Electronic Supplementary Material
A porous silicon immunoassay platform for fluorometric determination of α-synuclein in human cerebrospinal fluid
Sangwook Lee1,4*, Edina Silajdžić2, Hon Yang3, Maria Björkqvist2, Soyon Kim4, Ok Chan Jeong5, Oskar Hansson6, Thomas Laurell3,4*
1 Bioengineering Laboratory, Riken Institute, 351 0198, Saitama, Japan
2Department of Experimental Medical Science, Lund University, 221 00, Lund, Sweden
3Dept. Measurement Technology and Industrial Electrical Engineering, 221 00, Lund University, Lund, Sweden
4Department of Biomedical Engineering, Dongguk University, 100 715, Seoul, Korea
5Department of Mechanical Engineering, Inje University,633 165, Busan, Korea
6Department of Clinical Sciences Malmö, Lund University, 205 02, Malmö, Sweden
To determine the limit of detection (LOD), we performed the immunoassay down to 500fg mL-1 of a-synuclein, and assayed fluorescent microarray images were compared to that of the negative control. As shown in figure S-1, to determine the LOD we marked 2s and 3s above the negative signal. Around 5pg mL-1 and 50 pg mL-1 can be defined as LOD using 2s and 3s, respectively. We selected 3 standard deviations above the negative signal as the LOD in this assay.
The dynamic range was defined as the signal from lowest detectable level of -synuclein to highest one before signal became saturated. The microarray signal became saturated above 500 ng mL-1 a-synuclein, Figure S-2 shows the assay titration series up to 5 µg mL-1 a-synuclein. Over 500 ng mL-1, the signal reached a plateau.
Figure S-1. Determination of limit of detection. Level of 2s and 3s are plotted.
Figure S-2. Saturation curve of α-synuclein, The spot signals are saturated over 500 ngmL-1 α-synuclein.
Figure S-3 shows the comparison of assay performance between a conventional α-synuclein ELISA and our developed P-Si microarray platform. The detection limit of the standard ELISA was subsequently compared with the P-Si microarray assay (Figure S-1), and a two order of magnitude lower α-synuclein level could be detected in our system (LOD was 2.5 ng mL-1 and 50 pg mL-1 by conventional ELISA and our P-Si microarray system, respectively).
Figure S-3. Comparison of assay performance between a conventional α-synuclein ELISA and our P-Si microarray immunoassay platform
Figure S-4 Titration series of asynuclein in CSF. asynuclein was spiked into human CSF and two independent measurement series were performed. Final concentrations of asynuclein in CSF were 35pg mL-1 to 500 ng mL-1 at the first trial, and 17pg mL-1 to 333 ng mL-1 at the second trial.
Figure S-4 shows the assay performance between two repeated experiments, where good correlation between samples was observed. The first trial was performed at α-synuclein titrations ranging from 35 pg mL-1 to 500 ng mL-1 and the second from 17 pg mL-1 to 500 ng mL-1. Spiked samples were initially prepared at a high concentration (over ten µg mL-1) and diluted by CSF sample to a final concentration ranging from a few tens of pg mL-1 to a few hundred ng mL-1. As a negative control we assayed unspiked CSF to compare the signal between the negative control and spiked sample. The fluorescent signal in the lowest concentration of the spiked samples was at least two standard deviations higher than that of the negative control giving an LOD in the order of 101 pg mL-1. Figure 3 also shows a broad dynamic range over 4 orders of magnitude, from 17 pg mL-1 to 500 ng mL-1. The intensity difference of each observed titration was less than 15%. The CV of each assay on CSF samples was less than 20 %, indicating a performance that is sufficient for future clinical studies. It was also noted that the signal intensities of α-synuclein spiked CSF samples were significantly lower (about 40%) than the same α-synuclein levels spiked in PBS buffer.