DNA Robots for CRISPR/Cas12a Activity Management and Universal Platforms for Biosensing.

The CRISPR/Cas12a system is a revolutionary genome editing technique that is widely employed in biosensing and molecular diagnostics. However, there are few reports on precisely managing the trans-cleavage activity of Cas12a by simple modification since the traditional methods to manage Cas12a often require difficult and rigorous regulation of core components. Hence, we developed a novel CRISPR/Cas12a regulatory mechanism, named DNA Robots for Enzyme Activity Management (DREAM), by introducing two simple DNA robots, apurinic/apyrimidinic site (AP site) or nick on target activator. First, we revealed the mechanism of how the DREAM strategy precisely regulated Cas12a through different binding affinities. Second, the DREAM strategy was found to improve the selectivity of Cas12a for identifying base mismatch. Third, a modular biosensor for base excision repair enzymes based on the DREAM strategy was developed by utilizing diversified generation ways of DNA robots, and a multi-signal output platform such as fluorescence, colorimetry, and visual lateral flow strip was constructed. Furthermore, we extended logic sensing circuits to overcome the barrier that Cas12a could not detect simultaneously in a single tube. Overall, the DREAM strategy not only provided new prospects for programmable Cas12a biosensing systems but also enabled portable, specific, and humanized detection with great potential for molecular diagnostics.

Intelligent Cell Profiling and Precision Release: Multimolecular Marker-Activated Transmembrane DNA Computing Nanosystem.

Accurate classification of tumor cells is of importance for cancer diagnosis and further therapy. In this study, we develop multimolecular marker-activated transmembrane DNA computing systems (MTD). Employing the cell membrane as a native gate, the MTD system enables direct signal output following simple spatial events of "transmembrane" and "in-cell target encounter", bypassing the need of multistep signal conversion. The MTD system comprises two intelligent nanorobots capable of independently sensing three molecular markers (MUC1, EpCAM, and miR-21), resulting in comprehensive analysis. Our AND-AND logic-gated system (MTDAND-AND) demonstrates exceptional specificity, allowing targeted release of drug-DNA specifically in MCF-7 cells. Furthermore, the transformed OR-AND logic-gated system (MTDOR-AND) exhibits broader adaptability, facilitating the release of drug-DNA in three positive cancer cell lines (MCF-7, HeLa, and HepG2). Importantly, MTDAND-AND and MTDOR-AND, while possessing distinct personalized therapeutic potential, share the ability of outputting three imaging signals without any intermediate conversion steps. This feature ensures precise classification cross diverse cells (MCF-7, HeLa, HepG2, and MCF-10A), even in mixed populations. This study provides a straightforward yet effective solution to augment the versatility and precision of DNA computing systems, advancing their potential applications in biomedical diagnostic and therapeutic research.

Self-Assembled DNA Nanospheres Driven by Carbon Dots for MicroRNAs Imaging in Tumor via Logic Circuit.

DNA nanostructures with diverse biological functions have made significant advancements in biomedical applications. However, a universal strategy for the efficient production of DNA nanostructures is still lacking. In this work, a facile and mild method is presented for self-assembling polyethylenimine-modified carbon dots (PEI-CDs) and DNA into nanospheres called CANs at room temperature. This makes CANs universally applicable to multiple biological applications involving various types of DNA. Due to the ultra-small size and strong cationic charge of PEI-CDs, CANs exhibit a dense structure with high loading capacity for encapsulated DNA while providing excellent stability by protecting DNA from enzymatic hydrolysis. Additionally, Mg2+ is incorporated into CANs to form Mg@CANs which enriches the performance of CANs and enables subsequent biological imaging applications by providing exogenous Mg2+. Especially, a DNAzyme logic gate system that contains AND and OR Mg@CANs is constructed and successfully delivered to tumor cells in vitro and in vivo. They can be specifically activated by endogenic human apurinic/apyrimidinic endonuclease 1 and recognize the expression levels of miRNA-21 and miRNA-155 at tumor sites by logic biocomputing. A versatile pattern for delivery of diverse DNA and flexible logic circuits for multiple miRNAs imaging are developed.

