This work investigated the functional changes in whey proteins obtained from goat milk subject to various temperature treatments. Ultra-high temperature instantaneous sterilization (UHTIS) caused less damage than the common low-temperature, whereas spray-drying treatment had the opposite effect. A total of 426 proteins were identified in UHTIS and control treatment groups, including 386 common proteins and 16 and 14 unique proteins. The UHTIS treatment upregulated 55 whey proteins while down-regulated 98. The UHTIS-treated whey proteins may upregulate three metabolic pathways but downregulate one. Overall, UHTIS only slightly impacted the composition and functions of whey proteins from goat milk compared to the common low-temperature treatments.

BACKGROUND: Rapid and sensitive detection of foodborne pathogens in food plays a crucial role in controlling outbreaks of foodborne diseases, of which Listeria monocytogenes and Salmonella typhimurium are representative and notable pathogens. Thus, it\'s of great importance to achieve the effective detection of these pathogens. However, the most common detection methods (culture-based technique, Polymerase Chain Reaction and immunological methods) have disadvantages that cannot be ignored, such as time-consuming, laborious, complex sample preparation process, and the possibility of cross-reaction. Hence, it is essential to develop a facile detection method for the pathogens with high sensitivity and specificity to avoid the above-mentioned disadvantages.
RESULTS: We report a label-free visual platform for the simultaneous capture and detection of Listeria monocytogenes and Salmonella typhimurium. For the first time, we have prepared polydimethylsiloxane-Chromotrope 2R membrane which serves as the substrate for bacterial capture and enrichment through the formation of specific recognition sites. The positively charged Pt-covalent organic framework combines with the pathogens through surface charge interaction, thereby the label-free sandwich platform is formed. Remarkable peroxidase activity of Pt-covalent organic framework converts the conversion of bacterial quantity into amplified color signal by catalyzing 3,3\',5,5\'-Tetramethylbenzidine to oxidized 3,3\',5,5\'-Tetramethylbenzidine. The platform demonstrates the capability to identify two representative food-borne pathogens within a time frame of 100 min, exhibiting high sensitivity and excellent specificity without the interference from non-target bacteria. The limit of detection of the visual platform toward Listeria monocytogenes and Salmonella typhimurium was 1.61 CFU mL-1 and 1.31 CFU mL-1, respectively. And the limit of quantification toward Listeria monocytogenes and Salmonella typhimurium was 4.94 CFU mL-1 and 2.47 CFU mL-1, respectively. The relative standard derivations of the visual platform for both bacteria were lower than 4.9 %. Furthermore, our proposed platform has obtained reliable and satisfactory results on analyzing diverse food samples.
CONCLUSIONS: This research expands the application of a label-free platform combined with unlabeled nanocomponents in the rapid isolation and detection of diverse of food-borne pathogens. The platform possesses the advantages of simple operation and real-time monitoring, without complicated sample pretreatment process. The whole detection process can realize the simultaneous monitoring of Listeria monocytogenes and Salmonella typhimurium within 100 min. Furthermore, it is also of reference significance for the detection of other common pathogens.

G-quadruplex/thioflavin T (G4/THT) is one of the ideal label-free fluorescent light-emitting elements in the field of biosensors due to its good programmability and adaptability. However, the unsatisfactory luminous efficiency of single-molecule G4/THT limits its more practical applications. Here, we developed a G4 embedded semi-catalytic hairpin assembly (G4-SCHA) reaction by rationally modifying the traditional CHA reaction, and combined with the invasive reaction, supplemented by magnetic separation technology, for label-free sensitive detection of single nucleotide polymorphisms (SNPs). The invasive reaction enabled specific recognition of single-base mutations in DNA sequences as well as preliminary signal cycle amplification. Then, magnetic separation was used to shield the false positive signals. Finally, the G4-SCHA was created for secondary amplification and label-free output of the signal. This dual-signal amplified label-free biosensor has been shown to detect mutant targets as low as 78.54 fM. What\'s more, this biosensor could distinguish 0.01 % of the mutant targets from a mixed sample containing a large number of wild-type targets. In addition, the detection of real and complex biological samples also verified the practical application value of this biosensor in the field of molecular design breeding. Therefore, this study improves a label-free fluorescent light-emitting element, and then proposes a simple, efficient and universal label-free SNP biosensing strategy, which also provides an important reference for the development of other G4/THT based biosensors.

