The point-of-care-testing of nucleic acids by chip,cartridge and paper sensors

Point-of-care nucleic acid testing (POCNAT) has played an important role in the outbreak of infectious diseases (e.g., COVID-19) over recent years. POCNAT aims to realize the rapid, simple and automatic detection of nucleic acid. Thanks to the development of manufacturing technology, electronic information technology, artificial intelligence technology, and biological information technology in recent years, the development of the POCNAT device has led to significant advancement. Instead of the normal nucleic acid detection methods used in the laboratory, some novel experimental carriers have been applied, such as chips, cartridges and papers. The application of these experimental carriers has realized the automation and integration of nucleic acid detection. The entire process of nucleic acid detection is normally divided into three steps (nucleic acid extraction, target amplification and signal detection). All of the reagents required by the process can be pre-stored on these experimental carriers, without unnecessary manual operation. Furthermore, all of the processes are carried out in this experimental carrier, with the assistance of a specific control device. Although they are complicated to manufacture and precise in design, their application provides a significant step forwards in nucleic acid detection and realizes the integration of nucleic acid detection. This technology has great potential in the field of point-of-care molecular diagnostics in the future. This paper focuses on the relevant content of these experimental carriers.     

Rapid isolation and detection of cancer cells by utilizing integrated microfluidic systems

The present study reports a new three-dimensional (3D) microfluidic platform capable of rapid isolation and detection of cancer cells from a large sample volume (e.g. ~1 mL) by utilizing magnetic microbead-based technologies. Several modules, including a 3D microfluidic incubator for the magnetic beads to capture cancer cells, a microfluidic control module for sample transportation and a nucleic acid amplification module for genetic identification, are integrated into this microsystem. With the incorporation of surface-modified magnetic beads, target cancer cells can be specifically recognized and conjugated onto the surface of the antibody-coated magnetic microbeads by utilizing a swirling effect generated by the new 3D microfluidic incubator, followed by isolating and purifying the magnetic complexes via the incorporation of an external magnet and a microfluidic control module, which washes away any unbound waste solution. Experimental results show that over 90% of the target cancer cells can be isolated from a large volume of bio-samples within 10 min in the 3D microfluidic incubator. In addition, the expressed genes associated with ovarian and lung cancer cells can also be successfully amplified by using the on-chip nucleic acid amplification module. More importantly, the detection limit of the developed system is found to be 5 × 10(1) cells mL(-1) for the target cancer cells, indicating that this proposed microfluidic system may be adapted for clinical use for the early detection of cancer cells. Consequently, the proposed 3D microfluidic system incorporated with immunomagnetic beads may provide a promising automated platform for the rapid isolation and detection of cancer cells with a high sensitivity.     

Sample preparation: a challenge in the development of point-of-care nucleic acid-based assays for resource-limited settings

Currently available nucleic acid testing (NAT)-based assays are complex and time-consuming, and they require expensive instrumentation and dedicated laboratory spaces for sample preparation as well as for amplification and detection of the nucleic acid target. Reagents required for these tests are also expensive and must be transported and stored refrigerated or frozen. These characteristics have limited the use of such assays for point-of-care (POC) testing, especially in resource-poor settings. Efforts to develop simple and rapid NAT-based assays have focused predominantly on the amplification and detection steps, with sample preparation and nucleic acid extraction remaining the bottleneck in the development of NAT systems suitable for POC applications or resource-limited settings. A review of NAT platforms and technologies currently under development and validation for rapid field testing revealed that, in addition to requiring expensive and complex instrumentation, many of these systems also require off-line sample preparation and reagent handling. In their current format, they are therefore not appropriate for POC testing in resource-limited settings. We evaluated several commercially available technologies and procedures for the isolation of nucleic acid with the extraction of HIV-1 RNA from human plasma as a model system. Our results indicate that solid-phase extraction with silica or glass in the presence of a chaotropic salt provides the highest extraction efficiency. However, none of the existing methods and technologies is readily adaptable to a POC system. The integration of sample preparation procedures well suited to NAT-based assays in resource-limited settings therefore remains a challenge.     

