Abscisic acid-induced transcription factor PsMYB306 negatively regulates tree peony bud dormancy release.

Bud dormancy is a crucial strategy for perennial plants to withstand adverse winter conditions. However, the regulatory mechanism of bud dormancy in tree peony (Paeonia suffruticosa) remains largely unknown. Here, we observed dramatically reduced and increased accumulation of abscisic acid (ABA) and bioactive gibberellins (GAs) GA1 and GA3, respectively, during bud endodormancy release of tree peony under prolonged chilling treatment. An Illumina RNA sequencing study was performed to identify potential genes involved in the bud endodormancy regulation in tree peony. Correlation matrix, principal component, and interaction network analyses identified a downregulated MYB transcription factor gene, PsMYB306, the expression of which positively correlated with 9-CIS-EPOXYCAROTENOID DIOXYGENASE 3 (PsNCED3) expression. Protein modeling analysis revealed 4 residues within the R2R3 domain of PsMYB306 to possess DNA binding capability. Transcription of PsMYB306 was increased by ABA treatment. Overexpression of PsMYB306 in petunia (Petunia hybrida) inhibited seed germination and plant growth, concomitant with elevated ABA and decreased GA contents. Silencing of PsMYB306 accelerated cold-triggered tree peony bud burst and influenced the production of ABA and GAs and the expression of their biosynthetic genes. ABA application reduced bud dormancy release and transcription of ENT-KAURENOIC ACID OXIDASE 1 (PsKAO1), GA20-OXIDASE 1 (PsGA20ox1), and GA3-OXIDASE 1 (PsGA3ox1) associated with GA biosynthesis in PsMYB306-silenced buds. In vivo and in vitro binding assays confirmed that PsMYB306 specifically transactivated the promoter of PsNCED3. Silencing of PsNCED3 also promoted bud break and growth. Altogether, our findings suggest that PsMYB306 negatively modulates cold-induced bud endodormancy release by regulating ABA production.

Electrically Oriented Antibodies on Transistor for Monitoring Several Copies of Methylated DNA.

An antibody transistor is a promising biosensing platform for the diagnosis and monitoring of various diseases. Nevertheless, the low concentration and short half-life of biomarkers require biodetection at the trace-molecule level, which remains a challenge for existing antibody transistors. Herein, we demonstrate a graphene field-effect transistor (gFET) with electrically oriented antibody probes (EOA-gFET) for monitoring several copies of methylated DNA. The electric field confines the orientation of antibody probes on graphene and diminishes the distance between graphene and methylated DNAs captured by antibodies, generating more induced charges on graphene and amplifying the electric signal. EOA-gFET realizes a limit of detection (LoD) of ∼0.12 copy µL-1, reaching the lowest LoD reported before. EOA-gFET shows a distinguishable signal for liver cancer clinical serum samples within ∼6 min, which proves its potential as a powerful tool for disease screening and diagnosis.

Highly Bionic Neurotransmitter-Communicated Neurons Following Integrate-and-Fire Dynamics.

In biological neural networks, chemical communication follows the reversible integrate-and-fire (I&F) dynamics model, enabling efficient, anti-interference signal transport. However, existing artificial neurons fail to follow the I&F model in chemical communication, causing irreversible potential accumulation and neural system dysfunction. Herein, we develop a supercapacitively gated artificial neuron that mimics the reversible I&F dynamics model. Upon upstream neurotransmitters, an electrochemical reaction occurs on a graphene nanowall (GNW) gate electrode of artificial neurons. Charging and discharging the supercapacitive GNWs mimic membrane potential accumulation and recovery, realizing highly efficient chemical communication upon use of acetylcholine down to 2 × 10-10 M. By combining artificial chemical synapses with axon-hillock circuits, the output of neural spikes is realized. With the same neurotransmitter and I&F dynamics, the artificial neuron establishes chemical communication with other artificial neurons and living cells, holding promise as a basic unit to construct a neural network with compatibility to organisms for artificial intelligence and deep human-machine fusion.

