Surface Plasmon Resonance System Research Articles

Point-of-care personalized rapid diagnosis of allergies using peptide epitopes and SPR multiplexed detection

Allergy is the most common chronic disease in Europe. Up to 20 % of patients with allergies live with a severe debilitating form of their condition, struggling with the fear of a possible asthma attack, anaphylactic shock, or even death from an allergic reaction. Allergic reactions vary among different patients, due to the presence of different antibodies, which may also define the severity of the allergic reaction. Diagnostic tools are still suboptimal, as they require an oral food challenge for diagnostic, which is personnel, time, and cost consuming and bear the risk of severe allergic reactions for the patient. Also, there is a major gap in food allergy diagnosis to easily distinguish between sensitization and allergy. Therefore, a fast point-of-care multiplexed analytical platform for allergy assessment, supporting low-risk minimally invasive personalized diagnostics and treatment monitoring is advanced. The novel portable Surface Plasmon Resonance system integrates microfluidics and a low-fouling, multiple-ligand biosensor based on recently identified peptide epitopes for appraising allergen-specific serum antibodies. Aiming at personalized diagnosis of allergy, we successfully tested and validated the platform on hazelnuts and peanuts, two of the most threatening and frequent food allergens. Moreover, in contrast to the conventional SPR analysis and ELISA, by using an innovative analysis of the entire sensing area where the peptides are immobilized, it was even possible to discern different sera related to the same allergen.

Preliminary Study of Resonance Systems States in Surface Plasmon Resonance Systems

Abstract Resonance between light and collective electrons is known as Surface Plasmon Resonance or SPR. The development of SPR-based sensors can generate the use of a Kretschmann configuration prism. Otto configuration dielectric medium between prism and gold thin film. For the Kretschmann configuration, a gold thin film between the prism and dielectric medium. So, this research focuses on the use of the Kretschmann configuration in the SPR system because the setup of the half-cylindrical prism is less complicated than using the Otto configuration. In this research, we compare the reflectance of an uncoated half-cylindrical prism and a half-cylindrical prism coated with a gold thin film to see the resonance effect. The beam reflected by the back surface of the prism was detected using a photodiode sensor (OPT-101, Texas Instruments). The photodiode sensor is connected to Arduino Uno, and the voltage reading results are processed with Arduino Ide software. Adjusting the He-Ne laser light’s angle of incidence and the prism’s angle of impact 30° – 50°. The experimental results show that the TIR (total internal reflection) phenomenon occurs in half-cylindrical prisms without a gold coating. Meanwhile, in the half-cylindrical prism coated with gold, a resonance phenomenon can be seen, this case, at a particular angle of incidence, can be detected through a reduction in the beam’s intensity of the reflected light. The photodiode sensor detects a decrease in voltage value, which indicates an intensity decrease, which was originally 4.34 volts to 2.52 volts when the incident angle of the light beam was 45°.

Development of Unique Signature to Define the Extent of Adulteration in Virgin Coconut Oil (VCO) by Localized Surface Plasmon Resonance (LSPR) System

Development of Unique Signature to Define the Extent of Adulteration in Virgin Coconut Oil (VCO) by Localized Surface Plasmon Resonance (LSPR) System

The investigation of the interaction between fluorescent carbon dots labeling silk fibroin using a fluorescence microscope–surface plasmon resonance system

The use of fluorescent carbon dots (CDs) as highly precise biolabeling probes has been widespread in the fields of live cell imaging and protein labeling due to their small size and excellent photoluminescence ability to accurately target specific molecules with surface chemical properties. However, there was a lack of research on the interaction between CDs and labeled molecules. In this work, we presented a novel investigation strategy, the fluorescence microscopy-surface plasmon resonance (FM-SPR) system, which combined the use of fluorescence microscopy and wavelength modulation surface plasmon resonance to study the interaction between CDs and labeled molecules in real-time. Using this system, simultaneously recorded the SPR signals and the fluorescence images on the surface of the FM-SPR sensor chip. We observed the dynamic curve and fluorescence images of the interaction between green emissive nitrogen-doped carbon dots (N-CDs) and silk fibroin (SF) in real-time. The kinetic parameters, the quantitative analysis, and the investigation of the binding could be achieved. The results showed a strong linear relationship between the change in SPR signals and the concentration of N-CDs, with a linear coefficient of 0.99913. The linear detection range was 2.5 μg/mL–100 μg/mL, and the real lowest detection limit reached 0.5 μg/mL. Additionally, the green fluorescence points in the imaging region on the FM-SPR sensor chip increased with the concentration of N-CDs, which was consistent with the change in SPR signals. Using this system we also acquired the association rate and dissociation rate of N-CDs to SF which were 2.65 × 10−5/s and 1.52 × 10−5/s, respectively. This demonstrated the effectiveness of our method in quantitatively analyzing SF labeled with N-CDs.

