Single-Nucleus RNA-Seq: Open the Era of Great Navigation for FFPE Tissue.

Single-cell sequencing (scRNA-seq) has revolutionized our ability to explore heterogeneity and genetic variations at the single-cell level, opening up new avenues for understanding disease mechanisms and cell-cell interactions. Single-nucleus RNA-sequencing (snRNA-seq) is emerging as a promising solution to scRNA-seq due to its reduced ionized transcription bias and compatibility with richer samples. This approach will provide an exciting opportunity for in-depth exploration of billions of formalin-fixed paraffin-embedded (FFPE) tissues. Recent advancements in single-cell/nucleus gene expression workflows tailored for FFPE tissues have demonstrated their feasibility and provided crucial guidance for future studies utilizing FFPE specimens. In this review, we provide a broad overview of the nuclear preparation strategies, the latest technologies of snRNA-seq applicable to FFPE samples. Finally, the limitations and potential technical developments of snRNA-seq in FFPE samples are summarized. The development of snRNA-seq technologies for FFPE samples will lay a foundation for transcriptomic studies of valuable samples in clinical medicine and human sample banks.

Simultaneously responsive microfluidic chip aptasensor for determination of kanamycin, aflatoxin M1, and 17ß-estradiol based on magnetic tripartite DNA assembly nanostructure probes.

The authors describe a microfluidic chip-based aptasensor platform combined with magnetic tripartite DNA structure-functionalized nanocomposites to achieve simultaneous determination of kanamycin (KANA), aflatoxin M1 (AFM1), and 17ß-estradiol (E2) in milk. The two-duplex tripartite DNA nanostructure was first assembled on the surface of magnetic beads. When the aptamer on the probes recognized the specific target, the aptamer-target would be released into the supernatant. The pre-primer@circular DNA template structure initiates rolling circle amplification (RCA) by phi29 polymerase. After magnetic separation, the magnetic nanocomposites were added into a solution containing three different lengths of complementary strands to the RCA products. The number of complementary strands significantly decrease, and this can be quantitated by the microfluidic chip. Further, the employment of magnetic nanocomposites and microfluidic chip not only resolve the complex matrix interference, but also dramatically enhances the determination selectivity and sensitivity. This aptasensor allows for determination of KANA, AFM1, and E2 with limits of detection as low as 0.32 pg mL-1, 0.95 pg mL-1, and 6.8 pg mL-1, respectively. This novel method exhibits the advantages of excellent stability and fast response time (< 3 min on microfluidic chip platform) for simultaneous determination of KANA, AFM1, and E2 in milk samples and ensures food safety. Graphical abstract.

A microfluidic chip based ratiometric aptasensor for antibiotic detection in foods using stir bar assisted sorptive extraction and rolling circle amplification.

A ratiometric and sensitive microfluidic chip based aptasensor was developed for antibiotic detection with kanamycin (Kana) as a model analyte. A novel stir bar assisted sorptive extraction and rolling circle amplification strategy was designed to largely amplify the signal and overcome complex matrix interference in food samples. The detection mechanism was as follows: firstly, many duplex DNA probes (a single-stranded DNA as a primer hybrid with an aptamer sequence) were modified on a stir bar. In the presence of Kana, the probes on the bar could specifically capture Kana and release the primer to trigger RCA in the presence of a circular DNA template (CDT). As the reaction proceeds, the amount of CDT decreased and the number of RCA products increased. It is worth mentioning that they can be efficiently separated and detected using a microfluidic chip. The signal ratio of RCA products and CDT (IR/IC) can be employed to qualify Kana in a wide linear range from 0.8 pg mL-1 to 10 ng mL-1 with a low detection limit of 0.3 pg mL-1. This method exhibited excellent sensitivity and selectivity and can obviously reduce the matrix interference through a ratiometric strategy combined with stir bar extraction. The aptasensor was successfully tested in milk and fish samples, confirming that it can be applied for on-site quantitation of antibiotic residues in foods.

Spatial transcriptomics: recent developments and insights in respiratory research.

The respiratory system's complex cellular heterogeneity presents unique challenges to researchers in this field. Although bulk RNA sequencing and single-cell RNA sequencing (scRNA-seq) have provided insights into cell types and heterogeneity in the respiratory system, the relevant specific spatial localization and cellular interactions have not been clearly elucidated. Spatial transcriptomics (ST) has filled this gap and has been widely used in respiratory studies. This review focuses on the latest iterative technology of ST in recent years, summarizing how ST can be applied to the physiological and pathological processes of the respiratory system, with emphasis on the lungs. Finally, the current challenges and potential development directions are proposed, including high-throughput full-length transcriptome, integration of multi-omics, temporal and spatial omics, bioinformatics analysis, etc. These viewpoints are expected to advance the study of systematic mechanisms, including respiratory studies.

The universal dual-mode aptasensor for simultaneous determination of different bacteria based on naked eyes and microfluidic-chip together with magnetic DNA encoded probes.

