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Label-free Nuclease Assay with Long-term Stability Rui Liu, Jianyu Hu, Yongxin Chen, Min Jiang, and Yi Lv Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.9b02467 • Publication Date (Web): 05 Jun 2019 Downloaded from http://pubs.acs.org on June 6, 2019

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Analytical Chemistry

Label-free Nuclease Assay with Long-term Stability

Rui Liu,† Jianyu Hu,† Yongxin Chen, † Min Jiang, † Yi Lv†,‡,*

†Key

Laboratory of Green Chemistry & Technology, Ministry of Education, College of Chemistry, Sichuan

University, Chengdu 610064, China ‡Analytical

& Testing Center, Sichuan University, Chengdu 610064, China.

*Email: [email protected]; Tel. & Fax +862885412798

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Analytical Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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ABSTRACT Nature endows us a unique toolbox of highly specific enzymes, whilst their detection is of great importance in biological process. The label-free assay based on DNA-templated CuNPs is widely accepted for enzyme assay owning to its simple procedure, fast kinetic, high quantum yield, and large Stokes shift. A challenge in the application of them is the low fluorescent signal stability of DNAtemplated CuNPs, whose signal sharply decreases in a few minutes after formation. In this work, a long-term stable nuclease assay is proposed by utilizing the elemental mass spectrometry detection of CuNPs. The high sensitivity was also realized, thanks to a great number of copper isotopes (63Cu and 65Cu)

intrinsically incorporated in CuNPs. The experimental conditions, including the length of polyT

ssDNA template, the concentration of polyT template, the concentration of Cu2+, the sodium ascorbate concentration, the copper reduction reaction time, and the Exonuclease I (Exo I) digestion reaction time, were investigated in detail. The dynamic range of the Exo I concentration from 0.1 U/mL to 20 U/μL was obtained using inductive coupled plasma mass spectrometry (ICPMS) 63Cu signal, with a detection limit (3σ) of 0.029 U/mL. The ICPMS 63Cu signal remained unchanged for at least 18 days. The spiked-recovery assay in RPMI 1640 cell medium also demonstrated the reliability of the proposed nuclease assay.


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Nature endows a unique toolbox of highly specific enzymes, such as ligase, telomerase, endonucleases, nicking enzymes, and other DNA modifying enzymes, which allow manipulating and molding DNA structures with remarkable resolution.1,2 Developing assays for enzymes is of great importance, due to their special functions in DNA replication, recombination, repair, genotyping, mapping and molecular cloning etc.3-9 In the past few years, many analytical methods for nuclease have been delicately designed and successfully developed.10-13 Among them, the label-free enzyme assay based on DNAtemplated CuNPs intrigued great attention,14-18 thanks to their simple procedure, fast formation kinetic, high quantum yield, and large Stokes shift.19-22 For instance, Ouyang et al. successfully developed a label-free assay for DNase I after AT sequences dsDNA-templated formation of CuNPs.23 High sensitivity and fast-response were obtained by fluorescence detection. Dai et al. realized a label-free fluorescent alkaline phosphatase assay based on polyT-templated CuNPs, with a low limit of detection of 3.5 × 10-2 U/L.24 Label-free assays for enzymes such as S1 nuclease,25 T4 polynucleotide kinase,26 protein kinase,27 deoxyribonuclease I28 etc. have been extensively investigated and successfully developed. Despite great success, a challenge in the application of these label-free assays is the vulnerable signal stability.14 The fluorescence signal of DNA-templated CuNPs could be only stable for a few minutes and then decrease quickly (and lost more than 80% intensity in 1 h),29 which greatly limit its applications for long-time and real-time monitoring research. To overcome this issue, Wang et al. developed a concatemeric dsDNA-templated CuNPs strategy by applying the rolling circle replication (RCR) technique.30 The concatemeric dsDNA-templated CuNPs showed a lower fluorescence decreasing rate. After a 16 h RCR pretreatment, the fluorescent signal intensity was improved and reserved 60% in 2.5 h. Li et al. found DNA-histone interaction could largely increase the fluorescence



