Viability of a Serum Infliximab Concentration-Detecting Reagent as a Qualitative Assay for an Infliximab Biosimilar.

The efficacy of infliximab in treating rheumatoid arthritis depends on its serum trough concentration, which must be maintained at a minimum of 1 µg/mL to achieve the desired effects. However, Japan's National Health Insurance system does not cover tests for rheumatoid arthritis patients undergoing treatment with biosimilar infliximab because its performance as a biosimilar remains unclear. This study aimed to investigate whether the Remi-check Q qualitative assay yields comparable results for biosimilar infliximab and the originator product. Infliximab BS 100 "NK" and Remicade 100® were separately diluted in pooled human serum to yield test samples at the following concentrations: 0.30, 0.70, 1.20, and 3.00 µg/mL. Prepared samples were quantitatively assessed using an enzyme-linked immunosorbent assay (ELISA) and qualitatively using Remi-check Q, and the results obtained for the originator and biosimilar product were compared. For both originator and biosimilar infliximab, Remi-check Q yielded a negative result for all 0.30 and 0.70 µg/mL samples and a positive result for all 3.00 µg/mL samples. However, negative results were obtained with a fraction of the 1.20 µg/mL samples (biosimilar, 4/15; originator, 3/15). Concurrence rates between the results of quantitative ELISA and qualitative Remi-check Q analyses were comparable between originator and biosimilar infliximab at all tested concentrations. These results indicate that Remi-check Q yields comparable results for biosimilar infliximab and the originator product on being used as a qualitative assay for trough serum levels.

Optical Single Cell Resolution Cytotoxicity Biosensor Based on Single Cell Adhesion Dot Arrays.

Low cost, easy to use cell viability tests are needed in the pharmaceutical, biomaterial, and environmental industries to measure adverse cellular effects. We present a new methodology to track cell death with high resolution. Adherent cells commonly detach from the surface when they die, but some toxic compounds promote cell adhesion. A methodology that enables both dynamic detachment monitoring but also rapid detection of toxic effects of compounds that promote cell adhesion would constitute a step forward toward high-throughput cytotoxicity measurements. We achieved dynamic digital quantification of cell viability by simple optical imaging using "single cell adhesion dot arrays" (SCADA), fibronectin (FN) dot arrays designed to accommodate a single cell on each fibronectin dot. For cytotoxicity measurements, cell-filled SCADA substrates were exposed to K2CrO4, HgSO4 salts, and dimethyl sulfoxide (DMSO). The toxic effect of DMSO and K2CrO4 was dynamically monitored by measuring the cell detachment rate during more than 30 h by quantifying the number of occupied dots in the SCADA array. HgSO4 inhibited cellular detachment from the surface, and cytotoxicity was monitored using the trypan blue life/death assay directly on the surface. In all cases, the cytotoxicity effects were easily monitored with single cell resolution, and the results were comparable to previous reports. SCADA enabled dynamic measurements at the highest resolution due to the digital measuring in this method. The integration of SCADA substrates into microfluidic platforms will provide a practical tool that will extend to fundamental research and commercial applications.

Rapid lipid bilayer membrane formation on Parylene coated apertures to perform ion channel analyses.

We present a chip design allowing rapid and robust lipid bilayer (LBL) membrane formation using a Parylene coated thin silicon nitride aperture. After bilayer formation, single membrane channels can be reconstituted and characterized by electrophysiology. The ability for robust reconstitution will allow parallelization and enhanced screening of small molecule drugs acting on or permeating across the membrane channel. The aperture was realized on a microfabricated silicon nitride membrane by using standard clean-room fabrication processes. To ensure the lipid bilayer formation, the nitride membrane was coated with a hydrophobic and biocompatible Parylene layer. We tested both Parylene-C and Parylene-AF4. The contact angle measurements on both Parylene types showed very good hydrophobic properties and affinity to lipids. No precoating of the Parylene with an organic solvent is needed to make the aperture lipophilic, in contradiction to Teflon membranes. The chips can be easily placed in an array utilizing a 3D printed platform. Experiments show repetitive LBL formation and destruction (more than 6 times) within a very short time (few seconds). Through measurements we have established that the LBL layers are very thin. This allows the investigation of the fusion process of membrane proteins i.e. outer membrane protein (OmpF) in the LBL within a few minutes.

