RESUMEN
PURPOSE: This study aimed to evaluate the usefulness of the LDN-PSA (LacdiNAc-glycosylated-prostate specific antigen) in detecting clinically significant prostate cancer in patients suspected of having clinically significant prostate cancer on multiparametric magnetic resonance imaging. MATERIALS AND METHODS: Patients with prostate specific antigen levels ranging between 3.0 ng/mL and 20 ng/mL and suspicious lesions with PI-RADS (Prostate Imaging-Reporting and Data System) category ≥3 were included prospectively. The LDN-PSA was measured using an automated 2-step Wisteria floribunda agglutinin lectin-anti-prostate specific antigen antibody sandwich immunoassay. RESULTS: Two hundred four patients were included. Clinically significant prostate cancer was detected in 105 patients. On multivariable logistic regression analysis, prostate specific antigen density (OR 1.61, P = .010), LDN-PSAD (OR 1.04, P = .012), highest PI-RADS category (3 vs 4, 5; OR 14.5, P < .0001), and location of the lesion with highest PI-RADS category (transition zone vs peripheral zone) (OR 0.34, P = .009) were significant risk factors for detecting clinically significant prostate cancer. Among the patients with the highest PI-RADS category 3 (n=113), clinically significant prostate cancer was detected in 28 patients. On multivariable logistic regression analysis to predict the detection of clinically significant prostate cancer in patients with the highest PI-RADS category 3, age (OR 1.10, P = .026) and LDN-PSAD (OR 1.07, P < .0001) were risk factors for detecting clinically significant prostate cancer. CONCLUSIONS: LDN-PSAD would be a biomarker for detecting clinically significant prostate cancer in patients with prostate specific antigen levels ≤20 ng/mL and suspicious lesions with PI-RADS category ≥3. The use of LDN-PSAD as an adjunct to the use of prostate specific antigen levels would avoid unnecessary biopsies in patients with the highest PI-RADS category 3. Multi-institutional studies with large population are recommended.
Asunto(s)
Antígeno Prostático Específico , Neoplasias de la Próstata , Humanos , Masculino , Imagen por Resonancia Magnética , Neoplasias de la Próstata/diagnóstico por imagenRESUMEN
Wisteria floribunda agglutinin (WFA)-reactive ceruloplasmin (CP) is a candidate marker for ovarian clear cell carcinoma (CCC) reported in our previous paper. Herein, a new measurement system was developed to investigate its potential as a serum marker for CCC. Site-specific glycome analysis using liquid chromatography/mass spectrometry showed that WFA-CP from CCC binds to WFA via the GalNAcß1,4GlcNAc (LDN) structure. We used mutant recombinant WFA (rWFA), which has a high specificity to the LDN structure, instead of native WFA, to increase the specificity of the serum sample measurement. To improve the sensitivity, we used a surface plasmon field-enhanced fluorescence spectroscopy immunoassay system, which is approximately 100 times more sensitive than the conventional sandwich enzyme-linked immunosorbent assay system. With these two improvements, the specificity and sensitivity of the serum rWFA-CP measurement were dramatically improved, clearly distinguishing CCC from endometrioma, from which CCC originates. This rWFA-CP assay can be used clinically for the serodiagnosis of early-stage CCC, which is difficult to detect with existing serum markers.
