Homogeneous immunoassay based on gold nanoparticles and visible absorption detection

A sensitive homogeneous immunoassay, using human serum albumin (HSA) as a model analyte coupled with simple visible absorption detection, has been developed. The new assay is based on the use of gold nanoparticles functionalized with the target protein, which compete with the analyte for the binding of a specific polyclonal antibody. The binding of antibodies to the functionalized nanoparticles determines a shift of the visible absorption maximum of the gold colloid, and quantification of the analyte could be obtained as the competitive inhibition of the binding of antibodies to the nanoparticles. The proposed immunoassay has been optimized and successfully applied to measuring HSA in human urine samples, in which results agreed well with those obtained by a nephelometric reference method.     

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.     

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%.     

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.

     

Homogeneous immunoassay based on aggregation of antibody-functionalized gold nanoparticles coupled with light scattering detection

A universal platform of homogeneous noncompetitive immunoassay, using human immunoglobulin (IgG) as a model analyte, has been developed. The assay is based on aggregation of antibody-functionalized gold nanoparticles directed by the immunoreaction coupled with light scattering detection with a common spectrofluorimeter. In phosphate buffer (pH 7.0) solution, the light scattering intensity of the gold nanoparticles functionalized with goat-anti-human IgG can be greatly enhanced by addition of the human IgG. Based on this phenomenon, a wide dynamic range of 0.05-10 microg ml(-1) for determination of human IgG can be obtained, and the detection limit can reach 10 ng ml(-1). The proposed immunoassay can be accomplished in a homogeneous solution with one-step operation within 10 min and has been successfully applied to the determination of human IgG in serum samples, in which the results are well consistent with those of the enzyme-linked immunosorbent assay (ELISA), indicating its high selectivity and practicality. Therefore, the gold nanoparticle-based light scattering method can be used as a model to establish the general methods for protein assay in the fields of molecular biology and clinical diagnostics.     

Homogeneous fluorescence-based immunoassay via inner filter effect of gold nanoparticles on fluorescence of CdTe quantum dots

Homogeneous immunoassays are becoming more and more attractive for modern medical diagnosis because they are superior to heterogeneous immunoassays in sample and reagent consumption, analysis time, portability and disposability. Herein, a universal platform for homogeneous immunoassay, using human immunoglobulin (IgG) as a model analyte, has been developed. This assay relies upon the inner filter effect (IFE) of gold nanoparticles (AuNPs) on CdTe QDs fluorescence. The immunoreaction of antigen and antibody can induce the aggregation of antibody-functionalized AuNPs, and after aggregation the IFE of AuNPs on CdTe QDs fluorescence is greatly enhanced, resulting in a decrease of fluorescence intensity in the system. Based on this phenomenon, a wide dynamic range of 1-100 pg mL(-1) for determination of IgG can be obtained. The proposed method shows a detection limit of 0.3 pg mL(-1) for human IgG, which is much lower than the corresponding absorbance-based approach and compares favorably with other reported fluorescent methods. This immunoassay method is simple, rapid, cheap, and sensitive. The proposed method has been successfully applied to measuring IgG in serum samples, and the obtained results agreed well with those of the enzyme-linked immunosorbent assay (ELISA).     

PDMS microfludic device for optical detection of protein immunoassay using gold nanoparticles

A simple but highly specific immunoassay system for goat anti-human IgG has been developed using gold nanoparticles and microfluidic techniques. The assay is based on the deposition of gold nanoparticles that are coated with protein antigens in the presence of their corresponding antibodies to microfluidic channel surface. The effects of time accumulation, the flow velocity, and the concentration of antibodies to the red light absorption percentage (RAP) of deposition were investigated with an ordinary optical microscope. By controlling the reaction time and flow velocity, a dynamic range of 3 orders of magnitude and a detection sensitivity of 10 ng ml(-1) of goat anti-human IgG were achieved. Because of its simplicity and flexibility, this new technique should be useful for fast, highthroughput screening of antibodies in clinical diagnostic applications.     

