Bioluminescence immunoassay for cortisol using recombinant aequorin as a label

The analysis of hormones in saliva is a powerful tool in the assessment of a patient's endocrine function, since it allows multiple noninvasive samplings. Since salivary levels of most hormones are 10 to 50 times lower than plasma levels, accurate and highly sensitive assays are needed for saliva measurements. Herein, we describe the development of a solid-phase competitive immunoassay for cortisol in saliva, in which a mutant of the photoprotein aequorin has been used as a label. We have chemically conjugated cortisol to aequorin at different molar ratios. The various cortisol-aequorin conjugates were characterized in terms of bioluminescent activity and affinity for the anti-cortisol antibody. The conjugate that gave the best analytical performance was used for the development of the immunoassay and the analysis of cortisol in saliva samples. The conjugates were stable for at least 6 months when stored at 4 degrees C. The method fulfilled all the standard requirements of precision and accuracy. The optimized immunoassay gave a detection limit of 300 fmol/tube, corresponding to 3 nmol/L, with a linear dynamic range of 10-1000 nmol/L. Therefore, cortisol can be detected down to 0.1 ng in 100 microl of saliva sample using this assay, without any sample pretreatment. This detection limit is almost one order of magnitude lower than the physiological levels of salivary cortisol, which are reported to be 10-25 nmol/L. This allows the quantification of salivary cortisol to be performed in the linear range of the calibration curve, which is most reliable for quantification purposes.     

A simple radioimmunoassay for plasma cortisol and 11-deoxycortisol (17, 21-dihydroxy-4-pregnene-3, 20-dione).

A simple procedure for the measurement of cortisol and 11-deoxycortisol in plasma or serum is described. One-half ml. of plasma is extracted with methylene chloride. Separation of cortisol and deoxycortisol is achieved by a Sephadex LH-20 mini-column. The assay itself is achieved by the use of a commercially available cortisol kit employing rabbit anti-cortisol coated tubes. This antibody exhibits a 20% crossreactivity with 11-deoxycortisol. This procedure is extremely useful in the assessment of a pituitary-adrenal reserve in the Metyrapone test.     

The production and assessment of monoclonal antibodies to cortisol

In an extensive series of experiments, Balb/C mice and Lou rats were immunised with 3-O-(carboxymethyl)oximinocortisol conjugated to bovine serum albumin. The spleen cells from selected animals were fused with cells from mouse or rat plasmacytoma lines. Out of many hundreds of hybridomas screened, more than seventy produced antibody that bound 125I-labeled cortisol. These cultures were investigated further for stability of antibody production, affinity for cortisol and cross-reactivity with other steroids. An unexpected but consistent finding was that immunised rats produced antibody which cross-reacted with 11-deoxycortisol to a level greater than 100% and this characteristic was reproduced by rat-rat hybridomas. Strategies designed to improve the chances of generating non-cross-reactive anti-cortisol monoclonal antibodies did not appear to be successful. Nevertheless, several monoclonals were identified with properties that suggest they may be useful for the development of sensitive and specific cortisol assays.     

Covalently coupling the antibody on an amine-self-assembled gold surface to probe hyaluronan-binding protein with capacitance measurement

Hyaluronan-binding proteins (HABPs), the important structural components of extracellular matrices, served important structural and regulatory functions during development and in maintaining adult tissue homestats. A sensitive, specific and rapid-responsing immunosensor to probe hyaluronan-binding cartilage protein was presented in this work. The novel immunosensor supplied a label-free detection method for HABP, which was based on measuring the capacitance change in-between the unlabeled HABP (antigen) and rabbit-anti-HABP (Ra-HABP, antibody). The HABP immunosensor was prepared by covalently coupling Ra-HABP on an amine-self-assembled gold surface with glutaraldehyde. The capacitance change corresponding to the concentration of HABP, the target antigen, was evaluated by an electrochemical approach called potentiostatic-step in microseconds. The immunosensor showed a specific response to HABP in the range 10-1000 ng/ml. The presented work supplied a promising clinical screening method.     

A mediatorless and label-free amperometric immunosensor for detection of h-IgG

A novel experimental methodology for studying a mediatorless and label-free immunosensor is proposed by immobilizing antibody on gold nanoparticle/L-cysteine coated electrode (nano-Au/L-cysteine electrode). Differential pulse voltammograms (DPV) resulting from the assembled immunosensor indicate that the immunosensor shows excellent electrochemical response to dopamine so that the electrochemical response is utilized for the signal generation step of the immunosensor. Therefore, by means of unenzymatic-labeling procedure combined with the amperometric detection using dopamine as substrate, the immunological reaction can be detected. After the immunosensor is incubated with h-IgG solution, the access of electrocatalytic behavior center of the immunosensor to dopamine is partly inhibited, which leads to a linear decrease in amperometric response of the immunosensor with h-IgG concentration over a range 0.82-90 ng mL(-1) by DPV.     

