Immunomultiple PCR-based electrochemical and lateral flow strategy for the simultaneous detection of liver cancer tumor markers.

The current use of the single serum biomarker α-fetoprotein (AFP) in clinical practice has limitations in terms of specificity and sensitivity. We propose a strategy that combines antigen capture polymerase chain reaction (AC-PCR), lateral flow assay (LFA), and electrochemical biosensors to detect both AFP and circulating tumor cells (CTCs) in liver cancer serum. First, we used the AC-PCR technique to achieve target separation, purification, signal conversion, and amplification, eliminating target heterogeneity. Then, we achieved rapid results through the LFA and electrochemical biosensor platforms. As a result, the proposed assay has limits of 5 cells/mL for CTCs and 5 µg/L for AFP. The proposed method was applied effectively to simulated blood samples. This method has the potential to play a role in early liver cancer and provide a potential application for the diagnosis and precision treatment of liver cancer.

Liver fibrosis-derived exosomal miR-106a-5p facilitates the malignancy by targeting SAMD12 and CADM2 in hepatocellular carcinoma.

The mechanism of hepatocellular carcinoma (HCC) development induced by liver fibrosis is obscure. The objective of this study is to establish miRNAs from exosomes associated with liver fibrosis, and to identify potential biomarkers for the prediction of personalized clinical management effectiveness in HCC. Our research focused on miRNAs from exosomes and mRNA from liver fibrosis, which we found in the gene expression omnibus (GEO) database. Weighted gene co-expression network analysis (WGCNA) evaluated miRNAs from exosomes associated with liver fibrosis, and Wilcoxon analysis assessed differentially expressed mRNAs (DEGs) across liver fibrosis/normal tissues. Following that, DEGs were assessed through gene set enrichment analysis (GSEA), gene ontology (GO), and the Kyoto Encyclopedia of Genes and Genomes (KEGG). In addition, based on the screened targeted genes, including SAMD12 and CADM2, we further elucidated their correlation in HCC patients from the BEST database. The Kaplan-Meier Plotter platform was applied to evaluate the prognostic values of miRNA in HCC. In vitro and vivo experiments validated our findings. Six miRNAs associated with liver fibrosis were evaluated in our investigation. In-depth research presented exosome-derived miR-106a-5p, SAMD12 and CADM2 could exert valuable predictive implications for HCC treatment and illness assessment. Serum miR-106a-5p derived from liver fibrosis was decreased compared with healthy individuals. SAMD12 and CADM2 were diminished in liver cancer cell lines, and their knockdown of them exacerbated the proliferation capacities of liver cells in vitro. Exosome-derived miRNA of liver fibrosis modulated tumorigenesis by targeting SAMD12 and CADM2 in HCC.

XNA probe and CRISPR/Cas12a-powered flexible fluorescent and electrochemical dual-mode biosensor for sensitive detection of m6A site-specific RNA modification.

N6-methyladenosine (m6A) in RNAs is closely related to various biological progresses, but the specific regulatory mechanisms are still unclear. The existing m6A single-base resolution analysis techniques have problems of specificity and sensitivity to be improved, which can hardly meet the urgent needs of basic research and clinical applications. This work proposes a new strategy based on xeno nucleic acid (XNA) probe and CRISPR/Cas12a signal amplification for the sensitive detection of site-specific m6A modifications. According to the difference in the thermodynamic stability of hybridization between XNA probe with m6A-RNA and A-RNA, XNA was designed as a block probe to mediate m6A-RNA specific reverse transcription polymerase chain reaction (MsRT-PCR). Therefore, m6A can be specifically distinguished by converting difficult-to-test m6A modifications into easily detectable dsDNA fragments. Integration of CRISPR/Cas12a technology, skilfully designed sequences of crRNAs targeting m6A site-specific amplification dsDNA. The specificity was significantly improved through dual specific recognition of XNA probe and crRNA. Furthermore, the sensitivity of the assay was also greatly increased by the combined signal amplification of PCR and CRISPR/Cas12a. Additionally, we extend the application of CRISPR/Cas12a to flexible fluorescent and electrochemical biosensing system, which can accurately detect m6A modifications with different ranges of methylation fractions. The analysis results of m6A sites in MALAT1, ACTB and TPT1 further demonstrated the feasibility of the constructed biosensor for the accurate detection of hypomethylated samples in cells. The implementation of this work will provide strong technical support to promote the in-depth research on m6A in disease regulation mechanisms and in vitro molecular diagnosis.

Specific and robust hybridization based on double-stranded nucleic acids with single-base resolution.

