EQCM and in situ FTIR spectroelectrochemistry study on the electrochemical oxidation of TMB and the effect of large-sized anions

Journal of Electroanalytical Chemistry 622 (2008) 184–192

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EQCM and in situ FTIR spectroelectrochemistry study on the electrochemical oxidation of TMB and the effect of large-sized anions Meiling Liu a,b, Youyu Zhang a,b,*, Yuandao Chen c, Qingji Xie a,b, Shouzhuo Yao a,b a

Key Laboratory of Chemical Biology and Traditional Chinese Medicine Research, Ministry of Education, China College of Chemistry and Chemical Engineering, Hunan Normal University, Changsha 410081, PR China c Department of Chemistry and Chemical Engineering, Hunan University of Arts and Science, Changde 415000, PR China b

a r t i c l e

i n f o

Article history: Received 13 December 2007 Received in revised form 17 April 2008 Accepted 3 June 2008 Available online 7 June 2008 Keywords: In situ piezoelectric Fourier transform infrared Electrochemical quartz crystal microbalance Fourier transform reflection absorption infrared spectroscopy 3,30 ,5,50 -tetramethylbenzidine (TMB) Large-sized anions

a b s t r a c t In situ piezoelectric FTIR (Fourier transform infrared) spectroelectrochemistry, a combination technique of in situ FTIR and electrochemical quartz crystal microbalance (EQCM), was used to study the electrochemical oxidation of 3,30 ,5,50 -tetramethylbenzidine (TMB) and the effect of some large-sized anions on the oxidation process. A V-shaped frequency response curve was observed during the electrochemical oxidation of TMB. The formation of TMB cation free radical and charge transfer complex (CTC) were experimentally found in the process of TMB electro-oxidation. In cyclic voltammetric test, the deposition of CTC onto the electrode surface from TMB solution was observed after several scanning cycles, even though the CTC is dissolvable in the solution. Further investigations suggested that the CTC was formed by TMB and TMB2+. Some representative large-sized anions, such as heparin, alizarin red and DNA, can affect the electro-oxidation of TMB. Ó 2008 Published by Elsevier B.V.

1. Introduction A non-carcinogenic chemical compound, 3,30 ,5,50 -tetramethylbenzidine (TMB) as a new analytical reagent has been widely used for swift detection of blood sugar, urine glucose or sugar, hypothermia, peroxide enzyme and some antibodies in clinic or criminal samples [1]. TMB could undergo almost completely reversible electro-oxidation reaction under appropriate conditions [2]. It was reported that TMB in Britton–Robinson (BR) solution underwent a one-step-two-electron electro-oxidation to yield quinonediimine in pH 2.0  4.0. While the pH shifted to 4.0  7.0, TMB electro-oxidation proceeded via a two successive one-electron electro-oxidation processes and yielded TMBfree radical as the mediate and quinonediimine as the final product. At pH 8.4, the electro-oxidation of TMB was also a quasi-reversible two-electron-transfer process and yielded an azo compound [3]. TMB molecule is an organic electron donor and can form charge transfer complex (CTC) cation when it meet electron acceptor molecules under proper condition [4]. The formation and applications of CTCs based on TMB have

* Corresponding author. Address: College of Chemistry and Chemical Engineering, Hunan Normal University, Changsha 410081, PR China. Tel./fax: +86 731 8865515. E-mail addresses: [email protected] (M. Liu), [email protected] (Y. Zhang). 0022-0728/$ - see front matter Ó 2008 Published by Elsevier B.V. doi:10.1016/j.jelechem.2008.06.002

been paid extensive attention in the development of analytical techniques. A spectrophotometric method based on the formation of CTC of copper substituted tungstophosphate with TMB, a colored compound which was stabilized and sensitized by addition of polyvinyl alcohol (PVA) in aqueous solution, was used for the determination of copper [5]. The photo-induced electron transfer between TMB and C70 in various solvent was studied with in situ electron paramagnetic resonance (EPR) spectroscopy. The formation of CTC of C70 with TMB in benzonitrile was reported [6]. Another novel CTC ((TMB)3 PMo12O40) was synthesized by Wu et al. They found that it was a strong electronic interaction between organic donor TMB and PMo12 O3 40 heteropoly anion. They also found that the irradiation of ultraviolet light on (TMB)3PMo12O40 can cause the intramolecular electron transfer between the TMB and the heteropoly anion of the complex [7]. However, the investigations about TMB have been chiefly focused on the applications of TMB in bio-detection and bio-assays [1,8]. And to our knowledge, the research on the formation and mechanism of TMB CTC has been only carried out in organic solutions by resonance and absorption spectroscopic and studied the mechanism of the formation of CTC in the flowed and conventional cell in detail [9]. Clarifying the effects of potential range and large-sized anions on the electro-oxidation of TMB is an essential and meaningful work to the development of TMB applications in bio-electrochemical analysis area, and it is still remained to be done up to now.

