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Abstract
Laser trapping (LT) of metallic nanoparticles (NPs) is an approach that has the potential to enhance Raman spectroscopy in aqueous media. In this paper, we report the LT of multiple 60-nm Ag NPs using a tightly focused 1064-nm Gaussian laser beam. The dynamic process (trapping and escaping) of the individual Ag NPs were recorded using a charge coupled device (CCD) camera in backscattering illumination mode. We found that up to four Ag NPs could be simultaneously trapped; however, they were unstable in the laser trap due to Brownian motion and NP-NP interactions. However, after mixing Ag NPs with Bacillus subtilis, more of the Ag NPs could be trapped together with the bacteria. Furthermore, a 532-nm solid-state laser beam was used to activate Raman scattering of the Ag NPs + Bacillus subtilis sample. Based on repetitive measurements, the Raman spectra of the Ag NPs + Bacillus subtilis sample were enhanced and the results were consistent. Our work suggests that LT of metallic NPs can be used to enhance Raman spectroscopy in aqueous media. We believe that the enhanced Raman spectroscopy will be useful for real-time biological assays.
© 2019 Optical Society of America under the terms of the OSA Open Access Publishing Agreement
1. Introduction
Since A.Ashkin et al. [1–3] observed the confinement, trapping, and acceleration of small particles with a tightly focused laser beam in 1970, laser trapping (also known as laser tweezers (LT)) has become a well-established technique to manipulate and characterize particles ranging from nanometer to micrometer in size for various applications ranging from physics to biology [4,5]. LT is primarily based on the balance between the gradient forces of the light field and the scattering forces from the photons affecting the particles in the light field. The particles are always trapped around the focal point with frequent Brownian motions. LT offers the advantage of capturing and manipulating particles without any physical contact. LT is an optical tool that can be easily integrated with other optical measurement techniques such as imaging or spectroscopy in order to acquire the images or spectra of the captured particles of interest in their native condition.
Ever since its development as an optical tool, LT has been typically used to directly trap bacteria and single living cells, as well as dielectric particles such as polystyrene beads in order to manipulate biomacromolecules such as protein, deoxyribonucleic acid (DNA), ribonucleic acid (RNA). However, it has been believed for a long time that trapping nontransparent particles by LT is impossible owing to their reflecting nature. In the classical ray optics model, most of the light reflected by these particles is scattered, which leads to the absence of gradient forces, which are the primary forces for LT, while the scattering forces increase. In 1994, Svoboda and Block [6] were the first to experimentally trap 36-nm Au nanoparticles (NPs) using an infrared (IR) laser source. The frequency of the trapping laser source was far from the Au resonance and the NPs essentially behaved as dielectric particles. Recent evidence from theory and experiments [7–9] showed that when the particle size was significantly smaller than the incident wavelength, the NPs were optically trappable with a higher trapping force compared with dielectric particles of the same size. Nowadays, trapping NPs is commonly seen in studies related to NP-NP interactions, which forms the basis for constructing functional nanosystems using a bottom-up approach [10–12].
Raman spectroscopy (RS) is a label-free, noninvasive measurement technique, which detects molecular vibrational signals [13–16]. RS is widely used to analyze biological samples including proteins, cellular organelles, and single living cells, which provide information on the chemical composition of samples [17–20]. However, because of the intrinsic low scattering cross sections of RS, the user needs to acquire the Raman spectra for tens of seconds or even a few minutes. Light toxicity and bleaching may degrade the samples, which limits the practicability of RS. One of the methods used to enhance RS signals is to exploit the effects of surface plasmon resonance (SPR) of metallic NPs or nanostructured surfaces. SPR-based RS enhancement is also known as surface-enhanced Raman scattering (SERS). SERS significantly enhances Raman scattering and provides single-molecule sensitivity [21,22]. SERS is a well-known technique that has been used in various fields such as quantification of DNA bases [23,24], identification of viruses and microbes [25,26], and characterization of cancer cells [27,28] with elaborate experimental designs. However, SERS is rather time-consuming because of its tedious sample preparation. To guarantee a satisfactory enhancement rate, most of the samples are dried with metallic NPs prior to the measurements. This approach can cause detriment to living biological samples because most of these samples are naturally found in aqueous media. Alternatively, SERS can be realized by mixing the biological samples with colloidal NPs before measurements [29]. However, the enhancement rate and repeatability of the measurements cannot be ensured with this approach because of the random distribution of NPs in the aqueous media.
