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Article

In Situ Tracking of Nanoparticles During Electrophoresis in Hydrogels Using a Fiber-Based UV-Vis System

by
Matthäus Barasinski
1,2,
Valentin Jasper
1,
Marion Görke
1,2 and
Georg Garnweitner
1,2,*
1
Institute for Particle Technology (iPAT), Technische Universität Braunschweig, Volkmaroder Straße 5, 38104 Braunschweig, Germany
2
Laboratory for Emerging Nanometrology (LENA), Technische Universität Braunschweig, Langer Kamp 6a, 38106 Braunschweig, Germany
*
Author to whom correspondence should be addressed.
Powders 2025, 4(1), 3; https://doi.org/10.3390/powders4010003
Submission received: 22 November 2024 / Revised: 5 January 2025 / Accepted: 7 January 2025 / Published: 10 January 2025
(This article belongs to the Collection Multidimensional Particle Properties: Characterization, Description, Separation)
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Abstract

:
Gel electrophoresis is a powerful method for the separation of nanoparticulate suspensions into several fractions with distinct particle properties. To monitor particle migration through the three-dimensional net structure of the gel and gain insights about the separation process, this study introduces a self-designed fiber-based UV-Vis measurement system equipped with five probes for the sequential in situ recording of absorption spectra. The system was employed to investigate the migration and separation of Au and Fe3O4 particles within hydrogels of varying agarose concentrations (0.15–0.50 wt.-%), revealing an increase in scattering with higher agarose content. The identification of specific particle fractions with a spherical or rod-shaped morphology was successfully achieved within the gels due to characteristic absorption peaks, allowing the real-time observation of particle separation. For the separation of a binary mixture, an adequate migration distance is needed according to the difference in the electrophoretic mobility of the two samples. The particle tracking and an additional mathematical deconvolution allowed the analysis of mixed particle samples within the gel so that their weight ratio could be determined. Finally, the system was calibrated for the determination of the particle concentration within the gel matrix, quantitatively revealing the particle concentration at a specific position in the gel.

1. Introduction

The continuous development of nanoparticle (NP) syntheses with increasing controllability of dimensional parameters (e.g., size, morphology or porosity), and surface chemistry [1,2,3] enables the preparation of NPs tailored for specific applications, e.g., narrow- or broad-band-emitting NPs at defined energies for light emitting devices (LEDs), sensors and photovoltaic systems [4,5,6]. Furthermore, having an adequate level of a specific particle property (e.g., size) is crucial for applications with systemic boundaries, e.g., intracellular drug delivery or molecular imaging [7,8,9]. Unfortunately, increased controllability goes usually hand in hand with a more complex synthesis and, e.g., commonly used precipitation methods lead to broad particle size distribution. For NP syntheses at the industrial scale, the standard deviation in size and morphology is typically even larger than at the laboratory scale, leading to the necessity of post-synthetic purification steps [10,11]. Among techniques such as dielectrophoresis [12], ultracentrifugation [13] and chromatography [14], it has been shown that gel electrophoresis (GE) possesses great potential in classifying nanomaterials in regard to the parameters discussed above [15,16,17,18]. The possibility for continuous flow operation and the possibility to obtain specific fractions as well as the prevention of a remixing of already separated NP fractions make it advantageous in comparison to, e.g., the common density gradient centrifugation. The extraction of the NPs can be performed by elution or the so-called pool method, as we have demonstrated previously [12]. Thereby, a gel fragment containing the separated particles is cut out and placed near an electrode of a second electrophoretic chamber. The chamber is partially refilled with buffer and the applied voltage causes the particles to accumulate in a small pool [12]. A great advantage of the use of agarose gels for gel electrophoresis is the possible variation in the mesh size, which can be adjusted by the agarose concentration [19,20]. To design a gel electrophoresis process for the separation of a specific system, an understanding and modeling of the migration of NPs with different properties is vital. Generally, the particle migration velocity is expressed by the electrophoretic mobility µE of the particles, which is usually influenced by the particle and mesh size, particle morphology and surface chemistry (charge and any corona around the particles), temperature, the pH and ionic strength of the buffer solution, and electric field strength, which is spatially heterogeneous and changes over the experimental time [16,21,22,23,24]. Consequently, the true electrophoretic mobility and the calculated zeta potential from this value are extrinsic values, which are strongly dependent on the system and experimental time and therefore difficult to access [25]. For electrophoretic separation experiments, it was observed that in addition to nonsteric effects such as the electroosmotic flow and the interaction between the particle surface (ligands) with the gel or buffer solution [24,26], the relation between the mesh size and particle size is the key parameter for the successful separation of differently sized particles. Especially for metal oxide NPs with high zeta potentials and a negligible corona of organic ligands on their surface, this relation leads to two distinct general regimes of the particle migration [17]. If both sizes are similar, the migration of particles is restricted or sterically completely prevented. In contrast, with a particle size much smaller than the mesh size, the migration is unrestricted so that particles migrate according to their zeta potential independently of their particle size. Using such agarose gels, several studies showed the migration of metal NPs (e.g., Au, Ag, etc.) and their purification or separation according to their particle size, morphology or surface chemistry due to their facile traceability because of their optical properties [15,27,28]. In contrast, the migration of metal oxide NPs, typically obtained as products that are more inhomogeneous and used more often in industrial applications than metal NPs, has not been investigated systematically. Usually, the purification is monitored qualitatively, but for application at an industrial scale, a quantitative consideration of the separated fractions is important to evaluate the separation process. This is, however, more difficult for metal oxides as they do not exhibit distinct size-dependent colors with strong intensity like metal NPs or quantum dots. Nevertheless, many metal oxides show UV and/or Vis absorption, and larger NPs also lead to scattering effects. Hence, for the in situ measurement and quantification of NPs in gel electrophoresis, a self-designed fiber-based UV-Vis system with five measurement positions is introduced in this study, which is able to record the absorption of Au and Fe3O4 NPs used as model systems for comparison within the gel matrix at several positions. The objective is to recognize different particle bands within the agarose gels due to differences in the particle size and shape at a given position in the gel and infer the separation quality. Furthermore, the general influence of the particle and gel concentrations on the UV-Vis absorption signals was studied. Therefore, spherical and rod-shaped NPs with different particle sizes were synthesized and introduced into the gels for separation. The envisioned use case in industry is the separation of one synthesized sample into several fractions with, e.g., narrower particle size distributions. To achieve a systematic investigation and to be able to draw clear conclusions from the results, the separation experiments in this work were conducted with binary mixtures of the model particle samples, where the samples differ in shape, size or material so that each one can be clearly recognized according to these properties. Our results show that the separation process of particles can be traced with the fiber-based system so that the separation position as well as the separation sharpness/purity of individual parts of a sample can be determined online within the gel. Furthermore, it was possible to determine the particle concentration of a running particle band within the gel after prior calibration, thus enabling its quantification.

