Semisynthetic Bioluminescent Sensor Proteins for Direct Detection of Antibodies and Small Molecules in Solution

Abstract

Single-step immunoassays that can be performed directly in solution are ideally suited for point-of-care diagnostics. Our group recently developed a new platform of bioluminescent sensor proteins (LUMABS; LUMinescent AntiBody Sensor) that allow antibody detection in blood plasma. Thus far, LUMABS has been limited to the detection of antibodies recognizing natural peptide epitopes. Here, we report the development of semisynthetic LUMABS sensors that recognize nonpeptide epitopes. The non-natural amino acid para-azidophenylalanine was introduced at the position of the original antibody-recognition sites as a chemical handle to enable site-specific conjugation of synthetic epitope molecules coupled to a dibenzocylcooctyne moiety via strain-promoted click chemistry. The approach was successfully demonstrated by developing semisynthetic LUMABS sensors for antibodies targeting the small molecules dinitrophenol and creatinine (DNP-LUMABS and CR-LUMABS) with affinities of 5.8 pM and 1.3 nM, respectively. An important application of these semisynthetic LUMABS is the detection of small molecules using a competitive assay format, which is demonstrated here for the detection of creatinine. Using a preassembled complex of CR-LUMABS and an anti-creatinine antibody, the detection of high micromolar concentrations of creatinine was possible both in buffer and in 1:1 diluted blood plasma. The use of semisynthetic LUMABS sensors significantly expands the range of antibody targets and enables the application of LUMABS sensors for the ratiometric bioluminescent detection of small molecules using a competitive immunoassay format.

Recent Progress in Optical Sensors for Biomedical Diagnostics

“…It has been used to develop microfluidic paper-based assays for simultaneous nanomolar antibody detection in whole blood (Figure 10e) [202] using a novel platform called LUMinescent AntiBody Sensor (LUMABS) [203]. LUMABS with furimazine substrates compares well against ELISA [204] and is compatible with several different antibodies [205]. Furimazine systems have also been employed for intracellular ATP sensing [206], voltage indication in live cells [207] as well as metal ion detection and bioimaging [207][208][209].…”

Coelenterazine-Dependent Luciferases as a Powerful Analytical Tool for Research and Biomedical Applications

“…Recently, nanoBRET sensor platforms for detection of small-molecule drugs (LUCiferase-based Indicators of Drugs, LUCIDs) and antibodies (LUMinescent AntiBody Sensor, LUMABS) have been developed by the groups led by M. Merkx. Sensing mechanisms of LUCIDs and LUMABS sensors are described in detail in [ 212 , 213 ]. These sensors allow measurement of antibodies directly in blood plasma, eliminating the need for the complex liquid handling steps associated with conventional immunoassays [ 214 ].…”

“…Bioluminescence can be easily registered with a smartphone digital camera [ 215 ]. The developed sensors were applied to detect anti-HIV1, anti-hemagglutinin, and anti-dengue virus type I antibodies [ 212 , 214 , 215 ] as well as methotrexate, dinitrophenol, creatinine, and theophylline molecules [ 212 , 213 , 216 ]. In principal, the sensor’s modular design allows the easy adaptation to many other analytes.…”

RAPPID: a platform of ratiometric bioluminescent sensors for homogeneous immunoassays

“…The Johnsson lab pioneered the development of semisynthetic sensor proteins based on bioluminescence resonance energy transfer (BRET) for the detection of small-molecule drugs such as methotrexate and digoxin, and important metabolites such as NAD + and NADPH [10][11][12][13] . Alternatively, the LUMABS platform was developed to enable detection of specific antibodies directly in blood plasma with the use of a smartphone camera [14][15][16][17] . Both sensor platforms employ ratiometric bioluminescent detection, which allows quantitative measurements and enables integration in paper-and thread-based analytical devices for low-cost point-of-care detection 11,18,19 .…”