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Fourier transform infrared reflection absorption spectroscopy (FTIR-RAS) is a powerful tool to investigate the orientation of chemical functional groups and bonding mode of the absorbent on electrode surfaces [10], and EQCM can provide multiple information of the electrochemical processes on the piezoelectric quartz crystal (PQC) electrode surface [11]. Therefore, combination FTIR-RAS with EQCM yields a versatile combination technique. And applying the combined technique to study the electrochemical reaction on electrodes allows one to conveniently get insight into the electrochemical processes. This combined technique has been used to study the polymerization mechanism of aniline and aniline-co-o-aminophenol in detail [12]. The present work was aimed to elucidate the electrochemical oxidation process of TMB in aqueous solution and to clarify the effects of potential and large-sized anions on the electro-oxidation of TMB through in situ observations of FTIR-RAS and EQCM responses by using the versatile combination technique of FTIR-RAS with EQCM. 2. Experimental section 2.1. Apparatus and chemicals The experimental device was illustrated schematically in our previous study [12] and shown in Fig. 1A. Electrochemical experiments were performed with a CHI660A electrochemical workstation (CH Instruments Co., USA). AT-cut 9 MHz piezoelectric quartz crystals (PQC) (12.5 mm in diameter) were adopted. One side of the PQC gold electrode with diameter of 6 mm was in contact with solution and served as work electrode, while the PQC gold electrode on the other side located in the waterproof air compartment served as the oscillation electrode. A Pt foil and a saturated KCl Ag/AgCl electrode were used as counter and reference electrode, respectively. All the potentials are related to this reference electrode. A research quartz crystal microbalance (RQCM, Maxtek Inc., USA) controlled by Maxtek RQCM software was used to accurately record the resonant frequency (f0) and the motional resistance (R1) of the crystal via its high performance phase lock oscillator circuit, as given in the operation manual provided by the manufacturer. The Sauerbrey equation describes a frequencymass relationship for loading or removal of a rigid and thin film [13] is given as follows: 2 Df0 ¼ 2f0g Dm=½Aðqq lq Þ1=2  ¼ 2:264  106 f0g 2Dm=A

ð1Þ

where Df0 is the measured frequency shift (Hz), f0g is the parent frequency of QCM (9 MHz), Dm is the adsorption amount (ng), qq is the density of quartz (2.648 g cm3), lq is the shear modulus of quartz (2.947  1011 g cm1 s2), and A is the effective area of the electrode (0.28 cm2). In addition, it is known that Eq. (1) can be used for the solution system under specific conditions where the influ-

A

ence of the viscous/elasticity of the polymer is negligible [13]. A net liquid-loading effect for a PQC with one side contacting solution can be characterized by the following equation:

2pL1q dDfG1=2L  DR1L ¼ 2pf DL1L ¼ 4pL1q Df0L

qffiffiffiffiffiffiffiffi qffiffiffiffiffiffiffiffiffiffiffiffi f lq = f0g c66

 4pL1q Df0L

ð2Þ

where dDfG1/2L, Df0L, DR1L and DL1L are changes in DfG1/2 (the half peak width of the conductance curve), f0L (the resonant frequency, and f0 = 1/[2p(L1C1)1/2]), R1 and L1 due to the variations of solution density and viscosity, respectively. f0g is the resonant frequency in air, L1q is the motional inductance for the PQC in air, and c66 (2.957  1010 N m2) is the lossy piezoelectrically stiffened quartz elastic constant. According to this equation, the characteristic slope value of Df0/DR1L for a net density/viscosity effect on the 9 MHz PQC resonance is 10 Hz/X. Obviously, for an investigated system, the larger the absolute value of Df0/DR1L, the weaker the viscous effect and the stronger the mass effect. In situ FTIR spectra were collected on Nicolet Nexus 670 FTIR spectrometer (Nicolet Instrument Co., Madison, WI). And the spectrometer was equipped with an external reflection accessory and a liquid nitrogen cooled mercury–cadmium–telluride (MCT) detector. A disk of calcium fluoride (CaF2) was used as IR transparent window and the incidence angle on the CaF2 window was about 58°. The setups of the thin layer of spectroelectrochemical cell used in the experiment was the same as reported in our former publication [12] and shown in Fig. 1B. The work electrode was faced to the CaF2 window and contacts the solution, and the oscillation electrode contacted the air. TMB, heparin sodium, alizarin red and DNA were purchased from Sigma and used as received. All other chemicals were of analytical grade and used as received without further purification. 2.2. Method The PQC gold work electrode was firstly polished with Al2O3 powder to obtain a mirror-like surface and then washed with doubly distilled water. To remove surface oxides or possible surface contamination, the PQC electrode was treated by nitric acid, and then cyclically scanned between 0 and 1.5 V in 0.2 M HClO4 solution till reproducible cyclic voltammograms were obtained [14]. Electrochemical oxidation of TMB on the pretreated PQC electrodes were conducted in the electrochemical cell in pH 5.0 BR solution containing 0.5 mM TMB in the absence or presence of different large-sized molecule such as heparin sodium, alizarin red or DNA. And highly purified nitrogen gas was bubbled through all the sample solutions for at least 15 min in order to purge oxygen prior to electrochemical oxidation. The potential ranges were controlled in 0–0.4, 0–0.6 and 0–0.75 V in the potential-dependent

