Electrochemical Determination of Ca2+

Communication Cite This: Bioconjugate Chem. XXXX, XXX, XXX−XXX

pubs.acs.org/bc

Electrochemical Determination of Ca2+ Based On Recycling Formation of Highly Selective DNAzyme and Gold NanoparticleMediated Amplification Li Li,† Xiaoyi Ma,†,‡ Wenfei Dong,† Peng Miao,*,†,‡ and Yuguo Tang† †

Suzhou Institute of Biomedical Engineering and Technology, Chinese Academy of Sciences, Suzhou 215163, People’s Republic of China ‡ University of Science and Technology of China, Hefei 230026, People’s Republic of China S Supporting Information *

ABSTRACT: Calcium ion (Ca2+) plays a critical and indispensable role in many physiological and biochemical processes in the human body. In this report, we demonstrate a novel electrochemical method for the determination of the Ca2+ level aided by three functional DNA probes and gold nanoparticles (AuNPs). It affords high selectivity in sensing Ca2+ over other metal cations, which is due to the adoption of the DNAzyme at the electrode interface with exceptionally high binding ability. This method also integrates recycling formation of DNAzyme and AuNPs-mediated amplification; thus, high sensitivity is promised. Therefore, this work provides a favorable way to probe Ca2+ for biomedical applications.

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Recently, Ca2+-dependent DNAzyme has attracted great attention in biomedical applications.18,19 DNAzyme, also named deoxyribozyme, is able to perform specific chemical reactions similar to biological enzymes.20 DNAzyme can be exploited as a signal amplification element instead of a hybridization chain reaction,21,22 rolling circle amplification,23 and exonuclease-aided amplification.24 In this Communication, we have designed a DNA modified electrode and constructed specific RNA-cleaving DNAzyme tautologically in the presence of Ca2+ and DNA−gold nanoparticle (AuNPs) nanoconjugates. The principle for the sensitive and selective detection of Ca2+ is illustrated in Scheme 1. Generally, three DNA probes are designed (Table S1). DNA probe a is first immobilized on the surface of the gold electrode via gold−sulfur chemistry.25 Next, DNA probe b is added, forming the DNAzyme by partial hybridization, which has the potential to be activated by Ca2+. The specific DNAzyme contains a single RNA linkage as the cleavage site, and is reported to cooperatively bind two Ca2+ ions.26 After the introduction of Ca2+, DNA probe a is cleaved. On the other hand, AuNPs are modified with DNA probe c. The concentrations of DNA probes used for modifications are according to a previous report.27 The sequence of DNA probe c is complementary to the rest of DNA probe a on the electrode surface; thus, DNA probe b coupled with the two Ca2+ ions could be released by strand displacement reaction and initiate

alcium is one of the most essential elements in the human body.1 It not only comprises 1−2% of body weight, but also participates in metabolism and mineral homeostasis.2,3 Ionized calcium (Ca2+) levels in certain biofluids have been demonstrated to modulate numerous physiological and pathological processes, affecting different functions of organs or systems, such as neural signaling, biomineralization, acid− base balance, and muscle action.4−6 Excessive alternation of Ca2+ may lead to diverse disorders including hyperparathyroidism, osteoporosis, renal disorder, and muscle tightening.7−9 The level of Ca2+ can even be used for clinical estimation.10 Thus, it is necessary to develop reliable and sensitive methods for the detection of Ca2+. So far, several sophisticated analytical methods have been explored like flame atomic absorption spectroscopy (FAAS) and nuclear magnetic resonance (NMR) analysis.11,12 There are also some cost-effective techniques applied.13 For example, Heidari’s group fabricated a paperbased microfluidic device for colorimetric assay of Ca2+ and Mg2+ based on sticker templates with specific patterns and a highly accessible waterproof eye pencil.14 Javey’s group invented a wearable electrochemical platform for noninvasive and continuous monitoring of Ca2+ with a flexible printed circuit board.15 Wu et al. expanded the palette of genetically encoded fluorescent Ca2+ indicators based on protein engineering.16 However, the limit of detection are relatively high with current electrochemical, colorimetric, or fluorescent techniques, which may also require consuming manipulations.17 Thus, novel convenient and sensitive analytical methods for Ca2+ sensing in biological samples are still highly desired. © XXXX American Chemical Society

