Ultrafast RNA extraction-free SARS-CoV-2 detection by direct RT-PCR using a rapid thermal cycling approach.

The COVID19 pandemic has underlined the need for quick and high-throughput SARS-CoV-2 detection assays. Here we report the development of a direct RT-PCR detection method that can reliably detect SARS-CoV-2 gRNA in nasopharyngeal swab samples in under 27 minutes without needing nucleic acid extraction. Fluorescence readouts were highly linear, robust, and sensitive with a LoD95% of determined at 1.46 copies/µL as determined by RT-PCR on a surrogate sample panel containing clinical samples with varying SARS-CoV-2 viral load. We benchmarked our direct RT-PCR method against a reference qPCR method in 368 nasopharyngeal swab samples, confirming a sensitivity score of 99.4% and a specificity score of 98.5% as compared to the reference method. In summary, we here describe a novel rapid direct RT-PCR method to detect SARS-CoV-2 gRNA in clinical specimens, which can be completed in significantly less time compared to conventional PCR methods making it ideal for large-scale screening applications.

Ultrafast Plasmonic Nucleic Acid Amplification and Real-Time Quantification for Decentralized Molecular Diagnostics.

Point-of-care real-time reverse-transcription polymerase chain reaction (RT-PCR) facilitates the widespread use of rapid, accurate, and cost-effective near-patient testing that is available to the public. Here, we report ultrafast plasmonic nucleic acid amplification and real-time quantification for decentralized molecular diagnostics. The plasmonic real-time RT-PCR system features an ultrafast plasmonic thermocycler (PTC), a disposable plastic-on-metal (PoM) cartridge, and an ultrathin microlens array fluorescence (MAF) microscope. The PTC provides ultrafast photothermal cycling under white-light-emitting diode illumination and precise temperature monitoring with an integrated resistance temperature detector. The PoM thin film cartridge allows rapid heat transfer as well as complete light blocking from the photothermal excitation source, resulting in real-time and highly efficient PCR quantification. Besides, the MAF microscope exhibits close-up and high-contrast fluorescence microscopic imaging. All of the systems were fully packaged in a palm size for point-of-care testing. The real-time RT-PCR system demonstrates the rapid diagnosis of coronavirus disease-19 RNA virus within 10 min and yields 95.6% of amplification efficiency, 96.6% of classification accuracy for preoperational test, and 91% of total percent agreement for clinical diagnostic test. The ultrafast and compact PCR system can decentralize point-of-care molecular diagnostic testing in primary care and developing countries.

Nanotechnology for ultrafast nucleic acid amplification

Nucleic acid detection has been one of the most valued tools in point-of-care diagnostics from life science, agriculture, food safety and environmental surveillance, because of its high sensitivity, great specificity and simple operation. Since polymerase chain reactions (PCR) were discovered, more and more researchers attach importance to exploring ultrafast nucleic acid amplification methods for further expediting the process of detection and curbing infectious diseases' high spread rate, especially after the coronavirus disease 2019 (COVID-19) worldwide pandemic event. Nowadays, nanotechnology as one of the most cut-ting-edge technologies has aroused growing attention. In this review, we describe new advances in na-notechnology research for ultrafast nucleic acid amplification. We have introduced commonly used nanotechnologies, namely nanofluidics, nanoporous materials, nanoparticles and so on. Recent advances in these nanotechnologies for ultrafast sample pretreatments, accelerated enzymatic amplification and rapid heating/cooling processes was summarized. Finally, challenges and perspectives for the future applications of ultrafast nucleic acid amplification are presented.(c) 2022 Elsevier Ltd. All rights reserved.

