Development of ZN_R-calcium biosensor application

Semiconductor ${\rm Z}{{{\rm N}}_{{\rm R}}}$ were obtained by depositing two different layers by means of two methods: the sol-gel and the aqueous chemical growth (ACG) methods.

2.1.1. Pretreatment of the substrate.

2.1.2. Seed solution prepared by sol-gel method.

2.1.3. Growth of ZN_R by aqueous chemical growth method.

Zinc oxide nanorods on all substrates ${\rm Z}{{{\rm N}}_{{\rm R}}}{\rm P}$, ${\rm Z}{{{\rm N}}_{{\rm R}}}{\rm S}$, ${\rm Z}{{{\rm N}}_{{\rm R}}}{\rm W}$ and ${\rm Z}{{{\rm N}}_{{\rm R}}}{\rm T}$ were coated with calcium ionophore polymeric membrane by procedure as follow, 120 mg of powdered polyvinyl chloride (PVC) was dissolved in 5 ml tetrahydrofuran together with 10 mg of dibutylphtalate (DBP) plasticizer and 10 mg of ${\rm C}{{{\rm a}}^{2+}}$ specific ionophore (DB18C6). All chemicals were from Sigma-Aldrich-Fluka. After preparing the solution, the all mentioned ZnO-coated substrate were dipped twice into the solution until membrane thin film were attached to their surfaces then they will dried at room temperature.

All calcium biosensors working electrodes (${\rm Z}{{{\rm N}}_{{\rm R}}}{\rm P}$, ${\rm Z}{{{\rm N}}_{{\rm R}}}{\rm S}$, ${\rm Z}{{{\rm N}}_{{\rm R}}}{\rm W}$ and ${\rm Z}{{{\rm N}}_{{\rm R}}}{\rm T}$) were stored at ${\rm 4}\ {}^\circ {\rm C}$ in dry condition when not in use.

After finishing these steps, the biosensors were initially checked potentiometrically in two solutions, the former solution is the ${\rm CaC}{{{\rm l}}_{2}}$ with different concentration of calcium ranging from 100 mM to 100 nM and the latter is human blood cells serum.

The electrochemical potential cell (electromotive force) changed when the test solution composition was altered. These changes related to the calcium concentration ions in the test solution via a calibration procedure. The actual electrochemical potential cell can be described by the diagrams for each kind of substrates as follows

• For conducting plastic substrate: ${\rm Z}{{{\rm N}}_{{\rm R}}}{\rm P}$ $\parallel$ ${\rm CaC}{{{\rm l}}_{2}}$ | ${\rm C}{{{\rm l}}^{-}}~$ $\parallel$ AgCl | Ag,
• For silicon substrate: ${\rm Z}{{{\rm N}}_{{\rm R}}}{\rm S}$ $\parallel$ ${\rm CaC}{{{\rm l}}_{2}}$ | ${\rm C}{{{\rm l}}^{-}}~$ $\parallel$ AgCl | Ag,
• For silver wire: ${\rm Z}{{{\rm N}}_{{\rm R}}}{\rm W}$ $\parallel$ ${\rm CaC}{{{\rm l}}_{2}}$ | ${\rm C}{{{\rm l}}^{-}}~$ $\parallel$ AgCl | Ag,
• For tube tip: ${\rm Z}{{{\rm N}}_{{\rm R}}}{\rm T}$ $\parallel$ ${\rm CaC}{{{\rm l}}_{2}}$ | ${\rm C}{{{\rm l}}^{-}}~$ $\parallel$ AgCl | Ag.

${\rm Z}{{{\rm N}}_{{\rm R}}}$-based ${\rm C}{{{\rm a}}^{2+}}$ biosensor electrochemical response against an Ag/AgCl reference electrode was measured at temperature (about $25\ {}^\circ {\rm C}$). The pH meter (model 3510 Metrohm) was used to measure prepared calcium biosensors potentiometric output voltage. This pH meter has the advantage and the possibility of measuring: (i) pH of the solution, (ii) the temperature, and (iii) the output voltage of the biosensors.

