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1.
Chem Commun (Camb) ; 2020 May 22.
Artigo em Inglês | MEDLINE | ID: mdl-32441293

RESUMO

We incorporate three conceptual components including luminescence-concentrating upconversion nanoparticles, optical tweezers, and DNA walkers into bead carriers to establish a new imaging analysis.

2.
Artigo em Inglês | MEDLINE | ID: mdl-32246736

RESUMO

Comprehensive phenotypic profiling of heterogeneous circulating tumor cells (CTCs) at single-cell resolution has great importance for cancer management. Herein, a novel spectrally combined encoding (SCE) strategy was proposed for multiplex biomarker profiling of single CTCs using a multifunctional nanosphere-mediated microfluidic platform. Different cellular biomarkers uniquely labeled by multifunctional nanosphere barcodes, possessing identical magnetic tags and distinct optical signatures, enabled isolation of heterogeneous CTCs with over 91.6 % efficiency and in situ SCE of phenotypes. By further trapping individual CTCs in ordered microstructures on chip, composite single-cell spectral signatures were conveniently and efficiently obtained, allowing reliable spectral-readout for multiplex biomarker profiling. This SCE strategy exhibited great potential in multiplex profiling of heterogeneous CTC phenotypes, offering new avenues for cancer study and precise medicine.

3.
Anal Chim Acta ; 1105: 112-119, 2020 Apr 08.
Artigo em Inglês | MEDLINE | ID: mdl-32138909

RESUMO

Perturbation of thiol homeostasis in biological fluids are thought to be associated with several diseases, and reliable analytical methods for the determination of low molecular weight (LMW) thiols in human plasma or serum are thus required. In this study, a matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) method is described for high throughput determination of four LMW thiols (glutathione, cysteine, homocysteine and cysteinylglycine) in human serum. It is based on the use of a bromoacetyl functionalized C60 (Br-C60) as a derivatization reagent to label thiols. The Br-C60 labeling can add an 832-Da tag to thiols, which moves thiol signals to high mass region and effectively avoids the signal interference generated by the traditional MALDI matrix below 800 Da. The labeling can be completed within 5 min under microwave-assisted condition. Thereby, the Br-C60 labeling based MALDI-TOF MS analytical method can achieve high throughput analysis of LMW thiols in serum. Good linearities of the method for the thiols in human serum were obtained in the range of 0.5-500.0 µM with correlation coefficient (R) greater than 0.9960. The limit of detection is in the range of 0.07-0.18 µM for the investigated thiols in human serum with relative standard deviations of lower than 13.5% and recoveries ranging from 81.9 to 117.1%. Using the method, four thiols in microliter serum samples of breast cancer (BC) patients were determined. The result showed that the contents of the four thiols in BC serum samples significantly changed compared to the healthy control (HC).

4.
Anal Chem ; 92(7): 5258-5266, 2020 Apr 07.
Artigo em Inglês | MEDLINE | ID: mdl-32156113

RESUMO

To enhance signal acquisition stability and diminish background interference for conventional flow bead-based fluorescence detection methods, we demonstrate here an exceptional microfluidic chip assisted platform by integrating near-infrared optical tweezers with upconversion luminescence encoding. For the former, a single 980 nm laser is employed to perform optical trapping and concurrently excite upconversion luminescence, avoiding the fluctuation of the signals and the complexity of the apparatus. By virtue of the favorable optical properties of upconversion nanoparticles (UCNPs), the latter is carried out by employing two-color UCNPs (Er-UCNPs and Tm-UCNPs) with negligible spectral overlaps. With the assistance of the double key techniques, we fabricated complex microbeads referred to a UCNPs-miRNAs-microbead sandwich construct by a one-step nucleic acid hybridization process and then obtained uniform terrace peaks for the automatic and simultaneous quantitative determination of miRNA-205 and miRNA-21 sequences with a detection limit of pM level on the basis of a special home-built flow bead platform. Furthermore, the technique was successfully applied for analyzing complex biological samples such as cell lysates and human tissue lysates, holding certain potential for disease diagnosis. In addition, it is expected that the flow platform can be utilized to investigate many other biomolecules of single cells and to allow analysis of particle heterogeneity in biological fluid by means of optical tweezers.

