Colorimetric semi-quantitative measurement of pyrophosphate by functionalised SPPS resin in biological media

https://doi.org/10.1016/j.snb.2016.12.057Get rights and content

Highlights

  • Colorimetric detection of pyrophosphate using polymer beads.

  • Analysis using only a smartphone or optical microscope.

  • Detection of pyrophosphate in tris buffer, artificial plasma and human urine.

Abstract

Inorganic pyrophosphate is a molecule of significant biological importance, which has roles in metabolism and disease diagnosis. In this communication we demonstrate a novel platform for the colorimetric naked eye detection of pyrophosphate using a threshold test in both aqueous solutions and biological fluids. Our system uses peptide synthesis resin as a solid support for a bis-Zn(II)dipicolylamino mediated indicator displacement assay. Our threshold tests are able to detect pyrophosphate in concentrations as low as 1 mM.

Introduction

Inorganic pyrophosphate (ppi) is a molecule of significant biological importance. It is produced in the hydrolysis of adenosine triphosphate (ATP) to produce energy within living cells. Concentrations of ppi in plasma, synovial fluid and urine can also be diagnostic for diseases such as vascular calcification [1], calcium ppi deposition disease [2], and kidney stones [3], respectively. As monitoring the concentrations of ppi can play a crucial role in diagnosis of these diseases, rapid, affordable and reliable sensing technology is needed.

The field of supramolecular chemistry, and in particular host-guest chemistry, have provided numerous methods for detecting ppi (See references [4], [5], [6] for reviews of ppi chemosensors). One particularly promising method for detecting ppi is through a coordination strategy using bis-Zn(II)dipicolylamino (ZnDPA) furnished hosts; binding of ppi by these groups has been shown to be both specific and strong [7], [8], [9], [10], [11], [12], [13], [14], [15], [16]. However, many of these hosts provide little measurable response to binding, and so indicator displacement assays have been developed using these selective receptors in order to provide a measurable response. Using Pyrocatechol Violet in conjunction with a bis-ZnDPA functionalised host molecule has proven to be an excellent strategy for the detection of ppi in solution. In addition, numerous scaffolds for arranging binding groups have been explored in the literature (See reference [17] for an excellent review), one such scaffold which can be readily assembled and functionalised are peptide based scaffolds [18], [19], [20]. Combining the above aspects Jolliffe et al., have used a peptide scaffold furnished with ZnDPA in a colorimetric indicator displacement assay for the detection of ppi with high levels of selectivity for ppi over other anions and a strong colorimetric response were reported [7], [8].

While there have been significant advances in the field of host-guest and supramolecular chemistries in recent years, relatively little attention has been given to the recognition of ionic analytes at a solid-solution interface [21]. Modern examples of immobilised ion recognition scaffolds are essentially limited to calixarene [22], [23], [24], [25], [26] and calixpyrole [27], [28], [29] based scaffolds, and some nanoparticle/nanopore immobilised chemistries [11], [30], [31], or formulation rather than true immobilisation [32], [33], [34]. The use of a solid support to anchor specific anion recognition groups to solids or gels has potential to become a selective filtration media, such as membranes or selective ion exchange resins.

In this communication we show that an indicator displacement assay, that combines a polymer bead functionalised with ZnDPA with a colorimetric catechol dye (pyrocatechol violet) is able to selectively detect ppi in a threshold test. The resin beads require minimal sample preparation and enable analysis with minimal scientific equipment requirements, using only a standard microscope or smartphone equipped with a macro lens. Our colorimetric threshold test has been shown to work in tris buffer, simulated bodily fluids (artificial plasma) and real biological fluids (human urine).

Section snippets

Results and discussion

To provide an easily accessible and scalable platform, construction of the functional polymer beads was completed using well-known solid phase peptide synthesis methodology using well-known and easily accessible starting materials. A key goal was that the bis-ZnDPA functionalised beads (Fig. 1) could be easily and cost-effectively [35] produced using commercially available materials.

The functional component was based on a small peptide based receptor originally reported by Jolliffe et al. [7],

Conclusion

In this communication we have constructed a new polymer based functional material for the selective detection of ppi in biological fluids. The material provides a fast (∼5 min), cost-effective (USD$ 0.15 per threshold test [35], [37]) method for detecting high levels of ppi above 1 mM in tris buffer without the need for complex scientific equipment. In biological fluids the development time in increased to 1 h with comparable detection limits of 5 mM and 2 mM ppi in artificial plasma and human

Acknowledgements

BL would like to acknowledge funding support from The School of Life and Environmental Sciences and The Centre for Chemistry and Biotechnology, Deakin University. Ethics approval for the use of human urine samples was obtained (STEC-22-2016-LONG).

Benjamin M. Long obtained his PhD from Deakin University in 2014 before undertaking Postdoctoral Research and Teaching Fellowships at the University of Sydney working with Professor Katrina Jolliffe. In 2016 he returned to Deakin University as a lecturer and research fellow. Research interests include organic, supramolecular and materials chemistries. Current focus is on applied anion recognition chemistry, characterisation of supramolecular polymers and thermosetting polymers.

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  • Cited by (0)

    Benjamin M. Long obtained his PhD from Deakin University in 2014 before undertaking Postdoctoral Research and Teaching Fellowships at the University of Sydney working with Professor Katrina Jolliffe. In 2016 he returned to Deakin University as a lecturer and research fellow. Research interests include organic, supramolecular and materials chemistries. Current focus is on applied anion recognition chemistry, characterisation of supramolecular polymers and thermosetting polymers.

    Fred M. Pfeffer graduated with PhD from Deakin University in 2001 and then worked at Trinity College Dublin; first as a lecturer then as postdoctoral fellow in the group of professor Thorfinnur Gunnlaugsson. He was appointed as a lecturer at Deakin in 2004, senior lecturer in 2010 and associate professor in 2017. Research interests include many aspects of organic, medicinal and supramolecular chemistry with a key focus on anion recognition and sensing and also the synthesis and use of fused [n]polynorbornane scaffolds as preorganising elements for host:guest chemistry and the construction of metallosupramolecular cages.

    Colin J. Barrow is Alfred Deakin Professor, Chair of Biotechnology and Director of the Centre for Chemistry and Biotechnology (CCB) at Deakin University. Professor Barrows research is primarily focused on food biotechnology and the application of nanomaterials for industrial purposes. Professor Barrow has a Ph.D. in chemistry from the University of Canterbury in New Zealand and an MBA from Penn State in the USA. He has more than 200 peer-reviewed publications, several patents, and has presented at numerous conferences and workshops. He has held senior roles in both Industry and Academia and has served as a member of the Expert Advisory Committee for Canadian Natural Health Product Directorate (NHPD) and is a founding member of International Society for Nutraceuticals and Functional Foods (ISNFF).

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