IGSF10 inhibits the metastasis of lung adenocarcinoma via the Spi-B/Integrin-ß1 signaling pathway.

IGSF10, a protein that belongs to the immunoglobulin superfamily, is involved in regulating the early migration of neurons that produce gonadotropin-releasing hormone and performs a fundamental function in development. Our previous study confirmed that the mRNA expression level of IGSF10 may be a protective prognosis factor for lung adenocarcinoma (LUAD) patients. However, the specific mechanisms of IGSF10 are still unclear. In this research, it was shown that the protein level of IGSF10 was down-modulated in LUAD tissues and had a link to the clinical and pathological characteristics as well as the patient's prognosis in LUAD. Importantly, IGSF10 regulates the metastatic ability of LUAD cells in vitro and in vivo. It was proven in a mechanistic sense that IGSF10 inhibits the capacity of LUAD cells to metastasize through the Spi-B/Integrin-ß1 signaling pathway. These findings gave credence to the premise that IGSF10 performed a crucial function in LUAD.

A CRISPR-Cas12a-based electrochemical biosensor for the detection of microphthalmia-associated transcription factor.

A novel electrochemical biosensor that combines the CRISPR-Cas12a system with a gold electrode is reported for the rapid and sensitive detection of microphthalmia-associated transcription factor (MITF). The biosensor consists of a gold electrode modified with DNA1, which contains the target sequence of MITF and is labeled with ferrocene, an electroactive molecule. The biosensor also includes hairpin DNA, which has a binding site for MITF and can hybridize with helper DNA to form a double-stranded complex that activates CRISPR-Cas12a. When MITF is present, it binds to hairpin DNA and prevents its hybridization with helper DNA, thus inhibiting CRISPR-Cas12a activity and preserving the DPV signal of ferrocene. When MITF is absent, hairpin DNA hybridizes with helper DNA and activates CRISPR-Cas12a, which cleaves DNA1 and releases ferrocene, thus reducing the DPV signal. The biosensor can detect MITF with high sensitivity (with an LOD of 8.14 fM), specificity, and accuracy in various samples, such as cell nuclear extracts and human serum. The biosensor can also diagnose and monitor melanocyte-related diseases and melanin production. This work provides a simple, fast, sensitive, and cost-effective biosensor for MITF detection and a valuable tool for applications in genetic testing, disease diagnosis, and drug screening.

Electrochemical biosensor strategy combining DNA entropy-driven technology to activate CRISPR-Cas13a activity and triple-stranded nucleic acids to detect SARS-CoV-2 RdRp gene.

By merging DNA entropy-driven technology with triple-stranded nucleic acids in an electrochemical biosensor to detect the SARS-CoV-2 RdRp gene, we tackled the challenges of false negatives and the high cost of SARS-CoV-2 detection. The approach generates a CRISPR-Cas 13a-activated RNA activator, which then stimulates CRISPR-Cas 13a activity using an entropy-driven mechanism. The activated CRISPR-Cas 13a can cleave Hoogsteen DNA due to the insertion of two uracil (-U-U-) in Hoogsteen DNA. The DNA tetrahedra changed on the electrode surface and can therefore not construct a three-stranded structure after cleaving Hoogsteen DNA. Significantly, this DNA tetrahedron/Hoogsteen DNA-based biosensor can regenerate at pH = 10.0, which keeps Hoogsteen DNA away from the electrode surface, allowing the biosensor to function at pH = 7.0. We could use this technique to detect the SARS-CoV-2 RdRp gene with a detection limit of 89.86 aM. Furthermore, the detection method is very stable and repeatable. This technique offers the prospect of detecting SARS-CoV-2 at a reasonable cost. This work has potential applications in the dynamic assessment of the diagnostic and therapeutic efficacy of SARS-CoV-2 infection and in the screening of environmental samples.

In situ biosensor for detection miRNA in living cells based on carbon nitride nanosheets with catalytic hairpin assembly amplification.

