Advances in point-of-care genetic testing for personalized medicine applications

A S de Olazarra; S X Wang

doi:10.1063/5.0143311

. 2023 May 3;17(3):031501. doi: 10.1063/5.0143311

Advances in point-of-care genetic testing for personalized medicine applications

A S de Olazarra ¹, S X Wang ^1,2,^1,2,^a)

PMCID: PMC10163839 PMID: 37159750

Abstract

Breakthroughs within the fields of genomics and bioinformatics have enabled the identification of numerous genetic biomarkers that reflect an individual's disease susceptibility, disease progression, and therapy responsiveness. The personalized medicine paradigm capitalizes on these breakthroughs by utilizing an individual's genetic profile to guide treatment selection, dosing, and preventative care. However, integration of personalized medicine into routine clinical practice has been limited—in part—by a dearth of widely deployable, timely, and cost-effective genetic analysis tools. Fortunately, the last several decades have been characterized by tremendous progress with respect to the development of molecular point-of-care tests (POCTs). Advances in microfluidic technologies, accompanied by improvements and innovations in amplification methods, have opened new doors to health monitoring at the point-of-care. While many of these technologies were developed with rapid infectious disease diagnostics in mind, they are well-suited for deployment as genetic testing platforms for personalized medicine applications. In the coming years, we expect that these innovations in molecular POCT technology will play a critical role in enabling widespread adoption of personalized medicine methods. In this work, we review the current and emerging generations of point-of-care molecular testing platforms and assess their applicability toward accelerating the personalized medicine paradigm.

I. INTRODUCTION

Medicine has begun to deviate away from the traditional “one size fits all” approach. Major breakthroughs in genomics, bioinformatics, and molecular analysis methods have facilitated the discovery of novel biomarkers and genetic signatures that reflect an individual's disease susceptibility, disease progression, and/or therapy responsiveness.^1,2 The personalized medicine paradigm seeks to leverage these findings to improve health outcomes by implementing an individualized, genomics-based approach to clinical decision-making and therapy selection.

Pharmacogenetic (PGx) testing and “theranostics” are among the most well-known embodiments of the personalized medicine paradigm. Studies regarding the genetic basis of adverse drug responses have revealed several polymorphic variations, such as single nucleotide polymorphisms (SNPs), that impact the metabolization of common medications, hence producing significant inter-individual therapy response variability.^1,3–6 Currently, more than 500 drugs contain pharmacogenetic information in their labels, and the United States Food and Drug Administration (FDA) has approved or cleared at least 13 DNA-based PGx tests to evaluate personal drug metabolizing enzyme activity, as well at least 30 additional nucleic acid based tests for precision or personalized medicine applications.^7–9

PGx testing and theranostics translate these findings into practice by utilizing an individual's genotypic information to guide therapeutic drug selection and dosing optimization.^1,10–12 For example, CYP2C19 polymorphisms have been implicated in increased rates of major adverse events in patients receiving clopidogrel after percutaneous coronary intervention (PCI),^13,14 and contribute to about 12% of the variability in the drug's antiplatelet effect.¹⁵ Accordingly, several point-of-care (POC) CYP2C19 genotyping platforms, including the Spartan RX CYP2C19 System, have been developed in order to personalize antiplatelet treatment and reduce the risk of major adverse events.^13,14 As knowledge surrounding the genetic basis of therapy responsiveness continues to grow, platforms that offer rapid and straightforward implementation of genotyping/PGx testing at the POC will become invaluable, serving as tools to improve treatment selection and outcomes.

Along a similar vein, developments in bioinformatics and computational biology have facilitated the discovery of gene expression signatures that provide insights into disease pathogenesis or predicted treatment outcomes.^16,17 Applications of these transcriptional signatures include diagnosing highly prevalent infectious diseases,^16,18–21 adapting/modulating treatment assignments for common cancers,^21–24 and discriminating between infectious etiologies.²⁵ For example, Sweeney et al. reported an 18-gene panel to distinguish patients with bacterial and viral infections as well as those experiencing inflammation unrelated to infection.²⁵ The ability to properly diagnose and discriminate between pathogens causing similar clinical presentations has the potential to improve triaging decisions and treatment selection (e.g., prescription of antibiotics vs. anti-viral medication).¹⁶ Furthermore, measuring the host immune response to disease has the potential to provide valuable information regarding disease severity, disease progression, or clinical management (e.g., antiviral dosage).¹⁹ As such, demand for broadly accessible gene expression analysis tools has begun to grow.²⁶

Despite the marked benefits and broad potential impact of personalized medicine, widespread adoption of these methods remains limited. Some reasons for this include insufficient provider training, continued dispute over clinical function, and, perhaps most importantly, a scarcity of rapid genetic testing tools to support genetic analysis at the point-of-care.^6,8,27 Discussing all of these issues is beyond the scope of this article, but recent developments in point-of-care genetic testing platforms and the role they may play in increasing the viability of near-patient genetic analysis for personalized medicine applications are reviewed. Special attention is paid to those platforms that present highly integrated, POC-friendly solutions, such as those that deploy microfluidics to ensure low reagent and sample consumption, device miniaturization, and rapid analysis.

Today, nucleic acid amplification tests (NAATs)—particularly those that are based on polymerase chain reaction (PCR) and its constituent reverse transcription-PCR (RT-PCR)—are considered the gold standard for most forms of genetic testing, including genotyping^28–32 and gene expression analysis.^{18,19,33–36} While NAATs are highly effective in performing sensitive and specific detection of low-abundance analytes from matrix samples,³⁷ most require complex sample preparation procedures,³⁸ sophisticated thermal cycling instrumentation, and trained personnel that are unlikely to be available outside of clinical laboratory settings.^39–41 Consequently, genetic testing is often restricted to centralized facilities,^37,39 which protracts turnaround times (TATs) and limits accessibility, rendering NAATs largely impractical for rapid genetic analysis at the POC.^27,42 While alternative approaches to traditional genetic testing—including next-generation sequencing (NGS)—are becoming increasingly common,^43–48 these methods suffer from similar drawbacks, including high complexity, cost, and prohibitively long TATs.^3,6 As such, the unavailability of prompt, user-friendly genetic testing platforms continues to be one of the major barriers to widespread implementation of personalized medicine methods.^3,6,8

Fortunately, the last several years have been characterized by tremendous progress with respect to POC genetic testing technologies. Much of this progress has been facilitated by advancements in microfluidics, which have enabled the integration and miniaturization of complex fluid handling and detection procedures.⁴⁹ Furthermore, uptake of these tools has been materially accelerated by the SARS-CoV-2 pandemic.⁵⁰ In this review, we discuss the latest advancements in POC NAAT development and the role that such technologies are expected to play in enabling genetic testing for personalized medicine applications. We begin by discussing many of the key technologies that address the challenges associated with each of the key stages of a typical NAAT assay. Then, we present and discuss several platforms that offer fully integrated, sample-to-answer solutions for ready or near-ready deployment at the POC.

II. POINT-OF-CARE NUCLEIC ACID AMPLIFICATION TESTS: CHALLENGES AND DEVELOPMENTS

An ideal POC testing platform should be low-cost, rapid, and simple-to-use for minimally trained personnel (i.e., highly automated) while still maintaining a degree of sensitivity and specificity that is comparable with laboratory-grade tests.^8,51,52 This has traditionally been difficult to achieve for NAATs, which require the consolidation of three major operations into a single, robust platform: (1) sample preparation (i.e., nucleic acid extraction and purification); (2) amplification; and (3) detection (Fig. 1).^8,53 Herein, we describe some of the key technologies that have been employed to overcome the challenges associated with each of these three key phases.

FIG. 1. — An illustration of the typical workflow associated with a nucleic acid amplification test.

A. Sample preparation

Sample preparation is one of the major bottlenecks in POC NAAT development.^51,54 The process typically comprises two stages: (1) cell lysis, wherein nucleic acids (NAs) are released by disrupting the cell wall and/or cellular membrane; (2) purification/isolation, wherein NAs are separated from the lysate to remove impurities and inhibitors.^54–56 In laboratory settings, sample preparation is typically performed using a liquid/liquid extraction protocol.^54,57 A phenol or chloroform mixture is used in combination with centrifugal force to aqueously separate NAs; alcohol or salt precipitation and rehydration are then implemented to isolate the DNA or RNA.^18,54,57 This process is laborious, time-consuming, and requires hazardous chemicals that are not suitable for use among untrained users.⁵⁴ Accordingly, notable efforts have been made to enable simple, effective, and POC-friendly extraction methods.^38,54,56,58

1. Commercial sample preparation kits

Major life science companies, including Qiagen and Roche, have developed a range of sample preparation kits that vary in complexity and user involvement (Table I).¹⁸ Many of these kits employ chemical lysis followed by solid-phase extraction (SPE) methods, wherein analytes are selectively transferred to the solid-phase by passing cell lysate through solid-phase materials such as filter papers,^59,60 magnetic beads,^61,62 or silica membranes.⁵⁴ SPE methods are among the most promising extraction/isolation techniques as they not only obviate many of the hazards and challenges associated with traditional liquid/liquid extraction protocols but they are also amenable to integration with miniaturized and automated POC devices.^54,56

TABLE I.

Summary of commercial nucleic acid isolation kits.

Company	Extraction platform	Kit	Lysis method	Purification/Isolation method	Total time	Automated?
Qiagen	QIAcube Connect	QIAamp DNA Micro Kit	Lysis buffer	Silica-membrane-based spin column	∼30 min	Partially, Hands-on time: 20 min
Roche	MagNA Pure LC 2.0 System	MagNA Pure LC Kit	Lysis buffer and Proteinase K	Magnetic glass particles	∼1 h.	Completely
Roche	MagNA Pure Compact Instrument	MagNA Pure Compact Isolation Kit	Lysis buffer and Proteinase K	Magnetic glass particles	∼30 min	Completely
LGC, Biosearch Technologies	N/A	QuickExtract™ DNA Extraction Solution	Heat treatment	Heat treatment	3–8 min	No, but amenable to automation

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Though less common, several non-SPE based sample preparation kits are also available on the commercial market. For example, LGC Biosearch Technologies' QuickExtract™ DNA Extraction Solution involves a protocol wherein samples are mixed directly with an extraction solution, then heated to achieve lysis and purification. The protocol is simple, rapid (3–8 min) and requires only a few user steps.⁶³ Further, it has been successfully employed as a rapid, POC-friendly sample preparation technique in prior publications.^64–67

While these kits considerably simplify the sample preparation process, many either fail to provide fully automated solutions or require costly auxiliary instrumentation to minimize user involvement. Further, most are not directly integrated with downstream amplification and detection technologies. While this may grant a degree of flexibility during development stages, it complicates deployment among untrained users. Further integration, automation, and miniaturization of these sample preparation processes is likely needed to ensure their broad use for POC applications.

