Real-time profiling Anti-EpCAM Based Immune capture, from

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Real-time profiling Anti-EpCAM Based Immune capture, from Molecules to Cells using MP-SPR Su Gao, Shuangshuang Chen, and Qinghua Lu Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.8b03898 • Publication Date (Web): 03 Jan 2019 Downloaded from http://pubs.acs.org on January 5, 2019

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Figure 1. A typical full-SPR angular spectra. 296x209mm (300 x 300 DPI)

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Figure 2. Schematic of anti-EpCAM immobilization process and cancer-cell capture on a gold-chip surface. (a) Pre-cleaned gold chip, (b) biotin-PEG400-thiol functionalization, (c) streptavidin attachment, (d) biotinylated anti-EpCAM modification, (e) immune capture of MCF-7 cells. 500x300mm (300 x 300 DPI)


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Figure 3. Sensorgram of biotin-PEG400-thiol binding to gold-chip surface in PBS buffer. Point a was the moment of biotin-PEG400-thiol injection, point b was the moment of biotin-PEG400-thiol solution substitution by PBS buffer. For equilibrium adsorption, the biotin-PEG400-thiol solution was injected three times. 296x209mm (300 x 300 DPI)


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Figure 4. Kinetics and thermodynamics of streptavidin binding to biotin-terminated SAM. (a) Sensorgram of streptavidin binding to biotin-terminated SAM via biotin–streptavidin interaction in PBS buffer. (b) Linear relationship between 1/Req and 1/C. Streptavidin with a series of concentrations (6.25, 12.5, 25, 50, 75, 100 μg mL-1, or 0.104, 0.208, 0.417, 0.833, 1.25 and 1.67 μM) bound to biotin-terminated SAM. Streptavidin has a molecular weight of 60000 g mol-1. 209x72mm (300 x 300 DPI)


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Figure 5. Kinetics and thermodynamics of biotinylated anti-EpCAM binding to streptavidin. (a) Sensorgram of biotinylated anti-EpCAM interaction with immobilized streptavidin in PBS buffer. (b) Linear relationship plot of 1/Req versus 1/C. Biotinylated anti-EpCAM with a series of concentrations (3.13, 6.25, 12.5, 25, 37.5 and 50 μg mL-1, or 0.0783, 0.156, 0.312, 0.625, 0.938 and 1.25 μM) bound to streptavidin-immobilized surface to determine binding affinity. Biotinylated anti-EpCAM has a molecular weight of 40000 g mol-1. 209x75mm (300 x 300 DPI)


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Figure 6. Sensorgram of anti-EpCAM-based immune capture of MCF-7 cells. 296x209mm (300 x 300 DPI)


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Real-time profiling Anti-EpCAM Based Immune capture, from Molecules to Cells using MP-SPR Su Gao,a Shuangshuang Chen b* and Qinghua Lu a* a

School of Chemistry and Chemical Engineering, The State Key Laboratory of

Metal Matrix Composites, Shanghai Jiao Tong University, Shanghai, 200240, China. b

School of Chemical Science and Engineering, Tong Ji University, Shanghai,

200092, China.

ABSTRACT: Antibodies of epithelial cell-adhesion-molecule (anti-EpCAM)based interfaces have proven to be highly efficient at capturing circulating tumor cells (CTCs). To achieve the bonding of anti-EpCAM to the interface, biotin and streptavidin are used to modify the surface. These processes are critical to subsequent cell-capture efficiencies. However, quantitative research on the interactions between biotin, streptavidin and biotinylated anti-EpCAM on the interface is lacking. In this work, the thermodynamics and kinetics of biomolecular interactions were determined by using surface plasmon 1


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resonance. The equilibrium binding affinities for biotinylated anti-EpCAM to streptavidin and streptavidin to biotin (illustrated by biotin-PEG400-thiol) were found to be 2.75×106 M-1 and 8.82×106 M-1, respectively. Each streptavidin can bind up to 2.30 biotinylated anti-EpCAM under thermodynamic equilibrium. The findings provide useful information to optimize the modification of anti-EpCAM and improve the capture efficiency of CTCs.

