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

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

Comparative study of some synthesised and commercial fluorogenic substrates for horseradish peroxidase and its mimetic enzyme hemin by a flow injection method

Four 3,4-dihydroquinoxalin-2(1H)-one derivatives, i.e., 3,4-dihydroquinoxalin-2(lH)-one (DHQ), 3-methyl-3,4-dihydroquinoxalin-2(1H)-one (MDHQ), 3,4-dihydroquinoxalin-2(1H)-one-6-acid (DHQ-6-A) and 3-methyl-3,4-dihydroquinoxalin-2(1H)-one-6-acid (MDHQ-6-A), and N,N′-dicyanomethyl-o-phenylenediamine (DCM-OPA) were synthesised as potential substrates for horseradish peroxidase (HRP). Of these compounds DCM-OPA, DHQ and MDHQ can be prepared by very simple methods in a pure form in large quantities. Their properties for use as fluorogenic substrates for HRP and its mimetic enzyme hemin were compared with commercially available substrates, i.e., p-hydroxyphenylacetic acid (p-HPA), p-hydroxyphenylpropionic acid (p-HPPA), homovanillic acid (HVA) and tyramine, by a flow injection method. The results showed that DCM-OPA and MDHQ were the best among the five synthesised substrates and p-HPPA and p-HPA are better than HVA and tyramine. Substrates p-HPPA, p-HPA, DCM-OPA and MDHQ showed comparable ability for H₂O₂ detection in HRP and hemin catalysed reaction systems, with detection limits in the nmol l⁻¹ region. The stability of DCM-OPA is better than MDHQ, but both are stable for at least a month in a refrigerator.     

Apoferritin protein nanoparticles dually labeled with aptamer and horseradish peroxidase as a sensing probe for thrombin detection

A novel and ultrasensitive sandwich-type electrochemical aptasensor has been developed for the detection of thrombin, based on dual signal-amplification using HRP and apoferritin. Core/shell Fe₃O₄/Au magnetic nanoparticles (AuMNPs) loading aptamer1 (Apt1) was used as recognition elements, and apoferritin dually labeled with Aptamer2 (Apt2) and HRP was used as a detection probe. Sandwich-type complex, Apt1/thrombin/Apt2–apoferritin NPs–HRP was formed by the affinity reactions between AuMNPs–Apt1, thrombin, and Apt2–apoferritin–HRP. The complex was anchored on a screen-printed carbon electrode (SPCE). Differential pulse voltammetry (DPV) was used to monitor the electrode response. The proposed aptasensor yielded a linear current response to thrombin concentrations over a broad range of 0.5–100 pM with a detection limit of 0.07 pM (S/N = 3). The detection signal was amplified by using apoferritin and HRP. This nanoparticle-based aptasensor offers a new method for rapid, sensitive, selective, and inexpensive quantification of thrombin, and offers a promising potential in protein detection and disease diagnosis.     

Performance of an aqueous two‐phase‐based countercurrent chromatographic system for horseradish peroxidase purification

Countercurrent chromatography (CCC) purification of horseradish peroxidase (HRP) from Armoracia rusticana root extracts was achieved by employing polymer‐phosphate aqueous two‐phase systems (ATPS). By using preparative columns at 1000 rpm, a 25–30% retention of the top phase of an ATPS composed of 10% w/w PEG 1540 and 14.8% w/w phosphate – with added 2 mol/kg sodium chloride – was obtained. The retention level was stable during the standard separation running time (4 h). Horseradish root extract samples were injected into the system (10–25 mL; 200–250 U/mL peroxidase; 2.0–4.0 mg/mL total protein). Retention of HRP in the CCC “column” during the chromatographic run was attained in the selected ATPS, where the partition coefficient K for the enzyme was ≥ 8. Replacement of the mobile phase with a fresh one but in the absence of added salt brought about product elution. Recovery of HRP in this fraction accounts for ≥ 45% of the total activity loaded, with a purification factor of 6. Enzyme activity was also found in the pass‐through fraction and in the remaining liquid (stationary) phase, a fact that should be ascribed to the existence of multiple peroxidase isoforms. SDS‐PAGE of the active fraction showed a protein band at 44 kDa, compatible with the presence of HRP. Thus, the optimised CCC system allowed the separation of HRP directly from a complex biological material. These results open up the possibility of achieving protein separation with CCC/ATPS and of scaling‐up processes in industrial separators.     

Electrochemical studies of o-tilidine as a hydrogen donor substrate for peroxidase and its application in enzyme immunoassay

A highly sensitive electrochemical method is described for assay of horseradish peroxidase (HRP) using o-tilidine as substrate. Under the optimal conditions, the detection limit for HRP is 2.5 mU/l and the calibration range is from 5.0 to 1000 mU/l. The relative standard deviation of 11 measurements is 6.3% for 10.0 mU/l HRP. This new electrochemical system was further combined with an indirect enzyme immunoassay using direct antigen-coating format for the detection of cucumber mosaic virus (CMV). The results show an improved sensitivity over the traditional o-phenylenediamine (OPD) spectrophotometric enzyme-linked immunosorbent assay (ELISA) method. Using this technique, a minimum detectable level of 2.0 ng/ml of purified CMV and 1:12500 dilution of CMV in infected leaf extractions can be achieved.     