AE

B

RE WE

OE1 OE2

IR AE

CHI 660A

RQCM Analog output

Analog output

SEC cell FTIR

OE2 OE1

A/D RE Personal computer Fig. 1. The EQCM-FTIR setup (A) and its spectroelectrochemistry cell, (B). WE, work electrode; RE: Ag/AgCl reference electrode; AE, auxiliary electrode; OE1, work electrode (Au, contacting solution); OE2, oscillation electrode (Au, in air).

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cyclic voltammetry. In situ piezoelectric FTIR spectroelectrochemistry experiments were carried out with simultaneous recording frequency, resistance response and collecting the in situ FTIR spectra during potential scanning. The pretreated PQC electrode was pushed against the CaF2 window and vertical to the plane of CaF2 window to form a thin electrolyte layer. The thickness of the thin layer was about 10 lm calculated from the cyclic voltammograms 3 of FeðCNÞ2 6 =FeðCNÞ6 in aqueous solution [15]. Therefore, the PQC electrode will not be distorted and can reflect the real electrochemical process. Each spectrum of the in situ FTIR spectra resulted from co-addition of 40 interferograms with resolution of 8 cm1. After spectra processing, every spectrum represented the average spectrum of every 100 mV when performed at a scan rate of 5 mV/s. And we chose the open circle potential as the reference potential and the spectrum collected at this potential as reference spectrum. The spectra collected at sampling potential were subtractively normalized based on the reference spectrum as DR/R = (Rs–Rr)/Rr, where Rs and Rr are the IR reflected intensity measured at the sampling and the reference potential, respectively [16,17]. Fig. 1B was the experimental setup. Therefore, the response of IR spectra was the products from the electrode reaction in the thin layer, owing to slower transfer of the species between the inner and outer of the thin layer. In all experiments, the sample was placed on the sampling device and aligned according to the manufacturer’s recommendation. Ominic E. S. P. software of version 6.0a (Nicolet Instrument Co.) was used for processing the spectra data. According to the definition, the upward and downward peaks in the DR/R spectra refer to the loss and gain of IR absorbable species at the potential Es, respectively. 3. Results and discussion 3.1. Electrochemical oxidation of TMB Fig. 2 (left) shows the typical responses of frequency, resistance and current density of the first 3 cycles during the cyclic voltammetric scan in a conventional electrochemical cell in pH 5.0 BR buffer solution containing 0.5 mM TMB. Two pairs of oxidation– reduction peaks are visible in the CV curve of TMB in aqueous solu-

tion. The oxidation peak potentials are 0.33 and 0.49 V, respectively. These results indicate that the electro-oxidation of TMB may proceed via two successive one-electron oxidation processes. And it is supposed that the electro-oxidation of TMB in aqueous solution yields semiquinione-imine, a cation radical (TMB+) firstly and diimine TMB2+ secondarily as shown in Scheme 1. During the cyclic voltammogram scan, blue species appeared near electrode with the simultaneously notable decrease in the PQC frequency at around 0.37 V. And they dissolved at 0.52–0.63 V in the positive scan with the accompanying of increase in PQC frequency. While in the negative scan, the PQC frequency increased within the potential range of 0.20–0 V. After scanned for 3 cycles, the PQC frequency decreased about 8 Hz, which means some species deposited onto the electrode surface. The V-shaped PQC frequency response observed in the potential range of 0.37–0.63 V during the positive scan, as showed in Fig. 2 (left) is an interesting and meaningful phenomenon. It is well known that the PQC frequency shift reflects to the change of mass of the PQC surface loading. Considering that only a very small PQC frequency response (<1 Hz) and no V-shaped PQC frequency response was observed in the blank test (cyclic voltammetric scan conducted in TMB-free solution under the same conditions), we inferred from the above observations that the frequency response was dominated by the electro-oxidation of TMB and the deposition of the resultant blue species on the PQC electrode surface. The above experimental evidence is also a direct demonstration of the formation of TMB CTC during the electro-oxidation of TMB in aqueous solution. We will further discuss the issue in the following section. In addition, Fig. 2 (left) shows that the resistance decreased notably during the first positive scan and then decreased slightly with scan number. The notable decrease in the resistance at the beginning might be ascribed to the density/viscosity change of the solution near the electrode and the change of the arrangement pattern of the adsorbed TMB molecules on the electrode surface when the potential changed from the open circuit potential to 0 V. In order to clarify the oxidation mechanism of TMB, in situ piezoelectric FTIR spectroelectrochemical investigations about the reaction was conducted in a thin layer cell. Fig. 3 shows the simultaneous responses of Df, DR and j (left) and the representative FTIR

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Fig. 2. (Left) Current density (j), frequency shift (Df) and resistance change (DR) of PQC electrode during the electrochemical oxidation of TMB in pH 5 BR solution in conventional electrolyte cell. Scan rate: 50 mV/s. (right) The IR spectrum of TMB.

