The pad was then pasted on the backing card in a way that overlapped the nitrocellulose membrane (High Flow Plus 120 Membrane Card; Millipore). A glass fiber sample pad was placed at the bottom of the backing card to overlap the conjugate pad to facilitate the flow of sample from a specimen vial to the strip. To accelerate migration of the samples through the strip, we used cellulose fiber as an absorbent pad pasted on the backing card opposite the conjugate pad. All pads were cut to make the desired strip shape by using a guillotine cutter (CT300 and ZQ2000; Kin Biotech Co., China). Based on a paper platform, the lateral flow assay is for the detection and quantification of analytes in complex mixtures. In LFA, the sample is added on a test pad and then the results will show up in 5 to 30 minutes.
The stability of colloidal solutions for C-GNPs and their conjugated derivatives depended significantly on their size. Visible precipitates occurred for the average diameter of C-GNPs, which was equal to 47.5 after one to two months of storage . This effect may create worse sensitivity in the assay with these GNPs as a label. This finding is in accordance with earlier presented data about C-GNPs for large diameters that needed additional surface modifications to provide stability . The S-GNPs conjugated with antibodies possess long-time stability of colloidal solutions based on spectral and DLS data in a range of diameters up to 64.5 nm.
Quantitative Tests
In Fig 3 representative gold nanoparticle LFA and phage construct LFA strips for a dilution series of Norwalk VLPs are compared. The top line on each strip is the control line, used to confirm correct flow of liquid and detection agent along the membrane. The lower line is the test line used for the detection of the Norwalk VLPs. The assay specificity was tested using 109 MS2 virus particles instead of VLPs, and also with NeutrAvidin phage with no antibody attached, on captured VLPs. Those negative controls showed no background signal and the strips were indistinguishable from the zero VLPs samples .
The measurements were carried out at 25°C with a count rate of 193.7 kcps at a scattering angle of 173°. The average diameter of the prepared gold nanoparticles was 20 nm, as determined from the dynamic light scattering spectrum (Fig. 1). The performance of immunoassays depends critically upon the use of the optimal antibody sandwich pair with a specific orientation . A range of antibodies were initially checked, both in native and biotinylated form, to confirm binding to our VLPs. Thereafter, all binding antibodies were evaluated in all pairwise combinations in a sandwich ELISA.
Competitive Assays
These assays have the affinity molecule both conjugated to the reporter and immobilized on the test strip. If the analyte is present in the sample, then both the test strip and reporter will bind to it, giving a high contrast line indicative of a positive test. If no analyte is present in the sample, then the reporters do not accumulate on the test line on the strip, indicating a negative test. One type of competition assay will conjugate the affinity molecule on the reporter . If the test analyte exists in the sample, then the reporters will not bind to the test line on the strip indicating a positive test.
- It also ensures better control of gold nanoparticle surface area and consistent flow dynamics across membranes along with high sensitivity from even binding of antibody or multiplexing modifications.
- The results of this study were similar to those recorded by Fusco et al. using a recombinant antigen based ELISA.
- The former format is an “open” system while the latter is a “closed” system.
- We also found that a minimum of 12 μg of goat anti-human IgG or 16 μg of goat anti-human IgA was required to stabilize 1 ml of colloidal gold solution (Fig. 3).
- The present genotype was assigned by a single biosensor, and the results are shown in Figure 6.
Recently, in some researches of LFAs development, fluorescent nanoparticles (quantum dots, fluorescent quenching material, lanthanide, up-converting particles, etc.) are applied rather than colorimetric markers and low detection limits are obtained. The hCG assay had previously been developed using colloidal gold DCN for internal use and demonstration purposes. The same assay materials were used with the Anteo Mix&Go Coupling Kit, 200 nm Magnetic Particles and covalently conjugated magnetic particles. Mix&Go technology helps overcome these issues by creating an activated surface that gently yet strongly binds proteins using metal chelation rather than passive binding and/or covalent chemistry. Mix&Go was developed, through a screening process, to bind antibodies to particles.
LFIA’s components selection was based on manufacturer’s advices and visual inspection of test and control lines results . To avoid unspecific antibody binding to the AuNP-Msg and AuNP-Kex1 conjugates, bovine serum albumin (AppliChem®) and Casein (Sigma®) were studied as blocking agents. A BSA and Casein stock solution at 1 mg.mL–1 were added to 0.06 nM AuNP-RSA conjugates in solution at increased molar ratios ranging from 0 to 10 with AuNP-Kex1 conjugates and from 0 to 50 with AuNP-Msg conjugates, producing AuNP-RSA-BSA and AuNP-RSA-Casein conjugates. The incubation was performed during 90 min at 4°C, the non-bound blocking agents were removed by centrifugation and the pellets prepared for agarose gel electrophoresis. Similarly to what was done with the AuNP-RSA conjugates, the molar ratio plateau was selected through duplicate experiments for each blocking agent.
