Study on an amperometric immunosensor based on Nafion-cysteine composite membrane for detection of carcinoembryonic antigen

In this work, protonated l-cysteine was entrapped in Nafion (Nf) membrane by cation exchange function, forming Nf-Cys (cysteine) composite membrane, which was more stable, compact, biocompatible, and favorable for mass and electron transfer compared with Nf film solely. Then gold (Au) nanoparticles were adsorbed onto the electrode surface by thiol groups on the composite membrane. After that, nano-Au monolayer was formed, onto which carcinoembryonic antibody was loaded to prepare carcinoembryonic antigen (CEA) immunosensor. The results indicated that the immunosensor had good current response for CEA using potassium ferricyanide as the redox probe. A linear concentration range of 0.01 to 100 ng/ml with a detection limit of 3.3 pg/ml (signal/noise = 3) was observed. Moreover, the morphology of the modified Au substrates was investigated with atomic force microscopy, and the electrochemical properties and performance of modified electrodes were investigated by cyclic voltammograms and electrochemical impendence spectroscopy. The results exhibited that the immunosensor has advantages of simple preparation, high sensitivity, good stability, and long life expectancy. Thus, the method can be used for CEA analysis.     

Magnetic nanoparticle-based immunosensor for electrochemical detection of hepatitis B surface antigen

An electrochemical immunosensor was developed for the detection of hepatitis B surface antigen (HBsAg). The biotinylated hepatitis B surface antibody was immobilized on streptavidin magnetic nanoparticles and used for targeting the HBsAg. By the addition of horseradish peroxidase conjugated with secondary antibody (HRP–HBsAb), a sandwich-type immunoassay format was formed. Aminophenol as substrate for conjugated HRP was enzymatically changed into 3-aminophenoxazone (3-APZ). This electroactive enzymatic production (3-APZ) was transferred into an electrochemical cell and monitored by cyclic voltammetry. Under optimal conditions, the cathodic current response of 3-APZ, which was proportional to the HBsAg concentration, was measured by a glassy carbon electrode. The immunosensor response was linear toward HBsAg in the concentration range from 0.001 to 0.015 ng/ml with a detection limit of 0.9 pg/ml at a signal/noise ratio of 3.     

Comparison of electrochemical immunosensors based on gold nano materials and immunoblot techniques for detection of histidine-tagged proteins in culture medium

In this work, the direct electrochemical determination of poly-histidine tagged proteins using immunosensor based on anti-His (C-term) antibody immobilized on gold electrodes modified with 1,6-hexanedithiol, gold colloid particles or gold nanorods is described. The recombinant histidine-tagged silk proteinase inhibitor protein (rSPI2-His(6)) expressed in Pichia system selected as antigen for this immonosensor. An electrochemical impedance spectroscopy was used as label free detection technique for immune conjugation. The gold nanorods modified electrode layer showed better analytical response than gold nano particles. The linear calibration range was observed between 10pg/ml and 1ng/ml with limit of detection 5pg/ml (S/N=3). Up to four successive assay cycles with retentive sensitivity were achieved for the immunosensors regenerated with 0.2M glycine-HCl buffer, pH 2.8. The performance of this immnosensor were compared with immuoblotting techniques.     

Amperometric immunosensor based on multiwalled carbon nanotubes/Prussian blue/nanogold-modified electrode for determination of α-fetoprotein

