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1.
Biosens Bioelectron ; 145: 111730, 2019 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-31590074

RESUMO

Single-cell analysis is critical to understanding its heterogeneity and biological processes, such as stem cell differentiation, and elucidating the underlying mechanisms of cellular metabolism. New tools to promote intercellular variability studies help elucidate cellular regulation mechanisms. Here an impedance measurement and analysis system was built to monitor the osteogenic differentiation of single bone marrow mesenchymal stem cells (BM-MSCs) in droplets. The biochip including a microelectrode array was designed based on droplet microfluidics and fabricated. A novel theoretical electrical model was proposed to simulate the electrical properties of cells in the droplets. Impedance measurements showed that single cells are substantially heterogeneous during osteoblast differentiation at different stages (days 0, 7, 14 and 21) and different cell passages (passages 6, 7 and 11). This result was consistent with the appearance of two biomarkers (alkaline phosphatase and calcium nodules), which are the gold standard biomarkers of osteoblastogenesis and differentiation. The device enabled highly efficient single-cell trapping, accurate positioning, and sensitive, label-free and noninvasive impedance measurements of individual cells with multiple channels. This system provides a strategy for exploring the processes of osteoblastogenesis and differentiation at the single-cell level and has substantial potential for applications in the biomedical field.

2.
J Exp Bot ; 70(20): 5943-5958, 2019 Oct 24.
Artigo em Inglês | MEDLINE | ID: mdl-31365744

RESUMO

Plant-parasitic nematodes secrete numerous effectors to facilitate parasitism, but detailed functions of nematode effectors and their plant targets remain largely unknown. Here, we characterized four macrophage migration inhibitory factors (MIFs) in Meloidogyne incognita resembling the MIFs secreted by human and animal parasites. Transcriptional data showed MiMIFs are up-regulated in parasitism. Immunolocalization provided evidence that MiMIF proteins are secreted from the nematode hypodermis to the parasite surface, detected in plant tissues and giant cells. In planta MiMIFs RNA interference in Arabidopsis decreased infection and nematode reproduction. Transient expression of MiMIF-2 could suppress Bax- and RBP1/Gpa2-induced cell death. MiMIF-2 ectopic expression led to higher levels of Arabidopsis susceptibility, suppressed immune responses triggered by flg22, and impaired [Ca2+]cyt influx induced by H2O2. The immunoprecipitation of MiMIF-2-interacting proteins, followed by co-immunoprecipitation and bimolecular fluorescence complementation validations, revealed specific interactions between MiMIF-2 and two Arabidopsis annexins, AnnAt1 and AnnAt4, involved in the transport of calcium ions, stress responses, and signal transduction. Suppression of expression or overexpression of these annexins modified nematode infection. Our results provide functional evidence that nematode effectors secreted from hypodermis to the parasite cuticle surface target host proteins and M. incognita uses MiMIFs to promote parasitism by interfering with the annexin-mediated plant immune responses.

3.
Biosens Bioelectron ; 142: 111523, 2019 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-31336224

RESUMO

Emerging evidence indicates that extracellular vesicle (EV) long non-coding ribonucleic acids (lncRNAs) in lung cancer may be clinically useful biomarkers for early diagnosis using liquid biopsy. However, the extremely low quantities of EV-lncRNAs in peripheral blood are a major challenge for multi-target detection. In this study, we developed a new multi-colour fluorescence digital PCR EV-lncRNA (miDER) analysis chip, and then demonstrated its ability to quickly and accurately analyse the levels of two target genes and one reference gene from peripheral blood. Under the miDER assay, the limit of detection of the target gene from peripheral blood was 10 copies/µL. Based on multiplex assay, the expression levels of two lung cancer-related genes (SLC9A3-AS1 and PCAT6) in patients with lung cancer (n = 32) and healthy controls (n = 30) showed a significant difference between the two groups (P < 0.001; two-tailed t-test). A receiver operating characteristic (ROC) curve analysis was used to evaluate the discrimination ability of these lncRNAs. The combination of two lncRNAs in the miDER assay yielded a higher area under curve (AUC) value of 0.811 (95% CI = 0.705-0.918). Moreover, to determine the absolute quantitation capacity of the miDER assay, we compared the results to those obtained by quantitative real-time polymerase chain reaction (qPCR), demonstrating that the miDER assay is more sensitive than qPCR. The multiplex assay based on the miDER could provide a new solution for the multi-index combined detection of trace EV-lncRNAs in body fluids and demonstrate the use of EV-lncRNAs as biomarkers for lung tumour biopsy.

