The intracellular microenvironment involves vital physiological characteristics of various cellular compartments. The fabrication of nanoprobes for subcellular measurement is important to fully characterize cellular function. Single-cell interrogation at the nanoscale is carried out mainly via optical approaches, such as the use of fluorescent dyes,1^–3 quantum dots or nanoparticles,4^–11 nanofiber probes,12^,13 single nanowire probes,14^–19 and planar photonic crystal nanowaveguides;20 mechanical approaches, such as the use of atomic force microscopy probes21^–23 and nanobeam arrays;24 and electrochemical approaches, such as the utilization of single metal nanowires,25^,26 pillars, or tube electrodes.27^–31 Dyes and quantum-dot-based probes have attracted much attention in biology, attributable to their photoluminescence (PL), and achieved great success in measuring the temperature of cells and their organelles,32^,33 as they can be easily phagocytosed by cells due to their ultrasmall dimensions and are quite robust against external disturbances. However, these techniques suffer from some limitations, such as fluorescence bleaching and background fluorescence interruption. Optical nanofibers have become increasingly popular owing to their excellent optical waveguiding property, high flexibility, and ease of integration34 and thus have been applied for the detection of intracellular pH14 and ionic concentrations.15 Notably, the existing fiber probes are passive in nature and serve only as conduits to guide light signals into/from a cell but not as a complete optical functional module for physical or chemical label-free sensing. Accordingly, most nanoprobe techniques still have to modify other nanoparticles, resulting in the low functional integration of structures.

The miniaturization of optical modules based on fibers is continuously pursued by researchers. Limited by manufacturing techniques, the scale of all-fiber functional modules is on the order of tens or hundreds of micrometers,35^,36 which is not applicable compared to the sizes of most common biological cells. Fortunately, one-dimensional nanowires compensate for this deficiency. Their smaller dimensions and high robustness make them suitable for optical resonators,37 couplers,38 gratings,39 and lasers.40^,41 However, most of the current optical nanodevices are fixed on substrates, lacking portability and the plug-and-play feature. Moreover, local far-field incident light also implies low-energy efficiency. For an indeed label-free device, the excitation, transmission, detection, and collection of light signals should be integrated in the same device without any chemical modification. Only in this way can the integration of the device, the convenience of manipulation, and the efficiency of sensing be greatly improved. Under these circumstances, the combination of nanowire optical devices and fiber probes provides a novel configuration. Adhered to the tip of a tapered fiber, the nanodevice can be freely moved, can be flexibly directed to anywhere, and can safely penetrate the plasma membrane and enter biological cells, which is potentially useful in single-cell interrogation with high resolution and efficiency.

In this paper, we develop a compact label-free nanosensor based on a complete functional module composed of a fiber taper integrated with zinc oxide (ZnO) nanogratings for the early monitoring of apoptosis in living cells, as illustrated in Fig. 1(a). Cellular apoptosis, which is involved in cell turnover in many healthy adult tissues, is responsible for the focal elimination of cells during normal embryonic development. The visualization and monitoring of early apoptosis and the real-time tracking of cell status with high sensitivity in living cells are highly desired. We choose a single ZnO nanowire not only because of its low toxicity and high biological compatibility, but also because it can serve as a waveguiding matrix and has low transmission loss and a relatively high refractive index (RI, n=1.9831 at 655 nm), which are required for strong light confinement in the nanowire, especially in aqueous environments (n=1.3310 at 655 nm). Moreover, the uniform diameter of the nanowire results in less physical damage to cellular structures and functions than caused by a conical optical fiber probe when penetrating a cell. With optical nanogratings etched on the ZnO nanowire, the integrated fiber sensor is functionalized with the capability of simultaneously sending and receiving optical signals, being sensitive to changes in the surrounding environment, and can realize rapid, accurate, and real-time sensing by being physically located in a single living cell. In our study, we find that the RI of the nucleus is higher than that of the cytoplasm in HeLa cells. More importantly, a gradual increase in nuclear RI during the apoptosis process after the invasion of hydrogen peroxide is observed, which illustrates an increase in molecular density and a decrease in cell volume.42 Overall, the quantitative probing and analysis of the RI naturally present in single living cells during apoptosis may offer insight into the life activities within cells. Additionally, our strategy creates a precedent that allows a passive fiber nanodevice for in situ, dynamic intracellular monitoring and may provide new ideas for a number of novel studies combining photonics with biochemistry.

