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Electrochemical Non-Enzymatic Cholesterol Sensor

Based on Cu and Ni Catalysts

Haris Nurhidayat1, Rahmat Wibowo1*, and Dita Arifa Nurani2*

Department of Chemistry, Faculty of Mathematics and Natural Sciences Universitas Indonesia, Depok 16424,
Indonesia

*Corresponding author: rwibowo73@gmail.com

Abstract. Cholesterol has a wide range of physiological function in the human body, but if the concentration
exceeds the normal limit, it can trigger diseases such as arteriosclerosis. Therefore a rapid and accurate method
of determining cholesterol is developed. Of all the methods, enzymatic sensors have received a lot of attention,
but these sensors is easily denatured. Therefore, the determination of cholesterol using non-enzymatic sensors is
widely developed. In this study, we developed electrochemical non-enzymatic cholesterol sensor using Cu/SPE,
Ni/SPE, CuNi/SPE electrode combined with FIA system at flow rate of 0.5 mL/min and KOH concentration of 1
M. Cu/SPE, Ni/SPE, and CuNi/SPE in FIA systems have LODs of 1.08, 13.59, 1.24 M and sensitivity of
3,584.97, 465.98, 3.258.54 A mM-1 cm-2 respectively. Cu/SPE, Ni/SPE, CuNi/SPE sensors have excellent
repeatability, reproducibility, and stability. Sensors are also selective against cholesterol from common
interfering substances such as glucose, sucrose, fructose, ascorbic acid, and NaCl. Cu/SPE, Ni/SPE, and
CuNi/SPE succeeded in determining cholesterol in packaging milk samples and showing 1.88%, 3.59% and
3.05% yield differentials and 99.79 %, 98.61 % and 99.02% recovery.

Keyword : non-enzymatic sensor, SPE, electrodeposition, cholesterol oxidation, a flow system

INTRODUCTION

Cholesterol is an important component in the intracellular membranes of mammalian


1
cells. 67% of cholesterol was esterified with fatty acids and 33% of cholesterol in the form of
sterols in human blood serum, which contained cholesterol around 5.17 mM (or 200 mg/dl)
for normal people. In particular, higher cholesterol concentrations may cause health problems
such as arteriosclerosis because the vessels may become blocked by cholesterol, thereby
weakening the capability of the vessels.2
Currently cholesterol analysis in clinical laboratories takes a long time and
expensive. The development of sensitive and accurate methods, and being able to determine
various substances with different properties in real-life samples, becomes a major challenge
today. Electrochemical sensors for measuring analytes are concern in clinical chemistry
because they improve the performance of conventional analytical tools, eliminate long sample
preparations and the use of expensive reagents, provide cheap, easy operative analytical tools,
high sensitivity and selectivity and rapid response.3 Of all the electrochemical methods,
amperometric cholesterol biosensors receive much attention because of their simple, high
selectivity, and cost effectiveness.4
Previously, much research was done to make enzyme-based sensors due to their high
sensitivity and good selectivity. However, enzyme activity decreases with use and enzymes
are easily denatured during the immobilization process because enzyme activity is affected by
pH, temperature, and toxic chemicals.5 To overcome the disadvantages of enzymatic
cholesterol sensors, the non-enzymatic cholesterol sensor is designed and manufactured
because it has attractive advantages, such as good stability, simple manufacturing, good
reproduction, and low cost.6
In non-enzymatic cholesterol sensors, a nanomaterial is required which can increase
the electrocatalytic activity of the working electrode employed. Nanomaterials commonly
used in non-enzymatic cholesterol sensors are nickel (Ni) and copper (Cu) in alkaline
medium. Nickel and Copper have an incomplete electronic configuration of d orbitals and
unpaired electronic orbitals d configuration showing excellent electrocatalytic activity. In
detecting cholesterol, the involved species are the redox couples Cu (II)/Cu (III) and Ni (II)/Ni
(III).7
The development of cheap, fast, accurate, and simple non-enzymatic cholesterol
sensors is continuously underway. Screen Printed Electrode (SPE) is an analytical tool that is
currently widely used for biosensors. The advantages of using SPE in biosensors are the
working electrode, comparator, and counter are not separate so they can be combined with the
Flow Injection Analysis (FIA) system, reducing the sample volume, and easily modifiable
SPE surfaces.8 Flow Injetion Analysis (FIA) is a simple, fast, cheap and versatile technique
made with extensive applications in quantitative chemical analysis.9
In this study, we developed electrochemical non-enzymatic cholesterol sensors using
Screen Printed Electrode which have been deposited nickel and copper combined with the
Flow Injection Analysis system. Screen Printed Electrode which have been deposited nickel
and copper are characterized by scanning electron microscopy and Energy Dispersion X-ray
spectroscopy. Electrochemical studies, used to evaluate the electrocatalytic performance of
composite electrodes. In addition, we tested the effect of common interfering agents on
cholesterol detection..