In this article, a conspicuously simple and highly sensitive amperometric immunosensor based on the sequential electrodeposition of Prussian blue (PB) and gold nanoparticles (GNPs) on multiwalled carbon nanotube (MWCNT)-modified glassy carbon electrode (GCE) surface is proposed for the detection of α-fetoprotein (AFP). By comparison with PB, the MWCNT/PB composite film had been proven to show much better electrochemical stability and a larger response current. The electrodeposited GNP film can be used not only to immobilize biomolecules but also to avoid the leakage of PB and to prevent shedding of MWCNT/PB composite film from the electrode surface. The performance and factors influencing the performance of the immunosensor were investigated. Under optimal experimental conditions, the proposed immunosensor for AFP was observed with an ultralow limit of detection (LOD) equal to 3 pg/ml (at 3δ), and the linear working range spanned the concentrations of AFP from 0.01 to 300 ng/ml. Moreover, the immunosensor, as well as a commercially available kit, was examined for use in the determination of AFP in real human serum specimens. More significant, the assay mentioned here is simpler than the traditional enzyme-linked immunosorbent assay (ELISA), and an excellent correlation of levels of AFP measured was obtained, indicating that the developed immunoassay could be a promising alternative approach for detection of AFP and other tumor markers in the clinical diagnosis.     

A signal-amplified electrochemical immunosensor for aflatoxin B1 determination in rice

A sensitive and simple electrochemical immunosensor based on enzymatic silver deposition amplification was constructed for the detection of aflatoxin B₁ (AFB₁) in rice. The immunosensor was based on an indirect competitive format between free AFB₁ and aflatoxin B₁-bovine serum albumin (AFB₁-BSA) conjugate immobilized on the electrode surface for binding to a fixed amount of anti-AFB₁ antibody. Then the alkaline phosphatase (ALP)-labeled anti-mouse immunoglobulin G (IgG) secondary antibody was bound to the electrode surface through reaction with primary antibody. Finally, ALP catalyzed the substrate, ascorbic acid 2-phosphate, into ascorbic acid that reduced silver ions in solution to metal silver deposited onto the electrode surface. Linear sweep voltammetry was carried out to quantify the metal silver, which indirectly reflected the amount of the analyte. The experimental parameters, such as the dilution ratio of antibody and the concentration of AFB₁-BSA conjugate, have been evaluated and optimized. At the optimal conditions, the working range of the electrochemical immunosensor was from 0.1 to 10 ng/ml with a detection limit of 0.06 ng/ml. Good recoveries were obtained for the detection of spiked rice samples. So, the proposed method in this article could find a good use for screening AFB₁ in real samples.     

Surface plasmon resonance assay for chloramphenicol without surface regeneration

A surface plasmon resonance (SPR) assay without surface regeneration was developed for rapid and sensitive detection of chloramphenicol (CAP). A CAP-amine derivative was synthesized using a polyethylene glycol chain attached to the CAP through a carbamate linkage and immobilized onto a Biacore dextran surface. This chemically modified surface significantly changed the binding behavior between antibody and CAP, shown by both fast association and fast dissociation rates, and created a rapid and sensitive SPR immunoassay of the CAP without any regeneration. The limits of detection achieved for CAP were 32.2 pg/ml in aqueous buffer and 42.4 pg/ml in honey-spiked samples.     

Development of surface plasmon resonance imaging biosensors for detection of ubiquitin carboxyl-terminal hydrolase L1

We have developed a new method for highly selective determination of the ubiquitin carboxyl-terminal hydrolase L1 (UCH-L1) concentration using a surface plasmon resonance imaging (SPRI) technique and two different biosensors. UCH-L1 was captured from a solution by immobilized specific rabbit monoclonal antibody or specific LDN-57444 inhibitor due to formation of receptor–UCH-L1 complex on the biosensor surface. The analytically useful dynamic response range of both biosensors is between 0.1 and 2.5 ng/ml. The detection limit is 0.06 ng/ml for the biosensor with antibody and 0.08 ng/ml for the biosensor with inhibitor. Biosensors based on both antibody and inhibitor were found to be suitable for quantitative determination of the UCH-L1 and exhibit good tolerance to the potential interferents. Both biosensors gave comparable results in the range of 0 to 0.20 ng/ml for plasma samples and 0.30 to 0.49 ng/ml for cerebrospinal fluid samples. To validate the new methods, comparative determination of UCH-L1 by the commercial enzyme-linked immunosorbent assay (ELISA) kit was performed. In general, in terms of UCH-L1 concentration, a good correlation between SPRI and ELISA was found. The developed biosensors can be used successfully for the determination of UCH-L1 in body fluids.     

