Synthetic biology for the food industry: advances and challenges.

As global environmental pollution increases, climate change worsens, and population growth continues, the challenges of securing a safe, nutritious, and sustainable food supply have become enormous. This has led to new requirements for future food supply methods and functions. The use of synthetic biology technology to create cell factories suitable for food industry production and renewable raw material conversion into: important food components, functional food additives, and nutritional chemicals, represents an important method of solving the problems faced by the food industry. Here, we review the recent progress and applications of synthetic biology in the food industry, including alternatives to: traditional (artificial pigments, meat, starch, and milk), functional (sweeteners, sugar substitutes, nutrients, flavoring agents), and green (green fiber, degradable packing materials, green packaging materials and food traceability) foods. Furthermore, we discuss the future prospects of synthetic biology-based applications in the food industry. Thus, this review may serve as a reference for research on synthetic biology in the: food safety, food nutrition, public health, and health-related fields.

Development and perspectives of rapid detection technology in food and environment.

Food safety become a hot issue currently with globalization of food trade and food supply chains. Chemical pollution, microbial contamination and adulteration in food have attracted more attention worldwide. Contamination with antibiotics, estrogens and heavy metals in water environment and soil environment have also turn into an enormous threat to food safety. Traditional small-scale, long-term detection technologies have been unable to meet the current needs. In the monitoring process, rapid, convenient, accurate analysis and detection technologies have become the future development trend. We critically synthesizing the current knowledge of various rapid detection technology, and briefly touched upon the problem which still exist in research process. The review showed that the application of novel materials promotes the development of rapid detection technology, high-throughput and portability would be popular study directions in the future. Of course, the ultimate aim of the research is how to industrialization these technologies and apply to the market.

Stimuli-responsive DNA-based hydrogels for biosensing applications.

The base sequences of DNA are endowed with the rich structural and functional information and are available for the precise construction of the 2D and 3D macro products. The hydrogels formed by DNA are biocompatible, stable, tunable and biologically versatile, thus, these have a wide range of promising applications in bioanalysis and biomedicine. In particular, the stimuli-responsive DNA hydrogels (smart DNA hydrogels), which exhibit a reversible and switchable hydrogel to sol transition under different triggers, have emerged as smart materials for sensing. Thus far, the combination of the stimuli-responsive DNA hydrogels and multiple sensing platforms is considered as biocompatible and is useful as the flexible recognition components. A review of the stimuli-responsive DNA hydrogels and their biosensing applications has been presented in this study. The synthesis methods to prepare the DNA hydrogels have been introduced. Subsequently, the current status of the stimuli-responsive DNA hydrogels in biosensing has been described. The analytical mechanisms are further elaborated by the combination of the stimuli-responsive DNA hydrogels with the optical, electrochemical, point-of-care testing (POCT) and other detection platforms. In addition, the prospects of the application of the stimuli-responsive DNA hydrogels in biosensing are presented.

CRISPR/Cas12a-based technology: A powerful tool for biosensing in food safety.

BACKGROUND: In the context of the current pandemic caused by the novel coronavirus, molecular detection is not limited to the clinical laboratory, but also faces the challenge of the complex and variable real-time detection fields. A series of novel coronavirus events were detected in the process of food cold chain packaging and transportation, making the application of molecular diagnosis in food processing, packaging, transportation, and other links urgent. There is an urgent need for a rapid detection technology that can adapt to the diversity and complexity of food safety. SCOPE AND APPROACH: This review introduces a new molecular diagnostic technology-biosensor analysis technology based on CRISPR-Cas12a. Systematic clarification of its development process and detection principles. It summarizes and systematically organizes its applications in viruses, food-borne pathogenic bacteria, small molecule detection, etc. In the past four years, which provides a brand-new and comprehensive solution for food detection. Finally, this article puts forward the challenges and the prospects for food safety. KEY FINDINGS AND CONCLUSIONS: The novel coronavirus hazards infiltrated every step of the food industry, from processing to packaging to transportation. The biosensor analytical technology based on CRISPR-Cas12a has great potential in the qualitative and quantitative analysis of infectious pathogens. CRISPR-Cas12a can effectively identify the presence of the specific nucleic acid targets and the small changes in sequences, which is particularly important for nucleic acid identification and pathogen detection. In addition, the CRISPR-Cas12a method can be adjusted and reconfigured within days to detect other viruses, providing equipment for nucleic acid diagnostics in the field of food safety. The future work will focus on the development of portable microfluidic devices for multiple detection. Shao et al. employed physical separation methods to separate Cas proteins in different microfluidic channels to achieve multiple detection, and each channel simultaneously detected different targets by adding crRNA with different spacer sequences. Although CRISPR-Cas12a technology has outstanding advantages in detection, there are several technical barriers in the transformation from emerging technologies to practical applications. The newly developed CRISPR-Cas12a-based applications and methods promote the development of numerous diagnostic and detection solutions, and have great potential in medical diagnosis, environmental monitoring, and especially food detection.

