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Abstract
Doxorubicin (DOX) is an important drug for cancer treatment, but its clinical application is limited due to its toxicity and side effects. Therefore, detecting the concentration of DOX during treatment is crucial for enhancing efficacy and reducing side effects. In this study, the authors developed a biophotonic fiber sensor based on localized surface plasmon resonance (LSPR) with the multimode fiber (MMF)-four core fiber (FCF)-seven core fiber (SCF)-MMF-based direct-taper and anti-taper structures for the specific detection of DOX. Compared to other detection methods, it has the advantages of high sensitivity, low cost, and strong anti-interference ability. In this experiment, multi-walled carbon nanotubes (MWCNTs), cerium-oxide nanorods (CeO2-NRs), and gold nanoparticles (AuNPs) were immobilized on the probe surface to enhance the sensor's biocompatibility. MWCNTs and CeO2-NRs provided more binding sites for the fixation of AuNPs. By immobilizing AuNPs on the surface, the LSPR was stimulated by the evanescent field to detect DOX. The sensor surface was functionalized with DOX aptamers for specific detection, enhancing its specificity. The experiments demonstrated that within a linear detection range of 0-10 µM, the sensitivity of the sensor is 0.77 nm/µM, and the limit of detection (LoD) is 0.42 µM. Additionally, the probe's repeatability, reproducibility, stability, and selectivity were evaluated, indicating that the probe has high potential for detecting DOX during cancer treatment.
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1. Introduction
Doxorubicin (DOX) is an antibiotic drug belonging to the anthracycline class. It has a broad spectrum of antitumor activity and is used in the treatment of acute leukemias (lymphocytic and myelogenous), malignant lymphomas, breast cancer, and other cancers [1–4]. Doxorubicin is a broad-spectrum antitumor drug that produces a wide range of biochemical effects in the body, exerting strong cytotoxic effects. Its primary mechanism of action involves embedding within DNA to inhibit nucleic acid synthesis, thereby affecting the functionality of normal cells [5,6]. This can result in irreversible damage to organs such as the heart and brain, posing a life-threatening risk in severe cases [7,8]. Given the hazardous side effects of DOX and individual variations among patients [9]. When the concentration of DOX exceeds 0.8 µM, it induces apoptosis and autophagy, and when the concentration exceeds 2.67 µM, it inhibits DNA synthesis. It is crucial to develop sensitive and reliable methods for detecting DOX in order to monitor its concentration in the body. This approach aims to achieve the desired therapeutic effects while avoiding overdosing and minimizing toxicity.
In recent years, various methods for the detection of DOX have emerged through research, including liquid chromatography, high-performance liquid chromatography [10], fluorescence spectroscopy [11], and electrochemical methods [12]. However, some of these methods require expensive instruments, complex sample processing, specialized expertise, and considerable time, and they’re less reproducible. Biophotonic fiber sensors, as an alternative approach, have garnered widespread attention due to their strong specificity, high sensitivity, rapid response, and robust interference resistance [13,14]. It utilizes light as an information carrier and optical fibers as transmission media, offering excellent biocompatibility and finding extensive applications in the field of biosensing [15,16].
