Jieon Lee

Quantitative and Multiplexed MicroRNA Sensing in Living Cells Based on Peptide Nucleic Acid and Nano Graphene Oxide (PANGO)

MicroRNA (miRNA) is an important small RNA which regulates diverse gene expression at the post-transcriptional level. miRNAs are considered as important biomarkers since abnormal expression of specific miRNAs is associated with many diseases including cancer and diabetes. Therefore, it is important to develop biosensors to quantitatively detect miRNA expression levels. Here, we develop a nanosized graphene oxide (NGO) based miRNA sensor, which allows quantitative monitoring of target miRNA expression levels in living cells. The strategy is based on tight binding of NGO with peptide nucleic acid (PNA) probes, resulting in fluorescence quenching of the dye that is conjugated to the PNA, and subsequent recovery of the fluorescence upon addition of target miRNA. PNA as a probe for miRNA sensing offers many advantages including high sequence specificity, high loading capacity on the NGO surface compared to DNA and resistance against nuclease-mediated degradation. The present miRNA sensor allowed the detection of specific target miRNAs with the detection limit as low as ~1 pM and the simultaneous monitoring of three different miRNAs in a living cell.

A New Assay for Endonuclease/Methyltransferase Activities Based on Graphene Oxide

A new endonuclease/methyltransferase activity assay method based on graphene oxide (GO) is developed. Substrate DNA is designed to possess a double-stranded part to serve as a nuclease substrate and a single-stranded part for anchoring the DNA to the GO surface via strong noncovalent binding. Nuclease-mediated DNA hydrolysis induces the recovery of fluorescence intensity of the dye attached to the end of the double-stranded DNA region. This GO-based method allows real-time measurement and quantitative assay for endonuclease/methyltransferase activities in short time.

Laser desorption/ionization mass spectrometric assay for phospholipase activity based on graphene oxide/carbon nanotube double-layer films.

A new method for quantitative phospholipase activity assays using mass spectrometry (MS) and a supported thin film consisting of a graphene oxide (GO)/carbon nanotube (CNT) double layer as a substrate for laser desorption ionization (LDI) has been developed. Phospholipids were very efficiently analyzed by LDI-time-of-flight (TOF) MS on the GO/CNT films, presumably because of the affinity of phospholipids for the GO/CNT surface. Therefore, the rate of lipid hydrolysis was conveniently measured using LDI-TOF mass spectra obtained from a GO/CNT surface on which the phospholipid hydrolysis reaction mixtures had been spotted by comparing the mass-peak intensities of reactants and products. The present platform for phospholipase assays based on MS and GO/CNT double-layer films enables quantitative measurements at low cost, allows assays to be performed in a short time, and is compatible with an array format, unlike conventional assay methods.

Biosensors based on graphene oxide and its biomedical application

Abstract Graphene oxide (GO) is one of the most attributed materials for opening new possibilities in the development of next generation biosensors. Due to the coexistence of hydrophobic domain from pristine graphite structure and hydrophilic oxygen containing functional groups, GO exhibits good water dispersibility, biocompatibility, and high affinity for specific biomolecules as well as properties of graphene itself partly depending on preparation methods. These properties of GO provided a lot of opportunities for the development of novel biological sensing platforms, including biosensors based on fluorescence resonance energy transfer (FRET), laser desorption/ionization mass spectrometry (LDI-MS), surface-enhanced Raman spectroscopy (SERS), and electrochemical detection. In this review, we classify GO-based biological sensors developed so far by their signal generation strategy and provide the comprehensive overview of them. In addition, we offer insights into how the GO attributed in each sensor system and how they improved the sensing performance.

Cytoprotective effects of graphene oxide for mammalian cells against internalization of exogenous materials

To date, graphene oxide (GO), an oxidized version of graphene, has been utilized in many research areas including bioapplications such as drug delivery and bioanalysis. Unlike other spherical or polygonal nanomaterials, GO exhibits a sheet-like structure, which in itself suggests interesting applications based on its shape. Here we show that GO can protect cells from internalization of toxic hydrophobic molecules, nanoparticles, and nucleic acids such as siRNA and plasmid DNA by interacting with cell surface lipid bilayers without noticeably reducing cell viability. Furthermore, the cytoprotective effect of GO against the internalization of extracellular materials enabled spatial control over gene transfection through region-selective gene delivery only into GO-untreated cells, and not into the GO-treated cells.

Graphene oxide for fluorescence-mediated enzymatic activity assays

Graphene oxide (GO), an oxidized 2-dimensional carbon material, has been studied actively in both academia and industry due to its unique physicochemical properties including high surface area, excellent aqueous dispersibility, quenching capability of contiguous fluorophores and adsorption affinity with diverse biomolecules. Particularly, GO has been widely used in biological applications involving small molecule biosensors, enzymatic reaction monitoring, drug delivery and therapeutic applications. Among these rapidly developing fields, we focused on the current advancements of fluorescence mediated activity assay platforms for enzymes including nucleases, methyltransferases, protein kinases, caspases and helicases by using affinity and fluorescence quenching capability of GO with fluorophore labelled biomolecule substrates in the aqueous solution phase, and its important applications with future perspectives in this article.