Exploring ultrafast flow chemistry by autonomous self-optimizing platform

Automated microreactor platform with Bayesian optimization achieves efficient exploring the unstable intermediate and optimizing the reaction conditions for ultrafast chemistry. [Display omitted] • Fully automated microreactor platform (AMP) was developed and demonstrated. • AI-integrated AMP efficiently optimized reaction conditions of ultrafast chemistry. • Thioquinazolinone derivatives were automatically synthesized within 20 min. The rapid development of novel synthetic routes for pharmaceutical compounds is highly attractive for overcoming pandemic and epidemic-prone diseases like COVID-19. Herein, we report an automated microreactor platform (AMP) with Bayesian optimization (BO) that can autonomously explore the optimal conditions for ultrafast synthesis of biologically active thioquinazolinone. First, AMP operation is successfully demonstrated with full control of quantitative variables, specifically reaction volume, temperature, and flow rate, allowing to sequentially conduct a total of 80 experiments planned by the user. Next, BO enables the AMP to autonomously self-optimize the reaction conditions, demonstrating the high efficiency of the fully automated AMP. The fully automated approach is extended to optimize more complex variables including a categorical variable (i.e. the type of organolithium for synthesis), revealing that phenyllithium (PhLi) gives superior yield for synthesizing thioquinazolinone. In addition, the autonomous AMP is utilized for combinatorial chemistry to sequentially synthesize a library composed of nine types of S-benzylic thioquinazolinone under autonomously optimized conditions within only 20 min. [ FROM AUTHOR]

Ultrafast Nucleic Acid Detection Equipment with Silicon-Based Microfluidic Chip.

Recently, infectious diseases, such as COVID-19, monkeypox, and Ebola, are plaguing human beings. Rapid and accurate diagnosis methods are required to preclude the spread of diseases. In this paper, an ultrafast polymerase chain reaction (PCR) equipment is designed to detect virus. The equipment consists of a silicon-based PCR chip, a thermocycling module, an optical detection module, and a control module. Silicon-based chip, with its thermal and fluid design, is used to improve detection efficiency. A thermoelectric cooler (TEC), together with a computer-controlled proportional-integral-derivative (PID) controller, is applied to accelerate the thermal cycle. A maximum of four samples can be tested simultaneously on the chip. Two kinds of fluorescent molecules can be detected by optical detection module. The equipment can detect viruses with 40 PCR amplification cycles in 5 min. The equipment is portable, easily operated, and low equipment cost, which shows great potential in epidemic prevention.

System "Person-State-Society" in Period of Social Turbulence and Big Challenges (Case Study: Tomsk City, the Russian Federation)

The article reflects the role of society in an era of uncertainty and people's behavior in response to big challenges. The aim is to consider the responsibility for resolving crisis situations by state power. Comprehending is possible on the theory formed by the concepts of social turbulence and aggravated regimes, which are based on such characteristics of processes as nonlinearity, spontaneity, uncertainty, and high speeds. This study offers the hypothesis about the shift in the value orientations of the population from the rational to the irrational area in the face of growing uncertainty and turbulence in the environment, which should become the subject of managerial influence when forming a corrective or anti-crisis policy, and about the formation public demand for "strong" state intervention, protecting the population from the negative consequences of regimes with escalations. The article concludes the practical significance and applicability of the research, but also as a theoretical basis for the development of methods and technologies for diagnostics of public demand within the framework of information and analytical support of public administration.

Nanotechnology for ultrafast nucleic acid amplification

Nucleic acid detection has been one of the most valued tools in point-of-care diagnostics from life science, agriculture, food safety and environmental surveillance, because of its high sensitivity, great specificity and simple operation. Since polymerase chain reactions (PCR) were discovered, more and more researchers attach importance to exploring ultrafast nucleic acid amplification methods for further expediting the process of detection and curbing infectious diseases' high spread rate, especially after the coronavirus disease 2019 (COVID-19) worldwide pandemic event. Nowadays, nanotechnology as one of the most cutting-edge technologies has aroused growing attention. In this review, we describe new advances in nanotechnology research for ultrafast nucleic acid amplification. We have introduced commonly used nanotechnologies, namely nanofluidics, nanoporous materials, nanoparticles and so on. Recent advances in these nanotechnologies for ultrafast sample pretreatments, accelerated enzymatic amplification and rapid heating/cooling processes was summarized. Finally, challenges and perspectives for the future applications of ultrafast nucleic acid amplification are presented.

Exploring ultrafast flow chemistry by autonomous self-optimizing platform

The rapid development of novel synthetic routes for pharmaceutical compounds is highly attractive for overcoming pandemic and epidemic-prone diseases like COVID-19. Herein, we report an automated microreactor platform (AMP) with Bayesian optimization (BO) that can autonomously explore the optimal conditions for ultrafast synthesis of biologically active thioquinazolinone. First, AMP operation is successfully demonstrated with full control of quantitative variables, specifically reaction volume, temperature, and flow rate, allowing to sequentially conduct a total of 80 experiments planned by the user. Next, BO enables the AMP to autonomously self-optimize the reaction conditions, demonstrating the high efficiency of the fully automated AMP. The fully automated approach is extended to optimize more complex variables including a categorical variable (i.e. the type of organolithium for synthesis), revealing that phenyllithium (PhLi) gives superior yield for synthesizing thioquinazolinone. In addition, the autonomous AMP is utilized for combinatorial chemistry to sequentially synthesize a library composed of nine types of S-benzylic thioquinazolinone under autonomously optimized conditions within only 20 minutes.

