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Koloid dan Permukaan B: Biointerfaces 88 (2011)

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Daftar Konten Tersedia di ScienceDirect

Koloid dan Permukaan B:


Biointerfaces

Penerapan elektroda berdinding nanotube karbon yang dimodifikasi


karbon ionik cair untuk oksidasi elektrokatalitik dopamin
Yonghong Li a , Xinsheng Liu b , Xiaoying Liu a , Nannan Mai a , Yuandong Li a , Wanzhi Wei a,,
Qingyun Cai a
a
State Key Laboratory of Chemo/Biosensing and Chemometrics, College of Chemistry and Chemical Engineering, Hunan University, Hunan, Changsha
410082, PR China
b
Hunan Nonferrous Xiangxiang Fluoride Chemical Co. Ltd., Hunan, Xiangxiang 411400, PR China

info artikel abstrak


Riwayat artikel: Deteksi selektif
Diterima pada 18 April 2011 Sebuah metode sederhana, sensitif dan dapat diaplikasikan berdasarkan nanotube karbon
Diterima setelah direvisi pada berdinding banyak yang dapat dimodifikasi untuk elektroda karbon cair ionik telah berhasil
6 Juli 2011 dikembangkan untuk mengetahui keberadaan dopamin dalam keberadaan asam askorbat. Asam
Tersedia online pada 20 July askorbat pada metode ini direaksikan dengan gugus fungsi asam karboksilat agar terjadi transfer
2011 elektron pada penentuan dopamin dan menghambat respon voltametri asam askorbat.
Berdasarkan kemampuan cairan ionik yang baik, respon elektrokimia dari penentuan dopamin
Kata Kunci: dengan metode ini lebih baik dari metode modifikasi elektroda lain. Dibawah kondisi optimal, plot
Elektroda karbon ionik cair kalibrasi linear yang didapatkan berada pada rentang 5.0 10 8 ke 2.0 104 mol L1 dan
Nanotube karbon berdinding
limit deteksinya sebesar 1.0 108 mol L1.
banyak
Voltametri gelombang persegi
Dopamin 2011 Elsevier B.V. All rights
reserved.

1. Pendahuluan

Baru-baru ini, penentuan dopamin menarik minat yang tersebut bisa dipisahkan dengan baik pada CILE dan tidak akan
cukup besar karena memiliki peran penting dalam fungsi saling mengganggu.
saraf pusat, ginjal, hormon dan sistem kardiovaskular [1].
Namun, ada banyak senyawa dalam matriks biologi . Asam

askorbat merupakan senyawa pengganggu utama dalam Corresponding author. Tel.: +86 731 88821960; fax: +86 731 88821967.
penentuan dopamin [24] karena potensial oksidasi E-mail addresses: lyh21230@163.com (W. Wei), qycai0001@yahoo.com.cn (Q.
Cai).
dopamin mirip dengan potensial oksidasi asam askorbat .
Sehingga masalah utama dalam penentuan dopamin
adalah solusi penentuan dopamin dan senywa
pengganggunya.
Selama beberapa tahun terakhir, sebuah elektroda pasta
karbon baru yang disebut elektroda cair ion karbon (CILE)
telah berkembang dengan pesatnya. CILE dibuat
menggunakan konduktif cairan ionik sebagai pengikat
paste di tempat pengikat organik non konduktif paste.
Perilakunya dalam eketrokimia lebih baik dibandingkan
dengan material karbon lainnya. Jenis elektoda ini memiliki
kelebihan seperti memiliki konduktiitas tinggi, transfer
elektron cepat, dan memiliki stabilitas yang baik. Sun et al
menyebutkan bahwa cairan ionik untuk memodifikasi
elektroda pasta karbon menunjukan perkembangan respon
elektrokimia dan aktifitas analitik yang kuat terhadap
oksidasi dopamin. Safai et al telah membuat CILE untuk
penentuan dopamin secara simultan, asam askorbat dan
asam urat. Hasilnya menunjukan bahwa tiga senyawa
Dalam rangka meningkatkan selektiitas dan sensitivitas menyerupai nanotube karbon berdinding banyak dipelajari
sensor, berbagai materi digunakan untuk memodifikasi berdasarkan aktivitas elektrokatalitik terhadap asam askorbat
elektroda. Karbon nanotube (CNT) menarik perhatian dan dopamin . Zhang et al menggunakan asam yang
banyak ilmuan karena strukturnya yang unik dan direaksikan dengan nanotube karbon berdinding banyak untuk
kemampuan hantar listrik yang baik serta mekanika dan memodifikasi elektroda emas yang menawarkan kemajuan
komposisi kimianya yang baik. Baru-baru ini, karbon pesat pada kesensitifan dan keselektifan voltametri dalam
nanotube untuk memodifikasi elektroda telah banyak menentukan dopamin dengan keberadaan konsentrasi asam
diaplikasikan untuk penentuan dopamin dengan askorbat yang tinggi. Pada kondisi yang ditentukan, arus
keberadaan asam askorbat dengan hasil yang baik. puncak yang koresponden linear dengan konsentrasi
Contohnya pada Britto nanotube karbon berdinding penentuan dopamin berada pada kisaran 5 10-7 ke 4 10-4
banyak dimasukkan ke dalam elektroda karbon gelas mol L-1 dengan batas deteksi 2 10-7 mol L-1 . Hasil yang
untuk membentuk nanotube karbon yang dimodifikasi dilaporkan menunjukkan nanotube karbon berdinding banyak
sensor elektroda telah berhasil digunakan untuk yang diolah dengan asam adalah elektroda yang sangat baik
mengoksidasi dopamin. Kemudian Zhang et al untuk mendeteksi dopamin .
menggunakan metode lapisan demi lapisan untuk

