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.     

Recent advances in the development of colorimetric analysis and testing based on aggregation-induced nanozymes

Colorimetric sensing strategies as a powerful point-of-care testing(POCT) tool have attracted significant interest in various chem/biosensing applications.Taking the excellent bare-eye-detectable signaling feature,nanozymes-based colorimetric sensors enable more potential applications and have been a new forefront in the colorimetric POCT analysis toward different target analytes.However,the low catalytic activity of nanozymes in most cases limits their practical application.Recent efforts demonstrate that the aggregation-induced nanozymes provide a general means to modulate nanozymes activity and enhance colorimetric sensing performances of some nanozymes-based colorimetric sensors.But there are few reports are explored to discuss and review such aggregation-induced nanozymes and their colorimetric sensing applications.To highlight the advances and progress in aggregation-induced nanozymes based colorimetric assays,we herein summary the fundamentals,classify and applications of this newlydeveloping field,focusing on the aggregation-induced activity enhancement of nanozymes(AIAEnanozymes) with a significant "signal-on" feature and aggregation-induced activity inhibition of nanozymes(AIAI-nanozymes) with a dramatical "signal-of" characteristics.Finally,we also propose the current challenges and the future prospects on both AIAE-nanozymes and AIAI-nanozymes.     

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.     

Quality and timeliness in medical laboratory testing

In terms of testing, modern laboratory medicine can be divided into centralized testing in central laboratories and point-of-care testing (POCT). Centralized laboratory medicine offers high-quality results, as guaranteed by the use of quality management programs and the excellence of the staff. POCT is performed by clinical staff, and so such testing has moved back closer to the patient. POCT has the advantage of shortening the turnaround time, which potentially benefits the patient. However, the clinical laboratory testing expertise of clinical staff is limited. Consequently, when deciding which components of laboratory testing must be conducted in central laboratories and which components as POCT (in relation to quality and timeliness), it will be medical necessity, medical utility, technological capabilities and costs that will have to be ascertained. Provided adequate quality can be guaranteed, POCT is preferable, considering its timeliness, when testing vital parameters. It is also preferred when the central laboratory cannot guarantee the delivery of results of short turn-around-time (STAT) markers within 60 or (even better) 30 min. POCT should not replace centralized medical laboratory testing in general, but it should be used in cases where positive effects on patient care have been clearly demonstrated.     

Miniaturized electrochemical sensors and their point-of-care applications

Most of the current analytical methods depend largely on laboratory-based analytical techniques that require expensive and bullky equipment,potentially incur costly testing,and involve lengthy detection processes.With increasing requirements for point-of-care testing(POCT),more attention has been paid to miniaturized analytical devices.Miniaturized electrochemical(MEC)sensors,including different material-based MEC sensors(such as DNA-,paper-,and screen electrode-based),have been in strong demand in analytical science due to their easy operation,portability,high sensitivity,as well as their short analysis time.They have been applied for the detection of trace amounts of target through measuring changes in electrochemical signal,such as current,voltage,potential,or impedance,due to the oxidation/reduction of chemical/biological molecules with the help of electrodes and electrochemical units.MEC sensors present great potential for the detection of targets including small organic molecules,metal ions,and biomolecules.In recent years,MEC sensors have been broadly applied to POCT in various fields,including health care,food safety,and environmental monitoring,owing to the excellent advantages of electrochemical(EC)technologies.This review summarized the state-of-the-art advancements on various types of MEC sensors and their applications in POCT.Furthermore,the future perspectives,opportunities,and challenges in this field are also discussed.     

Point-of-care testing: Implementation and practice of cost-effective total quality management

In the United States of America, point-of-care testing (POCT) generally is defined as laboratory testing performed at or near the patient. The objective is to have results immediately available to clinicians for timely medical intervention. The widespread use of POCT is, in part, a response to advances in technology and increased patient acuity. Theoretically, in the context of the entire health care system, POCT improves "quality" by promoting cost through quicker diagnosis and treatment, which in turn leads to faster recovery, reduced length of stay, more efficient clinicians, and overall better utilization of resources. Total quality management (TQM) generally is associated with improving processes and, therefore in this context, improving patient outcomes. The TQM philosophy focuses on creating products or services, which meet or exceed customer expectations. The successful implementation of POCT in a manner consistent with TQM principles requires assessment of direct, measurable benefits including cost-effectiveness to the health care system.     

