key: cord-1013266-2k2c00fj
authors: Zhou, Yaofeng; Chen, Yuan; Liu, Yang; Fang, Hao; Huang, Xiaolin; Leng, Yuankui; Liu, Zhengqiong; Hou, Li; Zhang, Wei; Lai, Weihua; Xiong, Yonghua
title: Controlled copper in situ growth-amplified lateral flow sensors for sensitive, reliable, and field-deployable infectious disease diagnostics
date: 2020-10-21
journal: Biosens Bioelectron
DOI: 10.1016/j.bios.2020.112753
sha: 3848bcb8c584c3e2bee9615ef60468010fd54e48
doc_id: 1013266
cord_uid: 2k2c00fj

A polyethyleneimine (PEI)-assisted copper in-situ growth (CISG) strategy was proposed as a controlled signal amplification strategy to enhance the sensitivity of gold nanoparticle-based lateral flow sensors (AuNP-LFS). The controlled signal amplification is achieved by introducing PEI as a structure-directing agent to regulate the thermodynamics of anisotropic Cu nanoshell growth on the AuNP surface, thus controlling shape and size of the resultant AuNP@Cu core–shell nanostructures and confining free reduction and self-nucleation of Cu(2+) for improved reproducibility and decreased false positives. The PEI-CISG-enhanced AuNP-LFS showed ultrahigh sensitivities with the detection limits of 50 fg mL(−1) for HIV-1 capsid p24 antigen and 6 CFU mL(−1) for Escherichia coli O157:H7. We further demonstrated its clinical diagnostic efficacy by configuring PEI-CISG into a commercial AuNP-LFS detection kit for SARS-CoV-2 antibody detection. Altogether, this work provides a reliable signal amplification platform to dramatically enhance the sensitivity of AuNP-LFS for rapid and accurate diagnostics of various infectious diseases.

Infectious diseases are usually caused by various pathogens, such as bacteria (Chen et In this work, we report the use of a polymer-type SDA (PEI) to assist in copper 132 in-situ growth (PEI-CISG) on the surface of AuNP probes for significantly amplifying 133 the detection signal of traditional AuNP-LFS. In this design, Cu 2+ solution was pre-134 incubated with PEI to form the PEI@Cu 2+ complex via the strong chelation of Cu 2+ 135 with the amino groups of PEI. The as-prepared PEI@Cu 2+ complex was adsorbed on 136 the surface of AuNP probes. Then, PEI was used as the template skeleton to control 137 the copper reduction and growth in the presence of mild reducing agent, that is, Anti-p24 cAbs (5.0 mg mL −1 ) and dAbs (6.4 mg mL −1 ) were provided by Abcam 177 (Cambridge, MA). Anti-E. coli O157:H7 dAbs (2 mg mL −1 ) and cAbs (2 mg mL −1 ), 178 goat anti-mouse IgG (10 mg mL −1 ), and BSA were purchased from Meridian Life (Figure 1b and 1c) . Energy-dispersive X-ray (EDX) spectroscopy shown in Figure   269 1d indicates the presence of Cu element, showing the successful growth and (Figure 1g) . Powder X-ray diffraction pattern ( Figure S3a ) and X-ray AuNP@BSA on T line with R 2 of 0.9922 (Figure 1h) , implying that the PEI-CISG-297 amplified strip is suitable for the accurate quantitative detection of targets. 354 The proposed PEI-CISG-enhanced strip was first used for the ultrasensitive detection 355 of p24 antigen (Figure 3a) , which is the earliest emerging protein biomarker in HIV- coli O157:H7 determination in milk (Figure 4a) . The dAbs and cAbs against E. coli 418 O157:H7 were used for the preparation of AuNP@dAb probes and sprayed on the NC 419 membrane as the T line. The optimization of AuNP-LFS for E coli O157:H7 detection 420 is described in Figure S11 and Table S4 . Under optimum conditions, the strips before 421 and after PEI-CISG treatment were employed to detect E. coli O157:H7 in various 422 standard solutions with concentrations from 0 CFU mL −1 to 9.60 × 10 5 CFU mL −1 .

