# Hemostasis-On-a-Chip: Impedance Spectroscopy Meets Microfluidics for Hemostasis Evaluation

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## Abstract

**:**

## 1. Introduction

## 2. Materials and Methods

#### 2.1. Microflow Chamber Design and Manufacturing

#### 2.2. Biomimetic Coatings and Sample Preparation

^{2}and 0.95 ng/cm

^{2}, respectively, as previously described [17], and were flushed with saline prior to perfusion in order to eliminate the remaining collagen over the surface.

#### 2.3. Flow Assays

^{−}

^{1}was achieved on the glass surface, applying a flow rate of 0.1 mL/h to the microchannel through a syringe pump.

#### 2.4. Image Capture and Analysis

#### 2.5. Impedance Characterization

_{E}) can be described by different components according to [21]:

_{s}is the resistance of the solution, ${\mathrm{Z}}_{\mathrm{dl}}=\left({\mathrm{R}}_{\mathrm{ct}}+{\mathrm{Z}}_{\mathrm{w}}\right)\Vert \left(\frac{1}{{\mathrm{j}\mathsf{\omega}\mathrm{C}}_{\mathrm{dl}}}\right)$ is the double-layer impedance and C

_{g}is the dielectric capacitance and Z

_{p}the parasitic capacitance of the substrate.

_{s}is related to the conductivity of the solution, $\mathsf{\sigma}={\mathrm{qn}}_{\mathrm{i}}\text{}\left({\mathsf{\mu}}_{\mathrm{p}}+{\mathsf{\mu}}_{\mathrm{n}}\right)$, where $\mathrm{q}$ is the electric charge, ${\mathsf{\mu}}_{\mathrm{p}}$ and ${\mathsf{\mu}}_{\mathrm{n}}$ are the ionic mobilities of the dominant positive and negative ions in the solution, and ${\mathrm{n}}_{\mathrm{i}}$ is the ionic concentration, which can vary during coagulation.

_{dl}originates from the adsorbed charge layer and diffuse layer charge. For electrode separation higher than Debye length ($\lambda \sim 1\text{}\mathsf{\mu}\mathrm{m}$), C

_{dl}can be described by diffuse layer capacitance ${C}_{dl}={C}_{dif}=A\sqrt{\frac{2\mathsf{\epsilon}{n}_{i}{q}^{2}}{kT}}\mathrm{cosh}\left(\frac{q\text{}{V}_{ac}}{2kT}\right)$, where A is the area of the electrode (A = wL), V

_{ac}is the voltage applied, q is the electric charge, k is the Boltzmann constant, T is the temperature of the solution, and $\mathsf{\epsilon}$ is the permittivity of the medium separating the electrodes.

- For low frequencies ${\mathrm{f}}_{\mathrm{low}}=\frac{2}{2{\mathsf{\pi}\mathrm{R}}_{\mathrm{s}}{\mathrm{C}}_{\mathrm{dl}}}$, ${\mathrm{C}}_{\mathrm{dl}}$ dominates the impedance measured.
- For frequencies ${\mathrm{f}}_{\mathrm{low}}<f<{\mathrm{f}}_{\mathrm{high}}=\frac{2}{2{\mathsf{\pi}\mathrm{R}}_{\mathrm{s}}{\mathrm{C}}_{\mathrm{g}}}$, ${\mathrm{R}}_{\mathrm{s}}$ is the dominant impedance.
- For frequencies $\mathrm{f}>{\mathrm{f}}_{\mathrm{high}}$, ${\mathrm{C}}_{\mathrm{g}}$ is the dominant impedance.

_{s}is depending upon the concentration of the ions in the solution, since during coagulation different ions are involved. R

_{s}will change during the coagulation process; basically, if the concentration is increased, the conductivity will increase and R

_{s}decreases.

_{dl}will also increase if the concentration of ions is increased, and finally, the C

_{g}is independent on the ion concentration. C

_{g}changes could be related to changes in the permittivity of the solution, that could change if there were volume changes of the sample, but since our system is inflow, the volume covering the electrodes is constant.

