Quantitation of Circulating Mycobacterium tuberculosis Antigens by Nanopore Biosensing in Children Evaluated for Pulmonary Tuberculosis in South Africa

Abstract

Nanopore sensing of proteomic biomarkers lacks accuracy due to the ultralow abundance of targets, a wide variety of interferents in clinical samples, and the mismatch between pore and analyte sizes. By converting antigens to DNA probes via click chemistry and quantifying their characteristic signals, we show a nanopore assay with several amplification mechanisms to achieve an attomolar level limit of detection that enables quantitation of the circulating Mycobacterium tuberculosis (Mtb) antigen ESAT-6/CFP-10 complex in human serum. The assay's nonsputum-based feature and low-volume sample requirements make it particularly well-suited for detecting pediatric tuberculosis (TB) disease, where establishing an accurate diagnosis is greatly complicated by the paucibacillary nature of respiratory secretions, nonspecific symptoms, and challenges with sample collection. In the clinical assessment, the assay was applied to analyze ESAT-6/CFP-10 levels in serum samples collected during baseline investigation for TB in 75 children, aged 0−12 years, enrolled in a diagnostic study conducted in Cape Town, South Africa. This nanopore assay showed superior sensitivity in children with confirmed TB (94.4%) compared to clinical "gold standard" diagnostic technologies (Xpert MTB/RIF 44.4% and Mtb culture 72.2%) and filled the diagnostic gap for children with unconfirmed TB, where these traditional technologies fell short. We envision that, in combination with automated sample processing and portable nanopore devices, this methodology will offer a powerful tool to support the diagnosis of pulmonary TB in children.

Nanopore sensing specific enhancement technique for single molecule detection

“…帕金 森病目前仍然依赖于临床症状进行判断, 很难做到 提早的诊断 [91] , α-突触核蛋白(α突触核蛋)浓度被 发现在健康人群和帕金森病患者中存在显著差 异 [92] . 如图20所示, Liu等 [93] 将α-突触核蛋白的 图 19 基于夹心法分析, 点击化学以及纳米孔传感来检测生物标志物的方法示意图 [85] Fig. 19.…”

“…19. Schematic diagram of a method for detecting biomarkers based on sandwich assay, click chemistry, and nanopore sensing [85] .…”

“…特异性 增强技术 目标生物分子 纳米孔类型 检测极限 表面官能化 α-thrombin g-thrombin SiN x N/A [39] miRNA-21 Quartz 0.1 pmol/L [42] HAdV SARS-CoV-2 PET 6 pfu/mL [43] 10 4 copies/mL [43] N protein Nanopipette 73.204 pg/mL [47] Neuraminidase ClyA 38 nmol/L [49] SARS-CoV-2 Spike Protein ClyA 2.3 nmol/L [50] Barstar t-FhuA 12.6 nmol/L [51] Streptavidin PET N/A [52] L-His, D-His PET N/A [58] L-Trp PI N/A [59] norepinephrine epinephrine MspA 1 μmol/L [61] ribonucleotide MspA N/A [62] 分子探针 miR155 SiN x 10 nmol/L [68] C-reactive protein SiN x 0.3 ng/μL [72] alpha-1 antitrypsin Tau 381 BACE1 Aerolysin 77.9 fmol/L [73] 6.79 fmol/L [73] 86.4 fmol/L [73] miRNA-21 Glass 5 nmol/L [83] Carcinoembryonic-antigen Quartz 0.01 nmol/L [84] alpha fetoprotein α-HL 1 fmol/L [85] ESAT-6/CFP-10 α-HL 10 amol/L [86] α-Syn Quartz 2.2 pmol/L [93] Thrombin AChE Quartz N/A [65] 物 理 学 报 Acta Phys. Sin.…”

Narrowing Signal Distribution by Adamantane Derivatization for Amino Acid Identification Using an α-Hemolysin Nanopore

“…Furthermore, using ADIC as a modifier, similar to adamantane, can provide more possibilities for improving the specificity of nanopore signals. For instance, the interaction between adamantane and CB[6] produces unique oscillation signals in the nanopore, − which could lead to innovative strategies for AA discrimination and protein sequencing. Building on these advantages, a viable approach to protein sequencing could entail enzymatic digestion to peptide fragments, size-based separation of peptides, anchoring the C-terminus of a peptide on a solid phase, ADIC labeling, and enzymatic cleavage of the N -terminal AAs into a mobile phase, followed by subsequent identification of each released AA via nanopore to sequentially decode the peptide’s structure.…”