Low Sample Volume Research Articles

Experimental infection of bovine spongi-form encephalopathy in guinea pigs: clinical symptoms, pathohistology and immunohistochemistry

Prion diseases are infectious neurodegenerative diseases affecting humans and animals that are invariably fatal and have no currently available prevention or treatment methods. These diseases are also known as transmissible spongiform encephalopathies (TSE), characterized by similar neurological disorders, vacuolization of the neuroparenchyma, accumulation of the pathogenic isoform of the prion protein in it, the absence of an immunological response to the pathogen, and the inevitable death. The most famous of them is the classic bovine spongiform encephalopathy, because. has unprecedented social and economic consequences. C-BSE of cattle is also dangerous for humans, causing one of the forms of Creutzfeldt-Jakob disease, leading to a gradual progression of irreversible C-BSE lesions, leading to the death of a sick person. Laboratory animal models make a huge contribution to the study of the biological properties of prions and the diseases they cause. Thus, mouse models of prion infections have played a major role in deciphering fundamental questions of prion biology and assessing the potential transmissibility of neurodegenerative diseases. However, despite the economic acceptability and other positive properties of this model, it has a number of disadvantages. It produces low-volume tissue samples, and it is very difficult to obtain the required volume of serum and cerebrospinal fluid for many studies. Therefore, the search for and study of the disease in new models of laboratory animals sensitive to bovine C-BSE is of great scientific importance in the study of prion diseases. This work shows that during experimental intracerebral infection of guinea pigs with the causative agent of classical bovine spongiform encephalopathy, they are 100% sensitive to the pathogen. The clinical picture of the disease, developing severe pathomorphological changes in all parts of the brain with vacuolization of neurons and neropil and massive deposition of the prion protein PrPSc are described. Guinea pigs can be a highly sensitive object for laboratory study of the causative agent of bovine spongiform encephalopathy.

Closed EWOD-based low-cost portable thermal detection system for point-of-care applications

Electrowetting-on-Dielectric (EWOD) is a powerful technology for biological and chemical diagnostics. It offers advantages of low sample volume, high sensitivity, high throughput and the possibility of automation. Of special interest is a miniaturization of a thermal detection system by utilizing the EWOD technique since it can minimize effort while reducing cost and the system size. The present study describes the development of a closed EWOD device for droplet actuation that is integrated with thermal cycling and optical diagnostics. Important steps, including sample and reagent transportation, merging and mixing, and color detection, are performed on the integrated platform. The thermal module uses heating as well as Peltier elements and K-type thermocouples with a PID controller to perform precise thermal cycling needed in clinical and medical diagnostics. Experimentally recorded temperature distribution that is realized in the closed EWOD configuration is numerically validated using the COMSOL simulator. The efficacy of the integrated system is experimentally demonstrated by performing Fehling’s test for glucose detection in a glycerin solution. Furthermore, the device is used to demonstrate the transportation of a liquid drop of blood mimicking liquid at a relatively high temperature of 90 °C. The system thus developed is reliable, providing real-time proof-of-concept and the feasibility of building a low-cost point-of-care EWOD device. • A portable closed EWOD based medical diagnostic system with thermal cycling is demonstrated. • Thermal management using heating and cooling element is integrated with the closed EWOD device. • Temperature distribution within the device is measured experimentally and validated against simulation. • Droplet motion is successfully realized over the temperature range studied, providing real-time proof-of-concept. • The system developed is tested by performing Fehling’s test for glucose detection.

Liquid Chromatography-Tandem Mass Spectrometry with Online, In-Source Droplet-Based Phenylboronic Acid Derivatization for Sensitive Analysis of Saccharides.

