DNA Logical Device Combining an Entropy-Driven Catalytic Amplification Strategy for the Simultaneous Detection of Exosomal Multiplex miRNAs In Situ.

Exosomal miRNAs are considered promising biomarkers for cancer diagnosis, but their accuracy is severely compromised by the low content of miRNAs and the large amount of exosomal miRNAs released from normal cells. Here, we presented a dual-specific miRNA's logical recognition triggered by an entropy-driven catalysis (EDC)-enhanced system in exosomes for accurate detection of liver cancer-cell-derived exosomal miR-21 and miR-122. Taking advantage of the accurate analytical performance of the logic device, the excellent membrane penetration of gold nanoparticles, and the outstanding amplification ability of the EDC reaction, this method exhibits high sensitivity and selectivity for the detection of tumor-derived exosomal miRNAs in situ. Moreover, due to its excellent performance, this logic device can effectively distinguish liver cancer patients from healthy donors by determining the amount of cancer-cell-derived exosomal miRNAs. Overall, this strategy has great potential for analyzing various types of exosomes and provides a viable tool to improve the accuracy of cancer diagnosis.

A dual-mode system based on molybdophosphoric heteropoly acid and fluorescent microspheres for the reliable and ultrasensitive detection of alkaline phosphatase.

A novel colorimetric and fluorescent dual-mode sensing system based on molybdophosphoric heteropoly acid (PMA) and fluorescent microspheres (FMs) was established for monitoring the activity of alkaline phosphatase (ALP). In the presence of ALP, L-ascorbic acid-2-phosphate (AAP) could be hydrolyzed catalytically to ascorbic acid (AA), which could reduce PMA to phosphorus molybdenum blue (PMB), accompanied by the generation of colorimetric signals depending on the level of ALP. Meanwhile, the fluorescence of FMs was quenched markedly by the PMB produced due to the inner-filter effect, which constituted the response mechanism for the dual-mode sensing systems of ALP. On this basis, a PMA-FMs based dual-mode sensing system was used for the detection of ALP, which not only possessed remarkable sensitivity, with a limit of detection of 0.27 U L-1 and 0.11 U L-1, but also exhibited good analytical performance in biological samples with satisfactory results. Moreover, a simple and portable test kit for the visual detection of ALP in real serum samples was fabricated utilizing a smartphone with image-recognition and data-processing capabilities as a visual-detection platform.

DNA Dendrimer-Based Directed 3D Walking Nanomachine for the Sensitive Detection and Intracellular Imaging of miRNA.

Taking advantage of the remarkable processivity and membrane penetrability, the gold nanoparticle (AuNP)-based three-dimensional (3D) DNA walking nanomachine has induced tremendous promise in molecular diagnostics and cancer therapy, whereas the executive ability of this nanomachine was eventually limited because of the disordered assembly between the walker and the track. Therefore, we developed a well-directed 3D DNA walking nanomachine by employing a DNA dendrimer as the track for intracellular imaging with high directionality and controllability. The nanomachine was constructed on a DNA dendrimer decorated with a substrate strand serving as the DNA track and a DNAzyme restrained by a locking strand as the walker. In this system, the distribution of the substrate strand and DNAzyme on the DNA dendrimer could be precisely regulated to achieve expected goals because of the specificity and predictability of the Watson-Crick base pairing, paving an explicit route for each walker to move along the track. Moreover, such a DNA dendrimer-based nanomachine owned prominent stability and anti-interference ability. By choosing microRNA-21 as a model analyte, the nanomachine was applied for the imaging of microRNA-21 in different cell lines and the monitoring of the dynamic microRNA-21 expression level in cancer cells. Therefore, we believe that this directed DNA walking nanomachine will have a variety of applications in molecular diagnostics and biological function modulation.

Spatial Confinement-Derived Double-Accelerated DNA Cascade Reaction for Ultrafast and Highly Sensitive In Situ Monitoring of Exosomal miRNA and Exosome Tracing.