Nucleic acid hybridization plays a critical role in medical diagnostics and nanotechnology, but its selectivity and robustness remain to be improved. Here, focusing on double-stranded nucleic acid-based hybridization, we present a series of related strategies. Above all, two simple strategies for enriching toehold-included double-stranded nucleic acids have been proposed. On this basis, two universal hybridization methods with higher selectivity than typical toehold exchange reaction and a long target detection method using short probes to extend the detectable length range are realized. We also provide a double-stranded nucleic acids-catalyzed cycle amplification reaction to improve sensitivity, which has superior interference resistance and excellent discrimination for single-base mismatches. Besides, double-stranded nucleic acids with forked toeholds are used as essential elements to construct a series of logic gates that can evaluate different input combinations. Given the unique advantages of double-stranded nucleic acids, we expect the current work to advance the application of double-stranded nucleic acid-based hybridization in medical diagnostics and nanotechnology.

Xeno nucleic acid probes mediated methylation-specific PCR for single-base resolution analysis of N6-methyladenosine in RNAs.

Reliable and cost-effective quantification of RNA modifications at a specific gene locus is essential to elucidate the pathogenic mechanism encoded by RNA epigenetics. Current methods to quantify N6-methyladenosine (m6A) at specific sites can hardly satisfy the requirement of clinical application because epigenetic information is easily lost through polymerase chain reaction (PCR) assay or other isothermal amplification methods unless tedious pretreatment is applied. Herein, we propose a simple xeno nucleic acid (XNA) as a blocker probe to mediate the methylation specific reverse transcription quantitative polymerase chain reaction (MsRT-qPCR) assay to directly magnify the minor differences between epigenetic bases and unmodified bases in RNA. Strand displacement reactions selectively initiated between the reverse transcription primer (RT-primer) and the XNA probe at the m6A template given the affinity differences between the blocker probes and the m6A-modified RNA (m6A-RNA) and unmodified RNA (A-RNA). Thus, preferential amplification of m6A-RNA was allowed. Integration of a well-established oligo-modified Fe3O4@UiO-66-NH4 allowed purification of mRNA and lncRNA from cellular total RNA samples and greatly reduced the non-specific interference of m6A detection in real samples. Multiple specific sites of m6A in mRNA and lncRNA samples are also successfully quantified. The XNA probe-based m6A assay required only common and available lab equipment and materials, which can be applied in m6A-related fundamental studies and clinical diagnosis.

DNAzyme based three-way junction assay for antibody-free detection of locus-specific N6-methyladenosine modifications.

N6-methyladenosine (m6A) is the most abundant post-transcriptional modification in RNA and has important implications in physiological processes and tumor development. However, sensitive and specific quantification of locus-specific m6A modification levels remains a challenging task. In the present work, a novel m6A-sensitive DNAzyme was utilized to directly detect m6A by coupling with a three-way junction-mediated isothermal exponential CRISPR amplification reaction for the first time. This method was built on the fact that the binding arm of the DNAzyme bound to the specific site and its core structure catalyzed the selective cleavage of unmodified adenine instead of methylated adenines. Subsequently, the intact RNA was identified by the proximity effect of the three-way junction. Enormous amounts of single-stranded DNA products were generated through a combination of SDA and EXPAR for signal amplification. The specific real-time curve of products was recorded through detecting the fluorescence intensity triggered by CRISPR Cas12a. As a result, methylation target of abundance down to 1% was successfully identified. In addition, this strategy could be used for the analysis of cell RNA extracts. Combined with an electrochemical sensor for quantitative detection of RNA methylation, we demonstrated the generality of as-proposed strategy. We envision the present method would provide a new platform for the analysis of m6A in RNA and promote its application in clinical diseases.

An electrochemical biosensor based on hemin/G-quadruplex DNAzyme and PdRu/Pt heterostructures as signal amplifier for circulating tumor cells detection.

Metastasis due to circulating tumor cells (CTCs) shed from the original tumor accounts for the majority of cancer-related death. Efficient CTCs detection is pivotal to the diagnosis of early cancer metastasis. In this work, Platinum nanoparticles (PtNPs) decorated hyperbranched PdRu nanospines (PdRu/Pt) hierarchical structures were firstly synthesized to detect CTCs with the assistance of DNAzyme. Meanwhile, Super P and gold nanoparticles (AuNPs) acted as sensing medium to improve electrical conductivity and immobilization of anti-EpCAM antibody to specifically capture model CTCs. After immune-conjugation of anti-EpCAM-MCF-7-signal probes on the gold electrode, PtNPs, PdRu nanospines (PdRuNSs) and hemin/G-quadruplex co-catalyzed substrate H2O2 to realize multiplexed signal amplification, which significantly improves the analytical performance of the electrochemical biosensor. As-proposed biosensor reached a limit of detection (LOD) down to 2 cells mL-1 and showed a wide detection range of 2 to 106 cells mL-1. Application of the biosensor to detect MCF-7 cells spiked human blood samples further demonstrated the feasibility for early cancer evaluation in clinic.