5.
Anal Chim Acta ; 1098: 56-65, 2020 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-31948587

RESUMO

RNA molecules carry diverse modifications that exert important influences in many cellular processes. In addition to the single modification occurring in either nucleobase or 2' hydroxyl of ribose in RNA, some dual modifications occur in both the nucleobase and 2' hydroxyl of ribose in RNA. 2'-O-methyl-5-methylcytidine (m5Cm), the dual modifications of cytidine, was first discovered from the tRNA of archaea. Recent studies identified that 2'-O-methyl-5-hydroxymethylcytidine (hm5Cm) and 2'-O-methyl-5-formylcytidine (f5Cm) were present in the anticodon of cytoplasmic tRNA of mammals. Similar to the series of single modification of cytidines of 5-methylcytosine (m5C), 5-hydroxymethylcytidine (hm5C), 5-formylcytidine (f5C), and 5-carboxylcytidine (ca5C) in nucleic acids, the dual modifications of m5Cm, hm5Cm, f5Cm and 2'-O-methyl-5-carboxylcytidine (ca5Cm) may also constitute the series of cytidine modifications in mammals. However, it is normally challenging to detect these modifications because of their low endogenous levels. Here, we established a method by chemical labeling-assisted liquid chromatography - electrospray ionization - tandem mass spectrometry (LC-ESI-MS/MS) analysis for the sensitive and simultaneous determination of all these four cytidine dual modifications, i.e., m5Cm, hm5Cm, f5Cm and ca5Cm. Three different labeling reagents (2-bromo-1-(3,4-dimeth oxyphenyl)-ethanone, BDMOPE; 2-bromo-1-(4-methoxyphenyl)-ethanone, BMOPE; 2-bromo-1-(4-diethylaminophenyl)-ethanone, BDEPE) were used for the chemical labeling. The results showed that the detection sensitivities of m5Cm, hm5Cm, f5Cm and ca5Cm increased up to 462 folds after chemical labeling. With the developed method, we achieved the simultaneous detection of m5Cm, hm5Cm and f5Cm in RNA of mammals. In addition, we found these cytidine dual modifications mainly exist in small RNA (<200 nt) and barely detected in other types of RNA. Moreover, we found that the levels of m5Cm in RNA of human lung carcinoma tissues significantly increased, while hm5Cm and f5Cm significantly decreased compared to tumor adjacent normal tissues. The significant changes of m5Cm, hm5Cm and f5Cm levels may serve as indicator for the detection and prognosis of lung cancer.

6.
Anal Chem ; 92(2): 2301-2309, 2020 01 21.
Artigo em Inglês | MEDLINE | ID: mdl-31845797

RESUMO

Ribonucleotide analogues and their related phosphorylated metabolites play critical roles in tumor metabolism. However, determination of the endogenous ribonucleotides from the complex biological matrix is still a challenge due to their high structural similarity and high polarity that will lead to the low retention and low detection sensitivities by liquid chromatogram mass spectrometry analysis. In this study, we developed the diazo reagent labeling strategy with mass spectrometry analysis for sensitive determination of ribonucleotides in the living organism. A pair of light and heavy stable isotope labeling reagents, 2-(diazomethyl)-N-methyl-N-phenyl-benzamide (2-DMBA) and d5-2-(diazomethyl)-N-methyl-N-phenyl-benzamide (d5-2-DMBA), were synthesized to label ribonucleotides. 2-DMBA showed high specificity and high efficiency for the labeling of ribonucleotides. Our results demonstrated that the detection sensitivities of 12 ribonucleotides increased by 17-174-fold upon 2-DMBA labeling. The obtained limits of detection (LODs) of ribonucleotides ranged from 0.07 fmol to 0.41 fmol. Using this method, we achieved the sensitive and accurate detection of ribonucleotides from only a few cells (8 cells). To the best of our knowledge, this is the highest detection sensitivity for ribonucleotides ever reported. In addition, we found that the contents of almost all of these ribonucleotides were significantly increased in human breast carcinoma tissues compared to tumor-adjacent normal tissues, suggesting that endogenous ribonucleotides may play certain functional roles in the regulation of cancer development and formation. This method also can be potentially applied in the analysis of phosphorylated compounds.