Exosomal microRNAs (miRNAs) are reliable biomarkers of disease progression, allowing for non-invasive detection. However, detection of exosomal miRNAs in situ remains a challenge due to low abundance, poor permeability of the lipid bilayers, and slow kinetics of previous methods. Herein, an accelerated DNA nanoprobe was implemented for fast, in situ monitoring of miRNA in exosomes by employing a spatial confinement strategy. This nanoprobe not only detects miRNA in exosomes but also distinguishes tumor exosomes from those derived from normal cells with high accuracy, paving the way toward exosomal miRNA bioimaging and disease diagnosis. Furthermore, the fast response allows for this nanoprobe to be successfully utilized to monitor the process of exosomes endocytosis, making it also a tool to explore exosome biological functions.

Multivalent self-assembled nano string lights for tumor-targeted delivery and accelerated biomarker imaging in living cells and in vivo.

Highly efficient monitoring of microRNA is of great significance for cancer research. By attaching aptamers to DNA nanowires through base pairing, here we designed a multivalent self-assembled DNA nanowire for fast quantification of intracellular target miRNAs in special cancer cells. In this work, an aptamer AS1411 and a microRNA-21 anti-sequence labeled with Cy5 were fixed on DNA nanowires, and then a short DNA strand with black hole quencher 2 (BHQ2) hybridizes with the microRNA-21 anti-sequence to quench Cy5. With the aid of AS1411, the probe can recognize and enter the target special cells efficiently. In addition, because of the banding between microRNA-21 and microRNA-21 anti-sequence, short DNA strands with BHQ2 are detached from the DNA nanowire and result in the recovery of Cy5 fluorescence signals. Ultimately, the fluorescence of Cy5 was activated quickly due to the high local concentration of recognition units on the nanowire, resulting in a large number of activated Cy5 dyes in a short time just like DNA nano string lights. Experimental results revealed that the designed DNA nanowire probe shows great performance for specifically and quickly monitoring microRNA-21 in living cells and in vivo. This developed strategy may become a general platform for detecting targets in living cells and possess great potential for biological and diagnostic research.

DNA origami-based protein networks: from basic construction to emerging applications.

Natural living systems are driven by delicate protein networks whose functions are precisely controlled by many parameters, such as number, distance, orientation, and position. Focusing on regulation rather than just imitation, the construction of artificial protein networks is important in many research areas, including biomedicine, synthetic biology and chemical biology. DNA origami, sophisticated nanostructures with rational design, can offer predictable, programmable, and addressable scaffolds for protein assembly with nanometer precision. Recently, many interdisciplinary efforts have achieved the precise construction of DNA origami-based protein networks, and their emerging application in many areas. To inspire more fantastic research and applications, herein we highlight the applicability and potentiality of DNA origami-based protein networks. After a brief introduction to the development and features of DNA origami, some important factors for the precise construction of DNA origami-based protein networks are discussed, including protein-DNA conjugation methods, networks with different patterns and the controllable parameters in the networks. The discussion then focuses on the emerging application of DNA origami-based protein networks in several areas, including enzymatic reaction regulation, sensing, bionics, biophysics, and biomedicine. Finally, current challenges and opportunities in this research field are discussed.

Programmable DNAzyme Computing for Specific In Vivo Imaging: Intracellular Stimulus-Unlocked Target Sensing and Signal Amplification.

Molecular probe that enables in vivo imaging is the cornerstone of accurate disease diagnosis, prognostic estimation, and therapies. Although several nucleic acid-based probes have been reported for tumor detection, it is still a challenge to develop programmable methodology for accurately identifying tumors in vivo. Herein, a reconfigurable DNA hybridization-based reaction was constructed to assemble DNAzyme computing that contains an intracellular miRNA-unlocked entropy-driven catalysis module and an endogenous metal ion-responsive DNAzyme module for specific in vivo imaging. By reasonable design, the programmable DNAzyme computing can not only successfully distinguish tumor cells from normal cells but also enable tumor imaging in living mice. Due to its excellent operation with high specificity and sensitivity, this design may be broadly applied in the biological study and personalized medicine.

Entropy-driven amplification strategy-assisted lateral flow assay biosensor for ultrasensitive and convenient detection of nucleic acids.

