Electrophoretic-deposited HAP nano-layer as a QCM-D sensor coating : effects of suspension concentration and electric-field strength

This study investigates the fabrication of hydroxyapatite (HAP) nano-coating on a gold–quartz crystal sensor used for quartz crystal microbalance with dissipation (QCM-D) measurement using an electrophoretic deposition technique. Surface morphology and thickness of the HAP coating are examined via scanning electron microscopy and nano-indention testing. Its repeatability is verified via QCM-D testing. Results show that electrophoretic deposition with ultrasonic treatment is feasible and cost-effective for fabricating nano-thick HAP coatings on a QCM-D gold–quartz crystal sensor surface. Both suspension concentration and electric-field strength influence the compactness of HAP coatings. There exists a non-linear relationship between HAP coating compactness and the suspension concentration/electric-field strength. When the HAP suspension concentration is 30 g/l and the applied electric-field strength is 150 V/cm, the HAP coating on the QCM-D gold–quartz crystal sensor surface is uniform and compact with a thickness of 35 nm and is tightly bonded to the sensor surface. The obtained HAP-coated sensor is thus suitable for QCM-D measurement.


Introduction
Quartz crystal microbalance with dissipation (QCM-D) is widely used in the analysis of biomembrane adsorption kinetics on different substrates such as metals, polymers and functionalised coatings, owing to its high sensitivity and real-time monitoring [1][2][3].There are many types of sensors used for QCM-D measurements such as gold, chrome, silver and titanium.The choice of sensor type depends on the testing purpose.
Owing to its excellent biocompatibility, hydroxyapatite (HAP) is one of the most promising biomaterials.Recently, HAP has been widely used in dental and orthopaedic surgeries such as bone replacements and implants.It is inevitable for artificial implants to contact various proteins in the human body, and the high biocompatibility of HAP is partly explained by its ability to adsorb 'mild' proteins [4,5].Thus, understanding and controlling protein adsorption and its mechanism on the HAP surface are of great importance for clinical application and further improvement of HAP.QCM measurements have been applied to study the biosorption of HAP surfaces in simulated body fluids, and the HAP sensor is the best choice.However, commercial HAP sensors are really expensive currently because its fabrication is limited by special processes and equipment.Gold-quartz crystal sensors are the most common and cheapest among the commercial QCM-D sensors.Thus, many studies concerning the bio-adsorption of HAP have been conducted using gold-quartz crystal sensors rather than HAP sensors [1,6,7].In fact, proteins exhibit different adsorption behaviours on different substrates [8].Given that properties of gold and HAP are different, there should exist differences between protein adsorption behaviours on the two surfaces.It has been demonstrated that HAP-coated gold sensors can replace commercial HAP sensors to study the biosorption of HAP materials [5].Therefore, it is necessary to investigate how to prepare cost-effective HAP coatings on gold-quartz crystal sensors.
Technologies such as the sol-gel method, electrochemical precipitation, laser cladding, plasma spraying and electrophoretic deposition have been used to coat metal substrates with thin layers of HAP [3,5,[9][10][11][12].Among them, plasma spraying and laser cladding are linear coating processes that cannot be used to prepare a coating with a uniform thickness on a complex substrate surface, whereas sol-gel or electrochemical technology is timeconsuming, and the thickness of the obtained coating is mostly micron-scale.By comparison, electrophoretic deposition technology is simple and mild, and it can quickly deposit high-quality coatings on complex surfaces.In 2006, Monkawa et al. [5] first used electrophoretic deposition technology to prepare HAP nano-coatings on QCM-D gold-quartz crystal sensors.However, there has been little news on the influence of deposition parameters on the morphology and thickness of coatings.Therefore, electrophoretic deposition technology is not widely used to coat QCM-D gold-quartz crystal sensors with ultra-thin layers of HAP.
In this paper, nano-thick HAP coatings are prepared on QCM-D gold-quartz crystal sensor surfaces using the electrophoretic deposition method with ultrasonic treatment.The effects of deposition parameters such as suspension concentrations and electric-field strengths on the compactness of HAP coatings are investigated.The surface morphology, thickness and adsorption repeatability of the coating obtained with the optimum process parameters are characterised.