2. Microfluidic and cartridge-based sample preparation

Owing to their high degree of integrability and miniaturization, as well as their ability to perform complex fluid handling steps on small scales, microfluidic technologies have gained quite a bit of attention as a sample preparation solution with potential advantages over benchtop extraction methods.⁶⁷ Several fully automated cartridge- and microfluidic-based sample preparation platforms have been developed in recent years, serving as a promising means of processing/purifying crude samples prior to downstream amplification, analysis, and/or detection.^51,68 One of the earliest and most commercially successful examples is Cepheid's GeneXpert system (Fig. 2). A novel cartridge mechanically lyses cells via sonification, then uses a series of microtubes, pumps, and rotary drives to transfer liquids to different chambers where washing and extraction take place.^51,56,69 After the nucleic acids are purified and concentrated, they are transferred to a separate chamber wherein PCR amplification and detection are performed.⁵⁶ As a highly effective, fully integrated solution, the GeneXpert cartridge has widely been considered a breakthrough technology in sample preparation for nucleic acid testing.

FIG. 2. — A schematic of Cepheid GeneXpert's cartridge.⁶⁹ The cartridge implements nucleic acid extraction and reaction preparation prior to PCR amplification. A plunger engages the syringe barrel, drawing the sample and the necessary reagents into a cavity that contains beads for solid-phase extraction. Purified nucleic acids are then combined with lyophilized PCR reagents, and transferred to the PCR “tube,” wherein amplification and real-time fluorescence-based detection are performed. Figure reproduced with permission from Raja *et al*., Clin. Chem. 51, 882 (2005). Copyright 2005 Oxford University Press.

Other highly integrated microfluidics-based sample preparation solutions that are similarly amenable to integration with downstream amplification technologies have been presented in literature. For instance, Zhang et al. developed a microfluidic chip to isolate nucleic acids from hepatitis B virus (HBV) and human immunodeficiency virus (HIV) in under 1 min.⁷⁰ The chip contained pre-stored reagents and a reaction chamber, wherein ultrasonic cell lysis and silica-membrane-based SPE were performed to isolate NAs from serum samples.⁷⁰ Yang et al. reported a cartridge that contained lysis buffer, magnetic beads, binding buffer, and washing buffer to perform chemical lysis and NA isolation from salivary samples. However, a control instrument was required to automatically dispense and transfer reagents, effectively mimicking a desktop magnetic bead-based SPE protocol.⁷¹ Several other cartridge- and microfluidics-based sample preparation platforms continue to emerge regularly and have been reviewed accordingly.^{38,56,68,72,73}

Microfluidics-based devices have also been proposed as tools to enable sample preparation prior to analysis via next-generation sequencing.^74,75 Most existing methods of configuring DNA samples for sequencing-ready libraries rely on benchtop procedures that are slow, labor-intensive, and complex, hence limiting the applicability of NGS at the POC.^74–76 Digital microfluidic (DMF) technologies have recently emerged as a promising solution for expediting and optimizing these processes.^76,77 For example, Kim et al. developed a DMF platform that functioned as a fluid distribution hub for multiple modules that performed sub-processes of a larger NGS library sample preparation procedure.⁷⁶ By using a capillary interface, the group was able to show highly reproducible transfer of liquid between the DMF hub and external fluidic modules, hence enabling continuous-flow and droplet-based sample manipulations using a single integrated platform. Moreover, they demonstrated that their platform facilitated the automation of two key steps in the NGS library sample preparation pipeline: fraction collection of target analytes and buffer exchange/sample clean-up.⁷⁶ Separately, Kim et al. designed a high-density, two-layer microarchitecture that integrated all of the key steps of sample preparation for whole-genome sequencing into an automated microfluidic device.⁷⁴ The resulting device was capable of processing batch sizes of up to 96 samples and facilitated reductions in reagent consumption, input requirements, and also curtailed risk of contamination.⁷⁶ These works—combined with others being reviewed on a regular basis^75,77—demonstrate the potential for microfluidic technologies to not only simplify sample preparation for sequencing-based analysis but also to optimize the cost and throughput of NGS technologies, hence increasing their amenability to POC applications.

Overall, microfluidic technologies show great promise toward easing the sample preparation bottleneck at the POC. Their scalability, integrability, and amenability to automation are complemented by their low sample input and reagent requirements.^67,68 However, their frequent dependence on external pumping and fluidic handling systems continues to limit their widespread adoption.⁶⁸ Fortunately, these issues have begun to be addressed through the development of hand-operated, capillary force-driven, and vacuum-driven microfluidic devices among other innovations.⁶⁸ Together, these innovations, and the advantages boasted by microfluidics at large, will likely enable microfluidic technologies to significantly reduce the cost and complexity of sample preparation at the POC, hence enabling downstream amplification, analysis, and/or detection technologies to become more prevalent in near-patient settings.

3. Paper-based sample preparation

Owing to their simplicity, low cost, speed, and relative independence from external instrumentation, paper-based devices are another popular POC sample preparation technology.^38,54,59 Representative examples include Whatman Fast Technology Analysis (FTA) cards (GE Healthcare),⁵⁴ as well as Fusion 5 membrane-,^60,78 nitrocellulose-,⁷⁹ and polyethersulfone (PES) membrane-based devices.⁸⁰ Whatman FTA cards are currently among the most widely used paper-based sample preparation devices.^53,81 FTA cards function by using chemically treated filter matrices to lyse cells, denature proteins, and bind nucleic acids from the cell lysate.^54,59,60,82 Subsequent wash steps are typically required prior to downstream analysis to ensure that cellular debris, stabilizing chemicals, and/or inhibitors are removed.⁸³ However, overall, the FTA card extraction process is simpler, cheaper, and faster (<40 min) than conventional phenol-chloroform extraction methods.^53,84 Owing to these advantages, FTA cards have been integrated into the workflows of several POCT devices.^53,84,85

For example, Trinh et al. integrated FTA cards into a foldable, all-in-one POC molecular diagnostic device.⁸⁴ The device comprised three layers of polymethyl methacrylate (PMMA) [Fig. 3(a)]. The top layer was connected to the middle layer in a hinge-like fashion using a thin film tape, which also functioned to affix paper discs that were pre-soaked in PCR reagents to the upper PMMA layer. The middle layer included an array of six through-holes that, when attached to the bottom layer, formed six chambers that FTA cards could be inserted into. By using FTA cards, the group was able to successfully purify DNA from bacterial cells at room temperature in about 30 min. After serial washing, DNA captured on the FTA cards was used as template for PCR reactions, which could be initiated by folding the top layer down onto the bottom layer. Colorimetric detection of the PCR targets was then performed by adding a silver nitrate solution to the chambers. Using this approach, Trinh et al. were able to extract, amplify, and detect three foodborne pathogens in a relatively simple fashion.⁸⁴ While these sorts of examples do underscore the promise of FTA cards as POC sample preparation tools, the serial wash steps that FTA cards tend to require prior to downstream analysis evidence the need to streamline or automate procedures to ensure seamless integration with simple, fast, POC-friendly workflows.

FIG. 3. — (a) A schematic of Trinh *et al*.'s three-layer foldable POC molecular diagnostic device.⁸⁴ The upper PMMA layer was connected to the middle PMMA layer in a hinge-like fashion and contained a series of paper disks pre-soaked with PCR reagents. The middle layer contained a series of through-holes that, when coupled with the bottom layer, created six chambers wherein FTA cards were placed to enable nucleic acid extraction. After completion of the nucleic acid extraction procedure, the top layer was folded onto the bottom layer, and the device was inserted into a commercial thermal cycler with a custom heat block that enabled PCR amplification. Colorimetric detection of the PCR targets was then performed by adding a silver nitrate solution to the main chambers and illuminating them with a UV light. (b) An illustration of Gan *et al*.'s filter paper-based nucleic acid analysis device. The device was constructed by sandwiching a Fusion 5 filter paper and a PDMS membrane between two PMMA layers. Automated DNA extraction was performed by directly adding the sample to the Fusion 5 filter membrane for a specified period of time, then sequentially aspirating NaOH, HCl, and water through the filter using a syringe pump. The filter containing the extracted DNA was then directly used as template in downstream PCR reactions. Image (a) reproduced with permission from Trinh *et al*., Sens. Actuators B Chem. **314**, 128057 (2020). Copyright 2020 Elsevier. Image (b) reproduced with permission from Gan *et al*., Lab Chip, 14(19), 3719 (2014). Copyright 2014 Royal Society of Chemistry.

Fusion 5 membranes have also gained widespread attention among the POC community.^53,78,86,87 Characterized as single layer matrix membranes, Fusion 5 membranes are simple and inexpensive to manufacture.^88,89 One of the most popular nucleic acid extraction methods involving Fusion 5 membranes is known as filtration isolation of nucleic acids (FINA).^54,89 In this method, whole blood is added directly to the Fusion 5 membrane, which traps blood cells. A single wash with sodium hydroxide (NaOH)—or some other alkaline solution—is then performed to lyse the cells and remove cellular debris and other inhibitors.^54,89 By averting the serial washing and purification procedures associated with FTA cards, Fusion 5 membrane-based FINA procedures facilitate faster (<10 min) and simpler nucleic acid extractions.⁸⁹ These favorable qualities have similarly led many POCT developers to employ Fusion 5 membranes for sample preparation.

For example, Gan et al. developed a filter paper-based microdevice for DNA extraction and amplification at the POC.⁹⁰ The device was fabricated by inserting a Fusion 5 filter and a polydimethylsiloxane (PDMS) membrane between two polymethyl methacrylate (PMMA) layers [Fig. 3(b)]. Using a FINA-like method, the group showed that they were able to successfully extract nucleic acids by directly adding whole blood to the Fusion 5 filter surface, then sequentially washing the membrane with a NaOH solution followed by a diluted hydrochloric (HCl) acid solution using a syringe pump. The group also showed comparable results by deploying a similar method to other raw samples, such as buccal swabs and saliva. By integrating Fusion 5 membranes into a highly integrated microfluidic device and by showing that the device could be used to efficiently obtain nucleic acids from a broad range of samples in a timely fashion, Gan et al.'s work emphasizes the promise of Fusion 5 membranes as a means with which to perform nucleic acid extraction at the POC.

As evidenced by the FTA card- and Fusion 5 membrane-based devices discussed in this review, as well as other paper-based sample preparation tools being reported and reviewed on a regular basis,^59,86,89,91 paper-based nucleic acid extraction devices offer rapid and simple solutions for sample preparation at the POC. However, their use of chemical buffers introduces inhibitors that impact downstream amplification efficiency, which is often required for further analysis.^72,92 To overcome this inadequacy, many paper-based sample preparation tools require wash steps to ensure removal of inhibitors prior to downstream processing.⁸⁹ This can draw out the overall sample preparation procedure and also necessitate the use of microfluidic apparatuses/fluidic handling tools to ensure automated implementation, as has been seen in works that employ Whatman FTA cards.⁸⁹ While Fusion 5 membranes offer improvements over FTA cards by requiring fewer wash steps—and, hence, less time—to ensure sufficient purification and inhibitor removal, they still require at least one wash step.^54,89 Fortunately, some progress has been made toward mitigating the effects of chemical buffers to reduce washing requirements, with several prior works reporting the use of chitosan to meet this need; however, further innovation with respect to inhibitor reduction is likely needed to overcome this commonly cited challenge.^72,92 Even so, paper-based extraction methods remain among the most promising solutions to relieve the POC sample preparation bottleneck.

