Direct electron transfer reactions between human ceruloplasmin and electrodes

In an effort to find conditions favouring bioelectrocatalytic reduction of oxygen by surface-immobilised human ceruloplasmin (Cp), direct electron transfer (DET) reactions between Cp and an extended range of surfaces were considered. Exploiting advances in surface nanotechnology, bare and carbon-nanotube-modified spectrographic graphite electrodes as well as bare, thiol- and gold-nanoparticle-modified gold electrodes were considered, and ellipsometry provided clues as to the amount and form of adsorbed Cp. DET was studied under different conditions by cyclic voltammetry and chronoamperometry. Two Faradaic processes with midpoint potentials of about 400 mV and 700 mV vs. NHE, corresponding to the redox transformation of copper sites of Cp, were clearly observed. In spite of the significant amount of Cp adsorbed on the electrode surfaces, as well as the quite fast DET reactions between the redox enzyme and electrodes, bioelectrocatalytic reduction of oxygen by immobilised Cp was never registered. The bioelectrocatalytic inertness of this complex multi-functional redox enzyme interacting with a variety of surfaces might be associated with a very complex mechanism of intramolecular electron transfer involving a kinetic trapping behaviour.     

Determination of ceruloplasmin in human serum by SEC-ICPMS

This paper describes an analytical method for the determination of ceruloplasmin (Cp) in human serum. The method uses immunoaffinity chromatography and size-exclusion chromatography (SEC) to “purify” the serum sample prior to analysis of ⁶³Cu and ⁶⁵Cu by inductively-coupled plasma mass spectrometry (ICPMS). By removing the six most abundant proteins from serum with immunoaffinity chromatography and by using SEC to separate Cu bound by Cp from any free Cu that might be present in the serum sample, we demonstrated that SEC-ICPMS can accurately and reproducibly measure Cp in the ERM DA470 reference serum. Cp identification is based on retention time match of the unknown in the serum sample with the Cp external standard and the presence of ⁶³Cu and ⁶⁵Cu at a ratio of 2.2±0.1. This method was used to analyze a reference serum certified for Cp, 47 serum samples from four different diseases and a set of normal controls. The reference serum and a serum sample from a patient with myocardial infarction, as well as a Cp standard, were also analyzed by electrospray mass spectrometry to confirm the presence of Cp in the SEC fraction known to contain ⁶³Cu.     

锌与Wilson氏病

用锌剂治疗18例Wilson氏病患者，83％的患者获得满意效果，尤以说话不清、流涎、肢体震颤改善较明显，但血清铜氧化酶无明显改善，Wilson氏病患者的发锌与正常人对照、治疗前后自身对用均无差异，推测高浓度锌可能通过在肠道阻止铜的吸收和促使内源性铜排出面起到治疗作用，但不能根治Wilson氏病，由于锌的毒性小，口服锌剂可作为改善Wilson氏病临床症状的治疗方法之一。     

Electrochemical Magnetic Immunosensors for the Determination of Ceruloplasmin

Electrochemical immunosensors for ceruloplasmin (Cp) are reported for the first time. Two configurations involving magnetic beads (MBs) functionalized with Protein A or Streptavidin for immobilization of Cp antibodies were compared, using competitive immunoassay with synthesized alkaline phosphatase‐Cp conjugate. Upon capturing MBs‐immunoconjugates onto screen‐printed carbon electrodes, quantification of Cp was accomplished by DPV measurement of 1‐naphthol generated after 1‐naphthylphosphate addition. Linear ranges of calibration curves and detection limits were 0.1–1000 µg/mL and 0.040 µg/mL (Protein A‐MBs), and 0.025–20 µg/mL and 0.018 µg/mL (Strept‐MBs). Good results were obtained in the determination of Cp in spiked human serum samples.     

The selective immobilization of ceruloplasmin onto the chromatographic material used for its purification

Summary When mammalian plasma was passed through a chromatographic material containing aminoethyl functional groups, ceruloplasmin was selectively retained. At a specific ionic strength of the eluant buffer, a single chromatographic peak corresponding to the electrophoretically homogeneous purified ceruloplasmin was eluted. This single-step procedure is easy to perform and gave a purification yield of more than 60%. The direct immobilization of the ceruloplasmin, while it was still adsorbed and concentrated at the basal part of the gel bed (last stage of the purification), was achieved by carbodiimide treatment, with coupling yields of 50–70%.The immobilized ceruloplasmin retained about 100% of its enzymatic activity. Kinetic studies have shown a decreased affinity of the immobilized protein for the substrate and a maximal velocity of 81% as compared to the free protein. The immobilized ceruloplasmin was much more resistant to proteolytic attack than the free enzyme which is highly protease sensitive. Using pronase and thermolysine proteases, the activity of free ceruloplasmin was entirely lost in few hours. However, under similar conditions, the immobilized ceruloplasmin exhibited a high stability, maintaining its integral activity even after 24 hours of proteolytic attack.     

A reusable piezo-immunosensor with amplified sensitivity for ceruloplasmin based on plasma-polymerized film

A reusable piezoelectric immunosensor with amplified sensitivity has been developed for the detection of ceruloplasmin (CP) in human serum. The quartz crystal microbalance (QCM) was deposited with plasma-polymerized n-butyl amine film with the surface topology further characterized by using atomic force microscopy (AFM). Anti-ceruloplasmin antibody (CP-Ab) was electrostatically adsorbed on the PPF-modified crystal via an oppositely charged polyelectrolyte layer of alginate. It was found that the alginate-mediated immobilization interface could allow for antibodies to be largely immobilized with well-retained immunoactivity. In particular, a simple regeneration process for the sensor produced, i.e. by shifting the pH, can also be realized. Moreover, an optimized assay medium containing polyethylene glycol (PEG) was tested with enhanced immunosensing response (sensitivity). A dynamic concentration range of two orders of magnitude and a detection limit of 0.15 μg ml⁻¹ of CP were observed. Analytical results of clinical samples show that the developed immunoassay is comparable with the enzyme-linked immunosorbent assay (ELISA) method. However, it presents some superior advantages over the traditional sandwich format in that the analyzing performances are direct, rapid and simple without multiple separation and labeling steps.