2. Materials and Methods

2.1. Synthesis and Modification of Au NPs

Spherical Au NPs with varied particle sizes below 100 nm were synthesized using a seeded growth technique according to Zheng et al. [29], as performed in our previous work [30]. Thus, gold(III)-chloride trihydrate (HAuCl4∙3H2O), cetyltrimethylammonium bromide (CTAB), cetyltrimethylammonium chloride (CTAC), sodium borohydride (NaBH4) and 11-mercaptoundecanoic acid (MUA) were procured from Sigma Aldrich. The syntheses and subsequent functionalization resulted in Au NPs coated with MUA on their surface, which were stable in the used 0.5× TBE buffer.
Initially, spherical seeds (<5 nm) were formed at room temperature by adding CTAB to a HAuCl4 solution after a prior dissolution step at 50 °C and NaBH4 as a reducing agent, causing a color change in the solution from yellow to brownish. After a brief and gentle stirring treatment, the suspension was left unstirred for 30 min. Next, this seed dispersion was utilized for the growth step by introducing it into a stirred gold precursor solution using a syringe pump. This led to an increase in the diameter of the NPs, dependent on the volume of the precursor solution and the seed concentration. Next, the NPs were washed via centrifugation (SIGMA 3-30KS, 30 min at 20,000 RCF, Sigma Laborzentrifugen GmbH, Osterode, Germany), resulting in two phases. The supernatant was removed, whereas the concentrated dispersion of NPs either was used directly or subjected to additional growth steps following the same procedure to increase the particle diameter.
The produced Au NPs were dispersed in water and exhibited stability against agglomeration, attributed to the adsorbed CTAC double layer. However, when introduced into 0.5× TBE buffer (pH = 8.3) for electrophoresis, their zeta (ζ-) potential experienced a significant decrease, leading to unstable suspensions. To address this issue, a ligand exchange is usually carried out with thiols due to their strong coordination properties to replace the CTAC on the Au NP surface [31,32,33]. This was conducted directly after synthesis in a 1:1 mixture of water and ethanol with dissolved MUA using a one-pot protocol. The pH was maintained at a value of around 9 by the addition of NaOH, which was close to the pH of the 0.5× TBE buffer (pH~8.3), so that the carboxylic groups of the MUA molecules became deprotonated, leading to electrostatic repulsion forces and thus stable suspensions in the buffer solution [34]. Based on the assumption of full synthesis yield and no particle loss during subsequent processing steps, a solid content of 0.01 mol/L for the Au NP sample (suspension) was estimated. Under the assumption that an MUA molecule occupies 0.2 nm2 on the particle surface and to ensure a monolayer of the MUA on the surface, a 2-fold excess of the stabilization agent was used. Therefore, a 0.01 mol/L MUA solution in 1:1 (v/v) ethanol and water mixture was added in a ratio of 1:1 (v/v) to the sample suspension for the ligand exchange, ensuring an excess of MUA for the complete surface coverage of the Au NPs.
The excess MUA was removed via dialysis (Carl Roth, ZelluTrans, Karlsruhe, Germany) with a molecular weight cut-off of 12,000–14,000 mol/g. For each purification treatment, three samples with volumes around 5–10 mL were placed in a 2 L beaker, whereby the medium was changed every day for four days. This medium consisted of a 1:1 mixture of 1× TBE and EtOH so that a final concentration of 0.5× TBE was reached to ensure an alkaline pH value (~8.3) and hence, a deprotonated state of the stabilization agent.

2.2. Synthesis and Modification of Fe3O4 NPs

The synthesis of spherical Fe3O4 NPs was based on the method reported by Dehsari et al. [35] and on the results of our recent study [36]. After synthesis, the NPs were stabilized in an aqueous dispersion, and the surface was refunctionalized with tiron, as adapted from Akbulut et al. [37]. These tiron molecules have been reported to bind to the surface of the particles with their catechol group, and therefore, the polar sulfonic acid groups can interact with the aqueous medium. For the functionalization, the obtained dispersion was added to a solution of 100 mg tiron (0.302 mmol) in 2 mL dist. water and sonicated for 2 h. After this time, 5 mL water was added to dilute the dispersion of tiron-coated NPs, which shifted the equilibration and therefore increased the yield. The mixture was sonicated for another 2 h, and afterwards, the phases were separated with a centrifuge (6000 rpm, 2 min). The aqueous phase turned dark purple, which indicated the presence of tiron-coated Fe3O4 NPs, and was isolated for further usage. The organic phase turned brown-orangish and was discarded.

