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Electronic Supplementary Material (ESI) for Analyst

A 8-minute colorimetric paper-based reverse phase vertical flow serum microarray for screening of Hyper IgE syndrome

Philippa Reuterswärd, Jesper Gantelius and Helene Andersson Svahn

Division of Proteomics and Nanobiotechnology, Science for Life Laboratory, KTH Royal Institute of Technology, Sweden

Address correspondence to: Prof. Helene Andersson Svahn Division of Proteomics and Nanobiotechnology KTH Royal Institute of Technology Science for Life Laboratory Box 1031 SE-171 65 Solna Sweden Tel: +46852480096 Email: helene.andersson.svahn@scilifelab.se A B C

Supplementary Figure 1. IgE dilution array 1A) Layout of the IgE dilution array and sample orientation. Red spots are layout spots. Blue spots are serum samples spiked with IgE in dilution series and the light blue sample is IgE negative serum without spiked IgE. 1B) Color scan of the IgE dilution array of the same array at optimized conditions. 1C) Inverted image of the IgE serum array used for image analysis.

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Layout spot IgE dilution IgE negative serum

Electronic Supplementary Material (ESI) for Analyst.This journal is © The Royal Society of Chemistry 2015

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A B

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Supplementary Figure 2. Variability gold nanoparticle dilution 2A) Variability no gold nanoparticle dilution. 2B) Variability 1:2 nanoparticle dilution. 2C) Variability 1:4 gold nanoparticle dilution. 2D) Variability 1:6 gold nanoparticle dilution. 2E) Variability 1:8 gold nanoparticle dilution. 2F) Variability 1:10 gold nanoparticle dilution.

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Undiluted detection gold nanoparticle

Concentration IgE [µg/mL]

MC

I [A

U]

0.976 1.95 3.9 7.81 15.63 31.25 62.5 125 250

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10000

15000

1:2 dilution detection gold nanoparticle

Concentration IgE [µg/mL]

MC

I [A

U]

0.976 1.95 3.9 7.81 15.63 31.25 62.5 125 250

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1:4 dilution detection gold nanoparticle

Concentration IgE [µg/mL]

MC

I [A

U]

0.976 1.95 3.9 7.81 15.63 31.25 62.5 125 250

05000

10000

15000

1:6 dilution detection gold nanoparticle

Concentration IgE [µg/mL]

MC

I [A

U]

0.976 1.95 3.9 7.81 15.63 31.25 62.5 125 250

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1:8 dilution detection gold nanoparticle

Concentration IgE [µg/mL]

MC

I [A

U]

0.976 1.95 3.9 7.81 15.63 31.25 62.5 125 250

02000

4000

6000

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10000

1:10 dilution detection gold nanoparticle

Concentration IgE [µg/mL]

MC

I [A

U]

0.976 1.95 3.9 7.81 15.63 31.25 62.5 125 250

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Supplementary Figure 3. IgE serum array with different assay times Inverted colorimetric scan in black and white of the IgE array assay run. A) 2 minutes total assay time. B) 5 minutes total assay time. C) 10 minutes total assay time. A B

Supplementary Figure 4. Comparison of VFM assay with time Intensity of the IgE dilution experiment run at 8, 11 and 16 minute total VFM assay time run at 1:4 dilution of gold nanoparticles and 1 mL/min flow rate. A) MCI for different assay times. B) S4B. Signal over noise for different assay times.

Supplementary Figure 5. IgE dilution in serum and PBS MCI signal intensity of the IgE serum dilution in PBS.

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Concentration IgE [..g/mL]

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I [AU

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VFM assay time8 min11 min16 min

1.953 3.906 7.813 15.625 31.25 62.5 125 250

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Concentration IgE [..g/mL]

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VFM assay time8 min11 min16 min

1.953 3.906 7.813 15.625 31.25 62.5 125 250

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Dilution serum IgE in PBS

Concentraion [µg/mL]

MC

I [A

U]

0.030 0.122 0.488 1.953 7.813 31.25 125

PBS dilutionUndiluted1:21:41:61:81:10

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Supplementary Figure 6. Unisart array Fluorescent scan of the Unisart serum array for the 113 serum samples including dilutions of IgE and control spots.

Supplementary Figure 7. MFI IgE dilution Intensity of the IgE serum dilution serie from the Unisart reference array.

Supplementary Figure 8. ROC curves ROC curves for VFM showed an AUC of 0.86 and the Unisart array showed an AUC of 0.87.

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Unisart:IgE dilution

Concentration IgE [µg/mL]

MFI

[AU

]

0.98 1.95 3.91 7.81 15.6 31.3 62.5 125 250

False positive rate

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0.0 0.2 0.4 0.6 0.8 1.0

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AUCVFM 0.86Unisart 0.87