Highly sensitive and selective demethylase FTO detection using a DNAzyme-mediated CRISPR/Cas12a signal cascade amplification electrochemiluminescence biosensor with C-CN/PCNV heterojunction as emitter.

Fat mass and obesity-associated protein (FTO) has gained attention as the first RNA N6-methyladenosine (m6A) modification eraser due to its overexpression being associated with various cancers. In this study, an electrochemiluminescence (ECL) biosensor for the detection of demethylase FTO was developed based on DNAzyme-mediated CRISPR/Cas12a signal cascade amplification system and carboxylated carbon nitride nanosheets/phosphorus-doped nitrogen-vacancy modified carbon nitride nanosheets (C-CN/PCNV) heterojunction as the emitter. The biosensor was constructed by modifying the C-CN/PCNV heterojunction and a ferrocene-tagged probe (ssDNA-Fc) on a glassy carbon electrode. The presence of FTO removes the m6A modification on the catalytic core of DNAzyme, restoring its cleavage activity and generating activator DNA. This activator DNA further activates the trans-cleavage ability of Cas12a, leading to the cleavage of the ssDNA-Fc and the recovery of the ECL signal. The C-CN/PCNV heterojunction prevents electrode passivation and improves the electron-hole recombination, resulting in significantly enhanced ECL signal. The biosensor demonstrates high sensitivity with a low detection limit of 0.63 pM in the range from 1.0 pM to 100 nM. Furthermore, the biosensor was successfully applied to detect FTO in cancer cell lysate and screen FTO inhibitors, showing great potential in early clinical diagnosis and drug discovery.

Electrochemiluminescence aptasensing method for ultrasensitive determination of lipopolysaccharide based on CRISPR-Cas12a accessory cleavage activity.

In this study, an ultrasensitive electrochemiluminescence (ECL) aptasensing method was developed for lipopolysaccharide (LPS) determination based on CRISPR-Cas12a accessory cleavage activity. Tris (2,2'-bipyridine) dichlororuthenium (II) (Ru(bpy)32+) was adsorbed on the surface of a glassy carbon electrode (GCE) coated with a mixture of gold nanoparticles (AuNPs) and Nafion film via electrostatic interaction. The obtained ECL platform (Ru(bpy)32+/AuNP/Nafion/GCE) exhibited strong ECL emission. Thiol-functionalized single-stranded DNA (ssDNA) was modified with a ferrocenyl (Fc) group and autonomously assembled on the ECL platform of Ru(bpy)32+/AuNP/Nafion/GCE via thiol-gold bonding, resulting in the quenching of ECL emission. After hybridization of the LPS aptamer strand (AS) with its partial complementary strand (CS), the formed double-stranded DNA (dsDNA) could activate CRISPR-Cas12a to indiscriminately cleave ssDNA-Fc on the surface of Ru(bpy)32+/AuNP/Nafion/GCE, resulting in recovery of the ECL intensity of Ru(bpy)32+ due to the increasing distance between Fc and the electrode surface. The combination of LPS and AS suppressed the formation of dsDNA, inhibited the activation of CRISPR-Cas12a, and prevented further cleavage of ssDNA-Fc. This mechanism aided in upholding the integrity of ssDNA-Fc on the surface of the electrode and was combined with ECL quenching induced by the target. The ECL intensity decreased linearly as the concentration of LPS increased from 1 to 50,000 pg/mL and followed a logarithmic relationship. This method exhibited a remarkably low detection limit of 0.24 pg/mL, which meets the requirement for low-concentration detection of LPS in the human body. The proposed method demonstrates the capacity of CRISPR-Cas12a to perform non-specific cutting of single-stranded DNA and transform the resultant cutting substances into changes in the ECL signal. By amalgamating this approach with the distinct identification abilities of LPS and its aptamers, a simple, responsive, and discriminatory LPS assay was established that holds immense significance for clinical diagnosis.

Homogeneous Electrochemiluminescence for Highly Sensitive Determination of Demethylase FTO Based on Target-Regulated DNAzyme Cleavage and Host-Guest Interaction.

