Development of a Cell-Free, Toehold Switch-Based Biosensor for Rapid and Sensitive Zika Virus Detection
开发用于快速、灵敏检测寨卡病毒的无细胞、基于 toehold Switch 的生物传感器

Yongbin Chen, Wenhao Xia, Ziwei Pan, Fuping Lu, Yihan Liu, Mingfeng Cao,* and Ning He*
陈永斌、夏文浩、潘紫薇、卢福平、刘逸涵、曹明峰*和何宁*

Cite This: Anal. Chem. 2025, 97, 3486-3494
引用此： Anal. Chem. 2025， 97， 3486-3494

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Abstract 抽象

The need for rapid and sensitive diagnostic tools is emphasized by the significant impact of infectious diseases on global health. This study presents a cell-free biosensor utilizing toehold switch technology, combined with nucleic acid sequencebased amplification (NASBA), for high specificity and sensitivity in Zika virus detection. The toehold switch, a denovo-designed regulator of gene expression, forms the crux of our detection system, offering a versatile and programmable approach to nucleic acid-based diagnostics. The cell-free system based on Escherichia coli extract served as the platform for sensor expression, enabling real-time monitoring and optimization of the reaction conditions for minimal background leakage and maximal activation efficiency. The performance of the toehold switch sensor was rigorously evaluated through a series of tests, revealing that switch S23 demonstrated the most promising activation effects and sequence specificity. Notably, the integration of NASBA technology significantly enhanced the detection sensitivity, achieving a remarkable limit of 2.9 aM , thus addressing the intrinsic limitation of toehold switches in detecting low-abundance targets. The detection system’s low cost, simplicity, and adaptability to various pathogens render it a valuable asset in the global health toolkit. This study presents a significant advancement in the field of synthetic biology, offering a robust, sensitive, and rapid diagnostic solution for Zika virus detection.
传染病对全球健康的重大影响强调了对快速、灵敏诊断工具的需求。本研究提出了一种利用 toehold 开关技术的无细胞生物传感器，结合基于核酸序列的扩增（NASBA），可在寨卡病毒检测中实现高特异性和灵敏度。toehold 开关是 Denovo 设计的基因表达调节因子，构成了我们检测系统的关键，为基于核酸的诊断提供了一种多功能且可编程的方法。基于大肠杆菌提取物的无细胞系统用作传感器表达平台，能够实时监测和优化反应条件，以实现最小的背景泄漏和最大的活化效率。通过一系列测试对 toehold 开关传感器的性能进行了严格评估，结果表明开关 S23 表现出最有希望的激活效应和序列特异性。值得注意的是，NASBA 技术的集成显著提高了检测灵敏度，达到了 2.9 aM 的显着极限，从而解决了脚趾固定开关在检测低丰度目标方面的固有局限性。该检测系统成本低、简单且对各种病原体的适应性使其成为全球健康工具包中的宝贵资产。本研究展示了合成生物学领域的重大进展，为寨卡病毒检测提供了一种稳健、灵敏和快速的诊断解决方案。

1. INTRODUCTION 1. 引言

Infectious disease outbreaks are usually large-scale events that can significantly disrupt agricultural systems, including livestock production, and present a major threat to public health. Many infectious diseases are caused by viruses,

^{2}

many of which have had a profound impact on human societies, such as the severe acute respiratory syndrome coronavirus 2 (SARS-CoV2 ), avian influenza virus, Zika virus, and norovirus. The untimely outbreaks of Zika and norovirus in underdeveloped areas including Africa have significantly impacted the productive lives of the local people.

^{3, 4}

The most important and primary strategy in the prevention and treatment of infectious diseases is to control the source of infection, which also means early detection, isolation, and treatment of infected patients.

