- Email: [email protected]
Microcantilever aptasensor for detecting epithelial tumor marker Mucin 1 and diagnosing human breast carcinoma MCF-7 cells
Sensors & Actuators: B. Chemical 297 (2019) 126759
Contents lists available at ScienceDirect
Sensors and Actuators B: Chemical journal homepage: www.elsevier.com/locate/snb
Microcantilever aptasensor for detecting epithelial tumor marker Mucin 1 and diagnosing human breast carcinoma MCF-7 cells ⁎
Chen Lia,b, Miaomiao Zhanga,b, Zhe Zhanga, Jilin Tanga,b, , Bailin Zhanga,b, a b
T
⁎
State Key Laboratory of Electroanalytical Chemistry, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun, 130022, PR China University of Science and Technology of China, Hefei, 230026, PR China
A R T I C LE I N FO
A B S T R A C T
Keywords: Microcantilever aptasensor MUC1 MCF-7 cells Dynamic process Diagnose
In this paper, a simple and novel method based on microcantilever for detecting tumor biomarker MUC1 and diagnosing human breast carcinoma MCF-7 cells was proposed. With thiolated aptamers of MUC1 self-assembled on the sensing microcantilever, the targets interact with aptamers resulting in the change of surface stress on the microcantilever. As a result, MUC1 and MCF-7 cells were successfully detected. Among them, the differential deflection of the microcantilever is linear with the logarithm of the MUC1 concentration in the range of 5–500 nM with low detection limit of 0.9 nM. However, for MCF-7 cells, the linear relationship between cantilever deflection and cell concentration ranges from 2.0*103 to 5.4*104 cells/mL with low detection limits of 213 cells/mL. In the meantime, according to the characteristics of real-time recording the change of microcantilever deflection over time, we have speculated the dynamic process of the binding of MUC1 to the aptamers. This work has confirmed that the aforementioned microcantilever aptasensor could sensitively detect other biomarkers and provide dynamic information of binding, and also could be used to identify tumor cells.
1. Introduction Protein biomarkers are generally specific tumor-related antigen molecules secreted by a tumor or a specific organ in serum, whose levels can be associated with the stages of tumors [1]. They can be regarded as indicators for tumor diagnosis. Mucin 1 (MUC1) is a highmolecular-weight protein (> 400 kDa), that is widely distributed on various mucosal surfaces of normal organisms. It is a type I transmembrane protein, which can reduce the adhesion between tumor cells and thereby enhance the tumor cells and vascular endothelial cells. The adhesion makes it easy to penetrate the blood vessel wall, thereby promoting the metastasis of tumor cells [2]. As MUC1 is over-expressed and aberrantly glycosylated in adenocarcinomas and in hematological malignancies, it is reported as a cancer biomarker for ovarian [3], gastric [4], colorectal [5,6], bladder [7], prostate [8], lung [9,10] and pancreatic [11] carcinomas, specifically for breast cancers [12]. At present, the predominant methods for MUC1 detection mainly include aptamer-antibody hybrid sandwich enzyme-linked immunosorbent assay (ELISA) [13], electrochemical aptasensors based on enzymelinked gold nanoparticles [14], paper chip on graphene oxide (GO)based fluorescence resonance energy transfer (FRET) [15]. These methods still have disadvantages such as expensive reagents,
sophisticated preparation and vulnerable to the environment which cannot satisfy the requirements of low-expression MUC1 detection [13]. Thus, a simple sensing platform for the sensitive and accurate evaluation of MUC1 in organism is urgently needed. Microcantilever sensor is a very highly attractive biochemical analysis tool with low-cost, label-free and detecting in nanoscale unit in the recent years [16–21]. In addition, the sensor can easily and compatibly integrate into “lab-on-a-chip” devices [22]. In general, the device consists of a flexible beam which can swing up and down freely when the surface stress changes. The other end normally is the body of microcantilever for supporting. Specific capture molecules can be immobilized on one side of flexible beam as receptor. Aptamers are the most commonly one due to its small size, easy to modify, chemical stability and high affinity and selectivity to target molecules. Biological interaction between target molecules and receptor can change the surface stress of microcantilever and then directly transduced into gradual bending of a microcantilever [23], which is referred to the “static mode”. Compared with other conventional sensor, microcantilever aptasensor enables the target to be determined with free-label and high precision and specificity [24]. Up to now, this sensor has been applied in the determination all kinds of analytes, such as toxic chemicals
⁎ Corresponding authors at: State Key Laboratory of Electroanalytical Chemistry, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun, 130022, PR China. E-mail addresses: [email protected] (J. Tang), [email protected] (B. Zhang).
https://doi.org/10.1016/j.snb.2019.126759 Received 6 May 2019; Received in revised form 13 June 2019; Accepted 28 June 2019 Available online 02 July 2019 0925-4005/ © 2019 Elsevier B.V. All rights reserved.
Sensors & Actuators: B. Chemical 297 (2019) 126759
C. Li, et al.
thickness of 1 μm for each one, on top of which coated by a 20 nm gold layer. Before functionalization, the array was immersed in a piranha solution (Caution! the piranha solution is submitted to specific safety rules due to its dangerousness) for 1 min. Then the microcantilever array was cleaned under ultraviolet ozone environment for 30 min to remove any contaminants. Four cleaned sensing microcantilevers were functionalized in parallel by inserting separately into four microcapillaries filled with 1 μM aptamers solution for 3 h at room temperature. While the other four microcantilevers were left as the reference ones. After that, the whole microcantilever array was immersed in 1 mM MCH for 1 h to eliminate the nonspecific adsorption and environment interference. Finally the array was dried under a stream of nitrogen gas. After each step, the whole microcantilever array was washed three times by Milli-Q water and ethanol, respectively.
2.2. Deflection measurement of microcantilever aptasensor All of the experiments were carried out on a commercial Cantisens sensor platform (Concentris GmbH, Switzerland). The test system is composed of a laser oscillator, a charge-coupled device (CCD) camera, a sample cell with volume of 250 μL and a four-quadrant photodiode as PSD as shown in Scheme 1. Firstly, the microcantilever array was fixed on a holder inserting into the sample cell. Then a beam from the laser oscillator was modulated to the free end of the microcantilever, which could be monitored by CCD. Subsequently, buffer was flowed past the sample cell under a peristaltic pump driving with a constant flow rate of 0.42 μL s−1 until the system reach equilibrium with a stable baseline. Then a 250 μL MUC1 solution of different concentrations was injected into the flowing buffer, and the nanomechanical deflection of each microcantilever was recorded in real time. The detection of MCF-7 cell and specific experiments were conducted in the same conditions. Furthermore, the temperature of all the experiments was kept a constant at 25 ± 0.01 °C. The deflection caused by compressive surface stress, i.e., the microcantilever bending downward the silicon side, is defined as negative. Conversely, the positive deflection represents the bending of microcantilever upward the gold side, which is caused by the tensile surface stress.
Scheme 1. The skeleton diagram of the experimental device on microcantilever aptasensor for detecting MUC1. The inset is the enlarged microcantilever when MUC1 was injected into a sample cell along flowing path and induced the bending of sensing microcantilevers.
[25–27], heavy metal ions [28,29], biochemical [30,31] and microorganisms [16,32–35]. In recent year, Chen et al [36] also used this sensor measuring liver cancer cell HepG 2 with high sensitivity and selectivity. In this paper, we developed a simple and novel microcantilever aptasensor to measure epithelial tumor marker MUC1. The basic principle was illustrated in Scheme 1. The sensing microcantilevers (yellow ones) were functionalized with self-assembled monolayers (SAMs) of MUC1 aptamers, which was previously modified with a thiol group and could easily react with gold surface via Au-S bond. For reference microcantilevers (pink ones), 6-mercapto-1-hexanol (MCH) were immobilized onto the gold side to remove nonspecific adsorption and the influence of temperature or perturbation. Except that, MCH can interpenetrate into the residual sites among the aptamers, rendering the aptamer to stand up and be more accessible to targets [37]. When MUC1 was captured by aptamers to form MUC1-aptamers complexes, surface stress of sensing microcantilever was changing. And then the change was translated into a bending of microcantilever, which could be monitored by a position sensitive detector (PSD). Since the microcantilever is very sensitive to the system, the interaction between MUC1 and aptamers on the microcantilever is recorded in real time. After successful detection of MUC1, we also measured human breast carcinoma MCF-7 cells by utilizing MUC1 as a tumor biomarker for further application. It is demonstrated that this aptamer-based microcantilever sensor has a promising application prospects in biomolecule detection and tumor cell recognition.