Electrochemical competitive immunodetection of messenger RNA modified with N6-methyladenosine by using DNA-modified mesoporous PtCo nanospheres.

N6-Methyladenosine (m6A) is the most abundant RNA modification in eukaryotic messenger RNA (mRNA). A highly sensitive electrochemical immunosensor is described for the determination of m6A-RNA. The method is based on the use of antibody (anti-m6A) and PtCo mesoporous nanospheres (MPNs). The analogously modified probe of type m6A-DNA-PtCo competes with m6A-RNA for antibodies on the gold electrode as an electrical signal probe. The electrical signal, best acquired at a working potential of -0.37 V (vs. Ag/AgCl) reflects the concentration of m6A. The PtCo MPNs catalyze the reduction of H2O2, and this amplifies the current and enhances sensitivity. The detection time of the assay is <1.5 h. Under optimal conditions, response is linear in the 0.005 to 100 nM m6A RNA concentration range, and the detection limit is 2.1 pM. The results obtained by this immunoassay with human cell lines are comparable to those obtained with a commercial kit. Graphical abstractSchematic representation of a method for electrochemical determination of m6A-modified mRNA. Anti-m6A Ab: antibody against m6A; BSA: bovine serum albumin; PtCo: PtCo mesoporous nanospheres; SH-m6A-DNA: DNA modified with both m6A and thiol groups; DPV: differential pulse voltammetry.

Fluorometric determination of microRNA by using an entropy-driven three-dimensional DNA walking machine based on a catalytic hairpin assembly reaction on polystyrene microspheres.

An entropy-driven 3-D DNA walking machine is presented which involves catalytic hairpin assembly (CHA) for detection of microRNA. A 3-D DNA walking machine was designed that uses streptavidin-coated polystyrene microspheres as track carriers to obtain reproducibility. The method was applied to microRNA 21 as a model analyte. Continuous walking on the DNA tracks is achieved via entropy increase. This results in a disassembly of ternary DNA substrates on polystyrene microspheres and leads to cycling of microRNA 21. The release of massive auxiliary strands from ternary DNA substrates induces the CHA. This is accompanied by in increase in fluorescence, best measured at excitation/emission wavelengths of 480/520 nm. On account of entropy-driven reaction, the assay is remarkably selective. It can differentiate microRNA 21 from homologous microRNAs in giving a signal that is less than 5% of the signal for microRNA 21 except for microRNA-200b. The assay works in the 50 pM to 20 nM concentration range and has a 41 pM detection limit. The method displays good reproducibility (between 1.1 and 4.2%) and recovery (from 99.8 to 104.0%). Graphical abstract An entropy-driven 3-D DNA walking machine is described. It is based on the use of polystyrene microspheres and of a catalytic hairpin assembly reaction for sensitive microRNA detection. Figure Notes: AS represents auxiliary strand; S represents substrate strand; LS represents link strand; F represents fuel nucleic acid; RepF represents nucleic acid labeled with FAM; RepQ represents nucleic acid labeled with BHQ1.

Simultaneous colorimetric determination of acute myocardial infarction biomarkers by integrating self-assembled 3D gold nanovesicles into a multiple immunosorbent assay.

An improved enzyme-free immunosorbent assay is described for the simultaneous detection of the myocardial infarction biomarkers N-terminal pro B type natriuretic peptide (NT-proBNP), creatine kinase-MB (CK-MB), and cardiac muscle troponin T (cTnT). The assay integrates 3D gold nanovesicles (GNVs) and three allochroic agents (phenolphthalein, methyl red, bromothymol blue). The pH regulated allochroic agents were enwrapped in GNVs to acts as ultrasensitive nanoprobes. Loading can be controlled by adjusting the temperature to efficiently load and release the allochroic agents. This bare-eye multicolor assay has limits of detection of 70 pg·mL-1 for NT-proBNP, 910 pg·mL-1 for CK-MB, and 7.8 pg·mL-1 for cTnT. Other features include (a) a linear range that extends over a wide range and sometimes is better than conventional HRP-based immunoassays, and (b) a precision that is comparable to immunofluorescence assays as used in the clinical laboratory. Graphical abstract Schematic presentation of an improved enzyme-free immunosorbent assay (EFISA). It integrates 3D gold nano-vesicles (GNVs) and allochroic agents for the simultaneous detection of acute myocardial infarction (AMI) biomarkers (N-terminal prohormone of brain natriuretic peptide (NT-proBNP), kinase-muscle/brain test (CK-MB), and cardiac muscle troponin (cTnT)).

Analogous modified DNA probe and immune competition method-based electrochemical biosensor for RNA modification.