In this study, an ultrasensitive fluorescence turn-on assay for in situ sensing of intracellular microRNA (miRNA) was developed utilizing a carbon nitride nanosheet (CNNS) and a catalytic hairpin assembly (CHA). The CHA showed favourable signal amplification for low-level biomarkers, and CNNS was an excellent candidate as a fluorescence quencher and gene vector. Moreover, the hairpin DNA of CHA could be adsorbed onto the surface of CNNS. An enzyme-free fluorescence biosensor for ultrasensitive sensing of intracellular miRNA in cells based on CHA and CNNS was designed. When faced with target miRNA, the fluorescence was recovered due to the miRNA, which could trigger cycling of CHA circuits, leading to the production of a marked enhanced fluorescence signal. Compared with traditional methods, the proposed method is convenient, with low cytotoxicity, and high specificity and ultrasensitivity. It has promising potential for detection low-level biomarkers.

Ultra-sensitive fluorescent sensor for intracellular miRNA based on enzyme-free signal amplification with carbon nitride nanosheet as a carrier.

A novel ultra-sensitive fluorescent sensor for monitoring microRNA (miRNA) in living cells was constructed by utilizing a hybridization chain reaction (HCR) as the signal amplification with a carbon nitride nanosheet (CNNS) as a carrier. The Cy5-labeled hairpin DNA could be adsorbed onto the surface of CNNS, resulting in fluorescence quenching of Cy5. When treated with complementary miRNA, the fluorescence was recovered because miRNA could efficiently trigger an HCR, which led to the release of the HCR products from the CNNS. This intracellular HCR strategy can be used for ultra-sensitive monitoring of intracellular miRNA. The main advantages of the proposed method are its simplicity, high sensitivity, high specificity and low toxicity for monitoring low-level biomarkers.

A peptide nucleic acid-functionalized carbon nitride nanosheet as a probe for in situ monitoring of intracellular microRNA.

A novel probe for recognition of both cancer cells and intracellular microRNA (miRNA) is designed by functionalizing a carbon nitride nanosheet (f-CNNS) with a Cy5-labeled peptide nucleic acid (Cy5-PNA) and folate. The interaction between Cy5-PNA and CNNS quenches the fluorescence of Cy5, and the presence of folate endows the probe with good specificity to folate acceptor overexpressed cells. The probe can be specifically taken up by cancer cells with an incubation step. Upon the recognition of the PNA to complementary miRNA, the hybridization product is released from the CNNS surface, which leads to the fluorescence recovery and provides a specific method for sensing of miRNA. Thus, this probe can be used for cell-specific intracellular miRNA sensing with a confocal microscope. Using miRNA-18a as a target model, the dynamic changes of its expression level inside living cells can be monitored with the proposed method. This method possesses promising applications in the study of miRNA related bioprocesses and biomedicine.

Electrochemical biosensing for E.coli detection based on triple helix DNA inhibition of CRISPR/Cas12a cleavage activity.

BACKGROUND: Escherichia coli (E.coli) is both a commensal and a foodborne pathogenic bacterium in the human gastrointestinal tract, posing significant potential risks to human health and food safety. However, one of the major challenges in E.coli detection lies in the preparation and storage of antibodies. In traditional detection methods, antibodies are indispensable, but their instability often leads to experimental complexity and increased false positives. This underscores the need for new technologies and novel sensors. Therefore, the development of a simple and sensitive method for analyzing E.coli would make significant contributions to human health and food safety. RESULTS: We constructed an electrochemical biosensor based on triple-helical DNA and entropy-driven amplification reaction (EDC) to inhibit the cleavage activity of Cas12a, enabling high-specificity detection of E.coli. Replacing antibodies with nucleic acid aptamers (Apt) as recognition elements, we utilized the triple-helical DNA generated by the binding of DNA2 and DNA5/DNA6 double-helical DNA through the entropy-driven amplification reaction to inhibit the collateral cleavage activity of clustered regularly interspaced short palindromic repeats gene editing system (CRISPR) and its associated proteins (Cas). By converting E.coli into electrical signals and recording signal changes in the form of square wave voltammetry (SWV), rapid detection of E.coli was achieved. Optimization of experimental conditions and data detection under the optimal conditions provided high sensitivity, low detection limits, and high specificity. SIGNIFICANCE: With a minimal detection limit of 5.02 CFU/mL and a linear range of 1 × 102 - 1 × 107 CFU/mL, the suggested approach was successfully verified to analyze E.coli at various concentrations. Additionally, after examining E.coli samples from pure water and pure milk, the recoveries ranged between 95.76 and 101.20%, demonstrating the method's applicability. Additionally, it provides a feasible research direction for the detection of pathogenic bacteria causing other diseases using the CRISPR/Cas gene editing system.