2.3. Atomic force microscopy assay In order to observe the actual modification for the microcantilever surface, we investigated the morphology before and after immobilizing the aptamers (1 μmol/L, 3 h) in binding buffer by atomic force microscope (AFM). AFM imaging was carried out on Bioscope Resolve AFM (Bruker Nano Surface, Santa Barbara, CA, USA). The images were operated in the ScanAsyst mode by using commercial Si3N4 tips (ScanAsyst-air, 0.4 N/m, Bruker). Single molecular recognition force spectroscopy (SMFS) was used to reveal the real process of the binding of MUC1 to aptamers in the sample cell. MUC1 was covalently attached to AFM tip as a molecular recognition sensor by the PEG cross-linker. AFM tips (MSCT, Bruker, USA) with a nominal spring constant of 20–30 pN/nm were functionalized as described in the literature [38]. Firstly, AFM tip, cleaned by ethanol and chloroform, was amino-functionalized by triethylamine and APTES. Then, the tip was coupled with 2 mg mL−1 PEG cross-linker and incubated in freshly prepared NaCNBH3 solution containing 0.1 mg/mL MUC1. Finally the tip was passivated the unoccupied aldehyde groups by ethanolamine and stored in PBS buffer at 4 °C. Forcedistance curves were collected on a Bioscope Resolve AFM (Bruker Nano Surface, Santa Barbara, CA, USA) in phosphate buffer solution (PBS) at room temperature. The spring constants of the microcantilevers were determined by the thermal-noise method. Both AFM images and quantitative analysis of force-distance curves were performed with NanoScope Analysis v1.9 (Bruker, USA).
2. Experiment section All materials and reagents, sequence and structure of aptamer probes and apparatus and cells cultures were described in the Supplementary material in detail. 2.1. Functionalization of microcantilevers The rectangular microcantilever array used in this assay consisted of eight microcantilevers with a length of 500 μm, width of 90 μm and 2
Sensors & Actuators: B. Chemical 297 (2019) 126759
C. Li, et al.
Fig. 1. AFM topography images of gold surface of microcantilever before (A) and after (B) modifying aptamers (1 μM), and corresponding cross-sectional profiles (below) along the lines.
injected into the sample cell, both sensing and reference microcantilever have a prominent sharply negative deflection appeared at ˜200 s and then the deflection returned soon. These results mean that both aptamer layer on sensing microcantilever and MCH layer on reference one have a great "attractive interaction" with MUC1, which may be related to nature and structure of MUC1 itself. This "attraction" was further proved by single molecular recognition force spectroscopy (SMFS) in Fig. 2C. In PBS buffer solution, we used the AFM tip modified by MUC1 to gradually approach the aptamer layer or MCH layer and recorded the approaching force-distance curve. (The typical force-distance cycle with a single molecular recognition event was displayed in Fig. S2). As shown in Fig. 2C, there both have an about 75 pN negative peak with the proximity of MUC1 to the substrate modified by aptamer or MCH, which means that molecular layer has an attractive effect the MUC1. Because MUC1 is a kind of transmembrane proteins that exhibits hydrophobic sequences or “transmembrane domains” responsible for their anchoring in the lipid bilayer and C-terminal peptides enter the cytosol [39]. We inferred that the MUC1 molecule also has an "attraction" similar anchoring effect on the molecular layer on the microcantilever. When the free MUC1 is squeezed into the molecular layer, an acting force is exerted on the cantilever, resulting in a negative deflection of the cantilever. This effect is non-specific compared to the interaction of the aptamer with MUC1 as it occurs on both reference and sensing cantilever. After the cantilever surface is saturated with MUC1, the force on the cantilever disappears, so the cantilever will recover. Besides, since this molecular monolayer on the cantilever is unlike the phospholipid layer of the cell membrane, this anchoring is unstable and the cantilever equally recovers. Wherein, the negative deflection value of the sensing microcantilever was slightly larger than the reference microcantilever. This is because in addition to the anchored attraction, there also has other interaction such as electrostatic repulsion, etc. between MUC1 and aptamer layer of sensing microcantilever. Then the MUC1 continued to produce specific and non-specific interactions with the molecules on the microcantilever, causing the microcantilever negative deflection again. Finally the deflection value of
2.4. Immunofluorescence assay For imaging the distribution of MUC1 on the cell surface, the three cells (MCF-7, Hela, HL7702) were cultured on glass bottom dishes. Firstly, the cells were washed three times by PBS preheated at 37 °C, and then fixed with 4% (w/v) paraformaldehyde in PBS for 10 min. After washing three times with PBS, the cells were incubated with 1 μM FAM-aptamers (FAM: 5(6)-carboxylfluorescein) for 1 h to visualize MUC1. Then the cells were washed with PBS three times and incubated in PBS. Fluorescence imaging was performed with a confocal microscope (TCS SP2, Leica, Germany) using a 100x/1.45 NA immersion oil objective. 3. Results and discussion 3.1. The characterization for the microcantilever surface Firstly we investigated the adsorption of the aptamers on the microcantilever surface by AFM. Fig. 1A was showed topography of the bare microcantilever, which only coated by a layer gold on the silicon surface. Fig. 1B was the topography of microcantilever probe after modifying aptamers (1 μM). Compare with bare microcantilever, we can easily see that the roughness of microcantilever surface after modifying aptamers changed significantly and there also had many little bumps. It is estimated that the height of the bump was approximately 5˜6 nm through the comparison for the two cross-section line images (below the Fig. 1A and B), which indicates the self-assemble aptamer monolayer on the surface of microcantilever was successful established. 3.2. Detection of MUC1 3.2.1. The average deflection of sensing and reference microcantilevers The average deflection signal of four sensing and reference microcantilevers monitored in real time was shown in Fig. 2A. The shaded area was the period when MUC1 was flowing through the sample cell after recording baseline about 200 s. We can see that when MUC1 was 3
Sensors & Actuators: B. Chemical 297 (2019) 126759
C. Li, et al.
Fig. 2. (A) The average deflection curves of reference and sensing microcantilevers as function of time. (B) The differential deflection curve deducted the reference one from sensing one. The shaded area was that 250 μL 25 nM MUC1 was injected into the fluid cell when the phosphate buffer was flowing about 200 s. (C) The force-distance curves recording interactions of the aptamers or MCH with MUC1 during approaching to surface. (D) The dynamic model of interaction between MUC1 and its aptamers on the microcantilever.
circular dichroism (CD) spectrum of aptamer before and after interact with MUC1(see Fig. S4 in Supporting Information). The volume of the MUC1-aptamer complex is much bigger than that of aptamer, causing the molecular layer on the microcantilever to expand and produce a compressive stress. On the other hand, the formation of MUC1-aptamer might increase repulsive force between the neighboring chains and change Gibbs free energy associated with adsorption processes. As a result, to minimize the surface free energy, the active surface tried to expand [42] accompanied by the changes of the interfacial compressive stress. So the microcantilever could be deflected further and eventually reach a new balance. Combining the experimental results, we constructed a dynamic model of microcantilever deflection for detection MUC1 shown in Fig. 2D. As the sample enters the sample cell, the bending of the microcantilever undergoes four stages: (i) Initially the microcantilever functionalized by aptamers bends negatively a bit whereas the deflection curve is defined as zero (baseline), which is corresponded to stage I. (ii) When MUC1 is introduced, the electrostatic repulsion between MUC1 and aptamer is preferentially exhibited among other interactions, which induced negative deflection of the microcantilever as marked in stage II. (iii) Then, the aptamer conformation changes to be well-defined in order to bind MUC1, at which point the microcantilever recovers to some extent (bending positively) as shown in stage III. (iv) Finally, the specific interaction between MUC1 and aptamers dominates in all the interaction, resulting in the microcantilever continues to bend negatively and greatly in stage IV. In summary, the microcantilever can produce an evaluable deformation due to the addition of MUC1, which demonstrated that the MUC1 can be determined by aptamer-based microcantilever sensor.
sensing microcantilever could reach maximum at -84.04 nm in the shading area, which of reference microcantilever was -37.54 nm. While the signal was recorded more than 800 s, the negative deflections of both sensing and reference microcantilever had a slight rebound (bending positively) about ˜18 nm and then stabilized. The appearance of rebound may be that the flowing buffer washed nonspecific adsorbates.
