Analysis of ribonuclease activity in sub-nanoliter droplets by label-free fluorescence measurements

Choi, Jung‐Do; Vasamsetti, Bala Murali Krishna; Kim, Kyuwan; Seo, Seung Hwan; Lee, Dong Hun; Chang, Soo-Ik; Choo, Jaebum; Kim, Hee Young

doi:10.1039/c6an02724e

Cited by 11 publications

(6 citation statements)

References 28 publications

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“…The enzyme inhibition assay is essential in high-throughput drug screening since several samples must be screened. Droplet-based microfluidic system can be used as a platform to perform enzyme inhibition assays due to their advantages of rapid mixing of reagents and low reagent/sample consumption [ 24 , 25 ]. Here, dPIA was demonstrated through a simple dose–response assay using ethyl-3,4-dephostatin as the phosphatase inhibitor ( Figure 4 a).…”

Section: Resultsmentioning

confidence: 99%

“…The microfluidic device was designed and prepared based on our previous reports [ 24 , 25 ]. A simple T-junction PDMS microfluidic device featuring an oil inlet, a sample inlet, and an outlet was fabricated using standard soft lithography techniques.…”

Section: Methodsmentioning

confidence: 99%

“…Lab-on-a-chip technologies have been successfully demonstrated in the fields of pharmacology, cell biology, and biochemistry [ 21 ]. Using droplet-based microfluidic system, we have previously reported protein–protein interactions analysis [ 22 ], SNP detection [ 23 ], RNase activity assay [ 24 ], and DNase activity assay [ 25 ]. Therefore, to identify phosphatases and screen for phosphatase inhibitors, droplet-based microfluidics has the potential to be a low-cost, high-throughput screening technology.…”

Section: Introductionmentioning

confidence: 99%

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Analysis of Phosphatase Activity in a Droplet-Based Microfluidic Chip

et al. 2022

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We report analysis of phosphatase activity and inhibition on droplet-based microfluidic chips. Phosphatases are such attractive potential drug targets because abnormal phosphatase activity has been implicated in a variety of diseases including cancer, neurological disorders, diabetes, osteoporosis, and obesity. So far, several methods for assessing phosphatase activity have been reported. However, they require a large sample volume and additional chemical modifications such as fluorescent dye conjugation and nanomaterial conjugation, and are not cost-effective. In this study, we used an artificial phosphatase substrate 3-O-methylfluorescein phosphate as a fluorescent reporter and dual specificity phosphatase 22. Using these materials, the phosphatase assay was performed from approximately 340.4 picoliter (pL) droplets generated at a frequency of ~40 hertz (Hz) in a droplet-based microfluidic chip. To evaluate the suitability of droplet-based platform for screening phosphatase inhibitors, a dose–response inhibition study was performed with ethyl-3,4-dephostatin and the half-maximal inhibitory concentration (IC50) was calculated as 5.79 ± 1.09 μM. The droplet-based results were compared to microplate-based experiments, which showed agreement. The droplet-based phosphatase assay proposed here is simple, reproducible, and generates enormous data sets within the limited sample and reagent volumes.

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Analysis of Phosphatase Activity in a Droplet-Based Microfluidic Chip

et al. 2022

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“…It is of great significance to develop new approaches for cost-effective detection of RNase A. Ribonucleotide cytosine and uracil (rC and rU) are the enzyme cutting sites for RNase A, − so a chimeric DNA probe that contains rC or rU base can be specifically hydrolyzed into small fragments by RNase A too. According to such a biochemical mechanism, some biosensors based on electrochemical and fluorescent technology were developed. − For example, Takenaka et al designed a rC containing chimeric DNA probe which was immoblized on the gold electrode; the presence of RNase A would hydrolyze the probe and release ferrocenyl-labeled fragment, leading to a decreasing current response for quantification.…”

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confidence: 99%

Homogeneous Electrochemiluminescence Biosensor for the Detection of RNase A Activity and Its Inhibitor

Lin

Yang

et al. 2019

Anal. Chem.