0927-7765/$ see front matter 2011 Elsevier B.V. All rights reserved.
doi:10.1016/j.colsurfb.2011.07.021
Y. Li et al. / Colloids and Surfaces B: Biointerfaces 88 (2011) 402406 403

.
In this paper, the acid-treated MWNTs were modied on the
CILE (MWNTs/CILE) for the selective determination of DA by
using square-wave voltammetry techniques (SWVs). The
SWVs exhibits the advantages of large speed and high
sensitivity. The sensitivity is generally larger than the one
of differential pulse voltammetry (DPV), in which the
reverse current is not measured. A comparison between
SWV and DPV for both, reversible and irreversible system,
shows that, for analogous experimental conditions, the
peak cur- rents obtained by SWV are about 24 times
larger than those ones obtained by DPV [15]. In addition, it
is easier to implement and is less time consuming [16].
The high sensitivity of this method allows low detection
limits and good precision in the detection of DA. The
effective but simple method proposed in this paper may
have the potential for the practical analysis of DA.

2. Experimental

Apparatus andchemicals

The electrochemical measurements were carried Electrode preparation


out with CHI660A electrochemical workstation
(CHInstruments, Shanghai Chenhua Instruments Carbonionicliquidelectrode(CILE)waspreparedbymixi
Corporation, China). A conventional three electrode ngthe graphite powder and OPPF6 with a ratio of 50/50
system was employed comprising a multi-walled (w/w) and then a portion of the resulting paste was packed
carbon nanotubes modiedcarbon ionic liquidelectrode rmly into the cav- ity (3mm in diameter) of a Teon holder.
(MWNTs/CILE) (3.0 mm in diameter) as working The electrode was then heated simply by using a hair drier
electrode, a platinum wire as counter electrode, and a for 2 min. It was then left to
Ag/AgCl (3M KCl) electrode as reference electrode. All
potentials were reported with respect to the refer-
ence electrode. A magnetic stirrer (Model 79-1) was
used to stir the testing solution during the pre-
concentration step. All the exper- iments were carried
out at room temperature. Scanning electron
micrographs were obtained with a Hitachi S-4800
scanning elec-
tronmicroscope(HitachiLtd.,Tokyo,Japan).
The ionic liquid N-octylpyridium hexauorophosphate
(OPPF6) was purchasedfrom Shanghai Chengjie
Chemical Co., Ltd.(Shang- hai, China), graphite
powder (extra pure) were obtained from Shanghai
Chemical Reagents (Shanghai, China). Shortened
multi- walled carbon nanotubes (MWNTs) with a
diameter of about 1030 nm (95% purity) were
purchased from Shenzhen Nanotech Port Co., Ltd.
(Shenzhen, China). Dopamine (DA) and ascorbic acid
(AA) were purchased from SigmaAldrich.
0.1Mphosphate buffer solutions (PBS) of various pH
values were used as the support- ing electrolytes. All
other chemicals were of analytical grade, and
usedwithout further purication. Double-distilledwater
was used throughout.

