Immobilization of Thai Population-specific Human Leukocyte Antigens on Magnetic Nanoparticles Integrated with Nuclear Magnetic Resonance Technology

Kidney disease, as a global health problem, can progress to kidney failure. Kidney transplantation is a treatment option for end-stage disease and an alternative to dialysis. Complement-dependent cytotoxicity tests and flow cytometry are methods used to test for compatibility between autoantibodies and donor recipient. Antibodies against donor human leukocyte antigens (HLAs), linked to hyperacute or severely acute rejection, frequently result in graft loss. Currently, fluorescent bead assay (Luminex ® ) technology is used in Thailand for HLA–antibody typing tests. However, each test requires specific location and time, expensive equipment, and high costs. The goal of such research is that, HLA–antibody typing tests can be performed wherever it is the most convenient for recipients and hospitals. A further advantage is that the surgeon can perform a final test for confirmation before the surgery. Numerous technologies are currently available for this purpose. The focus of this study was on the Fe 3 O 4 magnetic nanoparticles (MNPs) immobilized with HLAs frequently found in the Thai population. These were examined using nuclear magnetic resonance (NMR) to measure HLA antibody typing. MNPs have recently attracted considerable attention because of their strong magnetization and large surface areas. Immobilization reactions with antigens and the straightforward magnetic separation of MNPs are low-cost techniques. Hence, the commercialization of Fe 3 O 4 MNPs is favorable. In this study, MNPs were functionalized with 3-aminopropyl triethoxysilane (APTES) in toluene and ethanol, and the nanoparticles were streptavidin activated after APTES treatment. A specific biotinylated HLA for the Thai population was used and immobilized on streptavidin. The MNPs were effectively immobilized. The magnetic properties can be modified by adding other ions to Fe 3 O 4 . We discovered that the shape and size variations of Fe 3 O 4 MNPs may have an impact on the amount of functionalized and immobilized surface area.


Introduction
Kidney disease (KD) is a significant global health problem. Recently, the number of people with renal illnesses has increased worldwide. Dialysis is the ultimate treatment for KD and helps patients extend their lives. Chronic kidney disease (CKD) is the most prevalent factor leading to kidney (renal). The Thai Transplant Society requires patients to register for KT. Over time, the number of transplants has gradually increased. Kidneys are obtained from both live and deceased donors (LD and DD, respectively) [1][2][3][4]. Data indicate that the number of DD has increased at a greater rate than LD. Prior to surgery, transplant patients must take a blood test to determine if they match with the a LD or a DD. Kidney transplant recipients are divided into groups according to their age (if over 18) [2]. A blood test determines the tissue typing and ABO compatibility required for successful transplantation. The donor and recipient must share the same or compatible blood type.
Human leukocyte antigens (HLAs) and HLA antibodies are the essential components for the success of kidney transplantation. Before receiving a kidney transplant, a patient or recipient carrying donor-specific HLA antibodies (DSA) is at risk of experiencing HLA antibody-mediated rejection and kidney wasting [5][6][7]. Currently, a single antigen bead multiplex fluorescence-based solid-phase immunoassay (Luminex assay) with high sensitivity and specificity is used for the detection of antibodies recognized by HLA. However, this technique has the potential to produce false-positive and false negative results owing to variations in density, shape, and antigen on the microparticle beads. Dyed microparticle beads were used with the Luminex assay. A single-coated HLA bead is called a singleantigen bead (SAB). Owing to its excellent sensitivity and specificity, it can identify a particular serum in the recipient. The specificity of the antibody used in the Luminex assay is compared with the donor's HLA to determine whether the recipient carries DSA [8].
Nuclear magnetic resonance (NMR) technology is superior to existing techniques. Current methods for HLA detection are complement-dependent cytotoxicity (CDC), flow cytometry, and solid phase assays. These are more expensive and timeconsuming than biosensor technologies. Currently, numerous biosensor technologies are available, including NMR, surface plasmonic resonance (SPR) [9-14,], and ion-sensitive field-effect transistor (ISFET) technology [8]. NMR has the potential to reduce the cost of testing and processing time required for HLA antibody typing tests. Some individuals forego the HLA antibody typing procedure because of financial hardships. Using NMR technology, testing costs can be reduced, enabling patients to obtain their HLA antibody results.
The first prototype of a micro-NMR device and/or magnetic nanoparticles (MNPs) antigen bead for the HLA antibody typing test, based on its prevalence in the Thai population, was developed in this study. Both small and large hospitals and community health centers can use this technology. Additionally, it lowers costs to the patient over the course of the normal testing process that occurs every three months. It also decreases the cost of the equipment, time, and samples used. In the Thai population, we identified the prevalence of HLA, MHC class I and II. HLA-A2, having the highest prevalence of MHC class I in the Thai population, was selected for this study. The MNP, Fe 3 O 4 , is considered due to its ability to direct a magnetic field, low toxicity, strong biocompatibility, and high relaxivity [16-19] in a biosensing system based on micro-NMR. Owing to the instability of iron in a strongly acidic environment, MNPs may shorten the lifespan of a material. Strong magnetic interactions between the particles lead to particle agglomeration, which reduces the surface energy and particle dispersion in aqueous solutions and matrices. A large surface area leads to limitations. The magnetic activity decreases if proteins and/or enzymes are exposed to these interfaces. When conducting an HLA antibody typing test, MNP beads should be highly sensitive, specific, and require minimal amounts of sample. To overcome these limitations, various techniques have been used to modify surfaces by loading them with additional target chemicals or biological components during or after the production process. These techniques increase the biocompatibility, dispersibility, and biodegradability of Fe 3 O 4 MNPs on their surface. This study focused on directly functionalizing the surfaces of Fe 3 O 4 MNPs with 3-aminopropyl triethoxysilane (APTES) to create amine (NH 2 ) groups that can bind to other biomolecules. Streptavidin-biotin technology, which employs biologically mediated immobilization, is one of the most examined and utilized methods. In this study, streptavidin (SA) had already been functionalized with an amine group and immobilized with HLA-A2, was activated before immobilization. MNPs are affected by both physical and chemical factors. Understanding the characteristics, chemical compositions, and distribution of functional groups on the surfaces was a goal of this study.

