A ddPCR platform based on a microfluidic chip with a dual-function flow-focusing structure for sample-to-result DNA quantification analysis.

Droplet digital PCR (ddPCR) is a powerful method for absolute nucleic acid quantification with high precision and accuracy. However, complicated operational steps have hampered the use and diffusion of ddPCR. Therefore, an automated, easy-to-use, low-sample-consumption, and portable ddPCR platform is urgently needed. This paper proposes a microfluidic ddPCR platform based on a microfluidic chip that can realize the sample-to-result function by switching the rotary valve, achieving the dual function of the flow-focusing structure for droplet generation and readout. Sample, generation oil, and analysis oil were pre-added to the reservoirs. Droplets were generated due to focusing flow, and after passing through the integrated temporary storage bin in the rotary valve, the droplets and oil subsequently entered the collecting tube, improving the droplet-to-oil volume ratio for enhanced thermal cycle performance. Droplets with an average diameter of 107.44 µm and a CV of 2.38% were generated using our chip under the optimal pressures. High-performance thermal cycling was achieved through improvements of the droplet-to-oil volume ratio of the sample, the integrated heating lid, the pure copper heating base, and the temperature-controlling algorithm. Gradient quantification experiments were conducted for the HER2 and CEP17 genes extracted from breast cancer cells, yielding strong linear correlations with R2 values of 0.9996 for FAM and 0.9989 for CY5. Moreover, pronounced linearity was obtained between the detected concentrations of HER2 and CEP17, indicated by a slope of 1.0091 and an R2 of 0.9997, signifying consistent HER2 : CEP17 ratios across various sample dilutions. The outcomes of the quantitative analysis, encompassing the dynamic range and the consistency of the HER2 : CEP17 ratio using our ddPCR platform, meet the standards required for breast cancer assessment and therapy. Our ddPCR platform is automated, portable, and capable of stable droplet generation, high-efficiency amplification, realization of the sample-to-result function based on dual-function flow-focusing structure, and accuracy absolute quantification, underscoring its significant potential for ddPCR analysis in clinical diagnostics.

Deep-Orga: An improved deep learning-based lightweight model for intestinal organoid detection.

PROBLEM: Organoids are 3D cultures that are commonly used for biological and medical research in vitro due to their functional and structural similarity to source organs. The development of organoids can be assessed by morphological tests. However, manual analysis of organoid morphology requires intensive labor from professionals and is prone to observer discrepancies. AIM: Computer-assisted methods alleviate the pressure of manual labor, especially with the development of deep learning, the performance of morphological detection has been further improved. The aim of this paper is to automate the assessment of organoid morphology using deep learning techniques to reduce the labor pressure of professionals. METHODS: Based on the lightweight model YOLOX, a lightweight intestinal organoid detection model named Deep-Orga is proposed. First, the performance of the Deep-Orga model is compared with other classical models on the intestinal organoids dataset. Then, ablation experiments are used to validate the improvement of the model detection performance by the improved module. Finally, Deep-Orga is compared with other methods. RESULTS: Deep-Orga achieves optimal organoid detection with a partial increase in computational effort. Using Deep-Orga to replace the manual analysis process provides a new automated method for organoid morphology evaluation. CONCLUSION: Deep-Orga proposed in this paper is able to accurately assess organoid development, effectively relieving the labor pressure of professionals and avoiding the subjectivity of assessment. This paper demonstrates the potential application of deep learning in the field of organoid morphology analysis.

Establishment and Validation of an Integrated Microfluidic Step Emulsification Chip Supporting Droplet Digital Nucleic Acid Analysis.

Uniform and stable droplet generation is critical for accurate and efficient digital nucleic acid analysis (dNAA). In this study, an integrated microfluidic step emulsification device with wide-range droplet generation capability, small device dimensions, convenient fabrication strategy, low contamination and high robustness was developed. A tree-shaped droplet generation nozzle distribution design was proposed to increase the uniformity of droplet generation by equating flow rates, and the flow field in the design was numerically simulated. Theoretical analysis and comparative experiments on droplet size were performed regarding the influences of nozzle dimensions and surface properties. With incubation and hydrophobic reagent treatment, droplets as small as 73.1 µm were generated with multiplex nozzles of 18 µm (h) × 80 µm (w). The droplets were then collected into a standard PCR tube and an on-chip monolayer droplet collection chamber, without manual transfer and sample contamination. The oil-to-sample volume ratio in the PCR tube was recorded during collection. In the end, the droplets generated and collected using the microfluidic device proved to be stable and uniform for nucleic acid amplification and detection. This study provides reliable characteristic information for the design and fabrication of a micro-droplet generation device, and represents a promising approach for the realization of a three-in-one dNAA device under a step emulsification method.

Synthesis of uniform Pickering microspheres doped with quantum dot by microfluidic technology and its application in tumor marker.