Leveraging the fluorescence enhancement effect of the G-triplex (G3)/thioflavin T (ThT) catalyzed by the adjacent double-stranded DNA positioned at the 5\' terminus of the G3, the G3-specific oligonucleotide (G3MB6) was utilized to facilitate the rapid detection of mercury (Hg(II)) through thymine-Hg(II)-thymine (T-Hg(II)-T) interactions. G3MB6 adopted a hairpin structure in which partially complementary strands could be disrupted with the presence of Hg(II). It prompted the formation of double-stranded DNA by T-Hg(II)-T, inducing the unbound single strand of G3MB6 to spontaneously form a parallel G3 structure, producing a solid fluorescence signal by ThT. Conversely, fluorescence was absent without Hg(II), since no double strand and formation of G3 occurred. The fluorescence intensity of G3MB6 exhibited a positive correlation with Hg(II) concentrations from 17.72 to 300 nM (R2 = 0.9954), boasting a notably low quality of limitation (LOQ) of 17.72 nM. Additionally, it demonstrated remarkable selectivity for detecting Hg(II). Upon application to detect Hg(II) in milk samples, the recovery rates went from 100.3% to 103.2%.

In the field of biomedicine, efficiently and non-invasively isolating target cells has always been one of the core challenges. Optical fiber tweezers offer precise and non-invasive manipulation of cells within a medium and can be easily integrated with microfluidic systems. Therefore, this paper investigated the mechanism of cell manipulation using scattering force with optical fiber tweezers. We employed flat-ended single-mode fiber to drive and sort cells and derived the corresponding scattering force formula based on the T-matrix model. A single-mode optical tweezers system for cell sorting was developed, and an optofluidic experimental platform was constructed that effectively integrates the optical system with microfluidic chips. The chip, featuring an expanded cross-channel design, successfully achieved continuous separation of yeast cells (8~10 µm in diameter) and polystyrene microspheres (15~20 µm in diameter), with a sorting efficiency of up to 86% and maintaining viability in approximately 90% of the yeast cells. Compared to other sorting systems, this system does not require labeling and can achieve continuous sorting with cell viability at a lower cost of instrumentation.

Cell dielectric property measurement holds significant potential for application in cell detection and diagnosis due to its label-free and noninvasive nature. In this study, we developed a biosensor designed to measure the permittivity of liquid samples, particularly cell suspensions at the nanoliter scale, utilizing microwave and millimeter wave coplanar waveguides in conjunction with a microchannel. This biosensor facilitates the measurement of scattering parameters within a frequency domain ranging from 1 GHz to 110 GHz. The obtained scattering parameters are then converted into dielectric constants using specific algorithms. A cell capture structure within the microchannel ensures that cell suspensions remain stable within the measurement zone. The feasibility of this biosensor was confirmed by comparison with a commercial Keysight probe. We measured the dielectric constants of three different cell suspensions (HepG2, A549, MCF-7) using our biosensor. We also counted the number of cells captured in multiple measurements for each cell type and compared the corresponding changes in permittivity. The results indicated that the real part of the permittivity of HepG2 cells is 0.2-0.8 lower than that of the other two cell types. The difference between A549 and MCF-7 was relatively minor, only 0.2-0.4. The fluctuations in the dielectric spectrum caused by changes in cell numbers during measurements were smaller than the differences observed between different cell types. Thus, the sensor is suitable for measuring cell suspensions and can be utilized for label-free, noninvasive studies in identifying biological cell suspensions.

Three-dimensional (3D) network provide a promising platform for construction of high sensitive electrochemical immunosensor due to the benefits of high specific surface area and electron mobility. Herein, a sensitive label-free electrochemical immunosensor based on Au nanoparticles modified Ni-B nanosheets/graphene matrix was constructed to detect diethylstilbestrol (DES). The 3D network not only could increase the electron transport rate and surface area, but also could provide confinement area, which is conducive to increases the collision frequency with the active site. Moreover, Au NPs also have good biocompatibility, which is beneficial for ligating antibodies. Benefiting from the 3D network structure and Au collective effect, the electrochemical immunosensor possess sterling detection ability with wide linear response range (0.00038-150 ng/mL) and low detection limit (31.62 fg/mL). Moreover, the constructed immunosensor can also be extend to detect DES in Tap-water and river water. This work may provide a novel material model for the construction of high sensitive immunosensor.

