Low Sample Volume Research Articles

Biomimetic nanoplasmonic sensor for rapid evaluation of neutralizing SARS-CoV-2 monoclonal antibodies as antiviral therapy

Monoclonal antibody (mAb) therapy is one of the most promising immunotherapies that have shown the potential to prevent or neutralize the effects of COVID-19 in patients at very early stages, with a few formulations recently approved by the European and American medicine agencies. However, a main bottleneck for their general implementation resides in the time-consuming, laborious, and highly-specialized techniques employed for the manufacturing and assessing of these therapies, excessively increasing their prices and delaying their administration to the patients. We propose a biomimetic nanoplasmonic biosensor as a novel analytical technique for the screening and evaluation of COVID-19 mAb therapies in a simpler, faster, and reliable manner. By creating an artificial cell membrane on the plasmonic sensor surface, our label-free sensing approach enables real-time monitoring of virus-cell interactions as well as direct analysis of antibody blocking effects in only 15 min assay time. We have achieved detection limits in the 102 TCID50/mL range for the study of SARS-CoV-2 viruses, which allows to perform neutralization assays by only employing a low-volume sample with common viral loads. We have demonstrated the accuracy of the biosensor for the evaluation of two different neutralizing antibodies targeting both Delta and Omicron variants of SARS-CoV-2, with half maximal inhibitory concentrations (IC50) determined in the ng/mL range. Our user-friendly and reliable technology could be employed in biomedical and pharmaceutical laboratories to accelerate, cheapen, and simplify the development of effective immunotherapies for COVID-19 and other serious infectious diseases or cancer.

Magnetic hydrogel particles improve nanopore sequencing of SARS-CoV-2 and other respiratory viruses

Presented here is a magnetic hydrogel particle enabled workflow for capturing and concentrating SARS-CoV-2 from diagnostic remnant swab samples that significantly improves sequencing results using the Oxford Nanopore Technologies MinION sequencing platform. Our approach utilizes a novel affinity-based magnetic hydrogel particle, circumventing low input sample volumes and allowing for both rapid manual and automated high throughput workflows that are compatible with Nanopore sequencing. This approach enhances standard RNA extraction protocols, providing up to 40 × improvements in viral mapped reads, and improves sequencing coverage by 20–80% from lower titer diagnostic remnant samples. Furthermore, we demonstrate that this approach works for contrived influenza virus and respiratory syncytial virus samples, suggesting that it can be used to identify and improve sequencing results of multiple viruses in VTM samples. These methods can be performed manually or on a KingFisher automation platform.

A Bioanalytical Method for Quantification of N-nitrosodimethylamine (NDMA) in Human Plasma and Urine with Different Meals and following Administration of Ranitidine

Control of N-nitrosoamine impurities is important for ensuring the safety of drug products. Findings of nitrosamine impurities in some drug products led FDA to develop new guidance providing recommendations for manufacturers towards prevention and detection of nitrosamine impurities in pharmaceutical products. One of these products, ranitidine, also had a published in vivo study, which has since been retracted by its authors, suggesting a potential for in vivo conversion of ranitidine to the probable human carcinogen, N-nitrosodimethylamine (NDMA). FDA subsequently initiated a randomized, double-blind, placebo-controlled, crossover clinical investigation to assess the potential for in vivo conversion of ranitidine to NDMA with different meals. A bioanalytical method toward characterization of NDMA formation was needed as previously published methods did not address potential NDMA formation after biofluid collection. Therefore, a bioanalytical method was developed and validated as per FDA's Bioanalytical Method Validation guidance. An appropriate surrogate matrix for calibration standards and quality control sample preparation for both liquid matrices (human plasma and urine) was optimized to minimize the artifacts of assay measurements and monitor basal NDMA levels. Interconversion potential of ranitidine to NDMA was monitored during method validation by incorporating the appropriate quality control samples. The validated methods for NDMA were linear from 15.6 pg/mL to 2000 pg/mL. Low sample volumes (2 mL for urine and 1 mL for plasma) made this method suitable for clinical study samples and helped to evaluate the influence of ranitidine administration and meal types on urinary excretion of NDMA in human subjects.

Microfluidic formation and manipulation of OLA-based liposomes for synthetic biology research.

