An electronic platform for real-time detection of bovine serum albumin by means of amine-functionalized zinc oxide microwires

Abstract

We report the fabrication of a customized electronic platform for biosensing, integrating a single functionalized microwire between gold microelectrodes as a sensing element, including a custom microelectronic chip for signal readout. As a proof-of-concept, the platform was validated for the real-time detection of Bovine Serum Albumin (BSA) binding onto an NH₂-functionalized zinc oxide (ZnO-NH₂) microwire. The ZnO-NH₂ microwires were deposited between two gold electrodes by means of a dielectrophoresis (DEP) technique. A Quasi-Digital Impedance Converter (QDIC) was conceived to constantly and instantaneously readout ZnO microwire impedance and transfer data to a laptop. Microelectrodes, a QDIC, a DEP generator, and data analysis were integrated in a stacked-card PCB configuration for a better noise reduction and usability. The system was able to distinguish between different BSA concentrations and to give real-time information about the binding process.

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1 Introduction

Sensing devices based on nano- and micrometer-scale structures can provide novel tools for the investigation of physical parameters as well as of a broad range of molecules. Research activity has already been devoted to produce sensors able to detect extremely small amounts of analytes, which are particularly important in the context of biosensing. In this field, there is a great demand for low concentration detection and high specificity towards the analyte of interest. The ultimate goal of nano- and microscale-based biosensors is to detect any biochemical and biophysical signal associated with a specific disease at the level of a single molecule or cell. This technology can improve conventional medical practices by enabling the early diagnosis of chronic debilitating diseases, ultrasensitive detection of pathogens and long-term monitoring of patients using biocompatible integrated medical instrumentation.¹ Moreover, the future impact of nano- and micro-biosensor systems will move to point-of-care diagnostics. Indeed, as soon as nano- and micro-biosensor technology becomes more refined and reliable, it is expected that it will make its way from the lab to the clinic, where future lab-on-a-chip devices incorporating an array of nano- and micro-biosensors could be used for rapid screening of a wide variety of analytes at low cost, using small amounts of patient materials and fluids.

Among all biomolecules, protein detection is more challenging than nucleic acid detection for two reasons: (i) protein amplification (like PCR for DNA) does not yet exist, thus requiring in many cases for very small concentrations to be detected; (ii) protein sensing needs a very controlled environment (e.g., temperature, contamination, pH) which is difficult to reproduce, thus a high accuracy measurement system is required to detect low concentrations. To overcome these challenges, the detection of proteins with biosensors can be implemented by directly detecting the bioanalyte as soon as it binds to two electrode surfaces separated by a nanometer-sized distance (i.e. a nanogap). In this case, effective protein detection in nanogap-based² bioelectronic devices strongly depends on the binding process of the biomolecule onto the electrode surface. Actually, electron transfer between the protein and the electrode is influenced by four parameters: the biomolecule binding process, the material and surface properties of the electrode and the interface between the bioanalyte and the metal electrode, which behaves as an inherent barrier for electron transfer. All these factors can modify the orientation of the protein and its distance from the electrode, thus influencing the biosensor response. However, a strong limitation of this detection process is that the nanogap dimensions should match the biomolecule size, which is in most cases a few nanometers, and the biomolecule has to bridge the two facing electrodes to be successfully detected.

