ABSTRACT
Nucleic acid analysis using ultrasensitive and simple methods is critically important for the early-stage diagnosis and treatment of diseases. The CRISPR/Cas proteins, guided by a single-stranded RNA have shown incredible capability for sequence-specific targeting and detection. Herein, in order to improve and expand the application of CRISPR/Cas technology to the electrochemical interface-based nucleic acids analysis, the authors develop a CRISPR/Cas12a powered DNA framework-supported electrochemical biosensing platform via the cis and trans cleavage of Cas12a on the heterogeneous carbon interface (the existing publications which commonly adopted trans-cleavage). Their solid-liquid interface is first immobilized by 3D tetrahedral framework nucleic acids (FNAs) with specific DNA recognition probe. Based on the recognition of the complementary target through protospacer adjacent motif (PAM) confirmation and CRISPR-derived RNA (crRNA) matching, the easily formed Cas12a/crRNA duplex can get access to the interface, and the cis and trans cleavage of Cas12a can be easily activated. In combination with the enzyme catalyzed reaction, they achieved an ultralow limit of detection (LOD) of 100 fm in HPV-16 detection without pre-amplification. Furthermore, the platform is compatible with a spike-in human serum sample and has superior stability. Thus, their reported platform offers a practical, versatile, and amplification-free toolbox for ultrasensitive nucleic acid analysis.
Subject(s)
Bacterial Proteins/metabolism , Biosensing Techniques/methods , CRISPR-Associated Proteins/metabolism , Endodeoxyribonucleases/metabolism , Nucleic Acids/analysis , Biosensing Techniques/instrumentation , CRISPR-Cas Systems , DNA, Viral/analysis , Human papillomavirus 16/genetics , Human papillomavirus 16/isolation & purification , Limit of DetectionABSTRACT
Epigenetic alterations hold great promise as biomarkers for early stage cancer diagnosis. Nevertheless, direct identification of rare methylated DNA in the genome remains challenging. Here, we report an ultrasensitive framework nucleic acid-based electrochemical sensor for quantitative and highly selective analysis of DNA methylation. Notably, we can detect 160 fg of methylated DNA in million-fold unmethylated DNA samples using this electrochemical methylation-specific polymerase chain reaction (E-MSP) method. The high sensitivity of E-MSP enables one-step detection of low-abundance methylation at two different genes in patient serum samples. By using a combination test with two methylation alterations, we achieve high accuracy and sensitivity for reliable differentiation of prostate cancer and benign prostate hypertrophy (BPH). This new method sheds new light on translational use in early cancer diagnosis and in monitoring patients' responses to therapeutic agents.
Subject(s)
DNA Methylation , Prostatic Neoplasms , Biomarkers, Tumor/genetics , DNA/genetics , DNA Methylation/genetics , Humans , Male , Polymerase Chain Reaction , Prostatic Neoplasms/diagnosis , Prostatic Neoplasms/geneticsABSTRACT
BACKGROUND: The current study aims to investigate the relationship between plasma levels of miR-513a-5p and lipid metabolism and insulin resistance in patients with type 2 diabetes mellitus (T2DM) and nonalcoholic fatty liver disease (NAFLD). METHODS: Two hundred patients with T2DM were selected, including 104 patients with NAFLD diagnosed by ultrasound and 96 patients without NAFLD. They were divided into combined group (T2DM/NAFLD) and control group (T2DM). Height, weight, blood lipid, and blood sugar were measured. Additionally, body mass index (BMI) and homeostasis model assessment of insulin resistance (HOMA-IR) index were calculated. RT-PCR was carried out to analyze the level of plasma miR-513a-5p. The correlation between plasma miR-513a-5p level and clinical indicators was analyzed by Pearson's correlation assay. RESULTS: Compared with the T2DM group, BMI, AST, ALT, TG, GGT, LDL-C, 2hPBG, Fins, 2hIns, HbAIc (%), and HOMA-IR were significantly increased in the T2DM/NAFLD group, and plasma miR-513a-5p levels were significantly decreased. Pearson's correlation analysis showed that miR-513a-5p was negatively correlated with ALT, LDL-C, 2h Ins, and HomA-IR. Receiver operating characteristic (ROC) analysis showed plasma miR-513a-5p could differentiate T2DM/NAFLD patients from NAFLD patients. CONCLUSIONS: Decreased plasma miR-513a-5p level may act as a potential biomarker for diagnosis of NAFLD in T2DM patients.
Subject(s)
Diabetes Mellitus, Type 2/complications , MicroRNAs/blood , Non-alcoholic Fatty Liver Disease , Adult , Biomarkers/blood , Case-Control Studies , Female , Humans , Insulin Resistance , Lipid Metabolism , Male , Middle Aged , Non-alcoholic Fatty Liver Disease/complications , Non-alcoholic Fatty Liver Disease/diagnosis , Young AdultABSTRACT
DNA hydroxymethylation (5-hmC) is a kind of new epigenetic modification, which plays key roles in DNA demethylation, genomic reprogramming, and the gene expression in mammals. For further exploring the functions of 5-hmC, it is necessary to develop sensitive and selective methods for detecting 5-hmC. Herein, we developed a novel multiplexing electrochemical (MEC) biosensor for 5-hmC detection based on the glycosylation modification of 5-hmC and enzymatic signal amplification. The 5-hmC was first glycosylated by T4 ß-glucosyltransferase and then oxidated by sodium periodate. The resulting glucosyl-modified 5-hmC (5-ghmC) was incubated with ARP-biotin and was bound to avidin-HRP. The 5-hmC can be detected at the subnanogram level. Finally, we performed 5-hmC detection for mouse tissue samples and cancer cell lines. The limit of detection of the MEC biosensor is 20 times lower than that of commercial kits based on optical meaurement. Also, the biosensor presented high detection specificity because the chemical reaction for 5-hmC modification can not happen at any other unhydroxymethylated nucleic acid bases. Importantly, benefited by its multiplexing capacity, the developed MEC biosensor showed excellent high efficiency, which was time-saving and cost less.
Subject(s)
Biosensing Techniques , DNA/chemistry , DNA/metabolism , Deoxycytidine/analogs & derivatives , Electrochemical Techniques , Genomics , Animals , Bacteriophage T4/enzymology , Biosensing Techniques/economics , Cell Line, Tumor , DNA Methylation , Deoxycytidine/analysis , Deoxycytidine/genetics , Deoxycytidine/metabolism , Electrochemical Techniques/economics , Epigenesis, Genetic , Glucosyltransferases/metabolism , Glycosylation , Humans , Limit of Detection , Mice , Oxidation-Reduction , Periodic Acid/chemistryABSTRACT
In recent years, graphene has received widespread attention owing to its extraordinary electrical, chemical, optical, mechanical and structural properties. Lately, considerable interest has been focused on exploring the potential applications of graphene in life sciences, particularly in disease-related molecular diagnostics. In particular, the coupling of functional molecules with graphene as a nanoprobe offers an excellent platform to realize the detection of biomarkers, such as nucleic acids, proteins and other bioactive molecules, with high performance. This article reviews emerging graphene-based nanoprobes in electrical, optical and other assay methods and their application in various strategies of molecular diagnostics. In particular, this review focuses on the construction of graphene-based nanoprobes and their special advantages for the detection of various bioactive molecules. Properties of graphene-based materials and their functionalization are also comprehensively discussed in view of the development of nanoprobes. Finally, future challenges and perspectives of graphene-based nanoprobes are discussed.