EXPERIMENTAL METHOD

Materials. Cholesterol, ascorbic acid, sucrose, fructose, glucose, NaCl, K3Fe(CN)6,


KCl, CuSO4.5H2O, H2SO4, Ni(NO3)2.6H2O, Na2HPO4.7H2O, NaH2PO4.H2O, CH3COONa,
and KOH were purchased from Sigma Aldrich. While glacial CH3COOH, isopropanol, and
triton X-100 were purchased from Merck.
Synthesis Cu/SPE, Ni/SPE, and CuNi/SPE composite. The first step of making
Cu/SPE, Ni/SPE, and CuNi/SPE is by scanning the potential vs Ag/AgCl using 0.01 M
CuSO4 solution in 0.1 M H2SO4, Ni(NO3)2 0.01 M in acetate buffer pH 5 0.1 M, and a
mixture of 0.01 M CuSO4 and Ni(NO3)2 0.01 M in acetate buffer pH 5 0.1 M with a scan rate
of 100 mV/s. The second stage is electrodeposition at a potential of 200 mV after the peak
potential of Cu, Ni, and CuNi reduction.
Preparation of the cholesterol sample. Cholesterol solution was prepared by
dissolving 0.388 grams of cholesterol with 100 mL phosphate buffer pH 6.8 0.05 mM
containing 2 mL isopropanol and 10 ml Triton X-100. The solution was heated at 60C for 1
hour.
Electrochemical Analysis. Electrochemical experiments were performed using a
three-electrode cell system. The 1 M KOH solution was used as an electrolyte, Cu/SPE,
Ni/SPE, and CuNi/SPE were used as working electrodes. The platinum and Ag/AgCl layers of
the SPE act as counter electrodes and comparators.
Characterization. SEM images were recorded using a Hitachi S-4300 microscope
equipped with an energy-dispersive X-ray detector (EDX), which was operated at an
accelerating voltage of 15-20 kV.
RESULTS AND DISCUSSION

Electrochemical cholesterol is studied by looking at cyclic voltammograms using


SPE electrodes that have been modified with deposits of Cu and Ni. First the two deposits are
activated in 1 M KOH solution with a potential range of 0 to +0.8 V. Here are the reactions
that occur when activation between Cu and Ni with KOH :
M + 2OH- M(OH)2 + 2e-
M(OH)2 + OH- MOOH + H2O + e- (1)
M = Cu dan Ni
Figure 1 is a cyclic voltamogram of Cu/SPE, Ni/SPE, and CuNi/SPE with different
cholesterol concentrations at a scan rate of 100 mV/s in KOH 1 M. The cyclic voltamogram
shows the peak oxidation current lies at a +0.45 V potential indicating the occurrence of
adsorption and oxidation of cholesterol on the surface of the electrode. Electroxidation
cholesterol mechanism at Cu/SPE, Ni/SPE, and CuNi/SPE surface are described by the
following equation:
CuOOH or NiOOH + cholesterol choleston + CuO or NiO (2)

a b c

d e f

Figure 1. Cyclic Voltametry of composite electrode (a) Cu/SPE, (b) Ni/SPE and (c) CuNi/SPE in KOH 1M
at different cholesterol concentration (scan rate = 100 mV/s). Plot of peak current vs. concentration for (d)
Cu/SPE, (e) Ni/SPE, and (f) CuNi/SPE

The SEM EDX analysis of the CuNi/SPE composite electrode is shown in Figure 6.
From Figure 6, it appears that the Ni deposit is quite evenly distributed whereas the Cu
deposit is stacked at several points. In addition, the amount of Cu deposit is much more than
Ni because Cu is much more easily reduced than Ni. Besides Cu and Ni, there are also other
elements such as carbon (C), oxygen (O), chloride (Cl), and gold (Au). The presence of the C
atom comes from the SPE electrode material. The presence of the O atom comes from the
deposit oxidized by oxygen in the air, but the oxidation is not too large. The presence of Cl
atoms may be derived from the used distilled or comparative electrode Ag/AgCl. While Au
atoms are derived from the coating process when measuring SEM EDX because the sample is
less conductive.
Figure 2. SEM images, elemental mapping, and EDX data of CuNi/SPE composite electrodes

Figure 3 shows the amperometric current response of the Cu/SPE, Ni/SPE, and
CuNi/SPE composite electrodes to the addition of a cholesterol solution of varying
concentrations. The resulting current response is then divided by the amount of loading to see
the efficiency of the catalyst used. Figure 7 shows the higher concentration of injected
cholesterol solution giving rise to cholesterol oxidation current. Linearity, LOD, and
sensitivity for Cu/SPE, Ni/SPE, and CuNi/SPE summarized in Table 1 indicate the three
sensors have small LOD values and high sensitivity. Of the three electrodes, Cu/SPE has the
smallest LOD value and the greatest sensitivity because the amount of Cu loading in SPE is
the largest. However, when viewed from the peak per-loading ratio, Ni/SPE is the most
efficient composite electrode because its peak per-loading ratio is the largest.

