CRISPR-Cas Biochemistry and CRISPR-Based Molecular Diagnostics.

Polymerase chain reaction (PCR)-based nucleic acid testing has played a critical role in disease diagnostics, pathogen surveillance, and many more. However, this method requires a long turnaround time, expensive equipment, and trained personnel, limiting its widespread availability and diagnostic capacity. On the other hand, the clustered regularly interspaced short palindromic repeats (CRISPR) technology has recently demonstrated capability for nucleic acid detection with high sensitivity and specificity. CRISPR-mediated biosensing holds great promise for revolutionizing nucleic acid testing procedures and developing point-of-care diagnostics. This review focuses on recent developments in both fundamental CRISPR biochemistry and CRISPR-based nucleic acid detection techniques. Four ongoing research hotspots in molecular diagnostics-target preamplification-free detection, microRNA (miRNA) testing, non-nucleic-acid detection, and SARS-CoV-2 detection-are also covered.

Bioanalytical approaches for the detection, characterization, and risk assessment of micro/nanoplastics in agriculture and food systems.

This review discusses the most recent literature (mostly since 2019) on the presence and impact of microplastics (MPs, particle size of 1 µm to 5 mm) and nanoplastics (NPs, particle size of 1 to 1000 nm) throughout the agricultural and food supply chain, focusing on the methods and technologies for the detection and characterization of these materials at key entry points. Methods for the detection of M/NPs include electron and atomic force microscopy, vibrational spectroscopy (FTIR and Raman), hyperspectral (bright field and dark field) and fluorescence imaging, and pyrolysis-gas chromatography coupled to mass spectrometry. Microfluidic biosensors and risk assessment assays of MP/NP for in vitro, in vivo, and in silico models have also been used. Advantages and limitations of each method or approach in specific application scenarios are discussed to highlight the scientific and technological obstacles to be overcome in future research. Although progress in recent years has increased our understanding of the mechanisms and the extent to which MP/NP affects health and the environment, many challenges remain largely due to the lack of standardized and reliable detection and characterization methods. Most of the methods available today are low-throughput, which limits their practical application to food and agricultural samples. Development of rapid and high-throughput field-deployable methods for onsite screening of MP/NPs is therefore a high priority. Based on the current literature, we conclude that detecting the presence and understanding the impact of MP/NP throughout the agricultural and food supply chain require the development of novel deployable analytical methods and sensors, the combination of high-precision lab analysis with rapid onsite screening, and a data hub(s) that hosts and curates data for future analysis.

Nanofibrillar cellulose/Au@Ag nanoparticle nanocomposite as a SERS substrate for detection of paraquat and thiram in lettuce.

A nanocomposite based on nanofibrillar cellulose (NFC) coated with gold-silver (core-shell) nanoparticles (Au@Ag NPs) was developed as a novel surface-enhanced Raman spectroscopy (SERS) substrate. SERS performance of NFC/Au@Ag NP nanocomposite was tested by 4-mercaptobenzoic acid. The cellulose nanofibril network was a suitable platform that allowed Au@Ag NPs to be evenly distributed and stabilized over the substrate, providing more SERS hotspots for the measurement. Two pesticides, thiram and paraquat, were successfully detected either individually or as a mixture in lettuce by SERS coupled with the nanocomposite. Strong Raman scattering signals for both thiram and paraquat were obtained within a Raman shift range of 400-2000 cm-1 and a Raman intensity ~ 8 times higher than those acquired by NFC/Au NP nanocomposite. Characteristic peaks were clearly observable in all SERS spectra even at a low concentration of 10 µg/L of pesticides. Limit of detection values of 71 and 46 µg/L were obtained for thiram and paraquat, respectively. Satisfactory SERS performance, reproducibility, and sensitivity of NFC/Au@Ag NP nanocomposite validate its applicability for real-world analysis to monitor pesticides and other contaminants in complex food matrices within a short acquisition time. Graphical abstract.

Synthesis of polyhedral gold nanostars as surface-enhanced Raman spectroscopy substrates for measurement of thiram in peach juice.

