A new red-region substrate, tetra-substituted amino aluminium phthalocyanine, for the fluorimetric determination of H2O2 catalyzed by mimetic peroxidases

A new red-region fluorogenic substrate, tetra-substituted amino aluminium pthalocyanine, was developed for the selective determination of H2O2 based on the catalytic effect of mimetic peroxidases, viz., hemin or iron tetrasulfonatophthalocyanine (FeTSPc). Under the optimum conditions, the linearity of the calibration graph for the determination of H2O2 with hemin (or FeTSPc) as the catalyst was in the range from 0.0 to 3.0 x 10(-7) mol L-1 (or from 0.0 to 2.0 x 10(-6) mol L-1). The detection limits were 3.7 x 10(-9) and 4.9 x 10(-9) mol L-1 H2O2, respectively. The relative standard deviation (n = 7) was within 1.5% in the middle of the linear range. The peroxidase activity of the mimetic enzymes hemin and FeTSPc, the effects of some experimental conditions and the influence of foreign substances were investigated. With this substrate, 0.0-7.5 x 10(-8) mol L-1 hemin and 0.0-2.0 x 10(-6) mol L-1 FeTSPc can be determined with an accuracy and precision of about 1.3%. The potential application of the reagent was tested by the determination of H2O2 in rainwater.     

Characterization of G-quadruplex/hemin peroxidase: substrate specificity and inactivation kinetics

Recently, G-quadruplex/hemin (G4/hemin) complexes have been found to exhibit peroxidase activity, and this feature has been extensively exploited for colorimetric detection of various targets. To further understand and characterize this important DNAzyme, its substrate specificity, inactivation mechanism, and kinetics have been examined by comparison with horseradish peroxidase (HRP). G4/hemin DNAzyme exhibits broader substrate specificity and much higher inactivation rate than HRP because of the exposure of the catalytic hemin center. The inactivation of G4/hemin DNAzyme is mainly attributed to the degradation of hemin by H(2)O(2) rather than the destruction of G4. Both the inactivation rate and catalytic oxidation rate of G4/hemin DNAzyme depend on the concentration of H(2)O(2), which suggests that active intermediates formed by G4/hemin and H(2)O(2) are the branch point of catalysis and inactivation. Reducing substrates greatly inhibit the inactivation of G4/hemin DNAzyme by rapidly reacting with the active intermediates. A possible catalytic and inactivation process of G4/hemin has been proposed. These results imply a potential cause for the hemin-mediated cellular injury and provide insightful information for the future application of G4/hemin DNAzyme.     

Oxygen-Binding Profile of the Iron Porphyrin Complex Embedded in Polymerized Liposome: Effect of the Polymerized Liposome on the Stability and the Oxygen-Binding Ability of the Iron Porphyrin Complex

5,10,15,20-Tetra(α,α,α,α, -o-(2′, 2′-dimethyl-20′-(2′ α-trimethylammonioethyl)phosphonatoxyeicosanamido)phenyl)porphinatoiron(II) (lipidheme) complex embedded in polymerized liposome was prepared by polymerizing l-(9-(p-vinylbenzoyl)nonanoyl)-2-O-octadecyl-rac-glycero-3-phosphocholine in the presence of lipid-heme under ultraviolet irradiation. The polymerization proceeded rapidly, and the reduction of the hemin to the heme occurred spontaneously during the polymerization. The lipid-heme complex embedded in the polymerized liposome bound molecular oxygen reversibly under physiological conditions (pH 7, 37°C) and was chemically, physically, and mechanically stable during storage for a long period and even in a high-speed flow system. The oxygen-binding affinity was not affected by the type of medium due to the effect of the rigid polymerized liposome.     

Mimicry of peroxidase by co-immobilization of 1-allylimidazole and hemin on N-isopropylacrylamide-based hydrogel

Hemin and 1-allylimidazole (AI) were copolymerized covalently to N,N′-methylene-bis-acrylamide (MBA) cross-linked N-isopropylacrylamide (NIPAAm) hydrogel to form a hemin-based mimetic enzyme of peroxidase, i.e., poly(NIPAAm/MBA/hemin/imidazole). Its catalytic activity was studied in comparison with hemin and a similar hydrogel, poly(NIPAAm/MBA/hemin), that was prepared in the absence of AI. The evaluation was based on their activities in catalyzing oxidation of fluorogenic substrate p-hydroxyphenylacetic (p-HPA) with H₂O₂ as the oxidant. The results showed that the activity of poly(NIPAAm/MBA/hemin/imidazole) is 21% higher than that of poly(NIPAAm/MBA/hemin) and 102% higher than hemin. This mimetic enzyme can be used for the determination of hydrogen peroxide with a detection limit (S/N = 3) of 26 nmol l⁻¹.     

