Introduction

Tumor markers are substances existing in tumor cells or host body fluids, which are produced by tumors associated with cancers, and can be used for diagnosis of patients and clinical therapy according to their biochemical or immune characteristics [1]. Some tumor markers are associated with many types of cancer, others, with as few as one [2]. Some tumor markers are always elevated in specific cancers, and most are less predictable. For example, an elevation of alpha-fetoprotein (AFP) level in human serum can give rise to liver cancer and epithelial ovarian tumors, while carcinoembryonic antigen (CEA) can engender liver cancer and colorectal cancer. Hence, searching for a sensitive, rapid, simple method for simultaneous detection of multiple tumor markers in a single run would be of great interest in clinical diagnosis.

For a multianalyte testing (dual assays, simultaneous assays), two or more analytes are simultaneously measured in a single assay [3, 4]. The advantages of using multianalyte testing are working simplification (fewer assay tubes and fewer pipetting operations), an increase in test throughput, and possible reduction in the overall cost per test [5–7]. Various methods and schemes have been reported for simultaneous determination of multianalyte. Morgan and Wians in the early used two radio-isotopic labels (131I and 125I or 125I and 57Co) for simultaneous immunoassay of two analytes, respectively [8, 9]. Despite high sensitivity, they have some limitations such as the short shelf life of 125I-labeled antibody, radiation hazards, a complicated wash procedure, and a relatively long assay time. Following that, Hemmila and Saarma utilized the fluorophore labels as a broad range of distinguishable fluorescence signals, e.g. europium/terbium and europium/samarium, which can be effectively combined for dual assays [10, 11]. Later finding the disadvantages of traditional fluorescent chromophores are that they tend to have poor photostability, brightness, and a short Stokes shift. This causes significant problems when working with samples with a naturally high background fluorescence. Recently, Cooper group used a multianalyte immunosensor for simultaneous determination of follicle stimulating hormone and luteinizing hormone [12]. Wu et al. designed a disposable multianalyte electrochemical immunosensor array for automated simultaneous determination of tumor markers [13]. Niessner group constructed a chemiluminescence multianalyte chip immunoassays for simultaneous determination of four different antibiotic residues in honey [14]. Among these methods, electrochemical immunoassays are suitable for sensor miniaturization and automated detection, since it is highly sensitive, low cost, low power requirements, and has high compatibility with advanced micromachining technologies [15–17].

Typically, the electrochemical multianalyte immunoassays are usually performed by using the parallel electrodes or two/more enzyme labels [18–20]. In the past, we designed an arrayed immunosensor comprising five working electrode and a reference electrode for the detection of four tumor markers [21]. The electrochemical signals of five working electrodes were recorded by coupling a potentiometry with a changeover switch. Hence, the assay process was relatively tedious. Recently, we constructed a sandwich-type electrochemical immunoassay protocol for simultaneous multiplexed immunoassays of two tumor markers by using thionine and ferrocene as distinguishable signal tags [22]. Although two types of antibodies were simultaneously immobilized on the same probe, the assay involved in twice immunoreactions and multiple washing. Moreover, the preparation of the immunosensing probes and signal tags were complicated. To tackle this point, our motivation in this work is to devise a new one-spot electrochemical immunosensor for simultaneous detection of two tumor markers by using one-step immunoassay format.

Thionine and ferrocene derivatives have often been used as electrochemical signaling probes for immunoassays. Labeling of thionine or ferrocene derivatives to biomolecules has been intensively studied and used as mediators in biosensors [23, 24]. Herein, we combined with the merits to construct a multiplexed electrochemical immunoassay. Initially, thionine and ferrocene derivatives were covalently conjugated onto two types of antibodies (anti-AFP and anti-CEA here used in this case), respectively. The formed conjugates were simultaneously immobilized on nanogold-modified glassy carbon electrode. Finally, horseradish peroxidase (HRP) was immobilized onto the resulting electrode. The assay was carried out based on the immobilized HRP toward the catalytic reduction of H2O2 at both peak potentials of thionine and ferrocene. With the progress of the antigen-antibody reaction, the formed immunocomplex was coated on the surface of the immunosensor, which partly shielded the active centre of the enzyme, resulting in the decrease of the corresponding peak currents.