Fluorometric Assay of Tyrosinase and Atrazine Based on the Use of Carbon Dots and the Inhibition of Tyrosinase Activity.

Sensitive and convenient strategy of tyrosinase (TYR) and its inhibitor atrazine is in pressing demand for essential research as well as pragmatic application. In this work, an exquisite label-free fluorometric assay with high sensitivity, convenience and efficiency was described for detecting TYR and the herbicide atrazine on the basis of fluorescent nitrogen-doped carbon dots (CDs). The CDs were prepared via one-pot hydrothermal reaction starting from citric acid and diethylenetriamine. TYR catalyzed the oxidation of dopamine to dopaquinone derivative which could quench the fluorescence of CDs through a fluorescence resonance energy transfer (FRET) process. Thus, a sensitive and selective quantitative evaluation of TYR can be constructed on the basis of the relationship between the fluorescence of CDs and TYR activity. Atrazine, a typical inhibitor of TYR, inhibited the catalytic activity of TYR, leading to the reduced dopaquinone and the fluorescence was retained. The strategy covered a broad linear range of 0.1-150 U/mL and 4.0-80.0 nM for TYR and atrazine respectively with a low detection limit of 0.02 U/mL and 2.4 nM/mL. It is also demonstrated that the assay can be applied to detect TYR and atrazine in spiked complex real samples, which provides infinite potential in application of disease monitoring along with environmental analysis.

A metal-organic framework and quantum dot-based ratiometric fluorescent probe for the detection of formaldehyde in food.

A green and sensitive ratio fluorescence strategy was proposed for the detection of formaldehyde (FA) in food based on a kind of metal-organic frameworks (MOFs), MIL-53(Fe)-NO2, and nitrogen-doped Ti3C2 MXene quantum dots (N-Ti3C2 MQDs) with a blue fluorescence at 450 nm. As a type of MOFs with oxidase-like activity, MIL-53(Fe)-NO2 can catalyze o-phenylenediamine (OPD) into yellow fluorescent product 2,3-diaminophenazine (DAP) with a fluorescent emission at 560 nm. DAP has the ability to suppress the blue light of N-Ti3C2 MQDs due to inner filter effect (IFE). Nevertheless, Schiff base reaction can occur between FA and OPD, inhibiting DAP production. This results in a weakening of the IFE which reverses the original fluorescence color and intensity of DAP and N-Ti3C2 MQDs. So, the ratio of fluorescence intensity detected at respective 450 nm and 560 nm was designed as the readout signal to detect FA in food. The linear range of FA detection was 1-200 µM, with a limit of detection of 0.49 µM. The method developed was successfully used to detect FA in food with satisfactory results. It indicates that MIL-53(Fe)-NO2, OPD, and N-Ti3C2 MQDs (MON) system constructed by integrating the mimics enzyme, enzyme substrate, and fluorescent quantum dots has potential application for FA detection in practical samples.

A dual-emission ratiometric fluorescence strategy with enzyme-based inhibition for organophosphorus pesticides determination.

A fast, eco-friendly and accurate ratiometric fluorescent strategy is presented for the determination of organophosphorus pesticides (OPs) using intrinsic dual-emission silica nanoparticles modified with Rhodamine 6G (SiNPs-Rho6G). SiNPs-Rho6G had intrinsic dual-emission at 410 and 550 nm. The substrate acetylcholine was catalyzed by acetylcholinesterase (AChE) to produce thiocholine (TCh). TCh triggered the specific reaction of Ellman's reagent 5, 5-dithiobis (2-nitrobenzoic acid) to obtain 5-thio-2-nitrobenzoic acid, which caused the decrease in fluorescence intensity of SiNPs-Rho6G at 410 nm by the inner filter effect, while the fluorescence intensity of SiNPs-Rho6G at 550 nm was not significantly changed. OPs caused the recovery of the fluorescence at 410 nm by inhibiting the activity of AChE. Thus, the quantitative detection of OPs could be achieved through utilizing the catalytic characteristic of AChE. The linear curve from 0.010 to 0.250 µg mL-1 with a detection limit of 7 ng mL-1 was obtained for the determination of chlorpyrifos (Cpf). The ratiometric probe was used to detect the spiked Cpf in environmental and food samples with good recoveries. Therefore, combined with the dual emission characteristics of SiNPs-Rho6G and the specificity of the enzyme, the ratio fluorescence sensing platform has potential application prospects in OPs determinations.