DNA Logical Computing on a Transistor for Cancer Molecular Diagnosis.

Given the high degree of variability and complexity of cancer, precise monitoring and logical analysis of different nucleic acid markers are crucial for improving diagnostic precision and patient survival rates. However, existing molecular diagnostic methods normally suffer from high cost, cumbersome procedures, dependence on specialized equipment and the requirement of in-depth expertise in data analysis, failing to analyze multiple cancer-associated nucleic acid markers and provide immediate results in a point-of-care manner. Herein, we demonstrate a transistor-based DNA molecular computing (TDMC) platform that enables simultaneous detection and logical analysis of multiple microRNA (miRNA) markers on a single transistor. TDMC can perform not only basic logical operations such as "AND" and "OR", but also complex cascading computing, opening up new dimensions for multi-index logical analysis. Owing to the high efficiency, sensing and computations of multi-analytes can be operated on a transistor at a concentration as low as 2×10-16 M, reaching the lowest concentration for DNA molecular computing. Thus, TDMC achieves an accuracy of 98.4% in the diagnosis of hepatocellular carcinoma from 62 serum samples. As a convenient and accurate platform, TDMC holds promise for applications in "one-stop" personalized medicine.

Catalytic Hairpin Assembly-Enhanced Graphene Transistor for Ultrasensitive miRNA Detection.

MicroRNAs (miRNAs) have emerged as powerful biomarkers for disease diagnosis and screening. Traditional miRNA analytical techniques are inadequate for point-of-care testing due to their reliance on specialized expertise and instruments. Graphene field-effect transistors (GFETs) offer the prospect of simple and label-free diagnostics. Herein, a GFET biosensor based on tetrahedral DNA nanostructure (TDN)-assisted catalytic hairpin assembly (CHA) reaction (TCHA) has been fabricated and applied to the sensitive and specific detection of miRNA-21. TDN structures are assembled to construct the biosensing interface, facilitating CHA reaction by providing free space and preventing unwanted entanglements, aggregation, and adsorption of probes on the graphene channel. Owing to synergistic effects of TDN-assisted in situ nucleic acid amplification on the sensing surface, as well as inherent signal sensitization of GFETs, the biosensor exhibits ultrasensitive detection of miRNA-21 down to 5.67 × 10-19 M, approximately three orders of magnitude lower than that normally achieved by graphene transistors with channel functionalization of single-stranded DNA probes. In addition, the biosensor demonstrates excellent analytical performance regarding selectivity, stability, and reproducibility. Furthermore, the practicability of the biosensor is verified by analyzing targets in a complex serum environment and cell lysates, showing tremendous potential in bioanalysis and clinical diagnosis.

Artificial Nucleotide Aptamer-Based Field-Effect Transistor for Ultrasensitive Detection of Hepatoma Exosomes.

An aptamer-based field-effect transistor (Apta-FET) is a well-developed assay method with high selectivity and sensitivity. Due to the limited information density that natural nucleotide library holds, the Apta-FET faces fundamental restriction in universality to detect various types of analytes. Herein, we demonstrate a type of Apta-FET sensors based on an artificial nucleotide aptamer (AN-Apta-FET). The introduction of an artificial nucleotide increases the diversity of the potential aptamer structure and expands the analyte category of the Apta-FET. The AN-Apta-FET specifically detects hepatoma exosomes, which traditional Apta-FET fails to discriminate from other tumor-derived exosomes, with a limit of detection down to 242 particles mL-1. The AN-Apta-FET distinguishes serum samples of hepatocellular carcinoma patients within 9 min from those of healthy people, showing the potential as a comprehensive assay tool in future disease diagnosis.

Triple-Probe DNA Framework-Based Transistor for SARS-CoV-2 10-in-1 Pooled Testing.