Accurate, sensitive and rapid nucleic acid tests are important to implement timely treatment measures and control the spread of disease. Herein, we developed a novel portable platform for highly sensitive and specific detection of nucleic acids by integrating an entropy-driven amplification strategy into lateral flow assay (LFA) biosensor. We find that introducing an entropy-driven amplification strategy yields bright intensities on the test line of LFA stirp, which results in improved sensitivity for targeted nucleic acid detection. The developed LFA biosensor showed good reproducibility, specificity and sensitivity for target DNA and H1N1-RNA detection with a low detection limit of 1.43 pM and 2.02 pM, respectively. Its practical potential was also verified by detecting the target nucleic acid in human serum. More importantly, the design of an entropy-driven amplification strategy in this portable platform retained the convenient, rapid and low-cost characterizations of LFA biosensor due to the compact amplification principle and the elimination of enzyme use. Thus, we believe that this assay biosensor will certainly report its own position in the timely detection of nucleic acid, especially when the medical environment and resources are fewer.

2D Co-MOF nanosheet-based nanozyme with ultrahigh peroxidase catalytic activity for detection of biomolecules in human serum samples.

A two-dimensional (2D) Co-MOF nanosheet-based nanozyme was developed for colorimetric detection of disease-related biomolecules. The prepared 2D Co-MOFs exhibited ultrahigh peroxidase catalytic activity. 2D Co-MOFs can catalyze the oxidation of colorless 3,3',5,5'-tetramethylbenzidine (TMB) to the blue product oxTMB, accompanying an obvious change of absorption value at 652 nm. However, alkaline phosphatase can catalyze the hydrolysis of L-ascorbic acid-2-phosphate to produce ascorbic acid which can reduce the oxTMB to TMB, resulting in an obvious color fading. Therefore, by recording the change of absorption value at 652 nm, the 2D Co-MOF nanosheets were used to detect ascorbic acid (AA) and alkaline phosphatase (ALP). The limit of detection for AA and ALP was 0.47 µM and 0.33 U L-1, respectively. The limit of quantification for AA and ALP was 1.56 µM and 1.1 U L-1, respectively. The developed nanozyme was successfully used to determine alkaline phosphatase in clinical human serum samples and the results were consistent with those provided by the hospital. Furthermore, by integrating 2D Co-MOF nanosheets with image recognition and data processing function fixed on a smartphone, a portable test of ascorbic acid was reached. Schematic presentation of the preparation of two-dimensional Co-MOF nanosheet-based nanozyme and their application in portable detection of biomolecules.

A DNAzyme-based label-free fluorescent probe for guanosine-5'-triphosphate detection.

Guanosine-5'-triphosphate (GTP) plays a key role in many important biological processes of cells. It is not only a primer for DNA replication and one of the four essential nucleoside triphosphates for mRNA synthesis, but also an energy source for translation and other important cellular processes. It can be converted to adenine nucleoside triphosphate (ATP), and the intracellular GTP level is closely related to the specific pathological state, so it is crucial to establish a simple and accurate method for the detection of GTP. Deoxyribozymes have unique catalytic and structural properties. One of the deoxyribozymes which is named DK2 with self-phosphorylation ability can transfer a phosphate from GTP to the 5' end in the presence of manganese(ii), while lambda exonuclease (λexo) catalyzes the gradual hydrolysis of double-stranded DNA molecules phosphorylated at the 5'-end from 5' to 3', but cannot cleave the 5'-OH end. The fluorescent dye SYBR Green I (SG I) can bind to dsDNA and produce significant fluorescence, but it can only give out weak fluorescence when it is mixed with a single strand. Here, we present a novel unlabeled fluorescence assay for GTP based on the self-phosphorylation of deoxyribozyme DK2 and the specific hydrolysis of λexo. Owing to the advantages of simple operation, high sensitivity, good specificity, low cost and without fluorophore (quenching group) labeling, this method has great potential in biological applications.

A fluorescent nanosphere-based immunochromatography test strip for ultrasensitive and point-of-care detection of tetanus antibody in human serum.

Tetanus still possesses a high infection risk and leads a number of human deaths in poor nations. Point-of-care and ultrasensitive detection of tetanus antibody levels in serum is the key to decrease the risk of tetanus infection and improve the health of people. In this work, by using ultra bright fluorescent nanospheres (FNs) and portable lateral flow test strip (LFTS), a point-of-care and ultrasensitive sensing method has been developed for the detection of tetanus antibodies in human serum. This assay works quite well for tetanus antibodies in the concentration range from 0.0002 to 0.0220 IU/mL with a low detection limit of 0.00011 IU/mL, which is 100-fold lower than conventional gold-based LFTSs. The high sensitivity makes this method suitable for use to detect the low-abundant target in real samples. Besides, this cost-effective FN-based LFTS assay possesses good selectivity, high accuracy, and satisfactory reliability, which holds great potential as a robust candidate for routine medical diagnosis and rapid home testing. Graphical abstract.