In this work, we realized the identification of 60-nm Ag NPs using backscattered imaging. Next, we realized trapping of the Ag NPs in an aqueous medium using a 1064-nm convergent Gaussian laser beam. The dynamic processes (trapping and escaping) of the individual Ag NPs were recorded using a charge-coupled device (CCD) camera. With our simple backscattered illumination scheme, we were able to maintain a high trapping stiffness for the LT system, where up to four Ag NPs could be trapped simultaneously. When we prepared a sample composed of Ag NPs and Bacillus subtilis bacteria, we found that more Ag NPs could be simultaneously trapped with the bacteria. The increase in the number of trapped Ag NPs can be attributed to the higher trapping forces, resulting from the interactions of the trapped bacteria cells due to their higher trapping gradient forces. Following this, we used a 532-nm laser beam to excite the Raman scattering of the Ag NPs + Bacillus subtilis samples and the results showed that the RS was enhanced repetitively. We believe that the laser trapped Ag NPs can be used for in situ RS enhancement with minimal sample pretreatment.
2. Materials and methods
2.1. Optical system
Figure 1 shows the schematic of the optical system used in this study. The optical system consisted of an inverted microscope (Axiovert-200, Carl Zeiss Microscopy GmbH, Germany) with an oil-immersion objective lens with a magnification of 100 × and numerical aperture of 1.25 (A-Achroplan, Carl Zeiss Microscopy GmbH, Germany). A linearly polarized continuous wave 2-W Nd:YAG laser beam operating at a wavelength of 1064 nm (Coherent Inc., USA) was coupled to the microscope. The maximum trapping laser power at the focus is ~500 mW with a beam size of ~1 . Another 532-nm solid-state laser beam (Excelsior 532-200, Spectra Physics, USA) was introduced the microscope, which was used as the Raman excitation laser source. The excitation laser power at the focus is ~20 mW with a beam size of ~500 nm. The Raman spectra were recorded using a spectrometer with a 1200 g⋅mm−1 grating blazed at 500 nm (SpectraPro 2300i, Teledyne Princeton Instruments (formerly Acton Research Corporation), USA) with a liquid-nitrogen cooled spectroscopic CCD (Spec-10: 400BR/LN, Teledyne Princeton Instruments, USA) having a working temperature of −120°C. A 100-μm diameter pinhole was used to block stray light from the environment. A notch filter (NF533-17, Thorlabs Inc., USA) with a center wavelength of 532 nm was used to eliminate the Rayleigh scattering light. The Raman scattering wavelength and intensity were calibrated using a Hg-Ar calibration light source (HG-1, Ocean Optics, USA) and polystyrene beads, respectively.
Fig. 1 Schematic of the optical system. The 1064-nm linearly polarized continuous wave Nd:YAG laser beam and the 532-nm solid-state laser beam were coupled into the inverted microscope as the sources of LT and RS, respectively. Raman spectra were recorded by the spectrometer and liquid nitrogen-cooled spectroscopic CCD (CCD2). Here, L1–L2 and L3–L4 represent two pairs of beam expanders, R1, R2, and R3 represent reflecting mirrors, and D1, D2, and D3 represent dichroic mirrors.
The focal position of the laser was adjusted using a coupling mirror (L5) and monitored based on the reflection spot on the glass substrate of the chamber using a CCD (CCD1) (MH15, Fuzhou Tucsen Photonics Co., Ltd., China). The same CCD was used to record the dynamic process of the trapped particles at an acquisition rate of 50 Hz. The trapping laser power was measured at the back focal plane of the objective using an optical power meter (842-PE, Spectra Physics, USA). A Hg light source was coupled via the fluorescence path of the microscope to illuminate the NPs in backscattering mode (not shown in the figure), which was commonly used for fluorescence excitation. It shall be noted that the 532-nm solid-state laser beam and Hg light source could not be used simultaneously because they shared the same light path. During Raman spectra acquisition the 532 nm laser beam was coupled into the microscope through the same path of the mercury lamp and the mercury light was removed.
2.2. Characterization of the Ag NPs
The Ag NPs (0.02 mg⋅mL−1 in aqueous buffer) with a particle size of 60 nm were purchased from Sigma-Aldrich, USA, and stored at a temperature of 4°C. A scanning electron microscope (SEM) (Quanta 600F, FEI Company, USA) was used to confirm that the Ag NPs were well-dispersed with a uniform sharpness, as shown in Fig. 2. The Ag NPs were stabilized using a citrate buffer and the zeta potential was found to be negative, indicating that the Ag NPs were covered with negative charge [30].
2.3. Sample preparation
A Petri dish with a diameter of 35 mm was used as the sample chamber, which was sealed with a cover slip by surface tension after the aqueous sample was injected into the Petri dish. The sample chamber was filled with ~500 μL of sample with a depth of 1 mm. For the experimental group, the Ag NPs were mixed with the Bacillus subtilis bacteria prior to the experiments without any further pretreatment. The bacteria were freshly cultivated and then stored at a temperature of 4°C.
