Liposomes in the cosmetics: present and outlook.

Liposomes are small spherical vesicles composed of phospholipid bilayers capable of encapsulating a variety of ingredients, including water- and oil-soluble compound, which are one of the most commonly used piggybacking and delivery techniques for many active ingredients and different compounds in biology, medicine and cosmetics. With the increasing number of active cosmetic ingredients, the concomitant challenge is to effectively protect, transport, and utilize these substances in a judicious manner. Many cosmetic ingredients are ineffective both topically and systemically when applied to the skin, thus changing the method of delivery and interaction with the skin of the active ingredients is a crucial step toward improving their effectiveness. Liposomes can improve the delivery of active ingredients to the skin, enhance their stability, and ultimately, improve the efficacy of cosmetics and and pharmaceuticals. In this review, we summarized the basic properties of liposomes and their recent advances of functionalities in cosmetics and and pharmaceuticals. Also, the current state of the art in the field is discussed and the prospects for future research areas are highlighted. We hope that this review will provide ideas and inspiration on the application and development of cosmetics and pharmaceuticals.

CRISPR-Cas12a coupled with universal gold nanoparticle strand-displacement probe for rapid and sensitive visual SARS-CoV-2 detection.

Point of care (POC) diagnosis of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are particularly significant for preventing transmission of coronavirus disease 2019 (COVID-19) by any user at any given time and place. CRISPR/Cas-assisted SARS-CoV-2 assays are viewed as supplemental to RT-PCR due to simple operation, convenient use and low cost. However, most current CRISPR molecular diagnostics based on fluorescence measurement increased the difficulty of POC test with need of the additional light sources. Some instrument-free visual detection with the naked eye has limitations in probe universality. Herein, we developed a universal, rapid, sensitive and specific SARS-CoV-2 POC test that combines the outstanding DNase activity of Cas12a with universal AuNPs strand-displacement probe. The oligo trigger, which is the switch the AuNPs of the strand-displacement probe, is declined as a result of Cas12a recognition and digestion. The amount of released AuNPs produced color change which can be visual with the naked eye and assessed by UV-Vis spectrometer for quantitative detection. Furthermore, a low-cost hand warmer is used as an incubator for the visual assay, enabling an instrument-free, visual SARS-CoV-2 detection within 20 min. A real coronavirus GX/P2V instead of SARS-CoV-2 were chosen for practical application validation. After rapid virus RNA extraction and RT-PCR amplification, a minimum of 2.7 × 102 copies/mL was obtained successfully. The modular design can be applied to many nucleic acid detection applications, such as viruses, bacteria, species, etc., by simply modifying the crRNA, showing great potential in POC diagnosis.

Pharmacovigilance in China: Evolution and future challenges.

Drug-related adverse reactions are among the main reasons for harm to patients under care worldwide and even their deaths. The pharmacovigilance system has been proven to be an effective method of avoiding or alleviating such adverse events. In 2019, after two decades of implementation of the drug-related adverse reaction reporting system, China formally implemented a pharmacovigilance system with the Pharmacovigilance Quality Management Standards and a series of supporting technical documents created to improve the safety of medication given to patients. China's pharmacovigilance system has faced many problems and challenges during its implementation. This spontaneous reporting system is the main source of data for China's medication vigilance activities, but it has not provided sufficiently powerful evidence for regulatory decision-making. In conformity with the health-centred drug regulatory concept, the Chinese government has accelerated the speed of examination and approval of urgently needed clinical drugs and orphan drugs along with the requirement to improve the safety supervision of these drugs after their listing. China's marketing authorization holders (MAHs) must strengthen their pharmacovigilance capabilities as the primary responsible departments for drug safety. Chinese medical schools generally lack professional courses on pharmacovigilance. The regulatory authorities have recognized such problems and have made efforts to improve the professional capacity of pharmacovigilance personnel and to strengthen cooperation with stakeholders through the implementation of an action plan of medication surveillance and the establishment of a patient-based adverse events reporting system and active surveillance systems, which will help China bridge the gap to bring its pharmacovigilance practice up to standards.

Association of necrotizing enterocolitis with antimicrobial exposure in preterm infants <32 weeks gestational age:A multicenter prospective case-control study.

