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Advanced Techniques for the Analysis of Protein and RNA

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A special issue of Chemosensors (ISSN 2227-9040). This special issue belongs to the section "Analytical Methods, Instrumentation and Miniaturization".

Deadline for manuscript submissions: closed (31 December 2023) | Viewed by 21067

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Special Issue Editor

Prof. Dr. Xiaolong Yang

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Guest Editor

School of Medicine, Queen's University, Kingston, ON, Canada
Interests: molecular biology; cell biology; cancer biology; drug resistance; signal transduction; RNAseq; proteomics; bioluminescent biosensors

Special Issue Information

Dear Colleagues,

Protein and RNA are of great functional and structural importance in life activities and play important roles in diverse cellular functions. They are involved in a wide variety of different biological processes in growth and differentiation at various stages of the cell cycle. Therefore, the rapid and accurate detection of proteins and RNAs plays a vital role in the biomedical field.

With the development of many new analytical techniques in the last few years, this field has experienced rapid progress. Exploring the roles of protein and RNA in life activities using mass spectrometry, spectral analysis, microfluidic analysis, biosensors technologies and other new analytical techniques has the tremendous potential to revolutionize healthcare. It will greatly improve the quality of life of millions of people suffering from a wide variety of diseases.

This Special Issue aims to provide a comprehensive collection of the latest advances in this technology. We cordially invite you to submit an article to this Special Issue. We welcome short communications, full research articles, and timely reviews, focusing on advanced in protein/RNA analysis techniques.

Prof. Dr. Xiaolong Yang
Guest Editor

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Keywords

protein
RNA
mass spectrometry
spectral analysis
microfluidic device
biosensors
new analysis techniques

Published Papers (11 papers)

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Editorial

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4 pages, 190 KiB

Open AccessEditorial

Advanced Techniques for the Analysis of Proteins and RNAs

by Xiaolong Yang

Chemosensors 2024, 12(1), 12; https://doi.org/10.3390/chemosensors12010012 - 10 Jan 2024

Cited by 1 | Viewed by 1406

Abstract

Proteins and RNAs, as fundamental components of cellular machinery, play pivotal roles in the intricate landscape of life [...] Full article

(This article belongs to the Special Issue Advanced Techniques for the Analysis of Protein and RNA)

Research

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15 pages, 3538 KiB

Open AccessArticle

Development of a New HiBiT Biosensor Monitoring Stability of YAP/TAZ Proteins in Cells

by Liqing Wu, Anni Ge, Yawei Hao and Xiaolong Yang

Chemosensors 2023, 11(9), 492; https://doi.org/10.3390/chemosensors11090492 - 6 Sep 2023

Cited by 2 | Viewed by 1516

Abstract

The Hippo signaling cascade is frequently dysregulated in a variety of cancers, such as breast cancer (BC), which is one of the most commonly diagnosed malignancies in women. Among BC subtypes, triple-negative BC (TNBC) stands out due to its poor prognosis and high metastatic potential. Despite extensive research aimed at establishing treatment options, existing therapies demonstrate limited efficacy for TNBC. Recently, it has been recognized that targeting the core components of the Hippo pathway (YAP and its paralog TAZ) is a promising strategy for developing anti-cancer treatment. However, no YAP/TAZ inhibitors have been approved by the FDA as anti-TNBC treatments, and only a few compounds have been identified that directly affect YAP and TAZ activity and stability to enhance the prospect of innovative HiBiT biosensors for monitoring of YAP and TAZ in cells. Employing these biosensors, we conducted a small-scale drug screen involving 279 compounds, leading to the identification of several small molecule inhibitors (SMIs) capable of inducing YAP/TAZ degradation in diverse TNBC cell lines. It is worth noting that some drugs may indirectly affect the protein stability following prolonged treatment, and a shorter exposure can be included in the future to identify drug candidates with more direct effects. Nevertheless, our study introduces a novel approach for assessing YAP and TAZ levels, which can have significant implications for developing anti-TNBC targeted therapies. Full article

