scholarly journals Optimization of PTFE Coating on PDMS Surfaces for Inhibition of Hydrophobic Molecule Absorption for Increased Optical Detection Sensitivity

Sensors ◽  
2021 ◽  
Vol 21 (5) ◽  
pp. 1754
Author(s):  
Junyi Yao ◽  
Yiyang Guan ◽  
Yunhwan Park ◽  
Yoon E Choi ◽  
Hyun Soo Kim ◽  
...  
Keyword(s):  
Fluorescent Dyes ◽  
Lipid Production ◽  
Optical Detection ◽  
Nile Red ◽  
Porous Matrix ◽  
Detection Sensitivity ◽  
Microfluidic Chips ◽  
Fluorescent Intensity ◽  
Lipid Signal ◽  
Significant Difference

Polydimethylsiloxane (PDMS) is a polymer widely used for fabrication and prototyping of microfluidic chips. The porous matrix structure of PDMS allows small hydrophobic molecules including some fluorescent dyes to be readily absorbed to PDMS and results in high fluorescent background signals, thereby significantly decreasing the optical detection sensitivity. This makes it challenging to accurately detect the fluorescent signals from samples using PDMS devices. Here, we have utilized polytetrafluoroethylene (PTFE) to inhibit absorption of hydrophobic small molecules on PDMS. Nile red was used to analyze the effectiveness of the inhibition and the absorbed fluorescence intensities for 3% and 6% PTFE coating (7.7 ± 1.0 and 6.6 ± 0.2) was twofold lower compared to 1% and 2% PTFE coating results (17.2 ± 0.5 and 15.4 ± 0.5). When compared to the control (55.3 ± 1.6), it was sevenfold lower in background fluorescent intensity. Furthermore, we validated the optimized PTFE coating condition using a PDMS bioreactor capable of locally stimulating cells during culture to quantitatively analyze the lipid production using Chlamydomonas reinhardtii CC-125. Three percent PTFE coating was selected as the optimal concentration as there was no significant difference between 3% and 6% PTFE coating. Intracellular lipid contents of the cells were successfully stained with Nile Red inside the bioreactor and 3% PTFE coating successfully minimized the background fluorescence noise, allowing strong optical lipid signal to be detected within the PDMS bioreactor comparable to that of off-chip, less than 1% difference.

En face dual fluorescence staining of fatty streaks allows observation of initiation and progression of atherosclerotic lesions

1995 ◽  
Vol 53 ◽  
pp. 864-865
Author(s):  
Anne M. Klinkner ◽  
Crystal R. Waites ◽  
Peter J. Bugelski ◽  
William D. Kerns
Keyword(s):  
Fluorescent Dyes ◽  
Nile Red ◽  
Dimethyl Formamide ◽  
High Cholesterol Diet ◽  
Methods Development ◽  
Atherosclerotic Lesions ◽  
En Face ◽  
Biochemical Evaluation ◽  
Stock Solutions ◽  
Dual Staining

A primary effort in the understanding of the progression of atherosclerotic disease has been methods development for visualization of the atherosclerotic plaque. We introduce a new method for the qualitative analysis of lipids in atherosclerotic fatty streaks which also retains those lipids for biochemical evaluation. An original aspect of the process is the ability to view an entire fatty streak en face, selectively stained for specific lipid classes within the lesion.New Zealand white rabbits were fed a high cholesterol diet(0.15%-0.3% for 14 wks). The aorta was removed and fixed in Carson's phosphate buffered formaldehyde followed by dual staining in the fluorescent dyes Nile red and filipin. Stock solutions of nile red(0.5mg/ml acetone) and filipin(2.5mg/ml dimethyl formamide) were prepared and kept at -20°C; all subsequent steps were at RT. 0.5cm × 1.0cm pieces of aorta were trimmed and adventitia removed. The pieces were then washed 3×15 min in PBS w/o CaMg, soaked in Nile red(NR)/filipin(Fl) stain(100(il NR stock + 200μl Fl stock in 10 ml PBS for 30 min, washed in PBS 3×30 min, rinsed with distilled water, mounted(Crystal Mount, Biomedia) and coverslipped and viewed by fluorescence microscopy.

scholarly journals Monolithic integration of nanorod arrays on microfluidic chips for fast and sensitive one-step immunoassays

2021 ◽  
Vol 7 (1) ◽  
Author(s):  
Ye Wang ◽  
Jiongdong Zhao ◽  
Yu Zhu ◽  
Shurong Dong ◽  
Yang Liu ◽  
...  
Keyword(s):  
Troponin I ◽  
Specific Antigen ◽  
Monolithic Integration ◽  
Detection Sensitivity ◽  
Quantitative Detection ◽  
Plasmonic Nanostructures ◽  
Nanorod Arrays ◽  
Microfluidic Chips ◽  
One Step ◽  
Flow Through