B. Nucleic acid amplification

1. Polymerase chain reaction-based amplification

PCR, and its derivative RT-PCR, are the most common nucleic acid amplification technology. In this method, nucleic acids are amplified via a thermocycling protocol that effects temperature-dependent DNA melting and polymerase-driven replication. Despite its relatively simple principles, the sample preparation requirements, thermocycling instrumentation, and turnaround times required to perform PCR assays have historically limited their use at the POC.⁵⁸ Consequently, both industry and academic researchers have placed a strong focus on developing devices that automate, accelerate, and miniaturize PCR assays, with microfluidic technologies often serving as the basis for many of these efforts.

Microfluidics-based PCR amplification devices generally belong in one of two categories: temporal domain devices or space domain devices.⁹³ In temporal domain PCR, the reaction remains stationary while the temperature of the surrounding system is modulated. Advantages of this approach include a smaller footprint and reduced risk of nonspecific surface adsorption; however, total assay times tend to be longer, and the microfluidic mechanisms required to keep the reaction stationary during amplification can complicate integration with upstream and downstream companion technologies.⁹³ In space domain PCR, the reaction is continuously moved through microchannels located atop individual thermostable “blocks,” and the reaction temperature is modulated according to its position along the channel.^93–95 Microchannel configurations vary based on application, but include serpentine,^96,97 spiral,^98,99 oscillating-flow-based,^100,101 or straight geometries.^102–104 Advantages of this approach include rapid assay times and ready integration with “sample-in-answer-out” processes; however, enabling continuous flow often necessitates external instrumentation or microfluidic geometries with variable channel widths, which are more difficult to fabricate.^94,95

Fortunately, several groups have demonstrated microfluidic platforms that leverage the advantages of space domain PCR while obviating major fabrication and deployment challenges. For example, some groups have demonstrated “lab-on-a-disk” (LOAD) devices that utilize centrifugal forces to enable pump-free reaction transport on space domain PCR devices.^105,106 That is, rather than relying on external pumps and instrumentation to enable fluid handling, LOAD devices rely on rotationally controlled liquid handling.¹⁰⁷ Others have demonstrated convective PCR, wherein temperature-induced density changes enable passive movement between thermostable regions. For example, Khodakov et al. developed a portable convective PCR cassette that comprised two thermostable zones: a 60 °C annealing region in the upper part of the chamber and a 95 °C denaturation region in the lower part of the chamber (Fig. 4). Continuous, instrument-free flow of the reaction was achieved by leveraging the changes in fluid density caused by changes in the reaction temperature. Using this approach, Khodakov et al. were able to perform highly multiplexed amplification and precise quantitative nucleic acid detection with single nucleotide discrimination in under 20 min.¹⁰⁸

FIG. 4. — An illustration of Khodakov *et al*.'s convective PCR system.¹⁰⁸ The PCR reaction was loaded into the ring-shaped reaction chamber using the loading port on the bottom of the chip. Two independent heaters controlled the denaturation and annealing temperatures, respectively. Thermal cycling was achieved via autonomous fluid flow enabled by temperature-induced changes in fluid density. Figure reproduced with permission from Khodakov *et al*., Nat. Biomed. Eng. 5, 702 (2021). Copyright 2021 Springer Nature.

A major consideration for any of these approaches is how heat is delivered to the system. Selection of an appropriate heat source is critical, as it can impact final form factor, heating and cooling efficiency, total assay times, and power consumption. Integrated heaters (e.g., thin metallic resistive heaters) are among the most promising methods of heat delivery for POC applications as they can enable straightforward miniaturization while also dramatically reducing amplification times.¹⁰⁹ For example, Neuzil et al. developed a silicon micromachined real-time PCR chip that utilized integrated thin film resistive heating for thermal cycling. The miniaturized chip, which measured 24.2 × 24.2 mm in size, boasted heating and cooling rates of +175 and −125 °C/s, respectively. A single optimized thermal cycle took just 8.5 s, which corresponded to a < 6 min 40-cycle PCR time.¹¹⁰

Aside from the microfluidic-based devices delineated above, several other point-of-care PCR platforms have been reported in literature, including micro-electromechanical systems (MEMS),¹¹¹ polymer-based devices,¹¹² and digital droplet-based devices.¹¹³ Because these approaches have previously been evaluated, they will not be discussed in great detail here. However, taken in aggregate, these technologies demonstrate the tremendous progress being made toward simplifying, miniaturizing, and integrating PCR-based assay platforms for applications at the POC.

2. Isothermal amplification

Isothermal amplification approaches have emerged as an attractive alternative to conventional PCR-based assays. By operating at uniform temperatures (typically between 30 and 65 °C), isothermal technologies obviate the need for sophisticated thermocyclers, hence reducing system complexity while increasing portability and integrability with upstream (e.g., sample preparation) and downstream (e.g., biosensing/detection) companion technologies.¹¹⁴ Further, isothermal approaches tend to be more tolerant of inhibitors than their PCR-based counterparts, which partially relieves sample preparation bottlenecks.¹¹⁵ Representative approaches include loop-mediated isothermal amplification (LAMP), recombinase-polymerase amplification (RPA), nucleic acid sequence-based amplification (NASBA), strand displacement amplification (SDA), helicase dependent amplification (HDA), and nicking and extension amplification reaction (NEAR). Table II provides a summary of the operating principles, advantages, and disadvantages associated with these various methods. Because several reviews have provided detailed discussions of these technologies, as well as additional isothermal-based amplification mechanisms, this work will simply provide an overview of two of the most widely used methods, LAMP and RPA, and their relevance to genetic testing at the POC.^51,115–118

TABLE II.

Summary of major features of selected isothermal amplification technologies.

Amplification technology	Operating principle	Operating temperature (°C)	Primer design	Sample preparation requirements	Pre-heating requirement?	Amplification time	Selected applications
LAMP	Strand displacement polymerase	60–65	Difficult (6–8 primers/target)	Amplification from crude sample possible	No	30–60 min	Pathogen detection,^119–121 pathogen genotyping,¹²² SNP detection^123,124
RPA	Recombinase-polymerase complexes, single-stranded binding protein	37–42	Simple-Moderate (2 (long) primers/target)	Amplification from crude sample possible	No	<25 min	Pathogen detection,^125–127 pathogen genotyping,^128,129 SNP detection¹³⁰
NASBA	Reverse transcription, strand displacement polymerase	∼41	Simple (2 primers/target)	Requires nucleic acid extraction	Yes (65 °C for RNA, 95 °C for DNA)	∼1.5–2 h	Pathogen detection,¹³¹ pathogen genotyping,¹³² SNP detection¹³³
SDA	Strand displacement polymerase	37	Moderate (2 chimeric primers/target + 2 bumper primers)	Amplification from crude sample possible	Yes	30–60 min	Pathogen detection,^134,135 SNP detection¹³⁶
HDA	Helicase, strand displacement polymerase	60–65	Simple (1 primer/target)	Amplification from crude sample possible	No	∼60 min	Pathogen detection,¹³⁷ SNP detection^138,139
NEAR	Nicking-enzymes, strand displacement polymerase	55	Simple (2 DNA/RNA chimeric primers/target)	Amplification from crude sample possible	No	∼10 min	Pathogen detection^140,141

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LAMP is a sensitive and specific NA amplification technology that functions at a fixed temperature of about 60–70 °C.¹⁴² Exponential target amplification relies on Bst DNA polymerase, an enzyme that displays strong strand displacement activity.¹⁴³ The highly specific nature of this amplification mechanism, combined with Bst polymerase's tolerance of many common sample matrix inhibitors, enables direct amplification of crude samples. By virtue of these redeeming qualities, LAMP has received notable mainstream attention and has been applied to a broad range of applications including pathogen detection,^{119,144–146} genetic analysis,¹⁴⁷ and gene expression profiling.^26,148 For example, Liao et al. developed a minimally instrumented POC molecular diagnostic device that utilized LAMP for target pathogen amplification. The platform leveraged a phase-change material (PCM) that underwent a water-triggered exothermic chemical reaction to supply heat to the system. A smartphone flashlight was used to excite fluorescent dyes in the reaction, hence enabling real-time measurements of fluorescence emission during the amplification process. Using this approach, Liao et al. were able to detect and quantify herpes simplex virus 2 (HSV-2) viral DNA in under 1 h [Fig. 5(a)].¹⁴⁹

RPA is another competitive isothermal alternative to PCR. By leveraging a phage recombinase, single-stranded DNA binding protein, and a strand displacement polymerase, RPA offers exponential isothermal amplification of a broad range of nucleic acids in under 25 min.^150,151 Similarly to LAMP, RPA chemistries are tolerant to many of the impurities and inhibitors that traditionally reduce PCR efficiency, allowing for direct amplification from crude samples.^150,152 Further, RPA has the added benefit of reduced operating temperatures (37−42 °C).¹⁵² Because of these favorable qualities, interest in RPA has grown dramatically in recent years, and current application spaces include pathogen detection^153–156 and genetic analysis.^157–159

For example, Law et al. reported an automated lab-on-a-disc platform based on RPA and fluorescence detection [Fig. 5(b)]. A custom-designed microfluidic disc was pre-loaded with all necessary reagents; resuspension, mixing, and fluid transport were performed using centrifugation. Heating during amplification was achieved using resistive heating elements that were embedded on a printed circuit board (PCB) on the disc and controlled by a wireless data communication module. Real-time monitoring of amplification was achieved using fluorescent indicators. Using this approach, Law et al. demonstrated real-time detection of drug-resistant tuberculosis in under 15 min.¹⁶⁰ Due to the low operating temperatures of RPA, there have also been reports of assays that leverage body heat for thermal incubation/amplification, eliminating the need for external thermal instrumentation altogether.¹⁶¹ For example, Kong et al. developed a wearable microfluidic RPA-based diagnostic platform capable of detecting HIV-1 DNA in under 24 min [Fig. 5(c)].¹⁶² Owing to the flexibility of RPA assays, these platforms have the potential to be repurposed for the rapid amplification and detection of other genetic targets.

With the advantages of isothermal amplification technologies in mind, it is also important to consider their limitations. LAMP assays require the design of six to eight primers that recognize distinct regions of each individual target DNA sequence.¹⁴² This not only complicates assay design but also increases the risk of non-specific amplification and false positives due to primer–primer interactions, hence reducing multiplexing capabilities.^163–165 Similar challenges are routinely encountered with RPA assays. Because of the natural function of the enzymes used in RPA (performing homology directed repair), a greater tolerance of probe and target sequence mismatches is often observed, hence reducing discrimination specificity between closely related sequences.^152,156,166 The long optimal primer lengths (∼30−35 nucleotides) associated with RPA can further contribute to non-specific amplification and false positives by increasing the risk of primer–primer and homo–dimer interactions.^115,152 Together, these weaknesses complicate the use of isothermal amplification technologies for genetic testing applications, which often require multiplexing or single-base discrimination for SNP detection.

