All‐in‐one platform: Versatile, Easy, and User‐friendly System (VEUS) based on automated and expert‐independent antibody immobilization and immunoassay by utilizing customized movement of magnetic particles

The ELISA is the most worldwide method for immunoassay. However, the ELISA is losing ground due to low reproducibility of manual experimental processes in both R&D and IVD areas. An automated platform is a good solution, but there are still limitations owning to extremely high cost and requiring large space to set up especially for a small size laboratory. Here, we present a novel all‐in‐one platform called “VEUS” settable on the laboratory table that offers comprehensive automation of the entire multiplex immunoassay process by exploiting antibody conjugated magnetic particles, quality control and then immunoanalytical reaction, thereby enhancing detection sensitivity and high reproducibility. As a proof of concept, the system exhibits a sensitive LOD of 0.6 and 3.1 pg mL−1 within 1 h run, comparable precision that of molecular diagnostic systems based on PCR method, enabling rapid multiplex diagnosis of Influenza A, Influenza B, and COVID‐19 viruses with similar symptoms. Through automation by the all‐in‐one system, it can be used by novice users, something innovative for immunoassays, relying heavily on user experience. Furthermore, it can contribute to streamline entire immunoassay processes of diverse biomarkers with high reproducibility and convenience in laboratories.


INTRODUCTION
The ELISA [1][2][3] stands as a globally prominent immunoassay method within both IVD and R&D domains.[16] A rapid and effective global containment was achieved through collaborative efforts between research institutes and industry, [17,18] underscoring ELISA's stature as a world-class immunoassay.However, despite its versatility, ELISA, like any method, entails inherent trade-offs.
[21][22] Recognizing the need to minimize human errors associated with manual techniques, the automation of ELISA is generally deemed necessary.Typically, separate automated systems for antibody immobilization and immunoassay are employed in conventional ELISA setups, with their large footprint presenting challenges in terms of cost and convenience for small to medium-sized laboratories. [23,24]Despite efforts to enhance reproducibility through the design and development of magnetic bead assays, [25][26][27][28][29][30] these methods still incur high costs and demand significant space, posing obstacles to widespread automation.However, as experienced a global pandemic, government and researchers have become keenly aware of the need for automatic platforms that can quickly develop diagnostic kit to diagnose the causative viruses and develop treatments.3][34] Herein, we introduce "VEUS," an innovative and miniaturized allin-one platform designed to autonomously execute antibody immobilization (conjugation), quality control of the immobilized antibody on RSMPs, [35,36] and subsequent their immunoassays, thereby mitigating human errors and elevating experimental reproducibility.The VEUS system leverages chemically treated RSMPs for full automation steps of both antibody immobilization and immunoassays.Through automated control of RSMPs using strong magnetic forces on a large scale (hundreds of micrometers), we have automated all processes, including RSMPs' conjugation with capture antibodies, quality control to ensure successful immobilization, and multiplex assay analysis for type and quantity diagnosis.The system exhibits a sensitive LOD of 0.6 and 3.1 pg mL   work. [35,36]

2.2
Fully automated all-in-one system

Manual antibody immobilization process
The materials used in the entire manual immobilization process are the equal as those used in the automated process, but, only the process of moving the RSMPs is different.In order to move RSMPs smoothly in the manual process, a rotator (Multi Bio RS-24, BIOSAN, 25 rpm) is utilized.The RSMPs moved up to 8 mm by a single of movement action in the automated process, whereas, the RSMPs are reacted in a microcentrifuge tube (Eppendorf, 2 mL) in the manual process.Therefore, the RSMPs are manipulated to move as much as the length of the tube up to 30 mm at once.

