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Ultrasensitive electrochemical DNA biosensor by exploiting hematin as efficient biomimetic catalyst toward in situ metallization
Biosensors and Bioelectronics 63 (2015) 269–275
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Biosensors and Bioelectronics journal homepage: www.elsevier.com/locate/bios
Ultrasensitive electrochemical DNA biosensor by exploiting hematin as efficient biomimetic catalyst toward in situ metallization Qiong Hu a, Weiwen Hu a, Jinming Kong a,n, Xueji Zhang a,b,nn a b
School of Environmental and Biological Engineering, Nanjing University of Science & Technology, Nanjing 210094, PR China Chemistry Department, College of Arts and Sciences, University of South Florida, East Fowler Ave, Tampa, FL 33620-4202, United States
art ic l e i nf o
a b s t r a c t
Article history: Received 16 May 2014 Received in revised form 7 July 2014 Accepted 12 July 2014 Available online 22 July 2014
In this work, we presented a novel signal amplification approach to construct an electrochemical DNA biosensor for the ultrasensitive determination of sequence-specific DNA by exploiting hematin as biomimetic catalyst toward in situ metallization. Briefly, thiolated peptide nucleic acid (PNA) probes were firstly immobilized onto gold electrode through the formation of self-assembled monolayer (SAM) and then hybridization was accomplished in the ensuing step. After that, hematin molecules were introduced to the hybridized PNA/DNA heteroduplexes by employing phosphate–zirconium–carboxylate coordination chemistry. Next, the attached hematin molecules acted as catalyst in accelerating the reduction of silver ions in the presence of catechol, leading to the in situ deposition of silver particles onto the electrode. Finally, the deposited silver particles were electrochemically stripped into KCl solution and measured by square wave voltammetry (SWV). Under optimal conditions, the hematin-based electrochemical DNA biosensor presented a good linear relationship between the stripping peak currents and logarithm of single-stranded DNA (ssDNA) concentrations in the range from 0.1 fM to 0.1 nM with a low detection limit of 62.41 aM, and it rendered satisfactory analytical performance for the determination of ssDNA in serum samples. Furthermore, it exhibited good reproducibility and stability, meanwhile, it also showed excellent specificity toward single-nucleotide polymorphism (SNP). Therefore, the hematinbased signal amplification approach has great potential in clinical applications and is also suitable for quantification of biomarkers at ultralow level. & Elsevier B.V. All rights reserved.
Keywords: Electrochemical DNA biosensor Hematin Biomimetic catalyst Metallization Metalloporphyrin Signal amplification
1. Introduction Porphyrins and metalloporphyrins are universally distributed in functional proteins, such as hemoproteins, cytochromes, peroxidases, etc., which are of critical importance for the fulfillment of various biological functions including respiration, photosynthesis, and other enzymatic reactions associated with metabolisms of living organisms (Smith, 1975; Shelnutt et al., 1998; Sen and Poon, 2011). Porphyrins, especially metalloporphyrins, are the essential cofactors for the conservation of versatile biological activities of these functional proteins, and metalloporphyrins are the actual catalytic centers of many existed redox enzymes like horseradish peroxidase (HRP), catalases, and methylreductases (Smith, 1975; Shelnutt et al., 1998). Although the immediate surroundings, especially the tightly conjugated axial coordination moieties as well as the specialized microenvironments created by the bulk n
Corresponding author. Corresponding author at: School of Environmental and Biological Engineering, Nanjing University of Science & Technology, Nanjing 210094, PR China. E-mail addresses: [email protected] (J. Kong), [email protected] (X. Zhang). nn
http://dx.doi.org/10.1016/j.bios.2014.07.034 0956-5663/& Elsevier B.V. All rights reserved.
proteins, have significant influence on regulating the catalytic activity of metalloporphyrins (Ringe and Petsko, 2008), the free metalloporphyrins in vitro can also independently exhibit excellent catalytic activity and distinct substrate selectivity under optimum conditions (Gross and Nimri, 1994; Rutkowska-Zbik and Witko, 2006; Shelnutt et al., 1998; Sen and Poon, 2011). Compared with their corresponding natural enzymes, metalloporphyrins are low molecular weight and show superior thermal stability along with higher pH tolerance, thus implicating a great potential in practical applications (Smith, 1975). Consequently, metalloporphyrins have now been extensively investigated as biomimetic catalysts in the fields involving analytical chemistry, life science, and chemical synthesis (Breslow et al., 1996; Biesaga et al., 2000; Imahori, 2004; Umile and Groves, 2011; Wertz et al., 1998; Watanabe et al., 2013). Recently, using various metalloporphyrins as efficient biomimetic catalysts has attracted considerable interests in the field of selective catalytic oxidation of diverse substrates (de Groot et al., 2005; Meunier, 1992; Monnereau et al., 2010; Nimri and Keinan, 1999; Wang et al., 2007). For example, hemin, a stable iron-containing porphyrin, has now been widely exploited as a highly active biomimetic catalyst toward the
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oxidation of pyrogallol, reduced nicotinamide adenine dinucleotide (NADH), and other metabolic intermediates (Golub et al., 2011; Xue et al., 2012; Yuan et al., 2012), and the obtained results revealed that both of the catalytic activity and substrate binding affinity were approximately equal to that of natural enzymes. Moreover, metalloporphyrins have also been extensively investigated as biomimetic catalysts for the highly sensitive determination of inorganic ions, e.g. Hg2 þ , Ge4 þ , and Cu2 þ , small molecules, e.g. ascorbic acid, dopamine, and hydrogen peroxide, and even biomacromolecules, e.g. DNA, RNA, and proteins (Grabowska et al., 2014; Pelossof et al., 2011; Wu et al., 2012; Wang et al., 2013; Xu et al., 2013; Zhang et al., 2013). In the past decade, there has been an increasing demand for the ultrasensitive determination of sequence-specific DNA, which is of great importance for molecular diagnostics, clinical medicine, forensic investigations, and environmental monitoring (Drummond et al., 2003; Hansen et al., 2006; Liu et al., 2013; Sassolas et al., 2008). Compared with conventional methods such as fluorescence spectrometry (Xiang et al., 2010), ultraviolet absorption spectrophotometry (Holden et al., 2009), and capillary electrophoresis (Fujii et al., 2010), electrochemical methods have drawn considerable attentions because of their high sensitivity, uncomplicated instrumentation, low cost, and good portability (Hansen et al., 2006; Liu et al., 2008; Sassolas et al., 2008). Considering the requirement of highly sensitive methods to accurately determine sequence-specific DNA at ultralow levels, intensive attentions are concentrated on the development of effective signal amplification approaches to improve the electrochemical response to a detectable level (Liu et al., 2008; Song et al., 2014). For example, given the outstanding catalytic activity of HRP, great efforts have been devoted to exploit it as an efficient biocatalyst in the procedures of signal amplification for the highly sensitive determination of low-abundant target analytes (Möller et al., 2005; Schüler et al., 2009). Nevertheless, the practical application of the well-established HRP-based signal amplification approach currently suffered from several inevitable drawbacks. For example, apart from the complicated preparation procedures to introduce HRP onto supporting substrates provided for the determination of target analytes, the high cost and unsatisfactory longterm stability of HRP in solution have largely inhibited its widespread application in analytical fields. Meanwhile, the relative large dimensions of HRP limit its maximum loading amount, which is unfavorable