^{5}

传染病爆发通常是大规模事件，可以严重破坏农业系统，包括畜牧生产，并对公共卫生构成重大威胁。许多传染病是由病毒引起的，

^{2}

其中许多病毒对人类社会产生了深远的影响，例如严重急性呼吸系统综合症冠状病毒 2 （SARS-CoV2）、禽流感病毒、寨卡病毒和诺如病毒。寨卡病毒和诺如病毒在包括非洲在内的欠发达地区不合时宜地爆发，严重影响了当地人民的生产生活。

^{3, 4}

预防和治疗传染病最重要和首要的策略是控制感染源，这也意味着早期发现、隔离和治疗感染患者。

^{5}

However, traditional viral diagnostic technologies are unable to meet the needs of large-scale rapid screening. Clinical diagnosis typically involves isolating and characterizing pathogens by detecting specific substances or their nucleic acid sequences.

^{5}

This process is complex and time-consuming, requiring multiple procedural steps. The target for detection is
然而，传统的病毒诊断技术已无法满足大规模快速筛查的需求。临床诊断通常包括通过检测特定物质或其核酸序列来分离和表征病原体。

^{5}

此过程复杂且耗时，需要多个程序步骤。检测对象为
commonly an unidentified pathogen. Although traditional methods such as enzyme-linked immunosorbent assay (ELISA), polymerase chain reaction (PCR), and quantitative real-time PCR (qPCR) exhibit high accuracy, they require specialized equipment and laboratory support, which limit their capacity to provide large-scale, on-site rapid detection in resource-limited areas.

^{6 - 8}

Point-of-care testing (POCT) has the advantages of fast, low cost, and simple operation, which is suitable for application in underdeveloped areas to realize the rapid screening and diagnosis of diseases.

^{9, 10}

The outbreak of COVID-19 has brought POCT closer to the public, accelerating innovation in the in vitro diagnostic industry and promoting the development of molecular POCT. The use of biosensors as detection elements is one of the directions of
通常是一种未识别的病原体。尽管酶联免疫吸附测定（ELISA）、聚合酶链反应（PCR）和定量实时 PCR （qPCR）等传统方法具有很高的准确性，但它们需要专门的设备和实验室支持，这限制了它们在资源有限地区提供大规模现场快速检测的能力。

^{6 - 8}

即时检测（POCT）具有速度快、成本低、作简单等优点，适用于欠发达地区应用，实现疾病的快速筛查和诊断。

^{9, 10}

COVID-19 的爆发使 POCT 更接近公众，加速了体外诊断行业的创新，促进了分子 POCT 的发展。使用生物传感器作为检测元件是

POCT technology development.

^{11}

The main mode of action of biosensors is to use biological molecules such as enzymes, antibodies, nucleic acids, etc., to serve as recognition elements, converting the results into readable electrical signals or light signals through signal transduction devices,

^{12 - 14}

but how to prolong cell viability and enhance the selectivity of specific detections remains challenging.

^{15}

Therefore, it is crucial to develop a new rapid, sensitive, and portable method for the POCT.
POCT 技术开发。

^{11}

生物传感器的主要作用方式是利用酶、抗体、核酸等生物分子作为识别元件，通过信号转导装置将结果转化为可读的电信号或光信号，

^{12 - 14}

但如何延长细胞活力和增强特异性检测的选择性仍然具有挑战性。

^{15}

因此，为 POCT 开发一种新的快速、灵敏和便携的方法至关重要。

Fortunately, cell-free synthetic biology is an emerging technology that can achieve the genetic central dogma of biology in vitro. As a platform technology without living cells, the cell-free system is expected to overcome the limitations of the cell membrane and directly modulate the reaction system. When the cell-free system is used for bioengineering, more carbon and energy can flow to the target product without considering the impact of excessive product accumulation on cell survival.

^{16, 17}

Moreover, cell-free systems enable more effective real-time monitoring of reaction processes compared to traditional cell-based systems, ensuring optimal conditions are maintained through timely adjustments and optimizations as the reaction progresses.

^{18}

By harnessing the transcriptiontranslation (TX-TL) mechanisms, cell-free systems can be engineered into open, modular biosensors. Specifically, the recognition mechanism for a target analyte is incorporated into the cell-free system, coupled with specific molecular structures and binding regulators.