Automated method for precise control and customized movement of magnetic particle and antibody immobilization
The RSMPs used in this all-in-one platform have a size on the order of hundreds of micrometers and their ferromagnetic core, making them significantly more responsive to magnetic forces compared to nanoparticles or microparticles of smaller sizes.Figure 1A illustrates the RSMPs utilized in the system.Due to their strong magnetic response, RSMPs move towards the permanent magnet and are attracted to it, while losing their magnetic influence and falling to the bottom when they move away from the permanent magnet.Consequently, by thorough control of the magnetic forces acting on RSMPs and the presence of suitable auxiliary equipment that can completely separate them from the permanent magnet, it is feasible to achieve a more effective separation of RSMPs.As depicted in Figure 1B, a plastic T-tip positioned between the permanent magnet and the RSMPs allows for their complete separation.The RSMPs are caught when the permanent magnet approaches the T-tip, and they are released when the magnetic force exerted on them decreases by separating the permanent magnet from the T-tip.Furthermore, we conducted experiments to verify whether RSMPs can be precisely controlled even during repetitive operations.
We repeatedly performed the action of catching and releasing 1000, 3000, 6000, and 10,000 RSMPs for 10,000 cycles, and all RSMPs were consistently caught and released.This result demonstrates that RSMPs can be thoroughly manipulated and moved by magnetic forces.
RSMPs controlled by the permanent magnet and T-tip can perform movements and operations not only within a single well but also across multiple wells.To tailor the entire process to the user's requirements, we designed a protocol for automation (Note S1, Figure S1-S5).The fully automated system consists of 8-well strips (cartridges) capable of containing various solutions.Within each well, the movement of RSMPs can be precisely controlled, allowing for easy execution of activation, washing, and blocking processes.As shown in Figure 1C, we utilized a continuous automated process involving multiple wells that fulfilled the user's requirements to carry out the antibody immobilization process for Influenza A, Influenza B, and COVID-19 with unified symptomatic patterns that can demonstrate the capabilities of the platform.By ensuring that the RSMPs in each well underwent rapid movements approximately 34 times per minute, we created an environment conducive to sufficient reactions (Note S2).The detailed steps are as follows: firstly, we immersed chemically treated RSMPs in MES solution for preparation.In the subsequent well, we performed a 30-min activation process using EDC and NHS.To wash away the remaining EDC and NHS, the RSMPs were moved in MES solution for 2 min in total, with each movement lasting 1 min.Next, we introduced capture antibodies along with HEPES buffer to attach them to the surface of RSMPs, and the RSMPs were moved during a 2-h reaction period to immobilize a sufficient amount of antibodies.After the completion of the reaction, the RSMPs were washed with HEPES buffer alone for 1 min in the next well to remove residual capture antibodies.Subsequently, in the following well, a 1-h movement process with blocking buffer was performed to prevent nonspecific binding.As a result, using the fully automated system designed according to the user's specifications, even novice users can easily perform the somewhat complex and cumbersome antibody immobilization process.

Method for automated quality check of antibody-conjugated RSMPs
While automating the antibody immobilization process significantly improves accessibility, manual quality control processes can undermine the achieved improvements.To address this, we developed an automated quality control process, enabling easy utilization by anyone.obtained through the automated system showed efficient conjugation, with equal or greater amounts compared to manually produced acRSMPs.Additionally, the acRSMPs exhibited good reproducibility based on the less than 7% of coefficient of variation value (Influenza A: 6.7%, Influenza B: 2.9%, and COVID-19: 4.7%).Overall, our automated system allows even beginners to easily perform the complex and laborious antibody immobilization process, yielding conjugation results comparable to those achieved by expert researchers.This approach enables anyone to dependably conduct the antibody immobilization process and verify quality, facilitating the production and preparation.

F I G U R E 2
Automated quality control process and evaluation of Influenza A, Influenza B, and COVID-19 acRSMPs.(A) Total process of automatic run of the quality control process by inserting the acRSMPs obtained through the manual or automated antibody immobilization process and the solutions together into the system is depicted as an illustration.(B) Quality comparison of acRSMPs generated through manual and automated antibody immobilization process.

Automated assay for infectious disease diagnosis using acRSMPs
acRSMPs, specifically designed for the identification of a particular antigen or antibody, can be stored in a blocking buffer for extended periods and directly employed in disease diagnosis.Figure 3A illustrates the automated assay process, which is outlined as follows.
Initially, Influenza A, Influenza B, and COVID-19 acRSMPs are prepared by adding them to the first well.Subsequently, the lysed antigen is added to the subsequent wells, allowed to react with acRSMPs for 30 min, and unbound residues are eliminated by washing with a clean buffer for 1 min.In the subsequent wells, the detection antibody is added and allowed to react with the antigen-conjugated acRSMPs for 5 min.Following this, SA-PE attached to the detection antibody is introduced in the next wells and allowed to react for 5 min.The wells are then washed twice for 1 min each, totaling 2 min, to remove any remaining residues.This assay process enables fluorescence imaging of Influenza A, Influenza B, and COVID-19 acRSMPs, with the intensity being confirmed through image processing.Finally, the absolute amount of antigen is calculated within 40 s per well by comparing the observed intensity with a previously analyzed standard curve.The entire assay process takes less than an hour to generate the final result.
A fully automated all-in-one system was developed to facilitate conjugation, quality control, and the assay process, making it accessible to any user.The accuracy of this system was evaluated, as depicted in Figure 3B.The standard curves obtained from the manual and automated production of acRSMPs for Influenza A, Influenza B, and COVID-19 were compared.The manually produced acRSMPs demonstrated not only high fluorescent intensity but also a coefficient of determination (R 2 ) of 0.984 or higher, indicating a significant linear relationship.The standard curves obtained from the automatic method outperformed those from the manual method, exhibiting a higher coefficient of determination (R 2 ≥ 0.995) and increased fluorescent intensity.This suggests that acRSMPs produced through the automated antibody immobilization process can influence the assay process, thereby enabling even untrained personnel to obtain accurate results.concentrations, signifying better system performance.Figure 3C demonstrates the LOD results achievable with the automated all-inone system.The system exhibited an LOD value of 3.1 pg mL −1 for Influenza A and B, and 0.6 pg mL −1 for COVID-19.Even enzyme-based diagnostic techniques, known for their relatively high accuracy, typically possess LOD values ranging from several to several tens of pg mL −1 .Therefore, the relatively precise detection limit demonstrated by the automated system is noteworthy.

