Enhanced carbohydrate-based plastic performance by incorporating cerium-based metal-organic framework for food packaging application.

The development of biodegradable active packaging films with hydrophobic characteristics is vital for extending the shelf life of food and reducing the reliance on petroleum-based plastics. In this study, novel hydrophobic cerium-based metal-organic framework (Ce-MOF) nanoparticles were successfully synthesized. The Ce-MOF nanoparticles were then incorporated into the cassava starch matrix at varying concentrations (0.5 %, 1.5 %, 3 %, and 4 % w/w of total solid) to fabricate cassava-based active packaging films via the solution casting technique. The influence of Ce-MOF on the morphology, thermal attributes, and physicochemical properties of the cassava film was subsequently determined through further analyses. Biomedical analysis including antioxidant activity and the cellular morphology evaluation in the presence of the films was also conducted. The results demonstrated that the consistent dispersion of Ce-MOF nanofillers within the cassava matrix led to a significant enhancement in the film's crystallinity, thermal stability, antioxidant activity, biocompatibility, and hydrophobicity. The introduction of Ce-MOF also contributed to the film's reduced water solubility. Considering these outcomes, the developed cassava/Ce-MOF films undoubtedly have significant potential for active food packaging applications.

Advanced technologies in biodegradable packaging using intelligent sensing to fight food waste.

The limitation of conventional packaging in demonstrating accurate and real-time food expiration dates leads to food waste and foodborne diseases. Real-time food quality monitoring via intelligent packaging could be an effective solution to reduce food waste and foodborne illnesses. This review focuses on recent technological advances incorporated into food packaging for monitoring food spoilage, with a major focus on paper-based sensors and their combination with smartphone. This review paper offers a comprehensive exploration of advanced macromolecular technologies in biodegradable packaging, a general overview of paper-based probes and their incorporation into food packaging coupled with intelligent sensing mechanisms for monitoring food freshness. Given the escalating global concerns surrounding food waste, our manuscript serves as a pivotal resource, consolidating current research findings and highlighting the transformative potential of these innovative packaging solutions. We also highlight the current intelligent paper-based food freshness sensors and their various advantages and limitations. Examples of implementation of paper-based sensors/probes for food storage and their accuracy are presented. Finally, we examined how intelligent packaging can be an alternative to reduce food waste. Several technologies discussed here have good potential to be used in food packaging for real-time food monitoring, especially when combined with smartphone diagnosis.

Fluorometric and colorimetric platforms for rapid and sensitive hydroxychloroquine detection in aqueous samples.

The detection of pharmaceuticals has been an active area of research with numerous application areas ranging from therapeutic and environmental monitoring to pharmaceutical manufacturing and diagnostics. And, the emergence of COVID-19 pandemic has increased the demand for detection of certain active pharmaceutical ingredients such as Hydroxychloroquine (HCQ) mainly due to their increased manufacturing and usage. In this study, we present two optical, fluorometric and colorimetric, detection platforms for the rapid and sensitive detection of HCQ. These platforms take advantage of the interactions between the highly fluorescent dye Thioflavin T (ThT) and Tel24 G-quadruplex (G4) DNA structure, as well as the salt-induced aggregation behavior of negatively charged citrate-capped silver nanoparticles (Cit-AgNPs) in the presence of HCQ. In the fluorometric method, the addition of HCQ led to a significant and rapid decrease in the fluorescence signal of the ThT + Tel24 probe. In the colorimetric method, HCQ induced the aggregation of Cit-AgNPs in the presence of NaCl, resulting in a noticeable color change from yellowish-gray to colorless. Under the optimized conditions, the colorimetric platform exhibited a linear range of 18.0-240.0 nM and a detection limit of 9.2 nM, while the fluorometric platform showed a linear range of 0.24-5.17 µM and a detection limit of 120 nM. The selectivity of the proposed optical methods towards the target analyte was demonstrated by evaluating the response to other structurally similar small molecules. Finally, the practical applicability of both detection systems was confirmed by analyzing HCQ-spiked human urine samples that yielded average recoveries ranging from 75.4 to 110.2 % for the fluorometric platform and 86.9-98.2 % for the colorimetric platform. These results indicate the potential of the developed methods for HCQ detection in complex matrices.

Improving the functionality of biodegradable food packaging materials via porous nanomaterials.

