Adsorption and isolation of nucleic acids on cellulose magnetic beads using a three-dimensional printed microfluidic chip

While advances in genomics have enabled sensitive and highly parallel detection of nucleic acid targets, the isolation and extraction of the nucleic acids remain a critical bottleneck in the workflow. We present here a simple 3D printed microfluidic chip that allows for the vortex and centrifugation free extraction of nucleic acids. This novel microfluidic chip utilizes the presence of a water and oil interface to filter out the lysate contaminants. The pure nucleic acids, while bound on cellulose particles, are magnetically moved across the oil layer. We demonstrated efficient and rapid extraction of spiked Human Papillomavirus (HPV) 18 plasmids in specimen transport medium, in under 15 min. An overall extraction efficiency of 61% is observed across a range of HPV plasmid concentrations (5 × 10¹ to 5 × 10⁶ copies/100 μl). The magnetic, interfacial, and viscous drag forces inside the microgeometries of the chip are modeled. We have also developed a kinetics model for the adsorption of nucleic acids on cellulose functionalized superparamagnetic beads. We also clarify here the role of carrier nucleic acids in the adsorption and isolation of nucleic acids. Based on the various mechanistic insights detailed here, customized microfluidic devices can be designed to meet the range of current and emerging point of care diagnostics needs.     

Drop-to-drop liquid–liquid extraction of DNA in an electrowetting-on-dielectric digital microfluidics

Recent advancements in microfluidics and lab-on-a-chip technologies enabled miniaturization and automation of many downstream nucleic acid analysis steps such as PCR. However, DNA extraction/isolation protocol remains a stand-alone sample preparation step. For a quick sample-to-result solution, downstream protocols and sample preparation protocols need to be seamlessly integrated into a single lab-on-a-chip platform. As a step toward such integration, this paper introduces microfluidic DNA isolation using the liquid–liquid extraction (LLE) method in the drop-to-drop (DTD) format. The electrowetting-on-dielectric digital microfluidic platform is capable of handling a two-phase liquid system easily, which enables DTD LLE. In this study, the extraction of plasmid DNA (pDNA) from an aqueous sample to an ionic liquid is demonstrated. Prior to pDNA extraction study, the DTD LLE protocol was developed and optimized using organic dyes as solutes. The selective extraction of pDNA in the presence of proteins as interfering molecules is also demonstrated. This work implies that DTD LLE can substitute for magnetic beads steps in standard DNA isolation protocols.     

Integration of programmable microfluidics and on-chip fluorescence detection for biosensing applications

We describe the integration of an actively controlled programmable microfluidic sample processor with on-chip optical fluorescence detection to create a single, hybrid sensor system. An array of lifting gate microvalves (automaton) is fabricated with soft lithography, which is reconfigurably joined to a liquid-core, anti-resonant reflecting optical waveguide (ARROW) silicon chip fabricated with conventional microfabrication. In the automaton, various sample handling steps such as mixing, transporting, splitting, isolating, and storing are achieved rapidly and precisely to detect viral nucleic acid targets, while the optofluidic chip provides single particle detection sensitivity using integrated optics. Specifically, an assay for detection of viral nucleic acid targets is implemented. Labeled target nucleic acids are first captured and isolated on magnetic microbeads in the automaton, followed by optical detection of single beads on the ARROW chip. The combination of automated microfluidic sample preparation and highly sensitive optical detection opens possibilities for portable instruments for point-of-use analysis of minute, low concentration biological samples.     

