Analytical Methods最新文献

Food safety has become a critical global concern, with foodborne diseases affecting approximately 600 million people annually and causing 420 000 deaths each year, posing significant risks to human health and well-being. Rapid, efficient, and reliable detection methods are essential to mitigate these risks. Traditional detection methods, such as PCR and culture-based assays, while widely used, often face challenges related to speed, accuracy, and portability. Over the past 5 years (2020–2025), the (CRISPR)/Cas system has emerged as a powerful tool for food safety detection due to its high sensitivity, specificity, and versatility. This review highlights recent advances in CRISPR/Cas-based biosensors and their applications in food safety. First, we discuss the key challenges in food safety detection and the design principles of CRISPR/Cas biosensors. Next, we comprehensively summarize their applications in detecting foodborne pathogens (viruses and bacteria), food fraud, genetically modified organisms (GMOs), toxins, heavy metals, antibiotic residues, and pesticides. Finally, we address the current limitations and future prospects of CRISPR/Cas biosensors, providing insights into their potential for next-generation food safety solutions.

Surface-enhanced Raman spectroscopy (SERS) is a powerful analytical tool for the observation, the detection and the identification of chemical or biological species at low concentrations due to its high sensitivity, specific fingerprinting capability and real-time detection. However, a key challenge lies in establishing a suitable and reliable measurement protocol to ensure both reproducibility and repeatability when SERS is used as a sensing nanoplatform. In this paper, we propose a specific methodology to investigate the performance of SERS substrates, with a particular focus on their reproducibility and repeatability. Furthermore, we validate our approach on one commercial SERS Hamamatsu substrate from Hamamatsu Photonics by using diluted solution of 4-mercaptobenzoic acid (MBA) at an excitation wavelength of 633 nm. This proposed protocol consists in recording 25 SERS maps equally distributed on the whole surface substrate. For each map, 16 spectra have been acquired and averaged to provide a representative SERS signal. In total, 400 spectra have been collected and analyzed by using the integrated intensities of characteristic MBA bands to determine both reproducibility and repeatability. This approach enables us to quantify signal variations, at the micrometer scale, as well as across the entire substrate. We demonstrated that while the SERS response is highly reproducible locally, it becomes less consistent when evaluated across the full surface. However, the SERS signal is not repeatable at the local scale but it can be repeatable at the whole substrate scale as the average SERS intensity is identical for both SERS measurements. Furthermore, we demonstrated that this method can also be applied to DNA strands thereby demonstrating its effectiveness in evaluating biosensors. Finally, the proposed methodology and protocol can then be used as a standard to precisely evaluate the sensing performances of other substrates.

Detection of matrix metalloproteinase-2 (MMP-2) is of great significance for clinical diagnosis and therapy of cancer in the early stage. In this study, a novel magnetic-fluorescent nanocomposite probe for simple, fast and sensitive detection of MMP-2 was innovatively developed by self-assembly of a specifically designed MMP-2 substrate peptide and cobalt-nitrilotriacetate (Co²⁺-NTA) modified magnetic beads via the chelation mechanism. The specifically designed peptide consisted of green fluorescein (FITC) that acted as the fluorescence signal donor, and the MMP-2 cleavage sequence fused with the hexa-histidine (6× His) tag. In the presence of MMP-2, the His-tagged fluorescent peptides conjugated to the Co²⁺-NTA modified magnetic beads would be cleaved by MMP-2 due to specific substrate recognition, releasing the FITC-labeled peptide segment into the solution. After the nanocomposite probe was removed from the reaction solution by magnetic separation, high fluorescence signal intensity of the supernatant would be obtained. The proposed nanoprobe demonstrated a wide linear range and a high sensitivity for MMP-2 with a detection limit of 0.3 ng mL^-1. It also exhibited satisfactory performance in cell samples. Additionally, the novel nanoprobe could be effectively separated and recycled using a magnet, which allows it to exhibit reusable characteristics. This method not only provides a novel strategy for MMP-2 assay but also offers potential application in biomedical and clinical studies.