Accurate and population-scale screening technology is crucial in the control and prevention of COVID-19, such as pooled testing with high overall testing efficiency. Nevertheless, pooled testing faces challenges in sensitivity and specificity due to diluted targets and increased contaminations. Here, we develop a graphene field-effect transistor sensor modified with triple-probe tetrahedral DNA framework (TDF) dimers for 10-in-1 pooled testing of SARS-CoV-2 RNA. The synergy effect of triple probes as well as the special nanostructure achieve a higher binding affinity, faster response, and better specificity. The detectable concentration reaches 0.025-0.05 copy µL-1 in unamplified samples, lower than that of the reverse transcript-polymerase chain reaction. Without a requirement of nucleic-acid amplification, the sensors identify all of the 14 positive cases in 30 nasopharyngeal swabs within an average diagnosis time of 74 s. Unamplified 10-in-1 pooled testing enabled by the triple-probe TDF dimer sensor has great potential in the screening of COVID-19 and other epidemic diseases.

Electrochemical Detection of a Few Copies of Unamplified SARS-CoV-2 Nucleic Acids by a Self-Actuated Molecular System.

The existing electrochemical biosensors lack controllable and intelligent merit to modulate the sensing process upon external stimulus, leading to challenges in analyzing a few copies of biomarkers in unamplified samples. Here, we present a self-actuated molecular-electrochemical system that consists of a tentacle and a trunk modification on a graphene microelectrode. The tentacle that contains a probe and an electrochemical label keeps an upright orientation, which increases recognition efficiency while decreasing the pseudosignal. Once the nucleic acids are recognized, the tentacles nearby along with the labels are spontaneously actuated downward, generating electrochemical responses under square wave voltammetry. Thus, it detects unamplified SARS-CoV-2 RNAs within 1 min down to 4 copies in 80 µL, 2-6 orders of magnitude lower than those of other electrochemical assays. Double-blind testing and 10-in-1 pooled testing of nasopharyngeal samples yield high overall agreement with reverse transcription-polymerase chain reaction results. We fabricate a portable prototype based on this system, showing great potential for future applications.

Rapid SARS-CoV-2 Nucleic Acid Testing and Pooled Assay by Tetrahedral DNA Nanostructure Transistor.

Direct SARS-CoV-2 nucleic acid testing with fast speed and high frequency is crucial for controlling the COVID-19 pandemic. Here, direct testing of SARS-CoV-2 nucleic acid is realized by field-effect transistors (FETs) with an electro-enrichable liquid gate (LG) anchored by tetrahedral DNA nanostructures (TDNs). The applied gate bias electrostatically preconcentrates nucleic acids, while the liquid gate with TDNs provides efficient analyte recognition and signal transduction. The average diagnosis time is ∼80 s, and the limit of detection approaches 1-2 copies in 100 µL of clinical samples without nucleic acid extraction and amplification. As such, TDN-LG FETs solve the dilemma of COVID-19 testing on mass scale that diagnosis accuracy and speed undergo trade-off. In addition, TDN-LG FETs achieve unamplified 10-in-1 pooled nucleic acid testing for the first time, and the results are consistent with PCR. Thus, this technology promises on-site and wide population COVID-19 screening and ensures safe world-reopening.

Ultraprecise Antigen 10-in-1 Pool Testing by Multiantibodies Transistor Assay.

Effective screening of infectious diseases requires a fast, cheap, and population-scale testing. Antigen pool testing can increase the test rate and shorten the screening time, thus being a valuable approach for epidemic prevention and control. However, the overall percent agreement (OPA) with polymerase chain reaction (PCR) is one-half to three-quarters, hampering it from being a comprehensive method, especially pool testing, beyond the gold-standard PCR. Here, a multiantibodies transistor assay is developed for sensitive and highly precise antigen pool testing. The multiantibodies capture SARS-CoV-2 spike S1 proteins with different configurations, resulting in an antigen-binding affinity down to 0.34 fM. The limit of detection reaches 3.5 × 10-17 g mL-1SARS-CoV-2 spike S1 protein in artificial saliva, 4-5 orders of magnitude lower than existing transistor sensors. The testing of 60 nasopharyngeal swabs exhibits ∼100% OPA with PCR within an average diagnoses time of 38.9 s. Owing to its highly precise feature, a portable integrated platform is fabricated, which achieves 10-in-1 pooled screening for high testing throughput. This work solves the long-standing problem of antigen pool testing, enabling it to be a valuable tool in precise diagnoses and population-wide screening of COVID-19 or other epidemics in the future.