Bottom-up synthetic biology aims to create biomimetic systems from the basic elements of cellular life. Such synthetic cell analogs may support scientists in understanding the fundamental biological behaviors at the cellular level. As microscopic aqueous compartments confined within a lipid bilayer, liposomes have been widely used as simplistic cellular mimics for synthetic cell studies. Here, we describe the manufacturing protocol for octanol-assisted liposome assembly (OLA) to produce unilamellar, monodispersed, cell-sized, and biocompatible liposomes as well as in situ liposome experiments. We use maskless, direct-write lithography, which allows for rapid prototyping, to fabricate PDMS-based lab-on-a-chip devices. OLA makes use of the double emulsion generation and dewetting purification approach, with the membrane composition of the formed liposomes and the nature of the encapsulated solution widely adjustable. Using highly responsive pressure-driven flow, the liposome size is tuneable with a steady liposome generation, and the experiment requires very low sample volumes (∼50 µl). We enrich the liposomes from the waste products using density-based on-chip separation techniques, which allows us to conduct experiments on the same device with real-time observation, right after production. As a proof-of-principle synthetic biology application, we demonstrate the capability of adjusting the microenvironment by inducing pH-controlled coacervation between ATP and poly-Llysine molecules inside the liposomes via transmembrane proton flux. Such liquid-liquid phase separation, creating multiple distinct phases from a single homogeneous mixture, is known to play a key role in a variety of cellular processes, including the formation of membraneless organelles as well as numerous other supramolecular assemblies. Our technique shows promising potential for synthetic cell research, particularly in terms of step-by-step construction and experimental control.

Lab-on-a-Chip Electrochemical Biosensors for Foodborne Pathogen Detection: A Review of Common Standards and Recent Progress.

Foodborne pathogens are an important diagnostic target for the food, beverage, and health care industries due to their prevalence and the adverse effects they can cause to public health, food safety, and the economy. The standards that determine whether a given type of food is fit for consumption are set by governments and must be taken into account when designing a new diagnostic tool such as a biosensor platform. In order to meet these stringent detection limits, cost, and reliability standards, recent research has been focused on developing lab-on-a-chip-based approaches for detection devices that use microfluidic channels and platforms. The microfluidics-based devices are designed, developed, and used in different ways to achieve the established common standards for food pathogen testing that enable high throughput, rapid detection, low sample volume, and minimal pretreatment procedures. Combining microfluidic approaches with electrochemical biosensing could offer affordable, portable, and easy to use devices for food pathogen diagnostics. This review presents an analysis of the established common standards and the recent progress made in electrochemical sensors toward the development of future lab-on-a-chip devices that will aid 'collection-to-detection' using a single method and platform.

An automated micro solid phase extraction gas chromatography–mass spectrometry (μSPE-GC–MS) detection method for geosmin and 2-methylisoborneol in drinking water

Geosmin and 2-methylisoborneol (2-MIB) are amongst the most common earthy and musty taste and odour (T&O) compounds found in drinking water. With low odour threshold detection limits below 10 ng L−1, and the complexity of raw water matrices, these two compounds provide a significant challenge for water companies globally. In this research, for the first time, a novel and fully automated micro-solid phase-extraction (μSPE) method coupled with gas chromatography (GC)–mass spectrometry (MS) has been developed for the detection of geosmin and 2-MIB for drinking water analysis. The new automated method described herein is environmentally friendly requiring low raw water sample volumes, of 25 mL, and only 50 μL of elution solvent. Our μSPE-GC–MS method exhibits excellent linearity for both compounds (R2 > 0.999) and low limits of detection of 2.0 ng L−1 and 4.3 ng L−1 for geosmin and 2-MIB, respectively. The method showed excellent recovery rates (95.1–100.1%) and good precision (RSD < 7%) in raw sample matrices. Our approach is fully automated onto a robotic workstation which can be readily integrated into a laboratory workflow for routine water analysis. Furthermore, the method has excellent potential to be incorporated within a portable system for onsite analysis.

