Biosens Bioelectron. 2017 Mar 15;89(Pt 2):795-801.

A rapid and visual aptasensor for lipopolysaccharide detection based on the bulb-like triplex turn-on switch coupled with HCR-HRP nanostructures

Wentao Xu1, 2*, Jingjing Tian2, Xiangli Shao1, Longjiao Zhu2, Kunlun Huang1, 2, , Yunbo Luo1*

1Beijing Advanced Innovation Center for Food Nutrition and Human Health, College of Food Science & Nutritional Engineering, China Agricultural University, Beijing 100083, China

2Beijing Laboratory for Food Quality and Safety, College of Food Science and Nutritional Engineering, China Agricultural University, Beijing 100083, China

* To whom correspondence should be addressed.

Tel/Fax: +86 010 62736479; Email:xuwentao@cau.edu.cn

https://doi.org/10.1016/j.bios.2016.10.012

 

Abstract

For previously reported aptasensor, the sensitivity and selectivity of aptamers to targets were often suppressed due to the reporter label of single-stranded molecular beacon or hindrance of the duplex DNA strand displacement. To solve the affinity declining of aptamers showed in traditional way and realize on-site rapid detection of Lipopolysaccharides (LPS), we developed an ingenious structure-switching aptasensor based on the bulb-like triplex turn-on switch (BTTS) as the effective molecular recognition and signal transduction element and streptavidin-horseradish peroxidase modified hybridization chain reaction (HCR-HRP) nanocomposites as the signal amplifier and signal report element. In the presence of LPS, the bulb-like LPS-aptamer (BLA) and LPS formed the LPS/aptamer complex, while the BTTS disassembled and liberated the dissociative bridge probes (BP) to achieve molecular recognition and signal transduction. Immobilized BP, captured by immobilized capture probes (CP), triggered hybridization chain reactions (HCR) to amplify the switching signal, and the HCR products were then modified with streptavidin-horseradish peroxidase (SA-HRP) to form HCR-HRP nanostructures to output colorimetric signals. In less than four hours, the proposed biosensor showed a detection limit of 50 pg/mL of LPS quantitatively with the portable spectrophotometer and the observation limit of 20 ng/mL semi-quantitatively with the naked eye, opening up new opportunities for LPS detection in future clinical diagnosis, food security and environment monitoring.

PMID: 27816585

 

Supplement

A turn-on sensor based on a bulb-like triplex turn-on switch (BTTS) was developed.

As illustrated in Fig. 1, BTTS was designed as bulb-like and was composed of a bulb-like LPS-aptamer (BLA) in the center to capture LPS from E. coli O111:B4 flanked by mirror sequences to hybridize with the bridge probe (BP) to form a triplex nucleic acid stem by Watson-Crick base pairing and Hoogsteen base pairing. As the molecular recognition element, the nudity of the bulb-like aptamers promoted effective binding to LPS. Compare with two conformational alteration strategies, neither signal reporter labels nor hybridization of double strands interfere the sensitivity and affinity of aptamers to targets. When LPS was introduced, BTTS turned on, which means that the triplex nucleic acid stem disassembled and liberated LPS-combined aptamers and dissociative BP. After dissociative BP was anchored by an immobilized capture probe (CP) on the surface, Biotin-H1 and Biotin-H2 were initiated to proceed HCR via cross-opening linkage. Upon SA-HRP addition, HCR-HRP nanocomposites were formed, serving as the signal amplifier and signal report element, which catalyzed hydrogen peroxide (H2O2) via TMB to generate an obvious green color and turned yellow after sulfuric acid termination with optical absorption at 450 nm. The chromogenic output was able to be semiquantitatively monitored by the naked eye and quantitatively inspected by a portable spectrophotometer. In our design, turning on the BTTS was equivalent to the occurrence of the aptamer-target binding event, converting LPS content to visual output accompanied by the generation and amplification of the detection signal. Because the BTTS and immobilized CP could be prepared in advance, the actual total detection time, including the LPS sample introduction for 0.5 h, BP anchoring for 1 h, and the generation and amplification of the detection signal for less than 2 h (composed of HCR for 1 h, the formation of HCR-HRP nanocomposites for 40 min and chromogenic reaction via TMB for 5 min), was less than 4h, indicating great promise for on-site rapid analysis.