Objective: To assess the risk of necrotizing enterocolitis (NEC) and explore the relationship between antibiotic overexposure and disease occurrence in a large prospective birth cohort. Methods: Based on a prospective birth cohort, the study collected hospitalization data of very preterm infants (VPIs) having gestational age of less than 32 weeks from January 1, 2018, to June 30, 2021 via the China Northern Neonatal Network. Infants diagnosed with NEC ≥ stage II were included in the case group, and each case was matched for GA and birth weight for the control group. Furthermore, the risk factors for NEC were determined by statistical analyses. Results: A total of 6425 VPIs were included in this study, and 167 (2.6%) of these subjects were diagnosed with NEC ≥ stage II. The study also included 984 extremely preterm infants (gestational age <28 weeks), including 50 (5.1%) infants diagnosed with NEC ≥ stage II. In the matched case-control study, subjects had a total of antibiotic days-of-therapy for 9015 days, of which broad-spectrum antibiotics (BSAs) accounted for 77%. The antibiotic spectrum index per antibiotic day in the case group was significantly higher and was an independent risk factor for the occurrence of NEC (p = 0.001, OR = 1.13). Conclusion: The cohort of VPIs was overexposed to antiboitics. Unreasonable combination of antibiotics and overexposure to BSAs may increase the risk of NEC in preterm infants.

Fluorescence Signal-Readout of CRISPR/Cas Biosensors for Nucleic Acid Detection.

The CRISPR/Cas system is now being used extensively in nucleic acid detection applications, particularly after the trans-cleavage activity of several Cas effectors was found. A CRISPR/Cas system combined with multiple signal-readout techniques has been developed for various molecular diagnostics applications. Fluorescence is now a widely utilized dominant read-out technique in CRISPR biosensors. An in-depth understanding of various fluorescence readout types and variables affecting the fluorescence signals can facilitate better experimental designs to effectively improve the analytical performance. There are the following two commonly used types of CRISPR/Cas detection modes: the first is based on binding activity, such as Cas9 and dCas9; the second is based on cleavage activity, such as Cas12a, Cas12b, Cas13, and Cas14. In this review, fluorescence signal-readout strategies from the last 5 years based on the binding activity and cleavage activity of the CRISPR/Cas system with fundamentals and examples are fully discussed. A detailed comparison of the available fluorescent reporter sequences and design principles is summarized. Current challenges and further applications of CRISPR-based detection methods will be discussed according to the most recent developments.

Systematically investigating the fluorescent signal readout of CRISPR-Cas12a for highly sensitive SARS-CoV-2 detection.

The CRISPR/Cas system is widely used for molecular diagnostics after the discovery of trans-cleavage activity, especially now with the COVID-19 outbreak. However, the majority of contemporary trans-cleavage activity-based CRISPR/Cas biosensors exploited standard single-strand DNA (ssDNA) reporters, which were based on the FRET principle from pioneering research. An in-depth comparison and understanding of various fluorescent readout types are essential to facilitate the outstanding analytical performance of CRISPR probes. We investigated various types of fluorescent reporters of Cas12a comprehensively. Results show that trans-cleavage of Cas12a is not limited to ssDNA and dsDNA reporters, but can be extended to molecular beacons (MB). And MB reporters can achieve superior analytical performance compared with ssDNA and ds DNA reporters at the same conditions. Accordingly, we developed a highly-sensitive SARS-CoV-2 detection with the sensitivity as low as 100 fM were successfully achieved without amplification strategy. The model target of ORF1a could robustly identify the current widespread emerging SARS-CoV-2 variants. A real coronavirus GX/P2V instead of SARS-CoV-2 were chosen for practical application validation. And a minimum of 27 copies/mL was achieved successfully. This inspiration can also be applied to other Cas proteins with trans-cleavage activity, which provides new perspectives for simple, highly-sensitive and universal molecular diagnosis in various applications.

Nucleic Acids Detection for Mycobacterium tuberculosis Based on Gold Nanoparticles Counting and Rolling-Circle Amplification.

Tuberculosis (TB) is a common infectious disease caused by Mycobacterium tuberculosis, which usually disturbs the lungs, and remains the second leading cause of death from an infectious disease worldwide after the human immunodeficiency virus. Herein, we constructed a simple and sensitive method for Mycobacterium tuberculosis-specific DNA detection with the dark-field microscopic imaging of gold nanoparticles (AuNPs) counting strategy and rolling-circle amplification (RCA). Taking advantage of RCA amplification, one target molecule produced hundreds of general oligonucleotides, which could form the sandwich structure with capture-strand-modified magnetic beads and AuNPs. After magnetic separation, AuNPs were released and detected by dark-field imaging; about 10 fM Mycobacterium tuberculosis-specific DNA target can still be differentiated from the blank. No significant change of the absorbance signals was observed when the target DNA to genomic DNA ratio (in mass) was from 1:0 to 1:106. The spike recovery results in genomic DNA from human and Klebsiella pneumoniae suggested that the proposed method has the feasibility for application with biological samples. This proposed method is performed on an entry-level dark-field microscope setup with only a 6 µL detection volume, which creates a new, simple, sensitive, and valuable tool for pathogen detection.