Due to the unique optical properties and biocompatibility of AuNPs, they are widely used in the development of localized surface plasmon resonance (LSPR)-based biophotonic fiber sensors [17–21]. Compared to various other metals, AuNPs exhibit the highest sensitivity to changes in the surrounding refractive index (RI). Virk et al. [22] developed a D-shaped optical fiber probe with AuNPs immobilized in the sensing region for the detection of GaHIgG. By varying the antigen concentration, they achieved a limit of detection (LoD) of 0.6 µg/mL, enhancing the detection sensitivity of the sensor. Wang et al. [23] developed a structure of single mode fiber (SMF)-multimode fiber (MMF)-single mode fiber (SMF), i.e., an SMS-based optical fiber sensor for the quantitative detection of cardiac troponin I (cTnI) solution. They utilized AuNPs and cerium dioxide nanoparticles (CeO2-NPs) fixed on the fiber structure, achieving a LOD of 108.15 ng/mL, thereby improving the sensing capacity and sensitivity of the optical fiber. Zhang et al. [16] developed a humanoid-shaped fiber optic waveflex biosensor functionalized with graphene oxide/multi-walled carbon nanotubes (MWCNTs) for histamine detection. The sensor exhibited a sensitivity of 5.5 nm/mM and a LoD of 59.45 µM, contributing to advancements in the field of biosensing. MWCNTs have the advantages of high strength and toughness, good electrical conductivity, high surface area, good chemical stability and thermal stability. It can be applied to many complex environments, and has a wide range of application prospects in materials science, electronics and other fields [24]. Additionally, MWCNTs possess adsorption and photocatalytic capabilities [25,26], which play an essential role in immobilizing AuNPs and enhancing the sensitivity of the sensor. Cerium dioxide nanorods (CeO2-NRs) possess the advantages of fine particle size, large surface area, high biochemical activity, and good optical properties [27]. Moreover, CeO2-NPs have been prepared into heterojunction structures with other materials and applied in the biosensors field, showing good cascading ability with other low-dimensional materials [28]. CeO2 is often employed to stabilize and disperse the metal particles on the load, generating a strong metal-carrier interaction and enhancing the cascade of CeO2-NRs and AuNPs. A compact integrated composite structure can be formed on the surface of the fiber probe by combing CeO2-NRs and MWCNTs. While increasing the contact area with AuNPs, the LSPR effect of AuNPs is also enhanced, and the sensitivity of the sensor is improved.
Currently, specialty optical fiber fusion, such as multicore optical fiber, has been widely used in the field of biosensing [29]. Due to its unique structural characteristics, multi-core fibers exhibit excellent performance, simplicity, and high sensitivity to various external parameters [30]. Weihs et al. in “All-fiber three-path Mach–Zehnder interferometer” that the multi-core diameter can improve the sensitivity to phase changes, have shown that four-core fiber can improve the accuracy of measurement data retrieval [31]. The seven-core optical fibers offer greater waveguide flexibility and Zhu et al. illustrated in “Highly Sensitive Balloon-Like Temperature Fiber Sensor Based on a Seven-Core Fiber” that they utilize a composite-core mode structure to excite more higher-order modes, which can improve the sensitivity of the fiber sensor [32]. In this work, the four-core fiber and seven-core fiber were used comprehensively, and the developed fiber probe had the advantages of both kinds of fiber, so that the performance of the developed fiber sensor can be improved. In addition, the sensing area of the fusion structure of two kinds of fiber will cause the mode mismatch of the fiber probe at the fusion, resulting in more light leakage, and further improve the sensitivity of the fiber sensor.
Thus, this study proposed an LSPR sensor probe with a multimode fiber (MMF)-four core fiber (FCF)-seven core fiber (SCF)-MMF-based direct-taper and anti-taper structure for the detection of DOX [33]. A 40 µm taper was pulled on FCF and SCF to leak more light from the fiber structure [34], enhancing the interaction between light and the detected object. FCF and SCF were fusion-spliced using a counter-taper structure to ensure that each fiber core had sufficient light field to excite higher-order cladding modes. MWCNTs [35], CeO2-NRs [36], and AuNPs were sequentially fixed on the sensing region to enhance the signal. MWCNTs, with their excellent planar conductivity and high surface area, along with the strong interaction between CeO2 and metal particles, provided additional binding sites for AuNPs and ligands, enhancing the LSPR effect and improving the sensitivity of the sensor. The developed fiber probe detected DOX in the linear range of 0 to 10 µM. The fiber optic sensor demonstrated practical capabilities for DOX detection through assessments of its other sensing performance, such as, repeatability, reproducibility, stability, and selectivity.