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H

N

H

H

H3C

CH3

0.33V -e

N

H

H3C

H CH3

H3C

CH3 H

N

N

H3C

0.5V -e

CH3 H

H

A H

CH3

N

H3C

H

CH3 H

B

H

H CH3

H3C

H

+e

+e H3C

N

N

H

C

H

H3C

N

H CH3

H3C

CH3

H3C

N

H

H CH3

H3C

CH3

H3C

N

H CH3

+

H3C H

H3C

CH3 N

H

H

N

H

N

H

H

H

CH3 N

H

Scheme 1. Oxidation of TMB and formation of CTC in BR solution, and (A) TMB; (B) TMB+; (C) TMB2+

0.00

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Pred1 2000

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1052

1385 1570 1458

0.0005

-2

j /mA cm

1416

B

1600

1400

1200

1000

Wavenumbers (cm-1)

Fig. 3. Simultaneously responses of j, Df and DR of the PQC electrode (left) and in situ FTIR spectra (right) during the electrochemical oxidation of TMB in pH 5 BR solution in the thin layer electro-cell. Scan rate: 5 mV/s. A and B: the spectra collected at 0.3–0.4 V and 0.5–0.6 V.

spectra (right) during the oxidation of TMB in the thin-layer cell. There are also two pairs of oxidation and reduction peaks in the cyclic voltammogram, but the peak current was lower than that of the conventional cell. The trend of the PQC frequency shift and the resistance change were similar to that in the conventional cell though the PQC frequency shift were larger (about 12 Hz) after scanned 3 cycles. The difference lying between the observed peak currents in the thin layer cell and conventional cell should be ascribed to the decreased migration rate of electrolyte in the thin layer cell at different scan rate. The larger PQC frequency shift in the thin layer cell might result from the increased diffusion difficulty of the electro-oxidation products. Thus, it could be concluded that TMB is actually oxidized in the same way both in the thin

layer cell and conventional cell, if only the reaction conditions, i.e. the solution pH and potential, are the same. The observations about TMB oxidation performed in the thin layer cell certainly reflect the TMB oxidation behavior in the conventional cell. In order to illustrate oxidation and reduction mechanism of TMB, the IR spectrra of TMB was also collected as shown in Fig. 2 (right) as reference. In Fig. 3 (right A), many IR peaks appeared in the simultaneously obtained in situ FTIR spectrum in the potential range of 0.3–0.4 V. It is known that the IR absorbance at 1350 and 1236 cm1 are related to the stretch vibration of C-N bond and the IR absorbance at 1540 cm1 and 1575 may be relevant to the stretch vibration of C@C in phenyl ring of aromatic amines [18] and the scissor vibration of N–H bonds in secondary amines [19].

M. Liu et al. / Journal of Electroanalytical Chemistry 622 (2008) 184–192

Table 1 Typical IR vibrational bands in the range of 1800–900 cm1 for TMB CTC Wavenumber (cm1)

Assignment

1350, 1236 1462 1575 1514 1144, 1052 1550, 1332 1261 1570 1293

C–N stretch vibration of TMB in solution CH2 deformation vibration of TMB in solution scissor vibration of N–H C@C stretching vibration C–H bending + C–H twisting of TMB C@N stretching vibration of TMB+ C–N stretching vibration of TMB+ C@N stretching vibration of TMB2+ C@N and C–N stretching vibration of TMB2+