A ratiometric lanthanide-free fluorescent probe based on two-dimensional metal-organic frameworks and carbon dots for the determination of anthrax biomarker.

A lanthanide-free fluorescent probe has been constructed for the first time based on two-dimensional metal-organic frameworks (2D MOFs) and carbon dots (CDs) for ratiometric determination of dipicolinic acid (DPA), the biomarker of Bacillus anthracis. The fluorescence intensity at 659 nm increased due to the release of organic ligands TCPP resulting from the selective interaction between DPA and Zn2+ of 2D MOFs. CDs provided a reference signal at 445 nm which was almost unaffected, realizing self-calibration DPA sensing. F659/F445 versus the concentration of DPA shows good linear relationships in the range 0.01-0.2 µM and 0.2-10 µM under 390-nm excitation, with a detection limit of 7 nM. The ratiometric probe was prepared from 2D lanthanide-free MOFs so that the drawbacks of lanthanide-based probes were overcome. The proposed sensing system was successfully applied to the determination of DPA in spiked biological samples. These results suggest that a novel, simple, and selective strategy of determining DPA with 2D lanthanide-free MOFs is implemented. Graphical abstract Zn-TCPP nanosheets and a blue carbon dots (b-CDs) are synthesized to construct the ratiometric probe, which can exhibit fluorescence at 445and 659 nm with 390-nm excitation. Dipicolinic acid (DPA) can deprive the junction ions of Zn-TCPP nanosheets, triggering the collapse ofZn-TCPP nanosheets. The fluorescence at 659 nm is enhanced due to the release of TCPP, while the peak of b-CDs at 445 nm is almost not affected. Thus, the fluorescence intensity ratio (F659/F445) can serve as the response signal for sensitive DPA sensing.

A nanoplatform based on metal-organic frameworks and coupled exonuclease reaction for the fluorimetric determination of T4 polynucleotide kinase activity and inhibition.

A nanoplatform based on metal-organic frameworks (MOFs) and lambda exonuclease (λ exo) for the fluorimetric determination of T4 polynucleotide kinase (T4 PNK) activity and inhibition is described. Fe-MIL-88 was selected as the nanomaterial because of its significant preferential binding ability to single-stranded DNA (ssDNA) over double-stranded DNA (dsDNA) and its quenching property. The synthesized Fe-MIL-88 was characterized by transmission electron microscope, scanning electron microscope, and X-ray photoelectron spectroscopy. In the presence of T4 PNK, FAM-labeled dsDNA (FAM-dsDNA) is phosphorylated on its 5'-terminal. λ exo then recognizes and cleaves the phosphorylated strand yielding FAM-labeled ssDNA (FAM-ssDNA). The fluorescence of the produced FAM-ssDNA is quenched due to Fe-MIL-88's absorbing on FAM-ssDNA. On the contrary, in the absence of T4 PNK, the phosphorylation and cleavage processes cannot take place. Therefore, the fluorescence of FAM-dsDNA still remains. The fluorescence intensity is detected at the maximum emission wavelength of 524 nm using the maximum excitation wavelength of 488 nm. The assay of T4 PNK based on the fluorescence quenching of FAM-ssDNA achieves a linear relationship in the range 0.01-5.0 U mL-1 with a detection limit of 0.0089 U mL-1 in buffer. The assay exhibits excellent performance for T4 PNK activity determination in a complex biological matrix. The results also reveal the ability of the assay for T4 PNK inhibitor screening. Graphical abstract Schematic presentation of a nanoplatform based on Fe-MIL-88 and coupled exonuclease reaction for the fluorimetric determination of T4 polynucleotide kinase activity. FAM-ssDNA, FAM-labeled single-stranded DNA; cDNA, complementary DNA; λ exo, lambda exonuclease;T4 PNK, T4 polynucleotide kinase.

A redox modulated ratiometric fluorometric method based on the use of dual-color carbon dots for determination of the activity of enzymes participating in ascorbic acid-related reactions.

