Development of peptide nucleic acid-based bead array technology for Bacillus cereus detection.

Numerous novel methods to detect foodborne pathogens have been extensively developed to ensure food safety. Among the important foodborne bacteria, Bacillus cereus was identified as a pathogen of concern that causes various food illnesses, leading to interest in developing effective detection methods for this pathogen. Although a standard method based on culturing and biochemical confirmative test is available, it is time- and labor-intensive. Alternative PCR-based methods have been developed but lack high-throughput capacity and ease of use. This study, therefore, attempts to develop a robust method for B. cereus detection by leveraging the highly specific pyrrolidinyl peptide nucleic acids (PNAs) as probes for a bead array method with multiplex and high-throughput capacity. In this study, PNAs bearing prolyl-2-aminocyclopentanecarboxylic acid (ACPC) backbone with groEL, motB, and 16S rRNA sequences were covalently coupled with three sets of fluorescently barcoded beads to detect the three B. cereus genes. The developed acpcPNA-based bead array exhibited good selectivity where only signals were detectable in the presence of B. cereus, but not for other species. The sensitivity of this acpcPNA-based bead assay in detecting genomic DNA was found to be 0.038, 0.183 and 0.179 ng for groEL, motB and 16S rRNA, respectively. This performance was clearly superior to its DNA counterpart, hence confirming much stronger binding strength of acpcPNA over DNA. The robustness of the developed method was further demonstrated by testing artificially spiked milk and pickled mustard greens with minimal interference from food metrices. Hence, this proof-of-concept acpcPNA-based bead array method has been proven to serve as an effective alternative nucleic acid-based method for foodborne pathogens.

Cyanostilbene-based fluorescent paper array for monitoring fish and meat freshness via amino content detection.

The detection of biogenic amines released from degraded meats is an effective method for evaluating meat freshness. However, existing traditional methods like titration are deemed tedious, while the use of sophisticated analytical instruments is not amenable to field testing. Herein, a cyanostilbene-based fluorescent array was rapidly fabricated using macroarray synthesis on a cellulose paper surface to detect amines liberated from spoiled beef, fish, and chicken. The fluorescence changes of immobilized molecules from the interaction with gaseous amines were used to monitor changes in freshness levels. Thanks to the high-throughput nature of macroarray synthesis, a set of highly responsive molecules such as pyridinium and dicyanovinyl moieties were quickly revealed. Importantly, this method offers flexibility in sensing applications including (1) sensing by individual sensor molecules, where the fluorescence response correlated well with established titration methods, and (2) collective sensing whereby chemometric analysis was used to provide a cutoff of freshness with 73-100% accuracy depending on meat types. Overall, this study paves the way for a robust and cost-effective tool for monitoring meat freshness.

Paper spray mass spectrometry as an effective tool for differentiating coffees based on their geographical origins.

With the rising trend of valuing flavor complexity of coffees, means to distinguish the properties of individual coffee sources is vital to the sustainable growth of the coffee industry. Herein, paper spray mass spectrometry (PS-MS), a simple technique with little sample preparation, was used to collect mass data from aqueous extracts of coffees from various sources. Thereafter, principal component analysis and linear discriminant analysis were used to successfully classify coffee samples (with 80-100 % accuracy) from various studies including the differentiations of Arabica and Robusta coffees, Arabica coffees from different countries, Robusta coffees from different geographical locations, and Arabica coffees from different locations within the same province in Thailand. With further insight from significant test via Fisher weight determination, this method was proved to be practical for differentiating coffees based on types and geographical origins, thus paving the way for broader applications.

Paper-based sensor from pyrrolidinyl peptide nucleic acid for the efficient detection of Bacillus cereus.