The ability to identify abnormalities in the body's saccharide profile is a promising means for early disease detection but requires analytical tools capable of detecting saccharides at low concentrations and/or for volume-limited samples. The preferred analysis approach for these compounds, liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS), often lacks sensitivity due to poor ionization efficiency. In this work, we employ a modified electrospray interface-termed contained-electrospray (contained-ESI) to couple accelerated droplet chemistry to conventional LC-MS for the online and automated separation, derivatization, and detection of saccharides. The chromatographic component enables complex sample and mixtures analysis with low sample volume requirements, while the enhanced reaction kinetics afforded by electrosprayed microdroplets facilitates rapid, on-the-fly derivatization to boost sensitivity. Derivatization occurs during ion formation as analytes elute from the column, eliminating the need for superfluous post-column derivatization hardware or complicated benchtop protocols. A grounded coupler was incorporated to shield the LC from the high-voltage ion source, and method conditions were optimized to accommodate the low flow rates preferred for microdroplet reactions. The new LC-contained-ESI-MS/MS platform was demonstrated for the analysis of several mono-, di-, and oligosaccharides using in-source droplet-based phenylboronic acid derivatization. Femtomole limits of detection were achieved for a 1 μL injection, representing sensitivity enhancement of 1-2 orders of magnitude over conventional LC-ESI-MS/MS without derivatization. In addition, isobaric saccharides that are difficult to differentiate by MS alone were easily distinguished. Method precision, accuracy, and linearity were established, and the ability to detect oligosaccharides at trace levels in human urine and plasma was demonstrated.

Microbial community profiling by next-generation DNA sequencing of adenocarcinoma of the prostate with evidence of ochratoxin A producing fungi

BackgroundProstatic carcinomas are a leading cancer and leading cause of mortality in the developed world. The etiology is diverse with underlying patient genetics, environmental factors, and microbial associations. Sequencing DNA for microbes allows the detection of potential disease relationships. ObjectiveTargeted 16S (prokaryotic) and 18S (eukaryotic) rDNA sequencing was performed to map the tumor microbial flora. DesignTwelve patients undergoing elective laparoscopic prostatectomy for biopsy proven adenocarcinoma of the prostate were enrolled. PCR and amplicon based sequencing was conducted; a portion of the sequencing results were confirmed by special stains. SettingPatients were recruited by the urologist were prospectively scheduled for radical prostatectomy by ‘Da Vinci’ robotically assisted procedure in an outpatient setting. Samples were portioned in the hospital surgical suite at the time of prostatectomy. ParticipantsMale patients were requested to enter the study on a first come basis. Outcome Measurement and Statistical Analysis: Average age of the 12 participants was 64.3 years. Results and limitationsDNA reads were detected and by ‘best match’ were identified belonging to Perkinsus, Hydrurus, Diversispora and Funneliformis genera, few samples displayed bacteria. Out of the 12 total patients, 11 patients had detectable DNA sequences matching arbuscular mycorrhizal fungi in the Glomeromycetes Class; Funneliformis mosseae and Diversasporum versiformis. Specific PCR for arbuscular mycorrhizal fungi failed to confirm Glomeromycetes Class; in-depth taxonomic analysis suggests a newer fungal grouping, not falling within an accepted Phylum of fungi. Calcoflour white staining of histological sections confirmed potential fungal markers in all 12 cases. Ochratoxin A antigen was identified by immunofluorescence in all 12 patient samples. The study was limited by the low sample volume and disease free normal controls. ConclusionsFungi may play a significant role in adenocarcinoma of the prostate.

A Direct Infusion Probe for Rapid Metabolomics of Low-Volume Samples.

Targeted and nontargeted metabolomics has the potential to evaluate and detect global metabolite changes in biological systems. Direct infusion mass spectrometric analysis enables detection of all ionizable small molecules, thus simultaneously providing information on both metabolites and lipids in chemically complex samples. However, to unravel the heterogeneity of the metabolic status of cells in culture and tissue a low number of cells per sample should be analyzed with high sensitivity, which requires low sample volumes. Here, we present the design and characterization of the direct infusion probe, DIP. The DIP is simple to build and position directly in front of a mass spectrometer for rapid metabolomics of chemically complex biological samples using pneumatically assisted electrospray ionization at 1 μL/min flow rate. The resulting data is acquired in a square wave profile with minimal carryover between samples that enhances throughput and enables several minutes of uniform MS signal from 5 μL sample volumes. The DIP was applied to study the intracellular metabolism of insulin secreting INS-1 cells and the results show that exposure to 20 mM glucose for 15 min significantly alters the abundance of several small metabolites, amino acids, and lipids.

AACC Guidance Document on the Use of Point-of-Care Testing in Fertility and Reproduction.

