Biosensor for determining biochemical index of bone status from body fluids

Abstract

The present invention relates to a biosensor that enables the detection of biomarkers related to bone metabolism through saliva samples. This biosensor, which uses a saliva sampling method and offers an economical and user-friendly solution by adopting a non-invasive approach, can detect biomarkers related to bone formation and resorption such as Procollagen Type I N-terminal Propeptide (PINP) and β-C- terminal Telopeptide (β-CTX) in saliva with high sensitivity. The present invention increases patient comfort while also providing rapid analysis, making the diagnosis and treatment processes of diseases related to bone metabolism more efficient.

Description

[0001] DESCRIPTION

[0002] BIOSENSOR FOR DETERMINING BIOCHEMICAL INDEX OF BONE STATUS FROM BODY FLUIDS

[0003] Field of the Invention

[0004] The present invention relates to a biosensor that enables the measurement of biomarkers related to bone metabolism from saliva samples. Non-invasive and cost-effective saliva sampling is used as an alternative to serum and urine samples used in traditional methods with the biosensor of the present invention. Bone formation marker Procollagen Type I N-terminal Propeptide (PINP) and bone resorption marker |3-C-terminal telopeptide ([3-CTX) biomarkers are measured with high sensitivity in saliva samples with the mentioned biosensor. This biosensor, which increases patient comfort and provides a fast, practical solution in clinical applications, facilitates the diagnosis and treatment monitoring of diseases related to bone metabolism.

[0005] State of the Art

[0006] The human skeletal system not only provides mechanical support but also plays a vital role in calcium-phosphate homeostasis, hematopoiesis and metabolic processes. However, the functionality of this system can be impaired over time by various diseases and pathological conditions. The most common bone disorders include osteoporosis, osteomalacia, Paget's disease, osteogenesis imperfecta, primary hyperparathyroidism, and bone metastases. In addition, conditions resulting from pharmacological agents such as glucocorticoid-induced osteoporosis are also common. Early detection of these diseases not only prevents bone mass loss and structural deformations but also reduces the risk of fractures and improves quality of life. For example, early diagnosis of osteoporosis allows patients to begin treatment before fractures, reducing morbidity and mortality rates. Detection of early biochemical changes in bone metabolism through biomarkers plays a critical role in preventing these disorders. In this regard, the widespread and effective use of biomarkers in diagnostic processes is of great importance for individual patient health and public health services.

[0007] Bone metabolism is maintained by a delicate balance of bone formation (osteoblastic activity) and bone resorption (osteoclastic activity) processes. The imbalance of theseprocesses constitutes the fundamental pathophysiology of pathological bone disorders. Biomarkers, as molecules reflecting bone metabolism, are an important tool in monitoring these processes. Biomarkers are basically divided into two categories: bone formation biomarkers (e.g., Procollagen Type I N-terminal Propeptide [PINP], osteocalcin, alkaline phosphatase) and bone resorption biomarkers (e.g., [3-C-terminal telopeptide [[3-CTX], deoxypyridinoline, tartrate-resistant acid phosphatase). Detection of these biomarkers enables the evaluation of dynamic changes in bone metabolism by non-invasive methods and provides critical information in the diagnosis, prognosis and treatment management of bone diseases.

[0008] Procollagen Type I N-terminal Propeptide (PINP) and [3-C-terminal telopeptide ([3-CTX) biomarkers play an important role in the diagnosis and monitoring of a number of bone disorders. PINP is a biomarker of Type I collagen synthesis and reflects osteoblastic activity, while [3-CTX represents collagen degradation and osteoclastic activity. These biomarkers are used in the diagnosis and management of some diseases. For example, PINP and [3-CTX are used as basic biomarkers in the evaluation of bone turnover in osteoporosis disease and in monitoring the response to osteoporosis treatment. Accurate and sensitive detection of these two biomarkers enables the determination of effective treatment strategies in common diseases such as osteoporosis. Paget's disease is characterized by increased bone turnover, and biomarkers Procollagen Type I N-terminal Propeptide (PINP) and [3-C-terminal telopeptide ([3-CTX) provide critical information in assessing the activity of this disease. Similarly, increased bone turnover resulting from the spread of malignancies to bone tissue, i.e. bone metastases, can be monitored by PINP and [3-CTX measurements. In addition, in conditions with increased osteoclastic activity, such as primary hyperparathyroidism, elevated [3-CTX levels and PINP measurements are an important tool for assessing the effects of the disease on bone metabolism.