Electrochemical impedimetric immunosensor for insulin like growth factor-1 using specific monoclonal antibody-nanogold modified electrode

An electrochemical impedimetric immunosensor was developed for ultrasensitive determination of insulin-like growth factor-1 (IGF-1) based on immobilization of a specific monoclonal antibody on gold nanoparticles (GNPs) modified gold electrode. Self-assembly of colloidal gold nanoparticles on the gold electrode was conducted through the thiol groups of 1,6-hexanedithiol (HDT) monolayer as a cross linker. The redox reactions of [Fe(CN)(6)](4-)/[Fe(CN)(6)](3-) on the electrode surface was probed for studying the immobilization and determination processes, using cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). The interaction of antigen with grafted antibody recognition layer was carried out by soaking the modified electrode into antigen solution at 37°C for 3 h. The immunosensor showed linearity over 1.0-180.0 pg mL(-1) and the limit of detection was 0.15 pg mL(-1). The association constant between IGF-1 and immobilized antibody was calculated to be 9.17×10(11) M(-1). The proposed method is a useful tool for screening picogram amounts of IGF-1 in clinical laboratory as a diagnostic test.     

Fluorescein-labeled “arch-like” DNA probes for electrochemical detection of DNA on gold nanoparticle-modified gold electrodes

In the present study, a gold nanoparticle-modified gold electrode (nanogold electrode) was used to develop a novel fluorescein electrochemical DNA biosensor based on a target-induced conformational change. The nanogold electrode was obtained by electrodepositing gold nanoparticles onto a bare gold electrode. This modification not only immobilized probe oligonucleotides, but also adsorbed fluorescein onto the surface of the gold nanoparticles to form an “arch-like” structure. This article compares the electrochemical signal changes caused by the hybridization of “arch-like” DNA on nanogold electrode and linear DNA on bare gold electrode. The results showed that the adsorption effect of nanogold can enhance the sensitivity of the sensor. The linear range of target ssDNA is from 2.0 × 10⁻⁹ M to 2.0 × 10⁻⁸ M with a correlation coefficient of 0.9956 and detection limit (3σ) of 7.10 × 10⁻¹⁰ M. Additionally, the specificity and hybridization response of this simple sensor were investigated.     

Spectroelectrochemical characterization of pain biomarkers

This article reports the first electrochemical characterization of pain biomarkers that include arachidonic acid (AA), prostaglandin G₂ (PGG₂), and cyclooxygenase 2 (COX-2). These biomarkers are mediators of pathophysiology of pain, inflammation, and cell proliferation in cancer. The article also reports the development of an electrochemical immunosensor for monitoring these pain biomarkers. The results revealed that direct electron transfer between AA metabolites and the electrode could be easily monitored and that an enzyme-modified electrode dramatically enhanced bioelectrocatalytic activity toward AA. Cyclic voltammetric analysis of AA revealed a concentration-dependent anodic current with a slope of 2.37 and a limit of detection (LOD) of 0.25 nM. This unique AA/gold electrode electron transfer provides a good electrochemical sensing platform for prostaglandin H₂ (PGH₂) as the basis for quantitation of pain. An amperometric signal intensity of a COX-2 antibody-modified gold electrode was linear with COX-2 concentration in the range of 0.1-0.5 μg/ml and an LOD of 0.095 μg/ml. The results also revealed a linear correlation of the concentration of PGG₂ with an LOD of 0.227 μM.     

