Investigation of biological cell–small molecule interactions with a gold surface plasmon resonance sensor using a laser scanning confocal imaging-surface plasmon resonance system

Abstract

In our work, we investigated the interactions between a small molecule, folic acid, and biological cells through the interaction of folic acid and folate receptors using a laser scanning confocal imaging-surface plasmon resonance (LSCI-SPR) system. The changes of SPR peaks and cell concentration had good linear relationships, and fluorescence imaging provided further data. The detection limit was as low as 1.0 × 10³ cells per mL, and linear coefficients were 0.95206, 0.95454, 0.94287, 0.98711, and 0.99228 for mouse lymphoma (L5178Y TK+/−) cells, mouse lymphoma (EL4) cells, mouse T lymphocytes (Cl.Ly 1+2−/9) cells, human lung cancer (A549) cells, and human oral epidermis carcinoma (KB) cells, respectively. The results indicated that the LSCI-SPR system has potential future application in analyzing small molecule–biological cell affinity and in acquiring quantitative parameters.

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Introduction

Folic acid (FA) is an important vitamin for cell growth, differentiation, and homeostasis.^1–6 For existing pre-neoplastic and neoplastic lesions in animals, FA supplementation increases tumor burden, and different amounts of folic acid receptor (FAR) proteins exist on the cell surface.^7,8 FAR proteins have low expression in normal tissues but have high expression in malignant epithelial cells, including ovary, brain, kidney, breast, colon, lung, and myeloid cells, which have high affinity with FA (K_d < 1 nM).⁹ Hence, exploiting these unique characteristics of differential FAR overexpression through strong FA–FAR protein interactions on the surfaces of living cells is important in the fields of cancer diagnosis, therapy, and imaging.^10–14 Many studies have been conducted on the interaction mechanism between FA and FAR proteins. Results show that FAR proteins have a globular structure stabilized by eight disulfide bonds and contain a deep, open folate-binding pocket that comprises residues conserved in all receptor subtypes; therefore, FAR proteins have high affinity with FA.^15,16 Many researchers have investigated the effects of anti-cancer drugs by labeled and label-free methods.^17,18 Normally, an in-depth understanding of the interaction between cells and biomolecules is the key to identifying a favorable method in physiological processes to cure and control diseases, including cancers, diabetes, and some genetic ailments in biological processes of organisms.^19,20 With the advancement in proteomics, a wide range of methods have been developed for investigating biomolecular interactions, such as enzyme-linked immunosorbent assays, fluorescence resonance energy transfer, laser scanning confocal imaging (LSCI) microscopy, chromatography, and co-immunoprecipitation.^21–24 These methods not only require label markers but also destroy biomolecular structures and properties easily, which limit their applications in practice.^25–27 Surface plasmon resonance (SPR) and surface plasmon resonance imaging have been the most widely-used techniques for in situ and real-time measurements of surface probe–target interactions based on gold metal nano-structure surfaces.^28–31 Commercial instruments based on SPR theory have been developed and extensively used to trace contaminants in water, food, and air because of the highly sensitive optical reflectivity of gold film to dielectric changes in the environment.^32–35 Biological cell action has previously been monitored by SPR.^36,37 However, small molecule–biological cell interactions are difficult to investigate using a label-free method because of their low molecular weight, although interactions of a few small molecules have been achieved through special modification with large molecules or nanoparticles.^38,39 Detecting small molecule–biological cell interactions directly and effectively is still a challenge. In our previous work, we developed an LSCI-SPR system to monitor big molecule–biological cell interactions. We achieved quantitative results by identifying real-time nonspecific adsorption to detect biological cell–protein interactions.^25,40

In this paper, an LSCI-SPR system is used to monitor small molecule FA–biological cell interactions, where cells are DiO-labeled and include suspended and adherent cancer cells with different FAR protein expression levels. Due to specific interactions between FA and FAR proteins on the cancer cell membrane, cancer cells can be assembled on an FA-modified SPR sensor chip. With the change in cell concentration, SPR signals and fluorescence images are synchronously recorded in real time. FA reactions with different FAR cells through the binding process are observed and ascertained from the SPR signal. Binding rate values are also acquired through FA–FAR protein interactions on membranes of different adherent and suspended biological cells using a simple and rapid-preparation process. Green fluorescent points on the SPR sensor chip increased with increasing cancer cell concentration and fitted well with the changes in SPR signals. This method has potential applications for direct detection of cancer cells using small molecule–biological cell interactions.