Bacillus cereus is one of the most common foodborne pathogens found in various kinds of staple foods such as rice and wheat. A rapid and accurate detection method for this pathogen is highly desirable for the sustainable production of relevant food products. While several classical and molecular-based detection methods are available for the identification of B. cereus, they suffered one or more limitations such as the requirement for a tedious and time-consuming process, less than ideal specificity, and the lack of portability. Herein, we developed the first paper-based sensing device that exhibits high species specificity with sufficiently low limit of detection for the visual detection of specific DNA sequences of B. cereus. The success is attributed to the strategic planning of fabrication in various dimensions including thorough bioinformatics search for highly specific genes, the use of the pyrrolidinyl peptide nucleic acid (PNA) probe whose selectivity advantage is well documented, and an effective PNA immobilization and DNA-binding visualization method with an internal cross-checking system for validating the results. Testing in rice matrices indicates that the sensor is capable of detecting and distinguishing B. cereus from other bacterial species. Hence, this paper-based sensor has potential to be adopted as a practical means to detect B. cereus in food industries.

Rapid geographical indication of peppercorn seeds using corona discharge mass spectrometry.

With increasing demands for more rapid and practical analyses, various techniques of ambient ionization mass spectrometry have gained significant interest due to the speed of analysis and abundance of information provided. Herein, an ambient ionization technique that utilizes corona discharge was applied, for the first time, to analyze and categorize whole seeds of black and white peppers from different origins. This setup requires no solvent application nor gas flow, thus resulting in a very simple and rapid analysis that can be applied directly to the sample without any prior workup or preparation. Combined with robust data pre-processing and subsequent chemometric analyses, this analytical method was capable of indicating the geographical origin of each pepper source with up to 98% accuracies in all sub-studies. The simplicity and speed of this approach open up the exciting opportunity for onsite analysis without the need for a highly trained operator. Furthermore, this methodology can be applied to a variety of spices and herbs, whose geographical indication or similar intellectual properties are economically important, hence it is capable of creating tremendous impact in the food and agricultural industries.

Investigation of key chemical species from durian peduncles and their correlations with durian maturity.

The popularity and high price of durian make quality control in terms of ripeness very important, which in turn depends heavily on harvesting at an appropriate maturity stage. To date, reports on data-driven methods for maturity prediction are scarce, with many rather focusing on ripeness prediction. Herein, we report the first disclosure of key molecular markers in the liquid extract of durian peduncle that can be a predictive tool for maturity. Multiple chromatographic and spectroscopic techniques including TLC, HPLC, PS-MS, LC-MS/MS, and NMR, were used to characterize chemical profiles of the aqueous extracts from peduncles at different ages. Four compounds that show positive correlations with maturity were identified as sucrose, asparagine, arginine, and pipecolic acid, with asparagine as the most abundant species. This finding paves the way for more research of high impact such as the relationship between biochemical reactions in peduncle and pulp, and the development of accurate and non-destructive sensors for maturity prediction.

Accelerated nucleophilic substitution reactions of dansyl chloride with aniline under ambient conditions via dual-tip reactive paper spray.

Paper spray ionization (PSI) mass spectrometry (MS) is an emerging tool for ambient reaction monitoring via microdroplet reaction acceleration. PSI-MS was used to accelerate and monitor the time course of the reaction of dansyl chloride with aniline, in acetonitrile, to produce dansyl aniline. Three distinct PSI arrangements were explored in this study representing alternative approaches for sample loading and interaction; conventional single tip as well as two novel setups, a dual-tip and a co-axial arrangement were designed so as to limit any on-paper interaction between reagents. The effect on product abundance was investigated using these different paper configurations as it relates to the time course and distance of microdroplet travel. It was observed that product yield increases at a given distance and then decreases thereafter for all PSI configurations. The fluorescent property of the product (dansyl aniline) was used to visually inspect the reaction progress on the paper substrate during the spraying process. Amongst the variety of sample loading methods the novel dual-tip arrangement showed an increased product yield and microdroplet density, whilst avoiding any on-paper interaction between the reagents.

Flexible Drift Tube for High Resolution Ion Mobility Spectrometry (Flex-DT-IMS).

