NiCoBP-doped carbon nanotube hybrid: A novel oxidase mimetic system for highly efficient electrochemical immunoassay

Analytica Chimica Acta 851 (2014) 49–56

Contents lists available at ScienceDirect

Analytica Chimica Acta journal homepage: www.elsevier.com/locate/aca

NiCoBP-doped carbon nanotube hybrid: A novel oxidase mimetic system for highly efficient electrochemical immunoassay Bing Zhang, Yu He *, Bingqian Liu, Dianping Tang * Institute of Nanomedicine and Nanobiosensing, Department of Chemistry, Fuzhou University, Fuzhou 350108, People’s Republic of China

H I G H L I G H T S

G R A P H I C A L A B S T R A C T


We report a new oxidase mimetic system for highly efficient electrochemical immunoassay.
NiCoBP-doped carbon nanotube hybrids were used as the nanocatalysts.
NiCoBP-doped carbon nanotube hybrids were used as the mimic oxidase.

A R T I C L E I N F O

A B S T R A C T

Article history: Received 19 July 2014 Received in revised form 12 August 2014 Accepted 14 August 2014 Available online 20 August 2014

NiCoBP-doped multi-walled carbon nanotube (NiCoBP–MWCNT) was first synthesized by using induced electroless-plating method and functionalized with the biomolecules for highly efficient electrochemical immunoassay of prostate-specific antigen (PSA, used as a model analyte). We discovered that the as-synthesized NiCoBP–MWCNT had the ability to catalyze the glucose oxidization with a stable and well-defined redox peak. The catalytic current increased with the increment of the immobilized NiCoBP–MWCNT on the electrode. Transmission electron microscope (TEM) and energy dispersive X-ray spectrometry (EDX) were employed to characterize the as-prepared NiCoBP–MWCNT. Using the NiCoBP–MWCNT-conjugated anti-PSA antibody as the signal-transduction tag, a new enzyme-free electrochemical immunoassay protocol could be designed for the detection of target PSA on the capture antibody-functionalized immunosensing interface. Experimental results revealed that the designed immunoassay system could exhibit good electrochemical responses toward target PSA, and allowed the detection of PSA at a concentration as low as 0.035 ng mL1. More importantly, the NiCoBP-MWCNT-based oxidase mimetic system could be further extended for the monitoring of other low-abundance proteins or disease-related biomarkers by tuning the target antibody. ã 2014 Elsevier B.V. All rights reserved.

Keywords: Electrochemical immunoassay Carbon nanotube Oxidase mimetic system NiCoBP nanoparticles Prostate-specific antigen

1. Introduction The important role for the determination of disease-related proteins in the fields of medical diagnostics [1], drug discovery [2], environmental monitoring [3], and food safety [4] has driven the ever-increasing demand for developing simple, sensitive,

* Corresponding authors. Tel.: +86 591 2286 6125; fax: +86 591 2286 6135. E-mail address: [email protected] (D. Tang). http://dx.doi.org/10.1016/j.aca.2014.08.026 0003-2670/ ã 2014 Elsevier B.V. All rights reserved.

highly selective, and cost-effective biosensors. Immunoassay, based on the specific reaction of the antigen–antibody recognition, opens up the new avenue for the detection of biomarkers [5–7]. In recent years, ongoing effort has been made worldwide to provide new insights into the application of immunoassays using some sophisticated techniques, e.g. plasma atomic emission spectrometry [8], fluorescence [9], chemiluminescence [10], mass spectrometry [11], and electrochemical methods [12]. Among these methods, electrochemistry holds great potential as the next-generation detection strategy because of its high sensitivity

50

B. Zhang et al. / Analytica Chimica Acta 851 (2014) 49–56

and simple instrumentation. Despite many advances in this field, there is still the need for exploiting new protocols and strategies to improve the sensitivity and simplicity of the immunoassays. The development of nanotechnology has brought a great momentum. In this case, scientists are always enthusiastic about finding materials with good biocompatibility to improve the behavior of biosensors [13–15]. To this end, biosensors based on enzyme-mimetic inorganic materials have emerged as a new class of ideal and important electrochemical detection tools for biosensing [16,17], owing to their high stability, easy preparation, controllable structure and composition, and tunable catalytic activity. Carbon materials such as carbon felt and carbon cloth have many advantages, including good stability, favorable conductivity, large specific surface area, and low cost, and thus have been widely used as the anode materials of biosensors. However, bare carbon felt or carbon cloth shows its poor biocompatibility and electrocatalytic activity. The biocompatibility can be improved by using carbon nanotube (CNT) [18,19]. Functionalized CNT provides large surface area with oxygen-containing groups and thus exhibit excellent biocompatibility. The functionalization of CNT has been carried out in various ways for numerous applications in biotechnology, including for the preparation of sensors [20], as scaffolds for cell growth [21], imaging reagents [22], and transporters for drug delivery [23]. One way is to synthesize CNT-based hybrids with iron oxide, cobalt oxide, or nickel oxide for improvement of electrochemical behaviors. Nickel-based nanomaterials are substantially undergoing a great deal of attention to be one of the most attractive candidates for functionalizing the CNT owing to their low-cost, environment benignity and outstanding catalytic behavior [24]. However, recent reports on Ni-doped nanostructures were usually synthesized under harsh conditions and required cumbersome post-treatments. Moreover, addition of Co, Fe, P, or B in the nanostructures has been used for improvement of catalytic activity of Ni-based nanostructures [25]. Herein, our motivation is to combine the advantages of CNT with electroactive metal ions to construct a new hybrid nanostructure with redox activity by using a simple and feasible method. We imagined that the doped metal Co can facilitate the growth of NiCoBP nanocrystals in the multi-walled carbon nanotubes. The nanocomplexes have a well-defined morphology, controllable particle size, and high specific surface areas. The fabricated nanocomplexes deliver excellent rate capability and cycle performance when used as electrode materials and the NiCoBP–MWCNT nanocomplexes exhibit outstanding catalytic behavior for the glucose oxidation. Further, we employed NiCoBP–MWCNT as the nanolabel application in immunoassay. As expected, the immunosensors exhibit excellent performance for the detection of PSA.