Electrochemical DNA base pairs quantification and endonuclease cleavage detection

A label-free multiplexed immunoassay strategy was proposed for the simultaneous detection of two tumor markers, carcinoembryonic antigen (CEA) and α-fetoprotein (AFP). Monoclonal antibody of CEA was co-immobilized with ferrocenecarboxylic acid (FCA) inside the channels of mesoporous silica (MPS) to prepare the label-free probe for CEA. Also, monoclonal antibody of AFP was co-immobilized with horseradish peroxidase (HRP) inside the channels of MPS to prepare the label-free probe for AFP by using o-phenylenediamine (OPD) and H(2)O(2) as the electrochemical substrates. Thus, the multianalyte immunosensor was constructed by coating the probes of CEA and AFP respectively onto the different areas of indium-tin oxide (ITO) electrode. When the immunosensor was incubated with sample antigens, CEA and AFP antigens were introduced into the mesopores of MPS after the immunoassay reaction. Because all of the Si-OH groups on the external surface of MPS were blocked with Si(CH(3))(3), the proteins and substrates were limited to be embedded on the internal pore walls. Therefore, the electric response transfer was confined inside the pore channels. The nonconductive immunoconjugates blocked the electron transfer and the peak responses changed on the corresponding surface respectively. Then, the simultaneous detection of CEA and AFP achieved. The linear ranges of CEA and AFP were 0.5-45ngmL(-1) and 1-90ngmL(-1) with the detection limits of 0.2ngmL(-1) and 0.5ngmL(-1) (S/N=3), respectively. The fabricated immunosensor shows appropriate sensitivity and offers an alternative to the multianalyte detection of antigens or other bioactive molecules.     

A novel label-free amperometric immunosensor for carcinoembryonic antigen based on redox membrane

A label-free immunosensor was developed to detect the presence of an antigen. This immunosensor was based on the modulation of the electrochemistry of the surface bound redox species K(3)Fe(CN)(6) (FC). The model antigen was carcinoembryonic antigen (CEA) and the model epitope was the antibody of CEA (anti-CEA). Glassy carbon (GC) electrode surfaces were first drop-coated with a mixture of FC and chitosan and air-dried. The electrode surface was then covered with nafion membrane, which contained gold nanoparticles. After binding with polyethyleneimine (PEI), glutaraldehyde (GA) was used to cross-link PEI and anti-CEA. Binding of CEA to the surface bound epitope resulted in attenuation of the FC electrochemistry. Under optimal conditions, the response of the label-free immunosensor had a linear range of 0.01-150 ng mL(-1) with a detection limit of 3 pg mL(-1) (S/N = 3). Its response was better than those of radioimmunoassays, enzyme-linked immunosorbent assays, and chemiluminescence assays.     

An efficient method for the synthesis and purification of trans-[14C]geranylgeranyl pyrophosphate.

A new and highly sensitive enzyme immunoassay of cortisol was established using horseradish peroxidase as the label. Separation of free and bound cortisol was effected by insolubilized anti-cortisol antibody which was prepared by coupling the purified immunoglobulin G of antiserum with Sepharose 4B. The enzyme activity was measured by the chemiluminescence reaction using luminol and hydrogen peroxide as substrate. The faint chemiluminescence was measured by a photon counter. Comparison of assay results obtained by radioimmunoassay and this enzyme immunoassay showed excellent agreement of results in all cases (r = 0.913). The detection limit of cortisol was about 10 pg per assay tube. This enzyme immunoassay is applicable to the routine determination of plasma cortisol.     

The Relationship between Dehydroepiandrosterone (DHEA), Working Memory and Distraction – A Behavioral and Electrophysiological Approach

Dehydroepiandrosterone (DHEA) and dehydroepiandrosterone-sulphate (DHEAS) have been reported to have memory enhancement effects in humans. A neuro-stimulatory action and an anti-cortisol mechanism of action may contribute to that relation. In order to study DHEA, DHEAS and cortisol relations to working memory and distraction, we recorded the electroencephalogram of 23 young women performing a discrimination (no working memory load) or 1-back (working memory load) task in an audio-visual oddball paradigm. We measured salivary DHEA, DHEAS and cortisol both before each task and at 30 and 60 min. Under working memory load, a higher baseline cortisol/DHEA ratio was related to higher distraction as indexed by an enhanced novelty P3. This suggests that cortisol may lead to increased distraction whereas DHEA may hinder distraction by leading to less processing of the distractor. An increased DHEA production with consecutive cognitive tasks was found and higher DHEA responses attributed to working memory load were related to enhanced working memory processing as indexed by an enhanced visual P300. Overall, the results suggest that in women DHEA may oppose cortisol effects reducing distraction and that a higher DHEA response may enhance working memory at the electrophysiological level.     