[0009] Among the methods used for the detection of PINP and [3-CTX, immunoassay-based technologies such as ELISA (Enzyme-Linked Immunosorbent Assay), ECLIA (Electrochemiluminescence Immunoassay) and SIMOA (Single Molecule Array) stand out. Each method has its own advantages and disadvantages. For example, while ELISA offers high sensitivity and specificity, its long processing time and cost are disadvantages. Although ECLIA provides faster results, it is a costly option due to theneed for specialized equipment. SIMOA, on the other hand, has the capacity to detect low biomarker levels with its ultra-sensitivity, but can be used in limited laboratories and is quite costly. Body fluid-based biosensors, which stand out as a non-invasive alternative, increase patient comfort and facilitate sample collection; however, low biomarker concentrations in these liquid samples may negatively affect measurement sensitivity. In addition, lack of standardization in biomarker analysis limits the comparability of results between laboratories and creates problems in terms of reliability.

[0010] In the state of the art, a study by Kerschan-Schindl et al. focused on the detection of PINP and CTX biomarkers from body fluids [1], In this study, the detectability of bone turnover biomarkers such as PINP and [3-CTX in saliva was investigated and the usability of this method as a non-invasive alternative to serum analysis was evaluated. In addition, it also investigates the potential to assess bone metabolism by analyzing the correlation of salivary levels of these biomarkers with bone mineral density (BMD). However, this study on the detection of PINP and [3-CTX biomarkers from saliva has important shortcomings and limitations. First, the concentrations of PINP and [3-CTX in saliva are quite low compared to serum levels, limiting the sensitivity of the measurement. In addition, only one saliva collection method was used in the study, and this method was not adequately evaluated compared to other analysis techniques, weakening the reliability of the results. Other important shortcomings include the lack of a complete understanding of biomarker transfer mechanisms across the bloodsaliva barrier and the inability to detect high molecular weight proteins such as PINP. Issues such as molecular stability and biological variability in saliva complicate measurement processes. Finally, the lack of correlation between salivary biomarkers and serum levels indicates that current biomarkers are inadequate in saliva analysis, and this reveals the need for more effective biomarkers and improved analysis techniques.

[0011] The study conducted by Brescia et.al, which is in the state of the art, aims to evaluate the detectability of bone turnover biomarkers (including PINP and [3-CTX) in saliva samples and to assess the feasibility of using these biomarkers as a non-invasive alternative in clinical follow-up by analyzing their biological variability in saliva [2], However, the study reveals that the low concentrations of PINP and [3-CTX in salivalimit the measurement sensitivity, and the levels of these biomarkers in saliva do not show a significant correlation with clinical parameters such as bone mineral density (BMD). In addition, the lack of standardization of the methods used in saliva sampling and analysis processes, the risk of blood contamination affecting measurement accuracy, and the high intra-individual and inter-individual variability of biomarkers limit the generalizability and reliability of this method. It is stated that metrics such as critical difference (RCV) and individuality index (II) used in the clinical interpretation of salivary biomarkers are insufficient for PINP and [3-CTX and reference intervals are limited in terms of clinical evaluation. In addition, factors such as the gland from which saliva samples are collected, stimulation status, and blood contamination lead to large variability in measurements, thus creating a problem with the lack of standardization. In conclusion, the detection of PINP and [3-CTX biomarkers from saliva has several limitations in terms of monitoring bone metabolism with current methods, and better standardization, sensitivity-enhancing technologies, and development of alternative biomarkers are required.

