The construction of ofloxacin detection in fish matrix based on a shark-derived single-domain antibody.

BACKGROUND: Due to the serious issue of ofloxacin (OFL) abuse, there is an increasingly urgent need for accurate and rapid detection of OFL. Immunoassay has become the "golden method" for detecting OFL in complex matrix beneficial to its applicability for a large-scale screening, rapidity, and simplicity. However, traditional antibodies used in immunoassay present challenges such as time-consuming preparation, unstable sensitivity and specificity, and difficulty in directional evolution. In this paper, we successfully developed an OFL detection method based on a shark-derived single-domain antibody (ssdAb) to address these issues. RESULTS: Using phage display technology and a heterologous expression system, OFL-specific clones 1O11, 1O13, 1O17, 1O19, 1O21, and 2O26 were successfully isolated and expressed in soluble form. Among all OFL-specific ssdAbs, the 1O17 ssdAb exhibited the highest binding affinity to OFL in a concentration-dependence manner. The limit of detection (IC10) of 1O17 ssdAb was calculated as 0.34 ng/mL with a detection range of 3.40-1315.00 ng/mL, and its cross reactivity with other analogs was calculated to be less than 5.98 %, indicating high specificity and sensitivity. Molecular docking results revealed that 100Trp and 101Arg located in the CDR3 region of 1O17 ssdAb were crucial for OFL binding. In fish matrix performance tests, the 1O17 ssdAb did not demonstrate severe matrix interference in OFL-negative fish matrix, achieving satisfactory recovery rates ranging from 83.04 % to 108.82 % with high reproducibility. SIGNIFICANCE: This research provides a new and efficient OFL detection recognition element with significant potential in immunoassay applications, broadening the application scenarios of ssdAbs. It offers valuable insights into the structure-activity relationship between ssdAbs and small molecules, laying a theoretical foundation for the further directional modification and maturation of ssdAbs in subsequent applications.

Multifunctional metal-organic framework-enhanced sodium alginate-based intelligent indicator: Mechanism and application for freshness monitoring.

Intelligent food packaging has recently gained significant attention due to the heightened consumer awareness regarding food quality. Although anthocyanins avoid safety issues, the instability and leakage of anthocyanins restrict their utilization in freshness indicator labels. In this study, we introduced an innovative metal-organic framework (UiO-66-NH2) synergistic pH-colorimetric label with fast ammonia-responsive, incorporating sodium alginate, red cabbage anthocyanin, and UiO-66-NH2. The cross-linked sodium alginate substrate enabled the label to possess superior insolubility. The microscopic morphology of the labels was intricately analyzed, while their sensitivity was rigorously tested utilizing ammonia as a representative gas. Due to the remarkable UV absorption capability of UiO-66-NH2 and various molecular interactions with anthocyanins, the label exhibited good UV absorption, enhanced stability, and optimized performance in reducing anthocyanin leakage, ensuring the stability and effectiveness of the labels in practical applications. The prepared label exhibited good specificity for volatile amines and ammonia gases, and robust anti-interference properties, enabling visualization and early detection of shrimp spoilage during storage at different temperatures. The strategy employed in this study presents promising new possibilities for developing intelligent packaging solutions for food products.

Anti-Inflammatory Efficacy of Nanobody Specific to ß-Glucan on a Fungal Cell Wall in a Murine Model of Fungal Keratitis.

Purpose: to explore the anti-inflammatory effects of a nanobody (Nb) specific to ß-glucan on fungal keratitis (FK). Methods: in order to verify the therapeutic and anti-inflammatory efficacy of Nb in FK, the severity of inflammation was assessed with inflammatory scores, hematoxylin-eosin (HE) staining, and myeloperoxidase (MPO) assays. In corneas of mice of FK model and human corneal epithelial cells stimulated by fungal hyphae, real-time reverse transcriptase polymerase chain reaction, Western blot, and enzyme-linked immunosorbent assay were used to detect the expression levels of inflammatory cytokines and pattern recognition receptors (PRRs). In vivo, macrophages and neutrophils infiltration in the cornea stroma was detected by immunofluorescence (IFS) staining. Results: In murine models infected with Aspergillus fumigatus (A. fumigatus), Nb treatment could reduce the inflammatory scores. HE staining and MPO results showed Nb significantly alleviated corneal edema and reduced inflammatory cell infiltration 3 days post-infection. In addition, the expression levels of LOX-1 and Dectin-1 were significantly decreased in the Nb group in vivo. The expression of chemokines CCL2 and CXCL2 also decreased in the Nb group. Compared with the PBS group, the number of macrophages and neutrophils in the Nb group was significantly decreased, which was shown in IFS results. Moreover, Nb attenuated the expression of Dectin-1, LOX-1, and inflammatory mediators, including IL-6 and IL-8 in vitro. Conclusion: our study showed that Nb could alleviate FK by downregulating the expression of PRRs and inflammatory factors as well as reducing the infiltration of macrophages and neutrophils.

