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1.
Cytometry A ; 99(11): 1067-1078, 2021 11.
Artigo em Inglês | MEDLINE | ID: mdl-34328262

RESUMO

Neurological disorders affect hundreds of millions of people around the world, are often life-threatening, untreatable, and can result in debilitating symptoms. The high prevalence of these disorders, which feature biochemical or structural abnormalities in neuronal systems, has spurned innovations in both rapid and early detection to assist in the selection of appropriate treatment strategies to improve the patients' quality of life. Plasmonic nanoparticles (PNPs), a versatile and promising class of nanomaterials, are widely utilized in numerous imaging techniques, drug delivery systems, and biomarker detection methods. Recently, PNP-based nanoprobes have attracted considerable attention for the early diagnosis of neurological disorders. Gold nanoparticles (AuNPs), with high local surface plasmon resonance (LSPR) signals, have been particularly well exploited as probes for dynamic biomarker detection, with quantification sensitivity demonstrated down to the single-molecule level. In this review, we will discuss the possibilities of PNPs in the methodological development for rapid neurological disease identification. In addition, we will also describe a new digital cytometry method that combines dark-field imaging and machine learning for precise biomarker enumeration on single cells. The aim of this review is to attract researchers working on the future development of new plasmonic nanoprobe-based strategies for the diagnosis of neurological disorders.


Assuntos
Doenças do Sistema Nervoso Central , Nanopartículas Metálicas , Biomarcadores , Ouro , Humanos , Qualidade de Vida , Ressonância de Plasmônio de Superfície
2.
Int J Mol Sci ; 22(4)2021 Feb 10.
Artigo em Inglês | MEDLINE | ID: mdl-33578653

RESUMO

We present a template-assisted method for synthesizing nanogap shell structures for biomolecular detections based on surface-enhanced Raman scattering. The interior nanogap-containing a silver shell structure, referred to as a silver nanogap shell (Ag NGS), was fabricated on silver nanoparticles (Ag NPs)-coated silica, by adsorbing small aromatic thiol molecules on the Ag NPs. The Ag NGSs showed a high enhancement factor and good signal uniformity, using 785-nm excitation. We performed in vitro immunoassays using a prostate-specific antigen as a model cancer biomarker with a detection limit of 2 pg/mL. To demonstrate the versatility of Ag NGS nanoprobes, extracellular duplex surface-enhanced Raman scattering (SERS) imaging was also performed to evaluate the co-expression of cancer biomarkers, human epidermal growth factor-2 (HER2) and epidermal growth factor receptor (EGFR), in a non-small cell lung cancer cell line (H522). Developing highly sensitive Ag NGS nanoprobes that enable multiplex biomolecular detection and imaging can open up new possibilities for point-of-care diagnostics and provide appropriate treatment options and prognosis.


Assuntos
Carcinoma Pulmonar de Células não Pequenas/diagnóstico , Neoplasias Pulmonares/diagnóstico , Nanopartículas Metálicas/química , Receptor ErbB-2/análise , Prata/química , Biomarcadores Tumorais/análise , Linhagem Celular Tumoral , Receptores ErbB/análise , Humanos , Nanopartículas Metálicas/ultraestrutura , Análise Espectral Raman/métodos
3.
J Ovarian Res ; 15(1): 28, 2022 Feb 26.
Artigo em Inglês | MEDLINE | ID: mdl-35219339