Miniaturized isothermal nucleic acid amplification, a review

Micro-Total Analysis Systems (μTAS) for use in on-site rapid detection of DNA or RNA are increasingly being developed. Here, amplification of the target sequence is key to increasing sensitivity, enabling single-cell and few-copy nucleic acid detection. The several advantages to miniaturizing amplification reactions and coupling them with sample preparation and detection on the same chip are well known and include fewer manual steps, preventing contamination, and significantly reducing the volume of expensive reagents. To-date, the majority of miniaturized systems for nucleic acid analysis have used the polymerase chain reaction (PCR) for amplification and those systems are covered in previous reviews. This review provides a thorough overview of miniaturized analysis systems using alternatives to PCR, specifically isothermal amplification reactions. With no need for thermal cycling, isothermal microsystems can be designed to be simple and low-energy consuming and therefore may outperform PCR in portable, battery-operated detection systems in the future. The main isothermal methods as miniaturized systems reviewed here include nucleic acid sequence-based amplification (NASBA), loop-mediated isothermal amplification (LAMP), helicase-dependent amplification (HDA), rolling circle amplification (RCA), and strand displacement amplification (SDA). Also, important design criteria for the miniaturized devices are discussed. Finally, the potential of miniaturization of some new isothermal methods such as the exponential amplification reaction (EXPAR), isothermal and chimeric primer-initiated amplification of nucleic acids (ICANs), signal-mediated amplification of RNA technology (SMART) and others is presented.     

A novel cartridge for nucleic acid extraction,amplification and detection of infectious disease pathogens with the help of magnetic nanoparticles

Nucleic acid detection (NAD) based on real-time polymerase chain reaction (real-time PCR) is gold standard for infectious disease detection. Magnetic nanoparticles (MNPs) are widely used for nucleic acid extraction (NAE) because of their excellent properties. Microfluidic technology makes automated NAD possible. However, most of the NAD microfluidic chips are too complex to be applied to point-of-care (POC) testing. In this paper, a simple-structure cartridge was developed for POC detection of infectious diseases. This self-contained cartridge can be divided into a magnetic-controlled NAE part, a valve-piston combined fluidic control part and a PCR chip, which is able to extract nucleic acid from up to 500 µL of liquid samples by MNPs and finish the detection process from “sample in” to “answer out” automatically. Performance tests of the cartridges show that it met the demands of automated NAD. Results of on-cartridge detection of hepatitis B virus (HBV) demonstrated that this system has good uniformity and no cross-contamination between different cartridges, and the limit of detection (LOD) of this system for HBV in serum is 50 IU/mL. Multiplex detections of severe acute respiratory syndrome coronaviruses 2 (SARS-CoV-2) with a concentration of 500 copies/mL were carried out on the system and 100% positive detection rate was achieved.     

稀土掺杂上转换纳米材料在兽药残留检测领域的应用

食品和环境中兽药残留问题时有发生,对人类健康构成很大的潜在威胁。随着人们对美好生活和同一健康的向往和不断追求,对微量甚至痕量兽药残留的分析检测已显得日益迫切和重要。因此,构建对兽药残留进行灵敏、准确、稳定、简便、快速检测的方法已成为一个热点研究领域。稀土掺杂上转换纳米材料(REEs-UCNPs)作为一种新兴的纳米荧光材料,具有独特的反斯托克斯发光性质,由于其具有荧光寿命长、光散射小、激发光生物组织穿透深度大且对组织损伤小等显著优点,在分析检测领域逐步凸显出巨大优势。本文重点介绍了基于REEs-UCNPs构建荧光共振能量转移核酸适配体传感器、磁性纳米颗粒结合核酸装配体传感器、荧光免疫探针以及现场快速检测等在兽药残留检测方面的研究进展,并对其应用前景进行了探讨和展望。     

Isothermal nucleic acid amplification technologies for point-of-care diagnostics: a critical review

Nucleic Acid Testing (NAT) promises rapid, sensitive and specific diagnosis of infectious, inherited and genetic disease. The next generation of diagnostic devices will interrogate the genetic determinants of such conditions at the point-of-care, affording clinicians prompt reliable diagnosis from which to guide more effective treatment. The complex biochemical nature of clinical samples, the low abundance of nucleic acid targets in the majority of clinical samples and existing biosensor technology indicate that some form of nucleic acid amplification will be required to obtain clinically relevant sensitivities from the small samples used in point-of-care testing (POCT). This publication provides an overview and thorough review of existing technologies for nucleic acid amplification. The different methods are compared and their suitability for POCT adaptation are discussed. Current commercial products employing isothermal amplification strategies are also investigated. In conclusion we identify the factors impeding the integration of the methods discussed in fully automated, sample-to-answer POCT devices.     

