Toward Rapid and Accurate Molecular Diagnostics at Home

The global outbreaks of infectious diseases have significantly driven an imperative demand for rapid and accurate molecular diagnostics. Nucleic acid amplification tests (NAATs) feature high sensitivity and high specificity; however, the labor‐intensive sample preparation and nucleic acid amplification steps remain challenging in order to carry out rapid and precision molecular diagnostics at home. This review discusses the advances and challenges of automatic solutions of sample preparation integrated with on‐chip nucleic acid amplification for effective and accurate molecular diagnostics at home. The sample preparation methods of whole blood, urine, saliva/nasal swab, and stool on chip are examined. Then, the repurposable integrated sample preparation on a chip using various biological samples is investigated. Finally, the on‐chip NAATs that can be integrated with automated sample preparation are evaluated. The user‐friendly approaches with combined sample preparation and NAATs can be the game changers for next‐generation rapid and precision home diagnostics.


DOI: 10.1002/adma.202206525
methicillin-resistant Staphylococcus aureus (MRSA), [7] has threatened public health worldwide, particularly in low-income countries. [8] Regardless of the significant advancements in the basic research of infectious diseases and therapeutic developments, the preventive controls of these diseases face substantial challenges. It is time to reflect and find the best solutions to improve biosecurity by gaining the wisdom of preventative medicine and healthcare.
Rapid and accurate molecular diagnostics are crucial in preventing the global pandemic of infectious diseases, [9] and other diseases. [10] Nucleic acid amplification tests (NAATs) possess high specificity and sensitivity for the confirmatory analysis of infectious diseases by amplifying and identifying pathogen-originated nucleic acids. [11] However, NAAT-based molecular diagnostics undergoes a series of complicated processing steps, including the collection and transportation of clinical samples to centralized laboratories, lysis/digestion, nucleic acid binding, buffer wash, and nucleic acid elution to extract pathogenic DNA/RNA, reverse transcription of RNA, followed by polymerase chain reaction (PCR), as shown in Figure 1. These processes require trained personnel to operate the expensive apparatuses with the time-consuming procedure. Besides, manual operations, especially the multiple steps of reopening the tube and pipetting the reagent, may lead to laboratory-acquired infection by accident or the contamination of aerosol from sample to sample. [12] Thus, there is an enormous demand for user-friendly sample preparation and NAAT techniques for rapid and accurate molecular diagnostics at home. Integrated molecular diagnostic system on chip, which combines all DNA/RNA extraction and amplification steps into a single device, offers the best solution for preventive medicine and public health via home diagnostics. The system requires a fluidic device to extract DNA/RNA from the raw biological samples in an automated manner and nucleic acid (NA) amplification unit. Regardless of the continuous progress of sample preparations toward point-of-care (POC) molecular diagnostics, [13] the reported methods show limited capabilities as fully integrated sample preparation techniques for NA-based molecular diagnostics from raw biological samples remain challenging.
In this review, we evaluate the advancements in integrated sample preparations with NA amplifications reported in literature research for rapid and accurate home diagnostics. Specifically, biological samples such as blood, urine, nasal swab, saliva, and stool are highlighted as the most widely used clinical

Introduction
The outbreak of infectious diseases, including coronavirus disease 2019 (COVID-19), [1] malaria, [2] dengue, [3] tuberculosis, [4] monkeypox, [5] acquired immunodeficiency syndrome, [6] and samples, which contain a series of disease-related endogenous genes and foreign microorganisms as sources to extract NAs for molecular diagnostics. We also investigate the repurposable integrated sample preparation chips with various biological samples to encourage innovative solutions and realize rapid and accurate molecular diagnostics at home. Finally, we evaluate the on-chip NA amplification methods, which can be integrated with automated sample preparation for molecular diagnostics at home. We expect this review will provide a framework of the recent advancements in integrated sample preparation and nucleic acid amplification on-chip, and present new insights into designing fully integrated molecular diagnostic systems for home diagnostics.

Integrated Whole Blood Sample Preparations
Blood sample contains a variety of disease-related biomarkers with higher concentrations. Thus, it is employed as one type of primary biofluids, liquid biopsy for clinical diagnostics. Blood samples are usually collected in invasive approaches, such as venipuncture, to acquire a large volume and finger prick to obtain a small volume (Figure 2a). One of the conventional materials for blood collection is the FTA card which is pretreated with a proprietary mixture of chemicals to collect NAs, retard bacterial growth, inhibit nuclease activity, and release NAs from lysed cells within the card's matrix, [14] which is favorable to the transportation.
Low-cost integrated microfluidic sample preparation devices are excellent candidates for POC systems. For example, integrated microcapillary loop-mediated isothermal amplification (icLAMP) was established to extract DNA from 200 nL of whole blood (Figure 2b). [15] The reagent droplets were first stored in the glass microcapillary, followed by the insertion of the FTA membrane. For nucleic acid extraction, blood samples were collected using a finger-pricking device and immersed with the FTA membrane in the microcapillary for chemical lysis, protein digestion, and nucleic acid purification. End users conducted the process with a piston by driving the preloaded reagent droplets across the FTA membrane in one direction. The optimized structure of the microcapillary ensured the reliability and robustness of the preloaded reagents suitable for storage and transportation. Pure water segments were injected to avoid cross-contamination by separating adjacent reagent droplets Step 1: sample collection and transportation. Biological specimen types are collected by various collection procedures, such as a nasopharyngeal swab, nasal swab, and oropharyngeal swab, and transported to the centralized laboratories as soon as possible after collection. Of particular interest for molecular epidemiology analysis are those by which the samples can be collected most conveniently and effectively at the lowest cost.
Step 2: heat deactivation. The viruses are deactivated by incubating the clinical sample at an elevated temperature to destabilize viral proteins and assemblies, rendering them incapable of infection during the downstream manual operation.
Step 3: lysis/digestion. Lysis buffer contains a high concentration of chaotropic salts and detergents. Chaotropes disrupt hydrogen interactions and lead to the destabilization of proteins and nucleases. Organic amphipathic detergents break up the cell membrane structure by separating membrane proteins with the hydrophobic part of detergents from membranes. Proteases can be included in the lysis buffer to digest the contaminating proteins and degrade the nucleases. The lysis buffer shows higher efficiency at elevated temperatures.