KEYWORDS: cell capture, surface plasmon resonance, antibodies of epithelial cell-adhesion-molecule,

biotin–streptavidin

interaction,

kinetics,

thermodynamics Circulating tumor cells (CTCs) are cancer cells that detach from primary tumors or metastatic sites and circulate in the peripheral blood as the cellular origin of metastasis.1 CTCs are regarded as a prognostic cancer “biomarker”, and their capture and detection reveal mysteries of cancer development and provide critically important information on cancer therapy. However, CTC separation has been technically challenging owing to the extremely low concentration (a few to hundreds per milliliter) of CTCs among a large number of hematologic cells (billions of red blood cells and millions of white blood cells per milliliter) in the blood.2 CTC capture is a continually active research field with thousands of publications focused on the design of highly efficient 2


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diagnosis protocols. Based on various physical principles, the enrichment of CTCs has been proposed for cancer identification and prognosis, such as microfiltration,3 centrifugation,4 electrophoresis5,

6

and optical tweezers.7

Despite the convenient operation and low financial cost, these approaches are still critically limited because of the low purity of captured cells, which interferes with follow-up analysis. To address these concerns, the introduction of specific antigen-ligand interactions is considered a promising solution to improve the efficiency and precision of CTC capture. Among various antibodies, the antibody of epithelial cell-adhesion molecules (EpCAM) or CD 326 has been applied universally in numerous CTC capture techniques. EpCAM is described as a weak adhesion molecule and disrupts the interaction between cadherin and cytoskeleton, which leads to neoplastic characterizations, such as cellular depolarization and contact inhibition.8-10 EpCAM forms a tight junction with claudin and develops a signal pathway in cancer progression and metastasis. In these cases, EpCAM overexpression has been found in most cancer cells and exhibits a close relationship with the first stage of cell proliferation, such as CTCs, which can be treated as CTC biomarkers.8 According to the mechanism, EpCAM antibodies have bound covalently or non-covalently to various surfaces to capture CTCs

3


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selectively.11-17 To date, many EpCAM-based CTCs diagnosis methods have been developed (CellSearch®, VeridexTM, Warren, PA).2, 18-20 In previously reported assays, biotinylated anti-EpCAM was bound biologically to the streptavidin pre-modified interfaces by the biotin–streptavidin interaction to recognize CTCs.11-16,

21-23

Despite the ubiquitous use of

streptavidin and biotinylated anti-EpCAM interaction, it is surprising that, until recently, detailed information on the successful modification of antibodies on the interface has largely been missing. X-ray photoelectron spectroscopy (XPS) measurements have been used to detect the modification of anti-EpCAM.16, 23 However, they provide only information on the emergence of new elements (nitrogen) or changes in elemental (oxygen or nitrogen) contents. Detailed information on the anti-EpCAM binding process towards the interface are still unknown. Hence, the development of a suitable method to study the kinetic and thermodynamic processes of biotinylated anti-EpCAM and streptavidin interactions is vital to improve the CTC capture efficiency.

4


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Figure 1. Typical full-SPR angular spectra. Surface plasmon resonance (SPR) was developed in the 1990s, and can be used as a label-free and highly sensitive biosensor for the real-time monitoring of biomolecular interactions, such as antigens and antibodies, receptors and ligands.24-28 As illustrated in Figure 1, a typical SPR spectrum is characterized by two angular positions, namely total internal reflection (TIR) and the minimum SPR angle.29 TIR provides refractive information on the running buffer, and changes in the position and intensity of the SPR angle represent binding events on the sensor surface. The refractive index and thickness of targeted layers on the sensor surface can be obtained by calculation. Compared with common SPR, multi-parameter SPR (MP-SPR) allows for the use of two wavelengths and the simultaneous recording of several parameters during an experiment, including TIR, the real-time change in the position and intensity of the SPR angle, the layer thickness or the mass density. Kinetics and dynamics processes can also be monitored and studied. In this paper, MP-SPR was used as a visual tool to quantify the antibody modification. As illustrated in Figure 2, biotin-PEG400-thiol was immobilized on a SPR gold chip by forming an Au–S self-assembly monolayer (SAM). Streptavidin was bound to biotin-terminated SAM via streptavidin-biotin interaction. Biotinylated anti-EpCAM was immobilized via streptavidin-biotin 5


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interaction. Finally, the anti-EpCAM functionalized surface was applied to follow cancer-cell capture. As far as we know, this is the first time that real-time monitoring of anti-EpCAM immobilization has been achieved by a visual window. The detailed thermodynamic and kinetic parameters provide critical information to optimize the modification of anti-EpCAM, and improve the capture efficiency of the CTCs.