Experiment

Materials and reagents

FA, dimethyl sulfoxide (DMSO), and cysteamine were purchased from Sigma (St. Louis, MO, USA). 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) were obtained from Huamei Biotechnology Co., China. 3,3′-Dioctadecyloxacarbocyanine perchlorate (DiO) was purchased from Molecular Probe, Inc. All biological reagents were kept in a refrigerator at 4 °C. All other chemicals were of analytical reagent grade. All solutions were prepared with ultrapure water (18.4 MΩ cm, Millipore Corp.). Exactly 0.3 mol L⁻¹ citrate buffer (pH 2.7) and 0.1 mol L⁻¹ phosphate-buffered saline (PBS, pH 7.4) were prepared. Mouse lymphoma (L5178Y TK+/−) cells, mouse lymphoma (EL4) cells, mouse T lymphocytes (Cl.Ly 1+2−/9), human lung cancer (A549) cells, and human oral epidermis carcinoma (KB) cells were provided by the Center of Cells, Peking Union Medical College.

LSCI-SPR instrument

The LSCI-SPR experimental setup (integrated with an angle modulated SPR with a laser scanning confocal microscope) and the biosensor substrates are described in Fig. S1.† A 10× objective (Nikon CFI Plan Apochromat, N.A. 0.30, W.D. 16 mm, Spring-loaded) was used for imaging, and DiO was excited by a sapphire solid laser at 488 nm with 50.1% intensity ratio and M pin hole. The biosensor substrates were prepared by sputtering 2 nm Cr and 48 nm Au onto LaSFN9 (n = 1.83) slides with mainly (111) planes with some (222) planes (Fig. S2†). The side of the slide with the Au film was immersed in the flow cell with a 0.13 mm-thick bottom, and the blank side was attached to the prism using a refractive index matching liquid. The prism, substrate, and flow cell were fixed on the stage of the inverted microscope of the LSCM (Nikon C1 Si, Japan) system. The focal plane was positioned on the gold film to detect the fluorescence image when the cancer cells were inserted in the flow cell. Two rotary stages (KS432-75, Japan) were vertically mounted coaxially with all the incident optics components and the detection devices were fixed on two Alum boards separately. The angle of incidence was freely varied by adjusting the rotation arms on the instrument by two rotary stages, and the resonance minimum at a given angle was positioned accordingly. The samples were injected into the flow cell by a spring pump (Harvard 33 Twin Syringe Pump, USA). A He–Ne laser was focused into a polarization maintaining fiber, and the fiber was fixed on one of the Alum boards. The incident light went into a collimator. After polarization, the light possessed P-polarity. A chopper (SR540, USA) was put before the LaSFN9 prism, and the reflected light signal from the photodiode (Dsi300, China) was analyzed by a lock-in amplifier (SR830, USA). The samples were then injected into the flow cell through a spring pump (Harvard 33 Twin Syringe Pump, USA). The LSCI-SPR instrument can successfully monitor the process of special interaction in real time and in situ by combining the virtues (identifying the interaction between the biomolecule and biological cell and imaging) of SPR and LSCI.

Cell culture and preparation of cell solutions

L5178Y and EL4 were grown in RPM1640 media supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin, and incubated under 5% CO₂ at 37 °C. Cl.Ly was grown in RPM1640 supplemented with 10% fetal bovine serum, 100 U mL⁻¹ IL-2, and 1% penicillin/streptomycin, and incubated under 5% CO₂ at 37 °C. Adherent cells A549 and KB were grown in McCoy’s 5A and RPM1640, respectively, supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin, and incubated under 5% CO₂ at 37 °C. A549 and KB cells were dissociated by trypsin for suspension.

