Surface Plasmon Resonance Imaging Research Articles

Abstract LB030: Building a platform of validated high throughput screening approaches for molecular glue degrader identification

Abstract Over recent years, Induced proximity therapeutics (IPT) and targeted protein degradation (TPD) approaches have become increasingly popular. Whilst significant initial efforts focused upon heterobifunctional degraders, molecular glue degraders (MGDs) represent the next generation proximity-based drugs, but their rational discovery remains challenging. Here, we describe unbiased and broadly applicable approaches for molecular glue hit ID through the development of a suite of orthogonal and highly complementary screening approaches, relying on Homogeneous Time-Resolved Fluorescence (HTRF), Surface Plasmon Resonance (SPR) and High Content Imaging (HCI). To demonstrate the utility of these assays, we have focussed on the therapeutically relevant protein SOS1 as a model target. SOS1 is the major guanine nucleotide exchange factor (GEF) for KRAS, where inhibition of the SOS1/KRAS interaction reduces tumor growth in preclinical settings, and small molecule inhibitors of the SOS1/KRAS interaction are currently undergoing clinical testing for KRAS driven cancers. Our HTRF approach measures the induced interaction between the ubiquitin ligase CRBN/DDB1 and the SOS1 protein. The assay was successfully developed and validated in a miniaturized format utilizing a bifunctional tool compound as a positive control. Complementing our HTRF based approach, we also developed a highly sensitive SPR assay for detecting compounds that generate a ternary complex with CRBN/DDB1. These assay formats are amenable for large scale HTS screening, and as proof-of-concept we have established workflows and screened compound collections from the Sygnature LeadFinder and fragment libraries, in each of these assay platforms. Our combined biochemical/biophysical approach to glue identification using HTRF and SPR demonstrates the successful and scalable use of these methodologies for HTS, with the exciting potential for identification of novel MGD candidates for SOS1. To further complement these strategies for glue identification, we have additionally developed a functional cell-based approach utilizing HCI. HCI offers a medium-high throughput approach to monitor endogenous protein abundance and localization in complex cellular models. One major advantage of imaging assays is that they are amenable to multiplexing, allowing simultaneous detection of target engagement, biomarker analysis and compound toxicity. Here, we showcase HCI as a robust, high throughput method of screening for induced protein degradation at the endogenous level, again focusing on the KRAS pathway. Using this approach we quantified endogenous protein abundance in response to compound treatment, as well as downstream effects in the RAS pathway. Together, these approaches demonstrate multiple, complementary options for identifying molecular glue degraders. Further, the described assays are also applicable for screening and characterization of bifunctional degrader compounds. This platform is poised to identify high quality lead compounds across a range of oncology targets in areas of unmet clinical need. Citation Format: Allan Jordan, Philip Addis, Toby Allen, Tarun Ayra, Sarah Beck, Roslyn Brant, Roland Hjerpe, Benoit Gourdet, Martin Jennings, Rachel Lawrence, Sigrun Campbell-Maurer, Pei Cing Ng, Lyn Parkinson, Eva Rejnowicz, Gonzalo Robles, Elizabeth Rosethorne, Joshua Shaw, Duncan Smith, Denise Swift, Daniel Tait, Stuart Thomson, Chris Tomlinson, Ailsa Townley, Stephanie Ward, Clare Wilson. Building a platform of validated high throughput screening approaches for molecular glue degrader identification [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB030.

Is the leptin/BMI ratio a reliable biomarker for endometriosis?

The aim of this study was to analyze the concentration of leptin in peritoneal fluid and plasma and to assess their role as potential biomarkers in the diagnosis of endometriosis. Leptin adjusted for BMI (leptin/BMI ratio) was measured using surface plasmon resonance imaging (SPRI) biosensors. Patients with suspected endometriosis were included in the study. Plasma was collected from 70 cases, and peritoneal fluid from 67 cases. Based on the presence of endometriosis lesions detected during laparoscopy, patients were divided into a study group and a control group (patients without endometriosis). Leptin/BMI ratio in plasma did not differ between women with endometriosis and the control group (0.7159 ± 0.259 vs 0.6992 ± 0.273, p= 0,7988). No significant differences were observed in peritoneal leptin/BMI ratio levels in patients with and without endometriosis (0.6206 ± 0.258 vs 0.6215 ± 0.264, p= 0,9896). Plasma and peritoneal leptin/BMI ratios were significantly lower in women with endometriosis - related primary infertility compared to women with endometriosis without primary infertility (0.640 ± 0.502 vs 0.878 ± 0.623, p < 0.05). The difference was observed in case of primary infertility, but not in terms of the secondary one. No significant differences were noted between leptin/BMI ratio in the proliferative phase and the secretory phase (0.716 ± 0.252 vs 0.697 ± 0.288, p= 0,7785). The results of present study do not support the relevance of leptin concentration determination as a biomarker of the endometriosis. Due to the limited number of samples in the tested group, further studies are needed to confirm its role.