Ochratoxin A in human blood serum – retrospective long-term data

In a long-term study (1990–1997) on ochratoxin A (OTA) in human blood serum, 102 serum samples from 36 persons of the Munich Institute for Hygiene and Technology of Food of Animal Origin were analysed by enzyme immunoassay (EIA), and by high performance liquid chromatography (HPLC) for control. Detection limits were at 50 pg/ml (EIA) and 50–70 pg/ml (HPLC), recoveries were 80–120% (EIA) and 30–60% (LC). OTA was detected in 98% (EIA, 368 ± 217 pg/ml) and 93% (HPLC, 271 ± 170 pg/ml) of samples (maximum 1,290 pg/ml). Using published conversion factors for serum/intake estimates (1.34 or 1.97), the mean daily OTA intake of these 36 persons was 493–725 pg/kg bw. Long-term individual mean OTA levels of nine persons ranged from 162 ± 80 pg/ml to 549 ± 172 pg/ml. Our data were compared with published OTA serum levels (1985–2008) for apparently healthy persons from a total of 30 countries. On a worldwide basis, the mean of means for OTA in human serum was estimated to be 700 pg/ml, corresponding to a mean daily OTA intake of 940–1380 pg/kg bw. This level, which was relatively stable over the last decades, is well below published tolerable daily intake values (14,000–18,000 pg/kg bw).     

Detection of glucose using immobilized bienzyme on cyclic bisureas–gold nanoparticle conjugate

A highly sensitive electrochemical glucose sensor has been developed by the co-immobilization of glucose oxidase (GOx) and horseradish peroxidase (HRP) onto a gold electrode modified with biocompatible cyclic bisureas–gold nanoparticle conjugate (CBU–AuNP). A self-assembled monolayer of mercaptopropionic acid (MPA) and CBU–AuNP was formed on the gold electrode through a layer-by-layer assembly. This modified electrode was used for immobilization of the enzymes GOx and HRP. Both the HRP and GOx retained their catalytic activity for an extended time, as indicated by the low value of Michaelis–Menten constant. Analytical performance of the sensor was examined in terms of sensitivity, selectivity, reproducibility, lower detection limit, and stability. The developed sensor surface exhibited a limit of detection of 100 nM with a linear range of 100 nM to 1 mM. A high sensitivity of 217.5 μA mM⁻¹ cm⁻² at a low potential of −0.3 V was obtained in this sensor design. Various kinetic parameters were calculated. The sensor was examined for its practical clinical application by estimating glucose in human blood sample.     

Sandwich electrochemical immunoassay for the detection of Staphylococcal enterotoxin B based on immobilized thiolated antibodies

A new approach for the sensitive detection of Staphylococcal enterotoxin B (SEB) is presented based upon an electrochemical enzymatic immunoassay that utilizes thiolated antibodies immobilized on a gold surface. This method relies on the use of amine- or sulfhydryl-reactive heterobifunctional cross-linkers for the introduction of 2-pyridyl-disulfide groups to the antibody. The disulfide-containing linkages are subsequently cleaved with a suitable reducing agent, such as dithiothreitol (DTT), and the thiolated antibody–gold bond is covalently formed on a gold working electrode. Various cross-linking agents for immobilization of the capture antibody onto the gold electrode were investigated and compared. Factors influencing the thiolation and immobilization were investigated and optimized. The feasibility of such antibody immobilization and the subsequent sandwich enzyme immunoassay is demonstrated for the sensitive detection of SEB. The detection limit estimated from a representative dose–response curve is 1 ng/mL, corresponding to 5 pg in a 5-μL sample. Coupling the specificity of immunoassays with the sensitivity and low detection limits of electrochemical detection shows real promise for future sensing technology in enabling the development of single-use disposable devices.     

Impedimetric immunosensor for the specific label free detection of ciprofloxacin antibiotic

Impedance spectroscopy approaches combined with the immunosensor technology have been used for the determination of trace amounts of ciprofloxacin antibiotic belonging to the fluoroquinolone family. The sensor electrode was based on the immobilization of anti-ciprofloxacin antibodies by chemical binding onto a poly(pyrrole-NHS) film electrogenerated on a solid gold substrate. The electrode surface was modified by electropolymerization of pyrrole-NHS, antibody grafting and ciprofloxacin immunoreaction. The sensitive steps of surface modification, cyclic voltammetry (CV) and atomic force microscopy (AFM) imaging have been used for electrode surface characterization. The immunoreaction of ciprofloxacin on the grafted anti-ciprofloxacin antibody directly triggers a signal via impedance spectroscopy measurements which allows the detection of extremely low concentration of 10 pg/ml ciprofloxacin.     