[0012] Due to the limitations and inadequacies of current technical solutions, the lack of standard methods for detecting PINP and [3-CTX biomarkers from saliva samples, the fact that PINP and [3-CTX biomarkers are present in saliva at concentrations too low to be detected, and the need to resort to invasive methods for the detection of PINP and [3-CTX biomarkers, development in this field has become necessary.

[0013] Brief Description and Objects of the Invention

[0014] The present invention discloses a biosensor that allows the measurement of biomarkers associated with bone metabolism from saliva samples. This biosensor uses saliva sampling method, which provides a non-invasive and economical solution. The abovementioned biosensor can detect the biomarkers Procollagen Type I N-terminal Propeptide (PINP), a bone formation marker, and [3-C-terminal Telopeptide ([3-CTX), a bone resorption marker, from saliva with high sensitivity. This innovative technology, which increases patient comfort and provides a fast and practical solution in clinical applications, facilitates the diagnosis and treatment processes of diseases related to bone metabolism.

[0015] The main object of the present invention is to provide a biosensor that measures bone metabolism markers non-invasively. The invention aims to change the structure oftraditional invasive methods that reduce patient comfort and enables the use of saliva samples as a non-invasive biomaterial. In this regard, the biosensor of the present invention both increases patient comfort and facilitates the sampling process by sensitively detecting bone metabolism markers in saliva. The biosensor of the present invention enables the evaluation of bone formation and destruction processes by detecting Procollagen Type I N-terminal Propeptide (PINP) and [3-C-terminal Telopeptide ([3-CTX) biomarkers. The biosensor enables the detection of these biomarkers through biomarker-bioreceptor binding and holistic monitoring of bone metabolism processes by analysis of the obtained signals. In addition, the biosensor of the present invention makes healthcare services more efficient by accelerating diagnosis and treatment processes, while also providing advantages in early diagnosis and treatment monitoring processes with low-cost and sensitive analyses. The above mentioned biosensor provides results within minutes, thus enabling rapid analysis at the patient's bedside and contributing to clinical decision-making processes. This biosensor, which reduces laboratory costs with the help of the non-invasive and economical structure of the saliva sampling method, can detect bone metabolism markers even at low concentrations with its high sensitivity. In addition, this innovative biosensor, developed as a domestic and national technology, provides applicability in various fields such as osteoporosis, dental health, sports health and veterinary medicine, creating a portable and practical alternative for both home health monitoring and field studies.

[0016] Brief Description of Figures

[0017] Figure 1 : Cyclic voltammogram of the biosensor prepared for PINP determination Figure 2: PINP Optimum pH Chart

[0018] Figure 3: PINP Temperature Chart

[0019] Figure 4: Cyclic voltammogram of the biosensor prepared for [3-CTX determination Figure 5: [3-CTX Optimum pH Graph

[0020] Figure 6: [3-CTX Temperature Graph

[0021] Figure 7: Comparison of ELISA and autoanalyzer methods (A: Bland-Altman plot of PINP results between ELISA and autoanalyzer methods, B: Scatter plot of ELISA and autoanalyzer data, C: Regression plot of ELISA and autoanalyzer)Figure 8: Comparison of ELISA and biosensor methods (A: Bland-Altman plot of PINP results between ELISA and biosensor methods, B: Scatter plot of ELISA and biosensor data, C: Regression plot of ELISA and biosensor)

[0022] Figure 9: Comparison of autoanalyzer and biosensor (A: Bland-Altman plot of PINP results between autoanalyzer and biosensor methods, B: Scatter plot of autoanalyzer and biosensor data, C: Regression plot of autoanalyzer and biosensor)

[0023] Figure 10: Comparison of autoanalyzer and biosensor (A: Bland-Altman plot of PINP results between autoanalyzer and biosensor methods, B: Scatter plot of autoanalyzer and biosensor data, C: Regression plot of autoanalyzer and biosensor)