RESUMO

BACKGROUND: Measurement of serum CA125, an antigenic fragment of human mucin 16 (MUC16), is used to monitor the clinical progression of epithelial ovarian cancer (EOC). However, rather than simply a passive marker reflecting tumor burden, MUC16 may have a more active role by binding to immune cells and altering their tumor response. We developed a research tool to measure MUC16-binding to the surfaces of peripheral blood mononuclear cell (PBMC) subtypes and tested its research value using specimens collected serially from a woman being treated for high grade serous EOC. METHODS: Cryopreserved PBMCs were mixed with anti-CA125 antibody-labeled plasmonic gold nanoparticles (PNPs) to detect cell surface MUC16-binding along with fluorescent stains to identify B cells, NK cells, NK-T cells, T cells, and monocytes. From 3D darkfield images, a computer algorithm was applied to enumerate PNP-binding and fluorescence microscopy to identify cell lineage. Average MUC16-binding was determined by fitting a Poisson distribution to PNP-counts across similar cell types. MUC16-binding to cell types was correlated with treatment details, CA125 levels, and complete blood count (CBC) data. RESULTS: Over a 21-month period, monocytes had the highest level of MUC16-binding which was positively correlated with serum CA125 and inversely correlated with circulating monocyte and lymphocyte counts. Fluctuations of PNP-binding to NK cells were associated temporally with types of chemotherapy and surgical events. Levels of MUC16 bound to NK cells were positively correlated with levels of MUC16 bound to T and NK-T cells and inversely correlated with circulating platelets. CONCLUSIONS: Assessment of MUC16-binding among cryopreserved PBMC cell types can be accomplished using darkfield and fluorescence microscopy. Correlations observed between level of binding by cell type with serum CA125, CBC data, and treatment details suggest that the new techniques may offer novel insights into EOC's clinical course.


Assuntos
Antígeno Ca-125/sangue , Carcinoma Epitelial do Ovário/sangue , Leucócitos Mononucleares/metabolismo , Proteínas de Membrana/sangue , Neoplasias Ovarianas/sangue , Algoritmos , Anticorpos , Antígeno Ca-125/imunologia , Carcinoma Epitelial do Ovário/patologia , Carcinoma Epitelial do Ovário/terapia , Feminino , Corantes Fluorescentes , Ouro , Humanos , Células Matadoras Naturais/metabolismo , Contagem de Linfócitos , Proteínas de Membrana/imunologia , Microscopia de Fluorescência/métodos , Monócitos/metabolismo , Nanopartículas , Células T Matadoras Naturais/metabolismo , Gradação de Tumores , Neoplasias Ovarianas/patologia , Neoplasias Ovarianas/terapia , Contagem de Plaquetas
4.
Cancers (Basel) ; 13(9)2021 Apr 25.
Artigo em Inglês | MEDLINE | ID: mdl-33922973

RESUMO

MUC16, a sialomucin that contains the ovarian cancer biomarker CA125, binds at low abundance to leucocytes via the immune receptor, Siglec-9. Conventional fluorescence-based imaging techniques lack the sensitivity to assess this low-abundance event, prompting us to develop a novel "digital" optical cytometry technique for qualitative and quantitative assessment of CA125 binding to peripheral blood mononuclear cells (PBMC). Plasmonic nanoparticle labeled detection antibody allows assessment of CA125 at the near-single molecule level when bound to specific immune cell lineages that are simultaneously identified using multiparameter fluorescence imaging. Image analysis and deep learning were used to quantify CA125 per each cell lineage. PBMC from treatment naïve ovarian cancer patients (N = 14) showed higher cell surface abundance of CA125 on the aggregate PBMC population as well as on NK (p = 0.013), T (p < 0.001) and B cells (p = 0.024) compared to circulating lymphocytes of healthy donors (N = 7). Differences in CA125 binding to monocytes or NK-T cells between the two cohorts were not significant. There was no correlation between the PBMC-bound and serum levels of CA125, suggesting that these two compartments are not in stoichiometric equilibrium. Understanding where and how subset-specific cell-bound surface CA125 takes place may provide guidance towards a new diagnostic biomarker in ovarian cancer.