Step 4: binding. The lysate is transported to a spin column. The chaotropic salts provide favorable conditions for nucleic acid transfer to the silica membrane of the spin column by creating a hydrophobic environment to break down the association between NAs and water. Meanwhile, chaotropes provide positively charged cations to saturate the silica membrane, thus improving the absorption of negatively charged phosphate backbones of NAs under hydrophobic conditions. Since NAs are insoluble in ethanol, the addition of ethanol will enhance nucleic acid precipitation to the silica membrane.
Step 5: wash the washing buffer, with ethanol as the dominant component, removes impurities such as protein polysaccharides residues. Multiple washing steps are usually conducted to thoroughly remove residual contaminations and buffer solutes. Residual ethanol should be avoided after the washing step since it may prevent the sequent elution of NAs and inhibit nucleic acid amplification.
Step 6: elution. The elution buffer or pure water at pH 8-9 is typically used to release the NAs from the silica membrane to the bottom of the centrifuge tube during centrifugation.
Step 7: reverse transcription-polymerase chain reaction (RT-PCR). The purified RNAs are reverse-transcribed to complementary DNA (cDNA), followed by cDNA amplification and fluorescence detection. and reducing aerosol evaporation during thermal reactions. [20] Apart from the glass microcapillaries, plastic micropipette tips were also used to extract NAs from whole blood with a similar procedure. [21] Microcapillary-and micropipette-tip-based systems feature good merits, such as low cost, easy operation, disposability, and reduced sample volume. Another benefit is the availability of parallelization for multiplexed assays or high throughput detection. [22] Silica-coated magnetic beads are also excellent candidates for extracting NAs in automatic apparatus. Similar to the silica-membrane-based spin column, silica beads have been used for solid-phase nucleic acid extraction based on reversibly absorbing and desorption of NAs at specific conditions of buffers. [23] By replacing the core material of silica beads with magnetic ferrites, the silica-coated magnetic beads can realize buffer exchange with the actuation of external magnetic fields, thus eliminating the need for centrifugation. For example, magnetic beads were loaded onto a lab-on-a-disk (LOAD) platform, forming a stand-alone and sample-to-answer molecular diagnostic system (AnyMDx) for malaria screening from 10 µL of blood collected by finger prick (Figure 2c). [16] Unlike the popular centrifugal-force-based LOAD platforms, [24] the palm-sized AnyMDx utilized noncentrifugal magnetic interaction methods for nucleic acid extraction. The multichambers were preloaded with alternating reagent buffers and oil phases to form "virtual walls" between each chamber by the modified surface tension and the structural pinning effect of teeth-shaped passive valves. Simultaneously, magnetic beads were pulled smoothly through each chamber by programmable actuation of the magnetic beads against the relative stationary reagent solution using auxiliary electrical equipment. Antibody-functionalized immunomagnetic beads can also be used to extract DNA/RNA by binding to the nucleoproteins in a native complex with the pathogenic DNA/RNA. [25] The design of a self-powered microfluidic device is a practical way to facilitate fluid flow passively without the necessity of auxiliary electrical equipment for sample preparations. Conventional self-powered flow actuation methods depend on the capillary force, [26] evaporation, [27] gravity-driven flow, [28] or hand-powered flow. [29] Hand-powered centrifugal microfluidic platforms have been demonstrated to realize blood separation, reagent mixing, and pathogen concentration in electricity-free systems inspired by whirligig toys, [30] spinning top, [31] and fidget spinner, [32] respectively. Another promising approach is to employ the gas-permeable poly(dimethylsiloxane) (PDMS) as a vacuum battery to control the fluid flow. [33] The PDMS device is positioned in a vacuum container to evacuate the air molecules from the bulk material, thus forming a vacuum battery. After the vacuum PDMS bulk is exposed to atmospheric pressure, air molecules diffuse from the ambient into the vacuum PDMS bulk to actuate the fluid to flow in the microfluidic channels. [34] Researchers reported a self-powered integrated microfluidic point-of-care low-cost enabling (SIMPLE) chip for the molecular diagnostics of S. aureus (MRSA) and HIV infection from 10 µL of blood samples (Figure 2d). [17] A PDMS reservoir was adopted as the vacuum battery to allow the on-chip integration of sample preparations, including reagent prepatterning, plasma separation, and sample compartmentalization, with minimal manual operation in 10 min. The lung-like vacuum structure allowed efficient air diffusion from microchannels to the vacuum battery via gas-permeable PDMS walls, facilitating the plasma separation and sample compartmentalization by the designed microcliff gap. The flow velocity could be optimized by tuning the PDMS reservoir's negative pressure and varying the PDMS membrane's diffusion resistance.
Point-of-care kit for the entire test (POCKET) platform was reported for analyzing multiple DNA types from peripheral venous blood without auxiliary electrical equipment ( Figure 2e). [18] The platform comprises an integrated device for sample preparation and DNA amplification and a foldable box for colorimetric readout by cellphone. The sample preparation unit was composed of a basket module for DNA binding to the extraction disk at the bottom, a rotary valve for controlling the connection of the microfluidic amplification unit, and a block module containing a wash chamber for DNA purification and a reaction chamber for DNA amplification. DNA was preconcentrated using an extraction disk at the basket bottom, followed by the wash buffer purification. Reagents were preloaded into a tandem tube and separated by an air gap to streamline the reagent delivery by a syringe. A cellphone application was programmed to enable the intense operations of the central processing unit, which leads to a temperature rise of the cellphone as a heater for isothermal amplification between 37 and 42 °C.
Paper folding, or origami, is a novel platform for integrating sample preparations onto paper-based devices. The origami method allows the fabrication of the entire device on a single sheet of flat paper in one step. It proceeds with the sample preparations by folding the layers in succession. [35] The simple fabrication processes using laser cutting and hot wax printing enable the mass production and assembly of origami devices at a low cost. An origami-based microfluidic device was developed as a simple visualization system to enable multiplexed assays for DNA diagnostics of malaria from whole blood finger-prick samples (Figure 2f). [36] This device can perform vertical-flow extraction of DNA, isothermal amplification, and lateral-flow detection of DNA amplicons. For the deployment in rural communities, colorimetric visualization [19] and dry storage of reagents [35a] are readily incorporated with origami-based sample preparations. Similarly, sample preparations can be integrated into a sliding-strip device for POC tests in resource-limited settings. [37] This handheld and disposable device adopted a central patterned paper strip to slide in and out of a fluidic path, allowing minimal pipetting for loading blood samples and reagents.