An Overview of Wearable and Implantable Electrochemical Glucose Sensors

Electrochemical glucose sensors have garnered considerable attention because of their attractive prospect in point-of-care testing (POCT). In this review, we firstly introduce the principles and challenges of electrochemical glucose sensors. Subsequently, we present an overview of the application of electrochemical glucose sensors and discuss their advantages and drawbacks. Wearable and implantable devices based on diverse target biofluid and platforms provide a considerable prospect of accurate and continuous monitoring. Thus, we believe that the future development direction of electrochemical glucose sensors is non-invasive, wearable devices and implantable devices with minimally invasive for continuous glucose monitoring in real time.     

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.     

A colorimetric assay for cholesterol based on the encapsulation of multienzyme in leaf-shape crossed ZIF-L

The serum cholesterol level is an important indicator of healthy and there is a great necessity for frequent cholesterol monitoring to some cardiovascular-related diseases, which puts forward higher requirements for point-of-care testing (POCT) of cholesterol. In this work, a cascade catalytic system of cholesterol is developed by encapsulation of cholesterol oxidase (ChOx) and PdCuAu nanoparticles into zeolitic imidazolate framework-L (ChOx/PCA@ZIF-L). Results indicate that ZIF-L carrier can significantly increase the catalytic activity of single or multiple enzymes, due to its high loading capacity and efficient molecular transport. Under the optimal conditions, the absorbance of reaction system performs linear relationships with the concentration of cholesterol in two intervals from 0.0005 mmol/L to 1.0000 mmol/L, with a limit of detection of 0.2176 µmol/L. The proposed colorimetric strategy based on ChOx/PCA@ZIF-L performs a good agreement with the results provided by chemiluminescence method for the serum cholesterol detection. Interestingly, a simple paper-based sensing system is constructed through a pre-reaction-transfer operation, which gets rid of the complex pre-processing requirements of traditional operations on filter paper. The presented strategy allows for the sensitive, convenient, costless assay of serum cholesterol, and paves a new way to design the POCT device for daily monitoring of healthy.     

An Aptasensing Strategy Using the Phosphatase-mimic Nanozyme and pH Meter as Signal Readout

The demand for point-of-care testing (POCT) is growing dramatically, especially for district where health facilities are poorly staffed, poorly skilled and ill-equipped. As a commercialized portable device, pH meters can be used for detection of various targets, relying on bioactive enzymes. The nanozymes, as the alternative of the natural enzymes, have rarely been used for pH-metric POCT strategies. Herein, we developed an ultrasensitive pH-metric sandwich-type aptasensor based on the CeO₂ nanorods (CeO₂ NRs) as phosphatase-mimic and sodium monofluorophosphate (MFP) as catalytic substrate. Under optimal conditions, such strategy yields a detection limit of 1.17 nM with eligible selectivity for detecting thrombin.     

Immune fluorescence test strips based on quantum dots for rapid and quantitative detection of carcino-embryonic antigen

A QDs-based immune fl uorescence test strips was built up for carcino-embryonic antigen detection to realize cancers POCT diagnostic, with a sensitivity of 0.72 ng/mL in 25 min.     

Paper-based electrochemiluminescent 3D immunodevice for lab-on-paper, specific, and sensitive point-of-care testing

Recent research on microfluidic paper-based analytical devices (μPADs) has shown that paper has great potential for the fabrication of low-cost diagnostic devices for healthcare and environmental monitoring applications. Herein, electrochemiluminescence (ECL) was introduced for the first time into μPADs that were based on screen-printed paper-electrodes. To further perform high-specificity, high-performance, and high-sensitivity ECL on μPADs for point-of-care testing (POCT), ECL immunoassay capabilities were introduced into a wax-patterned 3D paper-based ECL device, which was characterized by SEM, contact-angle measurement, and electrochemical impedance spectroscopy. With the aid of a home-made device-holder, the ECL reaction was triggered at room temperature. By using a typical tris(bipyridine)ruthenium-tri-n-propylamine ECL system, this paper-based ECL 3D immunodevice was applied to the diagnosis of carcinoembryonic antigens in real clinical serum samples. This contribution further expands the number of sensitive and specific detection modes of μPADs.     

pH Readout enhanced ELISA for point-of-care testing of cardiac troponin I

In this study, we described a pH ELISA using synthetic melanin nanoparticles (SMNPs) for the co-immobilization of glucose oxidase and second antibody as signal labels, portable pH meter as signal readout device for detecting biomarker of myocardial injury. This assay is easy-to-use, portable, sensitive and able to realize point-of-care testing (POCT), which was demonstrated the signifi cant promising in the early diagnosis and screening of acute myocardial infarction.     

