One‐Step Nucleic Acid Purification and Noise‐Resistant Polymerase Chain Reaction by Electrokinetic Concentration for Ultralow‐Abundance Nucleic Acid Detection

Nucleic acid amplification tests (NAATs)integrated on a chip hold great promise for point‐of‐care diagnostics. Currently, nucleic acid (NA) purification remains time‐consuming and labor‐intensive, and it takes extensive efforts to optimize the amplification chemistry. Using selective electrokinetic concentration, we report one‐step, liquid‐phase NA purification that is simpler and faster than conventional solid‐phase extraction. By further re‐concentrating NAs and performing polymerase chain reaction (PCR) in a microfluidic chamber, our platform suppresses non‐specific amplification caused by non‐optimal PCR designs. We achieved the detection of 5 copies of M. tuberculosis genomic DNA (equaling 0.3 cell) in real biofluids using both optimized and non‐optimal PCR designs, which is 10‐ and 1000‐fold fewer than those of the standard bench‐top method, respectively. By simplifying the workflow and shortening the development cycle of NAATs, our platform may find use in point‐of‐care diagnosis.     

Rapid Absolute Determination Platform of Nucleic Acid for Point-of-care Testing

Nowadays nucleic acid tests are promising to be considered as point-of-care testing(POCT). However, no such devices are currently available that can perform all the functions, including absolute nucleic acid determination, worldwide on-site detection, rapid analysis and real-time results reporting via ubiquitous mobile networks simultaneously with full package and automated means of measurement. In this study, we presented a compact low-cost portable POC automated testing platform with all attributes mentioned above. A disposable self-priming compartmentalization(SPC) microfluidic chip is used to conduct isothermal amplification. The platform also includes a micro-computer controlled heating unit, an inexpensive optical imaging setup, and a mobile device with customized software. It may become a useful tool for the rapid on-site detection of infectious diseases as well as other pathogens.     

集成核酸提取的实时荧光PCR微全分析系统

集成核酸提取的实时荧光PCR微全分析系统将核酸提取、PCR扩增与实时荧光检测进行整合,在同一块微流控芯片上实现了核酸分析过程的全自动和全封闭,具有试剂用量少、分析速度快、操作简便等优点。本研究采用微机械加工技术制作集成核酸提取微流控芯片的阳极模,使用组合模具法和注塑法制作具有3D通道的PDMS基片,与玻璃基底通过等离子体键合封装成集成核酸提取芯片。构建了由微流体速度可调节(0~10 mL/min)的驱动控制装置、温控精度可达0.1℃的TEC温控平台、CCD检测功能模块等组成的微全分析系统。以人类血液裂解液为样品,采用硅胶膜进行芯片上核酸提取。系统根据设置好的时序自动执行,以2 mL/min的流体驱动速度完成20μL裂解液上样、清洗;以1 mL/min的流体驱动速度完成DNA洗脱,抽取PCR试剂与之混合注入到反应腔。提取的基因组DNA以链上内参基因GAPDH为检测对象,并以传统手工提取为对照,在该系统平台上进行PCR扩增和熔解曲线分析实验。片上PCR扩增结果显示,扩增曲线明显,Ct值分别为25.3和26.9。扩增产物进行熔解曲线分析得到的熔解温度一致,均为89.9℃。结果表明,此系统能够自动化、全封闭的在微流控芯片上完成核酸提取、PCR扩增与实时定量分析。     