4.
Biosens Bioelectron ; 137: 140-147, 2019 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-31096080

RESUMO

We propose the first black phosphorus (BP) - fiber optic biosensor for ultrasensitive diagnosis of human neuron-specific enolase (NSE) cancer biomarkers. A novel optical-nano configuration has been exploited by integrating BP nanosheets with a largely tilted fiber grating (BP-TFG), where the BP is bio-functionalized by the poly-L-lysine acting as a critical cross-linker to facilitate bio-nano-photonic interface with extremely enhanced light-matter interaction. BP nanosheets are synthesized by a liquid ultrasonication-based exfoliation and deposited on fiber device by an in-situ layer-by-layer method. The BP-induced optical modulation effects in terms of thickness-tunable feature, polarization-dependence and enhanced light-matter interaction are experimentally investigated. The anti-NSE immobilized BP-TFG biosensor has been implemented to detect NSE biomarkers demonstrating ultrahigh sensitivity with limit of detection down to 1.0 pg/mL, which is 4 orders magnitude lower than NSE cut-off value of small cell lung cancer. The enhanced sensitivity of BP-TFG is 100-fold higher than graphene oxide or AuNPs based biosensors. We believe that BP-fiber optic configuration opens a new bio-nano-photonic platform for the applications in healthcare, biomedical, food safety and environmental monitoring.


Assuntos
Biomarcadores Tumorais/isolamento & purificação , Técnicas Biossensoriais , Neoplasias/diagnóstico , Fosfopiruvato Hidratase/isolamento & purificação , Biomarcadores Tumorais/química , Tecnologia de Fibra Óptica , Ouro/química , Grafite/química , Humanos , Limite de Detecção , Nanopartículas Metálicas/química , Fosfopiruvato Hidratase/química , Fósforo/química
5.
Biosens Bioelectron ; 139: 111326, 2019 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-31129389

RESUMO

In view of their critical function in metastasis, characterization of single circulating tumor cells (CTCs) can provide important clinical information to monitor tumor progression and guide personal therapy. Single-cell genetic analysis methods based on microfluidics have some inherent shortcomings such as complicated operation, low throughput, and expensive equipment requirements. To overcome these barriers, we developed a simple and open micro-well array containing 26,208 units for either nuclear acids or single-cell genetic analysis. Through modification of the polydimethylsiloxane surface and optimization of chip packaging, we addressed protein adsorption and solution evaporation for PCR amplification on a chip. In the detection of epidermal growth factor receptor (EGFR) exon gene 21, this micro-well array demonstrated good linear correlation at a DNA concentration from 1 × 101 to 1 × 105 copies/µL (R2 = 0.9877). We then successfully integrated cell capture, lysis, PCR amplification, and signal read-out on the micro-well array, enabling the rapid and simple genetic analysis of single cells. This device was used to detect duplex EGFR mutation genes of lung cancer cell lines (H1975 and A549 cells) and normal leukocytes, demonstrating the ability to perform high-throughput, massively parallel duplex gene analysis at the single-cell level. Different types of point mutations (EGFR-L858R mutation or EGFR-T790M mutation) were detected in single H1975 cells, further validating the significance of single-cell level gene detection. In addition, this method showed a good performance in the heterogeneity detection of individual CTCs from lung cancer patients, required for micro-invasive cancer monitoring and treatment selection.