[0024] Figure 11: Comparison of ELISA and biosensor methods (A: Bland-Altman plot of [3-CTX results between ELISA and biosensor methods, B: Scatter plot of ELISA and biosensor data, C: Regression plot of ELISA and biosensor)

[0025] Figure 12: Comparison of autoanalyzer and biosensor (A: Bland-Altman plot of [3-CTX results between autoanalyzer and biosensor methods, B: Scatter plot of autoanalyzer and biosensor data, C: Regression plot of autoanalyzer and biosensor)

[0026] Figure 13: ROC curve comparing the results of the samples measured in the biosensor prepared for PINP determination with the autoanalyzer results

[0027] Figure 14: ROC curve comparing the results of the samples measured in the biosensor prepared for [3- CTX determination with the autoanalyzer results

[0028] Detailed Description of the Invention

[0029] The present invention relates to a biosensor that enables the measurement of biomarkers related to bone metabolism from saliva samples. This biosensor uses saliva sampling method, which offers an economical and user-friendly solution by adopting a non-invasive approach. The developed biosensor has the capacity to detect bone formation and resorption biomarkers such as Procollagen Type I N-terminal Propeptide (PINP) and [3-C-terminal Telopeptide ([3-CTX) found in saliva with high sensitivity. This technology, which increases patient comfort and enables rapid analysis with its innovative structure, makes the diagnosis and treatment processes of diseases related to bone metabolism more effective.

[0030] A biosensor for monitoring bone status indices, primarily detecting Procollagen Type I N-terminal Propeptide (PINP) and [3-C-terminal Telopeptide ([3-CTX) biomarkers from saliva samples, comprising• biosensor surface on which biomarker-specific bioreceptors are immobilized,

[0031] • a transducer that detects the biochemical signal resulting from the biomarker-bioreceptor binding and converts the same into an electrical or optical signal,

[0032] • an analysis unit that processes the signal from the transducer and produces digital data on the concentration of biomarkers,

[0033] • screen where data from the analysis unit can be displayed.

[0034] A biosensor working method for monitoring bone status indices, primarily detecting Procollagen Type I N-terminal Propeptide (PINP) and [3-C-terminal Telopeptide ([3-CTX) biomarkers from saliva samples, comprising the process steps of;

[0035] i. collecting saliva samples by non-invasive methods,

[0036] ii. placing saliva on the biosensor surface whose surface has been modified with gold nanoparticles, carbon nanotubes or graphene and bioreceptors have been immobilized thereon,

[0037] iii. Binding of PINP and [3-CTX biomarkers to bioreceptors immobilized on the biosensor surface,

[0038] iv. converting the biochemical signal resulting from the biomarker-bioreceptor binding into an electrical or optical signal in the transducer,

[0039] v. processing this signal converted in the transducer by the analysis unit to determine the biomarker concentration,

[0040] vi. presenting the analysis results to the user digitally on the screen.

[0041] Biomarkers related to bone metabolism are detected non-invasively using saliva with the help of the biosensor of the present invention. The biosensor of the present invention not only measures bone formation and resorption markers, but also monitors bone status indices by covering the entire metabolic state. This enables a more comprehensive assessment in the diagnosis and treatment processes of bone health-related diseases, thereby facilitating improvements to existing methods. In addition, this method provides significant advantages in terms of both cost and time by detecting substances with high sensitivity even at low concentrations. Analyses performed using traditional methods in the state of the art are generally invasive, requiring the collection of samples such as blood, urine, or tissue biopsies. This invasive process negativelyimpacts patient comfort and typically involves complex and costly analytical devices and long-term procedures. In addition, existing systems have significant limitations, such as low sensitivity or an inability to detect biomarkers at low concentrations. The invention addresses these shortcomings by detecting Procollagen Type I N-terminal Propeptide (PINP) and [3-C-terminal Telopeptide ([3-CTX) bone formation and resorption biomarkers with a non-invasive method. In particular, the use of an easily accessible and non-invasive matrix such as saliva increases patient comfort and accelerates the diagnosis and treatment processes. The biosensor of the present invention can provide rapid results in emergency situations with the ability to perform tests at the patient's bedside and facilitates the early diagnosis and follow-up of diseases related to bone metabolism.