The AACC Academy revised the reproductive testing section of the Laboratory Medicine Practice Guidelines: Evidence-Based Practice for Point-of-Care Testing (POCT) published in 2007. A panel of Academy members with expertise in POCT and laboratory medicine was formed to develop guidance for the use of POCT in reproductive health, specifically ovulation, pregnancy, premature rupture of membranes (PROM), and high-risk deliveries. The committee was supplemented with clinicians having Emergency Medicine and Obstetrics/Gynecology training. Key recommendations include the following. First, urine luteinizing hormone (LH) tests are accurate and reliable predictors of ovulation. Studies have shown that the use of ovulation predicting kits may improve the likelihood of conception among healthy fertile women seeking pregnancy. Urinary LH point-of-care testing demonstrates a comparable performance among other ovulation monitoring methods for timing intrauterine insemination and confirming sufficient ovulation induction before oocyte retrieval during in vitro fertilization. Second, pregnancy POCT should be considered in clinical situations where rapid diagnosis of pregnancy is needed for treatment decisions, and laboratory analysis cannot meet the required turnaround time. Third, PROM testing using commercial kits alone is not recommended without clinical signs of rupture of membranes, such as leakage of amniotic fluid from the cervical opening. Finally, fetal scalp lactate is used more than fetal scalp pH for fetal acidosis due to higher success rate and low volume of sample required. This revision of the AACC Academy POCT guidelines provides recommendations for best practice use of POCT in fertility and reproduction.

Homemade Pipette Tip Solid-Phase Extraction for the Simultaneous Determination of 40 Drugs of Abuse in Urine by Liquid Chromatography–Tandem Mass Spectrometry

Pipette tip solid-phase extraction facilitates the handling of low-volume samples and organic solvents in order to achieve more environmentally friendly pre-treatment sample techniques. The use of pipette tip extraction was examined for the quick and simple determination of a heterogeneous group of 40 drugs of abuse and some of their metabolites in urine by liquid chromatography coupled to tandem mass spectrometry. Several parameters were studied and optimized, including those which can affect extraction efficiencies, such as the amount of sorbent and the volumes and number of aspirating/dispensing cycles of the sample and organic solvents. The linear range of this method was between the quantification limit and 75 or 100 ng mL−1. Detection limits between 0.025 and 0.500 ng mL−1 and quantification limits from 0.100 to 1.500 ng mL−1 were achieved, which are adequate to determine the studied compounds in urine from drug users. Finally, in order to prove its suitability in toxicological and forensic analyses, the method was successfully applied to 22 urine specimens from women who were starting a detoxification program. Cocaine was the most frequently detected substance, as its presence or the presence of its main metabolite was found in 86% of the analyzed samples.

Automating DUID methods using robotics for rapid high throughput sample preparation – Section 4 and Section 5A UK Road Traffic Act Compounds in Whole Blood