Precision miRNA profiling: Electrochemiluminescence powered by CRISPR-Cas13a and hybridization chain reaction.

The article details a groundbreaking platform for detecting microRNAs (miRNAs), crucial biomolecules involved in gene regulation and linked to various diseases. This innovative platform combines the CRISPR-Cas13a system's precise ability to specifically target and cleave RNA molecules with the amplification capabilities of the hybridization chain reaction (HCR). HCR aids in signal enhancement by creating branched DNA structures. Additionally, the platform employs electrochemiluminescence (ECL) for detection, noted for its high sensitivity and low background noise, making it particularly effective. A key application of this technology is in the detection of miR-17, a biomarker associated with multiple cancer types. It exhibits remarkable detection capabilities, characterized by low detection limits (14.38 aM) and high specificity. Furthermore, the platform's ability to distinguish between similar miRNA sequences and accurately quantify miR-17 in cell lysates underscores its significant potential in clinical and biomedical fields. This combination of precise targeting, signal amplification, and sensitive detection positions the platform as a powerful tool for miRNA analysis in medical diagnostics and research.

A novel electrochemical biosensor for B-type natriuretic peptide detection based on CRISPR/Cas13a and chain substitution reaction.

B-type natriuretic peptide (BNP) is a biomarker for heart failure, a serious and prevalent disease that requires rapid and accurate diagnosis. In this study, we developed a novel electrochemical biosensor for BNP detection based on CRISPR/Cas13a and chain substitution reaction. The biosensor consists of a DNA aptamer that specifically binds to BNP, a T7 RNA polymerase that amplifies the signal, a CRISPR/Cas13a system that cleaves the target RNA, and a two-dimensional DNA nanoprobe that generates an electrochemical signal. The biosensor exhibits high sensitivity, specificity, and stability, with a detection limit of 0.74 aM. The biosensor can also detect BNP in human serum samples with negligible interference, demonstrating its potential for clinical and point-of-care applications. This study presents a novel strategy for integrating CRISPR/Cas13a and chain substitution reaction into biosensor design, offering a versatile and effective platform for biomolecule detection.

Proximity hybridization induced molecular machine for signal-on electrochemical detection of α-synuclein oligomers.

α-synuclein oligomer is a marker of Parkinson's disease. The traditional enzyme-linked immunosorbent assay for α-synuclein oligomer detection is not conducive to large-scale application due to its time-consuming, high cost and poor stability. Recently, DNA-based biosensors have been increasingly used in the detection of disease markers due to their high sensitivity, simplicity and low cost. In this study, based on the DNAzyme-driven DNA bipedal walking method, we developed a signal-on electrochemical sensor for the detection of α-syn oligomers. Bipedal DNA walkers have a larger walking area and faster walking kinetics, providing higher amplification efficiency compared to conventional DNA walkers. The DNA walker is driven via an Mg2+-dependent DNAzyme, and the binding-induced DNA walker will continuously clamp the MB, resulting in the proliferation of Fc confined near the GE surface. The linear range and limit of detection were 1 fg/mL to 10 pg/mL and 0.57 fg/mL, respectively. The proposed signal-on electrochemical sensing strategy is more selective. It will play a significant role in the sensitive and precise electrochemical analysis of other proteins.

DNAzyme-driven bipedal DNA walker for label-free and signal-on electrochemical detection of amyloid-ß oligomer.