Then, cell suspension concentrations (4 mL) were adjusted to 1.0 × 10³, 3.3 × 10³, 1.0 × 10⁴, 3.3 × 10⁴, 1.0 × 10⁵, 3.3 × 10⁵, and 1.0 × 10⁶ cells per mL. An appropriate volume of cell culture was concentrated by centrifugation at 1000 × g for 3 min for all the cells. The supernatant was removed; cells were resuspended in a 4 mL culture medium that contained 1 μg mL⁻¹ DiO. Then, samples were incubated under 5% CO₂ at 37 °C for 20 min. All cells were cultured in a carbon dioxide incubator until the concentration reached more than 1.0 × 10⁶ cells per mL. After the medium was removed, cells were rinsed once by PBS and then resuspended in PBS. Cell solutions were diluted to 1.0 × 10³, 3.3 × 10³, 1.0 × 10⁴, 3.3 × 10⁴, 1.0 × 10⁵, 3.3 × 10⁵, and 1.0 × 10⁶ cells per mL by plate counting.

LSCI-SPR sensor-chip fabrication and characterization

Biosensor substrates were rinsed with ultrapure water, treated with ethanol for 5 min, and then rinsed with ultrapure water again. Fig. 1 shows the cell modification process and sensing principle. Treated substrates were soaked in 10 mmol L⁻¹ cysteamine solution for 24 h to form a self-assembled monolayer on the gold surface, because the sulfide bond of cysteamine can adhere firmly to gold. 200 mg FA was added to 15 mL DMSO and dissolved completely by ultrasonic heating treatment for 20 min. Then, 20 mg NHS and 32 mg EDC were added to the FA solution. The mixed solution was diluted to 20 mL with ultrapure water. The carboxyl group of FA was activated by NHS and EDC for 30 min. Then, the activated FA solution was pumped from the sensor chip surface, which was covered with cysteamine at a 1 μL min⁻¹ reaction rate to be assembled on the surface.

Fig. 1 Schematic of SPR chip modification and interaction with biological cells.

Fourier transform infrared (FTIR) spectra confirmed the presence of cysteamine and folic acid on the gold surface. A comparison of the FTIR spectra of bare gold film, cysteamine assembled on the gold film, and FA that reacted with cysteamine on the gold surface is shown in Fig. S3.† The FTIR spectra of cysteamine-covered gold film exhibited absorption peaks at 3556 and 2933 cm⁻¹ (black curve in Fig. S3†), which corresponded to N–H functional groups and C–H bonds of cysteamine according to a previous study.⁴¹ The most characterized peaks of FA at 1690 (amide I) and 1572 (amide II) became more prominent and intense in the Au–cysteamine–FA conjugate (red curve in Fig. S3†). This result may provide evidence for extra amide bond formation during FA attachment, as described in previous work.⁴² FA assembly was carried out for 3 h to organize the processing on the solid surface and form a stable biosensor membrane. Then, PBS buffer was used to wash off the non-covalently bound FA until the SPR signal reached a steady state (Fig. S3†), thereby indicating that excess EDC, NHS, and FA were removed from the sensor surface. DiO-labeled cells of different kinds and concentrations were injected into the flow cell at a rate of 200 μL min⁻¹ and bonded to FA because of the strong FA–FAR protein interaction at 37 °C. Cysteamine coverage on the gold film was estimated to be approximately 200 ng cm⁻² (2.592 × 10⁻⁹ mol cm⁻²), as shown in Fig. S4,† by using the method mentioned by Wang (100 mdegree changes were induced by 100 ng cm⁻² coverage on the SPR sensor surface).⁴³

SPR measurements

SPR signal data were collected with LabVIEW program. SPR peaks were denoted in degrees. Different SPR signals according to changes in cell concentration and type were achieved by setting the degree of PBS without cells as a reference and fixed on a point of resonance peak with maximum slope. All experiments were conducted under the same conditions.

