Enzymatically amplified electrochemical detection for lipopolysaccharide using ferrocene-attached polymyxin B and its analogue

Ferrocene-attached polymyxin B (PMB-Fc) was prepared by the reaction of polymyxin B with ferrocenoyl chloride in a toluene/pyridine mixture. An electrochemical detection of lipopolysaccharide (LPS) was carried out using a combination of PMB-Fc and an enzyme-modified electrode constructed from a glassy carbon electrode modified with a bovine serum albumin membrane containing glucose oxidase. The ferrocene units of the PMB-Fc molecules were oxidized on the electrode, and then reduced to the original neutral form by a glucose oxidase-catalyzed reaction in the presence of D-glucose. The consumption/regeneration cycle for PMB-Fc resulted in a chemically amplified current response. The current response for PMB-Fc decreased in association with its complexation with LPS, and the magnitude of this current decrease caused by LPS was also amplified by the recycling process. The enzyme-modified electrode exhibited a rapid response of 5 min for LPS with the detection limit as low as 50 ng ml(-1). Further, the addition of D-solbitol or poly(vinyl alcohol) of high concentration over 1 mg ml(-1) substantially induced no response, and three kinds of LPS from different strains exhibited similar magnitudes of current response for the same concentrations; these results suggest the advantages of this detection system for practical applications. Ferrocene-attached colistin, an analogue of PMB-Fc, was also effective for the LPS detection using the glucose oxidase-modified electrode.     

Internal standard method for the measurement of choline and acetylcholine by capillary electrophoresis with electrochemical detection

An internal standard method has been developed for the determination of the neurotransmitter acetylcholine and/or its metabolic precursor choline. This approach couples the high separation efficiency of capillary electrophoresis with the sensitivity and selectivity of electrochemical detection at an enzyme-modified electrode. Indirect electrochemical detection is accomplished at a 25 microm platinum electrode modified by cross-linking the enzymes choline oxidase and acetylcholinesterase with glutaraldehyde. Although in this simple form of electrode fabrication there is a gradual loss of response from the electrochemical detector with time, accurate quantitation is achieved by the addition of butyrylcholine, which is also a substrate for acetylcholinesterase, as an internal standard. A linear response is achieved between 0 and 125 microM with a limit of detection of 2 microM (25 fmol). The utility of this method was demonstrated by monitoring the kinetics of choline uptake in synaptosomal preparations.     

Selective detection of L-glutamate using a microfluidic device integrated with an enzyme-modified pre-reactor and an electrochemical detector

A microfluidic device integrated with a nanoliter volume enzyme pre-reactor and an enzyme-modified electrode was developed for the highly selective continuous measurement of glutamate (Glu). The device consists mainly of two glass plates. One plate incorporates an electrochemical cell that consists of working electrode (WE), reference electrode (RE) and counter electrode (CE). The WE is modified with a bilayer film of Os-polyvinylpyrridine-based mediator containing horseradish peroxidase (Os-gel-HRP). The WE was operated at -50 mV versus Ag. The other plate has a thin layer flow channel integrated with a pre-reactor. The reactor has a number of micropillars (20 microm in diameter, 20 microm high and separated from each other by a 20 microm gap) modified with ascorbate oxidase (AAOx) to eliminate L-ascorbic acid (AA). The enzymatic oxidation of AA is superior to that obtained with our previously reported pre-electrolysis type micro-reactor since electrochemically reversible transmitters such as catecholamines do not provide a cathodic current at the WE. In addition, the high operation potential of the pre-reactor causes unknown electroactive species, which also cause interference at the detection electrode. As a result, we were able to detect 1 microM Glu continuously at a low flow rate even when AA concentration was 100 microM.     

