Miniaturized nucleic acid amplification systems for rapid and point-of-care diagnostics: A review

Point-of-care (POC) genetic diagnostics critically depends on miniaturization and integration of sample processing, nucleic acid amplification, and detection systems. Polymerase chain reaction (PCR) assays have extensively applied for the diagnosis of genetic markers of disease. Microfluidic chips for microPCR with different materials and designs have been reported. Temperature cycling systems with varying thermal masses and conductivities, thermal cycling times, flow-rates, and cross-sectional areas, have also been developed to reduce the nucleic acid amplification time. Similarly, isothermal amplification techniques (e.g., loop-mediated isothermal amplification or LAMP), which are still are emerging, have a better potential as an alternative to PCR for POC diagnostics. Isothermal amplification techniques have: (i) moderate incubation temperature leading to simplified heating and low power consumption, (ii) yield high amount of amplification products, which can be detected either visually or by simple detectors, (iii) allow direct genetic amplification from bacterial cells due to the superior tolerance to substances that typically inhibit PCR, (iv) have high specificity, and sensitivity, and (v) result in rapid detection often within 10–20 min. The aim of this review is to provide a better understanding of the advantages and limitations of microPCR and microLAMP systems for rapid and POC diagnostics.     

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.     

Emerging optofluidic technologies for point-of-care genetic analysis systems: a review

This review describes recently emerging optical and microfluidic technologies suitable for point-of-care genetic analysis systems. Such systems must rapidly detect hundreds of mutations from biological samples with low DNA concentration. We review optical technologies delivering multiplex sensitivity and compatible with lab-on-chip integration for both tagged and non-tagged optical detection, identifying significant source and detector technology emerging from telecommunications technology. We highlight the potential for improved hybridization efficiency through careful microfluidic design and outline some novel enhancement approaches using target molecule confinement. Optimization of fluidic parameters such as flow rate, channel height and time facilitates enhanced hybridization efficiency and consequently detection performance as compared with conventional assay formats (e.g. microwell plates). We highlight lab-on-chip implementations with integrated microfluidic control for “sample-to-answer” systems where molecular biology protocols to realize detection of target DNA sequences from whole blood are required. We also review relevant technology approaches to optofluidic integration, and highlight the issue of biomolecule compatibility. Key areas in the development of an integrated optofluidic system for DNA hybridization are optical/fluidic integration and the impact on biomolecules immobilized within the system. A wide range of technology platforms have been advanced for detection, quantification and other forms of characterization of a range of biomolecules (e.g. RNA, DNA, protein and whole cell). Owing to the very different requirements for sample preparation, manipulation and detection of the different types of biomolecules, this review is focused primarily on DNA–DNA interactions in the context of point-of-care analysis systems.     

Membrane technologies for remediating contaminated soils: a critical review

Regulatory compliance requires that soils contaminated with toxic organic and metallic compounds be cleaned up. Several chemical and thermal detoxification technologies are commercially available for directly treating the contaminated soils excavated from contaminated sites. In contrast, soil washing with aqueous solutions transfers the contaminants from the solid matrix to the aqueous fraction which, then, needs to be further treated. Membranes have not been used much for treating these effluents. However, several membrane techniques appear promising. In these techniques, the main objective is to find ways of concentrating the contaminants with much higher volume reduction than is customarily available. Applications of membranes to soil washings should primarily be looked upon as an inexpensive aid to destruction, disposal, or recovery technologies that determine the ultimate fate of the contaminants.     

Engineering innovative interfaces for point-of-care diagnostics

The ongoing Coronavirus disease 2019 (COVID-19) pandemic illustrates the need for sensitive and reliable tools to diagnose and monitor diseases. Traditional diagnostic approaches rely on centralized laboratory tests that result in long wait times to results and reduce the number of tests that can be given. Point-of-care tests (POCTs) are a group of technologies that miniaturize clinical assays into portable form factors that can be run both in clinical areas —in place of traditional tests— and outside of traditional clinical settings —to enable new testing paradigms. Hallmark examples of POCTs are the pregnancy test lateral flow assay and the blood glucose meter. Other uses for POCTs include diagnostic assays for diseases like COVID-19, HIV, and malaria but despite some successes, there are still unsolved challenges for fully translating these lower cost and more versatile solutions. To overcome these challenges, researchers have exploited innovations in colloid and interface science to develop various designs of POCTs for clinical applications. Herein, we provide a review of recent advancements in lateral flow assays, other paper based POCTs, protein microarray assays, microbead flow assays, and nucleic acid amplification assays. Features that are desirable to integrate into future POCTs, including simplified sample collection, end-to-end connectivity, and machine learning, are also discussed in this review.     

