Dual-Aptamer Recognition of DNA Logic Gate Sensor-Based Specific Exosomal Proteins for Ovarian Cancer Diagnosis.

Clinical diagnosis of ovarian cancer lacks high accuracy due to the weak selection of specific biomarkers along with the circumstance biomarkers localization. Clustering analysis of proteins transported on exosomes enables a more precise screening of effective biomarkers. Herein, through bioinformatics analysis of ovarian cancer and exosome proteomes, two coexpressed proteins, EpCAM and CD24, specifically enriched, were identified, together with the development of an as-derived dual-aptamer targeted exosome-based strategy for ovarian cancer screening. In brief, a DNA ternary polymer with aptamers targeting EpCAM and CD24 was designed to present a logic gate reaction upon recognizing ovarian cancer exosomes, triggering a rolling circle amplification chemiluminescent signal. A dynamic detection range of 6 orders of magnitude was achieved by quantifying exosomes. Moreover, for clinical samples, this strategy could accurately differentiate exosomes from healthy persons, other cancer patients, and ovarian cancer patients, enabling promising in situ detection. By accurately selecting biomarkers and constructing a dual-targeted exosomal protein detection strategy, the limitation of insufficient specificity of traditional protein markers was circumvented. This work contributed to the development of exosome-based prognosis monitoring in ovarian cancer through the identification of disease-specific exosome protein markers.

Metal-organic framework-based ratiometric point-of-care testing for quantitative visual detection of nitrite.

Nitrite (NO2-) is categorized as a carcinogenic substance and is subjected to severe limitations in water and food. To safeguard the public's health, developing fast and convenient methods for determination of NO2- is of significance. Point-of-care testing (POCT) affords demotic measurement of NO2- and shows huge potential in future technology beyond those possible with traditional methods. Here, a novel ratiometric fluorescent nanoprobe (Ru@MOF-NH2) is developed by integrating UiO-66-NH2 with tris(2,2'-bipyridyl)ruthenium(II) ([Ru(bpy)3]2+) through a one-pot approach. The special diazo-reaction between the amino group of UiO-66-NH2 and NO2- is responsible for the report signal (blue emission) with high selectivity and the red emission from [Ru(bpy)3]2+ offers the reference signal. The proposed probe shows obviously distinguishable color change from blue to red towards NO2- via naked-eye. Moreover, using a smartphone as the detection device to read color hue, ultra-sensitive quantitative detection of NO2- is achieved with a low limit of detection at 0.6 µΜ. The accuracy and repeatability determined in spiked samples through quantitative visualization is in the range of 105 to 117% with a coefficient of variation below 4.3%. This POCT sensing platform presents a promising strategy for detecting NO2- and expands the potential applications for on-site monitoring in food and environment safety assessment.

Responsive calcium-derived nanoassemblies induce mitochondrial disorder to promote tumor calcification.

Physiological calcification of the treated tumor area is considered to be a predictor of good prognosis. Promoting tumor calcification by inducing mitochondrial metabolic disorder and destroying calcium equilibrium has a potential inhibitory effect on tumor proliferation. Here, by promoting calcification by inducing mitochondrial dysfunction combined with triggering a surge of reactive oxygen species, we construct a bioresponsive calcification initiator, termed CaP-AA, using CaHPO4 covalently doped l-ascorbic acid. CaHPO4 releases Ca2+ within the cytoplasm of tumor cells to trigger calcium overload. Meanwhile, exogenous l-ascorbic acid indirectly enhances metabolic balance disruption via pro-oxidant effects. Such Ca2+ overload increases the likelihood of tumor calcification in vivo for tumor inhibition by perturbing mitochondrial homeostasis. The introduction of responsive calcium sources that would, in turn, trigger intratumoral calcification mediated by perturbing mitochondrial homeostasis would be an effective regulatory strategy for tumor therapy.

Compute-and-Release Logic-Gated DNA Cascade Circuit for Accurate Cancer Cell Imaging.

