Split crRNA Precisely Assisted Cas12a Expansion Strategy for Simultaneous，Discriminative，and Low－Threshold Determination of Two miRNAs Associated with Multiple Sclerosis
《精确辅助 Cas12a 扩展策略的分离 crRNA 用于同时、区分性和低阈值测定与多发性硬化症相关的两种 miRNA》

Cite This：https：／／doi．org／10．1021／acs．analchem．4c05388
Cite This：https://doi.org/10.1021/acs.analchem.4c05388

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Multiple sclerosis（MS）is an organ－specific autoimmune neurodegenerative disease characterized by chronic inflamma－ tory demyelination of the central nervous system，axon damage，and impaired neuromuscular function．${}^{1,2}\mathrm{MS}$${}^{1,2}\mathrm{MS}$^(1,2)MS{ }^{1,2} \mathrm{MS} is one of the leading causes of non－traumatic neurological disorders in young people and occurs more frequently in the ages of 20－ $40{.}^3$$40{.}^3$40.^(3)40 .^{3} Studies have shown that females have a higher prevalence， which may be related to hormonal，genetic，or environmental factors．The disease is clinically heterogeneous，and approx－ imately $85\mathrm{\%}$$85\mathrm{\%}$85%85 \% of patients progress to relapsing－remitting MS （RRMS）．More than $50\mathrm{\%}$$50\mathrm{\%}$50%50 \% of RRMS patients eventually transmit into secondary progressive MS，which is characterized by a steady decline in neurological function within 10 years．In addition，about $15\mathrm{\%}$$15\mathrm{\%}$15%15 \% of MS patients are primary progressive MS，with deterioration of neurological function from the onset of symptoms．${}^{4,5}$${}^{4,5}$^(4,5){ }^{4,5} Currently，the clinical diagnosis methods of MS include oligoclonal bands in cerebrospinal fluid，${}^6$${}^6$^(6){ }^{6} the white matter／gadolinium－enhancing lesions detected by magnetic resonance imaging，${}^7$${}^7$^(7){ }^{7} and the John Cunningham virus antibody titers，${}^8$${}^8$^(8){ }^{8} which can further identify the possible risk of developing progressive multifocal leukoencephalopathy in these patients．However，these methods have problems with
多发性硬化症（MS）是一种器官特异性自身免疫性神经退行性疾病，其特征是中枢神经系统慢性炎症性脱髓鞘、轴突损伤和神经肌肉功能受损。 ${}^{1,2}\mathrm{MS}$${}^{1,2}\mathrm{MS}$^(1,2)MS{ }^{1,2} \mathrm{MS} 是年轻人非创伤性神经系统疾病的主要原因之一，常见于 20- $40{.}^3$$40{.}^3$40.^(3)40 .^{3} 岁之间。研究表明，女性患病率更高，这可能与激素、遗传或环境因素有关。该病临床表现异质性，大约 $85\mathrm{\%}$$85\mathrm{\%}$85%85 \% 的患者进展为复发-缓解型 MS（RRMS）。超过 $50\mathrm{\%}$$50\mathrm{\%}$50%50 \% 的 RRMS 患者最终转变为继发进展型 MS，其特征是在 10 年内神经系统功能持续下降。此外，约 $15\mathrm{\%}$$15\mathrm{\%}$15%15 \% 的 MS 患者为原发进展型 MS，从症状出现时起神经系统功能逐渐恶化。 ${}^{4,5}$${}^{4,5}$^(4,5){ }^{4,5} 目前，MS 的临床诊断方法包括脑脊液中的寡克隆带、 ${}^6$${}^6$^(6){ }^{6} 通过磁共振成像检测到的白质/钆增强病变、 ${}^7$${}^7$^(7){ }^{7} 以及约翰·坎宁安病毒抗体滴度， ${}^8$${}^8$^(8){ }^{8} 这些可以进一步识别可能的发展风险 这些患者中的进行性多灶性白质脑病。然而，这些方法存在一些问题
the necessity for advanced equipment，intricate operational procedures，and skilled technicians，which are not conducive to the portable diagnosis of MS．Therefore，considering the significance of point－of－care testing for human analytes，which can save limited medical resources and assist in clinical diagnosis，${}^{9,10}$${}^{9,10}$^(9,10){ }^{9,10} there is a pressing need to devise a portable and precise diagnostic approach for MS，thus propelling advance－ ments in clinical diagnosis．
对先进设备的需求、复杂的操作程序以及需要熟练技术人员，这些都不利于多发性硬化症（MS）的便携式诊断。因此，考虑到床旁检测人类分析物的重要性，这可以节省有限的医疗资源并辅助临床诊断， ${}^{9,10}$${}^{9,10}$^(9,10){ }^{9,10} 迫切需要设计一种便携且精确的多发性硬化症诊断方法，从而推动临床诊断的进步

MicroRNAs（miRNAs）are a class of small endogenous noncoding RNAs（about 22 nucleotides in length）that regulate post－transcriptional gene expression by binding to target genes，resulting in degradation or transcriptional inhibition of target genes，and have emerged as key participants in gene expression regulation．${}^{11}$${}^{11}$^(11){ }^{11} Dysregulated miRNAs expression can be a useful diagnostic，prognostic，or
MicroRNAs（miRNAs）是一类小型的内源性非编码 RNA（长度约为 22 个核苷酸），通过结合目标基因来调控转录后的基因表达，导致目标基因的降解或转录抑制，已成为基因表达调控中的关键参与者。 ${}^{11}$${}^{11}$^(11){ }^{11} miRNAs 表达的失调可以作为有用的诊断、预后或

therapeutic marker in the development of diseases. ${}^{12}$${}^{12}$^(12){ }^{12} Current research has shown that neurological diseases such as MS, Alzheimer’s disease, Parkinson’s disease, brain tumors and so on have a strong correlation with miRNA disorders. ${}^{13-15}$${}^{13-15}$^(13-15){ }^{13-15} Beyond modulating expression levels, these RNAs possess the capacity to induce disease by altering target gene mutations. ${}^{16}$${}^{16}$^(16){ }^{16} Extensive expression profiling studies have scrutinized miRNA profiles in both active and inactive MS lesions, ${}^{5,17}$${}^{5,17}$^(5,17){ }^{5,17} revealing significantly elevated levels of miRNA-155 and miRNA-326 in active lesions compared to inactive ones or normal white matter. Animal model studies have also demonstrated these two miRNAs to be promising novel biomarkers and therapeutic targets, sparking growing interest in this rapidly evolving field. ${}^{18-20}$${}^{18-20}$^(18-20){ }^{18-20} Currently, numerous studies have utilized miRNAs as potential disease biomarkers in developing detection methods, but these efforts face challenges in striking the right balance between detection efficiency and accuracy across various miRNA targets. ${}^{21,22}$${}^{21,22}$^(21,22){ }^{21,22} Notably, accurately identifying low-threshold miRNA levels or ensuring high specificity for multiple analytes concurrently in complex biological environments remains a formidable task. ${}^{23,24}$${}^{23,24}$^(23,24){ }^{23,24} miRNAs, as biomarkers of MS, face the same problem. Therefore, the development of a biosensor that is capable of simultaneous, discriminative, and low-threshold detection of these two miRNAs is crucial for the clinical diagnosis of MS.
治疗标志物在疾病发展中的作用。 ${}^{12}$${}^{12}$^(12){ }^{12} 当前研究表明，多发性硬化症、阿尔茨海默病、帕金森病、脑肿瘤等神经系统疾病与 miRNA 紊乱有强烈相关性。 ${}^{13-15}$${}^{13-15}$^(13-15){ }^{13-15} 除了调节表达水平外，这些 RNA 还能通过改变靶基因突变来诱导疾病。 ${}^{16}$${}^{16}$^(16){ }^{16} 广泛的表达谱研究已经详细分析了活跃和不活跃 MS 病变中的 miRNA 谱， ${}^{5,17}$${}^{5,17}$^(5,17){ }^{5,17} 发现与不活跃病变或正常白质相比，活跃病变中 miRNA-155 和 miRNA-326 的水平显著升高。动物模型研究也表明，这两种 miRNA 是有前景的新型生物标志物和治疗靶点，引发了这一快速演变领域的日益关注。 ${}^{18-20}$${}^{18-20}$^(18-20){ }^{18-20} 目前，许多研究已将 miRNA 作为潜在疾病生物标志物用于开发检测方法，但这些努力在平衡各种 miRNA 靶标的检测效率和准确性方面面临挑战。 ${}^{21,22}$${}^{21,22}$^(21,22){ }^{21,22} 值得注意的是，在复杂的生物环境中准确识别低阈值的 miRNA 水平，或确保对多种分析物同时具有高特异性，仍然是一项艰巨的任务。 ${}^{23,24}$${}^{23,24}$^(23,24){ }^{23,24} 作为多发性硬化症（MS）的生物标志物，miRNA 也面临同样的问题。因此，开发一种能够同时、区分且低阈值检测这两种 miRNA 的生物传感器，对于多发性硬化症的临床诊断至关重要。