Rapid detection of food contaminants using novel analytical methods in combination with nanomaterials has received much attention. This study aimed to synthesize polyhedral gold nanostars (AuNS) with multi-angled corners and develop a surface-enhanced Raman spectroscopy (SERS) method coupled with AuNS to detect pesticide residues in juice products. AuNS are multi-branched three-dimensional metal nanostructures with rough surface features which can induce surface plasmon resonance. A facile synthesis of AuNS was achieved using a two-step method and as-prepared AuNS had much cleaner surfaces than gold nanoparticles. A Raman reporter molecule (4-aminothiophenol) was used to evaluate the performance of the SERS method, yielding fingerprint-like Raman spectra and the sensitivity of the SERS method could reach 10 ppb (µg kg-1). SERS coupled with AuNS was used to detect thiram residues in peach juice and the detection limit was 50 ppb, and is 100 times more sensitive than using normal gold nanoparticles. These results demonstrate that AuNS are excellent substrates for SERS measurement, which has great potential for rapid detection of chemical contaminants in food products.

Eggshell membrane-templated gold nanoparticles as a flexible SERS substrate for detection of thiabendazole.

The authors describe a three-dimensional (3D) flexible interconnected porous nanocomposite membrane for use in surface-enhanced Raman scattering (SERS). It was obtained via in -situ deposition of gold nanoparticles (AuNPs, ca. 10 nm) on eggshell membranes (ESM). The AuNP/ESM nanocomposites were used as a SERS substrate for detection of the pesticide thiabendazole (TBZ) with prominent Raman bands at 1180, 1280, and 1580 cm-1. The abundant "hot spots" are generated by the closely arranged AuNPs in the 3D geometry of the ESM networks. This makes the SERS substrate highly sensitive because of remarkable signal amplification. The substrates were applied to the rapid detection of TBZ in Oolong tea. The limit of detection for TBZ is 0.1 ppm. Graphical abstract Schematic presentation of a three-dimensional flexible interconnected porous nanocomposite membrane as surface-enhanced Raman scattering (SERS) substrate for detection of thiabendazole (TBZ) in tea.

Use of aminothiophenol as an indicator for the analysis of silver nanoparticles in consumer products by surface-enhanced Raman spectroscopy.

Silver nanoparticles (Ag NPs) are one of the top five engineered nanoparticles that have been used in various products. Current methods for the measurement of Ag NPs are time consuming and expensive. Therefore, it is of critical importance to develop novel strategies to detect the presence of Ag NPs at low concentrations in different matrices. This study aimed at detecting and measuring Ag NPs in consumer products using surface-enhanced Raman spectroscopy (SERS) coupled with aminothiophenol (PATP) as an indicator molecule that binds strongly with Ag NPs. Quantification and qualification of Ag NPs were achieved using this method of acquiring SERS signals from Ag NP-PATP complexes. Four dietary supplement products and one nasal spray were selected to evaluate the performance of SERS in the detection of Ag NPs. Inductively coupled plasma optical emission spectrometry (ICP-OES) and transmission electron microscopy (TEM) were utilized to measure the physical properties of Ag NPs in the samples. The results demonstrate that distinctive Raman peaks of PATP can be used to distinguish Ag NPs from silver bulk particles and silver nitrate. SERS is able to detect Ag NPs with different sizes ranging from 20 to 100 nm, with the highest intensity for ∼30 nm Ag NPs. A partial least squares method was used to develop quantitative models for the analysis of spectral data (R = 0.94). These results indicate that the conjugation of Ag NPs with PATP can be measured by SERS. These results demonstrate that SERS is a simple and rapid method and has great potential to detect Ag NPs in various products.

DNA-embedded Au-Ag core-shell nanoparticles assembled on silicon slides as a reliable SERS substrate.