Accurate identification of cancer cells is an essential prerequisite for cancer diagnosis and subsequent effective curative interventions. The logic-gate-assisted cancer imaging system that allows a comparison of expression levels between biomarkers, rather than just reading biomarkers as inputs, returns a more comprehensive logical output, improving its accuracy for cell identification. To fulfill this key criterion, we develop a compute-and-release logic-gated double-amplified DNA cascade circuit. This novel system, CAR-CHA-HCR, consists of a compute-and-release (CAR) logic gate, a double-amplified DNA cascade circuit (termed CHA-HCR), and a MnO2 nanocarrier. CAR-CHA-HCR, a novel adaptive logic system, is designed to logically output the fluorescence signals after computing the expression levels of intracellular miR-21 and miR-892b. Only when miR-21 is present and its expression level is above the threshold CmiR-21 > CmiR-892b, the CAR-CHA-HCR circuit performs a compute-and-release operation on free miR-21, thereby outputting enhanced fluorescence signals to accurately image positive cells. It is capable of comparing the relative concentrations of two biomarkers while sensing them, thus allowing accurate identification of positive cancer cells, even in mixed cell populations. Such an intelligent system provides an avenue for highly accurate cancer imaging and is potentially envisioned to perform more complex tasks in biomedical studies.

Activatable Near-Infrared Fluorescent and Photoacoustic Dual-Modal Probe for Highly Sensitive Imaging of Sulfatase In Vivo.

Sulfatase is an important biomarker closely associated with various diseases. However, the state-of-the-art sulfatase probes are plagued with a short absorption/emission wavelength and limited sensitivity. Developing highly sensitive fluorescent probes for in vivo imaging of sulfatase remains a grand challenge. Herein, for the first time, an activatable near-infrared fluorescence/photoacoustic (NIRF/PA) dual-modal probe (Hcy-SA) for visualizing sulfatase activity in living cells and animals is developed. Hcy-SA is composed of a sulfate ester moiety as the recognition unit and a NIR fluorophore hemicyanine (Hcy-OH) as the NIRF/PA reporter. The designed probe exhibits a rapid response, excellent sensitivity, and high specificity for sulfatase detection in vitro. More importantly, cells and in vivo experiments confirm that Hcy-SA can be successfully applied for PA/NIRF dual-modal imaging of sulfatase activity in living sulfatase-overexpressed tumor cells and tumor-bearing animals. This probe can serve as a promising tool for sulfatase-related pathological research and cancer diagnosis.

Promoting sensitive colorimetric detection of hydroquinone and Hg2+ via ZIF-8 dispersion enhanced oxidase-mimicking activity of MnO2 nanozyme.

Reducing the agglomeration and improving the dispersibility in water of two-dimensional (2D) nanozymes is one of the effective ways to improve their enzyme-like activity. In this work, we propose a method by constructing zeolitic imidazolate framework-8 (ZIF-8)-dispersed 2D manganese-based nanozymes to achieve the specific regulated improvement of oxidase-mimicking activity. By in-situ growth of manganese oxides nanosheets of MnO2(1), MnO2(2) and Mn3O4 on the surface of ZIF-8, the corresponding nanocomposites of ZIF-8 @MnO2(1), ZIF-8 @MnO2(2), and ZIF-8 @Mn3O4 were prepared at room temperature. The Michaelis-Menton constant measurements indicated that ZIF-8 @MnO2(1) exhibits best substrate affinity and fastest reaction rate for 3,3',5,5'-tetramethylbenzidine (TMB). The ZIF-8 @MnO2(1)-TMB system was exploited to detection of trace hydroquinone (HQ) based on the reducibility of phenolic hydroxyl groups. In addition, by employing the fact that the cysteine (Cys) with the excellent antioxidant capacity can bind the Hg2+ based on the formation of "S-Hg2+" bonds, the ZIF-8 @MnO2(1)-TMB-Cys system was applied to detection of Hg2+ with high sensitivity and selectivity. Our findings not only provide a better understanding of the relationship between dispersion of nanozyme and enzyme-like activity, but also provide a general method for the detection of environmental pollutants using nanozymes.