Therefore, a more effective alternative method for biomolecule detection is based on indirect measurement which involves the monitoring of the electrical properties of functionalized sensing nano- or micromaterials during the binding of target biomolecules.^3,4 Among these materials, zinc oxide (ZnO) has received great attention, having several favorable properties including high electron mobility and a wide band gap (3.3 eV), as well as different sensing abilities toward the pH of solutions,^5,6 gas molecules,⁷ ultraviolet (UV) light,⁸ and temperature variation.⁹ The advantages associated with a large band gap include high breakdown voltages, robustness to large electric fields, and high-temperature and high-power operation, which are quite desirable properties in view of integration of the material into electronic circuits. In addition, ZnO can be easily synthesized using wet-chemical methods in various morphologies and shapes, including nanoparticles,^10,11 nano- and micro-wires,^11,12 multipods,¹³ nanotubes,¹⁴ flower-like particles^15,16 and other structures.¹⁷ Complementing its unique chemical qualities, ZnO offers significantly lower fabrication costs using wet-chemical methods when compared to other semiconductors used in nanotechnology. The external surface of the ZnO crystalline structure, particularly when synthesized using wet-chemical low temperature processes,¹⁸ exposes several hydroxyl groups (–OH), which can be further used to chemically anchor molecules and functional groups. ZnO crystallizes in an anisotropic wurtzite hexagonal phase, which leads to polar crystalline end surfaces. Particularly, monocrystalline ZnO micro- and nanowires show an intrinsic polarization due to an oxygen plane (negatively charged) and zinc plane (positively charged) placed at the edges of the nanostructure.¹⁹ This intrinsic dipole allows the easy alignment of ZnO micro- and nanowires suspended in a liquid medium exploiting the well-known dielectrophoresis (DEP) process.^8,20 The DEP technique consists of generating a non-uniform electric field E⃑ between the metal electrodes where the micro- and nanoparticle has to be assembled. Indeed, the non-uniform E⃑ induces an attractive force in the ZnO microwires, leading to them moving towards the electrodes (i.e., where E⃑ is higher), and aligns them following the electric field lines. Other semiconductor nano- or microwires are good candidates for such biosensing but not all of them can be as eligible for such a task as ZnO materials. Titania nanowires, for example, show similar semiconductor and wide band-gap properties but they can not be guided on electrical contacts with the DEP technique, losing the advantage of easy integration. Other types of exotic materials are too difficult or sometimes impossible to chemically functionalize, for further anchoring and detecting biomolecules. On the other hand, silicon nano- and microwires (SiNW) have been extensively studied and exploited. There is a large number of papers in the literature reporting the use of SiNWs between two electrodes as chemical and biomolecule sensors.^4,21

For these reasons and because of the previously mentioned properties of ZnO, following the state-of-the-art in bio-nanosensors, this paper studies real-time protein detection based on functionalized ZnO microwires assembled on a gold electrode. A custom microelectronic chip, called Micro-for-Nano (M4N),²² continuously measures ZnO microwire impedance and transfers data to a laptop that plots and stores them for further analysis. The whole system is designed to be easy-to-use and reliable. In fact, an Arduino²³ board is the interface between the laptop and the customized chip while the integration of the electrodes on the same board of the chip assures low noise and precise measurement. The custom microchip also provides an AC signal to enable the DEP process for ZnO microwire integration. We used this integrated system for the real time detection of Bovine Serum Albumin (BSA) protein, a well-known plasma protein able to bind and transport a range of hydrophilic molecules which shows no electroactive role in its action.

2 Indirect measurement of BSA protein concentration

2.1 ZnO synthesis and functionalization

The ZnO microwires were prepared using a hydrothermal method according to ref. 5 and 6. In detail, 1.48 g of zinc nitrate hexahydrate Zn(NO₃)₂·6H₂O (5 mmol, Sigma-Aldrich S.r.l. Milan, Italy) in 10 ml of bi-distilled water (Direct Q, Millipore Co., Billerica, MA, USA) was slowly dropped into a solution of 3.35 g of potassium hydroxide (60 mmol, Merck KGaA, Darmstadt, Germany) in 10 ml of water under vigorous stirring. The obtained solution was then heated at 70 °C for 5 h in a closed Teflon vessel. The formed ZnO microwires were then collected by filtration, washed thoroughly with water until neutral pH was reached, and dried in air at 60 °C.