Integrated Urine Sample Preparations
Although urine sample is primarily associated with home pregnancy tests, it is also an excellent source for the diagnostics of specific diseases like urinary tract infections. [38] Meanwhile, urine sample has been proven to reflect pathophysiology and provide a biological understanding of human-organ-related dysfunction by analyzing transrenal DNA fragments, [39] or tracking the urinary exosomal genes. [40] The middle part of the firstmorning urine sample is typically collected in a sterile cup and transferred to a storage bag or vial (Figure 3a). Since urine samples can be collected in a large volume, it maximizes the biomarker availability and reduces the possibility of false-negative results.
A magnetic-bead-in-a-tube method was demonstrated to extract DNA from human urine for tuberculosis diagnostics (Figure 3b). [41] 1 mL of urine spiked with a short tuberculosis DNA sequence was first mixed within a pipette bulb containing lyophilized reagents and silica-coated magnetic beads. Lyophilized reagents are suitable for long-term storage on a chip and long-distance transportation, and the liquid sample can dissolve during the bioassays. Target DNA sequences bound to the surface of magnetic beads, followed by the transfer from the pipette bulb to the plastic tube. The reagent solutions were preloaded in the plastic tube and separated by air gaps or mineral oil with surface tension. A magnet was pulled along the plastic tube to wash and release the target DNA. In another report, urine samples were injected into a microfluidic channel where the spiked bacteria were captured and concentrated by ion-exchange magnetic beads (Figure 3c). [42] In this assay, a magnet was fixed underneath the valveless channel to catch the magnetic-bead-bacteria complex, followed by thermal lysis of bacteria and DNA extraction within 10 min. The long serpentine channel was fabricated as a hydraulic resistance to control the flow rate.
GeneXpert system is a commercialized pioneering product for integrated molecular diagnostics. The analyzer automatically performs sample preparations in a disposable cartridge, including flow control, filter capture, and ultrasonic lysis. [46] However, there is only one reaction chamber in the cartridge, thus limiting its ability for multiplex diagnostics. Besides, the costly and complex system hinders its widespread application for personalized medicine. To solve these issues, a fully integrated microfluidic system with multiple detection chambers was demonstrated for the multiplex assay of pathogens in 200 µL of urine samples within 100 min (Figure 3d). [43] This system employed a rotary valve and a syringe plunger to automatically actuate the urine sample to flow in a designed cartridge containing six reagent chambers for nucleic acid extraction using silica-coated magnetic beads and eight amplification chambers for multiplex detection of urinary tract infection.
Since biomarkers of interest in urine are often at low concentrations, the enrichment of biomarkers from large volumes is usually required to avoid "false negatives" during urinalysis. Low-cost amine-functionalized diatomaceous earth was used to capture negatively charged pathogens from 10 mL of urine via electrostatic adsorption in a Teflon syringe filter ( Figure 3e). [44] The large-volume sample capture efficiency was enhanced with high sensitivity. This low-cost, hand-powered, self-contained sample preparation system can extract highquality NAs from large-volume samples for downstream diagnostics.
Simplicity is one of the principal characteristics of developing integrated sample preparations. It can be accomplished by optimizing sample preparation systems, including adopting high-efficient materials and structures, eliminating unnecessary steps, and merging multiple steps. For example, a nanophotonic light-driven integrated cell lysis and PCR on a chip with gravity-driven cell enrichment health technology (LIGHT) was present for rapid and precise identification of Escherichia coli from urine to diagnose urinary tract infections ( Figure 3f). [45] The unique character of the simplified LIGHT was the merging of three steps: 1) pathogen enrichment, 2) thermal lysis, and 3) photonic thermal cycling on a nanoplasmonic porous membrane. The sample gravity and the absorbent pad's capillary force drive the urine sample to flow automatically through the porous membrane filter, where E. coli was isolated and enriched. After the enrichment, E. coli was thermally lysed, and the released NAs were amplified through the ultrafast photothermal conversion of the nanoplasmonic membrane.

Integrated Saliva/Nasal Sample Preparations
Since the human oral and nasal cavity harbors abundant disease-related microorganisms, saliva and nasal tests are usually used to diagnose diseases, especially infectious respiratory diseases. Saliva is also an alternative to blood for disease diagnostics since it covers one-third of the biomolecule types found in a blood sample. [47] Since saliva is collected by either drool pooling or swab/sponge in a noninvasive way, and the nasal sample is collected by swab in a minimum invasive way (Figure 4a), saliva/nasal samples possess the promising potential as two typical sources for the home diagnostics.
Clinical studies reveal that saliva samples from patients with a high load of plasma HIV RNA contain detectable HIV RNA. [53] The result indicates that saliva can be used as an alternative to blood for molecular diagnostics in the middle stage. An integrated microfluidic cartridge was developed to extract HIV RNA from saliva samples within 10 min (Figure 4b). [48] The polycarbonate cartridge consisted of pouches for reagent storage, a group of diaphragm valves, a mixing chamber, a silica membrane embedded in a nucleic acid extraction chamber, an amplification chamber stored with dried reagents, an amplicon dilution trap, a waste chamber, and a lateral flow strip. Step 1: DNA is captured by the silica-coated magnetic beads in the pipette bulb and transferred to the plastic tube.
Step 2: DNA is extracted by pulling a magnet along the plastic tube for washing, precipitation, and elution. b) Reproduced with permission. [41] Copyright 2013, Public Library of Science. c) Magnetic-bead-on-chip-based method. Top view (left) and cross-section view (right) of the microfluidic chip. Reproduced with permission. [42] Copyright 2016, Springer Science+Business Media. d) Self-contained microfluidic cassette. The cassette is composed of a Luer syringe to drive liquid and a rotary valve to regulate the flow direction for DNA extraction. Reproduced with permission. [43] Copyright 2020, Royal Society of Chemistry. e) Handheld syringe filter method based on amine-functionalized diatomaceous earth in conjunction with homo-bifunctional imidoesters. Reproduced with permission. [44] Copyright 2019, Springer Nature. f) Integrated cell lysis and PCR on a chip with gravity-driven cell enrichment health technology (LIGHT) for rapid precision detection of pathogens. The nanoplasmonic porous membrane is used for pathogen enrichment, photothermal lysis, and nucleic acid amplification. Reproduced with permission. [45] Copyright 2019, American Chemical Society.