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stability of DNA-templated CuNPs.31 The three-dimensional structure initiated by the DNA-histone interaction provided a confined microenvironment to stabilize CuNPs. After 8 h, the fluorescence signal of the histone/DNA -templated CuNPs reserved about 40%, while the fluorescence signal of the original DNA template CuNPs significantly decreased to 6%. Following these delicate attempts, simple stabilization methods without multi-step pretreatment, and longtime consumption still urgently needed to facilitate further applications. In this work, a long-term stable and label-free enzyme assay is developed by utilizing inductively coupled plasma mass spectrometry (ICPMS) detection of CuNPs. As a method of choice for longtime stable analysis, ICPMS is prevalent in metal elements and metal nanoparticles research, thanks to its longtime signal stability, high sensitivity, wide dynamic range, and absolute quantification feature traceable to primary SI units.32 ICPMS has been widely used for the measurement of metal containing nanoparticles, metal isotopes, metal elements, and biomolecules after metal stable isotope tagging3348.

Besides excellent luminescent properties, the DNA-templeted CuNPs intrinsically comprising a

large amount of copper isotopes (63Cu and

65Cu),

which can be efficiently and long-term stably

detected by elemental mass spectrometry.21 Exonuclease I (Exo I), a widely used 3′-termini specific exonulease for ssDNA,49 was selected as a modal analyte. After digestion of CuNPs, the copper isotopes were accurately and sensitively analyzed by ICPMS, resulting a long-term stable response for Exo I nuclease.

EXPERIMENTAL Reagents DNA oligonucleotides were bought from Sangon Biotech (Shanghai, China) Co., Ltd. The sequences


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of the polyT DNA oligonucleotides are as following: T12, 5′-H2N-TTT TTT TTT TTT-3′, T20, 5′H2N-TTT TTT TTT TTT TTT TTT TT-3′; T30 5′-H2N-TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT-3′; T48, 5′-H2N-TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT-3′. The oligonucleotides were diluted in 0.1 M imidazole before use. Exo Ⅰ (20 U/μL) were purchased from Thermo Scientific (Shanghai, China). NEBuffer 3 was purchased from New England Biolabs (Beijing, China) for nuclease dilution. Imidazole was purchased from Sangon Biotech (Shanghai) Co., Ltd. and prepared as 0.1M imidazole solution (pH 7.0). 1-(3-Dimethylaminopropyl)3-Ethylcarbodiimide hydrochloride (EDC) was bought from Adamas Reagent, Ltd. (Shanghai, China) and prepared as 0.1 M EDC in 0.1 M imidazole. Tris-(hydroxymethyl) methyl amino methane was bought from Huishi Biochemical Reagent Co. Ltd. (Shanghai, China) and prepared as washing buffer (7 mM Tris, 0.17 M NaCl, 0.05% Tween 20, pH 8.0). Bovine serum albumin (BSA) were purchased from Biosharp Co. Ltd. (Hefei, China) and prepared as 1% m/v BSA solution in DIW. 3-(Nmorpholino) propanesulfonic acid (MOPS) were purchased from Solarbio Reagent Co. Ltd. (Beijing, China) and prepared as MOPS buffer (10 mM MOPS，2 mM MgCl2 ，150 mM NaCl，pH 7.6). Ascorbic acid was purchased from Kemiou Chemical Reagent Co., Ltd. (Tianjin, China) and prepared as a solution of 20 mM. Carboxyl-terminated magnetic beads (Affimag SLE 3112, 1-2μm, 10 mg/mL, SiO2 matrix) were bought from Baseline Chromtech Research Centre (Tianjin, China). Instruments A NexION 350 ICPMS instrument from Perkin-Elmer Co., LTD. was employed for sample analysis. Enzymolysis and inactivation of enzymes were performed in the K960 PCR instrument for Likang Biomedical Technology Co. LTD. A MS-100 adjustable temperature mixer from Hangzhou Aosheng Instrument Co. Ltd. was used for sample reaction. A water purification system from Sichuan Ultrapure