Fat mass and obesity-associated protein (FTO) is the first reported N6-methyladenosine (m6A) RNA demethylase. The dysregulation of FTO demethylation is strongly associated with various human cancers in a m6A-dependent manner. Herein, a homogeneous electrochemiluminescence (ECL) method for the determination of FTO was proposed based on the target-regulated DNAzyme cleavage. Moreover, the ECL signal was highly enhanced by host-guest interaction between ß-cyclodextrin (ß-CD) and tri-n-propylamine (TPrA). The m6A caged DNAzyme 17E-Me acted as a padlock, while the FTO served as the corresponding key. As the key, FTO could specifically remove m6A modification, restoring the cleavage activity of DNAzyme 17E. With the assistance of the Zn2+ cofactor, the substrate strand was cleaved at a specific site, and the ECL indicator of Ru(phen)32+ was discharged to produce an ECL signal. On the contrary, 17E-Me was blocked and no cleavage reaction occurred without the key. For the ECL detection, the electrode modification of ß-CD@AuNPs concentrated Ru(phen)32+ species through electrostatic adsorption and gathered TPrA molecules through host-guest interaction with ß-CD, which resulted in an intense ECL response. The results demonstrated the ECL intensity linearly correlated with the logarithm of the FTO concentration (from 0.0001 to 100 nM) with a low detection limit (30 fM). The IC50 value for FTO inhibitors rhein and meclofenamic acid were 35.6 µM and 20.3 µM, respectively. The strategy was further validated for FTO detection in MCF-7 cell lysates and Hela cell lysates. This work reveals that this strategy is promising for developing homogeneous ECL method for detection of FTO and screening of the demethylase inhibitors.

Washing-Free Electrochemiluminescence Biosensor for the Simultaneous Determination of N6 Methyladenosines Incorporating a Tri-Double Resolution Strategy.

We propose a novel washing-free electrochemiluminescence (ECL) biosensor for the simultaneous detection of two types of N6 methyladenosines-RNAs (m6A-RNAs), which are potential cancer biomarkers, on the basis of binding-induced DNA strand displacement (BINSD). The biosensor integrated a tri-double resolution strategy that combined spatial and potential resolution, hybridization and antibody recognition, and ECL luminescence and quenching. The biosensor was fabricated by separately immobilizing two ECL reagents (gold nanoparticles/g-C3N4 nanosheets and ruthenium bipyridine derivative/gold nanoparticles/Nafion) and the capture DNA probe on the two sections of glassy carbon electrode. As a proof of concept, m6A-Let-7a-5p and m6A-miR-17-5p were chosen as model analytes, while m6A antibody-DNA3/ferrocene-DNA4/ferrocene-DNA5 was designed as an m6A-binding probe and DNA6/DNA7 was designed as a hybridization probe with DNA3 to release the quenching probes ferrocene-DNA4/ferrocene-DNA5. The recognition process led to the quenching of the ECL signals from both probes via BINSD. The proposed biosensor has the advantage of being washing-free. The ECL methods using the fabricated ECL biosensor with the designed probes exhibited a low detection limit of 0.03 pM for two m6A-RNAs and high selectivity. This work reveals that this strategy is promising for developing an ECL method for the simultaneous detection of two m6A-RNAs. The proposed strategy could be expanded to develop the analytical methods for the simultaneous detection of other RNA modifications by changing the antibody and hybridization probe sequences.

The Effects of Rso2 and PI Monitoring Images on the Treatment of Premature Infants Based on Deep Learning.

In recent years, due to the combined effects of individual behavior, psychological factors, environmental exposure, medical conditions, biological factors, etc., the incidence of preterm birth has gradually increased, so the incidence of various complications of preterm infants has also become higher and higher. This article is aimed at studying the therapeutic effects of preterm infants and proposing the application of rSO2 and PI image monitoring based on deep learning to the treatment of preterm infants. This article introduces deep learning, blood perfusion index, preterm infants, and other related content in detail and conducts experiments on the treatment of rSO2 and PI monitoring images based on deep learning in preterm infants. The experimental results show that the rSO2 and PI monitoring images based on deep learning can provide great help for the treatment of preterm infants and greatly improve the treatment efficiency of preterm infants by at least 15%.

Effects of early vitamin D supplementation on the prevention of bronchopulmonary dysplasia in preterm infants.

