scholarly journals Self-Assembly of Functional Nucleic Acid-Based Colorimetric Competition Assay for the Detection of Immunoglobulin E

Sensors ◽  
2019 ◽  
Vol 19 (10) ◽  
pp. 2224 ◽  
Author(s):  
Xuexia Lin ◽  
Caiyun Yu ◽  
Honggui Lin ◽  
Cui Wang ◽  
Jianlong Su ◽  
...  
Keyword(s):  
Self Assembly ◽  
Immunoglobulin E ◽  
Limit Of Detection ◽  
Solid Phase ◽  
Colorimetric Assay ◽  
Cost Effective ◽  
Enzyme Linked Immunosorbent Assay ◽  
Cost Effective Approach ◽  
G Quadruplex ◽  
Functional Nucleic Acids

In this work, we have developed a simple and rapid colorimetric assay for the detection of immunoglobulin E (IgE) using functional nucleic acids (FNAs) and a solid-phase competition enzyme-linked immunosorbent assay (ELISA). The FNAs including aptamer of recombinant IgE, G-quadruplex and its complementary fragments were immobilized on 96-well microplates to achieve recognition and detection of IgE in biological samples. The G-quadruplex DNAzyme catalyzed 2,2′-Azino-bis (3-ethylbenzothiazoline-6-sulfonic acid (ABTS)-hemin-H2O2 system was used to improve the sensitivity of colorimetric assay. In the presence of IgE, the hairpin structure and G-quadruplex would be destroyed, resulting in the inactivation of DNAzyme and subsequent reduction of its absorbance. This cost-effective approach detected IgE in the linear range from 5.0 pg/mL to 500 ng/mL, with the limit of detection (LOD) of 2.0 pg/mL, under optimal conditions. Moreover, the developed method was successfully applied to the rapid detection of IgE in human urine, indicating a great potentiality of this approach in clinical diagnosis and other biomedical applications.

scholarly journals Sensitive colorimetric assay using insulin G-quadruplex aptamer arrays on DNA nanotubes coupled with magnetic nanoparticles

2018 ◽  
Vol 5 (3) ◽  
pp. 171835 ◽  
Author(s):  
A. Rafati ◽  
A. Zarrabi ◽  
S. Abediankenari ◽  
M. Aarabi ◽  
P. Gill
Keyword(s):  
Magnetic Nanoparticles ◽  
Limit Of Detection ◽  
Clinical Performance ◽  
Colorimetric Assay ◽  
Enzyme Linked Immunosorbent Assay ◽  
G Quadruplex ◽  
Human Sera ◽  
Dna Nanotubes ◽  
Performance Statistics ◽  
Detection Of Biomolecules

Described here is a methodology for fabrication of a sensitive colorimetric nanoassay for measurement of insulin using G-quadruplex aptamer arrays on DNA nanotubes (DNTs) coupled with magnetic nanoparticles. The spectroscopic findings (e.g. visible spectra, velocity assay and limit of detection determination) indicated a highly sensitive performance of this new nanoassay in comparison to those results obtained from the insulin assay with non-arrayed aptamers. The clinical performance statistics (i.e. paired sample t -test, Bland–Altman plot and scatter diagram) from the newly developed assay and the enzyme-linked immunosorbent assay suggested its reliable precision and its acceptable repeatability for measurement of insulin in human sera. This is, to our knowledge, the first study for the application of magnetic nanoparticle-coupled DNTs for carrying G-quadruplex aptamers for detection of biomolecules (such as insulin) in human serum.

scholarly journals A Fluorescent Aptasensor Based on Assembled G-Quadruplex and Thioflavin T for the Detection of Biomarker VEGF165