The 2008 Benjamin Franklin Medal in Chemistry is presented to Albert Eschenmoser, Dr. sc.nat

The Franklin Institute, Philadelphia, Pennsylvania, awards the 2008 Benjamin Franklin Medal in Chemistry to Professor Albert Eschenmoser for his seminal efforts in the chemical etiology of nucleic acid structure, which through systematic exploration employing chemical synthesis of potentially natural structural alternatives to DNA, demonstrated that Watson-Crick base pairing is not unique to the ribofuranosyl system, and that DNA’s structure represents an optimization of several factors rather than a maximization of base-pairing strength.In his early scientific career, Professor Albert Eschenmoser established himself as one of the premier synthetic organic chemists in the world, with the monumental achievement of two total syntheses of Vitamin B₁₂, the first in collaboration with Professor Robert B. Woodward of Harvard University.However, since the 1980s, Professor Eschenmoser has concentrated his research interest in establishing the area of chemical etiology of nucleic acid structures to understand better the structural and functional uniqueness of the molecular basis of nature’s genetic information carrier—DNA and RNA. This systematic investigation of potential natural nucleic acid alternatives has demonstrated experimentally that Watson-Crick base pairing is not a unique property of DNA and RNA. Moreover, his research on homo-DNA revealed that the helicality of the famous double-stranded DNA is a direct outcome of the 5-membered ring nature of the deoxyribofuranose structure unit, while the study on p-RNA demonstrated that nature did not choose her genetic system by the standard of maximal base pairing strength, but instead optimization. Of equal significance, his recent design and synthesis of TNA (α-threofuranosyl nucleic acid), found to possess extraordinary base-paring properties, led to the hypothesis suggesting TNA as a possible precursor to the “RNA World”. Finally, his research on the correlation between the differences in the pKa of nucleic bases and their base-pairing strength led to a novel principal for the selection of nucleic base alternatives with proper bonding strength.     

Isolation of target DNA using synergistic magnetic bead transport and electrokinetic flow

The advent and dissemination of next-generation sequencing (NGS) technologies such as Illumina''s sequencing platforms has brought forth vast reductions in the cost, time, and technical difficulties associated with DNA and RNA sequencing. Despite this trend, the workflow required to generate nucleic acid libraries for sequencing remains time-consuming and laborious. The following research proposes a method for simplifying and streamlining this process by replacing the manual washing steps of the common magnetic bead-based cleanup with a novel microfluidic method by integrating magnetic separation and electrokinetic purification (MSEP). Requiring no pumps, pipette mixing, vortexing, or centrifugation, MSEP relies on selective adsorption of target DNA onto the magnetic beads with subsequent transport of beads through a microchannel undergoing an antiparallel electroosmotic flow. The synergetic flow conditions were optimized using a simple electrohydrodynamic flow model. This work demonstrates that MSEP is as effective in eliminating adapter-dimers from the post-ligation library mix as the manual method while also greatly reducing the hands-on time and amount of pipetting required. Although MSEP has been applied specifically toward NGS library preparation at this time, it has the potential to be adapted and employed for any bead-based separation scheme, namely, solid phase extraction, sequence-specific hybridization, and immunoprecipitation on a microscale.     

DNA纳米技术与生物编程

DNA纳米技术是设计和构建具有一定用途的人工核酸纳米结构。DNA纳米技术中的一个重要挑战就是如何有效设计和构建具有明确功能的纳米尺度的结构和器件。计算机辅助工具是预测、设计、建模以及描绘DNA结构的最强有力的工具。计算机辅助设计DNA结构以及识别有效的自组装路径让DNA作为一种独特的材料在构建纳米结构领域中显露锋芒。文章总结了DNA纳米结构设计的原理,介绍了一些用于DNA结构设计的算法以及一些用户友好软件。同时,对DNA纳米技术中构建DNA逻辑门以及DNA计算方面的最新研究进展做了介绍。     

Rapid microfluidic solid-phase extraction system for hyper-methylated DNA enrichment and epigenetic analysis

Genetic sequence and hyper-methylation profile information from the promoter regions of tumor suppressor genes are important for cancer disease investigation. Since hyper-methylated DNA (hm-DNA) is typically present in ultra-low concentrations in biological samples, such as stool, urine, and saliva, sample enrichment and amplification is typically required before detection. We present a rapid microfluidic solid phase extraction (μSPE) system for the capture and elution of low concentrations of hm-DNA (≤1 ng ml⁻¹), based on a protein-DNA capture surface, into small volumes using a passive microfluidic lab-on-a-chip platform. All assay steps have been qualitatively characterized using a real-time surface plasmon resonance (SPR) biosensor, and quantitatively characterized using fluorescence spectroscopy. The hm-DNA capture/elution process requires less than 5 min with an efficiency of 71% using a 25 μl elution volume and 92% efficiency using a 100 μl elution volume.     