BACKGROUND: Bronchopulmonary dysplasia (BPD) is a respiratory dysfunction caused by poor lung bronchial development, which may lead to long-term lung disease, threatening the lives of children. Studies have shown that premature infants with low vitamin D are highly associated with BPD. In this study, we aim to obtain insights into whether early vitamin D supplementation could prevent BPD in preterm infants. METHODS: A total of 112 preterm infants were randomly divided into two groups: the control and vitamin D supplementation (VD) group. The VD group received vitamin D (800 IU/day) within 48 h at birth for consecutively 28 days. The serum levels of 25(OH)D3 and C-reactive protein (CRP), IL6, and TNF-α were measured using ELISA assay. The arterial partial pressure of oxygen (PaO2 ) and carbon dioxide (PaCO2 ) was measured using an i-STAT analyzer. RESULTS: The occurrence of BPD was decreased in the VD group compared with the control. The decreased serum 25(OH)D3 was significantly elevated by supplementation with vitamin D. In addition, the serum inflammation factors (CRP, IL6, and TNF-α) were significantly reduced by vitamin D supplementation. CONCLUSION: We demonstrated that early vitamin D supplementation could significantly reduce BPD incidence in preterm infants. We showed that early vitamin D supplementation could significantly increase serum level of 25(OH)D3 and reduce inflammatory response thereby preventing and reducing neonatal BPD. LIMITATION: Firstly, a larger sample size will be needed to be included to gain a comprehensive understanding of the protective effects of vitamin D and BPD mechanistically in preterm infants. Secondly, the pathophysiological process of BPD will need to be studied. In addition, the pathways that vitamin D is responsible for, need to be further researched.

The association of vitamin D and vitamin E levels at birth with bronchopulmonary dysplasia in preterm infants.

BACKGROUND: Despite improvements made in neonatal care, bronchopulmonary dysplasia (BPD) is still the most common respiratory disease in preterm infants. The relationship between the blood contents of vitamin D/E in premature infants and BPD is still controversial. METHODS: Preterm infants were recruited as the research subjects. On the basis of the inclusion and exclusion criteria, a total of 133 eligible cases were finally included. A total of 63 preterm infants with a clear diagnosis of BPD and 5 preterm infants who died before the diagnosis of BPD were in the case group, and 65 non-BPD preterm infants with equivalent baseline characteristics were in the control group. The BPD group included 38 cases in Grade Ⅰ, 18 cases in Grade Ⅱ, and 12 cases in Grade Ⅲ. The contents of vitamin D and E in the cord blood of different groups were detected by high-performance liquid chromatography and enzyme-linked immunosorbent assay. Correlation analysis adopted the Pearson correlation analytic method. RESULTS: The serum vitamin D and E levels at birth were remarkably lower in the BPD group than the non-BPD group, both of which were also correlated with the severity of BPD. The vitamin D and E contents were negatively correlated with the oxygen support duration required for premature infants with BPD. CONCLUSION: This study deepens our understanding of the field of BPD pathogenesis by demonstrating an association between vitamin D/E deficiency and BPD severity, suggesting that vitamin D and E might have potential clinical value in the prognosis and treatment of BPD.

Electrochemical non-enzymatic glucose sensors: recent progress and perspectives.

The detection of glucose has important significance in clinical medicine and the food industry, especially in the diagnosis of diabetes. In recent years, electrochemical non-enzymatic glucose sensors have attracted intensive attention to detect the glucose level with great progress. In this review, we summarize a variety of non-enzymatic glucose sensor materials, including precious metals Pt, Au and their alloy metals, non-precious transition metals and their metal oxides, composites and other functional materials. Moreover, fundamental insights into the reaction mechanism and influencing factors of materials are given. Finally, this review discusses the perspectives and challenges of future developments in electrochemical non-enzymatic glucose detection.

High-performance non-enzymatic glucose detection: using a conductive Ni-MOF as an electrocatalyst.

Conductive metal-organic frameworks (MOFs) have been studied extensively in applications like water electrolysis, gas storage, and supercapacitors due to their high conductivity and large pore volume. In this communication, we report the first use of a conductive Ni-MOF as a non-noble-metal catalyst for efficient electro-oxidation of glucose in alkaline electrolyte. As an electrochemical sensor for glucose detection, this Ni-MOF shows a fast response time of less than 3 s, a low detection limit of 0.66 µM (S/N = 3), and a high sensitivity of 21 744 µA mM-1 cm-2. This glucose sensor also displays excellent selectivity, stability and reproducibility, and its application for the detection of glucose in real samples is also demonstrated successfully.

Dendritic Cu: a high-efficiency electrocatalyst for N2 fixation to NH3 under ambient conditions.