2021 ◽  
Vol 9 ◽  
Author(s):  
Xin Zheng ◽  
Shunxiang Gao ◽  
Jihong Wu ◽  
Xiaobo Hu
Keyword(s):  
Limit Of Detection ◽  
Cost Effective ◽  
Disease Diagnosis ◽  
Serum Samples ◽  
Signal Molecule ◽  
Diagnosis And Prognosis ◽  
Cost Effective Approach ◽  
G Quadruplex ◽  
Diabetic Eye Disease ◽  
Fluorescent Aptasensor

VEGF165, a regulator of angiogenesis, has been widely used as a serum biomarker for a number of human diseases, including cancer, rheumatoid arthritis, bronchial asthma, and diabetic eye disease. The rapid, accurate, and convenient detection of VEGF165 is a crucial step in effective healthcare monitoring, disease diagnosis, and prognosis assessment. In this study, a fluorescent aptasensor based on an assembled G-quadruplex and the signal molecule ThT was developed for VEGF165 detection. First, G-rich DNA fragments were assembled at both ends of the anti-VEGF165 aptamer, and the B-DNA form was converted into a G-quadruplex structure aptamer (G4-Apt). Then, ThT was introduced, and the G-quadruplex significantly enhanced the fluorescence intensity of the bound ThT. When VEGF165 was present, the higher affinity of the aptamer to the target protein allowed the G4-Apt/VEGF165 complex to form and release ThT, which emitted only weak fluorescence in the free state. Therefore, the aptasensor exhibited a good linear detection window from 1.56 to 25 nM VEGF165, with a limit of detection of 0.138 nM. In addition, the aptasensor was applied to detect VEGF165 in clinical serum samples, showing good accuracy, reproducibility, and stability. These results indicate that our developed fluorescent aptasensor can potentially be a reliable, convenient, and cost-effective approach for the sensitive, specific, and rapid detection of the VEGF165 biomarker.

scholarly journals Pitfalls in the Immunochemical Determination of β‑Lactam Antibiotics in Water

Antibiotics ◽  
2021 ◽  
Vol 10 (3) ◽  
pp. 298
Author(s):  
Alexander Ecke ◽  
Rudolf J. Schneider
Keyword(s):  
Limit Of Detection ◽  
Cost Effective ◽  
Enzyme Linked Immunosorbent Assay ◽  
Degree Of Hydrolysis ◽  
Site Analysis ◽  
Antibody Recognition ◽  
Immunochemical Determination ◽  
Key Factor ◽  
Lactam Ring

Contamination of waters with pharmaceuticals is an alarming problem as it may support the evolution of antimicrobial resistance. Therefore, fast and cost-effective analytical methods for potential on-site analysis are desired in order to control the water quality and assure the safety of its use as a source of drinking water. Antibody-based methods, such as the enzyme-linked immunosorbent assay (ELISA), can be helpful in this regard but can also have certain pitfalls in store, depending on the analyte. As shown here for the class of β-lactam antibiotics, hydrolysis of the β‑lactam ring is a key factor in the immunochemical analysis as it influences antibody recognition. With the antibody used in this study, the limit of detection (LOD) in the immunoassay could be significantly reduced by hydrolysis for the five tested penicillins, with the lowest LOD for carbenicillin (0.2 nmol/L) and the greatest impact on penicillins G and V (reduction by 85%). In addition to enhanced quantification, our strategy also provides access to information about the degree of hydrolysis in water samples as shown for the most abundant penicillin amoxicillin.

A Simple and Rapid Light-Initiated Chemiluminescence Assay for Quantitation of Artemisia-Specific Immunoglobulin E

2021 ◽  
pp. 1-8
Author(s):  
Dandan Liu ◽  
Bei Zhang ◽  
Lina Zhu ◽  
Lisheng Zheng ◽  
Shaoshen Li ◽  
...  
Keyword(s):  
Food Allergen ◽  
Clinical Guideline ◽  
Immunoglobulin E ◽  
Limit Of Detection ◽  
Enzyme Linked Immunosorbent Assay ◽  
Serum Samples ◽  
Chemiluminescence Assay ◽  
Limit Of Quantitation ◽  
Specific Immunoglobulin E ◽  
Specific Immunoglobulin