Study of cell and drug interactions based on dual-mode detection using SPR and fluorescence imaging

The investigation of the interactions between cells and drugs forms a crucial aspect of biological and clinical medical studies. Generally, single-cell or local-cellular studies require a microscopic imaging system with high magnifications, which suffers from low detection throughputs and poor time responses. The study presented in this paper combined SPR and fluorescence to achieve cell localization, real-time monitoring of cell images and quantitative analysis of drugs. In order to obtain more comprehensive, accurate and real-time data, a dual-mode system based on surface plasmon resonance (SPR) and fluorescence was constructed based on a 4× magnification lens. This enables simultaneous studies of an entire cell and a specific region of the cell membrane. An adaptive adjustment algorithm was established for distorted SPR images, achieving temporal and spatial matching of the dual-mode detection. The combination of SPR and fluorescence not only achieved micro-detection but also complemented the qualitative or quantitative limitations of SPR or fluorescence method alone. In system characterization, the response signal of SPR was noticed to increase with the increasing concentration of EGF in stimulated cells. It indicated that this platform could be employed for quantitative detection of the cell membrane region. Upon addition of EGF, a peak in the SPR curve was observed, and the cells in the corresponding SPR image turned whiter. This indicated that the platform can simultaneously monitor the SPR response signal and image changes. The response time of fluorescence in EGF testing was several seconds earlier than SPR, revealing that signal transduction first occurred in the whole cell and then propagated to the cell membrane region. The inhibitory ability of Gefitinib on cells was verified in a fast and real-time manner within 20 min. The results indicated that the detection limit of this method was 20 IU/mL for EGF and 10 µg/mL for Gefitinib. In conclusion, this study demonstrates the advantages of SPR and fluorescence dual-mode techniques in the analysis of cell-drug interactions, as well as their strong potential in drug screening.

Microstructuring of Thermoresponsive Biofunctional Hydrogels by Multiphoton Photocrosslinking

AbstractA pioneering method is reported for creating thermoresponsive biofunctional hydrogel microstructures using maskless multiphoton lithography. Departing from conventional multiphoton‐triggered polymerization‐based techniques, this approach relies on simultaneous photocrosslinking and attachment of already pre‐synthesized polymer chains onto solid substrates. The method allows improving control over polymer network characteristics and enables facile integration of additional functionalities through postmodification with biomolecules at specific sites. Exploring two distinct benzophenone‐ and anthraquinone‐based photocrosslinkers incorporated into specially designed poly(N‐isopropyl acrylamide)‐based co‐ and terpolymers, the photocrosslinking efficacy is scrutinized with the use of a custom femtosecond near‐infrared laser lithographer. Comprehensive characterization via surface plasmon resonance imaging, atomic force microscopy, and optical fluorescence microscopy reveals swelling behavior and demonstrates postmodification feasibility. Notably, within a specific range of multiphoton photocrosslinking parameters, the surface‐attached microstructures exhibit a quasiperiodic topography akin to wrinkle‐pattern formation. Leveraging the capabilities of established multiphoton lithographer systems that offer fast pattern writing with high resolution, this approach holds great promise for the versatile fabrication of multifunctional 3D micro‐ and nanostructures. Such tailored responsive biofunctional materials with spatial control over composition, swelling behavior, and postmodification are particularly attractive in the areas of bioanalytical and biomedical technologies.

Surface Plasmon Resonance Biosensors: A Review of Molecular Imaging with High Spatial Resolution.

