Magnetic Graphene Nanosheet-Based Microfluidic Device for Homogeneous Real-Time Electronic Monitoring of Pyrophosphatase Activity Using Enzymatic Hydrolysate-Induced Release of Copper Ion

Abstract

A novel flow-through microfluidic device based on a magneto-controlled graphene sensing platform was designed for homogeneous electronic monitoring of pyrophosphatase (PPase) activity; enzymatic hydrolysate-induced release of inorganic copper ion (Cu(2+)) from the Cu(2+)-coordinated pyrophosphate ions (Cu(2+)-PPi) complex was assessed to determine enzyme activity. Magnetic graphene nanosheets (MGNS) functionalized with negatively charged Nafion were synthesized by using the wet-chemistry method. The Cu(2+)-PPi complexes were prepared on the basis of the coordination reaction between copper ion and inorganic pyrophosphate ions. Upon target PPase introduction into the detection system, the analyte initially hydrolyzed pyrophosphate ions into phosphate ions and released the electroactive copper ions from Cu(2+)-PPi complexes. The released copper ions could be readily captured through the negatively charged Nafion on the magnetic graphene nanosheets, which could be quantitatively monitored by using the stripping voltammetry on the flow-through detection cell with an external magnet. Under optimal conditions, the obtained electrochemical signal exhibited a high dependence on PPase activity within a dynamic range from 0.1 to 20 mU mL(-1) and allowed the detection at a concentration as low as 0.05 mU mL(-1). Coefficients of variation for reproducibility of the intra-assay and interassay were below 7.6 and 9.8%, respectively. The inhibition efficiency of sodium fluoride (NaF) also received good results in pyrophosphatase inhibitor screening research. In addition, the methodology afforded good specificity and selectivity, simplification, and low cost without the need of sample separations and multiple washing steps, thus representing a user-friendly protocol for practical utilization in a quantitative PPase activity.

Surface Charge Density Determination in Water Based Magnetic Colloids: a Comparative Study

“…Figure 4 exhibits the typical pH-dependence of the surface charge density determined with both methods (sample A). The curve related to the SPM was evaluated from equations 3, 4 and 5 while that of the PCM was obtained from equation 6.…”

“…These materials have been widely used in many different fields, especially in nanobiotechnology 1,2 . Among their properties, the surface charge density plays a key role not only in colloidal stability, but also in many applications such as cellular uptake 3,4 , drug delivery 5 , biosensing 6,7 and removal of pollutants from wastewater 8 . In water dispersions of magnetic nanoparticles based on metal oxides, the nanoparticle surface presents an amphoteric behavior and becomes charged from protonation/ deprotonation reactions according to 9 …”

“…Thus, it is possible to determine the total concentration of the surface sites C T using the mass balance and the volume of titrant reagent used to titrate the nanoparticle surface. The pH-dependence of the surface charge density is achieved from a Two-pK Model according to: (6) where pK 1 and pK 2 corresponds to the thermodynamical constants (pK = -logK) related to the protonation/deprotonation mechanism. These constants are determined from the Henderson-Hasselbalch equation using the potentiometric data.…”

Microfluidics‐Implemented Biochemical Assays: From the Perspective of Readout

“…Magnetic beads can be simultaneously employed for labelling, separation, and detection. Lin et al introduced a microfluidic device integrated with a magnetic graphene nanosheet sensor for the real‐time monitoring of the pyrophosphatase activity electrically . This approach consisted of a magnetocontrolled microfluidic device, Cu 2+ ‐coordinated pyrophosphate ions (Cu 2+ ‐PPi) complex, and Nafion‐modified magnetic graphene nanosheets.…”

Electrochemiluminescence Immunosensor Based on Functionalized Graphene/Fe3O4‐Au Magnetic Capture Probes for Ultrasensitive Detection of Tetrodotoxin

“…In addition, graphene can be decorated with nanoparticles efficiently to obtain desirable properties. For example, GO can easily load magnetic iron oxide nanoparticles and form the graphene/Fe 3 O 4 hybrid materials, which can provide an effective separation and enrichment tool in analytical science . The core/shell Fe 3 O 4 ‐Au nanoparticles, one of the widely used nanoparticles in the immunoassay, combine the excellent characteristic of Fe 3 O 4 and Au nanoparticles .…”