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Biosensors & Bioelectronics Oct 2024Exosomes have been considered as promising biomarkers for cancer diagnosis due to their abundant information from originating cells. However, sensitive and reliable...
Exosomes have been considered as promising biomarkers for cancer diagnosis due to their abundant information from originating cells. However, sensitive and reliable detection of exosomes is still facing technically challenges due to the lack of a sensing platform with high sensitivity and reproducibility. To address the challenges, here we propose a portable surface plasmon resonance (SPR) sensing of exosomes with a three-layer Au mirror/SiO spacer/Au nanohole sensor, fabricated by an economical polystyrene nanosphere self-assembly method. The SiO spacer can act as an optical cavity and induce mode hybridization, leading to excellent optimization of both sensitivity and full width at half maximum compared with normal single layer Au nanohole sensors. When modified with CD63 or EpCAM aptamers, a detection of limit (LOD) of as low as 600 particles/μL was achieved. The sensors showed good capability to distinguish between non-tumor derived L02 exosomes and tumor derived HepG2 exosomes. Additionally, high reproducibility was also achieved in detection of artificial serum samples with RSD as low as 2%, making it feasible for clinical applications. This mode hybridization plasmonic sensor provides an effective approach to optimize the detection sensitivity of exosomes, pushing SPR sensing one step further towards cancer diagnosis.
Topics: Exosomes; Humans; Surface Plasmon Resonance; Gold; Limit of Detection; Silicon Dioxide; Aptamers, Nucleotide; Epithelial Cell Adhesion Molecule; Tetraspanin 30; Hep G2 Cells; Biosensing Techniques; Reproducibility of Results; Equipment Design; Nanospheres; Nucleic Acid Hybridization; Biomarkers, Tumor
PubMed: 38870828
DOI: 10.1016/j.bios.2024.116492 -
PloS One 2024Quantitative transcription regulation studies in vivo and in vitro often make use of reporter proteins. Here we show that using Broccoli aptamers, quantitative study of...
Quantitative transcription regulation studies in vivo and in vitro often make use of reporter proteins. Here we show that using Broccoli aptamers, quantitative study of transcription in various regulatory scenarios is possible without a translational step. To explore the method we studied several regulatory scenarios that we analyzed using thermodynamic occupancy-based models, and found excellent agreement with previous studies. In the next step we show that non-coding DNA can have a dramatic effect on the level of transcription, similar to the influence of the lac repressor with a strong affinity to operator sites. Finally, we point out the limitations of the method in terms of delay times coupled to the folding of the aptamer. We conclude that the Broccoli aptamer is suitable for quantitative transcription measurements.
Topics: Aptamers, Nucleotide; Transcription, Genetic; Brassica; Thermodynamics; Gene Expression Regulation
PubMed: 38870160
DOI: 10.1371/journal.pone.0304677 -
Analytical Chemistry Jun 2024Herein, we, for the first time, synthesize silver nanoparticles (Ag NPs) within the nanochannels of amino group-functionalized vertically ordered mesoporous silica films...
Silica Nanochannels as Nanoreactors for the Confined Synthesis of Ag NPs to Boost Electrochemical Stripping Chemiluminescence of the Luminol-O System for the Sensitive Aptasensor.
Herein, we, for the first time, synthesize silver nanoparticles (Ag NPs) within the nanochannels of amino group-functionalized vertically ordered mesoporous silica films (NH-VMSF) and investigate their coreaction accelerator role in the luminol-dissolved oxygen (O) electrochemical stripping chemiluminescence (ESCL) system. The synthesized Ag NPs are capable of electrocatalytic reduction of O to superoxide radicals, and meanwhile, sliver ions (Ag) electrochemically stripped from Ag NPs can promote the amount of luminol anion radicals, generating the boosted ECL intensity of the luminol-dissolved O system. This proposed Ag NPs@NH-VMSF on the indium tin oxide electrode was applied to construct the ESCL aptasensor for quantitative determination of prostate-specific antigen (PSA), yielding a low detection limit [0.19 pg/mL (S/N = 3)] and a broad linear dynamic range (1 pg/mL to 100 ng/mL). Furthermore, good analytical performance of PSA in serum with satisfactory recoveries and low relative standard deviation values is achieved by our developed ESCL aptasensor, rendering it a convenient and sensitive method for PSA determination in clinical applications and further broadening the strategy of ESCL techniques.