Pretreatment ofMWNTs

Pretreatment of MWNTs was progressed according to the


previ- ous literature [17]. Firstly, MWNTs were heated in
air at 600 C for
2 h, and then soaked in 6 M HCl solution for 24 h and
centrifuged. The precipitate was rinsed with deionized
water and dried in air. The MWNTs were chemically
functionalized by ultrasonic agita- tion in a mixture of
sulfuric acid and nitric acid (3:1) for 10 h. Then MWNTs
were washed with deionized water (until pH 7.0 was
obtained) and separated by centrifuging three times and
then dried.
electrode is denoted as MWNTs/CILE.

Analytical procedure

The sensor was immersed in 0.1 mol L1 PBS (pH 7.0)


containing a known concentration of DA for 120 s
accumulation at open circuit potential with stirring. The
square wave voltammograms (SWVs)
were then recorded from 0.2 to 0.4 V, experiment
parameters: 4 mV step height, 20 mV pulse height, 2 Hz
frequency, and 10 s quiet time.

3. Results and discussion


cool to room temperature. The electric contact was
established via a stainless steel handle. A new 3.1 Surface morphologies of different electrodes
surface was obtained by smoothing the electrode
onto a weighing paper. The morphologies of CILE and MWNTs/CILE were
Multi-walled carbon nanotubes modied carbon ionic liquid characterized bySEM.AsshowninFig.1A,anuniform
electrode (MWNTs/CILE) was prepared in the following surfacetopographyandno
procedure. separatedcarbonlayerscouldbeobservedatthesurfaceof
theCILE. Theuniformity of the surface showed the
First, 1 mg MWNTs was dispersed with the aid of
ultrasonic agi- tation in 1 mL double-distilled water to good adherence of OPPF6 to graphite due to its high
give a 1 mg mL1 black suspension. Then, 5 L of 1 mg viscosity. After the MWNTs were cast on the CILE, it
mL1 MWNTs was dropped on the could be seen that the MWNTs were distributed homo-
surface of CILE and dried under an infrared lamp. This modied geneously on the surface of CILE with special three-
dimensional
404 Y. Li et al. / Koloid dan Permukaan B: Biointerfaces 88 (2011) 402406

Gambar 2. Siklik voltammogram dari 5 mM Fe(CN) 63/4 mengandung 0.1 M KCl pada (a) CILE
(b) MWNTs/CILE, v = 50 mV s1. Fig. 4. Cyclic voltammograms obtained at different scan rates from the
MWNTs/CILE in 0.1 mol L1 PBS (pH 7.0) containing 10 M DA. Scan rates
from inner to outer (mV s1): 20, 50, 100, 150, 200, 250, 300.

Karena MWNTs memiliki gugus asam karboksilat pada


3.2 Karakterisasi Elektrokimia MWNTs/CILE and CILE permukaan CILE. Larutan tak netral atau lingkungan
Perilaku elektrokimia dari elekroda modifikasi dalam larutan
biologi, DA keluar sebagai kation sedangkan AA keluar
5.0mmolL1 [Fe(CN)6]3-/4 and0.1molL1 KCl sebagai anion [18]. DA dengan muatan positif dapat
diinvestigasi dengan siklik voltametri dan hasil tertarik ke permukaan elektroda modifikasi,
ditunjukkan pada Gambar.2. Sepasang puncak redoks sementara AA dengan muatan negatif akan dicegah
semi-reversibel secara terpisah diamati pada Cile dan untuk mendekati permukaan. Oleh karena itu, MWNTs
sebagai pemodifikasi dapat meningkatkan selektivitas
MWNTs / Cile, tetapi redoks arus puncak meningkat dan
sensor dan mempromosikan kecepatan transfer
potensi pemisahan puncak ke puncak (6EP) mengalami
elektron.
penurunan pada MWNTs / Cile (kurva b) dibandingkan
Selain itu, potensi pemisahan puncak antara AA dan
dengan dengan Cile (a). Hasil penelitian menunjukkan DA di MWNTs / Cile adalah 230 mV, yang meningkat
bahwa MWNTs diam pada permukaan Cile bisa sekitar 20 mV dibandingkan dengan Cile kosong. The
mempromosikan kecepatan transfer elektron dan lebih MWNTs / Cile menunjukkan resolusi sangat luar biasa
meningkatkan respon, karena konduktivitas elektronik antara puncak voltametri AA dan DA, dan itu membuat
yang baik dan area permukaan besar. mudah untuk mendeteksi DA tanpa gangguan dari AA.

