Transcriptome analysis of drought-responsive and drought-tolerant mechanisms in maize leaves under drought stress.

Maize is a major crop essential for food and feed, but its production is threatened by various biotic and abiotic stresses. Drought is one of the most common abiotic stresses, causing severe crop yield reduction. Although several studies have been devoted to selecting drought-tolerant maize lines and detecting the drought-responsive mechanism of maize, the transcriptomic differences between drought-tolerant and drought-susceptible maize lines are still largely unknown. In our study, RNA-seq was performed on leaves of the drought-tolerant line W9706 and the drought-susceptible line B73 after drought treatment. We identified 3147 differentially expressed genes (DEGs) between these two lines. The upregulated DEGs in W9706 were enriched in specific processes, including ABA signaling, wax biosynthesis, CHO metabolism, signal transduction and brassinosteroid biosynthesis-related processes, while the downregulated DEGs were enriched in specific processes, such as stomatal movement. Altogether, transcriptomic analysis suggests that the different drought resistances were correlated with the differential expression of genes, while the drought tolerance of W9706 is due to the more rapid response to stimulus, higher water retention capacity and stable cellular environment under water deficit conditions.

Rapid, Highly Sensitive, and Label-Free Pathogen Assay System Using a Solid-Phase Self-Interference Recombinase Polymerase Amplification Chip and Hyperspectral Interferometry.

Recombinase polymerase amplification (RPA) is a useful pathogen identification method. Several label-free detection methods for RPA amplicons have been developed in recent years. However, these methods still lack sensitivity, specificity, efficiency, or simplicity. In this study, we propose a rapid, highly sensitive, and label-free pathogen assay system based on a solid-phase self-interference RPA chip (SiSA-chip) and hyperspectral interferometry. The SiSA-chips amplify and capture RPA amplicons on the chips, rather than irrelevant amplicons such as primer dimers, and the SiSA-chips are then analysed by hyperspectral interferometry. Optical length increases of SiSA-chips are used to demonstrate RPA detection results, with a limit of detection of 1.90 nm. This assay system can detect as few as six copies of the target 18S rRNA gene of Plasmodium falciparum within 20 min, with a good linear relationship between the detection results and the concentration of target genes (R2 = 0.9903). Single nucleotide polymorphism (SNP) genotyping of the dhfr gene of Plasmodium falciparum is also possible using the SiSA-chip, with as little as 1% of mutant gene distinguished from wild-type loci (m/wt). This system offers a high-efficiency (20 min), high-sensitivity (6 copies/reaction), high-specificity (1% m/wt), and low-cost (∼1/50 of fluorescence assays for RPA) diagnosis method for pathogen DNA identification. Therefore, this system is promising for fast identification of pathogens to help diagnose infectious diseases, including SNP genotyping.

Single cell capture, isolation, and long-term in-situ imaging using quantitative self-interference spectroscopy.

Single cell research with microfluidic chip is of vital importance in biomedical studies and clinical medicine. Simultaneous microfluidic cell manipulations and long-term cell monitoring needs further investigations due to the lack of label-free quantitative imaging techniques and systems. In this work, single cell capture, isolation and long-term in-situ monitoring was realized with a microfluidic cell chip, compact cell incubator and quantitative self-interference spectroscopy. The proposed imaging method could obtain quantitative and dynamic refractive index distribution in living cells. And the designed microfluidic chip could capture and isolate single cells. The customized incubator could support cell growth conditions when single cell was captured in microfluidic chip. According to the results, single cells could be trapped, transferred and pushed into the culture chamber with the microfluidic chip. The incubator could culture single cells in the chip for 120 h. The refractive index sensitivity of the proposed quantitative imaging method was 0.0282 and the relative error was merely 0.04%.

Tissue-level transcriptomic responses to local and distal chilling reveal potential chilling survival mechanisms in maize.

Chilling is a major stress to plants of subtropical and tropical origins including maize (Zea mays L.). To reveal molecular mechanisms underlying chilling tolerance and survival, we investigated transcriptomic responses to chilling stress in differentiated leaves and roots as well as in crowns with meristem activity in maize. Chilling stress on shoots and roots is found to each contributes to seedling lethality in maize. Comparison of maize lines with different chilling tolerance capacities reveals that chilling survival is highly associated with upregulation of abscisic acid biosynthesis and response as well as transcriptional regulators in leaves and crowns. It is also associated with the downregulation of translation in leaves and heat response in crowns. Chilling treatment on whole or part of the plants reveals that response to distal-chilling is very distinct from, and sometimes opposite to, response to local- or whole-plant chilling in both leaves and roots, suggesting a communication between shoots and roots in environmental response. This study thus provides transcriptomic responses in leaves, roots and crowns under differential chilling stresses in maize and reveals potential chilling tolerance and survival mechanisms which lays ground for improving chilling tolerance in crop plants.

