10-甲基吖啶苯酚酯衍生物的合成、表征及化学发光特性

吖啶-9-羧酸苯酚酯是一类效率较高的化学发光试剂.其结构一般有两部分组成,吖啶环发光部分和9-羧酸苯酚酯离去基团.本文对离去基团进行修饰,分别在离去基团苯酚部分的2,5或2,6位引入取代基CF₃、NO₂、Br、CH₃,合成了4个新吖啶酯衍生物,并对它们的分子结构进行了表征.4个新化合物和模型化合物(无取代基)比较,均表现出较好的化学发光效率;发光动力学基本符合文献报道规律.而且化学发光法对其水解稳定性考察显示,取代基的电性和位阻是影响稳定性的两大因素.     

Detection and identification of the designer benzodiazepine flubromazepam and preliminary data on its metabolism and pharmacokinetics

The appearance of pyrazolam in Internet shops selling ‘research chemicals’ in 2012 marked the beginning of designer benzodiazepines being sold as recreational drugs or ‘self medication’. With recent changes in national narcotics laws in many countries, where two uncontrolled benzodiazepines (phenazepam and etizolam), which were marketed by pharmaceutical companies in some countries, were scheduled, clandestine laboratories seem to turn to poorly characterized research drug candidates as legal substitutes. Following the appearance of pyrazolam, it comes with no surprise that recently, flubromazepam (7‐bromo‐5‐(2‐fluorophenyl)‐1,3‐dihydro‐2H‐1,4‐benzodiazepin‐2‐one), a second designer benzodiazepine, was offered on the market. In this article, this new compound was characterized using nuclear magnetic resonance, gas chromatography‐mass spectrometry (GC–MS), liquid chromatography–mass spectrometry (LC–MS/MS) and liquid chromatography quadrupole time‐of‐flight MS (LC–Q–ToF–MS). Additionally, a study was carried out, in which one of the authors consumed 4 mg of flubromazepam to gain preliminary data on the pharmacokinetic properties and the metabolism of this compound. For this purpose, serum as well as urine samples were collected for up to 31 days post‐ingestion and analyzed applying LC–MS/MS and LC–Q‐ToF‐MS techniques. On the basis of this study, flubromazepam appears to have an extremely long elimination half‐life of more than 100 h. One monohydroxylated compound and the debrominated compound could be identified as the predominant metabolites, the first allowing a detection of a consumption for up to 28 days post‐ingestion when analyzing urine samples in our case. Additionally, various immunochemical assays were evaluated, showing that the cross‐reactivity of the used assay seems not to be sufficient for safe detection of the applied dose in urine samples, bearing the risk that it could be misused in drug‐withdrawal settings or in other circumstances requiring regular drug testing. Furthermore, it may be used in drug‐facilitated crimes without being detected. Copyright © 2013 John Wiley & Sons, Ltd.     

Automated,high performance,flow-through chemiluminescence microarray for the multiplexed detection of phycotoxins

A novel multiplexed immunoassay for the analysis of phycotoxins in shellfish samples has been developed. Therefore, a regenerable chemiluminescence (CL) microarray was established which is able to analyze automatically three different phycotoxins (domoic acid (DA), okadaic acid (OA) and saxitoxin (STX)) in parallel on the analysis platform MCR3. As a test format an indirect competitive immunoassay format was applied. These phycotoxins were directly immobilized on an epoxy-activated PEG chip surface. The parallel analysis was enabled by the simultaneous addition of all analytes and specific antibodies on one microarray chip. After the competitive reaction, the CL signal was recorded by a CCD camera. Due to the ability to regenerate the toxin microarray, internal calibrations of phycotoxins in parallel were performed using the same microarray chip, which was suitable for 25 consecutive measurements. For the three target phycotoxins multi-analyte calibration curves were generated. In extracted shellfish matrix, the determined LODs for DA, OA and STX with values of 0.5 ± 0.3 μg L⁻¹, 1.0 ± 0.6 μg L⁻¹, and 0.4 ± 0.2 μg L⁻¹ were slightly lower than in PBS buffer. For determination of toxin recoveries, the observed signal loss in the regeneration was corrected. After applying mathematical corrections spiked shellfish samples were quantified with recoveries for DA, OA, and STX of 86.2%, 102.5%, and 61.6%, respectively, in 20 min. This is the first demonstration of an antibody based phycotoxin microarray.     