Acetylcholine biosensor involving entrapment of acetylcholinesterase and poly(ethylene glycol)-modified choline oxidase in a poly(vinyl alcohol) cryogel membrane

A bienzymatic sensor for the determination of acetylcholine was prepared by physical coimmobilization of acetylcholinesterase and poly(ethylene glycol)-modified choline oxidase in a poly(vinyl alcohol) cryogel membrane obtained by a cyclic freezing-thawing process. The enzyme-modified polymer was applied on a platinum electrode to form an amperometric sensor, based on the electrochemical detection of enzymatically developed hydrogen peroxide. The analytical characteristics of this sensor, including calibration curves for choline and acetylcholine, pH, and temperature effects, and stability are described.     

DNA-Cu(II) poly(amine) complex membrane as novel catalytic layer for highly sensitive amperometric determination of hydrogen peroxide

A novel hydrogen peroxide biosensor was fabricated by using a DNA-Cu(II) complex as a novel electrocatalyst for the reduction of hydrogen peroxide (H2O2). A polyion complex (PIC) membrane composed of DNA and poly(allylamine) (PAA) functioned as a support matrix for immobilization of electrocatalytic element-copper ion. The circular dichroism (CD) spectrum of the DNA-Cu(II)/PAA membrane in wet state showed that the DNA exists in B-like form within the membrane. Electrochemical measurements of the DNA-Cu(II)/PAA membrane-modified glassy carbon (GC) electrode revealed that the copper ion embedded in the DNA/PAA layer exhibits good electrochemical behaviors, and the electrochemical rate constant between the immobilized copper ion and the GC electrode surface was estimated to be 26.4 s(-1). The resulting DNA-Cu(II)/PAA/GC electrode showed an excellent electrocatalytic activity for the H2O2 reduction. The sensitivity of the sensor for the determination of H2O2 was affected by the amount of each component, such as copper ion, DNA and PAA in the DNA-Cu(II)/PAA membrane. Effects of applied potential, pH, temperature, ionic strength and buffer concentrations upon the response currents of the sensor were also investigated for an optimum analytical performance. Even in the presence of dissolved oxygen, the sensor exhibited highly sensitive and rapid (response time, less than 5 s) response to H2O2. The steady-state cathodic current responses of the sensor obtained at -0.2 V versus Ag/AgCl in air-saturated 50 mM phosphate buffer (pH 5.0) increased linearly up to 135 microM with the detection limit of 50 nM. Interference by ascorbic acid and uric acid due to the reduction of Cu(II) was effectively cancelled by further modification of outermost layer of polyion complex film. In addition, the sensor exhibited good reproducibility and stability.     

Design of a sandwich-mode amperometric biosensor for detection of PML/RARα fusion gene using locked nucleic acids on gold electrode

In this study, a novel DNA electrochemical probe (locked nucleic acid, LNA) was designed and involved in constructing an electrochemical DNA biosensor for detection of promyelocytic leukemia/retinoic acid receptor alpha (PML/RARα) fusion gene in acute promyelocytic leukemia for the first time. This biosensor was based on a 'sandwich' sensing mode, which involved a pair of LNA probes (capture probe immobilized at electrode surface and biotinyl reporter probe as an affinity tag for streptavidin-horseradish peroxidase (streptavidin-HRP). Since biotin can be connected with streptavidin-HRP, this biosensor offered an enzymatically amplified electrochemical current signal for the detection of target DNA. In the simple hybridization system, DNA fragment with its complementary DNA fragment was evidenced by amperometric detection, with a detection limit of 74 fM and a linear response range of 0.1-10 pM for synthetic PML/RARα fusion gene in acute promyelocytic leukemia (APL). Otherwise, the biosensor showed an excellent specificity to distinguish the complementary sequence and different mismatch sequences. The new pattern also exhibited high sensitivity and selectivity in mixed hybridization system.     

