Magnetic graphene oxide nanocomposite as dual-mode genosensor for ultrasensitive detection of oncogenic microRNA (2023)

Table of Contents
Microchemical Journal Abstract Graphical abstract Introduction Section snippets Materials Detection mechanism and fabrication of the genosensor Conclusion CRediT authorship contribution statement Declaration of Competing Interest Acknowledgements References (44) Highly sensitive and simultaneous detection of microRNAs in serum using stir-bar assisted magnetic DNA nanospheres-encoded probes Biosens. Bioelectron. Electrochemical determination of miRNA-155 using molybdenum carbide nanosheets and colloidal gold modified electrode coupled with mismatched catalytic hairpin assembly strategy Microchem. J. Electrochemical detection of microRNAs based on AuNPs/CNNS nanocomposite with Duplex-specific nuclease assisted target recycling to improve the sensitivity Talanta Bio-Inspired Nacre-like Layered Hybrid Structure of Calcium Carbonate under the Control of Carboxyl Graphene CrstEngComm Influence of graphene microstructures on electrochemical performance for supercapacitors Prog. Nat. Sci.: Mater. Int. Au-SH-SiO2 nanoparticles supported on metal-organic framework ([emailprotected]) as a sensor for electrocatalytic oxidation and determination of hydrazine Electrochim Acta Synergistic removal of arsanilic acid using adsorption and magnetic separation technique based on [emailprotected] graphene nanocomposite J. Ind. Eng. Chem. Polyaniline-nanofiber-modified screen-printed electrode with intermediate dye amplification for detection of endocrine disruptor bisphenol A Microchem. J. Laser induced self-N-doped porous graphene as an electrochemical biosensor for femtomolar miRNA detection Carbon Methylene blue embedded duplex DNA as an efficient signal stimulator of petal-like BiVO4 for ultrasensitive photoelectrochemical bioassay Anal. Chim. Acta Ultrasensitive electrochemical sensing platform for microRNA based on tungsten oxide-graphene composites coupling with catalyzed hairpin assembly target recycling and enzyme signal amplification Biosens. Bioelectron. Electrochemical determination of microRNA-21 based on graphene LNA integrated molecular beacon, AuNPs and biotin multifunctional bio bar codes and enzymatic assay system Biosensors & bioelectronics Electrochemical immunosensor for serum parathyroid hormone using voltammetric techniques and a portable simulator Anal. Chim. Acta Current review about design's impact on analytical achievements of magnetic graphene oxide nanocomposites TrAC Trends Anal. Chem. Synthesis, properties, and applications of graphene oxide/reduced graphene oxide and their nanocomposites, Nano Mater. Sci. Design, development and evaluation of microRNA-199a-5p detecting electrochemical nanobiosensor with diagnostic application in Triple Negative Breast Cancer Talanta PAMAM dendrimer modified screen printed electrodes for impedimetric detection of miRNA-34a Microchem. J. Pd nanoparticles-DNA layered nanoreticulation biosensor based on target-catalytic hairpin assembly for ultrasensitive and selective biosensing of microRNA-21 Sens. Actuators B Sensors for diagnosis of prostate cancer: Looking beyond the prostate specific antigen Biosens. Bioelectron. Surface-enhanced Raman scattering biosensors for detection of oncomiRs in breast cancer Drug Discov. Today Recent trends in application of nanomaterials for the development of electrochemical microRNA biosensors Microchim. Acta Magnetic Beads-Based Sensor with Tailored Sensitivity for Rapid and Single-Step Amperometric Determination of miRNAs Int. J. Mol. Sci. Cited by (0) Recommended articles (6) Carbon quantum dot fluorescent probe for labeling and imaging of stellate cell on liver frozen section below freezing point High fluorescent nitrogen−doped carbon dots derived from Sanghuangporus lonicericola for detecting tetracyclines in aquaculture water and rat serum samples A light-up fluorescence probe for wash-free analysis of Mu-opioid receptor and ligand-binding events Bi-functional antibody-CRISPR/Cas12a ribonucleoprotein conjugate for improved immunoassay performance Theoretical analysis of surface plasmon resonance in capillary sensors Ultrasensitive visual detection of the food-borne pathogen via MOF encapsulated enzyme