5.
Sci Rep ; 10(1): 5360, 2020 03 24.
Artigo em Inglês | MEDLINE | ID: mdl-32210332

RESUMO

Understanding a drug candidate's pharmacokinetic (PK) parameters is a challenging but essential aspect of drug development. Investigating the penetration and distribution of a topical drug's active pharmaceutical ingredient (API) allows for evaluating drug delivery and efficacy, which is necessary to ensure drug viability. A topical gel (BPX-05) was recently developed to treat moderate to severe acne vulgaris by directly delivering the combination of the topical antibiotic minocycline and the retinoid tazarotene to the pilosebaceous unit of the dermis. In order to evaluate the uptake of APIs within human facial skin and confirm accurate drug delivery, a selective visualization method to monitor and quantify local drug distributions within the skin was developed. This approach uses fluorescence lifetime imaging microscopy (FLIM) paired with a multicomponent phasor analysis algorithm to visualize drug localization. As minocycline and tazarotene have distinct fluorescence lifetimes from the lifetime of the skin's autofluorescence, these two APIs are viable targets for distinct visualization via FLIM. Here, we demonstrate that the analysis of the resulting FLIM output can be used to determine local distributions of minocycline and tazarotene within the skin. This approach is generalizable and can be applied to many multicomponent fluorescence lifetime imaging targets that require cellular resolution and molecular specificity.


Assuntos
Microscopia de Fluorescência/métodos , Minociclina/farmacocinética , Ácidos Nicotínicos/farmacocinética , Pele/efeitos dos fármacos , Administração Tópica , Algoritmos , Fármacos Dermatológicos/administração & dosagem , Fármacos Dermatológicos/farmacocinética , Combinação de Medicamentos , Face , Fluorescência , Géis/administração & dosagem , Humanos , Processamento de Imagem Assistida por Computador , Minociclina/administração & dosagem , Imagem Molecular/métodos , Ácidos Nicotínicos/administração & dosagem , Pele/química , Pele/diagnóstico por imagem , Espectrometria de Fluorescência
6.
ACS Sens ; 5(9): 2772-2782, 2020 09 25.
Artigo em Inglês | MEDLINE | ID: mdl-32847358

RESUMO

Although levels of the circulating ovarian cancer marker (CA125) can distinguish ovarian masses that are likely to be malignant and correlate with severity of disease, serum CA125 has not proved useful in general population screening. Recently, cell culture studies have indicated that MUC16 may bind to the Siglec-9 receptor on natural killer (NK) cells where it downregulates the cytotoxicity of NK cells, allowing ovarian cancer cells to evade immune surveillance. We present evidence that the presence of MUC16 can be locally visualized and imaged on the surface of peripheral blood mononuclear cells (PBMCs) in ovarian cancer via a novel "digital" cytometry technique that incorporates: (i) OC125 monoclonal antibody-conjugated gold nanoparticles as optical nanoprobes, (ii) a high contrast dark-field microscopy system to detect PBMC-bound gold nanoparticles, and (iii) a computational algorithm for automatic counting of these nanoparticles to estimate the quantity of surface-bound MUC16. The quantitative detection of our technique was successfully demonstrated by discriminating clones of the ovarian cancer cell line, OVCAR3, based on low, intermediate, and high expression levels of MUC16. Additionally, PBMC surface-bound MUC16 was tracked in an ovarian cancer patient over a 17 month period; the results suggest that the binding of MUC16 on the surface of immune cells may play an early indicator for recurrent metastasis 6 months before computational tomography-based clinical diagnosis. We also demonstrate that the levels of surface-bound MUC16 on PBMCs from five ovarian cancer patients were greater than those from five healthy controls.


Assuntos
Nanopartículas Metálicas , Neoplasias Ovarianas , Apoptose , Antígeno Ca-125 , Linhagem Celular Tumoral , Feminino , Ouro , Humanos , Leucócitos Mononucleares , Proteínas de Membrana
7.
Adv Drug Deliv Rev ; 153: 147-168, 2020 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-32217069

RESUMO

Understanding the delivery and diffusion of topically-applied drugs on human skin is of paramount importance in both pharmaceutical and cosmetics research. This information is critical in early stages of drug development and allows the identification of the most promising ingredients delivered at optimal concentrations to their target skin compartments. Different skin imaging methods, invasive and non-invasive, are available to characterize and quantify the spatiotemporal distribution of a drug within ex vivo and in vivo human skin. The first part of this review detailed invasive imaging methods (autoradiography, MALDI and SIMS). This second part reviews non-invasive imaging methods that can be applied in vivo: i) fluorescence (conventional, confocal, and multiphoton) and second harmonic generation microscopies and ii) vibrational spectroscopic imaging methods (infrared, confocal Raman, and coherent Raman scattering microscopies). Finally, a flow chart for the selection of imaging methods is presented to guide human skin ex vivo and in vivo drug delivery studies.