<b><i>Background:</i></b> Light-initiated chemiluminescence assay (LICA) is a homogeneous assay that has been successfully used for the quantitation of food allergen-specific immunoglobulin E (sIgE), but not inhaled allergen-sIgE. Simultaneously, current assays used to detect allergen-sIgE are serum consuming and/or time consuming. Hence, we established a method for the quantitation of <i>Artemisia</i>-sIgE based on LICA and verified its performance according to the clinical guideline documents, laying a foundation for the quantitation of inhaled and food allergen-sIgE in parallel on LICA. <b><i>Methods:</i></b> The assay was established after optimizing the first incubation time and the dilutions of <i>Artemisia</i>-coated chemibeads, biotinylated goat anti-human IgE, and serum. In order to quantitate <i>Artemisia</i>-sIgE, the calibration curve was established with a high positive serum of known concentration. The assay performance was confirmed per the clinical guideline documents. In addition, the correlation between the results of LICA and capture enzyme-linked immunosorbent assay was evaluated. <b><i>Results:</i></b> The developed LICA’s coefficients of variation of repeatability and intermediate precision were 3.20%, 2.14%, and 3.85% and 4.30%, 4.00%, and 4.40%, respectively. The limit of detection was 0.10 kU<sub>A</sub>/L, and the limit of quantitation was 0.11 kU<sub>A</sub>/L. The range of linearity was from 0.27 kU<sub>A</sub>/L to 97.53 kU<sub>A</sub>/L (<i>r</i> = 0.9968). The correlation coefficient (<i>r</i>) for the correlation analysis between results of LICA and capture ELISA was 0.9087. This assay was successfully applied in 64 human serum samples, showing good sensitivity (82.20%) and specificity (100%). <b><i>Conclusion:</i></b> An <i>Artemisia</i>-sIgE quantitation assay based on LICA was successfully established. Its performance satisfied the clinical requirements and could be widely used in clinical laboratories.

scholarly journals Molecularly Imprinted Nanoparticles Based Sensor for Cocaine Detection

Biosensors ◽  
2020 ◽  
Vol 10 (3) ◽  
pp. 22 ◽  
Author(s):  
Roberta D’Aurelio ◽  
Iva Chianella ◽  
Jack A. Goode ◽  
Ibtisam E. Tothill
Keyword(s):  
Electrochemical Impedance ◽  
Limit Of Detection ◽  
Solid Phase ◽  
Cost Effective ◽  
Cross Reactivity ◽  
P Value ◽  
Sensing Element ◽  
Molecularly Imprinted ◽  
Cost Effective Method ◽  
Cocaine Detection

The development of a sensor based on molecularly imprinted polymer nanoparticles (nanoMIPs) and electrochemical impedance spectroscopy (EIS) for the detection of trace levels of cocaine is described in this paper. NanoMIPs for cocaine detection, synthesized using a solid phase, were applied as the sensing element. The nanoMIPs were first characterized by Transmission Electron Microscopy (TEM) and Dynamic Light Scattering and found to be ~148.35 ± 24.69 nm in size, using TEM. The nanoMIPs were then covalently attached to gold screen-printed electrodes and a cocaine direct binding assay was developed and optimized, using EIS as the sensing principle. EIS was recorded at a potential of 0.12 V over the frequency range from 0.1 Hz to 50 kHz, with a modulation voltage of 10 mV. The nanoMIPs sensor was able to detect cocaine in a linear range between 100 pg mL−1 and 50 ng mL−1 (R2 = 0.984; p-value = 0.00001) and with a limit of detection of 0.24 ng mL−1 (0.70 nM). The sensor showed no cross-reactivity toward morphine and a negligible response toward levamisole after optimizing the sensor surface blocking and assay conditions. The developed sensor has the potential to offer a highly sensitive, portable and cost-effective method for cocaine detection.

