Microsatellite Enrichment from AFLP Fragments by Magnetic Beads

A simple and efficient method was presented for isolating microsatellite DNA markers from peanut (Arachis hypogaea L.) genome. The genomic DNA was converted into pre-amplified AFLP fragments and hybridized with biotin-labeled SSR probes. Then the hybrid mixture was used to incubate with magnetic beads coated with streptavidin. After washing to remove the non-SSR fragments, the eluted single-strand DNA, which was cloned and sequenced, was largely enriched for microsatellites. Primers can then be designed according to the sequence flanking the repeat motifs and used for polymorphism analysis. The whole experiment can be completed within one week and can be employed as a reliable option for any molecular laboratory to develop SSR markers.

Highly efficient generation of GGTA1 knockout pigs using a combination of TALEN m RNA and magnetic beads with somatic cell nuclear transfer

The transcription activator-like effector nuclease （TALEN） technique combined with the somatic cel nuclear transfer （SCNT） method has been successfuly applied for creating geneticaly modiifed pigs. However, methods for isolating cels with bialelic indels requires further improvement because of the relatively low enrichment efifciency of mutated somatic cels. Moreover, little is known regarding the off-target effects of the TALEN system and the heredity of TALEN-modiifed pigs. In this study, an efifcient method to increase the enrichment efifciency of TALEN-mediated bialelic knockout （KO） cels was established, and corresponding geneticaly modiifed pigs with the expected genotype were generated whose off-target effect, fertility and heredity characteristics were aslo evaluated. Two TALEN pairs were constructed to target the porcine α-1,3-galactosyltransferase （GGTA1） gene locus. TALEN mRNA was transfected into the ear ifbroblasts folowed by the enrichment of α-Gal nul cels of minipigs using isolectin B4 （IB4） lectin and magnetic beads. A total of 115 cel colonies were formed and validated to beGGTA1 KO cels by sequencing and 10 bialelic KO cel colonies were used as nuclear donors for SCNT. ThirtyGGTA1 bialelic KO piglets were successfuly delivered and grew normaly. Seventeen potential off-target sites were investigated, and no off-target events were detected in the live piglets. To determine the fertility and heredity characteristics of TALEN-modiifed pigs, 10 mature founders were mated with each other and the mutations were determined to be transmitted to the F1 piglets. We established a robust and safe technology for developing geneticaly modiifed pig lines with expected genotypes for agricultural breeding and biomedical application.

Effects of Magnetic Particles Entrance Arrangements on Mixing Efficiency of a Magnetic Bead Micromixer

In this study, a computer code is developed to numerically investigate a magnetic bead micromixer under different conditions. The micromixer consists of a microchannel and numerous micro magnetic particles which enter the micromixer by fluid flows and are actuated by an alternating magnetic field normal to the main flow. An important feature of micromixer which is not considered before by researchers is the particle entrance arrangement into the micromixer. This parameter could effectively affect the micromixer efficiency. There are two general micro magnetic particle entrance arrangements in magnetic bead micromixers: determined position entrance and random position entrance. In the case of determined position entrances, micro magnetic particles enter the micromixer at specific positions of entrance cross section. However, in a random position entrance,particles enter the microchannel with no order. In this study mixing efficiencies of identical magnetic bead micromixers which only differ in particle entrance arrangement are numerically investigated and compared.The results reported in this paper illustrate that the prepared computer code can be one of the most powerful and beneficial tools for the magnetic bead micromixer performance analysis. In addition, the results show that some features of the magnetic bead micromixer are strongly affected by the entrance arrangement of the particles.

Suppressing of humping bead using an external magnetic field in high-speed gas metal arc welding

During high-speed gas metal arc welding （GMAW）, the backward flowing molten jet with high momentum in the weld pool is considered to be responsible for the occurrence of humping bead. To suppress humping bead, an electromagnetic device is developed and coupled with the welding system. By adjusting the conditions of external magnetic field, forward electromagnetic force is obtained to reduce the momentum of the backward flow of molten metal in weld pool. Consequently, the humping bead can be suppressed by adjusting the external magnetic field. Bead-on-plate welding experiment was conducted on mild steel plates, and the influence of magnetic flux density on the arc deflection angle and weld bead quality is investigated. It is found that external magnetic field can remarkably adjust the momentum of backward flow jet and significantly improve the quality of weld bead.