Multiplex detection with the capability of discriminating different bacterial species or infecting subtypes is essential to carry out accurate diagnostics and the best treatment rapidly. Conventional multiplex molecular diagnostics involves multiple groups of chemicals to amplify multiple targets in a simple single step, leading to the complexity of the assay development and a decrease in the sensitivity of each target. The design of multiple amplification zones on a device can avoid these issues and amplify DNA/RNA. For example, a multiplex autonomous disposable nucleic acid amplification test (MAD NAAT) was designed using paper networks to realize NA extraction and multiplex diagnostics of MRSA bacteria from a nasal swab in less than 1 h (Figure 4c). [49] The nasal sample was collected and incubated in the sample chamber for bacterial lysis and enzyme deactivation. An automated valve then transferred the lysate to a 2D paper network, where it was split and transported to two independent amplification zones. After the isothermal amplification, a second valve released the PCR amplicons to the lateral flow detection zone. Both valves were made of wax, and heaters melted the wax to allow fluid transportation. The dried reagents were stored in the device and rehydrated by the stored buffer.
Microfluidic paper-based technology comprises a broad branch of sample preparation devices for nucleic acid extraction from saliva samples. A recent report demonstrated a self-contained paper-based device for fully integrated nucleic acid extraction from 30 µL of saliva samples within 2 min (Figure 4d). [50] It integrated all necessary components to facilitate reagent storage, cell lysis, and nucleic acid extraction into one paper machine. For DNA capture, paper-based valves and channels with different lengths autonomously directed the reagents and samples to the positively charged glass fiber paper. The lysis and washing buffers were separately stored in two reservoirs using sponges to retain them and sealing rubbers to avoid evaporation. On pressing the button, the buffers were squeezed out from the sponges and flowed to the paper-based channels, enabling the nucleic acid extraction processes. An absorbent pad was embedded under the paper strip for waste absorption.
A series of processing steps can be accomplished within one chamber to simplify the sample preparations. Researchers have designed a multifunctional reactor that combines solidphase nucleic acid enrichment and extraction, reagent storage, and temperature-triggered reagent release for human papillomavirus-16 (HPV-16) DNA detection (Figure 4e). [51] A silica membrane was embedded in the reactor's inlet to extract NAs from saliva samples. All lyophilized reagents were encapsulated : lysis valve to 2D paper network connector; 6: 2D paper network tray; 7: sample delivery and IAC pad; 8: amplification and detection dry reagent pad; 9: amplification valve; 10: 2D paper network cover; 11: salt pad; 12: lateral flow strip; 13: waste pad. Reproduced with permission. [49] Copyright 2016, Royal Society of Chemistry. d) Paper-based DNA extraction device comprising a top cover, a reservoir module, and a paper-based module supported by the substrate. Reproduced with permission. [52] Copyright 2019, Springer Nature.
with wax in the reaction chamber. The wax melted and floated up when the chip was heated to its operating temperature, allowing the reagent hydration for nucleic acid amplification. To further simplify the operation, the extracted DNAs were directly amplified in the reactor by eliminating the separate step of DNA elution.
The logical design of simplicity also includes the reliable storage of all the reagents in the self-contained format and the automatic release of the stored reagents in sequence without requiring multiple labor-intense steps. A simplified device was developed for nucleic acid extraction from saliva by passing a polymer-coated swab through a series of membranes between multiple chambers preloaded with reagents ( Figure 4f). [52] The device comprised a group of 3D-printed chambers to store reaction buffers and elastic gel-type membranes to separate each chamber. The polymer interacted as a pH-sensitive material to capture and release DNA with each buffer inside the chambers when the swab passes through the chambers, yielding an isolation efficiency comparable with conventional silica-membrane-based and magnetic-bead-based methods. Apart from the chambers, blisters [54] and syringes [55] are also good choices for liquid reagent storage on the chip, and pressure can be applied to release the liquid reagent from the blisters and syringes for further processing.

Integrated Stool Sample Preparations
Since the gastrointestinal tract contains a large number of microorganisms, stool sample is an excellent source to identify a variety of pathogens relevant to lower digestive tract dysfunction. Conventionally, a specific amount of fresh stool is collected using a small spatula and transferred into a sterile container (Figure 5a). Different from the blood, urine, and saliva/nasal samples, the stool sample shows large variations in the configuration, ranging from liquid consistency with no solid pieces to separate hard lumps. Therefore, a thorough homogenization and proper centrifugation of stool sample are required to release NAs before nucleic acid extraction. [56] The strong negative polarity of cellulose/nitrocellulose fiber is adopted to extract NAs from potential amplification inhibitors. Due to the electrostatic repulsion between negatively charged cellulose/nitrocellulose and strongly negatively charged nucleic acids, NAs migrate at a fast rate from the sample loading area to the other side of the paper along with the capillary-force-induced bulk liquid flow. By contrast, the positively charged or weakly negatively charged proteins and cell debris are slowed down with the increased retention time. Based on this mechanism, a cellulose/nitrocellulose  [57] Copyright 2014, Royal Society of Chemistry. c) Glass-fiber-paper-based method for DNA/RNA extraction. Reproduced with permission. [58] Copyright 2018, Elsevier. d) Immiscible filtration assisted by surface tension. 1: Sample loading and cell lysis; 2: mixing of superparamagnetic particles with sample for DNA binding; 3: transfer of superparamagnetic particles through the immiscible phase for washing; 4: elution of DNA from the superparamagnetic particles followed by off-chip analysis. Reproduced with permission. [59a] Copyright 2016, Royal Society of Chemistry. e) Fully integrated microfluidic cartridge. The stool sample is homogenized by heating and magnetic beating and filtered to remove impurities at the sample chamber, followed by the lysis, purification, and elution to the elution chamber. The reagents are moved to the designated chamber by applying air pressure. Reproduced with permission. [60] Copyright 2017, Springer Nature. f) Sharp-edge-based acoustofluidic device. The stool samples are homogenized by the strong microvortex streaming and filtered by an array of parallel microchannels. Reproduced with permission. [61] Copyright 2019, Royal Society of Chemistry. paper strip was used for nucleic-acid-based molecular diagnostics of human Salmonella typhimurium infections from stool samples within 5 min (Figure 5b). [57] The paper strip was comprised of the sample loading region, elution region, and an absorbent pad. The sample loading area was first immersed into the aliquots of 10% diluted stool samples, followed by the addition of lysis buffer and elution buffer for nucleic acid extraction. Apart