Construction Strategy of Functionalized Liposomes and Multidimensional Application.

Liposomes are widely used in the biological field due to their good biocompatibility and surface modification properties. With the development of biochemistry and material science, many liposome structures and their surface functional components have been modified and optimized one by one, pushing the liposome platform from traditional to functionalized and intelligent, which will better satisfy and expand the needs of scientific research. However, a main limiting factor effecting the efficiency of liposomes is the complicated environmental conditions in the living body. Currently, in order to overcome the above problem, functionalized liposomes have become a very promising strategy. In this paper, binding strategies of liposomes with four main functional elements, namely nucleic acids, antibodies, peptides, and stimuli-responsive motif have been summarized for the first time. In addition, based on the construction characteristics of functionalized liposomes, such as drug-carrying, targeting, long-circulating, and stimulus-responsive properties, a comprehensive overview of their features and respective research progress are presented. Finally, the paper critically presents the limitations of these functionalized liposomes in the current applications and also prospectively suggests the future development directions, aiming to accelerate realization of their industrialization.

G-quadruplex based biosensors for the detection of food contaminants.

G-quadruplex (G4) is a very interesting DNA structure, commonly associated with cancer and its treatment. With flexible binding ability, G4 has been extended as a significant component in biosensors. On account of its simple operation, high sensitivity and low cost, G4-based biosensors have attracted considerable interest for the detection of food contaminants. In this review, research published in recent 5 years is collated from a principle perspective, that is target recognition and signal transduction. Contaminants with G4 binding capacity are illustrated, emerging G4-based biosensors including colorimetric, electrochemical and fluorescent sensors are also elaborated. The current review indicates that G4 has provided an efficient and effective solution for the rapid detection of food contaminants. A distinctive feature of G4 as recognition unit is the simple composition, but the selectivity is still unsatisfactory. As signal reporter, G4/hemin DNAzyme has not only achieved amplified signals, but also enabled visualized detection, which offers great potential for on-site measurement. With improved selectivity and visualized signal, the combination of aptamer and G4 seems to be an ideal strategy. This promising combination should be developed for the real-time monitor of multiple contaminants in food matrix.

SERS-fluorescence dual-mode nanoprobe for the detection and imaging of Bax mRNA during apoptosis.

A dual-mode nanoprobe was constructed to detect Bax messenger RNA (mRNA), consisting of gold nanotriangles (AuNTs), a Cy5-modified recognition sequence, and a thiol-modified DNA sequence. Bax mRNA is one of the key pro-apoptotic factors in the apoptosis pathway. Raman enhancement and fluorescence quenching of the signal group Cy5 were performed using AuNTs as substrates. The thiol-modified nucleic acid chain is partially complementary to the Cy5-modified nucleic acid chain to form a double strand and is linked to the AuNTs by the Au-S bond. When Bax mRNA is present, the Cy5-modified strand specifically binds to it to form a more stable duplex, making Cy5 far away from AuNTs, and SERS signal is weakened while fluorescence signal is enhanced. The nanoprobe can be used for the quantitative detection of Bax mRNA in vitro. Combined with the high sensitivity of SERS and the visualization of fluorescence, this method has good specificity and can be used for in situ imaging and dynamic monitoring of Bax mRNA during deoxynivalenol (DON) toxin-induced apoptosis of HepG2 cells. DON plays a pathogenic role mainly by inducing cell apoptosis. The results confirmed that the proposed dual-mode nanoprobe has good versatility in various human cell lines.

An RNA-Cleaving DNAzyme That Requires an Organic Solvent to Function.

Engineering functional nucleic acids that are active under unusual conditions will not only reveal their hidden abilities but also lay the groundwork for pursuing them for unique applications. Although many DNAzymes have been derived to catalyze diverse chemical reactions in aqueous solutions, no prior study has been set up to purposely derive DNAzymes that require an organic solvent to function. Herein, we utilized in vitro selection to isolate RNA-cleaving DNAzymes from a random-sequence DNA pool that were "compelled" to accept 35 % dimethyl sulfoxide (DMSO) as a cosolvent, via counter selection in a purely aqueous solution followed by positive selection in the same solution containing 35 % DMSO. This experiment led to the discovery of a new DNAzyme that requires 35 % DMSO for its catalytic activity and exhibits drastically reduced activity without DMSO. This DNAzyme also requires divalent metal ions for catalysis, and its activity is enhanced by monovalent ions. A minimized, more efficient DNAzyme was also derived. This work demonstrates that highly functional, organic solvent-dependent DNAzymes can be isolated from random-sequence DNA libraries via forced in vitro selection, thus expanding the capability and potential utility of catalytic DNA.