Clustered regularly interspaced short palindromic repeats (CRISPR) technology is regarded as a revolutionary genome editing tool that has garnered significant interest for its immense potential in nucleic acid-based molecular diagnostics. ${}^{25,26}$${}^{25,26}$^(25,26){ }^{25,26} In the CRISPR-Cas family, Cas12a is ingeniously utilized in the field of analytical detection due to its unique DNA nuclease activity. ${}^{27-29}$${}^{27-29}$^(27-29){ }^{27-29} Cas12a forms a ribonucleoprotein complex with CRISPR RNA (crRNA) that is able to identify, unwind, and produce highly specific double-strand breaks in DNA targets complementary to the spacer of crRNA, known as cis-cleavage activity. ${}^{30}$${}^{30}$^(30){ }^{30} This feature occurs in all activated Cas12a, but the accessibility of target sequence is relatively poor due to the limitation of crRNA’s inherent sequence, so signal output in this way is rarely reported. Besides, the activated Cas12a can also indiscriminately degrade singlestranded DNA (ssDNA) substrates efficiently with a turnover rate of $1250/\mathrm{s}$$1250/\mathrm{s}$1250//s1250 / \mathrm{s}, which is defined as trans-cleavage capability. ${}^{31,32}$${}^{31,32}$^(31,32){ }^{31,32} Currently, Cas12a has generally been considered to be active only against DNA targets and has been widely used in the field of clinical diagnostics. Few studies of its RNAactivated ability to directly detect RNA are based on high concentrations of different Cas12a subtypes or remote RNA recognition via protospacer adjacent motif (PAM)-proximal seed DNAs. ${}^{33,34}$${}^{33,34}$^(33,34){ }^{33,34} Notably, RNA activation of Cas12a can also be achieved by splitting the crRNA and utilizing the target RNA as part of its division. ${}^{35}$${}^{35}$^(35){ }^{35} However, all these designs have not been able to overcome the constraints of indistinguishable signal output caused by the undifferentiated nature of Cas12a’s trans-cleavage, which hinders its application in the field of simultaneous detection. Currently, this limitation of Cas12a can be removed by combining with Cas13a, which can specifically recognize and cleave single-stranded RNA (ssRNA), or developing devices that can physically separate different components of the Cas12a system, but there exist problems of high enzyme cost, multi-crRNA requirements and cumbersome device design. ${}^{36,37}$${}^{36,37}$^(36,37){ }^{36,37} Therefore, the realization of single-Cas12a-based simultaneous detection in one pot remains challenging. Overcoming this obstacle is a significant
成簇规律性间隔短回文重复序列（CRISPR）技术被认为是一种革命性的基因编辑工具，因其基于核酸的分子诊断巨大潜力而备受关注。 ${}^{25,26}$${}^{25,26}$^(25,26){ }^{25,26} 在 CRISPR-Cas 家族中，Cas12a 因其独特的 DNA 核酸酶活性而在分析检测领域被巧妙应用。 ${}^{27-29}$${}^{27-29}$^(27-29){ }^{27-29} Cas12a 与 CRISPR RNA（crRNA）形成核糖核蛋白复合物，能够识别、解旋并产生与 crRNA 间隔区互补的 DNA 靶标的高度特异性双链断裂，这一特性被称为顺式切割活性。 ${}^{30}$${}^{30}$^(30){ }^{30} 这一特性出现在所有激活的 Cas12a 中，但由于 crRNA 固有序列的限制，靶序列的可接近性相对较差，因此通过这种方式输出的信号很少被报道。此外，激活的 Cas12a 还能无差别地高效降解单链 DNA（ssDNA）底物，其转换率为 $1250/\mathrm{s}$$1250/\mathrm{s}$1250//s1250 / \mathrm{s} ，这被定义为反式切割能力。 ${}^{31,32}$${}^{31,32}$^(31,32){ }^{31,32} 目前，Cas12a 普遍被认为仅对 DNA 靶标具有活性，并已广泛应用于临床诊断领域。 很少有研究基于高浓度的不同 Cas12a 亚型或通过原型间隔基序（PAM）附近的种子 DNA 进行远程 RNA 识别，来直接检测其 RNA 激活能力。值得注意的是，Cas12a 的 RNA 激活也可以通过分割 crRNA 并利用目标 RNA 作为其分割的一部分来实现。然而，所有这些设计都未能克服 Cas12a 的跨切割未分化特性所导致的不可区分信号输出的限制，这阻碍了其在同时检测领域的应用。目前，这一 Cas12a 的局限性可以通过与能够特异性识别和切割单链 RNA（ssRNA）的 Cas13a 结合，或开发能够物理分离 Cas12a 系统不同组件的设备来消除，但存在酶成本高、多-crRNA 需求和设备设计繁琐的问题。因此，实现基于单一 Cas12a 的一锅式同时检测仍然具有挑战性。克服这一障碍具有重要意义。
milestone for the development of Cas12a in the field of simultaneous, discriminative, and low-threshold detection of multiple miRNAs, which may potentially impact clinical diagnosis and treatment.
Cas12a 在多 miRNA 的同时、区分性和低阈值检测领域发展的里程碑，这可能对临床诊断和治疗产生潜在影响。

Hence, our research was committed to construct a universal detection platform based on the Cas12a system, which was named “split crRNA precisely assisted Cas12a expansion (SPACE)”. The platform pioneered simultaneous, discriminative, and low-threshold detection of dual MS-related miRNAs in one pot and one step. First, the limitation of Cas12a’s inability to directly detect miRNAs could be circumvented with particularly engineered crRNA. Studies have demonstrated that crRNA split at the appropriate site could also activate Cas12a efficiently. ${}^{38}$${}^{38}$^(38){ }^{38} Inspired by this property, the dexterous design that miRNA-155 and miRNA326 served as spacer parts of crRNA and combined with scaffold to form an integrated crRNA could break through the constraints of Cas12a-specific DNA recognition and lay a foundation for subsequent multi-miRNAs detection. Second, regarding the problem that Cas12a could not produce distinguishable signal outputs, the SPACE strategy designed the corresponding activators according to miRNAs and integrated them with the reporter. Then, the cis-cleavage activity of Cas12a was utilized to realize the integrative recognition and discriminative signal output of miRNA-155 and miRNA-326. To further expand the application dimension, trans-cleavage with better turnover efficiency was also jointly employed in the SPACE strategy to advance the prospect of this strategy in calculating the total amount of target miRNAs in low thresholds, thus successfully realizing the identification of healthy individuals and MS patients. In conclusion, the SPACE strategy pioneered a novel Cas12a simultaneous detection strategy and applied it to discriminative and lowthreshold quantification of dual MS-related miRNAs, significantly promoting the diversification of Cas12a applications in the field of analytical detection and clinical diagnosis.
因此，我们的研究致力于构建一个基于 Cas12a 系统的通用检测平台，该平台被命名为“分离 crRNA 精确辅助 Cas12a 扩展策略（SPACE）”。该平台首创了在单次反应中同时、区分性和低阈值检测两种与 MS 相关的 miRNA。首先，通过特别设计的 crRNA 可以克服 Cas12a 无法直接检测 miRNA 的局限。研究表明，在适当位置分裂的 crRNA 也能有效激活 Cas12a。 ${}^{38}$${}^{38}$^(38){ }^{38} 受此特性启发，巧妙地将 miRNA-155 和 miRNA326 作为 crRNA 的间隔部分，并与支架结合形成完整的 crRNA，能够突破 Cas12a 特异性 DNA 识别的限制，为后续的多 miRNA 检测奠定基础。其次，针对 Cas12a 无法产生可区分信号输出的问题，SPACE 策略根据 miRNA 设计了相应的激活剂，并将其与报告基因整合。 然后，利用 Cas12a 的顺式切割活性实现了对 miRNA-155 和 miRNA-326 的综合识别和区分信号输出。为进一步拓展应用维度，SPACE 策略中还联合采用了具有更好周转效率的反式切割，以推进该策略在低阈值下计算目标 miRNA 总量的前景，从而成功实现了健康个体和 MS 患者的识别。总之，SPACE 策略开创了一种新型的 Cas12a 同步检测策略，并将其应用于区分和低阈值量化双重 MS 相关 miRNA，显著促进了 Cas12a 在分析检测和临床诊断领域的应用多样化。

Materials and Reagents. All oligonucleotides (as shown in Supporting Information Tables S1-S3) were synthesized and purified by Shanghai Sangon Biotechnology Co.; Ltd. (Shanghai, China). LbaCas12a nuclease (Cpf1) was purchased from Novoprotein Scientific Inc. (Suzhou, China). AsCas12a nuclease (Cpf1) was purchased from Ezassay Biotechnology Inc. (Shenzhen, China). RNAiso Blood was purchased from Takara Biomedical Technology Co.; Ltd. (Beijing, China). miRNA first Strand cDNA Synthesis Kit (by stem-loop) and ChamQ Blue Universal SYBR qPCR Master Mix were purchased from Vazyme Biotech Co.; Ltd. (Nanjing, China). All experimental water was obtained through the Milli-Q purification system. All other chemicals were of analytical reagent grade.
材料和试剂。所有寡核苷酸（如支持信息表 S1-S3 所示）均由上海桑戈生物技术有限公司（上海，中国）合成和纯化。LbaCas12a 核酸酶（Cpf1）购自苏州诺沃蛋白质科学有限公司（苏州，中国）。AsCas12a 核酸酶（Cpf1）购自深圳易赛生物技术有限公司（深圳，中国）。RNAiso Blood 购自宝日医生物技术（北京）有限公司（北京，中国）。miRNA 第一链 cDNA 合成试剂盒（通过茎环法）和 ChamQ Blue 通用 SYBR qPCR 预混液均购自南京诺唯赞生物科技有限公司（南京，中国）。所有实验用水均通过 Milli-Q 纯化系统获得。所有其他化学试剂均为分析纯。

Preparation of Double-Strand DNA Substrates. The fluorophores (TAMRA and Cy5)-labeled target strands (TS) and quenchers (BHQ2 and BHQ3)-labeled non-target strands (NTS) were first denatured for 5 min at ${95}^{\circ }\mathrm{C}$${95}^{\circ }\mathrm{C}$95^(@)C95^{\circ} \mathrm{C} and then subsequently annealed by slowly cooling to room temperature naturally to fold into perfect and stable double-strand DNA (dsDNA) structures (dsDNA-T and dsDNA-C), respectively.
双链 DNA 底物的制备。首先将荧光素（TAMRA 和 Cy5）标记的目标链（TS）和淬灭剂（BHQ2 和 BHQ3）标记的非目标链（NTS）在 ${95}^{\circ }\mathrm{C}$${95}^{\circ }\mathrm{C}$95^(@)C95^{\circ} \mathrm{C} 下变性 5 分钟，然后通过自然缓慢冷却至室温，分别折叠成完美且稳定的双链 DNA（dsDNA）结构（dsDNA-T 和 dsDNA-C）。