Outdoor Air Contaminants-Heavy Metals and Associated Health Risks in Duhok-Iraq

Toxic elements in the atmosphere can cause a variety of health and environmental problems. The current work aims to assess the concentrations of heavy metals associated with the air in the Duhok city-Kurdistan Region of Iraq. For the first time a low volume sampling pump with Glass Microfiber Filter paper was used. Forty samples of air filter aerosol particles were collected from several areas in the Duhok city, during the dry and rainy seasons. Samples of air filters were prepared by a microwave digestion system and assessed by inductively coupled plasma Optical-Emission Spectrometry (ICP-OES). The average concentrations of Cr, Zn, Ni, Cd, Fe, and Pb found in 20 sites in the study area for the dry season were 0.004 mg/m3, 0.007 mg/m3, 0.003 mg/m3, 0.002 mg/m3, 0.142 mg/m3, and 0.402 mg/m3 respectively. Also, the average levels of the mentioned elements were 0.003 mg/m3, 0.004 mg/m3, 0.012 mg/m3, 0.003 mg/m3, 0.138 mg/m3, and 0.645 mg/m3 respectively in wet season. Seasonal results confirmed no significant differences in the concentrations of elements measured at all study sites, with the exception of lead, which doubles during the rainy season. Results showed that the levels of metal concentrations in the present study exceeded the standard limits of WHO. The results of the health risk assessment showed that the population in Duhok city had a higher lifetime chance of developing cancer as a result of these air concentrations and their heavy metal content.

Partition Management of Soil Nutrients Based on Capacitive Coupled Contactless Conductivity Detection

A method based on capacitively coupled contactless conductivity detection (C4D), which has been proven effective for the rapid detection of available soil potassium content, was firstly proposed to apply to soil nutrient detection. By combining a detection signal spectrum analysis, geographic information system (GIS) data, and a cluster analysis, a soil nutrient management system to match the detection device was developed. This system included six modules: soil sample information management, electrophoresis analysis, quantitative calculation, nutrient result viewing, cluster analysis, and nutrient distribution map generation. The soil samples, which were collected from an experimental field in Xuchang City of Henan Province, were analyzed using the C4D and flame photometer methods. The results showed that the detection results for the soil samples obtained via the two methods were in good agreement. C4D technology was feasible for the detection of the soil available nutrients and had the advantages of a high timeliness, low sample volume, and low pollution. The soil nutrient management system adopted the hierarchical clustering method to classify the grid cells of the experimental field according to the nutrient detection results. A soil nutrient distribution map displayed the spatial difference in nutrients. This paper provides a systematic solution for soil nutrient zone management that includes nutrient detection, signal analysis, data management for the nutrient zone, and field nutrient distribution map generation to support decision making in variable fertilization.

Flexible Label-Free Platinum and Bio-PET-Based Immunosensor for the Detection of SARS-CoV-2.

The demand for new devices that enable the detection of severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) at a relatively low cost and that are fast and feasible to be used as point-of-care is required overtime on a large scale. In this sense, the use of sustainable materials, for example, the bio-based poly (ethylene terephthalate) (Bio-PET) can be an alternative to current standard diagnostics. In this work, we present a flexible disposable printed electrode based on a platinum thin film on Bio-PET as a substrate for the development of a sensor and immunosensor for the monitoring of COVID-19 biomarkers, by the detection of L-cysteine and the SARS-CoV-2 spike protein, respectively. The electrode was applied in conjunction with 3D printing technology to generate a portable and easy-to-analyze device with a low sample volume. For the L-cysteine determination, chronoamperometry was used, which achieved two linear dynamic ranges (LDR) of 3.98-39.0 μmol L-1 and 39.0-145 μmol L-1, and a limit of detection (LOD) of 0.70 μmol L-1. The detection of the SARS-CoV-2 spike protein was achieved by both square wave voltammetry (SWV) and electrochemical impedance spectroscopy (EIS) by a label-free immunosensor, using potassium ferro-ferricyanide solution as the electrochemical probe. An LDR of 0.70-7.0 and 1.0-30 pmol L-1, with an LOD of 0.70 and 1.0 pmol L-1 were obtained by SWV and EIS, respectively. As a proof of concept, the immunosensor was successfully applied for the detection of the SARS-CoV-2 spike protein in enriched synthetic saliva samples, which demonstrates the potential of using the proposed sensor as an alternative platform for the diagnosis of COVID-19 in the future.