Non-biodegradability and disposal problems are the major challenges associated with synthetic plastic packaging. This review article discusses a new generation of biodegradable active and smart packaging based on porous nanomaterials (PNMs), which maintains the quality and freshness of food products while meeting biodegradability requirements. PNMs have recently gained significant attention in the field of food packaging due to their large surface area, peculiar structures, functional flexibility, and thermal stability. We present for the first time the recently published literature on the incorporation of various PNMs into renewable materials to develop advanced, environmentally friendly, and high-quality packaging technology. Various emerging packaging technologies are discussed in this review, along with their advantages and disadvantages. Moreover, it provides general information about PNMs, their characterization, and fabrication methods. It also briefly describes the effects of different PNMs on the functionality of biopolymeric films. Furthermore, we examined how smart packaging loaded with PNMs can improve food shelf life and reduce food waste. The results indicate that PNMs play a critical role in improving the antimicrobial, thermal, physicochemical, and mechanical properties of natural packaging materials. These tailor-made materials can simultaneously extend the shelf life of food while reducing plastic usage and food waste.

Electrochemical detection of Oxaliplatin induced DNA damage in G-quadruplex structures.

Oxaliplatin (OXP) is a platinum-based chemotherapeutic agent that induces DNA damage by forming intra- and interstrand crosslinks, mainly at the N7s of adenine (A) and guanine (G) bases. In addition to double-stranded DNA, G-rich G-quadruplex (G4)-forming sequences can also be targeted by OXP. However, high doses of OXP can lead to drug resistance and cause serious adverse effects during treatment. To better understand the targeting of G4 structures by OXP, their interactions as well as the molecular mechanisms underlying OXP resistance and adverse effects, there is a need for a rapid, quantitative, and cost-effective method to detect OXP and the damage it causes. In this study, we successfully fabricated a graphite electrode biosensor modified with gold nanoparticles (AuNPs) to investigate the interactions between OXP and the G4-forming promoter region (Pu22) of Vascular endothelial growth factor (VEGF). The overexpression of VEGF is known to be associated with tumor progression and the stabilization of VEGF G4 by small molecules is shown to suppresses VEGF transcription in different cancer cell lines. Differential pulse voltammetry (DPV) was used to investigate the interactions between OXP and Pu22-G4 DNA by monitoring the decrease in the oxidation signal of guanine with increasing OXP concentration. Under the optimized conditions (37 °C, 1:2 v/v AuNPs/water as electrode surface modifier, and 180 min incubation time) the developed probe showed a linear dynamic range of 1.0-10.0 µM with a detection limit of 0.88 µM and limit of quantification of 2.92 µM. Fluorescence spectroscopy was also used to support the electrochemical studies. We observed a decrease in the fluorescence emission of Thioflavin T in the presence of Pu22 upon addition of OXP. To our knowledge, this is the first electrochemical sensor developed to study OXP-induced damage to G4 DNA structures. Our findings provide new insights into the interactions between VEGF G4 and OXP, which could aid in targeting VEGF G4 structures and the development of new strategies to overcome OXP resistance.

Plant protein-based nanocomposite films: A review on the used nanomaterials, characteristics, and food packaging applications.

Consumer demands to utilize environmentally friendly packaging have led researchers to develop packaging materials from naturally derived resources. In recent years, plant protein-based films as a replacement for synthetic plastics have attracted the attention of the global food packaging industry due to their biodegradability and unique properties. Biopolymer-based films need a filler to show improved packaging properties. One of the latest strategies introduced to food packaging technology is the production of nanocomposite films which are multiphase materials containing a filler with at least one dimension less than 100 nm. This review provides the recent findings on plant-based protein films as biodegradable materials that can be combined with nanoparticles that are applicable to food packaging. Moreover, it investigates the characterization of nanocomposite plant-based protein films/edible coatings. It also briefly describes the application of plant-based protein nanocomposite films/coating on fruits/vegetables, meat and seafood products, and some other foods. The results indicate that the functional performance, barrier, mechanical, optical, thermal and antimicrobial properties of plant protein-based materials can be extended by incorporating nanomaterials. Recent reports provide a better understanding of how incorporating nanomaterials into plant protein-based biopolymers leads to an increase in the shelf life of food products during storage time.

The control of fungi and mycotoxins by food active packaging: a review.

