Separation Of Nucleic Acids Research Articles

Rheology and Viscoelasticity of Proteins and Nucleic Acids Condensates.

Phase separation is as familiar as watching vinegar separating from oil in vinaigrette. The observation that phase separation of proteins and nucleic acids is widespread in living cells has opened an entire field of research into the biological significance and the biophysical mechanisms of phase separation and protein condensation in biology. Recent evidence indicates that certain proteins and nucleic acids condensates are not simple liquids and instead display both viscous and elastic behaviors, which in turn may have biological significance. The aim of this Perspective is to review the state-of-the-art of this quickly emerging field focusing on the material and rheological properties of protein condensates. Finally, we discuss the different techniques that can be employed to quantify the viscoelasticity of condensates and highlight potential future directions and opportunities for interdisciplinary cross-talk between chemists, physicists, and biologists.

Measuring the velocity profile of spinning particles and its impact on Cr(VI) sequestration

The evolution and development of magnetic nanoparticles (MNPs) are of great interest to researchers owing to their novel properties and wide-ranging applications. Essentially, MNPs coupled with magnetic fields (MF) have been used in biomedical separation, catalysis, cell separation, magnetic target drug delivery, hyperthermia, nucleic acid separation and wastewater treatment. In that regard, this paper reports the investigation of the sorption phenomena of chromium six (Cr(VI)) to MNPs interaction spun under the influence of alternating MF utilizing this unique particle shadow velocimetry technique. Under an alternating MF, the velocity field of spinning particles was observed to increase with magnetic field intensification. The determined mean velocity field for the spinning particles in the Cr(VI) solution increases from 0.0075-0.0125 m/s at a MF range of 16.54-25.54 mT. The rotating MF was observed to improve the magnetic force and torque induced on the particles, with particle aggregates reorienting with the magnetic moment of the field. The variability observed at the liquid-particle interface led to a fast mixing, with chaotic flow behaviour in the channel wall induced by the magnetic force observed. The induced cluster particle rotation and broad magnetic velocity distributions also led to cluster particle collisions hence the enhanced sorption process.

Simple Electric Device to Isolate Nucleic Acids from Whole Blood Optimized for Point of Care Testing of Brain Damage.

Detection or monitoring of brain damage is a clinically crucial issue. Nucleic acids in the whole blood can be used as biomarkers for brain injury. Polymerase chain reaction (PCR) which is one of the most commonly used molecular diagnostic assays requires isolated nucleic acids to initiate amplification. Currently used nucleic acid isolation procedures are complicated and require laboratory equipments. In this study, we tried to develop a simple and convenient method to isolate nucleic acids from the whole blood sample using a tiny battery-powered electric device. The quality of the isolated nucleic acids should be suitable for PCR assay without extra preparation. A plastic device with separation chamber was designed and printed with a 3D printer. Two platinum electrodes were placed on both sides and a battery was used to supply the electricity. To choose the optimal nucleic acid isolation condition, diverse lysis buffers and separation buffers were evaluated, and the duration and voltage of the electricity were tested. Western blot analysis and PCR assay were used to determine the quality of the separated nucleic acids. 2ul of whole blood was applied to the cathode side of the separation chamber containing 78 ul of normal saline. When the electricity at 5 V was applied for 5 min, nucleic acids were separated from segment 1 to 3 of the separation chamber. The concentration of nucleic acids peaked around 7~8 mm from cathode side. PCR assay using the separation buffer as the template was performed successfully both in conventional and realtime PCR methods. The hemoglobin in the whole blood did not show the inhibitory effect in our separation system and it may be due to structural modification of hemoglobin during electric separation. Our simple electric device can separate nucleic acids from the whole blood sample by applying electricity at 5 V for 5 min. The separation buffer solution taken from the device can be used for PCR assay successfully.

Novel Viral DNA Polymerases From Metagenomes Suggest Genomic Sources of Strand-Displacing Biochemical Phenotypes.