CRISPR/dCas9-surface-enhanced Raman scattering for the detection of drug resistance gene macB.

Antibiotics have brought many benefits to public health systems worldwide since their first use in the last century, yet with their overuse in clinical treatment and livestock farming, new public health issues have arisen. Previously, we found in our experiments that the levels of macB genes in bovine raw milk ranked among the top of many drug resistance genes. In this paper, we present an analysis of regularly interspaced clustered short palindromic repeats (CRISPR) combined with surface-enhanced Raman scattering (SERS) technology for the detection of the drug resistance gene macB. The analysis was accomplished through the collaboration of the CRISPR system's ability to specifically identify genes and the more sensitive performance of the SERS. The analysis detects the drug resistance gene macB and does not yet require complex steps such as nucleic acid amplification. This method may prove to be an effective method for accurate detection of the drug-resistant gene macB, thus enabling more effective prevention of contamination of drug-resistant genes in food hygiene.

Fabrication of Magnetic Al-Based Fe3O4@MIL-53 Metal Organic Framework for Capture of Multi-Pollutants Residue in Milk Followed by HPLC-UV.

The efficient capture of multi-pollutant residues in food is vital for food safety monitoring. In this study, in-situ-fabricated magnetic MIL-53(Al) metal organic frameworks (MOFs), with good magnetic responsiveness, were synthesized and applied for the magnetic solid-phase extraction (MSPE) of chloramphenicol, bisphenol A, estradiol, and diethylstilbestrol. Terephthalic acid (H2BDC) organic ligands were pre-coupled on the surface of amino-Fe3O4 composites (H2BDC@Fe3O4). Fe3O4@MIL-53(Al) MOF was fabricated by in-situ hydrothermal polymerization of H2BDC, Al (NO3)3, and H2BDC@Fe3O4. This approach highly increased the stability of the material. The magnetic Fe3O4@MIL-53(Al) MOF-based MSPE was combined with high-performance liquid chromatography-photo diode array detection, to establish a novel sensitive method for analyzing multi-pollutant residues in milk. This method showed good linear correlations, in the range of 0.05-5.00 µg/mL, with good reproducibility. The limit of detection was 0.004-0.108 µg/mL. The presented method was verified using a milk sample, spiked with four pollutants, which enabled high-throughput detection and the accuracies of 88.17-107.58% confirmed its applicability, in real sample analysis.

Dual Sensitization Smartphone Colorimetric Strategy Based on RCA Coils Gathering Au Tetrahedra and Its Application in the Detection of CK-MB.

In recent years, the combination of DNA nanotechnology and biosensing has been extensively reported. Herein, we attempted to develop a dual sensitization smartphone colorimetric strategy based on rolling circle amplification (RCA) coils gathering Au tetrahedra and explore its application. The dual sensitization effect of this strategy was achieved by rolling circle amplification (RCA) and Au tetrahedra. Under the initiation of the complementary DNA, a large number of ssDNA were generated, achieving amplification of the reaction signal. At the same time, due to the formation of Au tetrahedra, more gold nanoparticles could be gathered under the same conditions, and the signal would be amplified again. Using software ImageJ, the gray value of the reaction solution can be analyzed, detecting the target timely under the practical conditions of lack of equipment. By selecting aptamers with strong binding affinity, we applied this strategy to detect creatine kinase isoenzymes (CK-MB), showing a limit of detection of 0.8 pM, which performed well in actual detection and can meet the needs for real-time detection of CK-MB. Therefore, a universal detection platform was developed, which has broad application prospects in biosensing, clinical diagnosis, food detection, and other fields.