一种可绝对定量核酸的数字PCR微流控芯片

构建了一种新型的可进行核酸单分子扩增和核酸绝对定量的数字聚合酶链式反应(数字PCR)微流控芯片. 应用多层软光刻技术, 以聚二甲基硅氧烷(PDMS)作为芯片材料, 盖玻片作为基底制作了具有3层结构以及微阀控制功能的微流控芯片. 芯片的大小与载玻片相当, 可同时检测4个样品, 每个样品通入芯片后平均分配到640个反应小室, 每个小室的体积为6 nL. 以从肺癌细胞A549中提取的18sRNA为样品检测了该芯片的可行性. 将样品稀释数倍后通入芯片, 核酸分子随机分布在640个小室中并扩增. 核酸分子在芯片中的分布符合泊松分布原理, 当样品中待测核酸分子平均拷贝数低于0.5个/小室时, 则每个反应小室包含0个或1个分子. 经过PCR扩增后, 有模板分子的小室检测结果为阳性反应, 而无模板分子的小室为阴性反应, 最后通过计数阳性反应室的个数, 可绝对定量原始待测样品中的目标DNA分子拷贝数. 实验结果表明, 该数字 PCR芯片可实现DNA单分子反应和核酸绝对定量, 具有成本低、 灵敏度高、 节省时间和试剂以及操作简单等优点, 为数字PCR方法在普通实验室的应用提供了一种新途径, 可用于癌症及感染性疾病的早期诊断、 单细胞分析、 产前诊断以及各种细菌病毒的核酸检验等研究.     

Programming Non-Nucleic Acid Molecules into Computational Nucleic Acid Systems

Nucleic acid (NA) computation has been widely developed in the past years to solve kinds of logic and mathematic issues in both information technologies and biomedical analysis. However, the difficulty to integrate non-NA molecules limits its power as a universal platform for molecular computation. Here, we report a versatile prototype of hybridized computation integrated with both nucleic acids and non-NA molecules. Employing the conformationally controlled ligand converters, we demonstrate that non-NA molecules, including both small molecules and proteins, can be computed as nucleic acid strands to construct the circuitry with increased complexity and scalability, and can be even programmed to solve arithmetical calculations within the computational nucleic acid system. This study opens a new door for molecular computation in which all-NA circuits can be expanded with integration of various ligands, and meanwhile, ligands can be precisely programmed by the nuclei acid computation.     

Light-Start CRISPR-Cas12a Reaction with Caged crRNA Enables Rapid and Sensitive Nucleic Acid Detection

The clustered regularly interspaced short palindromic repeats (CRISPR) system is a promising platform for nucleic acid detection. Regulating the CRISPR reaction would be extremely useful to improve the detection efficiency and speed of CRISPR diagnostic applications. Here, we have developed a light-start CRISPR-Cas12a reaction by employing caged CRISPR RNA (crRNA). When combined with recombinase polymerase amplification, a robust photocontrolled one-pot assay is achieved. The photocontrolled one-pot assay is simpler and is 50-fold more sensitive than the conventional assay. This improved detection efficiency also facilitates the development of a faster CRISPR diagnostic method. The detection of clinical samples demonstrated that 10–20 min is sufficient for effective detection, which is much faster than the current gold-standard technique PCR. We expect this advance in CRISPR diagnostics to promote its widespread detection applications in biomedicine, agriculture, and food safety.     

Enhancement of the Photolysis of Nucleic Acid Monomers by Phosphates

Abstract— In the tentative simulation of prebiotic synthesis, it was found that the photolysis yield of nucleic acid bases, nucleosides and nucleotides (NA) undergoing UV irradiation was sharply enhanced by added orthophosphate. Filter experiments demonstrated that the enhancement is due to UV quanta of wavelength less than 215 nm in the emission spectrum of a medium pressure mercury lamp. The phosphate-induced enhancement of the photolysis of NA constitutes an additional channel of nucleic acid monomer degradation through their interaction with photoionized phosphate radicals.     

A magnetic bead-based assay for the rapid detection of methicillin-resistant Staphylococcus aureus by using a microfluidic system with integrated loop-mediated isothermal amplification

This study reports a new diagnostic assay for the rapid detection of methicillin-resistant Staphylococcus aureus (MRSA) by combing nucleic acid extraction and isothermal amplification of target nucleic acids in a magnetic bead-based microfluidic system. By using specific probe-conjugated magnetic beads, the target deoxyribonucleic acid (DNA) of the MRSA can be specifically recognized and hybridized onto the surface of the magnetic beads which are then mixed with clinical sample lysates. This is followed by purifying and concentrating the target DNA from the clinical sample lysates by applying a magnetic field. Nucleic acid amplification of the target genes can then be performed by the use of a loop-mediated isothermal amplification (LAMP) process via the incorporation of a built-in micro temperature control module, followed by analyzing the optical density (OD) of the LAMP amplicons using a spectrophotometer. Significantly, experimental results show that the limit of detection (LOD) for MRSA in the clinical samples is approximately 10 fg μL(-1) by performing this diagnostic assay in the magnetic bead-based microfluidic system. In addition, the entire diagnostic protocol, from bio-sample pre-treatment to optical detection, can be automatically completed within 60 min. Consequently, this miniature diagnostic assay may become a powerful tool for the rapid purification and detection of MRSA and a potential point-of-care platform for detection of other types of infections.     