Conventionally used petrochemical-based plastics are poorly degradable and cause severe environmental pollution. Alternatively, biopolymers (e.g., polysaccharides, proteins, lipids, and their blends) are biodegradable and environment-friendly, and thus their use in packaging technologies has been on the rise. Spoilage of food by mycotoxigenic fungi poses a severe threat to human and animal health. Hence, because of the adverse effects of synthetic preservatives, active packaging as an effective technique for controlling and decontaminating fungi and related mycotoxins has attracted considerable interest. The current review aims to provide an overview of the prevention of fungi and mycotoxins through active packaging. The impact of different additives on the antifungal and anti-mycotoxigenic functionality of packaging incorporating active films/coatings is also investigated. In addition, active packaging applications to control and decontaminate common fungi and mycotoxins in bakery products, cereal grains, fruits, nuts, and dairy products are also introduced. The results of recent studies have confirmed that biopolymer films and coatings incorporating antimicrobial agents provide great potential for controlling common fungi and mycotoxins and enhancing food quality and safety.

Application of bacterial nanocellulose decorated with zeolitic imidazolate framework (ZIF-L) as a platform for food freshness monitoring.

Recently, the food freshness indicator (FFI) has garnered great interest from consumers and food producers. A novel FFI based on bacterial nanocellulose (BNC)/zeolitic imidazolate framework-L (ZIF-L) and grape anthocyanins was developed and characterized using field emission scanning electron microscopy, Fourier-transform infrared, X-ray diffraction, water contact angle, and BET techniques. The results confirmed that the BNC fibrils were decorated by in situ growth of ZIF-L, with a 3D flower-shaped structure and randomly multiple sharp-edged petals, and hydroxyl and oxygenated heterocycle aromatic ring functional groups on its surface. The reversibility, color stability performance, and moisture sorption of FFI were studied and its applicability in a two-layer arrangement as a visual freshness monitoring of shrimp and minced beef was evaluated. The FFI was able to distinguish (ΔE > 5) the fresh, medium fresh, and spoiled minced meat and shrimp visually during 10 and 4 days of storage at 4 °C, respectively. Also, monitoring of food chemical and microbiological parameters approved the correlation of food spoilage with the color parameters of FFI. These results confirmed the function of ZIF-L in the fabrication of highly pH-sensitive food intelligent packaging material.

Renewable and Recyclable Polymeric Materials for Food Packaging: A New Open Special Issue in Materials.

"Renewable and Recyclable Polymeric Materials for Food Packaging" is a new open Special Issue of Materials that will publish original and review papers on new scientific and applied research, and the articles it contains will make a contribution to the discovery and understanding of biodegradable and recyclable materials, their functional properties, characterization and applications [...].

Coumarin 6H-fused fluorescent probe for highly sensitive detection of coralyne using oligonucleotide-modified silver nanoparticles.

In this study, a novel, rapid, and sensitive fluorescence sensing platform was developed for the detection of coralyne (COR) by the conjugation of coumarin 6H (C6H) fluorescent dye with oligonucleotide-modified silver nanoparticles [(dT)32-AgNPs]. In the presence of COR, a remarkable and rapid decrease in the fluorescence signal of the probe with a quenching efficiency of around 62% was observed. The quenching response of the system towards COR was possibly due to the displacement of thymidine-rich deoxyoligonucleotides by COR on the surface of AgNPs. The complementary experiments with an adenine-rich single strand as well as with two different secondary structures (i.e., duplex and triplex) revealed a favorable sequence specificity of the sensing platform. The influence of key parameters including the incubation time and temperature was evaluated and optimized to achieve the highest performance. The linear range of 10-183 nM with a correlation coefficient of R = 0.9982 and a limit of detection of 5.24 nM were obtained under the optimized conditions. The selectivity of the proposed probe towards COR was revealed by the evaluation of its response to other small molecules that have molecular structures similar to COR. Finally, the successful applicability of the system was shown with the obtained average recoveries in the range of 87.28-104.52% in human urine samples.

The role of genetic manipulation and in situ modifications on production of bacterial nanocellulose: A review.