Ultrafast and absolute quantification of SARS-CoV-2 on food using hydrogel RT-LAMP without pre-lysis.

With rapid growing of environmental contact infection, more and more attentions are focused on the precise and absolute quantification of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus on cold chain foods via point-of-care test (POCT). In this work, we propose a hydrogel-mediated reverse transcription loop-mediated isothermal amplification (RT-LAMP) for ultrafast and absolute quantification of SARS-CoV-2. Cross-linked hydrogel offers opportunities for digital single molecule amplification in nanoconfined spaces, facilitating the virus lysis, RNA reverse transcription and amplification process, which is about 3.4-fold faster than conventional bulk RT-LAMP. Ultrafast quantification of SARS-CoV-2 is accomplished in 15 min without virus pre-lysis and RNA extraction. The sensitivity can accurately quantify SARS-CoV-2 down to 0.5 copy/µL. Furthermore, the integrated system has an excellent specificity, reproducibility and storage stability, which can be also used to test SARS-CoV-2 on various cold chain fruits. The developed ultrafast and simple hydrogel RT-LAMP will be an enormous potential for surveillance of virus or other hazardous microbes in environmental, agricultural and food industry.

ULTRA-RAPID REAL-TIME MICROFLUIDIC POLYMERASE CHAIN REACTION INSTRUMENT FOR CLINICAL AND FORENSIC APPLICATIONS

The SARS-CoV-2 pandemic has elevated the development of novel diagnostic solutions, including rapid nucleic acid amplification tests (NAATs), to a global priority to meet the high demand for accurate, timely viral detection and diagnosis. However, ubiquitously implemented NAATs, such as polymerase chain reaction (PCR), consume hours of testing. We report a field-forward instrument capable of ultra-fast real-time PCR for amplification-based nucleic acid detection in a custom-designed microfluidic chip. Prudent selection and unconventional positioning of thermal cyclers relative to the microfluidic chip and a fluorescent detector permit ultra-fast simultaneous amplification and detection, with 40 cycles complete in under 10 minutes. © 2021 MicroTAS 2021 - 25th International Conference on Miniaturized Systems for Chemistry and Life Sciences. All rights reserved.

HANDHELD AND ULTRAFAST PHOTOTHERMAL QPCR SYSTEM FOR RAPID DETECTION OF SARS-COV-2

Rapid polymerase chain reaction (PCR) utilizing plasmon-driven photothermal cycling requires real-time quantification of amplicons during PCR and miniaturization of real-time PCR (qPCR) system for point-of-care (POC) diagnostics. In this work, we have demonstrated handheld photothermal qPCR system with disposable aluminum PCR chips for the ultrafast amplification and real-time quantification of plasmids expressing SARSCoV-2 envelope protein within 5 min. This novel system provides stable and useful point-of-care diagnostic platform for prevention of fast-spreading pandemic in airport and harbor. © 2021 MicroTAS 2021 - 25th International Conference on Miniaturized Systems for Chemistry and Life Sciences. All rights reserved.

Performance Evaluation of a Novel Ultrafast Molecular Diagnostic Device Integrated With Microfluidic Chips and Dual Temperature Modules.

