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Posted by Sumler on April 22nd, 2021

Accordingly, the corresponding LOD values , dynamic detection range, and Hook effect point are summarized in Table 1. The results indicated that the AuNP120-LFIA strip exhibited the lowest LOD value of 0.97 mIU/mL, which was ca. 20.1-, 4.02-, and 2.01-fold lower than those of AuNP40 (19.5 mIU/mL), AuNP80 (3.9 mIU/mL), and AuNP180 (1.95 mIU/mL), respectively. The linear detection of AuNP120-LFIA strip ranged from 1.9 mIU/mL to 1000 mIU/mL. Thus, we conclude that the LFIA sensitivity increased when AuNP size was increased from 40 nm to 120 nm. However, when AuNP size was further increased to 180 nm, the sensitivity decreased despite the increased optical signal. We speculate that this result may be due to the significantly enhanced Qsca rather than Qabs for increased Qext of AuNP180, conflicting with absorption-dominated signal output of AuNP-LFIA.

In the case of less hydrophobic antibodies or a more hydrophilic surface (i.e. –COOH modified), attachment by both ionic interactions and hydrophobic interactions can occur. Small changes in pH can alter the association dynamics and affect the efficiency of conjugation, so a pH titration and a sweep of the antibody to gold ratio should be performed to identify the optimal conditions for antibody adsorption. It is recommended that the pH of the adsorption buffer be slightly above the isoelectric pointof the protein, which varies from antibody to antibody. The constant region of the antibody is generally more hydrophobic and therefore more likely to be adsorbed as compared to theFab portion, offering some control over binding orientation. A large excess of antibody with respect to nanoparticle surface area may be required to ensure dense surface binding and high salt stability post conjugation. Please keep in mind that every antibody requires slightly different conditions which must be optimized according to the considerations described above.

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GNPs with immobilized antibodies were separated from unreacted antibodies by centrifugation for 15 min at the accelerations indicated in Table 3. After the supernatant liquid was discarded, the sediment was resuspended in 0.02 M Tris–HCl buffer (pH 7.6) containing 1.0% BSA, 1.0% sucrose, 1.0% Tween 20, and 0.1% sodium azide (all w/v). Immunochromatographic assay for serodiagnosis of tuberculosis using an antigen-colloidal gold conjugate. Gel electrophoretic analysis of differently shaped interacting and non-interacting bioconjugated nanoparticles. Serum antibody levels to the Pneumocystis jirovecii major surface glycoprotein in the diagnosis of P. jirovecii pneumonia in HIV+ patients.

The results demonstrate the optimization studies for a rapid single-step assay, which requires low amount of the analyzed sample and provides simultaneous amplification and genotyping of nodavirus DNA in a single, closed-tube methodology. The assay was optimized in terms of the biosensors’ preparation and the detection assay parameters, demonstrating attractive characteristics with respect to specificity and reproducibility.

Thus, J. Dong et al. described a technique that decreased the RSD of the GNPs diameter to 8–10% with an aspect ratio of 1.10–1.22 . Kimimg et al. modified the Turkevich–Frens method, providing an RSD of the GNP diameter from 13% to 16% for a preparation range of up to 40 nm. Shiba described a finely dispersed preparation with an RSD of 7.6%, but this was only the case where the diameter was equal to 14 nm. et al. reached the gain in homogeneity with a decrease in RSD from 8% to 3%, but only for small GNPs with a diameter of 12 nm. Xia et al. described an improved synthesis of citrate GNPs (C-GNPs) in the 12–36 nm range characterized by an RSD of 9% or higher.

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From bottom to top, strips were composed by the sample pad, the conjugate pad, the nitrocellulose ballya strip cutter lateral flow membrane with the test and control lines and the absorbent pad. Quantification of color intensity of the control and test lines present in all replicates, where the intensity shown in each line corresponds to the maximum height of the Gaussian line fitted by eReuss software and the error bars represent the standard deviation values.