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Carcinoma , Endometriosis , Antígenos de Neoplasias , Biomarcadores , Ceruloplasmina/metabolismo , Endometriosis/diagnóstico , Humanos , Cirrosis Hepática/diagnóstico , Lectinas de Plantas/química , Receptores N-Acetilglucosamina/metabolismoRESUMEN
To reduce unnecessary prostate biopsies (Pbx), better discrimination is needed. To identify clinically significant prostate cancer (CSPC) we determined the performance of LacdiNAc-glycosylated prostate-specific antigen (LDN-PSA) and LDN-PSA normalized by prostate volume (LDN-PSAD). We retrospectively measured LDN-PSA, total PSA (tPSA), and free PSA/tPSA (F/T PSA) values in 718 men who underwent a Pbx in 3 academic urology clinics in Japan and Canada (Pbx cohort) and in 174 PC patients who subsequently underwent radical prostatectomy in Australia (preop-PSA cohort). The assays were evaluated using the area under the receiver operating characteristics curve (AUC) and decision curve analyses to discriminate CSPC. In the Pbx cohort, LDN-PSAD (AUC 0.860) provided significantly better clinical performance for discriminating CSPC compared with LDN-PSA (AUC 0.827, P = 0.0024), PSAD (AUC 0.809, P < 0.0001), tPSA (AUC 0.712, P < 0.0001), and F/T PSA (AUC 0.661, P < 0.0001). The decision curve analysis showed that using a risk threshold of 20% and adding LDN-PSA and LDN-PSAD to the base model (age, digital rectal examination status, tPSA, and F/T PSA) permitted avoidance of even more biopsies without missing CSPC (9.89% and 18.11%, respectively vs 2.23% [base model]). In the preop-PSA cohort, LDN-PSA values positively correlated with tumor volume and tPSA and were significantly higher in pT3, pathological Gleason score ≥ 7. Limitations include limited sample size, retrospective nature, and no family history information prior to biopsy. LacdiNAc-glycosylated PSA is significantly better than the conventional PSA test in identifying patients with CSPC. This study was approved by the ethics committee of each institution ("The Study about Carbohydrate Structure Change in Urological Disease"; approval no. 2014-195).
Asunto(s)
Lactosa/análogos & derivados , Próstata/metabolismo , Próstata/patología , Neoplasias de la Próstata/metabolismo , Neoplasias de la Próstata/patología , Anciano , Glicosilación , Humanos , Lactosa/metabolismo , Masculino , Persona de Mediana Edad , Clasificación del Tumor/métodos , Antígeno Prostático Específico , Curva ROC , Estudios Retrospectivos , Carga Tumoral/fisiologíaRESUMEN
Wisteria floribunda agglutinin (WFA) preferably binds to LacdiNAc glycans, and its reactivity is associated with tumor progression. The aim of this study to examine whether the serum LacdiNAc carrying prostate-specific antigen-glycosylation isomer (PSA-Gi) and WFA-reactivity of tumor tissue can be applied as a diagnostic and prognostic marker of prostate cancer (PCa). Between 2007 and 2016, serum PSA-Gi levels before prostate biopsy (Pbx) were measured in 184 biopsy-proven benign prostatic hyperplasia patients and 244 PCa patients using an automated lectin-antibody immunoassay. WFA-reactivity on tumor was analyzed in 260 radical prostatectomy (RP) patients. Diagnostic and prognostic performance of serum PSA-Gi was evaluated using area under the receiver-operator characteristic curve (AUC). Prognostic performance of WFA-reactivity on tumor was evaluated via Cox proportional hazards regression analysis and nomogram. The AUC of serum PSA-Gi detecting PCa and predicting Pbx Grade Group (GG) 3 and GG ≥ 3 after RP was much higher than those of conventional PSA. Multivariate analysis showed that WFA-reactivity on prostate tumor was an independent risk factor of PSA recurrence. The nomogram was a strong model for predicting PSA-free survival provability with a c-index ≥0.7. Serum PSA-Gi levels and WFA-reactivity on prostate tumor may be a novel diagnostic and pre- and post-operative prognostic biomarkers of PCa, respectively.