Surface plasmon resonance (SPR) is a powerful tool for determining molecular interactions quantitatively. SPR imaging (SPRi) further improves the throughput of SPR technology and provides the spatially resolved capability for observing the molecular interaction dynamics in detail. SPRi is becoming more and more popular in biological and chemical sensing and imaging. However, SPRi suffers from low spatial resolution due to the imperfect optical components and delocalized features of propagating surface plasmonic waves along the surface. Diverse kinds of approaches have been developed to improve the spatial resolution of SPRi, which have enormously impelled the development of the methodology and further extended its possible applications. In this minireview, we introduce the mechanisms for building a high-spatial-resolution SPRi system and present its experimental schemes from prism-coupled SPRi and SPR microscopy (SPRM) to surface plasmonic scattering microscopy (SPSM); summarize its exciting applications, including molecular interaction analysis, molecular imaging and profiling, tracking of single entities, and analysis of single cells; and discuss its challenges in recent decade as well as the promising future.

Phospho-Tau 181 quantification method for Alzheimer's disease based on an array 2D biosensor combined with surface plasmon resonance imaging

Alzheimer's disease is among the neurodegenerative diseases for which there is a lack of rapid, effective, and non-invasive diagnostic methods. The development of a phospho-Tau 181 assay biosensor is therefore a response to the need for methods to diagnose AD. The present work was aimed at developing a fast, selective, and repeatable method for the quantitative determination of phospho-Tau 181, which could be used even during routine blood tests. Our method is a form of what is called liquid biopsy. The developed method underwent validation, as a result of which its analytical parameters were determined.An LOQ of 3.35 pg mL−1 was obtained, confirming the possibility of trace analysis of phospho-Tau 181 in human plasma. Relative percentage error values below 15 % and CVs in the range 1.47–7.09 % attest to the high accuracy and precision of the presented method. Also, the sample matrix was not found to significantly affect the results obtained for phospho-Tau 181 concentrations. The new SPRi biosensor provides reproducible measurements of the analyte under study (CV = 3.18–4.26 %). Although the method requires absolute adherence to the recommendations of the analytical procedure protocol, it achieves high selectivity and provides 90 % certainty of the correctness of the diagnosis based on measurements of phospho-Tau 181 concentration.

Optimization of OpenCV based spot identification method for surface plasmon resonance imaging

In this work, we focus on the OpenCV based microarray recognition method for Surface Plasmon Resonance Imaging (SPRi), proposing the hit-ratio of global light pixels and coverage of the potential spots in a microarray as the criteria for identification evaluation in SPRi data. We optimized the design of the ellipse fitting strategy by analyzing the impact of different parameters in the method. After optimization of the parameters, the accuracy of microarray recognition was successfully increased to over 90%. This work not only contributes to reducing errors in microarray signal extraction and improving signal processing quality but also has significant implications for applying computer graphic technology in high-throughput biochemical analysis.

Localized Surface Plasmon Resonance Imaging to Detect Protein Secretions from a Single Cell

Localized Surface Plasmon Resonance Imaging to Detect Protein Secretions from a Single Cell

Localized Surface Plasmon Resonance Imaging to Detect Protein Secretions from a Single Cell

Localized Surface Plasmon Resonance Imaging to Detect Protein Secretions from a Single Cell

Anti-allergic drug screening in single living mast cells by means of a novel surface plasmon resonance imaging system

In recent years, phenotypic drug screening has achieved renewed prominence, because it can develop drugs for some rare diseases with unknown etiology and without effective therapeutic drugs. Combined with biochip microarray technology, surface plasmon resonance imaging (SPRi) technology based on changes in reflected light intensity has been widely used to analyze interactions between biomolecules, especially for in vitro drug screening. However, changes in reflected light intensity are merely derivative of changes in surface plasmon resonance angles, so current SPRi technology suffers from low accuracy, poor sensitivity, and narrow linear range. In this study, we built an improved SPR microscopic imaging system capable of directly detecting SPR resonance angle (SPR-dip) shifts, with a sensitivity of 8.14 × 10−6 RIU, and a stability less than ± 2 m°. By optimizing the optical path system, the linear correlation coefficient of the system within the scanning range of 3800 m° is 0.9975. Based on this imaging system, we established a reliable screening system for single living mast cell allergy drugs. By developing small molecule microarray chips, we achieved high-throughput screening of 2400 sample points at one time. Our SPR microscopic imaging system integrates the comparative advantages of existing angle-scanning SPRi systems and opens the possibility of new applications in phenotypic drug screening.

