Research ArticleHEALTH AND MEDICINE

Point-of-care biomarker quantification enabled by sample-specific calibration

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Science Advances  25 Sep 2019:
Vol. 5, no. 9, eaax4473
DOI: 10.1126/sciadv.aax4473
  • Fig. 1 Proposal and development of parallel calibration approach for matrix-specific biomarker quantification.

    (A) Schematic of standardization method to account for matrix effects. An array of standard reactions will have saturated biomarker concentrations and varied regulator concentrations. The test reaction will have a set regulator concentration and no added biomarker. The sample to be analyzed will be added to both the standard and test reactions so that all reactions run in the same sample matrix. After a set incubation time, the color of the test reaction can be matched to the color of the standard reactions to determine biomarker concentration in the test reaction. (B) Schematic of a zinc-responsive circuit used to control β-galactosidase production. On one plasmid, ZntR is expressed from a T7 promoter, and on a second plasmid, β-galactosidase is expressed from the ZntR-activated promoter PzntA. (C) Pictures of visible colors from reactions corresponding with different absorbance measurements. The lowest A580 readings correspond with yellow reactions (the color of CPRG), and as the A580 increases, and the reaction color turns different shades of orange, red, and purple (the color of CPR). (D) Quantitative colorimetric response to added zinc. At early time points, there is no detectable absorbance of the purple substrate CPR at tested [Zn2+], and reactions appear yellow (the color of CPRG). As the reactions proceed, they produce CPR at different rates based on the concentration of Zn2+ in the reaction, with the maximal differences visible between 60 and 70 min. An ideal assay readout time would yield outputs spanning a wide range of absorbances across as much of the [Zn2+] range as possible. (E) Fluorescent response to zinc in 25% human serum, demonstrating substantial matrix effects in serum. AU, arbitrary units. (F) Selected time course readings of test and standard reactions run in 25% serum, demonstrating the mapping between the two reaction designs.

  • Fig. 2 Quantification approach provides high prediction accuracy at micromolar resolution of raw sample concentrations.

    (A) Quantification of serum zinc concentrations for all four single-donor samples tested, evaluated at 56 min. Zinc concentration in chelex-treated samples was measured with inductively coupled plasma–mass spectrometry (ICP-MS), and defined zinc standards were added to each sample to achieve a range of concentrations. Serum from donors 1 to 3 was used in initial calibration, and serum from donor 4 was only used in test validation, indicated by the asterisk. Symbols falling inside the horizontal bars that correspond with the binned prediction ranges for each y-axis level indicate that the CFE test accurately quantified zinc in the serum sample. (B) Error quantification of all individual donor samples via the QEM. Low QEM for correct predictions and high QEM for incorrect predictions indicate unambiguously interpretable test results for a given time. (C) Error quantification in nonideal reaction conditions with serum from donor 4.

  • Fig. 3 Addition of small molecules reverses color shifts due to serum albumin.

    (A) Reactions run in serum appear different from reactions run in the absence of serum. Reactions with complete conversion of CPRG to CPR appear more purple in a 25% serum matrix. In 25% serum, the colored orange and red reaction intermediates initially observed are no longer visible. (B) Whereas the presence of serum shifts the absorbance peak of CPR approximately 10 nm, the addition of small molecules that bind albumin reverses this spectrum shift. (C) Visualization of standard reference and test reactions in 25% serum. Standard reference reactions contain a saturated amount of zinc and predetermined concentrations of the pZntR plasmid. Test reactions contain a set amount of the pZntR plasmid and specified zinc concentrations. All reactions were run for 50 min and then visualized either with or without 40 mM naproxen addition. With added naproxen, the color of test reactions matches the color of the appropriate standard reference reaction.

  • Fig. 4 Quantification approach is generalizable to toehold switches.

    (A) Schematic of toehold switch used to control β-galactosidase production. Trigger RNA unfolds the toehold switch and enables β-galactosidase translation. Increasing amounts of trigger RNA correspond with increased β-galactosidase translation. (B) Test quantification of trigger concentrations, evaluated at 40 min. Purified RNA was added to each sample at different known concentrations. Symbols falling inside the horizontal bars that correspond with the binned prediction ranges for each y-axis level indicate that the CFE test accurately quantified RNA in the sample. (C) Quantification error for toehold sensing. (D) Visualization of test and standard reactions, evaluated at 40 min. The color of test reactions matches that of the appropriate standard reference reaction.

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/5/9/eaax4473/DC1

    Supplementary Text

    Definition of QEM

    Computational model of parallel calibration approach

    Computational model of trigger response and standardization approach

    Table S1. Species and their initial conditions in the model of zinc response in cell-free reactions.

    Table S2. Zinc model parameters and estimated values.

    Table S3. Equations used to model the zinc response in cell-free reactions.

    Table S4. Species and their initial conditions in the model of toehold switch circuit in cell-free reactions.

    Table S5. Toehold switch model parameters and estimated values.

    Table S6. Equations used to model the toehold switch in cell-free reactions.

    Table S7. Approximate cost analysis for zinc diagnostic tools.

