Lateral flow immunoassays

  • 文章类型: Journal Article
    双酚S(BPS)是环境中常见的污染物,对水生动物和人类健康构成潜在威胁。为了准确评估BPS的污染水平和生态风险,迫切需要建立简便、灵敏的BPS检测方法。在这项研究中,通过引入4-溴丁酸甲酯和偶联牛血清白蛋白(BSA)成功制备了BPS完全抗原。以BPS完全抗原为基础,研制出高亲和力(1:256000)的抗BPS单克隆抗体,显示与BPS结构类似物的低交叉反应性。然后,以多壁碳纳米管和金纳米花复合材料为信号放大元件,以抗BPSmAb为探针,构建了电化学免疫传感器来检测BPS。电化学免疫传感器的线性范围为1至250ng·mL-1,检测限(LOD)低至0.6ng·mL-1。此外,基于氧化铱纳米颗粒,开发了一种更稳定、更灵敏的BPS侧流免疫分析法(LFIA),视觉检测限为1ng·mL-1,比经典Au-NPLFIA低10倍。在评估其稳定性和特异性后,通过测量水和鱼组织中的BPS浓度进一步验证了这两种方法的可靠性。因此,这项研究提供了敏感的,在环境和生物中检测BPS的稳健和快速的方法,为环境污染物监测提供方法参考。
    Bisphenol S (BPS) is a common pollutant in the environment and has posed a potential threat to aquatic animals and human health. To accurately assess the pollution level and ecological risk of BPS, there is an urgent need to establish simple and sensitive detection methods for BPS. In this study, BPS complete antigen was successfully prepared by introducing methyl 4-bromobutyrate and coupling bovine serum albumin (BSA). The monoclonal antibody against BPS (anti-BPS mAb) with high affinity (1: 256,000) was developed based on the BPS complete antigen, which showed low cross-reactivity with BPS structural analogues. Then, an electrochemical immunosensor was constructed to detect BPS using multi-walled carbon nanotubes and gold nanoflower composites as signal amplification elements and using anti-BPS mAb as the probe. The electrochemical immunosensor had a linear range from 1 to 250 ng⋅mL-1 and a limit of detection (LOD) down to 0.6 ng⋅mL-1. Additionally, a more stable and sensitive lateral flow immunoassay (LFIA) for BPS was developed based on iridium oxide nanoparticles, with a visual detection limit of 1 ng⋅mL-1, which was 10 times lower than that of classical Au-NPs LFIA. After evaluation of their stability and specificity, the reliability of these two methods were further validated by measuring BPS concentrations in the water and fish tissues. Thus, this study provides sensitive, robust and rapid methods for the detection of BPS in the environment and organisms, which can provide a methodological reference for monitoring environmental contaminants.
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  • 文章类型: Journal Article
    细菌感染对人类健康构成巨大威胁。侧流免疫测定(LFIA)具有低成本的优点,快速筛选,现场检测是细菌检测的竞争性技术,但是它们的检测限取决于所采用的纳米标签的光学性能。在这里,我们提出了使用聚多巴胺(PDA)功能化的Au纳米颗粒(表示为Au@PDA)作为纳米标签进行细菌检测的LFIA平台。PDA的引入可以增强Au的光吸收,以及许多用于共轭的官能团。小识别分子,即万古霉素(Van)和对巯基苯基硼酸(PMBA)共价锚定在Au@PDA上,并选择作为针对革兰氏阳性(G+)和革兰氏阴性(G-)细菌的特异性探针,分别。服用金黄色葡萄球菌(S.金黄色葡萄球菌)和大肠杆菌(E.大肠杆菌)作为G+和G-细菌的代表靶标,基于免疫夹心原理成功构建了两个LFA条。他们可以定量检测金黄色葡萄球菌和大肠杆菌都低至102cfu/mL,与其他基于比色或发光探针的LFIA相比,这是一个非常有竞争力的检测限。此外,提出的两个条带用于定量,准确,并快速检测食品和人体尿液样品中的金黄色葡萄球菌和大肠杆菌,获得了良好的分析结果。此外,它们被整合为一个筛选平台,在3小时内快速评估各种抗菌剂,与细菌培养和平板计数两种传统方法相比,明显缩短。