6.
Talanta ; 200: 169-176, 2019 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-31036170

RESUMO

Circulating tumor cells (CTCs) are rare cancer cells that are shed from the tumors into the peripheral blood and are instrumental in distant metastasis. Early detection of CTCs can therefore improve prognoses and help design patient-specific treatment regimen. However, the current CTC isolation techniques have poor efficacy and selectivity, owing to the rarity and heterogeneity of the CTCs. We designed a microchip for integrated single-cell isolation of CTCs - based on cell size and immuno-phenotype - and analysis. Each isolation unit consisted of a trap channel, a bypass channel, and a release channel. The larger cells were preferentially captured at the trap channels and flushed out selectively via release microvalves according to their immuno-phenotype. The average recovery rate and purity of lung cancer cells isolated from a spiked WBC population were respectively 92.5% and 94% using the microchip, which were significantly higher compared to that obtained using anti-CD45 magnetic beads. In addition, the isolated cancer cells were analyzed on chip for the surface markers of epithelial mesenchymal transition. Taken together, the integrated microchip is a promising tool for the isolation and analysis of CTCs in the clinical setting.


Assuntos
Separação Celular/instrumentação , Separação Celular/métodos , Neoplasias Pulmonares/patologia , Técnicas Analíticas Microfluídicas/instrumentação , Células Neoplásicas Circulantes/patologia , Análise de Célula Única/instrumentação , Análise de Célula Única/métodos , Linhagem Celular Tumoral , Humanos
7.
Oncol Lett ; 15(5): 7639-7648, 2018 May.
Artigo em Inglês | MEDLINE | ID: mdl-29725463

RESUMO

DNA methylation is closely associated with aberrant epigenetic changes. Previous studies have identified various genes associated with non-small cell lung cancer (NSCLC), but the precise combination responsible for its etiology is still debated. The aim of the present study was to select a new set of NSCLC-related genes using methylation-sensitive high-resolution melting. The promoter methylation status of six selected genes, consisting of protocadherin γ subfamily B, 6 (PCDHGB6), homeobox A9 (HOXA9), O6-methylguanine-DNA methyltransferase (MGMT), microRNA (miR)-126, suppressor of cytokine signaling 3 (SOCS3) and Ras association domain family member 5, also termed NORE1A, was evaluated in 54 NSCLC patients. From these samples, genome-wide DNA was extracted and bisulfite conversion was performed along with fluorogenic quantitative polymerase chain reaction to detect methylation values of the six selected promoters. The present results revealed frequent methylation on PCDHGB6, HOXA9 and miR-126, which contrasted with infrequent methylation on MGMT. The results indicated no methylation on either SOCS3 or NORE1A. The sensitivity and specificity of the methylation assessment were 85.2 and 81.5%, respectively, and the analysis results were validated by pyrosequencing. Furthermore, minute comparison of the association between DNA methylation and clinical features was performed. Overall, these results may provide potential information for the development of better clinical diagnostics and more targeted and effective therapies for NSCLC.

8.
Talanta ; 185: 229-236, 2018 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-29759193

RESUMO

Circulating tumor cells (CTCs) have become an important biomarker for liquid biopsy to monitor tumor progression and indicate response to therapies. Many epithelial cellular adhesion molecule (EpCAM) dependent CTC isolation methods have been developed, which have a limitation for low EpCAM expressed tumor cells. In an effort to overcome these drawbacks, we developed combined immunomagnetic beads (EpCAM, Mucin1 and epidermal growth factor receptor) to sensitively isolate CTCs for immunofluorescence analysis and genetic characterization. With this combined immunomagnetic beads, 93.35% H446 cells from spiked blood sample can be recovered. We were able to detect CTCs in 127 among 143 patients included in the study (88.8%). Some CTC clusters were captured with the combined magnetic beads system. In 17 of them, CTCs after chemotherapy significantly decreased compared to that before chemotherapy (4.42 (±â€¯3.94) vs. 12 (±â€¯7)/mL, P = 0.002). For subsequent genetic characterization of CTCs, 2 of 6 samples, using a droplet digital PCR (ddPCR) chip, have detectable EGFR L858R mutation in the cells enriched with the combined immunomagnetic beads. In conclusion, this method integrating the combined immunomagnetic beads and the ddPCR chip for CTCs detection can be of potential application in terms of diagnosis, therapeutic evaluation and personalized medicine in lung cancer.