Experimental

Reagents and chemicals

Monoclonal mouse anti-human anti-CEA antibody (clone C6G9) and monoclonal mouse anti-human anti-AFP antibody (clone 1G7), CEA and AFP standards were purchased from Biocell Biotechnol. Co., Ltd. (Zhengzhou, China, www.chinbiocell.com). Peroxidase from horseradish (HRP, EC 1.11.1.7, 250–330 units mg−1), N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC), N-Hydroxysuccinimide (NHS), N-2-hydroxyethylpiperazine-N′-(2-ethanesulfonic acid), thionine acetate salt (Thi), and ferrocenecarboxylic acid (Fc) were obtained from Sigma-Aldrich (USA, www.signaaldrich.com). HAuCl4·4H2O and hydrogen peroxide (30 wt.%) were provided from Sinopharm Chem. Re. Co. Ltd. (Shanghai, China, www.cncm.cn). All other reagents were of analytical grade and used without further purification. Ultrapure water obtained from a Millipore water purification system (≥ 18 MΩ, Milli-Q, Millipore, www.millipore.com) was used in all runs. Phosphate buffer saline (PBS, 0.1 mol L−1) solution with various pHs were prepared by mixing the stock solutions of 0.1 mol L−1 NaH2PO4 and 0.1 mol L−1 Na2HPO4, and 0.1 mol L−1 KCl was added as the supporting electrolyte.

Preparation of ferrocene-anti-CEA and thionine-anti-AFP conjugates

Ferrocene-labeled anti-CEA (designated as Fc-anti-CEA) and thionine-anti-AFP (designated as Thi-anti-AFP) were prepared through a typical carbodiimide coupling [25], as shown in Scheme 1a–b. Initially, 2.76 mg of ferrocenecarboxylic acid was dissolved thoroughly in 700 μL of N-2-hydroxyethylpiperazine-N′-(2-ethanesulfonic acid) buffer (50 mmol L−1, pH 9.3), and the pH was then adjusted to 7.3 with 3 mmol L−1 HCl. 11.0 g of NHS nd 15.0 mg of EDC were dissolved in the solution followed with continuous stirring for 45 min at room temperature (RT). Following that, 300 μL of 1.0 mg mL−1 anti-CEA antibody dissolved in 50 mmol L−1 pH 7.3 N-2-hydroxyethylpiperazine-N′-(2-ethanesulfonic acid) buffer was added drop by drop to the mixture under continuous stirring at 150 rpm, and leaved at RT for 20 h. After completion of the incubation, the conjugates were centrifuged for 10 min at 5,000 rpm to remove the precipitates. Finally, the obtained conjugates were dialyzed in a dialysis bag against 0.1 mol L−1 pH 7.4 PBS at RT for 24 h by changing the buffer every 6 h to remove non-conjugated ferrocenecarboxylic acid. The as-prepared Fc-anti-CEA conjugates were dispersed into 500 μL PBS (0.1 mol L−1, pH 7.4) (conc. 0.5 mg mL−1). In contrast, Thi-anti-AFP conjugates were also prepared by using the similar method.