The development of sunscreens incorporating nanostructured lipid carriers (NLCs) is gaining traction in the cosmeceutical industry to enhance photoprotection. These innovative systems require robust and precise analytical methods to accurately quantify the chemical filters encapsulated within the lipid matrix. This study presents a fully validated RP-HPLC method specifically designed for the simultaneous quantification of three UV filters: diethylamino hydroxybenzoyl hexyl benzoate (DHHB), bis-ethylhexyloxyphenol methoxyphenyl triazine (BEMT), and ethylhexyl triazone (EHT), which were encapsulated in nanostructured lipid carriers (NLCs). The methods reported in the literature lack validation for nanostructure systems as they usually focus on exploring methods for free filters. Here, our approach was to optimize and validate an analytical method for NLCs, which demonstrated robustness, high sensitivity (LOD as low as 0.6 µg mL⁻¹), and compliance with the ICH guidelines. The method enabled the accurate and precise determination of filter extracted content, based on the extraction method, and encapsulation efficiency, supporting formulation optimization and industrial scalability by identifying a pool of interchangeable prototypes. Encapsulation efficiency of the UV-filters as the main response evaluated, which was interpreted through the critical quality attributes (CQAs) of values above 70%. Hence, the present study addresses a critical gap in the analytical methodologies for emerging nanoformulation technologies.

Saracatinib (AZD-0530; SRB) is a pharmaceutical agent produced by AstraZeneca and is currently undergoing clinical studies. It is classified as a dual-kinase inhibitor, exhibiting selective activity both as an Src inhibitor and a Bcr–Abl tyrosine kinase inhibitor. No metabolic stability study for SRB has been reported; hence, so the goal of the present study was to establish an ultra-fast, green, sensitive, and validated UPLC-MS/MS method for the quantification of SRB levels in human liver microsomes (HLMs) using different in silico software to support the practical outcomes. The validated approach was used to estimate the SRB metabolic stability in HLMs. In silico software tools were employed to predict the potential sites of metabolic lability and toxicity within the SRB structure. SRB and baricitinib, used as an internal standard (IS), were isolated from HLMs using protein precipitation with acetonitrile (ACN) as the extracting agent. Chromatographic separation was conducted utilizing a Luna 3 µm HILIC column (200 Å: 50 × 2 mm, Ea), with the mobile phase comprising 0.1% formic acid in ACN (85%) and 10 mM ammonium formate in water (15% at pH 3.2), and the total run time was 1.0 min. SRB and IS were analyzed utilizing the MRM mass analyzer mode. The approach was validated according to the latest FDA guidelines for bioanalytical method validation. The SRB calibration curve demonstrated significant sensitivity, with a range of statistical linearity from 1 to 4000 ng mL⁻¹. The intraday and interday accuracies of the four quality controls varied from −4.17% to 12.25% and −3.92% to 13.50%, respectively. The metabolic stability parameters, including the in vitro half-life (t_1/2) and intrinsic clearance (Cl_int) of SRB, were assessed at 17.24 min and 47.02 mL min⁻¹ kg⁻¹, respectively. In silico research indicated that slight structural modifications to the N-methyl piperazine ring in the drug design may enhance metabolic stability and safety compared with those of SRB.

Insertion/deletion (indel) polymorphisms in the promoter region (23 bp) and intron 1 (12 bp) of the bovine PRNP gene influence gene expression and susceptibility to bovine spongiform encephalopathy (BSE). Conventional detection strategies dependent on DNA sequencing are cumbersome and costly. A tetra-primer amplification refractory mutation system PCR (tetra-ARMS PCR) assay was developed which can enable efficient and cost-effective genotyping of each indel locus. Optimized tetra-ARMS PCR primers and multiplex conditions allowed electrophoretic genotyping of the 23 bp indel via the size and presence of 422 bp, 259 bp, and 193 bp amplicons, whereas the 12 bp indel was genotyped based on the size and presence of 598 bp, 472 bp, and 153 bp fragments. Furthermore, the compatibility of these primer sets was preliminarily investigated, demonstrating the potential for co-amplification in a single-tube multiplex format. Validation against Sanger sequencing using 62 randomly selected cattle-derived retail samples demonstrated complete concordance. This straightforward, specific and cost-effective method requires only conventional PCR instrumentation, thereby establishing a robust and accessible platform for PRNP-assisted breeding and cattle product genotyping.