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Biomarcadores de Tumor/sangre , Lectinas de Plantas/metabolismo , Polisacáridos/metabolismo , Antígeno Prostático Específico/metabolismo , Neoplasias de la Próstata/sangre , Neoplasias de la Próstata/diagnóstico , Receptores N-Acetilglucosamina/metabolismo , Adulto , Anciano , Anciano de 80 o más Años , Área Bajo la Curva , Biopsia , Glicosilación , Humanos , Inmunohistoquímica , Masculino , Persona de Mediana Edad , Análisis Multivariante , Clasificación del Tumor , Recurrencia Local de Neoplasia/patología , Pronóstico , Próstata/patología , Próstata/cirugía , Antígeno Prostático Específico/sangre , Prostatectomía , Hiperplasia Prostática/sangre , Hiperplasia Prostática/patología , Neoplasias de la Próstata/patología , Neoplasias de la Próstata/cirugía , Curva ROC , Factores de RiesgoRESUMEN
Two types of plasmonic silver- and gold-coated grating biosensor chips (plasmonic chip) were applied in the detection of α-fetoprotein (AFP) with a sandwich imunoassay and surface plasmon field-enhanced fluorescence. On the plasmonic chip, unlabeled marker in the sandwich immunoassay was first quantitatively detected over a wide range between 10(-12) and 10(-8) g/mL. The affinity constants between AFP and anti-AFP antibody, which were obtained by fitting the experimental data to the Langmuir isotherm adsorption curve, were 1 × 10(8) g(-1) mL regardless of the kind of metal in the plasmonic chips. Although the fluorescence intensity on the silver plasmonic chip was 5 times larger than that on the gold plasmonic chip, the limit of detection (LOD) was on the order of 10(-11) g/mL and not improved with a silver plasmonic chip. Herein, we used a new setup that generated less dispersions of both the fluorescence intensity for nonspecific adsorption and the background (optical blank) signal and improved the LOD of AFP to 4 pg/mL (55 fM) with the silver plasmonic chip. With the highly sensitive detection in the sandwich immunoassay, the development of a plasmonic chip for clinical diagnosis by a blood test is promising.
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Biomarcadores de Tumor/análisis , Técnicas Biosensibles/instrumentación , alfa-Fetoproteínas/análisis , Adsorción , Fluorescencia , HumanosRESUMEN
A high-sensitivity immunoassay system with surface plasmon field-enhanced fluorescence spectrometry (SPFS) was constructed using a plastic sensor chip and then applied to the detection of total prostate-specific antigen (total PSA) and GalNAcß1-4GlcNAc-linked prostate-specific antigen (LacdiNAc-PSA) in serum, to discriminate between prostate cancer (PC) and benign prostate hyperplasia (BPH). By using this automated SPFS immunoassay, the detection limit for total PSA in serum was as low as 0.04 pg/mL, and the dynamic range was estimated to be at least five digits. A two-step sandwich SPFS immunoassay for LacdiNAc-PSA was constructed using both the anti-PSA IgG antibody to capture PSA and Wisteria floribunda agglutinin (WFA) for the detection of LacdiNAc. The results of the LacdiNAc-PSA immunoassay with SPFS showed that the assay had a sensitivity of 20.0 pg/mL and permitted the specific distinction between PC and BPH within the PSA gray zone. These results suggested that high-sensitivity automated SPFS immunoassay systems might become a powerful tool for the diagnosis of PC and other diseases.
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Inmunoensayo/instrumentación , Lactosa/análogos & derivados , Antígeno Prostático Específico/sangre , Neoplasias de la Próstata/diagnóstico , Diseño de Equipo , Humanos , Lactosa/análisis , Lactosa/sangre , Límite de Detección , Masculino , Antígeno Prostático Específico/análisis , Neoplasias de la Próstata/sangre , Espectrometría de Fluorescencia/instrumentaciónRESUMEN
Three-dimensional cell spheroids are superior cell-administration form for cell-based therapy which generally exhibit superior functionality and long-term survival after transplantation. Here, we nondestructively measured the oxygen consumption rate of cell spheroids using an on-chip electrochemical device (OECD) and examined whether this rate can be used as a marker to estimate the quality of cell spheroids. Cell spheroids containing NanoLuc luciferase-expressing mouse mesenchymal stem cell line C3H10T1/2 (C3H10T1/2/Nluc) were prepared. Spheroids of high or low quality were prepared by altering the medium change frequency. After transplantation into mice, the high-quality C3H10T1/2/Nluc spheroids exhibited a higher survival rate than the low-quality ones. The oxygen consumption rate of the high-quality C3H10T1/2/Nluc spheroids was maintained at high levels, whereas that of the low-quality spheroids decreased with time. These results indicate that OECD-based measurement of the oxygen consumption rate can be used to estimate the quality of cell spheroids without destructive analysis of the spheroids.
