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.

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

scholarly journals Immunofluorometric Assay of Human Kallikrein 10 and Its Identification in Biological Fluids and Tissues

2001 ◽  
Vol 47 (2) ◽  
pp. 237-246 ◽  
Author(s):  
Liu-Ying Luo ◽  
Linda Grass ◽  
David J C Howarth ◽  
Pierre Thibault ◽  
Huy Ong ◽  
...  
Keyword(s):  
Biological Fluids ◽  
Expression System ◽  
Specific Antigen ◽  
Cross Reactivity ◽  
Yeast Expression System ◽  
Disease States ◽  
Pichia Pastoris Yeast ◽  
Sandwich Type ◽  
Kallikrein 2 ◽  
Concentration Changes

Abstract Background: The human kallikrein 10 gene [KLK10, also known as normal epithelial cell-specific 1 gene (NES1)] is a member of the human kallikrein gene family. The KLK10 gene encodes for a secreted serine protease (hK10). We hypothesize that hK10 is secreted into various biological fluids and that its concentration changes in some disease states. The aim of this study was to develop a sensitive and specific immunoassay for hK10. Methods: Recombinant hK10 protein was produced and purified using a Pichia pastoris yeast expression system. The protein was used as an immunogen to generate mouse and rabbit polyclonal anti-hK10 antisera. A sandwich-type immunofluorometric assay was then developed using these antibodies. Results: The hK10 immunoassay has a detection limit of 0.05 μg/L. The assay is specific for hK10 and has no detectable cross-reactivity with other homologous kallikrein proteins, such as prostate-specific antigen (hK3), human glandular kallikrein 2 (hK2), and human kallikrein 6 (hK6). The assay was linear from 0 to 20 μg/L with within- and between-run CVs <10%. hK10 is expressed in many tissues, including the salivary glands, skin, and colon and is also detectable in biological fluids, including breast milk, seminal plasma, cerebrospinal fluid, amniotic fluid, and serum. Conclusions: We report development of the first immunofluorometric assay for hK10 and describe the distribution of hK10 in biological fluids and tissue extracts. This assay can be used to examine the value of hK10 as a disease biomarker.

Enzyme linked immunosorbent assay for PAT protein detection in genetically modified rape

2006 ◽  
Vol 3 (3) ◽  
pp. 177-181 ◽  
Author(s):  
Xu Wen-Tao ◽  
Huang Kun-Lun ◽  
Deng Ai-Ke ◽  
Luo Yun-Bo
Keyword(s):  
Immunosorbent Assay ◽  
Polyclonal Antibody ◽  
Genetically Modified ◽  
Protein A ◽  
Protein Detection ◽  
Cross Reactivity ◽  
Enzymic Activity ◽  
Enzyme Linked Immunosorbent Assay ◽  
Sepharose 4B ◽  
Pat Protein

AbstractWe have developed and applied an immunoassay method to detect genetically modified (GM) rape containing phosphinothricin acetyltransferase (PAT). The purified PAT was identified by Western blotting and enzymic activity analysis. The polyclonal antibody against purified PAT protein was obtained and purified by both a saturated ammonium sulphate method and protein A-Sepharose 4B. The sensitivity and cross-reactivity of the polyclonal antibody has been demonstrated in an enzyme-linked immunosorbent assay (ELISA). The result of the ELISA for antiserum sensitivity was about 2×10−5mg/ml and the cross-reactivity determined experimentally showed a high degree of specificity for the antiserum used, because values were all less than 0.1%. Detection of transgenic plants was evaluated using two transgenic rape lines (MS1/RF1 and MS8/RF3) which could be easily distinguished by ELISA.

scholarly journals Assessing the Reliability of Commercially Available Point of Care in Various Clinical Fields

2019 ◽  
Vol 12 (1) ◽  
pp. 342-368
Author(s):  
Federica Pezzuto ◽  
Antonio Scarano ◽  
Carlotta Marini ◽  
Giacomo Rossi ◽  
Roberta Stocchi ◽  
...  
Keyword(s):  
Biological Fluids ◽  
Point Of Care ◽  
Turnaround Time ◽  
Sample Volume ◽  
Good Reliability ◽  
Disease Identification ◽  
Great Relevance ◽  
Key Features ◽  
Fast Turnaround Time

a Updated and precise molecular diagnostics are essential in disease identification, treatment and management. Conventional technologies are limited to laboratories, which are expensive, require moderate to great volumes of biological fluids and generally create great discomfort among patients. This review discusses some key features of commercially available point of care (POC) devices, such as time to provide results, accuracy and imprecision, in several medical and veterinary fields. We searched Pubmed/Medline using the keywords “point” “of” “care” “device”, selected papers from 1984 to 2019 on the basis of their content and summarized the features in tables. Fast turnaround time and overall good reliability, in terms of accuracy and imprecision, were observed for most of POCs included in the research. POC devices are particularly useful for clinicians since they hold the potential to deliver rapid and accurate results in an inexpensive and less invasive way with an overall improvement of patients' quality of life in terms of time spent at the point-of-care and sample volume withdrawn. These features gain great relevance also in the veterinary practice, where patients’ compliance is generally poor, available sample volumes are quite far from the human ones and analysis costs are higher.

