Detection of [Ca2+]I Changes In Sub-Plasma Membrane Micro Domains in A Single Living Cell By an Optical Fiber-Based Nanobiosensor

Research Article

Austin J Nanomed Nanotechnol. 2014;2(4): 1022.

Detection of [Ca2+]I Changes In Sub–Plasma Membrane Micro Domains in A Single Living Cell By an Optical Fiber–Based Nanobiosensor

Wang S1,2, Ye F3, Lang X3, Fei D4, Turner APF2,5 and Ge Y2*

1College of Life Science and Technology & Advanced Biomaterials and Tissue Engineering Center, Huazhong University of Science and Technology, China

2Centre for Biomedical Engineering, Cranfield University, UK

3Division of BioEngineering, School of Chemical and Biomedical Engineering & Center for Advanced Bionanosystems, Nanyang Technological University, Singapore

4Leicester School of Pharmacy, Hawthorn Building, De Montfort University, UK

5Biosensors and Bioelectronics Centre, IFM-Linköping University, Sweden

*Corresponding author: Ge Y, Centre for Biomedical Engineering, School of Engineering, Cranfield University, Cranfield, Bedfordshire, MK43 0AL, UK

Received: May 26, 2014; Accepted: May 28, 2014; Published: May 29, 2014


An optical fiber-based nanobiosensor, for advanced detection of [Ca2+]i (i.e. intracellular Ca2+ concentration) changes in sub–plasma membrane microdomains in a single living smooth muscle cell and a single living cardiomyocyte, was successfully prepared by coating silver and then immobilizing Calcium Green–1 Dextran, a calcium ion sensitive dye, on the distal end of the nanoprobe. The constructed nanobiosensor was capable of detecting ultra–low and local intracellular calcium ion concentration within the nanomolar range, which is around the physiological level of free cytosolic calcium ion in a single living cell. The response time was less than milliseconds enabling the detection of transient elementary calcium ion signaling events associated with calcium ion microdomains. The effects of stimulants such as high potassium buffer solution and norepinephrine solution were also investigated. The resulting system could thus greatly facilitate the development of an advanced nano–diagnostic platform for in vivo and real–time sensing/diagnosing of [Ca2+]i at the single cell level.

Keywords: Nanobiosensor; Optical fiber; Intracellular Ca2+ Concentration; Nano–diagnosis; Single cell level detection


Calcium ion (Ca2+) is broadly recognized as one of the most versatile and universal signaling agents in the human body. It serves as an intracellular messenger, relaying information within cells to regulate their activity [1–3]. The evaluation of Ca2+ level is imperative not only because of its importance in signaling but also due to its lethality when prolonged increase of Ca2+ concentration ([Ca2+]) is presented.

In the spatial aspect of calcium ion channels, Ca2+ is organized into distinct microdomains (i.e. localized regions with a [Ca2+] significantly different from the bulk cytoplasm) to regulate various cellular processes in localized regions within single cells. Although Ca2+ microdomains are a key element of Ca2+ signaling, there remains a great challenge for the research community to develop a monitoring methodology with very high spatial and temporal resolution for more efficient studies of Ca2+ microdomains [4,5]. It becomes more challenging for the detection since the residing and moving area of Ca2+ in the cellular organelle and membrane microdomains is only about 200–300 nm diameter. In addition, it is of great interest to detect Ca2+ level by employing the technology of single–cell analysis, which is important not only to complement conventional bulk cell assays but also to perform dynamic analysis of interactions within individual living cells that are critical for mapping and deciphering cell signalling pathways and networks.

Many types of biosensors, such as the genetically encoded biosensors (chimeric proteins) [6], green fluorescent protein–based biosensors [7], and FRET (fluorescence resonance energy transfer)– based biosensor [8], have been designed and successfully applied to detect Calcium. More recently, by taking advantage of the unique properties of nanomaterials, nanobiosensors have been developed and exploited at an amazing rate, showing a much faster and sensitive performance as well as affordable, robust and reproducible properties [9,10]. Zamaleeva and his co–workers very recently reported a smart cell–penetrating nanobiosensor for pointillistic intracellular Ca2+ transient detection [11].

Optical fiber–based nanobiosensors, evolved from optical fiberbased mircrobiosensors, [12] have recently been developed to monitor apoptosis, [13,14] lactate release [15] and telomerase over–expression [16] demonstrating some distinct advantages such as submicron probe size, dramatically reduced sample volume, absolute detection limit and response time. In this paper, we describe the development of an optical fiber–based nanobiosensor, to detect [Ca2+]i (i.e. intracellular Ca2+ concentration) changes in sub–plasma membrane microdomains in a single living smooth muscle cell and a single living cardiomyocyte for the first time. With a temporal resolution of less than 1 millisecond, the constructed nanobiosensor was demonstrated to be well suited for intracellular monitoring of [Ca2+] in individual living cells.

