Preparation of Surface-enhanced Raman Scattering（SERS）-active Optical Fiber Sensor by Laser-induced Ag Deposition and Its Application in Bioidentification of Biotin/Avidin

A surface-enhanced Raman scattering（SERS） optical fiber sensor was prepared by the laser-induced deposition ofAg nanoparticle membrane on a silica optical fiber tip, which was applied to the real time SERS spectral monitoring on the biorecognition of biotin/avidin. The bioidentification of biotin/avidin was carried out through a indirect method, in which the bioidentification is based on the SERS response signal of a labeled dye（Atto610） after its fluorescence has been quenched totally by the deposited Ag nanoparticle membrane. By SERS monitoring the bioidentification process of biotin/avidin, it has been found that this recognition process is finished in 40 min. The lowest detection concentration of biotin is 1.0 × 10^-7 mg/mL. This research is promising in the application of immunoassays on line and in vivo.

IgG-Biotin-Avidin-HRP信号转导探针结合免疫比色法快速检测克罗诺阪崎肠杆菌

本文建立了一种快速检测克罗诺阪崎肠杆菌的新型免疫比色法。用抗体修饰的磁球(MNPs-IgG)作为捕获探针分离、富集克罗诺阪崎肠杆菌,再通过免疫夹心法分别结合生物素标记的克罗诺阪崎肠杆菌抗体(Biotin-IgG)和亲和素标记的辣根过氧化物酶(Avidin-HRP),形成的复合物与底物四甲基联苯胺(3,3′,5,5′-Tetramethylbenzidine,TMB)发生显色反应,经硫酸终止后,有色产物的吸光度值与克罗诺阪崎肠杆菌浓度的对数有良好的线性关系,在103~107CFU/mL范围内,线性方程为y=0.2353x-0.2307。本研究成功设计优化了基于比色法的克罗诺阪崎肠杆菌定量检测新方法,检测限为8.68×10^2CFU/mL(S/N=3),加标样品检出率96%,检测时间不超过2.5 h,为实际应用与商品化提供了理论支撑。

Binding of Streptavidin to Surface-attached Biotin with Different Spacer Thicknesses

The specific binding of receptor to ligand covalently attached to surface with different surface densities was studied using streptavidin-biotin model pair. Biotinylated substrates with different spacer thicknesses as formed through a simple reaction between amine immobilized surfaces and N-hydroxysucciimide groups at the end of biotin modifi ed PEG in anhydrous organic solutions（＂grafting to＂ technique）. The amount of the specifi cally adsorbed protein was measured as a function of spacer thickness between hard surface and biotin moieties. It has been shown that the amount of specifically adsorbed streptavidin decreases with the increase spacer thickness and the protein adsorbs onto the functionalized surfaces in a single molecular manner. It provides an interesting model system for studying single molecular interactions.

Application of Fuzzy Logic Algorithm to Single-Molecule Force Spectroscopy of the Streptavidin-Biotin System

The rupture force of the streptavidin-biotin complex was investigated using atomic force microscopy (AFM). The most frequently observed rupture force (MFOF), which is essential for the evaluation of the potential landscape, was evaluated by processing 22,500 force curves using two methods. One method is a conventional method, which is usually built in commercial AFM systems, i.e., difference between the baseline value and the minimum force value in the force curve. The other is a detection of rupture events based on a fuzzy logic algorithm to detect the rupture event from analyzing the shape of the force curves. Our statistical analysis revealed that the conventional method exhibited a significant artifact, which is the increase in the population of small forces comparable to thermal noise of cantilevers, resulting in a smaller MFOF. Based on this finding, we discuss the choice of a method and its effecton the illustrated potential landscapes of ligand-receptor complexes.

The biodistribution and kinetics of the Samarium-153 labeled avidin,streptavidin and biotin

Objective:To labelavidin(Av)or streptavidin(SA)with 153 Sm by takingadvantageof thehighbindingaffin-ityof biotinto Av or SA.Methods:A biotinderivative(DTPA-biotin)wasradiolabelledwith 153 Sm andthenboundto Av or SA.Thein vivo kineticsandbiodistributionof 153 Sm-labeledAv,SA andDTPA-biotinwerestudiedinratsandmice.Results:153 Sm-Avwascharacterizedby rapidclearancefromthebloodwithhighliverandrenaluptake;153 Sm-SAwas clearedfromthebloodslowlywithhighretentionintheliver,spleenandkidney,whereas 153 Sm-DTPA-biotinmetabolismwas accelerated,anditsexcretionwasmainlythroughthekidney.Conclu sion:Thebiodistributiondifferenceof SAandAvmay providean experimentalbasisfor theselectionof differentcomponentsof avidin-biotinsystemin pretagetingradioim-munoimagingandradioimmunotherapy.