Quantifying late-stage host-seeking behaviour of Anopheles gambiae at the insecticidal net interface using a baited-box bioassay.

BACKGROUND: Insecticide-treated nets (ITNs) are losing efficacy against pyrethroid-resistant malaria vector populations throughout Africa. Safeguarding bed net efficacy, vital for effective malaria control, requires greater knowledge of mosquito-ITN interactions and how this impacts on the mosquito. METHODS: A purpose-built benchtop apparatus with a closed 10 cm cubic chamber (the 'Baited-box') was used to video record behaviour of individual free-flying female Anopheles gambiae during approach and blood-feeding on a human hand through untreated nets and ITNs at close range. Time and duration of defined behavioural events, and knockdown and mortality at 1- and 24-h post-exposure respectively, were recorded for pyrethroid susceptible and resistant mosquitoes. RESULTS: Using three human volunteers differing in relative attractiveness to mosquitoes, 328 mosquitoes were individually tested. There were no significant differences between response rates to ITNs and untreated nets (P > 0.1) or between resistant (Tiassalé) and susceptible (Kisumu) mosquito strains, at untreated nets (P = 0.39) or PermaNet 2.0 (P = 1). The sequence of behavioural events from host-seeking to completion of blood-feeding was consistent in all tests but duration and start time of events involving net contact were reduced or delayed respectively with ITNs. Blood-feeding durations at untreated nets (means from 4.25 to 8.47 min (95% confidence interval (CI) = 3.39-9.89) at 3 human volunteers) were reduced by 37-50% at PermaNet 2.0, in susceptible (mean 2.59-4.72 min, 95% CI = 1.54-5.5, P = < 0.05) and resistant (mean 4.20 min, 95% CI = 3.42-4.97, P = 0.01) strains. Total accumulated net contact was approximately 50% lower at PermaNet and Olyset ITNs (P < 0.0001) in susceptible (two of the three volunteers) and resistant mosquitoes. Times prior to first net contact were similar at untreated nets and ITNs (P > 0.2), and neither ITN type showed detectable spatial repellency. After initial contact, blood-feeding commenced later at Olyset (mean 2.76 min, 95% CI = 1.74-3.76, P = 0.0009) and PermaNet (mean 2.4 min, 95% CI = 1.52-3.33, P = 0.0058) than untreated netting (mean 0.68 min, 95% CI = 0.42-0.94). CONCLUSIONS: The baited box offers a simple method for detailed characterization of mosquito behavioural responses to insecticidal nets, for comparing entomological modes of action between nets and for defining the behavioural responses of particular mosquito strains or populations. The device has potential as a screening assay in the search for novel net treatments and for investigations into behavioural resistance mechanisms.

Chromatographic methods development for clinical practice: requirements and limitations.

Development of a chromatographic method in bioanalysis is a challenging and complex procedure with many pitfalls and often unexpected reversals that can require several months to accomplish. Even an experienced analytical team must contend many limitations mainly in connection with the strict requirements imposed on current clinical research. These restrictions typically persist throughout the whole development process, from clinical trial assignment, across optimization of extraction of biological materials and chromatographic separation, to validation and data interpretation. This paper describes questions and their possible answers raised during the pre-analytical phase such as use of modern sample preparation techniques in clinical methods, application of internal standards, as well as selection of stationary phases and detection techniques in the analytical phase. Validation problems and interpretation of results are demonstrated with three typical examples of characteristics to be considered, i.e. recovery, matrix effect, and limit of detection vs. lower limit of quantification.

Method to quantify cytokines and chemokines in mouse brain tissue using Bio-Plex multiplex immunoassays.