Results and discussion

Real-time monitoring of cell-biosensor chip preparation

To identify FA from a self-assembled monolayer on the surface of gold, the real-time modification processes were recorded at every modifying step (Fig. S4†). First, when cysteamine flowed over the gold chip, the SPR peak began to move from 58.58° to 58.82° during the modification process. Moreover, the peak increased by 0.20° after FA flowed over the gold chip. After L5178Y cells flowed over the gold chip, the SPR peak reached 59.33°, and the angle changed by 0.75° compared with the bare gold chip. Hence, the strongest response occurred when cells flowed over the chip surface, which is attributed to cells self-assembled on the gold surface by the FA–FAR protein interaction.^44–46 Results indicate that FA was successfully modified on the gold surface. Cells were specifically bound on the gold surface through the strong interaction of FA and FAR proteins (green line in Fig. S5†). Without FA, cells cannot self-assemble on the gold surface (black and red lines in Fig. S5†).

FA and L5178Y cell interaction

FA has a high-binding affinity for tumor cells because of the large amount of FAR proteins on the tumor cell membrane. First, we chose L5178Y cell to study the interaction between the small molecule FA and the cell. When the cell sensor chip was prepared, DiO-labeled L5178Y cells with different concentrations were flowed over the FA-modified gold chip. Real-time interaction processes were recorded by SPR (Fig. 2). With increasing cell concentration, the SPR-absorbing peak increased in angle (Fig. 2a). As shown in Fig. 2b, the SPR response peak changed from 59.00° to 59.52° in the stable situation with L5178Y cell concentration changing from 1.0 × 10³ cells per mL to 1.0 × 10⁶ cells per mL. A L5178Y cell concentration as low as 1.0 × 10³ cells per mL could even be detected easily within half an hour. The calibration curve of cell concentration and SPR peak angle is shown in Fig. 3. Cell concentration and SPR peak angle had a good linear relationship, as indicated by a linear coefficient of 0.95206.

Fig. 2 L5178Y capture process by the SPR sensor chip: (a) moving angle of the SPR peak with changes in concentration (1.0 × 10³, 3.3 × 10³, 1.0 × 10⁴, 3.3 × 10⁴, 1.0 × 10⁵, 3.3 × 10⁵, and 1.0 × 10⁶ cells per mL in PBS buffer); (b) real-time dynamic interaction curve between FA and L5178Y cells at different concentrations from 1.0 × 10³ cells per mL to 1.0 × 10⁶ cells per mL.

Fig. 3 SPR response with different L5178Y cell concentrations from 1.0 × 10³ cells per mL to 1.0 × 10⁶ cells per mL in PBS solution. Error bars represent the standard deviations taken from at least three independent measurements.

To identify FA–cell specific interactions, L5178Y cell suspensions flowed over the bare gold chip, cysteamine-modified gold chip, and FA-modified gold chip at a rate of 200 μL min⁻¹. After flowing for 1000 seconds, PBS solution was injected into the flow cell using a 400 μL min⁻¹ flow rate. The SPR signal returned to the same level as the initial level for the two previous chips. Only the third chip maintained a high-level SPR signal, which means that the cells could interact with FA specifically (Fig. S5†).

We also collected fluorescence images at 1.0 × 10³, 1.0 × 10⁴, 1.0 × 10⁵, and 1.0 × 10⁶ cells per mL concentration in the PBS solution. A clear green fluorescent image with high brightness and contrast was observed because DiO-labeled cells are bound to the sensor chip (Fig. 4). With the increased cell concentration, the number of green points in the view region also increased, thereby indicating that L5178Y cells attached to the surface of the SPR chip by FA–FAR protein interaction on the L5178Y cell membrane. When the cell concentration reached 1.0 × 10⁶ cells per mL, the cells congregated and formed much brighter fluorescent blocks on the sensor chip surface to induce a strong SPR response.

