Journal: Analytical chemistry
A microfluidic device was developed to separate heterogeneous particle or cell mixtures in a continuous flow using acoustophoresis. In this device, two identical surface acoustic waves (SAWs) generated by interdigital transducers (IDTs) propagated toward a microchannel, which accordingly built up a standing surface acoustic wave (SSAW) field across the channel. A numerical model, coupling a piezoelectric effect in the solid substrate and acoustic pressure in the fluid, was developed to provide a better understanding of SSAW-based particle manipulation. It was found that the pressure nodes across the channel were individual planes perpendicular to the solid substrate. In the separation experiments, two side sheath flows hydrodynamically focused the injected particle or cell mixtures into a very narrow stream along the centerline. Particles flowing through the SSAW field experienced an acoustic radiation force that highly depends on the particle properties. As a result, dissimilar particles or cells were laterally attracted toward the pressure nodes at different magnitudes, and were eventually switched to different outlets. Two types of fluorescent microspheres with different sizes were successfully separated using the developed device. In addition, E. coli bacteria pre-mixed in peripheral blood mononuclear cells (PBMCs) were also efficiently isolated using the SSAW-base separation technique. Flow cytometric analysis on the collected samples found that the purity of separated E. coli bacteria was 95.65%.
The recent outbreak of Zika virus (ZIKV) infection in the Americas and its devastating impact on fetal development have prompted WHO to declare the ZIKV pandemic as a Public Health Emergency of International Concern. Rapid and reliable diagnostics for ZIKV are vital since ZIKV-infected individuals display no symptoms or nonspecific symptoms similar to other viral infections. Since immunoassays lack adequate sensitivity and selectivity and are unable to identify active state of infection, molecular diagnostics are an effective means to detect ZIKV soon after infection and throughout pregnancy. We report on a highly sensitive reverse transcription-loop mediated, isothermal amplification (RT-LAMP) assay for rapid detection of ZIKV and on the assay implementation in a simple, easy to use, inexpensive, point of care (POC), disposable cassette that carries out all the unit operations from sample introduction to detection. For thermal control of the cassette, we use a chemically-heated cup without a need for any electrical power. Detection is carried out with leuco crystal violet (LCV) dye by eye, thus eliminating the need for any instrumentation. We demonstrated the utility of our POC diagnostic system by detecting ZIKV in oral samples with sensitivity of 5 plaque-forming units (PFU) in less than 40 min. Our system is particularly suitable for resource poor settings, where centralized laboratory facilities, funds, and trained personnel are in short supply, and for use in doctors' offices and at home.
Earthquakes are lethal natural disasters frequently burying people alive under collapsed buildings. Tracking entrapped humans from their unique volatile chemical signature with hand-held devices would accelerate urban search and rescue (USaR) efforts. Here, a pilot study is presented with compact and orthogonal sensor arrays to detect the breath- and skin-emitted metabolic tracers acetone, ammonia, isoprene, CO2and relative humidity (RH), all together serving as sign of life. It consists of three nanostructured metal-oxide sensors (Si-doped WO3, Si-doped MoO3and Ti-doped ZnO), each specifically tailored at the nanoscale for highly sensitive and selective tracer detection along with commercial CO2and humidity sensors. When tested on humans enclosed in plethysmography chambers to simulate entrapment, this sensor array rapidly detected sub-ppm acetone, ammonia and isoprene concentrations with high accuracies (19, 21 and 3 ppb, respectively) and precision, unprecedented by portable sensors but required for USaR. These results were in good agreement (Pearson’s correlation coefficients ≥ 0.9) with bench-top selective reagent ionization time-of-flight mass spectrometry (SRI-TOF-MS). As a result, an inexpensive sensor array is presented that can be integrated readily into hand-held or even drone-carried detectors for first responders to rapidly screen affected terrain.