_{2}(5mM)) to induce coagulation. Electrical impedance across the electrodes between 10 Hz and 1 MHz was measured while a sinusoidal voltage of 250 mV was applied.

## 3. Results

#### 3.1. Flow Assay Results

#### 3.2. Impedance Spectroscopy Assay Results

_{dl}dominates). The impedance is high at low frequencies and decreases gradually. Around 10

^{5}Hz, the impedance decreases to the behavior of becoming more resistive (R

_{s}dominates). Both samples PPP and PRP show the same trend, but the impedance of PRP is always higher due to the increased amount of cellular component which increases the resistivity of the solution, especially when CaCl

_{2}is added. Figure 5 shows a clear effect of the coagulation on the impedance modulus during the coagulation process, which results in an increase of impedance. This increase is remarkable for low and high frequencies in PRP samples, while in PPP samples the impedance increases at frequencies higher than 10

^{5}Hz.

## 4. Discussion

## 5. Conclusions

## Supplementary Materials

## Author Contributions

## Funding

## Conflicts of Interest

## References

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**Figure 1.**3D schematics and the picture of the biomimetic microfluidic channel. (

**a**) the cross-sectional view of the microfluidic channel. (

**b**) 3D assembly of the different parts of the microfluidic channel. (

**c**) Real image of the microfluidic channel.

**Figure 2.**(

**a**) Pictures of the test chamber assembled and open. (

**b**) Pictures of the microfluidic channel inside the test chamber with the thrombogenic surface with embedded electrodes. (

**c**) Impedance model of the electrode inside the channel.

**Figure 3.**Confocal image labeled by immunofluorescence for morphometric analysis from microfluidic studies. (

**a**) Confocal image showing platelets labeled byanti-CD36 Alexa Fluor 488 and Fibrin labeled by anti-fibrin(ogen) Alexa Fluor 594. The thrombogenic surface is a biomimetic combination with type-I fibrillar collagen (30.9 mg/cm

^{2}) and tissue factor (0.95 ng/cm

^{2}). Scale bar = 20 µm. (

**b**) The plot shows the quantification of platelet aggregates (green) and fibrin masses (red) interacting with the collagen/tissue factor surface. The bar graphs in the right panel summarize the results as percentages of the total surface exposed.

**Figure 4.**Real and Imaginary part of Impedance for plasma poor in platelets (PPP) and plasma rich in platelets (PRP) over time.

**Figure 5.**Impedance module at different frequencies after 90 s interaction of the electrode with (

**a**) PPP and PPP + CaCl

_{2}. (

**b**) PRP and PRP + CaCl

_{2}. (

**c**) Image of the platelets attached on the electrodes. Scale bar = 10 µm.

[APIX] ng/mL | Platelets | Fibrin |
---|---|---|

0 | 23.0 ± 3.0 | 43.4 ± 4.8 |

10 | 17.9 ± 0.9 | 42.1 ± 1.9 |

40 | 14.0 ± 5.3 | 23.4 ± 7.7 |

160 | 5.4 ± 2.2 *# | 14.1 ± 4.9 *# |

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**MDPI and ACS Style**

Karimi, S.; Farré-Lladós, J.; Mir, E.; Escolar, G.; Casals-Terré, J.
Hemostasis-On-a-Chip: Impedance Spectroscopy Meets Microfluidics for Hemostasis Evaluation. *Micromachines* **2019**, *10*, 534.
https://doi.org/10.3390/mi10080534

**AMA Style**

Karimi S, Farré-Lladós J, Mir E, Escolar G, Casals-Terré J.
Hemostasis-On-a-Chip: Impedance Spectroscopy Meets Microfluidics for Hemostasis Evaluation. *Micromachines*. 2019; 10(8):534.
https://doi.org/10.3390/mi10080534

**Chicago/Turabian Style**

Karimi, Shadi, Josep Farré-Lladós, Enrique Mir, Ginés Escolar, and Jasmina Casals-Terré.
2019. "Hemostasis-On-a-Chip: Impedance Spectroscopy Meets Microfluidics for Hemostasis Evaluation" *Micromachines* 10, no. 8: 534.
https://doi.org/10.3390/mi10080534