A Simple Colorimetric Au-on-Au Tip Sensor with a New Functional Nucleic Acid Probe for Food-borne Pathogen Salmonella typhimurium.

An Au-on-Au tip sensor is developed for the detection of Salmonella typhimurium (Salmonella), using a new synthetic nucleic acid probe (NAP) as a linker for the immobilization of a DNA-conjugated Au nanoparticle (AuNP) onto a DNA-attached thin Au layer inside a pipette tip. In the presence of Salmonella, RNase H2 from Salmonella (STH2) cleaves the NAP and the freed DNA-conjugated AuNP can be visually detected by a paper strip. This portable biosensor does not require any electronic, electrochemical or optical equipment. It delivers a detection limit of 3.2×103  CFU mL-1 for Salmonella in 1 h without cell-culturing or signal amplification and does not show cross-reactivity with several control bacteria. Further, the sensor reliably detects Salmonella spiked in food samples, such as ground beef and chicken, milk, and eggs. The sensor can be reused and is stable at ambient temperature, showing its potential as a point-of-need device for the prevention of food poisoning by Salmonella.

Functional Xeno Nucleic Acids for Biomedical Application.

Functional nucleic acids(FNAs) refer to a type of oligonucleotides with functions over the traditional genetic roles of nucleic acids, which have been widely applied in screening, sensing and imaging fields. However, the potential application of FNAs in biomedical field is still restricted by the unsatisfactory stability, biocompatibility, biodistribution and immunity of natural nucleic acids(DNA/RNA). Xeno nucleic acids(XNAs) are a kind of nucleic acid analogues with chemically modified sugar groups that possess improved biological properties, including improved biological stability, increased binding affinity, reduced immune responses, and enhanced cell penetration or tissue specificity. In the last two decades, scientists have made great progress in the research of functional xeno nucleic acids, which makes it an emerging attractive biomedical application material. In this review, we summarized the design of functional xeno nucleic acids and their applications in the biomedical field.

Recent Advances on Functional Nucleic-Acid Biosensors.

In the past few decades, biosensors have been gradually developed for the rapid detection and monitoring of human diseases. Recently, functional nucleic-acid (FNA) biosensors have attracted the attention of scholars due to a series of advantages such as high stability and strong specificity, as well as the significant progress they have made in terms of biomedical applications. However, there are few reports that systematically and comprehensively summarize its working principles, classification and application. In this review, we primarily introduce functional modes of biosensors that combine functional nucleic acids with different signal output modes. In addition, the mechanisms of action of several media of the FNA biosensor are introduced. Finally, the practical application and existing problems of FNA sensors are discussed, and the future development directions and application prospects of functional nucleic acid sensors are prospected.

Studies on the preferred uracil-adenine base pair at the cleavage site of 10-23 DNAzyme by functional group modifications on adenine.

10-23 DNAzyme is capable of catalytically cleaving RNA substrates with the preferred cleavage sites rAU and rGU, in which the common base pair U-dA0 forms between the substrate and the DNAzyme in the cleavage reaction. Here its conservation was studied with base modifications on dA and extra functional groups introduced. The nitrogen atom at 7- or 8-position of adenine was demonstrated to be equally important for the cleavage reaction, although it is not related to the thermal stability of the base pair. Deletion of 6-amino group led to decreased stability of the base pair and a slight slower reaction rate. Extra functional groups through 6-amino group were not favorably accommodated in the cleavage site. From these modifications at the level of functional groups, it demonstrated that the base pair U-dA0 not only contributes to the recognition and binding stability, but also it is involved in the active catalytic center by its functional groups and base stacking. This kind of chemical modifications with 7-substituted 8-aza-7-deaza-2'-deoxyadenosine at dA0 is favorable for the introduction of signal molecules for mechanistic studies and biological applications, without significant loss of the catalytic function and structural destruction.

Autonomous enzymatic synthesis of functional nucleic acids for sensitive measurement of long noncoding RNA in human lung tissues.