^{19}

Subsequently, the TX-TL machinery activates the expression of reporter genes, enabling detection of the analyte and generation of readable signals, thereby constructing a functional cell-free biosensor for analytical and diagnostic applications.
幸运的是，无细胞合成生物学是一项新兴技术，可以在体外实现生物学的遗传中心法则。作为一种没有活细胞的平台技术，无细胞系统有望克服细胞膜的限制，直接调节反应系统。当无细胞系统用于生物工程时，更多的碳和能量可以流向目标产物，而无需考虑过度产物积累对细胞存活的影响。

^{16, 17}

此外，与传统的基于细胞的系统相比，无细胞系统能够更有效地实时监测反应过程，确保在反应过程中通过及时调整和优化来保持最佳条件。

^{18}

通过利用转录翻译（TX-TL）机制，可以将无细胞系统设计成开放的模块化生物传感器。具体来说，目标分析物的识别机制与特定的分子结构和结合调节因子相结合，被整合到无细胞系统中。

^{19}

随后，TX-TL 机制激活报告基因的表达，能够检测分析物并产生可读信号，从而构建用于分析和诊断应用的功能性无细胞生物传感器。
Toehold switches can serve both as genetic circuit components in vivo and as nucleic acid diagnostic tools in cell-free systems in vitro. As a detection element in cell-free biosensors, it represents a programmable component that is based on ribosome regulation. Its sequence flexibility enables researchers to efficiently modify it for various pathogens.

^{20}

Nicolaas et al. synthesized and characterized a dataset covering 23 viral genomes and 906 human transcription factors using toehold switches in vivo.

^{21}

Importantly, the toehold switch enables controlled initiation of gene expression in response to specific RNA triggers, providing a versatile tool for gene regulation and biosensing applications. This system offers notable advantages, including high specificity and a straightforward design. To improve the sensitivity of toehold switches, enzyme-mediated target preamplification is essential for signal amplification.

^{22, 23}

Nucleic acid sequence-based amplification (NASBA) technology is an enzymatic reaction process for isothermal amplification of specific nucleotide sequences. It can amplify the template RNA up to

10^{13}

times at a constant temperature in

2 h^{24}

and has been applied to the detection of various pathogenic microorganisms.

^{25, 26}

However, the limitations associated with its detection threshold constrain its utility in special detection scenarios.
Toehold 开关既可以用作体内的遗传电路组件，也可以用作体外无细胞系统中的核酸诊断工具。作为无细胞生物传感器中的检测元件，它代表了基于核糖体调节的可编程元件。其序列灵活性使研究人员能够针对各种病原体对其进行有效修饰。

^{20}

Nicolaas 等人在体内使用脚趾开关合成并表征了一个涵盖 23 个病毒基因组和 906 个人类转录因子的数据集。

^{21}

重要的是，脚趾固定开关能够响应特定 RNA 触发物的基因表达的受控起始，为基因调控和生物传感应用提供了多功能工具。该系统具有显著的优势，包括高特异性和简单的设计。为了提高 toehold 开关的灵敏度，酶介导的靶标预扩增对于信号放大至关重要。

^{22, 23}

基于核酸序列的扩增（NASBA）技术是一种用于特定核苷酸序列等温扩增的酶促反应过程。它可以在恒温下扩增模板 RNA 长达

10^{13}

数倍

2 h^{24}

，并已应用于各种病原微生物的检测。

^{25, 26}

但是，与其检测阈值相关的限制限制了它在特殊检测场景中的实用性。

In this study, the Zika virus was employed as the target analyte for detection purposes. A simple and cost-effective cellfree system based on Escherichia coli cell extract was used as an in vitro TX-TL platform. A toehold switch was introduced to develop a cell-free biosensor for the visual detection of the virus. To enhance the sensitivity of the detection method, we combined with NASBA technology optimized from multiple angles, resulting in a significant enhancement of the detection limit of the toehold switch to 2.9 aM , which demonstrates the
在这项研究中，寨卡病毒被用作检测目的的目标分析物。使用一种基于大肠杆菌细胞提取物的简单且经济高效的无细胞系统作为体外 TX-TL 平台。引入了一个脚趾固定开关来开发一种用于视觉检测病毒的无细胞生物传感器。为了提高检测方法的灵敏度，我们结合多角度优化的 NASBA 技术，导致脚趾固定开关的检测限显着提高到 2.9 aM，这表明
capability of cell-free biosensors based on the toehold switch to visualize and detect trace amounts at single-digit attomole levels. This study effectively merges the cell-free system with advanced molecular biology techniques to achieve portable, on-site testing without the need for expensive equipment, offering a promising avenue for the advancement of future diagnostic tools.
基于 ToeHold 开关的无细胞生物传感器能够可视化和检测个位数阿托摩尔水平的痕量。这项研究有效地将无细胞系统与先进的分子生物学技术相结合，无需昂贵的设备即可实现便携式现场检测，为未来诊断工具的发展提供了一条有前途的途径。