To fully automate a 96-well plate sample preparation technique using liquid handling robotics for the purpose of significantly increasing sample throughput for whole blood samples taken under the UK Road Traffic Act (RTA). Introduction: demand for analysis of whole blood samples taken under the Road Traffic Act 1988 has increased in recent years due to UK Policing and Government policy on tackling driving under the influence of drugs (DUID). Previous extraction techniques utilised by our laboratory include manual or semi-automated approaches that are lengthy and labour intensive. Both approaches require time consuming human input, to precipitate blood and prepare calibrators and quality controls. Recent developments have sped up chromatographic methods through use of techniques such as ultra-high performance liquid chromatography. However, there has been little progress with speeding up sample preparation and extraction processes, particularly for whole blood to compliment increased LC-MS/MS capacity. Use of 96-well plate formats for protein precipitation and extraction provide a platform that can be readily coupled to automated liquid handling (ALH) instruments. This provides a solution to the increase in demand by allowing for extraction of more samples, with minimal human input, in a much shorter amount of time. Here we present development, validation and implementation of an automated robotic sample preparation method for whole blood for analysis of drugs in RTA cases. Aliquots (200 μL) of blank blood and casework samples were dispensed into a 96-well plate using a robotic pipetting system before being loaded onto a Hamilton ALH. Casework samples and blank blood were spiked with internal standard. Additional blank blood samples were further spiked to produce calibrants and quality controls. The plate was mixed with the addition of a cell lysis solution and the aliquots transferred to a protein/lipid depletion (PPT) plate. A positive pressure manifold was used to draw the solution through the PPT plate into a collection plate where the precipitate was dried. The precipitate was reconstituted and loaded by the Hamilton onto a microelution SPE plate. Following extraction, the samples were reconstituted and the collection plate placed in the LC-MS/MS autosampler for analysis. The LC-MS/MS method consists of a 2.1 mm × 30 mm, 1.7 μm, C18 column running a 4 minute gradient of water and methanol with 0.1% formic acid. Quantitation of 34 compounds (including common drugs of abuse and their metabolites) was successfully validated with CVs ranging from 3.0%–9.0% and accuracies of 85.8%–111.7%. Within batch precision had CVs of < 10.0%. The robotic platform was able to undertake sample pre-treatment and extraction of 40 case samples in 3.5 hours. The method was implemented into casework following UKAS accreditation and in 2021, 12,400 samples were analysed using the method. Introduction of this method to our laboratory has resulted in a significant increase in speed for a sample to be processed and results obtained. Each ALH is capable of extracting two batches per day; equating to 160 case sample versus 28 cases on semi-automated methods. This, along with a reduced sample volume requirement and reduced solvent consumption has had a very beneficial impact in terms of cost, turnaround times and ability to use low sample volume cases. Full automation of sample preparation of whole blood samples coupled with high speed chromatographic techniques can significantly increase throughput, decrease turnaround times and reduce solvent consumption.

Wastewater surveillance for different classes of pharmaceutical drugs: Focus on psychotropic drugs and their metabolites

Wastewater screening analysis for the determination of pharmaceutical drugs belonging to different classes, with particular focus on psychotropic drugs and their metabolites. Wastewater based epidemiology (WBE) recently emerged as an essential and complementary methodology for the evaluation of pharmaceutical and illicit drugs prevalence, by means of quantification of either the parent drugs or their human-specific metabolites in wastewater. Furthermore, water pollution by pharmaceutical drugs is becoming an unavoidable environmental issue of emerging concern, making the water quality monitoring crucial to safeguard its healthy supply to citizens. The most commonly used pharmaceuticals include non-steroidal anti-inflammatory drugs, cardiovascular drugs, anti-depressant and antipsychotic drugs; their Active Pharmaceutical Ingredients (APIs) and some of their metabolites are known to partially survive the conventional process of wastewater treatment. Our study involves a wide screening analysis on wastewater samples entering and leaving various purification plants in the Piedmont region (Italy) to identify the prevalent pharmaceutical drugs and metabolites present in it and so to provide important information about the diffusion of compounds of toxicological interest. A total of 140 target compounds (pharmaceuticals of various therapeutic classes, benzodiazepines and some of their metabolites) were investigated in wastewater samples using only 30 mL aliquots instead of the typical 500 mL reported in the literature. The filtered samples (0.22 μm filters) were extracted and purified by SPE and subsequently 5 μL of the concentrated samples was analyzed with UHPLC-QTOF-HRMS and UHPLC-MS/MS protocols. Thanks to the combined use of the two instruments, a full scan on the exact mass and MS/MS fragmentation data were collected for parent drugs and their main metabolites. 61 out of 119 targeted pharmaceutical drugs and 6 out of 21 targeted metabolites of psychotropic pharmaceutical drugs were detected over a wide concentration interval, ranging from ng/L to μg/L. The highest concentration was 62 μg/L detected for paracetamol. The classes of antidepressants, benzodiazepines, antiepileptics, and antipsychotic required particular attention as they are known to be harmful to aquatic flora and fauna. Among these, citalopram (mean concentration 25.8 ng/mL), lorazepam (mean concentration 32.2 ng/mL), trazodone (mean concentration 17.0 ng/mL) and carbamazepine (mean concentration 134 ng/mL) were detected in both the purifier's affluent and effluent waters. Since some environmental studies have shown that carbamazepine is one of the most frequently detected drugs in wastewater treatment plants, the presence of its main metabolites was explored. In particular, the presence of 10,11-dihydro-10-hydroxycarbamazepine, 10,11-dihydro-10,11-dihydroxycarbamazepine, oxarbamazepine and 10,11-dihydro-10,11-epoxycarbamazepine was revealed. The latter compound (approximately three times more concentrated than carbamazepine) proved to possess antiepileptic properties similar to carbamazepine, possibly producing neurotoxic effects. A reliable wastewater method of analysis involving a low sample volume, its SPE purification, and UHPLC-QTOF-HRMS and UHPLC-MS/MS targeted detection was developed for the simultaneous determination of 119 pharmaceutical drugs and 21 metabolites. The collected data are consistent with those available in the literature and confirm that many pharmaceutical drugs are present in wastewater at a level that pose some risk to flora and fauna, taking into account also the health issues associated with long-term simultaneous exposure to a large number of pharmaceutical products. In conclusion, wastewater surveillance is essential not only to identify the pharmaceutical drugs used in the area but also to monitor the purity of waters. In the future, analyses will be carried out to study the variation in the substances (including NPS) found over time and in the different territories at both intra- and inter-regional levels.