Data fusion of near-infrared and Raman spectroscopy: An innovative tool for non-destructive prediction of the TVB-N content of salmon samples.

Total volatile basic nitrogen (TVB-N) serves as a crucial indicator for evaluating the freshness of salmon. This study aimed to achieve accurate and non-destructive prediction of TVB-N content in salmon fillets stored in multiple temperature settings (-20, 0, -4, 20 °C, and dynamic temperature) using near-infrared (NIR) and Raman spectroscopy. A partial least square support vector machine (LSSVM) regression model was established through the integration of NIR and Raman spectral data using low-level data fusion (LLDF) and mid-level data fusion (MLDF) strategies. Notably, compared to a single spectrum analysis, the LLDF approach provided the most accurate prediction model, achieving an R2P of 0.910 and an RMSEP of 1.922 mg/100 g. Furthermore, MLDF models based on 2D-COS and VIP achieved R2P values of 0.885 and 0.906, respectively. These findings demonstrated the effectiveness of the proposed method for precise quantitative detection of salmon TVB-N, laying a technical foundation for the exploration of similar approaches in the study of other meat products. This approach has the potential to assess and monitor the freshness of seafood, ensuring consumer safety and enhancing product quality.

Improving the detection accuracy of the dual SERS aptasensor system with uncontrollable SERS "hot spot" using machine learning tools.

BACKGROUND: Simultaneous detection of food contaminants is crucial in addressing the collective health hazards arising from the presence of multiple contaminants. However, traditional multi-competitive surface-enhanced Raman scattering (SERS) aptasensors face difficulties in achieving simultaneous accurate detection of multiple target substances due to the uncontrollable SERS "hot spots". In this study, using chloramphenicol (CAP) and estradiol (E2) as two target substances, we introduced a novel approach that combines machine learning methods with a dual SERS aptasensor, enabling simultaneous high-sensitivity and accurate detection of both target substances. RESULTS: The strategy effectively minimizes the interference from characteristic Raman peaks commonly encountered in traditional multi-competitive SERS aptasensors. For this sensing system, the Au@4-MBA@Ag nanoparticles modified with sulfhydryl (SH)-CAP aptamer and Au@DTNB@Ag NPs modified with sulfhydryl (SH)-E2 aptamer were used as signal probes. Additionally, Fe3O4@Au nanoflowers integrated with SH-CAP aptamer complementary DNA and SH-E2 aptamer complementary DNA were used as capture probes, respectively. When compared to linear regression random forest, and support vector regression (SVR) models, the proposed artificial neural network (ANN) model exhibited superior precision, demonstrating R2 values of 0.963, 0.976, 0.991, and 0.970 for the training set, test set, validation set, and entire dataset, respectively. Validation with ten spectral groups reported an average error of 244 µg L-1. SIGNIFICANCE: The essence of our study lies in its capacity to address a persistent challenge encountered by traditional multiple competitive SERS aptasensors - the interference generated by uncontrollable SERS "hot spots" that hinders simultaneous quantification. The accuracy of the predictive model for simultaneous detection of two target substances was significantly improved using machine learning tools. This innovative technique offers promising avenues for the accurate and high-sensitive simultaneous detection of multiple food and environmental contaminants.

Seafood waste derived carbon nanomaterials for removal and detection of food safety hazards.