Preparation of MNPs for micro-NMR detection
350 mg of Fe 3 O 4 MNPs was sonicated in a mixture of EtOH and toluene (1:2 volume fraction). The Fe 3 O 4 MNPs and APTES at ratios of 1:1, 1:2, and 1:3 were reacted at 25 °C and stirred for 4 h. The precipitate was dried in air at 25 °C after being thoroughly rinsed with ethanol several times until all residues had been removed, as indicated in Fig. 1.

SA coating of MNPs
The Fe 3 O 4 MNP treatment with APTES included additional SA in two different concentrations (20 and 40 μg/mL). At 4 °C, the mixture was incubated for 20 h. Reconstituted SA-Fe 3 O 4 MNPs were added to 0.2 mL of PBS (pH = 7.4) containing 1.0% BSA. Subsequently, samples were kept in a refrigerator for several days at 2-8 °C as shown in Fig. 1.

Immobilization of MNPs
Prior to use, the vortexed Fe 3 O 4 MNPs/APTES/SA was mixed for 20 s. A suitable amount of Fe 3 O 4 MNPs/APTES/SA was added to a tube along with one antigen test volume (10 μL) of biotin-labeled HLA antigen. The tube was incubated on ice for 30 min while being mixed. The tube volume was increased to 2 mL with wash buffer (NaN 3 and BSA), and the tube was then placed in a magnetic particle separator. After 3-5 min, the fraction containing the Fe 3 O 4 /APTES/SA MNPs:HLA antigen complexes was washed three times with buffer before use, as depicted in Fig. 1. Scientific. To perform the measurements, the sample was diluted with potassium bromide (KBr) and acquired over a range of 400-4 000 cm -1 .

Field emission scanning electron microscope (FE-SEM)
Data were obtained on a QUORUM Q150R ES using gold. The solid surface of the MNPs was determined using SEM. This SEM renders images in threedimensional (3D) and is used to examine the morphology and specificity of the sample surface. Crystal alignment was checked using a backscattering electron diffraction-reception system. EDS was used to assess changes in the extracted sample. The SEM X-ray detector enabled analysis of the elements of the sample.

Vibrating sample magnetometer (VSM)
An in-house developed vibrating sample magnetometer (VSM, calibrated with a 3-mm-diameter Ni sphere, model 730908 LakeShore) was used. The instrument analyzes the magnetic characteristics of low-magnetic moments and nanostructured materials. To display a hysteresis curve, the magnetization of the sample is monitored, and the resonance frequency of the vibrationdetection device is scanned. was selected because it showed the best. O−Si−O and Fe−Si occur as bands in the spectra at around 1 003 cm -1 in Fig. 2(c) and 1 107 cm -1 in Fig. 2(d), respectively, in symmetric stretching vibration.  Fig. 3(c). In Fig. 3(d), the surface of MNPs functionalized with SA at 40 μg exhibits results comparable to those of SA at 20 μg, but with less density than other surfaces. Biotinlabeled HLA antigens were immobilized at both SA concentrations.

Magnetic measurements
The magnetic characteristics of the

Conclusion
The HLA-A2 antigen, occurring in the highest frequency specific to the