Sensitive sandwich immunoassay based on single particle mode inductively coupled plasma mass spectrometry detection

A sensitive sandwich type immunoassay has been proposed with the detection by inductively coupled plasma mass spectrometry (ICP-MS) in a single particle mode (time resolved analysis). The signal induced by the flash of ions (¹⁹⁷Au⁺) due to the ionization of single Au-nanoparticle (Au-NP) label in the plasma torch can be measured by the mass spectrometer. The frequency of the transient signals is proportional to the concentration of Au-NPs labels. Characteristics of the signals obtained from Au-NPs of 20, 45 and 80 nm in diameters were discussed. The analytical figures for the determination of Au-labeled IgG using ICP-MS in conventional integral mode and single particle mode were compared in detail. Rabbit-anti-human IgG was used as a model analyte in the sandwich immunoassay. A detection limit (3σ) of 0.1 ng mL⁻¹ was obtained for rabbit-anti-human IgG after immunoreactions, with a linear range of 0.3-10 ng mL⁻¹ and a RSD of 8.1% (2.0 ng mL⁻¹). Finally, the proposed method was successfully applied to spiked rabbit-anti-human IgG samples and rabbit-anti-human serum samples. The method resulted to be a highly sensitive ICP-MS based sandwich type immunoassay.     

A novel immunoassay based on the dissociation of immunocomplex and fluorescence quenching by gold nanoparticles

This study reports a novel, simple and sensitive immunoassay using fluorescence quenching caused by gold nanoparticles coated with antibody. The method is based on a non-competitive heterogeneous immunoassay of human IgG conducted by the typical procedure of sandwich immunocomplex formation. Goat anti-human IgG was first adsorbed on polystyrene microwells, and human IgG analyte was captured by the primary antibody and then sandwiched by antibody labeled with gold nanoparticles. The sandwich-type immunocomplex was subsequently dissociated by the mixed solution of sodium hydroxide and trisodium citrate, the solution obtained, which contains gold nanoparticles coated with antibody, was used to quench fluorescence. The fluorescence intensity of fluorescein at 517 nm was inversely proportional to the logarithm of the concentration of human IgG in the dynamic range of 10-5000 ng mL⁻¹ with a detection limit of 4.7 ng mL⁻¹. The electrochemical experiments and the UV-vis measurements were applied to demonstrate whether the immunoglod was dissociated completely and whether the gold nanoparticles aggregated after being dissociated, respectively. The proposed system can be extended to detect target molecules such as other kinds of antigen and DNA strands, and has broad potential applications in disease diagnosis.     

A novel lipase-based stationary phase in liquid chromatography

A streptavidin functionalized capillary immune microreactor was designed for highly efficient flow-through chemiluminescent (CL) immunoassay. The functionalized capillary could be used as both a support for highly efficient immobilization of antibody and a flow cell for flow-through immunoassay. The functionalized inner wall and the capture process were characterized using scanning electron microscopy. Compared to conventional packed tube or thin-layer cell immunoreactor, the proposed microreactor showed remarkable properties such as lower cost, simpler fabrication, better practicality and wider dynamic range for fast CL immunoassay with good reproducibility and stability. Using α-fetoprotein as model analyte, the highly efficient CL flow-through immunoassay system showed a linear range of 3 orders of magnitude from 0.5 to 200 ng mL⁻¹ and a low detection limit of 0.1 ng mL⁻¹. The capillary immune microreactor could make up the shortcoming of conventional CL immunoreactors and provided a promising alternative for highly efficient flow-injection immunoassay.     

Flow injection chemiluminescent immunoassay with para-phenylphenol and sodium tetraphenylborate as synergistic enhancers

A new approach of flow injection chemiluminescent immunoassay (FICLIA) with synergistic enhancement was developed. The synergistic action was significant in the chemiluminescence (CL) system of luminol-H₂O₂-HRP with two enhancers, para-phenylphenol (PPP) and sodium tetraphenylborate (NaTPB). The relative CL intensity was 128.84% stronger (compared to NaTPB alone) and 122.66% stronger (compared to PPP alone) using both enhancers than any one alone (both P<0.001). While the present approach was applied to the determination of rabbit IgG as a model analyte, satisfactory results were obtained in the dynamic range of 2-60 μg l⁻¹ with a detection limit of 0.68 fmol per injection and a precision of 4.7-9.3%, and the recoveries were 92.5-99.4%. The results of rabbit IgG determination accorded with those of radio immunoassay (RIA). The lifetime of the immuno-column was considerably long: one column was applied to more than 200 determinations in 1 month without significant loss of activity.     