A turn-on ratiometric fluorescent assay is described for the determination of the activity of enzymes participating in ascorbic acid-forming reactions. Blue-emitting carbon dots (bCDs; with excitation/emission wavelength at 380/450 nm) serve as fluorescent indicator. Their fluorescence is reduced by Fe3+ ions via an inner filter effect. Yellow-emitting CDs (yCDs; with excitation/emission wavelength at 380/550 nm) serve as internal reference because their fluorescence is insensitive to Fe3+. Upon exposure to ascorbic acid (AA), Fe3+ is reduced to Fe2+. Hence, the fluorescence of the bCDs is restored. Thus, enzymes participating in AA-related reactions such as α-glucosidase (α-Glu) and alkaline phosphatase (ALP) can be determined. α-Glu activity was quantified in the range from 0.13 to 6.70 U mL-1, and ALP activity was determined between 2.0 and 130 U L-1. Endowed with excellent sensitivity, selectivity and low background signals, the method may also be used to screen the inhibitors of α-Glu and ALP. Graphical abstractSchematic representation of a redox modulated ratiometric fluorometric method based on the use of dual-color carbon dots for determination of the activity of enzymes participating in ascorbic acid-related reactions. Blue-emitting carbon dots (bCDs) serve as fluorescent indicator while yellow-emitting CDs (yCDs) serve as internal reference.

Carbon dots based immunosorbent assay for the determination of GFAP in human serum.

Glial fibrillary acidic protein (GFAP) is expressed in the central nervous system and the level of GFAP normally rises with brain injury and astroglial tumors. So, serum GFAP is used as a marker for diagnosing various types of brain damage and astroglial tumors. In this study, a new sensor based on carbon dots (CDs) linked with antibodies to specifically detect GFAP in human serum was developed. Anti-GFAP (Ab1) linked with protein A/G agarose resin (PA/G) as a capture antibody (PA/G-Ab1) and anti-GFAP (Ab2) labeled with CDs as a detection antibody (CDs-Ab2) were prepared firstly. Then the CD-linked antibody immunosorbent assay (CLAISA) method was constructed based on the sandwich conjunction reaction among PA/G-Ab1, GFAP, and CDs-Ab2. CLAISA, using the fluorescence of PA/G-Ab1-GFAP-Ab2-CDs as the direct signal, enabled the proposed immunosensor to detect GFAP sensitively with a linear range of 0.10-8.00 ng ml-1 and a detection limit of 25 pg ml-1. This method was applied to the determination of GFAP in human serum by the standard addition method, and the results showed high accuracy and precision. Considering the easy synthetic process and excellent performance of CLAISA, this method has great potential to be used to monitor GFAP in the clinic.

Aptamer based fluorometric determination of ATP by exploiting the FRET between carbon dots and graphene oxide.

The authors describe a fluorometric aptamer based assay for adenosine triphosphate (ATP). It is based on the use of carbon dots (CDs) and graphene oxide (GO). The resultant CD-aptamer is adsorbed on the surface of GO via π-stacking and hydrophobic interaction, and the fluorescence of CD-aptamer is quenched via fluorescence resonance energy transfer (FRET) between CDs and GO. If ATP is present, it will bind to the aptamer and the CD-aptamer will be desorbed from GO. This will suppress FRET and the fluorescence of the CDs is restored. Under the optimal conditions and at typical excitation/emission wavelengths of 358/455 nm, the assay has a 80 pM detection limit and a linear range that extends from 0.10 to 5.0 nM concentrations of ATP. The method was successfully applied to the determination of ATP in yogurt samples. This method can also be conceivably applied to the detection of other analytes for which appropriate aptamers are available. Graphical abstract Schematic of a novel fluorometric ATP assay based on the fluorescence resonance energy transfer (FRET) between aptamer modified carbon dots (CD-aptamer) and graphene oxide (GO). CD-aptamer was used as the energy donor and molecular recognition probe, and GO acted as energy acceptor. This assay exhibits high sensitivity and selectivity with a detection limit as low as 80 pM.

Aptamer-based fluorescent platform for ultrasensitive adenosine detection utilizing Fe3O4 magnetic nanoparticles and silver nanoparticles.

