One-step and label-free ratiometric fluorescence assay for the detection of plasma exosome towards cancer diagnosis.

Exosomes are closely associated with tumor development and are regarded as viable biomarkers for cancer. Here, a ratiometric fluorescence method was proposed for the one-step and label-free detection of plasma exosomes. A bicolor streptavidin magnetic beads were specifically created with an immobilized Cy5-labeled hairpin aptamer for CD63 (Cy5-Apt) on its surface to identify exosome, and a green color SYBR Green I (SGI) embedded in the stem of Cy5-Apt to respond to exosomes. After exosome capture, the Cy5-Apt could undergo a conformational shift and release the encapsulated SGI, allowing exosome measurement based on the fluorescence ratio of Cy5 and SGI. The enrichment, separation and detection of exosomes in proposed method could be completed in one step (30 min), which is a significant improvement over previous method. Furthermore, the use of ratiometric fluorescence and magnetic separation allows for exosome enrichment and interference elimination from complex matrices, improving accuracy and sensitivity. Particularly, the assay could detect exosomes in plasma and has potential to distinguish lung cancer patients from healthy volunteers with an area under the receiver operator characteristic curve of 0.85. Besides, the study provided an efficient method for analyzing the various divisions of exosomes by merely modifying the aptamer, which holds great promise for point-of-care applications.

Spatial Control of Receptor Dimerization Using Programmable DNA Nanobridge.

Receptor dimerization is an essential mechanism for the activation of most receptor tyrosine kinases by ligands. Thus, regulating the nanoscale spatial distribution of cell surface receptors is significant for studying both intracellular signaling pathways and cellular behavior. However, there are currently very limited methods for exploring the effects of modulating the spatial distribution of receptors on their function by using simple tools. Herein, we developed an aptamer-based double-stranded DNA bridge acting as "DNA nanobridge", which regulates receptor dimerization by changing the number of bases. On this basis, we confirmed that the different nanoscale arrangements of the receptor can influence receptor function and its downstream signals. Among them, the effect gradually changed from helping to activate to inhibiting as the length of DNA nanobridge increased. Hence, it can not only effectively inhibit receptor function and thus affect cellular behavior but also serve as a fine-tuning tool to get the desired signal activity. Our strategy is promising to provide insight into the action of receptors in cell biology from the perspective of spatial distribution.

A SERS bioassay based on vancomycin-modified PEI-interlayered nanocomposite and aptamer-functionalized SERS tags for synchronous detection of Acinetobacter baumannii and Klebsiella pneumoniae.

Klebsiella pneumoniae (KP) and Acinetobacter baumannii (AB) are two important gram-negative bacteria that cause pneumonia and have been recently known to be associated with food. The rapid detection of these pathogens in food is important to minimize their colonization of the gut and stop new threats of the disease from spreading across the food chain. Herein, a double-edged sword aptasensor was developed for the synchronous detection of KP and AB in food and clinical samples. A highly sensitive, selective, specific, and synchronous detection of the target bacteria was achieved, and the limit of detection (LOD) was 10 cells/mL with a liner range of 50 to 105 cells/mL. The total assay time was 1.5 h. This study does not only provide a new tool for the detection of the target bacteria, but also serves as a promising tool for food safety and pneumonia diagnosis.

A review of sensors for classification and subtype discrimination of cancer: Insights into circulating tumor cells and tumor-derived extracellular vesicles.

Liquid biopsy can reflect the state of tumors in vivo non-invasively, thus providing a strong basis for the early diagnosis, individualized treatment monitoring and prognosis of tumors. Circulating tumor cells (CTCs) and tumor-derived extracellular vesicles (tdEVs) contain information-rich components, such as nucleic acids and proteins, and they are essential markers for liquid biopsies. Their capture and analysis are of great importance for the study of disease occurrence and development and, consequently, have been the subject of many reviews. However, both CTCs and tdEVs carry the biological characteristics of their original tissue, and few reviews have focused on their function in the staging and classification of cancer. In this review, we focus on state-of-the-art sensors based on the simultaneous detection of multiple biomarkers within CTCs and tdEVs, with clinical applications centered on cancer classification and subtyping. We also provide a thorough discussion of the current challenges and prospects for novel sensors with the ultimate goal of cancer classification and staging. It is hoped that these most advanced technologies will bring new insights into the clinical practice of cancer screening and diagnosis.

