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In recent years, numerous platforms have been developed for the detection of DNA sequences due to their great importance in biological analysis and application in biomedical fields. In recent two decades, gold nanoparticles (GNPs) have shown great application value in bioanalytical fields owing to their facile preparation methods and excellent optical properties. These properties are utilized to develop many analytical methods including colorimetry, light scattering, scanometry, surface enhanced Raman scattering and chemiluminescence. Colorimetry has received increasing attention due to its incomparable advantages such as simplicity, low cost and amenability. The oligonucleotide-modified GNPs-based colorimetric method for detection of oligonucleotides was first reported by Mirkin and his co-workers. In their system, dispersed oligonucleotidemodified GNPs were assembled into aggregated polymeric networks via hybridization events with complementary target oligonucleotides. An obvious change in the SPR absorption peak between dispersed and aggregated GNPs was observed and led to a significant red-to-blue color change which can be easily visualized by the naked eye. In the utilization of largediameter GNPs to detect targets of lower concentration, the efficiency was limited by the aggregation sedimentation. We also noticed that GNPs have also been widely used for tag amplification by silver staining for the quantitative analysis of biomolecules. Although the staining method opened a new window to ultra-sensitive observation of the assay signals on glass chips or nitrocellulose strips by the naked eye or a light scattering instrument, it indeed increased the complexity of the detection. To this regard, we are now seeking a simple, rapid and homogeneous colorimetric approach without a staining process. Herein, we report a non-aggregated GNPs-based colorimetric strategy that relies on oligonucleotide-modified GNPs and magnetic beads (MBs). We assume that the supernatant of dispersed GNPs is an ideal signal source for a simple colorimetric strategy. In the previously reported aggregated GNPs-based colorimetric assays, the supernatant containing both dispersed and aggregated GNPs was studied. Nevertheless, it has been shown that efforts by using large-diameter GNPs for lower concentration detection of oligonucleotide were limited because of the sedimentation of aggregates that is extremely sensitive to ionic strength. In the previous report, ionic strength must be carefully controlled to avoid the sedimentation of massive aggregates which would cause a non-static endpoint. Furthermore, both the non-aggregated and aggregated GNPs contribute to the absorption from 520 nm to 640 nm, which may interfere with experimental determination. This, to a certain extent, limited their application. Comparatively, taking into account that the distinct absorbance and stability of dispersed GNPs are greatly superior to GNPs aggregates, we believe that using non-aggregated GNPs instead of aggregated GNPs to indicate the existence of target molecules in colorimetry could solve the problem mentioned above. In addition, we have noticed that DNAmodified MBs have been used to detect DNA and protein targets combined with noble metallic nanoparticles. Magnetic particles can be easily removed from the solution and therefore do not take part in the subsequent colorimetric detections. In this study, we make an attempt to achieve higher sensitivity and better linear fit results, along with establishing a more stable GNPs-based colorimetric detection system. Compared with the previous methods, our strategy shows the potential for simultaneous determination of multiple targets in a multi-color homogeneous system. The bioterrorism viruses have brought serious concerns to our society due to their inherent high level of lethality, thus there is an increasing demand for rapid detection of such infectious viruses. As a proof-of-concept experiment, an oligonucleotide sequence associated with the variola virus (smallpox, VV 50 AGTTGTAACGGAAGA–TGCAATAGTAATCAG 30, the fifth G is the mutational site) was chosen as a model target. This sequence has vital application and significance in bioterrorism, and it has been studied in the literature. In this study, we have addressed a simple, rapid, and cost-effective method based on the non-aggregated GNPs for the variola virus detection. Two probes were designed to recognize the target DNA. The first probe was 30 nm GNPs (TEM image shown in Fig. S1, ESIw) Key Laboratory of Analytical Chemistry for Biology and Medicine (Ministry of Education), College of Chemistry & Molecular Sciences, Wuhan University, Wuhan, 430072, China. E-mail: [email protected] w Electronic supplementary information (ESI) available: Experimental details. See DOI: 10.1039/c2cc16741g z These authors contributed equally to the work. ChemComm Dynamic Article Links