Natural polysaccharides are well-known biomaterials because of their availability and low-cost, with applications in diverse fields. Cellulose, a renowned polysaccharide, can be obtained from different sources including plants, algae, and bacteria, but recently much attention has been paid to the microorganisms due to their potential of producing renewable compounds. In this regard, bacterial nanocellulose (BNC) is a novel type of nanocellulose material that is commercially synthesized mainly by Komagataeibacter spp. Characteristics such as purity, porosity, and remarkable mechanical properties made BNC a superior green biopolymer with applications in pharmacology, biomedicine, bioprocessing, and food. Genetic manipulation of BNC-producing strains and in situ modifications of the culturing conditions can lead to BNC with enhanced yield/productivity and properties. This review mainly highlights the role of genetic engineering of Komagataeibacter strains and co-culturing of bacterial strains with additives such as microorganisms and nanomaterials to synthesize BNC with improved functionality and productivity rate.

Highly-sensitive and fast detection of human telomeric G-Quadruplex DNA based on a hemin-conjugated fluorescent metal-organic framework platform.

The formation of G-quadruplex (G4) structures in Human telomeric DNA (H-Telo) has been demonstrated to inhibit the activity of telomerase enzyme that is associated with the proliferation of many cancer cells. Accordingly, G-quadruplex structures have become one of the well-established targets in anticancer therapeutic strategies. And, the development of simple and selective detection platforms for G4 structures has become a significant focus of research in recent years. In this study, a simple "off-on" fluorometric method was developed for the selective detection of picomolar quantities of H-Telo G4 DNA based on a fluorescent cerium-based metal organic framework (Ce-MOF) conjugated with hemin to form the sensing probe, Hemin@Ce-MOF. The solvothermal synthesis of the Ce-MOF took advantage of 5-aminoisophtlalic acid (5AIPA) as the organic bridging ligand, (Ce2(5AIPA)3(DMF)2). Characterization of Ce-MOF and Hemin@Ce-MOF was performed by XRD, XPS, TEM, SEM, BET and FTIR techniques. The detection and quantification of the H-Telo was carried out through the adsorption/incorporation of hemin molecules on the pores and surface of Ce-MOF resulting in the fluorescent quenching of the system followed by the restoration of the fluorescence upon addition of H-Telo probably due to a competition between H-Telo and Ce-MOF to bind to hemin. The impact of the key variables including MOF quantity, hemin concentration and detection time was investigated and optimized. Under the optimized conditions, the developed probe provides a limit of detection (LOD) of 665 pM, linear dynamic range (LDR) of 1.6-39.7 nM and excellent selectivity towards H-Telo. Taken together, these results present a simple, novel and superior platform for the selective detection of H-Telo G4 DNA.

Novel Fluorescent Azacyanine Compounds: Improved Synthesis and Optical Properties.

Benzothiazoles are known to possess a number of biological activities and therefore are considered to be an important scaffold in the design and synthesis of pharmacophores. In this study, an improved synthesis method for novel fluorescent benzothiazole-based cyclic azacyanine (CAC) dyes bearing different electron-donating/withdrawing groups on their scaffold is presented. The improved method enabled us to increase the synthesis yield for the previously reported CACs. More importantly, it allowed us to synthesize new CAC dyes that were not synthesizable with the previously reported method. The synthesized dyes were characterized by 1H and 13C NMR spectroscopy, elemental analysis, and mass spectrometry and their optical (absorption and fluorescence) properties were investigated. All of the synthesized CACs were found to be displaying strong absorption within the range of 387-407 nm. The spectral shifts observed in the absorption and fluorescence measurements suggested that the spectroscopic and optical properties of CACs can be directly modulated by the nature of the electron-donating/withdrawing substituents. The fluorescence quantum yields (QYs) of the unsubstituted (parent CAC) and substituted CACs were also measured and compared. The fluorescence QY of CACs with electron-donating substituents (methoxy or ethoxy) was found to be at least four times higher than that of the parent CAC with no substitutions.

A conjugated gold nanoparticle-azacyanine off-on-off fluorescence probe for sensitive and selective detection of G-quadruplexes.