AbstractHere, we present integrated nanorod arrays on microfluidic chips for fast and sensitive flow-through immunoassays of physiologically relevant macromolecules. Dense arrays of Au nanorods are easily fabricated through one-step oblique angle deposition, which eliminates the requirement of advanced lithography methods. We report the utility of this plasmonic structure to improve the detection limit of the cardiac troponin I (cTnI) assay by over 6 × 105-fold, reaching down to 33.9 fg mL−1 (~1.4 fM), compared with an identical assay on glass substrates. Through monolithic integration with microfluidic elements, the device enables a flow-through assay for quantitative detection of cTnI in the serum with a detection sensitivity of 6.9 pg mL−1 (~0.3 pM) in <6 min, which was 4000 times lower than conventional glass devices. This ultrasensitive detection arises from the large surface area for antibody conjugation and metal-enhanced fluorescent signals through plasmonic nanostructures. Moreover, due to the parallel arrangement of flow paths, simultaneous detection of multiple cancer biomarkers, including prostate-specific antigen and carcinoembryonic antigen, has been fulfilled with increased signal-to-background ratios. Given the high performance of this assay, together with its simple fabrication process that is compatible with standard mass manufacturing techniques, we expect that the prepared integrated nanorod device can bring on-site point-of-care diagnosis closer to reality.

Detection of Ureteral Stones in Kidney Ureter Bladder Radiography: Usefulness of Digital Post-processing

2021 ◽  
Vol 17 ◽  
Author(s):  
Sang Lim Choi ◽  
Sung Bin Park ◽  
Seungwook Yang ◽  
Eun Sun Lee ◽  
Hyun Jeong Park ◽  
...  
Keyword(s):  
Detection Sensitivity ◽  
Ureteral Stones ◽  
Data Sets ◽  
Post Processing ◽  
Stone Size ◽  
Data Set ◽  
Detection Rates ◽  
Exact Test ◽  
Significant Difference ◽  
Exact Size

Purpose: Kidney, ureter, and bladder radiography (KUB) has frequently been used in suspected urolithiasis, but its performance is known to be lower than that of computed tomography (CT). This study aimed to investigate the diagnostic performance of digitally post-processed kidney ureter bladder radiography (KUB) in the detection of ureteral stones. Materials And Methods: Thirty patients who underwent digital KUB and CT were included in this retrospective study. The original digital KUB underwent post-processing that involved noise estimation, reduction, and whitening to improve the visibility of ureteral stones. Thus, 60 digital original or post-processed KUB images were obtained and ordered randomly for blinded review. After a period, a second review was performed after unblinding stone laterality. The detection rates were evaluated at both initial and second review, using CT as reference standard. The objective (size) and subjective (visibility) parameters of ureteral stones were analyzed. Fisher’s exact test was used to compare the detection sensitivity between the original and post-processed KUB data set. Visibility analysis was assessed with a paired t-test. Correlation of stone size between CT and digital KUB data sets was assessed with Pearson’s correlation test. Results: The detection rate was higher for most reviewers once stone laterality was provided and was non-significantly better for the post-processed KUB images (p > 0.05). There was no significant difference in stone size among CT and digital KUB data sets. In all reviews, visibility grade was higher in the post-processed KUB images, irrespective of whether stone laterality was provided. Conclusion: Digital post-processing of KUB yielded higher visibility of ureteral stones and could improve stone detection, especially when stone laterality was available. Thus, digitally post-processed KUB can be an excellent modality for detecting ureteral stones and measuring their exact size.

scholarly journals Visualizing the Integrity of Chloroplast Envelope by Rhodamine and Nile Red Staining

2021 ◽  
Vol 12 ◽  
Author(s):  
Jinjie An ◽  
Xin Miao ◽  
Lulu Wang ◽  
Xu Li ◽  
Xiaomin Liu ◽  
...  
Keyword(s):  
Fluorescent Dyes ◽  
Gradient Centrifugation ◽  
Nile Red ◽  
Middle Layer ◽  
Chloroplast Envelope ◽  
The Past ◽  
Biochemical Methods ◽  
Percoll Density Gradient Centrifugation ◽  
Percoll Density Gradient ◽  
Isolated Chloroplasts