A SERS-based immunoassay for porcine circovirus type 2 using multi-branched gold nanoparticles

We report on a facile immunoassay for porcine circovirus type 2 (PCV2) based on surface enhanced Raman scattering (SERS) using multi-branched gold nanoparticles (mb-AuNPs) as substrates. The mb-AuNPs in the immunosensor act as Raman reporters and were prepared via Tris base-induced reduction and subsequent reaction with p-mercaptobenzoic acid (pMBA). They possess good stability and high SERS activity. Subsequently, the modified mb-AuNPs were covalently conjugated to the monoclonal antibody (McAb) against the PCV2 cap protein to form SERS immuno nanoprobes. These were captured in a microtiterplate via a immunoreaction in the presence of target antigens. The effects of antibody concentration, reaction time and temperature on the sensitivity of the immunoassay were investigated. Under optimized assay conditions, the Raman signal intensity at 1,076 cm^?1 increases logarithmically with the concentrations of PCV2 in the concentration ranging from 8?×?10² to 8?×?10⁶ copies per mL. The limit of detection is 8?×?10² copies per mL. Compared to conventional detecting methods such as those based on PCR, the method presented here is rapid, facile and very sensitive.

A SERS-based immunoassay for porcine circovirus type 2 using multi-branched gold nanoparticles

We report on a facile immunoassay for porcine circovirus type 2 (PCV2) based on surface enhanced Raman scattering (SERS) using multi-branched gold nanoparticles (mb-AuNPs) as substrates. The mb-AuNPs in the immunosensor act as Raman reporters and were prepared via Tris base-induced reduction and subsequent reaction with p-mercaptobenzoic acid (pMBA). They possess good stability and high SERS activity. Subsequently, the modified mb-AuNPs were covalently conjugated to the monoclonal antibody (McAb) against the PCV2 cap protein to form SERS immuno nanoprobes. These were captured in a microtiterplate via a immunoreaction in the presence of target antigens. The effects of antibody concentration, reaction time and temperature on the sensitivity of the immunoassay were investigated. Under optimized assay conditions, the Raman signal intensity at 1,076 cm⁻¹ increases logarithmically with the concentrations of PCV2 in the concentration ranging from 8 × 10² to 8 × 10⁶ copies per mL. The limit of detection is 8 × 10² copies per mL. Compared to conventional detecting methods such as those based on PCR, the method presented here is rapid, facile and very sensitive.

     

Highly sensitive determination of hydroxylamine using fused gold nanoparticles immobilized on sol-gel film modified gold electrode

We are reporting the highly sensitive determination of hydroxylamine (HA) using 2-mercapto-4-methyl-5-thiazoleacetic acid (TAA) capped fused spherical gold nanoparticles (AuNPs) modified Au electrode. The fused TAA-AuNPs were immobilized on (3-mercaptopropyl)-trimethoxysilane (MPTS) sol-gel film, which was pre-assembled on Au electrode. The immobilization of fused TAA-AuNPs on MPTS sol-gel film was confirmed by UV-vis absorption spectroscopy and atomic force microscopy (AFM). The AFM image showed that the AuNPs retained the fused spherical morphology after immobilized on sol-gel film. The fused TAA-AuNPs on MPTS modified Au electrode were used for the determination of HA in phosphate buffer (PB) solution (pH = 7.2). When compared to bare Au electrode, the fused AuNPs modified electrode not only shifted the oxidation potential of HA towards less positive potential but also enhanced its oxidation peak current. Further, the oxidation of HA was highly stable at fused AuNPs modified electrode. Using amperometric method, determination of 17.5 nM HA was achieved for the first time. Further, the current response of HA increases linearly while increasing its concentration from 17.5 nM to 22 mM and a detection limit was found to be 0.39 nM (S/N = 3). The present modified electrode was also successfully used for the determination of 17.5 nM HA in the presence of 200-fold excess of common interferents such as urea, NO₂⁻, NH₄⁺, oxalate, Mn²⁺, Na⁺, K⁺, Mg²⁺, Ca²⁺, Ba²⁺ and Cu²⁺. The practical application of the present modified electrode was demonstrated by measuring the concentration of HA in ground water samples.     

