Analytical Chemistry最新文献

In proteomics, postproline cleaving enzymes (PPCEs), such as Aspergillus niger prolyl endopeptidase (AnPEP) and neprosin, complement proteolytic tools because proline is a stop site for many proteases. But while aiming at using AnPEP in online proteolysis, we found that this enzyme also displayed specificity to reduced cysteine. By LC-MS/MS, we systematically analyzed AnPEP sources and conditions that could affect this cleavage preference. Postcysteine cleavage was blocked by cysteine modifications, including disulfide bond formation, oxidation, and alkylation. The last modification explains why this activity has remained undetected so far. In the same experimental paradigm, neprosin mimicked this cleavage specificity. Based on these findings, PPCEs cleavage preferences should be redefined from post-Pro/Ala to post-Pro/Ala/Cys. Moreover, this evidence demands reconsidering PPCEs applications, whether cleaving Cys-rich proteins or assessing Cys status in proteins, and calls for revisiting the proposed enzymatic mechanism of these proteases.

This work designed a DNA conformation-regulated hemin switch for rapid chemiluminescent (CL) detection of a monoclonal antibodies. This switch was performed with an affinity probe and an inhibition probe, which were conveniently prepared by hybridizing hemin-labeled DNA1 with KHL peptide-labeled DNA2 and binding biotin-labeled DNA3 to streptavidin, respectively. In the absence of the target antibody, streptavidin-DNA3 could hybridize with hemin-DNA1/KHL-DNA2 to release KHL-DNA2, which led to the loss of hemin activity due to the affinity hindrance of streptavidin-DNA3. After the KHL peptide was recognized by the target antibody, the strand replacement hybridization could be inhibited by the bound antibody, which retained the high catalytic activity of hemin overhung on the antibody-bound affinity probe for a CL reaction, leading to a "signal-on" process for CL antibody detection. Using a KHL-specific antibody, anti-proprotein convertase subtilisin/kexin type 9 antibody (PCSK9-Ab), as a target model and common L012-1,2,4-triazole-H₂O₂ CL system, the designed switch showed a detection range of 10 ng mL^-1 to 1 μg mL^-1 with a detection limit of 4.16 ng mL^-1 (56.2 pM) and a short analytical time of 6.5 min. The proposed quick method could simply be used for lab-on-chip CL detection of PCSK9-Ab in situ-secreted from PCSK9-6E3 hybridoma cells, which showed an accuracy of 90.2% compared with the statistical results from general fluorescence imaging, providing a potential technique for screening specific hybridoma cells.

Spherical biosamples such as immunobeads, cells, and cell aggregates have been widely used in bioapplications. The bioactivity of individual spherical biosamples in highly sensitive assays and individual analyses must be evaluated in a high-throughput manner. Electrochemiluminescence (ECL) imaging was recently proposed for the high-throughput analysis of diffusive molecules from spherical biosamples. ECL imaging involves the placing of spherical biosamples on a flat electrode filled with a solution. The biosamples produce (or consume) biological/chemical molecules such as H₂O₂ and O₂, which diffuse to form a concentration gradient at the electrode. The ECL signals from the molecules are then measured to obtain the concentration profile, which allows the flux to be estimated, from which their bioactivities can be successfully calculated. However, no studies on theoretical approaches for spherical biosamples on flat surfaces have been conducted using ECL imaging. Therefore, this paper presents a novel spherical diffusion theory for spherical biosamples on a flat surface, which is based on the common spherical diffusion theory and was designated as the extended spherical diffusion theory. First, the concepts behind this theory are discussed. The theory is then validated by comparison with a simulated analysis. The resulting equation successfully expresses the concentration profile for the entire area. The glucose oxidase activity in the hydrogel beads is subsequently visualized using ECL imaging, and the enzymatic product flux is calculated using the proof-of-concept theory. Finally, a time-dependent simulation is conducted to fill the gap between the theoretical and experimental data. This paper presents novel guidelines for this analysis.