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Technology Co. Ltd. was applied to produce deionized water (DIW). Energy Dispersive Spectrum (EDS) was characterized by scanning electron microscopy (SEM, Hitachi, S3400). Transmission electron microscopy (TEM) images was investigated by JEM-2010 microscope (JEOL Co., Japan) at accelerating voltage of 200 kV. Immobilization of PolyT DNA on MBs Briefly, 10 μL of 10 mg mL-1 carboxyl-modified MBs was transferred into a 600 μL centrifuge tube. The MBs were washed three times with 200 μL of 0.1 M imidazole solution, suspended to a final volume of 200 μL in the same buffer solution containing 0.1 M EDC, and incubated at 37 oC for 30 min. Then polyT DNA was added and incubated for 2 h. The MBs-captured probe was washed three times with washing buffer, and incubated in 1% BSA for 1 h to decrease the nonspecific binding. The resulting polyT DNA labeled MBs were separated magnetically, washed, resuspended in 80 μL MOPS buffer, and kept at 4 oC for the further study. Nuclease Assay Typically, Exo I nuclease was serially diluted to the working concentrations by the nuclease buffer. Thirty microliters reaction solution comprising 20 μL Exo I nuclease of various concentrations and 10 μL MBs-polyT was used to perform the nuclease induced cleavage. After incubated in the PCR tube at 37 °C for 2h, the nuclease induced cleavage was stopped by heating for 5 min at 95 oC. After cooled down to room temperature, 20 mM ascorbic acid was added followed by stirring for 1 min. Subsequently, 1 mM copper sulfate was added and kept for 10 min with stirring. After magnetic separation and washing for twice, 200 μL 20% nitric acid was added. After several minutes stirring, the supernatant was magnetically separated and diluted with 4 mL DIW for ICPMS measurement.


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RESULTS AND DISCUSSION Establishment of Nuclease Assay Exo I is a widely used sequence-independent 3’-5’ exonuclease in biochemical study. Exo I can initiate the step-by-step cleavage of mononucleotides from single strand DNA from recessed or blunt 3’terminal to 5’-terminal. Fig. 1 illustrates the schematic of the label-free ICPMS assay for Exo I activity measurement. The analytical system comprises a single strand poly-thymine DNA and magnetic beads for matrix separation. The polyT ssDNA was act as both substrate of Exo I and templates for CuNPs formation. Without Exo I, after the addition of sodium ascorbate and copper ions, the synthesized polyT-templated CuNPs are formed through the reduction from Cu2+ to Cu+, the disproportionation from Cu+ to Cu2+ and elementary Cu, and the aggregation of the elementary Cu on DNA templates to form CuNPs. The formed CuNPs can be sensitively and accurately detected by ICPMS. On the contrary, with the addition of Exo I, the polyT ssDNA is split to fragments because of the high activity of exodeoxyribonuclease on ssDNAs. Therefore, the addition of sodium ascorbate and Cu2+ ions could not produce CuNPs due to the absence of polyT DNA templates. Accordingly, low Cu signal was monitored by ICPMS. Thus, the Exo I activity was obtained via the alteration of the ICPMS signal intensity. The TEM image (Fig. 2a) and EDS image (Fig. 2b) demonstrated the production of CuNPs on the polyT ssDNA coated MBs. After digestion of formed CuNPs by diluted nitric acid, two Cu isotopes with respective abundances of 30.83% (65Cu) and 69.17% (63Cu) were recorded by ICPMS. The both isotopes of Cu are free of isobaric interference. Besides, polyatomic interferences (31P16O2+, 47Ti16O+, 40Ar23Na+, 46Ca16O1H+, 23Na40Ca+, 14N12C37Cl+, 36Ar12C14N1H+, 40Ar25Mg+,

32S16O21H+,

36Ar14N 1H+, 2

40Ca16O1H+,

and16O12C35Cl+ for

32S16O17O+,


32S33S+,

63Cu; 49Ti16O+,

12C16O37Cl+,

33S16O2+,


31P16O18O+,

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and 12C18O35Cl+ for 65Cu) are found to be negligible, thanks to the simple matrix of high

purity deionized water and reagents. Either of the Cu isotopes is suitable for our application. As illustrated in Fig. 2c, 63Cu was selected as the analyte for further investigations, due to the about twofold higher abundance over 65Cu. To investigate the feasibility of the present strategy for measuring 3’-5’ exonuclease activity,