Assuntos
Fármacos Dermatológicos/farmacocinética , Sistemas de Liberação de Medicamentos/métodos , Imagem Óptica/métodos , Absorção Cutânea/fisiologia , Análise Espectral/métodos , Animais , Fármacos Dermatológicos/administração & dosagem , Humanos , Modelos Animais , Modelos Biológicos , Imagem Óptica/normas , Pele/metabolismo , Análise Espectral/normas
8.
Adv Drug Deliv Rev ; 153: 137-146, 2020 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-31778729

RESUMO

In this two-part review we present an up-to-date description of different imaging methods available to map the localization of drugs on skin as a complement of established ex-vivo absorption studies. This first part deals with invasive methods which are grouped in two classes according to their underlying principles: i) methods using radioactivity such as autoradiography and ii) mass spectrometry methods such as MALDI and SIMS. For each method, a description of the principle is given along with example applications of imaging and quantifying drug delivery in human skin. Thanks to these techniques a better assessment of the fate of drugs is obtained: its localization on a particular skin structure, its potential accumulation, etc. A critical comparison in terms of capabilities, sensitivity and practical applicability is included that will help the reader to select the most appropriate technique depending on the particular problem to be solved.


Assuntos
Autorradiografia/métodos , Fármacos Dermatológicos/farmacocinética , Sistemas de Liberação de Medicamentos/métodos , Espectrometria de Massas/métodos , Absorção Cutânea/fisiologia , Administração Cutânea , Autorradiografia/normas , Fármacos Dermatológicos/administração & dosagem , Humanos , Espectrometria de Massas/normas , Modelos Biológicos , Pele/metabolismo , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/normas
9.
Chem Commun (Camb) ; 55(18): 2700-2703, 2019 Feb 26.
Artigo em Inglês | MEDLINE | ID: mdl-30756101

RESUMO

We developed a ready-to-read on-bead peptide encoding method for high-throughput screening bioassays. With two-dimensional surface-enhanced Raman scattering nano-identifiers (2D-SERS IDs) which are concurrently labelled with two SERS codes (coupling steps and kinds of amino acid), we could possibly generate more than 10 trillion codes with only 30 Raman label compounds.


Assuntos
Peptídeos/análise , Análise Espectral Raman/métodos , Aminoácidos/química , Ensaios de Triagem em Larga Escala , Nanopartículas/química , Peptídeos/química , Dióxido de Silício/química
10.
Adv Healthc Mater ; 8(15): e1900035, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31165556

RESUMO

Efficient and timely delivery of vaccine antigens to the secondary lymphoid tissue is crucial to induce protective immune responses by vaccination. However, determining the longitudinal biodistribution of injected vaccines in the body has been a challenge. Here, the near-infrared (NIR) fluorescence imaging is reported that can efficiently enable the trafficking and biodistribution of vaccines in real time. Zwitterionic NIR fluorophores are conjugated on the surface of model vaccines and tracked the fate of bioconjugated vaccines after intradermal administration. Using an NIR fluorescence imaging system, it is possible to obtain time-course imaging of vaccine trafficking through the lymphatics, observing notable uptake in lymph nodes with minimal nonspecific tissue interactions. Flow cytometry analysis confirmed that the uptake in lymph nodes by antigen presenting cells was highly dependent on the hydrodynamic diameter of vaccines. These results demonstrate that the combination of a real-time NIR fluorescence imaging system and zwitterionic fluorophores is a powerful tool to determine the fate of vaccine antigens. Since such non-specific vaccine uptake causes serious adverse reactions, this method is not only useful for optimization of vaccine design, but also for safety evaluation of clinical vaccine candidates.