Sensitive amplification immunoassay for human IgG and anti-human IgG by using an enzyme cascade system

A sensitive heterogeneous immunoassay for human IgG and anti-human IgG was developed using an enzyme cascade system in limulus amoebocyte as a signal amplification system. Lipopolysaccharide (LPS) was conjugated to human IgG and anti-human IgG was adsorbed on polystyrene beads. The LPS-labelled human IgG mixed with unlabelled human IgG was allowed to react in a competitive manner with the immobilized anti-IgG on the polystyrene bead surface. After B/F separation, the LPS activity in the supernatant (free) and LPS activity on the bead (bound) were measured by using the chromogenic limulus test. IgG could be measured in the range 10^?7-10^?11 g ml^?1. LPS-labelled anti-IgG and IgG absorbed on polystyrene beads were prepared, and LPS-labeled anti-IgG mixed with unlabelled anti-IgG was allowed to react again in a competitive manner with solid-phase IgG. The LPS activity specifically bound to the bead was then measured. Anti-IgG could be measured in the range 10^?7-10^?11 g ml^?1.     

Radially oriented cellulose triacetate chains on gold nanoparticles

Cellulose triacetate (CTA) derivatives having a disulfide group at the reducing-end (CTA2S, CTA13S, CTA41S), with number average degrees of polymerization (DP_ns) of 2, 13 and 41, respectively, were prepared. The CTA-self-assembled gold nanoparticles (CTA2Au, CTA13Au, and CTA41Au) were obtained through the reduction of gold salt (HAuCl₄) with CTASs. The diameters (d) and the interparticle distances (L) of the gold cores were analyzed by transmission electron microscopy (TEM) observations. The d values of CTA2Au, CTA13Au, and CTA41Au, were 8.7, 7.9, and 13.4 nm respectively. The L values of CTA2Au, CTA13Au, and CTA41Au, were 2.8, 6.3, and 20.9 nm, respectively, and agreed well with the molecular length (l) of CTAS chains (ls of CTA2S, CTA13S, CTA41S = 2.0, 7.5, 21.5 nm, respectively). The hydrodynamic diameters (D) of CTAAu nanoparticles in chloroform solution, measured by dynamic light scattering (DLS), were larger than the d values and increased with the increase in the molecular length of the CTA chains. The CTAS chain was found to work as an excellent stabilizer of the gold nanoparticles in both solid state and solution. The molecular length of CTA chains controlled the size and the alignment of the gold nanoparticles. As a result, the radially oriented CTA chains on the gold nanoparticles were successfully prepared.     

Water-soluble low-molecular-weight cellulose chains radially oriented on gold nanoparticles

Cellulose chains bearing N-lipoyl group at the reducing-end as a sulfide linker, self-assembled on the surface of gold nanoparticles (CELL2Au, CELL13Au, and CELL41Au with the number average degrees of polymerization (DP_n) of 2, 13, and 41, respectively) were prepared. CELL2Au, CELL13Au, and CELL41Au were obtained via deprotection of the cellulose triacetate (CTA) self-assembled on the surface of gold nanoparticles that are consisting of CTA chains with corresponding DP_n organized in a radial manner with head-to-tail orientation, where a head is the reducing-end, and a tail is the non-reducing-end. CELL2Au and CELL13Au were well-dispersed in water including a trace of methanol, whereas CELL41Au was not. The transmission electron microscopy (TEM) observation of CELLAus deposited on copper grids revealed that the diameters (d) of the gold cores of CELL2Au, CELL13Au, and CELL41Au were 6.1, 6.1, and 11.5 nm, respectively. Wide angle X-ray diffractgram showed that cellulose chains of CELL13Au had quite low crystallinity and exhibited additional faint diffraction pattern of cellulose II. Cellulose chains of CELL41Au were amorphous. The UV–vis measurements revealed that CELL2Au and CELL13Au were well-dispersed in water. The hydrodynamic diameters (D) of CELL2Au and CELL13Au in water were 21.8 and 55.9 nm, respectively, according to dynamic light scattering (DLS) measurements, suggesting that cellulose chains on the gold were organized in a radial manner with head-to-tail orientation. ¹H-NMR measurement revealed that low-molecular-weight cellulose chains (DP_n = 13) on the gold dissolved in water, whereas low-molecular-weight cellulose (DP_n = 13) itself did not.     

