A comprehensive review of Interferometric Reflectance Imaging Sensor as a sensitive detection platform and its application areas Monireh Bakhshpour-Yucel a,*, Nese Lortlar Unlu b,c, Elif Seymour d, Adil Denizli e a Department of Chemistry, Faculty of Arts and Science, Bursa Uludag University, 16059, Bursa, Turkiye b Faculty of Medicine, Histology and Embryology, Atlas University, 34408, İstanbul, Turkiye c Photonics Center, Department of Biomedical Engineering, Boston University, Boston, MA, 02215, USA d Boston University Business Incubation Center, Boston, MA, 02215, USA e Department of Chemistry, Faculty of Science, Hacettepe University, 06800, Ankara, Turkiye A R T I C L E I N F O Keywords: Interferometric Reflectance Imaging Sensor Detecting molecular interactions Biomedical research Biomedical diagnostics A B S T R A C T The Interferometric Reflectance Imaging Sensor (IRIS) technology represents a significant advancement in bio sensing, providing a label-free, selective, sensitive, and high-throughput platform for detecting molecular in teractions. This review explores the underlying principles, instrumentation, and diverse applications of IRIS, with a focus on its efficacy for real-time monitoring of DNA-protein and protein-protein interactions, as well as virus detection. IRIS can measure DNA hybridization kinetics and identify pathogens without labeling, highlighting its versatility and reliability in biomedical research and diagnostics. IRIS achieves enhanced sensitivity and speci ficity by leveraging spectral reflectivity as a transduction mechanism and employing a 3D polymeric surface chemistry for bioreceptor immobilization. The review underscores IRIS’s potential to revolutionize clinical di agnostics, biomolecular screening, and the study of biomolecular binding affinities, establishing it as a powerful tool for future research and medical applications. 1. Introduction Diagnostic studies of diseases and their causes have traditionally been a key area for medical and technological advancements. Histori cally, diagnoses were largely based on patient history and physical ex aminations. However, the 20th century brought about a significant transformation with the introduction of highly sensitive sensing tech nologies and rapid measurement biotechnologies. These advancements have enabled the detection of even minute quantities of substances in bodily fluids, such as blood, urine, and cerebrospinal fluid, which has greatly improved diagnostic capabilities (Gubala et al., 2012). In vitro tests, ranging from traditional bacterial cultures to modern DNA-based chips and protein microarrays, have become critical in clinical settings. Despite these advancements, recent outbreaks of novel infectious diseases and epidemics have displayed the limitations of the current diagnostic tools. Rapid detection plays a crucial role in di agnostics. For example, cancer can spread through the bloodstream and progressively affect human physiology. As the immune system weakens, cancer gains ground. Advanced techniques have been developed to rapidly identify cancer cells, offering significant advancements in early detection and treatment. This emphasizes the urgent need for ongoing innovation and refinement of diagnostic technologies to address emerging health threats (Kazemi et al., 2022; Sierra et al., 2020) effectively. Numerous sensing methods have been used to detect biological markers, which are pivotal for providing healthy physiological condi tions and diagnosing diseases that cause significant threats to human health, such as cardiovascular diseases, cancer, or infectious diseases. Diagnostics can involve a wide range of biomarkers detection, such as viruses, bacteria, yeasts, and toxins, and can be used to determine various biological processes and health conditions. This comprehensive approach to biomarker detection plays a significant role in under standing disease mechanisms, simplifying early diagnosis, predicting disease progression, and guiding effective treatment strategies (Stoeva et al., 2006; Li et al., 2011; Seymour et al., 2023a). In recent years, important progress has been made in the area of biomarker detection, particularly in detecting biomarkers at low con centrations. These biomarkers are crucial for the diagnosis and pro gression of diseases and have the potential to revolutionize clinical research. The future of diagnostic technology will focus on achieving * Corresponding author. Bursa Uludag University, Department of Chemistry, Faculty of Arts and Science, Bursa, Turkiye. E-mail address: myucel@uludag.edu.tr (M. Bakhshpour-Yucel). Contents lists available at ScienceDirect Biosensors and Bioelectronics: X journal homepage: www.journals.elsevier.com/biosensors-and-bioelectronics-x https://doi.org/10.1016/j.biosx.2025.100574 Received 31 July 2024; Received in revised form 26 December 2024; Accepted 2 January 2025 Biosensors and Bioelectronics: X 22 (2025) 100574 Available online 7 January 2025 2590-1370/© 2025 Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license ( http://creativecommons.org/licenses/by-nc-nd/4.0/ ). mailto:myucel@uludag.edu.tr www.sciencedirect.com/science/journal/25901370 https://www.journals.elsevier.com/biosensors-and-bioelectronics-x https://doi.org/10.1016/j.biosx.2025.100574 https://doi.org/10.1016/j.biosx.2025.100574 http://crossmark.crossref.org/dialog/?doi=10.1016/j.biosx.2025.100574&domain=pdf http://creativecommons.org/licenses/by-nc-nd/4.0/ highly selective, sensitive, specific, multiplexed, and quantitative detection of biomarkers, signifying a significant evolution in diagnostics for the next generation (Özkumur et al., 2008, 2009, 2010; Daaboul et al., 2010). Various amplification and transduction mechanisms, including me chanical devices, optical techniques, and electrochemical sensors, have been widely utilized for serum biomarkers detection to achieve these goals. These techniques play an important role in sensitive and selective biomolecule detection in biological samples, supporting advances in clinical diagnostics and biomedical research (Lopez et al., 2011; Yurt et al., 2012; Daaboul et al., 2012). This review aims to release a detailed overview of an optical sensor system based on interferometric sensing without labeling and its appli cations. This approach directly monitors molecular binding interactions without labeling (Ünlü et al., 2017). This innovation aims to simplify and expand the application of high-throughput and real-time detection methods (Ünlü, 2018). The IRIS is a highly efficient detection system that offers high- throughput capabilities, sensitivity, and real-time and rapid moni toring of molecular binding events on solid surfaces without labeling (Chiodi et al., 2020a; Needham et al., 2019; Ünlü et al., 2018). The effectiveness of IRIS has been demonstrated in the real-time detection of binding interactions, showcasing its high level of sensitivity and reli ability. Furthermore, IRIS allows for effortless, label-free, and rapid measurements of DNA hybridization kinetics and detection of viruses (Yurdakul and Ünlü, 2020; Özkumur et al., 2019). IRIS is a significant breakthrough in sensing technology and offers versatile tools for research and clinical applications (Marn et al., 2021; Lortlar Ünlü et al., 2024; Bakhshpour-Yucel et al., 2024). 