3.2.2. The differential deflection by subtracting the reference ones from the sensing microcantilevers Fig. 2B showed the differential deflection by subtracting the reference ones from the sensing microcantilevers. After subtracting nonspecific interaction, we noticed that there still exists a peak of ˜−20 nm initially as marked by the dotted square. This phenomenon also occurred at other concentrations of MUC1 (see Fig. S3 in Supporting Information). Considering that the aptamer modified on the microcantilever is negatively charged and MUC1 is also negatively charged at pH 7.5 (The isoelectric point of MUC1 is 6.18), we conclude that due to the electrostatic repulsion, the compressive stress on the active surface increases and causes the microcantilever bent downward at the initial stage of MUC1 entering the sample cell. However, when the target gradually reacts with SAMs, driven by the specific interaction between each other, the conformation of aptamer changes to be well-defined from more random (disorder), which is similar to the mechanism proposed by the induced-fit process [40,41]. Therefore, the interaction force between the aptamers is decreased and the compressive stress on the surface of the microcantilever is also reduced, showing a positive signal. Finally, since the target is completely combined with the aptamers, this interaction prevails over other ones and the compressive stress increases causing the microcantilever bending negatively sharply. We hypothesized that there exists two aspects for inducing microcantilever deflection by the interaction between aptamer and MUC1. On the one hand, MUC1 was tightly wrapped by aptamer to form MUC1-aptamer complexes on the microcantilever surface, resulting in a change in the conformation of the aptamers which can be verified by
3.3. The analytical performance of this microcantilever sensor The real time deflection response induced by different amounts MUC1-aptamer was presented in Fig. 3A. As displayed in the figure, with increasing the concentration of MUC1, the deflection of 4
Sensors & Actuators: B. Chemical 297 (2019) 126759
C. Li, et al.
in inset. The related regression equation of differential deflection (DD) signals to the logarithm of concentration of MUC1 (cMUC1) was DD = -39.917 log cMUC1 + 7.134 with a correlation coefficient of 0.995. The limit of detection at a signal-to-noise ratio of 3 was 0.9 nM. The error bar represented the relative standard deviation (RSD) of four identical microcantilevers within an array. The comparison of microcantilevers sensors with other methods for the detection of MUC1 was shown in Table S1. Compared to other previous reported biosensors, this method offered acceptable sensitivity for detection of MUC1. The selectivity of the microcantilever sensor for detecting MUC1 was assessed in Fig. 3C. Some possible interferences, for example, α-1fetoprotein (AFP), carcino-embryonic antigen (CEA), prostate specific antigen (PSA), human serum albumin (HSA), bovine serum albumin (BSA) and histone were introduced. In this assay, the same experimental procedures and target concentration (50 nM) was implemented with our aforementioned experimental section. As shown in the Fig. 3C, although the same concentration of interference analytes was injected, there was no a significant deflection appeared. Namely, only MUC1 could be recognized by this microcantilever sensor. Hence, our protocol was proposed to be sufficiently selective for the determination of target MUC1. 3.4. Diagnosis of human breast carcinoma MCF-7 cells As we known, MUC1 was overexpressed on the surface of human breast carcinoma MCF-7 cells [12]. By assessing the concentration of breast cancer cells, breast cancer can be diagnosed. Some studies using MUC1 as the tumor marker to detect breast cancer cells have been reported [43,44]. We also explored detecting MCF-7 breast cancer cells by using MUC1 as tumor biomarker based on microcantilever sensor. The aptamers modified on the cantilever is capable to interact with MUC1 expressing on MCF-7 cells. On the one hand, since the size of cells is much larger than MUC1, they can cause greater steric repulsive force, impelling the microcantilevers to produce bigger compress stress and deflect more negatively. On the other hand, the negative charges on the cell membrane also generated electrostatic repulsion with aptamers, inducing the sensing microcantilevers bending negatively severely. As a result, the cantilever can produce large deflection for determining MCF7 cells. Fig. 4 showed the analytical performance of measuring the MCF7 cells. Fig. 4A was the differential deflection induced by MCF-7 cells at different concentration. The deflection increased proportionally to the number of MCF-7 cells in the range of 2.0*103 - 5.4*104 cells/mL. The regression equation was DD = -0.01137 cMCF-7 + 2.439, with a regression coefficient of 0.99. The limit of detection was 213 cells/mL at S/N of 3. The comparison of microcantilevers sensors with other methods for recognition MCF-7 cells was shown in Table S2. Compared to other previous reported biosensors, this method offered acceptable sensitivity for detection of MCF-7. To evaluate the selectivity of detecting MCF-7 cells, we explored deflection induced by Hela and HL7702, which one is cancer cell and the other is normal cell. Different cells versus different concentrations generated deflection signal was shown in Fig. 4C. The differential deflection of MCF-7 was −118.095 nm, −344.649 nm and −598.682 nm at 1.1*104, 2.7*104 and 5.4*104 cells/mL, respectively. While for Hela and HL7702, the deflections were −42.349 nm, −142.477 nm, −304.119 nm and −31.936 nm, −51.690 nm, −110.953 nm, respectively. We compared the deflection values caused by MCF-7 cells with those caused by Hela and HL7702 cells and the multiple relationships were shown in in Table S3. Compared with normal cell HL7702, the deflection signal of MCF-7 cells is much larger at any concentration, which suggests that MUC1 over-expressed in MCF-7 cells and this method can differentiate between normal and cancer cells. While compared with Hela cells, the specificity of this method is significantly reduced. This is because that the aptamer we used is specific for MUC1 and MUC1 is also expressed on other tumor cells. Finally, we validated the results by laser confocal fluorescence
Fig. 3. (A) Differential deflection curves as a function of time for different concentrations of MUC1 from 0.5 nM to 500 nM. (B) Differential deflection of microcantilevers as a function of MUC 1 concentrations when the deflection reached an equilibrium value. The inset was the linear relationship of differential deflection (at 1000s) versus MUC1 concentration. (C) The differential deflection with injection of 50 nM AFP, CEA, PSA, BSA, HSA, histone and MUC1, respectively. The error bars represent the standard deviation of data (n = 3).
microcantilevers increased gradually. Fig. 3B was plots of microcantilever differential deflection as a function of logarithm of MUC1 concentration when the bending was stabilized after recording 1000s. The linear range between differential deflection and logarithm of MUC1 concentration was 5 nM - 500 nM and the calibration curve was shown 5
Sensors & Actuators: B. Chemical 297 (2019) 126759
C. Li, et al.
Fig. 4. (A) Differential deflection curves as a function of time with different concentrations of MCF-7 from 2.0*103 to 7.2*105 cells/mL. (B) The linear relationship of differential deflection values as a function of MCF-7 concentrations at equilibrium. (C) The ability for diagnosing tumor cell MCF-7. The error bars represent the standard deviation of data (n = 3). (D) Laser confocal fluorescence imaging of these three kinds of cells under dark and bright field. Excitation wavelength: 492 nm, scale bar: 30 μm.
imaging. When MCF-7, Hela, HL7702 cells were stained with FAM and imaging, MCF-7 cells showed a bright green fluorescence; however, Hela cells showed weaker fluorescence and even HL7702 cells displayed no fluorescence, which was consistent with experimental results by microcantilever. These results further indicate that the developed microcantilever sensor has pretty good efficiency in the diagnosis of MCF-7 cells.
679–688. [2] T.L. Thirkill, T. Cao, M. Stout, T.N. Blankenship, A. Barakat, G.C. Douglas, MUC1 is involved in trophoblast transendothelial migration, Biochimica et Biophysica Acta (BBA) – Mol. Cell Res. 1773 (2007) 1007–1014. [3] C. Van Elssen, P.W.H. Frings, F.J. Bot, K.K. Van de Vijver, M.B. Huls, B. Meek, et al., Expression of aberrantly glycosylated Mucin-1 in ovarian cancer, Histopathology 57 (2010) 597–606. [4] N.W. Toribara, A.M. Roberton, S.B. Ho, W.L. Kuo, E. Gum, J.W. Hicks, et al., Human gastric mucin. Identification of a unique species by expression cloning, J. Biol. Chem. 268 (1993) 5879–5885. [5] R. Aoki, S. Tanaka, K. Haruman, M. Yoshihara, K. Sumii, G. Kajiyama, et al., MUC-1 expression as a predictor of the curative endoscopic treatment of submucosally invasive colorectal carcinoma, Dis. Colon Rectum 41 (1998) 1262–1272. [6] W.M.C. Mulder, M.J. Stukart, E. de Windt, J. Wagstaff, R.J. Scheper, E. Bloemena, Mucin-1-related T cell infiltration in colorectal carcinoma, Cancer Immunol. Immunother. 42 (1996) 351–356. [7] M. Retz, J. Lehmann, C. Röder, B. Plötz, J. Harder, J. Eggers, et al., Differential mucin MUC7 gene expression in invasive bladder carcinoma in contrast to uniform MUC1 and MUC2 gene expression in both normal urothelium and bladder carcinoma, Cancer Res. 58 (1998) 5662–5666. [8] O. Andrén, K. Fall, S.O. Andersson, M.A. Rubin, T.A. Bismar, M. Karlsson, et al., MUC-1 gene is associated with prostate cancer death: a 20-year follow-up of a population-based study in Sweden, Br. J. Cancer 97 (2007) 730. [9] A. Ohgami, T. Tsuda, T. Osaki, T. Mitsudomi, Y. Morimoto, T. Higashi, et al., MUC1 mucin mRNA expression in stage I lung adenocarcinoma and its association with early recurrence, Ann. Thorac. Surg. 67 (1999) 810–814. [10] J. Gao, M.J. McConnell, B. Yu, J. Li, J.M. Balko, E.P. Black, et al., MUC1 is a downstream target of STAT3 and regulates lung cancer cell survival and invasion, Int. J. Oncol. 