Simultaneous and Discriminative Detection of Multiple MS-Related miRNAs. All reactions were performed in a volume of $100\mu \mathrm{L}$$100\mu \mathrm{L}$100 muL100 \mu \mathrm{~L} in DNase/RNase-free tubes. Cas12a ( 80 $\mathrm{nM})$$\mathrm{nM})$nM)\mathrm{nM}), scaffold ( 30 nM ), dsDNA-T ( 100 nM ), dsDNA-C ( 150
同时和区分性检测多种 MS 相关 miRNA。所有反应均在 $100\mu \mathrm{L}$$100\mu \mathrm{L}$100 muL100 \mu \mathrm{~L} 体积的 DNase/RNase-free 管中进行。Cas12a（80 $\mathrm{nM})$$\mathrm{nM})$nM)\mathrm{nM}) ，支架（30 nM），dsDNA-T（100 nM），dsDNA-C（150

Scheme 1. Illustration of the Principle of SPACE Strategy for Simultaneous, Discriminative, and Low-Threshold Detection of Multiple MS-Related miRNAs. (A) Traditional Pattern for the Activation of Cas12a. (B) Schematic Diagram of Split crRNA for the Activation of Cas12a. © Schematic Diagram of the SPACE Strategy. Cas12a in Gray Represents Inactivated and Purple Represent Activated, Respectively
Scheme 1. SPACE 策略用于同时、区分性和低阈值检测多种 MS 相关 miRNA 的原理图示。(A) Cas12a 激活的传统模式。(B) 用于激活 Cas12a 的分裂 crRNA 示意图。© SPACE 策略的示意图。灰色表示未激活的 Cas12a，紫色表示已激活的 Cas12a。

$\mathrm{nM})$$\mathrm{nM})$nM)\mathrm{nM}), and ssDNA substrates labeled with a fluorophore (FAM) and quencher (BHQ1) pair (FQ probe) ( 100 nM ) and different concentrations of miRNA-155 and miRNA-326 were conducted with $1\times$$1\times$1xx1 \times NEBuffer 2.1 at $37{}^{\circ }\mathrm{C}$$37{}^{\circ }\mathrm{C}$37^(@)C37{ }^{\circ} \mathrm{C} for 2 h . The fluorescence intensities of the reaction system were then measured and the specific fluorescence detection parameters are listed in the Supporting Information.
$\mathrm{nM})$$\mathrm{nM})$nM)\mathrm{nM}) ，以及标记有荧光团（FAM）和猝灭剂（BHQ1）对的 ssDNA 底物（FQ 探针）（100 nM）和不同浓度的 miRNA-155 和 miRNA-326 在 $1\times$$1\times$1xx1 \times NEBuffer 2.1 中于 $37{}^{\circ }\mathrm{C}$$37{}^{\circ }\mathrm{C}$37^(@)C37{ }^{\circ} \mathrm{C} 进行 2 小时的反应。然后测量反应系统的荧光强度，具体的荧光检测参数列在支持信息中。

Working Principle for Determining Multiple MSRelated miRNAs through the SPACE Strategy. The typical Cas12a system is a ternary complex composed of Cas12a protein, crRNA, and activator dsDNA (Scheme 1A). Among them, the required crRNA is a complete ssRNA consisting of an identical “scaffold” motif which folds into a pseudoknot recognized by Cas12a and a variable “spacer” motif that complements the “protospacer” motif in the activator. ${}^{39}$${}^{39}$^(39){ }^{39} Cas12a can identify the thymidine (T)-rich PAM in target dsDNA activator through shape and base readout mechanisms, progressively unwinding the activator dsDNA with the hybridization of crRNA and TS to form a complete Rloop, thus catalytically activating Cas12a, which licenses ciscleavage of the target dsDNA. ${}^{40}$${}^{40}$^(40){ }^{40} The cis-cleavage mediated by Cas12a is staggered and the cleavage sites occur after the 18th base on the NTS and the 23rd base on TS from PAM. ${}^{41,42}$${}^{41,42}$^(41,42){ }^{41,42} Furthermore, it has been reported that the integrated crRNA could still work when it was split between the scaffold and
通过 SPACE 策略确定多种 MS 相关 miRNA 的工作原理。典型的 Cas12a 系统是由 Cas12a 蛋白、crRNA 和激活剂 dsDNA 组成的三元复合物（方案 1A）。其中，所需的 crRNA 是一个完整的 ssRNA，包含一个相同的“支架”基序，该基序折叠成被 Cas12a 识别的假结，以及一个可变的“间隔”基序，该基序与激活剂中的“前间隔”基序互补。 ${}^{39}$${}^{39}$^(39){ }^{39} Cas12a 可以通过形状和碱基读取机制识别目标 dsDNA 激活剂中的胸腺嘧啶（T）丰富的 PAM，逐步解开激活剂 dsDNA，并与 crRNA 和 TS 的杂交形成完整的 R 环，从而催化激活 Cas12a，使其能够顺式切割目标 dsDNA。 ${}^{40}$${}^{40}$^(40){ }^{40} 由 Cas12a 介导的顺式切割是错开的，切割位点发生在 PAM 后的 NTS 第 18 个碱基和 TS 第 23 个碱基处。 ${}^{41,42}$${}^{41,42}$^(41,42){ }^{41,42} 此外，有报道称，当 crRNA 在支架和间隔之间分裂时，整合的 crRNA 仍然可以发挥作用。
spacer (Scheme 1B). ${}^{38}$${}^{38}$^(38){ }^{38} As shown in Figure S1, Cas12a could be effectively activated by ssDNA or dsDNA regardless of whether the crRNA was intact or split, but the activation effects of intact crRNA were stronger. Moreover, for different variants of Cas12a, ssDNA with split crRNA had a stronger activation capacity to AsCas12a than LbaCas12a, while dsDNA with split crRNA had a similar ability to activate AsCas12a as LbaCas12a. Leveraging this characteristic, miRNA can be ingeniously engineered as a spacer to break through the inherent limitations that Cas12a cannot be used to detect RNA directly. This innovation laid the foundation for the development and application of the subsequent SPACE strategy.
间隔区（图 1B）。 ${}^{38}$${}^{38}$^(38){ }^{38} 如图 S1 所示，无论 crRNA 是完整的还是分裂的，Cas12a 都可以被 ssDNA 或 dsDNA 有效激活，但完整 crRNA 的激活效果更强。此外，对于不同变体的 Cas12a，带有分裂 crRNA 的 ssDNA 对 AsCas12a 的激活能力比 LbaCas12a 强，而带有分裂 crRNA 的 dsDNA 激活 AsCas12a 的能力与 LbaCas12a 相似。利用这一特性，miRNA 可以被巧妙地设计为间隔区，突破 Cas12a 不能直接用于检测 RNA 的固有局限。这一创新为后续 SPACE 策略的开发和应用奠定了基础。

Taking advantage of the cis-cleavage activity of Cas12a, a novel SPACE strategy that could realize simultaneous, discriminative, and low-threshold detection of dual MS-related miRNAs was constructed (Scheme 1C). The SPACE strategy consisted of following five components: two activators (dsDNA-T and dsDNA-C) corresponding to two different miRNAs, inactive Cas12a, reporter FQ probe, and scaffold portion of crRNA. According to the sequences of miRNA-155 and miRNA-326, the activators were designed with fluorophores (TAMRA and Cy5) modified at the $3^{\prime }$$3^{\prime }$3^(')3^{\prime} end of ciscleavage site on the TS chain, respectively, and quenchers (BHQ2 and BHQ3) modified at the $5^{\prime }$$5^{\prime }$5^(')5^{\prime} end of cis-cleavage site on the NTS chain, respectively. In the absence of target miRNA-155 and miRNA-326, only the scaffold portion of crRNA was present, and Cas12a could not be activated. The
利用 Cas12a 的顺式切割活性，构建了一种新型的 SPACE 策略，该策略能够实现双 MS 相关 miRNA 的同时、区分性和低阈值检测（方案 1C）。SPACE 策略包括以下五个组成部分：两个对应于两种不同 miRNA 的激活剂（dsDNA-T 和 dsDNA-C）、非活性 Cas12a、报告 FQ 探针和 crRNA 的支架部分。根据 miRNA-155 和 miRNA-326 的序列，激活剂被设计为在 TS 链上顺式切割位点的 $3^{\prime }$$3^{\prime }$3^(')3^{\prime} 端分别修饰有荧光团（TAMRA 和 Cy5），在 NTS 链上顺式切割位点的 $5^{\prime }$$5^{\prime }$5^(')5^{\prime} 端分别修饰有猝灭剂（BHQ2 和 BHQ3）。在没有目标 miRNA-155 和 miRNA-326 的情况下，只有 crRNA 的支架部分存在，Cas12a 无法被激活。