Nanomaterial-based multiplex optical sensors.

The drive for a simultaneous analysis of multiple targets with excellent accuracy and efficiency, which is often required in both basic biomedical research and clinical applications, demands the development of multiplexed bioassays with desired throughput. With the development of nanotechnologies, innovative multiplex optical bioassays have been achieved. Nanomaterials exhibit unique physical and chemical properties such as easily tunable size, large surface-to-volume ratio, excellent catalysis and the desired signal transduction mechanism, which makes them excellent candidates for the fabrication of novel optical nanoprobes. This mini review summarizes nanomaterial-based optical multiplex sensors from the last 5 years. Specific optical techniques covered in this review are fluorescence, surface-enhanced Raman scattering (SERS), localized surface plasmon resonance (LSPR), chemiluminescence (CL), and the multimodality with fundamentals and examples.

Competitive aptasensor for the ultrasensitive multiplexed detection of cancer biomarkers by fluorescent nanoparticle counting.

Cancer biomarker quantification in human serum is of great importance for accurate patient diagnosis and informed clinical management. To date, ultrasensitive multiplexed detection of proteins without amplification is still a major challenge. Herein, we proposed a competitive aptasensor strategy for ultrasensitive multiplexed cancer biomarker detection by fluorescent nanoparticle (FNP) counting. The sequences are designed such that the binding abilities of linker DNA (L-DNA) with DNA-functionalized FNPs (DNA-FNPs) and aptamer are comparable. As long as one target binds with one molecule of aptamer, a signalling FNP forms a sandwich-structured nanocomposite, which was subsequently observed and enumerated with a fluorescence microscope. This 1 : 1 target-to-signal FNP production assured an improved sensitivity, benefiting from the reasonably good brightness and photostability of FNPs. For both singleplexed and multiplexed detection, this proposed strategy achieved an approximately 1000-fold improved limit of detection than the conventional method with the detection volume of 3.2 µL. Notably, the results for carcinoembryonic antigen (CEA) detection obtained directly from 9 human serum samples (colorectal/lung/healthy individuals) were consistent with that obtained by ELISA, showing potential application in clinical diagnosis.

Colocalized Particle Counting Platform for Zeptomole Level Multiplexed Quantification.

For multiplexed detection, it is important yet challenging to simultaneously meet the requirement of sensitivity, throughput, and implementation convenience for practical applications. Using the detection of DNAs and miRNAs for illustration, we present a colocalized particle counting platform that can realize the separation-free multiplexed detection of 6 nucleic acid targets with a zeptomole sensitivity and a dynamic range of up to 5 orders of magnitude. The presence of target induces the formation of a sandwich nanostructure via hybridization; thus, there is an occurrence of colocalization of two microbeads with two different colors. The sequence specific coding is realized by an arbitrary combination of two fluorescence channels with different emitting colors. The platform presents robustness in detecting multiple nucleic acid targets with a minimal cross talk and matrix effect as well as the ability to distinguish the specific miRNA from members of the same family. The results of simultaneous detection of 3 miRNAs in 3 different cell lines present straight consistency with that of the standard qRT-PCR. This platform can be adapted to other multiplexing designs such as the "turn-off" mode, in which the proportion of colocalized microbeads is decreased due to the strand-displacement reaction initiated by the specific target. This separation-free platform offers the possibility to achieve the on-site multiplexed detection with compatibility to different experimental designs and extensibility to other signal sources for enumeration.

Ultraspecific Multiplexed Detection of Low-Abundance Single-Nucleotide Variants by Combining a Masking Tactic with Fluorescent Nanoparticle Counting.