Laser-induced sublimation extraction for centimeter-resolution multi-species greenhouse gas analysis on ice cores

Abstract. Precision, accuracy, and temporal resolution are key to making full use of atmospheric trace gas records in ice cores. These aspects will become especially crucial for ice cores that aim to extend the ice core record to the last 1.5 Myr, i.e., across the Mid-Pleistocene Transition (as currently drilled within the European project Beyond EPICA – Oldest Ice Core (BE-OIC)). The ice from this period is expected to be close to bedrock and, due to glacier flow, extremely thinned with 15 000 years of climate history contained in only 1 m of ice. Accordingly, for a century-scale resolution, the sample vertical extent must be reduced to a few centimeters containing only about 1–2 mL air STP. We present a novel combined system for the extraction and the simultaneous measurement of CO2, CH4, and N2O concentrations, as well as δ13CO2, which achieves a vertical resolution of 1–2 cm (3.5×3.5 cm cross section) with precisions of 0.4 ppm, 3 ppb, 1 ppb, and 0.04 ‰, respectively, in sublimation tests with standard gas over gas-free ice. This is accomplished by employing a directional and continuous laser-induced sublimation followed by analysis of the sample gas by a quantum cascade laser absorption spectrometer (QCLAS). Besides the low sample volume requirements and the vertical resolution capabilities, the described method holds additional advantages over previous methods, including the immunity of the highly specific QCLAS analysis to drilling fluid contamination as well as the non-destructive nature of the spectroscopic gas analysis. The combined extraction and analysis system was extensively tested by sublimating gas-free ice with introduction of a standard gas to determine the accuracy and characterize potential artifacts. Moreover, Antarctic ice samples were measured to confirm the measurement performance, covering the range of variability expected in Pleistocene ice and highlighting the vertical resolution capabilities critical for its application within BE-OIC.

Effective assay technologies fit for large-scale population screening of type 1 diabetes.

While worldwide prevention efforts for type 1 diabetes (T1D) are underway to abrogate or slow progression to diabetes, mass screening of islet autoantibodies (IAbs) in the general population is urgently needed. IAbs, the most reliable biomarkers, play an essential role in prediction and clinical diagnosis of T1D. Through laboratory proficiency programs and harmonization efforts, a radio-binding assay (RBA) has been well established as the current 'gold' standard assay for all four IAbs. However, in view of the need for large-scale screening in the non-diabetic population, RBA consistently faces two fundamental challenges, cost-efficiency and disease specificity. While all four IAbs are important for disease prediction, the RBA platform, with a separate IAb test format is laborious, inefficient and expensive. Furthermore, the majority of IAb positivity in screening, especially from individuals with single IAb were found to be low risk with low affinity. It is well documented from multiple clinical studies that IAbs with low affinity are low risk with less or no disease relevance. At present, two non-radioactive multiplex assays, a 3-assay ELISA combining three IAbs and a multiplex ECL assay combining all four IAbs, have been successfully used as the primary methods for general population screenings in Germany and the US, respectively. Recently, the TrialNet Pathway to Prevention study has been organizing an IAb workshop which aims to analyze the 5-year T1D predictive values of IAbs. A T1D-specific assay with high efficiency, low cost and requiring low volume of sample will definitely be necessary to benefit general population screening.

High-throughput analysis of the transcriptional patterns of sexual genes in malaria

