IgG, rabbit anti-human IgG, carcinoembryonic (CEA), α-fetoprotein (AFP), human serum albumin (HSA) and bovine serum albumin (BSA) did not significantly interfere with the assay for human IgG. The analytical result of HgS NPs with AFS-based immunoassay technology for the quantification of human IgG in human serum from patients is in good agreement with the result obtained by conventional immunoturbidimetric method. The consequence shows that the novel immunosensor possessed satisfactory precision, extremely high sensitivity, high selectivity and could be applied for the quantification analysis of real samples.     

Hollow mesoporous silica microspheres as sensitive labels for immunoassay of prostate-specific antigen

A novel electrochemical immunosensor for sensitive detection of cancer biomarker prostate-specific antigen (PSA) has been reported based on nanoporous gold (NPG) and hollow mesoporous silica microspheres (HSMs). The immunoassay was conducted by following the typical procedure for sandwich-type immunoreaction. Nanoporous gold (NPG) foils were immobilized onto a glassy carbon electrode (GCE) for primary anti-PSA (Ab(1)) anchoring. Making use of the unique properties of NPG, antibodies could be self-assembled onto the support-free NPG via amine-Au affinity with a high loading amount and reserve high immunological activity. HSMs were used as a secondary anti-PSA antibody (Ab(2)) label. For the preparation of the label, mediator thionine (TH) was first conjugated onto amino-functionalized HSMs (NH(2)-HSMs) via crosslinking with glutaraldehyde, and then the amino group of TH was used to immobilize horseradish peroxidase (HRP) and Ab(2). Due to the large specific surface area of NH(2)-HSMs, the loading of antibodies as well as mediator and enzyme onto HSMs was substantially increased, which increased the sensitivity of the immunosensor. The immunosensor exhibited a high sensitivity and showed a linear response within the range of 0.01-10 ng mL(-1) PSA, low detection limit (6.00 pg mL(-1)), good reproducibility and stability. Significantly, the new method may be quite promising, with potential broad applications for clinical immunoassays.     

Heterogeneous immunoassay for soy protein determination using nile blue-doped silica nanoparticles as labels and front-surface long-wavelength fluorimetry

A long-wavelength fluoroimmunoassay for the determination of soy protein is reported for the first time using a conjugate composed of anti-soy protein antibodies bound to nile blue-doped silica nanoparticles (NPs). These NPs have been synthesized by a reverse-micelle microemulsion method and functionalized by using 3-(aminopropyl)triethoxysilane (APS) and 3-(trihydroxysilyl)propyl methylphosphonate (THPMP) to avoid NP aggregation. The tracer has been obtained by linking the functionalized NPs with anti-soy protein antibodies previously oxidised with sodium periodate. The immunoassay has been developed in 96-well microplates using a heterogeneous competitive format with antibody capture. Soy proteins are immobilised onto the wells and bovine serum albumin is added to block the surface, thus minimising non-specific binding. After washing, the microplates can be stored ready to use. At the analysis time, soy protein standards or sample and tracer are added and incubated and, after the corresponding washing and drying steps, the fluorescence is measured onto the solid surface at λ_ex 620 and λ_em 680 nm. The method features a dynamic range of 0.1–10 mg L⁻¹ and a detection limit of 0.05 mg L⁻¹. The precision of the method has been assayed at 0.5 and 5 mg L⁻¹ protein concentrations, obtaining the values of relative standard deviation of 9.6% and 6.1%, respectively. This new immunoassay has been applied to the analysis of food containing soy protein and the results obtained have been compared to those provided by a commercial ELISA kit with no statistically differing results. Also, a recovery study has been performed, providing percentages in the range of 81.5–111.0%.     

Homogeneous immunoassay for soy protein determination in food samples using gold nanoparticles as labels and light scattering detection

A homogeneous aggregation immunoassay involving the use of gold nanoparticles (AuNPs) and light scattering detection is described for soy protein determination in food samples. AuNPs act as enhancers of the precipitate that appears when the antigen-antibody complex is formed. The AuNPs-antibody conjugate has been synthesized by physical adsorption of polyclonal anti-soy protein antibodies onto the surface of commercial AuNPs with a nominal diameter of 20 nm. The direct assay is based on the reaction of the conjugate with soy protein, which reaches the equilibrium in about 10 min, and the measurement of the light scattering intensity at 530 nm, which is proportional to the analyte concentration. The dynamic range of the calibration graph is 0.2-20 μg mL⁻¹ and the detection limit value is 65 ng mL⁻¹. The precision, expressed as relative standard deviation, has been assayed at two different concentrations, 0.2 and 1 μg mL⁻¹, giving values ranging from 4.7 to 5.9%. The interference of other proteins has been assayed. The usefulness of this method has been shown by its application to the analysis of fruit juice and “nonmilk yoghourt” samples. The results obtained with the proposed method are similar to those obtained by using a commercial ELISA kit, but the assay time is significantly shorter and the detection limit was about 10 times lower. A recovery study has been also performed, giving values in the range of 84.0-119.3%.     

