Effective ligand selection and optimisation of bioconjugation conditions is vital for the development of a successful lateral flow assay. Kimialys have utilised their expertise in Surface Plasmon Resonance imaging (SPRi), combined with their K-One surface coating to develop a protocol to screen ligand candidates and their immobilisation on gold biochip surfaces before transfer to a lateral flow format. This protocol enables both time and material savings to be made during Lateral Flow Assay (LFA) development.
The orientation, and therefore the accessibility, of ligands such as antibodies, proteins, peptides or oligonucleotides in SPRi or LFA is a key factor in order to enhance sensitivity and specificity of the assay. Orientation of the immobilised ligands on a biosensor surface depends largely on the immobilisation conditions, which often differ from one ligand to another.
This proof-of-concept study demonstrates that an SPRi screening approach could be directly implemented in the preparation of gold nanoparticle bioconjugates for LFA, due to a direct correlation between the optimised immobilisation conditions on K-One pre-functionalised SPRi biochips and on K-One functionalised gold nanoparticle surfaces used in LFA.
SPRi – ligand immobilisation and detection of the target molecule Three monoclonal antibodies, mAb1, mAb2 and mAb3 were immobilised on a K-One gold biochip surface under four different conditions. The three antibodies were diluted to 50 µg/ml in four different solutions (C1, C2, C3 and C4) and deposited in a microarray format on a single EDC/NHS activated K-One SPRi biochip (Figure 1).
Figure 1 – Captured microarray map image of the biochip surface after ligand immobilisation under conditions C1, C2, C3 and C4 (A), and differential image taken during injection of the target molecule (B).
The level of antibody density on the biochip surface was assessed on a microarray map (A) by comparing the brightness of the antibody spots under the various conditions. Conditions C1 and C2 were the most effective in enabling high antibody density, whereas condition C3 showed a poorer spot contrast with the background, and C4 showed no contrast demonstrating lower antibody density on the chip. The contrasts observed for mAb1 and mAb3 in conditions C1 and C2 were similar, while the surface density of mAb2 appeared to be significantly lower.
After injection of the target molecule, its interaction with the three different antibodies was visualised on the second image (B). In this format, the greater the contrast between the spot and background, the greater the capture efficiency of the target by the antibodies. The capture efficiency was highest when immobilisation was performed under conditions C1 and C2, indicating these antibody surface densities are optimal compared to conditions C3 and C4. Visually, the capture efficiency of mAb1 was slightly higher than mAb2, with mAb2 giving rise to a stronger contrast than mAb3. Whilst the surface density of mAb3 was higher than for mAb2 (A), the capture efficiency of mAb3 could not be optimised in the tested conditions, as demonstrated by the feint contrast in image (B). Whilst the surface density of mAb2 in conditions C1 and C2 is lower than that of mAb3, its orientation on the surface appears to be optimal as the capture efficiency is higher than that of mAb3. Therefore, the combined surface density and orientation of mAb1 and mAb2 in conditions C1 and C2 led to a high capture efficiency of the target molecule.
Figure 2 – SPRi sensorgrams showing injection of 0.5 ug/ml of the target molecules across the biochip surface where mAb1, mAb2 and mAb3 were immobilised under conditions C1, C2, C3 and C4.
The SPRi sensorgram signal intensity (Figure 2) is directly proportional to the capture efficiency of the target molecule, therefore conditions C1 and C2 displayed a higher capture efficiency for the 3 antibodies. In conditions C1 and C2 the orientation of mAb1 and mAb2 was optimal. Lower signal intensity in the SPRi sensorgram was demonstrated by mAb3, confirming that the tested immobilisation conditions were not optimal for this antibody. Immobilisation with condition C4 led to a very weak signal intensity with all 3 antibodies, confirming poor immobilisation under this condition.
Conclusions from the SPRi experiment
- Conditions C1 and C2 presented the most efficient immobilisation conditions in terms of both surface density and orientation on SPRi biochip. It is therefore anticipated that these conditions will lead to more efficient bioconjugation on gold nanoparticles.
- When effectively immobilised, mAb1 showed a greater capture efficiency than mAb2. Capture efficiency with mAb3 was lower, almost certainly due to less optimal antibody orientation. It is anticipated that mAb1 and mAb2 immobilised on gold nanoparticles will lead to better detection in a dipstick rapid test.
Ligand immobilisation on K-One gold nanoparticles (K-One AuNP)
The three ligands mAb1, mAb2 and mAb3 were immobilised on K-One 40 nm AuNP under the same conditions (C1, C2, C3 and C4). Bioconjugation was performed by mixing 5 µg of antibody with 1 ml of K-One 40 nm carboxylated AuNP at OD=1. A stable colloidal AuNP solution characterised visually by a pink-red colour was observed using conditions C1 and C2. However, conditions 3 and 4 appeared to cause aggregation of the particles, as determined by their purple appearance. This observation was confirmed by UV-visible spectrophotometry where aggregation of AuNP is characterised by a red shift and broadening of the plasmon peak (Figure 3).
AuNP K-One bioconjugates: UV-visible spectrophotometry
Figure 3 – Normalised absorbance spectra of mAb1, mAb2 and mAb3 bioconjugate solutions prepared under conditions C1, C2, C3 and C4.
The 3 bioconjugates prepared under conditions C1 and C2 did not lead to a red shift of the plasmon peak. In addition, negligible broadening of the plasmon peak was observed, which indicated good stability of the bioconjugates in solution. Conversely, the bioconjugates prepared with conditions C3 and C4 both demonstrated a red shift and broadening of the plasmon peak, confirming the aggregation of nanoparticles visually observed. The optimum conditions C1 and C2 for immobilisation of the 3 ligands were identical for both SPRi and gold nanoparticle bioconjugation.
Characterization of target/bioconjugate interaction in a dipstick assay
Further investigation was carried out using conditions C1 and C2 only, as conditions C3 and C4 had resulted in unstable bioconjugates. In a sandwich dipstick format, the test line was composed of an antibody able to interact with the bioconjugate-target complex. When the target and bioconjugates were premixed and applied to the dipstick, the same result was achieved, with mAb3 displaying a weaker interaction (Figure 4, A). When the intensity of the test lines was measured on an iPeak reader (Figure 4, B) it was possible to discriminate between C1 and C2 and mAb1 and mAb2. Condition C2 led to a slightly higher signal, while mAb1 gave a stronger band compared to mAb2, in agreement with the SPRi results.
Figure 4 – Sandwich dipstick membranes after migration of the bioconjugates for the detection of 1 ng/ml of the target molecule (A) and intensity of the test lines measured with an iPeak reader (B).
Through this short study, Kimialys demonstrated that the screening of ligand and immobilisation conditions using SPRi could be directly translated to the preparation of gold nanoparticle bioconjugates for LFA applications.
This SPRi screening approach can be extended to screen several dozen ligands and conditions simultaneously, permitting optimisation of bioconjugates for any LFA test in a single day. This proof of concept sets the basis for larger scale experiments, where the generated savings in time and reagents would be substantial in comparison to lengthy traditional approaches.
Kimialys provide services and ready-to-use biochips and nanoparticles, coated with customized surface chemistries, to SPR users and LFA developers looking for innovative immobilisation methods. Contact us for more information on how we can help.