New highly sensitive enzyme immunoassay for the determination of pravastatin in human plasma

New highly sensitive enzyme immunoassay (EIA) has been developed and validated for the determination of pravastatin (PRV) in human plasma samples. PRV was coupled to keyhole limpt hemocyanin (KLH) and bovine serum albumin (BSA) via its terminal carboxylic acid group by carbodiimide reagent. PRV-KLH conjugate was used as an immunogen for raising anti-PRV polyclonal antibody in rabbits. The generated anti-PRV antibody recognized PRV with high affinity and selectivity. PRV-BSA conjugate was immobilized onto microwell plates and used as a solid phase. The assay involved a competitive binding reaction between PRV, in plasma sample, and the immobilized PRV-BSA for the binding sites on a limited amount of the anti-PRV antibody. The anti-PRV antibody bound to the plate wells was quantified with horseradish peroxidase-labeled anti-immunoglobulin second anti-rabbit IgG antibody and 3,3′,5,5′-tetramethylbenzidine as a substrate for the peroxidase enzyme. The concentration of PRV in the sample was quantified by its ability to inhibit the binding of the anti-PRV antibody to the immobilized PRV-BSA and subsequently the color development in the assay wells. The conditions of the proposed EIA were investigated and the optimum conditions were employed in the determination of PRV in plasma samples. The assay limit of detection was 0.2 ng mL⁻¹ and the effective working range at relative standard deviation (RSD) of ≤5% was 0.5-20 ng mL⁻¹. The mean analytical recovery of PRV from spiked plasma was 100.9 ± 2.98%. The precision of the assay was satisfactory; RSD was 2.61-3.70 and 3.96-4.17% for intra- and inter-assay precision, respectively. The analytical procedure is convenient, and one can analyze ∼200 samples per working day, facilitating the processing of large-number batch of samples. The proposed EIA has a great value in the routine analysis of PRV in plasma samples for its therapeutic monitoring and pharmacokinetic studies.     

A dendrimer-based immunosensor for improved capture and detection of tumor necrosis factor-α cytokine

A dendrimer-based sandwich type enzyme-linked immunosorbent assay (ELISA) was developed for the improved detection of recombinant human tumor necrosis factor-alpha (TNF-α) for early diagnosis of perinatal diseases. Hydroxyl-terminated generation four poly(amidoamine) dendrimer (G4-OH) was used for the development of a solid phase bio-sensing platform. The surface of the ELISA plate was modified with polyethylene-glycol (PEG) and thiol-functionalized G4-OH was immobilized on the PEG-functionalized plate. A capture antibody was oxidized and covalently immobilized onto the dendrimer-modified ELISA plate, which provides favorable orientation for the antigen binding sites toward the analyte. The dendrimer-modified plate showed enhanced sensitivity, and the detection limit for TNF-α was found to be 0.48 pg mL⁻¹, which is significantly better than the commercially available ELISA kit. The selectivity of the dendrimer-modified ELISA plate was further evaluated with a mixture of cytokines, which showed results for similar to that of TNF-α alone. The modified plate provides a greater opportunity for the detection of a wide range of cytokines and biomarkers.     

Improved porous silicon microarray based prostate specific antigen immunoassay by optimized surface density of the capture antibody

Enriching the surface density of immobilized capture antibodies enhances the detection signal of antibody sandwich microarrays. In this study, we improved the detection sensitivity of our previously developed P-Si (porous silicon) antibody microarray by optimizing concentrations of the capturing antibody. We investigated immunoassays using a P-Si microarray at three different capture antibody (PSA – prostate specific antigen) concentrations, analyzing the influence of the antibody density on the assay detection sensitivity. The LOD (limit of detection) for PSA was 2.5 ng mL⁻¹, 80 pg mL⁻¹, and 800 fg mL⁻¹ when arraying the PSA antibody, H117 at the concentration 15 μg mL⁻¹, 35 μg mL⁻¹, and 154 μg mL⁻¹, respectively. We further investigated PSA spiked into human female serum in the range of 800 fg mL⁻¹ to 500 ng mL⁻¹. The microarray showed a LOD of 800 fg mL⁻¹ and a dynamic range of 800 fg mL⁻¹ to 80 ng mL⁻¹ in serum spiked samples.     