The ${\rm Z}{{{\rm N}}_{{\rm R}}}{\rm P}$, ${\rm Z}{{{\rm N}}_{{\rm R}}}{\rm S}$, ${\rm Z}{{{\rm N}}_{{\rm R}}}{\rm W}$ and ${\rm Z}{{{\rm N}}_{{\rm R}}}{\rm T}$ surface morphologies were investigated by using field emission scanning electron microscopy (FESEM) as shown in figures 1–4, respectively.

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The obtained ${\rm Z}{{{\rm N}}_{{\rm R}}}{\rm P}$ FESEM photos were employed to explore the nanorods size and alignment at 12 000×  and 24 000  ×  as showing in figures 1(a) and (b), respectively, while figure 2 shows the ${\rm Z}{{{\rm N}}_{{\rm R}}}{\rm S}$ FESEM image at constant magnification equal to 60 000×. It is clearly seen from the mentioned figures that the nanorods were distributed uniformly in diameters range between 26 and 38 nm.

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Figures 3 and 4 show the ${\rm Z}{{{\rm N}}_{{\rm R}}}{\rm W}$ and ${\rm Z}{{{\rm N}}_{{\rm R}}}{\rm T}$ FESEM images of panoramic view for ZnO nanorods and by focusing in small area for both of them. It is displaying from the figures that the nanorods were found to be perpendicular to the substrate and well alignment, however its diameter is in the range between 22 and 24.2 nm for ${\rm Z}{{{\rm N}}_{{\rm R}}}{\rm T}$.

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We can conclude that the figures of the ${\rm Z}{{{\rm N}}_{{\rm R}}}{\rm P}$, ${\rm Z}{{{\rm N}}_{{\rm R}}}{\rm S}$, ${\rm Z}{{{\rm N}}_{{\rm R}}}{\rm W}$ and ${\rm Z}{{{\rm N}}_{{\rm R}}}{\rm T}$ are in rodshapes, hexagonal cross section and have homogeneous distribution perpendicular to the substrate with small diameter nano-rods [1, 11–13].

The four fabricated calcium biosensor kinds ${\rm Z}{{{\rm N}}_{{\rm R}}}{\rm P}$, ${\rm Z}{{{\rm N}}_{{\rm R}}}{\rm S}$, ${\rm Z}{{{\rm N}}_{{\rm R}}}{\rm W}$ and ${\rm Z}{{{\rm N}}_{{\rm R}}}{\rm T}$ were used for ${\rm C}{{{\rm a}}^{2+}}$ concentrations measurement in extracellular solutions. They were used to study calcium ion selectivity by using biosensors based on ${\rm Z}{{{\rm N}}_{{\rm R}}}$ coating with ionophore membrane. The working electrode (Ca²⁺) potentiometric response was evaluated by immersing it in CaCl₂ aqueous solutions having concentration range between 100 nM and 0.1 M.

Figures 5 shows the calibration curves for the potential difference between ${\rm Z}{{{\rm N}}_{{\rm R}}}{\rm P}$, ${\rm Z}{{{\rm N}}_{{\rm R}}}{\rm S}$, ${\rm Z}{{{\rm N}}_{{\rm R}}}{\rm W}$ and ${\rm Z}{{{\rm N}}_{{\rm R}}}{\rm T}$ as calcium biosensor working electrodes with reference electrode (Ag/AgCl) versus ${\rm log}\,{\rm C}{{{\rm a}}^{2+}}$ concentrations in ${\rm CaC}{{{\rm l}}_{2}}$ solution with concentration ranging from 100 nM to 0.1 M [14]. From the curves we detected the calcium concentration in human blood serum (dashed lines). All these curves show the linear dependence. Moreover, the electrodes are found to be very sensitive to calcium ions giving a slope calculated from linear equations curve and biosensors efficiency ${{R}^{2}}$ (regression coefficient), as depicted in table 1.

Table 1. The linear equation and efficiency for ${\rm Z}{{{\rm N}}_{{\rm R}}}{\rm P}$, ${\rm Z}{{{\rm N}}_{{\rm R}}}{\rm S}$, ${\rm Z}{{{\rm N}}_{{\rm R}}}{\rm W}$ and ${\rm Z}{{{\rm N}}_{{\rm R}}}{\rm T}$ calcium biosensors.

