Extraction and Purification of Nicotine from Tobacco Rhizomes by Supercritical CO2.

Currently, in the ongoing development of the tobacco industry, a large amount of tobacco rhizomes is discarded as waste. These wastes are usually disposed of through incineration or burial. However, these tobacco wastes still have some economic value. High-purity nicotine has a promising market outlook as the primary raw material for electronic cigarette liquid. Nicotine is not only found in tobacco leaves but also in the rhizomes of tobacco plants. This study presents a method for treating tobacco waste and extracting high-purity nicotine from it. After mixing the raw material powder and entrainer in specific ratios, as much of the nicotine in tobacco roots can be extracted as possible using supercritical carbon dioxide extraction. The effects of temperature, the ratio of the entrainer, and the volume fraction of ethanol in the entrainer on the nicotine yield in supercritical fluid extraction (SFE) at 25 MPa for 120 min were discussed. By using 90% ethanol (a raw material mass-to-volume ratio of 1:5) as the entrainer, we obtained the highest nicotine yield of 0.49% at 65 °C. Meanwhile, the purity of the crude extract was 61.71%, and after purification, it increased to 97.57%. In this way, we can not only obtain nicotine with market value but also further reduce the harm to the environment caused by tobacco waste disposal.

Long-term monitoring of ultratrace nucleic acids using tetrahedral nanostructure-based NgAgo on wearable microneedles.

Real-time and continuous monitoring of nucleic acid biomarkers with wearable devices holds potential for personal health management, especially in the context of pandemic surveillance or intensive care unit disease. However, achieving high sensitivity and long-term stability remains challenging. Here, we report a tetrahedral nanostructure-based Natronobacterium gregoryi Argonaute (NgAgo) for long-term stable monitoring of ultratrace unamplified nucleic acids (cell-free DNAs and RNAs) in vivo for sepsis on wearable device. This integrated wireless wearable consists of a flexible circuit board, a microneedle biosensor, and a stretchable epidermis patch with enrichment capability. We comprehensively investigate the recognition mechanism of nucleic acids by NgAgo/guide DNA and signal transformation within the Debye distance. In vivo experiments demonstrate the suitability for real-time monitoring of cell-free DNA and RNA with a sensitivity of 0.3 fM up to 14 days. These results provide a strategy for highly sensitive molecular recognition in vivo and for on-body detection of nucleic acid.

Binding Activity of Prokaryotic Argonaute for Background-Suppressed Exponential Isothermal Amplification.

Prokaryotic Argonautes (pAgos) have been recently used in many nucleic acid biosensing applications but have rarely been used for regulating the isothermal amplification system. Herein, we reported Thermus thermophilus Argonaute (TtAgo)-mediated background-suppressed exponential isothermal amplification (EXPAR) as the first example to explore the binding activity of pAgos toward regulation of the amplification template. It was demonstrated that thermophilic pAgos efficiently eliminated nonspecific hybridization between templates by their binding affinity with the template, resulting in greatly enhancing the specificity of EXPAR. TtAgo-mediated, background-suppressed EXPAR was employed to detect miRNA with a detection limit of 10-15 M, which was 1000 times and 100 times more sensitive than that of traditional RT-PCR and EXPAR, respectively. This method further showed good performance in discriminating cancer patients from healthy individuals, indicating its potential for practical clinical applications.

Electro-Optical Multiclassification Platform for Minimizing Occasional Inaccuracy in Point-of-Care Biomarker Detection.

On-site diagnostic tests that accurately identify disease biomarkers lay the foundation for self-healthcare applications. However, these tests routinely rely on single-mode signals and suffer from insufficient accuracy, especially for multiplexed point-of-care tests (POCTs) within a few minutes. Here, this work develops a dual-mode multiclassification diagnostic platform that integrates an electrochemiluminescence sensor and a field-effect transistor sensor in a microfluidic chip. The microfluidic channel guides the testing samples to flow across electro-optical sensor units, which produce dual-mode readouts by detecting infectious biomarkers of tuberculosis (TB), human rhinovirus (HRV), and group B streptococcus (GBS). Then, machine-learning classifiers generate three-dimensional (3D) hyperplanes to diagnose different diseases. Dual-mode readouts derived from distinct mechanisms enhance the anti-interference ability physically, and machine-learning-aided diagnosis in high-dimensional space reduces the occasional inaccuracy mathematically. Clinical validation studies with 501 unprocessed samples indicate that the platform has an accuracy approaching 99%, higher than the 77%-93% accuracy of rapid point-of-care testing technologies at 100% statistical power (>150 clinical tests). Moreover, the diagnosis time is 5 min without a trade-off of accuracy. This work solves the occasional inaccuracy issue of rapid on-site diagnosis, endowing POCT systems with the same accuracy as laboratory tests and holding unique prospects for complicated scenes of personalized healthcare.