The material analyzed in this study is probably the most ancient archaeological solid residue of cheese ever found to date. The sample was collected during the Saqqara Cairo University excavations in the tomb of Ptahmes dated to XIX dynasty. Our biomolecular proteomic characterization of this archaeological sample shows that the constituting material was a dairy product obtained by mixing sheep/goat and cow milk. The interactions for thousands of years with the strong alkaline environment of the incorporating soil rich in sodium carbonate and the desertic conditions did not prevent the identification of specific peptide mark-ers which showed high stability under these stressing conditions. Moreover, the presence of Brucella melitensis has been attested by specific peptide markers providing a direct biomolecular evidence of the presence of this infection in the Ramesside period for which only indirect paleopathological evidence has been so far provided. Finally, it’s worth noting that, although proteomic approaches are successfully and regularly used to characterize modern biological samples, their application in ancient materials is still at an early stage of progress, only few results being reported about ancient food samples. In the absence of previous rel-evant evidences of cheese production and/or use, this study, undoubtedly has a clear added value in different fields of knowledge ranging from archaeometry, anthropology, archaeology, medicine history to the forensic sciences.
In March 2020, the SARS-CoV-2 virus outbreak was declared as a world pandemic by the World Health Organization (WHO). The only measures for controlling the outbreak are testing and isolation of infected cases. Molecular real-time polymerase chain reaction (PCR) assays are very sensitive but require highly equipped laboratories and well-trained personnel. In this study, a rapid point-of-need detection method was developed to detect the RNA-dependent RNA polymerase (RdRP), envelope protein (E), and nucleocapsid protein (N) genes of SARS-CoV-2 based on the reverse transcription recombinase polymerase amplification (RT-RPA) assay. RdRP, E, and N RT-RPA assays required approximately 15 min to amplify 2, 15, and 15 RNA molecules of molecular standard/reaction, respectively. RdRP and E RT-RPA assays detected SARS-CoV-1 and 2 genomic RNA, whereas the N RT-RPA assay identified only SARS-CoV-2 RNA. All established assays did not cross-react with nucleic acids of other respiratory pathogens. The RT-RPA assay’s clinical sensitivity and specificity in comparison to real-time RT-PCR (n = 36) were 94 and 100% for RdRP; 65 and 77% for E; and 83 and 94% for the N RT-RPA assay. The assays were deployed to the field, where the RdRP RT-RPA assays confirmed to produce the most accurate results in three different laboratories in Africa (n = 89). The RPA assays were run in a mobile suitcase laboratory to facilitate the deployment at point of need. The assays can contribute to speed up the control measures as well as assist in the detection of COVID-19 cases in low-resource settings.
We present a proof-of-concept demonstration of an all-printed temporary tattoo-based glucose sensor for noninvasive glycemic monitoring. The sensor represents the first example of an easy-to-wear flexible tattoo-based epidermal diagnostic device combining reverse iontophoretic extraction of interstitial glucose and an enzyme-based amperometric biosensor. In-vitro studies reveal the tattoo sensor’s linear response toward physiologically relevant glucose levels with negligible interferences from common coexisting electroactive species. The iontophoretic-biosensing tattoo platform is reduced to practice by applying the device on human subjects and monitoring variations in glycemic levels due to food consumption. Correlation of the sensor response with that of a commercial glucose meter underscores the promise of the tattoo sensor to detect glucose levels in a noninvasive fashion. Control on-body experiments demonstrate the importance of the reverse iontophoresis operation and validate the sensor specificity. This preliminary investigation indicates that the tattoo-based iontophoresis-sensor platform holds considerable promise for efficient diabetes management and can be extended toward noninvasive monitoring of other physiologically relevant analytes present in the interstitial fluid.
Molecular analysis of exhaled breath aerosol (EBA) with simple procedures represents a key step in clinical and point-of-care applications. Due to the crucial health role, a face mask now is a safety device that helps protect the wearer from breathing in hazardous particles such as bacteria and viruses in the air; thus exhaled breath is also blocked to congregate in the small space inside of the face mask. Therefore, direct sampling and analysis of trace constituents in EBA using a face mask can rapidly provide useful insights into human physiologic and pathological information. Herein, we introduce a simple approach to collect and analyze human EBA by combining a face mask with solid-phase microextraction (SPME) fiber. SPME fiber was inserted into a face mask to form SPME-in-mask that covered nose and mouth for in vivo sampling of EBA, and SPME fiber was then coupled with direct analysis in real-time mass spectrometry (DART-MS) to directly analyze the molecular compositions of EBA under ambient conditions. The applicability of SPME-in-mask was demonstrated by direct analysis of drugs and metabolites in oral and nasal EBA. The unique features of SPME-in-mask were also discussed. Our results showed that this method is enabled to analyze volatile and nonvolatile analytes in EBA and is expected to have a significant impact on human EBA analysis in clinical applications. We also hope this method will inspire biomarker screening of some respiratory diseases that usually required wearing of a face mask in daily life.