2.3. Characterization of Au and Fe3O4 NPs by UV-Vis, SEM and TEM

To ensure the reliability of the assembled fiber-based UV-Vis device (details are presented in Section 3.1 and Section 3.2), initial absorption measurements in quartz cuvettes of various commercial Au suspensions capped with citric acid at an optical density of 1 (BBI solutions, Crumlin, UK) were compared to measurements performed with a commercial UV-Vis device (SPECORD 210 PLUS, Analytik Jena AG, Jena, Germany). Suspensions of spherical Au NPs with varied particle sizes exhibit one characteristic localized surface plasmon resonance (LSPR) peak at a wavelength of approx. 500–600 nm. For particles stemming from an ideal synthesis, the particle size distribution (PSD) is narrow and no aggregates are present. In such a case, larger particle sizes cause a red-shift to larger wavelengths, opening up the possibility of calculating a mean particle size using the wavelength obtained at the LSPR [38,39]. In contrast, if the synthesis is non-ideal, the resulting particles can be polydisperse and polymorphic, also leading to a red-shift due to wide PSD and aggregation [40], showing the importance of additional measurements such as TEM or DLS.
Furthermore, the particle sizes and morphology were investigated by microscopic methods. On the one hand, scanning electron microscopy (SEM) was used (Helios 5 UX DualBeam, Thermo Fischer Scientific Inc., Waltham, MA, USA) for the characterization of Au NPs and, on the other hand, transmission electron microscopy (TEM, Tecnai G2 F20 TMP, FEI Company, Hillsboro, OR, USA) was performed for the smaller Fe3O4 NPs. To accomplish both measurements, woven copper nets with a carbon film (Plano GmbH, Wetzlar, Germany) were dipped into diluted suspensions and dried at room temperature.

2.4. Preparation of Agarose Gels Followed by Electrophoretic Experiment

The procedure for preparing agarose gels involved mixing agarose powder (UltraPure, Thermo Fisher Scientific Inc., Waltham, MA, USA) with 0.5× TBE buffer (Tris, borate, EDTA; Rotiphorese, Carl Roth, Karlsruhe, Germany), previously diluted with deionized water. To create a 0.15 wt.-% agarose gel, 0.075 g of agarose powder was dissolved in 50 mL of the 0.5× TBE buffer at 90 °C under stirring, covering the flask to prevent evaporation. The solution was maintained at 90 °C for at least 10 min to ensure the complete dissolution of the agarose powder. Once transparent, the solution was cooled to 55 °C before being poured into a horizontal electrophoretic chamber (MINIeasy, Carl Roth, Karlsruhe, Germany). After polymerization for 30 min, an additional 50 mL of buffer solution was added to ensure electrode contact, and the gel was left to stand for another 30 min.
Before the electrophoretic experiment, the density of each sample was increased by mixing with a saturated sucrose solution at a ratio of 4:1 (v/v) to facilitate sinking into the gel pocket (V = 35 μL). A direct current (DC) electric field with a voltage of 100 V was applied, causing particles to migrate through the gel (electrode distance d = 10 cm). Images were captured from the top every 60 s using a camera (acA2040–90uc, Basler AG, Ahrensburg, Germany), displaying each sample as a colorful band within the gel. Generally, the contrast and brightness of the images were slightly adjusted. It was assumed that the spatial and temporal dependence of the electric field was negligible.

3. Results

3.1. Set-Up of the Fiber-Based UV-Vis Measurement System

Figure 1 presents the fiber-based UV-Vis measurement system with five measurement positions, which can be changed flexibly. In Figure 1A, a schematic set-up is depicted consisting of (1) a light source (SL5 deuterium halogen fiber optical light source; StellarNet Inc., Tampa, FL, USA), which is used to illuminate the sample in a spectral range of 190–2500 nm. This light is passed to (2) a multiplexer (JMA-Multiplexer-2×5-SMA, Laser2000, Berlin, Germany) and further on to five measuring points consecutively. From each point the irradiation is led to (4) a UV-Vis spectrometer (fiber optic spectrometer: Black-Comet; StellarNet Inc., Tampa, FL, USA), measuring the absorption signals in the wavelength range of 220–1100 nm. Several fiber optic cables with a core diameter of 600 µm are used to connect the individual components as well as collimation optics to parallelize the light before and after traveling through (3) the gel. Figure 1B shows a photograph of the fiber-based UV-Vis system, where the red arrows symbolize the way of the light before reaching the gel and, on the opposite side, the green arrows show the way of the attenuated light due to the absorption of the sample. Additionally, the voltage source is visible in the bottom part of the image and the associated power cables connect this source with the gel chamber. A detailed photograph of the gel chamber can be found in Figure 1C, with the chamber being placed between two perforated plates with integrated screw threads (see Figure 1D). For a higher flexibility of the system, the optical fibers can be screwed into various threads of these plates to change the measurement positions. Moreover, the plates and the chamber are aligned vertically on a rail, ensuring that the light propagates from the optical fiber at the top through the gel into the optical fiber at the bottom. The SpectraWiz® spectroscopy software (StellarNet Inc., Tampa, FL, USA) and a simple LabVIEW (National Instruments Corp., Austin, TX, USA) program are used to obtain the measured absorption data at the five measurement positions.