(This article belongs to the Special Issue Advanced Techniques for the Analysis of Protein and RNA)

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15 pages, 2092 KiB

Open AccessArticle

Trypsin-Based Chemoenzymatic Assay for Detection of Pollutants and Safety Assessment of Food Additives

by Elena N. Esimbekova, Irina G. Torgashina, Elena V. Nemtseva, Anna A. Antashkevich, Polina Yu. Sasova and Valentina A. Kratasyuk

Chemosensors 2023, 11(4), 237; https://doi.org/10.3390/chemosensors11040237 - 10 Apr 2023

Cited by 3 | Viewed by 1552

Abstract

Chemoenzymatic assay systems are widely used to detect toxicants in various samples, including food and environment specimens. These methods are based on the ability of various types of toxicant to specifically inhibit/activate the functions of individual enzymes or enzyme systems. The present study examines the possibility of using the proteolytic enzyme trypsin as a specific marker to detect protease inhibitors in different samples. The study shows that trypsin activity is not affected by various heavy metals, pesticides, or quinones at levels considerably greater than their maximum allowable concentrations (MACs) in water bodies. At the same time, the IC₅₀ value for the food preservative potassium sorbate (E202) is 15 mg/L, which is substantially lower than its acceptable daily intake (ADI). The quenching of trypsin fluorescence in the presence of potassium sorbate suggests that inhibition could occur due to the binding of the preservative to the enzyme in the region adjacent to the active center. The trypsin was immobilized in starch gel to ensure its stability in the enzyme inhibition based assay. Single-use reagents were prepared as dry starch disks that could be stored over long periods. Their sensitivity to copper (II) chloride, potassium sorbate, and chromium (III) chloride was similar to the sensitivity of the free trypsin. Full article

(This article belongs to the Special Issue Advanced Techniques for the Analysis of Protein and RNA)

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15 pages, 6087 KiB

Open AccessArticle

Design of Peptide Ligand for Lactoferrin and Study of Its Binding Specificity

by Tatiana Zimina, Nikita Sitkov, Vladimir Karasev, Yury Skorik, Alexey Kolobov, Alexander Kolobov, Nikolay Bunenkov and Viktor Luchinin

Chemosensors 2023, 11(3), 162; https://doi.org/10.3390/chemosensors11030162 - 27 Feb 2023

Cited by 4 | Viewed by 1361

Abstract

The in silico modelling of peptides complementary to lactoferrin was carried out using the Protein 3D software package and replication of the natural bonding site between pneumococcal surface protein (PSP) and lactoferrin (LF). The modeling was based on analysis of the conjugated ion–hydrogen bond systems between these proteins (CIHBS). The oligopeptide EEVAPQAQAKIAELENQVHRLE was proposed via computer modelling and synthesized using the solid phase synthesis technique, purified, and analyzed with MS and HPLC methods to confirm >95% purity. The peptide was then studied by capillary electrophoresis (CE). The CE experiments demonstrated the split of peptide zone in the presence of LF, due to complex formation and subsequent mobility change of the system peptide-protein. The reference experiments with homomyeloperoxidase and myoglobin did not show binding with LETI-11. Full article

(This article belongs to the Special Issue Advanced Techniques for the Analysis of Protein and RNA)

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11 pages, 1988 KiB

Open AccessArticle

Highly Efficient, Non-Covalent Functionalization of CVD-Graphene via Novel Pyrene-Based Supporter Construct

by Misbah Shahzadi, Sobia Nisar, Deok-Kee Kim, Nasir Sarwar, Aamir Rasheed, Waqas Ahmad, Amir Muhammad Afzal, Muhammad Imran, Mohammed Ali Assiri, Zafar M. Shahzad and Ghulam Dastgeer

Chemosensors 2023, 11(2), 83; https://doi.org/10.3390/chemosensors11020083 - 21 Jan 2023