Rapid and inexpensive method of PCR ready DNA isolation from human peripheral blood and saliva

BackgroundThe isolation of nucleic acids is a frequently performed procedure in the molecular biology area. Although several rapid DNA isolation techniques from human peripheral blood and saliva have been developed, there are still some disadvantages – volume, time, cost, and yield are a few notable ones. ObjectiveWe aim to develop a rapid and inexpensive method to isolate high-molecular-weight genomic DNA from human peripheral blood and saliva that can be used for molecular biology experiments. MethodsFive DNA isolation methods with slightly varying protocols were used. High-quality DNA obtained from one specific method was further amplified by PCR and the template with good amplification was further used for performing RFLP and sequencing. ResultsOut of 5 different isolation methods (R1 to R5), DNA obtained from the R4 was of good quality (molecular weight is > 10 kb and 260/280 ratio is 1.89 ± 0.2), which allows successful PCR amplification and good separation in Restriction Fragment Length Polymorphism analysis. Sequencing by the Sanger Sequencing produced a good readable sequence of an amplified fragment from Method R4 DNA. ConclusionIn the present study we have developed a simple, rapid, and cost-effective DNA isolation method, which uses low sample volume and yields good quantity and high-quality product. The DNA obtained is highly fit for molecular genetics research applications.

Comparative Assessment of Filtration- and Precipitation-Based Methods for the Concentration of SARS-CoV-2 and Other Viruses from Wastewater.

ABSTRACTWastewater-based epidemiology (WBE) has been widely used to track levels of SARS-CoV-2 infection in the community during the COVID-19 pandemic. Due to the rapid expansion of WBE, many methods have been used and developed for virus concentration and detection in wastewater. However, very little information is available on the relative performance of these approaches. In this study, we compared the performance of five commonly used wastewater concentration methods for the detection and quantification of pathogenic viruses (SARS-CoV-2, norovirus, rotavirus, influenza, and measles viruses), fecal indicator viruses (crAssphage, adenovirus, pepper mild mottle virus), and process control viruses (murine norovirus and bacteriophage Phi6) in laboratory spiking experiments. The methods evaluated included those based on either ultrafiltration (Amicon centrifugation units and InnovaPrep device) or precipitation (using polyethylene glycol [PEG], beef extract-enhanced PEG, and ammonium sulfate). The two best methods were further tested on 115 unspiked wastewater samples. We found that the volume and composition of the wastewater and the characteristics of the target viruses greatly affected virus recovery, regardless of the method used for concentration. All tested methods are suitable for routine virus concentration; however, the Amicon ultrafiltration method and the beef extract-enhanced PEG precipitation methods yielded the best recoveries. We recommend the use of ultrafiltration-based concentration for low sample volumes with high virus titers and ammonium levels and the use of precipitation-based concentration for rare pathogen detection in high-volume samples.IMPORTANCE As wastewater-based epidemiology is utilized for the surveillance of COVID-19 at the community level in many countries, it is crucial to develop and validate reliable methods for virus detection in sewage. The most important step in viral detection is the efficient concentration of the virus particles and/or their genome for subsequent analysis. In this study, we compared five different methods for the detection and quantification of different viruses in wastewater. We found that dead-end ultrafiltration and beef extract-enhanced polyethylene glycol precipitation were the most reliable approaches. We also discovered that sample volume and physico-chemical properties have a great effect on virus recovery. Hence, wastewater process methods and start volumes should be carefully selected in ongoing and future wastewater-based national surveillance programs for COVID-19 and beyond.