Biochemical basis of HLA-DR and CR3 modulation on human peripheral blood monocytes by lipopolysaccharide

The biochemical events leading to enhanced membrane expression of HLA-DR and CR3 by human peripheral blood monocytes (MO) following exposure to bacterial lipopolysaccharide (LPS) were examined. In a previous study we demonstrated that an increase in intracellular calcium was necessary, but not sufficient, for MO to increase membrane expression of both antigens within 1 hr of addition of LPS. The present study was initiated to examine the other biochemical requirements which lead to the MO response to LPS. Enhanced expression of both antigens following addition of LPS was dependent on microfilament function, but independent of microtubule function and of protein synthesis. Inhibition of formation of cyclooxygenase or lipoxygenase metabolites of arachidonic acid had no effect on HLA-DR or CR3 modulation by LPS. A role for phosphatidylinositol metabolism was suggested by the inhibition of the MO response to LPS by dibutyryl cAMP and theophylline and by the enhanced expression of both antigens following addition of phorbol diesters. However, H-7, a putative inhibitor of protein kinase C, did not alter the MO response to LPS or phorbol diesters. These results suggest that LPS enhances expression of HLA-DR and CR3 by inducing redistribution of these antigens from an intracellular pool. The data also support a role for the generation of hydrolysis products of phosphatidylinositol, leading to calcium redistribution and activation of protein kinase C or other kinases, in the MO response to LPS.     

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.     

Amperometric biosensor based on tyrosinase-conjugated polysaccharide hybrid film: selective determination of nanomolar neurotransmitters metabolite of 3,4-dihydroxyphenylacetic acid (DOPAC) in biological fluid

The amperometric detection of neurotransmitters metabolite of 3,4-dihydroxyphenylacetic acid (DOPAC) was achieved at a tyrosinase-chitosan composite film-modified glassy carbon (GC) electrode. The optimal conditions for the preparation of the biosensor were established. This bio-composite film was characterized by scanning electron microscopy (SEM) and Fourier transformed infrared (FT-IR) spectra, suggesting that chitosan covalently connected to chitosan chains. Electrochemical characterization of the bio-hybrid membrane-covered electrodes were also performed in 0.05 M phosphate buffer solution (pH 6.52) containing neurotransmitters or their derivatives by using cyclic voltammetry (CV), linear sweep voltammetry (LSV), square wave voltammetry (SWV) and amperometry. This simply-prepared protein-polysaccharide hybrid film provides a microenvironment friendly for enzyme loading. The sensor was operated at -0.15 V with a short response time. The current linearly increased with the increasing concentration of DOPAC over the concentration of 6 nM-0.2 mM. The lower detection limit for DOPAC is 3 nM (S/N=3). The sensitivity of the sensor is 40 microA mM(-1). A physiological level of neurotransmitters and their derivatives including dopamine, l-dopa, adrenaline, noradrenaline and homovanillic acid as well as ascorbic acid, uric acid and acetaminophen do not affect the determination of DOPAC.     

A Highly Sensitive and Selective Hydrogen Peroxide Biosensor Based on Gold Nanoparticles and Three-Dimensional Porous Carbonized Chicken Eggshell Membrane

A sensitive and noble amperometric horseradish peroxidase (HRP) biosensor is fabricated via the deposition of gold nanoparticles (AuNPs) onto a three-dimensional (3D) porous carbonized chicken eggshell membrane (CESM). Due to the synergistic effects of the unique porous carbon architecture and well-distributed AuNPs, the enzyme-modified electrode shows an excellent electrochemical redox behavior. Compared with bare glass carbon electrode (GCE), the cathodic peak current of the enzymatic electrode increases 12.6 times at a formal potential of −100mV (vs. SCE) and charge-transfer resistance decreases 62.8%. Additionally, the AuNPs-CESM electrode exhibits a good biocompatibility, which effectively retains its bioactivity with a surface coverage of HRP 6.39×10⁻⁹ mol cm⁻² (752 times higher than the theoretical monolayer coverage of HRP). Furthermore, the HRP-AuNPs-CESM-GCE electrode, as a biosensor for H₂O₂ detection, has a good accuracy and high sensitivity with the linear range of 0.01–2.7 mM H₂O₂ and the detection limit of 3μM H₂O₂ (S/N = 3).     