HAP coating preparation
Commercial QCM-D gold-quartz crystal sensors are used in this paper.They are cleaned for 5-10 min with 70-75°C mixed solution consisting of deionised water, 25% ammonia (w/w) and 30% hydrogen peroxide (w/w) (volume ratio 5:1:1), and then it is rinsed several times with deionised water.After drying, the sensors are irradiated with ultraviolet/ozone for 10 min.
HAP suspensions at different concentrations (10, 20, 30 and 40 g/l) are made by dispersing HAP nano-particles in ethanol.The suspensions are ultrasonically oscillated for 1 h (40 kHz, 100 W) and static aged for 2 days.They are ultrasonically oscillated for 1 h again before usage.
The nano-thick HAP coating is deposited by electrophoretic deposition methods using the gold-quartz crystal sensor as the cathode and the HAP suspension at different concentrations as electrolytes.The space of two electrodes is 1 cm and the electrophoretic deposition lasts 5 min.Four DC voltages, 50, 100, 150 and 200 V/cm, are applied.After deposition, the sensor is ultrasonically treated in ethanol for 1 min to remove the surplus HAP from the deposited layer.Fig. 1 gives the schematic diagram of the electrophoretic deposition device.

HAP coating characterisation
HAP coating surface morphology is observed via scanning electron microscopy (SEM) (QUANTA200, FEI, Holland), and the chemical composition and crystal characteristics of the coating are examined by X-ray diffractometer (XRD) (D1, BeDe, UK).
During nano-indentation testing of material surfaces under a continuous stiffness mode, there exists a phase shift between the load and the displacement oscillation, which is called phase angle.Generally, the phase angle gradually decreases to a constant with the increase of indentation depth [13].The initial variation of phase angle with indentation depth is very responsive and is considered to result from the unstable press-in frequency which is closely associated with the surface properties of materials.Recently, it has been pointed out that the thickness of nano-film can be obtained by measuring the indentation depth corresponding to the starting point of unchanged phase angle [13].Thus, nano-indentation tests under a continuous stiffness mode are conducted on the original and HAP-coated surfaces of gold-quartz crystal sensors using a nano-indentation tester (G200, Agilent Technologies Inc., USA).A Berkovich diamond tip with a radius of 20 nm is used, and the maximum indentation depth is 500 nm.Given that ambient condition, tip radius of curvature and the contact condition of tip with substrate can significantly affect test data at the beginning of nano-indentation testing, especially when the indentation depth is <15 nm, the thickness of HAP coating is obtained by calculating the difference between the indentation depths of the original and coated surfaces corresponding to the starting point of unchanged phase angle.
The adsorption repeatability of the HAP coating is evaluated using QCM-D (Q-Sense Explorer, Biolin Scientific, Sweden), and human saliva is used as an adsorption medium.The saliva was provided by a 25-year-old healthy male volunteer.The collection and treatment of saliva are described in detail in [14].The adsorption testing process is as follows: first, deionised water flows to the QCM-D with a flow rate of 200 μl/min to establish a baseline; second, human saliva flows to the QCM-D with a flow rate of 80 μl/min for 25 min; and third, deionised water flows again with a flow rate of 80 μl/min for 10 min.