Regulation of the activity of maize glutamate dehydrogenase by ammonium and potassium.

Glutamate dehydrogenase (GDH) is an important enzyme in ammonium metabolism, the activity of which is regulated by multiple factors. In this study, we investigate the effects of ammonium and potassium on the activity of maize GDH. Our results show that both ammonium and potassium play multiple roles in regulating the activity of maize GDH, with the specific roles depending on the concentration of potassium. Together with the structural information of GDH, we propose models for the substrate inhibition of ammonium, and the elimination of substrate inhibition by potassium. These models are supported by the analysis of statistic thermodynamics. We also analyze the binding sites of ammonium and potassium on maize GDH, and the conformational changes of maize GDH. The findings provide insight into the regulation of maize GDH activity by ammonium and potassium and reveal the importance of the dose and ratio of nitrogen and potassium in crop cultivation.

Transcriptomic analysis reveals somatic embryogenesis-associated signaling pathways and gene expression regulation in maize (Zea mays L.).

KEY MESSAGE: Transcriptome analysis of maize embryogenic callus and somatic embryos reveals associated genes reprogramming, hormone signaling pathways and transcriptional regulation involved in somatic embryogenesis in maize. Somatic embryos are widely utilized in propagation and genetic engineering of crop plants. In our laboratory, an elite maize inbred line Y423 that could generate intact somatic embryos was obtained and applied to genetic transformation. To enhance our understanding of regulatory mechanisms during maize somatic embryogenesis, we used RNA-based sequencing (RNA-seq) to characterize the transcriptome of immature embryo (IE), embryogenic callus (EC) and somatic embryo (SE) from maize inbred line Y423. The number of differentially expressed genes (DEGs) in three pairwise comparisons (IE-vs-EC, IE-vs-SE and EC-vs-SE) was 5767, 7084 and 1065, respectively. The expression patterns of DEGs were separated into eight major clusters. Somatic embryogenesis associated genes were mainly grouped into cluster A or B with an expression trend toward up-regulation during dedifferentiation. GO annotation and KEGG pathway analysis revealed that DEGs were implicated in plant hormone signal transduction, stress response and metabolic process. Among the differentially expressed transcription factors, the most frequently represented families were associated with the common stress response or related to cell differentiation, embryogenic patterning and embryonic maturation processes. Genes include hormone response/transduction and stress response, as well as several transcription factors were discussed in this study, which may be potential candidates for further analyses regarding their roles in somatic embryogenesis. Furthermore, the temporal expression patterns of candidate genes were analyzed to reveal their roles in somatic embryogenesis. This transcriptomic data provide insights into future functional studies, which will facilitate further dissections of the molecular mechanisms that control maize somatic embryogenesis.

New Derivatives from Microbial Transformation of ent-Kaur-16-en-19-oic Acid by Cunninghamella echinulata.

Biotransformation of ent-kaur-16-en-19-oic acid using fungus Cunninghamella echinulata resulted in two novel hydroxylated metabolites together with five known compounds. Their structures were elucidated by means of extensive NMR and HR-ESI-MS data analysis. The eight compounds were measured for their cytotoxicity against the human breast carcinoma (MCF-7) and human hepatoblastoma (HepG-2) cell lines. Seven compounds showed no cytotoxicity to the two cell lines. One compound displayed moderate cytotoxicity against HepG-2 and MCF-7 with the IC50 values of 12.6 and 27.1 µM, respectively.

iTRAQ-Based Proteomic Analysis Reveals Several Strategies to Cope with Drought Stress in Maize Seedlings.