2. IRIS The IRIS signal depends on the interference of reflected fields from a layered substrate, which commonly comprises a silicon dioxide (SiO2) layer deposited on the silicon surface by thermal processes. The IRIS technology offers two distinct sensitive detection methodologies. High-throughput Label-Free Biomass Measurement: This method allows for rapid and precise biomass measurement without labeling. It detects changes in the interference patterns caused by molecular binding events on the solid surface. In the biosensing methodology employed by IRIS, detection is based on spectral reflectivity as the transduction mechanism. The upper layer’s thickness increases as biomass accumu lates on the layered substrate’s surface (Vedula et al., 2010). This growth changes the optical path difference (OPD) between the top surface and the Si-SiO2 interface. Consequently, there is a perceptible shift in spec tral reflectivity, as illustrated in Fig. 1. In the IRIS optical system, the reflection coefficient of an oxide layer can be calculated as: R = |r|2 = r2 1 + r2 2 + 2r1r2 cos 2 ϕ 1 + r2 1 + r2 2 + 2r1r2 cos 2 ϕ where r1 = n2 − n1 n2 + n1 , r1 = n3 − n2 n3 + n2 and ϕ = 2πd λ n2 cos θ where r1 and r2 are the Fresnel reflection coefficients for the air-SiO2 and Si-SiO2 interfaces, respectively, and, n1, n2, and n3 show the refractive indices of air, SiO2, and Si, respectively. The wavelength of incident light is shown by λ, and θ represents the angle of incidence. Given the use of a low-numerical aperture (NA) objective (~0.1, i.e., θ < 6◦), the polarization sensitivity of the incident light is minimal. This setup ensures that the optical path remains insensitive to the polariza tion state of the incoming light, which is crucial for maintaining the consistency and accuracy of spectral reflectivity measurements across different wavelengths. During the capture process, reflectivity curves are generated across diverse wavelengths using a charge-coupled device (CCD) sensor. These curves are then analyzed by fitting them to the spectral reflectance signatures derived from Fresnel equations. These equations define how light interacts with materials and interfaces within the sensor system. Fig. 1. (a) Shows the operational principle of IRIS, depicting the interference of light reflected from the reference plane (Si-SiO2 interface) and the top surface. (b) Changes in wavelength-dependent reflectance characteristics following the introduction of biomass. (c) Detection of the target using the IRIS system. IRIS consists of an illumination source, transduction platform, and optical detector. Sensor chip, integrated into a microfluidic flow cell, utilizes biorecognition elements, such as antibodies, aptamers, peptides, nucleic acids, proteins, and enzymes, on the transducer surface to selectively bind to target substances. This setup enabled precise detection and analysis in various biomedical and research fields. M. Bakhshpour-Yucel et al. Biosensors and Bioelectronics: X 22 (2025) 100574 2 This analytical approach enables the precise quantification of biomole cular binding events on the sensor surface, thereby increasing the sensitivity of the IRIS system to show molecular affinities. Single- particle interferometric reflectance imaging sensor (SP-IRIS): This sys tem provides high-magnification digital detection of single nano particles. It is particularly useful for applications requiring detailed analysis of single molecules or particles and demonstrates high sensi tivity and resolution compared to traditional methods. Both methods present the interference-based optical sensing capability of the IRIS system, showing useful performance in the rapid and real-time detection of molecular interactions and virus/bacteria determination. These ad vancements in label-free detection have fostered significant growth in research and innovation within the fields of biomedical research and clinical diagnostics (Ünlü, 2014). 2.1. Instrumentation 2.1.1. IRIS system IRIS is an imaging system composed of a microscope and a Si-SiO2 substrate. It uses a light-emitting diode (LED) to illuminate the surface of the chip. Light reflects off both silicon and oxide layers and produces an interference pattern that can be observed as an intensity change relative to the thickness of the oxide layer. This system has basic components, such as LEDs, turn mirrors, stages, a camera, and an objective. Four colors are housed in an integrating sphere to achieve uniform light output in the setup: red, green, blue, and yellow. The Chip on board-light-emitting diode (COB-LED) incorporates λD = 630, 525, 460, and 594 nm for red, green, blue, and yellow, respectively (LED Engin LZ4-00MA00). This configuration is essential for precision lighting techniques without introducing unwanted LED patterns on the camera sensor. First, four-color images of the chip surface are used to capture and fit spectral curves for defining the layer of oxide thickness, after which the blue color is commonly utilized for subsequent testing steps. The turn mirror setup contains an elliptical mirror that is coated silver (PFE10-P01, Thorlabs, 45-degree elliptical shape). It is attached to a kinematic mount deliberately to rotate the collection path by 90◦. This elliptical mirror has a clear aperture of 45◦, which reduces the possi bility of clipping the collected light beam. The stage works as a platform to hold the chip in position, enables contact points for tubing and O-rings to the chip interface, and imple ments pressure to consubstantiate the fluidic cartridge by compressing an antireflective (AR) glass slide matched by gasket together. The camera is a 5-megapixel (MP) (2448 pixels × 2048 pixels) global shutter CMOS camera. It has a pixel pitch of 3.45 μm and a sensor size of 8.445 × 7.065 mm. The IRIS system can utilize a 2x/0.06 NA or a 5x/0.1 NA objective (Nikon). The user has significant control over the noise observed in the analyses. Switching from a 2x to a 5x objective increases the "pixels per spot," potentially reducing the noise floor. Moreover, using a larger spot size to print the ligands of interest can further lower undesired noise levels (Ünlü, 2014; Daaboul et al., 2011; Reddington et al., 2013a, 2013b; Monroe et al., 2011). 