Viruses are the most abundant and diverse biological entities on the planet and constitute a significant proportion of Earth’s genetic diversity. Most of this diversity is not represented by isolated viral-host systems and has only been observed through sequencing of viral metagenomes (viromes) from environmental samples. Viromes provide snapshots of viral genetic potential, and a wealth of information on viral community ecology. These data also provide opportunities for exploring the biochemistry of novel viral enzymes. The in vitro biochemical characteristics of novel viral DNA polymerases were explored, testing hypothesized differences in polymerase biochemistry according to protein sequence phylogeny. Forty-eight viral DNA Polymerase I (PolA) proteins from estuarine viromes, hot spring metagenomes, and reference viruses, encompassing a broad representation of currently known diversity, were synthesized, expressed, and purified. Novel functionality was shown in multiple PolAs. Intriguingly, some of the estuarine viral polymerases demonstrated moderate to strong innate DNA strand displacement activity at high enzyme concentration. Strand-displacing polymerases have important technological applications where isothermal reactions are desirable. Bioinformatic investigation of genes neighboring these strand displacing polymerases found associations with SNF2 helicase-associated proteins. The specific function of SNF2 family enzymes is unknown for prokaryotes and viruses. In eukaryotes, SNF2 enzymes have chromatin remodeling functions but do not separate nucleic acid strands. This suggests the strand separation function may be fulfilled by the DNA polymerase for viruses carrying SNF2 helicase-associated proteins. Biochemical data elucidated from this study expands understanding of the biology and ecological behavior of unknown viruses. Moreover, given the numerous biotechnological applications of viral DNA polymerases, novel viral polymerases discovered within viromes may be a rich source of biological material for further in vitro DNA amplification advancements.

Spatiotemporal modulations in heterotypic condensates of prion and \u03b1-synuclein control phase transitions and amyloid conversion

Biomolecular condensation via liquid-liquid phase separation of proteins and nucleic acids is associated with a range of critical cellular functions and neurodegenerative diseases. Here, we demonstrate that complex coacervation of the prion protein and α-synuclein within narrow stoichiometry results in the formation of highly dynamic, reversible, thermo-responsive liquid droplets via domain-specific electrostatic interactions between the positively-charged intrinsically disordered N-terminal segment of prion and the acidic C-terminal tail of α-synuclein. The addition of RNA to these coacervates yields multiphasic, vesicle-like, hollow condensates. Picosecond time-resolved measurements revealed the presence of transient electrostatic nanoclusters that are stable on the nanosecond timescale and can undergo breaking-and-making of interactions on slower timescales giving rise to a liquid-like behavior in the mesoscopic regime. The liquid-to-solid transition drives a rapid conversion of complex coacervates into heterotypic amyloids. Our results suggest that synergistic prion-α-synuclein interactions within condensates provide mechanistic underpinnings of their physiological role and overlapping neuropathological features.

Functionalized cryogel monoliths for fast and selective separation of nucleic acids directly from crude lysate.

The fast and selective separation of nucleic acids has been attractive recently because of their wide number of applications in the biomedical field such as the development of vaccines for infectious diseases, gene therapy, and diagnosis. Traditional approaches of nucleic acids separation are costlier, lengthy, and associated with possible denaturation because of the use of organic solvents in the elution step. Under this perspective, cryogels represent an attractive choice as a monolith stationary phase in column chromatography, which have proven efficient in recent chromatographic studies. Cryogels are the macroporous hydrogels with interconnecting properties between the pores. They allow the easy flow of large biomolecules with minimum mass transfer resistance. They are spongy in nature and possess good mechanical strength. Current article represents different developed functionalized cryogel monoliths for nucleic acids separation, their separation strategies, and challenges associated with further advancement in separation science.

A Liquid State Perspective on Dynamics of Chromatin Compartments.

The interior of the eukaryotic cell nucleus has a crowded and heterogeneous environment packed with chromatin polymers, regulatory proteins, and RNA molecules. Chromatin polymer, assisted by epigenetic modifications, protein and RNA binders, forms multi-scale compartments which help regulate genes in response to cellular signals. Furthermore, chromatin compartments are dynamic and tend to evolve in size and composition in ways that are not fully understood. The latest super-resolution imaging experiments have revealed a much more dynamic and stochastic nature of chromatin compartments than was appreciated before. An emerging mechanism explaining chromatin compartmentalization dynamics is the phase separation of protein and nucleic acids into membraneless liquid condensates. Consequently, concepts and ideas from soft matter and polymer systems have been rapidly entering the lexicon of cell biology. In this respect, the role of computational models is crucial for establishing a rigorous and quantitative foundation for the new concepts and disentangling the complex interplay of forces that contribute to the emergent patterns of chromatin dynamics and organization. Several multi-scale models have emerged to address various aspects of chromatin dynamics, ranging from equilibrium polymer simulations, hybrid non-equilibrium simulations coupling protein binding and chromatin folding, and mesoscopic field-theoretic models. Here, we review these emerging theoretical paradigms and computational models with a particular focus on chromatin’s phase separation and liquid-like properties as a basis for nuclear organization and dynamics.