BSCI-02 CSF PROTEOMICS AS A MINIMALLY-INVASIVE STRATEGY FOR DISTINGUISHING BRAIN METASTASES FROM OTHER PRIMARY BRAIN MALIGNANCIES

BACKGROUNDAccurate diagnosis and prognostication of intra-axial brain tumors hinges on invasive brain sampling, which carries risk of morbidity. Minimally invasive sampling of proximal fluids, also known as liquid biopsy, can mitigate this risk. Although the cerebrospinal fluid (CSF) is the ideal liquid biopsy source, the traditionally high volumes required for impactful analyses have deterred progress. The objective of this study was to identify diagnostic and prognostic CSF proteomic signatures in glioblastoma (GBM), brain metastases (BM), and central nervous system lymphoma (CNSL).METHODSCSF samples were retrospectively retrieved from the Penn State Neuroscience Biorepository and profiled using shotgun proteomics with low sample volumes. Proteomic signatures were identified using machine learning classifiers and survival analyses.RESULTSUsing 30µL CSF volumes, we recovered 800 unique peptides across 73 samples [20 normal pressure hydrocephalus (NPH, non-tumor control), 22 GBM, 17 BM, and 14 CNSL]. Externally-validated proteomic-based classifiers identified malignancy with AUROC of 0.94 and distinguished individual tumor entities from others with AUROC ≥0.96. More clinically relevant triplex classifiers, comprised of just 3 peptides, distinguished individual tumor entities with AUROC ≥0.90. Novel biomarkers were identified among the top classifiers, including TFF3 and CACNA2D2, and characterized using single-cell RNA sequencing data. Survival analyses validated previously implicated prognostic signatures, including blood brain barrier disruption.DISCUSSIONReliable classification of intra-axial malignancies using low CSF volumes is feasible, which has ramifications for longitudinal tumor surveillance. Novel biomarkers identified here necessitate future validation. Based on emerging evidence, upfront implantation of CSF reservoirs in brain tumor patients warrants consideration.

Microwave Measurement of Bovine Serum Albumin Solutions Based on High-Q/High-Resolution Resonator

Progressive techniques that enable reagent-free detection and analysis of low-concentration and low-volume chemical samples are of great interest for scientific and technological investigations in biochemical and physiological areas. Highly sensitive microwave-based sensors are promising approaches among such techniques. In this work, we present a novel microwave-based noncontact sensing method to determine the concentration of standard bovine serum albumin (BSA) solution. It is demonstrated that the modal suppression technique can excite a split-ring resonator by two independent microstrip lines with reversed phase, resulting in high quality factor and resolution. The quality factor exceeding <inline-formula xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink"> <tex-math notation="LaTeX">$5.5\times 10^{5}$ </tex-math></inline-formula> with lossy-liquid loaded can be achieved at <inline-formula xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink"> <tex-math notation="LaTeX">$\sim 4.3$ </tex-math></inline-formula> GHz. The obtained high resolution in frequency is <inline-formula xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink"> <tex-math notation="LaTeX">$\sim 20$ </tex-math></inline-formula> kHz for the concentration of BSA from 0 to 1 mg/mL. The measured concentration level ranges from 1 to 200 mg/mL in 0.9% sodium chloride, which covers most commercial standard BSA solutions. An equivalent circuit model is built to study and explain its working principle. Using the fit premeasurement data of standard solutions, the concentration and permittivity of an unknown BSA solution can be estimated.