Chemical functionalization with aminopropyl groups was carried out with 250 mg (3.075 mmol) of ZnO microwires after outgassing them for 2 h in a glass round flask. Then 0.307 mmol of aminopropyltrimethoxysilane (APTMS; 55.04 mg), corresponding to 10 mol% of the ZnO molar amount, was added together with 10 ml of toluene, and the solution was refluxed for 24 h under a nitrogen atmosphere. The functionalized microwires (ZnO-NH₂ microwires) were then washed with toluene to remove unbound molecules and then dried at 60 °C overnight.

2.2 Reaction mechanism

The EDC/sulfo-NHS system has probably been the most successful method of creating zero length crosslinks for decades.²⁴ Facilitated by a reactive carbodiimide (EDC) and N-hydroxysulfosuccinimide (sulfo-NHS) as an EDC stabilizer, this coupling procedure is a highly efficient choice for crosslinking proteins or immobilizing proteins to a support. 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) is the most readily available and commonly used carbodiimide. Two of the main advantages of EDC coupling are the room temperature reaction, which enables it to operate with all biological molecules, and the water solubility of EDC, which allows direct bioconjugation without dissolution in organic solvents. The addition of sulfo-NHS stabilizes the amine-reactive intermediate by converting it to an amine-reactive NHS ester, thus increasing the efficiency of EDC-mediated coupling reactions. In this work we constantly and instantaneously (in the order of few ms) monitored each required step for the coupling of carboxyl groups of the BSA protein to the amine-functionalized ZnO microwire already deposited on the gold electrodes. In particular we divided the whole EDC amidation process into four steps to clarify the different measured electric impedance outputs:

• measurement of the ZnO-NH₂ microwire–gold junction in air for 30 minutes;

• addition of 250 μl of bi-distilled water and 30 minutes real-time monitoring;

• addition of equimolar amounts of EDC and sulfo-NHS reagent (5 μmol) + 125 μl of bi-distilled water and 30 minutes real-time monitoring;

• injection of 15 or 150 nmol of BSA + 125 μl of bi-distilled water and monitoring the electric impedance for several hours.

Every solution was prepared a few minutes before addition to the previous step.

The mean value of the current was used as a reference to normalize the measurements, since each microwire has its own impedance value that has to be known for comparing different microwires. All measurements were normalized using the mean value of the measured impedance of each ZnO microwire assembled onto a different gold microelectrode. This was needed to compare the results obtained from different ZnO microwires. The selection of this quite long time step is arbitrarily defined in order to test the stability over time of the proposed whole system. However, the M4N chip constantly measures microwire impedance with an acquisition time of a few milliseconds, thus leading to a real-time output.