This protocol describes how to prepare mouse brain tissue for quantification of multiple inflammatory mediators using a multiplex bead-based immunoassay. It is important to have methods that allow quantification of multiple analytes from small amounts of tissue. Bio-Plex is a Luminex xMAP-based multiplex bead-based immunoassay technology that permits simultaneous analysis of up to 100 analytes from a single tissue sample. This assay has been used extensively to investigate analytes in plasma and serum samples as well as cultured and primary cells. Here, we describe a method for simultaneous analysis of 33 different inflammatory cytokines and chemokines from mouse brain tissue using the Bio-Plex Pro Mouse Chemokine Panel 33-Plex.

Comparison of bacterial endotoxin testing methods in purified pharmaceutical water matrices.

Current bacterial endotoxin testing systems can be labor-intensive and time-consuming, involving several manual pipetting steps. In our quality control laboratory, annually, we test about 15,000 samples of different grades of purified water, WFI and water samples taken to validate cleaning procedures for endotoxins. We are currently using the Kinetic-QCL™ assay which is a pharmacopeia method that provides reliable results. We compared this assay with another Limulus amebocyte lysate (LAL)-based assay (Endosafe®-MCS) and an alternative endpoint fluorescent recombinant Factor C (rFC) assay (ENDOZYME II GO®). Both these assays have been developed to reduce analyst preparation time. Our objective was to assess if they could increase the throughput of our testing while maintaining low rates of invalid results. The results demonstrated that the two most appropriate methods for rapid endotoxin detection in water are our current assay, K-QCL, and the rFC-based assay, ENDOZYME II GO. This latter assay was found to be less sensitive to interference than our current assay, particularly in cleaning validation water samples. It also showed better performance, accuracy, repeatability and had a shorter time-to-results. ENDOZYME II GO assay allows quick testing of large numbers of samples with reliable results and is a good alternative for conventional LAL assays.

Reference intervals established using indirect method for serum ferritin assayed on Abbott Architect i2000SR analyzer in Chinese adults.

BACKGROUND: Serum ferritin (SF) test has been widely used in clinical practice. However, its reference intervals (RIs) vary depending on the analytical method and ethnic origin. This study was to establish the RIs using indirect method for SF in Chinese adults. METHODS: SF was assayed on Abbott i2000SR analyzer. The SF test results of all health examinees (8913 males aged 18-93 years and 5397 females aged 18-90 years) between December 2010 and April 2019 were obtained from our laboratory information system. After Box-Cox transformation of raw data and exclusion of outliers, parametric and non-parametric approaches were used to calculate 95% RIs. The correlation between SF levels and ages, and the differences in SF levels between subgroups were also analyzed. RESULTS: SF levels in females were significantly different from those in males (Z = 88.96, Z* = 23.17; Z > Z*) and showed a weak positive correlation with age (r = .466, P < .0001). The RIs based on parametric approach in males were 66.12-561.58 µg/L, whereas in all females were 3.59-269.59 µg/L, females aged <50 years 3.26-148.02 µg/L and those aged ≥50 years 17.28-303.27 µg/L. The RIs based on non-parametric approach in males were 65.00-571.37 µg/L whereas in all females were 4.00-254.00 µg/L, females aged <50 years 4.00-152.00 µg/L and those aged ≥50 years 16.00-304.05 µg/L. CONCLUSIONS: Our indirect RIs for SF were markedly different from the manufacturer's recommended RIs and might be more suitable for Chinese adults, which would be helpful in interpreting laboratory data and clinical decision-making.

Resistance to deltamethrin in Triatoma infestans: microgeographical distribution, validation of a rapid detection bioassay and evaluation of a fumigant canister as control alternative strategy.