To be able to detect simultaneously multiple single-nucleotide variants (SNVs) with both ultrahigh specificity and low-abundance sensitivity is of pivotal importance for molecular diagnostics and biological research. In this contribution, we for the first time developed a multiplex SNV detection method that combines the masking tactic with fluorescent nanoparticle (FNP) counting based on the sandwich design. The method presents a rivaling performance due to its advantageous features: the masking reagent was designed to hybridize with an extremely large amount of the wild-type sequence to render the assay with high specificity; FNP counting provides a sensitive multiplexed SNV detection; the sandwich design facilitates an easy separation to make the detection free of interferences from the matrix. For single SNV target discrimination, including the 6 most frequently occurring DNA KRAS gene mutations and 2 possible RNA KRAS gene mutations as well as 11 artificial mutations, the discrimination factor ranged from 204 to 1177 with the median being 545. Among the tested 19 SNVs, abundances as low as 0.05% were successfully identified in 14 cases, and an abundance as low as 0.1% was identified for the remaining 5 cases. For multiplexed detection of SNVs in the KRAS gene, abundances as low as 0.05-0.1% were achieved for multiple SNVs occurring at the same and different codons. As low as 0.05% low-abundance detection sensitivity was also achieved for PCR amplicons of human genomic DNA extracted from cell samples. This proposed method presents the potential for ultrahigh specific multiplexed detection of SNVs with low-abundance detection capability, which may be applied to practical applications.

Multiplexed Detection of Attomoles of Nucleic Acids Using Fluorescent Nanoparticle Counting Platform.

The sensitive multiplexed detection of nucleic acids in a single sample by a simple manner is of pivotal importance for the diagnosis and therapy of human diseases. Herein, we constructed an automatic fluorescent nanoparticle (FNP) counting platform with a common fluorescence microscopic imaging setup for nonamplification multiplexed detection of attomoles of nucleic acids. Taking the advantages of the highly bright, multicolor emitting FNPs and magnetic separation, the platform enables sensitive multiplexed detection without the need for extra fluorescent labels. Quantification for multiplex DNAs, multiplex microRNAs (miRNA), as well as a DNA and miRNA mixture was achieved with a similar dynamic range, a limit of detection down to 5 amol (5 µL detection volume), and a 81-115% spike recovery from different biological sample matrices. In particular, the sensitivity for multiplex miRNA is by far among the highest without using amplification or the lock nucleic acid hybridization enhancement strategy. Results regarding miRNA-141 from four different cell lines were agreeable with those of the quantitative reverse transcription polymerase chain reaction. Simultaneous detection of miRNA-141 and miRNA-21 in four different cell lines yielded consistent results with publications, indicating the potential for monitoring multiplex miRNA expression associated with the collaborative regulation of important cellular events. This work expands the rule set of multiplex nucleic acid detection strategies and shows promising potential application in clinical diagnosis.

Proton-detected solid-state NMR detects the inter-nucleotide correlations and architecture of dimeric RNA in microcrystals.

We report a novel proton-detected MAS solid-state NMR strategy based on 15N-15N proton assisted recoupling to detect the inter-nucleotide NHN hydrogen bonds within the Watson-Crick base pairs of micro-crystallized dimeric RNA and to confirm the kissing-loop structure. This would contribute to advances in the structural determination of RNA using solid-state NMR.

A universal and enzyme-free immunoassay platform for biomarker detection based on gold nanoparticle enumeration with a dark-field microscope.

Developing an enzyme-free, non-amplification strategy for biomarker detection with universality and easy implementation is of central importance in clinical diagnosis and therapeutic monitoring. Herein, we report for the first time a universal and enzyme-free magnetic bead-based sandwich-format immunoassay platform for biomarker detection by combining secondary antibody functionalized AuNPs and automatic AuNP counting readout. For the prostate specific antigen (PSA), the detection limit is found to be 1 ng mL-1, and the spike recoveries (n = 3) with 10% fetal bovine serum are 113.5% for 2 ng mL-1 and 107.7% for 10 ng mL-1. The assay also presents reasonable repeatability as indicated by the coefficient of variance of 13.1% with 5 measurements in 60 days. This strategy has been successfully applied to the determination of carcinoembryonic antigen (CEA) and alpha-fetoprotein (AFP), demonstrating the universality of this strategy. Our proposed non-amplification platform presents sensitivity comparable to that of the enzyme-linked immunosorbent assay (ELISA) with better repeatability; and more importantly, our method has better simplicity than most of the amplification-based methods, and thus is more suitable for routine analysis. The highlights of our work suggest that it is a promising method and would be potentially an alternative for ELISA in laboratories where routine analyses are intensively performed.

