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.
Recent advancements in integrated sample preparations and nucleic acid (NA) detection are reported for rapid and accurate home diagnostics. Automated sample preparation methods of blood, urine, nasal swabs, saliva, or stool that can be integrated with NA amplification on a single device are highlighted. Recent progress, challenges, and perspectives of user‐friendly integrated molecular diagnostics at home are discussed.
Conspectus The global outbreaks of deadly infectious diseases caused by pathogenic microorganisms have threatened public health worldwide and significantly motivated scientists to satisfy an urgent ...need for a rapid and accurate detection of pathogens. Traditionally, the culture-based technique is considered as the gold standard for pathogen detection, yet it has a long turnaround time due to the overnight culturing and pathogen isolation. Alternatively, nucleic acid amplification tests provide a relatively shorter turnaround time to identify whether pathogens exist in individuals with high sensitivity and high specificity. In most cases, nucleic acid amplification tests undergo three steps: sample preparation, nucleic acid amplification, and signal transduction. Despite the explosive advancement in nucleic acid amplification and signal transduction technologies, the complex and labor-intensive sample preparation steps remain a bottleneck to create a transformative integrated point-of-care (POC) molecular diagnostic device. Researchers have attempted to simplify and integrate the sample preparations for nucleic acid-based molecular diagnostic devices with innovative progress in integration strategies, engineered materials, reagent storages, and fluid actuation. Therefore, understanding the know-how and obtaining truthful knowledge of existing integrated POC molecular diagnostic devices comprising sample preparations, nucleic acid amplification, and signal transduction can generate innovative solutions to achieve personalized precision medicine and improve global health. In this Account, we discuss the challenges of automated sample preparation solutions integrated with nucleic acid amplification and signal transduction for rapid and precise home diagnostics. Blood, nasal swab, saliva, urine, and stool are emphasized as the most commonly used clinical samples for integrated POC molecular diagnostics of infectious diseases. Even though these five types of samples possess relatively correlated biomarkers due to the human body’s circulatory system, each shows unique properties and exclusive advantages for molecular diagnostics in specific situations, which are included in this Account. We examine different integrated POC devices for sample preparation, which includes pathogen isolation and enrichment from the crude sample and nucleic acid purification from isolated pathogens. We present the promising on-chip integration approaches for nucleic acid amplification. We also investigate the on-chip integration methods for reagent storage, which is crucial to simplify the manual operation for end-users. Finally, we present several integrated POC molecular diagnostic devices for infectious diseases. The integrated sample preparation and nucleic acid amplification approach reviewed here can potentially impact the next generation of POC molecular home diagnostic chips, which will significantly impact public health, emergency medicine, and global biosecurity.
Prompt on‐site diagnosis of SARS‐CoV‐2 with other respiratory infections will have minimized the global impact of the COVID‐19 pandemic through rapid, effective management. However, no such multiplex ...point‐of‐care (POC) chip has satisfied a suitable sensitivity of gold‐standard nucleic acid amplification tests (NAATs). Here, a rapid multiplexed ultrasensitive sample‐to‐answer loop‐mediated isothermal amplification (MUSAL) chip operated by simple LED‐driven photothermal amplification to detect six targets from single‐swab sampling is presented. First, the MUSAL chip allows ultrafast on‐chip sample preparation with ≈500‐fold preconcentration at a rate of 1.2 mL min−1. Second, the chip enables contamination‐free amplification using autonomous target elution into on‐chip reagents by photothermal activation. Finally, the chip accomplishes multiplexed on‐chip diagnostics of SARS‐CoV‐2 and influenza viruses with a limit of detection (LoD) of 0.5 copies µL−1. The rapid, ultrasensitive, cost‐effective sample‐to‐answer chip with a multiplex capability will allow timely management of various pandemics situations that may be faced shortly.
An ultrasensitive sample‐to‐answer chip operated by LED is developed to detect SARS‐CoV‐2 and influenza viruses simultaneously. The ultrafast target enrichment (≈500×) followed by autonomous elution and amplification via photothermal activation enables limit‐of‐detection of 0.5 copies µL−1. The sensitive detection in point‐of‐care settings comparable to standard polymerase chain reaction allows timely management of various pandemic situations that may be faced shortly.
