Require Complex Instrumentation Research Articles

Dual signal amplification platform based on recombinant polymerase amplification and fluorescence-enhanced nanoparticles for sensitive detection of Salmonella

Salmonella is a common foodborne pathogen, and its detection is essential for managing public food safety. Lateral flow assay (LFA) strips are well suited for the purpose as they are inexpensive and easy to use. However, the sensitive detection of low-concentration targets requires the use of signal amplification strategies. This paper presents a LFA for the ultrasensitive detection of Salmonella based on recombinase polymerase amplification (RPA) and fluorescence-enhanced composite nanospheres dSiO2@CD@SiO2 (dCDS). RPA is capable of rapid (∼20 min) exponential amplification of bacterial DNA and does not require complex instrumentation. Mesoporous silica (dSiO2) has a large specific surface area and radial pore size, making it an excellent structural material for internal assembly. Nanoparticles obtained by loading carbon dots (CDs) had a strong fluorescence signal and stability. We used dCDSs as reporter molecules, which captured a large number of DNA-dCDS complexes on the test line of the LFA. With double signal amplification, the RPA and dCDS-based LFA showed greatly improved sensitivity: The limits of detection for Salmonella in buffer and milk were 3.8 CFU/mL and 36 CFU/mL, respectively. This study provides an inexpensive strategy for the sensitive detection of pathogenic bacteria.

CRISPR-powered microfluidic biosensor for preamplification-free detection of ochratoxin A

The CRISPR technology, which does not require complex instruments, expensive reagents or professional operators, has attracted a lot of attention. When utilizing the CRISPR-Cas system for detection, the pre-amplification step is often necessary to enhance sensitivity. However, this approach tends to introduce complexity and prolong the time required. To address this issue, we employed Pd@PCN-222 nanozyme to label single-stranded DNA, referred to as Pd@PCN-222 CRISPR nanozyme, which serves as the reporter of the CRISPR system. Pd@PCN-222 nanozyme possess exceptional catalytic activity for the reduction of H2O2. Compared with traditional electrochemical probe ferrocene and methylene blue without catalytic activity, there is a significant amplification of the electrochemical signal. So the need for pre-amplification was eliminated. In this study, we constructed a CRISPR-Cas system for ochratoxin A, utilizing the Pd@PCN-222 CRISPR nanozyme to amplified signal avoiding pre-amplification with outstanding detection of 1.21 pg/mL. Furthermore, we developed a microfluidic electrochemical chip for the on-site detection of ochratoxin A. This achievement holds significant promise in establishing a practical on-site detection platform for identifying food safety hazards.

THE FORMATION OF RESEARCH COMPETENCЕ OF THE 7TH GRADE PUPILS BY MEANS OF THE SECTION “ANIMAL BEHAVIOR”

The article highlights the problem of formation of research competences of 7th grade pupils by means of the school course of biology, in particular its section “Animal Behavior”.The role of observation among the methods used in biology is revealed. It does not require complex instruments and tools, and that is why it can be used with great success by pupils at school and become a mean of developing their research skills.The State Standard of Basic Secondary Education is analyzed in relation to the expected learning outcomes of pupils, where research competences are identified and united into groups: intellectual, practical, valuable, self-organization and self-control.Research competence formation program of pupils in extracurricular activities by means of the section “Animal behavior” is offered. The purpose of the program is to ensure the formation of research competence of 7th grade pupils based on awareness of the biological basis of behavioral reactions, knowledge of the mechanisms of animal and human behavior, behavior research methodology, mastering skills and abilities of research activities.Criteria are defined to assess the formation of research competence of pupils. Based on these criteria, the levels of formation of pupils’ skills of research competence are developed.The results of conducting a formative experiment on the development of the 7th grade pupils’ research competences by means of the section “Animal behavior” are highlighted. Ways of further development of the problem of pupils’ research competence formation taking into account the State standard of basic secondary education are outlined.It is found out that in the process of pupils’ research competence formation during the biology course mastering it is important to take into account the availability of forms and methods of research conducted by pupils; correspondence of the research topic to the age peculiarities and personal interests of pupils; pupils’ need for knowledge and understanding of the value of research activities.
 Keywords: 7th grade biology course, research competence, State standard of basic secondary education, section “Animal behavior”, observation as a research method, components of research competence, Research competence formation program.

