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%.

Reconfiguration of chemical sensors, intended as the capacity of the sensor to adapt to novel operational scenarios, e.g., new target analytes, is potentially game changing and would enable rapid and cost-effective reaction to dynamic changes occurring at healthcare, environmental, and industrial levels. Yet, it is still a challenge, and rare examples of sensor reconfiguration have been reported to date. Here, we report on a reconfigurable label-free optical sensor leveraging the versatile immobilization of metal ions through a chelating agent on a nanostructured porous silica (PSiO2) optical transducer for the detection of different biomolecules. First, we show the reversible grafting of different metal ions on the PSiO2 surface, namely, Ni2+, Cu2+, Zn2+, and Fe3+, which can mediate the interaction with different biomolecules and be switched under mild conditions. Then, we demonstrate reconfiguration of the sensor at two levels: 1) switching of the metal ions on the PSiO2 surface from Cu2+ to Zn2+ and testing the ability of Cu2+-functionalized and Zn2+-reconfigured devices for the sensing of the dipeptide carnosine (CAR), leveraging the well-known chelating ability of CAR toward divalent metal ions; and 2) reconfiguration of the Cu2+-functionalized PSiO2 sensor for a different target analyte, namely, the nucleotide adenosine triphosphate (ATP), switching Cu2+ with Fe3+ ions to exploit the interaction with ATP through phosphate groups. The Cu2+-functionalized and Zn2+-reconfigured sensors show effective sensing performance in CAR detection, also evaluated in tissue samples from murine brain, and so does the Fe3+-reconfigured sensor toward ATP, thus demonstrating effective reconfiguration of the sensor with the proposed surface chemistry.

Polymerase β (POLB), with dual functionality as a lyase and polymerase, plays a critical role in the base excision repair (BER) pathway to maintain genomic stability. POLB knockout and rescue studies in BRCA1/2-mutant cancer cell lines revealed that inhibition of lyase and polymerase activity is required for the synthetic lethal interaction observed with PARP inhibitors, highlighting POLB as a valuable therapeutic target. Traditional biochemical assays to screen for enzyme inhibitors focus on a single substrate to product relationship and limit the comprehensive analysis of enzymes such as POLB that utilize multiple substrates or catalyze a multi-step reaction. This report describes the first high-throughput mass spectrometry-based screen to measure the two distinct biochemical activities of POLB in a single assay using a duplexed self-assembled monolayer desorption ionization (SAMDI) mass spectrometry methodology. A multiplexed assay for POLB dual enzymatic activities was developed optimizing for kinetically balanced conditions and a collection of 200,000 diverse small molecules was screened in the duplexed format. Small molecule modulators identified in the screen were confirmed in a traditional fluorescence-based polymerase strand-displacement assay and an orthogonal label-free binding assay using SAMDI affinity selection mass spectrometry (ASMS). This work demonstrates the flexibility of high-throughput mass spectrometry approaches in drug discovery and highlights a novel application of SAMDI technology that opens new avenues for multiplexed high-throughput screening.

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.

Immunoglobulin G (IgG) and immunoglobulin M (IgM) testing are commonly used to determine infection status. Typically, the detection of IgM indicates an acute or recent infection, while the presence of IgG alone suggests a chronic or past infection. However, relying solely on IgG and IgM antibody positivity may not be sufficient to differentiate acute from chronic infections. This limitation arises from several factors. The prolonged presence of IgM can complicate diagnostic interpretations, and false positive IgM results often arise from antibody cross-reactivity with various antigens. Additionally, IgM may remain undetectable in prematurely collected samples or in individuals who are immunocompromised, further complicating accurate diagnosis. As a result, additional diagnostic tools are required to confirm infection status. Avidity is a measure of the strength of the binding between an antigen and antibody. Avidity-based assays have been developed for various infectious agents, including toxoplasma, cytomegalovirus (CMV), SARS-CoV-2, and avian influenza, and are promising tools in clinical diagnostics. By measuring the strength of antibody binding, they offer critical insights into the maturity of the immune response. These assays are instrumental in distinguishing between acute and chronic or past infections, monitoring disease progression, and guiding treatment decisions. The development of automated platforms has optimized the testing process by enhancing efficiency and minimizing the risk of manual errors. Additionally, the recent advent of real-time biosensor immunoassays, including the label-free immunoassays (LFIA), has further amplified the capabilities of these assays. These advances have expanded the clinical applications of avidity-based assays, making them useful tools for the diagnosis and management of various infectious diseases. This review is structured around several key aspects of IgG avidity in clinical diagnosis, including: (i) a detailed exposition of the IgG affinity maturation process; (ii) a thorough discussion of the IgG avidity assays, including the recently emerged biosensor-based approaches; and (iii) an examination of the applications of IgG avidity in clinical diagnosis. This review is intended to contribute toward the development of enhanced diagnostic tools through critical assessment of the present landscape of avidity-based testing, which allows us to identify the existing knowledge gaps and highlight areas for future investigation.

This work describes the development of a label-free (LF) biosensing platform for the direct detection of targets based on diffractive structures fabricated with acrylamide-based hydrogels biofunctionalized with proteins and antibodies. Hydrogels containing Bovine Serum Albumin protein (BSA) with different crosslinking degrees were synthesized and characterized to find the optimal conditions for the suitable fabrication of surface relief gratings (SRGs). The bioavailability of BSA-functionalized hydrogels for the specific recognition of anti-BSA antibodies was verified by fluorescence detection. After the hydrogel-based SRG fabrication, diffraction efficiency measures at two different laser wavelengths were used for the direct LF detection of anti-BSA antibodies. The limit of detection in the sub mg L-1 range was read. Additionally, SRGs were prepared with hydrogels biofunctionalized with anti-rabbit antibodies for the direct detection of IgGs from rabbit serum, obtaining similar analytical performance without the necessity of labeling or applying amplification strategies.

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.