Sensitive aptasensing of ATP based on a PAM site-regulated CRISPR/Cas12a activation.

The combination of CRISPR/Cas12a and functional DNA provides the possibility of constructing biosensors for detecting non-nucleic-acid targets. In the current study, the duplex protospacer adjacent motif (PAM) in the activator of CRISPR/Cas12a was used as a molecular switch, and a sensitive adenosine triphosphate (ATP) detection biosensor was constructed using an allosteric probe-conjugated PAM site formation in hybridization chain reaction (HCR) integrated with the CRISPR/Cas12a system (APF-CRISPR). In the absence of ATP, an aptamer-containing probe (AP) is in a stem-loop structure, which blocks the initiation of HCR. In the presence of ATP, the structure of AP is changed upon ATP binding, resulting in the release of the HCR trigger strand and the production of long duplex DNA with many PAM sites. Since the presence of a duplex PAM site is crucial for triggering the cleavage activity of CRISPR/Cas12a, the ATP-dependent formation of the PAM site in HCR products can initiate the FQ-reporter cleavage, allowing ATP quantification by measuring the fluorescent signals. By optimizing the sequence elements and detection conditions, the aptasensor demonstrated superior detection performance. The limit of detection (LOD) of the assay was estimated to be 1.16 nM, where the standard deviation of the blank was calculated based on six repeated measurements. The dynamic range of the detection was 25-750 nM, and the whole workflow of the assay was approximately 60 min. In addition, the reliability and practicability of the aptasensor were validated by comparing it with a commercially available chemiluminescence kit for ATP detection in serum. Due to its high sensitivity, specificity, and reliable performance, the APF-CRISPR holds great potential in bioanalytical studies for ATP detection. In addition, we have provided a proof-of-principle for constructing a CRISPR/Cas12a-based aptasensor, in which the PAM is utilized to regulate Cas12a cleavage activity.

Application of Foley balloon catheter in palliative surgery for pulmonary atresia with an intact ventricular septem, with additional cases of pulmonary atresia with ventricular septal defect and tetralogy of Fallot.

BACKGROUND: Pulmonary atresia and tetralogy of Fallot can require palliative surgery in the neonatal period due to severe hypoxia; however, limitations of established techniques include high failure rate and need for cardiopulmonary bypass. Herein, right ventricular outflow tract reconstruction on a beating heart using a Foley balloon catheter is described. METHODS: A retrospective review of patients who underwent right ventricular outflow tract reconstruction on a beating heart using a Foley balloon catheter at our institution between September 2018 and March 2022 was completed. During the procedure, a Foley balloon catheter was used to occlude the blood from the right ventricular inflow tract. RESULTS: Eight patients with pulmonary atresia and intact ventricular septum underwent an off-pump right ventricular outflow tract reconstruction. One patient with pulmonary atresia and ventricular septal defect, and two patients with tetralogy of Fallot underwent an on-pump right ventricular outflow tract reconstruction on a beating heart. The procedures were successful in all patients. Patent ductus arteriosus ligation without modified Blalock-Taussig shunt placement was performed in three patients with pulmonary atresia with intact ventricular septum and two patients with tetralogy of Fallot, ductus arteriosus was left open in four patients with pulmonary atresia with intact ventricular septum. All patients remained clinically well without serious complications. CONCLUSIONS: Right ventricular outflow tract reconstruction on a beating heart using a Foley balloon catheter for pulmonary atresia and tetralogy of Fallot is a feasible alternative to catheter-based interventions or traditional surgical treatment, especially in patients with muscular infundibular stenosis or hypoplastic pulmonary annulus. Further studies with more cases are needed to verify feasibility and superiority of this approach.

Development of a DNAzyme-based colorimetric biosensor assay for dual detection of Cd2+ and Hg2.