CdSe quantum dots as labels for sensitive immunoassay of cancer biomarker proteins by electrogenerated chemiluminescence

A sensitive and specific immunoassay method for detecting α-fetoprotein (AFP) based on electrogenerated chemiluminescence (ECL) was described. ECL could perform detection for a series of different concentrations of AFP. CdSe quantum dots (QDs) were used as labels and were linked to AFP antibody (anti-AFP, the secondary antibody, Ab2*). Immunoassay was carried out on a modified electrode using a sandwich assay approach, where anti-AFP (Ab1) was covalently bound to the surface of an Au electrode to be allowed to capture AFP specifically. Afterwards, Ab2* was allowed to bind selectively to the captured AFP. The non-specific adsorption was negligible. In the presence of H(2)O(2), the ECL intensity increased with the increase of AFP, which indicated that an immunosensor for AFP was constructed. The detection of AFP based on measuring the ECL intensity of CdSe without the enzyme and mediator can promote the stability of the immunosensor. The linear range of the AFP assay was from 0.002 to 32 ng mL(-1). Furthermore, the immunosensor showed high sensitivity, good precision, stability, and reproducibility and could be used for the detection of real samples with consistent results in comparison with those obtained by the enzyme-linked immunosorbent assay (ELISA) method. The strategy was successfully demonstrated as a simple, cost-effective, specific, and potential method to detect AFP in practical samples.     

Highly sensitive electrochemical immunoassay for human IgG using double-encoded magnetic redox-active nanoparticles

A new sandwich-type electrochemical immunoassay was developed for the detection of human IgG using doubly-encoded and magnetic redox-active nanoparticles as recognition elements on the surface of a glassy carbon electrode modified with anti-IgG on nanogold particles. The recognition elements were synthesized by coating magnetic Fe₃O₄ nanoparticles with Prussian blue nanoparticles and then covered with peroxidase-labeled anti-IgG antibodies (POx-anti-IgG) on Prussian blue nanoparticles. The immunoelectrode displays very good electrochemical properties towards detection of IgG via using double-encoded magnetic redox-active nanoparticles as trace and hydrogen peroxide as enzyme substrate. Its limit of detection (10 pmol·L^?1) is 10-fold better than that of using plain POx-anti-IgG secondary antibodies. The method was applied to the detection of IgG in serum samples, and an excellent correspondence with the reference values was found.     

Chiroplasmonic assemblies of gold nanoparticles as a novel method for sensitive detection of alpha-fetoprotein

Microchimica Acta - Serum alpha-fetoprotein (AFP) is present in enhanced concentrations in patients with liver cancer. Thus, sensitive detection of AFP plays an important role in the early...     

Rapid and sensitive lateral flow immunoassay for influenza antigen using fluorescently-doped silica nanoparticles

We report on a lateral flow immunoassay (LFIA) for influenza A antigen using fluorescently-doped silica nanoparticles as reporters. The method is taking advantage of the high brightness and photostability of silica nanoparticles (doped with the dye Cy5) and the simplicity and rapidity of LFIA. The nucleoprotein of influenza A virion (one of its most abundant structural proteins) was used as a model to demonstrate a performance of the LFIA. Under optimized conditions and by using a portable strip reader, the fluorescence-based LFIA is capable of detecting a recombinant nucleoprotein as low as 250 ng?·?mL^-1 using a sample volume of 100 μL, within 30 min, and without interference by other proteins. The successful detection of the nucleoprotein in infected allantoic fluid demonstrated the functionality of the method. By comparison with a commercial influenza A test based on gold nanoparticles as reporters, the system provides an 8-fold better sensitivity.