This paper describes, in detail, the development of a novel, low-cost, and flexible drift tube (DT) along with an associated ion mobility spectrometer system. The DT is constructed from a flexible printed circuit board (PCB), with a bespoke "dog-leg" track design, that can be rolled up for ease of assembly. This approach incorporates a shielding layer, as part of the flexible PCB design, and represents the minimum dimensional footprint conceivable for a DT. The low thermal mass of the polyimide substrate and overlapping electrodes, as afforded by the dog-leg design, allow for efficient heat management and high field linearity within the tube-achieved from a single PCB. This is further enhanced by a novel double-glazing configuration which provides a simple and effective means for gas management, minimizing thermal variation within the assembly. Herein, we provide a full experimental characterization of the flexible DT ion mobility spectrometer (Flex-DT-IMS) with corresponding electrodynamic (Simion 8.1) and fluid dynamic (SolidWorks) simulations. The Flex-DT-IMS is shown to have a resolution >80 and a detection limit of low nanograms for the analysis of common explosives (RDX, PETN, HMX, and TNT).

Visual genotyping of thalassemia by using pyrrolidinyl peptide nucleic acid probes immobilized on carboxymethylcellulose-modified paper and enzyme-induced pigmentation.

A simple probe pair was designed for the detection of hemoglobin E (HbE) genotype, a single-point mutation that leads to abnormal red blood cells commonly found in South East Asia. The key to differentiation is the use of a conformationally constrained peptide nucleic acid (PNA) that was immobilized on carboxymethylcellulose-modified paper. This was then used for target DNA binding and visualization by an enzyme-catalyzed pigmentation. The biotinylated target DNA bound to the immobilized probe was visually detected via alkaline phosphatase-linked streptavidin. This enzyme conjugate catalyzed the dephosphorylation of the substrate 5-bromo-4-chloro-3-indolyl phosphate, leading to a series of reactions that generate an intense, dark blue pigment. The test was validated with 100 DNA samples, which shows good discrimination among different genotypes (normal, HbE, and heterozygous) with 100% accuracy when optimal conditions of analysis were applied. The method does not require temperature control and can be performed at ambient temperature. This is an attractive feature for diagnosis in primary care, which accounts for a large part of affected population. Graphical abstract Schematic representation of a paper-based sensor for the detection of the gene Hemoglobin E. The interaction between an immobilized peptide nucleic acid and a DNA target leads to enzymatic pigmentation, allowing simple visual readout with up to 100% accuracy.

Enhanced Lipid Production and Molecular Dynamics under Salinity Stress in Green Microalga Chlamydomonas reinhardtii (137C).

Microalgal lipids are a source of valuable nutritional ingredients in biotechnological industries, and are precursors to biodiesel production. Here, the effects of salt-induced stresses, including NaCl, KCl, and LiCl stresses, on the production of lipid in green microalga Chlamydomonas reinhardtii (137c) were investigated. NaCl stress dramatically increased saturated fatty acids (SFAs), which accounted for 70.2% of the fatty acid methyl ester (FAMEs) under stress. In contrary, KCl stress led to a slight increase in SFAs (47.05%) with the remaining being polyunsaturated fatty acids (PUFAs) (45.77%). RT-PCR analysis revealed that the genes involved in FA biosynthesis, such as PDH2, ACCase, MAT and KAS2, were up-regulated by NaCl-induced stress. Conversely, the genes responsible for the Kennedy pathway were suppressed. The alteration of FA homeostasis was further assessed by overexpressing MAT, the enzyme responsible for the production of malonyl-ACP, a key building block for FA biosynthesis, in the cyanobacterium Synechococcus elongatus PCC 7942. Intracellular FA composition was affected, with a predominant synthesis of SFAs in transformed cells. Owing to the diversity and relative abundance of SFAs, monounsaturated fatty acid (MUFAs) and PUFAs enable the feasibility of using microorganisms as a source of microalgal lipids or valuable nutritional ingredients; salt-induced stress and expression of MAT are useful in providing precursors for enhanced lipid production.