Ultrafast, portable, and inexpensive molecular diagnostic platforms are critical for clinical diagnosis and on-site detection. There are currently no available real-time polymerase chain reaction (PCR) devices able to meet the demands of point-of-care testing, as the heating and cooling processes cannot be avoided. In this study, the dual temperature modules were first designed to process microfluidic chips automatically circulating between them. Thus, a novel ultrafast molecular diagnostic real-time PCR device (approximately 18 and 23 min for DNA and RNA detection, respectively) with two channels (FAM and Cy5) for the detection of 12 targets was developed. The device contained three core functional components, including temperature control, optics, and motion, which were integrated into a portable compact box. The temperature modules accurately control temperature in rapid thermal cycles with less than ±0.1 °C, ±1 °C and ±0.5 °C for the temperature fluctuation, uniformity, and error of indication, respectively. The average coefficient of variation (CV) of the fluorescence intensity (FI) for all 12 wells was 2.3% for FAM and 2.7% for Cy5. There was a good linear relationship between the concentrations of fluorescent dye and the FIs of FAM and Cy5(R 2 = 0.9990 and 0.9937), and the average CVs of the Ct values calculated by the embedded software were 1.4% for FAM and Cy5, respectively. The 100 double-blind mocked sputum and 249 clinical stool samples were analyzed by the ultrafast real-time PCR device in comparison with the DAAN Gene SARS-CoV-2 kit run on the ABI 7500 instrument and Xpert C. difficile/Epi, respectively. Among the 249 stool samples, the ultrafast real-time PCR device detected toxigenic C. difficile in 54 samples (54/249, 21.7%) with a specificity and positive predictive values of 99.0 and 96.3%, which were higher than the Xpert C. difficile/Epi values of 94.4 and 88.1% (p > 0.05). The ultrafast real-time PCR device detected 15 SARS-CoV-2 positive samples, which has a 100% concordance with that obtained by the DAAN Gene SARS-CoV-2 kit. This study demonstrated that the ultrafast real-time PCR device integrated with microfluidic chips and dual temperature modules is an ultrafast, reliable, easy-to-use, and cost-effective molecular diagnostic platform for clinical diagnosis and on-site testing, especially in resource-limited settings.

Evaluation of enterovirus nucleic acids detection method based on ultra-fast real-time fluorescence RT-PCR technology-A pilot study.

The outbreak of COVID-19 epidemic has enabled the establishment and application of various rapid detection methods. It is particularly important to establish a fast and accurate detection method for enterovirus, which will be beneficial for clinical diagnosis, epidemic prevention and control, and timely traceability. Through establishing an ultra-fast reverse transcription-polymerase chain reaction (RT-PCR) equipment, this study aimed to evaluate the sensitivity and specificity of the testing method of enterovirus nucleic acids based on ultra-fast real-time fluorescence RT-PCR technology. A total of 61 cases were sampled, which were then transported and preserved. After the nucleic acid extraction, the nucleic acids of the same sample were tested with the enterovirus nucleic acid detection kit produced by Guangzhou Da An Gene Company and the ultra-fast RT-PCR equipment system established in this study. ABI7500Fast and Ahram biosystems S1 fast equipment were used for amplification detection. If the sample had an S-shaped amplification curve in the FAM channel and the Ct value ≤40.00, the result was positive. The sensitivity, precision, and accuracy of the detection method were then verified. This study established a novel testing method to achieve enterovirus nucleic acid detection within 24 min. The sensitivity detection limit of the method was 1.0 × 102 copies/ml. The coefficients of variation for repeated detection of the high, medium, and low concentration samples were 2.644%, 1.674%, and 4.281%, respectively, with good detection repeatability. In addition, a total of 29 cases were positive by the ultra-fast RT-PCR detection method in 61 suspected samples, which was consistent with the conventional fluorescent RT-PCR method. The established rapid detection method can greatly shorten the time for providing a detection report, which may greatly improve the efficiency of diagnosis and treatment.

Establishment and evaluation of a 30-minute detection method for SARS-CoV-2 nucleic acid using a novel ultra-fast real-time PCR instrument.

BACKGROUND: The coronavirus disease 2019 (COVID-19) pandemic is still raging worldwide. Efficient, fast and low-cost severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) nucleic acid detection methods are urgently needed. METHODS: A rapid PCR temperature change mode was explored by moving the reaction tube between the independent temperature modules with large temperature differences and a portable ultra-fast real-time PCR instrument were developed. We established a rapid SARS-CoV-2 test method using the ultra-fast real-time PCR instrument, a China Food and Drug Administration-certified SARS-CoV-2 reagent and optimized reaction condition. The analytical and clinical performances of the rapid tests were evaluated by comparing with the standard SARS-CoV-2 tests. RESULTS: The new temperature change mode can effectively shorten the amplification reaction time and be successfully used in the development of the ultra-fast real-time PCR instrument. The rapid SARS-CoV-2 test method was established and the time to yield results were greatly shortened from 81 min of the standard test to 31 min. Specificity of the rapid test was assessed and no non-specific amplification (0/63) was observed. The limits of detection of the rapid and standard tests were similar. Clinical performance was evaluated using 184 respiratory specimens from patients with suspected SARS-CoV-2 infection. The positive agreement between the rapid and standard tests was 100% (67/67), the negative agreement was 97.4% (114/117), and the kappa statistic was 0.965 (P<0.001). No significant differences in the Ct values for each target gene were observed between the rapid test and the standard test (P>0.05). CONCLUSIONS: We had developed a 30-minute detection method for SARS-CoV-2 nucleic acid using a novel ultra-fast real-time PCR instrument. The rapid test method may impact on patient management.

