Elucidation and control of low and high active populations of alkaline phosphatase molecules for quantitative digital bioassay

Abstract

Alkaline phosphatase (ALP), a homo-dimeric enzyme has been widely used in various bioassays as disease markers and enzyme probes. Recent advancements of digital bioassay revolutionized ALP-based diagnostic assays as seen in rapid growth of digital ELISA and the emerging multiplex profiling of single-molecule ALP isomers.However, the intrinsic heterogeneity found among ALP molecules hampers the ALP-based quantitative digital bioassays. This study aims quantitative analysis of single-molecule activities of ALP from Escherichia coli and reveals the static heterogeneity in catalytic activity of ALP with two distinct populations: half-active and fully-active portions. Digital assays with serial buffer exchange uncovered singlemolecule Michaelis-Menten kinetics of ALP; half-active molecules have halved values of the catalytic turnover rate, k cat , and the rate constant of productive binding, k on , of the fully active molecules. These findings suggest that half-active ALP molecules are heterogenic dimers composed of inactive and active monomer units, while fully active ALP molecules comprise two active units. Static heterogeneity was also observed for ALP with other origins: calf intestine or shrimp, showing how the findings can be generalized across species. Cell-free expression of ALP with disulfide bond enhancer and spiked zinc ion resulted in homogenous population of ALP of full activity, implying that inactive monomer units of ALP are deficient in correct disulfide bond formation and zinc ion coordination. These findings provide basis for further study on molecular mechanism and biogenesis of ALP, and also offer the way to prepare homogenous and active populations of ALP for highly quantitative and sensitive bioassays with ALP.

Assessment of Colorimetric Reporter Enzymes in the PURE System

“…While we focused on the PURE CFE system, the same enzyme/substrate pairs could be useful in cell-based or lysate-based CFE applications depending on endogenous enzyme activity. We note that optimization of the expression conditions could improve the performance of some these pairs; as an example, recent work showed expression of active alkaline phosphatase (PhoA) in PURE by addition of a disulfide bond enhancer and zinc . For applications in sensing, our results show that simply optimizing the concentration of the substrate used can play a significant role in signal strength and response time.…”

“…We note that optimization of the expression conditions could improve the performance of some these pairs; as an example, recent work showed expression of active alkaline phosphatase (PhoA) in PURE by addition of a disulfide bond enhancer and zinc. 22 For applications in sensing, our results show that simply optimizing the concentration of the substrate used can play a significant role in signal strength and response time. The demonstration of potential multiplexing of CFE sensors with an eye-readable signal increases the range of field-forward applications possible with CFE.…”

Enzyme-based digital bioassay technology – key strategies and future perspectives

“…2d). Owing to the versatility and stability of this system, FRAD has been utilized in various types of digital bioassays, including single-molecule enzyme analyses [10][11][12]26 and enzyme screening. 25 In addition, a cyclic olefin polymer (COP)-based device for droplet arrays has been developed (Fig.…”

“…β-gal, 7,26,30,41,42 β-glucuronidase (β-gluc) 8,43 and alkaline phosphatase (ALP) 11,26,44,45 are often used in digital bioassays because the chemistry of their fluorogenic substrates has been well established, thus providing several types of fluorogenic substrates for each enzyme. Horseradish peroxidases (HRPs) have also been studied in digital bioassays.…”

“…Distinctive heterogeneities in enzyme activity have been found in enzymes with oligomeric structures, for example, β-gal with a homo-tetramer 56 and ALP with a homodimer, 11 both of which are often used in digital ELISAs or related assays. Li et al reported a non-Gaussian distribution of the catalytic activity of engineered hetero-tetrameric β-gal molecules composed of active monomer(s) and mutated inactive monomerIJs).…”