High-performance portable grating-based surface plasmon resonance sensor using a tunable laser at normal incidence

Surface plasmon resonance (SPR) sensors are among the most sensitive sensors. In such devices, a grating is a compelling alternative to a prism for the excitation of a surface plasmon, especially in the development of sensors for point-of-care applications due to their compactness and cost-effectiveness. Here, we demonstrate the use of a tunable laser in a grating-based SPR system for further miniaturization and integration. The tunable laser working at normal incidence replaces spectral and moving components, while also simplifying the optical setup. Normal incidence is conventionally avoided due to the complexity of the control of degenerated SPR modes. We investigate, both computationally and experimentally, the splitting of the SPR modes at small nonzero incidences, which is lacking in previously reported studies. By optimizing the grating configuration, we were able to diminish the SPR mode splitting phenomenon when the excitation was feasible with the normal incidence configuration. The fabricated sensor showed a high sensitivity of 1101.6 nm/RIU. Notably, the figure of merit of the sensor, defined as the ratio between the sensitivity and bandwidth of the SPR dip, was 229.5. The experimental results were consistent with the simulation results. We also demonstrate its capability for detecting low concentrations of glucose and creatinine with the limit of detection of 14.2 and 19.1 mmol/L, respectively.

A dual-target SPR screening system for simultaneous ligand discovery of SARS-CoV-2 spike protein and its receptor ACE2 from Chinese herbs

Traditional Chinese Medicine (TCM) is a supremely valuable resource for the development of drug discovery. Few methods are capable of hunting for potential molecule ligands from TCM towards more than one single protein target. In this study, a novel dual-target surface plasmon resonance (SPR) biosensor was developed to perform targeted compound screening of two key proteins involved in the cellular invasion process of the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2): the spike (S) protein receptor binding domain (RBD) and the angiotensin-converting enzyme 2 (ACE2). The screening and identification of active compounds from six Chinese herbs were conducted taking into consideration the multi-component and multi-target nature of Traditional Chinese Medicine (TCM). Puerarin from Radix Puerariae Lobatae was discovered to exhibit specific binding affinity to both S protein RBD and ACE2. The results highlight the efficiency of the dual-target SPR system in drug screening and provide a novel approach for exploring the targeted mechanisms of active components from Chinese herbs for disease treatment.

Prediction of quality markers of Angong Niuhuang Pills based on LC-MS and pull-down with SPR chips

Elucidating the quality markers(Q-markers) of traditional Chinese medicines is essential for understanding the mechanisms of action and promoting the rational use of traditional Chinese medicines as well as for developing traditional Chinese medicine-derived drugs. Studies have shown that surface plasmon resonance(SPR) is promising in this field. This study proposed a method based on pull-down with SPR chips to predict the Q-markers of Angong Niuhuang pills(AGNHP). Firstly, 71 main chemical components of AGNHP were analyzed by UPLC-Q-TOF-MS, and then network pharmacology was employed to predict the potential targets of AGNHP against stroke. Secondly, the STAT3 protein chip was constructed, and the extract of AGNHP was recovered by pull-down of the SPR system for STAT3 ligand. The potential active ingredients were collected, enriched, and identified as coptisine, palmatine, epiberberine, berberine, worenine, demethyleneberberine, jatrorrhizine, tetrahydrocoptisine, baicalein, and baicalin methyl ester. Next, the affinity constants of the 10 active ingredients were determined as 44.7, 44, 58.1, 51.3, 39.7, 32.1, 49.2, 69.1, 19.7, and 24.9 μmol·L~(-1), respectively. The molecular docking results showed that the 10 compounds could compete for binding with STAT3. This is the first report that SPR combined with UPLC-Q-TOF-MS is reliable and feasible for determining the active ingredients of AGNHP at the molecular level from complex systems. STAT3 could be used as a potential target for the biological quality evaluation of AGNHP.