Microchemical Journal

Volume 191,

August 2023

, 108775

Author links open overlay panel, , , ,

Abstract

MicroRNA (miRNA) is a noncoding RNA that controls cellular functions and gene expression. Several oncogenic miRNAs that aberrantly expressed in prostate cancer have the potential to be used as biomarkers. We designed multifunctional nanosheets that can capture, detect, and quantify miRNA 183-5p from prostate cancer cells with the aid of a disposable printed electrode and a portable potentiostat. Magnetic reduced graphene oxide (MrGO) has been used as the starting nanocomposite to analyze miRNA. Three cationic dyes—toluidine blue (TBO), thionine, and neutral red—were used to modify MrGO and evaluate its impact on the electron transfer rate. MrGO modified with TBO had the fastest conductivity and a large electrochemically active surface area. Two strategies were used to detect miRNA. One used peroxidase-labeled amplification and the other used TBO as the redox probe intercalating in the miRNA-capture probe duplex. The intercalator method reduced the complications of using peroxidase-labeled probes and exhibited superior performance. The limits of miRNA detection in human serum and urine were 3.73 and 0.86 aM, respectively, with a linear range from 0.1nM to over 1 aM. The assay time of the intercalator method, including wash, was less than 16min, and only one sample droplet (5μL) was needed for analysis. We provided dual-mode genosensors for miRNA detection, which might be used for point-of-care testing. The incorporation of MrGO, screen-printed carbon electrodes, and portable potentiostat can accelerate biomarker detection, simplify analysis, and reduce the time and cost of analysis.

Introduction

MicroRNAs (miRNAs) control the posttranscriptional expression of gene, and they affect several cellular processes. The overexpression of oncogenic miRNAs (oncomiRs) is related to the initiation, promotion, transformation, and metastasis of cancer by downregulating tumor-suppressor genes [1]. Since miRNAs are useful biomarkers in clinical practice, sensing technologies to quantitatively measure the levels of their expression are crucial. Generally, the methods for miRNA analysis are reverse transcription polymerase chain reaction (rt-PCR), PCR array, and northern blotting. However, these traditional technologies require expertise, expensive instruments, and long procedures [2]. Nanomaterial-based biosensors and portable electrochemical devices could provide rapid and sensitive detection of miRNAs, and they may also be used for point-of-care testing (POCT) [3]. Several electrochemical methods have been used to detect miRNA, including cyclic voltammetry (CV) [4], differential pulse voltammetry (DPV) [5], square wave voltammetry (SWV) [6], and electrochemical impedance spectroscopy (EIS) [7].

EIS has been applied to detect miRNA-199a-5p in serum from breast cancer patients by using a glassy carbon electrode modified with graphene oxide (GO) and a gold nanorod. Thiolated capture probes immobilized on the electrode can form DNA–RNA hybrids. The EIS biosensor offered a 4.5 fM detection limit in serum samples, with a linear range of detection between 15 fM and 148pM [8]. GO possesses abundant hydroxyl, epoxide, and carboxyl groups [9], [10] with advantages such as facile functionalization and low cost [11]. Reduced GO (rGO) offers further advantages, such as good conductivity, thermal stability, and large surface area [12]. The GO/rGO-modified electrodes have gradually replaced gold and mercury electrodes because of their good conductivity, low price, and easy modification. In addition, the use of magnetic nanocomposites in sensing has significantly reduced analysis time and simplified extraction by using an external magnet to enrich targets. Magnetic rGO (MrGO) has the sensing advantages of a large conductive surface area, rapid magnetic separation and enrichment of targeted compounds [13]. With their superparamagnetism and reduced size, MrGO effectively capture and concentrate targets using capture molecules and magnets.