教师桌面发布与共享实验教学软件的设计与实现

程序设计、软件开发类的实验教学要求越来越高,教学环境一般在网络机房。而学生掌握着自用电脑的控制权,甚至可以随意上网,网络教学环境使学生的思想不容易集中于教学,导致效率不高,教学效果不好。教师桌面发布与共享使教师的电脑桌面在必要时取代学生电脑桌面,迫使学生集中于教学内容,减少了外界环境对学生的吸引。借助于网络控制,教师和学生还可以实现互动式的教学,方便教师演示操作,协助学生提升教学效果。     

Free DNA – new potential analyte in clinical laboratory diagnostics?

The existence of cell free DNA in the human circulatory system has been known since the 1950s, however, intensive research in this area has been conducted for the last ten years. This review paper brings a short overview of the existing literature concerning the cell free DNA research in various clinical fields and pathological states and considers the application possibilities of this new analyte in clinical laboratory diagnostics. At the moment, cell free DNA is most widely used for the purpose of non-invasive prenatal diagnosis of fetal sex or fetal RhD status. The recent discovery of epigenetic changes in placental/fetal DNA and the detection of fetal/placental-specific RNAs have made it possible to use this technology in all pregnancies irrespective of the gender of the fetus. With the application of new techniques such as next generation sequencing, digital PCR and mass spectrometry, it is now possible to detect very small amounts of specific DNA in the presence of excess of other nonspecific nucleic acids. Second most probable application is in oncology, where detection and monitoring of tumors is now possible by the detection of tumor-derived nucleic acids. Third promising field for near future implementation of this analyte is transplantation medicine, where free DNA level could serve as a marker of transplant rejection. Before any further utilization of this new biomarker, pre-analytical and analytical aspects of free DNA analysis remain to be standardized. In the field of noninvasive prenatal diagnosis, important ethical, legal and social questions remain to be discussed.     

“纳米-生物”会聚技术的测度及启示

运用专利计量与信息可视化技术手段,对会聚技术进行测度与可视化分析,是一个较新的研究课题。对纳米-生物(NB)会聚技术测度与可视化分析的结果显示,2000-2009年间,NB会聚技术呈现出快速发展的势头;NB会聚技术共涉及到4 998个不同的技术小类,其中"细菌、真菌、病毒等的测试与探测技术"是最重要的领域;NB会聚技术的热点主题,主要集中在核酸、碳纳米管、制备方法、生物标本、癌症治疗、靶标分子等方面。中国政府和科学技术界,应通过加强纳米与生物技术领域的合作,拓展纳米与生物技术的合作领域和合作主题,培养更多的NB会聚技术领域人才等途径,推动中国NB会聚技术的不断发展。NB会聚技术的测度与可视化分析方法,可应用于其他会聚技术领域。     

An easy and robust method for the isolation of high quality RNA from coconut tissues

BackgroundCoconut tissues consist of a complex network of polysaccharides, proteins, polyphenols, and lipids that can bind to nucleic acids and pose difficulty in isolation. Certainly, a vigorous method is required to isolate high quality and quantity of RNA from such tissues for the purpose of downstream experiments. In this paper, we discuss a newly developed method for the Isolation of RNA from Complex Matrices (IRCM) method from coconut tissues.ResultsThe method is robust, cheap, and efficient for the extraction of quality RNA in high quantities from the solid endosperm of stored and fresh coconut (150 μg/g FW with A260/280 = 1.89 and 247.5 μg/g FW with A260/280 = 1.91), coconut apple (263.8 μg/g FW with A260/280 = 1.97), and coconut bud (1052.5 μg/g FW with A260/280 = 2.00). The other well established methods, such as Method of RNA Isolation from Palm (MRIP), Cetyl Trimethyl Ammonium Bromide (CTAB), TRIZOL, and RNA plant kit failed to isolate quality RNA in appreciable quantities from the coconut tissues. Furthermore, the resultant RNA performed well in the downstream experiment, that is, RT-PCR for the production and amplification of cDNA.ConclusionsFrom the study, we concluded that the present method will play a vital role in the extraction of high quality RNA from complex matrices in a short time.How to cite: Iqbal A, Yang Y, Wu Y, et al. An easy and robust method for the isolation of high quality RNA from coconut tissues. Electron J Biotechnol 2020;48. https://doi.org/10.1016/j.ejbt.2020.09.008     