E /V 0.00

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Δf /Hz

3

0

-3

0.4

ΔR /Ω

0.0

-0.4

-0.8 0.02 -2

We are convinced that these change in IR spectra in Fig. 3 shows the disappearance or decrease of C–N and C@C. The downward peak at 1550 cm1 in Fig. 3 is relevant to the characteristic stretch vibration of C@N bond which will shift to a lower wavenumber when conjugated with benzene rings in the test system [20]. This refers to the emergence and increase in amount of C@N bonds. For the IR absorbance relevant to the stretch vibration of C@N bond of quiniod rings containing C@N bond, we may conclude that the downward peaks at 1332 and 1261 cm1 [21] in Fig. 3 are the direct experimental evidence for the formation of quiniod rings containing C@N and C–N bond. Because the IR absorbance at 1144, 1052, 1115 and 1068 cm1 is relevant to the vibration of C–H bond of benzene rings, we could assign the upward peaks at 1144 and 1052 cm1 and the downward peaks at 1115 and 1068 cm1 to the change of the conjugative structure of TMB benzene ring [22]. Sum up the above discussions about the in situ FTIR observations (see Table 1), we are believed that TMB+, a quinonediimine alike cation radical was formed during the electro-oxidation of TMB in aqueous solution under the adopted conditions. When scanning to the potential range of 0.5–0.6 V, new upward peaks at 1556 cm1 and downward peak at 1570, 1293 and 1074 cm1 appeared in the simultaneously obtained in situ FTIR spectrum. The other peaks lying between 1400 and 1050 cm1 exhibited red-shift, which may refer to the formation of TMB2+ or CTC formed by TMB and the oxidation product at higher potential. After scanned several cycles, the character IR absorbance peaks corresponding to the CTC did not disappear even if the potential scanned back to 0 V. In addition, simultaneously collecting the in situ FTIR spectrum for the of TMB-free system in the same way, no peak was observed in the same potential region during the cyclic voltammetric scan. These facts imply that part of the CTC molecules were deposited onto the electrode surface in the electro-oxidation of TMB process. Combining the above investigation with the reported results in Ref. [9], it should be a reasonable conclusion that the blue species yielded from TMB oxidation is CTC. To clarify the electro-oxidation property of TMB CTC, a TMB CTC modified PQC electrode was prepared by conducting cyclic voltammetric scan on a bare PQC electrode in 0.5 mM TMB solution for 50 cycles. As a result, visible blue TMB CTC was electrodeposited on the PQC electrode surface. Then, cyclic voltammetric scan was performed with the CTC-modified electrode in TMB-free BR buffer solution. The frequency, resistance and j responses of the CTCmodified electrode in BR were recorded and shown in Fig. 4. The resultant CV curve showed a pair of nearly symmetrical oxidation–reduction peak at around 0.4 V, which implied that TMB CTC is of electroactivity in aqueous solution and its oxidation– reduction reaction is nearly reversible. It is notable that the oxidation potential of TMB CTC is different from that of TMB monomer (shown in Figs. 2 or 3 (left)). The simultaneously obtained PQC frequency response during the cyclic voltammogram scan shown in Fig. 4. exhibited a relatively small decrease in PQC frequency (no

j /mA/cm

188

Pox1 0.01

0.00

-0.01

Pred1 0.00

0.15

0.30

0.45

0.60

0.75

E /V Fig. 4. Responses of j, Df and DR of the CTC modified PQC electrode (prepared for cycling 50 segments as in Fig. 1) in pH 5 BR solution.

more than 3 Hz) in the positive scan and a same scale increase in the reverse scan, which should be resulted from the electrolyte ions incorporated into and expulsed from the TMB CTC film loaded on the PQC electrode surface during the oxidation and reduction of TMB CTC, respectively. 3.2. Effect of potential In order to further investigate the mechanism of TMB electrooxidation and the effect of potential on the formation of TMB CTC, cyclic voltammetric scan in different potential range were performed. Fig. 5 shows the CV curves and the frequency responses in the first 10 cycles of TMB oxidation in the potential range of 0– 0.4, 0–0.6 and 0–0.75 V. When confined the CV scanning potential range within 0–0.4 V, only one symmetrical pair of redox peak at 0.32 and 0.25 V could be obtained. The PQC frequency decreased in the positive scan, increased in the reverse scan and returned to its initial level in the end. These facts indicate that TMB could undergo reversible electro-redox reaction with no additional electro-deposition on the electrode surface within this potential range. While enlarging the CV scan potential range to 0–0.6 V or 0–0.75 V, two pairs of redox peak appeared on the CV curve, which implied that there are two successive oxidation steps in the electro-oxidation of TMB. The oxidation peak potential relevant to the two successive steps was 0.33 and 0.52 V, respectively. These results further confirmed the reaction route of TMB electro-oxidation is accorded with Scheme 1. In the potential range of 0–0.6 V or 0–0.75 V, the PQC frequency decrease notably at about 0.33 V with the proceeding of the positive scan and started to increase at 0.55 V. Furthermore, the PQC frequency could not turn back to its original level but to a lowered value at the end of CV scan tests, quite different from the case performed within the potential range of 0–0.4 V. And the final PQC frequency shift in the case of CV scan within 0–0.75 V was larger than

M. Liu et al. / Journal of Electroanalytical Chemistry 622 (2008) 184–192 E /V 0.12

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0

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60

120 180 time /s

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300

Fig. 5. Simultaneously responses of j and Df of PQC electrode during electrooxidization of TMB in BR solution. The potential range: 0–0.4 V (solid line), 0–0.6 V (dashed line) and 0–0.75 V (dotted line), respectively.