    Table S8. Description of plasmids constructed and used in this study.

    Table S9. Sequences of primers used in this study.

    Table S10. Sequences of promoters used in this study.

    Table S11. Sequences of genes used in this study.

    Table S12. Sequences of plasmids used in this study.

    Fig. S1. Fluorescent response to zinc addition in CFE systems.

    Fig. S2. Validation of knockout strains.

    Fig. S3. Qualitative colorimetric response to added zinc.

    Fig. S4. Zinc response in lyophilized reactions.

    Fig. S5. Effect of reporter plasmid concentration on the ability to distinguish between different zinc concentrations.

    Fig. S6. Quantitative colorimetric response to reactions run in serum.

    Fig. S7. Response to zinc saturates in 25% serum.

    Fig. S8. Zinc concentrations in individual donor serum.

    Fig. S9. Relationship between regulator in standard reactions and zinc in test reactions.

    Fig. S10. Validation of zinc quantification without added serum.

    Fig. S11. Validation of direct protein addition to modulate zinc response.

    Fig. S12. Validation of zinc quantification with direct protein addition.

    Fig. S13. Comparison of parameterized model and experimental data in zinc-response circuit.

    Fig. S14. Simulated zinc and regulator mapping.

    Fig. S15. Perturbation of parameter values to identify the most sensitive parameters in the zinc model.

    Fig. S16. Color change caused by serum corresponds with a shift in the visible spectrum of CPR.

    Fig. S17. Human serum albumin replicates the spectrum shift caused by serum.

    Fig. S18. Naproxen concentration necessary to restore colored intermediates.

    Fig. S19. Response to trigger RNA addition saturates.

    Fig. S20. Selected time course readings of test and standard reactions for a toehold sensor.

    Fig. S21. Relationship between trigger concentration in the test reaction and the switch concentration in the calibration reaction.

    Fig. S22. Comparison of parameterized model to experimental data in toehold switch circuit.

    Fig. S23. Simulated trigger RNA and switch plasmid mapping.

    Fig. S24. Perturbation of parameter values to identify the most sensitive parameters in toehold switch model.

    Fig. S25. Overview schematic of test manufacture and use.

  • Supplementary Materials

    This PDF file includes:

    • Supplementary Text
    • Definition of QEM
    • Computational model of parallel calibration approach
    • Computational model of trigger response and standardization approach
    • Table S1. Species and their initial conditions in the model of zinc response in cell-free reactions.
    • Table S2. Zinc model parameters and estimated values.
    • Table S3. Equations used to model the zinc response in cell-free reactions.
    • Table S4. Species and their initial conditions in the model of toehold switch circuit in cell-free reactions.
    • Table S5. Toehold switch model parameters and estimated values.
    • Table S6. Equations used to model the toehold switch in cell-free reactions.
    • Table S7. Approximate cost analysis for zinc diagnostic tools.
    • Table S8. Description of plasmids constructed and used in this study.
    • Table S9. Sequences of primers used in this study.
    • Table S10. Sequences of promoters used in this study.
    • Table S11. Sequences of genes used in this study.
    • Table S12. Sequences of plasmids used in this study.
    • Fig. S1. Fluorescent response to zinc addition in CFE systems.
    • Fig. S2. Validation of knockout strains.
    • Fig. S3. Qualitative colorimetric response to added zinc.
    • Fig. S4. Zinc response in lyophilized reactions.
    • Fig. S5. Effect of reporter plasmid concentration on the ability to distinguish between different zinc concentrations.
    • Fig. S6. Quantitative colorimetric response to reactions run in serum.
    • Fig. S7. Response to zinc saturates in 25% serum.
    • Fig. S8. Zinc concentrations in individual donor serum.
    • Fig. S9. Relationship between regulator in standard reactions and zinc in test reactions.
    • Fig. S10. Validation of zinc quantification without added serum.
    • Fig. S11. Validation of direct protein addition to modulate zinc response.
    • Fig. S12. Validation of zinc quantification with direct protein addition.
    • Fig. S13. Comparison of parameterized model and experimental data in zinc-response circuit.
    • Fig. S14. Simulated zinc and regulator mapping.
    • Fig. S15. Perturbation of parameter values to identify the most sensitive parameters in the zinc model.
    • Fig. S16. Color change caused by serum corresponds with a shift in the visible spectrum of CPR.
    • Fig. S17. Human serum albumin replicates the spectrum shift caused by serum.
    • Fig. S18. Naproxen concentration necessary to restore colored intermediates.
    • Fig. S19. Response to trigger RNA addition saturates.
    • Fig. S20. Selected time course readings of test and standard reactions for a toehold sensor.
    • Fig. S21. Relationship between trigger concentration in the test reaction and the switch concentration in the calibration reaction.
    • Fig. S22. Comparison of parameterized model to experimental data in toehold switch circuit.
    • Fig. S23. Simulated trigger RNA and switch plasmid mapping.
    • Fig. S24. Perturbation of parameter values to identify the most sensitive parameters in toehold switch model.
    • Fig. S25. Overview schematic of test manufacture and use.

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