Clinical trial results from a fully automated all-in-one system
While RSMPs manufactured with a unified single length can detect  this, RSMP must be produced with the correct length.Figure S6A illustrates the precise fabrication of RSMPs cut into 200, 300, and 400 µm sizes using an automated fabrication system.The measured lengths of the RSMPs were found to be 199.5908µm, 300.8131 µm, and 400.4747 µm, respectively, with narrow standard deviations of 13.1523 µm, 13.6987 µm, and 13.2982 µm, resulting in CV values below 7% (6.59%, 4.55%, and 3.32%).Even if the length of the RSMP is manufactured accurately, it may be recognized as an incorrect length due to overlapping of the RSMP, long line arrangement of the RSMP due to the bar shape, and so on.When this situation occurs, RSMP classification may be incorrect, resulting in inaccurate results.
To solve this, we introduced artificial intelligence-based analysis technology, and as a result, our system can perfectly distinguish RSMPs manufactured with a length difference of 100 µm.Figure S6B shows that artificial intelligence-based RSMP classification algorithm distinguished each RSMP well and there is no overlap among 36 RSMPs.
The multiple immunodiagnosis results with 95% confidence interval (CI) derived from the all-in-one system within 1 h exhibited high sensitivity, specificity, and accuracy for each virus.Specifically, for all-in-one system offers a user-friendly solution, providing results with rapid PCR-like sensitivity, owing to its exceptionally low LOD.
Consequently, the VEUS system has achieved less than 1 h immunoassay through automation with accuracy comparable to molecular diagnostics.
These results indicate that individuals without professional training can easily perform the automated antibody immobilization process and injection of the solution using the provided protocol.Furthermore, the quality control process ensures the reliable manufacturing of acRSMPs.The utilization of acRSMPs with variable lengths in a multiplex assay, combined with a sensitive limit of detection, allows for accurate and rapid diagnosis.This achievement is attributed to the fully automated protocol from the coupling to multiplex analysis processes, making it a versatile all-in-one platform as summarized in Table S2.

3.5
All-in-one platform utilization in R&D and IVD areas Figure 4 illustrates the applicability of a fully automated all-in-one system within the realms of R&D and IVD fields.This integrated platform streamlines the entire process, encompassing antibody immobilization, quality checks, and multiplex assays, thereby mitigating human error and concurrently enhancing reproducibility.
The singular device facilitates the seamless execution of these processes, occupying a compact volume.This compactness offers novel prospects for exhaustive investigations involving multiple biomarkers, even within constrained laboratory spaces.This innovation holds promise for advancing multibiomarker research within the R&D domain.Furthermore, the system demonstrates sensitivity, specificity, and accuracy of equivalent with established molecular diagnostic equipment, elevating its status as a sophisticated analytical tool deployable with urgency in the analysis field.Beyond its analytical prowess, the system contributes to a nuanced understanding of diverse diseases, ultimately fostering clinical applications.Consequently, its active integration into the IVD area is anticipated, thereby offering valuable insights and applications.