Fig. 4 Fluorescence images obtained from the covering of the L5178Y cells by interacting with folic acid that self-assembled on the gold film at different concentrations in the PBS solution. Letters a, b, c, and d show the L5178Y cell conditions at 1.0 × 10³, 1.0 × 10⁴, 1.0 × 10⁵, and 1.0 × 10⁶ cells per mL interacting with modified FA on the chip surface. (The scale bar of all fluorescence images is 50 μm.)

Binding process of FA and other cells

To determine the response of the SPR sensor chip to other cells, A549, Cl.Ly, KB, and EL4 cell suspensions were prepared with different concentrations to flow over the SPR sensor chip. SPR spectra were recorded in real time by the LSCI-SPR system (Fig. S6†). Under the same conditions with L5178Y, cell concentration and SPR peak had good linear relationships. Linear coefficients for A549, Cl.Ly, KB, and EL4 cells were 0.99228, 0.98711, 0.95494 and 0.94287, respectively (Fig. 5). After the SPR signals were recorded, fluorescence images were collected under the same conditions (Fig. 6). With the increase of concentration from 1.0 × 10³ cells per mL to 1.0 × 10⁶ cells per mL, an increasing number of green dots appeared in the fluorescence images for A549, KB, Cl.Ly, and EL4 cells.

Fig. 5 SPR response with different concentrations from 1.0 × 10³ cells per mL to 1.0 × 10⁶ cells per mL for A549, KB, Cl.Ly, and EL4 in PBS buffer. Error bars represent the standard deviations taken from at least three independent measurements.

Fig. 6 Fluorescence images obtained from the covering of the sensor chip with different cells at different concentrations in PBS buffer. The first line to the last line represent the changing concentrations of A549, KB, Cl.Ly, and E14 cells from 1.0 × 10³ to 1.0 × 10⁶ cells per mL, respectively. (The scale bar of all fluorescent images is 50 μm.)

To determine the affinity between FA and different cells, binding rates were estimated according to a real dynamic process at 1.0 × 10⁶ cells per mL for L5178Y, A549, KB, Cl.Ly, and EL4 cells (Fig. S6†) using d(degree)/dt; the results are 3.02 × 10⁻⁴/s, 8.05 × 10⁻⁵/s, 1.99 × 10⁻⁴/s, 3.69 × 10⁻⁶/s, and 8.09 × 10⁻⁵/s, respectively. For adherent cells, the KB cells had a larger SPR response peak and binding rate value than those of the A549 cells at the same concentration, which means that the KB cell membrane has a higher FAR protein expression than A549 cells; this finding is consistent with the findings of Parker.⁶ For suspended cells, the L5178Y cell membrane has the highest FAR protein expression, thereby enabling the LSCI-SPR system to identify the FAR protein expression of suspended cells. Some types of suspended cell may have higher FAR protein expression than adherent cells. Further experiments should be conducted in the future by using biological methods to verify these results.^6,7

Conclusions

We demonstrated a new method for monitoring the affinity of small molecules and suspended and adherent cells in PBS solution by using an angle-modulated LSCI-SPR system. Cells were bound to the surface of the SPR plate through interactions between FA and their receptor on the cell membrane. The sensor chip exhibited an obvious response to cells and could even detect 1.0 × 10³ cells per mL concentration in real time. Under the same condition, these cells exhibited different interactions with the self-assembled FA sensor chip, and the binding rate values were estimated according to the dynamic line. This study is the first to identify suspended cell–small molecule interactions. The proposed method has potential applications in cancer cell recognition for further development of specific receptors.

Acknowledgements

This work has been supported by the National Nature Science Foundation of China (Grant No. 61378044, 61227008 and 60978034) and the Instrument Developing Project of the Chinese Academy of Sciences (Grant No. YZ201235).

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Footnote

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c6ra10396k

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