Assuntos
Nanopartículas/química , Espectroscopia de Luz Próxima ao Infravermelho/métodos , Vacinas/farmacocinética , Animais , Células Apresentadoras de Antígenos/citologia , Células Apresentadoras de Antígenos/metabolismo , Corantes Fluorescentes/metabolismo , Íons , Linfonodos/metabolismo , Linfonodos/patologia , Camundongos , Modelos Biológicos , Ovalbumina/química , Ovalbumina/imunologia , Compostos de Amônio Quaternário/química , Dióxido de Silício/química , Ácidos Sulfônicos/química , Distribuição Tecidual , Vacinas/química , Vacinas/imunologia
11.
Biomed Opt Express ; 9(7): 3434-3448, 2018 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-29984108

RESUMO

Acne vulgaris is a common chronic skin disease in young adults caused by infection of the pilosebaceous unit, resulting in pimples and possibly permanent scarring on the skin. Minocycline, a common antibiotic, has been widely utilized as a systemic antimicrobial treatment for acne via oral administration. Recently, a topical minocycline gel (BPX-01) was developed to directly deliver minocycline through the epidermis and into the pilosebaceous unit to achieve localized treatment with lower doses of drug. As the effectiveness of the drug is directly related to its successful delivery, there is a need to evaluate the pharmacokinetics at the cellular level within tissue. Advantageously, minocycline is naturally fluorescent and can be directly visualized using microscopy-based approaches. Due to high endogenous autofluorescence, however, imaging of weakly emitting fluorescent molecules such as minocycline in skin tissue can be challenging. Here, we demonstrate a method for the selective visualization of minocycline within human skin tissue by utilizing two-photon excitation fluorescence (TPEF) microscopy and fluorescence lifetime imaging microscopy (FLIM). To demonstrate the feasibility of this approach, ex vivo human facial skin samples treated with various concentrations of BPX-01 were investigated. From the TPEF analysis, we were able to visualize relatively high levels of drug uptake within facial skin. However, minocycline fluorescence could be overwhelmed by endogenous fluorescence that complicates TPEF quantitative analysis, making FLIM more advantageous for visualizing drug uptake. Importantly, we found a unique signature of minocycline uptake via FLIM analysis that enabled the successful differentiation of the drug and enabled the extraction of drug local distribution from the endogenous fluorescence using a non-Euclidean phasor analysis method. Based on these results, we believe that the drug local distribution visualization method using TPEF and FLIM with phasor analysis can play an important role in studying the pharmacokinetics and pharmacodynamics of a topically applicable drug.

12.
Biomed Opt Express ; 9(11): 5400-5418, 2018 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-30460136

RESUMO

In pharmacokinetic studies of topical drugs, fluorescence microscopy methods can enable the direct visualization and quantification of fluorescent drugs within the skin. One potential limitation of this approach, however, is the strong endogenous fluorescence of skin tissues that makes straightforward identification of specific drug molecules challenging. To study this effect and quantify endogenous skin fluorescence in the context of topical pharmacokinetics, an integrating sphere-based screening tool was designed to collect fluorescence yield data from human skin specimens. Such information could be utilized to select specific donors in the investigation of drug uptake and distribution. Results indicated human facial skin specimens from a group of more than 35 individuals exhibited an at least 6-fold difference in endogenous fluorescence. In visualizing drug distributions, the negative impact of autofluorescence could be exacerbated in cases where there are overlapping spatial distributions or spectral emission profiles between endogenous fluorophores and the exogenous fluorophore of interest. We demonstrated the feasibility of this approach in measuring the range of tissue endogenous fluorescence and selecting specimens for the study of drug pharmacokinetics with fluorescence microscopy.