An Alternating Current Electroosmotic Flow-Based Ultrasensitive Electrochemiluminescence Microfluidic System for Ultrafast Monitoring, Detection of Proteins/miRNAs in Unprocessed Samples.

Early diagnosis of acute diseases is restricted by the sensitivity and complex process of sample treatment. Here, an ultrasensitive, rapid, and portable electrochemiluminescence-microfluidic (ECL-M) system is described via sandwich-type immunoassay and surface plasmonic resonance (SPR) assay. Using a sandwich immunoreaction approach, the ECL-M system employs cardiac troponin-I antigen (cTnI) as a detection model with a Ru@SiO2 NPs labeled antibody as the signal probe. For miR-499-5p detection, gold nanoparticles generate SPR effects to enhance Ru(bpy)3 2+ ECL signals. The system based on alternating current (AC) electroosmotic flow achieves an LOD of 2 fg mL-1 for cTnI in 5 min and 10 aM for miRNAs in 10 min at room temperature. The point-of-care testing (POCT) device demonstrated 100% sensitivity and 98% specificity for cTnI detection in 123 clinical serum samples. For miR-499-5p, it exhibited 100% sensitivity and 97% specificity in 55 clinical serum samples. Continuous monitoring of these biomarkers in rats' saliva, urine, and interstitial fluid samples for 48 hours revealed observations rarely documented in biotic fluids. The ECL-M POCT device stands as a top-performing system for ECL analysis, offering immense potential for ultrasensitive, rapid, highly accurate, and facile detection and monitoring of acute diseases in POC settings.

Innovative Highly Specific Nucleic Acid Isothermal Detection Assay Based on the Polymerization-Coupled Endonuclease Activity of Prokaryotic DNA Polymerase I.

In this study, we developed an innovative highly specific nucleic acid isothermal detection assay based on prokaryotic DNA polymerase I with exquisitely designed fluorescent probes, achieving high sensitivity and 100% specificity within 30 min. The fluorescent nucleic acid probe was designed and constructed based on the specific flap cleavage endonuclease activity of prokaryotic DNA polymerase I (including the Bst, Bsu, Bsm, and Klenow DNA polymerases). The flap endonuclease activity depends on the length of the flap DNA and polymerization activity, which greatly reduces the false-positive rate caused by primer dimerization. This robust assay was also validated by the detection of rotavirus with great specificity and sensitivity. It could be a great alternative to qPCR in the field of point-of-care detection of pathogens.

Methylene-Blue-Encapsulated Metal-Organic-Framework-Based Electrochemical POCT Platform for Multiple Detection of Heavy Metal Ions in Milk.

Considering the high risk of heavy metal ions (HMIs) transferring through the food chain and accumulating in milk, a flexible and facile point-of-care testing (POCT) platform is urgently needed for the accurate, sensitive, and highly selective on-site quantification of multiple HMIs in milk. In this work, a cost-effective disk with six screen-printed electrodes (SPEs) was designed for hand-held electrochemical detection. Metal organic frameworks (MOFs) were adopted to amplify and enhance the electrochemical signals of methylene blue (MB). Using differential pulse voltammetry (DPV) methods, low limits of detection for four HMIs (Cd2+, 0.039 ppb; Hg2+, 0.039 ppb; Pb2+, 0.073 ppb; and As3+, 0.022 ppb) were achieved within four minutes. Moreover, the quantitative POCT system was applied to milk samples. The advantages of low cost, ease of on-site implementation, fast response, and accuracy allow for the POCT platform to be used in practical monitoring applications for the quantitation of multiple HMIs in milk samples.

Programmable Clostridium perfringens Argonaute-Based, One-Pot Assay for the Multiplex Detection of miRNAs.