Topics: Silicon Dioxide; Luminol; Silver; Metal Nanoparticles; Electrochemical Techniques; Luminescent Measurements; Aptamers, Nucleotide; Oxygen; Humans; Biosensing Techniques; Prostate-Specific Antigen; Limit of Detection; Electrodes; Luminescence
PubMed: 38869321
DOI: 10.1021/acs.analchem.4c01033 -
BMC Genomics Jun 2024Reverse transcription quantitative PCR (RT-qPCR) with intercalating dyes is one of the main techniques to assess gene expression levels used in basic and applied...
BACKGROUND
Reverse transcription quantitative PCR (RT-qPCR) with intercalating dyes is one of the main techniques to assess gene expression levels used in basic and applied research as well as in diagnostics. However, primer design for RT-qPCR can be complex due to the high demands on primer quality. Primers are best placed on exon junctions, should avoid polymorphic regions, be specific to the target transcripts and also prevent genomic amplification accurately, among others. Current software tools manage to meet all the necessary criteria only insufficiently. Here, we present ExonSurfer, a novel, user-friendly web-tool for qPCR primer design.
RESULTS
ExonSurfer combines the different steps of the primer design process, encompassing target selection, specificity and self-complementarity assessment, and the avoidance of issues arising from polymorphisms. Amplification of potentially contaminating genomic DNA is avoided by designing primers on exon-exon junctions, moreover, a genomic alignment is performed to filter the primers accordingly and inform the user of any predicted interaction. In order to test the whole performance of the application, we designed primer pairs for 26 targets and checked both primer efficiency, amplicon melting temperature and length and confirmed the targeted amplicon by Sanger sequencing. Most of the tested primers accurately and selectively amplified the corresponding targets.
CONCLUSION
ExonSurfer offers a comprehensive end-to-end primer design, guaranteeing transcript-specific amplification. The user interface is intuitive, providing essential specificity and amplicon details. The tool can also be used by command line and the source code is available. Overall, we expect ExonSurfer to facilitate RT-qPCR set-up for researchers in many fields.
Topics: Exons; Software; DNA Primers; Internet; Humans; Reverse Transcriptase Polymerase Chain Reaction
PubMed: 38867172
DOI: 10.1186/s12864-024-10456-2 -
Mikrochimica Acta Jun 2024The combination of CRISPR/Cas12a and functional DNA provides the possibility of constructing biosensors for detecting non-nucleic-acid targets. In the current study, the...
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.
Topics: Adenosine Triphosphate; Biosensing Techniques; Aptamers, Nucleotide; CRISPR-Cas Systems; CRISPR-Associated Proteins; Limit of Detection; Bacterial Proteins; Nucleic Acid Hybridization; Endodeoxyribonucleases
PubMed: 38867016
DOI: 10.1007/s00604-024-06477-z -
Scientific Reports Jun 2024Antibodies have been extensively used in numerous applications within proteomics-based technologies, requiring high sensitivity, specificity, a broad dynamic range for...
Antibodies have been extensively used in numerous applications within proteomics-based technologies, requiring high sensitivity, specificity, a broad dynamic range for detection, and precise, reproducible quantification. Seeking alternatives to antibodies due to several inherent limitations of antibodies is an area of active research of tremendous importance. Recently, aptamers have been receiving increasing attention, because they not only have all of the advantages of antibodies, but also have unique advantages, such as thermal stability, low cost, and unlimited applications. Aptamers are gaining importance in immunological studies and can potentially replace antibodies in immunoassays. B7H3, an immunoregulatory protein belonging to the B7 family, is an attractive and promising target due to its overexpression in several tumor tissues while exhibiting limited expression in normal tissues. This study employed hybrid-SELEX with next-generation sequencing to select ssDNA aptamers specifically binding to the B7H3 protein. These aptamers demonstrated versatility across various assays, including flow cytometry, dot-blot, and immunohistochemistry. Effective performance in sandwich dot-blot assays and western blot analysis suggests their potential for diagnostic applications and demonstrates their adaptability and cost-effectiveness in diverse protein detection techniques.