The nanosensor was fabricated by bonding ZnO nanogratings on the tip of an optical fiber taper (see Sec. S1 in the Supplementary Material). The manufactured fiber taper had a conical transition region generally several millimeters in length and a long pigtail to allow prompt linking to other optical fiber components. Light traveling along the fiber can be effectively coupled into the nanogratings, where part of the light is reflected back. Predictably, thinner nanowires cause fewer injuries to cell membranes during the puncture process; however, a larger diameter is more desirable in terms of light confinement and mechanical robustness. Therefore, we prioritized nanowires with diameters of 500 to 800 nm. The microscope image [see Fig. 1(c)] of a typical probe shows that a single ZnO nanowire with gratings in the visible band (∼660nm) etched on the front end was successfully attached onto the tip of the fiber taper. The scanning electron microscopy (SEM) micrograph of the nanostructured probe [see Fig. 1(b)] shows that the gratings on the ZnO nanowire included 40 shallow grooves with a period Λ=169nm, resulting in a total length <7μm, which is two orders of magnitude shorter than gratings fabricated in conventional optical fibers. Each groove was ∼100nm deep and ∼80nm wide and located at a position with a local radius of ∼400nm. The configuration has good manufacturing reproducibility and stability.

For a highly efficient nanowire probe, the optical output is closely confined to the ZnO nanowire, offering highly localized illumination. Owing to the excellent waveguiding properties of both the optical fiber and ZnO nanowire, the optical transmission loss is generally concentrated in the coupling region between them. As illustrated in our previous research,43 the normalized optical power flow coupled into the nanowire depends on the propagating constants of the two waveguides, which are related to the diameters of the two waveguides in the coupling region. To improve the coupling efficiency, we designed and fabricated probes with appropriate sizes. Figure 1(b) clearly shows that most light transmitted in the fiber probe is coupled into the nanowire. Benefitting from the high RI of ZnO, the external environmental RI hardly affects the optical coupling and light propagation.

The setup used to test the nanogratings sensor is illustrated in Sec. S3 in the Supplementary Material. Figure 2(a) shows the typical reflectance spectrum of the fiber probe immersed in deionized water (see Sec. S3 in the Supplementary Material). To investigate the function of the ZnO nanogratings, we measured the reflectance spectra of the fiber taper without (black curve) and with (blue curve) the ZnO nanogratings. It is evident that the sole fiber taper reflects little light, while by contrast, the ZnO nanogratings have a characteristic reflection wavelength at ∼658nm. The ratio of reflectance is ∼30% compared to the end face of a sliced fiber. The extinction ratio, which indicates the ratio of the maximum to minimum reflection intensities, is ∼11dB, and the full-width at half-maximum is ∼8nm.

A distinctive feature of optical gratings is that they are very sensitive to the RI of the external environment.44 With the change in RI, we discovered an apparent redshift in the peak reflection wavelength [see Fig. 2(b) and Sec. S5 in the Supplementary Material]. Figure 2(c) (black line) demonstrates the linear relationship between the peak reflection wavelength of the nanosensor and the surrounding RI, with a sensitivity of ∼83nm/RIU. The experimental result is in good agreement with the theoretical simulation (see Sec. S2 in the Supplementary Material). After calibration, the RI of pure phosphate buffered saline (PBS, HyClone) was measured to be 1.3345, again similar to previously reported data.45