7.
Anal Chem ; 91(17): 11440-11446, 2019 Sep 03.
Artigo em Inglês | MEDLINE | ID: mdl-31397147

RESUMO

Chiral carboxylic acids play important roles in energy metabolism and signal transduction in the human body. These enantiomers usually possess different bioactivities and are also associated with the development of some diseases. Therefore, simultaneous determination of multiple chiral carboxylic acids is vital for study of the pathogenesis of related diseases. However, it is still challenging to simultaneously detect the enantiomers of multiple chiral carboxylic acids in biological samples. Here, we developed a novel 4-plex chemical labeling strategy based on 4 analogues of cinchona alkaloid-derived primary amines (CAPAs) for simultaneous determination of 16 enantiomers of 8 chiral carboxylic acids by liquid chromatography-mass spectrometry (LC-MS). To achieve high-throughput analysis, one CAPA analogue was used to label chiral carboxylic acid standards and served as internal standards (ISs), while the other 3 CAPA analogues were used to label endogenous chiral carboxylic acids in 3 different biological samples. After CAPAs labeling, the 16 chiral carboxylic acid enantiomers could be detected by LC-MS, and their detection sensitivity was greatly enhanced by up to 3 orders of magnitude compared to intact analytes. Further, the developed method for the determination of 16 chiral carboxylic acid enantiomers was validated in human serums and mammalian cells. Finally, the proposed method was applied to the determination of chiral carboxylic acids in the serum samples from type 2 diabetes mellitus (T2DM) and colorectal cancer (CRC) patients. We found that 5 chiral carboxylic acid enantiomers in T2DM serum samples and 4 chiral carboxylic acid enantiomers in CRC serum samples exhibited significant change compared to the healthy control (HC).

8.
Anal Chim Acta ; 1081: 103-111, 2019 Nov 12.
Artigo em Inglês | MEDLINE | ID: mdl-31446947

RESUMO

Both DNA cytosine methylation (5-methyl-2'-deoxycytidine, m5dC) and RNA cytosine methylation (5-methylcytidine, m5rC) are important epigenetic marks that play regulatory roles in diverse biological processes. m5dC and m5rC can be further oxidized by the ten-eleven translocation (TET) proteins to form 5-hydroxymethyl-2'-deoxycytidine (hm5dC) and 5-hydroxymethylcytidine (hm5rC), respectively. 2'-O-methyl-5-hydroxymethylcytidine (hm5rCm) was recently also identified as a second oxidative metabolite of m5rC in RNA. Previous studies showed that the dysregulation of cytidine modifications in both DNA and RNA are closely related to a variety of human diseases. These cytidine modifications are generally excreted from cell into urine. If these cytidine modifications exhibit specific features related to certain diseases, determination of the cytidine modifications in urine could be utilized as non-invasive diagnostic of diseases. Here, we established a solid-phase extraction in combination with liquid chromatography-mass spectrometry (LC-MS/MS) analysis for simultaneous detection of these cytidine modifications in human urine samples. The developed method enabled the distinct detection of these cytidine modifications. We reported, for the first time, the presence of hm5rCm in human urine. Furthermore, we found that compared to the healthy controls, the contents of hm5dC, hm5rC, and hm5rCm showed significant increases in urine samples of cancer patients, including lymphoma patients, gastric cancer patients, and esophageal cancer patients. This study indicates that the urinary hydroxylmethylation modifications of hm5dC, hm5rC, and hm5rCm may serve as potential indicator of cancers.