scholarly journals Proposal of De Novo Antigen Test for COVID-19: Ultrasensitive Detection of Spike Proteins of SARS-CoV-2

Diagnostics ◽  
2020 ◽  
Vol 10 (8) ◽  
pp. 594 ◽  
Author(s):  
Yuta Kyosei ◽  
Mayuri Namba ◽  
Sou Yamura ◽  
Rikiya Takeuchi ◽  
Noriko Aoki ◽  
...  
Keyword(s):  
De Novo ◽  
Limit Of Detection ◽  
False Negative ◽  
Cost Effective ◽  
Detection Sensitivity ◽  
Enzyme Linked Immunosorbent Assay ◽  
Antigen Test ◽  
Virus Rna ◽  
Antigen Tests ◽  
Spike Proteins

Polymerase chain reaction (PCR)-based antigen tests are technically difficult, time-consuming, and expensive, and may produce false negative results requiring follow-up confirmation with computed tomography. The global coronavirus disease 2019 (COVID-19) pandemic has increased the demand for accurate, easy-to-use, rapid, and cost-effective antigen tests for clinical application. We propose a de novo antigen test for diagnosing COVID-19 using the combination of sandwich enzyme-linked immunosorbent assay and thio-nicotinamide adenine dinucleotide (thio-NAD) cycling. Our test takes advantage of the spike proteins specific to the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus. The limit of detection of our test was 2.3 × 10−18 moles/assay. If the virus has ~25 spike proteins on its surface, our method should detect on the order of 10−20 moles of virus/assay, corresponding to ~104 copies of the virus RNA/assay. The detection sensitivity approaches that of PCR-based assays because the average virus RNA load used for PCR-based assays is ~105 copies per oro- or naso-pharyngeal swab specimen. To our knowledge, this is the first ultrasensitive antigen test for SARS-CoV-2 spike proteins that can be performed with an easy-to-use microplate reader. Sufficient sensitivity can be achieved within 10 min of thio-NAD cycling. Our antigen test allows for rapid, cost-effective, specific, ultrasensitive, and simultaneous multiple measurements of SARS-CoV-2, and has broad application for the diagnosis for COVID-19.

scholarly journals Low temperature isothermal amplification of microsatellites drastically reduces stutter artifact formation and improves microsatellite instability detection in cancer

2019 ◽  
Vol 47 (21) ◽  
pp. e141-e141 ◽  
Author(s):  
Antoine Daunay ◽  
Alex Duval ◽  
Laura G Baudrin ◽  
Olivier Buhard ◽  
Victor Renault ◽  
...  
Keyword(s):  
Low Temperature ◽  
Microsatellite Instability ◽  
Tandem Repeats ◽  
Limit Of Detection ◽  
Cost Effective ◽  
Isothermal Amplification ◽  
Dna Repeat ◽  
Cost Effective Approach ◽  
Living Organisms ◽  
Artifact Formation

Abstract Microsatellites are polymorphic short tandem repeats of 1–6 nucleotides ubiquitously present in the genome that are extensively used in living organisms as genetic markers and in oncology to detect microsatellite instability (MSI). While the standard analysis method of microsatellites is based on PCR followed by capillary electrophoresis, it generates undesirable frameshift products known as ‘stutter peaks’ caused by the polymerase slippage that can greatly complicate the analysis and interpretation of the data. Here we present an easy multiplexable approach replacing PCR that is based on low temperature isothermal amplification using recombinase polymerase amplification (LT-RPA) that drastically reduces and sometimes completely abolishes the formation of stutter artifacts, thus greatly simplifying the calling of the alleles. Using HT17, a mononucleotide DNA repeat that was previously proposed as an optimal marker to detect MSI in tumor DNA, we showed that LT-RPA improves the limit of detection of MSI compared to PCR up to four times, notably for small deletions, and simplifies the identification of the mutant alleles. It was successfully applied to clinical colorectal cancer samples and enabled detection of MSI. This easy-to-handle, rapid and cost-effective approach may deeply improve the analysis of microsatellites in several biological and clinical applications.