Magnetic-nanowaxberry-based microfluidic ExoSIC for affinity and continuous separation of circulating exosomes towards cancer diagnosis.

Exosomes are seen as promising biomarkers for minimally invasive liquid biopsies and disease surveillance. However, the complexity of body fluids, inherent heterogeneity, and tiny size of exosomes impede their extraction, consequently restricting their clinical application. In this study, in order to efficiently isolate exosomes from clinical samples, an irregular serpentine channel microfluidic chip (ExoSIC) was designed to continuously separate exosomes from plasma based on a magnetic-nanowaxberry (MNWB). In the ExoSIC, irregular serpentine microchannels are utilized to increase fluid chaotic mixing, hence improving exosome capture efficiency. In comparison to commonly used spherical magnetic particles, the designed MNWB can not only enhance the capture efficiency of exosomes, but also possess a size-exclusion effect to improve exosome purity. Consequently, the ExoSIC exhibited a large yield (24 times higher than differential centrifugation), optimum purity (greater than precipitation and similar to differential centrifugation), and high specificity. Furthermore, the ExoSIC was utilized for plasma-based cancer diagnosis by multiplex monitoring of five exosomal biomarkers (exosomal concentration, EGFR, EpCAM, SAA1 and FV), and the AUC reached 0.791. This work provides a comprehensive framework for exosome-based cancer diagnostics in order to meet clinical requirements for exosome isolation and downstream analysis.

Locus-Specific Detection of DNA Methylation: The Advance, Challenge, and Perspective of CRISPR-Cas Assisted Biosensors.

Deoxyribonucleic acid (DNA) methylation is one of the epigenetic characteristics that result in heritable and revisable phenotype changes but without sequence changes in DNA. Aberrant methylation occurring at a specific locus was reported to be associated with cancers, insulin resistance, obesity, Alzheimer's disease, Parkinson's disease, etc. Therefore, locus-specific DNA methylation can serve as a valuable biomarker for disease diagnosis and therapy. Recently, Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas systems are applied to develop biosensors for DNA, ribonucleic acid, proteins, and small molecules detection. Because of their highly specific binding ability and signal amplification capacity, CRISPR-Cas assisted biosensor also serve as a potential tool for locus-specific detection of DNA methylation. In this perspective, based on the detection principle, a detailed classification and comprehensive discussion of recent works about the latest advances in locus-specific detection of DNA methylation using CRISPR-Cas systems are provided. Furthermore, current challenges and future perspectives of CRISPR-based locus-specific detection of DNA methylation are outlined.

New horizons in the identification of circulating tumor cells (CTCs): An emerging paradigm shift in cytosensors.

Circulating tumor cells (CTCs) are cancer cells that are shed from a primary tumor into the bloodstream and function as seeds for cancer metastasis at distant locations. Enrichment and identification methods of CTCs in the blood of patients plays an important role in diagnostic assessments and personalized treatments of cancer. However, the current traditional identification methods not only impact the viability of cells, but also cannot determine the type of cancer cells when the disease is unknown. Hence, new methods to identify CTCs are urgently needed. In this context, many advanced and safe technologies have emerged to distinguish between cancer cells and blood cells, and to distinguish specific types of cancer cells. In this review, at first we have briefly discussed recent advances in technologies related to the enrichment of CTCs, which lay a good foundation for the identification of CTCs. Next, we have summarized state-of-the-art technologies to confirm whether a given cell is indeed a tumor cell and determine the type of tumor cell. Finally, the challenges for application and potential directions of the current identification methods in clinical analysis of CTCs have been discussed.

A digital immuno-PCR assay for simultaneous determination of 5-methylcytosine and 5-hydroxymethylcytosine in human serum.