RESUMEN
BACKGROUND: Serum LacdiNAc-glycosylated prostate-specific antigen (LDN-PSA) and LDN-PSA density together with PSA and PSA density (PSAD) were measured as a diagnostic tool for prostate cancer (PCa). PATIENTS AND METHODS: We included 150 patients with PCa without hormonal therapy and 41 patients without PCa obtained from the Kyoto University Hospital between 2012 and 2017. LDN-PSA levels were measured through a WFA-anti-PSA antibody sandwich immunoassay using a highly sensitive surface plasmon field-enhanced fluorescence spectroscopy (SPFS) system. Diagnostic performance of serum LDN-PSA and LDN-PSAD was evaluated by measuring the area under the receiver-operating characteristic curve (AUC). RESULTS: The AUCs of LDN-PSA, LDN-PSAD, and PSAD levels (0.780, 0.848, and 0.835, respectively) detected in patients with PCa were significantly higher (P = .0001, P < .0001, and P < .0001, respectively) than that of PSA (0.590). Moreover, among 143 patients with PCa who received radical prostatectomy (RP), the AUCs of LDN-PSA, LDN-PSAD, and PSAD levels (0.750, 0.812, and 0.769, respectively) detected in patients with a pathologic Gleason grade group ≥ 2 were significantly higher (P = .0170, P = .0028, and P = .0003, respectively) than that of PSA (0.578). In the group comprising 35 patients who received RP with a Gleason grade group 1-graded biopsy, the LDN-PSA, LDN-PSAD, and PSAD levels were significantly different (P = .0097, P = .0024, and P = .0312, respectively). However, PSA alone could not discriminate cases with adverse features (P = .454). CONCLUSIONS: LDN-PSAD is a potential marker for detecting PCa and selecting candidates for RP.
Asunto(s)
Calicreínas/sangre , Antígeno Prostático Específico/sangre , Neoplasias de la Próstata/diagnóstico , Anciano , Biopsia , Glicosilación , Humanos , Calicreínas/metabolismo , Lactosa/análogos & derivados , Lactosa/metabolismo , Masculino , Persona de Mediana Edad , Clasificación del Tumor , Estadificación de Neoplasias , Próstata/patología , Próstata/cirugía , Antígeno Prostático Específico/metabolismo , Prostatectomía , Neoplasias de la Próstata/sangre , Neoplasias de la Próstata/patología , Neoplasias de la Próstata/cirugía , Curva ROC , Estudios RetrospectivosRESUMEN
We have investigated the effectiveness of surface-adsorbed silver half-shells for inducing the surface-enhanced fluorescence phenomenon. Our simple structure consists of a dense monolayer of monodispersive latex sphere covered by thermally evaporated silver, some tens of nm thick. In order to increase its effectiveness as a platform for immunoassay, we explored three physical parameters; the diameter of the latex sphere, the deposition thickness and the adsorption density of the latex sphere. The maximum enhancement of 25.5 was achieved using the sphere with 56 nm diameter with 10 nm silver deposited. As for the adsorption density, the maximum fluorescence signal was achieved with a relatively sparse adsorption density, 12.5 per microm(2) vs. 105.9 per microm(2), corresponding to the full coverage. With the adsorption density also optimized, the enhancement was further increased by a factor of three.
