Science Advances

Supplementary Materials

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  • Discussion S1. Increased hydrophilicity of HCCA-Phos and its contribution to the increased reliability of microdevice-based assay.
  • Discussion S2. Optimization of photo-induced electron transfer in sTG.
  • Discussion S3. Assignment of the activity states of ALPs from P. pastoris.
  • Discussion S4. Preparation of probes with distinctive reactivities to monitor phosphatase activities.
  • Discussion S5. Assignment of ALPI clusters in SEAP of phosphatases.
  • Discussion S6. Evaluation of ALPI activity in conventional biochemical assay.
  • Discussion S7. Optimization of the assay conditions.
  • Discussion S8. Assignment of ENPP3 in SEAP of ENPPs.
  • Scheme S1. Preparation of HCCA-Phos.
  • Scheme S2. Preparation of 2,4-dimethoxybenzenesulfonic acid– or 2,5-dimethoxybenzenesulfonic acid–substituted TGs.
  • Scheme S3. Preparation of sTG-Phos.
  • Scheme S4. Preparation of sTM and sTM-Phos.
  • Scheme S5. Preparation of HCCA-mPhos, sTG-mPhos, and sTM-mPhos.
  • Scheme S6. Preparation of sTG-qmPhos.
  • Scheme S7. Preparation of ENPP probes.
  • Fig. S1. Suitability of coumarin derivatives for microchamber.
  • Fig. S2. Comparison of the reactivity of HCCA-Phos and 4MU-Phos against ALP.
  • Fig. S3. Optical properties of developed TG derivatives.
  • Fig. S4. Optical properties of developed fluorescence dyes.
  • Fig. S5. Optical properties of developed fluorescence probes.
  • Fig. S6. Stability of fluorescent signals of sTG and sTM in microchamber.
  • Fig. S7. Linearity of fluorescent dye concentration and fluorescence intensity of wells.
  • Fig. S8. Suitability of sTG-Phos for single-molecule microdevice analysis of ALP activity.
  • Fig. S9. Linearity of enzyme kinetics analyzed using the microdevice-based activity detection of ALP.
  • Fig. S10. Comparison of the reactivity of sTG-Phos and FDP in microdevice-based activity detection of ALP.
  • Fig. S11. Michaelis-Menten analyses of the reactivities of developed fluorescent probes toward different ALP isozymes.
  • Fig. S12. Scatter plots of the activities of the proteins in the experiment shown in Fig. 2D (right).
  • Fig. S13. Detection limit of ALPI in microdevice-based assay.
  • Fig. S14. Detection limit of TNAP in the microdevice-based assay.
  • Fig. S15. Limit of detection of ALPI in mixture.
  • Fig. S16. Distributions of single-molecule enzyme activities measured by microdevice-based assay.
  • Fig. S17. Scatter plots of the activities of the proteins in the experiment shown in Fig. 2 (D and E).
  • Fig. S18. pH-dependent changes of clusters of phosphatase activities in human serum.
  • Fig. S19. Fluorescence overlay image of microdevice prepared in the same condition as in Fig. 3A.
  • Fig. S20. All serum measurement data of 10 individuals.
  • Fig. S21. Detection of fractions of phosphatases with unique reactivity against sTG-qmPhos.
  • Fig. S22. Comparison of the reactivity of HCCA-mPhos, sTG-qmPhos, and sTM-Phos against TNAP and PTP1B.
  • Fig. S23. Assignment of spots for ALPI at high activity states in pH 7.4 condition.
  • Fig. S24. Detection of TNAP and ALPI activities in serum samples.
  • Fig. S25. Reactivity of ENPPs in human serum compared to recombinant ENPP2.
  • Fig. S26. Reactivity of ENPPs toward probes based on sTG and sTM with diverse nucleotide reactive sites.
  • Fig. S27. Choice of sets of fluorescent probes to monitor ENPP activities in plasma.
  • Fig. S28. Assignment of spots for ENPP3.
  • Fig. S29. Limit of detection of ENPP3 in microdevice-based assay.
  • Fig. S30. The result of Western blotting analysis of ENPP3 detection in serum.
  • Fig. S31. Analysis of ENPP3 activities based on cancer stages and receiver operating characteristic curves and AUC values for diagnosis of patients with pancreatic cancer.
  • Table S1. Fluorescence quantum yields of developed dyes.
  • Table S2. Photophysical properties of developed probes.
  • Table S3. The number of chambers in each peak in the experiment shown in fig. S8.
  • Table S4. kcat and Km values of developed probes with different ALP isozymes.
  • Table S5. Evaluation of ALPI activity in serum samples of patients with diabetes.
  • Table S6. Optimization of assay conditions for SEAP of phosphatases.
  • References (41, 42)

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