scholarly journals Multiplexed Detection of Sepsis Markers in Whole Blood using Nanocomposite Coated Electrochemical Sensors

2020 ◽  
Author(s):  
Uroš Zupančič ◽  
Pawan Jolly ◽  
Pedro Estrela ◽  
Despina Moschou ◽  
Donald E. Ingber
Keyword(s):  
Electrochemical Detection ◽  
Whole Blood ◽  
Electrochemical Sensors ◽  
Point Of Care ◽  
Sample Volume ◽  
Nanocomposite Coating ◽  
Cross Reactivity ◽  
Detection Methods ◽  
Clinical Samples ◽  
Undiluted Serum

ABSTRACTSepsis is a leading cause of mortality worldwide that is difficult to diagnose and manage because this requires simultaneous analysis of multiple biomarkers. Electrochemical detection methods could potentially provide a way to accurately quantify multiple sepsis biomarkers in a multiplexed manner as they have very low limits of detection and require minimal sensor instrumentation; however, affinity-based electrochemical sensors are usually hampered by biological fouling. Here we describe development of an electrochemical detection platform that enables detection of multiple sepsis biomarkers simultaneously by incorporating a recently developed nanocomposite coating composed of crosslinked bovine serum albumin containing a network of reduced graphene oxide nanoparticles that prevents biofouling. Using nanocomposite coated planar gold electrodes, we constructed a procalcitonin sensor and demonstrated sensitive PCT detection in undiluted serum and clinical samples, as well as excellent correlation with a conventional ELISA (adjusted r2 = 0.95). Sensors for two additional sepsis biomarkers — C-reactive protein and pathogen-associated molecular patterns — were developed on the same multiplexed platform and tested in whole blood. Due to the excellent antifouling properties of the nanocomposite coating, all three sensors exhibited specific responses within the clinically significant range without any cross-reactivity in the same channel with low sample volume. This platform enables sensitive simultaneous electrochemical detection of multiple analytes in human whole blood, which can be expanded further to any target analyte with an appropriate antibody pair or capturing probe, and thus, may offer a potentially valuable tool for development of clinical point-of-care diagnostics.GRAPHICAL ABSTRACT

Comparison of 2 glucose analytical methodologies in immature Kemp’s ridley sea turtles: dry chemistry of plasma versus point-of-care glucometer analysis of whole blood

2021 ◽  
pp. 104063872110018
Author(s):  
Justin R. Perrault ◽  
Michael D. Arendt ◽  
Jeffrey A. Schwenter ◽  
Julia L. Byrd ◽  
Kathryn A. Tuxbury ◽  
...  
Keyword(s):  
Whole Blood ◽  
Point Of Care ◽  
Sea Turtles ◽  
Sample Volume ◽  
Specific Reference ◽  
Volume Data ◽  
Analytical Methodology ◽  
Glucose Determination ◽  
Kemp's Ridley Sea Turtles ◽  
Kemp's Ridley

Blood glucose measurements provide important diagnostic information regarding stress, disease, and nutritional status. Glucose analytical methodologies include dry chemistry analysis (DCA) of plasma and point-of-care (POC) glucometer analysis of whole blood; however, these 2 methods differ in cost, required sample volume, and processing time. Because POC glucometers use built-in equations based on features of mammalian blood to convert whole blood measurements to plasma equivalent units, obtained glucose data must be compared and validated using gold-standard chemistry analytical methodology in reptiles. For in-water, trawl-captured, immature Kemp’s ridley sea turtles ( Lepidochelys kempii) from Georgia, USA, we observed significant, positive agreement between the 2 glucose determination methods; however, the glucometer overestimated glucose concentrations by 1.4 mmol/L on average in comparison to DCA and produced a wider range of results. The discordance of these results suggests that POC glucometer glucose data should be interpreted in the context of methodology- and brand-specific reference intervals along with concurrent packed cell volume data.

