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 500fgmL-1 of 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 2 and 3above the negative signal. Around 5pgmL-1 and 50

pgmL-1 can be defined as LOD using 2and 3respectivelyWe 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 ngmL-1 synuclein, Figure S-2 shows the assay titration

series up to 5 µgmL-1 synuclein. Over 500 ngmL-1, the signal reached a plateau.

Figure S-1. Determination of limit of detection. Level of 2 and 3are 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 ngmL-1 and 50 pgmL-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 synuclein in CSF. synuclein was spiked into

human CSF and two independent measurement series were performed. Final

concentrations of synuclein in CSF were 35pgmL-1 to 500 ngmL-1 at the first

trial, and 17pgmL-1 to 333 ngmL-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 pgmL-1 to 500 ngmL-1 and the second from

17 pgmL-1 to 500 ngmL-1. Spiked samples were initially prepared at a high

concentration (over ten µgmL-1) and diluted by CSF sample to a final concentration

ranging from a few tens of pgmL-1 to a few hundred ngmL-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 pgmL-1. Figure 3 also shows a broad dynamic

range over 4 orders of magnitude, from 17 pgmL-1 to 500 ngmL-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.