Miniature magnetic resonance system for point-of-care diagnostics

We have developed a next generation, miniaturized platform to diagnose disease at the point-of-care using diagnostic magnetic resonance (DMR-3). Utilizing a rapidly growing library of functionalized magnetic nanoparticles, DMR has previously been demonstrated as a versatile tool to quantitatively and rapidly detect disease biomarkers in unprocessed biological samples. A major hurdle for bringing DMR to the point-of-care has been its sensitivity to temperature variation. As an alternative to costly and bulky mechanisms to control temperature, we have implemented an automated feedback system to track and compensate for the temperature drift, which enables reliable and robust DMR measurements in realistic clinical environments (4-50 °C). Furthermore, the new system interfaces with a mobile device to facilitate system control and data sharing over wireless networks. With such features, the DMR-3 platform can function as a self-contained laboratory even in resource-limited, remote settings. The clinical potential of the new system is demonstrated by detecting trace amounts of proteins and as few as 10 bacteria (Staphylococcus aureus) in a short time frame (<30 min).     

Isothermal cycling and cascade signal amplification strategy for ultrasensitive colorimetric detection of nucleic acids

Microchimica Acta - We have designed a novel isothermal cascade signal-amplification strategy for ultrasensitive colorimetric determination of nucleic acids. It is based on double-cycling...     

Trends in the development of nucleic acid biosensors for medical diagnostics

Some of the recent advances in the field of biosensors for nucleic acid analysis in medical diagnostic applications are highlighted. Particular attention is paid in this review to the progress made in two key areas of development: (i) enhancements achieved in device selectivity, and (ii) enhancements achieved in device sensitivity.     

Integrated microelectronic device for label-free nucleic acid amplification and detection

We present an integrated microelectronic device for amplification and label-free detection of nucleic acids. Amplification by polymerase chain reaction (PCR) is achieved with on-chip metal resistive heaters, temperature sensors, and microfluidic valves. We demonstrate a rapid thermocycling with rates of up to 50 degrees C s(-1) and a PCR product yield equivalent to that of a bench-top system. Amplicons within the PCR product are detected by their intrinsic charge with a silicon field-effect sensor. Similar to existing optical approaches with intercalators such as SYBR Green, our sensing approach can directly detect standard double-stranded PCR product, while in contrast, our sensor does not require labeling reagents. By combining amplification and detection on the same device, we show that the presence or absence of a particular DNA sequence can be determined by converting the analog surface potential output of the field-effect sensor to a simple digital true/false readout.     

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.     

An integrated portable hand-held analyser for real-time isothermal nucleic acid amplification

A compact hand-held heated fluorometric instrument for performing real-time isothermal nucleic acid amplification and detection is described. The optoelectronic instrument combines a Printed Circuit Board/Micro Electro Mechanical Systems (PCB/MEMS) reaction detection/chamber containing an integrated resistive heater with attached miniature LED light source and photo-detector and a disposable glass waveguide capillary to enable a mini-fluorometer. The fluorometer is fabricated and assembled in planar geometry, rolled into a tubular format and packaged with custom control electronics to form the hand-held reactor. Positive or negative results for each reaction are displayed to the user using an LED interface. Reaction data is stored in FLASH memory for retrieval via an in-built USB connection. Operating on one disposable 3 V lithium battery >12, 60 min reactions can be performed. Maximum dimensions of the system are 150 mm (h) × 48 mm (d) × 40 mm (w), the total instrument weight (with battery) is 140 g. The system produces comparable results to laboratory instrumentation when performing a real-time nucleic acid sequence-based amplification (NASBA) reaction, and also displayed comparable precision, accuracy and resolution to laboratory-based real-time nucleic acid amplification instrumentation. A good linear response (R² = 0.948) to fluorescein gradients ranging from 0.5 to 10 μM was also obtained from the instrument indicating that it may be utilized for other fluorometric assays. This instrument enables an inexpensive, compact approach to in-field genetic screening, providing results comparable to laboratory equipment with rapid user feedback as to the status of the reaction.     