奥曲肽-葡聚糖-亲和素的偶联及其与^(177)Lu-DTPA-BIS-BIOTIN的体外结合

以葡聚糖为载体,奥曲肽为导向分子合成了生长抑素配体化合物奥曲肽-葡聚糖-亲和素(Tyr3-oct-reotide-dxtran 40-avidin,TOC-Dx40-Av);在体外模拟肿瘤预定位二步法以177Lu-DTPA-BIS-BIOTIN对TOC-Dx40-Av培养的PANC-1细胞的结合特性进行了研究。将长满人源胰腺癌细胞PANC-1的24孔板置于含有TOC-Dx40-Av的缓冲液中培养,2 h后洗去上清液,再用含不同浓度177Lu-DTPA-BIS-BIOTIN(177Lu-DT-PA-BIS-BIOTIN的摩尔质量范围为48.8~391 pmol)的缓冲液继续培养细胞,使177Lu-DTPA-BIS-BIOTIN与细胞上的亲和素结合,测定细胞上结合的放射性计数,考察TOC-Dx40-Av与177Lu-DTPA-BIS-BIOTIN的结合特性以评价合成的大分子亲和素连接物的活性。实验结果显示,奥曲肽-葡聚糖-亲和素的化学纯度>99%,其中亲和素的含量为6.46 g/L。体外细胞结合实验结果表明,177Lu-DTPA-BIS-BIOTIN能快速与细胞上连接的亲和素结合,生物素与亲和素的摩尔比约为1∶1达到平衡。

^(177)Lu-DTPA-BIS-BIOTIN的制备及正常鼠体内生物分布

研究了DTPA-BIS-BIOTIN的177Lu标记方法,优化了标记条件,并进行了标记物在正常小鼠体内分布实验。在最佳标记条件下(DTPA-BIS-BIOTIN 25μg,标记介质pH=4.5,80℃反应20 min),177Lu-DTPA-BIS-BIOTIN标记率大于99.0%,室温下放置96 h,标记物体外稳定性良好。正常小鼠体内分布实验结果表明,177Lu-DTPA-BIS-BIOTIN在血液中清除快,主要浓集于肝、脾和肾,经肾脏排泄。本研究为进一步采用177Lu-DTPA-BIS-BIOTIN进行肿瘤预定位显像及治疗研究提供了实验基础。

生物素靶向显影探针Biotin-S-S-Rhodol对乳腺癌细胞的靶向显影作用

目的对新型生物素靶向显影探针(Biotin-S-S-Rhodol,3a)的靶向显影性能进行分析。方法采用紫外吸收光谱和荧光光谱分析谷胱甘肽(GSH)处理后3a的光谱特性;采用不同浓度GSH处理3a,荧光光谱分析3a对GSH的敏感性;采用流式细胞术和荧光倒置显微镜观察生物素预处理前后MCF-7细胞、MDA-MB-231细胞和Hs 578Bst细胞对3a的摄取率和靶向显影性能;四甲基偶氮唑盐比色法(MTT)初步分析3a对MCF-7细胞、MDA-MB-231细胞和Hs 578Bst细胞的毒性。结果 3a的最强吸收峰在510 nm处,在544 nm处荧光发射信号最强;荧光光谱结果显示随着GSH浓度(0~6 mmol/L)处理浓度提高,544 nm波长处的荧光强度升高;MDA-MB-231、MCF-7细胞对3a的摄取率明显高于Hs 578Bst细胞;3a能实现乳腺癌细胞专一性成像,成像清晰;2~20μmol/L 3a对人正常乳腺细胞无明显毒性,可在一定程度上抑制MCF-7细胞和MDA-MB-231细胞存活。结论 3a能通过生物素介导专一靶向乳腺癌细胞,成像清晰,对正常乳腺细胞毒性极低,有一定的抑制乳腺癌细胞存活作用。

