Carbon Dot-Decorated Polystyrene Microspheres for Whispering-Gallery Mode Biosensing

Abstract

Whispering gallery mode (WGM) resonators doped with fluorescent materials find impressive applications in biological sensing. They do not require special conditions for the excitation of WGM inside that provide the basis for in vivo sensing. Currently, the problem of materials for in vivo WGM sensors are substantial since their fluorescence should have stable optical properties as well as they should be biocompatible. To address this we present WGM microresonators of 5–7 $\mathsf{\mu }$m, where the dopant is made of carbon quantum dots (CDs). CDs are biocompatible since they are produced from carbon and demonstrate bright optical emission, which shows different bands depending on the excitation wavelength. The WGM sensors developed here were tested as label-free biosensors by detecting bovine serum albumin molecules. The results showed WGM frequency shifting, with the limit of detection down to ${10}^{-16}$ M level.

1. Introduction

In biological research and clinical diagnosis, it is crucial to detect and identify various proteins accurately. This process requires highly sensitive and selective detection methods. Many modern methods, which are well-developed, provide information about large ensembles of molecules and structures; however, sometimes it is required to detect unlabelled single proteins. Doped microresonators supporting whispering gallery modes (WGM) have gained attention as biosensors due to their biocompatibility, compact size, high Q-factor, and narrow spectral width of resonance lines [1,2]. They represent a logical way of further advancing WGM sensors and WGM-based optoplasmonic sensors, which are capable of detecting not just single molecules but also single atomic ions [3,4], tracking molecular movements during biochemical reactions [2], and collecting information about the spectral properties of molecules [5,6]. It is relatively easy to achieve lasing in such resonators [7]; however, biosensors based on WGM microlasers have been the focus for researchers for the last 10–15 years. Similarly to WGM sensors, WGM fluorescent sensors are highly responsive to even the slightest changes in the refractive index of the environment. This sensitivity enables the detection of frequency shifts caused by attaching molecules as it is in WGM sensors; while in WGM fluorescent sensors the number of detecting parameters can be extended. WGM fluorescent sensors have a wide range of applications, from the label-free detection of proteins and enzymes [2,8] to imaging in biological tissue [9]. In addition, WGM-based sensors can be used to study drug interactions [10] and sensing at the cellular level [11], where WGM sensors embed in or interact with cells, enabling new applications for the in vivo sensing and tracking of physiological parameters of individual cells [12]. The sensitivity of WGM devices, coupled with their structural diversity and compatibility with existing infrastructures such as traditional chip-based technologies has led to the development of a wide range of sensor applications based on them.

Quantum dots are a popular choice for fluorescent dopants in the development of emitting WGM cavities. However, their use in biomedical applications may be limited due to potential toxicity issues arising from their composition of heavy metals. A non-toxic alternative to quantum dots is carbon dots (CDs), which are quasi-spherical nanoparticles less than 10 nm in size consisting of sp²- or sp³-carbon domains and rich in oxygen- and nitrogen-containing functional groups [13]. CDs are characterized by high biocompatibility, chemical inertness, photostability, and high photoluminescence quantum yield, which makes them most suitable for use in biological applications [14,15,16]. Carbon dots are water-soluble and have low toxicity, making them suitable for various medical applications, such as biosensing, drug delivery, and cell analysis [17,18]. When embedded into polystyrene microspheres, the carbon dots retain their biocompatibility. In our previous work on carbon dots, utilizing LIVE/DEAD and AlamarBlue assays we demonstrated the high biocompatibility of CDs on cell lines THP-1 (monocytes isolated from human peripheral blood), B16–F10 (mouse skin melanoma epithelial cell line), and K562 (human myelogenous leukemia cell line) [19]. CDs have been used as a gain medium for WGM lasers where the WGM resonator was made of conventional glass fiber [20]; the cavity sizes were up to 70 $\mathsf{\mu }$m, which is barely suitable for in vivo biosensing. One of the first demonstrations of WGM lasers with CDs was made with CDs incorporated in NaCl; the Q-factor of such lasers was rather low—447 [21]. Recently, our group demonstrated a microfluidic approach to WGM cavities fabrication for another type of nanocrystals—AIS quantum dots [22]. Although a significant reduction in the emission lifetime was observed, there were no WGM resonances detected. Polystyrene microparticles decorated with CDs were used in [23], where their enhanced luminescence was observed. Although carbon dots are promising for biosensing and bioimaging [18], there are no reports about their use for WGM biosensors yet. So, in contrast to previously published works, here we focus on making WGM decorated with CDs for biosensing applications.