The orphan nuclear receptor Nur77 plays a vital role in BPA-induced PC12 cell apoptosis.

Bisphenol A (BPA) is a typical environmental endocrine disruptor that can migrate into organisms through skin contact, breathing, diet and various other approaches. The reproductive toxicity and neurotoxicity of BPA has been confirmed by several toxicological studies. However, the neurotoxicity of BPA is still controversial. In the present study, we used PC12 cells as a model to investigate the mechanism of BPA-induced neuronal apoptosis. BPA exposure reduced cell viability, altered cell morphology and aggravated intracellular Lactate dehydrogenase (LDH) release, intracellular Ca2+ concentration, Reactive oxygen species (ROS) levels, apoptosis and the reduction in the mitochondrial transmembrane potential (ΔΨm). Moreover, the results of the Western blot (WB) and Real-time quantitative polymerase chain reaction (RT-qPCR) assays indicated that the expression levels of Nur77 in the BPA group were down-regulated and accompanied by the downregulation of the NF-κb/Bcl-2 proteins and the upregulation of cleaved-caspase 3, which is a marker of apoptosis. However, these changes were significantly reversed with the upregulation of the Nur77 protein by introducing plasmids carrying the nur77 gene. These results indicated that BPA-induced apoptosis was closely related to Nur77-mediated inhibition of the NF-κb/Bcl-2 pathway.

Micro-/nanostructures for surface-enhanced Raman spectroscopy: Recent advances and perspectives.

Surface-enhanced Raman spectroscopy (SERS) has great potential for the analysis of molecules adsorbed on metals with rough surfaces or substrates with micro-/nanostructures. Plasmonic coupling between metal nanoparticles and the morphology of the rough metal surface can produce "hot spots" that enhance Raman scattering by adsorbed molecules, typically at micro- to nanomolar concentrations, although high enhancement factors can also facilitate single-molecule detection. This phenomenon is widely applicable for chemical analysis and sensing in various fields. In this review, the latest research progress on SERS micro-/nanosensors is evaluated, and the sensors are classified according to their individual functions. Furthermore, the design principles and working mechanisms of reported SERS-active micro-/nanostructured substrates are analyzed, and the design features adopted to overcome the difficulties associated with precision detection are explored. Finally, challenges and directions for future development in this field are discussed. This review serves as a design guide for novel SERS-active substrates.

A facile dual-mode immunosensor based on speckle Ag-doped nanohybrids for ultrasensitive detection of Ochratoxin A.

Ochratoxin A (OTA) is a potent carcinogen, and is among the most dangerous mycotoxins in agricultural products. In this study, an ultrasensitive dual-mode immunosensor was developed for naked-eye and fluorescence detection of OTA based on Ag-doped core-shell nanohybrids (Ag@CSNH). Complete antigen-labeled Ag@CSNH (CA-Ag@CSNH) were used as a competitive bind and dual-mode probe. The diffused doping structure of CA-Ag@CSNH provided improved stability, color and fluorescence quencher performance. Antibodies modified magnetic beads were used as a capture probe. The competitive binding between OTA and CA-Ag@CSNH produced both color change and fluorescence quenching. Ultraviolet and fluorescence intensitie correlated linearly with OTA concentration ranges of 0.03-3 ng/mL and 10-10000 pg/mL, and limits of detection of 0.0235 ng/mL and 0.9921 pg/mL, respectively. The practical applicability of proposed strategy was demonstrated by analysis of OTA in spiked corn, soybean and flour samples. This study offers a new insight on multi-mode platforms for various applications.

Amplification-free detection of HBV DNA mediated by CRISPR-Cas12a using surface-enhanced Raman spectroscopy.