2. Experiments
2.1 Materials
The sensor probe utilizes multi-mode fiber (MMF, 62.5/125 µm), four-core fiber (FCF, 8.0/125 µm), and seven-core fiber (SCF, 6.1/125 µm). MMF fibers were purchased from EB-Link Technologies Co, Shenzhen. FCF and SCF fibers were purchased from FIBERCORE, UK. A solution of AuNPs was synthesized using trisodium citrate, tetra-chloroauric acid (HAuCl4), and deionized water (DI). To clean the optical fibers and plate nanomaterials, a combination of acetone, hydrogen peroxide solution (H2O2, 30%), concentrated sulfuric acid (H2SO4, 98%), (3-mercaptopropyl) trimethoxysilane (MPTMS), and ethanol were used. For functionalization steps, streptavidin (SA), tris(2-carboxyethyl) phosphine (TECP), tween 20, an aptamer sequence for DOX (5'-SH-(CH2)6-ACC-ATC-TGT-GTA-AGG-GGT-AAG-GGG-TGG-T), and phosphate-buffered saline (1×PBS, pH = 7.4) were utilized. MWCNTs were purchased from Xianfeng Nano, Nanjing, and their solution was prepared using a dimethylformamide (DMF) solution. CeO2-NRs was obtained from Xianfeng Nano, Nanjing, and its solution was prepared using an N-methyl-2-pyrrolidone (NMP) solvent. To validate the probe's selectivity, various biomolecules such as doxorubicin (DOX), glucose (GLU), tetracycline (TET), dactinomycin (DTIC), histidine (His), and ciprofloxacin (CIP) were used. These reagents were purchased from local vendors in China.
2.2 Instruments and measurements
The fabrication of the sensing probe involved the use of a special optical fiber fusion splicer machine (FSM-100, Fujikura, Japan) and a combiner manufacturing system (CMS, USA). Quantitative cutting of fiber lengths was performed using a specialized cleaver from Fujikura, Japan. The absorption spectra of AuNPs were measured using a UV-visible spectrophotometer (Hitachi-3310, Japan) to estimate the particle diameter. Scanning electron microscopy (SEM, Carl Zeiss Microscopy, Germany) was used to scan samples with a high-energy electron beam, observing the nanomaterial coating on the surface of the sensing probe and studying the overall structure of the probe. High-resolution transmission electron microscopy (HR-TEM, Talos L120C, Thermo-Fisher Scientific, USA) was used to confirm the microscopic distribution of nanomaterials in the synthesized solution. The experimental setup included a broadband-emitting tungsten-halogen light source (HL-2000, Ocean Optics, USA) and a spectrometer (USB2000+, Ocean Optics, USA) to collect experimental data and investigate the light transmission characteristics of the sensing probe.
2.3 Fabrication of fiber probe
Multicore optical fibers possess characteristics such as low splicing loss and significant sensitivity to changes in the RI of the surrounding medium. The SCF used in the experiment was seven-core fiber. The seven-core fiber consisted of seven identical germanium-doped silicon cores, with one core in the middle surrounded by six hexagonally arranged cores. The existence of multiple cores improved the freedom of the waveguide and made the probe based on SCF fiber possess good sensing performance [37]. The FCF has strong anti-interference ability, good chemical stability and high non-conductive safety performance, and combines FCF and SCF with reverse cone technology to better play their advantages [20]. In this study, a combination of multicore and multimode optical fibers was employed to enhance the sensitivity of the probe. The diameter of the tapered region significantly influences the characteristics of the tapered fiber structure. By tapering, more light is leaked, enhancing the depth of evanescent waves (EWs) and better exciting the LSPR effect. Considering practical factors, a too-small diameter of the tapered region may lead to breakage. After repeated testing, a diameter of 40 µm was determined, ensuring minimal breakage while maximizing the excitation of LSPR. The fusion of multicore optical fibers utilized an anti-tapering method, allowing more-light to enter the fiber core, better preserving light intensity, and ensuring sufficient intensity for exciting the LSPR effect in the second tapered region. The structural scan of the fiber and its repeatability test results are shown in Fig. 1 (a) and (b). Figures 1(c) and (d) show the cross-sectional view of FCF and SCF.