BackgroundPlasmodium falciparum (Pf) is the leading protozoan causing malaria, the most devastating parasitic disease. To ensure transmission, a small subset of Pf parasites differentiate into the sexual forms (gametocytes). Since the abundance of these essential parasitic forms is extremely low within the human host, little is currently known about the molecular regulation of their sexual differentiation, highlighting the need to develop tools to investigate Pf gene expression during this fundamental mechanism.MethodsWe developed a high-throughput quantitative Reverse-Transcription PCR (RT-qPCR) platform to robustly monitor Pf transcriptional patterns, in particular, systematically profiling the transcriptional pattern of a large panel of gametocyte-related genes (GRG). Initially, we evaluated the technical performance of the systematic RT-qPCR platform to ensure it complies with the accepted quality standards for: (i) RNA extraction, (ii) cDNA synthesis and (iii) evaluation of gene expression through RT-qPCR. We then used this approach to monitor alterations in gene expression of a panel of GRG upon treatment with gametocytogenesis regulators.ResultsWe thoroughly elucidated GRG expression profiles under treatment with the antimalarial drug dihydroartemisinin (DHA) or the metabolite choline over the course of a Pf blood cycle (48 h). We demonstrate that both significantly alter the expression pattern of PfAP2-G, the gametocytogenesis master regulator. However, they also markedly modify the developmental rate of the parasites and thus might bias the mRNA expression. Additionally, we screened the effect of the metabolites lactate and kynurenic acid, abundant in severe malaria, as potential regulators of gametocytogenesis.ConclusionsOur data demonstrate that the high-throughput RT-qPCR method enables studying the immediate transcriptional response initiating gametocytogenesis of the parasites from a very low volume of malaria-infected RBC samples. The obtained data expand the current knowledge of the initial alterations in mRNA profiles of GRG upon treatment with reported regulators. In addition, using this method emphasizes that asexual parasite stage composition is a crucial element that must be considered when interpreting changes in GRG expression by RT-qPCR, specifically when screening for novel compounds that could regulate Pf sexual differentiation.Graphical

Assessment of Oxidative Stress Biomarkers in Rat Blood.

Redox status assessments are time-consuming, require a large volume of samples and great reagent amounts, and are not adequately described for methodological reproducibility. Here, the objective was to standardize redox balance determination, based on previously described spectrophotometric tests in pregnant rats, to improve precision, time dispensed, and the volume of samples and reagents, while maintaining accuracy and adequate cost benefits. This protocol summarizes oxidative stress markers, which focus on spectrophotometric tests for the assessment of thiobarbituric acid-reactive substances, reduced thiol groups, and hydrogen peroxide, as well as the antioxidant activity of superoxide dismutase, glutathione peroxidase, and catalase in washed erythrocyte and serum samples from full-term pregnant rats. For non-pregnant rats and other species, it is necessary to standardize these determinations, especially the sample volume. All measurements were normalized by the estimated protein concentrations in each sample. To establish optimum conditions for the reproducibility of the proposed methods, we describe all changes made in each assay's steps based on the reference method reassessed for the new standardizations. Furthermore, the calculations of the concentrations or activities of each marker are presented. Thus, we demonstrate that the analysis of serum samples is easier and faster, but it is impossible to detect catalase activity. Furthermore, the proposed methods can be applied for redox balance determination, especially using smaller reagent amounts and lower sample volumes in lesser time without losing accuracy, as is required in obtaining samples during rat pregnancy.

Analysis of microsamples by miniaturized magnetic-based pipette tip microextraction: determination of free cortisol in serum and urine from very low birth weight preterm newborns.

Miniaturized magnetic-based pipette tip microextraction is presented as a sample preparation approach for microsamples. It involves quick dispersion of a diminutive amount of a magnetic sorbent material in a low-volume sample (10 μL) to entrap the target analytes. Next, the dispersion is aspirated using a (semi)automatic pipette through a pipette tip with a small cubic neodymium magnet inside, which retrieves the magnetic sorbent containing the analytes. After discarding the rest of the sample, the sorbent is properly rinsed by aspirating/dispensing deionized water, and then, the analytes are eluted by aspirating/dispensing an appropriate solvent. This approach was employed for the determination of free cortisol in serum and urine from very low birth weight preterm newborns, a vulnerable patient group who present low availability for sampling biological fluids. A magnetic immunosorbent made of a cortisol antibody was employed for the selective extraction, followed by liquid chromatography-tandem mass spectrometry. Good analytical features were obtained, such as limits of detection and quantification of 0.08 and 0.27 ng mL-1, respectively, linearity up to 50 ng mL-1 (R2 > 0.999), RSD values under 15% and relative recoveries between 91 and 111%. The cross-reactivity with other glucocorticoids (i.e., cortisone and prednisolone) was evaluated to show the selectivity of the extraction. Finally, the method applicability was demonstrated towards the determination of free cortisol in the serum and urine samples from low birth weight preterm newborns.

Microfluidic Acoustic Method for High Yield Extraction of Cell-Free DNA in Low-Volume Plasma Samples.