Holographic detection of nanoparticles using acoustically actuated nanolenses

The optical detection of nanoparticles, including viruses and bacteria, underpins many of the biological, physical and engineering sciences. However, due to their low inherent scattering, detection of these particles remains challenging, requiring complex instrumentation involving extensive sample preparation methods, especially when sensing is performed in liquid media. Here we present an easy-to-use, high-throughput, label-free and cost-effective method for detecting nanoparticles in low volumes of liquids (25 nL) on a disposable chip, using an acoustically actuated lens-free holographic system. By creating an ultrasonic standing wave in the liquid sample, placed on a low-cost glass chip, we cause deformations in a thin liquid layer (850 nm) containing the target nanoparticles (≥140 nm), resulting in the creation of localized lens-like liquid menisci. We also show that the same acoustic waves, used to create the nanolenses, can mitigate against non-specific, adventitious nanoparticle binding, without the need for complex surface chemistries acting as blocking agents.

Isolation of methylmercury using distillation and anion-exchange chromatography for isotopic analyses in natural matrices

The development of mercury (Hg) stable isotope measurements has enhanced the study of Hg sources and transformations in the environment. As a result of the mixing of inorganic Hg (iHg) and methylmercury (MeHg) species within organisms of the aquatic food web, understanding species-specific Hg stable isotopic compositions is of significant importance. The lack of MeHg isotope measurements is due to the analytical difficulty in the separation of the MeHg from the total Hg pool, with only a few methods having been tested over the past decade with varying degrees of success, and only a handful of environmentally relevant measurements. Here, we present a novel anion-exchange resin separation method using AG 1-X4 that further isolates MeHg from the sample matrix, following a distillation pretreatment, in order to obtain ambient MeHg stable isotopic compositions. This method avoids the use of organic reagents, does not require complex instrumentation, and is applicable across matrices. Separation tests across sediment, water, and biotic matrices showed acceptable recoveries (98 ± 5%, n = 54) and reproducible δ202Hg isotope results (2 SDs ≤ 0.15‰) down to 5 ng of MeHg. The measured MeHg pools in natural matrices, such as plankton and sediments, showed large deviations from the non-speciated total Hg measurement, indicating that there is an important isotopic shift during methylation that is not recorded by typical measurements, but is vital in order to assess sources of Hg during bioaccumulation. Graphical abstract.

Supercritical fluid chromatography as a rapid single-step method for the determination of mineral oil saturated and aromatic hydrocarbons in purified mineral oils for food and cosmetics applications

Mineral oil hydrocarbons are used in the consumer goods sector for the elaboration of a wide range of foods and cosmetics. Traditional methods for determining their levels and composition are time consuming and laborious, besides requiring complex instrumentation. Here a simple and fast method was developed that uses columns packed with silver-modified silica in supercritical fluid chromatography with flame ionization and UV detection (SFC-FID/UV) for the determination of mineral oil saturated hydrocarbons (MOSH) and mineral oil aromatic hydrocarbons (MOAH) in purified mineral oil samples. The method requires no sample preparation apart from dilution. Direct quantification of MOSH and MOAH was possible for samples with MOSH/MOAH ratios around one. For other samples deconvolution of the MOSH and MOAH humps in the FID chromatogram using the UV signal was needed since baseline separation of the two fractions could not be obtained. Validation of the method performance showed an excellent linearity (R2 > 0.9995) in the range of concentrations tested (2.5–100 mgmL−1) and a better repeatability than the standard methods (<5%). MOAH detection limits were better than 0.36% MOAH, which makes the method sufficiently sensitive for analysis of all but the highest purity mineral oils. The proposed SFC-FID/UV method was suitable for the analysis of mineral oils with viscosities and molecular weights below approximately 56 mm2s−1 and 450 gmol−1. The quantitative results of the new method were not statistically significantly different from those obtained with the standard SPE-GC-FID method where the new method has the advantages of a better repeatability, simpler operation and a significantly shortened analysis time. This new method could potentially also be used for the analysis of mineral oil contaminations in consumer products such as foods. However, in this case additional sample clean-up and preconcentration steps are needed for reducing matrix interferences from e.g. triglycerides and olefins and for improving the detection limits.