in the case of CV scan within 0–0.60 V. The above facts indicated that additional electro-deposition on the PQC electrode surface occurred in the positive scan and more deposit was loaded on the electrode in the case of CV scan within 0–0.75 V than in the case of scan within 0–0.60 V. Combining these results with the above mentioned reaction route of TMB electro-oxidation, it is inferred that the electro-deposition on the PQC electrode only occurred behind the first electro-oxidation step of TMB. It is to say that the electro-deposition on the PQC electrode during the electro-oxidation of TMB in aqueous solution would not take place till TMB+ is converted into TMB2+. Therefore, the deposit should be the CTC formed by TMB2+ and TMB as shown in Scheme 1. This deduction and conclusion is solidly supported by the IR spectra shown in Fig. 3 (right). 3.3. Electrochemical oxidation of TMB in the presence of different large-sized anions As mentioned in the introduction section of this article, TMB have been widely utilized in the area of bio-detection and bio-assays. In bio-detection and bio-assay systems, large-sized anions are very common components. Therefore, to clarify the effects of large-sized anions on the electro-oxidation of TMB is indispensable for the development of analytical approaches in which the electrooxidation of TMB is involved. In the present work, heparin sodium, alizarin red and DNA were selected as the representatives of largesized anions. 3.3.1. Effect of heparin Fig. 6 (left) shows the CV curves and the simultaneous PQC frequency and resistance response during the electrochemical oxidation of TMB in the presence of 0.3 g/L heparin sodium in thin layer cell. The CV curve significantly differed from the case of the heparin sodium free system (shown in Fig. 3 (left)). The first oxidation peak current decreased with scan number and the reduction peak negatively shifted. In addition, the PQC frequency rapidly decreased about 800 Hz with the proceeding of potential scanning from 0.32 to 0.48 V and then increased about the same scale with

189

the proceeding of potential scanning from 0.48 to 0.75 V. The similar phenomenon was observed by Xie et al. when they performed CV scan in o-tolidine and heparin coexisting system [23]. For comparing, CV scan and simultaneous in situ PQC frequency recording for a heparin solution without TMB were performed under the same conditions except for the absence of TMB. The results showed that only a pair of small redox peak was observed on the CV curve at around 0.5 V and the maximum PQC frequency shift scale was no more than 3 Hz. This fact suggests that the oxidation of heparin does not result in remarkable PQC frequency shift and any sensible electro-deposition during the CV scan. And the Dfmax in Fig. 3 in TMB-BR solution is also relatively small. Therefore, the very remarkable PQC frequency shift during CV scan in the heparin and TMB coexisting system should not be the individual result of heparin or TMB oxidation. Nevertheless, the TMB electro-oxidation products TMB+, TMB2+ and TMB–CTC are all positive charged species and heparin is a negatively charged macromolecular anion. Therefore, when they met, heparin can dope into TMB–TMB2+ complex to make the final product neutral and then deposited on the electrode at proper condition. With the increasing of potential, the CTC dissolved, resulting in de-doping of heparin and further increasing in frequency. The response of frequency shift became smaller with the increase of scan cycles. It may result from the thin film on the electrode or the free cation radicals near electrode held back further formation of CTC. When the potential swept back to 0 V after 3 cycles, the change in frequency were small, indicating that little additional species loaded on the electrode. The resistance change was similar to that of in the absence of heparin at the beginning, but then increased with the decrease of frequency and changed significantly in the range of potential from 0.35 to 0.6 V. This may be relevant to the changes in viscosity/density of the solution and the properties of possible foreign films on the electrode owing to the formation, deposition of CTC. Therefore, the frequency shift was a coordinate factor of viscosity/density of the solution and the viscous properties of possible foreign films on the electrode owing to the large resistance change in this case. The characteristic slope value of Df0/DR1L is much larger than 10 Hz/X and the viscosity/density of the solution can be neglected. Therefore, the maximum mass of the film can be calculated according to Sauerbrey Equation, given the value of 1.26 lg. The molar ratio of the CTC to heparin in the CTC-heparin adduct (x) deposited on the gold electrode can be evaluated according to reference [23] using linear scan voltammetry and EQCM, which was close to the anticipated full electrical neutralization CTC-heparin adduct. The average negative charge of heparin and the average positive charge of TMB2+ are 75 and +2, and the mole mass of them are 280 and 15,000, respectively, and therefore the maximum amount of anion incorporated into the CTC can be evaluated to be about 4.94  1011 mol according to the mass change. Fig. 6 (right) shows the FTIR spectra collected simultaneously with the experiment shown in Fig. 6 (left). The IR spectra reflected the concentration change of the species in the thin layer, owing to the concentration of heparin in the thin layer was much larger than on the electrode (and the amount of doping ions was little). Therefore, the IR spectra reflected the species change in the thin layer and the upward peaks mean the decrease of concentration of heparin in the thin layer. The new upward peaks at 1251 and 1096 cm1 were the characteristics of sulfuric (S@O) and C–O–C, 850 and 820 cm1 were ascribed to the vibration of C–O–S in heparin [24]. And these peaks in Fig. 6 (right A) are smaller than that in Fig. 6 (right B), which may explain the doping process of heparin in Fig. 6 (left). And these peaks increased in the oxidation process and decreased in the reduction process, and did not disappeared at last. At the same time, the peaks related to the oxidation of TMB were also red-shifted, which was also ascribed to the formation of CTC. Therefore, combined the IR spectra with the PQC responses, trace

