Introduction

Recent developments in nanotechnology have led to the development of fiber-optic nanosensors with nanoscale dimensions suitable for intracellular measurements. The possibility of monitoring in-vivo biological processes within single living cells could greatly improve our understanding of cellular function, thereby revolutionizing cell biology. An advantage of fiber-optic sensors is the small size of optical fibers; this enables intracellular sensing of physiological and biological processes in nano-environments. Biosensors, which use biological probes coupled to a transducer, have been developed during the last two decades for environmental, industrial, and biomedical diagnostics. These biosensors usually consists of a bio-sensitive layer that can contain either biological recognition elements or be made of biological recognition elements covalently attached to the transducer. The interaction between the target analyte and the bioreceptor is designed to produce a physicochemical perturbation that can be converted into a measurable effect such as an electrical signal. Bioreceptors are important elements providing specificity for biosensor technology because they enable binding of the specific analyte of interest to the sensor for the measurement with minimum interference from other components in complex sampled mixtures. Biological sensing elements can be either a biological molecular species (e.g. an antibody, an enzyme, a protein, or a nucleic acid) or a living biological system (e.g. cells, tissue, or whole organisms) that utilizes a biochemical mechanism for recognition.Extensive research and development activity in our laboratory have been devoted to the development of a variety of fiber-optic chemical sensors and biosensors [1–3] which have paved the way for the development of fiber-optic nanosensors.

Fiber optical nanosensors can generally be defined as nanometer (nm) scale (nm=10−9 m) measurement devices that consist of a biologically or chemically sensitive layer. This layer can either contain biological recognition elements or be made of chemical recognition elements covalently attached to an optical transducer. Interaction between the target analyte and the sensing layer is designed to produce a physicochemical perturbation that can be converted into a measurable effect such as an electrical signal. The development and application of submicron fiber-optic chemical sensors for chemical analysis has been reported [4–7] with distal diameters between 20 and 500 nm. They have been used to study the submicron spatial resolution achievable using near-field scanning optical microscopy (NSOM). The combination of NSOM and surface-enhanced Raman scattering (SERS) has been demonstrated to detect biochemicals on solid substrates with sub-wavelength 100-nm spatial resolution [8, 9]. On the other hand, submicron fiber-optic biological sensors, nanobiosensors, have been developed and used to detect biochemical targets inside single cells [10–14]. Single-cell analysis (SCA) technology is important not only to complement conventional bulk cell assays but also to perform dynamic analysis of interactions within individual living cells, which are critical for mapping and deciphering cell-signaling pathways and networks. SCA is also especially critical when primary cells are obtained using surgical procedures and cannot be propagated for study, because of the number available or the type of cell, and therefore have to be analyzed with a technology capable of SCA. Conventional techniques for intracellular analysis require “fixing” cell samples before analysis. The fixing process often destroys cellular viability and may significantly alter intracellular architecture. Fiber-optic nanobiosensors could provide the tools to investigate important biological processes at the cellular level in vivo. Not only can antibodies be developed against specific epitopes, but also an array of antibodies can be established to investigate the overall structural architecture of a given protein. Finally, the most significant advantage of nanosensors for cell monitoring is the minimal invasiveness of the technique. The integration of these advances in biotechnology and nanotechnology could lead to a new generation of nanosystems with unprecedented sensitivity and selectivity for probing sub-compartments of living cells at the molecular level. In this article we provide an overview of the principle, development and applications of fiber-optic nanobiosensors. The article provides a description of the fabrication method for fiber-optic nanobiosensors and detection systems, and applications in single-cell analysis.

Efforts to track the distribution, localization and movement of specific cellular components in cellular signaling pathways without adversely perturbing the physiological equilibrium of a cellular system has led to the development and investigation of fiber-optic nanosensors with dimensions suitable for performing intracellular measurements in single living cells. Fiber-optic nanosensors can be classified into one of two broad categories, fiber optic chemical nanosensors and fiber-optic nanobiosensors, depending on the probe used [15–18]. Both types of sensor have been used to provide a reliable method for monitoring various biochemicals in microscopic environments. They have also been used to facilitate the detection and analysis of different species within single cells. Such fundamental analysis of biological processes within living cells is important because they can dramatically improve our understanding of dynamic cellular function. These nanosensors employ biological sensing elements, which can be either a biological molecular species (e.g. an antibody, an enzyme, a protein, or a nucleic acid) or a living biological system (e.g. cells, tissue, or whole organisms) that utilizes a biochemical mechanism for recognition or chemical sensing element, for example a pH-sensitive dye.