Drought stress, especially during the seedling stage, seriously limits the growth of maize and reduces production in the northeast of China. To investigate the molecular mechanisms of drought response in maize seedlings, proteome changes were analyzed. Using an isotopic tagging relative quantitation (iTRAQ) based method, a total of 207 differentially accumulated protein species (DAPS) were identified under drought stress in maize seedlings. The DAPS were classified into ten essential groups and analyzed thoroughly, which involved in signaling, osmotic regulation, protein synthesis and turnover, reactive oxygen species (ROS) scavenging, membrane trafficking, transcription related, cell structure and cell cycle, fatty acid metabolism, carbohydrate and energy metabolism, as well as photosynthesis and photorespiration. The enhancements of ROS scavenging, osmotic regulation, protein turnover, membrane trafficking, and photosynthesis may play important roles in improving drought tolerance of maize seedlings. Besides, the inhibitions of some protein synthesis and slowdown of cell division could reduce the growth rate and avoid excessive water loss, which is possible to be the main reasons for enhancing drought avoidance of maize seedlings. The incongruence between protein and transcript levels was expectedly observed in the process of confirming iTRAQ data by quantitative real-time polymerase chain reaction (qRT-PCR) analysis, which further indicated that the multiplex post-transcriptional regulation and post-translational modification occurred in drought-stressed maize seedlings. Finally, a hypothetical strategy was proposed that maize seedlings coped with drought stress by improving drought tolerance (via. promoting osmotic adjustment and antioxidant capacity) and enhancing drought avoidance (via. reducing water loss). Our study provides valuable insight to mechanisms underlying drought response in maize seedlings.

iTRAQ-Based Quantitative Proteomic Analysis of Embryogenic and Non-embryogenic Calli Derived from a Maize (Zea mays L.) Inbred Line Y423.

Somatic embryos (SE) have potential to rapidly form a whole plant. Generally, SE is thought to be derived from embryogenic calli (EC). However, in maize, not only embryogenic calli (EC, can generate SE) but also nonembryogenic calli (NEC, can't generate SE) can be induced from immature embryos. In order to understand the differences between EC and NEC and the mechanism of EC, which can easily form SE in maize, differential abundance protein species (DAPS) of EC and NEC from the maize inbred line Y423 were identified by using the isobaric tags for relative and absolute quantification (iTRAQ) proteomic technology. We identified 632 DAPS in EC compared with NEC. The results of bioinformatics analysis showed that EC development might be related to accumulation of pyruvate caused by the DAPS detected in some pathways, such as starch and sucrose metabolism, glycolysis/gluconeogenesis, tricarboxylic acid (TCA) cycle, fatty acid metabolism and phenylpropanoid biosynthesis. Based on the differentially accumulated proteins in EC and NEC, a series of DAPS related with pyruvate biosynthesis and suppression of acetyl-CoA might be responsible for the differences between EC and NEC cells. Furthermore, we speculate that the decreased abundance of enzymes/proteins involved in phenylpropanoid biosynthesis pathway in the EC cells results in reducing of lignin substances, which might affect the maize callus morphology.

Isolation and functional characterization of a cold responsive phosphatidylinositol transfer-associated protein, ZmSEC14p, from maize (Zea may L.).

KEY MESSAGE: A Sec14-like protein, ZmSEC14p , from maize was structurally analyzed and functionally tested. Overexpression of ZmSEC14p in transgenic Arabidopsis conferred tolerance to cold stress. Sec14-like proteins are involved in essential biological processes, such as phospholipid metabolism, signal transduction, membrane trafficking, and stress response. Here, we reported a phosphatidylinositol transfer-associated protein, ZmSEC14p (accession no. KT932998), isolated from a cold-tolerant maize inbred line using the cDNA-AFLP approach and RACE-PCR method. Full-length cDNA that consisted of a single open reading frame (ORF) encoded a putative polypeptide of 295 amino acids. The ZmSEC14p protein was mainly localized in the nucleus, and its transcript was induced by cold, salt stresses, and abscisic acid (ABA) treatment in maize leaves and roots. Overexpression of ZmSEC14p in transgenic Arabidopsis conferred tolerance to cold stress. This tolerance was primarily displayed by the increased germination rate, root length, plant survival rate, accumulation of proline, activities of antioxidant enzymes, and the reduction of oxidative damage by reactive oxygen species (ROS). ZmSEC14p overexpression regulated the expression of phosphoinositide-specific phospholipase C, which cleaves phosphatidylinositol 4,5-bisphosphate (PIP2) and generates second messengers (inositol 1,4,5-trisphosphate and 1,2-diacylglycerol) in the phosphoinositide signal transduction pathways. Moreover, up-regulation of some stress-responsive genes such as CBF3, COR6.6, and RD29B in transgenic plants under cold stress could be a possible mechanism for enhancing cold tolerance. Taken together, this study strongly suggests that ZmSEC14p plays an important role in plant tolerance to cold stress.

