The design of a peptide linker group to enhance the SERS signal intensity of an atto680 dye‐nanoparticle system

The Surface enhanced resonance Raman spectroscopy (SERRS) spectra of three modified atto680 dyes were recorded using Au nanoparticles and an excitation laser operating at 670 nm. The dyes were modified with linker groups based on the small peptides, Cys, Cys–Gly and Cys–Gly–Gly. The Cys thiol group acted as the coupling point to the Au surface and the Gly  NH₂ group used to attach the dye. The maximum signal was recorded for the Cys–Gly linker. This gave a signal intensity for the 577 cm⁻¹ Raman peak of the atto680 dye that was more than 27 times greater than the unmodified dye. The Au nanoparticles used had a diameter of 49.8 ± 1.2 nm and were synthesised by the citrate reduction method. The Raman dye‐AuNP probes were also used in an immunoassay to detect mouse IgG in the femto mole range. Copyright © 2010 John Wiley & Sons, Ltd.     

Peptide mimotopes of phomopsins: Identification, characterization and application in an immunoassay

Peptide mimotopes of plant-associated toxins offer the potential for improving analytical and diagnostic methodologies as well as providing candidates for potential protective vaccines against plant poisoning diseases. Monoclonal antibody (mAb) C₃C₁₁, which recognizes the antimicrotubule phomopsin mycotoxins, was used to isolate peptide mimics of phomopsin A from a random 15-mer phage display peptide library. A total of 46 clones were isolated that showed specific reactivity with the mAb. Amino acid sequence analysis revealed four different types of mimotope sequences, all of which contained a common motif V-A-L/V-C. Of the 46 clones isolated, 44 contained the motif V-A-L-C while 2 contained the V-A-V-C motif. All four types of phage clones inhibited the reactivity of the mAb with phomopsin A in a competition ELISA. The clone with the mimotope sequence CT VALCNMYFGAKLD demonstrated the strongest binding. It was further shown that synthetic peptides containing these mimotope amino acid sequences were able to inhibit the mAb-phomopsin A interaction, indicating that the peptide mimotopes were responsible for the specific binding, independent of the phage framework. The results also suggest that the mimotope peptides bind to mAb C₃C₁₁ at the same site as phomopsin A. The application of recombinant phage particles carrying phomopsin mimotopes in immunoassay was evaluated and the results demonstrated approximately 100-fold increase in sensitivity in comparison with a conventional immunoassay using a chemically linked phomopsin-horseradish peroxidase conjugate.     

Highly sensitive room-temperature method of non-invasive in vivo detection of magnetic nanoparticles

Methods of non-invasive in vivo quantification of magnetic nanoparticles (MP) have been proposed and realized. The methods are based on non-linear MP magnetization at two frequencies and measuring the response at combinatorial frequencies. The first method is developed for real-time study of MP dynamics and their clearance from the blood system of animals. High sensitivity of 3 ng of Fe₃O₄ in 0.1 ml was achieved for MP detection in mice tail veins. The second technique is proposed for MP detection inside animal tissues by an external probe. The proposed methods could essentially widen capabilities of biomedical research which involves magnetic nanoparticles.     

荧光偏振免疫分析测定磺胺甲基嘧啶研究

磺胺甲基嘧啶(sulfamerazine,SMR)的芳伯胺基与荧光素异硫氰酸钠异构体Ⅰ(fluorescein isothiocyanate, FITC)的异硫氰基在常温下可以发生偶联反应,经薄层色谱分离之后,可以得到SMR的FITC荧光标记物(Tracer)SMR-FITC。应用荧光偏振免疫分析(fluorescence polarization immunoassay, FPIA),以SMR单克隆抗体为竞争免疫试剂,优化了检测条件,建立了SMR的FPIA检测方法。在选定的条件下,SMR的半数抑制量(IC₅₀)为23.4 ng·mL-1,最低检测限量为2.3 ng·mL-1,检测范围为5.4到218.8 ng·mL-1,可以达到食品中SMR的最高残留限量要求。同时对16种磺胺类药物的交叉反应做了初步研究,结果显示对SMR、磺胺二甲基嘧啶(sulfamethazine, SMZ)、磺胺嘧啶(sulfadiazine, SDZ)交叉反应率分别为100%,25%和8.6%,对其他磺胺类药物的交叉反应率均低于5%。     