Direct SARS-CoV-2 Nucleic Acid Detection by Y-Shaped DNA Dual-Probe Transistor Assay.

Rapid screening of infected individuals from a large population is an effective means in epidemiology, especially to contain outbreaks such as COVID-19. The gold standard assays for COVID-19 diagnostics are mainly based on the reverse transcription polymerase chain reaction, which mismatches the requirements for wide-population screening due to time-consuming nucleic acid extraction and amplification procedures. Here, we report a direct nucleic acid assay by using a graphene field-effect transistor (g-FET) with Y-shaped DNA dual probes (Y-dual probes). The assay relies on Y-dual probes modified on g-FET simultaneously targeting ORF1ab and N genes of SARS-CoV-2 nucleic acid, enabling high a recognition ratio and a limit of detection (0.03 copy µL-1) 1-2 orders of magnitude lower than existing nucleic acid assays. The assay realizes the fastest nucleic acid testing (∼1 min) and achieves direct 5-in-1 pooled testing for the first time. Owing to its rapid, ultrasensitive, easily operated features as well as capability in pooled testing, it holds great promise as a comprehensive tool for population-wide screening of COVID-19 and other epidemics.

"Off-On"switching electrochemiluminescence biosensor for mercury(II) detection based on molecular recognition technology.

A novel "off-On" electrogenerated chemiluminescence (ECL) biosensor has been developed for the detection of mercury(II) based on molecular recognition technology. The ECL mercury(II) biosensor comprises two main parts: an ECL substrate and an ECL intensity switch. The ECL substrate was made by modifying the complex of Ruthenium(II) tris-(bipyridine)(Ru(bpy)32+)/Cyclodextrins-Au nanoparticles(CD-AuNps)/Nafion on the surface of glass carbon electrode (GCE), and the ECL intensity switch is the single hairpin DNA probe designed according to the "molecular recognition" strategy which was functionalized with ferrocene tag at one end and attached to Cyclodextrins (CD) on modified GCE through supramolecular noncovalent interaction. We demonstrated that, in the absence of Hg(II) ion, the probe keeps single hairpin structure and resulted in a quenching of ECL of Ru(bpy)32+. Whereas, in the presence of Hg(II) ion, the probe prefers to form the T-Hg(II)-T complex and lead to an obvious recovery of ECL of Ru(bpy)32+, which provided a sensing platform for the detection of Hg(II) ion. Using this sensing platform, a simple, rapid and selective "off-On" ECL biosensor for the detection of mercury(II) with a detection limit of 0.1 nM has been developed.

The material basis of bitter constituents in Carbonized Typhae Pollen, based on the integration strategy of constituent analysis, taste sensing system and molecular docking.

The discovery of bitter constituents is of great significance to the exploration of medicinal substances for they have potential physiological effects. Carbonized Typhae Pollen (CTP), which is a typical example of carbonized Traditional Chinese Medicine (TCM), has a bitter taste and hemostatic effect after carbonized processing. The objective of this study is to elucidate the material basis of bitter constituents in CTP. Firstly, the constituents of CTP extracts with 7 different solvents were characterized by UPLC-Q-TOF-MS. Then, multivariate statistical analysis was used to visualize the CTP extracts from 7 solvents. A total of 37 constituents were tentatively identified and 17 constituents were considered as the key constituents in differentiating 7 different solvent extracts. Subsequently, the bitter evaluation of extracts from different polar parts was investigated by using an electronic tongue. As a result, the order of bitterness of the extracts was as follows: ethanol > methanol > water > n-butyl alcohol > petroleum ether > butyl acetate > isopropanol. There were statistically significant differences in the bitter degree of extracts. By correlation analysis of bitter information and chemical constituents with partial least squares regression (PLSR), 8 potential bitterness constituents were discovered, including phenylalanine, valine, chlorogenic acid, isoquercitrin, palmitic acid, citric acid, quercetin-3-O-(2-α-L-rhamnosyl)-rutinoside, and typhaneoside. Additionally, molecular docking analysis was conducted to reveal the interaction of these constituents with the bitter taste receptor. The docking result showed that these constituents could be embedded well into the active pocket of T2R46 and had significant affinity interactions with critical amino acid residues by forming hydrogen bonds. This study provided a reliable theoretical basis for future research on biological activity of bitterness substances.

