Analysis of solid-phase immobilized antibodies by atomic force microscopy

Antibody adsorption to solid surfaces creates a number of constraints that may interfere with epitope recognition and ligand-antibody interaction. By optimizing the conditions of adsorption, one may minimize these constraints. We have studied several factors that affect the antibody adsorption using atomic force microscopy (AFM) as a readout mechanism. AFM provides a highly sensitive, label-free method for detecting and analyzing molecular interactions. In this report, AFM was used to study antibody properties, the efficiency of particle capture and ligand-antibody interaction using anti-bacteriophage fd antibodies in a solid phase assay format. The capture efficiencies of anti-fd preparations adsorbed onto gold surfaces under various conditions including pH and antibody concentration were determined and compared. The relative sensitivities of each antibody for the capture of phage fd as a function of applied phage concentrations was evaluated. The collective data indicates that AFM is effective as an analytical instrument for studying the functionality of surface adsorbed antibodies in particle capture assays. This method of analysis can be extended to rapidly screen and select antibodies or other ligands with a specific set of characteristics. As the number and complexity of chip-based analytical platforms in proteomics increases, rapid selection/screening processes such as that described here will become invaluable.     

High-throughput SNP genotyping based on solid-phase PCR on magnetic nanoparticles with dual-color hybridization

Single-nucleotide polymorphisms (SNPs) are one-base variations in DNA sequence that can often be helpful when trying to find genes responsible for inherited diseases. In this paper, a microarray-based method for typing single nucleotide polymorphisms (SNPs) using solid-phase polymerase chain reaction (PCR) on magnetic nanoparticles (MNPs) was developed. One primer with biotin-label was captured by streptavidin coated magnetic nanoparticles (SA-MNPs), and PCR products were directly amplified on the surface of SA-MNPs in a 96-well plate. The samples were interrogated by hybridization with a pair of dual-color probes to determine SNP, and then genotype of each sample can be simultaneously identified by scanning the microarray printed with the denatured fluorescent probes. The C677T polymorphisms of methylenetetrahydrofolate reductase (MTHFR) gene from 126 samples were interrogated using this method. The results showed that three different genotypes were discriminated by three fluorescence patterns on the microarray. Without any purification and reduction procedure, and all reactions can be performed in the same vessel, this approach will be a simple and labor-saving method for SNP genotyping and can be applicable towards the automation system to achieve high-throughput SNP detection.     

A protein binds to a satellite DNA repeat at three specific sites that would be brought into mutual proximity by DNA folding in the nucleosome

Using a generally applicable assay for specific DNA-binding proteins in crude extracts, we have detected and purified an HMG-like nuclear protein from African green monkey cells that preferentially binds to the 172 bp repeat of alpha-satellite DNA (alpha-DNA). DNAase I footprinting with the purified protein detects three specific binding sites (I-III) per alpha-DNA repeat. Site II is 145 bp (one core nucleosome length) from site III on the adjacent alpha-DNA repeat, while site I lies midway between sites II and III. In the alpha-nucleosome phasing frame corresponding with this arrangement, sites I-III would be brought into mutual proximity by DNA folding in the nucleosome. This phasing frame is identical with the preferred frame detected previously in isolated chromatin. Our results suggest that this new and abundant protein recognizes a family of short, related nucleotide sequences found not only in alpha-DNA but also throughout the genome, and that functions of this protein are mediated through its nucleosome-positioning activity. Such nucleosome-positioning proteins may underlie the sequence specificity of both nucleosome arrangements and higher order chromatin structures.     