Piezoelectric immunoassay of thyroidal triiodothyronine

A re-utilizable piezoelectric crystal (PC) immunosensor for thyroidal triiodothyronine (T3) has been developed. The crystal was coated with protein A and then reacted with T3 antibody. T3 could be detected in the range of 0.5×10⁻⁶ g/L to 10.0×10⁻⁶g/L by the, system. Crystal was regenerated by saturating with T3 and subsequent binding of a new anti-T3 antibody layer. The T3 assay could be repeated up to 5 times using each crystal. Supported by the National Natural Science Foundation of China Zha Hongying: born May, 1969, Ph. D.     

抗人凝血VIII因子的单克隆抗体制备及其鉴定

经凝胶过滤和亲和层析分离得到的人抗血友病A因子——VIII因子抗原VIII:CAg,在用一期法和火箭免疫电泳证实其纯度后,免疫Balb/c小鼠和包被免疫测定板。采用常规鼠杂交瘤技术经间接ELISA法筛选、克隆化得到四个稳定分泌抗VIII:C的单克隆抗体的细胞株,即DC8,DE4,DE7和DE8,腹水效价为1:1×10~4～1:1×10~5,一期法抑制反应表明,单克隆抗体对VIII:C活性有显著的抑制作用,但其抑制是不完全的,对抗体进行了纯化及分析,并将单抗用于血友病A多态性的分析。     

Latex piezoelectric immunoassay: Effect of interfacial properties

Latex piezoelectric immunoassay is a technique for detecting agglutination of antibody- or antigen-bearing latex by an immunoreaction using a piezoelectric quartz crystal; the agglutination decreases the oscillation frequency of the crystal. This is advantageous in that immobilization of antibody or antigen on the crystal surface is unnecessary. In this report, different kinds of chemical functional groups were immobilized on the electrode surface, allowing us to consider the effect of interfacial structure on the frequency change. Electrode modifications such as self-assembly of alkanethiol and aminoalkoxysilane monolayers, and polyethylenimine-glutaraldehyde coating as well as plasma treatment were examined. The sensitivity of the system was found to imitate the interfacial properties so that modification of the electrode surface could improve the response. Among the methods examined for this electrode surface modification, the polyethylenimine-glutaraldehyde modification had the advantages of high reproducibility, fast operation and simplicity. It was also suggested that the frequency change originated primarily from the immunoreaction at the interface.     

Electrochemical Enzyme Immunoassay of Tumor Marker CA15-3 with Capillary Electrophoresis

CA15-3 is a circulating antigen that is relatively specific for human breast tissue. CA15-3 is significantly more sensitive than carcinoembryonic antigen in the evaluation of patients with both primary and metastatic breast cancer. CA15-3 levels are often measured by using radioimmunoassay and enzyme immunoassay (EIA) in clinic. These methods are carried out manually. Tedious processes, slow reaction, poor reproducibility are their major problems. Capillary electrophoretic immunoassay …     

Preparation of Functional Magnetic Nanoparticles and its Application in Diagnostic Analysis

The magnetic nanoparticles modified with carboxyl functional group were synthesized and characterized. These nanoparticles covalently bound with hepatitis B surface antibody（HBsAb）, were used to detect hepatitis B surface antigen （HBsAg） in immunovoltammetry. The detection limit was found to be 0.06 ng/mL, which is much higher than that of enzyme-linked immunosorbent assay （ELISA） used in clinical analysis.