[0042] The biosensor of the present invention comprises bioreceptor, transducer, signal processing and analysis unit and sampling and preparation system. The bioreceptor used in the invention consists of antibodies that recognize PINP and [3-CTX biomarkers and can specifically bind to these markers. This bioreceptor is immobilized on the biosensor surface and this surface selectively binds target biomarkers present in the saliva matrix. This selectivity ensures accurate and precise measurement. A transducer system is used that converts the biochemical signals resulting from the biomarker-bioreceptor binding into a measurable electrical or optical signal. This transducer is capable of making precise measurements to detect the presence and concentration of biomarkers. The signals generated by the transducer are processed in an electronic system and reflected on the screen of the signal processing and analysis unit in a format that the user can understand.

[0043] Non-invasive saliva collection is another important part of this system. Samples are easily taken and made ready for analysis by using saliva sampling containers or apparatus. This process is much faster and more user-friendly than invasive blood sampling procedures. These elements come together to provide the biosensor of the present invention with high sensitivity, portability and ease of use.

[0044] The working principle of the invention is based on the biosensor selectively detecting biomarkers in saliva. The analysis process begins with collecting the saliva sample using non-invasive methods. Saliva samples can be collected using specially designed collection cups or sterile swabs. Since the sampling process does not require anyinvasive procedures, it increases patient comfort and ensures that the process is carried out quickly and easily. Collected samples are applied to the biosensor surface. The biosensor surface is modified before saliva samples are placed thereon. During this modification, the electrochemical sensor surface of the biosensor is modified with nanomaterials (gold nanoparticles, carbon nanotubes or graphene). This modified surface provides increased bioaffinity for target biomarkers and optimizes the conductivity of the sensor. Said modified surface allows for higher binding capacity and faster response of biomarkers with its large surface area; it also reduces the detection limit by increasing electrical conductivity. Bioreceptors (specific antibodies) immobilized on said surface bind to the PINP and [3-CTX biomarkers found in saliva. Said bioreceptors are specific structures for target biomarkers such as antibodies, PINP and CTX immobilized on the electrode surface and have high binding capacity. Said bioreceptors provide high specificity and sensitivity and minimization of false positive / negative results.

[0045] Bioreceptors are the most critical components of biosensors and interact specifically with the target biomarker. These bioreceptors have the ability to recognize biomolecules. These structures consist of enzymes, antibodies, nucleic acids (DNA, RNA), cells or tissue and aptamers. The biomarker-bioreceptor interaction creates a biochemical signal, which is converted into an electrical or optical signal via the transducer. For example, this signal is measured as a current change in electrochemical sensors, while in optical sensors it can be detected as a light wavelength change. The biosensor of the present invention quickly analyzes this signal and transmits the same to the signal processing unit. This unit converts signals into digital format and displays them in a user-friendly interface. This process is usually completed within minutes, allowing for rapid diagnosis and treatment decisions. In addition, since the biosensor device is portable, testing can be done at the patient's bedside and real-time results can be obtained. The detection of biomarkers, especially at low concentrations, proves the high sensitivity level of the biosensor and its effectiveness in the saliva matrix.

[0046] The biosensor of the present invention performs analysis by detecting biomarkers in saliva. The analysis process takes place by binding biomarkers to bioreceptors and converting this binding into a signal via a transducer. The biochemical signal producedduring the biomarker-bioreceptor interaction is measured to determine the presence and concentration of the biomarkers. This signal is processed by sensitive electronic units and precise results regarding the concentration of biomarkers are obtained. The LOD (Limit of Detection) value of the analysis expresses the ability of the biosensor to detect the lowest biomarker concentrations. This biosensor can detect low concentrations that current conventional methods cannot reach. In particular, this biosensor can successfully detect biomarkers when existing methods for the CTX biomarker cannot exceed the LOD limits. Low LOD values offer a significant advantage in terms of early diagnosis and treatment follow-up.