Scheme 1
scheme 1

a Fabrication process of ferrocene-labeled anti-CEA and thionine-labeled anti-AFP antibody, and b schematic illustration of one-step electrochemical immunoassay

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Preparation of electrochemical immunosensor

A glassy carbon electrode (GCE, 3 mm in diameter) was polished repeatedly with 1.0 and 0.3-μm alumina slurry, followed by successive sonication in distilled water and ethanol for 5 min and dried in air. Gold nanoparticles (AuNPs) were electrochemically deposited on the pretreated GCE by a potential-step electrolysis from +1.1 to 0 V in 0.5 M H2SO4 containing 1.0 mM HAuCl4 with different pulse times, i.e., 10, 30 and 60 s [26]. The resulting AuNP/GCE was taken out from the solution and thoroughly rinsed with water. Following that, 3 μL of 0.5 mg mL−1 Fc-anti-CEA and 3 μL of 0.5 mg mL−1 Thi-anti-AFP were simultaneously cast on the AuNP/GCE, which were incubated for 2 h at 4 °C. During this process, Fc-anti-CEA and Thi-anti-AFP were conjugated onto the AuNP surface through Au–S or Au–NH bonds. Subsequently, the resulting electrode was incubated with 0.5 mg mL−1 HRP solution for 1 h at RT to eliminate non-specific binding effect and block the remaining active sites. Finally, the as-prepared immunosensor was stored at 4 °C for further use.

Electrochemical measurement

The preparation process and measurement principle of the electrochemical immunosensor are schematically illustrated in Scheme 1. All electrochemical measurements were carried out by using a conventional three-electrode system with a modified GCE as working electrode, a platinum foil as auxiliary electrode, and a saturated calomel electrode (SCE) as reference electrode. The assay was based on the inhibition of the bioelectrocatalytic activity of the immobilized HRP after the formation of the immunocomplex on the electrode surface. AFP/CEA standards or serum samples with various concentrations were initially prepared with distilled water, and then the immunosensor was incubated for 30 min with the samples at RT. After the residual was removed with doubly distilled water, the immunosensor was placed into 3 mL of pH 6.0 PBS containing 6 mmol L−1 H2O2. Meanwhile, the signals were assayed by using differential pulse voltammetry (DPV) from +600 to −600 mV (vs SCE) with a pulse amplitude of 50 mV and a pulse width of 50 ms. The DPV peak currents at various potentials were collected and registered as the signal of the immunosensor relative to the concentration of AFP and CEA (Note: All the currents were calculated relative to the base line throughout the text, unless specifically stated).

Results and discussion

Characteristics of Fc-anti-CEA and Thi-anti-AFP conjugates

Scheme 1 gives the fabrication process of the electrochemical immunosensor. During the process, gold nanoparticles were electrochemically deposited on the GCE by reduction of Au(III) to the zero-valent gold nanoparticles [26–28]. The formed gold nanoparticles could be utilized as an affinity support for the immobilization of Thi-anti-AFP, Fc-anti-CEA and HRP due to the strong interaction between nanogold particles and proteins. Significantly, the electron mediators comprising thionine and ferrocene usually have the different peak potentials during the redox process in the same supporting electrolyte. Therefore, the difference in the peak position could be used for simultaneous determination of two tumor markers. Use of HRP is expected to enhance the sensitivity of the electrochemical immunosensors.

Figure 1 shows UV–vis absorption spectra of various components before and after the formation of Thi-anti-AFP and Fc-anti-CEA, respectively. Two absorption peaks at 310 and 450 nm were observed for pure ferrocenecarboxylic acid (curve ‘a’ of Fig. 1a), while one characteristic peak at 278 nm was obtained at anti-CEA antibody (curve ‘b’ of Fig. 1a). When ferrocenecarboxylic acid was labeled onto anti-CEA antibody, however, we only observed one absorption peak at 288 nm (curve ‘c’ of Fig. 1a). The peak might be derived from the peak overlapping of ferrocenecarboxylic acid (310 nm) and anti-CEA (278 nm). Importantly, the absorbance of Fc-anti-CEA was obviously lower than those of ferrocenecarboxylic acid and anti-CEA alone. By the same token, we also investigated UV–vis absorption spectra of Thi-anti-AFP before and after the formation of the conjugates (Fig. 1b). Similar results were acquired. Hence, the formed Thi-anti-AFP and Fc-anti-CEA conjugates could provide a precondition for determination of AFP and CEA.