Figure 2. Schematic of anti-EpCAM immobilization process and cancer-cell capture on a gold-chip surface. (a) Pre-cleaned gold chip, (b) biotin-PEG400thiol functionalization, (c) streptavidin attachment, (d) biotinylated anti-EpCAM modification, (e) immune capture of MCF-7 cells. Experimental section Materials Biotin-PEG400-thiol was from Ponsure Biotechnology. Streptavidin was from Sigma-Aldrich. Biotinylated anti-human EpCAM monoclonal antibody was from 6


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eBioscience. Fetal bovine serum (FBS) and Dulbecco’s modified Eagle medium (DMEM) were from Gibco company. All other cell-culture reagents were from the Beyotime Institute of Biotechnology. The proteins were dissolved in PBS to the desired working concentrations. All chemicals were used without further purification. Water used in the experiments was purified with a Hitech system to a minimum resistivity of 18.2 MΩ cm. MP-SPR instrument Biotinylated anti-EpCAM-based immune capture of cancer cells was carried out on a multi-parameter SPR device (MP-SPR Navi 200, BioNavis Ltd, Tampere, Finland) with gold chips (BioNavis Ltd, Finland). All sensor slides were made of glass and were coated with a 50 nm Au layer with a 2 nm Cr layer as the adhesive metal layer. SPR measurements were performed in angular scan mode to record the position changes of the minimum SPR peak in realtime. The analyses of biomolecular interactions were performed at a 670 nm wavelength. Prior to the SPR experiments, the sample injection system and flow channels were washed and filled with running buffer (PBS). Throughout the SPR measurement, the flow rate was 10 μL min-1 and the sensor temperature was 25°C. Attachment of biotin-PEG400-thiol via SAM

7


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For the accuracy and reproducibility of SPR measurements, a bare goldsensor surface was cleaned with ammonia/hydrogen peroxide solution. The gold chip was immersed in a mixture of NH4OH/H2O2/H2O (v/v/v = 1:1:5) at 85– 90°C for 10 min, removed from solution, rinsed with ultrapure water and dried under a stream of nitrogen. The cleaned bare gold chip was placed in a SPR slide holder for the in-situ SAM of biotin-PEG400-thiol. After the baseline had stabilized for approximately 10 min, a 500 μL sample of 1 mM biotin-PEG400thiol in PBS was injected and flowed through the sample channel for 25 min, after which excess biotin was removed by the PBS buffer. The biotin-terminated SAM can be immobilized on the gold chip via an Au–S covalent bond. Binding of streptavidin via biotin–streptavidin interaction The streptavidin can be attached to biotin-terminated SAM via a biotin– streptavidin interaction. After the monolayer of the biotin-PEG400-thiol being self-assembled, the interaction between the streptavidin and biotin on its surface was detected. Their kinetic processes were tracked by measuring the minimum SPR angle changes versus time. A sample of 500 μL of 20 μg mL-1 (333 nM) streptavidin was injected. After the sample flowed through the flow cell, excess streptavidin was removed by PBS buffer. A continuous scan was performed during the biotin–streptavidin interaction. To obtain the binding affinity from equilibrium thermodynamics, a series of streptavidin PBS solutions 8


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with concentrations from 6.25 μg mL-1 to 100 μg mL-1 were dropped onto the biotin-terminated SAM chip surface. At each streptavidin concentration, the molecular interaction process was incubated at 25℃ for 12 h, which allowed it to reach equilibrium, and a PBS wash was used to remove excess protein. The gold chip was monitored with SPR for 30 min until the response had stabilized. All measurements can be controlled and monitored by MP-SPR NaviTM Control software. Immobilization of biotinylated anti-EpCAM via streptavidin-biotin interaction Biotinylated anti-EpCAM can be immobilized on the modified gold sensor via a streptavidin-biotin interaction. To determine the kinetic parameters of biotinylated anti-EpCAM and streptavidin, a 500 μL sample of 5 μg mL-1 (125 nM) biotinylated anti-EpCAM was injected and flowed through the streptavidinimmobilized chip surface, after which excess anti-EpCAM was removed by PBS buffer. For the binding-affinity measurements, increasing concentrations of biotinylated anti-EpCAM from 3.13 μg mL-1 to 50 μg mL-1 were dropped onto the streptavidin-immobilized chip surface. Similar to the streptavidin attachment procedure, a molecular interaction process was maintained for 12 h to reach equilibrium and washed with PBS at each anti-EpCAM concentration. The gold chip was monitored by SPR for 30 min until the response had stabilized. All