2.1.2. IRIS chip The IRIS chip was created using a silicon-silicon dioxide chip affixed to an anti-reflective-coated (AR-coated) glass slide using a thin silicone gasket as the channel layer. This cartridge forms a small fluidic chamber simplified by through-silicon via (TSV) holes. Buffers and samples can easily pass through this chamber as needed. The setup contains a silicon chip (25.2 mm × 12.5 mm) with a 110 nm thermally grown oxide layer, through-silicon-via (TSV) holes for fluid flow control, and an AR-coated glass slide used with a gasket, or optionally, an optically transparent coverslip with pressure-sensitive adhesive (PSA). The PSA coverslip consists of two protective layers: one for the transparent upper surface and the other for the adhesive. Precision tweezers are used to remove the adhesive layer from the coverslip carefully. A coverslip is placed over the IRIS chip by applying gentle pressure to adhere it securely. After placing the coverslip, the chip was placed on a flat surface, and a clean, small polypropylene test tube was used to roll over the cover, ensuring even pressure across the chip surface. Once the adhesive is fully attached, the protective upper layer of the coverslip is peeled off. Finally, a fluidic chamber is created. The characteristics of the obtained chamber are as follows: 50–500 μm channel with a volume of 2.0–50 μL. The assembly and loading are effortless and easy (Sevenler and Ünlü, 2015; Avci et al., 2015). 2.2. Protocol of IRIS system In the IRIS platform, silicon chip surfaces are functionalized prior to specific ligand spotting via either epoxy silane or a copolymer N,N- dimethylacrylamide, 3-(trimethoxysilyl)propyl methacrylate, N-acryl oyloxysuccinimide, copoly(DMA-MAPS-NAS) from Lucidant Polymers. Epoxysilanes, such as 3-glycidoxipropyl and trimethoxysilane, create a hydrophobic 2D coating that enables rapid ligand immobilization but requires a complex spotting process. In addition, its bi-dimensional na ture may not adequately preserve the structure of immobilized re ceptors. In contrast, MCP, an NHS-based reactive polymer, forms a 3D polymeric matrix on the silicon/silicon oxide surfaces. This three- dimensional coating elevates receptors, effectively maintaining their molecular structure and offering advantages over epoxy silane for microarray applications. The multifunctional copoly-DMA-MAPS-NAS (MCP copolymer) incorporates DMA for substrate adhesion, MAPS for covalent bonding to oxide groups, and NAS for probe immobilization via amide bonds with proteins or amino-modified oligonucleotides. Ver sions such as MCP-2 and MCP-4 differ in monomer ratios, influencing active site availability, with MCP-4 offering significantly more amine- reactive sites than MCP-2. MCP copolymers outperform 2D silane coatings by enhancing the probe density and reducing nonspecific mo lecular binding and interactions, proving their important properties and usability for bioreceptor immobilization in microarray technologies. An automatic spotter is used to create the spots on the IRIS chip. Antibodies and control molecules (Bovine serum albumin) are typically spotted on the chip surface, commonly at concentrations ranging be tween 0.5 and 2 mg/mL, 1%; v/v glycerol added to enhance spot morphology. Then, the IRIS chips are kept in a humidity chamber for 12 h to allow antibodies to attach to the chip surface. Finally, the IRIS chip loading into the sensor system easily occurs, and the binding process is performed with the following protocol, which is typically the same in each experiment. 1x PBST with 1% BSA flows over the chip for approximately 10–20 min at a 100–200 μL/min flow rate. After equilibrating the IRIS chip surface, an analyte at different concentrations is flowed over the chip for 20–30 min (the flow time and rate can be changed for different experiments). 3. Applications of the IRIS system The IRIS system can be utilized in various applications, such as di agnostics, biomolecular screening, and research. Specifically, it is useful for detecting microorganisms, proteins, and other targets. In diagnostics, the IRIS has great potential for identifying pathogens and disease markers with no labeling and high sensitivity. Biomolecular screening enables rapid and real-time analysis of protein interactions and helps in drug development. IRIS facilitates the easy characterization of bio molecules and virus and bacteria detection, supporting advancements in the biotechnology and biomedical fields. IRIS system has demonstrated significant utility across various ap plications, particularly in diagnostics, biomolecular screening, and biomedical research. This innovative technology excels in detecting microorganisms, proteins, nucleic acids, and other biomolecular targets with high sensitivity and accuracy without labeling. IRIS can accurately identify bacterial and viral pathogens in complex samples, significantly reducing the time and complexity associated with traditional diagnostic M. Bakhshpour-Yucel et al. Biosensors and Bioelectronics: X 22 (2025) 100574 3 methods (Seymour et al., 2023a). Additionally, the system aids in the early detection of disease biomarkers (Ekiz Kanik et al., 2022), allowing for timely diagnosis and treatment of conditions such as cancer and infectious diseases (Kazemi et al., 2022). For biomolecular screening, IRIS enables the real-time study of protein interactions, facilitating un derstanding complex biological processes and aiding in drug discovery (Ünlü et al., 2017). It also effectively analyzes interactions of DNA and RNA molecules, crucial for genetic research, including identifying ge netic mutations and variations (Özkumur et al., 2008). In biomedical research, IRIS is employed to monitor cellular be haviors and interactions, providing insights into cell biology and the mechanisms of diseases. The technology supports screening potential drug candidates by assessing their interaction with target biomolecules, thereby streamlining the drug development process (Seymour et al., 2023b). In environmental monitoring, IRIS detects environmental pol lutants, including toxins and pathogens, ensuring environmental safety and public health (Kazemi et al., 2022; Bakhshpour-Yucel et al., 2024). It also monitors water quality by identifying harmful microorganisms and chemical contaminants (Gubala et al., 2012). The IRIS system has several significant advantages. It provides pre cise measurements, enabling the detection of minute quantities of bio molecular targets (Monroe et al., 2011). By eliminating the need for fluorescent or radioactive labels, IRIS simplifies the detection process and reduces potential sources of error (Özkumur et al., 2008). Further more, IRIS allows for the continuous monitoring of biomolecular in teractions, providing immediate feedback and enabling dynamic studies (Kazemi et al., 2022). The versatility of this technology allows it to be adapted to various fields, including medical diagnostics, pharmaceutical research, and environmental monitoring (Seymour et al., 2023b). 