to signal amplification (Wang et al., 2013). Furthermore, the catalytic activity of a natural enzyme is highly dependent upon the protein conformation, leading to vulnerability to the pH and temperature of the external conditions (Song et al., 2010). Hematin, the hydroxylated derivative of the metalloporphyrin center of HRP (Akkara et al., 2000), has been confirmed to be a promising alternative to replace HRP for the polymerization, hydrogelation, or degradation of phenols, aromatic amines, and their derivatives because of its excellent peroxidatic activity in vitro (Akkara et al., 2000; Nagarajan et al., 2009; Pirillo et al., 2010; Sakai et al., 2010). Accordingly, from the view points of green chemistry, hematin shows great value as a possible environmentally friendly biomimetic catalyst for the synthesis or modification of versatile polymeric materials, together with the cost-effective disposal of pigment-rich wastewater discharged from textile industry, papermaking industry as well as printing industry. In addition, several attempts have been made to exploit hematin as a peroxidase substitute in the determination of hydrogen peroxide (Zhang and Dasgupta, 1992) as well as in the ultrasensitive electrical detection of nucleic acids in a device based sub-microgapped biosensor (Kong et al., 2008). Thus, the application of hematin as a substitute for HRP shows great applicability to circumvent the aforementioned drawbacks on account of its excellent thermal stability and predominant pH tolerance, while
its excellent peroxidatic activity like HRP is still preserved (Akkara et al., 2000; Carvalhal et al., 2005). Herein, for the first time, we presented a novel signal amplification approach to construct an electrochemical DNA biosensor for the ultrasensitive determination of sequence-specific DNA by exploiting hematin as efficient biomimetic catalyst toward in situ metallization. In this work, thiolated peptide nucleic acid (PNA) probes were firstly immobilized onto the surface of the pretreated gold electrode and subsequently used as the capture probes for the specific recognition of target ssDNA to be determined. After hybridization, hematin molecules were introduced to the hybridized PNA/DNA heteroduplexes by employing zirconium–phosphate and zirconium–carboxylate coordination interactions (Gao et al., 2007; Kong et al., 2008). Then, the attached hematin molecules acted as biomimetic catalyst in promoting the reduction and conversion of free silver ions into the deposited metallic silver. Owing to its excellent catalytic capability of hematin molecules in vitro, the electron exchange between silver ions and catechol could be significantly enhanced. Thus, the amount of the deposited silver particles on the electrode could be rapidly accumulated to a detectable level. Finally, the deposited silver particles were electrochemically stripped into KCl solution and measured by square wave voltammetry (SWV). Due to its significant signal amplification of the hematin-catalysed silver deposition and its favorable immunity to the elimination of background current of square wave voltammetric technique (Christie et al., 1977; O’Dea et al., 1981), the sensitivity of the proposed biosensor was improved with a remarkable level, resulting in ultrasensitive electrochemical DNA biosensing with a detection limit much lower than those previously reported (Liu et al., 2008; Lepage et al., 2011; Kong et al., 2008; Wang et al., 2011). Results also demonstrated that it exhibited excellent specificity in differentiating mismatched oligonucleotide fragments, suggesting great potential in the genotyping of single-nucleotide polymorphism (SNP).
2. Material and methods 2.1. Materials and reagents. Thiolated peptide nucleic acid with sequence 5′-HS-(CH2)11 -AAC CAT ACA ACC TAC TAC CTC A-3′ (PNA) were custom-made by Panagene Inc. (South Korea). Target complementary ssDNA 5′-TGA GGT AGT AGG TTG TAT GGT T-3′ (cDNA), single-base mismatched ssDNA (mismatch underlined) 5′-TGA GGT AGT AGG TTG TGT GGT T-3′ (SBM), three bases mismatched ssDNA (mismatches underlined) 5′-TGA GGT ATT AGA TTG TGT GGT T-3′ (TBM), and completely non-complementary ssDNA 5′-ACT TAC CTT TGC TCA TTG ACG A-3′ (Control) were all purchased from Invitrogen Biotechnology Co., Ltd. (Shanghai, China). Zirconium dichloride oxide octahydrate (ZrOCl2 8H2O), hematin, 6-mercapto-1-hexanol (MCH), and silver nitrate (AgNO3) were purchased from SigmaAldrich (St. Louis, MO). Catechol was purchased from Aladdin Industrial Inc. (Shanghai, China). Fetal bovine serum (FBS, Defined) was purchased from Shanghai YiJi Industrial Co., Ltd. (Shanghai, China). N,N-dimethylformamide (DMF) and all other chemical reagents were purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China) and used without further purification. Ultrapure water obtained from a Millipore Milli-Q water purification system (Z 18.25 MΩ) was used in all assays. Tris–EDTA buffer (TE, 10 mM Tris–HCl, 1 mM EDTA, pH ¼ 8.0) was prepared and used as the stocking solution and washing buffer for ssDNA, and various concentrations of ssDNA samples were prepared by serial dilution. NaAc/HAc buffers (0.1 M, pH ¼5.2, 5.4, 5.6, 5.8, 6.0, 6.2, 6.4) were prepared by mixing
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0.1 M NaAc solution and 0.1 M HAc solution and used as the buffers for the in situ deposition of metallic silver. 2.2. Apparatus All electrochemical experiments were performed on a CHI 760D electrochemical workstation (Chenhua, Shanghai, China) with the conventional three-electrode system consisted of modified gold electrode (Φ¼2 mm) as working electrode, saturated calomel electrode (SCE) as reference electrode and platinum wire as auxiliary electrode. The scanning electron microscopy (SEM) images were recorded with a Quanta FEG 250 field emission scanning electron microscopy (FEI, USA) at an acceleration voltage of 30.0 kV. 2.3. Pretreatment of the gold electrode and immobilization of capture probes Prior to modification, the gold electrode was mechanically polished to a mirror-like surface with 1.0, 0.3 and 0.05 μm alumina slurry, and then successively washed with absolute ethanol and ultrapure water under ultrasonication. Then, it was chemically cleaned in freshly prepared piranha solution (a mixture of 98% H2SO4 and 30% H2O2, 3:1 v/v, CAUTION: piranha solution is strongly corrosive and must be prepared and handled with extreme care) under ultrasonication. Afterwards, it was electrochemically cleaned in a fresh 0.5 M H2SO4 solution by cycling the electrode potential between 0.2 V and 1.5 V with a scan rate of 100 mV/s until a reproducible cyclic voltammogram was achieved to remove any remaining impurities. Finally, it was rinsed thoroughly with ultrapure water and dried with nitrogen prior to modification. Immobilization of capture probes was accomplished by incubating the clean electrode in 1 μM PNA aqueous solution to allow the formation of SAM. After washing with ultrapure water, it was immersed in 2 mM MCH solution (dissolved in 70% ethanol solution) to block the nonspecific binding sites, and then successively rinsed with 70% ethanol solution and ultrapure water to remove excessive MCH. 2.4. DNA hybridization and in situ metallization Hybridization was performed in 10 μL of TE buffer that contained certain concentration of ssDNA at 37 °C for 1.5 h, followed by washing with TE buffer to remove the unhybridized ssDNA. After incubating in 5 mM ZrOCl2 8H2O solution (dissolved in 60% ethanol solution) for 1.0 h, 20 μL of freshly prepared hematin solution (dissolved in DMF) was added onto the electrode and kept for 1.0 h. After that, the electrode was moderately rinsed with DMF and ultrapure water to remove the excessive hematin molecules. 30 μL of freshly prepared mixed solution of AgNO3 and catechol with the stoichiometric ratio of 2:1 (both freshly dissolved in 0.1 M NaAc/HAc buffer) was subsequently added on the electrode and then incubated at 25 °C for 10 min for the deposition of metallic silver. Finally, the resultant electrode was moderately washed with ultrapure water to remove the remaining reactants.
