Supporting Information

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1 Supporting Information Wiley-VCH Weinheim, Germany

2 A Simple and Sensitive Dip Stick Test in Serum Based on Lateral Flow Separation of Aptamer-Linked Nanostructures Juewen Liu, Debapriya Mazumdar and Yi Lu Department of Chemistry, Beckman Institute for Advanced Science and Technology University of Illinois at Urbana-Champaign Urbana, IL, 61801, USA 1

3 1. Assembly of a lateral flow device For all experiments, the Millipore Hi-Flow TM Plus Assembly Kit (Millipore Corporation, Bedford, MA) was used. The kit contains a Hi-Flow Plus Cellulose Ester Membrane with a nominal capillary flow time of 90 seconds/4 cm and a nominal membrane thickness of 135 µm directly cast onto 2 mil polyester backing and placed on an adhesive card. The length of the membrane along the flow direction is 2.5 cm on the backing. The absorption pad and wicking pad were cut from Millipore cellulose fiber sample pads, and the conjugation pad was cut from the Millipore glass fiber conjugate pad. The absorption pad, wicking pad, and conjugation pad were attached to the adhesive card of the membrane in a way as shown in Figure S1. The overlap for each pad was ~2 mm, and the width was ~8 mm cut by a paper cutter. 8 mm Absorption pad 15 mm Membrane 25 mm Conjugation pad 13 mm Wicking pad 15 mm Figure S1. Assembly of a lateral flow device. 2

4 2. Optimization of NaCl concentration for drying the adenosine aptamer-linked aggregates Because the nanoparticle aggregates were stabilized by DNA base pairing interactions, ionic strength may play a very important role on the properties of the aggregates. Therefore, we studied was the effect of NaCl concentration during drying. The aggregates were dispersed in varying concentration of NaCl (200, 300, or 500 mm, all with 8% sucrose), and the devices were tested with either no adenosine or 0.5 mm adenosine. An untested device is also presented in Figure S2 for comparison. At all the three salt concentrations, adenosine induced a red band; while no band was observed in the absence of adenosine. 500 mm NaCl gave the lowest band intensity, and 200 mm was determined to be the optimal ionic strength. Figure S2. The effect of NaCl concentration during drying. 3. Test the sensor stability on storage After storing at room temperature in air for a week, the device has retained its function because no red band was observed in the absence of adenosine (Figure S3, left panel), and red bands were formed in the presence of adenosine (right panel). It appeared that two bands were present in the positive test. The cause of dual bands formation is unclear and will be a subject for future investigations. 3

5 Figure S3. Performance of the device after storing at room temperature for a week. 4. Understand the reason for failure to produce a red color when dried in the absence of sucrose In Figure 2a of the paper, if the untested strips were compared (i.e., the first strip in each group), increasing the sucrose concentration led to a more intense color on the conjugation pads, even though the same amount of aggregates were applied on each pad. This result indicated that with sucrose the nanoparticles were still in their native states because strong individual and coupled surface plasmons still existed, and the aggregates did not collapse due to drying. To identify the reason why no signal was observed for the directly dried samples (without sucrose), distilled water was used to soak the conjugation pad. Under the soaking condition, the NaCl concentration of the system would be so low (~20 mm) that any DNA-linked structures will be disassembled. However, no red color was produced (Figure S4, left). Instead, only purple color was observed, suggesting that the nanoparticles already formed irreversible aggregates by touching each other (like many small particles melted into a large aggregate). As a control, the aggregates dried with sugar showed a red color upon addition of water (Figure S4, right). Unprotected colloidal gold particles tend to aggregate irreversibly upon addition of electrolytes. The salt stability of the nanoparticles increased significantly by coating with DNA. [1,2] During drying, with the evaporation of water, the salt concentration drastically increased (from 200 mm to infinity), and even DNA-functionalized nanoparticles could be damaged. 4

6 Figure S4. Left: directly dried (no sucrose) soaked in water. Right: dried with 8% sucrose and soaked in water. 5. Comparison between the aptamer-based and antibody-based lateral flow detections Although the same device components were used here as the traditional antibody-based test strips, the detection mechanism behind the scene is completely different. In a typical antibodybased pregnancy test, for example, the target protein (HCG) is first captured on the conjugation pad by a colloidal gold attached antibody. The whole complex then moves alone the membrane and is finally captured by a second antibody for HCG on the membrane to form a red line. As a result, HCG is sandwiched between the two antibodies. For such an assay format to work, the target has to be able to move along the membrane. In contrast, the aptamer-based system described here allows all recognition reactions to occur on the conjugation pad, and the color reporting group (biotinylated nanoparticles) did not contain target molecules. Therefore, even for some molecules that cannot migrate along the membrane (i.e. too large in size), the aptamerbased method should still be applicable for their detection. References [1] C. A. Mirkin, R. L. Letsinger, R. C. Mucic, J. J. Storhoff, Nature 1996, 382, 607. [2] Z. Li, R. Jin, C. A. Mirkin, R. L. Letsinger, Nucleic Acids Res. 2002, 30,