A graphene quantum dot-based multifunctional two-photon nanoprobe for the detection and imaging of intracellular glutathione and enhanced photodynamic therapy.

A multifunctional nanosystem, which integrates biosensing, bioimaging, and therapeutic functions into a single nanoprobe, is of great significance for biosensing and biomedicine. Near-infrared (NIR) graphene quantum dots (GQDs) have emerged as an attractive bioimaging and therapy tool for exploring biological events because they can provide deep imaging penetration and low fluorescence background and produce 1O2 for PDT. Here, we reported a GQD-based multifunctional two-photon nanoprobe for intracellular tumor-related glutathione (GSH) detection and enhanced photodynamic therapy by reducing GSH levels in cancer cells. By taking the excellent quenching property of MnO2 nanosheets and the reduction ability of GSH, a GQD@MnO2 nanoprobe was developed through adsorption of MnO2 nanosheets onto the surface of GQDs for sensing intracellular tumor-related GSH. The nanoprobe shows a highly sensitive response to GSH in aqueous solutions with a detection limit of 83 nM. It also exhibits a high selectivity toward GSH relative to other biomolecules and electrolytes. In addition, once endocytosed, the MnO2 nanosheets are reduced by intracellular GSH, simultaneously releasing GQDs and decreasing the level of GSH for highly efficient PDT.

Efficient Two-Photon Fluorescent Probe for Glutathione S-Transferase Detection and Imaging in Drug-Induced Liver Injury Sample.

Drug-induced liver injury (DILI) is a potential complication of any prescribed medication. So far, the diagnosis of DILI is still a clinical challenge due to the lack of efficient diagnosis method. Glutathione S-transferase (GST), with a high concentration in liver cytosol, can reduce toxicity and facilitate urinary excretion by catalyzing the conjugation of glutathione (GSH) with reactive metabolites in liver. When liver is seriously damaged, GST and GSH will be released into plasma from liver cytosol, which caused a lower GST activity in liver cytosol. Therefore, monitoring the level of GST activity in liver tissue may be a potential strategy for diagnosis of DILI. Here, we reported a two-photon probe P-GST for GST activity detection for the first time. In the proposed design, a donor-π-acceptor (D-π-A) structured naphthalimide derivative with efficient two-photon properties was chosen as the fluorescent group, and a 2,4-dinitrobenzenesulfonate group was employed as the GST recognition unit, which also acted as the fluorescence quencher. In the present of GST and GSH, the recognition unit was removed and the fluorophore was released, causing a 40-fold enhancement of fluorescence signal with a detection limit of 35 ng/mL. At last, P-GST was successfully applied in two-photon imaging of GST in cells and DILI samples, which demonstrated its practical application in complex biosystems as a potential method for diagnosis of DILI.

Aptamer-integrated DNA nanostructures for biosensing, bioimaging and cancer therapy.

The combination of nanostructures with biomolecules leading to the generation of functional nanosystems holds great promise for biotechnological and biomedical applications. As a naturally occurring biomacromolecule, DNA exhibits excellent biocompatibility and programmability. Also, scalable synthesis can be readily realized through automated instruments. Such unique properties, together with Watson-Crick base-pairing interactions, make DNA a particularly promising candidate to be used as a building block material for a wide variety of nanostructures. In the past few decades, various DNA nanostructures have been developed, including one-, two- and three-dimensional nanomaterials. Aptamers are single-stranded DNA or RNA molecules selected by Systematic Evolution of Ligands by Exponential Enrichment (SELEX), with specific recognition abilities to their targets. Therefore, integrating aptamers into DNA nanostructures results in powerful tools for biosensing and bioimaging applications. Furthermore, owing to their high loading capability, aptamer-modified DNA nanostructures have also been altered to play the role of drug nanocarriers for in vivo applications and targeted cancer therapy. In this review, we summarize recent progress in the design of aptamers and related DNA molecule-integrated DNA nanostructures as well as their applications in biosensing, bioimaging and cancer therapy. To begin with, we first introduce the SELEX technology. Subsequently, the methodologies for the preparation of aptamer-integrated DNA nanostructures are presented. Then, we highlight their applications in biosensing and bioimaging for various targets, as well as targeted cancer therapy applications. Finally, we discuss several challenges and further opportunities in this emerging field.