磁珠法快速提取基因组DNA的实验研究

针对临床标本基因组DNA的提取方法缺乏广泛适用性,提取步骤繁琐,需要进行离心操作,并且提取过程中会用到苯酚、氯仿等有机试剂,会对操作人员有一定的危害性等不足,本研究拟建立一种适用于临床标本的基因组DNA快速提取方法。在传统的基因组DNA提取试剂和方法的基础上,本研究采用二氧化硅修饰的超顺磁珠设计了一种基因组DNA快速提取方法;探讨磁珠用量、裂解液p H、盐酸胍浓度等因素对基因组DNA提取效率的影响并采用凝胶电泳实验进行验证。当磁珠用量在50~100μg/100 mg样品,裂解液p H约为6,盐酸胍的浓度为6 mol/L时基因组DNA提取效果好、效率高,且磁珠的用量与吸附表面积成正比,但达到一定用量后不会增加提取量,对于100 mg肺组织,适宜的磁珠用量为80μg。此基因组DNA提取方法高效省时,简便快捷,性价比高,适用临床大量样本基因组DNA提取。     

Reversible immobilization of antibodies on magnetic beads.

A streptavidin-biotin system was utilized to prepare an antibody-polyadenylic acid conjugate which was subsequently attached to commercially available magnetic beads, Dynabeads oligo(dT)25. Biotinylated polyadenylic acid was combined with streptavidin and the resulting polyadenylic acid-streptavidin was conjugated with an antibody-biotin derivative. The immobilized antibody-polyadenylic acid conjugate was separated from the reaction mixture by hybridization with complementary oligonucleotide immobilized on the surface of Dynabeads oligo(dT)25. The immobilized antibody-polyadenylic acid can be released from the carrier, utilizing low-ionic-strength buffers. The system is intended to be utilized in cell sorting, using immobilized antibodies against cell surface antigens. Dissociation of antibody-containing conjugate from magnetic beads is essential for the isolation of viable cells via positive cell sorting.     

Identifying FGA peptides as nasopharyngeal carcinoma-associated biomarkers by magnetic beads

Early diagnosis and treatment is known to improve prognosis for nasopharyngeal carcinoma (NPC). The study determined the specific peptide profiles by comparing the serum differences between NPC patients and healthy controls, and provided the basis for the diagnostic model and identification of specific biomarkers of NPC. Matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry (MALDI‐TOF‐MS) can be used to detect the molecular mass of peptides. Mass spectra of peptides were generated after extracting and purification of 40 NPC samples in the training set, 21 in the single center validation set and 99 in the multicenter validation set using weak cationic‐exchanger magnetic beads. The spectra were analyzed statistically using FlexAnalysis? and ClinProt? bioinformatics software. The four most significant peaks were selected out to train a genetic algorithm model to diagnose NPC. The diagnostic sensitivity and specificity were 100% and 100% in the training set, 90.5% and 88.9% in the single center validation set, 91.9% and 83.3% in the multicenter validation set, and the false positive rate (FPR) and false negative rate (FNR) were obviously lower in the NPC group (FPR, 16.7%; FNR, 8.1%) than in the other cancer group (FPR, 39%; FNR, 61%), respectively. So, the diagnostic model including four peptides can be suitable for NPC but not for other cancers. FGA peptide fragments identified may serve as tumor‐associated biomarkers for NPC. J. Cell. Biochem. 113: 2268–2278, 2012. © 2012 Wiley Periodicals, Inc.     

Highly sensitive and selective colorimetric genotyping of single-nucleotide polymorphisms based on enzyme-amplified ligation on magnetic beads

Herein we report a new strategy for highly sensitive and selective colorimatric assay for genotyping of single-nucleotide polymorphisms (SNPs). It is based on the use of a specific gap ligation reaction, horseradish peroxidase (HRP) for signal amplification, and magnetic beads for the easy separation of the ligated product. Briefly, oligonucleotide capture probe functionalized magnetic beads are first hybridized to a target DNA. Biotinylated oligonucleotide detection probes are then allowed to hybridize to the already captured target DNA. A subsequent ligation at the mutation point joins the two probes together. The introduction of streptavidin-conjugated HRP and a simple magnetic separation allow colorimetric genotyping of SNPs. The assay is able to discriminate one copy of mutant in 1000 copies of wild-type KRAS oncogene at 30 picomolar. The detection limit of the assay is further improved to 1 femtomolar by incorporating a ligation chain reaction amplification step, offering an excellent opportunity for the development of a simple and highly sensitive diagnostic tool.     