Comparison of copper labeling followed by liquid chromatography-inductively coupled plasma mass spectrometry and immunochemical assays for serum hepcidin-25 determination

Hepcidin-25 has been defined as the key biomarker in iron metabolism. This peptide binds to the iron transporter ferroportin to cause its degradation. Therefore, the need for specific, accurate and precise methods for the quantification of hepcidin-25 in biological fluids is dramatically increasing. In this regard, the use of rapid immunochemical methods that provide low limit of quantification is desired for routine clinical use. However, such fast methodologies should be first analytically evaluated and compared with alternative strategies to check for their advantages and limitations. Here we compare the use of a commercial immunochemical assay for hepcidin determination with a novel analytical approach based on Cu-labeling of the peptide followed by Cu determination using liquid chromatography (HPLC) and plasma mass spectrometry (ICP-MS). The figures of merit of both systems reveal similar analytical characteristics and both seem to be adequate for the determination of the peptide at biologically relevant concentrations in human serum samples. The analysis of a larger number of samples (n = 50) by both techniques showed a good agreement in the concentrations found. Such finding permits to address the hepcidin recovery in the sample preparation procedure necessary for the HPLC-ICP-MS analysis in human serum that turn out to be 76–85%. Additionally, limitations due to cross-reactivity issues of the ELISA method could be addressed in some of the samples by using LC-ICP-MS and were confirmed by LC-Electrospray-MS.     

An ultrasensitive quantum dots fluorescent polarization immunoassay based on the antibody modified Au nanoparticles amplifying for the detection of adenosine triphosphate

In this work, an ultrasensitive fluorescent polarization immunoassay (FPIA) method based on the quantum dot/aptamer/antibody/gold nanoparticles ensemble has been developed for the detection of adenosine triphosphate (ATP). DNA hybridization is formed when ATP is present in the PBS solution containing the DNA-conjugated quantum dots (QDs) and antibody-AuNPs. The substantial sensitivity improvement of the antibody-AuNPs-enhanced method is mainly attributed to the slower rotation of fluorescent unit when QDs-labeled oligonucleotides hybridize with antibody modified the gold nanoparticle. As a result, the fluorescent polarization (FP) values of the system increase significantly. Under the optimal conditions, a linear response with ATP concentration is ranged from 8 × 10⁻¹² M to 2.40 × 10⁻⁴ M. The detection limit reached as low as 1.8 pM. The developed work provides a sensitive and selective immunoassay protocol for ATP detection, which could be applied in more bioanalytical systems.     

Development of colloidal gold-based immumochromatographic assay for the rapid detection of medroxyprogesterone acetate residues in biological materials

A competitive colloidal gold-based immunoassay in lateral-flow format for the rapid detection of medroxyprogesterone acetate (MPA) in biological materials was developed. A nitro-cellulose membrane strip was separately coated with goat anti-rabbit IgG (control line) and MPA hapten-OVA conjugate (test line). Anti-MPA polyclonal antibody labelled with colloidal gold particles was first incubated with MPA. The limit of detection for lateral flow was 5?ng?g^?1 for detecting an MPA standard solution, and the limit of detection was 10?ng?mL^?1 for detecting the MPA spiked in pig urine and 10?ng?g^?1 for spiked in pig liver. The results were confirmed by high-performance liquid chromatography tandem mass spectrometry (HPLC/MS/MS) and indicated that there was a good agreement between both methods (R ²?=?0.976). The assay time for the test was less than 5?min, suitable for rapid testing on site.     

Point‐of‐Care Amperometric Testing of Diabetic Marker (HbA1c) Using Specific Electroactive Antibodies

Specific immune detection of glycated hemoglobin is still a great challenge owing to the small epitopic difference between Hemoglobin (Hb) and HbA1c. We report a new electrochemical immunoassay format for point of care testing of HbA1c. A conducting self‐assembled monolayer of mercaptophenyl boronic acid (MPBA) was used as a capture layer for binding of glycated proteins and ferrocene tagged anti‐HbA1c antibody (FcAb) as a tracer molecule on a gold screen printed electrode. Validation of the new HbA1c assay was carried out using 6 clinical samples with known HPLC values and a correlation coefficient of 98 % was observed.     