Chronic liver disease, a long-term condition resulting from various causes such as alcohol abuse, metabolic disorders, and viral hepatitis, is becoming a significant global health challenge. Gypenosides (GPs), derived from the traditional Chinese medicine Gynostemma pentaphyllum (Thunb.) Makino, exhibited hepatoprotective properties in recent years, yet the precise therapeutic mechanism remains unclear. In this study, label-free and parallel reaction monitoring (PRM) proteomics were used to elucidate the hepatoprotective mechanism of GPs in liver injury rats. Through label-free proteomics, we identified 2104 differentially expressed proteins (DEPs) associated with liver injury, along with 1974 DEPs related to the effects of GPs. Bioinformatics analysis revealed that GPs primarily restored metabolic processes involving valine, leucine, and isoleucine degradation, as well as propanoate and butanoate metabolism, and steroid hormone biosynthesis during liver injury. Subsequently, overlapping the two groups of DEPs identified 1508 proteins reversed following GPs treatment, with key targets further validated by PRM. Eight target proteins were identified for GPs treatment of liver injury, including Lgals3, Psat1, Phgdh, Cyp3a9, Cyp2c11, Cyp4a2, Glul, and Ces1d. These findings not only elucidated the hepatoprotective mechanism of GPs, but may also serve as potential therapeutic targets of chronic liver disease.

A label-free fluorescent sensing strategy for the rapid and highly sensitive detection of Pb2+ was developed by integrating Pb2+ DNAzyme-specific cleavage activity and a tetrahedral DNA nanostructure (TDN)-enhanced hyperbranched hybridization chain reaction (hHCR). This strategy provides accelerated reaction rates because of the highly effective collision probability and enriched local concentrations from the spatial confinement of the TDN, thus showing a higher detection sensitivity and a more rapid detection process. Moreover, a hairpin probe based on a G-triplex instead of a G-quadruplex or chemical modification makes hybridization chain reaction more controlled and flexible, greatly improving signal amplification capacities and eliminating labeled DNA probes. The enhanced reaction rates and improved signal amplification efficiency endowed the biosensors with high sensitivity and a rapid response. The label-free detection of Pb2+ based on G-triplex combined with thioflavin T can be achieved with a detection limit as low as 1.8 pM in 25 min. The proposed Pb2+-sensing platform was also demonstrated to be applicable for Pb2+ detection in tap water, river water, shrimp, rice, and soil samples, thus showing great potential for food safety and environmental monitoring.

Carcinoembryonic Antigen (CEA), an acidic glycoprotein with human embryonic antigen properties, is found on the surface of cancer cells that have differentiated from endodermal cells. This paper presents a label-free electrochemical immunoassay for the dual amplification detection of CEA using gold nanoparticles loaded with polypyrrole polydopamine (Au/PPy-PDA) and polymerized polycaprolactone (Ng-PCL) prepared by ring-opening polymerization (ROP). First, the composite Au/PPy-PDA was adhered to the electrode surface. Then, gold nanoparticles form a Au-S bond with the sulfhydryl group in Apt1 to secure it on the electrode surface. Subsequently, the non-specific binding sites on the electrodes surface are closed by bovine serum albumin (BSA). Next, CEA is dropped onto the electrode surface, which is immobilized by antigen-antibody specific recognition, and the carboxyl-functionalized Apt2 forms a \"sandwich structure\" of antibody-antigen-antibody by specific recognition. Polymeric Ng-PCL is adhered to the electrode surface, leading to an increase in the electrochemical impedance signal, resulting in a complete chain of signal analysis. Finally, the response signal is detected by electrochemical impedance spectroscopy (EIS). Under optimal experimental conditions, the method has the advantages of high sensitivity and wide linear range (1 pg mL-1∼100 ng mL-1), and the lower limit of detection (LOD) is 0.234 pg mL-1. And it has the same high sensitivity, selectivity and interference resistance for the real samples detection. Thus, it provides a new way of thinking about biomedical and clinical diagnosis.