Assuntos
Receptores ErbB/genética , Separação Imunomagnética , Neoplasias Pulmonares/diagnóstico , Células Neoplásicas Circulantes/patologia , Reação em Cadeia da Polimerase , Humanos , Neoplasias Pulmonares/genética , Mutação , Tamanho da Partícula
9.
Nanotechnology ; 29(13): 135501, 2018 Apr 03.
Artigo em Inglês | MEDLINE | ID: mdl-29322943

RESUMO

In this paper, ultra-sensitive and highly selective Hg2+ detection in aqueous solutions was studied by free-standing silicon nanowire (SiNW) sensors. The all-around surface of SiNW arrays was functionalized with (3-Mercaptopropyl)trimethoxysilane serving as Hg2+ sensitive layer. Due to effective electrostatic control provided by the free-standing structure, a detection limit as low as 1 ppt was obtained. A linear relationship (R 2 = 0.9838) between log(CHg2+ ) and a device current change from 1 ppt to 5 ppm was observed. Furthermore, the developed SiNW sensor exhibited great selectivity for Hg2+ over other heavy metal ions, including Cd2+. Given the extraordinary ability for real-time Hg2+ detection, the small size and low cost of the SiNW device, it is expected to be a potential candidate in field detection of environmentally toxic mercury.

10.
New Phytol ; 217(2): 687-699, 2018 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-29034957

RESUMO

Root-knot nematodes, Meloidogyne spp., are obligate endoparasites that maintain a biotrophic relationship with their hosts. They infect roots as microscopic vermiform second-stage juveniles, and establish specialized feeding structures called 'giant-cells', from which they withdraw water and nutrients. The nematode effector proteins secreted in planta are key elements in the molecular dialogue of parasitism. Here, we compared Illumina RNA-seq transcriptomes for M. incognita obtained at various points in the lifecycle, and identified 31 genes more strongly expressed in parasitic stages than in preparasitic juveniles. We then selected candidate effectors for functional characterization. Quantitative real-time PCR and in situ hybridizations showed that the validated differentially expressed genes are predominantly specifically expressed in oesophageal glands of the nematode. We also soaked the nematodes in siRNA to silence these genes and to determine their role in pathogenicity. The silencing of the dorsal gland specific-Minc18876 and its paralogues resulted in a significant, reproducible decrease in the number of mature females with egg masses, demonstrating a potentially important role for the small glycine- and cysteine-rich effector MiSGCR1 in early stages of plant-nematode interaction. Finally, we report that MiSGCR1 suppresses plant cell death induced by bacterial or oomycete triggers of plant defense.

11.
Nutr Neurosci ; 21(2): 123-131, 2018 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-28277184

RESUMO

Domoic acid (DA) is one of the best known marine toxins, causative of important neurotoxic alterations. DA effects are documented both in wildlife and experimental assays, showing that this toxin causes severe injuries principally in the hippocampal area. Accumulating evidence indicates that mitochondrial dysfunction and oxidative stress are involved in DA-induced cognitive functional impairment. Therefore, therapeutics targeted to improve mitochondrial function and increase oxidative stress defence could be beneficial. Quercetin, a bioflavanoid, has been reported to have potent neuroprotective effects and anti-oxidative ability, but its preventive effects on DA-induced mitochondrial dysfunction and cognitive impairment have not been well characterised. In this study, we evaluated the effects of quercetin on DA-induced cognitive deficits in mice and explored its potential mechanism. Our results showed that the oral administration of quercetin to DA-treated mice significantly improved their behavioural performance in a novel objective recognition task and a Morris water maze task. These improvements were mediated, at least in part, by a stimulation of PPARγ coactivator 1α-mediated mitochondrial biogenesis signalling and an amelioration of mitochondrial dysfunction. Moreover, quercetin activated nuclear factorerythroid-2-related factor-2 (Nrf2)-mediated phase II enzymes and decreased reactive oxygen species and protein carbonylation. Furthermore, the AMP-activated protein kinase (AMPK) activity significantly increased in the quercetin-treated group. Taken together, these findings suggest that a reduction in mitochondrial dysfunction through the increase of AMPK activity, coupled with an increase in Nrf2 pathway mediated oxidative defence, may be one of the mechanisms by which quercetin improves cognitive impairment induced by DA in mice.