63Cu

signal intensity were recorded under different conditions. As

illustrated in Fig. 2d, the polyT-30 successfully served as templates for the production of CuNPs thus high 63Cu signal intensity was recorded. However, while Exo I was added, the polyT-30 was cleaved into segments from the 3’-terminal. For the absence of polyT ssDNA templates, CuNPs could not be produced, leading to low 63Cu intensity. Besides, the denatured Exo I nuclease (heating at 95 °C for 5 min) was also tested for polyT30 digestion, the resulted solution displayed high 63Cu signal intensity, which was similar to that without Exo I. Optimization of the Nuclease Assay To obtain the optimum analytical performance, we investigated the effects of the length of polyT ssDNA template, the concentration of polyT template, the concentration of Cu2+, the concentration of sodium ascorbate, the copper reduction reaction time, and the Exo I nuclease digestion reaction time. Because polyT ssDNA served as templates for the production of CuNPs, we first investigated the influence of the length of polyT ssDNA template, the concentration of polyT template on the signal intensity. As illustrated in Fig. 3a, the

63Cu

63Cu

signal raised with the increased of the length of

polyT-20 to polyT-30, and then decreased by using polyT-48. The length of polyT was reported to have positive effect for CuNPs growth.20,50 However, the steric hindrance of ssDNA as long as polyT48 may affect the immobilization of them onto MBs, and lower down the 63Cu signal intensity. Thus polyT-30 was selected as ssDNA template for CuNPs formation. As illustrated in Fig. 3b, the 63Cu


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signal intensity was raised with the increase of polyT-30 concentration from 0.5 μM to 1.5 μM, and then leveled off. To save the reagents, 1.5 μM polyT-30 was selected during the immobilization of polyT DNA on MBs. As the source of the formed CuNPs, the Cu2+ concentration and sodium ascorbate concentration were important factors for 63Cu signal intensity. The 100 μM Cu2+ and 2 mM sodium ascorbate were selected for further study after optimization. The effect of reaction times of Cu2+ reduction process and polyT DNA digestion process were also investigated. Without Exo I, the reaction time was investigated after initiating the reduction of Cu2+ ions with ascorbate on polyT-30. As illustrated in Fig. 3c, the 63Cu signal raised with the increase of reaction time and reached maximum within 7 min, indicating the production of CuNPs was completed. Thus 7 min Cu2+ reduction time was selected for further study. After the addition of Exo I, a digestion time is required to complete the 3’ terminal to 5’ terminal digestion of polyT DNA. As shown in Fig. 3d, 120 min is required to complete polyT DNA digestion process, thus is selected for the further study. Analytical Performance Under optimum conditions, the relationship between the concentration of target Exo I nuclease and ICPMS signal was investigated (Fig. 4a and Fig. 4b). As illustrated in Fig. 4b, the dynamic range of the Exo I concentration from 0.1 U/mL to 20 U/μL was realized on ICPMS 63Cu signal. The correlation equation was Y = 2.23E4Log[X] + 1.07E5, with a correlation coefficient R2 of 0.9814. And the limit of detection (LOD, 3σ) was calculated to be 0.029 U/mL, which comparable with, if not better than the existing label-free nuclease assays28,51,52. The specificity of the target Exo I by the developed mass spectrometric assay was also investigated. The results are shown in Fig. 4c. It can be seen that relatively low signal decrease was obtained for Exo III (20 U/μL), Exo VI (20 U/μL), Bst (20 U/μL), Thrombin (20 U/μL), and BSA (0.05% m/v)



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while the signal intensity was decreased significant in the presence of Exo I (5 U/μL), demonstrating an excellent specificity of the proposed nuclease assay. The long-term stability was examined for the polyT30-templated CuNPs. As shown in Fig. 4d, the fluorescent signal decrease sharply in a few minutes, while ICPMS

63Cu

signal remained unchanged for more than 18 days, demonstrating the

superior stability of the proposed nuclease assay. In order to validate the proposed method in complex biological fluids, the Exo I-spiked RPMI 1640 cell medium was applied to study the applicability for nuclease measurement. As shown in Table 1, the relative standard deviation (RSD) and the spiked recovery for Exo I of different concentrations were satisfactory, demonstrating the reliability of the proposed nuclease assay for real sample matrix.

