Hepialiamides A-C: Aminated Fusaric Acid Derivatives and Related Metabolites with Anti-Inflammatory Activity from the Deep-Sea-Derived Fungus Samsoniella hepiali W7.

A systematic investigation combined with a Global Natural Products Social (GNPS) molecular networking approach, was conducted on the metabolites of the deep-sea-derived fungus Samsoniella hepiali W7, leading to the isolation of three new fusaric acid derivatives, hepialiamides A-C (1-3) and one novel hybrid polyketide hepialide (4), together with 18 known miscellaneous compounds (5-22). The structures of the new compounds were elucidated through detailed spectroscopic analysis. as well as TD-DFT-based ECD calculation. All isolates were tested for anti-inflammatory activity in vitro. Under a concentration of 1 µM, compounds 8, 11, 13, 21, and 22 showed potent inhibitory activity against nitric oxide production in lipopolysaccharide (LPS)-activated BV-2 microglia cells, with inhibition rates of 34.2%, 30.7%, 32.9%, 38.6%, and 58.2%, respectively. Of particularly note is compound 22, which exhibited the most remarkable inhibitory activity, with an IC50 value of 426.2 nM.

Penidihydrocitrinins A-C: New Polyketides from the Deep-Sea-Derived Penicillium citrinum W17 and Their Anti-Inflammatory and Anti-Osteoporotic Bioactivities.

Three new polyketides (penidihydrocitrinins A-C, 1-3) and fourteen known compounds (4-17) were isolated from the deep-sea-derived Penicillium citrinum W17. Their structures were elucidated by comprehensive analyses of 1D and 2D NMR, HRESIMS, and ECD calculations. Compounds 1-17 were evaluated for their anti-inflammatory and anti-osteoporotic bioactivities. All isolates exhibited significant inhibitory effects on LPS-stimulated nitric oxide production in murine brain microglial BV-2 cells in a dose-response manner. Notably, compound 14 displayed the strongest effect with the IC50 value of 4.7 µM. Additionally, compounds 6, 7, and 8 significantly enhanced osteoblast mineralization, which was comparable to that of the positive control, purmorphamine. Furthermore, these three compounds also suppressed osteoclastogenesis in a dose-dependent manner under the concentrations of 2.5 µM, 5.0 µM, and 10 µM.

Mitochondria-Targeted Thymidylate Synthase Inhibitor Based on Fluorescent Molecularly Imprinted Polymers for Tumor Antimetabolic Therapy.

Antimetabolites targeting thymidylate synthase (TS), such as 5-fluorouracil and capecitabine, have been widely used in tumor therapy in the past decades. Here, we present a strategy to construct mitochondria-targeted antimetabolic therapeutic nanomedicines based on fluorescent molecularly imprinted polymers (FMIP), and the nanomedicine was denoted as Mito-FMIP. Mito-FMIP, synthesized using fluorescent dye-doped silica as the carrier and amino acid sequence containing the active center of TS as the template peptide, could specifically recognize and bind to the active site of TS, thus inhibiting the catalytic activity of TS, and therefore hindering subsequent DNA biosynthesis, ultimately inhibiting tumor growth. The imprinting factor of FMIP reached 2.9, and the modification of CTPB endowed Mito-FMIP with the ability to target mitochondria. In vitro experiments demonstrated that Mito-FMIP was able to efficiently aggregate in mitochondria and inhibit CT26 cell proliferation by 59.9%. The results of flow cytometric analysis showed that the relative mean fluorescence intensity of Mito-FMIP accumulated in the mitochondria was 3.4-fold that of FMIP. In vivo experiments showed that the tumor volume of the Mito-FMIP-treated group was only one third of that of the untreated group. In addition, Mito-FMIP exibited the maximum emission wavelength at 682 nm, which allowed it to be used for fluorescence imaging of tumors. Taken together, this study provides a new strategy for the construction of nanomedicines with antimetabolic functions based on molecularly imprinted polymers.

Biomaterials promote in vivo generation and immunotherapy of CAR-T cells.