Microfluidic Isolation of Nucleic Acids

The detection of nucleic acids (NAs) within micro total analysis systems (μTASs) for point‐of‐care use is a rapidly developing research area. The efficient isolation of NAs from a raw sample is crucial for these systems to be maximally effective. The use of microfluidics assists in reducing sample sizes and reagent consumption, increases speed, avoids contamination, and enables automation. Through miniaturization into microchips, new techniques have been realized that would be unfavorable and inconvenient to use on a macroscopic scale, but provide an excellent platform for the purification of NAs on a microscopic scale. This Review considers the complexities of NA isolation with miniaturized and microfluidic devices, as well as the considerations when choosing a technique for microfluidic NA isolation, along with their advantages, disadvantages, and potential applications. The techniques presented include using silica‐based surfaces, functionalized paramagnetic beads, oligonucleotide‐modified polymer surfaces, pH‐dependent charged surfaces, Al₂O₃ membranes, and liquid‐phase isolation. This Review provides a basis to develop the chemistry to improve NA isolation and move it toward achieving 100 % efficiencies.     

Mismatches Improve the Performance of Strand‐Displacement Nucleic Acid Circuits

Catalytic hairpin assembly (CHA) has previously proven useful as a transduction and amplification method for nucleic acid detection. However, the two hairpin substrates in a CHA circuit can potentially react non‐specifically even in the absence of a single‐stranded catalyst, and this non‐specific background degrades the signal‐to‐noise ratio. The introduction of mismatched base pairs that impede uncatalyzed strand exchange reactions led to a significant decrease of the background signal, while only partially damping the signal in the presence of a catalyst. Various types and lengths of mismatches were assayed by fluorimetry, and in many instances, our MismatCHA designs yielded 100‐fold increased signal‐to‐background ratios compared to a ratio of 4:1 with the perfectly matched substrates. These observations could be of general utility for the design of non‐enzymatic nucleic acid circuits.     

A Pyrene-Modified Serinol Nucleic Acid Nanostructure Converts the Chirality of Threoninol Nucleic Acids into Circularly Polarized Luminescence Signals

Herein is reported a circularly polarized luminescent (CPL) probe that can respond to the chirality of nucleic acids. An achiral nanostructure was prepared by the hybridization of symmetric serinol nucleic acid (SNA) containing pyrene-modified residues. When chiral oligomers that were complementary to the SNA were added, they induced helicity into the SNA nanowire. Efficient circular dichroism (CD) signal amplification was observed when pyrene was attached to uracil bases through a rigid alkynyl linker. Both CPL and CD signals were observed; they depended on the chirality of the added acyclic threoninol nucleic acid (aTNA) oligomer. This system can be used to convert the chirality of chiral biomolecules into chiroptical signals.     

一种用于核酸绝对定量检测的高鲁棒性液滴式数字PCR芯片

为满足液滴式数字聚合酶链式反应（PCR）技术对扩增反应过程中稳定保存液滴以及反应后高效检测的核心需求,构建了一种具有过滤气泡和增强荧光信号功能的液滴式数字聚合酶链式反应芯片.该芯片可在10 min内产生20多万个半径约为21 μm的液滴.利用“玻璃天花板”的方式构建了独立于芯片主体材料的液滴收集腔,为液滴提供稳定的保存与反应环境;还构建了过滤结构,可有效过滤混入液相中的空气,提高芯片鲁棒性.同时,在液滴收集腔中引入反射层,增强荧光信号,使单个视野荧光成像时间缩短约40%,提高了检测效率.利用该芯片定量检测EGFR基因第21号外显子,检测信号与DNA浓度在10¹~10⁵ copies/μL范围内呈现良好的线性关系（R²=0.998）.该方案在载玻片大小的芯片上实现了液滴产生、PCR扩增和荧光信号读取,并具有较高的鲁棒性与检测效率,在核酸检测等方面具有应用潜力.     