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measurement processes were controlled and monitored by MP-SPR NaviTM Control software. Anti-EpCAM-based immune capture of cancer cells To check the anti-EpCAM capture of CTCs, human breast cancer cell line MCF-7 from the China Center for Type Culture Collection in Wuhan was selected as a model. MCF-7 cells were cultured in DMEM with 10% FBS and 1% penicillin-streptomycin. Cells were incubated at 37℃ and 5% CO2. Prior to each experiment, cells were harvested using trypsin-EDTA and suspended in PBS at desired concentrations to obtain mimics of CTCs in human blood. A 500 μL sample of cancer-cell suspension was injected and flowed through the antiEpCAM-modified sensor surface, after which uncaptured cells were removed by PBS buffer. A continuous scan was performed in a liquid range and recorded by MP-SPR NaviTM Control software. MP-SPR data analysis All data can be extracted and analyzed via MP-SPR NaviTM Dataviewer software after completion of the measurements. The kinetic constants and binding affinity of the biotinylated anti-EpCAM modification process were obtained through TraceDrawer for MP-SPR NaviTM. The immune capture of MCF-7 cells on an anti-EpCAM-modified sensor was obtained via MP-SPR NaviTM Dataviewer. 10


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Results and discussion Attachment of biotin-PEG400-thiol on gold sensor via SAM The binding affinity and dynamics between protein and ligand may be influenced by the binding-site density. To understand the adsorption capacity of the Au chip, biotin-PEG400-thiol was fed three times until equilibrium. During this process, the minimum SPR angle was recorded as the sensorgram in Figure 3. The minimum SPR increased sharply once biotin-PEG400-thiol was injected (point a in Figure3), which indicates a rapid self-assembly on the surface of the Au chip that benefits from the strong coordination interaction between Au-thiol pairs, and the self-assembly process reached equilibrium in a short time. When biotin-PEG400-thiol was substituted with PBS buffer (starting at point b in Fig. 3), the minimum SPR angle decreased slightly owing to the removal of unbound biotin-PEG400-thiol. The entire binding and desorption process was repeated three times. Figure 3 shows that there was no obvious change in minimum SPR angle during the latter two injections, which means that the adsorption was almost saturated during the first injection.

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Figure 3. Sensorgram of biotin-PEG400-thiol binding to gold-chip surface in PBS buffer. Point a was the moment of biotin-PEG400-thiol injection, point b was the moment of biotin-PEG400-thiol solution substitution by PBS buffer. For equilibrium adsorption, the biotin-PEG400-thiol solution was injected three times. To quantitatively evaluate the adsorbed biotin-PEG400-thiol, detailed changes in minimum SPR angle for three cycles of molecular adsorption were read as 0.568°, 0.033° and –0.013°. By multiplying the angle changes with 1000 ng cm-2 (conversion factor given by the manufacturer for protein in angular scan mode), the biotin-terminated-SAM coverage was found to be Γbiotin-1 = 568, Γbiotin-2 = 33 and Γbiotin-3

≈

0 ng cm-2, respectively.30 Because there was less

molecular adsorption in the latter two injections compared with the first injection, we assumed that saturated adsorption had been reached after three injection cycles.

