Rapid signal enhancement method for nanoprobe-based biosensing

The introduction of nanomaterials as detection reagents has enabled improved sensitivity and facilitated detection in a variety of bioanalytical assays. However, high nanoprobe densities are typically needed for colorimetric detection and to circumvent this limitation several enhancement protocols have been reported. Nevertheless, there is currently a lack of universal, enzyme-free and versatile methods that can be readily applied to existing as well as new biosensing strategies. The novel method presented here is shown to enhance the signal of gold nanoparticles enabling visual detection of a spot containing <10 nanoparticles. Detection of Protein G on paper arrays was improved by a 100-fold amplification factor in under five minutes of assay time, using IgG-labelled gold, silver, silica and iron oxide nanoprobes. Furthermore, we show that the presented protocol can be applied to a commercial allergen microarray assay, ImmunoCAP ISAC sIgE 112, attaining a good agreement with fluorescent detection when analysing human clinical samples.


-Dynamic light scattering
Dynamic light scattering (DLS) measurements were performed on a Delsa Nano Beckman Coulter instrument at 25°C. Each sample was diluted in H 2 O, and was measured three times, combining 10 runs per measurement ( Figure S2). IONPs before modification with IgG; (h) after modification with IgG. Each sample was diluted in water at pH 7 and was measured three times, combining 10 runs per measurement.
The effect on the diameter of the different sets of NPs was studied with dynamic light scattering (DLS). Prior the antibody modification, the AuNPs were modified with PEG-COOH molecules 3 (5 kDa), to provide anchor points for the antibody coupling. As observed by TEM, the synthesised AuNPs had an average diameter of 35 nm that shifted to approximately 50 nm after the decoration with PEG molecules ( Figure S2a). The modification of these NPs with IgG resulted in their average diameter to increase to approximately 60 nm, as expected 1 ( Figure S2b) Similarly, for the other sets of NPs an average increase of approximately 10 nm in diameter was observed after the modification with the IgG (Figure S2c-h)."

Characterisation and optimisation of enhancement solution
For gold growth to occur primarily in a defined region of the arrays where NPs are present it was required to optimize conditions for the reduction of Au 3+ to Au 0 . There are three main components involved in promoting the reduction of the gold salt: HAuCl 4 (gold precursor), H 2 O 2 and MES buffer (reduction agents). The concentration of all three and the pH at which the reduction was allowed to occur was characterized. Optimising the system allowed gold colloid formation in solution prior to 5 minutes of incubation to be avoided since this would turn the solution from colourless to dark purple, compromising the signal/noise ratio. While spectrometric analysis was used to follow the arise of a SPR peak, reduction of HAuCl 4 could also be determined by observation of the colour change of the solution. From optimizations, conditions where no SPR peak was observed within 5 minutes were considered for further study.
This time mark allowed us to assure that the conditions were favourable for gold nanoparticle formation while still being slow enough to favour the deposition of reduced Au 0 onto already existing nanoparticle seeds. HAuCl4, 10mM MES at pH 6 (b) and 5mM HAuCl4, 10mM MES at pH 11 (c) measured every 5 minutes for a total incubation time of 30 minutes. Figure S3c shows that at pH 11 gold clusters start to form in solution. At pH 3 ( Figure S3a) and pH 6 ( Figure S3b) no SPR was detected, demonstrating that no gold nanoparticles were formed. Due to incompatibility with normal assay environments where the method could be applied, pH 11 conditions were not evaluated further.   The suggested enhancement effect was required to be rapid while minimizing the gold growth outside sensor regions already harboring nanoparticle detection probes. For proof of concept the following sets of conditions were allowed to incubate with a paper array where printed protein G spots had been detected by incubation with IgG-AuNPs ( Figure S6).  The change in colour from red to purple observed in the arrays was also observed when a solution of AuNPs was incubated with the enhancement solution ( Figure S7). The SPR was observed to red-shift, characteristic of the formation of aggregates and/or increase in size of the nanoparticles. Although the absorbance intensity decreases with the formation of these larger, anisotropic nanoparticles, optically the intensity of the colour increases. This effect is due to the higher light scattering capability of these anisotropic nanoparticles.

Number of nanoparticles quantification
The volume and concentration of the gold nanoparticles' solution printed onto the paper support were taken into account for the estimation of the approximate number of nanoparticles.

Proof-of-concept with commercial IgE detection kit
Four serum samples were analysed with Thermo-Fischer's ImmunoCap ISAC array using fluorescent detection (ISAC-F) and using the presented gold enhancement detection (ISAC-G) to detect allergen-specific IgE.