Intracellular Biosynthesis of Fluorescent CdSe Quantum Dots in Bacillus subtilis: A Strategy to Construct Signaling Bacterial Probes for Visually Detecting Interaction Between Bacillus subtilis and Staphylococcus aureus.

In this work, fluorescent Bacillus subtilis (B. subtilis) cells were developed as probes for imaging applications and to explore behaviorial interaction between B. subtilis and Staphylococcus aureus (S. aureus). A novel biological strategy of coupling intracellular biochemical reactions for controllable biosynthesis of CdSe quantum dots by living B. subtilis cells was demonstrated, through which highly luminant and photostable fluorescent B. subtilis cells were achieved with good uniformity. With the help of the obtained fluorescent B. subtilis cells probes, S. aureus cells responded to co-cultured B. subtilis and to aggregate. The degree of aggregation was calculated and nonlinearly fitted to a polynomial model. Systematic investigations of their interactions implied that B. subtilis cells inhibit the growth of neighboring S. aureus cells, and this inhibition was affected by both the growth stage and the amount of surrounding B. subtilis cells. Compared to traditional methods of studying bacterial interaction between two species, such as solid culture medium colony observation and imaging mass spectrometry detection, the procedures were more simple, vivid, and photostable due to the efficient fluorescence intralabeling with less influence on the cells' surface, which might provide a new paradigm for future visualization of microbial behavior.

Liquid Metal Memory.

Storage systems are vital components of electronic devices, while significant challenges persist in achieving flexible memory due to the limitations of existing storage methodologies. Inspired by the polarization and depolarization mechanisms in the human brain, here a novel class of storage principles is proposed and achieve a fully flexible memory through introducing the oxidation and deoxidation behaviors of liquid metals. Specifically, reversible electrochemical oxidation is utilized to modulate the overall conductivity of the target liquid metals, creating a substantial 11-order resistance difference for binary data storage. To obtain the best storage performance, systematic optimizations of multiple parameters are conducted. Conceptual experiments demonstrate the memory's stability under extreme deformations (100% stretching, 180° bending, 360° twisting). Further tests reveal that the memory performs better when its unit size gets smaller, warranting superior integrability. Finally, a complete storage system achieves remarkable performance metrics, including rapid storage speed (>33 Hz), long data retention capacity (>43200 s), and stable repeatable operation (>3500 cycles). This groundbreaking method not only overcomes the inherent rigidity limitations of existing electronic storage units but also opens new possibilities for innovating neuromorphic devices, offering fundamental and practical avenues for future applications in soft robotics, wearable electronics, and bio-inspired artificial intelligence systems.

Multi-stimulus perception and visualization by an intelligent liquid metal-elastomer architecture.

Multi-stimulus responsive soft materials with integrated functionalities are elementary blocks for building soft intelligent systems, but their rational design remains challenging. Here, we demonstrate an intelligent soft architecture sensitized by magnetized liquid metal droplets that are dispersed in a highly stretchable elastomer network. The supercooled liquid metal droplets serve as microscopic latent heat reservoirs, and their controllable solidification releases localized thermal energy/information flows for enabling programmable visualization and display. This allows the perception of a variety of information-encoded contact (mechanical pressing, stretching, and torsion) and noncontact (magnetic field) stimuli as well as the visualization of dynamic phase transition and stress evolution processes, via thermal and/or thermochromic imaging. The liquid metal-elastomer architecture offers a generic platform for designing soft intelligent sensing, display, and information encryption systems.

In Situ Fabricated Liquid Metal Capacitors for Plant Sensing.

Capacitive sensors are essential to promoting modernization and intelligence in agriculture. With the continuous advancement of this sensor technology, the demand for materials with high conductivity and flexibility is rapidly increasing. Herein, we introduce liquid metal as a solution for the in-site fabrication of high-performance capacitive sensors for plant sensing. As a comparison, three pathways have been proposed for the preparation of flexible capacitors inside plants, as well as on their surfaces. Specifically, concealed capacitors can be constructed by directly injecting liquid metal into the plant cavity. Printable capacitors are prepared via printing Cu-doped liquid metal with better adhesion on plant surfaces. A composite liquid metal-based capacitive sensor is achieved by printing liquid metal on the plant surface and injecting it into the interior of the plant. While each method has limitations, the composite liquid metal-based capacitive sensor provides an optimal trade-off between signal capture capability and operability. As a result, this composite capacitor is chosen as a sensor for monitoring water changes within plants and demonstrates the desired sensing performance, making it a promising technology for monitoring plant physiology.