Efficient Two-Photon Fluorescence Nanoprobe for Turn-On Detection and Imaging of Ascorbic Acid in Living Cells and Tissues.

Ascorbic acid (AA) serves as a key coenzyme in many metabolic pathways, and its abnormal level is found to be associated with several diseases. Therefore, monitoring AA level in living systems is of great biomedical significance. In comparison with one-photon excited fluorescent probes, two-photon (TP) excited probes are more suitable for bioimaging, as they could afford higher imaging resolution with deeper imaging depth. Here, we report for the first time an efficient TP fluorescence probe for turn-on detection and imaging of AA in living cells and tissues. In this nanosystem, the negatively charged two-photon nanoparticles (TPNPs), which were prepared by modifying the silica nanoparticles with a two-photon dye, could adsorb cobalt oxyhydroxide (CoOOH) nanoflakes which carried positive charge by electrostatic force, leading to a remarkable decrease in their fluorescence intensity. However, the introduction of AA could induce the fluorescence recovery of the nanoprobe because it could reduce CoOOH into Co(2+) and result in the destruction of the CoOOH nanoflakes. The nanosystem exhibits a high sensitivity toward AA, with a LOD of 170 nM observed. It also shows high selectivity toward AA over common potential interfering species. The nanoprobe possessed both the advantages of TP imaging and excellent membrane-permeability and good biocompatibility of the silica nanoparticles and was successfully applied in TP-excited imaging of AA in living cells and tissues.

Multiple functional nanoprobe for contrast-enhanced bimodal cellular imaging and targeted therapy.

Many one-photon fluorescence-based theranostic nanosystems have been developed for simultaneous therapeutic intervention/monitoring for various types of cancers. However, for early diagnosis of cancer, two-photon fluorescence microscopy (TPFM) can realize deep-tissue imaging with higher spatial resolution. In this study, we first report a multiple functional nanoprobe for contrast-enhanced bimodal cellular imaging and targeted therapy. Components of the nanoprobe include (1) two-photon dye-doped mesoporous silica nanoparticles (TPD-MSNs); (2) MnO2 nanosheets that act as a (i) gatekeeper for TPD-MSNs, (ii) quencher for TP fluorescence, and (iii) contrast agent for MRI; (3) cancer cell-targeting aptamers. Guided by aptamers, TPD-MSNs are rapidly internalized into the target cells. Next, intracellular glutathione reduces MnO2 to Mn(2+) ions, resulting in contrast-enhanced TP fluorescence and magnetic resonance signal for cellular imaging. Meanwhile, preloaded doxorubicin and Chlorin e6 are released for chemotherapy and photodynamic therapy, respectively, with a synergistic effect and significantly enhanced therapeutic efficacy.

Activatable two-photon fluorescence nanoprobe for bioimaging of glutathione in living cells and tissues.

Glutathione (GSH) serves vital cellular biological functions, and its abnormal levels are associated with many diseases. To better understand its physiological and pathological functions, efficient methods for monitoring of GSH in living systems are desired. Although quite a few small molecule-based and nanomaterial-based one photon fluorescence probes have been reported for GSH, two-photon (TP) probes, especially nanoprobes with good membrane permeability, are more favorable for bioimaging applications, since TP fluorescence imaging can provide improved spatial localization and increased imaging depth. In this work, we for the first time reported a "turn-on" TP fluorescence nanoprobe for efficient detection of GSH in aqueous solutions and TP excited fluorescence imaging of GSH in living cells and tissues. The nanoprobe consists of two-photon mesoporous silica nanoparticles (TP-MSNs) with a large TP excitation action cross-section (Φδ) value of 103 GM and MnO2 nanosheets, which show intense and broad optical absorption and could act as efficient quenchers for TP fluorescence. In the sensing system, the negatively charged MnO2 nanosheets are adsorbed on the positively charged MSNs through electrostatic interaction, resulting in efficient quenching of their fluorescence, with very low background fluorescence observed. The addition of GSH could reduce MnO2 into Mn(2+), lead to the decomposition of the MnO2 nanosheets, and thereby result in remarkable enhancement of both one photon and TP excited fluorescence of the nanosystem. The nanoprobe shows a highly sensitive response to GSH in aqueous solutions, with a detection limit of 200 nM achieved. It also exhibits a high selectivity toward GSH relative to other biomolecules and electrolytes, with good membrane permeability and excellent biocompatibility. The nanoprobe was successfully applied in monitoring the change of the intracellular GSH in living cells and tissues via TP fluorescence imaging, demonstrating its value of practical application in biological systems.