Topics: Aptamers, Nucleotide; B7 Antigens; Humans; SELEX Aptamer Technique; High-Throughput Nucleotide Sequencing; Cell Line, Tumor; Antibodies
PubMed: 38866941
DOI: 10.1038/s41598-024-64559-7 -
Nature Communications Jun 2024Radio-immunotherapy exploits the immunostimulatory features of ionizing radiation (IR) to enhance antitumor effects and offers emerging opportunities for treating...
Radio-immunotherapy exploits the immunostimulatory features of ionizing radiation (IR) to enhance antitumor effects and offers emerging opportunities for treating invasive tumor indications such as melanoma. However, insufficient dose deposition and immunosuppressive microenvironment (TME) of solid tumors limit its efficacy. Here we report a programmable sequential therapeutic strategy based on multifunctional fusogenic liposomes (Lip@AUR-ACP-aptPD-L1) to overcome the intrinsic radio-immunotherapeutic resistance of solid tumors. Specifically, fusogenic liposomes are loaded with gold-containing Auranofin (AUR) and inserted with multivariate-gated aptamer assemblies (ACP) and PD-L1 aptamers in the lipid membrane, potentiating melanoma-targeted AUR delivery while transferring ACP onto cell surface through selective membrane fusion. AUR amplifies IR-induced immunogenic death of melanoma cells to release antigens and damage-associated molecular patterns such as adenosine triphosphate (ATP) for triggering adaptive antitumor immunity. AUR-sensitized radiotherapy also upregulates matrix metalloproteinase-2 (MMP-2) expression that combined with released ATP to activate ACP through an "and" logic operation-like process (AND-gate), thus triggering the in-situ release of engineered cytosine-phosphate-guanine aptamer-based immunoadjuvants (eCpG) for stimulating dendritic cell-mediated T cell priming. Furthermore, AUR inhibits tumor-intrinsic vascular endothelial growth factor signaling to suppress infiltration of immunosuppressive cells for fostering an anti-tumorigenic TME. This study offers an approach for solid tumor treatment in the clinics.
Topics: Liposomes; Aptamers, Nucleotide; Animals; Mice; Cell Line, Tumor; Immunotherapy; Melanoma; Humans; Tumor Microenvironment; Matrix Metalloproteinase 2; Gold; Mice, Inbred C57BL; Female; B7-H1 Antigen; Adenosine Triphosphate
PubMed: 38866788
DOI: 10.1038/s41467-024-49482-9 -
Analytical Chemistry Jun 2024Exosomes, as an emerging biomarker, have exhibited remarkable promise in early cancer diagnosis. Here, a highly sensitive, selective, and automatic...