Assays for the molecular detection of miRNAs are typically constrained by the level of multiplexing, especially in a single tube. Here, we report a general and programmable diagnostic platform by combining mesophilic Clostridium perfringens Argonaute (CpAgo) with exponential isothermal amplification (EXPAR), which is a dual-signal amplification strategy, allowing for the rapid and sensitive detection of multiple miRNAs with single-nucleotide discrimination in one pot. The CpAgo-based One-Pot (COP) assay achieved a limit of detection of 1 zM miRNA within 30 min of turnaround time and a wide concentration range. This COP assay was applied to simultaneously detect four miRNAs in a single tube from clinical serum samples, showing superior analytical performance in distinguishing colorectal cancer patients from healthy individuals. This programmable, one-pot, multiplex, rapid, and specific strategy offers great promise in scientific research and clinical applications.

Hybridized Chain Reaction-Amplified Alkaline Phosphatase-Induced Ag-Shell Nanostructure for the Sensitive and Rapid Surface-Enhanced Raman Scattering Immunoassay of Exosomes.

Exosomes are small extracellular vesicles that can be utilized as noninvasive biomarkers for diagnosis and treatment of cancer and other diseases. This study reports on a strategy involving a hybridized chain reaction-amplified chain coupled with an alkaline phosphatase-induced Ag-shell nanostructure for the ultrasensitive and rapid surface-enhanced Raman scattering immunoassay of exosomes. Exosomes from prostate cancer were captured using prostate-specific membrane antigen aptamer-modified magnetic beads; then, the hybridized chain reaction-amplified chain was released, incorporating a large number of functional moieties with signal amplification effects. Moreover, the steps of traditional immunoassay were simplified using magnetic materials, and the rapid, sensitive, and accurate detection of exosomes was achieved. Results could be obtained within 40 min with a detection limit of 19 particles/µL. Furthermore, the sera of human prostate cancer patients could be easily distinguished from those of healthy controls, highlighting the potential use of exosome analysis in clinical diagnostics.

Fluorescence and Colorimetric Analysis of African Swine Fever Virus Based on the RPA-Assisted CRISPR/Cas12a Strategy.

It is well-established that different detection modes are necessary for corresponding applications, which can effectively reduce matrix interference and improve the detection accuracy. Here, we reported a magnetic separation method based on recombinase polymerase amplification (RPA)-assisted clustered regularly interspaced short palindromic repeats (CRISPR)/Cas12a for dual-mode analysis of African swine fever virus (ASFV) genes, including colorimetry and fluorescence. The ASFV gene was selected as the initial RPA template to generate the amplicon. The RPA amplicon was then recognized by CRISPR-associated RNA (crRNA), activating the trans-cleavage activity of Cas12a and leading to the nonspecific cleavage of ssDNA as well as a significant release of alkaline phosphatase (ALP) in the ALP-ssDNA modified magnetic bead. The released ALP can catalyze para-nitrophenyl phosphate to generate para-nitrophenol, resulting in substantial changes in absorbance and fluorescence, both of which can be used for detection with the naked eye. This strategy allows the sensitive detection of ASFV DNA, with a 20 copies/mL detection limit; no cross-reactivity with other viruses was observed. A good linear relationship was obtained in serum. In addition, this sensor displayed 100% specificity and sensitivity for clinical sample analysis. This method integrates the high sensitivity of fluorescence with easy readout of colorimetry and enables a simple, low-cost, and highly sensitive dual-mode detection of viral nucleic acid, thereby providing a broad prospect for the practical application in the diagnosis of virus infection.

Real-Time Monitoring and Early Warning of a Cytokine Storm In Vivo Using a Wearable Noninvasive Skin Microneedle Patch.

A cytokine storm may be the last attack of various diseases, such as sepsis, cancer, and coronavirus disease 2019, that can be life threatening. Real-time monitoring of cytokines in vivo is helpful for assessing the immune status of patients and providing an early warning of a cytokine storm. In this study, a functional carbon nanotube biointerface-based wearable microneedle patches for real-time monitoring of a cytokine storm in vivo via electrochemical analysis are reported. This wearable system has sensitivity with a detection limit of 0.54 pg mL-1 , high specificity, and 5 days of stability with a coefficient of variation of 4.0%. The system also has a quick response of several hours (1-4 h) to increasing cytokines. This wearable microneedle patch may offer a promising route for real-time biomolecule wearables construction. The patch is also the first reported integrated capture and monitoring system that is capable of real-time measurement of protein markers in interstitial fluid.

Simultaneous Rapid Nucleic Acid and Protein Detection in a Lateral Chromatography Chip for COVID-19 Diagnosis.