scholarly journals Evaluation of mobile real-time polymerase chain reaction tests for the detection of severe acute respiratory syndrome coronavirus 2

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Chukwunonso Onyilagha ◽  
Henna Mistry ◽  
Peter Marszal ◽  
Mathieu Pinette ◽  
Darwyn Kobasa ◽  
...  
Keyword(s):  
Severe Acute Respiratory Syndrome ◽  
Real Time ◽  
Point Of Care ◽  
Middle Eastern ◽  
Turnaround Time ◽  
Cross Reactivity ◽  
Clinical Samples ◽  
Diarrhea Virus ◽  
Highly Sensitive ◽  
Transmissible Gastroenteritis

AbstractThe coronavirus disease 2019 (COVID-19) pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), calls for prompt and accurate diagnosis and rapid turnaround time for test results to limit transmission. Here, we evaluated two independent molecular assays, the Biomeme SARS-CoV-2 test, and the Precision Biomonitoring TripleLock SARS-CoV-2 test on a field-deployable point-of-care real-time PCR instrument, Franklin three9, in combination with Biomeme M1 Sample Prep Cartridge Kit for RNA 2.0 (M1) manual extraction system for rapid, specific, and sensitive detection of SARS-COV-2 in cell culture, human, and animal clinical samples. The Biomeme SARS-CoV-2 assay, which simultaneously detects two viral targets, the orf1ab and S genes, and the Precision Biomonitoring TripleLock SARS-CoV-2 assay that targets the 5′ untranslated region (5′ UTR) and the envelope (E) gene of SARS-CoV-2 were highly sensitive and detected as low as 15 SARS-CoV-2 genome copies per reaction. In addition, the two assays were specific and showed no cross-reactivity with Middle Eastern respiratory syndrome coronavirus (MERS-CoV), infectious bronchitis virus (IBV), porcine epidemic diarrhea virus (PEDV), transmissible gastroenteritis (TGE) virus, and other common human respiratory viruses and bacterial pathogens. Also, both assays were highly reproducible across different operators and instruments. When used to test animal samples, both assays equally detected SARS-CoV-2 genetic materials in the swabs from SARS-CoV-2-infected hamsters. The M1 lysis buffer completely inactivated SARS-CoV-2 within 10 min at room temperature enabling safe handling of clinical samples. Collectively, these results show that the Biomeme and Precision Biomonitoring TripleLock SARS-CoV-2 mobile testing platforms could reliably and promptly detect SARS-CoV-2 in both human and animal clinical samples in approximately an hour and can be used in remote areas or health care settings not traditionally serviced by a microbiology laboratory.

scholarly journals Analytical and Clinical Evaluation of the NOWDiagnostics ADEXUSDx Human Chorionic Gonadotropin Test Using Whole Blood

2016 ◽  
Vol 1 (1) ◽  
pp. 67-76 ◽  
Author(s):  
Robert D Nerenz ◽  
Jennifer R Bell ◽  
Nancy Montes de Oca ◽  
Joann Short ◽  
Theresa Mims ◽  
...  
Keyword(s):  
Chorionic Gonadotropin ◽  
Human Chorionic Gonadotropin ◽  
Whole Blood ◽  
Point Of Care ◽  
False Negative ◽  
Sample Volume ◽  
High Dose ◽  
Control Line ◽  
Intermediate Range ◽  
Pregnancy Status