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Portadores de Fármacos , Plata , Adsorción , Inmunoensayo/métodos , Microesferas , Tamaño de la Partícula , Propiedades de SuperficieRESUMEN
Grating-coupled surface plasmon field-enhanced fluorescence spectroscopy (GC-SPFS) with optical bound/free (B/F) separation technique was developed by employing a highly directional fluorescence with polarization of surface plasmon-coupled emission (SPCE) to realize highly sensitive immunoassay regardless of the ligand affinity. A highly sensitive immunoassay system with GC-SPFS was constructed using a plastic sensor chip reproducibly fabricated in-house by nanoimprinting and applied to the quantitative detection of an anti-lysozyme single-domain antibody (sdAb), to compare conventional washing B/F separation with optical B/F separation. Differences in the affinity of the anti-lysozyme sdAb, induced by artificial mutation of only one amino acid residue in the variable domain were attributed to higher sensitivity than that of the conventional Biacore surface plasmon resonance (SPR) system. The detection limit (LOD; means of six replicates of the zero standard plus three standard deviations) of the GC-SPFS immunoassay with optical B/F separation, was estimated to be 1.2 ng/ml with the low-affinity ligand (mutant sdAb Y52A: KD level was of the order of 10-7 ~ 10-6 M) and was clearly improved as compared to that (LOD: 9.4 ng/ml) obtained with the conventional washing B/F separation. These results indicate that GC-SPFS with the optical B/F separation technique offers opportunities to re-evaluate low-affinity biomaterials that are neither fully utilized nor widespread, and could facilitate the creation of novel and innovative methods in drug and diagnostic development.
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Espectrometría de Fluorescencia/métodos , Resonancia por Plasmón de Superficie/métodos , Inmunoensayo/instrumentación , Inmunoensayo/métodos , Límite de Detección , Muramidasa/inmunología , Anticuerpos de Dominio Único/análisis , Anticuerpos de Dominio Único/inmunología , Espectrometría de Fluorescencia/instrumentación , Resonancia por Plasmón de Superficie/instrumentaciónRESUMEN
Cyclo olefin polymer (COP) is an attractive plastic because it has low protein adsorption despite its hydrophobic chemical structure. Here, the adsorption of model proteins to the COP was evaluated in comparison with a representative plastic, polystyrene (PSt), using reflectometry interference spectroscopy (RIfS) technology. The effects of different salts on adsorption were then examined. The adsorption of bovine serum albumin onto COP increased in the presence of kosmotropic salts, whereas adsorption of IgG increased in the presence of chaotropic salts. By contrast, the adsorption of these 2 proteins to PSt was unaffected by these Hofmeister salts. Langmuir-Freundlich model of COP adsorption suggested that the COP surface is more homogeneous for protein binding than the PSt surface. Furthermore, RIfS and sum frequency generation analyses indicated that water molecules bind more weakly to COP than to PSt. Our data propose a novel viewpoint of the way protein binds to COP surface that is different from the way it binds to PSt.
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Polímeros/química , Albúmina Sérica Bovina/química , Adsorción , Interacciones Hidrofóbicas e Hidrofílicas , Poliestirenos/química , Unión Proteica , Sales (Química)/química , Análisis Espectral/métodos , Propiedades de Superficie , Agua/químicaRESUMEN
Electrochemical microbial chip for mutagen screening were microfabricated and characterized by scanning electrochemical microscopy (SECM). Salmonella typhimurium TA1535 with a plasmid pSK1002 carrying a umuC'-'lacZ fusion gene was used for the whole cell mutagen sensor. The TA1535/pSK1002 cells were exposed to mutagen solutions containing 2-(2-furyl)-3-(5-nitro-2-furyl)acrylamido (AF-2), mitomycin C (MMC) or 2-aminoanthracene (2-AA) and embedded in a microcavity (5nl) on a glass substrate using collagen gel. The beta-galactosidase expression on the microbial chip was electrochemically monitored using p-aminophenyl-beta-d-galactopyranoside (PAPG) as the enzymatic substrate. This system has several advantages compared with the conventional umu test: drastic reduction of the sample volume, less time-consuming for beta-galactosidase detection (free from substrate reaction time) and lower detection limit for the three mutagens (AF-2, MMC, 2-AA). Finally, a multi-sample assay was carried out using the microbial array chip with four microcavities.