[Fluorescence Anisotropy and Applications Based on Functional Nucleic Acid Recognition].

Based on the difference of the depolarization of the emitted light before and after the interaction of molecules, fluorescence anisotropy, also known as fluorescence polarization, can be beneficial for the study of interactions and the detection of the targets. In 1950s, Gregorio Weber first studied the interactions between dansyl chloride and bovine serum albumin or ovalbumin with fluorescence anisotropy method, which paved the way for fluorescence anisotropy in biochemical applications. Since the early 1990s, functional nucleic acids (FNAs, including aptamers, and nucleic acid enzymes) were discovered and synthesized, which have been widely used in functional nucleic acid-based sensing. Aptamers are oligonucleotides that can recognize and bind specifically to various molecular targets. The fluorescence anisotropy methods which are based on aptamer recognition have the advantages of high selectivity, sensitivity and through-put. They play an important role in the study of interaction with protein, nucleic acids and small molecules. However, the way of enhancing the fluorescence anisotropy change of small molecules associated with the binding events is challenging. This paper reviews the basic principles and designs of fluorescence anisotropy methods bases on functional nucleic acid recognition for study and detection of proteins, nucleic acids and small molecules that play an important role in the life activity.

Modulating fluorescence anisotropy of dye-labeled DNA without involving mass amplification.

Fluorescence anisotropy, known as a simple, homogeneous and cost-effective analytical technology, is an invaluable technique for studying the micro-environmental changes of the dye associated with the molecular interactions. An in-depth understanding of the variables affecting the fluorescence anisotropy signal can facilitate better experimental designs to effectively improve the analytical performance. This work is a follow-up effort in evaluating the factors that can significantly influence fluorescence anisotropy. We systematically studied fluorescence anisotropy of dsDNA with the changing length based on dye-DNA interactions, with the fluorophores in the end-labeling, the middle-site-labeling, and multiple number of labeling manners. The fluorescence anisotropy value and the base-pair response dynamic range could be expanded by labeling the fluorophores in the middle of dsDNA and increasing the number of labels on dsDNA. The C overhang configuration in the end-labeling manner could enhance the fluorescence anisotropy signal but not expand the base-pair response range. Results from all the labeling fluorophores reinforced the leveling-off effect, i.e., the fluorescence anisotropy signal does not response to the increased length of the DNA duplex when the length is larger than a critical number of base pairs. These findings provide perspectives about choosing appropriate fluorescent dyes and labeling sites for simple and universal fluorescence anisotropy designs in various applications.

Pre-Incubation of Auric Acid with DNA Is Unnecessary for the Formation of DNA-Templated Gold Nanoclusters.

The rationale for the preparation of DNA-templated gold nanoclusters (DNA-Au NCs) has not been well understood, thereby slowing down the advancement of the synthesis and applications of DNA-Au NCs. The interaction between metal ions and the DNA template seems to be the key factor for the successful preparation of DNA-templated metal nanoclusters. With the help of circular dichroism in this contribution, we put efforts into interrogating the necessity of pre-incubation of HAuCl4 with poly-adenine template in the formation of Au NCs by citrate reduction. Our results revealed that the pre-incubation of HAuCl4 with poly-adenine is not favorable for the formation of Au NCs, which is distinctly different from the formation process for silver nanoclusters. It is our hope that this study can provide guidance in the preparation of Au NCs with more DNA templates.

Phosphate-perylene modified G-quadruplex probes for the detection of Pb2+ using fluorescence anisotropy.

A simple and universal phosphate-perylene modification strategy was applied in the development of G-quadruplex probes with thrombin binding aptamer (TBA) and [d(TGGGT)4] (G4) sequences. A perylene moiety was inserted at different phosphate positions of oligonucleotides without a significant effect on the G-quadruplex structures. Upon binding with K+ or Pb2+, these probes showed different perylene fluorescence anisotropy responses due to the different labeling positions and G-quadruplex structures. Two probes (G4-9 and TBA-9) were successfully used in Pb2+ detection through fluorescence anisotropy. Once the complexes of Pb2+ with G4-9 or TBA-9 were formed, the rotational diffusion of the perylene moiety was limited, resulting in a significant increase in fluorescence anisotropy. Both probes showed good sensitivity to Pb2+ and their fluorescence anisotropy signals demonstrated good linear responses to the logarithm of Pb2+ concentrations and the detection limits were 24.5 nM and 30.0 nM for TBA-9 and G4-9, respectively.