scholarly journals Immobilization Techniques and Integrated Signal Enhancement for POC Nanocolor Microfluidic Devices

2015 ◽  
Vol 2015 ◽  
pp. 1-10 ◽  
Author(s):  
Marlies Schlauf ◽  
Saied Assadollahi ◽  
Roland Palkovits ◽  
Peter Pointl ◽  
Thomas Gerhard Maria Schalkhammer
Keyword(s):  
Microfluidic Devices ◽  
Thin Layers ◽  
Test Device ◽  
Silver Enhancement ◽  
Protein Conjugates ◽  
Enhanced Absorption ◽  
Optical Readout ◽  
Highly Sensitive ◽  
One Step ◽  
Immobilization Techniques

Resonance enhanced absorption (REA) nanocolor microfluidic devices are new promising bioassay platforms, which employ nanoparticle- (NP-) protein conjugates for the immunodetection of medically relevant markers in biologic samples such as blood, urine, and saliva. The core component of a REA test device is a PET chip coated with aluminum and SiO2thin layers, onto which biorecognitive molecules are immobilized. Upon addition of a sample containing the analyte of interest, a NP-protein-analyte complex is formed in the test device that is captured on the REA chip, for example, via streptavidin-biotin interaction. Thereby, a colored symbol is generated, which allows optical readout. Silver enhancement of the bound nanoparticles may be used to increase the sensitivity of the assay. Herein, we demonstrate that adsorptive immobilization via a cationic polymeric interlayer is a competitive and fast technique for the binding of the capture protein streptavidin onto planar SiO2surfaces such as REA biochips. Moreover, we report the development of a silver enhancement technology that operates even in the presence of high chloride concentrations as may be encountered in biologic samples. The silver enhancement reagents may be integrated into the microfluidic assay platform to be released upon sample addition. Hereby, a highly sensitive one-step assay can be realized.

scholarly journals Analysis and validation of a highly sensitive one-step nested quantitative real-time polymerase chain reaction assay for specific detection of severe acute respiratory syndrome coronavirus 2

2020 ◽  
Vol 17 (1) ◽  
Author(s):  
Yang Zhang ◽  
Chunyang Dai ◽  
Huiyan Wang ◽  
Yong Gao ◽  
Tuantuan Li ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Real Time ◽  
Quantitative Polymerase Chain Reaction ◽  
Clinical Samples ◽  
Chain Reaction ◽  
Pcr Assay ◽  
Qrt Pcr ◽  
Highly Sensitive ◽  
One Step ◽  
Polymerase Chain

Abstract Background Coronavirus disease 2019 (COVID-19), caused by SARS-CoV-2, is posing a serious threat to global public health. Reverse transcriptase real-time quantitative polymerase chain reaction (qRT-PCR) is widely used as the gold standard for clinical detection of SARS-CoV-2. Due to technical limitations, the reported positive rates of qRT-PCR assay of throat swab samples vary from 30 to 60%. Therefore, the evaluation of alternative strategies to overcome the limitations of qRT-PCR is required. A previous study reported that one-step nested (OSN)-qRT-PCR revealed better suitability for detecting SARS-CoV-2. However, information on the analytical performance of OSN-qRT-PCR is insufficient. Method In this study, we aimed to analyze OSN-qRT-PCR by comparing it with droplet digital PCR (ddPCR) and qRT-PCR by using a dilution series of SARS-CoV-2 pseudoviral RNA and a quality assessment panel. The clinical performance of OSN-qRT-PCR was also validated and compared with ddPCR and qRT-PCR using specimens from COVID-19 patients. Result The limit of detection (copies/ml) of qRT-PCR, ddPCR, and OSN-qRT-PCR were 520.1 (95% CI: 363.23–1145.69) for ORF1ab and 528.1 (95% CI: 347.7–1248.7) for N, 401.8 (95% CI: 284.8–938.3) for ORF1ab and 336.8 (95% CI: 244.6–792.5) for N, and 194.74 (95% CI: 139.7–430.9) for ORF1ab and 189.1 (95% CI: 130.9–433.9) for N, respectively. Of the 34 clinical samples from COVID-19 patients, the positive rates of OSN-qRT-PCR, ddPCR, and qRT-PCR were 82.35% (28/34), 67.65% (23/34), and 58.82% (20/34), respectively. Conclusion In conclusion, the highly sensitive and specific OSN-qRT-PCR assay is superior to ddPCR and qRT-PCR assays, showing great potential as a technique for detection of SARS-CoV-2 in patients with low viral loads.

scholarly journals One Step Highly Sensitive Piezoelectric Agglutination Method for Cholera Toxin Detection Using GM1 Incorporated Liposome