2. Experimental 2.1. Materials and reagents All reagents (analytical grade) were used without further purification. Multi-walled carbon nanotube (MWCNT) was pretreated before the active component NiCoBP was impregnated. Briefly, MWCNT (100 mg) was treated with 3:1 H2SO4/HNO3 and sonicated for 4 h to obtain carboxylic group-functionalized MWCNT [26]. Prostate-specific antibody (anti-PSA, 0.1 mg mL1) was purchased from Amyjet Scientific Inc. (Abcam product, Wuhan, China). PSA standards with various concentrations were obtained from Biocell Biotechnology Co., Ltd. (Zhengzhou, China). N-Hydroxysulfosuccinimide sodium salt (NHS), N-(3-dimethylaminopropyl)-N0 -ethylcarbodiimide hydrochloride (EDC),

HAuCl44H2O, Ni(NO3)26H2O, CoCl26H2O, Na3C6H5O72H2O, (NH4)2SO4, NaOH, and NaBH4 were achieved from Alfa Aesar1. Ultrapure water obtained from a Millipore water purification system (18 MV, Milli-Q, Millipore) was used in all runs. 2.2. Preparation of NiCoBP-doped multi-walled carbon nanotube (NiCoBP–MWCNT) NiCoBP–MWCNT was prepared by using the induced electroless-plating technique [27]. The acid-pretreated MWCNT was immersed in an aqueous solution containing Ni(NO3)26H2O (0.4 M) and CoCl26H2O (0.04 M), followed by chemical reduction in NaBH4 (2.0 M). The thin layer of NiCoB thus formed on the MWCNT can further induce deposition of NiCoBP on the MWCNT. Then, the NiCoB–MWCNT prepared above was transferred into an aqueous solution containing Na3C6H5O72H2O (0.068 M), (NH4)2SO4 (0.23 M), CoCl26H2O, (0.008 M), Ni(NO3)26H2O (0.04 M) and NaH2PO2H2O (0.7 M), and agitated for 1 h. NaBH4 (2.0 M) aqueous solution was added dropwise to the above solution under agitation. The pH of the NiCoBP plating solution was adjusted to 8–9 using NH3H2O. The NiCoBP was formed during the titration process. The titration process was operated at 50  C under N2 atmosphere and completed when no bubbles were observed in the solution. The resulting NiCoBP–MWCNT was washed repeatedly with deionized water and ethanol. Finally, the as-prepared NiCoBP–MWCNT was dispersed into distilled water with a concentration of 20 mg mL1, and stored at 4  C for further use. Next, NiCoBP–MWCNT was employed for the labeling of anti-PSA antibody (Ab2) by using the classical carbodiimide coupling method. Initially, 11 mg of NHS and 15 mg of EDC were dissolved into 1 mL of NiCoBP–MWCNT suspension, followed by continuous stirring for 45 min at room temperature (RT). Afterward, 500 mL of Ab2 (0.1 mg mL1) was added into the mixture, and gently stirred for 12 h at 150 rpm at 4  C. The excess chemicals and antibodies were removed by centrifugation. Finally, the as-prepared NiCoBP–MWCNT-Ab2 was dispersed into 1.0 mL PBS (0.1 M, pH 7.4) for the detection of PSA. 2.3. Preparation of different electrochemical sensors A glassy carbon electrode (GCE, 3 mm in diameter) was polished repeatedly with 0.3 and 0.05 mm alumina slurry, followed by successive sonication in bi-distilled water and ethanol for 5 min and dried in air. 5 mL of the above-prepared NiCoBP–MWCNT suspension was coated onto the GCE and dried at RT for 5 min to form the modified electrode: NiCoBP–MWCNT–GCE. The electrochemical immunosensor was constructed on a cleaned GCE as the following steps: (i) a layer of nanogold particles was initially electrodeposited on the GCE by using a potential of 0.2 V in 1.0 mM HAuCl4 for 60 s (designated as AuNP–GCE); (ii) 5 mL of anti-PSA antibody (Ab1) was dropped on the surface of AuNP–GCE, and incubated for 12 h at 4  C (designated as Ab1-AuNP–GCE); and (iii) the as-prepared Ab1-AuNP–GCE was incubated in 2.5 wt% BSA for 60 min at RT to eliminate non-specific binding effects and block the remaining active groups. Finally, the obtained electrode (Ab1-AuNP–GCE) was stored at 4  C while not in use. 2.4. Electrochemical measurement All electrochemical measurements were carried out with a CHI 430A Electrochemical Workstation (Shanghai, China) with a conventional three-electrode system using a modified GCE working electrode, a platinum auxiliary electrode, and an Hg/ HgCl reference electrode. Electrochemical impedance spectroscopy (EIS) measurements were carried out on the CHI 604D Electrochemical Workstation (Shanghai, China) at +0.2 V, with a