Research progress of the retrospective detection of organophosphorus nerve agent exposure using point-of-care testing techniqueEI北大核心CSCD

有机磷神经性毒剂(OPNA)是一类速杀性化学战剂,人员中毒后通过溯源检测来确定OPNA种类和剂量对于毒剂使用指证和人员救治非常重要。当前大部分溯源检测方法灵敏度高、特异性好,但因依赖大型设备限于实验室,不便于现场检测。随着现场快速检测(POCT)技术的发展和新识别分子的开发,大量OPNA中毒溯源现场检测技术被建立和应用。本文主要基于胆碱酯酶活性抑制、特异性识别和直接生化反应原理从3方面分析总结了OPNA中毒溯源POCT技术的研究进展和应用,展望了其未来发展趋势。     

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.     

重组酶聚合酶扩增:扩增原理及性能分析

脱氧核糖核酸(DNA)放大技术对于核酸检测(NAT)至关重要. 聚合酶链式反应(PCR)虽然是核酸检测的基准扩增技术, 但其主要适用于条件较好的中心实验室. 重组酶聚合酶扩增(RPA)是一种非常有潜力的等温扩增技术, 对仪器设备依赖性小, 适用于资源贫乏地区. 因此, 该技术在核酸检测时不受实验场所限制, 非常适合即时检测(POCT). 作为一种正在快速发展的扩增技术, RPA也存在阻碍其进一步发展的缺陷. 本文对RPA的扩增原理和扩增性能进行了总结, 重点讨论了对扩增性能至关重要的引物重组和ATP动态平衡调控过程, 并概述了RPA存在的缺陷和潜在的解决方向.     

Point-of-care testing (POCT): Current techniques and future perspectives

Point-of-care testing (POCT) is a laboratory-medicine discipline that is evolving rapidly in analytical scope and clinical application. In this review, we first describe the state of the art of medical-laboratory tests that can be performed near the patient. At present, POCT ranges from basic blood-glucose measurement to complex viscoelastic coagulation assays. POCT shortens the time to clinical decision-making about additional testing or therapy, as delays are no longer caused by transport and preparation of clinical samples, and biochemical-test results are rapidly available at the point of care. Improved medical outcome and lower costs may ensue.Recent, evolving technological advances enable the development of novel POCT instruments. We review the underlying analytical techniques. If new instruments are not yet in practical use, it is often hard to decide whether the underlying analytical principle has real advantage over former methods. However, future utilization of POCT also depends on health-care trends and new areas of application. But, even today, it can be assumed that, for certain applications, near-patient testing is a useful complement to conventional laboratory analyses.     

Quality in point-of-care testing: taking POC to the next level

Point-of-care testing (POCT) is a complex system with many opportunities for error. Delivering quality POCT requires multidisciplinary coordination and an understanding of the preanalytic, analytic, and postanalytic processes that are necessary to deliver a test result and take clinical action. Most errors in laboratory testing occur in the pre and postanalytical phases and many mistakes that are referred to as lab error are actually due to poor communication, actions by others involved in the testing process, or poorly designed processes outside the laboratory's control. POCT requires significant operator interaction with analysis and documentation of calibration and quality control, unlike other medical devices. Clinicians often interpret POCT as equivalent to core laboratory testing, only faster, and mistakenly utilize the results interchangeably despite the differences in test methodologies. Taking quality of POCT to the next level involves looking beyond the analytical phase and integration of POCT into the entire pathway of patient care to understand how POCT relates to medical decision-making at specific points during the patient's care. A systematic review of the literature by the National Academy of Clinical Biochemistry is currently being conducted to draft guidelines for best practice that link the use of POCT to improved patient outcomes.Presented at the 10th Conference Quality in the Spotlight, March 2005, Antwerp, Belgium.     

A Sensitive Signal-off Electrochemical Aptasensor for Thrombin Detection using PB−Au@MoS2 Nanomaterial as Both Platform and Signal Reporter

Human serum is one of the effective samples for point-of-care testing (POCT). Sensitive and quick determination of thrombin content in human serum samples is important. An electrochemical aptasensor based on Prussian blue and Au nanoparticles loaded MoS₂ nanoflowers (PB−Au@MoS₂) hybrid was constructed. By using PB−Au@MoS₂ as both a substrate and a signal reporter, this aptasensor could demonstrate excellent performance for thrombin detection with a detection linear range from 0.01 pM to 30 nM and detection limit down to 1 fM. This work may provide a strategy to establish effective and sensitive sensing devices for thrombin in clinical diagnosis.