13.
Biomed Opt Express ; 9(12): 6425-6443, 2018 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-31065440

RESUMO

The most superficial layer of the epidermis, the stratum corneum, plays a crucial role in retaining hydration; if its structure or composition is compromised, dry skin may result as a consequence of poor water retention. Dry skin is typically treated with topical application of humectant agents that attract water into the skin. Corneometry, the industry standard for measuring skin hydration, works by assessing the bulk electrical properties of skin. However, this technique samples a large volume of tissue and thus does not resolve the biochemical changes that occur at the cellular level that may underlie mechanisms of dry skin. These limitations can be addressed using coherent Raman scattering (CRS) microscopy to probe the intrinsic vibrational modes of chemical groups such as lipids and water. In the present study, ex vivo human skin explants undergoing dehydration and humectant-induced rehydration were measured via CRS imaging and corneometry. Corneometry data and chemically specific images were obtained from the stratum corneum of each patient sample at each timepoint. The resulting data was statistically analyzed using linear mixed effect model regression analysis. The cellular imaging data revealed water loss in the stratum corneum during dehydration that was correlated with corneometer readings. Interestingly, the imaging data and corneometer readings show differences under the experimental rehydration conditions. The rehydration results suggest that hydration restored by the humectant agents may not be retained by the corneocytes in the ex vivo model system. Given the complementary nature of corneometry, a bulk assessment tool, and CRS microscopy, a modality with subcellular resolution implemented here in an en-face tissue imaging setup, these techniques can be used to measure uptake and efficacy of topical compounds in order to better understand their mode of action and improve therapeutic applications.

14.
Adv Healthc Mater ; 7(4)2018 02.
Artigo em Inglês | MEDLINE | ID: mdl-29195032

RESUMO

Immunotargeting ability of antibodies may show significant difference between in vitro and in vivo. To select antibody leads with high affinity and specificity, it is necessary to perform in vivo validation of antibody candidates following in vitro antibody screening. Herein, a robust in vivo validation of anti-tetraspanin-8 antibody candidates against human colon cancer using ratiometric quantification method is reported. The validation is performed on a single mouse and analyzed by multiplexed surface-enhanced Raman scattering using ultrasensitive and near infrared (NIR)-active surface-enhanced resonance Raman scattering nanoprobes (NIR-SERRS dots). The NIR-SERRS dots are composed of NIR-active labels and Au/Ag hollow-shell assembled silica nanospheres. A 93% of NIR-SERRS dots is detectable at a single-particle level and signal intensity is 100-fold stronger than that from nonresonant molecule-labeled spherical Au NPs (80 nm). The result of SERRS-based antibody validation is comparable to that of the conventional method using single-photon-emission computed tomography. The NIR-SERRS-based strategy is an alternate validation method which provides cost-effective and accurate multiplexing measurements for antibody-based drug development.


Assuntos
Anticorpos/química , Neoplasias do Colo/diagnóstico , Corantes Fluorescentes/química , Pontos Quânticos/química , Animais , Linhagem Celular Tumoral , Neoplasias do Colo/diagnóstico por imagem , Ouro/química , Humanos , Radioisótopos do Iodo/química , Camundongos , Camundongos Nus , Tomografia por Emissão de Pósitrons combinada à Tomografia Computadorizada , Dióxido de Silício/química , Prata/química , Análise Espectral Raman
15.
Nanoscale ; 9(7): 2548-2555, 2017 Feb 16.
Artigo em Inglês | MEDLINE | ID: mdl-28150822

RESUMO

Antibody-conjugated nanoparticles (NPs) have attracted great attention in diagnostic and therapeutic applications due to their high sensitivity and specificity for biotargets, as well as their wide applicability. Unfortunately, these features are significantly affected by antibody conjugation methods in terms of conjugation efficiency, orientation of the target binding site in the antibody, and denaturation during chemical conjugation reactions. Furthermore, the number of conjugated antibodies on each NP and the overall targeting efficacy are critical factors for a quantitative bioassay with antibody-conjugated NPs. Herein, we report a versatile and oriented antibody conjugation method using copper-free click chemistry. Moreover, the number of conjugated antibodies and their binding capacity were quantitatively and experimentally evaluated using fluorescently-labeled antibodies and antigens. The strong binding capability of antibody-conjugated NPs prepared using the copper-free click chemistry-based conjugation strategy was 8 times superior to the binding capability seen following the use of the EDC/NHS-coupling method. Additionally, the versatility of the developed antibody conjugation method was also demonstrated by conjugation of the antibody to three kinds of silica-encapsulated NPs.