Fluorescent and colorimetric determination of glutathione based on the inner filter effect between silica nanoparticle-gold nanocluster nanocomposites and oxidized 3,3',5,5'-tetramethylbenzidine.

Determination of glutathione (GSH) is closely related to the clinical diagnosis of many diseases. Thus, a fluorescent and colorimetric dual-readout strategy for the sensitive determination of glutathione was proposed. The mesoporous silica nanoparticle-gold nanocluster (MSN-AuNC) nanocomposites with significantly enhanced emission and effectively improved photostability characteristics were used as fluorescent probes. Based on the inner filter effect (IFE), the fluorescence of MSN-AuNCs at 570 nm can be effectively quenched by oxidized 3,3',5,5'-tetramethylbenzidine (oxTMB) with absorption in the wavelength ranges of 330-470 nm and 500-750 nm. However, the addition of GSH could cause the reduction of blue oxTMB to colorless TMB, resulting in the inhibition of IFE and the recovery of the fluorescence of MSN-AuNCs. Therefore, using oxTMB as both quencher and color indicator, a dual-readout oxTMB/MSN-AuNC sensing system for the sensitive determination of GSH was constructed. As signal amplification is caused by the fluorescence enhancement of MSN-AuNCs, the detection limits as low as 0.12 µM and 0.34 µM can be obtained for fluorescent and colorimetric assay, respectively. This method may not only offer a new idea for the sensitive and effective determination of GSH, but also broaden the applications of AuNCs in fluorescent and colorimetric dual-readout bioanalysis.

In-situ synthesis of 3D Cu2O@Cu-based MOF nanobelt arrays with improved conductivity for sensitive photoelectrochemical detection of vascular endothelial growth factor 165.

Construction of novel photoelectrochemical (PEC) materials with unique structures can effectively improve the photoelectric conversion efficiency. Here, a self-supported Cu2O@Cu-MOF/copper mesh (CM) nanobelt arrays with high specific surface area, high orientation, and high photoelectric conversion performance is obtained by in-situ grown strategy. Such PEC aptasensor is constructed based on the Cu2O@Cu-MOF/CM combined with rolling circle amplification and enzymatic biocatalytic precipitation for vascular endothelial growth factor 165 analysis. This strategy achieves excellent cooperative signal amplification, which greatly improves the detection sensitivity. The PEC aptasensor exhibited a wide calibration ranged from 10 to 1 × 108 fM with a detection limit down to 2.3 fM (S/N = 3). The construction of semiconductor@MOFs has developed the potential application of MOFs in photoelectrochemical and found a reliable path for ultrasensitive detection of biomarkers.

Hg2+-mediated stabilization of G-triplex based molecular beacon for label-free fluorescence detection of Hg2+, reduced glutathione, and glutathione reductase activity.

G-triplexes have been reported recently with the similar function to G-quadruplex that can combine with thioflavin T (ThT) and emit strong fluorescence but easier to be controlled and excited. In this work, we report an Hg2+-mediated stabilization of G-triplex based functional molecular beacon (G3TMB) sensing system for the label-free detection of Hg2+, reduced glutathione (GSH), and glutathione reductase (GR) activity. In the presence of Hg2+, the extended G-triplex sequence containing the "T" bases can form a stable hairpin structure due to the strong interactions of "T-Hg2+-T", resulting in the locking of G-tracts in the stem of the G3TMB effectively. However, the hairpin structure of the G3TMB can be opened by the introduction of GSH through the stronger "GSH-Hg2+" interaction. Therefore, by employing the fact that GR can catalyze the reduction of oxidized glutathione (GSSG) into GSH, this concept can be applied to fluorescence "off-on" detection of GR activity, with a linear range of 0.02-30 mU/mL and detection limit of 0.01 mU/mL. This work may expand a new perspective of G-triplex based functional molecular beacon as the label-free fluorescent probes in the detection of small biomolecule and enzyme activity.

