Analytical biochemistry最新文献

Promoter prediction is critical for deciphering transcriptional regulatory mechanisms. However, traditional one-hot encoding strategies suffer from dimensionality explosion with vocabulary expansion, while single-domain knowledge constraints limit predictive performance. Therefore, we propose a promoter prediction method (Word2Vec-ResNet) that innovatively integrates natural language processing (NLP) techniques with cross-domain transfer learning. By pretraining word embeddings on source domain data and transferring the pretrained embedding table to the target domain, the method effectively reduces the dimensionality of nucleotide sequence encoding while leveraging inter-domain knowledge to enhance model generalization. Comprehensive experiments on promoter datasets of four representative organisms (Bacillus subtilis, Escherichia coli, Saccharomyces cerevisiae, and Drosophila melanogaster) demonstrate that the proposed method achieves significant performance improvements: compared with one-hot encoding, its average encoding dimension is reduced by 97.6%; compared with baseline methods, the prediction accuracy is increased by an average of 18.12% (with the ratio of the training set to the test set being 8:2).

Higenamine (HM) is a naturally occurring benzylisoquinoline alkaloid found in plants and is classified as an S3 prohibited substance in the 2020 World Anti-Doping Agency report. To prevent doping violations in competitive sports, it is essential to develop timely and accurate detection methods. In this study, we propose, for the first time, a detection method that combines artificial intelligence (AI)-based image recognition technology with lateral flow immunoassay (LFIA). This method utilizes gold nanoparticles (AuNPs) conjugated with HM-specific antibodies for the rapid detection of HM in urine via LFIA. It integrates AI-based image recognition to quantitatively analyze color intensity of the test strip's detection line. Experimental results demonstrated that the proposed method achieves a limit of detection of 0.49 ng/mL, enabling accurate identification within the concentration range of 2-8 ng/mL. For model training, an Image Classification of HM Test strip dataset was created. To address the limitations of this dataset, namely its small size (n = 304) and limited feature diversity, an advanced solution tailored to these constraints was proposed. The use of Contrast Limited Adaptive Histogram Equalization (CLAHE), which enables finer-grained feature extraction, ensured excellent recognition accuracy of the model in this dataset. During actual urine detection, the model achieved a prediction accuracy of 96.88% on the test set. This approach provides an efficient and reliable technical solution for on-site rapid detection of HM, addressing the critical need for timely monitoring in anti-doping efforts.

Early screening for colorectal cancer (CRC) is crucial for reducing its mortality. This study developed a amplification-free, highly sensitive sensing system based on localized surface plasmon resonance (LSPR) technology integrated with a multi-channel microfluidic chip platform for the in situ detection of the key CRC biomarker microRNA-451 (miRNA-451) using gold nanoparticles (AuNPs), achieving a limit of detection (LOD) of 19.2 fM. The core of this system is a multi-channel microfluidic chip serving as the detection substrate, where single-stranded DNA (ssDNA) probes are immobilized on the AuNPs surfaces to construct the sensor. In the presence of the target miRNA-451, it hybridizes specifically with two types of ssDNA probes, inducing the formation of AuNPs dimers and consequently causing a red shift in their LSPR scattering spectra. Unlike conventional methods, this study employed a precise single-nanoparticle tracking strategy, quantifying the target by statistically analyzing the proportion of AuNPs exhibiting significant spectral red shifts within the same field of view (FOV). This approach effectively mitigates spatial sampling errors arising from FOV inconsistencies. Furthermore, the system decouples the "hybridization" and "detection" functions, significantly enhancing detection efficiency and accuracy. This work provides a robust technical platform for the amplification-free and highly sensitive detection of miRNA-451.

Progastrin-releasing peptide is a crucial serum biomarker for the diagnosis, differential diagnosis, therapeutic monitoring, and prognostic evaluation of small cell lung cancer. In this study, a double-antibody sandwich immunoassay for the biomarker was developed. Key factors in the method development were systematically investigated, including magnetic particle coating, conjugate labeling, concentrations of magnetic particles and conjugates, and serum sample volume. The established assay has a total analysis time of approximately 13 minutes, with a limit of blank of 3.53 pg/mL. The repeatability and within-laboratory precision coefficients of variation were less than 8%. Assay accuracy was unaffected by common endogenous interferents at pathological concentrations or by the presence of gastrin-releasing peptide in the serum. Additionally, thermal accelerated stability testing over 7 days confirmed the robust stability of the reagents. Method comparison further demonstrated that the clinical results obtained by the established method showed high consistency with those of the Roche assay, fully meeting the requirements for clinical application.