from the cellulose/nitrocellulose paper, glass fiber paper also can extract NAs from the stool. The glass fiber matrix captures NAs in high-salt and low-pH buffer (Figure 5c). [58] Meanwhile, the potential amplification inhibitors in the lysed sample were eluted along with the washing buffer by capillary forces automatically. Assembly of a group of paper materials with distinguishing binding affinity to NAs was also used for nucleic acid transportation, concentration, and purification by lateral flow separation. [62] Immiscible filtration assisted by surface tension (IFAST) was developed to extract NAs from 400 µL liquid stool samples with minimal setup and materials (Figure 5d). [59] IFAST chip comprised a large-volume sample chamber and several washing and elution chambers, which enabled the enrichment of NAs from crude biological samples. Reagent buffers were discretely preloaded in chambers based on the surface tension between alternating aqueous and oil phases. Solid chaotropic salt and dried magnetic particles were preloaded on the chip and reconstituted upon sample loading into the sample chamber. DNA extraction was achieved based on the nucleic acid binding to magnetic particles and the handheld magnet drawing from the washing chamber to the elution chamber, where NAs were released into aqueous media. An oil-immersed lossless total analysis system was further developed for RNA extraction and detection based on the integration of underoil droplet microfluidic technology, IFAST-like solid-phase analyte extraction method, and isothermal amplification with colorimetric readout. [63] Currently, most research on stool sample preparations has been focusing on detecting infectious agents that lead to diarrhea, which results in the liquid phase of stool samples that is favorable to transfer into the microfluidic chip. Solid stool samples receive less attention for integrated sample preparations due to the necessity of off-chip homogenization and liquefaction. To address this issue, an integrated microfluidic cartridge was reported to extract NAs from solid stools to diagnose pathogen-induced gastroenteritis (Figure 5e). [60] The homogenization of stool samples was accomplished by heating the pretreatment chamber and beating the preloaded magnet with the electromagnetic field. In a recent report, researchers fabricated a sharp-edge-based acoustofluidic device for onchip homogenization, liquefaction, and purification of stool samples in the microfluidic channel (Figure 5f). [61] The robust microvortex streaming force generated by oscillated sharpedge structures actively homogenized stool samples and phosphate buffer without destroying pathogens. Meanwhile, an array of parallel 100 µm wide microchannels removed large stool debris from homogenized stool samples. This acoustofluidic chip provides a feasible pathway to adopting microfluidics in a continuous manner for integrated on-chip preparations of stool samples.

Repurposable Integrated On-Chip Sample Preparation with Various Biological Samples
Apart from the blood, urine, saliva/nasal swab, and stool samples, various biological samples have been adopted by researchers to demonstrate the advanced design of integrated sample preparation chips and their potential applications in practical nucleic-acid-based molecular diagnostics. By repurposing and optimizing these integrated sample preparation techniques, more innovative solutions will emerge for rapid and accurate molecular diagnostics at home.
Silica bead immobilization within the microfluidic channel is a novel clog-free method for nucleic acid extraction in a microfluidic platform. After the plain silica bead solution is driven into the PDMS microfluidic channel and left to dry at room temperature, the silica beads can be immobilized via bonding to the PDMS walls using UV ozone treatment. Based on silica bead immobilization, a monolithic microfluidic platform was demonstrated to realize transfer-messenger RNA purification, nucleic-acid-sequence-based amplification (NASBA), and fluorescence detection of pathogens in crude lysate within 30 min (Figure 6a). [64] The microfluidic device comprised a RNA purification chamber packed with silica beads and a real-time NASBA chamber. The sample mixture was driven to the RNA purification chamber, followed by the isopropyl alcohol washing to remove the unbound substances. The waste was drained through the waste output. Then, the RNA was eluted with the solution containing primers from the RNA purification chamber to the NASBA chamber. Afterward, NASBA enzymes were introduced through the NASBA port and mixed with reagents by applying manual pressure pulses on the PDMS surface over the center of the NASBA chamber. Finally, the mixture was heated up to initiate the amplification, and the quantitative information was derived with a fluorescence microscope.
To realize the miniaturization of POC diagnostic devices, a stand-alone self-powered integrated microfluidic blood analysis system (SIMBAS) was proposed to integrate flow propulsion, plasma separation from whole blood, multiple assays, and sample volume metering into a disposable microfluidic platform for blood detection with the volume of 5 µL in 10 min (Figure 6b). [65] The integrated gas-permeable PDMS suction chambers provided low pressure to actuate the fluid flow in the dead-end channel. The self-powered plasma separation region of SIMBAS comprised a round filter trench to filter out the erythrocytes and leukocytes from whole blood via sedimentation. This structure design reduced the fabrication complexity and avoided clogging. The detection region consisted of a sample channel with a group of immobilized specific-capture molecules for multiplex detection. Because SIMBAS was a closed system with no outlets, the total fluid volume could be controlled by controlling the suction chamber volume. Later, the SIMBAS was further developed as a microfluidic biomolecular amplification reader (µBAR) to detect the HIV-1 integrase gene using the LAMP assay (Figure 6c). [66] The µBAR controlled the assay temperature through an integrated indium tin oxide substrate as the resistive heater and recorded real-time fluorescence signals from an array of individual reaction chambers using light-emitting diodes (LEDs) and phototransistors. It featured blue excitation LEDs at the side of the PDMS chip, and the light was coupled to the PDMS chip by optical waveguides.
The logical design of simplicity also includes the reliable storage of all the reagents in the self-contained format and automatic release of the stored reagents in sequence without the necessity of labor-intense steps. Reservoirs and blisters can be employed to store liquid reagent on the chip, and pressure can be applied to release the liquid reagent from the reservoirs or blisters for further processing. A valve-assisted microfluidic device was reported to realize the automation of fluid control for reagent delivery (Figure 6d). [67] The on-chip valves contain six individual cylinders, and each cylinder has a punched hole whose position gradually ascends to neighboring cylinders. A peristaltic pump drives the reagents to flow through the reaction area when the microfluidic chip is located in an automated machine. The open and close status of the valves is controlled by the downward movement of the cylinders so that the reagents in the different reservoirs can flow through the reaction area sequentially. The liquid reagents can also be preloaded into the tube or the chamber and separated by the air or oil, [16a,18,41,46] followed by the sequential release to the reaction area.