Droplet Microarray on Patterned Butterfly Wing Surfaces for Cell Spheroid Culture.

Three-dimensional (3D) cell spheroids have a demonstrated value for in vitro biological research and therapeutics development. Attempts to this technique focus on the development of effective methods for fabricating cell spheroids. Here, inspired by the heterogeneously textured wettability bumps (with hydrophilic peaks and hydrophobic bases) of Stenocara beetle, we present a biotemplated substrate with wettable hydrogel arrays for culturing the cell spheroids. The biotemplates were Morpho butterfly wings with chitin and protein components, which could provide a natural superhydrophobic surface without any modification. The droplet microarrays could be formed for cell spheroid culture on this bioinspired wing substrate by using the hydrogel patterns to hanging droplets. The hanging drop culture method on hydrogel-covered wings has the advantages of high speed, uniform size, and controllable diameter for the formation of 3D cell spheroids. It was demonstrated that drugs produced distinct responses in the 3D cell spheroids compared to conventional two-dimensional cell cultures. As the presented system does not require complex instruments and chemical modifications, our method can simply construct the desired wettability substrates with high biocompatibility for cell culture, drug testing, and other biomedical applications.

Development of a rapid and sensitive recombinase polymerase amplification-lateral flow assay for detection of Burkholderia mallei.

Burkholderia mallei, a potential biothreat agent is the aetiological agent of glanders, a zoonotic disease primarily affecting equines. B. mallei shares close genetic proximity with B. pseudomallei, the aetiological agent of melioidosis. Hence, molecular detection of B. mallei and its differentiation from B. pseudomallei has always been challenging. Early diagnosis of glanders is critical for timely treatment in humans and disease containment in animals. In this study a recombinase polymerase amplification-lateral flow (RPA-LF) assay has been developed for early and accurate detection of B. mallei. RPA-LF assay was found to be highly sensitive and detected as low as 10fg genomic DNA of B. mallei. The assay was highly specific and could differentiate B. mallei and B. pseudomallei. The assay also detected B. mallei in artificially spiked blood, tap water and garden soil. The established assay is simple, rapid and does not require complex instrumentation. The field deployable assay can have better implications in rapid glanders diagnosis and environmental detection of B. mallei over PCR-based detection tools in glanders endemic areas with limited laboratory resources.

Assessment of a system for the prediction of water permeability coefficient in pervious concretes

Abstract Proposing a system which makes it possible to determine the water permeability coefficient in pervious concretes in a practical way, without the need of devices which require complex instrumentation, is one of the aims of this work. An experimental matrix was proposed for the evaluation of the measurement system of the coefficient of permeability into pervious concrete considering three different pervious concretes obtained from different aggregate granulometric sizes, under the condition of dry sample or water saturated. The pervious concretes were characterized by their void contents, mechanical properties and later compared with the properties of a reference, control concrete made up of coarse and fine aggregates. The different properties exhibited in this study were assessed aiming at validating the experimental matrix developed for pervious concretes to provide a basis for comparison with information available in the literature. As a noticeable result, it was evidenced that the permeability coefficient of 0.21 cm.s-1 obtained for concrete with aggregate dimensions of 2.4 to 9.5 mm. It is possible to measure the coefficient of permeability of pervious concrete with the system developed in this study; the obtained results agree with the description of ACI522-06 for pervious concretes.

Immunohistochemistry Microarrays.