Comparison of infrared-excited up-converting phosphors and europium nanoparticles as labels in a two-site immunoassay

Research in the field of immunoassays and labels used in the detection has been recently focused on particulate reporters, which possess very high specific activity that excludes the label as a sensitivity limiting factor. However, the large size and shape of the particulate labels may produce additional problems to immunoassay performance. The aim of this work was to study with two identical non-competitive two-site immunoassays whether up-converting phosphor (UCP) particles are comparable in performance with europium(III) chelate-dyed nanoparticles as particulate labels. In addition we strived to verify the common assumption of the photostability of up-converting phosphor particles supporting their potential applicability in imaging. Detection limits in two-site immunoassay for free prostate-specific antigen (free-PSA) were 0.53 ng L⁻¹ and 1.3 ng L⁻¹ using two different up-converting phosphors and 0.16 ng L⁻¹ using europium(III) nanoparticle. Large size distribution and non-specific binding of up-converting phosphor particles caused assay variation in low analyte concentrations and limited the analytical detection limit. The non-specific binding was the major factor limiting the analytical sensitivity of the immunoassay. The results suggests the need for nanoscaled and uniformely sized UCP-particles to increace the sensitivity and applicability of up-converting phosphor particles. Anti-Stokes photoluminescence of up-converting phosphor particles did not photobleach when measured repeatedly, on the contrary, the time-resolved fluorescence of europium nanoparticles photobleached relatively rapidly.     

Enzyme-catalyzed silver deposition on irregular-shaped gold nanoparticles for electrochemical immunoassay of alpha-fetoprotein

A new and disposable electrochemical immunosensor was designed for detection of alpha-fetoprotein (AFP), as a model analyte, with sensitivity enhancement based on enzyme-catalyzed silver deposition onto irregular-shaped gold nanoparticles (ISGNPs). The assay was carried out with a sandwich-type immunoassay protocol by using ISGNP-labeled anti-AFP antibodies conjugated with alkaline phosphatase (ALP–Ab₂) as detection antibodies. The enzymatically catalytic deposition of silver on the electrode could be measured by stripping analysis in KCl solution due to the Ag/AgCl solid-state voltammetric process. Several labeling protocols including spherical gold nanoparticle-labeled ALP–Ab₂ and ISGNP-labeled ALP–Ab₂ were investigated for determination of AFP, and improved analytical properties were achieved with the ISGNP labeling. With the ISGNP labeling method, the effects of incubation time and incubation temperature for antigen-antibody reaction, and deposition time of silver on the current responses of the electrochemical immunosensors were also monitored. Under optimal conditions, the electrochemical immunosensor exhibited a wide dynamic range from 0.01 ng mL⁻¹ to 200 ng mL⁻¹ with a detection limit of 5.0 pg mL⁻¹ AFP. The immunosensor displayed a good stability and acceptable reproducibility and accuracy. No significant differences at the 95% confidence level were encountered in the analysis of 10 clinical serum samples between the developed immunoassay and the commercially available electrochemiluminescent method for determination of AFP.     

Sandwich immunoassay for alpha-fetoprotein in human sera using gold nanoparticle and magnetic bead labels along with resonance Rayleigh scattering readout

We describe a sensitive sandwich immunoassay for alpha-fetoprotein (AFP). It is making use of gold nanoparticles (GNPs) and magnetic beads (MBs) as labels, and of resonance Rayleigh scattering for detection. Two antibodies were labeled with GNPs and MBs, respectively, and MB-antigen-GNP complexes were formed in the presence of antigens. The MB labels also serve as solid phase carriers that can be used to magnetically separate the immuno complex. The GNP labels are used as optical probes, and Rayleigh scattering was used to determine the concentration of free GNPs-antibody after separation of the MB-antigen-GNP complexes. The concentration of AFP is related to the intensity of light scattered by free GNPs in the 13.6 pM to 436 pM concentration range, and the limit of detection is 13.6 pM. The method was applied to the determination of AFP in sera of cancer patients, and the results agree well with those obtained by conventional ELISA.

Chemiluminescent detection of DNA hybridization using gold nanoparticles as labels

A chemiluminescent (CL) detection method has been developed for DNA hybridization. The assay relies on a sandwich-type DNA hybridization in which gold nanoparticles modified with alkylthiol-capped oligonucleotide strands are used as probes to monitor the presence of the specific target DNA. The , which is the dissolving product of the gold nanoparticles anchored on the DNA hybrids, serves as an analyte in the H₂O₂–luminol– CL reaction for the indirect measurement of the target DNA. The combination of the remarkable sensitivity of the CL analysis with the large number of released from each DNA hybrid allows a detection limit at levels as low as 0.1 pM of the target DNA. Moreover, with a further silver amplification step, the detection limit will be pushed down to the femtomolar domain.      