Usefulness of gold nanoparticles as labels for the determination of gliadins by immunoaffinity chromatography with light scattering detection

A simple and fast immunoaffinity method is proposed for the determination of gliadins for the first time using gold nanoparticles (AuNPs) as labels. The tracer used consists in a gliadin-AuNP conjugate prepared by the adsorption of gliadins onto the nanoparticle surface. Two AuNP sizes with diameters of 10 nm and 20 nm were assayed to compare the behaviour of the corresponding tracer in the assay. The method relies on the injection in a commercial Protein G column of a preincubated mixture containing gliadins, polyclonal anti-gliadin antibodies, and the gliadin-AuNP tracer. This approach allows the separation of free and bound tracer fractions without any additional elution step, and the direct measurement of the resonance light scattering intensity of the free tracer through the peak height of the immunochromatogram, which is proportional to the analyte concentration. The immunocolumn can be used up to 25 times without eluting and it can be regenerated for at least 20 times. The dynamic ranges of the calibration graphs and the detection limits are 0.5-15.0 and 1.5-15.0 μg mL⁻¹ gliadins, and 0.2 μg mL⁻¹ and 0.8 μg mL⁻¹ gliadins, using 20-nm and 10-nm Au-NPs as labels, respectively. The precision, expressed as relative standard deviation, ranges between 2.7% and 2.9% using 20-nm AuNPs and 4% and 6.1% for 10-nm AuNPs. The method has been applied to the determination of the prolamin fraction in beer samples, obtaining recovery values in the range 71.2% and 101.7%.     

Silver deposition directed by self-assembled gold nanorods for amplified electrochemical immunoassay

A novel electrochemical immunoassay was developed based on the signal amplification strategy of silver deposition directed by gold nanorods (AuNRs), which was in-situ assembled on the sandwich immunocomplex. The superstructure formed by the self-assembly of AuNRs provided abundant active sites for the nucleation of silver nanoparticles. In this pathway, the stripping current of silver was greatly enhanced. Using human immunoglobulin G (HIgG) as a model analyte, the ultrasensitive immunoassay showed a wide linear range of six orders of magnitude from 0.1 fg mL⁻¹ to 100 pg mL⁻¹, with the low detection limit down to 0.08 fg mL⁻¹. The practicality of this electrochemical immunoassay for detection of HIgG in serum was validated with the average recovery of 93.9%. In addition, this enzyme-free immunoassay also has the advantages of acceptable reproducibility and specificity, and thus this immunosensing protocol can be extended to the detection of other low-abundant protein biomarkers.     

Electrochemical stripping voltammetry of gold ions for development of ultra-sensitive immunoassay for chlorsulfuron

The paper reports a highly sensitive enzyme free electrochemical immunoassay (EFEIA) for the detection of herbicide chlorsulfuron. The assay is based upon oxidative gold nanoparticle (GNP) dissolution in an acidic solution. The consequent release of large amounts of gold (Au) metal ions after dissolution of gold nanoparticles tagged to antibody leads to the development of sensitive stripping voltammetry based immunoassay. The detection is made possible by the reduction of Au^3 + ions at the screen printed electrode surface followed by metal analysis by using the square wave voltammetry technique. The sensitivity of chlorsulfuron detection by competitive assay procedure was 6.7 pg mL^− 1 for EFEIA in marked contrast to optical detection using Standard ELISA procedure that gives a sensitivity of 4.97 ng mL^− 1.     