G-quadruplex secondary structures have gained significant recognition due to the discovery of their involvement in regulation of gene expression and their association with many diseases such as cancer and neurological disorders. Consequently, the need for the recognition and characterization of G-quadruplex structures has increased considerably. Here, we present a rapid, facile and sensitive off-on-off in vitro platform for G-quadruplex detection, based on the gold nanoparticle-azacyanine5 (AuNP-Aza5) conjugated fluorescence probe. The conjugated probe is governed by Fluorescence Resonance Energy Transfer (FRET) mechanism between the fluorophore molecule, Aza5, and AuNPs. The fluorescence of Aza5 that was hindered by AuNPs (off), was restored in the presence of L-cysteine (on) until the addition of a G-quadruplex structure (off). The developed sensing platform selectively responds to G-quadruplex structures formed within the promoter regions of VEGF-Pu22, K-RAS, C-myc and BCL-2. It doesn't exhibit a similar response to the other secondary structures such as single, double or triple stranded nucleic acid structures. The detailed investigation of the probe with VEGF-Pu22 as a model G-quadruplex structure revealed a linear response between the concentration range of 0.032-0.347 µM with a detection limit of 12.66 nM.

Silver nanoparticles-tragacanth gel as a green membrane for effective extraction and determination of capecitabine.

A novel eco-friendly and effective electromembrane extraction method combining high-performance liquid chromatography with UV detection was developed for the enrichment and determination of capecitabine. Tragacanth-silver nanoparticles conjugated gel was prepared by dissolving the tragacanth powder in synthesized silver nanoparticles solution and was used as a green membrane in electromembrane extraction. The porosity and presence of silver nanoparticles in the gel were characterized by field emission scanning electron microscopy. This new electromembrane extraction approach uses neither organic solvent nor carrier agents to extract the target analyte. The best electromembrane extraction efficiency was obtained by using 4.0 mm membrane gel thickness containing 2.5% w/v of tragacanth gum, donor phase pH = 5.0, acceptor phase pH = 3.0, applied voltage 50 V, extraction time 20 min, and agitation rate 500 rpm. During method validation under the optimized conditions, good linearity dynamic range between 1 and 500 ng/mL with the coefficient of determination (R2 ) = 0.998 was obtained. Limit of detection and Limit of quantitation were estimated to be 0.84 and 1.0 ng/mL, respectively. Finally, the applicability of this method in real samples was confirmed by an acceptable performance in extraction and determination of capecitabine in human plasma samples.

Development of an easy-to-use colorimetric pH label with starch and carrot anthocyanins for milk shelf life assessment.

An intelligent freshness indicator was developed by immobilizing anthocyanins of black carrot (ABC) within the starch matrix (total anthocyanins content of 10 mg/100 mL) to monitor freshness/spoilage of milk. The microstructural, spectral, swelling and solubility properties as well as color stability (as a function of time, temperature and light) of the indicator at different pHs were characterized. The incorporation of ABC did not change the swelling index and water solubility. The prepared label showed visible color changes as a function of pH and excellent color stability after one month storage at different conditions. The total color difference (TCD) value of the indicator corresponded to the pH, acidity, and microbial growth of the pasteurized milk. The Pearson correlation coefficient showed a high correlation between TCD and pH (R = -0.979), while a high and positive correlation between TCD and acidity as well as TMC (R = 0.983 and 0.968, respectively) was observed. The developed label can discriminate fresh milk form the milk entered into the initial (TCD: 7.8 after 24 h) and final (TCD: 34.8 after 48 h) steps of spoilage. The fabricated label opens a new perspective to use anthocyanins-incorporated biopolymers in the milk intelligent packaging as a simple and easy-to-use freshness indicator.

A novel pH-sensing indicator based on bacterial cellulose nanofibers and black carrot anthocyanins for monitoring fish freshness.

A novel intelligent pH-sensing indicator based on bacterial nanocellulose (BC) and black carrot anthocyanins (CA) was developed and characterized to monitor the freshness/spoilage of rainbow trout and common carp fillet during the storage at 4 °C. The indicator displayed wide color differences from red to gray over the 2-11 pH range, which was clearly discerned by the naked eye. The fabricated pH-sensing indicator showed distinguishable color changes during fresh (deep carmine color), best to eat (charm pink color), and spoiled (jelly bean blue and khaki colors) stages of both fish fillets. Moreover, a strong and positive correlation was obtained between the total color differences values of the indicator and bacterial count (R = 0.952 and 0.991) and total volatile basic nitrogen (R = 0.815 and 0.92) in rainbow trout and common carp samples. The results of this work demonstrated a significant correspondence of fish shelf life and color changes of a nanocellulose-based pH-sensing indicator.