In this work, we present new composite WGM fluorescent sensors made of carbon quantum dots and polystyrene microspheres. We tested their sensing properties by applying bovine serum albumin (BSA) molecules—a protein that is commonly utilized as a reference protein for examining interactions with drugs, biomolecules, and nanoparticles. Its structural similarity to human serum albumin makes it a valuable tool for research in drug development and medical diagnostics [24,25]. Sensitive detection of BSA is crucial in a variety of applications; in biomedicine, BSA is used as a stabilizer of enzymes and proteins [26,27]; in pharmaceuticals, BSA is used as a drug carrier [28,29] due to its ability to bind and transport various compounds. Among numerous biomolecule detection methods, carbon-dot-doped WGM-based sensors offer a highly relevant approach for BSA protein detection. Their high sensitivity, ease of fabrication, low cost, biocompatibility, and selectivity make them valuable tools for various biomedical and analytical applications.

2. Materials and Methods

2.1. Synthesis of CDs

Multicolored carbon dots were synthesized by the solvothermal method described in [14], from citric acid and urea in dimethylformamide. Chemicals were sourced from reputable suppliers such as Sigma-Aldrich and LCC Vekton. To synthesize CDs, 1 g of citric acid and 2 g of urea were dissolved in 10 mL of dimethylformamide and heated in a Teflon-lined autoclave at 180 °C for 8 h. The colloidal solution obtained was purified by dialysis against deionized water using a dialysis tube with a 3.5 kDa molecular cut-off tube for 24 h and then freeze-dried.

2.2. Synthesis of Polystyrene Microspheres

Polystyrene microspheres with diameters ranging from 5 to 7 $\mathsf{\mu }$m were synthesized via emulsifier-free emulsion and dispersion copolymerization using a combination of styrene and 4-styrenesulfonic acid sodium salt (NaSS) [30,31]. Styrene was purified using a 30% aqueous solution of potassium hydroxide followed by distillation under vacuum. Emulsifiers including sodium dodecyl sulfate, Tween 80, Tween 65, dioctyl sulfosuccinate sodium salt, and poly(vinyl alcohol) were used without prior purification. Bidistilled water, free from surface active impurities as indicated by specific conductivity and surface tension measurements, was utilized for solution preparation and polymerization. The synthesis was conducted in a 100 mL flask equipped with standard laboratory apparatuses for temperature control and agitation. A mixture of styrene and NaSS in appropriate solvents was heated to specified temperatures to initiate polymerization. After completion, the residual monomer was removed by steam distillation, and water-soluble impurities were eliminated through centrifugation and redispersion cycles.

2.3. Impregnation of CDs into Microspheres

To decorate microspheres with CDs, we adopted the impregnation method. This method was selected due to its cost-effectiveness and simplicity, making it an attractive choice for modifying the properties of loose polymeric and mesoporous materials [32,33]. Impregnation involves saturating the microspheres with a solution containing carbon dots, allowing for their uniform distribution throughout the microsphere matrix without compromising their structural integrity. To 100 $\mathsf{\mu }$L of an aqueous solution with a ${10}^{-3}$ M concentration of CDs, 600 $\mathsf{\mu }$L of an aqueous solution of polystyrene particles (0.5 wt%) was added, with the resulting solution occasionally shaken over 20 h. It was then washed with deionized water after which the solution was centrifuged three times for 10 min at 5000× g. Upon aggregation of the microspheres, the solution was held in an ultrasonic bath until the particles dissolved completely in water (1–2 min).

2.4. WGM Sensing

To study the sensor properties of CD-decorated microspheres supporting WGM in relation to the presence of BSA, they were deposited from an aqueous buffer onto a glass substrate and subsequently dried under ambient conditions. Preliminary CD concentration in the aqueous buffer was reduced to ensure that any droplet of the solution contains from two to five microspheres. The stock solution of BSA with a concentration of 1.5 · ${10}^{-5} \mathrm{M}$ was prepared by dissolving 10 mg powder in 10 mL of distilled water. Then, a range of BSA solutions with concentrations from ${10}^{-12}$ to ${10}^{-18} \mathrm{M}$ were prepared by dilution of the stock solution. During the experiment, the BSA solution was added to the same slide where polystyrene microspheres with CDs were already deposited and the same particles were examined for changes in their emission.