Nanobiotechnology and the engineering of nanomaterials are currently the main focus of many researches. Seafood waste carbon nanomaterials (SWCNs) are a renewable resource with large surface area, porous structure, high reactivity, and abundant active sites. They efficiently adsorb food contaminants through π-π conjugated, ion exchange, and electrostatic interaction. Furthermore, SWCNs prepared from seafood waste are rich in N and O functional groups. They have high quantum yield (QY) and excellent fluorescence properties, making them promising materials for the removal and detection of pollutants. It provides an opportunity by which solutions to the long-term challenges of the food industry in assessing food safety, maintaining food quality, detecting contaminants and pretreating samples can be found. In addition, carbon nanomaterials can be used as adsorbents to reduce environmental pollutants and prevent food safety problems from the source. In this paper, the types of SWCNs are reviewed; the synthesis, properties and applications of SWCNs are reviewed and the raw material selection, preparation methods, reaction conditions and formation mechanisms of biomass-based carbon materials are studied in depth. Finally, the advantages of seafood waste carbon and its composite materials in pollutant removal and detection were discussed, and existing problems were pointed out, which provided ideas for the future development and research directions of this interesting and versatile material. Based on the concept of waste pricing and a recycling economy, the aim of this paper is to outline current trends and the future potential to transform residues from the seafood waste sector into valuable biological (nano) materials, and to apply them to food safety.

Bimetallic Fe/Ni metal organic framework-based hypoxanthine biosensor for early monitoring of freshness changes of aquatic products.

The timely detection of freshness changes of aquatic products is crucial. In this study, we have developed a reliable, cost-effective, and user-friendly method for rapidly detecting hypoxanthine using a xanthine oxidase (XOD)/nanozyme enzymatic cascade system. The nanozyme, derived from the Fe7/Ni3 metal-organic framework (Fe7Ni3MOF), exhibited good peroxidase-mimetic activity and stability. Our proposed XOD/Fe7Ni3MOF enzymatic cascade system demonstrated a linear response to hypoxanthine in the range of 3-70 µM, with a low detection limit of 1.39 µM. We also analyzed hypoxanthine in actual aquatic products, achieving spiked recoveries ranging from 90.04 % to 107.37 %. The correlation coefficient between our developed colorimetric method and the HPLC method was 0.98. Importantly, our proposed method holds several advantages over alternative techniques, particularly in terms of cost-effectiveness, precision, and speed. Consequently, this methodology shows great promise for the early detection of freshness changes in aquatic samples.

Spectral data fusion in nondestructive detection of food products: Strategies, recent applications, and future perspectives.

In recent years, the food industry has shown a growing interest in the development of rapid and nondestructive analytical methods. However, the utilization of a solitary nondestructive detection technique offers only a constrained extent of physical or chemical insights regarding the sample under examination. To overcome this limitation, the amalgamation of spectroscopy with data fusion strategies has emerged as a promising approach. This comprehensive review delves into the fundamental principles and merits of low-level, mid-level, and high-level data fusion strategies within the domain of food analysis. Various data fusion techniques encompassing spectra-to-spectra, spectra-to-machine vision, spectra-to-electronic nose, and spectra-to-nuclear magnetic resonance are summarized. Moreover, this review also provides an overview of the latest applications of spectral data fusion techniques (SDFTs) for classification, adulteration, quality evaluation, and contaminant detection within the purview of food safety analysis. It also addresses current challenges and future prospects associated with SDFTs in real-world applications. Despite the extant technical intricacy, the ongoing evolution of online data fusion platforms and the emergence of smartphone-based multi-sensor fusion detection technology augur well for the pragmatic realization of SDFTs, endowing them with formidable capabilities for both qualitative and quantitative analysis in the realm of food analysis.

Transformation of arsenic species from seafood consumption during in vitro digestion.