As a common technique for detecting AßO, the enzyme-linked immunosorbent assay (ELISA) method is time-consuming, high in cost, and poor in stability. Therefore, it is necessary to develop a highly sensitive, method-simple and low-cost method for the selective detection of AßO. Here, we created a novel signal-on and label-free electrochemical aptamer sensor for the detection of AßO based on a DNAzyme-driven DNA bipedal walking strategy. Compared with common DNA walkers, bipedal DNA walkers exhibit larger walking areas and faster walking kinetics, and provide higher amplification efficiency. The DNAwalker is powered by an Mg2+-dependent DNAzyme, and the binding-induced DNAwalker continuously clamps the MB, unlocking several active G-quadruplex-forming sequences. These G-quadruplexes can be further combined by hemin to generate a G-quadruplex/heme complex, resulting in an amperometric signal, resulting in a broad proportional band from 0.1 pM to 1 nM and an excellent detection range of 46 fM. A bipedal DNA walker aptamer sensor can detect human serum AßO with remarkable specificity, high reproducibility and practical application value.

Inverting the DNA tetrahedron: A novel strategy for sensitive and stable detection of aging-related enzyme MMP2.

In the current study, a novel electrochemiluminescence biosensor based on the entropy-driven DNA tetrahedron for the detection of matrix metalloproteinase 2 (MMP2), an enzyme that regulates extracellular matrix remodeling and affects aging was reported. The biosensor utilizes an inverted DNA tetrahedron structure, which exposes three vertices to the solution, as molecular recognition units for capturing specific biomolecules. The biosensor also employs a ratiometric method and an entropy-driven reaction, which enhance the response rate and sensitivity of the detection. The biosensor can detect MMP2 with a detection limit of 55.2 fM, which is lower than that of conventional sensors. The biosensor also exhibits excellent stability and reproducibility, and can accurately measure MMP2 levels in complex samples, such as human serum. The paper demonstrates the feasibility and effectiveness of using the "inverted" DNA tetrahedron structure and the entropy-driven process to construct interfacial biosensors. The paper also discusses the potential applications of the biosensor in clinical diagnosis and anti-aging research, where MMP2 plays a crucial role in tissue damage and repair. The paper provides a valuable contribution to the field of biosensor development, and opens up new possibilities for using DNA nanotechnology for sensitive and reliable detection of various biomolecules.

Entropy-driven assisted T7 RNA polymerase amplification-activated CRISPR/Cas13a activity for SARS-CoV-2 detection in human pharyngeal swabs and environment by an electrochemiluminescence biosensor.

In this study, we introduce an electrochemiluminescence (ECL) sensing platform based on the "Entropy-driven triggered T7 amplification-CRISPR/Cas13a system" (EDT-Cas). This platform combines a programmable entropy-driven cycling strategy, T7 RNA polymerase, and the CRISPR/Cas13a system to amplify the determination of the SARS-CoV-2 RdRp gene. The Ti3C2Tx-compliant ECL signaling molecule offers unique benefits when used with the ECL sensing platform to increase the assay sensitivity and the electrode surface modifiability. To obtain the T7 promoter, the SARS-CoV-2 RdRp gene may first initiate an entropy-driven cyclic amplification response. Then, after recognizing the T7 promoter sequence on the newly created dsDNA, T7 RNA polymerase starts transcription, resulting in the production of many single-stranded RNAs (ssRNAs), which in turn trigger the action of CRISPR/Cas13a. Finally, Cas13a/crRNA identifies the transcribed ssRNA. When it cleaves the ssRNA, many DNA reporter probes carrying -U-U- are cleaved on the electrode surface, increasing the ECL signal and allowing for the rapid and highly sensitive detection of SARS-CoV-2. With a detection limit of 7.39 aM, our method enables us to locate the SARS-CoV-2 RdRp gene in clinical samples. The detection method also demonstrates excellent repeatability and stability. The SARS-CoV-2 RdRp gene was discovered using the "Entropy-driven triggered T7 amplification-CRISPR/Cas13a system" (EDT-Cas). The developed ECL test had excellent recoveries in pharyngeal swabs and environmental samples. It is anticipated to offer an early clinical diagnosis of SARS-CoV-2 and further control the spread of the pandemic.

DNAzyme-driven bipedal DNA walker triggered to hybridize silver nanoparticle probes for electrochemical detection of amyloid-ß oligomer.

