A portable/miniaturized analytical kit for on-site analysis: Chemical vapor generation-visual colorimetric and smartphone RGB dual-mode for detection of sulfide ion in water and food additives.

Chemical vapor generation (CVG) was used as a gaseous sample introduction technique for the visual/smartphone RGB readout colorimetric system, with the advantages of efficient matrix elimination and high vapor generation efficiency, this analytical system exhibits a good selectivity and sensitivity. Sulfide ion (S2-) in solution was transformed to its volatile form (H2S), the generated H2S reacted with a silver-containing metal organic framework (Ag-BTC) selectively, Ag2S was thus generated. Ag-BTC (fabricated on paper sheet) changed from white to dark brown, the color variance was identified by smartphone and naked-eye simultaneously. Under the optimized conditions, a limit of detection of 0.02 µg/mL was obtained by naked-eye. Several water samples and commercial food additives were analyzed for confirming its accuracy and potential application for on-site detection, recoveries ranging 94-110 % were obtained. To meet the demand of on-site analysis of S2-, this colorimetric system was integrated in a portable/miniaturized analytical kit. It is an easy-used, affordable and portable analytical kit for S2- detection in field.

A label-free cyclic amplification strategy for microRNA detection by coupling graphene oxide-controlled adsorption with superlong poly(thymine)-hosted fluorescent copper nanoparticles.

Herein, based on a terminal deoxynucleotidyl transferase (TdT)-mediated superlong poly-T-templated-copper nanoparticles (poly T-CuNPs) strategy, a simple, universal and label-free fluorescent biosensor for the detection of miRNA was constructed by employing graphene oxide (GO) and DNase I. In this strategy, GO and DNase I were used as a switch and amplifier of the signal generation pathway, respectively, and the fluorescence of poly T-CuNPs was used as the signal output. In the presence of target miRNA, the DNA dissociated from the GO surface by forming a miRNA/DNA duplex and was degraded by DNase I. The short oligos with 3'-OH, the product of DNase I degradation, could be recognized by the TdT and added to a long poly-T tail. Finally, the fluorescence signal was output through the synthesis of poly T-CuNPs. As a proof of concept, let-7a was analyzed. The method showed good sensitivity and selectivity with a linear response in the 50 pM-10,000 pM let-7a concentration range and a 30 pM limit of detection (LOD = 30 pM, R2 = 0.9954, the relative standard deviation were 2.79%-5.30%). It was also successfully applied to the determination of miRNA in spiked human serum samples. It showed good linearity in the range of 500-10000 pM (R2 = 0.9969, the relative standard deviation were 1.61%-3.85%). Moreover, both the adsorption of GO and the degradation of DNase I are DNA sequence-independent; thus, this method can be applied to the detection of any miRNA simply by changing the assisted-DNA sequence.

A Cu2+-assisted fluorescence switch biosensor for detecting of coenzyme A employing nitrogen-doped carbon dots.

Herein, a simple and sensitive Cu2+-assisted fluorescence switch biosensor for the detection of coenzyme A (CoA) was proposed by employing nitrogen-doped carbon dots (N-CDs). N-CDs were successfully synthesized by sodium alginate and melatonin via pyrolysis. The as-prepared N-CDs were spherical with an average diameter of 2.8 nm and exhibited blue emission (λem = 480 nm, λex = 360 nm) with a high fluorescence quantum yield of 50.2%. The intense blue emission of the N-CDs could be effectively quenched by copper ions through the formation of the N-CDs/Cu2+ complex. With the introduction of CoA, a more stable CoA/Cu2+ complex formed, leading to the fluorescence recovery of N-CDs. Based on this strategy, CoA could be sensitively and selectively detected with a good linear relationship in the range of 0.02-5.00 µM and with a detection limit of 12 nM. In addition, this sensor was applied for CoA detection in human serum samples with satisfactory recovery. The results showed great potential towards advancing applications in CoA-dependent bioresearch.

Association of sirtuin-1 and vascular endothelial growth factor expression with tumor progression and poor prognosis in liposarcoma.