This study aimed at developing a sensitive and reliable SERS substrate by assembling DNA-embedded Au-Ag core-shell nanoparticles (NPs) on silicon slides. First, a monolayer of well separated DNA-functionalized Au NPs (40 nm) was decorated on (3-aminopropyl)triethoxysilane modified silicon slides. The DNA-embedded Au-Ag core-shell NPs were assembled on the 40 nm Au-DNA NPs to form a core-satellite structure through DNA hybridization. Using 4-MBA as a Raman dye, the SERS performance of the substrates was evaluated after being cleaned by low oxygen and argon plasma. The Raman intensity of the assembly using DNA-embedded Au-Ag core-shell NPs was 8-10 times higher than the intensity of the assembly using Au NPs as satellites. In addition, the signal-to-noise ratio of the assembly was 2.6 times higher than that of a commercial substrate (Klarite™) when a 785 nm laser was used. The SERS enhancements of the assembled substrates were 2.2 to 2.8 times higher than the Klarite when an acquisition time of 5 s was used at an excitation wavelength of 633 nm. The assembled substrates also show a good spot-to-spot and substrate-to-substrate reproducibility at the excitation wavelengths of 633 and 785 nm. These results demonstrate that the fabrication process is simple and cost-effective for assembling DNA-embedded Au-Ag core-shell NPs on silicon slides that can be used as a reliable SERS substrate.

Engineered Nanoscale Food Ingredients: Evaluation of Current Knowledge on Material Characteristics Relevant to Uptake from the Gastrointestinal Tract.

The NanoRelease Food Additive project developed a catalog to identify potential engineered nanomaterials (ENMs) used as ingredients, using various food-related databases. To avoid ongoing debate on defining the term nanomaterial, NanoRelease did not use any specific definition other than the ingredient is not naturally part of the food chain, and its dimensions are measured in the nanoscale. Potential nanomaterials were categorized based on physical similarity; analysis indicated that the range of ENMs declared as being in the food chain was limited. Much of the catalog's information was obtained from product labeling, likely resulting in both underreporting (inconsistent or absent requirements for labeling) and/or overreporting (inability to validate entries, or the term nano was used, although no ENM material was present). Three categories of ingredients were identified: emulsions, dispersions, and their water-soluble powdered preparations (including lipid-based structures); solid encapsulates (solid structures containing an active material); and metallic or other inorganic particles. Although much is known regarding the physical/chemical properties for these ingredient categories, it is critical to understand whether these properties undergo changes following their interaction with food matrices during preparation and storage. It is also important to determine whether free ENMs are likely to be present within the gastrointestinal tract and whether uptake of ENMs may occur in their nanoform physical state. A practical decision-making scheme was developed to help manage testing requirements.

Facile synthesis of gold nanostars for the duplex detection of pesticide residues in grapes using SERS.

In recent years, concerns have been raised regarding the contamination of grapes with pesticide residues. As consumer demand for safer food products grows, regular monitoring of pesticide residues in food has become essential. This study sought to develop a rapid and sensitive technique for detecting two specific pesticides (phosmet and paraquat) present on the grape surface using the surface-enhanced Raman spectroscopy (SERS) method. Gold nanostars (AuNS) particles were synthesized, featuring spiky tips that act as hot spots for localized surface plasmon resonance, thereby enhancing Raman signals. Additionally, the roughened surface of AuNS increases the surface area, resulting in improved interactions between the substrate and analyte molecules. Prominent Raman peaks of mixed contaminants were acquired and used to characterize and quantify the pesticides. It was observed that the SERS intensity of the Raman peaks changed in proportion to the concentration ratio of phosmet and paraquat. Moreover, AuNS exhibited superior SERS enhancement compared to gold nanoparticles. The results demonstrate that the lowest detectable concentration for both pesticides on grape surfaces is 0.5 mg/kg. These findings suggest that SERS coupled with AuNS constitutes a practical and promising approach for detecting and quantifying trace contaminants in food. PRACTICAL APPLICATION: This research established a novel surface-enhanced Raman spectroscopy (SERS) method coupled with a simplified extraction protocol and gold nanostar substrates to detect trace levels of pesticides in fresh produce. The detection limits meet the maximum residue limits set by the EPA. This substrate has great potential for rapid measurements of chemical contaminants in foods.