Voltammetry is a popular and cost-effective technology used in electrochemical biosensors by adjusting the decay rates of charging and faradaic currents to obtain a lower detection limit and higher sensitivity [12], [14]. Sandwich-type biosensors using capture probes and detection probes labeled with peroxidase such as horseradish peroxidase (HRP) or phosphatase to catalyze substrate and record electrical signals using voltammetry have been well reported [15], [16]. The hybridization of target genes to the capture probe on the electrode surface also can change the electrochemical impedance, which can be used to detect the target gene. Another method to report the hybridization of target miRNA with the capture probe immobilized on the electrode surface was to use a small redox-active mediator molecule that could be intercalated into the duplex formed. In this detection of miRNAs, complementary DNA/RNA probes first hybridize with the target miRNA. The redox probes intercalated into the double helixes can then help generate currents that are proportional to the amount of miRNA. The major forces that the indicators react with the duplex nucleotides are π-π and hydrophobic interaction, groove binding, and electrostatic adsorption [17]. By inserting planar and aromatic rings between the nearby G-C pairing bases, these redox chemicals can reversibly intercalate into the DNA helix [18]. Several redox intercalators have been applied to electrochemical biosensors for miRNA and gene detection. For instance, an electrochemical genosensor that used methylene blue as the intercalator was able to detect miRNA-21 down to 0.3fM with a linear range of 5.0fM to 10nM [19]. Methylene blue can electrochemically detect TP-53 gene using the affinity of heterocyclic ring and the bases of the DNA sequence [20]. Wan et al. detected miRNA-486-5p with a nitrogen-doped graphene electrode using magnetic materials and ferricyanide as the redox probe. The detection of miRNA was shown to be as low as 10 fM with excellent reproducibility [21]. Breast and prostate cancers exhibit dysregulation of the miRNA-183 family, which includes miRNA-96, −182, and −183. These miRNAs may be exploited as biomarkers for prostate cancer [22]. Redox mediators like methylene blue, neutral red, and thionine also can enhance the electron transfer rate and reduce working potential [23], [24].

For this work, we designed and synthesized dual-mode MrGO that could target, detect, and quantify miR-183-5p. Two methods were systematically compared: the use of peroxidase-labeled probe (the HRP method) and the use of redox-active intercalators inserted in the duplex formed (the intercalator method). The magnet-aided screen printed electrode (SPE) integrated with MrGO have several benefits. The wash process is easy since the removal of centrifugation step. Due to the quick capture and detection using MrGO, the detection time can be reduced to few minutes. Less chemical reagents and tiny sample size are needed, which can greatly reduce the cost of detection.

Section snippets

Materials

Graphene, hydroquinone (HQ), 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC), N-hydroxysuccinimide (NHS), sodium phosphate dibasic, sodium phosphate monobasic, bovine serum albumin (BSA), potassium ferricyanide, TBO, neutral red (NR), thionine acetate (Thi), sodium dodecyl sulfate (SDS), Trizol, isopropanol, and human serum were from Sigma-Aldrich (St. Louis, MO, USA). Hydrogen peroxide (30%) was from Fluka (Charlotte, NC, USA). phosphate buffer solution (PBS, 0.1M and pH

Detection mechanism and fabrication of the genosensor

The detection mechanism and fabrication steps of the developed genosensor for detecting miRNA are shown in Fig. 1. MrGO was used as the matrix, with TBO modification, to further increase the transfer rate of electrons and act as the platforms for probe immobilization and electrochemical reactions. Cp is complementary to miRNA-185, and it contains the amine group that forms an amide bond with the carboxyl group, activated by EDC/NHS on TBO/MrGO. When the target was present, the miRNA could be

Conclusion

In this study, MrGO was developed with the functions of capturing, detecting, and quantifying miRNA 183-5p using a screen-printed electrode and a portable potentiostat. The magnet-aided SPE features an external magnet on the rear that can quickly gain magnetic forces through the insertion of external magnets. The magnet-aided SPE can simultaneously concentrate and analyze by target-captured MrGO. After detection, magnetic nanocomposites can be washed away from the electrode by detaching the

CRediT authorship contribution statement

Pinpinut Kabinsing: Data curation, Investigation, Writing – original draft. Pravanjan Malla: Data curation, Investigation. Chi-Hsien Liu: Investigation, Methodology, Resources, Writing – original draft. Wei-Chi Wu: Methodology, Resources. Paiboon Sreearunothai: Data curation, Writing – original draft.

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgements

We express gratitude to the Ministry of Science and Technology (MOST 111-2221-E-182-016) and Chang Gung Memorial Hospital (CMRPD 1M0681, 1M0601) for funding and supporting this research. The authors thank the Instrumentation Center and Microscopy Center @CGU for technical assistance.

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