2. MATERIALS AND METHODS
2. 材料和方法

2.1. Toehold Switch Design. The Zika virus genome sequences were obtained from the NCBI database (https:// www.ncbi.nlm.nih.gov/). BLAST was used to conduct sequence alignment analysis on the Zika virus genome sequences from various regions to determine the conserved sequence. To design the toehold switches, the target sequences were selected from the conserved sequences, and the B-series sensor sequences,

^{25}

originally designed by Pardee et al., served as the conserved sequences for the hairpin structure (which can be adjusted during the actual design process). The universal sequences used in toehold switches are shown in Table S2. Bioinformatics analysis software Nupack was utilized to analyze and screen the toehold switch components. Following the Nupack software manual, the nucleic acid sequences were converted into a format recognizable by the software.

^{27}

After script writing, the script was run to obtain the theoretical structure of the toehold switch and the ensemble defect value data,

^{28}

thus completing the sequence design and initial screening.
2.1. 脚趾开关设计。寨卡病毒基因组序列来自 NCBI 数据库（https:// www.ncbi.nlm.nih.gov/）。BLAST 用于对来自不同区域的 Zika 病毒基因组序列进行序列比对分析，以确定保守序列。为了设计脚趾固定开关，从守恒序列中选择目标序列，

^{25}

最初由 Pardee 等人设计的 B 系列传感器序列用作发夹结构的守恒序列（可以在实际设计过程中进行调整）。脚趾固定开关中使用的通用序列如表 S2 所示。生物信息学分析软件 Nupack 用于分析和筛选脚趾固定开关组件。按照 Nupack 软件手册，将核酸序列转换为软件可识别的格式。

^{27}

脚本编写后，运行脚本，得到 toehold 开关的理论结构和集成缺陷值数据，

^{28}

从而完成序列设计和初步筛选。

2.2. Cell Extract Preparation. E. coli BL21-Star (DE3)
2.2. 细胞提取物制备。大肠杆菌 BL21-Star （DE3）

was used for the preparation of cell extract. Initially, a single colony was picked from a solid LB medium plate and inoculated into a LB liquid culture for overnight incubation to prepare the primary seed culture. Subsequently,

5 %

of the primary seed culture was inoculated into the YTP liquid medium to prepare the secondary culture. The

{OD}_{600}

value postinoculation was approximately 0.1 and was monitored regularly until it reached

0.6 - 0.8

. To induce the production of T7 RNA polymerase, 1 M IPTG solution was added to the culture to achieve a final IPTG concentration of 1 mM . The

{OD}_{600}

was periodically measured until it reached 3.0. The bacterial culture was then transferred to prechilled centrifuge tubes and centrifuged to collect the cells (

5000 g, 4^{\circ} C, 10

\min)

. The collected cells were washed 3 times with prechilled S30A buffer ( 14 mM magnesium glutamate; 60 mM potassium glutamate; 50 mM Tris, pH 7.7 ). The harvested cells could be flash-frozen in liquid nitrogen and stored at

- 80^{\circ} C

or used immediately for subsequent steps.
用于制备细胞提取物。最初，从固体 LB 培养基板中挑选单个菌落，并接种到 LB 液体培养物中过夜孵育，以制备原代种子培养物。随后，

5 %

将初级种子培养物接种到 YTP 液体培养基中以制备次级培养物。接种后

{OD}_{600}

的值约为 0.1，并定期监测直至达到

0.6 - 0.8

。为了诱导 T7 RNA 聚合酶的产生，向培养物中加入 1 M IPTG 溶液，以达到 1 mM 的最终 IPTG 浓度。定期测量直到

{OD}_{600}

达到 3.0。然后将细菌培养物转移到预冷的离心管中并离心以收集细胞（

5000 g, 4^{\circ} C, 10

\min)