B. Zhang et al. / Analytica Chimica Acta 851 (2014) 49–56

disturbance potential of 5 mV and a frequency range from 1 Hz to 1 105 Hz. After the immunosensor was incubated with various PSA concentrations for 30 min at RT, the resulting immunosensor was submerged into NiCoBP–MWCNT-Ab2 suspension for another 30 min at RT. After rinsing with deionized water, the electrochemical signal were recorded by using differential pulse voltammetry (DPV) from 200 to 600 mV (potential step: 4 mV; frequency: 25 Hz; amplitude: 25 mV). All electrochemical measurements were done at RT. Analyses are always made in triplicate. Scheme 1 represents the immunoassay process and assay protocol toward target PSA by coupling with the oxidase mimetic system. 3. Results and discussion 3.1. Characterization of NiCoBP–MWCNT In this work, the NiCoBP–MWCNT hybrids were synthesized by the induced electroless-plating technique. To prepare the NiCoBP– MWCNT, the long MWCNT was initially shortened by using the H2SO4 and HNO3 mixture. It is well known that upon treatment in acid, numerous carboxylate groups were generated on the surface of MWCNT [28]. Upon addition of metal ions, the positively charged metal precursors could be adsorbed on the functionalized MWCNT with negative charge [29]. The adsorbed metal ions could be in situ reduced by the strong reduction reagent to form the hybrid nanostructures. During the reduction reaction, some boron and phosphorous atoms could be simultaneously migrated into the CoNiBP, and coated to the MWCNT [30]. Typically, the redox reaction between metal ion and reducing agent produces gas bubble that can adsorb on the substrate surface to prevent further deposition of metal on the surface sites during the electrolessplating process, thus causing the formation of voids or pits on the substrate surface. First, we used transmission electron microscope (TEM) to characterize the as-prepared NiCoBP–MWCNT (Fig. 1). Before metal precursors were introduced, holes and defects could be observed on the pretreated MWCNT (Fig. 1A). The presence of holes and defects was expected to not only improve the interaction between MWCNT and metal precursors, but also facilitate the electron transport from liquid phase to the active sites inside the MWCNT. Fig. 1B represents typical TEM image of the as-synthesized NiCoBP–MWCNT, and a large number of nanoparticles were dispersed on the surface of MWCNT. The size

51

of NiCoBP–MWCNT was about 50 nm in the diameter and 450 nm in the length. Fig. 1C shows the wide-angle X-ray diffraction (XRD) patterns of the MWCNT before (pattern ‘a’) and after (pattern ‘b’) modification with CoNiBP. The CoNiBP–MWCNT displayed a XRD pattern similar to pure MWCNT. The diffraction peak at 2u = 26.03 was corresponded to the graphic carbon. In contrast, a broadening peak at 2u = 44.5 was appeared after formation of the CoNiBP– MWCNT (pattern ‘b’), indicating that the CoNiBP on the MWCNT was an amorphous structure [28,30]. To further verify the formation of the hybrid nanostructures, the as-prepared CoNiBP–MWCNT was characterized by using X-ray photoelectron spectroscopy (XPS). Fig. 1D represents the Ni2p, Co2p, B1s, P2p, O1s, and C1s core level regions of the CoNiBP–MWCNT, respectively. The existence of the new bands at 857, 781, 190 and 133 eV for the hybrids indicated the presence of Ni2p, Co2p, B1s, and P2p. In addition, the bulk compositions of the CoNiBP–MWCNT determined by inductively coupled plasma (ICP) displayed the hybrid nanostructures containing 81.2 wt% metal (73.1 wt% Co, 8.1 wt% Ni), 3.2 wt% B and 9.2 wt% P, respectively. The results preliminarily revealed that the NiCoBP–MWCNT could be successfully synthesized by using our designed route. 3.2. Electrochemical characteristics of NiCoBP–MWCNT To investigate the possible application of the NiCoBP–MWCNT for subsequent electrochemical immunoassay, the electrochemical behaviors of the as-synthesized NiCoBP–MWCNT were evaluated by using cyclic voltammetry (0.1–0.6 V, vs. Hg/HgCl) in 0.1 M NaOH. The modified electrode was prepared by directly dropping the NiCoBP–MWCNT on a cleaned GCE. As shown from curve ‘c’ in Fig. 2A, at a scan rate of 0.1 V s1, a couple of well-defined redox peaks was observed with an anodic peak at +0.45 V and a cathodic peak at + 0.35 V (E = Epa + Epc/2 = 0.4 V), a high peak current response (Ip) of 130 mA and a low peak potential separation (DEp) of 100 mV. This pair of redox peaks was ascribed to the redox reaction of NiIII/NiII couple on the electrode surface [31]: Ni + 2OH ! Ni(OH)2 + 2e (1) Ni(OH)2 + OH $ NiO(OH) + e (2) Unfavorably, the absence of Ni (curve ‘a’ in Fig. 2A) or Co (curve ‘b’ in Fig. 2A) in the nanocomposites only caused weak

Scheme 1. Schematic illustration of (a) CoNiBP–MWCNT-based oxidase mimetic system and (b) the enzyme-free electrochemical immunoassay by coupling with the CoNiBP–MWCNT oxidase mimetic system (Note: CoNiBP–MWCNT: CoNiBP-doped multi-walled carbon nanotube).