Direct Plasmonic Solar Cell Efficiency Dependence on Spiro-OMeTAD Li-TFSI Content.

The proliferation of the internet of things (IoT) and other low-power devices demands the development of energy harvesting solutions to alleviate IoT hardware dependence on single-use batteries, making their deployment more sustainable. The propagation of energy harvesting solutions is strongly associated with technical performance, cost and aesthetics, with the latter often being the driver of adoption. The general abundance of light in the vicinity of IoT devices under their main operation window enables the use of indoor and outdoor photovoltaics as energy harvesters. From those, highly transparent solar cells allow an increased possibility to place a sustainable power source close to the sensors without significant visual appearance. Herein, we report the effect of hole transport layer Li-TFSI dopant content on semi-transparent, direct plasmonic solar cells (DPSC) with a transparency of more than 80% in the 450-800 nm region. The findings revealed that the amount of oxidized spiro-OMeTAD (spiro+TFSI-) significantly modulates the transparency, effective conductance and conditions of device performance, with an optimal performance reached at around 33% relative concentration of Li-TFSI concerning spiro-OMeTAD. The Li-TFSI content did not affect the immediate charge extraction, as revealed by an analysis of electron-phonon lifetime. Hot electrons and holes were injected into the respective layers within 150 fs, suggesting simultaneous injection, as supported by the absence of hysteresis in the I-V curves. The spiro-OMeTAD layer reduces the Au nanoparticles' reflection/backscattering, which improves the overall cell transparency. The results show that the system can be made highly transparent by precise tuning of the doping level of the spiro-OMeTAD layer with retained plasmonics, large optical cross-sections and the ultrathin nature of the devices.

An Ultrafast One-Step Quantitative Reverse Transcription-Polymerase Chain Reaction Assay for Detection of SARS-CoV-2.

We developed an ultrafast one-step RT-qPCR assay for SARS-CoV-2 detection, which can be completed in only 30 min on benchtop Bio-Rad CFX96. The assay significantly reduces the running time of conventional RT-qPCR: reduced RT step from 10 to 1 min, and reduced the PCR cycle of denaturation from 10 to 1 s and extension from 30 to 1 s. A cohort of 60 nasopharyngeal swab samples testing showed that the assay had a clinical sensitivity of 100% and a clinical specificity of 100%.

Ultrafast physical bacterial inactivation and photocatalytic self-cleaning of ZnO nanoarrays for rapid and sustainable bactericidal applications.

Various nanostructured surfaces have been developed recently to physically inactivate bacteria, for reducing the rapidly spreading threat of pathogenic bacteria. However, it generally takes several hours for these surfaces to inactivate most of the bacteria, which greatly limits their application in the fields favoring rapid bactericidal performance. Besides, the accumulated bacteria debris left on these surfaces is rarely discussed in the previous reports. Herein we report the nanotip-engineered ZnO nanoarrays (NAs) with ultrafast physical bactericidal rate and the ability to photocatalytically remove the bacteria debris. Neither chemical (Zn2+ or reactive oxygen species) nor photocatalytic effect leads to the ultrafast bactericidal rate, where 97.5% of E. coli and 94.9% of S. aureus are inactivated within only 1 min. The simulation analysis further supported our proposed mechanism attributing the ultrafast bactericidal activity to the great stress enabled by the uneven topography. Moreover, the re-exposure of the ZnO NAs nanotips can be achieved in only 10 min under a mild UV light source. This study not only presents an ultrafast physical bactericidal activity, but also demonstrates the potential of the recyclable and photocatalytic self-cleaning functions of theses surfaces for applications that desire rapid and sustainable bactericidal performance.