Khlebtsov B.N., Tumskiy R.S., Burov A.M., Pylaev T.E., Khlebtsov N.G. Quantifying the gold nanoparticle numbers in the test zone of lateral flow immunoassay strips. Li J., Duan H., Xu P., Huang X.L., Xiong Y.H. Effect of different-sized spherical gold nanoparticles grown layer by layer on the sensitivity of an immunochromatographic assay. As can be seen in the case of using C-GNPs as labels, the most effective binding of conjugates in the test zone was obtained for C-GNP3 particles with an average size of 33.7 nm. Note that the adsorption immobilization of antibodies was more effective than the covalent binding.
The infected sample was positive with low signal intensity, and it was correctly classified as RGNNV. The genotype of the sample was previously determined by direct sequencing by an independent research group. Optimization studies for assessment of the oligonucleotide probe impact in the hybridization reaction mixtures were performed. The oligonucleotide probes were tested in amounts of 0.5–4 pmol/1 pmol of target (Figures 4 and 4). Both test zones resulted in optimum signals when 1 pmol of probe was used and decreased with higher amounts of probe. When higher amounts of biotinylated probes are used, the amount of nanoparticles that bind to the free probe is increasing. Even though these nanoparticles move along the LFB, they cannot be immobilized from the deposited antibodies in the test zones, and the red bands become fainter strip cutter .
The anti-digoxigenin antibody performance for test zone construction was evaluated in the previously mentioned study , as well as other independent studies . Therefore, the same type of anti-digoxigenin antibody was utilized for the TZ-S construction in the present study. However, the use of the polyclonal fluorescein antibody for the construction of TZ-R zone was only evaluated in our previous study.
Bioreadytm Gold Nanoparticles
Visual and reader results of Mix&Go and covalently conjugated magnetic particle hCG assays with urine. The amount of antibody conjugated to the different particles and the amount of particles used per test were optimized to produce the highest sensitivity. The Mix&Go conjugate was tested with an antibody loading of 25 ug of anti-hCG antibody per mg of particles at 0.05% solids. The covalently conjugated particle was tested with an antibody loading of 50 ug of anti-hCG per mg particles at 0.05% solids.
Monodispersed production of AuNPs with adequate size was confirmed by UV/Vis spectrophotometry (Thermo Scientific™ Evolution 60S). Spherically shaped (ca. 40 nm in diameter) and deep red colored AuNPs were synthesized. The size of these unmodified AuNPs was estimated by using the Beer–Lambert law at 530 ± 2 nm and transmission electron microscopy (Fig. 2) (JEOL Ltd. JEM-1011).
Overview Universal Lateral Flow Assay Kit is designed to enable the easy development of customized sandwich lateral flow assays, by combining Ulfa-Tag and GOLD conjugation technologies with an immunochromatography test performed on Universal-LFA strips. The current immunochromatographic assay and the previously reported ELISA-based TPTest are both based on detection of antibodies secreted ex vivo by activated lymphocytes recovered from the peripheral circulation during acute infection . These lymphocytes have been stimulated by the recent infection and require no ex vivo stimulation. Removing the plasma component of blood limits the confounding influence of preexisting circulating antibodies that reflect prior exposure. These circulating antibodies can affect assay specificity and have markedly limited the utility of plasma antibody-based assays in areas of the world where enteric fever and salmonellosis are endemic. After making colloidal gold, we determined the size of the gold nanoparticles by differential light scatterings using a Zetasizer Nano ZS90 instrument (Malvern Instrument, Ltd.).
Preparation Of Lfa Strips
After 30 min of the reaction, the resulting 10-nm GNPs were centrifuged at least thrice at 20,000 g for 60 min. Finally, 10-nm GNPs were resuspended in 10 mL of 0.1 M CTAC. Then, these 10 nm GNPs were overgrown to a designed size. To this end, 0.1 M CTAC, 10 mM AA and the 10-nm GNPs were mixed, as indicated in Table 2, in a 200-mL flask. Further, 2 mM HAuCl4 was added by using a syringe pump at the injection rate 10 mL/h.