Colorimetric Sensing of Methyl Parathion Using Nanozymatic Activity of Gold Nanoparticles

2020 ◽  
Vol 12 (2) ◽  
pp. 232-241
Author(s):  
Anjana Pandey ◽  
Saumya Srivastava ◽  
Gayatri ◽  
Priya Rai ◽  
Ashutosh Pandey
Keyword(s):  
Gold Nanoparticles ◽  
Methyl Parathion ◽  
Limit Of Detection ◽  
Colorimetric Assay ◽  
Cost Effective ◽  
Colorimetric Sensing ◽  
Electron Transfer Mechanism ◽  
Au Nps ◽  
New Approach ◽  
Cost Effective Technique

In this study, we have used new approach for detection of the methyl parathion residues i.e., by inhibiting half of the peroxidase-like activity of gold nanoparticles. We have investigated the peroxidase like activity of gold nanoparticles by colorimetric assay and optimized the pH, temperature, incubation time and different concentrations of H2O2 by using TMB as substrate as well as organophosphates effect on their enzyme mimetic activity. Kinetic study of gold nanoparticles has shown better catalytic activity than horseradish peroxidase at pH 3.5. The peroxidase substrate TMB (3,3,5,5-tetramethylbenzidine) can be oxidized by H2O2 by the enzymatic action of the gold nanoparticles resulting in a blue-coloured product, oxidized TMB. The principle involved behind the inhibition of enzymatic activity of nanozyme is due to hindrance of electron transfer mechanism TMB-H2O2-Au NPs system by the methyl parathion. The peroxidase activity is selectively reduced with increasing methyl parathion concentration. This sensing method has lowest limit of detection of 78.95 nM. This study can be used for development of sensitive and cost effective technique for sensing of harmful pesticides.

scholarly journals Use of Bispecific Antibodies in Molecular Velcro Assays Whose Specificity Approaches the Theoretical Limit of Immunodetection for Bordetella pertussis

2004 ◽  
Vol 11 (4) ◽  
pp. 752-757 ◽  
Author(s):  
X. L. Tang ◽  
M. S. Peppler ◽  
R. T. Irvin ◽  
M. R. Suresh
Keyword(s):  
Bordetella Pertussis ◽  
Whooping Cough ◽  
Limit Of Detection ◽  
Solid Phase ◽  
High Sensitivity ◽  
Enzyme Linked Immunosorbent Assay ◽  
Nasopharyngeal Swab ◽  
Theoretical Limit ◽  
Benzhydroxamic Acid ◽  
New Generation

ABSTRACT A bispecific monoclonal antibody (bsMAb) that detects Bordetella pertussis, the causative agent of whooping cough, and horseradish peroxidase (HRPO) has been developed by use of the quadroma technology. A quadroma, P123, was produced by fusing two well-characterized hybridomas against the bacterium and the enzyme and was subcloned to obtain a stable bsMAb-secreting cell line. The quadroma was theoretically expected to produce up to 10 different molecular species of immunoglobulins, so secreted bispecific antibody was complexed with excess HRPO and the HRPO-bsMAb complex was purified in one step by benzhydroxamic acid-agarose affinity cochromatography. An ultrasensitive homosandwich molecular “velcro” enzyme-linked immunosorbent assay for the detection of B. pertussis whole bacteria with HRPO-bsMAb was established in both microplate and nasopharyngeal swab formats. This assay demonstrates a high sensitivity that approaches the theoretical limit of detection of one bacterium. This new nanoprobe can be used to develop a new generation of assays that are simple, inexpensive alternatives to quantitative PCR and that can be used by clinical laboratories. This strategy of homosandwich assays with solid-phase monospecific antibodies and solution-phase bsMAb with specificity for the same repeating surface determinants can be applied to generate ultrasensitive immunodiagnostic assays for viruses and bacteria.

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