A Sensitive Electrochemical Immunosensor for α‐Fetoprotein Detection with Colloidal Gold‐Based Dentritical Enzyme Complex Amplification

A sensitive and specific electrochemical immunosensor was developed with α‐fetoprotein (AFP) as the model analyte by using gold nanoparticle label for enzymatic catalytic amplification. A self‐assembled monolayer membrane of mercaptopropionic acid (MPA) was firstly formed on the electrode surface through gold‐sulfur interaction. Monoclonal mouse anti‐human AFP was covalently immobilized to serve as the capture antibody. In the presence of the target human AFP, gold nanoparticles coated with polyclonal rabbit anti‐human AFP were bound to the electrode via the formation of a sandwiched complex. With the introduction of goat anti‐rabbit IgG conjugated with alkaline phosphatase, the dentritical enzyme complex was formed through selective interaction of the secondary antibodies with the colloidal gold‐based primary antibody at the electrode, thus affording the possibility of signal amplification for AFP detection. Current response arising from the oxidation of enzymatic product was significantly amplified by the dentritical enzyme complex. The current signal was proportional to the concentration of AFP from 1.0 ng mL^?1 to 500 ng mL^?1 with a detection limit of 0.8 ng mL^?1. This system could be extended to detect other target molecules with the corresponding antibody pairs.     

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.     

An ultrasensitive streptavidin-functionalized carbon nanotubes platform for chemiluminescent immunoassay

An ultrasensitive chemiluminescent (CL) immunoassay system was developed for the detection of tumor marker. This sandwich CL assay method was for the first time designed based on a highly efficient streptavidin-functionalized multi-walled carbon nanotubes (MWCNTs) platform. The glass slide was firstly silylanized with 3-gycidoxypropyltrimethoxysilane (GPTMS) to generate surface epoxy group functionality. Subsequently, the MWCNTs/chitosan solution was mixed with streptavidin solution, and a certain amount of the resulting suspension was dropped on the surface of the epoxy-activated glass substrate to form a firm streptavidin-functionalized MWCNTs platform. The biofunctionalized-MWCNTs platform shows large reactive surface area and excellent biocompatibility. The capture antibody can be efficiently immobilized on the biosensing platform surface based on the highly selective recognition of streptavidin to biotinylated antibody. Using α-fetoprotein (AFP) as model analyte, the proposed method exhibits wide linear range of 0.001–0.1 ng mL⁻¹ with a low detection limit down to 0.52 pg mL⁻¹. The CL immunoassay system displays 7.9-fold increase in the detection sensitivity compared to the immunosensor without using MWCNTs. Moreover, the resulting immunosensor demonstrates excellent specificity, good reproducibility, and acceptable stability. This streptavidin-functionalized MWCNTs platform opens a novel and promising avenue for fabricating ultrasensitive CL immunoassay system.     

Flow injection chemiluminescence determination of naproxen based on KMnO4-Na2SO3 reaction in neutral aqueous medium

A simple, rapid and sensitive flow injection chemiluminescence (CL) method is described for the determination of naproxen. It was found that strong CL signal was generated when naproxen was mixed with KMnO₄ and Na₂SO₃ in neutral aqueous medium. Under the optimum experimental conditions, the CL intensity was linearly related to the concentration of naproxen from 4.0 × 10⁻⁹ to 1.0 × 10⁻⁶ g mL⁻¹ (r = 0.9993). The detection limit was 2 × 10⁻⁹ g mL⁻¹ naproxen, the relative standard deviation for 1.0 × 10⁻⁷ g mL⁻¹ naproxen solution was 1.5% (n = 11) and the sampling frequency was 120 h⁻¹. The method was applied to the determination of naproxen in pharmaceutical preparation with satisfactory results. The mechanism of CL reaction was discussed briefly.     