The authors describe an aptamer-based fluorescent assay for adenosine (Ade). It is based on the interaction between silver nanoparticles (AgNPs) and CdTe quantum dots (QDs). The beacon comprises a pair of aptamers, one conjugated to Fe3O4 magnetic nanoparticles, the other to AgNPs. In the presence of Ade, structural folding and sandwich association of the two attachments takes place. After magnetic separation, the associated sandwich structures are exposed to the QDs. The AgNPs in sandwich structures act as the signaling label of Ade by quenching the fluorescence of QDs (at excitation/emission wavelengths of 370/565 nm) via inner filter effect, electron transfer and trapping processes. As a result, the fluorescence of QDs drops with increasing Ade concentration. The assay has a linear response in the 0.1 nM to 30 nM Ade concentration range and a 60 pM limit of detection. The assay only takes 40 min which is the shortest among the aptamer-based methods ever reported. The method was successfully applied to the detection of Ade in spiked biological samples and satisfactory recoveries were obtained. Graphical abstract Schematic of a highly efficient and convenient adenosine (Ade) fluorometric assay. It is based on the interaction between Ag nanoparticles (NPs) and CdTe quantum dots (QDs). Ade aptamers (ABA1 and ABA2) are used as recognition unit and Fe3O4 magnetic nanoparticles act as magnetic separator. The assay exhibits superior sensitivity and speediness.

Core-Shell-Shell Multifunctional Nanoplatform for Intracellular Tumor-Related mRNAs Imaging and Near-Infrared Light Triggered Photodynamic-Photothermal Synergistic Therapy.

A multifunctional nanoplatform, which generally integrates biosensing, imaging diagnosis, and therapeutic functions into a single nanoconstruct, has great important significance for biomedicine and nanoscience. Here, we developed a core-shell-shell multifunctional polydopamine (PDA) modified upconversion nanoplatform for intracellular tumor-related mRNAs detection and near-infrared (NIR) light triggered photodynamic and photothermal synergistic therapy (PDT-PTT). The nanoplatform was constructed by loading a silica shell on the hydrophobic upconversion nanoparticles (UCNPs) with hydrophilic photosensitizer methylene blue (MB) entrapped in it, and then modifying PDA shells through an in situ self-polymerization process, thus yielding a core-shell-shell nanoconstruct UCNP@SiO2-MB@PDA. By taking advantages of preferential binding properties of PDA for single-stranded DNA over double-stranded DNA and the excellent quenching property of PDA, a UCNP@SiO2-MB@PDA-hairpin DNA (hpDNA) nanoprobe was developed through adsorption of fluorescently labeled hpDNA on PDA shells for sensing intracellular tumor-related mRNAs and discriminating cancer cells from normal cells. In addition, the fluorescence resonance energy transfer from the upconversion fluorescence (UCF) emission at 655 nm of the UCNPs to the photosensitizer MB molecules could be employed for PDT. Moreover, due to the strong NIR absorption and high photothermal conversion efficiency of PDA, the UCF emission at 800 nm of the UCNPs could be used for PTT. We demonstrated that the UCNP@SiO2-MB@PDA irradiated with NIR light had considerable PDT-PTT effect. These results revealed that the developed multifunctional nanoplatform provided promising applications in future oncotherapy by integrating cancer diagnosis and synergistic therapy.

Aptamer based fluorometric ß-lactoglobulin assay based on the use of magnetic nanoparticles and carbon dots.

The authors describe a fluorometric aptamer based assay for detecting ß-lactoglobulin by using carbon dots (C-dots) as a signal indicator. The aptamer was immoblized on magnetite (Fe3O4) nanoparticles (MNPs), and the C-dots served as a label for the complementary oligonucleotide (cDNA). The assay is based on the hybridization that takes place between aptamer and cDNA. In the presence of ß-lactoglobulin (ß-LG), the aptamer preferentially binds to ß-LG, and this leads to a partial release of the C-dots-cDNA into the solution. After magnetic separation, the supernatant of the solution contains the released C-dots-cDNA which are quantified by fluorometry, best under excitation/emission wavelengths of 354/447 nm. Under the optimal conditions, the fluorescence intensity is proportional to the logarithm of the ß-LG concentration in the 0.25 to 50 ng mL-1 range, with a 37 pg mL-1 detection limit. The method was successfully applied to the determination of ß-LG in hypoallergenic formulations, and the results demonstrated that this assay is a promising tool in food quality control. Conceivably, it also provides the opportunity for detection of other analytes. Graphical abstract Schematic of a novel aptamer based fluorometric ß-lactoglobulin assay based on the use of magnetite (Fe3O4) nanoparticles (MNPs) and carbon dots (C-dots). C-dots were used as a signal indicator and Fe3O4 MNPs acted as a magnetic separator. This assay exhibits high sensitivity and selectivity with a detection limit as low as 37 pg mL-1.