3. Outlook on nucleic acid amplification for genetic testing

Given that both PCR and isothermal methods have their own respective advantages and disadvantages, neither technology is decidedly better than the other for rapid genetic testing applications. Both are likely to play a major role in increasing the feasibility of genetic testing for personalized medicine at the POC; however, selection of one over the other should be guided by the qualities that a given assay or POC platform is optimizing for.

In the case of gene expression assays that require highly multiplexed amplification, or genotyping assays that require single-base differentiation, PCR appears more attractive as it offers greater specificity and ease of multiplexing than its isothermal counterparts. Additionally, the cyclical nature of PCR makes it amenable to straightforward quantitation for applications including differential gene expression analysis. That said, PCR's stringent sample preparation and thermocycling requirements increase total system complexity and assay times. However, recent innovations in microfluidics have begun to overcome those challenges by proposing highly integrated and miniaturized device architectures that enable high-speed, low-consumption PCR assays.

Isothermal methods offer reduced system complexity and faster assays; however, performing highly multiplexed assays and/or single-base differentiation remains challenging. Consequently, companion technologies are likely to be instrumental in accelerating the development of isothermal amplification-based genetic testing platforms. For instance, implementation of microfluidic geometries that enable independent, parallel processing might relieve multiplexing limitations by facilitating simultaneous single-plex reactions that can be analyzed downstream using a multiplexable biosensing platform. Similarly, clustered regularly interspaced short palindromic repeat (CRISPR)/Cas-mediated systems, which are lauded for their high specificity, can be used alongside isothermal technologies to overcome issues with single-base specificity while still appreciating the benefits of increased sensitivity.

C. Nucleic acid detection

The final step in an NAAT assay involves the qualitative or quantitative detection of amplicons. Proper selection of a detection method is crucial, as it not only impacts the efficiency, sensitivity, and accuracy of a test but it also impacts its ability to be deployed at the POC. Many detection methods have been demonstrated for POCT applications, including fluorescence,^167,168 chemiluminescence,^169,170 surface-enhanced Raman spectroscopy (SERS),^171–174 surface plasmon resonance (SPR),^175–178 pH-based,^179–181 CRISPR/Cas-mediated,^182–184 optical,¹⁶⁸ electrochemical,^185,186 magnetic, paper-based,^{39,61,134,187–196} and colorimetric approaches.^51,197 Discussing all of these methods in detail is beyond the scope of this review, but we will focus on a few of the most common and promising approaches, namely, fluorescence, electrochemical, magnetic, paper-based, and CRISPR/Cas-mediated detection. For more information regarding alternative detection technologies, see prior reviews.^{39,134,168,179,185}

1. Fluorescence detection

Fluorescence-based detection is common in both real-time and endpoint nucleic acid biosensing. This form of detection is typically facilitated by intercalating dyes, oligonucleotide probes that are cleaved during reactions or fluorescent nanomaterials.^51,197 With the exception of a few systems, most commercial POC NAATs utilize desktop analyzers to perform real-time fluorescence detection (Table III); however, these analyzers tend to be bulky, complex, and costly, which is due—in large part—to the associated optical instrumentation.⁵¹ Commercial analyzers developed for real-time analysis of isothermal amplification are somewhat more cost-effective and simpler because they do not require integration with PCR thermocycling equipment. Axxin's T8-ISO and T16-ISO are portable instruments that report real-time fluorescence measurements of two and three fluorophores, respectively, during RPA reactions.^198,199 Optigene's Genie II is a portable instrument that is designed to detect targets by fluorescence measurement while running any isothermal amplification reaction.²⁰⁰

TABLE III.

Examples of PCR-based POC NAATs in literature.

Author	Platform architecture	Extraction method	Amplification method	Detection method	Total assay time	Multiplexing demonstrated?	Selected application
Trick et al.²⁰¹	Disposable assay cartridge + reusable instrument with magnetic actuation arm, heat block, and detection instrumentation	Magnetic bead-based	PCR	Real-time fluorescence	<15 min	Yes (Duplex)	N. gonorrhoeae detection and genotyping for antimicrobial resistance
Huang et al.²⁰²	Integrated microfluidic chip + Onestart microchip analyzer	Magnetic bead-based	PCR	Real-time fluorescence	<1.5 h.	Yes (21-plex)	Detection of multiple respiratory tract infection pathogens
Shu et al.²⁰³	Handheld system comprising sample preparation module, real-time convective PCR module, and fluorescence imaging module	Magnetic bead-based	PCR	Real-time fluorescence	∼30 min	No	Detection of bacterial pathogens
Shin et al.²⁰⁴	Disposable cartridge + portable multiaxial magnetofluidic instrument with PCR thermal control faceplate and fluorescence detection module	Magnetic bead-based	PCR	Real-time fluorescence	∼1 h	No, but duplex detection is possible	Detection of hepatitis C virus (HCV)
Zhu et al.²⁰⁵	Integrated microfluidic chip + thermoelectric unit for thermal cycling + charge coupled device (CCD) camera for endpoint detection	Hyperthermic pyrolysis	Solid-phase PCR	Fluorescence	<1 h	Yes (5-plex)	Human papillomavirus (HPV) detection and genotyping
Czilwik et al.²⁰⁶	Integrated microfluidic disk + external instrument with rotary motor and optical detection module	Magnetic bead-based	Two-step PCR	Fluorescence	<4 h	Yes	Detection of Escherichia coli, Staphylococcus warneri, Streptococcus agalctiae, Haemophilus influenzae

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There are also reports of real-time and endpoint fluorescence-based detection platforms in academic publications. For example, Obahiagbon et al. developed a portable, low-cost, quantitative, and multiplexed fluorescence biosensing platform (Fig. 6). The prototype comprised five modules: (1) an excitation module with a 2 × 2 array of inorganic light emitting diodes; (2) a sample chamber module; (3) an emission and signal readout module; (4) a microcontroller module; (5) an optional display and data connectivity module. To interrogate the fluorescence signal, the excitation LEDs were turned on sequentially at each position in the array. The LED broadband spectrum was filtered using a low-cost excitation interference filter, and the biorecognition fluorescence signal was passed through an emission filter before being detected by coaxially aligned photodiodes.¹⁶⁷ Similar portable fluorescence detection systems have been demonstrated in other works.^168,207 Most can be characterized as platforms that similarly incorporate numerous external optical components into closed systems, making them highly elaborate. As an alternative to external optical equipment, some groups have reported completely on-chip fluorescent detection systems.^208–210 However, monolithic integration of whole optoelectronic systems is costly, particularly for single-use assay devices.¹⁶⁸

FIG. 6. — Schematic of Obahiagbon *et al*.'s portable fluorescence-based biosensing platform. A circuit schematic shows the platform's proposed charge-integration amplifier read-out circuit. The two pictured printed circuit boards represent the photodiode and amplifiers (top), and the soldered LEDs (bottom). Figure reproduced with permission from Obahiagbon *et al*., Biosens. Bioelectron. 17, 153 (2018). Copyright 2018 Elsevier.

Though fluorescence biosensing technologies are highly useful—particularly in the context of real-time, quantitative analyte detection—their dependence on bulky, delicate, costly, and/or elaborate optical systems limit their viability for limited-resource POC applications. Further, the limited number of filters available for distinguishing multiple unique fluorophores reduces the utility of fluorescence-based sensing for highly multiplexed assays.^18,211 Accordingly, these characteristics currently make fluorescence-based biosensing most well-suited to limited-analyte assays that require fast turnaround times in POC settings with few resource limitations. The real-time analysis capabilities afforded by these technologies make them faster than many alternatives that require endpoint detection; however, their cost and complexity are often incompatible with the needs of limited-resource settings. Further optimization and innovation with respect to these disadvantages are likely required to broaden the applicability of this technology.

2. Electrochemical detection

Electrochemical biosensors function by coupling a biological recognition element to an electrode transducer, then converting corresponding biological recognition events into detectable electrical signals.^185,186 Many detection technologies fall under the electrochemical umbrella, including voltametric/amperometric,^212–214 impedimetric,^215,216 potentiometric,^217,218 and field-effect transistor (FET)-based biosensors.^{197,219–221} Each of these technologies has been discussed at length in prior reviews.^{185,186,197,217} Advantages of electrochemical detection approaches include economical fabrication and deployment, simplicity, low instrumentation needs, and amenability to miniaturization.^{185,186,222,223} Commonly cited disadvantages include increased risk of non-specific adsorption and reduced detection limits for electrochemical bio-affinity assays.¹⁸⁵

Perhaps the most commercially successful example is the home-use glucometer.^179,224 The device contains a test strip that is functionalized with glucose oxidase or glucose dehydrogenase enzymes and an electrode.²²⁴ When plasma that is separated from a drop of blood diffuses across the test strip, the enzymes catalyze the conversion of glucose to gluconic acid, and the electrons generated during this reaction induce a current that can be measured to determine the concentration of glucose in the specimen.²²⁴ More recently, Cue Health developed an integrated molecular diagnostic system that utilizes microfluidic technology in combination with isothermal nucleic acid amplification and electrochemical biosensing.²²⁵

Other, lesser-known examples have also been demonstrated. Kim et al. reported a combined RPA and electrochemical biosensing platform that was characterized as a wearable multi-microelectrode array [Fig. 7(a)].²²⁶ Temperature-regulated incubation for the RPA-based amplification was achieved by leveraging body heat delivered to the wearable device. During the reaction, amplicons hybridized with thiol-modified primers that were immobilized on the working electrodes, causing a measurable change in current density that could be measured using differential pulse voltammetry. To ensure stable and reproducible performance from the working electrodes and to ensure biocompatibility, the group elected to fabricate the multi-array electrodes using gold (Au). With multiple electrodes, Kim et al. were able to facilitate the detection of several different targets, hence realizing multiplexed genetic analysis. Using this approach, the group demonstrated SARS-CoV-2 diagnosis via multiplex target gene detection in under 20 min.²²⁶ With certain adaptations and further development with respect to integrating sample preparation protocols, an ultra-compact wearable device like Kim et al.'s might be useful for applications including at-home disease or therapy monitoring, as it would provide clinicians with more frequent and decisive information regarding a patient's disease state or therapy responsiveness.

Pennisi et al. developed a combined LAMP and electrochemical detection platform that performed host-based gene expression analysis [Fig. 7(b)].²²⁷ The lab-on-a-chip (LOC) device comprised a PCB for signal acquisition and communication, and a cartridge containing a CMOS microchip arrayed with ion-sensitive field-effect transistors (ISFETs).²²⁷ A microfluidic chamber was assembled on top of the CMOS microchip to contain the sample during DNA amplification and detection.^227,228 After sample loading, the cartridge was placed on top of a commercial thermal cycler and heated to enable LAMP amplification.²²⁸ As exponential amplification of the target occurred, the sample's pH was altered, inducing a proportional electronic (voltage) change that was measured by the ISFETs.^227,228 Using this method, Pennisi et al. demonstrated amplification and detection of Herberg et al.'s two-transcript signature that distinguished bacterial and viral infections.^227,229 This device, and similar platforms,^130,230 demonstrate the promise of electrochemical biosensors as gene expression analysis tools. However, for true POC applicability, sample preparation and isothermal heat delivery should be integrated into the devices.