Highly Stable Duplex Formation by Artificial Nucleic Acids Acyclic Threoninol Nucleic Acid (aTNA) and Serinol Nucleic Acid (SNA) with Acyclic Scaffolds

The stabilities of duplexes formed by strands of novel artificial nucleic acids composed of acyclic threoninol nucleic acid (aTNA) and serinol nucleic acid (SNA) building blocks were compared with duplexes formed by the acyclic glycol nucleic acid (GNA), peptide nucleic acid (PNA), and native DNA and RNA. All acyclic nucleic acid homoduplexes examined in this study had significantly higher thermal stability than DNA and RNA duplexes. Melting temperatures of homoduplexes were in the order of aTNA>PNA≈GNA≥SNA?RNA>DNA. Thermodynamic analyses revealed that high stabilities of duplexes formed by aTNA and SNA were due to large enthalpy changes upon formation of duplexes compared with DNA and RNA duplexes. The higher stability of the aTNA homoduplex than the SNA duplex was attributed to the less flexible backbone due to the methyl group of D ‐threoninol on aTNA, which induced clockwise winding. Unlike aTNA, the more flexible SNA was able to cross‐hybridize with RNA and DNA. Similarly, the SNA/PNA heteroduplex was more stable than the aTNA/PNA duplex. A 15‐mer SNA/RNA was more stable than an RNA/DNA duplex of the same sequence.     

Bioanalytical applications of isothermal nucleic acid amplification techniques

The most popular in vitro nucleic acid amplification techniques like polymerase chain reaction (PCR) including real-time PCR are costly and require thermocycling, rendering them unsuitable for uses at point-of-care. Highly efficient in vitro nucleic acid amplification techniques using simple, portable and low-cost instruments are crucial in disease diagnosis, mutation detection and biodefense. Toward this goal, isothermal amplification techniques that represent a group of attractive in vitro nucleic acid amplification techniques for bioanalysis have been developed. Unlike PCR where polymerases are easily deactivated by thermally labile constituents in a sample, some of the isothermal nucleic acid amplification techniques, such as helicase-dependent amplification and nucleic acid sequence-based amplification, enable the detection of bioanalytes with much simplified protocols and with minimal sample preparations since the entire amplification processes are performed isothermally. This review focuses on the isothermal nucleic acid amplification techniques and their applications in bioanalytical chemistry. Starting off from their amplification mechanisms and significant properties, the adoption of isothermal amplification techniques in bioanalytical chemistry and their future perspectives are discussed. Representative examples illustrating the performance and advantages of each isothermal amplification technique are discussed along with some discussion on the advantages and disadvantages of each technique.     

Nucleic Acid Based Logical Systems

Researchers increasingly visualize a significant role for artificial biochemical logical systems in biological engineering, much like digital logic circuits in electrical engineering. Those logical systems could be utilized as a type of servomechanism to control nanodevices in vitro, monitor chemical reactions in situ, or regulate gene expression in vivo. Nucleic acids (NA), as carriers of genetic information with well‐regulated and predictable structures, are promising materials for the design and engineering of biochemical circuits. A number of logical devices based on nucleic acids (NA) have been designed to handle various processes for technological or biotechnological purposes. This article focuses on the most recent and important developments in NA‐based logical devices and their evolution from in vitro, through cellular, even towards in vivo biological applications.     

A Self-immolative Molecular Beacon for Amplified Nucleic Acid Detection**

Fluorogenic hybridization probes allow the detection of RNA and DNA sequences in homogeneous solution. Typically, one target molecule activates the fluorescence of a single probe molecule. This limits the sensitivity of nucleic acid detection. Herein, we report a self-immolative molecular beacon (iMB) that escapes the one-target/one-probe paradigm. The iMB probe includes a photoreductively cleavable N-alkyl-picolinium (NAP) linkage within the loop region. A fluorophore at the 5’-end serves, on the one hand, as a reporter group and, on the other hand, as a photosensitizer of a NAP-linker cleavage reaction. In the absence of target, the iMB adopts a hairpin shape. Quencher groups prevent photo-induced cleavage. The iMB opens upon hybridization with a target, and both fluorescent emission as well as photo-reductive cleavage of the NAP linker can occur. In contrast to previous chemical amplification reactions, iMBs are unimolecular probes that undergo cleavage leading to products that have lower target affinity than the probes before reaction. Aided by catalysis, the method allowed the detection of 5 pm RNA target within 100 min.     