Cell-free DNA has many applications in clinical medicine, in particular in cancer diagnosis and cancer treatment monitoring. Microfluidic-based solutions could provide solutions for rapid, cheaper, decentralized detection of cell-free tumoral DNA from a simple blood draw, or liquid biopsies, replacing invasive procedures or expensive scans. In this method, we present a simple microfluidic system for the extraction of cell-free DNA from low volume of plasma samples (≤500μL). The technique is suitable for either static or continuous flow systems and can be used as a stand-alone module or integrated within a lab-on-chip system. The system relies on a simple yet highly versatile bubble-based micromixer module whose custom components can be fabricated with a combination of low-cost rapid prototyping techniques or ordered via widely available 3D-printing services. This system is capable of performing cell-free DNA extractions from small volumes of blood plasma with up to a tenfold increase in capture efficiency when compared to control methods.

A novel point-of-care diagnostic prototype system for the simultaneous electrochemiluminescent sensing of multiple traumatic brain injury biomarkers.

Traumatic brain injuries (TBI) are typically acquired when a sudden violent event causes damage to the brain tissue. A high percentage (70-85%) of all TBI patients are suffering from mild TBI (mTBI), which is often difficult to detect and diagnose with standard imaging tools (MRI, CT scan) due to the absence of significant lesions and specific symptoms. Recent studies suggest that a screening test based on the measurement of a protein biomarker panel directly from a patient's blood can facilitate mTBI diagnosis. Herein, we report a novel prototype system designed as a precursor of a future hand-held point-of-care (POC) diagnostic device for the simultaneous multi-biomarker sensing, employing a microarray-type spatially resolved electrochemiluminescence immunoassay (SR-ECLIA). The small tabletop prototype consists of a screen-printed electrode compartment to conduct multi-analyte ECL sandwich assays, a potentiostat module and a light collection module, all integrated into a compact 3D-printed housing (18.2 × 16.5 × 5.0 cm), as well as an sCMOS detector. Based on this design concept, further miniaturization, system integration, performance optimization and clinical evaluation shall pave the way towards the development of a portable instrument for use at the site of accident and healthcare. To demonstrate the system's feasibility, current performance and efficiency, the simultaneous detection of three mTBI biomarkers (GFAP, h-FABP, S100β) in 50% serum was achieved in the upper pg mL-1 range. The proposed device is amenable to the detection of other biomarker panels and thus could open new medical diagnostic avenues for sensitive multi-analyte measurements with low-volume biological sample requirements.

A thermally robust method of sample sealing for capillary DLS

Capillary Dynamic Light Scattering (DLS), has recently been introduced as a simple and enabling technique that increases the measurement range of traditional DLS analysis with minimized sample volumes (Ruseva et al., 2018). The previously published protocol for the preparation of samples for analysis within a capillary called for sealing of the capillary end using a clay compound (Ruseva et al., 2019). This material is not, however, compatible with organic solvents, nor with elevated sample temperatures. To extend the uses of capillary DLS to more complex assays like thermal aggregation studies, a new sealing method is demonstrated using a UV curing compound. This further motivates the use of capillary DLS to minimize volumes of destroyed precious samples in pharmaceutical development assays to study thermal kinetics.•Use of UV curing compound to seal capillaries used in DLS to preserve low volumes of sample.

Modular 3D-printed fluorometer/photometer for determination of iron(ii), caffeine, and ciprofloxacin in pharmaceutical samples

The demand for the development of portable and low-cost analytical devices has encouraged studies employing additive manufacturing techniques, such as 3D-printing. This method can be used to produce components such as printed electrodes, photometers, and fluorometers for low-cost systems that provide advantages including low sample volume, reduced chemical waste, and easy coupling with LED-based optics and other instrumental devices. In the present work, a modular 3D-printed fluorometer/photometer was designed and applied for the determination of caffeine (CAF), ciprofloxacin (CIP), and Fe(ii) in pharmaceutical samples. All the plastic parts were printed separately by a 3D printer, using Tritan as the plastic material (black color). The final size of the modular 3D-printed device was 12 × 8 cm. The radiation sources were light-emitting diodes (LEDs), while a light dependent resistor (LDR) was used as a photodetector. The analytical curves obtained for the device were: y = 3.00 × 10-4 [CAF] + 1.00 and R 2 = 0.987 for caffeine; y = 6.90 × 10-3 [CIP] - 3.39 × 10-2 and R 2 = 0.991 for ciprofloxacin; and y = 1.12 × 10-1 [Fe(ii)] + 1.26 × 10-2 and R 2 = 0.998 for iron(ii). The results obtained using the developed device were compared with reference methods, with no statistically significant differences observed. The 3D-printed device was composed of moveable parts, providing flexibility for adaptation and application as a photometer or fluorometer, by only switching the photodetector position. The LED could also be easily switched, permitting application of the device for different purposes. The cost of the device, including the printing and electronic components, was lower than US$10. The use of 3D-printing enables the development of portable instruments for use in remote locations with a lack of research resources.