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It has been reported previously that too high a biotin density in the SPR measurement produced steric hindrance and reduced the accessibility of individual biotin as binding sites for protein binding, which resulted in a lower streptavidin binding amount. 31 If we consider the two factors of binding capacity and

steric

hindrance,

biotin-PEG400-thiol

coverage

was

chosen

as

(359.0±12.7) ng cm-2, which could be achieved by regulating the injection concentration of biotin-PEG400-thiol (1mM).32 Because the biotin-PEG400thiol has a molecular weight of 703 g mol-1, the number of adsorbed molecules was calculated to be (3.07±0.11) ×1014 cm-2. Binding of streptavidin to biotin-terminated SAM Figure 4a shows the SPR sensorgram of streptavidin binding to biotinterminated SAM. To obtain the thermodynamic parameters of this process, a Langmuir model was used to fit the data. The dynamic equilibrium can be expressed as:

where A is the analyte (streptavidin), L is the ligand (biotin-PEG400-thiol) immobilized on the gold chip surface, and AL is the resulting complex. The forward and reverse reaction rates are described by the association rate constant (ka) and the dissociation rate constant (kd), respectively. In SPR

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measurements, C is the concentration of streptavidin, which is a constant; Rmax is the saturation response of streptavidin and R is the real-time response of streptavidin to biotin SAM. Thus, we find that the association rate is equal to kaC(Rmax-R), and the dissociation rate is equal to kdR. At equilibrium, the rates of association and dissociation are equal. Therefore, the final Langmuir equation that is used to fit the SPR measurements data in our research is: k𝑎C(R𝑚𝑎𝑥 ― R𝑒𝑞) = k𝑑R𝑒𝑞

(1)

The solution to Eq. (1) yields: 1 R𝑒𝑞

=

1 R𝑚𝑎𝑥

+

1 1 K𝐴R𝑚𝑎𝑥

1

×C

(2)

In Eq. (2), KA is the affinity constant, which is defined as ka/kd. 1/Req and 1/C have a linear relationship. Consequently, we can calculate the affinity constant KA from the slope of the linear relationship of 1/Req and 1/C (Figure 4).

Figure 4. Kinetics and thermodynamics of streptavidin binding to biotinterminated SAM. (a) Sensorgram of streptavidin binding to biotin-terminated SAM via biotin–streptavidin interaction in PBS buffer. (b) Linear relationship 14


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between 1/Req and 1/C. Streptavidin with a series of concentrations (6.25, 12.5, 25, 50, 75, 100 μg mL-1, or 0.104, 0.208, 0.417, 0.833, 1.25 and 1.67 μM) bound to biotin-terminated SAM. Streptavidin has a molecular weight of 60000 g mol1.

After data fitting, KA was 2.75106 M-1 and Rmax was 1479.29 ng cm-2 with a goodness above 97.9%. Therefore, the maximum coverage of streptavidin is 1479.29 ng cm-2. Streptavidin has a molecular weight of 60000 g mol-1, and so, the saturated adsorption capacity of streptavidin is 1.48×1013 per cm2. The KA of streptavidin binding to biotin in this study is consistent with the value of (7.30.2)106 M-1 reported previously by Tang et al.33 In another study, the affinity constant of streptavidin binding to 4 mol% density biotin measured by second-harmonic generation (SHG) intensity was (4.30.9)107 M-1 , which is slightly higher than the KA obtained here.31 The difference in the KA of streptavidin binding to a biotin-immobilized surface may be attributed to the difference in density of the biotin sites. To answer this question, immobilized biotins were calculated quantitatively. As mentioned above, the adsorption number of biotin-PEG400-thiol was (3.07±0.11) ×1014 cm-2. The corresponding biotin density of 4 mol% biotin on 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) was approximately 6.44×1011 cm-2 (Supporting information). We realized that the biotin density used in our experiment was approximately 477 15


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times that of the second-harmonic generation. The high biotin density used in the SPR study may produce steric hindrance, which reduces streptavidin binding. In addition, polyethylene glycol (PEG) can make surfaces resistant to non-specific adsorption.34 The intrinsic hydrophilicity and flexibility of the PEG segment of the biotin-PEG400-thiol may lead to an embedding of biotin and reduce the possibility of biotin as a protein binding site. These contributions are responsible for the low streptavidin binding. In the practical application of cell capture, the adsorption of streptavidin can be increased by controlling the immobilization amount of biotin-PEG400-thiol. The data from Figure 4a were analyzed to evaluate the detailed bound streptavidin quantitively. An abnormal sensorgram of streptavidin binding was displayed in this process: a pseudomorphic decrease appeared when streptavidin was injected but disappeared when PBS buffer flowed again. The pseudomorphic fluctuation would be shielded, which has been re-verified by quartz crystal microbalance (QCM) (Supporting Information). As shown in Figure 4, 20 μg mL-1 (333nM) streptavidin was injected and presented a rapid response to biotin. Similar to the attachment process of biotin-PEG400-thiol, a slight decrease was also found when PBS was injected, which was caused by an elimination of non-bound adhesion. The corrected responses for streptavidin adsorption were (0.58±0.07) minimum SPR angle changes, based on the angle 16