 

 

Fig. 1 Schematic of the design of the rapid and visual aptasensor for LPS detection based on the BTTS as a selective molecular recognition and signal transduction element and HCR-HRP nanostructures as effective signal amplifier and signal report element.

 

The sensor is based on HCR-HRP nanostructures.

In the presence of BP, Biotin-H1 was opened from the sticky end and paired with BP, undergoing an unbiased strand-displacement interaction. Immediately following this event, the newly released sticky end of the Biotin-H1 hairpin hybridized with the Biotin-H2 hairpin from its sticky end via a strand displacement reaction, generating new single-stranded linker sequences that were identical to BP. In this way, the opening of hairpins continued, accompanied by strand displacement reactions, until the formation of a long linear nanostructure labeled with biotin group. Upon SA-HRP addition, HCR-HRP nanocomposites were formed via biotin-streptavidin interactions. As shown in Fig.2A, OD450 of the HCR-based system was obviously higher than that of the non-HCR system, and the sensitivity was noticeably improved, clearly indicating that the signal amplification effect of HCR process, which was at least 4-fold greater than that in the non-HCR method. In addition, transmission electron microscope (TEM) was further employed to observe the morphology of the assembled HCR-HRP nanostructures. From Fig.2B, morphology with helical nanocomposites was observed. The result suggested the successful formation of the HCR-HRP nanostructures.

 

 

Fig. 2 (A, left) Comparison of OD450 with different concentrations of LPS (ng/mL) between with and without HCR-HRP. (B, right) Transmission electron microscope (TEM) of HCR-HRP nanostructures

 

Outstanding sensitivity and selectivity.

By virtue of the optimal conditions, the sensitivity and quantitative range of the BTTS aptasensor were evaluated for the detection of a series of different concentrations of LPS from E. coli O111:B4. As shown in Fig. 3A, the plot of optical intensity at 450 nm versus the concentration of LPS displayed a good linear relationship in the range from 1 to 150 ng/mL with a regression equation expressed as y = 0.016x + 0.6226, and the correlation coefficient was 0.9981. The limitation of detection (LOD) was calculated to be 50 pg/mL in terms of the rule of three times the standard deviation over the blank response. Moreover, the analytical performances of different techniques for LPS analysis are summarized and the sensitivity of the BTTS aptasensor is competitive compared to these techniques, which should be attributed to the nudity of the aptamers and HCR-HRP nanostructures for signal amplification.

The specificity of the BTTS turn-on aptasensor was evaluated by challenging the system against other interfering agents; e.g., D-mannose, BSA, peptidoglycan from S. aureus, hemoglobin (Hb), glucose, human serum albumin (HSA) and LPS from E.coli O55:B5. As indicated in Fig. 6B, in contrast to significant responses observed for LPS and the mixture containing the above seven interferences, other agents showed very low interference to LPS detection. Such an excellent selectivity might be attributed to the high specificity of the aptamer of LPS from E. coli O111:B4. Hence, the developed BTTS turn-on aptasensor could be used for specific detection of LPS.

 

 

Fig. 3 Analytical performance of the BTTS aptasensor for LPS detection. (A) Calibration plot of optical density vs. LPS detection. The dotted line represents the linear fit to the data. The inset shows semi- quantitatively color chart (upper) and chromogenic signal output (middle), the concentrations from left are 1, 10, 20, 50, 100 and 150 ng/mL of LPS. (B) Specificity of the BTTS aptasensor toward (a) blank control, (b) 50 ng/mL D-mannose, (c) 50 ng/mL BSA, (d) 50 ng/mL peptidoglycan from S. aureus, (e) 50 ng/mL Hb, (f) 50 ng/mL glucose, (g) 50 ng/mL HAS, (h) 50 ng/mL LPS from E.coli O55:B5, (i) 50 ng/mL D-mannose + 50 ng/mL BSA + 50 ng/mL peptidoglycan from S. aureus + 50 ng/mL Hb + 50 ng/mL glucose + 50 ng/mL HAS + 50 ng/mL LPS from E.coli O55:B5 + 50 ng/mL LPS from E.coli O111:B4, (j) 50 ng/mL LPS from E. coli O111:B4. Error bars represent the standard deviation (N = 3).

 

 

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