鸡蛋清粉制品残存抗生物素蛋白(Avidin)的分离和分析

对市售冲饮用的鸡蛋清粉中残存的抗生物素蛋白进行了分离和鉴定,结果表明鸡蛋清粉中仍具有一定比活性10U/mg)的抗生物素蛋白,含量达0．040~0．055 mg/g。

Effects of a rumen protected B vitamin blend substituted for biotin upon milk production and component yield in lactating dairy cows

Results from 4 switchback field trials involving 608 cows were combined to assess the effects of a protected B vitamin blend (BVB) vs 10 mg of unprotected biotin upon milk yield (kg), fat %, protein %, fat yield (kg) and protein yield (kg) in primiparous and multiparous cows. Trials consisted of 3 DHIA periods executed in the order control-test-control. Cows from 45 to 300 days in milk (DIM) at the start of the experiment that were available for all 3 periods were included in the analysis. No diet changes other than the substitution of 3 grams/cow/day of the BVB for 10 mg of biotin during the test period occurred. Results from the two control periods were compared to results obtained during the test period by individual cow using a paired T test. Results for all cows showed that the provision of the BVB resulted in increased (P < 0.05) milk, fat percentage (%), protein %, fat yield and protein yield. Analysis by age revealed that milk production and milk protein yield were only improved in mature cows. Milk production had a negative effect upon the magnitude of the increase in milk components. The change in milk yield was greatest in early lactation and declined with DIM. Protein % and fat % increased with DIM in mature cows, but not in first lactation cows. Differences in fat yields between test and control feeding periods did not change with DIM, but the improvement in protein yield in mature cows declined with DIM. These results indicate that the BVB provided economically important advantages throughout lactation beyond those witnessed with biotin, but expected results would vary with cow age and stage of lactation.

Avidin-dendrimer对Hela细胞的靶向研究

树状大分子对肿瘤有显著的EPR效应,本文在5代聚酰胺-胺类树状大分子表面连接对肿瘤有主动靶向作用的抗生物素蛋白(亲和素),然后用放射性同位素99mTc标记这种兼具被动靶向与主动靶向作用的纳米粒子。Hela细胞体外摄取实验表明连接靶向分子亲和素(Avidin)的Biotin-PAMAM G5-1B4M-99mTc纳米复合物对Hela细胞的摄取远高于未连接靶向分子Avidin的同类纳米粒子。如进一步在小鼠SPECT成像试验中表现出良好的显像效果,这种同位素标记的纳米粒子将有望用于肿瘤的早期诊断或治疗。

A Simple Method Designed for the Separation of Biotinylated Ligate from Free Biotin

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Establishment of Cell Free Conversion System With Biotin-labelled Recombinant PrP^(sen) Expressed in E.coli

Objective To report a protocol using biotin-labelled PrP protein in cell free conversion assay instead of isotope. Methods A hamster PrP protein （HaPrP） was expressed in E. coli and purified with HIS-tag affinity chromatograph. After being labelled with biotin, HaPrP was mixed with PrP^sen preparation from scrapie strain 263K. Results Protease-resistant bands were detected after four-day incubation. Conclusion The new conversion model provides a reliable, easily handling, and environment-friendly method for studies of prion and transmissible spongiform encephalopathies.

Post-Marketing Surveillance of Fixed Dose Combination of Methylcobalamin, Alpha Lipoic Acid, Folic Acid, Biotin, Benfotiamine &Vitamin B6-Nutripathy for the Management of Peripheral Neuropathy