CONCLUSIONS A long-term stable and label-free nuclease assay has been proposed by monitoring Cu isotopes of the CuNPs. The CuNPs are templated formed on polyT30 ssDNA within 7 minutes. The

63Cu

signal

intensity from CuNPs are stable for at least 18 days by ICPMS detection. The analytical procedure is easy to operate, low cost, and simple. The unique performances make it an attractive platform for the measurement of enzymes and possibly other biomolecules. Thanks to the high multiplex and absolute quantification ability of the metal stable isotope detection-based assay, the proposed label-free nuclease assay may also demonstrate the great multiplex and absolute quantification potential for multi-nuclease analysis.

Supporting Information


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The Supporting Information is available free of charge on the ACS Publications website at DOI: XXX. Linear relationship between Cu concentration and ICPMS signal intensity; Sequence of nucleic acid in Exo III assay; Linear relationship of ICPMS 63Cu intensity and Exo III.

AUTHOR INFORMATION Corresponding authors Email: [email protected]; Tel. & Fax +86-28-8541-2798 Notes The authors declare no competing financial interest.

ACKNOWLEDGMENTS The National Natural Science Foundation of China is gratefully acknowledged (No. 21505008, &21575093). This work is also supported by the Recruitment Program of Global Experts (Thousand Talents Program) of Sichuan Province (No.903), Sichuan Science and Technology Program (19CXRC0047), and the Fundamental Research Funds for the Central Universities. Dr. Peng Wu from Analytical & Testing Center, Sichuan University, and Dr. Chunxia Wang from College of Chemistry, Sichuan University, are gratefully thanked for helpful discussion and/or technical assistance.

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For TOC only



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Figure 1. Schematic diagram of the proposed mass spectrometric nuclease assay.

Figure 2. Characterization of polyT-templated copper nanoparticles. The TEM image (a) and EDS (b) of MBs after CuNPs formation; (c) The ICPMS signal of Cu isotopic pattern; and (d) CuNPs formed in the presence of Exo I + T30, Exo I(denatured) + T30, and T30, respectively. ( 20 U μL-1 Exo I and 1.5μM T30)


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Figure. 3. The effect of experimental parameters to the 63Cu signal intensity. (a) The length of polyT DNA; (b) the concentration of polyT-30 DNA; (c) the reduction reaction time of polyT-30 DNA from 0 to 10 min, respectively; and (d) the digestion reaction time of polyT-30 DNA from 0 to 180 min. (20 U μL-1 Exo I and 1.5μM T30)



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Figure. 4 The analytical figures of the proposed nuclease assay. (a) The relation between ICPMS 63Cu intensity and the concentration of Exo I nucleaseFigure. 4 The analytical figures of the proposed nuclease assay. (a) The relation between ICPMS 63Cu intensity and the concentration of Exo I nuclease (0.0001, 0.0002, 0.0004, 0.0005, 0.0008, 0.001, 0.002, 0.005, 0.02, 0.05, 0.1, 0.2, 0.5, 1, 2, 5 and 20 U/μL); (b) the calibration curve; (c) the specificity of label-free nuclease assay; (d) the long-term stability. ( 20 U μL-1 Exo I and 1.5μM T30)

Table 1. Measurement of Exo I in RPMI 1640 Cell Medium.

Samples

Exo Ⅰ added (U/μL)

total Exo Ⅰ detected (U/μL)

recovery (%)

SD (%), n=3

1 2 3 4

5×10-4 1×10-3 5×10-3 1×10-2

4.86×10-4 1.13×10-3 4.91×10-3 1.09×10-2

97 113 98 109

8.7 9.2 11.2 9.7


Label-free Nuclease Assay with Long-term Stability | Analytical

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