A highly sensitive and specific polyclonal antibody-based enzyme immunoassay for therapeutic monitoring and pharmacokinetic studies of atorvastatin

This study describes the development and validation of a highly sensitive and specific enzyme immunoassay (EIA) for therapeutic monitoring and pharmacokinetic studies of atorvastatin (ATR). The assay employs a polyclonal antibody that recognizes ATR with high specificity and affinity, and ATR conjugated to bovine serum albumin (ATR-BSA) immobilized onto microwell plates as a solid phase. The assay involved a competitive binding reaction between ATR and the immobilized ATR-BSA for the binding sites on a limiting amount of the anti-ATR antibody. The bound anti-ATR antibody was quantified with horseradish peroxidase-labeled anti-immunoglobulin secondary antibody and 3,3′,5,5′-tetramethylbenzidine as a substrate for the peroxidase enzyme. The concentration of ATR in the sample was quantified by its ability to inhibit the binding of the anti-ATR antibody to the immobilized ATR-BSA and subsequent color development in the assay wells. The conditions for the EIA were investigated and optimized for the determination of ATR in plasma samples. The limit of detection was 0.04 ng mL^?1 and the effective working range at relative standard deviations (RSD) of ≤5% was 0.1–10 ng mL^?1. Mean analytical recovery of ATR from spiked plasma was 99.3?±?2.8%. The precision of the assay was satisfactory; RSD were 2.7–4.6 and 3.3–5.7% for intra- and inter-assay precision, respectively. The reliability of the EIA was confirmed by HPLC. The EIA is convenient, and one can analyze ～ 200 samples per working day, facilitating the processing of large-number of samples of ATR.     

Ultrasensitive one-step rapid detection of ochratoxin A by the folding-based electrochemical aptasensor

A one-step electrochemical aptasensor using the thiol- and methylene blue- (MB-) dual-labeled aptamer modified gold electrode for determination of ochratoxin A (OTA) was presented in this research. The aptamer against OTA was covalently immobilized on the surface of the electrode by the self-assembly effect and used as recognition probes for OTA detection by the binding induced folding of the aptamer. Under the optimal conditions, the developed electrochemical aptasensor demonstrated a wide linear range from 0.1 pg mL⁻¹ to 1000 pg mL⁻¹ with the limit of detection (LOD) of 0.095 pg mL⁻¹, which was an extraordinary sensitivity compared with other common methods for OTA detection. Moreover, as a practical application, this proposed electrochemical aptasensor was used to monitor the OTA level in red wine samples without any special pretreatment and with satisfactory results obtained. Study results showed that this electrochemical aptasensor could be a potential useful platform for on-site OTA measurement in real complex samples.     

Development of an immunosensor for the detection of testosterone in bovine urine

In the presented work, a disposable immunosensor for the detection of testosterone, an endogenous steroid hormone, in bovine urine has been developed using screen-printed electrodes (SPEs). Due to concerns over the use of steroid hormones as growth promoters, the EU prohibits their use in food producing animals. Consequently, rigorous screening procedures have been implemented in all member states to detect the illegal administration of such compounds. Competitive immunoassays were developed, initially by enzyme linked immunosorbent assay (ELISA), and subsequently transferred to an electrochemical immunosensor format using disposable screen-printed carbon electrodes. Horseradish peroxidase (HRP) was the enzyme label of choice and chronoamperometric detection was carried out using a tetramethylbenzidine/hydrogen peroxide (TMB/H₂O₂) substrate system, at +100 mV. The EC₅₀ values obtained for the assay in buffer and urine gave relatively comparable results, 710 pg mL⁻¹ and 960 pg mL⁻¹, respectively. The linear range obtained for the assay in buffer extended from 0.03 ng mL⁻¹ to 40 ng mL⁻¹; while that in urine ranged from 0.03 ng mL⁻¹ to 1.6 ng mL⁻¹. The corresponding limits of detection (LOD) in buffer and urine were 26 pg mL⁻¹ and 1.8 pg mL⁻¹. Cross reactivity profiles of the antibody have been examined, with notable cross reactivities with 19-nortestosterone (11.6%) and boldenone (9.86%). Precision studies for the sensor demonstrated adequate reproducibility (CV < 13%, n = 3) and repeatability (CV < 9%, n = 3). Recovery data obtained showed good agreement between spiking studies and known concentrations of analyte. Sensors showed stability for 4 days at +4 °C. A sensitive, highly specific, inexpensive, disposable immunosensor, showing excellent overall performance for the detection of testosterone in bovine urine, has been developed.     