35 (2009) 337–345. [11] D.M. Besmer, J.M. Curry, L.D. Roy, T.L. Tinder, M. Sahraei, J.L. Schettini, et al., Pancreatic Ductal Adenocarcinoma (PDA) mice lacking Mucin 1 have a profound defect in tumor growth and metastasis, Cancer Res. 71 (2011) 4432. [12] D.F. Hayes, R. Mesa-Tejada, L.D. Papsidero, G.A. Croghan, A.H. Korzun, L. Norton, et al., Prediction of prognosis in primary breast cancer by detection of a high molecular weight mucin-like antigen using monoclonal antibodies DF3, F36/22, and CU18: a cancer and leukemia group b study, J. Clin. Oncol. 9 (1991) 1113–1123. [13] C.S.M. Ferreira, K. Papamichael, G. Guilbault, T. Schwarzacher, J. Gariepy, S. Missailidis, DNA aptamers against the MUC1 tumour marker: design of aptamer–antibody sandwich ELISA for the early diagnosis of epithelial tumours, Anal. Bioanal. Chem. 390 (2008) 1039–1050. [14] R. Hu, W. Wen, Q. Wang, H. Xiong, X. Zhang, H. Gu, et al., Novel electrochemical aptamer biosensor based on an enzyme–gold nanoparticle dual label for the ultrasensitive detection of epithelial tumour marker MUC1, Biosens. Bioelectron. 53 (2014) 384–389. [15] H. Li, X. Fang, H. Cao, J. Kong, Paper-based fluorescence resonance energy transfer assay for directly detecting nucleic acids and proteins, Biosens. Bioelectron. 80 (2016) 79–83. [16] B. Anja, D. Søren, K. Stephan Sylvest, S. Silvan, T. Maria, Cantilever-like micromechanical sensors, Rep. Prog. Phys. 74 (2011) 036101. [17] P.A. Rasmussen, J. Thaysen, O. Hansen, S.C. Eriksen, A. Boisen, Optimised
4. Conclusion In conclusion, a simple and novel strategy for detecting epithelial tumor marker MUC1 based on microcantilever array sensor has been demonstrated successfully. This sensor was based on changes of surface stress on microcantilevers induced by target analyst and aptamers. In the method, MUC1 was been determined simply and label-free with good sensitivity and selectivity. In the meantime, the dynamic process of the binding of MUC1 to the aptamer according to the relationship of the deflection signal of the microcantilever over time was been displayed simultaneously. Furthermore, it is also can be used to diagnose human breast carcinoma MCF-7 cells with MUC1 as the cancer biomarker. Therefore, it is worth pointing out that the successful use of microcantilever aptasensor in biomarker in the early stage of cancer, even in extending to disease diagnosis and biological analysis. Acknowledgments This work was supported by the National Natural Science Foundation of China (No. 21375122 and No. 21775144). Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.snb.2019.126759. References [1] J. Wu, Z. Fu, F. Yan, H. Ju, Biomedical and clinical applications of immunoassays and immunosensors for tumor markers, TrAC Trends Anal. Chem. 26 (2007)
6
Sensors & Actuators: B. Chemical 297 (2019) 126759
C. Li, et al.
[18] [19] [20] [21]
[22] [23]
[24] [25]
[26]
[27] [28]
[29] [30]
[31]
4860–4865. [32] R.L. Gunter, W.G. Delinger, K. Manygoats, A. Kooser, T.L. Porter, Viral detection using an embedded piezoresistive microcantilever sensor, Sens. Actuators A Phys. 107 (2003) 219–224. [33] V. Shrotriya, G. Li, Y. Yao, C.-W. Chu, Y. Yang, Transition metal oxides as the buffer layer for polymer photovoltaic cells, Appl. Phys. Lett. 88 (2006) 073508. [34] N. Nugaeva, K.Y. Gfeller, N. Backmann, M. Düggelin, H.P. Lang, H.-J. Güntherodt, et al., An antibody-sensitized microfabricated cantilever for the growth detection of aspergillus niger spores, Microsc. Microanal. 13 (2007) 13–17. [35] A. Subramanian, P.I. Oden, S.J. Kennel, K.B. Jacobson, R.J. Warmack, T. Thundat, et al., Glucose biosensing using an enzyme-coated microcantilever, Appl. Phys. Lett. 81 (2002) 385–387. [36] X. Chen, Y. Pan, H. Liu, X. Bai, N. Wang, B. Zhang, Label-free detection of liver cancer cells by aptamer-based microcantilever biosensor, Biosens. Bioelectron. 79 (2016) 353–358. [37] S. Zheng, J.H. Choi, S.M. Lee, K.S. Hwang, S.K. Kim, T.S. Kim, Analysis of DNA hybridization regarding the conformation of molecular layer with piezoelectric microcantilevers, Lab Chip 11 (2011) 63–69. [38] J. Zhang, L.A. Chtcheglova, R. Zhu, P. Hinterdorfer, B. Zhang, J. Tang, Nanoscale organization of human GnRH-R on human bladder cancer cells, Anal. Chem. 86 (2014) 2458–2464. [39] F.-G.H.S. Müller, MUC1: the polymorphic appearance of a human mucin, Glycobiology 10 (2000) 439–449. [40] D.E. Koshland, Application of a theory of enzyme specificity to protein synthesis, Proc. Natl. Acad. Sci. U. S. A. 44 (1958) 98–104. [41] G.R. Bishop, J. Ren, B.C. Polander, B.D. Jeanfreau, J.O. Trent, J.B. Chaires, Energetic basis of molecular recognition in a DNA aptamer, Biophys. Chem. 126 (2007) 165–175. [42] J. Tamayo, P.M. Kosaka, J.J. Ruz, Á. San Paulo, M. Calleja, Biosensors based on nanomechanical systems, Chem. Soc. Rev. 42 (2013) 1287–1311. [43] Y. Li, Y. Zhang, M. Zhao, Q. Zhou, L. Wang, H. Wang, et al., A simple aptamerfunctionalized gold nanorods based biosensor for the sensitive detection of MCF-7 breast cancer cells, Chem. Commun. 52 (2016) 3959–3961. [44] C. Li, Y. Meng, S. Wang, M. Qian, J. Wang, W. Lu, et al., Mesoporous carbon nanospheres featured fluorescent aptasensor for multiple diagnosis of cancer in vitro and in vivo, ACS Nano 9 (2015) 12096–12103.
cantilever biosensor with piezoresistive read-out, Ultramicroscopy 97 (2003) 371–376. X. Yu, J. Thaysen, O. Hansen, A. Boisen, Optimization of sensitivity and noise in piezoresistive cantilevers, J. Appl. Phys. 92 (2002) 6296–6301. J. Fritz, Cantilever biosensors, Analyst 133 (2008) 855–863. S.K. Vashist, A review of microcantilevers for sensing applications, J. Nanotechnol. 3 (2007) 1–15 online. H.P. Lang, C. Gerber, Microcantilever sensors, in: P. Samorì (Ed.), STM and AFM Studies on (Bio)Molecular Systems: Unravelling the Nanoworld, Springer, Berlin Heidelberg, Berlin, Heidelberg, 2008, pp. 1–27. M. Alvarez, L.M. Lechuga, Microcantilever-based platforms as biosensing tools, Analyst 135 (2010) 827–836. N. Khemthongcharoen, W. Wonglumsom, A. Suppat, K. Jaruwongrungsee, A. Tuantranont, C. Promptmas, Piezoresistive microcantilever-based DNA sensor for sensitive detection of pathogenic Vibrio cholerae O1 in food sample, Biosens. Bioelectron. 63 (2015) 347–353. K.M. Goeders, J.S. Colton, L.A. Bottomley, Microcantilevers: sensing chemical interactions via mechanical motion, Chem. Rev. 108 (2008) 522–542. C. Karnati, H. Du, H.-F. Ji, X. Xu, Y. Lvov, A. Mulchandani, et al., Organophosphorus hydrolase multilayer modified microcantilevers for organophosphorus detection, Biosens. Bioelectron. 22 (2007) 2636–2642. X. Yan, Y. Tang, H. Ji, Y. Lvov, T. Thundat, Detection of organophosphates using an acetyl cholinesterase (AChE) coated microcantilever, Instrum. Sci. Technol. 32 (2004) 175–183. J. Yang, L.R. Roberts, Hepatocellular carcinoma: a global view, Nat. Rev. Gastroenterol. Hepatol. 7 (2010) 448–458. S. Cherian, R.K. Gupta, B.C. Mullin, T. Thundat, Detection of heavy metal ions using protein-functionalized microcantilever sensors, Biosens. Bioelectron. 19 (2003) 411–416. S. Velanki, S. Kelly, T. Thundat, D.A. Blake, H.-F. Ji, Detection of Cd(II) using antibody-modified microcantilever sensors, Ultramicroscopy 107 (2007) 1123–1128. Y. Arntz, J.D. Seelig, H.P. Lang, J. Zhang, P. Hunziker, J.P. Ramseyer, et al., Labelfree protein assay based on a nanomechanical cantilever array, Nanotechnology 14 (2003) 86. G.A. Baker, R. Desikan, T. Thundat, Label-free sugar detection using phenylboronic acid-functionalized piezoresistive microcantilevers, Anal. Chem. 80 (2008)
7
Contents lists available at ScienceDirect
Sensors and Actuators B: Chemical journal homepage: www.elsevier.com/locate/snb
Microcantilever aptasensor for detecting epithelial tumor marker Mucin 1 and diagnosing human breast carcinoma MCF-7 cells ⁎
Chen Lia,b, Miaomiao Zhanga,b, Zhe Zhanga, Jilin Tanga,b, , Bailin Zhanga,b, a b
T
⁎
State Key Laboratory of Electroanalytical Chemistry, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun, 130022, PR China University of Science and Technology of China, Hefei, 230026, PR China
A R T I C LE I N FO
A B S T R A C T
Keywords: Microcantilever aptasensor MUC1 MCF-7 cells Dynamic process Diagnose
In this paper, a simple and novel method based on microcantilever for detecting tumor biomarker MUC1 and diagnosing human breast carcinoma MCF-7 cells was proposed. With thiolated aptamers of MUC1 self-assembled on the sensing microcantilever, the targets interact with aptamers resulting in the change of surface stress on the microcantilever. As a result, MUC1 and MCF-7 cells were successfully detected. Among them, the differential deflection of the microcantilever is linear with the logarithm of the MUC1 concentration in the range of 5–500 nM with low detection limit of 0.9 nM. However, for MCF-7 cells, the linear relationship between cantilever deflection and cell concentration ranges from 2.0*103 to 5.4*104 cells/mL with low detection limits of 213 cells/mL. In the meantime, according to the characteristics of real-time recording the change of microcantilever deflection over time, we have speculated the dynamic process of the binding of MUC1 to the aptamers. This work has confirmed that the aforementioned microcantilever aptasensor could sensitively detect other biomarkers and provide dynamic information of binding, and also could be used to identify tumor cells.