Abstract Background Point-of-care (POC) urine qualitative human chorionic gonadotropin (hCG) devices are used to rapidly assess pregnancy status, but many of these devices are susceptible to false-negative results caused by increased concentrations of hCG β core fragment (hCGβcf) that does not contain hCGβcf. Methods Purified hCG was added to hCG-negative heparinized whole blood to generate samples with known hCG concentrations, and the resulting samples were used to evaluate device sensitivity, low-end reproducibility, high-dose hook effect, intermediate range performance, acceptable sample volume, acceptable hematocrit range, and lot-to-lot variation. Device performance was also prospectively evaluated in 40 pregnant and 40 nonpregnant women aged 18–44 years in a hospital-based clinic or an academic hospital emergency department. Results All device observations were positive using a whole blood sample containing a plasma hCG concentration of 2.2 × 106 IU/L, and all device observations were positive from18 IU/L to 1.2 × 103 IU/L and from 2.5 × 104 IU/L to 2.2 × 106 IU/L. Three invalid results were observed in the intermediate range because of decreased control line intensity. The minimum sample volume was 30 μL, and maximum hematocrit was 46%. In 40 pregnant and 40 nonpregnant women aged 18–44 years, the device generated 100% concordance with urine qualitative and plasma quantitative test results. Conclusions The ADEXUSDx™ hCG test demonstrates acceptable performance for the determination of pregnancy status using capillary fingerstick samples.

scholarly journals OC 8173 RAPID DETECTION OF MYCOBACTERIUM ULCERANS BY RECOMBINASE POLYMERASE AMPLIFICATION

2019 ◽  
Vol 4 (Suppl 3) ◽  
pp. A2.1-A2
Author(s):  
Michael Frimpong ◽  
Hubert Ahor ◽  
Francisca Sarpong ◽  
Ken Laing ◽  
Mark Wansbrough-Jones ◽  
...  
Keyword(s):  
Sensitivity And Specificity ◽  
Point Of Care ◽  
Clinical Signs ◽  
Buruli Ulcer ◽  
Cross Reactivity ◽  
Current Control ◽  
Mycobacterium Ulcerans ◽  
Recombinase Polymerase Amplification ◽  
Clinical Samples ◽  
Clinical Sensitivity

BackgroundThere are no primary measures to prevent people from contracting Buruli ulcer, mainly due to poor understanding of its epidemiology. The current control strategy emphasises early diagnosis and prompt treatment, with the goal of avoiding the complications associated with advanced stages of the disease. There is no diagnostic test for the disease appropriate for use at the primary health care level where most cases are detected and treated. Diagnosis based on clinical signs is unreliable in inexperienced hands and complicated by infections that have similar presentations. This study was to develop and evaluate the use of recombinase polymerase amplification (RPA) assay for the detection of Mycobacterium ulcerans at the point of patient care.MethodsA specific fragment of IS2404 of M. ulcerans was amplified in 15 min at a constant temperature of 42°C, using the RPA assay and analysed on a portable fluorometre. The’method was tested for sensitivity and specificity with molecular standard of IS2404 DNA fragment, various M.’ulcerans strains, other mycobacteria and environmentally associated bacteria. Additionally, the assay performance as a diagnostic tool was tested with archived DNA from symptomatic patients. All results were compared with that of a highly sensitive IS2404 PCR.ResultsThe detection limit was 50 copies of IS2404 in 15 min using plasmid standard and 125 fg with genomic Mu DNA equivalent 25 genomic copies. The assay was highly specific in detecting all strains of M. ulcerans with no observed cross reactivity with other mycobacteria and common skin colonising bacteria. The clinical sensitivity and specificity of the BU-RPA assay using clinical samples was 86% and 100% respectively.ConclusionWe have developed a real-time isothermal RPA assay for the detection of M. ulcerans as a cheaper alternative to PCR. Combining this assay with a simple extraction protocol will maximise its use as point-of-care test for Buruli ulcer.

scholarly journals Ultrarapid, Ultrasensitive One-Step Kinetic Immunoassay for C-Reactive Protein (CRP) in Whole Blood Samples: Measurement of the Entire CRP Concentration Range with a Single Sample Dilution

2002 ◽  
Vol 48 (2) ◽  
pp. 269-277 ◽  
Author(s):  
Piia Tarkkinen ◽  
Tom Palenius ◽  
Timo Lövgren
Keyword(s):  
Whole Blood ◽  
Solid Phase ◽  
Point Of Care ◽  
High Sensitivity ◽  
Clinical Samples ◽  
Cardiac Complications ◽  
Fluorescence Signal ◽  
C Reactive Protein ◽  
Reactive Protein ◽  
One Step