Determination of human serum 8-hydroxy-2′-deoxyguanosine (8-OHdG) by HPLC-ECD combined with solid phase extraction (SPE)

Human serum 8-hydroxy-2′-deoxyguanosine (8-OHdG) was measured by HPLC-ECD method combined with solid phase extraction (SPE) developed by our group: (our proprietary kit, named 8-OHdG Pre-treatment Kit (TANITA Corporation)). The major interfering substances and proteins in serum were removed by 8-OHdG Pre-treatment Kit. This measurement method was highly reproducible (CV = 2.2–7.1%) and demonstrated the lower detection limit for control serum sample of less than 10 pg/ml without the sample evaporation. The other hand 8-OHdG concentration in serum for healthy people was in the range of 0–70 pg/ml (25.5 ± 13.8 pg/ml, n = 37). Secondary a relationship between the HPLC-ECD and ELISA methods was investigated. ELISA method could not detect 8-OHdG concentration in serum for healthy people, because the detection limit of 130 pg/ml was higher than the normal range for healthy people. These results show our SPE method has high sensitivity and quantitative accuracy for 8-OHdG analysis.     

Sensitive detection of multiplex toxins using antibody microarray

Using a newly developed fluorescent nanoparticle (NP) that gives rise to a high-intensity and stable fluorescent light, a sensitive antibody (Ab) microarray assay system has been developed for specific detection of bioterrorism agents, as exemplified by ricin, cholera toxin (CT), and staphylococcal enterotoxin B (SEB). The Ab microarray uses a sandwich format that consists of capture Abs, analytes (toxins), biotinylated detection Abs, and avidin-conjugated NP. In all three cases, polyclonal Abs (pAbs) displayed superiority over monoclonal antibodies (mAbs) in capturing toxins on microarray slides even when the pAbs and mAbs had similar affinity as determined by enzyme-linked immunosorbent assay (ELISA). The detection system was successfully used to detect toxins spiked in milk, apple cider, and blood samples. We were able to detect ricin at 100 pg/ml in buffer and at 1 ng/ml in spiked apple cider or milk, whereas CT and SEB were detected at 10 pg/ml in buffer and 100 pg/ml in spiked apple cider or milk. High specificities were also demonstrated in the detection of mixed toxin samples with similar sensitivities. The matrix effect of blood samples on the detection of mixed toxins seems to be minimal when the toxin concentration is at or above 100 ng/ml. The current study highlights the significant role of pAb and NP in increasing selectivity and sensitivity of toxin detection in a microarray format.     

Electrochemical assay of plasmin activity and its kinetic analysis

A sensitive and convenient electrochemical assay of plasmin activity and its kinetic analysis are described. Thus, a ferrocenyl peptide substrate (FcPS) having a plasmin-specific substrate sequence, Lys-Thr-Phe-Lys, and a Cys residue was prepared and immobilized on a gold electrode through the sulfur-gold linkage. The obtained electrode showed a redox signal based on the ferrocene moiety, suggesting the immobilization of FcPS on the electrode. After treatment of this electrode with plasmin, its electrochemical signal was decreased in proportion to an increase of the amount of plasmin. The detection limit for plasmin in this assay system was as low as 50 ng/ml or 0.15 mU/ml. Real-time monitoring of plasmin reaction on the electrode could also be achieved, and the kinetic parameters of this enzymatic reaction could be determined; for example, the k_cat/K_m value was 0.063 μM⁻¹ s⁻¹. Furthermore, a quantitative assay for streptokinase as a plasminogen activator was also demonstrated by using this system.     