Green Synthesis of Magnetite Nanoparticles Using <i>Moringa oleifera</i> and their Electro-Optic Surface Plasmon Resonance Properties

The application of an external electric field to the surface plasmon resonance (SPR) system of green-synthesized magnetite (Fe3O4) nanoparticles (MNPs) is very promising for increasing the SPR detection signal. Electro-optic surface plasmon resonance (EOSPR) behavior of MNPs has been successfully carried out. The EOSPR system was investigated using the Kretschmann configuration with the prism/Au thin film/MNPs/air layer arrangement and applying an electric voltage of 0 V, 2 V, 4 V, and 6 V. In this study, we synthesized MNPs using the green synthesis approach from moringa oleifera extract. The benefits of green synthesis include being safe, affordable, clean, and ecologically friendly processes. X-ray diffraction results obtained crystal size of the MNPs is about 9.2 nm with inverse spinel face-centered cubic crystal structure. Fourier transforms infrared characterization showed the presence of Fe-O bonds at wave numbers 569 cm-1 and 629 cm-1, indicating that MNPs were successfully formed. The saturation magnetization of the samples is 55.3 emu/g. The SPR angle of the SPR system Prism/Au thin film/air without the addition of MNPs is 44.66°. After being deposited by MNPs and induced by a voltage of 0 V, 2 V, 4 V, and 6 V, the SPR angles changed to 44.87°, 44.90°, 44.95° and 45.12°. The addition of MNPs and an external electric field causes the SPR angle to increase. The results of this study can provide new insights into the development of optical devices that can be manipulated electrically and have the potential for future biosensor applications.

Design and numerical-phase analysis of an SPR system for direct detection of SARS-CoV-2 virus in pharyngeal swab solution

An ultrasensitive differential-phase SPR biosensor has been successfully established, capable of direct detection of SARS-CoV-2 virus.

Surface Plasmon Resonance Binding Study on the Interaction of Acetazolamide and Bovine Serum Albumin

Objective: Serum albumins are major plasma proteins in systemic blood circulation and act as transport proteins for endogenous and exogenous compounds such as drugs. In pharmaceutical applications, it is essential to characterize how drugs bind to serum albumin in the evaluation of drug candidates. Surface plasmon resonance (SPR) is fast, real-time, label-free optical based detection technique that offers the monitoring of molecular interactions, analyzing binding reactions and determining the affinity constants with real-time and high sensitivity. Acetazolamide (AZA) is used in the treatment of epilepsy and glaucoma.
 Methods: To determine the binding kinetics of AZA-Bovine serum albumin (BSA) interaction, (i) SPR gold sensor surface was functionalized, (ii) amine coupling procedure was applied to activate the surface group and BSA was immobilized on functionalized sensor surface, (iii) the concentration series of AZA (10, 25, 50, 75, 100, 150, 200 and 250 µM) was injected to SPR system and (iv) kinetic values were measured using the software of SPR system. 
 Results: 5 mM MUA was coated for surface functionalization. 250 µg/mL BSA as ligand, 30 µL/min flow rate, 1X PBS buffer (pH 7.4) and 10 mM acetate buffer (pH 5.2) as running and coupling buffers, respectively, were performed for SPR binding study. According to result, equilibrium constant (KD) of AZA-BSA was determined as 67.72 µM.
 Conclusion: In this study, we investigated the AZA-BSA binding interaction using SPR system based on Kretchmann configuration. The study designed with fast, label-free and real-time approach will provide valuable knowledge for pharmaceutical and clinical applications.

Customizable miniaturized SPR instrument

Prism-based surface Plasmon resonance (SPR) system, as one of the leading candidate concepts for scale application and commercial solution, has good stability, high-sensitivity and greater theoretical/technical maturity. Therefore, to take advantage of prism-based SPR system fully, and break up limitations of complicated and bulky traditional prism-based SPR system, optimal and compact design of optical system is an effective solution. Herein, a customizable miniaturized prism-based SPR system is developed by optical system optimization and integrated design, combining portable data acquisition and processing technology (FPGA-based multifunctional data processing). This proposed prism-based SPR system can achieve a miniaturized SPR system, thus, it also can meet the requirements of flexibility configuration and customizable performance to accommodate the various needs of different users and application scenes. Additionally, the customizable features can make it to achieve the best performance optimization and differentiation.