Fig. 1. (a) Diameter scan of fabricated fiber structures, (b) repeatability analysis of structures, (c) cross-sectional view of FCF and (d) SCF.
The above structure was simulated using RSoft simulation software, and as shown in Fig. 2, it can be observed that a significant amount of light leaks when the light passes through the tapered region, indicating that this structure can generate strong evanescent waves (EWs). The light is recoupled back into the core through the anti-tapered region, ensuring that there is still strong energy when reaching the second tapered region, which also results in a substantial amount of light leakage. Therefore, the feasibility of the structure was validated through RSoft software.
The coating layers of MMF, FCF, and SCF are removed from the fiber. The FCF and SCF are fused separately with the MMF using an FSM, forming MMF-FCF and MMF-SCF structures. The fabrication process of the optical fiber is shown in Fig. 3(a). The FCF and SCF sections are retained at a length of 1.5 cm using the specialized cleaver. Subsequently, the MMF-FCF and SCF-MMF are fused together using the FSM, creating a convex structure. The fusion of the convex structure is set to automatic mode with a discharge time of 3000 ms, a discharge power of 357 units, an overlap distance of 160 µm, and a subsequent discharge power of 512 units. During the electrode discharge process, the optical fiber softens at high temperatures and gradually expands under the thrust of the motor, forming a convex structure with a diameter increase of approximately 160 µm. The tapered structures on FCF and SCF are produced by CMS, and the thermal repeatability of the heat-stable plasma in CMS is more than 10 times that of existing arc technologies. This unique heating method generates a highly adjustable plasma field, completely surrounding the optical fiber to achieve a highly uniform heat distribution. It can also achieve a highly symmetrical, gradually tapered structure with ultra-low loss. The entire process of CMS tapering is controlled by a calibrated computer program. First, the CMS platform is calibrated, and then the preprocessed optical fiber is placed on the workbench and clamped with a fixture.
Fig. 3. (a) Fiber optic structure fabrication process, (b) internal structure of CMS instrument, (c) internal structure of FSM instrument.
The waist diameter is set to 40 µm [38], and the program is started, ensuring a stable production process and allowing for the precise fabrication of multiple probe structures. The structures of CMS and FSM are shown in Fig. 3(b) and (c). Optical signal propagation in the proposed fiber sensor structure is shown in Fig. 5.
Fig. 4. MWCNTs/CeO2-NRs/AuNPs-immobilization and DOX aptamer functionalization over sensor structure.
2.4 Immobilization of MWCNTs/CeO2-NRs/AuNPs over the fiber probe
The performance of sensors is significantly influenced by the immobilization of nanoparticles and the functionalization of enzymes. Thorough cleaning of the optical fiber surface is required before nanomaterials are fixed to the probe surface. In the first step, the optical fiber sensing region was washed in acetone for 20 minutes to eliminate residual particles on its surface. Subsequently, it was rinsed with deionized water and dried with nitrogen gas. A mixture of 98% sulfuric acid and 30% hydrogen peroxide (piranha solution, volume ratio of 7:3) was used to immerse the probe for 30 minutes. This step aims to completely remove almost all organic matter from the substrate and create hydroxyl groups on the surface, facilitating better fixation of other nanomaterials. The probe was then rinsed with deionized water and dried at 60°C for 30 minutes. Following the drying process, MWCNTs are fixed to the optical fiber surface through quenching phenomena. The optical fiber was immersed in a freshly prepared MWCNTs solution for 10 minutes, and then it was dried for 20 minutes at 80°C. This process was repeated three times to establish a uniform coating on the probe surface. A similar method was employed for the coating of CeO2-NRs on the probe surface. The probe was subsequently immersed in a 1% MPTMS (3-mercaptopropyltrimethoxysilane) ethanol solution for 12 hours. Then, the immersed fiber was rinsed with ethanol and dried with nitrogen gas. MPTMS, acting as a coupling agent, is capable of modifying a uniform monolayer of thiol on the probe surface. The methoxy group (CH3O-) of MPTMS undergoes alcoholysis with silicon-based hydroxyl groups, leading to the formation of a self-assembled monolayer of terminated thiol on the tapered fiber surface. Then, synthesized AuNPs solution was used to completely immerse the probe for 48 hours. This process was followed by washing with an ethanol solution and drying with nitrogen gas to remove the unbounded AuNPs. A chemical bond is formed as one end of the coupling agent of MPTMS reacts with AuNPs. A stable Au-S covalent bond is easily formed as gold interacts with thiol groups (-SH), promoting the strong adhesion of AuNPs. After the fixation of nanomaterials, the functionalization of enzymes becomes the focus.