Assuntos
Cromatografia Líquida/métodos , Citidina/análogos & derivados , Citidina/urina , Extração em Fase Sólida/métodos , Espectrometria de Massas em Tandem/métodos , Adulto , Idoso , Idoso de 80 Anos ou mais , DNA/química , Metilação de DNA , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , RNA/química
9.
Anal Chem ; 91(16): 10477-10483, 2019 Aug 20.
Artigo em Inglês | MEDLINE | ID: mdl-31318193

RESUMO

RNA molecules harbor diverse chemical modifications that play important regulatory roles in a variety of biological processes. Up to date, more than 150 modifications have been identified in various RNA species. Most of these modifications occurring in nucleic acids are the methylation of nucleic acids. It has been demonstrated that many of these methylation are reversible and undergo dynamic demethylation. Previous studies established that the demethylation of the two most important and prevalent modifications of 5-methylcytidine (m5C) and N6-methyladenosine (m6A) in nucleic acids is through the hydroxylation of m5C and m6A, forming 5-hydroxymethylcytidine (hm5C) and N6-hydroxymethyladenosine (hm6A), respectively. This indicates the hydroxylation of the methylated nucleosides may be a general pathway for the demethylation of nucleic acid methylation. However, few other hydroxylmethylation modifications have yet to be reported in existence in mammals. In the current study, we developed a neutral enzymatic digestion method for the mild digestion of nucleic acids, followed by liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) analysis. With the established method, we reported the existence of a new hydroxylmethylated nucleosides, N2-hydroxymethylguanosine (hm2G), in mammalian RNA. In addition, we found that the contents of hm2G, as well as N2-methylguanosine (m2G), showed significant differences between thyroid carcinoma tissues and tumor-adjacent normal tissues, indicating that m2G and hm2G in RNA may play certain roles in the carcinogenesis of thyroid carcinoma. Collectively, our study suggests that RNA hydroxylmethylation may be a new prevalent group of modifications existing in RNA, which expands the diversity of nucleic acid modifications and should exert regulatory functions in living organisms.

10.
Anal Chem ; 91(12): 7950-7957, 2019 Jun 18.
Artigo em Inglês | MEDLINE | ID: mdl-31117416

RESUMO

Herein, a conceptual approach for significantly enhancing a bead-supported assay is proposed. For the fluorescence imaging technology, optical tweezers are introduced to overcome the fluid viscosity interference and immobilize a single tested bead at the laser focus to guarantee a fairly precise imaging condition. For the selection of fluorescent materials and the signal acquisition means, a type of innovative luminescence confined upconversion nanoparticle with a unique sandwich structure is specially designed to act as an efficient energy donor to trigger the luminescent resonance energy transfer (LRET) process. By further combining the double breakthrough with a molecular beacon model, the newly developed detection strategy allows for achieving a pretty high LRET ratio (≈ 88%) to FAM molecules and offering sound assay performance toward miRNA analysis with a detection limit as low as the sub-fM level, and is capable of well identifying single-base mismatching. Besides, this approach not only is able to accurately qualify the low-abundance targets from as few as 30 cancer cells but also can be employed as a valid cancer early warning tool for performing liquid biopsy.