DISCUSSION AND CONCLUSION
The implementation of an all-in-one system offers improved accessibility by automating the entire process, from antibody immobilization to the multiplex assay.In this study, experiments and analyses were conducted using acRSMPs of up to three different lengths, specifically focusing on respiratory infectious diseases.However, the versatility of this system extends beyond respiratory diseases, as it can be tailored to meet diverse needs in various fields.By modifying the biomarkers, solutions, and protocols employed in the antibody immobilization and assay process, multiple diagnostics can be performed, such as F I G U R E 4 Summary illustration of an all-in-one system designed for the automation of antibody immobilization, quality control, and multiplex assay processes.The miniaturized platform diminishes human errors and enhances overall reproducibility.The integrated platform holds significant promise for applications in R&D and IVD fields.The comprehensive system not only explores the application of various biomarkers in R&D but also presents a fast and accurate diagnosis in IVD.
traumatic brain injury or brain disease diagnosis utilizing GFAP, UCH-L1, pTau181, pTau231, and NFL biomarkers in the field of neurology.
Moreover, at the laboratory level, this automated system can serve as a valuable tool for conducting related research involving a wide range of biomarkers.
The implementation of automated systems not only enhances accessibility to rapid and accurate diagnostics with higher reproducibilities far from human errors, but also opens up opportunities for comprehensive investigations using multiple biomarkers in laboratories.This advancement will undoubtedly contribute to multiplexed biomarker studies in both R&D and IVD areas and improve our understanding of various diseases, ultimately benefiting both research and clinical applications.The all-in-one system can be easily set up in small and used for multiplexed immunoassay studies in laboratories because it is all automated in the processes of both antibody immobilization to increase accuracy and reproducibility far from human errors and the size of its analyzer (53 × 49 × 47 cm) is settable on the laboratory table.
In this study, we present a fully automated all-in-one platform called "VEUS" that utilizes RSMPs with sizes in the hundreds of micrometers range.This system automates all stages of the diagnostic process, including the antibody immobilization process, quality control process, and multiplex assay analysis.The automated antibody immobilization process, tailored to meet specific requirements, achieved comparable or superior efficiency to that of experts, as validated by the automated quality control process.Furthermore, acRSMPs produced by the automated system demonstrated a remarkably low LOD, enabling precise detection comparable to rapid PCR equipment.
The detection results exhibited excellent performance, both in single detection and multisample diagnosis of Influenza A, Influenza B, and COVID-19 with similar symptoms, using acRSMPs of different lengths.Sensitivity ranging from 92% to 96%, specificity between 95% and 100%, and accuracy of 95% to 97% were achieved.Notably, this automated all-in-one system facilitated rapid diagnosis within an hour, eliminating the requirement for specialized personnel.By employing this system, we can effectively contribute to the containment and treatment of emerging diseases to detect the pathogens rapidly and accurately through multiplexed immunoassays, even at the small-scale level, mitigating the associated social and economic challenges.

Figure
Figure 2A illustrates the automated quality control process for assessing the quality of Influenza A, Influenza B, and COVID-19 acRSMPs obtained through the automated antibody immobilization process.We prepared different solutions in advance and utilized permanent magnets and T-tips to facilitate the movement of acRSMPs within or between wells.The quality control process involved the following steps: (1) preparing acRSMPs in a blocking buffer, (2) diluting biotinylated secondary antibody (2 µg mL −1 ) in blocking buffer and reacting for 60 min to allow binding to the capture antibody, (3) washing the well to remove residual secondary antibody (two washes for 1 min each), (4) diluting fluorescent material (SA-PE, 2 µg mL −1 ) in blocking buffer and mixing for 30 min to react with the secondary antibody, (5) rinsing the well to remove excess fluorescent substance (two washes for 1 min each), (6) observing the reacted acRSMPs using brightfield and fluorescent imaging, and (7) analyzing target identification and intensity values within 40 s per well.This fully automated process enhances accessibility in both the antibody immobilization and quality control steps.Moreover, we assessed whether the quality of acRSMPs produced by beginners using the automated system was comparable to acRSMPs manually produced by trained researchers.If beginners can achieve equivalent quality results, it would enable the production of highquality acRSMPs continuously without the need for dedicated research personnel.Figure 2B presents the results of the automated quality control process comparing manually and automatically produced Influenza A, Influenza B, and COVID-19 acRSMPs.The acRSMPs Among the performance indicators used to evaluate the system, the LOD value indicates the precision of antigen determination.A lower LOD indicates the ability to detect lower antigen F I G U R E 3 Automated assay process by acRSMPs and performance of the automated system.(A) Illustration of automated assay process in the system.(B) Influenza A, Influenza B, and COVID-19 assay comparison between automatically and manually produced acRSMPs in accordance with fluorescent intensities.(C) Automated system's LOD for the respiratory diseases, Influenza A (3.1 pg mL −1 ), Influenza B (3.1 pg mL −1 ), and COVID-19 (0.6 pg mL −1 ).