Background: Peripheral neuropathy is a commonly encountered troublesome condition which is often disabling & worsens when left untreated. Traditional neuropathic pain medications primarily provide symptomatic relief;however, the pathogenesis of nerve damage remains unresolved. Extensive literature survey reveals that patients with peripheral neuropathy experience significant benefits with the use of B-vitamins like methylcobalamin (B12), folic acid (B9), biotin (B7), benfotiamine (B1) and pyridoxine (B6). The other well documented antineuropathic agents include alpha lipoic acid, glutathione, omega fatty acids, myoinositol, certain trace elements, etc. Materials and Methods: A multicentre, prospective, open-label, non-comparative clinical study was carried out in 497 patients with peripheral neuropathy. A fixed dose combination of methylcobalamin, alpha lipoic acid (ALA), folic acid, biotin, benfotiamine & vitamin B6 capsule was orally administered once daily for 12 weeks. Results: Treatment led to significant reduction from baseline score in various neuropathy symptoms from the 4th week itself. After 12 weeks of treatment, the mean pain score declined by 78.0%, numbness by 92.1% and muscle weakness by 96.9%. Also, there was 96.0% & 99.2% reduction in tingling & burning sensation respectively. No serious adverse events were reported.Conclusion: The current study confirms that fixed dose combination of methylcobalamin, ALA, folic acid, biotin, benfotiamine & vitamin B6 is effective & well tolerated in the management of peripheral neuropathy.

Avidin chase reduces side effects of radioimmunotherapy in nude mice bearing human colon carcinoma

AIM: To evaluate the influence of avidin chase on the side effects of radioimmunotherapy (RIT) in nude mice bearing human colon carcinoma and therapeutic outcome.METHODS: Purified anti-CEA monoclonal antibody (McAb)was biotinylated with NHS-biotin, and then radiolabeled with 188Re by the direct method. 188Re-labeledbiotinylated anti-CEA McAb (188Re-CEA McAb-Bt) was intravenously injected followed by intravenous injection of avidin after 24 h. SPECT imaging and biodistribution study were performed at 28-48 h after the injection of 188Re-CEA McAb-Bt. Three groups of nude mice subcutaneously grafted with human colon carcinoma were treated 7 d after the graft. Mice in the avidin chase group received intravenous injection of 188Re-CEA McAb-Bt (11.1 MBq/20 μg) followed by intravenous injection of cold avidin (80 μg) after 24 h. Mice in the control group (treated group without avidin chase) only received the injection of 188Re-CEA McAb-Bt (11.1 MBq/20 μg), another control group (non-treated group) only received 0.1 mL normal saline solution. Toxicity was evaluated on the basis of change of body weight and peripheral WBC counts, and therapy effects were determined by variation in tumor volume. Histological analysis of tumors was also performed.RESULTS: Avidin chase markedly accelerated the clearance of 188Re-CEA McAb-Bt from the blood and normal tissues. The tumor uptakes of 188Re-CEA Mc Ab-Bt at 28 h were 5.90 and 6.42% ID/g, respectively, in chase group and in non-chase group, while the tumor-to-background (T/NT) ratios were 3.19 and 0.56, respectively. The tumor uptake was slightly decreased by avidin chase, but the T/NT ratios were increased. In treated groups the growth rate of body weight and the number of WBC decreased after injection of 188Re-CEA McAb-Bt, and the WBC counts recovered earlier in the group with avidin chase than in the group without avidin chase. Compared to the nontreated group, treated groups with and without avidin chase showed significant anti-tumor effects.CONCLUSION: Avidin chase can effectively reduce the side effects of RIT, and improve therapeutic efficacy.

A Sensitive Competitive ELISA for Determination of Biotin in Transformed Yeast Culture Media

Aim To develop a sensitive competitive ELBA for the determination of biotinin transformed yeast culture media. Methods The ELBA plate was firstly coated with Mycoplasmahyopneumoniae, and then successively incubated with rabbit anti-Mycoplasma hyopneumoniae serum andgoat anti-rabbit IgG-biotin to form the solid biotin, which competed with the biotin in the solution(standard or sample) for the limited streptavidin-horse radish peroxidase conjugate. The standardcalibration curve for biotin analysis was constructed in the range of 50 - 2000 ng·L^(-1). ResultsThe detection limit for biotin was found to be 83 ng·L^(-1), which was about 1000 times lower thanthe lowest determination concentration in the reported ELISA for biotin analysis. The relativestandard deviations for the spiked samples at biotin concentrations of 200 ng·L^(-1), 500ng·L^(-1), and 1000 ng·L^(-1) were 24.87%, 6.15% , and 7.86% , respectively, with the averagerecovery of 101.13% . The wild yeast and its sixty-three transformed yeast culture media wereapplied to the developed ELBA for the determination of biotin. It was found that the biotinconcentrations in more than 85% of the tested samples were enhanced with different increase factorsafter transformation. Conclusion Utilization of Mycoplasma hyopneumoniae as the coating proteinimproves the precision and accuracy of the ELBA assay, which might be used for the biotin assay inother media.