3.3 Perilaku Elektrokimia dari DA dan AA di elektroda 3.4 Optimisasi Kondisi


modifikasi
3.4.1 Laju Pindai
Perilaku elektrokimia dari DA dan AA di Cile dan Dalam rangka untuk mengevaluasi pengaruh dari
proses adsorpsi pada oksidasi elektrokimia DA di MWNTs
MWNTs / Cile dieksplorasi oleh SWVs di 0,1 M PBS pada
/ Cile, efek laju pindai pada sinyal voltametri dipelajari
pH 7,0. Seperti yang ditunjukkan pada Gambar. 3, arus
dari 20-300 mV s1 (ditunjukkan pada Gambar.4). Hasil
puncak dari DA di MWNTs / Cile sekitar 11 kali lipat lebih menunjukkan bahwa pada anodik (ipa) dan katodik (ipc)
tinggi daripada Cile kosong, tapi puncak arus dari AA arus puncak sebanding dengan laju pindai. Persamaan
hanya berubah sedikit. Hal ini menunjukkan bahwa 1
regresi linear yang didapat, ipa (A) = 0.0609 v (mV s )
MWNTs / Cile memiliki aktivitas katalitik selektif untuk 2 1
+ 1.9168 (r = 0.9978) dan ipc (A) = 0.0535 v (mV s )
DA dibandingkan dengan Cile kosong. Permukaan 0.3556 (r2 = 0.9996), menunjukkan bahwa reaksi
MWNTs-modifiasi elektroda bermuatan negative elektroda adalah proses adsorpsi yang dikendalikan.

3.4.2 Efek dari pH


Dalam rangka untuk menyelidiki mekanisme reaksi-
reaksi elektrokimia dari DA pada MWNTs / Cile,
pengaruh pH pada puncak potensial dan arus
diselidiki dengan SWVs di dalam larutan yang
mengandung 20 M DA di kisaran 5,0-9,0. Seperti
yang ditunjukkan pada Gambar. 5A, potensi puncak
bergeser ke arah potensi negatif dengan
meningkatnya pH larutan. Hubungan Ep dan pH dapat
dijelaskan dengan persamaan berikut: Ep (V) =
0,5336-0,0472 pH (r2 = 0,9972), yang menunjukkan
bahwa redoks dari DA menjalani proses proton-
elektron yang sama, yang konsisten dan dilansir
dalam literatur [19]. Di sisi lain, dari Gambar. 5B.
meningkatnya pH 5,0-7,0 akan menyebabkan
peningkatan tajam dari puncak arus, tetapi puncak
arus menurun dengan peningkatan lebih lanjut dari
Gambar.3. SWVs dari 200 M AA dan 20 M DA di 0.1 M PBS (pH 7.0) pH. Oleh karena itu, PBS 7,0 terpilih sebagai elektrolit
untuk (a) CILE, (b) MWNTs/CILE. pendukung dan itu dekat dengan nilai pH dari kondisi
fisiologis.
Karena oksidasi DA pada permukaan elektroda modifikasi adalah
3.4.3 Efek akumulasi proses adsorpsi-kendali, akumulasi waktu akan sangat
waktu memengaruhi respon dari DA.
Y. Li et al. / Colloids and Surfaces B: Biointerfaces 88 (2011) 402406 405
Fig. 7Ashows the SWVs of an
MWNTsmodiedelectrode to the variation of DA
concentration in the presence of 0.5 mM AA. Under

Fig. 5. Effects of pH on the (A) the potential and (B) the peak current
of 20 M DA at the MWNTs/CILE.

Fig. 6. Effect of accumulation time on the peak current of 10 M DA at


the MWNTs/CILE.

lation time will remarkably inuence the current


response of DA. Thus, the effect of accumulation time
in 10 M DA solution on peak current at the
MWNTs/CILE was investigated. It was clear from Fig. 6
that the peak current of DA was almost constant after
120 s, indicating an accumulation time of 120 s was
sufcient to achieve DA saturation of the electrode. An
accumulation time of 120 s was therefore adopted in
subsequent experiments.