Assuntos
Disfunção Cognitiva/tratamento farmacológico , Fármacos Neuroprotetores/farmacologia , Quercetina/farmacologia , Animais , Cognição/efeitos dos fármacos , Disfunção Cognitiva/induzido quimicamente , Modelos Animais de Doenças , Hipocampo/efeitos dos fármacos , Hipocampo/metabolismo , Ácido Caínico/análogos & derivados , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo
12.
Biosens Bioelectron ; 96: 339-344, 2017 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-28525852

RESUMO

Hypermethylation of CpG islands in the promoter region of many tumor suppressor genes downregulates their expression and in a result promotes tumorigenesis. Therefore, detection of DNA methylation status is a convenient diagnostic tool for cancer detection. Here, we reported a novel method for the integrative detection of methylation by the microfluidic chip-based digital PCR. This method relies on methylation-sensitive restriction enzyme HpaII, which cleaves the unmethylated DNA strands while keeping the methylated ones intact. After HpaII treatment, the DNA methylation level is determined quantitatively by the microfluidic chip-based digital PCR with the lower limit of detection equal to 0.52%. To validate the applicability of this method, promoter methylation of two tumor suppressor genes (PCDHGB6 and HOXA9) was tested in 10 samples of early stage lung adenocarcinoma and their adjacent non-tumorous tissues. The consistency was observed in the analysis of these samples using our method and a conventional bisulfite pyrosequencing. Combining high sensitivity and low cost, the microfluidic chip-based digital PCR method might provide a promising alternative for the detection of DNA methylation and early diagnosis of epigenetics-related diseases.


Assuntos
Caderinas/genética , Metilação de DNA , Proteínas de Homeodomínio/genética , Dispositivos Lab-On-A-Chip , Reação em Cadeia da Polimerase/instrumentação , Regiões Promotoras Genéticas , Adenocarcinoma/genética , Adenocarcinoma de Pulmão , Ilhas de CpG , DNA/genética , Desenho de Equipamento , Humanos , Neoplasias Pulmonares/genética
13.
Biosens Bioelectron ; 94: 200-206, 2017 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-28285197

RESUMO

We explore graphene oxide (GO) nanosheets functionalized dual-peak long period grating (dLPG) based biosensor for ultrasensitive label-free antibody-antigen immunosensing. The GO linking layer provides a remarkable analytical platform for bioaffinity binding interface due to its favorable combination of exceptionally high surface-to-volume ratio and excellent optical and biochemical properties. A new GO deposition technique based on chemical-bonding in conjunction with physical-adsorption was proposed to offer the advantages of a strong bonding between GO and fiber device surface and a homogeneous GO overlay with desirable stability, repeatability and durability. The surface morphology of GO overlay was characterized by Atomic force microscopy, Scanning electron microscope, and Raman spectroscopy. By depositing the GO with a thickness of 49.2nm, the sensitivity in refractive index (RI) of dLPG was increased to 2538nm/RIU, 200% that of non-coated dLPG, in low RI region (1.333-1.347) where bioassays and biological events were usually carried out. The IgG was covalently immobilized on GO-dLPG via EDC/NHS heterobifunctional cross-linking chemistry leaving the binding sites free for target analyte recognition. The performance of immunosensing was evaluated by monitoring the kinetic bioaffinity binding between IgG and specific anti-IgG in real-time. The GO-dLPG based biosensor demonstrates an ultrahigh sensitivity with limit of detection of 7ng/mL, which is 10-fold better than non-coated dLPG biosensor and 100-fold greater than LPG-based immunosensor. Moreover, the reusability of GO-dLPG biosensor has been facilitated by a simple regeneration procedure based on stripping off bound anti-IgG treatment. The proposed ultrasensitive biosensor can be further adapted as biophotonic platform opening up the potential for food safety, environmental monitoring, clinical diagnostics and medical applications.