Photovoltaic nanocells for high-performance large-scale-integrated organic phototransistors.

A high-performance large-scale-integrated organic phototransistor needs a semiconductor layer that maintains its photoelectric conversion ability well during high-resolution pixelization. However, lacking a precise design for the nanoscale structure, a trade-off between photoelectric performance and device miniaturization greatly limits the success in commercial application. Here we demonstrate a photovoltaic-nanocell enhancement strategy, which overcomes the trade-off and enables high-performance organic phototransistors at a level beyond large-scale integration. Embedding a core-shell photovoltaic nanocell based on perovskite quantum dots in a photocrosslinkable organic semiconductor, ultralarge-scale-integrated (>221 units) imaging chips are manufactured using photolithography. 27 million pixels are interconnected and the pixel density is 3.1 × 106 units cm-2, at least two orders of magnitude higher than in existing organic imaging chips and equivalent to the latest commercial full-frame complementary metal-oxide-semiconductor camera chips. The embedded photovoltaic nanocells induce an in situ photogating modulation and enable photoresponsivity and detectivity of 6.8 × 106 A W-1 and 1.1 × 1013 Jones (at 1 Hz), respectively, achieving the highest values of organic imaging chips at large-scale or higher integration. In addition, a very-large-scale-integrated (>216 units) stretchable biomimetic retina based on photovoltaic nanocells is manufactured for neuromorphic imaging recognition with not only resolution but also photoresponsivity and power consumption approaching those of the biological counterpart.

One-base-mismatch CRISPR-based transistors for single nucleotide resolution assay.

An effective strategy for accurately detecting single nucleotide variants (SNVs) is of great significance for genetic research and diagnostics. However, strict amplification conditions, complex experimental instruments, and specialized personnel are required to obtain a satisfactory tradeoff between sensitivity and selectivity for SNV discrimination. In this study, we present a CRISPR-based transistor biosensor for the rapid and highly selective detection of SNVs in viral RNA. By introducing a synthetic mismatch in the crRNA, the CRISPR-Cas13a protein can be engineered to capture the target SNV RNA directly on the surface of the graphene channel. This process induces a fast electrical signal response in the transistor, obviating the need for amplification or reporter molecules. The biosensor exhibits a detection limit for target RNA as low as 5 copies in 100 µL, which is comparable to that of real-time quantitative polymerase chain reaction (PCR). Its operational range spans from 10 to 5 × 105 copy mL-1 in artificial saliva solution. This capability enables the biosensor to discriminate between wild-type and SNV RNA within 15 min. By introducing 10 µL of swab samples during clinical testing, the biosensor provides specific detection of respiratory viruses in 19 oropharyngeal specimens, including influenza A, influenza B, and variants of SARS-CoV-2. This study emphasizes the CRISPR-transistor technique as a highly accurate and sensitive approach for field-deployable nucleic acid screening or diagnostics.

Ultra-Fast Single-Nucleotide-Variation Detection Enabled by Argonaute-Mediated Transistor Platform.

"Test-and-go" single-nucleotide variation (SNV) detection within several minutes remains challenging, especially in low-abundance samples, since existing methods face a trade-off between sensitivity and testing speed. Sensitive detection usually relies on complex and time-consuming nucleic acid amplification or sequencing. Here, a graphene field-effect transistor (GFET) platform mediated by Argonaute protein that enables rapid, sensitive, and specific SNV detection is developed. The Argonaute protein provides a nanoscale binding channel to preorganize the DNA probe, accelerating target binding and rapidly recognizing SNVs with single-nucleotide resolution in unamplified tumor-associated microRNA, circulating tumor DNA, virus RNA, and reverse transcribed cDNA when a mismatch occurs in the seed region. An integrated microchip simultaneously detects multiple SNVs in agreement with sequencing results within 5 min, achieving the fastest SNV detection in a "test-and-go" manner without the requirement of nucleic acid extraction, reverse transcription, and amplification.