From an economic viewpoint, it is highly recommended to develop a universal platform capable of carrying out sample preparations with different types of biological samples and multiplex detection of biomarkers on a single platform. Such a universal platform facilitates researchers to apply their specific design to this platform without requiring adjustments. An open-platform system was developed for bioassays from different types of biological samples on one instrument with a universal cartridge for the automated sample-in answer-out diagnostics (Figure 6e). [69] This universal diagnostic platform allows both the Mycobacterium tuberculosis detection from sputum sample with a molecular diagnostic cartridge and the alanine aminotransferase assay from whole blood with a clinical chemistry cartridge. Take the molecular diagnostic cartridge as an example, a sputum vessel was used to collect 1-6 mL sputum samples from patients. Then, the user snaps the sputum vessel onto the cartridge and inserts it into the instrument for automatic processing with an assay time of 90 min. Specifically, the sputum is digested by injecting a soluble reagent comprising a solution in the syringe and a lyophilized reagent on the cartridge, followed by homogenization using an integrated mixing paddle. Then, the M. tuberculosis cells are enriched on Figure 6. Repurposable integrated on-chip sample preparation with various biological samples. a) Integrated microfluidic RNA purification chamber and real-time NASBA device. 1: Sample load; 2: buffer wash; 3: RNA elution; 4: denaturation of secondary and tertiary RNA structures; 5: NASBA enzyme load; 6: amplification process. Reproduced with permission. [64] Copyright 2008, Royal Society of Chemistry. b) Stand-alone self-powered integrated microfluidic blood analysis system. The blood is driven into the microchannel by degas-driven flow, and the plasma is separated by trapping red and white blood cells in an integral trench structure. Reproduced with permission. [65] Copyright 2011, Royal Society of Chemistry. c) Microfluidic biomolecular amplification reader (µBAR), including the microfluidic cartridge and the monitor. Reproduced with permission. [66] Copyright 2013, Public Library of Science. d) On-chip valve-assisted microfluidic chip demonstrating the automation of reagent control. A mechanical slider controls the downward movement of the on-chip valves to open and close the channels, and the reagents flow through the chamber of interest in sequence. Reproduced with permission. [67a] Copyright 2018, Royal Society of Chemistry. e) A universal microfluidic cartridge for M. tuberculosis (MTB) diagnostics from sputum sample. Reproduced with permission. [68] Copyright 2017, Humana Press. f) i-STAT microfluidic cartridge comprising thin-film electrode sensors on silicon chips to detect targets. The air bladder is depressed by the motor in the analyzer to push all fluids along defined fluidic paths within the cartridge. Reproduced from cliawaived.com with permission from CLIAwaived. a size-exclusion membrane, washed using the blisters containing buffers, and lysed by ultrasound. The elute is driven to PCR chambers containing lyophilized reagents for nucleic acid amplification and fluorescence detection. The i-STAT device is one of the most successful on-chip diagnostic products for multiplex assays of blood chemistries, electrolytes, hematocrit, and hemoglobin in ≈2 min with a sample volume of 95 µL (Figure 6f). [70] The disposable plastic cartridge of i-STAT contains a silicon microchip with an array of thin-film electrodes functionalized with specific ionophores or enzymes for electrochemical detection. It also includes a buffered aqueous calibrant solution with known concentrations of analytes and preservatives. A dispensing tip is used to streamline the bloodtransfer process to the cartridge, and the end of the dispensing tip aligns with the cartridge port to assist the user in dispensing the desired amount of blood. A motor in the analyzer depresses an air bladder in the cartridge to force air to actuate the calibrant solution and blood sample along the defined microfluidic channels to the sensing electrodes. We summarize the reviewed technologies and present the comparative analysis in Table 1 to illustrate better the pioneering advances of integrated sample preparation strategies for molecular diagnostics.

On-Chip Nucleic Acid Amplification
On-chip nucleic acid amplification approaches offer many advantages over conventional bench-top nucleic acid amplification technologies, including portability and automation, less consumption of biological samples and reagents, better single-molecule detection performance, and shortened analysis time. Although amplification-free approaches with novel assays like clustered regularly interspaced short palindromic repeats (CRISPR)-Cas13a are developed for detecting DNA or RNA, [78] their sensitivity is generally not as high as the nucleic acid amplification technologies. [79] We have discussed several on-chip nucleic acid amplification technologies integrated with automatic sample preparation in the above sections. In this section, we will evaluate repurposable on-chip nucleic acid amplification approaches, which offer a new opportunity for rapid and accurate diagnostics at home. We summarize the reviewed pioneering techniques for on-chip nucleic acid amplification and show the comparative analysis in Table 2.
On-chip nucleic acid amplification generally utilizes the localized heating of a small volume of reagents in the microfluidic chamber by the heating elements. The heating block is widely used as the contact-heating element for on-chip nucleic acid amplification. [84] Three thermostated metallic blocks were employed for continuous-flow microfluidic PCR chips based on the Joule heating effect. [85] The sample repeatedly flowed through three temperature regions in a 2D microfluidic channel for the PCR. The 3D structure comprising stationary cylindrical heaters surrounded by a spiral microchannel is an alternative for continuous-flow microfluidic PCR. Compared with heating blocks, the on-chip thin film resistive heater shows outstanding performances in both low power assumption and on-chip integration for contact-heating-based nucleic acid amplification. The chemical or physical process, such as exothermic phase change crystallization of supersaturated sodium acetate of heat pack, [17] can also be used as the heating element for nucleic acid amplification.
Despite the advantageous merits of on-chip nucleic acid amplification technologies, there are still several bottlenecks in designing an efficient on-chip nucleic acid amplification device. Due to the high surface-to-volume ratio of the microfluidic chamber, the PCR efficiency can be reduced by the nonspecific binding between the reagents and the chip surface. Therefore, a proper surface treatment is required to minimize the nonspecific binding during the on-chip nucleic acid amplification. The surface treatment can be achieved by precoating the chip surface with a passivation layer using materials such as bull serum albumin, poly(ethylene glycol), hydrogen silsesquioxane, and silicon dioxide. Meanwhile, air trapped in the reaction chamber can expand at increased temperatures, thus leading to bubble generation. Reagent evaporation can also lead to bubble generation at high temperatures, especially when the reaction chambers are made of gas-permeable materials. To avoid bubble generation, the vacuum channels were patterned at the periphery of the gas-permeable fluidic channels by rollto-roll thermal imprinting (Figure 7a). [71] The injected solution fills the dead-end reaction chambers by degas-driven flow without bubble generation. The Al-coated paper substrate of the chip can enhance the fluorescence signal by reflecting the fluorescence signal and excitation light. In another design, a thin impermeable polyethylene (PE) was introduced as the barrier layer of the microfluidic PCR chip to avoid bubble generation by inhibiting mass transport along a vertical direction and minimizing evaporation (Figure 7b). [72] The bubble-free microfluidic chip completed the 35 cycles of 20 nL PCR mixture by a high-powered Peltier heater and realized the detection of the cMET gene in less than 3 min.