Chimeric antigen receptor-T (CAR-T) cell therapy based on functional immune cell transfer is showing a booming situation. However, complex manufacturing processes, high costs, and disappointing results in the treatment of solid tumors have limited its use. Encouragingly, it has facilitated the development of new strategies that fuse immunology, cell biology, and biomaterials to overcome these obstacles. In recent years, CAR-T engineering assisted by properly designed biomaterials has improved therapeutic efficacy and reduced side effects, providing a sustainable strategy for improving cancer immunotherapy. At the same time, the low cost and diversity of biomaterials also offer the possibility of industrial production and commercialization. Here, we summarize the role of biomaterials as gene delivery vehicles in the generation of CAR-T cells and highlight the advantages of in-situ construction in vivo. Then, we focused on how biomaterials can be combined with CAR-T cells to better enable synergistic immunotherapy in the treatment of solid tumors. Finally, we describe biomaterials' potential challenges and prospects in CAR-T therapy. This review aims to provide a detailed overview of biomaterial-based CAR-T tumor immunotherapy to help investigators reference and customize biomaterials for CAR-T therapy to improve the efficacy of immunotherapy.

A Novel Hexokinases Inhibitor Based on Molecularly Imprinted Polymer for Combined Starvation and Enhanced Photothermal Therapy of Malignant Tumors.

The heat tolerance of tumor cells induced by heat shock proteins (HSPs) is the major factor that seriously hinders further application of PTT, as it can lead to tumor inflammation, invasion, and even recurrence. Therefore, new strategies to inhibit HSPs expression are essential to improve the antitumor efficacy of PTT. Here, we prepared a novel nanoparticle inhibitor by synthesizing molecularly imprinted polymers with a high imprinting factor (3.1) on the Prussian Blue surface (PB@MIP) for combined tumor starvation and photothermal therapy. Owing to using hexokinase (HK) epitopes as the template, the imprinted polymers could inhibit the catalytic activity of HK to interfere with glucose metabolism by specifically recognizing its active sites and then achieve starvation therapy by restricting ATP supply. Meanwhile, MIP-mediated starvation downregulated the ATP-dependent expression of HSPs and then sensitized tumors to hyperthermia, ultimately improving the therapeutic effect of PTT. As the inhibitory effect of PB@MIP on HK activity, more than 99% of the mice tumors were eliminated by starvation therapy and enhanced PTT.

Multimode Sensing Platform Based on Turn-On Fluorescent Silicon Nanoparticles for Monitoring of Intracellular pH and GSH.

Various physiological activities and metabolic reactions of cells need to be carried out under the corresponding pH environment. Intracellular GSH as an acid tripeptide and an important reducing substance also plays an important role in maintaining cellular acid-base balance and redox balance. Therefore, developing a method to monitor pH and GSH and their changes in cells is necessary. Herein, we developed a novel turn-on fluorescent silicon nanoparticles (SiNPs) using N-(2-aminoethyl)-3-aminopropyltrimethoxysilane as the silicon source and dithiothreitol as the reducing agent via a one-pot hydrothermal method. It was worth mentioning that the fluorescence intensity of the SiNPs increased along with the acidity increase, making the SiNPs have excellent pH and GSH sensing capability. Furthermore, the pH and GSH sensing performance of the SiNPs in the cell was verified by confocal imaging and flow cytometry experiment. Based on the above, the prepared SiNPs had the potential to be used as an intracellular pH and GSH multimode fluorescent sensing platform and exhibited the ability to distinguish between normal cells and cancer cells.

Bimetal MOFs catalyzed Fenton-like reaction for dual-mode detection of thiamphenicol.

In this work, we used a simple ultrasonic stripping method to synthesize a bimetal MOFs at room temperature as a nanoenzyme with peroxidase-like (POD-like) activity. Through bimetal MOFs catalytic Fenton-like competitive reaction, thiamphenicol can be quantitatively dual-mode detected by fluorescence and colorimetry. It realized the sensitive detection of thiamphenicol in water, and the limits of detection (LOD) were 0.030 nM and 0.031 nM, and the liner ranges were 0.1-150 nM and 0.1-100 nM, respectively. The methods were applied to river water, lake water and tap water samples, and with satisfactory recoveries between 97.67% and 105.54%.

Dual-reverse-signal ratiometric fluorescence method for malachite green detection based on multi-mechanism synergistic effect.

