Label-Free and Sensitive Determination of Cadmium Ions Using a Ti-Modified Co₃O₄-Based Electrochemical Aptasensor

Abstract

The current work demonstrates an electrochemical aptasensor for sensitive determination of Cd²⁺ based on the Ti-modified Co₃O₄ nanoparticles. In this unlabeled system, Ti-modified Co₃O₄ nanoparticles act as current signal amplifiers modified on the screen-printed carbon electrode (SPCE) surface, while the derivative aptamer of Cd²⁺ works as a target recognizer. In addition, the sensing is based on the increase in electrochemical probe thionine current signal due to the binding of aptamer to Cd²⁺ via specific recognition. In the current study, key parameters, including aptamer concentration, pH, and incubation time were optimized, respectively, to ensure sensing performance. Cyclic voltammetry was used not only to characterize each preparation and optimization step, but also to profile the bindings of aptamer to Cd²⁺. Under optimal conditions, Cd²⁺ can be determined in a linear range of 0.20 to 15 ng/mL, with a detection limit of 0.49 ng/mL, significantly below the maximum concentration limit set by the U.S. Environmental Protection Agency. Based on comparative analysis and the results of recovery test with real samples, this simple, label-free but highly selective method has considerable potential and thus can be used as an in-situ environmental monitoring platform for Cd²⁺ testing.

1. Introduction

Cadmium (Cd²⁺) is a bioaccumulative, non-biodegradable and highly toxic heavy metal, which causes a variety of human health disorders [1,2,3]. To minimize the associated potential health risks, the maximum concentration in drinking water for Cd²⁺ is set at 5 ng/mL, as defined by the U.S. Environmental Protection Agency (EPA). Likewise, due to the high concern for determination and monitoring of Cd²⁺, a range of highly sensitive conventional techniques have been used, including electrothermal atomization atomic absorption spectroscopy (ET-AAS) [4,5], graphite furnace atomic absorption spectrometry (GFAAS) [6,7] and inductively coupled plasma mass spectroscopy (ICP-MS) [8,9,10]. All these conventional techniques are sensitive and accurate, but there are some limitations, such as laborious, time-consuming operation, and limited applicability for in-situ determination. In this regard, it is of paramount importance to develop a simple, convenient, and robust method for Cd²⁺ determination.

In recent decades, there has been a high level of attention for single-stranded oligonucleotide—aptamer since they were selected for the first time through the systematic evolution of ligands by exponential enrichment (SELEX) system [11,12]. Additionally, due to its high specificity, compatibility, environment-friendly properties, reliability and ease of synthesis and modification, aptamer opens up novel insights into both qualitative and quantitative determination of organic and inorganic targets such as pesticides [13,14], proteases [15], toxins [16,17], antibiotics [18], and heavy metals [19,20,21,22]. For instance, heavy metals including Pb²⁺, Hg²⁺, and Ag⁺ can be sensed and quantified through specific dimensional structures of aptamer/target complex as “G-quadruplex”, “T-Hg²⁺-T”, or “C-Ag⁺-C”, respectively [23,24,25]. Currently, two different aptamers of Cd²⁺ have been respectively selected by two independent groups [26,27]. Then, colorimetric [28] and fluorescent methods [26,29] were exploited to determine Cd²⁺. As for electrochemical methods, they are sensitive, and some portable devices are already commercial and available, including pocketSTAT2 (Ivium Technologies, Netherlands), PalmSens4 (Palmsens, Netherlands), μStat-i 400 (Metrohm, Switzerland) and CHI1200C (CH Instruments, China). In particular, electrochemical biosensors benefit a lot from screen-printed carbon electrode (SPCE), due to disposability, low cost, portability, customized fabrication and mass production [30,31]. They have considerable potential for environmental monitoring of targets in field. However, there is very limited data available in the literature regarding electrochemical biosensors based on SPCE and unlabeled aptamer for Cd²⁺ determination.