Fast and ultrafast thermal contrast amplification of gold nanoparticle-based immunoassays

For highly sensitive point-of-care (POC) diagnostics, we explored the limit of thermal contrast amplification (TCA) reading of gold nanoparticles (GNPs/mm2) at test regions in immunoassays. More specifically, we built and compared fast (minute scale) and ultrafast (seconds scale) TCA setups using continuous-wave (CW) and ms pulsed lasers, respectively. TCA improved the limit of detection (LoD) for silica-core gold nanoshells (GNSs) preloaded in nitrocellulose (NC) membrane as model lateral flow immunoassays (LFAs) by 10- to 20-fold over visual reading. While the ultrafast TCA led to higher thermal signals, this came with a twofold loss in LoD vs. fast TCA primarily due to noise within the infrared sensor and a necessity to limit power to avoid burning. To allow higher laser power, and therefore amplification fold, we also explored transparent glass coverslip substrate as a model microfluidic immunoassay (MIA). We found the ultrafast TCA reading of GNS-coated coverslips achieved a maximal signal amplification (57-fold) over visual reading of model LFAs. Therefore, ultrafast TCA-MIA is promising for ultrasensitive and ultrafast diagnostics. Further advantages of using TCA in MIA vs. LFA could include lower sample volume, multiplexed tests, higher throughput, and fast reading. In summary, TCA technology is able to enhance the sensitivity and speed of reading GNPs (GNPs/mm2) within both LFAs and MIAs.

Rapid immunomagnetic co-capture assay for quantification of lung cancer associated exosomes

Exosomes derived from biological fluids have the potential to serve as a biomarker for the early detection of various cancers. However, the lack of reliable enrichment and detection methods posed a challenge for its clinical utility. In this work, we designed a rapid co-capture-based approach for targeted enrichment and detection of lung cancer-derived exosomes from human plasma. This method relies on the formation of a sandwich complex around the exosomes that involves magnetic nanoparticles coupled to CD151 to assist in the immunomagnetic selection of lung-derived exosomes and a secondary detection antibody (CD81) coupled to horseradish peroxidase for signal amplification. The performance of the co-capture method to detect exosomes has been optimized with known exosome concentrations in human plasma and exhibited good linearity (108–105 exosomes mL−1) with a detection limit of 60.4 exosomes μL−1. This study further investigated the potential of the developed assay to differentiate healthy and lung cancer patients using 18 clinical samples by quantifying the CD151+/ CD81+ lung-derived exosomes. In conclusion, this study demonstrated a rapid co-capture-based approach that offers simultaneous isolation and detection of exosomes compatible with low sample volume for detecting lung cancer patients.

Plasmonic Fluor-Enhanced Antigen Arrays for High-Throughput, Serological Studies of SARS-CoV-2.

Serological testing for acute infection or prior exposure is critical for patient management and coordination of public health decisions during outbreaks. Current methods have several limitations, including variable performance, relatively low analytical and clinical sensitivity, and poor detection due to antigenic drift. Serological methods for SARS-CoV-2 detection for the ongoing COVID-19 pandemic suffer from several of these limitations and serves as a reminder of the critical need for new technologies. Here, we describe the use of ultrabright fluorescent reagents, Plasmonic Fluors, coupled with antigen arrays that address a subset of these limitations. We demonstrate its application using patient samples in SARS-CoV-2 serological assays. In our multiplexed assay, SARS-CoV-2 antigens were spotted into 48-plex arrays within a single well of a 96-well plate and used to evaluate remnant laboratory samples of SARS-CoV-2 positive patients. Signal-readout was performed with Auragent Bioscience’s Empower microplate reader, and microarray analysis software. Sample volumes of 1 μL were used. High sensitivity of the Plasmonic Fluors combined with the array format enabled us to profile patient serological response to eight distinct SARS-CoV-2 antigens and evaluate responses to IgG, IgM, and IgA. Sensitivities for SARS-CoV-2 antigens during the symptomatic state ranged between 72.5 and 95.0%, specificity between 62.5 and 100%, and the resulting area under the curve values between 0.76 and 0.97. Together, these results highlight the increased sensitivity for low sample volumes and multiplex capability. These characteristics make Plasmonic Fluor-enhanced antigen arrays an attractive technology for serological studies for the COVID-19 pandemic and beyond.