Fiber-optic nanobiosensors

Development of fiber-optic nanobiosensors

Because of the complexity of biological systems and the number of possible interferences with chemical nanosensors, there is an obvious need for added specificity. This specificity can be achieved by the development of nanobiosensors. Like their larger counterparts, biosensors, the added specificity provided by biological receptor molecules has enabled for the analysis of complex environments with minimal effects from other species. Nanobiosensors that use biological probes coupled to a transducer have been developed and investigated extensively by the Vo-Dinh Group [1–3, 19–21]. Biological probes provide nanobiosensors with the important property of specificity to facilitate sensing in complex matrices such as that of a single cell. They enable binding of the specific analyte of interest to the nanosensor for measurement, with minimum interference from other components in a complex matrix.

The fabrication of fiber-optic nanosensors is a crucial prerequisite for their successful development and application. A fairly well characterized method for fabricating the nanofiber tips is the so-called “heat and pull” technique. This method involves pulling nanotips from a larger diameter (600 μm) silica optical fiber using a special fiber-pulling device (Sutter Instruments P-2000). It is based on local heating of a glass fiber using a CO2 laser or a heat filament and subsequently pulling the fiber apart. The resulting tip shapes depend strongly on the temperature and the timing of the procedure. First, the fiber is then secured into the fiber-pulling device and the laser-heating source is focused on to the median point of the fiber. The optical fiber is then pulled to finally obtain two fibers with submicron tip diameters. Figure 1a shows a scanning electron microscopy (SEM) photograph of one of the fiber probes fabricated for our preliminary studies. The scale on the SEM photograph of this sample indicates that the distal end of the fiber is approximately 50 nm. As can be seen from the SEM photograph, this reproducible method yields uniform fibers with submicron tip diameters.

Fig. 1
figure 1

Scanning electron photograph of an optical nanofiber. a The tip diameter of the drawn optical fiber is approximately 50 nm. b The tip coated with 200 nm of silver metal using a thermal vacuum deposition system, achieving a final diameter of ~250 nm

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The second step of the nanofabrication process involves coating the tapered side walls of the optical fiber with a thin layer of silver, aluminium or gold (100–200 nm) by using thermal vapor deposition. This coating serves to restore the refractive index and enables propagation of the excitation light down the tapered sides of the nanofiber. Because the fiber tip is pointed away from the metal source, it remains free from any metal coating. The coating procedure is designed to leave the distal end of the fiber free from silver for subsequent derivatization, to enable covalent immobilization of biological sensing elements on the exposed silica nanotip. The nanofibers are then secured on to a rotating stage inside thermal vacuum evaporation chamber to ensure uniform silver coating. The fiber axis and the evaporation direction should form an angle of approximately 45° to the normal. While the nanofibers are rotated the metal is heated and allowed to evaporate on to the tapered sides of the nanofiber tips forming a thin uniform highly reflective coating. With the metal coating, the final tip diameter size is approximately 150–250 nm (Fig. 1b).

The next step involves derivatization of the nanotips to facilitate covalent immobilization of biorecognition molecules. The derivatization process involves silanization with glycidoxypropyltrimethoxy silane (GOPS) and activation with 1,1′-carbonyldiimidazole (CDI). This process facilitates covalent binding of biorecognition molecules such as antibodies or synthetic peptides coupled to a fluorescent molecular probe. The freshly fabricated fiber-optic nanobiosensors can then be stored in PBS at 4°C for later use.