2.4. Spectroscopic measurements and analysis
The Raman spectra were acquired and analyzed using a commercial software (Spectra Sense, Version 5.0, Acton, USA). The Raman spectra were acquired and corrections were made for cosmic spikes. Each spectrum was integrated for 30 s and repeated 50 times for the experimental group (Ag NPs + Bacillus subtilis samples), where the samples were selected randomly in the sample chamber. The excitation laser power for the samples was 20 mW.
The background of the Raman spectrum for each sample was removed by subtracting the Raman spectrum of the control group (pure water) from the Raman spectrum of the sample. Following this, the Raman spectrum for each sample was smoothed using the Savitzky-Golay filtering algorithm. A seventh-order polynomial fit was used to further reduce the background fluorescence. Finally, the Raman spectrum for each sample was averaged and truncated to within a wavenumber range of 640–1700 cm−1 for comparison.
3. Results and discussion
3.1. Observation of individual Ag NPs in backscattering illumination mode
Individual 60-nm NPs are too small to be observed directly in bright field illumination unless if dark field illumination is used. However, the use of dark field imaging will reduce the LT efficiency because this approach requires an objective with a smaller numerical aperture to match the illumination condenser. In this study, we used backscattering illumination with the light source from the fluorescent excitation Hg lamp, which was connected to the rear port of the microscope. With this approach, we successfully identified individual NPs in an aqueous medium with a large numerical aperture of the microscope objective. The individual Ag NPs dispersed in aqueous media appeared as individual bright dots, as shown in Fig. 3. The scattered light from the Ag NPs looked like sparklets because their brightness varies with respect to time due to Brownian motion. The sparklets were widely observed at different depths. We consider that the backscattering light was mostly Rayleigh scattering light, whose intensity is inversely proportional to the power of the light wavelength. Meanwhile, the spectrum of Hg light source is largely composed of shorter-wavelength components with strong light intensities, effectively enhanced the scattered light from the Ag NPs, therefore we were able to clearly identify the individual NPs in the aqueous medium. The advantage of our backscattering illumination mode is that we are able to use an objective with a large numerical aperture, which promotes the trapping stiffness.
Fig. 3 Image of the Ag NPs (particle size: 60 nm) in aqueous medium in backscattering illumination mode. The individual Ag NPs can be clearly identified from the bright spots in the image.
3.2. Laser trapping of Ag NPs
To investigate the ability of our optical system in trapping the Ag NPs, we recorded the dynamic process of the Ag NPs during the LT and we characterized the LT process by analyzing the recorded images. The LT was focused at 10 μm above the bottom of the sample chamber in order to eliminate interference from the bottom surface. We observed that the Ag NPs were dragged into the laser trap and then captured, which confirms that the highly convergent IR Gaussian laser beam is capable of trapping metallic NPs. Once an Ag NP is trapped, the scattered light intensity from the Ag NP becomes relatively stable compared with those from the freely moving Ag NPs. Figure 4 shows the typical results obtained from 1-min recording of LT of the Ag NPs. Figure 4(a) shows the dynamic process of the backscattered light from the trapped Ag NPs within a 2 μm × 2μm region of interest (ROI), centered in the laser trap. The scattered light intensity changes in a stepwise manner, indicating that the number of trapped Ag NPs varies with respect to time. Based on the intensity, we could directly estimate the number of Ag NPs trapped by LT. We perceived that the continuous increase in the scattered light intensity is likely because the Ag NPs gradually become closer and move towards the focal plane. In contrast, the stepwise change in the scattered light intensity indicates that an individual Ag NP enters or exits the laser trap. During the 1-min recording, up to four particles were trapped simultaneously. When multiple Ag NPs were trapped together, this made it more difficult to directly identify the number of trapped Ag NPs. However, the number of trapped Ag NPs can be clearly identified from the intensity profile in Fig. 4(c).
Fig. 4 Dynamic process of trapping Ag NPs in the aqueous medium: (a) Variation of the scattered light intensity with respect to the trapping time within a 2 μm × 2 μm ROI in the LT focus spot; (b, c) images and surface plots of the dynamic process corresponding to the labeled times in (a).