Canine parvovirus-2 remains a major threat to canine health, with maternal antibody interference and limitations of traditional diagnostic methods posing challenges to effective disease control. Maternal-derived antibodies can persist at levels that interfere with the development of active immunity following vaccination. Therefore, accurate detection of existing antibody levels prior to vaccination is essential to ensure induction of protective immunity. Hemagglutination inhibition tests are labor-intensive and highly susceptible to external variability, emphasizing the need for reliable and cost-effective alternatives. In this study, we developed and optimized an indirect ELISA using a full-length, recombinant VP2-2b protein expressed in E. coli to detect CPV-2 antibodies. To our knowledge, this is the first ELISA developed around a soluble full-length CPV-2b protein, with comprehensive optimization and clinical validation. VP2-2b was selected for its unique bidirectional neutralization kinetics, enhancing the diagnostic accuracy of CPV-2 serology. The ELISA demonstrated excellent sensitivity and specificity, effectively distinguishing between seropositive and seronegative samples. A gray zone (OD450 = 0.26-0.30) was defined to represent borderline antibody titers, where retesting is recommended to determine accurate vaccination timing. Optimal assay conditions were established as 7.5 μg antigen coating concentration and 1:300 serum dilution, yielding a high signal-to-noise ratio (SNR) of 5.6. Validation against HI tests showed strong correlation with minimal non-specific binding. This ELISA system provides a reliable and economical alternative to traditional methods, helping overcome the limitations of the HI test in field settings and supporting informed decisions in vaccination planning.

The incidence of hyperuricemia is rising globally; however, its molecular mechanisms and reliable progression biomarkers remain unclear. This study characterized temporal changes in serum metabolites and lipids in hyperuricemic rabbit models using untargeted metabolomic and lipidomic profiling with ultra-high performance liquid chromatography coupled with quadrupole time-of-flight-mass/mass spectrometry (UHPLC-QTOF-MS/MS). Serum samples collected at baseline (0 h), 48 h, and 84 h (n = 3 per time point) were analyzed using both hydrophilic interaction chromatography (HILIC) and reversed-phase C18 columns to achieve broad metabolite and lipid coverage. HILIC analysis yielded 9,813 peaks with 65 annotated compounds, whereas C18 analysis identified 7,333 peaks with 122 annotated compounds across both positive and negative ion modes. Significant monotonic increases were observed in the normalized feature intensities of 4-aminohippuric acid, L-alanine, N-isobutyrylglycine, and uric acid, whereas the normalized feature intensity of inosine decreased relative to baseline. These metabolites satisfied two selection criteria, [|log₂(fold change, FC)| ≥ 0.58, Nemenyi p < 0.05] and variable importance in projection (VIP) > 1.0 using orthogonal partial least squares-discriminant analysis (OPLS-DA). Among them, inosine and 4-aminohippuric acid, which showed a strong correlations with uric acid, emerged as the most exploratory candidate biomarkers for hyperuricemia progression. The C18-based lipidomic profiling identified the phosphatidylinositols, PI(16:0_20:4) and PI(18:1_18:2), as the only lipid species that met the two selection criteria. In particular, PI(16:0_20:4) was strongly correlate with uric acid. Collectively, our study reveals the metabolomic and lipidomic changes for hyperuricemia and suggests exploratory candidate biomarkers for its early detection and monitoring.

A wide variety of assay chemistries are routinely employed to determine cell health within an in vitro test population. Each method relies on the consistent and robust measurement of specific biological surrogates for cell viability or cytotoxicity. Unfortunately, the precise cellular origin or identity of many of these biomarkers remain poorly characterized or unknown, and thus subject to a host of undetermined biological and chemical interferences. This work details efforts to pinpoint the enzymatic sources of a set of proteolytic activity profiles previously discovered in a phenotypic activity screen and measured in a multiplexed viability ("live cell") and cytotoxicity ("dead cell") assay. First, Clustered Regularly Interspaced Short Palindromic Repeats gene editing (CRISPR) was utilized to knockout (KO) genes encoding candidate enzymes in a human cell background to create clones for identity testing. Next, clones demonstrating discrete reduction of either the live or dead cell signals were further characterized by Western blot analysis for presence of immunogenic protein and by Sanger Sequencing of the targeted edit site. The KO data directed the sourcing of potent and selective inhibitors for orthogonal activity studies of the proteases in a parental population. Last, the utility of the multiplexed assay reagent was further explored in both non-human and human primary cell lines to characterize the universality of the application. Collectively, the positive identification of Cathepsin C (CatC) and Tripeptidyl peptidase II (TPP II) informs assay users about potential modulators of activity leading to possible interferences. Last, the work provides new information about assay performance in previously untested cell types.