Preparation of Immuno-magnetic Beads and Their Separation & Detection to Ovary Cancer Cells

The organic monomer-molecule with nanometer magnetic powder by means of reforming the surface of nanometer magnetic powder have been synthesized. Magnetic beads in diameter of 2 mum or so am obtained by controlling conditions. Ovary cancer cells of ascites are separated and ovary cancer cells of blood arc detected by using immuno-magnetic beads linked with ovary cancer cell mono-antibodies. Results show that the specificity is 85% sensitivity is 87%, accuracy is 84%, cells acquiring purity is 90%, cells activity is 92% and detection sensitivity is 25 x 10(-7).

Applied Study on Magnetic Nanometer Beads in Preparation of Genechip Samples

A protocol for enrichment and adsorption of karyocyte from whole blood by using magnetic nanometer beads as solid phase absorbents was presented. The PCR amplification could be accomplished by using the nanobeads with karyocyte as template directly and the PCR products were applied on an oligonucleotide array to do gene typing. The HLA A PCR amplification system and a small HLA A oligonucleotide microarray were applied as the platform and an experiment protocol of separating karyocyte from whole blood using the magnetic nanometer beads (Fe 2O 3) were set up. The experimental conditions were also discussed. It showed that pH level of PBS eluent, Taq enzyme quantity and fragment length of products could influent the amplification results, and the magnetic nano beads could succeed in sample preparation in microarray to provide a promising way in automatic detection and lab on a chip.

A preparation strategy for protein-oriented immobilized silica magnetic beads with Spy chemistry for ligand fishing

Due to the complexity of bioactive ingredients in biological samples,the screening of target proteins is a complex process.Herein,a feasible strategy for directing protein immobilization on silica magnetic beads for ligand fishing based on SpyTag/SpyCatcher(ST/SC)-mediated anchoring is presented.Carboxyl functional groups on the surface of silica-coated magnetic beads(SMBs)were coupled with SC using the 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride/N-hydroxysulfosuccinimide method,named SC-SMBs.The green fluorescent protein(GFP),as the capturing protein model,was ST-labeled and anchored at a specific orientation onto the surface of SC-SMBs directly from relevant cell lysates via ST/SC self-ligation.The characteristics of the SC-SMBs were studied via electron microscopy,energy dispersive spectroscopy,and Fourier transform infrared spectroscopy.The spontaneity and site-specificity of this unique reaction were confirmed via electrophoresis and fluorescence analyses.Although the alkaline stability of ST-GFP-ligated SC-SMBs was not ideal,the formed isopeptide bond was unbreakable under acidic conditions(0.05 M glycine-HCl buffer,pH 1e6)for 2 h,under 20%ethanol solution within 7 days,and at most temperatures.We,therefore,present a simple and universal strategy for the preparation of diverse protein-functionalized SMBs for ligand fishing,prompting its usage on drug screening and target finding.

Preliminary Application of WCX Magnetic Bead-Based Matrix-Assisted Laser Desorption Ionization Time-of-Flight Mass Spectrometry in Analyzing the Urine of Renal Clear Cell Carcinoma

Objective To evaluate the application of weak cation exchange (WCX) magnetic bead-based Matrix-Assisted Laser Desorption Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS) in detecting differentially expressed proteins in the urine of renal clear cell carcinoma (RCCC) and its value in the early diagnosis of RCCC.Methods Eleven newly diagnosed patients (10 males and 1 female, aged 46-78, mean 63 years) of renal clear cell carcinoma by biopsy and 10 healthy volunteers (all males, aged 25-32, mean 29.7 years) were enrolled in this study. Urine samples of the RCCC patients and healthy controls were collected in the morning.Weak cation exchange (WCX) bead-based MALDI-TOF MS technique was applied in detecting differential protein peaks in the urine of RCCC. ClinProTools2.2 software was utilized to determine the characteristic proteins in the urine of RCCC patients for the predictive model of RCCC.Results The technique identified 160 protein peaks in the urine that were different between RCCC patients and health controls; and among them, there was one peak (molecular weight of 2221.71 Da) with statistical significance (P=0.0304). With genetic algorithms and the support vector machine, we screened out 13 characteristic protein peaks for the predictive model.Conclusions The application of WCX magnetic bead-based MALDI-TOF MS in detecting differentiallyexpressed proteins in urine may have potential value for the early diagnosis of RCCC.