We show six typical images of the dynamic process of LT of Ag NPs at different times and their corresponding surface plots in Figs. 4(b) and (c), respectively. It can be seen from Fig. 4(a) that the scattered light intensity continuously increases during the first 10 s whereas the scattered light intensity increases in a stepwise manner at the numbers 1, 2, 3, and 5 (shown in red). In contrast, the scattered light intensity decreases abruptly at the numbers 4 and 6. It can be observed from Figs. 4(b2) and (c2) that two Ag NPs are simultaneously trapped at ~20 s from the start of LT. In Figs. 4(b3) and (c3), there were three trapped Ag NPs whereas in Figs. 4(b5) and (c5), there were four trapped Ag NPs. In Figs. 4(b4) and (c4), one Ag NP escaped from the laser trap whereas in Figs. 4(b6) and (c6), multiple Ag NPs escaped simultaneously, leaving only one Ag NP in the laser trap. In general, the numerical aperture of the microscope objective, laser intensity and relative refractive index of trapped particles will determine the confinement stiffness or potential depth of the laser trap. We could not trap more than four NPs steadily in the laser trap over a long period because of the electrostatic repulsion of the Ag NPs. This scenario is analogous to the saturation of the number of protons in atoms. We believe that this is likely because the Ag NPs escaping from the laser trap have relatively weak vertical gradient forces, large reflections of laser radiation, and strong thermophoretic forces as a result of laser heating, all of which promote the likelihood of the Ag NPs escaping from the laser trap.
3.3. Trapping of Ag NPs and Bacillus subtilis bacteria
We repeated the LT experiments using a mixed sample composed of Ag NPs and Bacillus subtilis. After adding the Bacillus subtilis bacteria into the colloidal Ag NPs we recorded the scattering light images immediately. We found that the scattered light intensity was significantly stronger for the mixed sample even though we used the same LT power. In the previous experiment (in which the sample consisted of only Ag NPs), we cropped a 2 μm × 2 μm ROI in the images for further analysis. However, in this experiment (in which the sample was composed of Ag NPs and Bacillus subtilis), we observed that the scattered light intensity was rapidly saturated for the same ROI size. Hence, we used a larger ROI (4 μm × 4 μm) and the results are shown in Fig. 5(b). It can be seen that more Ag NPs are captured. Figures 5(a1, b1) and (a2, b2) correspond to trapping times of 1.6 s and 9.5 s, respectively. Figures 5(a3) and (b3) are the scattered light results for Bacillus subtilis in aqueous medium without Ag NPs and it is evident that the bacteria do not reflect sufficient light. Figure 5(a4) shows the image of the mixed sample under transmitted white light illumination mode. In this case, the Ag NPs cannot be identified even though several bacteria are trapped in the laser trap and they can be clearly seen. Based on the results shown in Figs. 5(a) and (b), we deduced that multiple Ag NPs and Bacillus subtilis bacteria aggregated in the laser trap.
Fig. 5 (a1, a2) Images of the trapped Ag NPs and Bacillus subtilis in backscattering illumination mode recorded at 1.6 s and 9.5 s, respectively; (b1, b2) Surface plots within a 4 μm × 4 μm ROI of images a1 and a2, respectively; (a3, b3) Trapped Bacillus subtilis without Ag NPs and its surface plot, respectively; (a4) Trapped Ag NPs and Bacillus subtilis under transmitted white light illumination mode.
We believe that more NPs can be trapped with bacteria from several reasons. The laser can trap both NPs and bacteria simultaneously and aggregate them. Moreover, both NPs and bacteria are polarized under the linear polarized laser filed, and generate induced dipoles. Thus attractive dipolar-dipolar interactions occur between NPs and bacteria. Considering bacteria (micron size) are much larger than NP, one bacterium can interact with multiple NPs. Similarly, some literatures reported that 60 nm metallic NPs and 2-11 um dielectric particles were trapped by 1064 nm laser [2,31–33]. Also, the attractive interactions promote NPs to be adsorbed on bacteria surface and form a cluster. Such micro-sized clusters are easy to be trapped due to their relative high trapping stiffness compared to individual NP. Multiple clusters are trapped together and formed a group as shown in Fig. 5(a4). In a word, bacteria effectively aggregate NPs and increase equivalent tapping stiffness of NPs in this case. Therefore, more NPs can be trapped with bacteria. However, as more Ag NPs and Bacillus subtilis bacteria are trapped in the laser trap their gravitational effect is no longer negligible, also there is complicated electrostatic interaction. Hence, there is maximum limit in the amount of Ag NPs and Bacillus subtilis bacteria that can be trapped.