Comparison of the OD values by spraying the AuNPs or GSPs as the T lines at the same molar concentrations. Citrate-capped gold nanoparticles were synthesized via the Frens method with modifications according to our paper . An aqueous solution of HAuCl4 was added to deionized water, as indicated in Table 1, and the mixture was brought to a boil. The mixtures were boiled for 25 min, and then cooled and stored at 4–6 °C. Determination of size and concentration of gold nanoparticles from UV-Vis spectra. Recently, studies showed that a technology based on synthetic amino acid sequences, designed to hold more than one reactive region of the selected antigens, could enhance the immunological diagnosis of Toxoplasma gondii (Dai et al., 2012, 2013). Therefore, in our previous study, this research group designed a recombinant synthetic antigen with three antigenic regions of the Msg protein, in order to standardize and enhance the detection of reactive antibodies against P. jirovecii (Tomás et al., 2016).

Colloidal Gold

Even though the minimal protective amount was determined to be 10 μg, 2.5 times MPA of anti goat IgG, used for effective conjugation with gold nanoparticles. Contagious agalactia is a notifiable disease listed by World Organisation for Animal Health (WOAH/OIE) and has been responsible for causing severe economic losses to goat and sheep industries. The disease has been reported from India (Vihan, 1989; Srivastava et al., 1996; Mondal et al., 2004; Kumaret al., 2009, 2014) but the prevalence of disease is overlooked due to lack of a rapid field diagnostic test. The isolation and biochemical identification of the organism is more tedious and time consuming (Aluotto et al., 1970; Poveda, 1998).

Determination of minimum amount of anti-human IgG and anti human-IgA required for conjugation of 1 ml of colloidal gold solution. Optical density ratios of values at 520 nm to 580 nm and at 600 nm to 520 nm represent stability and polydispersity, respectively.

Both rows encompass the diameter range of 30–40 nm that is typically recommended for LFIA. However, the protocol for obtaining S-GNPs provides the possibility of extending the particle diameters to 90 nm, whereas C-GNPs of such size are known to be unstable. Besides, S-GNPs are characterized by a unified spherical shape, with minimal variation in the ellipticity index . Thus, the chosen approach leads to essential unification for geometrical parameters of the obtained GNPs. Images of the C-GNPs and S-GNPs are given in the Supporting Information, Figures S1 and S2.

• Both test zones resulted in optimum signals when 1 pmol of probe was used and decreased with higher amounts of probe.
• For example, the curvature of the nanoparticle surface can play an important role in the affinity of the GNP–antibody complex .
• To assess stability of the conjugate and to define the optimum pH and minimum concentration of antibody required for conjugating the colloidal gold, we used an aggregation assay (20–22).
• After reaction for 3 h, the hydrophobic AuNPs were precipitated by adding 50 mL of ethyl alcohol and then collected by centrifugation.

Development of lateral flow assay based on size-controlled gold nanoparticles for detection of hepatitis B surface antigen. Finally, triplicates of the optimized LFIA strips were tested with a pool of serum specimens from patients with PcP and a pool of serum specimens from patients without P. jirovecii infection in the selected dilutions . During these assays, it was established that 3 min are enough for the sample to elute completely until the absorbing pad, giving a LFIA final result. The digital pictures and the color intensity analysis of the final results showed that in strips tested with the negative pool, only a colored line was visible on the control zone and detected by the color quantification software in all replicates.

Gold Nanosphere Labels

Two different IgG and IgM ELISA were developed, according to the protocols presented in Table 1, using Kex1 RSA and Msg RSA as coating antigens. jirovecii levels across patients with PcP and without P. jirovecii infection are represented in Figure 3. For synthesis and functionalization of gold nanospheres, all glassware was washed with aqua regia and rinsed thoroughly with deionized water followed by ultrapure water (18.2 MΩ⋅cm–1) before use. The fungus Pneumocystis jirovecii is a pathogen able to cause a fatal pneumonia in immunocompromised patients worldwide (Barry and Johnson, 2001; Huang et al., 2011; Esteves et al., 2014; Matos et al., 2017). Likewise, the rising number of immunocompromised non-HIV-infected patients susceptible to P. jirovecii infection in these countries, warrants the need for improved diagnostic and treatment strategies (Hughes, 2005; Roux et al., 2014). This is an open-access article distributed under the terms of the Creative Commons Attribution License . The use, distribution or reproduction in other forums is permitted, provided the original author and the copyright owner are credited and that the original publication in this journal is cited, in accordance with accepted academic practice.

The obtained Au superstructures show closely packed nanocrystal configurations and unexpected physicochemical and optical properties