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.

[Design of a dynamic measurement system for Bl factor spatial distribution of resonant blood viscoelastic sensor].

Bl factor is a key system parameter of the resonant blood viscoelastic sensor. In this paper, a dynamic measurement system for the spatial distribution of Bl factor based on velocity amplitude and motional impedance was designed. The system extracted the velocity amplitude and motional impedance of the coil under the dynamic condition of driving the sensor to generate simple harmonic oscillations using laser displacement and impedance analysis combined with in-phase/quadrature demodulation algorithm, and controlled the equilibrium position of the coil by adjusting the direct current component of the excitation current to realize the position scanning. In the position interval of [-240, 240] µm, the maximum coefficient of variation of the measurement results was 0.077 3%, and the maximum relative error to the simulation results was 2.937 9%, with a linear fitting correlation coefficient R 2 = 0.996 8. The system can be used to accurately measure the spatial distribution of Bl factor of the resonant blood viscoelastic sensor, which provides a technical support for the verification of the design of the sensor magnetic circuit.

Prediction of Sleep Apnea Events Using a CNN-Transformer Network and Contactless Breathing Vibration Signals.

It is estimated that globally 425 million subjects have moderate to severe obstructive sleep apnea (OSA). The accurate prediction of sleep apnea events can offer insight into the development of treatment therapies. However, research related to this prediction is currently limited. We developed a covert framework for the prediction of sleep apnea events based on low-frequency breathing-induced vibrations obtained from piezoelectric sensors. A CNN-transformer network was utilized to efficiently extract local and global features from respiratory vibration signals for accurate prediction. Our study involved overnight recordings of 105 subjects. In five-fold cross-validation, we achieved an accuracy of 85.9% and an F1 score of 85.8%, which are 3.5% and 5.3% higher than the best-performed classical model, respectively. Additionally, in leave-one-out cross-validation, 2.3% and 3.8% improvements are observed, respectively. Our proposed CNN-transformer model is effective in the prediction of sleep apnea events. Our framework can thus provide a new perspective for improving OSA treatment modes and clinical management.

[Design and experiment of online monitoring system for long-term culture of embryo].

In the study of embryo development process, the morphological features at different stages are essential to evaluate developmental competence of the embryo, which can be used to optimize and improve the system for in-vitro embryo culture. In this paper, an online monitoring system was designed for long-term culture of embryos, based on a monitoring strategy of low-magnification search and high-magnification observation. Three optical modules of 4× phase contrast, 10× and 20× Hoffman modulation phase contrast were configured in this system to meet the requirements of different fields of view, especially when the size of the embryo increases during the culture. Using an optomechanical system matching design, an error control and alignment test, the resolution of optical imaging was guaranteed, and a relief stereoscopic imaging with high contrast of embryos was obtained. Through low-magnification field of view to identify and locate embryos and high-magnification field of view to capture the details, the system realized online tracking and monitoring of embryos. In addition, we developed and verified an embryo identifying and locating algorithm based on image contour area and definition evaluation. The online monitoring system of in-vitro embryo culture proposed in this paper can track and record the morphological features of embryos without affecting the embryo development, providing a basis for the assessment of embryo development and the optimization of in-vitro culture system.

[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.

Identification of glutathione by voltammetric analysis with rolling circle amplification.

Glutathione (GSH), a common tripeptide, plays an essential role in a variety of cellular functions. GSH level is reported to be closely related to human health. In this study, we fabricate an ultrasensitive electrochemical biosensor for GSH quantification. DNA probes are firstly modified on the electrode surface and thymine-Hg2+-thymine is formed. Since GSH is able to chelate Hg2+ from the DNA mismatched sites effectively, which leads to DNA structural switching from hairpin to linear strand, rolling circle amplification (RCA) could be initiated with the released linear primer probe. The RCA product with multiple repeating sequences further captures numerous DNA modified silver nanoparticles (AgNPs) by the hybridization of complementary sequences. Stripping voltammetric responses of AgNPs are then detected to reveal GSH concentration. The linear detection range is from 0.1 pM to 10 nM and the limit of detection is 0.1 pM, which is lower than most current analytical methods. This method is also highly selective and functions well against a series of interferents. Additionally, the proposed method has been successfully utilized in human serum samples, which shows fairly good potential in clinical applications.

