Development and optimization of thermal contrast amplification lateral flow immunoassays for ultrasensitive HIV p24 protein detection

Li Zhan,Jeff Johnson,Ae Youngpairoj,Timothy Granade,Yilin Liu,Vickie Sullivan,John Bischof,Xierong Wei

doi:10.1038/s41378-020-0168-9

Abstract

Detection of human immunodeficiency virus (HIV) p24 protein at a single pg/ml concentration in point-of-care (POC) settings is important because it can facilitate acute HIV infection diagnosis with a detection sensitivity approaching that of laboratory-based assays. However, the limit of detection (LOD) of lateral flow immunoassays (LFAs), the most prominent POC diagnostic platform, falls short of that of laboratory protein detection methods such as enzyme-linked immunosorbent assay (ELISA). Here, we report the development and optimization of a thermal contrast amplification (TCA) LFA that will allow ultrasensitive detection of 8 pg/ml p24 protein spiked into human serum at POC, approaching the LOD of a laboratory test. To achieve this aim, we pursued several innovations as follows: (a) defining a new quantitative figure of merit for LFA design based on the specific to nonspecific binding ratio (BR); (b) using different sizes and shapes of gold nanoparticles (GNPs) in the systematic optimization of TCA LFA designs; and (c) exploring new laser wavelengths and power regimes for TCA LFA designs. First, we optimized the blocking buffer for the membrane and running buffer by quantitatively measuring the BR using a TCA reader. The TCA reader interprets the thermal signal (i.e., temperature) of GNPs within the membrane when irradiated by a laser at the plasmon resonance wavelength of the particle. This process results in higher detection and quantitation of GNPs than in traditional visual detection (i.e., color intensity). Further, we investigated the effect of laser power (30, 100, 200 mW), GNP size and shape (30 and 100 nm gold spheres, 150 nm gold-silica shells), and laser wavelength (532, 800 nm). Applying these innovations to a new TCA LFA design, we demonstrated that 100 nm spheres with a 100 mW 532 nm laser provided the best performance (i.e., LOD = 8 pg/ml). This LOD is significantly better than that of the current colorimetric LFA and is in the range of the laboratory-based p24 ELISA. In summary, this TCA LFA for p24 protein shows promise for detecting acute HIV infection in POC settings.

Highlights

There were 1.7 million new human immunodeficiency virus (HIV) infection cases, leading to 37.9 million people infected in total by the end of 2018, predominantly in resource-limited areas[1]
To maximize the performance of thermal contrast amplification (TCA) lateral flow immunoassays (LFAs), we studied the effect of laser power, laser wavelength, and gold nanoparticles (GNPs) size and shape on the limit of detection (LOD)
We showed that 8 pg/ml p24 protein spiked in human serum can be detected with TCA LFAs using 100 nm gold spheres and a 100 mW 532 nm laser

Summary

NSB between GNP conjugates and capture antibody

The poor limit of detection (LOD) for analytes of conventional LFAs (i.e., on the order of ng/ml) restricts their ability to detect p24 protein during acute infection (i.e., on the order of pg/ml); more complicated laboratory tests such as enzyme-linked immunoassay (ELISA) are required[11,12]. Inside the test line area, additional NSB between antibody-GNP conjugates and the capture antibody leads to higher signals for negative samples. Analytes can nonspecifically bind to the nitrocellulose membrane, which could capture GNP conjugates To reduce these NSBs, blocking of the nitrocellulose membrane, surface blocking of GNP conjugates and optimization of the running buffer need to be performed. After testing various membrane blocking buffers, researchers choose those with “clean” backgrounds and strong test and control lines based on the visual color intensity for further iterations This approach fails to provide definitive conclusions and rule out cases with subvisual signals, leading to excessive experimentation and suboptimal final performance. This TCA LFA with ultrasensitive p24 protein detection holds great promise in improving acute HIV infection diagnosis at POC

Results and discussion

Average TL signal

Materials and methods