3.1. Virus detection Microbes are usually identified by their individual size, shape, and staining features in clinical diagnosis via light microscopes. Unlike mi crobes, viruses are too tiny to be seen under regular light microscopes. Therefore, electron or atomic force microscopy is commonly utilized to visualize individual virus particles. However, these methods require particle purification and specialized knowledge and are ineffective for high-volume testing. Virus particles can only be detected under a light microscope when a specific labeling is used, which requires purification or genetic modification. Daaboul et al. present an innovative and highly promising approach to the label-free, rapid, and sensitive detection of virus particles and nanoparticles in complex biological samples, such as blood or serum, using the Single Particle Interferometric Reflectance Imaging Sensor (SP-IRIS). Their work addresses a critical challenge in detecting viruses in real-world clinical samples, where contaminants such as bacteria and other biomolecules can interfere with diagnostic assays. The SP-IRIS system provides a robust and efficient solution, demonstrating high sensitivity and specificity in detecting viruses like wild-type Vesicular Stomatitis Virus (VSV), defective VSV, and Ebola- and Marburg- pseudotyped VSV. They show that the SP-IRIS system can accurately identify virus particles in complex biological matrices, such as serum or whole blood, which are difficult to analyze due to high background noise levels (e.g., proteins, cells, and bacteria). Their study demonstrates the system’s ability to detect replication-competent and pseudotyped viruses with impressive sensitivity and specificity. This is a key advan tage for real-world diagnostic applications, where rapid and reliable identification of viruses in clinical samples is critical for timely decision- making. The authors report a 5 × 10³ pfu/mL LOD for the Ebola and Marburg pseudotypes, a significant achievement for virus detection in complex media. The low LOD demonstrates the high sensitivity of SP- IRIS, allowing for detecting viruses even at low concentrations. This is a critical feature for early diagnosis, especially in cases where viral loads may be low, such as in the early stages of infection. Another important contribution of this work is the demonstration of simultaneous detection of multiple viruses in a single sample. The SP-IRIS system is designed for high-throughput and rapid sizing of many biological nanoparticles on an antibody microarray. This capability makes it an excellent tool for research and diagnostic applications where quick and scalable testing of virus particles is required. The system’s ability to provide real-time sizing and identification of individual viral particles can support large- scale virus screening, helping to address the challenges of epidemic monitoring and outbreak detection (Daaboul et al., 2014). In another study, they developed the SP-IRIS, which detects, counts, and sizes individual virus particles directly from complex solutions. This technology significantly advances the capabilities of traditional light microscopy, which has long been limited in detecting and analyzing individual virus particles due to their small size. By overcoming these limitations, SP-IRIS opens new avenues for virus detection in biological fluids and for vaccine development, all while simplifying the process and reducing the need for sophisticated equipment typically required for virus analysis. Traditional light microscopy has long been invaluable for studying larger microorganisms such as parasites, fungi, and bacteria. However, visualizing individual virus particles has remained challenging due to their extremely small size (typically <100 nm), below the resolution limit of conventional visible light microscopes. So, they leverage the SP- IRIS technique, which utilizes reflectance interference imaging to visu alize unlabeled virus particles directly from complex samples without requiring purification or advanced sample preparation techniques. This approach represents a significant advancement, enabling real-time automated counting and sizing of thousands of virus particles. One of the most remarkable aspects of SP-IRIS is its ability to visualize various virus types, including flaviviruses, vesicular stomatitis virus (VSV), vaccinia virus, and even filamentous ebolavirus particles. By adjusting the illumination wavelength, SP-IRIS can detect viruses of different sizes. For example, violet/UV light illumination is used to visualize flavivirus particles (~40 nm), while green light enables the differenti ation between larger viruses, such as VSV and vaccinia virus (~360 nm). This wavelength-specific detection allows for clear differentiation be tween various viruses based on size and morphology, which is typically difficult to achieve with traditional microscopy. A particularly compel ling feature of the SP-IRIS system is its ability to identify virus-like particles (VLPs), often used in vaccine development and production. The technology can differentiate and quantify these particles, making it an invaluable tool for vaccine research. This capability to analyze VLPs without complex purification steps provides a significant advantage over traditional methods that often require electron microscopy or atomic force microscopy, which are time-consuming, expensive, and require specialized expertise. The ability to detect and quantify indi vidual virus particles directly from complex biological fluids, such as serum, plasma, or even whole blood, positions SP-IRIS as a powerful tool for rapid diagnostics. Additionally, its application to vaccine develop ment is particularly noteworthy, as it can be used to monitor the pres ence of virus-like particles (VLPs) during production. The non-invasive, label-free nature of the technique offers significant advantages for both basic research and industrial applications, such as vaccine manufacturing (Daaboul et al., 2017). Scherr et al. showed an innovative and highly promising approach for the label-free imaging of individual viruses and nanoparticles directly in complex biological solutions, such as serum, using a combi nation of single-particle reflectance imaging sensors and microfluidics. This work represents a significant step forward in virology research and biosensing, offering a rapid, sensitive, and cost-effective method for the real-time detection and visualization of viral particles without the need for traditional labeling or advanced microscopy techniques. The authors showed the system’s capability to detect vesicular stomatitis virus (VSV) particles as small as 100 nm in undiluted fetal bovine serum, with LOD as low as 100 PFU/mL. This is a significant achievement, as detecting low viral concentrations in complex, undiluted samples are often a major challenge in virology and diagnostics. The system’s ability to M. Bakhshpour-Yucel et al. Biosensors and