Spatiotemporal control of DNAzyme activity for fluorescent imaging of telomerase RNA in living cells.

BACKGROUND: Human telomerase is a ribonucleoprotein complex that includes proteins and human telomerase RNA (hTR). Emerging evidence suggested that the expression level of hTR was high related with the development of tumor, so it is important to accurately detect the content of hTR. Optical control of DNAzyme activity shows a promising strategy for precise biosensing, biomedical imaging and modulation of biological processes. Although DNAzyme-based sensors can be controlled spatiotemporally by light, its application in the detection of hTR in living cells is still rare. Therefore, designing DNAzyme activity spatiotemporal controllable sensors for hTR detection is highly needed. RESULTS: We developed a UV light-activated DNAzyme-based nanoprobe for spatially accurate imaging of intracellular hTR. The proposed nanoprobe was named MDPH, which composed of an 8-17 DNAzyme (D) inactivated by a protector strand (P), a substrate strand (H), and MnO2 nanosheets. The MnO2 nanosheets can enhance the cellular uptake of DNA strands, so that MDPH probe can enter cells autonomously through endocytosis. Under the high concentration of GSH in cancer cells, MnO2 nanosheets can self-generate cofactors to maintain the catalytic activity of DNAzyme. When exposing UV light and in presence of target hTR, DNAzyme could cleave substrate H, resulting in the recovery of fluorescence of the system. The cells imaging results show that MDPH probe could be spatiotemporally controlled to image endogenous hTR in cancer cells. SIGNIFICANCE: With this design, telomerase RNA-specific fluorescent imaging was achieved by MDPH probe in both cancer and normal cells. Our probe made a promising new platform for spatiotemporal controllable intracellular hTR monitoring. This current method can be applied to monitor a variety of other biomarkers in living cells and perform medical diagnosis, so it may has broad applications in the field of medicine.

Aptamer-Based DNA Allosteric Switch for Regulation of Protein Activity.

Allostery is a fundamental way to regulate the function of biomolecules playing crucial roles in cell metabolism and proliferation and is deemed the second secret of life. Given the limited understanding of the structure of natural allosteric molecules, the development of artificial allosteric molecules brings a huge opportunity to transform the allosteric mechanism into practical applications. In this study, the concept of bionics is introduced into the design of artificial allosteric molecules and an allosteric DNA switch with an activity site and an allosteric site based on two aptamers for selective inhibition of thrombin activity. Compared with the single aptamer, the allosteric switch possesses a significantly enhanced inhibition ability, which can be precisely regulated by converting the switch states. Moreover, the dynamic allosteric switch is further subjected to the control of the DNA threshold circuit for realizing automatic concentration determination and activity inhibition of thrombin. These compelling results confirm that this allosteric switch equipped with self-sensing and information-processing modules puts a new slant on the research of allosteric mechanisms and further application of allosteric tactics in chemical and biomedical fields.

In situ coupling of carbon dots with zeolitic imidazolate frameworks enabling highly red emission in solid state.

In this work, an extraordinary solid red emissive phosphor was prepared based on red-emitting carbon dots (R-CDs). The synthesis was conducted under an in-situ strategy, with assistance of zeolitic imidazolate frameworks. The obtained phosphor possesses a stronger red emission located at 630 nm in solid state, with CIE coordinate of (0.63, 0.35) and quantum yield of âˆ¼ 45 %. As a consequence, not only aggregation-induced fluorescence quenching of R-CDs is avoided in solid state, but also an enhanced emission with high quantum yield is presented. Fluorescence properties were further explored in detail. The emission is found to be responsive to temperature and applied pressure. Based on the excellent emissive performance, the material has great potentials in anti-counterfeiting, latent fingerprint imaging, and temperature/pressure-sensing. This work provides a facile and promising way of preparing solid carbon-based phosphors for special applications.