acs.analchem.7b00411

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Ultrasensitive Detection of DNA and Ramos Cell Using In-Situ Selective Crystallization Based Quartz Crystal Microbalance Li-Shang Liu, Congcong Wu, and Shusheng Zhang Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.7b00411 • Publication Date (Web): 17 Mar 2017 Downloaded from http://pubs.acs.org on March 19, 2017

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Analytical Chemistry

Ultrasensitive Detection of DNA and Ramos Cell Using In-Situ Selective Crystallization Based Quartz Crystal Microbalance Li-shang Liu1, Congcong Wu1,2, Shusheng Zhang1* 1 Shandong Province Key Laboratory of Detection Technology of Tumor Markers, Linyi University, Linyi 276005, China 2 Collaborative Innovation Center of Functionalized Probes for Chemical Imaging in Universities of Shandong, Shandong Normal University, Jinan 250014, China ABSTRACT: Herein we introduce the first successful assay of biomolecule by in-situ selective crystallization based quartz crystal microbalance (QCM). Selective crystallization of CaCO3 on QCM sensor surface was utilized as an efficient mass amplification strategy and enhanced the sensitivity of QCM significantly. High specificity is guaranteed by the cooperation of two functional groups: –N(CH3)3 and –COOH. Passivation of sensor surface is realized by self-assemble of –N(CH3)3 which effectively inhibited the nonspecific crystallization. The DNA target is detected through hybridization of probe DNA labelled with –COOH, which can effectively promot the in-situ surface crystallization of CaCO3. The concentration of target DNA is reflected by the frequency shift of QCM which is directly induced by the surface crystallization. The selective crystallization based QCM platform is simple, straightforward, high sensitive and high specific. We demonstrate the excellent LOD (2 aM DNA) and a linear range of 10aM to 1nM for DNA. Detection of Ramos cells are also realized with a LOD of 5 cells and a linear range of 5 cells to 6000 cells.

Ultrasensitive and cost-effective assay of DNA is of great significance for clinical diagnostics, gene analysis and forensic application1. Many technologies based on optical, electrochemical and mechanical instruments for the sensitive detection of DNA have been explored 2-4. Quartz crystal microbalance (QCM) is a mass-sensitive sensor that has been broadly applied in fields like charity discrimination, air pollution detection, biomolecule detection, kinetics monitoring, and solubility determination and so on5-8. Also QCM based DNA sensors have been explored recently. To further enhance the detection sensitivity, efforts have been made to improve the sensitivity by mass amplification such as conjugation with nanoparticles, by bio-catalyzed precipitation, by enzymatic amplification, by silver reduction etc 9-11. However each method has its demerit such as time-consuming, multi-step, high cost or enzyme dependent12. The development of simple, cost effective and bio-amplification-free mass amplification design would be a breakthrough for QCM. In this work, we proposed a novel effective signal amplification strategy for QCM: in-situ selective crystallization based QCM. The idea was enlightened by the selective crystallization controlled by self-assembled monolayers (SAMs) of ω-terminated alkanethiols (HS(CH2)nX) supported on metal films. Previously we found sulfamerazine can nucleate on –COOH SAM surface, while not on –NH2 SAM surface13. Also Aizenberg’s group demonstrated that functional groups like –COOH and -OH can obviously enhance the crystal growth, conversely, groups like –CH3 and –N(CH3)3 can significantly inhibited the CaCO3 crystal growth.14. Herein, we designed a mass amplification strategy by in-situ selective crystallization on QCM surface through modification of different functional groups.

Scheme 1. In-situ selective crystallization

The nucleation of CaCO3 on QCM sensor is expected to generate considerable amount of mass expected to cause large QCM response. In this report, for the first time, we rationalized that the in-situ selective nucleation could work as an effective signal amplification method for QCM to quantify biomolecules. The design is illustrated in Scheme 1. Thiolated capture DNAs (cDNA) are immobilized on QCM sensor surface first. Then, a thiolate –N(CH3)3 is utilized instead of MCH and functioned as blocker of CaCO3 nucleation. With the existence of target DNA (tDNA), probe DNA (pDNA) labeled with –COOH hybridizes on sensor surface and provide nucleation cites for CaCO3. For the nucleation process, the CaCl2 was feed in onto the QCM sensor surface to realize the Ca2+ capture by –COOH. The supplied CO2 from added

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(NH4)2CO3 triggered the surface selective nucleation of CaCO3 on QCM sensor. The whole process including the capture of target DNA, the hybridization of pDNA and the in-situ crystallization is recorded in real time by QCM.