The DOX aptamer solution was prepared, and then 62.4 µL of 100 µM TCEP solution was used at room temperature for 1 hour to reduce 50-disulfide bonds. The adapted concentration is subsequently reduced to 300 nM using PBS. The probe is then transferred to a 0.05 mg/mL of SA (streptavidin) solution for 3 hours, exploring the interaction between different surface densities of biotin and SA in a biotin-SA model. Different surface densities of biotin biomimetic matrices are prepared through the reaction of biotin-modified functionalized polyethylene glycol with self-assembled amine functional groups on the surface, achieving the specific adsorption of SA on the biotin-modified solid surface [39]. The probe was washed with PBS + 0.05% tween20 (PBST) and then placed in the DOX aptamer solution (300 nM) for 1 hour. Following a wash with PBST, the fiber probe was immersed in a 1% BSA (bovine serum albumin) solution for 1 hour to block the non-specific binding sites. At this point, the functionalization steps are complete. Figure 4 depicts the nanomaterial immobilization and aptamer functionalization processes over the optical fiber structure.
2.5 Sensing principle
Light propagation mainly occurs in the core of an optical fiber. As shown in Fig. 5, when the angle of incidence is greater than the critical angle between the optical fiber and the external medium, a portion of the light energy will penetrate the surface of the optical fiber and enter the external medium, forming a phenomenon of electromagnetic waves known as EWs. The generated EWs can excite the collective oscillation of valence electrons on the metal surface. LSPR occurs when light interacts with nanoparticles made of noble metals. A probe with a matching ligand is then immobilized, and when the target analyte binds to the ligand on the nanoparticles, it changes the RI around them. This shift in RI causes a change in the resonance peak position in the LSPR spectrum, resulting in an observable wavelength shift in the absorption peak. This allows for biochemical detection through spectral analysis. The electrons in AuNPs, under the influence of EWs, collectively undergo repetitive oscillations, giving rise to surface plasmon waves (SPW). Due to the size limitation of AuNPs, SPW is confined to the nanoscale, resulting in the phenomenon known as LSPR. Light within the optical fiber propagates forward through total internal reflection. EWs appear perpendicular to the interface direction and exhibit rapid decay characteristics, with the propagation range defined as the penetration depth $({{\textrm{d}_\textrm{p}}} )$:
here, λ represents the excitation wavelength, $\theta $ is the angle of incidence between the core-cladding interface, and nco and ncl are the refractive indices of the core and cladding, respectively. After tapering the optical fiber, the core and cladding can be approximated as a new core, with the outer part serving as the cladding. This change increases the penetration depth of EWs on the fiber surface, leading to LSPR induced by AuNPs in the presence of EWs. When the aptamers functionalized on the nanomaterials specifically binds to the analyte to be measured, the RI near the probe changes, resulting in a peak resonance wavelength shift. When the effective RI of an external medium change, the resonance wavelength shift can be expressed as [40]: where Δλ is the change in wavelength, m and Δnm represent the sensitivity of AuNPs to electromagnetic fields and the change of RI, respectively, and dp and d are the penetration depth of the evanescent wave (EW) and the sample thickness, respectivel. The essence of the developed fiber optic biosensor is to detect RI response of different concentrations of solution to the sensors. To be specific, the resonance conditions of LSPR are highly dependent on the dielectric characteristics of