Figure 1. Evaluating the feasibility of the SPACE strategy. (A) Agarose gel electrophoresis verification of the Cas12a activation under different reaction conditions. Lane 1 represents DNA markers. Fluorescence spectra obtained with different substrates for miRNAs analysis through channel (B) TAMRA, © Cy5, and (D) FAM, respectively.
Figure 1. 评估 SPACE 策略的可行性。(A) 不同反应条件下 Cas12a 活化的琼脂糖凝胶电泳验证。泳道 1 代表 DNA 标记物。通过通道(B) TAMRA、© Cy5 和(D) FAM 分别获得的不同底物的荧光光谱用于 miRNA 分析。
fluorescence of TAMRA and Cy5 were quenched since the fluorophores in the intact activator dsDNA were close to the quenchers to result in the fluorescence resonance energy transfer between fluorophores and quenchers. When the target miRNA-155 or miRNA-326 was present, they were regarded as a spacer of crRNA and could combine with the scaffold to form an integrated crRNA. After assembly with Cas12a, the complex could recognize PAM in dsDNA-T or dsDNA-C, and progressive dsDNA unwinding and RNA hybridization resulted in the formation of an R-loop. The formation of Rloop symbolized the successful activation of Cas12a, exerting the staggered cis-cleavage activity. PAM-distal cleavage products were released, and fluorophores were consequently away from the quenchers, thus recovering the fluorescence signal. As the dsDNA modified with a specific fluorophore exactly corresponded to its target miRNA, miRNA-155 and miRNA-326 selectively induced fluorescence recovery of the respective TAMRA or Cy5, thus realizing discriminative detection of different miRNAs within the Cas12a system. When both miRNA-155 and miRNA-326 were present, cleavage of dsDNA-T and dsDNA-C occurred, leading to the simultaneous signal recovery of TAMRA and Cy5. The specific cleavage process is shown in Figure S2. In addition to the differentiated signal outputs of TAMRA and Cy 5 through ciscleavage activity, the SPACE strategy also combined Cas12a’s trans-activity, which had a higher efficiency, ${}^{31}$${}^{31}$^(31){ }^{31} to obtain a faster response of FAM signal at a low threshold. Due to the nonselectivity of trans-cleavage, the presence of any target miRNA could lead to the recovery of FAM fluorescence, making it impossible to accurately distinguish between miRNA-155 and miRNA-326. However, the presence of either miRNA-155 or miRNA-326 could activate Cas12a to produce the FAM signal, which was independent of TAMRA and Cy5 and could be used to quantify the total concentration of miRNA-155 and
TAMRA 和 Cy5 的荧光被猝灭，因为完整的激活剂双链 DNA 中的荧光团靠近猝灭剂，导致荧光团与猝灭剂之间的荧光共振能量转移。当存在目标 miRNA-155 或 miRNA-326 时，它们被视为 crRNA 的间隔区，并能与支架结合形成完整的 crRNA。与 Cas12a 组装后，该复合物能够识别 dsDNA-T 或 dsDNA-C 中的 PAM，逐步的双链 DNA 解旋和 RNA 杂交导致 R 环的形成。R 环的形成象征着 Cas12a 的成功激活，发挥错开的顺式切割活性。PAM 远端的切割产物被释放，荧光团因此远离猝灭剂，从而恢复荧光信号。由于用特定荧光团修饰的双链 DNA 恰好对应其目标 miRNA，miRNA-155 和 miRNA-326 选择性地诱导各自 TAMRA 或 Cy5 的荧光恢复，从而实现在 Cas12a 系统内对不同 miRNA 的区分检测。 当 miRNA-155 和 miRNA-326 同时存在时，dsDNA-T 和 dsDNA-C 被切割，导致 TAMRA 和 Cy5 信号的同步恢复。具体的切割过程如图 S2 所示。除了通过顺式切割活性区分 TAMRA 和 Cy5 的信号输出外，SPACE 策略还结合了 Cas12a 的反式活性，其效率更高， ${}^{31}$${}^{31}$^(31){ }^{31} 以在低阈值下获得 FAM 信号的快速响应。由于反式切割的非选择性，任何目标 miRNA 的存在都会导致 FAM 荧光的恢复，使得无法准确区分 miRNA-155 和 miRNA-326。然而，无论是 miRNA-155 还是 miRNA-326 的存在，都能激活 Cas12a 产生 FAM 信号，该信号独立于 TAMRA 和 Cy5，可用于量化 miRNA-155 和
miRNA-326. Besides, the trans-cleavage efficiency of Cas12a was much higher than that of cis-cleavage, which itself gave a certain amplification effect to FAM signal, so the FAM signal was significantly recovered even under the ultra-low threshold where TAMRA and Cy 5 could not respond. In particular, when miRNA-155 and miRNA-326 existed at the same time, they could recognize their corresponding activators and activate Cas12a. Since these two miRNAs functioned in a similar manner, they could generate certain FAM signals, respectively, and then the signal intensity resulting from their combined action was superimposed, further reducing the detection threshold range and expanding the application prospect of this SPACE strategy. Overall, the integrated design of the SPACE strategy revolutionized a one-pot, one-step Cas12a-based detection process and successfully realized the simultaneous, discriminative, and low-threshold determination of dual-MS related miRNAs.
miRNA-326。此外，Cas12a 的跨切割效率远高于顺式切割，这本身就对 FAM 信号产生了一定的放大效果，因此即使在 TAMRA 和 Cy 5 无法响应的超低阈值下，FAM 信号也能显著恢复。特别是当 miRNA-155 和 miRNA-326 同时存在时，它们能够识别各自对应的激活剂并激活 Cas12a。由于这两种 miRNA 的作用方式相似，它们可以分别产生一定的 FAM 信号，然后它们联合作用产生的信号强度叠加，进一步降低了检测阈值范围，并扩展了 SPACE 策略的应用前景。总体而言，SPACE 策略的集成设计革新了一锅式、一步法的 Cas12a 检测过程，并成功实现了对双 MS 相关 miRNA 的同时、区分性和低阈值测定。

Proof-of-Concept. Considering that the activators used in the SPACE strategy were dsDNA, and there was no difference in the activation effects between AsCas12a and LbaCas12a (Figure S1), LbaCas12a was chosen, as it was more economical. To verify the feasibility of this SPACE sensing strategy for MS-related miRNAs, agarose gel electrophoresis was first performed. As shown in Figure 1A, dsDNA-T (lane 2) and dsDNA-C (lane 3 ) were introduced, serving as exquisite reference bands. Clearly, when dsDNA-T and dsDNA-C were mixed, brighter bands could be observed due to their comparable molecular weight (lane 4). Following the addition of Cas12a and scaffold, the band brightness remained unchanged, indicating the stable existence of dsDNA-T and dsDNA-C without signal leakage (lane 5). In the presence of either miRNA-155 (lane 6) or miRNA-326 (lane 7), shallow bands of dsDNA were observed, suggesting that Cas12a was successfully activated to exert its cis-cleavage potential. It is
概念验证。考虑到 SPACE 策略中使用的激活剂为双链 DNA，且 AsCas12a 和 LbaCas12a 在激活效果上没有差异（图 S1），因此选择了更为经济的 LbaCas12a。为了验证 SPACE 传感策略对 MS 相关 miRNA 的可行性，首先进行了琼脂糖凝胶电泳。如图 1A 所示，引入了 dsDNA-T（泳道 2）和 dsDNA-C（泳道 3），作为精细的参考条带。显然，当 dsDNA-T 和 dsDNA-C 混合时，由于它们的分子量相近，可以观察到更亮的条带（泳道 4）。在加入 Cas12a 和支架后，条带亮度保持不变，表明 dsDNA-T 和 dsDNA-C 稳定存在，没有信号泄漏（泳道 5）。在存在 miRNA-155（泳道 6）或 miRNA-326（泳道 7）的情况下，观察到浅色的 dsDNA 条带，表明 Cas12a 成功激活并发挥其顺式切割潜力。

Figure 2. Evaluating the signal accumulation effect of SPACE strategy on trans-cleavage. (A) Agarose gel electrophoresis verification of the Cas12a’s trans-cleavage under different reaction conditions. Lane 1 represents DNA markers. Fluorescence spectra obtained from different miRNAs at the concentrations of (B) 0.5 and © 2.5 nM .
Figure 2. 评估 SPACE 策略对转切割信号积累效应的影响。(A) 不同反应条件下 Cas12a 转切割的琼脂糖凝胶电泳验证。泳道 1 代表 DNA 标记物。在不同 miRNA 浓度下获得的荧光光谱：(B) 0.5 nM 和© 2.5 nM。
worth noting that the band in lane 7 corresponding to miRNA326 was slightly brighter, which could be attributed to two points. First, the number of complementary bases of miRNA326 and dsDNA-C ( 20 nt ) was less than that between dsDNAT and miRNA-155 (23 nt). Second, the melting temperature of dsDNA-C ( $78.2{}^{\circ }\mathrm{C}$$78.2{}^{\circ }\mathrm{C}$78.2^(@)C78.2{ }^{\circ} \mathrm{C} ) was higher than that of dsDNA-T ( ${72.6}^{\circ }\mathrm{C}$${72.6}^{\circ }\mathrm{C}$72.6^(@)C72.6^{\circ} \mathrm{C} ), so the stability of dsDNA-C was stronger, which was not conducive to Cas12a unwinding dsDNA to form a complete R-loop. Therefore, Cas12a/Scaffold/miRNA-326 had a weaker ability to bind dsDNA-C, resulting in a weaker cleavage capacity and brighter corresponding band in lane 7 compared to lane 6. When both miRNA-155 and miRNA-326 were present, the band of dsDNA in lane 8 became the shallowest, which was contributed to the stronger cleavage brought by dual activation.
值得注意的是，第 7 泳道中对应 miRNA326 的条带略微更亮，这可以归因于两点。首先，miRNA326 与 dsDNA-C（20 nt）的互补碱基数少于 dsDNAT 与 miRNA-155（23 nt）之间的互补碱基数。其次，dsDNA-C（ $78.2{}^{\circ }\mathrm{C}$$78.2{}^{\circ }\mathrm{C}$78.2^(@)C78.2{ }^{\circ} \mathrm{C} ）的熔解温度高于 dsDNA-T（ ${72.6}^{\circ }\mathrm{C}$${72.6}^{\circ }\mathrm{C}$72.6^(@)C72.6^{\circ} \mathrm{C} ），因此 dsDNA-C 的稳定性更强，不利于 Cas12a 解旋 dsDNA 以形成完整的 R 环。因此，Cas12a/Scaffold/miRNA-326 对 dsDNA-C 的结合能力较弱，导致其切割能力较弱，与第 6 泳道相比，第 7 泳道中对应的条带更亮。当 miRNA-155 和 miRNA-326 同时存在时，第 8 泳道中 dsDNA 的条带变得最浅，这是由双重激活带来的更强切割能力所致。