Micro/nanofluidic devices for DNA/RNA detection and separation.

The development and research have ramped up at a greater speed than ever in the field of diseases diagnosis. Still there is struggle in developing early detection techniques which uses complex biomolecules like RNA, DNA and proteins in order to detect diseases caused by bacteria, viruses or fungi. Until now separation techniques used before detection rely on traditional techniques like electrophoresis etc. which often require centralized services. Although efforts are made in developing devices that is capable enough on carrying out separation and detection based on microfluidic (MF) and nanofluidic (NF) or lab on chip. Hence, in this chapter, we have discussed about the advancement, limitations and future steps that needs to be taken to flourish the field of NF and MF for the detection and separation of nucleic acid.

Visualizing carboxyl-terminal domain of RNA polymerase II recruitment by FET fusion protein condensates with DNA curtains.

Many recent references show that living cells can form some membrane-less organelles by liquid-liquid phase separation (LLPS) of biomolecules, like proteins and nucleic acids. LLPS has been confirmed to link with many important biological functions in living cells, and one of the most important functions of biomolecular condensates is in the field of RNA transcription. Many studies confirm that mammalian RNA polymerase II (Pol II) molecules containing the CTD with different phosphorylation level are purposed to shuttle between initiation condensates and elongation condensates of RNA transcription. Traditional ensemble assays often experience difficulties in quantitively and directly recording the transient recruitment of Pol II CTD. Novel single-molecule approach - DNA curtains can be used to directly visualize biomolecular condensates formation and also recruitment of RNA polymerase II (Pol II) carboxyl-terminal domain (CTD) at the target sites in solution and in real time. This method can offer the potential for new insights into the mechanism of gene transcription. Here, we highlight the detailed protocol of DNA curtains method for studying LLPS.

Preparation of seaweed polysaccharide based hydrophobic composite membranes for the separation of oil/water emulsion and protein

Agarose is a seaweed-based polysaccharide and is widely used for the separation of nucleic acids in molecular biology. Cross-linked agarose beads are also used as solid-phase matrices in size exclusion chromatography for the separation of proteins. To find the application of agarose for the separation of oil/water emulsion and protein, herein hydrophobic derivative of the seaweed biopolymer [MW (1.27 ± 0.17) × 10 5 g/mol; sulphate content (0.29 ± 0.09) %, gel strength (2242 ± 21) g/cm2] is prepared by reacting the biopolymer with stearic acid and was used to prepare a composite membrane on polyester fabric. The oil and BSA rejection performance of the composite membrane was greater than 98%. The rejection rate increased with the increase in polymer content in the respective membranes for both oil/water and protein separation. The composite membrane showed a stable oil/water emulsion and protein separation performance over a period of six hours. Due to the biodegradable nature of the major components of the membrane, it has the potential for industrial applications.

Fabrication of chitosan-coated mixed spinel ferrite integrated with graphene oxide (GO) for magnetic extraction of viral RNA for potential detection of SARS-CoV-2.

Genetic variants of the COVID-19 causative virus have been arising and circulating globally. In many countries, especially in developing ones with a huge population, vaccination has become one of the major challenges. SARS-CoV-2 variants’ fast transmission rate has an upsurge in the COVID cases, leading to more stress on health systems. In the current COVID-19 scenario, there is the requirement of more adequate diagnostic approaches to check the COVID-19 spread. Out of many diagnostic approaches, a magnetic nanoparticle-based reverse transcription polymerase chain reaction could be nontrivial. The use of magnetic nanoparticles is to separate nucleic acid of SARS-CoV-2 from the patient samples and apply for SARS-CoV-2 detection in an easy and more effective way. Herein, the magnetic nanoparticles are synthesized using the solgel autocombustion methods and then successfully coated with biopolymer (chitosan) using ultrasonication. Chitosan-coated nanoparticles are successfully integrated into the graphene oxide sheets to introduce carboxyl groups. Crystallite size calculation, morphological and magnetic studies of synthesized magnetic nanoparticles, and multifunctional magnetic nanoparticles are done using XRD, SEM, TEM, and VSM, respectively. Besides, the potentiality of the fabricated nanocomposites in RNA extraction protocol is also discussed with schematic representation.