Arsenic (As) species analysis is important for the risk evaluation of seafood. Until now, there has been limited information on the change of As species during digestion. Here, the As species in different types of seafood before and after in vitro digestion were investigated. Although inorganic As was not detected in digested fish samples, As(V) contents in digested crabs and scallops were 17.12 ± 1.76 and 138.69 ± 7.53, respectively, which were approximately 2-3 times greater than those of the pre-digestion samples. In further experiments, arsenocholine, dimethylarsinate, arsenobetaine, and monomethylarsonate were all convertible to As(V) during in vitro digestions with different rates. The transformation demonstrates a complex process and could be affected by many factors, such as pH, time, and digestion juice composition, of which pH seemed to be particularly important. Free radicals were responsible for the oxidation in the transformation reactions. Unlike arsenobetaine, arsenocholine seemed to be able to directly transform to monomethylarsonate without the intermediate dimethylarsinate. This study reveals and validates the potential of other species (oAs or/and unknown species) to convert to iAs, identifies the main factors affecting this process, and proposes a reaction pathway. There is an important implication for promoting a more accurate risk assessment of arsenic in foodstuffs.

Efficient Hapten-Specific Biopanning Strategy Based on the Fe3O4@ENR-Functionalized Core-Shell Magnetic Nanoparticles.

The biopanning of target-specific phages is one of the most critical steps in the preparation of single-domain antibodies. In the traditional biopanning of haptens, the nonspecific binding of library phages to macromolecular proteins is one of the most challenging problems in preparing single-domain antibodies. In this research, Fe3O4@ENR-functionalized core-shell magnetic nanoparticles (FMNPs) were silylated and aminated by tetraethyl orthosilicate and (3-aminopropyl)triethoxysilane, and target enrofloxacin was coupled onto the surface by the carbodiimide method. The magnetic nanoparticles were characterized by Fourier transform infrared spectroscopy, particle size distribution, zeta potential, transmission electron microscopy observation, and indirect enzyme-linked immunosorbent assay (ELISA). A biopanning strategy based on Fe3O4@ENR FMNPs was then established to solve the problem in the traditional solid-phase biopanning process. The results showed that a considerable number of enrofloxacin (ENR)-positive phages were screened by only one round of biopanning. Finally, two ENR-specific shark-derived single-domain genes were identified and validated by monoclonal phage ELISA, gene sequencing, and biolayer interferometry technology. Our study provides a new biopanning strategy based on Fe3O4@ENR FMNPs for efficiently providing phages specific to haptens.

The preparation and therapeutic effects of ß-glucan-specific nanobodies and nanobody-natamycin conjugates in fungal keratitis.

Fungal keratitis (FK) is a severe infectious corneal disease. Since traditional eye drops exhibit poor dissolution and high corneal toxicity, the efficacy of current treatments for FK remains limited. It is needed to develop new approaches to control the cornea damage from FK. In this study, a nanobody (Nb) specific to ß-glucan in the fungal cell wall was prepared. The conjugate of the Nb with natamycin (NAT), a traditional antifungal drug, was synthesized. Firstly, we found the Nb specific to ß-glucan inhibited fungal growth by disrupting cell wall and biofilm formation.. In addition, the content of ß-glucan in the fungal cell wall decreased after Nb treatment. The Nb also reduced the adhesion ability of fungal conidia to human corneal epithelial cells (HCECs). Further, we examined the difference between NAT and Nb-NAT in antifungal growth. Nb-NAT showed better antifungal effects than NAT which was caused by the interaction between Nb and ß-glucan. Moreover, Nb concentration below 0.5 mg/mL did not affect the viability of HCECs. Nb-NAT had less cytotoxicity and ocular surface irritation than NAT. Nb specific to ß-glucan attenuated Aspergillus fumigatus (A. fumigatus) virulence and relieved inflammatory responses in FK. Nb-NAT treatment of the cornea improved therapeutic effects compared with NAT. It decreased clinical scores and expression level of inflammatory factors. To our knowledge, this study is the first to report a Nb specific to ß-glucan and Nb-NAT for the treatment of FK. These unique functions of the Nb specific to ß-glucan and Nb-NAT would render it as an alternative molecule to control fungal infections including FK. STATEMENT OF SIGNIFICANCE: Fungal keratitis is a corneal disease with a high rate of blindness. Due to the poor dissolution and high corneal toxicity exhibited by traditional eye drops, the efficacy of current therapeutic treatments for fungal keratitis (FK) remains limited. To enhance the therapeutic effect of natamycin in treating fungal keratitis, this study developed an innovative approach by preparing a ß-glucan-specific nanobody and loading it with the antifungal drug natamycin. The ß-glucan-specific nanobody has the ability to control both fungal pathogen invasion and inflammation, which can cause damage to the cornea in FK. The conjugation with the ß-glucan-specific nanobody significantly increased the antifungal capacity of natamycin and reduced its toxicity. The further application of natamycin conjugated with the ß-glucan-specific nanobody could be expanded to other diseases caused by fungal pathogen infections.