A Photothermally Enhanced Vancomycin-Coated Liquid Metal Antimicrobial Agent with Targeting Capability.

The targeted antimicrobial efficacy of Vancomycin decreases significantly over time due to bacterial resistance, whereas Ga-based liquid metals, which are less prone to inducing bacterial resistance, face challenges in achieving targeted antimicrobial effects. To tackle these issues, a highly efficient antimicrobial agent with targeting properties has been developed by combining Ga-based liquid metals and Vancomycin. Moreover, the performance of this antimicrobial agent can be greatly enhanced through the use of near-infrared light. Microscopic observations reveal that Vancomycin can be effectively encapsulated on the surface of liquid metal, facilitated by the presence of the oxide layer. The resulting core-shell structured antimicrobial agent demonstrates notable targeted antimicrobial effects against S. aureus. Antibacterial tests indicate that Vancomycin effectively improves the antibacterial properties of pure liquid metal. Additionally, this study unveils the excellent photothermal conversion capabilities of liquid metal, enabling the antimicrobial agent exposed to 808nm near-infrared light to exhibit significantly strengthened bactericidal performance. In this scenario, the antimicrobial agent can achieve nearly 100% effectiveness. This work enriches the investigation of integrating Ga-based antimicrobial agents with traditional antibiotics, showcasing promising antibacterial effects and establishing the groundwork for subsequent clinical applications.

Transcriptomic response for revealing the molecular mechanism of oat flowering under different photoperiods.

Proper flowering is essential for the reproduction of all kinds of plants. Oat is an important cereal and forage crop; however, its cultivation is limited because it is a long-day plant. The molecular mechanism by which oats respond to different photoperiods is still unclear. In this study, oat plants were treated under long-day and short-day photoperiods for 10 days, 15 days, 20 days, 25 days, 30 days, 40 days and 50 days, respectively. Under the long-day treatment, oats entered the reproductive stage, while oats remained vegetative under the short-day treatment. Forty-two samples were subjected to RNA-Seq to compare the gene expression patterns of oat under long- and short-day photoperiods. A total of 634-5,974 differentially expressed genes (DEGs) were identified for each time point, while the floral organ primordium differentiation stage showed the largest number of DEGs, and the spikelet differentiation stage showed the smallest number. Gene Ontology (GO) analysis showed that the plant hormone signaling transduction and hormone metabolism processes significantly changed in the photoperiod regulation of flowering time in oat. Moreover, Kyoto Encyclopedia of Genes and Genomes (KEGG) and Mapman analysis revealed that the DEGs were mainly concentrated in the circadian rhythm, protein antenna pathways and sucrose metabolism process. Additionally, transcription factors (TFs) involved in various flowering pathways were explored. Combining all this information, we established a molecular model of oat flowering induced by a long-day photoperiod. Taken together, the long-day photoperiod has a large effect at both the morphological and transcriptomic levels, and these responses ultimately promote flowering in oat. Our findings expand the understanding of oat as a long-day plant, and the explored genes could be used in molecular breeding to help break its cultivation limitations in the future.

Responsive Liquid Metal Droplets: From Bulk to Nano.

Droplets exist widely in nature and play an extremely important role in a broad variety of industrial processes. Typical droplets, including water and oil droplets, have received extensive attention and research, however their single properties still cannot meet diverse needs. Fortunately, liquid metal droplets emerging in recent years possess outstanding properties, including large surface tension, excellent electrical and thermal conductivity, convenient chemical processing, easy transition between liquid and solid phase state, and large-scale deformability, etc. More interestingly, liquid metal droplets with unique features can respond to external factors, including the electronic field, magnetic field, acoustic field, chemical field, temperature, and light, exhibiting extraordinary intelligent response characteristics. Their development over the past decade has brought substantial breakthroughs and progress. To better promote the advancement of this field, the present article is devoted to systematically summarizing and analyzing the recent fundamental progress of responsive liquid metal droplets, not only involving droplet characteristics and preparation methods, but also focusing on their diverse response behaviors and mechanisms. On this basis, the challenges and prospects related to the following development of liquid metal droplets are also proposed. In the future, responsive liquid metal droplets with a rapid development trend are expected to play a key role in soft robots, biomedicine, smart matter, and a variety of other fields.

A nature-inspired hierarchical branching structure pressure sensor with high sensitivity and wide dynamic range for versatile medical wearables.