Ultra-sensitive label-free electrochemical detection of the acute leukaemia gene Pax-5a based on enzyme-assisted cycle amplification.

Accurate and sensitive detection of the Pax-5a gene is of great importance in the early diagnosis and prognosis of acute leukaemia. Herein, a label-free electrochemical sensing system was proposed for the detection of the acute leukaemia Pax-5a gene based on enzyme-assisted signal amplification to generate abundant G-quadruplex/hemin DNAzyme. The presence of Pax-5a can open the hairpin probe (HP), which acts as a template. Under the action of the restriction enzymes Nt.BbvCI and Klenow fragment polymerase, the target gene Pax-5a is cycled to open the HP; On the other hand, a large number of G-quadruplex sequences are produced. The resulting G-quadruplex sequence is capable of forming the G-quadruplex/hemin complex on the surface of the electrode in the presence of hemin. The ultrasensitive label-free electrochemical detection of Pax-5a can be realized via the G-quadruplex/hemin complex-catalysed reduction of H2O2, and the detection limit was estimated to be as low as 4.6 fM. In addition, the biosensor has good specificity and stability, and also has excellent detection capabilities in a complex substrate environment. Therefore, the sensor shows great potential in bioanalysis and clinical diagnosis.

A G-triplex based molecular beacon for label-free fluorescence "turn-on" detection of bleomycin.

Since bleomycins (BLMs) play a prominent role in the clinical treatment of various cancers, the development of convenient and sensitive detection assays for BLM is of great significance in cancer therapy and related biological mechanism research. Here, taking advantage of the easily controllable and excitation of the G-triplex DNA structure, we reported a facile, label-free G-triplex based functional molecular beacon (G3MB) sensing system for fluorescence "turn-on" detection of BLM based on BLM-Fe(ii) mediated DNA strand scission. In the presence of BLM, the stable hairpin structure of G3MB undergoes an irreversible cleavage in the loop region that contains a 5'-GT-3' recognition site for BLM. The released G-tract DNA fragment self-assembles into a G-triplex-ThT complex showing a strong fluorescence. Owing to the effective locking of G-tracts in the stem of the G3MB and the specific DNA strand scission by BLM which is like a key for the release of G-tracts, the assay shows high sensitivity and selectivity with a detection limit of 0.2 nM. In addition, satisfactory results were obtained for the detection of BLM in human serum samples. Critically, the convenient "mix-and-detect" protocol, fast response and no need for modifying DNA offered a potential application of the proposed strategy for BLM assay in biomedical and clinical studies.

Sensitive fluorescence detection of heparin based on self-assembly of mesoporous silica nanoparticle-gold nanoclusters with emission enhancement characteristics.

Heparin (Hep) is widely used as a major anticoagulant in surgery. Simple and sensitive methods capable of quantitative detection of Hep are desired for better regulating its clinical use. Herein, a novel nanoassembly of amino-functionalized mesoporous silica nanoparticle-gold nanoclusters (MSN-AuNCs) with remarkable emission enhancement characteristics for sensitive fluorescence detection of Hep is developed. The electrostatic interaction between the positively charged amino-functionalized MSNs and the AuNC-stabilizing surface ligands triggers the self-assembly of MSN-AuNC nanocomposites which exhibit more than 5-fold fluorescence emission enhancement. However, the presence of negatively charged Hep inhibits the emission enhancement phenomenon due to the effective wrapping of Hep on the surface of MSNs, which blocks the interaction between AuNCs and MSNs. Benefitting from the remarkable emission enhancement and the competing binding of Hep, facile and ultrasensitive detection of Hep can be realized with a detection limit as low as 2 nM. Moreover, the successful application of the proposed method for detection of Hep in human serum samples shows promise for clinical applications.

Label-free fluorescence turn-on aptasensor for prostate-specific antigen sensing based on aggregation-induced emission-silica nanospheres.

