Point-of-Care Solution for Osteoporosis Management

What is osteoporosis? Why early diagnosis plays an important role in lowering the burden of this disease on the patient and society? How does a point-of-care device improve osteoporosis management? This chapter deals with the answers to these questions.

This chapter provides the results of a through literature review on three main topics:

1. Biochemical markers of bone turnover and their role in osteoporosis diagnosis.

2. Bone biosensors: knowing the present and predicting the future.

3. Protein immobilization strategies for biosensing purposes

This chapter aims to introduce the scientific and technical principles applied in the experimental chapters of this work. Three major subjects will be discussed.

• The first subject is an overview on biosensors and its types. A special focus is dedicated to electrochemical sensors.

• The second part deals with different electrochemical techniques. The definition of these techniques will be described.

• The last section embraces main principles of microfluidic devices.

As the first step, through the inherent interaction between gold nanoparticles (AuNPs) and antibody biomolecules, a novel gold nanoprobe was developed to be used in the non-enzymatic electrochemical immunoassay. This one-pot method not only provides a simple method for loading high-content antibody on nanoparticles but also greatly improves the repeatability and controllability of the nanoprobe preparation. Combined with a disposable electrode, it could be used for a single/multiplexed electrochemical immunosensing method. In other words, using this nanoprobe, high throughput instrumentation applying (single/multiplexed sensors) could be developed to screen a variety and vast quantities of samples for choice markers.

Since most electrochemical reactions occur in close proximity of the electrode surface, the electrodes play a crucial role in the performance of electrochemical biosensors. In other words, a biosensor is created on a solid electrode surface by chemically or electrostatically attaching bio(macro)molecules such as proteins and nucleic acids. If the surface is chemically modified, the biochemical molecules bind in a layer such that they cover the electrode surface. This layer is also known as the “recognition layer,” as its molecules are specific to a target in the analyte. When the electrode is placed into a sample solution, the analyte and recognition layer interact, and an electrical signal, characteristic of the analyte, is obtained [44].

This chapter therefore explains the attempts made to develop the most appropriate electrode that fulfilled the above-mentioned characteristics for our purposes and reports its reproducibility, reusability, and stability characteristics.

As mentioned in Chap. 3, while immunoassays have an extensive application in both clinic and research, the immunosensor concept is yet to be implemented in routine diagnostics [23, 24]. There are certain issues in this regard, the most important of which is the coupling chemistry of proteins on surfaces, which is critical for optimal functioning. In other words, the optimal orientation of Ab molecules on the surface is of great importance in selective and sensitive detection in a reproducible way without affecting its activity [25]. The relative lack of long-term stability of biological molecules is the most important limitation in commercializing biosensors.

Therefore, this chapter deals with the immobilization of two main BTMs (Oc and CTX) on the glass-based gold electrode fabricated as mentioned in Chap. 5 and coated with AuNPs. To our knowledge, a biosensor to assess serum levels of Oc has not been developed before. A stepwise protocol to develop these sensors is explained here.

As nowadays, microfluidic technologies are widely employed in developing point-of-care diagnostics to address global health issues because of their potential advantages of low sample and reagent consumption, high throughput and sensitivity, large surface-to-volume ratio, and other benefits related to miniaturization, we decided to integrate our electrochemical chip into a microfluidic system. The fabrication of microfluidic channels is commonly costly and requires laboratory-intensive cleaning, photolithography, and etching or baking steps in cleanroom environments, making it difficult to modify. Besides, proper channel enclosure without deforming small features or without clogging of the channel during the bonding process is challenging. We therefore developed a cheap, reliable, and rapid method for the fabrication of microfluidic channels using double-sided tapes, enabling not only highly uniform cross-sectional dimensions along the channels but also proper adhesion in hybrid systems, composed of different layers.

Unstable immobilization of antibody on the electrode surface and non-specific binding are the main factors that determine the detection limit for an immunoassay [3]. It is known that adsorption of proteins onto bulk metal surfaces leads to their denaturation and loss of bioactivity [4]. Therefore, as mentioned earlier, the gold electrodes were modified with AuNPs to overcome these concerns. The AuNP layer also improved the signals because of their large surface area and efficient electron conducting features. This chapter deals with surface modification of electrodes used in Osteokit and its validation process.

We demonstrated the implementation, feasibility, and specificity of this platform (Osteokit) in assaying serum levels of Oc and CTX. In other words, the developed system was sensitive and specific for serum Oc and could detect serum levels of the marker within the range of 2.5–100 ng/mL. This is while the normal reference of the marker is 9–42 ng/mL, suggesting that the system can acceptably detect Oc. Similarly, CTX levels were successfully measured from 1 to 2500 pg/mL. This is while the normal reference of the marker is 50–450 pg/mL, suggesting that the system can acceptably detect CTX.

Our system showed no cross-reactivity for other biomarkers (b-CrossLaps and parathyroid hormone (PTH) for Oc system and Oc and PTH for CTX system). The good correlation between the ECLIA and Osteokit showed that they can be used in the clinically relevant range and other macromolecules available in serum do not affect our results.

To our knowledge, this is the first such device fabricated to measure bone turnover markers (BTMs). Our results also showed the sensitivity of Osteokit to be comparable with the current state of the art, ELISA, and electrochemiluminescence. The developed electrochemical chips showed acceptable sensitivity, specificity, stability, and reliability, thus providing a highly promising potential for clinical applications. The total assay time for this system is about 10 min (loading of antigen, incubation time, flushing with PBS, and testing), while ECLIA needs several hours to be performed.

In this work, only two major BTMs were studied, but the assay system could easily be adapted to other biomarkers in future experiments. These results require further validation but may suggest a direction towards which the field of diagnostic biomarkers is moving.