Supplemental Data. Methods- Different concentrations of substrate solutions (final concentrations during incubation- 10, 3,

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1 Supplemental Data Michaelis-Menten Kinetics Methods- Different concentrations of substrate solutions (final concentrations during incubation- 10, 3, 1, 0.3 and 0.1 mmol/l) were used and enzymatic analysis was performed on the digital microfluidic cartridges for both enzymes (GAA and GLA) with an incubation time of 1 hour. These data were used to generate Lineweaver-Burk plots which were further used to calculate the Michaelis-Menten constant (K m ) to determine the working substrate concentration. Results and Discussion- Supplemental Figure 1 depicts the enzyme activity with various concentrations of the substrate for both Pompe and Fabry assays obtained on the digital microfluidic cartridges. Each data point is the average obtained from 6 replicate measurements and the error bars represent standard deviation. Lineweaver-Burk plots for GLA and GAA enzymes are shown in Supplemental Figures 2a and 2b respectively. K m values of 2 and 1 mmol/l were obtained for GLA and GAA respectively. To ensure substrate concentrations higher than K m, working substrate concentrations of 5 and 2.5 mmol/l were used for GLA and GAA enzymatic assays respectively for digital microfluidic assays. Linearity and Kinetics Methods- Optimal substrate concentrations determined using the Lineweaver-Burk plot was used to obtain reaction kinetics for both Pompe and Fabry assays. QCH and QCBP samples obtained from CDC were extracted using 100μL of extraction buffer. Pompe and Fabry assays were performed for 0.5, 1, 1.5 and 2 hours respectively. Raw fluorescence units are converted to μmoles of 4-MU per liter of blood. Results and Discussion- Supplemental Figures 3a and 3b show the kinetic data for QCH and QCBP samples for Pompe and Fabry assays (GAA and GLA enzymes respectively). Each data point is the average obtained from 10 replicate measurements and the error bars represent standard deviation. Kinetic profiles show that both the assays are in the linear range for 2 hours of incubation time. Also,

2 there is significant separation between QCBP and QCH suggesting that there is very minimal nonenzymatic hydrolysis. CDC QC Spots Materials and Methods- QC DBS (L, M and H) samples were obtained from CDC. Using the optimal substrate concentrations and 1 hour incubation time, we obtained data using multiple instruments, multiple cartridges, and multiple operators over a period of several months. All QC DBS samples were stored at -80 C during the entire time. Results and Discussion- Enzyme activities and coefficients of variation obtained for each of the QC samples are included in Supplemental Table 1 for both the enzymes. The CVs obtained for the 3 levels of QC spots are in good agreement with the published results. SAMPLE GLA (N=100) GAA (N=100) AVG CV% AVG CV% QCL % % QCM % % QCH % % Supplemental Table 1- Enzyme activity and coefficients of variation for CDC QC samples Effect of hematocrit on fluorescence at different levels of 4-MU on the microfluidic platform Materials and Methods- Extracts were obtained from QCBP using the standard extraction procedure as described in the manuscript. Solutions with different concentrations of hematocrit were obtained by mixing DBS extract, known concentration of 4-MU and extraction buffer. This process is repeated to obtain a matrix of solutions with different concentrations of hematocrit and different concentrations of 4-MU. The concentrations of 4-MU were chosen so that the fluorescence is equivalent to that obtained from an affected sample, a normal sample with low enzymatic activity and a normal sample with high enzymatic activity. All these solutions were loaded onto the microfluidic cartridge and the fluorescence

3 for each of these solutions was measured and tabulated. Fluorescence values obtained for the solutions with zero hematocrit was considered to be 100% and all other fluorescence values were normalized accordingly. Results and Discussion- Supplemental Figures 4(a) and 4(b) represent plots for the raw fluorescence units and normalized signals respectively for the aforementioned 4-MU solutions with varying amounts of hematocrit. In the digital microfluidic protocol to perform Pompe and Fabry assays, the DBS extract (obtained by extracting a 3mm DBS punch in 100μL extraction buffer) is further diluted 4 totally by the reagent droplet (2 ) and the stop buffer droplet (2 ) serially before the finally presented to the fluorometer for the detection of 4-MU. Hematocrit levels in neonatal samples generally vary from 30% to 70%, and are diluted to 7.5% to 17.5% during the enzymatic assay in the digital microfluidic protocol. The highlighted portion in the two plots (Figure 4a and 4b) represents this variation, which amounts to ~10% variation. The effect of increasing amounts of hematocrit on the fluorescence signal is the same irrespective of the amount of 4-MU present in the droplets.

4 Supplemental Figure 1- Effect of substrate concentration on activity for GAA and GLA enzymes

5 Supplemental Figure 2- Lineweaver-Burk plots for (a) GLA and (b) GAA

6 Supplemental Figure 3- Kinetics for QCH and QCBP samples for (a) GLA and (b) GAA enzymes

7 Supplemental Figure 4- Effect of hematocrit on fluorescence at different concentrations of 4-MU (a) Rfu levels (b) Normalized fluorescence signal