Rapid automatic nucleic acid purification system based on gas–liquid immiscible phase

The continuous expansion of nucleic acid detection applications has resulted in constant developments in rapid, low-consumption, and highly automated nucleic acid extraction methods. Nucleic acid extraction using magnetic beads across an immiscible phase interface offers significant simplification and parallelization potential. The gas–liquid immiscible phase valve eliminates the requirement for complicated cassettes and is suitable for automation applications. By analyzing the process of magnetic beads crossing the gas-liquid interface, we utilized a low magnetic field strength to drive large magnetic bead packages to cross the gas-liquid interface, providing a solution of high magnetic bead recovery rate for solid-phase extraction with a low-surfactant system based on gas-liquid immiscible phase valve. The recovery rate of magnetic beads was further improved to 90%–95% and the carryover of the reagents was below 1%. Consequently, a chip and an automatic system were developed to verify the applicability of this method for nucleic acid extraction. The Hepatitis B virus serum standard was used for the extraction test. The extraction of four samples was performed within 7 minutes, with nucleic acid recovery maintained above 80% and good purity. Thus, through analysis and experiments, a fast, highly automated, and low-consumption nucleic acid recovery method was proposed in this study.     

Amplified electrochemical detection of nucleic acid hybridization via selective preconcentration of unmodified gold nanoparticles

The common drawback of optical methods for rapid detection of nucleic acid by exploiting the differential affinity of single-/double-stranded nucleic acids for unmodified gold nanoparticles (AuNPs) is its relatively low sensitivity. In this article, on the basis of selective preconcentration of AuNPs unprotected by single-stranded DNA (ssDNA) binding, a novel electrochemical strategy for nucleic acid sequence identification assay has been developed. Through detecting the redox signal mediated by AuNPs on 1, 6-hexanedithiol blocked gold electrode, the proposed method is able to ensure substantial signal amplification and a low background current. This strategy is demonstrated for quantitative analysis of the target microRNA (let-7a) in human breast adenocarcinoma cells, and a detection limit of 16 fM is readily achieved with desirable specificity and sensitivity. These results indicate that the selective preconcentration of AuNPs for electrochemical signal readout can offer a promising platform for the detection of specific nucleic acid sequence.     

An integrated microfluidic device for the simultaneous detection of multiple antibiotics

Residual antibiotics in food pose a serious long-term threat to human health. Therefore, an on-site visualization method for antibiotic detection is required. However, the requirements of traditional antibiotic testing methods in terms of operator proficiency and equipment cost hinder the rapid point-of-care-testing detection of suspected samples. Herein, we reported an integrated microfluidic device combining a microfluidic chip containing cruciform valves with immunochromatographic strips for the rapid detection of multiple antibiotics in milk. The rapid qualitative and quantitative analysis of four types of antibiotics (sulfonamides, β-lactams, streptomycin, and tetracyclines) was performed using mobile phone photography and mobile phone application analysis. The detection time was maintained at 10 min. The limits of detection (LODs) for the four antibiotics were 0.15, 0.12, 0.25, and 0.29 ng/mL, respectively, and the selectivity for the different antibiotics was observed even in a highly complex matrix. This device successfully integrated separation and real-time detection onto a chip and might provide a promising perspective for the detection of multiple antibiotics in milk.     

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。     

便携式激光诱导击穿光谱仪测定土壤中重金属

土壤重金属污染对农作物生长和人体健康都有严重危害,现场快速检测对土壤重金属污染调查、应急监测具有重要意义。采用自主研发的土壤重金属激光诱导击穿光谱现场快速检测仪对矿区周边土壤进行现场检测分析,以835个不同基质土壤的光谱数据建立定标数据库,通过支持向量机建立回归模型对土壤重金属元素含量进行定量反演。现场检测获取的全波段光谱波动在15%以内,Cd、Cr、Cu、Ni、Pb和Zn等6种元素光谱强度相对标准偏差平均值为6.31%。将检测结果与实验室电感耦合等离子体质谱法分析对比,6种元素的皮尔逊相关系数r在0.850 1～0.982 9,检测结果80%以上处于±30%相对误差区间分布。对比结果表明自主研发的土壤重金属激光诱导击穿光谱现场检测仪可以满足现场快速检测需求。     

Analysis of free amino acids in Amur sturgeon by ultra‐performance liquid chromatography using pre‐column derivatization with 6‐aminoquinolyl‐carbamyl

A rapid, sensitive, and reliable ultra‐performance liquid chromatography (UPLC) coupled with photodiode array detection method was developed for the amino acid analysis of Amur sturgeon (Acipenser schrenckii Brandt). The method uses minimal sample volume and automated online precolumn derivitization of amino acids with fluorescent 6‐aminoquinolyl‐carbamyl reagent. The chromatographic separation was achieved by UPLC, which used a column with 1.7 μm particle packing that enabled higher speed of analysis, peak capacity, greater resolution, and increased sensitivity. Amino acid derivatives obtained under optimal conditions were separated on a Waters UPLC BEH C₁₈ column with Acetonitrile–acetate buffer as mobile phase. Matrix effects were investigated and good linearities with correlation coefficients better than 0.9949 were obtained over a wide range of 5–1000 μmol/L for all amino acids. The simple sample preparation and minimal sample volume make the method useful for the quantitation of 17 amino acids in Amur sturgeon samples. It is concluded that a rapid and robust platform based on UPLC was established, and a total of 17 amino acids of Amur sturgeon were tentatively detected. This method showed good accuracy and repeatability that can be used for the quantification of amino acids in real samples.     