Selective precipitation of RNA with linear polyacrylamide

Selective precipitation of RNA is often used in molecular biology as one of the methods for separation of nucleic acids to obtain samples enriched with DNA or RNA molecules alone or for purification of RNA samples. In the present study a simple and fast approach for selective precipitation of RNA with linear polyacrylamide is proposed for the first time. The method is based on the different predispositions of the DNA and RNA molecules to bind with the polyacrylamide. In this process, the linear polyacrylamide is used as the flocculant, collecting RNA particles to form aggregate, which then precipitated at low alcohol concentration. During and after precipitation the temperature is adjusted to maintain high solubility of DNA and other contaminates at given pH, salt and alcohol concentrations on the one hand, and globular state of polyacrylamide, preventing solubility of the RNA-LPA aggregate, on the other hand. The precipitated RNA can be used directly for RT-qPCR assay. The principal advantage of the present approach is the fast and quantitative precipitation of most RNA species from very dilute solutions. This makes it possible to obtain both almost DNA-free RNA and RNA-free DNA samples in one process. Supplemental data for this article is available online at https://doi.org/10.1080/15257770.2021.2007397 .

Solid-phase extraction methods for nucleic acid separation. A review.

The separation and purification of biomacromolecules such as nucleic acid is a perpetual topic in separation processes and bioengineering (fine chemicals, biopharmaceutical engineering, diagnostics, and biological characterization). In principle, the solid-phase extraction for nucleic acid exhibits efficient phase separation, low pollution risk, and small sample demand, compared to the conventional liquid-phase extraction. Herein, solid-phase extraction methods are systematically reviewed to outline research progress and explore additional solid-phase sorbents and devices for novel, flexible, and high-efficiency nucleic acid separation processes. The functional materials capture nucleic acid, magnetic and magnetic-free solid-phase extraction methods, separation device design and optimization, and high-throughput automatable applications based on high-performance solid-phase extraction are summarized. Finally, the current challenges and promising topics are discussed.

Looping-in complexation and ion partitioning in nonstoichiometric polyelectrolyte mixtures.

A wide variety of intracellular membraneless compartments are formed via liquid-liquid phase separation of charged proteins and nucleic acids. Understanding the stability of these compartments, while accounting for the compositional heterogeneity intrinsic to cellular environments, poses a daunting challenge. We combined experimental and theoretical efforts to study the effects of nonstoichiometric mixing on coacervation behavior and accurately measured the concentrations of polyelectrolytes and small ions in the coacervate and supernatant phases. For synthetic polyacrylamides and polypeptides/DNA, with unequal mixing stoichiometry, we report a general "looping-in" phenomenon found around physiological salt concentrations, where the polymer concentrations in the coacervate initially increase with salt addition before subsequently decreasing. This looping-in behavior is captured by a molecular model that considers reversible ion binding and electrostatic interactions. Further analysis in the low-salt regime shows that the looping-in phenomenon originates from the translational entropy of counterions that are needed to neutralize nonstoichiometric coacervates.

Pressure and Temperature Phase Diagram for Liquid-Liquid Phase Separation of the RNA-Binding Protein Fused in Sarcoma.

Liquid-liquid phase separation (LLPS) of proteins and nucleic acids to form membraneless cellular compartments is considered to be involved in various biological functions. The RNA-binding protein fused in sarcoma (FUS) undergoes LLPS in vivo and in vitro. Here, we investigated the effects of pressure and temperature on the LLPS of FUS by high-pressure microscopy and high-pressure UV/vis spectroscopy. The phase-separated condensate of FUS was obliterated with increasing pressure but was observed again at a higher pressure. We generated a pressure-temperature phase diagram that describes the phase separation of FUS and provides a general understanding of the thermodynamic properties of self-assembly and phase separation of proteins. FUS has two types of condensed phases, observed at low pressure (LP-LLPS) and high pressure (HP-LLPS). The HP-LLPS state was more condensed and exhibited lower susceptibility to dissolution by 1,6-hexanediol and karyopherin-β2 than the LP-LLPS state. Moreover, molecular dynamic simulations revealed that electrostatic interactions were destabilized, whereas cation-π, π-π, and hydrophobic interactions were stabilized in HP-LLPS. When cation-π, π-π, and hydrophobic interactions were transiently stabilized in the cellular environment, the phase transition to HP-LLPS occurred; this might be correlated to the aberrant enrichment of cytoplasmic ribonucleoprotein granules, leading to amyotrophic lateral sclerosis.

Emerging Roles of Liquid-Liquid Phase Separation in Cancer: From Protein Aggregation to Immune-Associated Signaling.