Fluorescence spectra were also employed to confirm the viability of this SPACE strategy. The fluorescence emission spectra of the mixed system showed that the fluorescence of FAM, TAMRA, and Cy5 did not interfere with each other (Figure S3). In addition, the fluorescence measurement indicated that no discernible fluorescence signal could be observed when any components of Cas12a, scaffold, dsDNA, and miRNAs were absent (Figure $1\mathrm{B}-\mathrm{D}$$1\mathrm{B}-\mathrm{D}$1B-D1 \mathrm{~B}-\mathrm{D}, line $\mathrm{a}-\mathrm{d}$$\mathrm{a}-\mathrm{d}$a-d\mathrm{a}-\mathrm{d} ). Conversely, in the presence of Cas12a, scaffold, dsDNA, and miRNAs, a distinct fluorescence signal was observed (line e), suggesting that miRNAs could serve as the spacer portion of crRNA to activate Cas12a, thereby exerting both cis- and transcleavage activity. These results clearly demonstrated that the SPACE sensing strategy could successfully distinguish MSassociated miRNA-155 and miRNA-326.
荧光光谱也被用于确认 SPACE 策略的可行性。混合系统的荧光发射光谱显示，FAM、TAMRA 和 Cy5 的荧光互不干扰（图 S3）。此外，荧光测量结果表明，当缺少 Cas12a、支架、dsDNA 和 miRNA 中的任何一种成分时，无法观察到明显的荧光信号（图 $1\mathrm{B}-\mathrm{D}$$1\mathrm{B}-\mathrm{D}$1B-D1 \mathrm{~B}-\mathrm{D} ，线 $\mathrm{a}-\mathrm{d}$$\mathrm{a}-\mathrm{d}$a-d\mathrm{a}-\mathrm{d} ）。相反，在存在 Cas12a、支架、dsDNA 和 miRNA 的情况下，可以观察到明显的荧光信号（线 e），这表明 miRNA 可以作为 crRNA 的间隔部分来激活 Cas12a，从而发挥顺式和反式切割活性。这些结果清楚地证明了 SPACE 传感策略能够成功区分 MS 相关的 miRNA-155 和 miRNA-326。

Furthermore, to validate the signal superposition of FAM achieved through the trans-cleavage of Cas12a caused by dual miRNAs, a 40 -nucleotide ssDNA (T40) was chosen as the substrate for electrophoretic analysis (Figure 2A). In a singletarget system, where either miRNA-155 (lane 4) or miRNA326 (lane 5) was present alone, a relatively shallower band indicative of partial cleavage of T40 was observed, and the band in lane 4 was shallower, which was also due to the stronger binding force between dsDNA-T and miRNA-155. However, when both miRNA-155 and miRNA-326 were present simultaneously, an almost complete absence of the T40 band was observed (lane 6), confirming the synergistic enhancement of Cas12a activation by dual miRNAs. Fluorescence spectra were also used for more accurate verification of the FAM signal superposition achieved by
此外，为了验证通过 Cas12a 在双 miRNA 诱导下的转切割作用所实现的 FAM 信号叠加，选择了一个 40 核苷酸的单链 DNA（T40）作为电泳分析的底物（图 2A）。在单目标系统中，当单独存在 miRNA-155（泳道 4）或 miRNA-326（泳道 5）时，观察到一条相对较浅的条带，表明 T40 部分被切割，且泳道 4 的条带更浅，这也是由于 dsDNA-T 与 miRNA-155 之间更强的结合力所致。然而，当 miRNA-155 和 miRNA-326 同时存在时，几乎完全看不到 T40 条带（泳道 6），证实了双 miRNA 对 Cas12a 激活的协同增强作用。荧光光谱也被用于更准确地验证通过双 miRNA 实现的 FAM 信号叠加。

Cas12a’s trans-cleavage induced by dual miRNAs. Within the low-threshold range ( 0.5 nM ), neither miRNA-155 nor miRNA-326 alone was capable of eliciting a notable recovery of the FAM fluorescence signal (Figure 2B). Nevertheless, upon combining these two miRNAs, a cumulative increase in FAM intensity was achieved, which was about the sum of the intensity caused by the single miRNA. In the high concentration range ( 2.5 nM ), a similar fluorescence accumulation of FAM intensity was also observed (Figure 2C). All the above results indicated that the FAM signal caused by double miRNAs was the superposition of a single miRNA. This was attributed to the fact that miRNA-155 and miRNA326 functioned in a similar manner, and each could generate a certain FAM signal, thereby obtaining the superimposed signal intensity and further reducing the detection threshold range for detecting total miRNAs. Then, with a further increase of the target miRNA, the superposition effect would be masked due to the limited amount of substrate FQ (Figure S4). Of course, the range of the superposition effect could be further expanded by increasing the amount of FQ and optimizing the concentration of other components. Besides, the FAM signal was caused by ultra-efficient trans-cleavage, which itself had a certain amplification effect, so it could appear earlier in the low threshold range. Overall, these results demonstrated the potential of the SPACE strategy in promoting the sensitivity and applicability of detection methods at low thresholds.
Cas12a 由双 miRNA 诱导的跨切割作用。在低阈值范围内（0.5 nM），单独的 miRNA-155 或 miRNA-326 均无法显著恢复 FAM 荧光信号（图 2B）。然而，当这两种 miRNA 结合时，FAM 强度呈现累积增加，大约是单一 miRNA 引起的强度的总和。在高浓度范围（2.5 nM）内，也观察到了类似的 FAM 荧光强度累积（图 2C）。以上所有结果表明，由双 miRNA 引起的 FAM 信号是单一 miRNA 信号的叠加。这归因于 miRNA-155 和 miRNA-326 以相似的方式发挥作用，每种都能产生一定的 FAM 信号，从而获得叠加的信号强度，并进一步降低检测总 miRNA 的阈值范围。随后，随着目标 miRNA 的进一步增加，由于底物 FQ 的量有限，叠加效应将被掩盖（图 S4）。 当然，通过增加 FQ 的量并优化其他组分的浓度，可以进一步扩大叠加效应的范围。此外，FAM 信号是由超高效转位切割引起的，其本身具有一定的放大效应，因此在低阈值范围内可以更早出现。总体而言，这些结果表明 SPACE 策略在提高低阈值检测方法的灵敏度和适用性方面具有潜力。

Optimization and Analytical Performance. The concentrations of Cas12a and the scaffold were critical for detection performance, so these two components were optimized (Figure 3). The fluorescence intensities of TAMRA and Cy5 reached the plateau at 80 nM for Cas12a and 30 nM for scaffold, respectively. The fluorescence signal stabilized with the further increase of concentration, indicating that the optimal reaction concentrations were obtained. Additionally, the concentration of dsDNA and reaction time were also optimized (Figures S5 and S6). At 100 and 150 nM , obtained dsDNA-T and dsDNA-C exhibited a lower background fluorescence effect, a better fluorescence recovery, and the best recovery ratio. The ideal reaction time was 2 h , because excessive reaction time could result in background interference.
优化与分析性能。Cas12a 和支架的浓度对检测性能至关重要，因此对这两个组分进行了优化（图 3）。TAMRA 和 Cy5 的荧光强度分别在 Cas12a 浓度为 80 nM 和支架浓度为 30 nM 时达到平台期。随着浓度的进一步增加，荧光信号趋于稳定，表明已获得最佳反应浓度。此外，还对 dsDNA 的浓度和反应时间进行了优化（图 S5 和 S6）。在 100 和 150 nM 时，获得的 dsDNA-T 和 dsDNA-C 表现出较低的背景荧光效应、更好的荧光恢复以及最佳的恢复比例。理想的反应时间为 2 小时，因为过长的反应时间可能导致背景干扰。
Under optimal experimental conditions, the sensitivity of the SPACE strategy was investigated. The fluorescence intensity of TAMRA, Cy5, and FAM increased substantially when the miRNAs concentration increased (Figure 4A,B). As shown in
在最佳实验条件下，研究了 SPACE 策略的灵敏度。当 miRNA 浓度增加时，TAMRA、Cy5 和 FAM 的荧光强度显著增强（图 4A,B）。如图所示，

Figure 3. Optimization of Cas12a reporting reaction. Optimization of the concentration of (A) Cas12a and (B) scaffold in the sensing system in the presence of 30 nM miRNA-155 and 30 nM miRNA326. The radar chart extended several rays from the center point, with each ray representing the different reactant concentrations and different fluorescence intensities by the position of the points on the rays relative to the circumference. Data are expressed as mean $\pm$$\pm$+-\pm SD ( $n=3$$n=3$n=3n=3 ).
图 3. Cas12a 报告反应的优化。在存在 30 nM miRNA-155 和 30 nM miRNA326 的传感系统中，优化（A）Cas12a 和（B）支架的浓度。雷达图从中心点延伸出若干射线，每条射线代表不同的反应物浓度，射线上的点相对于圆周的位置表示不同的荧光强度。数据以均值 $\pm$$\pm$+-\pm 标准差（ $n=3$$n=3$n=3n=3 ）表示。