Pressure-sensing capability is essential for flexible electronic devices, which require high sensitivity and a wide detection range to simplify the system. However, the template-based pressure sensor is powerless to detect high pressure due to the rapid deformation saturation of microstructures. Herein, we demonstrated that a nature-inspired hierarchical branching (HB) structure can effectively address this problem. Finite element analysis demonstrates that the HB structure permits a step-by-step mobilization of microstructure deformation, resulting in a dramatically improved sensitivity (up to 2 orders of magnitude) when compared with the traditional monolayer structure. Experiments show that the HB structure enables pressure sensors to have a lower elastic modulus (1/3 of that of monolayer sensors), a high sensitivity of 13.1 kPa-1 (almost 14 times higher than the monolayer sensor), and a wide dynamic range (0-800 kPa, the minimum detection pressure is 1.6 Pa). The maximum frequency that the sensor can detect is 250 Hz. The response/recovery time is 0.675/0.55 ms respectively. Given this performance, the HB sensor enables high-resolution detection of the weak radial artery pulse wave characteristics in different states, indicating its potential to noninvasively reveal cardiovascular status and the effectiveness of related interventions, such as exercise and drug intervention. As a proof of concept, we also verified that the HB sensor can serve as a versatile platform to support diverse applications from low to high pressure.

Mutation in Mg-Protoporphyrin IX Monomethyl Ester (Oxidative) Cyclase Gene ZmCRD1 Causes Chlorophyll-Deficiency in Maize.

Chlorophyll molecules are non-covalently associated with chlorophyll-binding proteins to harvest light and perform charge separation vital for energy conservation during photosynthetic electron transfer in photosynthesis for photosynthetic organisms. The present study characterized a pale-green leaf (pgl) maize mutant controlled by a single recessive gene causing chlorophyll reduction throughout the whole life cycle. Through positional mapping and complementation allelic test, Zm00001d008230 (ZmCRD1) with two missense mutations (p.A44T and p.T326M) was identified as the causal gene encoding magnesium-protoporphyrin IX monomethyl ester cyclase (MgPEC). Phylogenetic analysis of ZmCRD1 within and among species revealed that the p.T326M mutation was more likely to be causal. Subcellular localization showed that ZmCRD1 was targeted to chloroplasts. The pgl mutant showed a malformed chloroplast morphology and reduced number of starch grains in bundle sheath cells. The ZmCRD1 gene was mainly expressed in WT and mutant leaves, but the expression was reduced in the mutant. Most of the genes involved in chlorophyll biosynthesis, chlorophyll degradation, chloroplast development and photosynthesis were down-regulated in pgl. The photosynthetic capacity was limited along with developmental retardation and production reduction in pgl. These results confirmed the crucial role of ZmCRD1 in chlorophyll biosynthesis, chloroplast development and photosynthesis in maize.

Miniaturized microfluidic-based nucleic acid analyzer to identify new biomarkers of biopsy lung cancer samples for subtyping.

Identifying new biomarkers is necessary and important to diagnose and treat malignant lung cancer. However, existing protein marker detection methods usually require complex operation steps, leading to a lag time for diagnosis. Herein, we developed a rapid, minimally invasive, and convenient nucleic acid biomarker recognition method, which enabled the combined specific detection of 11 lung cancer typing markers in a microliter reaction system after only one sampling. The primers for the combined specific detection of 11 lung cancer typing markers were designed and screened, and the microfluidic chip for parallel detection of the multiple markers was designed and developed. Furthermore, a miniaturized microfluidic-based analyzer was also constructed. By developing a microfluidic chip and a miniaturized nucleic acid analyzer, we enabled the detection of the mRNA expression levels of multiple biomarkers in rice-sized tissue samples. The miniaturized nucleic acid analyzer could detect ≥10 copies of nucleic acids. The cell volume of the typing reaction on the microfluidic chip was only 0.94 µL, less than 1/25 of that of the conventional 25-µL Eppendorf tube PCR method, which significantly reduced the testing cost and significantly simplified the analysis of multiple biomarkers in parallel. With a simple injection operation and reverse transcription loop-mediated isothermal amplification (RT-LAMP), real-time detection of 11 lung cancer nucleic acid biomarkers was performed within 45 min. Given these compelling features, 86 clinical samples were tested using the miniaturized nucleic acid analyzer and classified according to the cutoff values of the 11 biomarkers. Furthermore, multi-biomarker analysis was conducted by a machine learning model to classify different subtypes of lung cancer, with an average area under the curve (AUC) of 0.934. This method shows great potential for the identification of new nucleic acid biomarkers and the accurate diagnosis of lung cancer.