On-site detection of chloramphenicol in fish using SERS-based magnetic aptasensor coupled with a handheld Raman spectrometer.

In recent years, the rapid detection of chloramphenicol (CAP) has become a market demand due to its high toxicity. In this study, for the first time, a portable surface-enhanced Raman scattering (SERS) aptasensor for the rapid and on-site detection of chloramphenicol (CAP) residues in fish was developed. Fe3O4@Au nanoflowers combined with sulfhydryl (SH)-CAP aptamer complementary DNA acted as capture probes. SH-CAP aptamer modified Au@Ag nanoparticles (Au@Ag NPs) embedded with 4-mercaptobenzoic acid (4-MBA) were served as reporter probes. The strongest Raman intensity was produced due to the coupling of Fe3O4@Au nanoflowers (Fe3O4@Au NFs) and Au@Ag NPs. For CAP detection, a wide linear range from 0.001 to 1000 µg/L, with an R2 of 0.9805, was obtained. The limit of detection was determined to be 0.87 ng/L. The SERS aptasensor showed excellent performance for analytical applications for real fish samples. Compared with the conventional HPLC method, the developed SERS aptasensor coupled with a handheld Raman spectrometer had flexible application and avoided the limitations of complex operating conditions. It should be a promising portable analytical tool for analysis of drug residues in the field.

Structural and expression analysis of the dopamine receptors reveals their crucial roles in regulating the insulin signaling pathway in oysters.

Dopamine performs its critical role upon binding to receptors. Since dopamine receptors are numerous and versatile, understanding their protein structures and evolution status, and identifying the key receptors involved in the modulation of insulin signaling will provide essential clues to investigate the molecular mechanism of neuroendocrine regulating the growth in invertebrates. In this study, seven dopamine receptors were identified in the Pacific oysters (Crassostrea gigas) and were classified into four subtypes according to their protein secondary and tertiary structures, and ligand-binding activities. Of which, DR2 (dopamine receptor 2) and D(2)RA-like (D(2) dopamine receptor A-like) were considered the invertebrate-specific type 1 and type 2 dopamine receptors, respectively. Expression analysis indicated that the DR2 and D(2)RA-like were highly expressed in the fast-growing oyster "Haida No.1". After in vitro incubation of ganglia and adductor muscle with exogenous dopamine and dopamine receptor antagonists, the expression of these two dopamine receptors and ILPs (insulin-like peptides) was also significantly affected. Dual-fluorescence in situ hybridization results showed that D(2)RA-like and DR2 were co-localized with MIRP3 (molluscan insulin-related peptide 3) and MIRP3-like (molluscan insulin-related peptide 3-like) in the visceral ganglia, and were co-localized with ILP (insulin-like peptide) in the adductor muscle. Furthermore, the downstream components of dopamine signaling, including PKA, ERK, CREB, CaMKK1, AKT, and GSK3ß were also significantly affected by the exogenous dopamine and dopamine receptor antagonists. These findings confirmed that dopamine might affect the secretion of ILPs through the invertebrate-specific dopamine receptors D(2)RA-like and DR2, and thus played crucial roles in the growth regulation of the Pacific oysters. Our study establishes the potential regulatory relationship between the dopaminergic system and insulin-like signaling pathway in marine invertebrates.

Controlling the amount of coupling agents on the synthesis of coating antigens to enhance the sensitivity of fluoroquinolone immunodetection.