    Bacterial infection is a great threat to human health. Lateral flow immunoassays (LFIAs) with the merits of low cost, quick screening, and on-site detection are competitive technologies for bacteria detection, but their detection limits depend on the optical performance of the adopted nanotags. Herein, we presented a LFIA platform for bacteria detection using polydopamine (PDA) functionalized Au nanoparticles (denoted as Au@PDA) as the nanotag. The introduction of PDA could provide enhanced light absorption of Au, as well as numerous functional groups for conjugation. Small recognition molecules i.e. vancomycin (Van) and p-mercaptophenylboronic acid (PMBA) were covalently anchored to Au@PDA, and selected as the specific probes towards Gram-positive (G+) and Gram-negative (G-) bacteria, respectively. Taken Staphylococcus aureus (S. aureus) and Escherichia coli (E. coli) as the representative targets of G+ and G- bacteria, two LFA strips were successfully constructed based on the immuno-sandwich principle. They could quantitatively detect S. aureus and E. coli both down to 102 cfu/mL, a very competitive detection limit in comparison with other colorimetric or luminescent probes-based LFIAs. Furthermore, the proposed two strips were applied for the quantitative, accurate, and rapid detection of S. aureus and E. coli in food and human urine samples with good analytical results obtained. In addition, they were integrated as a screening platform for quick evaluation of diverse antibacterial agents within 3 h, which is remarkably shortened compared with that of the two traditional methods i.e. bacterial culture and plate-counting.
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  • 文章类型: Journal Article
    抗体检测是追踪包括COVID-19在内的许多疾病传播的关键的第一步。侧流免疫分析法(LFIA)是最常用的快速抗体检测方法,因为它易于使用且便宜。然而,当金纳米颗粒(AuNP)用作信号时,LFIA具有有限的灵敏度。在这项研究中,在LFIA中将枯草芽孢杆菌的内生孢子与AuNP联合用于检测抗体。内生孢子用作信号放大器。抗β半乳糖苷酶抗体检测的检测限约为10-8M,而常规LFIA的检测限约为10-6M。与传统的LFIA相比,所提出的方法没有额外的用户步骤。这种方法,因此,在不损害LFIA的任何优势的情况下提高灵敏度100倍。我们相信所提出的方法将用于检测抗HIV抗体,寨卡病毒,SARS-CoV-2等等。
    Antibody detection is the critical first step for tracking the spread of many diseases including COVID-19. Lateral flow immunoassay (LFIA) is the most commonly used method for rapid antibody detection because it is easy-to-use and inexpensive. However, LFIA has limited sensitivity when gold nanoparticles (AuNPs) are used as the signals. In this study, the endospores of Bacillus subtilis were used in combination with AuNP in a LFIA to detect antibodies. The endospores serve as a signal amplifier. The detection limit was about 10-8 M for anti-beta galactosidase antibody detection whereas the detection limit of conventional LFIA is about 10-6 M. Furthermore, the proposed methods have no additional user steps compared with the traditional LFIA. This method, therefore, improved the sensitivity 100-fold without compromising any advantages of LFIA. We believe that the proposed method will be useful for detection of antibodies against HIV, Zika virus, SARS-CoV-2, and so on.