The Exercise aNd hEArt transplant (ENEA) trial – a registry-based randomized controlled trial evaluating the safety and efficacy of cardiac telerehabilitation after heart transplant

BackgroundHeart transplant (HTx) is gold-standard therapy for patients with end-stage heart failure. Cardiac rehabilitation (CR) is a multidisciplinary intervention shown to improve cardiovascular prognosis and quality of life. The aim in this randomized controlled trial is to explore the safety and efficacy of cardiac telerehabilitation after HTx. In addition, biomarkers of rehabilitation outcomes will be identified, as data that will enable treatment to be tailored to patient phenotype. MethodsPatients after HTx will be recruited at IRCCS S. Maria Nascente – Fondazione Don Gnocchi, Milan, Italy (n = 40). Consenting participants will be randomly allocated to either of two groups (1:1): an intervention group who will receive on-site CR followed by 12 weeks of telerehabilitation, or a control group who will receive on-site CR followed by standard homecare and exercise programme. Recruitment began on 20th May 2023 and is expected to continue until 20th May 2025. Socio-demographic characteristics, lifestyle, health status, cardiovascular events, cognitive function, anxiety and depression symptoms, and quality of life will be assessed, as well as exercise capacity and muscular endurance. Participants will be evaluated before the intervention, post-CR and after 6 months. In addition, analysis of circulating extracellular vesicles using Surface Plasmon Resonance imaging (SPRi), based on a rehabilomic approach, will be applied to both groups pre- and post-CR. ConclusionThis study will explore the safety and efficacy of cardiac telerehabilitation after HTx. In addition, a rehabilomic approach will be used to investigate biomolecular phenotypization in HTx patients.Trial registration number:ClinicalTrials.gov Identifier: NCT05824364.

An Array SPRi Biosensor for the Determination of Follicle-Stimulating Hormone in Blood Plasma.

Follicle-stimulating hormone (FSH) regulates the development, growth, pubertal maturation and reproductive processes of the human body. The determination of serous FSH concentration is significant as an alternative to testicular biopsy in the case of boys suffering from cryptorchidism after orchidopexy, and as a means of determining the menopausal stage in women. The aim of this investigation is to develop a specific array surface plasmon resonance imaging (SPRi) biosensor for the determination of FSH in body liquids such as blood plasma, obtaining sufficient sensitivity to determine FSH at levels characteristic for that hormone in blood plasma, without any signal enhancement. The biosensor consists of a mouse monoclonal anti-FSH antibody attached to the gold surface of a chip via a cysteamine linker. Its linear response range is from 0.08 mIU mL-1 (LOQ) to 20 mIU mL-1, and well covers most of the range of FSH activities found in blood without dilution. The precision of measurement is between 3.2% and 13.1% for model samples, and between 3.7% and 5.6% for spiked plasma samples. Recoveries are in the range from 94% to 108%. The biosensor has good selectivity, and is validated by comparison with ECLE, with good agreement of the results.

Development of an SPRi Test for the Quantitative Detection of Cadherin 12 in Human Plasma and Peritoneal Fluid

A new method for the determination of cadherin 12 (CDH12)—an adhesive protein that has a significant impact on the development, growth, and movement of cancer cells—was developed and validated. The method is based on a biosensor using surface plasmon resonance imaging (SPRi) detection. A quartz crystal microbalance was used to analyze the characteristics of the formation of successive layers of the biosensor, from the linker monolayer to the final capture of CDH12 from solution. The association equilibrium constant (KA = 1.66 × 1011 dm3 mol−1) and the dissociation equilibrium constant (KD = 7.52 × 10−12 mol dm−3) of the anti-CDH12 antibody–CDH12 protein complex were determined. The determined analytical parameters, namely the values determining the accuracy, precision, and repeatability of the method, do not exceed the permissible 20% deviations specified by the aforementioned institutions. The proposed method is also selective with respect to possible potential interferents, occurring in up to 100-fold excess concentration relative to the CDH12 concentration. The determined Limit of Quantification (LOQ = 4.92 pg mL−1) indicates the possibility of performing quantitative analysis in human plasma or peritoneal fluid without the need to concentrate the samples; however, particular attention should be paid to their storage conditions, as the analyte does not exhibit high stability. The Passing–Bablok regression model revealed good agreement between the reference method and the SPRi biosensor, with ρSpearman values of 0.961 and 0.925.