Apart from organic materials such as elastomeric PDMS and thermoplastic polymethyl methacrylate (PMMA), inorganic silicon is another promising material to fabricate an all-in-one nucleic acid amplification system with the merit of full integration of different functional modules onto a miniaturized silicon chip by the industry-standard semiconductor manufacturing process. A disposable NAAT module was developed using an integrated complementary metal-oxidesemiconductor (CMOS) biochip to identify and quantify multiple distinct nucleic acid sequences within 2 h (Figure 7c). [73] The CMOS biochip comprises a 40 µL flow-through chamber to receive the nucleic acid extract, an on-chip thin film resistive heater, several temperature sensors to enable the multiplex PCR and solid-phase melting curve analysis, and a solid-phase DNA microarray containing oligonucleotide sequences complementary to specific regions of PCR amplicons. A photodiode array was also implemented in the CMOS die to optically detect DNA hybridization events by the inverse fluorescence transduction method at every addressable pixel. Researchers also developed a cleanroom-free and low-cost method to fabricate an all-in-one micro-quantitative-PCR chip comprising an electrical heater for nucleic acid amplification, a thermistor for temperature measurement, and an electrochemical sensor for target NA detection. [86] In another report, a silicon-based microfluidic chip comprising an etched 1.3 µL meandering microreactor, integrated aluminum heaters, thermal insulation trenches, and resistive temperature detector was able to perform the on-chip a) Recovery rate/capture efficiency is generally evaluated by the ratio of the amount of NAs or NA-containing analytes after the sample preparation to the amount of NAs or NA-containing analytes before the sample preparation. The definition of recovery rate/capture efficiency may vary slightly regarding different reported technologies. For example, in ref. [41], the DNA recovery rate was calculated by dividing the total number of copies extracted by the initial number of copies present in the sample and multiplying by 100%; in ref. [44], capture efficiency was evaluated by comparing cycle threshold values obtained from the system with those obtained from kit-extracted 100 µL samples containing the same number of pathogens as for RNA testing (B. ovis in phosphate-buffered saline, 10 5 copies mL −1 ), which were used as absolute reference values; in ref. [45], the capture efficiency was quantified per area of the platform as the following: capture efficiency (η) = (total number of injected cells − total number of cells that passed through device)/total number of injected cells; in [50a], the DNA recovery rate is evaluated by the ratio of the amount of extracted DNA to the original amount of DNA; in ref.
[59a], the DNA extraction efficiency was measured by adding known amounts of DNA (using cultured E. coli cells as a model Gram-negative specimen) into the sample chamber on the 3-chamber IFAST device and comparing this to the amount of DNA recovered from the elution chamber. PCR assay within 25 min with an efficiency similar to the bench-top quantitative PCR. [81] Plasmonic nanostructures have recently attracted particular attention as noncontact heat sources to realize ultrafast photonic PCR based on their photothermal effect. Gold nanoparticles have been used as nanoheaters for thermal cycling in the PCR tube on exposure to light. [87] The gold film was also deposited as the heating element on the PMMA substrate for on-chip nucleic acid amplification in 5 min under the blue LED illumination (Figure 7d). [74] An optical cavity composed of two thin gold films with designed thicknesses can improve the uniformity of temperature distribution in the PCR chamber. [82] The nanoplasmonic pillar arrays, composed of gold nanoislands deposited on the surface of glass nanopillar arrays, provide numerous electromagnetic hotspots between the nanoislands to increase light absorption over the visible broadband range. [83] The plasmonic nanoporous film possesses the unique advantage of integration with automatic sample preparation, including the enrichment and photothermal lysis of pathogens and the on-chip photonic PCR. [45] Digital PCR is a promising absolute single-molecule counting technique with higher sensitivity and precision than quantitative PCR. The diluted sample is randomly partitioned into many separated reactions with Poisson distribution so that each reaction contains one or no copies of the sequence of interest, followed by nucleic acid amplification and fluorescence detection of NAs in each reaction as the positive or negative partition. [88] The more partitions, the greater resolution. Droplets and microwells are two typical formats of small-volume reactors for digital PCR analysis. Advancements in nano-/ microfabrication and microfluidics have promoted the production of digital PCR chips with hundreds to millions of microwells for the compartmentalization of digital PCR, [76] digital isothermal amplification methods, such as LAMP, [63,89] NASBA, [90] recombinase polymerase amplification (RPA), [17,91] rolling circle amplification, [92] helicase-dependent amplification, [93] transcription-mediated amplification, [94] multiple displacement amplification, [95] and strand displacement amplification (SDA). [96] For digital PCR or other NA amplifications with sample preparation on-chip, all complex layers should be simplified for functional operation and massive manufacturing.