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  • 文章类型: Journal Article
    侧流免疫测定法(LFIA)由于其简单性,在现场护理测试领域是成熟且广泛商业化的工具,快速性,成本效益,对用户和设备要求低。然而,与传统LFIA相关的不敏感和产生不准确结果的可能性阻碍了它们的广泛实施,特别是监测超痕量水平的分析物。此外,抗体(Ab)表面氨基酸的异质性分布导致缺乏对其方向的精确控制,这最终导致不令人满意的检测性能。为了解决这些问题,在本文中,我们提供了从纳米探针或膜上的固定化Ab的取向操作的角度来制备良好建立的LFIA的新兴努力的概述。制备优良的纳米探针与Abs定向固定化,由纳米探针类型组成,Ab类型,和它们的共轭化学,被审查。随后的介绍强调了在膜上定向固定Ab的重要性。Ab方向对LFIA平台灵敏度分析性能的影响,特异性,快速性,可靠性,成本效益,并对稳定性进行了总结。最后,还讨论了面向Ab的固定化辅助LFIA的未来发展和挑战。
    Lateral flow immunoassays (LFIAs) are well-established and broadly commercialized tools in the field of point-of-care testing due to their simplicity, rapidity, cost-effectiveness, and low requirements for users and equipment. However, the insensitivity and the possibility of producing inaccurate results associated with conventional LFIAs have impeded their wide-ranging implementation, especially for monitoring ultra-trace level of analytes. Moreover, the heterogeneous distribution of amino acids on the surface of antibody (Ab) results in a lack of precise control over their orientation, which ultimately leads to unsatisfactory detection performance. To address those concerns, herein we provide an overview of the emerging efforts to prepare well-established LFIAs from the perspective of orientation manipulation of immobilized Abs on the nanoprobes or membranes. The preparation of excellent nanoprobes with Abs being oriented immobilized, consisting of the nanoprobe types, Ab types, and their conjugation chemistries, are reviewed. Followed by the introduction of efforts highlight the importance of directionally immobilized Ab on the membrane. The effects of Ab orientation on the analytical performance of LFIA platforms in terms of sensitivity, specificity, rapidity, reliability, cost-effectiveness, and stability are also summarized. Finally, the future development and challenges of Ab-oriented immobilization-assisted LFIAs are also discussed.
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  • 文章类型: Journal Article
    背景:芬太尼试纸(FTS)是药物检查中常用的工具,用于在食用前测试街头药物样本中是否存在芬太尼。以前的报道表明,除了芬太尼,FTS还可以检测芬太尼类似物,如乙酰芬太尼和丁酰芬太尼,关于它们检测芬太尼类似物如卡芬太尼和呋喃基芬太尼的能力的报道相互矛盾。然而数百种已知的芬太尼类似物,目前还没有一项大规模的研究来合理化FTS对不同芬太尼类似物的反应性.
    方法:在本研究中,在两种品牌的芬太尼试纸上筛选了251种合成阿片类药物,包括214种芬太尼类似物,以(1)评估两种品牌的芬太尼试纸检测芬太尼相关化合物的能力差异,以及(2)确定芬太尼类似物化学结构中的哪些部分对于FTS检测最关键。在这项研究中评估了两个FTS品牌:BTNX快速反应和WHPMDanceSafe。
    结果:在评估的251个筛选化合物中,通过BTNX和DanceSafeFTS可检测到121种化合物的浓度为或低于20,000ng/mL,任何一个品牌都检测不到50个,一个品牌检测到80个,但另一个品牌检测不到(n=52个BTNX,n=28DanceSafe)。筛选的芬太尼类似物的结构分析显示,一般来说,对苯乙基部分的大量修饰抑制BTNXFTS的检测,而对羰基部分的大量修饰抑制DanceSafeFTS的检测。
    结论:不同的“盲点”是由用于引发这些不同条带的抗体的不同半抗原引起的。通过在常规药物检查中利用两个品牌的FTS,用户可以增加在一个品牌的“盲点”中检测芬太尼类似物的机会。
    Fentanyl test strips (FTS) are a commonly deployed tool in drug checking, used to test for the presence of fentanyl in street drug samples prior to consumption. Previous reports indicate that in addition to fentanyl, FTS can also detect fentanyl analogs like acetyl fentanyl and butyryl fentanyl, with conflicting reports on their ability to detect fentanyl analogs like Carfentanil and furanyl fentanyl. Yet with hundreds of known fentanyl analogs, there has been no large-scale study rationalizing FTS reactivity to different fentanyl analogs.
    In this study, 251 synthetic opioids-including 214 fentanyl analogs-were screened on two brands of fentanyl test strips to (1) assess the differences in the ability of two brands of fentanyl test strips to detect fentanyl-related compounds and (2) determine which moieties in fentanyl analog chemical structures are most crucial for FTS detection. Two FTS brands were assessed in this study: BTNX Rapid Response and WHPM DanceSafe.
    Of 251 screened compounds assessed, 121 compounds were detectable at or below 20,000 ng/mL by both BTNX and DanceSafe FTS, 50 were not detectable by either brand, and 80 were detectable by one brand but not the other (n = 52 BTNX, n = 28 DanceSafe). A structural analysis of fentanyl analogs screened revealed that in general, bulky modifications to the phenethyl moiety inhibit detection by BTNX FTS while bulky modifications to the carbonyl moiety inhibit detection by DanceSafe FTS.