the surrounding medium, which enabling AuNPs to be employed as sensors for detecting changes in the RI. Due to the presence of adapter, the RI of the surrounding medium varies with the concentration of the measured substance. When DOX solution is added, the DOX solution binds to the aptamer in an intercalation, non-covalent manner. The RI of the solution will change. According to Formula (2), the change of RI will lead to the shift of peak resonance wavelength [40]. With the increase of DOX concentration, the absorption intensity increases, and the peak resonance wavelength is redshifted.2.6 Experimental setup
The light signal was generated by a tungsten halogen light source (HL-2000), and the spectrometer detected a wavelength shift within the range of 200–1000 nm. When light passed through the sensing area of fiber probes, the LSPR would be excited due to the presence of AuNPs. Therefore, when the ligand on the nanomaterials reacted with DOX, the RI around AuNPs changed, and a wavelength shift occurred due to LSPR. To minimize errors, the sample concentrations were tested from low to high concentrations. Before each new concentration test, the sensor probe was cleaned with a PBS solution. The chemical reaction mechanism of DOX and effective optical test equipment for detecting different concentrations of DOX solutions are shown in Fig. 6.
Fig. 6. DOX reaction process and experimental setup for detection of DOX solution using a developed sensor.
3. Results and discussions
3.1 Characterization of nanomaterials and fiber structure
MWCNTs, CeO2-NRs, and AuNPs were immobilized on the sensing region of fiber probes to enhance the sensing performance. The AuNPs were employed to generate LSPR, and the sensing performance of fiber probes was greatly influenced by the size of the AuNPs. Firstly, the absorption spectrum of AuNPs was determined by a UV-visible spectrophotometer, as shown in Fig. 7(a). The absorption peak was located at 520 nm, indicating the diameter of AuNPs was approximately 10 nm. The previous work has demonstrated that the LSPR fiber biosensors with AuNPs with a diameter of 10 nm can obtain a good sensing result [15]. In addition, HR-TEM was used to characterize the NPs solution, as shown in Fig. 7(b). The synthesized AuNPs have a spherical shape with a uniform diameter. MWCNTs and CeO2-NRs immobilized on the fiber probes were used to increase the sites of aptamer. The HR-TEM image of MWCNTs shown in Fig. 7(c) displays a compact and fine linear tube-like structure. The HR-TEM picture of CeO2-NRs in Fig. 7(d) confirms the dense rod-like structure of the CeO2 nanomaterial. The combination of MWCNTs and CeO2-NRs is conducive to the binding of aptamers and the addition of sites. In addition, CeO2-NRs can generate a strong metal-carrier interaction and improve the cascade of CeO2-NRs and AuNPs.
The microscope was utilized to characterize the fiber structure based on a combination of MMF, SCF, and FCF by employing direct tapering and anti-tapering techniques. It can be observed from Fig. 8(a) that the diameter of the anti-tapered structure is 160 µm and the tapered region has a diameter of 40 µm. The SEM characterization of nanomaterials on the fiber probe surface are shown in Fig. 8(b)-(d). The information in Fig. 8(b) indicates that MWCNTs are immobilized on the fiber probes uniformly. Figure 8(c) provides SEM information on CeO2-NRs, indicating that CeO2-NRs were successfully deposited on top of MWCNTs. The SEM information of MWCNTs/CeO2-NRs/AuNPs on the sensing area is presented in Fig. 8(d), demonstrating that three nanomaterials have been successfully fixed to the surface of the fiber probe. The result of EDS shown in Fig. 8(e) confirms the presence of C, Si, O, Au, and Ce. This can also confirm that MWCNTs/CeO2-NRs/AuNPs have been immobilized on the probe surface successfully.