Aptasensors for full body health checkup

In the last couple of decades, aptamers have been one of the preferred biomolecular recognition elements in biosensors. The platform of aptasensors (aptamer-based biosensors) is demonstrated to solve practical research problems in various areas. Aptamers and biosensors altogether gift a specific, selective, sensitive, affordable, easy to operate, robust, rapid, stable, and low sample volume analytical platform. In diagnostics, numerous review articles are published discussing the detection of various types of non-infectious and infectious diseases using aptasensors, but to the best of our knowledge, no one has reviewed the available information from the perspective of the utility of aptasensors for a full body health checkup. Therefore, to bridge this existing gap in knowledge in terms of the utility of aptasensors for full body health checkup and to provide a new perspective to the aptasensor community, in this review article, we are reviewing a battery of aptasensors, which has been utilized to detect biomarkers that correlate well with an individual's health status. Our motive in this review is to demonstrate the expansion of utility of aptasensors in routine full body checkups. Systematic Evolution of Ligands by Exponential Enrichment (SELEX), design configurations, and various detection methods employed for aptasensors are also discussed. Along with, the existing gap in the field, current challenges, and future of aptamers in diagnostics with concluding remarks are also presented in this review article.

Selective Extraction of Biomolecules Using a Bidirectional Flow Filter.

We present a microfluidic device for selective separation and extraction of molecules based on their diffusivity. The separation relies on electroosmotically driven bidirectional flows in which high-diffusivity species experience a net-zero velocity and lower diffusivity species are advected to a collection reservoir. The device can operate continuously and is suitable for processing low sample volumes. Using several model systems, we show that the extraction efficiency of the system is maintained at more than 90% over tens of minutes with a purity of more than 99%. We demonstrate the applicability of the device to the extraction of genomic DNA from short DNA fragments.

CRISPR-Based Programmable Nucleic Acid-Binding Protein Technology Can Specifically Detect Fatal Tropical Disease-Causing Pathogens.

Diagnostic approaches capable of ultrasensitive pathogen detection from low-volume clinical samples, running without any sophisticated instrument and laboratory setup, are easily field-deployable, inexpensive, and rapid, and are considered ideal for monitoring disease progression and surveillance. However, standard pathogen detection methods, including culture and microscopic observation, antibody-based serologic tests, and primarily polymerase chain reaction (PCR)-oriented nucleic acid screening techniques, have shortcomings that limit their widespread use in responding to outbreaks and regular diagnosis, especially in remote resource-limited settings (RLSs). Recently, clustered regularly interspaced short palindromic repeats (CRISPR)-based programmable technology has emerged to challenge the unmet criteria of conventional methods. It consists of CRISPR-associated proteins (Cas) capable of targeting virtually any specific RNA or DNA genome based on the guide RNA (gRNA) sequence. Furthermore, the discovery of programmable trans-cleavage Cas proteins like Cas12a and Cas13 that can collaterally damage reporter-containing single-stranded DNA or RNA upon formation of target Cas-gRNA complex has strengthened this technology with enhanced sensitivity. Current advances, including automated multiplexing, ultrasensitive single nucleotide polymorphism (SNP)-based screening, inexpensive paper-based lateral flow readouts, and ease of use in remote global settings, have attracted the scientific community to introduce this technology in nucleic acid-based precise detection of bacterial and viral pathogens at the point of care (POC). This review highlights CRISPR-Cas-based molecular technologies in diagnosing several tropical diseases, namely malaria, zika, chikungunya, human immunodeficiency virus and acquired immunodeficiency syndrome (HIV-AIDS), tuberculosis (TB), and rabies.

Analysis of opiates in urine using microextraction by packed sorbent and gas Chromatography- Tandem mass spectrometry

Opiates recreational consumption has always been a concern in society, public health, and in clinical toxicology analysis. The aim of this study was to develop and fully validate an analytical method, which was simple and rapid for the determination of tramadol, codeine, morphine, 6- acetylcodeine, 6-monoacetylmorphine and fentanyl using gas chromatography coupled to tandem mass spectrometry. The procedure includes the use of microextraction by packed sorbent for sample clean-up. A mixed mode sorbent was used, allowing the minimal use of solvents. The method was validated in urine samples, with the ability to detect and quantify all analytes with satisfactory linearity (in the range of 1 – 1000 ng/mL for all analytes, except for fentanyl (10–1000 ng/mL)). Extraction efficiency varied from 17 to 107%, which did not impair sensitivity, taking into account the low LLOQs obtained (1 ng/ mL for all analytes; and 10 ng/mL for fentanyl). The developed procedure proved to be fast, selective, and accurate for use in routine analysis, with a low volume of sample (250 µL).