Cited by 11 | Viewed by 2113

Abstract

Ultra-thin two-dimensional (2D) materials have attained huge interest for biosensing applications because of their strong electrostatic coupling with target molecules such as spike proteins and DNA. One such 2D material is graphene, which is extremely thin and flexible and has a strong non-covalent interaction with the supporting constructs needed to detect biomolecules. This work aimed to develop a way to efficiently functionalize the surface of 2D material using a pyrene-based supporter construct to detect the target protein. For this purpose, high-quality, pristine graphene was grown via the chemical vapor deposition (CVD) method and transferred over the Si/SiO₂ substrate for its functionalization using our engineered pyrene–lysine-based supporter construct (PLB). The construct was synthesized using the solid-phase peptide synthesis (SPPS) method and utilized to functionalize the graphene-channel-based field-effect transistor (FET) device via non-covalent π−π stacking interaction. The optimum concentration of the functionalized PLB was evaluated via atomic force microscopy (AFM), Raman spectroscopy, and real-time electrical measurements. The characterization techniques successfully provide an overview of the effect of the concentration of PLB used for functionalization. Moreover, the performance was tested and compared in terms of the percentage response of the device generated after the detection of various concentrations of the streptavidin protein. This research could be useful in determining how to functionalize any 2D material by designing a supporter construct without material degradation and owing to over-stacking or bypassing surface screening effects. Full article

(This article belongs to the Special Issue Advanced Techniques for the Analysis of Protein and RNA)

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20 pages, 3683 KiB

Open AccessArticle

Fast Protein and Metabolites (Nucleotides and Nucleosides) Liquid Chromatography Technique and Chemical Sensor for the Assessment of Fish and Meat Freshness

by Artur Kuznetsov, Aleksandr Frorip, Alar Sünter, Nensi Kasvand, Vadim Korsakov, Georgii Konoplev, Oksana Stepanova, Linda Rusalepp, Dea Anton, Tõnu Püssa, Mati Roasto, Liubov Abramova, Andrey Kozin, Lauri Toom, Soeren Hirsch and Nikolay Mukhin

Chemosensors 2023, 11(1), 69; https://doi.org/10.3390/chemosensors11010069 - 14 Jan 2023

Cited by 7 | Viewed by 2355

Abstract

Fast protein and metabolite liquid chromatography (FPLMC) was introduced years ago to enable the easy separation of high-molecular compounds such as proteins from small molecules and the identification of the low-molecular substances. In this paper, the method is applied for the rapid evaluation of freshness and monitoring the aging of animal meat and fish. A novel chromatographic sensor was developed with a deep UV LED-based photometric detection unit (255–265 nm), an original flow cuvette and registration scheme; the processing of a chromatogram with the sensor takes approximately 15 min. Strict isochronism between the elution of ATP metabolites, mainly hypoxanthine (Hx) and inosine monophosphate (IMP), and the time of maturation of meat or fish, was discovered. A new freshness index H* = [Hx]/[IMP] was introduced, which is proportional to the instrumental delay time in the FPMLC chromatograms: the H* index < 0.5 indicates the presence of inosine monophosphate (IMP) and the high quality of the meat or fish. Reasonably strong correlations were revealed between data obtained by FPMLC and total volatile basic nitrogen TVB-N (for fish) or volatile fatty acids VFA (for meat) content. Moreover, putative nucleotide salvage and an increase in the concentration of IMP were observed in fish after heat treatment using the FPMLC sensor and NMR technique. Full article

(This article belongs to the Special Issue Advanced Techniques for the Analysis of Protein and RNA)

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17 pages, 5166 KiB

Open AccessArticle

Impedimetric Biosensor Coated with Zinc Oxide Nanorods Synthesized by a Modification of the Hydrothermal Method for Antibody Detection

by Nikita Sitkov, Andrey Ryabko, Alexey Kolobov, Alexsandr Maximov, Vyacheslav Moshnikov, Stanislav Pshenichnyuk, Alexei Komolov, Andrey Aleshin and Tatiana Zimina

Chemosensors 2023, 11(1), 66; https://doi.org/10.3390/chemosensors11010066 - 13 Jan 2023

Cited by 2 | Viewed by 1827

Abstract

Impedimetric biosensors are used for detecting a wide range of analytes. The detection principle is a perspective for the development of new types of analytical devices for biomolecular diagnosis of diseases. Of particular interest are biosensors with very high sensitivities, capable of detecting trace amounts of biomarkers or drugs in biological fluids. Impedimetric biosensors possess a potential for increased sensitivity, since their electrodes can be modified with nanostructured materials, in particular zinc oxide. In this work, a miniature biosensor with an array of zinc oxide nanorods synthesized by the hydrothermal method has been created. Protein A was immobilized on the resulting structure, which was previously tested for binding to omalizumab by capillary electrophoresis. Using impedance spectroscopy, it was possible to detect the binding of omalizumab at concentrations down to 5 pg/mL. The resulting structures are suitable for creating reusable biosensor systems, since ZnO-coated electrodes are easily cleaned by photocatalytic decomposition of the bound molecules. The biosensor is promising for use in Point-of-Care systems designed for fast, multimodal detection of molecular markers of a wide range of diseases. Full article

(This article belongs to the Special Issue Advanced Techniques for the Analysis of Protein and RNA)

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8 pages, 1765 KiB

Open AccessCommunication

Comparison of Colorimetric and Fluorometric Chemosensors for Protein Concentration Determination and Approaches for Estimation of Their Limits of Detection

by Anastasiya A. Mamaeva, Vladimir I. Martynov, Sergey M. Deyev and Alexey A. Pakhomov

Chemosensors 2022, 10(12), 542; https://doi.org/10.3390/chemosensors10120542 - 17 Dec 2022

Cited by 2 | Viewed by 1526

Abstract

Here, we present a direct comparison of different dyes and assays for the determination of protein concentrations. We compared the classical Bradford assay with two modern assays based on the fluorogenic dyes QuDye and ProteOrange and showed that the Bradford reagent achieved excellent results in the determination of protein concentrations as compared with more modern rivals. We also showed that standard approaches for determining the limit of detection (LoD) and limit of quantification (LoQ) may not work correctly with the tested dyes. We proposed a new approach that extends the standard algorithm for LoD and LoQ determination. This approach works well with both classical colorimetric and fluorogenic dyes, as well as with nontrivial fluorescent probes. Full article

(This article belongs to the Special Issue Advanced Techniques for the Analysis of Protein and RNA)

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14 pages, 2194 KiB

Open AccessArticle

Multi-Endpoint Toxicity Tests and Effect-Targeting Risk Assessment of Surface Water and Pollution Sources in a Typical Rural Area in the Yellow River Basin, China

by Fangxu Li, Jisui Tan, Qian Yang, Miao He, Ruozhen Yu, Chun Liu and Xiaohong Zhou

Chemosensors 2022, 10(12), 502; https://doi.org/10.3390/chemosensors10120502 - 28 Nov 2022

Cited by 3 | Viewed by 1346

Abstract

Multi-endpoint toxicity tests were used to evaluate the acute toxicity, estrogenic activity, neurotoxicity, genotoxicity, and ecological risks of surface water and sewage from possible pollution sources in rural areas of the Yellow River (China). Toxicity testing results showed that the luminescence inhibition rates of acute toxicity ranged from not detected (ND) to 38%, the 17β-estradiol equivalent (E2-EQ) values of estrogenic activity ranged from 4.8 to 131.0 ng·L⁻¹, neurotoxicity was not detected, and the protein effect level index (PELI) values of genotoxicity ranged from 1 to 6.06. Neither acute toxicity nor genotoxicity were detected in the tributaries of the Yellow River (River 2) flowing through the investigated rural area. The distribution of high estrogenic activity sites was relatively scattered, but mainly located in the tributaries of River 2. Industrial, domestic, and livestock and poultry breeding sewage were all possible sources of toxicity, and the contribution of livestock and poultry to environmental estrogens in the surface water was significant. Furthermore, the potential effect-targeting risks of toxic substances in the surface water for aquatic organisms were assessed using the risk quotient method, by considering the toxic equivalent concentration. The results indicated that the risk of estrogenic activity was the main ecological risk in the surface water of this rural area. Except for the reservoir site, the other sampling sites showed a moderate to high estrogenic activity risk, especially in the tributaries of River 2. Full article

(This article belongs to the Special Issue Advanced Techniques for the Analysis of Protein and RNA)

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Review

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28 pages, 9468 KiB

Open AccessReview

MicroRNA Biosensors for Early Detection of Hepatocellular Carcinoma

by Xiaogang Lin, Ke Wang, Chunfeng Luo, Mengjie Yang and Jayne Wu

Chemosensors 2023, 11(9), 504; https://doi.org/10.3390/chemosensors11090504 - 16 Sep 2023

Cited by 2 | Viewed by 1658

Abstract

Hepatocellular carcinoma (HCC) is the main pathological type of liver cancer. Due to its insidious onset and the lack of specific early markers, HCC is often diagnosed at an advanced stage, and the survival rate of patients with partial liver resection is low. Non-coding RNAs (ncRNAs) have emerged as valuable biomarkers for HCC detection, with microRNAs (miRNAs) being a particularly relevant class of short ncRNAs. MiRNAs play a crucial role in gene expression regulation and can serve as biomarkers for early HCC detection. However, the detection of miRNAs poses a significant challenge due to their small molecular weight and low abundance. In recent years, biosensors utilizing electrochemical, optical, and electrochemiluminescent strategies have been developed to address the need for simple, rapid, highly specific, and sensitive miRNA detection. This paper reviews the recent advances in miRNA biosensors and discusses in detail the probe types, electrode materials, sensing strategies, linear ranges, and detection limits of the sensors. These studies are expected to enable early intervention and dynamic monitoring of tumor changes in HCC patients to improve their prognosis and survival status. Full article

(This article belongs to the Special Issue Advanced Techniques for the Analysis of Protein and RNA)

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21 pages, 2237 KiB

Open AccessReview

Paper-Based Loop Mediated Isothermal Amplification (LAMP) Platforms: Integrating the Versatility of Paper Microfluidics with Accuracy of Nucleic Acid Amplification Tests

by Debayan Das, Manaswini Masetty and Aashish Priye

Chemosensors 2023, 11(3), 163; https://doi.org/10.3390/chemosensors11030163 - 28 Feb 2023

Cited by 6 | Viewed by 3402

Abstract

Paper-based diagnostics offer a promising alternative to traditional diagnostic methods for point-of-care use due to their low cost, ease of use, portability, rapid results, versatility, and low environmental impact. While paper-based serology tests in the form of lateral flow assays can provide rapid test results for past pathogen exposure, they currently lack the accuracy and sensitivity offered by molecular diagnostic tests such as the polymerase chain reaction (PCR). Loop-mediated isothermal amplification (LAMP)—an isothermal nucleic acid amplification test (NAAT)—provides PCR-like performance while simultaneously reducing the instrumentation and assay complexity associated with PCR. In this review, we discuss a newly emerging class of paper-based LAMP platforms that integrates the versatility of paper microfluidics with the accuracy of NAATs. Since its first adoption in 2015, we have discussed all paper-based LAMP platforms in terms of the paper substrates, reagent incorporation techniques, paper platform design, heating hardware, detection methods, and sensitivity and specificity of paper-based LAMP assays. We conclude by identifying the current challenges and future prospects of paper-based NAATs. Full article

(This article belongs to the Special Issue Advanced Techniques for the Analysis of Protein and RNA)

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