Fluorescent light-up probes based on aggregation-induced emission (AIE)-active molecules have recently attracted great research interest due to the intelligent fluorescence activation mechanism and high sensitivity. In this work, an AIE-silica nanosphere (SiO2 NP)-based label-free fluorescent aptasensor for the sensitive "turn-on" detection of prostate-specific antigen (PSA) is reported for the first time. The positively charged amino-functionalized SiO2 NPs were used as efficient nanocapturer to electrostatically adsorb single-stranded PSA aptamer (PA) to form SiO2 NP-PA nanocomposite as well as adsorb negatively charged tetraphenylethylene derivative 3 (TPE3) to form AIE-SiO2 NP nanocomposite. The binding of the aptamer to the target PSA could induce a rigid aptamer conformation, resulting in the release of the PA away from the surface of SiO2 NPs. This made the AIE molecules TPE3 aggregate on the SiO2 NP surface and emit high fluorescence. With the advantages of simple design and rapid responses, the proposed aptasensor showed high sensitivity and selectivity for PSA with a detection limit of 0.5 ng/mL. The aptasensor was further applied in human serum samples with satisfactory results. Given its versatility, high selectivity, and sensitivity, the proposed method could be extended to other targets by varying the recognition probes. Graphical abstract An AIE-SiO2 NP-based label-free fluorescent aptasensor for the sensitive "turn-on" detection of PSA is reported for the first time.

A novel aptamer-functionalized MoS2 nanosheet fluorescent biosensor for sensitive detection of prostate specific antigen.

Prostate specific antigen (PSA) is a significant and the most widely used biomarker for the early diagnosis of prostate cancer and its subsequent treatment. A MoS2 nanosheet is a two-dimensional (2D) layered nanomaterial analogous to graphene. However, a MoS2 nanosheet has a higher fluorescence-quenching ability than graphene when applied to a dye-labeled single-stranded DNA probe. In this work, we propose a novel aptamer-functionalized MoS2 nanosheet fluorescent biosensor that detects PSA. The binding of the aptamer to the target PSA induces a rigid aptamer structure which makes the integration with the MoS2 nanosheet very weak. This results in the release of the aptamer probe from the nanosheet surface and restores the quenched fluorescence. This approach has the advantage of simple design and rapid detection of PSA. The biosensor has the merits of high sensitivity and high selectivity with a detection limit for the PSA of 0.2 ng/mL. The biosensor was also successfully applied to the detection of PSA in human serum samples with satisfactory results. The foregoing indicates its promising application to real-life biological samples.

A ligation-triggered DNAzyme cascade for amplified fluorescence detection of biological small molecules with zero-background signal.

Many types of fluorescent sensing systems have been reported for biological small molecules. Particularly, several methods have been developed for the recognition of ATP or NAD(+), but they only show moderate sensitivity, and they cannot discriminate either ATP or NAD(+) from their respective analogues. We have addressed these limitations and report here a dual strategy which combines split DNAzyme-based background reduction with catalytic and molecular beacon (CAMB)-based amplified detection to develop a ligation-triggered DNAzyme cascade, resulting in ultrahigh sensitivity. First, the 8-17 DNAzyme is split into two separate oligonucleotide fragments as the building blocks for the DNA ligation reaction, thereby providing a zero-background signal to improve overall sensitivity. Next, a CAMB strategy is further employed for amplified signal detection achieved through cycling and regenerating the DNAzyme to realize the true enzymatic multiple turnover (one enzyme catalyzes the cleavage of several substrates) of catalytic beacons. This combination of zero-background signal and signal amplification significantly improves the sensitivity of the sensing systems, resulting in detection limits of 100 and 50 pM for ATP and NAD(+), respectively, much lower than those of previously reported biosensors. Moreover, by taking advantage of the highly specific biomolecule-dependence of the DNA ligation reaction, the developed DNAzyme cascades show significantly high selectivity toward the target cofactor (ATP or NAD(+)), and the target biological small molecule can be distinguished from its analogues. Therefore, as a new and universal platform for the design of DNA ligation reaction-based sensing systems, this novel ligation-triggered DNAzyme cascade method may find a broad spectrum of applications in both environmental and biomedical fields.