Nucleic acid markers have been widely used in the detection of various virus-related diseases, including hepatitis B virus (HBV), which is spreading worldwide. The trans-activated CRISPR-Cas system has shown excellent sensitivity and specificity in nucleic acid detection. However, nucleic acid testing usually requires amplification of the target nucleic acid for more accurate and specific detection; furthermore, current nucleic acid assays are time-consuming, costly, and are limited by non-specific cross-reactivity. We developed an amplification-free viral DNA biosensor-based diagnostic method that uses a clustered regularly interspaced short palindromic repeats-associated system (CRISPR/Cas)-based approach with surface enhanced Raman spectroscopy. This method can specifically identify the target site by changing the crRNA sequence. In addition, the incubation period and development of the disease can be determined by quantitative detection of viral DNA. This system could achieve rapid and highly sensitive detection of HBV DNA within 50 min and vast detection range from 0.1 pM to 1 nM. Therefore, a combined CRISPR/Cas12a-SERS-based assay would improve the sensitivity of detection in assays using multiple biomarkers. In conclusion, our CRISPR/Cas12a-based biosensor would enable rapid, simple, and sensitive detection of HBV nucleic acids.

Wearable biosensors for human fatigue diagnosis: A review.

Fatigue causes deleterious effects to physical and mental health of human being and may cause loss of lives. Therefore, the adverse effects of fatigue on individuals and the society are massive. With the ever-increasing frequency of overtraining among modern military and sports personnel, timely, portable and accurate fatigue diagnosis is essential to avoid fatigue-induced accidents. However, traditional detection methods require complex sample preparation and blood sampling processes, which cannot meet the timeliness and portability of fatigue diagnosis. With the development of flexible materials and biosensing technology, wearable biosensors have attracted increased attention to the researchers. Wearable biosensors collect biomarkers from noninvasive biofluids, such as sweat, saliva, and tears, followed by biosensing with the help of biosensing modules continuously and quantitatively. The detection signal can then be transmitted through wireless communication modules that constitute a method for real-time understanding of abnormality. Recent developments of wearable biosensors are focused on miniaturized wearable electrochemistry and optical biosensors for metabolites detection, of which, few have exhibited satisfactory results in medical diagnosis. However, detection performance limits the wide-range applicability of wearable fatigue diagnosis. In this article, the application of wearable biosensors in fatigue diagnosis has been discussed. In fact, exploration of the composition of different biofluids and their potential toward fatigue diagnosis have been discussed here for the very first time. Moreover, discussions regarding the current bottlenecks in wearable fatigue biosensors and the latest advancements in biochemical reaction and data communication modules have been incorporated herein. Finally, the main challenges and opportunities were discussed for wearable fatigue diagnosis in the future.

The development of a fluorescence/colorimetric biosensor based on the cleavage activity of CRISPR-Cas12a for the detection of non-nucleic acid targets.

Nano-biosensors are of great significance for the analysis and detection of important biological targets. Surprisingly, the CRISPR-Cas12a system not only provides us with excellent gene editing capabilities, it also plays an important role in biosensing due to its high base resolution and high levels of sensitivity. However, most CRISPR-Cas12a-based sensors are limited by their recognition and output modes, are therefore only utilized for the detection of nucleic acids using fluorescence as an output signal. In the present study, we further explored the potential application of CRISPR-Cas12a and developed a CRISPR-Cas12a-based fluorescence/colorimetric biosensor (UCNPs-Cas12a/hydrogel-MOF-Cas12a) that provides an efficient targeting system for small molecules and protein targets. These two sensors yield multiple types of signal outputs by converting the target molecule into a deoxyribonucleic acid (DNA) signal input system using aptamers, amplifying the DNA signal by catalyzed hairpin assembly (CHA), and then combining CRISPR-Cas12a with various nanomaterials. UCNPs-Cas12a/hydrogel-MOF-Cas12a exhibited prominent sensitivity and stability for the detection of estradiol (E2) and prostate-specific antigen (PSA), and was successfully applied for the detection of these targets in milk and serum samples. A major advantage of the hydrogel-MOF-Cas12a system is that the signal output can be observed directly. When combined with aptamers and nanomaterials, CRISPR-Cas12a can be used to target multiple targets, with a diverse array of signal outputs. Our findings create a foundation for the development of CRISPR-Cas12a-based technologies for application in the fields of food safety, environmental monitoring, and clinical diagnosis.

AuNP/Cu-TCPP(Fe) metal-organic framework nanofilm: a paper-based electrochemical sensor for non-invasive detection of lactate in sweat.