3.2. Comparison Between Commercial and Fiber-Based UV-Vis Measurement Systems

First, UV-Vis measurements are performed to compare the absorption spectra recorded by the commercial and the fiber-based UV-Vis measurement systems to prove the functional capability of the fiber-based system. To this end, Figure 2 shows the absorption spectra of three commercial Au NP suspensions with varied particle sizes (see Supporting Information Figure S1 for the respective PSD obtained from dynamic light scattering (DLS)) measured by the commercial UV-Vis device (dashed lines) and the fiber-based one (solid lines) using quartz cuvettes. As mentioned above, Au NPs possess a size-dependent LSPR peak, where peaks at larger wavelengths indicate larger particle diameters. A distinct peak can be observed for all three Au suspensions, although for the sample with a hydrodynamic mean volumetric particle diameter of x50,3 = 213 nm, the peak is not considerably pronounced. The suspension of this sample is not as clear as the other suspensions (x50,3 = 36 and 91 nm) but rather opaque; hence, it can be concluded that the particles within the sample are aggregated, resulting in a rather polydisperse particle size distribution. This is supported by the absorption spectrum with its wide peak. Moreover, the particles of this sample are not in the nm regime anymore but in the submicron meter range (<500 nm, Figure S1), leading to stronger light scattering due to larger particle diameters. Comparing the curve progression and the position of the peak of one specific sample measured by the two devices, no large deviations are visible. The signal-to-noise ratio in the spectra recorded by the commercial device (dashed lines) are equal or only slightly improved compared to the spectra measured by the fiber-based device (solid lines), underlining the high quality of the data obtained from the fiber-based measurement system. However, the spectra of the fiber-based device contain a few spikes, e.g., next to the LSPR peak of the sample with a particle size of 91 nm at a wavelength of around 550 nm. These are considered as artifacts or noise stemming from the measurement routine because several glass cuvettes are used with no fixed measurement points and the raw data are not smoothened. Overall, the obtained data from the fiber-based UV-Vis measurement system are sufficiently in accordance with the data obtained from a commercial device; therefore, the fiber-based system can be considered as equivalent to the commercial one and can be used for further experiments.

3.3. UV-Vis Measurements with Varied Gel and Particle Concentrations in Quartz Cuvettes

Next, the fiber-based UV-Vis system is used to measure the absorption spectra of commercial Au NPs with an x50,3-value of 36 nm in hydrogels with various concentrations. The samples are prepared as artificial mixtures of liquid gel with varied agarose concentrations and Au suspensions, always with the same concentration (see Figure 3A). Afterwards, the prepared cuvettes are placed between the perforated plates for the UV-Vis measurements, as shown in Figure 3B. First, a pure gel with the corresponding concentration (0.2 and 0.5 wt.-%) is measured to determine the background for subtraction for the samples containing the particles. Figure 3C clearly shows that the absorption intensity is strongly dependent on gel concentration, as expected. With higher gel concentrations, the gel structure is denser, and thus, scattering is increased, resulting in higher noise and lower absorption signals. Again, the signals are not perfectly smooth because no correction of the obtained raw data is performed and the measurement positions of the quartz cuvettes are different. Despite this, all samples show an LSPR peak centered at a wavelength of 526 nm, which corresponds to the LSPR peak of the same sample measured as a suspension in Figure 2.
Further experiments were performed to investigate the influence of the particle concentration of commercial small Au NPs (x50,3 = 36 nm) in 0.15 wt.-% (see Figure 4A) and commercial large Au NPs (x50,3 = 91 nm) in 0.50 wt.-% agarose gels (see Figure 4B). The sample preparation was executed as mentioned before, and the obtained spectra were normalized at a wavelength of 900 nm (where there were no expected signals from Au NPs or the gel) for better comparability because each sample required a different measurement time. Again, the results show that higher particle concentrations lead to increased absorption peaks and higher gel concentrations result in enlarged background noise levels. Both aspects, particle and gel concentration, need to be considered if absorption signals are interpreted. It can be concluded that concentrations of large Au NPs (x50,3 = 91 nm) ≥ 1.2 × 10 − 5 wt.-% (sample J) are high enough to measure reliably absorption signals in gels with agarose concentrations of 0.50 wt.-% (or sample P for 0.30 wt.-%, see Figure S2). For the 0.15 wt.-% agarose gel with small Au NPs (x50,3 = 36 nm), even lower particle concentrations can be detected confidently, e.g., sample E with 5.8 × 10 − 6 wt.-%. Summarizing the previous investigations, it can be concluded that the fiber-based system is able to measure absorption spectra in hydrogels with agarose concentrations (≤0.50 wt.-%) that are typically used for gel electrophoresis for NP separation processes.