Scheme 1. Schematic illustration of the preparation of the proposed electrochemical DNA biosensor based on hematin-induced in situ metallization and square wave voltammetric technique.
3. Results and discussion 3.1. Electrochemical characterization of the attached hematin molecules As zirconium ions and hafnium ions are able to form stable conjugates with free carboxyl groups and phosphate groups through the robust coordination interactions (Buscher et al., 1996; Li et al., 2010), thus they are applicable to be utilized as simple, cost-effective, and versatile linkers for the implementation of various desired functions. The hematin molecule contains two free carboxyl groups, consequently, it can be firmly conjugated to the deoxyribose phosphate backbone of DNA via the robust zirconium–phosphate and zirconium–carboxylate coordination interactions (Kong et al., 2008). In this work, zirconium ions were adopted as the coupling linkers to introduce free hematin molecules to the hybridized PNA/DNA heteroduplexes. To confirm hematin molecules have been successfully attached to the hybridized heteroduplexes, linear sweep voltammetry (LSV) was used to characterize the existence of hematin molecules on the electrode. As shown in Fig. 1, an apparent reduction peak was visible at the potential around 0.683 V, and it could be attributed to the electrochemical reduction of the conjugated Fe(III) into Fe(II). The peak current increased obviously with the increasing concentration of hematin, which in turn demonstrated that
2.5. Electrochemical measurement To carry out the electrochemical measurement, the gold electrode was finally immersed in 10 mL of 1.0 M KCl solution, and then SWV was performed under the potential from 0.15 V to 0.25 V to record the stripping current of the deposited metallic silver. The preparation process of the proposed hematin-based electrochemical DNA biosensor and the subsequent electrochemical measurement are illustrated in Scheme 1.
Fig. 1. Linear sweep voltammograms of the attached hematin molecules toward 0.1 pM cDNA in 1.0 M KNO3.
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hematin molecules had been successfully attached to the hybridized heteroduplexes via the robust phosphate–zirconium–carboxylate coordination interaction.
3.2. Characterization of the catalytic activity of hematin toward the deposition of metallic silver In the presence of catechol as the reductant, the free silver ions in solution can be reduced into metallic silver and subsequently deposit onto the electrode in the form of silver particles, while the catechol molecules are oxidized into benzoquinones, and each catechol molecule could react with two silver ions. Inspired by its excellent peroxidase-like activity in vitro, herein hematin was exploited as the biomimetic catalyst in catalyzing the reduction of silver ions into metallic silver in the electrochemical determination sequence-specific DNA. Theoretically, the amount of silver deposits aggregated on the electrode was proportionally correlated with the concentration of target ssDNA in solution, thus providing a quantitative measurement. To evaluate the catalytic activity of the attached hematin molecules in enhancing the electron exchange between silver ions and catechol molecules, the size and morphology changes of the deposited silver particles resulting from the catalysis of hematin molecules were characterized by SEM. The size and morphology of the naturally deposited silver particles, namely, without the catalysis of hematin, are nonuniform and disorganized, and they are sparsely distributing on the electrode (Fig. 2A). However, the distribution density of the deposited silver particles on the electrode would increase remarkably, meanwhile they would become more uniform at the average particle diameter as well as the particle morphology under the condition that hematin molecules have been previously introduced to the hybridized heteroduplexes (Fig. 2B). The results demonstrated that hematin could significantly promote the in situ deposition of metallic silver, and thus can be exploited as efficient biomimetic catalyst for the amplification of hybridization signal.
3.3. Optimization of detection conditions of the proposed electrochemical DNA biosensor The deposited metallic silver on the electrode could be easily stripped into KCl solution and accurately measured by square wave voltammetric technique, and the resulting voltammogram
showed a well-defined stripping sharp peak due to the favorable Ag/AgCl solid-state voltammetric process (Ting et al., 2009). In addition, SWV exhibits favorable immunity to the elimination of background current (Christie et al., 1977; O’Dea et al., 1981), thus it is a preferable electrochemical technique in the highly sensitive determination of low-abundant electroactive species. The representative stripping potential of silver deposits on the electrode consistently located at 0 V (vs. SCE), which was in agreement with the obtained value reported previously (Ting et al., 2009). Thus, the electrochemical stripping analysis based on SWV was favorable for the achievement of high detection precision. To achieve the best analytical performance of the proposed biosensor, several experimental parameters associated with the deposition rate of silver particles were investigated. Except for the pH value of the external conditions and the concentration of AgNO3, the concentration of hematin was also inspected. The amount of the in situ deposited metallic silver largely depended upon the pH value of the external conditions, as it could significantly affect the catalytic activity of the attached hematin molecules. Therefore, the effect of pH on stripping current was firstly investigated. As shown in Fig. 3A, after silver deposition for 10 min in 0.1 M NaAc/HAc buffer containing 1.2 mM AgNO3 and 0.6 mM catechol, the stripping peak current increased drastically with the pH varying from 5.2 to 5.8, and then it encountered a remarkable decrease with the further increasing of pH. The obtained result demonstrated that the hematin molecules showed maximum catalytic activity at pH 5.8 toward the reduction of silver ions into metallic silver. As a result, the 0.1 M NaAc/HAc buffer with pH 5.8 was adopted for the hematin-induced in situ metallization in the subsequent experiments. The effect of AgNO3 concentration on the stripping current was further investigated. After introducing hematin molecules (hematin concentration at 500 μM was applied) to the hybridized heteroduplexes, the modified electrode was incubated for 10 min in 0.1 M NaAc/HAc buffer (pH ¼ 5.8) containing various concentration of AgNO3 and catechol with their stoichiometric ratio kept at 2:1. As shown in Fig. 3B, the stripping peak current increased significantly with the AgNO3 concentration varying from 0.6 mM to 1.2 mM, afterwards, it increased slightly and even reached a plateau after 1.4 mM. As the hematin concentration applied here was high enough to maximize its loading amount, thus the concentration of AgNO3 at 1.4 mM could be regarded as the saturated concentration for the silver deposition. Taking this into account, the optimum concentration of AgNO3 was 1.4 mM. Obviously, the concentration of hematin was another important parameter that would significantly affect the deposition rate of
Fig. 2. SEM images of the in situ deposited silver particles on the electrode. Conditions: cDNA: 0.1 pM; pH: 6.0; Hematin: 200 μM; Deposition time: 10 min; AgNO3: 1.0 mM; Catechol: 0.5 mM. Scale bar: 5 μM.