Synthesis of an enzyme-like imprinted polymer with the substrate as the template, and its catalytic properties under aqueous conditions

Transition state analogues (TSAs) have long been regarded as ideal templates for the preparation of catalytically active synthetic imprinted polymers. In the current work, however, a new type of molecularly imprinted polymer (MIP) was synthesized with the substrate (homovanillic acid, HVA) as the template and hemin introduced as the catalytic center, with the use of plural functional monomers to prepare the active sites. The MIP successfully mimicked natural peroxidase, suggesting that it may not be imperative to employ a TSA as the template when preparing enzyme-like imprinted polymers and that the imprinted polymer matrix provided an advantageous microenvironment around the catalytic center (hemin), essentially similar to that supplied by apo-proteins in natural enzymes. Significantly, by taking advantage of the special structure of hemin and multiple-site interactions provided by several functional monomers, the intrinsic difficulties for MIPs in recognizing template molecules in polar solutions were overcome. The newly developed polymer showed considerable recognizing ability toward HVA, catalytic activity, substrate specificity and also stability, which are the merits lacked by the natural peroxidase. Meanwhile, the ease of recovery and reuse the MIP implies the potential for industrial application.     

Protein G-liposomal nanovesicles as universal reagents for immunoassays

To improve the antigen-binding activity of liposome-coupled antibodies and to develop universal liposomal nanovesicles for immunoassays, protein G was conjugated to dye-loaded liposomal nanovesicles for the preparation of immunoliposomes. Sulfosuccinimidyl-4-(N-maleimidomethyl) cyclohexane-1-carboxylate (sulfo-SMCC), a heterobifunctional cross-linker, was used to modify protein G for conjugation to the liposomal nanovesicles. Liposome immunosorbent assays were used to evaluate the binding ability of protein G after sulfo-SMCC modification, to optimize the protein G density on the liposome surface and to determine the amount of IgG binding to the protein G-liposomal nanovesicles. Test strips coated with a narrow zone of antibodies were used to show the successful conjugation. Immunomagnetic beads were used to demonstrate the feasibility of protein G-tagged universal liposomal nanovesicles for immunoassays. Results indicate that the Fc-binding capacity of protein G decreased by only 5.3% after sulfo-SMCC modification. Antibodies were easily conjugated to universal protein G-liposomal nanovesicles in 30 min. The conjugates (protein G-immunoliposomes) were successfully used in immunomagnetic bead assays for the detection of Escherichia coli O157:H7 with a detection limit of approximately 100 CFU/ml. This work demonstrated that protein G-liposomal nanovesicles are a successful universal reagent for easily coupling antibodies in an active orientation on the liposome surface for use in immunoassays.     

Synthesis and Characteristics of a New Fluorogenic Substrate for Horseradish Peroxidase

N,N′-Dicyanomethyl-o-phenylenediamine was synthesized with a 90% yield by a reaction ofo-phenylenediamine with chloroacetonitrile in triethylamine. Our experimental results showed that it was the effective fluorogenic substrate for horseradish peroxidase (HRP) and hemin. TheK_mfor the HRP system was 48 μM,and that for hemin was 1.3 μM.Properties of the substrate were evaluated from the detection limits of enzymes and H₂O₂. The linear ranges for the determination of HRP and hemin were 21–150 pMand 2–20 nM,respectively. The linear range for the determination of H₂O₂using HRP or hemin was 18–140 and 60–1000 nM,respectively. The structural elucidation of the fluorescent product using NMR and mass spectral techniques was proposed to be 1,2-dihydro-2-imido-imidazo[1,2-a] quinoxaline. Based on the product structure and earlier reports, the possible reaction mechanism of HRP and the substrate was also proposed, i.e., two steps for ring closures, one step of isomerization, and a final step of oxidative dehydrogenation.     

A G‐Quadruplex/Hemin Complex with Switchable Peroxidase Activity by DNA Hybridization

A hemin‐binding DNA G‐quadruplex (also known as a hemin aptamer or DNAzyme) has been previously reported to be able to enhance the peroxidase activity of hemin. In this work, we described a DNAzyme structure that had an effector‐recognizing part appearing as a single stranded DNA linkage flanked by two split G‐quadruplex halves. Hybridization of the single stranded part in the enzyme with a perfectly matched DNA strand (effector) formed a rigid DNA duplex between the two G‐quadruplex halves and thus efficiently suppressed the enzymatic activity of the G‐quadruplex/hemin complex, while the mismatched effector strand was not able to regulate the peroxidase activity effectively. With 2,2′‐azinobis(3‐ethylbenzthiazoline)‐6‐sulfonic acid (ABTS) as an oxidizable substrate, we were able to characterize the formation of the re‐engineered G‐quadruplex/hemin complex and verify its switchable peroxidase activity. Our results show that the split G‐quadruplex is an especially useful module to design low‐cost and label‐free sensors toward various biologically or environmentally interesting targets.     