Results
Fig. 2 shows the SEM morphologies of QCM-D gold-quartz crystal sensor surfaces subjected to electrophoretic deposition applying 100 V DC voltage and using the HAP suspensions at different concentrations.The sensor surface morphology varies with the concentration of HAP suspension.When the suspension concentration is 10 g/l, a layer of uneven HAP nano-particle deposits appear on the gold-quartz crystal sensor.When the suspension concentration is increased to 30 g/l, a uniform and dense nano-particle layer appear on the surface of the gold substrate.However, with the suspension concentration increasing to 40 g/l, the uniformity of deposits decreases and gaps appear between the nano-particles.Obviously, HAP suspension concentrations influence the compactness of the deposited nano-particles.
Fig. 3 shows the SEM morphologies of QCM-D gold-quartz crystal sensor surfaces subjected to electrophoretic deposition using 10 g/l HAP suspensions under different electric-field strengths.Electric-field strength plays an important role in the compactness of the deposited nano-particles.With the electric-field strengths increasing from 50 to 150 V/cm, the compactness of the deposits gradually increases.However, as the electric-field strength increases further to 200 V/cm, gaps appear between the deposited nanoparticles, then the surface compactness decreases.
Fig. 4 shows surface morphologies of QCM-D gold-quartz crystal sensors before and after electrophoretic deposition with 30 g/l HAP suspension and 150 V/cm electric-field strength.The chemical compositions and crystal characteristics of the two surfaces are shown in Fig. 5.After electrophoretic deposition and ultrasonic treatment, a layer of uniform and compact nano-particles is formed on the sensor surface.The XRD spectrum of deposited coating is consistent with the standard spectrum of HAP (PDFCard09-432), indicating that no chemical changes occurred to HAP nano-particles during electrophoretic deposition.
Fig. 6 shows the variations of gold-quartz crystal sensor surface phase angles before and after electrophoretic deposition (30 g/l HAP suspension and 150 V/cm electric-field strengths) with indentation depth.The phase angle of each surface is obtained from an average of five tests.With the indentation depth increasing, the phase angle decreases gradually and then remains constant.The indentation depth corresponding to the starting point of the unchanged phase angle is 60 nm for the original sensor and 95 nm for the coated sensor.The difference between them is approximately the thickness of the HAP coating on the gold-quartz crystal sensor [13,15].Thus, the coating thickness is about 35 nm.
Saliva adsorption tests are repeated three times on the same HAP-coated gold-quartz crystal sensor (30 g/l HAP suspension and 150 V/cm electric-field strength), and the corresponding variations of frequency shift (ΔF ) and dissipation shift (ΔD) of salivary pellicles with adsorption times are shown in Fig. 7. Variations of ΔF and ΔD curves with adsorption times are approximately the same for the three repeated tests.This means that few HAP nano-particles are detached from the sensor.That is, the HAP coating prepared in this paper has a high binding strength with the gold matrix.Thus, the HAP-coated QCM-D sensor has better reusability.