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Fig. 3. Effects of pH (A), AgNO3 concentration (B), and hematin concentration (C) on the analytical performance of the proposed electrochemical DNA biosensor in 1.0 M KCl toward 0.1 pM cDNA.
metallic silver, namely, the response performance of the proposed biosensor could be further optimized by choosing a suitable hematin concentration. Meanwhile, the deposition time could be shortened accordingly. As shown in Fig. 3C, the stripping peak
current increased rapidly with the increasing of hematin concentration until 250 μM and then it trended to a plateau until 300 μM. From the result mentioned above, it could be concluded that the optimum concentration of hematin was 300 μM.
Fig. 4. Square wave voltammograms (A), and calibration plot (B) of the proposed electrochemical DNA biosensor toward varying concentrations of cDNA.
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Fig. 5. SWV responses of the proposed electrochemical DNA biosensor toward 0.1 pM cDNA (A), SBM (B), TBM (C), and Control (D).
3.4. Analytical performance of the proposed electrochemical DNA biosensor Under the optimal conditions, the sensitivity and dynamic range of the proposed electrochemical DNA biosensor were determined with varying cDNA concentrations. As shown in Fig. 4, the stripping peak currents increased with the increasing concentration of cDNA. The calibration plot showed a good linear relationship between the stripping peak currents and the logarithm of the concentrations of cDNA in the range from 0.1 fM to 0.1 nM. The linear regression equation was I (μA)¼ 1101.364 þ52.868 lg [CDNA/M] with a correlation coefficient of 0.9986. The limit of detection at a signal-to-noise ratio of 3 was calculated to be 62.41 aM, which was much lower than those reported previously (Liu et al., 2008; Lepage et al., 2011; Kong et al., 2008; Wang et al., 2011). The improvement of sensitivity and the wide dynamic range of the biosensor might be attributed to the significant signal amplification based on exploiting hematin as the efficient biomimetic catalyst and the favorable immunity to the elimination of background current of SWV as well as the predominant selectivity of PNA. Compared with the HRP-based signal amplification approach, the proposed approach had distinctive advantages of low cost, easy operation, and favorable stability. The wide linear range over 7 orders of magnitude was also very important for clinical applications. 3.5. Specificity, repeatability, reproducibility, and stability of the proposed electrochemical DNA biosensor The specificity was investigated by using three types of ssDNA including SBM, TBM, and Control. As shown in Fig. 5, the stripping peak currents from SBM (B), TBM (C), and Control (D) were approximately 86.4%, 72.1%, and 20.1% of the value obtained from cDNA (A), respectively. The obvious difference could be attributed to the hybridized efficiency of the four types of ssDNA and the predominant selectivity of PNA, as the cDNA could effectively and robustly bind to the complementary PNA while the others can't. In addition, the strong background current could be ascribed to the spontaneous redox process, which was inevitable because the silver ions could be naturally reduced into metallic silver in the presence of catechol acted as the reductant. The obtained results demonstrated that the novel biosensor described here could effectively differentiate complementary and mismatched oligonucleotide fragments, indicating that it was highly specific and even showed great potential for the genotyping of SNP. The excellent
Fig. 6. SWV responses of the proposed electrochemical DNA biosensor toward 0.1 pM cDNA in TE buffer (A), 5% serum sample (B), and 1% serum sample (C).
specificity could be attributed to the use of the highly specific PNA as the capture probes. The repeatability and reproducibility were evaluated by the relative standard deviation (RSD) of intra- and inter-assays (n ¼8). The intra-assays were conducted by measuring two samples containing 0.1 pM and 10 pM cDNA. Each sample was repeatedly measured 8 times using 8 parallel prepared biosensors, and the resultant RSD values were 3.7% and 3.3%, respectively. The interassays were conducted under the same conditions, and the obtained RSD values were 4.3% and 4.1%, respectively. The results indicated that it had acceptable repeatability and reproducibility. In addition, the modified electrode could be stored in a moisture-saturated environment at 4 °C, and over 95% of the initial response remained after a storage period of four weeks. The results demonstrated that it possessed acceptable stability. 3.6. Application in analysis of serum samples To evaluate the analytical reliability and application potential of the proposed electrochemical DNA biosensor, the interference effect of complex serum components on analytical performance was investigated. The stripping peak currents originated from 0.1 pM cDNA in serum samples were compared with the reference value obtained from 0.1 pM cDNA in TE buffer. The assay results from 8 repeated experiments were displayed in Fig. 6, the stripping peak currents from 5% (B) and 1% (C) serum samples were approximately 88.61% and 92.60% of that from TE buffer (A), respectively. In addition, the stripping currents attributed to the naturally deposited metallic silver, namely, without the catalysis of hematin, were almost kept at the same level. All these obtained results highlighted that the proposed electrochemical DNA biosensor showed acceptable analytical reliability and application potential for clinical applications, allowing for its good detectable capability in complex systems such as samples in serum.
4. Conclusion In summary, an ultrasensitive electrochemical DNA biosensor based on exploiting hematin molecules as efficient biomimetic catalyst toward in situ metallization was developed. To our knowledge, we are the first one to exploit hematin-induced in situ metallization into the construction of electrochemical DNA biosensor. Using the thiolated PNA as the immobilized capture probes
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significantly improved the selectivity, leading to the great potential in the genotyping of SNP. The application of the robust phosphate–zirconium–carboxylate coordination chemistry to introduce the cost-effective hematin molecules to the hybridized PNA/DNA heteroduplexes was convenient and feasible. More importantly, the integration of hematin-based signal amplification approach and square wave voltammetric technique could synergistically improve the sensitivity, enabled a low detection limit of 62.41 aM achieved under optimal conditions. In addition, it excluded complicated preparation procedures and could be successfully applied for the quantification of sequence-specific DNA in serum samples. In consideration of the outstanding analytical performance including wide linear range, low cost, satisfactory repeatability and reproducibility, acceptable stability and reliability, the developed biosensor showed great potential in clinical applications. Furthermore, it could integrate with standard microfabrication techniques to produce electrochemical sensor array and realize multiplexed detection capabilities, while the hematinbased signal amplification approach could be easily extended to detect other low-abundant biomarkers after appropriate modifications.
