RESUMO
Nondestructive separation/enrichment and reliable detection of extremely rare circulating tumor cells (CTCs) in peripheral blood are of considerable importance in tumor precision diagnosis and treatment, yet this remains a big challenge. Herein, a novel strategy for nondestructive separation/enrichment and ultra-sensitive surface-enhanced Raman scattering (SERS)-based enumeration of CTCs is proposed via aptamer recognition and rolling circle amplification (RCA). In this work the magnetic beads modified with "Aptamer (Apt)-Primer" (AP) probes were utilized to specifically capture CTCs, and then after magnetic separation/enrichment, the RCA-powered SERS counting and benzonase nuclease cleavage-assisted nondestructive release of CTCs were realized, respectively. The AP was assembled by hybridizing the EpCAM-specific aptamer with a primer, and the optimal AP contains 4 mismatched bases. The RCA enhanced SERS signal nearly 4.5-fold, and the SERS strategy has good specificity, uniformity and reproducibility. The proposed SERS detection possesses a good linear relationship with the concentration of MCF-7 cells spiked in PBS with the limit of detection (LOD) of 2 cells/mL, which shows good potential practicality for detecting CTCs in blood with recoveries ranging from 100.56% to 116.78%. Besides, the released CTCs remained good cellular activity with the normal proliferation after re-culture for 48 h and normal growth for at least three generations. The proposed strategy of nondestructive separation/enrichment and SERS-based sensitive enumeration is promising for reliable analysis of EpCAM-positive CTCs in blood, which is expected to provide a powerful tool for analysis of extremely rare circulating tumor cells in complex peripheral blood for liquid biopsy.
Assuntos
Aptâmeros de Nucleotídeos , Técnicas Biossensoriais , Células Neoplásicas Circulantes , Humanos , Molécula de Adesão da Célula Epitelial , Células Neoplásicas Circulantes/patologia , Reprodutibilidade dos TestesRESUMO
The molecular diagnosis of disease by high-sensitively and specifically detecting extremely trace amounts of nucleic acid biomarkers in biological samples is still a great challenge, and the powerful sensing strategy has become an urgent need for basic researches and clinical applications. Herein, a novel one-pot cascade signal amplification strategy (Cas13a-bHCR) integrating CRISPR/Cas13a system (Cas13a) and branched hybridization chain reaction (bHCR) was proposed for ultra-highly sensitive and specific SERS assay of disease-related nucleic acids on SERS-active silver nanorods sensing chips. The Cas13a-bHCR based SERS assay of gastric cancer-related miRNA-106a (miR-106a) can be achieved within 60 min and output significantly enhanced SERS signal due to the multiple signal amplification, which possesses a good linear calibration curve from 10 aM to 1 nM with the limit of detection (LOD) low to 8.55 aM for detecting gastric cancer-related miR-106a in human serum. The Cas13a-bHCR based SERS sensing also shows good specificity, uniformity, repeatability and reliability, and has good practicability for detection of miR-106a in clinical samples, which can provide a potential powerful tool for SERS detection of disease-related nucleic acids and promise brighter prospects in the field of clinical diagnosis of early disease.
RESUMO
The visualization of protein dimerization on live cells is an urgent need and of vital importance for facile monitoring the signal transduction during intercellular communication. Herein, a highly sensitive and specific SERS strategy for simultaneously imaging dual homodimerizations of membrane proteins on single live cells was proposed by networking of AuNPs-based dual-recognition probes (dual-RPs) and SERS tags via proximity ligation-assisted catalytic hairpin assembly (CHA). The dual-RPs were prepared by comodifying hairpin-structured ssDNAs H1-Met and H1-TßRII on 50 nm AuNPs and two SERS tags for membrane proteins Met and TßRII were prepared respectively by labeling their corresponding Raman molecules and hairpin-structured single-stranded DNAs H2-Met or H2-TßRII on 15 nm AuNPs. The membrane proteins were ligated proximally by specific aptamers, and the dimerizations of proteins resulted in the proximity ligation-assisted CHA-based networking of dual-RPs and SERS tags to form 15Au-50Au network nanostructures with significantly enhanced SERS effect. The SERS strategy for visualizing the membrane protein dimerization was established and the good performance on simultaneously SERS imaging dual dimerizations of membrane proteins (i.e., Met-Met and TßRII-TßRII) was confirmed. Furthermore, the membrane protein dimerization-based signaling pathways between cancer cells and stromal cells or stem cells were observed by SERS, which indicates that the proposed SERS strategy is a good method for high-sensitivity monitoring of membrane proteins dimerizations-based multiple intercellular signal transductions in a natural and complex cellular microenvironment.