- Monodispersed production of AuNPs with adequate size was confirmed by UV/Vis spectrophotometry (Thermo Scientificâ„¢ Evolution 60S).
- The assay employs both biotinylated anti-norovirus antibodies and gold-labeled anti-norovirus antibodies; when target noroviruses are present in the sample, virions associate with the antibodies while flowing through the strip.
- However, such improvements result in the loss of the main advantage of LFIA as a simple point-of-care test.
On the other hand, we recently demonstrated that increasing the nanoparticle size up to 115 nm reduced the LoD . Moreover, conclusions about the capabilities of a label can be made only by comparing the analytical characteristics achieved for its conjugates with antibodies of different compositions . However, changes in the antigen-binding properties of antibodies with variation in their surface density on a nanoparticle are nontrivial and, to date, have not been described by a single, universally recognized model. For example, the curvature of the nanoparticle surface can play an important role in the affinity of the GNP–antibody complex . The results from Figures 6A,C show that visually the test and control lines became easier to see with the naked eye and the results from Figures 6B,D confirmed this by color quantification.
Alexandria Engineering Journal Impact
Encouraged by the improved optical property, we investigated the feasibility of the customized GSPs in highly sensitive colorimetric detection. In this case, the GSPs were utilized as visual contrast labels in LFIA strip. HCG, a key diagnostic marker of pregnancy and an important risk biomarker of certain diseases, was selected as a model analyte .
After reaction for 90 min at room temperature, BSA was added into the above mixture solution and allowed to react for 1 h to block the unreacted carboxyl group. The resulting GSP270 probes were then purified via centrifugation; resuspended PB solution (200 μL, 0.01 M, pH 7.4) containing 25% sucrose, 1% BSA, and 0.1% sodium azide; and stored at 4 °C for subsequent use. Summary of the detection performance of AuNP- and GSP-LFIA strip in detecting HCG. The theoretical Optical performance of GSPs and AuNPs; The illustration of sandwich LFIA platform; The detection sensitivity of GSP-LFIA and AuNP-LFIA.
Microplasma-assisted synthesis of colloidal gold nanoparticles and their use in the detection of cardiac Troponin i (cTn-I). / Wang, Ruixue; Zuo, Shasha; Wu, Dong; Zhang, Jue; Zhu, Weidong; Becker, Kurt H.; Fang, Jing.
Size Optimized For Lateral Flow
(LSPR peak wavelength of 533.5nm) have a lighter red appearance which trades contrast efficiency for increased optical absorbance per particle. This means 60 nm Gold NanoSpheres are ideal for immunoassays with low target analyte concentration samples, or when the targeting moiety is very expensive. Another type of competition assay will conjugate the analyte to the reporter.
To demonstrate the versatility of our strategy, we further extended the GSP-LFIA strip for the sensitive detection of HBsAg, an important serological biomarker for hepatitis B virus infection diagnosis . In this case, GSP270 was selected as visual label for the fabrication of GSP270-LFIA strip because GSP270 possesses large optical absorbance and good diffusivity on the NC membrane. For direct comparison, the AuNP40-LFIA strip was developed at the same time. Several key parameters that influence the sensitivity of AuNP40-LFIA and GSP270-LFIA strip were systematically investigated and optimized .
How Lateral Flow Assays Work
The Anteo Mix&Go particles come pre-activated and are ready for the antibody conjugation. The covalent conjugation procedure requires steps to wash and activate the particles before the addition of the antibody. Five replicates for each concentration were tested and results were recorded 15 minutes after the addition of the sample using DCN’s visual grading scale and an ESEQuant reflectance reader. The metal chelating principle behind Mix&Go is not specific to antibodies and can be applied universally to bind other proteins.