Label-free electronic probing of nucleic acids and proteins at the nanoscale using the nanoneedle biosensor

Detection of proteins and nucleic acids is dominantly performed using optical fluorescence based techniques, which are more costly and timely than electrical detection due to the need for expensive and bulky optical equipment and the process of fluorescent tagging. In this paper, we discuss our study of the electrical properties of nucleic acids and proteins at the nanoscale using a nanoelectronic probe we have developed, which we refer to as the Nanoneedle biosensor. The nanoneedle consists of four thin film layers: a conductive layer at the bottom acting as an electrode, an oxide layer on top, and another conductive layer on top of that, with a protective oxide above. The presence of proteins and nucleic acids near the tip results in a decrease in impedance across the sensing electrodes. There are three basic mechanisms behind the electrical response of DNA and protein molecules in solution under an applied alternating electrical field. The first change stems from modulation of the relative permittivity at the interface. The second mechanism is the formation and relaxation of the induced dipole moment. The third mechanism is the tunneling of electrons through the biomolecules. The results presented in this paper can be extended to develop low cost point-of-care diagnostic assays for the clinical setting.     

基于核酸的信息安全技术研究现状及发展建议

由于具有巨大并行计算能力、海量信息存储密度及超低能耗等优势,近年来,核酸分子在分子计算、数据存储以及信息安全等研究领域广受关注。特别作为基于数学难题的、传统的密码理论与技术的有益补充,以核酸为信息载体的数据隐藏、认证、加密等信息安全技术极富发展前景。文章在介绍基于核酸的加密、隐藏及认证技术原理的基础上,详细论述了该领域国内外的最新研究成果,并对我国如何发展基于核酸的信息安全技术的研究提出了建议。     

Viral satellites: parasitic nucleic acids capable of modulating disease expression

Viral satellites are small nucleic acids that can replicate only in conjunction with the nucleic acids of certain viruses in certain hosts. In spite of the absence of any known nucleotide sequence relationships between a viral satellite, its helper virus, and its host, the trilateral interaction that ensues is usually extremely specific. This specificity is often expressed as a highly characteristic modulation of the disease symptom(s) normally associated with a viral presence in a particular host. This opens up very interesting possibilities for the control of viral plant diseases.     

Continuous separation of blood cells in spiral microfluidic devices

Blood cell sorting is critical to sample preparation for both clinical diagnosis and therapeutic research. The spiral inertial microfluidic devices can achieve label-free, continuous separation of cell mixtures with high throughput and efficiency. The devices utilize hydrodynamic forces acting on cells within laminar flow, coupled with rotational Dean drag due to curvilinear microchannel geometry. Here, we report on optimized Archimedean spiral devices to achieve cell separation in less than 8 cm of downstream focusing length. These improved devices are small in size (<1 in.²), exhibit high separation efficiency (∼95%), and high throughput with rates up to 1 × 10⁶ cells per minute. These device concepts offer a path towards possible development of a lab-on-chip for point-of-care blood analysis with high efficiency, low cost, and reduced analysis time.     