It was critically important to develop some sensitive, convenient and on-site methods for simultaneous assay of different pathogenic bacteria in foods. In this work, a dual-mode aptasensor was established for fulfilling above aims combing colorimetry with microfluidic chip. This as-prepared dual-mode aptasensor not only realized rapid screening by naked eye on-site, but also the simultaneous quantification of multiple bacteria. Namely, the presence of pathogenic bacteria was firstly judged by naked eyes with Salmonella typhimurium (S.T) and Vibrio parahaemolyticus (V.P) as models. And then, S.T and V.P in positive samples were simultaneously quantified by microfluidic chip. In order to obtain the multiple signals, a series of magnetic DNA encoded-probes (MDEs) was fabricated containing rolling cycle amplified long DNA chain (RCA-DNA) rich in G-quadruplex sequences. They can combine with hemin as DNAzyme to catalyze 3,3'-5,5'-Tetramethyl benzidine (TMB)-H2O2 system for color development and be cleaved by EcoRV endonuclease to produce DNA fragments with different lengths. The microfluidic chip was employed to separate and quantify the fragments for quantifying S.T and V.P simultaneously. For this protocol, 100 CFU·mL-1 of V.P or S.T could be observed by the naked eye and as low as 32 S.T and 30 CFU·mL-1 V.P could be detected by the chip within 3 min. The dual-mode aptasensor could quickly screen positive samples, and simultaneously perform quantitative detection of the bacteria in positive samples. Our protocol demonstrated its potential in on-site qualification & simultaneous quantification of foodborne bacteria in foods.

Highly sensitive and simultaneous detection of microRNAs in serum using stir-bar assisted magnetic DNA nanospheres-encoded probes.

There are critical interests in the detection of microRNA (miRNA) because it can be a blood-borne biomarker, but analytical strategies are still limited by its small size, high sequence homology among family members and low abundance. In this work, three-dimensional magnetic DNA nanospheres were synthesized and immobilized on a gold stir-bar as encoded probes for miRNA capture and signal amplification. Electrochemical tags-labeled DNAs were immobilized on gold coated magnetic nanospheres via a hyperbranched hybridization chain reaction (HHCR). Subsequently, the magnetic DNA nanospheres were immobilized on the gold stir-bar as encoded probes. Target miRNAs were captured on the surface of the stir-bar by replacing the magnetic DNA nanospheres-encoded probes, and the probes were magnetically enriched for highly sensitive and selective electrochemical detection. The gold stir-bar assisted magnetic DNA nanospheres-encoded probes possess dual functions: They are as a nanocarrier to increase the loading amounts of HHCR products, and they are also a platform for efficient electrochemical signal amplification via magnetic enrichment. The method was successfully applied for the detection of miRNA21 and miRNA155 in a wide linear range of 5 fM to 2 nM, and with detection limits of 1.5 fM and 1.8 fM, respectively. The preliminary application of the method suggests that it has great potential in the detection of miRNAs in serum samples.

Performance characterization of integral imaging systems based on human vision.

The perceptual contrast threshold (PCT) surface is proposed for characterizing the systematic performance of integral imaging (InI) systems. The method to determine the PCT surface of InI systems is presented first. The theoretical model of the PCT surface is then derived by considering the integral contribution of an InI system as well as the human visual system. Preliminary simulated results show that the PCT can correctly describe the InI properties related to the lateral resolution, contrast, depth distance, and their trade-off relationships, which can be used to compare the systematic performance of InI systems with different design parameters. To the best of our knowledge, this is the first study of the relationship of the resolvable InI contrast with the lateral resolution and the object depth.

Zero background and triple-signal amplified fluorescence aptasensor for antibiotics detection in foods.

It's important to eliminate matrix interference for accurate detecting antibiotic residues in complex food samples. In this study, we designed a zero-backgrounded fluorescence aptasensor to achieve on-site detection of antibiotic residues, with chloramphenicol (CAP) as representative analyte. Moreover, a three stir-bars assisted target recycling system (TSBTR) was designed to achieve triple signal amplification and increase the sensitivity. The bars included one magnetic stir-bar modified with two kinds of long DNA chains, and two gold stir-bars modified with Y shape-duplex DNA probes respectively. In the presence of CAP, the target could recurrently react with the probes on the bars and replace a large amount of long DNA chains into supernatant. After then, the bars were taken out and SYBR green dye was added to the solution. The dye can specifically intercalate into the duplex structures of DNA chains to emit fluorescence while not emitting a signal in its free state. Under the optimized experimental conditions, a wide linear response range of 5 orders of magnitude from 0.001 ng mL-1 to 10 ng mL-1 was achieved with a detection limit of 0.033 pg mL-1 CAP. The assay was successfully employed to detect CAP in food samples (milk & fish) with consistent results with ELISA's. High selectivity and sensitivity were attributed to the zero background signal and triple signal-amplification strategy. Moreover, the detection time can be shortened to 40 min due to that three signal amplified process can occur simultaneously. The fluorescent aptasensor was also label- and enzyme-free. All these ensure the platform to be rapid, cost-effective, easily-used, and is especially appropriate for detection antibiotics in food safety.

