New Sample Prep Products and Accessories for 2023

[...]instrument-based sample preparation technologies are presented. [...]attention is turned to other sample preparation accessories and supporting technologies. First is the Swift HLB-DPX tips. First is the Thermo Scientific AccelerOme sample preparation platform automating sample preparation for liquid chroma-tography-mass spectrometry (LC-MS) proteomics.

Detection of Leptospira DNA among Post-Mortem Specimens during Covid-19 Pandemic

Intro: Leptospirosis is an emerging zoonosis with a global health concern. In Malaysia, leptospirosis incidence remains significant, since its first gazettement as a compulsorily notifiable disease in 2010. However, the prevalence of this disease among local forensic cases is unknown. Therefore, the present study aimed to determine the frequency of human leptospirosis among post-mortem specimens. Method(s): Archived forensic specimens referred to the Institute for Medical Research (IMR), Malaysia between January 2020 and December 2021 were retrieved. DNA from the specimens were extracted using an automated MagNA Pure 96 instrument and subjected to in-house qPCR targeting LipL32 gene and 16S rRNA gene of the pathogenic group of Leptospira spp. Amplification of RNaseP gene was included as internal amplification control (IAC). Finding(s): A total of 408 forensic specimens from 365 patients were received during the study period. Majority of the specimens were blood (n = 195, 47.8%), followed by tissue (n = 136, 33.3%) and liver (n = 59, 14.5%). Of the tested specimens, 2.2% (n = 9) were positive for leptospiral DNA. These positive specimens belonged to 9 different patients, of which the vast majority were male (n = 8, 88.9%), with an average age of 37.5 years. Conclusion(s): Albeit low detection of leptospiral DNA among forensic specimens in Malaysia, this study highlighted that majority of the positive patients were males of productive age.Copyright © 2023

Atomic spectrometry update: review of advances in the analysis of clinical and biological materials, foods and beverages

This update covers publications from the second half of 2021 to the middle of 2022. Advances in the application of atomic spectrometry techniques to clinical and biological materials, foods and beverages are reviewed in the text, highlighting their key features. Technical details of sample collection and preparation, as well as progresses with analytical techniques are considered and three tables complement the text, summarising details of a larger spectrum of publications. During this period, the trend toward the application of multi-element techniques, such as EDXRFS, ICP-MS and LIBS continued, in particular for food authenticity studies. Triple quadrupole ICP-MS is becoming increasing popular, as it is less affected by interferences, as well as LIBS and XRF, that require minimal sample preparation. However, AAS is still considered a valid alternative for single or a limited number of elements: as in previous years, numerous pre-concentration techniques were presented, some of which explored "greener” reagents. The interest in NPs continued, both as a potential exposure risk and for their application as tags of biological materials, and led to a wider application of spICP-MS. Chromium speciation in food received more attention than usual during this period, providing evidence that the carcinogenic species CrVI was not present. A number of studies covered the application of atomic spectrometry techniques for the indirect determination of biological macromolecules, including an interesting application of LIBS for the rapid detection of the immune response to SARS-CoV-2. © 2023 The Royal Society of Chemistry.

INTEGRATION OF MINIVALVES WITH RNA AMPLIFICATION DEVICE FOR SIMULTANEOUS DETECTION OF SARS-COV-2 AND INFLUENZA VIRUSES

We report a point-of-care (POC) device for simultaneous detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and influenza A viruses. The device carries out sample preparation using ball-based valves for sequential delivery of reagents. A microfluidic paper-based analytical device (µPAD) in the detection unit enables RNA isolation and enrichment, followed by reverse transcription loop-mediated isothermal amplification (RT-LAMP) and colorimetric detection. The device integrates all the necessary steps for the sample preparation, including virus lysis, RNA enrichment and purification of two virus samples. The device enabled simultaneous detection of SARS-CoV-2 and Influenza A N1H1 viruses in 50 min., with limit of detection of 2 and 6 genome equivalents (GEs), respectively. The device was also capable of detecting environmental sample of the two viruses. Copyright © 2022 by ASME.