Many of the electrochemical-based diagnostics discussed in this work, as well as those reviewed in other works,^223,231 show great promise toward enabling fast, cost-effective genetic testing at the POC. Their independence from costly and complex optical instrumentation, combined with their low cost of manufacturing, favorably differentiate them from many of their optical biosensing counterparts.^222,231 Moreover, many recent efforts have focused on imparting electrochemical sensors with anti-fouling or non-biofouling properties, making them amenable to direct analysis with untreated or scarcely treated samples.^223,231,232 These qualities make electrochemical sensors highly attractive for limited-resource and rapid turnaround-time applications, as they offer users with the ability to perform biosensing at low cost, without stringent sample preparation requirements. That said, most electrochemical sensors currently remain limited in their multiplexing capabilities, with most only being able to detect up to two or three analytes per sensor.²³¹ As such, electrochemical sensors are currently better suited to limited-analyte assays; however, their ready compatibility with microfluidic technologies is likely be instrumental improving multiplexability by enabling parallel processing.^222,231

3. Magnetic detection

Magnetic biosensors generally function by employing paramagnetic or super-paramagnetic particles to detect biorecognition events by measuring magnetically induced effects (e.g., changes in resistance, magneto-optical properties, etc.) or changes to magnetic properties.^233–236 Representative examples of magnetic biosensing technologies include giant magnetoresistive (GMR) biosensors, magnetic tunnel junction (MTJ) biosensors, and magnetic particle spectroscopy (MPS) biosensors.¹⁹⁷ Due to their relative popularity, GMR biosensors and their applications will be the focus of this review.

Giant magnetoresistive biosensors function via localized proximity magnetic sensing, which is enabled by the giant magnetoresistive (GMR) effect: a quantum mechanical phenomenon in which changes to a local magnetic field are transduced into detectable changes in electrical resistance through spin-dependent scattering.²³⁷ By functionalizing GMR sensors with surface-bound molecules that are complementary to analytes of interest, then labeling those target analytes with magnetic nanoparticles (MNPs), GMR sensors are able to detect the presence of target analytes (e.g., DNA^{18,189,233,238}) on surface-bound probes in real-time.^18,237 Advantages of GMR biosensing include a high dynamic range, low background signals, low limits of detection, tolerance to matrix impurities, and highly multiplexed detection capabilities.^{18,190,193,239–242}

There are many reports of genetic testing assays using GMR biosensors.^18,239,243 Ravi et al. showed successful quantification of cDNA on GMR biosensors¹⁸ and later demonstrated a PCR and GMR-based assay that enabled multiplexed quantification and differential gene expression analysis of ten influenza-activated genes.¹⁹ While promising from the standpoint of enabling multiplexed transcriptional signature analysis, these assays were not demonstrated on a POC-friendly platform.¹⁹ However, a number of portable/POC GMR-based platforms have recently been reported, which promises to increase their applicability.^{192,244–246} Recently, de Olazarra et al. reported a portable RPA and GMR-based platform that performed multiplex, non-invasive, qualitative genotyping of four SNPs along the catechol-O-methyltransferase gene (COMT) (Fig. 8).⁶⁶ The platform comprised three major elements: a fluidic system characterized by a custom-designed cartridge and peristaltic micropumps; a printed circuit board that enabled automation, signal acquisition, and GMR analysis; and a temperature-controlled PCB that was used for RPA amplification and post-amplification denaturation. The assay required very little user involvement and provided users with a smartphone interface that could be used to electronically share results. Further, they showed that sample preparation could be easily integrated into the automated assay using Biosearch Technologies' Lucigen QuickExtract™ DNA Extraction Solution on direct salivary samples.⁶⁶ While the reported platform did demonstrate sample-to-answer capabilities as well as portability, further integration and miniaturization would likely be achievable through the use of microfluidic technology, particularly for fluid handling.

FIG. 8. — Overview of de Olazarra *et al*.'s RPA- and GMR-based SNP analysis tool. Throughout the assay, automated fluidic control was achieved using a custom cartridge (shown left) and a series of peristaltic pumps. Signal acquisition, conditioning, and control were achieved using a custom control printed circuit board. RPA-based amplification and post-amplification denaturation were implemented using a temperature-controlled PCB. Figure reproduced from de Olazarra *et al*., Lab Chip 22, 2131 (2022). Copyright 2022 Royal Society of Chemistry.

Given their strengths, GMR biosensors are likely to be most impactful for assays requiring highly multiplexed qualitative or quantitative analysis. Already, they have been indicated for use in multiplexed SNP detection⁶⁶ and gene expression analysis.¹⁹ However, it is worth noting that most of these GMR-based genetic analysis assays perform endpoint detection following upstream genetic amplification, which causes total assay times to be longer than for modalities that enable real-time quantification during amplification (i.e., optical modalities).^18,19,66 As such, GMR biosensors are best suited for applications that do not require extremely rapid sample-to-answer analysis. Additionally, many existing magnetic detection platforms lack fully integrated sample-to-answer capabilities, hence limiting their use among untrained individuals. Advancements in automated and miniaturized sample preparation methods, such as those discussed above, are likely to be instrumental in increasing assay integration for magnetic biosensing platforms. For instance, GMR biosensors have been shown to be compatible with on-chip microfluidic architectures;²⁴⁷ expanding these on-chip architectures to include microfluidic sample preparation mechanisms would likely broaden the applications of GMR biosensing at the POC. Innovations like these will be critical to enabling GMR biosensors to realize their potential as highly agile POC molecular diagnostic tools.

4. Paper-based detection

The central feature defining paper-based detection devices is their use of porous substrate materials, which include chromatography paper, nitrocellulose membranes, filter paper, and paper-based composites.^40,248 The porous quality of these materials facilitates fluid transport via wicking, hence eliminating the need for bulky external pumps/support equipment, which increases portability while decreasing cost and complexity.¹³⁴ Additionally, the characteristically white nature of many paper-based materials enables straightforward integration with low-cost visual detection mechanisms that require high contrast for improved sensitivity, such as colorimetric and/or fluorescence-on-paper-based assays.¹³⁴

The most commercially successful paper-based POCTs are lateral flow devices (LFDs), which function on the principle of sandwich-based immunoassays or competitive format assays, wherein detection of target analytes is realized via colorimetric changes that are typically observed with the naked eye, though smartphones are also occasionally used.^134,249 Common examples of these devices include at-home pregnancy tests and rapid COVID-19 antigen tests.^134,250,251 While these well-known examples rely on protein detection, many LFDs have also been developed to detect nucleic acids for applications such as infectious disease diagnostics^134,252 and SNP identification for genetic analysis.²⁵³ Examples of these devices will not be described in detail, as they have been reported and evaluated in several prior reviews.^{53,91,134,249,254} However, we will discuss the viability of paper-based devices for genetic testing.

Like any other technology, paper-based devices suffer from drawbacks. Recent efforts have focused on enhancing paper-based assay performance by increasing sensitivity and improving multiplexing capabilities.⁴⁰ Several signal amplification strategies, including integration of nanomaterials and novel device engineering methods, are currently being explored and have been discussed in detail in prior reviews.²⁵⁵ Limited multiplexing capabilities are of greater concern. Because of the way that many LFDs are structured, there is significant risk of cross-reactivity between targets and test strip probes.^248,256 This compromises quantitative and multiplexed detection capabilities, which is particularly troubling in the context of genetic testing for personalized medicine applications.^248,256 Accordingly, very few paper-based assay devices that perform nucleic acid analysis have achieved commercial success. While there are a few reports of traditional LFDs enabling multiplexed detection,^171,257 microfluidic technologies will likely be instrumental in helping to realize highly multiplexed paper-based detection assays by facilitating separate but parallel processing or by creating dedicated reagent transport channels that would prevent cross-reactivity/cross-contamination. Until these improvements are realized, paper-based devices are likely best suited for low-cost, limited-analyte qualitative assays (e.g., SNP detection) among minimally trained individuals rather than highly multiplexed quantitative assay (e.g., gene expression analysis).

5. CRISPR/Cas-mediated detection

Clustered regularly interspaced short palindromic repeat (CRISPR) systems were first identified as a foundational facilitator of adaptive immunity in microbial species.^258–260 These systems confer immunity by recognizing foreign nucleic acid sequences that are stored as “spacers” among CRISPR loci, then eliminating them via endonuclease activity enabled by CRISPR-associated (Cas) enzymes.^258,261 While the operations and functions of CRISPR/Cas systems vary,^262,263 all depend on CRISPR RNA (crRNA) as guides for effector proteins to recognize and specifically cleave target sequences.²⁶¹ These crRNA sequences can be artificially synthesized to guide endonuclease activity toward certain sequences of interest and have hence been repurposed for several applications including gene editing,²⁶⁴ nucleic acid imaging,²⁶⁵ and recording of cellular events.²⁶⁶ More recently, CRISPR/Cas systems have also been leveraged as powerful nucleic acid detection/diagnostic tools.¹⁸³ As previous reviews^{182–184,261,267–270} have provided detailed evaluations of CRISPR/Cas-mediated nucleic acid detection, Sec. II C 5 will provide a cursory overview of these technologies with references to significant works that showcase their powerful diagnostic capabilities for genetic testing at the POC.

The mechanisms that facilitate CRISPR/Cas-mediated nucleic acid detection vary across assays and are ultimately governed by the specific Cas effector protein(s) being used. Early CRISPR/Cas-based diagnostic assays utilized Cas9 variants, which perform crRNA-guided cleavage of double stranded DNA (dsDNA) sequences.^{46,258,270,271} Because these systems are guided by RNA, they offer single-nucleotide specificity, making them useful for a wide variety of applications.²⁶¹ Groundbreaking progress in this field was further accelerated by the discovery of the protein collateral activity of Cas12,²⁷² Cas13,²⁷³ and Cas14²⁷⁴ effectors. In short, these systems have the ability to trigger multi-turnover non-specific collateral cleavage (trans-cleavage) events upon target recognition.^183,268 When used in conjunction with non-specific reporter complexes (ssRNA for Cas13; ssDNA for Cas12 and Cas14), this collateral cleavage activity can be leveraged to act as a signal amplifier, hence dramatically improving detection sensitivity while maintaining the benefits of single-nucleotide specificity.⁴⁶ By leveraging these systems, many CRISPR/Cas-based diagnostic platforms have been successfully demonstrated, with applications ranging from infectious disease diagnostics/pathogen detection^144,182,268 to genotyping.^268,275

While CRISPR/Cas-mediated detection of unamplified DNA targets is possible (e.g., Hajian et al.'s CRISPR-Chip technology²⁷⁶), most highly sensitive (attomolar range) platforms report use of nucleic acid amplification prior to detection.¹⁸³ Due to their low operating temperatures and ease of integrability, isothermal mechanisms are typically favored. The first report of a comprehensive CRISPR/Cas-based nucleic acid biosensing platform, known as SHERLOCK, employed isothermal RPA-based amplification and Cas13a effector proteins to detect DNA via the collateral trans-cleavage of quenched fluorescent ssRNA reporters.²⁷³ Several other NA detection platforms that similarly leveraged isothermal amplification and the collateral cleavage activity of Cas13 and Cas12 effector proteins were subsequently reported (e.g., HOLMES,²⁷⁷ HOLMESv2,²⁷⁸ DETECTR²⁷⁹).