3.4. In Situ UV-Vis Measurements

For the in situ UV-Vis investigations of nanoparticles within hydrogels, synthesized and subsequently functionalized spherical and rod-shaped Au NPs and irregular-shaped Fe3O4 NPs were used. Initially, their aqueous dispersions were measured with solid contents of 1.5–1.8 wt.-%. Due to their different shapes and materials, these particles can be distinguished by their characteristic UV-Vis absorption spectra. As already mentioned, spherical Au NPs show one LSPR peak, whilst rod-shaped Au NPs have two LSPR peaks, whereas the first peak correlates with the diameter and the second one with the length of the rod [41,42]. The Fe3O4 NPs show one rather wide peak at around 550 nm. Figure 5 shows the absorption spectra of each particulate suspension in water, proving that a differentiation is possible based on the shape of the curve or an assignment of the respective curve to a specific particle system, which is relevant here for a clear distinction between the model particle systems/particles of different types, sizes and shapes during particle separation. Besides this, the chemical structure of the respective ligand is presented as insets; MUA is attached to the particle surface of the Au NPs and tiron to the surface of the Fe3O4 NPs. Furthermore, the size and morphology of each sample were investigated by STEM and TEM measurements, and representative microscopic images are included in Figure 5. The spherical Au NPs are nearly monodisperse and monomorphic, with a particle diameter of around 38 nm according to SAXS measurements (see Figure S3 and Supporting Information as well as [43] for details). In contrast, the rod-shaped Au NP sample consists mostly of rod-shaped particles with a variation in mean particle length (~40 nm) and mean diameter (~26 nm), but also, some cubic and spherical particles can be seen in the STEM images. The Fe3O4 NPs possess a smaller particle size (~10 nm) and are irregularly shaped, as additional TEM images show in Figure S3.
For the in situ measurement experiment, a 0.15 wt.-% agarose gel was prepared and the particle dispersions were introduced into the gel pockets individually or as binary mixtures (see Figure 6A at t = 0 min). The individual samples were diluted with H2O in a volumetric ratio of 1:1 so that they possessed the same particle concentration as one sample in the binary mixture. The lower image in Figure 6A depicts the gel samples after the migration or separation of the binary mixtures after t = 6 min. Additional photographs, which were taken every minute, can be seen in Figure S4. All samples migrate completely out of the gel pocket and through the gel, indicating that they migrate under an unrestricted migration mechanism, with the mesh size being larger than the particle size, which is favored over the restricted mechanism (the mesh size similar to the particle size) for a desired continuous separation [17,44]. Additionally, it can be concluded that the particles are colloidally stable on a primary particle level due to sufficiently high charges on their surface stemming from the MUA and tiron ligands. The separation of the binary mixtures is based mainly on the difference in particle size, with the smaller Fe3O4 NPs (particle size ~ 10 nm) moving faster through the three-dimensional gel structure as they can pass more easily through the pore network and undergo, e.g., lower frictional forces. Moreover, the smaller NPs can take a rather straight path through the gel, whilst larger NPs need to avoid meshes that are smaller than the particle size, resulting in a snake-like motion (as described in reptation theory) and therefore longer migration distances. Here, the colorful bands already indicate the separation of the binary mixtures, and hence, we can infer that a successful separation by gel electrophoresis was achieved. Nevertheless, the fiber-based UV-Vis system was employed to verify whether individual bands could be assigned to the different initial components. Figure 6B shows the absorption spectra determined at the positions indicated by the circles in Figure 6A. First, it can be seen that, overall, three different types of spectra resulted: (i) red curves with one sharp and narrow LSPR peak around 520 nm, which can be assigned to spherical Au NPs; (ii) blue curves with two LSPR peaks, which can be assigned to rod-shaped Au NPs; and (iii) black curves with one wide LSPR peak, which can be assigned to the Fe3O4 NPs. This indicates that a successful separation has been achieved at this point, with the bands within the gel no longer overlapping or being in contact and each corresponding to a single initial component. However, complete separation cannot be proven by UV-Vis as absorption signals of a small content of one component (e.g., Au NPs) are overlaid by the signals of the main component (e.g., Fe3O4). Hence, the in situ measurements within the hydrogel are shown to permit the assignment of the absorption spectrum of a certain band to an individual component so that their tracking is enabled. For the detection of separated bands, the particle concentration, however, is a crucial parameter, and dilution effects should be considered such as the sample preparation (e.g., the addition of sugar solution for increased density) or the widening of a band during migration, where the particle concentration might be even locally decreased within the gel. The latter aspect leads to the impossible determination of the particle concentration at any position within the gel. Therefore, prior calibration is needed to determine the concentration, as often carried out for filtration processes to quantify the product mass (see Section 3.5).
Next, in situ UV-Vis investigations of nanoparticles were performed along one band after gel electrophoresis with a binary NP mixture, whereby the experiment was executed as described above. Figure 7A shows a photograph of a gel with the bands obtained from a mixture of the synthesized rod-shaped Au and the Fe3O4 NPs in a 0.15 wt.-% agarose gel after a six-minute runtime. The numbers indicate the measurement positions for the UV-Vis spectra, which can be seen in Figure 7B. Again, it is possible to assign the absorption spectra to the specific components of the sample. The spectra of positions 1 and 6 (golden lines) show no absorption peaks; therefore, no detectable particles are present at these positions after the experiment. Moreover, the spectra are very similar; therefore, it can be concluded that all particles have migrated past position 1, which demonstrates the unrestricted migration of the particles, with the particle diameter being smaller than the mesh size. The absorption spectra at positions 2 and 3 (blue lines) can be assigned to the rod-shaped Au NPs by their two LSPR peaks (see also Figure 6). Furthermore, the spectrum of position 3 shows higher absorption intensity compared to position 2, indicating that the particle concentration at 3 is higher and that most particles migrate rather quickly through the gel. Considering the spectra at positions 4 and 5 (black lines), they can be analogously assigned to the Fe3O4 NPs due to their one wide peak (see also Figure 6). Again, the largest proportion of this sample migrates faster, and therefore, the absorption signal at position 5 is larger. Whilst there might be a small content of Au nanorods present at positions 4 and 5, this cannot be detected in the measured UV-Vis spectra.
Figure 8 shows the in situ UV-Vis tracking of an electrophoretic separation experiment of an NP binary mixture in dependence of time at one position. This means that the NPs migrated through the measurement position (the dashed white line in Figure 8A), and the spectra were recorded at regular time intervals (Figure 8B). Again, a 0.15 wt.-% agarose gel and a mixture of the synthesized rod-shaped Au and the Fe3O4 NPs were used. The absorption spectrum after a migration time of 1.0 min (black line) can be correlated with the Fe3O4 NPs due to the one wide peak around 550 nm, although a minimal proportion of Au NPs might be suspected (considering the minimal shoulder at the wavelength of around 650 nm). This corresponds to our previous observation that the small Fe3O4 NPs migrate faster than the larger Au rods. In contrast, the absorption spectrum after a migration time of 5.0 min (golden line) can be assigned to the rod-shaped Au NPs due to the presence of the two characteristic LSPR peaks around 540 and 670 nm. In this case, the latter peak is clearly larger than the first one, as is expected for rod-shaped Au nanoparticle dispersions [41,42]. For the absorption spectra after a migration time of 2.0, 3.0, 3.5 and 4.0 min, a similar shape with two peaks can be seen. However, for these spectra, the first peak around 540 nm is higher than or very similar to the second one at around 670 nm, and thus, the spectra cannot be attributed to only the rod-shaped Au particles. Considering the photograph at t = 3 min in Figure 8A, the two bands (rod-shaped Au NPs and Fe3O4 NPs) seem to overlap at this time at the measurement position so that both bands contribute to the absorption spectra. Over time, obviously, the fastest particle band passed the measurement point first. The obtained spectrum could be assigned to one individual sample (Fe3O4), whilst the absorption spectra obtained for bands after a longer runtime changed because a mixture of both particle types (Fe3O4 and Au rods) was present. The sample obtained after a runtime of 5 min showed an absorption signal that can again be assigned to one individual sample (Au rods). The absorption spectra after 6 min are caused by very few Au rods, which is supported by the recorded image (Figure 8A) showing that the migrating sample apparently passes the measuring point (the dashed white line). Considering this, it can be concluded that the separation process is somehow diffuse in the interim timespan, where two samples overlap. To elucidate and quantify these interim spectra, a basic mathematical deconvolution was performed, assuming that the absorption signal after 1 min could be traced fully back to the Fe3O4 NPs and the one after 5 min to the Au rods (see Figure S5). These two absorption spectra were added in various ratios and multiplied with factor A to fit the measured absorption spectra of the mixtures at intermediate runtimes via the mathematical expression F ( λ ) = A • ( B • F e 3 O 4 ( λ ) + C • A u _ r o d s ( λ ) , with B and C being fitting factors between 0 and 1 corresponding to the ratio of the two particle types and A being a factor corresponding to the overall particle concentration at the measurement position. Figure 8C presents the calculated proportions of Fe3O4 and Au rods, showing a steady increase in the Au rod proportion from minute 1 to 5. Therefore, a proper separation time is crucial to prevent the overlapping of particle samples, and this can be determined in situ using the fiber-based UV-Vis system.