52

B. Zhang et al. / Analytica Chimica Acta 851 (2014) 49–56

Fig. 1. (A, B) TEM images of (A) MWCNT and (B) NiCoBP–MWCNT, (C) XRD patterns of (a) MWCNT and (b) NiCoBP–MWCNT, and (D) XPS analysis of NiCoBP–MWCNT.

electrochemical signal. The results could also be further verified by using electrochemical impedance spectroscopy (EIS) (right inset of Fig. 2A). The EIS data were fitted to the Randles equivalent circuit, which consisted of electrolyte resistance (RS), the capacitance (Cdl), charge transfer resistance (Ret) and Warburg element (Zw) [32,33]. In EIS, the semicircle diameter of EIS equals the electron transfer resistance, Ret [34]. As seen from inset in Fig. 2A, the resistance of NiCoBP–MWCNT-modified GCE was obviously lower than that of pure GCE, implying that the as-synthesized NiCoBP–MWCNT could facilitate the electron transfer and communication between the solution and the electrode. More importantly, the peak currents (Ip) of NiCoBP–MWCNTmodified GCE were proportional to the square root of the scan rate (n1/2) as shown from the inset in Fig. 2B, following the Randles– Sevcik equation:   nF nD 1=2 (3) Ip ¼ 0:4463 nFAC RT The results suggested the electrochemical reaction of NiCoBP– MWCNT-modified GCE was a surface-controlled process. On the other hand, the anodic peaks shifted to more positive potentials and the cathodic peaks shifted to more negative potentials, resulting in an increase of the peak separation between the anodic and cathodic peaks. The peak separation could be used to estimate the heterogeneous electron transfer rate constant (ks). The ks as well as the transfer coeffcient (a) were calculated based on the following Laviron equation [35]:

Epa ¼ E þ

RTln n ð1  aÞnF

(4)

Epc ¼ E 

RTln n anF

(5)

  ð1  aÞanF DEp RT  ln ks ¼ alnð1  aÞ þ ð1  aÞln a  ln nF n 2:3RT

(6)

where n is the number of electron transfer, DEp is the peak to peak potential separation, R, T and F are symbols that have conventional meanings. The a and ks were estimated as 0.507 and 0.501 s1, respectively, indicating this was a faster electron transfer process. The value of ks was significantly larger than the values obtained from NiBP–MWCNT (0.347 s1), Nafion–MWCNT (0.332 s1) [36], TiO2 (0.137 s1) [37], and HSG–SN–CNT (0.410 s1) [38]. Therefore, those results suggested that the presence of NiCoBP–MWCNT could effectively accelerate the interfacial electron transfer. For a surface-controlled process, the integration of oxidation peaks gives nearly constant charge (Q) at different scan rates. The average surface coverage (G *) of the electroactive Ni on the modified electrodes was estimated at a slow scan rate according to Faraday’s law [39]: Q ¼ nAF G

(7)

where Q is the charge involved in the reaction, n is the number of electron transferred, F is Faraday’s constant, and A is the effective

B. Zhang et al. / Analytica Chimica Acta 851 (2014) 49–56

53

Fig. 3. (A) Cyclic voltammograms of NiCoBP–MWCNT–GCE 0.1 M NaOH toward different-concentration glucose at 100 mV s1 (Insets: cyclic voltammograms of (a) CoBP– MWCNT–GCE and (b) NiBP–MWCNT–GCE in the absence of presence of 2 mM glucose). Chronoamperometric plots of (B) NiCoBP–MWCNT–GCE, (C) CoBP–MWCNT–GCE and (D) NiBP–MWCNT–GCE toward different-concentration glucose in 0.1 M NaOH recorded at +0.5 V.

surface area of the electrode (7.065 mm2 used in this case). The G * value of NiCoBP–MWCNT was calculated as 7.68  109 mol cm2, which was significantly higher than that of NiBP–MWCNT (2.54  109 mol cm2), Ppy–CA–IL (5.29  109 mol cm2) [39], F-NiO–IL–Hb-CPE (4.98  109 mol cm2) [40], and NiO (1.73  109 mol cm2) [41]. Thus, the electroactive Ni were entrapped in the NiCoBP–MWCNT and participated in the electron transfer process. 3.3. Monitoring of oxidase-like activity using NiCoBP–MWCNT To monitor the oxidase-like activity of NiCoBP–MWCNT, the as-synthesized nanocomposites were used for detection of glucose with various concentrations in 0.1 M NaOH by using cyclic

voltammetry. As shown in Fig. 3A, the anodic current increased with the increasing glucose concentration in the detection solution. The catalytic behavior might be originated from the doped NiIII toward the electro-oxidation of glucose as the following equation [42]: NiO(OH) + glucose ! Ni(OH)2 + glucolactone (8) To highlight the advantages of the as-prepared NiCoBP–MWCNT, another two nanostructures including CoBP–MWCNT and NiBP– MWCNT were used for the detection of the same-concentration glucose (Note: the amount of Co, Ni and MWCNT was almost the same within three nanostructures). The evaluation was implemented by comparing the shift in the anodic peak current before and after addition of glucose in 0.1 M NaOH. As seen from Fig. 3A,

Fig. 2. (A) Cyclic voltammograms of differently modified electrodes with various nanostructures (a) CoBP–MWCNT, (b) NiBP–MWCNT and (c) NiCoBP–MWCNT (Inset: Nyquist grams of (d) bare GCE and (e) NiCoBP–MWCNT–GCE in pH 7.4 PBS containing 5 nM Fe(CN)64/3 and 0.1 M KCl, and equivalent circuit). (B) Cyclic voltammograms of NiCoBP–MWCNT–GCE in 0.1 M NaOH at different scan rates from 50 mV s1 to 200 mV s1 (Insets: the relationship between current vs. n1/2 and potential vs. ln n).