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0.00

0.15

E /V 0.30

0.45

0.60

0.75

A

Δf /Hz

0

1716

-300

ΔR/R 1456 1545

0.0004

-600 -900

1146

1318 1417 1164

1744

6

1121

970

850

1649

0

820

ΔR/ Ω

3

982

B 1351

1716

-3 0.0005

0.015

1579

-2

850

1230 1120 1165

1291

1642

j /mA cm

839

1096

1748

0.010

897

1379

ΔR/R

Pox2

Pox1

1251

1510

0.005 981

0.000 -0.005 0.00

0.15

945 898

Pred2

Pred1

0.30

0.45

0.60

2000

0.75

1800

1600

1400

1200

1000

Wavenumbers (cm-1)

E /V

Fig. 6. Simultaneously responses of j, Df and DR of the PQC electrode (left) and in situ FTIR spectra (right) during the electrochemical oxidation of TMB in the presence of 0.3 g/ L heparin sodium in pH 5 BR solution in the thin-layer electrolyte cell. Scan rate: 5 mV/s. A and B: the spectra collected at 0.3–0.4 V and 0.5–0.6 V.

heparin in solution may be detected through studying the oxidation of TMB, and sensor for heparin can be made. 3.3.2. Effect of alizarin red Fig. 7 (left) shows the frequency shift, resistance and j responses during the cyclic potential scanning in the range of 0–0.75 V in the presence of 0.3 g/L alizarin red in the thin layer cell. It can be seen that the CV curve changed in the presence of alizarin red. The first

oxidation peak current remained almost unchanged and the second oxidation peak current increased significantly compared with CV in Fig. 3 (left). This maybe come from the catalysis effect of the alizarin red, which can accelerate the oxidization of TMB+ into TMB2+, or the oxidation of alizarin red itself in the potential range. We also conducted the experiment of the oxidation of alizarin red in the TMB-free solution in the same condition, and there was a pair of small peaks near 0.55 V, which was much smaller than that

E /V 0.00

0.15

0.30

0.45

0.60

0.75 1594

Δf /Hz

0

A

1716 1146

1384 1461 1515 1310 1552

-15

ΔR/R -30

1127 1169

1646

0.0005 1745

0

889 936

0.08

-3

Pox2

ΔR/ Ω

-2

1592

B

-1

-2

1260

1355

1098 906 871

1718 1458

ΔR/R

1568 0.0010

j /mA cm

1479

0.04

1745

Pox1

1293 1513

1384

1125 1167 890

1647

0.00

Pred2

Pred1 -0.04 0.00

0.15

0.30

0.45

0.60

0.75

2000

1800

1600

1400

1200

1000

Wavenumbers (cm-1)

E /V

Fig. 7. Responses of j, Df and DR of the PQC electrode (left) and in situ FTIR spectra (right) during the electrochemical oxidation of TMB in the presence of 0. 3 g/L alizarin red in pH 5 BR solution in the thin-layer electrolyte cell. Scan rate: 5 mV/s. A and B: the spectra collected at 0.3–0.4 V and 0.5–0.6 V.

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of the CV curve in Fig. 6 (left). Therefore, the current response may be the overlapping of the oxidation peak currents of the two species and the catalysis effect of alizarin red. And the catalysis effect may have relevant with the nature of alizarin red itself. The frequency shift of the PQC electrode in Fig. 3 (left) decreased about 8 Hz and the resistance decreased about 1.5 X after 3 cycles scan. And the frequency response may be ascribed to the oxidation of TMB, the formation of CTC and doping of alizarin red into the CTC. The characteristic slope value of Df/DR was about 5 Hz X1, which indicated that the resistance change was a co-effect of the changes of viscosity and density of the solution and the viscous properties of possible foreign films on the electrode. Owing to the smaller molecular mass of alizarin red, the response of frequency was smaller than that in the presence of heparin though alizarin red can catalysis TMB+ into TMB2+. Fig. 7 (right) was the collected FTIR spectra during potential scanning in the first cycle. The peak at 1594 may result from the absorption of C@O in alizarin red and 1355 cm1 can be ascribed to the vibration of C–O. 1260 and 1098 cm1 were the characteristics of sulfuric in alizarin red, and the peaks at 939, 871, 836 cm1 were the aromatic rings of alizarin red [25]. Therefore, it indicated that the alizarin red could dope into the complex and affect the oxidation of TMB.