Assuntos
Técnicas Biossensoriais , Nanopartículas Metálicas , Técnicas Biossensoriais/métodos , Dimerização , Ouro/química , Limite de Detecção , Proteínas de Membrana , Nanopartículas Metálicas/química , Transdução de Sinais , Análise Espectral Raman/métodosRESUMO
Identification and detection of extreme rare circulating tumor cells (CTCs) in peripheral blood can precisely monitor cancer recurrence and metastasis, however, how to ultra-sensitively and reliably detect CTCs is a big challenge. In this work, a ratiometric surface-enhanced Raman spectroscopy (SERS)-based strategy for ultra-sensitively and nondestructively detecting CTCs was proposed via CTCs-triggered DNA walker-assisted assembly of plasmonic nanostructure networks consisting of Walker probes and SERS tags. The Walker probes were prepared by modifying Fe3O4@SiO2@Au nanoparticles (GMNPs) with ROX-labeled EpCAM aptamer-blocked Zn2+-specific DNAzyme and hairpin-structured single-stranded DNAs H1, and the SERS tags were constructed by co-labelling hairpin-structured single-stranded DNAs H2 and Raman molecules (DTNB) on Au NPs. The aptamers can recognize EpCAM-positive CTCs via the specific binding to EpCAM, so that the activity of DNAzymes is activated with the assistance of Zn2+ to launch the DNA walker to move around for the cleavage of H1 on GMNPs. The residual fragments of H1 on GMNPs can hybridize with H2 on SERS tags and result in the formation of Walker probe-SERS tag network nanostructures (Nw NSs) with rich SERS hot spots. The reliable SERS detection of CTCs is achieved by the stable ratiometric SERS signals of DTNB and ROX generated from the Nw NSs, and a good linear relation between ratiometric SERS signal and MCF-7 cells concentration was obtained with the detection limit low to 1 cell/mL. The recovery rate of MCF-7 cells in peripheral blood is in the range of 94.0%-104.5%, which indicates a good application prospect of the novel ratiometric SERS cytosensor in the clinic detection of EpCAM-positive CTCs.
Assuntos
Técnicas Biossensoriais , Nanopartículas Metálicas , Células Neoplásicas Circulantes , Técnicas Biossensoriais/métodos , DNA , Ácido Ditionitrobenzoico , Molécula de Adesão da Célula Epitelial , Ouro/química , Humanos , Limite de Detecção , Nanopartículas Metálicas/química , Oligonucleotídeos , Dióxido de Silício , Análise Espectral Raman/métodosRESUMO
Development of theranostic nanosystems integrating cascaded surface-enhanced Raman scattering (SERS) imaging and gene silencing therapy for accurate cancer diagnosis and treatment is still a big challenge and rarely reported. Herein, a novel Au nanoparticles (AuNPs)-based theranostic nanosystem containing AuNP-Ys and AuNP-Ds for highly sensitive and specific cancer diagnosis and treatment was proposed for cascaded SERS imaging of intracellular cancer-related miR-106a and miR-106a-triggered DNAzyme-based dual gene-silencing therapy of cancer cells. The AuNP-Ys were prepared by modifying the AuNPs with specially designed Y-motifs, and the AuNP-Ds were obtained by colabeling Raman molecules and dsDNA linkers on AuNPs. When identifying the intracellular cancer-related miRNAs, the Y-motifs and dsDNA linkers undergoes miRNA-triggered ATP-driven conformational transitions and releases the miRNA for recycling, which results in the formation of AuNP network nanostructures to generate significantly enhanced SERS signals for sensitive identification of the cancer cells as well as the amplification and specific activation of DNAzymes to catalyze the Mg2+-assisted cleavage of the Survivin and c-Jun mRNAs for effective dual gene-silencing therapy of cancer cells. The AuNP-based theranostic nanosystem achieves the synergism of target-triggered SERS imaging and DNAzyme-based dual gene-silencing therapy with enhanced specificity, sensitivity, and curative effect, which can be a powerful tool for accurate diagnosis and efficient treatment of cancers.