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Evaluation of the Analytical Bounds of the DETECT Diagnostic System

for Antimicrobial Resistant Urinary Tract Infection

Emily Harari, Quality Control Biochemistry Intern

BioAmp Diagnostics

Abstract

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The purpose of this research is to assess the performance of BioAmp’s diagnostic system (DETECT) under a range of physiological conditions. �

Background

� DETECT stands for Dual Enzyme Trigger-Enabled Cascade Technology, in which a a probe designed to mimic beta-lactam antibiotics is synthesized to include a thiol triggering unit. In the presence of antimicrobial resistant bacteria (AMR), the probe is reduced, releasing thiols and triggering a cascade of reactions. The thiols react with papain, and the chemical product reacts with a color probe, Na-benzoyl-L-arginine-p-nitroaniline (BAPA). Absorption is measured to determine the presence of antibiotic resistant bacteria.�� This use case for DETECT is in urinary tract infection (UTI). Synthetic urine was formulated at a range of pH values for testing the system. Urine can be acidic after consuming certain foods is more basic during infection.��

Figure 1: DETECT diagnostic system includes a beta-lactam probe configured with a thiol that triggers a cascade, ultimately ending in a photoreaction to measure the presence of AMR bacteria.

Methods

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Urine Formulation

  • Prepare beta-lactam and substrate reactions for experimental control
  • Formulate synthetic urine at various pHs and test their buffering capacities.

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Antimicrobial Assays

  • Prepare buffered solutions in an 8x12 well plate.
  • With a multichannel pipette, dispense cysteine to catalyze the DETECT reaction and begin recording immediately in a UV spectrophotometer.
  • Measure the absorbance over time every 2 min. For 20 min.

Figure 3: Solution of every pH and a standard 50:50 buffer:reagent are dispensed in their respective rows, each with their own control well.

Results

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Ten trials were run for eight different formulations. UV absorption was measured and percent variance was calculated. Below are results from trials 4 & 5. (A) Sample Activity at Different pHs. (B) Corrected Activity at Different pHs. (C)% Variance Activity at Different pHs.

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Conclusions

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DETECT system activity is directly proportional to pH, with the exception of pH 5.03, which shows relatively high activity. It is suspected that pH 5.03 is the activity threshold, at and below which the oxyanion tetrahedral intermediate is protonated and destroyed.

Acknowledgements

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Qb3 Program, UC Berkeley�Tara DeBoer, BioAmp Diagnostics�Illumina Accelerator Program�

Future Plans

BioAmp Diagnostics should further investigate which probe intermediates are stabilized at various pHs. The DETECT system should be further tested in more acidic solutions, as it may be unreliable at or below pHs of 5.03.

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pH before substrate-1

pH after substrate-1

Abs(%Var)

A (pH 5.03 ©)

5.52

5.55

0.5435%

B (pH 5.03)

5.43

5.55

2.2099%

C (pH 6.31 ©)

6.3

6.3

0.0000%

D (pH 6.31)

6.31

6.31

0.0000%

E (pH 8.23 ©)

7.3

7.2

1.3699%

F (pH 8.23)

7.64

7.24

5.2356%

G (standard ©)

6.57

6.54

0.4566%

H (standard)

6.47

6.52

0.7728%

Ingredient

Range of Concentrations

1. Urea

~16.3 g/L

2. Chloride

53 mM - 237 mM

3. Sodium

51 mM - 191 mM

4. Potassium

19.2 mM - 66.8 mM

5. Creatinine

~12.5 mM - 19 mM

6. Sulfur (inorganic)

4 mM - 56 mM

7. Hippuric Acid

0.279 mM - 9.32 mM

8. Phosphorus

15.2 mM - 34.5 mM

9. Citric Acid

0.469 mM - 4.84 mM

10. Glucuronic Acid

0.361 mM - 4.53 mM

11. Ammonia

11.76 mM - 42.9 mM

12. Uric Acid

0.238 mM - 3.99 mM

13. Uropepsin

0.386 mM - 3.09 mM

14. Bicarbonate

0.328 mM - 9.18 mM

Figure 2: Urine formulation based on the urine metabolome. Consult documentation for additional compounds based on acidic, basic, or neutral formulations and their respective buffers.

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