The development of fast, simple, and visual methods for detecting adenosine triphosphate (ATP) is crucial for point-of-care diagnostics and environmental monitoring. Colorimetric assays based on the integration of DNA aptamer-triggered hybridization chain reaction (HCR) with gold nanoparticles (AuNPs) offer high potential, but existing methods often suffer from prolonged detection times (e.g., >130 min). To address this limitation, this study systematically investigated and optimized the core parameters of the aptamer-triggered HCR system and its subsequent mixing conditions with AuNPs to achieve a rapid visual detection platform. The HCR kinetics were accelerated by the high concentrations of single-stranded DNA (ssDNA) components, particularly the H0 initiator, as well as by the critical presence of divalent magnesium ions (Mg²⁺), which also support the functionality of the aptamer. This optimization allowed for a degree of HCR progression comparable to the traditional 24-hour incubation to be achieved within just 60 minutes. Subsequently, recognizing the inherent trade-off between the high ssDNA concentration required for fast HCR and the AuNP dispersion stability, we optimized the mixing ratio between the HCR product and the AuNP solution. Under these optimized conditions, the assay demonstrated the capability for rapid visual detection of ATP at the 100 µM level within a total assay time of 15 minutes, representing a significant acceleration compared to previously reported methods. Further improvements in sensitivity are anticipated through future fine-tuning of AuNP parameters and the use of post-reaction salt aggregation enhancers.

As a core medication for the prevention and treatment of cerebral vasospasm, especially after aneurysmal subarachnoid hemorrhage, real-time monitoring of nimodipine (NMD) concentration helps evaluate the adequacy of drug therapy and holds great significance for ensuring patient life and health. Herein, based on dual functional monomers, a molecularly imprinted electrochemical sensor (MIECS) for the detection of NMD with nitrogen-doped multi-walled carbon nanotubes (N-CNTs) and Fe-MOFs was developed. Fe-MOFs provided a large specific surface area, offering more space for generating imprinting sites, while N-CNTs enhanced the conductivity of the electrode. 2-Amino-5-mercapto-1,3,4-thiadiazole (AMT) and o-phenylenediamine (o-PD) served as dual functional monomers and NMD as the template molecule. A molecularly imprinted polymer (MIP) membrane was prepared on the electrode surface by electropolymerization. Compared to single functional monomers, the dual functional monomers exhibited better selectivity and specificity in NMD recognition. Under optimal experimental conditions, the response of the MIECS to NMD showed a linear relationship ranging from 10⁻¹⁴ M to 10⁻⁸ M, with a detection limit of 2.97 × 10⁻¹⁵ M. Satisfactory recovery rates were obtained in the detection of human serum and tablets. This multi-parametric enhancement establishes a new paradigm for therapeutic drug monitoring in clinical neurology and pharmaceutical quality control.

A novel fluorescent probe L for DNA detection was designed by linking a pyrene fluorophore to a naphthalimide-imidazole unit via a benzene spacer. The probe operates through a π–π stacking-driven intercalation and excimer formation mechanism, offering an immediate “turn-off” fluorescence response with high selectivity and a detection limit of 4.41 nM under physiologically relevant pH conditions. In cellular environments, Ca²⁺ enables probe release from DNA, restoring cytoplasmic fluorescence and thereby allowing indirect visualization of nuclear DNA via enhanced cytoplasmic signals. This strategy demonstrates the potential of probe L for effective bioimaging in living cells.

Zearalenone (ZEN) is a carcinogenic mycotoxin commonly found in grains, cereals, and dairy products, posing cancer risks to humans and animals. Chronic high-dose ZEN exposure can cause liver disease, immunosuppression, kidney damage, and tumorigenesis. Sensitive and efficient ZEN detection is therefore essential for protecting human and animal health. To meet the need for sensitive, selective, and low-cost mycotoxin sensors in real food samples, we developed a novel ratiometric electrochemical aptamer sensor for ZEN detection. The sensor design utilizes β-cyclodextrin-functionalized graphene (CG) to effectively enhance electron transfer efficiency, combined with host–guest recognition between ferrocene (Fc) and β-cyclodextrin (β-CD), as well as Au–S covalent bonding. The 5′-SH and 3′-MB-modified dual-labeled ZEN aptamer (MB-Apt-SH) was immobilized via β-CD/Fc inclusion, with signal amplified by AuNPs binding to –SH. Without ZEN, the aptamer stays on the electrode, giving high I_MB and low I_Fc. With ZEN, it binds the target, pulling it away. Adding Fc-COOH fills the β-CD cavities, increasing I_Fc and decreasing I_MB, thus raising the I_Fc/I_MB ratio proportionally to ZEN concentration. The sensor exhibits a wide linear range from 1 pg mL⁻¹ to 1000 ng mL⁻¹, with a detection limit of 0.4344 pg mL⁻¹. Furthermore, the method was successfully applied to the determination of ZEN in wine samples, yielding recovery rates between 94.00% and 104.60%, demonstrating excellent accuracy and applicability. These results indicate that the MCH/AuNPs/SH-Apt-MB/CG/GCE platform is a highly sensitive and selective electrochemical sensor for ZEN detection.