2.5. Characterization

To measure the absorption spectra of solutions over a wide spectral range with a step of 1 nm, a spectrophotometer SF-56 (St. Petersburg, Russia) was used; luminescence spectra were measured using a spectrofluorometer Shimadzu RF-5301PC (Kyoto, Japan). The laser scanning confocal microscope Zeiss LSM 710 (Jena, Germany) was used to obtain luminescent images of microspheres doped with CDs. An objective 20×/0.4 was selected to study the samples with excitation wavelengths of 405 and 488 nm. To detect emission and whispering gallery modes, a confocal Raman microspectrometer inVia Renishaw (New Mills, UK) was used, with sample emission excited by a laser with a wavelength of 488 nm. A microscopic image of microspheres was obtained by scanning electron microscope (SEM) Merlin (Zeiss).

3. Results and Discussion

The absorption spectrum of CDs in water (Figure 1) shows two distinct bands. The short-wavelength band in the ultraviolet region represents a combination of transitions involving $\pi$–${\pi }^{*}$ transitions in the sp²-hybridized carbon core and n–${\pi }^{*}$ transitions of carboxyl groups [14,34,35]. The absorption peak at 535 nm corresponds to the transitions related to the surface states. Given BSA’s limited absorption in the visible spectrum, the optical behavior of the mixture solution of BSA and CDs primarily reflects the inherent properties of carbon dots.

CDs exhibit predominant photoluminescence in the mixture’s spectra (Figure 1b), overshadowing the luminescent bands of bovine serum albumin. These bands, with peaks at 548 nm and 583 nm, are only faintly discernible. The fluorescence spectrum of carbon dots changes when they are embedded into polymer microspheres (Figure 2a) from the solution, which is explained by characteristic mechanisms such as surface passivation, aggregation effects, confinement effects, charge transfer, and/or polarity changes (Figure 2b) [36,37,38,39]. Meanwhile, the fluorescence spectra of CDs exhibit excitation-dependent emission due to the coexistence of multiple emissive centers within carbon dots.

The emission spectra of the obtained microspheres were measured with the micro-Raman spectrometer inVia, with 50 pm resolution (Figure 3a). They have two peculiarities. First, the emission spectra of CDs doped microspheres have similar bands to the solution of CDs (Figure 2b); however, we observe emission peaks corresponding to the whispering-gallery modes circulating inside the microsphere. This is the result of coupling between CD emission and cavity modes and amplified spontaneous emission. To check for possible lasing, an input–output characteristic was measured at different excitation levels. Figure 3b shows that the peak intensities are sublinear, with the characteristic saturation, corresponding to typical quantum dots [40].

For sensing experiments, 4 $\mathsf{\mu }$L of the aqueous BSA solution was added to the test sample; a concentration series of experiments with BSA (${10}^{-12}$ M, ${10}^{-15}$ M, ${10}^{-16}$ M, ${10}^{-18}$ M) was conducted to determine the limit of detection, that is reflected on Figure 4. The mode shifting is caused by changing the surrounding medium refractive index, so-called reactive sensing [5]. Note, that wavelength shifts values are huge; even for ${10}^{-15}$ M it is about 1.5 nm (Figure 4b), and it became smaller with a reduced concentration of BSA (Figure 4c). The second feature we observed is related to reducing the Q-factor of the resonances under the attachment of BSA molecules, which is also related to sensing [41]. This is because the adsorption of molecules on the surface of the resonator can increase the energy dissipated by the resonator, leading to a lower Q-factor. Indeed, as seen in Figure 4a, the value of Q-factors is changing; according to our assessments, it was reduced from 2820 to 1650 on average (please see Appendix A). However, the Q-factor is still high enough for observing amplified emission.

As a further step, we tried to find a limit of detection. For this, the resulting BSA concentration was reduced to ${10}^{-18}$ M and applied to samples of CDs doped WGM microspheres. The results are presented in Figure 4d. The normalized spectra show the reproducible character of frequency shifts; however, at ${10}^{-18}$ M it is becoming unresolvable with our sensors. Thus, we define the limit of detection as ${10}^{-16}$ M and the dynamic range as four orders of magnitude of concentrations at least (Figure 5).

4. Conclusions

We have developed a simple and reproducible method for making biosensors based on fluorescent whispering gallery mode resonators. This method exploits the decorating of polystyrene microspheres with carbon quantum dots. Such particles demonstrate narrow emission lines corresponding to whispering gallery modes. The method allows the production of particles for label-free sensing of low concentrations of biomolecules such as bovine serum albumin by WGM frequency shifting. The detection limit is defined as low as ${10}^{-16}$ M concentrations and the dynamic range is four orders of magnitude.