Absolute quantification of microRNAs based on mass transport limitation under a laminar flow SPR system

As an important biomarker for diagnostics and therapeutics of various diseases, the low-cost, quantitative detection method of microRNAs (miRNAs) has recently caught broad attention. However, their small size and low abundance still derive challenges to quantification detection. In this study, we developed an ultrasensitive and multiplexed surface plasmon resonance (SPR) biosensor for quantifying miRNAs without standard. We introduced the mass transport limitation (MTL) strategy for the absolute quantification of miRNAs. We first explore the mechanism of DNA capture and the condition for triggering MTL on the SPR biosensor. We demonstrated that probes of 22–25 nt in length with fewer influences of the secondary structure provide better triggering of MTL. For proof of concept studies, let-7a, miR-155 and miR-21 were selected as candidate targets. Based on the structure and kinetics analysis, we demonstrate the best capture probe efficiency, and this biosensor’s limit of detection (LOD) is 500 fM without any signal amplification. Furthermore, our biosensor achieves multiplex detection, which could detect three targets simultaneously. The quantitative results of miRNA indicated the great prospects of our biosensor in nucleic acid-related early diagnosis and biosensing.

Localized surface plasmon resonance properties dependence of green-synthesized Fe3O4/Ag composite nanoparticles on Ag concentration and an electric field for biosensor application

The use of green-synthesized Fe3O4/Ag composites nanoparticles (NPs) on the surface plasmon resonance (SPR) system induced by the electric field generates the effect of electrooptic-localized surface plasmon resonance (EO-LSPR). EO-LSPR is the promising method to increase dispersibility, generate plasmons, bind to biomolecular targets, modify the refractive index, and increase the SPR signal. Green synthesis of Fe3O4/Ag NPs has several advantages, including being environmentally friendly, cost-effective, and sustainable. This research successfully investigated the EO-LSPR properties of green-synthesized Fe3O4/Ag NPs with various Ag concentrations. Green synthesis of Fe3O4/Ag composites NPs was prepared utilizing Moringa oleifera by an aqueous solution method. The EO-LSPR phenomenon was investigated by applying various voltages in the Kretschmann configuration with a layer arrangement of a prism/Au thin film/NPs/air with a wavelength of 632.8 nm. Transmission electron microscope results show that the average size of Fe3O4/Ag particles is around 16.72 ± 7.30 nm. The scanning electron microscopy-energy dispersive x-ray results showed that Ag was distributed on the surface of Fe3O4. The addition of Ag concentration decreased the saturation magnetization while the coercivity field increased. The SPR angle of the prism/Au thin film/air layer structures is 44.66°. After depositing with Fe3O4/Ag with an Ag concentration of 60 millimolar, the LSPR angle shifted by 0.98°. Under an electric field, the LSPR angle shifted to 1.00°, 1.17°, and 1.22° of 2 volts, 4 volts, and 6 volts, respectively. The results show that applying the electric field induces the LSPR angle of Fe3O4/Ag NPs to shift to a larger angle. Applying an electric field causes a change in the material's refractive index. The greater the applied electric field, the more significant the LSPR angle shifts. The significant shifts in the LSPR angle due to the application of an electric field indicate that the EO-LSPR system using green-synthesized Fe3O4/Ag composites NPs could be a promising alternative to increase the performance of SPR biosensors in the future.

Ultrasensitive Screening of Endocrine-Disrupting Chemicals Using a Surface Plasmon Resonance Biosensor with Polarization-Compensated Laser Heterodyne Feedback.