Real-time quantification of proteins secreted of conditioned media from mesenchymal stromal cells (MSC) in co-culture with hematopoietic progenitor cells

The interaction of mesenchymal stromal cells (MSC) in co-cultures with hematopoietic progenitor cells (HPC) in ex-vivo models induce the secretion of pro-inflammatory protein factors and influences HPC cell proliferation and differentiation processes. Different studies have shown the quantification of these biomarkers in other conditioned media using an ELISA and Luminex™ type immunoassay format. In this paper, we developed real-time quantification of biomarkers in conditioned media of these cells in co-cultures from bone marrow, umbilical cord blood, and Wharton's jelly sources using surface plasmon resonance imaging (SPRi). We show the detection and quantification of CCL2, CCL5, bFGF, IL-6, IL-8 and VEGF proteins with high sensitivity and specificity. SPRi can be used with a real-time analysis methodology and could be considered a tool for biomarker assays.

Simultaneous detection of CA-125 and mesothelin by gold nanoparticles in surface plasmon resonance

The early-stage diagnosis and monitoring of disease evolution is still one of the most challenging tasks in the field of cancer research. Several research have been driven toward the discovery of new cancer biomarkers as well as to the development of biosensors able to detect these biomarkers with high specificity and sensitivity. Many types of cancer have been detected at an advanced stage and, in particular, epithelial ovarian cancer (EOC) has a high incidence of diagnosis in the metastatic stage. This cancer is considered as the most aggressive type of gynecological cancer with a 5-year survival and until now, no specific biomarkers have been identified. Nevertheless, some studies have indicated that mesothelin, a protein overexpressed by tumour cells in the ovary, interacts with the CA-125 biomarker present in the blood system accelerating the metastatic process. Therefore, the simultaneous presence of CA-125 and mesothelin in the blood during the evolution of EOC could be used to diagnose this disease before the metastasis. Therefore, with this motivation, this work aimed at the development of a biosensor capable of simultaneously detecting mesothelin and CA-125 at low concentrations. A biosensor based on the surface plasmon resonance imaging (SPRi) technique was developed and gold nanoparticles (AuNPs) were used in order to enhance the sensitivity. The study analysed successive injections of the biomarkers CA-125 and mesothelin, both in solution (analytical range of 9–120 nM). The limits of detection were obtained for CA-125 and mesothelin, being of 3.03 nM and 13.62 nM, respectively. The biosensor detected mesothelin at a concentration close to the cutoff point for EOC (3 nM) and was able to detect the biomarker CA-125 at 9 nM. Furthermore, the biosensor interacted preferentially and simultaneously with the biomarkers CA-125 and mesothelin incubated in fetal bovine serum. Finally, the use of AuNPs increased the sensor signal for CA-125 detection compared to its direct detection and showed greater selectivity for both biomarkers. Therefore, the biosensor has important characteristics that allow testing with real samples. In a future project, it could be applied in the research of diagnosis and prognosis of EOC.

Surface plasmon resonance sensor based on polarization parameter SPR imaging

Using polarization surface plasmon resonance (SPR) imaging as a sensor has the advantage of large throughput in detection, but its sensitivity has always been inferior to other SPR sensors. The high contrast of the two polarization parameters’ images related to scattering determines the high sensitivity of this new polarization SPR imaging sensor. It provides a new direction for solving the issue of low sensitivity in polarization SPR imaging. The sensor system was optimized by numerical simulation, whilst the baseline noise and sensitivity of the system were obtained by saline solution and virus detection. When the reflective index of the NaCl solution is within the range of 1.3331 to 1.36, the average sensitivity can reach 9300 RIU-1, and the maximum sensitivity can reach 13000 RIU-1. Using this new polarization SPR imaging sensor, the H1N1 virus was differentiated, showing its promising application potential within the field of biomedicine.

Thickness measurement of bimetallic film using surface plasmon resonance holographic microscopy.