Acknowledgments We are grateful to Nanjing University of Science and Technology for its start-up funding, National Natural Science Foundation of China funding (No. 21345002) for this project.
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Biosensors and Bioelectronics journal homepage: www.elsevier.com/locate/bios
Ultrasensitive electrochemical DNA biosensor by exploiting hematin as efficient biomimetic catalyst toward in situ metallization Qiong Hu a, Weiwen Hu a, Jinming Kong a,n, Xueji Zhang a,b,nn a b
School of Environmental and Biological Engineering, Nanjing University of Science & Technology, Nanjing 210094, PR China Chemistry Department, College of Arts and Sciences, University of South Florida, East Fowler Ave, Tampa, FL 33620-4202, United States
art ic l e i nf o
a b s t r a c t
Article history: Received 16 May 2014 Received in revised form 7 July 2014 Accepted 12 July 2014 Available online 22 July 2014
In this work, we presented a novel signal amplification approach to construct an electrochemical DNA biosensor for the ultrasensitive determination of sequence-specific DNA by exploiting hematin as biomimetic catalyst toward in situ metallization. Briefly, thiolated peptide nucleic acid (PNA) probes were firstly immobilized onto gold electrode through the formation of self-assembled monolayer (SAM) and then hybridization was accomplished in the ensuing step. After that, hematin molecules were introduced to the hybridized PNA/DNA heteroduplexes by employing phosphate–zirconium–carboxylate coordination chemistry. Next, the attached hematin molecules acted as catalyst in accelerating the reduction of silver ions in the presence of catechol, leading to the in situ deposition of silver particles onto the electrode. Finally, the deposited silver particles were electrochemically stripped into KCl solution and measured by square wave voltammetry (SWV). Under optimal conditions, the hematin-based electrochemical DNA biosensor presented a good linear relationship between the stripping peak currents and logarithm of single-stranded DNA (ssDNA) concentrations in the range from 0.1 fM to 0.1 nM with a low detection limit of 62.41 aM, and it rendered satisfactory analytical performance for the determination of ssDNA in serum samples. Furthermore, it exhibited good reproducibility and stability, meanwhile, it also showed excellent specificity toward single-nucleotide polymorphism (SNP). Therefore, the hematinbased signal amplification approach has great potential in clinical applications and is also suitable for quantification of biomarkers at ultralow level. & Elsevier B.V. All rights reserved.
Keywords: Electrochemical DNA biosensor Hematin Biomimetic catalyst Metallization Metalloporphyrin Signal amplification
1. Introduction Porphyrins and metalloporphyrins are universally distributed in functional proteins, such as hemoproteins, cytochromes, peroxidases, etc., which are of critical importance for the fulfillment of various biological functions including respiration, photosynthesis, and other enzymatic reactions associated with metabolisms of living organisms (Smith, 1975; Shelnutt et al., 1998; Sen and Poon, 2011). Porphyrins, especially metalloporphyrins, are the essential cofactors for the conservation of versatile biological activities of these functional proteins, and metalloporphyrins are the actual catalytic centers of many existed redox enzymes like horseradish peroxidase (HRP), catalases, and methylreductases (Smith, 1975; Shelnutt et al., 1998). Although the immediate surroundings, especially the tightly conjugated axial coordination moieties as well as the specialized microenvironments created by the bulk n
Corresponding author. Corresponding author at: School of Environmental and Biological Engineering, Nanjing University of Science & Technology, Nanjing 210094, PR China. E-mail addresses: [email protected] (J. Kong), [email protected] (X. Zhang). nn
http://dx.doi.org/10.1016/j.bios.2014.07.034 0956-5663/& Elsevier B.V. All rights reserved.
proteins, have significant influence on regulating the catalytic activity of metalloporphyrins (Ringe and Petsko, 2008), the free metalloporphyrins in vitro can also independently exhibit excellent catalytic activity and distinct substrate selectivity under optimum conditions (Gross and Nimri, 1994; Rutkowska-Zbik and Witko, 2006; Shelnutt et al., 1998; Sen and Poon, 2011). Compared with their corresponding natural enzymes, metalloporphyrins are low molecular weight and show superior thermal stability along with higher pH tolerance, thus implicating a great potential in practical applications (Smith, 1975). Consequently, metalloporphyrins have now been extensively investigated as biomimetic catalysts in the fields involving analytical chemistry, life science, and chemical synthesis (Breslow et al., 1996; Biesaga et al., 2000; Imahori, 2004; Umile and Groves, 2011; Wertz et al., 1998; Watanabe et al., 2013). Recently, using various metalloporphyrins as efficient biomimetic catalysts has attracted considerable interests in the field of selective catalytic oxidation of diverse substrates (de Groot et al., 2005; Meunier, 1992; Monnereau et al., 2010; Nimri and Keinan, 1999; Wang et al., 2007). For example, hemin, a stable iron-containing porphyrin, has now been widely exploited as a highly active biomimetic catalyst toward the
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oxidation of pyrogallol, reduced nicotinamide adenine dinucleotide (NADH), and other metabolic intermediates (Golub et al., 2011; Xue et al., 2012; Yuan et al., 2012), and the obtained results revealed that both of the catalytic activity and substrate binding affinity were approximately equal to that of natural enzymes. Moreover, metalloporphyrins have also been extensively investigated as biomimetic catalysts for the highly sensitive determination of inorganic ions, e.g. Hg2 þ , Ge4 þ , and Cu2 þ , small molecules, e.g. ascorbic acid, dopamine, and hydrogen peroxide, and even biomacromolecules, e.g. DNA, RNA, and proteins (Grabowska et al., 2014; Pelossof et al., 2011; Wu et al., 2012; Wang et al., 2013; Xu et al., 2013; Zhang et al., 2013). In the past decade, there has been an increasing demand for the ultrasensitive determination of sequence-specific DNA, which is of great importance for molecular diagnostics, clinical medicine, forensic investigations, and environmental monitoring (Drummond et al., 2003; Hansen et al., 2006; Liu et al., 2013; Sassolas et al., 2008). Compared with conventional methods such as fluorescence spectrometry (Xiang et al., 2010), ultraviolet absorption spectrophotometry (Holden et al., 2009), and capillary electrophoresis (Fujii et al., 2010), electrochemical methods have drawn considerable attentions because of their high sensitivity, uncomplicated instrumentation, low cost, and good portability (Hansen et al., 2006; Liu et al., 2008; Sassolas et al., 2008). Considering the requirement of highly sensitive methods to accurately determine sequence-specific DNA at ultralow levels, intensive attentions are concentrated on the development of effective signal amplification approaches to improve the electrochemical response to a detectable level (Liu et al., 2008; Song et al., 2014). For example, given the outstanding catalytic activity of HRP, great efforts have been devoted to exploit it as an efficient biocatalyst in the procedures of signal amplification for the highly sensitive determination of low-abundant target analytes (Möller et al., 2005; Schüler et al., 2009). Nevertheless, the practical application of the well-established HRP-based signal amplification approach currently suffered from several inevitable drawbacks. For example, apart from the complicated preparation procedures to introduce HRP onto supporting substrates provided for the determination of target analytes, the high cost and unsatisfactory longterm stability of HRP in solution have largely inhibited its widespread application in analytical fields. Meanwhile, the relative large dimensions of HRP limit its maximum loading amount, which is unfavorable to signal