OBJECTIVES: A relationship exists between sirtuin-1 expression and growth and survival of malignant tumors. This study aimed to investigate the prognostic value of sirtuin-1 and vascular endothelial growth factor (VEGF) expression in patients with liposarcoma by examining associations between their expression levels and clinical outcomes. METHODS: Clinical and histopathological characteristics and follow-up and survival information were retrospectively reviewed for 42 liposarcoma cases. Sirtuin-1 and VEGF protein expression levels were evaluated by immunohistochemistry and their associations with clinical parameters were analyzed using the Spearman-rho test. Univariate and multivariate Cox regression analyses were performed to identify potential prognostic factors. Kaplan-Meier analysis was performed to analyze overall survival. RESULTS: Sirtuin-1 and VEGF protein expression levels were significantly associated with histological grade, metastasis, and American Joint Committee on Cancer stage. A significant positive correlation was observed between sirtuin-1 and VEGF expression levels (R = 0.677). In univariate analysis, sirtuin-1 and VEGF expression were correlated with shorter overall survival, but the association was significant only for sirtuin-1 (hazard ratio = 3.752, 95% confidence interval 1.553-9.062) in multivariate analysis. CONCLUSION: Sirtuin-1 and VEGF expression levels are significantly correlated with progression of liposarcoma, and sirtuin-1 expression significantly predicts a poor prognosis in patients with liposarcoma.

Label-free and sensitive microRNA detection based on a target recycling amplification-integrated superlong poly(thymine)-hosted copper nanoparticle strategy.

Poly(thymine)-hosted copper nanoparticles (poly T-CuNPs) have emerged as a promising label-free fluorophore for bioanalysis, but its application in RNA-related studies is still rarely explored. Herein, by utilizing duplex-specific nuclease (DSN) as a convertor to integrate target recycling mechanism into terminal deoxynucleotidyl transferase (TdT)-mediated superlong poly T-CuNPs platform, a specific and sensitive method for microRNA detection has been developed. In this strategy, a 3'-phosphorylated DNA probe can hybridize with target RNA and then be cut by DSN to produce 3'-hydroxylated fragments, which can be further tailed by TdT with superlong poly T for fluorescent CuNPs synthesis. As proof of concept, an analysis of let-7d was achieved with a good linear correlation between 20 and 1000 pM (R2 = 0.9965) and a detection limit of 20 pM. Moreover, both homologous and heterologous microRNAs were also effectively discriminated. This strategy might pave a brand-new way for designing label-free and sensitive microRNA assays.

Label-free and sensitive assay for deoxyribonuclease I activity based on enzymatically-polymerized superlong poly(thymine)-hosted fluorescent copper nanoparticles.

Deoxyribonuclease I (DNase I) is an important physiological indicator and diagnostic biomarker, but traditional methods for assessing its activity are time-consuming, laborious, and usually radioactive. Herein, by effectively combining the special functions of DNase I and terminal deoxynucleotidyl transferase (TdT), a simple, green, cost-effective, label-free and ultrasensitive assay for DNase I activity has been constructed based on superlong poly(thymine)-hosted copper nanoparticles (poly T-CuNPs). In this strategy, a 3'-phosphorylated DNA primer is designed to block TdT polymerization. After addition of DNase I, the primer could be digested to release 3'-hydroxylated fragments, which could further be tailed by TdT in dTTP pool with superlong poly T ssDNA for CuNPs formation. Fluorescence measurements and gel electrophoresis demonstrated its feasibility for DNase I analysis. The results indicated that with a size of 3-4nm, the CuNPs templated by TdT-polymerized superlong poly T (>500 mer) had several advantages such as short synthetic time (<5min), large Stokes shift (~275nm) and intense red fluorescence emission. Under the optimal conditions, quantitative detection of DNase I was realized, showing a good linear correlation between 0.02 and 2.0U/mL (R2=0.9928) and a detection limit of 0.02U/mL. By selecting six other nucleases or proteins as controls, an excellent specificity was also verified. Then, the strategy was successfully applied to detect DNase I in diluted serum with a standard addition method, thus implying its reliability and practicability for biological samples. The proposed strategy might be promising as a sensing platform for related molecular biology and disease studies.

Nature-Inspired Smart DNA Nanodoctor for Activatable In Vivo Cancer Imaging and In Situ Drug Release Based on Recognition-Triggered Assembly of Split Aptamer.