Peptidome profiling of human serum of uveal melanoma patients based on magnetic bead fractionation and mass spectrometry

AIM： To find new biomarkers for uveal melanoma （UM） by analyzing the serum peptidome profile. METHODS： Proteomic spectra in patients with UM before and after operation were analyzed and compared with those of healthy controls. Magnetic affinity beads were used to capture serum peptides and matrix-assisted laser desorption/ionization time-of-flight （MALDI-TOF） mass spectrometer were used to compile serum peptide profiles. RESULTS： A panel of 49 peptides were differentially expressed between UM patients and controls, of which 33 peptides were of higher intensities in patient group and 16 peptides were of higher intensities in control group. Based on combined use of these potential markers, peptides with mean molecular masses of 1467 and 9289.0 Da provide high sensitivity （83.3%）, specificity （100%） and accuracy rate （93.0%） together to differentiate melanoma patients from healthy controls. At the time point of 6mo postoperatively, the levels of many peptides differentially expressed before surgery showed no more statistical difference between the patients and the control group. Fibrinogen o-chain precursors were identified as potential UM markers. CONCLUSION： We have shown that a convenient and fast proteomic technique, affinity bead separation and MALDI- TOF analysis combined with bioinformatic software, facilitates the identification of novel biomarkers for UM.

Selective adsorption behavior of ion-imprinted magnetic chitosan beads for removal of Cu(Ⅱ) ions from aqueous solution

Heavy metal ion is one of the major environmental pollutants.In this study,a Cu(Ⅱ)ions imprinted magnetic chitosan beads are prepared to use chitosan as functional monomer,Cu(Ⅱ)ions as template,Fe_(3)O_(4) as magnetic core and epichlorohydrin and glutaraldehyde as crosslinker,which can be used for removal Cu(Ⅱ)ions from wastewater.The kinetic study shows that the adsorption process follows the pseudosecond-order kinetic equations.The adsorption isotherm study shows that the Langmuir isotherm equation best fits for the monolayer adsorption processes.The selective adsorption properties are performed in Cu(Ⅱ)/Zn(Ⅱ),Cu(Ⅱ)/Ni(Ⅱ),and Cu(Ⅱ)/Co(Ⅱ)binary systems.The results shows that the ⅡMCD has a high selectivity for Cu(Ⅱ)ions in binary systems.The mechanism of ⅡMCD recognition Cu(Ⅱ)ions is also discussed.The results show that the ⅡMCD adsorption Cu(Ⅱ)ions is an enthalpy controlled process.The absolute value of DH(Cu(Ⅱ))and DS(Cu(Ⅱ))is greater than DH(Zn(Ⅱ),Ni(Ⅱ),Co(Ⅱ))and DS(Zn(Ⅱ),Ni(Ⅱ),Co(Ⅱ)),respectively,this indicates that the Cu(Ⅱ)ions have a good spatial matching with imprinted holes on ⅡMCD.The FTIR and XPS also demonstrates the strongly combination of function groups on imprinted holes in the suitable space position.Finally,the ⅡMCD can be regenerated and reused for 10 times without a significantly decreasing in adsorption capacity.This information can be used for further application in the selective removal of Cu(Ⅱ)ions from industrial wastewater.

Usefulness of Immuno-Magnetic Beads Conjugated with Anti-EpCAM Antibody for Detecting Endometrial Cancer Cells

A simple and non-invasive method for detecting endometrial cancer in women with abnormal uterine bleeding is required. For this purpose, we prepared immuno-magnetic beads conjugated with anti-human EpCAM rat monoclonal antibody (mAb) for isolating exfoliated endometrial cells including endometrial cancer cells in vaginal discharge. The affinities of the anti-human EpCAM rat mAbs were analyzed by flow cytometry and immunocytochemistry and then magnetic beads were conjugated with the mAbs. The rate of retrieval of endometrial cells using the immuno-magnetic beads was calculated. Endometrial cells were isolated using the immuno-magnetic beads from the vaginal discharges of 22 patients with endometrial cancer and 16 non-malignant controls. The isolated cells were stained using endometrial cancer specific-mAbs and analyzed by flow cytometry and imaging cytometry. The immuno-magnetic beads conjugated with high-affinity mAb (clone 1456) appeared to have very low auto-fluorescence. Sufficient enrichment of Ep-CAMpositive cells using immuno-magnetic beads was observed in both simulation and clinical samples. The overall sensitivities of flow cytometry and imaging cytometry to detect endometrial cancer cells were 72.7% and 45.5%, respectively. Meanwhile, the overall specificities of flow cytometry and imaging cytometry for healthy controls were 75.0% and 81.3%, respectively. Our immuno-magnetic beads have very low auto-fluorescence, so they could be useful for fluorescent analysis, such as fluorescent immunochemical staining. In the future, these novel immuno-magnetic beads could be used for cytological study.