Analytical approaches for determination of COVID-19 candidate drugs in human biological matrices.

Since the outbreak of the COVID-19 pandemic, the use of antiviral and other available drugs has been considered to combat or reduce the clinical symptoms of patients. In this regard, it would be necessary to choose sensitive and selective analytical techniques for pharmacokinetic and pharmacodynamic studies, monitoring of drug concentration in biological fluids, and determination of the most appropriate dose to achieve the desired effect on the disease. In the present study, the analytical techniques based on spectroscopy and chromatography with different detectors for diagnosis and determination of candidate drugs in the treatment of COVID-19 in human biological fluids are reviewed during the period 2015-2022. Moreover, various sample preparation and extraction techniques, are being used for this purpose, such as protein precipitation (PP), solid-phase extraction (SPE), liquid-liquid extraction (LLE), and QuEChERS (quick, easy, cheap, effective, rugged, and safe) are investigated.

Advances in RT-LAMP for COVID-19 testing and diagnosis.

INTRODUCTION: The SARS-CoV-2 pandemic, and the subsequent limitations on standard diagnostics, has vastly expanded the user base of Reverse Transcription Loop-mediated isothermal Amplification (RT-LAMP) in fundamental research and development. RT-LAMP has also penetrated commercial markets, with emergency use authorizations for clinical diagnosis. AREAS COVERED: This review discusses the role of RT-LAMP within the context of other technologies like RT-qPCR and rapid antigen tests, progress in sample preparation strategies to enable simplified workflow for RT-LAMP directly from clinical specimens, new challenges with primer and assay design for the evolving pandemic, prominent detection modalities including colorimetric and CRISPR-mediated methods, and translational research and commercial development of RT-LAMP for clinical applications. EXPERT OPINION: RT-LAMP occupies a middle ground between RT-qPCR and rapid antigen tests. The simplicity approaches that of rapid antigen tests, making it suitable for point-of-care use, but the sensitivity nears that of RT-qPCR. RT-LAMP still lags RT-qPCR in fundamental understanding of the mechanism, and the interplay between sample preparation and assay performance. Industry is now beginning to address issues around scalability and usability, which could finally enable LAMP and RT-LAMP to find future widespread application as a diagnostic for other conditions, including other pathogens with pandemic potential.

Recent advances in microchip electrophoresis for analysis of pathogenic bacteria and viruses.

Life-threatening diseases, such as hepatitis B, pneumonia, tuberculosis, and COVID-19, are widespread due to pathogenic bacteria and viruses. Therefore, the development of highly sensitive, rapid, portable, cost-effective, and selective methods for the analysis of such microorganisms is a great challenge. Microchip electrophoresis (ME) has been widely used in recent years for the analysis of bacterial and viral pathogens in biological and environmental samples owing to its portability, simplicity, cost-effectiveness, and rapid analysis. However, microbial enrichment and purification are critical steps for accurate and sensitive analysis of pathogenic bacteria and viruses in complex matrices. Therefore, we first discussed the advances in the sample preparation technologies associated with the accurate analysis of such microorganisms, especially the on-chip microfluidic-based sample preparations such as dielectrophoresis and microfluidic membrane filtration. Thereafter, we focused on the recent advances in the lab-on-a-chip electrophoretic analysis of pathogenic bacteria and viruses in different complex matrices. As the microbial analysis is mainly based on the analysis of nucleic acid of the microorganism, the integration of nucleic acid-based amplification techniques such as polymerase chain reaction (PCR), quantitative PCR, and multiplex PCR with ME will result in an accurate and sensitive analysis of microbial pathogens. Such analyses are very important for the point-of-care diagnosis of various infectious diseases.