Aberrant long noncoding RNA (lncRNA) expression is linked to varied pathological processes and malignant tumors, and lncRNA can serve as potential disease biomarkers. Herein, we demonstrate the autonomous enzymatic synthesis of functional nucleic acids for sensitive measurement of lncRNA in human lung tissues on the basis of multiple primer generation-mediated rolling circle amplification (mPG-RCA). This assay involves two padlock probes that act as both a detection probe for recognizing target lncRNA and a domain for producing complementary DNAzyme. Two padlock probes can hybridize with target lncRNA at different sites, followed by ligation to form a circular template with the aid of RNA ligase. The circular template can initiate mPG-RCA to generate abundant Mg2+-dependent DNAzymes that can specifically cleave signal probes to induce the recovery of Cy3 fluorescence. The inherent characteristics of ligase-based ligation reaction and DNAzymes endow this assay with excellent specificity, and the introduction of multiple padlock probes endows this assay with high sensitivity. This strategy can rapidly and sensitively measure lncRNA with a wide linear range of 1 fM - 1 nM and a detection limit of 678 aM within 1.5 h, and it shows distinct advantages of simplicity and immobilization-free without the need of precise temperature control and tedious procedures of nanomaterial preparation. Moreover, it enables accurate measurement of lncRNA level in normal cells and malignant tumor cells as well as differentiation of lncRNA expressions in tissues of non-small cell lung cancer (NSCLC) patients and normal individuals, with promising applications in biomedical studies and disease diagnosis.

Recent advances in functional nucleic acid decorated nanomaterials for cancer imaging and therapy.

Nanomaterials possess unusual physicochemical properties including unique optical, magnetic, electronic properties, and large surface-to-volume ratio. However, nanomaterials face some challenges when they were applied in the field of biomedicine. For example, some nanomaterials suffer from the limitations such as poor selectivity and biocompatibility, low stability, and solubility. To address the above-mentioned obstacles, functional nucleic acid has been widely served as a powerful and versatile ligand for modifying nanomaterials because of their unique characteristics, such as ease of modification, excellent biocompatibility, high stability, predictable intermolecular interaction and recognition ability. The functionally integrating functional nucleic acid with nanomaterials has produced various kinds of nanocomposites and recent advances in applications of functional nucleic acid decorated nanomaterials for cancer imaging and therapy were summarized in this review. Further, we offer an insight into the future challenges and perspectives of functional nucleic acid decorated nanomaterials.

Construction of a Functional Nucleic Acid-Based Artificial Vesicle-Encapsulated Composite Nanoparticle and Its Application in Retinoblastoma-Targeted Theranostics.

Retinoblastoma (RB) is an aggressive tumor of the infant retina. However, the ineffective targeting of its theranostic agents results in poor imaging and therapeutic efficacy, which makes it difficult to identify and treat RB at an early stage. In order to improve the imaging and therapeutic efficacy, we constructed an RB-targeted artificial vesicle composite nanoparticle. In this study, the MnO2 nanosponge (hMNs) was used as the core to absorb two fluorophore-modified DNAzymes to form the Dual/hMNs nanoparticle; after loaded with the artificial vesicle derived from human red blood cells, the RB-targeted DNA aptamers were modified on the surface, thus forming the Apt-EG@Dual/hMNs complex nanoparticle. The DNA aptamer endows this nanoparticle to target the nucleolin-overexpressed RB cell membrane specifically and enters cells via endocytosis. The nanoparticle could release fluorophore-modified DNAzymes and supplies Mn2+ as a DNAzyme cofactor and a magnetic resonance imaging (MRI) agent. Subsequently, the DNAzymes can target two different mRNAs, thereby realizing fluorescence/MR bimodal imaging and dual-gene therapy. This study is expected to provide a reliable and valuable basis for ocular tumor theranostics.

A ratiometric dual-fluorescent paper-based synthetic biosensor for visual detection of tetracycline on-site.

The excessive use of tetracycline poses a threat to human health, making it essential to monitor and regulate its usage. While whole-cell biosensors offer a simple and cost-effective method, their utility is constrained by limitations in sensitivity, portability, and robustness, hindering real-time measurements within complex environmental contexts. In this study, a ratiometric i/cTetR synthetic biosensing test strip with an engineered modified dual-fluorescence reporting was developed for detecting Tet antibiotics in water and food. First, the standardized unidirectional promoter PtetR by tailoring and screening TetR transcription factor binding sites and verified by molecular docking, shortening the detection time. Secondly, decoupling the sensing and reporting modules enhances the biosensor's performance, eliminating genetic background leakage and tripling the output signal. Thirdly, a ratiometric dual fluorescence signal i/cTetR biosensing test strip was designed. Under the light box LED/UV light source, the dual signal output method significantly reduced false negative results and enhanced the anti-interference capability of the biosensor. The i/cTetR strips can detect Tet in tap water (5-1280 µg/mL) and milk (50-3200 µg/kg) within 45 min in high volume on-site without separation and purification. This study provides a standardized and universal sensing method for the field detection of antibiotic contaminants.

Enzyme-free dual amplification biosensor based on functional nucleic acid and CDs/CoOOH for detection of leukemia fusion gene.