[0047] In order to determine the PINP working range of the biosensor which is the subject of the invention, bovine serum albumin-Gelatin (BSA-Gelatin) was used and one end of the poles in the biosensor was fixed to the electrode and the other end was immersed in the measurement cells (Figure 1). BSA-gelatin is a frequently used biomaterial in biosensors. BSA facilitates the immobilization of desired biomolecules on the surface with its biocompatibility and protein adsorption properties. Gelatin stabilizes these biomolecules, ensuring that the bioactive layer gains a homogeneous and stable structure. This structure enables specific binding and detection of target molecules such as PINP. They also support the preservation and long-term use of bioreceptors on the electrode surface.

[0048] In the biosensor, the use of a fixed structure where one end of the electrodes is connected to the electrode and the other end is immersed in the measurement cell also means that the electrodes function as working electrodes in this section and represent the active surface that detects biomolecule binding. Immersing the other ends into the measuring cells completes the circuit necessary for electrochemical measurement by providing ionic conductivity in the solution. This structure ensures linear and reliable results in electrochemical measurements such as voltammograms (current-potential graphs). By examining the change between the potential (V) and current (pA) values, the Anodic Peak Current (oxidation signal) or Cathodic Peak Current can be evaluated. The detection range and limits of PINP are determined based on the concentration dependence of this peak. In addition, measurements were carried out using phosphate buffers to determine the working buffer and optimum pH value (Figure 2). Measurements made in phosphate buffer solutions are critical fordetermining the optimal operating conditions of the biosensor. The optimum pH value maximizes PINP detection performance by increasing the strength of the electrochemical signal. This step ensures that the biosensor is optimized in terms of sensitivity, accuracy, and stability.

[0049] Temperature optimization experiments are a critical step in determining the conditions under which the biosensor operates at maximum efficiency. The 25°C - 40°C range provides the most suitable conditions for the stability of bioreceptors and their interaction with the target molecule, while sensor performance begins to decline above 40°C. These findings provide a fundamental reference for developing working protocols to enhance the stability and sensitivity of the biosensor. When current difference values were measured using a biosensor in the 20°C - 60°C range to determine the optimal temperature value for these measurements and graphically plotted against temperature, the 25-40°C range was found to be optimal in terms of binding efficiency. The maximum temperature value was determined to be 40°C (Figure 3).

[0050] Similarly, in order to determine the working range of CTX, BSA-Gelatin was used and one end of the poles in the biosensor was fixed to the electrode and the other end was immersed in the measurement cells and the cyclic voltammogram was taken (Figure 4). Measurements were carried out using phosphate buffers to determine the working buffer and optimum pH value of CTX (Figure 5). In studies to determine the optimum temperature value to be used in said measurements, when measurements are taken with the biosensor in the range of 20°C-60°C and the current difference values are shown graphically against the temperature, it is observed that it is more stable in the range of 20-30°C. The maximum temperature value was determined to be 30°C (Figure 6).