More recently, groups have combined CRISPR/Cas systems with other biosensing technologies to develop extremely powerful diagnostic platforms. Xiong et al. developed a lateral flow assay that employed reverse transcription-RPA (RT-RPA) and CRISPR/Cas9-mediated detection to enable dual-gene diagnosis of SARS-CoV-2 using a single strip test.²⁸⁰ Li et al. developed HOLMESv2 to specifically discriminate single nucleotide polymorphisms and quantify target NA sequences using a one-step system that combined LAMP amplification with CRISPR/Cas12b-mediated detection.²⁷⁸ Multiple other systems with combined isothermal amplification and CRISPR/Cas-mediated detection architectures have also been reported.^{144,268,281–283}

The highly sensitive and specific results obtained from platforms that combine isothermal amplification and CRISPR/Cas-mediated detection are extremely compelling from the standpoint of developing POC genetic testing platforms, particularly for applications requiring single-nucleotide discrimination. As prior discussions of amplification technologies acknowledged, isothermal methods offer greater integrability and ease of POC device development than their PCR counterparts; however, they suffer from reduced specificity, making them less suitable for certain genetic testing applications, such as genotyping for PGx. The impressive specificity afforded by CRISPR/Cas systems may help to overcome these challenges,²⁸⁴ hence providing an opportunity to develop powerful and timely genetic testing tools that leverage the sensitivity and integrability of isothermal amplification without compromising single-nucleotide discrimination capabilities. That said, limited multiplexing capabilities continue to be one of the central limitations associated with CRISPR/Cas-mediated nucleic acid diagnostics.¹⁸³ Further integration with microfluidic architectures comprising microchannels that enable parallel single-plex reactions will likely be needed in order to holistically meet POC genetic testing needs. However, for the time being, CRISPR/Cas-mediate biosensing technologies are likeliest to be most impactful for limited-analyte genetic testing assays that require extremely high sensitivity and specificity. Additionally, owing to their signal amplification capabilities and their compatibility with a broad array of reporter probes (e.g., fluorophore/quencher, enzyme-labeled reporters, horseradish peroxidase-labeled oligonucleotides, etc.), we predict that CRISPR/Cas complexes will grow in popularity as an integral part of many high-sensitivity genetic testing assays that do not require amplification prior to detection.^46,285

III. SAMPLE-TO-ANSWER POC NAAT DEVELOPMENT

Owing to their broad generalizability, particularly among users with minimal technical expertise, nucleic acid analysis platforms that offer fully integrated sample-in-answer-out capabilities are likely to be most effective in motivating widespread adoption of genetic testing for personalized medicine at the point-of-care. Accordingly, in Sec. III, we will review reports of platforms that consolidate nucleic acid extraction/isolation, amplification, and detection into highly integrated, user-friendly devices. We will begin by reviewing platforms that are still at the proof-of-concept or prototype stage and will finish by discussing fully developed devices that are available on the commercial market.

A. Sample-to-answer NAATs in literature

Numerous sample-to-answer NAAT prototypes have been reported in the literature. Most of these systems leverage microfluidic technologies, which are a promising solution for the realization of fully automated (i.e., hands-off) and portable NAATs due to their capacity to incorporate complex laboratory procedures onto miniaturized devices.^286–288 Examples of such systems include lab-on-a-chip (LOC) and lab-on-a disk (LOAD) devices, as well as microfluidic paper-based analytical devices (μPADs).^{134,228,287,289–293} All of these architectures have been leveraged to perform both PCR-based and isothermal amplification-based assays.

1. Fully integrated PCR-based platforms

Several examples of fully integrated PCR-based POC NAAT platforms are outlined in Table III. Most of these systems perform magnetic bead-based solid-phase extraction followed by fluorescence-based detection and offer multiplexed amplification and detection capabilities. Despite these similarities, device architectures and approaches to thermal cycling vary widely.

Trick et al. recently developed a rapid genetic testing platform for rapid detection and genotyping of Neisseria gonorrhoeae. The platform, coined “PROMPT” (portable, rapid, on-cartridge magnetofluidic purification and testing), comprised a disposable thermoplastic cartridge; a smartphone; and a reusable instrument that contained a magnetofluidic actuation apparatus, an aluminum heat block, and optical instrumentation (Fig. 9). The reported assay protocol required very little user involvement, making it ideal for at-home and point-of-care testing. After just three manual steps (elution of a swab, combining swab eluate with a magnetic bead solution, loading combined solution into the cartridge), the cartridge was mounted onto the faceplate of the reusable instrument, and sample preparation was initiated.

FIG. 9. — An overview of Trick *et al*.'s PROMPT system.²⁰¹ After the user preliminarily processes the patient sample, they insert it into the assay cartridge. Nucleic acid extraction and purification are performed within the cartridge, which is pre-loaded with the necessary buffers and reagents. The cartridge is then loaded onto the PCR heat block for amplification. A fluorescence detector measures the presence of the analyte and then sends the acquired signals to a smartphone or tablet for further analysis using Bluetooth communication. Figure reproduced with permission from Trick *et al*., Sci. Transl. Med. 13, eabf6356 (2021). Copyright 2021 The American Association for the Advancement of Science.

Nucleic acid extraction was performed in under 2.5 min using bacterial lysis (via an acidic binding solution) and magnetic bead-based SPE. The reaction was then transferred to an extruded well in the cartridge wherein reaction reagents were spatially isolated for targeted thermal control. An optimized aluminum heat block was used to rapidly modulate the reaction temperature during PCR amplification (heating and cooling rates were reported to be 4.2–10.4 °C/s and 10–18.5 °C/s, respectively). Notably, amplification times were further reduced by increasing the concentrations of primer, Mg²⁺, and DNA polymerase, which, in turn, reduced the temperature hold time requirements in their PCR protocol. Real-time fluorescence was monitored using an integrated two-color epifluorescence detector, which measured signal from hydrolysis probes that targeted both the pathogen and a wild-type mutation that was shown to be predictive of antimicrobial resistance. These signals were transmitted to a smartphone app for further analysis. Using this approach, the group was able to achieve a sample-to-answer time of 30 min.²⁰¹

Trick et al.'s platform shows great promise as a potential point-of-care genetic testing tool. The group already demonstrated that their PROMPT system could be used to perform multiplexed detection and genotyping of Neisseria gonorrhoeae, an assay which could improve therapy outcomes by providing clinicians with information regarding potential antimicrobial resistance. Beyond this proof-of-concept assay, the capacity to perform single-nucleotide discrimination on a highly automated, rapid TAT testing platform could also be useful for pharmacogenomic testing, wherein genomic polymorphisms are assessed to better understand potential variability in drug metabolization and therapy responsiveness on a more personalized basis. While this would require some adaptation, Trick et al. already demonstrated the versatility of their platform by repurposing it to perform two separate respiratory pathogen assays. The first detected and differentiated two SARS-CoV-2 variants of concern in duplex, while the second performed multiplexed detection of SARS-CoV-2 and Influenza A/B.²⁹⁴

Using a different approach, Shu et al. reported sample-to-answer, real-time convective PCR platform for the detection of bacterial pathogens (Fig. 10). The platform comprised three primary modules: (1) a sample preparation module; (2) a triangular closed-loop convective PCR system; (3) a wireless fluorescence imaging module. Sample preparation was performed using a magnetic bead-assisted photothermolysis method. The DNAs of the lysed bacteria were then mixed with PCR reagents and transferred to the triangular loop channel. Convective thermocycling was performed within the triangular loop using a single-heater thermal gradient. Optical instrumentation included within the platform (i.e., filter set, LED) enabled real-time fluorescence-based amplification monitoring by a wireless video camera equipped with a CMOS sensor. All these components, as well as the requisite thermal/optical/mechanical control electronics, were conveniently contained in a low-cost, handheld housing. Shu et al. were able to demonstrate sample-to-answer analysis in <30 min.²⁰³

FIG. 10. — An overview of Shu *et al*.'s sample-to-answer, closed-loop convective PCR platform.²⁰³ Major components include a module for magnetic bead-assisted photothermolysis sample preparation, a closed-loop triangular convective PCR reactor, and a wireless video camera used for real-time fluorescence detection. Figure reproduced with permission from Shu *et al*., Biosens. Bioelectron. 97, 360 (2017). Copyright 2017 Elsevier.

As Table III indicates, many of the fully integrated PCR-based platforms reported in the literature were specifically designed to enable molecular diagnosis of infectious pathogens. However, with thoughtful adaptations, many of these platforms may be useful for personalized medicine applications as well. Integration with downstream detection technologies that enable higher multiplexing capabilities will likely be essential to realizing this potential. For example, the thermal cycling method proposed by Trick et al. could be coupled with previously reported portable GMR biosensing technologies to enable highly multiplexed host gene expression analysis or SNP detection at the POC.^19,201 Such unions of state-of-the-art nucleic acid analysis technologies has the potential to greatly improve the generalizability of PCR-based assays at the point-of-care.

2. Fully integrated isothermal amplification-based platforms

Many fully integrated isothermal amplification-based NAATs have also been reported in literature in recent years, several of which are outlined in Table IV. By eliminating complex thermocycling requirements, these platforms are not only more amenable to integration with a greater number of upstream and downstream analysis technologies but they can also effectively function with far less instrumentation than their PCR counterparts. Standard low-cost incubators,²⁹⁵ hot plates,⁸⁰ or simple handheld/portable heaters⁵³ can be used in place of sophisticated thermocyclers or complex space domain approaches to temperature modulation. Additionally, high inhibitor tolerance can be leveraged to dramatically simplify and accelerate assay protocols by eliminating the need for complex sample preparation protocols.²⁹⁶ Taken together, these advantages have enabled the development of several promising sample-to-answer isothermal-based NAATs.

TABLE IV.

Examples of isothermal amplification based POC NAATs in the literature.