Biosensors	Linear equation	Slope	Efficiency ${{R}^{2}}$
${\rm Z}{{{\rm N}}_{{\rm R}}}{\rm P}$	$y=1. 221x+67.11$	67.11 mV/decade	0.96
${\rm Z}{{{\rm N}}_{{\rm R}}}{\rm S}$	$\newcommand{\e}{{\rm e}} \begin{array}{@{}cccccccccccccccccccc@{}} y=-2.442x+88.98 \\\end{array}$	88.98 mV/decade	0.96
${\rm Z}{{{\rm N}}_{{\rm R}}}{\rm W}$	$\newcommand{\e}{{\rm e}} \begin{array}{@{}cccccccccccccccccccc@{}} y=5.960x+136.3~ \\\end{array}$	136.3 mV/decade	0.98
${\rm Z}{{{\rm N}}_{{\rm R}}}{\rm T}$	$\newcommand{\e}{{\rm e}} \begin{array}{@{}cccccccccccccccccccc@{}} y=1.396 x+120.3 \\\end{array}$	136.3 mV/decade	0.98

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After successfully using the ${\rm Z}{{{\rm N}}_{{\rm R}}}{\rm P}$, ${\rm Z}{{{\rm N}}_{{\rm R}}}{\rm S}$, ${\rm Z}{{{\rm N}}_{{\rm R}}}{\rm W}$ and ${\rm Z}{{{\rm N}}_{{\rm R}}}{\rm T}$ as ${\rm C}{{{\rm a}}^{2+}}$ biosensors for calcium concentration measurements in ${\rm CaC}{{{\rm l}}_{2}}$ solution, we can use them for the same measurement in the human blood serum.

For ${\rm Z}{{{\rm N}}_{{\rm R}}}{\rm P}$, ${\rm Z}{{{\rm N}}_{{\rm R}}}{\rm S}$, ${\rm Z}{{{\rm N}}_{{\rm R}}}{\rm W}$ and ${\rm Z}{{{\rm N}}_{{\rm R}}}{\rm T}$ calcium biosensors, the measured potential responses were found to be equal to 63.9, 95.7, 121 and 116.8 mV in $y$ axis corresponding to  −2.63 in $x$ axis as depicted in table 2, which all are equivalent to $2.3{\rm mmol}\times {{{\rm l}}^{-1}}$ calcium concentration as detected from figures 5(a)–(d), respectively.

Table 2. The experimental calcium concentration in human blood serum value by ${\rm Z}{{{\rm N}}_{{\rm R}}}{\rm P}$, ${\rm Z}{{{\rm N}}_{{\rm R}}}{\rm S}$, ${\rm Z}{{{\rm N}}_{{\rm R}}}{\rm W}$ and ${\rm Z}{{{\rm N}}_{{\rm R}}}{\rm T}$ calcium biosensors.

Biosensors	Measured potential (mV)	${\rm log}\,{{{\rm C}}_{{\rm Ca}}}$	${\rm C}{{{\rm a}}^{{\rm 2+}}}$ concentration $\left({\rm mmol}\times {{{\rm l}}^{-1}} \right)$
${\rm Z}{{{\rm N}}_{{\rm R}}}{\rm P}$	63.9	−2.63	2.3
${\rm Z}{{{\rm N}}_{{\rm R}}}{\rm S}$	95.7	−2.63	2.3
${\rm Z}{{{\rm N}}_{{\rm R}}}{\rm W}$	121	−2.63	2.3
${\rm Z}{{{\rm N}}_{{\rm R}}}{\rm T}$	116.8	−2.63	2.3

From the obtained results we noted that the experimental calcium concentration in human blood serum value measured by the mentioned four fabricated ${\rm C}{{{\rm a}}^{2+}}$ biosensors are located at a normal range of calcium human concentration, where the normal value is ranging from 2.2 to $2.6\,\,{\rm mmol}\times {{{\rm l}}^{-1}}$.