Using the shift in the spectrum is a common method in optical sensing,46 whereas the increase in temperature resulting from the high light power cannot be neglected, as it may cause predictable thermal damage to the cells. To make the experimental conditions more rigorous, a low-power laser at 655 nm was used as the light source to conduct single-cell sensing. Figure 2(c) (red line) shows the reflection power of 655 nm derived from Fig. 2(b) (red dashed line) with increasing RI. The power sensitivity is ∼1260%/RIU. The linear relationship indicates that measuring the power change of a single wavelength is another feasible optical sensing approach. As illustrated in Fig. 3(a), the laser (∼800nW) was injected into the fiber nanosensor and the reflected signal was collected with a power meter (see Sec. S4 in the Supplementary Material). The optical power passing through the fiber taper was hundreds of nanowatts, but the light was not completely incident into the cell because nearly half the light was lost in the coupling region between the fiber taper and the nanowire. The power threshold before cell damaging and death involves the exposure time, the size of the cell, and the volume of the solution,47 but the power we used is far lower than the threshold. Such low optical power can have little impact on cell activities, as heat dissipates quickly in PBS solution. The calibration indicates that with increasing RI, the reflection power at 655 nm will gradually decrease within a certain range [see Fig. 2(d), black line]. Therefore, the RI information can also be obtained by measuring the light power at a single wavelength, with the experimental sensitivity of the nanosensor being ∼1280%/RIU for an RI ranging from 1.330 to 1.350 (red line). This result is generally consistent with the experimental results presented in Fig. 2(c) (red line).

To demonstrate that the nanowire probes incurred negligible cell membrane damage during insertion and extraction, fluorescent dyes were used to verify whether the cell membranes were ruptured (see Sec. S8 in the Supplementary Material). Optical fiber probes with and without a ZnO nanowire were manipulated to penetrate HeLa cells. HeLa was the first human cell line established in culture and has since become the most widely used human cell line in biological research.48Figure 3(b) shows the tested cell (red circle) after insertion of a fiber probe with an ∼500nm tip. But for the conical structure, the actual damaged area of the membrane was larger than the tip diameter. The PL intensity decreased significantly compared with that of other cells 3 min after puncture, which indicated that the cell membrane was likely to be damaged, resulting in the outflow of dyes. Figure 3(c) shows the tested cell (red circle) after insertion of the ZnO nanowire-integrated fiber probe with an ∼800nm tip. Owing to the uniform diameter of the nanowire, the PL intensity of the tested cell showed no apparent differences compared with the surrounding cells within 3 min after puncture, indicating that the cell membrane was still relatively intact. The observation of fluorescence from the cells shows that the cells continued to hydrolyse fluorescein diacetate to generate fluorescein inside the cells. Therefore, it is feasible to use the ZnO nanowire-integrated fiber sensor to implement intracellular measurements.

Figures 3(d) and 3(e) show that the nucleus and cytoplasm regions of single HeLa cells are clearly visible in the microscope images even after insertion of the ZnO nanograting-integrated fiber sensor (see Sec. S7 in the Supplementary Material). The nanowire is robust against cell penetration, with no obvious mechanical deformation. As illustrated in Fig. 3(f), the normalized reflection power declined by ∼0.5% when the probe was transferred from PBS into the cytoplasm and decreased by ∼7% when the probe entered the nucleus. The tiny fluctuations (∼0.08%) of real-time power stemmed from the variations of the light source power and the unevenness of intracellular microenvironments. Notably, the experiment was carried out in a constant temperature environment to eliminate the influence of temperature fluctuations. The probe did not stay in the cytoplasm after measuring the RI of the nucleus, so the information from nucleus-cytoplasm-PBS was not apparent. If we extract the probe out of the cell slower, the information will be basically similar to the former. We tested four single HeLa cells, and the probe was cleaned with PBS solution before and after each test to avoid contamination. The experimental results in Table 1 and Sec. S9 in the Supplementary Material indicate that the mean RI of the cytoplasm and the nucleus in the HeLa cells was 1.3348 and 1.3402, respectively. The distribution of substances in the cells is possibly nonuniform, and it can be inferred that the RI results of the single HeLa cells relate to the position of the probe. Since the RI is generally proportional to the molecular concentration, which is related to the viscosity of solutions, the molecular density of the cytoplasm is deduced to be slightly higher than that of PBS and the molecular density of the nucleus is much higher than that of the cytoplasm. The relatively stable fluctuation of the reflection power also indicated that the signal light had little thermal effect on the cells. Previous studies using optical diffraction tomography also drew similar conclusions.42 Compared with traditional far-field detection techniques, our method is more energy efficient, as only very low-power probe light is required.