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.     

Development and validation of a sensitive enzyme immunoassay for surveillance of Cry1Ab toxin in bovine blood plasma of cows fed Bt-maize (MON810)

The increasing global adoption of genetically modified (GM) plant derivatives in animal feed has provoked a strong demand for an appropriate detection method to evaluate the existence of transgenic protein in animal tissues and animal by-products derived from GM plant fed animals. A highly specific and sensitive sandwich enzyme immunoassay for the surveillance of transgenic Cry1Ab protein from Bt-maize in the blood plasma of cows fed on Bt-maize was developed and validated according to the criteria of EU-Decision 2002/657/EC. The sandwich assay is based on immuno-affinity purified polyclonal antibody raised against Cry1Ab protein in rabbits. Native and biotinylated forms of this antibody served as capture antibody and detection antibody for the ELISA, respectively. Streptavidin-horseradish peroxidase conjugate and TMB substrate provided the means for enzymatic colour development.The immunoassay allowed Cry1Ab protein determination in bovine blood plasma in an analytical range of 0.4-100 ng mL⁻¹ with a decision limit (CCα) of 1.5 ng mL⁻¹ and detection capability (CCβ) of 2.3 ng mL⁻¹. Recoveries ranged from 89 to 106% (mean value of 98%) in spiked plasma.In total, 20 plasma samples from cows (n = 7) fed non-transgenic maize and 24 samples from cows (n = 8) fed transgenic maize (collected before and, after 1 and 2 months of feeding) were investigated for the presence of the Cry1Ab protein. There was no difference amongst both groups (all the samples were below 1.5 ng mL⁻¹; CCα). No plasma sample was positive for the presence of the Cry1Ab protein at CCα and CCβ of the assay.     

Determination of oxytocin in milk of cows administered oxytocin

To address people's concerns of exogenous oxytocin (OT) administration to lactating bovines, a study was undertaken to (a) establish an enzyme immunoassay (EIA) for OT determination in milk, (b) quantify OT in milk of cows administered OT, and (c) study influence of pasteurization on OT stability in milk. A sensitive EIA validated according to the criteria of European Union—Decision 2002/657/EC was developed for OT in skim milk in an analytical range of 10-250 pg mL⁻¹ with a decision limit (CCα) of 30 pg mL⁻¹ and detection capability (CCβ) of 41.5 pg mL⁻¹. Milk samples collected from cows (n = 38) administered either 25 or 50 IU OT prior to milking were investigated for the presence of OT. There was no significant difference among both groups with the mean concentrations of OT being 15.8 and 14.9 pg mL⁻¹ for cows subjected to 25 and 50 IU OT administration, respectively. The OT levels in skim milk of control cows (n = 30; untreated) were basal (around 10 pg mL⁻¹). All the analyzed milk samples were below the CCα value of 30 pg mL⁻¹. Pasteurization of OT spiked milk samples at different temperature and sample holding conditions reduced the immunological activity of OT to 43% at 110 °C. However, no further decline occurred in the immunological activity with increased pasteurization temperature and time. It was concluded that the milk OT concentrations after OT administrations were minimal and below the assay decision limit. However, OT was quite stable to pasteurization in OT spiked milk.     