Picogram-detection of estradiol at an electrochemical immunosensor with a gold nanoparticle|Protein G-(LC-SPDP)-scaffold

Low picograms of the hormone 17β-estradiol were detected at an electrochemical immunosensor. This immunosensor features a gold nanoparticle|Protein G-(LC-SPDP)¹-scaffold, to which a monoclonal anti-estradiol capture antibody was immobilised to facilitate a competitive immunoassay between sample 17β-estradiol and a horseradish peroxidase-labelled 17β-estradiol conjugate. Upon constructing this molecular architecture on a disposable gold electrode in a flow cell, amperometry was conducted to monitor the reduction current of benzoquinone produced from a catalytic reaction of horseradish peroxidase. This current was then quantitatively related to 17β-estradiol present in a sample. Calibration of immunosensors in blood serum samples spiked with 17β-estradiol yielded a linear response up to ∼1200 pg mL⁻¹, a sensitivity of 0.61 μA/pg mL⁻¹ and a detection limit of 6 pg mL⁻¹. We attribute these favourable characteristics of the immunosensors to the gold nanoparticle|Protein G-(LC-SPDP) scaffold, where the gold nanoparticles provided a large electrochemically active surface area that permits immobilisation of an enhanced quantity of all components of the molecular architecture, while the Protein G-(LC-SPDP) component aided in not only reducing steric hindrance when Protein G binds to the capture antibody, but also providing an orientation-controlled immobilisation of the capture antibody. Coupled with amperometric detection in a flow system, the immunosensor exhibited excellent reproducibility.     

Development of a gold nanoparticles based chemiluminescence imaging assay and its application

In this paper, a novel gold nanoparticles based protein immobilization method was designed. Biocomposites of gold nanoparticles and proteins were successfully coated on poly(methyl methacrylate) (PMMA) plates and polystyrene microtiter plates. The proteins could be immobilized on solid materials with high density and better bioactivity. Based on above design, chemiluminescence (CL) imaging assay for determination of H₂O₂ and recombinant human interleukin-6 (rHu IL-6) was developed. The linear range and the loading capability were greatly improved when compared with imaging assay performed with direct proteins immobilization. Under the selected experimental conditions, a linear relationship was obtained between the CL intensity and the concentration of H₂O₂ in the range of 1.0 × 10⁻⁶ to 1.0 × 10⁻⁴ mol L⁻¹, and rHu IL-6 in the range of 2.0-312.0 pg mL⁻¹. The detection limits were 2 × 10⁻⁷ mol L⁻¹ (3σ) for H₂O₂ and 0.5 pg mL⁻¹ for rHu IL-6 with relative standard deviation of 3.8% for 3.0 × 10⁻⁵ mol L⁻¹ H₂O₂, and 4.4% for 39.0 pg mL⁻¹ rHu IL-6. This method has been applied to the determination of rHu IL-6 in human serum with satisfactory results.     

A label-free electrochemical immunoassay for IgG detection based on the electron transfer

In this study, a highly selective, label-free electrochemical immunoassay strategy based on the charge transport through the multilayer films associated with the electrocatalytic reduction of [Fe(CN)₆]³⁻ is proposed using human immunoglobulin G (human IgG) as the model analyte. The antibody-antigen complex formed on the sensing interface can efficiently induce change of the surface charge characteristics, the conductivity of multilayer film and/or electron transfer distance, resulting in an immunoreaction signal. The current reduction is proportional to the amount of analyte. Under the optimized experimental conditions, the proposed sensing strategy provides a linear dynamic range from 10 to 10⁴ ng mL⁻¹ and a detection limit of 3 ng mL⁻¹, indicating an improved analytical performance. This possibly makes it a potential alternative in bioanalysis of proteins and other molecules.     

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 0.0007 mIU L⁻¹ and became 0.003 mIU L⁻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 100 mIU L⁻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 2 h. Thus, we developed a novel, sensitive, quantitative and simple nanoparticle label-based TrIFA assay, suitable for routine application in hTSH screening of neonatal hypothyroidism.     