Bioelectronics: X 22 (2025) 100574 4 detect viral particles at such low concentrations in biologically relevant fluids underscores its high sensitivity and potential for clinical di agnostics, particularly in situations where viral load is low, such as in the early stages of infection or asymptomatic individuals (Scherr et al., 2016). Scherr and co-workers developed and used a single-use cartridge system for sensitive viral hemorrhagic fever virus detection (Scherr et al., 2017). Previously, they showed the effectiveness of SP-IRIS (Single-Particle IRIS), a light microscopy technique utilizing interfero metric reflectance. This method allows the individual to detect virions directly in serum without virus labeling. SP-IRIS employs signal enhancement via interference of the scattered light from the captured particles on the sensor with the reflected light from the silicon-silicon dioxide interface (Daaboul et al., 2010). In follow-up work, they presented a novel rapid virus detection method using a single-use paper fluidic cartridge system. This innova tive approach allows rapid counting and analysis of viruses in complex samples employing SP-IRIS. They showed excellent, sensitive virus detection within 20 min. These innovations result in a high-performance system utilizing a disposable test format with capillary-based passive fluid handling. This method removes the need for pressure controllers or complex incubation equipment, making the SP-IRIS platform ideal for point-of-care diagnostic applications. In addition, these cartridges reduce exposure risks by containing the sample securely. It minimizes sample preparation time and eliminates the need for cold chain storage, making it a practical and efficient so lution for virus detection in various settings. Although paper-based lateral flow immunoassays are widely used to produce disposable and inexpensive diagnostic tests, they often face challenges in ensuring adequate sensitivity and robust fluid control for conducting multiplexed tests. These limitations underline the need for alternative techniques to emerge. Scherr et al., aimed to eliminate dependence on external equipment by incorporating paper-based liquid handling. To obtain a constant and controlled 1–10 μL min⁻1 flow rate for at least 20 min, they designed a 270◦ fan-shaped single-layer absorbent material to be located in the channel after the sensor. This shape allows experimentally adjusting the width and length of the pad’s stem to control the flow rate. Depending on the fluid mechanism of this channel, a quasi-constant flow rate is maintained throughout the test period. They compared their results with the Corgenix ReEbov quick antigen test, a blood-based, rapid, and sensitive diagnostic test for Ebola, a lateral flow assay. This test specifically detects the VP40 matrix protein of the Ebola virus and requires an additive to break down the viral capsid before testing. In contrast, the SP-IRIS test captures whole viruses by targeting and binding to the surface glycoprotein on the Ebola virus capsid. They used gamma-irradiated Ebola diluted in cell culture media to compare the SP-IRIS disposable cartridge with the ReEbov rapid antigen test. Both tests were conducted for 20 min on the same samples. In another study, Aslan et al. have made significant advancements in diagnostic technologies by developing an innovative label-free digital detection platform based on IRIS technology. Their latest work in troduces the Pixel-Diversity IRIS (PD-IRIS), which builds on their earlier Single-Particle IRIS (SP-IRIS) platform. One of the key improvements of PD-IRIS is that it eliminates the time-consuming and costly z-scan pro cess required by SP-IRIS, making it more suitable for point-of-care (POC) applications. This breakthrough platform utilizes light interference from an optically transparent thin film to enhance signal detection without complex optical resonances. PD-IRIS allows for constructing an optical signature of target nanoparticles, such as whole viruses, from a single image, simplifying the process and making it more efficient. They demonstrated the effectiveness of PD-IRIS for the quantitative detection of the Monkeypox virus (MPXV). They achieved a limit of detection (LOD) of 200 PFU/mL, which significantly outperformed the laboratory- based ELISA’s LOD of 1800 PFU/mL in terms of sensitivity. Additionally, the specificity of PD-IRIS was validated by testing against other viruses, including the Herpes simplex virus (HSV-1) and Cowpox virus (CPXV), further confirming its reliability for viral diagnostics. This study estab lishes PD-IRIS as a promising tool for rapid, accurate, and cost-effective pathogen detection, especially in resource-limited or field-based diag nostic settings. Its modular design enables multiplex pathogen detec tion, highlighting its potential for future clinical diagnostics and infectious disease monitoring applications, particularly in POC Fig. 2. (A) Diagram outlining the steps for preparing bacterial samples. (B) Detection of the initial culture concentration through OD600 measurements via spec trophotometer. (C) Culture plates images. (D) Illustration of the sequential experimental protocol steps (Zaraee et al., 2020). M. Bakhshpour-Yucel et al. Biosensors and Bioelectronics: X 22 (2025) 100574 5 environments (Aslan et al., 2025). One of the main features of PD-IRIS is its ability to detect particles from a single image without the need for a z-scan. This not only reduces costs but also simplifies data analysis. Considering these studies, it is evident that IRIS technology has been continuously developed to enable more precise and sensitive virus detection. 3.2. Bacteria detection Bacterial infections cause significant risks to vulnerable groups like children, the elderly, and individuals with compromised health. Contaminated food and water are primary sources, with Escherichia coli (E. coli) being a major risk, causing severe illnesses like bloody diarrhea and potentially fatal kidney failure if untreated. The most conventional methods for detecting pathogenic bacteria are culture-based techniques, immunological tests like ELISAs, and molecular methods like PCR. However, conventional methods for detecting pathogenic bacteria have disadvantages such as complexity, limited sensitivity, high costs, lengthy procedural times, and the need for specialized facilities and trained personnel. Zaraee and coworkers have developed a sensitive, rapid, selective, cost-effective, and real-time method for detecting E. coli bacteria with minimal sample preparation. They used the SP-IRIS technique, providing sensitive, label-free detection and rapid imaging capabilities (Zaraee et al., 2020). They used Si/SiO2 IRIS substrate modified with MCP-4 polymer and then immobilized antibodies for detecting E. coli. The polymer coating allows long-term storage under vacuum and facilitates strong