Integrating transformer-based machine learning with SERS technology for the analysis of hazardous pesticides in spinach.

This study introduces an innovative strategy for the rapid and accurate identification of pesticide residues in agricultural products by combining surface-enhanced Raman spectroscopy (SERS) with a state-of-the-art transformer model, termed SERSFormer. Gold-silver core-shell nanoparticles were synthesized and served as high-performance SERS substrates, which possess well-defined structures, uniform dispersion, and a core-shell composition with an average diameter of 21.44 ± 4.02 nm, as characterized by TEM-EDS. SERSFormer employs sophisticated, task-specific data processing techniques and CNN embedders, powered by an architecture features weight-shared multi-head self-attention transformer encoder layers. The SERSFormer model demonstrated exceptional proficiency in qualitative analysis, successfully classifying six categories, including five pesticides (coumaphos, oxamyl, carbophenothion, thiabendazole, and phosmet) and a control group of spinach data, with 98.4% accuracy. For quantitative analysis, the model accurately predicted pesticide concentrations with a mean absolute error of 0.966, a mean squared error of 1.826, and an R2 score of 0.849. This novel approach, which combines SERS with machine learning and is supported by robust transformer models, showcases the potential for real-time pesticide detection to improve food safety in the agricultural and food industries.

Microencapsulation of highly concentrated polyphenolic compounds from purple corn pericarp by spray-drying with various biomacromolecules.

Colored corn pericarp contains unusually high amounts of industrially valuable phytochemicals, such as anthocyanins, flavanols, flavonoids, and phenolic acids. Polyphenols were extracted in an aqueous solution and spray-dried to produce microencapsulates using four carrier materials, namely, maltodextrin (MD), gum arabic (GA), methylcellulose (MC), and skim milk powder (SMP) at three concentrations (1, 2, and 3 %, respectively). The encapsulates were evaluated for their polyphenolic contents using spectrophotometric techniques and HPLC analyses, and their antioxidant properties were evaluated using four different assays. The physicochemical properties of encapsulates were analyzed by measuring the zeta potential (ZP), particle size distribution, water solubility index (WSI), water absorption index (WAI), and color parameters. Structural and thermal properties were evaluated using Fourier transform infrared spectroscopy (FTIR), optical profilometry, and differential scanning calorimetry (DSC) analyses. Comparative analysis of structural characteristics, particle size distribution, zeta potential, WSI, WAI, and aw of the samples confirmed the successful formulation of encapsulates. The microencapsulates embedded with 1 % concentrations of MD, MC, GA, or SMP retained polyphenolic compounds and exhibited noteworthy antioxidant properties. The samples encapsulated with GA or MD (1 %) demonstrated superior physicochemical, color, and thermal properties. Comprehensive metabolomic analysis confirmed the presence of 38 phytochemicals in extracts validating the spray-drying process.

Dicyclohexylcarbodiimide and disodium succinate induce the change of energy metabolism in relation to longan pulp breakdown.

Compared to control longan, DCC-treated longan had higher pulp breakdown index, lower ATP, ADP and EC levels, and lower H+, Ca2+ and Mg2+-ATPase activities. On day 6, DCC-treated longan presented 18% higher pulp breakdown index, with 44%, 9% and 31% lower levels of ATP, ADP and EC, respectively. Additionally, DCC-treated longan showed 29%, 53%, 37% lower activity of H+-ATPase, 34%, 54%, 4% lower activity of Ca2+-ATPase, and 13%, 21%, 6% lower activity of Mg2+-ATPase in the membranes of plasma, vacuole, and mitochondria, respectively. Whereas, DS-treated longan manifested the opposite trends of DCC treatment. These results suggest that the accelerated pulp breakdown in DCC-treated longan was linked to energy deficiency and reduced energy production. However, DS treatment restrained pulp breakdown occurrence in fresh longan by maintaining a higher energy level through the elevated energy production and ATPase activity.

DNP and ATP modulate the pulp softening and breakdown in fresh longan by acting on the antioxidant system and the metabolisms of membrane lipids and cell wall polysaccharides.