Multiplexed electrochemical aptasensor for antibiotics detection using metallic-encoded apoferritin probes and double stirring bars-assisted target recycling for signal amplification.

Simultaneous and sensitive detection of various antibiotic residues in one sample is essential to evaluation of food safety status. Herein, a multiplexed electrochemical aptasensor for multiplex antibiotics detection, with kanamycin (KANA) and ampicillin (AMP) as representative analytes, was designed by using metal ions encoded apoferrtin probes and double stirring bars-assisted target recycling for signal amplification. The encoded probes were prepared by apoferritin loading Cd2+ and Pb2+ ions and labeling with duplex DNAs (aptamers corresponding to KANA and AMP hybrid with its complementary DNA sequence), respectively. In the presence of KANA and AMP, the targets can recurrently react with the probes on the bars, and then replace a lot of Apo-Mencoded signal tags into supernatant. The peak currents of Cd2+and Pb2+from the tags corresponding with the concentrations of KANA and AMP were detected by square wave voltammetry in one run. As a result, KANA and AMP can be detected simultaneously within the range from 0.05 pM to 50 nM. And the detection limits were 18 fM KANA and 15 fM AMP (S/N = 3). The assay was testified to detect KANA and AMP residues with consistent results of ELISA in samples, e.g. milks and fishes. The assay was highly-sensitive, selective, cost-effective and easy-to-operate due to Apo-M encoded probes with high loading capacity of signal source substances. Moreover, double stirring bar-assisted target recycling, which was enzyme-free and could overcome matrix interference, was fabricated for signal amplification. Thus, the assay showed potential advantages for sensitively screening of antibiotic residues in foods.

Fibronectin Adsorption on Hydroxyapatite Nanosensors and the Effect of Fibronectin Preadsorption on Biological Apatite Growth.

In this study, hydroxyapatite (HA) nanoparticles were prepared at 30 °C by the chemical precipitation method. The adsorption behavior of fibronectin (Fn) on Au and HA surfaces at different Fn concentrations (50, 100, and 200 µg mL-1 and the effect of fibronectin preadsorption on the apatite growth process were investigated by the quartz crystal microbalance technique. The surface properties of Au and HA were characterized by atomic force microscopy and field emission scanning electron microscopy. Fn adsorption experiments show that the adsorption capacity of Fn on Au and HA surfaces is in the order Au-Fn200> Au-Fn100≈ HA-Fn100≈ HA-Fn200> HA-Fn50> Au-Fn50, where the number after Fn corresponds to the concentration of the Fn solution. The order of the viscoelasticity of the adsorbed layer is HA-Fn200> HA-Fn100> Au-Fn200> HA-Fn50> Au-Fn50> Au-Fn100. These results suggest that the Fn concentration and the chemical composition and topography of the substrate surface affect Fn adsorption and the viscoelasticity of the adsorbed Fn layer. A high Fn concentration is not conducive to Fn adsorption on the HA surface and formation of a viscoelastic Fn layer. Based on the experimental results, the states and orientations of Fn adsorbed on Au and HA are rationally discussed. Mineralization experiments show that the order of the mass of the apatite layer is HA > HA-Fn50 > Au-Fn50¼ Au. The apatite growth rate on the Au surface is essentially zero, whereas the apatite growth rate on the Au-Fn50 surface linearly increases. For the HA and HA-Fn50 surfaces, the apatite growth rate can be divided into several stages. An adsorbed Fn layer affects apatite nucleation on both the Au and HA surfaces and decreases the apatite growth rate on the HA surface.

Preparation of a beta-tricalcium phosphate nanocoating and its protein adsorption behaviour by quartz crystal microbalance with dissipation technique.

Beta-tricalcium phosphate (ß-TCP) nanocoatings, which can be analysed using a quartz crystal microbalance with dissipation technique (QCM-D), were fabricated on a gold surface by electrophoretic deposition. The influences of electric field intensity and electrophoresis time were investigated. The adsorption behaviours of bovine serum albumin (BSA) and lysozyme (LSZ) on Au and ß-TCP surfaces were observed in real time by QCM-D. The homogeneous ß-TCP nanocoating with moderately sized particles on gold surface was fabricated at 25V/cm for 5min, and it met the requirements for the QCM-D experiment. The adsorption behaviour of BSA was different from that of LSZ, which was caused by the differences of protein properties. The adsorption quantity of BSA on a ß-TCP surface was higher than that on a gold surface. However, the adsorption amount of LSZ on a ß-TCP surface was lower than that on a gold surface. The electrostatic force was the major factor affecting the adsorption quantities of BSA and LSZ on Au and ß-TCP surfaces based on the investigation of various factors. The findings reported here will be useful for understanding the mechanism of the interaction between biomaterials and proteins.