.用预冷的 S30A 缓冲液（14 mM 谷氨酸镁;60 mM 谷氨酸钾;50 mM Tris，pH 7.7）洗涤收集的细胞 3 次。收获的细胞可以在液氮中快速冷冻并储存或

- 80^{\circ} C

立即用于后续步骤。

The bacterial cells were thoroughly mixed with 1 mL of prechilled S30A buffer per gram of cells. The mixture was then sonicated with the energy adjusted to

800 - 900 J

per 1.4 mL of bacterial suspension. The sonicated solution was centrifuged at

18, 000 g, 4^{\circ} C

, for 10 min . The supernatant was collected into a prechilled, clean centrifuge tube, 3 mM DTT was added, and incubated at

37^{\circ} C, 200 rpm

, for 60 min . Following incubation, the solution was centrifuged at

10, 000 g, 4^{\circ} C

, for 10 min . The supernatant was then transferred to a prechilled, clean centrifuge tube, flash-frozen in liquid nitrogen, and stored at

- 80^{\circ} C

for subsequent use.
将细菌细胞与每克细胞 1 mL 预冷的 S30A 缓冲液充分混合。然后对混合物进行超声处理，能量调整为

800 - 900 J

每 1.4 mL 细菌悬浮液。将超声溶液在

18, 000 g, 4^{\circ} C

下离心 10 min。将上清液收集到预冷、干净的离心管中，加入 3 mM DTT，并在

37^{\circ} C, 200 rpm

下孵育 60 分钟。孵育后，将溶液在

10, 000 g, 4^{\circ} C

下离心 10 分钟。然后将上清液转移至预冷、干净的离心管中，在液氮中快速冷冻，并储存

- 80^{\circ} C

以备后续使用。
2.3. Cell-Free Reaction and Sensor Screening. The cell-free reaction system included 14 mM Mg -glutamate, 80 mM K-glutamate, 1.5 mM ATP, 1.5 mM CTP, 1.2 mM GMP, 1.4 mM UMP,

0.2 mg / mL

yeast tRNA, 0.26 mM CoA, 0.33 mM NAD, 0.75 mM cAMP, 0.067 mM folinic acid, 1 mM
2.3. 无细胞反应和传感器筛选。无细胞反应系统包括 14 mM Mg-谷氨酸、80 mM K-谷氨酸、1.5 mM ATP、1.5 mM CTP、1.2 mM GMP、1.4 mM UMP、

0.2 mg / mL

酵母 tRNA、0.26 mM CoA、0.33 mM NAD、0.75 mM cAMP、0.067 mM 亚叶酸、1 mM

Figure 1. Recognition process of the toehold switch sensor. The toehold switch operates through RNA hybridization, allowing the switch to respond to target RNA sequences with high specificity due to precise base-pairing between the trigger RNA and the toehold domain. Upon the binding of the trigger RNA to the target region within the toehold switch, it causes a strand displacement and gradually unfolds the stem-loop structure, exposing the ribosome-binding site (RBS) and the start codon (AUG) that were previously occluded, thereby enabling translation of the reporter gene sfGFP. This results in a detectable fluorescence signal, indicating the presence of the target.
图 1.脚趾固定开关传感器的识别过程。toehold 开关通过 RNA 杂交起作用，由于触发 RNA 和 toehold 结构域之间的精确碱基配对，允许开关以高特异性响应靶标 RNA 序列。当触发 RNA 与脚趾保持开关内的目标区域结合时，它会导致链位移并逐渐展开茎环结构，暴露出先前被封闭的核糖体结合位点（RBS）和起始密码子（AUG），从而能够翻译报告基因 sfGFP。这会产生可检测的荧光信号，表明目标的存在。
spermidine, 5.7 mM each of 20 amino acids,

0.9 μ L

40 %

PEG6000, and 34 mM glyceraldehyde 3-phosphate.
亚精胺、20 个氨基酸各 5.7 mM、

0.9 μ L

40 %

PEG6000 和 34 mM 3-磷酸甘油醛。
The switch sequences of the sensors, target trigger sequences, and primers were synthesized by Shanghai Sangon Biotech Co., Ltd. onto the pIVEX 2.4c plasmid or in the form of linear fragments. In the cell-free reaction system,