2.3 Real-time electronic acquisition interface

Considering that the final purpose of this study is the development of an integrated system for the real-time measurement of protein binding to functionalized ZnO nano- and microwires, we conceived a custom microelectronic chip⁸ able to both apply a dielectrophoretic signal to attract the microwire between two electrodes and readout the microwire signal (i.e. a current in the pA to μA range). We integrated the M4N chip on a PCB interfaced with an Arduino board to transfer the measured data to a workstation (i.e. a laptop) for data storage and real-time visualization. An Arduino platform was chosen due to the large availability of different tools that make the development of an intuitive user interface, a robust data transfer protocol and accessible documentation for future platform improvements easier. Fig. 1 shows a block diagram of the whole system. The current flowing through the ZnO microwire depends on the intrinsic characteristics of the material, here represented by a simple electrical model including the resistance R_μwire and the capacitance C_μwire in parallel. These electrical parameters are converted using a Quasi-Digital Impedance Converter (QDIC)⁸ to a digital signal, V_QD, compliant with microcontroller I/Os. The time-to-zero T_0,μwire and time-to-one T_1,μwire of the signal, V_QD, carry sensing information since they are directly proportional to C_μwire and R_μwire. As reported in ref. 8, the average values of T_0,μwire and T_1,μwire are accurately calculated in real-time by implementing a Time-to-Digital Converter (TDC) using the internal timers of the microcontroller (μcontroller). T_0,μwire and T_1,μwire are used in eqn (1) and (2),where a is a constant linked to the design parameter, C_base and R_par are estimated during a calibration step, C_ref is an off-chip component, and R_μwire and C_μwire are the calculated electrical properties of the ZnO microwire. The calibration algorithm and these simple equations are also implemented in the microcontroller firmware, so that the device can only transmit the final values to the workstation for real-time plot and data storage. Considering that the creation of chemical links between the BSA protein and ZnO microwire involves additional localized charges and the sharing of electrons in the conduction band of the ZnO microwire, an increase of R_μwire and C_μwire is expected during experiments.²⁵ The designed QDIC covers a wide range of resistance and capacitance loading, which is needed to support large process variation between different microwires. It achieves a measurement accuracy of 1%, and therefore can detect small interactions between the microwire and a low quantity of anchored bio-molecules. Since the measurement is represented in the time-domain, the measurement noise of quasi-digital converters corresponds to the jitter of the characteristic oscillation period T_μwire = T_0,μwire + T_1,μwire, which can be caused by coupling noise and external interference. In order to detect a small quantity of BSA protein and considering that the ZnO microwires are also sensitive to temperature and UV light irradiance variation, a controlled test environment is thus required. Hence, the system has been encapsulated into a bulky Faraday cage with two benefits: a shield for noise interference and protection from ambient light irradiance. Fig. 2 shows the experimental setup which includes the developed measurement system and a PC workstation for real-time graph visualization. The experiment needs several hours to evaluate the complete binding process between the BSA protein and ZnO-NH₂ microwire. Hence, the system was evaluated by performing a 24 hour measurement of a known stable impedance in order to identify the presence of output drift of the ROC during measurement. The results (ESI Fig. S1†) show a stable output with SNR ≥ 44 dB, assuring reliable data during the experiment that involves the ZnO microwire and BSA protein. The total time duration of the experiment is chemistry-driven since each single measurement can take at most a few milliseconds. Thus the system is able to monitor very fast to really slow reaction kinetics in real-time. In the next section the results of a preliminary experiment using two different concentrations of BSA protein are presented and discussed in detail.

Fig. 1 Block diagram of the electronic acquisition system. The M4N chip can apply a DEP signal to attract a microwire between two electrodes as shown in the scanning electron microscopy image. Such a microwire is modeled as a variable RC read by the Read Out Circuit (ROC). Such a value is transferred to a microcontroller that elaborates the signal and sends it to a laptop for further analysis. TDC: Time-to-Digital Converter, ALU: Arithmetic Logic Unit, cfg_regs: configuration registers.

Fig. 2 Picture and schematic drawing of the acquisition system. An Arduino board, ROC and sensor chips are integrated in a multi-stack PCB and encapsulated in a Faraday cage. Measured data are transmitted to a laptop through a USB cable. Data are then plotted in real-time and stored for further data analysis.

2.4 Experimental setup overview

Fig. 3 shows the complete experimental setup. An Arduino board controls the M4N chip described in Section 2.3, which is mounted onto an interface board (Fig. 3a). This board also hosts the sensing device composed of four replicas of two facing gold electrodes (thickness 20 nm) deposited by photolithography on a Si wafer with a Ti adhesion layer, as previously reported,⁵ with two other side electrodes not used in this work (Fig. 3c). A polypropylene plastic tube equipped with a cap was sealed on the sensor chip (Fig. 3a) to provide a reservoir for chemicals and to avoid possible contamination, as one experiment lasts for several hours. The functionalized ZnO-NH₂ microwires, suspended in isopropanol, were positioned between the sensing electrodes (Fig. 3f) by means of the DEP technique (3V_pp@1 MHz). Once solvent has completely evaporated, an I–V curve was recorded to check whether the microwire was well positioned or not. A typical ZnO microwire I–V curve (Fig. 3g) represents a double Schottky barrier where the threshold voltage changes for every microwire due to many factors such as contact resistance, microwire characteristics and the metal–semiconductor interface. After these integration steps the setup was ready for proof-of-concept real-time detection experiments, described in the next Section 3.1.