In this work, we report a fast, portable, and economical microfluidic platform for the simultaneous detection of nucleic acid and proteins. Using SARS-CoV-2 as a target, this microfluidic chip enabled to simultaneously detect the SARS-CoV-2 RNA (N gene) antigen (or specific IgG antibody) with respective detection limits of 1 copy/µL for nucleic acid, 0.85 ng/mL for antigen, and 5.80 ng/mL for IgG within 30 min with high stability and anti-interference ability. The capability of this system in clinical applications was further evaluated using clinical samples, displaying 100% sensitivity and 100% specificity for COVID-19 diagnosis. These findings demonstrate the potential of this method to be used for the detection and subsequent control of pathogens.

Rapid quantification of thiocyanate in milk samples using a universal paper-based SERS sensor.

Sodium thiocyanate (NaSCN) is a naturally antibacterial component in milk, but excessive consumption of thiocyanate may cause potential risks to human health. Currently available methods for the detection of thiocyanate have some disadvantages such as poor sensitivity and high price. Here, we report a robust and cost-effective method to detect NaSCN based on paper substrates deposited with in situ reduced Ag nanoparticles by surface-enhanced Raman spectroscopy (SERS). Densely packed multilayer AgNPs provide uniform narrow nanogaps, which exponentially enhance the Raman signals. Moreover, these homogeneous narrow hotspots ensure that this method has high sensitivity (the limit of detection is 10-12 mol L-1), a wide linear range (from 10-9 mol L-1 to 10-4 mol L-1), and remarkable reproducibility (the coefficient of variation within a SERS sensor is 6.5%). Spiked milk samples were detected and the recovery rates of NaSCN were in the range of 95.1%-108.0%. This paper-based SERS sensor offers great potential for sensitive NaSCN detection and milk analysis.

Programmable Analysis of MicroRNAs by Thermus thermophilus Argonaute-Assisted Exponential Isothermal Amplification for Multiplex Detection (TEAM).

The simultaneous analysis of the levels of multiple microRNAs (miRNAs) is critical to the early diagnosis of cancer. However, this analysis is challenging because of the low concentrations of miRNAs and their high sequence homology. Here, we report a general and programmable diagnostic strategy for miRNA analysis: Thermus thermophilus Argonaute (TtAgo)-assisted exponential isothermal amplification for multiplex detection (TEAM). This system combines exponential isothermal amplification (EXPAR), for target amplification, with programmable TtAgo cleavage, for the generation of the reporting signal. The TEAM assay achieved attomolar sensitivity with a rapid turnaround time (30-35 min). Because of the single-nucleotide precision of TtAgo, the system demonstrated robust multiplex capability in the simultaneous detection of four miRNA targets and the classification of let-7 family members. The TEAM assay was superior in differentiating colorectal cancer patients from healthy individuals relative to the conventional EXPAR and reverse transcription polymerase chain reaction (RT-PCR) methods. This tunable and scalable approach is a powerful nucleic acid analysis tool that holds promise in scientific and clinical applications.

Programmable CRISPR-Cas9 microneedle patch for long-term capture and real-time monitoring of universal cell-free DNA.

Recent advances in biointerfaces have led to the development of wearable devices that can provide insights into personal health. As wearable modules, microneedles can extract analytes of interest from interstitial fluid in a minimally invasive fashion. However, some microneedles are limited by their ability to perform highly effective extraction and real-time monitoring for macromolecule biomarkers simultaneously. Here we show the synergetic effect of CRISPR-activated graphene biointerfaces, and report an on-line wearable microneedle patch for extraction and in vivo long-term monitoring of universal cell-free DNA. In this study, this wearable system enables real-time monitoring of Epstein-Barr virus, sepsis, and kidney transplantation cell-free DNA, with anti-interference ability of 60% fetal bovine serum, and has satisfactory stable sensitivity for 10 days in vivo. The experimental results of immunodeficient mouse models shows the feasibility and practicability of this proposed method. This wearable patch holds great promise for long-term in vivo monitoring of cell-free DNA and could potentially be used for early disease screening and prognosis.

Accurate identification of SARS-CoV-2 variant delta using graphene/CRISPR-dCas9 electrochemical biosensor.