Cellulose/chitosan pH-responsive indicator incorporated with carrot anthocyanins for intelligent food packaging.

In this study, the possible use of anthocyanins of black carrot (ABC) as a chemo-responsive dye to fabricate a colorimetric pH indicator in a cellulose-chitosan matrix was investigated in order to the monitoring of spoilage in pasteurized milk. Cellulose paper was impregnated with a chitosan solution prepared by a sol-gel method containing ABC (total anthocyanins content of 10 mg/100 mL) and characterized. The swelling and water solubility increased by incorporation of ABC into the chitosan-cellulose film. The colorimetric pH indicator showed an obvious color variation from pink to khaki at different pH values (pH 2-11). Stability tests revealed that the indicator had acceptable color stability during one-month storage at 20 °C. The results also confirmed the immobilization of ABC into the matrix of the polymeric indicator with no significant effect on the chemical and super-molecular structure of the samples. In food trial, fresh pasteurized milk was entirely discerned through a perceptible color change from blue to violet rose color after 48 h storage at 20 °C, which was comfortably observable by the naked eye. The results proved that the fabricated indicator could be used as food grade biomaterials to monitor freshness/spoilage of milk.

Characterization of cellulosic paper coated with chitosan-zinc oxide nanocomposite containing nisin and its application in packaging of UF cheese.

A new antimicrobial bilayer film was developed using chitosan, cellulose, and nisin. Chitosan solution containing nisin (500 and 1000µg/mL) was prepared by sol-gel method and then the solution was coated on cellulose paper by dip coating method. A chitosan-cellulose film without antimicrobial had no inhibitory effect on Listeria monocytogenes, whereas, the incorporation of nisin made a significant increase (P<0.05) in antimicrobial characteristics of the films. Moreover, no significant differences were shown on antimicrobial activity of developed films during the storage at 4°C for one month. However, the addition of nisin showed a significant increase in the swelling index and solubility of bilayer film. Scanning electron microscope images revealed a uniform coating of chitosan solution on cellulose paper. The FTIR analysis also confirmed successful introducing and binding of the nisin in double layer film. Films with 1000µg/mL of nisin completely inactivated the initial (∼5log10 CFU/g) counts of L. monocytogenes on the surface of Ultra-filter white cheese after storage at 4°C for 14 days. We concluded that nanocomposite film of chitosan-cellulose containing nisin has novel antibacterial activity and can be used for packaging in cheese.

Rapid ionic liquid-supported nano-hybrid composite reinforced hollow-fiber electromembrane extraction followed by field-amplified sample injection-capillary electrophoresis: An effective approach for extraction and quantification of Imatinib mesylate in human plasma.

The focus of this study is development of a new, convenient, rapid and sensitive electromembrane extraction approach (based on an ionic liquid-supported MWCNTs/ZnO reinforced hollow fiber, for the first time) as an off-line sample clean-up/preconcentration method coupled with capillary electrophoresis (CE-UV) using field-amplified sample injection (FASI) for quantification of Imatinib mesylate in human plasma. The nano-hybrid sorbent, coated by 1-octyl-3-methylimidazolium bromide ionic liquid ([OMIm]Br) in this research, was prepared by a feasible basic catalyzed sol-gel method. Then, it was immobilized (supported by capillary forces and sonication) in pores of a segment of a polypropylene hollow fiber membrane as the extraction unit after dispersing in 2-nitrophenyl octyl ether (NPOE) solvent and subsequently served as the supported liquid membrane (SLM) composition. Significant variables affecting the proposed method were evaluated and optimized to achieve the maximum extraction performance. Optimal conditions were obtained by NPOE with 4mgmL-1 nano-sorbent as the SLM composition, 105V as the driving force, pH 2 and 1.8 of the donor and acceptor phases, respectively, an extraction time of 15min and an agitation rate of 800rpm. The developed method was validated according to FDA guidelines. Regarding the validation results, the method is proved to be linear (R2=0.998) over concentrations ranging from 25 to 1500ngmL-1 (LOD=6.24ngmL-1). The mean RSD values for intra- and inter-day precision studies were 6.83 and 7.71%, respectively and the mean recoveries ranged between 98 and 106%. Finally, the validated method was successfully applied for sensitive, selective and rapid determination of Imatinib in patient's plasma samples.