11.
Anal Chem ; 90(17): 10518-10526, 2018 09 04.
Artigo em Inglês | MEDLINE | ID: mdl-30089203

RESUMO

Profiling the heterogeneous phenotypes of individual circulating tumor cells (CTCs) from patients is a very challenging task, but it paves new ways for cancer management, especially personalized anticancer therapy. Herein, we propose a chip-assisted multifunctional-nanosphere system for efficient and reliable biomarker phenotype analysis of individual heterogeneous CTCs. Red fluorescent magnetic biotargeting multifunctional nanospheres and green fluorescent biotargeting nanospheres targeting to two kinds of CTC biomarkers are used for convenient dual-fluorescence labeling of CTCs along with magnetic tags. By integrating magnetic enrichment with a size-selective single-cell-trapping microfluidic chip (SCT-chip), over 90% of CTCs, even when the concentrations is as low as 10 CTCs per milliliter of blood, can be individually trapped at highly ordered micropillars, spatially separated from the minimal residual blood cells. Such single CTCs offer easy-readout fluorescence signals, facilitating efficient identification and reliable phenotype analysis in accordance with their biomarker expressions. Therefore, the phenotypes of breast tumor cells in terms of the expression level of human epidermal-growth-factor receptor 2, an important target of clinical anticancer drugs, are accurately assessed, and over 82% of them can be classified into corresponding cell subpopulations. Furthermore, this system demonstrates successful detection and subpopulation analysis of heterogeneous CTCs from seven breast cancer patients, which provides a promising new means for single-cell profiling of CTC-biomarker phenotypes and guiding of personalized anticancer therapy.


Assuntos
Biomarcadores Tumorais/metabolismo , Nanosferas , Nanoestruturas , Células Neoplásicas Circulantes/metabolismo , Análise de Célula Única , Neoplasias da Mama/sangue , Neoplasias da Mama/metabolismo , Feminino , Genes erbB-2 , Humanos
12.
Anal Chem ; 90(4): 2639-2647, 2018 02 20.
Artigo em Inglês | MEDLINE | ID: mdl-29364660

RESUMO

Establishment of a stable analytical methodology with high-quality results is an urgent need for screening cancer biomarkers in early diagnosis of cancer. In this study, we incorporate holographic optical tweezers with upconversion luminescence encoding to design an imageable suspension array and apply it to conduct the detection of two liver cancer related biomarkers, carcinoembryonic antigen and alpha fetal protein. This bead-based assay is actualized by forming a bead array with holographic optical tweezers and synchronously exciting the upconversion luminescence of corresponding trapped complex beads fabricated with a simple one-step sandwich immunological recognition. Owing to the fact that these flowing beads are stably trapped in the focal plane of the objective lens which tightly converges the array of the laser beams by splitting a 980 nm beam using a diffraction optical element, a fairly stable excitation condition is achieved to provide reliable assay results. By further taking advantage of the eminent encoding capability of upconversion nanoparticles and the extremely low background signals of anti-Stokes luminescence, the two targets are well-identified and simultaneously detected with quite sound sensitivity and specificity. Moreover, the potential on-demand clinical application is presented by employing this approach to respond the targets toward complex matrices such as serum and tissue samples, offering a new alternative for cancer diagnosis technology.


Assuntos
Biomarcadores Tumorais/análise , Neoplasias Hepáticas/diagnóstico por imagem , Luminescência , Imagem Óptica , Pinças Ópticas , Corantes Fluorescentes/síntese química , Corantes Fluorescentes/química , Humanos , Nanopartículas/química , Imagem Óptica/instrumentação , Tamanho da Partícula
13.
Talanta ; 176: 344-349, 2018 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-28917760

RESUMO

Monitoring the concentration of blood glucose in patients is a key component of good medical diagnoses. Therefore, developing an accurate, rapid and sensitive strategy for monitoring blood glucose is of vital importance. We proposed a strategy for serum glucose determination combining 2-(4-boronobenzyl) isoquinolin-2-ium bromide chemical labeling with black phosphorus assisted laser desorption ionization-time of flight mass spectrometry (CL-BP/ALDI-TOF MS). The entire analytical process consisted of 1min of protein precipitation and 3min of chemical labeling in a microwave oven prior to the BP/ALDI-TOF MS analysis. The analysis can be completed in 5min with high throughput and extremely low sample consumption. Good linearity for glucose was obtained with a correlation coefficient (R) of 0.9986. The limit of detection (LOD) and limit of quantification (LOQ) were 11.5 fmol and 37.5 fmol, respectively. Satisfied reproducibility and reliability were gained by evaluation of the intra- and inter-day precisions with relative standard deviations (RSDs) less than 7.2% and relative recoveries ranging from 87.1% to 108.1%, respectively. The proposed strategy was also applied for the analysis of endogenous glucose in various serum samples and the results were consistent with those obtained using the hexokinase method in a clinical laboratory. Considering the results, the proposed CL-BP/ALDI-TOF MS strategy has proven to be reliable, fast, and sensitive for quantitative analysis of serum glucose.