Multiplexed Ultrasensitive Sample-to-Answer RT-LAMP Chip for the Identification of SARS-CoV-2 and Influenza Viruses.

Prompt on-site diagnosis of SARS-CoV-2 with other respiratory infections would have minimized the global impact of the COVID-19 pandemic through rapid, effective management. However, no such multiplex point-of-care (POC) chip has satisfied a suitable sensitivity of gold-standard nucleic acid amplification tests (NAATs). Here, we present a rapid multiplexed ultrasensitive sample-to-answer LAMP (MUSAL) chip operated by simple LED-driven photothermal amplification to detect six targets from single-swab sampling. First, our MUSAL chip allows ultrafast on-chip sample preparation with ∼500-fold preconcentration at a rate of 1.2 mL min-1 . Second, our chip enables contamination-free amplification using autonomous target elution into on-chip reagents by photothermal activation. Finally, our chip accomplishes multiplexed on-chip diagnostics of SARS-CoV-2 and influenza viruses with a limit of detection (LoD) of 0.5 copies µL-1 . Our rapid, ultra-sensitive, cost-effective sample-to-answer chip with a multiplex capability will allow timely management of various pandemics situations we may face shortly. This article is protected by copyright. All rights reserved.

Pitfalls in Poteomics: Avoiding Problems That Can Occur Before Data Acquisition Begins

The analysis of peptides using liquid chromatography-mass spectrometry (LC-MS) for proteomics applications is powerful and can yield tremendously rich data sets. However, the exquisite sensitivity and global applicability of MS detection also makes it prone to contaminants that can easily and seriously compromise the quality of a data set. A short list of tips and tricks can increase the likelihood of consistently producing good LC-MS data in this context and streamline the troubleshooting process when problems do eventually occur.

Rapid Diagnostic Tests for the Detection of the Four Dengue Virus Serotypes in Clinically Relevant Matrices.

The efficient and accurate diagnosis of dengue, a major mosquito-borne disease, is of primary importance for clinical care, surveillance, and outbreak control. The identification of specific dengue virus serotype 1 (DENV-1) to DENV-4 can help in understanding the transmission dynamics and spread of dengue disease. The four rapid low-resource serotype-specific dengue tests use a simple sample preparation reagent followed by reverse transcription-isothermal recombinase polymerase amplification (RT-RPA) combined with lateral flow detection (LFD) technology. Results are obtained directly from clinical sample matrices in 35 min, requiring only a heating block and pipettes for liquid handling. In addition, we demonstrate that the rapid sample preparation step inactivates DENV, improving laboratory safety. Human plasma and serum were spiked with DENV, and DENV was detected with analytical sensitivities of 333 to 22,500 median tissue culture infectious doses (TCID50)/mL. The analytical sensitivities in blood were 94,000 to 333,000 TCID50/mL. Analytical specificity testing confirmed that each test could detect multiple serotype-specific strains but did not respond to strains of other serotypes, closely related flaviviruses, or chikungunya virus. Clinical testing on 80 human serum samples demonstrated test specificities of between 94 and 100%, with a DENV-2 test sensitivity of 100%, detecting down to 0.004 PFU/µL, similar to the sensitivity of the PCR test; the other DENV tests detected down to 0.03 to 10.9 PFU/µL. Collectively, our data suggest that some of our rapid dengue serotyping tests provide a potential alternative to conventional labor-intensive RT-quantitative PCR (RT-qPCR) detection, which requires expensive thermal cycling instrumentation, technical expertise, and prolonged testing times. Our tests provide performance and speed without compromising specificity in human plasma and serum and could become promising tools for the detection of high DENV loads in resource-limited settings. IMPORTANCE The efficient and accurate diagnosis of dengue, a major mosquito-borne disease, is of primary importance for clinical care, surveillance, and outbreak control. This study describes the evaluation of four rapid low-resource serotype-specific dengue tests for the detection of specific DENV serotypes in clinical sample matrices. The tests use a simple sample preparation reagent followed by reverse transcription-isothermal recombinase polymerase amplification (RT-RPA) combined with lateral flow detection (LFD) technology. These tests have several advantages compared to RT-qPCR detection, such as a simple workflow, rapid sample processing and turnaround times (35 min from sample preparation to detection), minimal equipment needs, and improved laboratory safety through the inactivation of the virus during the sample preparation step. The low-resource formats of these rapid dengue serotyping tests have the potential to support effective dengue disease surveillance and enhance the diagnostic testing capacity in resource-limited countries with both endemic dengue and intense coronavirus disease 2019 (COVID-19) transmission.

