Analytical Chemistry最新文献

Nowadays, aggregation-caused quenching (ACQ) of organic molecules in aqueous media seriously restricts their analytical and biomedical applications. In this work, hydrogen bond (H-bond) was utilized to resist the ACQ effect of 2,5,8-triamino-1,3,4,6,7,9,9b-heptaazaphenalene (Melem) as an advanced electrochemiluminescence (ECL) luminophore, whose ECL process was carefully studied in an aqueous K₂S₂O₈ system coupled with electron paramagnetic resonance (EPR) measurements. Notably, the H-bond-induced Melem assemblies (Melem-H) showed 16.6-fold enhancement in the ECL signals as compared to the Melem aggregates (Melem-A), combined by elaborating the enhanced mechanism. On such basis, the effective ECL signal transduction was in situ achieved through the specific recognition of the double-stranded DNA embedded in Melem-H assemblies (Me-dsDNA) with spike protein (SP) of coronavirus disease 2019 (COVID-19). For that, such an ECL biosensor showed a wider linear range (1.0-125.0 pg mL^-1) with a lower limit of detection (LOD) down to 0.45 pg mL^-1, which also displayed acceptable results in analysis of human nasal swab samples. Therefore, the work provides a distinctive insight on addressing the ACQ effect and broadening the application scope of the organic emitter and offers a simple platform for biomedical detection.

Noninvasive fluorescence detection of tumor-associated biomarker dynamics provides immediate insights into tumor biology, which are essential for assessing the efficacy of therapeutic interventions, adapting treatment strategies, and achieving personalized diagnosis and therapy evaluation. However, due to the absence of a single biomarker that effectively reflects tumor development and progression, the currently available optical diagnostic agents that rely on "always-on" or single pathological activation frequently show nonspecific fluorescence responses and limited tumor accumulation, which inevitably compromises the accuracy and reliability of tumor imaging. Herein, based on intramolecular charge transfer (ICT) and twisted intramolecular charge-transfer (TICT) hybrid mechanisms, we report a tandem-locked probe, NTVI-Biotin, for simultaneously specific imaging-guided tumor resection and ferroptosis-mediated tumor ablation evaluation under the coactivation of nitro reductase (NTR)/viscosity. The dual-stimulus-responsive design strategy ensures that NTVI-Biotin exclusively activates near-infrared (NIR) fluorescence signals upon interaction with both NTR and elevated viscosity levels through triggering ICT on while inhibiting the TICT process. Meanwhile, functionalization with a tumor-targeting hydrophilic biotin-poly(ethylene glycol) moiety enhances tumor accumulation. The probe's dual-response and tumor-targeting design minimizes nonspecific tissue activation, allowing for precise tumor identification and lesion removal with a superior tumor-to-normal tissue (T/N > 6) ratio. More importantly, NTVI-Biotin was capable of evaluating ferroptosis-mediated chemotherapeutics by real-time monitoring of the alternations of NTR/viscosity levels. The results reveal that the increased tumor signals of NTVI-Biotin following the combination of ferroptosis and chemotherapy correlate well with the tumor growth inhibition, demonstrating the potential of NTVI-Biotin to assess therapeutic efficacy.

Electrofusion is an effective method for fusing two cells into a hybrid cell, and this method is widely used in immunomedicine, gene recombination, and other related fields. Although cell pairing and electrofusion techniques have been accomplished with microfluidic devices, the purification and isolation of fused cells remains limited due to expensive instruments and complex operations. In this study, through the optimization of microstructures and electrodes combined with buffer substitution, the entire cell electrofusion process, including cell capture, pairing, electrofusion, and precise separation of the targeted fused cells, is achieved on a single chip. The proposed microfluidic cell electrofusion achieves an efficiency of 80.2 ± 7.5%, and targeted cell separation could be conveniently performed through the strategic activation of individual microelectrodes via negative dielectrophoresis, which ensures accurate release of the fused cells with an efficiency of up to 91.1 ± 5.1%. Furthermore, the survival rates of the cells after electrofusion and release are as high as 94.7 ± 0.6% and 91.7 ± 1.2%, respectively. These results demonstrate that the in situ cell electrofusion and separation process did not affect the cell activity. This chip offers integrated multifunctional manipulation of cells in situ, and can be applied to multiple fields in the future, thus laying the foundation for the field of precise single-cell analysis.

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.

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.