Assuntos
Anticorpos/química , Imunoconjugados/química , Nanopartículas/química , Química Click , Dióxido de Silício
16.
Sci Rep ; 7(1): 1035, 2017 04 21.
Artigo em Inglês | MEDLINE | ID: mdl-28432289

RESUMO

Fluorescence endomicroscopy provides quick access to molecular targets, while Raman spectroscopy allows the detection of multiple molecular targets. Using a simultaneous fluorescence-Raman endoscopic system (FRES), we herein demonstrate its potential in cancer diagnosis in an orthotopically induced colorectal cancer (CRC) xenograft model. In the model, epidermal growth factor receptor (EGFR) and vascular endothelial growth factor (VEGF) were targeted with antibody-conjugated fluorescence and surface-enhanced Raman scattering (F-SERS) dots. FRES demonstrated fast signal detection and multiplex targeting ability using fluorescence and Raman signals to detect the F-SERS dots. In addition, FRES showed a multiplex targeting ability even on a subcentimeter-sized CRC after spraying with a dose of 50 µg F-SERS dots. In conclusion, molecular characteristics of tumor cells (EGFR in cancer cell membranes) and tumor microenvironments (VEGF in the extracellular matrix) could be simultaneously investigated when performing a colonoscopy.


Assuntos
Neoplasias Colorretais/diagnóstico por imagem , Endoscopia Gastrointestinal/métodos , Receptores ErbB/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo , Animais , Neoplasias Colorretais/metabolismo , Células HT29 , Humanos , Camundongos , Microscopia de Fluorescência , Imagem Molecular/métodos , Transplante de Neoplasias , Análise Espectral Raman
17.
Rev Sci Instrum ; 86(5): 055004, 2015 May.
Artigo em Inglês | MEDLINE | ID: mdl-26026551

RESUMO

Surface-enhanced Raman scattering techniques have been widely used for bioanalysis due to its high sensitivity and multiplex capacity. However, the point-scanning method using a micro-Raman system, which is the most common method in the literature, has a disadvantage of extremely long measurement time for on-chip immunoassay adopting a large chip area of approximately 1-mm scale and confocal beam point of ca. 1-µm size. Alternative methods such as sampled spot scan with high confocality and large-area scan method with enlarged field of view and low confocality have been utilized in order to minimize the measurement time practically. In this study, we analyzed the two methods in respect of signal-to-noise ratio and sampling-led signal fluctuations to obtain insights into a fast and reliable readout strategy. On this basis, we proposed a methodology for fast and reliable quantitative measurement of the whole chip area. The proposed method adopted a raster scan covering a full area of 100 µm × 100 µm region as a proof-of-concept experiment while accumulating signals in the CCD detector for single spectrum per frame. One single scan with 10 s over 100 µm × 100 µm area yielded much higher sensitivity compared to sampled spot scanning measurements and no signal fluctuations attributed to sampled spot scan. This readout method is able to serve as one of key technologies that will bring quantitative multiplexed detection and analysis into practice.


Assuntos
Nanoestruturas/química , Análise Espectral Raman/métodos , Imunoensaio/instrumentação , Razão Sinal-Ruído
18.
Sci Rep ; 5: 9455, 2015 Mar 30.
Artigo em Inglês | MEDLINE | ID: mdl-25820115

RESUMO

Optical endoscopic imaging, which was recently equipped with bioluminescence, fluorescence, and Raman scattering, allows minimally invasive real-time detection of pathologies on the surface of hollow organs. To characterize pathologic lesions in a multiplexed way, we developed a dual modal fluorescence-Raman endomicroscopic system (FRES), which used fluorescence and surface-enhanced Raman scattering nanoprobes (F-SERS dots). Real-time, in vivo, and multiple target detection of a specific cancer was successful, based on the fast imaging capability of fluorescence signals and the multiplex capability of simultaneously detected SERS signals using an optical fiber bundle for intraoperative endoscopic system. Human epidermal growth factor receptor 2 (HER2) and epidermal growth factor receptor (EGFR) on the breast cancer xenografts in a mouse orthotopic model were successfully detected in a multiplexed way, illustrating the potential of FRES as a molecular diagnostic instrument that enables real-time tumor characterization of receptors during routine endoscopic procedures.