Because the diameter of the optical fiber’s tip is significantly less than the wavelength of light used to excite the target analyte, the photons cannot escape from the tip of the fiber by conventional optics. Instead, in a nanobiosensor, after the photons have traveled as far down the fiber as possible, evanescent fields continue to travel through the remainder of the tip, providing excitation for the fluorescent species of interest present in the vicinity of the biosensing layer. Therefore, only species that are in extremely close proximity to the fiber’s tip (i.e. antigens bound to the antibody probes) can be excited, thereby precluding excitation of interfering fluorescent species within other locations of the sample.

Optical measurement system

An evident advantage of fiber-optic nanosensors is their nanoscale size, which enables intracellular sensing of physiological and biological processes in cellular and sub-cellular compartments in individual living cells. Because of the small sampling volume probed by nanosensors, however, the amount of target analyte in the excitation volume is very small, making it important to use a sensitive optical spectroscopic technique, for example fluorescence, for the analysis. In addition to using a sensitive spectroscopic technique, it is also pertinent to design and employ a sensitive measurement and detection system. Such a system would consist of an excitation source (typically a laser), a fluorescent microscope, imaging detector intensified charge-coupled device (ICCD), a photon-counting detector (PMT), and computer to integrate the measurement system for data acquisition, analysis, and processing. Figure 2 shows a schematic diagram of the optical measurement system used for monitoring single cells using the nanobiosensors. This system has been described previously [12, 19]. Briefly, the experimental setup is adapted to this purpose from a standard micromanipulation/microinjection apparatus. A Nikon (Melville, NY, USA) Diaphot 300 inverted microscope with Diaphot 300/Diaphot 200 Incubator was used to maintain the cell cultures at ~37°C on the microscope stage. The micromanipulation equipment used consisted of Narishige (Tokyo, Japan) MN-2 three-dimensional manipulators for coarse adjustment and Narishige MMW-23 three-dimensional hydraulic micromanipulators for final movements. The fiber-optic nanosensor is mounted on a micropipette holder (World Precision Instruments, Sarasota, FL, USA). To record the fluorescence of target molecules binding to antibodies at the fiber tip, a Hamamatsu PMT detector assembly (HC125-2) is mounted in the front port of the Diaphot 300 microscope, and fluorescence is collected via this optical path (80% of available light at the focal plane can be collected through the front port). The fiber-optic nanobiosensor is coupled to the delivery fiber through the subminiature Version A (SMA) connector and is secured to the micromanipulators on the fluorescence microscope. The appropriate laser-line (depending on target molecule) is focused on to a 600-μm delivery fiber which is coupled to the nano-biosensor via an SMA connector. The fluorescence emitted at the nanobiosensor–cytosol interface is collected by the microscope objective and focused on to a PMT or ICCD for detection.

Fig. 2
figure 2

Schematic representation of the components of the optical measurement system used for making fluorescence measurements within single living cells using nanobiosensors

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Application of fiber-optic nanobiosensors

Fiber-optic nanobiosensors for measuring benzopyrene tetrol and benzo[a]pyrene

Fiber-optic nanobiosensors consisting of antibodies, as biorecognition molecules, coupled to an optical transducer element, have been developed and used to detect biochemical targets, benzopyrene tetrol (BPT), and benzo[a]pyrene (BaP), inside single cells [10–14]. For BPT detection the antibody used is targeted against BPT, an important biological compound, which is used as a biomarker of human exposure to the carcinogen benzo[a]pyrene (BaP). BaP is a polycyclic aromatic hydrocarbon (PAH) of great environmental and toxicological interest because of its mutagenic/carcinogenic properties and its ubiquitous presence in the environment [22].