Author	Platform architecture	Extraction method	Amplification method	Detection method	Total assay time	Multiplexing demonstrated?	Selected application
Choi et al.⁵³	Paper-based assay device + handheld battery-powered heating device	FTA card and glass fiber integrated on lateral flow strip	LAMP	Colorimetric on lateral flow strip (quantification via smartphone)	∼1 h.	No	Escherichia coli detection
Ning et al.²⁹⁶	Chip + smartphone fluorescence microscope device	Extraction-free protocol	RPA	CRISPR-fluorescence via smartphone-read	<15 min	No	SARS-CoV-2 detection
Zhang et al.²⁹⁷	Integrated microcapillary + fluorescent reader	FTA card	RT-LAMP	Real-time fluorescence	<2.5 h.	No	Screening of SNPs in CYP2C19 gene
Yin et al.²⁹⁸	Integrated microfluidic chip + external fluorescent imaging system (Maestro Ex, CRI Maestro, USA)	Magnetic bead-based	RPA	Fluorescence	<45 min	Yes	Detection of foodborne bacterial pathogens
Kim et al.²⁹⁹	Integrated microfluidic disk	Laser irradiation	RPA	Colorimetric on lateral flow strip	<30 min	No	Salmonella typhimurium detection
Deng et al.³⁰⁰	Disposable cartridge + reusable control unit	Boiling of diluted samples	RT-LAMP	Colorimetric	∼35 min	No	SARS-CoV-2 detection

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Choi et al. developed a fully integrated paper-based biosensing platform that leveraged LAMP amplification and paper-based extraction and detection methods to enable point-of-care nucleic acid analysis [Fig. 11(a)]. The biosensing platform comprised four hydrophobic polyvinyl chloride (PVC) layers that controlled the flow of the reaction between the nucleic acid extraction region, the amplification region, and the lateral flow strip detection region. Sample preparation was performed using an FTA card located on the third layer, while an absorbent pad on the fourth layer purified the sample and absorbed the waste. After extraction was complete, the fourth layer was discarded, the second and third layers were combined, and the paper-based device was inserted into the covered heating compartment of a handheld, battery-powered heating device. The reaction was then incubated at 60 °C to enable LAMP-based amplification. The product was then briefly denatured, and targets were detected using a typical lateral flow assay architecture. Colorimetric results could be qualitatively observed with the naked eye or quantitatively analyzed using smartphone captures coupled with a custom-built app. Using this approach, Choi et al. demonstrated sample-to-answer detection of E. coli and Streptococcus pneumonia in about 1 h, albeit several hands-on steps were required in order to complete the assay properly.³⁰¹

FIG. 11. — (a) An overview of Choi *et al*.'s paper-based sample-to-answer nucleic acid analysis platform.³⁰¹ The four hydrophobic PVC layers comprising the device controlled sample flow from the nucleic acid extraction area, to the amplification area, and finally to the lateral flow strip. A schematic of the experimental procedure is included for reference. (b) A schematic of Batule *et al*.'s paper-based nucleic acid testing system. A lateral flow-based platform was used for the extraction of viral RNA. Extracted viral RNAs were then directly added to the prepared RT-LAMP buffer, and the mixture was added to the sample hole on the paper chip. Detection was performed by measuring the fluorescence intensity of the paper chip using a ChemiDoc MP imaging system. Figure (a) reproduced with permission from Choi *et al*., Lab Chip 16, 611 (2016). Copyright 2016 Royal Society of Chemistry. Figure (b) reproduced with permission from Batule *et al*., Biosens. Bioelectron. **151**, 111998 (2020). Copyright 2020 Elsevier.

Ning et al. reported the development of a portable platform that leveraged isothermal RT-RPA amplification and CRISPR/Cas-mediated fluorescence detection to enable highly sensitive quantification of SARS-CoV-2 present in saliva. An optimized sample preparation procedure was developed to allow for viral lysis samples to be directly amplified and analyzed without any complex RNA isolation/purification steps. QuickExtract DNA Extraction Solution (Lucigen) was mixed with saliva samples (1:1) and heated for 5 min. The lysate was then added directly to a compact assay chip, which comprised multiple reactions wells containing an RT-RPA and CRISPR/Cas12a mixture. The reactions were then incubated at room temperature (for ≥10 min) to enable isothermal amplification and CRISPR/Cas12a-mediated fluorescence signal amplification. The assay chip was then inserted into a smartphone reader station, wherein low-angle laser illumination and a smartphone microscope were used to measure the fluorescence signal. Using this approach, the group was able to demonstrate sample-to-answer quantitative detection of SARS-CoV-2 from saliva samples in as little as 15 min.²⁹⁶

These highly integrated isothermal amplification-based nucleic acid analysis platforms show great promise for the future of widespread genetic testing, and recent developments with respect to improved specificity and multiplexing capabilities are likely to accelerate the adoption of these technologies for mainstream applications. For example, combining isothermal amplification and CRISPR/Cas-mediated detection has already been shown to enable single-nucleotide specificity for SNP detection.^268,275 Application of these principles on Ning et al.'s portable RPA and CRISPR fluorescence detection platform might enable ultra-sensitive, ultra-fast SNP detection for pharmacogenomic testing at the POC. Additionally, constant improvements in the field of microfluidics can be leveraged to improve multiplexing capabilities by enabling parallel processing. For example, Batule et al. developed a paper-chip device that contained four wax-printed reaction zones, each of which could be used to perform a separate RT-LAMP and colorimetric detection assay [Fig. 11(b)]. Viral RNA was obtained prior to the RT-LAMP reaction by using a lateral flow extraction platform that contained a binding pad to capture the RNA molecules. Using this approach, the group was able to demonstrate multiplex detection of three mosquito-borne viruses in under 1 h.²⁹⁵ While this particular work was oriented toward multiplexed pathogen detection, adaptations would allow for this architecture to be used for other genetic testing applications that might be useful toward enabling personalized medicine.

B. Commercial sample-to-answer point-of-care tests (POCTs)

Several fully integrated POC NAATs have been realized in commercial settings. Table V provides a summary of a few of these platforms. Additionally, Nguyen et al.³⁰² and Petralia and Conoci⁵⁸ have written comprehensive reviews that discuss several fully developed POC NAATs, as well as the factors that have influenced their commercialization. Most of these platforms comprise a single-use cartridge or cassette for individual sample processing and a reusable benchtop instrument for microfluidic/thermal actuation and detection.³⁰² Amplification and detection approaches vary, but PCR and real-time fluorescence tend to be the most common methods, respectively. Due to the relatively high upfront acquisition costs associated with many of the benchtop analyzers, adoption of these platforms is largely restricted to point-of-care/clinical care settings rather than at-home testing settings.³⁰³

TABLE V.

Examples of POC NAAT platforms that are commercially available or close to market.

Platform (Manufacturer)	Platform architecture	Integrated sample preparation?	Amplification method	Detection method	Total assay time	Cost	Sample assays
GeneXpert, Xpert Xpress (Cepheid)	Single-use plastic cartridge + benchtop instrument with microfluidic/thermal actuation & detection module	Yes (Magnetic bead extraction)	PCR	Real-time fluorescence	<1.5 h (Xpert), <45 min (Xpert Xpress)	€56 000/GeneXpert analyzer;³⁰⁴ $9.98/MTB RIF test cartridge³⁰⁵	Xpert MTB/RIF, Xpert Xpress SARS-CoV-2
FilmArray (bioMerieux)	Benchtop instrument + microfluidic/thermal actuation and detection module	Yes (Magnetic bead extraction)	Nested PCR	Real-time fluorescence	<1 h	$35 550/BioFire FA platform; $214.44/ME panel³⁰³	Respiratory panel, blood culture panel, GI panel
Cobas Liat (Roche)	Single-use cartridge + benchtop instrument with microfluidic/thermal actuation & detection module	Yes (Chaotropic lysis and silica-coated magnetic beads)	PCR	Real-time fluorescence	<20 min	$25 000/cobas Liat analyzer; $100/influenza test³⁰⁶	Influenza A/B & RSV, Strep A
Spartan RX (Spartan Bioscience Inc.)	Single-use cartridge + benchtop instrument with microfluidic/thermal actuation & detection module	Yes	PCR	Real-time fluorescence	<1 h	£17 000/analyzer; £9.98/CYP2C19 test¹⁴	CYP2C19 SNP genotyping assay
Accula (MESA BioTech)	Single-use cassette + reusable Accula dock	Yes	RT-OSCAR PCR	Paper-based colorimetric detection	<30 min	$63.16/SARS-CoV-2 test³⁰⁷	Influenza A/B, SARS-CoV-2
ML (Enigma)	Single-use plastic cartridge + benchtop instrument with microfluidic/thermal actuation & detection module	Yes (Magnetic bead extraction)	PCR	Real-time fluorescence	∼1.5 h.	…	Influenza A/B & RSV
ID Now (Abbott)	Single-use plastic cartridge + benchtop instrument with microfluidic/thermal actuation & detection module	Yes	NEAR (isothermal)	Real-time fluorescence	<15 min	…	Influenza A/B, Strep A, RSV, SARS-CoV-2
Cue (Cue Health)	Single-use cartridge inserted into Cue reader	Yes	LAMP (isothermal)	Electrochemical	<25 min	$249/analyzer; $65/SARS-CoV-2 test²²⁵	SARS-CoV-2

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Recently, however, Visby Medical reported the Visby Medical Sexual Health Test: a rapid, single-use nucleic acid-based test for the qualitative detection of Chlamydia trachomatis, Neisseria gonorrhoeae, and Trichomonas vaginalis.³⁰⁸ The platform comprises a fully integrated, handheld device that is equipped with all of the reagents and instrumentation necessary to automatically perform the entire assay. Printed circuit boards are used to control the temperature and the flow of the reagents/reaction throughout the assay. After the sample-containing media is inserted into the device input port, the instrument begins processing the sample using a combination of heat and chemical lysis. The processed sample is then mixed with pre-stored PCR reagents, and the genes of interest (C. trachomatis, N. gonorrhoeae, and T. vaginalis genes) are amplified with biotin-labeled PCR primers using a continuous-flow PCR approach. Specifically, the PCR reaction travels through a plastic-molded, serpentine-shaped fluidic circuit that enables highly efficient temperature modulation. After amplification, the PCR product is transported to a detection flow cell that is functionalized with oligonucleotide capture probes that hybridize to the amplified pathogenic target(s). Horseradish peroxidase-linked streptavidin then binds any biotin-labeled amplicon and surface-bound capture probe pairs, catalyzing a reaction that results in a visually readable colorimetric signal. Using this approach, Visby Medical has been able to demonstrate a highly sensitive, specific, accurate, and clinically useful assay for the detection of three pathogens related to sexually transmitted infections (STIs) in under 30 min.³⁰⁸ This device not only shows major potential for rapid detection and diagnosis of STIs in clinical settings but also for applications in the personalized medicine space should the platform be repurposed appropriately.

Along a similar vein, Cue Health recently released a portable, intuitive, and fully automated/integrated device that is suitable for use in both at-home and point-of-care settings. The platform, coined Cue, comprises a palm-sized processor/reader station that is designed for use with Cue Test kits and the Cue Health App. Automated sample preparation, loop-mediated isothermal amplification, and electrochemical detection are employed to provide results in under 20 min. While Cue is only offering SARS-CoV-2 diagnostics at the moment, a range of tests in other areas are currently under development, including women's health, respiratory health, sexual health, and cardiometabolic health.²²⁵ Increasing access to highly automated and intuitive genetic testing platforms such as the one offered by Cue Health promises to revolutionize both POC and at home testing for personalized medicine applications.

IV. FUTURE INNOVATIONS FOR POINT-OF-CARE GENETIC TESTING

A. Next-generation sequencing

While sequencing has traditionally been viewed as a prohibitively costly diagnostic technology, advancements in the field of NGS have driven the cost of whole-genome sequencing down to as low as a few thousand US dollars.^309,310 As these technologies continue to develop, whole-exome and whole-genome sequencing are projected to become increasingly relevant as regularly deployable genetic analysis tools.³⁰⁹ Further, translation of these technological advancements into platforms that can be readily deployed at the point-of-care is already under development.