Compared to the control longan, DNP treatment elevated pulp breakdown index, reduced the values of pulp firmness, CSP, ISP, cellulose, and hemicellulose by enhancing the activities of PE, PG, Cx, XET, and ß-Gal. Additionally, DNP treatment increased the levels of PLD, lipase, LOX, PA, and SFA, and decreased the values of PC, PI, USFA, U/S, and IUFA, displaying higher cell membrane permeability and more severe cell membrane damage in longan pulp. Furthermore, DNP treatment weakened the levels of SOD, CAT, APX, AsA, GSH, TP, and TF, thereby exacerbating ROS outbreak and MDA production. These results indicate that DNP treatment destroyed the antioxidant system to cause ROS eruption. This disruption further disturbed the metabolisms of membrane lipids and cell wall polysaccharides, leading to the breakdown of cell membrane and cell wall, and eventually aggravated longan pulp softening and breakdown. However, ATP treatment exhibited the opposite effects of DNP treatment.

Comparative transcriptome and metabolome reveal the role of acidic electrolyzed oxidizing water in improving postharvest disease resistance of longan fruit.

Acidic electrolyzed oxidizing water (AEOW) was applied to suppress disease development and maintain good quality of fresh fruit. However, the involvement of AEOW in improving disease resistance of fresh longan remains unknown. Here, transcriptomic and metabolic analyses were performed to compare non-treated and AEOW-treated longan during storage. The transcriptome analysis showed AEOW-induced genes associated with phenylpropanoid and flavonoid biosynthesis. The metabolome analysis found the contents of coumarin, phenolic acid, and tannin maintained higher levels in AEOW-treated longan than non-treated longan. Moreover, the weighted correlation network analysis (WGCNA) was performed to identify hub genes, and a gene-metabolite correlation network associated with AEOW-improved disease resistance in longan was constructed by the co-analysis of transcriptomics and metabolomics. These findings identified a series of important genes and metabolites involving in AEOW-induced disease resistance of longan fruit, expanding our knowledges on fruit disease resistance and quality maintenance at the transcript and metabolic levels.

Acidic electrolyzed-oxidizing water treatment mitigated the disease progression in Phomopsis longanae Chi-infected longans by modulating ROS and membrane lipid metabolism.

Postharvest harmful pathogenic infestation leads to rapid decay in longan fruit. Compared with P. longanae-infected longans, AEOW alleviated fruit disease severity and diminished the O2-. production rate and MDA content. It also increased APX, CAT, and SOD activities, delayed the decrease in the levels of GSH and AsA, as well as the reducing power and DPPH radical scavenging ability, which resulted in a decline in membrane lipid peroxidation in P. longanae-infected longans. Additionally, AEOW reduced LOX, lipase, PI-PLC, PC-PLC, and PLD activities, maintained higher levels of PC, PI, IUFA, USFAs, and U/S, while reducing levels of PA, DAG, SFAs, and CMP. These effects alleviated membrane lipid degradation and peroxidation in P. longanae-infected longans. Consequently, AEOW effectively maintained membrane integrity via improving antioxidant capacity and suppressing membrane lipid peroxidation. This comprehensive coordination of ROS and membrane lipid metabolisms improved fruit resistance and delayed disease development in longans.

Quantitative detection of nitrate in water and wastewater by surface-enhanced Raman spectroscopy.

The presence of inorganic nitrogen species in water can be unsuitable for drinking and detrimental to the environment. In this study, a surface-enhanced Raman spectroscopy (SERS) method coupled with a commercially available gold nanosubstrate (a gold-coated silicon material) was evaluated for the detection of nitrate and nitrite in water and wastewater. Applications of SERS coupled with gold nanosubstrates resulted in an enhancement of Raman signals by a factor of ∼10(4) compared to that from Raman spectroscopy. The new method was able to detect nitrate with linear ranges of 1-10,000 mg NO3(-)/L (R(2)= 0.978) and 1-100 mg NO3(-)/L (R(2)= 0.919) for water and wastewater samples, respectively. Among the common anions, phosphate appeared to be the major interfering anion affecting nitrate measurement. Nevertheless, the percentage error of nitrate measurement in wastewater by the proposed SERS method was comparable to that by ion chromatography. The nitrate detection limits in water and wastewater samples were about 0.5 mg/L. The SERS method could simultaneously detect sulfate, which may serve as a reference standard in water. These results suggested that the SERS coupled with nanosubstrates is a promising method to determine nitrate concentrations in water and wastewater.