EXPERIMENTAL SECTION Chemicals and Reagents. DNA oligonucleotides were from Sangon Biotech, and their sequences are listed in Table S-1. HS-C11-NMe3Cl and HS-C11-COOH were from Prochimia. CaCl2, (NH4)2CO3 and tris(2-carboxyethyl) phosphine hydrochloride (TCEP) were from Aladdin. COOH-Fe3O4 nanoparticles were from Tianjin Unibead Scientific Co., LTD. All aqueous solutions were prepared using ultrapure water (Milli-Q, Millipore). Apparatus. QCM of Q-sense E1 with sensors of basic frequency of 5MHz were from Biolin Scientific. SEM imaging was performed by Hitachi 3400, Japan. Polymorphism of crystal was detected by Renishaw’s inVia Raman. Preparation of QCM sensor. QCM sensors were boiled with solution H2O:H2O2:NH4H2O (5:1:1) for 15min, washed with water thoroughly and dried with N2 gas. Clean sensors were dipped in cDNA (1μM in 10 mM PBS, pH 7.4) for 12 hours. After thoroughly washed, the sensors were modified with –N(CH3)3 by immersing in 10mM HS-C11-NMe3Cl. Capture of TDNA. The prepared QCM sensors were dipped into tDNA prepared in hybridization buffer(10mM Tris, 10mM EDTA, 1M NaCl, pH 7.4) of 37 ℃ for 1 hour. After thoroughly washed with washing buffer (10mM NaCl, 5mM Tris, pH7.4), the sensors were dipped in excess pDNA for 1 hour. After thoroughly washed, the sensors were dried with nitrogen gas. In-situ Crystallization of CaCO3 on QCM Surface. A 37℃ CaCl2 solution was injected in the QCM open model cell with a cover. After the resonant frequency stabilized at ±2Hz, solid 0.05g (NH4)2CO3 (overweight) was added in a disk loacated near the sensor surface to supply CO2. The in-situ crystallization was monitored by QCM in real time. Optimization of CaCl2 concentration. With excess (NH4)2CO3, the concentration of CaCl2 was optimized. CaCl2 solutions with concentration from 1 mM to 7 mM were used to conduct the crystallization. Analysis of Ramos cell. COOH-MNPs were washed with imidazole-HCl (0.1M, pH6.8) for 3 times and then treated with EDC and NHS at 37 ℃ for 30 min to activate the –COOH on MNPs. Aptamer NH2-TDO5 was added to the activated MNPs, incubated at 37 ℃ for 12 hours and washed with PBS. TDO5-MNPs were added to Ramos cells solution at 37 ℃ for 30 min after hybridized with a complementary oligonucleotide (tDNA’). After magnetic separation, the released tDNA’ was used as tDNA to trigger the in-situ selective crystallization.

modified surface. We found that the QCM response to the crystallization on –N(CH3)3 & cDNA modified surface was negligible, similar with the response observed in the case of only –N(CH3)3 modified (Figure. S1). It’s indicated that the coexistence of DNA doesn’t affect the nucleation block ability of –N(CH3)3. The first SEM picture in Figure 3 showed none crystals were observed on –N(CH3)3 & cDNA surface, which also supported the statement. The result demonstrated that the modification of –N(CH3)3 can totally prevent the surface nucleation even with the existence of nucleic acid, guaranteed the specificity of surface crystallization to –COOH group. Optimization of CaCl2 Concentration. In order to achieve the highest amplification effect, the concentration of CaCl2 should be the higher the better. Form the Figure 1 we can see no obvious frequency shift was observed until the concentration of CaCl2 achieved 7 mM. That is to say the –N(CH3)3 can no longer totally prevent the surface crystallization if the concentration of CaCl2 is higher than 7 mM. Therefore 6 mM is believed to be the optimized concentration for CaCl2 at which surface crystallization can be totally blocked and highest mass amplification effect can be achieved when –COOH is introduced.

Figure 1 Optimization of CaCl2 concentration for in-situ selective crystallization of CaCO3 on QCM surface.