Bimetallic film with high stability and sensitivity is often used to excite surface plasmon resonance (SPR). The thicknesses of the bimetallic film play an important role in quantitative retrieval of the sample's parameters, and a precise measurement method is not available until now. In this paper, we propose a method for measuring the thicknesses of bimetallic film using surface plasmon resonance holographic microscopy (SPRHM). Considering that the refractive index of the dielectric upon the bimetallic film sensitively modulates the SPR phase response, the two thickness parameters of bimetallic film can be calculated by two phase-contrast SPR images with two different liquid dielectrics. The capability of this method was verified with several Ag-Au film couples by using a compact SPRHM setup. Our work provides a precise characterization method for the parameters of SPR configuration and may find wide applications in the research fields of SPR sensing and imaging.

Quantitatively sensitive and resolution-enhancing strategies in optical signal conversion of SPR images using extensive angle-scanning and image-reconstruction

Surface plasmon resonance imaging (SPRi) is a technology developed from SPR that has emerged in response to the growing need for high-throughput and for discovering diverse applications across multiple domains. Despite its potential, SPRi has encountered limitations in sensitivity when handling low-molecular-mass materials, which emphasizes the need for the quantitative processing of SPRi signals. In this study, an SPRi signal conversion process was developed to enhance the contrast of SPR images and convert them into quantitatively analyzed signals. At first, an optimum incident angle of light was determined as an angle that maximized the difference between sums of the brightness % of functionalized regions and the brightness % of background region in the SPR image, which enabled to obtain a distinct SPR image. In addition, the enhancement of contrast and reduction of noise in the SPR image was achieved using Contrast Limited Adaptive Histogram Equalization (CLAHE) method. Then various pixel domains of the SPR images were simultaneously quantified and visualized the multi-pixel processing technique. For validating the process, an SiO2/Au thin film was modified with four different functionalities and applied to the detection of gas molecules. The SPR images processed using the proposed process exhibited sensitive and quantitative information about the interactions of the functionalities with the target gas in the distinct multiple regions, thus, were applicable to the quantitative monitoring of various phenomena occurring on the plasmonic interface via the advantages of SPRi.

Intensity Interrogation-Based High-Sensitivity Surface Plasmon Resonance Imaging Biosensor for Apoptosis Detection in Cancer.

Intensity interrogation-based surface plasmon resonance imaging (ISPRi) sensing has a simple schematic design and is the most widely used surface plasmon resonance technology at present. In this study, we report the successful development of a novel high-sensitivity ISPRi biosensor and its application for apoptosis detection in cancer cells. By optimizing the excitation wavelength and excitation angle, we achieved a refractive index resolution (RIR) of 5.20 × 10-6 RIU. Importantly, the biosensor has been tested and validated for high-throughput and label-free detection of activated caspase-3 with its specific inhibitor Z-DEVD-FMK in apoptotic cells. Therefore, this study describes a novel molecular imaging system to monitor apoptosis in cancers for disease diagnosis and/or evaluation of therapeutic efficacy of anti-cancer drugs.

A New Approach to the Quantification of Fibroblast Growth Factor 23-An Array Surface Plasmon Resonance Imaging Biosensor.

A new biosensor based on the "surface plasmon resonance imaging (SPRi)" detection technique for the quantification of "fibroblast growth factor 23 (FGF23)" has been developed. FGF23 is mainly produced in bone tissues as a phosphaturic hormone that forms a trimeric complex with "fibroblast growth factor receptor 1 (FGFR1)" and αKlotho upon secretion. FGF23 stimulates phosphate excretion and inhibits the formation of active vitamin D in the kidneys. FGF23 has been shown to play a role in bone carcinogenesis and metastasis. The newly developed method, based on the array SPRi biosensor, was validated-the precision, accuracy, and selectivity were acceptable, and yielded less than ±10% recovery. The rectilinear response of the biosensor ranges from 1 to 75 pg/mL. The limit of detection was 0.033 pg/mL, and the limit of quantification was 0.107 pg/mL. The biosensor was used to determine FGF23 concentrations in the blood plasma of healthy subjects and patients with "clear cell" renal cell carcinoma (ccRCC). The obtained results were compared with those measured through an "enzyme-linked immunosorbent assay (ELISA)". The determined Pearson correlation coefficients were 0.994 and 0.989, demonstrating that the newly developed biosensor can be used as a competitive method for the ELISA.