DNA-based activatable theranostic nanoprobes are still unmet for in vivo applications. Here, by utilizing the "induced-fit effect", a smart split aptamer-based activatable theranostic probe (SATP) was first designed as "nanodoctor" for cancer-activated in vivo imaging and in situ drug release. The SATP assembled with quenched fluorescence and stable drug loading in its free state. Once binding to target proteins on cell surface, the SATP disassembled due to recognition-triggered reassembly of split aptamers with activated signals and freed drugs. As proof of concept, split Sgc8c against CEM cancer was used for theranostic studies. Benefiting from the design without blocking aptamer sequence, the SATP maintained an excellent recognition ability similar to intact Sgc8c. An "incubate-and-detect" assay showed that the SATP could significantly lower background and improve signal-to-background ratio (∼4.8 times of "always on" probes), thus affording high sensitivity for CEM cell analysis with 46 cells detected. Also, its high selectivity to target cells was demonstrated in analyzing mixed cell samples and serum samples. Then, using doxorubicin as a model, highly specific drug delivery and cell killing was realized with minimized toxicity to nontarget cells. Moreover, in vivo and ex vivo investigations also revealed that the SATP was specifically activated by CEM tumors inside mice. Especially, contrast-enhanced imaging was achieved in as short as 5 min, thus, laying a foundation for rapid diagnosis and timely therapy. As a biocompatible and target-activatable strategy, the SATP may be widely applied in cancer theranostics.

Label-Free Carbon-Dots-Based Ratiometric Fluorescence pH Nanoprobes for Intracellular pH Sensing.

Measuring pH in living cells is of great importance for better understanding cellular functions as well as providing pivotal assistance for early diagnosis of diseases. In this work, we report the first use of a novel kind of label-free carbon dots for intracellular ratiometric fluorescence pH sensing. By simple one-pot hydrothermal treatment of citric acid and basic fuchsin, the carbon dots showing dual emission bands at 475 and 545 nm under single-wavelength excitation were synthesized. It is demonstrated that the fluorescence intensities of the as-synthesized carbon dots at the two emissions are pH-sensitive simultaneously. The intensity ratio (I475 nm/I545 nm) is linear against pH values from 5.2 to 8.8 in buffer solution, affording the capability as ratiometric probes for intracellular pH sensing. It also displays that the carbon dots show excellent reversibility and photostability in pH measurements. With this nanoprobe, quantitative fluorescence imaging using the ratio of two emissions (I475 nm/I545 nm) for the detection of intracellular pH were successfully applied in HeLa cells. In contrast to most of the reported nanomaterials-based ratiometric pH sensors which rely on the attachment of additional dyes, these carbon-dots-based ratiometric probes are low in toxicity, easy to synthesize, and free from labels.

Nucleic acid tool enzymes-aided signal amplification strategy for biochemical analysis: status and challenges.

Owing to their highly efficient catalytic effects and substrate specificity, the nucleic acid tool enzymes are applied as 'nano-tools' for manipulating different nucleic acid substrates both in the test-tube and in living organisms. In addition to the function as molecular scissors and molecular glue in genetic engineering, the application of nucleic acid tool enzymes in biochemical analysis has also been extensively developed in the past few decades. Used as amplifying labels for biorecognition events, the nucleic acid tool enzymes are mainly applied in nucleic acids amplification sensing, as well as the amplification sensing of biorelated variations of nucleic acids. With the introduction of aptamers, which can bind different target molecules, the nucleic acid tool enzymes-aided signal amplification strategies can also be used to sense non-nucleic targets (e.g., ions, small molecules, proteins, and cells). This review describes and discusses the amplification strategies of nucleic acid tool enzymes-aided biosensors for biochemical analysis applications. Various analytes, including nucleic acids, ions, small molecules, proteins, and cells, are reviewed briefly. This work also addresses the future trends and outlooks for signal amplification in nucleic acid tool enzymes-aided biosensors.

Iodide-Responsive Cu-Au Nanoparticle-Based Colorimetric Platform for Ultrasensitive Detection of Target Cancer Cells.