Amyloid-ß oligomer has been considered as a promising molecular biomarker for the diagnosis of Alzheimer's disease due to their significant neural synapse toxicity. Therefore, it is essential to create an easy approach for the selective detection of Amyloid-ß oligomer that has high sensitivity and cheap cost. In this work, we developed an innovative enzyme-free electrochemical aptasensor based on the DNAzyme-driven DNA bipedal walker tactics for sensing Amyloid-ß oligomer. Bipedal DNA walkers demonstrate a wider walking region, better walking kinetics, and higher amplification effectiveness than typical DNA walkers. The Mg2+-dependent DNAzyme drove the DNA walker, and the binding-induced DNA walker can sequentially shear MBs and form MB fragment structure. Finally, the detection probes modified AgNPs hybridized with the MB fragment structure, resulting in the multiplication of AgNPs on the electrode surface. Electrochemical stripping of AgNPs was used to test the performance of the obtained electrochemical sensor. In particular, a low detection limit of 5.94 fM and a wide linear range of 0.01 pM-0.1 nM were attained. The detection of Amyloid-ß oligomer in human serum was then carried out using this bipedal DNA walker biosensor, which shown good selectivity and outstanding reproducibility, indicating its usefulness in bioanalysis.

Proximity hybridization induced bipedal DNA walker for label-free electrochemical detection of apolipoprotein A4 based on DNA meditated Ag nanoparticles growth.

Apolipoprotein A4 (Apo-A4) is considered as a prospective molecular biomarker for diagnosis of depression due to its neurosynaptic toxicity. Here, we propose a neighboring hybridization induced catalyzed hairpin assembly (CHA) driven bipedal DNA walker that mediates hybridization of Ag nanoparticles (Ag NPs) with DNA probes for highly sensitive electrochemical quantitative detection of Apo-A4. Driven by CHA, this bipedal DNA walker can spread all over the surface of the sensor, induce the HP1-HP2 double chain structure, make the surface of the sensor negatively charged, and adsorb a large number of Ag ions. After chemical reduction with hydroquinone, the Ag NPs formed provide signal tracers for electrochemical dissolution analysis of the target. The Ag NPs formed by chemical reduction of hydroquinone can provide signal traces for electrochemical stripping analysis of target thrombin. The linear range of this method is from 10 pg mL-1 to 1000 ng mL-1, and the detection limit is 5.1 pg mL-1. This enzyme-free and labeling detection method provides a new strategy for rapid clinical detection of Apo-A4 and accurate identification of depression.

Proximity hybridization induced DNA assembly for label-free surface-enhanced Raman spectroscopic detection of carcinoembryonic antigen.

In our research, label-free and surface-enhanced Raman dyes-free Raman spectroscopy which was used to detect carcinoembryonic antigen (CEA) according to poly adenine (Poly A)-regulated self-assembly methods was developed and studied. CEA induced partial hybridization of Ab-H2 and Ab-H1, and Ab-H1-CEA-Ab-H2 (a sandwich proximity CEA-DNA complex) was formed, which unfolded molecular beacon 1 (MB1) and modified the substrate. Subsequently, MB2-AuNPs were hybridized with MB1, and Ab-H1-CEA-Ab-H2 was released via toehold regulated displacements of DNA strands. Therefore, hybridization processes of MB2 and MB1 were induced and promoted by CEA-DNA complexes which worked as catalysts. The misplaced target then induced a next round of strand exchange, and the signals for determination of CEA were amplified by AuNPs absorbed on the substrate. It was indicated that the spectral characteristics of adenine at 736 cm-1 were consistent with the SERS spectrum of DNA. Adenine acted as an internal marker for label-free SERS detection of CEA. Moreover, satisfactory stability and reproducibility were found. Meanwhile, the antibody could specifically recognize the corresponding antigen. Since adenine was dominant in SERS spectra, which was also proximal to Au surface, the sensitivity of the novel method was high without modifications. The analytical performance of this method in determining serum CEA was satisfactory.

Proximity hybridization induced rolling circle amplification for label-free SERS detection of the depression marker human apolipoprotein A4.