Fig. 8. (a) SEM image of fiber structure, (b) MWCNTs, (c) MWCNTs and CeO2-NRs (d) MWCNTs, CeO2-NRs and AuNPs, (e) EDS image of nanomaterials-immobilized fiber probe.
3.2 Test of analytes
The developed probes were used to measure DOX solutions at different concentrations. To reduce experimental errors, three different probes were used for detection in our work. The testing process was carried out in order from low concentration to high concentration. It needed to be thoroughly washed with PBS solution and dried after one concentration of DOX solution was finished. Due to the presence of aptamers on the fiber sensor probes, the RI of the external environment will be changed when the fiber probes are placed in DOX solutions of different concentrations. The RI change can be reflected in the LSPR spectrum. Therefore, the change in DOX concentration can be determined by recording the drift of the spectral wavelength. By employing three probes individually and normalizing the transmission intensity of the LSPR spectrum, experimental results can be obtained. As shown in Fig. 9(a), the peak wavelength shifts towards a longer wavelength with the increase in DOX concentration. The linear fitting curve of the peak wavelength is shown in Fig. 9(b) over the range of 0-10 µM. The fitting coefficient of the fiber probe is 0.9933 (R2 = 0.9933), indicating a good linear relationship. The variation of peak wavelength with DOX solution concentration can be expressed as: λ=0.770C + 590.22, where λ is the peak wavelength and C is the concentration of DOX solution.
Fig. 9. The measurement results of the DOX sensor probe. (a) transmitted normalized intensity spectrum, (b) linearity of the developed fiber probe.
In the application of biosensors, stability is an important and unavoidable characteristic. The stability of the fiber probe is measured by using a sensor to measure the PBS solution ten times. The peak wavelength was tested, and its standard deviation (SD) was calculated, as shown in Fig. 10. The peak wavelength of the probe is almost the same, and a SD of 0.11 indicates that the stability of the probe is reliable. The LoD is a fundamental aspect of evaluating the sensor, representing the lowest detectable concentration. The formula is expressed as:
After calculation, the LoD of the developed DOX fiber biosensor is 0.42 µM.3.3 Repeatability and reproducibility test
Repeatability and reproducibility are important factors in determining whether the sensor can be applied in practice. Reproducibility refers to whether probes with the same structure have the same sensing parameters. Three different probes were used to detect a 10 µM concentration of DOX solution. The sensing data were recorded after the transmission spectrum was stabilized. As shown in Fig. 11(a), three different probes have the same peak wavelength under the same concentration of DOX solution, indicating good reproducibility of the developed fiber probes.
Fig. 11. Sensing performance analysis, (a) reproducibility, and (b) reusability test of fiber probes.
Using one probe for two repeated experiments on 1 µM and 10 µM concentrations of DOX solution, First, we tested the DOX with a 1 µM concentration and recorded the data after the LSPR spectrum stabilized. After recording, rinsed the probe surface thoroughly with PBS, dried the probe, and then measured the 1 µM DOX again. Similarly, 10 µM DOX solution was measured twice in a similar manner, and results were plotted as shown in Fig. 11(b). The spectra remain basically unchanged in both tests, indicating good repeatability for this probe.