A novel,integrated forensic microdevice on a rotation‐driven platform: Buccal swab to STR product in less than 2 h

This work describes the development of a novel microdevice for forensic DNA processing of reference swabs. This microdevice incorporates an enzyme‐based assay for DNA preparation, which allows for faster processing times and reduced sample handling. Infrared‐mediated PCR (IR‐PCR) is used for STR amplification using a custom reaction mixture, allowing for amplification of STR loci in 45 min while circumventing the limitations of traditional block thermocyclers. Uniquely positioned valves coupled with a simple rotational platform are used to exert fluidic control, eliminating the need for bulky external equipment. All microdevices were fabricated using laser ablation and thermal bonding of PMMA layers. Using this microdevice, the enzyme‐mediated DNA liberation module produced DNA yields similar to or higher than those produced using the traditional (tube‐based) protocol. Initial microdevice IR‐PCR experiments to test the amplification module and reaction (using Phusion Flash/SpeedSTAR) generated near‐full profiles that suffered from interlocus peak imbalance and poor adenylation (significant ?A). However, subsequent attempts using KAPA 2G and Pfu Ultra polymerases generated full STR profiles with improved interlocus balance and the expected adenylated product. A fully integrated run designed to test microfluidic control successfully generated CE‐ready STR amplicons in less than 2 h (<1 h of hands‐on time). Using this approach, high‐quality STR profiles were developed that were consistent with those produced using conventional DNA purification and STR amplification methods. This method is a smaller, more elegant solution than current microdevice methods and offers a cheaper, hands‐free, closed‐system alternative to traditional forensic methods.     

核酸探针技术

本文对核酸探针技术进行了较全面的综述，介绍了核探针的制备及非放射性标记方法，并对核酸的Ｓｏｕｔｈｅｒｎ转印杂交，Ｎｏｒｔｈｅｒｎ转印杂交，ＩｎＳｉｔｕ转印杂交及斑点杂交法的原理及应用进行了简明的评述。     

A Hydrogel Microneedle Assay Combined with Nucleic Acid Probes for On-Site Detection of Small Molecules and Proteins

Point-of-care testing (POCT) of clinical biomarkers is critical to health monitoring and timely treatment, yet biosensing assays capable of detecting biomarkers without the need for costly external equipment and reagents are limited. Blood-based assays are, specifically, challenging as blood collection is invasive and follow-upprocessing is required. Here, we report a versatile assay that employs hydrogel microneedles (HMNs) to extract interstitial fluid (ISF), in a minimally invasive manner integrated with graphene oxide-nucleic acid (GO.NA)-based fluorescence biosensor to sense the biomarkers of interest in situ. The HMN-GO.NA assay is supplemented with a portable detector, enabling a complete POCT procedure. Our system could successfully measure four clinically important biomarkers (glucose, uric acid (UA), insulin, and serotonin) ex vivo, in addition, to accurately detecting glucose and UA in vivo.     

Nucleic Acid Integrated Technologies for Electrochemical Point-of-Care Diagnostics: A Comprehensive Review

The need for routine and immediate healthcare monitoring has inspired “near-patient testing” or in other words “point-of-care testing (POCT)”. Therefore, POCT can be defined as laboratory tests that are performed at the patient's bedside or in the immediate vicinity of the incident. Among many POCTs, nucleic acid-based testing has attracted enormous attention for the diagnosis of important genetic, inherited and infectious diseases such as cancer and coronavirus. In this review, we outline the integration of nucleic acids into the remarkable electrochemical point-of-care diagnostics including microfluidic, paper and smartphone-based approaches, CRISPR/Cas and liquid biopsy related systems and DNA damage monitoring.