Generally, there are two essential indicators for a robust aptasensor, i.e., selectivity and sensitivity. The former one is basically ensured by high affinity of aptamer. As far as sensitivity is concerned, it is usual to have electrodes modify with functional nanomaterials. Their large surface area and unique nanostructure could boost electrons transfer rate, amplify electrical response, and enhance performance of biosensors [32,33]. Co₃O₄, a transition metal oxide with attractive electrocatalytic, electronic, and electrochemical properties, has been widely used in a wide range of applications [34]. In addition, most of the Co₃O₄-based nanocomposites developed to date have shown great potential in applications of Li-ion batteries [35], supercapacitor [36], electrocatalysts [37], and electrochemical sensors [38]. It has also been reported that their electron transfer and other electrochemical properties can be further promoted through modification with other transition metal oxides [35,39]. Anatase TiO₂, on the other hand, is the most widely explored functional metal oxide [40], which has demonstrated importance in many electrochemical applications due to its large specific surface area and uniform nanopore distribution [41,42]. Its nanohybrids have also presented great advantages in electrical biosensors with high performance [43]. However, the synergetic effect of Ti-modified Co₃O₄ on electrochemical aptasensor was insufficiently investigated.

Based on the above, we have proposed a label-free electrochemical aptasensor for highly selective as well as sensitive determination of Cd²⁺, which consists of the derived aptamer and Ti-modified Co₃O₄ as a recognizer and a signal amplifier, respectively. The electrochemical properties and selectivity of the aptasensor are characterized under optimal conditions. Furthermore, aptamer-based methods and a recovery test with environmental samples will be compared and performed, respectively, to assess its practical feasibility at field level. Scheme 1 shows the architecture and signaling mechanism of the label-free electrochemical aptasensor.

2. Materials and Methods

2.1. Chemicals and Reagents

Ti-modified Co₃O₄ was synthesized as per the following steps. Briefly, Co(NO₃)₂·6H₂O and Ti[O(CH₂)₃CH₃]₄ were dissolved in 80 mL of ultrapure water and 20 mL ethanol, respectively. Then, they were mixed with different molar ratios of Co:Ti (1:1 and 2:1) by stirring continuously for 3 h. Afterward, 10 mL of ammonia water was added dropwise and continuously stirred for 2 h. The resulting precipitates were finally rinsed three times with ultrapure water and separated using suction filtration followed by 10 h drying at 90 °C and 3 h calcination at 500 °C. Co₃O₄ was also prepared from Co(NO₃)₂·6H₂O by following the same procedures with the omission of Ti[O(CH₂)₃CH₃]₄. According to the initial molar ratios of Co:Ti (1:1 and 2:1) mixed, samples obtained were marked as Co₁Ti₁ and Co₂Ti₁, respectively. [44]. The cadmium aptamer sequence originated from its parent [27] by subtly adding 5 nucleotides at 3′: 5′-GGA CTG TTG TGG TAT TAT TTT TGG TTG TGC AGT CC-3′, which was synthesized by Sangon Biotech Co. Standard solution of Cd²⁺ (1000 mg/L) was purchased from Merck Co., Inc. (Darmstadt, Germany). Thionine was purchased from Sinopharm Chemical Reagent Co. (Beijing, China). The detection buffer, a 0.1 M HAc-NaAc solution (containing 1.0 mM thionine), consisted of different volumes of thionine, sodium acetate and glacial acetic acid. Ammonia water was purchased from Shanghai Lingfeng Reagent Co. (Shanghai, China). All other reagents purchased from Sigma Aldrich (St. Louis, MO, USA) were used (in the current study) without further purification.

