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Multiplexed single-molecule enzyme activity analysis for counting disease-related proteins in biological samples

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Science Advances  11 Mar 2020:
Vol. 6, no. 11, eaay0888
DOI: 10.1126/sciadv.aay0888
  • Fig. 1 Schematic view of SEAP.

    In this assay, a mixture of enzymes from biosamples were diluted and separately loaded into microwells of the device, and the numbers of targeted enzyme species were counted using multiplexed single-molecule enzymatic assays, in which the reactivities toward differentially colored and structure-wide substrates are used to characterize the enzyme species in each well.

  • Fig. 2 Development of a set of fluorogenic probes to discriminate various phosphatases.

    (A) Fluorescence images of a microdevice containing ALP (from P. pastoris, λ = 0.1, where λ is the existence probability of a protein molecule per well) with fluorescence probes (4MU-Phos and HCCA-Phos, 100 μM) in tris-HCl buffer [100 mM (pH 7.4), containing 1 mM MgCl2 and 0.1% (w/w) CHAPS] after 50-min incubation. (B) Top: Fluorescence spectra of sTG-Phos and sTM-Phos (1 μM) after incubation with or without ALP (from P. pastoris) in diethylamine (DEA) buffer [1 M, containing 1 mM MgCl2 (pH 9.3)]. Excitation wavelengths were 490 nm (for sTG) and 590 nm (sTM). Bottom: Fluorescence images of a microdevice containing ALP (from P. pastoris, λ = 0.1, where λ is the existence probability of a protein molecule per well) obtained with fluorogenic probes (sTG-Phos and sTM-Phos, 100 μM) in DEA buffer [1 M (pH 9.3), containing 1 mM MgCl2 and 0.1% (w/w) CHAPS] after 50-min incubation. a.u., arbitrary units. (C) Structures of phosphatase probes developed for the assay. (D) Left: Scatter plot of the activities of the proteins read out by HCCA-mPhos, sTG-Phos, and sTM-mPhos (100 μM) in DEA buffer [1 M (pH 9.3), containing 1 mM MgCl2 and 0.1% (w/w) CHAPS] in the presence of recombinant TNAP, ALPI (λ = 0.01), and their mixtures (1:1). Plots for the activities toward sTG-Phos (green, horizontal axis) and sTM-mPhos (red, vertical axis) are shown. Right: Experiments were performed with mixtures of different ratios of ALPI and TNAP (horizontal axis), and the percentages of spots that appeared in the area of ALPI were counted (vertical axis). Experiments were performed three times. Error bar, SD (n = 3). The whole dataset is shown in fig. S8. (E) Left: Ratiometric (red/green) fluorescence image of a microdevice containing 1:3000 diluted human serum with fluorogenic probes (sTG-mPhos and sTM-Phos, 30 μM) in DEA buffer [1 M (pH 9.3), containing 1 mM MgCl2 and 0.1% (w/w) CHAPS] after 50-min incubation at 25°C. Middle: Scatter plot of the activities of the proteins in the experiment shown in (E) (left). Each point was assigned to the ALPI or TNAP cluster by discrimination analysis (see Materials and Methods). Right: The comparison of the integral of the activities calculated from the spots assigned as ALPI and the ALPI activity monitored by conventional biochemical assay (see discussion S6). The analysis with serum from 10 individuals is shown.

  • Fig. 3 Applications of SEAP methodology.

    (A) Representative fluorescence overlay image of a microdevice containing 1:3000 diluted serum with fluorogenic probes [30 μM; HCCA-mPhos (blue), sTG-qmPhos (green), and sTM-Phos (red)] in tris-HCl buffer [100 mM (pH 7.4), containing 1 mM MgCl2, 100 mM dithiothreitol, and 0.1% (w/w) CHAPS] after 2-hour incubation. The larger device image is shown in fig. S11. (B) Scatter plots of activity acquired for five samples from healthy individuals and five from patients with diabetes [exemplified by (A)]. Data were subjected to cluster analysis as described in discussion S5 and Materials and Methods. The points assigned as ALPI are surrounded by red rectangles. The whole datasets are shown in fig. S12. (C) Left: Overlay fluorescence image of a microdevice containing 1:500 diluted serum with fluorogenic probes [sTG-mdTMP (green) and sTM-dCMP (red), 100 μM] in tris-HCl buffer [100 mM (pH 9.3), containing 1 mM MgCl2 and 0.5% (w/w) CHAPS] after 40-min incubation. Right: The chemical structures of sTG-mdTMP and sTM-dCMP. (D) Scatter plots of activity data for plasma samples derived from 14 healthy individuals and 31 patients with cancer [exemplified by (C)]. Points were assigned to three clusters by cluster analysis (see discussion S8 and Materials and Methods). (E) The dot plot shows the number of plots assigned to cluster 1 in (D). P value was analyzed using Mann-Whitney U test.

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/6/11/eaay0888/DC1

    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)

  • Supplementary Materials

    This PDF file includes:

    • 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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