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changes and by multiplying with 1000 ng cm-2, the streptavidin coverage was found to be Γstreptavidin = 582.3±67.8 ng cm-2. If we consider that the molecular weight of streptavidin is 60 kDa, the adsorption number of streptavidin was calculated to be (5.84±0.68) ×1012 cm-2. Compared with (3.07±0.11) ×1014 biotin per cm2, we can see that one among every 53 biotin molecules can bind to streptavidin. Immobilization of biotinylated anti-EpCAM to streptavidin. Figure 5a shows the SPR sensorgram of 5 μg mL-1 biotinylated anti-EpCAM binding to immobilized streptavidin. Similarly, from a linear relationship plot of 1/Req versus 1/C (Figure 5b), we calculated the binding constant KA of biotinylated anti-EpCAM to streptavidin that covered the gold chip surface to be 8.82106 M-1. The Rmax was calculated to be 2257.37 ng cm-2. That is, the maximum coverage of biotinylated anti-EpCAM was 2257.37 ng cm-2 or 3.40×1013 per cm2 (The molecular weight of the anti-EpCAM is 40000). Compared with streptavidin with a saturated adsorption capacity of 1.48×1013 per cm2 (measured by equilibrium thermodynamics), each streptavidin is estimated to bind 2.30 biotinylated anti-EpCAM. A streptavidin protein binds at most four biotins. In this work, one or two subunits in each streptavidin molecule was already occupied by biotin-PEG400-thiol immobilized on the gold chip

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surface, and the residual biotins have been used to immobilize biotinylated antiEpCAM.

Figure 5. Kinetics and thermodynamics of biotinylated anti-EpCAM binding to streptavidin. (a) Sensorgram of biotinylated anti-EpCAM interaction with immobilized streptavidin in PBS buffer. (b) Linear relationship plot of 1/Req versus 1/C. Biotinylated anti-EpCAM with a series of concentrations (3.13, 6.25, 12.5, 25, 37.5 and 50 μg mL-1, or 0.0783, 0.156, 0.312, 0.625, 0.938 and 1.25 μM) bound to streptavidin-immobilized surface to determine binding affinity. Biotinylated anti-EpCAM has a molecular weight of 40000 g mol-1. The binding kinetics of biotinylated anti-EpCAM is another key factor when designing an anti-EpCAM-functionalized surface for cancer-cell capture. The kinetic process was analyzed based on the data of Figure 5a. For most SPR systems, when performing kinetic measurements, the diffusion of analyte (A) from the bulk solution to the surface of the gold chip cannot be ignored. In this case, the binding of biotinylated anti-EpCAM can be described as follows: 18


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This equation describes two processes. First, analyte diffuses from the bulk solution to the surface of the gold chip. Second, analyte–ligand interaction occurs. In the equation, Abulk is the analyte in bulk solution, Asurface is the analyte that diffuses to the sensor chip surface and km is the diffusion rate constant. When the diffusion of analyte from the bulk to the surface is slower than the binding rate of the analyte to the ligand, a shortage of analyte occurs at the surface. In this situation, ka and kd are limited by mass transport. For kinetic measurements, a mass-transport limitation is undesired and should be minimized as much as possible. The affinity determines how many complexes form at equilibrium, which is measured by the equilibrium dissociation constant (KD). KD can be calculated from: K𝐷 =

k𝑑 k𝑎

(3)

The kinetic parameters (ka, kd and KD) obtained through the TraceDrawer software for MP-SPR NaviTM are given in Table 1. To obtain more realistic kinetic parameters, we chose two different models provided by the software to fit the curve. One-to-one is a simple model without considering the influence of mass transport, whereas another model is a diffusion-control model. Table 1 shows that the differences in kinetic parameters obtained by the two models is 19