Calibration curve
the selected conditions, the oxidative peak currents
increased lin- early with the concentrations of DA in the
range of 5.0 108 to
2.0 104 mol L1 with a correlation coefcient of 0.9937
(shown
in Fig. 7B). The linear regression equation was
expressed as Ip
(A) = 0.8514 + 0.2819c (M). When the concentration
of DA was
higher than 2.0 104 mol L1, the working curve
tended to level off. When the signal-to-noise ratio
(SNR) was 3, the detection limit was 1.0 108 mol
L1.
Theproposed MWNTs/CILE for DAdetermination
wascompared
with other CNTs modied electrodes and the results are listed
in Table 1. It can be seen that this method can provide
a comparable

Table 1
Comparison of different modied electrodes for DA determination.

Electrodes Method Linear Detection Reference


range limit (M)
(M)
MWNTs/Au It 0.5400 0.2 [14]
EBNBHCNPE DPV 0.1900 0.087 [20]
PA-SWNTs/Pt DPV 0.210 0.08 [21]
MWNTs/SPE ASV 0.051 0.015 [22]
MWNTs/GCE DPV 0.5100 0.2 [23]
MWNTs/CILE SWV 0.05200 0.01 This work
Au, gold electrode; MWNTs, multi-walled carbon nanotube; EBNBHCNPE,
2,2r-[1,2- ethanediylbis(nitriloethylidyne)]-bis-hydroquinone modied
Fig. 7. (A) SWVs at MWNTs/CILE in 0.1 M pH 7.0 PBS containing 5
carbon nanotube paste electrode; It, amperometric currenttime response
104 M AA in the presence different concentrations of DA (from curve; DPV, differential pulse voltammetry; Pt, platinum electrode;
bottom to upper): 5.0 108, 5.0 107, 5.0 106, 1.0 105, SWNTs, single-walled carbon nanotube; PA, phytic acid; SPE, screen-
5.0 105, 1.0 104, 2 104 M. (B) The plot of printed electrodes; GCE, glassy carbon electrode; CILE, carbon ionic
current versus concentration of DA. liquid electrode; ASV, adsorptive stripping voltammetry; SWV, square
wave voltammetry.
406 Y. Li et al. / Colloids and Surfaces B: Biointerfaces 88 (2011) 402406

interfering substances in human blood serum, such as


proteins and glucose did not interfere with the
determination of DA. The recov- ery and RSD were
acceptable, indicating that the proposed methods could be
efciently used for the determination of DA in practical
applications.

4. Conclusion

In summary, the acid-treated MWNTs could been


stably assem- bled on carbon ionic liquid electrode, and the
modied electrode showed large peak separations
between DA, AA and UA by using CV or SWV.
Whatsmore, a remarkable enhancement in the current
response of DA was observed at the modied electrode
in neutral pH. Therefore, selective and sensitive
detection of dopamine could beachievedat the
modiedelectrode. Under the chosen conditions, the
peak currents were correspondent linearly to the
Fig. 8. Cyclic voltammograms of 200 M AA, 50 M DA and 20 M concentra- tions of DA in the range of 5.0 108 to
UA in 0.1 M PBS (pH 7.0) at the MWNTs/CILE.
2.0 104 mol L1 with
a limit of detection of 1 108 mol L1. Whats more, the
Table 2 sensor
Experimental results for the determination of DA in human blood serum. exhibited good reproducibility and stability. All these
properties are
Samples DA spiking (M) DA found (M) Recovery (%) RSD (%)
benecial for fabricating a simple, sensitive and selective biosensor
1 1.0 1.02 102 2.2 for DA.
2 5.0 4.89 97.8 1.9
3 10.0 9.65 96.5 1.8
Acknowledgements

linear range and detection limit by SWVs with a


The nancial support from National Outstanding Youth
simpleelectrode preparation procedure.
Foundations of China, National Science Foundation of
China (50725825) and Special Research Found for the
Reproducibility, stability and selectivity of the Doctoral Program of Higher Education of China
MWNTs/CILE (20060532006) is acknowledged.