amplification (Wang et al., 2013). Furthermore, the catalytic activity of a natural enzyme is highly dependent upon the protein conformation, leading to vulnerability to the pH and temperature of the external conditions (Song et al., 2010). Hematin, the hydroxylated derivative of the metalloporphyrin center of HRP (Akkara et al., 2000), has been confirmed to be a promising alternative to replace HRP for the polymerization, hydrogelation, or degradation of phenols, aromatic amines, and their derivatives because of its excellent peroxidatic activity in vitro (Akkara et al., 2000; Nagarajan et al., 2009; Pirillo et al., 2010; Sakai et al., 2010). Accordingly, from the view points of green chemistry, hematin shows great value as a possible environmentally friendly biomimetic catalyst for the synthesis or modification of versatile polymeric materials, together with the cost-effective disposal of pigment-rich wastewater discharged from textile industry, papermaking industry as well as printing industry. In addition, several attempts have been made to exploit hematin as a peroxidase substitute in the determination of hydrogen peroxide (Zhang and Dasgupta, 1992) as well as in the ultrasensitive electrical detection of nucleic acids in a device based sub-microgapped biosensor (Kong et al., 2008). Thus, the application of hematin as a substitute for HRP shows great applicability to circumvent the aforementioned drawbacks on account of its excellent thermal stability and predominant pH tolerance, while
its excellent peroxidatic activity like HRP is still preserved (Akkara et al., 2000; Carvalhal et al., 2005). Herein, for the first time, we presented a novel signal amplification approach to construct an electrochemical DNA biosensor for the ultrasensitive determination of sequence-specific DNA by exploiting hematin as efficient biomimetic catalyst toward in situ metallization. In this work, thiolated peptide nucleic acid (PNA) probes were firstly immobilized onto the surface of the pretreated gold electrode and subsequently used as the capture probes for the specific recognition of target ssDNA to be determined. After hybridization, hematin molecules were introduced to the hybridized PNA/DNA heteroduplexes by employing zirconium–phosphate and zirconium–carboxylate coordination interactions (Gao et al., 2007; Kong et al., 2008). Then, the attached hematin molecules acted as biomimetic catalyst in promoting the reduction and conversion of free silver ions into the deposited metallic silver. Owing to its excellent catalytic capability of hematin molecules in vitro, the electron exchange between silver ions and catechol could be significantly enhanced. Thus, the amount of the deposited silver particles on the electrode could be rapidly accumulated to a detectable level. Finally, the deposited silver particles were electrochemically stripped into KCl solution and measured by square wave voltammetry (SWV). Due to its significant signal amplification of the hematin-catalysed silver deposition and its favorable immunity to the elimination of background current of square wave voltammetric technique (Christie et al., 1977; O’Dea et al., 1981), the sensitivity of the proposed biosensor was improved with a remarkable level, resulting in ultrasensitive electrochemical DNA biosensing with a detection limit much lower than those previously reported (Liu et al., 2008; Lepage et al., 2011; Kong et al., 2008; Wang et al., 2011). Results also demonstrated that it exhibited excellent specificity in differentiating mismatched oligonucleotide fragments, suggesting great potential in the genotyping of single-nucleotide polymorphism (SNP).
2. Material and methods 2.1. Materials and reagents. Thiolated peptide nucleic acid with sequence 5′-HS-(CH2)11 -AAC CAT ACA ACC TAC TAC CTC A-3′ (PNA) were custom-made by Panagene Inc. (South Korea). Target complementary ssDNA 5′-TGA GGT AGT AGG TTG TAT GGT T-3′ (cDNA), single-base mismatched ssDNA (mismatch underlined) 5′-TGA GGT AGT AGG TTG TGT GGT T-3′ (SBM), three bases mismatched ssDNA (mismatches underlined) 5′-TGA GGT ATT AGA TTG TGT GGT T-3′ (TBM), and completely non-complementary ssDNA 5′-ACT TAC CTT TGC TCA TTG ACG A-3′ (Control) were all purchased from Invitrogen Biotechnology Co., Ltd. (Shanghai, China). Zirconium dichloride oxide octahydrate (ZrOCl2 8H2O), hematin, 6-mercapto-1-hexanol (MCH), and silver nitrate (AgNO3) were purchased from SigmaAldrich (St. Louis, MO). Catechol was purchased from Aladdin Industrial Inc. (Shanghai, China). Fetal bovine serum (FBS, Defined) was purchased from Shanghai YiJi Industrial Co., Ltd. (Shanghai, China). N,N-dimethylformamide (DMF) and all other chemical reagents were purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China) and used without further purification. Ultrapure water obtained from a Millipore Milli-Q water purification system (Z 18.25 MΩ) was used in all assays. Tris–EDTA buffer (TE, 10 mM Tris–HCl, 1 mM EDTA, pH ¼ 8.0) was prepared and used as the stocking solution and washing buffer for ssDNA, and various concentrations of ssDNA samples were prepared by serial dilution. NaAc/HAc buffers (0.1 M, pH ¼5.2, 5.4, 5.6, 5.8, 6.0, 6.2, 6.4) were prepared by mixing
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0.1 M NaAc solution and 0.1 M HAc solution and used as the buffers for the in situ deposition of metallic silver. 2.2. Apparatus All electrochemical experiments were performed on a CHI 760D electrochemical workstation (Chenhua, Shanghai, China) with the conventional three-electrode system consisted of modified gold electrode (Φ¼2 mm) as working electrode, saturated calomel electrode (SCE) as reference electrode and platinum wire as auxiliary electrode. The scanning electron microscopy (SEM) images were recorded with a Quanta FEG 250 field emission scanning electron microscopy (FEI, USA) at an acceleration voltage of 30.0 kV. 2.3. Pretreatment of the gold electrode and immobilization of capture probes Prior to modification, the gold electrode was mechanically polished to a mirror-like surface with 1.0, 0.3 and 0.05 μm alumina slurry, and then successively washed with absolute ethanol and ultrapure water under ultrasonication. Then, it was chemically cleaned in freshly prepared piranha solution (a mixture of 98% H2SO4 and 30% H2O2, 3:1 v/v, CAUTION: piranha solution is strongly corrosive and must be prepared and handled with extreme care) under ultrasonication. Afterwards, it was electrochemically cleaned in a fresh 0.5 M H2SO4 solution by cycling the electrode potential between 0.2 V and 1.5 V with a scan rate of 100 mV/s until a reproducible cyclic voltammogram was achieved to remove any remaining impurities. Finally, it was rinsed thoroughly with ultrapure water and dried with nitrogen prior to modification. Immobilization of capture probes was accomplished by incubating the clean electrode in 1 μM PNA aqueous solution to allow the formation of SAM. After washing with ultrapure water, it was immersed in 2 mM MCH solution (dissolved in 70% ethanol solution) to block the nonspecific binding sites, and then successively rinsed with 70% ethanol solution and ultrapure water to remove excessive MCH. 2.4. DNA hybridization and in situ metallization Hybridization was performed in 10 μL of TE buffer that contained certain concentration of ssDNA at 37 °C for 1.5 h, followed by washing with TE buffer to remove the unhybridized ssDNA. After incubating in 5 mM ZrOCl2 8H2O solution (dissolved in 60% ethanol solution) for 1.0 h, 20 μL of freshly prepared hematin solution (dissolved in DMF) was added onto the electrode and kept for 1.0 h. After that, the electrode was moderately rinsed with DMF and ultrapure water to remove the excessive hematin molecules. 30 μL of freshly prepared mixed solution of AgNO3 and catechol with the stoichiometric ratio of 2:1 (both freshly dissolved in 0.1 M NaAc/HAc buffer) was subsequently added on the electrode and then incubated at 25 °C for 10 min for the deposition of metallic silver. Finally, the resultant electrode was moderately washed with ultrapure water to remove the remaining reactants.