A colorimetric biosensor assay has been developed for Cd2+ and Hg2+ detection based on Cd2+-dependent DNAzyme cleavage and Hg2+-binding-induced conformational switching of the G-quadruplex fragment. Two types of multifunctional magnetic beads (Cd-MBs and Hg-MBs) were synthesized by immobilizing two functionalized DNA sequences on magnetic beads via avidin-biotin chemistry. For Cd2+ detection, Cd-MBs are used as recognition probes, which are modified with a single phosphorothioate ribonucleobase (rA) substrate (PS substrate) and a Cd2+-specific DNAzyme (Cdzyme). In the presence of Cd2+, the PS substrate is cleaved by Cdzyme, and single-stranded DNA is released as the signal transduction sequence. After molecular assembly with the other two oligonucleotides, duplex DNA is produced, and it can be recognized and cleaved by FokI endonuclease. Thus, a signal output component consisting of a G-quadruplex fragment is released, which catalyzes the oxidation of ABTS with the addition of hemin and H2O2, inducing a remarkably amplified colorimetric signal. To rule out false-positive results and reduce interference signals, Hg-MBs modified with poly-T fragments were used as Hg2+ accumulation probes during the course of Cd2+ detection. On the other hand, Hg-MBs can perform their second function in Hg2+ detection by changing the catalytic activity of the G-quadruplex/hemin DNAzyme. In the presence of Hg2+, the G-quadruplex structure in Hg-MBs is disrupted upon Hg2+ binding. In the absence of Hg2+, an intensified color change can be observed by the naked eye for the formation of intact G-quadruplex/hemin DNAzymes. The biosensor assay exhibits excellent selectivity and high sensitivity. The detection limits for Cd2+ and Hg2+ are 1.9 nM and 19.5 nM, respectively. Moreover, the constructed sensors were used to detect environmental water samples, and the results indicate that the detection system is reliable and could be further used in environmental monitoring. The design strategy reported in this study could broadly extend the application of metal ion-specific DNAzyme-based biosensors.

LncRNA-HNF1A-AS1 functions as a competing endogenous RNA to activate PI3K/AKT signalling pathway by sponging miR-30b-3p in gastric cancer.

BACKGROUND: Accumulating evidence demonstrated that long noncoding RNAs (lncRNAs) played important regulatory roles in many cancer types. However, the role of lncRNAs in gastric cancer (GC) progression remains unclear. METHODS: RT-qPCR assay was performed to detect the expression of HNF1A-AS1 in gastric cancer tissues and the non-tumourous gastric mucosa. Overexpression and RNA interference approaches were used to investigate the effects of HNF1A-AS1 on GC cells. Insight into competitive endogenous RNA (ceRNA) mechanisms was gained via bioinformatics analysis, luciferase assays and an RNA-binding protein immunoprecipitation (RIP) assay, RNA-FISH co-localisation analysis combined with microRNA (miRNA)-pulldown assay. RESULTS: This study displayed that revealed expression of HNF1A-AS1 was associated with positive lymph node metastasis in GC. Moreover, HNF1A-AS1 significantly promoted gastric cancer invasion, metastasis, angiogenesis and lymphangiogenesis in vitro and in vivo. In addition, HNF1A-AS1 was demonstrated to function as a ceRNA for miR-30b-3p. HNF1A-AS1 abolished the function of the miRNA-30b-3p and resulted in the derepression of its target, PIK3CD, which is a core oncogene involved in the progression of GC. CONCLUSION: This study demonstrated that HNF1A-AS1 worked as a ceRNA and promoted PI3K/AKT signalling pathway-mediated GC metastasis by sponging miR-30b-3p, offering novel insights of the metastasis mechanism in GC.

FXR Acts as a Metastasis Suppressor in Intrahepatic Cholangiocarcinoma by Inhibiting IL-6-Induced Epithelial-Mesenchymal Transition.