In order to examine the reproducibility of the modied elec- References


trode, repeated cyclic voltammetric experiment was run
in 10 M DA in 0.1 M pH 7.0 PBS. The relative standard [1] P. Damier, E.C. Hirsch, Y. Agid, A.M. Graybiel, Brain 122 (1999)
deviation (RSD) was about 3.1% after 50 successive 1437.
CVs, indicating that the proposed sensor had an [2] P. Cappella, B. Ghasemzadeh, K. Mithchell, R.N. Adams,
Electroanalysis 2 (1990) 175.
excellent reproducibility. After measurement, the [3] R.M. Wightman, L.J. May, A.C. Michael, Anal. Chem. 60
modied electrode was cleaned with voltammetric cycles (1988) 769A. [4] G. Mihaela, W.M.A. Damien, Electrochim.
in pH Acta 49 (2004) 4743.
[5] A. Safavi, N. Maleki, F. Tajab adi, E. Farjami, Electrochem.
7.0 PBS to eliminate the adsorption and stored in air. Commun. 9 (2007) 1963.
The modied electrode retained 94% of its initial [6] N. Maleki, A. Safavi, F. Tajabadi, Anal. Chem. 78 (2006) 3820.
response after 2 weeks. These results showed that [7] W. Sun, M.X. Yang, K. Jiao, Anal. Bioanal. Chem. 389 (2007) 1283.
[8] A. Safavi, N. Maleki, O. Moradlou, F. Tajabadi, Anal. Biochem.
the MWNTs/CILE was stable and the voltam- metric
359 (2006) 224.
studies could be replicated for the sensitive [9] K. Balasubramanian, M. Burghard, Anal. Bioanal. Chem. 385
determination of DA. (2006) 452.
[10] P.M. Ajayan, Chem. Rev. 99 (1999) 1787.
Ascorbic acid (AA) and uric acid (UA) are the main
[11] M. Musameh, J. Wang, A. Merkoci, Y.H. Lin, Electrochem. Commun.
coexisting substance in real samples, so the 4 (2002) 743.
electrochemical response of DA in thepresence of AAand [12] P.J. Britto, K.S.V. Santhanam, P.M. Ajayan, Bioelectrochem. Bioenerg.
UAonthe MWNTs/CILE is investigated. Fig. 8 shows cyclic 41 (1996) 121.
[13] M.N. Zhang, K.P. Gong, H.W. Zhang, L.Q. Mao, Biosens.
voltammograms of 200 M AA, 50 M DA and 20 M UA Bioelectron. 20 (2005) 1270.
in 0.1 M PBS (pH 7.0) at the MWNTs/CILE. Three [14] P. Zhang, F.H. Wu, G.C. Zhao, X.W. Wei, Bioelectrochemistry
independent 67 (2005) 109.
[15] P. Qiu, Y.N. Ni, Chin. Chem. Lett. 19 (2008) 1337.
oxidation peaks at 0.002 V, 0.172 V and 0.292 V were
[16] J.Y. Tilquin, J. Glibert, P. Claes, Electrochim. Acta 38 (1993) 479.
observed at the MWNTs/CILE, indicating that their [17] B. Kim, W.M. Sigmund, Langmuir 20 (2004) 8239.
oxidation took place inde- pendently at the MWNTs/CILE. [18] F. Malem, D. Mandler, Anal. Chem. 65 (1993) 37.
Therefore, the modied electrode was suitable for the [19] C.R. Raj, T. Ohsaka, J. Electroanal. Chem. 496 (2001) 44.
selective detection of DA. [20] M. Mazloum-Ardakani, H. Beitollahi, B. Ganjipour, H. Naeimi, M.
Nejati, Bio- electrochemistry 75 (2009) 1.
[21] S. Jo, H. Jeong, S.R. Bae, S. Jeon, J. Microchem. 88 (2008) 1.
Analytical applications [22] M. Moreno, A.S. Arribas, E. Bermejo, M. Chicharro, A. Zapardiel,
M.C. Rodrguez,
Y. Jalit, G.A. Rivas, Talanta 80 (2010) 2149.
In order to examine the reliability of the proposed [23] L.Y. Jiang, C.Y. Liu, L.P. Jiang, Z. Peng, G.H. Lu, Anal. Sci. 20 (2004)
1055.
sensor in practical applications, it was appliedfor the
determination of DA in human blood serum. The
results are shown in Table 2. Some other