Thermal processes are widely used in small molecule chemical analysis and metabolomics for derivatization, vaporization, chromatography, and ionization especially in gas chromatography mass spectrometry (GC/MS). In this study the effect of heating was examined on a set of 64 small molecule standards and, separately, on human plasma metabolites. The samples, either derivatized or underivatized, were heated at three different temperatures (60, 100, and 250°C) at different exposure times (30s, 60s, and 300s). All the samples were analyzed by liquid chromatography coupled to electrospray ionization mass spectrometry (LC/MS) and the data processed by XCMS Online (xcmsonline.scripps.edu). The results showed that heating at an elevated temperature of 100°C had an appreciable effect on both the underivatized and derivatized molecules, and heating at 250°C created substantial changes in the profile. For example, over 40% of the molecular peaks were altered in the plasma metabolite analysis after heating (250°C, 300s) with a significant formation of upregulated, degradation and transformation products. Derivatized samples were similarly affected by thermal degradation. The analysis of the 64 small molecule standards validated the temperature-induced changes observed on the plasma metabolites, where most of the small molecules degraded at elevated temperatures even after minimal exposure times (30s). For example, tri- and di-organophosphates (e.g., adenosine triphosphate and adenosine diphosphate) were readily degraded into a mono-organophosphate (e.g., adenosine monophosphate) during heating. Nucleosides and nucleotides (e.g., inosine and inosine monophosphate) were also found to be transformed into purine derivatives (e.g., hypoxanthine). A newly formed transformation product, oleoyl ethyl amide, was also identified in both the underivatized and derivatized of the plasma metabolites and small molecule standard mixture, and was likely generated from reaction(s) with oleic acid. Overall these analyses show that small molecules and metabolites undergo significant time-sensitive alterations when exposed to elevated temperatures, especially those conditions consistent with GC/MS experiments.
Synthetic cannabinoid receptor agonists (SCRAs), termed “Spice” or “K2”, are molecules that emulate the effects of the active ingredient of marijuana, and they have gained enormous popularity over the past decade. SCRAs are Schedule 1 drugs that are highly prevalent in the U.K. prison system and among homeless populations. SCRAs are highly potent and addictive. With no way to determine the dose/amount at the point-of care, they pose severe health risks to users, including psychosis, stroke, epileptic seizures, and they can kill. SCRAs are chemically diverse, with over a hundred compounds used as recreational drugs. The chemical diversity of SCRA structures presents a challenge in developing detection modalities. Typically, GC-MS is used for chemical identification; however, this cannot be in place in most settings where detection is critical, e.g., in hospital Emergency Departments, in custody suites/prisons, or among homeless communities. Ideally, real time, point-of-care identification of SCRAs is desirable to direct the care pathway of overdoses and provide information for informed consent. Herein, we show that fluorescence spectral fingerprinting can be used to identify the likely presence of SCRAs, as well as provide more specific information on structural class and concentration (∼1 μg mL-1). We demonstrate that that fluorescence spectral fingerprints, combined with numerical modeling, can detect both parent and combusted material, and such fingerprinting is also practical for detecting them in oral fluids. Our proof-of-concept study suggests that, with development, the approach could be useful in a range of capacities, notably in harm reduction for users of Spice/K2.
The monitoring of genetically modified organisms (GMOs) is a primary step of GMO regulation. However, there is presently a lack of effective and high throughput methodologies for specifically and sensitively monitoring most of the commercialized GMOs. Herein, we developed a Multiplex Amplification on a Chip with Readout on an Oligo microarray (MACRO) system specifically for convenient GMO monitoring. This system is composed of a microchip for multiplex amplification and an oligo microarray for the readout of multiple amplicons, containing a total of 91 targets (18 universal elements, 20 exogenous genes, 45 events, and 8 endogenous reference genes) that covers 97.1% of all GM events that have been commercialized up to 2012.We demonstrate that the specificity of MACRO is ~100%, with a limit of detection (LOD) that is suitable for real-world applications. Moreover, the results obtained of simulated complex samples and blind samples with MACRO were 100% consistent with expectations and the results of independently performed real-time PCRs, respectively. Thus, we believe MACRO is the first system that can be applied for effectively monitoring the majority of the commercialized GMOs in a single test.