Review of single-molecule immunoassays: Non-chip and on-chip Assays.

Enhancing the sensitivity of immunoassays is an important requirement in the field of immunology, especially in light of rapid developments in genetic testing, making the detection of low-abundance protein biomarkers crucial. Therefore, innovations in highly sensitive immunoassays are imperative. This demand has led to the emergence of single-molecule immunoassays (SMIs), driving advancements in early diagnostic techniques, and ushering in a new era of immunoassays. This review begins by tracing the development of immunoassays and offers a detailed discussion of SMI technology across two distinct pathways: non-chip (SMI without microfluidic chips) and on-chip (SMI with microfluidic chips). Furthermore, we evaluated and compared these methods using two pathways. In addition, this review discusses the significance of SMI techniques in the diagnosis of various diseases and their current applications in laboratory and clinical settings. The progress of SMI in commercial applications and suggestions for innovative directions are also summarized. Despite the considerable potential of SMI, these technologies face challenges in practical application, particularly in developing countries and economically disadvantaged regions. The final section of this review addresses the challenges and prospects of these technologies.