Determination of perfluorocarboxylic acids in water by ion-pair dispersive liquid–liquid microextraction and gas chromatography–tandem mass spectrometry with injection port derivatization

A novel technique for derivatization in a gas chromatograph injection port after a one-step extraction of trace perfluorocarboxylic acids (PFCAs) in water with ion pair formation during dispersive liquid–liquid microextraction (DLLME) was investigated. Tetrabutylammonium hydrogen sulfate (TBAHS) was used as the ion pair reagent. PFCA butyl ester derivatives were formed in the GC injection port and then analyzed using gas chromatography coupled to tandem mass spectrometry with negative chemical ionization. According to our analysis, the operative linear range for PFCA detection from 250 pg mL⁻¹ to 2 μg mL⁻¹ with a relative standard derivation (RSD) below 13%. Detection limits were achieved at the level of 37–51 pg mL⁻¹. This method was successfully applied for the analyzing of PFCAs in river water samples from urban and industrial areas without tedious pretreatment. The concentration range over which PFCAs were detected is from 0.6 ng mL⁻¹ to 604.9 ng mL⁻¹.     

Simple approach based on ultrasound-assisted emulsification-microextraction for determination of polibrominated flame retardants in water samples by gas chromatography–mass spectrometry

A simple, efficient, innovative and environmentally friendly analytical technique was successfully applied for the first time for the extraction and preconcentration of polybrominated diphenyl ethers (PBDEs) from water samples. The PBDEs selected for this work were those most commonly found in the literature in natural water samples: 2,2′,4,4′-tetraBDE (BDE-47), 2,2′,4,4,5-pentaBDE (BDE-99), 2,2′,4,4,6-pentaBDE (BDE-100) and 2,2,4,4′,5,5′-hexaBDE (BDE-153). The extracted PBDEs were separated and determined by gas chromatography–mass spectrometry (GC–MS). The extraction/preconcentration technique is based on ultrasound-assisted emulsification-microextraction (USAEME) of a water-immiscible solvent in an aqueous medium. Several variables including, solvent type, extraction time, extraction temperature and matrix modifiers were studied and optimized over the relative response the target analytes. Chloroform was used as extraction solvent in the USAEME technique. Under optimum conditions, the target analytes were quantitatively extracted achieving enrichment factors (EF) higher than 319. The detection limits (LODs) of the analytes for the preconcentration of 10 mL sample volume were within the range 1–2 pg mL⁻¹. The relative standard deviations (RSD) for five replicates at 10 pg mL⁻¹ concentration level were <10.3%. The calibration graphs were linear within the concentration range of 5–5000 pg mL⁻¹ for BDE-47 and BDE-100; and 5–10,000 pg mL⁻¹ for BDE-99 and BDE-153, respectively. The coefficients of estimation were ≥0.9985. Validation of the methodology was performed by standard addition method at two concentration levels (10 and 50 pg mL⁻¹). Recovery values were ≥96%, which showed a successful robustness of the analytical methodology for determination of picogram per milliliter of PBDEs in water samples. Significant quantities of PBDEs were not found in the analyzed samples.     

A streptavidin functionalized graphene oxide/Au nanoparticles composite for the construction of sensitive chemiluminescent immunosensor

In this work, a novel streptavidin functionalized graphene oxide/Au nanoparticles (streptavidin/GO/AuNPs) composite is prepared and for the first time used to construct sensitive chemiluminescent immunosensor for the detection of tumor marker. The streptavidin/GO/AuNPs composite and the immunosensor are characterized using scanning electron microscopy, static water contact angle measurement and electrochemical impedance spectroscopy. The biofunctionalized composite has large reactive surface area and excellent biocompatibility, thus the capture antibody can be efficiently immobilized on its surface based on the highly selective recognition of streptavidin to biotinylated antibody. Using α-fetoprotein (AFP) as a model, the proposed chemiluminescent immunosensor shows a wide linear range from 0.001 to 0.1 ng mL⁻¹ with an extremely low detection limit down to 0.61 pg mL⁻¹. The resulting AFP immunosensor shows high detection sensitivity, fast assay speed, acceptable detection and fabrication reproducibility, good specificity and stability. The assay results of serum samples with the proposed method are in an acceptable agreement with the reference values. This work provides a promising biofunctionalized nanostructure for sensitive biosensing applications.     