Received: February 8, 2018 Revised: March 5, 2018

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DOI: 10.1021/acs.bioconjchem.8b00096 Bioconjugate Chem. XXXX, XXX, XXX−XXX

Communication

Bioconjugate Chemistry Scheme 1. Schematic Outline of the Electrochemical Biosensor for Sensitive and Selective Detection of Ca2+

Figure 2. Only in the case of Ca2+ triggered cleavage reaction can AuNPs with ferrocene be localized, which is reflected by

new cycles of DNAzyme cleavage after hybridizing with other complete DNA probe a on the electrode. Since DNA probe c is labeled with ferrocene, a remarkable electrochemical response could be recorded owing to the cleavage recycles and AuNPs with huge specific area (Figure S1), which is related to the initial concentration of Ca2+.

Figure 2. SWVs for (a) bare electrode, after immobilization of DNAzyme and subsequent AuNPs in the (b) absence and (c) presence of 1 mM Ca2+.

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the significant peak around 0.25 V. The incubation time of Ca2+ is also optimized to be 90 min based on the comparison of SWV peaks (Figure S2). Quantitative information on Ca2+ can also be determined by the SWV technique. After challenging different levels of Ca2+, corresponding voltammetric curves are recorded. As shown in Figure 3a, with the increase of the concentration of Ca2+, SWV peak increases gradually, which is evidence of the Ca2+ dependent DNAzyme cleavage and the subsequent localization of AuNPs. The detailed relationship between the peak current and the concentration of Ca2+ is depicted in Figure 3b. A linear range from 10−7 to 10−3 M is achieved with the fitting equation as follows:

RESULTS AND DISCUSSION The DNA assembly, DNAzyme construction, and further localization of AuNPs have been successfully confirmed by electrochemical impedance spectroscopy (EIS). As shown in Figure 1, a straight line is achieved for bare gold electrode,

y = 33.720 + 4.499x

(R2 = 0.993)

in which y stands for the value of the peak current, and x is the logarithmic of the concentration of Ca2+. The limit of detection is calculated to be 10−7 M (signal-to-noise = 3:1), which is lower than most previously reported Ca2+ sensors (Table S2). Reproducibility and stability of the proposed method are also checked. Five independent working electrodes are modified with DNAzyme, which are then used for the detection of Ca2+ with the concentrations of 1 μM and 1 mM. The obtained peak currents are in excellent agreements (Figure S3). To evaluate the high selectivity of this sensor, we have investigated the effect of various metal ions on the SWV peak intensity including Na+, K+, Mg2+, Zn2+, Cd2+, Ni2+, Co2+, Cu2+, Fe2+, Fe3+, and Pb2+. As shown in Figure 4, it is found that the SWV peak cannot be enhanced by the interfering ions except Pb2+ since most RNA-cleaving DNAzymes can be activated by Pb2+. Therefore, the sensor is demonstrated to be able to discriminate target ion from most interfering ions. We have also employed complicated biological fluids to study the practical utility of this sensor. Human sweat and urine samples are first collected, the Ca2+ concentrations in which are then validated by the proposed sensor and inductively coupled plasma-mass spectrometry (ICP-MS). Detailed results are listed in Table 1. The obtained Ca2+ levels by the two methods are in good agreement and the all relative errors are less than 4%.