Digital immunoassays enable the detection of protein biomarkers with very low concentrations, but the analysis stringently requires single-bead encapsulation. Low bead density has been adopted to minimize multiple-bead encapsulations, but the trade-off is the low droplet effectiveness (∼10%) in droplet-based assays. Here we report the method of inclusive droplet digital ELISA (iddELISA) that embraces all types of encapsulations by factoring in their varied "on-off" probabilities in the statistical inference. We derived the statistical model, optimized the bead encapsulation and immunoreaction, and developed an image analysis pipeline for accurate droplet and bead recognition, showing that approximately 40% of the droplets could be used in the analysis. Using the detection of SARS-CoV-2 nucleocapsid protein as a demonstration, the iddELISA achieved a limit of detection of 0.71 fg/mL, which was much lower than conventional ELISA as well as droplet digital ELISA. By effectively incorporating multiple bead encapsulations, the iddELISA simplified the digital immunoassay while improving the counting efficiency and sensitivity, representing a unique concept in digital immunoassays.

Solution and gas-phase measurements can provide valuable insights into biomolecular conformational dynamics. By comparing the data from such experiments, it is possible to elucidate the nature of the interactions governing a biomolecule's stability. Here, we measured human, bovine, and porcine hemoglobin stability in solution and the gas phase using collision-induced dissociation, collision-induced unfolding, surface-induced dissociation, and temperature-controlled nanoelectrospray mass spectrometry. Hemoglobin dimer and tetramer stability in solution and gas phases did not correlate, likely due to differences in the composition of positive and negative amino acids on the surface of these molecules. Specifically, the absence of Lys-116 on the β-subunit makes it easier for the human hemoglobin dimer to dissociate in the gas phase. However, the presence of Lys-60 makes the subunit more rigid thus it cannot unfold to the same extent as the other hemoglobin. Hemoglobin tetramers of different origins had similar stability in the gas phase, as there was no difference in the composition of charged amino acids at the tetramer interface. These results highlight how temperature-controlled mass spectrometry and collision-induced unfolding can elucidate the structural reasons behind differences in the gas-phase and solution stability of protein complexes.

Immunoassays have become essential tools for detecting infectious viruses. However, traditional monoclonal antibody-dependent immunoassays are costly, fragile, and unstable, especially in complex media. To overcome these challenges, we have developed cost-effective, robust, and high-affinity nanobodies as alternatives to monoclonal antibodies for rapid detection applications. We engineered dual-epitope nanobody (NB) pairs and incorporated them into a sandwich immunosensor design to detect transmitted rotaviruses in rectal swabs and wastewater samples. To further enhance sensitivity, we synthesized an advanced two-dimensional material, MXenes@CNTs@AuNPs, which offers an extensive specific surface area that supports the enrichment and immobilization of NBs. This integration with catalase-modified magnetic probes facilitates signal generation. Subsequently, our sensor achieved a detection limit of 0.0207 pg/mL for the rotavirus VP6 antigen, significantly outperforming commercial antigen kits with a sensitivity enhancement of 3.77 × 10⁵-fold. The exceptional sensor performance extended to specificity, repeatability, stability, and accuracy across various sample types, establishing it as a promising tool for rotavirus detection. This research outlines a viable strategy for creating a robust and ultrasensitive analytical nanoprobe, thereby addressing the critical need for efficient and reliable viral detection methods in various environments.

Therapeutic drug monitoring (TDM), which involves measuring drug levels in patients' body fluids, is an important procedure in clinical practice. However, the analysis technique currently used, i.e. liquid chromatography-tandem mass spectrometry (LC-MS/MS), is laboratory-based, so does not offer the short response time that is often required by clinicians. We suggest that techniques based on Fourier transform infrared spectroscopy (FTIR) offer a promising alternative for TDM. FTIR is rapid, highly specific and can be miniaturized for near-patient applications. The challenge, however, is that FTIR for TDM is limited by the strong mid-IR absorption of endogenous serum constituents. Here, we address this issue and introduce a versatile approach for removing the background of serum lipids, proteins and small water-soluble substances. Using phenytoin, an antiepileptic drug, as an example, we show that our approach enables FTIR to precisely quantify drug molecules in human serum at clinically relevant levels (10 μg/mL), providing an efficient analysis method for TDM. Beyond mid-IR spectroscopy, our study is applicable to other drug sensing techniques that suffer from the large background of serum samples.