The fabrication of a label-free electrochemical immunosensor using Nafion/carbon nanotubes/charged pyridinecarboxaldehyde composite film

A label-free electrochemical immunosensor based on Nafion/carbon nanotubes (CNTs)/charged pyridinecarboxaldehyde composite film was developed for the detection of hepatitis B surface antigen. Nafion/CNTs/charged pyridinecarboxaldehyde nanocomposites were prepared by dispersing charged pyridinecarboxaldehyde and CNTs in Nafion solution. The nanocomposites were cast on the electrode surface to form aldehyde-terminated composite film that can covalently bind antibody on the film without using other reagent. The immunosensor response was linearly changed with hepatitis B surface antigen concentration in the range from 0.1 to 25 ng ml⁻¹ with a detection limit (signal/noise ratio = 3) of 0.04 ng ml⁻¹. Some important advantages such as simple preparation, good stability, reproducibility, and selectivity of the immunosensor were achieved.     

Determination of bradykinin in rat urine by coupled-column high pressure liquid chromatography with precolumn derivatization with a water-soluble fluorogenic reagent

The green fluorescent protein (GFP) and its mutants have been extensively used to study various cellular processes and, more recently, as labels in binding assays. We have employed a mutant of GFP, an enhanced GFP (EGFP), in the development of homogeneous assays for biotin and cortisol. To demonstrate the feasibility of using EGFP as a label with different kinds of binders in the development of homogeneous assays, we employed the biotin-avidin and an antigen-antibody as the binding pairs. Biotin and cortisol were chemically conjugated to EGFP. A quenching of fluorescence intensity of EGFP was observed upon binding of avidin to the EGFP-biotin conjugate. The percentage fluorescence quenching observed decreased as the concentration of free biotin in the sample increased due to the fewer binding sites on avidin available for binding to the EGFP-biotin conjugate. A dose-response curve for biotin was generated by relating percentage fluorescence quenched with free biotin in the sample. To determine whether EGFP can undergo a similar type of homogeneous change when used as a label for antigen-antibody type of binding, cortisol was selected as a model analyte. In the presence of an anti-cortisol antibody the fluorescence signal of the EGFP-cortisol conjugate was quenched. A dose-response curve for cortisol was generated by relating the quenching in the fluorescence signal with varying amounts of free cortisol in the sample. This is the first time that GFP or one of its mutants has been employed as a label in homogeneous assays, thus enhancing the versatility of employing GFP or its mutants in a number of bioanalytical applications, such as clinical analysis and high-throughput screening systems.     

Biomolecules/gold nanowires-doped sol-gel film for label-free electrochemical immunoassay of testosterone

A direct, rapid, and label-free electrochemical immunoassay method for testosterone has been described based on encapsulating testosterone antibody into polyvinyl butyral sol–gel film doped with gold nanowires. Gold nanowires prepared by using nanopore polycarbonate membrane were used to conjugate testosterone antibody onto the probe surface. The presence of gold nanowires provided a biocompatible microenvironment for biomolecules, greatly amplified the immobilized amount of biomolecules on the electrode surface, and improved the sensitivity of the immunosensor. In comparison with gold nanoparticle-conjugating probe, the gold nanowire-functionalized probe could avoid the leakage of biomolecules from the composite film, and enhanced the stability of the sensor. The performance and factors influencing the performance of the resulting immunosensor were investigated in detail. Under optimal conditions, the developed immunosensor exhibited a good linear relationship with testosterone ranging from 1.2 to 83.5 ng mL^− 1 with a detection limit of 0.1 ng mL^− 1 (at 3δ). Moreover, the proposed immunosensor exhibited high sensitivity, good reproducibility and long-term stability. The as-prepared immunosensors were used to analyze testosterone in human serum specimens. Analytical results suggest that the developed immunoassay has a promising alternative approach for detecting testosterone in the clinical diagnosis. Compared with the conventional ELISAs, the proposed immunoassay method was simple and rapid without multiple labeling and separation steps. Importantly, the route provides an alternative approach to incorporate gold nanowires into the solid matrix for biosensing application.     