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small, which means that the mass-transfer effect is insignificant during the biotin–streptavidin interaction. This conclusion is consistent with previous literature by Tang et al.33 The one-to-one model was used to fit the data, and yielded a ka of (7.38±0.22)×104 M-1s-1and a kd of (3.20±0.62)×10-4 s-1. Table 1. Apparent association rate (ka), apparent dissociation rate constants (kd) and equilibrium constant (KD) of biotinylated anti-EpCAM binding to streptavidin fitted with one-to-one model and diffusion-corrected one-to-one model (DC one-to-one). Fit model

𝐤𝒂 (M-1s-1)

𝐤𝒅 (s-1)

𝐊𝑫 (M)

One-to-one

(7.38±0.22)×104

(3.20±0.62)×10-

(4.34±0.97)×10-

4

9

(3.26±5.85)×10-

(4.28±0.11)×10-

4

9

DC one-to-one

(7.61±2.97)×104

Immune capture of cancer cells In the above analyses, SPR exhibited a convenient and visual tool to understand the interaction between antigen and ligand, and the formation of the SAM of a biomolecule. SPR was also applied as a sensor for cancer-cell capture. The sensorgram of anti-EpCAM-based immune capture of MCF-7 cells 20


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is shown in Figure 6. SPR signal changes should be related closely to the morphology and mass changes of adsorbed cancer cells and refractive index changes in the medium solution.35 Because there were differences between the refractive index of cell and the surrounding buffer, the minimum SPR angle decreased first once the cell was injected, and then increased rapidly. The final minimum SPR angle shifts are 0.048° for a high concentration of MCF-7 cells and 0.023° for a low concentration. When washed with PBS, the minimum SPR angle increased sharply (right shift) due to the change in the refractive index, then remained stable after a slight decrease. This result indicates that when the cells flow through the surface modified by anti-EpCAM, the antibody interacts with the epithelial cell-adhesion molecules which overexpress on the cancer cell surface, and the MCF-7 cells are captured onto the sensor chip.

Figure 6. Sensorgram of anti-EpCAM-based immune capture of MCF-7 cells.

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Table 2. Summary of surface coverage, number of molecules and KA of antiEpCAM functionalization pathway.

Surface coverage (ng cm-2)

Number of KA (M-1) molecules (cm-2)

Biotin-PEG 400-thiol Streptavidin

359.0±12.7

AntiEpCAM

18.5±2.5

(3.07±0.11) ×1014 (5.84±0.68) ×1012 (2.83±0.38) ×1011

Step

582.3±67.8

Saturated surface coverage (ng cm-2)

Saturated number of molecule (cm-2)

2.75×106

1479.29

1.48×1013

8.82×106

2257.37

3.40×1013

Conclusions We developed a new method to track the reaction of biomolecules and the interaction of receptor–ligand on a golden-chip interface visually to study cancer-cell capture. A series of important thermodynamic and kinetic parameters on the SAM formation of biotin-PEG400-thiol on the gold-chip surface and the interaction of biotin–streptavidin and streptavidin-anti-EpCAM were obtained (see Table 2). By calculating the number of saturation-adsorbed streptavidin and anti-EpCAM in the thermodynamic equilibrium state, we found that four biotins in each streptavidin were fully occupied by biotin-PEG400-thiol and anti-EpCAM. The experimental results showed that the immobilization amount of streptavidin was determined by the pre-fixed biotin on the gold-sensor surface, which directly affected the subsequent adsorption of anti-EpCAM and the ability to capture 22


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CTCs. In order to improve the adsorption quality of anti-EpCAM, reasonable control of the immobilization amount of biotin-PEG400-thiol is necessary. The MP-SPR technology proved to be efficient for the real-time tracking of cancer-cell capture on anti-EpCAM-immobilized chip. In conclusion, our study fills the gaps in the tracking of anti-EpCAM immobilization and provides a visual window for the immune capture of cancer cells. The SPR is reliable and accurate in determining binding kinetics and thermodynamics. Association Content Supporting Information list: full SPR angular spectra for anti-EpCAM functionalization pathway and immune capture of MCF-7 cells; lowest-energy conformation of DOPC; QCM measurements of biotin-PEG400-thiol and streptavidin and SPR sensorgram of 1 mM biotin binding to streptavidin. Author Information Corresponding Authors *E-mail: [email protected] [email protected] Notes The authors declare no competing financial interest. Acknowledgment 23


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For TOC only

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Real-time profiling Anti-EpCAM Based Immune capture, from

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