Endonuclease IV cleaves apurinic/apyrimidinic sites in single-stranded DNA and its application for biosensing.

Endonuclease IV (Endo IV), as a DNA repairing enzyme, plays a crucial role in repairing damaged DNA comprising abasic sites to maintain genomic integrity. The cleaving capability of Endo IV to apurinic/apyrimidinic sites (AP) in single-stranded DNA (ssDNA) was demonstrated. It was found that Endo IV has considerably high cleaving activity to AP sites in ssDNA compared with that in double-stranded DNA (dsDNA). The unique feature of Endo IV in cleaving AP sites in ssDNA was further applied to construct a novel dual signal amplified sensing system for highly sensitive enzyme and protein detection by a combination of exonuclease III (Exo III)-aided cyclic amplification reaction and a rolling circle replication (RCR) technique, which showed a good sensing performance with a detection limit of 0.008 U mL(-1) for Endo IV and 2.5 pM for streptavidin. In addition, the developed method had considerably high specificity for Endo IV and streptavidin over other potential interferences. The developed strategy indeed provides a novel platform for protein and enzyme assays and may find a broad spectrum of applications in bioanalysis, disease diagnosis, and drug development.

Phospholipid-modified upconversion nanoprobe for ratiometric fluorescence detection and imaging of phospholipase D in cell lysate and in living cells.

Phospholipase D (PLD) is a critical component of intracellular signal transduction and has been implicated in many important biological processes. It has been observed that there are abnormalities in PLD expression in many human cancers, and PLD is thus recognized as a potential diagnostic biomarker as well as a target for drug discovery. We report for the first time a phospholipid-modified nanoprobe for ratiometric upconversion fluorescence (UCF) sensing and bioimaging of PLD activity. The nanoprobe can be synthesized by a facile one-step self-assembly of a phospholipid monolayer composed of poly(ethylene glycol) (PEG)ylated phospholipid and rhodamine B-labeled phospholipid on the surface of upconversion nanoparticles (UCNPs) NaYF4: 20%Yb, 2%Er. The fluorescence resonance energy transfer (FRET) process from the UCF emission at 540 nm of the UCNPs to the absorbance of the rhodamine B occurs in the nanoprobe. The PLD-mediated hydrolysis of the phosphodiester bond makes rhodamine B apart from the UCNP surface, leading to the inhibition of FRET. Using the unaffected UCF emission at 655 nm as an internal standard, the nanoprobe can be used for ratiometric UCF detection of PLD activity with high sensitivity and selectivity. The PLD activity in cell lysates is also determined by the nanoprobe, confirming that PLD activity in a breast cancer cell is at least 7-fold higher than in normal cell. Moreover, the nanoprobe has been successfully applied to monitoring PLD activity in living cells by UCF bioimaging. The results reveal that the nanoprobe provides a simple, sensitive, and robust platform for point-of-care diagnostics and drug screening in biomedical applications.

Moral courage and its influencing factor among oncology specialist nurses: A multi-centre cross-sectional study.