Abstract Background: Recently, measurement of very low concentrations of C-reactive protein (CRP) has gained popularity as a potential new means for predicting the risk of future cardiac complications. In this study, we demonstrate the feasibility of a kinetic, one-step microparticle assay for quantitative determination of extremely low and high CRP concentrations in the limited timeframe typical for point-of-care testing. Methods: A noncompetitive, kinetic CRP immunoassay was developed that uses individual, porous microparticles as the solid phase. The microparticles were covalently coated with a monoclonal capture antibody, and the monoclonal detection antibody was labeled with europium. The one-step binding reaction was stopped by washing after 2 min of incubation, and the fluorescence signal of individual particles was measured. Results: The analytical detection limit (mean of zero calibrator + 3 SD) was 0.00016 mg/L CRP. Clinical samples were diluted 400-fold before assay to cover the CRP concentration range of 0.064–1200 mg/L. The assay correlated well with the Dade Behring N High Sensitivity CRP assay (for 0–10 mg/L, r = 0.969, Sy|x = 0.68, n = 54; for 0–350 mg/L, r = 0.969, Sy|x = 11.7, n = 100). The within- and between-run CVs based on calculated concentrations were, respectively, 9–16% and 14% at 0.11 mg/L, 4.5–12% and 8.2% at 4.2 mg/L, and 3.5–6.3% and 4.4% at 105 mg/L, with a CV <15% at 0.2 mg/L and above. Conclusions: Use of the kinetic microparticle approach combined with time-resolved fluorometry allows ultrasensitive quantification of CRP in whole blood in 2 min with a linear assay range spanning more than four orders of magnitude.

scholarly journals Electrochemical DNA Detection Methods to Measure Circulating Tumour DNA for Enhanced Diagnosis and Monitoring of Cancer

Proceedings ◽  
2020 ◽  
Vol 60 (1) ◽  
pp. 15
Author(s):  
Bukola Attoye ◽  
Matthew Baker ◽  
Chantevy Pou ◽  
Fiona Thomson ◽  
Damion K. Corrigan
Keyword(s):  
Point Of Care ◽  
Low Cost ◽  
Detection Methods ◽  
Clinical Samples ◽  
Label Free ◽  
Ambient Conditions ◽  
Liquid Biopsies ◽  
Current Time ◽  
Electrochemical Approach ◽  
Label Free Detection

Liquid biopsies are becoming increasingly important as a potential replacement for existing biopsy procedures which can be invasive, painful and compromised by tumour heterogeneity. This paper reports a simple electrochemical approach tailored towards point-of-care cancer detection and treatment monitoring from biofluids using a label-free detection strategy. The mutations under test were the KRAS G12D and G13D mutations, which are both important in the development and progression of many human cancers and which have a presence that correlates with poor outcomes. These common circulating tumour markers were investigated in clinical samples and amplified by standard and specialist PCR methodologies for subsequent electrochemical detection. Following pre-treatment of the sensor to present a clean surface, DNA probes developed specifically for detection of the KRAS G12D and G13D mutations were immobilized onto low-cost carbon electrodes using diazonium chemistry and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride/N-hydroxysuccinimide coupling. Following the functionalisation of the sensor, it was possible to sensitively and specifically detect a mutant KRAS G13D PCR product against a background of wild-type KRAS DNA from the representative cancer sample. Our findings give rise to the basis of a simple and very low-cost system for measuring ctDNA biomarkers in patient samples. The current time to result of the system was 3.5 h with considerable scope for optimisation, and it already compares favourably to the UK National Health Service biopsy service where patients can wait weeks for their result. This paper reports the technical developments we made in the production of consistent carbon surfaces for functionalisation, assay performance data for KRAS G13D and detection of PCR amplicons under ambient conditions.