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.

Transcriptome Profiling Reveals a Petunia Transcription Factor, PhCOL4, Contributing to Antiviral RNA Silencing.

RNA silencing is a common antiviral mechanism in eukaryotic organisms. However, the transcriptional regulatory mechanism that controls the RNA silencing process remains elusive. Here, we performed high-depth transcriptome analysis on petunia (Petunia hybrida) leaves infected with tobacco rattle virus (TRV) strain PPK20. A total of 7,402 differentially expressed genes (DEGs) were identified. Of them, some RNA silencing-related transcripts, such as RNA-dependent RNA polymerases (RDRs), Dicer-like RNase III enzymes (DCLs), and Argonautes (AGOs), were induced by viral attack. Furthermore, we performed TRV-based virus-induced gene silencing (VIGS) assay on 39 DEGs encoding putative transcription factors (TFs), using green fluorescent protein (GFP) and phytoene desaturase (PhPDS) as reporters. Results showed that the down-regulation of PhbHLH41, PhbHLH93, PhZPT4-3, PhCOL4, PhHSF-B3A, PhNAC90, and PhWRKY75 led to enhanced TRV accumulation and inhibited PhPDS-silenced photobleaching phenotype. In contrast, silencing of PhERF22 repressed virus accumulation and promoted photobleaching development. Thus, these TFs were identified as potential positive and negative regulators of antiviral RNA silencing, respectively. One positive regulator PhCOL4, belonging to the B-box zinc finger family, was selected for further functional characterization. Silencing and transient overexpression of PhCOL4 resulted in decreased and increased expression of several RNA silencing-related genes. DNA affinity purification sequencing analysis revealed that PhCOL4 targeted PhRDR6 and PhAGO4. Dual luciferase and yeast one-hybrid assays determined the binding of PhCOL4 to the PhRDR6 and PhAGO4 promoters. Our findings suggest that TRV-GFP-PhPDS-based VIGS could be helpful to identify transcriptional regulators of antiviral RNA silencing.

Rapid and ultrasensitive electromechanical detection of ions, biomolecules and SARS-CoV-2 RNA in unamplified samples.

The detection of samples at ultralow concentrations (one to ten copies in 100 µl) in biofluids is hampered by the orders-of-magnitude higher amounts of 'background' biomolecules. Here we report a molecular system, immobilized on a liquid-gated graphene field-effect transistor and consisting of an aptamer probe bound to a flexible single-stranded DNA cantilever linked to a self-assembled stiff tetrahedral double-stranded DNA structure, for the rapid and ultrasensitive electromechanical detection (down to one to two copies in 100 µl) of unamplified nucleic acids in biofluids, and also of ions, small molecules and proteins, as we show for Hg2+, adenosine 5'-triphosphate and thrombin. We implemented an electromechanical biosensor for the detection of SARS-CoV-2 into an integrated and portable prototype device, and show that it detected SARS-CoV-2 RNA in less than four minutes in all nasopharyngeal samples from 33 patients with COVID-19 (with cycle threshold values of 24.9-41.3) and in none of the 54 COVID-19-negative controls, without the need for RNA extraction or nucleic acid amplification.

Short-wavelength ultraviolet dosimeters based on DNA nanostructure-modified graphene field-effect transistors.

Direct and sensitive short-wavelength ultraviolet (UVC) dosimeters could provide a safer disinfection environment against viruses. We developed direct, quantitative, specific and highly sensitive UVC dosimeters based on DNA nanostructure-modified graphene field-effect transistors. Detectable doses of the dosimeters range from 0.005 to 6 kJ m-2 and such dosimeters have at least 5 times better sensitivity than the current direct UV dosimeters.