1. Introduction Protein biomarkers are generally specific tumor-related antigen molecules secreted by a tumor or a specific organ in serum, whose levels can be associated with the stages of tumors [1]. They can be regarded as indicators for tumor diagnosis. Mucin 1 (MUC1) is a highmolecular-weight protein (> 400 kDa), that is widely distributed on various mucosal surfaces of normal organisms. It is a type I transmembrane protein, which can reduce the adhesion between tumor cells and thereby enhance the tumor cells and vascular endothelial cells. The adhesion makes it easy to penetrate the blood vessel wall, thereby promoting the metastasis of tumor cells [2]. As MUC1 is over-expressed and aberrantly glycosylated in adenocarcinomas and in hematological malignancies, it is reported as a cancer biomarker for ovarian [3], gastric [4], colorectal [5,6], bladder [7], prostate [8], lung [9,10] and pancreatic [11] carcinomas, specifically for breast cancers [12]. At present, the predominant methods for MUC1 detection mainly include aptamer-antibody hybrid sandwich enzyme-linked immunosorbent assay (ELISA) [13], electrochemical aptasensors based on enzymelinked gold nanoparticles [14], paper chip on graphene oxide (GO)based fluorescence resonance energy transfer (FRET) [15]. These methods still have disadvantages such as expensive reagents,
sophisticated preparation and vulnerable to the environment which cannot satisfy the requirements of low-expression MUC1 detection [13]. Thus, a simple sensing platform for the sensitive and accurate evaluation of MUC1 in organism is urgently needed. Microcantilever sensor is a very highly attractive biochemical analysis tool with low-cost, label-free and detecting in nanoscale unit in the recent years [16–21]. In addition, the sensor can easily and compatibly integrate into “lab-on-a-chip” devices [22]. In general, the device consists of a flexible beam which can swing up and down freely when the surface stress changes. The other end normally is the body of microcantilever for supporting. Specific capture molecules can be immobilized on one side of flexible beam as receptor. Aptamers are the most commonly one due to its small size, easy to modify, chemical stability and high affinity and selectivity to target molecules. Biological interaction between target molecules and receptor can change the surface stress of microcantilever and then directly transduced into gradual bending of a microcantilever [23], which is referred to the “static mode”. Compared with other conventional sensor, microcantilever aptasensor enables the target to be determined with free-label and high precision and specificity [24]. Up to now, this sensor has been applied in the determination all kinds of analytes, such as toxic chemicals
⁎ Corresponding authors at: State Key Laboratory of Electroanalytical Chemistry, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun, 130022, PR China. E-mail addresses: [email protected] (J. Tang), [email protected] (B. Zhang).
https://doi.org/10.1016/j.snb.2019.126759 Received 6 May 2019; Received in revised form 13 June 2019; Accepted 28 June 2019 Available online 02 July 2019 0925-4005/ © 2019 Elsevier B.V. All rights reserved.
Sensors & Actuators: B. Chemical 297 (2019) 126759
C. Li, et al.
thickness of 1 μm for each one, on top of which coated by a 20 nm gold layer. Before functionalization, the array was immersed in a piranha solution (Caution! the piranha solution is submitted to specific safety rules due to its dangerousness) for 1 min. Then the microcantilever array was cleaned under ultraviolet ozone environment for 30 min to remove any contaminants. Four cleaned sensing microcantilevers were functionalized in parallel by inserting separately into four microcapillaries filled with 1 μM aptamers solution for 3 h at room temperature. While the other four microcantilevers were left as the reference ones. After that, the whole microcantilever array was immersed in 1 mM MCH for 1 h to eliminate the nonspecific adsorption and environment interference. Finally the array was dried under a stream of nitrogen gas. After each step, the whole microcantilever array was washed three times by Milli-Q water and ethanol, respectively.
2.2. Deflection measurement of microcantilever aptasensor All of the experiments were carried out on a commercial Cantisens sensor platform (Concentris GmbH, Switzerland). The test system is composed of a laser oscillator, a charge-coupled device (CCD) camera, a sample cell with volume of 250 μL and a four-quadrant photodiode as PSD as shown in Scheme 1. Firstly, the microcantilever array was fixed on a holder inserting into the sample cell. Then a beam from the laser oscillator was modulated to the free end of the microcantilever, which could be monitored by CCD. Subsequently, buffer was flowed past the sample cell under a peristaltic pump driving with a constant flow rate of 0.42 μL s−1 until the system reach equilibrium with a stable baseline. Then a 250 μL MUC1 solution of different concentrations was injected into the flowing buffer, and the nanomechanical deflection of each microcantilever was recorded in real time. The detection of MCF-7 cell and specific experiments were conducted in the same conditions. Furthermore, the temperature of all the experiments was kept a constant at 25 ± 0.01 °C. The deflection caused by compressive surface stress, i.e., the microcantilever bending downward the silicon side, is defined as negative. Conversely, the positive deflection represents the bending of microcantilever upward the gold side, which is caused by the tensile surface stress.
Scheme 1. The skeleton diagram of the experimental device on microcantilever aptasensor for detecting MUC1. The inset is the enlarged microcantilever when MUC1 was injected into a sample cell along flowing path and induced the bending of sensing microcantilevers.
[25–27], heavy metal ions [28,29], biochemical [30,31] and microorganisms [16,32–35]. In recent year, Chen et al [36] also used this sensor measuring liver cancer cell HepG 2 with high sensitivity and selectivity. In this paper, we developed a simple and novel microcantilever aptasensor to measure epithelial tumor marker MUC1. The basic principle was illustrated in Scheme 1. The sensing microcantilevers (yellow ones) were functionalized with self-assembled monolayers (SAMs) of MUC1 aptamers, which was previously modified with a thiol group and could easily react with gold surface via Au-S bond. For reference microcantilevers (pink ones), 6-mercapto-1-hexanol (MCH) were immobilized onto the gold side to remove nonspecific adsorption and the influence of temperature or perturbation. Except that, MCH can interpenetrate into the residual sites among the aptamers, rendering the aptamer to stand up and be more accessible to targets [37]. When MUC1 was captured by aptamers to form MUC1-aptamers complexes, surface stress of sensing microcantilever was changing. And then the change was translated into a bending of microcantilever, which could be monitored by a position sensitive detector (PSD). Since the microcantilever is very sensitive to the system, the interaction between MUC1 and aptamers on the microcantilever is recorded in real time. After successful detection of MUC1, we also measured human breast carcinoma MCF-7 cells by utilizing MUC1 as a tumor biomarker for further application. It is demonstrated that this aptamer-based microcantilever sensor has a promising application prospects in biomolecule detection and tumor cell recognition.