This work aimed to develop an ultrasensitive and specific immunosorbent assay for simultaneous detection of double DNA methylation marks. Being considered the most important indicators in disease diagnosis, clinical treatment, and prognosis, 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) were chosen as the proof-of-concept targets. The described strategy consisted of Phos-tag Biotin anchoring at streptavidin-magnetic nanoparticles, specific immune recognition of anti-5mC antibody and anti-5hmC antibody and labeling of Barcode-antibody, signal amplification of immune PCR and digital PCR machine. Under optimal conditions, the digital immuno-PCR assay showed a board dynamic range from 2.7 × 10-13 mol/L to 2.7 × 10-9 mol/L and the detection limits were 61.7 fmol/L for 5mC, and of 0.111 pmol/L for 5hmC. A 16-fold and 186-fold improvement of LOD were obtained by the proposed approach for 5mC and 5hmC detection compared with real-time immune PCR. The approach also showed ideal specificity, repeatability and stability. The recovery test demonstrated that the digital immuno-PCR assay is a promising platform for the simultaneous determination of the two DNA methylation marks in human serum sample.

State-of-the-arts techniques and current evolving approaches in the separation and detection of circulating tumor cell.

Circulating tumor cells (CTCs) are cancer cells that shed from the primary tumor and then enter the circulatory system, a small part of which may evolve into metastatic cancer under appropriate microenvironment conditions. The detection of CTCs is a truly noninvasive, dynamic monitor for disease changes, which has considerable clinical implications in the selection of targeted drugs. However, their inherent rarity and heterogeneity pose significant challenges to their isolation and detection. Even the "gold standard", CellSearch™, suffers from high expenses, low capture efficiency, and the consumption of time. With the advancement of CTCs analysis technologies in recent years, the yield and efficiency of CTCs enrichment have gradually been improved, as well as detection sensitivity. In this review, the isolation and detection strategies of CTCs have been completely described and the potential directions for future research and development have also been highlighted through analyzing the challenges faced by current strategies.

Neutrophil-Dependent Immunity During Pulmonary Infections and Inflammations.

Rapid recruitment of neutrophils to an inflamed site is one of the hallmarks of an effective host defense mechanism. The main pathway through which this happens is by the innate immune response. Neutrophils, which play an important part in innate immune defense, migrate into lungs through the modulation actions of chemokines to execute a variety of pro-inflammatory functions. Despite the importance of chemokines in host immunity, little has been discussed on their roles in host immunity. A holistic understanding of neutrophil recruitment, pattern recognition pathways, the roles of chemokines and the pathophysiological roles of neutrophils in host immunity may allow for new approaches in the treatment of infectious and inflammatory disease of the lung. Herein, this review aims at highlighting some of the developments in lung neutrophil-immunity by focusing on the functions and roles of CXC/CC chemokines and pattern recognition receptors in neutrophil immunity during pulmonary inflammations. The pathophysiological roles of neutrophils in COVID-19 and thromboembolism have also been summarized. We finally summarized various neutrophil biomarkers that can be utilized as prognostic molecules in pulmonary inflammations and discussed various neutrophil-targeted therapies for neutrophil-driven pulmonary inflammatory diseases.

A simple "signal off-on" fluorescence nanoplatform for the label-free quantification of exosome-derived microRNA-21 in lung cancer plasma.