Analytical methods for the determination of antidepressants, antipsychotics, benzodiazepines and their metabolites through wastewater-based epidemiology

The wastewater-based epidemiology approach, popular for estimating illicit drug use, has been used to evaluate lifestyle habits such as alcohol, tobacco and caffeine consumption, health biomarkers including pharmaceuticals, plasticizers and flame retardants, and recently to track SARS-CoV-2 at the population level. Equally, the number of WBE studies investigating psychoactive pharmaceuticals such as antidepressants, antipsychotics and benzodiazepines has also increased, which can be connected to the overall growth of psychological disorders worldwide. This review aims to discuss novelties in sampling techniques and analytical methodologies, including sample preparation and analysis, developed for estimating the consumption of psychoactive pharmaceuticals in defined populations. Seventy-four peer-reviewed studies monitoring psychoactive pharmaceutical consumption published since 2010 have been systematically reviewed. Its findings show that a broad range of bioanalytical methodologies is used to simultaneously measure several antidepressants, antipsychotics, benzodiazepines, and their metabolites from influent wastewater samples in low concentrations and different time periods. The application of WBE commenting on the temporal and spatial variations worldwide, showing widespread consumption, is also discussed. Despite much progress and excellent studies, there remains a need for research, and deeper knowledge is needed to reduce method uncertainty, especially since excretion rates, their transformation, and in-sewer and in-sample stability for many psychoactive pharmaceutical biomarkers are not available.

Self-directed molecular diagnostics (SdMDx) system for COVID-19 via one-pot processing.

Rapid, sensitive, and specific detection of the severe acute respiratory syndrome coronavirus (SARS-CoV)- 2 during early infection is pivotal in controlling the spread and pathological progression of Coronavirus Disease 2019 (COVID-19). Thus, highly accurate, affordable, and scalable point-of-care (POC) diagnostic technologies are necessary. Herein, we developed a rapid and efficient self-directed molecular diagnostic (SdMDx) system for SARS-CoV-2. This system combines the sample preparation step, including virus enrichment and extraction processes, which involve dimethyl suberimidate dihydrochloride and diatomaceous earth functionalized with 3-aminopropyl(diethoxy)methylsilane, and the detection step using loop-mediated isothermal amplification-lateral flow assay (LAMP-LFA). Using the SdMDx system, SARS-CoV-2 could be detected within 47 min by hand without the need for any larger instruments. The SdMDx system enabled detection as low as 0.05 PFU in the culture fluid of SARS-CoV-2-infected VeroE6 cells. We validated the accuracy of the SdMDx system on 38 clinical nasopharyngeal specimens. The clinical utility of the SdMDx system for targeting the S gene of SARS-CoV-2 showed 94.4% sensitivity and 100% specificity. This system is more sensitive than antigen and antibody assays, and it minimizes the use of complicated processes and reduces contamination risks. Accordingly, we demonstrated that the SdMDx system enables a rapid, accurate, simple, efficient, and inexpensive detection of SARS-CoV-2 at home, in emergency facilities, and in low-resource sites as a pre-screening platform and POC testing through self-operation and self-diagnosis.