Rapid Discovery and Structure-Property Relationships of Metal-Ion Fluorescent Sensors via Macroarray Synthesis.

A macroarray immobilisation of fluorophores on filter papers for sensing metal ions by in-situ reductive amination and carbodiimide coupling is reported herein. Chemometric approaches resulted in a rapid discovery of sensors that can synergistically discriminate up to 12 metal ions with great prediction accuracies. Covalently bound on paper, sensoring scaffolds that were synthesised from the macroarray format can readily be adopted as practical paper-based sensors with great reusability and sensitivity, achieving the limit of detection at low nanomolar level with some repeating spotting. Lastly, the discovered scaffolds were also confirmed to be functional as unbound molecules, thus paving the way for more diverse applications.

Nutrient Deprivation-Associated Changes in Green Microalga Coelastrum sp. TISTR 9501RE Enhanced Potent Antioxidant Carotenoids.

The utilization of microalgae as a source of carotenoid productions has gained increasing popularity due to its advantages, such as a relatively fast turnaround time. In this study, a newly discovered Coelastrum sp. TISTR 9501RE was characterized and investigated for its taxonomical identity and carotenoid profile. To the best of our knowledge, this report was the first to fully investigate the carotenoid profiles in a microalga of the genus Coelastrum. Upon use of limited nutrients as a stress condition, the strain was able to produce astaxanthin, canthaxanthin, and lutein, as the major carotenoid components. Additionally, the carotenoid esters were found to be all astaxanthin derivatives, and ß-carotene was not significantly present under this stress condition. Importantly, we also demonstrated that this practical stress condition could be combined with simple growing factors, such as ambient sunlight and temperature, to achieve even more focused carotenoid profiles, i.e., increased overall amounts of the aforementioned carotenoids with fewer minor components and chlorophylls. In addition, this green microalga was capable of tolerating a wide range of salinity. Therefore, this study paved the way for more investigations and developments on this fascinating strain, which will be reported in due course.

Effects of Potassium Chloride-Induced Stress on the Carotenoids Canthaxanthin, Astaxanthin, and Lipid Accumulations in the Green Chlorococcal Microalga Strain TISTR 9500.

Microalgae are a diverse group of photosynthetic eukaryotic organisms that are widely distributed globally. They are prolific sources of highly valuable compounds with fascinating chemical structures. Due to their balanced nutritional compositions and health benefits, they are increasingly being used as functional food ingredients. Carotenoid-based pigments and polyunsaturated fatty acids (PUFAs) are examples of high-value nutrients that can be accumulated abundantly in microalgae. Here, the effects of potassium chloride-induced stress on the productions of lipids and carotenoids in the green microalga of the Chlorococcaceae family were investigated. Under normal BG11 medium, this green microalga strain TISTR 9500 accumulated high levels of PUFA and primary carotenoid lutein. Stress tests revealed that KCl enhanced and modulated lipid and carotenoid accumulation levels. The liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis revealed that secondary carotenoids astaxanthin and canthaxanthin were robustly produced under KCl stress with the similar content of lutein. Further, this stress led to a significant increase in the total FA amount with the higher proportion of unsaturated FA than saturated FA. Thus, this green microalga could be an attractive and alternative natural biosource for canthaxanthin and astaxanthin, as well as for functional lipids.

Rapid Detection of the Antibiotic Sulfamethazine in Pig Body Fluids by Paper Spray Mass Spectrometry.

We report herein a practical method for nonlethal detection of the antibiotic sulfamethazine in pig body fluids via the combination of simple extraction and paper spray mass spectrometry (PS-MS). This method requires minimal sample preparation while still providing high sensitivities and accuracies in complex matrices including pig whole blood (LOD = 7.9 µg/L; recovery = 95.4-103.7%), pig serum (LOD = 11.5 µg/L; recovery = 103.2-106.2%), and synthetic urine (LOD = 11.2 µg/L; recovery = 99.1-103.2%). Given a known correlation between the level of sulfamethazine in body fluids and edible tissues, this method shows great promise as a practical and nonlethal solution for rapid testing of the drug, which can substantially aid managerial decision in the livestock industry.