54

B. Zhang et al. / Analytica Chimica Acta 851 (2014) 49–56

the change in the anodic peak current of using NiCoBP–MWCNT was obviously larger than those of CoBP–MWCNT and NiBP– MWCNT alone. The phenomenon could be explained as a result of the fact that the doped Ni in the nanostructures could exhibit better electrocatalytic performance within the applied potential than that of Co alone. According to the Michaelis–Menten (MM) mechanism [43], we suspected that the possible reaction pathway for the oxidization of glucose by the NiCoBP–MWCNT is as shown in Scheme 2. Initially, the added glucose binds on the oxidized form of the biomimetic site reversibly (step 1), followed by the oxidization of the glucose with the simultaneous formation of reduced form of the biomimic, that is, NiCoBP–MWCNT through an inner-sphere electron-transfer mechanism (step 2). Then, the active site, NiCoBP–MWCNT is regenerated at the operating potential (step 3). The mechanism is almost in accordance with the Michaelis– Menten general equation [39]: Ipa

nFAK cat GE C ¼ ðK M þ CÞ

(9)

whereas the parameter of KM is obtained by using the Lineweaver– Burk approach: 1 KM 1 þ ¼ Ipa nFAK cat GE C nFAK cat GE

(10)

According to Eqs. (9–10), we figured out the KM = 0.163 for NiCoBP– MWCNT. Note that the KM value obtained in this work was less than that of CoBP–MWCNT (KM = 0.538) or NiBP–MWCNT (KM = 0.371) alone, thus suggesting the high catalytic activity of the as-synthesized NiCoBP–MWCNT toward glucose. Further, the NiCoBP–MWCNT-modified GCE was conducted by using chronoamperometry in the stationary solution to evaluate the catalytic rate constant kcat. Typically, when the anodic current is dominated by the rate of the electrocatalytic reaction, the catalytic current Icat can be written as follows [31]:

Icat ¼ p1=2 ðkcat C 0 tÞ1=2 IL

(11)

where Icat and IL are the currents in the presence and absence of glucose, respectively; kcat, C0 and t are the catalytic rate constant, the bulk concentration of glucose, and the time elapsed, respectively. Taking the glucose concentration C0 = 2 mM into account, the kcat was calculated to be 68.6 M1 s1 (Fig. 3B). In contrast, the kcat values were 32.4 M1 s1 for CoBP–MWCNT (Fig. 3C) and 44.7 M1 s1 for NiBP–MWCNT (Fig. 3D), respectively. The results revealed that the NiCoBP–MWCNT was more efficient for the electro-oxidation of glucose than CoBP–MWCNT and NiBP–MWCNT alone. 3.4. Evaluation of NiCoBP–MWCNT-based electrochemical immunoassay To further investigate the applicable potential of the as-prepared NiCoBP–MWCNT in the electrochemical immunoassay as the glucose oxidase mimic, the NiCoBP–MWCNT was utilized for the labeling of anti-PSA antibody (NiCoBP–MWCNT-Ab2). By using NiCoBP–MWCNT-Ab2 as the signal-transduction tag, a new sandwich-type immunoassay protocol was built for determination of PSA on the mouse anti-human monoclonal anti-PSA-functionalized glassy carbon electrode (Ab1-AuNP–GCE) (Note: the deposited gold nanoparticles were characterized and described in our previous work [44]). In a typical target PSA detection experiment, Ab1-AuNP–GCE and NiCoBP–MWCNT-Ab2 initially sandwiched the target PSA, generating an immunocomplex. Accompanying the NiCoBP–MWCNT-Ab2, the carried NiCoBP–MWCNT could catalytically oxidize the glucose in the detection solution, thereby causing the increasing anodic peak current. Scheme 1b represents the assay protocol of the electrochemical immunoassay. As shown from curve ‘d’ in Fig. 4A, a pair of well-defined and quasi-reversible redox peaks was obtained in 0.1 M NaOH when the Ab1-AuNP–GCE reacted with PSA and NiCoBP–MWCNT-Ab2. In contrast, no peaks of interest were

Scheme 2. Catalytic mechanism of the NiCoBR–MWCNT toward the oxidation of glucose.

Fig. 4. (A) Cyclic voltammograms of variously modified electrodes (a) GCE, (b) Ab1-AuNP–GCE, (c) PSA/Ab1-AuNP–GCE, (d) electrode ‘c’ after reaction with NiCoBP–MWCNTAb2, and (e) probe ‘d’ after incubation with 5 mM glucose in 0.1 M NaOH. (B) The calibration curve of the NiCoBP–MWCNT-based immunoassay protocol (Inset: the corresponding DPV curve toward different-concentration PSA). (C) The specificity of NiCoBP–MWCNT-based immunoassay.

B. Zhang et al. / Analytica Chimica Acta 851 (2014) 49–56

55

Table 1 Comparison of the results for clinical serum specimens obtained by using the NiCoBP–MWCNT-based immunoassay and the referenced ECLIA. Sample no.