Osmoprocessor for enabling highly sensitive biomarker detection via lateral flow assays

Lateral flow assays are low-cost devices suitable for point-of-care testing, particularly in low-resource settings. However, some of the lateral flow assays exhibit limited diagnostic utility because the assays can only sample <100uL specimen and the biomarker concentration is significantly lower than the assay detection limit, which compromise the sensitivity. To address the challenge, we have developed the osmoprocessor that statically and spontaneously concentrated biomarkers via osmosis. The specimen in the device interfaces with the aqueous polymer solution via a dialysis membrane. The polymer solution induces an osmotic pressure difference that extracts water from the specimen, while the membrane retains the biomarkers. The evaluation demonstrated that osmosis induced by various water-soluble polymers efficiently extracted water, ca. 15 mL/hr. The water transport kinetics can be adjusted by varying polymer molecular weights and mass concentrations. The osmoprocessor concentrated the specimens to improve the lateral flow assays' detection limits for the model analytes-human chorionic gonadotropin and SARS-CoV-2 nucleocapsid protein. The device processed a 10 mL specimen into a 100uL concentrated sample. Then, the lateral flow assays detected the corresponding biomarkers in the concentrated specimens. The test band intensities of the assays with the concentrated specimens were very similar to the reference assays with 100-fold concentrations. The mass spectrometry analysis estimated the SARSCoV- nucleocapsid protein concentration increased ca. 200-fold after the osmosis. With its simplicity and flexibility, this device demonstrates a great potential to be utilized in conjunction with the existing lateral flow assays for enabling highly sensitive detection of dilute target analytes.

A CENTRIFUGAL MICROFLUIDIC METHOD FOR ENRICHMENT AND ENZYMATIC EXTRACTION OF SARS-COV-2 RNA FROM CLINICAL SAMPLES

The requirement for diversification of methods for sample preparation of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) is increasingly important to circumvent analysis bottlenecks and continue widespread surveillance testing. We report a centrifugally-driven microfluidic platform for automated affinity nanoparticle enrichment and enzymatic RNA extraction of SARS-CoV-2. The microfluidic disc (μCD) and accompanying mechatronic system(s) are capable of sample preparation from up to six patient samples simultaneously and under 20 minutes. © 2021 MicroTAS 2021 - 25th International Conference on Miniaturized Systems for Chemistry and Life Sciences. All rights reserved.

MAGNETOFLUIDICS-ENABLED POINT-OF-CARE SARS-COV-2 DIAGNOSTICS

As the SARS-CoV-2 virus continues to mutate, global eradication of infections is unlikely, and COVID-19 is predicted to become a seasonal or endemic disease like influenza. Widespread detection of variant strains will be critical to inform policy decisions to mitigate further spread, and post-pandemic multiplexed screening of respiratory viruses will be necessary to properly manage patients presenting with similar respiratory symptoms. We have developed a portable, magnetofluidic platform for multiplexed PCR testing in <30 min. Cartridges were designed for multiplexed detection of SARS-CoV-2 with either distinctive variant mutations or with Influenza A and B and tested with clinical samples. © 2021 MicroTAS 2021 - 25th International Conference on Miniaturized Systems for Chemistry and Life Sciences. All rights reserved.

SURFACE TENSION-MEDIATED PROCESSING OF WASTEWATER SAMPLES FOR SARS-CoV-2

Wastewater testing for SARS-CoV-2 has emerged as a promising tool for disease surveillance in aggregate populations. We present a novel method to rapidly extract, concentrate, and amplify viral RNA from wastewater using Exclusion-based Sample Preparation (ESP) and RT-PCR. This technology identified potential outbreaks of SARS-CoV-2 at University of Kentucky dormitories, resulting in targeted clinical testing and quarantine procedures. © 2021 MicroTAS 2021 - 25th International Conference on Miniaturized Systems for Chemistry and Life Sciences. All rights reserved.