Assay of cell surface-bound immunoliposomes using monoclonal antibody reactive with a cross-linking reagent.

A monoclonal antibody (mAb) reactive with a crosslinking reagent, N-(m-maleimidobenzoyl)dipalmitoylphosphatidylethanolamine (mMBPE), in liposomes was produced from a hybridoma clone established by a fusion between P3X63Ag8.653 mouse myeloma cells and spleen cells from a BALB/c mouse hyperimmunized with the antibody-coated liposomes containing mMBPE. Using this mAb (termed AL-6), the quantity of immunoliposomes bound on target tumor cells was assessed by flow cytofluorometry. The results obtained using fluorescein isothiocyanate-coupled AL-6 allowed the enumeration not only of the immunoliposomes bound on all tumor cells but also those on individual target tumor cells. The relevance of this assay method was confirmed by a comparison with another assay method of cell-bound liposomes using immunoliposomes containing carboxyfluorescein in the vesicles.     

Flat hydrogel substrate for atomic force microscopy to observe liposomes and lipid membranes

In order to avoid denaturation of biomolecules due to strong adsorption on solid surfaces, a soft substrate has to be used for atomic force microscopy (AFM) observation. We propose a hydrophilic agarose gel surface as a soft substrate for AFM to observe liposomes and lipid membranes. Although our simple method does not require any delicate control at the molecular level, an agarose gel surface can be simply flattened to 0.3 nm in roughness using an atomically flat solid surface during gelation. The AFM images revealed that liposomes were unruptured on the gel surface at low liposome density, whereas an unruptured state was difficult to obtain on a solid surface like mica. This indicates that the weak interaction between the liposome and the soft surface inhibits the liposome from rupturing, and also that the surface rougher than the solid surface prevents lateral diffusion of the liposomes along the surface to be fused. Increasing the liposome density resulted in a lipid membrane at various thicknesses forming on the hydrogel surface by the fusion and rupture of liposomes. Using the soft substrate, it can be expected to promote investigations of structures and functions of biomolecules at the nanometer scale under physiological conditions with AFM.     

增强氯化血红素催化活性用于水胺硫磷比色检测研究

建立了一种基于增强氯化血红素(Hemin)过氧化物酶催化活性比色检测水胺硫磷的方法.在H2O2存在下, Hemin催化氧化底物3,3′,5,5′-四甲基联苯胺(TMB), 使其失去1个电子, 导致反应体系由无色变为蓝绿色.水胺硫磷的加入可提高Hemin对底物亲和力, 进一步增强其催化活性, 使底物氧化失去2个电子, 反应体系由蓝绿色变为黄色, 且颜色变化程度与水胺硫磷浓度成正比.在最优条件下, 本方法的动态检测范围为2～100 μg/L, 检出限为1.2 μg/L(3σ).其它有机磷农药对水胺硫磷检测无明显干扰, 实际样品中水胺硫磷的加标回收率为93.0％～113.0％. 本方法可应用于农产品中水胺硫磷残留的检测.     

Liposomes as signal amplification reagents for bioassays in microfluidic channels

Liposomes with encapsulated carboxyfluorescein were used in an affinity-based assay to provide signal amplification for small-volume fluorescence measurements. Microfluidic channels were fabricated by imprinting in a plastic substrate material, poly(ethylene terephthalate glycol) (PETG), using a silicon template imprinting tool. Streptavidin was linked to the surface through biotinylated-protein for effective immobilization with minimal nonspecific adsorption of the liposome reagent. Lipids derivatized with biotin were incorporated into the liposome membrane to make the liposomes reactive for affinity assays. Specific binding of the liposomes to microchannel walls, dependence of binding on incubation time, and nonspecific adsorption of the liposome reagent were evaluated. The results of a competitive assay employing liposomes in the microchannels are presented.     