Chloroplasts are essential organelles in plant cells with many important functions. Chloroplasts isolated by Percoll density gradient centrifugation are widely used in the study of chloroplasts. The intactness of isolated chloroplasts is necessary for many of the experiments. In the past, those isolated chloroplasts were either simply believed to be intact or had to be analyzed by indirect biochemical methods. Here we show a new method to check the intactness of isolated chloroplasts by staining their envelope with fluorescent dyes, Rhodamine or Nile red, and then observing them with a fluorescence microscope. With this method, broken chloroplasts and intact chloroplasts can be distinguished easily and their integrity can be checked in a few minutes. Results of this method agreed well with those of biochemical methods. Moreover, we have also found that sometimes the middle layer chloroplasts from the Percoll gradient centrifugation could be mostly broken, which could cause mistakes in the experiment. With our method, this problem can be easily found. This chloroplast envelope staining method can be used in the preparation of isolated chloroplasts to ensure the intactness.

Hardware Design for Multiple Gas Detection System Using Zeolite Coated with Nile Red

2011 ◽  
Vol 2011 (1) ◽  
pp. 000861-000867
Author(s):  
Son Nguyen ◽  
Z. Joan Delalic ◽  
David M. Kargbo ◽  
Joel B. Sheffield ◽  
Zameer Hasan
Keyword(s):  
Fluorescent Dyes ◽  
Detection System ◽  
Nile Red ◽  
Sensor Arrays ◽  
Gas Absorption ◽  
Fully Integrated ◽  
Vlsi Chip ◽  
Dye Sensing ◽  
Different Gases ◽  
Nanoporous Structures

The goal of this research is to develop a nanosensor that integrates a zeolite/dye sensing unit with an optoelectronic detector, fully integrated into a portable gas sensor. The device will detect and measure more than one target gas at the same time. Since nanoporous structures of zeolites are manipulated, the device is expected to be more accurate, more sensitive, is able to better differentiate and detect any one target in a mixture of different gases. This is achieved by incorporating fluorescent dyes into the zeolites’ cavities, measuring gas absorption, desorption and photo-chromic interaction of dye and gases, interfacing the zeolite/dye sensor arrays with light source and electronic detectors. The electronic part of the device is fully customized VLSI chip. The final device will be packaged into a portable unit. The designed and packaged prototype will be presented.

SERS Effect and SEF Effect of Silver Nanoparticles Applied in Optical Detection of Microfluidic Chips

2018 ◽  
Vol 39 (11) ◽  
pp. 1633-1638
Author(s):  
郭 威 GUO Wei ◽  
吴 坚 WU Jian ◽  
王春艳 WANG Chun-yan ◽  
陈 涛 CHEN Tao
Keyword(s):  
Silver Nanoparticles ◽  
Optical Detection ◽  
Microfluidic Chips

Ca2+ signals obtained with multiple indicators in mammalian vascular muscle cells

1987 ◽  
Vol 253 (6) ◽  
pp. H1456-H1461
Author(s):  
T. T. DeFeo ◽  
G. M. Briggs ◽  
K. G. Morgan
Keyword(s):  
Portal Vein ◽  
Calcium Concentration ◽  
Fluorescent Dyes ◽  
Contractile Function ◽  
Single Cells ◽  
Fluorescent Intensity ◽  
Fura 2 ◽  
Multiple Indicators ◽  
Calcium Storage ◽  
Vascular Muscle Cells

Enzymatically isolated single cells from ferret portal vein were loaded with the fluorescent dyes fura-2 and chlortetracycline. Ferret portal vein intact strips were loaded with the luminescent indicator aequorin. At short loading times, fura-2 loading resulted in relatively homogeneous images of labeled cells. At longer loading times, extremely heterogeneous images were obtained that were similar to those produced by chlortetracycline, an indicator recognized to enter calcium-storage organelles. A significant effect of fura-2 on contractile function was detected at the long but not at the short loading time. Caffeine, which is known to deplete calcium from sarcoplasmic reticulum, decreased the fura-2 fluorescent intensity when cells were incubated for a long loading time but caused no statistically significant change at the short loading time. Caffeine caused no drop in the aequorin signal but did cause a drop in the chlortetracycline fluorescence. These results are consistent with the idea that aequorin reports cytoplasmic intracellular calcium concentration ([Ca2+]i), chlortetracycline reports stored calcium, and fura-2 reports a mixed signal from the cytoplasm and calcium-storage organelles depending on incubation time.