Developing an ultrasensitive and reliable device for continuous monitoring of various endocrine-disrupting chemicals (EDCs) is in high demand, yet it remains a significant challenge. Traditional label-free surface plasmon resonance (SPR) sensing relies on the interaction of the surface plasmon wave and the sensing liquid via intensity modulation, endowed with simple structure and easy-to-miniaturization, however suffering from inferior sensitivity and stability. Here, we propose a novel optical structure in which the frequency-shifted light of different polarization returned to the laser cavity to stimulate laser heterodyne feedback interferometry (LHFI), hence amplifying the reflectivity change caused by the refractive index (RI) variations on the gold-coated SPR chip surface, and the s-polarized light could be further used as a reference to compensate for the noise of the LHFI-amplified SPR system, resulting in nearly three orders of magnitude enhancement of the RI sensing resolution (5.9 × 10-8 RIU) compared to the original SPR system (2.0 × 10-5 RIU). To further boost intense signal enhancement, custom-designed gold nanorods (AuNRs), which were optimized by the finite-difference time-domain (FDTD) simulation, were used to generate localized surface plasmon resonance (LSPR). By exploiting the estrogen receptor as the recognition material, estrogenic active chemicals were detected with a 17β-estradiol/L detection limit of 0.004 ng, which is nearly 180-fold lower than that of the system without introducing AuNRs. The developed SPR biosensor is expected to be capable of screening various EDCs with universality by using several nuclear receptors, such as the androgen receptor and thyroid receptor, and will substantially accelerate the assessment of global EDCs.

An Ultrasensitive and Universal Surface Plasmonic Biosensor for Detection of Micropollutants in Aquatic Environments.

Simple yet ultrasensitive and accurate quantification of a variety of analytical targets by virtue of a universal sensing device holds promise to revolutionize environmental monitoring, medical diagnostics, and food safety. Here, we propose a novel optical surface plasmon resonance (SPR) system in which the frequency-shifted light of different polarizations returned the laser cavity to stimulate laser heterodyne feedback interferometry (LHFI), hence amplifying the reflectivity change caused by the refractive index (RI) variations on the gold-coated SPR chip surface. In addition, the s-polarized light was further used as the reference to compensate the noise of the LHFI-amplified SPR system, resulting in nearly 3 orders of magnitude enhancement of RI resolution (5.9 × 10-8 RIU) over the original SPR system (2.0 × 10-5 RIU). By exploiting nucleic acids, antibodies, and receptors as recognition materials, a variety of micropollutants were detected with ultralow detection limits, ranging from a toxic metal ion (Hg2+, 70 ng/L) to a group of commonly occurring biotoxin (microcystins, 3.9 ng microcystin-LR/L) and a class of environmental endocrine disruptors (estrogens, 0.7 ng 17β-estradiol/L). This sensing platform exhibits several distinct characteristics, including dual improvement of sensitivity and stability and common-path optical construction without needing optical alignment, demonstrating a promising avenue toward environmental monitoring.

Two-dimensional material-enhanced surface plasmon resonance for antibiotic sensing

Two-dimensional (2D) materials attract attention from the academic community due to their excellent properties, and their wide application in sensing is expected to revolutionize environmental monitoring, medical diagnostics, and food safety. In this work, we systematically evaluate the effects of 2D materials on the Au chip surface plasmon resonance (SPR) sensor. The results reveal that 2D materials cannot improve the sensitivity of intensity-modulated SPR sensors. However, there exists an optimal real part of RI of 3.5–4.0 and optimal thickness when choosing nanomaterials for sensitivity enhancement of SPR sensors in angular modulation. In addition, the smaller the imaginary part of the nanomaterial RI, the higher the sensitivity of the proposed Au SPR sensor. The 2D material’s thickness needed for the highest sensitivity decreases with increasing real part and imaginary part of the RI. As a case study, we developed a 5 nm-thickness MoS2-enhanced SPR biosensor, which exhibited a low sulfonamides (SAs) detection limit of 0.05 μg/L based on a group-targeting indirect competitive immunoassay, nearly 12-fold lower than that of the bare Au SPR system. The proposed criteria help to shed light on the 2D material-Au surface interaction, which has greatly promoted the development of novel SPR biosensing with outstanding sensitivity.

Ultra-wide range and high-sensitivity temperature sensor based on a simple SPR system

In this work, a temperature sensor is proposed and experimentally demonstrated using a low cost surface plasmon resonance (SPR) system based on optical fiber. The sensor is formed by splicing a short length of no-core fiber (NCF) with two multimode fibers (MMFs). A silver film is deposited on the NCF to excite SPR effect. Overcome the defect of the complex fabrication process of the existing optical fiber sensor. The sensing characteristics at different deposition times have been studied. Experimental results show that for low and medium–high temperature, the sensitivity of the sensor is −3.01 and −1.425 nm/°C, respectively. It has a wide detection range from −40 to + 70 °C. The sensor also showed good reliability after repeated experiments. The temperature sensor covering low and medium–high temperature has important application value in agricultural production and soil temperature monitoring.