Tumor markers play a significant role in early cancer diagnosis, evaluation of the extent of the disease, and monitoring of therapy response. In this study, we described the Pickering emulsion polymerization method to synthesize uniform magnetic/fluorescent microspheres. A Pickering-structure composed of a lot silica nanoparticle closely covered onto the quantum dot-encoded magnetic microbeads is designed and synthesized. The uniform magnetic/fluorescent microspheres were prepared using a microfluidic device and the performance of the microspheres synthesized by the instruments was evaluated by flow cytometry. To avoid fluorescence quenching and intrinsic toxicity, CdSe/ZnS core-shell quantum dot and Fe3O4 nanoparticle were successfully encapsulated into MFM microspheres using the microfluidic technology. Using this structure enables the facile realization of a theoretical 4 × 4 barcoding matrix combining two colors and four fluorescence intensity levels. Then, different optical codes were prepared by simple changing the emission wavelength and the intensity of the quantum dots. The resulting microsphere are combined with flow cytometer using two lasers for decoding of multiplex tumor markers. Moreover, the stability testing of microspheres demonstrated good performance for further application in detection of tumor markers as well. When applied for the high-throughput ultrasensitive detection of three tumor markers (CEA, CA125 and CA199) in a single sample, the detection limits of 0.027 ng/mL for CEA, 1.48 KU/L for CA125 and 1.09 KU/L for CA199 are achieved, which exhibit superior detection performance. Thus, Pickering-structure magnetic/fluorescent microspheres are promising for application in tumor markers.

DNA-functionalized gold nanoparticles: Modification, characterization, and biomedical applications.

With the development of technologies based on gold nanoparticles (AuNPs), bare AuNPs cannot meet the increasing requirements of biomedical applications. Modifications with different functional ligands are usually needed. DNA is not only the main genetic material, but also a good biological material, which has excellent biocompatibility, facile design, and accurate identification. DNA is a perfect ligand candidate for AuNPs, which can make up for the shortcoming of bare AuNPs. DNA-modified AuNPs (DNA-AuNPs) have exciting features and bright prospects in many fields, which have been intensively investigated in the past decade. In this review, we summarize the various approaches for the immobilization of DNA strands on the surface of AuNPs. Representative studies for biomedical applications based on DNA-AuNPs are also discussed. Finally, we present the challenges and future directions.

Fluorescence DNA Switch for Highly Sensitive Detection of miRNA Amplified by Duplex-Specific Nuclease.

DNA is a type of promising material for the construction of sensors owing to its sequence programmability to control the formation of certain structures. MicroRNA (miRNA) can be applied as promising biomarkers for the diagnosis of a range of diseases. Herein, a novel fluorescent sensing strategy for miRNA is proposed combining duplex-specific nuclease (DSN)-mediated amplification and dumbbell DNA structural switch. Gold nanoparticles (AuNPs) are employed, which provide a 3D reaction interface. They also act as effective fluorescence quenchers. The proposed sensor exhibits high sensitivity (sub-femtomolar level) with a wide dynamic range. In addition, excellent selectivity to distinguish homology sequences is achieved. It also performs satisfactorily in biological samples. Overall, this fluorescent sensor provides a powerful tool for the analysis of miRNA levels and can be applied for related biological studies and clinical diagnosis.

[Design of Dyson Flow Cytometry System].

In order to meet the needs of the flow cytometry for the simultaneous analysis of multiple fluorescence wavelengths and small volume, the design method of flow cytometry spectrum analysis system is presented by analyzing the characteristics of Dyson structure. And according to the method, a flow cytometry spectrum analysis system is disigned with Dyson type.The system's spectral range is 400 nm to 800 nm, the defocused spot size is less than the pixel size 24µ mm, the ransfer function value is above 0.8 at the Nyquist cut-off frequency 21 lp/mm,the spectral resolution is less than 3 nm, and the overall size is 83.54 mm×85.60 mm.The system has good optical performance and small volume, which meets the needs of the flow cytometry fluorescence spectral analysis. The outstanding innovation of this system is the application of Dyson light splitting structure and EMCCD detector which is high speed and high sensitivity.

Electrochemical detection of aqueous Ag+ based on Ag+-assisted ligation reaction.

In this work, a novel strategy to fabricate a highly sensitive and selective biosensor for the detection of Ag(+) is proposed. Two DNA probes are designed and modified on a gold electrode surface by gold-sulfur chemistry and hybridization. In the presence of Ag(+), cytosine-Ag(+)-cytosine composite forms and facilitates the ligation event on the electrode surface, which can block the release of electrochemical signals labeled on one of the two DNA probes during denaturation process. Ag(+) can be sensitively detected with the detection limit of 0.1 nM, which is much lower than the toxicity level defined by U.S. Environmental Protection Agency. This biosensor can easily distinguish Ag(+) from other interfering ions and the performances in real water samples are also satisfactory. Moreover, the two DNA probes are designed to contain the recognition sequences of a nicking endonuclease, and the ligated DNA can thus be cleaved at the original site. Therefore, the electrode can be regenerated, which allows the biosensor to be reused for additional tests.

[High-precision 3D morphology measurement by digital gatling method based on structured light].

In order to measure the microscopic 3D morphology of the objects with high-precision, a 3D texture measurement system of digital gatling based on structured light was designed, which can calculate the 3D height information with the analytic phase method. First, the authors collected sixteen equal step phase images by the four-step equal step method, and calculated their main value by dividing them into four groups. Then, the authors found the average as the final phase main value. The pretreatment on the fringe was done by the adaptive Wiener filter and wavelet multi-threshold method to eliminate the various effects of noise, projector distortion and CCD camera distortion. Besides, gradient-oriented phase unwrapping algorithm based on multifrequency was introduced to avoid phase discontinuity point in the course phase unwrapping, and it was proven to be effective and stable. Experiments showed that the system's 3D resolution was 2.75 microm, and the high degree accuracy was better than 0.5 microm, when the system was running with the fringe parameter p0 = 22.7 mm(-1). In addition, the system has many advantages such as fast measuring, simple operation and non-contact, which can meet the need of the high precision measurement requirements for the microscopic 3D morphology.