scholarly journals Development of an Improved Whole Blood Assay for Diagnosis of Latent and Active Tuberculosis Cases

2021 ◽  
pp. 197-209
Author(s):  
Rupam R. Nashine ◽  
Amit R. Nayak ◽  
Aliabbas Husain ◽  
Gargi D. Mudey ◽  
Hatim F. Daginawala ◽  
...  
Keyword(s):  
Whole Blood ◽  
Cross Reactivity ◽  
Clinical Samples ◽  
Interferon Gamma Release Assay ◽  
Antigenic Peptides ◽  
Whole Blood Assay ◽  
Blood Assay ◽  
Variable Sensitivity ◽  
Latent Tb Infection

Background: Latent TB infection (LTBI) is an infection where the presence of disease causing organism M. tuberculosis is there without any sign and symptoms of the disease hence mostly remains undiagnosed, though Tuberculin skin test (TST) and Interferon Gamma Release Assay (IGRA) were used to diagnose the LTBI. They have their limitations, TST gives major cross-reactivity with BCG vaccine and gives inaccurate results in individuals who have taken BCG and IGRA are very costly and variable sensitivity is repeated in various populations hence the modifications are needed in the IGRA for proper diagnosis of LTBI. Objectives: In the proposed study we aimed to develop an improved whole blood assay                    towards a diagnosis of latent and active TB infection as an alternative to the Quantiferon QFT assay Methodology: Synthetic antigenic peptides against latency specific antigens will be designed and synthesized. Designed peptides will be screened for LTBI specific cytokine by in-vitro experiments. Development & production of Whole assay using selected peptides evaluation of developed assay through ELISA in clinical samples. Expected Results: Latent specific peptides will be identified and peptide-based whole blood assay for detection and diagnosis of tuberculosis will be developed as an indigenous alternative for the existing QFT assay. Conclusion: An improved whole blood assay will be developed for screening of LTBI in the Indian population.

scholarly journals Quantification of malaria antigens PfHRP2 and pLDH by quantitative suspension array technology in whole blood, dried blood spot and plasma

2019 ◽  
Author(s):  
Xavier Martiáñez-Vendrell ◽  
Alfons Jiménez ◽  
Ana Vásquez ◽  
Ana Campillo ◽  
Sandra Incardona ◽  
...  
Keyword(s):  
Whole Blood ◽  
Point Of Care ◽  
External Validation ◽  
Simultaneous Detection ◽  
Parasite Clearance ◽  
Dried Blood Spots ◽  
Clinical Samples ◽  
Lower Sensitivity ◽  
Suspension Array ◽  
Array Technology

ABSTRACTBackgroundMalaria diagnostics by rapid diagnostic tests (RDTs) relies primarily on the qualitative detection of Plasmodium falciparum histidine-rich protein 2 (PfHRP2) and Plasmodium sp lactate dehydrogenase (pLDH). As novel RDTs with increased sensitivity are being developed and implemented as point of care diagnostics, highly sensitive laboratory based assays are needed for evaluating RDTs performance. Here, a quantitative suspension array technology (qSAT) was developed, validated and applied for the simultaneous detection of PfHRP2 and pLDH in a variety of clinical samples (whole blood, plasma and dried blood spots) from different endemic countries.ResultsThe qSAT was specific for the target antigens, with analytical ranges of 6.8 to 762.8 pg/ml for PfHRP2 and 78.1 to 17076.6 pg/ml for P. falciparum (Pf-LDH). The assay detected P. vivax LDH (Pv-LDH) at a lower sensitivity than Pf-LDH (analytical range of 1093.20 to 187288.5 pg/ml). Both PfHRP2 and pLDH levels determined using the qSAT showed to positively correlate with parasite densities determined by quantitative PCR (Spearman r=0.59 and 0.75, respectively) as well as microscopy (Spearman r=0.40 and 0.75, respectively), suggesting the assay to be a good predictor of parasite density.ConclusionThis immunoassay can be used as a reference test for the detection and quantification of PfHRP2 and pLDH, and could serve for external validation of RDTs performance, to determine antigen persistence after parasite clearance, as well as a complementary tool to assess malaria burden in endemic settings.