TMB2+. Oxidation of DNA solution in the TMB-free solution in the same condition was conducted and it was found that a pair of small peaks appeared near 0.35 V with a small decrease in frequency shift in the process. It also showed that the frequency decreased in the positive scan and increased in the reduction process, which was similar to the frequency response of Fig. 8 (left). Hence, it supposed that the frequency shift was attributed to the simultaneous oxidations of TMB and DNA. The resistance decreased about 4 X in the initial and then decreased slowly owing to the arrangement mode change of TMB and the viscosity/density change in solution. The simultaneously collected FTIR spectra during the electrochemical experiment are shown in Fig. 8 (right). It shows that the spectra were changed compared with that in the absence of DNA. The downward peaks at 1595, 1291, 1123, 1016 and 947, 898 cm1 are characteristics of DNA [26]. Because the concentration of DNA in the thin layer was much larger than on the electrode, the downward IR spectra reflected that the DNA was oxidized in the thin layer in this experiment condition. Therefore, it indicated that the oxidation of TMB was overlapped with DNA and this result consisted with the EQCM response.

3.3.3. Effect of DNA Fig. 8 (left) shows the electrochemical oxidation of TMB in the presence of 0.3 g/L DNA in the thin layer cell. The isoelectric point of DNA is 4–4.5, and therefore, DNA was negative charged in this experiment condition. The oxidation peak current decreased to some extent compared with that in the absence of DNA. The frequency shift of the PQC electrode changed and the V-shaped response in the oxidation process disappeared. The frequency responses decreased during the oxidation of TMB and increased in its reduction process. Finally, the frequency shift was about 7 Hz after 3 cycles. These phenomena indicated that the oxidation of TMB was largely changed owing to the presence of DNA. We supposed that DNA is a larger anion with double-helical structure and long chain in solution and it was difficult to incorporate into the film and will hold back the combination of TMB and

By using in situ piezoelectric FTIR spectroelectrochemistry, the electrochemical oxidation of TMB was studied. In the process of TMB oxidation, an intermediate formed, which deposition and dissolving led to a V-shaped response in frequency shift in the positive scan. The frequency decreased at last with some blue species deposited on electrode. Potential can affect the oxidation of TMB and blue species was CTC which formed between TMB and TMB2+. Anions which have different size, charge, length and structures, such as heparin and alizarin red could dope/de-dope into the CTC and affected the oxidation of TMB. In the case of TMB/heparin sodium, a larger V-shape in frequency response appeared in the positive scan. The second oxidation peak current increased notably in the case of TMB/alizarin red owing to the catalysis effect of alizarin red. Therefore, based on the combination technique and the response of the frequency or current, trace heparin and alizarin

0.00

0.15

E /V 0.30 0.45

0.60

4. Conclusions

0.75

A

Δf /Hz

0

ΔR/R

1506 1545

0.0004

-10

1417

1318

1145

1456

1744

1164

-20

1016 982 952

0

-4

1251

1579

1379

1096

1230

1642

-2

868 1120

1291

1165

1670

0.005

897

1219

1450 1509

1748

-6

Pox2

Pox1

1595

0.0005

ΔR/R 0.010

1351

B

ΔR/ Ω

-2

j /mA cm

968

1123

1669

981

0.000

Pred2 -0.005

947

898

Pred1 0.00

0.15

0.30

0.45

E/V

0.60

0.75

2000

1800

1600

1400

1200

1000

Wavenumbers (cm-1)

Fig. 8. Responses of j, Df and DR of the PQC electrode (left) and in situ FTIR spectra (right) during the electrochemical oxidation of TMB in the presence of 0.3 g/L DNA in pH 5 BR solution in the thin-layer electrolyte cell. Scan rate: 5 mV/s. A and B: the spectra collected at 0.3–0.4 V and 0.5–0.6 V.

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red can be detected, and new biosensor may be prepared in future in our laboratory. But for the oxidation of TMB in the presence of DNA, the PQC response was changed notably which may be ascribed to the helical structure of DNA or the oxidation of DNA in this condition. Acknowledgments This work was supported by the National Natural Science Foundation of China (20675030), The Key Project of Chinese Ministry of Education (207076), and Scientific Research Fund of Hunan Provincial Education Department and Science and Technology Department (06A035, 06FJ3151), and Youth Scientific Research Fund of Hunan Normal University (070645) and the Opening Fund of Key Laboratory of Chemical Biology and Traditional Chinese Medicine Research (Ministry of Education of China), Hunan Normal University(KLCBTCMR2008-12). References [1] [2] [3] [4] [5] [6] [7]

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