2.2. Apparatus

CHI1030A Electrochemical Workstation (CH Instruments, Shanghai, China) was used for electrochemical characterizations with an SPCE system (Jiangsu Rongbin Biotech Co., Nanjing, China), which contained (i) a carbon auxiliary electrode, (ii) Ag/AgCl reference electrode and (iii) a 2.0 mm diameter working electrode, respectively. All the ultrapure water utilized in this work was prepared by a Millipore-MilliQ system (Millipore Inc., Bedford, MA, USA). Scanning electron microscope (SEM, NOVA NanoSEM 230, Hillsboro, OR, USA), transmission electron microscope (TEM, TALOS F200X, Thermo Fisher Scientific, Waltham, MA, USA) and energy dispersive spectroscopy (EDS, TALOS F200X, Thermo Fisher Scientific, USA) were employed for characterization of as-prepared Co₃O₄ and Ti-modified Co₃O₄. X-ray diffraction patterns (XRD) were conducted on a D8 Advance Da Vinci Poly-functional X-Ray Diffractometer (Bruker Corporation, Billerica, MA, USA).

2.3. Aptasensor Fabrication Procedures

For drop-coating evenly on a bare electrode, the homodisperse of nanoparticles in water was required. Thus, only Co₂Ti₁ was adequate and then used for the aptasensor fabrication. 5.0 mg Co₂Ti₁ were added into 5.0 mL of ultrapure water and then ultrasonicated for 45 min. From this homogeneous dispersion mixture, 2.5 μL was drop-coated on the working electrode, followed by drying at room temperature for 1~2 h. Afterward, 2.5 μL of 2.0 μM Cd²⁺ aptamer was evenly introduced on the surface of the resulting electrode and dried for another 1~2 h to form a sensing interface, which was subsequently rinsed with ultrapure water to remove unbound ssDNA. Eventually, the aptasensor for Cd²⁺ determination was prepared after drying.

2.4. Electrochemical Measurements

The electrochemical measurements were performed by cyclic voltammetry (CV) in detection buffer (pH 5.5), which was swept from −0.65 to 0.00 V with a scan rate of 100 mV/s. Each measurement was repeated at least three times.

2.5. Determination of Cd²⁺ in Environmental Samples

Freshwater samples from a tributary of the Huangpu River and tap water at Minhang campus at Shanghai Jiao Tong University were collected to assess the reliability and operational capability of the proposed aptasensor through a recovery test. These samples were filtered through 0.22 µm membranes after adding various concentrations of Cd²⁺, followed by target determination via the proposed apparatus and procedure. Meanwhile, a standard approach, AAS, was employed as a contrast. Each measurement was repeated at least three times.

3. Results and Discussion

3.1. Characterization of Co₃O₄ and Co₂Ti₁

The microscopic morphologies of Co₃O₄ and Co₂Ti₁ were respectively characterized by SEM, TEM, EDS, and XRD. As presented in Figure 1a,b, pure Co₃O₄ was up to 50 nm in size, while the size of Co₂Ti₁ varied from 10 to 20 nm after the incorporation of Ti (Figure 1c,d). As a consequence, the specific surface area enlarged remarkably, boosting both loading capacity and conductivity. The XRD patterns of Co₃O₄ and Co₂Ti₁ were also demonstrated in Figure 2a. It was easy to observe that the diffraction peaks of Co₂Ti₁ were weaker than those of pure Co₃O₄, indicating that the size of Co₃O₄ decreased obviously after the incorporation of Ti. Meanwhile, several weak peaks appeared in the diffraction pattern of Co₂Ti₁. Those peaks located at 27.4°, 32.7°, 48.9°, 63.6°, and 35.4°, 62.0° could be respectively assigned to CoTiO₃ and Co₂TiO₄ [45,46], suggesting the formation of Co-O-Ti solid solutions. Finally, as shown in the images of EDS elemental mapping (Figure 2b,c), these three elements were distributed homogeneously. However, the content of Ti in Co₂Ti₁ was lower than that of Co and O (Figure 2c), which might be ascribed to the initial molar ratio of Co:Ti mixed in preparation.