Figure 4C, the fluorescence intensity of TAMRA exhibited a linear correlation with the miRNA-155 concentration ranging from 0.07 to 30 nM . The correlation equation was $F=$$F=$F=F= $38.031c_{\text{miRNA-155}}+259(R^2=0.9936)$$38.031c_{\text{miRNA-155 }}+259\left(R^2=0.9936\right)$38.031c_("miRNA-155 ")+259(R^(2)=0.9936)38.031 c_{\text {miRNA-155 }}+259\left(R^{2}=0.9936\right), and the limit of detection (LOD) was as low as 21 pM according to the $3\sigma$$3\sigma$3sigma3 \sigma rule. Furthermore, a linear relationship ( $R^2=0.9961$$R^2=0.9961$R^(2)=0.9961R^{2}=0.9961 ) was obtained between the fluorescence intensity of Cy 5 and the miRNA- 326 concentration from 0.07 to 50 nM . The linear regression equation could be derived as $F=28.646c_{\text{miRNA-326}}+$$F=28.646c_{\text{miRNA-326 }}+$F=28.646c_("miRNA-326 ")+F=28.646 c_{\text {miRNA-326 }}+ 391 with a LOD of 25 pM (Figure 4D). Moreover, since either miRNA-155 or miRNA-326 could trigger the trans-cleavage of Cas12a to degrade FQ, FAM represented the concentration of these two miRNAs mixtures, expressed as “miRNA-155&326”. The fluorescence intensity displayed a linear dependence upon the total miRNA concentration ranging from 0.01 to 1 nM (Figure 4E), with the linear equation being $F=$$F=$F=F= $1101.2c_{\text{miRNA-}155\mathrm{\&}326}+107(R^2=0.9976)$$1101.2c_{\text{miRNA- }155\mathrm{\&}326}+107\left(R^2=0.9976\right)$1101.2c_("miRNA- "155&326)+107(R^(2)=0.9976)1101.2 c_{\text {miRNA- } 155 \& 326}+107\left(R^{2}=0.9976\right). The LOD was
图 4C 中，TAMRA 的荧光强度与 miRNA-155 浓度在 0.07 至 30 nM 范围内呈线性相关。相关方程为 $F=$$F=$F=F= $38.031c_{\text{miRNA-155}}+259(R^2=0.9936)$$38.031c_{\text{miRNA-155 }}+259\left(R^2=0.9936\right)$38.031c_("miRNA-155 ")+259(R^(2)=0.9936)38.031 c_{\text {miRNA-155 }}+259\left(R^{2}=0.9936\right) ，根据 $3\sigma$$3\sigma$3sigma3 \sigma 规则，检测限（LOD）低至 21 pM。此外，Cy 5 的荧光强度与 miRNA-326 浓度在 0.07 至 50 nM 范围内也呈现出线性关系（ $R^2=0.9961$$R^2=0.9961$R^(2)=0.9961R^{2}=0.9961 ）。线性回归方程可表示为 $F=28.646c_{\text{miRNA-326}}+$$F=28.646c_{\text{miRNA-326 }}+$F=28.646c_("miRNA-326 ")+F=28.646 c_{\text {miRNA-326 }}+ 391，检测限为 25 pM（图 4D）。此外，由于 miRNA-155 或 miRNA-326 均能触发 Cas12a 的跨切割以降解 FQ，FAM 代表了这两种 miRNA 混合物的浓度，表示为“miRNA-155&326”。荧光强度显示出对总 miRNA 浓度在 0.01 至 1 nM 范围内的线性依赖性（图 4E），线性方程为 $F=$$F=$F=F= $1101.2c_{\text{miRNA-}155\mathrm{\&}326}+107(R^2=0.9976)$$1101.2c_{\text{miRNA- }155\mathrm{\&}326}+107\left(R^2=0.9976\right)$1101.2c_("miRNA- "155&326)+107(R^(2)=0.9976)1101.2 c_{\text {miRNA- } 155 \& 326}+107\left(R^{2}=0.9976\right) 。检测限为
measured to be 3.6 pM . Evidently, compared to TAMRA and Cy5, the FAM signal associated with trans-cleavage could respond at a lower detection threshold and recover more rapidly, which was concretely manifested by a lower linear range and about an order of magnitude of LOD enhancement. This further illustrated the pivotal advantages of the dualmiRNA coresponsive SPACE strategy in enhancing sensitivity within the realm of low-threshold detection. Meanwhile, the analytical performance of the single-target detection system was also explored (Figure S7). The LOD of TAMRA and FAM channels for detecting single miRNA-155 was 21 and 4.6 pM , respectively. The LOD of Cy5 and FAM channels for detecting single miRNA- 326 was 30 and 12.4 pM , respectively. These results showed that the linearity, linear range, and LOD obtained by cis-cleavage in the single-target detection system were similar to those obtained in the multi-target detection system, indicating that the TAMRA and Cy 5 signals generated by the cis-cleavage of miRNA-155 and miRNA-326 would not interfere with each other. In addition, due to the ultra-high efficiency of trans-cleavage, the FAM signal could respond at a lower detection threshold and recover more rapidly than TAMRA or Cy5, which was concretely manifested by a lower linear range and better LODs. It was worth noting that the FAM signal in the multi-target detection system was the superposition of dual miRNAs, so its LOD was better than single-target detection system. Further, even if miRNA-155 and miRNA-326 were present in different proportions, only the corresponding fluorescence signals of TAMRA and Cy5 achieved through different channels could be detected, and the intensities were comparable to those of the single-target detection system (Figure S8). The performance of the constructed SPACE strategy was also compared in terms of strategy capability with some of the previous reports on simultaneous detection of miRNAs (Table S4). The proposed SPACE strategy demonstrated outstanding analytical performance for sensitive detection of multiple miRNAs. Meanwhile, the previous Cas12a-based simultaneous detection systems were inherently limited in distinguishing multiple targets
测量值为 3.6 皮摩尔。显然，与 TAMRA 和 Cy5 相比，与反式切割相关的 FAM 信号能够在更低的检测阈值下响应，并且恢复得更快，这具体表现为更低的线性范围和大约一个数量级的 LOD 提升。这进一步说明了双 miRNA 共响应 SPACE 策略在低阈值检测领域内提高灵敏度的关键优势。同时，也探讨了单目标检测系统的分析性能（图 S7）。TAMRA 和 FAM 通道检测单 miRNA-155 的 LOD 分别为 21 和 4.6 皮摩尔。Cy5 和 FAM 通道检测单 miRNA-326 的 LOD 分别为 30 和 12.4 皮摩尔。这些结果表明，在单目标检测系统中通过顺式切割获得的线性、线性范围和 LOD 与多目标检测系统中获得的结果相似，表明由 miRNA-155 和 miRNA-326 的顺式切割产生的 TAMRA 和 Cy5 信号不会相互干扰。 此外，由于跨切割的超高效率，FAM 信号能够在更低的检测阈值下响应，并且比 TAMRA 或 Cy5 恢复得更快，具体表现为更低的线性范围和更好的 LOD。值得注意的是，多目标检测系统中的 FAM 信号是双 miRNA 的叠加，因此其 LOD 优于单目标检测系统。进一步地，即使 miRNA-155 和 miRNA-326 以不同比例存在，也只能通过不同通道检测到相应的 TAMRA 和 Cy5 荧光信号，其强度与单目标检测系统相当（图 S8）。构建的 SPACE 策略在策略能力方面也与一些先前关于 miRNA 同时检测的报告进行了比较（表 S4）。提出的 SPACE 策略展示了对于多 miRNA 敏感检测的卓越分析性能。同时，先前的基于 Cas12a 的同时检测系统在区分多个目标方面存在固有的局限性。

Figure 4. Analytical performance of the SPACE strategy. (A) Fluorescence spectra of TAMRA/Cy5/FAM with increasing concentrations of miRNA-155, miRNA-326, and miRNA-155&326. The concentrations of miRNA-155 and miRNA-326 (proximal to distal) were all $0.07,0.1,0.2$$0.07,0.1,0.2$0.07,0.1,0.20.07,0.1,0.2, $0.5,1.0,5.0,10,20,30,50$$0.5,1.0,5.0,10,20,30,50$0.5,1.0,5.0,10,20,30,500.5,1.0,5.0,10,20,30,50, and 80 nM . The concentrations of miRNA-155&326 (proximal to distal) were all $0.01,0.07,0.1,0.2,0.5,1.0,5.0,10$$0.01,0.07,0.1,0.2,0.5,1.0,5.0,10$0.01,0.07,0.1,0.2,0.5,1.0,5.0,100.01,0.07,0.1,0.2,0.5,1.0,5.0,10, 20, 30, 50, and 80 nM . (B) Relationship between the fluorescent intensities of TAMRA/Cy5/FAM and the concentrations of miRNA-155, miRNA-326, and miRNA-155&326. (C-E) Linear relationship of the fluorescence intensity with the concentration of miRNA-155, miRNA-326, and miRNA-155&326. Data are expressed as mean $\pm$$\pm$+-\pm SD $(n=3)$$(n=3)$(n=3)(n=3).
图 4. SPACE 策略的分析性能。（A）随着 miRNA-155、miRNA-326 和 miRNA-155&326 浓度增加的 TAMRA/Cy5/FAM 荧光光谱。miRNA-155 和 miRNA-326 的浓度（从近端到远端）均为 $0.07,0.1,0.2$$0.07,0.1,0.2$0.07,0.1,0.20.07,0.1,0.2 、 $0.5,1.0,5.0,10,20,30,50$$0.5,1.0,5.0,10,20,30,50$0.5,1.0,5.0,10,20,30,500.5,1.0,5.0,10,20,30,50 和 80 nM。miRNA-155&326 的浓度（从近端到远端）均为 $0.01,0.07,0.1,0.2,0.5,1.0,5.0,10$$0.01,0.07,0.1,0.2,0.5,1.0,5.0,10$0.01,0.07,0.1,0.2,0.5,1.0,5.0,100.01,0.07,0.1,0.2,0.5,1.0,5.0,10 、20、30、50 和 80 nM。（B）TAMRA/Cy5/FAM 荧光强度与 miRNA-155、miRNA-326 和 miRNA-155&326 浓度之间的关系。（C-E）荧光强度与 miRNA-155、miRNA-326 和 miRNA-155&326 浓度的线性关系。数据表示为均值 $\pm$$\pm$+-\pm 标准差 $(n=3)$$(n=3)$(n=3)(n=3) 。