Fig. 3 Complete acquisition system. (a) Electronic control board underneath the sensor board with a polypropylene tube attached to the sensor to keep the chemicals in contact with the sensing element. (b–e) The sensor chip, bonded to a PCB, is composed of four sections of two electrodes plus two other side electrodes (not used in this work). (f) A functionalized ZnO microwire attracted between the two electrodes provides an electrical contact. (g) I–V curve of a ZnO microwire after deposition between the gold electrodes of the microchip.

3 Results and discussion

3.1 Real-time electrical monitoring

Fig. 4 shows capacitance trends over time for two different BSA concentrations (15 and 150 nmol ml⁻¹). Different color segments are used to highlight the continuous measurement of ZnO-NH₂ during the four experimental steps: (i) ZnO-NH₂ exposed to air (black line), (ii) bi-distilled water addition (green line), (iii) EDC and sulfo-NHS reagent addition (red line) and (iv) BSA addition (blue line). The behavior of water molecules when in contact with the ZnO-NH₂ surface, as also depicted in Fig. 5a, did not show any strong interaction, except weak hydrogen bond formation between the hydrogen from the amine group and the oxygen of water. The small capacitance increase, with respect to the previous black line recorded in air, was due to the different dielectric constant of water, creating a capacitor in parallel with the ZnO microwire. After about 30 minutes we added EDC and the sulfo-NHS reagent needed for EDC amidation. As expected, they did not react with the NH₂ groups present on the ZnO surface (red lines in Fig. 4b and the scheme in Fig. 5b). However, we still recorded a prompt capacitance increase due to the electrical double layer (EDL) formed at the electrode surface.²⁶ Such an abrupt capacitance change was no longer present when after about 30 min BSA protein dissolved in the remaining 50% of water solvent was added. Indeed, following the reaction mechanism already described in Section 2.2, sulfo-NHS, when in contact with a carboxyl group of BSA, formed a semi-stable amine-reactive NHS ester (Fig. 5c), which then reacted with primary amines, present on the ZnO surface, to form amide crosslinks (Fig. 5d and e). Amine reactions took place over a long time period because of many factors including two different reaction kinetics, diffusion coefficients, temperature and pH.^27,28 Moreover, the higher the number of –NH₂ groups already conjugated with the protein was, the more difficult the reaction of other BSA molecules was, which is likely due to steric hindrance of the bulky protein and depletion of reactants with time. Initially, since all –NH₂ sites were free to react, the capacitance of the ZnO microwire increased quickly (blue line in Fig. 4 and 5d) in a logarithmic trend up to a plateau due to complete site saturation (Fig. 5e). Validation of such a mechanistic interpretation came from a control experiment in which a ZnO microwire without NH₂ functionalization underwent the same procedure. In this case, air, water, EDC and sulfo-NHS reagent capacitance trends were consistent with the ZnO-NH₂ experiment but after BSA introduction the capacitance remained almost constant (ESI Fig. S2†).

Fig. 4 (a) Real-time measurement of ZnO-NH₂ capacitance measured during the amidation reaction of the BSA protein continuously observed for five hours. The different reaction steps are highlighted with different colors: ZnO is in air during the first step, then bi-distilled water, EDC and sulfo-NHS reagent, and BSA protein (two different concentrations) are added in sequence. (b) Magnification of the first three steps. An artificial offset is added to distinguish the curve trends.