    The different \"blind spots\" are caused by different haptens used to elicit the antibodies for these different strips. By utilizing both brands of FTS in routine drug checking, users could increase the chances of detecting fentanyl analogs in the \"blind spot\" of one brand.
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  • 文章类型: Journal Article
    背景:快速抗原测试(RAT)结果可直观地读取为是否存在彩色线。由于测试样品中分析物浓度较低,主观解释可能会错过检测弱线。需要训练。尽管常规测试经验提高了结果读出技能,它消耗时间和资源。因此,我们使用开源实验心理学软件创建了一种基于计算机的反馈培训方法,其中参与者通过反复对显示RAT结果的随机呈现的图片做出正/负响应来积累RAT结果读出经验;然后,他们收到反馈他们的答案是正确的或不正确的,并被要求盯着图片再次与正确答案的知识。本研究旨在研究培训在提高技能方面的效果,使用冠状病毒病2019(COVID-19)RAT。
    方法:将22名医疗技术人员随机分为两组:反馈培训组和测试体验组。使用几张图片显示COVID-19RAT的阳性和阴性结果,在检查了他们的初始结果读出技能之后,反馈培训组接受了反馈培训,而测试体验组进行了相同数量的测试,而没有反馈来积累测试经验,他们的技能再次被检查。“阳性”答案与显示阳性结果的图片的比率(即,命中率)进行统计分析。
    结果:反馈训练组在训练后显示出较高的命中率,而测试体验组没有。反馈训练效果表现在弱线检测中。
    结论:这种基于计算机的反馈训练方法可以成为提高RAT结果读出技能的有效工具。
    BACKGROUND: Rapid antigen testing (RAT) results are visually read as whether colored line is present or absent. The subjective interpretation potentially misses detecting weak lines due to lower analyte concentration in samples tested, requiring training. Although routine test experience has improved the result readout skills, it consumes time and resources. Therefore, we created a computer-based feedback training method using open-source experimental psychology software, wherein participants accumulate RAT result readout experience by repeatedly responding positive/negative to randomly presented pictures showing RAT results; then, they receive feedback on their answers as correct or incorrect and are asked to stare at the pictures again with the knowledge of correct answer. This study aimed to examine the training effects in improving the skills, using coronavirus disease 2019 (COVID-19) RAT.
    METHODS: Twenty-two medical technologists were randomly divided into two groups: the feedback-training and test-experience groups. Using several pictures showing positive and negative results of COVID-19 RAT, after examination of their initial result readout skills, feedback-training group received the feedback training, whereas test-experience group performed an equal number of tests without feedback to accumulate test experience, and their skills were examined again. The ratio of \"positive\" answers to the pictures showing positive results (i.e., hit rate) was statistically analyzed.
    RESULTS: The feedback-training group showed a significantly higher hit rate after their training, whereas the test-experience group did not. The feedback training effects were manifested in weak line detection.
    CONCLUSIONS: This computer-based feedback training method can be an effective tool for improving RAT result readout skills.
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  • 文章类型: Journal Article
    住院动物的医院沙门氏菌病是公认的危害,尤其是在大型动物诊所。用于检测沙门氏菌的标准化培养方案(SCP)。在使用48小时富集步骤的环境样品中,阴性结果的周转时间为5天。RapidChek®SELECT™沙门氏菌(RCSS)测试系统通过双重富集,然后进行侧流免疫测定,可在22-44小时内检测生物体。在48小时内报告阴性结果。为了确定最灵敏,最快速的检测沙门氏菌的方法。从大型动物普渡兽医院(LA-PVH)收集的环境样本中,一项初步研究比较了RCSS和SCP在测试人工加标和自然污染样品时的性能。一项扩大的研究分析了使用RCSS方法在12个月内测试872个环境样品所获得的结果。选择基质辅助激光解吸/电离飞行时间(MALDI-TOF)质谱作为RCSS推定阳性样品的确认方法。随机选择的样品子集通过实时PCR获得额外的确认。这里,我们报告了RCSS在灵敏度方面的性能数据,特异性,和使用MALDI-TOF结果作为比较参考的阳性预测值。我们还提供报告使用该系统获得的结果的指南。
    Nosocomial salmonellosis in hospitalized animals is a recognized hazard, especially in large animal clinics. A standardized culture protocol (SCP) for detecting Salmonella spp. in environmental samples using a 48-h enrichment step results in a 5-day turnaround time for negative results. The RapidChek® SELECT™ Salmonella (RCSS) test system offers detection of organisms in 22-44 h through double enrichment followed by a lateral flow immunoassay. Negative results are reported within 48 h. To determine the most sensitive and rapid method for detecting Salmonella spp. from environmental samples collected at the large animal Purdue Veterinary Hospital (LA-PVH), a preliminary study compared the performance of RCSS and a SCP when testing artificially spiked and naturally contaminated samples. An expanded study analyzed results obtained using the RCSS method to test 872 environmental samples over a 12-month period. Matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry was chosen as the confirmation method for RCSS-presumptive positive samples. A randomly selected subset of samples received additional confirmation by real-time PCR. Here, we reported the performance data of RCSS in terms of sensitivity, specificity, and positive predictive value using MALDI-TOF results as reference for comparison. We also provide guidelines for reporting results obtained using this system.