A highly sensitive enzyme immunoassay for evaluation of 2′-deoxycytidine plasma level as a prognostic marker for breast cancer chemotherapy

A highly sensitive competitive enzyme immunoassay (EIA) has been developed and validated for the determination of the plasma level of 2′-deoxycytidine (dCyd), the potential prognostic marker for breast cancer chemotherapy. This assay employed a monoclonal antibody that recognizes dCyd with a high specificity, and 5′-succinyl-dCyd (5′sdCyd) conjugate of bovine serum albumin (5′sdCyd-BSA) immobilized onto microplate wells as a solid phase. The assay involved a competitive binding reaction between dCyd, in plasma sample, and the immobilized 5′sdCyd-BSA for the binding sites of the anti-dCyd antibody. The bound antibody was quantified with horseradish peroxidase-labeled anti-immunoglobulin second antibody and 3,3′,5,5′-tetramethylbenzidine as a peroxidase substrate. The concentration of dCyd in the sample was quantified by its ability to inhibit the binding of the antibody to the immobilized 5′sdCyd-BSA and subsequently the color formation in the assay. The assay limit of detection was 8 nM and the effective working range at relative standard deviations (R.S.D.s) of ≤10% was 20-800 nM. No cross-reactivity from the structurally related nucleobases, nucleosides, and nucleotides was observed in the proposed assay. Mean analytical recovery of added dCyd was 98-100 ± 3.2-8.2%. The precision of the assay was satisfactory; R.S.D. was 3.4-4.2 and 4.3-8.9% for intra- and inter-assay precision, respectively. The proposed EIA was compared favorably with HPLC method in its ability to accurately measure dCyd spiked into plasma samples. The analytical procedure is convenient, and one can analyze 200 samples per working day, facilitating the processing of large-number batch of samples. The proposed EIA is expected to contribute in further evaluation of dCyd as a prognostic marker for breast cancer chemotherapy and elucidation of the role of dCyd in various biological and biochemical systems.     

Fast,selective, and sensitive analysis of low-abundance peptides in human plasma by electromembrane extraction

A totally new concept based on electrokinetic migration was evaluated for the extraction of three biologically active peptides from human plasma. Angiotensin 2, leu-enkephalin, and endomorphin 1 migrated from a diluted human plasma sample (2 mL, positive electrode), through a supported liquid membrane (SLM) of 1-octanol, di-isobutylketon, and di-(2-ethylhexyl) phosphate (DEHP) (55:35:10, w/w/w), and into an acidified acceptor solution (25 μL 50 mM HCl, negative electrode) by the application of an electrical potential (20 V) across the SLM. After only five min of extraction, the acceptor solution was injected and analyzed directly by liquid chromatography. The three peptides were quantified by tandem mass spectrometry, with acceptable linearity ranging from 100.0 to 1000.0 pg mL⁻¹ (r² in the range 0.9736–0.9988), and repeatability (RSD) ranging between 15% and 24% (n = 5), using plasma spiked with the three peptides in 100 pg mL⁻¹ concentration. The estimated detection limits (S/N ratio of 3:1) for angiotensin 2, leu-enkephalin, and endomorphin 1, were 60, 24, and 24 pg mL⁻¹, respectively. With this novel approach based on electromembrane extraction (EME) coupled to LC–MS/MS, endogenous concentrations of the peptides were detected in non-spiked human plasma samples, with a total analysis time less than 50 min. These experimental findings were highly interesting, and showed the opportunities for EME with regard to future peptide extractions.     