AIM: Moral courage among healthcare workers has been extensively studied. However, few studies have been conducted on oncology specialist nurses, who frequently encounter complex moral situations. This study aimed to describe the current situation regarding moral courage and explored its influence on oncology specialist nurses in China. DESIGN: This was an exploratory, descriptive study. METHODS: A convenience sample of 390 nurses was conducted from 15 hospitals in Sichuan Province, China, between March and May 2023. Participants were assessed using the Moral Distress Scale-Revised, Nurses' Moral Courage Scale and the Moral Sensitivity Questionnaire. RESULTS: The results demonstrated that moral courage was negatively associated with moral distress, and positively associated with moral sensitivity. Having a master's degree or above, an intermediate title or senior title, medical ethics training, moral distress or moral sensitivity contributed to explaining 54.1% of the variance in moral courage. CONCLUSIONS: Moral courage was associated with several factors. Developing clinical intervention strategies and effective teaching methods will be critical for improving moral courage. No Patient or Public Contribution.

[Opening experiments: synthesis, purification, and characterization of polyethyleneimine-modified carbon dots].

Open experiments are an effective means of cultivating top-notch innovative talents. Based on student interests, research hotspots and our laboratory conditions, an experimental scheme was designed. In this experiment, polyethyleneimine modified carbon dots (PEI-CDs) were prepared via a one-step hydrothermal method using citric acid (CA) as the carbon source and PEI as the surface passivator. First, CA and PEI were completely dissolved in 0.1 mol/L HCl and transferred into an autoclave. The autoclave was heated to 130 ℃ for 2 h. PEI-CDs solution was obtained. After cooling to room temperature, the solution was concentrated to 2 mL by rotary evaporation. Finally, the PEI-CDs were precipitated, washed with ethanol, and dried under vacuum at 70 ℃ for 12 h. The obtained PEI-CDs were characterized by fluorescence spectrophotometry, absorption spectrophotometry, infrared spectrometry, and transmission electron microscopy. The results indicated that anhydrous-ethanol precipitation is a simple, rapid, economical, and green purification method. The as-prepared PEI-CDs had unique properties, such as good water solubility, high luminescence, uniform particle sizes, and good stability. Through this open experiment, students can not only master the operation of large-scale instruments but also enhance their interest in scientific research.

A dual-emission fluorescent nanocomplex of gold-cluster-decorated silica particles for live cell imaging of highly reactive oxygen species.

A novel nanocomplex displaying single-excitation and dual-emission fluorescent properties has been developed through a crown-like assembly of dye-encapsulated silica particles decorated with satellite AuNCs for live cell imaging of highly reactive oxygen species (hROS), including •OH, ClO(-) and ONOO(-). The design of this nanocomplex is based on our new finding that the strong fluorescence of AuNCs can be sensitively and selectively quenched by these hROS. The nanocomplex is demonstrated to have excellent biocompatibility, high intracellular delivery efficiency, and stability for long-time observations. The results reveal that the nanocomplex provides a sensitive sensor for rapid imaging of hROS signaling with high selectivity and contrast.

CRISPR/Cas12a-based fluorescence aptasensor integrated with two-dimensional cobalt oxyhydroxide nanosheets for IFN-γ detection.

Cytokine storm (CS) is a risky immune overreaction accompanied by significant elevations of pro-inflammatory cytokines including interferon-γ (IFN-γ), interleukin and tumor necrosis factor. Sensitive detection of cytokine is conducive to studying CS progress and diagnosing infectious diseases. In this study, we developed a tandem system combining aptamer, strand displacement amplification (SDA), CRISPR/Cas12a, and cobalt oxyhydroxide nanosheets (termed Apt-SCN tandem system) as a signal-amplified platform for IFN-γ detection. Owing to the stronger affinity, target IFN-γ bound specifically to the aptamer from aptamer-complementary DNA (Apt-cDNA) duplex. The cDNA released from the Apt-cDNA duplex initiated SDA, resulting in the generation of double-stranded DNA products that could activate the trans-cleavage activity of CRISPR/Cas12a. The activated CRISPR/Cas12a further cleaved FAM-labeled single-stranded DNA probe, preventing it from adhering to the cobalt oxyhydroxide nanosheets and recovering the fluorescence signal. Sensitive fluorometric analysis of IFN-γ was successfully performed with detection limit as low as 0.37 nM. Unlike traditional protein analysis methods, Apt-SCN tandem system incorporates multiple signal amplification techniques and may also be applicable for other cytokines assay. This study was the initial study to utilize SDA and CRISPR/Cas12a to detect IFN-γ, showing great potential for cytokines clinical assay and CS prevention.