Assuntos
Anticorpos Anti-Idiotípicos/isolamento & purificação , Técnicas Biossensoriais/métodos , Grafite/química , Anticorpos/imunologia , Anticorpos/isolamento & purificação , Anticorpos Anti-Idiotípicos/química , Anticorpos Anti-Idiotípicos/imunologia , Antígenos/imunologia , Antígenos/isolamento & purificação , Óxidos/química
14.
Oncotarget ; 8(8): 12917-12928, 2017 Feb 21.
Artigo em Inglês | MEDLINE | ID: mdl-28039472

RESUMO

Circulating tumor cells (CTCs) have attracted pretty much attention from scientists because of their important relationship with the process of metastasis. Here, we developed a size-based microfluidic chip containing triangular pillar array and filter channel array for detecting single CTCs and CTC clusters independent of tumor-specific markers. The cell populations in chip were characterized by immune-fluorescent staining combining an epithelial marker and a mesenchymal marker. We largely decreased the whole time of detection process to nearly 1.5h with this microfluidic device. The CTCs were subsequently measured in 77 patients with lung cancer and 39 healthy persons. The microfluidic device allowed for the detection of CTCs with apparent high sensitivity and specificity (82.7% sensitivity and 100% specificity). Furthermore, the total CTC counts were found to be elevated in advanced patients with metastases when compared with those without (20.89±14.57 vs 8.428±5.858 cells/mL blood; P<0.01). Combined epithelial marker and mesenchymal marker analysis of CTCs could provide more information about metastasis in patients than only usage of epithelial marker. In conclusion, the development of the size-based microfluidic device for efficient capture of CTCs will enable detailed characterization of their biological properties and values in cancer diagnosis.


Assuntos
Dispositivos Lab-On-A-Chip , Neoplasias Pulmonares/patologia , Procedimentos Analíticos em Microchip/métodos , Técnicas Analíticas Microfluídicas/instrumentação , Células Neoplásicas Circulantes , Adulto , Idoso , Idoso de 80 Anos ou mais , Biomarcadores Tumorais/análise , Linhagem Celular Tumoral , Separação Celular , Feminino , Humanos , Neoplasias Pulmonares/sangue , Masculino , Técnicas Analíticas Microfluídicas/métodos , Pessoa de Meia-Idade
15.
Anal Chim Acta ; 958: 77-84, 2017 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-28110687

RESUMO

Assay of multiple serum tumor markers such as carcinoembryonic antigen (CEA), cytokeratin 19 fragment antigen (CYFRA21-1), and neuron specific enolase (NSE), is important for the early diagnosis of lung cancer. Dickkopf-1 (DKK1), a novel serological and histochemical biomarker, was recently reported to be preferentially expressed in lung cancer. Four target proteins were sandwiched by capture antibodies attached to microarrays and detection antibodies carried on modified gold nanoparticles. Optical signals generated by the sandwich structures were amplified by gold deposition with HAuCl4 and H2O2, and were observable by microscopy or the naked eye. The four tumor markers were subsequently measured in 106 lung cancer patients and 42 healthy persons. The assay was capable of detecting multiple biomarkers in serum sample at concentration of <1 ng mL-1 in 1 h. Combined detection of the four tumor markers highly improved the sensitivity (to 87.74%) for diagnosis of lung cancer compared with sensitivity of single markers. A rapid, highly sensitive co-detection method for multiple biomarkers based on gold nanoparticles and microarrays was developed. In clinical use, it would be expected to improve the early diagnosis of lung cancer.


Assuntos
Biomarcadores Tumorais/análise , Ouro , Neoplasias Pulmonares/diagnóstico , Nanopartículas Metálicas , Antígenos de Neoplasias/análise , Antígeno Carcinoembrionário/análise , Humanos , Peróxido de Hidrogênio , Queratina-19/análise
16.
Biosens Bioelectron ; 91: 482-488, 2017 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-28073028

RESUMO

In this work, a real-time assay for highly sensitive, label-free, multiplexed electrical detection of lung cancer biomarkers was developed by using silicon nanowire field-effect (SiNW-FET) devices. Highly responsive SiNW arrays were fabricated using a CMOS-compatible anisotropic self-stop etching technique with mass reproducibility and low cost character. The SiNW nanosensor was integrated with PDMS microfluidic device, which allows rapid analyte delivery, makes the analysis to be conducted using exceedingly small samples and enables potential multiplexed detection. The nanowire arrays allowed highly selective and sensitive multiplexed detection of microRNA (miRNA)-126 and CEA. Due to high surface-to-volume ratio that the nanowire dimensions confer, the detection floor of single molecule was achieved. The potential utility in identifying clinical samples for early diagnosis of cancer was demonstrated by analyzing biomarkers in clinical related samples. The developed nanosensor with capability for multiplexed real-time monitoring of biomarkers with high sensitivity and selectivity in clinically relevant samples is highly attractive for diagnosis and treatment of cancer and other diseases.