The proposition of ratiometric detection mode has demonstrated great superiority in improving analysis accuracy by forming self-calibration. Herein, the novel dual-reverse-signal ratiometric fluorescence detection for malachite green (MG) was first achieved based on synergistic effect of fluorescence resonance energy transfer (FRET) and inner filter effect (IFE). The ratiometric fluorescence probe (B-RCDs) was self-assembled via electrostatic attraction between blue-emission carbon dots (BCDs) and red-emission carbon dots (RCDs), followed with FRET effect from BCDs to RCDs and exhibited dual-emission at 450 nm and 627 nm. In the presence of MG, the IFE effect between MG and RCDs quenched the fluorescence at 627 nm and restored the fluorescence at 450 nm, sending out two reverse signals along with an obvious color change from pink to purple (302 nm UV lamp). This ratiometric method not only simplified the preparation process, but also improved the detection sensitivity, showing a low limit of detection (LOD) of 41.8 nM, which exhibited superiority than that of single-signal RCDs (157.3 nM). This method held a rapid response of 10 min and represented satisfactory recoveries (99.14%-109.08%) in real water samples, revealing it was a promising candidate in the fast, sensitive and practical detection of MG. Moreover, the design of synergistic effect supplied a new perspective for the development of ratiometric sensing in the future.

GSH-Responsive Drug Delivery System for Active Therapy and Reducing the Side Effects of Bleomycin.

The application of drug delivery system (DDS) has achieved breakthroughs in many aspects, especially in the field of tumor treatment. In this work, polyethylene glycol (PEG)-modified hollow mesoporous manganese dioxide (HMnO2@PEG) nanoparticles were used to load the anti-tumor drug bleomycin (BLM). When the DDS reached the tumor site, HMnO2@PEG was degraded and reduced to Mn2+ by the overexpression of glutathione in the tumor microenvironment, and the drug was released simultaneously. BLM coordinated with Mn2+ in situ, thereby greatly improving the therapeutic activity of BLM. The results of in vivo and in vitro treatment experiments showed that the DDS had excellent responsive therapeutic activation ability. In addition, Mn2+ exhibited strong paramagnetism and was used for T1-weighted magnetic resonance imaging in vivo. Furthermore, this therapeutic mode of responsively releasing drugs and activating in situ effectively attenuated pulmonary fibrosis initiated by BLM. In short, this DDS could help in avoiding the side effects of drugs.

Bimetallic molecularly imprinted nanozyme: Dual-mode detection platform.

Molecularly imprinted polymer nanozyme (MIL-101(Co,Fe)@MIP) with bimetallic active sites and high-efficiency peroxidase-like (POD-like) activity were synthesized for the ratiometric fluorescence and colorimetric dual-mode detection of vanillin with high selectivity and sensitivity. Compared with the monometallic nanozyme, the POD-like activity of bimetallic nanozyme was greatly enhanced by changing the electronic structure and surface structure. Ratiometric fluorescence and colorimetric dual-mode detection of vanillin in aqueous solution was realized by vanillin entering specific imprinted cavities and blocking the molecular channels on the surface of MIL-101(Co,Fe)@MIP and the dual-mode visual detection was also realized. The limits of detection were as low as 104 nM and 198 nM, respectively. The method proposed in this paper was applied to the real samples of ice cream and candy. And the recoveries were between 93.3% and 105.5%, which also reached a satisfactory degree. The further detection of dexamethasone and prednisone, two drugs belonging to glucocorticoid, proved that the nanozyme analysis method based on MIL-101(Co,Fe)@MIP could be developed into a sensing platform.

Two-photon-excited tumor cell fluorescence targeted imaging based on transferrin-functionalized silicon nanoparticles.

Transferrin-functionalized silicon nanoparticles (Trf-SiNPs) were fabricated and utilized for targeted fluorescence imaging in tumor cells. Silicon nanoparticles (SiNPs) was firstly synthesized by microwave irradiation method, and then coupled with transferrin in the presence of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS). The structural informations of Trf-SiNPs were measured by transmission electron microscope and Fourier transform infrared spectrometer. The optical properties of Trf-SiNPs were characterized by ultraviolet absorption spectrum, fluorescence emission spectrum, fluorescence quantum yield, fluorescence lifetime, photo-stability, and so on. MTT assay evidenced the low toxicity of Trf-SiNPs. Finally, Trf-SiNPs were successfully applied in HeLa cells and HepG2 cells for targeted fluorescence imaging under single-photon excitation and two-photon excitation.