Near-Infrared Ag2S Quantum Dots-Based DNA Logic Gate Platform for miRNA Diagnostics.

Dysregulation of miRNA expression is correlated with the development and progression of many diseases. These miRNAs are regarded as promising biomarkers. However, it is challenging to measure these low abundant molecules without employing time-consuming radioactive labeling or complex amplification strategies. Here, we present a DNA logic gate platform for miRNA diagnostics with fluorescence outputs from near-infrared (NIR) Ag2S quantum dots (QDs). Carefully designed toehold exchange-mediated strand displacements with different miRNA inputs occur on a solid-state interface, which control QDs release from solid-state interface to solution, responding to multiplex information on initial miRNAs. Excellent fluorescence emission properties of NIR Ag2S QDs certify the great prospect for amplification-free and sensitive miRNA assay. We demonstrate the potential of this platform by achieving femtomolar level miRNA analysis and the versatility of a series of logic circuits computation.

DNA tetrahedron and star trigon nanostructures for target recycling detection of nucleic acid.

Human immunodeficiency virus (HIV) is a retrovirus which attacks the human body's immune system and further leads to acquired immunodeficiency syndrome (AIDS). Nucleic acid detection is of great importance in the medical diagnosis of such diseases. Herein, we develop a simple and enzyme-free electrochemical method for the target recycling detection of nuclei acid. DNA tetrahedron and star trigon nanostructures are designed and constructed on the electrode interface for target capture and signal enrichment. This strategy is convenient and sensitive, with a limit of detection as low as 1 fM, and can also successfully distinguish single-base mismatched DNA. Therefore, the proposed method has a promising potential application for HIV DNA detection.

An elastography analytical method for the rapid detection of endotoxin.

We present a flexible analytical method for the study of coagulation systems by monitoring elastography (EG). The rapid detection of endotoxin is achieved by the EG analysis of endotoxin-induced limulus amebocyte lysate coagulation. This method is superior to other methods using the same reagents in not only sensitivity but also detecting time.

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.

Tetrahedral DNA nanostructure-based microRNA biosensor coupled with catalytic recycling of the analyte.

MicroRNAs are not only important regulators of a wide range of cellular processes but are also identified as promising disease biomarkers. Due to the low contents in serum, microRNAs are always difficult to detect accurately . In this study, an electrochemical biosensor for ultrasensitive detection of microRNA based on tetrahedral DNA nanostructure is developed. Four DNA single strands are engineered to form a tetrahedral nanostructure with a pendant stem-loop and modified on a gold electrode surface, which largely enhances the molecular recognition efficiency. Moreover, taking advantage of strand displacement polymerization, catalytic recycling of microRNA, and silver nanoparticle-based solid-state Ag/AgCl reaction, the proposed biosensor exhibits high sensitivity with the limit of detection down to 0.4 fM. This biosensor shows great clinical value and may have practical utility in early diagnosis and prognosis of certain diseases.

Ultrasensitive detection of microRNA through rolling circle amplification on a DNA tetrahedron decorated electrode.

MicroRNAs are a class of evolutionally conserved, small noncoding RNAs involved in the regulation of gene expression and affect a variety of biological processes including cellular differentiation, immunological response, tumor development, and so on. Recently, microRNAs have been identified as promising disease biomarkers. In this work, we have fabricated a novel electrochemical method for ultrasensitive detection of microRNA. Generally, a DNA tetrahedron decorated gold electrode is employed as the recognition interface. Then, hybridizations between DNA tetrahedron, microRNA, and primer probe initiate rolling circle amplification (RCA) on the electrode surface. Silver nanoparticles attached to the RCA products provide significant electrochemical signals and a limit of detection as low as 50 aM is achieved. Moreover, homology microRNA family members with only one or two mismatches can be successfully distinguished. Therefore, this proposed method reveals great advancements toward improved disease diagnosis and prognosis.