Portable rotary PCR system for real-time detection of Pseudomonas aeruginosa in milk.

The rapid quantitative detection of Pseudomonas aeruginosa in milk is of great significance to food safety. Quantitative real-time polymerase chain reaction (qPCR) technology is a good choice to meet this requirement. A good qPCR system should show the advantages of being low cost, having low-power consumption, having potential for miniaturization and be portable. However, most of the time-domain-based qPCR systems reported to date do not meet these requirements. In this study, we propose a novel real-time rotary PCR reaction system (RRP) that meets all the abovementioned specifications, and contains four modules: a heating control module, a disposable PCR capillary tube, a mechanical control module, and a photoelectric detection module. The volume of our homemade-PCR capillary tube is only 3 μL. The total manufacturing cost is cheaper than $200, and the capillary tube is about 1.4 cents. The size parameter of the RRP is less than 300 mm × 150 mm × 150 mm, using low mobile power sources to operate. All the features mean that the RRP meets the advantages of low sample volumes, enhanced thermal conductivity and being portable. Through conducting the experimental quantitative detection of Pseudomonas aeruginosa in milk and theoretical simulations by COMSOL, we prove the feasibility of this rotary PCR real-time detection system, which has broad application prospects in the rapid detection of bacteria and food safety.

Mixed-mode chromatography of mixed functionalized analytes as the homologues of benzalkonium chloride. Application to pharmaceutical formulations

In this work, a retention behavior based on mixed-mode reversed-phase (RP)/hydrophilic interaction liquid chromatography (HILIC) was observed for benzalkonium chloride (BAK) using a core-shell column functionalized with biphenyl groups. Although in the literature, the U-shaped retention was reported for polar compounds in mixed functionalized phases, in the present work, the behavior was dependent upon the chemical structure of the analyte with mixed functionality (ammonium group, a benzyl group and an alkyl chain) and on the high selectivity of the chromatographic column. The bimodal retention was observed for the four BAK homologues using a content of acetonitrile from 65 to 95% in the mobile phase. The data were adjusted to polynomial equations which allow for modeling and predicting the U-shaped retention. The salt concentration (50 and 100 mM), anion (formate and acetate) and cation (ammonium and triethylammonium) of the salt, pH (4 and 5) in the mobile phase were studied in order to understand their influence on the two retention modes. Significant electrostatic interactions were involved in the two retention modes, especially with a content of acetonitrile higher that 90%. Linear relationships between the retention factors of the four homologues were found in a wide range of %acetonitrile when the salt and triethylamine concentration, pH and nature of salt were changed. The differences found on the retention of the homologues, when increasing the alkyl chain length, were more significant in the RP mode due to predominant hydrophobic interactions. A pH decrease and a salt concentration increase caused a retention decrease for both modes. A decrease on of the retention was observed when acetate anion was replaced by formate anion. The different order of the polynomial equations according to the used mobile phase confirmed its relevant role in the interactions with the analytes and stationary phase. A mobile phase was selected (85% acetonitrile, pH 4 and 100 mM ammonium formate) for the BAK determination in cutaneous, otic and ophthalmic formulations with different active pharmaceutical ingredients and excipients. Low sample volume (500 μL) and short analysis time (<5 min) were some of the advantages of the proposed method. In addition, good analytical performance (R2 > 0.999, % RSD <4.5% for intra-day precision and <5.8% for inter-day precision, and recoveries in the 92–105% range) was obtained.