Figure 1. Nyquist diagram of EIS for (a) bare electrode, (b) DNA probe a modified electrode, (c) DNAzyme modified electrode, (d) after Ca2+ triggered cleavage, and (e) after further loading of AuNPs.

which is due to the fine conductivity of the unmodified electrode (curve a). After the immobilization of DNA probe a, the negatively charged phosphate backbone repels [Fe(CN)6]3‑/4‑, which results in a significant semicircle domain (curve b). The degree of the repellent increases after the formation of DNAzyme, which is reflected by the increase of the semicircle diameter (curve c). Nevertheless, Ca2+ is able to activate the DNAzyme cleavage; thus, the diameter decreases after the reaction (curve d). Since the cleaved DNA probe a is able to capture DNA probe c modified AuNPs, electronegativity is increased dramatically, coupled with the increase of the semicircle diameter (curve e). We have then confirmed the electrochemical responses of ferrocene in this system by square wave voltammetry (SWV), the curves of which are depicted in B

DOI: 10.1021/acs.bioconjchem.8b00096 Bioconjugate Chem. XXXX, XXX, XXX−XXX

Communication

Bioconjugate Chemistry

Therefore, the potential applicability of the method for the detection of Ca2+ in biological samples is well confirmed.

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CONCLUSIONS In summary, we have constructed a sensitive electrochemical sensor for the detection of Ca2+ based on the application of the DNAzyme with exceptionally high selectivity. Ca2+ triggers the DNAzyme cleavage and the recruitment of AuNPs helps the recycling formation of additional DNAzyme. The detection range is wide and the limit of detection is quite low. The developed Ca2+ sensor has also been successfully examined in the presence of interfering ions and biological samples. The encouraging results suggest great potential utility of this method for Ca2+ sensing in biomedical applications.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.bioconjchem.8b00096. Experimental details, TEM image of DNA modified AuNPs, optimization of reaction time, electrode-toelectrode reproducibility, DNA sequences, and comparison of analytical performances (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Tel.: +86-512-69588279. ORCID

Figure 3. (a) SWVs for the detection of Ca2+ with a series of concentration (10−7, 2 × 10−7, 10−6, 2 × 10−6, 10−5, 2 × 10−5, 10−4, 2 × 10−4, and 10−3 M, from top to bottom). (b) Calibration curve of the biosensor for the detection of Ca2+. Error bars represent standard deviations of three independent measurements.

Wenfei Dong: 0000-0003-1319-3166 Peng Miao: 0000-0003-3993-4778 Author Contributions

All authors have given approval to the final version of the manuscript. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the National Natural Science Foundation of China (Grant no. 81771929) and National Key Research and Development Program of China (Grant no. 2017YFF0108600, 2017YFC0211900 and 2016YFF0103800).

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ABBREVIATIONS AuNPs, gold nanoparticles; EIS, electrochemical impedance spectroscopy; FAAS, flame atomic absorption spectroscopy; ICP-MS, inductively coupled plasma-mass spectrometry; NMR, nuclear magnetic resonance; SWV, square wave voltammetry

Figure 4. Selectivity of the biosensor for the detection of Ca2+ against a range of other ions including Na+, K+, Mg2+, Zn2+, Cd2+, Ni2+, Co2+, Cu2+, Fe2+, Fe3+, and Pb2+. The concentrations of the ions are 100 μM (10 μM for Pb2+).

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Table 1. Detection Results for Ca2+ in Sweat and Urine Samples by the Proposed Biosensor and ICP-MS sample

sensor (mM)

standard deviation (mM)

ICP-MS (mM)

relative error (%)

sweat 1 sweat 2 urine 1 urine 2

0.71 0.89 1.15 2.53

0.03 0.01 0.05 0.04

0.72 0.91 1.18 2.44

1.39 2.20 2.54 3.69

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DOI: 10.1021/acs.bioconjchem.8b00096 Bioconjugate Chem. XXXX, XXX, XXX−XXX

Communication

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DOI: 10.1021/acs.bioconjchem.8b00096 Bioconjugate Chem. XXXX, XXX, XXX−XXX