One of the compartmentalization technologies for on-chip digital PCR relied on microfluidic valves created by overlapping a microfluidic channel network and a control channel network made of the elastomeric material PDMS. [97] Those networks are separated by a thin membrane that can be deformed into a microfluidic channel network by applying pneumatic or hydraulic pressure into the control channel network to create micromechanical valves and isolate the chambers from one another. Immiscible oil can also be used to isolate dead-end chambers filled with reagents. For example, a megapixel microfluidic PCR device was invented via a soft lithography process to perform a million PCRs in uniform arrays of picoliter-volume chambers for the single-molecule detection of human genomic DNA (Figure 7e). [75] The megapixel digital PCR chip was thermocycling on a thermocycler block with a wide dynamic range and single-nucleotide-variant detection. Figure 7f shows a simple microfluidic solution for efficient digital PCR on-chip by creating hundreds to millions of microwells with simple microfluidic networks that can be integrated with the sample preparation unit. [76] The microinjection molding of high optical clarity cyclo-olefin polymer simplified the manufacturing process of the digital PCR chip containing 4 arrays of 20 000 partitions each. Digital PCR or digital LAMP can be integrated with sample preparation microfluidics as demonstrated in digital RPA. [17] Another active partitioning method for digital PCR is the SlipChip device, which comprises a bottom plate and a top plate in contact with each other. [98] The fluidic path for introducing the sample combined with the PCR mixture was formed using elongated wells in the two plates of the SlipChip device designed to overlap during the sample loading. This fluidic path is broken up by simply slipping the two plates that remove the overlap among wells and brings each well in contact with a reservoir preloaded with oil to generate reaction compartments without complex pumps or valves. As we mentioned, self-powered compartmentalization by the vacuum battery made of air-permeability materials can simplify the onchip sample preparation. [17] Another method for self-powered compartmentalization is utilizing the controlled pinning of fluid at geometric discontinuities within an array of staggered chambers across a central channel, which allows alternate pinning between the two sides of the main channel. [99] Centrifugation can also distribute reagents into the wells located along a spiraling channel. [100]

Outlook
We have evaluated the advancements of integrated sample preparations with nucleic acid amplifications on a chip that allow rapid and accurate NA-based molecular diagnostics with different biological samples for home diagnostics. Considering the dynamics of the pathogens within the human body and the dynamics of the spread from person to person, the high sensitivity of the bench-top NAATs in the centralized lab can only marginally improve the effectiveness of preventing the global spread of infectious diseases. Instead, the accessibility of molecular diagnostics at home promotes effective large-population screening with high test frequency, which can radically break transmission chains and suppress the ongoing pandemic. [101] Nowadays, automatic platforms are adopted in some advanced laboratories for nucleic acid extraction from raw clinical samples with high throughput and high repeatability. Mixing multiple clinical samples in one tube for nucleic acid extraction using automatic platforms in laboratories can further accelerate the throughput, which is favorable to the large-population screen during the pandemic. However, laboratory-based automated platforms are not readily available in low-resource settings due to the limited sanitary condition, high cost, and requirement of highly trained personnel for platform operation and management. By contrast, the on-chip automated sample preparation solutions integrated with nucleic acid amplifications provide an opportunity for rapid home diagnostics with low cost and accessible population, showing the potential to meet the World Health Organization's affordable, sensitive, specific, userfriendly, rapid and robust, equipment-free, and deliverable to end users criteria for POC diagnostic tests. [102] As one of the most successful POC diagnostic test devices, the pregnancy test has been widely adopted by the end users for a preliminary test at home, followed by the confirmatory test in the hospital/laboratory. During the pandemic, on-chip nucleic acid amplifications with automated sample preparation can be applied for the precision screening of a large population. The repeatability of the current on-chip automated sample preparation solution integrated with nucleic acid amplification for DNA or RNA can be better than with the automatic platforms in the laboratory. During the pandemic period, many companies have already demonstrated simplified versions of automated laboratory PCR platforms. [103] Therefore, it is possible to create PCR home test similar platform like a lateral-flow-based COVID-19 Ag home test. Many groups and companies demonstrated DNA or RNA tests on chips, [104] so it takes time and effort to solve the challenges of integration and reagent storage. Convergence of life sciences, chemistry, physics, medicine, and engineering can find creative solutions for this task. Integrated with nucleic acid amplifications can minimize the operation discrepancy of the end users, and the standardization of the manufacturing process can also ensure the consistency of the chip performance, Figure 7. Repurposable PCR on-chip or other DNA/RNA amplification platforms. a) Integrated PDMS-paper microfluidic chip by the roll-to-roll manufacturing process. Reproduced with permission. [71] Copyright 2018, Royal Society of Chemistry. b) Bubble-free rapid PE-PDMS microfluidic PCR chip using a high-powered Peltier element for thermal cycling. Reproduced with permission. [72] Copyright 2019, Elsevier. c) Integrated semiconductor biochip for multiplex PCR and sequence analysis. The CMOS biochip composes a fluid chamber, a resistive heater, multiple temperature sensors, a DNA array, and an embedded photodiode array. Reproduced with permission. [73] Copyright 2018, Springer Nature. d) Ultrafast photonic PCR. Goldnanofilm-based (top) and optical-cavity-based (bottom) thermal cycling. Top: Reproduced with permission. [74] Copyright 2015, Springer Nature. Bottom: Reproduced with permission. [82] Copyright 2015, Wiley-VCH. e) Megapixel digital PCR chip fabricated using soft lithography process. Reproduced with permission. [75] Copyright 2011, Springer Nature. f) Two-layer microfluidic array partitioning digital PCR chip manufactured by high-volume injection molding. Reproduced with permission. [76] Copyright 2019, Springer Nature.
both of which will improve the repeatability of the on-chip automated sample preparation solution integrated with nucleic acid amplification. Simplification of the on-chip single-step sample preparation is the solution for the best molecular diagnostic device at home as Leonardo Da Vinci's teaching, "simplicity is the ultimate sophistication." We should remember this core value to play a critical role in our scientific and technical design that being simple is not ordinary; it is elegant. Nevertheless, it is inherently challenging to solve multiple challenges with a simple and reliable approach, which also can reflect the value of functional sample-to-answer chip for molecular diagnostics at home.
Integrating automatic sample preparation process and nucleic acid amplifications on a wearable biomolecular sensing system can be the next-generation personalized diagnostic technology. [105] Promoted by the material innovation from traditional metal and semiconductor materials to flexible/stretchable 2D materials, polymers, and biomaterials, wearable chemical sensors have attracted more attention to detecting the chemicals in biofluids such as sweat, tears, and saliva for personalized healthcare monitoring. [106] A face-mask-based wearable chemical sensing system is a great candidate for collecting pathogens in respiratory droplets and aerosols from exhaled breath and diagnosing respiratory infectious diseases. For instance, facemask-integrated biological and chemical sensors are developed for wearable and noninvasive SARS-CoV-2 detection in exhaled aerosols via automatic sample preparation and nucleic acid amplification at room temperature within 90 min. [107] The facemask-integrated chemical sensor comprises a water reservoir for hydration activated by a button, a droplet/aerosol collection pad, a wax-patterned microfluidic paper-based analytical device (µPAD) containing freeze-dried components, and a lateral flow assay strip for colorimetric readout. The virus accumulates inside masks due to coughing, talking, and normal respiration. Capillary action wicks the collected fluid containing viral particles from the sample collection pad to the wax-patterned µPAD for viral RNA extraction, reverse transcription-recombinase polymerase amplification (RT-RPA), and Cas 12a specific high-sensitivity enzymatic reporter unlocking (SHERLOCK) detection of the amplified double-stranded DNA amplicon. It is worth noting that the face-mask-integrated chemical sensor can also show potential to inform the end users about environmental safety by locating the sensor on the outside of masks and detecting pathogens from the surrounding environment.
In conclusion, the technique of integrated sample preparations with nucleic acid amplifications on a chip is a promising candidate to obtain both high sensitivity and accessibility to transformative preventive diagnostics, allowing us to improve human healthcare and global biosecurity.