A new label-free method was developed for SERS detection of human apolipoprotein A4. Rolling circle amplification (RCA) was used, which could induce the production of AuNPs (poly adenine and adsorption gold nanoparticles). When there were two DNA labeled antibodies and target protein, MB1 (molecular beacon 1) was unfolded and the substrate was modified in the homogeneous solution, and the proximate complex was formed. The unfolded molecular beacon worked as a primer in the hybridization with the RCA template to start RCA, which could produce many long sequences of DNA containing amounts of adenines. The AuNPs were bound with the long-repeated adenine in the RCA product, causing accumulation of AuNPs on the surface of the electrode. It was indicated that the spectral characteristics of adenine at 736 cm-1 strongly dominated the SERS spectrum of DNA. Adenine worked as an internal marker for detecting human apolipoprotein A4 by using label-free SERS method. When the conditions were optimal, the detection of human apolipoprotein A4 was carried out from 10 pg mL-1 to 1000 ng mL-1, and the detection limit was low (4.1 pg mL-1). Meanwhile, the specificity was also excellent because the antibody could specifically bind with the corresponding antigen. In addition, since adenine was dominant in SERS spectra and the affinity between AuNPs and poly adenine was high, the detection procedure could be performed without any sophisticated modification. This method might provide a promising strategy for diagnosis in clinical practice.

Hybridization chain reaction triggered poly adenine to absorb silver nanoparticles for label-free electrochemical detection of Alzheimer's disease biomarkers amyloid ß-peptide oligomers.

Amyloid ß-peptide oligomer (AßO) has received extensive attention from researchers because of its clinical therapeutic intervention targets and the value of reliable biological macromolecules markers for early diagnosis of Alzheimer's disease. We have developed a novel label-free electrochemical detection sensor for AßO based on hybridization chain reaction (HCR)-triggered poly adenine to absorb silver nanoparticles (AgNPs). In this method, we first use the "capture probe" to immobilize the aptamer 1 on the surface of the gold electrode (GE) via poly adenine-Au. Next, aptamer 2 and AßO were deposited on the electrode surface. The HCR process was initiated by the aptamer 2 fragment as a primer, producing a large number of long DNA sequences, which contained many adenines. Thereafter, the HCR product with long-repeated adenines could absorb many AgNPs on the surface of the electrode, which were used for subsequent electrochemical stripping of the AgNPs. The concentration range of the electrochemical signal of AßO was 1 pM-10 nM, and the detection limit was 430 fM, which indicated that that the detection system has high selectivity for the target protein.

Catalytic effect of ReAu nanoalloy on the Te particle reaction and its application to resonance scattering spectral assay of CA125.

ReAu nanoparticles with a molar ratio of 2:8 Re and Te nanoparticles were prepared by NaBH4 reduction. In HCl medium at 65°C, ultratrace Re, Te and ReAu bimetallic nanoparticles strongly catalyzed the slow reaction between Sn(II) and Te(VI) to form Te particles, which exhibited the strongest resonance scattering (RS) peak at 782 nm. As the amount of nanocatalyst increased, the RS intensity at 782 nm (I(782 nm) ) increased linearly, and the increase in intensity ΔI(782 nm) was linear to the ReAu, Re and Te concentrations in the ranges 0.07-9.0, 0.01-4.5 and 30-1200 nM, respectively. As a model, a ReAu immunonanoprobe catalytic Te-particle resonance scattering spectral (RSS) method was established for detection of CA125, using ReAu nanoparticle labeling CA125 antibody (CA125Ab) to obtain an immunonanoprobe (ReAuCA125Ab) for CA125. In pH 7.6 citric acid-Na2HPO4 buffer solution, ReAuCA125Ab aggregated nonspecifically. Upon addition of CA125, the immunonanoprobe reacted with it to form ReAuCA125Ab-CA125 dispersive immunocomplex in the solution. After the centrifugation, the supernatant containing the immunocomplex was used to catalyze the reaction of Te(VI)-Sn(II) to produce the Te particles that resulted in the I(782 nm) increasing. The ΔI(782 nm) was linear to CA125 concentration (C(CA125)) in the range 0.1-240 mU/mL. The regression equation, correlation coefficient and detection limit were ΔI(782 nm) = 1.61 C(CA125) + 1.5, 0.9978 and 0.02 mU/mL, respectively. The proposed method was applied to detect CA125 in serum samples, with satisfactory results.