Alternative messenger RNA (mRNA) splicing is a fundamental process of gene regulation, and errors in RNA splicing are known to be associated with a variety of different diseases. However, there is ...currently a lack of quantitative technologies for monitoring mRNA splice variants in cells. Here, we show that a combination of plasmonic dimer probes and hyperspectral imaging can be used to detect and quantify mRNA splice variants in living cells. The probes are made from gold nanoparticles functionalized with oligonucleotides and can hybridize to specific mRNA sequences, forming nanoparticle dimers that exhibit distinct spectral shifts due to plasmonic coupling. With this approach, we show that the spatial and temporal distribution of three selected splice variants of the breast cancer susceptibility gene, BRCA1, can be monitored at single-copy resolution by measuring the hybridization dynamics of the nanoplasmonic dimers. Our study provides insights into RNA and its transport in living cells, which could improve our understanding of cellular protein complexes, pharmacogenomics, genetic diagnosis and gene therapies.
2D transition‐metal dichalcogenides (TMDs) have been successfully developed as novel ubiquitous optoelectronics owing to their excellent electrical and optical characteristics. However, active‐matrix ...image sensors based on TMDs have limitations owing to the difficulty of fabricating large‐area integrated circuitry and achieving high optical sensitivity. Herein, a large‐area uniform, highly sensitive, and robust image sensor matrix with active pixels consisting of nanoporous molybdenum disulfide (MoS2) phototransistors and indium–gallium–zinc oxide (IGZO) switching transistors is reported. Large‐area uniform 4‐inch wafer‐scale bilayer MoS2 films are synthesized by radio‐frequency (RF) magnetron sputtering and sulfurization processes and patterned to be a nanoporous structure consisting of an array of periodic nanopores on the MoS2 surface via block copolymer lithography. Edge exposure on the nanoporous bilayer MoS2 induces the formation of subgap states, which promotes a photogating effect to obtain an exceptionally high photoresponsivity of 5.2 × 104 A W−1. A 4‐inch‐wafer‐scale image mapping is successively achieved using this active‐matrix image sensor by controlling the device sensing and switching states. The high‐performance active‐matrix image sensor is state‐of‐the‐art in 2D material‐based integrated circuitry and pixel image sensor applications.
Ultrasensitive and robust active‐matrix image sensor circuitry is demonstrated by integrating a wafer‐scale nanoporous molybdenum disulfide (MoS2) phototransistor array with high‐performance indium‐gallium‐zinc oxide (IGZO) switching transistors. A 4‐inch wafer‐scale 2D)MoS2 is synthesized and a periodic nanopore array is created on its surface to achieve exceptionally high photoresponsivity of 5.2 × 104 A W−1. The device shows a technically advanced form based on large‐area 2D material similar to a commercialized complementary metal–oxide–semiconductor (CMOS) image sensor consisting of a sensor and a driving transistor in the matrix pixel.
Acoustic tweezers provide an effective means for manipulating single cells and particles in a high-throughput, precise, selective and contact-free manner. The adoption of acoustic tweezers in ...next-generation cellular assays may advance our understanding of biological systems. Here we present a comprehensive set of instructions that guide users through device fabrication, instrumentation setup and data acquisition to study single cells with an experimental throughput that surpasses traditional methods, such as atomic force microscopy and micropipette aspiration, by several orders of magnitude. With acoustic tweezers, users can conduct versatile experiments that require the trapping, patterning, pairing and separation of single cells in a myriad of applications ranging across the biological and biomedical sciences. This procedure is widely generalizable and adaptable for investigations in materials and physical sciences, such as the spinning motion of colloids or the development of acoustic-based quantum simulations. Overall, the device fabrication requires ~12 h, the experimental setup of the acoustic tweezers requires 1-2 h and the cell manipulation experiment requires ~30 min to complete. Our protocol is suitable for use by interdisciplinary researchers in biology, medicine, engineering and physics.