Fiber-optic nanobiosensors for monitoring apoptosis

Several years involving research on antibody-based nanosensors [2, 23–25] culminated in the development of a unique application of these sensors for measurement of apoptosis. Conventional molecular biology procedures for measuring early events in apoptosis in intact living cells has usually been limited to the measurement of end-point biochemical and morphological markers of apoptosis. Apoptosis or programmed cell death is a process by which cells in our tissues and organs degenerate during normal development, aging, or in disease. When the process of apoptosis malfunctions, it can lead to clinical problems such as cancer, hydrocephalus, and neurodegenerative disease. Therefore, apoptosis is an important cellular process for maintaining the balance and proper functioning of organs and tissues in our bodies, for both developmental purposes and the control of diseases. In addition, apoptosis is of fundamental importance to the survival of multicellular organisms, as is proliferation. Fiber-optic nanobiosensors offer a strategy to monitor and measure apoptosis proteins early in the cell death cascade. Caspase specificity toward peptide substrates has been studied in detail as a means of developing both specific substrates and inhibitor probes of each enzyme and predicting preferences toward natural substrates and therefore their role [26, 27]. A unique application of fiber-optic nanosensors involves the monitoring of caspase-9, an apoptosis protein, in single living cells [28]. This study utilized human mammary carcinoma cells (MCF-7), a well-studied and characterized cell line that is significant because these cells are known to cause cancer in 8–10% of the female population in the United States. In addition, they are one of the most commonly used cell lines in breast cancer research. Monitoring apoptosis in single living MCF-7 cells is accomplished using a synthetic amino acid sequence conjugated to a molecular probe. This biorecognition molecule is immobilized on to the nanotips and used to probe individual MCF-7 cells.

Analysis of apoptosis in single living MCF-7 cells is accomplished using two types of fiber-optic nanobiosensor; the first is antibody-based and the second is one with a synthetic tetrapeptide-conjugated to a molecular probe. These biological recognition molecules facilitate the detection and visualization of cytochrome c [21] and caspase-9 [20] in single living cells during apoptosis, confirming the occurrence of proteins involved in the regulation of apoptosis at the single-cell level. These nanobiosensors were used to determine their role in response to a photodynamic therapy (PDT) agent, δ-aminolevulinic acid (ALA) [29] in MCF-7 cells. These cells were exposed to the photosensitizer ALA to explore ALA–PDT-induced apoptosis by monitoring cytochrome c and caspase-9.

Measuring cytochrome c

Cytochrome c is a very important protein in the process which produces cellular energy, in addition to being a well-known protein involved in apoptosis. In this application, the release of cytochrome c from the mitochondria to the cytoplasm of individual MCF-7 cells is monitored by a nanobiosensor inserted inside a single cell followed by an enzyme-linked immunosorbent assay (ELISA) outside the cell. Cytochrome c is detected in a single cell by using nanobiosensors with mouse and cytochrome c antibodies. After in-situ binding of cytochrome c to the antibody-based nanoprobe inserted inside a single cell, an enzyme-linked immunosorbent assay, which provides the enzymatic fluorescent amplification to give high detection sensitivity, is performed to detect cytochrome c in a single MCF-cell. The cells were treated with the PDT drug, ALA, which induces the mitochondrial pathway of apoptosis. Combination of the nanosensor with the ELISA immunoassay improves the detection sensitivity of the nanosensor, because of enzymatic amplification.

Measuring caspase-9

Activation of cysteine aspartate-dependent proteases (caspases) is one of the earliest biomarkers of apoptosis because caspases play a central role in the induction of this process [26, 27]. This makes caspases an early and ideal target for measuring apoptosis. Caspase-9 protease activity is assessed in single living MCF-7 cells with the known caspase-9 substrate, leucine–aspartic acid–histidine–glutamic acid 7-amino-4-methylcoumarin (LEHD-AMC), covalently immobilized on the nanotips of a nanobiosensor. On the induction of apoptosis using ALA, activated target caspases recognize the tetrapeptide sequence and specifically cleave it. Recognition of the substrate by caspases is immediately followed by a cleavage reaction yielding the fluorescent AMC, which can be excited with a helium–cadmium (HeCd) laser to generate a measurable fluorescence signal. By comparing the fluorescence signal generated from AMC within cells with activated caspases and those with inactive caspases, we can successfully detect caspase activity within a single living MCF-7 cell.