Immunohistochemistry (IHC) on tissue sections is widely used for quantifying the expression patterns of proteins and is part of the standard of care for cancer diagnosis and prognosis, but is limited to staining a single protein per tissue. Tissue microarray and microfluidics staining methods have emerged as powerful high throughput techniques, but they either only permit the analysis of a single protein per slide or require complex instrumentation and expertise while only staining isolated areas. Here, we introduce IHC microarrays (IHCμA) for multiplexed staining of intact tissues with preserved histological and spatial information. Droplets of a dextran solution containing antibodies were prespotted on a slide and snapped onto a preprocessed formalin-fixed, paraffin-embedded (FFPE) tissue section soaked in a polyethylene glycol solution. The antibodies are confined within the dextran droplets and locally stain the tissue below with a contrast similar to the one obtained by conventional IHC. The microarray of antibody droplets can be prespotted on a slide and stored, thus neither the preparation of the antibody solutions nor a sophisticated microarray spotter is needed. Sampling considerations with IHCμA were evaluated by taking three tissues with varying levels of cancer cells. A multiplex IHCμA with 180 spots targeting 8 cancer proteins was performed on a breast cancer tissue section to illustrate the potential of this method. This work opens the avenue of applying microarray technologies for conducting IHC on intact tissue slices and has great potential to be used in the discovery and validation of tissue biomarkers in human tumors.

Alkaline hydrolysis to increase the selectivity of colorimetric determination of polysorbate

Here, we describe a straightforward sample pretreatment step for the colorimetric cobaltthiocyanate determination of polysorbate, which circumvents the assay's shortcomings due to interference of protein and does not require complex instrumentation. Protein-containing test samples are hydrolyzed with strong alkali at 100 °C, neutralized and clarified by filtration before applying the colorimetric assay. The modified method performs with appropriate accuracy and precision, allowing specific polysorbate measurement in the presence of Triton X-100 during virus inactivation, determination of residual amounts of polysorbate in the final products and measurement of polysorbate 80 in final formulated products. The alkaline hydrolysis step, primarily designed to provide the assay's reliability in the presence of protein, also enhances its selectivity towards interference by the non-ionic detergent Triton X-100 and increases its robustness against changes in the fatty acid moiety of polysorbate as it released the fatty acid essentially contributing to the known heterogeneity of polysorbates. These results demonstrate that with sample pretreatment the handy colorimetric assay, not requiring complex instrumentation, can be used to measure polysorbate 80 concentrations in intermediates and final products of therapeutic protein solutions.

Microfluidic Platform with Multiplexed Electronic Detection for Spatial Tracking of Particles.

Microfluidic processing of biological samples typically involves differential manipulations of suspended particles under various force fields in order to spatially fractionate the sample based on a biological property of interest. For the resultant spatial distribution to be used as the assay readout, microfluidic devices are often subjected to microscopic analysis requiring complex instrumentation with higher cost and reduced portability. To address this limitation, we have developed an integrated electronic sensing technology for multiplexed detection of particles at different locations on a microfluidic chip. Our technology, called Microfluidic CODES, combines Resistive Pulse Sensing with Code Division Multiple Access to compress 2D spatial information into a 1D electrical signal. In this paper, we present a practical demonstration of the Microfluidic CODES technology to detect and size cultured cancer cells distributed over multiple microfluidic channels. As validated by the high-speed microscopy, our technology can accurately analyze dense cell populations all electronically without the need for an external instrument. As such, the Microfluidic CODES can potentially enable low-cost integrated lab-on-a-chip devices that are well suited for the point-of-care testing of biological samples.

Estimación del caudal a partir de la evolución del nivel del agua en un estuario con onda de marea estacionaria

A method to calculate the freshwater discharge in an estuary and the net exchange flow with the sea measuring the evolution of the water level in fixed monitoring stations is presented. It rises from the need to obtain reliable data of discharges under the particular morphological conditions that determine the hydrodynamic behaviour of the coastal plain Quequén Grande River estuary (Buenos Aires, Argentina), in which the tide behaves as a standing wave. The technique is calibrated with direct measurements of discharges performed with an acoustic Doppler current profiler in field studies during periods of poor and abundant rains. It is found that the exchange of water with the sea is proportional to the time derivative of the height of the free surface, and the fluvial discharge can be estimated from the height difference between monitoring stations. The results make it possible to quantify the river contribution during dry and wet seasons, and the flows generated by occasional and unusual tides. The method proposed is simple, does not require complex instruments to continuously measure velocity, and takes advantage of existing monitoring nets to provide the information of the estuarine flows in real time and of historic files to evaluate the changes produced in the past.