Capturing real-time electron transfer, enzyme activity, molecular dynamics, and biochemical messengers in living cells is essential for understanding the signaling pathways and cellular ...communications. However, there is no generalizable method for characterizing a broad range of redox-active species in a single living cell at the resolution of cellular compartments. Although nanoelectrodes have been applied in the intracellular detection of redox-active species, the fabrication of nanoelectrodes to maximize the signal-to-noise ratio of the probe remains challenging because of the stringent requirements of 3D fabrication. Here, we report an asymmetric nanopore electrode-based amplification mechanism for the real-time monitoring of NADH in a living cell. We used a two-step 3D fabrication process to develop a modified asymmetric nanopore electrode with a diameter down to 90 nm, which allowed for the detection of redox metabolism in living cells. Taking advantage of the asymmetric geometry, the above 90% potential drop at the two terminals of the nanopore electrode converts the faradaic current response into an easily distinguishable bubble-induced transient ionic current pattern. Therefore, the current signal was amplified by at least 3 orders of magnitude, which was dynamically linked to the presence of trace redox-active species. Compared to traditional wire electrodes, this wireless asymmetric nanopore electrode exhibits a high signal-to-noise ratio by increasing the current resolution from nanoamperes to picoamperes. The asymmetric nanopore electrode achieves the highly sensitive and selective probing of NADH concentrations as low as 1 pM. Moreover, it enables the real-time nanopore monitoring of the respiration chain (i.e., NADH) in a living cell and the evaluation of the effects of anticancer drugs in an MCF-7 cell. We believe that this integrated wireless asymmetric nanopore electrode provides promising building blocks for the future imaging of electron transfer dynamics in live cells.
Drug discovery and development are hampered by high failure rates attributed to the reliance on non-human animal models employed during safety and efficacy testing. A fundamental problem in this ...inefficient process is that non-human animal models cannot adequately represent human biology. Thus, there is an urgent need for high-content in vitro systems that can better predict drug-induced toxicity. Systems that predict cardiotoxicity are of uppermost significance, as approximately one third of safety-based pharmaceutical withdrawals are due to cardiotoxicty. Here, we present a cardiac microphysiological system (MPS) with the attributes required for an ideal in vitro system to predict cardiotoxicity: i) cells with a human genetic background; ii) physiologically relevant tissue structure (e.g. aligned cells); iii) computationally predictable perfusion mimicking human vasculature; and, iv) multiple modes of analysis (e.g. biological, electrophysiological, and physiological). Our MPS is able to keep human induced pluripotent stem cell derived cardiac tissue viable and functional over multiple weeks. Pharmacological studies using the cardiac MPS show half maximal inhibitory/effective concentration values (IC₅₀/EC₅₀) that are more consistent with the data on tissue scale references compared to cellular scale studies. We anticipate the widespread adoption of MPSs for drug screening and disease modeling.
Despite a growing focus from the academic community, the field of microfluidics has yet to produce many commercial devices for point-of-care (POC) diagnostics. One of the main reasons for this is the ...difficulty in producing low-cost, sensitive, and portable optical detection systems. Although electrochemical methods work well for certain applications, optical detection is generally regarded as superior and is the method most widely employed in laboratory clinical chemistry. Conventional optical systems, however, are costly, require careful alignment, and do not translate well to POC devices. Furthermore, many optical detection paradigms such as absorbance and fluorescence suffer at smaller geometries because the optical path length through the sample is shortened. This review examines the innovative techniques which have recently been developed to address these issues. We highlight microfluidic diagnostic systems which demonstrate practical integration of sample preparation, analyte enrichment, and optical detection. We also examine several emerging detection paradigms involving nanoengineered materials which do not suffer from the same miniaturization disadvantages as conventional measurements.
Newly developed acoustic technologies are playing a transformational role in life science and biomedical applications ranging from the activation and inactivation of mechanosensitive ion channels for ...fundamental physiological processes to the development of contact-free, precise biofabrication protocols for tissue engineering and large-scale manufacturing of organoids. Here, we provide our perspective on the development of future acoustic technologies and their promise in addressing critical challenges in biomedicine.
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