STUDY ON THE SEPARATION AND UTILIZATION TECHNOLOGY OF MAGNETIC BEAD IN FLY ASH

On the basis of study on physical and chemical properties of magnetic bead (MB) in fly ash (FA), the paper gives out the separation methods of MB and results of three separating process. The result of comparative test in size, density, stability, magnetic material content, specific magnetic susceptibility (SMS), medium recovery oxidation resistance and wear resistance between MB and magnetic fines currently used in dense medium separation leads to that using MB recovered from fly ash is used as medium solids in coal cleaning in stead of magnetic fines not only have no influence upon taryests of separation, but can bring good economic and social benefits.

The detection of tetrahydrofuran hydrate formation and saturation using magnetic resonance imaging technique

Tetrahydrofuran（THF） hydrate was formed in bulk as well as in glass beads pack with a mean diameter of 3.0 mm by controlling the temperature under ambient pressure.Images of THF hydrate formation procedure were obtained using the magnetic resonance imaging（MRI） technique.The experiment results showed that MRI is an effective method for the detection of hydrate formation.Saturation of hydrate formed both in bulk and glass beads can be confirmed by intensity integration of MRI images.

Study on Shigella Detection by ATP Bioluminescence Magnetic Enzyme Immunoassay

[Objective] The research aimed to establish a rapid detection method for Shigella. [Method] Combining immunomagnetic separation technology with ATP bioluminescence technology, a new kind of fast and accurate ATP bioluminescence magnetic enzyme immunoassay technique for Shigella was established. [Result] Using ATP bioluminescence magnetic enzyme immunoassay technique to detect standard solution for Shigella （ATCC 25931 ）, result showed that correlation coefficient between relative light intensity detected by instrument and bacteria concentration detec- ted by culture counting method was 0.981 1. Moreover, relation curve between relative light intensity and Shigella concentration was drawn. [ Conclusion] The method had a high detection speed and accuracy, and could be used for the rapid detection of pathogen in food and environment.

Immunomagnetic Indirect Positive Sorting of Precartilaginous Stem Cells from Neonatal Rat

To investigate the technique of sorting high-purity precartilaginous stem cells from rat＇s perichondrium, neonatal rat＇s perichondrium cells suspensions were incubated with monoclone antibody of anti-fibroblast growth factor receptor-3 （anti-FGFR-3）, and the labeled cells were separated from the suspension in the magnetic field by immuno-beads coated with the second antibody, Purity of the sorted neural stem cells was found to be 93.0 %-99.0 %, with living cells amounting to 80 % -85 %. The magnetic cell sorting system could effectively separate precartilaginous stem cells from perichondrium cell suspensions.

自动化磁珠法提取血清脂溶性维生素应用LC-MS/MS检测的性能评价

目的评估自动化磁珠法提取血清脂溶性维生素应用液相色谱串联质谱法(liquid chromatography-tandem mass spectrometry,LC-MS/MS)检测的性能。方法收集200例临床剩余血清样本,采用自动化磁珠法提取血清中脂溶性维生素A,D_(2),D_(3),E和K;同时联合LC-MS/MS检测脂溶性维生素A,D_(2),D_(3),E和K的线性、定量限、精密度、正确度、携带污染率等性能指标以及基质效应。并比较此方法与传统萃取法检测结果的一致性。结果自动化磁珠法提取脂溶性维生素A,D_(2),D_(3),E和K线性相关系数均>0.99;五种物质的定量限分别为5,0.25,0.25,125和0.025ng/ml;批内精密度和批间精密度分别为0.66%~4.83%,0.15%~3.70%;平均加标回收率为87.05%~111.11%;基质效应为95.43%~99.07%;高-低值样本循环进样结果均值与低-低值样本循环进样结果均值之差,均小于低-低值样本循环进样结果均值的3s;统计学结果显示自动化磁珠法和传统萃取法提取的脂溶性维生素结果相关性良好(r>0.99),两种方法的检测结果无显著偏倚。结论自动化磁珠法提取脂溶性维生素的检测性能良好,有望提高样品通量和分析效率。