Effective and real-time detection of lactate (LA) content in human sweat has attracted considerable attention from researchers. In this work, a novel electrochemical paper-based analysis device (ePAD) was developed for the non-invasive detection of LA in sweat. The electrocatalytic properties of AuNP/Cu-TCPP(Fe) hybrid nanosheets, which were prepared by an optimised synthetic method, were studied by CV and EIS electrochemical methods for the first time and the working electrode can be fabricated using a drip coating method. The lactate sensor was optimised and validated for usability, adoptability and interpretability. To the best of our knowledge, this was the fastest, lowest detection line and widest linear range method reported to date for the detection of lactate. It achieved the detection limit of 0.91 pM and a linear range from 0.013 nM to 100 mM. The dual catalytic effects of the hybrid NSs shortened the detection time by nearly two times and enhanced the sensitivity approximately two times, an accuracy unmatched until now. Furthermore, this sensor was employed for LA analysis and validated by high performance liquid chromatography (HPLC). The ePAD shows superior biocompatibility, accuracy, and high sensitivity and can be easily manufactured. Hence, it is applicable for the long-term monitoring of sweat LA concentrations in point-of-care testing, athletic testing of athletes and military personnel and other subjects in different extreme environments.

Sensitive colorimetric detection of antibiotic resistant Staphylococcus aureus on dairy farms using LAMP with pH-responsive polydiacetylene.

Rapidly and accurately detecting antibiotic-resistant pathogens in agriculture and husbandry is important since these represent a major threat to public health. While much attention has been dedicated to detecting now-common resistant bacteria, such as methicillin-resistant Staphylococcus aureus, fewer methods have been developed to assess resistance against macrolides in Staphylococcus aureus (SA). Here, we report a visual on-site detection system for macrolide resistant SA in dairy products. First, metagenomic sequencing in raw milk, cow manure, water and aerosol deposit collected from dairy farms around Tianjin was used to identify the most abundant macrolide resistance gene, which was found to be the macB gene. In parallel, SA housekeeping genes were screened to allow selective identification of SA, which resulted in the selection of the SAOUHSC_01275 gene. Next, LAMP assays targeting the above-mentioned genes were developed and interpreted by agarose gel electrophoresis. For on-site application, different pH-sensitive colorimetric LAMP indicators were compared, which resulted in selection of polydiacetylene (PDA) as the most sensitive candidate. Additionally, a semi-quantitative detection could be realized by analyzing the RGB information via smartphone with a LOD of 1.344 × 10-7 ng/µL of genomic DNA from a milk sample. Finally, the proposed method was successfully carried out at a real farm within 1 h from sample to result by using freeze-dried reagents and portable devices. This is the first instance in which PDA is used to detect LAMP products, and this generic read-out system can be expanded to other antibiotic resistant genes and bacteria.

Functionalized magnetic nanobeads for SERS-based detection of Staphylococcus aureus.

In this study, we introduced a Raman detection technique based on a combination of functionalized magnetic beads and surface-enhanced Raman scattering (SERS) tags to develop a rapid and sensitive strategy for the detection of Staphylococcus aureus (S. aureus), a typical foodborne pathogen. Polyethylene glycol (PEG) and bovine serum albumin (BSA) dual-mediated teicoplanin functionalized magnetic beads (TEI-BPBs) were prepared for separation of target bacteria. SERS tags were used to immobilize antibodies on gold surfaces with bifunctional linker proteins to ensure specific recognition of S. aureus. Under optimal conditions, the combination of TEI-BPBs and SERS tags showed reliable performance, exhibiting good capture efficiency even in the presence of 106 CFU mL-1 of non-target bacteria. The SERS tag provided an effective hot spot for subsequent Raman detection, presenting good linearity in the range of 102-107 CFU mL-1. Good performance has also been shown in detecting target bacteria in milk samples, where it has a recovery of 95.5-101.3%. Thus, the highly sensitive Raman detection technique combined with TEI-BPBs capture probes and SERS tags is a promising method for the detection of foodborne pathogens in food or clinical samples.

Biodegradable Microneedle Array-Mediated Transdermal Delivery of Dimethyloxalylglycine-Functionalized Zeolitic Imidazolate Framework-8 Nanoparticles for Bacteria-Infected Wound Treatment.