Protein modified- and membrane electrodes: strategies for the development of biomolecular sensors

Different procedures used for constructing protein/enzyme-modified electrodes are examined, in particular adsorption, covalent attachment and film deposition. The performances of such modified electrodes with electroactive proteins or enzymes attached to their active surface are examined, especially in the case of c-type cytochromes, hydrogenases and glucose oxidase. Another strategy presented in this review consists of the use of membrane electrodes with an electroactive protein imprisoned between a dialysis membrane and the electrode surface. The versatility and other advantages of such a procedure are underlined. Applications of membrane electrodes to the bioremediation of soils and effluents and as models for investigating interactions between proteins and soils are described.     

Electrochemical detection of uric acid via uricase-immobilized graphene oxide

Measurement of the uric acid level in the body can be improved by biosensing with respect to the accuracy, sensitivity and time consumption. This study has reported the immobilization of uricase onto graphene oxide (GO) and its function for electrochemical detection of uric acid. Through chemical modification of GO using 1-ethyl-3-(dimethylaminopropyl) carbodiimide (EDC) and N-hydroxysulfosuccinimide (NHS) as cross-linking reagents, the enzyme activity of the immobilized uricase was much comparable to the free enzyme with 88% of the activity retained. The modified GO-uricase (GOU) was then subjected to electrocatalytic detection of uric acid (UA) via cyclic voltammetry (CV). For that reason, a glassy carbon electrode (GCE) was modified by adhering the GO along with the immobilized uricase to facilitate the redox reaction between the enzyme and the substrate. The modified GOU/GCE outperformed a bare electrode through the electrocatalytic activity with an amplified electrical signal for the detection of UA. The electrocatalytic response showed a linear dependence on the UA concentration ranging from 0.02 to 0.49 mM with a detection limit of 3.45 μM at 3σ/m. The resulting biosensor also exhibited a high selectivity towards UA in the presence of other interference as well as good reproducibility.     

Endotoxin detection in a competitive electrochemical assay: synthesis of a suitable endotoxin conjugate

A biotin-lipopolysaccharide (biotin-LPS) conjugate was synthesized from LPS smooth from Salmonella minnesota, yielding a conjugate with a biotin/LPS ratio equal to 1:1 and endotoxic activity of 0.08 EU ng(-1). The conjugate was used in an amperometric competitive assay to determine endotoxins with endotoxin-neutralizing protein (ENP) as the recognition element. The assay is performed on a modified electrode, involving the covalent binding of carboxymethyl dextran (CMDex) to a cystamine-modified gold electrode and then the covalent binding of the recognition protein, ENP, to CMDex. The assay is carried out by incubating the modified electrode in an LPS sample to which biotin-LPS was added. Both species compete for the recognition sites on the modified surface. After the incubation stage and a careful rinsing, the electrode is immersed in a solution containing neutravidin-horseradish peroxidase conjugate (N-HRP), which binds to the sites containing biotin-LPS on the electrode. The system is rinsed and a current signal is generated by the addition of hydrogen peroxide and a redox mediator. The assay is able to detect LPS from Salmonella minnesota at concentrations as low as 0.1 ng ml(-1), equivalent to 0.07 EU ml(-1).     