Triatoma infestans (Klug) (Hemiptera: Reduviidae) is the main vector of Chagas disease in the Southern Cone of America and resistance to pyrethroid insecticides has been detected in several areas from its geographical distribution. Pyrethroid resistance presents a complex geographical pattern at different spatial scales. However, it is still unknown if the toxicological variability is a common feature within villages of the Gran Chaco were high resistance was descripted. The objectives of this study were to determine: (a) the microgeographical distribution of the deltamethrin-resistance in insects from Pampa Argentina village, (b) the performance of the insecticide impregnated paper bioassay to evaluate deltamethrin-resistance in field collected insects and (c) the lethal activity of the fumigant canister containing DDVP against insects resistant to deltamethrin. High survival of T. infestans exposed to discriminant dose was observed in the samples of all the evaluated dwellings, suggesting that the resistance to deltamethrin is homogeneous at the microgeographical level. Resistance determination by impregnated paper bioassay was similar to traditional topical determination, highlighting the use of this rapid methodology in field large-scale monitoring. The fumigant canister was not effective against resistant insects, remarking the need to develop suitable formulations that ensure minimal toxicological risk and high effectivity.

Diagnostic accuracy of the Troponin-only Manchester Acute Coronary Syndromes (T-MACS) decision aid with a point-of-care cardiac troponin assay.

OBJECTIVE: Point-of-care (POC) cardiac troponin (cTn) assays have a rapid turnaround time but are generally less sensitive than laboratory-based assays. Previous research found that the Abbott i-Stat cardiac troponin I (cTnI) assay has good diagnostic accuracy when used with the Troponin-only Manchester Acute Coronary Syndromes (T-MACS) decision aid and serial sampling over 3 hours. Accuracy of other assays may differ. We therefore evaluated the diagnostic accuracy of a different POC cTnI assay with serial sampling over 3 hours, both with T-MACS and when used alone. METHODS: In a prospective diagnostic accuracy study at eight EDs in England (July 2015-October 2017), we collected clinical data from consenting adults with suspected ACS at the time of assessment in the ED. Blood samples were drawn on arrival and 3 hours later for POC cTnI (Cardio 3 Triage, Alere). The target condition was an adjudicated diagnosis of acute myocardial infarction (AMI), based on reference standard serial laboratory-based cTn testing. We calculated test characteristics for POC cTnI using the limit of detection (LoD, 0.01 µg/L) and the T-MACS decision aid. RESULTS: Of 347 participants, 59 (14.9%) had AMI. With serial POC cTnI testing over 3 hours, POC cTnI at the LoD cut-off ruled out AMI in 193 (55.6%) patients with 98.1% sensitivity (95% CI 89.9% to 100.0%) and 99.5% negative predictive value (NPV, 95% CI 96.5% to 99.9%). T-MACS ruled out AMI in 117 (33.7%) patients with 98.1% sensitivity (95% CI 89.9% to 100%) and 99.2% NPV (95% CI 94.3% to 99.9%). T-MACS ruled in AMI with 97.9% specificity (95% CI 95.8% to 99.5%) and 83.7% positive predictive value (95% CI 70.6% to 91.7%). CONCLUSIONS: With serial sampling over 3 hours, the Alere Cardio 3 Triage cTnI assay has relatively high NPV for AMI using either the LoD cut-off alone or the T-MACS decision aid. However, wide CIs around the measures of diagnostic accuracy mean that further prospective testing of this strategy is required before clinical implementation. TRIAL REGISTRATION NUMBER: UKCRN 18000.

Barcoded point-of-care bioassays.

Barcode technology can deliver batched information for patient healthcare. For clinical examinations, barcodes serve as reporters for labeling multiple targets, and meanwhile, facilitate improved sensitivity and specificity, thus enabling barcode as a promising alternative to traditional labels for biomarker identification and signal amplification. However, faced with the stringent claims of point-of-care (POC) bioassays, efforts are needed to advance current technologies toward rapidity, robustness, affordability, and user-friendliness. In the past decades, chemists have succeeded in delicate fabrication of the barcode libraries for encoding. Nevertheless, the decoding technologies remain poorly discussed, especially simplified decoding strategies for POC bioassays. Recent emergence of portable cartridges and miniaturized signal-recording devices has brought a promise to merge barcodes-assisted bioassay with POC testing (POCT). This review provides a comprehensive summary on barcode encoding and decoding, with emphasis on their potential use in POCT, facilitated by improved manufacturing and portable devices. Future directions of barcoded bioassays for POCT and current challenges are also presented. We anticipate that this review will be beneficial to promoting barcodes toward broad applications.