Fig. 1
figure 1

A UV–vis absorption spectra of (a) ferrocenecarboxylic acid, (b) anti-CEA, and (c) Fc-anti-CEA, and B UV–vis absorption spectra of (a) thionine, (b) anti-AFP, and (c) Thi-anti-AFP

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EIS characteristics of variously modified electrodes

To monitor the modification process of the immunosensors, electrochemical impedance spectroscopy (EIS) was employed to monitor the interface properties of the electrode during stepwise modifications (Fig. 2). These EIS data were fitted to a Randles equivalent circuit (inset of Fig. 2a), consisting of electrolyte resistance (R s), the modified electrode/electrolyte capacitance (C dl), charge transfer resistance (R ct) and Warburg element (Z w). The complex impedance can be presented as the sum of the real, Z re and imaginary, Z im, components that originate mainly from the resistance and capacitance of the cell. The two components of the scheme, R s and Z w, represent bulk properties of the electrolyte solution and diffusion of the applied redox probe in solution, respectively. Thus, they are not affected by chemical transformations occurring at the electrode interface. The other two components of the circuit, C dl and R ct, depend on the dielectric and insulating features at the electrode/electrolyte interface [29]. In the fitting curve of EIS, the semicircle diameter of EIS equals the electron transfer resistance, R ct. This resistance controls the electron transfer kinetics of the redox-probe at electrode interface. Its value varies when different substances are immobilized on the electrode. Figure 2a shows the Nyquist diagrams of variously modified electrodes in 5 mmol L−1 Fe(CN) 3−/4−6 containing 0.1 mol L−1 KCl. As seen from curve ‘a’ in Fig. 2a, a relatively large resistance (R ct ≈ 125 Ω) was observed at bare GCE. When nanogold particles were electrochemically deposited on the GCE, the resistance greatly decreased, and seemed a straight line (curve ‘b’ in Fig. 2a), which was in accordance with the previous report [30]. In contrast, when Thi-anti-AFP and Fc-anti-CEA conjugates were immobilized onto the AuNP/GCE, the resistance gently increased (curve ‘c’ in Fig. 2a). This is most likely a consequence of the fact that thionine and ferrocene, as electron mediators, could favor for the transfer of Fe(CN) 3−/4−6 between the solution and the electrode. To further clarify this point, anti-CEA and anti-AFP antibody without thionine and ferrocene were directly immobilized on the AuNP/GCE. Figure 2b displays Nyquist diagrams of Thi-anti-AFP/Fc-anti-CEA and anti-AFP/anti-CEA-modified AuNP/GCE, respectively. As seen from curve ‘a’ in Fig. 2b, the simultaneous presence of thionine and ferrocene could obviously decrease the resistance of the modified electrode. In addition, the results also revealed that thionine and ferrocene could be covalently conjugated onto the antibodies.

Fig. 2
figure 2

a Nyquist diagrams for (a) GCE, (b) AuNP/GCE, and (c) Fc-anti-CEA(Thi-anti-AFP)/AuNP/GCE (inset: equivalent circuit); b Nyquist diagrams for (a) Fc-anti-CEA(Thi-anti-AFP)/AuNP/GCE and (b) anti-CEA(anti-AFP)/AuNP/GCE; c Nyquist diagrams for (a) Fc-anti-CEA(Thi-anti-AFP)/GCE and (b) Fc-anti-CEA(Thi-anti-AFP)/AuNP/GCE; conditions: in 5 mM Fe(CN) 4−/3−6 + 0.1 M KCl with the range from 10−2 Hz to 105 Hz at an alternate voltage of 5 mV