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  • 文章类型: Journal Article
    脓毒症是一种高致死率的急性全身性感染综合征。快速准确的诊断,监测,败血症的药物治疗是必不可少的。我们利用了荧光金属-AIEgen框架(MAF),并证明了蛋白质检测和细菌鉴定的双重功能:(i)脓毒症生物标志物的超灵敏即时(POC)检测(灵敏度提高100倍);(ii)革兰氏阴性/阳性细菌的快速POC鉴定(20分钟内的选择性聚集)。荧光侧流免疫测定(LFA)对于POC测试是方便且廉价的。MAF具有较大的表面积,优异的光稳定性,高量子产率(80%),和多个活性位点作为蛋白结合域用于超灵敏检测LFA上的脓毒症生物标志物(IL-6/PCT)。IL-6/PCT的检测限(LOD)为0.252/0.333pg/mL。快速评估感染性细菌对于指导药物使用至关重要。双功能荧光MAFs在临床诊断细菌感染的POC测试中具有巨大的潜力。
    Sepsis is an acute systemic infectious syndrome with high fatality. Fast and accurate diagnosis, monitoring, and medication of sepsis are essential. We exploited the fluorescent metal-AIEgen frameworks (MAFs) and demonstrated the dual functions of protein detection and bacteria identification: (i) ultrasensitive point-of-care (POC) detection of sepsis biomarkers (100 times enhanced sensitivity); (ii) rapid POC identification of Gram-negative/positive bacteria (selective aggregation within 20 min). Fluorescent lateral flow immunoassays (LFAs) are convenient and inexpensive for POC tests. MAFs possess a large surface area, excellent photostability, high quantum yield (∼80%), and multiple active sites serving as protein binding domains for ultrasensitive detection of sepsis biomarkers (IL-6/PCT) on LFAs. The limit of detection (LOD) for IL-6/PCT is 0.252/0.333 pg/mL. Rapid appraisal of infectious bacteria is vital to guide the use of medicines. The dual-functional fluorescent MAFs have great potential in POC tests for the clinical diagnosis of bacterial infections.
