In this study, we report the development of a paper-based platform with low amount of reagents for the detection of the histidine-rich protein 2 by an enzyme-based colorimetric assay aiming for the early diagnosis of Plasmodium falciparum malaria. A CO₂ laser cutter was used for the fabrication of the device in a simple layout suitable to perform the lateral flow assay, which consisted in a region for sample addition, a detection zone and an absorbent pad, all of which were linked by microfluidic channels. Investigations related to the performance of the sandwich immunoassay in the nitrocellulose membrane shown that the blocking and washing steps are essential to promote efficient antigen-antibody recognition and to generate reliable results, avoiding misinterpretations. Several parameters were also evaluated and the experimental conditions were optimized: concentration and incubation time with the capture antibody (50 μg mL⁻¹ and 5 minutes, respectively); BSA solution at 1.5% (w/v) containing 0.1% (v/v) Tween 20 for blocking the nitrocellulose and 10 minutes as the incubation time; 10 mmol L⁻¹ Tris-HCl buffer solution at pH 7.4 containing 0.15 mol L⁻¹ NaCl and 0.1% (v/v) Tween 20 for washing the device before adding the ready to use TMB substrate at the detection zone. The system demonstrated a good sensitivity and detectability reaching a visual and calculated limit of detection of 5.0 ng mL⁻¹ (65 parasites μL⁻¹) and 4.5 ng mL⁻¹ (59 parasites μL⁻¹) respectively, a clinically relevant concentration that should be sufficient to identify the disease in the appearance of the first symptoms and with the advantage of not needing to use nanostructures or other strategy for signal amplification beyond the enzyme.

中文翻译：

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一种简单，灵敏且成本低廉的纸质设备，使用基于酶的比色测定法可对恶性疟原虫疟疾进行早期诊断，且化学品含量低

在这项研究中，我们报告了一种基于纸质平台的开发，该平台具有少量试剂，可通过基于酶的比色测定法检测富含组氨酸的蛋白质2，目的是早期诊断恶性疟原虫疟疾。一氧化碳₂激光切割机以适合于执行侧向流动测定的简单布局用于设备的制造，其包括用于样品添加的区域，检测区域和吸收垫，所有这些区域均通过微流体通道连接。有关硝酸纤维素膜中夹心免疫测定性能的研究表明，阻断和洗涤步骤对于促进有效的抗原-抗体识别和产生可靠的结果，避免误解至关重要。还评估了几个参数并优化了实验条件：浓度和与捕获抗体（50μgmL ^-1的孵育时间）和5分钟）；1.5％（w / v）的BSA溶液含有0.1％（v / v）的Tween 20用于封闭硝酸纤维素，孵育时间为10分钟；含有0.15 mol L ^-1 NaCl和0.1％（v / v）Tween 20的10 mmol L ^-1 pH 7.4的Tris-HCl缓冲溶液，用于清洗设备，然后在检测区域添加现成的TMB底物。该系统显示出良好的灵敏度和可检测性，达到了视觉和计算上的检测极限，分别为5.0 ng mL ^-1（65个寄生虫μL ^-1）和4.5 ng mL ^-1（59个寄生虫μL ^-1）分别是一种临床相关浓度，该浓度应足以在最初症状出现时识别出该疾病，并具有不需要使用纳米结构或酶以外的其他信号放大策略的优势。