Optical Detection Systems on Microfluidic Chips

2011 ◽  
pp. 171-201 ◽  
Author(s):  
Hongwei Gai ◽  
Yongjun Li ◽  
Edward S. Yeung
Keyword(s):  
Optical Detection ◽  
Microfluidic Chips ◽  
Detection Systems

scholarly journals Improved Recognition of Hematogones From Precursor B-Lymphoblastic Leukemia by a Single Tube Flow Cytometric Analysis

2020 ◽  
Vol 153 (6) ◽  
pp. 790-798
Author(s):  
Michelle D Don ◽  
Washington Lim ◽  
Amanda Lo ◽  
Brian Cox ◽  
Qin Huang ◽  
...  
Keyword(s):  
Lymphoblastic Leukemia ◽  
Flow Cytometric Analysis ◽  
Marrow Transplant ◽  
Diagnostic Challenge ◽  
Fluorescent Intensity ◽  
Algorithmic Approach ◽  
Single Tube ◽  
Flow Cytometric ◽  
Significant Difference ◽  
Ancillary Studies

Abstract Objectives To improve diagnostic accuracy in differentiating hematogones from leukemic blasts in cases of precursor B-lymphoblastic leukemia/lymphoma (B-ALL), particularly those that are posttreatment or after bone marrow transplant, and to provide an algorithmic approach to this diagnostic challenge. Methods A seven-color antibody panel including CD10, CD19, CD45, CD38, CD34, CD58, and CD81 was generated to assess the feasibility of a single tube panel and provide an algorithmic approach to distinguish hematogones from B-ALL. Fifty-three cases were analyzed, and results were correlated with histology and ancillary studies. Results There was a significant difference in mean fluorescent intensity (MFI) for CD81 and CD58 when comparing hematogones and B-ALL populations (P &lt; .001). B-ALL cases had a mean (SD) MFI of 24.6 (27.5; range, 2-125) for CD81 and 135.6 (72.6; range, 48-328) for CD58. Hematogones cases had a mean (SD) MFI of 70.2 (19.2; range, 42-123) for CD81 and 38.8 (9.4; range, 23-58) for CD58. Conclusions The flow cytometry panel with the above markers and utilization of the proposed algorithmic approach provide differentiation of hematogones from B-ALL. This includes rare cases of hematogones and B-ALL overlap where additional ancillary studies are necessary.

Multicenter performance evaluation of the MultiDrugQuant assay kit.

2012 ◽  
Vol 30 (15_suppl) ◽  
pp. e21140-e21140
Author(s):  
Peter Krajcsi ◽  
Katalin Tauber Jakab ◽  
Sandor Barath ◽  
Edit Gyimesi ◽  
Zsuzsanna Hevessy ◽  
...  
Keyword(s):  
Multidrug Resistance ◽  
Mononuclear Cells ◽  
Fluorescent Dyes ◽  
Clinical Laboratory ◽  
Diagnostic Methods ◽  
Cytotoxic Agents ◽  
Efflux Transporters ◽  
Target Cells ◽  
Functional Assay ◽  
Fluorescent Intensity

e21140 Background: Multidrug resistance is the most frequent type of resistance to anticancer chemotherapy, which usually results from the overexpression of efflux transporters, such as the MDR1, MRP1 and BCRP. Unfortunatelly, neither the genetic polymorphisms nor the mRNA/protein expression levels correlate closely with the functional activity. On the other hand, although the functional methods separately gave promising results, standardization and reproducibility of these tests failed to conform with values required from routine diagnostic methods. MultiDrugQuant (MDQ) kit was developed as an improved functional assay system, which can measure the multidrug resistance activity of the three, clinically most relevant efflux transporters using flow cytometry in living tumor cells. The present study aimed to carry out the laboratory validation and to evaluate the performance of the MDQ-kit. Methods: Validation of the kit was carried out according to the standards of the Clinical Laboratory Standards Institute in three university centres. Mononuclear cells were separated using Ficoll gradient and tested at 2-5×106/ml within 6 hours after specimen collection. Activities of the multidrug transporters were calculated from the difference between the mean fluorescent intensity of cells w/o the specific inhibitors, respectively. Inaccuracy and comparative measurements were carried out using cell lines with low and high activity of the transporters. Results on different flow cytometers were compared using CD45 CD19 or CD3 monoclonal antibodies for gating the population of interest. Results: The assay proved to be specific and robust at various concentrations of the fluorescent dyes (10-100 % of the original) or inhibitors (50-150 % of the original). Both intraassay and interassay reproducibilities were <5 %. Multidrug resistance activity values determined on different flow cytometers were comparable and eligible. Conclusions: The MDQ assay provides quantitative results on the activity of the MDR1, MRP1 and BCRP in the target cells, which might be used to predict the resistance of these cells to particular cytotoxic agents. Recently, the MDQ-kit has been registered for in vitro diagnostic use in the EU.

Sign in / Sign up

Export Citation Format

Share Document