N6-methyladenosine (m6A), one of the most abundant RNA methylation which is ubiquitous in eukaryotic RNA, plays vital roles in many biological progresses. Therefore, the rapid and accurate quantitative detection of m6A is particularly important for its functional research. Herein, a label-free and highly selective electrochemical immunosensor was developed for the detection of m6A. The method is established on that the anti-m6A-Ab can recognize both m6A-RNA and m6A-DNA. An analogous modified DNA probe (L1) serves as a signal molecule, by competing with m6A-RNA for binding to Abs to broaden the linear range. The detection of m6A-RNA by this method is unaffected by the lengths and base sequences of RNA. Under optimal conditions, the proposed immunosensor presented a wide linear range from 0.05 to 200 nM with a detection limit as low as 0.016 nM (S/N = 3). The specificity and reproducibility of the method are satisfactory. Furthermore, the developed immunosensor was validated for m6A determination in human cell lines. Thus, the immunosensor provides a promising platform for m6A-RNA detection with simplicity, high specificity and sensitivity.

Double-loop hairpin probe and doxorubicin-loaded gold nanoparticles for the ultrasensitive electrochemical sensing of microRNA.

An electrochemical microRNA (miRNA) analysis platform by combining double-loop hairpin probe (DHP) and doxorubicin-loaded gold nanoparticles (AuNPs@Dox) for ultrasensitive miRNA detection is proposed. Firstly, we here report a DHP that is simultaneously engineered to incorporate a miRNA recognition sequence, an output segment and output's complementary fragment. The important aspect of this hairpin probe is that it would not be degraded by duplex specific nuclease (DSN) and circumvents elaborately chemical modification disadvantages encountered by classic molecular beacon. For the DHP-based DSN signal amplification system, DHP hybridizes with target miRNA to form DNA-miRNA heteroduplexes, and the DSN can hydrolyze the DNA in the heteroduplexes structure selectively, while released target miRNA strand can initiate another cycle resulting in a significant signal amplification and the accumulated output segments could be responsible for strand displacement on the electrode directly. Furthermore, a great deal of doxorubicin (Dox) are loaded on the gold nanoparticles (AuNPs) to fabricate the AuNPs@Dox biocomposites that could magnify the electrochemical signal and enable the ultrasensitive analysis of miRNA. As a result, the miRNA was capable of being detected in a limit of 0.17pM and other kinds of miRNA were discriminated facilely by this method. The described DHP as a toolbox and the nano-biocomposites as a novel signal material would not only promote the design of electrochemical biosensors but also open a good way to promote the establishment of test method in malignant tumors.

Universal ratiometric electrochemical biosensing platform based on mesoporous platinum nanocomposite and nicking endonuclease assisted DNA walking strategy.

The occurrence and development of many complex diseases are associated with various molecules, whose contents are rarely in the early stage of the disease. Thus a universal platform for the ultrasensitive detection of multilevel biomarkers should be developed. In this study, we introduced an electrochemical biosensing system based on nicking endonuclease (Nt.BbvCI) assisted DNA walking strategy. We successfully constructed a universal signal-off-on ratiometric electrochemical biosensor for various biomolecules, including small molecules, nucleic acids, and proteins, by progressively optimizing the schematics (schemes 1, 2, and 3). The MB-hairpin probes (MB-HPs) acted as a signal-off probe, and nanocomposites (MPNs@DOX@DNA2) acted as a conventional signal-on probe (scheme 3). With the aid of the MPNs@DOX@DNA2 and Nt.BbvCI assisted DNA walking mechanism, the designed ratiometric electrochemical biosensor showed a high sensitivity and broad detection range. In addition, the proposed method can be utilized to detect diverse targets quantitatively by changing the sequence of aptamers under optimum experimental conditions. Furthermore, it has been widely proved to realize well-accepted signal response in identifying complex samples, thereby resulting in an wide prospect for bioanalysis and clinical diagnosis.

Overexpression of Sirt3 inhibits lipid accumulation in macrophages through mitochondrial IDH2 deacetylation.

This study aims to explore the relationship between Sirt3 expression and lipid accumulation in macrophages by inducing mitochondrial IDH2 deacetylation. In this study, Sirt3 interference and overexpression lentiviral vectors were constructed. Macrophages collected from C57BL/6J mice by peritoneal lavage were used to construct Sirt3 gene interference and overexpression models, and cultured in medium containing 1 mg/ml ox-LDL for 72 h to observe the enrichment of ox-LDL. Reverse transcription PCR was used to detect the expression of Sirt3 mRNA, western blot to detect Sirt3 and acetylated IDH2 proteins, and Nile Red staining and flow cytometry to detect intracellular lipids in macrophages. The results indicated that as compared to Sirt3 overexpressed and normal groups, the acetylation of IDH2 and accumulation of ox-LDL were significantly higher in the Sirt3 inhibited group. In conclusion, the expression of Sirt3 can inhibit lipid accumulation in macrophages by inducing mitochondrial IDH2 deacetylation.