Novel electrochemical catalysis as signal amplified strategy for label-free detection of neuron-specific enolase

A label-free electrochemical immunoassay for neuron-specific enolase (NSE), a kind of lung cancer marker, was developed in this work via novel electrochemical catalysis for signal amplification. The new amplified strategy was based on the electrochemical catalysis of nickel hexacyanoferrates nanoparticles (NiHCFNPs) in the presence of dopamine (DA). NiHCFNPs, which were assembled on the porous gold nanocrystals (AuNCs) modified glassy carbon electrode (GCE), could exhibit a distinct pair of redox peaks corresponding to anodic and cathodic reactions of hexacyanoferrate (II/III). Subsequently, gold nanoparticles functionalized graphene nanosheets (Au-Gra) were coated on the surface of NiHCFNPs/AuNCs film. Then an enhanced amount of neuron-specific enolase antibody (anti-NSE) could be loaded to obtain a sensitive immunosensor of anti-NSE/Au-Gra/NiHCFNPs/AuNCs/GCE due to the strong adsorption capacity and large specific surface area of Au-Gra. More importantly, the oxidation peak current can be enormously enhanced towards the electrocatalytic oxidation of DA based on NiHCFNPs, resulting in the further improvement of the immunosensor sensitivity. Under optimal conditions, the electrochemical immunosensor exhibited a linear range of 0.001-100 ng/mL with a detection limit of 0.3 pg/mL (S/N=3). Thus, the proposed immunosensor provides a rapid, simple, and sensitive immunoassay protocol for NSE detection, which may hold a promise for clinical diagnosis.     

Label-free immunosensor for prostate-specific antigen based on single-walled carbon nanotube array-modified microelectrodes

We have fabricated a label-free electrochemical immunosensor using microelectrode arrays modified with single-walled carbon nanotubes (SWNTs). Label-free detection of a cancer marker, total prostate-specific antigen (T-PSA), was carried out using differential pulse voltammetry (DPV). The current signals, derived from the oxidation of tyrosine (Tyr), and tryptophan (Trp) residues, increased with the interaction between T-PSA on T-PSA-mAb covalently immobilized on SWNTs. The selectivity of our biosensor was challenged using bovine serum albumin (BSA) as the target protein. The detection limit for T-PSA was determined as 0.25 ng/mL. Since the cut-off limit of T-PSA between prostate hyperplasia and cancer is 4 ng/mL, the performance of our label-free electrochemical immunosensor seems promising for further clinical applications.     

Methods of collection for salivary cortisol measurement in dogs

Salivary cortisol has been increasingly used as a measure of stress response in studies of welfare, reaction to stress and human-animal interactions in dogs and other species. While it can be a very useful measure, there are a number of saliva collection issues made evident through studies in the human and animal fields which have not been investigated in the canine species. Collection materials and the volume of saliva that is collected; the use of salivary stimulants; and the effect of food contamination can all dramatically impact cortisol measurement, leading to spurious results. In order to further examine the limitations of the collection method and the effects of collection material and salivary stimulant on salivary cortisol levels, a series of clinical, in vitro and in vivo studies were performed. It was found that there is a large amount of inter- and intra-individual variation in salivary cortisol measurement. Beef flavoring of collection materials leads to unpredictable variability in salivary cortisol concentration. Using salivary stimulants such as citric acid also has the potential to affect cortisol concentration measurement in saliva. Hydrocellulose appears to be a useful collection material for salivary cortisol determination. Recommendations for collection materials and use of salivary stimulants are presented.     

A label-free immunosensor by controlled fabrication of monoclonal antibodies and gold nanoparticles inside the mesopores

A label-free immunosensor for the detection of α-fetoprotein (AFP) is proposed based on controlled fabrication of monoclonal antibodies of AFP (anti-AFP) and gold nanoparticles (GNPs) inside the pores of mesoporous silica (MPS). The silanol groups on the internal pore walls were grafted by aminopropyltriethoxyl silane, whereas the silanol groups on the external surface of MPS were blocked by trimethylchlorosilane (TMCS). Thus, anti-AFP and GNPs could be confined inside the mesopores of TMCS-MPS by the covalent linking with the amino groups. The prepared anti-AFP/GNPs/TMCS-MPS particles were used to modify glassy carbon electrode (GCE) to construct a label-free immunosensor. After incubating the sample AFP with the anti-AFP/GNPs/TMCS-MPS/GCE, the immunoconjugates were formed on the surface of GCE and the spatial block increased. Thus, the peak current decreased with increasing concentrations of AFP. GNPs inside the mesopores could promote the electron transportation through the pore channel. Under the optimal experimental conditions, the fabricated immunosensor could detect AFP in a linear range from 1.0 to 90 ng ml(-1) with a detection limit of 0.2 ng ml(-1) (3σ). It provided a novel alternative method for the label-free determination of other antigens.     