Colorimetric analysis is promising in developing facile, fast, and point-of-care cancer diagnosis techniques, but the existing colorimetric cancer cell assays remain problematic because of dissatisfactory sensitivity as well as complex probe design or synthesis. To solve the problem, we here present a novel colorimetric analytical strategy based on iodide-responsive Cu-Au nanoparticles (Cu-Au NPs) combined with the iodide-catalyzed H2O2-TMB (3,3,5,5-tetramethylbenzidine) reaction system. In this strategy, bimetallic Cu-Au NPs prepared with an irregular shape and a diameter of ∼15 nm could chemically absorb iodide, thus indirectly inducing colorimetric signal variation of the H2O2-TMB system. By further utilizing its property of easy biomolecule modification, a versatile colorimetric platform was constructed for detection of any target that could cause the change of Cu-Au NPs concentration via molecular recognition. As proof of concept, an analysis of human leukemia CCRF-CEM cells was performed using aptamer Sgc8c-modified Cu-Au NPs as the colorimetric probe. Results showed that Sgc8c-modified Cu-Au NPs successfully achieved a simple, label-free, cost-effective, visualized, selective, and ultrasensitive detection of cancer cells with a linear range from 50 to 500 cells/mL and a detection limit of 5 cells in 100 µL of binding buffer. Moreover, feasibility was demonstrated for cancer cell analysis in diluted serum samples. The iodide-responsive Cu-Au NP-based colorimetric strategy might not only afford a new design pattern for developing cancer cell assays but also greatly extend the application of the iodide-catalyzed colorimetric system.

Poly(thymine)-Templated Copper Nanoparticles as a Fluorescent Indicator for Hydrogen Peroxide and Oxidase-Based Biosensing.

Biomineralized fluorescent metal nanoparticles have attracted considerable interest in many fields by virtue of their excellent properties in synthesis and application. Poly(thymine)-templated fluorescent copper nanoparticles (T-CuNPs) as a promising nanomaterial has been exploited by us recently and displays great potential for signal transducing in biochemical analysis. However, the application of T-CuNPs is rare and still at an early stage. Here, a new fluorescent analytical strategy has been developed for H2O2 and oxidase-based biosensing by exploiting T-CuNPs as an effective signal indicator. The mechanism is mainly based on the poly(thymine) length-dependent formation of T-CuNPs and the probe's oxidative cleavage. In this assay, the probe T40 can effectively template the formation of T-CuNPs by a fast in situ manner in the absence of H2O2, with high fluorescent signal, while the probe is cleaved into short-oligonucleotide fragments by hydroxyl radical (·OH) which is formed from the Fenton reaction in the presence of H2O2, leading to the decline of fluorescence intensity. By taking advantage of H2O2 as a mediator, this strategy is further exploited for oxidase-based biosensing. As the proof-of-concept, glucose in human serum has been chosen as the model system and has been detected, and its practical applicability has been investigated by assay of real clinical blood samples. Results demonstrate that the proposed strategy has not only good detection capability but also eminent detection performance, such as simplicity and low-cost, holding great potential for constructing effective sensors for biochemical and clinical applications.

Multiple amplification detection of microRNA based on the host-guest interaction between ß-cyclodextrin polymer and pyrene.

MicroRNAs (miRNAs) participate in various biological processes during the course of life. The levels of miRNAs can be useful biomarkers for cellular events or cancer diagnosis, thus sensitive and accurate analysis of miRNA expression is crucial for better understanding its functions and the early diagnosis of human disease. Here, we developed a multiple amplification detection method for miRNA based on the host-guest interaction between ß-cyclodextrin polymer and pyrene, which takes advantage of the polymerase-aided strand displacement amplification and λ exonuclease-assisted cyclic enzymatic amplification. The proposed method allowed quantitative detection of miRNA-21 in a dynamic range of 1 pM to 5 nM with a detection limit of 0.3 pM and demonstrated good ability to discriminate the target sequence from the single-base mismatched miRNA sequence. Moreover, the assay was applied successfully in a complex biological matrix. We believe that this proposed sensitive and specific assay has great potential as a quantification method for miRNA detection in biomedical research and clinical diagnosis.

Masking agent-free and channel-switch-mode simultaneous sensing of Fe(3+) and Hg(2+) using dual-excitation graphene quantum dots.