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Another question to be answered is whether Thi-anti-AFP and Fc-anti-CEA were immobilized onto the GCE by nanogold electrodeposites. To demonstrate this point, the cleaned GCE and AuNP/GCE were directly immersed into the mixture containing Thi-anti-AFP and Fc-anti-CEA, and incubated for 12 h at 4 °C. Thereafter, the modified electrodes were investigated by using electrochemical impedance spectroscopy (Fig. 2c). As indicated from curve ‘a’ in Fig. 2c, the resistance of the Thi-anti-AFP/Fc-anti-CEA-modified GCE was almost the same as that of bare GCE (curve ‘a’ in Fig. 2a). The result suggested that the cleaned GCE could not be used for immobilization of Thi-anti-AFP and Fc-anti-CEA. In contrast, the presence of gold nanoparticles (i.e. Thi-anti-AFP/Fc-anti-CEA-modified AuNP/GCE) could obviously decrease the resistance (curve ‘b’ in Fig. 2c), indicated that Thi-anti-AFP and Fc-anti-CEA could be conjugated onto the GCE through gold nanoparticles. Based on these results, we might make a conclusion that (a) thionine and ferrocene could be labeled to the antibodies by using the carbodiimide coupling method, and (b) nanogold particles could be used for construction of the electrochemical immunosensors.

Voltammetric characteristics of the electrochemical immunosensors

In this work, the amplification of the electrochemical signal mainly derives from the immobilized HRP toward the catalytic reduction of H2O2 in the solution. In the presence of thionine and ferrocene, the redox behaviors are simultaneously occurred at the corresponding peak potentials. The response mechanism is simply summarized as follows:

$$ {{\text{H}}_{{2}}}{{\text{O}}_{{2}}} + {\text{HR}}{{\text{P}}_{{({\text{red}})}}} \to {{\text{H}}_{{2}}}{\text{O}} + {\text{HR}}{{\text{P}}_{{({\text{ox}})}}} $$
(1)
$$ {\text{HR}}{{\text{P}}_{{({\text{ox}})}}} + {\text{Thi}} \to {\text{HR}}{{\text{P}}_{{({\text{red}})}}} + {\text{Thi}}{\left( {\text{H}} \right)^{ + }} $$
(2)
$$ {\text{Thi}}{\left( {\text{H}} \right)^{ + }} + {{\text{H}}^{ + }} + {2}{{\text{e}}^{ - }} \to {\text{Thi}} $$
(3)

while

$$ {{\text{H}}_{{2}}}{{\text{O}}_{{2}}} + {\text{HR}}{{\text{P}}_{{({\text{red}})}}} \to {{\text{H}}_{{2}}}{\text{O}} + {\text{HR}}{{\text{P}}_{{({\text{ox}})}}} $$
(4)
$$ {\text{HR}}{{\text{P}}_{{({\text{ox}})}}} + {\text{F}}{{\text{c}}_{{({\text{red}})}}} \to {\text{HR}}{{\text{P}}_{{({\text{red}})}}} + {\text{F}}{{\text{c}}_{{({\text{ox}})}}} $$
(5)
$$ {\text{F}}{{\text{c}}_{{({\text{ox}})}}} + {{\text{e}}^{ - }} \to {\text{F}}{{\text{c}}_{{({\text{red}})}}} $$
(6)

Curve ‘a’ in Fig. 3a displays the cyclic voltammogram of the as-prepared immunosensors in pH 6.0 PBS at 50 mV s−1. Two pairs of redox waves were observed. One couple of redox peaks was appeared at −210 and −160 mV, which mainly originated from the labeled thionine. Another pair of redox peaks was achieved at +230 and +320 mV, which was mainly ascribed to the labeled ferrocene. The peak separation between two reduction peaks was ∼440 mV (ΔE pc). Thus, a differentiation of CEA and AFP was possible according to the position of the corresponding reduction peak. Upon addition of 6.0 mM H2O2 into pH 6.0 PBS, an obvious catalytic process with the decrease of anodic peak and the increase of cathodic peak was observed for the two couples of redox waves (Curve ‘b’ in Fig. 3a). The results indicated that the bioactivity of the immobilized HRP still remained.