Subnanomolar Detection of Oligonucleotides through Templated Fluorogenic Reaction in Hydrogels: Controlling Diffusion to Improve Sensitivity

Oligonucleotide‐templated reactions are valuable tools for nucleic acid sensing both in vitro and in vivo. They are typically carried out under conditions that make any reaction in the absence of template highly unfavorable (most commonly by using a low concentration of reactants), which has a negative impact on the detection sensitivity. Herein, we report a novel platform for fluorogenic oligonucleotide‐templated reactions between peptide nucleic acid probes embedded within permeable agarose and alginate hydrogels. We demonstrate that under conditions of restricted mobility (that is, limited diffusion), non‐specific interactions between probes are prevented, thus leading to lower background signals. When applied to nucleic acid sensing, this accounts for a significant increase in sensitivity (that is, lower limit of detection). Optical nucleic acid sensors based on fluorogenic peptide nucleic acid probes embedded in permeable, physically crosslinked, alginate beads were also engineered and proved capable of detecting DNA concentrations as low as 100 pm .     

CRISPR-Cas Biochemistry and CRISPR-Based Molecular Diagnostics

Polymerase chain reaction (PCR)-based nucleic acid testing has played a critical role in disease diagnostics, pathogen surveillance, and many more. However, this method requires a long turnaround time, expensive equipment, and trained personnel, limiting its widespread availability and diagnostic capacity. On the other hand, the clustered regularly interspaced short palindromic repeats (CRISPR) technology has recently demonstrated capability for nucleic acid detection with high sensitivity and specificity. CRISPR-mediated biosensing holds great promise for revolutionizing nucleic acid testing procedures and developing point-of-care diagnostics. This review focuses on recent developments in both fundamental CRISPR biochemistry and CRISPR-based nucleic acid detection techniques. Four ongoing research hotspots in molecular diagnostics-target preamplification-free detection, microRNA (miRNA) testing, non-nucleic-acid detection, and SARS-CoV-2 detection-are also covered.     

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.     

Rapid and high-throughput determination of cationic surfactants in environmental water samples by automated on-line polymer monolith microextraction coupled to high performance liquid chromatography-mass spectrometry

A rapid, high throughput and sensitive method was presented for automated determination of cationic surfactants in environmental water samples. The method was based on an automated analysis platform that was composed of on-line polymer monolith microextraction (PMME) and high performance liquid chromatography-mass spectrum (HPLC-MS) with an autosampler. A poly(methacrylic acid-co-ethylene dimethacrylate) (MAA-co-EDMA) monolith was selected as the sorbent for purification and enrichment of cationic surfactants in environmental water samples while a new mixed-mode chromatographic column packed with octyl and sulfonic acid co-bonded silica (OSS) was employed for separation and quantitative determination of cationic surfactants in water samples. By integrating sample preparation, chromatographic separation and MS detection into one automated platform, it makes the whole analysis procedure simple, accurate, and time and labor-saving. Several parameters affecting the extraction performance were investigated. Under the optimized conditions, the proposed method was applied to the analysis of seven cationic surfactants in environmental water samples. Good linearities were obtained for all cationic surfactants with R2 larger than 0.9895. The limits of detection were found to be in the range of 15-24 ng/L. The method recoveries of the cationic surfactants spiked in water samples were from 80.5% to 115.1%, with relative standard deviations less than 12.4%.     

Nanomechanical sensing of DNA sequences using piezoresistive cantilevers

A microfabricated cantilever with an internal piezoresistive component has been sensitized with thiol tethered ss-DNA strands and utilized for an in situ, label-free, highly specific, and rapid DNA detection assay. The generation of a differential surface stress onto the functionalized cantilever surface upon target recognition has allowed nanomechanical identification of 12-nucleotide complementary DNA probes with single base mismatch discrimination (sensitivity of 0.2 microM). Interestingly, utilization of an overhang extension distal to the surface enhanced the sensitivity to the 0.01 microM level. The cantilever was functionalized by inkjet printing technology. Replacing the capture probe with locked nucleic acid (LNA) resulted in a faster target probe capture kinetics compared to DNA-DNA hybridization. The capabilities of the piezoresistive cantilever indicate future ergonomic convenience via miniaturization alternative to the conventional laser-based detection method for portable on-site applications.