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. 2016 Aug;408(21):5757-5768.
doi: 10.1007/s00216-016-9678-6. Epub 2016 Jun 10.

Characterisation of the protein corona using tunable resistive pulse sensing: determining the change and distribution of a particle's surface charge

Emma L C J Blundell  1 , Matthew J Healey  1 , Elizabeth Holton  1 , Muttuswamy Sivakumaran  2 , Sarabjit Manstana  3 , Mark Platt  4
Affiliations

Characterisation of the protein corona using tunable resistive pulse sensing: determining the change and distribution of a particle's surface charge

Emma L C J Blundell et al. Anal Bioanal Chem. 2016 Aug.

Erratum in

Abstract

The zeta potential of the protein corona around carboxyl particles has been measured using tunable resistive pulse sensing (TRPS). A simple and rapid assay for characterising zeta potentials within buffer, serum and plasma is presented monitoring the change, magnitude and distribution of proteins on the particle surface. First, we measure the change in zeta potential of carboxyl-functionalised nanoparticles in solutions that contain biologically relevant concentrations of individual proteins, typically constituted in plasma and serum, and observe a significant difference in distributions and zeta values between room temperature and 37 °C assays. The effect is protein dependent, and the largest difference between the two temperatures is recorded for the γ-globulin protein where the mean zeta potential changes from -16.7 to -9.0 mV for 25 and 37 °C, respectively. This method is further applied to monitor particles placed into serum and/or plasma. A temperature-dependent change is again observed with serum showing a 4.9 mV difference in zeta potential between samples incubated at 25 and 37 °C; this shift was larger than that observed for samples in plasma (0.4 mV). Finally, we monitor the kinetics of the corona reorientation for particles initially placed into serum and then adding 5 % (V/V) plasma. The technology presented offers an interesting insight into protein corona structure and kinetics of formation measured in biologically relevant solutions, i.e. high protein, high salt levels, and its particle-by-particle analysis gives a measure of the distribution of particle zeta potential that may offer a better understanding of the behaviour of nanoparticles in solution. Graphical Abstract The relative velocity of a nanoparticle as it traverses a nanopore can be used to determine its zeta potential. Monitoring the changes in translocation speeds can therefore be used to follow changes to the surface chemistry/composition of 210 nm particles that were placed into protein rich solutions, serum and plasma. The particle-by-particle measurements allow the zeta potential and distribution of the particles to be characterised, illustrating the effects of protein concentration and temperature on the protein corona. When placed into a solution containing a mixture of proteins, the affinity of the protein to the particle's surface determines the corona structure, and is not dependent on the protein concentration.

Keywords: Biosensor; Protein corona; TRPS; Tunable pores; Zeta potential.

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Conflict of interest statement

The authors declare no competing interests. Human and animal rights and informed consent This work has been performed with the consent of healthy volunteers. The studies were approved by the ethics committee and performed in accordance of the ethical standards or Loughborough University.

Figures

Graphical Abstract
Graphical Abstract
The relative velocity of a nanoparticle as it traverses a nanopore can be used to determine its zeta potential. Monitoring the changes in translocation speeds can therefore be used to follow changes to the surface chemistry/composition of 210 nm particles that were placed into protein rich solutions, serum and plasma. The particle-by-particle measurements allow the zeta potential and distribution of the particles to be characterised, illustrating the effects of protein concentration and temperature on the protein corona. When placed into a solution containing a mixture of proteins, the affinity of the protein to the particle’s surface determines the corona structure, and is not dependent on the protein concentration
Fig. 1
Fig. 1
Particles in the presence of human plasma and serum showing the formation of both a ‘hard’ and ‘soft’ protein corona. I1.0, I0.8, I0.6, I0.4, I0.2 represent the position of the particle as it translocates the pore (where I1.0 is the narrow pore entrance) and are relative to T1.0, T0.8, T0.6, T0.4, T0.2, which represent the time taken (ms) for the particle to reach that position. T1.0, is equivalent to dRmax when the blockade event is at 100 % magnitude; T0.8, T0.6, T0.4, T0.2, correspond to when the blockade is 80, 60, 40, and 20 % of its dRmax and indicates the particle traversing the pore
Fig. 2
Fig. 2
Mean zeta potential (mV) versus the protein the particles were incubated with. The blue bars show results for a 10-min particle incubation at 25 °C and the red bars show the mean zeta potential values for particles incubated with the proteins for 10 min at 37 °C. The green lines represent the measured mean zeta potential for calibration particles of known zeta potential (−20 mV) in PBS that were run after each protein sample to show the protein samples were not having a direct effect on the pore walls themselves that may influence the recorded zeta potentials of future samples run on the same pore. Error bars are representative of the st.dev where n = 3
Fig. 3
Fig. 3
Zeta potential (mV) versus particle size (nm). The red, blue and green datasets are zeta potential distributions for CPC200s incubated for 10 min with fibrinogen, γ-globulin and albumin, respectively at (a) 25 °C and (b) 37 °C. The black data points represent CPC200s in PBS for both figure parts a and b
Fig. 4
Fig. 4
(a) Mean zeta potential (mV) versus incubation medium. Comparison of CPC200 particles incubated in PBS (green), plasma and serum for 10 min at 25 °C (blue) and 37 °C (red). Error bars are representative of the st.dev where n = 3. (b) Frequency (%) versus zeta potential (mV). Zeta potential distributions for CPC200s incubated for 10 min at 37 °C in plasma (purple) and serum (pink). Repeat datasets for CPC200s incubated in both plasma and serum at 37 °C for 10 min are illustrated in ESM Fig. S3 and are compared to a zeta potential distribution of CPC200s in PBS only
Fig. 5
Fig. 5
(a) Zeta potential distributions for CPC200s incubated in plasma for 10 min at 25 °C (red) and 37 °C (purple). (b) Zeta potential distributions for CPC200s incubated in serum for 10 min at 25 °C (green) and 37 °C (blue)
Fig. 6
Fig. 6
The effect of spiking a sample of CPC200s incubated in serum with plasma. (a) Visual representation of the effect of protein displacement and exchange within a protein corona system. (i) Protein corona formed by particle incubation in serum, (ii) introduction of plasma proteins to sample, (iii) displacement of hard corona proteins due to proteins of higher affinities and exchange of soft corona proteins, (iv) Depletion of soft corona layer as proteins dissociate from loose protein-protein interaction. (b) Particles were incubated in serum for 10 min and then spiked with 5 % (v/v) plasma. Zeta potentials were measured at 5, 10, 15, 20, 30 and 60 min. (i)–(iv) indicate the shift in zeta potential as a result of the effects described in (a)
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