Development of a Loop-Mediated Isothermal Amplification Assay for Rapid Detection of Mycoplasma hyorhinis

To establish a method for sensitive and rapid diagnosis of Mycoplasma hyorhinis in clinical specimens, a simple, sensitive loop-mediated isothermal amplification (LAMP) assay was designed and evaluated. Three sets of four special primers, recognizing distinct sequences of the target, were designed for sensitive, specific amplification of nucleic acid under isothermal conditions. The LAMP assay was carried out using 35 clinical specimens of bronchoalveolar lavage fluid (BALF) from pigs. For comparison, these specimens were also tested using conventional PCR, real-time PCR, and nested PCR assays. After optimization of the reaction condition and reaction system, the LAMP reaction successfully detected Mycoplasma hyorhinis within 40 minutes at 61 degrees C. The LAMP assay achieved a sensitivity of 10(1) copies per microL at 61 degrees C in 40 minutes, compared to real-time PCR and nested PCR, and was over 10(3) times more sensitive than conventional PCR. In the test for the specificity of the LAMP assay, only Mycoplasma hyorhinis genomic DNA was positive and no other microorganisms were positive with the primers, indicating that the LAMP assay is specific to Mycoplasma hyorhinis. Mycoplasma hyorhinis was detected in 32 samples using the LAMP and real-time PCR assays and in 27 and 11 samples using the nested PCR assay and conventional PCR assay, respectively. All the positive samples detected by real-time PCR, nested PCR and conventional PCR assays were positive in the LAMP assay. The LAMP assay is inexpensive, easy to perform, shows a rapid reaction and does not require complex instruments like PCR. Therefore, LAMP is a simple, accurate, fast, and economical assay suitable as an alternative in veterinary practices.

Use of Narrow Band Imaging (NBI) To Detect Esophagitis in Non Erosive Reflux Disease (NERD)

Purpose: Most patients with reflux symptoms do not have signs of mucosal injury on routine endoscopy. In patients with non erosive reflux disease (NERD) chromoendoscopy with Lugol's iodine and confocal laser endomicroscopy have been reported to identify lesions with histologic evidence of esophagitis. However, both procedures are time consuming and require the use of contrast agents. Narrow band imaging (NBI) is known to display macroscopic lesions such as ulcers with high contrast and clarity. We report our findings in an initial group of patients with persistent reflux symptoms and a normal esophagus on white light (WL) endoscopy to see if erosive lesions could be detected with high contrast NBI endoscopy. Methods: The Olympus GIF-H180 gastroscope was used in five patients; 3 male, 2 female, with a mean age of 49 (range 22–63) who underwent upper endoscopy for persistent reflux symptoms (heartburn at least 3 times per week for more than 3 months). On finding no evidence of esophagitis on WL endoscopy in all 5 patients, the NBI mode was switched on and the esophagus re-examined. Any discrete lesions were photographed and biopsies taken with cold forceps for histology. If no lesions were seen, biopsies were taken from the distal esophagus. Results: In asymptomatic individuals, NBI displays the normal esophagus with an even blue color. On switching to narrow band imaging (NBI) mucosal breaks or erosions were seen in 4 out of 5 patients (80%). These consisted of: 1) discrete, depressed erosions of up to 1 mm in diameter, surrounded by an even, bluish appearing normal squamous mucosa; 2) larger, flat, depressed, magenta colored patches of up to 10 mm in diameter, with visible raised margins. Targeted biopsies from these lesions showed microscopic evidence of reflux esophagitis in all 4 patients (100%). Biopsies from the distal esophagus of the patient without mucosal lesions were normal. Conclusion: These early findings suggest that high contrast imaging methods such as NBI may be useful in unmasking subtle erosive changes in the esophagus of patients with reflux symptoms that are not readily visible on routine white light endoscopy. High contrast imaging with NBI is a rapid and easy technique that does not require complex instrumentation or dyes. If these initial findings are verified by future studies, this may become a useful technique for identifying erosive lesions and targeting biopsies in patients with presumed NERD.