a b c

Figure 3. chronoamperometry data of composite electrode (a) Cu/SPE, (b) Ni/SPE, and (c) CuNi/SPE on
cholesterol addition of 0.1 mM, 0.5 mM, 1.0 mM, 2.0 mM, 3.0 MM, 4.0 mM, 5.0 mM, and 10.0 mM of 0.1
mL in KOH 1 M at 0.5 mL/min flow rate.
Tabel 1. Linearity, LOD (Limit of Detection), dan Sensitivity

Composite R2 LOD (M) Sensitivity


Electrode (A mM-1cm-2)
Cu/SPE 0,9998 1,09 3.584,97466
Ni/SPE 0,9998 13,59 465,981598
CuNi/SPE 0,9997 1,24 3.258,54654

Reproducibility and stability are two important parameters of non-enzymatic


cholesterol sensors. Reproducibility aims to determine the ability of sensor detection with the
same measurement parameters but at different time intervals of analysis whereas stability
aims to determine whether the sensors are made stable at different time intervals respectively.
The reproducibility of the sensor is done by measuring the peak current change in the first and
second weeks. Figure 8 and Table 2 show the three composite electrodes having good
reproducibility. While the sensor stability is done by measuring the peak change for five
consecutive days. As shown in Figure 5 and Table 3, the sensor shows good stability.

a b c

Figure 4. Chronoometerometric data of composite electrode (a) Cu/SPE, (b) Ni/SPE, and (c) CuNi/SPE on
addition of 5.0 mM cholesterol as much as 0.1 mL in KOH 1 M with a flow rate of 0.5 mL/min in the first
and second weeks

Table 2. Average peak current, standard deviation and% RSD reproducibility

Composite Peak current/loading (A/g Deviation


RSD
electrode cm-2) Standard
Cu/SPE 16,52 0,12 0,72
CuNi/SPE 19,64 0,18 0,91
Ni/SPE 150,04 2,64 1,76

a b c

Figure 5. Chronoamperometry data of composite electrode (a) Cu / SPE, (b) Ni / SPE, and (c) CuNi / SPE
on addition of cholesterol 5.0 mM of 0.1 mL in KOH 1 M at a flow rate of 0.5 ML / min for five
consecutive days
Table 3. Average peak, standard deviation and% RSD stability

Composite Peak current/Loading Deviation


RSD
electrode (A/g cm-2) standard
Cu/SPE 16,55 0,11 0,66
CuNi/SPE 19,63 0,18 0,93
Ni/SPE 148,62 2,28 1,54

Selectivity is a very important parameter of the non-enzymatic cholesterol sensor. It


is known that glucose, sucrose, fructose, ascorbic acid, and NaCl may interfere with
cholesterol detection. Therefore, in this study, glucose (5.0 mM), sucrose (5.0 mM), (fructose
5.0 mM), ascorbic acid (5.0 mM), and NaCl (5.0 mM) were added to mimic disorder. Based
on Figure 6, it appears that the sensor made only responds to the cholesterol solution. So it can
be concluded that the sensors are made to have good selectivity.

a b c
c

Figure 6. Results of selectivity test of composite electrode (a) Cu/SPE, (b) Ni/SPE, and (c) CuNi/SPE in
KOH 1 M with addition of 0.1 mL cholesterol solution 5.0 mM, glucose 5.0 MM, ascorbic acid 5.0 mM, 5.0
mM sucrose, 5.0 mM NaCl, and 5.0 mM fructose at a 0.5 mL/min flow rate

To find out how accurately the sensor is made, a recovery test is done. Prior to the
recovery test, a cholesterol spike was prepared by mixing the analytes of 5 ml standard
cholesterol solution with a sample solution with known concentration in a 100 mL flask. To
get% recovery, spike cholesterol levels are measured compared to calculated cholesterol
levels. The results summarized in Table 4 indicate that Cu/SPE, Ni/SPE, and CuNi/SPE with
FIA systems have a high accuracy of accuracy at the time of measurement.
Table 4. The results of recovery test (recovery)

Composite Spike count Spike Recovery


electrode (mM) measurable (%)
(mM)
Cu/SPE 0,3464 0,346 99,79
Ni/SPE 0,3447 0,339 98,61
CuNi/SPE 0,3452 0,342 99,02

CONCLUSIONS

Cu/SPE, Ni/SPE, and CuNi/SPE composite electrodes were successfully made by


electrodeposition technique. SEM analysis shows the amount of Cu deposit is much more than
the amount of Ni. Cu/SPE, Ni/SPE, and CuNi/SPE composite electrodes have LOD values of
1.09 M, 13.59 M, and 1.24 M. Cu/SPE, Ni/SPE, and CuNi/SPE sensors in FIA systems
have good reproducibility and stability. Cu/SPE, Ni/SPE, and CuNi/SPE sensors in the FIA
system have good accuracy and also selective against cholesterol from glucose, sucrose,
fructose, ascorbic acid and NaCl disruptors. But of the three electrodes, Ni/SPE is the most
efficient electrode because the peak current ratio vs. the largest loading amount.

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