scholarly journals Development of an Ultrasensitive Immunoassay for Human Glandular Kallikrein with No Cross-Reactivity from Prostate-specific Antigen

1999 ◽  
Vol 45 (6) ◽  
pp. 790-799 ◽  
Author(s):  
Margot H Black ◽  
Angeliki Magklara ◽  
Christina V Obiezu ◽  
Dimitrios N Melegos ◽  
Eleftherios P Diamandis
Keyword(s):  
Detection Limit ◽  
Prostate Specific Antigen ◽  
Seminal Plasma ◽  
Limit Of Detection ◽  
Biological Fluids ◽  
Specific Antigen ◽  
Cross Reactivity ◽  
Free Form ◽  
Glandular Kallikrein ◽  
Healthy Males

Abstract Background: Studies demonstrating that human glandular kallikrein (hK2) is increased in prostate cancer patients have prompted speculation that this marker may of use in addition to prostate-specific antigen (PSA). Methods: An ultrasensitive hK2 sandwich immunoassay was developed, and its detection limit, cross-reactivity, analytical recovery, precision, and linearity of dilution were evaluated. hK2 was measured in seminal plasma and sera from healthy males, females, and prostatectomized patients. Results: Our assay has an excellent detection limit (6 ng/L) and precision (>90%). Recovery studies indicated that hK2 binds to serum protease inhibitors. All sera from healthy males had measurable hK2 concentrations (median, 402 ng/L). Almost all female sera had undetectable hK2. Serum hK2 and PSA in males correlated positively (r = 0.44), but hK2 was present at concentrations ∼2.5-fold lower than PSA. The PSA/hK2 ratio in male sera was 0.1–34, with a median of 2.6. In seminal plasma, this ratio was 100–500. More than 94% of immunoreactive hK2 in serum was in the free form (∼30 kDa); traces of hK2 complexed to α1-antichymotrypsin were present. Conclusions: The limit of detection of the method for hK2 measurement described here (∼20-fold lower than any other reported assay for hK2) allows the generation of new clinical information. When combined with a previously described method for PSA measurement that has no cross-reactivity from hK2, this methods allows the relative proportions of hK2 and PSA in biological fluids to be measured.

scholarly journals Pgp3 Antibody Enzyme-Linked Immunosorbent Assay, a Sensitive and Specific Assay for Seroepidemiological Analysis of Chlamydia trachomatis Infection

2009 ◽  
Vol 16 (6) ◽  
pp. 835-843 ◽  
Author(s):  
Gillian S. Wills ◽  
Patrick J. Horner ◽  
Rosy Reynolds ◽  
Anne M. Johnson ◽  
David A. Muir ◽  
...  
Keyword(s):  
Chlamydia Trachomatis ◽  
Immunosorbent Assay ◽  
Chlamydia Pneumoniae ◽  
Specific Antigen ◽  
Cross Reactivity ◽  
Enzyme Linked Immunosorbent Assay ◽  
Chlamydia Trachomatis Infection ◽  
Female Patients ◽  
Lower Genital Tract ◽  
Significant Difference

ABSTRACT Understanding of the burden of Chlamydia trachomatis infection and its clinical sequelae is hampered by the absence of accurate, well-characterized tests using serological methods to determine past exposure to infection. An “in-house” immunoglobulin G (IgG) enzyme-linked immunosorbent assay (ELISA) based on the C. trachomatis-specific antigen Pgp3 was produced and evaluated against three commercial ELISAs derived from the major outer membrane protein: the Medac pELISA plus, the Savyon SeroCT-IgG ELISA, and the Ani Labsystems IgG enzyme immunoassay. Sensitivities and specificities were determined using sera from both male and female patients (n = 356) for whom C. trachomatis had been detected in the lower genital tract at least 1 month prior to the testing of the sample and from 722 Chlamydia-negative children aged 2 to 13 years. The Pgp3 ELISA was significantly more sensitive (57.9% [95% confidence interval {95% CI}, 52.7 to 62.9%]) than the Ani Labsystems (49.2% [95% CI, 44.0 to 54.3%]; P = 0.003), SeroCT (47.2% [95% CI, 42.1 to 52.4%]; P < 0.0005), and Medac (44.4% [95% CI, 39.3 to 49.6%]; P < 0.0005) ELISAs. The Pgp3, Ani Labsystems, and SeroCT assays, but not the Medac assay, had significantly higher sensitivity for female specimens than for male specimens (73.8 versus 44.2%, 59.8 versus 40.5%, 55.5 versus 40%, and 45.7 versus 43.7%, respectively). For female patients, the Pgp3 assay was 14.0% (95% CI, 5.5 to 22.5%) more sensitive than the next most sensitive ELISA, the Ani Labsystems assay (P = 0.001). There was no significant difference in specificity between the Pgp3 (97.6% [95% CI, 96.2 to 98.6%]), Ani Labsystems (99% [95% CI, 97.7 to 99.6%]), SeroCT (97.2% [95% CI, 95.7 to 98.2%]), and Medac (96% [95% CI, 94.3 to 97.2%]) ELISAs. None of the ELISAs showed evidence of cross-reactivity with antibodies to Chlamydia pneumoniae.