Sequencing solutions such as Illumina's MiSeq (99 000 USD) and MiniSeq (495 000 USD) are being explored and deployed for genotyping, targeted gene expression profiling, and targeted gene sequencing (i.e., amplicon-based);³¹¹ however, due to high upfront acquisition costs and sustained assay complexity, these solutions are typically still restricted to use in clinical laboratories or well-equipped medical centers and, hence, are not fully suitable for near-patient/POC applications. Further optimization with respect to cost, complexity, and turnaround times of emerging POC sequencing solutions is likely necessary before widespread adoption of these platforms can be realized.

Nanopore sequencing is a third-generation sequencing (TGS) technology that offers relatively low-cost, real-time single molecule DNA and RNA sequencing without the explicit need for pre-amplification or chemical labeling. These platforms contain arrays of protein nanopores that decode nucleic acid sequences by measuring changes in the electrical current across nanopores as nucleotides corresponding to nucleic acid strands are passed through the nanopore. Oxford Nanopore Technologies (ONT), one of the leading producers of nanopore sequencing platforms, offers a handheld sequencing device (MinION) for as low as 1000 USD.³¹² Several studies have already begun to develop POC-oriented sequencing assays using ONT's MinION.^313,314 For example, Pembaur et al. developed an assay wherein they amplified nearly the entire SARS-CoV-2 genome using PCR, then employed ONT's portable MinION Mk1C sequencer to sequence the amplicons.³¹³ The workflow that they developed was a simplified, less time-consuming adaptation of traditional sequencing pipelines. Accordingly, they were able to demonstrate completion of their pipeline in about 7 h for one specimen, and about 11 h for 12 multiplexed specimens.³¹³ Moreover, their proposed approach provided timely and valuable genomic insights for improved SARS-CoV-2 variant identification and outbreak surveillance. In a separate work, Liu et al. developed a digital microfluidic device for whole-genome amplification of low-abundance bacterial DNA and then used ONT's MinION sequencer to sequence the amplicons.³¹⁴ Using this approach, they were able to sequence microbes from extremely low-abundance bacterial DNA in under 3 h.³¹⁴

While these works, and others like them,^315–317 indicate the promise of MinION as a tool for genetic analysis at the POC, many reports cite relatively protracted assay times (typically ∼6−10 h/test) as a major deterrent.³¹⁷ Fortunately, work is already being done to address this obstacle. The recent development of a LamPORE test that combined isothermal amplification (LAMP) with nanopore sequencing exhibited dramatically shorter assay times, hence indicating the value of using nanopore sequencing as a companion technology to NAATs.³¹⁸ Additionally, as the microfluidics-based sample preparation section indicated, integrated and automated microfluidics-based solutions for sequencing library preparation are also likely to play a major role in driving down the time, cost, and complexity of sequencing. These sorts of developments, which continue to be made on a regular basis, will be critical to not only increasing the viability of sequencing platforms as tools for genetic analysis at the POC but also to helping us better understand the clinical utility of these tools.

B. Smartphone-assisted POCTs

As POC testing continues to expand beyond traditional clinical settings and into the home, smartphones are likely to play a critical role as standalone and companion diagnostic technologies. Their embedded sensors (e.g., cameras, microphones) and powerful processing capabilities equip them to perform on-device detection and analysis of biological signals with reproducible precision.^319,320 Their familiar displays enable straightforward interactions with otherwise complex diagnostic platforms and can be used to provide users with instruction as they perform diagnostic tests.^244,246,321 Their impressive data transmission capabilities, including wireless fidelity (Wi-Fi), universal serial bus (USB), and Bluetooth, facilitate the communication of diagnostic results with healthcare providers and centralized laboratory facilities for further analysis.³²²

Already, these devices are being leveraged as tools in several nucleic acid-based tests. For example, Choi et al.'s previously reviewed paper-based assay device employed smartphone-based fluorescence detection to perform quantitative nucleic acid analysis.⁵³ Ning et al.'s RT-RPA and CRISPR/Cas12a-mediated POCT platform utilized a smartphone microscope for fluorescence-based nucleic acid detection.²⁹⁶ Numerous other examples of smartphone-assisted nucleic acid-based tests have been reported^123,323,324 and discussed at length in other reviews.^319,323,325 As smartphone distribution and subscriptions continue growing, these devices are well-placed to facilitate greater uptake of, and accessibility to, POC NAATs. In doing so, they also have the potential to play a critical role in improving telemedicine and telehealth outcomes by helping clinicians to gather patient-specific diagnostic data while still performing remote evaluations. Access to more holistic information in off-site or remote care settings will likely improve diagnosis, therapy selection, and clinical decision-making.^321,326,327

C. Data management and connectivity

Effective management and documentation of patient data amidst an increasingly decentralized testing landscape has proven to be quite challenging.^328,329 The development of data management and connectivity solutions that ensure that a patient's medical information is collected, communicated, and stored in compliance with ethical and legal standards is critical to the efficient expansion of POC testing technologies. Several data management systems that enable interfacing between POCT devices and centralized databases have emerged accordingly.³²⁸ However, hospitals and medical care facilities tend to utilize them for regulatory compliance and/or quality assurance purposes.^328,330 For example, some hospital systems have utilized these databases alongside computerized algorithms to detect trends and errors in a continuous fashion.³³⁰ While this is useful for the assessment of operatory competency, instrument quality, and test validity, it does not facilitate effective reporting of personalized results.

Further integration of these data management systems with electronic medical records (EMRs) is needed to ensure proper documentation and communication of POCT results on an individual basis.^8,328 The importance of such documentation and communication will only grow as at-home testing and telehealth/telemedicine continue to gain traction.⁸ Hence, tremendous effort has been placed on developing digital support systems that fulfill these demands, and many of the ensuing technologies have been reviewed accordingly.^47,328–332

V. CONCLUSIONS

The last several years have been characterized by unparalleled advancements in point-of-care molecular testing platforms, and the SARS-CoV-2 pandemic has played a critical role in normalizing use of these systems in everyday clinical practice. Several fully developed systems that are available on the commercial market (Table V) have demonstrated that NAATs can be successfully performed in a timely fashion at the point-of-care. Multiple other fully integrated, sample-to-answer NAAT platforms are regularly being reported in literature (Table IV). Innovations in microfluidics and beyond have facilitated new approaches to performing minimally instrumented sample-to-answer NAATs using both PCR and isothermal amplification technologies. Furthermore, advancements in biosensing methods have markedly improved the sensitivity, specificity and multiplexability of these emerging POCTs. These developments show great promise for future generations of point-of-care nucleic acid-based testing platforms.

Although many of the platforms discussed in this review were developed with infectious disease diagnostics in mind, we posit that with thoughtful adaptations, they can be repurposed to enable a new generation of point-of-care genetic testing technologies. For example, there have been multiple reports of platforms that combine isothermal amplification and CRISPR/Cas-mediated detection to enable highly sensitive, rapid turnaround-time detection of nucleic acids at the POC.^{144,268,281–283} However, there are few reports of these systems being applied to perform single-nucleotide discrimination for SNP identification in genetic testing.^273,278 Further development and adaptation of POC diagnostics that marry these technologies may enable a new class of ultra-fast, highly specific and sensitive near-patient genotyping tools. Additionally, integrating these tools with microfluidic architectures may facilitate added advantages, such as robust multiplexing capabilities.

Many of the other POC NAAT platforms discussed in this review can also be adapted for genetic testing purposes. For instance, there were several reports of smartphones or other portable devices being coupled with amplification technologies to enable quantitative fluorescence-based detection of nucleic acids.^149,296,301 These platforms could potentially serve as a basis for the development of POC-friendly quantitative gene expression profiling/analysis tools. Work is already being done to realize these possibilities, with companies such as Inflammatix demonstrating a fluorescence and qRT-LAMP-based assay for discrimination of bacterial and viral etiologies at the point-of-care using host gene expression analysis.²⁶

As the acute phase of the SARS-CoV-2 pandemic begins to taper off, there will be ample opportunity to leverage the recent advancements in POC testing platforms, and the corresponding espousal of these technologies, to realize broader scale integration of genetic testing in routine clinical care, acute care, at-home testing, and telehealth/telemedicine settings. While the technological landscape shepherding point-of-care genetic testing is more propitious than ever before, widespread adoption and integration of these tests for personalized medicine methods will not be determined by access to these technologies, alone. Ultimately, synergistic efforts between healthcare providers, patients, regulators, and researchers/developers will be required to fully realize the potential benefits of the personalized medicine paradigm.^8,333

ACKNOWLEDGMENTS

A. S. de Olazarra would like to acknowledge support from the Affymetrix Bio-X Stanford Interdisciplinary Graduate Fellowship.

AUTHOR DECLARATIONS

Conflict of Interest

The authors have no conflicts of interest to disclose.

Author Contributions

A. S. de Olazarra: Conceptualization (equal); Investigation (equal); Writing – original draft (equal); Writing – review & editing (equal). S. X. Wang: Supervision (equal); Writing – review & editing (equal).

DATA AVAILABILITY

Data sharing is not applicable to this article as no new data were created or analyzed in this study.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Data Availability Statement

Data sharing is not applicable to this article as no new data were created or analyzed in this study.

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PERMALINK

Advances in point-of-care genetic testing for personalized medicine applications

A S de Olazarra

S X Wang

Abstract

I. INTRODUCTION

II. POINT-OF-CARE NUCLEIC ACID AMPLIFICATION TESTS: CHALLENGES AND DEVELOPMENTS

FIG. 1.

A. Sample preparation

1. Commercial sample preparation kits

TABLE I.

2. Microfluidic and cartridge-based sample preparation

FIG. 2.

3. Paper-based sample preparation

FIG. 3.

B. Nucleic acid amplification

1. Polymerase chain reaction-based amplification

FIG. 4.

2. Isothermal amplification

TABLE II.

FIG. 5.

3. Outlook on nucleic acid amplification for genetic testing

C. Nucleic acid detection

1. Fluorescence detection

TABLE III.

FIG. 6.

2. Electrochemical detection

FIG. 7.

3. Magnetic detection

FIG. 8.

4. Paper-based detection

5. CRISPR/Cas-mediated detection

III. SAMPLE-TO-ANSWER POC NAAT DEVELOPMENT

A. Sample-to-answer NAATs in literature

1. Fully integrated PCR-based platforms

FIG. 9.

FIG. 10.

2. Fully integrated isothermal amplification-based platforms

TABLE IV.

FIG. 11.

B. Commercial sample-to-answer point-of-care tests (POCTs)

TABLE V.

IV. FUTURE INNOVATIONS FOR POINT-OF-CARE GENETIC TESTING

A. Next-generation sequencing

B. Smartphone-assisted POCTs

C. Data management and connectivity

V. CONCLUSIONS

ACKNOWLEDGMENTS

AUTHOR DECLARATIONS

Conflict of Interest

Author Contributions

DATA AVAILABILITY

REFERENCES

Associated Data

Data Availability Statement

ACTIONS

PERMALINK

RESOURCES

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