Electrochemical enzyme immunoassay using immobilized antibody on gold film with monitoring of surface plasmon resonance signal

Sandwich immunoassay was conducted on a thin gold film set in a surface plasmon resonance (SPR) cell. Monochronal antibody (anti-IgG) was immobilized onto the gold film via 4,4′-dithiodibutyric acid (DDA) and avidin-biotin bonding. Next, IgG sample and alkaline phosphatase-conjugated anti-IgG (ALP anti-IgG) were introduced into the cell successively. Finally, p-aminophenyl phosphate (PAPP) was injected as an enzyme substrate, and the produced p-aminophenol (PAP) was electrochemically measured. Flow did not need to be stopped for incubation for the enzyme reaction, because of the thinness of the cell. In these processes, all the antigen-antibody reactions took place on the gold film. Therefore, the immobilization was performed quickly, and each process could be confirmed by SPR signal. This system had the advantage that the middle of the complicated process could be monitored. For example, the amount of antibody immobilized, which affected on the final electrochemical signal, could be confirmed in the course of immobilization. It was also convenient to investigate process conditions, such as removal of used antigens and labeled antibodies. Good correlation was obtained between the electrochemical current and the SPR signals due to the adsorption of IgG and ALP anti-IgG, and the sensitivity of the electrochemical measurement was much higher than the SPR’s.     

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.     

Multiple signal amplified electrochemiluminescent immunoassay for brombuterol detection using gold nanoparticles and polyamidoamine dendrimers-silver nanoribbon

Electrochemiluminescent (ECL) immunosensor with multiple signal amplification was designed based on gold nanoparticles (AuNPs), polyamidoamine dendrimers (PAMAM) and silver-cysteine hybrid nanoribbon (SNR). Low toxic l-cysteine capped CdSe QDs was chosen as the ECL signal probe. To verify the proposed ultrasensitive ECL immunosensor for β-adrenergic agonists (β-AA), we detected Brombuterol (Brom) as a proof-of-principle analyte. Therein, AuNPs as the substrate can simplify the experiment process, accelerate the electron transfer rate, and carry more coating antigen (Ag-OVA) to enlarge ECL signal. On one hand, SNR on the surface of electrode can avoid the aggregation of AuNPs, and SNR-PAMAM-AuNPs also can be acted as a good accelerator for electron transfer. On the other hand, PAMAM (16 -NH₂) functionalized SNR (SNR-PAMAM) with numerous amino groups could be employed to bond abundant actived QDs to further amplify ECL signal. The new immunosensor can offer a simple, reliable, rapid, and selective detection for Brom, which have a dynamic range of 0.005–700 ng mL⁻¹ with a low detection limit at 1.5 pg mL⁻¹. The proposed biosensor will extend the application of nanomaterials in ECL immunoassays and open a new road for the detection of Brom and other β-AA in the future.     

Impedimetric immunoassay for aflatoxin B1 using a cysteine modified gold electrode with covalently immobilized carbon nanotubes

The authors describe a label-free electrochemical biosensor based on CNTs for picogram range detection of the food carcinogen aflatoxin B1 (AFB1). A gold electrode was modified with a cysteine (Cys) self-assembled layer, and carboxyl-functionalized carbon nanotubes were covalently attached to Cys for the subsequent tethering of antibody against AFB1 (anti-AFB1). Topographical images of the biosensor surface were acquired by atomic force microscopy throughout the modification and assay procedure. Upon exposure to samples containing AFB1, its binding to anti-AFB1 will result in a change in electrical conductivity. The use of CNT warrants enhanced electrical properties, and the charge-transfer resistance (Rct) can be related to the concentration of AFB1. Biosensor selectivity was tested by using samples containing ochratoxin A. The electrode displays a limit of detection as low as 0.79 pg·g^?1, and response is linear in the 0.1 to 20 pg·g^?1 concentration range. The assay was applied to the determination of AFB1 in contaminated corn flour at concentrations so low that they cannot be quantified by established ELISAs. In our perception, this method represents a viable point-of-care probe for detection of AFB1.