Microarray-based amplification and detection of RNA by nucleic acid sequence based amplification

Nucleic acid sequence based amplification (NASBA) is a versatile in vitro nucleic acid amplification method. In this work, RNA amplification and labeling by NASBA and microarray analysis are combined in a one-step process. The NASBA reaction is performed in direct contact with capture probes. These probes are bound to surface-attached hydrogel spots generated at the chip surfaces by using a simple printing and UV irradiation process. Five gene expression and SNP parameters with known relevance in breast cancer diagnostics were chosen to demonstrate that multiplex NASBA-on-microarray analysis is possible. A minimum amount of 10 pg of total RNA was shown to be sufficient for the detection of the reference parameter RPS18, which demonstrates that the detection limit of the microarray-based NASBA assays theoretically allows single-cell assays to be performed.     

A portable and integrated nucleic acid amplification microfluidic chip for identifying bacteria

In this work, we developed a portable integrated microchip of loop-mediated isothermal nucleic acid amplification (LAMP). This chip, with sample-to-answer capability, could perform rapid DNA release, exponential signal amplification and naked-eye result read-out in single or multiplex format. We call it iμLAMP, namely integrated micro-LAMP, which was successfully used for point-of-care identification of bacteria.     

Picoliter droplet microfluidic immunosorbent platform for point-of-care diagnostics of tetanus

We have developed a sensitive, specific, rapid and low cost picoliter microsphere-based platform for bioanalyte detection and quantification. In this method, a biological sample, biosensing microspheres, and fluorescently labeled detection (secondary) antibodies are co-encapsulated to capture the analyte (here: human anti-tetanus immunoglobulin G) on the surface of the microsphere in microfluidic pL-sized droplets. The absorption of the analyte and detecting antibodies on the microsphere concentrate the fluorescent signal in correlation with analyte concentration. Using our platform and commercially available antibodies, we were able to quantify anti-tetanus antibodies in human serum. In comparison to standard bulk immunosorbent assays, the microfluidic droplet platform presented here reduces the reagent volume by four orders of magnitude, while fast reagent mixing reduces the detection time from hours to minutes. We consider this platform to be a major leap forward in the miniaturization of immunosorbent assays and to provide a rapid and low cost tool for global point-of-care.

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.     

Emerging biosensing and transducing techniques for potential applications in point-of-care diagnostics

With the deepening of our understanding in life science, molecular biology, nanotechnology, optics, electrochemistry and other areas, an increasing number of biosensor design strategies have emerged in recent years, capable of providing potential practical applications for point-of-care (POC) diagnosis in various human diseases. Compared to conventional biosensors, the latest POC biosensor research aims at improving sensor precision, cost-effectiveness and time-consumption, as well as the development of versatile detection strategies to achieve multiplexed analyte detection in a single device and enable rapid diagnosis and high-throughput screening. In this review, various intriguing strategies in the recognition and transduction of POC (from 2018 to 2021) are described in light of recent advances in CRISPR technology, electrochemical biosensing, and optical- or spectra-based biosensing. From the perspective of promoting emerging bioanalytical tools into practical POC detecting and diagnostic applications, we have summarized key advances made in this field in recent years and presented our own perspectives on future POC development and challenges.

POC diagnostics are driven by the rapid advances in CRISPR, electrochemical and optical biosensors. Related emerging strategies are described and discussed from the perspective of facilitating the practical application of biosensors in POC testing.     

Electrochemical aptamer-based assays coupled to isothermal nucleic acid amplification techniques: New tools for cancer diagnosis

Highly sensitive detection of cancer biomarkers in blood is key not only to find cancer at an early stage but also to help clinicians to decide the best treatment plan and to find how well treatment is working. To quantify the small changes in clinically validated biomarkers associated with carcinogenesis both selective receptors and signal amplification strategies of the recognition event between the receptor and the biomarker are highly in demand. This report covers the most recent developments in the integration of aptamer-based recognition of blood-circulating cancer biomarkers and isothermal nucleic acid amplification platforms with electrochemical readout, highlighting the potential of these novel tools, and the challenges to translate these assays to the clinical practice.     

核酸等温扩增技术在电化学生物传感器中的应用

生物分子检测在临床诊断、基因治疗、基因突变分析等方面变得日益重要,因而,建立简单、快速、灵敏的检测方法具有重要意义。近年,电化学生物传感器因其简单、便携、易操作、成本低等优势在生物分子检测的研究中备受关注。为了提高检测方法的灵敏度,不同的核酸等温扩增技术被应用于电化学生物传感器的构建中。本文简单介绍了电化学生物传感器的工作原理,着重综述了几种主要应用于电化学传感器中的核酸等温扩增技术,同时比较了各方法的优缺点。