Rapid signal enhancement method for nanoprobe-based biosensing

www.nature.com/scientificreports Correction: Author Correction OPEN Rapid signal enhancement method for nanoprobe-based biosensing Jorge T. Dias1, Gustav Svedberg Gantelius1 Received: 30 March 2017 Accepted: 21 June 2017 Published online: 28 July 2017 1 , Mats Nystrand2, Helene Andersson-Svahn1 & Jesper 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. Biosensing applications have benefited considerably from the introduction of nanomaterials such as gold nanoparticles (AuNPs) or iron oxide nanoparticles (IONPs)1. Optical properties such as surface plasmon resonance in the case of AuNPs or magnetic properties of the IONPs have contributed to improved sensitivity in biodetection applications2, 3. More generally, the large surface to volume ratio of affinity labelled nanomaterials together with the myriad of protocols for their surface decoration with various ligands have made such materials interesting tools for the development of more efficient and sensitive sensors4. Nonetheless, large nanoprobe numbers are typically still required to achieve detectable signals when used as biosensing tools. For instance, in the case of 40 nm AuNPs approximately 90 million nanoparticles (NPs) were required to obtain a colorimetric UV-vis spectroscopy detection5. To circumvent sensitivity limitations due to low nanoprobe densities, several strategies of signal amplification have been developed6. Such enhancement techniques are often based on either interparticle aggregation or staining of the nanoprobe with a material that allows improved signal acquisition. The former relies on the gathering of a higher number of nanoparticles on the nanoprobe site after the detection has occurred. Increasing the density of nanoparticles allows visual signal acquisition or UV-Vis measurement7. The gathering of extra nanoparticles on the detection site can be achieved by targeting a second set of nanoparticles to the initial detection nanoprobes. The effectiveness of the signal enhancement will be intrinsically associated with the recognition capacity of the nanoprobes by this second set of nanoparticles. Alternative enhancement strategies such as silver staining of either gold or silver nanoprobes8, 9 rely on the reduction of silver ions onto the surface of the nanoparticles creating a silver precipitation film detectable visually or by UV-Vis analysis10. Such techniques’ specificity is determined by the presence or absence of either AuNPs or silver nanoparticles (AgNPs), thus it is possible to apply it without the restrictions associated with an interparticle aggregation method. Thus far, silver staining techniques have only be shown to be applied to either AuNPs or AgNPs10, 11. Zayats et al.12 showed in 2005 that it was possible to potentiate the surface plasmon resonance signal of AuNPs by promoting the reduction of Au ions onto the surface of existing gold nanoprobes. In their work, hydrogen peroxide generated by glucose oxidase catalysis and cetyltrimethylammonium chloride were used to achieve the reduction of the chloroauric acid. However, only enhancement of gold nanoprobes was studied, and a spectrophotometer was required for signal acquisition. Wang et al.13 reported a colorimetric-based enhancement method where a sandwich based assay was developed for detection of human IgG using AuNPs. After detection, HAuCl4·4H2O and NH2OH·HCl were added to the system, forming a new gold layer on the surface of the AuNPs. These enlarged AuNPs showed a peroxidase-like catalytic ability against the substrate 1 Division of Proteomics and Nanobiotechnology, Science for Life Laboratory, KTH Royal Institute of Technology, Stockholm, Sweden. 2Global Research and Development, Thermo Fisher Scientific IDD, Uppsala, Sweden. Correspondence and requests for materials should be addressed to J.G. (email: ) Scientific Reports | 7:6837 | DOI:10.1038/s41598-017-07030-0 1 www.nature.com/scientificreports/ 3,3′,5,5′-tetramethylbenzidine (TMB) and a bright blue colour was observed. With this enhancement strategy, the authors were able to detect as low as 3 × 10−10 g·mL−1 of human IgG. More recently Stevens et al.14 developed a plasmonic ELISA-based assay where AuNPs allowed ultrasensitive visual detection of a prostate-specific antigen (PSA). The detection strategy consisted of a traditional ELISA followed by the labelling of the primary antibody with a secondary antibody previously modified with catalase. The sensor was immersed in a solution that contained H2O2 and chloroauric acid. If the secondary antibody carrying catalase did not detect the primary antibody, the H2O2 in solution was not consumed and the chloroauric acid was reduced at a fast rate. In the presence of analyte and formation of the complex primary antibody – secondary antibody modified with catalase, the enzyme could consume H2O2 and so decrease the reduction kinetics of the chloroauric acid. This difference in the speed at which chloroauric acid was reduced affected the morphology of the AuNPs formed. Higher concentrations of H2O2 allowed the formation of quasi-spherical monodispersed gold nanoparticles with a red colour, lower concentrations of H2O2 yielded ill-defined aggregate-like structures that showed a blue colour. This red/ blue colour dichotomy allowed the authors to detect as low as 10−18 g·mL−1 of PSA in whole serum. Similar to the work of Zayats et al., the enhancement effect demonstrated by Stevens et al. was promoted by the reduction of chloroauric acid to gold ions by hydrogen peroxide. Here, the concentration of available hydrogen peroxide was determined by the activity of catalase and subsequently correlated to the concentration of the analyte being detected. The need to modify a detection antibody with an enzyme and control of the catalytic activity are two limiting factors for the universality of the method proposed by Stevens et al. Furthermore, the conditions used do not account for the formation of gold clusters independently of the reporte (...truncated)