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Graphical abstract A gold electrode was modified with a cysteine self-assembled layer, and carboxyl-functionalized carbon nanotubes were covalently attached to cysteine for the subsequent tethering of antibody against AFB1. The assay was applied to the determination of AFB1 in contaminated corn flour.

     

Solid-state NMR structural studies of peptides immobilized on gold nanoparticles

In this paper we describe solid-state NMR experiments that provide information on the structures of surface-immobilized peptides. The peptides are covalently bound to alkanethiolates that are self-assembled as monolayers on colloidal gold nanoparticles. The secondary structure of the immobilized peptides was characterized by quantifying the Ramachandran angles phi and psi. These angles were determined in turn from distances between backbone carbonyl 13C spins, measured with the double-quantum filtered dipolar recoupling with a windowless sequence experiment, and by determination of the mutual orientation of chemical shift anisotropy tensors of 13C carbonyl spins on adjacent peptide planes, obtained from the double-quantum cross-polarization magic-angle spinning spectrum. It was found that peptides composed of periodic sequences of leucines and lysines were bound along the length of the peptide sequence and displayed a tight alpha-helical secondary structure on the gold nanoparticles. These results are compared to similar studies of peptides immobilized on hydrophobic surfaces.     

Ultrasensitive electrochemiluminescent immunoassay for morphine using a gold electrode modified with CdS quantum dots, polyamidoamine, and gold nanoparticles

We report on a novel electrochemiluminescent (ECL) immunoassay for the ultrasensitive determination of morphine by making use of a gold electrode which was modified with a nanocomposite film containing self-assembled polyamidoamine (PAMAM) CdS quantum dots and electrodeposited gold nanoparticles (Au-NPs). The highly uniform and well-dispersed quantum dots were capped with PAMAM dendrimers. Due to the synergistic effect of the modified quantum dots and the electrodeposited Au-NPs, the ECL response is dramatically enhanced. Under optimal experimental conditions, the immunoreaction between morphine and anti-morphine antibody resulted in a decrease of the ECL signal because of steric hindrance. The calibration plot is linear in the morphine concentration range from 0.2 to 180 ng?mL^?1, with a detection limit as low as 67 pg?mL^?1. The sensor was successfully applied to the determination of morphine in blood plasma. This kind of assay is expected to pave new avenues in label-free drug assays.

Fluorescence polarization immunoassay using IgY antibodies for detection of valnemulin in swine tissue

Immunoglobulin Y (IgY) is derived from egg yolk and has been identified as a cheap and high-yield immunoreagent. The application of IgY in immunoassays for the detection of chemical contaminants in food samples has rarely been reported. In this work, we describe a rapid and sensitive fluorescence polarization immunoassay (FPIA) for valnemulin (VAL) using IgY which was produced using a previously prepared immunogen. Three fluorophore-labeled VAL tracers were synthesized and the sensitivity of the best tracer (VAL–DTAF) in the optimized FPIA with antibody IgY₁₀₀ demonstrated an IC₅₀ value of 12 ng mL^?1 in buffer. After evaluation of several extraction procedures, acidified acetonitrile was selected to extract VAL from swine tissue. The recoveries of VAL in spiked swine tissue at three levels (50, 100, and 200 μg kg^?1) were higher than 79 % with coefficients of variation (CVs) lower than 12 %. The limit of detection (LOD) of the FPIA in swine tissue was 26 μg kg^?1 and was lower than the maximum residue limit (MRL) of VAL set by the European Union. The study showed that IgY could be a good substitute for IgG when developing a high-throughput assay for chemical residues.

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Graphical Abstract FPIA based on IgY for rapid detection of Valnemulin in swine tissue