3.4 pH and selective test
Due to the presence of DOX aptamer on the sensor probe surface, the fiber sensor has the ability to perform specific and selective recognition. The purpose of the selective test is to confirm whether the probe can specifically recognize DOX in the presence of other complex biological interfering substances. For this purpose, TET, GLU, DITC, CIP, and HIS solutions with concentrations of 0 µM and 10 µM were prepared for the selectivity test. Then, a negative control experiment was performed to test the samples detected in the selectivity test without two-dimensional materials. The results in Fig. 12 show that show that the peak wavelength corresponding to the detected sample shifts under the concentration changes of 0 µM and 10 µM. The observed wavelength shift was smaller than the results of fiber probes with 2D materials (shown in Fig. 13(a)), providing additional evidence that AuNPs and 2D material can enhance the sensitivity of the fiber sensors. As shown in Fig. 13(a), the wavelength shift of the fiber probes with aptamers is small when the sample is another substance to be measured. The results indicate that the fiber probes can specifically identify DOX. To further validate the specific binding of the adapter, the fiber probes without the adapter were prepared. The results of probes without aptamers show that the wavelength shift is almost same when testing any sample. By comparing the experimental results, it was found that only when the probe functionalized aptamer had the maximum wavelength drift, indicating that the aptamer had the ability to recognize DOX specifically.
Fig. 12. (a) Peak wavelength and (b) selectivity test of developed fiber probes without 2D materials.
The fiber probes are also designed to detect DOX levels in human fluids (blood/serum), so it is necessary to simulate the pH environment in humans. The pH was adjusted to 3, 6, 10, and 13 by mixing sodium hydroxide/acetic acid with DI water, respectively. The 0 µM and 10 µM DOX solutions were tested at different pH values, and the spectral redshift data were recorded. The results of Fig. 13(b) show that the largest redshift occurs at pH 7.4, which means that the developed probe can effectively test DOX levels in the human body. The DOX aptamers have better binding affinity and stability when detecting DOX with fiber probes at a pH of 7.4, resulting in stronger and more specific binding reactions. However, the aptamer may denature or change its structure at a lower or higher pH, which can lead to reduced binding efficiency and reduced detection sensitivity. The pH value of 7.4 is the same as the pH of most human fluids, thus confirming the suitability of the developed optical fiber sensors for practical clinical applications.
3.5 Sensor performance evaluation
The biophotonic fiber sensors were developed based on MMF-FCF-SCF-MMF in our work. The LSPR optical fiber biosensors possesses the advantages of high sensitivity, low cost, and compact structure. Table 1 summarizes the sensitivity, LoD, and detection range of different types of DOX sensors. Through comparison, the developed LSPR fiber sensors in this work exhibits higher sensitivity and a broader detection range, indicating its excellent capabilities in detecting DOX.
Table 1. Performance comparison of the proposed DOX biophotonic fiber sensor with existing ones
4. Conclusion
This study has developed a direct-tapered and anti-tapered DOX sensor probe based on the MMF-FCF-SCF-MMF structure. The surface of the probe was modified by fixing MWCNTs and CeO2-NRs to enhance the contact area and sensitivity of the probe. Additionally, fixing AuNPs on the probe amplified the LSPR effect. The presence of the DOX aptamer confers specific selectivity to the developed probe, mitigating the influence of other biomolecules. Using the developed probe, DOX solutions were tested within the range of 0–10 µM. Experimental results demonstrate a good linear relationship between DOX concentration and wavelength redshift within this range, with a fitting degree of 0.9933. The sensitivity and LoD were found to be 0.77 nm/µM and 0.42 µM, respectively. The experiments illustrate that the developed probe can effectively detect DOX within a broad range. Moreover, the sensor's stability, repeatability, selectivity, and pH were evaluated, meeting the required criteria, thereby proving the significant application potential of the developed sensor probe for DOX detection.
Funding
Natural Science Foundation of Shandong Province (ZR2022QF137); Double-Hundred Talent Plan of Shandong Province, China; Special Construction Project Fund for Shandong Province Taishan Mountain Scholars; Liaocheng University (318052205, 318052341); Science and Technology Support Plan for Youth Innovation of Colleges and Universities of Shandong Province of China (2022KJ107).
Disclosures
The authors declare no conflicts of interest.
Data availability
Data underlying the results presented in this paper are not publicly available at this time but may be obtained from the authors upon reasonable request.
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