Acute promyelocytic leukemia (APL) is an acute myeloid leukemia (AML) with a specific fusion gene target, PML/RARα fusion gene (PML/RARα), which is formed by the translocation of chromosomes 15 and 17. Detection of PML/RARα is the most reliable parameter for the diagnosis, treatment adjustment, efficacy evaluation, prognosis analysis and relapse prediction of APL. In this study, a novel biosensor was constructed for rapid enzyme-free detection of PML/RARα using DNAzyme and carbon dots/cobalt oxhydroxide nanosheet complexs (CDs/CoOOH). In the detection system, the separated DNAzymes could specifically recognize and bind together by the PML/RARα to form a complete DNAzyme for shearing hairpin probe (HP), then generated trigger, which was the first signal amplification. Then, trigger could hybridize with the capture probe (CP) anchored to streptavidin (SA) modified microplate as well as fluorescence quenching signal probe (SP@CDs/CoOOH). Finally, ascorbic acid (AA) was added to decompose CoOOH and the fluorescence of CDs was released, which was the second signal amplification. Through the dual signal amplification of DNAzyme and CDs/CoOOH, PML/RARα could be detected quickly and sensitively, which overcame the limitation of protein enzyme in traditional fluorescence methods, showing potential clinical application value in the diagnosis and treatment of leukemia.

Biosensors based on functional nucleic acids and isothermal amplification techniques.

In the past few years, with the in-depth research of functional nucleic acids and isothermal amplification techniques, their applications in the field of biosensing have attracted great interest. Since functional nucleic acids have excellent flexibility and convenience in their structural design, they have significant advantages as recognition elements in biosensing. At the same time, isothermal amplification techniques have higher amplification efficiency, so the combination of functional nucleic acids and isothermal amplification techniques can greatly promote the widespread application of biosensors. For the purpose of further improving the performance of biosensors, this review introduces several widely used functional nucleic acids and isothermal amplification techniques, as well as their classification, basic principles, application characteristics, and summarizes their important applications in the field of biosensing. We hope to provide some references for the design and construction of new tactics to enhance the detection sensitivity and detection range of biosensing.

Strategy of functional nucleic acids-mediated isothermal amplification for detection of foodborne microbial contaminants: A review.

Foodborne microbial contamination (FMC) is the leading cause of food poisoning and foodborne illness. The foodborne microbial detection methods based on isothermal amplification have high sensitivity and short detection time, and functional nucleic acids (FNAs) could extend the detectable object of isothermal amplification to mycotoxins. Therefore, the strategy of FNAs-mediated isothermal amplification has been emergingly applied in biosensors for foodborne microbial contaminants detection, making biosensors more sensitive with lower cost and less dependent on nanomaterials for signal output. Here, the mechanism of six isothermal amplification technologies and their application in detecting FMC is firstly introduced. Then the strategy of FNAs-mediated isothermal amplification is systematically discussed from perspectives of FNAs' versatility including recognition elements (Aptamer, DNAzyme), programming tools (DNA tweezer, DNA walker and CRISPR-Cas) and signal units (G-quadruplex, FNAs-based nanomaterials). Finally, challenges and prospects are presented in terms of addressing the issue of nonspecific amplification reaction, developing better FNAs-based sensing elements and eliminating food matrix effects.

Electrochemical DNAzyme-based biosensors for disease diagnosis.

DNAzyme-based electrochemical biosensors provide exceptional analytical sensitivity and high target recognition specificity for disease diagnosis. This review provides a critical perspective on the fundamental and applied impact of incorporating DNAzymes in the field of electrochemical biosensing. Specifically, we highlight recent advances in creating DNAzyme-based electrochemical biosensors for diagnosing infectious diseases, cancer and regulatory diseases. We also develop an understanding of challenges around translating the research in the field of DNAzyme-based electrochemical biosensors from labs to clinics, followed by a discussion on different strategies that can be applied to enhance the performance of the currently existing technologies to create truly point-of-care electrochemical DNAzyme biosensors.

Recent Progress in Functional-Nucleic-Acid-Based Fluorescent Fiber-Optic Evanescent Wave Biosensors.

