Immunoassays utilizing the antigen–antibody reaction are an essential technique for measuring trace amounts of target molecules due to high specificity and strong affinity of antibodies. Therefore, it is possible to specifically measure the target molecules from a sample containing complex components with simple pretreatment. Because of this feature, immunoassays have been used in medical testing, diagnosis, and mass screening. Then, aptamers are single-stranded DNA and RNA oligonucleotides that can fold into unique secondary or ternary structures, such as hairpin, bulge, pseudoknot, and G-quartet. They have the potential to capture target molecules in a manner similar to antibodies [1]. In this highlight, recent relevant reports have been introduced.

Iida and co-worker reported a light-induced acceleration of antigen–antibody reaction for trace amounts of proteins in microchannel by the laser irradiation [2]. Based on the proposed system, membrane proteins were detected at the attogram level only 3 min after laser irradiation. This is approximately 100 times more sensitive and shorter than the conventional method, which requires several hours. A reusable fluorescent biosensor for rapid and sensitive quantitative detection of trace pollutants was developed [3]. The proposed biosensor was integrated with a smartphone to enable on-site analysis, and a competitive immunoassay for bisphenol A and norfloxacin was measured in 15 min in a microfluidic system. A rapid quantitative microarray system for multiple SARS-CoV-2 (COVID-19) mutant protein-specific antibodies was developed using photo-reactive polymers [4]. Viral proteins were immobilized on a plastic plate with two types of photo-reactive polymers, phenyl-azide, and poly-oxy-ethylene. The automated assay was possible using 5 µL whole blood in 8 min by chemiluminescence detection. An ultrasensitive nanobody-based immunoassay was developed to monitor Tetrabromobisphenol A (TBBPA) as an environmental pollutant in sediment [5]. Nanobodies are immunoglobulins produced in vivo from camelid mammals, which consist only of heavy chains without light chains. First, the anti-TBBPA nanobody was conjugated with nanoluciferase, and a one-step bioluminescent enzyme immunoassay (BLEIA) was developed with high sensitivity for TBBPA. To further improve the performance of this one-step BLEIA, a self-assembling linker was inserted between the nanobody and nanoluciferase. The proposed dual-functional reagent significantly improves the sensitivity of one-step BLEIA due to its superior binding and signal amplification capabilities. A FRET-based homogeneous immunoassay was reported to monitor tetracycline in the environment [6]. In a homogeneous system, the Eu3+–cryptate complex and Cy5 were used as fluorescence donor and acceptor, respectively. Their reagents were conjugated with antigen and antibody, and FRET was observed with the formation of antigen–antibody complexes. The addition of hydrogen peroxide (H2O2) is an important step in the traditional detection system of immunoassays. A strategy of self-supplying H2O2 by copper peroxide nanodots encapsulated in metal–organic frameworks was proposed in an immunoassay for bisphenol A [7]. The improved ELISA was applied in a portable lab-in-a-tube device integrated with a smartphone sensing platform. Open sandwich assays for small molecules using antibody or aptamer were reported [8, 9]. Non-competitive assay systems of small antigens coupled with Au NP-based colorimetric detection were developed using variable region fragments (VH and VL) separated from a single antibody, or antigen-specific split aptamer. Finally, a label-free fluorescent aptamer sensor designed to fold triple-helix DNA and G-quadruplex was proposed for detection of carbohydrate antigen (CA15-3) [10]. The triple-helical DNA containing antigen-specific aptamer is locked state, and when the antigen as a key reacts with the aptamer, G-rich DNA is released to fold G-quadruplex, which is intercalated with N-methylisoporphyrin IX (NMM) and exhibits fluorescence.

It is strongly expected that immunoassays coupled and integrated with various techniques will be further developed in near future.

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