Assuntos
Técnicas Biossensoriais/instrumentação , Antígeno Carcinoembrionário/sangue , Dispositivos Lab-On-A-Chip , Neoplasias Pulmonares/sangue , MicroRNAs/sangue , Nanofios/química , Silício/química , Biomarcadores Tumorais/sangue , Dimetilpolisiloxanos/química , Desenho de Equipamento , Humanos , Nanofios/ultraestrutura
17.
Oncotarget ; 8(8): 13329-13337, 2017 Feb 21.
Artigo em Inglês | MEDLINE | ID: mdl-28076325

RESUMO

The aberrant expressions of long noncoding RNAs have been reported in numerous cancers, which have facilitated the cancer diagnosis. However, the expression profile of lncRNAs in early stage lung squamous cell carcinoma has not been well discussed. The present study aimed to examine the expression profile of lncRNAs in early stage lung squamous cell carcinoma and identify lncRNA biomarkers for diagnosis. Through high-throughput lncRNA microarray, we screened thousands of aberrantly expressed lncRNAs and mRNAs in early stage lung squamous cell carcinoma tissues compared to their corresponding adjacent nontumorous tissues. Bioinformatics analyses were used to investigate the functions of aberrantly expressed mRNAs and their associated lncRNAs. After that, in order to identify lncRNA biomarkers for early detection, candidate lncRNA biomarkers were selected based on our established filtering pipeline and validated by real-time quantitative polymerase chain reaction on a total of 63 pairs of tumor samples. Five lncRNAs were finally identified which were able to distinguish early stage tumor and normal samples with high sensitivity (92%) and specificity (83%). These results imply that lncRNAs may be powerful biomarker for early diagnosis.


Assuntos
Carcinoma de Células Escamosas/diagnóstico , Detecção Precoce de Câncer/métodos , Perfilação da Expressão Gênica/métodos , Neoplasias Pulmonares/diagnóstico , RNA Longo não Codificante/análise , Adulto , Idoso , Área Sob a Curva , Carcinoma de Células Escamosas/genética , Feminino , Redes Reguladoras de Genes , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Neoplasias Pulmonares/genética , Masculino , Pessoa de Meia-Idade , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Reação em Cadeia da Polimerase , Curva ROC , Sensibilidade e Especificidade
18.
Biosens Bioelectron ; 87: 701-707, 2017 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-27636559

RESUMO

A label-free immunosensor based on antibody-modified graphene field effect transistor (GFET) was presented. Antibodies targeting carcinoembryonic antigen (Anti-CEA) were immobilized to the graphene surface via non-covalent modification. The bifunctional molecule, 1-pyrenebutanoic acid succinimidyl ester, which is composed of a pyrene and a reactive succinimide ester group, interacts with graphene non-covalently via π-stacking. The succinimide ester group reacts with the amine group to initiate antibody surface immobilization, which was confirmed by X-ray Photoelectron Spectroscopy, Atomic Force Microscopy and Electrochemical Impedance Spectroscopy. The resulting anti-CEA modified GFET sufficiently monitored the reaction between CEA protein and anti-CEA in real-time with high specificity, which revealed selective electrical detection of CEA with a limit of detection (LOD) of less than 100pg/ml. The dissociation constant between CEA protein and anti-CEA was estimated to be 6.35×10-11M, indicating the high affinity and sensitivity of anti-CEA-GFET. Taken together, the graphene biosensors provide an effective tool for clinical application and point-of-care medical diagnostics.