BACKGROUND/AIMS: Intrahepatic cholangiocarcinoma (ICC) is a complicated condition, with difficult diagnosis and poor prognosis. The expression and clinical significance of the farnesoid X receptor (FXR), an endogenous receptor of bile acids, in ICC is not well understood. METHODS: Western blotting and immunochemical analyses were used to determine the levels of FXR in 4 cholangiocarcinoma cell lines, a human intrahepatic biliary epithelial cell line (HIBEpic) and 322 ICC specimens, respectively, while quantitative reverse transcription polymerase chain reaction was used to detect the mRNA levels of FXR in cholangiocarcinoma cell lines. We evaluated the prognostic value of FXR expression and its association with clinical parameters. We determined the biological significance of FXR in ICC cell lines by agonist-mediated activation and lentivirus-mediated silence. IL-6 expression was tested by an enzyme-linked immunosorbent assay and flow cytometry. In vitro, cell proliferation was examined by Cell Counting Kit-8, migration and invasion were examined by wound healing and transwell assays; in vivo, tumor migration and invasion were explored in NOD-SCID mice. RESULTS: FXR was downregulated in ICC cell lines and clinical ICC specimens. Loss of FXR was markedly correlated with aggressive tumor phenotypes and poor prognosis in patients with ICC. Moreover, FXR expression also had significant prognostic value in carbohydrate antigen 19-9 (CA19-9) negative patients. The expression of FXR was negatively correlated with IL-6 levels in clinical ICC tissues. FXR inhibited the proliferation, migration, invasion and epithelial mesenchymal transition (EMT) of ICC cells via suppression of IL-6 in vitro. Obeticholic acid, an agonist of FXR, inhibited IL-6 production, tumor growth and lung metastasis of ICC in vivo. CONCLUSIONS: FXR could be a promising ICC prognostic biomarker, especially in CA19-9 negative patients with ICC. FXR inhibits the tumor growth and metastasis of ICC via IL-6 suppression.

New Concept of X-Ray Repair Cross-Complementing Groups 1 Polymorphisms and Gynecologic Cancer Risk.

BACKGROUND: Several meta-analyses have been conducted to examine the possible link between X-ray repair cross-complementing groups 1 (XRCC1) Arg399Gln polymorphism and cervical cancer risk. However, the results are controversial. Therefore, we carried out a more comprehensive meta-analysis to examine whether XRCC1 polymorphisms are associated with general gynecologic cancer risk. METHODS: Twenty studies, comprising 4,230 cases and 5,458 controls that included analyses of XRCC1 polymorphisms (Arg194Trp, Arg280His, or Arg399Gln) were included in our study. RESULTS: Overall, no significant association between any of the studied XRCC1 polymorphisms and gynecologic cancer risk was observed. However, in further stratified analyses, the Arg399Gln was definitely associated with increased gynecologic cancer risk in Asians (A vs. G: OR 1.24; 95% CI 1.02-1.53), which was also associated with increased cervical cancer risk (A vs. G: OR 1.20; 95% CI 1.00-1.44). Similarly, the Arg194Trp was significantly associated with increased gynecologic cancer risk in Asians (TT vs. CC: OR 1.87; 95% CI 1.02-3.42) and endometrial cancer (T vs. C: OR 1.45; 95% CI 1.05-2.02). CONCLUSIONS: These findings provided evidence that XRCC1 Arg399Gln and Arg194Trp variants may modify the susceptibility to gynecologic cancers based on ethnicity and type. Further studies with large sample size are warranted to extend our findings.

Direct observation of positive supercoils introduced by reverse gyrase through atomic force microscopy.

Reverse gyrase is a hyperthermophilic enzyme that can introduce positive supercoiling in substrate DNA. It is showed in our studies that positive DNA supercoils were induced in both pBR322 vector and an artificially synthesized mini-plasmid DNA by reverse gyrase. The left-handed structures adopted by positively supercoiled DNA molecules could be identified from their right-handed topoisomers through atomic force microscopic examination. Additional structural comparisons revealed that positively supercoiled DNA molecule AFM images exhibited increased contour lengths. Moreover, enzymatic assays showed that the positively supercoiled DNA could not be cleaved by T7 endonuclease. Together, this suggests that the overwound structure of positive supercoils could prevent genomic duplex DNA from randomly forming single-stranded DNA regions and intra-stranded secondary structures.