Preparation and Characterization of Boza Enriched with Nonfat Dry Milk and Its Impact on the Fermentation Process.

Boza is an indigenous, traditional, low-alcohol and highly viscous beverage prepared by fermenting cereals. Its thick and gel-like consistency make it suitable for consumption via spoon. Although boza is a nutritious beverage, its protein content is very low (<2%). A new type of boza was developed by incorporating nonfat dry milk (NFDM) to elevate the protein content of the beverage. Different NFDM amounts (10 to 40% w/v) were added to determine the best concentration and fermentation time based on the refractive index and pH values at room temperature (0-48 h). The best sample was further characterized by rheological analyses and Fourier transform infrared (FTIR) spectroscopy. The sample with 10% NFDM was the best, as fermentation was successfully performed, and further addition of NFDM increased the initial pH. The refractive index and pH decreased from 21.9 ± 0.1 to 11.8 ± 0.1 and 5.77± 0.50 to 4.09 ± 0.35 during fermentation, respectively. The samples exhibited shear-thinning, solid-like behavior, and a gel-like structure. FTIR analysis by independent modeling of class analogy (SIMCA) demonstrated that unfermented slurry and the fermented product could be effectively differentiated. With the addition of 10% NFDM, the increase in the protein content of the boza medium became significant.

Comparison between two cultivars of longan fruit cv. 'Dongbi' and 'Fuyan' in the metabolisms of lipid and energy and its relation to pulp breakdown.

This work studied the difference in pulp breakdown between two cultivars of longan cv. 'Dongbi' and 'Fuyan' from the aspect of metabolisms of lipid and energy. The results reflected that, compared to 'Fuyan' longan, 'Dongbi' longan had higher levels of energy charge, U/S and IUFA, and higher amounts of USFA, PC, PI, ATP and ADP. Moreover, 'Dongbi' longan exhibited lower levels of SFA, PA, AMP and cell membrane permeability. Also, lower PLD, LOX and lipase activities, but higher ATPase activity, lower pulp breakdown index, and better pulp appearance were exhibited in 'Dongbi' longan. These data revealed that the mitigated pulp breakdown in 'Dongbi' longan was due to the comprehensive coordination of metabolisms in lipid and energy through maintaining a higher level of energy, a higher unsaturation degree of fatty acids, delaying the degradation of phospholipids, and better retaining the membrane structural integrity of microsome and entire cell.

DNP and ATP regulate the breakdown occurrence in the pulp of Phomopsis longanae Chi-infected longan fruit through modulating the metabolism of membrane lipid.

This study aimed to illustrate how DNP and ATP affected the pulp breakdown occurrence in P. longanae-infected longan and their relationship with the membrane lipid metabolism. Compared with P. longanae-inoculated samples, the pulp of DNP-treated P. longanae-infected longan exhibited higher cellular membrane permeability, breakdown index, activities of PI-PLC, PLD, PC-PLC, LOX, and lipase, and values of SFAs, PA, and DAG, while lower levels of PI, PC, USFAs, IUFA and U/S. However, the opposite findings were observed in ATP-treated P. longanae-infected longan. The data manifested that DNP-increased the pulp breakdown occurrence in P. longanae-inoculated samples was due to the elevated MLDEs activities that reduced the contents of phospholipids (PI, PC) and USFAs, disrupting the cell membrane structures. Nevertheless, ATP decreased the pulp breakdown occurrence in P. longanae-inoculated samples, which was ascribed to the reduced MLDEs activities that raised phospholipids (PI, PC) and USFAs contents, thus maintaining the cell membrane structures.