Scheme 1. Schematic illustration of the preparation of the proposed electrochemical DNA biosensor based on hematin-induced in situ metallization and square wave voltammetric technique.
3. Results and discussion 3.1. Electrochemical characterization of the attached hematin molecules As zirconium ions and hafnium ions are able to form stable conjugates with free carboxyl groups and phosphate groups through the robust coordination interactions (Buscher et al., 1996; Li et al., 2010), thus they are applicable to be utilized as simple, cost-effective, and versatile linkers for the implementation of various desired functions. The hematin molecule contains two free carboxyl groups, consequently, it can be firmly conjugated to the deoxyribose phosphate backbone of DNA via the robust zirconium–phosphate and zirconium–carboxylate coordination interactions (Kong et al., 2008). In this work, zirconium ions were adopted as the coupling linkers to introduce free hematin molecules to the hybridized PNA/DNA heteroduplexes. To confirm hematin molecules have been successfully attached to the hybridized heteroduplexes, linear sweep voltammetry (LSV) was used to characterize the existence of hematin molecules on the electrode. As shown in Fig. 1, an apparent reduction peak was visible at the potential around 0.683 V, and it could be attributed to the electrochemical reduction of the conjugated Fe(III) into Fe(II). The peak current increased obviously with the increasing concentration of hematin, which in turn demonstrated that
2.5. Electrochemical measurement To carry out the electrochemical measurement, the gold electrode was finally immersed in 10 mL of 1.0 M KCl solution, and then SWV was performed under the potential from 0.15 V to 0.25 V to record the stripping current of the deposited metallic silver. The preparation process of the proposed hematin-based electrochemical DNA biosensor and the subsequent electrochemical measurement are illustrated in Scheme 1.
Fig. 1. Linear sweep voltammograms of the attached hematin molecules toward 0.1 pM cDNA in 1.0 M KNO3.
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hematin molecules had been successfully attached to the hybridized heteroduplexes via the robust phosphate–zirconium–carboxylate coordination interaction.
3.2. Characterization of the catalytic activity of hematin toward the deposition of metallic silver In the presence of catechol as the reductant, the free silver ions in solution can be reduced into metallic silver and subsequently deposit onto the electrode in the form of silver particles, while the catechol molecules are oxidized into benzoquinones, and each catechol molecule could react with two silver ions. Inspired by its excellent peroxidase-like activity in vitro, herein hematin was exploited as the biomimetic catalyst in catalyzing the reduction of silver ions into metallic silver in the electrochemical determination sequence-specific DNA. Theoretically, the amount of silver deposits aggregated on the electrode was proportionally correlated with the concentration of target ssDNA in solution, thus providing a quantitative measurement. To evaluate the catalytic activity of the attached hematin molecules in enhancing the electron exchange between silver ions and catechol molecules, the size and morphology changes of the deposited silver particles resulting from the catalysis of hematin molecules were characterized by SEM. The size and morphology of the naturally deposited silver particles, namely, without the catalysis of hematin, are nonuniform and disorganized, and they are sparsely distributing on the electrode (Fig. 2A). However, the distribution density of the deposited silver particles on the electrode would increase remarkably, meanwhile they would become more uniform at the average particle diameter as well as the particle morphology under the condition that hematin molecules have been previously introduced to the hybridized heteroduplexes (Fig. 2B). The results demonstrated that hematin could significantly promote the in situ deposition of metallic silver, and thus can be exploited as efficient biomimetic catalyst for the amplification of hybridization signal.
3.3. Optimization of detection conditions of the proposed electrochemical DNA biosensor The deposited metallic silver on the electrode could be easily stripped into KCl solution and accurately measured by square wave voltammetric technique, and the resulting voltammogram
showed a well-defined stripping sharp peak due to the favorable Ag/AgCl solid-state voltammetric process (Ting et al., 2009). In addition, SWV exhibits favorable immunity to the elimination of background current (Christie et al., 1977; O’Dea et al., 1981), thus it is a preferable electrochemical technique in the highly sensitive determination of low-abundant electroactive species. The representative stripping potential of silver deposits on the electrode consistently located at 0 V (vs. SCE), which was in agreement with the obtained value reported previously (Ting et al., 2009). Thus, the electrochemical stripping analysis based on SWV was favorable for the achievement of high detection precision. To achieve the best analytical performance of the proposed biosensor, several experimental parameters associated with the deposition rate of silver particles were investigated. Except for the pH value of the external conditions and the concentration of AgNO3, the concentration of hematin was also inspected. The amount of the in situ deposited metallic silver largely depended upon the pH value of the external conditions, as it could significantly affect the catalytic activity of the attached hematin molecules. Therefore, the effect of pH on stripping current was firstly investigated. As shown in Fig. 3A, after silver deposition for 10 min in 0.1 M NaAc/HAc buffer containing 1.2 mM AgNO3 and 0.6 mM catechol, the stripping peak current increased drastically with the pH varying from 5.2 to 5.8, and then it encountered a remarkable decrease with the further increasing of pH. The obtained result demonstrated that the hematin molecules showed maximum catalytic activity at pH 5.8 toward the reduction of silver ions into metallic silver. As a result, the 0.1 M NaAc/HAc buffer with pH 5.8 was adopted for the hematin-induced in situ metallization in the subsequent experiments. The effect of AgNO3 concentration on the stripping current was further investigated. After introducing hematin molecules (hematin concentration at 500 μM was applied) to the hybridized heteroduplexes, the modified electrode was incubated for 10 min in 0.1 M NaAc/HAc buffer (pH ¼ 5.8) containing various concentration of AgNO3 and catechol with their stoichiometric ratio kept at 2:1. As shown in Fig. 3B, the stripping peak current increased significantly with the AgNO3 concentration varying from 0.6 mM to 1.2 mM, afterwards, it increased slightly and even reached a plateau after 1.4 mM. As the hematin concentration applied here was high enough to maximize its loading amount, thus the concentration of AgNO3 at 1.4 mM could be regarded as the saturated concentration for the silver deposition. Taking this into account, the optimum concentration of AgNO3 was 1.4 mM. Obviously, the concentration of hematin was another important parameter that would significantly affect the deposition rate of
Fig. 2. SEM images of the in situ deposited silver particles on the electrode. Conditions: cDNA: 0.1 pM; pH: 6.0; Hematin: 200 μM; Deposition time: 10 min; AgNO3: 1.0 mM; Catechol: 0.5 mM. Scale bar: 5 μM.