[0051] The biosensor which is the subject of the present invention has been compared with the ELIZA and autoanalyzer methods in the state of the art. In Figure 7, the fact that 98.67% of the variation in either of the ELIZA and autoanalyzer methods can be explained by the other method is demonstrating the agreement between both methods. The results shown in Figure 8 indicate that 90.79% of the variation in either the Eliza or biosensor methods can be explained by the other method, demonstratingagreement between the two methods. Finally, the fact that 98.13% of the variation in either the autoanalyzer or biosensor method can be explained by the other method, as shown in Figure 9, demonstrates agreement between the two methods. In the method developed for the quantification of PINP and [3-CTX in the invention, linearity, precision, recovery limits were determined and a comparison study was carried out with sensitivity, specificity and other traditionally used methods to test the usability of the biosensor method of the present invention. The biosensor method can be used as an alternative analytical method for measuring saliva PINP and [3-CTX levels, offering advantages such as lower cost, rapid results, high sensitivity, and information validity compared to chemiluminescence immunoassay and ELISA methods. The biosensor of the present invention is presented as an accurate, rapid, and economical alternative analytical method for the detection of PINP and [3-CTX levels. The high accuracy achieved with current techniques (90.79%-98.13%) demonstrates that the biosensor can be used safely in clinical and laboratory applications. This method, particularly in saliva samples, offers non-invasive, practical, and accurate measurement capabilities, making it a promising candidate for widespread future application.

[0052] The present invention, a non-invasive saliva sampling method, eliminates the need for invasive blood or urine sampling and significantly improves patient comfort. It offers an ideal solution, especially for sensitive groups such as children and the elderly. Secondly, the biosensor's high sensitivity level enables it to detect biomarkers at low concentrations. This situation plays a critical role, particularly in the early diagnosis and treatment follow-up of diseases related to bone metabolism. In addition, the biosensor enables rapid decision-making in clinical applications with its ability to provide results within minutes. Compared to traditional methods that take hours or days to analyze, this ability to deliver fast results is a major advantage. Tests can be performed at the patient's bedside and real-time data can be obtained with the help of its portable design. This situation speeds up the diagnosis and treatment process in emergencies and ensures more effective patient management.

[0053] Early diagnosis and monitoring of bone health-related diseases such as osteoporosis, bone metabolism disorders, Paget's disease, osteomalacia, and rickets are now possible with the help of the present invention. This enables diseases to be treated before they progress and allows treatment processes to be monitored. In addition, therapid detection of adverse changes in bone metabolism plays an effective role in disease management by enabling early initiation of treatment.REFERENCES

[0054] 1. Kerschan-Schindl K, Boschitsch E, Marculescu R, Gruber R, Pietschmann P.

[0055] “Bone turnover markers in serum but not in saliva correlate with bone mineral density”. Scientific Reports 10:11550. (2020).

[0056] 2. Brescia V, Cazzolla AP, Fontana A, Varraso L, Capobianco C, Lovero R, Lo Muzio L, Dioguardi M, Faienza MF, Crincoli V, Di Serio F, Ciavarella D. “Bone biomarkers measured on salivary matrix: study of biological variability in a cohort of young subjects”. Appl. Sci.13, 10234. (2023)

Claims

CLAIMS1. A biosensor for monitoring bone status indices, primarily detecting Procollagen Type I N-terminal Propeptide (PINP) and [3-C-terminal Telopeptide ([3-CTX) biomarkers from saliva samples, comprising• biosensor surface on which biomarker-specific bioreceptors are immobilized,• a transducer that detects the biochemical signal resulting from the biomarker-bioreceptor binding and converts the same into an electrical or optical signal,• an analysis unit that processes the signal from the transducer and produces digital data on the concentration of biomarkers, and • screen where data from the analysis unit can be displayed.

2. A biosensor working method for monitoring bone status indices, primarily detecting Procollagen Type I N-terminal Propeptide (PINP) and [3-C-terminal Telopeptide ([3-CTX) biomarkers from saliva samples, comprising the process steps of;i. collecting saliva samples by non-invasive methods,ii. placing saliva on the biosensor surface whose surface has been modified with gold nanoparticles, carbon nanotubes or graphene and bioreceptors have been immobilized thereon,iii. Binding of PINP and [3-CTX biomarkers to bioreceptors immobilized on the biosensor surface,iv. converting the biochemical signal resulting from the biomarker- bioreceptor binding into an electrical or optical signal in the transducer, v. processing this signal converted in the transducer by the analysis unit to determine the biomarker concentration, andvi. presenting the analysis results to the user digitally on the screen.

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