Asunto(s)
Bioensayo/instrumentación , Electroquímica/instrumentación , Escherichia coli/efectos de los fármacos , Escherichia coli/enzimología , Mutágenos/administración & dosificación , Mutágenos/análisis , Salmonella typhimurium/efectos de los fármacos , Bioensayo/métodos , Técnicas Biosensibles/instrumentación , Técnicas Biosensibles/métodos , Electroquímica/métodos , Diseño de Equipo , Análisis de Falla de Equipo , Reproducibilidad de los Resultados , Salmonella typhimurium/enzimología , Sensibilidad y Especificidad , beta-Galactosidasa/análisisRESUMEN
A microbial array chip with collagen gel spots entrapping living bacterial cells has been applied to investigate the metabolic regulation in Paracoccus denitrificans. Scanning electrochemical microscopy (SECM) was used to monitor the ferrocyanide production that reflects the electron flow in the respiratory chain located within the internal membrane of P. denitrificans. The ferrocyanide production from P. denitrificans largely depends on the types of the carbon source (glucose or lactate), suggesting that the electron flow rate in the respiratory chain depends on the activity of the metabolic pathway located up-stream of the respiratory chain. More importantly, it was found that the enzymes affecting glucose catabolic reactions were significantly up-regulated in cultures with a nutrient agar medium containing D-(+)-glucose as a sole carbon source. Enzyme assays using crude extracts of P. denitrificans were carried out to identify the enzymes expressed at a higher level in cultures supplemented with D-(+)-glucose. It was confirmed that the pyruvate kinase and enzymes of the overall Entner-Doudoroff pathway were highly induced in cultures containing D-(+)-glucose.
Asunto(s)
Electrofisiología , Paracoccus denitrificans/metabolismo , Electroquímica , Ferrocianuros/química , Ferrocianuros/metabolismo , Glucosa/metabolismo , Glucosa/fisiología , Microelectrodos , Microscopía Electrónica , Paracoccus denitrificans/enzimología , Paracoccus denitrificans/fisiología , Vía de Pentosa Fosfato/fisiologíaRESUMEN
A microbial chip was fabricated by filling the micropores on a glass substrate with collagen-embedded Escherichia coli(E. coli) cells, and characterized by scanning electrochemical microscopy (SECM) in a solution containing ferricyanide. The activity of the E. coli cells in the collagen gel microstructure was imaged and characterized with SECM by mapping the localized concentration of ferrocyanide produced by the respiration of the cells. The SECM-based activity measurement detected as low as approximately 100 E. coli cells. Furthermore, the optical-microscopic observation indicated that the E. coli cells on the chip proliferated during the incubation. The sequential SECM measurements were performed for the same E. coli chip to obtain the microbial growth curve for a small number of microorganisms.
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Colágeno/química , Escherichia coli/química , Microcomputadores , Electroquímica , Escherichia coli/crecimiento & desarrollo , Ferricianuros/metabolismo , Geles , Microscopía Electrónica de Rastreo , Nanotecnología , Oxidación-ReducciónRESUMEN
The respiratory activities of cultured HeLa cells were monitored at a single cell level using scanning electrochemical microscopy (SECM) that produces images of the localized distribution of oxygen around the cell. The change in the cellular activity was traced after exposures to KCN, ethyl alcohol and the antibiotic drug, Antimycin A. The results were compared with those from the conventional fluorescence monitoring using Calcein-AM that is sensitive to deformation of the cell membrane. The SECM-based measurement follows the decrease in the cellular activity upon exposure to KCN and Antimycin A more rapidly than the fluorescence-based measurements, demonstrating that SECM is suitable for studying the cellular influence of respiration inhibitors.