Intracellular measurement technique

Monitoring BPT and caspase activity in single cells using the nanosensor is conducted as follows. A culture dish with sparsely distributed MCF-7 cells is placed on the pre-warmed microscope stage set at 37°C. The nanobiosensor, mounted on a micropipette holder of a micromanipulation system, is moved into position, in the same plane as the cells, using bright-field phase-contrast microscopic illumination, so that the tip is just outside the cell to be probed. Total magnification is 600 times. A reading is taken with the nanoprobe outside the cell and the laser shutter closed. The fiber-optic nanobiosensor is then gently micromanipulated into the cell using the hydraulic fine adjustments, past the cell membrane and extending a short way into the cytoplasm. During these micromanipulations, great care is taken not to penetrate the nuclear envelope and compromise the integrity of the nucleus. Room light and microscope illumination light were switched-off, the laser shutter opened, and laser light allowed to illuminate the nanobiosensor with excitation light being transmitted into the nanotip. First, a signal reading is taken with the nanobiosensor inside the cell with the laser shutter closed, to record the dark signal. After 5 min the laser shutter is opened, enabling the excitation light to be transmitted to the nanoprobe tip, and fluorescence readings were recorded as a function of time. Figures 3a and 3b are images illustrating intracellular measurements performed in a single MCF-7 cell using the micro-positioning system on the inverted microscope. Once inserted through the cell membrane into the cytoplasm the nanosensor detects and identifies the protein of interest. Comparison of the fluorescence signal generated from AMC within cells with activated caspases and within those with inactive caspases enables detection and visualization of caspase-9 activity in single living MCF-7 cells during apoptosis.

Fig. 3
figure 3

a Image of a fiber-optic nanobiosensor before insertion and a single MCF-7 cell. b Image of a fiber-optic nanobiosensor inserted into the MCF-7 cell. (Adapted from Ref. [10])

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Fiber-optic chemical nanosensors

The development of chemical nanosensors made it possible to perform highly spatially localized analyses, for monitoring concentration gradients and spatial inhomogeneities in microenvironments (e.g. cells). Since the construction and use of the first fiber-optic nanosensors by Tan et al. [30, 31] several different optical fiber-based chemical nanosensors have been reported for measurement of pH [32–35], concentrations of various ions [36, 37], and other chemical species [38]. In this original work a micropipette puller was used to taper multimode fibers down to diameters of 100–1000 nm at the tip [30, 31]. These tapered fibers were then used in the production of pH nanosensors via a three-step process. The first step in the process is to apply a thick layer of aluminium to the walls of the fiber, using a vacuum evaporator, to ensure total internal reflection over the tapered region of the fiber. During this aluminium deposition process, it is important to ensure the tip of the fiber remains free from metal, thus providing a silica surface for binding of the probe or receptor molecules to the fiber’s tip. To achieve this the fibers are placed in the evaporator system and rotated with their tips facing away from the source of the evaporating metal. If the fibers are precisely angled their sides will shadow the tips from the evaporating metal. When the walls of the fiber are coated with aluminium, the next step is to silanize the fiber tip to enable cross-linking to a polymer coating. The final step in the fabrication of this pH nanosensor is to attach the pH-sensitive dye, acrylofluoresceinamine, to the silanized fiber tip by a variation of a photopolymerization process that has often been used for construction of larger chemical sensors [39, 40]. Because of the near-field excitation provided by these small fiber probes, however, the cross-linking of the polymer solution is restricted to the near field of the fiber. After fabrication of the sensor its properties are characterized. During this characterization, the sampling volume of the sensor is determined to be greater than six orders of magnitude smaller than conventional fiber-optic chemical sensors, making it ideal for sub-cellular measurements. The reduced sampling volume and response time of the sensor were evaluated by measuring the pH in 10-micrometer diameter pores in a polycarbonate membrane. This evaluation found that the response time of the sensor (300 ms) is 100-fold faster than conventional fiber-optic chemical sensors and that it is both stable and reversible with respect to pH changes.

The first reported application of fiber-optic nanosensors for biological measurements was on rat embryos [31]. In this study, pH nanosensors like those described above were inserted into the extra-embryonic space of a rat conceptus, with minimal damage to the surrounding visceral yolk sac, and pH measurements were made. Values of the pH in the extra-embryonic fluid of rat conceptuses ranging in age from 10 to 12 days old were then compared for any differences. In a similar study using the same pH sensor, indirect measurements of nitrite and chloride levels in the yolk sac of rat conceptuses were also performed [41]. Because of the minimally invasive nature of such measurements, the techniques provide much promise for biological analyses and may aid in furthering our understanding of the effect of environmental factors on embryonic growth.