基于杂交链式反应扩增检测奶粉中阪崎肠杆菌的适配体磁珠荧光传感器

构建一种基于杂交链式反应(hybridization chain reaction,HCR)扩增的适配体磁珠荧光传感器。巧妙设计序列HP和发卡序列H1、H2,其中HP是由适配体序列与触发序列结合而成的,并且序列互补形成稳定的二级结构。然后采用戊二醇反应和亲和素-生物素反应进行适配体功能化磁珠的制备。将阪崎肠杆菌与适配体磁珠一起孵育,HP中的适配体序列识别靶标,引起HP构象变化,露出触发序列,通过HCR触发H1和H2的链状组装,产生长双链DNA。荧光指示剂SYBR Green I以插层和小槽结合的方式与HCR产物的长双链结合。最后加入氧化石墨烯(graphene oxide,GO)后,游离的H1、H2和SYBR Green I将通过π-π堆积紧密吸附在GO表面,荧光信号被猝灭。HCR产物不能被吸附在GO表面,因此与HCR产物结合的SYBR Green I发出依赖于靶浓度的强荧光信号,从而实现阪崎肠杆菌的定量检测。本方法在纯培养条件下的检出限为2CFU/mL,对奶粉的检出限为8CFU/g,对奶粉样品的检测结果与传统微生物培养法具有良好的一致性。该方法具有无需DNA提取,快速、稳定性高、高特异性和高灵敏度等优点,因此为阪崎肠杆菌的现场快速检测提供了一种很有潜力的方法。

儿童消化道异物35例手术治疗分析

目的:回顾性分析需手术治疗的儿童消化道异物的临床资料,总结其临床诊治经验。方法:选取2016年1月—2021年12月深圳市儿童医院行手术治疗的35例消化道内异物患儿作为研究对象,分析不同类型异物的临床特点、诊治方法和预后情况。结果:所有病例中,男性28例(80%),女性7例(20%)。年龄范围从11月~9岁。临床症状主要为腹痛哭闹19例(54.29%)和呕吐17例(48.57%)。所有患者做X线检查,提示消化道异物阳性33例(94.29%),包括:多个磁珠30例,尖锐金属异物3例;提示消化道异物阴性2例(5.71%),包括泡发球1例、枣核1例。做超声检查共13例,提示消化道异物阳性10例(76.92%),其中有2例患儿在超声检查中提示消化道异物阳性,而在X线检查中则为阴性。全部患儿均接受手术治疗,术后随访3~6月,发生消化道梗阻并发症5例。结论:磁珠和尖锐金属异物是常见需手术治疗的儿童消化道内异物种类,腹痛哭闹和呕吐是该类疾病的主要症状,儿童消化道异物可引起较为严重的并发症,特别是多个磁珠更易引起的消化道并发症,家长及儿科医生需重点关注。

磁性浮珠VMB@Fe_(3)O_(4)@CS对鱼腥藻和微囊藻的去除

为了有效处理有害藻华,并避免化学物质二次污染,结合磁絮凝与浮珠浮选方法,以玻化微珠、Fe_(3)O_(4)、壳聚糖为原料合成磁浮珠VMB@Fe_(3)O_(4)@CS,对水华鱼腥藻和铜绿微囊藻进行去除实验.结果表明,磁浮珠对2种水华藻的最佳去除效率分别达到99.01%(0.12g/L)、98.6%(0.21g/L).VMB@Fe_(3)O_(4)@CS在较宽的pH值范围(4~10)和水温范围(5℃~35℃)内实现较高的去除率(>60%),离子强度的增加对微藻的去除起负面影响.通过形态学观察、表面疏水率及Zeta电位分析确定静电吸引、吸附架桥是磁浮珠对藻细胞主要的絮凝机理.制备的VMB@Fe_(3)O_(4)@CS去除有害藻华具有很大应用潜力.