Nanoporous gold film based immunosensor for label-free detection of cancer biomarker

Nanoporous gold (NPG) film modified electrode for the construction of novel label-free electrochemical immunosensor for ultrasensitive detection of cancer biomarker prostate specific antigen (PSA) is described. Due to its high conductivity, large surface area, and good biocompatibility, NPG film modified electrode was used for the adsorption of anti-PSA antibody (Ab). The sensing signal is based on the monitoring of the electrode's current response towards K(3)Fe(CN)(6), which is extremely sensitive to the formation of immunocomplex within the nanoporous film. Under optimum conditions, the amperometric signal decreases linearly with PSA concentration (0.05-26 ng/mL), resulting in a low limit of detection (3 pg/mL). We demonstrated the application of the novel immunosensor for the detection of PSA in real sample with satisfactory results.     

Electrochemical immunoassay for carcinoembryonic antigen based on signal amplification strategy of nanotubular mesoporous PdCu alloy

Interests in using nanoporous metals for biosensing applications have been increasing. Herein, nanotubular mesoporous PdCu (NM-PdCu) alloy is used to fabricate a novel label-free electrochemical immunosensor for cancer biomarker carcinoembryonic antigen (CEA). It operates through physisorption of anti-CEA on NM-PdCu and the mixture of sulfonated graphene sheets (HSO(3)-GS) and thionine (TH) functionalized glassy carbon electrode interface as the detection platform. In this study, chitosan (CS)-PdCu is bound very strongly to carcinoembryonic antibody (anti-CEA), because of the good electron conductivity, high surface area, and good biocompatibility. CS-PdCu is immobilized on electrodes by electrostatic interactions between the negatively charged sulfo group of HSO(3)-GS and the abundant positively charged amino groups of chitosan. TH acts as the redox probe. Under the optimized conditions, the electrochemical immunosensor exhibits a wide working range from 0.01 to 12 ng/mL with a low detection limit of 4.86 pg/mL. The accuracy, reproducibility, and stability of the immunosensor are acceptable. The assay is evaluated for real serum samples, receiving satisfactory results. The nanoporous metal materials-based immunoassay provides a promising approach in clinical application and thus represents a versatile detection method.     

Detection of human chorionic gonadotrophin hormone using a label-free epoxysilane-modified capacitive immunosensor

A new and label-free capacitive immunosensor based on antibody-functionalized epoxysilane on a glassy carbon electrode has been developed for quantitative detection of human chorionic gonadotropin (hCG). Monitoring the changes in the capacitance signals of antibodies before and after the binding of the antigen provides the basis for an immunoassay. The performance and factors influencing the immunosensor were also studied. Under the optimized conditions, the developed immunosensor quantitatively detected serum hCG in the range of 18–450 mIU/ml with a detection limit of 5.0 mIU/ml (at 3δ). Thirty-five patients’ sera were assayed by the proposed immunosensor, and the results agreed with those given by the commercial radioimmunoassay test kit, with correlation coefficient of 0.998. Further research about the intrinsic electroactivity of antibodies and their target molecules would surely provide new and sensitive screening assays as well as extensive data regarding their interaction mechanisms.     

Label-free electrochemical immunosensor based on Nile blue A-reduced graphene oxide nanocomposites for carcinoembryonic antigen detection

In this article, a novel, label-free, and inherent electroactive redox immunosensor for carcinoembryonic antigen (CEA) based on gold nanoparticles (AuNPs) and Nile blue A (NB) hybridized electrochemically reduced graphene oxide (NB–ERGO) is proposed. The composite of NB–graphene oxide (NB–GO) was prepared by π–π stacking interaction. Then, chronoamperometry was adopted to simultaneously reduce HAuCl₄ and nanocomposites of NB–GO for synthesizing AuNPs/NB–ERGO. The immunosensor was fabricated by capturing CEA antibody (anti-CEA) at this nanocomposite modified electrode. The immunosensor determination was based on the fact that, due to the formation of antigen–antibody immunocomplex, the decreased response currents of NB were directly proportional to the concentrations of CEA. Under optimal conditions, the linear range of the proposed immunosensor was estimated to be from 0.001 to 40 ng ml⁻¹ and the detection limit was estimated to be 0.00045 ng ml⁻¹. The proposed immunosensor was used to determine CEA in clinical serum samples with satisfactory results. The proposed method may provide promising potential application in clinical immunoassays with the properties of facile procedure, stability, high sensitivity, and selectivity.