1 2 3 4 5 6 7 8 9 10 11 12

Method; concentration (ng mL1, n = 3) Found by the developed immunoassay

Found by ECLIA

2.9 30.8 10.8 0.6 29.3 50.8 0.8 7.9 20.5 0.3 105.8 22.4

2.2 31.2 11.2 0.1 29.7 51.2 1.3 8.3 19.7 0.5 99.9 18.7

observed at bare glassy carbon electrode (curve ‘a’), Ab1-AuNP–GCE (curve ‘b’) and PSA/Ab1-AuNP–GCE (curve ‘c’). These results clearly indicated that the redox peak mainly derived from the labeling NiCoBP–MWCNT. Upon addition of 5 mM glucose in the NaOH, significantly, an obvious catalytic characteristic with an increase of the oxidation current was achieved (curve ‘e’ in Fig. 4A). Moreover, the catalytic current increased with the increment of target PSA concentration in the sandwich-type immunoassay (inset of Fig. 4B). A linear dependence relationship between DPV peak current and the logarithm of PSA concentration could be acquired within the dynamic range from 0.1 to 50 ng mL1 (Fig. 4B). The detection limit (LOD) was 0.035 ng mL1 estimated at the 3sB criterion. Although the system has not yet been optimized for maximum efficiency, the LOD of using NiCoBP–MWCNT-Ab2 was lower than that of commercialized PSA ELISA kit (0.5 ng mL1, Biocell Biotechnology Co., Ltd., Zhengzhou, China). Furthermore, the electrochemical immunoassay could also completely meet the requirement of clinical diagnosis because of the threshold value of PSA (4–10 ng mL1) in human serum. 3.5. Reproducibility, precision, stability and selectivity The precision and reproducibility of the NiCoBP–MWCNT-based electrochemical immunoassay was examined toward 10 ng mL1 PSA (as an example) by using identical batches of NiCoBP–MWCNTAb2. Initially, we investigated the precision by using the same-batch NiCoBP–MWCNT-Ab2 for the detection of 10 ng mL1 PSA, and the obtained coefficient of variation (CV) was 8.5% (n = 5). The batch-tobatch reproducibility was studied by using different-batch NiCoBP– MWCNT-Ab2 for the above-mentioned analyte, and the CV was 9.7% (n = 5). The results indicated good precision and acceptable fabrication reproducibility. Additionally, the as-prepared NiCoBP– MWCNT-Ab2 was stored at 4  C when not in use, and the signal at 60th day was 88.2% of the initial current toward 10 ng mL1 PSA. The specificity of the NiCoBP–MWCNT-based electrochemical immunoassay was also monitored toward other possible components in human serum samples, e.g. carcinoembryonic antigen (CEA), alpha-fetoprotein (AFP), luteinizing hormone (LH), thyroidstimulating hormone (TSH), and human IgG. As indicated from Fig. 4C, the strong electrochemical signal could be obtained with target PSA relative to other components. Significantly, the presence of interfering materials in the target PSA did not obviously change the signal of the developed immunoassay. These results clearly demonstrated the high specificity of the NiCoBP–MWCNT-based electrochemical immunoassay. 3.6. Analysis of real sample and evaluation of method accuracy To further evaluate the accuracy of NiCoBP–MWCNT-based electrochemical immunoassay system during the measurement

RSD (%)

texp

0.35 1.12 0.25 0.55 0.20 1.75 0.45 0.20 1.81 2.28 2.96 3.67

2.42 0.44 1.92 1.11 2.45 0.28 1.35 2.45 0.54 0.11 2.44 1.24

toward target PSA, we collected 12 human serum specimens from Fujian Provincial Hospital of China according to the rules of the local ethical committee. Initially, these serum samples were centrifuged for 10 min at 5000  g (at 4  C) to remove some possible precipitation. Then, these samples were detected by using our developed immunoassay. Following that, the concentrations of PSA in these samples were calculated according to the abovementioned regression equation. The results were compared with those of using commercialized PSA ELISA kit, which are summarized in Table 1. As shown from Table 1, the relative standard deviation (RSD) values in these cases were lower than 5.0%. Meanwhile, all the experimental values of t (texp) were less than the critical value of t (tcrit(4, 0.05) = 2.77). Therefore, the NiCoBP– MWCNT-based immunoassay method could be employed for the determination of target PSA in real samples. 4. Conclusions In summary, this work describes a new and enzyme-free electrochemical immunoassay for the detection of low-abundance protein (PSA used as a model) in biological fluids by using a novel glucose oxidase mimic, NiCoBP-doped multi-walled nanotube, as the signal-transduction labels (tags). Results revealed that the as-synthesized NiCoBP–MWCNT exhibited good electrocatalytic properties toward glucose oxidization without the need of natural enzyme. Compared with CoBP–MWCNT and NiBP–MWCNT alone, the catalytic efficiency by using NiCoBP–MWCNT hybrids could be improved and enhanced. Importantly, the NiCoBP–MWCNT could be used as the glucose oxidase mimic to conjugate the biomolecules, e.g. detection antibody, for the development of the sandwich-type immunoassay. In contrast with conventional enzyme-based immunoassays, the NiCoBP–MWCNT-based immunosensing system was not susceptible to interference and changes in assay conditions during the signal generation stage. These features, as well as its other advantages, such as convenience of operation, enzyme-free nature, and low cost, make it further applicable for other antigens or biomolecules by changing the target antibody. Acknowledgements Support by the National Natural Science Foundation of China (41176079), the National “973” Basic Research Program of China (2010CB732403), the National Science Foundation of Fujian Province (2011J06003), the Program for Changjiang Scholars and Innovative Research Team in University (IRT1116), and the National Key Technologies R&D Program of China during the 12th Five-Year Plan Period (Key Technology of Quality and Safety Control During Aquatic Product Processing) (2012BAD29B06) is gratefully acknowledged.