Liquid–liquid Phase Separation (LLPS) of proteins and nucleic acids has emerged as a new paradigm in the study of cellular activities. It drives the formation of liquid-like condensates containing biomolecules in the absence of membrane structures in living cells. In addition, typical membrane-less condensates such as nuclear speckles, stress granules and cell signaling clusters play important roles in various cellular activities, including regulation of transcription, cellular stress response and signal transduction. Previous studies highlighted the biophysical and biochemical principles underlying the formation of these liquid condensates. The studies also showed how these principles determine the molecular properties, LLPS behavior, and composition of liquid condensates. While the basic rules driving LLPS are continuously being uncovered, their function in cellular activities is still unclear, especially within a pathological context. Therefore, the present review summarizes the recent progress made on the existing roles of LLPS in cancer, including cancer-related signaling pathways, transcription regulation and maintenance of genome stability. Additionally, the review briefly introduces the basic rules of LLPS, and cellular signaling that potentially plays a role in cancer, including pathways relevant to immune responses and autophagy.

Functional Magnetite Nanoparticle: A Review on the Particles Lysis and Nucleic Acid Separation

The functional magnetite nanoparticles are one of the most important functional materials for nucleic acid separation. Cell lysis and magnetic separation are two essential steps involve in optimizing nucleic acid extraction using the magnetic beads method. Many coating materials, coupling agents, chemical cell lysis, and several methods have been proposed to produce the specific desired properties for nucleic acid extraction. The important properties, such as biocompatibility, stability, linking ability, hydrophobicity, and biodegradable, were considered. The appropriate coating material of magnetite core and coupling agent are necessary to give biomolecules a possibility to link with each other through chemical conjugation. In this review, progress in functional magnetite nanoparticles to optimize the high binding performance in nucleic acid extraction is discussed.

Complete genome sequence of hollyhock vein yellowing virus, a novel monopartite begomovirus infecting hollyhock in Pakistan.

Hollyhock (Alcea rosea, family Malvaceae) is an ornamental plant grown widely in gardens across South Asia. In a bed of ornamental plants near the village of Chakri (Punjab Province, Pakistan) in 2014, hollyhock plants showing two distinct symptom types were identified: yellow vein mosaic and leaf crumple. PCR amplification with universal primers amplified a begomovirus from separate nucleic acid extracts of single plants of each type but amplified a betasatellite only from the plant with the yellow vein mosaic symptoms. No potential begomovirus DNA B component or alphasatellite could be identified in either sample. After cloning, the genome sequences of two viruses, one from a plant of each symptom type, were determined and shown to share 99.9% nucleotide sequence identity with each other but less than 91% nucleotide sequence identity with all previously characterized begomoviruses, with the highest identity (90%) to an isolate of pedilanthus leaf curl virus (PeLCV). This indicates that the two hollyhock plants were infected with a newly identified begomovirus for which the name "hollyhock vein yellowing virus" (HoVYV) is proposed. HoVYV likely has a recombinant origin. The betasatellite showed the highest nucleotide sequence identity to an isolate of cotton leaf curl Multan betasatellite (CLCuMuB), a betasatellite associated with cotton leaf curl disease across Pakistan and northwestern India. These findings add to the diversity of known begomoviruses in South Asia and again highlight the role of hollyhock as a reservoir of the cotton leaf curl begomovirus betasatellite complex. The results also suggest that the yellow vein mosaic symptoms in hollyhock are due to the betasatellite rather than the virus.

(Invited) Fieldable Optical Biosensors with Integrated Sample Processing for Universal Surveillance and Diagnostics – Application to COVID-19