Robot-based Sample Tube Handling for Automated Pre- and Post-processing of SARS-CoV-2 PCR Tests

The SARS-CoV-2 pandemic worldwide has led to millions of infections with partly severe complications and death. The diagnosis is based on a PCR test of a saliva sample and is carried out in specialised laboratories. While the actual PCR analysis is usually automated, sample preparation and post-processing often involve manual handling steps. The paper presents an approach for robot-based pre- and post-processing of sample tubes. A seven-axis industrial robot is equipped with a multifunctional gripper and enabled for the application. In addition, machine-learning based image processing solutions are be integrated to increase flexibility and robustness. The implementation shows the feasibility of the approach in principle. The achieved cycle time does not fulfil the set requirements completely, but the implementation provides numerous approaches for optimisation. © VDE VERLAG GMBH ∙ Berlin ∙ Offenbach.

AN INTEGRATED RT-LAMP AND CRISPR ASSAY FOR NUCLEIC ACID DETECTION IN A SINGLE MICROFLUIDIC CHIP

We will present a microfluidic assay to detect SARS-CoV-2 RNA from nasopharyngeal swab samples. Our method leverages isotachophoresis (ITP) to integrate sample preparation, RT-LAMP, and CRISPR-based nucleic acid detection in an automatable chip. For the first time, we use ITP to purify, pre-concentrate and isothermally amplify target nucleic acids into a ~1 µL reaction volume on-chip. The device then transitions LAMP amplicons into an on-chip zone containing Cas12-gRNA complexes and reporter molecules to measure target-activated CRISPR activity. We will use our method to automatically detect COVID-19 from nasopharyngeal swab samples. © 2021 MicroTAS 2021 - 25th International Conference on Miniaturized Systems for Chemistry and Life Sciences. All rights reserved.

Advances in Nucleic Acid Amplification-Based Microfluidic Devices for Clinical Microbial Detection

Accurate and timely detection of infectious pathogens is urgently needed for disease treatment and control of possible outbreaks worldwide. Conventional methods for pathogen detection are usually time-consuming and labor-intensive. Novel strategies for the identification of pathogenic nucleic acids are necessary for practical application. The advent of microfluidic technology and microfluidic devices has offered advanced and miniaturized tools to rapidly screen microorganisms, improving many drawbacks of conventional nucleic acid amplification-based methods. In this review, we summarize advances in the microfluidic approach to detect pathogens based on nucleic acid amplification. We survey microfluidic platforms performing two major types of nucleic acid amplification strategies, namely, polymerase chain reaction (PCR) and isothermal nucleic acid amplification. We also provide an overview of nucleic acid amplification-based platforms including studies and commercialized products for SARS-CoV-2 detection. Technologically, we focus on the design of the microfluidic devices, the selected methods for sample preparation, nucleic acid amplification techniques, and endpoint analysis. We also compare features such as analysis time, sensitivity, and specificity of different platforms. The first section of the review discusses methods used in microfluidic devices for upstream clinical sample preparation. The second section covers the design, operation, and applications of PCR-based microfluidic devices. The third section reviews two common types of isothermal nucleic acid amplification methods (loop-mediated isothermal amplification and recombinase polymerase amplification) performed in microfluidic systems. The fourth section introduces microfluidic applications for nucleic acid amplification-based detection of SARS-CoV-2. Finally, the review concludes with the importance of full integration and quantitative analysis for clinical microbial identification.

New Gas Chromatography Products for 2021-2022

I am pleased to present our annual review of new products in gas chromatography (GC), introduced between spring 2021 and spring 2022. With the Covid-19 pandemic still impacting travel and conferences, the 72nd meeting of the Pittsburgh Conference on Analytical Chemistry and Applied Spectroscopy (Pittcon) from 8-12 March 2022, originally scheduled to take place in Atlanta, Georgia, USA, was cancelled at the last minute. Although many of the technical presentations were conducted virtually, there was not sufficient time to re-tool the exposition, so this review was conducted virtually for the second in a row.