A rapid and highly sensitive portable chemiluminescent immunosensor of carcinoembryonic antigen based on immunomagnetic separation in human serum

To detect a biomarker for lung cancer, carcinoembryonic antigen (CEA), a highly sensitive, selective, rapid and portable immunosensor based on immunomagnetic separation and chemiluminescence immunoassay was introduced. A sandwich scheme assay has been utilized with horseradish peroxidase (HRP) labeled anti-CEA antibody and immunomagnetic beads (IMBs). The presence of target protein CEA caused the formation of the sandwich structures (IMBs-CEA-HRP labeled antibody). IMBs were applied to capture CEA and immobilize CEA through the external magnetic field. The HRP at the surface of the antibody catalytically oxidized the luminescence substrate to generate optical signals which were detected by a portable home-made luminometer and which were directly proportional to the concentration of CEA in the samples. The signals were dependent on CEA concentrations in a linear range from 0 to 50 ng mL⁻¹. The limit of detection (LOD) of this method was as low as 5.0 pg mL⁻¹ (S/N = 3). The novel immunosensor was highly sensitive with an assay time of <35 min. The intra- and inter-assay coefficients of variation were <10%. The anti-CEA antibody can be bound to the bead efficiently with a conjugation rate of 73%. IMBs could be stored in 4 °C protecting from light for 2 months without obvious reduction of biological activity. Human reference sera mixed with various concentrations of CEA were tested with the proposed method and commercial enzyme-linked immunosorbent assay (ELISA) kit, and a good linear relationship was obtained. This proposed technique demonstrated an excellent performance for quantifying CEA and was expected to be used for clinical testing.     

Determination of dimethylselenide and dimethyldiselenide in milk and milk by-products by solid-phase microextraction and gas chromatography with atomic emission detection

A method based on solid-phase microextraction (SPME) followed by gas chromatography with microwave-induced plasma atomic emission detection for determining dimethylselenide (DMSe) and dimethyldiselenide (DMDSe) in milk and milk by-products is proposed. Parameters affecting the SPME, such as sample volume or mass, ionic strength, adsorption and desorption times and temperatures were optimized in the headspace mode. The matrix effect was evaluated for the different samples studied, concluding that standard additions calibration was required for quantification purposes. The detection limits ranged from 70 to 110 pg mL⁻¹ for DMSe and from 80 to 400 pg mL⁻¹ for DMDSe, depending on the sample under analysis. None of the twenty-three samples analyzed contained the studied compounds at concentrations above the corresponding detection limits.     

Use of 3-(4-hydroxyphenyl)propionic acid as electron donating compound in a potentiometric aflatoxin M1-immunosensor

We developed a potentiometric aflatoxin M₁-immunosensor which utilizes 3-(4-hydroxyphenyl)propionic acid (p-HPPA) as electron donating compound for horseradish peroxidase (HRP; EC 1.11.1.7). The assay system consists of a polypyrrole-surface-working electrode coated with a polyclonal anti-M₁ antibody (pAb-AFM₁), a Ag/AgCl reference electrode and a HRP-aflatoxin B₁ conjugate (HRP-AFB₁ conjugate).To optimize the potentiometric measuring system p-HPPA as well as related compounds serving as electron donating compounds were compared. Also the influence of different buffer systems, varying pH and substrate concentrations on signal intensity was investigated. Our results suggest that reaction conditions that favor the formation of Pummerer's type ketones lead to an increase in signal intensity rather than formation of fluorescent dye. Comparison with commercial ready-to-use HRP electron donating compounds such as 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS), o-phenylenediamine (OPD) or 3,3′,5,5′-tetramethylbenzidine (TMB) showed that only 34%, 77% and 49% of the signal intensity of p-HPPA were reached, respectively.The optimized assay had a detection limit of 40 pg mL⁻¹ and allowed detection of 500 pg mL⁻¹ (FDA action limit) aflatoxin M₁ (AFM₁) in pasteurized milk and UHT-milk containing 0.3-3.8% fat within 10 min without any sample treatment. The working range was between 250 and 2000 pg mL⁻¹ AFM₁.     

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.     

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.     

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.