2.3. Atomic force microscopy assay In order to observe the actual modification for the microcantilever surface, we investigated the morphology before and after immobilizing the aptamers (1 μmol/L, 3 h) in binding buffer by atomic force microscope (AFM). AFM imaging was carried out on Bioscope Resolve AFM (Bruker Nano Surface, Santa Barbara, CA, USA). The images were operated in the ScanAsyst mode by using commercial Si3N4 tips (ScanAsyst-air, 0.4 N/m, Bruker). Single molecular recognition force spectroscopy (SMFS) was used to reveal the real process of the binding of MUC1 to aptamers in the sample cell. MUC1 was covalently attached to AFM tip as a molecular recognition sensor by the PEG cross-linker. AFM tips (MSCT, Bruker, USA) with a nominal spring constant of 20–30 pN/nm were functionalized as described in the literature [38]. Firstly, AFM tip, cleaned by ethanol and chloroform, was amino-functionalized by triethylamine and APTES. Then, the tip was coupled with 2 mg mL−1 PEG cross-linker and incubated in freshly prepared NaCNBH3 solution containing 0.1 mg/mL MUC1. Finally the tip was passivated the unoccupied aldehyde groups by ethanolamine and stored in PBS buffer at 4 °C. Forcedistance curves were collected on a Bioscope Resolve AFM (Bruker Nano Surface, Santa Barbara, CA, USA) in phosphate buffer solution (PBS) at room temperature. The spring constants of the microcantilevers were determined by the thermal-noise method. Both AFM images and quantitative analysis of force-distance curves were performed with NanoScope Analysis v1.9 (Bruker, USA).
2. Experiment section All materials and reagents, sequence and structure of aptamer probes and apparatus and cells cultures were described in the Supplementary material in detail. 2.1. Functionalization of microcantilevers The rectangular microcantilever array used in this assay consisted of eight microcantilevers with a length of 500 μm, width of 90 μm and 2
Sensors & Actuators: B. Chemical 297 (2019) 126759
C. Li, et al.
Fig. 1. AFM topography images of gold surface of microcantilever before (A) and after (B) modifying aptamers (1 μM), and corresponding cross-sectional profiles (below) along the lines.
injected into the sample cell, both sensing and reference microcantilever have a prominent sharply negative deflection appeared at ˜200 s and then the deflection returned soon. These results mean that both aptamer layer on sensing microcantilever and MCH layer on reference one have a great "attractive interaction" with MUC1, which may be related to nature and structure of MUC1 itself. This "attraction" was further proved by single molecular recognition force spectroscopy (SMFS) in Fig. 2C. In PBS buffer solution, we used the AFM tip modified by MUC1 to gradually approach the aptamer layer or MCH layer and recorded the approaching force-distance curve. (The typical force-distance cycle with a single molecular recognition event was displayed in Fig. S2). As shown in Fig. 2C, there both have an about 75 pN negative peak with the proximity of MUC1 to the substrate modified by aptamer or MCH, which means that molecular layer has an attractive effect the MUC1. Because MUC1 is a kind of transmembrane proteins that exhibits hydrophobic sequences or “transmembrane domains” responsible for their anchoring in the lipid bilayer and C-terminal peptides enter the cytosol [39]. We inferred that the MUC1 molecule also has an "attraction" similar anchoring effect on the molecular layer on the microcantilever. When the free MUC1 is squeezed into the molecular layer, an acting force is exerted on the cantilever, resulting in a negative deflection of the cantilever. This effect is non-specific compared to the interaction of the aptamer with MUC1 as it occurs on both reference and sensing cantilever. After the cantilever surface is saturated with MUC1, the force on the cantilever disappears, so the cantilever will recover. Besides, since this molecular monolayer on the cantilever is unlike the phospholipid layer of the cell membrane, this anchoring is unstable and the cantilever equally recovers. Wherein, the negative deflection value of the sensing microcantilever was slightly larger than the reference microcantilever. This is because in addition to the anchored attraction, there also has other interaction such as electrostatic repulsion, etc. between MUC1 and aptamer layer of sensing microcantilever. Then the MUC1 continued to produce specific and non-specific interactions with the molecules on the microcantilever, causing the microcantilever negative deflection again. Finally the deflection value of
2.4. Immunofluorescence assay For imaging the distribution of MUC1 on the cell surface, the three cells (MCF-7, Hela, HL7702) were cultured on glass bottom dishes. Firstly, the cells were washed three times by PBS preheated at 37 °C, and then fixed with 4% (w/v) paraformaldehyde in PBS for 10 min. After washing three times with PBS, the cells were incubated with 1 μM FAM-aptamers (FAM: 5(6)-carboxylfluorescein) for 1 h to visualize MUC1. Then the cells were washed with PBS three times and incubated in PBS. Fluorescence imaging was performed with a confocal microscope (TCS SP2, Leica, Germany) using a 100x/1.45 NA immersion oil objective. 3. Results and discussion 3.1. The characterization for the microcantilever surface Firstly we investigated the adsorption of the aptamers on the microcantilever surface by AFM. Fig. 1A was showed topography of the bare microcantilever, which only coated by a layer gold on the silicon surface. Fig. 1B was the topography of microcantilever probe after modifying aptamers (1 μM). Compare with bare microcantilever, we can easily see that the roughness of microcantilever surface after modifying aptamers changed significantly and there also had many little bumps. It is estimated that the height of the bump was approximately 5˜6 nm through the comparison for the two cross-section line images (below the Fig. 1A and B), which indicates the self-assemble aptamer monolayer on the surface of microcantilever was successful established. 3.2. Detection of MUC1 3.2.1. The average deflection of sensing and reference microcantilevers The average deflection signal of four sensing and reference microcantilevers monitored in real time was shown in Fig. 2A. The shaded area was the period when MUC1 was flowing through the sample cell after recording baseline about 200 s. We can see that when MUC1 was 3
Sensors & Actuators: B. Chemical 297 (2019) 126759
C. Li, et al.
Fig. 2. (A) The average deflection curves of reference and sensing microcantilevers as function of time. (B) The differential deflection curve deducted the reference one from sensing one. The shaded area was that 250 μL 25 nM MUC1 was injected into the fluid cell when the phosphate buffer was flowing about 200 s. (C) The force-distance curves recording interactions of the aptamers or MCH with MUC1 during approaching to surface. (D) The dynamic model of interaction between MUC1 and its aptamers on the microcantilever.
circular dichroism (CD) spectrum of aptamer before and after interact with MUC1(see Fig. S4 in Supporting Information). The volume of the MUC1-aptamer complex is much bigger than that of aptamer, causing the molecular layer on the microcantilever to expand and produce a compressive stress. On the other hand, the formation of MUC1-aptamer might increase repulsive force between the neighboring chains and change Gibbs free energy associated with adsorption processes. As a result, to minimize the surface free energy, the active surface tried to expand [42] accompanied by the changes of the interfacial compressive stress. So the microcantilever could be deflected further and eventually reach a new balance. Combining the experimental results, we constructed a dynamic model of microcantilever deflection for detection MUC1 shown in Fig. 2D. As the sample enters the sample cell, the bending of the microcantilever undergoes four stages: (i) Initially the microcantilever functionalized by aptamers bends negatively a bit whereas the deflection curve is defined as zero (baseline), which is corresponded to stage I. (ii) When MUC1 is introduced, the electrostatic repulsion between MUC1 and aptamer is preferentially exhibited among other interactions, which induced negative deflection of the microcantilever as marked in stage II. (iii) Then, the aptamer conformation changes to be well-defined in order to bind MUC1, at which point the microcantilever recovers to some extent (bending positively) as shown in stage III. (iv) Finally, the specific interaction between MUC1 and aptamers dominates in all the interaction, resulting in the microcantilever continues to bend negatively and greatly in stage IV. In summary, the microcantilever can produce an evaluable deformation due to the addition of MUC1, which demonstrated that the MUC1 can be determined by aptamer-based microcantilever sensor.
sensing microcantilever could reach maximum at -84.04 nm in the shading area, which of reference microcantilever was -37.54 nm. While the signal was recorded more than 800 s, the negative deflections of both sensing and reference microcantilever had a slight rebound (bending positively) about ˜18 nm and then stabilized. The appearance of rebound may be that the flowing buffer washed nonspecific adsorbates.