2.5, 5

, and 7.5 nM switch plasmid were added, and both short-term ( 2 h ) and long-term ( 14 h ) monitoring were performed. The reaction mixtures were incubated in a

37^{\circ} C

incubator. After the reaction,

10 μ L

of the reaction solution was added to each well of a black 96-well plate containing

190 μ L

of deionized water, and the plate was read using a microplate reader (excitation wavelength: 488 nm ; emission wavelength: 520 nm).
传感器的开关序列、靶标触发序列和引物由上海桑贡生物科技有限公司合成到 pIVEX 2.4c 质粒上或以线性片段的形式合成。在无细胞反应系统中，

2.5, 5

加入和 7.5 nM 开关质粒，并进行短期（ 2 h ）和长期（ 14 h ）监测。将反应混合物在

37^{\circ} C

培养箱中孵育。反应结束后，

10 μ L

将反应溶液加入含有

190 μ L

去离子水的黑色 96 孔板的每个孔中，并使用酶标仪（激发波长：488 nm;发射波长：520 nm）读取板。

Based on the experimental results, toehold switch components with low background leakage were selected and subjected to a dual-plasmid activation test in the cell-free system (switch plasmid and trigger plasmid), with each plasmid added at a concentration of 2.5 nM . Both shortterm and long-term monitoring were performed. The specific operation and result collection methods were as described previously.
根据实验结果，选择具有低背景泄漏的 toehold 开关组分，并在无细胞系统（转换质粒和触发质粒）中进行双质粒激活试验，每个质粒添加浓度为 2.5 nM。进行了短期和长期监测。具体作和结果收集方法如前所述。

Switches with favorable activation effects were chosen for the subsequent tests: (1) increasing the concentration of switch plasmid ( 5 and 7.5 nM ); (2) assessing the detection capability of long conserved sequences at three different switch plasmid concentrations; (3) determining the detection limit of the toehold switch; and (4) examining the specificity of the toehold switch for the target sequence. Short-term monitoring

(2 h)

was conducted.
选择具有良好激活效果的开关进行后续测试：（1）增加开关质粒的浓度（5 和 7.5 nM）;（2）评估 3 种不同开关质粒浓度下长保守序列的检测能力;（3）确定 toehold 开关的检测限;（4）检查目标序列的 toehold 开关的特异性。进行了短期监测

(2 h)

。
2.4. RNA Template Preparation. High-fidelity RNA polymerase (P515, Vazyme) was used to amplify the gene sequence from the plasmid template containing part of the
2.4. RNA 模板制备。使用高保真 RNA 聚合酶（P515， Vazyme）从含有部分

Zika virus genome sequence, where the upstream primer contained the T7 promoter sequence. After confirmation of the size of the amplified band by

3 %

agarose gel electrophoresis, the fragment was recovered using the gel extraction kit (D2500, OMEGA). Finally, the T7 in vitro transcription kit (TR101, Vazyme) was used for in vitro transcription, and the RNA purification kit (TR205, Genstone) was used for RNA purification and recovery to obtain the RNA template required for NASBA system amplification.
寨卡病毒基因组序列，其中上游引物包含 T7 启动子序列。通过

3 %

琼脂糖凝胶电泳确认扩增条带的大小后，使用凝胶提取试剂盒（D2500， OMEGA）回收片段。最后，使用 T7 体外转录试剂盒（TR101， Vazyme）进行体外转录，使用 RNA 纯化试剂盒（TR205， Genstone）进行 RNA 纯化和回收，以获得 NASBA 系统扩增所需的 RNA 模板。
2.5. NASBA Amplification and Urea-PAGE Verification. The

20 μ

L NASBA system comprised

5 \times

NASBA buffer

(50 mM MgCl_{2}, 300 mM KCl, 50 mM

DTT, 400 mM Tris

HCl), 2 μ L

dimethyl sulfoxide (DMSO),

0.8 μ L

dNTP ( 25

mM), 1.6 μ L

NTP ( 25 mM ),

0.5 μ L

RNase inhibitor (

40 U /

μ L), 4 μ M

of each NASBA primer,

1 μ L

RNA template, and RNase-free water. This mixture was then incubated at

65^{\circ} C

for 5 min , followed by incubation at

41^{\circ} C

for 5 min . Immediately after, the enzyme solution containing 0.1 mg .