binding of biomolecules, preserving their native conformation and functionality throughout the biosensing process. The concentration of anti-E. coli antibodies was reported at 3.0 mg/mL to detect E. coli on an IRIS sensor chip. This study ran 10 CFU/mL to 106 CFU/mL E. coli concentrations in Fig. 3. a) Schematic of the optical setup and an IRIS chip. b) Step-by-step MATLAB algorithm for particle detection (i) IRIS chip surface image is acquired with low- magnification setup, which constitutes the algorithm’s input. (ii) Zoomed-in image of an antibody spot from (i), with E. coli particles showing as diffraction-limited spots (blue dashed circles) (iii) The high-magnification image of the same antibody spot in (ii) revealing the original rod shape of E. coli particles (blue dashed circles). The arrows 1, 2, and 3 correspond to the steps of the particle detection algorithm: 1. Scanning and registering the antibody and BSA spots on the chip. 2. Detection of the captured particles on the spots in low-magnification images using the initial size and intensity parameters determined by the user and comparing them to the high-magnification images of the same antibody spot. 3. Feedback loop to Step 2 for optimizing the detection parameters to detect only the E. coli particles using the size and shape information in high-magnification images (Zaraee et al., 2020). M. Bakhshpour-Yucel et al. Biosensors and Bioelectronics: X 22 (2025) 100574 6 PBS. Incubation occurred in 1.0 mL of these dilutions within a 24-well plate, with the IRIS chips resting at the well bottoms for 2 h on a shaker, as shown in Fig. 2. To prevent microbial agglomeration on the chip surface and reduce non-specific binding, 0.1% Tween-20 was added to each well as a surfactant with non-ionic features. Fig. 3a shows the IRIS chip surface using Köhler illumination setup with an LED light source emitting at a wavelength of 520 nm. Fig. 3b shows the images of the low-magnification-IRIS chips that were achieved using a numerical aperture (NA) objective through a specific optical configuration. This setup offers a substantial field of view, approximately 5.85 mm2 (2.83 mm × 2.1 mm), enabling quick scanning of the entire IRIS chip surface. They use a custom-developed MATLAB algorithm for digital analysis to detect and count the captured particles. The entire IRIS chip surface is analyzed, enabling morphological characterization and analysis of the captured particles. Fig. 4 displays a single field of view from the low- magnification imaging mode, showing two IRIS chips incubated with bacteria samples at concentrations of 3.2 × 104 and 3.2 × 10 CFU/mL, respectively. Selected antibody spots designated for high-magnification characterization are highlighted in blue and green squares. 3.3. Binding affinity determination The IRIS system detects microbes and facilitates studies on antibody- antigen interactions, as documented in the literature. In a study by Lortlar et al., a sensitive IRIS system was developed to study the Vascular Endothelial Growth Factor (VEGF) binding affinity of two wet Age- related macular degeneration treatments, aflibercept and monoclonal antibody bevacizumab (Lortlar Ünlü et al., 2024). Wet age-related macular degeneration (AMD) is the foremost cause of vision impairment in developed countries, frequently resulting in blindness. Biologics, which are treatments derived from biological sources, have proven effective in addressing this condition. Due to the high costs associated with biologics, it is essential to evaluate their binding affinity to ensure their efficacy and to make quantitative com parisons between different treatments. To examine the binding affinity of VEGF, both biologics were immobilized on the same platform using a microarray IRIS chip, allowing real-time and sensitive measurement of their binding to recombinant human VEGF (rhVEGF). The findings demonstrated that aflibercept had a higher VEGF binding affinity than bevacizumab. The IRIS system’s cost-effective and sensitive capabilities, which include silicon-based chips for enhanced signal detection and multiplexed analysis, highlight its potential as a valuable sensor technology. Given the constraints of traditional detection methods, recent research has shown the development of advanced sensors that provide highly selective, sensitive, real-time, label-free, cost-effective, and real- time biomolecular interaction detection. Bakhshpour et al. demon strated the detection of immunoglobulin G (IgG) in aqueous solutions using sensitive and selective, real-time, label-free measurements using the IRIS sensor (Bakhshpour et al., 2022) via immobilized protein A on the epoxy-coated IRIS chip. According to the results, the sensor’s per formance is thoroughly assessed, achieving a high correlation coefficient (>0.94) across an IgG 1.0– 50 μg/mL concentration. They showed a 1.67 nM detection limit with a highly sensitive system and reliable validation of binding events. This study highlighted its potential for direct biomarker detection in clinical settings. They optimized the chips through ellipsometry, contact angle measurements, and Atomic Force Microscopy. The thickness of protein A-immobilized IRIS chips was measured at 46.6 ± 1.4 nm using ellipsometry, whereas epoxy-modified IRIS chips showed a thickness of 20.2 ± 0.6 nm (Fig. 5.A1,B1,C1). Additionally, the root mean square values of IRIS chips were measured as 22.96 ± 2.8, 14.56 ± 3.6, and 1.12 ± 0.2 nm for the bare, epoxy-modified, and protein A-immobilized surfaces, respectively, using AFM measurements (Fig. 5A2,B2,C3). According to the results of water contact angles, the immobilization of protein A has led to increased hydrophobic properties on the surface of the IRIS chip (Fig. 5.A3,B3,C3). 3.4. MicroRNA detection MicroRNAs (miRNAs) are functional noncoding RNAs implicated in various diseases. Traditional miRNA detection methods, such as DNA microarrays, have limitations in specificity for diagnostic applications. A new digital microarray using plasmonic gold nanorods was developed to address these challenges by Kanik et al. They showed improved Fig. 4. A-B) Showing one field of the low-magnification image of two IRIS chips incubated in different concentrations of bacteria sample, C) Histogram showing the count of antibody spots on 4 different IRIS chips (Zaraee et al., 2020). M. Bakhshpour-Yucel et al. Biosensors and Bioelectronics: X 22 (2025) 100574 7 sensitivity and multiplexing capability compared to conventional assays by enabling real-time tracking of miRNA molecules on the IRIS sensor surface. The method achieved a 10 aM detection limit for miRNAs miR- 223 and miR-451, significantly enhancing sensitivity and reducing in cubation time from 5 h to 35 min (Ekiz Kanik et al., 2022). Microarrays on these chips consist of DNA probes immobilized on the chip surface with an ideal chemistry containing reactive functional