A novel glucose biosensor using bi-enzyme incorporated with peptide nanotubes

A novel amperometric glucose biosensor was developed using the bio-inspired peptide nanotube (PNT) as an encapsulation template for enzymes. Horseradish peroxidase (HRP) was encapsulated by the PNT and glucose oxidase (GO(x)) was co-immobilized with the PNT on a gold nanoparticle (AuNP)-modified electrode. A binary SAM of 3-mercaptopropionic acid (MPA) and 1-tetradecanethiol (TDT) was formed on the surface of the electrode to immobilize the PNT and GO(x). The resulting electrode appeared to provide the enzymes with a biocompatible nanoenvironment as it sustained the enhanced enzyme activity for an extended time and promoted possible direct electron transfer through the PNT to the electrode. Performance of the biosensor was evaluated in terms of its detection limit, sensitivity, pH, response time, selectivity, reproducibility, and stability in a lab setting. In addition the sensor was tested for real samples. The composite of AuNP-SAM-PNT/HRP-GO(x) to fabricate a sensor electrode in this study exhibited a linear response with glucose in the concentration range of 0.5-2.4mM with a R(2)-value of 0.994. A maximum sensitivity of 0.3mAM(-1)and reproducibility (RSD) of 1.95% were demonstrated. The PNT-encapsulated enzyme showed its retention of >85% of the initial current response after one month of storage.     

GPI-linked endothelial CD14 contributes to the detection of LPS

The inflammatory endothelial response to LPS is critical to the host's surviving a gram-negative bacterial infection. In this study we investigated whether human endothelial cells express the functional coreceptor for LPS, CD14, and most importantly whether it is glycosylphosphatidylinositol (GPI) linked. We also examined whether plasma proteins could reconstitute an LPS response in CD14-inhibited endothelium. RT-PCR- and CD14-specific MAbs demonstrated CD14 expression on primary human umbilical vein endothelial cells (HUVEC) but not passaged HUVEC. The amino acid sequence of endothelial CD14 was 99% homologous to CD14 on monocytes. Endothelium responded to relatively low levels of LPS in the absence of plasma, and this was entirely dependent on CD14. Removal of GPI-linked proteins with phosphatidylinositol-phospholipase C prevented LPS detection and subsequent protein synthesis (E-selectin expression). Endothelial CD14 was sufficient to initiate functional leukocyte recruitment, an event inhibited by blocking its LPS binding epitope and also by removing CD14 from the endothelial surface. Plasma proteins restored only approximately 30% of the LPS response in CD14-inhibited endothelium. In conclusion, our results strongly support an important role for endothelial membrane CD14 in the activation of endothelium for leukocyte recruitment.     

Real-time electrochemical imaging using an individually addressable multi-channel electrode.

We developed a real-time electrochemical imaging method that uses a multiple enzyme-modified microelectrode. The method will enable the investigation of the functions of biological materials and cells. To test its effectiveness, we imaged the two-dimensional concentration distribution for hydrogen peroxide and L-glutamate in a standard solution. The multiple electrode consists of an 8 x 8 array of 30 x 30 microm2 carbon micro electrode. Each electrode was connected to a 64-channel potentiostat that could apply a potential to all electrodes at the same time. The multiple electrode was coated with an Os-polyvinylpyridine based polymer (Os-gel) containing horse radish peroxidase (HRP) to detect hydrogen peroxide, which is a very common product of oxidase enzyme. When measuring glutamate, which is a well-known neurotransmitter in the mammalian central nerve system, we modified the electrode with a bilayer of Os-gel-HRP and GluOx. The detection limit of our method was 1 microM and images of the glutamate concentration-distribution changes induced by local injection of glutamate through microcapillary were obtained in real time.     

Iron(II) phthalocyanine-modified carbon-paste electrode for potentiometric detection of ascorbic acid

A chemically modified electrode constructed by incorporating iron(II) phthalocyanine [Fe(II)Pc] into carbon-paste matrix was used as a sensitive potentiometric sensor for detection of ascorbic acid. The resulting electrode exhibits catalytic properties for the electrooxidation of ascorbic acid, and lowers the overpotential for the oxidation of this compound. The faster rate of electron transfer results in a near-Nernstian behavior of the modified electrode, and makes it a suitable potentiometric sensor for detection of ascorbic acid. A linear response in concentration range from 10(-6) to 10(-2) M (0.18--1800 microg ml(-1)) was obtained with a detection limit of 5 x 10(-7) M for the potentiometric detection of ascorbic acid. The modified electrode was used for the determination of ascorbic acid in vitamin preparations. The recovery was 97.2--102.4% for the vitamin added to the preparations with a relative standard deviation of less than 5%. The modified electrode exhibited a fast response time (<10 s),had good stability, and had an extended lifetime.     