The E. coli O157:H7 standard solutions were prepared by adding E. coli O157:H7 424 stock solution into 10-fold diluted milk. The strip stereograms in Figure S12 reveal 425 that the vLOD of AuNP-LFS before PEI-CISG treatment was 3.84 × 10 3 CFU mL −1 . 426 After signal amplification, the vLOD of the enhanced strip remarkably decreased to 427 6.0 CFU mL −1 , whereas no bands were observed in the test of all three E. coli 428 O157:H7 negative samples. This LOD value is better than most reported protocols in 429 detecting E. coli O157:H7, as shown in Table S5 . Additionally, one of the three (Table S6) . (Figure 5c) . Notably, three randomly selected negative samples (S1, S2, and S10) 

Proc. Natl. Acad. Sci. 573 USA

or after (f) PEI-CISG. The inset of Figure 1e and 1f indicate high magnification of 657 individual AuNP and AuNP@Cu. Results demonstrate the formation of AuNP@Cu 658 core-shell nanostructure after PEI-CISG treatment. (g) HAADF-scanning TEM EDS 659 elemental mapping analysis of AuNP@Cu. (h) The correlative curve of OD value 660

Figure 2 Optimization of work conditions for PEI-CISG technology. Optimization for 666 the concentrations and ratio of PEI and Cu 2+ : (a) Cu 2+ concentration at a given PEI 667 concentration of 1 mg mL −1 , and (b) PEI concentrations under the optimal ratio of 668

c) Optimization for strip soaking time in PEI@Cu 2+ complex and 669 copper growth time. (d) Schematic representation of PEI-CISG technology for 670 enhanced AuNP-LFS via biotin-SA system. (e) Evaluation for signal amplification 671 potential of our proposed PEI-CISG on AuNP-LFS platform via biotin-SA system

Figure 3 A PEI-CISG enhanced AuNP-LFS for the detection of p24

The positive detection was conducted by using the sample 683 solution containing 3.0 ng mL −1 to run the strip, while the negative test was performed 684 using blank control solution without p24. (d) The calibration curves of AuNP-LFS 685 and PEI-CISG enhanced AuNP-LFS for detection of p24. These curves were plotted 686 by recording specific response against p24 concentration. (e) Reproducibility analysis 687 of PEI-CISG enhanced AuNP-LFS for the detection of p24. Three standard curves 688 were established by measuring a series of different concentrations of p24 in the 689 succeeding 3 days. (f) Specificity evaluation of the PEI-CISG enhanced AuNP-LFS 690 for p24 detection by collecting the signal response against p24 at 0.5 ng mL −1 , and 691 other protein markers of

Figure 4 Application of PEI-CISG enhanced AuNP-LFS for the detection of E. coli 700 O157:H7. (a) Schematic illustration of PEI-CISG enhanced AuNP-LFS for detecting

The curve was constructed by analyzing a series of 703 different concentrations of E. coli O157:H7 ranging from 10 0 CFU mL −1 to 10 6 CFU 704 mL −1 . (c) Reliability evaluation of the PEI-CISG enhanced AuNP-LFS for the 705 detection of E. coli O157:H7 at ultralow concentrations of 9.0 × 10 1 CFU mL −1 and 706 9.0 × 10 0 CFU mL −1 . (d) Specificity analysis for our enhanced AuNP-LFS

Figure 5 The integration of commercial AuNP-LFS strip with our proposed PEI-713 CISG technology for the detection of SARS-CoV-2 Abs. (a) Evaluation of the 714 detection ability of commercial AuNP-LFS strip with or without PEI-CISG treatment

CoV-2-positive serum sample was continuously diluted by different factors, 716 and then all diluted samples were simultaneously analyzed by using commercial

AuNP-LFS strip with or without PEI-CISG treatment. (b) Performance characteristics 718 of commercial AuNP-LFS strip. 132 clinical samples, including 69 positives and 63 719 negatives were tested using commercial AuNP-LFS strip. 7 PCR-confirmed SARS

CoV-2 positive samples were not successfully tested by commercial AuNP-LFS strip. 721 (c) Improved evaluation of the commercial AuNP-LFS strip combined with PEI-CISG 722 treatment for differentiating false negatives

☒ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. ☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: