MazF-rolling circle amplification combined MALDI-TOF MS for site-specific detection of N6-methyladenosine RNA.

N6-methyladenosine (m6A) is one of the most abundant chemical modifications in RNA and has vital significance in cellular processes and tumor development. However, the accurate analysis of site-specific m6A modification remains a challenge. In this work, a MazF endoribonuclease activated rolling circle amplification (MazF-RCA) combined MALDI-TOF MS assay is developed for the detection of site-specific m6A-RNA. MazF endoribonuclease can specifically cleave the ACA motif, leaving methylated (m6A)CA motif intact. The intact methylated RNA can then be amplified through rolling circle amplification, and the generated reporter oligonucleotides are detected by MALDI-TOF MS. The assay exhibits good quantification ability, presenting a wide linear range (100 fM to 10 nM) with the limit-of-detection lower than 100 fM. Additionally, the assay can accurately detect methylated RNA in the presence of large amount of non-methylated RNA with a relative abundance of methylated RNA down to 0.5%. The developed assay was further applied to detect m6A-RNA spiked in MCF-7 cell RNA extracts, with the recovery rates in the range of 90.64-106.93%. The present assay provides a novel platform for the analysis of site-specific m6A-RNA at high specificity and sensitivity, which can promote the study of RNA methylation in clinical and biomedical research.

Multiplexed discrimination of SARS-CoV-2 variants via duplex-specific nuclease combined MALDI-TOF MS.

The frequent mutations in SARS-CoV-2 significantly increase the virus's pathogenicity and transmissibility while also diminishing the effectiveness of vaccines. Consequently, assays capable of rapidly and simultaneously identifying multiple SARS-CoV-2 variants are essential for large-scale applications that aim to monitor the evolution of the virus. In this work, we propose a method combining duplex-specific nuclease (DSN)-assisted cyclic amplification with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) detection, enabling the simultaneous identification of multiple SARS-CoV-2 variants at high-throughput. Due to the high specificity of DSN, single-base mutations can be resolved by the method. With ultra-sensitive detection by MALDI-TOF MS, a limit of detection of 100 pM viral RNA fragment was demonstrated. The assay was used for simultaneous identification and typing of SARS-CoV-2 Alpha, Beta, and Delta variants. The whole assay can be accomplished within 3 h, and the amplification is performed under constant temperature, making the technique simple in operation and efficient. It is also feasible to extend the technique to the detection of many other variants of the virus. We expect that the method can add value to the rapid screening of viral variants and can play an important role in pandemic control.

Programmable Clostridium perfringens Argonaute-Based, One-Pot Assay for the Multiplex Detection of miRNAs.

Assays for the molecular detection of miRNAs are typically constrained by the level of multiplexing, especially in a single tube. Here, we report a general and programmable diagnostic platform by combining mesophilic Clostridium perfringens Argonaute (CpAgo) with exponential isothermal amplification (EXPAR), which is a dual-signal amplification strategy, allowing for the rapid and sensitive detection of multiple miRNAs with single-nucleotide discrimination in one pot. The CpAgo-based One-Pot (COP) assay achieved a limit of detection of 1 zM miRNA within 30 min of turnaround time and a wide concentration range. This COP assay was applied to simultaneously detect four miRNAs in a single tube from clinical serum samples, showing superior analytical performance in distinguishing colorectal cancer patients from healthy individuals. This programmable, one-pot, multiplex, rapid, and specific strategy offers great promise in scientific research and clinical applications.

Programmable Analysis of MicroRNAs by Thermus thermophilus Argonaute-Assisted Exponential Isothermal Amplification for Multiplex Detection (TEAM).

The simultaneous analysis of the levels of multiple microRNAs (miRNAs) is critical to the early diagnosis of cancer. However, this analysis is challenging because of the low concentrations of miRNAs and their high sequence homology. Here, we report a general and programmable diagnostic strategy for miRNA analysis: Thermus thermophilus Argonaute (TtAgo)-assisted exponential isothermal amplification for multiplex detection (TEAM). This system combines exponential isothermal amplification (EXPAR), for target amplification, with programmable TtAgo cleavage, for the generation of the reporting signal. The TEAM assay achieved attomolar sensitivity with a rapid turnaround time (30-35 min). Because of the single-nucleotide precision of TtAgo, the system demonstrated robust multiplex capability in the simultaneous detection of four miRNA targets and the classification of let-7 family members. The TEAM assay was superior in differentiating colorectal cancer patients from healthy individuals relative to the conventional EXPAR and reverse transcription polymerase chain reaction (RT-PCR) methods. This tunable and scalable approach is a powerful nucleic acid analysis tool that holds promise in scientific and clinical applications.

Isothermal gene amplification coupled MALDI-TOF MS for SARS-CoV-2 detection.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been spreading worldwide for more than a year and has undergone several mutations and evolutions. Due to the lack of effective therapeutics and long-active vaccines, accurate and large-scale screening and early diagnosis of infected individuals are crucial to control the pandemic. Nevertheless, the current widely used RT-qPCR-based methods suffer from complicated temperature control, long processing time and the risk of false-negative results. Herein, we present a three-way junction induced exponential rolling circle amplification (3WJ-eRCA) combined MALDI-TOF MS assay for SARS-CoV-2 detection. The assay can detect simultaneously the target nucleocapsid (N) and open reading frame 1 ab (orf1ab) genes of SARS-CoV-2 in a single test within 30 min, with an isothermal process (55 °C). High specificity to discriminate SARS-CoV-2 from other coronaviruses, like SARS-CoV, MERS-CoV and bat SARS-like coronavirus (bat-SL-CoVZC45), was observed. We have further used the method to detect pseudovirus of SARS-CoV-2 in various matrices, e.g. water, saliva and urine. The results demonstrated a great potential of the method for large scale screening of COVID-19, which is an important part of the pandemic control.

MALDI-TOF Mass Spectrometry in Clinical Analysis and Research.

In the decade after being awarded the Nobel Prize in Chemistry in 2002, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has been widely used as an analytical chemistry tool for the detection of large and small molecules (e.g., polymers, proteins, peptides, nucleic acids, amino acids, lipids, etc.) and for clinical analysis and research (e.g., pathogen identification, genetic disorders screening, cancer diagnosis, etc.). In view of the fast development of MALDI-TOF MS in clinical usage, this review systematically summarizes the most important applications of MALDI-TOF MS in clinical analysis and research by analyzing MALDI TOF MS-related reviews collected in the Web of Science database. On the basis of the analysis of keyword co-occurrence of over 2000 review articles, four themes consisting of "pathogen identification", "disease diagnosis", "nucleic acids analysis", and "small molecules analysis" were found. For each theme, the review further outlined their application implications, analytical methods, and systems as well as limitations that need to be addressed. Overall, the review summarizes and elaborates on the clinical applications of MALDI-TOF MS, providing a comprehensive picture for researchers embarking on MALDI TOF MS-related clinical analysis and research.

Silk sericin induced fabrication of reduced graphene oxide and its in-vitro cytotoxicity, photothermal evaluation.

Nowadays, photothermal agents have attained considerable attention in nanomedicine for the treatment of cancer after chemotherapy, surgery, biological therapy and radiotherapy. In this work, we showed a sericin-based, simple approach for synthesis of sericin functionalized reduced graphene oxide (SRGO) with low cytotoxicity and photo thermal efficiency. During the synthesis, the GO is deoxygenated in situ and functionalized by sericin, a low-cost, silk protein and concurrently forms SRGO. The subsequent SRGO disperse well in water with higher biocompatibility because of the decoration of sericin on graphene sheets. The prepared SRGO exhibited a good photothermal capacity with near-infrared laser irradiation (808 nm) for efficient killing of HeLa cells. Further, the synthesized SRGO could act as a promising material for photo thermal therapy applications in future.

Simple preparation of magnetic metal-organic frameworks composite as a "bait" for phosphoproteome research.

Phosphospecific enrichment techniques and mass spectrometry (MS) are primary tools for comprehending the cellular phosphoproteome. In this work, a rational and extremely facile route to synthesize the magnetic metal-organic frameworks (mMOFs) was employed and the prepared composite was first utilized as a "bait" for selective enrichment of phosphopeptides. Typically, the mMOFs was synthesized via electrostatic self-assembly between the negatively charged Fe3O4 magnetic nanoparticles (MNPs) and positively charged MIL-101(Fe). The obtained Fe3O4/MIL-101(Fe) composite possessed well-defined structures, rough surface, highly specific surface area and excellent magnetic property. To demonstrate their ability for enrichment of phosphopeptides, we applied Fe3O4/MIL-101(Fe) as a "bait" to capture the phosphopeptides from standard protein digestion and practical samples. The enriched phosphopeptides were analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). The MS results show that the Fe3O4/MIL-101(Fe) exhibits superior enrichment performance for phosphopeptides with low detectable concentration assessed to be 8 fmol, selectivity investigated to be 1:1000 using ß-casein/bovine serum albumin mixture and enrichment recovery evaluated to be 89.8%. Based on these excellent properties, the prepared composite was used to enrich the phosphopeptides from tilapia eggs biological samples for the first time. A total number of 51 phosphorylation sites were identified from the digest of tilapia eggs proteins, suggesting the excellent potential of Fe3O4/MIL-101(Fe) composite in the practical application.

MIL-101(Cr) as matrix for sensitive detection of quercetin by matrix-assisted laser desorption/ionization mass spectrometry.

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) has been proven as a useful and advanced technique in the identification of polymers and proteins. However, MALDI-TOF-MS still has the unavoidable drawback of self-signal interference with traditional organic matrices, which could suppress and overlap with the analyte signals in the low-mass region. In this work, MIL-101(Cr), a kind of metal-organic frameworks which possess high molecular weight, π-conjugated 3-D structure, coordinately unsaturated chromium sites (CUS) and strong absorption in the UV range, was employed to replace traditional organic matrices, and it was found that MIL-101(Cr) can dramatically eliminate the background peaks, showing high signal-to-noise level in the analysis of small molecules. As proof-of-concept, quercetin, daidzein, genistein and naringenin, members of flavonol family which widely exists in food and natural products, were successfully determined by utilizing MIL-101(Cr) as the surface-assisted matrix, and the detection of quercetin was sensitive with good salt tolerance and reproducibility. Under optimal conditions, the mass peak intensity exhibited good linear relationships in the range from 0.25µg/mL-7.00µg/mL for quercetin (R2=0.996) with detection limit 2.11ng/mL (3σ/k), making the identification of quercetin in sophora japonica successfully. With this strategy we have demonstrated the potentiality of MIL-101(Cr) nanomaterials as MALDI-MS matrix for the detection of small molecules.

Hydrophobic coating- and surface active solvent-mediated self-assembly of charged gold and silver nanoparticles at water-air and water-oil interfaces.

We report self-assembly of charge-stabilized gold and silver nanoparticles at water-air and water-oil interfaces, via manipulation of the interactions between the interfaces and the adsorbing nanoparticles. Nanoparticle adsorption from bulk colloids to an interface is an energy-favored, but finite sorption barrier-restrained (kinetics-controlled) process. Consequently, to successfully mediate self-assembly of nanoparticles, the finite sorption barrier should be decreased. That can be accomplished by manipulating its three controlling forces: the repulsive electrostatic force, the repulsive van der Waals force, and the attractive hydrophobic force between the interface and the adsorbing nanoparticles. It was found that hydrophobic coatings change nanoparticle hydrophobicity and greatly increase the attractive hydrophobic force. Surface active organic solvents (methanol, ethanol, isopropanol, and acetone) decrease the attractive hydrophobic force to some extent. However, they decrease the repulsive electrostatic force to a larger extent, via a "charge dilution" mechanism, due to their positive adsorption at the charged water-air and water-oil interfaces. Hydrophobic coatings and organic solvents consequently decrease the sorption barrier, facilitate nanoparticles overcoming the sorption barrier, and mediate the self-assembly of nanoparticles.

Total internal reflection fluorescence spectroscopy for investigating the adsorption of a porphyrin at the glass/water interface in the presence of a cationic surfactant below the critical micelle concentration.

Total internal reflection fluorescence (TIRF) spectroscopy was used to investigate the adsorption behavior of meso-tetrakis(p-sulfonatophenyl)porphyrin (TPPS) at the glass/water interface in the presence of a cationic surfactant (cetyltrimethylammonium bromide, CTAB) far below the critical micelle concentration. The adsorption model of TPPS at the glass/water interface in the presence of low concentration of CTAB was proposed, which was different from the adsorption of TPPS in the presence of micelles of CTAB at the glass/water interface. TPPS and CTAB did not form stable complex at the interface in dilute system. The interfacial species of TPPS were analyzed by comparing the spectra of TPPS at the glass/water interface and in the aqueous phase. The influences of the TPPS concentration, the CTAB concentration, and the pH values on the interfacial fluorescence spectra and intensities were studied. It was demonstrated that electrostatic interaction and hydrophobicity performed an important role on the adsorption of TPPS in the presence of CTAB. The different effects of TPPS concentration on the adsorption behaviour of TPPS at different pH were observed for the first time. It was found that the adsorption isotherms of TPPS at glass/water interface could fit Freundlich equation at pH 7.1.

Theoretical consideration on preparing silver particle films by adsorbing nanoparticles from bulk colloids to an air-water interface.

In our previous paper, a method for preparing enormous surface-enhanced Raman scattering (SERS) active substrates through the aggregation of silver particles trapped at an air-water interface was reported. Here, further efforts were devoted to investigate the origin of assembling silver particle films by adsorbing nanoparticles from bulk colloids to the air-water interface. It was revealed that it is thermodynamically favorable for a colloidal particle in bulk colloids to adsorb to the air-water interface; however, a finite sorption barrier between it and the nearby particles usually restrains the adsorption process. When an electrolyte such as KCl, which is commonly used as an activating agent for additional SERS enhancement, was added into silver colloids, it largely reduced the sorption barrier. Thus, silver nanoparticles can break through the sorption barrier, pop up, and be trapped at the air-water interface. The trapped silver particles are more inclined to aggregate at the interface than those in bulk colloids due to the increase of van der Waals forces and the reduction of electrostatic forces. The morphology of the as-prepared silver particle films was characterized by scanning electron microscope, and their SERS activity was tested using NaSCN as a probe molecule. The surface enhancement of the silver particle films is about 1-2 orders of magnitude higher compared with that of silver colloids, because most of the silver particles in the films are in the aggregation form that provides enormous SERS enhancement. Furthermore, the stability of such type of films is much better that of colloid solutions.