Assuntos
Glicemia/análise , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Humanos , Lasers , Fósforo
14.
ACS Appl Mater Interfaces ; 9(43): 37606-37614, 2017 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-28994579

RESUMO

As an emerging fascinating fluorescent nanomaterial, carbon nanodots (CDs) have attracted much attention owing of their unique properties such as small size, antiphotobleaching, and biocompatibility. However, its use in biomedical analysis is limited because of its low quantum yield. Herein, we constructed a dual amplification fluorescence sensor by incorporating immunohybridization chain reaction (immuno-HCR) and metal-enhanced fluorescence (MEF) of CDs for the detection of alpha fetal protein (AFP). The immunoplasmonic slide and detection antibodies-conjugated oligonucleotide initiator are served to capture and probe AFP molecules, respectively. Then, CD-tagged hairpin nucleic acids were introduced to trigger the HCR, in which the hairpin nucleic acid and oligonucleotide initiator are complementary. The interaction between CDs and the gold nanoisland film greatly improves the radiative decay rate, increases the quantum yield, and enhances the fluorescence emission of the CDs. Furthermore, the HCR provides secondary amplification of fluorescence intensity. Therefore, the MEF-capable immunohybridization reactions provide obvious advantages and result in exceptional sensitivity. In addition, the sandwich immunoassay method offers high specificity. The results show a wide linearity between the fluorescence intensity and AFP concentration over 5 orders of magnitude (0.0005-5 ng/mL), and the detection limit reaches as low as 94.3 fg/mL. This method offers advantages of high sensitivity and reliability, wide detection range, and versatile plasmonic chips, thus presenting an alternative for the technologies in biomedical analysis and clinical applications.


Assuntos
alfa-Fetoproteínas/química , Carbono , Ouro , Limite de Detecção , Nanoestruturas , Reprodutibilidade dos Testes
15.
J Chromatogr A ; 1493: 57-63, 2017 Apr 14.
Artigo em Inglês | MEDLINE | ID: mdl-28292517

RESUMO

A fully automated in-tube solid phase microextraction/liquid chromatography-post column derivatization-mass spectrometry (in-tube SPME/LC-PCD-MS) method was developed for the analysis of urinary hexanal and heptanal. Online in-tube SPME enabled effective enrichment of the low level aldehydes and elimination of matrix interferences. PCD could be simply realized by mixing the LC elute and hydroxylamine hydrochloride (HAHC) solution with just a tee. The peak broadening and loss in separation efficiency associated with post column dead-volume could be ignored and even completely eliminated by employing suitable PCD configuration. What's more, HAHC is commercially available and quite cheap, and shows no contaminations to MS. The entire procedure, including the extraction of aldehydes by in-tube SPME, LC separation, post column derivatization and MS detection were integrated together and completely automated, offering competitive advantages in terms of rapidity, economy, reproducibility and simplicity. The developed protocol was then successfully performed to determine lung cancer biomarkers (hexanal, heptanal) levels in urine samples.