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Ribonuclease A (RNase A) is increasingly considered as a biomarker for tumor diagnosis, and it is of great significance to develop an ultrasensitive, cost-effective assay for RNase A detection. Electrochemiluminescence (ECL) technology has distinctive advantages in the development of biosensors for diverse targets. However, most of the ECL biosensors require the complex process of electrode modification, which is laborious and time consuming. In this work, an immobilization-free homogeneous ECL assay was developed for the highly sensitive detection of RNase A activity for the first time. On the basis of the fact that RNase A can specifically hydrolyze RNA at the site of ribonucleotide uracil (rU), a rU-containing chimeric DNA probe is designed and labeled with Ru(bpy)3 2+ (act as ECL indicator). The chimeric DNA probe hardly diffuses to the surface of negatively charged indium tin oxide (ITO) electrode due to the strong electrostatic repulsion between the negatively charged DNA and ITO electrode, resulting in a weak ECL signal detected. When the RNase A is present, the chimeric DNA probe is hydrolyzed into small fragments, which contains little negative charge and can diffuse easily to the ITO electrode surface due to the decreased electrostatic repulsion. In this case, an enhanced ECL signal can be detected. Under the optimal conditions, there is a linear relationship between the ECL signal and the concentration of RNase A in the range of 0.001–0.10 ng/mL, and the detection limit is 0.2 pg/mL. In addition, the proposed ECL sensing system is also applied to detect the RNase A inhibitor, taking As3+ as an example. The proposed homogeneous ECL sensing system provides a new approach for the highly sensitive and convenient detection of RNase A as well as other ribonucleases only by redesigning a responding chimeric DNA probe.

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“…These widely used methods also showed inherent disadvantages of being time-consuming and having tedious operation, low sensitivity, and high cost. Recently, highly sensitive methods such as electrochemical method, , fluorescence method, , and EtBr-based label-free method for RNase A were developed , to overcome the shortcomings of traditional detection methods. However, these methods still have some limitations.…”

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confidence: 99%

Sensitive Detection of RNase A Activity and Collaborative Drug Screening Based on rGO and Fluorescence Probe

et al. 2018

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In addition to being an important object in theoretical and experimental studies in enzymology, RNase A also plays an important role in the development of many kinds of diseases by regulating various physiological or pathological processes, including cell growth, proliferation, differentiation, and invasion. Thus, it can be used as a useful biomarker for disease theranostics. Here, a simple, sensitive, and low-cost assay for RNase A was constructed by combining a fluorogenic substrate with reduced graphene oxide (rGO). The method with detection limit of 0.05 ng/mL was first applied for RNase A targeted drug screening, and 14 natural compounds were identified as activators of this enzyme. Then, it was applied to detect the effect of drug treatment and Hepatitis B virus (HBV) infection on RNase A activity. The results indicated that RNase A level in tumor cells was upregulated by G-10 and Chikusetsusaponin V in a concentration-dependent manner, while the average level of RNase A in the HBV infection group was significantly inhibited compared with that in the control group. Furthermore, the concentration-dependent inhibitory effect of heavy metal ions on RNase A was observed using the method and the results indicated that Ba, Co, Pb, As, and Cu inhibited RNase A activity with IC values of 93.7 μM (Ba), 90.9 μM (Co), 110.6 μM (Pb), 171.5 μM (As), and 165.1 μM (Cu), respectively. In summary, considering the benefits of rapidity and high sensitivity, the method is practicable for RNase A assay in biosamples and natural compounds screening in vitro and in vivo.

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Analysis of ribonuclease activity in sub-nanoliter droplets by label-free fluorescence measurements

Cited by 11 publications

References 28 publications

Analysis of Phosphatase Activity in a Droplet-Based Microfluidic Chip

Analysis of Phosphatase Activity in a Droplet-Based Microfluidic Chip

Homogeneous Electrochemiluminescence Biosensor for the Detection of RNase A Activity and Its Inhibitor

Sensitive Detection of RNase A Activity and Collaborative Drug Screening Based on rGO and Fluorescence Probe

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