There is now increasing demand to improve the sensitivity of various immunoassays for fluoroquinolones (FQs) and other food hazards. In this study, different coating antigens were prepared by adjusting the content of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) to explore its influence on the immunoassay sensitivity of FQs. The results indicated that, unlike traditional assumptions, a reasonable EDC dosage should be addressed to reach the best analytical efficiency, and excessive EDC could enhance the hapten-carrier conjugation but significantly reduce the detection sensitivity. For the FQs investigated, the hapten:EDC:BSA proportion of 20:2.5:50 (Mole ratio:74:34:1) seemed the best for preparation of coating antigens, and the sensitivity could be improved more than 1000 times both for indirect competitive enzyme linked immunosorbent assay ELISA (ic-ELISA) and gold immunochromatography assay (GICA) due to two key factors including coupling-ratios and amide bond groups. Such an improved efficiency was also validated well with different food samples, which indicated the reasonable optimization of EDC in coating antigen synthesis may be widely used as a new, simple and more effective strategy to improve the immunoassay for low molecular targets in medical, environment and food detection filed.

Characterization, specific recognition, and the performance in fish matrix of a shark-derived single-domain antibody against enrofloxacin.

The third generation of genetic engineering antibodies, single-domain antibodies, have been widely reported as potential biomaterials in recognizing small molecular hazards. In this study, a shark-derived single-domain antibody was used as the recognition element for the first time to detect enrofloxacin (ENR), one of the most representative hazards in aquaculture. An ENR-specific clone named 2E6 was isolated by phage display technology. Experimental results proved that 2E6 ssdAb showed high affinity to ENR-PEI complete antigen, with the highest OD450 value of 1.348 in binding ELISA. Through icELISA, it was determined that the IC50 of 2E6 ssdAb to ENR was 19.230 ng/mL, while the IC10 was 0.975 ng/mL, with rare recognition to other fluoroquinolones, which showed high sensitivity and specificity to ENR. The 2E6 ssdAb also performed excellently in fish matrix immunoassay. Results showed that the ENR-negative fish matrix did not seriously interfere with the recognition of 2E6 ssdAb to ENR-OVA, with the matrix index between 4.85% and 11.75%, while the results of icELISA in ENR-spiked fish matrix showed that 2E6 ssdAb could recognize the target ENR in different ENR-spiked concentrations of the fish matrix (10-1000 ng/mL), with the recovery between 89.30% and 126.38% and the RSD between 1.95% and 9.83%. This study broadens the application scenario of shark-derived single-domain antibodies as small molecule recognition biomaterials, providing a new recognition element on ENR detection for immunoassay.

Amide-containing neoepitopes: the key factor in the preparation of hapten-specific antibodies and a strategy to overcome.

For a long time, people have suffered from uncertainty, complexity, and a low success rate in generating and screening antibodies against small molecules, which have become the core bottlenecks of immunochemistry. Here, the influence of antigen preparation on antibody generation was investigated at both molecular and submolecular levels. Neoepitopes (amide-containing neoepitopes) formed in the preparation of complete antigens are one of the most important factors limiting the efficiency of generating hapten-specific antibodies, which was verified by different haptens, carrier proteins, and conjugation conditions. Amide-containing neoepitopes present electron-dense structural components on the surface of prepared complete antigens and, therefore, induce the generation of the corresponding antibody with much higher efficiency than target hapten. Crosslinkers should be carefully selected and not overdosed. According to these results, some misconceptions in the conventional anti-hapten antibody production were clarified and corrected. By controlling the content of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) during the synthesis of immunogen to limit the formation of amide-containing neoepitopes, the efficiency of hapten-specific antibody generation could be significantly improved, which verified the correctness of the conclusion and provided an efficient strategy for antibody preparation. The result of the work is of scientific significance in the preparation of high-quality antibodies against small molecules.

Biopanning, Heterologous Expression, and Characterization of a Shark-Derived Single-Domain Antibody Fusion Protein against Pancreatic Lipase.

Nowadays, obesity severely impacts human health and is the fifth leading risk factor that leads to death globally. Pancreatic lipase (PL) inhibitors have attracted extensive attention owing to their role in effective prevention and treatment of obesity. Here, a shark-derived single-domain antibody fusion protein was used to inhibit PL for the first time. After biopanning, the heterologous expression system pET28a-SUMO-D2 was constructed using the method of double restriction enzyme digestion and T4 ligase to achieve the soluble expression of the PL-specific antibody gene D2. According to the calculation of protein concentration, the final expression of fusion protein PL-D2S was 1.183 mg per liter of Luria Bertani broth. The binding ability of the soluble fusion protein PL-D2S to PL was identified. Enzyme-linked immunosorbent assay results showed that the fusion protein PL-D2S exhibited a strong binding affinity to PL. The experimental results of PL inhibition of PL-D2S in vitro showed that the fusion protein could significantly inhibit the activity of PL, with an IC50 of 404 µg/mL. Our study shows that the fusion protein PL-D2S is a potential PL inhibitor to prevent and treat obesity.