Assuntos
Aldeídos/urina , Cromatografia Líquida/métodos , Espectrometria de Massas/métodos , Microextração em Fase Sólida/métodos , Biomarcadores Tumorais/urina , Cromatografia Líquida/instrumentação , Humanos , Neoplasias Pulmonares/urina , Espectrometria de Massas/instrumentação , Reprodutibilidade dos Testes , Microextração em Fase Sólida/instrumentação
16.
Biosens Bioelectron ; 94: 219-226, 2017 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-28285199

RESUMO

Detecting viable circulating tumor cells (CTCs) without disruption to their functions for in vitro culture and functional study could unravel the biology of metastasis and promote the development of personalized anti-tumor therapies. However, existing CTC detection approaches commonly include CTC isolation and subsequent destructive identification, which damages CTC viability and functions and generates substantial CTC loss. To address the challenge of efficiently detecting viable CTCs for functional study, we develop a nanosphere-based cell-friendly one-step strategy. Immunonanospheres with prominent magnetic/fluorescence properties and extraordinary stability in complex matrices enable simultaneous efficient magnetic capture and specific fluorescence labeling of tumor cells directly in whole blood. The collected cells with fluorescent tags can be reliably identified, free of the tedious and destructive manipulations from conventional CTC identification. Hence, as few as 5 tumor cells in ca. 1mL of whole blood can be efficiently detected via only 20min incubation, and this strategy also shows good reproducibility with the relative standard deviation (RSD) of 8.7%. Moreover, due to the time-saving and gentle processing and the minimum disruption of immunonanospheres to cells, 93.8±0.1% of detected tumor cells retain cell viability and proliferation ability with negligible changes of cell functions, capacitating functional study on cell migration, invasion and glucose uptake. Additionally, this strategy exhibits successful CTC detection in 10/10 peripheral blood samples of cancer patients. Therefore, this nanosphere-based cell-friendly one-step strategy enables viable CTC detection and further functional analyses, which will help to unravel tumor metastasis and guide treatment selection.


Assuntos
Técnicas Biossensoriais/métodos , Separação Celular/métodos , Neoplasias/sangue , Células Neoplásicas Circulantes/metabolismo , Humanos , Nanosferas/química , Neoplasias/patologia , Células Neoplásicas Circulantes/patologia
17.
Anal Chem ; 89(7): 4153-4160, 2017 04 04.
Artigo em Inglês | MEDLINE | ID: mdl-28271879

RESUMO

5-Methylcytosine (5-mC) is an important epigenetic mark that plays critical roles in a variety of cellular processes. To properly exert physiological functions, the distribution of 5-mC needs to be tightly controlled in both DNA and RNA. In addition to methyltransferase-mediated DNA and RNA methylation, premethylated nucleotides can be potentially incorporated into DNA and RNA during replication and transcription. To exclude the premodified nucleotides into DNA and RNA, endogenous 5-methyl-2'-deoxycytidine monophosphate (5-Me-dCMP) generated from nucleic acids metabolism can be enzymatically deaminated to thymidine monophosphate (TMP). Therefore, previous studies failed to detect 5-Me-dCMP or 5-methylcytidine monophosphate (5-Me-CMP) in cells. In the current study, we established a method by chemical labeling coupled with liquid chromatography-electrospray ionization mass spectrometry (LC-ESI-MS/MS) for sensitive and simultaneous determination of 10 nucleotides, including 5-Me-dCMP and 5-Me-CMP. As N,N-dimethyl-p-phenylenediamine (DMPA) was utilized for labeling, the detection sensitivities of nucleotides increased by 88-372-fold due to the introduction of a tertiary amino group and a hydrophobic moiety from DMPA. Using this method, we found that endogenous 5-Me-dCMP and 5-Me-CMP widely existed in cultured human cells, human tissues, and human urinary samples. The presence of endogenous 5-Me-dCMP and 5-Me-CMP indicates that deaminases may not fully deaminate these methylated nucleotides. Consequently, the remaining premethylated nucleosides could be converted to nucleoside triphosphates as building blocks for DNA and RNA synthesis. Furthermore, we found that the contents of 5-Me-dCMP and 5-Me-CMP exhibited significant decreases in renal carcinoma tissues and urine samples of lymphoma patients compared to their controls, probably due to more reutilization of methylated nucleotides in DNA and RNA synthesis. This study is, to the best of our knowledge, the first report for detecting endogenous 5-Me-dCMP and 5-Me-CMP in mammals. The detectable endogenous methylated nucleotides indicate the potential deleterious effects of premodified nucleotides on aberrant gene regulation in cancers.