H2O2 self-supplying degradable epitope imprinted polymers for targeted fluorescence imaging and chemodynamic therapy.

Chemodynamic therapy (CDT), the ability to transform H2O2 into a highly toxic hydroxyl radical (˙OH) through a Fenton or Fenton like reaction to kill cancer cells, enables selective tumor therapy. However, the effect is seriously limited by the insufficiency of endogenous H2O2 in cancer cells. Additionally, the specific recognition of epitope imprinting plays an important role in targeting cancer cell markers. In this work, we prepared H2O2 self-supplying degradable epitope molecularly imprinted polymers (MIP) for effective CDT, employing fluorescent calcium peroxide (FCaO2) as an imaging probe and a source of H2O2, the exposed peptide in the CD47 extracellular region as the template, copper acrylate as one of the functional monomers and N,N'-bisacrylylcystamine (BAC) as a cross-linker. MIP with recognition sites can specifically target CD47-positive cancer cells to achieve fluorescence imaging. Under the reduction of glutathione (GSH), the MIP were degraded and the exposed FCaO2 reacted with water to continuously produce H2O2 in the slightly acidic environment in cancer cells. The self-supplied H2O2 produced ˙OH through a Fenton like catalytic reaction mediated by copper ions in the MIP framework, inducing cancer cell apoptosis. Therefore, the MIP nano-platform, which was capable of specific recognition of the cancer cell marker, H2O2 self-supply and controlled treatment, was successfully used for targeted CDT.

Phosphate-Degradable Nanoparticles Based on Metal-Organic Frameworks for Chemo-Starvation-Chemodynamic Synergistic Antitumor Therapy.

Chemodynamic therapy (CDT) was regarded as a promising approach for tumor treatment. However, owing to the insufficient amount of endogenous hydrogen peroxide (H2O2) in tumor cells, the efficacy of CDT was limited. In this study, we designed phosphate-responsive nanoparticles (denoted as MGDFT NPs) based on metal-organic frameworks, which were simultaneously loaded with drug doxorubicin (DOX) and glucose oxidases (GOx). The decorated GOx could act as a catalytic nanomedicine for the response to the abundant glucose in the tumor microenvironment, generating a great deal of H2O2, which would enhance the Fenton reaction and produce toxic hydroxyl radicals (·OH). Meanwhile, the growth of tumors would also be inhibited by overconsuming the intratumoral glucose, which was the "fuel" for cell proliferation. When the nanoparticles entered into tumor cells, a high concentration of phosphate induced structure collapse, releasing the loaded DOX for chemotherapy. Furthermore, the decoration of target agents endowed the nanoparticles with favorable target ability to specific tumor cells and mitochondria. Consequently, the designed MGDFT NPs displayed desirable synergistic therapeutic effects via combining chemotherapy, starvation therapy, and enhanced Fenton reaction, facilitating the development of multimodal precise antitumor therapy.

Targeted Mitochondrial Fluorescence Imaging-Guided Tumor Antimetabolic Therapy with the Imprinted Polymer Nanomedicine Capable of Specifically Recognizing Dihydrofolate Reductase.

As we all know, inhibiting the activity of dihydrofolate reductase (DHFR) has always been an effective strategy for folate antimetabolites to treat tumors. In the past, it mainly relied on chemical drugs. Here, we propose a new strategy, (3-propanecarboxyl)triphenylphosphonium bromide (CTPB)-modified molecularly imprinted polymer nanomedicine (MIP-CTPB). MIP-CTPB prepared by imprinting the active center of DHFR can specifically bind to the active center to block the catalytic activity of DHFR, thereby inhibiting the synthesis of DNA and ultimately inhibiting the tumor growth. The modification of CTPB allows the nanomedicine to be targeted and enriched in mitochondria, where DHFR is abundant. The confocal laser imaging results show that MIP-CTPB can target mitochondria. Cytotoxicity experiments show that MIP-CTPB inhibits HeLa cell proliferation by 42.2%. In vivo experiments show that the tumor volume of the MIP-CTPB-treated group is only one-sixth of that of the untreated group. The fluorescent and paramagnetic properties of the nanomedicine enable targeted fluorescence imaging of mitochondria and T2-weighted magnetic resonance imaging of tumors. This research not only opens up a new direction for the application of molecular imprinting, but also provides a new idea for tumor antimetabolic therapy guided by targeted mitochondrial imaging.