3.2.2. The differential deflection by subtracting the reference ones from the sensing microcantilevers Fig. 2B showed the differential deflection by subtracting the reference ones from the sensing microcantilevers. After subtracting nonspecific interaction, we noticed that there still exists a peak of ˜−20 nm initially as marked by the dotted square. This phenomenon also occurred at other concentrations of MUC1 (see Fig. S3 in Supporting Information). Considering that the aptamer modified on the microcantilever is negatively charged and MUC1 is also negatively charged at pH 7.5 (The isoelectric point of MUC1 is 6.18), we conclude that due to the electrostatic repulsion, the compressive stress on the active surface increases and causes the microcantilever bent downward at the initial stage of MUC1 entering the sample cell. However, when the target gradually reacts with SAMs, driven by the specific interaction between each other, the conformation of aptamer changes to be well-defined from more random (disorder), which is similar to the mechanism proposed by the induced-fit process [40,41]. Therefore, the interaction force between the aptamers is decreased and the compressive stress on the surface of the microcantilever is also reduced, showing a positive signal. Finally, since the target is completely combined with the aptamers, this interaction prevails over other ones and the compressive stress increases causing the microcantilever bending negatively sharply. We hypothesized that there exists two aspects for inducing microcantilever deflection by the interaction between aptamer and MUC1. On the one hand, MUC1 was tightly wrapped by aptamer to form MUC1-aptamer complexes on the microcantilever surface, resulting in a change in the conformation of the aptamers which can be verified by
3.3. The analytical performance of this microcantilever sensor The real time deflection response induced by different amounts MUC1-aptamer was presented in Fig. 3A. As displayed in the figure, with increasing the concentration of MUC1, the deflection of 4
Sensors & Actuators: B. Chemical 297 (2019) 126759
C. Li, et al.
in inset. The related regression equation of differential deflection (DD) signals to the logarithm of concentration of MUC1 (cMUC1) was DD = -39.917 log cMUC1 + 7.134 with a correlation coefficient of 0.995. The limit of detection at a signal-to-noise ratio of 3 was 0.9 nM. The error bar represented the relative standard deviation (RSD) of four identical microcantilevers within an array. The comparison of microcantilevers sensors with other methods for the detection of MUC1 was shown in Table S1. Compared to other previous reported biosensors, this method offered acceptable sensitivity for detection of MUC1. The selectivity of the microcantilever sensor for detecting MUC1 was assessed in Fig. 3C. Some possible interferences, for example, α-1fetoprotein (AFP), carcino-embryonic antigen (CEA), prostate specific antigen (PSA), human serum albumin (HSA), bovine serum albumin (BSA) and histone were introduced. In this assay, the same experimental procedures and target concentration (50 nM) was implemented with our aforementioned experimental section. As shown in the Fig. 3C, although the same concentration of interference analytes was injected, there was no a significant deflection appeared. Namely, only MUC1 could be recognized by this microcantilever sensor. Hence, our protocol was proposed to be sufficiently selective for the determination of target MUC1. 3.4. Diagnosis of human breast carcinoma MCF-7 cells As we known, MUC1 was overexpressed on the surface of human breast carcinoma MCF-7 cells [12]. By assessing the concentration of breast cancer cells, breast cancer can be diagnosed. Some studies using MUC1 as the tumor marker to detect breast cancer cells have been reported [43,44]. We also explored detecting MCF-7 breast cancer cells by using MUC1 as tumor biomarker based on microcantilever sensor. The aptamers modified on the cantilever is capable to interact with MUC1 expressing on MCF-7 cells. On the one hand, since the size of cells is much larger than MUC1, they can cause greater steric repulsive force, impelling the microcantilevers to produce bigger compress stress and deflect more negatively. On the other hand, the negative charges on the cell membrane also generated electrostatic repulsion with aptamers, inducing the sensing microcantilevers bending negatively severely. As a result, the cantilever can produce large deflection for determining MCF7 cells. Fig. 4 showed the analytical performance of measuring the MCF7 cells. Fig. 4A was the differential deflection induced by MCF-7 cells at different concentration. The deflection increased proportionally to the number of MCF-7 cells in the range of 2.0*103 - 5.4*104 cells/mL. The regression equation was DD = -0.01137 cMCF-7 + 2.439, with a regression coefficient of 0.99. The limit of detection was 213 cells/mL at S/N of 3. The comparison of microcantilevers sensors with other methods for recognition MCF-7 cells was shown in Table S2. Compared to other previous reported biosensors, this method offered acceptable sensitivity for detection of MCF-7. To evaluate the selectivity of detecting MCF-7 cells, we explored deflection induced by Hela and HL7702, which one is cancer cell and the other is normal cell. Different cells versus different concentrations generated deflection signal was shown in Fig. 4C. The differential deflection of MCF-7 was −118.095 nm, −344.649 nm and −598.682 nm at 1.1*104, 2.7*104 and 5.4*104 cells/mL, respectively. While for Hela and HL7702, the deflections were −42.349 nm, −142.477 nm, −304.119 nm and −31.936 nm, −51.690 nm, −110.953 nm, respectively. We compared the deflection values caused by MCF-7 cells with those caused by Hela and HL7702 cells and the multiple relationships were shown in in Table S3. Compared with normal cell HL7702, the deflection signal of MCF-7 cells is much larger at any concentration, which suggests that MUC1 over-expressed in MCF-7 cells and this method can differentiate between normal and cancer cells. While compared with Hela cells, the specificity of this method is significantly reduced. This is because that the aptamer we used is specific for MUC1 and MUC1 is also expressed on other tumor cells. Finally, we validated the results by laser confocal fluorescence
Fig. 3. (A) Differential deflection curves as a function of time for different concentrations of MUC1 from 0.5 nM to 500 nM. (B) Differential deflection of microcantilevers as a function of MUC 1 concentrations when the deflection reached an equilibrium value. The inset was the linear relationship of differential deflection (at 1000s) versus MUC1 concentration. (C) The differential deflection with injection of 50 nM AFP, CEA, PSA, BSA, HSA, histone and MUC1, respectively. The error bars represent the standard deviation of data (n = 3).
microcantilevers increased gradually. Fig. 3B was plots of microcantilever differential deflection as a function of logarithm of MUC1 concentration when the bending was stabilized after recording 1000s. The linear range between differential deflection and logarithm of MUC1 concentration was 5 nM - 500 nM and the calibration curve was shown 5
Sensors & Actuators: B. Chemical 297 (2019) 126759
C. Li, et al.
Fig. 4. (A) Differential deflection curves as a function of time with different concentrations of MCF-7 from 2.0*103 to 7.2*105 cells/mL. (B) The linear relationship of differential deflection values as a function of MCF-7 concentrations at equilibrium. (C) The ability for diagnosing tumor cell MCF-7. The error bars represent the standard deviation of data (n = 3). (D) Laser confocal fluorescence imaging of these three kinds of cells under dark and bright field. Excitation wavelength: 492 nm, scale bar: 30 μm.
imaging. When MCF-7, Hela, HL7702 cells were stained with FAM and imaging, MCF-7 cells showed a bright green fluorescence; however, Hela cells showed weaker fluorescence and even HL7702 cells displayed no fluorescence, which was consistent with experimental results by microcantilever. These results further indicate that the developed microcantilever sensor has pretty good efficiency in the diagnosis of MCF-7 cells.