Rapid and sensitive detection of Mycobacterium tuberculosis based on strand displacement amplification and magnetic beads

Tuberculosis is one of the main infectious diseases threatening public health, and the development of simple, rapid, and cost‐saving methods for tuberculosis diagnosis is of profound importance for tuberculosis prevention and treatment. The bacterium Mycobacterium tuberculosis (MTB) is the pathogen that causes tuberculosis, and assaying for MTB is the only criterion for tuberculosis diagnosis. A new enzyme‐free method based on strand displacement amplification and magnetic beads was developed for simple, rapid, and cost‐saving MTB detection. Under optimum conditions, a good linear relationship could be observed between fluorescence and MTB specific DNA concentration ranging from 0.05 to 150 nM with a correlation coefficient of 0.993 (n = 8) and a detection limit of 47 pM (3σ/K). The present method also distinguished a one base mismatch from MTB specific DNA, showing great promise for MTB genome single base polymorphism analysis. MTB specific DNA content in polymerase chain reaction samples was successfully detected using the new method, and recoveries were 97.8–100.8%, indicating that the present method had high accuracy and shows good potential for the early diagnosis of tuberculosis.     

基于磁珠富集法的绣线菊SSR分子标记分离与筛选

微卫星序列（SSR）具有多态性高、共显性遗传等特点,是一种极具价值的分子遗传标记。采用磁珠富集法从高山绣线菊基因组DNA中分离和筛选SSR标记。高山绣线菊基因组经限制性内切酶Mse I酶切后与接头连接,并与生物素标记SSR探针（AC）15和（AG）15杂交,然后通过链霉亲和素磁珠富集、洗脱、PCR扩增、克隆,完成微卫星文库构建。利用载体通用引物和探针序列引物进行PCR扩增,筛选重组克隆并测序,获得112条序列。随机挑选其中60条序列设计的引物,经初期筛选获得多态性引物16对。用所得16对引物对4个居群92个个体的蒙古绣线菊和高山绣线菊进行PCR扩增。统计分析PCR产物的毛细管电泳结果,发现4个居群的平均等位基因数、平均期望杂合度及平均观测杂合度都比较高。64个数据系列（4个居群×16个位点）中的26个显著偏离HardyWeinberg平衡,推测可能由于无效等位基因的存在所引起。分析显示研究开发的16对多态性SSR引物可以用于后续遗传多样性、物种进化与亲缘关系等方面研究,丰富了绣线菊遗传多样性研究的分子标记。     

DNA-binding peptides searched from the solid-phase combinatorial library with the use of the magnetic beads attaching the target duplex DNA

We have exhibited successful and rapid screening of DNA-binding peptide ligands from solid-phase library beads with the use of the target DNA-conjugated magnetic beads. The target duplex DNA (3) has a polyether linker between two complementary sequences (T4A3G-ether linker-CT3A4) and is stable in the duplex form during the selection procedure. Finally, 71 pentapeptide sequences were identified from the solid-phase pentapeptide library. From an analysis of the peptide sequences identified in this study, it has been revealed that peptide ligands contain hydrophobic amino acids as the major component. The synthetic peptides with identified sequences and a combination of the major components have exhibited moderate to high binding affinity to the duplex DNA in competition experiments with ethidium-DNA complexes.     

双磁珠法质粒DNA提取技术及应用

质粒是基因合成与测序领域中使用最为频繁的基因运载工具,然而传统的质粒DNA提取方法面临提取通量低、生产成本高等问题,无法满足日益增长的需求。本研究基于质粒提取原理,开发了双磁珠法(double-magnetic-bead method, DMBM)质粒提取技术,探究了磁珠投入量、质粒DNA片段大小、菌液投入量等因素对质粒提取的影响,并且对比了本技术与商业化质粒DNA提取试剂盒提取DNA质量、提取通量及提取成本。结果表明,双磁珠法质粒DNA提取技术可满足不同细胞密度、不同片段长度的质粒DNA提取。此外,该技术搭载96通道全自动核酸提取仪,提取的质粒DNA纯度更高、提取时间缩短80%、提取成本缩减57.1%,从而实现了质粒DNA提取的高通量、低成本,有效助力基因合成与测序。     