3.5. Determination of Particle Concentration Within the Gel by Calibrated UV-Vis Measurements

In order to gain quantitative information on the particle concentration with the performed UV-Vis measurements, a calibration curve of known solid contents of spherical Au NPs was generated in 0.15 wt.-% agarose gel samples (see Figure S6). Figure 9 shows a part of the electrophoretic experiment of Figure 6, which is used here to locally determine the solid content of the migrating sample within the gel, whilst the initially applied dispersion is diluted within the buffer and gel, and this dilution might even vary during its migration. During the electrophoresis, a maximum absorption intensity of 0.595 was measured by the fiber-based UV-Vis system at a wavelength of 525 nm for the Au NP system. Next, by using the calibration curve (inset in Figure 9B), it is possible to determine the solid content of this sample within the gel, which amounts to 0.21 wt.-%. It is important to understand that each NP system needs to be considered on its own because particle mixtures can lead to the addition of UV-Vis signals that would need to be deconvoluted (see above). A second important aspect is that with a varied gel concentration, a new calibration curve is needed because the agarose structure influences the UV-Vis absorption. Nevertheless, we showed that it is possible to determine the particle concentration of a specific sample during gel electrophoresis. Moreover, the fiber-based UV-Vis system can enable the in situ determination of particle concentrations at multiple positions over time, allowing for the determination of the weight distribution of the sample over the migration distance within the gel.

4. Conclusions

In this study, a self-designed fiber-based in situ UV-Vis measurement system with five measurement probes, which can record absorption spectra in succession, was introduced. The probes can be installed on various positions in perforated plates with screw threads, resulting in a variable measurement device. Before it was put in operation, a comparison with a commercial UV-Vis device was performed, whereas a similar signal-to-noise ratio was obtained in the absorption spectra of Au NPs with various particle sizes, proving the high signal quality obtained via the fiber-based device. Furthermore, the fiber-based system was used to record the absorption spectra of Au NPs in hydrogels with different concentrations, revealing that with higher agarose concentration, the scattering increased. Additional experiments with varied particle concentrations in specific gels showed that the particle concentration is a crucial parameter. After these preliminary tests, the fiber-based device was used for the detection of individual spherical and rod-shaped Au and Fe3O4 NP samples within the gel after separation by electrophoresis. Due to the characteristic peaks, each absorption spectrum could be assigned to one specific particle system. Finally, particle tracking was performed during gel electrophoresis, and supported by a basic mathematical deconvolution, we show that overlapping bands can also be analyzed and the composition roughly quantified. Further experiments focused on the determination of the particle concentration (solid content) within the gel matrix, which could be quantified by a prior calibration of the system. Overall, this study showed the use of a fiber-based UV-Vis system within a hydrogel matrix as an online measurement tool for gel electrophoresis. This system could be utilized to clearly identify various types of NPs by their different in absorption spectra. In practical separation applications for broadly distributed NP systems, it could be utilized under certain conditions to determine the average particle size, morphology or particle concentration of separated bands. Besides the identification of individual samples, an observation of the particle separation process is enabled during gel electrophoresis.
In conclusion, the presented fiber-based UV-Vis system with five probes allows in situ measurement within hydrogels and therefore (i) the differentiation of particle types based on unique characteristic absorption spectra, (ii) particle tracking during gel electrophoresis and (iii) the quantification of particle ratios and concentrations after prior calibration.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/powders4010003/s1, Figure S1: Particle size distribution of the commercial Au NPs capped with citric acid in deionized H2O measured by dynamic light scattering. Additionally the averaged volumetric diameters (x50,3) are displayed as well as the zeta potentials (ζ), which were measured by electrophoretic light scattering.; Figure S2: UV-Vis spectra of commercial Au NPs with a particle size of x50,3 = 91 nm in a 0.30 wt.-% gels. All spectra were measured using quartz cuvettes and the fiber-based UV-Vis system. The obtained data were normalized at a wavelength of 900 nm. Several outliers for sample F were removed for better overview, because there were values up to ±6 for wavelengths >700 nm.; Figure S3: Characterization of the synthesized Au NPs via SAXS (A) and Fe3O4 NPs via TEM (B). Figure S4: Photographs of different individual samples and binary mixtures of synthesized spherical and rod-shaped Au NPs as well as Fe3O4 NPs with an applied voltage of 100 V at various times. In the background, one can see printed lines on a paper with a 1 mm spacing. Figure S5: Basic mathematical deconvolution: Absorption spectra measured in-situ by the fiber-based UV-Vis system, normalized at a wavelength of 900 nm (see Figure 8) and smoothened by Savitzky-Golay filter (A). Measured absorption signals between 2 and 4 min were fitted with calculated curves F(λ) derived from F(λ) = A • (B • Fe2O3(λ) + C • Au_rods(λ)) using the measurements at minute 1 and 5 under the assumption that these curves represent the absorption signals of pure Fe3O4 (black curve) and Au rods NPs (gold curve), respectively (B–E). Figure S6: Absorption spectra of synthesized Au spheres with varied solid contents in agarose gels with 0.15 wt.-%. The particle diameter is 38 nm according to SAXS measurement in Figure S3 (see also Figure 5 for REM image) (A). The absorption values at a wavelength of 526 nm were used to derive the corresponding calibration curve (B).