The Preparation of CuInS2-ZnS-Glutathione Quantum Dots and Their Application on the Sensitive Determination of Cytochrome c and Imaging of HeLa Cells.

Cytochrome c (Cyt c), one of the most significant proteins acting as an electron transporter, plays an important role during the transferring process of the energy in cells. Apoptosis, one of the major forms of cell death, has been associated with various physiological regularity and pathological mechanisms. It was found that Cyt c can be released from mitochondria to cytosol under different pathological conditions, triggering subsequent cell apoptosis. Herein, we developed a fluorescence nanoprobe based on negatively charged CuInS2-ZnS-GSH quantum dots (QDs) for the sensitive determination of Cyt c. CuInS2-ZnS-GSH QDs with high photochemical stability and favorable hydrophilicity were prepared by a simple hot reflux method and emit a bright orange-red light. The electron-deficient heme group in Cyt c is affiliated with the electron-rich CuInS2-ZnS-GSH QDs through the photo-induced electron transfer process, resulting in a large decrease in fluorescence intensity of QDs. A good linearity for concentration of Cyt c in the range of 0.01-7 µmol L-1 is obtained, and the detection limit of Cyt c is as low as 1.1 nM. The performance on the detection of Cyt c in spiked human serum and fetal bovine serum samples showed good recoveries from 85.5% to 95.0%. Furthermore, CuInS2-ZnS-GSH QDs were applied for the intracellular imaging in HeLa cells showing an extremely lower toxicity and excellent biocompatibility.

Determination of Fe(â ¢) ion and cellular bioimaging based on a novel photoluminescent silicon nanoparticles.

The determination approaches of Fe (Ⅲ) in biological samples were developed by a novel water-soluble silicon nanoparticles (SiNPs). The SiNPs were synthesized by a facile microwave-assisted method, and simultaneously featured strong blue fluorescence (photoluminescence quantum yield: 25.2%), long lifetime (~13.29 ns) and good photo-stability. The fluorescence intensities of SiNPs were gradually quenched with Fe (Ⅲ) concentration increasing from 2.0 to 50 µmol/L. The detection limit of the established method was 0.56 µmol/L and the precision for eleven replicate detections of 20 µmol/L Fe (Ⅲ) was 3.2% (relative standard deviation, RSD). The spiked recoveries were 99.0%-104.5%. Results of the lifetime decay and cyclic voltammetry (CV) evidenced that the electron transfer was responsible for the fluorescence quenching mechanism of SiNPs and Fe (Ⅲ). Moreover, the SiNPs were successfully applied in the determination of Fe(Ⅲ) in different environmental waters and human serum. Finally, the resulting SiNPs exhibited the green fluorescence in HeLa cells as the optical probe.

Homochiral fluorescence responsive molecularly imprinted polymer: Highly chiral enantiomer resolution and quantitative detection of L-penicillamine.

In this work, we innovatively synthesized homochiral fluorescence nano molecularly imprinted polymers (D-MIP) with dual affinity (metal ion affinity and homochiral affinity) for the specific separation and detection of L-penicillamine (L-PA), which is a core-shell structure with a SiO2-covered CDs core and an imprinted layer with L-PA cavities. A switch for fluorescence response was built by chelating grafted Cu2+, what's more, the imprinted L-PA was pre immobilized by Cu2+ to form the directional imprinting with predetermined spatial structure. More importantly, the homochiral affinity of D-galactose in homochiral molecularly imprinted polymers (D-MIP) greatly enhanced the selective adsorption of L-PA, and D-MIP showed a high selectivity factor (α) of 3.45, which is 1.9 times that of the non-homochiral molecularly imprinted polymers (MIP). Meanwhile, D-MIP exhibited a high enantiomeric excess (ee) value of 56% for separation of racemic PA. Additionally, a high sensitive and selective method was established for the detection of L-PA.

Preparation of responsive "dual-lock" nanoparticles and their application in collaborative therapy based on CuS coordination.

