Osteoporosis is well-known as a disease causing the continuous loss of bone mineral density (BMD) which eventually leads to the deterioration of bones and increased risk of bone fracture. Globally over 200 million women have been affected and approximately 8.9 million fractures have been reported due to this disease [1
]. Currently the diagnosis of osteoporosis is highly dependent on the X-ray imaging of 2-D structure of bones, for example, bone densitometry or dual-energy X-ray absorptiometry (DEXA), which can be further interpreted as density of bone minerals [2
]. However, the progress of osteoporosis and frequent monitoring of efficacy of treatment using medicine is still dependent of changes in levels of the related bone turnover markers of bone formation and resorption which are tightly linked in BMD conditions [3
]. In addition, immunoassays for bone turnover markers provide complementary management of dynamic changes of BMD due to its easiness to access, convenient, and no risk of radiation. Among the number of related biomarkers, Osteocalcin (OC) and the N-terminal propeptide of procollagen type I (P1NP) have been used as a bone formation biochemical marker and the C-terminal cross-linked telopeptide of type I collagen (CTx) has been commonly used as bone resorption marker. The level of serum osteoporosis was measured to be relatively higher in post-menopausal woman with osteoporosis than the normal [8
]. Due to its high affinity to calcium, the increased level of osteocalcin present in serum is attributable to the deficiency of calcium and phosphorous on which the bone formation process is highly dependent [10
]. P1NP, the product of osteoblast, was also widely recommended as a bone formation marker because of its stability in serum and less variability between serum samples [11
]. The level of P1NP in serum is highly related with the rate of the bone formation because it is released before it is incorporated into the extracellular matrix. The fragment from C-telopeptide of type I collagen, CTx, has been studied for monitoring the resorption rate during osteoclasts. In contrary to the aforementioned bone formation markers, CTx is an indicator of the rate of degradation of the synthesized collagen. Therefore, the quantification of each of the bone formation and resorption markers has been used as indirect indicators of bone related diseases including osteoporosis. Other than those well-known markers, Parathyroid Hormone (PTH) is known as the regulator of serum calcium and phosphate metabolism in bone and can act as an osteoporosis-related biomarker in parathyroid disorders. In this regard, we believe that membrane-based electrochemical detection technology is a promising candidate for the simple method of multiplexed immunoassays. Electrochemical immunoassays have been studied in many ways to increase sensitivity, cost effectiveness, efficiencies in pretreatment, amplification, and simple handling [13
]. Recently, the electrochemical immunoassay of osteoporosis has been studied regarding the sensing electrode carrying gold nanoparticles [17
] or MoS2
-graphene composite [18
]. In this study, we developed a simple electrochemical immunoassay based on the microwave-mediated immobilization of antibodies and the antibody–antigen reaction. The use of a membrane for pretreatment and a custom-made electrode for measurement provide low cost, ease of use, and rapid preparation for the immunoassay of target proteins because the role of the membrane is for the pretreatment or formation of the antigen–antibody complex and that of the electrode is used only for measurement. Therefore, the same electrode can be used multiple times, which provides an advantage over the most commonly used electrodes that require target antibodies to be immobilized on it. A target conjugated electrode cannot be used more than once. In addition, reduced preparation time from immobilization of capture antibodies to last step of measurements gives better expandable applications than the use of precoated cartridges in that the kinds of target needed to be measured at the field can be easily applied.
The objectives of present work are: (i) to prepare a membrane disk (MD) in which target antibodies are placed; (ii) to fabricate custom-made electrodes to perform electrochemical assays via differential pulsed voltammetry (DPV); and (iii) to perform a multiplexed detection of such antigens and evaluate the method through comparison with results obtained from clinical instrumentation.