groups for covalent binding and minimal nonspecific interactions. They used variants of MCP polymers like MCP-2 and MCP-2F for increasing hydrophobicity, and an MCP-2 with an MCP-2F (5%, v/v) is utilized to improve the probe density and minimize non-specific binding. These polymers can easily swell in liquid solutions. Therefore, the accessibility of immobilized probes for surface reactions greatly increases. The op tical setup of the polarization-enhanced SP-IRIS system is shown in Fig. 6a, while the cartridge assembly is illustrated in Fig. 6b. After cleaning the IRIS chip, the complementary and control single- stranded DNA (ssDNA) surface probes were printed onto the chips. They incubated the spotted chips overnight at 67% humidity to allow the amine-modified ssDNA probes to immobilize on the polymer-coated surface. They prepared GNR-polyT conjugates. The homogeneous assay for miRNA detection uses a streamlined protocol with a single incubation step, where miRNA samples are mixed with polyT- conjugated gold nanorods (GNRs) before being applied to a micro array chip. This method offers rapid detection times, often within 30 min, due to continuous monitoring of particle binding rates. All binding events between miRNA-GNR complexes and their complementary sur face probes are tracked throughout the assay, focusing on total binding events to enhance sensitivity. The results for 100 fM miR-451 and 10 fM miR-223 target complexes are shown in Fig. 7a and (c), respectively. In Fig. 7 (b) and (d), the binding rates during the homogeneous assay incubation are depicted with respect to the concentration of the target miRNAs. These results highlight the assay’s ability to track specific binding events and quantify them in relation to target concentration. Fig. 5. Ellipsometry images (A1, B1, C1) illustrate the surfaces of bare, epoxy-modified, and protein A-immobilized IRIS chips, respectively, highlighting their thickness and characteristics. AFM images (A2, B2, C2) show the surface topography similarly. Contact angle images (A3, B3, C3) illustrate the hydrophobicity of the surfaces, with measurements from the corresponding chip (Bakhshpour et al., 2022). M. Bakhshpour-Yucel et al. Biosensors and Bioelectronics: X 22 (2025) 100574 8 3.5. Other applications of IRIS Exploring the binding interactions between small molecules and large ligands is crucial for drug development and agro-biotechnology, as drugs and toxins typically have low molecular weights. Chiodi et al. developed a label-free and highly multiplexed IRIS biosensor to improve the detection limits to ease small-molecule screening (Chiodi et al., 2020b). Label-free techniques are preferred for studying the kinetic Fig. 6. (a) Optical system of the polarization-enhanced SP-IRIS and (b) Assembly of the SP-IRIS cartridge. (c) Components for conducting a miRNA assay and the procedural steps involved in both heterogeneous and homogeneous assay methods(Ekiz Kanik et al., 2022) M. Bakhshpour-Yucel et al. Biosensors and Bioelectronics: X 22 (2025) 100574 9 behavior of toxins, drugs, and small targets with specific detecting probes. Surface plasmon resonance (SPR) is commonly used to detect various targets and is known for its good sensitivity to small and large molecules. SPR sensors also detect and analyze the interactions between biological and chemical molecules (Diken et al., 2019; Safran et al., 2021). SPR sensors usually analyze substances with low molecular weights in pharmaceutical research, theranostics, food safety, homeland security, and environmental monitoring (Chiodi et al., 2022; Akgönüllü and Denizli, 2023; Wang et al., 2010). These optical refractometers detect changes in the refractive index of media on SPR substrates by utilizing the collective oscillations of free electrons in a metal’s con duction band stimulated by an electromagnetic field at the metal/di electric interface. However, SPR measurements can be challenging due to solvent-induced refractive index changes, temperature fluctuations, or pH variations. IRIS demonstrated the characterization of small molecule binding to immobilized probes in a microarray format with a mass density detection limit of 1 pg/mm2. Table 1 compares the IRIS sensor’s performance with established commercial diagnostic techniques, including (Polymerase Chain Reac tion) PCR and (Enzyme-Linked Immunosorbent Assay) ELISA. This table outlines key performance metrics such as detection limits, sensitivity, specificity, accuracy, and cost, which help clarify the strengths and limitations of IRIS compared to these traditional methods. While the IRIS sensor exhibits strong sensitivity and specificity, its ability to pro vide rapid and cost-effective results in point-of-care settings offers notable advantages over PCR, particularly regarding operational costs and user-friendliness. This comparison aims to underscore the potential role of IRIS in clinical diagnostics, especially in situations where rapid, accessible, and affordable testing is crucial. Despite the promising po tential of the IRIS in diagnostics, several challenges remain that may limit its widespread application. One of the issues is non-specific bind ing, where molecules that are not the target of interest can adhere to the Fig. 7. (a, c) Cumulative binding events on each spot type during incubation with 10 fM miR-223 target complex and 100 fM miR-451 target complex. (b, d) Binding rates during the incubation relative to target concentration(Ekiz Kanik et al., 2022) Table 1 Comparative performance of IRIS with PCR, ELISA diagnostic techniques. Diagnostic Technique Sensitivity Detection Limits Detectable Molecules Accuracy Real Time Detection Label free Cost IRIS 85–95% Attomolar -nanomolar DNA, RNA, Proteins, viruses, bacteria, biomolecular interactions High (depends on sample quality and surface prep) Yes Yes Moderate initial setup cost, but low operational cost PCR 95–99% Femtomolar DNA, RNA, viral genomes, bacterial genomes Very high No No Moderate to high ELISA 80–95% Picomolar -nanomolar Proteins, antibodies, antigens Very high No No Low to moderate M. Bakhshpour-Yucel et al. Biosensors and Bioelectronics: X 22 (2025) 100574 10 sensor surface, leading to false positives and reduced assay accuracy. Appropriate planning and assay optimization can effectively prevent non-specific interactions, particularly in antigen-antibody interactions (Hosseini et al., 2018; Angelopoulou et al., 2024). By carefully designing the assay conditions and using selective sur face chemistry, non-specific binding can be minimized, ensuring that only the specific binding between the antigen and antibody is detected. Optimizing the concentration of the target antigen, antibody, and other assay components can further reduce the likelihood of non-specific in teractions. Additionally, surface modifications and proper blocking strategies can be employed to enhance the specificity