Antibacterial cotton fabrics based on hydrophilic amino-containing scaffolds.

A hydrophilic amino compound, 4,7,10-trioxatridecane-1,13-diamine, has been utilized in several chemical and biochemical studies. Among previous applications is its use as a flexible and economical spacer molecule to increase the length between two moieties of interest, one of which may be a solid-phase interface. In this study, we immobilized this molecule on cotton fabrics and showed that this modified surface (DA) exhibited significant antibacterial activities in both Gram-negative bacteria and a Gram-positive bacterium. Studies on the structure-activity relationship revealed that additional chemical modifications on DA usually led to lowered antibacterial activities, emphasizing an importance of having free amino groups. Further investigation by fluorescence microscope indicated that this modified surface likely interfered with the membrane integrity of bacteria, leading to cell lysis. In addition, this scaffold was also tested for its biocompatibility with mouse fibroblast cells, and exerted no detrimental effect to the cell growth, highlighting its potential as a practical antibacterial surface modifier.

Paper-based chemical reaction arrays as an effective tool for geographical indication of turmerics.

Geographical indications have gained increasing importance as a powerful marketing tool for highly valuable products especially foods. In this study, a unique and synergistic combination of chemical reaction arrays on paper and chemometric analysis was used to uncover geographical indication of turmerics, an important food ingredient in several cultures. The key to effective differentiation was based on the subtle differences in the compositions of compounds found in each sample, mainly curcumin and derivatives. When these compounds reacted with various reagents in the form of paper arrays, different optical and fluorescence profiles were generated, which can then be exploited by chemometrics. As a result, our strategy could provide up to 94% prediction accuracy without the need for any sophisticated instruments.

Chemical methods to interrogate bacterial quorum sensing pathways.

Bacteria frequently manifest distinct phenotypes as a function of cell density in a phenomenon known as quorum sensing (QS). This intercellular signalling process is mediated by "chemical languages" comprised of low-molecular weight signals, known as autoinducers, and their cognate receptor proteins. As many of the phenotypes regulated by QS can have a significant impact on the success of pathogenic or mutualistic prokaryotic-eukaryotic interactions, there is considerable interest in methods to probe and modulate QS pathways with temporal and spatial control. Such methods would be valuable for both basic research in bacterial ecology and in practical medicinal, agricultural, and industrial applications. Toward this goal, considerable recent research has been focused on the development of chemical approaches to study bacterial QS pathways. In this Perspective, we provide an overview of the use of chemical probes and techniques in QS research. Specifically, we focus on: (1) combinatorial approaches for the discovery of small molecule QS modulators, (2) affinity chromatography for the isolation of QS receptors, (3) reactive and fluorescent probes for QS receptors, (4) antibodies as quorum "quenchers," (5) abiotic polymeric "sinks" and "pools" for QS signals, and (6) the electrochemical sensing of QS signals. The application of such chemical methods can offer unique advantages for both elucidating and manipulating QS pathways in culture and under native conditions.

Synthesis and application of an N-acylated l-homoserine lactone derivatized affinity matrix for the isolation of quorum sensing signal receptors.

The design and synthesis of an agarose resin functionalized with a Gram-negative quorum sensing (QS) signaling molecule analogue is described. The modified resin was utilized in affinity pull-down assays to successfully isolate QscR, a LuxR-type QS receptor from Pseudomonas aeruginosa. This resin may facilitate the identification of novel QS signal receptors using affinity chromatography techniques.

Efficient synthesis and evaluation of quorum-sensing modulators using small molecule macroarrays.

A method for the synthesis of small molecule macroarrays of N-acylated L-homoserine lactones (AHLs) is reported. A focused library of AHLs was constructed, and the macroarray platform was found to be compatible with both solution and agar-overlay assays using quorum-sensing (QS) reporter strains. Several QS antagonists were discovered and serve to showcase the macroarray as a straightforward technique for QS research.