Use of self-assembled monolayers, metal ions and smectic liquid crystals to detect organophosphonates

Smectic liquid crystals form stable, micrometer-thick films when deposited onto the surfaces of solids. In this paper, we report that orientational transitions of thin films of the smectic liquid crystal (LC), 4′-octyl-4-biphenyl-carbonitrile (8CB) supported on surfaces presenting copper ions can be exploited to detect the presence of organophosphonate compounds at parts-per-billion (ppb) levels in gas phases. The orientational transition of the 8CB is driven by the competitive binding of the nitrile groups of 8CB and dimethyl methyl phosphonate (DMMP) for the surface-immobilized copper ions. Because the orientational transition of the LC can be observed by using a polarizing film and the naked eye, this detection scheme does not require complex instrumentation to transduce the signal. The film of LC responds to 10 ppbv of DMMP and the initial response time is approximately 20 s. The system is also demonstrated to exhibit molecular specificity. The LC does not respond to gaseous streams containing 1% of the saturated vapor concentrations of ethanol (770 ppm), hexane (1990 ppm) and acetone (3020 ppm) and 75% relative humidity of water.

The colorimetric determination of nitrate anion in aqueous and solid samples utilizing an aromatic derivative in acidic solvent

Ingestion of nitrates has been associated with cancer activity and other health disorders in humans. Presented here are colorimetric methods to detect and quantify nitrates from solids and liquids. A rapid spot test protocol determines nitrate anion to an amount of less than 0.10 µg. Applying a standard curve allows determination of in many samples by spectrometer. Assay by standard curve is sensitive to less than 1.00 mg/mL. In a strongly acidic mixture a brilliant blue color develops upon the reaction of nitrate anion with the secondary amine diphenylamine. An absorbance spectrum determined for the nitrated diphenylamine shows a broad absorbance peak at 597 nm and molar absorptivity of approximately 61 400 L/mole cm. The derivative that is formed is stable for more than 1 h. Inorganic salts such as phosphates, carbonates, sulfates, azides, and chlorides do not interfere with determination of nitrates. Groundwater, pond water, and dust particles were analyzed for nitrates. These methods are rapid, sensitive, relatively inexpensive, do not require complex instrumentation, and are useful for solid and liquid samples. The acidic solvent will dissolve sample components not soluble in water and results are easily discerned by eye. The reagent may easily be transported.

Principles for measurement of chemical exposure based on recognition-driven anchoring transitions in liquid crystals.

The competitive binding of a molecule forming a liquid crystal and a targeted analyte to a common molecular receptor presented at a solid surface possessing nanometer-scale topography is used to trigger an easily visualized surface-driven change in the orientation of a micrometer-thick film of liquid crystal. Diffusion of the targeted analyte from atmosphere to surface-immobilized receptor across the micrometer-thick film of liquid crystal is fast (on the order of seconds), and the competitive interaction of the targeted analyte and liquid crystal with the receptor provides a high level of tolerance to nontargeted species (water, ethanol, acetone, and hexanes). Systems that provide parts-per-billion (by volume) sensitivity to either organoamine or organophosphorus compounds are demonstrated, and their use for imaging of spatial gradients in concentration is reported. This approach does not require complex instrumentation and could provide the basis of wearable personalized sensors for measurement of real-time and cumulative exposure to environmental agents.

Simplified image processing for ultrasonic scans

A system is described which achieves dynamic cardiac imaging by digital data storage without requiring complex instrumentation, employing, with a minimum of modification, data processing facilities already available and being used for analysis and display of gamma camera images. Thus, a conventional ultrasound B-scanner has been interfaced to a Hewlett-Packard HP 2100A computer with a 16k store and both magnetic tape and disc peripherals, and the standard gamma camera software package (HP 5407) has required only minor modifications for application to ultrasonic scanning.