Fig. 5 EDC/sulfo-NHS/BSA-NH₂ reaction mechanism. (a) Only bi-distilled water does not interact with the ZnO microwire but slightly increases the parallel capacitance at the electrodes (not shown in the picture). (b) EDC and sulfo-NHS addition forms an electrical double layer on the electrode thus increasing the capacitance. The ZnO microwire is still not affected. (c) BSA addition triggers the EDC/sulfo-NHS reaction creating amine-reactive intermediate compounds. (d) As soon as the intermediate gets in contact with ZnO amine groups a reaction occurs linking the BSA carboxyl group to the ZnO amine. The ZnO capacitance starts to increase. (e) As long as the crosslinking reaction occurs, the capacitance continues to increase until all ZnO reaction sites are saturated.

The different capacitances recorded between the two experiments at different concentrations, i.e. 15 and 150 nmol ml⁻¹, better evidence the kinetics of the reaction and the related active site occupation by bulky BSA molecules. Actually the curve with 15 nmol ml⁻¹ BSA in Fig. 4a depicts a more gradual increase of the capacitance when BSA molecules are added, which takes more time to reach a saturation plateau with respect to the experiment using 150 nmol ml⁻¹ BSA. In the latter case, we attribute the rapid reaching of the plateau to the higher concentration of biomolecules, which tend to rapidly occupy all the available reactive sites on the ZnO surface. This is more clear if we plot each capacitance normalized with respect to the values of the respective reagents. In fact, in Fig. 6 the higher capacitance increment of the 150 nmol ml⁻¹ concentration is more visible in comparison with that of the 15 nmol ml⁻¹ one.

Fig. 6 ZnO normalized capacitances continuously measured during the EDC/sulfo-NHS/BSA-NH₂ reaction. A 150 nmol ml⁻¹ concentration shows an increase of about 100% over 4 hours. A 15 nmol ml⁻¹ concentration shows an increase of about 75% over the same period of time.

The two tested concentrations are in the same range as other BSA sensors using a different transduction mechanism such as surface acoustic waves (60 nmol ml⁻¹),²⁹ gold nanospheres (∼200 nmol ml⁻¹),³⁰ chemiluminescence (1.5 nmol ml⁻¹)³⁰ and cyclic voltammetry (∼0.5 nmol ml⁻¹).³¹ The available reactive sites on the ZnO microwire surface, i.e. the estimated maximum density of amino-groups, is about 1.78 molecules per nm². After combining the ZnO-NH₂ microwires with BSA protein in a concentration of 15 nmol ml⁻¹ in a batch reaction, the amount of protein coupled to the ZnO-NH₂ surface, was 0.75 molecules per nm² (ESI Fig. S3†). Thus, in the absence of any other organic contamination one could assume this value to be the maximum amount of protein anchored to the –NH₂ groups of the ZnO surface. This is reasonable if compared to the number of available amino-reacting sites and considering the steric hindrance of the bulky BSA protein. This value can be considered to be the maximum final amount of conjugated protein that can be reached at the curve plateau of our real-time experiment.

4 Conclusions

We propose an efficient and easy-to-use electronic platform which exploits a functionalized material integrated by dielectrophoresis between gold electrodes to measure in real-time a proof-of-concept protein binding process. The DEP process and real-time monitoring of protein binding onto the assembled ZnO-NH₂ microwire were carried out using a custom integrated electronic system directly connected to a PC for data storage. This can be considered the first step toward the smart and portable integration of micro- and nanomaterials (e.g., microwires), electrodes and a CMOS chip for the detection of biomolecules. Moreover, such a platform is suitable for multiparametric detection with different sensing materials. The platform is very sensitive thanks to the integration of the sensing chip and the readout circuit on the same PCB, and it can be easily combined with other commercial devices exploiting the well-established Arduino ecosystem. Future work will concern the parallel detection of different bio-probes by means of different functionalization or binding to increase the process selectivity and specificity.

Acknowledgements

The authors thank Dr Laura Simone for her intensive work and Professor M. Morelson for his support.

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Footnote

† Electronic supplementary information (ESI) available: Characterization data for electronic system, control experiment and estimation of protein amount procedure. See DOI: 10.1039/c5ra15787k

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