Materials and Methods

Reagents and chemicals

Glucose, (3–aminopropyl) triethoxysilane (APTES) and poly–LIntroduction lysine hydrochloride (15,000–30,000 Da) were purchased from Sigma (St.Louis, MO). 50 % (v⁄v) glutaraldehyde in water was purchased from Merck (Hohenbrunn, Germany). Silver nitrate (AgNO3) was obtained from Fisher Scientific (UK). Calcium Green–1 Dextran (3000 Da) was obtained from Molecular Probes (Eugene, OR). High potassium buffer solution (105 mM) contained a final concentration of 40 mM NaCl, 105 mM KCl, 1 mM MgCl2, 2 mM CaCl2, 10 mM glucose and 10 mM HEPES (titrated to pH 7.4). Norepinephrine (NPPH) solution (1μM) was prepared in PBS. The deionized water (18.2 MΩ•cm) was collected from a Millipore Milli–Q water purification system. All other reagents were of analytical grade.

Cell cultures

Smooth muscle cells were grown in Dulbecco’s Modified Eagle’s Medium (DMEM) (PAA Laboratories, Pasching, Austria) supplemented with 10 % heat inactivated fetal bovine serum (PAA laboratories) and 50 U⁄mL penicillin⁄streptomycin (Gibco, Grand Island, NY). Cell cultures were grown in a humidified incubator at 37 °C in an atmosphere with 5.0 % CO2. Cells were seeded at a density of 6 × 106 cells⁄cm2 onto poly–L–lysine (5μg⁄mL) coated coverslips for cell attachment prior to calcium detection by our optical fiber based nanobiosensor.

The hearts of 1 to 3–day–old Sprague–Dawley rats were quickly removed into chilled Hanks’ Balanced Salt Solution (HBSS buffer, Sigma), containing (per liter): 8 g NaCl, 0.048 g Na2HPO4, 1 g D–glucose, 0.4 g KCl, 0.06 g KH2PO4, 0.35 g NaHCO3, 0.14 g CaCl2, 0.1 g MgCl2·6 H2O, 0.1 g MgSO4·7H2O, with addition of penicillin 50 U⁄ml and streptomycin 50 U⁄ml. The ventricles were cut into 1 to 2 mm cubes and dissociated by the three–step treatment with (1) 0.1 % trypsin digestion for 20 minutes at 180 rpm at 37 °C; and (2) addition of fetal bovine serum in the digestion solution (20 %) for 1–2 minutes with gentle pipetting at 24 °C; (3) dissociated cells were collected in cold low glucose Dulbecco’s Modified Eagles Medium (DMEM) medium supplemented with 3% fetal bovine serum and 1 % Insulin–Transferrin–Selenium. The above treatment was usually repeated for several rounds to obtain sufficient number of cells. The collected cell suspension was then centrifuged at 1200 rpm and 4 °C for 5 minutes. The resulting cell pellet was subsequently suspended in the supplemented DMEM medium and plated in a Petri dish for 1–1.5 hours at 37 °C to allow fibroblast cells to attach onto the Petri dish. The myocyte–containing supernatant was then collected and centrifuged (4 °C and 1200 rpm for 5 minutes). The myocyte pellets were then re–suspended and plated onto coverslip at a density of 2.5×105 cells⁄ml and cultured in the supplemented DMEM mediumat 37 °C and 5 % CO2.

Fabrication of silver–coated optical fiber–based nanoprobes

Optical fiber based nanoprobes were fabricated from F–MCC–T multimode fibers with 200 μm diameter core (Newport Corporation, Irvine, CA) by heating and pulling method using a laser based micropipette pulling device (Model P2000, Sutter Instruments, Novato, CA). The lateral wall of pulled nanofiber was coated with silver via the silver mirror reaction as we reported previously [17]. The aperture at the nanosized tip was left uncoated.

Immobilization of calcium green–1dextran on the silvercoated optical fiber–based nanoprobes

As illustrated in Figure 1, the silver–coated nanoprobes were first cleaned by immersing in pure ethanol for 45 min followed by several rinsing with deionized water and then allowed to air dry at room temperature. The air dried nanoprobes were silanized by treating with 5% (v⁄v) (3–aminopropyl) triethoxysilane (APTES) in ethanol at 60 °C for 3 h. The silanized nanoprobes were washed with ethanol and dried in a vacuum oven at 135 °C for 3 h. After drying, the nanoprobes were activated by immersing in 200 μL of 5% (v⁄v) glutaldehyde [12] at 4 °C for 12 h followed by rinsing with deionized water. Finally, the activated nanoprobes were incubated in 100 μL saturated Calcium Green–1 Dextran in DMSO solution for 24 h at 25 °C.