Direct sensing of ligand-protein interaction by counting of bioaffinity beads with atomic force microscopy

Estradiol-displayed bioaffinity beads binding to the anti-estradiol antibody attached via the protein A-coated mica surface were examined by atomic force microscopy (AFM). The amount of specifically bound beads on the surface was directly proportional to the concentration of free estradiol in solution. Estradiol from 10 ng ml^–1 to 10 g ml^–1 could be determined. This suggested that direct counting of bioaffnity beads by AFM can be used to detect specific ligand for the target protein.     

Automated determination of rifampicin in plasma samples by in-tube solid-phase microextraction coupled with liquid chromatography

A sensitive and automated method is described for determination of rifampicin in plasma samples for therapeutic drug monitoring by in-tube solid-phase microextraction coupled with liquid chromatography (in-tube SPME/LC). Important factors in the optimization of in-tube SPME are discussed, such as coating type, sample pH, sample draw/eject volume, number of draw/eject cycles, and draw/eject flow rate. Analyte pre-concentrated in the polyethylene glycol phase was directly transferred to the liquid chromatographic column by percolation of the mobile phase, without carryover. The method was linear over the 0.1-100 μg/mL range, with a linear coefficient value (r(2)) of 0.998. The inter-assay precision presented coefficient of variation ≤ 1.7%. The effectiveness and practicability of the proposed method are proven by analysis of plasma samples from ageing patients undergoing therapy with rifampicin.     

Switch on or switch off: an optical DNA sensor based on poly(p-phenylenevinylene) grafted magnetic beads

There has been an enormous demand for commercial label-free DNA sensors in a diverse range of fields including pre-emptive medicine, diagnostics, environmental monitoring, and food industry. Addressing the need for sensitive, selective and facile DNA sensors, we demonstrate a novel switch on/off sensor design that utilizes sandwich hybridization between photoluminescent anionic conjugated polyelectrolyte (CPE) bound captureprobe coated onto magnetic beads, target and the signaling probe. The hybridization-readout in our sensor was monitored by either fluorescence resonance energy transfer (FRET, switch-on) or superquenching (switch-off) depending on the type of signaling probe used. Moreover recent designs that utilize beads for sensing DNA have been limited towards using electrostatic interactions or intercalation of dyes to observe FRET. To our knowledge this is the first report of a switch on/off sensor utilizing either FRET or superquenching thus providing flexibility for future development of such rapid, facile and sensitive DNA sensors. The FRET-based sensor was investigated by optimizing the reaction parameters and selectivity. A low detection limit of 240 fmol in 2 mL of SSC buffer was achieved.     

Aptamer‐based downstream processing of his‐tagged proteins utilizing magnetic beads

Aptamers are synthetic nucleic acid‐based high affinity ligands that are able to capture their corresponding target via molecular recognition. Here, aptamer‐based affinity purification for His‐tagged proteins was developed. Two different aptamers directed against the His‐tag were immobilized on magnetic beads covalently. The resulting aptamer‐modified magnetic beads were characterized and successfully applied for purification of different His‐tagged proteins from complex E. coli cell lysates. Purification effects comparable to conventional immobilized metal affinity chromatography were achieved in one single purification step. Moreover, we have investigated the possibility to regenerate and reuse the aptamer‐modified magnetic beads and have shown their long‐term stability over a period of 6 months. Biotechnol. Bioeng. 2011;108: 2371–2379. © 2011 Wiley Periodicals, Inc.     