Accurate measurement of aerosol pH is crucial for understanding atmospheric processes and mitigating haze pollution. However, online detection of aerosol pH is challenging due to the complex composition of single-particle matter and trace components. This study develops a sensitive and selective sensor for the online detection of aerosol pH using surface-enhanced Raman spectroscopy (SERS). A novel Fe₃O₄@SiO₂@Au-p-aminothiophenol (FA-pATP) sensor was fabricated using a layer-by-layer self-assembly method, achieving enhanced uniformity and increased density of SERS-active hotspots. Magnetic aggregation was employed to further amplify the Raman signal. This sensor was integrated into a 3D-printed microfluidic device to facilitate online monitoring of aerosol pH. The FA-pATP sensor exhibited a significant increase in peak intensity ratio with rising pH, demonstrating high sensitivity and responsiveness due to structural changes in the -NH₂ groups of pATP under different pH conditions. The sensor demonstrated a linear pH response ranging from 5 to 11. The 3D-printed microfluidic device, coupled with the FA-pATP sensor, demonstrated notable performance in various environmental media, indicating strong anti-interference capabilities. The proposed sensor shows great promise for real-time online monitoring of aerosol pH, with broad applications in environmental monitoring.

Peroxynitrite (ONOO^-) is a short-term reactive biological oxidant and plays an important role in cellular signal transduction and homeostatic regulation. However, excess ONOO^- is associated with neurodegenerative and cardiovascular diseases. Therefore, rapid, sensitive, and accurate assays for ONOO^- detection are essential for exploring its physiological and pathological function. In this work, a wavelength-shifted and ratiometric fluorescent sensing platform for ONOO^- is constructed by splitting green fluorescent carbon dots (G-CDs) and aggregating orange fluorescent carbon dots (O-CDs). The mixed G-CDs and O-CDs (M-CDs) show a fast and precise response to ONOO^- in the range of 0-250 μM, with a detection limit of 10 nM. In the linearity range within 3 μM ONOO^-, an obvious wavelength shift of G-CDs from 495 to 475 nm is observed owing to the oxidation and nitration of ONOO^- to the surface-state fluorescence of G-CDs, accompanied by the splitting of G-CDs. In the linearity range of 3-250 μM ONOO^-, the fluorescence of G-CDs remains constant, while the molecular-state fluorescence of O-CDs gradually quenches by the oxidation and nitration of ONOO^- through the fluorescence static process and induces their aggregation. Additionally, M-CDs show favorable intracellular imaging of endogenous and exogenous ONOO^-. This study not only presents a new fluorescence wavelength shift mechanism for ONOO^- sensing but also provides insights into CDs' fluorescence mechanism by exploring their morphology and structure via reacting with reactive oxygen species (ROS).

Almost all proteins on the cell surface are modified by glycosylation. Cell surface glycoproteins participate in various cellular pathways, such as cell adhesion, cell-cell communication, and immune response. Due to their functional importance, glycoproteins on the cell surface often serve as potential therapeutic targets. Recent advancements in mass spectrometry (MS) have facilitated the characterization of glycoproteins that are generally localized on the cell surface, secreted to the extracellular environment, or found in intracellular organelles such as the endoplasmic reticulum, Golgi apparatus, and peroxisome. However, the selective characterization of glycoproteins on the cell surface remains challenging. In this study, we applied enzymatic treatment to live cells, followed by MS-based glycoproteomics analysis, to assess changes in protein glycosylation at different treatment time points as a method to identify cell surface glycoproteins. To demonstrate this approach, a renal cell carcinoma cell line, A498, was treated with glycosidases, sialidase and PNGase F, over two treatment time intervals, 2 and 24 h. Glycoproteins were identified as cell surface glycoproteins from A498 cells when enzyme treatment altered the glycosylation of the glycoproteins. The results revealed the effectiveness of integrating enzymatic treatment with MS-based glycoproteomics for analyzing cell surface glycoproteins. Our established method has demonstrated the potential applications for assessing accessibility of therapeutic targets on the cell surface over time and supporting the development of new targeted therapies.