Discussion
The QCM-D technique has a high sensitivity and is widely used in the quantification research of biofilm formation.It is based on monitoring the change of the quartz crystal sensor's resonant frequency.Thus, there are strict requirements for the  sensor coating quality (e.g.thickness, compactness and bonding with substrates).Generally, the thickness of the sensor coating should be <100 nm.Otherwise, it difficult to obtain a stable resonant frequency [5].Additionally, the coating should bond tightly with substrates to prevent it from detaching during adsorption testing and cleaning treatment.Thus, the sensor can be used repeatedly.
Generally, HAP coatings prepared by electrophoretic deposition techniques are relatively thick, and a higher-quality coating with a micron thickness can be obtained only after certain sintering treatment [12,16,17].For an unsintered coating, visible cracks appear as the internal ethanol evaporates, and the particles in the coating easily fall off.Thus, ultrasonic treatment can remove particles and thin the coating.The HAP coating thickness on the sensor after ultrasonic treatment is ∼35 nm (Fig. 6), whereas the HAP nanopowder in the suspension used in this paper has a particle size ranging from 30 to 50 nm, indicating that the obtained HAP coating on the gold-quartz crystal sensor surface using electrophoretic deposition method with ultrasonic treatment is comprised of a single layer of HAP nano-particles.Hence, it can be inferred that there exist two stages during the HAP deposition process on the gold-quartz crystal sensor, as shown in Fig. 8. First, HAP nano-particles directly deposited on the sensor surface to form a nano-thick initial layer, and the initial layer has a high binding strength with the sensor.Subsequently, HAP nano-particles deposit on the initial layer to form a micrometre-thick covering layer, which has a weak bond with the initial layer and then is easily removed by ultrasonic cleaning.Thus, in this paper, a nanometre-thick coating with a high binding strength is obtained on the sensor surface using an electrophoretic deposition method in combination with ultrasonic treatment.
It is evident that the HAP suspension concentration has a significant influence on the compactness of the HAP nano-coating.As shown in Fig. 2, there exists a suitable suspension  However, an extra-high content of HAP nano-particles can lead to a decreased stability of the suspension, so that the particles agglomerate before moving directionally to the electrode.Thus, the HAP nano-coating turns increasingly uniform and compact as the concentration of the HAP suspension increases from 10 to 30 g/l.However, with the concentration increasing further to 40 g/l, the compactness of the coating decreases.
The compactness of the HAP nano-coating also relates to the applied electric-field strength.The HAP nano-particles not only move slowly but also bond weakly with the substrate under the action of a low electric-field strength.That is, the deposited particles on the sensor surface are easily removed by ultrasonic treatment.As the electric-field strength increases, the movements of HAP nano-particles accelerate, and the electric-field force applied to the particles increases and contributes to a tight bonding of deposits with substrates [5,18].However, under an extra-high electric-field strength, HAP nano-particles move too fast to deposit on the electrode surface under an optimal arrangement [19].Thus, as shown in Fig. 3, the obtained HAP nano-coating is noncompact under a low electric-field strength and its compactness gradually increases as the electric-field strength increases from 50 to 150 V/cm.As the electric-field strength increases further to 200 V/cm, the compactness of the coating decreases.
Whether the coated sensors are suitable for QCM-D testing depends on the compactness, thickness and binding strength of the coating.When applying 30 g/l HAP suspension and 150 V/cm electric-field strength, the obtained HAP nano-coating is uniform and compact, and its thickness is about 35 nm (Fig. 6).This meets the requirements of QCM-D testing for the thickness of the coating on the sensor surface.Additionally, the results of saliva adsorption testing demonstrated that the HAP nano-coatings obtained under the above conditions have a high binding strength with the gold matrix (Fig. 7).Obviously, the condition of 30 g/l HAP suspension and 150 V/cm electric-field strength is suitable for the deposition of HAP nano-particles on the sensor surface.

Conclusion
In this paper, HAP nano-coating was prepared on the QCM-D gold-quartz crystal sensor surface using the electrophoretic deposition method with ultrasonic treatment.Within the limitations of the present paper, the main conclusions can be summarised as follows: (i) Electrophoretic deposition in combination with ultrasonic treatment is a feasible and cost-effective method for fabricating nano-thick HAP coatings on a QCM-D gold-quartz crystal sensor surface.Both suspension concentration and electric-field strength influenced the compactness of HAP coating.There existed a non-linear relationship between HAP coating compactness and the suspension concentration/electric-field strength.(ii) When the HAP suspension concentration was 30 g/l and the applied electric-field strength was 150 V/cm, the HAP coating on the QCM-D gold-quartz crystal sensor surface had a thickness of about 35 nm thick, and it was uniform, compact and tightly bonded on the sensor surface.The obtained HAP-coated sensor was suitable for QCM-D testing.

Fig. 2
Fig. 2 SEM morphologies of QCM-D gold-quartz crystal sensor surfaces subjected to electrophoretic deposition using HAP suspensions at different concentrations a 10 g/l b 20 g/l c 30 g/l d 40 g/l

Fig. 4
Fig. 4 SEM micrographs of gold-quartz crystal sensors before and after electrophoretic deposition with 30 g/l HAP suspension and 150 V/cm electric-field strength a Before deposition b After deposition

Fig. 6 Fig. 7
Fig. 6 Variations of the phase angle of the gold-quartz crystal sensor surfaces before and after electrophoretic deposition with indentation depth

Fig. 8
Fig. 8 Coating preparation schematics by electrophoretic deposition and ultrasonic treatment