Transformation of a Thermostable G-Quadruplex Structure into DNA Duplex Driven by Reverse Gyrase.

Reverse gyrase is a topoisomerase that can introduce positive supercoils to its substrate DNA. It is demonstrated in our studies that a highly thermal stable G-quadruplex structure in a mini-plasmid DNA was transformed into its duplex conformation after a treatment with reverse gyrase. The structural difference of the topoisomers were verified and analyzed by gel electrophoresis, atomic force microscopy examination, and endonuclease digestion assays. All evidence suggested that the overwinding structure of positive supercoil could provide a driven force to disintegrate G-quadruplex and reform duplex. The results of our studies could suggest that hyperthermophiles might use reverse gyrase to manipulate the disintegration of non-B DNA structures and safekeep their genomic information.

[Absorption and metabolism of Chuanxiong Rhizoma decoction with multi-component sequential metabolism method].

The multiple components in Chinese herbal medicines (CHMS) will experience complex absorption and metabolism before entering the blood system. Previous studies often lay emphasis on the components in blood. However, the dynamic and sequential absorption and metabolism process following multi-component oral administration has not been studied. In this study, the in situ closed-loop method combined with LC-MS techniques were employed to study the sequential process of Chuanxiong Rhizoma decoction (RCD). A total of 14 major components were identified in RCD. Among them, ferulic acid, senkyunolide J, senkyunolide I, senkyunolide F, senkyunolide G, and butylidenephthalide were detected in all of the samples, indicating that the six components could be absorbed into blood in prototype. Butylphthalide, E-ligustilide, Z-ligustilide, cnidilide, senkyunolide A and senkyunolide Q were not detected in all the samples, suggesting that the six components may not be absorbed or metabolized before entering the hepatic portal vein. Senkyunolide H could be metabolized by the liver, while senkyunolide M could be metabolized by both liver and intestinal flora. This study clearly demonstrated the changes in the absorption and metabolism process following multi-component oral administration of RCD, so as to convert the static multi-component absorption process into a comprehensive dynamic and continuous absorption and metabolism process.

Caveolin-1 Confers Resistance of Hepatoma Cells to Anoikis by Activating IGF-1 Pathway.

BACKGROUND/AIMS: Anoikis resistance is a prerequisite for hepatocellular carcinoma (HCC) metastasis. The role of Caveolin-1 (CAV1) in anoikis resistance of HCC remains unclear. METHODS: The oncogenic effect of CAV1 on anchor-independent growth and anoikis resistance was investigated by overexpression and knockdown of CAV1 in hepatoma cells. IGF-1 pathway and its downstream signals were detected by immunoblot analysis. Caveolae invagination and IGF-1R internalization was studied by electron microscopy and (125)I-IGF1 internalization assay, respectively. The role of IGF-1R and tyrosine-14 residue (Y-14) of CAV1 was explored by deletion experiment and mutation experiment, respectively. The correlation of CAV1 and IGF-1R was further examined by immunochemical analysis in 120 HCC specimens. RESULTS: CAV1 could promote anchor-independent growth and anoikis resistance in hepatoma cells. CAV1-overexpression increased the expression of IGF-1R and subsequently activated PI3K/Akt and RAF/MEK/ERK pathway, while CAV1 knockdown showed the opposite effect. The mechanism study revealed that CAV1 facilitated caveolae invagination and (125)I-IGF1 internalization. IGF-1R deletion or Y-14 mutation reversed CAV1 mediated anchor-independent growth and anoikis resistance. In addition, CAV1 expression was positively related to IGF-1R expression in human HCC tissues. CONCLUSION: CAV1 confers resistance of hepatoma cells to anoikis by activating IGF-1 pathway, providing a potential therapeutic target for HCC metastasis.