A novel channel-switch-mode strategy for simultaneous sensing of Fe(3+) and Hg(2+) is developed with dual-excitation single-emission graphene quantum dots (GQDs). By utilizing the dual-channel fluorescence response performance of GQDs, this strategy achieved a facile, low-cost, masking agent-free, quantitative and selective dual-ion assay even in mixed ion samples and practical water samples.

A versatile activatable fluorescence probing platform for cancer cells in vitro and in vivo based on self-assembled aptamer/carbon nanotube ensembles.

Activatable aptamer probes (AAPs) have emerged as a promising strategy in cancer diagnostics, but existing AAPs remain problematic due to complex design and synthesis, instability in biofluids, or lack of versatility for both in vitro and in vivo applications. Herein, we proposed a novel AAP strategy for cancer cell probing based on fluorophore-labeled aptamer/single-walled carbon nanotube (F-apt/SWNT) ensembles. Through π-stacking interactions and proximity-induced energy transfer, F-apt/SWNT with quenched fluorescence spontaneously formed in its free state and realized signal activation upon targeting surface receptors of living cells. As a demonstration, Sgc8c aptamer was used for in vitro analysis and in vivo imaging of CCRF-CEM cancer cells. It was found that self-assembled Cy5-Sgc8c/SWNT held robust stability for biological applications, including good dispersity in different media and ultralow fluorescence background persistent for 2 h in serum. Flow cytometry assays revealed that Cy5-Sgc8c/SWNT was specifically activated by target cells with dramatic fluorescence elevation and showed improved sensitivity with as low as 12 CCRF-CEM cells detected in mixed samples containing ~100,000 nontarget cells. In vivo studies confirmed that specifically activated fluorescence was imaged in CCRF-CEM tumors, and compared to "always on" probes, Cy5-Sgc8c/SWNT greatly reduced background signals, thus resulting in contrast-enhanced imaging. The general applicability of the strategy was also testified by detecting Ramos cells with aptamer TD05. It was implied that F-apt/SWNT ensembles hold great potential as a simple, stable, sensitive, specific, and versatile activatable platform for both in vitro cancer cell detection and in vivo cancer imaging.

A label-free activatable aptamer probe for colorimetric detection of cancer cells based on binding-triggered in situ catalysis of split DNAzyme.

A novel label-free tailed hairpin-shaped activatable aptamer probe (THAAP) was developed by rationally integrating an aptamer and a split G-quadruplex into one sequence. Based on target recognition-triggered in situ catalysis of split DNAzyme, the THAAP strategy achieved a simple, fast, washing-free, specific and quantitative colorimetric assay of human leukemic CCRF-CEM cells.

Concatemeric dsDNA-templated copper nanoparticles strategy with improved sensitivity and stability based on rolling circle replication and its application in microRNA detection.

DNA-templated copper nanoparticles (CuNPs) have emerged as promising fluorescent probes for biochemical assays, but the reported monomeric CuNPs remain problematic because of weak fluorescence and poor stability. To solve this problem, a novel concatemeric dsDNA-templated CuNPs (dsDNA-CuNPs) strategy was proposed by introducing the rolling circle replication (RCR) technique into CuNPs synthesis. In this strategy, a short oligonucleotide primer could trigger RCR and be further converted to a long concatemeric dsDNA scaffold through hybridization. After the addition of copper ions and ascorbate, concatemeric dsDNA-CuNPs could effectively form and emit intense fluorescence in the range of 500-650 nm under a 340 nm excitation. In comparison with monomeric dsDNA-CuNPs, the sensitivity of concatemeric dsDNA-CuNPs was greatly improved with ~10,000 folds amplification. And their fluorescence signal was detected to reserve ~60% at 2.5 h after formation, revealing ~2 times enhanced stability. On the basis of these advantages, microRNA let-7d was selected as the model target to testify this strategy as a versatile assay platform. By directly using let-7d as the primer in RCR, the simple, low-cost, and selective microRNA detection was successfully achieved with a good linearity between 10 and 400 pM and a detection limit of 10 pM. The concatemeric dsDNA-CuNPs strategy might be widely adapted to various analytes that can directly or indirectly induce RCR.

Locked nucleic acid/DNA chimeric aptamer probe for tumor diagnosis with improved serum stability and extended imaging window in vivo.