A Facile Strategy for Immobilizing GOD and HRP onto Pollen Grain and Its Application to Visual Detection of Glucose.

Pollen grain was explored as a new carrier for enzyme immobilization. After being modified with boric acid-functionalized titania, the pollen grain was able to covalently immobilize glycosylated enzymes by boronate affinity interaction under very mild experimental conditions (e.g., pH 7.0, ambient temperature and free of organic solvent). The glucose oxidase and horse radish peroxidase-immobilized pollen grain became a bienzyme system. The pollen grain also worked as an indicator of the cascade reaction by changing its color. A rapid, simple and cost-effective approach for the visual detection of glucose was then developed. When the glucose concentration exceeded 0.5 mM, the color change was observable by the naked eye. The assay of glucose in body fluid samples exhibited its great potential for practical application.

Versatile Tool for Droplet Generation in Standard Reaction Tubes by Centrifugal Step Emulsification.

We present a versatile tool for the generation of monodisperse water-in-fluorinated-oil droplets in standard reaction tubes by centrifugal step emulsification. The microfluidic cartridge is designed as an insert into a standard 2 mL reaction tube and can be processed in standard laboratory centrifuges. It allows for droplet generation and subsequent transfer for any downstream analysis or further use, does not need any specialized device, and manufacturing is simple because it consists of two parts only: A structured substrate and a sealing foil. The design of the structured substrate is compatible to injection molding to allow manufacturing at large scale. Droplets are generated in fluorinated oil and collected in the reaction tube for subsequent analysis. For sample sizes up to 100 µL with a viscosity range of 1 mPa·s-4 mPa·s, we demonstrate stable droplet generation and transfer of more than 6 × 105 monodisperse droplets (droplet diameter 66 µm ± 3 µm, CV ≤ 4%) in less than 10 min. With two application examples, a digital droplet polymerase chain reaction (ddPCR) and digital droplet loop mediated isothermal amplification (ddLAMP), we demonstrate the compatibility of the droplet production for two main amplification techniques. Both applications show a high degree of linearity (ddPCR: R2 ≥ 0.994; ddLAMP: R2 ≥ 0.998), which demonstrates that the cartridge and the droplet generation method do not compromise assay performance.

An improved method of nasal swab analysis for assessing alpha internal radioactive contaminants.

When responding to nuclear and radiological emergencies, rapid and on-site detections of possible internal radioactive contaminants are required for early dose estimation and medical triage. Nasal swab analysis is an effective method to provide valuable information for early and fast estimates of alpha radionuclide inhalation intakes and resultant doses. In this study, to improve the quality of nasal swab measurements, a specialised double-detector alpha counter was designed. Various parameters including swab materials, sample pre-preparation, angle-dependence and vacuum dependence were investigated to optimise the reliability and convenience of the nasal swab method. An improved procedure of direct nasal swab measurement was eventually established, which could be used to obtain early data for initial dose assessment during the first response of nuclear and radiological emergencies.

3D Printing: The Second Dawn of Lab-On-Valve Fluidic Platforms for Automatic (Bio)Chemical Assays.