Determination of Biotin in Pharmaceutical Formulations by Potassium Permanganate-luminol-CdTe Nanoparticles Chemiluminescence System

A sensitive flow-injection chemiluminescence method was developed for the determination of biotin in the pharmaceutical formulations.The affinity between avidin and biotin was used to adsorb biotin on the polystyrene,with subsequent quantification of biotin based on its ability to enhance the chemiluminescence（CL） signal generated by the redox reaction of potassium permanganate-luminol-CdTe nanoparticles CL system.The investigations prove that apart from 3-aminophthalate,the CdTe quantum dots（QDs） play both catalytic and emitter roles.Under optimum conditions,the linear range for the determination of biotin was 0.01―25 ng/mL with a detection limit of 7.3×10-3 ng/mL（S/N=3）.The relative standard deviation of 5 ng/L biotin was 2.06%（n=7）.The proposed method was used to determine the biotin concentration in the pharmaceutical formulations and the recovery was between 96.4% and 104%.The proposed method is simple,convenient,rapid and sensitive.

Synthesis of Biotinylated Galiellalactone Analogues

Two biotinylated derivatives of the fungal metabolite galiellalactone (1) were synthesized in order to facilitate the investigation of the molecular mechanism of action of the galiellalactonoids. Galiellalactone is a STAT3-signaling inhibitor that inhibits growth in vitro as well as in vivo of prostate cancer cells expressing activated STAT3. To provide a suitable point of attachment for biotin, the 8-hydroxymethyl derivative (3) and its 7-phenyl analogue 4 were synthesized by a modified tandem Pd-catalysed carbonylation and intramolecular vinyl allene Diels-Alder procedure previously developed. The two primary alcohols obtained, 3 and 4, were coupled to biotin as the 6-aminohexanoic acid amide, activated as the acid chloride, yielding the derivatives 5 and 6.

Intracellular Self-assembly of TPE-biotin Nanoparticles Enables Aggregation-Induced Emission Fluorescence for Cancer-Targeted Imaging

Fluorogens with aggregation-induced emission (AIE) characteristics have recently been widely applied for studying biological events, and fluorogens with “smart” properties are especially desirable. Herein, we rationally designed and synthesized a biotinylated and reduction-activatable probe (Cys(StBu)-Lys(biotin)-Lys(TPE)-CBT (1)) with AIE properties for cancer-targeted imaging. The biotinylated probe 1 can be actively uptaken by the biotin receptor-overexpressing cancer cells, and then “smartly” self-assemble into nanoparticles inside cells and turn the fluorescence “On”. Employing this “smart” strategy, we successfully applied probe 1 for cancer-targeted imaging. We envision that this biotinylated intelligent probe 1 might be further developed for cancer-targeted imaging in routine clinical studies in the near future.

Enhanced Succinic Acid Production from Sake Lees Hydrolysate by Dilute Sulfuric Acid Pretreatment and Biotin Supplementation

Succinic acid is valued as a potential starting point for the production of chemicals of the C4 family or in the prepara-tion of biodegradable polymers. For sustainable development in this era of petroleum shortage, production of succinic acid by microbial fermentation of renewable feedstock has attracted great interest. In this study, pretreatment with sulfuric acid and biotin supplementation were used to enhance succinic acid production by Actinobacillus succinogenes 130Z from sake lees, a byproduct of Japanese rice wine. Pretreatment with sulfuric acid resulted in little change of glucose, total nitrogen and succinic acid content in the sake lees hydrolysate but had a positive effect on succinic acid fermentation, which caused a 25.0% increase in succinic acid yield in batch fermentation. Biotin supplementation was used to further enhance the fermentability of sake lees hydrolysate. As a result, a 30 h batch fermentation of 0.5% sulfuric acid pretreated sake lees hydrolysate with 0.2 mg/L biotin gave a succinic acid yield of 0.59 g/g from 61.6 g/L of glucose, with a productivity of 1.21 g/(L?h). A 22.9% increase in succinic acid yield and a 101.7% increase in succinic acid productivity were obtained compared with untreated sake lees hydrolysate.