{mL}^{- 1}

bovine serum albumin (BSA), 32 U T7 RNA Polymerase (2540A, Takara), 0.1 U RNase H (M0297S, NEB), and 6.4 U AMV reverse transcriptase (M0277S, NEB) was added to the mixture to bring the final volume to

20 μ L

. The mixture was further incubated at

41^{\circ} C

for

90 - 150 \min

. The obtained samples were verified using

8 %

Urea-PAGE gel. For validation with the cell-free system,

1 μ L

of NASBA sample was added to the cell-free system for reaction.
2.5. NASBA 扩增和 Urea-PAGE 验证。L

20 μ

NASBA 系统包括

5 \times

NASBA 缓冲液

(50 mM MgCl_{2}, 300 mM KCl, 50 mM

DTT、400 mM Tris

HCl), 2 μ L

二甲基亚砜（DMSO）、

0.8 μ L

dNTP （ 25

mM), 1.6 μ L

NTP （ 25 mM）、

0.5 μ L

RNase 抑制剂（

40 U /

μ L), 4 μ M

每个 NASBA 引物、

1 μ L

RNA 模板和不含 RNase 的水。然后将该混合物孵育

65^{\circ} C

5 分钟，然后

41^{\circ} C

孵育 5 分钟。紧接着，加入含有 0.1 mg .

{mL}^{- 1}

向混合物中加入牛血清白蛋白（BSA）、32 U T7 RNA 聚合酶（2540A， Takara）、0.1 U RNase H （M0297S， NEB）和 6.4 U AMV 逆转录酶（M0277S， NEB），使最终体积达到

20 μ L

。将混合物进一步孵育

41^{\circ} C

90 - 150 \min

。使用

8 %

Urea-PAGE 凝胶验证所获得的样品。为了使用无细胞系统进行验证，

1 μ L

将 NASBA 样品添加到无细胞系统中进行反应。

3. RESULTS AND DISCUSSION
3. 结果和讨论

3.1. Design and Screening of the RNA Toehold Switch Sensors. The functionality of RNA sensors is enabled through the programmable interaction between toehold switches and their complementary target sequences (trigger RNAs). This method can be used for the detection of
3.1. RNA Toehold Switch 传感器的设计和筛选。RNA 传感器的功能是通过 toehold 开关与其互补靶序列（触发 RNA）之间的可编程交互实现的。该方法可用于检测

Received: October 29, 2024
收稿日期： 2024-10-29
Revised: January 22, 2025
修订日期：2025 年 1 月 22 日
Accepted: January 28, 2025
录用日期： 2025-01-28
Published: February 10, 2025
发布时间：2025 年 2 月 10 日

Development of a Cell-Free, Toehold Switch-Based Biosensor for Rapid and Sensitive Zika Virus Detection 开发用于快速、灵敏检测寨卡病毒的无细胞、基于 toehold Switch 的生物传感器

Abstract 抽象

1. INTRODUCTION 1. 引言

2. MATERIALS AND METHODS2. 材料和方法

2.2. Cell Extract Preparation. E. coli BL21-Star (DE3)2.2. 细胞提取物制备。大肠杆菌 BL21-Star （DE3）

3. RESULTS AND DISCUSSION3. 结果和讨论

Development of a Cell-Free, Toehold Switch-Based Biosensor for Rapid and Sensitive Zika Virus Detection
开发用于快速、灵敏检测寨卡病毒的无细胞、基于 toehold Switch 的生物传感器

2. MATERIALS AND METHODS
2. 材料和方法

2.2. Cell Extract Preparation. E. coli BL21-Star (DE3)
2.2. 细胞提取物制备。大肠杆菌 BL21-Star （DE3）

3. RESULTS AND DISCUSSION
3. 结果和讨论