Evaluation of an Element-Tagged Duplex Immunoassay Coupled with Inductively Coupled Plasma Mass Spectrometry Detection: A Further Study for the Application of the New Assay in Clinical Laboratory

Background: Element-tagged immunoassay coupled with inductively coupled plasma mass spectrometry (ICP-MS) detection has the potential to revolutionize immunoassay analysis for multiplex detection. However, a further study referring to the standard evaluation and clinical sample verification is needed to ensure its reliability for simultaneous analysis in clinical laboratories. Methods: Carcinoembryonic antigen (CEA) and α-fetoprotein (AFP) were chosen for the duplex immunoassay. The performance of the assay was evaluated according to guidelines from the Clinical and Laboratory Standards Institute (CLSI). Moreover, reference intervals (RIs) of CEA and AFP were established. At last, 329 clinical samples were analyzed by the proposed method and results were compared with those obtained with electrochemiluminescent immunoassay (ECLIA) method. Results: The measurement range of the assay was 2–940 ng/mL for CEA and 1.5–1000 ng/mL for AFP, with a detection limit of 0.94 ng/mL and 0.34 ng/mL, respectively. The inter-assay and intra-assay imprecision were all less than 6.58% and 10.62%, respectively. The RI of CEA and AFP was 0–3.84 ng/mL and 0–9.94 ng/mL, respectively. Regarding to clinical sample detection, no significant difference was observed between the proposed duplex assay and the ECLIA method. Conclusions: The ICP-MS-based duplex immunoassay was successfully developed and the analytical performance fully proved clinical applicability. Well, this could be different with other analytes.     

Specific Recognition of β-Galactofuranose-Containing Glycans of Synthetic Neoglycoproteins by Sera of Chronic Chagas Disease Patients

Chagas disease (CD) can be accurately diagnosed by detecting Trypanosoma cruzi in patients’ blood using polymerase chain reaction (PCR). However, parasite-derived biomarkers are of great interest for the serological diagnosis and early evaluation of chemotherapeutic efficacy when PCR may fail, owing to a blood parasite load below the method’s limit of detection. Previously, we focused on the detection of specific anti-α-galactopyranosyl (α-Gal) antibodies in chronic CD (CCD) patients elicited by α-Gal glycotopes copiously expressed on insect-derived and mammal-dwelling infective parasite stages. Nevertheless, these stages also abundantly express cell surface glycosylphosphatidylinositol (GPI)-anchored glycoproteins and glycoinositolphospholipids (GIPLs) bearing nonreducing terminal β-galactofuranosyl (β-Galf) residues, which are equally foreign to humans and, therefore, highly immunogenic. Here we report that CCD patients’ sera react specifically with synthetic β-Galf-containing glycans. We took a reversed immunoglycomics approach that entailed: (a) Synthesis of T. cruzi GIPL-derived Galfβ1,3Manpα-(CH₂)₃SH (glycan G29_SH) and Galfβ1,3Manpα1,2-[Galfβ1,3]Manpα-(CH₂)₃SH (glycan G32_SH); and (b) preparation of neoglycoproteins NGP29b and NGP32b, and their evaluation in a chemiluminescent immunoassay. Receiver-operating characteristic analysis revealed that NGP32b can distinguish CCD sera from sera of healthy individuals with 85.3% sensitivity and 100% specificity. This suggests that Galfβ1,3Manpα1,2-[Galfβ1,3]Manpα is an immunodominant glycotope and that NGP32b could potentially be used as a novel CCD biomarker.     

氧化铈在生物分析与传感检测中的应用研究进展

铈是镧系元素中活性最高的一种稀土元素,氧化铈已广泛应用于抛光剂、脱色剂、催化剂、发光增强剂等领域。Ce3+/Ce4+之间自发的氧化还原循环可在氧化铈晶面上产生许多氧空位（V?）,加快反应中的电荷转移,使氧化铈具有很高的催化活性和储放氧能力。混合价态自发的氧化还原循环以及由此产生的氧空位对氧化铈的性质有着重大影响,使其表现出独特的抗菌、抗炎、抗肿瘤和模拟酶等生物医学活性,可用于多种疾病的诊疗。此外,氧化铈还可单独使用或与其他材料掺杂、复合来构建灵敏的传感材料,应用于多种物质的分析与检测,并已成为国内外研究的热点。该文首先阐明了氧化铈作为传感材料的理论基础,然后分别从电化学传感、比色传感、荧光传感和化学发光传感四个方面对国内外氧化铈传感检测领域的应用进展进行了归纳,最后对氧化铈传感器的研发现状进行总结并对未来发展进行了展望,为高性能传感器的研发提供了参考。