Author Contributions

Conceptualization: M.B. and G.G.; methodology: M.B., G.G. and V.J.; formal analysis: M.B.; investigation: M.B., V.J. and M.G.; data curation: M.B.; writing—original draft preparation: M.B.; writing—review and editing: M.G. and G.G.; visualization: M.B.; supervision: G.G.; project administration: M.B. and G.G.; funding acquisition: G.G. All authors have read and agreed to the published version of the manuscript.

Funding

This work was supported by the German Research Foundation (DFG), grant GA 1492/11-2, project no. 382141490, within the priority program SPP 2045 MehrDimPart “Highly specific and multidimensional fractionation of fine particle systems with technical relevance”.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The data presented in this study are available upon reasonable request from the corresponding author.

Acknowledgments

The authors would like to thank Julian Cedric Porsiel for their valuable discussions, Bogdan Semenenko for the SAXS analysis and Sherif Okeil for the SEM measurements.

Conflicts of Interest

The authors declare no conflicts of interest.

Abbreviations

AgNO3Silver nitrate
BEDibenzyl ether
CTABCetyltrimethylammonium bromide
CTACCetyltrimethylammonium chloride
EDTAEthylenediaminetetraacetic acid
Fe(acac)3Iron(III)acetylacetonate
HAuCl4Gold(III)chloride
HDD1,2-Hexadecanediol
LSPRLocalized surface plasmon resonance
MUA11-Mercaptoundecanoic acid
NPNanoparticle
PSDParticle size distribution
NaBH4Sodium borohydride
NaOLSodium oleate
OAcOleic acid
OAmOleylamine
SEMScanning electron microscopy
STEMScanning transmission electron microscopy
TBETris/borate/EDTA
TEMTransmission electron microscopy
TrisTris(hydroxymethyl)aminomethane
UV-VisUltraviolet–visible
x50,3Mean volumetric diameter [nm]