Localized transfection on arrays of magnetic beads coated with PCR products

High-throughput gene analysis would benefit from new approaches for delivering DNA or RNA into cells. Here we describe a simple system that allows any molecular biology laboratory to carry out multiple, parallel cell transfections on microscope coverslip arrays. By using magnetically defined positions and PCR product-coated paramagnetic beads, we achieved transfection in a variety of cell lines. Beads may be added to the cells at any time, allowing both spatial and temporal control of transfection. Because the beads may be coated with more than one gene construct, the method can be used to achieve cotransfection within single cells. Furthermore, PCR-generated mutants may be conveniently screened, bypassing cloning and plasmid purification steps. We illustrated the applicability of the method by screening combinatorial peptide libraries, fused to GFP, to identify previously unknown cellular localization motifs. In this way, we identified several localizing peptides, including structured localization signals based around the scaffold of a single C2H2 zinc finger.     

Degradation of carbazole by microbial cells immobilized in magnetic gellan gum gel beads

Polycyclic aromatic heterocycles, such as carbazole, are environmental contaminants suspected of posing human health risks. In this study, we investigated the degradation of carbazole by immobilized Sphingomonas sp. strain XLDN2-5 cells. Four kinds of polymers were evaluated as immobilization supports for Sphingomonas sp. strain XLDN2-5. After comparison with agar, alginate, and kappa-carrageenan, gellan gum was selected as the optimal immobilization support. Furthermore, Fe(3)O(4) nanoparticles were prepared by a coprecipitation method, and the average particle size was about 20 nm with 49.65-electromagnetic-unit (emu) g(-1) saturation magnetization. When the mixture of gellan gel and the Fe(3)O(4) nanoparticles served as an immobilization support, the magnetically immobilized cells were prepared by an ionotropic method. The biodegradation experiments were carried out by employing free cells, nonmagnetically immobilized cells, and magnetically immobilized cells in aqueous phase. The results showed that the magnetically immobilized cells presented higher carbazole biodegradation activity than nonmagnetically immobilized cells and free cells. The highest biodegradation activity was obtained when the concentration of Fe(3)O(4) nanoparticles was 9 mg ml(-1) and the saturation magnetization of magnetically immobilized cells was 11.08 emu g(-1). Additionally, the recycling experiments demonstrated that the degradation activity of magnetically immobilized cells increased gradually during the eight recycles. These results support developing efficient biocatalysts using magnetically immobilized cells and provide a promising technique for improving biocatalysts used in the biodegradation of not only carbazole, but also other hazardous organic compounds.     

Designing chitosan based magnetic beads with conocarpus waste-derived biochar for efficient sulfathiazole removal from contaminated water

The development of a simple method to synthesize highly efficient and stable magnetic microsphere beads for sulfathiazole (STZ) removal from contaminated aqueous media was demonstrated in this study. Conocarpus (Conocarpus erectus L.) tree waste (CW) derived biochar (BC) was modified to fabricate chitosan-BC (CBC) and magnetic CBC (CBC-Fe) microsphere beads. Proximate, chemical, and structural properties of the produced adsorbents were investigated. Kinetics, equilibrium, and pH adsorption batch trials were conducted to evaluate the effectiveness of the synthesized adsorbents for STZ removal. All adsorbents exhibited the highest STZ adsorption at pH 5.0. STZ adsorption kinetics data was best emulated using pseudo-second order and Elovich models. The equilibrium adsorption data was best emulated using Langmuir, Freundlich, Redlich–Peterson, and Temkin models. CBC-Fe demonstrated the highest Elovich, pseudo-second order, and power function rate constants, as well as the highest apparent diffusion rate constant. Additionally, Langmuir isotherm predicted maximum adsorption capacity was the highest for CBC-Fe (98.67 mg g⁻¹), followed by CBC (56.54 mg g⁻¹) and BC (48.63 mg g⁻¹). CBC-Fe and CBC removed 74.5%–108.8% and 16.2%–25.6% more STZ, respectively, than that of pristine BC. π-π electron-donor–acceptor interactions and Lewis acid-base reactions were the main mechanisms for STZ removal; however, intraparticle diffusion and H-bonding further contributed in the adsorption process. The higher efficiency of CBC-Fe for STZ adsorption could be due to its magnetic properties as well as stronger and conducting microsphere beads, which degraded the STZ molecules through generation of HO^• radicals.