RESULT AND DISCUSSION It’s previously demonstrated that –N(CH3)3 modified on gold surface could totally prevent the surface nucleation of CaCO3. However, in our design a layer of nucleic acid (cDNA) also exists. Therefore, it’s necessary to check whether the crystals can nucleate on –N(CH3)3 & DNA surface or not. Therefore we checked its blocking ability compared with pure –N(CH3)3

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Figure 2: Amplification effect of in-situ crystallization


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Figure 3： QCM sensor surface condition after in-situ selective crystallization to DNA target of different concentration. From left to right: blank, 10aM, 100aM, 1fM, 10fM, 100fM, 1pM, 10pM, 100pM, 1nM target DNA.

Figure 4 a Frequency shift of in-situ selective based QCM to a series concentration of DNA; b Calibration of frequency shift of in-situ selective based QCM to target DNA concentration.

Mass Amplification Effect of In-Situ Selective Crystallization Based QCM. The capture of target DNA, the hybridization of probe DNA and the in-situ crystallization of CaCO3 were monitored in real time by QCM. The frequency shift during the whole process was shown in figure 2. From the inset, we can see the capture of DNA with a concentration of 1nM didn’t cause any observable frequency shift due to its low mass, neither did the hybridization of pDNA, which is consistent with reported work15. It’s reported that DNA with concentration higher than 10nM can cause frequency shift about 8 Hz using sensor of 9 MHz, which is also quite small. On the contrary, large frequency shift was observed with the introduction of (NH4)2CO3 where nucleation took place. Frequency shift higher than 10,000 Hz was observed, which is thousands folds signal compared with the direct detection of DNA. The significant enhancement of frequency shift suggests the crystallization method is a good strategy to amplify the signal of QCM. Calibration of Frequency Shift of In-Situ Selective Crystallization Based QCM to DNA. The QCM response of the in-situ selective crystallization system to a series concentration of target DNA was recorded in real time and presented in Figure 4a. It was found that with DNA of higher concentration larger frequency shift was observed, which is highly consistent with the crystals amount found on QCM

sensor surface (SEM images in Figure. 3). With triple repeats, the calibration of QCM response to DNA concentration was presented in figure 4b. The linear regression follows an equation Y=-21044.13-117073lgc with a R2 0f 0.997, where Y is the resonant frequency shift of QCM and c is the DNA concentration. The linear range for DNA detection in our work is as wide as from 10aM to 1nM with a detected LOD of 2aM. A wide linear range is crucial for the practical application. Selectivity of the In-Situ Selective Crystallization Based QCM. We evaluated the sequence selectivity of the proposed QCM sensor by using 1-base and 2-base mismatched oligonucleotides. The QCM response by mismatched DNA were insignificant from that of the blank (Figure S2). In the presence of perfect matched target DNA, the frequency decreased significantly larger than that of mismatched DNA, for both concentration of 1*10-15 M and 1*10-9 M (Figure 5). The result suggests that the in-situ selective crystallization based sensor could discriminate perfect matched target DNA from mismatched DNA with high selectivity. The proposed sensor can be applied to analyze the single nucleotide polymorphism (SNP). Besides the specific hybridization between the oligonucleotides, the high specificity of the proposed system is believed to from the cooperation of the two functional groups: –COOH and - N(CH3)3.



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cells to 6000 cells with a detected LOD of 5 cells. The regression equation is Y=1523.69-3015.04lgN with a R2 of 0.999, where Y is the resonant frequency shift of QCM and N is the number of Ramos cells. Table 1. Comparison of QCM techniques based on mass amplification.

Figure 5. Selectivity of in-situ crystallization based QCM sensor. Scheme 2 Turn the Detection of Ramos cell to detection of DNA

Figure 6. Calibration of frequency shift of QCM to Ramos cell.

Application of In-Situ Selective Crystallization System as Cell Biosensor Using the proposed DNA detection strategy, Ramos cells were analyzed taking the advantage of its interaction with the aptamer TDO5. The principle is shown in scheme 2. Aptamers TDO5 are modified on the surface of Fe3O4 magnetic nanoparticles (MNPs) and form a duplex with complementary oligonucleotides (tDNA’). In the presence of Ramos cells, the complementary oligonucleotides are released from the MNPs as Ramos cells have stronger affinity with aptamer TDO5. Then the release oligonucleotides (tDNA’) go to the above DNA detection system as target DNA and trigger the in-situ selective crystallization on QCM sensor. The QCM response of the in-situ selective crystallization system to a series concentration of Ramos cells was summarized in Figure 6. The resonant frequency shift of QCM showed a linear relationship with the logarithm of Ramos cell number in the range from 5

Method

tDNA

Cell (Cells/ML)

