Enhancing Nanoparticle-Based Visible Detection by Controlling the Extent of Aggregation

SUBJECT AREAS: SENSORS MODELLING AND THEORY OPTICAL MATERIALS AND DEVICES Enhancing Nanoparticle-Based Visible Detection by Controlling the Extent of Aggregation Seokwon Lim1,2,3, Ok Kyung Koo4, Young Sang You2, Yeong Eun Lee2, Min-Sik Kim5, Pahn-Shick Chang1,2, Dong Hyun Kang1,2, Jae-Hyuk Yu6, Young Jin Choi1,2 & Sundaram Gunasekaran3 NANOBIOTECHNOLOGY 1 Received 4 April 2012 Accepted 23 May 2012 Published 13 June 2012 Correspondence and requests for materials should be addressed to Y.J.C.(. kr) or S.G. (guna@ wisc.edu) Center for Agricultural Biomaterials, Seoul National University, Seoul, 151-742, Republic of Korea, 2Department of Agricultural Biotechnology, Seoul National University, Seoul, 151-742, Republic of Korea, 3Department of Biological System Engineering, University of Wisconsin-Madison, Madison, WI 53706, USA, 4Center for Food Safety, Dept. of Food science, University of Arkansas, Fayetteville, AR 72704, USA, 5Division of Pharmaceutical sciences, school of Pharmacy, University of Wisconsin-Madison, Madison, WI 53705, USA, 6Department of Bacteriology, University of Wisconsin-Madison, Madison, WI 53706, USA. Visible indication based on the aggregation of colloidal nanoparticles (NPs) is highly advantageous for rapid on-site detection of biological entities, which even untrained persons can perform without specialized instrumentation. However, since the extent of aggregation should exceed a certain minimum threshold to produce visible change, further applications of this conventional method have been hampered by insufficient sensitivity or certain limiting characteristics of the target. Here we report a signal amplification strategy to enhance visible detection by introducing switchable linkers (SLs), which are designed to lose their function to bridge NPs in the presence of target and control the extent of aggregation. By precisely designing the system, considering the quantitative relationship between the functionalized NPs and SLs, highly sensitive and quantitative visible detection is possible. We confirmed the ultrahigh sensitivity of this method by detecting the presence of 20 fM of streptavidin and fewer than 100 CFU/mL of Escherichia coli. V isible indication based on the aggregation of colloidal nanoparticles (NPs) is highly advantageous for rapid on-site detection of biological entities, which even untrained persons can perform without specialized instrumentation1-9. However, since the extent of aggregation should exceed a certain minimum threshold to produce a visible change, further applications of this method have been hampered by insufficient sensitivity10 or certain limiting characteristics of the target11. Here we report a simple and facile biosensing strategy controlling the extent of NPs aggregation, which provides several strategic options to design and to enhance the visible detection of wide-ranging targets. Between the two well-known mechanisms for aggregating NPs12, interparticle crosslinking mechanism allows controlling the extent of aggregation via a quantitative relationship between the functionalized NPs (f-NPs) and the linkers13,14. Advances in nanotechnology help to ensure the reproducibility in controlling the extent of aggregation14. Further, the crosslinking mechanism is more specific12,15 and less influenced by the sample conditions than the non-crosslinking mechanism2,8,16,17, which obviates sophisticated sample preparation steps. The extent to which f-NPs aggregate via crosslinking is determined by the number of linkers, available binding sites, and NPs in the system (see supplementary information). For a given number of f-NPs with multiple binding sites, there is a range of linker concentration, which can induce their aggregation to an extent over a minimum threshold sufficient to exhibit a visible color change (Fig. 1a, Region 2). Otherwise, color change is imperceptible due to either the lack of sufficient linkers to cause aggregation (Region 1) or the presence of excess linkers (Region 3), which prevents the bridging of the NPs. When the number of effective linkers (nLK) available to bridge the fNPs is altered, the range of linker concentration exhibiting visual color change (REVC) takes different set of values from that for the control system. Thus, once the control REVC is well established, even a slight change in nLK can produce visually distinguishable difference, especially at the boundaries of the control REVC, and the extent of difference in REVC quantitatively indicates the change in nLK. Accordingly, the mechanism for producing an indication signal is independent from that for target recognition, which affords the opportunity to amplify the signal and allows designing quantitative colorimetric detection systems for wide-ranging targets. SCIENTIFIC REPORTS | 2 : 456 | DOI: 10.1038/srep00456 1 www.nature.com/scientificreports Figure 1 | (a) Linker concentration can be grouped into three regions for a given amount of NPs based on the observable visual color change. Numbers on the bottle lids represent the number of linkers. (b) Different possible SL designs (top row) and the corresponding effects of switching off SLs to reduce the nLK by 1 at low (middle row) and high (bottom row) concentration of SL. To attain detection based on the proposed scheme, we introduce the concept of switchable linkers (SLs) to alter the nLK based on the presence of target. The SL is an element allowing multiple specific bindings, which can be selectively enabled or disabled to bridge fNPs. When some SLs are disabled (switched off) to function as a linker, the nLK decreases changing the chance for the f-NPs to aggregate to an expected extent. The consequence of switching off SLs is that some binding sites on NPs, which could have been occupied by those SLs, become ‘‘vacant’’. Because the switched-off SLs remain in the system and can compete for the binding sites, the vacating effect varies according to the SL design (Fig. 1b). When nLK is less than a certain minimum, the reduced nLK by switching SL off should lower the extent of aggregation regardless of the SL design. Because the binding sites compete for linkers to bridge f-NPs, the nLK determines the extent of aggregation and the vacating effect is negligible. Therefore, at the low end of REVC, where the fewest number of SLs is present to induce visible change in the control system, the effect of switching off an SL is to reduce nLK by 1 lowering the possibility to bridge the f-NPs. However, when nLK exceeds a certain maximum, the linkers compete to occupy the binding sites for conjugating f-NPs. Thus, the number of binding sites available to bridge NPs determines the extent of aggregation, and the vacating effect of switching SL off rather than the change in nLK dictates the change in the extent of aggregation. Subsequently, at the high end of REVC, where the number of SLs present is just over the maximum to induce visible change, the effect of switching SL off on the e (...truncated)