2011 ◽  
Vol 8 ◽  
pp. 248-256 ◽  
Author(s):  
Hong-wei Yu ◽  
Yuan-sheng Wang ◽  
Yu li ◽  
Guo-Li Shen ◽  
Hai-long Wu ◽  
...  
Keyword(s):  
Cholera Toxin ◽  
Toxin Detection ◽  
Highly Sensitive ◽  
Agglutination Method ◽  
One Step

scholarly journals Optical Readout of Gold Nanoparticle-Based DNA Microarrays without Silver Enhancement

2006 ◽  
Vol 90 (1) ◽  
pp. L13-L15 ◽  
Author(s):  
Gerhard A. Blab ◽  
Laurent Cognet ◽  
Stéphane Berciaud ◽  
Isabelle Alexandre ◽  
Dieter Husar ◽  
...  
Keyword(s):  
Gold Nanoparticle ◽  
Dna Microarrays ◽  
Silver Enhancement ◽  
Optical Readout

Universal primers for rapid detection of six pospiviroids in Solanaceae plants using one-step RT-PCR and RT-LAMP

Plant Disease ◽  
2021 ◽  
Author(s):  
Yi-Wen Tseng ◽  
Chien-Fu Wu ◽  
Chia-Hwa Lee ◽  
Chung Jan Chang ◽  
Yuh-Kun Chen ◽  
...  
Keyword(s):  
Potato Spindle Tuber Viroid ◽  
Detection Methods ◽  
Universal Primers ◽  
Rt Pcr ◽  
Degenerate Primers ◽  
Highly Sensitive ◽  
One Step ◽  
Tomato Chlorotic Dwarf Viroid ◽  
Minimal Concentration ◽  
Solanaceous Plants

A number of viruses and viroids infect solanaceous plants causing severe yield losses. Several seed-borne viroids are currently listed as quarantine pathogens in many countries. Among them, columnea latent viroid (CLVd), pepper chat fruit viroid (PCFVd), potato spindle tuber viroid (PSTVd), tomato apical stunt viroid (TASVd), tomato chlorotic dwarf viroid (TCDVd), and tomato planta macho viroid (TPMVd) are of major concerns. The objective of this study was to design and test universal primers that could be used to detect six viroids in solanaceous plants using one-step RT-PCR and reverse transcription loop-mediated isothermal amplification (RT-LAMP). Results revealed that a pair of degenerate primers could be used in a one-step RT-PCR to amplify six pospiviroids from Solanaceae seeds and plants. Moreover, five primers were designed and used in RT-LAMP to amplify six pospiviroids. The minimal concentration of viroid RNA required for a successful detection varied, ranging from one femtogram to 10 nanograms, depending on the species of viroid and detection method. In general, RT-LAMP was more sensitive than RT-PCR but both assays were rapid and highly sensitive tools to detect six pospiviroids. Detection methods currently in use for these viroids require at least two different sets of primers. The assays developed in this research could facilitate to screen a large number of solanaceous plants and seeds intended for import and export.

scholarly journals Immobilization Techniques for Aptamers on Gold Electrodes for the Electrochemical Detection of Proteins: A Review

Biosensors ◽  
2020 ◽  
Vol 10 (5) ◽  
pp. 45
Author(s):  
Franziska V. Oberhaus ◽  
Dieter Frense ◽  
Dieter Beckmann
Keyword(s):  
Electrochemical Detection ◽  
Gold Electrodes ◽  
Dna Nanostructures ◽  
Profound Knowledge ◽  
Low Concentrations ◽  
Reduced Graphene ◽  
Highly Sensitive ◽  
The Subject ◽  
Immobilization Techniques ◽  
Direct Immobilization

The development of reliable biosensing platforms plays a key role in the detection of proteins in clinically and environmentally derived samples for diagnostics, as well as for process monitoring in biotechnological productions. For this purpose, the biosensor has to be stable and reproducible, and highly sensitive to detect potentially extremely low concentrations and prevent the nonspecific binding of interfering compounds. In this review, we present an overview of recently published (2017–2019) immobilization techniques for aptamers on gold electrodes for the electrochemical detection of proteins. These include the direct immobilization of thiolated aptamers and the utilization of short linkers, streptavidin/biotin interaction, as well as DNA nanostructures and reduced graphene oxide as immobilization platforms. Applied strategies for signal amplification and the prevention of biofouling are additionally discussed, as they play a crucial role in the design of biosensors. While a wide variety of amplification strategies are already available, future investigations should aim to establish suitable antifouling strategies that are compatible with electrochemical measurements. The focus of our review lies on the detailed discussion of the underlying principles and the presentation of utilized chemical protocols in order to provide the reader with promising ideas and profound knowledge of the subject, as well as an update on recent discoveries and achievements.