A simple nanoplatform based on molybdenum disulfide (MoS2) nanosheets, a fluorescence quencher (signal off), and a hybridization chain reaction (HCR) signal amplification (signal on) used for the enzyme-free, label-free, and low-background signal quantification of microRNA-21 in plasma exosome is reported. According to the sequence of microRNA-21, carboxy-fluorescein (FAM)-labeled hybridization probe 1 (FAM-H1) and hybridization probes 2 (FAM-H2) were designed with excitation maxima at 488 nm and emission maxima at 518 nm. MoS2 nanosheets could adsorb FAM-H1 and FAM-H2 and quenched their fluorescence signals to reduce the background signal. However, HCR was triggered when microRNA-21 was present. Consequently, HCR products containing a large number of FAM fluorophores can emit a strong fluorescence at 518 nm and could realize the detection of microRNA-21 as low as 6 pmol/L and had a wide linear relation of 0.01-25 nmol/L. This assay has the ability of single-base mismatch recognition and could identify microRNA-21 with high specificity. Most importantly, this approach was successfully applied to the detection of plasma exosomal microRNA-21 in patients with lung cancer, and it is proposed that other targets can also be detected by changing the FAM-H1 and FAM-H2 corresponding to the target sequence. Thus, a novel, hands-on strategy for liquid biopsy was proposed and has a potential application value in the early diagnosis of lung cancer.

In situ detection of plasma exosomal microRNA for lung cancer diagnosis using duplex-specific nuclease and MoS2 nanosheets.

MicroRNAs (miRNAs) encapsulated in tumor-derived exosomes are becoming ideal biomarkers for the early diagnosis and prognosis of lung cancer. However, the accuracy and sensitivity are often hampered by the extraction process of exosomal miRNA using traditional methods. Herein, this study developed a fluorogenic quantitative detection method for exosomal miRNA using the fluorescence quenching properties of molybdenum disulfide (MoS2) nanosheets and the enzyme-assisted signal amplification properties of duplex-specific nuclease (DSN). First, a fluorescently-labeled nucleic acid probe was used to hybridize the target miRNA to form a DNA/RNA hybrid structure. Under the action of the DSN, the DNA single strand in the DNA/RNA hybrid strand was selectively digested into smaller oligonucleotide fragments. At the same time, the released miRNA target triggers the next reaction cycle, so as to achieve signal amplification. Then, MoS2 was used to selectively quench the fluorescence of the undigested probe leaving the fluorescent signal of the fluorescently-labeled probe fragments. The fluorometric signals for miRNA-21 had a maximum excitation/emission wavelength of 488/518 nm. Most importantly, the biosensor was then applied for the accurate quantitative detection of miRNA-21 in exosome lysates extracted from human plasma and this method was able to successfully distinguish lung cancer patients from healthy people. This biosensor provides a simple, rapid, and a highly specific quantitative method for exosomal miRNA and has promising potential to be used in the early diagnosis of lung cancer.

Effect of vitamin D supplementation on fasting plasma glucose, insulin resistance and prevention of type 2 diabetes mellitus in non-diabetics: A systematic review and meta-analysis.

Increasing epidemiological studies suggest that there is an association between vitamin D deficiency and risk of type 2 diabetes mellitus (T2DM). Therefore, randomized clinical trials (RCTs) have been performed to observe the effect of vitamin D supplementation on preventing T2DM, decreasing fasting plasma glucose (FPG) and improving insulin resistance to confirm the association between vitamin D and T2DM. However, the results of RCTs on controlling FPG level, improving insulin resistance and preventing T2DM in non-diabetics are inconsistent. In the present study, a systematic meta-analysis considering individual variation and intervention strategy was conducted to establish an objective and definitive conclusion. The results suggested that vitamin D supplementation had no significant effect on controlling FPG level, improving insulin resistance or preventing T2DM in non-diabetics in a pooled meta-analysis of 23 articles (containing 28 RCTs). However, stratified analysis indicated that supplementation of vitamin D had differential effects on FPG control, insulin sensitivity improvement and T2DM prevention in individuals with different baseline states: FPG was decreased for those with BMI <25 (P=0.048) or 20≤ 25(OH)D <30 ng/ml (P=0.002); insulin resistance was improved for those with 25(OH)D ≥30 ng/ml (P=0.021); and risk of T2DM was lower for pre-diabetic individuals (P=0.047) or for those with 25≤ BMI <30 (P=0.032). Additionally, the effect on T2DM prevention was improved when the supplement dose was >2,000 IU/day (P=0.047) and with intervention without calcium (P=0.047). Thus, further trials should focus on individual baselines and the supplementation strategy of vitamin D in the prevention of T2DM.