Time-resolved fluorescence study of the single tryptophan in thiocyanate and azide derivatives of horseradish peroxidase: Implication for a p H-induced conformational change in the heme cavity

Detailed pH-dependent steady state and picosecond time-resolved tryptophan fluorescence studies on thiocyanate and azide complexes of horseradish peroxidase have been carried out. The fluorescence decay of the single tryptophan in these species was fitted to a discrete three exponential model. Maximum entropy method analysis also gave three distinct regions of lifetime distributions. The fast subnanosecond lifetime component was found to have > 97% amplitude contribution while other two longer lifetime components have small contributions. Small contributions from the nanosecond lifetime components possibly arise from apoprotein impurity or some small amount of disordered heme conformer of the protein. pH dependence of the fast picosecond lifetime components was found to show a systematic behavior which has been interpreted in the light of obligatory conformation change associated with activation of the enzyme at low pH.     

Fibre optic fluorometric enzyme sensors for hydrogen peroxide and lactate,based on horseradish peroxidase and lactate oxidase

An optical biosensor for the determination of hydrogen peroxide based on immobilized horseradish peroxidase is described. The fluorescence of the dimeric product of the enzyme catalysed oxidation of homovanillic acid is utilized to determine the concentration of H₂O₂. The membrane-bound enzyme is attached to a bifurcated fibre bundle permitting excitation and detection of the fluorescence by a fluorometer. The response of the sensor is linear from 1 to 130 M hydrogen peroxide; the coefficient of variation is 3%. The sensor is stable for more than 10 weeks. The operating pH for maximal sensor response is 8.15. This allows the sensor to be used in combination with oxidase reactions producing hydrogen peroxide, as is demonstrated with a co-immobilized lactate oxidase-horseradish peroxidase optode for the determination of L-lactate. The fluorescence intensity of this sensor depends linearly on the concentration of lactate between 3 and 200 M and a throughput of 10 samples per hour is possible. The precision is in the same range as that of the monoenzyme optode. The lifetime of the bienzyme sensor for lactate is considerably shorter than that of the peroxidase sensor; it is limited by the stability of the immobilized lactate oxidase enzyme. The sensor has been applied to the determination of lactate in control serum.     

Rapid measurement of fish freshness indices by an amperometric flow-injection system with a 16-way switching valve and immobilized enzyme reactors

A novel flow-injection system is proposed for the rapid measurement of the fish freshness indices K₁ and K₂: K₁=[([HxR+[Hx])×100/([IMP]+[HxR]+[Hx])] and K₂=[[Hx]×100/([HxR]+[Hx])], where [IMP], [HxR] and [Hx] are inosine-5′-monophosphate, inosine and hypoxanthine concentrations, respectively. For the estimation of index K₁, 5′-nucleotidase immobilized reactor and nucleoside phosphorylase (NP)/xanthine oxidase (XO) coimmobilized reactor were incorporated in series in the flow-injection line made up by a 16-way switching valve with two sample loops. For the estimation of index K₂, NP and XO immobilized reactors were also incorporated in the similar flow-line. Two sample portions passed through the flow-line with different residence times so that two peaks were obtained. The first and second peaks obtained in the K₁-determining system corresponded to the total of HxR and Hx and the total of Hx, HxR and IMP, respectively. Similarly, the first and second peaks obtained in the K₂-determining system corresponded to Hx and the total of Hx and HxR, respectively. Therefore, the indices K₁ and K₂ can be estimated by

where i₁ and i₂ present the peak current of the first and second peaks, respectively, and f the ratio of the peak currents of the first and second peaks for a Hx standard solution. A sea bream was selected as a model fish and it was stored at 4 °C after death. A good correlation was found between the index K₁ and the storage time over a period of 400 h, with a correlation coefficient of 0.992, but no correlation between the index K₂ and the storage time. The measurements could be performed at a rate of 22 samples per hour with satisfactory precision (0.6–1.2% R.S.D.), without calibration for each species, accurate weighing of fish meat and any interferences in fish meat.     

A novel method for time-resolved fluorimetric determination and imaging of the activity of peroxidase, and its application to an enzyme-linked immunosorbent assay

A new type of fluorescence assay for the determination of peroxidase (POx) activity is presented. The assay is based on the indication of the enzymatic consumption of H(2)O(2) (HP), using a fluorescent europium-tetracycline (Eu(3)TC) complex as indicator. On addition of HP, this complex forms a highly fluorescent adduct (Eu(3)TC-HP), which is decomposed in the presence of POx to form the weakly fluorescent europium-tetracycline (Eu(3)TC). Hence, the activity of the enzyme can be directly determined by means of the luminescent Eu(3)TC complex as indicator. The POx assay demonstrated herein was elaborated starting from a spectral characterization of the complex systems involved. Due to the long lifetime of lanthanide luminescence, both steady-state and time-resolved luminescence assays can easily be performed. The time-resolved assay can quantify POx in the range from 4.0 x 10(-5) to 5.9 x 10(-3) U mL(-1), with a limit of detection of 1.0 x 10(-5) U mL(-1). The effects of POx inhibitors such as cyanide, hydroxylamine, and azide have also been studied. In addition, a time-resolved fluorescent detection method for a POx-based enzyme-linked immunosorbent assay (ELISA) has been developed, which is demonstrated in a sandwich model assay with bovine IgG serving as analyte. Furthermore, a time-resolved fluorescent imaging method is demonstrated that makes use of a straightforward imaging set-up adjusted to the optical properties of the europium reagent.     