Fig. 3
figure 3

a Cyclic voltammograms of Fc-anti-CEA(Thi-anti-AFP)/AuNP/GCE in pH 6.0 PBS in the (a) absence and (b) presence of 6 mM H2O2 at 50 mV s−1; SWV curves of b Fc-anti-CEA(Thi-anti-AFP)/AuNP/GCE, c electrode ‘B’ after incubation with 5 ng mL−1 AFP, and d electrode ‘B’ after incubation with 5 ng mL−1 CEA in pH 6.0 PBS containing 6 mM H2O2

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Following that, we used differential pulse voltammetry (DPV) to evaluate the electrochemical properties of the as-prepared immunosensors for simultaneous detection of AFP and CEA (5 ng mL−1 as an example) in pH 6.0 PBS with a conventional three-electrode system. As shown from Fig. 3b, the as-prepared immunosensor exhibited two well-resolved peaks at −280 mV and 440 mV in pH 6.0 PBS toward zero analyte, and the peak separation (ΔE) was 720 mV. After the immunosensor was incubated with 5 ng mL−1 CEA, the peak current was decreased only at the ferrocene-based peak potential (Fig. 3c), and the peak current for thionine tags was almost the same as that of Fig. 3b. Similarly, when the as-prepared immunosensor was incubated with 5 ng mL−1 AFP, a remarkable decrease was obtained only at the thionine-based peak potential (Fig. 3d). These results demonstrated the capability of our strategy for simultaneous electrochemical determination of AFP and CEA.

Optimization of experimental conditions

Figure 4a shows the effect of H2O2 concentration in the assay solution on the current of the electrochemical immunosensor. To ensure the adequate reaction of the immobilized HRP on the electrode, the assay was performed in the absence of the analyte (i.e. zero analyte). As indicated from Fig. 4a, the DPV peak current increased with the increasing H2O2 concentration, and almost reached an optimal current at 6.0 mM H2O2. Higher concentration of H2O2 did not result in the significant increment of peak current. So, 6.0 mM H2O2 was used for detection of AFP and CEA.

Fig. 4
figure 4

Dependence of SWV peak currents on a H2O2 concentration, b pH of PBS and c incubation time toward a zero analyte, and b,c 1.0 ng mL−1 AFP and 1.0 ng mL−1 CEA

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Figure 4a represents the effect of pH of PBS on the electrochemical responses of the immunosensors in PBS containing 6.0 mM H2O2. The assay was carried out based on the responses of the newly prepared immunosensor toward 1.0 ng mL−1 AFP and CEA. Since thionine and ferrocene were two different mediators, they usually exhibit various electrochemical behaviors. As seen from Fig. 4b, the peak currents initially decreased, and then increased between pH 4.5 and pH 8.0. However, the difference between peak currents was the least at pH 6.0. Higher or lower pH values would result in the larger difference in the peak current, which were not conducive to determination at low concentration of the analyte. Therefore, pH 6.0 PBS was selected as the supporting electrolyte for detection of AFP and CEA.

Usually, the antigen-antibody reaction is adequately carried out at human normal body temperature (37 °C). Considering the possible application of the immunoassay in the future, we selected room temperature (25 ± 1.0 °C) for the antigen-antibody interaction throughout the experiment. At this condition, we investigated the effect of incubation time on current response of the immunosensor (Fig. 4c). The peak currents decreased with the increment of incubation time, and tended to level off after 30 min. Hence, an incubation time of 30 min was selected for sensitive determination of AFP and CEA at acceptable throughput.