Disintegration of cruciform and G-quadruplex structures during the course of helicase-dependent amplification (HDA).

Unlike chemical damages on DNA, physical alterations of B-form of DNA occur commonly in organisms that serve as signals for specified cellular events. Although the modes of action for repairing of chemically damaged DNA have been well studied nowadays, the repairing mechanisms for physically altered DNA structures have not yet been understood. Our current in vitro studies show that both breakdown of stable non-B DNA structures and resumption of canonical B-conformation of DNA can take place during the courses of isothermal helicase-dependent amplification (HDA). The pathway that makes the non-B DNA structures repairable is presumably the relieving of the accumulated torsional stress that was caused by the positive supercoiling. Our new findings suggest that living organisms might have evolved this distinct and economical pathway for repairing their physically altered DNA structures.

The association between c.1333C > T genetic polymorphism of MTHFR gene and the risk of congenital heart diseases.

This study aims to evaluate the influence of methylenetetrahydrofolate reductase (MTHFR) gene polymorphisms on congenital heart diseases (CHD) risk. The frequencies of allelic and genotypic in CHD patients were significantly different from non-CHD controls. Our data suggested that the c.1333C > T genetic polymorphism of MTHFR gene was statistically associated with the increased risk of CHD [TT versus CC: odds ratio (OR) = 2.70, 95% confidence interval (CI) 1.34-5.45, p = 0.005; T versus C: OR = 1.38, 95% CI 1.03-1.86, p = 0.032]. Our findings indicate that the c.1333C > T genetic polymorphism influences CHD risk in the studied population.

[Impacts of multicomponent environment on solubility of puerarin in biopharmaceutics classification system of Chinese materia medica].

To illustrate the solubility involved in biopharmaceutics classification system of Chinese materia medica (CMMBCS) , the influences of artificial multicomponent environment on solubility were investigated in this study. Mathematical model was built to describe the variation trend of their influence on the solubility of puerarin. Carried out with progressive levels, single component environment: baicalin, berberine and glycyrrhizic acid; double-component environment: baicalin and glycyrrhizic acid, baicalin and berberine and glycyrrhizic acid and berberine; and treble-component environment: baicalin, berberin, glycyrrhizic acid were used to describe the variation tendency of their influences on the solubility of puerarin, respectively. And then, the mathematical regression equation model was established to characterize the solubility of puerarin under multicomponent environment.

An optimized microRNA detection platform based on PAM formation-regulated CRISPR/Cas12a activation.

MicroRNAs (miRNAs) have emerged as biomarkers for the diagnosis and prognosis of various diseases, such as cancer. Recent advancements in CRISPR/Cas12a-based biosensors in combination with hybridization chain reaction (HCR) make it a promising approach for miRNA detection. To increase the compatibility of HCR and CRISPR/Cas12a, we compared two design strategies of hairpin DNA in HCR. The results showed that different arrangements of the protospacer sequence and protospacer adjacent motif (PAM) in the hairpin DNA could affect the sensing performance. The "PAM Formation" strategy, by which the duplex PAM sites are absent in the hairpin DNA and present in the long duplex DNA after HCR, exhibited advantages in detection sensitivity. By optimizing the probe sequences and reaction conditions, we developed a miRNA detection platform. With the same crRNA, this platform enables the identification of different miRNAs by simply replacing the loop region of the target recognition probe. In addition, the proposed platform can detect single-stranded DNA and distinguishing single or multiple base mutations in the target strand. The application of discriminating the target miRNA expression levels from different cell lines validated the reliability and practicability of the sensor platform, indicating its potential applications in early clinical accurate diagnosis of cancers.

Glycyrrhizae radix et rhizoma processing ameliorates adverse reactions of polygalae radix in zebra fish and rabbit models.