Use of biotin-streptavidin system for developing a viable, sensitive and specific antigen heterologous assay for hapten

The present study demonstrates improvement in sensitivity and specificity of hapten assay by using antigen heterology in conjunction with low molecular weight biotin label as compared to high molecular weight horseradish peroxidase (HRP) label. For generation of antiserum, cortisol-3-O-carboxylmethyl-oxime-bovine serum albumin (F-3-CMO-BSA) was used as immunogen whereas, for the preparation of primary label, corticosterone-3-carboxymethyl oxime (B-3-CMO) was coupled with biotinylcaproylhydrazide and HRP by employing N-hydroxysuccinimide mediated carbodiimide reaction. The data of the present study revealed that the antigen heterologous assay which employed high molecular weight HRP label showed 100% cross-reaction with corticosterone. On the contrary, when HRP was replaced with low molecular weight biotin label, less than 0.1% cross-reaction was observed with all analogous C₁₈, C₁₉, C₂₁ and C₂₇ steroids including corticosterone (0.2%). Moreover, the sensitivity of the later assay was 0.09 μg/dL, which is appreciable as compared to previously reported enzyme based assays. The recovery of the exogenously spiked serum pools lies in the range of 90.3-104.2%. The intra-assay and inter-assay coefficient of variation (CVs) ranged from 3.3% to 7.8% and 2.3% to 7.7%, respectively. The serum cortisol values obtained by this method correlated well with those obtained by radioimmunoassay; r = 0.9 (n = 50). The use of much stable biotin label in place of HRP has made the antigen heterologous enzyme linked immunosorbent assay (ELISA) of cortisol assay highly specific and sensitive.     

A rapid and precise assay for peroxide as 'active oxygen' in products, by flow injection analysis in a high pressure system with spectrophotometric detection

A simple, rapid and automated assay for ‘active oxygen’ originating from hydrogen peroxide, or other organic peroxides, in products is presented employing flow injection (FI) analysis. The product is dispersed and peroxide dissolved in a solvent of 5% (v/v) acetic acid, which constitutes the carrier stream. Ammonium molybdate can be added to this carrier stream to increase sensitivity as required. The sample solution is injected into the acid carrier stream, which is then merged with iodide ion in situ in a two-stream manifold. The ‘active oxygen’ in the product oxidises acidified iodide to iodine, which is detected spectrophotometrically at 350 nm. The closed conditions prevent interference from atmospheric oxygen and the short reaction time minimises the potential for interference from side reactions. Standard HPLC equipment is used throughout, employing a back-pressure to improve precision (high pressure flow injection). Conditions have been investigated using screening multivariate experimental design (two-level quarter fractional factorial design incorporating centre points) to identify and optimise the critical variables. The method has been fully validated (with sample solution R.S.D.s typically < 0.5%, LOQs of 0.04 or 0.006 μg ml⁻¹ as ‘active oxygen’ for acid or acid/molybdate carriers respectively) and is quicker and simpler than the currently employed manual titration approach. It should be applicable to a range of ‘active oxygen’ products.     

Preparative isolation and purification of ginsenosides Rf,Re, Rd and Rb1 from the roots of Panax ginseng with a salt/containing solvent system and flow step-gradient by high performance counter-current chromatography coupled with an evaporative light scattering detector

Ginseng is a popular herb worldwide and has had varied uses in traditional Asian medicine for thousands of years. There are several different species of the herb, but all share the same constituents. Ginsenosides, the most extensively studied chemical components of ginseng, are generally considered to be one of the most important active ingredients of the plant. In this study, we have developed fast and efficient methodology for isolation of four known ginsenosides Rf, Rd, Re and Rb1 from Ginseng by high performance counter-current chromatography (HPCCC) coupled with evaporative light scattering detection (ELSD). The crude sample for HPCCC was purified firstly from a ginseng extraction using macroporous resin. The enriched saponin fraction (480 mg) was separated by using methylene chloride–methanol–5 mM aqueous ammonium acetate–isopropanol (6:2:4:3, v/v,) as the two-phase solvent system and yielded 10.7 mg of Rf, 11.0 mg of Rd, 13.4 mg of Re and 13.9 mg of Rb1. The purity of these ginsenosides was 99.2%, 88.3%, 93.7% and 91.8%, respectively assessed by HPLC-DAD-ELSD, and their structures were characterized by electrospray ionization mass spectrometry (ESI-MS) and compared with standards. Ammonium acetate was used to shorten the separation time and eliminate emulsification together with a flow step-gradient. The salt can be removed by re-dissolving the sample using acetone.