Direct, sequence-specific detection of dsDNA based on peptide nucleic acid and graphene oxide without requiring denaturation

Sequence-specific detection of double stranded DNA (dsDNA) is important in various research fields. In general, denaturation of dsDNA into single strands is necessary for the sequence-specific recognition of probes to target DNA, posing several drawbacks which decrease the efficiency as a DNA sensor. Herein, we report a direct, sequence-specific dsDNA detection system without requiring any thermal denaturing step. Our strategy utilizes peptide nucleic acid (PNA) and graphene oxide (GO) as a probe and as a fluorescence quencher, respectively. The PNA first binds to the end of dsDNA strand due to the relatively easily dissociable terminal base pairs of DNA duplex. Next, superior binding affinity of PNA towards complementary DNA induces branch migration for gradual strand replacement, resulting in the formation of PNA/DNA duplex. Unlike other dsDNA sensors based on complementary DNA probes, PNA in combination with GO enabled hybridization with the target sequence hidden as a duplex form without denaturing step and thus, the formation of PNA/DNA duplex was translated into selective fluorescence signal. Moreover, it provided tighter turn-on signal control with very low background signal and high sensitivity and sequence selectivity even in the presence of serum proteins.

BSA as additive: A simple strategy for practical applications of PNA in bioanalysis

Application of peptide nucleic acid (PNA) in bioanalysis has been limited due to its nonspecific adsorption onto hydrophobic surface in spite of favorable properties such as higher chemical/biological stability, specificity and binding affinity towards target nucleic acids compared to natural nucleic acid probes. Herein, we employed BSA in PNA application to enhance the stability of PNA in hydrophobic containers and improve the sensing performance of the DNA sensor based on graphene oxide (GO) and PNA. Addition of 0.01% BSA in a PNA solution effectively prevented the adsorption of PNA on hydrophobic surface and increased the portion of the effective PNA strands for target binding without interfering duplex formation with a complementary target sequence. In the GO based biosensor using PNA, BSA interrupted the unfavorable adsorption of PNA/DNA duplex on GO surface, while allowing the adsorption of ssPNA, resulting in improvement of the performance of the DNA sensor system by reducing the detection limit by 90-folds.

A robust and quantitative assay platform for multiplexed, high throughput screening of protein kinase inhibitors

We present a new platform for multiplexed protein kinase activity assay using TiO2 decorated graphene oxide (GO), which is applicable to high throughput inhibitor screening. On the basis of the strong affinity of TiO2 for the phosphate group and the fluorescence quenching capability of GO, phosphorylation of substrates by protein kinases was quantitatively measured in a short time.

Evaluation of the pharmacokinetic and pharmacodynamic drug interactions between cilnidipine and valsartan, in healthy volunteers

Purpose Although cilnidipine and valsartan are widely coadministered to patients with hypertension, their drug–drug interaction potential has not been investigated. This study compared the pharmacokinetic (PK), pharmacodynamic (PD), and tolerability profiles of cilnidipine and valsartan, both alone and in combination, in healthy male subjects. Patients and methods Fifty-four subjects, enrolled into an open-label, single-dose, three-treatment, three-period crossover study, randomly received cilnidipine (10 mg), valsartan (160 mg), or both according to one of six sequences. Blood samples were collected at baseline and up to 24 hours after drug administration in each period. Plasma concentrations of cilnidipine and valsartan were determined by liquid chromatography with tandem mass spectrometry. Maximum plasma concentration (Cmax) and area under the concentration-time curve from 0 to the last measurable time (AUClast) were estimated using a noncompartmental method. Tolerability was evaluated by assessing adverse events (AEs), vital signs, electrocardiograms, and clinical laboratory tests. Blood pressure was also measured for PD assessment. Results A total of 51 subjects completed the study. The PK profile of cilnidipine was not significantly affected by coadministered valsartan; the geometric mean ratio and 90% confidence interval (90% CI) of AUClast for cilnidipine with and without valsartan was 1.04 (0.98–1.10). Likewise, cilnidipine did not affect the PK of valsartan; the geometric mean ratio (90% CI) of AUClast for valsartan with and without cilnidipine was 0.94 (0.83–1.07). Coadministration of cilnidipine and valsartan reduced blood pressure in an additive way. No serious AEs were reported, and both cilnidipine and valsartan were well tolerated. Conclusion Coadministered cilnidipine and valsartan do not cause a significant PK or PD interaction, and they are well tolerated.