Figure 5. Specificity evaluation of SPACE strategy. (A) Different miRNA sequences. The fluorescence spectra of the Cas12a system through channel (B) TAMRA, © Cy5, and (D) FAM. (E) Selectivity of SPACE strategy in response to let-7a, miRNA-21, miRNA-30e, miRNA-133a, and miRNA-499. (F) Heat map of multiplex detection of miRNAs. The $x$$x$xx-axis represents different detection systems, and the $y$$y$yy-axis represents different miRNAs. The amount of interference input was five times higher than that of the targets. Data are expressed as mean $\pm$$\pm$+-\pm SD ( $n=3$$n=3$n=3n=3 ).
图 5. SPACE 策略的特异性评估。（A）不同的 miRNA 序列。通过通道（B）TAMRA、© Cy5 和（D）FAM 的 Cas12a 系统的荧光光谱。（E）SPACE 策略对 let-7a、miRNA-21、miRNA-30e、miRNA-133a 和 miRNA-499 的选择性。（F）miRNA 多重检测的热图。 $x$$x$xx 轴代表不同的检测系统， $y$$y$yy 轴代表不同的 miRNA。干扰输入的量是目标物的五倍。数据表示为均值 $\pm$$\pm$+-\pm 标准差（ $n=3$$n=3$n=3n=3 ）。
within one pot solely by relying on Cas12a (Table S5). Only the SPACE strategy, which comprehensively utilized the cis-/ trans-cleavage activity of Cas12a, pioneered in realizing multisignal output in one pot with a simultaneous and discriminative manner.
在一个反应体系中仅依靠 Cas12a 即可实现（表 S5）。只有 SPACE 策略，全面利用了 Cas12a 的顺式/反式切割活性，率先实现了在一个反应体系中以同步且区分的方式输出多重信号。

To further scrutinize the specificity and selectivity of the SPACE strategy, we selected a set of nonspecific miRNAs to assess whether dsDNA-T and dsDNA-C would exhibit response beyond miRNA-155 and miRNA-326 (Figure 5A). The nonspecific miRNA group utilized five times concentrations ( 150 nM ) of miRNA-21, let-7a, miRNA-30e, miRNA133a, and miRNA-499, whereas the positive experimental group consisted of miRNA-155 and miRNA-326 ( 30 nM ). As illustrated in Figure 5B-E, these miRNAs with similar base sequences could not induce a fluorescence response, thus verifying the precision of the SPACE strategy. Furthermore, we conducted a comparative analysis between the SPACE multitarget detection strategy and the single-target detection systems specifically targeted at one miRNA, denoted as dsDNA-T (for miRNA-155) and dsDNA-T (for miRNA326), respectively (Figure 5F). In both single-target and SPACE systems, non-specific miRNAs were unable to induce any fluorescence signal. However, the presence of target miRNA-155 or miRNA-326 could trigger the cis-cleavage activity of Cas12a in their respective single-target detection system, which was visually manifested by the brightening of the TAMRA or Cy5 fluorophores. This initial event subsequently initiated the trans-cleavage, leading to the illumination of FAM.
为了进一步审视 SPACE 策略的特异性和选择性，我们选择了一组非特异性 miRNA 来评估 dsDNA-T 和 dsDNA-C 是否会在 miRNA-155 和 miRNA-326 之外表现出响应（图 5A）。非特异性 miRNA 组使用了五倍浓度（150 nM）的 miRNA-21、let-7a、miRNA-30e、miRNA-133a 和 miRNA-499，而阳性实验组则由 miRNA-155 和 miRNA-326（30 nM）组成。如图 5B-E 所示，这些具有相似碱基序列的 miRNA 无法诱导荧光响应，从而验证了 SPACE 策略的精确性。此外，我们对 SPACE 多目标检测策略与针对单一 miRNA 的单目标检测系统进行了比较分析，后者分别标记为 dsDNA-T（针对 miRNA-155）和 dsDNA-T（针对 miRNA-326）（图 5F）。在单目标和 SPACE 系统中，非特异性 miRNA 均无法诱导任何荧光信号。 然而，目标 miRNA-155 或 miRNA-326 的存在可以触发其各自单目标检测系统中 Cas12a 的顺式切割活性，这一现象通过 TAMRA 或 Cy5 荧光团的亮度增强得以直观显现。这一初始事件随后引发了反式切割，导致 FAM 的点亮。

In the SPACE multi-target detection system, the simultaneous introduction of miRNA-155 and miRNA-326 resulted in the fluorescence recovery of all three fluorophores (TAMRA, Cy5, and FAM), demonstrating the capability of SPACE to simultaneously respond to multiple analytes. Meanwhile, the selectivity of the SPACE system was also explored at a concentration of 2.5 nM where synergistic effects could be detected (Figure S9). Under this condition, although the signal response of TAMRA and Cy5 was weak, their intensities were still enhanced compared with non-specific miRNAs, indicating that the presence of nonspecific miRNAs would not affect simultaneous detection. Furthermore, the ability of the SPACE system to recognize base mismatch and resist interference under complex conditions was also fully verified (Figures S10 and S11). The intuitive results showed that the SPACE strategy could distinguish different miRNAs with high specificity and had strong applicability for simultaneous multi-target analysis.
在 SPACE 多目标检测系统中，同时引入 miRNA-155 和 miRNA-326 导致所有三种荧光团（TAMRA、Cy5 和 FAM）的荧光恢复，证明了 SPACE 能够同时响应多种分析物。同时，在 2.5 nM 的浓度下，也探讨了 SPACE 系统的选择性，在此浓度下可以检测到协同效应（图 S9）。在此条件下，尽管 TAMRA 和 Cy5 的信号响应较弱，但它们的强度仍比非特异性 miRNA 有所增强，表明非特异性 miRNA 的存在不会影响同时检测。此外，SPACE 系统在复杂条件下识别碱基错配和抗干扰的能力也得到了充分验证（图 S10 和 S11）。直观的结果显示，SPACE 策略能够以高特异性区分不同的 miRNA，并且对同时多目标分析具有强大的适用性。

Detection of miRNA-155 and miRNA-326 in Real Biological Samples. From theoretical concepts to practical applications, the reliability and applicability of the SPACE strategy were evaluated to further explore the feasibility of this system in complex blood samples. First, the normal operation of the SPACE strategy in the physiological environment was investigated. The blood samples of healthy individuals were diluted by $10\mathrm{\%}$$10\mathrm{\%}$10%10 \% and spiked with different concentrations of miRNA-155 and miRNA-326. The results of human blood sample analysis are illustrated in Table S6. The recoveries of
在真实生物样本中检测 miRNA-155 和 miRNA-326。从理论概念到实际应用，评估了 SPACE 策略的可靠性和适用性，以进一步探索该系统在复杂血液样本中的可行性。首先，研究了 SPACE 策略在生理环境中的正常运作。将健康个体的血液样本用 $10\mathrm{\%}$$10\mathrm{\%}$10%10 \% 稀释，并加入不同浓度的 miRNA-155 和 miRNA-326。人血液样本分析的结果如表 S6 所示。回收率分别为
miRNA-155 ranged from 96.4 to $109\mathrm{\%}$$109\mathrm{\%}$109%109 \%, and the relative standard deviation (RSD) was less than $5.2\mathrm{\%}$$5.2\mathrm{\%}$5.2%5.2 \%. The recoveries of miRNA-326 were from 94.5 to 108% with an RSD less than $9.5\mathrm{\%}$$9.5\mathrm{\%}$9.5%9.5 \%, indicating the reliability of the SPACE strategy in a complex environment. Subsequently, due to the low miRNAs level in human blood, endogenous miRNAs extracted from blood samples were analyzed through the FAM channel. The results of SPACE assay showed that the relative expression levels of miRNA-155 and miRNA-326 were higher in blood collected from MS patients than in healthy individuals, which were consistent with the traditional stem-loop RT-qPCR method (Figure 6A). Meanwhile, the signal changes between
miRNA-155 的范围在 96.4 到 $109\mathrm{\%}$$109\mathrm{\%}$109%109 \% 之间，相对标准偏差（RSD）小于 $5.2\mathrm{\%}$$5.2\mathrm{\%}$5.2%5.2 \% 。miRNA-326 的回收率在 94.5 到 108%之间，RSD 小于 $9.5\mathrm{\%}$$9.5\mathrm{\%}$9.5%9.5 \% ，这表明 SPACE 策略在复杂环境中的可靠性。随后，由于人血液中 miRNAs 水平较低，通过 FAM 通道分析了从血液样本中提取的内源性 miRNAs。SPACE 检测结果显示，MS 患者血液中 miRNA-155 和 miRNA-326 的相对表达水平高于健康个体，这与传统的茎环 RT-qPCR 方法一致（图 6A）。同时，

Figure 6. Analyzing the performance of the SPACE strategy for miRNA detection in blood samples. (A) Comparison of the detection results of SPACE and RT-qPCR for the relative expression of miRNA-155 and miRNA-326 in blood samples from healthy individuals and MS patients. (B) Scattered dot plot of significant expression difference of miRNA-155 and miRNA-326 in MS patients compared to the healthy individuals. Data are expressed as mean $\pm$$\pm$+-\pm SD ( $n=3$$n=3$n=3n=3 ). Statistical significance is calculated via one-way ANOVA ( $\ast \ast p<0.01$$\ast \ast p<0.01$****p < 0.01* * p<0.01 ).
图 6. 分析 SPACE 策略在血液样本中 miRNA 检测的性能。（A）比较 SPACE 和 RT-qPCR 对健康个体和 MS 患者血液样本中 miRNA-155 和 miRNA-326 相对表达水平的检测结果。（B）MS 患者与健康个体相比，miRNA-155 和 miRNA-326 显著表达差异的散点图。数据表示为均值 $\pm$$\pm$+-\pm 标准差（ $n=3$$n=3$n=3n=3 ）。统计学显著性通过单因素方差分析（ $\ast \ast p<0.01$$\ast \ast p<0.01$****p < 0.01* * p<0.01 ）计算。
healthy individuals and MS patients were statistically significant ( $p<0.01$$p<0.01$p < 0.01p<0.01 ) (Figure 6B). These findings demonstrate the strong potential for practical application of this SPACE approach in clinical sample analysis.
健康个体和 MS 患者的差异具有统计学意义（ $p<0.01$$p<0.01$p < 0.01p<0.01 ）（图 6B）。这些发现表明，SPACE 方法在临床样本分析的实际应用中具有巨大潜力。