As promising molecular probes for in vivo tumor imaging, aptamers without modification remain problematic due to insufficient serum stability and unabiding imaging window. To address this problem, a novel locked nucleic acid (LNA)/DNA chimeric aptamer probe was developed through proper LNA incorporation and supplemented 3'-3'-thymidine (3'-3'-T) capping. TD05, a DNA aptamer against lymphoma Ramos cells, being used as the model, a series of modification strategies were designed and optimized with different positions, numbers and combinations. It was revealed that the combined use of LNA and 3'-3'-T had a synergistic effect, and with the increase of LNA substitution in stem region, the serum stability of TD05 was gradually enhanced while its affinity and specificity were perfectly maintained to Ramos cells. Particularly, TD05.6 with 7-base pair-LNA substitution exhibited the significantly elevated detection stability half-life from ∼0.5 h of TD05 to 5-6h of TD05.6 for target cells in serum. Moreover, a much slower clearance rate in tumor-bearing mice was also observed for TD05.6, thus leading to the greatly extended tumor imaging window from <150 min of TD05 to >600 min of TD05.6. This strategy might be of great potentials to generate more aptamer probes that are stable and nuclease-resistant for tumor diagnosis in real biological systems.

Poly(thymine)-templated fluorescent copper nanoparticles for ultrasensitive label-free nuclease assay and its inhibitors screening.

Noble-metal fluorescent nanoparticles have attracted considerable interest on account of their excellent properties and potential applicable importance in many fields. Particularly, we recently found that poly(thymine) (poly T) could template the formation of fluorescent copper nanoparticles (CuNPs), offering admirable potential as novel functional biochemical probes. However, exploration of poly T-templated CuNPs for application is still at a very early stage. We report herein for the first example to develop a novel ultrasensitive label-free method for the nuclease (S1 nuclease as a model system) assay, and its inhibitors screening using the poly T-templated fluorescent CuNPs. In this assay, the signal reporter of poly T of 30 mer (T30) kept the original long state in the absence of nuclease, which could effectively template the formation of fluorescent CuNPs. In the presence of nuclease, poly T was digested to mono- or oligonucleotide fragments with decrease of fluorescence. The proposed method was low-cost and simple in its operation without requirement for complex labeling of probe DNA or sophisticated synthesis of the fluorescent compound. The assay process was very rapid with only 5 min for the formation of fluorescent CuNPs. The capabilities for target detection from complex fluids and screening of nuclease inhibitors were verified. A high sensitivity exhibited with a detectable minimum concentration of 5 × 10(-7) units µL(-1) S1 nuclease, which was about 1-4 orders of magnitude more sensitive than the developed approaches.

Label-free and turn-on aptamer strategy for cancer cells detection based on a DNA-silver nanocluster fluorescence upon recognition-induced hybridization.

We present here a label-free and turn-on aptamer strategy for cancer cell detection based on the recognition-induced conformation alteration of aptamer and hybridization-induced fluorescence enhancement effect of DNA-silver nanoclusters (DNA-Ag NCs) in proximity of guanine-rich DNA sequences. In this strategy, two tailored DNA probes were involved. One is designed as a hairpin-shaped structure consisting of a target specific aptamer sequence at the 3'-end, a guanine-rich DNA sequence, and an arm segment at the 5'-end (denote as recognition probe). The other, serving as a signal probe, contains a sequence for Ag NCs templated synthesis and a link sequence complementary to the arm segment of the recognition probe. Recognizing and binding of the aptamer to cancer cells enforces the recognition probe to undergo a conformational alteration and then initiates hybridization between the arm segment of the recognition probe and the link sequence of the signal probe. The Ag NCs are then close to the guanine-rich DNA, leading to an enhanced fluorescence readout. As proof-of-concept, the CCRF-CEM cancer cell detection were performed by using the specific aptamer, sgc8c. It was demonstrated that this strategy could specially image the CCRF-CEM cells. Determination by flow cytometry allowed for detection of as low as 150 CCRF-CEM cells in 200 µL binding buffer. The general applicability of the strategy is also achieved in the successful detection of Ramos cells. These results implied that this strategy holds considerable potential for simple, sensitive, universal, and specific cancer cell detection with no required washing and separation steps.