Amplified surface plasmon resonance based DNA biosensors, aptasensors, and Hg2+ sensors using hemin/G-quadruplexes and Au nanoparticles

Thiolated nucleic acid hairpin nanostructures that include in their stem region a "caged" G-quadruplex sequence, and in their single-stranded loop region oligonucleotide recognition sequences for DNA, adenosine monophosphate (AMP), or Hg(2+) ions were linked to bare Au surfaces or to Au nanoparticles (NPs) linked to Au surfaces. The opening of the hairpin nanostructures associated with the bare Au surface by the complementary target DNA, AMP substrate, or Hg(2+) ions, in the presence of hemin, led to the self-assembly of hemin/G-quadruplexes on the surface. The resulting dielectric changes on the surface exhibited shifts in the surface plasmon resonance (SPR) spectra, thus providing a readout signal for the recognition events. A similar opening of the hairpin nanostructures, immobilized on the Au NPs associated with the Au surface, by the DNA, AMP, or Hg(2+) led to an ultrasensitive SPR-amplified detection of the respective analytes. The amplification originated from the coupling between the localized surface plasmon associated with the NPs and the surface plasmon wave, an effect that cooperatively amplifies the SPR shifts that result from the formation of the hemin/G-quadruplexes. The different sensing platforms reveal impressive sensitivities and selectivities toward the target analytes.     

Liposome-based immunostrip for the rapid detection of Salmonella

Salmonellae are ubiquitous human pathogens, which pose a danger to the elderly and children. Due to the increased number of outbreaks of human illness associated with the consumption of contaminated products in the USA and many other countries, there is an urgent need to develop rapid assays to detect common food-borne pathogens. This study demonstrates the feasibility of using a detectable label comprising methyl blue (MB), a visible dye, entrapped inside liposomes. Immunoliposomes tagged with anti-Salmonella common structural antigens (CSA) antibody encapsulating MB dye were prepared and used as the signal amplifier for the development of a field-portable colorimetric immunoassay to detect Salmonellae. Tapping mode atomic force microscopy (TMAFM), a scanning probe technique, was utilized to demonstrate the presence of anti-Salmonella antibody at the thus-prepared liposome. A plastic-backed nitrocellulose strip with two immobilized zones formed the basis of a sandwich assay. The first zone was the antigen capture zone (AC zone), used in a sandwich (noncompetitive) assay format; the other was the biotin capture zone (BC zone), used as a quality control index for the strip assay. During the capillary migration of the wicking reagent containing 80 μL of immunoliposomes and 40 μL of the test sample (heat-killed S. typhimurium), sample pathogens with surface-bound immunoliposomes were captured at the AC zone, while the unbound immunoliposomes continued to migrate and bind to the anti-biotin antibodies coated on the BC zone. The color density of the AC zone was directly proportional to the number of Salmonella typhimurium in the test sample. The detection limit of the current assay with heat-killed Salmonella typhimurium was 1,680 cells. The cross-reactivity of the proposed immunoassay was also investigated, and pathogens including E. coli O157:H7 and Listeria genus specific caused no interference with the detection of Salmonella typhimurium. Shi-Chin Zeng and Wei-Hsiang Tseng contributed equally to this publication.     

DNA sensing on glassy carbon electrodes by using hemin as the electrochemical hybridization label

The electrochemical behavior of hemin, an iron complex of porphyrin, on binding to DNA at a glassy carbon electrode (GCE) and in solution, is described. Hemin, which interacts with covalently immobilized calf thymus DNA, was detected by use of a bare GCE, a double-stranded DNA-modified GCE (dsDNA-modified GCE), and a single-stranded DNA-modified GCE (ssDNA-modified GCE), in combination with differential pulse voltammetry (DPV). The structural conformation of DNA was determined from changes in the voltammetric signals acquired on reduction of hemin. As a result of its large steric structure and anionic substitution on its porphyrin plane, hemin intercalates between the base pairs of dsDNA. A scan-rate study for hemin and the dsDNA-hemin complex were also performed to determine the electrochemical behavior of the complex. The partition coefficient was obtained from the peak currents measured when different concentrations of hemin were in the presence of dsDNA. By observing the oxidation signals of guanine, damage to DNA after reaction with hemin at the GCE surface was also detected. The electrochemical detection of hybridization between the covalently immobilized probe and its target sequence was detected by use of hemin. These results demonstrate the use of DNA biosensors in conjunction with hemin for electrochemical detection of hybridization and damage to DNA.     