Assuntos
5-Metilcitosina/química , DNA/análise , RNA/análise , Células HEK293 , Células HeLa , Humanos , Espectrometria de Massas , Metilação , Estrutura Molecular
18.
Biosens Bioelectron ; 90: 146-152, 2017 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-27886601

RESUMO

Direct analysis of biomolecules in complex biological samples remains a major challenge for fluorescence-based approaches due to the interference of background signals. Herein, we report an analytical methodology by exploiting a single low-cost near-infrared sub-nanosecond pulse laser to synchronously actualize optical trapping and two-photon excitation fluorescence for senstive detection of carcinoembryonic antigen (CEA) in buffer solution and human whole serum with no separation steps. The assay is performed by simultaneously trapping and exciting the same immune-conjugated microsphere fabricated with a sandwich immunization strategy. Since the signal is strictly limited in the region of a three-dimensional focal volume where the microsphere is trapped, no obvious background signal is found to contribute the detected signals and thus high signal-to-background data are obtained. As a proof-of-concept study, the constructed platform exhibits good specificity for CEA and the detection limit reaches as low as 8pg/mL (45 fM) with a wide linear range from 0.01 to 60ng/mL in the both cases. To investigate the potential application of this platform in clinical diagnosis, 15 cases of serum samples were analyzed with satisfactory results, which further confirm the applicability of this method.


Assuntos
Técnicas Biossensoriais , Antígeno Carcinoembrionário/isolamento & purificação , Pinças Ópticas , Antígeno Carcinoembrionário/sangue , Fluorescência , Humanos , Limite de Detecção , Fótons
19.
Biosens Bioelectron ; 85: 633-640, 2016 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-27240010

RESUMO

Hepatocellular carcinoma (HCC) is an awful threat to human health. Early-stage HCC may be detected by isolation of circulating tumor cells (CTCs) from peripheral blood samples, which is beneficial to the diagnosis and therapy. However, the extreme rarity and high heterogeneity of HCC CTCs have been restricting the relevant research. To achieve an efficient isolation, reliable detection and subtype analyses of heterogeneous HCC CTCs, herein, we present a cell sorting strategy based on anti-CD45 antibody-modified magnetic nanospheres. By this strategy, leukocyte depletion efficiency was up to 99.9% within 30min in mimic clinical samples, and the purity of the spiked HCC cells was improved 265-317-fold. Besides, the isolated HCC cells remained viable at 92.3% and could be directly recultured. Moreover, coupling the convenient, fast and effective cell sorting strategy with specific ICC identification via biomarkers AFP and GPC3, HCC CTCs were detectable in peripheral blood samples, showing the potential for HCC CTC detection in clinic. Notably, this immunomagnetic cell sorting strategy enabled isolating more heterogeneous HCC cells compared with the established EpCAM-based methods, and further achieved characterization of three different CTC subtypes from one clinical HCC blood sample, which may assist clinical HCC analyses such as prognosis or personalized treatment.


Assuntos
Carcinoma Hepatocelular/patologia , Separação Celular/métodos , Separação Imunomagnética/métodos , Neoplasias Hepáticas/patologia , Células Neoplásicas Circulantes/patologia , Anticorpos Imobilizados/química , Carcinoma Hepatocelular/sangue , Células Hep G2 , Humanos , Células Jurkat , Procedimentos de Redução de Leucócitos/métodos , Neoplasias Hepáticas/sangue , Nanosferas/química
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