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  • 文章类型: Journal Article
    在分析化学仪器的发展中,作为关键的技术突破,应被视为电子学的共同引入及其在集成方面的所有进步,然后是微处理器,随后是广泛的计算机化。似乎可以将类似的作用归因于现代纳米技术的各种元素的引入,自本世纪初以来,进展迅速。它涉及分析化学应用的所有领域,还包括流量分析的进展,自20世纪中叶以来一直在发展。显然,它不应该省略早期开发和分析应用的平面结构,如脂质膜或自组装单层。它们在发现许多非凡的纳米颗粒如富勒烯之前具有重要的影响,碳纳米管和石墨烯,或纳米晶体半导体(量子点)。大多数情况下,由于催化作用,显着发展的表面和容易功能化的可能性,它们在流动分析程序的各个阶段的应用可以显着改善它们。新纳米材料的应用可用于在宏观流动设置以及微流体和侧流免疫测定测试中开发新的流动分析系统检测方法。还有利的是,测量的快速流动条件可以有助于防止纳米颗粒的不利聚集。已经发表了大量关于这一主题的文献(例如,关于碳纳米管和流动注射分析系统的近1000篇论文)意味着,对于这篇评论,有必要对这一趋势的报道例子进行任意选择。主要集中在最近十年报告的成就。
    In evolution of instrumentation for analytical chemistry as crucial technological breakthroughs should be considered a common introduction of electronics with all its progress in integration, and then microprocessors which was followed by a widespread computerization. It is seems that a similar role can be attributed to the introduction of various elements of modern nanotechnology, observed with a fast progress since beginning of this century. It concerns all areas of the applications of analytical chemistry, including also progress in flow analysis, which are being developed since the middle of 20th century. Obviously, it should not be omitted the developed earlier and analytically applied planar structures like lipid membranes or self-assembled monolayers They had essential impact prior to discoveries of numerous extraordinary nanoparticles such as fullerenes, carbon nanotubes and graphene, or nanocrystalline semiconductors (quantum dots). Mostly, due to catalytic effects, significantly developed surface and the possibility of easy functionalization, their application in various stages of flow analytical procedures can significantly improve them. The application of new nanomaterials may be used for the development of new detection methods for flow analytical systems in macro-flow setups as well as in microfluidics and lateral flow immunoassay tests. It is also advantageous that quick flow conditions of measurements may be helpful in preventing unfavorable agglomeration of nanoparticles. A vast literature published already on this subject (e.g. almost 1000 papers about carbon nanotubes and flow-injection analytical systems) implies that for this reviews it was necessary to make an arbitrary selection of reported examples of this trend, focused mainly on achievements reported in the recent decade.
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  • 文章类型: Journal Article
    多功能纳米材料(NP)的工程协作尺寸调节和形状工程为提高分析性能提供了非凡的机会。预计将解决区分由目标浓度的细微变化引起的颜色变化的困难。从而促进侧流免疫测定(LFIA)的高度灵敏分析。在这里,具有精确MnCl2调节的银耳样金锰氧化物(Au-MnOx)纳米颗粒是通过在环境温度下在碱性条件下轻松进行的一步氧化还原反应合成的,作为免疫信号示踪剂。形态的可调元素组成和各向异性的应用,黑色银耳状Au-MnOx表现出极好的比色信号亮度,增强抗体偶联效率,奇妙的光热性能,和不受限制的免疫识别亲和力,所有这些都有助于高度敏感的多信号转导模式。与手持式热读取器设备配合使用,通过将Au-MnOx与竞争性免疫反应相结合,实现了一种双峰型LFIA,该双峰型LFIA结合了大小调节和形状工程介导的比色-光热双响应测定(称为SSCPD测定),检测限为0.012ngmL-1,用于莱克多巴胺(RAC)监测。这项工作说明了该策略对建立高性能传感的有效性,和SCPD测定可能会扩展到未来的现场护理(POC)诊断应用的广泛范围。
    Engineered collaborative size regulation and shape engineering of multi-functional nanomaterials (NPs) offer extraordinary opportunities for improving the analysis performance. It is anticipated to address the difficulty in distinguishing color changes caused by subtle variations in target concentrations, thereby facilitating the highly sensitive analysis of lateral flow immunoassays (LFIAs). Herein, tremella-like gold-manganese oxide (Au-MnOx ) nanoparticles with precise MnCl2 regulation are synthesized as immuno signal tracers via a facile one-step redox reaction in alkaline condition at ambient temperature. Avail of the tunable elemental composition and anisotropy in morphology, black-colored tremella-like Au-MnOx exhibits superb colorimetric signal brightness, enhanced antibody coupling efficiency, marvelous photothermal performance, and unrestricted immunological recognition affinity, all of which facilitate highly sensitive multi-signal transduction patterns. In conjunction with the handheld thermal reader device, a bimodal-type LFIA that combines size-regulation- and shape-engineering-mediated colorimetric-photothermal dual-response assay (coined as the SSCPD assay) with a limit of detection of 0.012 ng mL-1 for ractopamine (RAC) monitoring is achieved by integrating Au-MnOx with the competitive-type immunoreaction. This work illustrates the effectiveness of this strategy for establishing high-performance sensing, and the SSCPD assay may be extended to a wide spectrum of future point-of-care (POC) diagnostic applications.
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