轴向配基对HRP模拟酶Hemin催化荧光及化学发光反应影响的研究

以氯化血红素作为辣根过氧化物酶（ＨＲＰ）的模拟物,对二十种氨基酸和其他含氮有机配体的轴向配位效应进行了研究。发现在氯化血红素催化的荧光和化学发光体系中,组氨酸和咪唑均使ｈｅｍｉｎ的催化活性显著提高,而酪氨酸,色氨酸,Ｌ－半胱氨酸及卤代烷基吡啶等具有淬灭效应。     

Investigation of the Photodecomposition of Hemin and Trace Amount Determination of Hemin with Flow Injection Chemiluminescence Method

Using the hemin‐H₂O₂‐Na₂CO₃‐NaOH chemiluminescence (CL) system, the study on the photodecomposition behavior of hemin under ultraviolet light and solar light were carried out and the determination of hemin was developed coupled with simple flow injection technique. The results showed that the decomposition reaction of hemin in different light irradiations corresponded with a first‐order reaction. And then the determination of hemin was completed by the CL emission from the reaction of hemin with H₂O₂ in aqueous carbonate. The linear range was 2.2 × 10^?10 to 6.88 × 10^?7 M and the detection limit was 2.2 × 10^?11 M (S/N = 3). The relative standard deviation (RSD) was 2.82% for ten independent detections of 1.72 × 10^?8 M hemin. As a preliminary application, the proposed method was successfully applied for the analysis of hemin in pharmaceutical formulations and animal blood with a recovery of 96?108%. A possible CL mechanism of the present system was discussed, and free radicals were suggested to be involved in this reaction.     

氯化血红素分子对牛血清白蛋白电子传输能级的调控

蛋白质分子的电子传输(ETp)性能,即导带(CB)和价带(VB)的能量差(带隙)是影响蛋白质电子器件性能的主要因素之一。因此,调控蛋白质ETp带隙是提高这些电子器件性能并扩展其应用领域的重要途径。本文报道一种通过外部分子结合调控蛋白质ETp带隙的方法。以氯化血红素(hemin)与牛血清白蛋白(BSA)结合为例,首先运用分子对接方法从理论上确定hemin分子能结合到BSA分子IIA域的疏水口袋中,位于Tpr213附近;然后实验(荧光光谱和吸收光谱)证实hemin与BSA结合后,能形成hemin-BSA复合物,并且没有改变BSA的原有结构;最后将hemin-BSA通过BSA分子表面Cys34的―SH固定在金电极表面,形成有序的分子层,研究其ETp性能;I–V结果表明,BSA表现出半导体的ETp特征,并且hemin的结合能使BSA的带隙由原来的~1.50±0.05e V降低到~0.93±0.05e V。本文的结果为调控蛋白质分子的ETp带隙提供了一种简单有效的方法,通过选择不同的结合分子能使蛋白质分子的带隙调控至所需要的范围,并且形成的蛋白质复合物还能用于各种电子器件的制作。     

Liposome-mediated enhancement of the sensitivity in immunoassay based on surface-enhanced Raman scattering at gold nanosphere array substrate

A novel immunoassay based on surface-enhanced Raman scattering (SERS) has been developed. The method exploits the SERS-derived signal from reporter molecules (crystal violet, CV) encapsulated in antibody-modified liposome particles. The antigen is firstly captured by the primary antibody immobilized in microwell plates and then sandwiched by secondary antibody-modified liposome. The CV molecules are released from the liposome and transferred to specially designed substrate of gold nanosphere arrays with sub-10-nm gaps. The concentration of the antigen is indirectly read out by the SERS intensity of the CVs. The substrate used could substantially improve the sensitivity and reproducibility of SERS measurement. The SERS intensity responses are linearly correlated to logarithm of antigen concentration in the range of 1.0 x 10(-8) to 1.0 x 10(-4) gm L(-1) with a detection limit of 8 ng mL(-1). To our knowledge, this is the first report describing liposome-mediated enhancement of the sensitivity in immunoassay based on surface-enhanced Raman scattering. Experimental results show that the proposed method illustrates a potential prospect of applications in immunoassay.     

Characterization of the enhanced peroxidatic activity of amyloid β peptide–hemin complexes towards neurotransmitters

Binding of heme to the amyloid peptides Aβ40/42 is thought to be an initial step in the development of symptoms in the early stages of Alzheimer’s disease by enhancing the intrinsic peroxidatic activity of heme. We found considerably higher acceleration of the reaction for the physiologically relevant neurotransmitters dopamine and serotonin than reported earlier for the artificial substrate 3,3′,5,5′-tetramethylbenzidine (TMB). Thus, the binding of hemin to Aβ peptides might play an even more crucial role in the early stages of Alzheimer’s disease than deduced from these earlier results. To mimic complex formation, a new surface architecture has been developed: The interaction between the truncated amyloid peptide Aβ1-16 and hemin immobilized on an aminohexanethiol spacer on a gold electrode has been analyzed by cyclic voltammetry. The resulting complex has a redox pair with a 25 mV more cathodic formal potential than hemin alone.