Linear range; LOD

Linear range; LOD

Enzymatic amplification

0.5nM-25nM; 0.1nM;

-

16

Magnet/magnetic beads

2.24uM-2.32nM; 0.4nM; 17

1*10^4-1.5*10^5; 8000; 18

Silver deposition

0.6-130nM; 0.1nM; 19

2*10^3-1*10^5; 1160; 20

Streptavidin-biotin/ Chitosan-Folic acid

1-75nM; 0.8nM; 21

4.5*10^2-1.01*10^ 5; 430; 22

In-situ selective crystallization

10aM-1nM; 2 aM;

this

5-6*10^3; 5; this work

work

The performance of QCM based on different mass amplification strategy as DNA and Ramos cell sensor is summarized in Table 1. The proposed in-situ selective crystallization QCM performed a wider linear range and a lower LOD compared with other mass amplification strategies. The reproducibility of the QCM sensor was estimated by determining the tDNA level with three sensors of different batches under the same experiment conditions. The relative standard deviation (RSD) of the inter-assay was 8.3% at the DNA concentration of 100pM indicating acceptable reproducibility. The selective crystallization based QCM sensor retained its QCM response after 2 weeks without obvious decline, indicating good stability. The Possible Mechanism of In-Situ Selective Crystallization System. By controlling the exposed functional group –COOH / N(CH3)3, we realized the selective crystallization of CaCO3 on gold surface. By quantifying the crystal amount, the amount of targets are be quantified. The group –COOH has been reported to be active in stimulating nucleation of CaCO3, whereas the -N(CH3)3 inhibits nucleation. As shown in scheme 3, when the CaCl2 solution is introduced, a layer of Ca2+ ions can be adsorbed on -COOH groups23. The Ca2+ ions adsorbed on gold surface function as the nucleation cites by attracting CO2 and ultimately result in the nucleation of CaCO3. As the amount of Ca2+ is determined by the amount of –COOH groups, therefore the amount of crystals are also determined by the amount of –COOH groups, which is ultimate determined by the amount of target DNA. The polymorphism of CaCO3 crystals formed on sensor surface was also investigated (Raman spectroscopy in Figure 7). The crystals formed on the surface of QCM sensor after DNA hybridization were exclusively calcite, while on surface of pure gold and in the bulk of solution are mixture of calcite and aragonite. The results proved that –COOH groups on QCM sensor surface played the significant role in reducing nucleation of calcite. Simultaneously, the existence of –N(CH3)3 prevented the sensor surface to allow the formation of any other polymorphs of CaCO3. The cooperation of the


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two groups realized the specific nucleation determined by the target DNA. Scheme 3: -COOH induced in-situ crystallization of CaCO3

The authors declare no competing financial interest.

ACKNOWLEDGMENT This work was supported by the National Natural Science Foundation of China (21675076, 21535002, 81502585), the Special Project of Leading Talent Prospective Research of Shandong Province (ZR2016QZ001). China Postdoctoral Science Foundation (2015M572420).

REFERENCES

Figure 7. Raman spectroscopy of CaCO3 crystals. Only calcite crystals are found on sensors of selective crystallization after DNA detection: I; Besides calcite, aragonite crystals are also found in bulk solution: II; Besides calcite, aragonite crystals are also found on bare gold sensor surface: III.

CONCLUSION To enhance the sensitivity of QCM for the detection of biomolecules, in-situ selective crystallization based QCM was constructed, where -N(CH3)3 group was applied as a nucleation blocker and –COOH group as a enhancer. As a mass amplification strategy, the in-situ selective crystallization system significantly enhanced the QCM signal for more than thousands folds, and improved the sensitivity of QCM for DNA detection. A detection limit of 2 aM and a wide linear range from 10aM to 1nM are achieved for DNA. The perfect matched DNA was discriminated from 1-base mismatched DNA with high selectivity. With introduction of aptamer, the detection of Ramos cell was also realized, with a LOD of 5 cells. In conclusion, the in-situ selective crystallization based QCM strategy is enzyme-free, straightforward, ultrasensitive and selective, and showed a promising prospect for early disease diagnosis.

ASSOCIATED CONCENT Supporting Information The supporting information is available free of charge on the ACS Publications website. Sequences of DNA used in this study (Table S1), Demonstration of availability of –N(CH3)3 (10mMol/l) & DNA modified sensor for prevention of surface crystallization (Figure S1), Investigation of selectivity (Figure S2).

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