Development of an automatic multi-channel ink-jet ejection chemiluminescence system and its application to the determination of horseradish peroxidase

In this work, an automatic multi-channel ink-jet for chemiluminescence (CL) analysis was developed. The four-channel ink-jet device was controlled by a home-made circuit. Differing from the classic flow injection CL, the whole procedure for CL analysis was automatically completed on a hydrophobic glass side. CL reaction of luminal and hydrogen peroxide for the determination of horseradish peroxidase (HRP) was selected as an application to automatic CL analysis platform. All solutions delivered by different channels were precisely ejected to the same position of the glass slide for the CL analysis. The consumption of reaction solution was reduced to nanoliter level. The whole CL analysis could be completed in less than 4 min, which was benefited from the prompt solution mixing in small size of droplet. The CL intensity increased linearly with HRP concentration in the range from 0.01 to 0.5 μg mL⁻¹. The limit of detection (LOD) (S/N = 3) was 0.005 μg mL⁻¹. Finally, the automatic CL system could also be used for the detection of HRP in HRP–protein conjugates, which showed its practical application in immunoassay.     

Flow Injection Chemiluminescence for the Determination of Estriol via a Noncompetitive Enzyme Immunoassay

A novel approach to the detection of estriol using a flow injection system coupled to enhanced chemiluminescent immunoassay was developed based on noncompetitive immunoassay formats. A conjugated estriol-ovalbumin immobilized immunoaffinity column was inserted into the flow system to trap the unbound horseradish peroxidase (HRP)-labeled antibody after an off-line incubation of estriol and HRP-labeled anti-estriol antibody. The trapped enzyme conjugate was detected by the injection of chemiluminescent substrates to produce enhanced chemiluminescence. The linear range for the determination of estriol is 10.0 to 400 ng · mL⁻¹ with a correlation coefficient of 0.996 and a detection limit of 5.0 ng · mL⁻¹. The total time for sampling and chemiluminescent detection of one sample is 400 seconds after 30 min of pre-incubation. The results for pregnancy serum samples obtained by this method are in good agreement with those obtained using ELISA.     

Antibodies conjugated with new highly luminescent Eu and Tb chelates as markers for time resolved immunoassays. Application to simultaneous determination of clenbuterol and free cortisol in horse urine

Highly luminescent Eu³⁺ and Tb³⁺ complexes of 10-[4-(3-isothiocyanatopropoxy)benzoylmethyl]-1,4,7,10-tetraazacyclododecane-1,4,7 triacetic acid Eu³⁺ ⊂ 1 and Tb³⁺ ⊂ 1 were conjugated with a goat anti-rabbit IgG and a rabbit anti-mouse IgG, respectively, and applied as markers in a time resolved immunoassay for simultaneous quantitative determination of anabolic compounds clenbuterol (CL) and hydrocortisone (HC). The assay was performed in horse urine, using a monoclonal antibody specific to CL and a rabbit polyclonal antibody specific to the free HC. These lanthanide chelates are very stable and highly luminescent in aqueous solution and allowed to reach 10 μg L⁻¹ and 40 μg L⁻¹ sensitivities for CL and for HC, respectively. Application to the horse urine, that is a very complex matrix, has a considerable interest in the control of illegal use of these compounds.     

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.     

Preparation and evaluation of an immunoaffinity sorbent with Fab′ antibody fragments for the analysis of opioid peptides by on-line immunoaffinity solid-phase extraction capillary electrophoresis–mass spectrometry

An immunoaffinity (IA) sorbent with antibody fragments was prepared for the analysis of opioid peptides by on-line immunoaffinity solid-phase extraction capillary electrophoresis–mass spectrometry (IA-SPE-CE–MS). The antibody fragmentation was evaluated by MALDI-TOF-MS. Fab′ fragments obtained from a polyclonal IgG antibody against Endomorphins 1 and 2 (End1 and End2) were covalently attached to succinimidyl silica particles to prepare the IA sorbent. An IA-SPE-CE–MS methodology was established analyzing standard solutions of End1 and End2 and acceptable repeatability, linearity ranges and LODs (0.5 and 5 ng mL⁻¹, respectively) were obtained. The LOD of End1 was slightly better than that previously obtained using an IA sorbent with intact antibodies (1 ng mL⁻¹). In human plasma samples, End1 and End2 could be detected at 1 and 50 ng mL⁻¹, respectively, which meant an improvement of 100 and 2-fold with regard to the LODs using an IA sorbent with intact antibodies (100 ng mL⁻¹).