It is difficult for drug delivery systems to release drugs as expected, often leading to undesired side effects. To solve this problem, a CuS@MSN/DOX@MnO2@membrane (CMDMm) was reasonably designed. It was introduced to release the drug by a double response, similar to using two keys to open two locks at the same time for one door. CuS@MSN was used as a photothermal therapy (PTT) material and carrier, and then the surface of CuS@MSN/DOX was sealed by MnO2 to prevent drug release in advance. MnO2 could be reduced and degraded in a tumor microenvironment. It was applied in MR imaging due to the T1 magnetism of Mn2+ following the reduction of MnO2. Finally, the 4T1 cell membrane was extracted and coated onto the surface of CuS@MSN/DOX@MnO2, which served as a target for 4T1 tumor cells. A noteworthy phenomenon was that the fluorescence of DOX was quenched by the coordination between DOX and CuS, and this greatly improved the interaction between DOX and CuS@MSN. However, the coordination was weakened when DOX was protonated in a tumor microenvironment (∼pH 5.0), leading to the release of DOX and fluorescence recovery. The drug release experiments showed that the release efficiency was higher at pH 5.0 with 10 mmol L-1 GSH. Through in vitro laser confocal imaging, it was successfully observed that DOX was reliably released in specific tumor cells according to the fluorescence recovery, and that there was no leakage in other cells. In short, effective double response drug release was successfully confirmed.

HA targeted-biodegradable nanocomposites responsive to endogenous and exogenous stimulation for multimodal imaging and chemo-/photothermal therapy.

Multimodal imaging-guided accurate tumor-targeting and efficient synergistic therapy are of great importance for cancer therapy in vitro and in vivo. In this study, a biocompatible, tumor-targeted, on-demand chemo-/photothermal therapeutic nanoplatform (HIDSiGdNPs@PDA-HA) based on hollow mesoporous organic silica nanoparticles (HMONs) was used for bimodal imaging and multi-factor stepwise response for drug release and treatment. Targeted molecule hyaluronic acid (HA) promoted the endocytosis of HIDSiGdNPs@PDA-HA in HeLa cancer cells. The gatekeeper pH-/light-sensitive PDA coating was stimulated by the endogenous tumor acidic microenvironment and exogenous NIR laser to release doxorubicin (DOX). Thereafter, HMONs containing S-S bonds were reduced and degraded by endogenous glutathione (GSH), and the drug was further released rapidly to kill cancer cells. Importantly, the photothermal reagent indocyanine green (ICG) was always retained in the carrier, improving the effectiveness of photothermal therapy. The loaded Gd-doped silicon nanoparticles (SiGdNPs) combined with DOX and ICG led to multi-color fluorescence imaging in vitro and magnetic resonance imaging in vivo to realize targeted diagnosis and track drug distribution. The treatment results of tumor-bearing mice also proved the excellent synergistic therapy. It is believed that the multifunctional nanomaterials with dual mode imaging capability and targeted and controlled collaborative therapy would provide an alternative for accurate diagnosis and efficient treatment.

Preparation of glycan-oriented imprinted polymer coating Gd-doped silicon nanoparticles for targeting cancer Tn antigens and dual-modal cell imaging via boronate-affinity surface imprinting.

Early and accurate detection of breast cancer plays an important role in improving the survival rates of patients. In this work, we designed and synthesized the Gal-NAc-imprinted nanoparticles (GIPs) via boronate-affinity glycan-oriented surface imprinting strategy. Molecularly imprinted polymers (MIPs) were hybridized with fluorescent silicon nanoparticles (SiNPs) to target Tn antigens. However, the single fluorescent imaging mode is not conducive to obtaining accurate diagnosis, due to its poor tissue penetration. To resolve this obstacle, doping gadolinium (Gd) into SiNPs was adopted to emerge an extra significant magnetic resonance (MR) signal, achieving highly sensitive fluorescence imaging and magnetic resonance imaging (MRI) with high spatial resolution. GIPs had uniform particle size around 31.8 nm, and exhibited satisfactory fluorescence stability. The maximum adsorption capacity of GIPs was 1.15 µM/g with a high imprinting factor (IF) of 7.5. Confocal laser scanning microscope imaging revealed that the GIPs had excellent specific recognition ability with a low cytotoxicity. GIPs also showed an outstanding MR performance on cancer cells. Therefore, the synthesized nanoparticles had desirable performance in dual-model imaging to specifically target recognition cancer cells. It may have a tremendous potential in real biological samples.