Electrochemical detection of cardiac troponin I using a microchip with the surface-functionalized poly(dimethylsiloxane) channel

A sensitive and rapid electrochemical microchip fabricated by assembling a surface-functionalized poly(dimethylsiloxane) (PDMS) microchannel with an interdigitated array (IDA) gold electrode was developed for the detection of human cardiac troponin I (cTnI) in the early diagnosis of acute myocardial infarction. Anti-cTnI was immobilized onto the internal surface of the PDMS channel on which protein G layer had been generated by silanization. To reduce electrode fouling, a PDMS channel was assembled with an IDA chip after surface treatment. The detection experiments were performed with successive injection of cTnI, alkaline phosphatase (AP)-labeled anti-cTnI, and p-aminophenylphosphate. Then, cyclic voltammograms were obtained by the oxidation peak current proportionally to the concentration of enzymatic product, p-aminophenol. The optimal packing density of anti-cTnI on the surface of the PDMS channel was determined at the anti-cTnI concentration of 30 microg/ml for the highest electrochemical signal. These demonstrate that the proper orientation and best packing density of antibody as well as no electrode fouling contributed to the low detection limit (148 pg/ml) of cTnI within 8 min.     

Electrochemical immunoanalysis of cardiac myoglobin

Methods of myoglobin determination based on electrochemical analysis by means of analysis of electrochemical parameters of modified electrodes have been proposed. The method of direct detection is based on interaction of myoglobin with anti-myoglobin with subsequent electrochemical registration of this hemoprotein. The electrode surface was modified by a membrane-like synthetic didodecyldimethylammonium bromide (DDAB), gold nanoparticles and antibodies to human cardiac myoglobin the electrochemical reduction of myoglobin heme was registered provided that the antigen (myoglobin) was present in the samples. The reaction of myoglobin binding to antibodies immobilized on the electrode surface was also registered using electrochemical impedance spectroscopy. The study of electro analytical characteristics revealed high specificity and sensitivity of the developed method. The biosensor was characterized by low detection limit and a high working range of the detected concentrations from 17.8 to 1780 ng/ml (from 1 to 100 nM). The method of myoglobin determination based on a signal of gold nanoparticles has also been proposed. The signal was detected with stripping voltammetry. There was a change in the cathodic peak area and the peak height of gold oxide reduction for the electrodes with antibodies and the electrodes with the antibody-myoglobin complex.     

Determination of lipoic acid in human plasma by HPLC-ECD using liquid–liquid and solid-phase extraction: Method development,validation and optimization of experimental parameters

A rapid, inexpensive, sensitive and specific HPLC-ECD method for the determination of lipoic acid in human plasma was developed and validated over the linearity range of 0.001–10 μg/ml using naproxen sodium as an internal standard (IS). Extraction of lipoic acid and IS from plasma (250 μl) was carried out with a simple one step liquid–liquid extraction using dichloromethane. Similarly solid-phase extraction was carried out using dichloromethane as extraction solvent. The separated organic layer was dried under the stream of nitrogen at 40 °C and the residue was reconstituted with the mobile phase. Complete separation of both lipoic acid and IS at 30 °C on Discovery HS C18 RP column (250 mm × 4.6 mm, 5 μm) was achieved in 6 min using 0.05 M phosphate buffer (pH 2.5 adjusted with phosphoric acid):acetonitrile (50:50, v/v) as a mobile phase pumped at the rate of 1.5 ml/min using electrochemical detector in DC mode at the detector potential of 1.0 V. The limit of detection and limit of quantification of lipoic acid were 200 pg/ml and 1 ng/ml, respectively. While on column limit of detection and limit of quantification of lipoic acid were 10 and 50 pg/ml, respectively. The absolute recoveries of lipoic acid with liquid–liquid and solid-phase extraction were 98.43, 95.65, 101.45, and 97.36, 102.73, 100.17% at 0.5, 1 and 5 μg/ml levels, respectively. Coefficient of variations for both intra-day and inter-day were between 0.28 and 4.97%. The method is validated and will be quite suitable for the analysis of lipoic acid in the plasma of human volunteers as well as patients with diabetes and cardiovascular diseases.