In this work, inexpensive manufacturing of unibody transparent mesofluidic platforms for pressure-driven Lab-On-a-Valve (LOV) methodologies is accomplished via rapid one-step 3D prototyping from digital models by user-friendly freeware. Multichannel architecture having 800-1800 µm cross-sectional features with unconventional 3D conduit structures and integrating optical and electrochemical detection facilities is for the first time reported. User-defined flow-programming capitalizing upon software control for automatic liquid handling is synergistically combined with additive manufacturing based on stereolithographic 3D printing so as to launch the so-called fourth generation of microflow analysis (3D-µFIA). Using an affordable consumer-grade 3D printer dedicated LOV platforms are 3D printed at will and prints are characterized in terms of solvent compatibility, optical and mechanical properties, and sorption of inorganic and organic species to prospect potentialities for the unfettered choice of chemistries. The unique versatility of the 3D-printed LOV device that is attached to a multiposition rotary valve as a central design unit is demonstrated by (i) online handling of biological materials followed by on-chip photometric detection, (ii) flow-through bioaccessibility tests in exposome studies of contaminated soils with miniaturized voltammetric detection, (iii) online phospholipid removal by TiO2-incorporated microextraction approaches using on-chip disposable sorbents, and (iv) automatic dynamic permeation tests mimicking transdermal measurements in Franz-cell configurations. A multipurpose LOV fluidic platform can be fabricated for less than 11 Euros.

Bioassay for Determining the Concentrations of Caffeine and Individual Methylxanthines in Complex Samples.

Caffeine and other methylxanthines are stimulant molecules found in formulated beverages, including sodas and energy drinks, and in brewed beverages, such as coffee and teas. Previously, we developed a bioassay for caffeine that involves monitoring the growth of a ΔguaB mutant of Escherichia coli defective in de novo guanine biosynthesis. When supplemented with a plasmid expressing the genes for an N-demethylation pathway from Pseudomonas putida CBB5, these bacteria demethylate caffeine (1,3,7-trimethylxanthine) and other methylxanthines into xanthine, which is then converted into guanine to support cell growth. A major limitation of this bioassay was that it could only measure the total concentration of all methylxanthines in a mixture. Therefore, it could not be used to measure the caffeine content of beverages like teas, which contain substantial quantities of multiple methylxanthines. To overcome this limitation, we created seven new plasmids containing all subsets of the three demethylase genes (ndmA, ndmB, and ndmC). We show that strains of ΔguaBE. coli containing each plasmid are able to demethylate specific subsets of methylxanthines and that they can be used to determine the concentrations of individual methylxanthines in complex mixtures containing multiple methylxanthines, including coffee doped with an additional methylxanthine. While validating this assay, we also discovered an unexpected demethylation event at the 1-methyl position when NdmB and NdmC were expressed in the absence of NdmA. The improved cell-based bioassay is inexpensive, is easy to use, and gives results comparable to standard high-performance liquid chromatography methods for measuring methylxanthine concentrations.IMPORTANCE Caffeine (1,3,7-trimethylxanthine) is the dominant neurostimulant found in coffee, teas, sodas, and energy drinks. Measuring the amount of caffeine and other methylxanthines in these beverages is important for quality assurance and safety in food science. Methylxanthines are also used in medicine and as performance-enhancing drugs, two contexts in which accurately determining their concentrations in bodily fluids is important. Liquid chromatography is the standard method for measuring methylxanthine concentrations in a sample, but it requires specialized equipment and expertise. We improved a previous bioassay that links E. coli growth to methylxanthine demethylation so that it can now be used to determine the amounts of individual methylxanthines in complex mixtures or beverages, such as coffee.

Lyophilization of chemiluminescent substrate reagents for high-sensitive microchannel-based lateral flow assay (MLFA) in point-of-care (POC) diagnostic system.

Over the last few years, lateral flow assay (LFA) devices have grown to be the most common point-of-care test (POCT) platform facilitating disease diagnostics in low-resource environments. However, the lack of consistency and the limited sensitivity of these devices often lead to misdiagnosis and generates the need for an alternate approach. A chemiluminescence based microchannel-based lateral flow assay (MLFA) in a POCT platform can result in a much higher sensitivity but involves multiple additional steps of liquid reagents for the sequential execution of the signal amplification protocol. One of the best ways to develop a sample-to-answer system with minimum user intervention is to dry reagents on a chip prior to sample addition and to control the flow of the biological fluid through the drying chambers resulting in the reconstitution of the reagents. This work reports the methods for the successful lyophilization of the chemiluminescent substrate and its reconstitution in artificial serum without any significant loss of functionality. The lyophilized reagents were reconstituted and incorporated into the reaction chambers of a designed polymer lab-on-a-chip to implement a sandwich assay for the detection of malarial biomarkers. The results report a limit of detection (LOD) of 5.75 ng mL-1 which is sensitive enough to detect active malarial infection. Successful lyophilization and reconstitution of the chemiluminescent substrate, as reported here, can pave the way towards developing an autonomous POCT system implementing chemiluminescence based sandwich ELISA for enhanced sensitivity, portability, and ease-of-use in resource limited settings.