Assuntos
Neoplasias da Mama/diagnóstico , Receptores ErbB/biossíntese , Patologia Molecular , Receptor ErbB-2/biossíntese , Animais , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Endoscopia , Receptores ErbB/genética , Feminino , Humanos , Camundongos , Receptor ErbB-2/genética , Espectrometria de Fluorescência , Análise Espectral Raman , Ensaios Antitumorais Modelo de Xenoenxerto
19.
Sci Rep ; 5: 10144, 2015 May 28.
Artigo em Inglês | MEDLINE | ID: mdl-26017924

RESUMO

Recently, preparation and screening of compound libraries remain one of the most challenging tasks in drug discovery, biomarker detection, and biomolecular profiling processes. So far, several distinct encoding/decoding methods such as chemical encoding, graphical encoding, and optical encoding have been reported to identify those libraries. In this paper, a simple and efficient surface-enhanced Raman spectroscopic (SERS) barcoding method using highly sensitive SERS nanoparticles (SERS ID) is presented. The 44 kinds of SERS IDs were able to generate simple codes and could possibly generate more than one million kinds of codes by incorporating combinations of different SERS IDs. The barcoding method exhibited high stability and reliability under bioassay conditions. The SERS ID encoding based screening platform can identify the peptide ligand on the bead and also quantify its binding affinity for specific protein. We believe that our SERS barcoding technology is a promising method in the screening of one-bead-one-compound (OBOC) libraries for drug discovery.


Assuntos
Peptídeos/análise , Análise Espectral Raman , Algoritmos , Ligantes , Nanopartículas/química , Biblioteca de Peptídeos , Peptídeos/química , Peptídeos/metabolismo , Ligação Proteica , Dióxido de Silício/química
20.
Biomaterials ; 45: 81-92, 2015 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-25662498

RESUMO

Au/Ag hollow nanoshells (AuHNSs) were developed as multifunctional therapeutic agents for effective, targeted, photothermally induced drug delivery under near-infrared (NIR) light. AuHNSs were synthesized by galvanic replacement reaction. We further conjugated antibodies against the epidermal growth factor receptor (EGFR) to the PEGylated AuHNS, followed by loading with the antitumor drug doxorubicin (AuHNS-EGFR-DOX) for lung cancer treatment. AuHNSs showed similar photothermal efficiency to gold nanorods under optimized NIR laser power. The targeting of AuHNS-EGFR-DOX was confirmed by light-scattering images of A549 cells, and doxorubicin release from the AuHNSs was evaluated under low pH and NIR-irradiated conditions. Multifunctional AuHNS-EGFR-DOX induced photothermal ablation of the targeted lung cancer cells and rapid doxorubicin release following irradiation with NIR laser. Furthermore, we evaluated the effectiveness of AuHNS-EGFR-DOX drug delivery by comparing two drug delivery methods: receptor-mediated endocytosis and cell-surface targeting. Accumulation of the AuHNS-EGFR-DOX on the cell surfaces by targeting EGFR turned out to be more effective for lung cancer treatments than uptake of AuHNS-EGFR-DOX. Taken together, our data suggest a new and optimal method of NIR-induced drug release via the accumulation of targeted AuHNS-EGFR-DOX on cancer cell membranes.


Assuntos
Sistemas de Liberação de Medicamentos , Liberação Controlada de Fármacos , Ouro/química , Hipertermia Induzida , Neoplasias Pulmonares/patologia , Fototerapia , Prata/química , Linhagem Celular Tumoral , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Doxorrubicina/farmacologia , Endocitose/efeitos dos fármacos , Receptores ErbB/metabolismo , Humanos , Nanoconchas/química , Nanoconchas/ultraestrutura , Polietilenoglicóis/química , Reprodutibilidade dos Testes , Espectrofotometria Infravermelho , Espectrofotometria Ultravioleta
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