Rapid and Sensitive Fluorescence Detection of Staphylococcus aureus Based on Polyethyleneimine-Enhanced Boronate Affinity Isolation.

There are increasing demands for fast and simple detection of pathogens in foodstuffs. Fluorescence analysis has demonstrated significant advantages for easy operation and high sensitivity, although it is usually hindered by a complex matrix, low bacterial abundance, and long-term bacterial enrichment. Effective enrichment procedures are required to meet the requirements for food detection. Here, boronate-functionalized cellulose filter paper and specific fluorescent probes were combined. An integrated approach for the enrichment of detection of Staphylococcus aureus was proposed. The modification of polyethyleneimine demonstrated a significant effect in enhancing the bacterial enrichment, and the boronate affinity efficiency of the paper was increased by about 51~132%. With optimized conditions, the adsorption efficiency for S. aureus was evaluated as 1.87 × 108 CFU/cm2, the linear range of the fluorescent analysis was 104 CFU/mL~108 CFU/mL (R2 = 0.9835), and the lowest limit of detection (LOD) was calculated as 2.24 × 102 CFU/mL. Such efficiency was validated with milk and yogurt samples. These results indicated that the material had a high enrichment capacity, simple operation, and high substrate tolerance, which had the promising potential to be the established method for the fast detection of food pathogens.

SERS detection of thiram using polyacrylamide hydrogel-enclosed gold nanoparticle aggregates.

The development of sensitive and long-term signal-stable plasmonic substrates is vital to the in-field application of the surface-enhanced Raman spectroscopy (SERS) technique. The colloidal gold nanoparticles (AuNPs) system is commonly used in SERS detection, but it shows less signal stability and reproducibility due to the uncontrollable aggregation of nanoparticles by adding aggregating agents in SERS detection. In this study, we developed a new SERS detection platform based on polyacrylamide hydrogel-enclosed plasmonic gold nanoparticle aggregates (PAH-AuANs). In the system, the formation of PAH can rapidly stabilize the gold nanoparticle aggregates, avoiding the over-aggregation or precipitation of AuNPs. With the PAH concentration in the range of 6-10 % and AuNPs at the concentration of 0.2 nM, the resulting PAH-AuNAs platform exhibited both sensitive SERS activity and excellent SERS signal stability. The relative standard deviation of the 4-MBA probe SERS signal collected from the PAH-AuNAs platform was lower than 3 %. The limit of detection for the pesticide thiram was down to 0.38 µg/L with a handheld Raman spectrometer. Moreover, the procedure for preparing the PAH-AuNAs platform was easy to handle, offering a new strategy for in-field detection of environmental contaminants with a handheld Raman spectrometer in the future.

The specific biopanning of single-domain antibody against haptens based on a functionalized cryogel.

Phage display technology is commonly applied for high-throughput screening of single-domain antibodies (sdAbs), and the problem of non-specific adsorption caused by carrier proteins seriously affects the biopanning of single-domain antibodies specific to haptens. In this paper, enrofloxacin (ENR)-functionalized cryogels were prepared by the ethylenediamine (EDA) and carbodiimide methods for application in the biopanning of ENR-specific phages. To improve the efficiency of biopanning, double blocking, a wash solution flow rate of 1 mL/min, and phage pre-incubation were applied to the biopanning process through single-factor experiments. Results of flat colony counting showed that the phage output of AG-ENR cryogels was 15 times higher than that of AG cryogels for the same input amount. And seven complete sequences of ENR-specific shark sdAbs were obtained by monoclonal phage ELISA and sequence alignment. All these results indicate that functionalized cryogels could be used as a novel and efficient method for phage biopanning for single-domain antibodies to haptens.