Helicobacter pylori, a Group I carcinogen that infects over 50% of the global population, is a major contributor to gastric cancer and the growing crisis of antibiotic resistance. However, vaccine development has remained challenging due to the poor immunogenicity of candidate antigens and limitations in delivery strategies. To address this, we engineered a novel Fc-fusion vaccine targeting antigen-presenting cells (APCs) by fusing H. pylori UreB₃₁₇-₃₂₉ and UreB₄₀₉-₄₂₁ epitopes with Lpp20 lipoprotein to the murine IgG2a Fc domain (UreB: Lpp20: mFcγ2a). This study presents a preliminary, proof-of-concept evaluation of an Fc-fusion epitope-based vaccine candidate, focusing on in vitro characterization and APC-targeting properties. The construct was codon-optimized, expressed in Pichia pastoris, and purified via affinity chromatography. Expression levels in the mg/mL range, as determined by biochemical analysis, with >95% purity confirmed by SDS-PAGE and Western blot. Co-localization assays demonstrated 4.2-fold higher APC uptake of the Fc-fusion protein versus a His-tagged control (p < 0.001, unpaired t-test), mediated by FcγRI (CD64) binding. The protein maintained its structural stability for 30 days at 4 °C (<5% batch variability), underscoring manufacturing feasibility. While in vitro results validate APC targeting, limitations include preclinical scope and murine Fc compatibility gaps for human translation. This study established P. pastoris as a scalable platform for Fc-fusion vaccines, bypassing adjuvants and enhancing immunogenicity through conserved epitopes. Future work must evaluate in vivo efficacy in infection models and human Fc adaptations. These findings offer a blueprint for next-generation vaccines against H. pylori and other intracellular pathogens, urgently needed in antimicrobial resistance containment strategies.

Acute hepatopancreatic necrosis disease (AHPND), caused by Vibrio parahaemolyticus (Vp) carrying pir^vpA/pir^vpB, continues to threaten shrimp aquaculture across Asia. This study demonstrates a laboratory proof-of-concept gold nanoparticle-based (AuNPs) colorimetric biosensor targeting the pir^vpA gene of Vp. Citrate-reduced AuNPs (∼15.54 nm; SPR ≈ 520 nm) were functionalized with thiolated ssDNA probes (AuNP-Probe) using a deoxyadenosine triphosphate-assisted immobilization strategy and subsequently stabilized with low molecular weight (LMW) chitosan (AuNP-Probe-Chit). Loop-mediated isothermal amplification (LAMP) generated the target DNA, while detection relied on salt-induced aggregation assessed by visual color/precipitate formation and UV-Vis spectral shifts (A₆₅0/A_λmax, λ_max ≈ 532 nm for AuNP-Probe-Chit). Optimization identified 1.0 μM probe and 0.01% (v/v) chitosan as the optimal conditions for forming stable conjugates. Upon hybridization, an 8:2 (v/v) AuNP-Probe-Chit:LAMP mixture, combined with 0.5 M NaCl, enabled clear discrimination between positive and negative samples. Positives retained a pink-red supernatant with minimal aggregation, whereas negatives formed precipitates and exhibited higher aggregation indices. The biosensor demonstrated strong specificity for pir^vpA-positive DNA, without cross-reacting with Vibrio vulnificus or Vibrio alginolyticus, and exhibited sensitivity approaching the LAMP amplification threshold, with reliable discrimination observed at ≥10^-1 ng of target DNA. These findings establish a robust, equipment-lean diagnostic platform that integrates sequence-specific hybridization with polymer-mediated colloidal stabilization. The AuNP-Probe-Chit system offers rapid, visible discrimination, highlighting its potential for future AHPND surveillance in resource-limited settings. Compared with previous works, the incorporation of thiolated ssDNA probe functionalization with chitosan stabilization within a single AuNPs-based platform tailored for aquaculture pathogens, such as Vp, is currently underexplored, highlighting a significant need that this study aims to address.

Background: Cirsium japonicum (CJ) and Cirsium setosum (CS) are two widely recognized medicinal crops in Chinese herbal medicine (CHM) that are listed as authentic herbal plants in the herbal pharmacopeia. Given their strikingly similar morphological characteristics, CJ and CS are particularly susceptible to adulteration in the herbal marketplace, raising significant concerns about product integrity and consumer safety.

Purpose: To ensure the safety and efficacy of CHM, proper authentication of these herbs is a critical step in maintaining high-quality pharmaceutical production.

Methods: In this study, we developed a species-specific loop-mediated isothermal amplification (LAMP) method for the authentication of CS and CJ.

Results: We designed specific LAMP primer sets for both species using the selected DNA barcode, the internal transcribed spacer (ITS) sequence, through in silico analysis. After validating the specificity of the primers, we successfully authenticated CS and CJ using the LAMP primer sets and visualized the detection results through gel electrophoresis. The sensitivity of the LAMP method for CJ and CS authentication was established, with a limit of detection (LOD) of 100 pg for CJ and 50 pg for CS, using direct SYBR Green staining. When we applied the LAMP method to commercial Cirsium products, we found that only 25% (5 out of 20) of the samples contained CJ, while none contained CS.

Conclusion: In conclusion, we successfully established a species-specific LAMP assay for authenticating CJ and CS. This method can be used for species verification and is applicable to commercial Cirsium products for authenticity testing, contributing to quality control in pharmaceutical manufacturing.