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 A Label and Probe-Free Zika Virus Immunosensor Prussian Blue@carbon Nanotube-Based for Amperometric Detection of the NS2B Protein

Biosensors ◽  
2021 ◽  
Vol 11 (5) ◽  
pp. 157
Author(s):  
Bárbara V. M. Silva ◽  
Marli T. Cordeiro ◽  
Marco A. B. Rodrigues ◽  
Ernesto T. A. Marques ◽  
Rosa F. Dutra
Keyword(s):  
Carbon Nanotube ◽  
Prussian Blue ◽  
Zika Virus ◽  
Point Of Care ◽  
Amperometric Detection ◽  
Cross Reactivity ◽  
Structural Protein ◽  
Serum Samples ◽  
Polypyrrole Composite ◽  
Congenital Disabilities

Zika virus (ZIKV) is a mosquito-borne infection, predominant in tropical and subtropical regions causing international concern due to the ZIKV disease having been associated with congenital disabilities, especially microcephaly and other congenital abnormalities in the fetus and newborns. Development of strategies that minimize the devastating impact by monitoring and preventing ZIKV transmission through sexual intercourse, especially in pregnant women, since no vaccine is yet available for the prevention or treatment, is critically important. ZIKV infection is generally asymptomatic and cross-reactivity with dengue virus (DENV) is a global concern. An innovative screen-printed electrode (SPE) was developed for amperometric detection of the non-structural protein (NS2B) of ZIKV by exploring the intrinsic redox catalytic activity of Prussian blue (PB), incorporated into a carbon nanotube–polypyrrole composite. Thus, this immunosensor has the advantage of electrochemical detection without adding any redox-probe solution (probe-less detection), allowing a point-of-care diagnosis. It was responsive to serum samples of only ZIKV positive patients and non-responsive to negative ZIKV patients, even if the sample was DENV positive, indicating a possible differential diagnosis between them by NS2B. All samples used here were confirmed by CDC protocols, and immunosensor responses were also checked in the supernatant of C6/36 and in Vero cell cultures infected with ZIKV.

scholarly journals Demonstration of a Label-Free and Low-Cost Optical Cavity-Based Biosensor Using Streptavidin and C-Reactive Protein

Biosensors ◽  
2020 ◽  
Vol 11 (1) ◽  
pp. 4
Author(s):  
Donggee Rho ◽  
Seunghyun Kim
Keyword(s):  
Limit Of Detection ◽  
Point Of Care ◽  
Low Cost ◽  
Optical Cavity ◽  
Sample Volume ◽  
Small Sample ◽  
Label Free ◽  
C Reactive Protein ◽  
Reactive Protein ◽  
Cavity Structure

An optical cavity-based biosensor (OCB) has been developed for point-of-care (POC) applications. This label-free biosensor employs low-cost components and simple fabrication processes to lower the overall cost while achieving high sensitivity using a differential detection method. To experimentally demonstrate its limit of detection (LOD), we conducted biosensing experiments with streptavidin and C-reactive protein (CRP). The optical cavity structure was optimized further for better sensitivity and easier fluid control. We utilized the polymer swelling property to fine-tune the optical cavity width, which significantly improved the success rate to produce measurable samples. Four different concentrations of streptavidin were tested in triplicate, and the LOD of the OCB was determined to be 1.35 nM. The OCB also successfully detected three different concentrations of human CRP using biotinylated CRP antibody. The LOD for CRP detection was 377 pM. All measurements were done using a small sample volume of 15 µL within 30 min. By reducing the sensing area, improving the functionalization and passivation processes, and increasing the sample volume, the LOD of the OCB are estimated to be reduced further to the femto-molar range. Overall, the demonstrated capability of the OCB in the present work shows great potential to be used as a promising POC biosensor.

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