56

B. Zhang et al. / Analytica Chimica Acta 851 (2014) 49–56

References [1] T. Tran, J. Cui, M. Hartman, S. Peng, H. Funabashi, F. Duan, D. Yang, J. March, J. Lis, H. Cui, D. Luo, A universal DNA-based protein detection system, J. Am. Chem. Soc. 135 (2013) 14008–14011. [2] W. Lohmann, H. Hayen, U. Karst, Covalent protein modification by reactive drug metabolites using online electrochemistry/liquid chromatography/mass spectrometry, Anal. Chem. 80 (2008) 9714–9719. [3] M. Simonova, T. Valyakina, E. Petrova, R. Komaleva, N. Shoshina, L. Samokhvalova, O. Lakhtina, I. Osipov, G. Philipenko, E. Singov, E. Grishin, Development of xMAP assay for detection of six protein toxins, Anal. Chem. 84 (2012) 6326–6330. [4] M. Su, M. Venkatachalam, C. Liu, Y. Zhang, K. Roux, S. Sathe, A murine monoclonal antibody based enzyme-linked immunosorbent assay for almond (Prunus dulcis L.) detection, J. Agric. Food Chem. 61 (2013) 10823–10833. [5] D. Liu, Z. Wang, A. Jin, X. Huang, X. Sun, F. Wang, Q. Yan, S. Ge, N. Xia, G. Niu, G. Liu, A. Walker, X. Chen, Acetylcholinesterase-catalyzed hydrolysis allows ultrasensitive detection of pathogens with the naked eye, Angew. Chem. Int. Ed. 52 (2013) 14065–14069. [6] Z. Qin, W. Chan, D. Boulware, T. Akkin, E. Butler, J. Bischof, Significantly improved analytical sensitivity of lateral flow immunoassays by using thermal contrast, Angew. Chem. Int. Ed. 51 (2012) 4358–4361. [7] K. Saha, S. Agasti, C. Kim, X. Li, V. Rotello, Gold nanoparticles in chemical and biological sensing, Chem. Rev. 112 (2012) 2739–2779. [8] N. Tung, M. Chikae, Y. Ukita, P. Viet, Y. Takamura, Sensing technique of silver nanoparticles as labels for immunoassay using liquid electrode plasma atomic emission spectrometry, Anal. Chem. 84 (2012) 1210–1213. [9] D. Tang, B. Liu, R. Niessner, P. Li, D. Knopp, Target-induced displacement reaction accompanying cargo release from magnetic mesoporous silica nanocontainers for fluorescence immunoassay, Anal. Chem. 85 (2013) 10589–10596. [10] H. Akhavan-Tafti, D. Binger, J. Blackwood, Y. Chen, R. Creager, R. Silva, R. Eickholt, J. Gaibor, R. Handley, K. Kapsner, S. Lopac, M. Mazelis, T. McLernon, J. Mendoza, B. Odegaard, S. Reddy, M. Salvati, B. Schoenfelner, N. Shapir, K. Shelly, J. Todtleben, G. Wang, W. Xie, A homogeneous chemiluminescent immunoassay method, J. Am. Chem. Soc. 135 (2013) 4191–4194. [11] R. Liu, X. Liu, Y. Tang, L. Wu, X. Hou, Y. Lv, Highly sensitive immunoassay based on immunogold-silver amplification and inductively coupled plasma mass spectrometric detection, Anal. Chem. 83 (2011) 2330–2336. [12] X. Jia, X. Chen, J. Han, J. Ma, Z. Ma, Triple signal amplification using gold nanoparticles: bienzyme and platinum nanoparticles functionalized graphene as enhancers for simultaneous multiple electrochemical immunoassay, Biosens. Bioelectron. 53 (2014) 65–70. [13] A. Huefner, W. Kuan, R. Barker, S. Mahajan, Intracellular SERS nanoprobes for distinction of different neuronal cell types, Nano Lett. 13 (2013) 2463– 2470. [14] L. Cui, Y. Song, G. Ke, Z. Guan, H. Zhang, Y. Lin, Y. Huang, Z. Zhu, C. Yang, Graphene oxide protected nucleic acid probes for bioanalysis and biomedicine, Chem. Eur. J. 19 (2013) 10442–10451. [15] D. Tang, R. Yuan, Y. Chai, H. An, Magnetic-core/porous-shell CoFe2O4/SiO2 composite nanoparticles as immobilized affinity supports for clinical immunoassays, Adv. Funct. Mater. 17 (2007) 976–982. [16] Y. Jiang, W. Wang, X. Li, X. Wang, J. Zhou, X. Mu, Enzyme-mimetic catalystmodified nanoporous SiO2-cellulose hybrid composites with high specific surface area for rapid H2O2 detection, ACS Appl. Mater. Interfaces 5 (2013) 1913–1916. [17] X. Dong, H. Xu, X. Wang, Y. Huang, M. Chan-Park, H. Zhang, L. Wang, W. Huang, P. Chen, 3D graphene–cobalt oxide electrode for high-performance supercapacitor and enzymeless glucose detection, ACS Nano 6 (2012) 3206–3213. [18] W. Yang, K. Ratinac, S. Ringer, P. Thordarson, J. Gooding, F. Braet, Carbon nanomaterials in biosensors: should you use nanotubes or graphene? Angew. Chem. Int. Ed. 49 (2010) 2114–2138. [19] D. Tang, J. Tang, B. Su, G. Chen, Ultrasensitive electrochemical immunoassay of staphylococcal enterotoxin B in food using enzyme-nanosilica-doped carbon nanotubes for signal amplification, J. Agric. Food Chem. 58 (2010) 10824– 10830. [20] J. Tang, D. Tang, B. Su, J. Huang, B. Qiu, G. Chen, Enzyme-free electrochemical immunoassay with catalytic reduction of p-nitrophenol and recycling of p-aminophenol using gold nanoparticles-coated carbon nanotubes as nanocatalysts, Biosens. Bioelectron. 26 (2011) 3219–3226. [21] S. Shin, H. Bae, J. Cha, J. Mun, Y. Chen, H. Tekin, H. Shin, S. Farshchi, M. Dokmeci, S. Tang, A. Khademhosseini, Carbon nanotube reinforced hybrid microgels as scaffold materials for cell encapsulation, ACS Nano 6 (2012) 362–372.