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Fig. 3. Effects of pH (A), AgNO3 concentration (B), and hematin concentration (C) on the analytical performance of the proposed electrochemical DNA biosensor in 1.0 M KCl toward 0.1 pM cDNA.
metallic silver, namely, the response performance of the proposed biosensor could be further optimized by choosing a suitable hematin concentration. Meanwhile, the deposition time could be shortened accordingly. As shown in Fig. 3C, the stripping peak
current increased rapidly with the increasing of hematin concentration until 250 μM and then it trended to a plateau until 300 μM. From the result mentioned above, it could be concluded that the optimum concentration of hematin was 300 μM.
Fig. 4. Square wave voltammograms (A), and calibration plot (B) of the proposed electrochemical DNA biosensor toward varying concentrations of cDNA.
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Fig. 5. SWV responses of the proposed electrochemical DNA biosensor toward 0.1 pM cDNA (A), SBM (B), TBM (C), and Control (D).
3.4. Analytical performance of the proposed electrochemical DNA biosensor Under the optimal conditions, the sensitivity and dynamic range of the proposed electrochemical DNA biosensor were determined with varying cDNA concentrations. As shown in Fig. 4, the stripping peak currents increased with the increasing concentration of cDNA. The calibration plot showed a good linear relationship between the stripping peak currents and the logarithm of the concentrations of cDNA in the range from 0.1 fM to 0.1 nM. The linear regression equation was I (μA)¼ 1101.364 þ52.868 lg [CDNA/M] with a correlation coefficient of 0.9986. The limit of detection at a signal-to-noise ratio of 3 was calculated to be 62.41 aM, which was much lower than those reported previously (Liu et al., 2008; Lepage et al., 2011; Kong et al., 2008; Wang et al., 2011). The improvement of sensitivity and the wide dynamic range of the biosensor might be attributed to the significant signal amplification based on exploiting hematin as the efficient biomimetic catalyst and the favorable immunity to the elimination of background current of SWV as well as the predominant selectivity of PNA. Compared with the HRP-based signal amplification approach, the proposed approach had distinctive advantages of low cost, easy operation, and favorable stability. The wide linear range over 7 orders of magnitude was also very important for clinical applications. 3.5. Specificity, repeatability, reproducibility, and stability of the proposed electrochemical DNA biosensor The specificity was investigated by using three types of ssDNA including SBM, TBM, and Control. As shown in Fig. 5, the stripping peak currents from SBM (B), TBM (C), and Control (D) were approximately 86.4%, 72.1%, and 20.1% of the value obtained from cDNA (A), respectively. The obvious difference could be attributed to the hybridized efficiency of the four types of ssDNA and the predominant selectivity of PNA, as the cDNA could effectively and robustly bind to the complementary PNA while the others can't. In addition, the strong background current could be ascribed to the spontaneous redox process, which was inevitable because the silver ions could be naturally reduced into metallic silver in the presence of catechol acted as the reductant. The obtained results demonstrated that the novel biosensor described here could effectively differentiate complementary and mismatched oligonucleotide fragments, indicating that it was highly specific and even showed great potential for the genotyping of SNP. The excellent
Fig. 6. SWV responses of the proposed electrochemical DNA biosensor toward 0.1 pM cDNA in TE buffer (A), 5% serum sample (B), and 1% serum sample (C).
specificity could be attributed to the use of the highly specific PNA as the capture probes. The repeatability and reproducibility were evaluated by the relative standard deviation (RSD) of intra- and inter-assays (n ¼8). The intra-assays were conducted by measuring two samples containing 0.1 pM and 10 pM cDNA. Each sample was repeatedly measured 8 times using 8 parallel prepared biosensors, and the resultant RSD values were 3.7% and 3.3%, respectively. The interassays were conducted under the same conditions, and the obtained RSD values were 4.3% and 4.1%, respectively. The results indicated that it had acceptable repeatability and reproducibility. In addition, the modified electrode could be stored in a moisture-saturated environment at 4 °C, and over 95% of the initial response remained after a storage period of four weeks. The results demonstrated that it possessed acceptable stability. 3.6. Application in analysis of serum samples To evaluate the analytical reliability and application potential of the proposed electrochemical DNA biosensor, the interference effect of complex serum components on analytical performance was investigated. The stripping peak currents originated from 0.1 pM cDNA in serum samples were compared with the reference value obtained from 0.1 pM cDNA in TE buffer. The assay results from 8 repeated experiments were displayed in Fig. 6, the stripping peak currents from 5% (B) and 1% (C) serum samples were approximately 88.61% and 92.60% of that from TE buffer (A), respectively. In addition, the stripping currents attributed to the naturally deposited metallic silver, namely, without the catalysis of hematin, were almost kept at the same level. All these obtained results highlighted that the proposed electrochemical DNA biosensor showed acceptable analytical reliability and application potential for clinical applications, allowing for its good detectable capability in complex systems such as samples in serum.
4. Conclusion In summary, an ultrasensitive electrochemical DNA biosensor based on exploiting hematin molecules as efficient biomimetic catalyst toward in situ metallization was developed. To our knowledge, we are the first one to exploit hematin-induced in situ metallization into the construction of electrochemical DNA biosensor. Using the thiolated PNA as the immobilized capture probes
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significantly improved the selectivity, leading to the great potential in the genotyping of SNP. The application of the robust phosphate–zirconium–carboxylate coordination chemistry to introduce the cost-effective hematin molecules to the hybridized PNA/DNA heteroduplexes was convenient and feasible. More importantly, the integration of hematin-based signal amplification approach and square wave voltammetric technique could synergistically improve the sensitivity, enabled a low detection limit of 62.41 aM achieved under optimal conditions. In addition, it excluded complicated preparation procedures and could be successfully applied for the quantification of sequence-specific DNA in serum samples. In consideration of the outstanding analytical performance including wide linear range, low cost, satisfactory repeatability and reproducibility, acceptable stability and reliability, the developed biosensor showed great potential in clinical applications. Furthermore, it could integrate with standard microfabrication techniques to produce electrochemical sensor array and realize multiplexed detection capabilities, while the hematinbased signal amplification approach could be easily extended to detect other low-abundant biomarkers after appropriate modifications.
Acknowledgments We are grateful to Nanjing University of Science and Technology for its start-up funding, National Natural Science Foundation of China funding (No. 21345002) for this project.
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