In summary, we established an innovative SPACE strategy based on Cas12a for simultaneous, discriminative, and lowthreshold quantification of dual-MS related miRNAs. First of all, taking advantage of the property that Cas12a could be activated by split crRNA, miRNA-155 and miRNA-326 were designed as the spacers for crRNA and could combine with the scaffold to form integrated crRNAs. These crRNA recognized the activators to activate Cas12a, thus breaking through the constraints of Cas12a’s inability to directly detect RNA. Besides, the SPACE strategy utilized the Cas12a system with staggered cis-cleavage activity and the dsDNA probe that deftly integrated activator and reporter to achieve simultaneous detection and distinct signal output of miRNA-155 and miRNA-326. Furthermore, through comprehensively capitalizing on the nuclease activity of Cas12a, the sensitivity of the SPACE assay at low thresholds could be further improved by synchronously utilizing the trans-cleavage with self-amplifying effects, and blood samples from healthy individuals and MS patients were successfully distinguished. In combination with the aforementioned designs, this work represented a first instance of simultaneous, discriminative, and low-threshold detection of dual MS-related miRNAs in one pot and one step based on Cas12a, which would be a significant milestone for the further development of Cas12a. Last but not least, the sequence of the dsDNA probe could be arbitrarily altered according to the target miRNA, which facilitated the
总之，我们建立了一种基于 Cas12a 的创新 SPACE 策略，用于同时、区分性和低阈值的定量双 MS 相关 miRNA。首先，利用 Cas12a 可以被分裂 crRNA 激活的特性，将 miRNA-155 和 miRNA-326 设计为 crRNA 的间隔区，并能与支架结合形成完整的 crRNA。这些 crRNA 识别激活剂以激活 Cas12a，从而突破了 Cas12a 无法直接检测 RNA 的局限。此外，SPACE 策略利用具有错位顺式切割活性的 Cas12a 系统以及巧妙整合激活剂和报告者的 dsDNA 探针，实现了 miRNA-155 和 miRNA-326 的同时检测和明显信号输出。进一步地，通过全面利用 Cas12a 的核酸酶活性，通过同步利用具有自放大效应的反式切割，可以进一步提高 SPACE 检测在低阈值下的灵敏度，并成功区分了健康个体和 MS 患者的血液样本。 结合上述设计，本研究首次实现了基于 Cas12a 的单管单步同时、区分性和低阈值检测两种 MS 相关 miRNA，这将是 Cas12a 进一步发展的重要里程碑。最后但同样重要的是，dsDNA 探针的序列可以根据目标 miRNA 任意改变，这有助于
construction of a universal detection platform. We anticipate that this multifunctional SPACE platform could be extended to the development of intracellular multiplexing detection systems, which hold great potential in pathological research and clinical diagnosis.
构建一个通用检测平台。我们预计这种多功能 SPACE 平台可以扩展到细胞内多重检测系统的开发，这在病理研究和临床诊断中具有巨大潜力。

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acs.analchem.4c05388.
支持信息可免费获取，网址为 https://pubs.acs.org/doi/10.1021/acs.analchem.4c05388。

Additional methods, sequences for DNA and RNA strands, optimization assay results, detection antiinterference, and real sample analysis (PDF)
附加方法、DNA 和 RNA 链序列、优化实验结果、检测抗干扰性及真实样本分析（PDF）

Qin Hu - School of Pharmacy, Nanjing Medical University, Nanjing, Jiangsu 211166, China; © orcid.org/0000-0002-4077-8760; Email: huqin@njmu.edu.cn
秦虎 - 南京医科大学药学院，江苏省南京市 211166，中国；© orcid.org/0000-0002-4077-8760；电子邮箱：huqin@njmu.edu.cn
Zheng Zhao - Clinical Translational Research Center of Aggregation-Induced Emission, School of Medicine, The Second Affiliated Hospital, School of Science and Engineering, Shenzhen Institute of Aggregate Science and Technology, The Chinese University of Hong Kong, Shenzhen (CUHKShenzhen), Shenzhen, Guangdong 518172, China; © orcid.org/0000-0002-5536-0439; Email: zhaozheng@ cuhk.edu.cn
郑赵 - 聚集诱导发光临床转化研究中心，医学院，香港中文大学（深圳）第二附属医院，科学与工程学院，深圳聚集科学技术研究院，香港中文大学（深圳），广东深圳 518172，中国；© orcid.org/0000-0002-5536-0439；邮箱：zhaozheng@ cuhk.edu.cn
Ben Zhong Tang - Clinical Translational Research Center of Aggregation-Induced Emission, School of Medicine, The Second Affiliated Hospital, School of Science and Engineering, Shenzhen Institute of Aggregate Science and Technology, The Chinese University of Hong Kong, Shenzhen (CUHKShenzhen), Shenzhen, Guangdong 518172, China; © orcid.org/0000-0002-0293-964X; Email: tangbenz@ cuhk.edu.cn
Ben Zhong Tang - 聚集诱导发光临床转化研究中心，医学院，第二附属医院，科学与工程学院，深圳聚集科学技术研究院，香港中文大学（深圳），广东省深圳市 518172，中国；© orcid.org/0000-0002-0293-964X；邮箱：tangbenz@ cuhk.edu.cn
Yao Cen - School of Pharmacy, Nanjing Medical University, Nanjing, Jiangsu 211166, China; Clinical Medical Laboratory Center, Department of Central Laboratory, The Affiliated Taizhou People’s Hospital of Nanjing Medical University, Taizhou, Jiangsu 225300, China; © orcid.org/ 0000-0002-7829-979X; Email: yaocen@njmu.edu.cn
Yao Cen - 南京医科大学药学院，江苏省南京市 211166；南京医科大学附属泰州人民医院临床医学检验中心，中心实验室，江苏省泰州市 225300；© orcid.org/ 0000-0002-7829-979X；邮箱：yaocen@njmu.edu.cn

Xinyi Xia - School of Pharmacy, Nanjing Medical University, Nanjing, Jiangsu 211166, China
夏新怡 - 南京医科大学药学院，江苏省南京市 211166
Zhigang Liang - School of Pharmacy, Nanjing Medical University, Nanjing, Jiangsu 211166, China
梁志刚 - 南京医科大学药学院，江苏省南京市 211166
Guanhong Xu - School of Pharmacy, Nanjing Medical University, Nanjing, Jiangsu 211166, China; Northern Jiangsu Institute of Clinical Medicine, The Affiliated Huaian No. 1 People’s Hospital of Nanjing Medical University, Huaian, Jiangsu 223300, China
徐冠宏 - 南京医科大学药学院，江苏省南京市 211166；南京医科大学附属淮安第一人民医院，江苏省淮安市 223300
Fangdi Wei - School of Pharmacy, Nanjing Medical University, Nanjing, Jiangsu 211166, China
Fangdi Wei - 南京医科大学药学院，江苏省南京市 211166
Jing Yang - School of Pharmacy, Nanjing Medical University, Nanjing, Jiangsu 211166, China
Jing Yang - 南京医科大学药学院，江苏省南京市 211166
Xiaolei Zhu - Department of Neurology, Drum Tower Hospital, Nanjing University, Nanjing, Jiangsu 210008, China
Xiaolei Zhu - 南京大学鼓楼医院神经内科，江苏省南京市 210008，中国
Chenglin Zhou - Clinical Medical Laboratory Center, Department of Central Laboratory, The Affiliated Taizhou
Chenglin Zhou - 临床医学检验中心，中心实验室 department，泰州附属医院

People’s Hospital of Nanjing Medical University, Taizhou, Jiangsu 225300, China
南京医科大学附属泰州人民医院，江苏泰州 225300，中国
Jun Ye - Clinical Medical Laboratory Center, Department of Central Laboratory, The Affiliated Taizhou People’s Hospital of Nanjing Medical University, Taizhou, Jiangsu 225300, China
叶俊 - 临床医学检验中心，中心实验室，南京医科大学附属泰州人民医院，江苏泰州 225300，中国

Complete contact information is available at:
完整联系信息可在以下网址获取：
https://pubs.acs.org/10.1021/acs.analchem.4c05388

Y.C., Q.H., Z.Z., and B.Z.T. conceived and supervised the project and provided financial support. X.X. performed the experiments and contributed the data analysis. Z. L., G.X., F.W., J.Y., X.Z., C.Z., and J.Y. participated the experiments and discussed the results. X.X. wrote the manuscript and Y.C., Q.H., Z.Z., and B.Z.T. revised the manuscript. All authors reviewed and approved the manuscript.
Y.C.、Q.H.、Z.Z.和 B.Z.T.构思并监督了该项目，并提供财务支持。X.X.进行了实验并贡献了数据分析。Z.L.、G.X.、F.W.、J.Y.、X.Z.、C.Z.和 J.Y.参与了实验并讨论了结果。X.X.撰写了手稿，Y.C.、Q.H.、Z.Z.和 B.Z.T.修订了手稿。所有作者审阅并批准了手稿。

The authors declare no competing financial interest.
作者声明无利益冲突。

This work was supported by the National Natural Science Foundation of China (nos. 81973283, 61775099, and 21705080), Natural Science Foundation of Jiangsu Province (nos. BK20221304, BK20171487, and BK20171043), “Blue Project” Foundation of the Higher Education Institutions of Jiangsu Province, Scientific Research Project of Jiangsu Provincial Health Commission (Z2021067), and College Student Innovation and Entrepreneurship Training Program of Jiangsu Province (no. 202310312003Z).
本研究得到了中国国家自然科学基金（项目编号：81973283、61775099 和 21705080）、江苏省自然科学基金（项目编号：BK20221304、BK20171487 和 BK20171043）、江苏省高等教育机构“蓝项目”基金、江苏省卫生健康委员会科研项目（Z2021067）以及江苏省大学生创新创业训练计划项目（项目编号：202310312003Z）的资助。

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