Generation of Tamm Plasmon Resonances for Light Confinement Applications in Narrowband Gradient-Index Filters Based on Nanoporous Anodic Alumina.

Gold-coated gradient-index filters based on nanoporous anodic alumina (Au-coated NAA-GIFs) were used as model platforms to elucidate how Tamm plasmons can be tailored by engineering the geometric features of the plasmonic and photonic components of these hybrid structures. NAA-GIFs with well-resolved, intense photonic stopbands at two positions of the visible spectrum were fabricated through sinusoidal pulse anodization. These model photonic crystals were used to assess how the quality of Tamm plasmon resonances can be enhanced by tuning the features of the dielectric mirror and the thickness of the porous gold coating layer. It is found that the highest value of the quality factor of Tamm resonance (Q Tamm = 237) is obtained for 11 nm of gold on a dielectric mirror with low porosity corresponding to the resonant spectral position of λTamm of ∼698 nm. Our analysis indicates that Tamm resonances in as-produced Au-coated NAA-GIFs are weak due to the constrained range of wavelengths (narrow bands) at which these photonic crystal structures reflect light. However, after broadening of their photonic stopband upon pore widening, Tamm resonances become better resolved, with higher intensity. It is also observed that the quality of light confinement worsens progressively with the thickness of the porous gold coating layer after a critical value. In contrast to conventional surface plasmon resonance systems, this hybrid Tamm porous system does not require complex coupling systems and provides a nanoporous structure that can be readily tailored for a range of photonic technologies such as sensing and lasing.

SPR and FBG sensors system combination for salinity monitoring: A feasibility test

This paper presents a novel optical fiber sensor system for salinity monitoring using data fusion in different optical fiber sensors approaches. In this case, a surface Plasmon resonance (SPR) sensor in a D-shaped optical fiber is developed in conjunction with a fiber Bragg grating (FBG). The SPR sensors are able to detect the liquid sample salinities through the refractive index variation of the different samples. In contrast, the FBG sensors estimate the salinity through the thermal dynamics of the samples. Results show a sensitivity of 164.0 nm/% and 12.5 pm/°C for the SPR and FBG sensors, respectively. Thus, the combination between the SPR and FBG sensors result in an improvement of the salinity monitoring of different samples, where a root mean squared error of 0.001% is obtained.

Non-filamentous sludge bulking induced by exopolysaccharide variation in structure and properties during aerobic granulation

The forming mechanism of non-filamentous sludge bulking during aerobic granulation were investigated basing on three feeding strategies (R1 direct aeration after fast feeding, R2 anaerobic stirring after fast feeding and R3 anaerobic plug-flow slow feeding). Results showed that strong selection stress (shortening settling time) led to a sharp flocs washout and the subsequent increase of food to microorganisms (F/M) in R1 and R3 reactors, but not found in R2 due to the different strategies of feeding modes. With the increase of F/M, zeta potential and hydrophobicity of sludge surfaces significantly decreased and thus leading to an enhanced repulsive force and energy barriers for sludge aggregation. Particularly, when F/M exceeded 1.2 kgCOD/(kgMLSS·d), non-filamentous sludge bulking was ultimately triggered in R1 and R3. Further analysis showed that massive extracellular exopolysaccharide (PS) accumulated on the surfaces of non-filamentous bulking sludge due to the increased abundance of the microorganisms related to PS secretion during sludge bulking. In addition, significantly increased intracellular second messenger (c-di-GMP), a key substance regulating PS biosynthesis, was confirmed via its concentration determination as well as microbial function prediction analysis, which played a critical role in sludge bulking. Combing with the systematic detection from surface plasmon resonance system, rheometer and size-exclusion chromatography-multiangle laser light detection-refractive index system, higher molecular weight, compact conformation, higher viscosity and higher hydrophilicity was determined in sludge bulking PS relative to PS extracted from non-filamentous bulking sludge. Clearly, the changes of PS (content, structures and properties) driven by c-di-GMP are the dominant mechanism for the formation of non-filamentous sludge bulking during aerobic granulation. This work could provide theoretical support for successful start-up and application of aerobic granular sludge technology.