For C-GNP, the position of the maximum extinction spectra depends on the amount of sodium citrate added during synthesis. It reached 518 nm for C-GNP-1, 520 nm for C-GNP-2, 527 nm for C-GNP-3, 532 nm for C-GNP-4, and 536 nm for C-GNP-5. The shift in the plasmonic peak to a longer wavelength range is indicative of a progressive increase in nanoparticle size. In the case of S-GNPs, extinction maximums reached values ranging from 522 to 563 nm, depending on the nanoparticle size. Note that the plasmonic peak of S-GNP had a lower width compared to C-GNP, which is indicative of higher sphericity and narrow size distribution. The lower end of a test strip was dipped into an aliquot of the sample (70 μL) for 1 min and then placed on a horizontal surface.
Gold Nanoparticle
These signals have low background noise since there are generally no magnetic materials in the environment or in the tested samples. The study confirmed the Anteo Mix&Go based hCG assay is significantly more sensitive than the covalently conjugated magnetic particle based test using the same critical reagents. The hCG assay conducted with the pad cutter Anteo Mix&Go particles used half the amount of antibody to achieve five times more sensitivity than the covalently conjugated assay. The limit of detection for the Anteo Mix&Go assay was ~25 mIU/mL in urine for the visual and reader based results. The limit of detection for the covalently conjugated hCG assay was ~100 mIU/mL in urine. As a reference, the limit of the detection for the colloidal gold assay is at ~25 mIU/mL in urine.
In general, Nanopartz is your one stop source for gold colloidal nanoparticles and gold nanomaterials. This book is a comprehensive review of the role of gold nanoparticles in analytical nanoscience and nanotechnology, with chapters devoted to their synthesis, physico-chemical characteristics, derivatization and potential toxicity. The main microscopic, spectroscopic and separation techniques for the characterization are reviewed as well as the developments for their determination in environmental, biological and agrifood samples.
The mixture was applied to the conjugate pad of the biosensor, which was then immersed into the developing solution. The solution migrated along the LFB by capillary action and rehydrated the anti-biotin-conjugated gold nanoparticles. The hybrids were captured from immobilized anti-digoxigenin or anti-fluorescein at the respective test zone (TZ-S/TZ-R) of the biosensor and interacted with the biotinylated probes.
80 nm gold nanoparticles or 150 nm gold nanoshells can provide increases in sensitivity compared to 40 nm gold nanoparticles. One important consideration for using 80 nm gold or the gold nanoshells is that because the per particle absorption is higher, the number of particles at a given OD is lower than the 40 nm colloids. This means that a larger volume or higher concentration of nanoshells may be necessary in a fully optimized assay (typically 2–5 times the volume or OD required for gold nanoshells, compared to the 40 nm gold). The scientists at NanoHybrids are available to answer any questions you might have related to choosing the right gold nanoparticles, buffers and antibodies for assay development.
The presented study demonstrated a significant improvement in lateral flow immunoassay sensitivity by using superspherical gold nanoparticles instead of the commonly used quasispherical citrate-capped gold nanoparticles via the Turkevich–Frens technique. The known modifications of C-GNPs synthesis do not give such monodispersity as the super-spherical preparation under consideration in this paper. The proposed superspherical GNPs have advantages in unified size and shape that are unattainable for alternative preparations. Therefore, these GNPs were compared with the common Turkevich–Frens C-GNPs. They caused a big gain in sensitivity in the immunochromatographic analysis. The freshly prepared S-GNPs and C-GNPs demonstrated good colloidal stability with reproducible adsorption spectra and the absence of visible precipitates, independent of their size.
In principle, any colored particle can be used, however latex or nanometer-sized particles of gold are most commonly used. The gold particles are red in color due to localized surface plasmon resonance. Fluorescent or magnetic labelled particles can also be used, however these require the use of an electronic reader to assess the test result. LFAs usually have a long shelf life and do not need to store in the refrigerator, which makes LFA ideal for use in developing countries. Besides, the visual result is usually clear and easily distinguished, which means no additional specific equipment is required.
Proportions of reagents for gold nanoparticles preparation using the Frens method. Despite the available wide range of GNP sizes, the question of the optimal size for LFIA is still under debate, including the impact of GNPs’ shape on this choice. Basically, the large sizes of GNPs allow a target molecule to be labeled with a large number of gold atoms. However, the literature does not give a reason to believe that the differences in detection limits of immunochromatography are determined primarily and exclusively by GNP size. The comparative consideration of GNPs with different diameters in immunochromatography indicates that the change in LFIA sensitivity with an increase in GNP size is nonmonotonic.