scholarly journals Nanoswitch-linked immunosorbent assay (NLISA) for fast, sensitive, and specific protein detection

2017 ◽  
Vol 114 (39) ◽  
pp. 10367-10372 ◽  
Author(s):  
Clinton H. Hansen ◽  
Darren Yang ◽  
Mounir A. Koussa ◽  
Wesley P. Wong
Keyword(s):  
Immunosorbent Assay ◽  
Biological Fluids ◽  
Point Of Care ◽  
Specific Antigen ◽  
Protein Detection ◽  
Basic Research ◽  
Specific Protein ◽  
Sample Volume ◽  
Cross Reactivity ◽  
Single Mutation

Protein detection and quantification play critical roles in both basic research and clinical practice. Current detection platforms range from the widely used ELISA to more sophisticated, and more expensive, approaches such as digital ELISA. Despite advances, there remains a need for a method that combines the simplicity and cost-effectiveness of ELISA with the sensitivity and speed of modern approaches in a format suitable for both laboratory and rapid, point-of-care applications. Building on recent developments in DNA structural nanotechnology, we introduce the nanoswitch-linked immunosorbent assay (NLISA), a detection platform based on easily constructed DNA nanodevices that change conformation upon binding to a target protein with the results read out by gel electrophoresis. NLISA is surface-free and includes a kinetic-proofreading step for purification, enabling both enhanced sensitivity and reduced cross-reactivity. We demonstrate femtomolar-level detection of prostate-specific antigen in biological fluids, as well as reduced cross-reactivity between different serotypes of dengue and also between a single-mutation and wild-type protein. NLISA is less expensive, uses less sample volume, is more rapid, and, with no washes, includes fewer hands-on steps than ELISA, while also achieving superior sensitivity. Our approach also has the potential to enable rapid point-of-care assays, as we demonstrate by performing NLISA with an iPad/iPhone camera for imaging.

Therapeutic monitoring of cyclosporine: impact of a change in standards on 125I-monoclonal RIA performance in comparison with liquid chromatography

1990 ◽  
Vol 36 (5) ◽  
pp. 804-807 ◽  
Author(s):  
P A Keown ◽  
J Glenn ◽  
J Denegri ◽  
U Maciejewska ◽  
D Seccombe ◽  
...  
Keyword(s):  
Whole Blood ◽  
Cross Reactivity ◽  
Recent Change ◽  
Therapeutic Monitoring ◽  
Clinical Samples ◽  
Marrow Graft ◽  
Standard Curve ◽  
The Impact ◽  
Control Samples

Abstract This study examines the measurement of cyclosporine (CsA) by 125I-monoclonal RIA, and describes the impact of the recent change in the standard curve provided. CsA concentrations in serum and whole-blood control samples measured by 125I-RIA were initially 8-18% higher than those by HPLC. During the first two months of 1989, a significant and sustained deviation in the 125I-RIA produced results that exceeded the HPLC results by 21-28% (P less than 0.001). Introduction of the new standard curve in March 1989 returned the concentration of the whole-blood controls to the previous range (11-12% above HPLC, P less than 0.001). Measurement of clinical samples from heart, liver, and bone-marrow graft recipients by 125I-RIA by both old and new kit standards produced a close linear correlation (y = 0.89 x - 19.02; r = 0.99; n = 75, range = 40-850 micrograms/L), with use of the new standards yielding results 82 (SD 8)% of those with the preceding assay. However, even with the new standard curve, CsA concentrations by 125I-RIA in the clinical samples exceeded those by HPLC by a factor of 1.37 (SD 0.18) to 1.52 (SD 0.19). Segregation for transplant type did not affect the RIA/HPLC ratio. The results suggest cross-reactivity of the 125I-RIA with material present in vivo.

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