3.4. Trapping of Ag NPs and Bacillus subtilis bacteria for RS enhancement
To explore the possibility of trapping NPs by LT in order to enhance RS of bacteria in aqueous media, we attempted to trap Ag NPs and Bacillus subtilis bacteria together. We measured the Raman spectrum of the mixed sample excited by 532-nm solid-state laser beam. Figure 6 shows the normal and enhanced Raman spectra of the Bacillus subtilis bacteria. The Raman spectrum of the Ag NPs in aqueous medium is also shown for comparison. We also analyzed RS region rich in bioinformation (also known as the “fingerprint region”) in Fig. 6. The main bands of Raman spectra of the samples investigated in this study are listed in Table 1.
Fig. 6 Raman spectra of Bacillus subtilis (black), Ag NPs + Bacillus subtilis sample (red), and Ag NP sample (blue). It is evident that the Ag NPs + Bacillus subtilis sample shows enhanced Raman scattering compared with the Bacillus subtilis in aqueous medium. The Ag NPs in aqueous medium are used as the control group for comparison.
Table 1. Bands assignment in the Raman spectra for the Ag NPs + Bacillus subtilis sample and comparison with those in previous studies [26,34–36] a
The bands in the Raman spectrum for Bacillus subtilis were assigned to amide I (1668 cm−1), amide II (1573 cm−1), saturated lipids (1448 cm−1), adenine (1330 cm−1), amide III (1120, 1248 cm−1), nucleic acid (1090 cm−1), phenylalanine (1004, 1604 cm−1), phosphate (952 cm−1), and tyrosine (831, 860 cm−1). In general, the Raman spectrum includes the entire cellular information of the bacteria while the enhanced Raman bands originate from the surface molecules, which are mostly close to the metallic NPs. It can be observed from the Raman spectrum for the Ag NPs + Bacillus subtilis sample that there is significant enhancement in the bands assigned to amide II (1573 cm−1) and cytosine (793 cm−1) whereas there is almost no enhancement in the bands assigned to amide I (1668 cm−1) and saturated lipids (1448 cm−1). The other Raman bands are enhanced by a factor of 2.
In our experiments, the surface charges of the Ag NPs and Bacillus subtilis bacteria were both negative. Hence, it is unlikely that the Ag NPs and bacteria are in direct contact. In other words, there is a physical distance between the Ag NPs and bacteria in aqueous medium even though they are trapped together in the laser trap. Hence, we can deduce that the enhancement of the Raman spectrum of the Ag NPs + Bacillus subtilis sample is relatively homogeneous. This also explains why the enhancement rate is not as pronounced as the enhancement rate of the RS assay, where the dried biological sample and Ag NPs are in direct contact. According to previous studies [37,38], the enhancement rate will further increase once metallic NPs are introduced into the cellular walls. However, because all of the samples used in our experiments are in aqueous media, our approach is more practical to analyze living bacteria, rendering it useful for single-cell sequencing for example. Our enhanced RS approach is also suitable for lab-on-a-chip applications in order to improve screening efficiency and prevent human exposure in studies involving pathogenic bacteria [39]. In the future, we intend to further enhance the RS signals by reducing electrostatic repulsion between the NPs and living cells by adding salt in the solution, and expand the application of our enhanced RS for other microorganism species and sub-cellular particles.
Owing to the randomness of the trapping of Ag NPs and Bacillus subtilis, we confirmed the stability and repeatability of the RS enhancement by comparing 10 randomly chosen Raman spectra and the results are shown in Fig. 7. It is apparent that the enhanced Raman spectra were consistent with acceptable variation.
Fig. 7 Ten randomly chosen Raman spectra of Ag NPs + Bacillus subtilis samples used to confirm the stability and repeatability of the RS enhancement.
4. Conclusions
In this study, we successfully trapped and analyzed the dynamic process of multiple 60-nm Ag NPs in aqueous medium by near-infrared LT. We identified the individual 60-nm Ag NPs by using backscattering illumination imaging. With our optical system, we simultaneously realized laser trapping and backscattering illumination imaging of Ag NPs while maintaining a high trapping stiffness. Moreover, we demonstrated that the scattered light intensity can be used to analyze the number of trapped Ag NPs, which is useful to study the interactions between metallic NPs. We found that the trapped Ag NPs became saturated at a certain threshold and observed that up to four Ag NPs could be simultaneously trapped. However, much more NPs could be trapped with the addition of Bacillus subtilis into the Ag NP colloidal solution. Furthermore, we demonstrated that the laser trapped Ag NPs with Bacillus subtilis can be used for in situ RS enhancement with minimal sample preparation. The simplified assay is suitable to rapidly identify and characterize environmental or pathogenic microorganism species.
Funding
National Key R&D Program of China (2017YFC0209504); National Natural Science Foundation of China (NSFC) (U1636110); State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences.
Acknowledgments
The authors thank Dr. Yu Fu’s group for providing the bacteria samples.
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