[22] S. Namgung, K. Baik, J. Park, S. Hong, Controlling the growth and differentiation of human mesenchymal stem cells by the arrangement of individual carbon nanotubes, ACS Nano 5 (2011) 7383–7390. [23] L. Minati, V. Antonini, M. Serra, G. Speranza, Multifunctional branched goldcarbon nanotube hybrid for cell imaging and drug delivery, Langmuir 28 (2012) 15900–15906. [24] C. Guo, H. Huo, X. Han, C. Xu, H. Li, Ni/CdS bifunctional Ti@TiO2 core–shell nanowire electrode for high-performance nonenzymatic glucose sensing, Anal. Chem. 86 (2014) 876–883. [25] W. Yang, Z. Gao, J. Wang, J. Ma, M. Zhang, L. Liu, Solvothermal one-step synthesis of Ni–Al layered double hydroxide/carbon nanotube/reduced graphene oxide sheet ternary nanocomposite with ultrahigh capacitance for supercapacitors, ACS Appl. Mater. Interfaces 5 (2013) 5443–5454. [26] L. Wang, J. Lei, R. Ma, H. Ju, Host–guest interaction of adamantine with a b-cyclodextrin-functionalized AuPd bimetallic nanoprobe for ultrasensitive electrochemical immunoassay of small molecules, Anal. Chem. 85 (2013) 6505–6510. [27] L. Li, C. Han, X. Han, Y. Zhou, L. Yang, B. Zhang, J. Hu, Catalytic decomposition of toxic chemicals over metal-promoted carbon nanotubes, Environ. Sci. Technol. 45 (2011) 726–731. [28] Q. Li, D. Tang, J. Tang, B. Su, J. Huang, G. Chen, Carbon nanotube-based symbiotic coaxial nanocables with nanosilica and nanogold particles as labels for electrochemical immunoassay of carcinoembryonic antigen in biological fluids, Talanta 84 (2011) 538–546. [29] S. Chen, X. Chen, B. Yi, T. Liu, W. Li, Zinc oxide nanoparticle decorated multiwalled carbon nanotubes and their optical properties, Acta Mater. 54 (2006) 5401–5407. [30] L. Li, C. Han, L. Yang, X. Yang, B. Zhang, The nature of PH3 decomposition reaction over amorphous CoNiBP alloy supported on carbon nanotubes, Ind. Eng. Chem. Res. 49 (2010) 1658–1662. [31] X. Niu, M. Lan, H. Zhao, C. Chen, Highly sensitive and selective nonenzymatic detection of glucose using three-dimensional porous nickel nanostructures, Anal. Chem. 85 (2013) 3561–3569. [32] A. Nechache, M. Cassir, A. Ringuede, Solid oxide electrolysis cell analysis by means of electrochemical impedance spectroscopy: a review, J. Power Sources 258 (2014) 164–181. [33] A. Bandarenka, Exploring the interfaces between metal electrodes and aqueous electrolytes with electrochemical impedance spectroscopy, Analyst 138 (2013) 5540–5554. [34] S. Park, J. Yoo, Electrochemical impedance spectroscopy for better electrochemical measurements, Anal. Chem. 75 (2003) 455A–461A. [35] E. Laviron, General expression of the linear potential sweep voltammogram in the case of diffusionless electrochemical systems, J. Electroanal. Chem. 101 (1979) 19–28. [36] W. Sun, X. Li, Y. Wang, X. Li, C. Zhao, K. Jiao, Electrochemistry of myoglobin in Nafion and multi-walled carbon nanotubes modified carbon ionic liquid electrode, Bioelectrochemistry 75 (2009) 170–175. [37] W. Li, A. Luo, J. Feng, Direct electron transfer for heme proteins assembled on nanocrystalline TiO2 film, Electroanalysis 13 (2001) 359–363. [38] Y. Liu, M. Hu, Facile preparation of magnetic core–shell Fe3O4@Au nanoparticle/myoglobin biofilm for direct electrochemistry, Biosens. Bioelectron. 25 (2010) 1447–1453. [39] S. Dong, N. Li, G. Suo, T. Huang, Inorganic/organic doped carbon aerogels as biosensing materials for the detection of hydrogen peroxide, Anal. Chem. 85 (2013) 11739–11746. [40] S. Dong, P. Zhang, H. Liu, N. Li, T. Huang, Direct electrochemistry and electrocatalysis of hemoglobin in composite film based on ionic liquid and NiO microspheres with different morphologies, Biosens. Bioelectron. 26 (2011) 4082–4087. [41] A. Salimi, E. Sharifi, A. Noorbakhsh, S. Soltanian, Direct voltammetry and electrocatalytic properties of hemoglobin immobilized on a glassy carbon electrode modified with nickel oxide nanoparticles, Electrochem. Commun. 8 (2006) 1499–1508. [42] Z. Gao, J. Guo, N. Shrestha, R. Hahn, Y. Song, P. Schmuki, Nickel hydroxide nanoparticle activated semi-metallic TiO2 nanotube arrays for non-enzymatic glucose sensing, Chem. Eur. J. 19 (2013) 15530–15534. [43] P. Gayathri, A. Kumar, An iron impurity in multiwalled carbon nanotube complexes with chitosan that biomimics the heme-peroxidase function, Chem. Eur. J. 19 (2013) 17103–17112. [44] K. Liu, R. Yuan, Y. Chai, D. Tang, H. An, [AuCl4] and Fe3+/[Fe(CN)6]3 ionsderived immunosensing interface for electrochemical immunoassay of carcinoembryonic antigen in human serum, Bioprocess Biosyst. Eng. 33 (2010) 179–185.