Ultrasensitive electrochemiluminescence immunoassay for tumor marker detection using functionalized Ru-silica@nanoporous gold composite as labels

In this work, we reported a simple and sensitive sandwich-type electrochemiluminescence (ECL) immunosensor for carcinoembryonic antigen (CEA) on a gold nanoparticles (AuNPs) modified glassy carbon electrode (GCE). The Ru-silica (Ru(bpy)(3)(2+)-doped silica) capped nanoporous gold (NPG) (Ru-silica@NPG) composite was used as an excellent label with amplification techniques. The NPG was prepared with a simple dealloying strategy, by which silver was dissolved from silver/gold alloys in nitric acid. The primary antibody was immobilized on the AuNPs modified electrode through l-cysteine and glutaraldehyde, and then the antigen and the functionalized Ru-silica@NPG composite labeled secondary antibody were conjugated successively to form a sandwich-type immunocomplex through the specific interaction. The concentrations of CEA were obtained in the range from 1 pg mL(-1) to 10 ng mL(-1) with a detection limit of 0.8 pg mL(-1). The as-proposed ECL immunosensor has the advantages of high sensitivity, specificity and stability and could become a promising technique for tumor marker detection.     

Horseradish peroxidase-loaded nanospheres attached to hollow gold nanoparticles as signal enhancers in an ultrasensitive immunoassay for alpha-fetoprotein

We report on a novel electrochemical signal amplification strategy for use in immunoassays. The highly responsive immunoelectrode was constructed in the following way: (1) The surface of a gold electrode was covered with a layer single-walled carbon nanotubes dispersed in chitosane functionalized with L-cysteine; (2) Gold nanoparticles containing protein A and anti-alpha-fetoprotein (anti-AFP) were then covalently attached to the surface via the thiol groups of the chitosane. The electrode is then exposed to the analyte (AFP) which then is bound by the antibody. In the next step, a gold-conjugated secondary antibody is added that was prepared in the following way: (1) Horseradish peroxidase was crosslinked and the resulting spheres were coated with hollow gold nanoparticles (hollow Au-NPs) to give nanospheres of ~100 nm in diameter. (2) These were the coated with thionine and, in a last step, with secondary antibody. The use of these materials has several attractive features: The HRP-NPs functionalized with hollow Au-NPs possess a large surface area that can load the large amount of secondary antibody. Thionine (Thi) is highly redox active and improves the intensity of the signal. Carbon nanotubes were used because they possess an excellent electron transfer rate and large surface area. Following incubation of the modified electrode (a) with a sample containing AFP, (b) then with the secondary antibody, and (c) with washing buffer, the electrode is placed in a solution containing H₂O₂. The HRP in the smart secondary antibody causes the catalytic decomposition of H₂O₂ and the results in an electrical current that is linearly related to the concentration of AFP in the 0.025 to 5.0 ng mL^?1 concentration range. The detection limit for AFP is as low as 8.3 pg mL^?1. We believe that this novel kind of immunoassay represents a promising tool for use in sensitive clinical assays.

A facile and sensitive immunoassay for the detection of alpha-fetoprotein using gold-coated magnetic nanoparticle clusters and dynamic light scattering

A facile and sensitive immunoassay protocol for the detection of alpha-fetoprotein (AFP) was developed using gold-coated iron oxide magnetic nanoclusters and dynamic light scattering (DLS) methods. The increase in the average particle size due to AFP-mediated aggregation was measured using DLS, and the detection limit was better than 0.01 ng mL(-1).     

Dye-doped silica nanoparticles as efficient labels for glycans

We report that dye-doped fluorescent silica nanoparticles (FSNPs) are highly efficient labels for glycans. Mono- and oligo-saccharides were conjugated to FSNPs using a general photocoupling chemistry. FSNP-labeled glycans were applied to image and detect bacteria, and to study carbohydrate-lectin interactions on a lectin microarray.     

基于多巴胺-量子点的比色免疫分析法对甲胎蛋白的高灵敏检测

基于抗原抗体识别特异性,以多巴胺-Mn/ZnS量子点(DA-QDs)为反应媒介,紫外可见分光光度计为检测手段,采用柠檬酸钠还原法制备金纳米颗粒(AuNPs),其表面用辣根过氧化物酶(HRP)和羊抗小鼠免疫球蛋白G (IgG)修饰获得功能化二抗(IgG-AuNPs-HRP)作为信号放大标签,构建一种简便高灵敏的检测方法....