Assuntos
Anticorpos Imobilizados/química , Técnicas Biossensoriais/instrumentação , Antígeno Carcinoembrionário/análise , Grafite/química , Biomarcadores Tumorais/análise , Técnicas Eletroquímicas/instrumentação , Desenho de Equipamento , Humanos , Imunoensaio/instrumentação , Limite de Detecção , Sistemas Automatizados de Assistência Junto ao Leito
19.
Langmuir ; 32(48): 12623-12631, 2016 12 06.
Artigo em Inglês | MEDLINE | ID: mdl-27934532

RESUMO

Two-dimensional graphene devices are widely used for biomolecule detection. Nevertheless, the surface modification of graphene is critical to achieve the high sensitivity and specificity required for biological detection. Herein, native bovine serum albumin (BSA) in inorganic solution is denatured on the graphene surface by heating, leading to the formation of nanoscale BSA protein films adsorbed on the graphene substrate via π-stacking interactions. This technique yields a controllable, scalable, uniform, and high-coverage method for graphene biosensors. Further, the application of such nanoscale heat-denatured BSA films on graphene as a universal graphene biosensor platform is explored. The thickness of heat-denatured BSA films increased with heating time and BSA concentration but decreased with solvent concentration as confirmed by atomic force microscopy. The noncovalent interaction between denatured BSA films and graphene was investigated by Raman spectroscopy. BSA can act as a p-type and n-type dopant by modulating pH-dependent net charges on the layered BSA-graphene surface, as assessed by current-voltage measurements. Chemical groups of denatured BSA films, including amino and carboxyl groups, were verified by X-ray photoelectron microscopy, attenuated total reflectance-Fourier transform infrared spectra, and fluorescent labeling. The tailoring of the BSA-graphene surfaces through chemical modification, controlled thickness, and doping type via noncovalent interactions provides a controllable, multifunctional biosensor platform for molecular diagnosis without the possibility of nonspecific adsorption on graphene.


Assuntos
Grafite/química , Soroalbumina Bovina/química , Adsorção , Animais , Técnicas Biossensoriais , Bovinos , Corantes Fluorescentes/química , Temperatura Alta , Concentração de Íons de Hidrogênio , Microscopia de Força Atômica , Nanoestruturas , Conformação Proteica , Desnaturação Proteica , Solventes , Espectroscopia de Infravermelho com Transformada de Fourier , Análise Espectral Raman , Propriedades de Superfície
20.
Talanta ; 161: 205-210, 2016 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-27769397

RESUMO

Carcinoembryonic antigen (CEA) is an important biomarker in cancer diagnosis. Here, we present an efficient, selective lateral-flow immunoassay (LFIA) based on magnetic nanoparticles (MNPs) for in situ sensitive and accurate point-of-care detection of CEA. Signal amplification mechanism involved linking of detection MNPs with signal MNPs through biotin-modified single-stranded DNA (ssDNA) and streptavidin. To verify the effectiveness of this modified LFIA system, the sensitivity and specificity were evaluated. Sensitivity evaluation showed a broad detection range of 0.25-1000ng/ml for CEA protein by the modified LFIA, and the limit of detection (LOD) of the modified LFIA was 0.25ng/ml, thus producing significant increase in detection threshold compared with the traditional LFIA. The modified LFIA could selectively recognize CEA in presence of several interfering proteins. In addition, this newly developed assay was applied for quantitative detection of CEA in human serum specimens collected from 10 randomly selected patients. The modified LFIA system detected minimum 0.27ng/ml of CEA concentration in serum samples. The results were consistent with the clinical data obtained using commercial electrochemiluminescence immunoassay (ECLIA) (p<0.01). In conclusion, the MNPs based LFIA system not only demonstrated enhanced signal to noise ratio, it also detected CEA with higher sensitivity and selectivity, and thus has great potential to be commercially applied as a sensitive tumor marker filtration system.


Assuntos
Biomarcadores Tumorais/sangue , Antígeno Carcinoembrionário/sangue , Anticorpos/imunologia , Biomarcadores Tumorais/imunologia , Biotina/química , Antígeno Carcinoembrionário/imunologia , DNA de Cadeia Simples/química , Humanos , Imunoensaio/métodos , Limite de Detecção , Fenômenos Magnéticos , Nanopartículas/química , Estreptavidina/química
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