679–688. [2] T.L. Thirkill, T. Cao, M. Stout, T.N. Blankenship, A. Barakat, G.C. Douglas, MUC1 is involved in trophoblast transendothelial migration, Biochimica et Biophysica Acta (BBA) – Mol. Cell Res. 1773 (2007) 1007–1014. [3] C. Van Elssen, P.W.H. Frings, F.J. Bot, K.K. Van de Vijver, M.B. Huls, B. Meek, et al., Expression of aberrantly glycosylated Mucin-1 in ovarian cancer, Histopathology 57 (2010) 597–606. [4] N.W. Toribara, A.M. Roberton, S.B. Ho, W.L. Kuo, E. Gum, J.W. Hicks, et al., Human gastric mucin. Identification of a unique species by expression cloning, J. Biol. Chem. 268 (1993) 5879–5885. [5] R. Aoki, S. Tanaka, K. Haruman, M. Yoshihara, K. Sumii, G. Kajiyama, et al., MUC-1 expression as a predictor of the curative endoscopic treatment of submucosally invasive colorectal carcinoma, Dis. Colon Rectum 41 (1998) 1262–1272. [6] W.M.C. Mulder, M.J. Stukart, E. de Windt, J. Wagstaff, R.J. Scheper, E. Bloemena, Mucin-1-related T cell infiltration in colorectal carcinoma, Cancer Immunol. Immunother. 42 (1996) 351–356. [7] M. Retz, J. Lehmann, C. Röder, B. Plötz, J. Harder, J. Eggers, et al., Differential mucin MUC7 gene expression in invasive bladder carcinoma in contrast to uniform MUC1 and MUC2 gene expression in both normal urothelium and bladder carcinoma, Cancer Res. 58 (1998) 5662–5666. [8] O. Andrén, K. Fall, S.O. Andersson, M.A. Rubin, T.A. Bismar, M. Karlsson, et al., MUC-1 gene is associated with prostate cancer death: a 20-year follow-up of a population-based study in Sweden, Br. J. Cancer 97 (2007) 730. [9] A. Ohgami, T. Tsuda, T. Osaki, T. Mitsudomi, Y. Morimoto, T. Higashi, et al., MUC1 mucin mRNA expression in stage I lung adenocarcinoma and its association with early recurrence, Ann. Thorac. Surg. 67 (1999) 810–814. [10] J. Gao, M.J. McConnell, B. Yu, J. Li, J.M. Balko, E.P. Black, et al., MUC1 is a downstream target of STAT3 and regulates lung cancer cell survival and invasion, Int. J. Oncol. 35 (2009) 337–345. [11] D.M. Besmer, J.M. Curry, L.D. Roy, T.L. Tinder, M. Sahraei, J.L. Schettini, et al., Pancreatic Ductal Adenocarcinoma (PDA) mice lacking Mucin 1 have a profound defect in tumor growth and metastasis, Cancer Res. 71 (2011) 4432. [12] D.F. Hayes, R. Mesa-Tejada, L.D. Papsidero, G.A. Croghan, A.H. Korzun, L. Norton, et al., Prediction of prognosis in primary breast cancer by detection of a high molecular weight mucin-like antigen using monoclonal antibodies DF3, F36/22, and CU18: a cancer and leukemia group b study, J. Clin. Oncol. 9 (1991) 1113–1123. [13] C.S.M. Ferreira, K. Papamichael, G. Guilbault, T. Schwarzacher, J. Gariepy, S. Missailidis, DNA aptamers against the MUC1 tumour marker: design of aptamer–antibody sandwich ELISA for the early diagnosis of epithelial tumours, Anal. Bioanal. Chem. 390 (2008) 1039–1050. [14] R. Hu, W. Wen, Q. Wang, H. Xiong, X. Zhang, H. Gu, et al., Novel electrochemical aptamer biosensor based on an enzyme–gold nanoparticle dual label for the ultrasensitive detection of epithelial tumour marker MUC1, Biosens. Bioelectron. 53 (2014) 384–389. [15] H. Li, X. Fang, H. Cao, J. Kong, Paper-based fluorescence resonance energy transfer assay for directly detecting nucleic acids and proteins, Biosens. Bioelectron. 80 (2016) 79–83. [16] B. Anja, D. Søren, K. Stephan Sylvest, S. Silvan, T. Maria, Cantilever-like micromechanical sensors, Rep. Prog. Phys. 74 (2011) 036101. [17] P.A. Rasmussen, J. Thaysen, O. Hansen, S.C. Eriksen, A. Boisen, Optimised
4. Conclusion In conclusion, a simple and novel strategy for detecting epithelial tumor marker MUC1 based on microcantilever array sensor has been demonstrated successfully. This sensor was based on changes of surface stress on microcantilevers induced by target analyst and aptamers. In the method, MUC1 was been determined simply and label-free with good sensitivity and selectivity. In the meantime, the dynamic process of the binding of MUC1 to the aptamer according to the relationship of the deflection signal of the microcantilever over time was been displayed simultaneously. Furthermore, it is also can be used to diagnose human breast carcinoma MCF-7 cells with MUC1 as the cancer biomarker. Therefore, it is worth pointing out that the successful use of microcantilever aptasensor in biomarker in the early stage of cancer, even in extending to disease diagnosis and biological analysis. Acknowledgments This work was supported by the National Natural Science Foundation of China (No. 21375122 and No. 21775144). Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.snb.2019.126759. References [1] J. Wu, Z. Fu, F. Yan, H. Ju, Biomedical and clinical applications of immunoassays and immunosensors for tumor markers, TrAC Trends Anal. Chem. 26 (2007)
6
Sensors & Actuators: B. Chemical 297 (2019) 126759
C. Li, et al.
[18] [19] [20] [21]
[22] [23]
[24] [25]
[26]
[27] [28]
[29] [30]
[31]
4860–4865. [32] R.L. Gunter, W.G. Delinger, K. Manygoats, A. Kooser, T.L. Porter, Viral detection using an embedded piezoresistive microcantilever sensor, Sens. Actuators A Phys. 107 (2003) 219–224. [33] V. Shrotriya, G. Li, Y. Yao, C.-W. Chu, Y. Yang, Transition metal oxides as the buffer layer for polymer photovoltaic cells, Appl. Phys. Lett. 88 (2006) 073508. [34] N. Nugaeva, K.Y. Gfeller, N. Backmann, M. Düggelin, H.P. Lang, H.-J. Güntherodt, et al., An antibody-sensitized microfabricated cantilever for the growth detection of aspergillus niger spores, Microsc. Microanal. 13 (2007) 13–17. [35] A. Subramanian, P.I. Oden, S.J. Kennel, K.B. Jacobson, R.J. Warmack, T. Thundat, et al., Glucose biosensing using an enzyme-coated microcantilever, Appl. Phys. Lett. 81 (2002) 385–387. [36] X. Chen, Y. Pan, H. Liu, X. Bai, N. Wang, B. Zhang, Label-free detection of liver cancer cells by aptamer-based microcantilever biosensor, Biosens. Bioelectron. 79 (2016) 353–358. [37] S. Zheng, J.H. Choi, S.M. Lee, K.S. Hwang, S.K. Kim, T.S. Kim, Analysis of DNA hybridization regarding the conformation of molecular layer with piezoelectric microcantilevers, Lab Chip 11 (2011) 63–69. [38] J. Zhang, L.A. Chtcheglova, R. Zhu, P. Hinterdorfer, B. Zhang, J. Tang, Nanoscale organization of human GnRH-R on human bladder cancer cells, Anal. Chem. 86 (2014) 2458–2464. [39] F.-G.H.S. Müller, MUC1: the polymorphic appearance of a human mucin, Glycobiology 10 (2000) 439–449. [40] D.E. Koshland, Application of a theory of enzyme specificity to protein synthesis, Proc. Natl. Acad. Sci. U. S. A. 44 (1958) 98–104. [41] G.R. Bishop, J. Ren, B.C. Polander, B.D. Jeanfreau, J.O. Trent, J.B. Chaires, Energetic basis of molecular recognition in a DNA aptamer, Biophys. Chem. 126 (2007) 165–175. [42] J. Tamayo, P.M. Kosaka, J.J. Ruz, Á. San Paulo, M. Calleja, Biosensors based on nanomechanical systems, Chem. Soc. Rev. 42 (2013) 1287–1311. [43] Y. Li, Y. Zhang, M. Zhao, Q. Zhou, L. Wang, H. Wang, et al., A simple aptamerfunctionalized gold nanorods based biosensor for the sensitive detection of MCF-7 breast cancer cells, Chem. Commun. 52 (2016) 3959–3961. [44] C. Li, Y. Meng, S. Wang, M. Qian, J. Wang, W. Lu, et al., Mesoporous carbon nanospheres featured fluorescent aptasensor for multiple diagnosis of cancer in vitro and in vivo, ACS Nano 9 (2015) 12096–12103.
cantilever biosensor with piezoresistive read-out, Ultramicroscopy 97 (2003) 371–376. X. Yu, J. Thaysen, O. Hansen, A. Boisen, Optimization of sensitivity and noise in piezoresistive cantilevers, J. Appl. Phys. 92 (2002) 6296–6301. J. Fritz, Cantilever biosensors, Analyst 133 (2008) 855–863. S.K. Vashist, A review of microcantilevers for sensing applications, J. Nanotechnol. 3 (2007) 1–15 online. H.P. Lang, C. Gerber, Microcantilever sensors, in: P. Samorì (Ed.), STM and AFM Studies on (Bio)Molecular Systems: Unravelling the Nanoworld, Springer, Berlin Heidelberg, Berlin, Heidelberg, 2008, pp. 1–27. M. Alvarez, L.M. Lechuga, Microcantilever-based platforms as biosensing tools, Analyst 135 (2010) 827–836. N. Khemthongcharoen, W. Wonglumsom, A. Suppat, K. Jaruwongrungsee, A. Tuantranont, C. Promptmas, Piezoresistive microcantilever-based DNA sensor for sensitive detection of pathogenic Vibrio cholerae O1 in food sample, Biosens. Bioelectron. 63 (2015) 347–353. K.M. Goeders, J.S. Colton, L.A. Bottomley, Microcantilevers: sensing chemical interactions via mechanical motion, Chem. Rev. 108 (2008) 522–542. C. Karnati, H. Du, H.-F. Ji, X. Xu, Y. Lvov, A. Mulchandani, et al., Organophosphorus hydrolase multilayer modified microcantilevers for organophosphorus detection, Biosens. Bioelectron. 22 (2007) 2636–2642. X. Yan, Y. Tang, H. Ji, Y. Lvov, T. Thundat, Detection of organophosphates using an acetyl cholinesterase (AChE) coated microcantilever, Instrum. Sci. Technol. 32 (2004) 175–183. J. Yang, L.R. Roberts, Hepatocellular carcinoma: a global view, Nat. Rev. Gastroenterol. Hepatol. 7 (2010) 448–458. S. Cherian, R.K. Gupta, B.C. Mullin, T. Thundat, Detection of heavy metal ions using protein-functionalized microcantilever sensors, Biosens. Bioelectron. 19 (2003) 411–416. S. Velanki, S. Kelly, T. Thundat, D.A. Blake, H.-F. Ji, Detection of Cd(II) using antibody-modified microcantilever sensors, Ultramicroscopy 107 (2007) 1123–1128. Y. Arntz, J.D. Seelig, H.P. Lang, J. Zhang, P. Hunziker, J.P. Ramseyer, et al., Labelfree protein assay based on a nanomechanical cantilever array, Nanotechnology 14 (2003) 86. G.A. Baker, R. Desikan, T. Thundat, Label-free sugar detection using phenylboronic acid-functionalized piezoresistive microcantilevers, Anal. Chem. 80 (2008)
7