A transparent paper-based platform for multiplexed bioassays by wavelength-dependent absorbance/transmittance.

This communication describes the rational design of a transparent paper-based chemosensing platform for multi-target detection by wavelength-dependent absorbance/transmittance. The platform was successfully applied in the examination of bovine serum albumin (BSA) and cholesterol in serum with a low detection limit of 0.1 µM and 0.1 mM, respectively. With low cost and high sensitivity, the paper-based platform shows great promise for multiplexed bioassays.

Selective recovery of RNAs from bacterial pathogens after their internalization by human host cells.

Selective RNA extractions are required when studying bacterial gene expression within complex mixtures of pathogens and human cells, during adhesion, internalization and survival within the host. New technologies should be developed and implemented to enrich the amount of bacterial RNAs since the majority of RNAs are from the eukaryotic host cells, requiring high read depth coverage to capture the bacterial transcriptomes in dual-RNAseq studies. This will improve our understanding about bacterial adaptation to the host cell defenses, and about how they will adapt to an intracellular life. Here we present an RNA extraction protocol to selectively enrich the lowest bacterial RNA fraction from a mixture of human and bacterial cells, using zirconium beads, with minimal RNA degradation. Zirconium beads have higher capacity to extract bacterial RNAs than glass beads after pathogen internalization. We optimized the beads size and composition for an optimal bacterial lysis and RNA extraction. The protocol was validated on two human cell lines, differentiated macrophages and osteoblasts, with either Gram-positive (Staphylococcus aureus) or -negative (Salmonella typhimurium) bacteria. Relative to other published protocols, yield of total RNA recovery was significantly improved, while host cell infection was performed with a lower bacterial inoculum. Within the host, bacterial RNA recovery yields were about six-fold lower than an RNA extraction from pure bacteria, but the quality of the RNA recovered was essentially similar. Bacterial RNA recovery was more efficient for S. aureus than for S. typhimurium, probably due to their higher protection by the Gram-positive cell walls during the early step of eukaryotic cell lysis. These purified bacterial RNAs allow subsequent genes expression studies in the course of host cell-bacteria interactions.

The Efficiency of Color Space Channels to Quantify Color and Color Intensity Change in Liquids, pH Strips, and Lateral Flow Assays with Smartphones.

Bottom-up, end-user based feed, and food analysis through smartphone quantification of lateral flow assays (LFA) has the potential to cause a paradigm shift in testing capabilities. However, most developed devices do not test the presence of and implications of inter-phone variation. Much discussion remains regarding optimum color space for smartphone colorimetric analyses and, an in-depth comparison of color space performance is missing. Moreover, a light-shielding box is often used to avoid variations caused by background illumination while the use of such a bulky add-on may be avoidable through image background correction. Here, quantification performance of individual channels of RGB, HSV, and LAB color space and ΔRGB was determined for color and color intensity variation using pH strips, filter paper with dropped nanoparticles, and colored solutions. LAB and HSV color space channels never outperformed the best RGB channels in any test. Background correction avoided measurement variation if no direct sunlight was used and functioned more efficiently outside a light-shielding box (prediction errors < 5%/35% for color/color intensity change). The system was validated using various phones for quantification of major allergens (i.e., gluten in buffer, bovine milk in goat milk and goat cheese), and, pH in soil extracts with commercial pH strips and LFA. Inter-phone variation was significant for LFA quantification but low using pH strips (prediction errors < 10% for all six phones compared). Thus, assays based on color change hold the strongest promise for end-user adapted smartphone diagnostics.