Biosensors capable of onsite and continuous detection of environmental and food pollutants and biomarkers are highly desired, but only a few sensing platforms meet the "2-SAR" requirements (sensitivity, specificity, affordability, automation, rapidity, and reusability). A fiber optic evanescent wave (FOEW) sensor is an attractive type of portable device that has the advantages of high sensitivity, low cost, good reusability, and long-term stability. By utilizing functional nucleic acids (FNAs) such as aptamers, DNAzymes, and rational designed nucleic acid probes as specific recognition ligands, the FOEW sensor has been demonstrated to be a general sensing platform for the onsite and continuous detection of various targets ranging from small molecules and heavy metal ions to proteins, nucleic acids, and pathogens. In this review, we cover the progress of the fluorescent FNA-based FOEW biosensor since its first report in 1995. We focus on the chemical modification of the optical fiber and the sensing mechanisms for the five above-mentioned types of targets. The challenges and prospects on the isolation of high-quality aptamers, reagent-free detection, long-term stability under application conditions, and high throughput are also included in this review to highlight the future trends for the development of FOEW biosensors capable of onsite and continuous detection.

Functional nucleic acids in glycobiology: A versatile tool in the analysis of disease-related carbohydrates and glycoconjugates.

As significant components of the organism, carbohydrates and glycoconjugates play indispensable roles in energy supply, cell signaling, immune modulation, and tumor cell invasion, and function as biomarkers since aberrance of them has been proved to be associated with the emergence and development of certain diseases. Functional nucleic acids (FNAs) have properties including easy-to-synthesize, good stability, good biocompatibility, low cost, and high programmability, they have attracted significant research attention and been incorporated into biosensors for detecting disease-related carbohydrates and glycoconjugates. This review summarizes the construction strategies and biosensing applications of FNAs-based biosensors in glycobiology in terms of target recognition and signal transduction. By illustrating the mechanisms and comparing the performances, the challenges and development opportunities in this area have been critically elaborated. We believe that this review will provide a better understanding of the role of FNAs in the analysis of disease-related carbohydrates and glycoconjugates, and inspire further discovery in fields that include glycobiology, chemical biology, clinical diagnosis, and drug development.

Methodologies in visualizing the activation of CRISPR/Cas: The last mile in developing CRISPR-Based diagnostics and biosensing - A review.

CRISPR/Cas-based analytical procedures have revolutionized the sensing platform to fulfill the requirements of the current era in terms of sensitivity, selectivity, robustness, user-friendly feature, and cost-effectiveness for the detection of nucleic acid as well as non-nucleic acid analytes. Molecular target monitoring and transduction of the signals is a crucial prerequisite for precise molecular sensing tools. Besides, the reporting systems have become the last milestone for fabricating Cas-based molecular probes to visualize the activation of CRISPR/Cas enzymes. In this review, we have highlighted various CRISPR/Cas reporters, their mechanisms, sensing strategies, merits, and demerits. Moreover, signal transducers, i.e. fluorescent, colorimetric, and electrochemical, have also been discussed in detail along with various sensing strategies to generate recordable signals. It was concluded that there is still a need to overcome issues offered by the reported sensing devices, such as off-target effect, target sequence limitation, multiplexed quantitative detection, the influence of the inhibitor, and reaction kinetic constraint. Additionally, it is required to make them available for commercial use by validating their stability, robustness, safety profile in an off-lab environment as most of the probes have been tested in the controlled atmosphere of the laboratories. We believe that this novel critical interpretation and summary will assist the researchers in designing and validating new CRISPR/Cas reporters and probes for practical applications on a commercial scale.

Development of a two-in-one integrated bioassay for simultaneous and rapid on-site detection of Pb2+ and Hg2+ in water.

Exposure to heavy metal pollution represents a serious health risk and requires rapid, sensitive, and accurate detection to protect human health. However, the existing methods had various limitations for simultaneous detection of heavy metals. Herein, a novel two-in-one integrated bioassay was constructed for simultaneous on-site determination of Hg2+ and Pb2+ through combining all-fiber bicolor fluorescence biosensor (ABFB), functional nucleic acids (FNAs), and FRET. Hg2+ biosensor, consisting of BQ-T14 and CY-A14, performed its detection based on specifical binding of Hg2+ and T bases to form T-Hg2+-T mismatch structure. GR-5 DNAzyme, consisting of GR-5S and GR-5E, is applied for Pb2+ biosensor. The higher concentration of Hg2+ and Pb2+ results in the more T-Hg2+-T mismatch structure formed and the GR-5E to cleave the more GR-5S, respectively, thus allowing higher detectable fluorescence intensities in two signal channels. Thanks to high selectivity of two FNAs-biosensors and excellent time-resolved effect of the ABFB, the Hg2+ and Pb2+ could be directly and simultaneously quantitated in 12 min, and had the LODs of 4.22 nM and 1.55 nM, respectively. The satisfied recovery rate of real samples was verified by ICP-OES. Our strategy could serve as a simple and cost-effective on-site high-frequency detection of heavy metal ions.