of the antigen- antibody interaction, improving the overall sensitivity and reliability of the IRIS system. These measures can be particularly useful in complex biological samples, ensuring more accurate results and expanding the potential applications of IRIS technology in diagnostic settings. Also, Table 2 compares the advantages and disadvantages of interferometric sensors, ELISA, and PCR. Noise reduction in the system was achieved through spatial and temporal averaging, as demonstrated by detecting biotin (MW = 244.3 Da) binding to a streptavidin-functionalized chip, optimizing parame ters to achieve a signal-to-noise ratio (SNR) of approximately 34. The optimized system was then used to screen a 20-multiplexed antibody chip against fumonisin B1 (MW = 721.8 Da), a mycotoxin in cereal grains, yielding an SNR of about 8. Additionally, five antibodies from the chip were tested against the toxin in a lateral flow assay, producing consistent results. The observed noise characteristics indicate that further sensitivity improvements are likely with advancements in cam era sensor technology. They present a sensitive optical method for detecting the affinity of very small structure molecule targets to their specific ligands. By integrating the IRIS technique, they improved the signal-to-noise ratio, achieving sensitivity for small molecules, as demonstrated by biotin, which is 244.3 Da molecule. Microarray technology is a crucial tool for displaying multiple bio molecular interactions used in various applications such as protein and gene expression profiling and point mutation analysis. The quality of printed microarray slides is crucial for providing the technology’s adoption in diagnostic applications. The poorly surface-bound probes in the microarray production reduce diagnostic sensitivity or, at worst, cause false negatives. Chiodi et al., present a reliable and straightfor ward quality control method for evaluating spotted probe properties in a microarray test using the IRIS system (Chiodi et al., 2021). The IRIS determines ligand mass accumulation on functionalized Si/ SiO2 chips with ligands in an array format. The IRIS generates pre- hybridization images of oligonucleotide microarrays and measures real-time hybridization label-free, quantifying bound mass. Addition ally, IRIS allows the comparison of different immobilization chemistries. They showed that the IRIS analysis of microarray chips immediately after probe immobilization can detect the absence of probes, which leads to a lack of signal in clinically relevant tests using fluorescence detec tion. Additionally, IRIS enables the determination of the optimal probe concentration to immobilize on the surface, maximizing target recog nition and signal while avoiding crowding effects. They showed the spotting scheme for 33 oncogene capture probes related to NRAS, BRAF, and KRAS, along with the initially immobilized mass for all the dry spotted probes in Fig. 8. The surface density was generally consistent across most spots (Fig. 8b and c), showing the MCP- 4 polymer’s effectiveness in stably immobilizing amine-modified probes; but, they reported that two probes, KRAS 12C and NRAS 61HT, did not immobilize correctly (Fig. 8b, red and green rectangles). This insufficiency could be due to the deterioration of the amine tail on the DNA strand. As they demonstrated in Fig. 8c, replacing the old probe with a fresh one improved immobilization significantly. In addition, lack of immobilization, as shown in Fig. 8b, would result in a negative detection for that specific mutation, compromising the assay’s diag nostic capability. 4. Conclusion The IRIS system has proven to be a highly effective tool for sensitive, and rapid detection of biomolecular interactions without needing la beling. Its ability to provide real-time, high-throughput analysis makes it invaluable for clinical diagnostics and biomedical research. The versa tility of IRIS demonstrated through applications ranging from virus detection to the study of binding affinities, underscores its potential to revolutionize diagnostic technologies. Incorporating high-binding ca pacity and antifouling polymers for bioreceptor immobilization has significantly improved the system’s performance, enhancing sensitivity and specificity. As a platform, IRIS offers robust and reliable measure ments that are critical for early diagnosis, disease monitoring, and the development of targeted therapies. Future advancements in IRIS tech nology will likely focus on increasing its multiplexing capabilities and integrating it with other diagnostic tools to create comprehensive and Table 2 A summary comparing the advantages and disadvantages of interferometric sensors, ELISA, and PCR. Feature PCR ELISA Interferometric Sensors Advantages Sensitivity High (95–100% sensitivity) High sensitivity for many analytes (proteins, hormones, antibodies) High sensitivity Versatility Can diagnose a wide range of human diseases (infectious diseases, cancer, genetic testing) Widely used in clinical diagnostics, food safety, and environmental testing Detects a variety of contaminants in food, and diagnose a wide range of human diseases Multiplexing Limited multiplexing capacity in standard setups Micro/nano fabrication and lab-on-chip systems enable multiplexing Excellent multiplexing ability, detecting multiple analytes simultaneously Speed Rapid results, especially for critical cases Moderate speed, results can take hours depending on the format Rapid results, can be real-time detection Portability Limited portability; requires a lab setup with advanced equipment Portable formats available (paper/fiber ELISA) Still in development but advancing towards portable, miniaturized devices Reusability Not reusable Typically, single-use, though miniaturization allows for more efficient use Sensors can be reused, especially with synthetic biorecognition elements Label-Free Detection No Often requires labeled antibodies or substrates Can perform label-free detection, reducing costs Cost High due to expensive equipment, reagents, and maintenance Relatively low, especially with paper/fiber- based formats Low-cost production using materials like Si, glass, or polymer Disadvantages Sample Handling Requires well-prepared samples (e.g., DNA extraction) Requires sample preparation (e.g., coating with antigen/antibody) Sample interference possible from the sample matrix such as food, serum matrix Sensitivity for Complex Samples Works well for complex samples (e.g., blood, tissues) Sensitivity can be insufficient for certain low-abundance biomolecules Can be affected by sample interference, requires treatment in some cases Time Consumption Relatively fast, but can vary depending on the protocol Time-consuming; results take hours Can be fast, especially with micro-devices; still depends on sample prep M. 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