scholarly journals Characterization of Hapten-Protein Conjugates: Antibody Generation and Immunoassay Development for Chlorophenoxyacetic Acid Pesticides

2009 ◽  
Vol 92 (6) ◽  
pp. 1773-1779 ◽  
Author(s):  
Robin C Boro ◽  
K Vikas Singh ◽  
C Raman Suri
Keyword(s):  
Inhibitory Concentration ◽  
Dichlorophenoxyacetic Acid ◽  
Protein Conjugation ◽  
Protein Conjugates ◽  
Highly Sensitive ◽  
Ic50 Values ◽  
Chlorophenoxyacetic Acid ◽  
2,4 Dichlorophenoxyacetic Acid

Abstract The generation of specific and sensitive antibodies against small molecules is greatly dependent upon the characteristics of the hapten-protein conjugates. In this study, we report a new fluorescence-based method for the characterization of hapten-protein conjugates. The method is based on an effect promoted by hapten-protein conjugation density upon the fluorescence intensity of the intrinsic tryptophan chromophore molecules of the protein. The proposed methodology is applied to quantify the hapten-protein conjugation density for two different chlorophenoxyacetic acid pesticides, 2,4-dichlorophenoxyacetic acid (2,4-D) and 2,4-dichlorophenoxybutyric acid (2,4-DB), coupled to carrier protein. Highly sensitive anti-2,4-D and anti-2,4-DB antibodies were obtained using these well-characterized hapten-protein conjugates. The generated antibodies were used in an immunoassay format demonstrating inhibitory concentration (IC50) values equal to 30 and 7 ng/mL for 2,4-D and 2,4-DB, respectively. Linearity was observed in the concentration range between 0.1500 ng/mL with LODs around 4 and 3 ng/mL for 2,4-D and 2,4-DB, respectively, in standard water samples. The proposed method was successfully applied for the determination of the extent of hapten-protein conjugation to produce specific antibodies for immunoassay development against pesticides.

Two-site enzyme immunoassay of CD4 and CD8 molecules on the surface of T lymphocytes from healthy subjects and HIV-1-infected patients

1994 ◽  
Vol 40 (1) ◽  
pp. 30-37 ◽  
Author(s):  
D Carriere ◽  
C Fontaine ◽  
A M Berthier ◽  
A M Rouquette ◽  
P Carayon ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
T Lymphocytes ◽  
Enzyme Immunoassay ◽  
Cd8 T Cells ◽  
Envelope Glycoprotein ◽  
Healthy Subjects ◽  
Gp 120 ◽  
Highly Sensitive ◽  
One Step ◽  
Hiv 1

Abstract A highly sensitive two-site enzyme immunoassay (Capcellia) was developed to determine the concentration of CD4 and CD8 molecules expressed on the surface of human T lymphocytes. This assay, performed in one step (20 min), involves the specific immunocapture of T lymphocytes and reaction of the CD4 or CD8 molecules with an enzyme-labeled monoclonal antibody (mAb). The results were expressed as molar concentrations of the T-cell markers on the basis of results obtained with calibrated CD4 and CD8 standards. The assay was sensitive enough to detect 0.4 pmol/L CD4 or 0.8 pmol/L CD8, which corresponded to approximately 20 x 10(6) CD4+ or CD8+ T cells per liter of blood. Mean concentrations in healthy adults were 17.2 pmol/L for CD4 and 22.1 pmol/L for CD8. The CD4 concentration was < 8 pmol/L in 50% of HIV-1-infected patients and in 95% of AIDS patients. Given the epitopic specificity of the mAb to CD4 we used, these values correspond to the concentration of CD4 molecules free of envelope glycoprotein (gp)120.

A highly sensitive dual-color lateral flow immunoassay for brucellosis using one-step synthesized latex microspheres

2019 ◽  
Vol 11 (22) ◽  
pp. 2937-2942 ◽  
Author(s):  
Mingsong Zhu ◽  
Yurui Jia ◽  
Lizhi Peng ◽  
Jifu Ma ◽  
Xiangru Li ◽  
...  
Keyword(s):  
Clinical Care ◽  
Lateral Flow ◽  
Lateral Flow Immunoassay ◽  
Screening Methods ◽  
Step Method ◽  
Dual Color ◽  
Highly Sensitive ◽  
One Step

A lateral flow immunoassay was developed to improve clinical care compared with conventional brucellosis screening methods. Detection is dual-color in format using dyed, carboxyl-functionalized latex microspheres synthesized with a one-step method.

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