ETHNOPHARMACOLOGICAL RELEVANCE: Polygala tenuifilia Willd (Polygalaceae), a traditional Chinese medicine, has been used for a long time to treat various illnesses with serious adverse reactions. Glycyrrhizae radix et rhizoma processing is generally used to reduce the adverse reactions. AIM OF THE STUDY: The aim of this study was to validate the irritation caused by raw Polygalaceae (RPA), to investigate whether processed Polygalaceae (PGA) was less irritating, and to screen and validate irritant properties of virgaureagenin G (polygala acid, PA), 3,6'-disinapoylsucrose (DSS), Tenuifolia (TEN) and polygalaxanthone III (POL), which had pharmacologically active in Polygalaceae. Zebrafish model, Draize test and High-Performance Liquid Chromatography (HPLC) were utilized to achieve the aim. MATERIALS AND METHODS: Scanning Electron Microscopy (SEM) and optical microscope were used to determine the presence of calcium oxalate needle crystal in RPA and PGA. Zebrafish egg spinning changes and zebrafish embryo behavior were used for irritation validation, irritation comparison and irritant screening. For additional evidence, the Draize test, HE staining of rabbit eyes and ELISA kit were used. Finally, changes in the composition of RPA and PGA were investigated using HPLC. RESULTS: SEM and optical microscopy revealed no calcium oxalate needle crystals in Polygalaceae. RPA, PGA, PA and DSS were able to accelerate the spinning of zebrafish eggs and the movement of embryos, while TEN and POL were not. RPA, PGA, DSS and PA may cause rabbit eyes to become hyperemic and swollen, resulting in damage to the iris, cornea and conjunctiva and increased levels of interleukin-6 (IL-6) and interleukin-10 (IL-10). Comparatively, the effects caused by PGA were less severe than those caused by RPA. In addition, compared to RPA, PGA had lower levels of DSS and PA. CONCLUSIONS: RPA, PGA, DSS, and PA were irritating. However, processing and curing could reduce the irritation by reducing the levels of DSS and PA. DSS and PA could be two potential irritants of Polygalaceae.

Confirmation of quinolone-induced formation of gyrase-DNA conjugates using AFM.

It was demonstrated in our studies that norfloxacin, a representative member of quinolone antibiotics, can indeed stabilize the gyrase-DNA complex formed during enzymatic cycle. In addition, the formation of the drug-induced complex has been firstly visualized through our atomic force microscopic examination.

Sutureless technique combined with vertical vein incision and pulmonary veins unroofed for correction of infracardiac total anomalous pulmonary venous connection.

Objective: We report a surgical method (sutureless technique), combined with vertical vein incision and pulmonary veins unroofed (semisutureless technique), to correct infracardiac total anomalous pulmonary venous connection (TAPVC). Materials and methods: The clinical characteristics of 21 patients, who were diagnosed with infracardiac TAPVS between February 2017 and March 2022, were retrospectively analyzed. These patients were divided into three groups according to different surgical methods: conventional surgery group, sutureless technique group, and semisutureless technique group. The conventional surgery group enrolled five patients with a median age of 16 days (interquartile range, 9-27 days) and a median weight of 3.25 kg (interquartile range, 3.1-3.42 kg). In this group, no preoperative pulmonary vein obstruction (PVO), preoperative ventilator support, or emergency surgery were reported. The sutureless technique group enrolled seven patients with a median age of 12 days (interquartile range, 5-16 days) and a median weight of 3.04 kg (interquartile range, 2.76-3.20 kg). In this group, two patients with preoperative PVO, four patients with preoperative ventilator support, and seven patients requiring emergency operation were found. The semisutureless technique group enrolled nine patients with a median age of 14 days (interquartile range, 7-24 days) and a median weight of 3.22 kg (interquartile range, 3.15-3.50 kg). In this group, four patients with preoperative PVO, two patients with preoperative ventilator support, and seven patients requiring emergency operation were noted. Results: In the conventional surgery group, two patients with postoperative supraventricular tachycardia, one patient with postoperative low cardiac output syndrome, one patient with PVO, and no case of postoperative death were reported. In the sutureless technique group, two patients with postoperative low cardiac output syndrome, one patient with postoperative supraventricular tachycardia, one patient with postoperative PVO, and no postoperative deaths were determined. In the semisutureless technique group, three patients had low cardiac output syndrome, two patients had supraventricular tachycardia after the operation, and one patient, who had been admitted to the hospital after cardiopulmonary resuscitation in the emergency room, died early after the operation. No case of death or PVO was noted after the operation. Conclusion: The semisutureless technique has positive effects. This surgery method can enlarge the anastomotic stoma, increase the volume of the left atrium, reduce the tension of the anastomotic stoma, fix the pulmonary vein to avoid distortion, and prevent postoperative hemorrhage.