Role of Intervention on Laboratory Performance: Evaluation of Quality Indicators in a Tertiary Care Hospital

Quality in laboratory has huge impact on diagnosis and patient management as 80–90% of all diagnosis is made on the basis of laboratory tests. Monitoring of quality indicators covering the critical areas of pre-analytical, analytical and post-analytical phases like sample misidentification, sample rejection, random and systemic errors, critical value reporting and TATs have a significant impact on performance of laboratory. This study was conducted in diagnostic laboratories receiving approximately 42,562 samples for clinical chemistry, hematology and serology. The list of quality indicators was developed for the steps of total testing process for which errors are frequent and improvements are possible. The trend was observed for all the QI before and after sensitisation of the staff over the period of 12 months. Incomplete test requisition form received in the lab was the most poor quality indicator observed (7.89%), followed by sample rejection rate (4.91%). Most significant improvement was found in pre- and post-analytical phase after sensitisation of staff but did not have much impact on analytical phase. Use of quality indicators to assess and monitor the quality system of the clinical laboratory services is extremely valuable tool in keeping the total testing process under control in a systematic and transparent way.     

Rapid detection of live methicillin-resistant Staphylococcus aureus by using an integrated microfluidic system capable of ethidium monoazide pre-treatment and molecular diagnosis

Methicillin-resistant Staphylococcus aureus (MRSA) is a bacterium resistant to all existing penicillin and lactam-based antimicrobial drugs and, therefore, has become one of the most prevalent antibiotic-resistant pathogens found in hospitals. The multi-drug resistant characteristics of MRSA make it challenging to clinically treat infected patients. Therefore, early diagnosis of MRSA has become a public-health priority worldwide. Conventionally, cell-culture based methodology and microscopic identification are commonly used for MRSA detection. However, they are relatively time-consuming and labor-intensive. Recently, molecular diagnosis based on nucleic acid amplification techniques, such as polymerase chain reaction (PCR), has been widely investigated for the rapid detection of MRSA. However, genomic DNA of both live and dead pathogens can be distinguished by conventional PCR. These results thus could not provide sufficient confirmation of an active infection for clinicians. In this study, live MRSA was rapidly detected by using a new integrated microfluidic system. The microfluidic system has been demonstrated to have 100% specificity to detect live MRSA with S. aureus and other pathogens commonly found in hospitals. The experimental results showed that the limit of detection for live MRSA from biosamples was approximately 10² CFU/μl. In addition, the entire diagnostic protocol, from sample pre-treatment to fluorescence observation, can be automatically completed within 2.5 h. Consequently, this microfluidic system may be a powerful tool for the rapid molecular diagnosis of live MRSA.     

托拉塞米中有关物质的HPLC测定

建立梯度洗脱反相高效液相色谱法测定托拉塞米中的有关物质。方法:采用高效液相色谱,色谱柱为十八烷基硅烷键合硅胶填充柱,以0.01mol/L磷酸二氢钾溶液(用磷酸调节pH值至3.0)为流动相A,1%四氢呋喃甲醇溶液为流动相B,进行梯度洗脱,进样量10μL,检测波长291nm。结果:各有关物质与主成分均达到基线分离,对托来赛米中4-(3-甲基苯氨基)-3-吡啶磺酰胺的含量进行定量分析。结论:本法灵敏、专属,可有效分离有关物质及降解产物,可用于有关物质及降解产物检查。     

After the double helix: Rosalind Franklin's research on Tobacco mosaic virus

Rosalind Franklin is best known for her informative X-ray diffraction patterns of DNA that provided vital clues for James Watson and Francis Crick's double-stranded helical model. Her scientific career did not end when she left the DNA work at King's College, however. In 1953 Franklin moved to J. D. Bernal's crystallography laboratory at Birkbeck College, where she shifted her focus to the three-dimensional structure of viruses, obtaining diffraction patterns of Tobacco mosaic virus (TMV) of unprecedented detail and clarity. During the next five years, while making significant headway on the structural determination of TMV, Franklin maintained an active correspondence with both Watson and Crick, who were also studying aspects of virus structure. Developments in TMV research during the 1950s illustrate the connections in the emerging field of molecular biology between structural studies of nucleic acids and of proteins and viruses. They also reveal how the protagonists of the "race for the double helix" continued to interact personally and professionally during the years when Watson and Crick's model for the double-helical structure of DNA was debated and confirmed.