A doubly amplified electrochemical immunoassay for carcinoembryonic antigen

An ultrasensitive electrochemical immunoassay (EIA) for the detection of carcinoembryonic antigen (CEA) is described in this report. The assay involves utilizing enzyme-catalyzed deposition of a redox polymer and electrocatalytic oxidation of ascorbic acid (AA) by the deposited redox polymer, a dual-amplification scheme to enhance analytical signals. Briefly, CEA capturing antibody and redox polymer anchoring agent were covalently immobilized on a gold electrode. After incubating with CEA, the electrode was treated in detection antibody-glucose oxidase conjugate solution. Thereafter, it was dipped into the redox polymer solution. Upon the addition of glucose, the redox polymer was enzymatically reduced and deposited on the electrode surface. The deposited redox polymer exhibits excellent electrocatalytic activity towards the oxidation of AA. Consequently, CEA could be quantified amperometrically. This electrochemical immunoassay combines the specificity of the immunological reaction with the sensitivity of the doubly amplified electrochemical detection.     

Use of a copper-phthalocyanine membrane electrode for rapid preliminary detection of polycyclic mutagens

Preliminary screening of polycyclic mutagens is achieved within 20 min by using a biomimetic electrode composed of an oxygen electrode and a copper-phthalocyanine membrane. When benzo[alpha]pyrene (0.05 mM) was added to the buffer solution in the presence of 0.98 M hydrogen peroxide, the current of the phthalocyanine electrode decreased. A linear relationship was obtained between the current decrease and the benzo[alpha]pyrene concentration over the range 0.19-0.60 mM. The minimum measurable concentration for benzo[alpha]pyrene was 0.01 mM. Such responses were not obtained for other organic compounds such as alcohol, ether, n-hexane and cyclohexane. The copper-phthalocyanine membrane electrode has selectively detected polycyclic mutagens such as amino acid pyrolysis products. The current decrease was 1.18-1.46 microA when 0.05 mM amino acid pyrolysis products were employed.     

Disposable amperometric immunosensing strips fabricated by Au nanoparticles-modified screen-printed carbon electrodes for the detection of foodborne pathogen Escherichia coli O157:H7

A disposable amperometric immunosensing strip was fabricated for rapid detection of Escherichia coli O157:H7. The method uses an indirect sandwich enzyme-linked immunoassay with double antibodies. Screen-printed carbon electrodes (SPCEs) were framed by commercial silver and carbon inks. For electrochemical characterization the carbon electrodes were coupled with the first E. coli O157:H7-specific antibody, E. coli O157:H7 intact cells and the second E. coli O157:H7-specific antibody conjugated with horseradish peroxidase (HRP). Hydrogen peroxide and ferrocenedicarboxylic acid (FeDC) were used as the substrate for HRP and mediator, respectively, at a potential +300 mV vs. counter/reference electrode. The response current (RC) of the immunosensing strips could be amplified significantly by 13-nm diameter Au nanoparticles (AuNPs) attached to the working electrode. The results show that the combined effects of AuNPs and FeDC enhanced RC by 13.1-fold. The SPCE immunosensing strips were used to detect E. coli O157:H7 specifically. Concentrations of E. coli O157:H7 from 10(2) to 10(7)CFU/ml could be detected. The detection limit was approximately 6CFU/strip in PBS buffer and 50CFU/strip in milk. The SPCE modified with AuNPs and FeDC has the potential for further applications and provides the basis for incorporating the method into an integrated system for rapid pathogen detection.