Ultrasensitive electrochemical detection of microRNA with star trigon structure and endonuclease mediated signal amplification.

MicroRNAs play important roles in gene regulation. They can be used as effective biomarkers for diagnosis and prognosis of diseases like cancers. Due to their intrinsic properties of short length, low abundance and sequence homology among family members, it is difficult to realize sensitive and selective detection with economical use of time and cost. Herein, we report an ultrasensitive electrochemical method for microRNA analysis employing two oligonucleotides and one endonuclease. Generally, a glassy carbon electrode is first covered with gold nanoparticles (AuNPs) mediated by poly(diallyldimethylammonium chloride) (PDDA). Then, thiolated capture probe (CP) with methylene blue (MB) labeled at 5' end is modified on the pretreated electrode. Hybridization occurs among target microRNA, CP and auxiliary probe (AP), forming a star trigon structure on the electrode surface. Subsequently, endonuclease recognizes and cleaves CP on CP/AP duplex, releasing microRNA and AP back to the solution. The two regenerated elements can then form another star trigon with other CP molecules, initiating cycles of CP cleavage and MB departure. Significant decrease of electrochemical signals is thus observed, which can be used to reflect the concentration of microRNA. This proposed method has a linear response to microRNA in a wide range from 100 aM to 1 nM and the sensitivity of attomolar level can be achieved. Moreover, it has high selectivity against single-base mismatch sequences and can be used directly in serum samples. Therefore, this method shows great feasibility for the detection of microRNA and may have potential applications in cancer diagnosis and prognosis.

Recent advances in carbon nanodots: synthesis, properties and biomedical applications.

Herein, a mini review is presented concerning the most recent research progress of carbon nanodots, which have emerged as one of the most attractive photoluminescent materials. Different synthetic methodologies to achieve advanced functions and better photoluminescence performances are summarized, which are mainly divided into two classes: top-down and bottom-up. The inspiring properties, including photoluminescence emission, chemiluminescence, electrochemical luminescence, peroxidase-like activity and toxicity, are discussed. Moreover, the biomedical applications in biosensing, bioimaging and drug delivery are reviewed.

An aptasensor for detection of potassium ions based on RecJ(f) exonuclease mediated signal amplification.

An electrochemical biosensor for potassium has been developed combining specific potassium-aptamer binding and RecJf exonuclease mediated signal amplification. Generally, the DNA probe with a stem-loop structure containing an anti-K(+) aptamer sequence is designed and modified on a gold electrode. K(+) can specifically bind to the aptamer and a G-quadruplex structure forms, which breaks the original stem-loop structure. The induced single-stranded 5' end can be further digested by RecJf exonuclease, releasing K(+) which can bind to another DNA probe on the electrode. After cycles of RecJf exonuclease cleavage initiated by K(+), the electrochemical signal intensity is significantly decreased, and can be used to determine the concentration of K(+). This aptasensor shows high sensitivity, selectivity as well as excellent stability and accuracy, which provides possibilities for further applications of K(+) assay in clinical diagnosis.

Melamine functionalized silver nanoparticles as the probe for electrochemical sensing of clenbuterol.

Clenbuterol, a member of ß-agonist family, has now been a serious threat to human health due to its illegal usage in the livestock feeding. Herein, we describe the application of melamine functionalized silver nanoparticles (M-AgNPs) as the electrochemical probe for simple, fast, highly sensitive and selective detection of clenbuterol. Generally, AgNPs are prepared and functionalized by melamine. After interacting with melamine modified gold electrode in the presence of clenbuterol, M-AgNPs can be immobilized on the surface of the electrode via the hydrogen-bonding interactions between clenbuterol and melamine. This sandwich structure permits sensitive and selective detection of clenbuterol. Since M-AgNPs can provide a couple of well-defined sharp silver stripping peaks, which stands for a highly characteristic solid-state Ag/AgCl reaction, a rather low detection limit of 10 pM can be achieved. The detection range is from 10 pM to 100 nM, which is quite wide. This developed biosensor can potentially be used for clenbuterol detection in biological fluids in the presence of various interferences.

[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.