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Figure 1. A schematic illustration (A) and a photograph (B) of the fiber-based UV-Vis measurement system. The multiplexer guides the irradiation into five optical fibers that lead it to five different positions at the gel (red arrows). When light passes the gel, it is partially absorbed, and the resulting signal is led back to the multiplexer and to the UV-Vis spectrometer for analysis (green arrows). Further detailed photographs of the electrophoretic chamber with the perforated plates (C) and the perforated plate with integrated screw threads (D).
Figure 1. A schematic illustration (A) and a photograph (B) of the fiber-based UV-Vis measurement system. The multiplexer guides the irradiation into five optical fibers that lead it to five different positions at the gel (red arrows). When light passes the gel, it is partially absorbed, and the resulting signal is led back to the multiplexer and to the UV-Vis spectrometer for analysis (green arrows). Further detailed photographs of the electrophoretic chamber with the perforated plates (C) and the perforated plate with integrated screw threads (D).
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Figure 2. Comparison between the commercial and the fiber-based UV-Vis measurement system with different Au NP suspensions in quartz cuvettes. The stated particle size values are determined as their mean volumetric diameters (x50,3) measured by dynamic light scattering in H2O.
Figure 2. Comparison between the commercial and the fiber-based UV-Vis measurement system with different Au NP suspensions in quartz cuvettes. The stated particle size values are determined as their mean volumetric diameters (x50,3) measured by dynamic light scattering in H2O.
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Figure 3. A photograph of a quartz cuvette filled with a mixture of commercial Au NPs and agarose gel as well as a pure gel sample for the blank measurement of the same gel concentration (A). A photograph of the cuvette between the perforated plates with connected optical fibers with the collimation optics (B). The absorption spectra of Au NPs in agarose gels with varied agarose concentrations using the fiber-based UV-Vis system (C).
Figure 3. A photograph of a quartz cuvette filled with a mixture of commercial Au NPs and agarose gel as well as a pure gel sample for the blank measurement of the same gel concentration (A). A photograph of the cuvette between the perforated plates with connected optical fibers with the collimation optics (B). The absorption spectra of Au NPs in agarose gels with varied agarose concentrations using the fiber-based UV-Vis system (C).
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Figure 4. The UV-Vis spectra of commercial Au NPs with a relatively small particle size (x50,3 = 36 nm) in 0.15 wt.-% gels (A) and a comparably large particle size (x50,3 = 91 nm) in 0.50 wt.-% gels (B). All spectra are measured using quartz cuvettes and the fiber-based UV-Vis system. The obtained data are normalized at a wavelength of 900 nm.
Figure 4. The UV-Vis spectra of commercial Au NPs with a relatively small particle size (x50,3 = 36 nm) in 0.15 wt.-% gels (A) and a comparably large particle size (x50,3 = 91 nm) in 0.50 wt.-% gels (B). All spectra are measured using quartz cuvettes and the fiber-based UV-Vis system. The obtained data are normalized at a wavelength of 900 nm.
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Figure 5. A characterization of the synthesized spherical and rod-shaped Au and irregular-shaped Fe3O4 NPs by the fiber-based UV-Vis spectroscopy in water (A) and microscopic images (B–D).
Figure 5. A characterization of the synthesized spherical and rod-shaped Au and irregular-shaped Fe3O4 NPs by the fiber-based UV-Vis spectroscopy in water (A) and microscopic images (B–D).
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Figure 6. Photographs of different individual samples and binary mixtures of synthesized spherical and rod-shaped Au NPs and Fe3O4 NPs in 0.15 wt.-% agarose gels. The upper photograph shows the samples within the gel pockets before the experiment at 0 min, whereas the lower photograph shows the samples after a 6 min runtime of the electrophoresis at an applied voltage of 100 V. In the background, one can see printed lines on paper with 1 mm spacing. The circles indicate the measurement positions of the optical fibers for the in situ UV-Vis measurement with the fiber-based device (A). Absorption spectra measured in situ by the fiber-based UV-Vis system and normalized at a wavelength of 900 nm (B).
Figure 6. Photographs of different individual samples and binary mixtures of synthesized spherical and rod-shaped Au NPs and Fe3O4 NPs in 0.15 wt.-% agarose gels. The upper photograph shows the samples within the gel pockets before the experiment at 0 min, whereas the lower photograph shows the samples after a 6 min runtime of the electrophoresis at an applied voltage of 100 V. In the background, one can see printed lines on paper with 1 mm spacing. The circles indicate the measurement positions of the optical fibers for the in situ UV-Vis measurement with the fiber-based device (A). Absorption spectra measured in situ by the fiber-based UV-Vis system and normalized at a wavelength of 900 nm (B).
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Figure 7. Photograph of a binary mixture of synthesized rod-shaped Au and Fe3O4 NPs after migration inside a 0.15 wt.-% agarose gel at an applied voltage of 100 V for 6 min. Numbers indicate the measurement positions of the optical fibers for the in situ UV-Vis measurement with the fiber-based device. In the background, printed lines on paper with 1 mm spacing can be seen (A). Absorption spectra measured in situ by the fiber-based UV-Vis system and normalized at a wavelength of 900 nm (B).
Figure 7. Photograph of a binary mixture of synthesized rod-shaped Au and Fe3O4 NPs after migration inside a 0.15 wt.-% agarose gel at an applied voltage of 100 V for 6 min. Numbers indicate the measurement positions of the optical fibers for the in situ UV-Vis measurement with the fiber-based device. In the background, printed lines on paper with 1 mm spacing can be seen (A). Absorption spectra measured in situ by the fiber-based UV-Vis system and normalized at a wavelength of 900 nm (B).
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Figure 8. Photographs of a binary mixture of synthesized rod-shaped Au and Fe3O4 NPs during migration within a 0.15 wt.-% agarose gel at an applied voltage of 100 V for various runtimes (A). The measurements of the absorption spectra took place at the same position of the gel indicated by the dashed white line. In the background, one can see printed lines on paper with 1 mm spacing. Absorption spectra measured in situ by the fiber-based UV-Vis system and normalized at a wavelength of 900 nm (B). Intensity proportions of Fe3O4 and Au rod samples dependent on time (C).
Figure 8. Photographs of a binary mixture of synthesized rod-shaped Au and Fe3O4 NPs during migration within a 0.15 wt.-% agarose gel at an applied voltage of 100 V for various runtimes (A). The measurements of the absorption spectra took place at the same position of the gel indicated by the dashed white line. In the background, one can see printed lines on paper with 1 mm spacing. Absorption spectra measured in situ by the fiber-based UV-Vis system and normalized at a wavelength of 900 nm (B). Intensity proportions of Fe3O4 and Au rod samples dependent on time (C).
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Figure 9. A photograph of synthesized spherical Au NPs in 0.15 wt.-% agarose gels at t = 0 and 6 min. The red circle in the lower image indicates the measurement position (not to scale) (A). The absorption spectrum of the Au NP sample smoothened by the Savitzky–Golay filter with an absorption signal of 0.595 at a wavelength of 525 nm. The inset shows the calibration curve, which is used to determine the corresponding solid content of 0.21 wt.-% (B).
Figure 9. A photograph of synthesized spherical Au NPs in 0.15 wt.-% agarose gels at t = 0 and 6 min. The red circle in the lower image indicates the measurement position (not to scale) (A). The absorption spectrum of the Au NP sample smoothened by the Savitzky–Golay filter with an absorption signal of 0.595 at a wavelength of 525 nm. The inset shows the calibration curve, which is used to determine the corresponding solid content of 0.21 wt.-% (B).
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Barasinski, M.; Jasper, V.; Görke, M.; Garnweitner, G. In Situ Tracking of Nanoparticles During Electrophoresis in Hydrogels Using a Fiber-Based UV-Vis System. Powders 2025, 4, 3. https://doi.org/10.3390/powders4010003

AMA Style

Barasinski M, Jasper V, Görke M, Garnweitner G. In Situ Tracking of Nanoparticles During Electrophoresis in Hydrogels Using a Fiber-Based UV-Vis System. Powders. 2025; 4(1):3. https://doi.org/10.3390/powders4010003

Chicago/Turabian Style

Barasinski, Matthäus, Valentin Jasper, Marion Görke, and Georg Garnweitner. 2025. "In Situ Tracking of Nanoparticles During Electrophoresis in Hydrogels Using a Fiber-Based UV-Vis System" Powders 4, no. 1: 3. https://doi.org/10.3390/powders4010003

APA Style

Barasinski, M., Jasper, V., Görke, M., & Garnweitner, G. (2025). In Situ Tracking of Nanoparticles During Electrophoresis in Hydrogels Using a Fiber-Based UV-Vis System. Powders, 4(1), 3. https://doi.org/10.3390/powders4010003

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