Bacteria-infected skin wounds caused by external injuries remain a serious challenge to the whole society. Wound healing dressings, with excellent antibacterial activities and potent regeneration capability, are increasingly needed clinically. Here, we reported a novel functional microneedle (MN) array comprising methacrylated hyaluronic acid (MeHA) embedded with pH-responsive functionalized zeolitic imidazolate framework-8 (ZIF-8) nanoparticles to treat bacteria-infected cutaneous wounds. Antibacterial activity was introduced into Zn-ZIF-8 to achieve sterilization through releasing Zn ions, as well as increased angiogenesis by dimethyloxalylglycine (DMOG) molecules that were distributed within its framework. Furthermore, biodegradable MeHA was chosen as a substrate material carrier to fabricate DMOG@ZIF-8 MN arrays. By such design, DMOG@ZIF-8 MN arrays would not only exhibit excellent antibacterial activity against pathogenic bacteria but also enhance angiogenesis within wound bed by upregulating the expression of HIF-1α, leading to a significant therapeutic efficiency on bacteria-infected cutaneous wound healing. Based on these results, we conclude that this new treatment strategy can provide a promising alternative for accelerating infected wound healing via effective antibacterial activity and ameliorative angiogenesis.

Raman spectroscopy-based adversarial network combined with SVM for detection of foodborne pathogenic bacteria.

Raman spectroscopy combined with artificial intelligence algorithms have been widely explored and focused on in recent years for food safety testing. It is still a challenge to overcome the cumbersome culture process of bacteria and the need for a large number of samples, which hinder qualitative analysis, to obtain a high classification accuracy. In this paper, we propose a method based on Raman spectroscopy combined with generative adversarial network and multiclass support vector machine to classify foodborne pathogenic bacteria. 30,000 iterations of generative adversarial network are trained for three strains of bacteria, generative model G generates data similar to the actual samples, discriminant model D verifies the accuracy of the generated data, and 19 feature variables are obtained by selecting the feature bands according to the Raman spectroscopy pattern. Better classification results are obtained by optimising the parameters of the multi-class support vector machine, etc. Our detection and classification method not only solves the problem of needing a large number of samples as training set, but also improves the accuracy of the classification model. Therefore, this GAN-SVM classification model provides a new idea for the detection of bacteria based on Raman spectroscopy technology combined with artificial intelligence algorithms.

Aptamer-based photonic crystals enable ultra-trace detection of staphylococcal enterotoxin B without labels.

Staphylococcal enterotoxin B (SEB) is one of the most common serotypes in staphylococcal food-poisoning cases. A rapid, sensitive, and simple method for SEB detection is crucial for public health. A photonic crystal (PC) sensing material for label-free detection of ultra-trace SEB was proposed in this study. Gold nanoparticle-doped silica microspheres were stacked to form an opal PC through self-assembly, and SEB aptamers, as the recognition element, were modified onto the PC. When the target protein of SEB came in contact with the PC sensing material, the reflection peak intensity of PCs decreased accordingly. The detection range was 1 × 10-6 to 1 ng mL-1, and the detection limit was 0.103 × 10-6 ng mL-1. Furthermore, the PC sensing material had great specificity and accuracy, which can be used for real sample monitoring. This PC sensing material achieved ultra-sensitive detection, which did not involve complicated preparation processes and reporter labelling.

Simple and programmed three-dimensional DNA tweezer for simultaneous one-step detection of ochratoxin A and zearalenone.

Three-dimensional (TD) deoxyribonucleic acid (DNA) tweezers were programmed for one-step identification and detection of ochratoxin A (OTA) and zearalenone (ZEN). The unfolding of the TD-DNA tweezers by aptamers specific to these two mycotoxins "turned" the fluorescent signals "on." The bonding of the aptamers to their corresponding targets in OTA and ZEN "turned" the fluorescent signals and the DNA tweezers "off." The detection limit of the TD-DNA tweezers for OTA and ZEN was 0.032 and 0.037 ng mL-1, respectively. The feasibility of this method was tested using two samples. Detection via this method increased the recovery of OTA and ZEN from 95.8% to 110.2%. Spike recovery and certified food products were used to detect applicability in actual situations. Analyte detection in complex samples using TD-DNA tweezers is rapid, as the process involves a single operational step. This proposed design has considerable potential for application in mycotoxin detection.