Delta (B.1.617.2), a highly infectious variant of SARS-CoV-2, has been sweeping the world, and threatening the safety of human life seriously. It is urgent to develop a highly selective and sensitive assay to accurately identify the SARS-CoV-2 variant Delta. In this work, we constructed a graphene/CRISPR-dCas9 electrochemical biosensor to accurately identify SARS-CoV-2 variant Delta, where the signal was further amplified by embedded electrochemical probe [Ru(phen)2dppz]BF4. This detection assay could be finished within 47 min totally, with the detection limit of 1.2 pM and good reproducibility with a C·V.% of 2.48% (n = 5). And the biosensor could selectively identify Delta among SARS-CoV-2 and other variants, including Alpha, Beta, Gamma. This assay was further validated by 26 real clinical samples, showing 100% clinical sensitivity and 100% clinical specificity, which provides a new direction for identifying other SARS-CoV-2 variants in the future.

Aptamer-Initiated Catalytic Hairpin Assembly Fluorescence Assay for Universal, Sensitive Exosome Detection.

Cancer-cell-derived exosomes are regarded as noninvasive biomarkers for early cancer diagnosis because of their critical roles in intercellular communication and molecular exchange. A robust aptamer-initiated catalytic hairpin assembly (AICHA) fluorescence assay is proposed for universal, sensitive detection of cancer-derived exosomes. The AICHA was verified with the specific detection of MCF-7 cell-derived exosomes with a wide calibration range of 8.4 particles/µL to 8.4 × 105 particles/µL and a low detection limit (LOD) of 0.5 particles/µL. The universality of the AICHA method was verified for PANC-1 cell-derived exosomes, the LOD of which was determined to be 0.1 particles/µL. The performances in serum samples were detected with a recovery rate range of 95.45-106.2%, which demonstrates its significant potential for protein biomarker analysis and cancer diagnosis.

Surface Plasmon Coupling Electrochemiluminescence Immunosensor Based on Polymer Dots and AuNPs for Ultrasensitive Detection of Pancreatic Cancer Exosomes.

Polymer dots (Pdots) have become attractive electrochemiluminescence (ECL) luminophores due to their facile synthesis, easy modification, and stable electrochemical and optical properties. However, their ECL efficiency is not high enough for practical applications. In this work, we proposed an ECL immunosensor based on localized surface plasmon resonance (LSPR) between AuNPs and Pdots for the determination of pancreatic cancer exosomes. Based on the finite-difference time-domain simulations and the band energy of Pdots and AuNPs, we proposed the possible LSPR mechanism. The hot electrons of plasmonic AuNPs were photoexcited to surface plasmon states by ECL emission of Pdots, and then the excited hot electrons were transferred to the conduction band of Pdots, which significantly improved the ECL efficiency of Pdots. The ECL immunosensor displayed a wide calibration range of 1.0 × 103 to 1.0 × 106 particles/mL with a detection limit of 400 particles/mL. Cancer-related protein profiling revealed high selectivity toward different expressions of exosomal surface proteins from PANC-01, HeLa, MCF-7, and HPDE6-C7 cell lines. The proposed ECL system exhibits a promising prospect for protein biomarker profiling and early cancer-related diagnosis.

An integrated flexible film as cathode for High-Performance Lithium-Sulfur battery.

Poor cycling stability and low volumetric capacity of sulfur cathode prevents practical application of Lithium-sulfur (Li-S) batteries. Herein, we demonstrate a strategy to address the two drawbacks of sulfur cathode by synthesizing a compact and flexible film cathode with bilayer structure using a two-step vacuum filtration method. Two layers make up the sulfur cathode, active layer (sulfur-acethlene black (SC) spheres) and barrier layer (three dimensional MnO2-graphene oxide-multi-walled carbon nanotubes (MnO2-GO-CNTs) composites), which are integrated together by reduced graphene oxide (rGO) through self-binding. The rGO sheets provide an electrical conductive framework and a stable architecture to accommodate volume changes of sulfur. SC spheres stacked orderly between the rGO layers facilitate fast Li+ storage and energy release. Polar MnO2-GO-CNTs composites with large specific surface area have not only afforded efficient sites for chemically binding polysulfides, but also provided fast electron transfer for accelerating polysulfides redox reaction. Consequently, the integrated film cathode exhibits an unprecedented cycling stability of ~0.0279% capacity decay per cycle over more than 600 cycles at 1C and high volumetric capacity of 1021.9 Ah L-1 at 2C. Meanwhile, a foldable Li-S battery based on this flexible cathode is fabricated and shows excellent mechanical and electrochemical properties. The integrated flexible sulfur cathode of this study sheds light on the design strategies for application in flexible high volumetric capacity system.