Cell apoptosis is an integral part of normal development and the maturation cycle. Despite the importance of this process, the mechanisms underlying cell death are still poorly understood. Previous work has shown the RI changes of cells can indirectly reflect different intracellular behaviors.42 Therefore, we hope to discover the connection between nuclear RI and cell apoptosis under external stimuli, which is helpful for further understanding of cell life events and development of diseases. The advantage of using nanogratings instead of fluorescence for single-cell detection is that the nanoconfiguration is more stable and reliable for long-term monitoring because fluorescence suffers from quenching and bleaching. After the probe punctured the nucleus of a HeLa cell, 10μL of 20 mmol/L H2O2 PBS solution was injected into the medium. Hydrogen peroxide (n=1.3350 at 655 nm) is a strong oxidant that breaks down cell membranes and is thus fatal to cells. The microscope images of the detected HeLa cells [see Fig. 4(a)–4(d)] show the variation in cell appearance. Simultaneously, a reduction in the reflection intensities was observed in the nucleus, indicating a continuous increase in RI with time [see Fig. 4(e)]. The gradual increase in nuclear RI went through three periods. During the first period, which lasted ∼20min, the process of apoptosis was slow. The cytoplasm was gradually dissolved under the corrosion of hydrogen peroxide, but the morphology of the nucleus did not have a notable impact [see Fig. 4(b)], and the RI of the nucleus increased slowly. During the second period, which lasted ∼10min, large areas of the cytoplasm dissolved, and the nucleus began to shrink [see Fig. 4(c)]. The nuclear RI rapidly increased by ∼0.0040 with decreasing nuclear volume. During the last period, the cells had little vitality, and the nuclear RI tended to become stable. We tested three single HeLa cells, and the mean nuclear RI after cell apoptosis was 1.3442, as indicated in Table 2 and Sec. S12 in the Supplementary Material. Although the exact reasons for the changes in the RI of the nucleus are still unknown, our results imply that the shape and volume of the cell are at least partly responsible. The numbers of pixels that the cell nucleus occupied in the microscope images [see Figs. 4(a)–4(d)] during the process of cell apoptosis were counted, which could roughly reflect the volume of the cell nucleus [see Fig. 4(e), blue circles]. The volume of the cell nucleus gradually contracted, leading to an increase in molecular density, which is one of the possible reasons for the increase in the nuclear RI.

A mature fluorescence technique was used to examine the cell viability of the tested cells and other normal cells during the process of apoptosis (see Secs. S10 and S11 in the Supplementary Material). The fluorescent dye we chose presents little toxicity to cells, can infiltrate through cell membranes, and is often used for nuclear staining and cell apoptosis tests (see Sec. S10 in the Supplementary Material).49 As shown in Fig. 4(e), the intensity of the PL signal underwent an apparent increase 40 min after H2O2 was injected, confirming that the cells had indeed been apoptotic. Moreover, the PL intensity of the tested cell (yellow dashed circle) had no obvious difference from that of other surrounding cells, which again indicated that the ZnO nanogratings had little influence on the cell life states.

We developed a versatile, biocompatible, portable, and reusable ZnO nanograting-integrated label-free nanosensor that enables in situ early monitoring of cellular apoptosis. The nanodevice integrates signal transmission, sensing, and collection, with high integration, operability, and sensitivity. By inserting the nanoscale functional optical module into single living HeLa cells, we can detect different features between the nucleus and cytoplasm. The highly flexible and robust nanosensor allowed us to reveal that the mean RI of the nucleus is 0.0054 higher than that of the cytoplasm in single HeLa cells, which to some extent verifies the higher molecular density of the nucleus than that of cytoplasm. Moreover, the stable and nondegradable configuration is promising for the long-term monitoring of cellular environmental variations, which are universally present in all cell life activities. We observed a decrease in the nuclear volume and detected the nuclear RI during cell apoptosis. The results show that the final mean RI is 0.0040 higher than that of normal living cells, linking the RI and the volume of the nucleus to the state of living cells. We consider that it is feasible to detect and compare the differences between normal living cells and cancer cells using the same sensing device. The application of the nanoprobe is only limited by the size of the cell and has no requirement for the type of cells. This study is the first to realize apoptosis sensing and tracking with nanogratings in living cells. Real-time probing of cellular apoptosis will not only attract more attention to cell life events but also broaden the application scope of fiber nanosensors.

Biographies of the authors are not available.