There is an urgent need for broad-based surveillance and diagnostic strategies that can be applied quickly and effectively to address emerging pathogens, and preferably at the point of need, as is evident with the current covid-19 crisis. Our innate immune system is an example of such a sensor – a recognition system that is capable of identifying all pathogens quickly and sensitively. Conserved pathogen associated molecular patterns (PAMPs) produced by the infectious agents are recognized by immune receptors (e.g. Toll like Receptors, TLRs) in an exquisite scheme of pattern recognition, culminating in universal early identification pipeline. Mimicking innate immune recognition in the laboratory can therefore provide a strategy for universal diagnosis of infection, and this has been the focus of our research for over a decade.There are three objectives to be addressed in order to develop a universal diagnostic platform based on innate immune recognition. They are: 1) understanding host-pathogen biology, and the development of tailored assays to address biochemically disparate pathogen associated molecular patterns in complex clinical samples, 2) development of multiplex detection assays and ultra-sensitive sensor platforms for their detection, 3) field adaptation of sensors and sample processing and 4) validation in blinded clinical samples. Herein, we present the strategy and progress in each of these areas of development, as outlined below.In bacterial pathogens, pathogen signatures recognized by our innate immune receptors are typically amphiphilic in biochemistry – lipoproteins or glycolipids. These lipidic molecules are intrinsically unstable in the aqueous milieu of blood, making them difficult to detect. Work from our team lead to the understanding that all bacterial amphiphilic pathogen associated molecular patterns associate with host lipoprotein carriers, which function as a biological taxi service for their transport in blood. Understanding this, we developed tailored sample processing pipeline and assays (membrane insertion and lipoprotein capture) for the detection of amphiphiles in serum samples.In viral pathogens, the pathogen signatures recognized by our innate immune receptors are nucleic acids – DNA and RNA. We developed a bioinformatic pipeline – FEVER- to capture the conserved aspects of these signatures and develop our recognition probes to target these pathogens, and then developed amplification-free assays for their detection using molecular beacon technology.Assays developed for both bacterial and viral signatures were applied to a novel, fieldable waveguide-based optical biosensor platform developed by our team. This platform uses single mode planar optical waveguides for bio-detection within an evanescent field, offering much greater sensitivity (10X or higher) than conventional immunoassay platforms. This fieldable sensor (<8Lbs) with integrated phone based controls allows for bio-detection in the field, as is required for surveillance technologies. We have developed a multiplex detection pipeline on this platform using photostable and tunable quantum dots as the fluorescence reporters – allowing for the simultaneous discriminatory detection of said signatures. This novel patented approach allows for the use of conventional platforms, in addition to our biosensor for such interrogations. For adaptation of the method to field detection, we developed the first ever microfluidic lipid and nucleic acid separation system – which allows for 5 min disposable separation of signatures from blood in the field. The performance of this platform has been validated for the detection of biomarkers not only in laboratory samples, but also in complex clinical samples from high-disease burden clinical cohorts for a variety of diseases such as COVID-19, tuberculosis, bacterial sepsis, food-borne diseases as well as biosecurity and national security applications. Figure: Schematic representing the fieldable universal diagnostic platform and approach. Blood is a universal sample – where all pathogens are recognized by innate immune receptors. Blood is collected and processed on site using a microfluidics sample processing cassette for biomarker extraction. The presence of the biomarker is then measured using novel tailored assays on a fieldable portable waveguide biosensor offering an answer within 15 min. Figure 1

3D printed integrated separator with hybrid micro-structures for high throughput and magnetic-free nucleic acid separation from organism samples

• 3D-printed integrated separator for nucleic acid (NA) extraction was fabricated. • The separator has ideal printing accuracy and stable structures, and can be reused. • Hybrid micro-structures enhanced NA extraction efficiency of 3D-printed separator. • Developed magnetic-free NA extraction method is fast, simple and high-throughput. • Developed NA extraction method is promising in routine PCR diagnosis of pathogens. Nucleic acid (NA) amplification is a powerful molecular biological tool. While, the high-efficient NA amplification can be only used in modern laboratory owing to the relatively time-consuming and multistep labor-intensive NA separation. Herein, an integrated polyacrylic acid (PAA) comb-like separator with hybrid micro-structures was fabricated via 3D printing technology for high throughput and convenient (magnetic-free) NA separation. It realizes the high-effective and fast NA separation from organism samples by simply transferring of the separator from different tubes containing lysate, washing solution and elution buffer, with enhanced throughput due to the feature of convenient operation on 8-tube strip or 96-well plate. Results showed the 3D printing PAA integrated separator has ideal printing accuracy and stability in the thread and groove structures, and can be reused with robust separation performance. The NA separation yield enhanced by the hybrid micro-structures on the interface of the integrated separator is comparable to that of classic magnetic nanoparticle (MNP) based NA separation method under optimized condition. The developed NA separation and purification process has the advantages of very short operation time (separation duration minimized to 1 min), no requirement of additional technical equipment (magnetic-free) and no intensive labors (no pipetting and centrifugation). The NA amplification results illustrated that NA isolated via the developed separator could meet the requirements for further biological applications, especially in the field of high-throughput molecular detection of pathogens. This novel separator can provide a promising pave for NA separation to satisfy the high-throughput and automation requirements of the related biological applications.