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.

在护理点 (POC) 环境中以单一 pg/ml 浓度检测人类免疫缺陷病毒 (HIV) p24 蛋白非常重要，因为它可以促进急性 HIV 感染诊断，其检测灵敏度接近基于实验室的检测。然而，最突出的 POC 诊断平台侧流免疫测定 (LFA) 的检测限 (LOD) 低于实验室蛋白质检测方法，如酶联免疫吸附测定 (ELISA)。在这里，我们报告了热对比放大 (TCA) LFA 的开发和优化，该 LFA 将允许在 POC 时对掺入人血清中的 8 pg/ml p24 蛋白进行超灵敏检测，接近实验室测试的 LOD。为了实现这一目标，我们进行了以下几项创新：(a) 基于特异性与非特异性结合比 (BR) 为 LFA 设计定义新的量化品质因数；(b) 在 TCA LFA 设计的系统优化中使用不同尺寸和形状的金纳米粒子 (GNP)；(c) 为 TCA LFA 设计探索新的激光波长和功率机制。首先，我们通过使用 TCA 阅读器定量测量 BR 来优化膜和运行缓冲液的封闭缓冲液。当用粒子的等离子体共振波长的激光照射时，TCA 读取器解释膜内 GNP 的热信号（即温度）。与传统的视觉检测（即颜色强度）相比，该过程导致 GNP 的检测和定量更高。此外，我们研究了激光功率（30、100、200 mW）的影响，GNP 大小和形状（30 和 100 nm 金球，150 nm 金-二氧化硅壳）和激光波长（532、800 nm）。将这些创新应用于新的 TCA LFA 设计，我们证明了 100 nm 球体和 100 mW 532 nm 激光可提供最佳性能（即 LOD = 8 pg/ml）。该 LOD 明显优于当前的比色 LFA，并且处于基于实验室的 p24 ELISA 的范围内。总之，p24 蛋白的这种 TCA LFA 显示出在 POC 环境中检测急性 HIV 感染的希望。该 LOD 明显优于当前的比色 LFA，并且处于基于实验室的 p24 ELISA 的范围内。总之，p24 蛋白的这种 TCA LFA 显示出在 POC 环境中检测急性 HIV 感染的前景。该 LOD 明显优于当前的比色 LFA，并且处于基于实验室的 p24 ELISA 的范围内。总之，p24 蛋白的这种 TCA LFA 显示出在 POC 环境中检测急性 HIV 感染的希望。