3.2. Electrochemical Characterization of the Aptasensor

Cyclic voltammetry is a convenient and effective technique for the characterization of modified electrode. Therefore, the behaviors of the electrochemical probe after each assembly procedure were studied by this technique. As shown in line a of Figure 3, the thionine presented an insignificant response on bare electrode because of poor conductivity. However, after the Co₂Ti₁ modification, the peak currents increased significantly (line b of Figure 3), due to its impact on specific surface area expansion and electron-transfer promotion. As a result, the sensitivity changed positively together with the detection limit of the aptasensor. The peak current increased slightly since the introduction of the aptamer (line c of Figure 3), indicating that it was successfully immobilized on working electrode. The possible reason for the current difference may be ascribed to the weak electrostatic attraction between the positively charged thionine and the negatively charged phosphate groups of the DNA backbone [47,48], resulting in its adsorption along with adhesion of aptamer to electrode, thus gently facilitating electron-transfer. Finally, after incubating the aptasensor with Cd²⁺ (line d of Figure 3), a significant peak current was observed, indicating the formation of Cd²⁺-aptamer complex through specific recognition. In short, a weak current signal could only be observed in absence of Cd²⁺, while a strong electrochemical signal could be detected in the presence of Cd²⁺ due to the Cd²⁺-aptamer complex, thereby confirming that the aptasensor was successfully prepared.

3.3. Optimization of Experimental Conditions

In addition to a profiling technique, CV also served as a sensitive electrochemical analysis method. In this regard, the experimental conditions including aptamer concentration, pH value and incubation time were optimized to achieve optimal performance. Respective data about the optimization results are presented in the Supplementary Material (Figure S1). In brief, the optimal conditions obtained were: (a) aptamer concentration: 2.0 μM; (b) pH value: 5.5; (c) incubation time: 40 min.

3.4. Analytical Performance

The analytical performance of the aptasensor was evaluated under the aforementioned optimal experimental conditions, where a series of Cd²⁺ determinations were performed to estimate the sensitivity. The ΔI value (variation of peak current signal before and after the introduction of Cd²⁺ to electrode) decreased as increasing concentration of Cd²⁺, which also presented a good linearity from 0.20 to 15 ng/mL (Figure 4b). The linear regression equations determined were:

∆I = −0.1105C + 3.8092 (R² = 0.99)

(1)

(C: the concentration of Cd²⁺; R²: correlation coefficient).

The limit of detection (LOD) was estimated as 0.49 ng/mL (3σ/slope), which is significantly lower than the maximum concentration limit set by EPA. Moreover, on the basis of the comparative analysis of aptamer-based approaches (

Table 1. Comparison of different aptamer-based approaches for Cd²⁺ determination.

Approaches	Linear Range (ng/mL)	LOD (ng/mL)	Reference
fluorometry	0.00~112.41	4.50	[26]
	100.00~10,000.00	0.04	[29]
colorimetry	1.00~400.00	1.00	[28]
	1.12~44.96	0.52	[27]
electrochemistry	28.10~112.41	10.34	[49]
	0.20~15.00	0.49	This work

), this aptasensor is more sensitive than most of the others. It is noteworthy that the LOD of this method is about 21-fold lower than that of another electrochemical aptasensor, demonstrating its high sensitivity. Although a fluorescent aptasensor has a significantly lower LOD, it is unable to quantify Cd²⁺ below 100 ng/mL, to which the current electrochemical aptasensor is well complementary.

3.5. Selectivity and Reproducibility

The selectivity performance of the current method was determined by recording current signal variations (ΔI) of potential interfering ions in the absence and presence of Cd²⁺. The concentrations of interferences (e.g., Pb²⁺, Cu²⁺, Mn²⁺, Ag⁺, Zn²⁺, Fe²⁺, As(III), As(V) and Hg²⁺) were 50.0 ng/mL, while Cd²⁺ concentration was 2.0 ng/mL. As shown in Figure 5, the ΔI values of sensing systems containing a higher concentration of interfering ions were significantly less than that added with Cd²⁺. Nevertheless, those recovered sharply after the introduction of Cd²⁺ to the sensing system containing interfering ions. These results enabled confirmation that the interfering ions could negligibly affect Cd²⁺ determination, even if their concentrations were 25 times higher than that of Cd²⁺, which confirmed excellent selectivity of the proposed aptasensor for Cd²⁺ determination.