As the field of fiber-optic nanosensors has evolved, the size of the environments they probe continues to become smaller. In fact, measurement of chemical species inside individual cells has even been reported using fiber-optic chemical nanosensors. In the first of these reports, opto-chemical nanosensors for sodium ions (Na+) were developed using a process similar to that described previously. These sensors were then used for measurement of Na+ concentrations in the cytoplasmic space inside a single mouse oocyte, one of the largest mammalian cells (~100 μm in diameter) [42]. These sensors enabled the monitoring of relative Na+ concentrations while ion channels were opened and closed by the external stimulant, kainic acid. In another application of fiber-optic nanosensors to biological measurements, calcium ion-sensitive nanosensors were developed and used to measure calcium ion fluctuations in vascular smooth muscle cells while the cells were stimulated [43]. These fluctuations were then directly correlated with the stimulation events that were being performed, providing another potential application of this nanometer sized fiber-optic sensor.

Discussion

The introduction of fiber-optic nanosensors into cells could raise questions about the effect of such treatment upon cell viability. The use of glass micropipettes to introduce dyes or perform gene-transfer experiments has been shown to have minimal effects on cell viability, assuming the attendant procedures do not cause too much trauma. Similarly, it has been observed and determined that introduction of a nanoprobe into the cell cytoplasm does not visibly seem to affect cellular function/viability [44]. This has been empirically established in several experiments in which, after nanoprobe penetration and equilibration for 5 min, the probe is withdrawn and the cells are monitored by video-microscopy. The probed cell was observed to undergo mitosis, indicating at least that one round of cell division could occur after probing. Figure 4 (I–IV) contains a series of time-lapse images from this experiment, showing the initial stages of mitosis in which the fiber-optic nanosensor is interrogating the cell, all the way through division of the cell into two identical daughter cells. This study demonstrated that nanosensors are minimally invasive tools appropriate for monitoring biomolecular processes inside living cells.

Fig. 4
figure 4

A series of time-lapse images which demonstrate the minimally invasive nature of fiber-optic nanobiosensors. This experimental study established that a cell was able to undergo mitosis after being probed by a fiber-optic nanobiosensor. (Adapted from Ref. [44])

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Conclusion

The development and investigation of analytical nanodevices [6, 10, 12, 45–49] capable of probing the nanometer world and will make it possible to characterize the chemical and mechanical properties of cells, discover novel phenomena and processes, and provide science with a wide range of tools, materials, devices, and systems with unique characteristics. Concrete knowledge of sub-cellular architecture and dynamic processes is important to fundamental biological understanding of cellular processes. Fiber-optic nanosensors provide us with the capability of single-cell analysis. This capability is useful to perform dynamic analyses of interactions within individual living cells, which are critical for mapping and deciphering cell-signaling pathways and networks. Single-cell analyses are also especially critical when primary cells are obtained by use of surgical procedures and cannot be propagated for study, because of the amount of tissue available or the type of cell, and therefore must be analyzed with a technology capable of SCA. Fiber-optic nanosensors for investigating intact living cells hold promise for minimally invasive dynamic analyses of biochemicals and metabolites in biochemical pathways at cellular and sub-cellular levels. Work involving the development and application of chemical and nanobiosensors demonstrates that the nanoscale size of fiber-optic sensors enables intracellular analysis, with minimal manipulation, of intact solitary living cells. These nanosensors provide a new method in cell-based assays offering highly miniaturized, nanoscaled devices that make cell-based analysis accessible at the single-cell level. Such work will hopefully inspire the development of new nanosensing technologies for single living cell analysis. The future direction of this work may be toward application of fiber-optic nanosensors for multi-analyte detection and for the analysis of protein–protein interactions and similar analyses of other proteins involved in cellular biochemical pathways.