Exosomes, as an emerging biomarker, have exhibited remarkable promise in early cancer diagnosis. Here, a highly sensitive, selective, and automatic electrochemiluminescence (ECL) method for the detection of cancerous exosomes was developed. Specific aptamer-(EK) peptide-tagged magnetic beads (MBs-(EK)-aptamer) were designed as a magnetic capture probe in which the (EK) peptide was used to reduce the steric binding hindrance of cancerous exosomes with a specific aptamer. One new universal ECL signal nanoprobe (CD9 Ab-PEG@SiOϵRu(bpy)) was designed and synthesized by using microporous SiO nanoparticles as the carrier for loading ECL reagent Ru(bpy), polyethylene glycol (PEG) layer, and anticluster of differentiation 9 antibody (CD9 Ab). A "sandwich" biocomplex was formed on the surface of the magnetic capture probe after mixing the capture probe, target exosomes, and ECL signal nanoprobe, and then it was introduced into an automated ECL analyzer for rapid and automatic ECL measurement. It was found that the designed signal nanoprobe shows a 270-fold improvement in the signal-to-noise ratio than that of the ruthenium complex-labeled CD9 antibody signal probe. The relative ECL intensity was proportional to MCF-7 exosomes as a model in the range of 10 to 10 particle/μL, with a detection limit of 11 particle/μL. Furthermore, the ECL method was employed to discriminate cancerous exosomes based on fingerprint responses using the designed multiple magnetic capture probes and the universal ECL signal nanoprobe. This work demonstrates that the utilization of a designed automated ECL tactic using the MBs-(EK)-aptamer capture probe and the CD9 Ab-PEG@SiOϵRu(bpy) signal nanoprobe will provide a unique and robust method for the detection and discrimination of cancerous exosomes.
Topics: Humans; Exosomes; Aptamers, Nucleotide; Electrochemical Techniques; Luminescent Measurements; MCF-7 Cells; Silicon Dioxide; Biosensing Techniques; Tetraspanin 29; Polyethylene Glycols
PubMed: 38866706
DOI: 10.1021/acs.analchem.4c01938 -
Biological & Pharmaceutical Bulletin 2024Long non-coding RNAs (lncRNAs) are sequences longer than 200 nucleotides, but they do not encode proteins. Nevertheless, they have significant roles in diverse...
Long non-coding RNAs (lncRNAs) are sequences longer than 200 nucleotides, but they do not encode proteins. Nevertheless, they have significant roles in diverse biological functions. It remains unclear how viral infections trigger the expression of lncRNAs. In our previous research, we revealed a distinct type of lncRNAs with a lifespan under 4 h in human HeLa cells. These short-lived lncRNAs might be associated with numerous regulatory roles. Given their potential impact on human physiology, these short-lived lncRNAs could be key indicators to measure polyinosinic:polycytidylic acid (poly I:C) stimulation. In our recent work, we discovered three lncRNAs: IDI2-AS1, OIP5-AS1, and LITATS1. After exposure to poly I:C, imitating viral assault in human A549 cells, IDI2-AS1 levels dropped significantly while OIP5-AS1 and LITATS1 levels rose markedly. Our results indicate that short-lived lncRNAs respond to poly I:C stimulation, exhibiting substantial changes in expression. This indicates that the understanding the role of lncRNAs in the host response to viral infection and the potential for these molecules to serve as novel therapeutic targets.
Topics: RNA, Long Noncoding; Humans; Poly I-C; A549 Cells; HeLa Cells
PubMed: 38866523
DOI: 10.1248/bpb.b24-00037 -
Science Advances Jun 2024Conventional directed evolution methods offer the ability to select bioreceptors with high binding affinity for a specific target in terms of thermodynamic properties....
Conventional directed evolution methods offer the ability to select bioreceptors with high binding affinity for a specific target in terms of thermodynamic properties. However, there is a lack of analogous approaches for kinetic selection, which could yield affinity reagents that exhibit slow off-rates and thus remain tightly bound to targets for extended periods. Here, we describe an in vitro directed evolution methodology that uses the nuclease flap endonuclease 1 to achieve the efficient discovery of aptamers that have slow dissociation rates. Our nuclease-assisted selection strategy can yield specific aptamers for both small molecules and proteins with off-rates that are an order of magnitude slower relative to those obtained with conventional selection methods while still retaining excellent overall target affinity in terms of thermodynamics. This new methodology provides a generalizable approach for generating slow off-rate aptamers for diverse targets, which could, in turn, prove valuable for applications including molecular devices, bioimaging, and therapy.
Topics: Aptamers, Nucleotide; SELEX Aptamer Technique; Kinetics; Thermodynamics
PubMed: 38865469
DOI: 10.1126/sciadv.adl3426