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Técnicas de Cultivo de Célula/métodos , Respiración de la Célula/fisiología , Electroquímica/métodos , Microelectrodos , Microscopía Confocal/métodos , Oxígeno/metabolismo , Espectrometría de Fluorescencia/métodos , Antimicina A/farmacología , Respiración de la Célula/efectos de los fármacos , Etanol/farmacología , Células HeLa , Humanos , Cianuro de Potasio/farmacologíaRESUMEN
[small beta]-Galactosidase expression in a small number of Escherichia coli cells has been measured in real time with an electrochemical sensor chip. E. coli cells were embedded using collagen gel within a micropore which was microfabricated onto a chip. The activity of the expressed [small beta]-galactosidase was determined using p-aminophenyl [small beta]-d-galactopyranoside (PAPG) as a substrate.
Asunto(s)
Electroquímica/métodos , Escherichia coli/genética , Genes Reporteros , beta-Galactosidasa/análisis , Aminofenoles/química , Colágeno , Escherichia coli/enzimología , Galactósidos/química , Galactósidos/metabolismo , Geles , Microcomputadores , Quinonas/química , Factores de Tiempo , beta-Galactosidasa/genética , beta-Galactosidasa/metabolismoRESUMEN
Surface-enhanced fluorescence has potentially many desirable properties as an analytical method for medical diagnostics, but the effect observed so far is rather modest and only in conjunction with fluorophores with low quantum yields. Coupled with the fact that preparation of suitable surfaces at low costs has been difficult, this has limited its utilities. Here we report a novel method for forming uniform and reproducible surfaces with respectable enhancement ratios even for high-quantum-yield fluorophores. Formation of dense surface-adsorbed latex spheres on a flat surface via partial aggregation, followed by evaporation of silver, results in a film consisting of cap-shaped silver particles at high densities. Binding of fluorescence biomolecules, either through physisorption or antigen-antibody reaction, was performed, and enhancements close to 50 have been observed with fluorophores such as R-phycoerythrin and Alexa 546-labeled, bovine serum albumin, both of which have quantum yields around 0.8. We attribute this to the unique shape of the silver particle and the presence of abundant gaps among adjacent particles at high densities. The effectiveness of the new surface is also demonstrated with IL-6 sandwich assays.
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Materiales Biocompatibles Revestidos/química , Coloides/química , Plata/química , Espectrometría de Fluorescencia/métodos , Animales , Bovinos , Fluorescencia , Colorantes Fluorescentes/química , Humanos , Microscopía Electrónica de Rastreo , Nanopartículas/química , Nanopartículas/ultraestructura , Sensibilidad y Especificidad , Albúmina Sérica Bovina/química , Propiedades de SuperficieRESUMEN
The metabolic activity of E. coli cells embedded in collagen gel microstructures in a cone-shaped well and in a cylindrical micropore was investigated using scanning electrochemical microscopy (SECM), based on the oxygen consumption rate and the conversion rate from ferrocyanide to ferricyanide. The analysis of the concentration profiles for oxygen and ferrocyanide afforded the oxygen consumption rate and the ferrocyanide production rate. A comparison indicated that the ferrocyanide production rates were larger than the oxygen consumption rate, and also that the rates observed in the cylindrical micropore were larger than those observed in the cone-shaped well. The ferrocyanide production rate of a single E. coli cell was calculated to be (5.4 +/- 2.6) x 10(-19) mol s(-1), using a cylindrical micropore system.
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Escherichia coli/fisiología , Electroquímica/métodos , Ferrocianuros/análisis , Microquímica/métodos , Microscopía Electrónica de Rastreo , Oxígeno/análisis , RespiraciónRESUMEN
The respiratory activity of collagen-embedded living cells was imaged by scanning electrochemical microscopy (SECM) with the objective to study anticancer drug sensitivity. Two kinds of cancer cells, the human erythroleukemia cell line (K562) and its adriamycin-resistant subline (K562/ADM), were immobilized at the array of microholes micromachined on a silicon wafer for comparative characterization of their sensitivity to the anticancer drug, ADM. The results obtained by the SECM method showed correspondence to a conventional colorimetric assay (SDI assay). Furthermore, since the SECM assay is based on the noninvasive measurement of the respiration activity, continuous monitoring of a dose response was possible.