Furthermore, according to possible secondary structures predicted preliminarily by UNAfold (https://sg.idtdna.com/UNAfold/) (Figure S2), the subtle addition with 5 nucleotides at 3′ of this aptamer successfully induced its preferential configuration of a stem-loop structure (Figure S2a) from a random coil sequence (Figure S2b) via conformational switching. Meanwhile, Cd²⁺ was incorporated and buried with the binding pocket of this structure, underlying its high sensitivity and selectivity. As structures of “G-quadruplex” and “T-Hg²⁺-T” have been sufficiently studied, we are able to develop various aptasensors for Pb²⁺ and Hg²⁺ detection. Unfortunately, the mechanism of how aptamer bind Cd²⁺ is not yet fully understood. To elucidate the fundamental and further development of Cd²⁺ aptasensor, a lot of effort is still needed.

Under the same conditions, reproducibility was also examined at a Cd²⁺ concentration of 2.0 ng/mL using 10 identically prepared SPCEs. The obtained results of the relative standard deviation (RSD) of 4.37% indicated that the reproducibility of this aptasensor was quite acceptable.

3.6. Application in Environmental Samples

As demonstrated in the selectivity investigation previously, the proposed aptasensor presented strong anti-interference capability against heavy metal ions. However, environmental samples were more complicated, in which various species of interferences might exist, e.g., microorganisms, biometabolites, and antibiotics. To assess the applicability of a new method to those complicated samples, a recovery test was widely adopted and accepted. Therefore, the recovery test with real samples was performed to analyze the validity of the current method, while AAS was employed as a contrast.

Table 2. Determination of Cd²⁺ in environmental samples (n = 3).

Sample	Background (ng/mL)	Spiked (ng/mL)	Proposed Electrochemical Aptasensor			AAS
			Found (ng/mL)	Recovery (%)	RSD (%)	Found (ng/mL)	Recovery (%)	RSD (%)
River water	0.02	3.00	3.18	105.24	8.17	3.09	102.17	0.66
		6.00	6.12	101.73	5.75	5.99	99.48	0.46
		12.00	13.03	108.43	6.11	12.75	106.03	1.50
Tap water	0.07	3.00	3.03	98.71	8.24	3.20	104.22	0.18
		6.00	6.22	102.43	7.43	6.29	103.69	0.82
		12.00	13.27	109.95	7.64	12.82	106.19	2.48

exhibited the mean recoveries of the Cd²⁺ and RSD values in two Cd²⁺ spiked samples obtained with the proposed aptasensor and AAS. The amounts of Cd²⁺ added were set according to the EPA limit (5 ng/mL). As shown in Table 2, the recoveries of this method ranged from 98.71% to 109.95% with RSD values of 5.75% to 8.24%. In consideration of its simplicity, convenience, analytical performance and application scenarios, these comparable recoveries with AAS were well acceptable. All of the above results substantiated its practicability and promising future for in-situ monitoring of Cd²⁺ in field.

4. Conclusions

In conclusion, the effectiveness of a robust electrochemical aptasensor based on the derivative aptamer and Ti-modified Co₃O₄ was summarized, which was designed for highly sensitive and selective determination of Cd²⁺ in environmental samples. The basic principle for Cd²⁺ determination underlies an increase in the current signal of electrochemical probe thionine due to the formation of Cd²⁺-aptamer complex through specific recognition. The detection limit obtained (0.49 ng/mL) is significantly lower than the limit set by the U.S. EPA, indicating its possible use as an environmental monitoring platform for Cd²⁺. Although the system possesses merits as aforementioned, there are still some limitations existing. For example, only one target can be detected in a measurement. Extra and tedious pretreatment is also needed for complex samples. Its availability can be improved if either simultaneous measurements of multiple heavy metal ions or integration with auto-pretreatment into a portable device is realized. Furthermore, the proposed aptasensor was based on an SPCE system, which meant each channel was one-off. The capability of being regenerated should be taken into serious consideration along with aptasensor development. Therefore, our future work will be carried out to address the above issues.