Analytical performance

Under optimal conditions, the sensitivity and dynamic range of the as-prepared immunosensors were evaluated with AFP and CEA standards. A differential pulse voltammetric (DPV) measurement was carried out in PBS, pH 6.0, containing 6.0 mM H2O2 after incubation with various analyte levels for 30 min at RT. As indicated in Fig. 5a, the DPV peak currents decreased with the increase of AFP and CEA concentrations. Both calibration plots displayed a good linear relationship between the reduction peak currents and the logarithm of the analyte concentration in the same range of 0.01–50 ng mL−1 for AFP (Fig. 5b) and CEA (Fig. 5c). The correlation coefficients were 0.9937 and 0.9883 for AFP and CEA (n = 18), respectively. The detection limit (LOD) for AFP and CEA were about 0.01 ng mL−1 for both analytes. Since the threshold values in normal human serum is ~10 ng mL−1 for AFP and 3 ng mL−1 for CEA, the electrochemical immunosensors could completely meet the requirements of clinical diagnosis. If the concentrations in the sample are higher than 50 ng mL−1, an appropriate dilution should be preferable.

Fig. 5
figure 5

a DPV responses and b,c calibration curves of the one-step multiplexed electrochemical immunoassay toward AFP and CEA standards in PBS, pH 6.0,containing 6.0 mM H2O2 (Note: The currents were obtained relative to the base line)

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The reproducibility of the electrochemical immunosensors was evaluated by assaying 1.0 ng mL−1 AFP (as an example) for three times with the same-batch immunosensors. The peak currents were −1.61 μA, −1.57 μA and −1.63 μA, respectively. The RSD value was 1.6 % (n = 3). The sensor-to-sensor reproducibility was monitored by using the various-batch immunosensors for determination of 1.0 ng mL−1 AFP. The peak currents were −1.64 μA, −1.59 μA and −1.62 μA, respectively, and the RSD value was 1.3 % (n = 3). Hence, the repeatability and intermediate precision of the electrochemical immunosensor was acceptable.

The stability of the electrochemical immunosensor was evaluated. The immunosensors were stored at 4 °C when not in use. The currents were assayed every 3–5 days. The currents of the immunosensor at 5th, 10th, 20th, and 30th could maintain 98.2 %, 96.1 %, 94.3 %, and 91.2 % of the initial current.

Evaluation of clinical specimens with the immunosensor

To investigate the possibility of the newly developed technique to be applied for clinical analysis, 15 serum specimens comprising AFP and CEA were examined by the developed immunosensors and enzyme-linked immunosorbent assay (ELISA). The measurement method of the immunosensor is as follows: 10 μL of serum sample was initially dropped on the immunosensor, and then incubated for 30 min at RT. After washing with distilled water, DPV measurement was carried out in pH 6.0 PBS. The results were compared with the reference values obtained from the ELISA method (Table 1). Statistical comparison of the experimental results of the electrochemical immunosensors with those of ELISA was performed using a t-test for comparison of means preceded by the application of an F-test. These data shows that there is no significant difference at the 0.05 significance level between the results given by two methods which are in concordance with the results obtained using the standard methods by ELISA (because the t exp were in all cases below than t crit (t crit[4, 0.05] = 2.77)), that is, the developed immunoassays may provide a promising alternative tool for simultaneous determining CEA and AFP in human serum in clinical laboratory.

Table 1 Comparison of the assay results for clinical serum specimens by using the electrochemical immunosensor herein and the referenced ELISA method
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Conclusions

In summary, this contribution devises a novel multiplexed electrochemical immunoassay for simultaneous detection of alpha-fetoprotein and carcinoembryonic antigen by using thionine and ferrocene as distinguishable signal tags on a one-spot immunosensor. The assay was performed by using one-step immunoreaction between the immobilized antibodies and the analytes. Compared with conventional sandwich-type or competitive-type immunoassays, the developed immunoassay can shorten the assay time, reduce the incubation times, and decrease the assay cost. Importantly, the preparation of the immunosensors is very simple, and does not require sophisticated fabrication. Just for the reason, the reproducibility and precision of the electrochemical immunosensors are pretty good. Although the linear range is relatively narrow, it completely meets the requirement of clinical diagnosis.