The artificial N2 fixation to NH3 is dominated by the traditional Haber-Bosch process, which consumes large amounts of energy and natural gas with low energy efficiency and large amounts of CO2 emissions. Electrochemical N2 reduction is a promising and environmentally friendly route for artificial N2-to-NH3 fixation under milder conditions. Herein, we report that dendritic Cu acts as a highly active electrocatalyst to catalyze N2 to NH3 fixation under ambient conditions. When tested in 0.1 M HCl, such an electrocatalyst achieves a high faradaic efficiency of 15.12% and a large NH3 yield rate of 25.63 µg h-1 mgcat.-1 at -0.40 V versus a reversible hydrogen electrode. Notably, this catalyst shows high electrochemical stability and excellent selectivity toward NH3 synthesis.

Greatly Improving Electrochemical N2 Reduction over TiO2 Nanoparticles by Iron Doping.

Titanium-based catalysts are needed to achieve electrocatalytic N2 reduction to NH3 with a large NH3 yield and a high Faradaic efficiency (FE). One of the cheapest and most abundant metals on earth, iron, is an effective dopant for greatly improving the nitrogen reduction reaction (NRR) performance of TiO2 nanoparticles in ambient N2 -to-NH3 conversion. In 0.5 m LiClO4 , Fe-doped TiO2 catalyst attains a high FE of 25.6 % and a large NH3 yield of 25.47 µg h-1 mgcat -1 at -0.40 V versus a reversible hydrogen electrode. This performance compares favorably to those of all previously reported titanium- and iron-based NRR electrocatalysts in aqueous media. The catalytic mechanism is further probed with theoretical calculations.

Imaging translational and rotational diffusion of single anisotropic nanoparticles with planar illumination microscopy.

Here we demonstrated a simple yet powerful method, planar illumination microscopy, to directly track the rotational and translational diffusion dynamics of individual anisotropic nanoparticles in solution and living cells. By illuminating gold nanorods (GNRs) with two orthogonal sheets of light and resolving the polarized scattering signal with a birefringent crystal, we readily achieved three-dimensional angular resolving capability for single GNRs in noisy surroundings. The rotational dynamics of individual GNRs dispersed in glycerol/water mixtures with different chemical modification were tracked, and the measured rotational diffusion coefficient was well fitted to a previously reported theoretical model (Torre, J. G. d. l.; Martinez, M. C. L. Macromolecules 1987, 20, 661-666; Tirado, M. M.; Torre, J. G. d. l. J. Chem. Phys. 1980, 73, 1986-1993). In addition, the translational and rotational movements of individual GNRs transported by kinesin motor protein on microtubules inside living cells were directly imaged. Compared to its motion in free solution, a GNR attached to motor-protein did not rotate significantly while moving forward. Our method can be further generalized to allow determination of three-dimensional orientation of single dipoles using many different illumination modes.

Separation of nanorods by density gradient centrifugation.

The experimental conditions necessary for the synthesis of well-defined nanoparticles are often difficult to control. There is thus a compelling need for post-synthesis separation of nanoparticles polydispersed in size and shape. We demonstrate here both theoretically and experimentally that gold nanorods with diverse aspect ratios can be separated using density gradient centrifugation. By analysing the force balance of a Brownian rod falling in a Stokes flow, we derive a rigorous and predictive model that reveals the quantitative dependency of the nanorod sedimentation rates on their mass and shape. The calculations show that while mass dependency is still the dominating factor during centrifugation, the shape factor is not insignificant. Relatively heavier but long and thin rods could sediment slower than certain size of lighter spheres, and some rods and spheres with different masses and shapes may never be separated. This mass and shape dependency is exploited to separate as-prepared gold nanorod colloids by sucrose gradient centrifugation. Two layers of nanorods with narrow aspect-ratio distributions are obtained.

Three dimensional orientational imaging of nanoparticles with darkfield microscopy.

The complete three-dimensional orientations of single gold nanorods (AuNR) were successfully resolved by using a standard optical darkfield microscope through deciphering the field distribution pattern in the slightly defocused darkfield images. The resulting images depend on the aspect ratio of the AuNR, the numerical aperture of the objective, the defocusing distance, and the polarization direction of the incident radiation. Interpretation of the observed images is facilitated by comparing them with a series of simulated images with different parameters. The experimental data matched well with the simulated results, and the reliability of this technique was further verified with polarization modulation experiments. Since deconvolution can be performed off-line after the images are recorded, this approach essentially allows video-rate data acquisition. The convenient, reliable and rapid angle-resolving capability should enable broad applications in imaging studies in many scientific fields.