An ultrasensitive Cd2+ detection biosensor based on DNAzyme and CRISPR/Cas12a coupled with hybridization chain reaction.

The detection of cadmium is essential because it poses a significant threat to human health and the environment. Recent advancements in biosensors that detect nonnucleic-acid targets using CRISPR/Cas12a in combination with aptamers or DNAzymes show promising performance. Herein, we integrated DNAzyme, hybridization chain reaction (HCR) and CRISPR/Cas12a into a single biosensor for the first time and realized the ultrasensitive detection of Cd2+. A single phosphorothioate ribonucleobase (rA)-containing oligonucleotide (PS substrate) and a Cd2+-specific DNAzyme (Cdzyme) are used for Cd2+ recognition, releasing short single-stranded DNA. Then, the HCR is triggered by the cleavage products for signal transduction and amplification. Next, the trans-cleavage activity of Cas12a is activated due to the presence of crRNA complementary strands and PAM sites in the HCR products. As a result, FQ-reporters are cleaved, and the fluorescence values can be easily read using a fluorometer, allowing Cd2+ quantification by measuring the fluorescent signal. The Cd2+ detection biosensor is ultrasensitive with a detection limit of 1.25 pM. Moreover, the biosensor shows great stability under different pH and various anion conditions. The proposed sensor was utilized for environmental water sample detection, demonstrating the dependability of the detection system. Considering the high sensitivity and reliable performance of the assay, it could be further used in environmental monitoring. In addition, the design strategy reported in this study could extend the application of CRISPR/Cas12a in heavy metal detection.

Observation of backbone self-crossings of organismal DNAs through atomic force microscopy.

It is demonstrated in our studies that intrinsic curvatures of organismal DNAs could lead to the generation of duplex backbone self-crossings in their relaxed forms. In addition, the backbone self-crossings of the organismal DNAs could be readily confirmed through atomic force microscopic examination.

Sensitive fluorescent aptasensing of tobramycin on graphene oxide coupling strand displacement amplification and hybridization chain reaction.

An ultrasensitive biosensor was designed and constructed for tobramycin detection. As a target recognition component, the DNA probe consists of an aptamer region for tobramycin binding and a template for amplification. In the absence of tobramycin, the probe was locked to form a stem-loop structure. In the presence of the target, the binding of tobramycin led to a conformational change in the probe. The released 3' end was used as a primer for the strand displacement amplification (SDA) to produce a large amount of single-stranded trigger DNA, which then efficiently initiated the following hybridization chain reaction (HCR) to produce a long duplex DNA with many fluorophores. The signals were detected after the addition of graphene oxide (GO) to quench the fluorescence from excess hairpin DNA. Through sequence and reaction condition optimization, the biosensor exhibited high selectivity for tobramycin. The linearity range and limit of detection (LOD) were 0.5-30 nM and 0.06 nM, respectively. Moreover, the application of detecting tobramycin in milk and lake water samples showed that this method is reliable and could be further used in food safety control and environmental monitoring.